iron-containing nanoparticles for the treatment of
TRANSCRIPT
University of New MexicoUNM Digital Repository
Nanoscience and Microsystems ETDs Engineering ETDs
Spring 4-15-2019
Iron-containing Nanoparticles for the Treatment ofChrionic Biofilm Infections in Cystic FibrosisLeisha M A MartinUniversity of New Mexico - Main Campus
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Recommended CitationMartin Leisha M A Iron-containing Nanoparticles for the Treatment of Chrionic Biofilm Infections in Cystic Fibrosis (2019)httpsdigitalrepositoryunmedunsms_etds52
i
Leisha Marie Martin Candidate
Nanoscience amp Microsystems Engineering
Department
This dissertation is approved and it is acceptable in quality and form for publication
Approved by the Dissertation Committee
Marek Osiński PhD Chairperson
Terefe Habteyes PhD
Erin Milligan PhD
Pavan Muttil PhD
ii
IRON-CONTAINING
NANOPARTICLES FOR THE TREATMENT OF
CHRONIC BACTERIAL BIOFILM INFECTIONS
IN CYSTIC FIBROSIS
by
LEISHA MARIE MARTIN
BS Biology University of New Mexico 2010
MS Nanoscience amp Microsystems University of New Mexico 2012
DISSERTATION
Submitted in Partial Fulfillment of the
Requirements for the Degree of
Doctor of Philosophy
Nanoscience amp Microsystems Engineering
The University of New Mexico
Albuquerque New Mexico
May 2019
iii
DEDICATION
To my children Jonathan and Isadora who have gone without many things so that this
project could be completed
사랑해
I love you very much
iv
ACKNOWLEDGEMENTS
Foremost I would like to thank my children Jonathan and Isadora for sacrificing after
school activities for hours in the lab thus solidifying their unwanted however thorough
advanced knowledge of materials science I want to also acknowledge the children I have
had or otherwise acquired between the time that this work was done and the time that this
paper was completed Tobias Hadassah Hannah and Joshua I would like to thank my
parents Robert and Anita Armijo for their continual support and of course their help with
the children I would also like to thank my advisor Dr Marek Osiński first of all for his
mentorship longsuffering and dedication to my work and also for teaching me what
veritable patience is Dr Maggie Werner-Washbourne for her above and beyond tireless
support lessons in truth and life Without her there is no way this work could have been
brought to completion She is truly the encompassment of a wise woman I kindly thank
Dr Sang Han for his mentorship and taking the time to personally review this dissertation
and keep me on track Another person who has contributed significantly to the completion
of this work Ms Linda Stewart who I sincerely thank for her hours of dedication and
overall generous contribution of personal time and effort committed to this project I thank
Dr Nathan Withers for sharing his wisdom and mentorship regarding everything science
Dr John Plumley for his assistance in mentoring students Dr Antonio Rivera and
Nathaniel Cook for their contribution in providing outstanding electron microscope
images Thank you to Dr Hugh Smyth for his mentorship and contributions to this work
Additionally I am grateful to Dr Yekaterina Brandt for her general mentorship in the field
of biology Many thanks to Stephen J Wawrzyniec not only for his perpetual support but
v
also for working alongside me over five exhausting 42-hour shifts in order to ensure that
our green chemistry solid-gas procedure was up and running I would also like to recognize
all of Dr Osińskirsquos lab members past and present whom have aided in one way or
another Dr Gennady Smolyakov Dr Erum Jamil Mallal Dr Farhana Anwar Brian
Akins Darcy Kruse and Shayla Nahar Bhuiya I must also acknowledge our collaborators
at the Sandia National Laboratories Center for Integrated Nanotechnology (CINT) Dr
Dale Huber Dr Todd Monsoon Dr Sergei Ivanov Dr Erika Cooley Vreeland and Dr
John Reno Also thank you to my students Jocelyn Baca Christian Carrillo Salomon
Maestas Cody Kamrowski Anna Sharma Shaheen Ahghar Alicia and Megan Williams
Madalyn Fetrow Michael Kopciuch Zuzia Olszoacutewka Qaiser Zaidi Allison Hayat F Zuly
Fornelli Annaka Westphal Abhyudai Nouni Surabhi Yadav Angelina Malagodi Gema
Alas Jane Nguyen Rana Chan Dennis Huang and all the other bright ambitious scientists
I have had the pleasure of working with over the years Thank you to the entire UNM
Neuroscience Department Dr Dan Savage Dr Martina Rosenberg and Dr Linda Saland
Many thanks to the Milligan lab and the previous Milligan lab members Wolfgang Scott-
Cohen Ellen Dengler Jenny Wilkerson and Audra Kerwin I thank Dr Natalie Adolphi
for her mentorship in physics electricity and magnetism and nanomagnetics I would like
to acknowledge my dissertation committee members and express my sincere thanks for
their review of this work Dr Terefe Habteyes chemist optics expert and recipient of the
National Science Foundationrsquos (NSF) Faculty Early Career Development (CAREER)
award for his project ldquoNear-Field Imaging for Nanoscale Visualization of Exciton-
Plasmon Energy Transferrdquo Dr Erin Milligan who mentored me during my time as an
vi
undergraduate student and taught me many things some of the most important being hard
work and attention to detail Dr Milligan was awarded the Regentrsquos Lectureship Award in
the Neurosciences Department in 2013 and has authored over 88 publications Dr Pavan
Muttil an expert in inhaled pharmaceuticals has authored over 50 publications disclosed
nine inventions and has two pending patent applications for his inhaled and oral vaccine
technologies I would like to thank all the scientists and staff at Lovelace Respiratory
Research Institute specifically Dr Phil Kuehl Dr Melanie Doyle Maurice Newton and
Aimee Kowell I also want to thank Dr Kevin Lind for his friendship and mentorship I
deeply thank my husband Joel Martin for his support and sacrifice Above all I thank God
for life for giving and taking away and for the fruits of the Spirit which are love joy
peace patience goodness gentleness faithfulness and self-control
This work was supported in part by the National Institutes of Health (NIH) under
the Grant No 1R21HL092812-01A1 ldquoMultifunctional Nanoparticles Nano-Knives and
Nano-Pullies for Enhanced Drug Delivery to the Lungrdquo Leisha Armijo was supported in
part by the NIH under the Grant No GM-060201 Initiatives to Maximize Student
Diversity (IMSD) the NSF IGERT program on ldquoIntegrating Nanotechnology with Cell
Biology and Neurosciencerdquo Grant No DGE-0549500 and by the More Graduate
Education Mountain States Alliance (MGEMSA) program through Arizona State
University This work was performed in part at CINTSNL under Project No U2010B1079
ldquoCharacterization of Multifunctional Nanoparticles for Enhanced Drug Delivery to the
Lungrdquo funded by DoE contract No DE-AC04-94AL85000
vii
IRON-CONTAINING NANOPARTICLES FOR THE
TREATMENT OF CHRONIC BIOFILM INFECTIONS
IN CYSTIC FIBROSIS
by
Leisha Marie Armijo
BS Biology
MS Nanoscience amp Microsystems
Doctor of Philosophy Nanoscience amp Microsystems Engineering
ABSTRACT
Cystic fibrosis (CF) is the most common genetic disease resulting in the morbidity and
mortality of Caucasian children and adults worldwide Due to a genetic mutation resulting
in malfunction of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)
protein CF patients produce highly viscous mucus in their respiratory tract This leads to
impairment of the mucociliary clearance of inhaled microbes In addition to reduced
microbial clearance anoxic environmental conditions in the lungs promote biofilm-mode
growth of the pathogenic bacterial species Pseudomonas aeruginosa Chronic infections of
P aeruginosa begin in early childhood and typically persist until respiratory failure and
viii
death result The average life-expectancy of CF patients is only about 40 years with
extensive treatment
Although the introduction of inhaled antibiotics has increased the life expectancy of
CF patients the thick mucus and biofilm formation contribute to the failure of inhaled
antibiotic drugs In order to address these issues we have synthesized and characterized
nanoparticles and nanoparticle-drug conjugates for magnetic gradient guided drug delivery
alone or in combination with medical magnetic hyperthermia to increase local temperature
and decrease the viscosity of these layers In the absence of the medical magnetic
hyperthermia application under static magnetic field the NP drug conjugates may be
gradient guided through the mucus and biofilm barriers to treat the P aeruginosa infection
directly We synthesized and characterized iron oxide (magnetite) and iron nitride
(martensite) nanoparticles as candidate nanomaterials for this application We synthesized
these materials using environmentally friendly green chemistry methods in multiple
nanoscale size ranges The NPs were synthesized using solvothermal methods and
characterized by transmission electron microscopy (TEM) energy dispersive x-ray
spectroscopy (EDS) x-ray diffraction (XRD) and direct current (DC) and alternating
current (AC) magnetometry These nanocomposites demonstrate observable bacterial
growth and biofilm inhibition even at surprisingly low (10 ngmL) concentrations making
them ideal candidates for incorporation into a low-cost treatment regime In vitro
cytotoxicity testing of the iron oxide nanoparticles shows low dosage dependent
cytotoxicity in human lung adenocarcinoma cells making the iron oxide nanoparticles an
ideal candidate material for this application
ix
Table of Contents
Dedicationhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellipiii
Acknowledgementshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip iv
Abstracthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellipvii
Table of Contentshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellipix
List of Figureshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellipxv
List of Tableshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellipxix
List of Abbreviationshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellipxx
Chapter 1 Introduction to Cystic Fibrosis Current Treatment Options and
Proposed Novel Treatment Method helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip1
11 The Epidemiology of Cystic Fibrosishelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip2
12 Inheritance of Cystic Fibrosis Disease Heterozygote Advantage and
Persistence in the Populationhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip6
13 The CFTR Gene and Different Mutation Typeshelliphelliphelliphelliphelliphelliphelliphelliphelliphellip8
14 Current Therapeutic Regimeshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip13
141 CFTR Mutation Types and Personalized Medicinehelliphelliphelliphelliphelliphellip14
142 Complications in Gene Therapyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip18
143 Summary of Treatment Failurehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip21
15 The Rode of Pseudomonas aeruginosa in the Morbidity and Mortality of
Cystic Fibrosis Patientshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip21
x
16 Antibiotic Drug Resistance and Biofilmshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip24
17 Proposed Universal Treatment Method Using Superparamagnetic
Nanoparticleshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip27
171 Particle Transporthelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip29
172 Biocompatibilityhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip32
173 Biofilm Considerationshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip34
174 Critical Parametershelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip35
18 Overview of Dissertationhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip36
Chapter 2 Synthesis and Characterization of Iron Oxide Nanoparticles38
21 Synthesis of Colloidal Magnetite Nanoparticleshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip40
211 Materialshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip40
212 Synthesis of iron oleate precursor complexhelliphelliphelliphelliphelliphelliphelliphelliphelliphellip41
213 Synthesis of cube-shaped and polymorphous nanoparticleshelliphelliphellip42
214 Synthesis of nanowireshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip43
215 Synthesis of spherical nanoparticleshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip44
216 Summary of green chemistry modificationshelliphelliphelliphelliphelliphelliphelliphelliphelliphellip46
217 Cost reductionhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip46
22 Structural Characterizationhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip47
23 Summary of Findingshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip53
Chapter 3 Magnetic Characterization of Iron Oxide Nanoparticles and
Magnetic Hyperthermia Experimentshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip55
31 Theoryhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip57
xi
32 Experimentalhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip59
321 SQUID Magnetic Characterization of Iron Oxide Nanoparticleshellip59
322 Magnetic Hyperthermia Experimentshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip64
323 AC Susceptometryhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip70
33 Summary of Findingshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip71
Chapter 4 Synthesis and Characterization of Iron Nitride (Fe16N2)
Nanoparticleshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip73
41 Introduction to Fe16N2helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip74
42 Theoryhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip75
43 Synthesis of Iron Nitride (Fe16N2) and Zero-Valent Iron (Fe0)
Nanoparticleshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip76
431 Materialshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip77
432 Synthesis of Iron Oleate Precursor Complexhelliphelliphelliphelliphelliphelliphelliphellip77
433 Synthesis of Iron Oxide Precursorhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip78
434 Removal of Oleic Acid Caphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip79
435 Production of Zero-valent Iron Nanoparticles helliphelliphelliphelliphelliphelliphellip79
436 Production of Iron Nitride Nanoparticleshelliphelliphelliphelliphelliphelliphelliphelliphelliphellip80
44 Structural Characterization of Iron Nitride Nanoparticleshelliphelliphelliphelliphellip80
45 Magnetic Characterization of Iron Nitride Nanoparticleshelliphelliphelliphelliphelliphellip82
46 Summary of Findingshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip85
Chapter 5 Hydrophilization and Bioconjugationhelliphelliphelliphelliphelliphelliphelliphellip87
51 Experimentalhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip88
xii
511 Materialshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip89
512 Removal of Oleic Acid Caphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip89
513 Citrate cappinghelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip93
514 Alginate Cappinghelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip93
515 Polyethylene Glycol (PEG) Succinylationhelliphelliphelliphelliphelliphelliphelliphelliphelliphellip94
516 Polyethylene Glycol (PEG) Capping of Iron Oxide
Nanoparticleshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip97
517 Conjugation to Tobramycinhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip98
52 Characterization of Functionalized Nanoparticleshelliphelliphelliphelliphelliphelliphelliphelliphelliphellip100
521 Size Determination helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip100
522 Zeta Potential Measurementshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip102
523 Fourier Transform Infrared (FTIR) Spectroscopyhelliphelliphelliphelliphelliphelliphellip103
53 Summary of Findingshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip103
Chapter 6 Determination of Minimum Inhibitory Treatment Concentrations
and Bacterial Sensitivitieshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip105
61 Microbiological Methodshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip109
611 Materialshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip109
612 Minimum Inhibitory Concentration of Tobramycin
Determinationhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip109
613 Establishment of Biofilm Communitieshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip110
614 Motility Testinghelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip112
615 Disk Diffusion Method helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip112
xiii
616 Biofilm and Mucus Model and Static Magnetic Field
Applicationhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip115
617 Determination of Minimum Inhibitory Concentration (MIC) of Test
Articleshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip116
618 Graphical and Statistical Analysishelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip119
62 Resultshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip119
621 Determination of Minimum Inhibitory Concentration (MIC) of
Tobramycinhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip119
622 Interpretation of Disk Diffusion Resultshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip121
623 Disk Diffusion Resultshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip124
624 Biofilm and Mucus Model and Static Magnetic Field Application
Resultshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip129
625 Motility Testing Resultshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip132
626 Comparison of Inhibition in Liquid Cultureshelliphelliphelliphelliphelliphelliphelliphelliphellip132
Chapter 7 Cytotoxicity of Iron Oxide Nanoparticleshelliphelliphelliphelliphelliphelliphellip138
71 Experimental Procedurehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip140
711 Materials and Reagentshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip141
712 Dynamic Light Scattering (DLS)helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip141
713 UV-vis-NIR Spectroscopyhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip142
714 Human Lung Adenoarcinoma Cell Growthhelliphelliphelliphelliphelliphelliphelliphelliphellip142
715 Cytotoxicity Assayhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip143
716 Viability Assayhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip144
xiv
717 Apoptosis Assayhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip145
718 Statistical Analysis Correction Factor and Mathematical
Methodshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip147
72 Resultshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip149
721 Dynamic Light Scattering (DLS) Size Distributionhelliphelliphelliphelliphellip149
722 UV-vis-NIR Spectroscopy Absorbance Measurementshelliphelliphelliphelliphellip150
723 Cytot oxicity Assay Resultshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip152
724 Viability Assay Resultshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip153
725 Apoptosis Assay Resultshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip155
73 Discussionhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip158
Chapter 8 Conclusions and Future Workhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip163
81 Importance of Green Methodologyhelliphelliphelliphelliphelliphelliphelliphelliphelliphellip164
82 Bacterial Sensitivity Discussionhelliphelliphelliphelliphelliphelliphelliphelliphelliphellip165
83 Conclusionshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip169
84 Future Workhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip170
Referenceshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip173
Publications (JournalsConferencesPatents) helliphelliphelliphelliphelliphelliphelliphelliphelliphellip214
APPENDIX I List of Chemicals Physical Properties and
Classificationhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip219
APPENDIX II Protocol for Drug Conjugationhelliphelliphelliphelliphelliphelliphelliphelliphellip229
APPENDIX III MagneThermtrade Inductive Heater Tunable Frequencies
Magnetic Field Capabilities and Derivation of Working Equation helliphellip231
xv
List of Figures
11 Statistical transmission of the CFTR gene from parents to offspringhellip7
12 Chromosomal location of CFTR genehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip9
13 Normal chloride ion channel function at the cell membranehelliphelliphelliphellip12
14 Barriers to drug diffusion surrounding biofilms of Pseudomonas
aeruginosa in the lungs of CF patientshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip24
15 Alginate moleculehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip26
21 Ion-exchange reaction between iron(iii) chloride and sodium oleatehellip41
22 Morphology alterations of iron oxide nanoparticles via additional
nucleation event(s)helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip43
23 Active nanoparticle synthesis in the Schlenk linehelliphelliphelliphelliphelliphelliphelliphellip44
24 Summary of morphology controlhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip45
25 TEM images of 250 nm cubic and 15 nm spherical NPshelliphelliphelliphelliphelliphellip47
26 TEM images of magnetite nanoparticles capped with oleic acid showing
different morphologieshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip47
27 TEM image of spherical magnetite nanoparticles capped with oleic
acidhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip49
28 HRTEM image and FFT of iron oxide monodisperse sphereshelliphelliphellip50
29 EDS spectrum of monodisperse spherical nanoparticleshelliphelliphelliphelliphellip51
210 X-ray diffraction (XRD) spectrum of 17 nm spherical
nanoparticleshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip52
xvi
211 XRD spectrum of iron oxide spherical nanoparticles using
monochromatorhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip53
31 Mechanisms of energy loss leading to heat production in magnetic
hyperthermiahelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip58
32 Magnetization vs temperature for polymorphous Fe3O4
nanoparticleshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip60
33 Ferromagnetic hysteresis loophelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip61
34 Superparamagnetic hysteresis loophelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip62
35 MagneThermtrade inductive heater setup in its entiretyhelliphelliphelliphelliphelliphelliphellip63
36 Inside of MagneThermtrade inductive heaterhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip63
37 Magnetic Hyperthermia Results for NPrsquos in the ferroferrimagnetic size
range at two frequencies and field
strengthshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip65
38 Hyperthermia results for superparamagnetic NPs in water and glycerol
mixturehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip69
41 HRTEM image of Fe16N2 nanoparticles showing excellent
crystallinityhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip81
42 XRD spectrum for iron nitride nanoparticleshelliphelliphelliphelliphelliphelliphelliphelliphelliphellip82
43 Magnetization vs temperature for Fe16N2 nanoparticleshelliphelliphelliphelliphellip83
44 Comparison of hysteresis loops of nanocrystalline samples of iron oxide
and iron nitride of similar grain sizehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip84
45 Close-up of hysteresis curvehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip85
51 Removal of oleate caphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip92
xvii
52 Citrate moleculehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip92
53 Monomer of alginic acidhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip94
54 Dialysis of succinylated PEG 5000helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip95
55 PEG succinylation overall reaction helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip95
56 Dried and purified succinylated PEG 5000helliphelliphelliphelliphelliphelliphelliphelliphelliphellip96
57 Absorbance spectra for succinylated PEGhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip97
58 Tobramycin Moleculehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip99
59 EDCSulfo-NHS crosslinking reaction schemehelliphelliphelliphelliphelliphelliphelliphelliphellip100
510 DLS size distribution histogramhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip101
61 Pyocyaninhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip108
62 Agar plates inoculated with P aeruginosa colonies taken from biofilm
cultures showing impregnated diskshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip114
63 Pole orientation for ring magnetshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip116
64 Illustration of serial dilution procedurehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip117
65 Schematic diagram of MIC determinationhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip118
66 MIC of tobramycin over timehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip120
67 Agar cultures for susceptibility testinghelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip122
68 Results of motility testhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip132
69 Optical density for liquid cultureshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip133
610 Percent bacterial inhibition vs treatment concentrationhelliphelliphelliphelliphellip134
xviii
71 Reduction of fluorescence signal in magnetite NPshelliphelliphelliphelliphelliphelliphellip148
72 DLS size distribution histogramhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip150
73 Absorbance spectrum for magnetite NPshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip151
74 Absorbance spectrum for succinylated PEGhelliphelliphelliphelliphelliphelliphelliphelliphelliphellip151
75 Cytotoxicityhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip152
76 Cell viability over timehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip154
77 Apoptosis luminescencehelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip156
78 Apoptosis time curve helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip157
81 Mechanisms of cell damage and response after exposure to iron-
containing nanoparticleshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip168
xix
List of Tables
11 Birth prevalence of cystic fibrosis worldwidehelliphelliphelliphelliphelliphelliphelliphelliphelliphellip3
12 Classes of cystic fibrosis transmembrane receptor (CFTR) mutations17
61 Guidelines for understanding susceptibility results using disk diffusion
methodhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip123
62 Comparison of 3-day old biofilm sensitivities to magnetic nanoparticles
(MNPs) capped with polyethylene glycol (PEG) tobramycin
ciprofloxacin and nanoparticle-drug conjugateshelliphelliphelliphelliphelliphelliphelliphellip124
63 Susceptibility of Pseudomonas aeruginosa biofilms to various treatments
after 3 and 60-days of growthhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip126
64 Results of cystic fibrosis (CF) biofilm model with applied magnetic
fieldhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip130
65 Results of cystic fibrosis (CF) biofilm model no magnetic field
appliedhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip130
66 Summary of biofilm modelhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip131
III1 Tunability specifications for magnetherm inductive heaterhelliphelliphelliphellip234
xx
List of Abbreviations
ABC adenosine triphosphate binding cassette
AI auto-inducer
ATP adenosine triphosphate
cAMP cyclic adenosine monophosphate
CDC Centers for Disease Control and Prevention
CF cystic fibrosis
CFTR cystic fibrosis transmembrane receptor
CLSI clinical and laboratory standards institute
DI deionized
DNA deoxyribonucleic acid
EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
EDC EDAC 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide
hydrochloride
EDS energy-dispersive x-ray spectroscopy
ENaC epithelial sodium channel
EPA Environmental Protection Agency
EPS extracellular polysaccharides
I intermediate
ICU intensive care unit
LB Luria-Bertani
LPS lipopolysaccharide
MIC minimum inhibitory concentration
MNP magnetic nanoparticle
m-PEG methyl-terminated polyethylene glycol
xxi
mRNA messenger ribonucleic acid
NABF nucleotide-binding
NABF+R nucleotide-binding and regulatory domain
NBD nucleotide binding domain
NBF nucleotide binding factor
NOAEL no observed adverse effects level
NP nanoparticle
OD optical density
PEG polyethylene glycol
PEG-OH hydroxyl-terminated polyethylene glycol
QS quorum sensing
R resistant
r radius
RNA ribonucleic acid
ROS reactive oxygen species
rpm revolutions per minute
rRNA ribosomal ribonucleic acid
S sensitive
SNP single nucleotide polymorphism
SPION superparamagnetic iron oxide nanoparticle
Sulfo-NHS N-hydroxyl sulfosuccinimide
TEM transmission electron microscope
TSCA toxic substance control act
UV-VIS-NIR ultraviolet-visible-near infrared
Vp-p peak-to-peak voltage
xxii
XRD x-ray diffraction
1
Chapter 1
INTRODUCTION TO CYSTIC FIBROSIS
CURRENT TREATMENT OPTIONS AND PROPOSED
NOVEL TREATMENT METHOD
The most common genetic disease resulting in the morbidity and mortality of Caucasian
children and adults worldwide is cystic fibrosis (CF) [Wood 1976] [Hodson 2007]
[Feuchtbaum 2012] CF results from a mutation on the gene that codes for a specific ion
channel in the epithelial cells The faulty ion channel alters normal function in multiple
organ systems most significantly affecting the respiratory system Thick viscous mucus
secreted by the goblet epithelial cells coat the cilia in the upper respiratory tract reducing
mechanical (ciliary) clearance of inhaled microbes (viruses bacteria and allergens) thus
resulting in a chronic inflammation condition The chronic inflammation state in CF is
believed to be caused in part by autophagy frustration due to reactive oxygen species
(ROS)-mediated sequestration of the beclin 1ndashclass III PI(3)K complex in perinuclear
aggregates which redirect it from the autophagy active site at the endoplasmic reticulum
(ER) [Luciani 2010] [Leivine 2011] Death occurs when pathogenic bacteria and viruses
set up residence in the respiratory mucus eventually leading to respiratory failure and
death
2
The first recorded case of CF was in the 1930rsquos [Davis 2006] at which time
abnormal mucus plugging of the exocrine ducts was believed to be the major cause of the
pathology [Davis 2006] Autopsies performed on malnourished babies showed abnormal
mucus plugging of the glandular ducts which is why the disease was initially called
ldquocystic fibrosis of the pancreasrdquo [Davis 2006] During that time the life expectancy for a
CF patient was only 6 months [Davis 2006] Despite this being the first recorded case a
genetic disorder as prevalent and widespread as CF has most likely existed in the human
gene pool for a considerable amount of time before being identified and classified
Evidence supporting this theory comes from documented European folklore [Busch 1989]
In 1838 it was held that a parent should lick across a childrsquos forehead a child who tasted
salty was believed to be ldquobewitchedrdquo and was feared to soon die [Busch 1989] Another
paper published elsewhere theorizes that CF is much older than that [Mateu 2002] The
age of the most common mutation resulting in CF (ΔF508) is debatable with estimates
ranging from more than 40000 years ago (upper Paleolithic era and pre-Neolithic era)
[Morral 1994] to 3000 years ago (post-Neolithic era) [Serre 1990] Significant evidence
exists that the disease we would one day be officially named ldquocystic fibrosisrdquo may have
existed for thousands of years prior to recorded history
11 The Epidemiology of Cystic Fibrosis
The description of the distribution and determinants of any disease frequency in the human
population is referred to as the disease epidemiology The incidence of the disease is
defined as the number of new cases per 100000 people The birth prevalence is defined as
3
the number of people born with CF per 10000 live births Population prevalence is defined
as the number of people with CF per 100000 It is important to note that an accurate
calculation of incidence and prevalence of CF depends strongly on the existence of a
complete and accurate registry [Hodson 2007] Population prevalence depends on both
birth prevalence and survival therefore birth prevalence will give the best estimate of the
incidence of CF in a population since survival depends on access to adequate medical care
Table 11
Birth Incidence of Cystic Fibrosis Worldwide Incident case per number of live births adapted from [Hodson 2007] [Farrell 2008] According to this table the lowest incidence in the world by country is Japan with only one
incident case per 355000 live births [Fredericksen 1996] The highest incidence in the world by
country is The Republic of Ireland with once case per 1353 live births [Farrell 2007] The
highest birth prevalence is reported for relatively isolated populations such as the Zuni tribe of
New Mexico and the Amish in Ohio [Wood 1976] [Stutman 2002]
Region CF Incidence Europe
Austria
13500 [Southern 2007]
Belgium
12850 [Lucotte 1995] [Chung 2002]
Bulgaria
12500 [Chung 2002]
Czech Republic
12833 [Romeo 1989] [Lucoette 1995]
Denmark 14760 [Batten 1965]
14700 [Lucoette 1995] [Klaassen 1998] [Nielsen 2002]
Faroe Islands
11775 [Kaplan 1968]
Finland
125000 [Denning 1968]
125000 [Kere 1994] [Klaassen 1998]
France
12667 West Brittany [Lev 1965] 12838 Brittany
11972 (including terminated pregnancies) [Danes 1968]
14700 [Southern 2007]
Germany
13300 [Romeo 1989] [Lucoette 1995] [Schulz 2006]
4
Italy
14238 [Siegel 1960]
14238 [Bossi 2004]
Italy (Milan)
13170 [Chernick 1959]
Netherlands
14750 [Spock 1967]
14750 [Slieker 2005]
Northern Ireland (UK)
11857 [Noblett 1969]
Norway
16574 [Johnson 1984]
Poland
15000 [Southern 2007]
Republic of Ireland
11353 [Farrell 2007]
Romania
12056 [Popa 1997]
Scotland
11984 [Hide 1969]
Slovakia
11800 [Kadasi 1997]
Spain
13750 [Lucotte 1995] [Chung 2002]
Sweden
12200-4500 [Rosan 1962]
15600 [Lannefors 2002]
United Kingdom
12415 [Gracey 1969]
12381 [Dodge 2007]
North America
United States
13200 Caucasian 110500 Native American
111500 Hispanic 114000-17000 African American
[Bowman 1969]
12380-2630 Caucasian 16800-27000 Hispanic
113300-114800 African American 11790-2880
Ashkenazi Jewish 113700-128000 Asian American
[Palomaki 2004]
Canada
12500 [Mearns 1974] 13608 [Dupuis 2005]
Saguenay-Lac St Jean
(Quebec)
1895 [Weaver 1994]
Amish OH USA
1569 [Stutman 2002]
5
Zuni Tribe NM USA
1333 Native American [Wood 1976]
Middle East
Ashkenazi Jews and Arabs
14000-18000 [Crozier 1974]
Bahrain
15800 [Corey 1988]
Jordan
12560 [Nielsen 1982]
Oceana
New Zealand
13179 (non-Maori) [Szaff 1983]
Australia
12021 (British) 13625 (Italian)13726 (Greek)
[Jensen 1987]
Other
Japan
1355000 [Frederiksen 1996]
South Africa
12000 (Caucasian) [Allan 1973] 1784-13924
(African) [Chase 1979]
Although the birth prevalence is higher in Caucasians than other ethnic groups
estimated to be roughly 1 in every 2500 people [Hodson 2007] statistics studies done in
2011 identified a considerable number of CF patients with mixed African-Caucasian
Mexican-Caucasian and Indian-Caucasian ancestries [Li 2011] The CFTR mutation has
been found in South Africans of pure African decent and did not arise from mixing with
European populations [Maek 1997] Another recent study compared CF prevalence in
newborns of various races and revealed somewhat surprisingly the highest prevalence in
Native Americans [Wood 1976] [Feuchtbaum 2012] In some populations the birth
prevalence is much higher than expected such as Ohio (Amish) and Saguernay-Lac St
Jean Qubec [Hodson 2007] This is attributed to the founder effect these populations are
6
culturally or geographically isolated Whereas in other populations the birth prevalence is
much lower than expected such as Norway and Finland [Hodson] Developing countries
such as Africa or India do not maintain sufficient records on CF incidence as detection of
CF is a low priority compared to other substantial health problems leading to infant
mortality Per the cystic fibrosis foundation there are approximately 30000 people in the
United States living with CF and 1000 new cases are diagnosed annually [Cystic Fibrosis
Foundation 2015]
A more recent publication ldquoGeographical distribution of cystic fibrosis the past 70
years of data analysisrdquo [Mirtajani 2017] also cites the same references for incidence due
to a lack of updated data in peer-reviewed scientific literature Mirtajani also notes that
African Asian and the South American continents have limited or no CF registry and
estimates that more than 50 of countries provide no data on CF incidence at present
[Mirtajani 2017] We have provided some relatively older and newer reported values for
CF incidence and some researchers have noted a slight decrease in CF cases [Massie
2010] these decreases are attributed to screening followed by pregnancy termination and
do not represent an actual decrease in the incidence of the CF mutation or mutations in the
population The rate of CF in the population has relatively remained consistent over time
as far as we can tell by considering and comparing the available published data
12 Inheritance of Cystic Fibrosis Disease Heterozygote Advantage and Persistence
in the Population
7
The gene that codes for the faulty receptor responsible for the pathology associated with
CF disease is the cystic fibrosis transmembrane receptor (CFTR) gene The CFTR gene is
recessive therefore in order to have clinically diagnosable CF disease a person must
inherit two defective copies of the CFTR gene one from the mother and one from the
father A carrier is a person who has inherited one defective gene and one normal gene and
would typically present as healthy although there are some exceptions depending on the
specific mutation type Carrier individuals also called heterozygotes exist in a much
higher frequency in a population as they only possess a single defective gene
Heterozygotes differ in phenotype from homozygotes in that they may be short in stature
[Aitken 2003] and they may exhibit increased upper respiratory inflammation than non-
carriers [Kilbourn 1968] Despite these documented features heterozygotes exhibit normal
life expectancies In order to be clinically diagnosed with CF disease a person must
possess two defective genes one from each parent (see Figure 11)
Figure 11 Statistical transmission of the
CFTR gene from parents to offspring Green
represents normal gene red represents the
defective gene Top left shows a carrier
father top right shows a carrier mother
Below are the statistically predicted
offspring each having a 25 chance of
inheriting CF Genotype is the gene
combination present for example
heterozygous homozygous recessive or
homozygous dominant and corresponding
phenotype or expression of the defective
gene of heterozygote parents and four
statistically predicted offspring Image by L
Armijo 2016
8
The life expectancy of a CF patient with extensive treatment at the time of this
publication is less than 40 years [Anderson 2016] Prior to the discovery of antimicrobial
drugs the life expectancy was much lower Recall the life expectancy of children
diagnosed with CF in the 1930rsquos was only six-months [Davis 2006] Accordingly it would
stand to reason that if CF sufferers died in childhood before reaching reproductive age
that the disease should have been completely eradicated by natural selection This
however has not been the case Before the late 1960rsquos scientists investigated possible
benefits of the defective gene which may have allowed it to persist in the population
Finally 1967 studies confirmed that the mean number of live offspring of the grandparents
of CF patients was higher than for the grandparents of the healthy control group [Knudson
1967] These findings suggest a heterozygote advantage A so-called heterozygote
advantage occurs when a carrier individual demonstrates a selective advantage over the
rest of the population Findings confirmed that heterozygote carriers are resistant to
cholera toxin [Gabriel 1994] Just four years after that another paper reported that the
single defective CF gene imparts resistance to typhoid fever [Pier 1998] Considering the
long history and persistence of CF from the consequences of these long-term selective
advantages CF is significantly likely to continue to persist in the population long into the
future
13 The CFTR Gene and Different Mutation Types
The CFTR gene encodes the instructions for the cell to manufacture the CFTR protein The
CFTR protein is a cyclic adenosine monophosphate (cAMP) regulated chloride ion
9
channel found virtually exclusively in the secretory epithelial cells [Frizzel 2012] The
CFTR protein is encoded by the DNA and transcribed into messenger RNA (mRNA) The
mRNA is translated and the CFTR protein is manufactured in the rough endoplasmic
reticulum of the cell The assembled CFTR migrates to the cell membrane where it exerts
its function A mutation in the DNA coding for the CFTR protein could lead to a premature
stop codon in which case the message would never be translated and a CFTR protein
would never be produced
Figure 12 Chromosomal location of the CFTR gene locus on the q arm of
chromosome 7 in region 3 band 1 and sub band 2 hence the location
designation 7q31_2 Image after [NIH 2016]
10
Alternatively a different mutation could change the code such that a different
protein is produced resulting in either a faulty or a non-functioning CFTR Even though the
defect is found on a single gene there are many different mutations on that gene that can
cause CF disease All the mutations documented correspond to the same location on
chromosome 7 the difference in the type of mutation is characteristic of the code that was
inserted in this region Evidence of a tight linkage between the CF locus and a DNA
sequence polymorphism in the center third of the long arm of chromosome 7 between
bands q21 and q31 was found in 1985 drawing more attention to this region [White 1985]
Others identified the CF locus more specifically on human chromosome 7ce-q22 in that
same year [Wainwright 1985]
It was not until much later when it was shown that several different mutations
could result in a faulty or missing CFTR protein (see Table 12) [Peebles 2005] This is
significant because variations in the type of mutation the presence of some type of
defective CFTR or the absence of a CFTR protein complicate current standard treatment
regimes Treatment is most effective when catered to the patientrsquos specific mutation type
For practicality specific mutations are given a class number corresponding to a recognized
treatment regime (see Section 14) Since different mutations result in different pathologies
and severities thereof optimization of treatment requires a personalized approach A
universal treatment method is needed
In all cases the major underlying issue is either a malfunctioning or non-
functioning chloride ion channel at the epithelial cell membrane resulting in a decreased
volume of periciliary fluid in the lower respiratory tract This in turn leads to impaired
11
mucociliary clearance of inhaled microbes which colonize and ravage the lungs causing
child-onset chronic infections chronic inflammation tissue damage and eventual
respiratory failure and death Therefore in order to increase the life-expectancy of CF
patients we must uncover a reliable method to annihilate the bacterial species that
overwhelms the already compromised respiratory system in these patients
The CFTR protein is an ATP-binding cassette (ABC) transporter-class ion channel
(Figure 13) ABC transporters are classified as proteins based on the sequence and
organization of their domain or domains For example the CFTR has Nucleotide Binding
and Regulatory Domains 1 and 2 (NBD1 and NBD2 +R respectively) areas where
nucleotides bind to regulate function In general the CFTR is simply a protein that
conducts chloride (Cl-) [Riordan 2008] and thiocyanate (SCN-) [Childers 2007] anions
across epithelial cell membranes A normally functioning CFTR protein acts as an ion
pump channeling chloride ions (Cl-) from inside the cell across the cell membrane and
into the extracellular space in order to maintain healthy salinity levels within the cell In
addition the CFTR protein can inhibit the epithelial sodium channel (ENaC) when
activation is triggered by nucleotide binding to NBF1 The ENaC is a separate channel
through which sodium ions (Na+) are transported A healthy CFTR protein influences a
low-level sodium intake by regulation of NBF1 [Annereau 2003] When a defective CFTR
protein or no CFTR protein is produced the Cl- concentration within the cells is
compromised ENaC is activated and a subsequent increase in sodium transport into the
cell results
12
Figure 13 Normal chloride ion channel function at the cell membrane CFTR is the
cystic fibrosis transmembrane receptor shown in active transport of chloride ions
through its channel NBD1 is the first nucleotide-binding domain and NBD2 is the
second nucleotide-binding domain where nucleotides can bind to regulate function
Image by Armijo L 2014
Mutations of the CFTR gene altering chloride ion channel function cause
dysregulation of epithelial fluid transport in the lung pancreas and other organ systems
Clinical pathologies include thick condensed mucus in the lungs and recurrent respiratory
infections causing chronic disability and reduced life expectancy CF patients also suffer
from pancreatic insufficiency which results in malnutrition and diabetes Abnormal ion
13
regulation also causes the salty epithelial excretions which promote bacterial colonization
of the lungs
14 Current Therapeutic Regimens Personalized Medicine and Investigational
Treatments
The discovery of antibiotics in 1928 was undoubtedly one of the most important
developments in medicine to date responsible for saving millions of lives by making
formerly deadly infections curable [Tan 2015] Antibiotic reliability is the foundation for
modern medicine and has facilitated the development of numerous formerly impossible
medical procedures Virtually every aspect of what we call modern medicine treatment of
autoimmune diseases and allergies therapeutic use of corticosteroids or other
immunosuppressant drugs chemo- and radiation therapy any and all surgical procedures
burn and wound treatment to include any procedures or accommodations in which stents
catheters orthodontic wires ventilators staples sutures bandages clamps belts implants
or virtually any procedure in which an inert object-biological interface exists they all put
the patient at risk for infection The development of antibiotic drugs made all this possible
On the other hand researchers and medical professionals alike continue to struggle with
the intensifying issue of antibiotic resistance especially prominent in healthcare
environments which threatens to collapse the crucial foundation on which modern
medicine was built
Since death from respiratory failure is preceded by bacterial colonization of the lungs
of CF patients most treatment regimens include the use of aminoglycoside antibiotics
14
[Peebles 2005] [De Boeck and Amaral 2016] The most common pathogenic bacterial
species having the ability to form biofilm colonies and causing respiratory failure in CF
is Pseudomonas aeruginosa [Govan 1996] The next most important adversary in the war
on morbidity and mortality in CF is Burkholderia cepacia Burkholderia cepacia is
believed by some to be the most significant and provocative new opportunistic pathogen to
torment the CF community [Govan 1996] Other microbiological organisms contributing to
pulmonary disease in CF lungs by predisposing the patient to Pseudomonas aeruginosa
colonization are often referred to as nonpseudomonal CF pathogens The following
nonpseudomonal pathogens are known Staphylococcus aureus and Haemophilus influenza
(common) Streptococcus pneumoniae Legionella species viruses (in particular
respiratory syncytial virus (RSV) various glucose nonfermenters mycobacteria fungal
agents (less-common) [Gilligan 1991 Govan 1996] Because of the infectious disease
aspect antibiotic therapy is a common component of the current CF treatment regime
141 CFTR Mutation Types and Personalized Medicine
CF is a genetic disease that can manifest differently depending on the type of
mutation of CFTR gene Therefore treatments are optimized when they are catered to a
specific CFTR gene mutation In the interest of treatment optimization the CFTR
mutations resulting in CF disease have been traditionally been separated into V classes
[Peebles 2005] A new class class VI was later described and is distinguished by rapid
CFTR turnover at the channel surface [Zielenski 2000] Even more recently a new
classification based on therapeutic strategies and accounting for the potential of
15
personalized medicine and targeted drugs was proposed [De Boeck and Amaral 2016] In
this model De Boeck and Amaral separated the class I mutations into class I (stop-codon)
and a new class class IV (no mRNA transcription) due to the differing successful
treatment options for the two [De Boeck and Amaral 2016]
One example of mutation type is caused by a single nucleotide polymorphism
(SNP) A SNP occurs when a single base (nucleotide) along the DNA ladder is replaced by
a different one Another mutation type called a nonsense mutation converts a codon (a
triplet of bases that codes for an amino acid) into a stop codon (a triplet of bases encoding
the termination of translation) A nonsense mutation is responsible for the pathology
described in a CF class I mutation For example an adenosine molecule replaces a cysteine
molecule resulting in synthesis of a faulty protein or no protein synthesis at all A
missense mutation as in CF mutation classes II III IV or V occurs when a SNP results in
the substitution of a different amino acid in the amino acid chain It should be noted that
overlaps between different classes of mutations can also exist For example the delta-F508
(ΔF508) mutation can cause reduced chloride channel opening time in addition to
abnormal CFTR processing Occasionally the CFTR mutation can be modified by another
mutation or polymorphism on the same allele (a modifier gene)
The most common therapeutic regime for class I mutations includes
aminoglycoside antibiotics Aminoglycosides are antibiotics traditionally used for the
treatment of gram-negative bacterial infections (such as P aeruginosa infection) They are
named as such because they contain as a portion of the molecule an amino-
modified glycoside an aminoglycoside This family of antibiotics consists of tobramycin
16
streptomycin gentamycin and the neomycins The drug tobramycin which we have
chosen for our investigations annihilates bacterial cells in a synergistic manner Initially it
electrostatically binds the negatively charged lipopolysaccharide bacterial membrane
compromising membrane integrity and thus resulting in its degradation [Shakil 2008]
Once internalized acting from the inside of the bacterial cell tobramycin inhibits
ribosomal translocation thus interfering with protein synthesis [Saiman 2004] This
treatment is used for the chronic bacterial infections of respiratory tract characteristic of
CF
For a class II mutation a faulty CFTR is produced in the endoplasmic reticulum
where it remains Butyrates are a popular treatment for class II mutations Butyrate is the
generic name for the conjugate base of hydrocarbons containing butanoic acid (C4H7O2minus)
somewhere in their structure These agents cause a reduction in CFTR current amplitude
suggesting a kinetically fast blocking mechanism [Linsdel 2001] thus artificially
regulating that component of the CFTR
In a class III mutation a faulty CFTR causes inappropriate activation and
regulation of ion transport Despite expression of the full-length protein at the apical
plasma membrane class III mutations change CFTR gating which results in decreased Clminus
transport [Kreindler 2010] Genistein supplementation has been recommended for class III
mutations Genistein has been demonstrated to alter the maturation cell surface expression
and single-channel function of CFTR protein [Schmidt 2008] Genistein is a phytoestrogen
(plant-derived xenoestrogen) belonging to the category of isoflavones Although it has
17
many uses in hormone modulation in this case it is exploited for its ability to modulate the
CFTR channel potentiating its opening at low concentration and inhibiting at higher doses
Table 12
Classes of CFTR Mutations This table summarizes the recognized classes of mutations that cause cystic fibrosis disease There
are IV mutation classes each resulting in a specific alteration to or absence of the CFTR protein
Due to the unique resulting manifestations each mutation class has a specific treatment regime
Proposed class [De Boeck and Amaral 2016] Approved therapy Adapted from [Peebles 2005]
and [De Boeck and Amaral 2016]
In class IV mutations a faulty CFTR reduces chloride conductance and transport is
altered Milrinone is used for the treatment of class IV mutations Milrinone marketed
Class
Effect on CFTR
Types of Mutation
Therapy Potential
Therapy
I Defective synthesis of
message (messenger RNA)
causing absence of CFTR
Premature stop codon
(nonsense or frame
shift)
Aminoglycosides
Gene transfer read-
through compounds
II Abnormal CFTR produced
which fails to leave
endoplasmic reticulum
Amino acid deletion
(∆ F508 or missense
mutation)
Correctors
Butyrates
Gene transfer
III Abnormal CFTR causing
disruption of activation and
regulation at cell membrane
impaired gating
Missense mutation
(ie G551D)
Potentiators
Genistein
Gene transfer
IV Abnormal CFTR reducing
chloride conductance
Missense mutation
(ie R117H or
R347P)
Milrinone
Potentiators
Gene transfer
V Reduced or absent synthesis
of CFTR due to decreased
splicing of normal CFTR
Missense mutation or
splice site mutation
(ie A445E or 5T)
Aminoglycosides
Antisense
oligonucleotides
Correctors Gene
transfer
VI Absence of CFTR No mRNA
transcription
Aminoglycosides
Stabilizers
Gene transfer
VII Absence of CFTR No mRNA Aminoglycosides
Bypass therapies
18
under the brand name Primacorreg Milrinone is an inhibitor of phosphodiesterase 3 a
vasodilator Although class V mutations can lead to the production of normal CFTR the
same mutation can also result in a reduced or absent CFTR A limitation of transcriptional
regulation results in a reduced quantity of the protein being produced As with the other
mutations resulting in an absent CFTR (I VI and VII) the only approved treatment is
aminoglycoside antibiotic and supportive therapy
Many of the identified CFTR gene mutations can be placed into one of the six
classes thus accounting for approximately 80 of all CF patients [Rogan 2011] However
of the gt1900 CFTR mutations that have been identified there are only roughly 20
mutations have a frequency greater than 01 [Rogan 2011] CF disease can result from
any one of those numerous mutations on a single gene Despite the considerable number of
mutations the encoded gene product is one and the same the CFTR protein The most
common mutation accounting for 70 of the disease alleles leads to a single amino acid
deletion (∆F508) [Zielenski 1995] As presented in Table 12 the class II mutation which
includes the ∆F508 deletion is responsible for 85 of cases in Europe [Peebles 2005] It is
important to note that the percentage of CF patients expressing the most common mutation
type varies among ethnic groups For example only 30 of Israelis with CF have the most
common mutation (∆F508) [Shoshani 1992]
142 Complications in Gene Therapy
Because CF is a genetic disease it was initially believed that gene therapy would
be the most effective treatment for all classes of CFTR mutations In gene therapy correct
19
copies of the CFTR gene are transferred to the respiratory epithelial cells where the gene
can be translated and a functional CFTR can be synthesized [Burney 2012] Previous
studies have focused on increased chloride secretion out of the cell demonstrating positive
results of some normal CFTR function however clinical efficacy has not yet been
achieved [Burney 2012] Despite the vast knowledge obtained by research focused on
understanding the genetic defect underlying CF this understanding has been referred to as
only ldquohalf the battlerdquo in finding the cure for this disease [Hearst 1995] Anxiously awaited
cures focused on the gene therapy approach have failed to materialize in spite of the
significant amount of research performed in this field Complementation of CF using gene
transfer or gene therapy methods specifically focusing on the delivery of a CFTR cDNA
to the airway epithelium seemed appealing initially since the proposed target cells are
accessible by aerosol delivery approaches (or other direct instillation) however since the
first human gene therapy trial in 1993 realization of this goal has proved challenging [Sinn
2011] The use of the previous gold standard in CF gene therapy adenoviral vectors has
decreased recently due to low transduction efficiency weak promoter activity and
incapability for re-administration due to the development of an anti-viral vector immune
response [Griesenbach 2006] The adenovirus package is also very small and packaging
the large CFTR gene has proven difficult One group attempted to package the gene by
cutting it in half and using two separate viral vectors (each carrying half the gene) [Song
2009] Another study pointed out immune responses to the viral vector may be enhanced if
the patient already has an established P aeruginosa infection [Tosi 2004] A similar
problem has been reported when the alternative Sendai virus (SeV) vector is used for gene
20
transfer Although the SeV is an efficient gene transfer agent the gene expression is
transient and requires repeat administration as with the adenovirus vectors re-
administration of SeV vectors also results in an immune response [Griesenbach 2006] A
more serious problem with SeV vectors is that they have demonstrated the induction of
oncogenesis in certain trials [Hacein-Bey-Abina 2008] The developments of novel non-
viral methods for gene transfer have been slow One report on NP-mediated gene transfer
did show increased chloride transport however vector-specific mRNA was could not be
detected [Konstan 2004] Another problem with non-viral gene transfer is caused by the
heightened inflammatory state further frustrated by the introduction of plasmid DNA
[Burney 2012] The unmethylated nucleotide sequence in the plasmid DNA is identified as
an antigen by the immune system thus causing further inflammation in the lower
respiratory tract [Zabner 1996] [Schwartz 1997] CRISPRCas9 has demonstrated the
ability to repair a single-gene hereditary defect causing CF in murine and human stem cells
[Schwank 2013] and this treatment may become available soon However a recent paper
published in Nature Communications shows that CRISPRCas9 causes numerous
unwanted insertions and deletions (up to 600 bp) in the mouse genome [Shin 2017] and
may need significantly more investigation before it is used to treat human patients It is
also unlikely that gene transfer would be a viable option for patients with more than a
single mutation or patients with a class VII mutation alone or in combination with other
mutations A class VII mutation results in the total absence of a CFTR as well as an
absence of mRNA
21
143 Summary of Treatment Failure
Yet another hurdle was realized after treatment data for a larger population was
available patients with the same CFTR mutation genotype often respond differently to
drug treatments [Amaral 2015] [Marson 2015] This data suggests an even deeper level of
personalization may be necessary to achieve sufficient efficacy of current therapeutics
Personalized medicine despite presenting significant benefits is also costly and may not
become available in developing countries for quite some time
15 The Role of Pseudomonas aeruginosa in the Morbidity and Mortality of Cystic
Fibrosis Patients
The most frequently reported pathogenic microbial species colonizing the lungs of
CF patients is Pseudomonas aeruginosa P aeruginosa has been cultured from the
respiratory tract of 61 of all patients (ranging from 21 of patients under 1 year of age
to ˃80 of patients 26 years or older) [FitzSimmons 1993] P aeruginosa is also the top
etiology of all gram-negative nosocomial (acquired in hospitals) infectious bacteria with a
striking mortality rate of 50 or more [Baltch 1994] [Hauser 2003]
Pseudomonas aeruginosa is one of the notorious ESKAPE pathogens (a group
consisting of Enterococcus faecium Staphylococcus aureus Klebsiella pneumoniae
Acinetobacter baumannii Pseudomonas aeruginosa and Enterobacter species) which
have developed resistance to the bulk of our current antimicrobial regimes and instead
ldquoescaperdquo the lethal action of antibiotics [Rice 2008] More specifically many highly
resistant Gram-negative bacteria from the ESKAPE group including P aeruginosa are
22
emerging as exceptionally noteworthy pathogens in threatening public health in United
States as well as other parts of the world [Boucher 2009] The ESKAPE bacteria are of
tremendous concern because they are responsible for causing the overwhelming majority
of nosocomial infections Several reports identify significant limitations in current
treatment options for these pathogens that force medical professionals to settle on the use
of previously discontinued drugs having documented toxicity and unclear dosage and
administration guidelines [Bradford 2004 Cardo 2004 Falgas 2007 Urban 2008] They
also provide complex models of pathogenesis transmission and drug resistance [Rice
2008 Boucher 2009] Treatment regimens found to exhibit success against the ESKAPE
bacteria can be applied to virtually any other species Successful treatment of these species
alone will result in significantly safer healthcare environments more suitable for treating
disease and illness
This member of the Gammaproteobacteria class and the Pseudomonadaceae
family is a deadly pathogen responsible for the morbidity and mortality of CF and
oncology patients as well as burn unit patients and infects up to two-thirds of ICU
patients with nosocomial pneumonia [Torres 1990] It is responsible for more than 90 of
respiratory failure cases in CF patients [Gilligan 1981] P aeruginosa has inherent as well
as acquired resistance to many drug classes In addition it possesses the ability to quickly
alter its genetics to impart resistance to the presence of new unrecognized treatments [Lee
2007] Despite its classification as an ldquoopportunistic pathogenrdquo [Fick 1992] [Campon
1993] it remains a major worldwide public health problem due to its ubiquity in the
environment its ability to colonize virtually all regions of the body and its overall vitality
23
which has allowed it to adapt to a wide range of environmental conditions The pathogen
possesses the ability to grow with limited nutrients and can metabolize some unusual
organic molecules as carbon sources some examples are acetate and citrate It can grow
without oxygen if NO3 is available as an electron acceptor for cellular respiration This
species is so robust that it is one of the few extremophiles that can colonize deionized
water
Many issues arise when attempting to treat P aeruginosa infections in the
respiratory tract of CF patients using conventional methods First CF sputum is highly
viscous interfering with normal oxygen diffusion thus hypoxic conditions exist The
hypoxic environment promotes biofilm formation by P aeruginosa [Worlitzsch 2002] as
low-oxygen conditions trigger the phenotypic switch to biofilm mode Once in the biofilm
mode of growth the bacterial colony produces a protective alginate layer around itself At
this point two barriers to drug diffusion exist physically blocking the aerosol antibiotics
from reaching the target the viscous mucus layer and the biofilm layer (Figure 14) No
antimicrobial agent can penetrate the biofilm unless the microorganisms form aggregates
that affect its diffusion [Stewart 2001]
While numerous microbial species can successfully colonize the respiratory tract of
CF patients P aeruginosa ultimately dominates the microbial flora becoming the chief
contributor to disease severity and respiratory failure The phenotypic switch of P
aeruginosa microcolonies from a planktonic (non-mucoid) to a biofilm (mucoid) state is
characterized by both antibiotic resistance and accelerated pulmonary decline [Govan
24
1996] Therefore an artificial active transport method is needed to deliver antibacterial
drugs to the bacterial cells
Figure 14 Biofilm and mucus barriers in a CF lung infection Illustration of
biofilm layer fixed to infected tissue protecting bacterial cells and CF mucus layer
inhibiting penetration of antibiotics and antibodies
16 Antibiotic Drug Resistance and Biofilms
Many bacterial species in response to the presence of antibiotics or bacteriophages or in
low oxygen or low nutrient conditions switch to the biofilm mode of growth These initial
bacteria release chemical signals inducing the switch in neighboring populations as well
Biofilm mode consists of a phenotypic switch from planktonic (free) cells by means of
gene regulation [An 2007] To form a biofilm planktonic cells first adhere to a surface via
van der Waals forces then by using flagella or cilia as an anchor Quorum sensing (QS) is
used to recruit other bacterial cells and promote expression of the genes necessary for cell
25
aggregation and subsequently biofilm production An inducer binds the bacterial QS
receptor triggering transcription and translation of necessary genes
Once a colony is established the anchor cells produce exopolysaccharides which
form the protective biofilm layer around the bacterial colonies N-acyl homoserine lactones
are signaling molecules called auto-inducers (AIs) used in QS [Smith 2002] It is
interesting to note that compounds with similar structures may be of interest for blocking
QS (receptor antagonists) [Sio 2006] Antibiotic resistance typically results from a transfer
of antibiotic resistance genes through bacterial conjugation gene regulation or other
modes of gene transfer However a major factor contributing to antibiotic drug resistance
in P aeruginosa is the production of biofilms The production of a biofilm results in a
slower growth combined with bacterial production of extracellular polysaccharides (EPS)
which form a physical barrier that limits the ability of antibiotic drugs to interact with the
bacteria The EPS biofilm is mainly composed of alginate a slimy anionic co-block
polymer which forms a viscous gum when in the presence of water hence the designation
ldquomucoidalrdquo Alginate or alginic acid is a linear copolymer consisting of homopolymeric
blocks of (1-4)-linked β-D-mannuronate (M) and its C-5 epimer α-L-guluronate (G)
residues [Gacesa 1990] (see Figure 15) The M and G residues are covalently linked
together in different sequences or blocks The monomers may be homopolymeric in
blocks of consecutive G-residues or consecutive M-residues co-block alternating M and
G-residues The known and proposed roles of alginate in biofilm infections include
generation of an alginate covering forming a direct barrier to phagocytosis and
26
opsonization immunomodulatory effects and other biofilm-related phenomena such as
bacterial adhesion and antibiotic resistance [Govan 1996]
Figure 15 Alginate molecule Carbon skeleton showing the homopolymeric
blocks of (1-4)-linked β-D-mannuronate (M) (upper ring) and its C-5 epimer α-
L-guluronate (G) (lower ring) Image created with MarvinSketchtrade
Bacterial biofilm infections in general are a significant public health problem
Specifically P aeruginosa biofilms cause infections in indwelling catheters burns open
wounds orthodontic wires CF lungs and stents and can infect virtually any part of the
body As discussed earlier bacterial biofilms reduce the efficacy of therapeutics due to
their physical interference with drug diffusion by blocking diffusion of the drug to the
target bacterial colonies [Govan 1996] In addition with regards to the more than 20 genes
that are differentially expressed in tobramycin-treated biofilms sheer existence in a biofilm
27
indicates moderate resistance to all antibiotic drugs [Whiteley 2001] Regarding the P
aeruginosa species interference of the alginate barrier with antibiotic penetration to the
strain and thus antibacterial action has also been thoroughly investigated [Kumon 1994]
Interestingly when bacterial cells are released from a biofilm they typically experience an
abrupt increased susceptibility to antibiotics This suggests that the antibiotic resistance of
biofilm bacteria was not acquired through mutations or incorporation of mobile genetic
elements into the bacterial genome [Anwar 1989] Since the most common cause death for
CF patients is respiratory failure from chronic bacterial infections and P aeruginosa is the
top etiology responsible for such infections annihilation of P aeruginosa is a fundamental
step in increasing the life expectancy of CF patients
17 Proposed Universal Treatment Method Using Superparamagnetic Nanoparticles
Significant improvements have been made in the treatment of CF over the past 30 years
Direct drug delivery via inhalation aerosols have increased the average life expectancy of
CF positive children born in developed countries to approximately 40 years [Elborn 1991]
[Staab 1998] Despite this the life expectancy of CF patients could still stand to improve
The efficacy of inhaled therapies still remains marginal due to the presence of the viscous
mucus barrier within the airways extensive degradation and metabolism of inhaled drug
prior to exerting its pharmacological action and the development of mucoid P aeruginosa
biofilm colonies Therefore an adequate active transport method is necessary to deliver
28
antibiotic drug to the bacterial colonies below the mucus layer within the protective
biofilm
The possibility of using magnetic gradient guided active transport of antibiotic
drug using superparamagnetic nanoparticles was investigated further since the barriers to
diffusion of therapeutic drug or gene through mucus and biofilm are the principal bases for
treatment failure Nanoparticle carrier mediated of drug or gene delivery based on passive
transport have demonstrated inadequate penetration efficiencies [Sanders 2000] Similar
passive transport-based nanocarrier methods perform insufficiently and are unlikely to
enhance the penetration efficiencies to clinically relevant levels Frequently drugs or gene
vectors are unable to reach the intended target prior to their activity being diminished or
eliminated Poor transport efficiencies in drug delivery have resulted in the inadequacy of
therapies since the mucus and biofilm barriers to drug diffusion result in sub-therapeutic
levels of drug at the infected area These low-levels of antibiotic drug near the bacterial
colonies further leads to drug resistant bacterial strains as the colonies become sensitized to
the drug Because the use of nanomagnetic materials bound to antibiotic drug would allow
us to guide the magnetic nanoparticles (MNPs) to the area of interest by using an external
magnetic field the particles could be guided deeper into the respiratory tract than
inhalation alone would allow Particularly of interest is the capability of MNP systems to
put forth robust influences on their local environment by means of heat under an
oscillating magnetic field In other words MNPs once guided via directed motion under
an inhomogeneous static magnetic field to an area of interest can be placed in an
oscillating magnetic field and raise the local temperature by means of inductive heating A
29
local temperature increase is anticipated to reduce the viscosity of the mucus and biofilm
layers facilitating delivery of the antibiotic drug We have demonstrated the ability of the
iron oxide NPs to increase local water temperature in vitro under AC magnetic field These
are attractive functional attributes for fostering transport and drug distribution in CF-
related lung infections Therefore utilizing the unique transport and inherent
superparamagnetic properties of selected nanoscale systems provides a promising strategy
for overcoming the biological mucus and biofilm barriers in CF lung disease
171 Particle Transport and Drug Delivery
Our group has previously demonstrated marked increases in particle transport of
nanoparticles can be attained using a static non-uniform magnetic field [Smyth 2008]
[McGill 2009a] in Chapter 3 we show that both ferromagnetic iron oxide NPs as well as
superparamagnetic iron oxide nanoparticles (SPIONs) can be heated using an external AC
magnetic field under which the SPION could cut through biopolymers such as alginate
and DNA which are responsible for the diffusion-limiting properties of the biofilm In
addition in Chapters 2 and 4 we demonstrate our ability to synthesize several different
types of magnetic nanoparticles (MNPs) to optimize the physical properties and chemical
stability We synthesized and characterized iron oxide NPs having various morphologies
iron nitride NPs and zero-valent iron NPs These particles are surface-biofunctionalized
for drug conjugation We then attach a model drug to the surface using a biocleavable
conjugation scheme (see Chapter 5) Drug release could potentially be triggered by
30
external magnetic fields in a non-invasive manner if necessary Many researchers have
reported the use of external magnetic fields to achieve controlled drug delivery using
hyperthermia via two general methods Hyperthermia-based controlled Drug delivery
through Bond Breaking (DBB) and Hyperthermia-based controlled Drug delivery through
Enhanced Permeability (DEP) [Kumar 2011] The first successful demonstration of DBB
was reported using radiofrequency EMF activation of release of fluorescein-labeled 18 bp
in a model tumor near the posterior mammary fat pad of mice [Derfus 2007] Our
laboratory later confirmed this concept by triggering the release of fluorophore bimane
amine from the surface of SPIONs under external oscillating magnetic fields [McGill
2009b] The first report was by Kost and others who demonstrated insulin release from a
magnetic composite of ethylene vinyl acetate under a low frequency magnetic field [Kost
1987] A commonly proposed approach is to use a composite carrier consisting of a
magnetic iron oxide core inside any thermally sensitive polymer having a temperature-
dependent drug release profile then when the core is self-heated drug release is triggered
[Liu 2008] [Liu 2008] reported the successful triggered delivery of Vitamin B12 within
minutes between 40-45 degC using poly(ethylene-oxide)-poly(propylene-oxide)-
poly(ethylene-oxide) block copolymers 4-nitrophenyl chloroformate gelatin and 1-ethyl-
3-(3- dimethylaminopropyl) carbodiimide self-assembled nanocapsules and magnetic iron
oxide NP cores which were responsible for the heating [Liu 2008] Triggered drug delivery
would be necessary if the required therapeutic dose is found to be higher than the dose
found to be cytotoxic to healthy cells In this case the overall environment could be kept
at a safe drug concentration while the highest concentration would be released specifically
31
at the infection site reducing collateral damage Finally when loaded with drug the MNPs
will be incorporated into inhalable microparticles suitable for lung targeting This will
initiate simultaneous highly efficient transport and highly specific lung deposition
Additionally these systems will transport inhibitory drug concentrations directly to the site
of action and will therefore facilitate improvements in drug and gene therapies in CF
prolonging survival and enhancing quality of life
The physics of particle delivery to the lower respiratory tract has been well
characterized Further engineering of the particle or particles into a stable micron-range
polymer matrix in a stable dry-powder form is necessary for successful pulmonary
delivery Many factors impact the performance of a particle system such as mass median
aerodynamic diameter (MMAD) particle size distribution dispersibility particle
morphology and thermodynamic stability [Chow 2007] [Hickey 2007b] The combination
of two specific parameters size and surface roughness greatly influence performance It is
known that the particles must be further engineered to increase the diameter from the
nanoscale to the microscale range to avoid deposition in the throat [Hickey 2003] Previous
research has also demonstrated that particles with MMADs 1-2 μm deposit in the smaller
(lower) airways and 5-10 μm deposit in the larger (upper) airways [Vehring 2007]
Particles having a high degree of surface roughness exhibit increased dispersibility due to
decreased interparticulate interactions consequently resulting in significantly decreased
particle aggregation resulting in a larger aerodynamic size (for the agglomerate) [Gilani
2005] Typically lactose [Kaialy 2012] or mannitol [Hamishehkar 2012] is used as a
carrier because it has a sweet taste Some other polymers which have been previously
32
investigated for this application are the FDA approved polymer poly(lactic-coglycolic)
(PLGA) [Tomoda 2009] poly(ethylene glycol)-co-poly(sebacic acid) (PEG-PSA) [Tang
2010] and dipalmitoylphosphatidylcholine (DPPC) with dipalmitoyl phosphatidylethanol
aminemethoxy-polyethylene glycol (DPPE-PEG) [Meenach 2013]
172 Biocompatibility
Previous work on biocompatible magnetic materials has focused on the iron oxides [Gupta
2005] [Xie 2009] [Xie 2010] iron core-iron oxide shell particles [Qiang 2006] cobalt
[Bao 2005] [Xu 2007] [Lukanov 2011] iron core gold shell particles [Chen 2003] or the
rare-earth elements [Meiser 2004] [Setua 2010] [Dobson 2006] However the iron oxides
have shown the greatest potential as biofilm inhibitors having low cytotoxicity [Johannsen
2007] Significant research on silver NPs as antimicrobial agents has been reported in the
literature [Sondi 2004 Morones 2005 Cho 2005 Kim 2007 Pal 2007 Shrivastava 2007
Duraacuten 2007 Martiacutenez-Castantildeoacuten 2008 Rai 2009 Chudasama 2010 Lara 2011 El-Kheshen
2012 Dong 2012 Prabhu 2012 Le 2012 Sadeghi 2012 Rai 2012 Emeka 2014 Losasso
2014 Agnihotri 2014 Franci 2015 Cavaliere 2015 Lara 2015 Giessen 2016 Russol
2017 Patra 2017 Shaker 2017] and much research has also been done on the efficacy of
silver NPs against P aeruginosa [Afreen 2011 Eid 2013 Palanisamy 2014 Singh 2014a
Anasari 2014 Mushin 2014 Singh 2014b Mapara 2015 Raza 2016 Haghighi 2016
Nasiri 2016 Kasitherar 2017] Due to their undisputable antibacterial properties silver
NPs are among the most commonly exploited nanomaterials in commercialized products
[Beer 2012] Although silver NPs have demonstrated antimicrobial properties against
many bacterial species silver is costly and is also known to exhibit toxicity in multiple
33
species [Asharani 2008] including in vitro cytotoxicity in various human cell lines
[Kawata 2009 Beer 2012 Foldbjerg 2011] Most researchers attribute the observed
toxicity either to silver ions [Asharani 2008] or the combination of silver NPs and silver
ions [Bilberg 2011 Foldbjerg 2011] An ideal bactericidal agent should be lethal to
bacteria but safe to human cells One such candidate is iron and its compounds Iron-oxide
NPs have been shown to be non-toxic [Sumanta 2008 Sun 2010 Prodan 2013 Grottone
2014] For example ferahemeferumoxytol containing superparamagnetic iron-oxide NPs
was approved by the US Food and Drug Administration as an iron supplement for
treatment of iron deficiency in patients with renal failure [Provenzano 2009 Coyne 2009
Lu 2010] According to a previous report iron-oxide in NP form is not only non-toxic but
its byproduct degraded iron from the cores apparently accumulates in natural iron stores
in the body [Weissleder 1989] Properly biofunctionalized iron-oxide NPs have been
shown to inhibit growth of Staphylococcus aureus [Tran 2010 Darwish 2015 Shi 2016]
and Escherichia coli [Darwish 2015 Chatterjee 2011] prevent biofilm formation by P
aeruginosa [Niemirowicz 2015] and Streptococcus mutans [Javanbakht 2016] and exhibit
bactericidal activity against a range of Gram-negative and Gram-positive bacterial species
[Behera 2012 Prodan 2013 Thukkaram 2014 Prabhu 2015 Arakha 2015 Nehra 2018]
While these are very encouraging results more work is necessary in the investigation of
iron-oxide NPs as a feasible alternative to silver NPs in the treatment of bacterial infections
and for biofilm disruption
34
173 Biofilm Considerations
According to a previous report there are no clinically effective inhibitors of biofilm
formation presently available [Musk 2005] However iron salts appeared to inhibit biofilm
formation in a concentration-dependent manner Investigations into the P aeruginosa
genetics show that elevated iron concentrations repress the expression of certain genes
essential for biofilm production in P aeruginosa [Musk 2005] To address the biofilm
problem we have synthesized and characterized iron oxide (magnetite) NPs capped with
biodegradable short-chain carboxylic acid derivatives conjugated to the most common
antibiotic arsenal for the treatment of gram-negative bacteria The functionalized
nanoparticles may carry the drug past the mucus and biofilm layers to target the bacterial
colonies via magnetic gradient-guided transport Additionally the magnetic ferrofluid may
be used under application of an oscillating magnetic field to raise the local temperature
causing biofilm disruption slowed growth and mechanical disruption P aeruginosa can
sustain normal growth at temperatures up to 42 ˚C therefore an increase in the local
temperature may increase the bacterial susceptibility to the antibiotic drugs if not
destroying them This temperature increase would not harm local healthy cells as a
temperature reached by natural fever does not harm healthy tissue It is well-known that
hyperthermia increases the penetration of cytostatic drugs into tissuecells [Witkamp 2001]
and may also increase penetration of drug into biofilms and bacterial colonies In this case
because the drug we are using is beta lactam antibiotic drug which works by interfering
with production of peptidoglycan cell walls increased influx of beta lactam antibiotics into
of healthy mammalian cells would have no effect as they do not have peptidoglycan cell
35
walls Caution must be used however if this technology was used in the delivery of a
chemotherapy agent The healthy tissue (along with the cancerous tissue) would become
more susceptible to the toxic effects of the chemotherapeutic agent [Witkamp 2001]
[Koning 2010] These abilities of the ferrofluid would also treat multi-drug resistant
strains which appear to be increasing in many nosocomial as well as acquired
opportunistic infections
174 Critical Parameters
Particle size prior to polymer engineering is a crucial parameter as polymer
coating and drug conjugation will increase particle diameter Previous studies have shown
that although conventional particles are often entrapped in mucus small sized particles
(120 nm) exceeded the rate of diffusion through mucus when compared to larger particles
(560 nm) [Sanders 2000] These findings are significant since it is now known that the
maximum pore size in CF sputum is 400 nm Therefore an ideal drug carrier would have
to be significantly smaller than 400 nm to enhance the rate of free diffusion of the particles
through mucus pores Our group has previously shown that superparamagnetic iron oxide
nanoparticles (SPIONS) exhibit enhanced diffusion through alginate biofilms using
magnetic field gradient guiding in vitro [McGill 2009a] In addition to magnetic field
guided transport capability MNPs are capable of releasing heat upon placement in an
external oscillating magnetic field [McGill 2009b] Three potential mechanisms are
implicated in heating in the frequency range suitable for human patient treatment Neacuteel
relaxation Brownian motion relaxation and hysteresis losses in the ferro (ferri) magnetic
36
size range This phenomenon is exploited in the application of hyperthermic tumor
destruction or thermotherapy an experimental cancer treatment in which heat released
from MNP placed in an AC magnetic field may be used to kill tumor cells We expect heat
released from MNP hyperthermia would further enhance the magnetic-field-guided particle
movement through the mucus and EPS matrix in the lower respiratory tract by reducing
their viscosity These methods would provide a viable universal treatment method which
would likely increase life expectancy for all CF sufferers without regard to the mutation
type or severity of the disease
18 Overview of Dissertation
In Chapter 2 we describe the synthesis and characterization of iron oxide nanoparticles
(NPs) of which we investigated several sizes and morphologies iron martensite NPs
(Fe16N2) and zero-valent iron NPs (Fe0) These samples were either uncapped or capped
with polyethylene glycol (PEG) for structural and magnetic characterization and either
uncapped or capped with alginate or PEG for in vitro bacterial sensitivity studies Capping
is done by attaching a water-soluble molecule or polymer to the positively-charged NP via
a negatively-charged terminal carboxyl group This is done to enhance solubility of NPs in
water When the iron oxide NPs come out of synthesis they are coated in the metal carrier
molecule oleic acid Oleic acid is a long chain hydrocarbon with a terminal carboxyl group
that attaches to the positively charged metal (Fe+) The long chain hydrocarbon which
remains surrounding the metal NP after its formation contains no other carboxyl carbonyl
or hydroxyl groups and is therefore hydrophobic In order to prevent NP oxidation in air
37
or aqueous solution prevent particle aggregation and allow for drug conjugation the NPs
should be coated with a passivation layer If the NPs are to be used in vivo it is necessary
to coat them with a water-soluble substance otherwise entropic forces would cause them
to aggregate in the aqueous environment of the body For these studies we chose alginate
citrate or polyethylene glycol coatings because they are FDA approved for human
consumption in food and pharmaceuticals The samples were characterized by transmission
electron microscopy (TEM) X-ray diffraction (XRD) and energy dispersive X-ray
spectroscopy (EDS) (Chapters 3 and 4) and tested for magnetic hyperthermia using the
NanoTherics Ltd MagneThermtrade as described in Chapter 4 Spherical magnetite (Fe3O4)
NPs having high iron content and a mean radius between 15 and 25 nm were found to
exhibit the best magnetic properties (Chapter 4) The NPs having a radius lt19 were
superparamagnetic The NPs were further functionalized and conjugated to tobramycin
using EDCsulfo-NHS cross-linking discussed in detail in Chapter 5 The drug-loaded NPs
as well as NP samples with different capping agents were investigated alone Antibiotic
drug was used to test the sensitivities of mucoidal colonies of P aeruginosa at time
intervals from 3-60 days to determine if growth time alters the dosage response the results
of these experiments are described in Chapter 6 Cytotoxicity viability and apoptosis
assays in a human adenocarcinoma cell line were performed on two concentrations of iron
oxide NPs and the results are described in chapter 7 Overall the iron oxide NPs did not
exhibit statistically significant cytotoxicity in this cell line
38
Chapter 2
SYNTHESIS AND CHARACTERIZATION OF IRON
OXIDE NANOPARTICLES
The iron oxides exist naturally the most common phases being hematite (α-Fe2O3)
maghemite (γ-Fe2O3) and magnetite (Fe3O4) [Cornell 2006] Magnetite exhibits the
strongest magnetic properties of all phases of iron oxide [Cornell 2006] [Majewski 2007]
[Teja 2009] which is why it was selected for this application The concept of magnetic-
field-guided drug delivery has existed for over 30 years [Indara 2010] Aside from our
antibacterial application colloidal suspensions of (SPIONs) called ferrofluids have been
proposed for a range of biomedical applications such as magnetic gradient-guided drug
carriers for targeted drug delivery [Sahoo 2003] [Veiseh 2010] cancer thermotherapy
[Hirsch 2003] [Thiesen 2008] and magnetic resonance imaging (MRI) contrast agents
[Kim 2005] [Alexiou 2006]
We have synthesized and characterized magnetic nanoparticles (MNPs) to
overcome the existing barriers and achieve critical improvements in CF therapy which will
increase the life expectancy of CF patients Antibiotic conjugated nanomaterial systems
will facilitate significant enhancement of the efficacy of model therapeutic agents due to
increased diffusion and penetration through mucus and biofilm barriers in cystic fibrosis
when administered directly to the lung as an inhalation aerosol Along with the numerous
39
applications numerous methods for synthesis of SPIONs have been previously published
[Laurent 2008] Various methods include thermal or sonochemical decomposition of iron
pentacarbonyl (Fe(CO)5) [Shafi 2001] [Hyeon 2003] [Wu 2008] microemulsions [Loacutepez
Peacuterez1997] [Santra 2001] [Chin 2007] sol-gel synthesis [Gash 2001] [Lu 2002]
hydrothermal reactions [Hu 2007] [Takami 2007] [Ge 2009] hydrolysis and thermolysis
of precursors [Iida 2007] flow injection syntheses [Salazar-Alvarez 2006] and
electrospray syntheses [Kruis 1998] [Basak 2007]
We have selected a green chemistry solvothermal method for our syntheses due to
the flawless crystallinity morphology control and monodispersity Green chemistry
applied to the practice of synthetic materials engineering focuses not only on minimizing
waste reducing energy use and recycling but also using natural water-soluble non-toxic
or reduced toxicity precursors and reagents When possible petroleum products are
replaced with natural lipids and toxic nitrates are replaced with chloride salts
Iron oxide NPs were synthesized in a high boiling point solvent consisting of inert
hydrocarbons The NP growth was facilitated and somewhat controlled by the organic
carrier molecule oleate At the end of synthesis the NPs remained capped with oleate
Later cap exchange may be performed using either alginate citrate PEG-OH (hydroxyl-
terminated polyethylene glycol) or PEG-COOH (carboxyl-terminated polyethylene
glycol) for water solubility as discussed further in Chapter 5 The synthesis is a
modification of a procedure published elsewhere [Park 2004] Our modifications to this
popular method yielded NPs of various sizes and morphologies achieved by changing the
boiling point of the solvent or reflux time These methods were not previously reported in
40
the literature Additional modifications were made to reduce cost while developing green
chemistry methods Spherical cube-shaped and polymorphous NPs as well as nanowires
were obtained by varying the reaction time and reflux temperature This was achieved by
using higher boiling point organic solvents for higher reaction temperatures In addition
green chemistry and lower-cost alternative chemicals were also investigated
21 Synthesis of Colloidal Magnetite Nanoparticles
The procedure consisted of two steps as described by [Park 2004] synthesis of the iron
oleate precursor complex and synthesis of the iron oxide NPs The precursor was iron
oleate (iron(II III) [(9Z)-9-octadecenoate] n) where n is the coordination number of iron
and could form a monomer dimer or trimer [Bronstein 2007] [Palchoudhury 2011]
produced in our laboratory using a modified procedure of Bronstein et al [Bronstein
2007] The iron oleate complex was formed from the combination of sodium oleate salt
(sodium (9Z)-9 octadecenoate) and iron(III) chloride hexahydrate (FeCl3middot6H2O) The
precursor preparation was modified by washing with water ethanol and acetone to
remove additional contaminants before aging in the oven overnight
211 Materials
FeCl3middot6H2O (97) was purchased from Sigma-Aldrich n-docosane (99) and n-eicosane
(99) were purchased from Alfa Aesar n-dodecane (gt99) was purchased from Fischer
Scientific sodium oleate (gt97) was purchased from Tokyo Chemical Industry Co
41
hexanes (95) ethanol (99) and acetone (99) were purchased from EMD Chemicals
Inc All chemicals and their physical properties may be referenced in Appendix I
212 Synthesis of Iron Oleate Precursor Complex
In a standard reaction 675 g of FeCl3middot6H2O was combined with 25 mL of deionized
water and vacuum-filtered through 022 μm filter paper The mixture was then combined
with 2435 g of sodium oleate in a three-neck round-bottom flask 150 mL of a stock
solution consisting of a 246 mixture of deionized water ethanol and hexane was added
to the flask Under argon flow the mixture was vented and filled
Figure 21 Ion exchange reaction between iron(III) chloride and sodium oleate producing
iron oleate and the byproduct sodium chloride Image by L Armijo 2012
42
for three one-minute intervals to remove all oxygen from the reaction flask The solution
was then slowly (5 degCmin) heated to 50 degC under vigorous stirring
Once the solid sodium oleate had completely melted and the reflux had begun
(around 50ndash60 degC) the temperature was further increased (3 degCmin) to 70 degC and the flask
was kept at this temperature for four hours ensuring that the total reflux time was 4 hours
The mixture was then cooled to 60 degC and washed three times with deionized water in a
separatory flask if necessary additional hexane was added to dissolve the organic layer
The product was then washed twice with 12 mL aliquots of acetone and ethanol The
organic layer was placed in a rotary evaporator (Rotovap) with the water bath set at 30 degC
until the hexane and ethanol were evaporated away The resulting waxy complex was then
dried in a vacuum oven for 24 hours at 70 degC The final product was a waxy dark-brown
solid The overall reaction is illustrated in Figure 21
213 Synthesis of Cubic Polymorphous and Spherical Nanoparticles
Using a 500 mL three-neck-flask attached to the Schlenk line (Figure 23) the reaction was
carried out In a standard reaction 5 g of iron oleate (washed with water for cubes and
water acetone and ethanol for spheres) was combined with 56 mL of oleic acid and
1315 g of n-eicosane (boiling point 3427 degC) The mixture was slowly heated (3 degCmin)
to 50 degC under argon flow and vigorous stirring Once the reactants had dissolved the
temperature was further increased to 342 degC at a heating rate of 30 degCmin For 19 nm
cubes the mixture was refluxed for 30 minutes For larger particles the reflux time was
extended with an average growth rate of 22 nm per minute The maximum size
43
achievable without adding additional reagents was 250 nm after 99 min If the solution was
allowed to cool below the nucleation temperature (~ 200 degC) [Bronstein 2007] for any
amount of time before being refluxed at the same maximum temperature again the NP
growth favored spherical morphology in which polymorphous NPs represented an
intermediate morphology (see Figure 22) It appears from these results that the spherical
morphology is thermodynamically favored exhibiting stability at high temperatures After
30 minutes the spheres were highly monodisperse
Figure 22 Morphology alterations of iron oxide nanoparticles via
additional nucleation event(s)
214 Synthesis of Iron Oxide Nanowires
In a standard reaction 5 g of iron oleate was combined with 16 mL of oleic acid and
1315 g of n-dodecane (boiling point 2162 degC) The mixture was slowly (3 degCmin)
heated to 50 degC under argon flow and vigorous stirring For ~55times2 nm wires once the
reactants had dissolved the temperature was further increased to 216 degC at a heating rate
44
of 3 degC per minute and the mixture was refluxed for 60minutes For smaller wires ~25times2
nm the reflux was carried out at 150 degC for the same time These findings confirm those
reported by [Palchoudhury 2011]
Figure 23 Active iron oxide nanoparticle synthesis in the Schlenk line
215 Synthesis of Spherical Nanoparticles
In a standard reaction 5 g of iron oleate was combined with 16 mL of oleic acid and
45
1315 g of n-docosane (boiling point 370 degC) The mixture was slowly heated to 50 degC at a
heating rate of 3degC per minute under argon flow and vigorous stirring Once the reactants
had dissolved the temperature was further increased to 370 degC at a heating rate of
3degCmin For ~20 nm particles the mixture was allowed to reflux for 32 minutes For
larger particles the reflux time was extended with an average growth rate of 16 nm per
minute The maximum size without adding additional reagents was 158 nm after 99 min
Figure 24 Summary of morphology control time and temperature
parameters established by this study for the synthesis of Fe3O4 NPs
46
216 Summary of Green Chemistry Modifications
The sustainability of novel materials is crucial to human progress Ensuring environmental
friendliness the engineering process and integrating natural compounds into the materials
was a priority in these studies Naturally existing molecules may have lower cytotoxicity
compared with synthetic products and are less likely to detrimentally affect the delicate
ecosystem upon disposal In addition the procedure for manufacturing many synthetic or
purified compounds is typically not environmentally friendly The Environmental
Protection Agency (EPA) standards for green chemistry [EPA 2015] are very clear reduce
waste maximize yield use less hazardous materials minimize accident risk By simply
replacing the popular metal nitrate precursors with water soluble chloride salts many
environmental benefits are suggested Our environmentally-friendly carrier molecule and
stabilizing agent oleic acid is a derivative of vegetable oil
217 Cost Reduction
A lower cost and environmentally sound modification may be made to the aforementioned
procedures by simply replacing the high molecular weight hydrocarbon solvent with
paraffin wax (bp gt370 ˚C) or the natural solvent beeswax Paraffin wax or beeswax may
be thermally separated for reuse as well rather than disposing of solvents after each
synthesis This simple green chemistry modification resulted in the same monodisperse
NPs above Docosane costs $7860 for 100 g or ~$079 per gram (Sigma-Aldrich)
paraffin wax costs $5 for 453 g (1 lb) This roughly corresponds to ~$001 per gram
compared to the cost of docosane thereby reducing the cost by 987 Beeswax costs ~$9
47
for 453 g (1 lb) or approximately $002 per gram resulting in a cost reduction of 974
In addition we have used re-distilled solvents and saved them for use in future NP
syntheses These solvents were processed and purified in-house to further reduce cost and
eliminate toxic waste
22 Structural Characterization
The transmission electron microscope (TEM) images in Figures 25 through 27 show the
various morphologies and sizes of Fe3O4 NPs we were able to obtain using this method
Cubic NPs having a maximum size of 250 nm were obtained after a 99-minute reflux
Figure 25 Transmission electron microscope image of 250 nm magnetite nanocube
(left image) formed after 99-minute reflux scale bar is 100 nm and ~15 nm spherical
NPs (right image) scale bar is 10 nm
Cubic and spherical NPs were easily produced with high monodispersity in sizes ranging
from 16 to 250 nm Polymorphous NPs shown in Figure 26a were obtained by allowing the
cubic NPs to cool below their nucleation temperature of 200 ˚C prior to refluxing above the
48
nucleation temperature Interestingly while attempting to measure particle size during
synthesis by taking aliquots of the NPs as time went on the temperature controller failed
triggering the power supply to shut-off and the particles cooled to room temperature In hopes
of salvaging the experiment we returned the temperature to 340 ordmC We found that the aliquot
taken after a 3-minute reflux performed after allowing the sample to cool below the nucleation
temperature was polymorphous
Figure 26 Transmission electron microscopy (TEM) images of magnetite
nanoparticles capped with oleic acid a) Polymorphous NPs scale bar is 100 nm
b) monodisperse spheres formed from refluxing of polymorphous NPs scale bar
is 100 nm c) monodisperse spherical NPs ~22 nm in diameter scale bar is 100
nm d) nanowires scale bar is 50 nm [Armijo 2012a]
49
A second aliquot taken after 30 minutes of refluxing consisted of monodisperse
spherical NPs These findings suggest that the spherical morphology may be favored at
higher temperatures due to growth on all faces Monodisperse spheres with a diameter
of ~30 nm in Figure26b formed from polymorphous NPs shown in Figure 26a
when the reaction mixture was allowed below the nucleation temperature of 200 ˚C
for approximately 30 minutes before being refluxed again Spheres of ~22 nm in
diameter (Figure 26c) and 55times2 nm nanowires (Figure 26d) were made in n-
docosane (boiling point 370 degC) and n-dodecane (boiling point 2162 degC)
respectively [Armijo 2012a] We performed high-resolution (HR) TEM to
characterize morphology and to confirm high crystallinity of the NPs
Figure 27 Transmission electron microscope (TEM) image of Fe3O4 spherical
superparamagnetic nanoparticles capped with oleic acid This sample was chosen for
bacterial sensitivity studies discussed in Chapter 6 due to its excellent monodispersity
and superparamagnetic properties scale bar is 50 nm
50
Figure 28 High-resolution transmission electron microscope
(TEM) image and its fast Fourier transform (FFT) of the iron
oxide monodisperse spheres (shown in Figure 26b above) scale
bar is 5 nm
The image in Figure 28 represents fringes observed for the monodisperse spheres from
Figure 26b The TEM images demonstrate the wide range of NP sizes and morphologies
attainable with minor time and temperature modifications to the procedure
Elemental composition of the Fe3O4 NPs was verified with energy dispersive x-ray
spectroscopy (EDS) and example is shown in Figure 29 Magnetite samples of all
morphologies gave the same spectrum in EDS therefore presented the same elemental
composition Iron and oxygen are present in the monodisperse spheres from Figure 26b
The carbon and copper peaks are due to the carbon-coated copper grid
The x-ray diffraction (XRD) data for iron oxide polymorphous nanoparticles
(Figure 210) and the XRD data for the ~17 nm spherical particles are similar and suggests
51
that the composition of the nanoparticles synthesized by this method to be ~70 (plusmn5)
magnetite Fe3O4 with space group Fd3mF41d32m due to a perfect card match to the
major peaks in the crystallography database
Figure 29 Energy dispersive x-ray spectroscopy (EDS) spectrum of magnetite
nanoparticles This particular spectrum was taken from the monodisperse spherical
NP sample imaged in Figure 26b
However it is important to note that the several of the peaks assigned [220] [311] [400]
[440] [422] and [511] which match magnetite in the database correspond to the spinel
phase Spinel phase peaks are present in XRD spectra of both γ-Fe2O3 and Fe3O4 as well
as multiphase crystals containing these phases [Casula 2006] [Bronstien 2007] who also
characterized SPIONS synthesized by this method attributed these peaks to (likely) being
Fe3O4 as do we The remaining 30 of the crystal appears to be composed of ferrous oxide
wuumlstite (Fe1-xO) where x can be between 005 and 017 and α-Fe2O3 The Wuumlstite is a
52
phase of iron(II) composing meteorites The presence of this highly dense highly
magnetic phase is typical of iron oxides produced under low oxygen conditions [Casula
2006] There are small peaks at ~56deg and 84deg which match to the [116] and [128] of α-
Fe2O3 possibly the result of surface oxidation Since the wuumlstite phase is metastable it is
known to convert to α-iron and magnetite or a mixture of wustite α-iron and magnetite
[Redl 2004] The α-iron is reported to accumulate on the shell where on exposure to
atmosphere it oxidizes [Bronstein 2007] which would explain why it is not detected on
the XRD however α-Fe2O3 is Wuumlstite and magnetite are structurally similar and likely
compatible in a multiphase crystal therefore it is not uncommon to observe both phases
together [Bronstein 2007] Magnetite and magemite are indistinguishable from one another
by XRD analysis [Bronstein 2007] It is important to note that due to the similarity in space
groups and lattice constant the oxidation state of iron oxide phases is difficult to determine
with absolute certainty using XRD
Figure 210 XRD spectrum of polymorphous nanoparticles (NPs) (pictured in
Fig 26a) The majority of the prominent peaks in this spectrum correspond to
magnetite or spinel phase iron oxide
53
12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
0
1000
2000
3000
4000
5000
Inte
nsity (
cps)
2-Theta (degree)
[111]
[110]
[311]
[220][422]
[011]
Figure 211 X-ray diffraction (XRD) spectrum of 17 nm spherical NPs taken
with monochromator attached for noise reduction This spectrum also shows
spinel phase hematite and magnetite peaks
23 Summary of Findings
We synthesized and characterized magnetite NPs having various sizes and morphologies
using green chemistry methods Our synthesis method significantly reduces cost while
producing superior nanomaterials while exercising environmental consciousness We were
able to synthesize iron oxide nanowires at a temperature that was not previously believed
to facilitate NP formation [Palchoudhury 2011] We have also shown that spherical
particles are the most thermodynamically stable
54
Although we thoroughly investigated the physical properties of all three NP
morphologies it was decided that the magnetic properties of spherical NPs in the
superparamagnetic size range (lt20 nm) were best for our specific application For
magnetic characterization refer to Chapter 3 In addition because this material will be
administered to the lung lower aspect ratios NPs (spheres rather than nanowires) are
anticipated to have lower cytotoxicity than high aspect ratio NPs Furthermore the
procedure for synthesizing spherical NPs is much simpler than the procedures for the
synthesis of other morphologies because the temperature range required for successful
synthesis is not as narrow Spherical NPs having an easily reproducible synthesis
procedure can be used to provide for further studies thus ensuring minimal variation
between batches For these reasons the 16-18 nm spherical NPs (Figure 27) were used in
the bacterial sensitivity studies discussed in Chapter 6
55
Chapter 3
MAGNETIC CHARACTERIZATION OF IRON OXIDE
NANOPARTICLES AND
MAGNETIC HYPERTHERMIA INVESTIGATIONS
The history of magnetism in medicine is extensive The first report of the use of magnetite
powder for in vivo medical treatment of iron poisoning was in the 11th century AD by the
Persian polymath Avicenna [Haumlfeli 1998] Since the dawn of the era of nanotechnology
thousands of papers have been published proposing uses of nanoscale grain sized magnetic
powders for many biomedical applications One important application which has arisen
only after the modern medicine acknowledged the fact that magnetic fields are not
especially contraindicated for humans is medical hyperthermia [Mornet 2004] also called
thermotherapyThe therapeutic potential of heat has been known for a very long time
beginning with the recognition that fever enhancement promotes faster recovery from
illness by homeopaths it is now known that heat can be used to cure a variety of different
diseases [Andrauml 2007] Heat use for cancer therapy and tumor cauterization has been
proposed since as early as 3000 BC [Strohbehn 1984] A more contemporary historical
account actually suggested the use of lower temperatures which would not cause damage
to healthy tissues (hyperthermia vs cauterization) [Busch 1866] In the past 150 years
much work has been done attempting to maximize heat effects in a local area of interest
56
with varying degrees of success [Streffer 1987] [Baronzio 2010] [Minev 2011] [Moros
2013]
In magnetic thermotherapy the response of MNPs to oscillating magnetic field
causes thermal energy to be dissipated into the surroundings killing the adjacent cells
Additionally hyperthermia can be used to enhance radiation and chemotherapy treatment
of cancer [Praetorius 2007] [Krishnan 2010] [Maier-Hauff 2011] As mentioned in
Chapter 1 one of our original aims was to use the hyperthermic heat evolved to thin the
alginate biofilm characteristic of chronic Pseudomonas aeruginosa lung infections and the
thick CF mucus barrier in conjunction with magnetic gradient guided drug delivery to
deliver antibiotic drug to the infected area Magnetic hyperthermia results from domain
switching upon AC electromagnetic (EM) radiation application Our group previously
investigated iron oxide nanoparticles for heating applications [Armijo 2012a] [Armijo
2012b] however the major mechanism involved in the temperature increases in these
specific nanomaterials have only now been uncovered Biomedical applications require a
material with a large magnetic moment as well as the control of the magnetic properties
imparted by superparamagnetism The attractive property of superparamagnetic materials
relates to the ability of the physician to induce their magnetic properties only after the
magnetic nanoparticles have arrived at the area of interest by application of an external
magnetic field This allows for venous delivery without agglomeration within the blood
vessels Iron-containing nanomaterials having high saturation magnetic moments in the
SPM size-range are attractive for in vivo use The iron oxides specifically have
demonstrated high biocompatibility and low systemic toxicity [Maier-Hauff 2011]
57
[Soenen 2010] [Soenen 2011] as well as having received FDA approval for use as contrast
agents in magnetic resonance imaging (MRI) [Shieh 2005] [Veiseh 2005] We have
investigated magnetic properties of iron oxide nanomaterials in the 15-30 nm size range for
this potential application This size range was chosen because it is close to the single-
domain multi-domain size limit for iron oxides 20-30 nm This size range has shown the
greatest temperature increase under oscillating magnetic field application at many of the
frequencies being investigated for medical hyperthermia in our case 1111 kHz with a
magnetic field of 25 mT [Hergt 2006]
31 Theory
Considering nanoscale colloidal suspensions of superparamagnetic NPs also called
ferrofluids the dominant relaxation mechanism resulting in heat generation could be due to
Brownian motion [Maier-Hauff 2011] or Neacuteel relaxation [Shieh 2005] Figure 31A
illustrates Neacuteel losses magnetic losses owing to domain wall displacements Figure 31B
(lower image) shows Brownian losses energy loss from mechanical rotation of the
particles in a colloidal suspension acting against viscous forces of medium Heat energy
generated results from the contribution of both energy loss effects Figure 31 is the
analysis of the AC data allows us to determine which of these mechanisms dominates
Brownian or Neacuteel relaxation of the particles
Single domain particles have a magnetic moment mp given by
mp = Msυ (31)
58
Where Ms denotes the saturation magnetization and υ is the magnetic volume of the
particle The Brownian relaxation time τB is given by
τB = 4πr3η=kBT (32)
Where r is the hydrodynamic radius η is the dynamic viscosity of the solvent kB is
Boltzmanrsquos constant and T is absolute temperature (K)
For uniaxial anisotropy the Neel relaxation energy barrier is given by Kυ where K
is the anisotropy value of the particle The associated magnetic moment direction reversal
or domain switching time is given by [Fannin 1989] [Fannin 1994] [Neel 1949] [Preacutevot
2001]
τN = τ0exp(σ) (33)
Where τ0 is a damping time having an average value of 10-9 s and σ=KυkT
Figure 31 Mechanisms of energy loss leading to heat production in magnetic
hyperthermia A) Models Neacuteel relaxation the magnetic field flipping within the
NP B) Models Brownian motion the entire particle moves within the solvent
59
For polydisperse samples combinations of the mechanisms respond to the heating in AC
fields This is why it is crucial to use a monodisperse sample for magnetic characterization
These samples were characterized using an effective relaxation time as follows
τeff=τNτB(τNτB) (34)
In which the dominant mechanism is the one with the shortest relaxation time [Fannin
1989] [Shliomis 1974] [Shliomis 1993] This is analogous to the dominant rate of a
chemical reaction being the slowest step
32 Experimental
A typical feature of magnetic nanocrystals is their irreversible ferromagnetic behavior
below the blocking temperature TB and reversible magnetization above it caused by
superparamagnetic behavior of the nanocrystals We investigated the magnetic
properties blocking temperature magnetic saturation and coercivity (hysteresis) using
a superconducting quantum interference device (SQUID) magnetometer We investigated
the heat evolved at two different frequencies and magnetic field strengths using the
nanoTherics Ltd MagneTherm trade Later we looked at the dominant loss mechanism
under AC field at room temperature using the DynoMagreg AC Susceptometer (IMEGO
AB Sweden)
321 SQUID Magnetic Characterization of Iron Oxide Nanoparticles
The blocking temperature can be found experimentally by measuring
magnetization under field-cooling (FC) and zero-field cooling (ZFC) conditions Below
60
TB the Neacuteel relaxation time τN is larger than the measurement time τm (typically 100 s)
and magnetization depends strongly on the field history Above TB magnetization is
strongly affected by thermal fluctuations (τmgtτN) making FC and ZFC curves coincide In
other words for a given measurement time τm hysteretic behavior observed below TB
would not be observed above TB
0 50 100 150 200 250 300 350
00004
00006
00008
00010
00012
00014
00016
Field-Cooled
Zero Field-Cooled
Mag
ne
tizatio
n (
em
u)
Temperature (K)
Figure 32 Magnetization vs temperature for polymorphous Fe3O4 nanoparticles
(NPs) zero-field cooled (circle symbols) and field cooled (square symbols) We
measured temperature dependence of magnetization for the Fe3O4 NP samples under
ZFC and FC conditions The DC (τm = 100 s) magnetization of the ferrofluid
samples was measured with a dc field of 100 Oe in the temperature range between 9
K and 350 K Data shown in Figure 32 for 22 nm spherical particles [Armijo
2012a]
In the entire temperature range up to 350 K the Fe3O4 NP samples demonstrated strong
ferriferromagnetic behavior as evidenced by the gap between the ZFC and FC curves
61
persisting even at 350 K (Figure 32) From the ZFC curve we can loosely estimate TB to
be ~175 K but even above that temperature equilibrium magnetization of the nanocrystal
sample was not reached and superparamagnetic behavior of the nanocrystals was not
observed
Figure 33 Ferromagnetic hysteresis loops for (a) Fe3O4 polymorphous nanocrystals
and (b) ~22 nm spherical Fe3O4 nanospheres τm= 100 s Left full sweep of magnetic
field measured at 293 K showing saturation Right enlarged loop measured at 293K
at low field [Armijo 2012a]
Strong ferromagnetic behavior of the Fe3O4 nanocrystal samples was confirmed in
magnetic hysteresis measurements Consistent with the results of dc magnetization
measurements magnetic hysteresis measurements at 293 K performed on Fe3O4
62
polymorphous NPs (Figure 33a) find large coercivity ~37 mT (~29 kAm) at 100 s
measurement time Even larger coercivity of ~119 mT (~947 kAm) was measured for ~22
nm Fe3O4 nanospheres
An additional sample which consisted of 17 nm spherical particles displayed no
hysteresis under full magnetization vs field strength (MH) sweep (Figure 34) This
demonstrates the significance of a 5 nm size difference on the magnetic properties Zero
coercivity is a typical feature of superparamagnetic materials [Cai 2007] Magnetite NPs in
this size range (10-20 nm) were the only samples shown to be truly superparamagnetic
and therefore are of the greatest interest for in vivo use
Figure 34 Superparamagnetic hysteresis loop for 17 nm spherical particles
showing no coercivity (hysteresis) thus superparamagnetic properties
63
Figure 35 MagneThermtrade inductive heater setup in its entirety Shows
MagneThermtrade function generator DC power supply oscilloscope and infrared
thermometer
Figure 36 Inside of MagneThermtrade inductive heater with front cover removed
shows inductor (coil) and capacitor (black box on right) clear hoses above and right carry
cooling water
64
322 Magnetic Hyperthermia Experiments
Magnetic hyperthermia for the Fe3O4 NP samples was tested using the nanoTherics Ltd
MagneThermtrade which operates at frequencies between 100 and 1000 kHz The
MagneTherm is frequency tunable changing capacitor and or inductors out The ranges of
frequencies and magnetic field strengths that may be achieved using this equipment were
calculated and may be referenced in Appendix III
In Figure 36 you can see the number of coils on the inductor by changing out coils
and capacitors you can tune to a range of frequencies (and magnetic field strengths) The
temperature of the sample was measured using the Omega HHTFO-A portable fiber optic
data logger thermometer version 1025 with 01 degC resolution Samples were prepared as
described in Chapters 2 and 4 coated with water soluble polymer as described in Chapter
5 and dispersed in deionized water All concentrations were 30 mgmL and sample
volumes were 5 mL The NPs compared in the following graphs were 22 nm spheres
polymorphous nanocrystals and 55 times 2 nm wires Later we investigated the heating of ~17
nm superparamagnetic spherical particles in water and in viscous (glycerol) media The
heating efficiency of the NP samples was tested at frequencies of 1111 kHz and 6292
kHz Data acquisition for hyperthermia was started at ambient temperature Figure 37a
shows the heating of NPs of various morphologies 22 nm spheres 25 nm polymorphous
NPs and 55x2 nm wires at a frequency of 1111 kHz (magnetic field of 25 mT) Figure
37b shows the heating of the same NPs at a frequency of 6292 kHz (magnetic field of 9
mT) The spherical and polymorphous particles follow a similar trend consistent with their
similar morphology and particle volume Although they do heat at the frequency of 1111
65
kHz the observed heating was relatively low Interestingly the total increase in
temperature after 40 minutes was 6 degC for spheres whereas it was only 1 degC for
Wire (black)
0 5 10 15 20 25 30 35 40 4515
18
21
24
27
30
33
36
39
42
45
48
Tem
pera
ture
(degC
)
Time (minutes)
Wires
Polymorphous
Spheres
Frequency f =1111 kHza)
0 5 10 15 20 25 30 35
24
26
28
30
32
34
36
38
40
42
Tem
pera
ture
(degC
)
Time (minutes)
Wires
Polymorphous
Spheres
Frequency f=6292 kHzb)
1111 kHz
6292 kHz
Spherical (blue)
Poloymorphus (red)
Figure 37
Magnetic hyperthermia
results for particles in the
ferriferromagnetic size
range in AC field comparing
the frequency dependence for
different MNP morphologies
22 nm spheres
polymorphous MNPs and
55x22 nm wires taken at at
the following frequencies
magnetic field strengths
(a) 1111 kHz 25 mT and
(b) 6292 kHz 9 mT
Adapted from [Armijo 2012a]
66
polymorphous NPs However as shown in Figure 38 the 17 nm spherical particles
performed significantly better at low frequency with a total temperature increase of 25 ˚C
When the frequency of the oscillating magnetic field was increased to 6292 kHz the
spheres and polymorphous NPs showed increased heating
This temperature increase can likely be attributed to a size effect However unlike
the data obtained at 1111 kHz the total temperature increase was similar for the
polymorphous NPs and NPs of spherical shape 16deg C for spheres and 15 degC for
polymorphous NPs As for the nanowires the observed trend was just the opposite The
total temperature increase at 1111 kHz after 40 minutes was a remarkable 30deg C and
notably saturation of the temperature was not reached in this timeframe At 6292 kHz
however the increase in temperature generated by the wires was much less than the one
obtained by spheres and polymorphous NPs representing the total temperature increase of
4 degC
It has been shown that the transition from ferromagnetic to superparamagnetic
behavior is associated with the change of the loss mechanism and accordingly of the
heating effect of magnetic NPs in hyperthermia experiments Hysteresis losses dominate in
ferromagnetic nanocrystals whereas heat production in superparamagnetic ones is due to
relaxation losses Since the blocking temperature TB explicitly depends on the
measurement time τm (inversely proportional to the frequency of the oscillating magnetic
field) superparamagnetic NPs as measured in DC magnetization experiments become
ferromagnetic at sufficiently high frequencies gt 1τN (or gt 1τ where τ = τΝτΒ (τΝ + τΒ) if
both Neacuteel and Brownian relaxation mechanisms are present) and generate heat due to
67
hysteresis losses With the Fe3O4 NP samples demonstrating strong ferromagnetic behavior
in DC magnetization and hysteresis measurements hysteresis losses are expected to be the
main mechanism of heating in the operating frequency range of our hyperthermia
experiments
We estimated the Neacuteel relaxation time τN at room temperature for the Fe3O4
polymorphous NPs (Figure 31) as follows
τN = τ0exp(EbkT) (34)
Where Eb is the magnetic anisotropy energy barrier k is the Boltzmann constant and
τ0=10minus10 s is the attempt time Eb is related to the blocking temperature TB as Eb =
kTBln(τmτ0) = 276 kTB and we arrive at τN = τ0exp(276 TBT) for the Neacuteel relaxation
time At T = 300 K τN asymp 0001 s At the frequencies of interest ω gtgt 1τN which is far
from the relaxation resonance the Neacuteel relaxation losses saturate at a level that is
negligible for large enough τN [Hergt 1998] Therefore we will interpret our results based
on the mechanism of hysteresis losses prevailing for NPs in this size range When
hysteresis losses are the main heating factor heating power is proportional to the area of
the hysteresis loop and to the frequency of the applied magnetic field An approximately 6-
fold increase in the heating power is expected when the frequency is changed from 1111
kHz to 6292 kHz The observed increase in heating power from the Fe3O4 polymorphous
NPs and nanospheres is not that large We note however that both the frequency and
amplitude of the magnetic field were changed in our experiments and the magnetic field
strength was reduced from 25 mT at 1111 kHz to 9 mT at 6292 kHz which can explain
68
the heating power increase not being proportional to the frequency for the Fe3O4
polymorphous NPs and nanospheres The higher temperature increase of 6 degC for the
spheres compared to 1 degC for the polymorphous NPs at 1111 kHz can be explained by a
significantly larger area of their hysteresis However the difference in the specific heat
production between the spherical and polymorphous NPs at 6292 kHz is not significantly
pronounced
We consider hyperthermia experiments with nanowires separately as their
morphology differs dramatically from that of polymorphous NPs and nanospheres and may
be the decisive factor Fine magnetite particles of needle shape with high aspect ratio have
been investigated previously by [Hergt 1998] High potential for hyperthermia was noted
there for the particles that possess very high shape anisotropy and hence high-energy
barrier for remagnetization resulting in a wide hysteresis and high hysteresis losses It was
concluded however that strong magnetic fields very often unacceptable for human
patients are required to fully utilize their potential Very strong nonlinear dependence of
the hysteresis loss on the strength of the applied magnetic field was reported Comparison
was made among particles of different shapes and it was found that needles were by far
superior when applied magnetic field exceeded ~35 kAm (4389 mT) while below that
value the magnetic field was not strong enough to open the hysteresis loop in needles and
their hysteresis losses were by several orders of magnitude lower compared to particles of
other shapes with low aspect ratio We expect similar effects to be observed in nanowires
that are characterized with even higher aspect ratios of their shape We believe that our
results for hyperthermia in nanowires can be explained by similar superlinear dependence
69
of their hysteresis loss on the magnetic field strength with that superlinear dependence
being much stronger than mere proportionality of the heating power to the frequency of the
applied magnetic field
Figure 38 Hyperthermia results for superparamagnetic NPs having
an average diameter of 17 nm in water and waterglycerol mixture
having high viscosity Data was taken at a frequency of 1111 kHz
with a magnetic field of 25 mT in the inductor
We note that the remarkable 30 degC temperature increase was observed in nanowire
sample at 111 kHz and magnetic field of 25 mT (196 kAm) which is very close to the
typical values used in medical treatments 100 kHz and 20 mT [Wust 2006] [Mehdaoui
2011] Smaller spherical particles having an average diameter of 17 nm were shown to
perform comparably (see Figure 38) at low frequency In order to verify our findings in
viscous media an additional sample consisting of 16-20 nm spherical magnetite particles
0 10 20 30 40 5020
30
40
50
Tem
per
ature
[d
egre
es C
elsi
us]
Time [min]
Fe3O
4 NPs in water
Fe3O
4 NPs in glycerol mixture
70
was characterized Figure 38 shows the summary of hyperthermia experiments with the
SPIONs dispersed in water and in aqueous glycerol (50 ww) mixture six times more
viscous than water alone
Additionally no temperature increase was observed for the control experiments
using DI water under the same AC field and under identical experimental conditions with
no NPs (not shown) With this sample we were able to achieve a total temperature increase
in excess of 25 degC and the initial fast heating rate of ~4 degCmin decreased to ~02 degCmin
after 12 minutes This confirms the heat-generation is a result of the magnetic AC energy
absorption by the magnetic component of the ferrofluid samples Due to the similar heating
trends in water and high viscosity aqueous glycerol we attributed most of the heating
losses to a Neacuteel process This further verifies our susceptometry findings (recall that NP
samples below 20 nm in size displayed no coercivity (hysteresis losses) in MH curves)
323 AC Susceptometry
Measurements of the frequency-dependent volume susceptibility in the frequency range
1 Hz to 100 kHz were performed using the DynoMagreg (IMEGOAB Sweden) with a
frequency range from 1 Hz to 200 kHz a resolution magnetic moment of 3times10-11 Am2 and
excitation amplitude of 05 mT The ferrofluid magnetite (Fe3O4) sample 1 and 2 in water
solvent at a concentration of 130 M was measured using a 200-microL sample Measurements
were performed on a sample which consisted of the base ferrofluid colloidal suspension of
magnetite (Fe3O4) particles having spherical morphology of mean particle diameter 15 nm
in deionized water solvent with succinylated PEG as a capping agent
71
Susceptometry data verify the magnetic hysteresis measurements in which we
found that the sample was superparamagnetic at room temperature The susceptometry
measurements demonstrate a single peak which we attribute to a Neacuteel process in which
τN=129x10-6 ms Assuming the superparamagnetism the Neacuteel relaxation time of moment
rotations activated by thermal fluctuation is given by Eq (43) and (44) with where V =
1767x10-24 m3 for the 15 nm diameter spherical SPIONs When kBT gt KuV the magnetic
moment flips during the measurement time demonstrating zero coercivity Presently the
effective anisotropy energy (Ku) of the iron oxide sample may be estimated to be 42 times 105
ergscc by the relation KuV = 25kBTB (assuming TB = 215 K) [Zhang 2010] higher than the
Ku of bulk Fe3O4 (Ku = 64 times 104) due to additional anisotropies which agrees with the
findings of [Zhang 2010] for particles of similar size The effective anisotropy energy of
the iron nitride sample was calculated to be 56 x105 ergscc A reference value for bulk
Fe16N2 is not presently available in the literature The real part of the susceptibility (χrsquo)
values for both samples was greater than zero a typical feature of ferriferromagnetic
materials Despite this the χrsquo value for iron nitride is two times higher than the value for
iron oxide As expected the real part of the susceptibility (χrsquo) curve remains above zero for
both materials a typical feature of ferriferromagnetic materials
33 Summary of Findings
We characterized the magnetic properties of iron oxide NPs of various morphologies in the
paramagnetic to ferromagnetic size range thus allowing for further functionalization and
drug conjugation DC magnetization and AC heating power (hyperthermia characteristics)
72
of the Fe3O4 NPs in water have been studied The Fe3O4 NPs samples having a mean
diameter gt20 nm demonstrated strong ferromagnetic behavior and hysteresis losses were
identified as the main mechanism of heating in hyperthermia experiments Whereas the
NP samples having a mean diameter of 15-17 nm demonstrated superparamagnetism and
Neacuteel relaxation appears to be the dominant heating mechanism Our hyperthermia data
shows that all three NP morphologies spheres polymorphous NPs and wires are good
candidates for thermotherapy Significant heating was observed well within the limits for
oscillating magnetic field parameters established for biological applications The observed
temperature increase for 22 nm Fe3O4 nanospheres at 1111 kHz and 25 mT after 40 min
was 6 degC If the corresponding temperature increase took place from normal human body
temperature (366 degC) as a starting point it would bring the local temperature up to 426
degC which is right within the desirable temperature limits for the applications of medical
hyperthermia (41ndash45 degC) [Hergt 2006] In the waterglycerol study we showed that the
initial fast heating rate of ~4 degCmin decreased to ~02 degCmin after 12 minutes (when the
temperature reached 45 degC) Although the heating rate is not linear and appears to slow
down as a function of temperature (when the slope between one point and the next is
considered) the data points considered to be within the initial fast heating rate are the data
points of interest for medical hyperthermia (36-42 degC) Of special interest for hyperthermia
applications the nanowires demonstrated a remarkable 30 degC temperature increase and the
superparamagnetic (~17 nm) spherical particles demonstrated a 25 degC temperature increase
under magnetic field conditions that were very close to (or lower than) the typical values
used in medical treatments
73
Chapter 4
SYNTHESIS AND CHARACTERIZATION OF HIGHLY
SUPERPARAMAGNETIC IRON NITRIDE
NANOPARTICLES (Fe16N2)
The Fe16N2 (martensite) phase is of interest for our application and many others not just
because it is in-fact the most magnetic material in the world [Kim 1972] [Ji 2010] but
also because it is free from toxic cobalt and the costly rare-earth elements Using a stronger
magnet has many benefits for gradient-guided drug delivery one obvious one being an
anticipated increased in the active transport rate due to a stronger interaction with the
external applied field Zero-valent iron is another highly magnetic phase which serves as
an intermediate in the synthesis of iron nitride described in Section 43 Samples of zero-
valent iron were taken from this procedure for use in bacterial sensitivity studies described
in Chapter 6
Over 20 years ago the iron nitride phase having the empirical formula Fe16N2 and
the specific phase of αrdquo was claimed to possess a giant saturation magnetization (Msat)
[Metzger 1994] [Ji 2010] At that time it was well established that iron cobalt having the
formula Fe65Co35 composed the strongest magnet in the world [OrsquoHandley 2000]
However it was suggested that Fe16N2 might possess a saturation magnetization far
exceeding the iron-cobalt alloy [Metzger 1994] The αrdquo-Fe16N2 phase was first discovered
74
prior to 1950 the procedure having been published in 1951 when researchers initially
characterize the temperature dependence on the formation of different phases of iron
nitrides [Jack 1951] In this paper phase diagrams for iron-nitrogen systems were
proposed and the αrdquo-Fe16N2 phase was described as a metastable crystal formed from rapid
quenching of γ-FeN (austenite) [Jack 1951] Unfortunately the magnetic properties were
not measured and it was not until 1972 after the giant saturation magnetization was
finally measured that interest in this material finally peaked [Kim 1972] Since then there
have been many attempts to synthesize this material as the sole phase in a crystal system
however typically these findings report the presence of a phase of αrdquo-Fe16N2 mixed in with
other phases of iron nitride iron oxide or alpha iron [Comstock 2002] Even now more
than 40 years later a need still existed to engineer single-phase crystals on the large-scale
This method was developed in the interest of solving that problem while promoting the use
of green chemistry methods
41 Introduction to Fe16N2
Iron nitride magnets offer a low cost however superior alternative to rare earth
magnets In addition the questionable stability of rare earth magnets on the nanoscale is
avoided in the binary iron phases It has been shown that the low nitrogen content phases
such as γ-Fe4N ε-Fe2ndash3N αrsquo-Fe8N and αrsquorsquo-Fe16N2 are ferromagnetic compounds having
exceptionally well characterized stoichiometry [Wang 2003] and electronic properties [Eck
1999] are attractive compounds for magnetic functional nanomaterials [Grachev 2001]
The synthetic routes for commercial production are also well-documented In order to
75
create a timeless protocol for large-scale manufacturing of these nanomaterials we must
anticipate the future regulations requiring green-chemistry procedures for the production of
all synthetic materials By making minor though profound modifications to known
methods using known physical and chemical properties we can be environmentally
conscious while continuing to engineer superior materials Fe16N2 being a phase of iron
nitride being a superior material reported to possess a very high magnetic moment even
greater than pure iron [Cadogan 1997] and iron cobalt [Hattori 2001] The saturation
magnetization of Fe16N2 powder with the largest specific surface area at room temperature
was previously reported to be a striking 200 emug with a maximum coercive force 2250
Oe [Hattori 2001]
42 Theory
The Fe16N2 phase is considered ferromagnetic [Wang 2003] meaning it consists of an
array of atomic moments exhibiting very strong interactions These interactions stem from
electronic exchange forces quantum mechanical phenomenon resulting from the relative
orientation of the electron spins These spin orientations result in either parallel or
antiparallel atomic moment alignment Exchange forces are exceptionally large in
magnitude on the order of 100 T or 1x108 times the strength of the Earthrsquos magnetic field
[Wang 2003] It is important to note that this field is detectable with simple low-cost
equipment Ferromagnetic materials exhibit parallel alignment of moments resulting in
large net magnetization even in the absence of a magnetic field The αrdquo-Fe16N2 phase in
particular being the most important new material of interest for high-density magnetic
76
recording due to its exceptionally high magnetic moment which as previously mentioned
is larger than α-iron [Sugita 1991] [Bao 1994] The coercivity and saturation
magnetization (Msat) of these phases incorporated into thin films have been investigated by
many researchers
The saturation magnetization Msat of the other ferromagnetic phases is generally
lower than that of the α-Fe except for the phases of αrdquo-Fe8N and αrdquo-Fe16N2 which have
been demonstrated by the above-mentioned researchers Although others appear to have
achieved a phase of αrdquo-Fe16N2 mixed with other phases we believe that their ultra-high
temperature synthesis to be unfavorable to the stability of the αrdquo-Fe16N2phase and the sole
parameter (aside from accidental oxidation) being responsible for the existence of mixed
phase crystals Producing these crystals at a temperature higher than 400deg C facilitates
formation of the more thermodynamically stable phases of iron nitride γ-Fe4N and ε-Fe3N
These other phases have signature saturation magnetizations lower than that of α-Fe which
makes the Msat measurement an essential tool for differentiating between phases [Wang
2003]
43 Synthesis of Iron Nitride (Fe16N2) and Zero-valent (Fe0) Iron Nanoparticles
This green-chemistry procedure consisted of five-steps 1) synthesis of the iron oleate
precursor complex 2) synthesis of the iron oxide NPs 3) oleic acid cap removal and
purification of iron oxide NPs 4) reduction to α-iron and 5) nitrogenation under ammonia
gas
77
431 Materials
FeCl3middot6H2O (97) was purchased from Sigma-Aldrich n-docosane (99) and n-eicosane
(99) were purchased from Alfa Aesar n-dodecane (gt99) and hydrochloric acid (1N
certified) were purchased from Fischer Scientific sodium oleate (gt97) was purchased
from Tokyo Chemical Industry Co UHP hydrogen gas (999999 ) and UHP ammonia
gas (999999 ) were purchased from Matheson Tri-Gas All chemicals were used as
received without purification Chemicals and their physical properties may be referenced
in Appendix I
432 Synthesis of Iron Oleate Precursor Complex
The precursor was iron oleate (iron(II III) [(9Z)-9-octadecenoate]n) where n is the
coordination number of iron and could form a monomer dimer or trimer [Bronstein
2007] [Palchoudhury 2011] as described in detail in Chapter 2 Iron oleate is produced in
our laboratory using a modified procedure published elsewhere [Bronstein 2005] The
formation of the complex was verified with UV-Vis-NIR spectroscopy The iron oleate
complex was formed from the combination of sodium oleate salt (sodium (9Z)-9-
octadecenoate) and iron(III) chloride hexahydrate (FeCl3middot6H2O) In a standard reaction
675 g of FeCl3middot6H2O was combined with 25 mL of deionized water and vacuum-filtered
through 022 μm filter paper The mixture was then combined with 2435 g of sodium
oleate in a three-neck round-bottom flask 150 mL of a stock solution consisting of a 246
mixture of deionized water ethanol and hexane was added to the flask Under argon flow
the mixture was vented and filled for three one-minute intervals in order to remove all
78
oxygen from the reaction flask The solution was the slowly (5deg Cmin) heated to 50deg C
under vigorous stirring Once the solid sodium oleate salt had completely melted and the
reflux had begun (around 50ndash60deg C) the temperature was further increased (3 degCmin) to
70deg C and the flask was kept at this temperature for four hours ensuring that the total
reflux time was 4 hours The mixture was then cooled to 60deg C and washed three times
with a 11 mixture of hexane and deionized water in a separatory flask The organic layer
was placed in a rotary evaporator (Rotovap) with the water bath set at 30deg C until the
hexane and ethanol were evaporated away Wet iron oleate complex (the hydrate form) as
obtained from the procedure described above was a reddish-brown highly viscous liquid
The precursor was further purified with ethanol acetone hexane and water washes and
dried in the oven at 70deg C for 24 hours After drying the product was a dark-brown waxy
solid
433 Synthesis of Iron Oxide Precursor
Subsequently iron oxide nanoparticles were prepared using a modification of a procedure
published previously [Park 2004] 148 mmol (5 g) of iron oleate were combined with 16
mL (50 mmol) of oleic acid and 1315 g (465 mmol) of n-docosane (for spherical
particles) or a molar equivalent of eicosane (for cubic morphology) The mixture was
slowly (3 degCmin) heated to 50 degC under argon flow and vigorous stirring Once the
reactants had dissolved the temperature was further increased to 370deg C with a heating
rate of 30deg Cmin For 20 nm particles (plusmn14 nm) the mixture was allowed to reflux for 30
79
minutes For larger particles the reflux time may be extended with an average growth rate
of 16 nm per minute The particles were washed three times with hexane and acetone
434 Removal of Oleic Acid Cap
As discussed in Chapter 2 the iron oxide NPs come out of synthesis capped with oleic
acid The presence of the cap may introduce unwanted contaminants into the new iron
nitride crystal and must be removed Either of two methods may be used to remove this
cap Chemically the coating is removed by adding 1M solution of hydrochloric acid drop-
wise until the carboxyl group of the oleic acid is protonated (pHlt5) and detaches from the
NPs The uncapped particles are then isolated using the standard methanol and hexanes
extraction An alternative method which does not require hazardous reagents is to simply
anneal the oleate coated NPs above the melting point of oleic acid allowing the capping
agent to evaporate off (Tgt 250ordm C) Annealing is typically done for 20-30 minutes
435 Production of Zero-valent Iron Nanoparticles
The iron oxide NP powder sample is reduced under UHP hydrogen gas overnight at 300-
350 degC Then the sample is exposed to ammonia gas for 2-24 hours at a temperature
between 250-400 degC This temperature is below the ammonia decomposition temperature
however recall that iron catalyses the decomposition of ammonia making the lower
temperature sufficient A sample of zero-valent iron NPs was preserved for bacterial
sensitivity studies reported in Chapter 6 For this study we produced zero-valent iron using
a hydrogen gas reduction (above) however other options exist for the synthesis of zero-
valent iron NPs Zero-valent iron nanoparticles may be produced from iron pentacarbonyl
80
in sonicated in a medium molecular weight alcohol under air-free conditions In addition
zero-valent iron NPs may be produced by mixing iron oxide NPs with a molar equivalent
of sodium borohydride then annealed in a high boiling point inert hydrocarbon under inert
gas for 30 minutes
436 Production of Iron Nitride Nanoparticles
Iron nitride NPs were produced using zero-valent iron nanoparticles as a precursor Any
capping agents are removed as described in Section 434 Then the sample is exposed to
ammonia gas overnight at a temperature between 250-400 degC for 2 to 24 hours
44 Structural Characterization of Iron Nitride Nanoparticles
Iron nitride NPs were characterized by XRD and TEM magnetic characterization was done
by SQUID magnetometry For structural characterization TEMEDS samples were
prepared by placing a drop of the colloidal solution onto a 200-mesh carbon-coated copper
grid The solvent was allowed to evaporate away thus fixing the sample on the grid The
JEOL-2010F transmission electron microscope was equipped with an OXFORD Link ISIS
energy dispersive spectroscopy (EDS) apparatus which determined elemental
composition The electron beam was focused on a single nanocrystal and the characteristic
X-ray peaks specific to each element were identified using the OXFORD Link ISIS
software EDS showed the presence of iron and a small peak corresponding to nitrogen
The iron binary phase and crystal structure were determined using a Rigaku Smartlabreg X-
Ray Diffractometer (XRD) with a Cu Kα source (0154 nm) and attached monochromator
81
It is important to note also that the TEM analysis was difficult due to the strong magnetic
interaction between the material and the electron beam The strong magnetic properties of
the sample caused the beam to oscillate interfering with the analysis Both the XRD and
TEM show a body centered tetragonal (BCT) crystal system This system would be
expected for Fe16N2 thus differentiating it from iron or iron oxide Excellent crystallinity
is demonstrated in the TEM image shown in Figure 41
Figure 41 High-resolution TEM image of Fe16N2 NP showing crystallinity
Figure 42 shows the XRD spectrum for the uncapped iron nitride NP sample The Jade
softward automatched the spectrum to the iron nitride (martinsite) phases Fe8N
ICDDICSD card number 01-070-6150 and Fe16N2 ICDDICSD card number 01-078-
1865 both tetragonal crystals with lattice constants a=571 Å b=571 Å c=6016 Å and
82
a=572 Å b=572 Å c=629 Å respectively The scan also reveals some magnetite
(Fe+2Fe2+3O4) ICDDICSD card number 00-019-0629 which is a cubic crystal with lattice
constants a=838 Å b=838 Å c=838Å This iron oxide likely resulted from surface
oxidation of the uncapped NP sample which was set onto the slide using ethyl alcohol
chloroform and heat
Figure 42 XRD spectrum for iron nitride NPs taken with CuKα having a 0154 nm
wavelength and using attached monochromator
45 Magnetic Characterization of Iron Nitride NPs
We measured temperature dependence of magnetization for the Fe16N2 NP samples under
zero-field cooled (ZFC) and field cooled (FC) conditions The DC (τm = 100 s)
magnetization of the samples was measured with a DC field of 100 Oe in the temperature
range between 10 K and 350 K In the entire temperature range up to 350 K the Fe16N2 NP
samples demonstrated strong ferromagnetic behavior as evidenced by the gap between the
83
ZFC and FC curves persisting even at 350 K From the ZFC curve we can loosely estimate
TB to be ~350 K but even above that temperature equilibrium magnetization of the NP
sample was not reached Superparamagnetic behavior of the nanocrystals was observed in
this sample but not observed in larger samples (gt20 nm)
Figure 43 Magnetization vs temperature for Fe16N2 NPs (blue) compared to magnetite
(red) Zero-field cooled (lower curves) and field cooled (upper curves) Magnetization measured
with a DC field of 100 s We measured temperature dependence of magnetization for the Fe16N2
NP samples under ZFC and FC conditions The DC (τm= 100 s) magnetization of the ferrofluid
samples was measured with a dc field of 100 Oe in the temperature range between 9 K and 350 K
Iron nitride appears to block around 350 K whereas iron oxide blocks around 210 K The
elevated blocking temperature of iron nitride makes it attractive for many applications that
presently require supercooling
Superparamagnetic behavior of the Fe16N2 NP samples was observed in magnetic
hysteresis measurements Consistent with the results of DC magnetization measurements
84
magnetic hysteresis measurements at 293 K performed on Fe16N2 NPs find no coercivity
verifying that the magnetic hyperthermia results from a Neacuteel process We were unable to
find saturation Msat with the field strengths presently attainable by the equipment (Figure
45) Extrapolating the line gives a loose estimate of Msat ~ 100 emug The DC (τm = 100
s) magnetization of the ferrofluid samples was measured with a dc field of 100 Oe in the
temperature range between 9 K and 350 K using a Quantum Designtrade magnetic property
measurement system (MPMS) superconducting quantum interference device (SQUID)
magnetometer
Figure 44 Comparison of
hysteresis loops of
nanocrystalline samples of
iron oxide (red) and iron
nitride (blue) of similar
grain size showing the
significantly stronger
magnetic properties of iron
nitride Upper image shows
entire hysteresis loop of iron
nitride Lower image is a
close-up of the same
showing hysteresis loop of
iron oxide
-50E4 00 50E4-1
0
1
(A
m2k
g)
H (mT)
Fe3O
4
Fe16
N2
-50E4 00 50E4-80
-60
-40
-20
0
20
40
60
80
(A
m2k
g)
H (mT)
Fe3O
4
Fe16
N2
85
00 20E4 40E40
20
40
60
80
100
(
Am
2k
g)
H (mT)
Figure 45 Close up of hysteresis curve (positive axis) showing
that Msat was not reached in the 50 T applied field at room
temperature
46 Summary of Findings
Iron nitride NPs were synthesized via solvothermal and solid-gas phase reaction in which
iron oxide powder as an intermediate The composition structure was characterized using
x-ray diffraction (XRD) Saturation magnetization (Msat) and coercivity of NPs was
determined using superconducting quantum interference device (SQUID) We found that
the successful formation of the Fe16N2 phase is strongly dependent on temperature and
reducing agent selection Fe16N2 exhibits saturation magnetizations larger than that of α-Fe
86
The highly magnetic Fe16N2 phase of iron nitride may be produced in high yields having
good resistance to oxidation exceptionally high blocking temperatures and depending on
the precursor some control of particle morphology [Armijo 2012a] This material has a
high magnetic moment though it contains no costly rare earth elements or toxic cobalt
Additionally the green chemistry procedure produces minimal toxic waste It still remains
unclear whether this material is safe for use in vivo
87
Chapter 5
HYDROPHILIZATION AND BIOCONJUGATION
All charged (metal) nanoparticles (NPs) require an organic or non-organic polymer shell
to prevent aggregation potential oxidation and allow for further conjugation In the case
of ferriferromagnetic NPs the coating of magnetic nanoparticles (MNPs) must also be
sufficient to prevent magnetic interactions between particles In general to keep the
particles from interacting magnetically in such a manner that they agglomerate the polymer
shell should have a thickness equal to at least half the radius of the magnetic NP In the
case of superparamagnetic NPs no magnetic interaction in the absence of an external
magnetic field occurs When the application is biomedical the organic coating or polymer
shell must be water-soluble in order to be used in the aqueous biological environment
Many FDA-approved polymers are available for use such as poly(lactic-co-glycolic acid)
(PLGA) and polyethylene glycol (PEG) are often chosen simply because of their
confirmed safety rather than their physical or chemical properties Some other attractive
options are natural polymers which are anticipated to be biocompatible simply due to their
existence in other biological systems Many are produced by plants algae or fungal
species and must simply be purified for use [Lehr 1992] [Dang 2006] These are typically
water-soluble and happen to possess many useful functional groups which allow for further
88
conjugation to a gene or drug Any organic molecule or polymer having a negatively
charged terminal functional group (OH- or COOH- are ideal) may be used to
electrostatically bind a positively charged NP The stronger charge on the carboxyl group
will hold stronger especially in high salinity The colloidal stability of the NPs depends on
the ability of the polymer to maintain a strong ionic interaction with the NP as charged salt
ions can easily electrostatically bind to the charged functional groups on an organic
molecule or polymer Shelf life as well as the systemic half-life of nanomaterials are
strongly dependent on and are highly controllable by this one parameter [Braatz 1993]
[Prencipe 2009]
NOTE The terms ldquoorganic moleculesrdquo or ldquoorganic polymersrdquo were not used to describe potential
NP passivation coatings because silicon-based polymers may also be used We use standard
chemistry terminology in which ldquoorganicrdquo refers to ldquocarbon-basedrdquo
51 Experimental
Prior to engineering polymer or other organic coatings the stabilizing agent oleic acid
must be removed from the surface of the NPs Afterwards water soluble polymers
presenting additional functional groups for bioconjugation may be attached We
investigated the FDA approved polymer PEG as well as the naturally occurring
biodegradable capping agents citrate and alginate for this application These capping
agents present carboxyl terminal groups for conjugation to the amine group of the
tobramycin molecule
89
511 Materials
m-PEG 5000 (methyl-terminated PEG) powder and sodium alginate from green algae
(medium molecular weight) succinic anhydride (gt99) phosphate buffered saline (PBS)
powder and TRIS hydrochloride (PharmaGrade) were purchased from Sigma Aldrich
anhydrous citric acid (995 ) chloroform (999 ) hexane (99) acetone (99) and
hexanes (99) pyridine (99) methanol (99)were purchased from EMD Chemicals
Inc 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (cat 22981)
and N-hydroxysulfosuccinimide Sulfo-NHS (cat 24510) were purchased from
ThermoFisher Scientific Chemicals and their physical and chemical properties can be
referenced in Appendix I
512 Removal of Oleic Acid Cap
The NPs come out of synthesis described in Chapter 2 capped with oleate (oleic acid) As
discussed earlier in Chapter 2 the major reagent is iron oleate an oleate molecule acting as
the organic carrier facilitating high temperature (solvothermal) crystal growth Since iron
oleate served as the organometallic (metal carbonyl) complex by which the iron was
delivered to the iron-oxide crystal [Bronstein 2007 Palchoudhry 2011 Armijo 2012a] The
oleic acid on the NP surface consists of a hydrocarbon chain and a single carboxyl group
that is electrostatically bound to the metal oxide NP (Figure 51) it has no functional
groups for drug conjugation and is not water soluble Due to these significant limitations
many researchers simply coat the NP with an additional water-soluble polymer (over the
existing oleate coating) [Liu 2012] in the case of Yb NPs Other groups have reported the
90
encapsulation of oleate-capped magnetite with a polystyrene layer [Ramirez 2003] or
chitosan [Shete 2014]
In order to ensure direct bacterial contact with the NP as well as sufficient charge
shielding for our application the oleic acid cap was removed using a hydrochloride
solution wash at a pH of 1 The carboxyl group of the oleic acid becomes protonated at a
pH around 5 because pKa is ~54 Fatty acids like oleic acid contain long carbon chains
and typically have Ka values near 1 times 10-5 (pKa ~ 5) The oleate cap was removed with a
hydrochloric acid wash The process of removing the cap is governed by the Henderson-
Hasselbalch equation [Henderson 1908a Henderson 1908b] which derives the pH as a
measure of acidity from pKa (the negative log of the dissociation constant) and the ratio of
the concentrations of an undissociated acid and its conjugate base [Brown 2012]
119901119867 = 119901119870119886 + 11989711990011989210([119860 minus]
[119867119860] (1)
where [A-] is the conjugate base (oleate anion) concentration and [HA] is the organic acid
(oleic acid in our case) concentration
The pKa is given by [Kanicky 2002]
119901119870119886 = minus11989711990011989210([1198673119874+][119860minus]
[119867119860] (2)
where [H3O+] is the hydronium ion concentration
When the pH is equal to the pKa there will exist in solution an equal amount of
protonated (acid) and deprotonated (conjugate base) molecules ([A-][HA] = 1 A typical
carboxylic acid has a pKa between 4 and 5 [154] however titration experiments have
shown that oleic acid has a much higher pKa of 985 [Kanicky 2002] An organic acid will
91
be significantly deprotonated in a solution if its pKa is two or more units lower than the pH
of the solution Although the reaction would have proceeded at a higher pH we used an
HCl solution having a pH of 1 to ensure a more rapid protonation and thus detachment of
oleate from the iron-oxide NP at 25 degC Inserting our pH value of 1 and the oleate pKa of
985 into equation 1 returns a value of 69743 for the ratio [HA][A-]
NOTE THIS PROCEDURE WAS DESIGNED USING BASIC
ORGANIC CHEMISTRY METHODS SPECIFICALLY THE
PKA OF THE OLEIC ACID MOLECULE WHICH IS KNOWN
AND CAN BE DETERMINED EXPERIMENTALLY WE CAN
DEDUCE FROM THE PKA OF THE TERMINAL CARBOXYL
GROUP THE PH AT WHICH IT IS PROTONATED AND WILL
DETACH FROM THE NANOPARTICLE THE METAL (M+)
BEING REPLACED BY THE HYDROGEN ION (H+) AND
BALANCING OUT THE NEGATIVE CHARGE ON THE
TERMINAL CARBOXYL GROUP
The oleate ion is the ionized form of oleic acid [Raymond 2010]
CH3 (CH2)7CH=CH(CH2)7CO2H + H2O CH3(CH2)7CH=CH(CH2)7CO2- + H3O+
oleic acid + water oleate ion + hydronium ion
119870119886 = [119900119897119890119886119905119890 119894119900119899][1198673119874+]
[119900119897119890119894119888 119886119888119894119889] = 1 times 10-5 pKa = 5
Upon reaching the pH which favors formation of the carboxylic acid group the
yellow-tinged transparent oleic acid could be visually observed to fall out of solution
Oleic acid is soluble in methanol so a standard methanolhexanes extraction removes the
oleic acid from the bare iron oxide NPs The NPs were separated in a 95
92
hexanesmethanol mixture in which the methanol solvated the oleic acid Acidic solution
slows oxidation dramatically so there is no need to use inert gas flow for this procedure
Once the two phases are separated the oleic acid is removed using a separatory funnel and
the NPs are isolated via centrifugation The NPs are then redispersed in chloroform
Capping should be done immediately to maintain passivation
Figure 51 Removal of oleate cap acid-wash facilitated removal of oleate cap
leading to uncapped NP and free oleic acid (the protonated form of the oleate
ion) Image by L Armijo 2014
Figure 52 Citric acid molecule the
protonated form of the citrate ion
3D image (upper image) and carbon
skeleton (lower image) drawn with
MarvinSketch
93
513 Citrate Capping
Citrate was the first biodegradable capping agent investigated The citrate molecule has 3
carboxyl groups and one hydroxyl group (Figure 52) available for attachment to the NP
and further conjugation to drug Attachment to a specific group cannot be controlled at
present Citrate was added to the NP solution in chloroform ensuring a (pH gt 6) by adding
drop-wise amounts of 1M sodium hydroxide solution to ensure that a free carboxyl group
is available for attachment to the colloidal NPs The sample was then sonicated and
washed The sample remained stable in water for more than one year when an excess of
citrate was used to ensure complete coverage The citrate cap may also be removed using
an acid wash which converts citrate to citric acid as in Figure 51
514 Alginate Capping
Because a major component of the P aeruginosa biofilm is alginate we anticipated that
alginate capping should facilitate transport through biofilms based on the principle that
ldquolike dissolves likerdquo The alginate monomer shown in Figure 53 has 2 carboxyl groups
and 4 hydroxyl groups contributing to electronegativity and facilitating attachment to the
positively-charged NP As with application of the other polymers alginate was added to
the NP solution in a basic aqueous chloroform solution (pH gt 6) to ensure a free carboxyl
group for attachment to the colloidal NPs The sample was then sonicated for 30 minutes
to an hour in order to keep the particles from agglomerating and then the NPs were
washed in water and removed via centrifugation This sample also remained stable in water
for more than one year when an excess of alginate was used to ensure complete coverage
94
Figure 53 Monomer of alginic acid the protonated form of the alginate ion
showing 3D image (left) and carbon skeleton (right) drawn with MarvinSketch
515 Polyethylene Glycol Succinylation
In order to enhance the binding affinity of PEG-OH to the NPs we further engineered
mPEG using a simple succinylation procedure mPEG-5000 was chosen as its use is
thoroughly documented for biomedical applications Succinylated PEG was produced in-
house from the PEG-OH terminal of mPEG (methyl terminated polyethylene glycol) in a
process during which the terminal hydroxyl group was converted by a small chain
extension to a more electronegative carboxyl group which enhances binding affinity and
thus promotes long-term colloidal stability even under increasing salinities In order to
keep a sealed pyridine bottle under close to atmospheric pressure 25 mL of nitrogen gas
were drawn up into a syringe through the septum of a nitrogen-filled three-neck flask
connected to the Schlenk line and injected into the pyridine bottle After injection 25 mL
of anhydrous pyridine (the solvent) were drawn up from the bottle and injected into the
nitrogen-filled flask The temperature controller was set to 50 degC the temperature at which
the solid mPEG dissolves Subsequently 25 g of succinic anhydride were added to the
three-neck flask This reaction process lasted for one hour at 50 degC The addition of
95
pyridine was repeated four more times using the same methodology as described above
and the reaction was allowed to continue for another 2 hours at 50 degC Pyridine was then
removed using three DI water washes using the rotary evaporator The material was then
re-dissolved in water and placed in 1 kDa cutoff dialysis tubing in a 1 L beaker of DI
water The DI water in the 1 L beaker was replaced after 2 4 and 8 hours The final
material is a light brown substance that originally was thought to be a contaminant of the
original synthesis
Figure 54 Dialysis of succinylated polyethylene 5000 glycol using
dialysis tubing (under stirring in deionized water at room temperature)
for removal of precursors and unreacted reagents
Figure 55 PEG Succinylation overall reaction Shows the initial PEG molecule
having the characteristic terminal hydroxyl group and the product of PEG
succinylation procedure now having a more electronegative terminal carboxyl
group
96
Figure 56 Dried and purified succinylated polyethylene glycol
(PEG) 5000 powder
In the first synthesis most of the succinylated PEG was lost through repeated
efforts to purify the material through crystallization and the use of activated charcoal
Once this was realized a second synthesis was performed by the original protocol that had
better results Shown in Figure 54 is the purification through dialysis with 1000 Dalton
(Da) dialysis bags The succinylated 5000 Da PEG is trapped inside of the dialysis tubing
while the lighter precursor materials are able to diffuse out of the bag into the surrounding
fluid called the dialyte After dialysis purification the mixture was dried with the rotary
evaporator with the water bath set to 50 degC the same temperature of synthesis The dried
succinylated polyethylene glycol was still liquid at this temperature and becomes a brown
waxy solid when cooled to room temperature as shown in Figure 57 Absorbance
measurements were performed on the succinylated polyethylene glycol using a Cary 5000
UV-VIS spectrophotometer It is known that iron oxide is a semiconductor having a band
gap of 22 eV The measurement of 1 by weight solution was performed from 200 to 700
97
nanometers which showed increasing absorption below 600 nm with a shoulder peak at
250 nm This data is especially important if this material is used in the future as a coating
for optically active materials such as quantum dots The graph of the absorbance curve is
shown below Figure 57
200 300 400 500 600 700
00
02
04
06
08
10
12
14
Abs
orba
nce
[OD
cm
]
Wavelength [nm]
1 Syccinylated mPEG 5000 in DI H20
Figure 57 Absorbance spectra for succinylated polyethylene
glycol (PEG) 5000
516 Polyethylene Glycol (PEG) Capping of Iron Oxide Nanoparticles
After succinylation PEG capping was performed using a modified procedure from
[Shtykova 2007] for our work we only used PEG as opposed to a combination of costly
polymers The iron oxide NPs were solvated in chloroform and combined with PEG using
a NP to PEG mass ratio of 12 The NP polymer solution was sonicated at 40 Hz for an
hour at room temperature The NPs were then washed three times with DI water via
centrifugation before being resuspended in DI water
98
517 Conjugation to Tobramycin
Drug conjugation to tobramycin was done using EDC with Sulfo-NHS Sulfo-NHS is a
chemical modification reagent used in the conversion of carboxyl groups to amine-reactive
esters in bioconjugation or crosslinking Sulfo-NHS is a charged analog of NHS (N-
hydroxysuccinimide) and like NHS facilitates control and alteration of carbodiimide
crosslinking reactions in which carboxylates (ndashCOOH) such as those present in the
polymer molecule are activated for conjugation with primary amines (ndashNH2) found on the
tobramycin molecule Such derivatives are synthesized by mixing the sulfo-NHS with a
carboxyl-containing molecule such as alginate citrate or carboxy-PEG with a dehydrating
agent such as the carbodiimide EDC (also abbreviated EDAC) EDC is a ldquozero-length
cross-linkerrdquo meaning that it acts by bringing the two molecules of interest together but
does not change the hydrodynamic size by increasing the polymer chain length In the first
step of the reaction the carboxylated particles are activated by addition of the EDC
followed by the formation of a reactive ester intermediate O-acylisourea After that the
ester will react with an amine group forming an amide however this amide is highly
unstable and will hydrolyze regenerating the carboxyl group if it does not encounter
another amine functional group Our procedure was adapted from a previous publication
[Hermanson 2013] In order to conjugate the SPIONs capped with any of the above-
mentioned organic molecules or polymers 100 mg of Fe3O4 NPs have been washed three
times with 10 mL of coupling buffer (50 mM phosphate buffered saline pH 72) and
removed by magnetic separation The purified NPs were then suspended in 5 mL of
coupling buffer To ensure an excess of the ligand 50 mg of tobramycin (50 mg
99
tobramycin per 100 mg NPs) was dissolved in coupling buffer thus making a 10 mgmL
tobramycin solution The detailed protocol for drug conjugation can be referenced in
Appendix II
Figure 58 Tobramycin molecule an aminoglycoside antibiotic having the formula
C18H37N5O9 shows 3D image (left) and carbon skeleton (right) drawn with
MarvinSketch
Under gentle stirring the NP solution was added drop-wise into a beaker containing
the tobramycin solution and allowed to sit for 2 min at 450 rpm 100 mg of EDC for each
100 mg of NPs were added to the reaction mixture under stirring until solvated The
conjugation reaction was allowed to proceed for 4 hours at room temperature under gentle
stirring Afterwards the NPs were washed twice with 5 mL of coupling buffer before
being resuspended in coupling buffer containing 35 mM Tris to block excess reactive sites
100
Afterwards the particles were washed twice again suspended in deionized water (DI
H2O) and stored in the refrigerator
Figure 59 EDCSulfo-NHS crosslinking reaction scheme in which the alginate coating
on the NP is conjugated to tobramycin thus binding drug to the NP via a new amide
linkage Image after [Conde 2014]
NOTE The sulfite in tobramycin sulfate completely ionizes in an aqueous environment it
is not covalently bound to the molecule and does not participate in the reaction
52 Characterization of Functionalized Nanoparticles
521 Size Determination
Hydrodynamic size distributions of the nanocrystals have been measured using a DynaPro
Titan Dynamic Light Scattering (DLS) module from Wyatt Technology Corporation In
101
order to reduce aggregation and maximize the accuracy of the measurement samples were
prepared for analysis by diluting the NPoleate stock solution to 50 microgmL in pure
chloroform The NPalginate stock solution was diluted in DI H2O The 1-mL samples
were vortexed then sonicated at 40 kHz for 5 minutes prior to analysis to separate
agglomerates and ensure that a more homogeneous solution was analyzed DLS results on
OA capped NPs right after synthesis returned an average diameter of ~16 nm (not shown)
in agreement with the TEM observations values PEG-5000 has a theoretical average
length of ~30 nm however it is important to note that the polymer length is just an average
value in addition the polymer chain can bend and twist resulting in a range of measured
Fig 510 DLS size distribution showing average hydrodynamic size of iron-oxide NPs
after alginate capping
102
In our experiments the succinylated PEG-5000 capping increased the hydrodynamic size
of the NPs from 16 to 4065 nm (not shown) Alginate capping using the natural alginate
also having a range of polymer lengths increased the hydrodynamic size of the NPs to
22971 nm (Fig 510) Tobramycin conjugation did not alter hydrodynamic size as
expected due to the small sizes of both the tobramycin molecule and the crosslinker
Tobramycin conjugation was confirmed by FTIR spectroscopy
522 Zeta potential measurements
Zeta potential measurements have been used to characterize the electrostatic potential at
the electrical double layer that forms at the interface of a colloidal NP and the dispersing
solvent Although the zeta potential measurement is often regarded as NP surface charge it
is not actually a measure of surface charge Zeta potential measures the potential difference
between the dispersion medium and the adsorbed layer of solvent ions surrounding the
particle This is not equal to the surface charge or the Stern potential [Kirby 2010] which
are defined at a different location Colloids with a zeta potential between -10 to +10 mV
are considered neutral while colloids with a zeta potential greater than 30 mV or smaller
than -30 mV are considered strongly cationic or anionic respectively [McNeil 2011]
Particles with a large measured value of zeta potential whether negative or positive are
electrostatically stabilized whereas particles with low absolute values of zeta potential
aggregate or flocculate [Greenwood 1999 McNeil 2011 Hanoar 2012] According to Liao
et al [Liao 2015] iron oxide NPs in water had a zeta potential of +161 mV (incipient
stability) which shifted to -601 mV (good-excellent stability) after capping with alginate
103
Because most cell membranes are negatively charged zeta potential is a key parameter in
membrane permeability and cationic particles tend to exhibit toxicity associated with
membrane disruption (lysis) [McNeil 2011] In our case the alginate coating will impart
the nanocomposites similar negatively charged electrostatic properties to the target
membrane and biofilm environment which should promote diffusion through the alginate
biofilms while also imparting the colloid significant stability at physiological pH
Additionally the average diameter of the functionalized NPs is still small enough to
diffuse through the pores in the mucus as long as they do not agglomerate
523 Forrier transform infrared (FTIR) spectroscopy
Was performed on tobramycin-conjugated NPs to confirm the successful conjugation of
the drug Since neither the tobramycin molecule nor the capping polymer have an amide
linkage preexisting in their structure the presence of an amide bond (1630-1681 cm-1) can
be used to verify a successful EDC conjugation The samples were dispersed in KBr pellets
for FTIR analysis The presence of an amide stretch visible on FTIR at 1630ndash1680thinspcmminus1
was used to verify the success of the crosslinking procedure Loading efficiency of
tobramycin calculated as mass of NP conjugates mass of alginate capped NPs was found
to be ~2
53 Summary of Findings
We report on a method for removal of the organic coating resulting from the carrier
molecule used in the solvothermal synthesis method We report a successful synthesis of
the more colloidally stable succinylated PEG from mPEG as well as a method for coating
104
uncapped NPs We have synthesized and water-solubilized magnetite NPs using various
organic shells We have conjugated drug (tobramycin) and verified conjugation to NP
samples capped with two biodegradable polymers alginate and citrate Using EDC
crosslinking in synergy with sulfo-NHS the NP samples were easily conjugated to the
amine groups on the tobramycin molecule The amide bond between the two groups is not
present on either organic molecule prior to conjugation and is visible on FTIR
spectroscopy We report an increase in colloidal stability and hydrodynamic size of
uncapped NPs (~60 nm) to ~230 nm for NPs capped in alginate
105
Chapter 6
DETERMINATION OF MINIMUM INHIBITORY
TREATMENT CONCENTRATIONS AND BACTERIAL
SENSITIVITY TESTING
Several different batches of NPs were used to thoroughly investigate against P aeruginosa
biofilms and liquid cultures uncapped magnetite NPs magnetite NPs capped with
alginate magnetite NPs capped with polyethylene glycol (PEG) zero-valent iron NPs
capped with alginate and magnetite NPs capped with alginate and conjugated to
tobramycin Citrate capped magnetite NPs were also produced however their use was
limited as they became oxidized and fell out of solution (lost colloidal stability) much
faster than the other compounds In addition initial investigations into the antimicrobial
properties of citrate-capped iron oxide NPs showed that they enhanced bacterial growth
[Brandt 2013] Iron oxide NPs were investigated in the uncapped form as well as with a
biodegradable (alginate) and a non-biodegradable (PEG) polymer coating because our
previous research showed that the PEG capped iron oxide did not inhibit bacterial growth
[Armijo 2014] and it was necessary to determine any inhibition without the contribution
of the cap The lack of inhibition observed in the PEG-capped sample is possibly due to the
non-biodegradable plastic PEG cap which kept the iron from ever interacting directly with
bacterial cells Despite the numerous investigations into the antibacterial effects of noble
106
metal and other nanomaterials described in the literature [Pal 2007] [Panaacutecek 2006]
[Shrestha 2009] and [Moritz 2013] not much focus has been placed on the role the
capping agent plays in the antibacterial properties Our findings demonstrate a major
dependence on the type of capping agent (if any) that is used We have investigated this
role by examining the same nanomaterial uncapped and capped with several different
polymers An ideal control would include a non-drug conjugated sample having the same
polymer cap used for drug conjugation Because it is well known that zero-valent iron
inactivates viruses [You 2005] gram negative E coli [Auffan 2008] [Lee 2008] and was
investigated in inactivating gram positive and gram-negative microbes as well as fungal
cells [Diao 2009] we investigated the antimicrobial properties of zero-valent iron NPs as a
positive control
Prior to the characterization of drug conjugates we investigated the bacterial
sensitivities to two FDA approved antibiotic drugs tobramycin (an aminoglycoside
antibiotic) and ciprofloxacin (a fluoroquinolone antibiotic) Proof of bacterial sensitivity to
tobramycin and determination of the minimum inhibitory concentration of tobramycin was
a necessary baseline establishment for the nanocomposite characterization studies since
tobramycin is the aminoglycoside antibiotic proposed for delivery by the nanosystem We
also investigated bacterial sensitivity to ciprofloxacin as an additional control in the event
that the bacterial colonies demonstrated resistance to tobramycin Although
fluoroquinolone antibiotics are not typically used in treatment due to their considerable
side-effects ciprofloxacin specifically is known to have a low MIC for the P aeruginosa
species Both the antibiotic drugs are known to be effective against P aeruginosa that has
107
not acquired resistance causing bacterial cell death via two different mechanisms of
action Therefore acquired genetic resistance to one mechanism should not impart
resistance to the other Aminoglycoside antibiotics possess several amino functional
groups becoming protonated in biological media thus resulting in a polycationic species
[Kotra 2000] The polycationic nature of the molecule imparts a high binding affinity to
negatively charged nucleic acids especially prokaryotic rRNA [Fourmy 1996] [Fourmy
1998] The simplest manifestation of resistance is posttranslational modification of the
rRNA target or to production of resistance enzymes [Kotra 2000]
It is important to note that P aeruginosa is a human pathogen therefore
appropriate biosafety practices need to be followed personal protective equipment used
and engineering controls must be in place and functional when working with this species
All experiments involving the use of live P aeruginosa must be performed in a biosafety
level 2 (BSL-2) laboratory Laboratory biosafety criteria detailing essential elements for
the BSL-2 and describing in detail required standard microbiological practices special
practices safety equipment and laboratory facilities can be obtained from the Centers for
Disease Control (CDC)
Although biomedical researchers typically limit their biofilm growth to 6 days
[Sauer 2012] [Benamara 2014] a previous report published [Moritz 2010] showed that
30-60-day-old biofilms have greater resistance to some stresses However another group
reports that after 7 days of biofilm formation the accumulation of biomass had not yet
reached a plateau [Klausen 2003] while a classic publication reports that 5 weeks of
growth was the optimal amount of time to achieve the maximum amount of biomass [Hays
108
1945] and yet another report in which the mucoid (biofilm) phenotype observed in P
aeruginosa typical of CF infections was investigated biofilm cultures were maintained for
90 days [Speert 1990] Because a typical P aeruginosa infection in CF patients is typically
thoroughly established we have chosen the extended growth period of 60-days We
anticipate that this model will more closely represent a classic CF infection Therefore
although much of the research on P aeruginosa is reported on biofilms which have
undergone shorter term growth it appears that short term cultures are only merited in in
vitro diagnostics as they were originally intended In diagnostic studies colonies are
allowed to differentiate just long enough to obtain diagnostic and sensitivity information
Figure 61 Pyocyanin
Image showing the
presence of pyocyanin
(blue-green) pigment
produced by P
aeruginosa cultures
grown on agar for
disk diffusion testing
Image by L Armijo
2014
Longer term studies although costlier are no doubt merited in research settings
due to the documented difference and robustness of established colonies Since
109
we are interested in modeling a typical P aeruginosa infection in CF patients
which is an established infection known to have more inherent resistance to
antibacterial agents we maintained our biofilms for a period of 60-days prior to
testing susceptibility to NPs and NP-tobramycin conjugates Established colonies
produced a thick alginate polymer matrix and secrete several pigments
characteristic of P aeruginosa pyocyanin (blue-green) pyoverdine (fluorescent
yellow-green) and pyorubin (red-brown) The optical color changes were noted in
the biofilm communities The blue-green pyocyanin can clearly be seen in figure
61
61 Microbiological Methods
611 Materials
Luria Bertani (LB) broth (cat 11006-004) and LB agar (cat 11006-001) were purchased
from IPM Scientific Inc Eldersburg Maryland USA Pseudomonas aeruginosa cultures
were purchased from ATCC (ATCC 27853)
612 Minimum Inhibitory Concentration of Tobramycin Determination
Because our proposed treatment method is based on the delivery of Tobramycin to P
aeruginosa colonies it was critical to first establish susceptibility to as well as the
inhibitory concentration of Tobramycin in this strain According to a previous report 1000
microg of tobramycin per mL was not sufficient to kill biofilm cells [Nickel 1985] however
others have reported minimum inhibitory concentrations (MICs) ranging from 05 microgmL
110
to 2 microgmL [Nichols 1981] Loosely considering these findings an initial range of 25-250
microgmL was selected for determination of the MIC of this strain To measure the MIC
tobramycin sulfate was first diluted with sterile DI H2O to 1 mgmL (stock solution)
Afterward tobramycin was serially diluted and added to the 1 mL aliquots of culture to
final concentrations between 25 and 250 microgmL with 25 microgmL increments 1 mL of sterile
DI H2O was added to the aliquot of the culture as a negative control The cultures were
then grown overnight on a rotary shaker at 37 ordmC and 150 rpm The next day 50 microL
aliquots of the cultures were diluted 12 with nutrient broth plated on the nutrient agar
plates and grown for 24 hours at 37 ordmC The next-day plates were examined for the
presence of bacterial colonies The MIC was accurately determined by using the dilution
series with 5 microgmL increments of tobramycin concentration ranging between its highest
concentration that still allowed the growth of P aeruginosa colonies on the plate and the
next lowest concentration that completely inhibited their growth [Brandt 2013] MIC of
tobramycin was measured over time beginning on day 1 after one overnight incubation (in
liquid culture without boiling stones) days 3 10 60 of biofilm growth
613 Establishment of Biofilm Communities
Cultures of Pseudomonas aeruginosa were maintained as a frozen stock (in 75 glycerol)
in a liquid nitrogen tank Two days before the experiment the broth medium was
inoculated and grown overnight on a rotary shaker at 37 ordmC and 150 rpm until the optical
density at 600 nm (OD600) reached 05-06 OD600 is a well-established method for
determination of bacterial cell concentration (mgmL) from the linear determination of
111
colony forming units (CFU) in the media The number of CFUs corresponding to the
optical density for P aeruginosa at an OD600=10 is 204x108 CFUmL which is equal to a
bacterial concentration of 2085 mgmL [Kim 2012] OD600 was determined using Cary
5000 UV-VIS-IR spectrophotometer against a blank cuvette containing the same volume
of the liquid medium This concentration was used to inoculate cultures in liquid media
P aeruginosa PAO1 biofilm communities were grown on sterile boiling stones in
liquid media for 60 days until firmly established P aeruginosa PAO1 biofilm
communities were grown on sterile boiling stones in liquid growth media for 60 days until
firmly established Other researchers have published protocols in which P aeruginosa was
grown for 3 days [Mandelbaum 1995] [Grassmeacute 2000] 6 days [Davey 2003] 8 days
[Suzuki 1973] or 7-10 days [Moreau-Marqiuis 2010] As mentioned above we not only
investigated these typical growth periods but investigated an extended growth period in
hopes of modeling an established (chronic) infection This significant amount of time for
biofilm establishment has not been previously reported Liquid cultures were grown in LB
broth at 37 ˚C for increments of 3 10 or 60 days Other researchers have reported
protocols for growth of P aeruginosa on sterile granite pebbles [Whiteley 2001] sterile
glass beads [Leboffe 2012] or glass wool [Benamara 2014] For our protocol biofilm
cultures were grown in LB broth on sterile boiling stones which have significant surface
area for nucleation events and conveniently biofilm attachment The liquid media was
decanted thus leaving only attached cells in the culture followed by replenishing cells
with fresh broth This method is a sufficient low-cost alternative to a flow chamber At the
end of the 60-day period the cultures were sonicated at 40 Hz for 15 minutes to remove
112
attached cells without damaging them similar protocols have been previously published
[Schmitt 1986] [Vandevivere 1993] The cells were then diluted to an optical density at a
600 nm wavelength (OD600) between 05 and 06 corresponding to about 102x107
CFUmL and a bacterial cell concentration of 10425 mgmL OD600 was determined using
the Cary 5000 UV-VIS-IR spectrophotometer against a blank cuvette which contained
only un-inoculated broth Once diluted the cultures were tested in liquid media or applied
to agar plates for susceptibility testing
614 Motility Testing
Motility testing was done on cultures after 1 3 and 60-days of growth Motility testing was
done by preparing agar in test tubes and inoculating the agar using the stab technique with
a sterile inoculation loop having a pointed end In this method the sharp end of the
inoculation loop is dipped into the cultures and stabbed into the agar inside of the test tube
one time The tubes are then incubated overnight and observed the next day Motile strains
can be seen to have disrupted the agar surrounding the place where the stab inoculation
was inserted into the agar This disruption of the agar is not detectable in non-motile
strains
615 Disk Diffusion Method
The disk diffusion method is one of the most popular approaches to bacterial sensitivity
testing due to its low cost and efficiency [Tendencia 2004] The disk impregnated with a
candidate antibiotic drug or compound of interest is placed on the inoculated agar which
contains a uniform layer of bacteria taken from liquid culture The disks are commercially
113
available containing the proper concentrations of antibiotic drugs based in moles per gram
As low-cost alternative disks may be prepared using filter paper soaked in the appropriate
aqueous concentrations of the antibiotic drugs of interest [Loo 1945] [Van Bijsterveld
1969] The underside of the plate is numbered for each sample to be tested The cultures
are distributed evenly onto a sterile agar plate using a sterile cotton swab to form a uniform
layer on the agar The disks containint the prescribed amount of antibiotic drug
recommended for susceptibility testing by the Clinical and Laboratory Standards Institute
(CLSI) the institution responsible for maintaining uniform standards for such research
Disks can also be made out of filter paper saturated in the standard dose of drug using a
known concentration and pipetting the corresponding volume onto the disk The underside
of the plate is numbered for each sample to be tested The method used was the agar disk
diffusion as described in CLSI where impregnated disks were applied to the cultured agar
plates overnight for 16-18 hours [CLSI 2014]
Approximately 108 CFUmL of bacterial cultures corresponding to an approximate 1
mgmL concentration determined by OD measurements was distributed evenly onto a
sterile agar plate using a sterile cotton swab to form a uniform layer on the agar The disks
impregnated with NPs drug or NP-drug conjugates were then placed on top of the agar
shown in Figure 62 A previous method of impregnation the dip method in which dry
disks were dipped into known concentrations by forceps and then placed onto the agar
cultures was found to produce inconsistent results because it was shown that the disks can
absorb different amounts of liquid introducing variability in the absorbed concentrations
[Simon 1970] Instead the more accurate drop method described by Sabath [Sabath 1976]
114
was used In this method the dry disks are placed on the agar plates then a known volume
The dry disks were placed atop the cultures and a 01 μL drop of the solution of interest at
the desired concentration was applied to the disk using a micropipette calibrated
micropipette
Figure 62 Agar plates inoculated with Pseudomonas aeruginosa colonies taken from
biofilm cultures showing disks impregnated with DI water NPs or NPs bound to
drug Image was taken prior to incubation [Armijo 2014]
This method eliminates variability in the total absorbed amount since a known volume is
applied Disk concentrations of tobramycin were initiated at the CLSI recommended disk
content for tobramycin corresponding to 10 μg absorbed into the disk when this mass
returned a negative susceptibility the concentrations were increased incrementally until a
susceptible mass was determined For the initial disk diffusion study investigating
115
tobramycin NPs and NP-conjugates the mass on the disk was determined from
concentration and applied volume For example a 01 μL aliquot of a solution having a
concentration of 100 mgmL corresponds to 10 μg in the disk (01 μL100 mg1 mL = 10
μg) a 50 mgmL concentration corresponds to 5 μg in the disk (01 μL100 mg1 mL = 5
μg) and a 25 mgmL concentration corresponds to 25 μg in the disk (01 μL25 mg1 mL
= 25 μg) and so on The cultures were grown under the previous conditions overnight (16-
18 hours) at 37 degC The diameter of zone of inhibition around the disc was observed and
recorded
616 Biofilm and Mucus Model and Static Magnetic Field Application
A CF biofilm mucus model was also investigated on the 60-day-old biofilms in order to
determine whether magnetic field application susceptibility For this model the cultures
were prepared on solid agar in a petri dish as described above however 1 mL of either
prepared pig mucin aqueous alginate or both were applied on top of the plated colonies
The drug or NP-drug impregnated disks were applied over the barriers Half of the agar
plates were placed on top of a ring magnet composed of sintered neodymium iron and
boron magnetic alloy blendgrade N45 having a Gauss rating of 13500 Gauss a pulling
force of 282 lbs an axial pole orientation a NiCuNi coating and a tolerance of 0002
inches The magnets were left below the agar plate in the incubator for the entire overnight
growth period
116
Figure 63 Pole orientation options for ring magnets Left Axial Right Radial
Ring magnets having axial pole orientation were used for this study
617 Determination of Minimum Inhibitory Concentration of Test Articles
The diluted bacterial cultures were treated with various treatment concentrations The
concentrations were attained by performing a standard serial dilution A 1735 mgmL
stock solution was serially diluted by removing 05 mL from the stock tube and moving it
to the next tube containing the same total volume and so on as shown below Twelve
117
dilutions were done in total Serial dilutions of tobramycin tobramycin bound NPs or NP
suspensions were prepared
Figure 64 Illustration of serial dilution procedure Starting
concentration in the first vial (red) was 1735 mgmL and 05 mL was
transferred from the previous vial to the subsequent vial in line all of
which contained the same final volume
For the MIC measurements the compound of interest (NPs tobramycin or NP-
conjugates) were serially diluted in liquid growth media as shown in figure 62 inoculated
from cultures grown for a specific period and incubated in sterile 2 mL vials overnight
The cultures were then grown overnight on a rotary shaker at 37 degC and 150 rpm Optical
density (OD) of liquid cultures was compared to a control cuvette containing only growth
118
media and ODs comparable to the growth media alone were considered inhibited growth
OD typically increased with decreasing treatment concentrations as the bacterial cells
were increasingly able to differentiate at the decreasing treatment concentrations The MIC
was narrowed down by using the dilution series with even smaller increments of
tobramycin concentration ranging between its highest concentration that still allowed the
growth of P aeruginosa colonies and the next lowest concentration that completely
inhibited their growth The MIC experiments are schematically illustrated in Figure 65
Figure 65 Schematic diagram of minimum inhibitory concentration (MIC) determination
of tobramycin iron-oxide NPs tobramycin-NP conjugates and zero-valent iron NPs in P
aeruginosa liquid cultures
To verify inhibition an inoculation loop was used to plate samples from liquid
cultures having been incubated overnight with a known treatment concentration and
119
having an OD comparable to growth media alone The bacteria were allowed to grow on
the agar plates overnight at 37 degC MIC was determined by complete inhibition defined by
negative growth on agar as well as no apparent growth in liquid cultures determined by
OD For the control sterile DI water was added to the aliquot of the culture as opposed to
an investigational compound Due to the potential for interference of NPs with OD
measurements NPs were removed from solution by magnetic separation after inoculates
were plated on agar but prior to OD measurement
618 Graphical and Statistical Analyses
Graphical and statistical analyses analysis of variance (ANOVA) were performed on
Microsoft Excel and GraphPad Prizmtrade Average values and standard deviations being
calculated on Microsoft Excelreg and ANOVA performed on GraphPad Prizmtrade
62 Results
621 Minimum Inhibitory Concentration of Tobramycin Results
Using the procedure described in Section 61 and a tobramycin concentration curve we
determined the MIC of this particular strain of P aeruginosa in the initial pilot study to be
between 10-15 microgmL for planktonic cultures and averaged 50 microgmL for established one-
week-old old biofilms in liquid media (plusmn5 microgmL) These findings are similar to the
previously reported MIC of 35-50 microgmL (plusmn5 microgmL) found in planktonic cultures [Brandt
2013] Despite the documented need for a significantly increased drug concentration for
the treatment of bacterial biofilm infections (if drug susceptible at all) A previous report
120
found that 1000 microg of tobramycin per mL was applied to an established biofilm and a
significant proportion of the bacterial cells within the biofilm were found to remain viable
after 12 h of exposure to this very high concentration [Nickel 1985] The same group
reported the MIC in another study was found to be several orders of magnitude lower only
04 microgmL so MIC may differ tremendously from strain to strain and among different
growth modes Others have reported MICs ranging from 05-2 microgmL [Nichols 1981]
Another previous investigation found that oxygen limitation and low metabolic activity in
the interior of the biofilm not poor antibiotic penetration to be factors contributing to the
antibiotic tolerance of the P aeruginosa biofilm system [Walters 2003]
Figure 66 Minimum inhibitory concentration (MIC) of tobramycin to P aeruginosa
colonies as a function of growth time Please note that the cutoff concentration for
susceptibility of P aeruginosa to tobramycin in liquid cultures is le4 μgmL therefore
none of the cultures are tobramycin susceptible by CLSI standards
121
The MIC of tobramycin in this strain of P aeruginosa determined at several time
points during biofilm growth increased over time and was found to be 32 μgmL for 3-day
old biofilm cells 50 microgmL for 10-day old biofilms and 937 mgmL for 60-day old
biofilms The MIC of tobramycin differs significantly from strain to strain when
comparing planktonic vs biofilm cells and biofilm growth time These trends were not
observed for shorter periods of growth These findings add merit to our longer-term growth
period for the establishment of biofilm colonies According to the breakpoints
recommended by the CLSI for determination of MIC inhibition at a concentration le 4
microgmL of tobramycin means the strain is susceptible inhibition at a concentration of 8
microgmL is intermediate and inhibition at concentrations ge 16 microgmL means the strain is
tobramycin resistant [CLSI 2019] Therefore according to the CLSI breakpoints for
interpretation of MIC the cultures taken from biofilm communities were never found to be
susceptible to concentration of tobramycin defining susceptibility This strain exhibited
intermediate susceptibility in some cases in planktonic colonies (liquid cultures grown
overnight) with a MIC of 10-15 microgmL The biofilm cultures were found to be
tobramycin-resistant in all cases beginning on day 3 and becoming more resistant over
time
622 Interpretation of Disk Diffusion Results
The impregnated disks diffuse antibiotic drug with the highest drug concentration
assumed to be at the center of the disk and decreasing with the distance from that center
point According to the Clinical and Laboratory Standards Institute (CLSI) the investigator
122
must use the standards provided for the organism and the corresponding infected tissue or
organ CLSI tables provide the drugs and corresponding concentration dose for
susceptibility testing using the disk diffusion method for many bacterial species including
Pseudomonas The doses recommended by the CLSI were used for the initial studies
(Table 61) the recommended dose used for the susceptibility determination of P
aeruginosa is 2 microgmL for tobramycin and the 02 microgmL for ciprofloxacin Since the
plates were streaked using a sterile inoculation loop dipped in the liquid culture (having an
OD between 05 and 06)
Figure 67 Agar cultures used for susceptibility testing A) Agar plate with impregnated
disks prior to overnight incubation B) Image shows zone of inhibition (ZOI) halo around
disk impregnated with antimicrobial agent of interest a positive susceptibility result C)
Motility testing results in agar stab cultures after incubation upper tube is a negative
motility result and lower tube is a positive motility result
123
The effective doses for the susceptibility testing are much lower than the MIC reported for
cultures in liquid media because the number of bacterial cells is much lower when a plate
is inoculated For example in a 1 mL liquid culture tube having a concentration of 104
mgmL corresponds to a total biomass of about 1 mg whereas an inoculation loop is
dipped into the tube and used for streaking the plate only contains about 50 microL
corresponding to a total biomass of 005 mg If we examine as mg per mass of bacterial
cells it is apparent that the CLSI dose of 2 microgmL on the disk for a biomass of about 005
mg is close to our initial experimentally determined average MIC of 35 microgmL applied to
a 1 mg biomass
After overnight incubation the agar plates were examined The presence of a ldquohalordquo
around the disk suggests some degree of bacterial susceptibility to the compound applied
The halo surrounding the disk is a positive result for sensitivity called the zone of
inhibition (ZOI) The diameter of the ZOI is used for interpretation of these results based
on CLSI breakpoints This represents a concentration gradient with the maximum drug
concentration at the center of the disk The diameter of the halo was measured and
susceptibility was based on this measurement as follows
Table 61
Guidelines for interpretation of disk diffusion results
Method Susceptible Intermediate Resistant
Disk diffusion
[mm]
ge 15 13-14 le 12
Where R is resistant S is susceptible and I is intermediate
124
623 Disk Diffusion Results
The results of disk diffusion susceptibility studies are reported The first table 62 shows
the results of Fe3O4 NPs capped with PEG-OH Fe2O3 capped with PEG-OH Fe3O4 NPs
capped with alginate and bound to tobramycin Fe3O4 NPs capped with citrate and bound
to tobramycin tobramycin ciprofloxacin citrate and a DI water negative control
Table 62
Comparison of 3-day old biofilm sensitivities to MNPs capped with
PEG tobramycin ciprofloxacin and NP-drug conjugates
The bacterial colonies were susceptible to the CLSI concentrations for the treatment of P
aeruginosa 02 microgmL for ciprofloxacin The colonies did not demonstrate antibiotic
Disk
number chemical or drug Radius of
inhibition Sensitivity
1 NPs alone (Fe3O4) capped
with PEG-OH 17 mm S
2 NPs alone (Fe2O3) capped
with PEG-OH 0 R
3 NP (Fe3O4)-alginate-
tobramycin 17 mm S
4 NP (Fe3O4)-citrate-tobramycin 295 S
5 Tobramycin alone 10 mm R
6 Ciprofloxacin alone 40 mm S
7 Deionized water 0 R
8 Aqueous citrate 1 mm R
125
resistance to ciprofloxacin It is important to note that despite the effectiveness of the drug
ciprofloxacin this drug is not typically included in the normal treatment regime for P
aeruginosa infections Ciprofloxacin has been given a black box warning by the FDA [US
Food and Drug Administration 2008] due to its potential to cause permanent damage to
muscles tendons joints nerves and the central nervous system Its use is recommended
only when there are no other treatment options
The results of overnight sensitivity studies comparing different capping agents are
summarized in Table 62 Due to our previous findings in which citrate capped NPs
slightly promoted bacterial growth [Brandt 2013] we also tested citrate alone Although no
explanation for this was described by [Brandt 2013] it is possible that the citrate on the NP
surfaces was used as a source for pyruvate synthesis by the bacterial cells We were not
able to characterize increased bacterial growth on this solid agar as was observed
previously in liquid cultures however we did not observe inhibition by citrate or citrate-
capped NPs
These results were obtained on 3-day-old biofilms plated on LB agar using the
standard dose described in the introduction In this result we can see that the P
aeruginosa biofilm colonies did have an intermediate sensitivity to PEG-OH capped
magnetite in this initial study suggesting incomplete coverage of the NP by the non-
biodegradable polymer There was no inhibition by maghemite NPs capped with PEG-OH
either due to the lower iron content of the material or due to complete coverage by the
capping agent It is important to note that we have observed PEG-OH capped NPs to be
less colloidally stable than a capping agent that is attached to the NP via a COO- group
126
Table 63 Susceptibility of P aeruginosa biofilms to various treatments after 3 and 60 days of
growth by disk diffusion
Sensitivity is described with S for sensitive I for intermediate and R for resistant DI
water was used as a negative control and no ZOI was observed for DI water
Material Dose on disk ZOI (mm)
day 3
ZOI (mm)
day 60
Fe3O4 NPsdagger 10 μg 22S 21S
5 μg 175S 16S
25 μg 11R 10R
Fe3O4PEG NPs 10 μg 0R 0R
5 μg 0R 0R
25 μg 0R 0R
Fe3O4ALG NPs 10 μg 22S 22S
5 μg 16S 15S
25 μg 10R 8R
Fe3O4ALGTOBRA NPs 10 μg 23S 22S
5 μg 11R 15I
25 μg 7R 5R
ZVFeALG NPs 10 μg 25S 24S
5 μg 21S 22S
25 μg 20S 20S
Tobramycin 10 μg 10R 0R
100 μg 25R 15R
1000 μg 35R 32R
Interpretation R ndash resistant I ndash intermediate S ndashsusceptible daggerUncapped NPs CLSI breakpoint for susceptibility of tobramycin by disk diffusion is 10 μg therefore all colonies are found to be tobramycin resistant by CLSI standards Higher tobramycin doses in the disk were investigated to determine whether any susceptibility existed at higher doses At present there are no CLSI valuesbreakpoints for NPs as antimicrobial agents ZOI zone of inhibition PEG polyethylene glycol ALG alginate TOBRA tobramycin ZVFe zero-valent iron
127
It is probable that a percentage of the polymer is protonated and detached from the NP in a
colloidal suspension at an undetermined equilibrium concentration As is well known
water at equilibrium contains H+ and OH- at pH dependent concentrations so it is possible
that some of the PEG-O- is protonated in water even at a physiological (neutral) pH
Table 63 shows results of sensitivity testing and determination of MIC for experiments
using NP-drug conjugates on the 60-day old established biofilm colonies These colonies
were also grown as described in Section 61 For this study succinylated PEG (PEG-
COOH) was used to ensure complete continuous coverage of the NP samples For this
study we also investigated the inhibitory properties of zero-valent iron which is known to
inactivate microbes
Disk diffusion results for tobramycin were interpreted based on the 2019 CLSI
breakpoints for tobramycin in P aeruginosa [CLSI 2019] in which the mass of
tobramycin on the disk is 10 microg and a disk diameter ge15 mm is susceptible (S) 13-14 mm
is intermediate (I) and le12 mm is resistant (R) Since there are no established standards for
the investigation of iron oxide nanoparticle susceptibility in any microbes we used the
same cutoff values as we used with tobramycin in order to maintain consistency We also
investigated a range of concentrations of both tobramycin NPs and NP-conjugates in order
to determine susceptibility range The disk diffusion results (Table 63) taken together
with the MIC results over time demonstrate that the tobramycin susceptibility decreases
and resistance increases as the colonies are allowed to grow in biofilm mode for longer
periods of time despite being tobramycin naiumlve Therefore this is not due to exposure-
related resistance development It is important to note that the observed increase in
128
resistance is not due to a larger initial amount of CFUrsquos in the 60-day old biofilms because
cultures were diluted and identical concentrations of CFUs were used for inoculation and
plating for all time periods These findings suggest that the age of the infection alone (ie
establishment of a chronic infection) contributes to resistance This is possibly due to
broader genetic diversity in the population No comparable increase in resistance over time
was observed for the NP samples investigated suggesting that a genetic resistance
mechanism to counter the action of the compound may not exist We can speculate that the
mechanism of action of the iron-oxide NPs is not based on inhibition of genes or bacterial
protein synthesis which implies the toxicity may not be prokaryote-specific
For the iron-oxide NPs alone we found that inhibition of established biofilms on agar
plates was observed for low concentrations When capped with alginate the inhibition
remained low even though part of the mass of this core-shell type NP consists of non-
bioactive alginate In the case of iron-oxide NPs capped with succinylated PEG no
inhibition was observed possibly because the non-biodegradable nature of the capping
agent may keep the iron from interacting directly with the bacteria (see Table 63) If the
iron ions contribute to the toxicity it may be possible that in this case they were not
distributed to the colonies and therefore could not inhibit bacterial growth
These findings demonstrate that the crucial role of the capping agent to the impartation
of antimicrobial properties Therefore the capping agent also contributes to or negates the
toxicity of this material We can speculate that a complete succinylated PEG cap may also
reduce the toxicity of NPs known to exhibit cytotoxic effects in vivo since it appears to
limit interaction with the cells at least in this short exposure time frame
129
Even at high concentrations we might expect to observe some inhibition due to
incomplete coverage however that is not the case In the case of iron-oxide NPs
conjugated to tobramycin we find that the bacterial inhibition at these concentrations
mirrors the inhibition trend of iron-oxide NPs alone It is important to note that these
findings are characteristic of this particular strain after this period of growth and its
susceptibility to tobramycin Recall the previous study which found that after a 1000
μgmL concentration of tobramycin was applied to established biofilms a significant
proportion of the bacterial cells were still viable after 12 hours [Nickel 1985] This group
also reported that planktonic cells taken from the same strain was completely killed by
only 50 μgmL Another relevant study reports the MIC from their clinical isolates to be 8
μgmL [Shawar 1999] These published findings suggest a huge theoretical therapeutic
dose ranging from 8 microgmL to more than 1000 μgmL MIC and susceptibilities appear to
differ dramatically from strain to strain and in planktonic vs biofilm communities
Therefore it is probable that these susceptibilities may also differ from strain to strain and
under different growth conditions
624 Biofilm and Mucus Model and Static Magnetic Field Application Results
The CF disk diffusion model grown on solid agar in petri dishes in which artificial mucin
and alginate barriers were applied over the bacterial colonies cultured from established 60-
day old biofilms reveal that the application of an external magnetic field enhances
susceptibility to the iron-oxide NPs and NP-drug conjugates possibly by promoting
transport across the two barriers For this study 50 mgmL concentrations of NP conjugate
130
and NP solution was applied to the disk such that each disk contained 50 microg of test article
The results with (Table 65) and without (Table 66) magnetic field application
demonstrate zero susceptibility to tobramycin alone
Table 64
Results of CF biofilm model (magnet applied)
A mucin barrier an alginate barrier or both- were applied to 60-day-old biofilm
colonies For this study a magnet was placed below the petri dish
Disk Number Compound Mucin
Barrier
Alginate
Barrier
Mucin +
Alginate
Barriers
1 Iron Oxide NPs dagger 30S 0R 20S
2 Zero-valent Iron NPs 5R 20S 20S
3 Iron Nitride NPs 30S 32S 15I
4 Iron Oxide NP-
Tobramycin
25S 19S 14I
5 Tobramycin (200 mg) 32R 30R 20R
Disk diffusion method was used Minimum concentrations demonstrating susceptibility in
previous disk diffusion studies were used for NPs and NP-tobramycin conjugates daggerUncapped
NPs Maximum CLSI cutoff concentration for susceptibility of tobramycin 10 μg absorbed onto
disk These doses of tobramycin shown are up to seven orders of magnitude higher than the CLSI
standard dose for disk diffusion therefore although inhibition was observed these colonies are
tobramycin resistant by CLSI standard
Table 65
Results of CF biofilm model
A mucin barrier an alginate barrier or both were applied to 60-day-old biofilm
colonies For this study a magnet was not applied
Disk Number Compound Mucin
Barrier
Alginate
Barrier
Mucin +
Alginate
Barriers
6 Iron Oxide NPs dagger 14I 0R 22S
7 Zero-valent Iron NPs 0R 0R 14I
8 Iron Nitride NPs 0R 0R 0R
9 Iron Oxide NP-
Tobramycin
0R 0R 0R
10 Tobramycin (200 mg) 30R 40R 40R Disk diffusion method was used Minimum concentrations demonstrating susceptibility in previous
disk diffusion studies were used for NPs and NP-tobramycin conjugates daggerUncapped NPs
Maximum CLSI cutoff concentration for susceptibility of tobramycin 10 μg absorbed onto disk
These doses of tobramycin shown are up to seven orders of magnitude higher than the CLSI
standard dose for disk diffusion therefore although inhibition was observed these colonies are
tobramycin resistant by CLSI standard
131
No CLSI breakpoints exist for NPs or NP conjugates at present however the CLSI
dose for susceptibility determination of P aeruginosa to tobramycin is 10 μg absorbed
onto disk with cutoff values are ge15 susceptible 13-14 intermediate and le12 resistant
[CLSI 2019] The same parameters were used for interpretation of the NP and NP
conjugate results Tables 64 and 65 demonstrate the highly statistically significant
contribution of the external magnetic field in enhancing susceptibility to the test articles
More work is needed to determine the exact role of the magnetic field in addition to
determining the minimum or maximum field strength necessary to achieve maximum
susceptibility It is possible that the pulling force of the magnet may relate in some way to
the thickness of the biofilm and mucus barriers
Table 66
Summary of biofilm model using alginate barrier mucin barrier or both
on 10-day-old biofilms Comparison between petri dishes in which a magnet was or was not applied
Alginate and
Mucin
(No magnet)
Alginate
(magnet)
Mucin
(magnet)
Alginate and
Mucin
(magnet)
DI Water R R R R
Tobramycin R S R S
NP-alginate-
drug
R I S S
NP-citrate-
drug
R I I I
A summary of the results of a pilot study presented in Table 66 summarizes the
results of a pilot study in which magnetite NPs conjugated to tobramycin was investigated
The results summarized in Table 66 shows that magnetic field application alone enhanced
132
susceptibility of biofilms to all the test articles including tobramycin Therefore magnetic
field may be acting as an antimicrobial facilitator by mechanism other than magnetic
gradient-guided transport It is interesting that this enhancement of the activity of
tobramycin by magnetic field application was not observed in the 60-day-old biofilms
More work is necessary to determine whether magnetic field application alone and what
rangeranges of field strengthduration interfere with biofilm growth
625 Motility Testing Results
Biofilm bacteria (3-days and older) tested positive for motility while the liquid cultures
(grown overnight) appeared to have minimal if any motile individuals This is a testimony
to the large genetic diversity of the bacteria composing a biofilm
Figure 68 Results of
motility test for P
aeruginosa grown in liquid
or biofilm cultures This
image was taken after a total
of 36 hours of growth
626 Comparison of Inhibition in Liquid Cultures
All cultures were inoculated in exactly the same manner with the same volume of bacteria
from the same liquid culture The OD600 of the negative control samples (containing only
133
inoculated broth) was determined to be 022 to 024 This result is slightly higher than the
lowest treatment concentration (8x10-6 mgmL) Since there is no CLSI breakpoint or
standard inhibitory concentration it was necessary to investigate a large range of
concentrations to determine MIC The range used was 1735 mgmL to 8x10-6 mgmL in a
consistent volume determined by serial dilution as the graph in figure 69 illustrates
Figure 69 Shows optical density (OD) at a 600 nm wavelength for liquid
cultures exposed to treatment with iron-oxide NPs zero-valent iron or
tobramycin-conjugated iron-oxide NPs The calculated average error for OD
measurements was plusmn001 Specific errors not the average error were used to
calculate statistical significance
134
Complete inhibition was observed for all materials at concentrations at 175 mgmL (or
higher) and various degrees of inhibition fall off somewhat linearly at concentrations
below 1735 mgmL (Fig 69) The inhibition by zero-valent iron was not surprisingly
higher than iron-oxide NPs and NP-drug conjugates We attribute this to the high reactivity
of zero-valent iron and its ability to increase reactive oxygen species (ROS) in the local
region [Hsueh 2017] Although speculative at this stage it is also possible that high levels
of iron contribute to cellular toxicity More work is necessary to determine toxic and non-
toxic dose ranges
Figure 610 Percent bacterial inhibition vs treatment concentration in liquid
cultures in cuvette All NP samples presented here are alginate capped
135
ANOVA results showed that while there was no statistically significant difference
between the zero-valent iron iron oxide or iron-oxide ndash tobramycin conjugates when
compared to control the results for all three NP treatments were found to be extremely
statistically significant (p lt 00001) The figures show that the inhibition of bacterial cells
was evident even at surprisingly low (8 ngmL) concentrations although the minimum
therapeutic dose would probably be much higher Speculation on a therapeutic dose for
targeted delivery would likely differ from the systemic dose and both will depend on
observed cytotoxicity in mammalian cell cultures at these concentrations Even higher
doses may be required for the treatment of chronic infections involving biofilms that have
been established for several years however more research is necessary to determine this
The MIC for different strains of P aeruginosa may differ as well According to
another report P aeruginosa (MTTC 1034) was not found to be susceptible to iron-oxide
NPs at 50 mgmL whereas our strain exhibited positive susceptibility [Behera 2012] It has
been shown previously that oxygen limitation and metabolic activity can alter MIC of
tobramycin in P aeruginosa [Walters 2003] Differences in zone diameter for
susceptibility testing have also been known to differ with different batches of growth agar
[Reller 1974] [Niemirowicz 2015] reported positive bacterial inhibition for P aeruginosa
PAO1 in agreement with our findings We attribute differences in susceptibilities to
genetic differences among strains in combination with the contribution of environmental
factors such as growth media and the use of different capping agents
The mechanism by which iron-oxide NPs exhibit antibacterial activity remains
unknown However according to the findings of [Musk 2005] iron may very well be the
136
bioactive component Zero-valent iron as predicted had a dramatic antibacterial effect
verifying the findings of [Diao 2009] Although zero-valent iron is too reactive for in vivo
use at present it may be a candidate for incorporation into antibacterial coatings Similarly
iron-oxide NPs having high biocompatibility may be a candidate material for
incorporation into polymer for use as antibacterial coatings on virtually any inert surface
used outside of the body as well as medical devices such as stents catheters and surgical
sutures as a low-cost alternative to silver NPs We anticipate that the combination of
tobramycin or other drugs with iron-oxide NPs incorporated into biodegradable polymers
may hold promise for the long-term control of biofilms and multidrug resistant microbial
strains More work is needed to determine antibacterial properties of these materials on
other microbial species
69 Summary of Sensitivity and Dosage Study Findings
We have shown that both drugs ciprofloxacin and tobramycin are effective against
biofilms and planktonic cells in a dosage-dependent manner Magnetic field application
may in some cases enhance drug susceptibility The drug seems to have exerted action
both in the free form as well as covalently bonded to a crosslinker chain There appears to
be no need for a drug release mechanism since the bound drug remains bioactive
Surprisingly the magnetite NPs alone inhibited bacterial growth and subsequent biofilm
formation We have examined standard models in addition to more accurate models using
inert surfaces for biofilm growth thus allows for purification of the bound colonies from
the planktonic cells Using this method we have also shown that the biofilm colonies
137
contain motile mutants previously undocumented evidence of the complex genetics
implied by such a rapid phenotypic switch
Although it appears that the iron oxide NPs inhibited growth better than drug-
conjugated iron oxide we must use caution in the interpretation of these results Recall that
conjugation was done which may have increased the mass of the non-active ingredients
Further characterization such as drug loading efficiency would allow the calculation of the
percentage by mass of iron oxide tobramycin and inert material Once those calculations
are done these parameters may be further understood as a function of active ingredients It
is apparent however that the iron oxide did inhibit bacterial growth via a presently
uncharacterized mechanism
Zero-valent iron had a dramatic antibacterial effect verifying the findings of [Diao
2009] Although zero-valent iron is too reactive for in vivo use at present it may be a
candidate for incorporation into antibacterial coatings Iron oxide alone may be a candidate
for antibacterial coatings on medical devices such as stents catheters and surgical sutures
as a low-cost alternative to silver NPs The drug tobramycin an aminoglycoside
annihilates bacterial cells in a synergistic manner It electrostatically binds the negatively
charged lipopolysaccharide bacterial membrane compromising membrane integrity and
thus resulting in its degradation [Shakil 2008] Once internalized acting from the inside of
the bacterial cell tobramycin inhibits ribosomal translocation thus interfering with protein
synthesis [Saiman 2004] We anticipate that the combination of tobramycin or other drugs
with iron oxide NPs incorporated into biodegradable polymers may hold promise for the
long-term control of multidrug resistant bacterial strains
138
Chapter 7
CYTOTOXICITY of IRON OXIDE NANOPARTICLEs
Not only is lung toxicity a crucial parameter to investigate due to the nature of our
application but also in acute inhalation exposure the organ system subjected to the
highest initial concentrations is the lungs Therefore a thorough investigation of the acute
toxicity of inhaled nanomaterials must begin with a baseline analysis of human lung cell
toxicity We have investigated the in vitro cytotoxicity of ~16 nm spherical magnetite
nanoparticles capped with succinylated polyethylene glycol on a human lung carcinoma (A
549) cell line at 6 12 and 24-hour exposure periods and at 05 mg mL and 1 mgmL
nanoparticle concentrations We investigated acute toxicity in a comprehensive study by
comparing overall cytotoxicity cell viability and apoptosis profiles against positive
controls We report a dose-dependent decrease in viability at the 12-hour time point
exhibiting a complete cell recovery by 24-hours as well as a dose independent time-
dependent alteration in cell proliferation rate No statistically significant deviation from
control in overall cytotoxicity or apoptosis was observed upon exposure to iron oxide
nanoparticles in this cell-line at the time points or concentrations investigated
Animal models have revealed a link between inhaled particles and murine lung
inflammation [Oberdoumlrster 2000] and lung cancer [Knappen 2004] [Borm 2004]
139
Although the dextran-coated iron oxide NP solution finding application as the IV-
administered MRI contrast agent Feridexreg had received FDA approval for human use in
the United States it was discontinued by the manufacturer [Anselmo 2016 Wei 2016] and
is no longer commercially available To date there still exists a significant lack of
knowledge regarding the effects of NPs in general but more specifically on the effects of
iron oxide (magnetite) NPs on cell viability and normal functionality [Sonen and De
Cuyper 2010] In fact many researchers have reported that the use of these particles can
exert severely detrimental actions on the living cell [Sonen and De Cuyper 2010] [Wei
2016] reports a SPION dosage-dependent iron overload linked to cirrhosis of the liver in a
murine systemic toxicity model Some other negative observations include LDH leakage
and abnormal IL-6 secretion at high (gt50 mgmL) concentrations [Mbeh 2012] significant
reductions in viability in murine and human cell lines [van den Bos 2003 Soto 2007]
[Pisanic 2007] decreased cell proliferation [Berry 2004 van den Bos 2003] and migration
[Berry 2004] Many of these studies reporting increased toxicity attribute toxic effects to
the failure of the dextran coating to remain bound to the cell Because of this we have
engineered the terminal hydroxyl group (OH-) on the FDA approved polymer polyethylene
glycol (PEG) to terminate in a more electronegative carboxyl group (COOH-) by
succinylation increasing binding efficiency to the metal oxide (M+) NP Due to the
association of uncapped iron oxide NP and toxicity in some cell types increased binding
efficiency is expected to reduce cytotoxicity of the iron oxide NPs
Regarding human inhalation exposure the occupational health literature abounds
with illustrations of aerosol-associated respiratory hazards and related lung pathologies
140
dating back many decades However the context of this prior research pertains specifically
to occupational exposure to nanoscale particulates formed accidentally as by-products
from processes such as welding smelting and combustion [Maynard and Kuempel 2005]
as opposed to engineered nanomaterials It is crucial that toxicity data on nanomaterials
having the potential to expose workers via the inhalation aerosol route be communicated
quickly to researchers so that they may cater future engineering design to reduce toxicity
At present there is limited data on the toxicity of these methodically engineered nanoscale
materials in the human respiratory tract Due to the exponential growth in the manufacture
and utilization of such nanomaterials which still remains largely unregulated we
anticipate an exponential increase in their presence in both the natural environment as well
as the workplace This rapid increased in commercialization of such novel materials
having unknown toxicity will merit an accurate determination of a safe exposure range
not only for a patient receiving nanomedical treatment but also for the employees
engineering transporting administering and disposing of these materials Toxicity profiles
are crucial for the determination of proper engineering controls proper personal protective
equipment (PPE) and emergency procedures for employees administering transporting
and manufacturing the material Dosage-dependent cytotoxicity will also be an important
parameter for determining the feasibility of purposely administering this material to the
lungs and determining and balancing dosages that are both safe and effective
71 Experimental Procedure
141
Succinylated PEG-capped iron oxide NPs were prepared as described in Chapters 2 and
capped using the methodology described in Chapter 5
711 Materials and Reagents
Iron(III) chloride hexahydrate (97) m-PEG 5000 (methyl-terminated PEG) powder
succinic anhydride (gt99) phosphate buffered saline (PBS) powder TRIS hydrochloride
(PharmaGrade) digitonin ionomycin and staurospirine were purchased from Sigma-
Aldrich n-docosane (99) was purchased from Alfa Aesar sodium oleate (gt97) was
purchased from Tokyo Chemical Industry Co hexanes (95) ethanol (99) and acetone
(99) chloroform (999 ) hexane (99) pyridine (99) methanol (99) were
purchased from EMD Chemicals Inc the ApoTox-Glotrade triplex assay (Catalog No
G6320) was purchased from Promegareg A 549 human alveolar epithelial carcinoma cells
(ATCCreg No CLL-185) and 025 Trypsin053 mM EDTA (ATCCreg No 30-2101) were
purchased from ATCCreg Hamrsquos F-12 Kaignrsquos modification (Catalog No 21127-022)
10 fetal bovine serum heat-inactivated (Catalog No 10082-147) and 100 unitmL pen-
strep (Catalog No 15140-122) were purchased from Invitrogen All chemicals were used
as received without purification
712 Dynamic Light Scattering (DLS)
Hydrodynamic size distributions of the nanocrystals have been measured using a DynaPro
Titan DLS module from Wyatt Technology Corporation In order to reduce aggregation
and maximize the accuracy of the measurement samples were prepared for analysis by
142
diluting the NP stock solution to 50 microgmL in pure chloroform The 1 mL sample was
vortexed then sonicated at 40 Hz for 5 minutes prior to analysis in order to separate
agglomerates and ensure that a more homogeneous solution was analyzed
713 UV-vis-NIR Spectroscopy
Light absorbance of iron oxide nanoparticles and succinylated PEG was characterized
using the Cary 5000 UV-vis-NIR Spectrometer Many published assay results fail to
report or even consider doing these measurements Nanomaterials or quantum dot are
known to have highly sought-after interactions with light It is important that we consider
these interactions when designing experiments using these kinds of assays that were not
developed with such considerations in mind By determining light absorbance we are able
to determine any possible interaction or interference of these materials with the assays
which are dependent on total light detection via the plate reader
714 Human Lung Adenocarcinoma Cell Growth
Cells were stored in liquid nitrogen in a cryostat until their use To initiate growth the
sample was thawed and centrifuged and then the culture medium was removed After that
the cells were rinsed with 025 Trypsin053 mM EDTA solution to remove any
remaining serum that may contain trypsin inhibitor Next 25 mL of Trypsin-EDTA
solution was added After 15 minutes the cells had dispersed into the solution and 7 mL of
complete growth medium (F-12K medium with 10 FBS) was combined with the cells by
gentile aspiration Cultures were incubated at 370 degC under 5 carbon dioxide weighted
with HEPA-filtered air
143
715 Cytotoxicity Assay
Bis-alanylalanyl-phenylalanyl-rhodamine 110 (bis-AAF-R110) is a fluorogenic cell-
impermeant peptide substrate marker for dead-cell protease activity This is used to
measure protease enzyme which has been released from cells that have lost membrane
integrity No signal from this marker is generated from viable (intact) cells because bis-
AAF-R110 is not cell-permeant and cannot cross the cell membrane Dead cells release
protease enzymes that will cleave the rhodamine 110 (R110) from the rest of the molecule
causing it to fluoresce R110 has an excitation peak at 498 nm and an emission peak at 520
nm
In growth medium 05 and 1 mgmL concentrations of NPs were incubated with
the cells for 12 or 24-hours exposure time Digitonin ionomycin and staurosporine are
known to elicit cytotoxic necrotic and apoptotic damage upon cells respectively and were
used as positive controls For the twelve (12) hour exposures cells in positive control wells
were treated with either 30 microgmL of digitonin for an incubation period of 15 minutes 100
microM of ionomycin or 10 microM of staurosporine both applied for 6-hour incubation periods
For the 24-hour measurements cells in positive control wells were treated with either 45
microgmL of digitonin for an incubation period of 30 minutes 150 microM of ionomycin or 15
microM for staurosporine for 6-hour incubation periods A 96 well-plate was used except for
background control wells which contained growth media alone each individual well was
seeded with 10000 A 549 cells dispersed in growth media Each well was filled to a
volume of 100 microL and cells were cultured for the respective time periods The background
readings from the wells containing no cells were averaged and subtracted from the
144
obtained averaged readings After the 6 12 or 24-hour period 20 μL of the
viabilitycytotoxicity reagent containing both GF-AFC substrate and bis-AAF-R110
substrate was added to all the wells Immediately after that the solutions were mixed by
orbital shaking at 300-500 rpm for ~30 sec The plate was incubated for 30 minutes at 37
degC Finally the samples were exposed to 485 nm light with a 20 nm bandwidth for
excitation fluorescence measurements were taken at 528 nm with a 20 nm bandwidth
Measurements were taken with a BioTech Flx800 Microplate Reader measuring
fluorescence from the bottom of the 96-well plate
716 Viability Assay
Glycylphenylalanyl-aminofluorocoumarin (GF-AFC) is a florigenic cell-permeant peptide
substrate which is used as a marker for live-cells Since live-cell proteases must be
detected from within the living cell having an intact membrane this substrate must cross
the cell-membrane and enter the cell Once inside the cell protease enzymes cleave the
AFC from the substrate triggering the fluorescence signal The AFC has an excitation
peak at 370 nm and a fluorescence emission peak at 490 nm Should the membrane rupture
while the substrate is inside the cell the fluorescence is quenched and the signal ceases
Therefore this substrate is able to give an accurate measure of viable cells
In growth medium 05 and 1 mgmL concentrations of NPs were incubated with
the cells for 6 12 or 24-hours exposure time Digitonin ionomycin and staurosporine are
known to elicit cytotoxic necrotic and apoptotic damage upon cells respectively and were
used as positive controls For the six (6) and twelve (12) hour exposures cells in positive
145
control wells were treated with either 30 microgmL of digitonin for an incubation period of 15
minutes 100 microM of ionomycin or 10 microM of staurosporine both applied for 6-hour
incubation periods For the 24-hour measurements cells in positive control wells were
treated with either 45 microgmL of digitonin for an incubation period of 30 minutes 150 microM
of ionomycin or 15 microM for staurosporine for 6-hour incubation periods A 96 well-plate
was used except for background control wells which contained growth media alone each
individual well was seeded with 10000 A 549 cells dispersed in growth media Each well
was filled to a volume of 100 microL and cells were cultured for the respective time periods
The background readings from the wells containing no cells were averaged and subtracted
from the obtained averaged readings After the 6 12 or 24-hour period 20μL of the
viabilitycytotoxicity reagent containing both GF-AFC substrate and bis-AAF-R110
substrate was added to all the wells Immediately after that the solutions were mixed by
orbital shaking at 300-500 rpm for ~30 sec The plate was incubated for 30 minutes at 37
degC Finally the samples were exposed to 360 nm light with a 40 nm bandwidth for
excitation fluorescence measurements were taken at 460 nm with a 40 nm bandwidth
Measurements were taken with a BioTech Flx800 Microplate Reader measuring
fluorescence from the bottom of the 96-well plate
717 Apoptosis Assay
In this assay cell apoptosis is measured by detecting the apoptosis biomarkers
caspase 3 and caspase 7 Cell lysis is followed by caspase cleavage of the substrate and
results in generation of a luminescent signal The fluorophore in this assay is luciferase
146
(aminoluciferin) a natural luminescent molecule borrowed from the firefly [Gould 1988]
Luminescence is proportional to the amount of caspase activity and thus apoptosis The
luminogenic caspase-37 substrate which contains the tetrapeptide sequence DEVD (Asp-
Glu-Val-Asp) in an optimized reagent (Caspase-Gloreg 37 Reagent Promegareg) optimized
for caspase activity luciferase activity and cell lysis
In growth medium 05 and 1 mgmL concentrations of NPs were incubated with
the cells for 6 12 or 24-hours exposure time Ionomycin is known to induce necrosis and
staurosporine is known to induce apoptosis therefore these compounds were used as
controls for this assay For the six (6) and twelve (12) hour exposures positive control
cells were treated with either 100 microM of ionomycin or 10 microM of staurosporine both
applied for 6-hour incubation periods For the 24-hour measurements cells in positive
control wells were treated with either 150 microM of ionomycin or 15 microM for staurosporine for
6-hour incubation periods A 96 well-plate was used except for background control wells
which contained growth media alone each individual well was seeded with 10000 A 549
cells dispersed in growth media Each well was filled to a volume of 100 microL and cells were
cultured for the respective time periods The background readings from the wells
containing no cells were averaged and subtracted from the obtained averaged readings
After the 6 12 or 24-hour period 20μL of the viabilitycytotoxicity reagent containing
both GF-AFC substrate and bis-AAF-R110 substrate was added to all the wells
Immediately after that the solutions were mixed by orbital shaking at 300-500 rpm for ~30
sec The plate was incubated for 30 minutes at 37 degC After fluorescence measurements
were taken 100μL of Caspase-Gloreg 37 Reagent was added to all wells and briefly mixed
147
by orbital shaking at 300ndash500 rpm for ~30 sec Measurements were taken with a BioTech
Flx800 Microplate Reader measuring luminescence from the bottom of the 96-well plate
718 Statistical Analysis Correction Factor and Mathematical Methods
This experiment was done in triplicate the median values presented and standard
deviations were calculated For comparison of the mean values for each test both a two-
way analysis of variance (ANOVA) was run for grouped values and to analyze trends over
time and a double-tailed t-test was run to compare single values to control All statistical
analyses were run in GraphPad Prismreg Values of plt05 (95 confidence interval) were
considered significant plt 001 (99 confidence interval) were considered very
significant and values of plt 0001 (999 confidence interval) were considered extremely
significant
In order to correct for the signal reduction caused fluorescence absorption by the
colloidal NPs a general correction was applied as follows The experimental findings of
[Doak 2009] in which fluorescence quenching by iron oxide NPs was measured at different
concentrations were plotted as a function of percent signal reduction The maximum
concentration of fluorescent dye used in the assay assuming 100 fluorophore activation
as calculated from the stock solution concentration and dilution factor is 5 microM Although
there is a slight difference in the percent reduction based on the difference between the 2
microM and 4 microM fluorophore concentrations we analyzed the mean collected values at each
NP concentration compared between the dye concentrations did not find them to be
statistically significant Although it is unlikely that 100 of the fluorophores were
148
activated in the assay the maximum concentration which is not likely to exceed 1
variance from the calculated value and should be encompassed by the correction factor
and corrected error The plot (Fig 71) demonstrates a nearly identical trend for both
concentrations of fluorescent dye which suggest that signal reduction is consistent over a
range of fluorophore concentrations and the values can be fit to the same trend line
Reduction of Fluorescence Intensity by Magnetite NPs
NP Concentration (gmL)
Perc
en
t In
ten
sit
y R
ed
ucti
on
co
ntr
ol
1x
10
-3
1x
10
-2
1x
10
-1 1
10
10
0
0
20
40
60
80 reduction 4 M dye
reduction 2 M dye
Figure 71 Reduction of fluorescence signal by magnetite NPs at two fluorescent dye
concentrations Based on the findings of [Doak 2009]
Extrapolating out one data point encompasses the two concentrations used for this study
The log transforms and linear curve calculations were run on GraphPad Prismreg The data
was fit to an exponential trend line in Microsoft Excelreg The exponential trend line
149
equations for the 4 microM and 2 microM concentrations of fluorescent dye were y=43311e03718x
and y=69758e03062x respectively According to this model the next data point
corresponding to a 1 mgmL concentration of magnetite NPs is between 585-595 At
this range the variation between the two fluorophore concentrations was found to be only
~1 Since the concentration of the fluorophores does contribute minimally to the
measurement this range was incorporated to the error margin Based on this model the
measured fluorescence values were reduced by ~59 for the 1 mgmL concentration and
~51 for the 05 mgmL concentration Since simply taking an increase by the percentage
of the measured value will not return the original value the measured values must be
adjusted according to
measured value
(100 minus reduction)
The collected values were included in the standard deviation for comprehensiveness
72 Results
721 Dynamic Light Scattering (DLS) Size Distribution
DLS results are shown in Figure 72 where the particles were measured for size in
chloroform solution Because polymer coating increases the NP hydrodynamic size this
measurement was done prior to polymer capping in order to verify NP sizes observed in
TEM measurements The colloidal NPs demonstrate some very minor aggregation
150
(responsible for the peaks at 30 and 35 nm) The average hydrodynamic diameter is 15946
nm with a standard deviation of 4393 nm in agreement with the TEM observations
Figure 72 DLS size distribution of colloidal magnetite nanoparticles
This graph shows an average hydrodynamic diameter of ~16 nm
722 UV-vis-NIR Spectroscopy Absorbance Measurements
Absorbance spectrum (Figure 73) of iron oxide NPs in colloidal suspension with
chloroform shows a strong absorbance peak at ~375 nm in the UV portion of the
spectrum The spectrum shows minimal absorption (05 AU) consistently throughout the
rest of the visible and near-infrared range These findings are in agreement with previously
demonstrated absorbance results for iron oxide NPs [Wang 2005] [Shi 2007] [Awwad
151
2012] and [Sathyanarayanan 2013] The succinylated PEG (capping agent) shows a strong
peak in the UV portion of the spectrum and no absorption throughout the visible range
Figure 73 Absorbance spectrum for magnetite NPs
Figure 74 Absorbance spectrum for succinylated polyethylene glycol (PEG)
152
723 Cytotoxicity Assay Results
Figure 75 Cytotoxicity results dead-cell marker fluorescence at 12- and 24-hours
exposure denotes statistical significance where plt05
A double-tailed t-test was conducted on the measured values compared to controls for
each time point None of the reagents applied to the cells demonstrated any statistically
significant effects at the 12-hour time point including digitonin the cytotoxicity positive
control These findings are important for future use of this assay in this cell type The
concentrations or periods of exposure for all three positive controls may need to be
153
increased in order to elicit a noteworthy response in this cell line The NPs did not exhibit
a statistically significant cytotoxic effect at this time point for either of the concentrations
investigated At the 24-hour time point the cytotoxicity positive control digitonin as well
as the necrosis positive control staurosporine demonstrated statistically significant
cytotoxic effects compared to untreated cells The magnetite NPs did not exhibit any
statistically significant cytotoxicity at various concentrations or time points
724 Viability Assay Results
A two-way ANOVA was run on the treatment results compared to control at all three time
points investigated The ANOVA showed a statistically significant time factor in all treated
and untreated cells attributed to the normal doubling time as expected a linear increase
was observed The normal doubling time of A549 cells is approximately 22 hours (ATCC)
this rate corresponds to our observed rate Statistical reductions in viability were found to
be very significant for the staurosporine positive control the 1 mgmL concentration of
magnetite NPs and the ionomycin positive control having p-values of 00083 00027 and
0001 respectively An initial increase in live-cells was observed in the magnetite NP
treated wells at the six-hour time point However a significant concentration-dependent
decrease in viable cells was observed at the 12-hour time point By the time the 24-hour
measurement was taken the magnetite NP-treated cells had more than recovered and the
viable-cell count was in the range of the untreated cells Taking into consideration the
range of measured values and the slope of the line it appears that the 05 mgmL NP
154
concentration may not have had lethal effects but simply inhibited cell differentiation over
the 6 to 12-hour time points
Viability
Time Point
Flu
ore
scen
ce (
RF
U)
6 hou
rs
12 h
ours
24 h
ours
0
20000
40000
60000
Digitonin
Staurosporine
Ionomycin
Magnetite NPs (1 mgmL)
Magnetite NPs (05 mgmL)
Untreated Cells
Figure 76 Cell viability over exposure time Image shows overall increase in live-cell
fluorescence over time for all exposures The ionomycin positive control and 01 mgmL
concentration of magnetite NPs demonstrate a statistically significant reduction in cell
viability
The 1 mgmL NP concentration seems to have had cytotoxic effects as demonstrated by
the negative slope between the 6- and 12-hour time points In both cases the growth rate of
the NP-treated cells seems to have demonstrated an overall increase compared to control
and the slope of the line corresponding to growth rate is identical for both NP treatment
concentrations A double-tailed t-test was performed on the individual treatments
compared to control at the 24-hour time point The ionomycin positive control was the
155
only treatment that exhibited an extremely statistically significant deviation from control at
this time point No statistically significant reduction in viability was observed in the NP
treated cells at the 24-hour time point
725 Apoptosis Assay Results
The apoptosis positive control staurosporine showed a statistically significant deviation
from control at all time points The 05 mgmL NP concentration exhibited nearly identical
results to the untreated cells at the 12 and 24-hour time points No statistically significant
deviation from control was noted in the NP treated cells at any time point The higher than
average mean values observed at the 6-hour time point were not only found to be not
statistically significant due to the range of values in which low values are very close to
those of control cells but are not verified by the results of the viability assay The
ionomycin (necrosis) marker showed even further reduced apoptosis signal than the other
four treatments The apoptosis observed in the ionomycin positive control wells were
reduced compared to controls this reduction was found to be statistically significant and
very statistically significant at the 6 and 12-hour time points respectively It is important
to note that cell death is occurring in the ionomycin treated wells as evidenced by the
reduced viability of cells exposed to this compound Ionomycin is known to induce cell
death by necrosis as opposed to apoptosis Therefore the low levels of caspase an enzyme
biomarker for apoptosis specifically are to be expected No statistically significant
increase (or decrease) in apoptosis was observed for NP-treated wells compared to control
at any of the time points investigated
156
Figure 77 Apoptosis luminescence The apoptosis positive control demonstrated
statistically significant results as expected The necrosis positive control ionomycin
demonstrates statistically significant results lower than the value for untreated cells
denotes significance in which plt001 and denotes significance in which plt0001
denotes extreme statistical significance (plt00001)
The apoptosis time curve shows a linear increase in all treatments and untreated
cells except the ionomycin-treated cells which show a slight increase from 6 to 12 hours
followed by a plateau from 12 to 24 hours The apoptosis rate over time is not sufficient to
exhibit a decrease or even steady plateau in cell differentiation as evidenced by the
viability over time The apoptosis increase likely demonstrates a percent of the total
number of cells as opposed to an increased incidence of apoptosis over time
157
Apoptosis Time Curve
Time Point
Lu
min
escen
ce (
RL
U)
6 hou
r
12 h
our
24 h
our
0
2100 5
4100 5
6100 5
Staurosporine
NPs (1 mgmL)
NPs (05 mgmL)
Untreated Cells
Ionomycin
Figure 78 Apoptosis time curve Shows relatively linear increase in apoptosis over all
time points for all exposures to include untreated cells No statistically significant
differences among iron oxide NPs treatment concentrations or untreated cells were
observed
This is because we observed the normal doubling rate in the viability studies for all treated
and untreated cells Therefore there are more cells at the later time points and apoptosis in
a constant percent of the population would be expected to follow the same linear increase
that the viability graph demonstrated Although it appears from the graph that the 05
mgmL concentration exhibited increased incidence of apoptosis the overlap of the error
bars reveals a similar range and thus no statistically significant difference between the
two concentrations Only the apoptosis positive control staruosporine was found to
exhibit a statistically significant deviation from the control cells in the apoptosis assay
158
73 Discussion
This study investigated the acute in vitro cytotoxicity of two concentrations of colloidal
magnetite NPs in a human lung carcinoma cell line (A549) by comparing cytotoxicity
viability and apoptosis profiles over time Although the reliability of assays used to
investigate nanomaterial toxicity has been called into question due to the potential for
fluorescent NPs to enhance the fluorescent signal or for other metal and metal oxide NPs
to absorb the fluorescent signal [Doak 2009 2012] [Monteiro-Riviere 2009] [Han 2011]
[Love 2012] [Darolles 2013] at this time the fluorescence signal reduction by both
magnetite and maghemite NPs has been well characterized [Doak 2009] We have
accounted for the fluorescence signal absorption by the magnetite NPs at the
concentrations investigated by producing a mathematical model and correction factor using
experimental data The absorbance data demonstrates that the absorption of visible light is
consistent throughout all the frequencies detected by the assay thereby affirming the
reliability of the method The combination of assays has previously been proposed to
verify findings of a single assay in the investigation of NP toxicity [Han 2011] [Alinovi
2015] therefore our use of three assays was justified as the comparison of viability and
cytotoxicity data add another dimension of quality control to the interpretation of the data
This investigation has revealed an overall low-toxicity of magnetite NPs when
capped with PEG Despite the dose-dependent decreZase in viability at the 12-hour time
point all other parameters did not show a statistically significant deviation from the control
values The measured viability decrease at the 12-hour time point may need to be
investigated further as the viability was nearly identical to controls by the 24-hour time
159
point In addition the cytotoxicity and apoptosis profiles at 12-hours do not demonstrate a
corresponding increase in apoptotoic or necrotic (lysed) cells this time point Although the
mean apoptosis values for the NP solutions were slightly higher than the control cells the
overlap of the error bars demonstrating the range of measured values makes this not
significant The slightly elevated apoptosis signal at the 6-hour time point is not
corroborated by the viability and cytotoxicity findings as the viability was slightly higher
than the control cells at the 6-hour time point and a low number of dead cells were
measured Taking into account the increased live cell count at 6-hours and the slope of the
viability over time line in NP-treated wells it appears that the doubling time (growth rate)
of the cells was altered initially being increased from 0-6 hours halted from 6-12 hours
then increased again from 12-24 hours If the iron oxide NP solution did in fact increase
the growth rate of the cells then the slightly increased apoptosis detection as a percentage
of the total number of viable cells as demonstrated by the viability results at 6-hours still
results in a low ratio comparable to the control wells The low viability measured at 12-
hours was not observed as apoptosis by this assay therefore either cell death having been
induced by necrosis cell proliferation was impaired or both Typically we would expect
viability and cytotoxicity to be inversely proportional However it has been well-
established that prototypical anticancer agents can exhibit antiproliferative effects
(specifically a reduction in cell division) for a prolonged period of time prior to
membrane rupture Until membrane rupture occurs it is difficult to detect cell stress using
this assay Further investigation such as detection of pro-inflammatory cytokines
characteristic of necrosis and present prior to cell death could be used to determine whether
160
this pathway has been activated and when Cell cycle arrest in A549 cells in response to
potential toxin exposure has been reported previously in G-1 phase (Chang et al 2004)
G-2M cell cycle arrest (Wu et al 2005 Lee et al 2011 Wu et al 2013) S-phase arrest
(Chairuangkittiet al 2013) by nanoparticles (Choudhury et al 2013 Wu et al 2013
Kansara et al 2015) and DNA breaks have been discovered along with cell cycle arrest
(Kansara et al 2015) in a nanoparticle toxicity study Since this cell cycle (growth) arrest
is commonly observed in this cell type we must at least take into consideration the
evidence which seems to suggest that the decrease in viability is due to cell cycle arrest
The time period of cell cycle arrest is observed as a decline in viability with no
concomitant increase in cytotoxic biomarker which is exactly what was observed Caspase
activation which would have been detectable by the apoptosis marker may or may not be
measurable during this period Conversely a measurable decline in apparent viability may
be paired with a substantially reduced or unmeasurable cytotoxicity biomarker if cells died
early (typically by primary necrosis) in the exposure period (Niles et al 2008)
Considering the lack of evidence of cytotoxicity of the NPs revealed by the
cytotoxicity assay in combination with the low level of observed apoptosis in addition to
the numerous evidence suggesting cell cycle arrest as a response to toxicity in A549 cells
the main contributing factor to the observation of reduced viability (a low measurement of
viable cells) at the 12- hour time point is probably reduced proliferation both preceded and
followed by increased proliferation as opposed to cell death Another study into
nanoparticle toxicity in this cell line showed that Ag NPs reduced cell viability and
modulated cell cycle distribution with an accumulation of cells at G2M and sub-G1 phases
161
(cell death) leading to a decrease in cells at G1 (Lee et al 2011) Results suggest that Ag
NPs induce strong toxicity and G2M cell cycle arrest by a mechanism involving PKCζ
downregulation in A549 cells (Lee et al 2011) It appears that the iron oxide NPs may
also be causing a cell cycle arrest as evidenced by the decrease in viability at 12-hours
More work is needed to investigate whether or not this is the case Superparamagnetic iron
oxide NPs have shown promote cell proliferation by effecting cyclins and cyclin-
dependent kinases in human stem-cells (Huang 2009) The effect on proliferation is
probably dosage-dependent and more dosages and time-points should be investigated in
the future Therefore such NPs may very likely have a complex effect on the proliferation
cycle in certain human cell lines This effect and the mechanism(s) thereof merit
significant further research
Since PEG is an FDA-approved polymer and it has been shown to cause no
significant adverse effects [Working 1997] we do not attribute any cytotoxicity or
alterations in cell proliferation to the PEG NP coating Previous studies on dextran-coated
NPs have shown that detrimental effects of magnetite NPs may be facilitated by the
biochemical modifications to dextran by biological systems as well as the weak interaction
between the dextran coating and the nanoparticle Dextran undergoes conformational
changes and may completely desorb from the nanoparticle surface [Sonen and De Cuyper
2010] Cellular uptake of magnetite NPs coated with dextran have been degraded in acidic
lysosomes leaving a rapidly degraded iron core This iron can then induce toxic reactive
oxygen species (ROS) intermediates by the Fenton reaction [Arbab 2003] [Idee 2007]
One of the causes for the weak interaction between dextran and the NP stems from the
162
functional groups binding to the hydrocarbon polymer to the metal oxide NP Dextran uses
a hydroxyl (OH-) functional group to bind the NP (M+) As a potential solution to this
problem we succinylated our PEG cap which changes the terminal functional group from a
hydroxyl to a carbonyl (COOH-) group increasing the negative character and thus
strengthening the bond between the polymer and the NP This capping method of
incorporation of a stronger bonding FDA-approved polymer is anticipated to reduce ROS-
mediated cytotoxicity
Comprehensive toxicity profiles should include data on toxicity in multiple cell
lines in addition to animal models to include investigations on developmental effects It is
important to translate cytotoxic effects revealed by exposure to a concentration in cell
culture to a no observed adverse effects level (NOAEL) systemic dose administration
which is not necessarily straightforward Even in cases where in vivo studies have
demonstrated a NOAEL localization in specific organ systems and subsequent toxicity to
those specific cell types may not yet have been identified In vitro cytotoxicity of NP
systems in specific cell types is also useful for identifying mechanisms of toxicity after
systemic toxicity is observed Also higher concentrations of the investigational
nanomaterial than could be feasibly systemically administered may be investigated in cell
culture This is important for materials that will be targeted to a specific cell type or
administered as inhalation aerosols as in our application
163
CHapter 8
Conclusions and future work
Nanotechnology being often described as an emerging technology brings with it what we
call ldquothe promise of nanotechnologyrdquo This promise of nanotechnology hopes to realize
novel batteries magnetic and semiconductor materials individualized medicine faster
computers in vivo genetic alterations non-invasive medical procedures and countless
others A recent article was published in the magazine ldquoRisk Managementrdquo entitled ldquoThe
big risk of small particleshelliprdquo I believe this title says it all [Piper 2013] We must use
caution when embarking on such exciting new scientific ventures The excitement over our
ability to make these materials must not take precedent over the fact that our knowledge of
the toxicity of both the nanomaterials as well as the procedures for engineering them is at
present still limited Nikola Tesla the famous electrical engineer proclaimed that there
was a difference between Progress and Technology ldquoProgress benefits mankind
Technology does not necessarily do that If you have a technology that is polluting the
planet thats not progress [Tesla 1891]rdquo Therefore in the interest of progress let us
examine our methodology
164
81 Importance of Green Methodology
In 1857 Michael Faraday discovered and demonstrating that nanostructured colloidal gold
under certain lighting conditions produces different-colored solutions [Thompson 2007] It
is not until 2005 that the EPA begins reviewing new chemical notices under the Toxic
Substance Control Act (TSCA) for nanoscale materials This nearly 150-year gap in
regulation of such materials is hardly surprising During this time it was the responsibility
of the researchers to ensure safety and environmental soundness Now despite the EPA
regulation and some minimal oversight we as researchers maintain much of the
responsibility for ensuring immediate safety while tailoring our methods for the long-term
benefit of humanity Now more than ever with the population of the planet well on its
way to reaching 8 billion people we must maintain consciousness regarding the long-term
effects of our work Green chemistry standards along with our adherence to them will
undoubtedly facilitate true progress Iron of course exists naturally in the environment in
a few phases the dissolved phase as ferric (Fe3+) or ferrous (Fe2+) salts (as in our
precursor) or in the solid phase iron oxides such as goethite magnetite or Wuumlstite (as in
our product) and hematite [Ponnamperuma 1972] [Klaine 2008] [Ševců 2011] In the
case of this work I can genuinely affirm that we have done our finest to ensure safety
while minimizing long-term risk to our delicate environment As described in Chapter 2
we have replaced harsh metal nitrate precursors with chloride salts in all cases We have
used as a hydrocarbon carrier for the facilitation of epitaxial (layer-by-layer) crystal
growth as well as our stabilizing agents with constituents of vegetable oil rather than
petroleum products We have committed to the use of natural and biodegradable polymers
165
not only due to the positive results realized by their utilization Additionally we have
redistilled our waste solvents for multiple uses reducing waste from 4 L per 200 mg of
product to less than 05 L By using paraffin wax as an alternative to high purity long chain
hydrocarbons we are able to reuse this wax solvent at least five times reducing waste and
cost It is also important to note that due to the use of these environmentally friendly
solvents and precursors that the biocompatibility of our engineered materials is
intrinsically amplified
82 Bacterial Sensitivity Discussion
Despite the fact that we have demonstrated the antibacterial properties of NP-drug or NPs
alone one issue that resists prediction is the cellular permeability of P aeruginosa by the
tobramycin-loaded Fe3O4 complexes Future work may overcome the realization that very
little is known regarding uptake NPs by the individual Pseudomonas aeruginosa cells It is
known that aminoglycoside antibiotics such as tobramycin enter the cell through porin
channels along with water and electrolytes It is also known that they are not only
somewhat actively transported but may also cause nonspecific membrane toxicity even to
the point of bacterial cell lysis [Frasier 1986] The outer membrane of gram-negative
microbes is composed of lipopolysaccharide which differs from the cell membrane of other
microbes The outer membranes produce something called periplasmic protrusions under
stress conditions or upon virulence requirements while encountering a host target cell and
thus such protrusions function as virulence organelles [Yash Roy 1999] It has been
demonstrated that P aeruginosa has a comparatively large exclusion limit the actual
166
molecular weight cutoff is 3 kDa allowing the passage low molecular weight organic acids
(amino acids) carbohydrates alcohols aliphatic molecules aromatics and nitrogenous
compounds used as nutrient sources [Nikaido 2012]
As presented in Chapter 6 it appears that a destructive consequence appears under
of greater concentrations of zero-valent iron and consequently reactive oxygen species
(ROS) induced by the presence of iron Experimentation regarding the bactericidal effects
of zero-valent iron and the theoretical mechanisms leading to cell death has been
thoroughly investigated and the established findings may be referenced in any of the
following notable publications The first of which reports significant disruption of the
Escherichia coli cell membrane by zero-valent iron NPs suggesting inactivation or
enhanced the biocidal effects of dissolved iron as well as oxidative stress as mechanisms of
cell death [Lee 2008] Another report [Chen 2011] investigated the use of zero-valent iron
NPs against gram negative Escherichia coli and gram-positive Bacillus subtilis showing
that B subtilis was more tolerant to zero-valent iron NPs than E coli but states that the
bactericidal mechanism has not yet been elucidated Lastly another report claims that zero-
valent iron had no deleterious effect on total bacterial abundance in the microcosms
Surprisingly zero-valent iron with a biodegradable polyaspartate cap actually increased
bacterial populations by an order of magnitude relative to controls [Kirschling 2010]
Perhaps once naturally oxidized this material will benefit symbiotic bacterial populations
in the environment by providing beneficial doses of iron
It is possible that iron NPs may indirectly generate ROS which subsequently damage
ironndashsulfur clusters located in an assortment of metalloproteins examples are the well-
167
known NADH dehydrogenase ferredoxins hydrogenases nitrogenase coenzyme Q and
succinate dehydrogenase [Lippard 1994] This combination leads to Fentonrsquos reagent a
solution of hydrogen peroxide and iron in which iron is a catalyst that is used to oxidize
contaminants In industrial applications Fentons reagent can be used to destroy organic
compounds by catalyzing the production of additional ROS ROS generated via this
reaction can easily diffuse into the cell cytoplasm triggering ROS-induced ROS release in
the mitochondria triggering death A known mechanism of bacterial cell death induced by
zero-valent iron NPs as we demonstrated in Chapter 6 and is illustrated in Figure 81 In
our case it also may occur that initial disruption of the outside membrane of bacteria by
tobramycin assists the subsequent penetration of NP-tobramycin complexes and or iron
ions into the bacterial cell via simple diffusion since it is known that one mechanism of
action of aminoglycoside antibiotics is cell wall disruption
It may also be possible that initial disruption of the outside membrane of bacteria
by tobramycin assists the subsequent penetration of NP-tobramycin complexes and or iron
ions into the bacterial cell via simple diffusion since it is known that one mechanism of
action of aminoglycoside antibiotics is interference with protein synthesis leading to cell
membrane disruption However if this is occurring it is not happening on a large scale
since no statistically significant difference in the MIC or susceptibility was noted in
tobramycin conjugated iron-oxide NPs compared to unconjugated NPs However more
work is needed to clarify the antibacterial mechanism(s) of action of iron-oxide NPs alone
and in combination with the aminoglycoside or other antibiotic drugs and to clarify the
overall role of the capping agent
168
Figure 81 Mechanisms of cell damage and response after exposure to iron-
containing NPs Iron ions released from NPs can cross the membrane via either
active cellular uptake or leakage through sites with reduced membrane integrity
Highly reactive hydroxyl radicals resulting from Fe2+ reaction with hydrogen
peroxide primarily cause oxidative damage Fe3+ could be reduced by NADH and
thus regenerating Fe2+ OHmiddot radicals could also cause damage to DNA proteins and
lipids Fe2+ may also directly damage DNA
It is apparent that the composition of the capping agent and possibly the interactions of
the capping agent with the NP surface the ROS and the cell surfaces are primarily
responsible for facilitating or negating the antimicrobial effects Since uncapped iron-oxide
NPs (~16 nm) had similar antibacterial effects as the alginate capped and alginate capped-
169
tobramycin conjugated NPs (~200 nm) whereas the PEG-capped NPs (~40 nm) were
ineffective we do not attribute these findings to size effects At least at this size range
83 Conclusions
We have presented an alternative method for the treatment of P aeruginosa biofilms in
cystic fibrosis potentially to be administered via the inhalation aerosol route Positive
inhibition of bacterial growth was observed for uncapped and alginate-capped iron-oxide
NPs and the corresponding MICs have been presented We have observed zero
susceptibility to iron-oxide NPs capped with polyethylene glycol (PEG) suggesting that
the capping agent plays a major role in enabling bactericidal ability in of the
nanocomposite Our findings suggest that the alginate-coated nanocomposites investigated
in this study have the potential to overcome the bacterial biofilm barrier possibly by
simple diffusion due to the favorable solubility of the alginate-coated NPs within the
alginate biofilm Magnetic field application increases the action likely via enhanced
diffusion of the iron-oxide NPs and NP-drug conjugates through mucin and alginate
barriers which are characteristic of CF respiratory infections We have demonstrated that
iron-oxide NPs coated with alginate as well as alginate-coated magnetite ndash tobramycin
conjugates inhibit P aeruginosa growth and biofilm formation in established colonies
which are often the most difficult to treat We have also determined that susceptibility to
tobramycin decreases for longer culture times as the colonies are allowed to differentiate
for longer periods of time However susceptibility to the iron-oxide NP compounds did
not demonstrate any comparable decrease with increasing culture time In addition these
170
findings imply that iron-oxide NPs are promising lower-cost alternatives to silver NPs in
antibacterial coatings solutions and drugs as well as other applications in which
microbial abolition or infestation prevention is sought
We report on alteration of a basic repeatable solvothermal green chemistry
synthesis method that can be used to produce iron oxide nanoparticles in various
monodispersed size ranges from 10-100 nm and in a variety of shapes (spherical
polymorphous cube wire) Taking the iron oxide NPs produced by these methods we can
convert them into zero-valent iron or iron nitride We have investigated several capping
agent compositions and provided proven methods for application of the cap We have also
demonstrated the importance of the capping agent in functionalization and antibacterial
properties of the nanocomposites
We have investigated the cytotoxicity of iron oxide NPs on a lung adenocarcinoma
cell line We have shown that in general the NPs did not exhibit a statistically significant
cytotoxic effect at the concentrations investigated We did observe a slight decrease in
viability at the 12-hour time point which was not observed at the 24-hour time point
84 Future Work
If future experiments do determine that the NPs do in fact enter the cell further issues
requiring clarification remain As discussed in Chapter 5 there are five amine groups
present on the tobramycin molecule representing the functional groups bound to the NP in
practice one of these functional groups must also bind to the molecule of the ribosomal
RNA stopping protein synthesis This property is one responsible for the bactericidal
171
effects of the drug Our drug conjugation does not allow for determination of which of the
amine groups bind to the NP capping agent however may pose an additional problem It
has been demonstrated that the loss of only one of these sterically unhindered functional
groups reduce binding affinity for RNA 10-fold [Wong 1998] Although we did not
directly witness a reduction in drug activity the antibacterial effects of the iron-containing
NP may have balanced out the loss Tobramycin also binds to a site on the bacterial 30S
and 50S ribosome preventing formation of the 70S complex inhibiting protein synthesis
in this manner Whether or not the entire drug conjugated nanocomposite remains attached
when the drug binds the binding to a site on the bacterial RNA or ribosome despite
significant steric hindrance remains to be uncovered
Despite this we have shown that iron oxide NPs zero-valent iron NPs and
tobramycin-coupled iron oxide NPs exhibit a marked antibacterial result against P
aeruginosa bacteria in planktonic and biofilm mode There is no need to ldquodetachrdquo the
drug from the NP in order to observe a bactericidal effect These findings imply that at
least a certain percentage of the bound tobramycin molecules remain active after delivery
In fact there does not appear to be a need to conjugate any drug at all as the iron oxide
NPs with biodegradable alginate coating or no coating also exhibit a significant
bactericidal effect
Some minor issues remain to be investigated regarding this work investigation into
the theory that iron oxide NPs produce ROS clarification regarding what a therapeutic
dose would be investigation regarding the feasibility of the use of these materials as
preventative medicine for CF patients and of course characterization in vivo An
172
additional more in-depth investigation into the cytotoxicity of all these materials would be
beneficial More work is needed to determine the dose-dependent cytotoxicity over a larger
range of concentrations and cell types A more straightforward method such as individual
livedead cell counting might work better due to the potential of iron oxide to absorb light
and potentially interfere with the fluorescence assay Largely our investigations into the
use of SPIONS for the treatment of chronic biofilm infections in cystic fibrosis shows
promising results for drug-susceptible as well as drug-resistant strains of Pseudomonas
aeruginosa and may in the future help to extend the life expectancy of cystic fibrosis in
both developing countries and the developed world
173
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[Afreen 2011] Afreen R V and E Ranganath ldquoSynthesis of monodispersed silver
nanoparticles by Rhizopus stolonifer and its antibacterial activity against MDR strains of
Pseudomonas aeruginosa from burnt patientsrdquo International Journal of Environmental
Sciences 1 no 7 (2011) 1583ndash92
[Agnihotri 2014] Agnihotri S S Mukherji S Mukherji ldquoSize-controlled silver
nanoparticles synthesized over the range 5ndash100 nm using the same protocol and their
antibacterial efficacyrdquo RSC Advances 4 no 8 (2014) 3974ndash83
[Alexiou 2006] Alexiou C R Jurgons C Seliger and H Iro ldquoMedical applications of
magnetic nanoparticlesrdquo Journal of Nanoscience and Nanotechnology 6 no 9-10 (2006)
2762-2768
[Alinovi 2015] Alinovi R M Goldoni S Pinelli M Campanini I Aliatis D Bersani P
Paolo Lottici S Iavicoli M Petyx P Mozzoni and Mutti A ldquoOxidative and pro-
inflammatory effects of cobalt and titanium oxide nanoparticles on aortic and venous
endothelial cellsrdquo Toxicology in Vitro 29 no 3 (2015) 426-437
[Allan 1973] Allan J D A Mason and A D Moss ldquoNutritional supplementation in
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[An 2007] An D and M R Parsek ldquoThe promise and peril of transcriptional profiling in
biofilm communitiesrdquo Current Opinion in Microbiology 10 no 3 (2007) 292-296
[Anderson 2016] Anderson C and C Flask ldquoID 63 rapid 3D preclinical quantitative
lung imaging with ultrashort-echo time (UTE) MRI in a mouse model of cystic fibrosis
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[Andrauml 2007] Andrauml W and H Nowak eds Magnetism in Medicine A Handbook John
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[Annereau 2003] Annereau J Y Ko and P Pedersen ldquoCystic fibrosis transmembrane
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[Ansari 2014] Ansari M A H M Khan A A Khan S S Cameotra Q Saquib and J
Musarrat ldquoGum arabic capped‐silver nanoparticles inhibit biofilm formation by multi‐drug
174
resistant strains of Pseudomonas aeruginosardquo Journal of Basic Microbiology 54 no 7
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[Anselmo 2016] Anselmo A C and S Mitragotri ldquoNanoparticles in the clinicrdquo
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[Anwar 1989] Anwar H T Van Biesen M Dasgupta K Lam and J W Costerton
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Pramanik B Mallick and S Jha ldquoAntimicrobial activity of iron oxide nanoparticle upon
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[Arbab 2003] Arbab A S L A Bashaw B R Miller E K Jordan B K Lewis H
Kalish and J A Frank ldquoCharacterization of biophysical and metabolic properties of cells
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MR imagingrdquo Radiology 229 no 3 (2003) 838-846
[Armijo 2012a] Armijo L M Y I Brandt D Mathew S Yadav S Maestas A C
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Huber H D C Smyth and M Osiński ldquoIron oxide nanocrystals for magnetic
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[Armijo 2012b] Armijo L M Y I Brandt N J Withers J B Plumley N C Cook J B
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Smyth and M Osiński ldquoMultifunctional superparamagnetic nanocrystals for imaging and
targeted drug delivery to the lungrdquo Colloidal Nanocrystals for Biomedical Applications
VII (W J Parak M Osiński and K Yamamoto Eds) SPIE International Symposium on
Biomedical Optics BiOS 2012 San Francisco California 21-23 January 2012
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[Armijo 2014] Armijo L M M Kopciuch Z Olszoacutewka and S J Wawrzyniec A C
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Smyth and M Osinski ldquoDelivery of tobramycin coupled to iron oxide nanoparticles
across the biofilm of mucoidal Pseudonomas aeruginosa and investigation of its efficacyrdquo
Colloidal Nanoparticles for Biomedical Applications IX (W J Parak M Osiński and K
Yamamoto Eds) SPIE International Symposium on Biomedical Optics BiOS 2014 San
Francisco California 1-3 February 2014 Proceedings of SPIE Vol 8955 Paper 9550I
(12 pp) doi101117122043340
175
[Asharani 2008] Asharani P V Y L Wu Z Y Gong and S Valiyaveettil ldquoToxicity of
silver nanoparticles in zebrafish modelsrdquo Nanotechnology 19 no 25 (2008) 255102 (8
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[Auffan 2008] Auffan M W Achouak J Rose M-A Roncato C Chaneacuteac D T Waite
A Masion J C Woicik M R Wiesner and J-Y Bottero ldquoRelation between the redox
state of iron-based nanoparticles and their cytotoxicity toward Escherichia colirdquo
Environmental Science amp Technology 42 no 17 (2008) 6730-6735
[Awwad 2012] Awwad A M and N M Salem ldquoA green and facile approach for
synthesis of magnetite nanoparticlesrdquo Nanoscience and Nanotechnology 2 no 6 (2012)
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[Baltch 1994] Baltch A L and R P Smith ldquoPseudomonas aeruginosa infections and
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[Bao 1994] Bao X H R M Metzger and M Carbucicchio ldquoSynthesis and properties of
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[Bao 2005] Bao Y A B Pakhomov and K M Krishnan ldquoA general approach to
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[Baronzio 2006] Baronzio G F and E D Hager eds Hyperthermia in Cancer
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[Basak 2007] Basak S D R Chen and P Biswas ldquoElectrospray of ionic precursor
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[Batten 1965] Batten John ldquoCystic fibrosis A reviewrdquo British Journal of Diseases of the
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[Bauernfeind 1987] Bauernfeind A K Rotter and C H Weisslein-Pfister ldquoSelective
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[Beer 2012] Beer C R Foldbjerg Y Hayashi D S Sutherland and H Autrup ldquoToxicity
of silver nanoparticlesmdashnanoparticle or silver ionrdquo Toxicology Letters 208 no 3 (2012)
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[Behera 2012] Behera S S J K Patra K Pramanik N Panda and H Thatoi
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176
oxide nanoparticlesrdquo World Journal of Nano Science and Engineering 2 no 4 (2012)
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[Benamara 2014] Benamara H C Rihouey I Mohamed AB Mlouka J Hardouin T
Jouenne and S Alexandre ldquoCharacterization of Membrane Lipidome Changes in
Pseudomonas aeruginosa during Biofilm Growth on Glass Woolrdquo (2014) e108478
[Berlyne 2000] Berlyne G S K Parameswaran D Kamada A Efthimiadis and F E
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[Berry 2004] Berry C C S Wells S Charles G Aitchison and A S Curtis ldquoCell
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[Bezeljak 2012] Bezeljak U A Golob M Jerala L Kandunc Z Lužnik F Pavlovec B
Pirš M Somrak M Stražar D Vucko U Zupancic M Bencina V Forstneric T Lebar
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[Bossi 2004] Bossi A G Casazza R Padoan and S Milani ldquoWhat is the incidence of
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Smolyakov H D C Smyth and M Osiński ldquoEffectiveness of tobramycin conjugated to
iron oxide nanoparticles in treating infection in cystic fibrosisrdquo Colloidal Nanoparticles for
Biomedical Applications VIII (W J Parak M Osiński and K Yamamoto Eds) SPIE
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[Buckley 2006] Buckley P R G H McKinley T S Wilson W Small IV W J Benett
J P Bearinger M W McElfresh and D J Maitland ldquoInductively heated shape memory
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[Burney 2012] Burney T J and J C Davies ldquoGene therapy for the treatment of cystic
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[Cai 2007] Cai W and J Q Wan ldquoFacile synthesis of superparamagnetic magnetite
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American Journal of Infection Control 32 no 8 (2004) 470ndash85
[Casula 2006] Casula M F Y-W Jun D J Zaziski E M Chan A Corrias A P
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[Cavaliere 2015] Cavaliere E S De Cesari G Landini E Riccobono L Pallecchi G M
Rossolini and L Gavioli ldquoHighly bactericidal Ag nanoparticle films obtained by cluster
beam depositionrdquo Nanomedicine 11 no 6 (2015) 1417ndash23
[Chairuangkitti 2013] Chairuangkitti P S Lawanprasert S Roytrakul S Aueviriyavit
D Phummiratch K Kulthong P Chanvorachote and R Maniratanachote ldquoSilver
nanoparticles induce toxicity in A549 cells via ROS-dependent and ROS-independent
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[Chase 1979] Chase H P M A Long and M H Lavin ldquoCystic fibrosis and
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[Childers 2007] Childers M G Eckel A Himmel and J Caldwell ldquoA new model of
cystic fibrosis pathology Lack of transport of glutathione and its thiocyanate conjugatesrdquo
Medical Hypotheses 68 no 1 (2007) 101-112
[Chin 2007] Chin A B and I I Yaacob ldquoSynthesis and characterization of magnetic
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[Cho 2005] Cho K-H J-E Park T Osaka and S-G Park ldquoThe study of antimicrobial
activity and preservative effects of nanosilver ingredientrdquo Electrochimica Acta 51 no 5
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[Choudhury 2013] Choudhury D P L Xavier K Chaudhari R John AK Dasgupta T
Pradeep and G Chakrabarti ldquoUnprecedented inhibition of tubulin polymerization directed
by gold nanoparticles inducing cell cycle arrest and apoptosisrdquo Nanoscale 5 no 10
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[Chow 2007] Chow A H L H H Y Tong P Chattopadhyay and B Y Shekunov
ldquoParticle engineering for pulmonary drug deliveryrdquo Pharmaceutical Research 24 no 3
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[Chudasama 2010] Chudasama B A K Vala N Andhariya N R V Mehta and R V
Upadhyay ldquoHighly bacterial resistant silver nanoparticles synthesis and antibacterial
activitiesrdquo Journal of Nanoparticle Research 12 no 5 (2010) 1677ndash85
[Chung 2002] Chung F N Barnes M Allen R Angus P Corris A Knox J Miles A
Morice J OReilly and M Richardson ldquoAssessing the burden of respiratory disease in the
UKrdquo Respiratory Medicine 96 no 12 (2002) 963-975
[Clancy 2012] Clancy J P S M Rowe F J Accurso M L Aitken R S Amin M A
Ashlock M Ballmann M P Boyle I Bronsveld P W Campbell K De Boeck S H
Donaldson H L Dorkin J M Dunitz P R Durie M Jain A Leonard K S McCoy R
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Botfield C L Ordontildeez G T Spencer-Green L Vernillet S Wisseh K Yen and M W
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Konstan ldquoResults of a phase IIa study of VX-809 an investigational CFTR corrector
compound in subjects with cystic fibrosis homozygous for the F508del-CFTR mutationrdquo
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[Corey 1988] Corey M F J McLaughlin M Williams and H Levison ldquoA comparison
of survival growth and pulmonary function in patients with cystic fibrosis in Boston and
Torontordquo Journal of Clinical Epidemiology 41 no 6 (1988) 583-591
[Cornell 2006] Cornell R M and U Schwertmann The Iron Oxides Structure
Properties Reactions Occurrences and Uses 2nd ed John Wiley amp Sons 2006
[Coyne 2009] Coyne D W ldquoFerumoxytol for treatment of iron deficiency anemia in
patients with chronic kidney diseaserdquo Expert Opinion on Pharmacotherapy 10 no 15
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[Crozier 1974] Crozier D N ldquoCystic fibrosis a not-so-fatal diseaserdquo Pediatric Clinics of
North America 21 no 4 (1974) 935
[Danes 1968] Danes B S and A G Bearn A genetic cell marker in cystic fibrosis of the
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[Dang 2006] Dang J M and K W Leong ldquoNatural polymers for gene delivery and
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[Darrolles 2013] Darolles C N Sage J Armengaud and V Malard V ldquoIn vitro
assessment of cobalt oxide particle toxicity identifying and circumventing interferencerdquo
Toxicology in Vitro 27 no 6 (2013) 1699-1710
[Darwish 2015] Darwish M S A N H A Nguyen A Ševců and I Stibor
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Staphylococcus aureusrdquo Journal of Nanomaterials (2015)416012 (10 pp)
[Davey 2003] Davey M E N C Caiazza and G A OToole ldquoRhamnolipid surfactant
production affects biofilm architecture in Pseudomonas aeruginosa PAO1rdquo Journal of
Bacteriology 185 no 3 (2003) 1027-1036
[Davis 2006] Davis P B ldquoCystic fibrosis since 1938rdquo American Journal of Respiratory
and Critical Care Medicine 173 no 5 (2006) 475-482
[De Boeck and Amaral 2016] De Boeck K and M D Amaral ldquoProgress in therapies for
cystic fibrosisrdquo The Lancet Respiratory Medicine 4 no 8 (2016) 662-674
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[Denning 1968] Denning C R S C Sommers and H J Quigley ldquoInfertility in male
patients with cystic fibrosisrdquo Pediatrics 41 no 1 (1968) 7-17
[Diao 2009] Diao M H and M S Yao ldquoUse of zero-valent iron nanoparticles in
inactivating microbesrdquo Water Research 43 no 20 (2009) 5243-5251
[Doak 2009] Doak S H S M Griffiths Bella Manshian N Singh P M Williams A P
Brown and G J S Jenkins ldquoConfounding experimental considerations in
nanogenotoxicologyrdquo Mutagenesis 24 no 4 (2009) 285-293
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Research 67 no 1 (2006) 55-60
[Dodge 2007] Dodge J A P A Lewis M Stanton and J Wilsher ldquoCystic fibrosis
mortality and survival in the UK 1947ndash2003rdquo European Respiratory Journal 29 no 3
(2007) 522-526
[Dong 2012] Dong P V C H Ha L T Binh and J Kasbohm ldquoChemical synthesis and
antibacterial activity of novel-shaped silver nanoparticlesrdquo International Nano Letters 2
no 1 (2012) 9 (9 pp)
[Dupuis 2005] Dupuis A D Hamilton D E C Cole and M Corey ldquoCystic fibrosis
birth rates in Canada a decreasing trend since the onset of genetic testingrdquo The Journal of
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[Duraacuten 2007] Duraacuten N PD Marcato G I H De Souza O L Alves and E Esposito
ldquoAntibacterial effect of silver nanoparticles produced by fungal process on textile fabrics
and their effluent treatmentrdquo Journal of Biomedical Nanotechnology 3 no 2 (2007) 203ndash
8
[Eck 1999] Eck B R Dronskowski M Takahashi and S Kikkawa ldquoTheoretical
calculations on the structures electronic and magnetic properties of binary 3d transition
metal nitridesrdquo Journal of Materials Chemistry 9 no 7 (1999) 1527-1537
[Eid 2013] Eid M and E Araby ldquoBactericidal effect of poly(acrylamideitaconic acid)-
silver nanoparticles synthesized by gamma irradiation against Pseudomonas aeruginosardquo
Applied Biochemistry and Biotechnology 171 no 2 (2013) 469ndash87
[Elborn 1991] Elborn J Stuart D J Shale and J R Britton ldquoCystic fibrosis current
survival and population estimates to the year 2000rdquo Thorax 46 no 12 (1991) 881-885
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[El-Kheshen 2012] El-Kheshen A A and S F G El-Rab ldquoEffect of reducing and
protecting agents on size of silver nanoparticles and their anti-bacterial activityrdquo Der
Pharma Chemica 4 no 1 (2012) 53ndash65
[Emeka 2014] Emeka E E O C Ojiefoh C Aleruchi L A Hassan O M Christiana
M Rebecca E O Dare and A E Temitope ldquoEvaluation of antibacterial activities of
silver nanoparticles green-synthesized using pineapple leaf (Ananas comosus)rdquo Micron 57
(2014) 1ndash5
[EPA 2015] EPA ldquoBasics of Green Chemistryrdquo Accessed February 18 2015
httpwww2epagovgreen-chemistrybasics-green-chemistrytwelve
[Falgas 2005] Falagas M E S K Kasiakou and L D Saravolatz ldquoColistin the revival
of polymyxins for the management of multidrug-resistant gram-negative bacterial
infectionsrdquo Clinical Infectious Diseases 40 no 9 (2005) 1333ndash41
[Falgas 2007] Falagas M E and I A Bliziotis ldquoPandrug-resistant Gram-negative
bacteria the dawn of the post-antibiotic erardquo International Journal of Antimicrobial
Agents 29 no 6 (2007) 630ndash6
[Fannin 1989] Fannin P C and S W Charles ldquoThe study of a ferrofluid exhibiting both
Brownian and Neacuteel relaxationrdquo Journal of Physics D Applied Physics 22 no 1 (1989)
187-191
[Fannin 1994] Fannin P C Y P Kalmykov and S W Charles ldquoOn the use of
frequency-domain measurements to investigate time-domain magnetization decay in a
ferrofluidrdquo Journal of Physics D Applied Physics 27 no 2 (1994) 194-197
[Farrell 2007] Farrell P S Joffe L Foley G J Canny P Mayne and M Rosenberg
ldquoDiagnosis of cystic fibrosis in the Republic of Ireland epidemiology and costsrdquo Irish
Medical Journal 100 no 8 (2007) 557-560
[Farrell 2008] Farrell P M ldquoThe prevalence of cystic fibrosis in the European Unionrdquo
Journal of Cystic Fibrosis 7 no 5 (2008) 450-453
[Feuchtbaum 2012] Feuchtbaum L J Carter S Dowray R J Currier and F Lorey
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raceethnicityrdquo Genetics in Medicine 14 no 11 (2012) 937-945
[Fick 1992] Fick Jr R B F Sonoda and D B Hornick ldquoEmergence and persistence of
Pseudomonas aeruginosa in the cystic fibrosis airwayrdquo In Seminars in Respiratory
Infections 7 no 3(1992)168-178
183
[Fishkum 1985] Fiskum G ldquoIntracellular levels and distribution of Ca2+ in digitonin-
permeabilized cellsrdquo Cell Calcium 6 no 1-2 (1985) 25-37
[FitzSimmons 1993] FitzSimmons S C ldquoThe changing epidemiology of cystic fibrosisrdquo
The Journal of Pediatrics 122 no 1 (1993) 1-9
[Foldbjerg 2011] Foldbjerg R D A Dang and H Autrup ldquoCytotoxicity and genotoxicity
of silver nanoparticles in the human lung cancer cell line A549rdquo Archives of Toxicology
85 no 7 (2011) 743ndash50
[Fourmy 1996] Fourmy D M I Recht S C Blanchard J D Puglisi ldquoStructure of the A-
site of Escherichia coli 16S ribosomal RNA complexed with an aminoglycoside
antibioticrdquo Science 274 (1996) 1367ndash1371
[Fourmy 1998] Fourmy D M I Recht and J D Puglisi ldquoBinding of neomycin-class
aminoglycoside antibiotics to the A-site of 16 S rRNArdquo Journal of Molecular Biology
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[Franci 2015] Franci G A Falanga S Galdiero L Palomba M Rai G Morelli M
Galdiero ldquoSilver nanoparticles as potential antibacterial agentsrdquo Molecules 20 no 5
(2015) 8856ndash74
[Fraser 1986] Fraser C M ed The Merck Veterinary Manual Sixth Edition Merck amp Co
1986
[Frederiksen 1996] Frederiksen B S Lanng C Koch and N Hoslashlby ldquoImproved survival
in the Danish center‐treated cystic fibrosis patients Results of aggressive
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[Frizzel 2012] Frizzell R A and J W Hanrahan ldquoPhysiology of epithelial chloride and
fluid secretionrdquo Cold Spring Harbor Perspectives in Medicine 2 no 6 (2012) a009563
[Gabriel 1994] Gabriel S E K N Brigman B H Koller R C Boucher and M J
Stutts ldquoCystic fibrosis heterozygote resistance to cholera toxin in the cystic fibrosis mouse
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[Gacesa 1990] Gacesa P and N J Russell ldquoThe Structure and Properties of Alginaterdquo
Pseudonomas Infection and Alginates Biochemistry Genetics and Pathology (P Gacesa
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[Gash 2001] Gash A E T M Tillotson J H Satcher J F Poco L W Hrubesh and R
L Simpson ldquoUse of epoxides in the sol-gel synthesis of porous iron (III) oxide monoliths
from Fe (III) saltsrdquo Chemistry of Materials 13 no 3 (2001) 999-1007
[Ge 2009] Ge S X Y Shi K Sun C P Li C Uher J R Baker Jr M M Banaszak
Holl and B G Orr ldquoFacile hydrothermal synthesis of iron oxide nanoparticles with
tunable magnetic propertiesrdquo The Journal of Physical Chemistry C 113 no 31 (2009)
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[Geelen 2005] Geelen Math JH ldquoThe use of digitonin-permeabilized mammalian cells for
measuring enzyme activities in the course of studies on lipid metabolismrdquo Analytical
Biochemistry 347 no 1 (2005) 1ndash9 doi101016jab200503032 PMID 16291302
[Giessen 2016] Giessen T W and P A Silver PA ldquoConverting a natural protein
compartment into a nanofactory for the size-constrained synthesis of antimicrobial silver
nanoparticlesrdquo ACS Synthetic Biology 5 no 12 (2016) 1497ndash504
[Gilani 2005] Gilani K A R Najafabadi M Barghi and M Rafiee‐Tehrani ldquoThe effect
of water to ethanol feed ratio on physical properties and aerosolization behavior of spray
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[Gilligan 1991] P H Gilligan ldquoMicrobiology of airway disease in patients with cystic
fibrosisrdquo Clinical Microbiology Reviews vol 4 (1) pp 35-51 Jan 1991
[Gould 1988] Gould S J and S Subramani ldquoFirefly luciferase as a tool in molecular and
cell biologyrdquo Analytical Biochemistry 175 no 1 (1988) 5-13
[Govan 1996] Govan J R and V Deretic ldquoMicrobial pathogenesis in cystic fibrosis
Mucoid Pseudomonas aeruginosa and Burkholderia cepaciardquo Microbiological Reviews
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[Gracey 1969] Gracey M V Burke and C M Anderson ldquoTreatment of abdominal pain
in cystic fibrosis by oral administration of n-acetyl cysteinerdquo Archives of Disease in
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[Grachev 2001] Grachev S D M Borsa S Vongtragool and D O Boerma ldquoThe
growth of epitaxial iron nitrides by gas flow assisted MBErdquo Surface Science 482 (2001)
802-808
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[Grassmeacute 2000] Grassmeacute H S Kirschnek J Riethmueller A Riehle G von Kuumlrthy F
Lang M Weller and E Gulbins ldquoCD95CD95 ligand interactions on epithelial cells in
host defense to Pseudomonas aeruginosardquo Science 290 no 5491 (2000) 527-530
[Greenwood 1999] Greenwood R and K Kendall ldquoSelection of suitable dispersants for
aqueous suspensions of zirconia and titania powders using acoustophoresisrdquo Journal of the
European Ceramic Society 19 no 4 (1999) 479ndash88
[Griesenbach 2006] Griesenbach U D M Geddes and E W F W Alton ldquoGene therapy
progress and prospects cystic fibrosisrdquo Gene Therapy 13 no 14 (2006) 1061-1067
[Grottone 2014] Grottone G T R R Loureiro J Covre E B Rodrigues J Aacute Pereira
Gomes ldquoARPE-19 cell uptake of small and ultrasmall superparamagnetic iron oxiderdquo
Current Eye Research 39 no 4 (2014) 403ndash10
[Gupta 2005] Gupta A K and M Gupta ldquoSynthesis and surface engineering of iron
oxide nanoparticles for biomedical applicationsrdquo Biomaterials 26 no 18 (2005) 3995-
4021
[Hacein-Bey-Albina 2008] Hacein-Bey-Abina S A Garrigue G P Wang J Soulier A
Lim E Morillon E Clappier L Caccavelli E Delabesse K Beldjord and V Asnafi
Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1rdquo
The Journal of Clinical Investigation 118 no 9 (2008) 3132-3142
[Haumlfeli 1998] Haumlfeli U ldquoThe history of magnetism in medicinerdquo Magnetism in Medicine
A Handbook Second Edition (1998) 1-25
[Haghighi 2016] Haghighi Pak Z H Abbaspour N Karimi and A Fattahi ldquoEco-friendly
synthesis and antimicrobial activity of silver nanoparticles using Dracocephalum
moldavica seed extractrdquo Applied Sciences 6 no 3 (2016) 69 (10 pp)
[Hamishehkar 2012] Hamishehkar H Y Rahimpour and Y Javadzadeh ldquoThe role of
carrier in dry powder inhalerrdquo INTECH Open Access Publisher 2012
[Han 2011] Han X R Gelein N Corson P Wade-Mercer J Jiang P Biswas J N
Finkelstein A Elder and G Oberdoumlrster G ldquoValidation of an LDH assay for assessing
nanoparticle toxicityrdquo Toxicology 287 no 1 (2011) 99-104
[Hanoar 2012] Hanaor D M Michelazzi C Leonelli and C C Sorrell ldquoThe effects of
carboxylic acids on the aqueous dispersion and electrophoretic deposition of ZrO2rdquo
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[Hattori 2001] Hattori T N Kamiya and Y Kato ldquoMagnetic properties of Fe16N2 fine
particlesrdquo Journal of the Magnetics Society of Japan 25 (2001) 927-930
[Hauser 2003] Hauser A R and J Rello Severe Infections Caused by Pseudomonas
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[Hays 1945] Hays EE IC Wells PA Katzman CK Cain FA Jacobs SA Thayer
EA Doisy WL Gaby EC Roberts RD Muir CJ Carroll ldquoAntibiotic Substances
produced by Pseudomonas aeruginosardquo Biological Chemistry 159 no 3 (1945) 725ndash50
[Hearst 1995] Hearst J E and K E Elliott ldquoIdentifying the killer in cystic fibrosisrdquo
Nature Medicine 1 no 7 (1995) 626
[Henderson 1908a] Henderson L J ldquoConcerning the relationship between the strength of
acids and their capacity to preserve neutralityrdquo American Journal of Physiology 21 no 2
(1908) 173ndash9
[Henderson 1908b] Henderson L J ldquoThe theory of neutrality regulation in the animal
organismrdquo American Journal of Physiology 21 no 4 (1908) 427ndash48
[Henle 1997] Henle E S and S Linn ldquoFormation prevention and repair of DNA
damage by ironhydrogen peroxiderdquo Journal of Biological Chemistry 272 no 31 (1997)
19095-19098
[Hergt 1998] Hergt R W Andra C G dAmbly I Hilger W A Kaiser U Richter and
H-G Schmidt ldquoPhysical limits of hyperthermia using magnetite fine particlesrdquo IEEE
Transactions on Magnetics 34 no 5 (1998) 3745-3754
[Hergt 2006] Hergt R S Dutz R Muumlller and M Zeisberger ldquoMagnetic particle
hyperthermia Nanoparticle magnetism and materials development for cancer therapyrdquo
Journal of Physics Condensed Matter 18 no 38 (2006) S2919-S2934
[Hermanson 2013] Hermanson G T Bioconjugate Techniques Second ed Academic
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[Hickey 2003] Hickey A J ed Pharmaceutical Inhalation Aerosol Technology CRC
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[Hickey 2007] Hickey A J H M Mansour M J Telko Z Xu H D C Smyth T
Mulder R McLean J Langridge D Papadopoulos ldquoPhysical characterization of
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of Pharmaceutical Sciences 571 no 96 (2007) 1302-1319
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[Hide 1969] Hide D W and D Burman ldquoAn infant with both cystic fibrosis and coeliac
diseaserdquo Archives of Disease in Childhood 44 no 236 (1969) 533
[Hirsch 2003] Hirsch L R R J Stafford J A Bankson S R Sershen B Rivera R E
Price J D Hazle N J Halas and J L West ldquoNanoshell-mediated near-infrared thermal
therapy of tumors under magnetic resonance guidancerdquo Proceedings of the National
Academy of Sciences 100 no 23 (2003) 13549-13554
[Hsueh 2017] Hsueh Y-H P-H Tsai K-S Lin W-J Ke C-L Chiang ldquoAntimicrobial
effects of zero-valent iron nanoparticles on gram-positive Bacillus strains and gram-
negative Escherichia coli strainsrdquo Journal of Nanobiotechnology 3 no 15 (2017)77 (12
pp)
[Hu 2007] Hu X L J C Yu J M Gong Q Li and G S Li ldquoα‐Fe2O3 nanorings
prepared by a microwave‐assisted hydrothermal process and their sensing
propertiesrdquo Advanced Materials 19 no 17 (2007) 2324-2329
[Huang 2009] Huang D-M J-K Hsiao Y-C Chen L-Y Chien M Yao Y-K Chen
B-S Ko S-C Hsu L-A Tai and H-Y Cheng ldquoThe promotion of human mesenchymal
stem cell proliferation by superparamagnetic iron oxide nanoparticlesrdquo Biomaterials 30
no 22 (2009) 3645-3651
[Hyeon 2003] Hyeon T G ldquoChemical synthesis of magnetic nanoparticlesrdquo Chemical
Communications 8 (2003) 927-934
[Idee 2007] Idee J M M Port I Raynal M Schaefer B Bonnemain P Prigent P
Robert C Robic and C Corot C ldquoSuperparamagnetic nanoparticles of iron oxides for
magnetic resonance imaging applicationsrdquo Nanotechnologies for the Life Sciences 10
(2007) 51-84
[Iida 2007] Iida H K Takayanagi T Nakanishi and T Osaka ldquoSynthesis of Fe3O4
nanoparticles with various sizes and magnetic properties by controlled
hydrolysisrdquo Journal of Colloid and Interface Science 314 no 1 (2007) 274-280
[Indira 2010] Indira T K and P K Lakshmi ldquoMagnetic nanoparticlesmdashA
reviewrdquo International Journal of Pharmarmaceutical Sciences and Nanotechnology 3 no
3 (2010) 1035-1042
[Jack 1951] Jack K H ldquoThe iron-nitrogen system The preparation and the crystal
structures of nitrogen-austenite (γ) and nitrogen-martensite (αrsquo)rdquo Proceedings of the Royal
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188
[Javanbakht 2016] Javanbakht T S Laurent D Stanicki and K J Wilkinson ldquoRelating
the surface properties of superparamagnetic iron oxide nanoparticles (SPIONs) to their
bactericidal effect towards a biofilm of Streptococcus mutansrdquo PLoS ONE 11 no 4
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[Jensen 1987] Jensen T S S Pedersen S Garne C Heilmann N Hoslashiby and C Koch
ldquoColistin inhalation therapy in cystic fibrosis patients with chronic Pseudomonas
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838
[Ji 2010] Ji N X Liu and J-P Wang ldquoTheory of giant saturation magnetization in α-
Fe16N2 Role of partial localization in ferromagnetism of 3d transition metalsrdquo New
Journal of Physics 12 no 6 (2010) 063032
[Johannsen 2007] Johannsen M U Gneveckow B Thiesen K Taymoorian C H Cho
N Waldoumlfner R Scholz A Jordan S A Loening and P Wust ldquoThermotherapy of
prostate cancer using magnetic nanoparticles Feasibility imaging and three-dimensional
temperature distributionrdquo European Urology 52 no 6 (2007) 1653-1662
[Johnson 1984] Johnson Sir R ldquoHistory of the Cystic Fibrosis Research Trustrdquo 20th
Anniversary Meeting Brighton (1984) pp 3-6
[Kadasi 1997] Kadasi L H Polakova A Zatkova and H Kayserova ldquoDistribution of 9
common mutations in the CFTR gene in Slovak cystic fibrosis patientsrdquo Gene Geography
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[Kaialy 2012] Kaialy W G P Martin H Larhrib M D Ticehurst E Kolosionek and
A Nokhodchi ldquoThe influence of physical properties and morphology of crystallised
lactose on delivery of salbutamol sulphate from dry powder inhalersrdquo Colloids and
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[Kanicky 2002] Kanicky J R and D O Shah ldquoEffect of degree type and position of
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Science 256 no 1 (2002) 201ndash7
[Kansara 2015] Kansara K P Patel D Shah R K Shukla S Singh A Kumar and
Dhawan ldquoTiO2 nanoparticles induce DNA double strand breaks and cell cycle arrest in
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[Kasithevar 2017] Kasithevar M P Periakaruppan S Muthupandian and M Mohan
ldquoAntibacterial efficacy of silver nanoparticles against multi-drug resistant clinical isolates
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[Kawata 2009] Kawata K M Osawa S Okabe ldquoIn vitro toxicity of silver nanoparticles
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[Kim 1972] Kim T K and M Takahashi ldquoNew magnetic material having ultrahigh
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[Kim 2005] Kim E H H S Lee B K Kwak and B-K Kim ldquoSynthesis of ferrofluid
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[Kim 2007] Kim J S E Kuk K N Yu J-H Kim S J Park H J Lee S H Kim Y K
Park Y H Park C-Y Hwang Y-K Kim Y-S Lee D H Jeong and M-H Cho
ldquoAntimicrobial effects of silver nanoparticlesrdquo Nanomedicine Nanotechnology Biology
and Medicine 3 no 1 (2007) 95ndash101
[Kim 2012] Kim D-J S-G Chung S-H Lee and J-W Choi ldquoRelation of microbial
biomass to counting units for Pseudomonas aeruginosardquo African Journal of Microbiology
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[Kirby 2010] Kirby Brian J Micro-and nanoscale fluid mechanics transport in
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[Kirschling 2010] Kirschling T L K B Gregory E G Minkley Jr G V Lowry and R
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[Klaine 2008] Klaine S J P J J Alvarez G E Batley T F Fernandes R D Handy D
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[Klausen 2003] Klausen M A Heydorn P Ragas L Lambertsen A Aaes‐Joslashrgensen S
Molin and T Tolker‐Nielsen ldquoBiofilm formation by Pseudomonas aeruginosa wild type
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[Knappen 2004] Knaapen A M P J Borm C Albrecht and R P Schins (2004)
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Hagen ldquoHyperthermia and thermosensitive liposomes for improved delivery of
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[Konstan 2004] Konstan M W P B Davis J S Wagener K A Hilliard R C Stern L
J H Milgram T H Kowalczyk S L Hyatt T L Flink C R Gedeon and S M Oette
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[Kosorok 1996] Kosorok M R W‐H Wei and P M Farrell ldquoThe incidence of cystic
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[Krishnan 2010] Krishnan S P Diagaradjane and S H Cho ldquoNanoparticle-mediated
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[Lannefors 2002] Lannefors Louise and Anna Lindgren ldquoDemographic transition of the
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[Lara 2011] Lara H H E N Garza-Trevintildeo L Ixtepan-Turrent and D K Singh ldquoSilver
nanoparticles are broad-spectrum bactericidal and virucidal compoundsrdquo Journal of
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[Lara 2015] Lara H H D G Romero-Urbina C Pierce J L Lopez-Ribot M J
Arellano-Jimeacutenez and M Jose-Yacaman ldquoEffect of silver nanoparticles on Candida
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[Laurent 2008] Laurent S D Forge M Port A Roch C Robic L Vander Elst and R
N Muller ldquoMagnetic iron oxide nanoparticles Synthesis stabilization vectorization
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[Le 2012] Le A-T T T Le V Q Nguyen H H Tran D A Dang Q H Tran and D L
Vu ldquoPowereful silver nanoparticles for the prevention of gastrointestinal bacterial
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[Leboffe 2012] Leboffe M J and B E Pierce Microbiology Laboratory theory and
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[Lee 2007] Lee Y C B J Ahn J S Jin J U Kim S H Lee D Y Song W K Lee
and J C Lee ldquoMolecular characterization of Pseudomonas aeruginosa isolates resistant to
all antimicrobial agents but susceptible to colistin in Daegu Koreardquo Journal of
Microbiology (Seoul Korea) 45 no 4 (2007) 358-363
[Lee 2008] Lee C J Y Kim W I Lee K L Nelson J Yoon and D L Sedlak
ldquoBactericidal effect of zero-valent iron nanoparticles on Escherichia colirdquo Environmental
Science amp Technology 42 no 13 (2008) 4927-4933
[Lee 2011] Lee YS DW Kim YH Lee JH Oh S Yoon MS Choi SK Lee JW
Kim K Lee and CW Song ldquoSilver nanoparticles induce apoptosis and G2M arrest via
PKCζ-dependent signaling in A549 lung cellsrdquo Archives of Toxicology 85 no 12 (2011)
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[Lehr 1992] Lehr C-M J A Bouwstra E H Schacht and H E Junginger ldquoIn vitro
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[Lev 1965] Lev R S S Spicer ldquoAn historical chemical comparison of human epithelial
mucins in normal and hypersecretory states including pancreatic cystic fibrosisrdquo American
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[Levine 2011] Levine B N Mizushima and H W Virgin ldquoAutophagy in immunity and
inflammationrdquo Nature 469 no 7330 (2011) 323-335
[Li 2011] Li W L Sun M Corey F Zou S Lee A L Cojocaru C Taylor S M
Blackman A Stephenson A J Sandford R Dorfman M L Drumm G R Cutting M
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[Liao 2015] Liao S H C H Liu B P Bastakoti N Suzuki Y Chang Y Yamauchi F
H Lin K C Wu ldquoFunctionalized magnetic iron oxidealginate core-shell nanoparticles
for targeting hyperthermiardquo International Journal of Nanomedicine 10 (2015) 3315ndash28
[Linsdell 2001] Linsdell P ldquoDirect block of the cystic fibrosis transmembrane
conductance regulator Clminus channel by butyrate and phenylbutyraterdquo European Journal of
Pharmacology 411 no 3 (2001) 255-260
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[Lippard 1994] Lippard S J and J M Berg Principles of Bioinorganic Chemistry Mill
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[Liu 2009] Liu T Y K H Liu D M Liu S Y Chen and I W Chen ldquoTemperature‐
sensitive nanocapsules for controlled drug release caused by magnetically triggered
structural disruptionrdquo Advanced Functional Materials 19 no 4 (2009) 616-623
[Liu 2012] Liu Y L K L Ai J H Liu Q H Yuan Y Y He and L H Lu ldquoA high‐performance ytterbium‐based nanoparticulate contrast agent for in vivo x‐ray computed
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[Loo 1945] Loo Y H P S Skell H H Thornberry J Ehrlich J M McGuire G M
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[Loacutepez Peacuterez 1997] Loacutepez Peacuterez J A M A Loacutepez Quintela J Mira J Rivas and S W
Charles ldquoAdvances in the preparation of magnetic nanoparticles by the microemulsion
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[Losasso 2014] Losasso C S Belluco V Cibin P Zavagnin I Micetić F Gallocchio
M Zanella L Bregoli G Biancotto and A Riccirdquo Antibacterial activity of silver
nanoparticles sensitivity of different Salmonella serovarsrdquo Frontiers in Microbiology 5
(2014) 227 (9 pp)
[Love 2012] Love S A M A Maurer-Jones J W Thompson Y-S Lin and C L
Haynes ldquoAssessing nanoparticle toxicityrdquo Annual Review of Analytical Chemistry 5
(2012) 181-205
[Lu 2002] Lu Y Y D Yin B T Mayers and Y N Xia ldquoModifying the surface
properties of superparamagnetic iron oxide nanoparticles through a sol-gel
approachrdquo Nano Letters 2 no 3 (2002) 183-186
[Lu 2010] Lu M M H Cohen D Rieves and R Pazdur ldquoFDA report Ferumoxytol for
intravenous iron therapy in adult patients with chronic kidney diseaserdquo American Journal
of Hematology 85 no 5 (2010) 315ndash9
[Luciani 2010] Luciani A V R Villella S Esposito N Brunetti-Pierri D Medina C
Settembre M Gavina L Pulze I Giardino M Poettoello-Mantovani M DrsquoApolito S
Guido E Masliah B Spencer S Quaratino V Raia A Ballabio and L Maiuri
ldquoDefective CFTR induces aggresome formation and lung inflammation in cystic fibrosis
through ROS-mediated autophagy inhibitionrdquo Nature Cell Biology 12 no 9 (2010) 863-
875
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[Lucotte 1995] Lucotte G S Hazout and M De Braekeleer ldquoComplete map of cystic
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[Ludwig 2005] Ludwig F S Maumluselein E Heim and M Schilling
ldquoMagnetorelaxometry of magnetic nanoparticles in magnetically unshielded environment
utilizing a differential fluxgate arrangementrdquo Review of Scientific Instruments 76 no 10
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[Lukanov 2011] Lukanov P V K Anuganti Y Krupskaya A‐M Galibert B Soula C
Tilmaciu A H Velders R Klingeler B Buumlchner and E Flahaut ldquoCCVD synthesis of
carbon‐encapsulated cobalt nanoparticles for biomedical applicationsrdquo Advanced
Functional Materials 21 no 18 (2011) 3583-3588
[Maek 1997] Macek M Jr Mackova A Hamosh A Hilman BC Selden RF
Lucotte G Friedman KJ Knowles MR Rosenstein BJ and GR Cutting
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(1997) 1122-1127
[Maier-Hauff 2011] Maier-Hauff K F Ulrich D Nestler H Niehoff P Wust B
Thiesen H Orawa V Budach and A Jordan ldquoEfficacy and safety of intratumoral
thermotherapy using magnetic iron-oxide nanoparticles combined with external beam
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oncology 103 no 2 (2011) 317-324
[Majewski 2007] Majewski P and B Thierry ldquoFunctionalized magnetite nanoparticlesmdash
Synthesis properties and bio-applicationsrdquo Critical Reviews in Solid State and Materials
Sciences 32 no 3-4 (2007) 203-215
[Mandelbaum 1995] Mandelbaum R T D L Allan and L P Wackett ldquoIsolation and
characterization of a Pseudomonas sp that mineralizes the s-triazine herbicide atrazinerdquo
Applied and Environmental Microbiology 61 no 4 (1995) 1451-1457
[Mapara 2015] Mapara N M Sharma V Shriram R Bharadwaj K C Mohite and V
Kumar ldquoAntimicrobial potentials of Helicteres isora silver nanoparticles against
extensively drug-resistant (XDR) clinical isolates of Pseudomonas aeruginosardquo Applied
Microbiology and Biotechnology 99 no 24 (2015) 10655ndash67
[Martiacutenez-Castantildeoacuten 2008] Martiacutenez-Castantildeoacuten G A N Nintildeo-Martiacutenez F Martiacutenez-
Gutierrez J R Martiacutenez-Mendoza and F Ruiz ldquoSynthesis and antibacterial activity of
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silver nanoparticles with different sizesrdquo Journal of Nanoparticle Research 10 no 8
(2008) 1343ndash8
[Massie 2010] Massie J L Curnow L Gaffney J Carlin and I Francis ldquoDeclining
prevalence of cystic fibrosis since the introduction of newborn screeningrdquo Archives of
Disease in Childhood 95 no 7 (2010) 531-533
[Mateu 2002] Mateu Eva Francesc Calafell Maria Dolors Ramos Teresa Casals and
Jaume Bertranpetit ldquoCan a place of origin of the main cystic fibrosis mutations be
identifiedrdquo The American Journal of Human Genetics 70 no 1 (2002) 257-264
[Maynard 2005] Maynard A D and E D Kuempel ldquoAirborne nanostructured particles
and occupational healthrdquo Journal of Nanoparticle Research 7 no 6 (2005) 587-614
[Mbeh 2012] Mbeh D R Franccedila Y Merhi X Zhang X T Veres E Sacher and L
Yahia ldquoIn vitro biocompatibility assessment of functionalized magnetite nanoparticles
Biological and cytotoxicological effectsrdquo Journal of Biomedical Materials Research Part
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[McGill 2009a] McGill S L C Cuylear N L Adolphi M Osiński and H D C Smyth
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Photonics Proceedings of SPIE (2009) 7189
[McGill 2009b] McGill S L C L Cuylear N L Adolphi M Osiński and H D C
Smyth ldquoMagnetically responsive nanoparticles for drug delivery applications using low
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[McNeil 2011] McNeil Scott E ed Characterization of nanoparticles intended for drug
delivery Vol 697 New York NY Humana press 2011
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[Meenach 2013] Meenach S A K W Anderson J Z Hilt R C McGarry and H M
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chemotherapeutic PEGylated phospholipid particles for dry powder inhalation delivery in
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[Mehdaoui 2011] Mehdaoui B A Meffre J Carrey S Lachaize L‐M Lacroix M
Gougeon B Chaudret and M Respaud ldquoOptimal size of nanoparticles for magnetic
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hyperthermia A combined theoretical and experimental studyrdquo Advanced Functional
Materials 21 no 23 (2011) 4573-4581
[Meiser 2004] Meiser F C Cortez and F Caruso ldquoBiofunctionalization of fluorescent
rare‐earth‐doped lanthanum phosphate colloidal nanoparticlesrdquo Angewandte Chemie
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[Minev 2011] Minev B R ed Cancer Management in Man Chemotherapy Biological
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[Mirtajani 2017] Mirtajani S B Poopak Farnia Maryam Hassanzad Jalaledin Ghanavi
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[Moritz 2010] Moritz M M H-C Flemming and J Wingender ldquoIntegration of
Pseudomonas aeruginosa and Legionella pneumophila in drinking water biofilms grown on
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[Moritz 2013] Moritz M and M Geszke-Moritz ldquoThe newest achievements in synthesis
immobilization and practical applications of antibacterial nanoparticlesrdquo Chemical
Engineering Journal 228 (2013) 596-613
[Mornet 2004] Mornet S S Vasseur F Grasset and E Duguet ldquoMagnetic nanoparticle
design for medical diagnosis and therapyrdquo Journal of Materials Chemistry 14 no 14
(2004) 2161-2175
[Morones 2005] Morones J R J L Elechiguerra A Camacho K Holt J B Kouri J T
Ramiacuterez M J Yacaman ldquoThe bactericidal effect of silver nanoparticlesrdquo Nanotechnology
16 no 10 (2005) 2346ndash53
[Moros 2013] Moros E G ed Physics of Thermal Therapy Fundamentals and Clinical
Applications CRC Press 2013
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[Mushin 2014] Muhsin T M and A K Hachim ldquoMycosynthesis and characterization of
silver nanoparticles and their activity against some human pathogenic bacteriardquo World
Journal of Microbiology and Biotechnology 30 no 7 (2014) 2081ndash90
[Musk 2005] Musk D J D A Banko and P J Hergenrother ldquoIron salts perturb biofilm
formation and disrupt existing biofilms of Pseudomonas aeruginosardquo Chemistry amp
Biology 12 no 7 (2005) 789-796
[Nasiri 2016] Nasiri A R Afsar Gharebagh S A Nojoumi SA M Akbarizadeh S
Harirchi M Arefnezhad S Sahraei M Hesaraki M Afshari F Javadian M Sheykhzade
Asadi Z Shahi and A Sargazi ldquoEvaluation of the antimicrobial activity of silver
nanoparticles on antibiotic-resistant Pseudomonas aeruginosardquo International Journal of
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[Neacuteel 1949] Neacuteel L Originally published in 1949 as ldquoTheacuteorie du traicircnage magneacutetique des
ferromagneacutetiques en grains fins avec application aux terres cuitesrdquo Annales de
Geacuteophysique 5 99-136 Nicholas Kurti ed Selected Works of Louis Neacuteel Gordon and
Breach Science Publishers 1988 pp 405ndash427ISBN 2-88124-300-2
[Nehara 2018] Nehra P R P Chauhan N Garg K Verma ldquoAntibacterial and antifungal
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Piktel X Gu Z Namiot A Kułakowska P B Savage and R Bucki ldquoBactericidal
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[NIH 2016] ldquoHow Do Geneticists Indicate the Location of a Gene - Genetics Home
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[Nikaido 1986] Nikaido H and R E W Hancock ldquoOuter membrane permeability of
Pseudomonas aeruginosardquo The Bacteria A treatise on structure and function Orlando
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TL Riss ldquoA homogeneous assay to measure live and dead cells in the same sample by
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[Noblett 1969] Noblett H R ldquoTreatment of uncomplicated meconium ileus by
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International Archives of Occupational and Environmental Health 74 no 1 (2000) 1-8
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[Palanisamy 2014] Palanisamy N K N Ferina A N Amirulhusni Z Mohd-Zain J
Hussaini L J Ping and R Durairaj ldquoAntibiofilm properties of chemically synthesized
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silver nanoparticles found against Pseudomonas aeruginosardquo Journal of
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R A Holler C H Turner and Y P Bao ldquoSynthesis and growth mechanism of iron oxide
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[Palomaki 2004] Palomaki G E S C Fitzsimmons and J E Haddow ldquoClinical
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K Sharma T Nevĕcnaacute and R Zbořil ldquoSilver colloid nanoparticles Synthesis
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[Park 2004] Park J K An Y Hwang J-G Park H-J Noh J-Y Kim J-H Park N-M
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[Patra 2017] Patra J K and K-H Baek ldquoAntibacterial activity and synergistic antibacterial
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[Piper 2013] Piper A ldquoThe big risk of small particles The threats and promise of
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[Pisanic 2007] Pisanic T R J D Blackwell V I Shubayev R R Fintildeones and S Jin
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[Prabhu 2012] Prabhu S and E K Poulose ldquoSilver nanoparticles mechanism of
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[Prabhu 2015] Prabhu Y T K V Rao B S Kumari V S S Kumar and T Pavani
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[Praetorius 2007] Praetorius N P and T K Mandal ldquoEngineered nanoparticles in cancer
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[Prencipe 2009] Prencipe G S M Tabakman K Welsher Z Liu A P Goodwin L
Zhang J Henry and H J Dai ldquoPEG branched polymer for functionalization of
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[Preacutevot 2001] Preacutevot M and D Dunlop ldquoLouis Neacuteel Forty years of magnetismrdquo Physics
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[Prodan 2013] Prodan A M S L Iconaru C S Ciobanu M C Chifiriuc M Stoicea
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by in vitro and in vivo assaysrdquo Journal of Nanomaterials (2013) 587021 (10 pp)
[Prodan 2013] Prodan A M S L Iconaru C M Chifiriuc C Bleotu C S Ciobanu M
Motelica-Heino S Sizaret D Predoi ldquoMagnetic properties and biological activity
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[Qiang 2006] Qiang Y J Antony A Sharma J Nutting D Sikes and D Meyer
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[Rai 2009] Rai M A Yadav and A Gade ldquoSilver nanoparticles as a new generation of
antimicrobialsrdquo Biotechnology Advances 27 no 1 (2009) 76ndash83
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[Rai 2012] Rai M K S D Deshmukh A P Ingle and A K Gade ldquoSilver nanoparticles
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[Ramirez 2003] Ramirez LP and K Landfester ldquoMagnetic polystyrene nanoparticles
with a high magnetite content obtained by miniemulsion processesrdquo Macromolecular
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[Raza 2016] Raza M A Z Kanwal A Rauf A N Sabri S Riaz and S Naseem ldquoSize-
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[Rice 2008] Rice L B ldquoFederal funding for the study of antimicrobial resistance in
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[Rosan 1962] Rosan R C H Shwachman and L L Kulczycki ldquoDiabetes mellitus and
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Pleurotus ostreatus and its inhibitory activity against pathogenic bacteriardquo Materials
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[Sabath 1976] Sabath LD ldquoThe assay of antimicrobial compoundsrdquo Human Pathology 7
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[Sahoo 2003] Sahoo S K and V Labhasetwar ldquoNanotech approaches to drug delivery
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[Samanta 2008] Samanta B H Yan N O Fischer J Shi D J Jerry V M Rotello
ldquoProtein-passivated Fe3O4 nanoparticles low toxicity and rapid heating for thermal
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N Sasaki S Boymans E Cuppen E C K van der Ent and E E Nieuwenhuis
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and M Rafailovich ldquoSonochemical synthesis of functionalized amorphous iron oxide
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[Shaker 2017] Shaker M A and M I Shaaban MI ldquoSynthesis of silver nanoparticles with
antimicrobial and anti-adherence activities against multidrug-resistant isolates from
Acinetobacter baumanniirdquo Journal of Taibah University Medical Sciences 12 no 4
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[Shakil 2008] Shakil S R Khan R Zarrilli and A U Khan ldquoAminoglycosides versus
bacteriandasha description of the action resistance mechanism and nosocomial battlegroundrdquo
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[Shete 2015] Shete P B R M Patil B M Tiwale and S H Pawar Water dispersible
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and Magnetic Materials 377 (2015) 406-410
[Shi 2007] Shi X T P Thomas L A Myc A Kotlyar and J R Baker Jr ldquoSynthesis
characterization and intracellular uptake of carboxyl-terminated poly (amidoamine)
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[Shi 2016] Shi S-F J-F Jia X-K Guo Y-P Zhao D-S Chen Y-Y Guo and X-L
Zhang ldquoReduced Staphylococcus aureus biofilm formation in the presence of chitosan-
coated iron oxide nanoparticlesrdquo International Journal of Nanomedicine 11 (2016) 6499ndash
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[Shieh 2005] Shieh D-B F-Y Cheng C-H Su C-S Yeh M-T Wu Y-N Wu C-Y
Tsai C-L Wu D-H Chen and C-H Chou ldquoAqueous dispersions of magnetite
nanoparticles with NH3+ surfaces for magnetic manipulations of biomolecules and MRI
contrast agentsrdquo Biomaterials 26 no 34 (2005) 7183-7191
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[Shin 2017] Shin H Y Wang C Lee H K Yoo K H Zeng X Kuhns T Yang C M
Mohr T Liu C and L Hennighausen ldquoCRISPRCas9 targeting events cause complex
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Tal H Seret L Yaar E Kerem and B Kerem ldquoAssociation of a nonsense mutation
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[Shrivastava 2007] Shrivastava S T Bera A Roy G Singh P Ramachandrarao D
Dash ldquoCharacterization of enhanced antibacterial effects of novel silver nanoparticlesrdquo
Nanotechnology 18 no 22 (2007) 225103 (9 pp)
[Shtykova 2007] Shtykova E V X Huang N Remmes D Baxter B Stein B Dragnea
D I Svergun and L M Bronstein ldquoStructure and properties of iron oxide nanoparticles
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[Shwachman 1965] Shwachman H L L Kulczycki and K-T Khaw ldquoStudies in cystic
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[Siegel 1960] Siegel B and S Siegel ldquoPregnancy and delivery in a patient with cystic
fibrosis of the pancreas Report of a caserdquo Obstetrics amp Gynecology 16 no 4 (1960) 438-
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[Simon 1970] Simon HJ EJ Yin ldquoMicrobioassay of antimicrobial agentsrdquo Applied
Microbiology 1970 Apr 119(4)573ndash9
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[Singh 2014a] Singh K M Panghal S Kadyan U Chaudhary and J P Yadav
ldquoAntibacterial activity of synthesized silver nanoparticles from Tinospora cordifolia
against multi drug resistant strains of Pseudomonas aeruginosa isolated from burn
patientsrdquo Journal of Nanomedicine amp Nanotechnology 5 no 2 (2014) 192 (6 pp)
[Singh 2014b] Singh K M Panghal S Kadyan U Chaudhary and J P Yadav ldquoGreen
silver nanoparticles of Phyllanthus amarus as an antibacterial agent against multi drug
resistant clinical isolates of Pseudomonas aeruginosardquo Journal of Nanobiotechnology 12
(2014) 40 (9 pp)
[Sinn 2011] Sinn P L R M Anthony and P B McCray ldquoGenetic therapies for cystic
fibrosis lung diseaserdquo Human molecular genetics 20 no R1 (2011) R79-R86
[Sio 2006] Sio C F L G Otten R H Cool S P Diggle P G Braun R Bos M
Daykin M Caacutemara P Williams and W J Quax ldquoQuorum quenching by an N-acyl-
homoserine lactone acylase from Pseudonomas aeruginosa PAO1rdquo Infection and
Immunology 74 no 3 (2006) 1673-1682
[Slieker 2005] Slieker M G C S P M Uiterwaal M Sinaasappel H G M Heijerman
J van der Laag and C K van der Ent ldquoBirth prevalence and survival in cystic fibrosis a
national cohort study in the Netherlandsrdquo Chest Journal 128 no 4 (2005) 2309-2315
[Smith 2002] Smith R S S G Harris R Phipps and B Iglewski ldquoThe Pseudonomas
aeruginosa quorum-sensing molecule N-(3-oxododecanoyl) homoserine lactone
contributes to virulence and induces inflammation in vivordquo Journal of Bacteriology 184
no 4 (2002) 1132-1139
[Smyth 2008] Smyth H D Marek Osinski and Shayna L McGill ldquoActive nanoparticles
and method of usingrdquo US Patent Application 12313847 filed November 25 2008
[Soenen 2010] Soenen S J H and M De Cuyper ldquoAssessing iron oxide nanoparticle
toxicity in vitro current status and future prospectsrdquo Nanomedicine 5 no 8 (2010) 1261-
1275
[Soenen 2011] Soenen S J H U Himmelreich N Nuytten and M De Cuyper
ldquoCytotoxic effects of iron oxide nanoparticles and implications for safety in cell labellingrdquo
Biomaterials 32 no 1 (2011) 195-205
[Sondi 2004] Sondi I B Salopek-Sondi ldquoSilver nanoparticles as antimicrobial agent a
case study on E coli as a model for Gram-negative bacteriardquo Journal of Colloid and
Interface Science 275 no 1 (2004) 177ndash82
207
[Song 2009] Song Y H H Lou J L Boyer M P Limberis L H Vandenberghe N R
Hackett P L Leopold J M Wilson and R G Crystal ldquoFunctional cystic fibrosis
transmembrane conductance regulator expression in cystic fibrosis airway epithelial cells
by AAV6 2-mediated segmental trans-splicingrdquo Human Gene Therapy 20 no 3 (2009)
267-281
[Soto 2007] Soto K K Garza and L Murr ldquoCytotoxic effects of aggregated
nanomaterialsrdquo Acta Biomaterialia 3 no 3 (2007) 351-358
[Southern 2007] Southern K W A Munck R Pollitt G Travert L Zanolla J Dankert-
Roelse C Castellani and ECFS CF Neonatal Screening Working Group ldquoA survey of
newborn screening for cystic fibrosis in Europerdquo Journal of Cystic Fibrosis 6 no 1
(2007) 57-65
[Speert 1990] Speert DP SW Farmer ME Campbell JM Musser RK Selander S
Kuo ldquoConversion of Pseudomonas aeruginosa to the phenotype characteristic of strains
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188ndash94
[Spock 1967] Spock A H M C Heick H Cress and W S Logan ldquoAbnormal serum
factor in patients with cystic fibrosis of the pancreasrdquo Pediatric Research 1 no 3 (1967)
173-177
[Staab 1998] Staab D K Wenninger N Gebert K Rupprath S Bisson M Trettin K
D Paul K M Keller and U Wahn ldquoQuality of life in patients with cystic fibrosis and
their parents what is important besides disease severityrdquo Thorax 53 no 9 (1998) 727-
731
[Streffer 2012] Streffer G ed Hyperthermia and the Therapy of Milignant Tumors
Volume 104 Springer 2012
[Strohbehn 1984] Strohbehn J W and Douple E B ldquoHyperthermia and cancer therapy
A review of biomedical engineering contributions and challengesrdquo IEEE Transactions on
Biomedical Engineering BME-31 no 12 (1984) 779-787
[Stutman 2002] Stutman H R J M Lieberman E Nussbaum M I Marks and the
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and young children with cystic fibrosis a randomized controlled trialrdquo The Journal of
Pediatrics 140 no 3 (2002) 299-305
208
[Sugita 1991] Sugita Y K Mitsuoka M Komuro H Hoshiya Y Kozono and M
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[Sun 2010] Sun C K Du C Fang N Bhattarai O Veiseh F Kievit Z Stephen D Lee
R G Ellenbogen B Ratner and M Zhang ldquoPEG-mediated synthesis of highly dispersive
multifunctional superparamagnetic nanoparticles their physicochemical properties and
function in vivordquo ACS Nano 4 no 4 (2010) 2402ndash10
[Suzuki 1973] Suzuki T Y Ichihara M Yamada and K Tonomura ldquoSome
characteristics of Pseudomonas 0ndash3 which utilizes polyvinyl alcoholrdquo Agricultural and
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[Szaff 1983] Szaff M N Hoslashiby and E W Flensborg ldquoFrequent antibiotic therapy
improves survival of cystic fibrosis patients with chronic Pseudomonas aeruginosa
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[Takami 2007] Takami S T Sato T Mousavand S Ohara M Umetsu and T Adschiri
ldquoHydrothermal synthesis of surface-modified iron oxide nanoparticlesrdquo Materials
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[Tan 2015] Tan S Y and Y Tatsumura ldquoAlexander Fleming (1881ndash1955) discoverer of
penicillinrdquo Singapore Medical Journal 56 no 7 (2015) 366-367
[Tang 2010] Tang B C J Fu D N Watkins and J Hanes ldquoEnhanced efficacy of local
etoposide delivery by poly (ether-anhydride) particles against small cell lung cancer in
vivordquo Biomaterials 31 no 2 (2010) 339-344
[Teja 2009] Teja A S and P-Y Koh ldquoSynthesis properties and applications of
magnetic iron oxide nanoparticlesrdquo Progress in Crystal Growth and Characterization of
Materials 55 no 1 (2009) 22-45
[Tendencia 2004] Tendencia E A ldquoDisk diffusion methodrdquo In Laboratory Manual of
Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from
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1891 Accessed February 19 2015 httpwwwancient-codecomnikola-tesla-secrets-
behind-genius
[Thiesen 2008] Thiesen B and A Jordan ldquoClinical applications of magnetic
nanoparticles for hyperthermiardquo International Journal of Hyperthermia 24 no 6 (2008)
467-474
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modern nanotechnologyrdquo Gold Bulletin 40 no 4 (2007) 267-269
[Thukkaram 2014] Thukkaram M S Sitaram S K Kannaiyan and G Subbiahdoss
ldquoAntibacterial efficacy of iron-oxide nanoparticles against biofilms on different
biomaterial surfacesrdquo International Journal of Biomaterials (2014) 716080 (6 pp)
[Thuret 2003] Thuret G C Chiquet S Herrag J M Dumollard D Boudard J Bednarz
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1427-1433
[Tran 2010] Tran N A Mir D Mallik A Sinha S Nayar T J Webster ldquoBactericidal
effect of iron oxide nanoparticles on Staphylococcus aureusrdquo International Journal of
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[Urban 2008] Urban C P A Bradford M Tuckman S Segal-Maurer W Wehbeh L
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care facilitiesrdquo Clinical Infectious Diseases 46 NO 11 (2008) e127ndash30
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release (marketed as Cipro XR and Proquin XR) gemifloxacin (marketed as Factive)
levofloxacin (marketed as Levaquin) moxifloxacin (marketed as Avelox) norfloxacin
210
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warning (2008)
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Archives of Ophthalmology 82 no 1 (1969) 10-14
[van den Bos 2003] van den Bos E J A Wagner H Mahrholdt R B Thompson
Morimoto Y Sutton B S Judd R M and D A Taylor ldquoImproved efficacy of stem
cell labeling for magnetic resonance imaging studies by the use of cationic liposomesrdquo
Cell Transplantation 12 no 7 (2003) 743-756
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exopolysaccharide synthesis by a bacteriumrdquo Applied and Environmental Microbiology
59 no 10 (1993) 3280-3286
[Vehring 2007] Vehring R W R Foss and D Lechuga-Ballesteros ldquoParticle formation
in spray dryingrdquo Journal of Aerosol Science 38 no 7 (2007) 728-746
[Veiseh 2005] Veiseh O C Sun J Gunn N Kohler P Gabikian D Lee N Bhattarai
R Ellenbogen R Sze A Hallahan J Olson and Miqin Zhang ldquoOptical and MRI
multifunctional nanoprobe for targeting gliomasrdquo Nano Letters 5 no 6 (2005) 1003-
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[Veiseh 2010] Veiseh O J W Gunn and M Q Zhang ldquoDesign and fabrication of
magnetic nanoparticles for targeted drug delivery and imagingrdquo Advanced Drug Delivery
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Law M Farrall H J Cooke H Eiberg and R Williamson ldquoLocalization of cystic
fibrosis locus to human chromosome 7cenndashq22rdquo Nature 318 no 6044 (1985) 384-385
[Walters 2003] Walters M C F Roe A Bugnicourt M J Franklin and P S Stewart
ldquoContributions of antibiotic penetration oxygen limitation and low metabolic activity to
tolerance of Pseudomonas aeruginosa biofilms to ciprofloxacin and tobramycinrdquo
Antimicrobial Agents and Chemotherapy 47 no 1 (2003) 317-323
[Wang 2003] Wang X W T Zheng H W Tian S S Yu W Xu S H Meng X D He
J C Han C Q Sun and B K Tay ldquoGrowth structural and magnetic properties of iron
nitride thin films deposited by dc magnetron sputteringrdquo Applied Surface Science 220 no
1 (2003) 30-39
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Disease in Childhood 70 no 2 (1994) 84-89
[Wei 2016] Wei Y M Zhao F Yang Y Mao H Xie and Q Zhou ldquoIron overload by
superparamagnetic iron oxide nanoparticles is a high risk factor in cirrhosis by a systems
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[Weissleder 1989] Weissleder R D D Stark B L Engelstad B R Bacon C C
Compton D L White P Jacobs J Lewis ldquoSuperparamagnetic iron oxide
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[White 1985] White R S Woodward M Leppert P OConnell M Holf J Herbstl J-M
Lalouel M Deanri and G V Woudei ldquoA closely linked genetic marker for cystic
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[Whiteley 2001] Whiteley M M G Bangera R E Bumgarner M R Parsek G M
Teitzel S Lory and E P Greenberg ldquoGene expression in Pseudomonas aeruginosa
biofilmsrdquo Nature 413 no 6858 (2001) 860-864
[Witkamp 2001] Witkamp A J E de Bree R Van Goethem and F A N Zoetmulder
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Cancer Treatment Reviews 27 no 6 (2001) 365-374
[Wong 1998] Wong C-H M Hendrix E S Priestley and W A Greenberg ldquoSpecificity
of aminoglycoside antibiotics for the A-site of the decoding region of ribosomal RNArdquo
Chemistry amp Biology 5 no 7 (1998) 397-406
[Wood 1976] Wood R E Boat T F Doershuk C F ldquoCystic fibrosis state of the artrdquo
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in airway Pseudomonas infections of cystic fibrosis patientsrdquo The Journal of Clinical
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[Wu 2005] Wu XJ F Kassie and V Mersch-Sundermann ldquoThe role of reactive oxygen
species (ROS) production on diallyl disulfide (DADS) induced apoptosis and cell cycle
arrest in human A549 lung carcinoma cellsrdquo Mutation ResearchFundamental and
Molecular Mechanisms of Mutagenesis 579 no 1-2 (2005) 115-124
[Wu 2008] Wu W Q G He and C Z Jiang ldquoMagnetic iron oxide nanoparticles
Synthesis and surface functionalization strategiesrdquo Nanoscale Research Letters 3 no 11
(2009) 397-415
[Wu 2013] Wu H H Zhu X Li Z Liu W Zheng T Chen B Yu and KH Wong
ldquoInduction of apoptosis and cell cycle arrest in A549 human lung adenocarcinoma cells by
surface-capping selenium nanoparticles an effect enhanced by polysaccharidendashprotein
complexes from Polyporus rhinocerosrdquo Journal of Agricultural and Food Chemistry 61
no 41 (2013) 9859-9866
[Wust 2006] Wust P C H Cho B Hildebrandt and J Gellermann ldquoThermal
monitoring Invasive minimal-invasive and non-invasive approachesrdquo International
Journal of Hyperthermia 22 no 3 (2006) 255-262
[Xie 2009] Xie J J Huang X Li S Sun and X Chen ldquoIron oxide nanoparticle platform
for biomedical applicationsrdquo Current Medicinal Chemistry 16 no 10 (2009) 1278-1294
[Xie 2010] Xie J K Chen J Huang S K Lee J H Wang J H Gao X G Li and X
Y Chen ldquoPETNIRFMRI triple functional iron oxide nanoparticlesrdquo Biomaterials 31 no
11 (2010) 3016-3022
[Xu 2007] Xu C J and S H Sun ldquoMonodisperse magnetic nanoparticles for biomedical
applicationsrdquo Polymer International 56 no 7 (2007) 821-826
[Yash Roy 1999] Yash Roy R C ldquoA structural Model for virulence organellae of gram-
negative organisms with reference to Salmonella pathogenicity in chicken ileumrdquo Indian
Journal of Poultry Science 34 no 2 (1999) 213-219
[You 2005] You Y W J Han P C Chiu and Y Jin ldquoRemoval and inactivation of
waterborne viruses using zerovalent ironrdquo Environmental Science amp Technology 39 no
23 (2005) 9263-9269
[Zabner 1996] Zabner J B W Ramsey D P Meeker M L Aitken R P Balfour R L
Gibson J Launspach R A Moscicki S M Richards and T A Standaert ldquoRepeat
administration of an adenovirus vector encoding cystic fibrosis transmembrane
213
conductance regulator to the nasal epithelium of patients with cystic fibrosisrdquo Journal of
Clinical Investigation 97 no 6 (1996) 1504
[Zelenski 2000] Zielenski J ldquoGenotype and phenotype in cystic fibrosisrdquo Respiration 67
no 2 (2000) 117-133
[Zhang 2010] Zhang X F S W Chen H-M Wang S-L Hsieh C-H Wu H-H Chou
and S C Hsieh ldquoRole of Neacuteel and Brownian relaxation mechanisms for water-based
Fe3O4 nanoparticle ferrofluids in hyperthermiardquo Biomedical Engineering Applications
Basis and Communications 22 no 05 (2010) 393-399
[Zielenski 1995] Zielenski J and L-C Tsui ldquoCystic fibrosis Genotypic and phenotypic
variationsrdquo Annual Review of Genetics 29 no 1 (1995) 777-807
214
PUBLICATIONS BY LEISHA MARIE MARTIN (ARMIJO)
Journal Papers
1 Savage D D J Chavez L Armijo and M Rosenberg ldquoPrenatal ethanol exposure
alters histamine H-3 receptor-mediated neurotransmission in adult offspringrdquo Alcoholism-
Clinical and Experimental Research 33 no 6 (2009) 133A Impact factor 3392
2 Wilkerson J L K R Gentry E C Dengler J A Wallace A A Kerwin L M
Armijo M N Kuhn G A Thakur A Makriyannis and E D Milligan ldquoIntrathecal
cannabilactone CB2R agonist AM1710 controls pathological pain and restores basal
cytokine levelsrdquo Pain 153 no 5 (2012) 1091-106 Impact factor 5836
2 Armijo L M Y I Brandt D Mathew S Yadav S Maestas A C Rivera N C
Cook N J Withers G A Smolyakov N L Adolphi T C Monson D L Huber H D
C Smyth and M Osiński ldquoIron oxide nanocrystals for magnetic hyperthermia
applicationsrdquo Nanomaterials 2 no 2 (2012) 134-146 Impact factor 3553
3 Dengler E C J Liu A Kerwin S Torres C M Olcott B N Bowman L Armijo
K Gentry J Wilkerson J Wallace X M Jiang E C Carnes C J Brinker and E D
Milligan ldquoMesoporous silica-supported lipid bilayers (protocells) for DNA cargo delivery
to the spinal cordrdquo Journal of Controlled Release 168 no 2 (2013) 209-224
Impact factor 7877
4 Rivera A C N N Glazener N C Cook B A Akins L M Armijo J B Plumley
N J Withers K Carpenter G A Smolyakov R D Busch and M Osiński
ldquoCharacterization of potassium bromide loaded with dysprosium fluoride nanocrystals for
neutron detectionrdquo International Journal of Nanotechnology 11 no 5678 (2014) 529-
538 Impact factor 1114
5 Armijo L M L A Ahureacute-Powell and N M Wereley ldquoRheological characterization
of a magnetorheological ferrofluid using iron nitride nanoparticles Journal of Applied
Physics 117 no 17 (2015) 17C747 Impact factor 2176
6 Armijo L M S J Wawrzyniec M Kopciuch Y I Brandt
A C Rivera N J Withers N C Cook D L Huber T C Monson H DC Smyth and
M Osiński ldquoAntibacterial activity of iron-oxide nanoparticles and tobramycin
nanoconjugates against Pseudomonas aeruginosa biofilmsrdquo Submitted to Journal of
Nanobiotechnology Spring 2019 Impact factor 5294
7 Armijo L M Y Brandt N J Withers J B Plumley P Jain A C Rivera N C
Cook H D C Smyth and M Osinski ldquoIn vitro Cytotoxicity of magnetite nanoparticles
215
in a human lung cell linerdquo Submitted to Toxicology in Vitro Spring 2019 Impact factor
3105
Conference Proceedings
1 M Osiński L M Armijo Y Brandt S R Maestas A C Rivera N C Cook J B
Plumley B A Akins G A Smolyakov N L Adolphi D L Huber S L McGill L
Gong and H D C Smyth ldquoMultifunctional nanoparticles for drug delivery in cystic
fibrosis (Invited Paper)rdquo Zing Nanomaterials Conference Xcaret Quintana Roo Mexico
28 Nov ndash 2 Dec 2011
2 Armijo L M Y Brandt D Mathew S Yadav S Maestas A C Rivera N C Cook
N J Withers G A Smolyakov N L Adolphi T C Monson H D C Smyth and M
Osiński ldquoIron oxide nanocrystals for magnetic hyperthermia applicationsrdquo Technical
Digest Zing Nanomaterials Conference Xcaret Quintana Roo Mexico 28 Nov ndash 2 Dec
2011 p 56
3 Rivera A C N N Glazener N C Cook L M Armijo J B Plumley B A Akins
K Carpenter G A Smolyakov R D Busch and M Osiński ldquoDysprosium-containing
nanocrystals for use as a neutron detector in a solvent suspensionrdquo Technical Digest Zing
Nanomaterials Conference Xcaret Quintana Roo Mexico 28 November ndash 2 December
2011 p 62
4 Armijo L M Y I Brandt N J Withers J B Plumley N C Cook A C Rivera S
Yadav G A Smolyakov T Monson D L Huber H D C Smyth and M Osiński
ldquoMultifunctional superparamagnetic nanocrystals for imaging and targeted drug delivery to
the lungrdquo Colloidal Nanocrystals for Biomedical Applications VII (W J Parak M
Osiński and K Yamamoto eds) SPIE International Symposium on Biomedical Optics
BiOS 2012 San Francisco CA 21-23 Jan 2012 Proceedings of SPIE Vol 8232 Paper
82320M (11 pp)
5 N J Withers Y I Brandt A C Rivera N C Cook L M Armijo G A Smolyakov
and M Osiński ldquoEffects of La02Ce06Eu02F3 nanoparticles capped with polyethylene
glycol on human astrocytoma cells in vitrordquo Colloidal Nanocrystals for Biomedical
Applications VII (W J Parak M Osiński and K Yamamoto eds) SPIE International
Symposium on Biomedical Optics BiOS 2012 San Francisco CA 21-23 Jan 2012
Proceedings of SPIE Vol 8232 Paper 82320R (9 pp)
6 Rivera A C N N Glazener N C Cook S Maestas B A Akins L M Armijo J B
Plumley N J Withers K Carpenter G A Smolyakov R D Busch and M Osiński
ldquoThermal neutron detection with PMMA nanocomposites containing dysprosium fluoride
nanocrystalsrdquo Chemical Biological Radiological Nuclear and Explosives (CBRNE)
Sensing XIII (A W Fountain III ed) SPIE Defense Security and Sensing Symposium
Baltimore MD 23-27 Apr 2012 Proceedings of SPIE Vol 8358 Paper 83581S (9 pp)
216
7 Cook N C A C Rivera N N Glazener B A Akins L M Armijo J B Plumley
N J Withers K Carpenter G A Smolyakov R D Busch and M Osiński ldquoPolyvinyl
tolueneGd2O310Ce scintillating nanocomposites for thermal neutron detectionrdquo
Technical Digest 7th International Conference on Quantum Dots Santa Fe New Mexico
13-18 May 2012 Paper Th-73
8 Armijo L M Y I Brandt S R Maestas A C Rivera N C Cook N J Withers G
A Smolyakov N L Adolphi T C Monson D L Huber H D C Smyth and M
Osiński ldquoMultifunctional nanocrystals for drug delivery in cystic fibrosisrdquo Technical
Digest 7th International Conference on Quantum Dots Santa Fe NM 13-18 May 2012
Paper Th-74
9 Rivera A C N N Glazener N C Cook S R Maestas B A Akins L M Armijo J
B Plumley N J Withers K Carpenter G A Smolyakov R D Busch and M Osiński
ldquoThermal neutron detection with Gd2O310Ce nanocrystals loaded into a polyvinyl
toluene matrixrdquo IEEE Symposium on Radiation Measurements and Applications SORMA
WEST 2012 Oakland CA 14-17 May 2012
10 Armijo L M Y I Brandt A C Rivera N C Cook J B Plumley N J Withers
M Kopciuch G A Smolyakov D L Huber H D C Smyth and M Osiński
ldquoMultifunctional superparamagnetic nanoparticles for enhanced drug transport in cystic
fibrosisrdquo Nanosystems in Engineering and Medicine (S H Choi J-H Choy U Lee and
V K Varadan eds) Incheon Korea 10-12 September 2012 Proceedings of SPIE Vol
8548 Paper 85480E (12 pp)
11 Armijo L M B A Akins J B Plumley A C Rivera N J Withers N C Cook G
A Smolyakov D L Huber H D C Smyth and M Osiński ldquoHighly efficient
multifunctional MnSeZnSeS quantum dots for biomedical applicationsrdquo Colloidal
Nanoparticles for Biomedical Applications VIII (W J Parak M Osiński and K
Yamamoto eds) SPIE International Symposium on Biomedical Optics BiOS 2013 San
Francisco California 2-4 Feb 2013 Proceedings of SPIE Vol 8595 Paper 859517 (7
pp)
12 Brandt Y I L M Armijo A C Rivera J B Plumley N C Cook G A
Smolyakov H D C Smyth and M Osiński ldquoEffectiveness of tobramycin conjugated to
iron oxide nanoparticles in treating infection in cystic fibrosisrdquo Colloidal Nanoparticles
for Biomedical Applications VIII (W J Parak M Osiński and K Yamamoto Eds) SPIE
International Symposium on Biomedical Optics BiOS 2013 San Francisco CA 2-4 Feb
2013 Proceedings of SPIE Vol 8595 Paper 85951C (9 pp)
13 Withers N J N N Glazener A C Rivera B A Akins L M Armijo J B
Plumley N C Cook J M Sugar R Chan Y I Brandt G A Smolyakov P H Heintz
and M Osiński ldquoEffects of La02Ce06Eu02F3 nanocrystals capped with polyethylene glycol
217
on human pancreatic cancer cells in vitrordquo Colloidal Nanoparticles for Biomedical
Applications VIII (W J Parak M Osiński and K Yamamoto Eds) SPIE International
Symposium on Biomedical Optics BiOS 2013 San Francisco CA 2-4 Feb 2013
Proceedings of SPIE Vol 8595 Paper 85951O (9 pp)
14Osiński M Y I Brandt L M Armijo N C Cook G A Smolyakov and H D C
Smyth ldquoEffectiveness of tobramycin conjugated to superparamagnetic nanoparticles in
treating cystic fibrosis (Invited Paper)rdquo Technical Digest Sixth International Conference
on Advanced Materials and Nanotechnology AMN-6 Auckland New Zealand 11-15 Feb
2013
15 Rivera A C N N Glazener N C Cook L M Armijo J B Plumley N J Withers
K Carpenter G A Smolyakov R D Busch and M Osiński ldquoCharacterization of
potassium bromide loaded with dysprosium fluoride nanocrystals for neutron detectionrdquo
Technical Digest Sixth International Conference on Advanced Materials and
Nanotechnology AMN-6 Auckland New Zealand 11-15 February 2013
16 Armijo L M A C Rivera J B Plumley N C Cook S Maestas G A Smolyakov
T C Monson D L Huber and M Osiński ldquoBasic mechanisms involved in the
magnetization reversal of magnetic single-domain nanoparticlesrdquo Technical Digest Sixth
International Conference on Advanced Materials and Nanotechnology AMN-6 Auckland
New Zealand 11-15 Feb 2013
17 Osiński M Y I Brandt L M Armijo M Kopciuch N J Withers N C Cook G
A Smolyakov and H D C Smyth ldquoHybrid multifunctional nanoparticles for drug
delivery to the lung in cystic fibrosis (Invited Paper)rdquo 21st Annual International
Conference on Composites Nano Engineering ICCE-21 Santa Cruz de Tenerife Spain
July 21-27 2013
18 Rivera A C N N Glazener N C Cook N J Withers L M Armijo D A Huang
J B Wright I Brener K Carpenter R D Busch G A Smolyakov and M Osiński
ldquoSynthesis and characterization of ytterbium-doped dysprosium fluoride nanocrystals for
use as neutron detectorsrdquo 21st Annual International Conference on Composites Nano
Engineering ICCE-21 Tenerife Spain 21-27 Jul 2013
19 Armijo L M Kopciuch B A Akins J B Plumley N J Withers A C Rivera N
C Cook Y I Brandt J M Baca S J Wawrzyniec G A Smolyakov D L Huber and
M Osiński ldquoLow-toxicity magnetic nanomaterials for biomedical applicationsrdquo 21st
Annual International Conference on Composites Nano Engineering ICCE-21 Tenerife
Spain 21-27 Jul 2013
20 Osiński M Y I Brandt L M Armijo M Kopciuch N J Withers N C Cook N
L Adolphi G A Smolyakov and H D C Smyth ldquoEfficacy of tobramycin conjugated to
superparamagnetic iron oxide nanoparticles in treating cystic fibrosis infections (Invited
218
Paper)rdquo Symposium 7E Low-Dimensional Semiconductor Structures (T V Torchynska
L Khomenkova G Polupan and G Burlak Eds) XXII International Material Research
Congress 2013 (IMRC 2013) Cancun Mexico 11-15 Aug 2013 MRS Proceedings Vol
1617 (11 pp) (Available online)
21 Rivera A C N N Glazener N C Cook N J Withers L M Armijo J Wright I
Brener K Carpenter R D Busch G A Smolyakov and M Osiński ldquoThermal neutron
detection using ytterbium-doped dysprosium fluoride nanocrystalsrdquo Zing Nanomaterials
2013 Conference Xcaret Mexico 13-17 Nov 2013
22 Armijo L M M Kopciuch Z Olszoacutewka S J Wawrzyniec A C Rivera J B
Plumley N C Cook Y I Brandt D L Huber G A Smolyakov N L Adolphi H D C
Smyth and M Osiński ldquoDelivery of antibiotics coupled to iron oxide nanoparticles across
the biofilm of mucoid Pseudonomas aeruginosa and investigation of their efficacyrdquo
Colloidal Nanoparticles for Biomedical Applications IX (W J Parak M Osiński and K
Yamamoto eds) SPIE International Symposium on Biomedical Optics BiOS 2014 San
Francisco CA 1-3 Feb 2014 Proceedings of SPIE Vol 8955 Paper 89550I (12 pp)
23 Osiński M Y I Brandt L M Armijo J B Plumley A C Rivera N C Cook G
A Smolyakov D L Huber and H D C Smyth ldquoSuperparamagnetic iron oxide
nanoparticles conjugated to tobramycin for treating cystic fibrosis infections (Invited
Paper)rdquo Technical Digest 4th Zing Bionanomaterials Conference Nerja Spain 6-9 Apr
2014 p 53
24 Armijo L M A Westphal P Jain A Malagodi F Fornelli A Hayat M French
H D C Smyth and M Osiński ldquoInhibition of bacterial growth by iron oxide
nanoparticles with and without attached drug Have we conquered the antibiotic resistance
problem in cystic fibrosis lung infectionsrdquo Colloidal Nanoparticles for Biomedical
Applications X (W J Parak M Osiński and Xing-Jie Liang eds) SPIE International
Symposium on Biomedical Optics BiOS 2015 San Francisco CA 7-9 Feb 2015
Proceedings of SPIE Vol 9338 Paper 1Q (11 pp)
Patent Applications
1 Armijo L M ldquoMethod of making magnetic iron nitride nanoparticlesrdquo US Patent
Application 13987912 filed 16 Sept 2013
2 Osiński M H D C Smyth L M Armijo and H M H Bandara ldquoMethods and
compositions for antimicrobial treatmentrdquo United States Provisional Patent Application
filed on 6 Feb 2015
219
APPENDIX I
List of Chemicals and Physical Properties
1 CAS No 67-64-1
Chemical Name Acetone
Synonyms 2-propanone
Molecular formula C3H6O
Molecular weight 5808
Melting point -94 degC
Boiling point 56 degC
Flash point -1722 degC
Density 0791 gmL
Vapor density 2
Vapor pressure 184 Torr
Refractive index 1359
EPA substance registry system 2-propanone (67-64-1)
Hazard codes F Xi T
2 CAS No 9005-32-7
Chemical name Alginic acid
Synonyms Alginate
Molecular formula (C6H8O6)n
Molecular weight 10000-600000
Melting point 300 degC
EPA substance registry system Alginic acid (9005-32-7)
Hazard codes Xi
3 CAS No 7664-41-7
Chemical name Ammonia
Synonyms Ammonia
Molecular formula NH3
Molecular weight 1703
Melting point -78 degC
Boiling point 60 degC
Flash point 1111
Density 1023 gmL
Vapor density 06
Vapor pressure 875 atm
220
EPA substance registry system Ammonia (7664-41-7)
Hazard codes F N T Xn
4 CAS No 67-66-3
Chemical name Chloroform
Synonyms Trichloromethane formyl trichloride
methane trichloride methyl trichloride
Molecular formula CHCl3
Molecular weight 11938 amu
Melting point -63
Boiling point 61
Flash point 1492
Density 075 gmL
Vapor density 41
Vapor pressure 160 Torr
Refractive index 1445
EPA substance registry system Methane trichloro-(67-66-3)
Hazard codes Xn F T Xi
Hazard codes Xi
5 CAS No 13754-17-1
Chemical name Citrate
Synonyms Citrate
Molecular formula C6H5O7
Molecular weight 1703
Melting point -78 degC
EPA substance registry system 123-Propanetricarboxylic acid 2-
hydroxy- ion(3-) (13754-17-1)
Hazard codes None
6 CAS No 64-17-5
Chemical name Ethanol
Synonyms Ethyl alcohol thanol grain alcohol
Molecular formula C2H6O
Molecular weight 460684 amu
Melting point -114
Boiling point 78
Flash point 12
221
Density 079
Refractive index 13614
EPA substance registry system Ethanol (64-17-5)
Hazard codes Xn F T N
Hazard codes Xi
EPA substance registry system Ethanol (64-17-5)
Hazard codes F T Xn N
CAS No 112-40-3
Chemical name n-dodecane
Synonyms Dodecane
Molecular formula C12H26
Molecular weight 17033 amu
Melting point -96 degC
Boiling point 215-217 degC
Flash point 83 degC
Density 075 gmL
Vapor density 596
Vapor pressure 1 Torr
Refractive index 1421
EPA substance registry system Dodecane (112-40-3)
Hazard codes Xn
CAS No 629-97-0
Chemical name n-docosane
Synonyms Docosane
Molecular formula C22H46
Molecular weight 3106 amu
Melting point 42-45 degC
Boiling point 369 degC
Flash point 95 degC
Density 0778 gmL
Vapor density 108
Refractive index 14455
Hazard codes Xi
CAS No 112-95-8
Chemical name n-eicosane
222
Synonyms Eicosane icosane
Molecular formula C20H42
Molecular weight 28255 amu
Melting point 35-37 degC
Boiling point 3431 degC
Flash point gt113 degC
Density 07886 gmL
Vapor density 98
Refractive index 14425
Hazard codes Xi
7 CAS No 106627-54-7
Chemical name N-hydroxysulfosuccinimide sodium
salt
Synonyms Sulfo-NHS sodium salt NHSS
Molecular formula C4H4NNaO6S
Molecular weight 21713 amu
Melting point 250 degC
8 CAS No 1333-74-0
Chemical name Hydrogen gas
Synonyms Hydrogen
Molecular formula H2
Molecular weight 202 amu
Melting point -2592 degC
Boiling point -2528 degC
Flash point lt-150 degC
Density 00899
Vapor density 007
EPA substance registry system Hydrogen (1333-74-0)
Hazard codes F+
9 CAS No 73513-42-5
Chemical name Hexanes
Synonyms Hexane cyclohexane
Molecular formula C6H14
Molecular weight 8617536
Melting point -95 degC
223
Boiling point 68-70 degC
Flash point -2277 degC
Density 0672 gmL
Vapor density 3
Refractive index 1379
Hazard codes F Xn N
EPA substance registry system Hydrochloric acid (7647-01-0)
Hazard codes T C F Xi F+ Xn
10 CAS No 1317-61-9
Chemical name Iron oxide NPs
Synonyms Black iron oxide magnetite iron(III)
oxide
Molecular formula Fe3O4
Molecular weight 23153 amu
Melting point 1538 degC
Density 48-51 gmL
Hazard codes Xi
11 CAS No 7439-89-6
Chemical name Iron
Synonyms Iron
Molecular formula Fe
Molecular weight 5585 amu
Melting point 1535 degC
Boiling point 2750 degC
Flash point gt110 degC
Density 105 gmL
EPA substance registry system Iron (7439-89-6)
Hazard codes F Xi
12 CAS No None
Chemical name Phosphate buffered saline
Synonyms PBS
Molecular formula O4P
Molecular weight 9497 amu
Hazard codes Xi
224
13 CAS No 25322-68-3
Chemical name Polyethylene glycol
Synonyms PEG poly(oxyethylene)
Molecular formula C2nH4n+2On+1
Molecular weight 1802 + 4405n gmol
Melting point 64-66 degC
Boiling point gt250 degC
Flash point 270 degC
Density 127 gmL
Vapor density gt1
Vapor pressure lt001 Torr
Refractive index 1469
EPA substance registry system Poly(oxy-12-ethanediyl) alpha-hydro-
omega-hydroxy (25322-68-3)
Hazard codes Xi T
14 CAS No 23335-74-2
Chemical name Iron oleate
Synonyms Iron(IIIII) oleate
Molecular formula C54H99FeO6
Molecular weight 90021 amu
Hazard codes none
15 CAS No 10025-77-1
Chemical name Iron chloride hexahydrate
Synonyms ferric chloride hyxahydrate iron(III)
chloride
Molecular formula Cl3FeH12O6
Molecular weight 2703 amu
Melting point 37 degC
Boiling point 280-285 degC
Flash point 280-285 degC
Density 182 gmL
Vapor pressure 1 Torr
Hazard codes Xn C
16 CAS No 110-86-1
Chemical name Pyridine