PROTOPORPHYRIN IX (PPIX)-CONJUGATED SELF-LIGHTING ...

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


, 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


2.3 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


2.3.4 Luminescence Property ................................................................................... 51

2.3.5 Stability of Ultimate NPs in Water ................................................................... 52

2.3.6 Detection of Singlet Oxygen Generation ......................................................... 52


2.3.8 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


, Dy3+

Powder .................................................................. 56


, 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


4.1.2 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


, Dy3+

powder (modified sol-gel method) in

basic environment (pH=10) measured by Spectrofluorophotometer ................................ 57


, Dy3+

powder (modified sol-gel method) .......... 58


, Dy3+

powder (modified sol-gel method) ..................................................................................... 59


, 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.

http://www.cancer.gov/Common/PopUps/popDefinition.aspx?term=lymph&version=Patient&language=English

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).

http://en.wikipedia.org/wiki/Protoporphyrin_IX

http://en.wikipedia.org/wiki/Heme

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].


, 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.


, 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

http://en.wikipedia.org/wiki/Absorbance

http://en.wikipedia.org/wiki/Wavelength

http://en.wikipedia.org/wiki/Spectrophotometer

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].


2.2.8.1 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.


2.3.3.1 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.

http://en.wikipedia.org/wiki/Organic_compound

http://en.wikipedia.org/wiki/Organic_compound

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.


2.3.7.1 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


, 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].


, Dy3+

powder

56

Figure 3-3 Luminescent decay after 5min X-ray irradiation of Sr2MgSi2O7: Eu

2+, Dy

3+ powder


, 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


, Dy3+

powder (modified sol-gel method) in acidic

environment (pH=2) measured by Spectrofluorophotometer


, 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.


, Dy3+


59


, 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].


, 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


, 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+


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+


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


Figure 3-14 The effect of temperature of calcination on XEOL and afterglow from Sr3MgSi2O8:

Eu2+

, Dy3+


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


Figure 3-15 The effect of the duration of calcination on XEOL and afterglow from Sr3MgSi2O8:

Eu2+

, Dy3+


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)


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.

http://en.wikipedia.org/wiki/Quantitative_analysis_(chemistry)

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.

.

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