design, synthesis and applications of polymer biomaterials
TRANSCRIPT
University of South FloridaScholar Commons
Graduate Theses and Dissertations Graduate School
2-24-2015
Design, Synthesis and Applications of PolymerBiomaterialsFrankie CostanzaUniversity of South Florida, [email protected]
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Scholar Commons CitationCostanza, Frankie, "Design, Synthesis and Applications of Polymer Biomaterials" (2015). Graduate Theses and Dissertations.https://scholarcommons.usf.edu/etd/5462
Design, Synthesis and Applications of Polymer Biomaterials
by
Frankie Costanza
A dissertation submitted in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
Department of Chemistry
College of Arts and Sciences
University of South Florida
Major Professor: Dr. Jianfeng Cai, Ph.D.
Kirpal Bisht, Ph.D.
Edward Turos, Ph.D.
Chuanhai Cao, Ph.D.
Date of Approval:
February 24th, 2015
Keywords: Antimicrobial polymers, drug delivery, amphiphilic, micelle
Copyright © 2015, Frankie Costanza
DEDICATION
To Michael and Victoria Costanza
ACKNOWLEDGMENTS
I would like to express my gratification to my parents Michael and Victoria Costanza
who have always been my beacon of light through my hardest of times over the past many years.
This Doctorate would not be possible without their love and support for me during this time of
my life. I would also like to express my gratification to my mentor, Dr. Jianfeng Cai, for
allowing me to work in his laboratory as well as providing me with guidance, support and
understanding as I have endured numerous personal issues over the years. Without his
compassion, I would have not accomplished this milestone in my life. It is through him that I can
see what a truly hard working mentor and researcher really is. He has accepted all members of
his group into his home, and into his family, thereby making everyone feel as though they belong
here. For these reasons I can say with great respect, that it has been an honor to work and learn in
your laboratory. I would also like to thank Dr. Kirpal Bisht, Dr. Edward Turos, and Dr. Chuanhai
Cao for their support and insights over the years as well as for the use of their laboratory
instruments. Next, I would like to thank the entire Cai group as my support system for the times
when research became the most frustrating. Without their professional help, I would not have
been able to conduct many experiments as efficiently. In particular I would like to thank Shruti
Padhee for the antibacterial assays and Yan Wang for the in vivo mouse studies. Lastly, I would
like to thank Zoya Khan for being the foundation of my social support system over the past 3
years. All of the mentioned above people have always stood by me and helped me achieve this
degree with their support and patience, and again, I would like to thank each and every one of
you.
F.C.
i
TABLE OF CONTENTS
LIST OF TABLES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .iii
LIST OF FIGURES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv
ABSTRACT. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .vi
CHAPTER 1: INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 Antibacterial Polymers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
1.2 Drug Delivery Polymers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.3 Outline of Dissertation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
1.4 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
CHAPTER 2: INVESTIGATION OF ANTIMICROBIAL PEG-POLY(AMINO ACID)s. . . . .10
2.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.2 Materials and Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12
2.3 Results and Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.4 Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.5 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
CHAPTER 3: PEG-POLY (AMINO ACID)s – ENCAPSULATED
……………TANSHINONE IIA AS POTENTIAL THERAPEUTICS FOR
…………... THE TREATMENT OF HEPATOMA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28
3.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3.2 Materials and Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31
3.3 Results and Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.4 Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3.5 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
CHAPTER 4: PEG-POLY (AMINO ACID)S/MICRORNA COMPLEX
…………… NANOPARTICLES EFFECTIVELY ARREST THE GROWTH AND
…………….METASTASIS OF HEPATOMA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
4.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
4.2 Materials and Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .55
4.3 Results and Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
4.4 Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
4.5 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
CHAPTER 5: CONCLUSIONS AND FUTURE DIRECTIONS. . . . . . . . . . . . . . . . . . . . . . . . .73
ii
APPENDIX A: SUPPORTING INFORMATION FOR INVESTIGATION OF PEG-
……………. POLY(AMINO ACID)S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
A1. Synthesis of PEG-poly(amino acid)s. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
A2. The Morphology of PEG-poly(amino acid)s in water. . . . . . . . . . . . . . . . . . . . . . . . 79
A3. Antimicrobial activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .80
A4. Hemolytic activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .81
A5. Fluorescence microscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
A6. SEM of bacteria after treatment with polymer P5. . . . . . . . . . . . . . . . . . . . . . . . . . . 82
A7. References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .83
iii
LIST OF TABLES
Table 2.1: The antimicrobial and hemolytic activities of PEG-poly(amino acid)s. . . . . . . . . . . .17
Table A1: Molecular weights of the polymers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
iv
LIST OF FIGURES
Figure 1.1: Difference between bacterial and mammalian cell membranes. . . . . . . . . . . . . . . . . . 3
Figure 1.2: Graphical representation of our drug delivery system used for miRNA-139. . . . . . . .6
Figure 2.1: Synthesis of PEG-poly(amino acid)s. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Figure 2.2: The nanomorphology of the PEG-poly(amino acid)s. . . . . . . . . . . . . . . . . . . . . . . . .17
Figure 2.3: Fluorescence micrographs of B. subtilis treated with polymer. . . . . . . . . . . . . . . . . .21
Figure 2.4: SEM pictures of bacteria treated with polymer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22
Figure 3.1: The structure of Tanshinone IIA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30
Figure 3.2: Synthesis and NMR of the PEG-poly (amino acid)s. . . . . . . . . . . . . . . . . . . . . . . . . .38
Figure 3.3: Nanomorphology of poly (amino acid)s. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39
Figure 3.4: In vitro cytotoxicity of nanoparticles in MHCC97-H hepatoma cells. . . . . . . . . . . . 40
Figure 3.5: Biodistribution of TanshinoneIIA NPs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Figure 3.6: Tumor inhibitory effect of TSIIA nanoparticles in vivo. . . . . . . . . . . . . . . . . . . . . . .43
Figure 3.7: (A) Kaplan-Meier overall survival analysis and (B) Life extended rate of the
MHCC97-H hepatoma-bearing mice treated with TSIIA nanoparticles. . . . . . . . . . . . . . . . . . . .44
Figure 3.8: Representative histopathological sections of the tumors from tumor-bearing mice. .45
Figure 3.9: Image of hepatoma-bearing mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Figure 4.1: Synthesis and NMR of PEG-poly(amino acid)s. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Figure 4.2: SEM pictures of prepared nanoparticles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .62
Figure 4.3: The tumor growth curves. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Figure 4.4: Survival time of hepatoma-bearing mice in different groups. . . . . . . . . . . . . . . . . . . 64
Figure 4.5: Image of orthotropic hepatoma-bearing mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .66
v
Figure 4.6: The immunohistochemical staining of CD31 antigen. . . . . . . . . . . . . . . . . . . . . . . . .67
Figure 4.7: Wound closure healing assay in miR-139 nanoparticles. . . . . . . . . . . . . . . . . . . . . . .68
Figure A1a: Synthesis of N-Carboxyanhydrides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .76
Figure A1b: Synthesis of PEG-OTs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .76
Figure A1c: Synthesis of PEG-N3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Figure A1d: Synthesis of PEG-NH2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Figure A1e: Synthesis of Polymer P3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Figure A2: SEM image showing the morphology of PEG-poly(amino acid)s . . . . . . . . . . . . . . .79
Figure A3: NMR of Polymer P2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .84
Figure A4: NMR of Polymer P3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Figure A5: NMR of Polymer P4. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Figure A6: NMR of Polymer P5. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Figure A7: NMR of Polymer P6. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
Figure A8: NMR of Polymer P7. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Figure A9: NMR of Polymer P8. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Figure A10: NMR of Polymer P9. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Figure A11: NMR of Polymer P10. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
vi
ABSTRACT
The emergence of antibiotic resistant bacteria has prompted the research into novel kinds
of antibacterial small molecules and polymers. Nature has solved this issue with the use of
cationic antimicrobial peptides, which act as nonspecific antibiotics against invading species.
Herein, we have tried to mimic this general mechanism in a biocompatible and biodegradable
polymer micelle based on the polymerization of naturally occurring amino acids lysine and
phenylalanine linked to a PEG tether. This amphiphilic structure allows for the spontaneous
collapse into stable nanoparticles in solution, which contains a hydrophilic outer layer and a
hydrophobic core. Our polymers have shown activity against clinically relevant strains including
Methicillin Resistant S. epidermidis, B. subtilis, K. pneumoniae, and P. aeruginosa.
To further the application of our biopolymers, we have used them as drug delivery
vehicles as well. First, we have used an anionic analogue based on glutamic acid to encapsulate a
super hydrophobic drug Tanshinone IIA, and use it against a hepatoma bearing mouse model.
Second, we have used a cationic analogue to form a complex with miRNA-139 and use it against
a hepatoma bearing mouse model as well. In both cases, our PEG poly(amino acids)s have
shown promising efficacy in drastically reducing the tumor size compared to the control only.
Taken together, our results show that our nanoparticles have the potential to be versatile
biomaterials as antibacterials as well as drug delivery vehicles in vivo.
1
CHAPTER 1: INTRODUCTION
1.1 Antibacterial Polymers
Clinics today are facing an increasing problem of infections associated with drug
resistance to conventional antibiotics1. One of the most common infections which account for
more than half of all reported skin infections, and causes an estimated 20,000 deaths annually is
from methicillin-resistant staphylococcus aureus and a similar strain from methicillin-resistant
staphylococcus epidermidis2. This problem becomes even more pronounced during surgery
because bacteria can easily colonize the surface of living and non-living substrate including the
raw patient’s tissue, as well as surgical devices, synthetic implants, orthopedics, and catheters,
etc3. However, this problem is not limited to the clinic and also applies to drinking water, food
packaging and agriculture4-6. This brings about an inherent need to develop new antibacterials
that are broad spectrum by design, but more importantly, are intrinsically difficult for the
bacteria to develop resistance.
In designing a new broad spectrum anti-bacterial material, there is one general structural
feature that has been employed by many different researchers to again activity against gram
positive and gram negative bacteria. Generally speaking, this involves having a proper balance of
cationic charge, which draws the material close to the negatively charged bacterial membrane,
and hydrophobic residues, which interact with the phospholipid bilayer3,7,8. This theology is
based on the natural defense mechanisms of many living organisms which synthesize cationic
antimicrobial peptides (CAPs) when under attack. While each peptide is different and unique to
2
the particular organism, they all generally consist of about 12-50 residues, in which
approximately 50% of them are hydrophobic. While the mechanism of these CAPs is still under
some debate, it is generally believed that it does not interact with any receptor on the cell
surface9. Instead, CAPs (as well as broad spectrum antimicrobial polymers [AMPs]6) target the
natural differences between bacterial and mammalian cells. When a bacterial cell is approached
by an AMP or CAP, hydrophobic interactions prevail and may sink the AMP or CAP into the
bacterial membrane, and thus places a torsional strain on the membrane bound phospholipids,
which can further lead to membrane disruption and subsequent cell lysis9.
Eukaryotic mammalian cells (including human erythrocytes) contain up to 25%
cholesterol9 in their lipid bilayers and are predominantly composed of zwitterionic (overall
neutral) phospholipids such as phosphatidylcholine, phosphatidyethanolamine, and
sphingomyelin10. Furthermore, most of the lipids in mammalian cells that do have a net negative
charge are actually located on the inner leaflet of the cell instead of the outer leaflet (Figure 1.1).
Bacteria on the other hand, have a structurally different outer membrane. First, they lack
cholesterol in their membranes, which is thought to act as a stabilization factor for mammalian
cells11. This alone can make bacterial membranes easier to disrupt compared to mammalian ones.
Second, the outer leaflet of the bacterial cell are generally composed of negatively charged
phospholipids such as phosphatidylglycine, cardiolipin and phosphatidylserine10. More
specifically in gram positive cells, some negative charge is thought to arise from lipoteichoic
acids (which links the outer thick peptidoglycan layer to the inner cell membrane) and its
associated negatively charged phosphate groups linked via phosphodiester bonds. In contrast,
gram-negative cells have only a thin peptidoglycan layer, but contain a second outer cell
membrane on top of this with the outer leaflet being composed mostly of lipopolysaccharides
3
that are highly anionic7. The outer membrane of gram negative cells do not contain teichoic
acids, but they do contain phosphate groups in a similar environment as the phosphates in
teichoic acids12. Discounting surface layer antigens, it can be said that the outer membranes of
gram positive and gram negative bacteria carry an overall net negative charge, which is greater
than that of mammalian cells which are relatively neutral. Therefore, a more electrostatically
favorable interaction exists between CAPs and AMPs with bacteria as compared to mammalian
cells.
Figure 1.1: Difference between bacterial and mammalian cell membranes.
4
In applying this theology to designing a functional biomaterial, we have developed
amphiphilic nanoparticles based on naturally occurring amino acids linked to a mono methyl
poly ethylene glycol (mPEG) tether. PEG is an FDA approved water soluble polymer for in vivo
use due to its excellent biocompatibility and enzyme degradability13. The use of PEG here is
highly advantageous in that it is highly hydrophilic and helps the ensuing polymer collapse into a
micelle as it has a hydrophobic core. Equally as important, PEG has non-adhesive antifouling
properties14, which prevent protein and cell adhesion to the nanoparticle. This prevents the
aggregation of debris on the outside of the particle which would hinder its activity due to the
particle being blocked from interacting with bacterial membranes. This most accepted reason for
this property is due to a layer of tightly bound water molecules that surrounds the PEG surface,
which effectively prevents protein adhesion15.
To ensure the polymer is fully biocompatible, the bulk of the material is made from poly
amino acids. Briefly, positively charged, negatively charged, and hydrophobic natural L-amino
acids (lysine, glutamic acid, and phenylalanine respectively) were cyclized via triphosgene into
N-carboxyanhydride (NCA) monomers and purified by recrystallization. Then, mPEG-NH2 was
used as the initiator for the polymerization and various equivalents of the amino acid monomers
were added to develop a library of polymers. To lessen the hydrophobicity of the core, we have
used a hybrid core that is composed of a random set of charged residues to hydrophobic residues.
In theory, by having the charged segments (which have favorable electrostatic interactions to
approach the bacterial membrane) next to the hydrophobic moieties, this allows the hydrophobic
phenylalanine rings to move closer to the bacterial membrane and increase the membrane
depolarization leading to cell lysis. Taken together, our compounds show broad spectrum activity
5
against both gram positive and gram negative strains and form nanoparticles in the sub 200nm
range.
1.2 Drug Delivery Polymers
Due to our polymers exhibiting excellent water solubility as well has having a defined
secondary nanoparticle structure with a hydrophobic core; we anticipated that they would also
make suitable drug delivery vehicles for hydrophobic drugs. As a proof of concept, we have used
a super hydrophobic drug, Tanshinone IIA, to be delivered by our PEG poly amino(acid)s in a
live mouse model. Tanshinone IIA is a small molecule isolated from salvia miltiorrhiza and
exhibits broad spectrum anti-tumor activity16-18. While its direct mechanism of action is
unknown, it is believed to have multiple intra- and extra- cellular targets19. For a molecule such
as Tanshinone which is highly insoluble in water and thus, unable to be administered in vivo, a
drug delivery vehicle must be used. We have used a glutamic acid analogue of our polymers for
this purpose. Its negative charge prevents it from aggregation with many human serum proteins,
many of which are also negatively charged, and our hybrid core is hydrophobic enough to ensure
that Tanshinone IIA is encapsulated and able to be administered in vivo. We have used a hybrid
core instead of a purely hydrophobic core because it is hydrophobic enough to drive the collapse
of the micelle and encapsulate the drug, but also contains less electrostatic interactions with the
drug which will aid in its release to occur more rapidly. Our results show that compared to
Tanshinone IIA alone, our encapsulated Tanshinone IIA has reduced the hepatoma tumor
volume by approximately 70%, which allows it to be a suitable carrier for this drug.
To further the clinical application of our polymers, we have also used a lysine analogue
of our nanoparticle to encapsulate and deliver a small interfering RNA, known as miRNA-139,
to be administered in a live mouse model as well. MicroRNAs are small non-coding RNA
6
molecules that regulate post-transcriptional gene expression. Since they can bind to any specific
mRNA sequence, they have potential for the regulation of virtually any gene, and have already
been implicated in many biological functions such as cell proliferation, metastasis,
differentiation and apoptosis20,21. For our delivery system of miRNA-139, we have used a lysine
analogue of our polymers due to its cationic nature, which has electrostatically favorable
interactions with the anionic charged miRNA. Again, we have used a hybrid core so as to limit
hydrophobic interactions, as well as facilitate the release of the miRNA due to the less tightly
packed hydrophobic core. Compared to miRNA-139 alone, our encapsulated miRNA-139 has
shown the ability to stunt the growth of a hepatoma tumor by approximately 75% in a live mouse
model (Figure 1.2). Taken together, these results show that our PEG poly amino(acid)s show
great versatility in delivering novel therapeutic agents, as well as inhibiting the proliferation of a
broad spectrum of bacterial strains.
Figure 1.2. Graphical representation of our drug delivery system used for miRNA-139 in
vivo.
7
1.3 Outline of the dissertation
In this dissertation, we discuss the design, synthesis, and applications of mPEG poly
amino (acid)s as well as testing of the compound in a live mouse model.
In chapter 2, we outline the synthetic design behind our positively charged polymer
library for use as broad spectrum antibacterial agents against both Gram positive and Gram
negative strains. The in vitro testing is also described.
In chapter 3, we describe the drug delivery application of a negatively charged polymer
for the delivery of a super hydrophobic drug, Tanshinone IIA. Its in vivo application is discussed
and data for its tumor suppression in a live mouse model is presented.
In chapter 4, we describe the drug delivery application of a positively charged polymer
for the delivery of miRNA-139. The rational of the design, techniques used, and data for the in
vivo tumor suppression of our delivery system is presented.
In chapter 5, we summarize the major findings of this dissertation and then conclude with
the future directions of this research.
1.4 References
(1) Alekshun, M. N.; Levy, S. B.: Molecular Mechanisms of Antibacterial Multidrug
Resistance. Cell 2007, 128, 1037-1050.
(2) Taubes, G.: The Bacteria Fight Back. Science 2008, 321, 356-361.
(3) Li, Y.; Fukushima, K.; Coady, D. J.; Engler, A. C.; Liu, S.; Huang, Y.; Cho, J. S.;
Guo, Y.; Miller, L. S.; Tan, J. P. K.; Ee, P. L. R.; Fan, W.; Yang, Y. Y.; Hedrick, J. L.: Broad-
Spectrum Antimicrobial and Biofilm-Disrupting Hydrogels: Stereocomplex-Driven
Supramolecular Assemblies. Angewandte Chemie 2013, 125, 702-706.
(4) Kang, D. H.; Gupta, S.; Rosen, C.; Fritz, V.; Singh, A.; Chander, Y.; Murray, H.;
Rohwer, C.: Antibiotic Uptake by Vegetable Crops from Manure-Applied Soils. Journal of
Agricultural and Food Chemistry 2013, 61, 9992-10001.
8
(5) Page, K.; Wilson, M.; Parkin, I. P.: Antimicrobial surfaces and their potential in
reducing the role of the inanimate environment in the incidence of hospital-acquired infections.
Journal of Materials Chemistry 2009, 19, 3819-3831.
(6) Kenawy, E.-R.; Worley, S. D.; Broughton, R.: The Chemistry and Applications of
Antimicrobial Polymers: A State-of-the-Art Review. Biomacromolecules 2007, 8, 1359-1384.
(7) Li, P.; Poon, Y. F.; Li, W.; Zhu, H.-Y.; Yeap, S. H.; Cao, Y.; Qi, X.; Zhou, C.;
Lamrani, M.; Beuerman, R. W.; Kang, E.-T.; Mu, Y.; Li, C. M.; Chang, M. W.; Jan Leong, S. S.;
Chan-Park, M. B.: A polycationic antimicrobial and biocompatible hydrogel with microbe
membrane suctioning ability. Nat Mater 2011, 10, 149-156.
(8) Zhou, C.; Qi, X.; Li, P.; Chen, W. N.; Mouad, L.; Chang, M. W.; Leong, S. S. J.;
Chan-Park, M. B.: High Potency and Broad-Spectrum Antimicrobial Peptides Synthesized via
Ring-Opening Polymerization of α-Aminoacid-N-carboxyanhydrides. Biomacromolecules 2009,
11, 60-67.
(9) Glukhov, E.; Stark, M.; Burrows, L. L.; Deber, C. M.: Basis for Selectivity of
Cationic Antimicrobial Peptides for Bacterial Versus Mammalian Membranes. Journal of
Biological Chemistry 2005, 280, 33960-33967.
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Antibacterial Activity of a Peptide-Based β-Hairpin Hydrogel. Journal of the American
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(11) Matsuzaki, K.: Why and How are Peptide–Lipid Interactions Utilized for Self-
Defense? Magainins and tachyplesins as archetypes. Biochimica et Biophysica Acta (BBA) -
Biomembranes 1999, 1462, 1-10.
(12) Jiang, W.; Saxena, A.; Song, B.; Ward, B. B.; Beveridge, T. J.; Myneni, S. C. B.:
Elucidation of Functional Groups on Gram-Positive and Gram-Negative Bacterial Surfaces
Using Infrared Spectroscopy. Langmuir 2004, 20, 11433-11442.
(13) Brandl, F. P.; Seitz, A. K.; Teßmar, J. K. V.; Blunk, T.; Göpferich, A. M.:
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(14) Glinel, K.; Jonas, A. M.; Jouenne, T.; Leprince, J. r. m.; Galas, L.; Huck, W. T.
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Bioconjugate Chemistry 2008, 20, 71-77.
(15) Li, L.; Chen, S.; Zheng, J.; Ratner, B. D.; Jiang, S.: Protein Adsorption on
Oligo(ethylene glycol)-Terminated Alkanethiolate Self-Assembled Monolayers: The Molecular
Basis for Nonfouling Behavior. The Journal of Physical Chemistry B 2005, 109, 2934-2941.
9
(16) Li, F. L.; Xu, R.; Zeng, Q. C.; Li, X.; Chen, J.; Wang, Y. F.; Fan, B.; Geng, L.; Li,
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Underlying Treatment Mechanism of Psoriasis. Evidence-Based Complementary and Alternative
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S. Y.; Chang, C. S.: The selective inhibitory effect of a synthetic tanshinone derivative on
prostate cancer cells. Prostate 2012, 72, 803-816.
(18) Yan, M. Y.; Chien, S. Y.; Kuo, S. J.; Chen, D. R.; Su, C. C.: Tanshinone IIA
inhibits BT-20 human breast cancer cell proliferation through increasing caspase 12, GADD153
and phospho-p38 protein expression. International Journal of Molecular Medicine 2012, 29,
855-863.
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Opinion on Therapeutic Patents 2013, 23, 149-153.
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2008, 9, 102-114.
10
CHAPTER 2: INVESTIGATION OF ANTIMICROBIAL PEG-POLY(AMINO ACID)S
Note to reader
This chapter has been previously published and has been reproduced by permission of The
Royal Society of Chemistry. Original citation:
Costanza, F.; Padhee, S.; Wu, H.; Wang, Y.; Revenis, J.; Cao, C.; Li, Q.; Cai, J.:
Investigation of antimicrobial PEG-poly(amino acid)s. RSC Advances 2014, 4, 2089-2095.
2.1 Introduction
The extensive use of antibiotics in recent decades for medical and agricultural purposes
has elicited the widely known issue of bacterial resistance.1 As such, the development of new
antimicrobial agents with novel mechanisms has been a constant area of research. This includes
the need for new antibacterial materials to prevent contamination in food packaging, storage,
medical supplies, and drinking water,2-4 in addition to drug discovery. One approach to
circumvent resistance could be the development of antimicrobial peptides (AMPs). 1,5,6 Cationic
AMPs exist in most organisms and are the first line of natural defense against infections.
Although they possess different secondary structures or sometimes even random structures in
solution, they are believed to adopt an amphipathic conformation when interacting with
negatively charged bacterial membranes, and subsequently disrupt the function of bacterial
membranes. 5 As this biophysical interaction lacks defined bacterial targets, this mechanism of
action is not expected to develop resistance as easily as traditional antibiotics. Based on such
11
recognition, many classes of AMP mimetics have been developed. 7-14 These small
peptidomimetics are capable of mimicking the function and mechanism of AMPs, and are shown
to have potent and broad spectrum antimicrobial activity. However, their step-by-step
preparation is tedious, and they are generally not economical to produce in substantial quantities,
limiting the potential of widespread use as affordable biomaterials in the future.
Recently, there have been some advances in polymer antimicrobial agents such as
polymers based on methacrylates, 15,16 norborenes, 17,18 amidoamines,19 and others. 3,20-23 These
polymers contain both hydrophobic components and cationic components, which are critical for
the disruption of bacterial membranes. Many of them have also been shown to kill both Gram-
positive and Gram-negative bacteria, including clinically relevant pathogens. In addition, they
are generally prepared through a one-pot polymerization, allowing the generation of products in
large amounts. As such, there is promising potential for these synthetic materials to be used as
antimicrobial agents to treat bacterial infections. However, the development of biodegradable
antimicrobial nanoparticles is very rare. Poly(amino acid)s,23 synthesized by ring-opening
polymerization, show attractive features for further exploration. They have been widely used for
drug delivery and other biomedical applications24-27 as they are completely biodegradable.
However, their antimicrobial development is rare. 23 In this study, we designed, synthesized and
studied the antimicrobial activity of biodegradable polymer nanoparticles - PEGylated
amphiphilic poly (amino acids). The PEG is introduced to the polymers because PEG
(polyethylene glycol) is essentially non-toxic and biocompatible.7,28-30 Meanwhile, PEGylation
promotes the formation of a corona which comprises the outer shell of the nanoparticles.
Additionally, PEG is widely available, inexpensive, highly water soluble and easily modifiable
by size and end group functionality. It has also been used in the development of peptide
12
nanoparticles with a hydrophilic corona which would enhance the stability of the micelle and
increase circulation time.31-33 Another attractive aspect of using PEG in a block type
antimicrobial nanoparticle is that it has non-adhesive antifouling properties which can prevent
protein and cell adhesion.29 This is a key feature of the material as any cellular debris that can
potentially adhere to the outer surface would likely inhibit its activity. Another report shows that
PEG does not reduce activity to bacteria, while also may potentially decrease toxicity to human
cells.19 As such, we also expected that introduction of a PEG segment to poly(amino acid)s will
promote the formation of nanoparticles, which may facilitate bacterial membrane disruption, and
therefore possess improved antimicrobial activity.
2.2 Materials and Methods
(All polymers were prepared by a similar method). 0.4 g of PEG-NH2 was dissolved in
10 ml of anhydrous dioxane and purged with N2. Separately 0.49 g (20 eq) of Z-lysine NCA was
dissolved in 5 ml anhydrous dioxane, passed through a 2 micron filter to remove any insoluble
decomposed NCA, thoroughly purged with N2 and then added via syringe. The reaction was then
allowed to proceed for 3 days under an active N2 atmosphere. Then 10 equivalents of Z-lysine
NCA and 15 equivalents of phenylalanine NCA were dissolved in 5 ml anhydrous dioxane,
passed through a 2 micron filter, purged, and added via syringe. Polymerization was allowed to
continue for another 3 days. The clear solution was then precipitated into ether and the product
was collected via filtration. The polymer was then dissolved in 10 ml of TFA and to this
solution, 10 equiv. of HBr in AcOH (33% v/v conc.) was added and stirred for 4 hours. The
product was then precipitated into ether and collected via filtration. Finally, the compounds were
13
dissolved in DMSO (dimethyl sulfoxide) and purified via dialysis (MWCO 3,500) for 3 days.
The water was changed daily and the final product was achieved after freeze dying.
The bacterial strains used for testing the efficacy of polymers were collected from the
American Type Culture Collection (ATCC) and were multi-drug resistant S. epidermidis
(RP62A), B. subtilis (BR151), K. pneumoniae (ATCC 13383) and multi-drug resistant P.
aeruginosa (ATCC 27853). The antimicrobial activities of the polymers developed were
determined in sterile 96-well plates by the 2-fold serial-dilution method. Bacterial cells were
grown overnight at 37 °C in 5 mL medium after which a bacterial suspension of approximately
106 CFU/mL in Luria broth or trypticase soy was prepared ensuring that the bacterial cells were
in the mid-logarithmic phase. Aliquots of 50 µL bacterial suspension were added to 50 µL of
medium containing different concentrations of polymers for a total volume of 100µL in each
well. The 96-well plates were incubated at 37 ºC for about 20 h. The Biotek microplate reader
was used to measure the optical density (OD) at a wavelength of 600 nm after about 20 h. The
experiments were carried out as three independent biological replicates, each in duplicate. The
lowest concentration at which complete inhibition of bacterial growth is observed is defined as
the minimum inhibitory concentration (MIC).
Freshly drawn human red blood cells (hRBC’s) were used for the assay. The blood
sample was washed with PBS buffer several times and centrifuged at 700g for 10 min until a
clear supernatant was observed. The hRBC’s were re-suspended in 1X PBS to get a 5% v/v
suspension which was used to perform the assay. 50 µL of different polymers solutions were
added to sterile 96-well plates. Then 50 µL of 5% v/v hRBC solution was added to make up a
total volume of 100 µL in each well. The 0% hemolysis point and 100% hemolysis point were
determined in 1 X PBS and 0.2% Triton-X-100 respectively. The 96 well plate was incubated at
14
37 °C for 1h and centrifuged at 3500 rpm for 10 min. The supernatant (30 µL) was then diluted
with 100 µL of 1XPBS and hemoglobin was detected by measuring the optical density at 360nm
by Biotek microtiter plate reader (Type: Synergy HT).
% hemolysis = (Abs sample -Abs PBS )/(Abs Triton
–Abs PBS ) x 100
A double staining method with DAPI (4’,6-diamidino-2-phenylindole
dihydrochloride,Sigma,>98%) and PI (propidium iodide, Sigma) as fluorophores was used to
visualize and differentiate the viable from the dead B. subtilis cells. DAPI being a double
stranded DNA binding dye, stains all bacterial cells irrespective of their viability. Ethidium
derivatives such as propidium iodide (PI) is capable of passing through only damaged cell
membranes and intercalates with the nucleic acids of injured and dead cells to form a bright red
fluorescent complex. The bacterial cells were first stained with PI and then with DAPI (2-(4-
Amidinophenyl)-6-indolecarbamidine dihydrochloride). The bacterial cells were grown until
they reached mid-logarithmic phase and then ~ 2x10 3 cells were incubated with 100 µg/mL
polymer P5 for 4 h. Then the cells were pelleted by centrifugation at 3000 g for 15 min in an
Eppendorf microcentrifuge. The supernatant was decanted and the cells were washed with 1X
PBS several times and then incubated with PI (5 µg/mL) in the dark for 15 min at 0 oC. The
excessive PI was removed by washing the cells several times with 1X PBS several times. Lastly,
the cells were incubated with DAPI (10 µg/mL in water) for 15 min in the dark at 0oC. Finally,
the excessive DAPI solution was removed by washing it with 1X PBS. The controls were
performed following the exact same procedure for bacteria without P5. The bacteria were then
examined by using the Zeiss Axio Imager Z1 optical microscope with an oil-immersion objective
(100X). 7,8
15
MRSE and B.subtilis were grown to an exponential phase and approximately about 2 x
106 cells were incubated with the polymer for about 20 h. The cells were then harvested by
centrifugation (3000 g) for 15 min. After pelleting the cells, they were washed three times with
DI water. The cells were then fixed with 2.5% (w/v) glutaraldeyhyde in nanopure water for about
30 min, followed by extensive wash with DI water to get rid of any excess glutaralhedyde.
Graded ethanol series (30%, 50%, 70%, 95% and 100%, 5 min each) were then used to
dehydrate the cells. Following the dehydration of cells, hexamethyldisilazane was added for
about 2 minutes. Then, about 2 µL of sample was added to a silicon wafer followed by Au/Pd
coating, and = the samples were observed at 25KV with a HITACHI S-800 scanning electron
microscope.
2.3 Results and Discussions
To test our hypothesis, we synthesized a series of PEGylated poly(amino acid)s
containing both hydrophobic groups and cationic groups (Figure 2.1) via ring-opening
polymerization of N-carboxyanhydrides (NCAs). 34 NCAs can easily be synthesized and attained
in large quantities from natural amino acids at low cost, which makes them attractive in
designing new materials. 35 The synthetic procedure was adapted from a previously published
protocol. 35 Briefly, Phe-NCA and Z-Lys-NCA (Figure 2.1) were prepared through the treatment
of amino acids phenylalanine or Z-Lysine with triphosgene, respectively. Next, CH3O-PEG-NH2
was used as the initiator to react with NCAs to generate poly(amino acid)s via the ring-opening
polymerization. In the synthesis of two polymers (P2 and P3), to ensure the formation of cationic
shell of nanoparticles, Z-Lysine-NCA was first added into the flask containing the initiator and
reacted for three days at the room temperature. Subsequently, Z-Lysine-NCA and Phe-NCA
were then added in one batch to form a hybrid segment, which forms the core structure of
16
nanoparticles in solution. Such a random core structure was expected to facilitate the interaction
with the bacterial membranes through both charge and hydrophobic forces. 23 The random core
was also designed to limit the packing density of the phenylalanine rings, which allows for a
more overall amphipathic material. As such, a series of polymers containing varying numbers of
hydrophobic and hydrophilic groups were prepared (Table 2.1). To test if the charge is critical to
the polymers’ antimicrobial activity, one negatively charged polymer was synthesized using both
Glu-NCA and Phe-NCA.
ONH Dioxane
N2, r.t., 3d
O
HN
NH
O
NHO
O
1. TFAHBr / AcOH
c equiv. Z-Lys NCA+ d equiv. Phe NCA
a
H
b
b equiv Z-Lys NCA
DioxaneN2, r.t., 3d
2. Dialysis O
HN
NH
O
NH2
ab
HNO
O
O
R
NCA
H
a
HN
NH2
O
NH
Od
Hc
Figure 2.1 Synthesis of PEG-poly(amino acid)s.
The structures of these polymers was characterized by the 1H NMR (Appendix A3-A11),
and their nanomorphology was evaluated by scanning electron microscopy. As expected, most of
these poly(amino acid)s form nanoparticles in water with sizes ranging from 50 to 200 nm
(Figure A2). The zoomed-in pictures (Figure 2.2b and 2.2c) clearly show that these nanoparticles
17
contain a hydrophobic core and a cationic shell including a PEG corona. As expected, polymer
P9 and P10 do not form nanoparticles due to the lack of the hydrophobic components. These
polymers were tested against a range of Gram-positive and Gram-negative bacteria. Their
antimicrobial activity is shown in Table 2.1.
Figure 2.2 The nanomorphology of the PEG-poly(amino acid)s P5. a-c, SEM pictures. a is a
zoomed-out view, while both b and c are the zoomed-in views. d is the schematic illustration of
PEG-poly(amino acid)s core-shell structure.
18
Table 2.1. The antimicrobial and hemolytic activities of PEG-poly(amino acid)s. The
microbial organisms used are K. pneumoniae (ATCC 13383), and P. aeruginosa (ATCC 27853),
methicillin-resistant S. epidermidis (RP62A), B. subtilis (BR151). The minimum inhibitory
concentration (MIC) is the lowest concentration that completely inhibits growth after 24 h. HC50
is the concentration causing 50% hemolysis. The antimicrobial peptide Magainin II was included
as a control.
O
HN
NH
O
NH2
ab
HN
NH2
O
NH
Od
Hc O
HN
NH
Oab
HN
O
NH
Od
Hc
O
O
HO
HO
P2-P10 P1
#
MR
SE
(µM
)
B.
Subtili
s
(µM)
K.
Pneum-
oniae
(µM)
P.
Aerug-
inosa
(µM)
mPE
G
(mw
)
Lysine
Content
b [(c
Phenyl-
alanine
Content
d)]
%
hydroph
obic
content
hemolytic
activity
HC50(µM)
MW
(Theor
etical)
Aver
age
size
of
nano
parti
cles
(nm)
P1 >100 >100 >100 >100 5000
Glu 20
(10 15) 33 - 10880
70-
80
P2 55
2.8-
5.5
27.5 -
55.3 >100 5000 10 (10 10) 33 9 9030
80-
90
P3
9-
22.6 4.5-9 45.3 >100 5000 20 (10 15) 33 25 11045
40-
50
P4
6.5-
13
1.6-
3.2 65 >100 5000 (10 10) 50 10 7750
60-
80
P5
5.3-
10.5
5.3-
10.5
26.3-
52.6 21-52.6 2000 (10 10) 50 6 4750
200-
250
P6
9.1-
18.2
18.2-
45.5
45.5-
91.6
45.5-
91.6 2000 (10 15) 60 5 5485
80-
100
P7
6.7-
13.3
3.4-
6.7 >100
33.3-
83.6 2000 (20 20) 50 3 7500
80-
100
P8
1.4-
2.8
5.7-
11.4 >100 28-56 2000 (30 20) 40 3 8780
100-
110
P9
5.5-
11 5.5-11 >100 >100 2000 20 0 0 >50 4560
P10
6.5-
12.8
6.5-
12.8 63.6-128 >100 2000 15 0 0 >50 3920
Mag
ainin
II11 30 16 40 40 2478
19
The negatively charged PEG-poly(amino acid)s P1, which contains glutamate residues,
did not show any antimicrobial activity under the tested conditions. This is consistent with the
hypothesis that negatively charged bacterial membranes interact weakly with molecules bearing
negative charge due to electrostatic repulsion. 36 The outer membrane of bacteria has a net
positive charge and thus the lack of activity of P1 is expected. The impact of PEGylation on the
hemolytic activity can be illustrated by P4, which contains a longer PEG corona than P5, but has
less hemolytic activity. It suggests that PEGylation does decrease the hemolytic activity of
polymers compared to the non-PEGylated analogues, although the impact is not significant.23
However, a long PEG corona may prevent the interaction of the polymer with the bacterial
membrane and thus less reactive against bacterial membranes. This is evidenced by the fact that
P4 is not as broad-spectrum active as P5. It must be noted that hemolytic activity is more tightly
related to the hydrophobic content of the polymers. Polymers P9 and P10 do not contain any
hydrophobic content, and they do not show discernible hemolytic activity even up to 50 µM.
Also, they are not active against Gram-negative bacterial membranes, indicating that the
existence of hydrophobic segments, and thus the formation of nanoparticles are important for the
disruption of membranes of Gram-negative bacteria. However, they are highly selective against
Gram-positive bacteria, including clinically-related multi-drug resistant S. epidermidis,
suggesting their potential application as novel antimicrobial biomaterials. This is consistent with
other research and is likely due to the inability of the material to penetrate the sturdy outer
membrane of Gram negative bacteria 37. Increasing hydrophobic contents lead to the most potent
antimicrobial polymers P5 and P6, which are active against all the tested bacteria. In fact, both
P5 and P6 have comparable antimicrobial activity to the antimicrobial peptide magainin II
against the tested strains. Our results also suggest that the selectivity between antimicrobial
20
activity and hemolytic activity can be tuned. P2 and P3 have similar hydrophobic content and
antimicrobial activity; however, P3 is less hemolytic than P2. It is possible that the cationic shell
(outer layer) of P3 is thicker than P2, which prevents the hydrophobic contents from non-
specifically interacting with the membrane due to increased hindrance. These effects are not
evident in the other synthesized polymers, in which the hydrophobic Phe-NCA and cationic Lys-
NCA were added simultaneously to produce hybridized core structures, and as such, their
selectivity is low. Therefore, we speculate the addition of a cationic shell (b portion), such as a
poly-lysine segment, into polymer P5 could allow for a more selective polymer to be obtained
with higher antimicrobial activity but less hemolytic activity.
We also examined the impact of particle size and molecular weight of poly(amino acid)s
on the antimicrobial activity. Although molecular weights of poly(amino acid)s do not have an
obvious impact on the antimicrobial activity and hemolytic activity, it seems that the size of
nanoparticles does affect these activities in some way. The smaller sized particles tend to have
lower toxicity to blood cells. For instance, P3 has the smallest particle size and it has the least
hemolytic activity. A similar trend is observed for other polymers, except for P5. We
hypothesize that the larger the particle size, the less specific interaction it will have with red
blood cells, thereby allowing the compound to be more hemolytic.
The ability of the polymers to damage bacterial membranes was first evaluated by
fluorescence microscopy using a double staining approach. In this assay, B. subtilis was co-
stained by DAPI (4',6-diamidino-2-phenylindole) and PI (propidium iodide). DAPI can pass
through the intact bacteria membranes irrelevant of bacterial viability; 8,10,11,36 however, PI can
only stain bacteria by intercalating nucleic acids in their nucleus after the membranes have been
disrupted. Figure 2.3 shows B. subtilis with intact cell membranes do not show fluorescence
21
after the incubation with PI. However, after 2 h treatment of B. subtilis with the most potent
polymer P5, a strong red fluorescence was observed inside the bacterium, indicating PI has
permeated into the cell’s nucleus. This demonstrates that the membranes of B. subtilis have been
disrupted.
Figure 2.3. Fluorescence micrographs of B. subtilis treated with 100 µg/ml polymer P5 for 2 h.
a1, control, no treatment, DAPI stained; a2, control, no treatment, PI stained; a3, control, no
treatment, the merged view. b1, P5 treatment, DAPI stained; b2, P5 treatment, PI stained; b3, P5
treatment, the merged view.
The ability of the polymer P5 to compromise the integrity of bacterial membranes was
further assessed by SEM (Figure 2.4). After treatment of B. subtilis with P5, the membranes of
B. subtilis are completely disrupted (Figure 2.4b). The same disruption is also observed after the
22
treatment of MRSE with P5. Figure 2.4d shows MRSE after incubation with P5 in which the
membranes of MRSE were damaged, leading to the aggregation of debris due to loss of
membrane zeta potential. 7-9 It is important to note that these polymers do not have any defined
targets and are interacting non-specifically, which would allow for some MRSE to survive once
they become shielded from nearby debris, as shown in figure 2.4d. Figure 2.4e clearly shows that
one core-shell nanoparticle from P5 is approaching the surface of a MRSE cell, while the other
nanoparticle is already merging into the membrane of the bacteria. These results support that the
polymer P5 kills bacteria via membrane disruption. Meanwhile, it is also possible that multiple
polymer nanoparticles interact with the membrane of the same bacteria cell simultaneously to
cause bacterial cell lysis.
Figure 2.4 SEM pictures. a, B. subtilis, no treatment; b, B. subtilis, treatment with 100 µg/mL
polymer P5. c, MRSE, no treatment; d, MRSE, treatment with 100 µg/mL polymer P5, a zoomed-
out review; e, MRSE, treatment with 100 µg/mL polymer P5, a zoomed-in view.
23
2.4 Conclusions
In summary, we have successfully synthesized a series of PEGylated poly (amino acids)
via ring-opening polymerization of NCAs. The reaction is run in one pot, and large quantities
can be produced easily. Our results show that these biodegradable nanoparticles have broad-
spectrum activity against both Gram-positive and Gram-negative bacteria. PEGylation can not
only promote the formation of nanoparticles, but also potentially decrease hemolytic activity to a
certain extent, even though both hemolytic activity and broad-spectrum activity are primarily
determined by the hydrophobic content. The selectivity of the polymers can be tuned by varying
both the ratio and numbers of hydrophobic and cationic groups. Both fluorescence microscopy
and SEM suggest that these PEGylated poly(amino acid)s kill bacteria by disrupting the integrity
of their membranes. Surprisingly, the polymers containing only PEG and lysine segments, show
remarkable selectivity against Gram-positive bacteria, and do not display any hemolytic activity
under our experimental conditions. It may suggest that these polymers can be not only used to
develop antimicrobial biomaterials to prevent contamination in food packaging, storage and
medical supplies, but also used as potential therapeutics to treat gram positive bacterial
infections. Furthermore, our core-shell PEG-poly(amino acid)s nanoparticles are also capable of
encapsulating drug molecules, making it possible to load small molecular antibiotics and to kill
bacteria more effectively and synergistically via membrane disruption as well as targeted
inhibition of bacterial functions. Such dual-functional antimicrobial nanoparticles are currently
being developed in our lab.
24
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28
CHAPTER 3: PEG-POLY (AMINO ACID)s – ENCAPSULATED TANSHINONE IIA AS
POTENTIAL THERAPEUTICS FOR THE TREATMENT OF HEPATOMA
Note to reader
This chapter has been previously published and has been reproduced by permission of
The Royal Society of Chemistry. Original citation:
Wang, Y*.; Costanza, F.*; Wu, H.; Song, D.; Cai, J.; Li, Q.: PEG-poly(amino acid)s-
encapsulated tanshinone IIA as potential therapeutics for the treatment of hepatoma. Journal
of Materials Chemistry B 2014, 2, 3115-3122.
3.1 Introduction
There have been many developments of drug delivery systems using amino acid
polymeric scaffolds including poly(sodium N-acryloyl-L-valinate-co-alkylacrylamide)1 as
carriers of nonsteroidal anti inflammatories, poly (L-lysine)-block-poly(L-phenylalanine)2, poly-
L-amino acid (homopolymer)3 as carriers for DNA delivery or doxorubicin as a model
encapsulated drug, and poly(L-lactide-β-γ-benzyl glutamate)4, among many others. However,
there are limitations to some of these such as inadequate water solubility and lack of a defined
amphiphilic water soluble structure in aqueous medium. This reduces circulation time as the
material is rapidly cleared by the kidneys during circulation. For those without a proper peptide
backbone, the materials are not biodegradable, which is very undesirable1.
There have been other kinds of drug delivery systems unrelated to peptides. One
intriguing example involves the emulsification of oleic acid as a surfactant in chloroform with
29
polystyrene allyl alcohol followed by a second emulsification with poly vinyl alcohol. A DNA
plasmid (hydrophilic model) or doxorubicin (hydrophobic model) nanocapsule is achieved after
evaporation of the organic solvent5. While this approach is certainly unique, its polymer
backbone is not ideal for a biological system. Other interesting delivery systems utilize
derivatives of chitin (one of the most abundant polysaccharides) to form nanoparticles from its
derivative chitosan, and have shown a model encapsulation of doxorubicin as well6,7. This
approach is valid as chitosan is non-toxic, biocompatible and very abundant, but there are issues
with water solubility at physiological pH levels.
Another class of drug delivery systems that have been gaining traction over the past
decade are those of liposomes, which are essentially made of phospholipid spheres in solution.
They have been used for aptamer delivery and labeled with FTIC8, and have also been used as a
delivery of the model drug doxorubicin,9 among many others10. However, a major limitation of
liposomes is their lack of stability as they are made up of small molecules and therefore, their
critical micelle concentration is higher than that of polymer based nanoparticles. Upon dilution in
a physiological system, they have a greater capacity to fall apart and the controlled release of the
drug is lost. One way of circumventing this issue is by attaching on PEG to prolong their
circulation time and in vivo stability upon delivering doxorubicin11.
While many groups have reported a novel kind of drug delivery system based on various
approaches already mentioned, often times only a model drug is used for encapsulation and for
proof of principle. Here, we have used our delivery system to encapsulate Tanshinone IIA and
test it in a live mouse model. Tanshinone IIA (Figure 3.1) is a lipo-soluble small molecule
isolated from salvia miltiorrhiza, and displays broad-spectrum anti-tumor activity.12-17 It is
believed to have multiple intra- and extra-cellular targets18 and therefore is less probable to
30
develop drug-resistance in tumor cells. Some known targets include inhibition of human
esterases and carboxylesterases19, inhibition of hypoxia inducible factor-120, dilation of coronary
blood vessels via activating potassium channels21, and induced maspin expression22 among many
more undiscovered targets. However, Tanshinone IIA (TSIIA) has some intrinsic drawbacks
such as poor water solubility and a short half-life.23 This has prompted the ongoing research to
develop new delivery systems to increase the solubility of TSIIA and to improve its anti-tumor
efficacy. 17,18,23 In order to advance the application and efficacy of TSIIA as a novel anti-cancer
therapeutic, the exploration of new polymer delivery systems are needed. As such, herein we
report the design and synthesis of amphiphilic PEG-poly(amino acid)s (PPAAs), which were
used as a novel nanocarrier to encapsulate and deliver TSIIA. The method of delivery is that of
passive diffusion out of the polymer and into circulation as the drug is noncovalently attached.
Many other drug delivery systems utilize polymer drug conjugates in which the drug is
covalently attached to the nanoparticle. Unless there is a clear method of release, this should be
avoided as it does decrease the activity of the drug by making it harder to reach its target24.
However, this also does limit some potential toxic effects the drug may have as well.
O
O
O
Figure 3.1 The structure of Tanshinone IIA.
31
Compared to other polymer delivery systems, poly (amino acid)s have many attractive
features. The synthesis of poly (amino acid)s is very straightforward, which can be achieved
through the ring-opening polymerization of N-carboxyanhydrides (NCAs).25 NCAs can be
obtained in one step upon treatment of side chain protected natural amino acids with phosgene or
triphosgene followed by crystallization as a primary purification method. Since the subsequent
polymerization is living, the monomers can be added batch by batch, which therefore allows for
easy synthesis of poly (amino acid)s with multiple blocks. As there are 20 natural amino acids,
the potential is limitless to obtain poly (amino acid)s that contain a diverse array of functional
groups with tunable charges. Additionally, they are biocompatible and completely
biodegradable26, and have low toxicities, which further augment their application for drug
delivery. 27-29 Furthermore, poly (amino acid)s can be easily modified with other functional
groups. For example, PEG can be attached to the terminus of poly (amino acid)s, which helps to
prevent self-aggregation and prolongs its half-life, etc.30 Therefore, we expect that PEG-
poly(amino acid)s will be an excellent carrier for the delivery of TSIIA and will improve its anti-
tumor efficacy.
3.2 Materials and Methods
Amino acids were purchased from Purebulk and Chemimpex. Other chemicals were
purchased either from Fisher Scientific or Sigma-Aldrich, and used directly without further
purification. Tanshinone IIA (98.4%) was purchased from Xian Guanyu Pharmaceutical Co,.
Ltd, China. MHCC97-H hepatoma cells, and BALB/c male athymic nude mice from Shanghai
Cancer Institute; and CCK-8 Cell Proliferation Assay Kit was purchased from Cayman
Chemical. D-Luciferin potassium salt was purchased from Synchem-Pharma Co., Ltd.
32
BALB/c male athymic nude mice (5 weeks old, body weight 14.8 ± 0.9 g) were
purchased from the Department of Experimental Animals of Shanghai Fudan University. All
animals were housed at 22°C on a 12-h light/dark cycle. All experiments were performed
according to the recommendations of the local animal protection legislation for conducting
animal studies approved by the Ethics Committee of Shanghai University of Traditional Chinese
Medicine (SYXK 2005-0008).
CH3O-PEG5000-NH2 (0.4g, 0.08 mmol) was dissolved in 10 ml of anhydrous dioxane
and purged with N2. Separately 20 equiv. (0.42g, 1.6 mmol) of Glu (OBz)-NCA was dissolved in
5 ml anhydrous dioxane, passed through a 2 μM filter to remove any decomposed NCA,
thoroughly purged with N2 and then added via syringe. The reaction was then allowed to proceed
for 3 days under an active N2 atmosphere. Then, 10 equiv. (0.21g, 0.8 mmol) of Glu (OBz)-NCA
and 15 equiv. of Phe-NCA (0.229g, 1.2 mmol) were dissolved in 5 ml anhydrous dioxane, passed
through a 2 μM filter, purged, and added via syringe. Polymerization was allowed to continue for
another 3 days. The clear solution was then precipitated into ether and the product was collected
via filtration.
The polymer was dissolved in 10 ml of TFA and to this solution, 10 equiv. of HBr in
AcOH (33% v/v conc.) was added and stirred for 4 hours. The product was then precipitated into
ether and collected via filtration.11,12
The polymer was dissolved in minimal DMSO and added to dialysis tubing (MWCO =
3,500) followed by immersion in water. Dialysis was carried out for 3 days, replacing the water
daily. Any precipitate was then filtered out, and the clear filtrates were lyophilized to afford the
final products.
33
5 mg TSIIA and 45 mg PEG-poly (amino acid)s were dissolved in 700 μL DMSO,
followed by drop-wise addition of 30 mL 30% tert-butanol/water. The resulting solution was
stirred for 3h at room temperature, and then lyophilized to produce a solid puffy cake. The solid
was re-dissolved in 30% tert-butanol/water, stirred for 3h and lyophilized. The resulting solid
was again dissolved in pure water, stirred for 3h, and the lyophilization provided the desired
TSIIA-loading poly (amino acid)s nanoparticles.
The cytotoxicity of nanoparticles against MHCC97-H hepatoma cells was determined
using CCK-8 cell viability assay19. Briefly, MHCC97-H hepatoma cells (1×104/well) were
seeded in 96 well and incubated overnight. The cells were then incubated for 24 h and 48h at
37°C/5% CO2 using different concentrations (5, 10, 20μg/mL)of TSIIA, (50, 100,
200μg/mL)PEG-poly(amino acid)s nanoparticles, and (50, 100, 200μg/mL; containing 5, 10,
20μg/mL TSIIA)PEG-poly (amino acid)s-encapsulated TSIIA nanoparticles. 10 µl cell counting
kit-8 (CCK-8; Dojindo Molecular Techinologies, Inc, Japan) was added to each well and the
plates were incubated for an additional 1h at 37°C/5%. Finally, the cell viability was measured as
the absorbance at 450 nm (A450) using a microplate reader (Bio-Rad, Model 680, Hercules, CA,
USA). All experiments were performed in triplicate.
The following study consisted of tissue distribution, targeted imaging and therapeutic
evaluation was carried out when the tumor grew to approximately 0.1 cm3. Four to six week old
Male BALB/c nude mice were subcutaneously injected with 2x106 human liver cancer cells
MHCC97-H. After 7-10 days when implanted tumors reached 1-1.2 cm in diameter, the mice
were sacrificed under sterilized condition, the xenografic tumors were dissected out, minced into
1mm3 pieces, and were then implanted into the right flanks of nude mice. When these tumors
reached 1 cm in diameter, they were minced to 1-mm3 chunks to establish the model of the
34
orthotopic liver cancer. The procedure was first done by making a 1-cm incision along the lower
edge of the left ribcage, which exposes the left lobe of liver. Then, a space was generated
between the liver and its capsule using forceps, into which tumor chunks were inserted.
Hemostatic measures were applied during surgery and no active bleeding was detected before
closure.
To evaluate TSIIA biodistribution, the six HCC-bearing mice (three mice per group)
were injected with TSIIA and TSIIA NPs at doses equivalent to 1 mg TSIIA per kg in 0.2 ml of
PBS via the caudal vein. The heart, liver, spleen, lung, kidney, colon, and tumors were collected
24 h post-injection. Biodistribution was calculated as the percentage of injected dose (ID) per
gram of tissue (%ID/g). Values are expressed as mean ± standard deviation (n = 3).
The forty-eight mice were classified into 4 groups on the basis of the solutions they were
administered: control group (normal saline, 13.5 mL/kg), blank nanoparticles group (blank NPs,
10mg/kg), TSIIA solution group (TSIIA, 1 mg/kg), and PEG-poly (amino acid)s-encapsulated
TSIIA nanoparticles group (TSIIA NPs, 10mg/kg, containing TSIIA 1mg/kg), by injection
through the vena caudalis at 1vic/2day. Fourteen days after the injection, six mice in each group
were randomly evaluated for survival time. Life extended rate (L) was calculated as follows:
L=(STtest-STcontrol)/STcontrol×100%
where STcontrol and STtest are the average survival time (days) for the mice administered with
normal saline and with tested agents respectively. The toxicities of the NPs were evaluated by
monitoring changes in body weight. The remaining six mice in each group were killed, and the
tumors were removed for examination. The longest (a) and the longest (b) vertical dimensions of
the tumor were measured. The size (V) of the tumor was calculated using the following equation:
35
Representative formalin-fixed, paraffin-embedded tissue blocks from each tumor were analyzed
by conventional hematoxylin-erosin (HE) staining. The degree of necrosis in each tumor was
visually graded as follows: +, no necrosis present or slight necrosis in fragments; ++, mid-range
necrosis, absence of nuclei from many cells with or without massive cytoplasmic damage; and
+++, severe necrosis, total loss of cytoplasm of the cancer cells.
The in vivo optical imaging in MHCC97-H hepatoma-bearing mice was evaluated using
CRI Maestro TM In vivo Fluorescence Imaging System from Cambridge Research &
Instrumentation (Massachusetts, USA). The twenty-four mice were classified into 4 groups:
control group (normal saline, 13.5 mL/kg), blank nanoparticles group (blank NPs, 10mg/kg),
TSIIA solution group (TSIIA, 1 mg/kg), and PEG-poly (amino acid)s-encapsulated TSIIA
nanoparticles group (TSIIA NPs, 10mg/kg, containing TSIIA 1mg/kg), by injection through the
vena caudalis at 1vic/2day. Prior to in vivo imaging, the mice were anesthetized with
phenobarbital sodium. D-luciferin solution (150 mg/kg) was injected i.p 5 min. before imaging.
Exposure times of the bioluminescence imaging ranged from 10 to 30s. Fluorescence images
were obtained at an excitation wavelength of 490 nm and emission wavelength of 535 nm.
Exposure times ranged from 1 to 2 min. and the fluorescence images were captured using CRI
Maestro TM In vivo Fluorescence Imaging System in vivo optical imaging.
3.3 Results and Discussion
To test our hypothesis, we synthesized a PPAA containing both hydrophilic and
hydrophobic groups (Figure 2) via ring-opening polymerization of N-carboxyanhydrides (NCAs).
2
2abV =
36
28 In an aqueous solution, the PEG-poly (amino acid)s are expected to form amphiphilic micelles
containing a hydrophobic core and a hydrophilic corona. In our attempt, we prepared PPAAs
containing glutamate and phenylalanine residues. In brief, Glu(OBz)-NCA and Phe-NCA were
synthesized by reacting amino acids phenylalanine or H-Glu(OBz)-OH with triphosgene (Figure
3.2a). 28 Then, CH3O-PEG5000-NH2 was used to initiate the ring-opening polymerization of the
NCAs (Figure 3.2b) to produce the desired poly (amino acid)s. The core-shell nanostructures were
achieved by first adding 20 equiv. Glu(OBz)-NCA and allowing the reaction to proceed for three
days at room temperature. Subsequently, 10 equiv. Glu(OBz)-NCA and 15 equiv. Phe-NCA were
added simultaneously in order to form an anionic-hydrophobic hybrid core in solution. This hybrid
core design is different from other previously reported PEG-poly(amino acid)s di-block
copolymers, in which the core contains only exclusively hydrophobic groups such as Phe. 28 We
anticipated the hybrid core to be advantageous as it is hydrophobic enough to encapsulate TSIIA,
yet hydrophilic enough (due to the presence of anionic charges) to aid in its water solubility. This
type of core can minimize the probability of aggregation, enhance the targeted delivery of TSIIA,
and aid in its release as it is more weakly bounded to the core compared to previously reported
literature31. The sequence of the poly (amino acid)s (poly dispersity Index: 1.05 by gel permeation
chromatography) was also confirmed by NMR (Figure 3.2c).
The PPAAs were then used to encapsulate TSIIA by adopting a previously reported
protocol. 32,33 Briefly, TSIIA and PEG-poly (amino acid)s were dissolved in DMSO, followed by
drop-wise addition of 30% tert-butanol/water and shaken overnight.. The solution was lyophilized
and re-dissolved in 30% tert-butanol/water and again shaken overnight. This solution was again
freeze dried and re-dissolved in water to be shaken overnight. Lastly, the solution was lyophilized
to produce the final TSIIA-loaded nanoparticles.
37
Figure 3.2. a, synthesis of Phe-NCA and H-Glu(OBz)-NCA; b, synthesis of the PEG-poly (amino
acid)s. c, 1H-NMR (800 MHz, DMSO-d6) of the PEG-poly (amino acid)s. Amino acid residues
shown in the bracket were added in one batch.
The nanomorphology of these NPs was confirmed by SEM. As expected, irrelevant of
encapsulation of TSIIA, poly (amino acid)s all form nanoparticles in water with sizes ranging from
50 to 100 nm (Figure 3.3). Interestingly, poly (amino acid)s containing TSIIA show a smaller size
38
distribution, probably due to the strong hydrophobic interactions between TSIIA and Phe residues
in the polymer, which leads to a tighter core in the nanoparticles.
Figure 3.3 Nanomorphology of poly (amino acid)s. a, SEM image of poly (amino acid)s; b, SEM
image of TSIIA-loading poly (amino acid)s. c, Size distribution of poly (amino acid)s obtained by
dynamic light scattering.
Cytotoxicity results showed that PEG-poly (amino acid)s-encapsulated TSIIA
nanoparticles effectively increase the cytotoxicity for MHCC97-H hepatoma cells as compared
with TSIIA alone (Figure 3.4). At 24 h post-incubation, the cell viability of MHCC97-H
hepatoma cells was higher than that of 48 h post-incubation. The MHCC97-H hepatoma cells
treated with increasing doses of PEG-poly(amino acid)s nanoparticles showed 94% to 98%
39
viability, respectively. This finding suggests that the blank PEG-poly(amino acid)s nanoparticles
were non-toxic at each of the tested concentrations. Specifically, the cytotoxicity of PEG-poly
(amino acid)s-encapsulated TSIIA nanoparticles was significantly improved over time, revealing
that the PEG-poly (amino acid)s-encapsulated TSIIA nanoparticles can enhance the cytotoxicity
of TSIIA for MHCC97-H hepatoma cells. This higher cytotoxicity is very beneficial for
antitumor therapy in vivo.
Figure 3.4. In vitro cytotoxicity of nanoparticles in MHCC97-H hepatoma cells. (A) 24 h-
treatment, and (B) 48 h-treatment. *p<0.05 when compared with TSIIA; **p<0.01 when
compared with TSIIA.
Biodistribution of free TSIIA and PEG-poly (amino acid)s-encapsulated TSIIA
nanoparticles in HCC-bearing mice in various tissues and organs is shown in Figure 3.5. After 24
h of injection, greater concentrations of free TSIIA were found in the heart, lung and kidney than
TSIIA NPs. In tissues, TSIIA concentration 24h post-TSIIA NPs injection followed the order of
tumor > liver > kidney > lung > spleen > colon > heart, whereas tumor accumulation of free
40
TSIIA was markedly lower. For TSIIA NPs, around 16.3% ID/g (injected dose / gram) was
found in tumor at 24 h post-injection whereas for free TSIIA found was around 1.2%ID/g. This
suggests that the TSIIA NPs does show mild selectivity for the tumor over other organ systems,
but the TSIIA itself is being widely distributed. Overall, there were significant differences in the
uptake of free TSIIA and TSIIA NPs by different tissues and organs. Therefore, with TSIIA
loaded NPs, a greater amount of the drug reached the tumor site than would have if injected
without a delivery system.
Figure 3.5. Biodistribution of TanshinoneIIA NPs. Data are shown as the percentage of injected
dose per gram of organ weight (%ID/g). **P < 0.01 compared to the other groups. Data is
presented as mean ± SD (n = 3).
**
41
To assess the potential of poly (amino acid)s with encapsulated TSIIA as a novel anti-
cancer agent, its in vivo therapeutic efficacy using a hepatoma-bearing mouse model was first
evaluated, as TSIIA was previously reported to have significant activity against hepatoma. 17,34-36
First, the ability of encapsulated TSIIA to prevent the growth of hepatic tumor was evaluated.
After administration of the polymer nanoparticles, the tumor volume was measured at
predetermined intervals so as to calculate the inhibition rate. As illustrated in Figure 3.6A,
compared to the control group in which only saline was administered, poly (amino acid)s alone
(denoted as blank NPs hereafter) did not stop tumor growth. Consistent with previous findings,
TSIIA is an effective anti-tumor agent, which suppressed the growth of the hepatic tumor by 40%.
However, as seen after 4 weeks of treatment, encapsulated TSIIA (denoted as TSIIA NPs) was a
superior therapeutic agent, as it successfully arrested the growth of the hepatic tumor by 75%.
Compared to TSIIA alone, the anti-tumor efficacy of TSIIA NPs inhibited another 50% tumor
growth. The results demonstrate that the delivery of TSIIA NPs by PPAAs is an effective approach
to treat hepatoma, suggesting the potential of further development of PPAAs for anti-cancer drug
delivery.
With regard to toxic side effects, the body weight of the animals showed no significant
difference between the control and treated groups (Figure 3.6B), but the difference of tumor size
is conspicuous, suggesting that the PPAAs effectively enhanced antitumor efficiency with minimal
toxicity. However, the slight decrease in body weight in the TSIIA only group shows that without
the drug delivery platform, the drug itself is likely quite toxic.
42
Figure 3.6 Tumor inhibitory effect of TSIIA nanoparticles in vivo. (A) The tumor growth curves
(mm3), (B) body weights (g). Data is represented as the mean ± standard deviation (n = 6).
The effect on the survival time of mice with hepatoma was also determined. As shown in
Figure 3.7A, all the mice eventually died of hepatoma. Among the four test groups, TSIIA NPs
exhibited the most promising results to improve the overall survival rate, with the longest survival
time of 37.50±1.87 days (P<0.01), compared with the saline control group that had the shortest
survival time of 24.83±2.56 days. Interestingly, TSIIA alone did not substantially improve the
survival time, which may be due to the insolubility and the short half-life. Blank NPs as well only
showed a very marginal effect on the mouse survival. The results indicate that among the four
groups, TSIIA NPs show the most potent anticancer activity, which further augments its potential
as an anti-cancer nanomedicine (Figure 3.7B). Meanwhile, the results further support that PPAAs
are excellent nanocarriers for therapeutic development. The TSIIA NPs have improved survival
time by more than double compared to the TSIIA alone proving that a drug delivery vehicle is
required for proper delivery and activity.
43
Figure 3.7. (A) Kaplan-Meier overall survival analysis and (B) Life extended rate of the
MHCC97-H hepatoma-bearing mice treated with TSIIA nanoparticles. **p<0.01vs the other
groups. Data is represented as the mean ± standard deviation (n = 6)
In addition, the degree of tumor putrescence in each group was also examined by electron
microscope with conventional HE staining as illustrated in Figure 3.8. The necrosis area in the
TSIIA NPs group was significantly greater than that of the other groups, which further
demonstrated the powerful capability of preventing the growth of the tumor. The TanshinoneIIA
group also displayed a certain extent of necrosis, which might be due to its high concentration in
solution and its intrinsic anti-tumor efficacy and cytotoxic effects.
44
Figure 3.8. Representative histopathological sections of the tumors from tumor-bearing mice (HE
Stained, magnification×200). (A) minimal necrosis after treatment with normal saline; (B)
minimal necrosis after treatment with blank nanoparticles; (C) mid-range necrosis after treatment
with free Tanshinone IIA; (D) severe necrosis after treatment with TSIIA nanoparticles.
Subcutaneous xenografting has been a widely used model to evaluate the tumor growth in
vivo. In our current report, we established an orthotopic murine liver cancer model by directly
implanting human hepatic cancer cell line MHCC97-H-Luc into the mouse liver. This model has
shown the ability to not only retain the morphology and biological characteristics of primary
human liver cancer, but also to mimic its microenvironment with the cancer cells capable of
metastasizing.37 To continuously observe and record in vivo tumor growth, we used a luciferase
labeled human hepatic cancer cell line MHCC97-H-Luc for this model. Twenty days after
implantation, when orthotopic tumors grew to an average size of 1 mm3, tumor-bearing animals
were randomly allocated into 4 different treatment/injection groups: saline control, blank NPs,
TSIIA, TSIIA NPs. Tumor size was determined on day 3, 6, 18, 24 and post-treatment. As shown
45
in Figure 3.9, on day 3, no significant difference of tumor size was observed among any
experimental group. On day 14, we found significantly smaller tumors in the mice treated with
TSIIA NPs compared to the controls (P<0.01). On day 24, tumors in the control groups grew to
considerable size and had detectable metastasis, while tumors in TSIIA treatment group were
smaller in spite of having some metastasis. In contrast, tumors were much smaller in the TSIIA-
nanoparticle group (P<0.01), and more interestingly, exhibited the smallest metastatic sites. The
results suggest that TSIIA NPs also inhibit tumor metastasis, as well as growth.
Figure 3.9. Image of hepatoma-bearing mice. The color bars (from blue to red) represent the
change of fluorescence intensity from low to high. Data is represented as the mean ± standard
deviation (n = 6)
46
3.4 Conclusion
In conclusion, we report the design and synthesis of PEG-poly (amino acid)s copolymer
nanoparticles. The nanoparticles encapsulated Tanshinone IIA and enhanced its antitumor
activity in vivo using a hepatoma-bearing mouse model. The increase in anti-tumor efficacy is
not due to the polymer nanoparticles themselves; instead, it comes from Tanshinone IIA after
encapsulated delivery. Our results suggest that PEG-poly (amino acid)s encapsulated Tanshinone
IIA are a potential novel nanomedicine for the treatment of hepatoma, and meanwhile, PEG-poly
(amino acid)s are an excellent class of nanocarriers for further development and delivery of
hydrophobic anti-cancer therapeutics.
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(18) Xu, S. W.; Liu, P. Q.: Tanshinone II-A: new perspectives for old remedies. Expert
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C.; Yan, B.; Potter, P. M.: Modulation of Esterified Drug Metabolism by Tanshinones from
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J.: Abietane Diterpenes from Salvia miltiorrhiza Inhibit the Activation of Hypoxia-Inducible
Factor-1. Journal of Natural Products 2007, 70, 1093-1097.
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Maspin Induction, and Antiprostate Cancer Activities of Tanshinone IIA and Its Novel
Derivatives with Modification in Ring A. Journal of Medicinal Chemistry 2011, 55, 971-975.
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M.: Pharmacokinetics and atherosclerotic lesions targeting effects of tanshinone IIA discoidal
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(30) Whitehead, K. A.; Langer, R.; Anderson, D. G.: Knocking down barriers:
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Amphiphilic poly(l-amino acids) - New materials for drug delivery. Journal of Controlled
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Tanshinone IIA, Isolated from Salvia miltiorrhiza Bunge, Exerts Anticancer Activity Through
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50
CHAPTER 4: PEG-POLY (AMINO ACID)S/MICRORNA COMPLEX
NANOPARTICLES EFFECTIVELY ARREST THE GROWTH AND METASTASIS OF
HEPATOMA
Note to reader
This chapter has been submitted for publication and is currently in the peer review
process.
4.1 Introduction
MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate post-
transcriptional gene expression.1,2 They function by specifically binding to their mRNA targets,
inhibiting protein translation, or destroying target mRNAs.3,4 MiRNAs have been found to play
key roles in various biological functions, including cell proliferation, metastasis, differentiation,
and apoptosis.5,6 The aberrant expression of miRNA is associated with a variety of diseases
including many types of cancers.7,8 Specifically, miRNAs can act as tumor suppressors or
oncogenes, which makes them promising targets for anti-cancer therapeutic development.9 In
contrast to small molecules, which generally act by hindering certain receptors, enzymes, ion
channels and metabolic pathways, interfering RNA can regulate a specific gene transcript
anywhere in the body10. This allows enormous specification and diversification when targeting
any kind of illness. Utilizing these natural processes of RNA interference, miRNAs show
considerable promise as they are quickly becoming a next generation of therapeutics for diseases
51
such as cancer11. Furthermore, since miRNAs are very small compared to a full size RNA, they
can easily be synthesized which allow them to become economically feasible for industrial
demands.
Despite the potential, there are significant challenges for the development of miRNA-
based therapeutics. One problem is the delivery of miRNAs, as they are not cell permeable and
are highly negatively charged.12-14 In addition, they also face obstacles such as short circulation
time, aggregation with serum proteins, and rapid enzymatic degradation.15,16 This elicits a need
to develop new kinds of nontoxic and biocompatible delivery systems that can further its clinical
application. In theory, a suitable carrier should protect the miRNA from enzymatic degradation,
so that it is transported it to the target of interest still intact. This should be followed by
internalization and subsequent release inside the cell so that it can intersect the mRNA at the
desired location.
As such, nanostructures such as polymeric micelles and liposomes have been widely used
as nanocarriers for targeted delivery of miRNAs to specific cells or tissues.15-18 These non-viral
transport mediums are less likely to provoke an immune response and are much less expensive to
produce compared to their viral counterparts. In order for successful delivery, the miRNA must
first form a stable complex with, and become shielded by the delivery system so as to prevent it
from degradation in the body. Both of these conditions are met by using a PEG tether as a
driving force for the collapsing of a stable nanoparticle as well as forming a protective shield
before reaching the target. Next, the nanocarrier must be internalized via endocytosis, which is
typically accomplished nonspecifically19. Finally, the cargo or miRNA must be released from the
nanocarrier before being degraded. Due to the less hydrophobic nature of our hybrid core, we
52
anticipate that our miRNA should be sufficiently released faster than endosomal enzymes can
degrade the miRNA.
To date, the most widely used delivery systems for miRNA are cationic lipid based
nanoparticles, many of which also use PEG as a shield and stabilizer20,21. More specifically,
liposomes have even shown the ability to directly target the hepatocytes for miRNA delivery10.
For this general purpose however, liposomes have many intrinsic drawbacks such as the
premature release of the cargo, rapid clearance from the blood and uptake by macrophages.22
Other popular approaches are cationic polymeric based particles such as poly(2-
(dimethylamino)ethyl methacrylate23 which has been used to form a complex with DNA. Here,
PEG was also used as a hydrator as well as to ameliorate the electrostatic interactions so as to
allow decomplexation inside the cell more readily. The polymeric nature of the nanoparticle
allows for greater stability compared to a lipid based liposome counterpart. However, it is
important to note that both liposome and polymeric based nanoparticles are produced from self-
assembly in solution, and as so, they are not exceptionally stable compared to a much more
robust delivery system such as an inorganic gold nanoparticle24. However, particles such as these
are inherently more toxic and less biodegradable and so; researches must fine tune their delivery
system to have a well-balanced profile of toxicity, stability, and biodegradability.
Among the different nanocarriers developed for drug delivery, poly (amino acid)s have
many unique advantages. Poly (amino acid)s are polypeptides that can be synthesized
conveniently using the ring-opening polymerization of N-carboxyanhydrides (NCAs).25 Since
NCAs can be easily synthesized on a large scale from natural amino acids, poly (amino acid)s
can be tuned straightforwardly with diverse functional groups and charges. Due to their intrinsic
low toxicity and full biodegradability, they have been widely used for drug delivery.26,27,28
53
Furthermore, the addition of a PEG segment to polymers has been shown to mitigate the problem
of non-specific interactions between cationic nanoparticle/RNA complexes and negatively
charged serum proteins due to its antifouling properties29, prevent self-aggregation, and evade
the immune system.15 However, to the best of our knowledge, the use of PEGylated poly (amino
acid)s for the delivery of miRNAs is not yet reported. Herein, we report the development of
PEG-poly (amino acid)s as a novel carrier for the delivery of miRNA-139. Low expression of
miR-139 has been linked to a few cancers including hepatocellular carcinoma, colorectal cancer,
leukemia, glioblastoma, etc.30-34 Up regulation of miRNA-139 has been shown to suppress tumor
growth, migration and metastasis by targeting the 3’-untranslated region of some oncogenic
mRNAs such as Rho-kinase 2, 32 insulin-like growth factor receptor 31 and MCl-1 mRNA.34 As
such, systemic delivery of miRNA-139 is expected to lead to novel anti-cancer therapeutics. In
this report, we show that the positively charged poly (amino acid)s with a PEG tail can form
defined nanoparticles in aqueous solution. Such nanoparticles can be used to form a complex
with the negatively charged miRNA-139. These PEG-poly (amino acid)s/miRNA-139 complex
nanoparticles (denoted as miR-139 NPs hereafter for simplification) effectively suppress the
growth of a hepatoma tumor in MHCC97-H hepatoma-bearing mice by significantly reducing
85% of the tumor size in four weeks, while subsequently also doubling their lifespan. In addition,
these nanoparticles have also been shown to prevent hepatic tumor cell migration and metastasis.
Our results suggest that PEG-poly (amino acid)s are promising non-viral gene vectors for the
delivery of miRNAs for the treatment of cancers.
54
4.2 MATERIALS AND METHODS
Amino acids used for the synthesis of poly (amino acid)s were purchased from purebulk
inc. The miRNA mimics and miRNA control were purchased from Biomics Biotechnologies
(Nantong, China). MHCC97-H hepatoma cells, and BALB/c male athymic nude mice from
Shanghai Cancer Institute; and CCK-8 Cell Proliferation Assay Kit was purchased from Cayman
Chemical. D-Luciferin potassium salt was purchased from Synchem-Pharma Co., Ltd.
CH3O-PEG2000-NH2 was dissolved in 10 ml of anhydrous dioxane and purged with N2.
Separately 20 eq of Z-lys-NCA was dissolved in 5 ml anhydrous dioxane, passed through a 2 µM
filter to remove any decomposed NCA, thoroughly purged with N2 and then added via syringe.
The reaction was then allowed to proceed for 3 days under an active N2 atmosphere. Then 10
equiv. of Z-Lys-NCA and 15 equiv. of Phe-NCA were dissolved in 5 ml anhydrous dioxane,
passed through a 2 μM filter, purged, and added via syringe. Polymerization was allowed to
continue for another 3 days. The clear solution was then precipitated into ether and the product
was collected via filtration.
The polymer was dissolved in 10 ml of TFA and to this solution, 10 equiv. of HBr in
AcOH (33% v/v conc.) was added and stirred for 4 hours. Product was then precipitated into
ether and collected via filtration.
The polymer was dissolved in minimal DMSO and added to dialysis tubing (MWCO =
3,500) followed by immersion in water. Dialysis was carried out for 3 days and the water was
replaced daily. Any precipitate was then filtered out, and the clear filtrates were lyophilized to
afford the final products.
55
To form the PEG-poly(amino acid)s/miR nanoparticle complex, 1 mg miRNA-139 and
15 mg PEG-poly (amino acid)s were dissolved in 200 μL DMSO, followed by drop-wise
addition of 10 mL water. The resulting solution was stirred for 3h at room temperature, and the
lyophilization provided the desired product.
The following targeted study consisting of tissue distribution, targeted imaging and
therapeutic evaluation was carried out when the tumor grew to approximately 0.1 cm3. Male
BALB/c nude mice, 4-6 weeks old, were subcutaneously injected with 2x106 human liver cancer
cells MHCC97-H. After 7 -10 days, when implanted tumors reached 1-1.2 cm in diameter, the
mice were sacrificed under sterilized condition, the xenographic tumors were dissected out,
minced into 1mm3 pieces, which were then implanted into the right flanks of nude mice. When
these tumors reached 1 cm in diameter, they were minced to 1-mm3 chunks for establishing the
orthotopic liver cancer model. The procedure was first done by making a 1-cm incision along
the lower edge of left ribcage, to expose the left lobe of liver. Then, a space was generated
between the liver and its capsule using forceps, into which tumor chunks were inserted.
Hemostatic measures were applied during surgery and no active bleeding was detected before
closing abdominal incision. No fasting was applied to the animals.
The mice were classified into 4 groups on the basis of the solutions they were
administered: control group(normal saline, 13.5 mL/kg), blank NPs(10mg/kg) group, miR-
control NPs(10mg/kg) group, miR-139 NPs (10mg/kg) group, by injection through the vena
caudalis at 1vic/2day. 7 days after the injection, six mice in each group were chosen randomly
for evaluation of survival time. Survival rate (SR) was calculated as follows:
56
where STcontrol and STtest are the average survival time (days) for the mice administered with
normal saline and with test agent respectively. The toxicities of the NPs were evaluated by
monitoring changes in body weight.
The remaining mice in each group were killed, and the tumors were removed for
examination. The longest (a) and the longest (b) vertical dimensions of the tumor were
measured. The size (V) of the tumor was calculated using the following equation:
The in vivo optical imaging of 4 groups in MHCC97-H hepatoma-bearing mice were
evaluated using CRI Maestro TM In vivo Fluorescence Imaging System from Cambridge
Research & Instrumentation (Massachusetts, USA).
Prior to in vivo imaging, the mice were anesthetized with Phenobarbital sodium. D-
luciferin solution (150 mg/kg) was injected i.p 5 min before imaging. Exposure times of
bioluminescence imaging ranged from 10 to 30 s. Fluorescence imaging was obtained with an
excitation wavelength of 490 nm and emission wavelength of 535 nm. Exposure times ranged
from 1 to 2 min. and the fluorescence images were captured using CRI Maestro TM In vivo
Fluorescence Imaging System In vivo optical imaging
Tissue sections of 5-μm in thickness were cut from formalin fixed and paraffin embedded
tissue blocks. After deparaffinization and rehydration, endogenous peroxidases were quenched in
0.3% H202 diluted in methanol. Tissue sections were then incubated overnight at room
temperature (RT) with mouse anti human CD31 antibody(1:400; Dako, Glostrup, Denmark).
100STtest STcontrol
SRSTcontrol
−= ×
2
2abV =
57
Immunodetection was performed incubating the slide with a goat anti-mouse antibody and
horseradish peroxidase (HRP)-streptavidin complex (both Dako) for 30 min at RT. Staining was
visualized using 0.05% 3,3’-diaminobenzidine containing 0.0038% H202. Representative
micrographs were taken under an Olympus BX-51TF microscope equipped with a DP23-3-5
camera. The CD31 positive microvessels in the tumor-bearing area were quantified by
computerized image analysis. Four representative areas in tumor for CD31 staining were selected
and photographed at 200 × magnification.
For cell motility assay, 8 × 105 cells were seeded onto 60-mm dishes and grown for 24 h.
A linear wound was made by scraping a pipette tip across the confluent cell monolayer. Cells
were rinsed with PBS and grown in 1640 supplemented with 10% FBS for additional 48 h. The
cell motility in terms of wound closure was measured by photographing at three random fields at
the time of wounding (time 0) and at 12, 24 and 48 h after wounding. 38
4.3 Results and Discussion
To test our hypothesis, we synthesized a PEGylated poly (amino acid)s containing both
cationic and hydrophobic groups (Figure 4.1) via ring-opening polymerization of N-
carboxyanhydrides (NCAs). 25 The synthetic procedure was adapted from a previously published
protocol.27 Briefly, Z-Lys-NCA and Phe-NCA were first synthesized through the treatment of
amino acids phenylalanine or Z-Lysine with triphosgene, respectively (Figure 4.1a). Next, CH3O-
PEG2000-NH2 was used as the initiator to react with both NCAs to produce poly (amino acid)s
via the ring-opening polymerization (Figure 4.1b). In order to generate defined core-shell
nanoparticles, 20 equiv. Z-Lysine-NCA was first added into the flask containing CH3O-PEG-NH2
and the reaction was allowed to proceed for three days at the room temperature. Subsequently, 10
58
equiv. Z-Lysine-NCA and 15 equiv. Phe-NCA were then added in one batch to form the cationic-
hydrophobic hybrid core of nanoparticles in solution. We hypothesized that such a hybrid core
would be superior to a pure hydrophobic core consisting of phenylalanines because the core is still
positively charged, which increases the solubility as well as the electrostatic attraction with
negatively charged miRNAs, but is still hydrophobic enough to cause the self-assembly into
nanostructures. The NMR of the poly (amino acid)s is shown in Figure 1c.
ONH Dioxane
N2, r.t., 3d
O
HN
NH
O
NHO
O
1. TFAHBr / AcOH
10 equiv. Z-Lys-NCA+ 15 equiv. Phe-NCA
45
H
20
20 equiv Z-Lys-NCA
DioxaneN2, r.t., 3d
2. Dialysis
O
HN
NH
O
NH2
4520
HNO
O
O
R
Z-Lys-NCA
HN
NH
O
NH2
H
15
H2N OH
O
R
R =
NH
O
O
or
Phe-NCA
a
b
45
Triphosgene
10
H
O
59
Figure 4.1. a, synthesis of Phe-NCA and Z-Lys-NCA; b, synthesis of PEG-poly (amino acid)s. c, 1H-NMR (800 MHz, DMSO-d6) of the PEG-poly (amino acid)s. Amino acid residues shown in
the bracket were added in one batch, and the residues were randomly mixed.
The PEG-poly (amino acid)s/miRNA-139 complex nanoparticles (miRNA-139 NPs) were
then prepared by adapting a previously reported protocol. 35,36 Briefly, 1 mg miRNA-139 and 15
mg PEG-poly (amino acid)s were dissolved in DMSO, followed by slow addition of water. The
nanoparticles (NPs) were obtained after lyophilization. Meanwhile, miR-67 (miR-control), a
miRNA that has a minimum sequence similarity to miRNAs in human and mouse, 37 was included
as a negative control.
The nanomorphology of these NPs was confirmed by SEM. The PEG-poly (amino acid)s
and their complexes with miRNAs form nanoparticles with sizes ranging from 50 to 200 nm
(Figure 4.2). PEG-poly (amino acid)s nanoparticles themselves tend to aggregate together (Figure
4.2a); however, after forming complexes with miRNAs, the nanoparticles are more defined and
60
segregated (Figure 4.2b and 4.2c). It is possible that the presence of miRNAs close to surface of
nanoparticles lead to electrostatic repulsion among nanoparticles.
Figure 4.2. SEM pictures of prepared nanoparticles. a, PEG-poly(amino acid)s (denoted as blank
NPs thereafter); b, miR-139 NPs; c, miR-control NPs. Bar = 50 nm.
To evaluate the in vivo anti-tumor effect of miR-139 NPs, we tested these particles in a
mouse xenograftic model of hepatoma cell line MHCC97-H. After four weeks, in comparison to
the control groups (only saline, miR-139, blank NPs, or miR-control NPs), the miR-139 NPs
treatment showed the most significant inhibition of the tumor growth, with treated tumors being
1/5 the average size of the ones in the control groups (P<0.05) (Figure 4.3). Our result further
indicated that the repressive effect on the tumor growth by miR-139 NPs was very specific, since
blank NPs or scramble miR-control NPs did not exhibit any inhibitory effect on tumors.
Although as anticipated, miR-139 alone did exhibit the ability to block the tumor growth, and the
efficacy is much less than that of miR-139 NPs. These results clearly demonstrate that the novel
miR-139 NPs are effective anti-tumor agents. It also indicates that PEG-poly(amino acid)s are
very effective nanocarriers for the delivery and the in vivo efficacy of miRNAs.
61
Figure 4.3. The tumor growth curves (mm3). Data is represented as the mean ± standard
deviation (n = 6).
The effect on the survival time of mice with hepatoma was also determined (Figure 4.4).
Although all the mice eventually died of hepatoma, administration of miR-139 NPs had greatly
improved the life-span of mice bearing hepatoma. As seen in Figure 4.4, among the tested
agents, the miR-139 NPs group showed that it had the most promising prospect to improve the
survival condition, coupled with the longest survival time of 50.33 ± 3.08 days (P<0.01),
compared with the saline control group with the shortest survival time of 25.50 ± 1.87 days.
Neither NPs alone nor miR-control NPs had virtually any effect in the improvement of the
survival time. Although as expected, miR-139 displayed certain antitumor efficacy, but it is far
inferior to that of miR-139 NPs. Overall, the results demonstrate that the complexation of PEG-
poly (amino acid)s with miRNAs greatly enhances the therapeutic efficacy of miRNAs.
62
Figure 4.4 Survival time (day) of hepatoma-bearing mice in different groups. Data is
represented as the mean ± standard deviation (n =10).
Subcutaneous xenograft has been a widely used in vivo tumor growth model of cancer
cells, but in many ways, has failed to retain and recapitulate characteristics of the primary
tumors. In our current report, we established an orthotopic murine liver cancer model by directly
implanting human hepatic cancer cell line, MHCC97-H, into a mouse liver. Not only does this
model retains the morphology and biological characteristics of a human liver cancer, but it also
mimics the microenvironment with which the cancer cells are capable of metastasizing in. To
continuously observe and record in vivo tumor growth, we used a luciferase labeled MHCC97-H
human hepatic cancer cell line and renamed it “MHCC97-H-Luc”. Twenty days after
implantation, when orthotopic liver tumors grew to an average size of 1 mm3, tumor-bearing
animals were randomly allocated into 5 different treatment/injection groups similar to Figure 4.4.
Tumor size was determined on day 7, 14, 21, and 28 post-treatment. As shown in Figure 4.5, on
63
day 7, no significant difference of tumor size was observed among mice, treated vs. controls. At
day 14 and beyond, significantly smaller tumors were observed in miR-139-NPs treated mice
compared to the controls (P<0.01). On day 28, tumors in saline, blank NPs and miR-control NPs
group grew to such a considerable size that they nearly took over the whole livers, and showed
detectable metastasis. Mice in miR-139 had smaller tumor sizes, however, the difference was
marginal compared to other control groups. In contrast, tumors were much smaller in miR-139
NPs treatment group (P<0.01), and more interestingly, no metastatic sites were observed.
Figure 4.5. Image of orthotropic hepatoma-bearing mice. The color bars (from blue to red)
represent the change of fluorescence intensity from low to high.
Since the loss of miR-139 was closely associated with aggressive pathologic features such
as metastasis in primary human hepatoma cases, we hypothesized that miR-139 NPs could
64
impede the migratory and invasive capabilities of hepatoma cells. As such, we also evaluated the
effect of miR-139 NPs on tumor angiogenesis. First, by immunohistochemistry staining, we
showed that the expression of CD31, a blood vessel endothelial cell marker, was present in the
control xenograft tumors, whereas its expression was weak or negative in the tumor treated with
miR-139 NPs (Figure 4.6). Semi-quantitative analysis of the microvessel area revealed that the
average MVD (microvessel density) value in miR-139 NPs treated tumor was significantly lower
than that of the other control groups. (p< 0.01).
Figure 4.6. The immunohistochemical staining of CD31 antigen showed that there were
micro-vessels around tumor cells (the brown parts). A, control group (saline, 13.5 mL/kg); B,
blank NPs (10mg/kg) group; C, miR-control NPs (10mg/kg) group; D, miR-139
(0.5mg/kg)group; E, miR-139 NPs (10mg/kg)group. Quantitative analysis was performed by
counting the number of tumor foci in 10 randomly selected high-power fields under microscope
(×200).
Next, we assessed the anti-metastasis effect of miR-139 NPs on MHCC97-H cells in vitro
by a wound healing assay. As shown in Figure 4.7, miR-139 NPs remarkably attenuated the
migratory ability of MHCC97-H hepatoma cells, as indicated by the decrease in the numbers of
65
migrated cells, compared to the controls. These results are consistent with the previous findings
in that miR-139 NPs played a significant role in hepatocarcinoma cell migration. 32
Figure 4.7. Wound closure was delayed in miR-139 nanoparticles transfected cells in
12h,24h and 36h time points in MHCC97-H cells
4.4 Conclusion
In conclusion, we designed and synthesized PEGylated poly (amino acid) nanoparticles,
for the purpose of using them as nanocarriers to form a miR-139 NP complex. Although the use
of nanoparticles for microRNA delivery is common, the employment of PEG-poly (amino acid)s
for such a purpose is rare. We believed that PEG-poly (amino acid)s would be versatile carriers
66
because they are amendable to further modification on their functional groups. Meanwhile, the
presence of a PEG tail is expected to minimize side reactions and to increase the bioavailability
of microRNAs. Indeed, miR-139 complex nanoparticles effectively suppress tumor growth in a
traditional xenograftic model, as well as in a newly established orthotopic liver cancer model of
human hepatoma cell line MHCC97-H, and displays significant inhibitory impacts on cancer cell
migration and metastasis. Our results suggest that PEG-poly (amino acid)s are potent non-viral
gene vectors for the delivery of therapeutic miRNAs for the treatment of cancers.
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70
CHAPTER 5: CONCLUSIONS AND FUTURE DIRECTIONS
In this dissertation, we have explored mPEG poly amino (acid)s as broad spectrum
antibacterial polymers as well as versatile drug delivery polymers. In this chapter, we want to
summarize our results and provide some perspective for future directions.
We have developed polymer micelles based on the ring opening polymerization of N-
carboxy anhydrides by mPEG-NH2. By using natural L-amino acids, our material is fully
biodegradable and biocompatible which makes it very suitable to be used in vivo. While here we
have only used three representative amino acids based on the cationic side chain of lysine,
anionic side chain of glutamic acid, and hydrophobic side chain of phenylalanine, in theory, any
natural amino acid can be used as long as the side chain is protected during the polymerization.
With 20 natural amino acids, and hundreds more unnatural amino acids being used in research,
this leads the way to develop new types of polymers with enormous diversity. Furthermore,
instead of using PEG as an initiator, any other polymer containing a nucleophilic primary amine
group can be used as an initiator to encourage even more diversity. Together, this allows one to
attach a different tether, use different equivalents of monomers to control the polymer size, and
use different amino acids which provide the polymer with different kinds of functionalization to
be used in post polymerization modification.
Our first application with our polymer micelles utilizes the differences in electrostatics
between bacterial membranes and eukaryotic membranes. Due to bacterial membranes having an
overall more net negative charge on their outer membrane compared to eukaryotic membranes
71
which are overall more neutral, this has allowed us to develop cationic polymer micelles that
selectively interact, and subsequently disrupt bacterial membranes more readily. The extent of
hemolysis against erythrocytes can be tuned by lowering the amount of hydrophobic content in
the polymers, but this also lessens its membrane disrupting ability. Also, our polymers allow for
the further functionalization to increase the antibacterial activity due to its reactive amine groups
that are still present. One could imagine attaching specific antibiotics or membrane targeting
ligands to the polymer in an effort to synergistically kill the microbes with greater activity. As
per our other two applications, antibacterial drugs could also be encapsulated by our polymers
for a more systemic effect.
Next, we have shown the ability of a negatively charged polymer to encapsulate a super
hydrophobic drug Tanshinone IIA and be delivered to a live liver cancer bearing mouse.
Polymeric micelles such as ours are gaining traction in the field of drug delivery due to their
small size, relatively narrow distribution of particles, ability to encapsulate hydrophobic drugs,
and their ability to prolong the circulation time of the drug. There were no additional side effects
noticed from our delivery method, and we observed a marked decrease in tumor size which
exemplifies our delivery method as safe and effective. Furthermore, the nanoparticles formed are
all less than 200 nanometers, which is thought of being the maximum size that a particle can be
in order to cross the blood brain barrier should one try and use this delivery system to target
brain chemistry. While unnecessary in this application, one could imagine loading a different
kind of novel therapeutic into our polymer with the end goal of delivering the cargo through the
blood brain barrier.
Lastly, we have used our system to show a proof of concept in delivering a negatively
charged miRNA with our cationic based polymer. Again, no significant side effects were
72
observed and the tumor size was drastically reduced, which shows the versatility in our
nanoparticles to deliver various kinds of cargo. However, the major limitation of our design is
that it lacks targeting efficiency and the cargo is not being delivered directly at the site of
interest. This drawback has spawned the latest research in our lab in which we are currently
testing our nanoparticles with Tanshinone IIA, but this time with FTIC as a fluorescent labeling
polymer, as well as with RGD as a targeting peptide. This is made possible by the many
available functional groups that the polymer still possesses. At the time of this writing, the
results of our site specific targeting and labeling have not yet arrived, and therefore are not
present in this dissertation. However, we do expect that with targeting peptides such as RGD, our
efficiency to deliver site specific cargo will increase.
Finally, we would like to conclude by stating that our research has further validated the
use of mPEG poly amino (acid)s in being able to exhibit broad spectrum antibacterial activity in
addition to acting as a versatile drug delivery nanoparticle. We anticipate that in research to
come, this type of methodology will become more site specific as in our targeting approach, and
that new types of functionalities will allow this class of materials to achieve its full potential in
becoming a new generation of safe and effective polymer biomaterials.
73
APPENDIX A
Supporting info for: Investigation of antimicrobial PEG-poly(amino acid)s
A1. Synthesis of PEG-poly(amino acid)s
Figure A1a. Synthesis of N-carboxyanhydrides (NCAs):1
HNO
O
O
R
H2N
O
OH
R
R =
HN
O
O
O
O
Typically, to a round bottom flask, 5 g of the amino acid was suspended in approximately 130 mL
of anhydrous THF and heated to 60°C. Separately, 0.34 equivalents of triphosgene was dissolved
in 20 mL of anhydrous THF and added dropwise to the solution under an active N2 atmosphere
(final concentration ~0.15 M). After the solution turned clear in ~4 h, any insoluble precipitates
were filtered off. The solution was concentrated under vacuum and crystallized from THF/hexane.
Three to four subsequent recrystallizations were performed until pure white compounds were
obtained.
74
A1b. Synthesis of PEG-OTs:
OO
H
a
OO
a
S
O
O
a = 45 or 113TsCl
DCM
Typically, to a solution of 10 g of mPEG (mw = 2000 or 5000) in 50 mL of DCM, 0.416
ml of TEA was added and stirred for 10 min. Then 3.8 g of p-toluensulfonyl chloride (10 eq.)
dissolved in DCM was added and stirred for 20 h at room temperature. The solution was washed
with citric acid to neutralize TEA and then dried over NaSO4. The solvent was removed and
product was precipitated into ether.
A1c. Synthesis of PEG-N3:
OO
a
S
O
O
ON3
aa = 45 or 113
NaN3
DMF
5 g of mPEG-OTs was dissolved in 40 ml of DMF and 1.89 g (30 eq.) of NaN3 was
added. The reaction was heated to 90°C for 20 h. Then 200 ml of brine was added and extracted
into DCM. The extracts were concentrated and then precipitated into ether.
A1d. Synthesis PEG-NH2:
Excess water/brine was used to move all salts into the water layer along with DMF. The
DCM extracts were dried over NaSO4, concentrated under reduced pressure and precipitated into
ether.
75
ON3
a
ONH
H
a
a = 45 or 113
THF
LAH
2 g of PEG-N3 was dissolved in 50 ml of THF (minimal) and 200 mg of LAH (2 eq.) was
added at 0 °C. The reaction was allowed to reach room temperature and was stirred overnight.
Then 5 eq of water was added from a 4M NaOH solution and stirred until all the LiOH and Al(OH)3
salts have precipitated out as a white solid. The salts were filtered off, and the solvent was removed
to be re-dissolved in DCM. It was then washed with water/brine to remove any excess salts. DCM
layers were collected, dried over NaSO4, and precipitated into ether.
ONH Dioxane
N2, r.t., 3d
O
HN
NH
O
NHO
O
1. TFAHBr / AcOH
c equiv. Z-Lys NCA+ d equiv. Phe NCA
a
H
b
b equiv Z-Lys NCA
DioxaneN2, r.t., 3d
2. Dialysis O
HN
NH
O
NH2
ab
HNO
O
O
R
NCA
H
a
HN
NH2
O
NH
Od
Hc
76
A1e. Synthesis of polymer P3
(The other polymers were prepared by a similar method). 0.4 g of PEG-NH2 was dissolved
in 10 ml of anhydrous dioxane and purged with N2. Separately 0.49 g (20 eq) of Z-lysine NCA
was dissolved in 5 ml anhydrous dioxane, passed through a 2 micron filter to remove any
decomposed NCA, thoroughly purged with N2 and then added via syringe. The reaction was then
allowed to proceed for 3 days under an active N2 atmosphere. Then 10 equivalents of Z-Lysine
NCA and 15 equivalents of Phenylalanine NCA were dissolved in 5 ml anhydrous dioxane, passed
through a 2 micron filter, purged, and added via syringe. Polymerization was allowed to continue
for another 3 days. The clear solution was then precipitated into ether and the product was collected
via filtration.
A1f. Removal of Z protecting groups:
Polymers were dissolved in 10 ml of TFA and to this solution, 10 equivalents of HBr in
AcOH (33% v/v conc.) was added and stirred for 4 hours. Product was then precipitated into
ether and collected via filtration.
A1g. Purification
The polymers were dissolved in minimal DMSO and added to dialysis tubing (MWCO =
3,500) followed by immersion in water. Dialysis was carried out for 3 days replacing the water
daily. Any precipitate was then filtered out, and the clear filtrates were lyophilized to afford the
final products.
77
Table A1. Molecular Weights of the polymers
Polymer # P1 P2 P3 P4 P5 P6 P7 P8 P9 P10
MW (calculated) 10880 9030 11045 7750 4750 5485 7500 8780 4560 3920
A2. The morphology of PEG-poly(amino acid)s in water.
Figure A2. SEM image showing the morphology of PEG-poly(amino acid)s
A3. Antimicrobial activity
The bacterial strains used for testing the efficacy of polymers were multi-drug resistant S.
epidermidis (RP62A), B. subtilis (BR151), K. pnuemoniae (ATCC 13383) and multi-drug resistant
P. aeruginosa (ATCC 27853). The antimicrobial activities of the polymers developed were
78
determined in sterile 96-well plates by serial dilution method. Bacterial cells were grown overnight
at 37 °C in 5 mL medium after which a bacterial suspension of approximately 106 CFU/mL in
Luria broth or trypticase soy was prepared ensuring that the bacterial cells were in the mid-
logarithmic phase. Aliquots of 50 µL bacterial suspension were added to 50 µL of medium
containing different concentrations of polymers for a total volume of 100µL in each well. The 96-
well plates were incubated at 37 0C for about 20 h. The Biotek microplate reader was used to
measure the optical density (OD) at a wavelength of 600 nm after about 20 h. The experiments
were carried out as three independent biological replicates, each in duplicate. The lowest
concentration at which complete inhibition of bacterial growth is observed is defined as the
minimum inhibitory concentration (MIC).
A4. Hemolytic activity
Freshly drawn human red blood cells (hRBC’s) were used for the assay. The blood sample
was washed with PBS buffer several times and centrifuged at 700g for 10 min until a clear
supernatant was observed. The hRBC’s were re-suspended in 1X PBS to get a 5% v/v suspension
which was used to perform the assay. 50 µL of different polymers solutions were added to sterile
96-well plates. Then 50 µL of 5% v/v hRBC solution was added to make up a total volume of 100
µL in each well. The 0% hemolysis point and 100% hemolysis point were determined in 1 X PBS
and 0.2% Triton-X-100 respectively. The 96 well plate was incubated at 37 °C for 1h and
centrifuged at 3500 rpm for 10 min. The supernatant (30 µL) was then diluted with 100 µL of
1XPBS and hemoglobin was detected by measuring the optical density at 360nm by Biotek
microtiter plate reader (Type: Synergy HT).
% hemolysis = (Abs sample -Abs PBS )/(Abs Triton
–Abs PBS ) x 100
79
A5. Fluorescence microscopy
A double staining method with DAPI (4’,6-diamidino-2-phenylindole dihydrochloride,
Sigma, >98%) and PI (propidium iodide, Sigma) as fluorophores was used to visualize and
differentiate the viable from the dead B. subtilis cells. DAPI being a double stranded DNA binding
dye, stains all bacterial cells irrespective of their viability. Ethidium derivatives such as propidium
iodide (PI) is capable of passing through only damaged cell membranes and intercalates with the
nucleic acids of injured and dead cells to form a bright red fluorescent complex. The bacterial cells
were first stained with PI and then with DAPI. The bacterial cells were grown until they reached
mid-logarithmic phase and then ~ 2x10 3 cells were incubated with 100 µg/mL polymer P5 for 4
h. Then the cells were pelleted by centrifugation at 3000 g for 15 min in an Eppendorf
microcentrifuge. The supernatant was decanted and the cells were washed with 1X PBS several
times and then incubated with PI (5 µg/mL) in the dark for 15 min at 0 oC .The excessive PI was
removed by washing the cells several times with 1X PBS several times. Lastly, the cells were
incubated with DAPI (10 µg/mL in water) for 15 min in the dark at 0oC. Then finally the excessive
DAPI solution was removed by washing it with 1X PBS. The controls were performed following
the exact same procedure for bacteria without P5. The bacteria were then examined by using the
Zeiss Axio Imager Z1optical microscope with an oil-immersion objective (100X). 2,3
A6. SEM of bacteria after treatment with polymer P5.
MRSE and B.subtilis were grown to an exponential phase and approximately about 2 x 106
cells were incubated with polymer P5 for about 20 h. The cells were then harvested by
centrifugation (3000 g) for 15 min. After pelleting the cells, the cells were washed three times with
80
DI water. The cells were then fixed with 2.5% (w/v) glutaraldeyhyde in nanopure water for about
30 min, followed by extensive wash with DI water to get rid of any excess glutaralhedyde. Graded
ethanol series (30%, 50%, 70%, 95% and 100%, 5 min each) was then used to dehydrate the cells.
Following the dehydration of cells hexamethyldisilazane was added for about 2 min. Then about
2 µL of sample was added to a silicon wafer followed by Au/Pd coating, and then the samples
were observed at 25KV with a HITACHI S-800 scanning electron microscope.
A7. References
(1) Koo, A. N.; Lee, H. J.; Kim, S. E.; Chang, J. H.; Park, C.; Kim, C.; Park, J. H.;
Lee, S. C.: Disulfide-cross-linked PEG-poly(amino acid)s copolymer micelles for glutathione-
mediated intracellular drug delivery. Chemical communications (Cambridge, England) 2008,
6570-2.
(2) Niu, Y.; Padhee, S.; Wu, H.; Bai, G.; Harrington, L.; Burda, W. N.; Shaw, L. N.;
Cao, C.; Cai, J.: Identification of gamma-AApeptides with potent and broad-spectrum
antimicrobial activity. Chemical communications (Cambridge, England) 2011, 47, 12197-12199.
(3) Niu, Y.; Padhee, S.; Wu, H.; Bai, G.; Qiao, Q.; Hu, Y.; Harrington, L.; Burda, W.
N.; Shaw, L. N.; Cao, C.; Cai, J.: Lipo-gamma-AApeptides as a New Class of Potent and Broad-
Spectrum Antimicrobial Agents. J Med Chem 2012, 55, 4003-4009.
81
O
HN
NH
HN
NH2
O
O
O
NH2
a b cd
NH2
A
B
B
B
CC
CD
E
E
F
G
G
Figure A3: NMR of Polymer P2
1H-NMR (DMSO-d6, 800 MHz)
82
O
HN
NH
HN
NH2
O
O
O
NH2
a b cd
NH2
A
B
B
B
CC
CD
E
E
F
G
G
Figure A4: NMR of Polymer P3
1H-NMR (DMSO-d6, 800 MHz)
83
O
NH
HN
NH2
O
O
NH2
a cd
A
B
B
CC
E
F
G
D
Figure A5: NMR of Polymer P4
1H-NMR (DMSO-d6, 800 MHz)
84
O
NH
HN
NH2
O
O
NH2
a cd
A
B
B
CC
E
F
G
D
Figure A6: NMR of Polymer P5
1H-NMR (DMSO-d6, 800 MHz)
85
O
NH
HN
NH2
O
O
NH2
a cd
A
B
B
CC
E
F
G
D
Figure A7: NMR of Polymer P7
1H-NMR (DMSO-d6, 800 MHz)
86
O
NH
HN
NH2
O
O
NH2
a cd
A
B
B
CC
E
F
G
D
Figure A8: NMR of Polymer P7
1H-NMR (DMSO-d6, 800 MHz)
87
O
NH
HN
NH2
O
O
NH2
a cd
A
B
B
CC
E
F
G
D
Figure A9: NMR of Polymer P8
1H-NMR (DMSO-d6, 800 MHz)
88
Oa
HN
O
NH2
NH2b
B
CD
E
G
Figure A10: NMR of Polymer P9
1H-NMR (DMSO-d6, 800 MHz)
89
Oa
HN
O
NH2
NH2b
B
CD
E
G
Figure A11: NMR of Polymer P10
1H-NMR (DMSO-d6, 800 MHz)