PROTOPORPHYRIN IX (PPIX)-CONJUGATED SELF-LIGHTING NANOPARTICLES
FOR PHOTODYNAMIC THERAPY: SYNTHESIS AND CHARACTERIZATION
by
HOMA HOMAYONI
Presented to the Faculty of the Graduate School of
The University of Texas at Arlington in Partial Fulfillment
of the Requirements
for the Degree of
DOCTOR OF PHILOSOPHY
THE UNIVERSITY OF TEXAS AT ARLINGTON
December 2013
ii
Copyright © by Homa Homayoni 2013
All Rights Reserved
iii
Acknowledgements
First and foremost I would like to extend my gratitude to my supervising mentor
Prof. Wei Chen who supported me during my PhD pursuit. It was a great pleasure to
have such a nice and caring advisor. I appreciate Prof. Jer-Tsong Hsieh, Prof. Liping
Tang, Prof. George Alexandrakis, and Prof. Yi Hong for helping to shape and guide the
direction of the work with their careful and instructive comments. Regarding
characterization, I thank “Characterization Center for Materials and Biology” and Dr.
Muhammed Yousufuddin for providing us with their facilities. I would like to acknowledge
the former scientists of Nano-Bio Physic group, Dr. Marus Hossu, Dr. Xiaoju Zou, and Dr.
Ke Jiang for all their guide, enthusiasm, intensity, and willingness to make my PhD
experience productive and stimulating. I am also thankful of Nao-Bio Physics group for all
their support.
I would like to express my heartfelt gratitude to former chair of Bioengineering
department Prof. Khosrow Behbehani. I appreciate all his contributions of time and ideas
to make Ph.D program of Bioengineering at UTA more productive.
Lastly, my sincere appreciation extends to my family for all their support, love,
and encouragement. For my parents who raised me with a love of research and
supported me unconditionally. For my son whose dreams were the best inspiration and
enthusiasm to remain loyal to my goal. Most of all for the presence of my caring and
encouraging brother here at UTA. I would not have fulfilled this goal without him who
supported me every single moment of this journey.
November 08, 2013
iv
Abstract
PROTOPORPHYRIN IX (PpIX)-CONJUGATED SELF-LIGHTINIG NANOPARTICLES
FOR PHOTODYNAMIC THERAPY: SYNTHESIS AND CHARACTERIZATION
Homa Homayoni, PhD
The University of Texas at Arlington, 2013
Supervising Professor: Wei Chen
In Photodynamic therapy (PDT) PDT, cancer destruction relies on applying a
photosensitizing drug (PS) followed by light. Absorbed light can activate the PS to
transfer energy to existing molecules and substrates or to oxygen to generate singlet
oxygen which are highly toxic to cells. Protophorphyrin IX (PpIX) is a photosensitizers
(PSs) which has FDA approval and an absorbance near the Soret band which is 10 times
stronger than the absorbance in Q-band (600nm). Our goal was to design a modality to
eliminate of external blue light in addition to increasing the drug’s water dispersion which
finally may result in enhancing PDT efficiency. The hypothesis of this study proposes an
enhanced PDT efficiency through the delivery of synthesized afterglow nanoparticles (AG
NPs), which may be excited by both X-ray and UV and emit blue light for a long time,
even after removing the energy source; Folic acid-PpIX-conjugated NPs could also
improve the water dispersion of PpIX. Afterglow alkaline earth silicates (Sr3MgSi2O8)
nanoparticles doped with rear earth elements (Eu, Dy) were synthesized through this
study; the best parameters to achieve a successful synthesis were investigated. the
silanol groups oriented outside of the AG NPs caused a net negative surface charge (-
38.52 mV). Alkaline wet grinding decreased the NP size from 809 ± 40.9 nm to 399.5 ±
v
117.5 nm. The surface silanization of synthesized AG NPs was induced to introduce an
NH2 functional group on the surface of AG NPs for further drug and FA conjugation. After
APTES coating, the surface charge changed to -4.28 mV because NH2 oriented out of the
NPs surface. Adding a new layer caused size increments to 458 ± 136.8 nm. Calculations
of Conjugation efficiency (CE) proved that 100 µg/ml of APTES-coated AG NPs was
containing of 43.043 ± 6.42 µg/ml of APTES. Protonated PpIX dicholoride with high
reactive COOH groups were successfully conjugated to the surface of APTES-AG NPs
which led to better water dispersion of PpIX-AG NPs and caused 20 times enhancement
of the red emission intensity of PpIX- AG NPs for the concentration equal to 6.25 µg/ml of
free PpIX compared to the same concentration of PpIX in water; in addition, 4 times
concentration decrement of drug was observed to get most intense red emission. The
results of the spectrofluorophotometer confirmed that FRET had happened between
APTES-AG NPs and PpIX which corresponds to the successful conjugation of PpIX and
NPs. The size decreased to 232 ± 1.3 nm. Raman spectroscopy results confirmed not
only PpIX but also FA were successfully conjugated to APTES-AG NPs. Ultimate NPs
(FA-PpIX-APTES-AG NPs) could improve the generation of singlet oxygen 2.4% more
than free PpIX for concentration of 1.5 µg/ml of free PpIX. Conjugation efficiency (CE)
calculation showed that in 100 µg/ml PpIX-APTES-AG NPs, there was a 2.050±0.207
µg/ml of conjugated PpIX and 100 µg/ml PpIX-APTES-AG NPs was containing
26.87±2.998 µg/ml of FA. Exposed PC3 cells to ultimate NPs (equal to 5µg/ml of free
PpIX) demonstrated 30% less dark toxicity and almost 15% more toxicity after exposure
to UV for 5 min compared to that of free PpIX. All mentioned results proved that the
fabrication of FA-PpIX-conjugated AG NPs may introduce an acceptable solution to
current challenges of PDT including weak penetration of blue light and low water
dispersion of PpIX in water.
vi
Table of Contents
Acknowledgements ............................................................................................................. iii
Abstract .............................................................................................................................. iv
List of Illustrations .............................................................................................................. xi
List of Tables ..................................................................................................................... xv
Chapter 1 Introduction ......................................................................................................... 1
1.1 Cancer ...................................................................................................................... 1
1.1.1 Treatment Types and Related Side Effects ...................................................... 1
1.2 Photodynamic Therapy (PDT) .................................................................................. 6
1.2.1 History of PDT ................................................................................................... 7
1.2.2 Mechanism of Action ......................................................................................... 8
1.2.3 The Effect of PDT on Tumors ............................................................................ 9
1.2.4 Photochemical Internalization ......................................................................... 14
1.2.5 Photosensitizer Drugs (PSs) ........................................................................... 15
1.2.6 Preferred PSs for the Current Study ............................................................... 20
1.3 Afterglow Nanoparticles (AG NPs) ......................................................................... 21
1.3.1 History of AG NPs ........................................................................................... 23
1.3.2 Afterglow Mechanism ...................................................................................... 24
1.3.3 Afterglow Host and Doped Materials ............................................................... 27
1.3.4 Methods of Afterglow Synthesis ...................................................................... 27
1.3.5 Preferred Host and Doped Materials for the Current Study ............................ 28
1.4 Objective of the Study ............................................................................................ 30
1.4.1 Background and Significance .......................................................................... 32
vii
1.4.2 Innovation ........................................................................................................ 35
Chapter 2 Materials and Methods ..................................................................................... 37
2.1 Aim I: Synthesis of Afterglow Nanoparticles (AG NPs) .......................................... 37
2.1.1. Synthesis of Sr2MgSi2O7: Eu2+
, Dy3+
by Solid State Reaction ....................... 37
2.1.2. Synthesis of Sr3MgSi2O8: Eu2+
, Dy3+
by Modified Sol-Gel Method ................ 37
2.1.3. Nanoparticles Characterization ...................................................................... 38
2.1.4. Luminescent and Afterglow (AG) Properties .................................................. 38
2.1.5 Affecting Parameters on the Luminescent and AG Properties ....................... 39
2.1.6 Improving the Size and Water Dispersion of NPs ........................................... 40
2.1.7 Stability of Eu2+
in Solution .............................................................................. 40
2.1.8 Surface Silanization of AG NPs to Prepare APTES-AG NPs ......................... 40
2.1.9 In Vitro Cell Study ............................................................................................ 42
2.2 Aim II: Surface Modification of PpIX ....................................................................... 42
2.2.1 Preparation of PpIX Dichloride ........................................................................ 43
2.2.2 Fabrication of APTES-Capped PpIX (Modified PpIX) ..................................... 43
2.2.3 Folic Acid Conjugated MPpIX (FA-MPpIX) ..................................................... 44
2.2.4 Characterization .............................................................................................. 45
2.2.5 Luminescent Properties ................................................................................... 46
2.2.6 Solubility and Stability ..................................................................................... 46
2.2.7 Singlet Oxygen Generation ............................................................................. 46
2.2.8 In Vitro Cell Study ............................................................................................ 47
2.3 Aim III: Conjugation of PpIX and FA to APTES-AG NPs and In Vitro
UV Treatment ............................................................................................................... 47
2.3.1 PpIX Conjugated APTES-AG NPs (PpIX-AG NPs) ......................................... 48
2.3.2 FA Conjugated APTES-AG NPs (FA-PpIX-AG NPs) ...................................... 49
viii
2.3.3 Characterization .............................................................................................. 50
2.3.4 Luminescence Property ................................................................................... 51
2.3.5 Stability of Ultimate NPs in Water ................................................................... 52
2.3.6 Detection of Singlet Oxygen Generation ......................................................... 52
2.3.7 In Vitro Cell Study ............................................................................................ 52
2.3.8 In Vitro Cancer Destruction (In Vitro PDT) ...................................................... 53
2.3.9 Statistical Analyses.......................................................................................... 53
Chapter 3 Results and Discussion, Aim I: Synthesis of Afterglow
Nanoparticles (AG NPs) .................................................................................................... 54
3.1 Luminescent Properties .......................................................................................... 54
3.1.1 of Sr2MgSi2O7: Eu2+
, Dy3+
Powder .................................................................. 54
3.1.2 of Sr3MgSi2O8: Eu2+
, Dy3+
Powder .................................................................. 56
3.1.3 of Sr3MgSi2O8: Eu2+
, Dy3+
Solution .................................................................. 59
3.2 Affecting Parameters on the Luminescent and AG Properties ............................... 60
3.2.1 The Effect of Temperature and pH .................................................................. 60
3.2.2 The Effect of Ratio of Eu/DY ........................................................................... 64
3.2.3 The Effect of Temperature of Calcination and the Duration of
Calcination ................................................................................................................ 65
3.3 Improving the Size and Water Dispersion of NPs .................................................. 67
3.3.1 The Effect of MgO Adding on Size and Water Dispersion of NPs .................. 67
3.3.2 The Effect of MgO Adding on X-ray Excited Optical Luminescence
(XEOL) ...................................................................................................................... 68
3.3.3 The Effect of APTES Coating on Afterglow and Water Dispersion ................. 69
3.4 Stability of Eu2+
in Solution ..................................................................................... 70
3.5 Characterization...................................................................................................... 71
ix
3.5.1 XRD Patterns ................................................................................................... 71
3.5.2 Raman ............................................................................................................. 72
3.5.3 Surface Charge ............................................................................................... 73
3.5.4 Conjugation Efficiency (CE) of APTES on the NPs Surface ........................... 73
3.6 In Vitro Cell Study ................................................................................................... 74
3.6.1 Cell Viability of PNT1A Cells Exposed to AG NPs .......................................... 74
.3.6.2 Cell Imaging, Nanoparticle Uptake by Cancer Cells ...................................... 74
Chapter 4 Results and Discussion, Aim II: Surface Modification of PpIX ......................... 76
4.1 Folic Acid Conjugated Modified PpIX (FA-MPpIX) ................................................. 76
4.1.1 Characterization .............................................................................................. 76
4.1.2 Conjugation Efficiency (CE) of APTES on the PpIX Surface .......................... 76
4.1.3 Conjugation Efficiency (CE) of FA on the Surface of MPpIX .......................... 77
4.2 Luminescent properties of MPpIX .......................................................................... 77
4.3 Solubility and Stability of MPpIX ............................................................................. 79
3.2.4 Detection of Singlet Oxygen Generation ......................................................... 79
4.5 In vitro Cell study .................................................................................................... 80
4.5.1 Cell Viability of PNT1A (Normal Prostate Epithelium) Exposed to
PpIX MPpIX .............................................................................................................. 80
4.5.2 Cell Imaging, Intensity Enhancement of FA-MPpIX Compared to
MPpIX ....................................................................................................................... 81
Chapter 5 Results and Discussion, Aim III: Conjugation of PpIX and FA to
APTES-AG NPs and In Vitro UV Treatment ..................................................................... 83
5.1 Characterization...................................................................................................... 83
5.1.1 Size and Surface Charge ................................................................................ 83
5.1.2 SEM and TEM ................................................................................................. 83
x
5.1.3 Raman Spectroscopy ...................................................................................... 84
5.1.4 Conjugation Efficiency (CE) of PpIX and FA on the Surface of
APTES-AG NPs ........................................................................................................ 85
5.2 Luminescence Property .......................................................................................... 86
5.2.1 Enhancement of Luminescent Intensity .......................................................... 86
5.2.2 FRET between APTES-AG NPs and PpIX ..................................................... 88
5.3 Stability of Ultimate NPs in Water ........................................................................... 89
5.4 Detection of Singlet Oxygen Generation ................................................................ 89
5.5 In Vitro Cell Study ................................................................................................... 90
5.5.1 Cell Viability of PNT1A Cells Exposed to PpIX-AG NPs and FA-
PpIX-AG NPs ............................................................................................................ 90
5.5.2 Cell Imaging and Intensity Enhancement of FA-PpIX-AG NPs
Compared to PpIX-AG NPs and Free PpIX ............................................................. 91
5.5.3 In Vitro Cancer Destruction (UV Treatment) ................................................... 92
Chapter 6 Conclusion and Future Work ............................................................................ 95
1.6 Conclusion .............................................................................................................. 95
6.2 Future Works ........................................................................................................ 103
References ...................................................................................................................... 104
Biographical Information ................................................................................................. 122
xi
List of Illustrations
Figure 1-1 Type I and type II reaction in photodynamic therapy (PDT) .............................. 8
Figure 1-2 The anti-tumor immunity response trigger by PDT .......................................... 14
Figure 1-3 Molecular structure of Photofrin....................................................................... 17
Figure 1-4 Molecular structure for ALA ............................................................................. 18
Figure 1-5 Molecular structure for Foscan ........................................................................ 19
Figure 1-6 Chemical structure of PpIX .............................................................................. 21
Figure 1-7 Persistent luminescence mechanism proposed by Aitasalo et al. for
CaAl2O4:Eu2+
,Dy3+
[72]. ................................................................................................... 26
Figure 1-8 Schematic illustration of nanoparticle–porphyrin conjugates for X-ray induced
PDT for cancer treatment .................................................................................................. 31
Figure 2-1 Synthesis of Sr3MgSi2O8: Eu2+
, Dy3+
by modified sol-gel method ................... 38
Figure 2-2 Surface Silanization of AG NPs by the help of APTES ................................... 41
Figure 2-3 Modified PpIX (MPpIX) fabricated by a two-step chemical process (OC: Oxalyl
Chloride) ............................................................................................................................ 44
Figure 2-4 FA conjugation to the surface of MPpIX (R can be both CH3 or siloxane) ...... 45
Figure 2-5 Conjugation of PpIX to AG NPs ....................................................................... 49
Figure 2-6 Conjugation of PpIX-AG NPs to FA ................................................................. 50
Figure 3-1 Photoluminescent excitation (PLE) and Photoluminescent emission (PL) of
Sr2MgSi2O7: Eu2+
, Dy3+
powder (solid state reaction) measured by
Spectrofluorophotometer................................................................................................... 54
Figure 3-2 XEOL from Sr2MgSi2O7: Eu2+
, Dy3+
powder .................................................... 55
Figure 3-3 Luminescent decay after 5min X-ray irradiation of Sr2MgSi2O7: Eu2+
, Dy3+
powder .............................................................................................................................. 56
xii
Figure 3-4 PLE and PL of Sr3MgSi2O8: Eu2+
, Dy3+
powder (modified sol-gel method) in
acidic environment (pH=2) measured by Spectrofluorophotometer ................................. 57
Figure 3-5 PLE and PL of Sr3MgSi2O8: Eu2+
, Dy3+
powder (modified sol-gel method) in
basic environment (pH=10) measured by Spectrofluorophotometer ................................ 57
Figure 3-6 XEOL from Sr3MgSi2O8: Eu2+
, Dy3+
powder (modified sol-gel method) .......... 58
Figure 3-7 Luminescent decay after 5min X-ray irradiation of Sr3MgSi2O8: Eu2+
, Dy3+
powder (modified sol-gel method) ..................................................................................... 59
Figure 3-8 Luminescent decay after 5min X-ray irradiation of Sr3MgSi2O8: Eu2+
, Dy3+
solution in water and ethanol (modified sol-gel method) .................................................. 60
Figure 3-9 Three different reaction during sol-gel process [106] ...................................... 60
Figure 3-10 SEM of Sr3MgSi2O8: Eu2+
, Dy3+
synthesized by modified sol-gel method ..... 62
Figure 3-11 The effect of pH on XEOL and afterglow from Sr3MgSi2O8: Eu2+
, Dy3+
powder
(modified sol-gel method).................................................................................................. 63
Figure 3-12 The effect of temperature on XEOL and afterglow from Sr3MgSi2O8: Eu2+
,
Dy3+
powder (modified sol-gel method) ............................................................................ 64
Figure 3-13 The effect of ratio of Eu/DY on XEOL and afterglow from Sr3MgSi2O8: Eu2+
,
Dy3+
powder (modified sol-gel method) ............................................................................ 65
Figure 3-14 The effect of temperature of calcination on XEOL and afterglow from
Sr3MgSi2O8: Eu2+
, Dy3+
powder (modified sol-gel method) ............................................... 66
Figure 3-15 The effect of the duration of calcination on XEOL and afterglow from
Sr3MgSi2O8: Eu2+
, Dy3+
powder (modified sol-gel method) ............................................... 67
Figure 3-16 The effect of MgO adding and Alkaline wet Grinding on water dispersion (A)
and Size of NPs (B): before adding MgO (C ): after adding MgO, (D) after adding MgO
and alkaline treatment ....................................................................................................... 68
xiii
Figure 3-17 XRD patterns of of Sr3MgSi2O8: Eu2+
, Dy3+
powder (modified sol-gel method)
before and after MgO addition .......................................................................................... 69
Figure 3-18 XEOL (A) and luminescent decay (B) from Sr3MgSi2O8: Eu2+
, Dy3+
powder
(modified sol-gel method) before and after MgO addition ................................................ 69
Figure 3-19 Luminescent decay after 5min photo luminescent irradiation (A) and Water
dispersion (B) of Sr3MgSi2O8: Eu2+
, Dy3+
powder (modified sol-gel method) before and
after MgO addition ............................................................................................................. 70
Figure 3-20 XEOL from AG NPs in water at different times(modified sol-gel method) (A)
and water dispersion of AG NPs in solution (modified sol-gel method) (B) ...................... 71
Figure 3-21 Raman spectra of AG samples : Sr2MgSi2O7: Eu2+
, Dy3+
by solid state
reaction with Eu/Dy=1/3(1), Sr3MgSi2O8: Eu2+
, Dy3+
NPs by sol gel metode with
Eu/Dy=1/3 at RT, pH=4 (3), at 600 C, pH=2.5 (4), at 80
0 C, pH=3.5 (5) and with
Eu/Dy=1/4 at 800 C, pH=4 (2). .......................................................................................... 72
Figure 3-22 Cell viability of PNT1A exposed to AG NPs for 24 hrs tested by MTT assay 74
Figure 3-23 Combined bright field microscopy and stained nucleus (A) and Uptake of AG
NPs by PC3 cancer cells (B). AG NPs (B) ........................................................................ 75
Figure 4-1 Raman Spectroscopy of PpIX, FA, and FA-MPpIX ......................................... 76
Figure 4-2 UV-visible absorption spectroscopy of FA-MPpIX and FA .............................. 77
Figure 4-3 Photoluminescence intensity of MPpIX (A) and PpIX (B) measured by
Spectrofluorophotometer................................................................................................... 78
Figure 4-4 Enhancement of photoluminescence intensity of MPpIX compared to PpIX .. 79
Figure 4-5 Improvement of water dispersion of MPpIX in watercomared to PpIX in Water
.......................................................................................................................................... 79
Figure 4-6 Singlet oxygen generation of PpIX and MPpIX ............................................... 80
xiv
Figure 4-7 Cell viability of PNT1A exposed to PpIX, and MPpIX for 24 hrs tested by MTT
.......................................................................................................................................... 81
Figure 4-8 Cell images of PC3 exposed to PpIX (A), and FA-MPpIX (B) taken by
fluorescent microscopy, Ex=405 nm, Em=420, 670 nm. Nuclei was stained with DAPI .. 82
Figure 5-1 SEM (A) and TEM (B) images of FA-PpIX-AG NPs ........................................ 84
Figure 5-2 Raman spectroscopy of FA-PpIX-AG NPs ...................................................... 85
Figure 5-3 Absorbance of AG NPs and conjugated compounds by UV-Vis ..................... 86
Figure 5-4 Photoluminescence intensity of PpIX-APTES-AG NPs measured by
Spectrofluorophotometry ................................................................................................... 87
Figure 5-5 Enhancement of photoluminescence intensity of PpIX-APTES-AG NPs
compared to PpIX ............................................................................................................. 87
Figure 5-6 PL of PpIX, APTES-AG NPs, and PpIX-APTES-AG NPs ............................... 88
Figure 5-7 Happened FRET Photoluminescence intensity of PpIX and AG NPs
(measured by Spectrofluorophotometer, Ex=400 nm) ...................................................... 88
Figure 5-8 Improvement of water dispersion at different concentrations (A) and PL (B) of
ultimate NPs ...................................................................................................................... 89
Figure 5-9 Singlet oxygen measurement of PpIX, PpIX-APTES-AG NPs, and FA-PpIX-
APTES-AG NPs (1.5 µg/ml water as a concentration of free drug) .................................. 90
Figure 5-10 Cell viability of PNT1A exposed to PpIX and its conjugated products by MTT
assay ................................................................................................................................. 91
Figure 5-11 Cell images of PC3 exposed to PpIX (A) and FA-PPIX-Ag NPs (B) taken by
fluorescent microscopy, Ex=405 nm, Em=420, 670 nm. Nuclei was stained with DAPI .. 92
Figure 5-12 In vitro UV treatment of exposed PC3 cells to PpIX, NPs, and FA-PpIX-AG
NPs .................................................................................................................................... 93
xv
List of Tables
Table 1-1 Summery of history of PDT ................................................................................. 7
Table 1-2 Subcellular effects of PS drug based on its accumulation site ......................... 21
Table 1-3 Classification of nanoparticles used for photodynamic therapy[71] ................. 23
Table 3-1 DLS results after and before alkaline wet grinding and APTES coating .......... 73
Table 5-1 DLS results after conjugation of PpIX and FA to APTES-AG NPs ................... 83
1
Chapter 1
Introduction
1.1 Cancer
Cancer is a term which is used for diseases arising from abnormal cells which are able
to not only divide without control but also to attack other tissues and organs. Metastasis is a
term used for a condition which the blood and lymph systems provide cancer cells the
opportunity to invade other parts of the body. The starting point of all cancers is cells and the
fact that how normal cells become cancer cells may guide researchers to figure out what is
cancer. Inside the body, there are very accurate and controllable monitoring pathways to govern
the growth and division of normal and healthy cells. The basis of the control is the need of body
to make more new normal cells to maintain the normal and healthy condition of the body. On
the other hand, those cells which are old or damaged must die and be replaced with new cells
while under controllable monitoring. However, any possible change or damage to the genetic
material (DNA) may results in mutations. Mutations may convert normal cell growth to abnormal
growth of cells which means the old cells do not die when they must, and the new ones are not
going to form when the body is in need of them. These extra cells may produce an unwanted
mass of tissue called a tumor. As a matter of fact, DNA mutation is able to disturb the available
balance between cell growth and cell death. Not only the uncontrolled cell growth but also the
lack of the ability to execute cell suicide, called apoptosis, is responsible for balance disruption
[1,2].
Cancer has been reported as the second leading cause of death in the United States.
Cancer Statistics has estimated a total of 1,529,560, and 1,638,910 new cancer cases and
569,490, 577,190 deaths from cancer in the United States in 2010 and 2012, respectively [3].
1.1.1 Treatment Types and Related Side Effects
There are currently several treatments to fight, kill, and damage cancer cells which
have been listed. Each treatment may result in its own side effects which have been mentioned.
2
1.1.1.1 Surgery
Surgery can play a great role in treatment of cancers which has not attacked other parts
of the body. In some kind of cancer, such as breast cancer, surgery is an essential part of
treatments. Although surgery is one of the standard treatmen,t it suffers from some serious
adverse effects. The surgeon’s experience in addition to the type of surgery is important
parameters to have a successful surgery. In addition, physical health condition of patients
undergone the surgery is also very important [4].
Possible risks during the surgery are arising from the respective diseases,, anesthesia
drugs, as well as surgery itself. The rate of risks is dependent to the operation complication. For
example, risks of biopsies (tissue samples) are not comparable to major surgery. Incision
inducing pain, incision infections, and local anesthesia inducing reactions are the most common
issues regarding the surgery [4, 5]. Some other serious side effects which are possible but not
common can be:
Bleeding during surgery that may result in blood transfusions. Blood
transfusions can cause problematic risks. This issue becomes serious when loss of blood is
unavoidable during some complicated operations. Sometimes autologous transfusion may be
offered as a solution [4, 6].
Allergy to anesthesia or other medicines can cause serious problems during
surgery such as low blood pressures [4, 7].
Damage to internal organs and blood vessels can be life threatening [4, 8].
Infection-induced incisions can happen after surgery,whileantibiotics are being
used to control and treat most infections [4, 9].
Problems with other organs during the operation can threat patients’ lives.
Although the rate of this adverse effect is not high but it can bring dangerous situation to those
patient who suffer from previous issue regarding their internal organs [4].
3
1.1.1.2 Chemotherapy
Chemotherapy focuses on killing or damaging cancer cells by the help of anti-cancer
drugs which target fast-growing cancer cells. Chemotherapy has great potential to destroy
certain normal, healthy cells which also grow quickly. The fast-growing normal cells affected by
anti-cancer treatments are blood-forming cells in the bone marrow, as well as cells in the
digestive track, reproductive system, and hair follicles. Side effects arisen from damage to these
cells are anemia, fatigue, infections, mouth sores, diarrhea, other digestive system problems,
and hair loss. Regarding long-term disadvantages, another form of cancer of white blood cells
such as leukemia or hodgkin's and non-hodgkin's lymphoma is threatening patients’ lives. Of
these concerns, damages to vital organs such as liver, kidneys, heart, nervous system and
brain are the other disturbing problems regarding the use of many anti-cancer treatments. In
addition, chemotherapy can damage sperm cells which would cause an increase in risk of
producing genetically defective babies [10, 11].
1.1.1.3 Radiation Therapy
Radiation therapy destroys or damages cancer cells by the help of high-energy particles
or waves. Radiation therapy itself, along with another treatment, is very common in North
America; more than half of all patients suffering from cancer have experienced radiation
therapy.
The serious issue related to radiation therapy is the energy of radiation which is needed
to damage DNA. A photon energies of several million electron-volts from outside the body is
needed to not to deposite in superficial structures and make it possible to penetrate enough
deep to reach cancer site. In addition to the cancer, this high dosage has potential to damage
DNA of surrounding healthy tissues also. Damage to DNA caused by radiation therapy may
result in secondary diseases and cancer because damaged DNA is responsible for the scenario
of loss of normal growth control [11-13].
4
1.1.1.4 Targeted Therapy
To identify and attack cancer cells more accurately, targeted therapy, which is a newer
type of cancer treatment, has been introduced for clinical approaches. It is believed that
targeted therapy does not damage healthy normal cells, and it has become a promising cancer
treatment. In targeted therapy, to shut down the cancer cell growing and dividing, the epidermal
growth factor receptor (EGFR) protein is targeted by the drug. This targeted drug is followed by
some skin problem because EGFR are abundant in normal skin, as well. They block the signal
monitoring the normal growth of skin cells. Some other drugs focus on limiting and blocking
blood supply containing nutrients and oxygen for cancer cell survival. To do this they target
vascular endothelial growth factor (VEGF). Unfortunately, VEGF located in tiny blood vessels
are targeted as well to cause Hand-foot syndrome (HFS). It is believed that leakage of drugs out
of the blood vessels may cause tissue damage too [14].
1.1.1.5 Immunotherapy
Immunotherapy focuses on fighting cancer cells with the help of the patients’ body
immune cells. Immunotherapy has several types which have been listed [4, 15].
Monoclonal antibodies: are man-made versions of immune system proteins.
Antibodies are very efficient weapon because based on their design very specific part of cancer
cells can be attacked. Side effects of this version are fever, chills, fatigue, headache, nausea,
vomiting, diarrhea, low blood pressure, and rashes
Cancer vaccines: to wake up the immune responses to fight diseases, vaccines
are designed. The most important roll of vaccines is to help a healthy body prevent diseases.
Interestingly, some vaccines help prevent or treat cancer. Cancer vaccines may cause some
adverse effects such as problems breathing and high blood pressure.
Non-specific immunotherapies: Helps the immune system awareness not very
specifically and able to fight cancer cells. Possible side effects can be abnormal heartbeat,
chest pain, flu-like symptoms (chills, fever, headache, fatigue, loss of appetite, nausea,
5
vomiting), low white blood cell counts (which increase the risk of infection), skin rashes, and
thinning hair.
1.1.1.6 Hyperthermia
This is an idea which focuses on using of heat to treat cancer. Although the early
results were not very promising, now improved technology provides this technique with more
accurate delivery of heat to increase its efficiency. The chosen technique for treatment as well
as the treated part of the body can monitor side effects of hyperthermia. Local treatment by
hyperthermia may result in damage to the skin, muscles, and nerves located near the treated
part. The other side effects can be listed as pain at the site, bleeding, infection, blood clots, and
burns. Vomiting, nausea, and diarrhea can be followed after exposure of whole body to
hyperthermia. Some serious problems including the heart, blood vessels, and other major
organs might be posed. One important challenge related to this technology is to control an exact
temperature range inside a tumor, which would then have no way to measure and monitor it. On
the other hand, maintaining the neighboring tissues to stay unaffected by the applied
temperature is very challenging. An accurate design of technology is needed because different
parts of the body have different sensitivity to heat. For example, the brain is very sensitive to
heat [4, 16].
1.1.1.7 Stem Cell Transplant
Stem cell (blood-forming stem cells) transplants focus on cancer treatment in the
condition which bone marrow has been destroyed by a disease. Bone marrow is the main first
home of stem cells. In bone marrow, stem cells divide to make new blood cells. This technique
may help restore the stem cells of patients’ body. When cells are mature, they enter the
bloodstream. Typically this treatment is used along with another treatment such as radiotherapy
or chemotherapy to get better efficiency. Stem cell transplants include different types which
depend on the source of the stem cells. These types are bone marrow transplant, a peripheral
blood stem cell transplant, or a cord blood transplant. Short term problems are listed as
6
bleeding and transfusions, infection, graft failure, graft-versus-host disease, and hepatic veno-
occlusive disease. Long term issues are relapse, organ damage (to the liver, kidneys, lungs,
heart and/or bones and joints), secondary cancers, infertility, abnormal growth of lymph tissues,
Hormone ( thyroid or pituitary gland) changes, and cataracts [4, 17].
1.1.1.8 Photodynamic Therapy
Photodynamic therapy, or PDT, is a combination of non-toxic drugs (photosensitizing)
and light and oxygen to generate the toxic singlet oxygen or free radical to damage or destroy
cancer cells. The drugs need to be activated by certain wavelengths of light to transfer energy
to oxygen. This modality has showed less side effects compared to other cancer treatments. All
of the adverse effects have been limited to photosensitivity reaction and swelling in the treated
area. Pain or trouble swallowing or breathing are followed by the swelling. But PDT suffers from
some challenging issues. So far PDT has had applications for cancer located near the skin or in
the lining of internal organs which light may penetrate and reach the diseased area. This
drawback of PDT can limit it to only the given area exposed to light and not for areas spread to
by metastasis,. Patients with certain blood diseases such as acute intermittent porphyria and
those who has allergy to porphyrins (drug) are not allowed to use PDT [18].
1.2 Photodynamic Therapy (PDT)
In PDT cancer destruction relies on applying a photosensitizing (PS) drug followed by
light. In fact, light activates the drug to transfer energy or electron to oxygen to generate
reactive oxygen species (ROS). ROS will react with vital biomolecules immediately to damage
cell organelles which would then result in cell death. PDT efficiency is dependent on a
successful combination of photosensitizing drugs, light energy, and oxygen [19]. PDT benefit
from several advantages over the conventional treatments, for example it shows high tumor
selectivity, low systemic toxicity, low possibility of secondary effects, and the possibility of
inducing the repeatable cycles of treatments and combination with other therapies such as
radiotherapy and chemotherapy [20].
7
1.2.1 History of PDT
First clinically application of PDT was limited to superficial conditions, such as skin
cancer and lupus vulgaris. In 1907 Von Tappeiner and Jodlbauer reported the first clinical
application of PDT. It was more than 100 years ago when scientists noticed and informed about
the toxicity of combined light and some specific drugs which may result in cell death. Firstly
Oscar Raab (German medical student) in 1900 observed and reported the possible toxicity and
lethality of combinations of certain wavelengths of light and acridine to treat infusoria including
Paramecium species. Probably Niels Finsen from Denmark took the beginning steps of light
therapy. When at the end of the nineteenth century, he observed that red-light exposure is able
to treat smallpox pustules. Indeed, he applied ultraviolet light (UV) of the sun to treat cutaneous
tuberculosis [21-22]. His effort to develop phototherapy was merited to win a Nobel Prize in
1903.
Table 1-1 shows the summary of PDT history and important steps taken toward
successful clinical application of this modality.
Table 1-1 Summery of history of PDT
Scientists’ name Used drug/light Cancer type Year
Oscar Raab acridine infusoria including
Paramecium species 1900
Niels Finsen red-light , ultraviolet light
(UV) smallpox pustules
Nobel Prize in 1903.
Herman Von Tappeiner and A.
Jesionek eosin and white light skin tumours 1903
W. Hausmann haematoporphyrin paramecium and red blood
cells 1911
Friedrich Meyer–Betz
Porphyrins (haematoporphyrin)
Skin 1913
Samuel Schwartz Synthesis of
haematoporphyrin derivative (HDP)
1955
Richard Lipson HDP and fluorescence tumor 1960s
Diamond haematoporphyrin Gliomas 1972
Thomas Dougherty HPD and red light mammary tumour growth 1975
Dougherty HPD skin tumours (first
controlled clinical study in humans)
1978
8
1.2.2 Mechanism of Action
One advantage of PDT is the possibility of regulating the obtained effects by
biodistribution changes as well as the timing of light exposure. A photosensitizer (PS) can be
delivered through an I.V. or by topical application to the skin. Since biodistribution is not stable
over time, the time of the light application may govern the effects of PDT. Absorbed light
(photons), can transform the PS state from its ground state (singlet state) into excited state
(triplet state). The excited triplet is followed by two different kinds of reactions. As shown in
Figure 1-1, firstly in a type I reaction, the direct reaction of the excited triplet with available
molecules or substrate (cell membrane) transfers an electron to form radicals which is followed
by the next step including oxygenated products. Secondly in type II reaction, direct energy
transfer from the triplet state of PS to a molecule of oxygen can generate singlet oxygen, which
is a highly toxic to cells. Both reactions happen simultaneously, and parametes affecting the
ratio of type I/ type II are the types of PS, the availability and concentrations of existing
substrate and oxygen at the site of treatment, as well as the PS binding affinity to molecule or
cell membranes [23-24].
Figure 1-1 Type I and type II reaction in photodynamic therapy (PDT)
It is believed that singlet oxygen cannot damage cells far from the area that is exposed
with light due to its high reactivity, short half-life (< 0.04 μs), and short distance diffusion (<0.02
μm) [25].
Some important parameters affecting and governing the efficiency of PDT are the type
of PS, the availability of oxygen, the dosage of PS and exposed light, its extracellular and
9
subcellular localization, and the interval time between the PS administration and light exposure.
[21].
1.2.3 The Effect of PDT on Tumors
Three main mechanisms have been recognized by which PDT induces its photo-
damage and toxicity to cancer cells. The first event arises from the direct-killing effect of
produced ROS at the site of the PS and light exposure. PDT also blocks cancer cells supply by
damaging its vasculature which would finally cause destruction. The last PDT-induced scenario
is awareness of an immune response to fight tumor cells. Although each mentioned mechanism
play important role in cell damage, a combinationof all events can execute a long term and
effective treatment.[21, 26].
The PDT-induced effect on tumor is an accumulation ofresults of both the direct-killing
effect and its effect on tumor stroma. The stroma includes the extracellular matrix, vasculature,
cellular components such as fibroblasts, endothelial cells (EC).The cells in the immune system
have close interactions with tumor cells. Research and studies have proven that the interruption
of stroma-cancer cell interaction is essential step for an efficient PDT [20, 26].
1.2.3.1 Tumor-Cell Killing Effects
As it was mentioned, the excited PS may result in two different types of reactions. Free
radical products from type 1 are very reactive. These free radicals are able to react not only with
oxygen molecule to produce reactive oxygen species (ROS) but also with available molecules
and substrates. Type 1 reactions end up with oxidative damage, which is capable to induce
biological damages. In a Type 2 reaction, energy transfer from an excited triplet state of the PS
and the ground-state of molecular oxygen causes singlet oxygen generation which possesses
very high reactivity to execute PDT cell killing effects. Although it is believed that the type 2
reaction plays a major role in PDT, with regards to oxygen dependency of type 2, it is obvious
that type 1 has a great governing role in oxygen-deprived areas such as cancer zones. Cell
10
damage can occur through either necrosis or apoptosis. Intracellular accumulation of PS can
dictate which kind of damage to happen after light exposure [24].
In addition, PDT induces both cell- killing effect and interruption effect on extra cellular
matrix (ECM) and tumor cells interactions. In fact, PDT can alter the ECM and the other cellular
components to change tumor survival condition. Cell adhesion proteins cause the adherence of
cellular components to ECM to induce and promote their proliferation and migration. So far, it
has been known that dosage of PS and light plays an important role in interrupting ECM- cell
adhesion, but not many studies have been done to fully understand the mentioned mechanism
[20, 27].
The interaction of the PDT-generated singlet oxygen and the available amino acids of
proteins at the site form free radicals which react with available molecules to induce new cross-
links. This new cross-linking formation in the collagen matrix may inactive available growth
factors in matrix. On the other hands, the new cross-linking can compensate matrix degradation
by metalloproteinases (MMPs). With regard to the fact that MMPs-inducing degradation of ECM
is a key role in metastasis the new formed cross-linking can hinder cell migration followed by
invasion [28].
The reduction of adventitial fibroblast migration as well as the reduction of invasive
smooth muscle cells is events followed by PDT. The other outcome of PDT is cell detachment,
which arises from structural cells and tissues adaption to their environment. It has been shown
that the alteration of the vascular wall matrix followed by in vivo PDT may result in the
decreasing of pepsin digestion of treated arteries and blockage of cell migration [20, 29]. It
should be mentioned that adhesion decrements of fibroblasts are able to change their survival
as well as their function related to ECM generation. PDT causes the inactivation of resident
growth factors and interruption in the integrin connections which induce not only the reduction of
ECM components but also the alteration of fibroblast survival. Integrin interruption can also
affect the survival of all the other cells of the tumor environment including cancer cells. ECM
11
damage as well as integrin protein damage followed by PDT can interrupt the cell–substrate
adhesion. In summary, photo-damage of adhesion proteins anchoring EC and tumor cells
interrupts their cell adhesion and affects their survivability and functions [20, 26, 27, 29].
1.2.3.2 Vascular Damage
Cancer cell survival depends on the availability of the essential oxygen and nutrient
supplies which blood vessels provide. On the other hand, some signals and related proteins
function are needed to send essential messages for new blood formation or angiogenesis. With
regards to the key role of blood vessels in maintaining cancer cell survival, it is reasonable to
target tumor vasculature to fight cancer cells. In this design, either EC or growth factors
stimulating angiogenesis can be a target, by which the former is by direct targeting and the
latter is by indirect targeting [29-30]. Therefore, targeting the local vascular microenvironment of
tumors is one strategy to destroy cancer cells. PDT is known as an ideal approach for this goal
because produced ROS can damage EC followed by shutting down the angiogenesis to block
nutrients supplies of cancer cells. The damage to tumor vasculature is considered as a
necrosis-leading factor of PDT. The photodamage of the wall of tumor vessels in tumor
subendothelial areas is the first event after PDT. The tumor subendothelial area is composed of
connective tissue and collagen fibers [20, 29, 30].
Microvascular damage during PDT which can cause hypoxia has been reported several
times. In the past 15 years. In 1989, Barbara Henderson and her team revealed PDT results
confirming vascular photodamages in a fibrosarcoma mouse model which led to oxygen
deprivation for cancer cells [31, 32].
In vivo studies showed that the photodamage to EC of a tumor can activate platelets.
Activated platelets send mediators to stimulate vasoconstriction, which is the first immediate
response after PDT. Three hours post-PDT, as the next step, aggregated platelets can cause
thrombus formation. Mentioned effects finally may contribute to impair tumor growth [21].
12
1.2.3.3 Immune Response
Many in vitro and in vivo researches have studied the role of the immune system’s
awareness in tumor destruction by different cancer treatment types. But between all reports,
there are no total agreements in what is to be the dominant factor for stimulating an immune
response. Although some results demonstrated that apoptotic tumor cells are the leader in
inducing an immune response, others presented results confirming the effective role of necrotic
tumor cells in activating an immune response. When necrosis is dominant it is believed that
photo damage to the cell membrane can expose the constituents of cytoplasm into the
extracellular space. These exposed materials can activate and stimulate inflammatory
responses, while during apoptosis, these constituents never split out of cell plasma until they
are phagocytized by macrophages. The acute inflammation which has been initiated by
necrosis in PDT is followed by the attraction of the host’s leukocyte into the tumor
microenvironment and antigen present to activate and prepare for immunity [33]. PDT-inducing
infiltration of leukocytes, lymphocytes, and macrophages into the tumor environment were
confirmed by studies done during the late 1980s and early 1990s. One mentioned an advantage
of PDT is its ability to induce damage to tumor tissues rather than to normal tissues, which
arises from the differences in the inflammatory reaction between these the two types of tissues.
Studies have confirmed that some immunoregulators mediate inflammatory responses
during PDT. Some listed regulators are cytokines, chemo attractants of leukocyte, growth
factors, ROS, vasoactive substances, complement and clotting cascades components, and
acute phase proteins. Up-regulation of cytokines interleukin (IL)-6 and IL -1 is the other
inflammatory response induced by PDT. Wil de Vree’s study in 1996 revealed that neutrophil
accumulation activated by PDT was able to shut down a tumor. This evidence was confirmed by
the tumor-bearing mice whose neutrophils were depleted; PDT did not induce tumor growth
decrement in this animal study [20, 21, 33].
13
In vitro studies have reported that PDT plays an effective role on monocyte/
macrophage and lymphocyte cell lineages. One of PDT’s roles contains lymphocyte-killing
effects which is able to kill activated lymphocytes as well. This result has proposed PDT as a
potential treatment for autoimmune diseases such as graft versus host disease, and cutaneous
T-cell lymphoma. On the other hand, a low dosage of PDT is able to activate macrophages.
Tumor-necrosis factor-α (TNFα) is secreted by activated macrophages during PDT. PDT
induces a release of lysophosphatidyl choline from lymphocytes followed by β-galactosidase
expression in B lymphocytes. β-galactosidase along with NEU1 sialidase from T lymphocytes
regulate macrophage-activating factor (MAF) by vitamin D3 binding protein mediator. Animal
studies examining analogous vitamin D3 binding protein in mouse serum confirmed MAF
production, as well. Macrophages also induce cytotoxicity to cancer cells exposing a low
dosage of PDT. Another study reported NK cell function decreasing [20, 21, 26, 29, 33].
PDT events can result in a development of a function leading to anti-tumor immunity.
Following PDT, the lysate of killed cells send danger signals to induce an expression of HSPs
and factors such as NF-κB and AP-1, which are able to express cytokines as well as other
immunologically genes. In addition, the photo damage of cell membranes initiate arachidonic
acid metabolites which itself is inflammatory mediators to activate immunity responses. Indeed,
the vasculature damage of a tumor’s microenvironment may result in a release of histamine and
serotonin which is followed by complement activation as well as neutrophils and other
inflammatory cells to attack to cancer cells [20, 32, 34].
In vitro studies have confirmed that activation the complement C3 protein is a strong
weapon of PDT to destroy tumor cells. Another animal study has emphasized the role of
complement C3 in regulating anti-glioma responses in mice [35]. Complement activation can
induce the secondary inflammatory response by releasing cytokines IL-1β, TNF-α, IL-6, IL-10,
histamine and coagulation factors, thromboxane, and granulocyte colony-stimulating factor [20,
33, 36].
14
The lysate from cancer cells in PDT-treated cancer cells can send signals to activate
and maturate dendritic cells (DCs). Followed by the migration to lymph nodes of tumor drainage
where they induce T-cell activation. Incubation of immature DCs with PDT-treated cancer cells
developed DC maturation and activation of T-cells. Indeed, activated DCs secrete IL-1α, IL-1β,
and IL-6. Danger signals of lysate are damage-associated molecular patterns (DAMPs) which
interact with pattern recognition receptors (PRRs) of innate immune cells to stimulate immune
responses. Indeed, research has revealed that PDT initiates danger signals which are followed
by the activation of antigen presenting cells [20, 37]. Figure 1-2 summarizes the anti-tumor
immunity response trigger by PDT.
Figure 1-2 The anti-tumor immunity response trigger by PDT
1.2.4 Photochemical Internalization
One of the important advantages of PDT is the endosomal escape of drug or
nanocarrier which lets drugs reach their targets after internalization. Internalization of
15
hydrophilic macromolecular drugs or those which suffer from limited penetration ability through
cell membrane are regulated by endocytosis. But the fate of penetrated drugs into the cell is
being trapped in endosome and lysosomes to be degraded hydrolytically. Photochemical
internalization (PCI)-based PDT facilitates entrapped drugs to photo damage and rupture of
lysosome and endosome to let drug reach their targets inside the cell.
PS retention in tumor cells due to a lack of ferrochelatase enzyme in addition to a
controlled delivery of light can limit PCI to cancer cell [38-39]
1.2.5 Photosensitizer Drugs (PSs)
PDT employs two non-toxic components to generate very toxic molecules to attack
cancer cells. The first element of PDT is the PS drug which can be accumulated in cancer cells
and can also absorb the exposed light. The second component is light which is applied to active
drug to transfer its energy to molecules of oxygen to generate toxic ROS [21].
So many natural and synthetic dyes are available which can function as a PS for PDT.
To be effective PDT-based PS, drugs must meet two important characteristics. First, they must
accumulate selectively at the site of treatment; second, the drug should possess the ability to
generate toxic ROS during PDT [24]. Typically, PSs are classified as porphyrins or non-
porphyrins drugs [29].
Being categorized by chemical structure introduces PSs in three different groups:
Porphyrin family, Chlorophyll family and Dyes [40].
The porphyrins are known as the first generation of PSs. Porphyrins was developed
during the 1970s and the early 1980s. Derivatives of porphyrin synthesized at late 1980s
introduced the second generation of PSs. Third generation PSs took advantages from antibody
conjugates, and biologic conjugates. The first PSs have been based on hematoporphyrin (Hp)
and its derivatives. Commercial products of synthesized hematoporphyrin derivative (HpD) are
named Photofrin, Photocan, Photosan which are different in ratio of porphyrin monomers,
dimers, and oligomers. New formulated PSs can be synthesized by changing on the porphyrins
16
ring such as adding, subtracting or substituting. Photosynthesis is a natural chemical process
which uses the energy of light. Chlorines are Chlorophyll-based PS with essential property
which has introduced both modified and synthesized commercial drugs [26, 29, 40, 41].
1.2.5.1 Porphyrin Family
This family includes Hematoporphyrin derivative (HpD), 5-Aminolevulinic acid (ALA),
Benzoporphyrin derivative (BPD), and Texaphyrins. Here only more information regarding two
mentioned is briefly explained.
1.2.5.1.1 Hematoporphyrin Derivative (HpD): Figure 1-3 shows the molecular structure
for Photofrin which is the oldest known PS drug in PDT. Photofrin has FDA approval in the U.S
for some diseases such as endobronchial lesions as well as Barrett’s esophagus and
esophagealobstructing lesions [40, 42]. Photofrin has been approved worldwide for bladder
cancer treatment [40, 43]. Photofrin is clinically applied at a dosage of 2 mg/kg. After 48 hours,
the site of disease is exposed to light by circumferential illumination (diffusing fiber) or
unidirectional illumination (micro lens). Different levels of light dosage of 150 J/cm2 (lens) or
200—300 J/cm2 (diffuser) is applied for clinical approaches. The clinical results related to
Photofrin-based PDT have been satisfactory. Of controlled cutaneous lesions, basal cell, Kaposi
sarcoma, and squamous cell can be mentioned [40, 44]. Indeed, Barrett’s mucosa and
obstructing esophageal lesions as well as late endobronchial disease revealed response to PDT
[45]. The other successfully PDT-treated diseases are rectal and anal tumors [46] brain tumors
[47] head and neck neoplasms [48] as well as Bladder tumors [49]. Although Photofrin is pain-
free, activatable, reliable, relatively safe and non-toxic, it suffers from some drawbacks. For
example, a dosage of 2 mg/kg does not show high selectivity at the site of diseases and its long
photosensitivity in normal tissues can pose serious problems. Patients have to avoid sunlight for
at least 4 weeks because normal skin tissue accumulated PS show high reactivity to exposed
light which is followed by skin swelling. Normal tissue reactions under light illumination can be
life threatening, especially necrotic tissue slough occured in airways [40].
17
Figure 1-3 Molecular structure of Photofrin
With regards to the accumulation of Photofrin at the site of disease more than in
surrounding normal tissue, researchers started figuring out the appropriate dosage of drug per
kilogram which can concentrate enough in malignant tissue to induce PDT-inducing toxic effects
while low concentrated drug in normal tissue keep them from adverse effects. Scientists
reported successful PDT for chest wall recurrence exposed to 0.8 mg/kg photofrin with minimal
or no adverse effect in normal tissue while 2—3 mg/kg of the same drug had caused fibrosis
and normal tissue slough [50]. Exposure of other cutaneous lesions, lesions of the oral cavity,
and head and neck malignancies to 1.2 mg/kg drug revealed controlled tumor growth with no
fibrosis. But bronchial treatment and Barrett’s esophagus treatment with 2 mg/kg Photofrin was
followed by tissue slough and fibrosis, respectively [40].
1.2.5.1.2 5-Aminolevulinic Acid (ALA): This naturally occurring amino acid can be found
in heme synthesis pathway of human body and ferrochelatase enzyme converts it to
protoporphyrin. Figure 1-4 shows the molecular structure for ALA. By topical administration of
ALA, it can offer a treatment without adverse effects on normal tissue. However, its systemic
administration does not have specific selectivity for concentrating at the site of disease [40, 51].
A wavelength of 630 nm can activate ALA and light at this wavelength is able to penetrate more
deeper compared to blue light, but it should be considered in the case of deep lesions
treatment that topical applied drug dose not penetrate deeply itself. So, nodular lesion scan not
18
be fully treated by ALA-based PDT [26, 40]. Although ALA is a naturally occurring PS, modifying
ALA through side chains alteration to enhance its absorption or activity cannot be named
natural PS. As a drawback, ALA is not very active and high light dosage or long term therapy is
essential for successful PDT.
Figure 1-4 Molecular structure for ALA
ALA-based PDT causes. Clinically, 20% ALA is administrated topically and after 4 hrs.
150 J/cm2 dosage of illumination is applied to treat diseases. Skin squamous cell and basal
cell cancers have demonstrated successful PDT [52]. DUSA Pharmaceuticals design that could
gain FDA approval was excitation of ALA concentrated at actinic keratosis by blue light which
resulted in a successful PDT [53]. In the case of multiple sessions, 70% of patients with oral
cavity leukoplakia could be treated by topical administration of 10% ALA. In a clinical approach
for superficial bladder cancers, 17% ALA was applied intravenously. After several hours post
injection, 100 J/cm2 of white light was delivered by the help of light catheter. Treatment was
undergone for 1—2 h. results confirmed that almost half the patients were cured [40].
1.2.5.2 Chlorophyll Family
Of the Chlorin family are mono-L-aspartyl chlorin e6, Temoporfin, talaporfin Sodium,
Purlytin (tin-ethyl-etiopurpurin), and HPPH (Photochlor). The following briefly explain about
Foscan and talaporfin Sodium.
1.2.5.2.1 Foscan: Foscan has several advantages over available PSs which have
highlighted its clinical application [29,40]. Figure 1-5 shows the molecular structure for Foscan.
Foscan PS has been reported with an excellent clinical outcome when bringing to treatment of
several diseases such as pulmonary [54], GI [55], esophageal [56], cutaneous lesions [57], and
19
especially head and neck tumors [58]. I.V administrated PS at a dosage of 0.15 mg/kg has
reported successful PDT. A four day interval between the drug injection and light exposure is
needed, which can limit its therapy and dose not let its application for emergency case. Light at
660 nm can activate drugs, and treatment for tumors located in deeper tissue is expected. Use
of m-THPC drug for head and neck tumors could damage a large number of blood vessels
supplying the cancer cells by the help of light that can penetrate tissue. Since the PS drug
shows a strong ability of light conversion, a dosage of only 20 J/cm2 is enough, which allows
rapid treatment about several minutes. However, therapy is not pain free and patients have to
experience a significant amount of pain during treatment [24, 29, 40].
Figure 1-5 Molecular structure for Foscan
With regards to high efficient light converting of this drug, light scattering toward the
normal surrounding tissue can be dangerous. Although this PS can be very useful, due to lack
of the knowledge of its dosimetry, its application is limited.
1.2.5.2.2 Talaporfin Sodium (LS11): LS11 has several absorption bands at 400 and 664
nm. Also, it is a water-soluble PS. LS 11 can be excreted quickly (half-life is 9 h) through the
bile, so its application for patients suffering liver disease must be done with extra caution [26,
40]. Interestingly, Light Sciences has developed a promising device in conjunction with LS11
[59]. Their innovated design includes a small sized (palm sized) energy source which can
facilitate light to LEDs attached to a flexible fiber. Interstitially, implantation of the source of
energy can provide the site of disease to a longer light exposure through an outpatient Image
20
Guided technique. This novel design may change clinical approaches of PDT. In a Phase I of a
related study, firstly, the source of energy was implanted via CT guidance and then drug was
administrated intravenously at the dose of 40 mg/m2 (not kg). After one hour, PDT was applied
in different durations from 83 to 664 min (250—2000 J/cm, respectfully). CT scans were applied
to monitor responses. Overall, a minimal toxicity and no photosensitivity were noticed among
treated patients. Longer light exposure in patients led to a good response. No clinical results
related to Phase II of this study are available [24, 29, 40].
1.2.6 Preferred PSs for the Current Study
Protoporphyrin IX (PpIX) with the chemical structure shown at Figure 1-6 has FDA
approval and has been recognized as the preferable drug because it shows good accumulation
in tumor cells. This is due to the reaction deficiency of ferrochelatase enzyme in tumor cells,
compared to normal cells which are in charge of converting PpIX into heme. PpIX is a precursor
in heme synthesis pathway inside the mitochondria of cells. On the other hands, PpIX can be
excreted in less than 24 hrs and surplus PpIX may be excreted into the intestine [60-65].
The other advantage of PpIX over the other PSs is that PpIX has very strong absorption
near 400 nm wavelength which is called the Soret band. Absorption at the Soret band is 10
times stronger than absorption at Q- band (around 630 nm) [66].
The subcellular location of endogenous PpIX is inside the mitochondria. But studies
have revealed that exogenous PpIX accumulates in the plasma membrane of cells. But
interestingly without regard to the accumulation site of PpIX, it is able to execute cell killing
following by tumor growth inhibition [25-27, 29] (Table 1-2).
21
Figure 1-6 Chemical structure of PpIX
Table 1-2 Subcellular effects of PS drug based on its accumulation site
Site of accumulation Subcellular effects
Mitochondria Cytochrome C release
Endoplasmic Reticulum (ER) Activation of the unfolded protein response (UPR)
Cytoplasm Destruction of the uniform architecture of actin filaments
Enabling actin depolymerization
Nucleus Ca2+
transport impairment Enlargement of the nuclear membrane
Plasma Membrane Inactivation of specific membrane-bound enzymes Inactivation of receptors and impairment of ion
channels
1.3 Afterglow Nanoparticles (AG NPs)
Although some diseases such as cancer are still challenging issues, current
researchare opening new promising avenues to effectively treat these diseases. One of the best
alternative therapies is the use of nanoparticles (NPs) to deliver drugs directly to the diseased
sites without affecting healthy (normal) cells, tissues, and/or organs. NPs are capable to
decrease the side effects of drugs by reducing the dosage of drugs through increasing their
therapeutic effect and specifying their sites to be delivered also known as targeted drug delivery
[67].
Afterglow materials which are also called long-lasting phosphors or persistent
luminescent phosphors are luminescent materials whose lifetimes prolong from a minutes to
22
hours [68]. Persistent luminescent materials can be found in daily life such as emergency
lighting, safe traffic, luminous paints, textiles ceramics, and wall painting [69].
Konan and related team in their publication back in 2001 have divided PS delivery for
PDT into passive and active process depending on the availability of the targeting molecule on
the surface of drug [70]. Based on their definitions, the active strategy includes targeted delivery
of PS to bind to diseased cell receptors, while any other delivery method can be termed as
passive.
But in PDT, a nanocarrier can play an important intermediate role in delivering light
and/or drug to induce photodamage at the site of disease. This point of view has not been taken
into consideration by Konan’s definition. The other group introduced a new definition that
matches better with PDT characterizations. They referred to PS activation and excitation as
ways to divide nanocarriers into active or passive terms. Based on their definition, passive
carriers are those who cannot excite PSs directly and includes biodegradable polymeric NPs
and non-polymeric NPs such as ceramic and metallic NPs. Active NPs are those with the ability
to transfer energy to PS drugs.Based on their activation mechanisms, these can be classified
into different groups (Table 1-3) [71].
23
Table 1-3 Classification of nanoparticles used for photodynamic therapy[71]
Category Material Size (nm)
Mechanism
Pa
ssiv
e
na
no
pa
rtic
les
Polymeric Biodegradabl
e
PLGA (50:50) PLGA (75:25)
PLA
58-670 118-167 121-988
Delivery of loaded PDTagents per degradation rate of NPs in a biological environment
Polymeric Non-
biodegradable
Polyacrylamide Ceramic (Silica)
Gold Iron oxide
20-30 20-350
2-4 11
NPs serve as multifunctional platformsfor the diagnosisand treatment, PSs are protected from the environment by NPs, PSs are encapsulated or covalently linked to NPs
Two-photon-active molecule are co-encapsulatedinto NPs
Active
nan
opa
rtic
les
Quantum dots
CdTe 2.26 3.09
Excited NPs transfer energy directly to surrounding oxygen
Self-illuminating
BaFBr:Eu+, Mn
+
20
NPs emit luminescence (after being excited) to active the photosensitizers so light deliveryto the PSs is not necessary
Radiotherapy and PDT can be combined and activated simultaneously
Lower doses of radiation leads an efficient therapeutic effect
Upconverting NaYF4:Yb,Er/Tm
28-40
Excitation of PSs in the near-IR wavelength results in an emission at a shorter wavelengthactivate associated PSs
Most afterglow phosphors host matrices have been doped with rare earth elements.
Rare earth elements are being used to create holes in host lattices. A good homogeneity of the
crystal lattice is essential to accessing an efficient structure for producing afterglow properties.
Lattice defects in the host lattice are responsible for decreasing the efficiency of luminescence
of afterglow materials [68].
1.3.1 History of AG NPs
Humans have known about the phenomenon of persistent luminescence for over 1000
years. Ancient Chinese artists had mixed colors and special kinds of pearl shells to create
afterglow properties on their paintings. In 1602, a shoemaker and alchemist Vincenzo
Casciarolo discovered Bologna stone, which had afterglow properties. Later on in 1640,
Fortunius Licetus explained this phenomenon scientifically. It was believed that available barium
sulfide in the rock had created the afterglow. Indeed, it was explained that existing natural
24
impurities of stone had prolonged its afterglow. Very little research has been guided on
afterglow phenomenon until the end of the 20th century. Zinc sulfide (ZnS) doped with copper
(and then co-doped with cobalt) was the most used afterglow for many years [72, 73]. Its
application was in commercial products such as glow-in-the-dark toys, luminous paints, and
watch dials. However, its short decay time and weak brightness had limited its practical
approaches. Traces of radioactive elements such as promethium or tritium were employed to
solve the mentioned issues [74]. But the proposed solution could not decrease the high
concentration of luminescent material used in commercial glow-in-the-dark objects to show
acceptable afterglow. In August 1996, at the same time, two different groups of Matsuzawa and
Takasaki [75, 76] independently synthesized a long lasting afterglow which was 10 times
brighter than ZnS:Cu,Co that was previously used widely. They introduced the rare earth
element dysprosium (Dy3+
) as a co-dopant into the green-emitting phosphor SrAl2O4:Eu2+
[72],
their afterglow was able to emit hours after removing the source of energy. This progress
encouraged researchers for different and better persistent afterglow materials. Alkaline earth
aluminates were mostly used materials for the mentioned studies for a long time. Afterglow
Sr2MgSi2O7:Eu2+
,Dy3+
was introduced by Lin and related research teams in 2001.
Sr2MgSi2O7:Eu2+
,Dy3+
demonstrated very bright and a long decay time [77]. Later on, other
doped (di) silicates were showing long afterglow and were synthesized. Research and studies
have been focused on the synthesis of new afterglow compounds. Although it has been 18
years since the first report of long lasting SrAl2O4: Eu2+
, Dy3+
afterglow particle, the number of
known afterglow compounds with long lasting decay time and good brightness is still limited
[72].
1.3.2 Afterglow Mechanism
The mechanism of afterglow occurs when energy is stored in trap center (created by
doped materials) of the host lattice during excitations under UV or X-ray photons, then this
energy can result in luminescence after removing the energy via photons. Research on
25
underlying mechanisms of afterglow was started seriously after the introduction of
SrAl2O4:Eu2+
,Dy3+
persistent luminescence. It was believed that after excitation, charge carriers
would be trapped in energy levels of the forbidden band gap, so this location is then called a
trap center. Charge carriers would later start to escape from traps to return their initial states to
create the afterglow effect. Different mechanisms such as basic conceptual models and
complicated models, including various types and depths of charge traps, had been proposed by
1996. Some important ones are the Matsuzawa model, the Aitasalo model, the Dorenbos
model, and the Clabau model. In the following paragraphs, we will try to give a brief and
adequate overview of the Aitasalo model [72].
1.3.2.1 Aitasalo Model
In 2003, Aitasalo proposed a different model from the Matsuzawa model (Figure 1-7)
[72]. Aitasalo explained that energy can excite electrons from the valence band to trap centers;
the produced holes can migrate into calcium vacancies to be captured. Thermal energy can
make electrons released from the trap centers to end up at an oxygen vacancy. The energy
level of an oxygen vacancy is far from conduction band and thermally transitions to condition
band are not possible. So they assumed that the release of energy on an electron and hole
recombination can be delivered to europium ions through energy transfer. They assumed that
vacancies and luminescent centers are close together. A europium electron can be excited to a
5d level and then the following emission can create luminescent property [78].
26
Figure 1-7 Persistent luminescence mechanism proposed by Aitasalo et al. for
CaAl2O4:Eu2+
,Dy3+
[72].
In this model, free charge carriers consists of only holes (in the valence band), which is
in agreement in Abbruscato and Matsuzawa model. This model introduced by Aitasalo was able
to explain some missing point in Matsuzawa model such as the observed afterglow in non-co-
doped SrAl2O4:Eu2+
. Because the Matsuzawa model was depended on the trivalent rare earth
co-dopants, Aitasalo considered the effect of adding co-dopants by assuming that the lattice
defect is increased by codopant, because divalent alkaline earth sites may be occupied by the
trivalent lanthanide causing a defect for charge compensation. Their model also could explain
the dominant effect of adding Sm3+
in creating afterglow. During the synthesis, Sm3+
is reduced
to Sm2+
, which can remove the cation vacancies, resulting in creating hole traps [72].
Another reason to reject Matsuzawa’s model is that the application of monovalent
europium and tetravalent dysprosium ions to explain the afterglow effect was found to be
implausible. Because Aitasalo believed that reduction of Eu2+
and oxidation of Dy3+
can result in
unstable ion formation. Other researchers later agreed with Aitasalo’s reasoning for rejecting
Matsuzawa model [80].
27
1.3.3 Afterglow Host and Doped Materials
Eu2+
-doped alkaline earth aluminates luminescent phosphors were a new generation of
afterglow products introduced In the 1990s. The Eu2+
-doped alkaline earth aluminates were the
most used afterglow phosphors which were enhanced by adding trivalent rare earth ions of Dy3+
and Nd3+
as co-dopants. These afterglow phosphors were able to emit visible luminescence,
which were recognizable by the naked eye after an excitation by UV radiation, fluorescent lamp,
or X-ray [68, 81].
The ratio of dopant and co-dopant to create the longest lifetime and brightest emission
was investigated. Wang reported 6.6% Eu2+
dopant in SrAl2O4 was enough to get optimal
fluorescence intensity [74]. Matsuzawa applied 1% of Eu2+
and 2% of Dy3+
as dopant and
codopant, respectively to obtain the best intensity [75]. Zhao described the brightest afterglow
in CaAl2O4 was obtained with 0.5% of Eu2+
and 1% of Nd3+
[82]. Later on, scientists explained
that the ratio of doping and codoping elements depends on the compounds as well as co-
dopants. For example, as it was mentioned, 1% of Eu2+
and 2% of the co-dopant was an
optimal ratio to create a long lifetime in SrAl2O4:Eu2+
with Dy3+
but it had been reported that the
optimal concentration of Nd3+
is around 1% the same as Eu2+
[75]. Although most researchers
picked the ratio of 1% Eu2+
and 1 or 2% RE3+
, not so much evidence has revealed that the
mentioned ratios are the optimal values. Lin noticed that the best results in Sr4Al14O25 was
related to 2/1 ratio of Dy/Eu ions [83], and Jiang reported Dy/Eu ratio of around 20/7 in
Ca2MgSi2O7: Eu2+
, Dy3+
, Nd3+
[84]. Sabbagh Alvani confirmed Dy/Eu ratio of 1/2 in
Sr3MgSi2O8:Eu2+
,Dy3+
as the optimal ratio [72, 85].
1.3.4 Methods of Afterglow Synthesis
Different, efficient, cheap, and simpler methods were applied to synthesize
MAl2O4:Eu2+
. A solid-state reaction which uses high temperature at 1300–1400 °C is the most
used method. The other methods such as sol-gel, microwave, Pechini, combustion, and laser
heated pedestal growth (LHPG) were good methods that were released. It is obvious that the
28
mentioned methods do not prepare identical crystallographic and afterglow properties.
SrAl2O4:Eu2+
,Dy3+
, synthesized by microwaves, revealed a decreased afterglow brightness and
a blue shift probably due to the small size of grain. The same blue shift was demonstrated for
SrAl2O4:Eu2+
,Dy3+
synthesized by sol-gel. Hölsä reported a hexagonal crystal structure instead
of the monoclinic one for CaAl2O4 prepared by sol-gel or combustion. It should be taken into
account that the composition of the starting materials plays an important role in afterglow
properties. For example, an alkaline earth’s deficit may result in improving the afterglow, while
excess of barium in BaAl2O4:Eu2+
,Dy3+
can ruin afterglow completely. Several articles have
revealed the magical effect of the flux agent of borate B2O3 on SrAl2O4:Eu2+
,Dy3+
. Samples
prepared without borate resulted in very weak if no afterglow, no matter whether or not perfect
SrAl2O4 was formed. It seems BO4 formation in the host can act as a substitutional defect
complexs with Dy3+
. This causes the depth decrement of the charge traps in SrAl2O4 from 0.79
eV to 0.65 eV, which is suitable for afterglow at room temperature [72].
Many researchers focus on the synthesis of alkaline earth silicates (Sr2MgSi2O7) doped
with rare earth elements by a high temperature solid-state reaction that prevents the synthesis
of the fine nano-sized particles [86-87]. To solve the size issue, synthesis of the Sr3MgSi2O8 by
the help of sol-gel method has attracted attention. Sabbagh Alvani et al. [85] reported the effect
of the ratio of Eu/Dy as dopant and co-dopant on XRD pattern, emission wavelength, and the
decay time on Sr3MgSi2O8 particles. Pan et al. applied the sol-gel method to prompt
Sr2MgSi2O7:Eu2+
, Dy3+
to Sr3MgSi2O8: Eu2+
, Dy3+
[88]. On the other study, Song et al.
demonstrated a novel modified method to synthesize Sr2MgSi2O7: Eu2+
, Dy3+
[86]. Brito et al.
[89] tried to reveal the nature of the traps to introduce the main trapping site for Sr2MgSi2O7
material.
1.3.5 Preferred Host and Doped Materials for the Current Study
Although aluminate-based doped rare earth ions phosphors has demonstrated unique
properties including long duration, excellent photoresistance, brightness, and environmental
29
capability, its dramatically decrement of mentioned properties after soaking in water limits its
applications [77]. Alkaline earth silicates, due to high chemical stability, heat stability, lower cost,
and good anti-hydrolysis in the water have been introduced as a proper host lattice. Being
doped with rare earth Eu ion results in the highly efficient emission, which its wavelength is
dependent on the surrounded host lattice, and it also provides the broad range of visible light
from blue through red [86, 90, 91]. Mostly, the 4f7 → 4f6 5d1 transitions cause the
phosphorescence of Eu2+
in hosts. Doping alkaline earth silicates with Eu gives luminescent
properties without any long afterglow [92]. To create long persistent luminescent property the
host needs to be co-doped with the other rare earth element like Dy to have the trap centers.
Strontium and Magnesium are most important host materials and Europium and Dysprosium
are doped and co-doped materials chosen for this study because it is believed they not only do
not introduce important health treat but also they can be helpful for body function at the low
concentration. From a toxicological point of view, for those materials which are already available
in the body, the body knows the way it should treat them. About Strontium it can be said that
[93]:
Its chemical similarity to calcium probably does not pose a significant health
threat.
Strontium is used as medicine in different forms.
Improvement on bone-building osteoblasts has been reported after taking
strontium as a medicine.
Strontium increases bone density, aids bone growth, and lessens vertebral,
peripheral, and hip fracture.
Regarding Magnesium it can be considered that [94]:
It is the eleventh most abundant element by mass in the human body.
Its compounds are used medicinally to treat common laxatives, antacids
abnormal nerve excitation, and blood vessel spasm.
30
Europium and Dysprosium have been reported to be relatively non-toxic
compared to other heavy metals. It is believed that these rare earth elements do not have any
significant biological role.
1.4 Objective of the Study
In photodynamic therapy (PDT), cancer destruction relies on applying a
photosensitizing (PS) drug followed by an application of light. In fact, light activates drug to
transfer energy or electron to oxygen to generate reactive oxygen species (ROS). ROS will
react with vital biomolecules immediately to damage cell organelles resulting in cell death. PDT
efficiency is dependent on the successful combination of photosensitizing drugs, light energy,
and oxygen [19]. Up to this point, all approved clinical drugs for PDT in the United States are
related to protoporphyrin IX (PpIX) [95]. The most challenging and unresolved issue related to
PpIX is the wavelength of light which is essential to activate it. Blue light is not able to penetrate
more than 10 µm through the tissue because most tissue chromophores, including
oxyhemoglobin, deoxyhemoglobin, melanin and fat absorb blue light limiting PDT efficiency.
Indeed, the hydrophobic PpIX shows very low solubility (only ~1 μg/ml) and low water
dispersion resulting in easily aggregation in aqueous media which ultimately undermines the
efficiency of singlet oxygen generation-a key role of managing of PDT efficiency. In addition,
aggregation limits intravenously injection (I.V) of drug [96, 97].
Our goal is to design a modality to eliminate of external blue light in addition to
increasing the drug’s water dispersion which finally may result in enhancing PDT efficiency. The
hypothesis of this study as illustrated at Figure 1-8 proposes enhanced PDT efficiency through
delivery of synthesized afterglow nanoparticles (AG NPs) which may be excited by X-ray and
emit blue light for a long time, even after removing the energy source; conjugated nanoparticles
can also improve the water dispersion of PpIX.
31
Figure 1-8 Schematic illustration of nanoparticle–porphyrin conjugates for X-ray induced PDT
for cancer treatment
Being successful to synthesize the proposed AG NPs can be a solution for weak
penetration of blur light, because after conjugation of the drug to AG NPs and injection NPs to
the site of tumor, exposure to X-ray can activate AG NPs to emit blue light. Then if the
conjugation step happens successfully, AG NPs will transfer its energy to oxygen to generate
singlet oxygen to kill cancer cells.
In addition, it is expected that water dispersion of drugs will improve due to not only
conjugation to NPs, but also conjugation to Folic Acid (FA), which make the final NPs (after
conjugation to drug and FA) more hydrophilic due to the NH2 and COOH group of it. Based on
our mentioned goal, our specific aims are:
Aim I: Synthesis of the AG NPs which can be excited by X-ray
Aim II: Surface modification of PpIX to improve its luminescent property
Aim III: Conjugation of modified PpIX and FA to AG NPs and in vitro UV treatment
32
If the proposed design works successfully, X-ray dosage will decrease. In fact,
functional combination of X-ray and PS drugs in addition to an improvement of water dispersion
of drugs may result in dosage decrement of drugs and X-ray radiation.
1.4.1 Background and Significance
The reasons to restrict this proposal to cancer treatment by PDT are straightforward: a)
as it was mentioned in the “cancer” section of this dissertation, cancer has been reported as the
second leading cause of death in the United States. Cancer Statistics has estimated a total of
1,529,560, and 1,638,910 new cancer cases and 569,490, 577,190 deaths from cancer in the
United States in 2010 and 2012, respectively [98], b) Although many anti-cancer treatments
(e.g., chemotherapy, radiation therapy) have been designed to kill and damage fast-growing
cancer cells, they have great potential to destroy certain normal, healthy cells which also grow
quickly.
As mentioned previously, all cancer treatments are going to bring adverse effects to
patients’ lifves, which can be disturbing. To avoid all mentioned side effects, PDT has been
introduced for clinical approaches [19, 95-97]. Light and light activated compounds have
attracted many attentions since ancient times. Niels Finsen won the 1903 Nobel Prize for his
phototherapy which could control skin manifestations of tuberculosis. Since then other
interested researchers such as Raab, Jesionek, and Tappeiner [21] introduced the use of
chemical photosensitizers to improve PDT. They figured out the systematical connection
between light activation of these dyes and therapeutic effects.
Although the development of phosphor nanoparticles introduced a new hope, most
researchers focused on those of phosphor nanoparticles which produce near-IR (NIR) light
after exposure to X-ray to image molecular tracers in vivo [99]. The absorption band of currently
approved PDT photosensitizers is located in the visible spectral regions below 700 nm which
causes very challenging limitations for an efficient PDT. Near infrared (NIR) spectral range
(700–1100 nm) can achieve the deepest penetration through body tissues while it is not
33
matched with most approved photosensitizers [19, 95-97]. In addition, the hydrophobicity of PS
drugs and its weak water dispersity play as a hindering role to be activated by light to generate
singlet oxygen. Photofrin-based family photosensitizers have FDA approval in the United States
for Barrett’s esophagus and esophageal obstructing lesions and early and late endobronchial
lesions [40-42].
Photosensitizers play a key role in the efficiency of PDT-known as a promising modality
of cancer treatment. Although the exact mechanism through which PDT induces cancer cell
death has not been fully understood, both apoptosis and necrosis are involved in this process.
Among all important factors monitoring the efficiency of PDT, handling photosensitizer-related
issues has attracted so much attention because it is believed the most important deficiency of
PDT is due to all unsolved problems pertaining to photosensitizer [96].
In spite the fact that PpIX has tunable characteristics, it suffers from some
disadvantages which results in reducing the PDT efficiency dramatically. Of all challenging
issues of PpIX, low purity and poor selectivity for tumor tissue are due to a complex mixture of
several partially unidentified porphyrins that PpIX is originated from. Low PDT efficiency that
comes up with high PpIX accumulation rate in skin causes a prolonged light sensitivity lasting
for up to weeks after PDT treatment. The administration of nearly large dosages of
photosensitizer is a current solution for aggregation and low solubility of PpIX in a physiological
medium to achieve satisfactory phototherapeutic windows and clinically relevant cellular levels
of PpIX [100-101]. This solution has not been introduced as an efficient resolution as it may
increase all drawbacks and side effects related to PpIX aggregation and toxicity such as
nausea, vomiting, hypotension combined with marked vasodilatation, and hypersensitivity of
both the skin and the eyes to light. Indeed, with regard to the kinetics of PpIX fluorescence
which is dose-dependent, the increased concentration of PpIX does not signify the maximum
fluorescence intensity or better light-induced cell death [102-103]. On the other hand, much
chemical and biological research has been done over the past 20 years to identify new
34
photosensitizers. However, most of these studies’ goals have been synthesizing chemically
purer photosensitizers that absorb more strongly at longer wavelengths, rather than placing a
high priority on the development of improved biological properties [105]. All issues related to
PpIX which PDT is facing confirm an essential and immediate need to achieve optimum
concentration of PpIX to access the efficient PDT. PDT potentially may become a major weapon
to end the struggle for cancer treatment if some barriers can be removed. Four vital and critical
characteristics of photosensitizers which are needed to be addressed are: 1) Wavelength: weak
tissue penetration of blue light has been introduced as an affecting challenge on PDT outcome.
Obviously, providing the photosensitizers’ activation with an appropriate wavelength that is able
to pass through the tissue is critical to get therapeutic effects of PDT. 2) Activation: to prevent
any accidental treatment an appropriate wavelength of light should be defined. 3) Reliability:
photosensitizer must be delivered at the exactly desired site with the capability of being
activated whenever is needed. The short lifetime of singlet oxygen species (less than 0.04 μs)
along with its limited diffusion distance (0.01 to 0.02 μm) can limit the initial extent of the
damage to the site of concentration of the PS molecule. So it is obvious to avoid any possible
normal tissue damage, and PS needs to be delivered selectively to tumor sites. 4) Integrative
ability: to be a clinically successful modality, photosensitizer should be able to be applied in
conjunction with other forms of treatment such as chemotherapy, radiation, and surgery [19,
96].
This design hypothesizes addressing all four mentioned issues to access an efficient
PDT. Specific aims of this study may improve PDT outcomes through elimination of external
blue light in addition to the modification of the PpIX molecule. Eventually, weak penetration of
blue light essential wavelength for activation of drugs, and low solubility of drug in a
physiological medium can be addressed and solved. Meanwhile, with this design tracking the
AG NPs may be possible through imaging as PpIX emits red light whose penetration in body
tissue is good and acceptable.
35
1.4.2 Innovation
The overall proposed approach for deep cancer treatment is aimed at studying
the possible solutions for low efficiency of PDT as a clinical alternative for current cancer
treatments which bring significant side effects to patients’ life. To achieve this goal,
improvements of the biological properties of the PpIX drug as well as providing internal blue
light to activate drug inside the tumor has been designed. The proposed study may benefit
cancer treatment through below listed novelties:
Treat tumors that are deeper under the skin or in body tissues.
A rand new area for afterglow nanoparticle applications which provide essential
light for activation of drug internally and can be excited by X-ray.
X-ray application to activate AG NPs, which allow deep cancer treatment due to
good penetration through the body.
Improvement of biological property of PpIX drug through conjugation to AG NPs
and FA which leads to lower therapeutic dosage and greater singlet oxygen generation.
Combination of PDT and current traditional X-ray radiation which may result in
lower dosage of X-ray.
Possible imaging achievement due to red emission of PpIX drugs.
On the other hand, improving the water dispersion of PS drug can result in
concentration decrement for biological application which may cause the following advantages:
Be selective for cancer cells as opposed to normal cells.
Collect in cancer cells more quickly, reducing the time needed between getting
the drug and the light treatment.
Be removed from the body more quickly, reducing the time people need to
worry about photosensitivity reactions.
The proposed PDT system will work more efficiently for i) activation of PPIX
because the intense absorption band at the UV-blue region of the photosensitizers will be used
36
for excitation instead of the weak absorption Q-bands in traditional PDT; ii) singlet oxygen
generation because the photosensitizers are closely attached to afterglow nanoparticles. In this
case, the risk of radiation damage to healthy tissues can be reduced because once the
afterglow nanoparticles are activated, the X-ray can be turned off.
37
Chapter 2
Materials and Methods
2.1 Aim I: Synthesis of Afterglow Nanoparticles (AG NPs)
To meet the first aim of this dissertation two different methods of synthesis of AG NPs
were examined which were solid state reaction and modified sol-gel method. The major goal
behind of synthesis of AG NPs was that to prepare in situ light source essentials for PDT which
does not suffer from weak penetration. So it may improve the PDT efficiency by providing PS
drugs with intense and appropriate wavelength followed by more efficient radical and singlet
oxygen generation which play a key role in PDT [23, 24].
2.1.1. Synthesis of Sr2MgSi2O7: Eu2+
, Dy3+
by Solid State Reaction
To obtain the AG characteristics for PDT, particles were first synthesized by solid state
reaction. Raw materials of SrCO3, 4MgCO3·Mg (OH)2·5H2O, SiO2, Eu2O3 and Dy2O3 were used.
H3BO3 was added as a flux. All starting materials were mixed homogeneously through very well
grinding; and alkaline earth silicate particles were prepared at 1050 0C for 3 hours in a weak
reductive atmosphere prepared by charcoal powder.
2.1.2. Synthesis of Sr3MgSi2O8: Eu2+
, Dy3+
by Modified Sol-Gel Method
To fabricate nanosized alkaline earth silicate particles, Sr3MgSi2O8: Eu2+
, Dy3+
was
synthesized by the modified sol-gel method. The purpose of comparison also was tracked. As
shown in Figure 2-1 all starting materials including SrCO3, 4MgCO3•Mg(OH)2•5H2O, Eu2O3,
Dy2O3 were suspended in stoichiometric ratio in equal volumes of tetraethyl orthosilicate
(TEOS) and ethanol. The deionized water was added dropwise while the solution into the 3
necked- flask was stirring vigorously under sonication. The molar ratio (R) of H2O/Si was
chosen equal 2 to have the optimum hydrolysis reaction [106]. The second chosen parameter to
achieve optimum hydrolysis degree of TEOS is pH value which was controlled by adding
appropriate hydrochloric acid (HCl) as a catalyst. The solution was heated and refluxed up to 80
0C till forming sol-gel. To conclude the optimum pH and temperature, different amount of acid or
38
temperature were examined and based on the results, the optimum values were reported.
Formed gel was dried at 150 0C at the oven for 3 hours in a weak reductive atmosphere
prepared by the charcoal powder. The dried powder was grinded and transformed to the
furnace for high temperature (1050 0C) calcination in a weak reductive atmosphere prepared by
charcoal powder.
1-Preparing sol-gel 2-Heating up to 80 0C 3-High temperature furnace
Figure 2-1 Synthesis of Sr3MgSi2O8: Eu2+
, Dy3+
by modified sol-gel method
2.1.3. Nanoparticles Characterization
The crystal structures of the phosphor nanoparticles were determined by X-ray
diffraction analysis (XRD). To observe the morphology and dimension of powder, scanning
electron microscope (SEM) was employed. To measure surface charge to make sure if NPs
possess the silanol group with minus value (for all future study) zeta potential measurements of
samples suspended in 2 mM DI water were done. To confirm all expected bonds Raman
spectroscopy was done. Raman spectra were taken with 514.5 nm laser excitation. The laser
was focussed with a 20X (N.A.=0.40) objective, and the laser power was approximately 3.6 mW
measured at the sample.
2.1.4. Luminescent and Afterglow (AG) Properties
Luminescent properties of both particles synthesized by different methods were sought
by using Spectrofluorophotometry. To see if X-ray was able to excite synthesized NPs, an X-ray
39
machine was employed for the same measurement. In addition, AG ability was determined by
X-ray. For Sr3MgSi2O8: Eu2+
, Dy3+
the measurements were done in both ethanol and water as
well as powder to determine if NPs were capable of keeping their properties even in aqueous
solutions to meet the requirement of biological application.
2.1.5 Affecting Parameters on the Luminescent and AG Properties
2.1.5.1 The Effect of Temperature and pH
Since pH and temperature are most important affecting parameters on the rate of
hydrolysis and condensation reactions during sol-gel method, the effect of different amount of
HCL between range of 300-2400 µl (1≤pH≤4) and different temperatures starting at room
temperature up to 800 C were examined. Then to seek the effect of mentioned variables,
synthesized particles were excited by X-ray.
2.1.5.2 The Effect of Ratio of Eu/DY
The dopant Dy3+
in the synthesized Sr3MgSi2O8: Eu2+
, Dy3+
acts as a trap-creating ion,
and considerably prolongs the afterglow [105]. Although Eu2+
and Dy3+
ions act as
luminescence and trap center to create the long afterglow phosphors [106] , the optimum ratio
of Eu/Dy was investigated through synthesis of Sr3MgSi2O8: Eu2+
, Dy3+
by the modified sol-gel
method with keeping all parameters constant but the ratio of Eu/Dy
2.1.5.3 The Effect of Temperature of Calcination and the Duration of Calcination
Shi et al. [87] reported the higher temperature at the time of PL measurement, the more
intensity of the emitted light. But here we were interested in seeking the effect of either
temperature during calcination or the duration of calcination process on the luminescence and
AG properties. For this goal, the temperature was varied between 600 0C-1050
0C at the
constant time (3 hours) and the duration time was varied from 1 hour to 3 hours at the constant
temperature (1050 0C), separately.
40
2.1.6 Improving the Size and Water Dispersion of NPs
Although the current applied method improved the size dramatically compared to solid
state reaction, still the size did not meet our criteria, so the effect of MgO -the reducing size
chemical- was investigated. On the other hand, as the water dispersion of NPs always has been
very important especially for any future biological application, the alkaline-wet grinding method
was applied to improve water dispersion through creating an OH barrier resulting in a stronger
repulsive force and better dispersion. Very simple and efficient alkaline wet grinding in 7.5 mM
NaOH solution was applied for 20 min and then NPs were soaked in 7.5 mM NaOH solution
overnight.
2.1.7 Stability of Eu2+
in Solution
Eu2+
is unstable in aqueous solutions as it reacts quickly with oxygen and is slowly
oxidized by water. This is a clear indication of Eu2+
oxidation to Eu3+
[107].
Since AG of particles is due to emission peaks related to Eu2+
, stability of Eu2+
in
solution is very critical; otherwise it will turn into Eu3+
and no afterglow can be expected. For this
goal, Sr3MgSi2O8: Eu2+
, Dy3+
particles synthesized by the modified sol-gel method were
dispersed in water and sonicated very well and their X-ray spectra were determined whether or
not Eu2+
is still stable in solution after 2, 4, and 24 hours.
2.1.8 Surface Silanization of AG NPs to Prepare APTES-AG NPs
The surface silanization of synthesized AG NPs was induced by a linking agent,
(aminopropyl)triethoxysilane (APTES), which introduces an NH2 functional group on the surface
of AG NPs for further drug and FA conjugation [108-112]. APTES was chosen because it has
good biocompatibility which has made its application possible for biofiled, in addition,
silanization can help bonds form between mineral component (AG NPs) and organic component
(PpIX). Silanization begins with the hydrolysis of ethoxy groups in APTES to form silanols, then
condensation of APTES silanols with surface silanols of AG NPs leads forming a monolayer of
APTES via a lateral siloxane network in which amino groups are oriented away from the
41
underlying silicon surface. Figure 2-2 shows the schematic illustration of APTES coating of AG
NPs.
Figure 2-2 Surface Silanization of AG NPs by the help of APTES
To make possible a surface modification of AG NPs with APTES, 100 mg of AG NPs
were resuspended in 25 ml of Toluene and then 1 ml of APTES was added to prepare 4 wt%
solution of APTES. 3-necked flask containing toluene, APTES, and AG NPs was simultaneously
heated, stirred, and refluxed under a dry nitrogen atmosphere at 90 0C overnight. Then solution
was washed by centrifuge 3 times in different solvents, DMF, ethanol, and water.
2.1.8.1 Conjugation Efficiency of APTES on the Surface of AG NPs
Since APTES does not show any absorbance in 405 nm but AG NPs does, Multiscan
reader was hired to calculate conjugation efficiency (CE) of APTES coating. The absorbance of
solution containing APTES-coated AG NPs can be quantified by measuring at a certain
wavelength (405 nm) by a multiplate reader. Because the measurement system was
concentration sensitive the measurement of different standard concentrations of AG NPs could
indicate the amount of APTES coated on the surface of AG NPs. Firstly 1 mg APTES coated
AG NPs were dispersed in 1ml DMSO, then different standard solutions of AG NPs was
42
prepared starting from 1 mg AG NPs/ml DMSO and then it was serially diluted for 6 times to
prepare lower concentrations. By the help of an obtained standard curve the percentage of
APTES conjugated to AG NPs was determined.
2.1.9 In Vitro Cell Study
2.1.9.1 Cell Viability of PNT1A (normal prostate epithelium) Exposed to AG NPs
To verify if AG NPs were biocompatible enough for biological application, cell viability
evaluation was tested by MTT assay. The PNT1A cells was cultured in RPMI 1640 medium at
370C and 5% CO2. The cells were plated into 96-well-plate inserts at 3000 cells per well and
allowed to grow for 1 days. The cells were washed with PBS following by replacement of the
medium, and then AG NPs were added at different concentrations up to 500 µg/ml and
incubated for 24 hours.
2.1.9.2 Cell Imaging, Nanoparticle Uptake by Cancer Cells
To observe the uptake of AG NPs, PC3 prostate cancer cells were grown on a
microscope cover slide and incubated overnight with 50µg/ml NPs in F-12K medium. The cells
were then fixed with formaldehyde (5% in PBS) and transferred to a microscope slide for
florescent microscopic studies. Images were captured with florescent microscope with excitation
at 350 nm and emission at 450 nm.
2.2 Aim II: Surface Modification of PpIX
The goal of this part of our study was the modification of the PpIX molecule to increase
its solubility in a physiological medium and eventually the improvement of its use in PDT. To
achieve the defined goal, PpIX was modified with organo-silane agent (aminopropyl)
triethoxysilane (APTES). Modified (MPpIX) were fabricated by a two-step chemical process.
Protoporphyrin IX (PpIX) was converted to high reactive protonated PpIX dichloride to keep its
COOH groups active. The reactive PpIX was covered and modified with the organo-silane agent
(3-Aminopropyl) triethoxysilane(APTES). Activated COOH group of protonated PpIX dichloride
reacts with NH2 groups of APTES to form amide bond. The applied method assures chemical
43
attachment of the PpIX to the APTES framework. The mentioned modification was expected to
improve PpIX properties especially water dispersion and stability of PpIX in the aqueous
solution. The increment of the water dispersion and stability of PpIX in the physiological media
may prevent aggregation, which can results in Pp IX and PDT efficacy that is ultimate goal and
hope. Longer singlet oxygen (1O2) lifetimes and improved
1O2 generation would be definite
results of better incorporated PpIX into cancer cells. Applying the potosentisizer drugs may
benefit drug delivery to cell through the scape from lysosome enzymatic degradation due to
their potential for photochemical internalization (PCI) [38, 39].
2.2.1 Preparation of PpIX Dichloride
Preparation of high reactive protonated PpIX dichloride was done by adding 1ml oxalyl
chloride to 10 mg of PpIX (1.65×10-5
mol). The solution was simultaneously stirred and refluxed
under a dry nitrogen atmosphere at 55 0 C overnight. Overnight reflux and stirring make
evaporation of the excess of oxalyl chloride possible to provide PpIX dichloride as a purple
powder.
2.2.2 Fabrication of APTES-Capped PpIX (Modified PpIX)
2 ml APTES was added to the purple powder of high reactive protonated PpIX
dichloride. Simultaneously stirring and reflux under a dry nitrogen atmosphere at room
temperature (RT) was applied overnight. Under a dry nitrogen atmosphere dimethyl-8,13-
divinyl-3,7,12,17-tetramethyl- 21H,23H-porphine-2,18-dipropyl-amidepropyltriethoxysilane was
obtained as a APTES-capped Pp IX which is called modified PpIx (MPpIX) through this study
(Figure 2-3). To remove any unreacted PpIX molecules, MPpIX product was dialyzed against
methanol/water solution by the help of a dialysis tube for 3 days (Molecular cutoff = 12-14 kDa).
44
Figure 2-3 Modified PpIX (MPpIX) fabricated by a two-step chemical process (OC: Oxalyl
Chloride)
2.2.3 Folic Acid Conjugated MPpIX (FA-MPpIX)
To prepare a tumor-targeted delivery of PpIX, folic acid (FA) was conjugated to MPpIX
as shown in Figure 2-4. It is believed that not all COOH functional groups of high reactive
protonated PpIX dichloride find opportunity to react with NH2 groups of APTES, so it is expected
some unreacted COOH of coated APTES-PpIX would be ready to conjugate to amine groups of
FA.
45
Figure 2-4 FA conjugation to the surface of MPpIX (R can be both CH3 or siloxane)
To prepare FA-conjugated MPpIX, firstly 23.39 mg FA (0.053 mmol) was dissolved in
3ml DMSO, then 27.47 mg EDC and 20.37 mg NHS (0.177 mmol of EDC and NHS) were
added, respectively . Modified PpIX (10 mg, 0.0177 mmol) was added into DMSO solution, and
let it stir for 3 days under a dry nitrogen atmosphere at RT. To get rid of unreacted chemicals,
the final solution was dialyzed against methanol/water solution by the help of a dialysis tube for
3 days (Molecular cutoff = 12-14 kDa,) and then was washed in water by centrifuge
(rpm=13500, 25min) 3 times.
2.2.4 Characterization
To make sure if preparation of MPpIX was done successfully Raman spectroscopy was
applied. To observe the expected peaks FA, PpIX, and FA-MPpIX were examined under
Raman machine which is able to measure both solution and powder. In addition, conjugation
efficiency (CE) of APTES on the surface of PpIX was determined by the absorption
measurement. Since PpIX shows absorption at 540 nm wavelength while APTES lacks an
absorption band in the wavelength region of interest, direct method was applied. To measure
46
CE, standard solutions with 6 different concentrations of PpIX starting at 100% were prepared.
Absorption of both standard solutions and samples (MPpIX) were measured by microplate
reader (Multiskan) equipment to calculate standard curve and determine the CE. In addition,
UV-visible absorption spectroscopic measurements were hired to determine the CE of FA on
the surface of MPpIX. The absorption intensity of FA around 280 nm can guide to measure the
CE of FA. To do this, firstly concentration of MpPIX solution in DMSO was set to get absorption
intensity equal or lower than 1, then based on the calculated concentration of MPpIX different
concentrations of FA solution in DMSO were prepared to calculate the standard curve and
determine the CE of FA [65, 113].
2.2.5 Luminescent Properties
The luminescence property of the obtained MPpIX was sought by
Spectrofluorophotometry to see if any alteration happened during all chemical and coating
processing. In addition, the effect of MPpIX concentration in the water (as a good and reliable
aqueous media) on luminescence property was measured and compared. Besides, to show the
effect of coupling PpIX with APTES on not only luminescence also stability in water, pictures
taken by camera was presented.
2.2.6 Solubility and Stability
To show and prove the improvement of solubility and stability of MPpIX compare to the
uncapped one, the same amount of PpIX and MPpIX were dispersed in water; the same
amount of PpIX was dissolved in DMF as a control. The solubility and stability status of all 3
samples were captured by camera as pictures.
2.2.7 Singlet Oxygen Generation
As discussed previously, singlet oxygen generation is a key role of photosensitizer
drugs to kill or damage cancer cells [114]. It was important to prove if mentioned modification
was able to improve singlet oxygen generation or not. A p-nitrosodimethylaniline RNO)-
imidazole(ID) method was used to detect singlet oxygen. Briefly, particles was added into 3 ml
47
of RNO (50 µM) and ID (8 mM) aqueous solution. The intensity of RNO absorption was
monitored with various X-ray doses (0, 1, 2, 4, 6, 8 Gy) using a UV-Vis spectrophotometer.
Meanwhile, as a reference, the same measurement was done in the absence of particles to
reveal the X-ray irradiation effect on RNO absorption [115].
2.2.8 In Vitro Cell Study
2.2.8.1 Cell Viability of PNT1A Cells Exposed to PpIX and MPpIX
To see the effect of coupling PpIX with APTES on cells, cell viability was done for
PNT1A cells exposed to different concentrations of PpIX and MPpIX [116]. Preparation steps
were the same as explained at 2.1.9.1, three different examined concentrations were 2.5, 5, and
10 µg/ml.
2.2.8.2 Cell Imaging, Intensity Enhancement of FA-MPpIX Compared to MPpIX
After getting all essential improvement done, in vitro imaging was performed to observe
the effect of modified drug on intensity of red emission [117]. If the modified product was evenly
dispersed in water with acceptable stability, it should be able to be excited by the light to emit
bright red emission. Then it can be concluded that it has cytotoxic effects inside the cells. To
observe the uptake of drug, PC3 prostate cancer cells were grown on a microscope cover slide
and incubated overnight with 5 µg/ml drug in F-12K medium. All preparation steps were the
same as cell imaging explained for AG NPs.
2.3 Aim III: Conjugation of PpIX and FA to APTES-AG NPs and In Vitro UV Treatment
Successful combination of light, photosensitizer drugs, and oxygen are governing PDT
efficiency [77, 91,115]. Although synthesized the AG NPs as a light source and improved PpIX
drug were achieved through Aim I (section 2.1) and Aim II (section 2.2), the power point of this
proposal relied on active interaction between AG NPs and the drug. The controlled synthesis of
afterglow nanoparticles with desired afterglow properties and surface functionalities was very
critical for the smooth progress of the whole project. In fact, not only does the drug have to be
conjugated to the surface of AG NPs, AG NPs must be able to transfer its energy to drug. In
48
addition drug must transfer its absorbed energy to available oxygen in cancer tissue to generate
toxic molecule of singlet oxygen. The nanoparticle emission spectra must match the
photosensitizer’s absorption spectra. This guarantees the high efficient energy transfer from
nanoparticles to photosensitizers and more efficient production of singlet oxygen. Efficient
energy transfer must meet three requirements. First, the emission band of the donor must
overlap effectively with the absorption band of the acceptor. Second, the spatial distance
between the donor and the acceptor must be close enough to permit energy transfer. The
distance at which fluorescence resonance energy transfer (FRET) is 50% efficient, namely, the
Förster distance, is typically 20–100 Å. It is obvious that, in order to have an efficient energy
transfer, the distance between the donor and the acceptor should be less than 10 nm. Third, the
decay lifetime of the donors must be longer than or comparable to the decay lifetime of the
acceptors [118]. These well-defined rules for efficient energy transfer will guide our materials
design and photosensitizer conjugation.
2.3.1 PpIX Conjugated APTES-AG NPs (PpIX-AG NPs)
PpIX will be activated following section of 2.2.2 and APTES-AG NPs are prepared per
section of 2.1.8 of this dissertation. To conjugate PpIX to APTES-AG NPs firstly 1.7×10-2
mM of
activated PpIX was added to the beaker followed by adding 0.21 mM of APTES-AG NPs. 1 ml
DMF was added as catalyst for amide formation between drug and APTES-AG NPs and let the
solution stir under a dry nitrogen atmosphere at RT for 3 days. Because PpIX is light sensitive,
the experiments were done in the dark [101, 119-126]. After two days stirring, products were
dialyzed against DI water /Dimethyl sulfoxide (DMSO): 10/1 to remove all unreacted chemicals.
Afterward 3 times centrifuge helped to get rid of any remaining unreacted chemicals. Collected
pellet was transferred to freeze-drying machine to prepare powder samples [127, 128]. Figure
2-5 illustrates conjugation of PpIX to APTES-AG NPs schematically.
49
Figure 2-5 Conjugation of PpIX to AG NPs
2.3.2 FA Conjugated APTES-AG NPs (FA-PpIX-AG NPs)
As shown in Figure 2-6 to conjugate FA to the surface of PpIX-AG NPs there are two
possible sites including NH2 groups of APTES which did not find opportunity to conjugate during
previous step or COOH of conjugated PpIX to APTES-AG NPs; both groups have potential to
conjugate to FA since FA has both groups as well.
50
Figure 2-6 Conjugation of PpIX-AG NPs to FA
For this goal, 1 mM of PpIX-AG NPs was add to beaker containing 3ml DMSO followed
by addition of 7 mM EDC and 7 mM NHS. After 30 min 3 mM FA was added and it was left to
stir under a dry nitrogen atmosphere at RT and darkness for 3 days [129, 130]. Again all steps
related to washing out all unreacted chemicals were followed as explained in the section of
2.3.1 and powder samples were obtained after freeze-drying.
2.3.3 Characterization
2.3.3.1 Size and Surface Charge
DLS was applied to confirm successful conjugation after each step. Change of surface
charge as well as size can be an indication of the formation of new layers. In addition, SEM and
TEM can confirm final morphology of NPs and layer by layer structure
51
2.3.3.2 Raman Spectroscopy
To confirm formation of expected bonds at each step, Raman spectroscopy was hired
to look for related peaks [131].
2.3.3.3 Ultraviolet–Visible Spectroscopy (UV-Vis)
UV/Vis has been previously applied for quantitative determination of organic
compounds due to the UV absorptivity at different compound [132]. For this goal, the same
concentration of AG NPs, PpIX, folic acid, and the final product of FA-PpIX-AG NPs were
dissolved in 4ml DMSO and the absorption peak of every sample was determined by UV-Vis to
not only confirm the successful conjugation but also to calculate the amount of conjugated PpIX
or FA to the surface of APTES-AG NPs. PpIX and FA absorbance peaks are around 403 and
298 nm, respectively.
As it was mentioned on the section of 2.2.4, conjugation efficiency of APTE on the
surface of AG NPs was determined by the Microplate reader.
2.3.4 Luminescence Property
Fluorescence resonance energy transfer (FRET) was determined by
Spectrofluorophotometry. As explained previously FRET is a strong indication of the successful
conjugation of PpIX on the modified surface of AG NPs. To FRET happen we expect the
luminescence intensity of donor (AG NPs) quenches and that of the acceptor (PpIX) decreases.
2.3.4.1 FRET between APTES-AG NPs and PpIX
To observe energy transfer, luminescence quenching method was used [133, 134]. In
this method it is expected that nanoparticle luminescence to disappear if the energy is
transferred from the nanoparticles to the photosensitizers attached to it, meanwhile the
luminescent intensity of acceptor will be increased. Based on this method, free PpIX and PpIX-
AG NPs were examined by the Spectrofluorophotometer to measure luminescent intensity of
them before and after conjugation. This step was essential to figure out if all taken steps to
nanofabricate conjugated permanent luminescent nanoparticles were in the right direction.
52
There is no hope for having effective PDT without efficient singlet oxygen generation. To
evaluate FRET, the same concentration of PpIX, NPs and its conjugated with PpIX were
prepared and its PL was measured. Decrement of intensity of PL of NPs (donor) in addition to
increment of PL of PpIX (acceptor) confirms energy transfer.
2.3.5 Stability of Ultimate NPs in Water
Not only PpIX but also AG NPs suffers from very weak water dispersion. It is logic
expectation to have better water dispersion of FA-PpIX-AG NPs (ultimate NPs) compared to
both free drugs and NPs. Very simple test was done to observe stability of solution in water.
Solutions of different concentrations of ultimate NPs (equal to 1.25, 2.5 and 5 µg/ml of free
PpIX) were prepared and let it stay without any movement for at least 2 hours to see how much
of NPs was settled down. The pictures were taken by camera to confirm water dispersion
improvement of ultimate NPs after conjugation with PpIX.
2.3.6 Detection of Singlet Oxygen Generation
To determine singlet oxygen generation as explained on the section of 2.2.7 A p-
nitrosodimethylaniline RNO)-imidazole(ID) method was used.
2.3.7 In Vitro Cell Study
2.3.7.1 Cell Viability of PNT1A Cells Exposed to PpIX-AG NPs and FA-PpIX-AG NPs
Cell viability of NPs was determined to measure biocompatibility of NPs. For this goal,
PNT1A cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum
and 100 units/ml penicillin at 37 0C and 5% CO2. The Cells were plated into 96-well-plate inserts
at 3000 cells per well and allow to grow for 1 days. The cells were washed with PBS following
by replacement of the medium, and then PpIX-AG NPs and its conjugated with FA were added
at concentrations starting at 10µg/ml which was diuluted 4 times to examine cell viability of cells
exposed to lower concentrations, all samples were incubated for 24 hours. Toxicity of free PpIX
and AG NPs was examined at the same well plate to compare their results [117, 128].
53
2.3.7.2 Cell Imaging, Intensity Enhancement of FA-PpIX-AG NPs Compared to PpIX-AG NPs
and Free PpIX
Since the main purpose of the proposed design was enhancement of red emission of
PpIX by the help of AG NPs, examination and verification of enhancement of red emission
intensity was done under fluorescent microscopy. To do cell imaging of free PpIX, PpIX- AG
NPs and its conjugated with FA, PC3 prostate cancer cells were grown on a microscope cover
slide and incubated overnight with nanoparticle solution (equal to 5 µg/ml of free PpIX). The
cells were then fixed with formaldehyde (5% in PBS) and transferred to a microscope slide for
florescent microscope studies. Images were captured with florescent microscope with excitation
at 350 nm and 405 nm and emission at 450 nm and 670 nm.
2.3.8 In Vitro Cancer Destruction (In Vitro PDT)
To ensure of having functional and efficient nanocarriers, the effect of cell exposure to
UV was examined to see if nanocarriers are able to play the new role to destroy cancer cells
more than free PpIX. For this goal, cell survival experiments was conducted using PC3 cancer
cells that were incubated with free drug and its conjugated with NPs and FA in different
concentration (starting at 5 µg/ml of free drugs calculated based on CE). The concentration was
diluted serially 2 times to see the effect of concentration, also. After exposure cells to PpIX or its
conjugated NPs, cells were washed with PBS and were exposed to the UV for 5 min. Then by
the help of MTT assay the percentage of survived cell were determined and compared.
2.3.9 Statistical Analyses
In vitro results were statistically analyzed by one-way ANOVA and two-way ANOVA
based on the numbers of sought parameters. Results will be expressed as mean ± standard
deviation.
54
Chapter 3
Results and Discussion, Aim I: Synthesis of Afterglow Nanoparticles (AG NPs)
3.1 Luminescent Properties
3.1.1 of Sr2MgSi2O7: Eu2+
, Dy3+
Powder
Figure 3-1 shows the excitation spectra of Sr2MgSi2O7: Eu2+
, Dy3+
with a broad band
from almost 250 to 430 nm while the emission wavelength was at 460 nm. It was clear that the
main emission peak was at 460 nm (excited at the range of 354- 420 nm) ascribing to the 4f-5d
transition of Eu2+
. Since there was no emission peaks of Eu3+
, it seems co-doped Dy3+
could be
transferred the energy to Eu2+
ions in the matrices crystal lattice and Eu3+
was completely
reduced to Eu2+
in the crystal matrix. The doped and co-doped materials (Eu2+
and Dy3+
) were
rare earth elements and to obtain Eu2+
, a one-step-reduction process was essential [19, 40].
Figure 3-1 Photoluminescent excitation (PLE) and Photoluminescent emission (PL) of
Sr2MgSi2O7: Eu2+
, Dy3+
powder (solid state reaction) measured by Spectrofluorophotometer
Not only UV, but X-ray radiation could excite the obtained particles. Figure 3-2 and
Figure 3-3 shows X-ray excited optical luminescent (XEOL) spectra from Sr2MgSi2O7: Eu2+
, Dy3+
powder. The spectra shows emission wavelength of 480 nm and longevity for excited powder by
X-ray, as well. The Sr2+
sites, or the Mg2+
sites, or the Si4+
sites are three available sites for
55
incorporating Eu2+
, and Dy3+
in Sr2MgSi2O7 lattice. Taking into account that ion radius of Mg2+
(0.72A°) and Si4+
(0.26A°) are small, only Sr2+
(1.26A°) has equal size match to Eu2+
(1.12A°)
and Dy3+
(1.03A°). So there is no possibility for incorporating Eu2+
, and Dy3+
ions into a
tetrahedral [MgO4] and [SiO4], but only into an [SrO8] anion complex in Sr2MgSi2O7. Therefore,
no significant lattice distortions were caused through the incorporation of Eu2+
, and Dy3+
ions
into the Sr2MgSi2O7 crystal lattice [86, 96].
Figure 3-2 XEOL from Sr2MgSi2O7: Eu2+
, Dy3+
powder
56
Figure 3-3 Luminescent decay after 5min X-ray irradiation of Sr2MgSi2O7: Eu
2+, Dy
3+ powder
3.1.2 of Sr3MgSi2O8: Eu2+
, Dy3+
Powder
Figure 3-4 and Figure 3-5 present the excitation and emission spectra of Sr3MgSi2O8:
Eu2+
, Dy3+
phosphors synthesized by the modified sol-gel method at different acidic and alkaline
environment. The characteristics and properties of a sol-gel inorganic network products strongly
depend on the parameters such as pH, temperature and time of reaction, reagent
concentrations, catalyst nature and concentration, H2O/Si molar ratio (R), aging temperature
and time. In addition, the drying process can change the rate of hydrolysis and condensation
reactions. For both environments the results were the same, except the gentle shift toward
longer wavelength for alkaline condition that was agree with its higher size as can be seen in
the SEM.
57
Figure 3-4 PLE and PL of Sr3MgSi2O8: Eu2+
, Dy3+
powder (modified sol-gel method) in acidic
environment (pH=2) measured by Spectrofluorophotometer
Figure 3-5 PLE and PL of Sr3MgSi2O8: Eu2+
, Dy3+
powder (modified sol-gel method) in basic
environment (pH=10) measured by Spectrofluorophotometer
58
The excitation spectra shown in Figure 3-4 and Figure 3-5 confirmed a broad band from
almost 280 to 400 nm and the emission wavelength around 450 nm. The broad bands of the
emission spectra under the ultraviolet excitation were due to transitions of Eu2+
between the
8S7/2 (
4f7) ground state and the excited configuration. Broad excitation band can provide the
particles with effective and complete energy absorption of natural light and consequently good
AG. Again no peaks related to emission of Dy3+ and Eu
3+ were observed in the spectra [87,
135].
Figure 3-6 and Figure 3-7 show the X-ray luminescence and luminescent decay spectra
of Sr3MgSi2O8: Eu2+
, Dy3+
synthesized by the modified sol-gel method excited by X-ray. Two
particular emission peaks around 406 and 480 nm confirmed that two different lattice sites may
be occupied in the host crystal lattice by Eu2+
with different coordinate numbers of 6 or 8 which
can be activated into different energy levels and may result in different emission peaks; it seems
that Dy3+
acts as trap centers causing long afterglow characteristics, rather than the
luminescent centers in the host lattice [86, 97, 136, 137]. As Figure 3-7 shows, the AG of
particles was due to the emission peak of 480 nm and the other peak was not able to create any
AG.
Figure 3-6 XEOL from Sr3MgSi2O8: Eu2+
, Dy3+
powder (modified sol-gel method)
59
Figure 3-7 Luminescent decay after 5min X-ray irradiation of Sr3MgSi2O8: Eu2+
, Dy3+
powder
(modified sol-gel method)
The excitation of Eu2+
due to 4f → 5d transition occurs after exposing the samples to
the ultraviolet lights or X-ray so as a result, a lot of holes are produced. Thermally release of
some of free holes to the valence band and simultaneously migration of part of released holes
through the valence band following by being traped by Dy3+
ia a chain of events which happen.
Dy3+
trap levels are located in between the excited state and the ground state of Eu2+
. Thermally
re-excitation of the trapped holes happens after the source of excitation is removed then the
holes migrate and its combination with the excited electrons results in the long afterglow [91].
3.1.3 of Sr3MgSi2O8: Eu2+
, Dy3+
Solution
Since biological application of synthesized NPs is one of the major goals of this study,
the luminescence behavior in solution especially the AG longevity is very critical. To evaluate if
the synthesized NPs are able to keep their AG in the solution, their longevity was measured in
both ethanol and water. It’s worth mentioning that, in spite the fact that other researchers’ NPs
suffer from very fast decay of luminescence [88] our NPs presented the good longevity in the
solutions as Figure 3-8 confirms.
60
Figure 3-8 Luminescent decay after 5min X-ray irradiation of Sr3MgSi2O8: Eu2+
, Dy3+
solution in
water and ethanol (modified sol-gel method)
3.2 Affecting Parameters on the Luminescent and AG Properties
3.2.1 The Effect of Temperature and pH
The sol-gel process is generally described at the functional group level by three
reactions as shown in Figure 3-9 hydrolysis, alcohol condensation, and water condensation.
Hydrolysis takes place regardless of pH by the help of the nucleophilic attack of the oxygen
contained in water on the silicon atom.
Figure 3-9 Three different reaction during sol-gel process [106]
Each reaction rate depends on the pH, both acidic and basic conditions would result in
faster hydrolysis reaction compared to a neutral pH, and pH from 6 to 14 can speed up the rate
of the condensation. Addition of an external catalyst, such as mineral acids (HCl) and ammonia
61
can speed up and complete hydrolysis. Acidic conditions make an alkoxide group protonated in
a rapid first step. Silicon atom withdraws the electron density and become more electrophilic
and makes water more easy and possible to attack it. At the same concentration of catalyst, the
speed of hydrolysis in a basic condition is much slower than in an acidic condition and the
nucleophile-OH is repelled by alkoxide oxygens. Dissociation of water is likely to produce
hydroxyl anions which later on attack the silicon atom. Either an alcohol-producing or a water-
producing condensation reaction results in formation of siloxane bonds. The rate of
condensation is dependent on pH; below pH=2, the [H+] concentration determines the
condensation rates, but between pH 2 to 6 condensation rates are determined by [-OH]
concentrations. Although condensation above pH 7 is the same as in the range of 2<pH<6,
condensed species are ionized and repulsive [106].
As previously mentioned, pH and temperature can play key role on the rate of
hydrolysis and condensation reactions that subsequently will affect the size of synthesized
particles. Obviously, SEM shown in Figure 3-10 reveals that the obtained size at acidic
condition (pH=2) was much lower than that of basic (pH=10), no matter before or after
calcination.
Figure 3-11 A and B, and Figure 3-12 present the effect of the mentioned variables on
the luminescence and afterglow. The decreasing of the pH resulted in the intensity increment of
emission peaks at 406 nm and the intensity decrement of emission peaks at 480 nm,
respectively. Since AG is only due to the wavelength of 480 nm, pH increment (up to 4) caused
its improvement. It seems the pH level can specify which lattice sites should be occupied by
Eu2+
in the host crystal lattice with coordinate numbers of 6 or 8 [136]. On the other hand, pH=4
may provide better active trap center to create the longer AG and more intense luminescence.
62
A: Acidic environment B: Alkaline environment B
efo
re C
alc
ina
tio
n
A
fte
r C
alc
ina
tio
n
Figure 3-10 SEM of Sr3MgSi2O8: Eu2+
, Dy3+
synthesized by modified sol-gel method
(A): in acidic (pH=2), and (B): in alkaline environment (pH=10)
63
A XEOL B Luminescent decay R
T
40
0C
60
0C
80
0C
Figure 3-11 The effect of pH on XEOL and afterglow from Sr3MgSi2O8: Eu
2+, Dy
3+ powder
(modified sol-gel method)
64
pH=4 pH=1.5
Figure 3-12 The effect of temperature on XEOL and afterglow from Sr3MgSi2O8: Eu2+
, Dy3+
powder (modified sol-gel method)
3.2.2 The Effect of Ratio of Eu/DY
Figure 3-13 A and B represent the luminescence spectrum and luminescent decay of
Sr3MgSi2O8: Eu2+
, Dy3+
NPs with different doped/co-doped ratio excited by X-ray. A rapid decay
and then long-lasting phosphorescence were common between all presented curves. The best
luminescence and afterglow referred to the value of Eu/Dy = 1/4. Because the amount of Dy ion
has to be optimum, at a ratio less or more than 1/4, both mentioned properties are not as well
as a ratio of 1/ 4. On one hand, if doped amount of Dy3+
is little it is not enough to form enough
trap defects in the matrix materials. Conversely, if the doped amount of Dy3+
is more than
enough, it may lead to concentration quenching and decreasing the luminescence effect [97].
The trap densities, i.e., the capacity to store energy into a particular trap, are significantly
influenced by the co-dopants [89].
65
A XEOL B Luminescent decay
Figure 3-13 The effect of ratio of Eu/DY on XEOL and afterglow from Sr3MgSi2O8: Eu
2+, Dy
3+
powder (modified sol-gel method)
3.2.3 The Effect of Temperature of Calcination and the Duration of Calcination
Figure 3-14 A and B show the strong temperature-dependency of XEOL and
luminescent decay from AG NPs. As Figure 3-14 B shows within the whole range of
investigated temperatures, Sr3MgSi2O8: Eu2+
, Dy3+
NPs presented the same behavior up to 900
°C. Only at 1050 °C noticeable longevity changes were observed presumably originating from
strong recrystallization effects. Assumingly, the solid state reaction process mainly plays the
role of creating all important changes [138].
Figure 3-15 A and B present the effect of the duration of calcination. As a general rule it
can be claimed that with the increment of duration of calcination the intensity of peak at 480 nm
related to Eu2+
was increasing as well as peak of Dy3+
at 570 nm. Because the peak of 480 nm
reflects the AG (as data at Figure 3-7 had previously proved), increasing the duration caused
subsequently the improvement of AG.
66
A XEOL B Luminescent decay
Figure 3-14 The effect of temperature of calcination on XEOL and afterglow from Sr3MgSi2O8:
Eu2+
, Dy3+
powder (modified sol-gel method)
Although it is hard to reveal the nature of the traps, to claim if they are because of
lattice defects as strontium or oxygen vacancies (that are present even in the non-doped
Sr2MgSi2O7 material) or the dopants as the main trapping sites, Brito et al. suggested that an
oxygen vacancy creates the main trap presenting in Sr2MgSi2O7: Eu2+
, Dy3+
materials and its
energy can be modified slightly by co-doping materials [89].
Eeckhout et al. pointed out the charge carrier traps as a major and crucial role in all
persistent luminescence mechanisms. They mentioned that the amount of energy needed to
activate a captured charge carrier so-called depth is a very important property [72].
To have a good afterglow at room temperature (RT), appropriate activation energy is
needed between shallow traps with a depth lower than around 0.4 eV (to be fully emptied at
room temperature) and very deep traps around 2 eV (to be emptied at high temperature). An
optimal trap depth is around 0.65 eV. Liu et al. reported 0.75 eV as the trap depths of
Sr2MgSi2O7: Eu2+
, Dy3+
[135].
67
A XEOL B Luminescent decay
Figure 3-15 The effect of the duration of calcination on XEOL and afterglow from Sr3MgSi2O8:
Eu2+
, Dy3+
powder (modified sol-gel method)
In summary, according to the other group research mentioned above and all gathered
observations in this study, it seems temperature and duration of calcination may vary the
appropriate depth to activate a captured charge carrier at room temperature. Apparently the
optimum parameters of the calcination process reduce unreacted phase and then cause
recrystallization; this results in more intense and longer luminescence properties. It is believed
the increment of the calcination temperature can increase and improve the crystallinity. On the
other hand, energy levels of the enhancing Dy3+
co-dopant is close to that of the intrinsic trap
resulting in the increment of the trap density. As other research suggests, apparently the Dy3+
-
defect interaction causes the modification of trap energies [72, 88, 89].
3.3 Improving the Size and Water Dispersion of NPs
3.3.1 The Effect of MgO Adding on Size and Water Dispersion of NPs
MgO addition makes the migration and the diffusion of elements limited during the
sintering by increasing the viscosity; in fact, MgO prevents the grain growth as barriers and
results in smaller NPs [19]. SEM shown in Figure 3-16 confirmed the mentioned effect of
increasing the MgO additive chemical.
Figure 3-16 also shows SEM related to NPs treated and soaked in NaOH 7.5 mM and
confirmed a dramatic size decrement and very good water dispersion which was stable up to 30
68
minutes, it should be mentioned that bigger observed NPs in Figure 3-16 D were aggregation of
very small NPs. As a result, it can be concluded that the introduction of alkaline wet grinding
along with hydroxylation acted as a very efficient method to decrease the size and improve the
water dispersion of NPs.
(A) (B)
( C) (D)
Figure 3-16 The effect of MgO adding and Alkaline wet Grinding on water dispersion (A) and
Size of NPs (B): before adding MgO (C ): after adding MgO, (D) after adding MgO and alkaline
treatment
3.3.2 The Effect of MgO Adding on X-ray Excited Optical Luminescence (XEOL)
Adding any new chemicals can change the structure and characteristics of fabricated
AG NPs. As Figure 3-17 confirmed MgO addition did not change the crystal structure of AG
NPs.
69
Figure 3-17 XRD patterns of of Sr3MgSi2O8: Eu2+
, Dy3+
powder (modified sol-gel method) before
and after MgO addition
On the other hand, it was important to ensure that adding MgO did not present any
unwanted effect on luminescence and longevity. The effect of MgO on the XEOL and
luminescent decay of AG NPs were sought and as Figure 3-18 A and B shows MgO addition
could enhance both luminescent intensity and decay time. The reason could be the smaller size
of AG NPs after adding MgO, which may improve the mentioned properties of AG NPs.
(A) (B)
Figure 3-18 XEOL (A) and luminescent decay (B) from Sr3MgSi2O8: Eu2+
, Dy3+
powder (modified
sol-gel method) before and after MgO addition
3.3.3 The Effect of APTES Coating on Afterglow and Water Dispersion
Keeping the afterglow is very critical for biological application of AG NPs. After surface
modification of Sr3MgSi2O8: Eu2+
, Dy3+
NPs with APTES, its longevity and water dispersion were
verified and as Figure 3-19 demonstrates, both mentioned properties were improved. It seems
70
APTES coating helped AG NPs improve their water dispersion, and resulted in improved
dispersion and longevity. To observe improvement of water dispersion of AG NPs after APTES
coating, the same concentration of AG NPs before and after coating were prepared in water and
after 30 min the settled down particles were observed and compared.
(A) after (Red) and before (Blue) APTES coating
(B): after (1) and before (2) APTES coating
Figure 3-19 Luminescent decay after 5min photo luminescent irradiation (A) and Water
dispersion (B) of Sr3MgSi2O8: Eu2+
, Dy3+
powder (modified sol-gel method) before and after
MgO addition
3.4 Stability of Eu2+
in Solution
As Figure 3-20 reports, XEOL from AG NPs not only confirmed existence of Eu2+
peak
of the AG NPs synthesized by sol-gel method after leaving particles in aqueous solution for 24
hours, but also did not show any decrement of peak related to Eu2+
. In fact, there is no emission
of Eu3+
in the spectra, which indicated that Eu3+
ions have been reduced as Eu2+
completely
[77,86]. Picture taken by camera shown in Figure 3-20 B confirmed good water dispersion as
well as good intense blue afterglow.
71
(A) (B) Figure 3-20 XEOL from AG NPs in water at different times(modified sol-gel method) (A) and
water dispersion of AG NPs in solution (modified sol-gel method) (B)
3.5 Characterization
3.5.1 XRD Patterns
The crystal structure of the phosphor nanoparticles shown in Figure 3-17 was
determined by X-ray diffraction analysis (XRD). The diffraction peaks were consistent with other
research, which indicated that the co-doped Eu and Dy ions had little influence on the structure
of luminescent materials, and all of the peaks were assigned to the phase of Sr3MgSi2O8:Eu2+
,Dy3+[77, 86].
As a result we may conclude that single-phased Sr3MgSi2O8 phosphors was
synthesized since the influence of Eu2+
and Dy3+
ions on the crystal structure of luminescent
materials was very little and no new phase was formed during the synthesis process [89].
72
Figure 3-21 Raman spectra of AG samples : Sr2MgSi2O7: Eu
2+, Dy
3+ by solid state reaction with
Eu/Dy=1/3(1), Sr3MgSi2O8: Eu2+
, Dy3+
NPs by sol gel metode with Eu/Dy=1/3 at RT, pH=4 (3),
at 600 C, pH=2.5 (4), at 80
0 C, pH=3.5 (5) and with Eu/Dy=1/4 at 80
0 C, pH=4 (2).
3.5.2 Raman
The Raman spectra of some of the investigated particles are presented in Figure 3-21.
Spectra confirmed three important bands that were on focus at this study. Bands related to
surface silanol group, Si-O or Si (O2) group, and Siloxane group were observed at 980 cm-1
, in
the range of 910-1080 cm-1
, and in the range of 450-810 cm-1
, respectively. The best results of
photoluminescence and afterglow was related to sol-gel environment with pH=4, and
interestingly, sample 3 which had synthesized at pH=4 in Figure 3-21 shows more intense
bands related to silanol and Si(O2) group on the surface compared to other samples prepared
by sol-gel method with different pH of processing condition. This indicated and confirmed the
fact that as mentioned previously acid-catalyzed hydrolysis of silicon alkoxides occurred much
faster and subsequently highly hydrolyzed silicones were obtained. The bands located in the
range of 450-810 cm-1
related to Siloxane group of NPs synthesized at different pH did not
73
show a big difference, which indicated that not only NPs prepared at higher pH did possess
greater amount of silanol groups (the products of hydrolysis), but they did not show more
siloxane groups (the products of condensation) [106, 139, 131].
3.5.3 Surface Charge
As shown in Table 3-1 DLS data of the synthesized NPs by sol-gel method revealed a
net negative surface charge (-38.52 mV) related to the silanol groups oriented out of the probe
and presented OH group on the surface. After the application alkaline wet grinding, the surface
charge decreased to -27.26 mV. It seems less negatively charge of NPs played the effective
role in size decrement as the size decreased from 809 ± 40.9 nm to 399.5 ± 117.5 nm after
physically grinding of NPs. After APTES coating, the surface charge changed to -4.28 mV as we
expected because NH2 oriented outside of the NPs surface. Adding a new layer caused size
increment to 458 ± 136.8 nm.
Table 3-1 DLS results after and before alkaline wet grinding and APTES coating
NPs Zeta Potential (mV) Size (nm)
AG NPs -38.42 809 ± 40.9
AG NPs after Hydroxylation -27.26 399.9 ± 117.5
APTES –AG NPs -4.28 458 ± 136.8
3.5.4 Conjugation Efficiency (CE) of APTES on the NPs Surface
As explained at the section of 2.1.8.1 a multiscan reader was hired to determine the
CE. The conjugation efficiency of APTES on the surface of AG NPs was calculated based on
absorption spectra of AG NPs within the framework of the concentration-dependent method
using a microplate reader device. To calculate of CE of APTES, 3 different concentrations of
APTES-coated AG NPs (1, 0.5, 0.25 µg/ml) were prepared and relative intensity at each
concentration was calculated based on the equation obtained from standard curves. Based on
the calculated equation of Y=2605.2X-77.308, 100 µg/ml of APTES-coated AG NPs was
containing of 43.043 ± 6.42 µg/ml of APTES.
74
3.6 In Vitro Cell Study
3.6.1 Cell Viability of PNT1A Cells Exposed to AG NPs
To verify if AG NPs are biocompatible enough for biological application, cell viability
evaluation was tested by MTT assay. The results shown in Figure 3-22 Cell viability of PNT1A
exposed to AG NPs for 24 hrs tested by MTT assay demonstrated almost 90% cell viability for
NPs with concentration up to 250 µg/ml. With regard to the high concentration of 500 µgr/ml of
NPs, it may be claimed that synthesized NPs met biocompatibility expectation. One way
ANOVA did not declare any significant differences between different concentrations and control
group.
Figure 3-22 Cell viability of PNT1A exposed to AG NPs for 24 hrs tested by MTT assay
.3.6.2 Cell Imaging, Nanoparticle Uptake by Cancer Cells
As results shown in Figure 3-23 demonstrates, internalization of AG NPs into cytoplasm
of cells was observed. The stronger blue shows the cell nucleus and the lighter blue shows
0
20
40
60
80
100
120
140
PBS 62.5 125 250 500
Ce
ll V
iab
ility
(%
)
Concentration (ug/ml)
Cell Viability AG NPs
75
(A): (B)
Figure 3-23 Combined bright field microscopy and stained nucleus (A) and Uptake of
AG NPs by PC3 cancer cells (B). AG NPs (B)
76
Chapter 4
Results and Discussion, Aim II: Surface Modification of PpIX
4.1 Folic Acid Conjugated Modified PpIX (FA-MPpIX)
4.1.1 Characterization
After surface modification of PpIX, Raman spectroscopy was hired to determine if all
expected bonds between each layer have been formed. After preparation of sample solutions
(PpIX, MPpIX, and FA-MPpIX) Raman measurement was performed. Raman spectroscopy
results shown in Figure 4-1 confirmed expected amide bonds in MPpIX and FA-MPpIX. The
peak located around 1200 cm-1
shown with the black arrow 1 is an indication of C-N bonding of
APTES. PpIX itself shows a peak at these regions due to (Cb-Cα) stretching [140, 141]. The
peak around 1500 and 3300 cm-1
(black arrows 2 and 3) of Figure 4-1, which are
representative of carbonyl and N–H band, respectively can be contributed to amide formation
between NH2 groups of APTES or FA to COOH groups of PpIX or FA. The peak at 1459 cm-1 is
a characteristic band of folic acid corresponding to the phenyl ring and was present in the
Raman spectrum of FA-MPpIX to confirm FA conjugation on the surface of MPpIX [142-144].
Figure 4-1 Raman Spectroscopy of PpIX, FA, and FA-MPpIX
4.1.2 Conjugation Efficiency (CE) of APTES on the PpIX Surface
The conjugation efficiency of APTES on the surface of PpIX was calculated based on
absorption spectra of organic PpIX within the framework of the concentration-dependent
method using microplate reader device. For this goal, first a standard curve was plotted to fit the
77
linear regression between the absorption intensity and percentage of PpIX. The conjugation
efficiency of MPpIX calculated based on the equation of Y=0.0295X (R2=0.89) confirmed that
29.59 µg/ml of APTES was coated successfully on the surface of 100 µg/ml of MPpIX.
4.1.3 Conjugation Efficiency (CE) of FA on the Surface of MPpIX
The photophysical and photochemical properties of the FA-MPpIX was investigated. As
Figure 4-2 demonstrates, FA-MPpIX showed a peak at 290 nm which attributed to the
conjugation of FA [145-148]. The same as explained for CE of APTES on the surface of PpIX,
first a standard curve was plotted and CE of FA on the surface of MPpIX calculated based on
the fitted linear regression of Y = 0.0081X + 0.0873 (R2=0.9954) confirmed that 43.70 µg/ml of
FA was conjugated successfully on the surface of 100 µg/ml of FA-MPpIX.
Figure 4-2 UV-visible absorption spectroscopy of FA-MPpIX and FA
4.2 Luminescent properties of MPpIX
To observe intensity enhancement of MPpIX, the luminescent intensity of both free
PpIX and MPpIX were determined by Spectrofluorophotometry. Different concentrations of free
PpIX and MPpIX in water were prepared and then their photoluminescence were measured by
Spectrofluorophotometer. As confirmed by Figure 4-3 A and B, intensity of MPpIX in water was
concentration dependent and the most intense luminescence was related to concentration of
12.5 µg/ml which was equal to 239.73 ± 28.97 and 145.066 ± 19.69 for emission peaks at 620
0
0.2
0.4
0.6
0.8
1
1.2
200 400 600 800
Ab
s.
Wavelength (nm)
UV-visible absorption spectra
FA-MPpIX
FA
78
nm and 670 nm, respectively, while the best intensity of PpIX in water was related to 25 µg/ ml
which was 10.25 ± 6.43 and 10.14 ± 6.85 for emission peaks at 620 nm and 670 nm,
respectively (Figure 4-4). These data revealed that the photoluminescence of MPpIX was
enhanced 10 times more than that of free PpIX. The reason of the observed enhancement of
luminescent intensity was improvement of solubility and stability of PpIX in water after APTES
coating which provided PpIX with functional NH2 group oriented out of its surface [101,102]. The
picture taken by camera in Figure 4-4 demonstrates improvement of photoluminescence of
MPpIX in water compared to free PpIX in water. One way ANOVA confirmed the significant
differences between PL of PpIX and MPpIX at both emission peaks of 620 and 670 nm.
(A)
(B)
Figure 4-3 Photoluminescence intensity of MPpIX (A) and PpIX (B) measured by
Spectrofluorophotometer
0
50
100
150
200
250
590 640 690 740
Inte
nsi
ty (
a.u
.)
Wavelength (nm)
PL of MPpIX MPpIX 50 ug/mlMPpIX 25 ug/mlMPpIX 12.5 ug/mlMPpIX 6.25 ug/ml
0
5
10
15
20
25
580 630 680 730
Inte
nsi
ty (
a.u
.)
Wavelength (nm)
PL of PpIX PpIX 50ug/mlPpIX 25 ug/mlPpIX 12.5 ug/mlPpIX 6.25 ug/ml
79
Figure 4-4 Enhancement of photoluminescence intensity of MPpIX compared to PpIX
4.3 Solubility and Stability of MPpIX
If all steps mentioned above have been done successfully, not only the red emission of
MPpIX but also its water dispersion and stability should be improved. Figure 4-5 confirms very
good and stable solution of high concentration of MPpIX (20 µg/ml) in water. To compare the
stability, PpIX was dissolved in its solution Dimethylformamide (DMF) at the same
concentration. As these results confirmed water dispersion of MPpIX were improved. Very weak
water dispersion of PpIX has limited its biological application. One reason for the improved
photoluminescence of MPpIX compared to free PpIX was its ability for better dispersion in water
which allowed it to absorb light and transfer its energy.
Figure 4-5 Improvement of water dispersion of MPpIX in watercomared to PpIX in Water
3.2.4 Detection of Singlet Oxygen Generation
From previous promising results related to enhancement of photoluminescence and
improvement of water dispersion of MPpIX in water, it was expected that MPpIX can generate
more singlet oxygen [103, 114]. Results shown in the Figure 4-6 illustrates 2.5% more singlet
0
50
100
150
200
250
300
MPpIX (12.5 ug/ml) PpIX 25 ug/ml
Inte
nsit
y (
a.u
.)
Different Drugs
Intensity improvement of MPpIX
Emission peak at 620nm
Emission peak at 670nm
80
oxygen generation for MPpIX (equal to 3.5 µg/ml of free PpIX) in water compared to the same
concentration of free PpIX. As it was mentioned, it seems that improving dispersion of MPpIX in
water played a key role in absorbing light and transfering it to molecule of oxygen.
Figure 4-6 Singlet oxygen generation of PpIX and MPpIX
4.5 In vitro Cell study
4.5.1 Cell Viability of PNT1A (Normal Prostate Epithelium) Exposed to PpIX MPpIX
As shown in Figure 4-7 PpIX was toxic at all examined concentrations with highest cell
survival around 50%. After APTES coating, cell viability of MPpIX was improved up to 80%. In
fact, the cell viability of MPpIX had improved by 30% compared to that of free PpIX. Since
based on our results related to luminescence intensity and the results of other research the
concentration equal to 5 µg/ml of free PpIX had an expected luminescence, this concentration
was considered for the next in vitro experiments [149]. It should be mentioned that
concentration of PpIX was calculated and constant to 5 µg/ml for all evaluated samples based
on CE of PpIX and FA. Two way ANOVA confirmed significant difference between cell viability
of cells exposed to PpIX and FA-MPpIX.
81
Figure 4-7 Cell Viability of PNT1A Exposed to PpIX, and MPpIX for 24 hrs Tested by MTT
4.5.2 Cell Imaging, Intensity Enhancement of FA-MPpIX Compared to MPpIX
As Figure 4-8 demonstrates that uptaken free PpIX was not able to emit intense red
emission under excitation 405 nm, while FA-MPpIX shows more intense red emission under the
same excitation. We believe modification by APTES and then coating with FA helped the water
dispersion improvement of PpIX to absorb energy under excitation. In addition, over expressed
FA receptors on the surface of cancer cells improved uptake rate; these results were expected
because of previous mentioned results and confirmed both enhancement of photoluminescence
and improvement of water dispersion of MPpIX [116].
*
*
82
(A)
(B)
Figure 4-8 Cell images of PC3 exposed to PpIX (A), and FA-MPpIX (B) taken by fluorescent
microscopy, Ex=405 nm, Em=420, 670 nm. Nuclei was stained with DAPI
83
Chapter 5
Results and Discussion, Aim III: Conjugation of PpIX and FA to APTES-AG NPs and In Vitro UV
Treatment
5.1 Characterization
5.1.1 Size and Surface Charge
After surface modification of AG NPs and preparing modified PpIX, PpIX-conjugated
APTES-AG NPs and its conjugated to FA were synthesized. As Table 5-1 reveals, the surface
charge changed from -4.28 mV related to APTES-AG NPs to -25.46 of PpIX-conjugated
APTES-AG NPs which indicated the presence of COOH on the surface of conjugated product.
Interestingly, the size decreased to 232 ± 1.3 nm which may be the outcome of better water
dispersion of new products which decreased NPs aggregation [159]. The surface charge of FA-
PpIX-NPs was -25.11 because FA ligands caused a negative charge due to ionization of the α-
carboxylic group [150]. After a new layer of FA, the size increased to 273 ± 5.5 nm; it seems the
conjugation of FA played an important role in improving water dispersion of FA-PpIX-APTES-
AG NPs not to let them aggregate. DLS results could confirm formation of a new conjugated
product at each step.
Table 5-1 DLS results after conjugation of PpIX and FA to APTES-AG NPs
NPs Zeta Potential (mV) Size (nm)
PpIX-AG NPs -25.46 232 ± 1.3
FA-PpIX-NPs -25.11 273 ± 5.5
5.1.2 SEM and TEM
To observe the sample surface morphology, a scanning electron microscope (SEM)
was hired. The transmission electron microscopy (TEM) image contrasts because of absorption
of electrons in the material; therefore different thicknesses and compositions of the material can
be detected by TEM indicative of formation of different layers [151. Figure 5-1 A shows SEM of
FA-PpIX-AG NPs with spherical morphology. Figure 5-1 B, which presents TEM of the same
84
NPs, confirmed the formation of different composition on the surface of NPs since different
contracts were observed.
(A) (B) Figure 5-1 SEM (A) and TEM (B) images of FA-PpIX-AG NPs
5.1.3 Raman Spectroscopy
FA and PpIX were conjugated to APTES-AG NPs based on the description at the
section of 2.3.1 and 2.3.2. After conjugation it was essential to assure the forming of all
expected bonds. At the conjugation of PpIX to APTES-AG NPs, amide formation between NH2
groups of coated APTES on the surface of AG NPs with activated COOH groups of PpIX
(protonated PpIX dichloride) was sought. The other expected amide bond was between
unreacted COOH groups of conjugated PpIX or NH2 groups of APTES (those which did not find
an opportunity to be conjugated) to NH2 or COOH groups of FA. As shown in Figure 5-2,
Raman spectroscopy results confirmed successful conjugation of FA and PpIX to AG NPs with
formation of mentioned amide.
85
Figure 5-2 Raman spectroscopy of FA-PpIX-AG NPs
The characteristic bands of FA including 1459 cm-1
and 1640 cm-1
corresponds to
asymmetric stretching vibration of - NH2 and C=O stretching in carboxyl acids, respectively were
observed in Raman spectrum of FA shown in Figure 5-2. The spectra related to PpIX revealed a
very slight shift of 1600 cm-1
which is corresponding to the stretching bands of carboxylic groups
from the free ligand. The Raman spectrum of final NPs (FA-PpIX-AG NPs) demonstrated bands
at 1600-1640 cm-1
and 1459 cm-1
related to C=O stretching in carboxyl acids of FA and PpIX,
and stretching vibration of - NH2 of FA, respectively. In addition, peaks around 1230-1310 cm-1
were indication of amide formation. The peaks of the stretching bands of carboxylic groups
(1600-1640 cm-1
) along with the stretching bands of N-H at 3300 cm-1
confirmed the formation
of amide bonding. Peaks located in 480-800 cm-1
corresponded to siloxane bridge (Si-O-Si)
which formed between AG NPs and APTES [131, 132, 141-143, 152-154].
5.1.4 Conjugation Efficiency (CE) of PpIX and FA on the Surface of APTES-AG NPs
As explained at 2.3.3.3 ultraviolet–visible spectroscopy (UV-Vis) was hired to
quantitatively evaluate the conjugation of PpIX and FA on the surface of AG NPs. As shown in
Figure 5-3 the UV absorptivity at 290 and 407 is commonly used to determine the relative
abundance of FA and PpIX, respectively [132, 155, 156]. As one can see, the absorbance of
AG NPs was changed after conjugation to PpIX and FA to represent an absorbance peak of
each conjugated compound.
86
Figure 5-3 Absorbance of AG NPs and conjugated compounds by UV-Vis
Conjugation efficiency of PpIX and FA was calculated based on UV absorptivity of
these chemicals. Standard curves of different PpIX and FA and the related linear equations
were Y= 0.1158X + 0.6599 (R2=0.9739) and Y= 0.0081X + 0.0873 (R
2=0.9954) for PpIX and
FA, respectively. Calculation showed that in 100 µg/ml PpIX-APTES-AG NPs there was
2.050±0.207 µg/ml of conjugated PpIX and 100 µg/ml FA-PpIX-APTES-AG NPs was contained
26.87±2.998 µg/ml of FA. It should be mentioned that the initial amount of PpIX was 10% of the
total mass of AG NPs and based on calculation, more than 20% of initial amount of drug has
been successfully conjugated to the surface of AG NPs. But the µg conjugated drug per 100 µg
of total PpIX-AG NPs seems not too much. Some previous studies have shown the same
results for different drugs with lower than 20 µg loaded drugs per 1000 µg of total NPs [157-
158].
5.2 Luminescence Property
5.2.1 Enhancement of Luminescent Intensity
As previous results confirmed photoluminescence of PpIX is concentration-dependent
[149], different concentrations of PpIX-APTES-AG NPs in water were prepared to be evaluated
its photoluminescence intensity. The starting concentration was equal to 50µg/ml of free PpIX in
water (calculated based on CE of PpIX). The concentration was serially diluted 4 times. Figure
0
0.2
0.4
0.6
0.8
1
1.2
200 300 400 500 600 700 800 900
Ab
sorb
ance
WAvelength (nm)
Absorbance of NPs and conjugated compounds
AG NPsPpIXFAPpIX-AG NPsFA-PpIX-AG NPs
87
5-4 confirmed that the most intense photoluminescence was pertaining to 6.25 µg/ml of free
PpIX in water (calculated based on CE of PpIX), while Figure 4-3 revealed that the most intense
peak was related to 25 µg/ml of free PpIX in water. With regard to Figure 5-4, it was clear that
luminescent intensity of PpIX was enhanced almost 20 times after conjugation to AG NPs.
Figure 5-5 confirmed the increment of PL of PpIX-APTES-AG NPs compared to PpIX.
Significant differences between PL of free PpIX and PL of PpIX-APTES-AG NPs for both
emission peaks at 620 nm and 670 nm was confirmed by One way ANOVA. Luminescent
enhancement of PpIX after conjugation was because conjugated PpIX to APTES-AG NPs could
resolve the aggregation and significantly enhance the red emission of the PpIX [160]
Figure 5-4 Photoluminescence intensity of PpIX-APTES-AG NPs measured by
Spectrofluorophotometry
Figure 5-5 Enhancement of photoluminescence intensity of PpIX-APTES-AG NPs compared to
PpIX
0
20
40
60
80
100
120
140
160
PpIX-APTES-AG NPs (5ug/ml) Free PpIX (25ug/ml)
Inte
nsi
ty (
a.u
.)
Intensity improvement of PpIX-APTES-AG NPs
Em @ 620 nm
Em @ 670 nm
88
5.2.2 FRET between APTES-AG NPs and PpIX
The results of Spectrofluorophotometry shown in Figure 5-6 and Figure 5-7 confirmed
that FRET had happened between APTES-AG NPs and PpIX which corresponded to successful
conjugation of PpIX and NPs as explained at the section of 2.3. Conjugated APTES-AG NPs
which possessed large absorption cross section and strong fluorescence provided an ideal
platform for FRET [160]. As it was obvious, the intensity of APTES-AG NPs as a donor was
quenched and the intensity of PpIX as an acceptor was increased [118]. One way ANOVA
showed significant difference between PL intensity of AG NPs and free PpIX before and after
conjugation for both emission peaks at 620 and 670 nm.
Figure 5-6 PL of PpIX, APTES-AG NPs, and PpIX-APTES-AG NPs
Figure 5-7 Happened FRET Photoluminescence intensity of PpIX and AG NPs (measured by
Spectrofluorophotometer, Ex=400 nm)
020406080
100120140160
AG NPs (450nm) PpIX (620 nm) PpIX (670 nm)
Inte
nsi
ty (
a.u
.)
Wavelength (nm)
PL intensity Before Conjugation to PpIXAfter Conjugation to PpIX
89
5.3 Stability of Ultimate NPs in Water
Images shown in Figure 5-8 A demonstrated good water dispersion of the ultimate NPs
(FA-PpIX-APTES AG NPs) for the high concentration equal to 5 µg/ml of free PpIX in water.
The water dispersion improvement of PpIX-AG NPs and successfully happened FRET may
explain the improved results related to intensity increment of luminescent [159-160]. Figure 5-8
B taken by the camera revealed that solution of FA- PpIX-APTES-AG NPs in water was able to
absorb and transfer light to PpIX to emit stronger red emission after exposure to UV, compared
to free PpIX which almost no red emission was observed after exposure to UV. The reason for
very week red emission of PpIX in water is its aggregation which along with the luminescent
dependency of PpIX does not let red emission produce [102, 103].
(A) (B) Figure 5-8 Improvement of water dispersion at different concentrations (A) and PL (B) of
ultimate NPs
5.4 Detection of Singlet Oxygen Generation
Improvement of water dispersion and subsequently enhancement of red emission of
conjugated PpIX to APTES-AG NPs and its conjugated-FA product resulted in increment of
singlet oxygen generation as Figure 5-9 proved. As it was clear the increment of singlet oxygen
generated by PpIX-APTES-AG NPs and ultimate NPs after exposing to 8 Gy X-ray radiations
was 1.2% and 2.4% more than that of PpIX, respectively for concentration equal to 1.5 µg/ml of
free PpIX. As explained, conjugation of PpIX as well as conjugation of FA decreased the
aggregation of PpIX and NPs. Since luminescent intensity of PpIX is concentration-dependent,
less aggregation may results in enhancement of red emission of PpIX after exposure to the
90
source of energy [102, 103]. Stronger red emission resulted in more energy transfer to PpIX to
generate more singlet oxygen [114].
Figure 5-9 Singlet oxygen measurement of PpIX, PpIX-APTES-AG NPs, and FA-PpIX-APTES-
AG NPs (1.5 µg/ml water as a concentration of free drug)
5.5 In Vitro Cell Study
5.5.1 Cell Viability of PNT1A Cells Exposed to PpIX-AG NPs and FA-PpIX-AG NPs
Figure 5-10 showed MTT assay results for PNT1A cells exposed to PpIX, PpIX-
APTES-AG NPs, and FA-PpIX-APTES-AG NPs. Concentration of PpIX was constant in all
samples and were calculated based on the determined CE of PpIX and FA. Concentration of
PpIX was calculated based on CE of PpIX and FA to have 5 µg/ml of free PpIX. MTT assay
revealed that PpIX was highly toxic even at low concentration of 1.25 µg/ml which was able to
kill almost 50% of cells [149]. After conjugation of PpIX and FA to AG NPs the amount of
surviving cells increased for all examined concentration. Data were suggesting that after
conjugation of PpIX, both AG NPs and FA were preventing PpIX from aggregation which
obviously could decrease its toxicity to healthy cells. Two-way ANAVA confirmed the significant
difference between FA-PpIX conjugated NPs and PpIX.
91
Figure 5-10 Cell viability of PNT1A exposed to PpIX and its conjugated products by MTT assay
5.5.2 Cell Imaging and Intensity Enhancement of FA-PpIX-AG NPs Compared to PpIX-AG NPs
and Free PpIX
All obtained results promised a stronger red emission of conjugated NPs in cell
imaging. Cell imaging taken by fluorescent microscopy in Figure 5-11 showed stronger red
emission of FA-PpIX-AG NPs compared to free PpIX.
It was clear that the amount of survived cells were very low for those cell exposed to
PpIX. In addition, PpIX did not show very distinguishable red emission after excitation at 405
nm, but FA-PpIX-AG NPs demonstrated strong and detectable red emission under excitation at
405 nm which we believe red emission enhancement is because of both water dispersion
improvement and conjugation of a source of energy to PpIX to make FRET happen [159-160,
102, 103]. So it seems that after being excited by the 405 nm wavelength, PpIX has an extra
excitation source attached to activate it. This dual source of energy as well as water dispersion
improvement may help the light activate the attached PpIX to emit red emission.
*
*
92
(A)
(B)
Figure 5-11 Cell images of PC3 exposed to PpIX (A) and FA-PPIX-Ag NPs (B) taken by
fluorescent microscopy, Ex=405 nm, Em=420, 670 nm. Nuclei was stained with DAPI
5.5.3 In Vitro Cancer Destruction (UV Treatment)
To do UV treatment 2 group studies were considered. Both the control group and the
study group included PC3 cells exposed to free PpIX, AG NPs, and FA-PpIX-AG NPs. But the
control group was not exposed to UV; while the study group was exposed to 5 min UV
treatment. Figure 5-12 demonstrated results of two group studies. Results confirmed that FA-
PpIX-AG NPs demonstrated not only better cell viability (more than 30%), but also better toxicity
under UV exposure (almost 15%). Accumulation of PpIX in aqueous media seems to be the
major reason of the inefficiency of UV treatment. Lack of proper water dispersion of PpIX
caused great aggregation followed by photoluminescent quenching because of its dose
dependency [159, 160, 102, 103]. But after conjugation of PpIX to AG NPs and then FA not only
water dispersion improvement helped drug be distributed more even inside the media but also
AG NPs generating blue light was providing the drugs with an attached extra source of energy
to excite conjugated drug to induce more efficient toxicity. Dose dependency of
photoluminescence was the main reason that FA-PpIX-AG NPs up to 5 µg/ml did not show an
93
efficient treatment. As it was shown in Figure 5-4, intensity quenching may happen noticeably
for PpIX concentrations lower than 3.25 µg/ml.
Figure 5-12 In vitro UV treatment of exposed PC3 cells to PpIX, NPs, and FA-PpIX-AG NPs
Regardless of concentration of PpX and AG NPs, two way ANOVA did not show any
significant differences before and after UV treatment for exposed PC3 cells to free PpIX and AG
NPs but did show significant difference for those cells which were exposed to FA-PpIX-AG NPs.
0
20
40
60
80
100
120
PBS 1.25 2.5 5
Ce
ll V
iab
ility
(%
)
Concentration of free PpIX (µg/ml)
NO UV PpIX
AG NPs
FA-PpIX-AG NPs
0
20
40
60
80
100
120
PBS 1.25 2.5 5
Ce
ll V
iab
ility
(%
)
Concentration of free PpIX (µg/ml)
5 min UV PpIX
AG NPs
FA-PpIX-AG NPs
* *
94
Again, statistical analysis confirmed that there was a significant difference between cell viability
of those cells which were exposed to free PpIX and FA-PpIX-AG NPs. Within each group not
only free PpIX but also AG NPs did not show any significant differences. FA-PpIX-AG NPs up to
5 µg/ml free PpIX did not show significant differences within each group; and only FA-PpIX-AG
NPs containing 5 µg/ml free PpIX showed a significant difference before and after UV
treatment. The results of UV treatment for those cells exposed to 5 µg/ml free PpIX and FA-
PpIX-AG NPs containing 5 µg/ml free PpIX, did show a significant difference.
95
Chapter 6
Conclusion and Future Work
1.6 Conclusion
This study focused on finding a solution for challenging issues related to PDT. PpIX is
a photosensitizer (PS) which has attracted much attention for clinical application due to all
mentioned benefits in chapter 1 [24]. Of those advantages, the absorption at the Soret band
acts as a double-edged sword; on one hand, the absorption there shows to be at least 10 times
stronger than that of the Q-band. [66], on the other hand, blue light is unable to penetrate
deeply through tissue, which results in limiting the application of this PS to superficial cancers
[19]. In addition, the extremely low water dispersity of PpIX is a serious issue for its biological
application [60, 65, 100, 101, 161]. To resolve the weak-penetration issue of blue light, AG-NPs
were synthesized by the modified sol-gel method from Aim I. To improve its dispersity in water,
surface modification of PpIX was investigated to determine the possible chemistries as
mentioned in Aim II. The step connecting Aim I and Aim II was APTES chemical which was
used for surface modification of AG NPs by silanization to create functional groups on the
surface of AG NPs. The results from Aim II proved that APTES bonding to PpIX is able to not
only improve its water dispersion but also enhance the red emission of PpIX after being excited
by the energy source.
To synthesize AG NPs, both solid state (Sr2MgSi2O7: Eu2+
, Dy3+
) and modified sol-gel
methods (Sr3MgSi2O8: Eu2+
, Dy3+
) were investigated. Spectrofluorophotometry showed that the
excitation spectra of Sr2MgSi2O7: Eu2+
, Dy3+
with a broad band from almost 250 to 430 nm while
the emission peak was at 460 nm. It was clear that the main emission peak was at 460 nm
(excited at the range of 354- 420nm) ascribes to the 4f-5d transition of Eu2+
. Not only UV but X-
ray could also excite the obtained particles to emit at blue wavelength, which showed longevity
after removing the source of energy [86, 97]. Despite all excellent results, due to the big size of
AG NPs fabricated by solid state reaction, modified sol-gel method was employed.
96
The results related to the synthesis of AG NPs (Sr3MgSi2O8: Eu 2+
, Dy3+
) demonstrated
that the particles are capable of being excited by both UV and X-ray to emit blue wavelength,
and they have the afterglow effect after removing the source of energy. The emission peak at
480nm was due the 4f-5d transition from Eu2+
excited at the range of 354- 420nm. Since there
was no emission peaks from Eu3+
, it seems the co-doped Dy3+
could transfer energy to Eu2+
ions in the crystal lattice while Eu3+
has been completely reduced to Eu2+
. [19, 40]. Eu2+
acted
as a luminescent center while Dy3+
perhaps acted as trap centers that caused long afterglow
characteristics instead of the host lattice being the main contributor to luminescence or
afterglow [136]. The excitation of Eu2+
due to 4f → 5d transition occurs after exposing the
samples to the ultraviolet lights or X-ray so as a result, a lot of holes are produced. Thermally
release of some of free holes to the valence band and simultaneously migration of part of
released holes through the valence band following by being traped by Dy3+
ia a chain of events
which happen. Dy3+
trap levels are located in between the excited state and the ground state of
Eu2+
. Thermally re-excitation of the trapped holes happens after the source of excitation is
removed then the holes migrate and its combination with the excited electrons results in the
long afterglow [91].
The effect of pH and temperature on the size of NPs and afterglow characteristics was
also investigated. The pH and temperature can played as key roles on the rate of hydrolysis
and condensation reactions during the sol-gel method and subsequently affected the size of
synthesized particles [106]. Decreasing the pH resulted in the intensity increase of emission
peaks at 406nm and the intensity decrease of emission peaks at 480nm. Since the 480 nm
wavelength was creating the AG property, pH increment (up to 4) caused its improvement. It
seems the pH level can specify which lattice sites should be occupied by Eu2+
in the host
crystal lattice with coordinate numbers of 6 or 8 [136]. On the other hand, a pH of 4 can help
provide a better active trap center to create the longer AG and a more intense luminescence.
97
The investigation of different Eu/Dy ratios revealed that the greatest intensities resulted
when the ration of Eu/Dy was 1/4. On the one hand, if the doped amount of Dy3+
is small, then it
is not enough to form enough trap defects in the matrix materials. Conversely, if the doped
amount of Dy3+
is more than enough, then it may result in concentration quenching and
decrease the luminescence [97]. The trap densities, i.e., the capacity to store energy to a
particular trap, were significantly influenced by the co-dopants [89].
The surface silanization of synthesized AG NPs was induced by a linking agent,
(aminopropyl) triethoxysilane (APTES) to create functional groups of NH2 on the surface of
inorganic NPs for further drug and FA conjugation [108-112]. APTES can initiate silanization to
help bonds form between the mineral component (AG NPs) and the organic component (PpIX).
After modifying the surface of Sr3MgSi2O8: Eu2+
, Dy3+
NPs with APTES, its luminescence
longevity and water dispersion were improved due to the new NH2 groups on the surface of
NPs. From the result of X-ray diffraction analysis (XRD), we may conclude that single-phased
Sr3MgSi2O8 phosphors was synthesized since there was little influence of Eu2+and Dy
3+ions on
the crystal structure of the luminescent material and no new phase was formed during the
synthesis process [77, 76,89]. Raman spectroscopy confirmed three important bands related to
surface silanol group, Si-O or Si (O2) group, and Siloxane group, which were observed at 980
cm-1
, in the range of 910-1080 cm-1
, and in the range of 450-810 cm-1
, respectively. NPs
prepared at higher pH did possess a greater amount of silanol groups (the products of
hydrolysis), but they did not show more siloxane groups (the products of condensation) [106,
139, 131].
DLS data of the synthesized NPs from the sol-gel method revealed a net negative
surface charge (-38.52 mV), which can be related to the silanol groups oriented outside of the
probe and presented OH group on the surface. After the application of alkaline wet grinding, the
surface charge increased up to -27.26 mV. It seems less negatively charge of NPs has played
the effective role in size decrement as the size decreased from 809 ± 40.9 nm to 399.5 ± 117.5
98
nm after physically grinding of NPs. After APTES coating, the surface charge changed to -4.28
mV as we expected because NH2 groups oriented outwards on the NPs surface. Adding a new
layer caused size increment to 458 ± 136.8 nm. Conjugation efficiency (CE) of APTES on the
NPs surface was calculated based on absorbency [132]. Based on the calculated equation of
Y=2605.2X-77.308, 100 µg/ml of APTES-coated AG NPs contained 43.043 ± 6.42 µg/ml of
APTES.
The cell viability assay using PNT1A cells exposed to the AG NPs demonstrated an
almost 90% cell viability for NPs with a concentration up to 250 µg/ml. With regards to the high
concentration of 500 µg/ml of NPs, it may be claimed that synthesized NPs meet
biocompatibility expectations. One way ANOVA did not declare any significant differences
between different concentrations as well as control group. In addition, internalization of AG NPs
into the cytoplasm of PC3 cancer cells was observed.
Since APTES was applied to modify the surface of AG NPs and the conjugation of PpIX
to the surface of APTES-AG NPs was the main goal of this research, the possibility of forming
chemical bonds between PpIX and APTESe as well as the effect of this possible bonding on the
water dispersion of PpIX were investigated. After surface modification of PpIX with APTES and
FA, Raman measurement was performed. Raman spectroscopy confirmed expected amide
bonds in MPpIX and FA-MPpIX [142-144]. The conjugation efficiency of APTES on the surface
of PpIX and FA on the surface of MPpIX was calculated based on the absorption of organic
PpIX within the framework of the concentration-dependent method using a microplate reader
device [65, 113]. The conjugation efficiency of APTES on the surface of PpIX based on the
equation of Y=0.0295X (R2=0.89) was confirmed that 29.59 µg/ml of APTES was coated
successfully on the surface of 100 µg/ml of MppIX. CE of FA on the surface of MPpIX
calculated based on the fitted linear regression of Y = 0.0081X + 0.0873 (R2=0.9954) confirmed
that 43.70 µg/ml of FA was conjugated successfully on the surface of 100 µg/ml of FA-MPpIX.
Spectrofluorophotometry demonstrated an intensity enhancement of MPpIX compared to free
99
PpIX. The intensity of MPpIX in water was concentration dependent and its concentration equal
to 12.5 µg/ml of free PpIX resulted in the most intense luminescence which was equal to 239.73
± 28.97 and 145.066 ± 19.69 for emission peaks at 620 nm and 670 nm, respectively while 25
µg/ml of PpIX in water resulted in the best intensity which was 10.25 ± 6.43 and 10.14 ± 6.85 for
emission peaks at 620 nm and 670 nm, respectively. These data revealed that the
photoluminescence of MPpIX was enhanced 20 times more than that of free PpIX. MPpIX in
water showed to be a good and stable solution of high concentration (20 µg/ml). Good stability
was seen in water as could be seen in a solution of PpIX in its organic solvent (DMF).
Singlet oxygen measurements revealed 2.5% more generated singlet oxygen for MPpIX
with concentration equal to 3.5 µg/ml of free PpIX in water compared to the same concentration
of free PpIX in water. It seems an improvement of the water dispersion of MPpIX played a key
role to absorb light and transfer it to oxygen [115].
Cell viability of PNT1A (normal prostate epithelium) exposed to free PpIX showed
highest cell survival around 50% while, when exposed to MPpIX, cell viability was improved up
to 80%. In fact, the cell viability of MPpIX had improved by 30% compared to that of free PpIX.
Like other research, our results confirmed that 5 µg/ml had good luminescence and is not low
concentration for PpIX [149].
Cell imaging demonstrated that free PpIX taken up was not able to give an intense red
emission under the excitation of 405nm, while MPpIX showed an intense red emission under
the same excitation, but not as strong as FA-MPpIX. We believed the modification by APTES
and then coating with FA helped the PPIX to disperse better in water and absorb energy better
under excitation. In addition, over expressed FA receptors on the surface of cancer cells have
increased the uptake rate. These results were expected because of previous mentioned results
which confirmed both enhancement of photoluminescence and improvement of water dispersion
of MPpIX in water [117].
100
All mentioned results confirmed that not only was there a possibility of conjugation of
PpIX to APTES-AG NPs, but, also, APTES was able to improve the cell viability as well as
enhance the red emission of PpIX in water.
In the next step, PpIX was conjugated to APTES-AG NPs. DLS revealed the surface
charge changed from -4.28 mV related to APTES-AG NPs to -25.46 of PpIX- APTES-AG NPs
which indicated the presence of COOH on the surface of conjugated product. Interestingly, the
size decreased to 232 ± 1.3 nm which may be an outcome of better water dispersion of new
products which decreased NPs aggregation. The surface charge of FA-PpIX-NPs was -25.11
because FA ligands caused a negative charge due to ionization of the α-carboxylic group [150].
After a new layer of FA, the size increased to 273 ± 5.5 nm, it seems the conjugation of FA
played an important role in improving water dispersion of ultimate NPs and not to letting them
aggregate.
Scanning electron microscope (SEM) revealed spherical morphology for FA-PpIX-AG
NPs. Since transmission electron microscopy (TEM) image contrast is used for the absorption
of electrons in the material, different thickness and composition of the material can be detected
by TEM indicating of formation of different layers [151]. TEM of FA-PpIX-AG NPs confirmed the
formation of different composition on the surface of NPs since different contracts were
observed.
Raman spectra demonstrated all expected bonds between different layers. The
characteristic bands of FA including 1459 cm-1
and 1640 cm-1
corresponded to asymmetric
stretching vibration of - NH2 and C =O stretching in carboxyl acids, respectively, were observed
in the Raman spectrum of FA. The Raman spectrum of PpIX revealed the slightly shift of 1600
cm-1
which was corresponding to the stretching bands of carboxylic groups from a free ligand.
Raman spectrum of final NPs (FA-PpIX-AG NPs) demonstrated bands at 1600-1640 cm-1
and
1459 cm-1
related to C =O stretching in carboxyl acids of FA and PpIX and stretching vibration
of - NH2 of FA, respectively. In addition, peaks around 1230-1310 cm-1
were indication of amide
101
formation. The peaks of the stretching bands of carboxylic groups (1600-1640 cm-1
) along with
the stretching bands of N-H at 3300 cm-1
confirmed the formation of amide bond. Peaks in the
range of 480-800 cm-1
corresponded to siloxane bridge (Si-O-Si) formation between AG NPs
and APTES [131, 132, 141-143, 152-154]. Conjugation efficiency (CE) of PpIX and FA on the
surface of APTES-AG NPs was calculated based on the UV absorptivity [132, 155, 156].
Standard curves of different PpIX and FA and the related linear equations were Y = 0.1158X +
0.6599 (R2=0.9739) and Y = 0.0081X + 0.0873 (R
2=0.9954) for PpIX and FA, respectively.
Calculation showed that 100 µg/ml APTES-AG NPs contained 2.050±0.207 µg/ml of PpIX and
100 µg/ml PpIX-APTES-AG NPs contained 26.87±2.998 µg/ml of FA. Spectrofluorophotometer
results confirmed that the most intense photoluminescence of PpIX-AG NPs was pertaining to
the concentration equal to 6.25 µg/ml of free PpIX (calculated based on CE of PpIX) in water.
While that most intense peak of free PpIX was obtained from 25 µg/ml free PpIX in water.
Spectrofluorophotometer results revealed that intensity was increased almost 20 times.
Fluorescence resonance energy transfer (FRET) could be seen, which is a strong
indication of the successful conjugation of PpIX on the modified surface of AG NPs [118, 133,
134]. As results demonstrated, the intensity of APTES-AG NPs as a donor were quenched and
the intensity of PpIX as an acceptor was increase.
Good water dispersion of the PpIX-APTES AG NPs for a concentration which was
equal to 5 ug/ml of free PpIX in water, was observed. This improvement of both PpIX and NPs
water dispersion and results related to FRET may explain intensity increment of PpIX-AG NPs.
A solution of FA- PpIX-APTES-AG NPs in water exposed to UV was able to absorb more
photons and transfer energy to PpIX to emit a more intense red emission compared to free
PpIX, which gave almost no red emission after being dispersed in water.
The improvement of water dispersion and subsequently the enhancement of the red
emission of conjugated PpIX to APTES-AG NPs and its conjugated-FA product resulted in an
increase of singlet oxygen generation. With both containing the same PPIX concentration of 1.5
102
µg/ml, FA-PpIX-AG NPs could be seen to generate 2.4% more singlet oxygen than free PpIX
in water. As results of singlet oxygen generation for both MPpIX and FA-PpIX-AG NPs
confirmed almost 2.33 less free drugs was needed to generate the same amount of singlet
oxygen (2.4%) when FA-PpIX-AG NPs were applied instead of MPpIX. From this result it can be
concluded that attaching the source of energy to the drug may lead to more efficient activation
of drug to generate more singlet oxygen.
MTT assay revealed that PpIX was highly toxic even at a low concentration of 2.5
µg/ml, which was able to kill almost 50% of cells. After conjugating AG NPs to PpIX and FA, the
amount of survived cells had increased significantly up to 90% for the same concentration of
free PpIX.
Cell imaging results showed the amount of survived cells was very low for those cell
exposed to PpIX. In addition, PpIX did not show a very distinguishable red emission after the
excitation at 405 nm, but FA-PpIX-AG NPs demonstrated a strong and detectable red emission
under the excitation at 405 nm, which we believe the red emission was enhanced because of
both the improvement of water dispersion and conjugation a source of energy to PpIX to make
FRET happen. So it seems after being excited by 405 nm wavelength, PpIX has an extra
excitation source attached to them to activate it. These dual factors of an additional source of
energy as well as the improvement of water dispersion may help photons to activate the
attached PpIX to give a red emission.
Results confirmed that FA-PpIX-AG NPs demonstrated not only a better cell viability,
which was more than 30%, but also a better toxicity under UV exposure of almost 15%. The
accumulation of PpIX in aqueous media seems to be the major reason of inefficiency of UV
treatment. The lack of proper water dispersion of PpIX causes great aggregation followed by
photoluminescent quenching because of dose dependency [100, 101, 149, 161]. But after
conjugating PpIX and FA to AG NPs, not only water dispersion improvement helped the drug to
be distributed better inside the media, but also, blue light generating AG NPs was providing the
103
drugs with an extra source of energy to excite the conjugated drug and induce a more efficient
toxicity. Dose dependency of photoluminescent was the main reason that FA-PpIX-AG NPs did
not show efficient treatment up to 5 µg/ml.
6.2 Future Works
Investigation of possibility of creating afterglow at lower calcination’s temperature and
shorter calcination’s treatment because of the direct effect of both mentioned parameters on the
size of NPs.
Investigation of the effect of other chemical additive (during calcination) on the size
decrement of NPs without any adverse effect on afterglow.
Seeking the possibility of increasing the CE of PpIX to the surface of NPs (to increase
the efficiency of in vitro UV treatment).
Completing in vitro X-ray treatment since the fabricated AG NPs had a potency of being
excited by both UV and X-ray and successfully conjugation of PpIX to the surface of AG NPs
was confirmed by FRET.
104
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Biographical Information
Homa Homayoni received her Master of Science (M.Sc ) degree in Textile
Engineering from Isfahan University of Technology (IUT) in 2007 with the highest grade
point average (4.0). She joined PhD program of Bioengineering department at UTA in
Spring 2010. During her M.Sc degree she accomplished two peer-reviewed journal
articles which one of them has been cited 68 times so far. In addition to several
conference papers she also had completed a certificate course from Switzerland
awarded by SULZER Company which is a very noteworthy achievement. Homa’s
academic outcome as a PhD student at UTA has been several conference
papers/presentations and several papers which are in progress. She is a member of
Biomedical Engineering Society (BMES), Controlled Release Society (CRS), Medical
Nanotechnology, Nano Material Society, Nanotechnology in Drug Delivery, and
Photodynamic therapy PDT.
.