Topical Clear Aqueous Nanomicellar Formulations for Age

TOPICAL CLEAR AQUEOUS NANOMICELLAR FORMULATIONS

FOR AGE-RELATED MACULAR DEGENERATION

A DISSERTATION IN

Pharmaceutical Sciences and Chemistry

Presented to the Faculty of University of Missouri-Kansas City in partial fulfillment of requirements for the degree

DOCTOR OF PHILOSOPHY

by Meshal Alshamrani

Pharm. D. King Abdul-Aziz University, Saudi Arabia, 2013

Kansas City, Missouri 2020

MESHAL ALSHAMRANI

TOPICAL CLEAR AQUEOUS NANOMICELLAR FORMULATIONS

FOR AGE-RELATED MACULAR DEGENERATION

Meshal Alshamrani, Candidate for the Doctor of Philosophy Degree

University of Missouri-Kansas City, 2020

ABSTRACT

Disorders affecting the posterior segment of eyes represent a significant burden to healthcare and patients’ wellbeing. Among the back of the eye disorders, age-related macular degeneration (AMD) is one of the major ailments responsible for blindness worldwide. There is currently no effective treatment available to cure AMD completely. Anti-VEGF therapy is currently considered the treatment of choice. However, it is invasive and does not offer substantial long-term relief to AMD patients. Other treatment options include thermal laser photocoagulation and surgery. None of these therapeutic approaches are safe and effective. Hence there is a real need for the development of an alternative therapeutic approach that is safe, effective and non-invasive.

The objective of this study is to develop a clear, aqueous drug-loaded nanomicellar formulation utilizing FDA approved polymers, namely Hydrogenated castor oil 40 (HCO-40) and

Octoxynol 40(OC-40) as a potential-therapeutics for AMD. Hydrophobic drugs such as curcumin

(CUR), celecoxib (CXB), and resveratrol (RSV) were successfully solubilized and entrapped in the core of the nanomicelles. These nanomicellar constructs efficiently utilize their hydrophilic corona and evade the wash-out into the systemic circulation from both the conjunctival and choroidal blood vessels and lymphatics, thus overcoming the dynamic barrier. A design of experiments (DOE) with JMP software was performed to optimize the formulation’s size, drug loading, entrapment efficiency, critical micellar concentration (CMC), polydispersity index

(PDI), zeta potential and delineate the effect of drug-polymer interactions. The formulation will be administered in the form of topical eye drops. iii

APPROVAL PAGE

The faculty listed below, appointed by the Dean of the School of Graduate Studies have examined a dissertation titled “Topical Clear Aqueous Nanomicellar Formulations for Age-

Related Macular Degeneration”, by Meshal Alshamrani, candidate for the Doctor of Philosophy degree and certify that in their opinion it is worthy of acceptance.

Supervisory Committee

Russell B. Melchert, Ph.D., R.Ph, Committee Chair Department of Pharmacology & Pharmaceutical Sciences

Hari K. Bhat, Ph.D. Department of Pharmacology & Pharmaceutical Sciences

Simon Friedman, Ph.D. Department of Pharmacology& Pharmaceutical Sciences

Zhonghua Peng, Ph.D. Department of Chemistry

Peter Silverstein, Ph.D. Department of Pharmacology & Pharmaceutical Sciences

TABLE OF CONTENTS

ABSTRACT ...... iii

LIST OF ILLUSTRATIONS ...... xiv

LIST OF TABLES ...... xvii

CHAPTER 1. OCULAR ANATOMY, DRUG DELEIVERY AND AGE-RELATED MACULAR DEGENERATION ......

1.1 Ocular Anatomy ...... 1

1.1.1 Anterior Segment of the Eye ...... 2

1.1.2 Posterior Segment of the Eye ...... 3

1.2 Ocular Drug Delivery Barriers ...... 8

1.2.1 Epithelial Tight Junction (ZO) ...... 9

1.2.2 Reflex Blinking ...... 9

1.2.3 Metabolism in Ocular Tissues ...... 9

1.2.4 Tear Turnover ...... 10

1.2.5 Nasolacrimal Drainage ...... 10

1.2.6 Efflux Pumps ...... 11

1.3 Ocular Drug Delivery Routes ...... 12

1.3.1 Systemic Route ...... 12

1.3.2 Topical Route ...... 13

1.3.3 Intravitreal Injection (IVT) and Implants ...... 15

1.3.4 Periocular Injection ...... 17

1.3.5 Sub-conjunctival Injection ...... 18

1.4 Age-Related Macular Degeneration (AMD) ...... 17

1.4.1 AMD Background ...... 18

1.4.2 AMD Pathogenesis ...... 19

1.4.3 AMD Statistics ...... 21

1.4.4 Dry AMD ...... 22

1.4.5 Wet AMD ...... 23

1.4.6 Therapies for AMD ...... 24

1.4.6.1 Nutritional Therapy ...... 24

1.4.6.2 Anti-VEGF Therapy ...... 25

1.4.7 Limitations of Anti-VEGF Therapy ...... 26

1.4.8 Intravitreal Implants ...... 27

1.5 The Use of Nanotechnology in AMD...... 28

1.5.1 Nanoparticles ...... 29

1.5.1.1 Emulsion-Solvent Evaporation Method ...... 30

1.5.1.2 Double Emulsion and Evaporation Method ...... 30

1.5.1.3 Salting-Out Method ...... 29

1.5.1.4 Nanoprecipitation Method ...... 29

1.5.2 Microparticles ...... 30

1.5.3 Core Materials ...... 34

1.5.4 Mechanism and Mechanics of Drug Release ...... 34

1.5.4.2 Dissolution ...... 34

1.5.4.3 Osmosis ...... 35

1.5.4.3 Erosion ...... 35

1.5.4.4 Applications of Microcapsules and Microspheres ...... 35

1.5.6 Nanomicelles ...... 36

1.5.7 Liposomes ...... 40

1.5.7.1 Liposomes Preparation ...... 40

CHAPTER 2. INTRODUCTION ...... 43

2.1 Overview ...... 43

2.2 Statement of the Problem and Hypothesis ...... 43

2.2.1 Rationale for Using Statistical Design of Experiments (DOE) ...... 44

2.2.2 Rational for Using Curcumin (CUR), Celecoxib (CXB), and Resveratrol

(RSV) ...... 45

2.3 Objectives ...... 46

2.3.1 Optimization of Curcumin (CUR) Nanomicellar Formulation: ...... 46

2.3.2 Optimization of Celecoxib (CXB) Nanomicellar Formulation: ...... 47

2.3.3 Preparation of Resveratrol (RSV) Nanomicellar Formulation: ...... 48

CHAPTER 3. SELF-ASSEMBLING TOPICAL NANOMICELLAR FORMULATION TO IMPROVE CURCUMIN ABSORPTION ACROSS OCULAR TISSUES ...... 48

3.1 Rationale ...... 48

3.2 Material and Methods ...... 51

3.3 Methods ...... 52

3.3.1 HPLC Analysis ...... 52

3.3.2 Experimental Design ...... 52 vii

3.3.3 Preparation of Curcumin Nanomicellar Formulation (CUR-NMF) ...... 55

3.3.4 Size, Polydispersity Index, and Surface Charge ...... 56

3.3.5 Viscosity ...... 56

3.3.6 Dilution Study ...... 57

3.3.7 Entrapment and Loading Efficiency ...... 57

3.3.8 Critical Micelle Concentration (CMC) Determination ...... 58

3.3.9 Transmission Electron Microscopy (TEM) ...... 58

3.3.10 Cytotoxicity Studies ...... 58

3.3.11 Lactate Dehydrogenase Assay (LDH) ...... 59

3.3.12 Antioxidant Activity Using ELISA ...... 60

3.3.13 ELISA (Enzyme-linked immunosorbent assay) ...... 60

3.3.14 In-Vitro Drug Release ...... 60

3.4 Results and Discussion ...... 61

3.4.1 Experimental Design ...... 61

3.4.2 Osmolarity and pH ...... 67

3.4.3 Size, PDI and Surface Charge ...... 67

3.4.4 Entrapment and Loading Efficiency ...... 67

3.4.5 Viscosity ...... 68

3.4.6 Dilution Effect ...... 68

3.4.7 Critical Micellar Concentration (CMC) ...... 68

3.4.8 1HNMR, FT-IR and XRD Characterizations ...... 71

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3.4.9 Transmission Electron Microscopy (TEM) ...... 74

3.4.10 Cytotoxicity Studies ...... 75

3.4.11 Cell Viability of D407 Cells Under Oxidative Stress by H2O2 ...... 77

3.4.12 Protective-Effect of CUR-NMF on D407 Cells Against H2O2-Induced

Oxidative Stress ...... 77

3.4.13 Oxidative Stress-Induced VEGF Secretion ...... 79

3.4.14 In-Vitro Drug Release ...... 81

3.5 Conclusion ...... 82

CHAPTER 4. SELF-ASSEMBLED BIOTINYLATED NANOMICELLES OF CELECOXIB FOR BACK OF THE EYE DISEASES ...... 84

4.1 Rationale ...... 84

4.2 Materials ...... 86

4.2.1 Cell Culture ...... 87

4.3 Methods ...... 87

4.3.1 Experimental Design ...... 87

4.3.2 Chemical Synthesis ...... 88

4.3.3 Characterization of The Synthesized Biotin Conjugates with 1H-NMRand FT-

IR ...... 88

4.3.4 Preparation of 0.1% CXB Loaded Biotinylated Nanomicelles ...... 89

4.3.5 Size, Polydispersity Index (PDI), Zeta Potential and Surface Morphology ..... 89

4.3.6 Entrapment Efficiency and Drug Loading...... 89

4.3.7 Osmolality and pH ...... 91

4.3.8 Clarity Examination...... 91 ix

4.3.9 Viscosity ...... 92

4.3.10 In-Vitro Cytotoxicity (MTT assay) ...... 93

4.3.11 Lactate Dehydrogenase Assay (LDH) ...... 93

4.3.12 Enzyme-Linked Immunosorbent Assay (ELISA) ...... 94

4.3.12.1 VEGF-A ...... 94

4.3.12.2 Pro-inflammatory Cytokines Analysis (TNF-α and IL-6) ...... 94

4.3.13 Drug Release Study ...... 95

4.3.14 LC-MS/MS Analytical Method Development ...... 95

4.4.15 Linearity, Sensitivity, Matrix effect, Recovery and Cell Uptake Study ...... 96

4.4.16 Cell Uptake Study by Confocal Microscopy ...... 98

4.5 Results and Discussion ...... 99

4.5.1 Experimental Design ...... 99

4.5.2 Chemical Synthesis ...... 103

4.5.3 1H-NMR and FT-IR ...... 103

4.5.4 Size, Polydispersity Index and Zeta Potential ...... 106

4.5.5 Entrapment Efficiency and Drug Loading...... 108

4.5.6 Osmolality and pH ...... 109

4.5.7 Clarity Examination...... 109

4.5.8 Viscosity ...... 110

4.5.9 Drug Release Study ...... 111

4.5.10 In-Vitro Cytotoxicity: MTT and LDH Assays ...... 112

4.5.11 ELISA ...... 117

4.5.11.1 VEGF-A Release from D407 ...... 117

4.5.11.2 Pro-inflammatory Cytokines TNF-α and IL-6 ...... 117

4.5.12 LC-MS/MS Chromatogram of Celecoxib with Internal Standard ...... 119

4.5.13 LC-MS/MS Method Validation ...... 120

4.5.14 Celecoxib Uptake Study into Retinal Cells from Nanomicelles ...... 121

4.5.15 Determination of Micellar Uptake by Confocal Microscopy ...... 122

4.6 Conclusion ...... 124

CHAPTER 5. RESVERATROL NANOMICELLES AS A POTENTIAL TRATEMENT FOR AGE RELATED MACULAR DEGENERATION ...... 125

5.1 Rationale ...... 125

5.2 Material ...... 127

5.3 Methods ...... 128

5.3.1 Cell Culture ...... 128

5.3.2 HPLC Analysis ...... 128

5.3.3 Preparation of RSV Nanomicelles ...... 128

5.3.4 Entrapment and Loading Efficiency ...... 129

5.3.6 Clarity Test ...... 130

5.3.7 Osmolality and pH ...... 130

5.3.8 Dilution Effect ...... 130

5.3.9 Viscosity ...... 131

5.3.11 1H-NMR and FT-IR ...... 131

5.3.12 Powder X-Ray Diffraction (XRD) ...... 132

5.3.13 Transmission Electron Microscopy (TEM) ...... 132

5.3.14 MTT Assay ...... 132

5.3.15 Lactate Dehydrogenase Assay (LDH) ...... 133

5.4 Results and Discussion ...... 135

5.4.1 Entrapment Efficiency and Drug Loading...... 135

5.4.2 Size, Polydispersity Index (PDI), and Surface Charge ...... 135

5.4.3 Clarity Testing ...... 136

5.4.4 Osmolality and pH ...... 137

5.4.5 Dilution Effect ...... 138

5.4.6 Viscosity ...... 138

5.4.7 Solubility Studies in Distilled Deionized Water (DDI) ...... 139

5.4.8 1HNMR, FT-IR and XRD Characterizations ...... 140

5.4.9 Transmission Electron Microscopy (TEM) ...... 143

5.4.10 Cytotoxicity Studies ...... 143

5.4.12 Protective Effect of RSV Nanomicelles on D407 Cells Against t...... 145

BHPS Induced Oxidative Stress ...... 145

5.4.13 Development and Optimization of a LC-MS/MS Method ...... 148

for Quantification of Resveratrol...... 148

5.4.14 Linearity and Sensitivity Study ...... 149

5.5 Discussion ...... 155

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5.6 Conclusion ...... 155

CHAPTER 6. SUMMARY AND RECOMMENDATIONS ...... 160

6.1 Summary ...... 160

6.2 Recommendations ...... 163

REFERENCES………………………………………………………………………………….164

APPENDIX……………………………………………………………………………………...194

VITA…………………………………………………………………………………………… 196

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LIST OF ILLUSTRATIONS

Figure 1. 1 Anatomy of the eye ...... 1

Figure 1. 2 Structure of the retina ...... 6

Figure 1. 3 Barriers for ocular drug delivery ...... 8

Figure 1. 4 Ocular and Systemic Pharmacokinetic Models for Drug Discovery and

Development ...... 12

Figure 1. 5 Schematic representation of the pathogenesis of AMD ...... 20

Figure 1. 6 Cequa (cyclosporine nanomicellar solution) approved by the FDA ...... 38

Figure 3. 1 Schematic representation of curcumin nanomicellar formulation (CUR-NMF ……………………………………………………………………………………………51

Figure 3. 2 (A) Pareto charts summarizes the effect of independent variables and their interaction on CUR-NMF size, PDI, %EE, %LD and % precipitation, respectively. Bar graphs extend past the tcrit cut off (blue vertical line) indicating statistical significance

(p<0.05) and (B) Surface profile for all the 5 responses...... 64

Figure 3. 3 Prediction profiler and desirability plot showing the effects of independent variables on CUR-NMF size, PDI, %EE, %LD and precipitation...... 66

Figure 3. 4 (A) Size distribution of the nanomicelles, (B) Zeta potential for CUR-NMF and (C) CMC: Plot of ultraviolet absorbance of I2 versus concentration of HCO-40/OC-

40 nanomicelles (2.5:0.1) in water. CMC was calculated by corresponding polymer concentration at which a sharp increase in absorbance was observed...... 71

Figure 3. 5 (A) Qualitative 1HNMR studies. 1HNMR spectrum for (i) blank nanomicelles, (ii) CUR-NMF and (iii) curcumin, (B) (i) Qualitative FT-IR spectrum for

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CUR-NMF, (ii) blank nanomicelles and (iii) curcumin alone, (C) XRD spectrums of

HCO-40, OC-40 and curcumin alone...... 74

Figure 3. 6 TEM micrograph of CUR-NMF suggests as spherical shape...... 75

Figure 3. 7 Cell cytotoxicity assays: (A) MTT (B) LDH conducted on D407 following

24h of CUR-NMF exposure. Triton-X 100 served as a positive control and culture medium as a negative control...... 77

Figure 3. 8(A) MTT assay on D407 cell line after exposing various H2O2 concentration for 24h. Data were expressed as mean ± SD (n=5), a p-value of <0.005 was considered to be statistically significant from the control and represented by *** and (B) Cells were pretreated for 4 hours with blank nanomicelles, 10 µM curcumin, 10 µM CUR-NMF, then all formulations were removed, rinsed with PBS, followed by the addition of 100

µM of H2O2. N=5, data are expressed as mean ±SD. A p-value of < 0.005 considers statistically significant from control and represented by ***...... 79

Figure 3. 9 (A) VEGF standard curve (B)Sandwich ELISA assay measuring VEGF concentration in D407 cell culture supernatant post exposure to H2O2 (24 h) in the absence or presence of 10 µM CUR-NMF. Values are represented as mean ± SD (n=4).

...... 81

Figure 3. 10 In vitro drug release in PBST (pH 7.4), at 37°C under sink conditions. Data are expressed in mean ± SD where n=3...... 82

Figure 4. 1 Schematic drawing image of self-assembled biotinylated nanomicelles loaded with CXB ...... 86

Figure 4. 2 Chemical structure of CXB (celecoxib) ...... 87

Figure 4. 3 Independent variables with coded, uncoded levels and their responses...... 88

Figure 4. 4 Pareto charts and surface profile for all responses summarizes the effect of independent variables and their interaction on CXB nanomicelles size (A), %EE (B), and

%LD (C), respectively. Bar graphs extend past the tcrit cut off (blue vertical line) indicating statistical significance (p<0.05) ...... 102

Figure 4. 5 Prediction profiler and desirability plot showing the effect of independent variables on biotinylated CXB nanomicelles size, %EE and %LD ...... 102

Figure 4. 6 Chemical synthesis schemes of biotinylated OC-40 ...... 103

Figure 4. 7 1H-NMR spectrums of (a) OC-40 in D2O, (b) biotin in d-DMSO and (c) biotinylated OC-40 in D2O ...... 104

Figure 4. 8 FT-IR spectrums of (a) OC-40 in D2O, (b) biotin in d-DMSO and (c) biotinylated OC-40 in D2O Preparation of the 0.1% CXB loaded biotinylated nanomicelles ...... 106

Figure 4. 9 (A) Size distribution, (B) intensity and (C) Zeta potential of biotinylated nanomicelles loaded CXB with DLS ...... 108

Figure 4. 10 TEM figure for CXB nanomicelles showing round smooth morphology .. 109

Figure 4. 11 Clarity test for CXB nanomicelles and DI water ...... 110

Figure 4. 12 Drug release study of naked CXB and CXB loaded nanomicelles carried out on PBST (pH 7.4) where n=3...... 112

Figure 4. 13 Cell viability assay MTT on (A) D407, (B) HCEC and (C) Conjunctival cells respectively, after 24 h of exposure to different concentration of CXB nanomicelles and CXB alone. (D) LDH release from of D407, CCL20.2 and HCEC cells after 24hr of exposure to blank and CXB loaded biotinylated nanomicelles. (Paired t-test) ...... 116

Figure 4. 14 ELISA analysis of (A) VEGF-A release from retinal cells D407 after exposure to different conc. of CXB nanomicelles; (B) TNF-α release in LPS-stimulated

RAW cells after exposure to blank and 30 µM CXB nanomicelles; (C) IL-6 release in

LPS-stimulated RAW cells after exposure to blank and CXB nanomicelles. The values are mean ± SD of triplicate. *p < 0.05, **p < 0.01 of blank vs biotinylated nanomicelles loaded CXB. Cells were exposed to treatment and control groups for 24 h...... 118 xvi

Figure 4. 15 (A) Standard curve and linearity characteristics of celecoxib; (B) LC-

MS/MS chromatogram of 10 µM celecoxib and 10 µM chlorzoxazone (IS)...... 120

Figure 4. 16 Celecoxib uptake study on HCEC cell line at 2, 6 and 24 h ...... 121

Figure 4. 17 Confocal localizations on conjunctival cells where Control = retinal cells with PBS; NT = non-targeted nanomicelles loaded coumarin as a hydrophobic fluorescent dye; T= biotinylated (targeted) nanomicelles; and T + B= biotinylated

(targeted) nanomicelles+ 1 mM biotin...... 123

Figure 5. 1 (A) Resveratrol (RSV) structure and (B) schematic representation of RSV nanomicelles ...... 127

Figure 5. 2 Average RT nanomicellar size and PDI using dynamic light scattering (DLS)

...... 136

Figure 5. 3 Clarity testing for RSV nanomicelles and DI water ...... 137

Figure 5. 4 Saturation solubility of resveratrol and resveratrol nanomicelles in water .. 139

1 Figure 5. 5 H-NMR spectrums of (A) RSV and (B) Blank nanomicelles in D2O ...... 141

Figure 5. 6 FT-IR spectra of (A) Blank nanomicelles, (B) Resveratrol, and (C)

Resveratrol nanomicelles ...... 142

Figure 5. 7 XRD analysis of (i) RSV nanomicelles, (ii) RSV and (iii) Blank nanomicelles

...... 143

Figure 5. 8 Cell viability assays conducted on D407 cells after 24 of exposure to different concentration of RSV alone and RSV nanomicelles (Paired t-test) ...... 144

Figure 5. 9 LDH assay conducted on D407 after 24 hr of exposure to different concentration of RSV alone and RSV nanomicelles. (Paired t-test) ...... 145

Figure 5. 10 Antioxidant Activity testing using tBHPS (500 µM) as a model of oxidative stress. Retinal cells were treated with tBHPS showed a significant decrease (p < 0.001) in viability compared to control (untreated cells). D407 cells pretreated with RSV nanomicelles and then tBHPS treated showed a significant increase (p < 0.05) in viability

xvii compared to tBHPS treated. All experiments were performed in triplicate and values were expressed as mean ± SD. Post hoc test (Bonferroni was used to compare tBHPS vs treated...... 147

Figure 5. 11 LC-MS/TOF of the RSV ...... 151

Figure 5. 12 Standard curve and linearity characteristics of Resveratrol ...... 152

Figure 5. 13 LC-MS/MS chromatogram of 10 µg/mL resveratrol and 1 µg/mL chlorzoxazone (IS) ...... 153

Figure 5. 14 Resveratrol uptake study on D407 cell line at 2, 6 and 24 h ...... 155

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LIST OFTABLES

Table 1. 1 List of current biologics used in AMD ...... 15

Table 1. 2 FDA approved intravitreal implants for AMD ...... 16

Table 3. 1 Independent variables with coded and uncoded levels. …………………...... 53

Table 3. 2 Summary of the independent factors and recorded dependent variables responses ...... 54

Table 3. 3 Checkpoint analysis...... 55

Table 3. 4 Dilution effect on CUR-NMF size and PDI...... 57

Table 4. 1 Size, loading and entrapment efficiency of CXB nanomicelles at varying wt. % ratio of polymers…………………………………………………………………………91

Table 4. 2 Physical properties of blank nanomicelles and CXB loaded biotinylated nanomicelles (F2) ...... 92

Table 4. 3 Mass spectrometric parameters for the most intense MS/MS transition of celecoxib and chlorzoxazone in negative ESI mode detection a...... 96

Table 4. 4 Recovery of celecoxib in retinal extract at three different concentration...... 98

Table 5. 1 Physicochemical properties of blank and RSV nanomicelles……………...135

Table 5. 2 Osmolality and pH of the Blank and RSV nanomicelles ...... 137

Table 5. 3 Dilution effect on RSV nanomicelles size and PDI...... 138

Table 5. 4Mass spectrometric parameters for the most intense MS/MS transition of

Resveratrol and Chlorzoxazone in negativeESI mode detectiona ...... 149

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ACKNOWLEDGEMENTS

I would like to express my immense gratitude towards my mentor Dean Melchert for his exceptional support and motivation during my graduate studies. His guidance was critical in solving a number of scientific challenges. I deeply appreciate him for encouragement and believing in me that helped me to develop critical thinking as a scientist. Special thanks to Dr. Gerald Wyckoff for his endless support and help. I would also like to extend my gratitude to members of my supervisory committee Drs. Friedman, Bhat, Silverstein and Peng for their constant support, discussions on my research and valuable critics on my dissertation. It is my honor to have such extraordinary scientists and researchers as a part of my supervisory committee. I thank my previous mentors Drs. Mitra and Pal. I am greatly thankful to Navid J Ayon, Dr. Coulibaly, Dr. Mary Joseph, and Dr. Holly for their timely help, their valuable suggestions on my research projects and creating such a cheerful environment in the lab throughout my studies. I am thankful to my junior Sadia, Vrinda and Mikel.

I sincerely appreciate constant support of Joyce Johnson, Sharon Self, Tamica Lige, and Nancy

Bahner in administrative assistance throughout my stay at UMKC. I appreciate support from Nancy

Hoover from School of Graduate Studies. I would also like to thank Dr. James B. Murowchick

(School of Geological Sciences) for helping in XRD analysis. My special gratitude towards my mother Hassanah Ali Hassan and stepmother Sahlah Hassan and my father Saleh Zuhair for their support. Without their endless love, support, I could never be the person who I am. They were always there for me and I never could ask for what they have given me. I would like to thank my wife Fatimah Mohammed, without her love, support and encouragement I would not be able to continue in this program, probably I would have quit from the program long back. Special thanks to my daughters Tulin and Seba

Dedicated to My Family

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CHAPTER1. OCULAR ANATOMY, DRUG DELEIVERY AND AGE-RELATED

MACULAR DEGENERATION

1.1 Ocular Anatomy

Efficient drug delivery to back of the eye remains a difficult approach due to its unique anatomical and physiological barriers1. The external and internal structures of the eye are protected by different barriers, such as dynamic and static, which lead to sub-therapeutic level of different drugs1-2.

The structure of the eye is divided into two segments: anterior and posterior. The anterior segment consists of cornea, conjunctiva, aqueous humor, and lens3. The posterior segment mainly consists of vitreous humor, retina, choroid, sclera, Bruch’s membrane, and macula (Fig. 1-1)4.

Figure 1. 1 Anatomy of the eye (modified from the reference) 5

1.1.1 Anterior Segment of the Eye

Cornea: The oval shaped cornea is avascular transparent tissue that helps the eye in two different ways. First, it blocks the passage of foreign particulates such as dust, water and bacteria to ocular tissues. Second, it has a large surface area which allows absorption of oxygen and nutrients from tear fluid which are then distributed to all layers of cornea6. cornea has a diameter of 12 mm (0.5 inch) with no blood vessels7. However, it has many nerves which help in pain sensation processes. Cornea is composed of five different layers. These layers include: (1) corneal epithelium, (2) corneal endothelium, (3) stroma, (4) Descement’s membrane and (5) Bowman’s membrane. Corneal epithelium is considered as the outermost layer8. Epithelium layer contains thousands of small nerve endings which plays a critical role in pain sensation especially when they get scratched or rubbed9.

Behind the corneal epithelium is Bowman’s membrane which is a transparent glassy film in nature and composed of protein fibers called collagen10. Stroma is located behind the Bowman’s layer and it’s considered as the thickest layer of the cornea. It primarily consists of water and collagen (connective tissue protein)11. Water provides the nourishment to the cells, while collagen provides the strength, elasticity and form. Collagen is synthesized in stroma in corneal corpuscles. Almost 90% of the cornea thickness arises from the stroma12.

Conjunctiva: It is a clear thin membrane that covers front and inner surface of eye13. The role of conjunctiva is to protect and lubricate ocular tissues by producing tears and mucus. Moreover, conjunctiva acts as a physical barrier by preventing entry of microbes into deep ocular tissues14. In addition, goblet cells which are located in conjunctiva secrete mucin which is a part of tear film.

Conjunctiva can be divided into three segments: tarsal conjunctiva, ocular conjunctiva, and conjunctival fronices. The thickness of conjunctiva is around 33 microns. The conjunctiva has 3 to 5 layers of epithelium15.

Aqueous humor: It is a clear transparent fluid that fills and helps to form anterior and posterior chambers of the eye. Clarity of lens and cornea allow light to transmit through them easily, and therefore cannot be invested within a vasculature16. One of the main functions of aqueous humor is to 2 provide nutrition and remove waste products of ocular tissues. Moreover, aqueous humor maintains homeostasis and stabilizes ocular tissues. Aqueous humor also allows inflammatory cells and mediators to diffuse in the eye in pathological conditions, as well as drugs to be circulated to different ocular structures17.

Lens: Anatomically, the lens is an almost transparent ocular structure that is biconvex in shape.

It remains suspended posterior to iris. The principal role of the lens is to focus rays of light onto the retina5. Lens is composed of unique cells that are elongated in nature with no blood supply of its own.

However, it fulfills its nutritional requirements via fluids flowing in the neighboring tissues. These fluids mostly consist of aqueous humor that flows in anterior segment of lens. Toxic waste removal is performed by these fluids as well5.

Ciliary muscles control shape of lens, and it may be changed by relaxation and contraction of surrounding ciliary muscles. This allows the eye to focus its vision on objects at widely differing distances clearly. The property of the lens to be able to properly adjust from far to a closer focus is referred to as accommodation which reduces with increasing age. This condition is referred to presbyopia, and most of the time it requires corrective action. Formation of cloud or the lens turning opaque, a condition referred to as cataract, may also occur with advancing age. Cataracts that cloud our vision may be corrected by surgical intervention, which involves removal of clouded lens with a replacement artificial lens18.

1.1.2 Posterior Segment of the Eye

Sclera: Sclera consists of densely packed connective tissue present in eyeball that creates the so called "white" portion of the ocular system. Thickness of sclera varies between 0.3 mm to 1.0 mm.

It consists of small fibrillary collagen19. This fibrillary collagen is packed in bundles of irregular and interlacing pattern. This kind of random and interweaving pattern of connective tissue fibers is responsible for a strong and flexible eyeball. The sclera lacks any dedicated supply of blood vessels.

Few blood vessels pass through sclera in order to bathe other tissues. However, they do not support sclera metabolically. Hence it is practically avascular (i.e they lack blood vessels)20. Nourishment of 3 scleral region is performed by blood vessels present in episclera tissues. Episclera is composed of a thinly spread loose connective tissue layer overlaying the scleral region and below optically transparent conjunctiva covering both sclera and episclera21. The other route of scleral nourishment is obtained from choroid present below sclera. Choroid is inundated with blood vessels and present between sclera and retina. Sclera is responsible for maintenance of eyeball shape along with intraocular shape. The tough, fibrous composition of sclera is also responsible for providing a degree of protection to the eye. The movement of eye is also somewhat controlled by sclera as it provides a strong attachment to extraocular muscles22.

Bruch’s Membrane: Bruch's membrane (BM) is a distinctive structural feature of eye composed of 5 laminar structures. It occupies a strategic location situated right between retinal pigment epithelium (RPE) and fenestrated choroidal capillaries23. BM is composed of elastin as well as collagen- rich extracellular matrix which serves the function of molecular sieve. BM is responsible for partial regulation of bidirectional transfer of biomolecules, nutrients, oxygen, fluids and metabolic waste products in-between retina and circulatory system of body24. Recent scientific literature points to the fact that age, genetic factors, environment, location of retina are the parameters that influence molecular, structural and functional properties of BM. Hence a segment of features of BM are unique to each person of a certain age. This directly correlates with development of normal vision as well as disorders of the ocular system25. With advancing age, BM undergoes change which involves enhanced calcification of elastic fibers, more cross-linkage of collagen fibers as well as higher production of

Glycosaminoglycan. Also, advanced glycation end products (AGEs) and fat deposition takes place26.

These age-induced modifications have effect on well-being of photoreceptor cells and also cause beginning and progression of disorders including retinitis pigmentosa(RP), age related macular degeneration (AMD) and similar debilitating diseases27. It is also the place where development of drusen occurs. Drusen with significant confluence and limitless activation of subsequent complement cascading pathway are initial symptoms of AMD development. The property of adhesive interactions that occurs between RPE and BM is responsible for retinal detachments and proliferative retinal 4 disease28. Finally, BM plays both an active or passive function in several other retinal diseases like

Pseudoxanthoma elasticum (PXE), Sorsby's Fundus Dystrophy and Malattia Leventinese. Hence BM plays an important role in retinal pathology, notably age-related macular degeneration29.

Macula: Middle portion of retina is referred to as macula. Macula is considered to be the functionally active part of retina. It is usually responsible for “20/20” vision and allows for optimum colorful vision. It is relatively small measuring about 5 mm in diameter. Located in the center of macula in a structure called fovea30. It is considered to be the most significant portion of human eye.

Commonly, complete vision loss does not occur until fovea gets affected by diseases like diabetic retinopathy (DR), age-related macular degeneration (AMD), etc. Macula is the region where light rays are focused by structures like cornea and lens present in anterior part of eye31. It is responsible for capturing image that is transmitted to brain, where the process of vision finds completion. Macula is also responsible for detailed visual and reading capacity while other parts of retina are responsible for peripheral vision32.

Macular Pigment and Its Contribution to Vision: Diabetic retinopathy is a major disease that has a profound effect on macula. Commonly it leads to leakage of fluids referred to as diabetic macular edema. This is one of the major causes of blindness due to diabetes. Blindness in the geriatric population usually occurs from cataracts, which may be taken care of by surgical intervention or age- related macular degeneration (AMD), which may not be as easily treated. Dry AMD has no treatment, while wet AMD may be treated with injections into vitreous body. Other significant disorders of macula include macular holes and macular pucker, diseases that may be treated surgically33.

Retina: The retina is a composed of photoreceptors sensitive cells within eye which obtains photons and passes them along neuronal pathways as both electrical and chemical signals to brain to perceive a visual image. The retina is located in posterior segment of eye. Retina is made up of millions of photoreceptor sensitive cells which play an important role in vision. The basic function of the retina is to translate the coming light to brain through optic nerve34. Under microscope, it is observed that retina is made up of ten layers of cells: (1) inner limiting membrane, (2) retinal nerve fiber layer, (3) 5 ganglion cell layer, (4) inner plexiform layer, (5) inner nuclear layer, (6) outer plexiform layer, (7) outer nuclear layer, (8) external limiting membrane, (9) photoreceptor layer, and (10) retinal pigmented epithelium cells (RPE)35.

The most important layers in retina are photoreceptor layer and retinal pigmented epithelial cells (RPE). Photoreceptor Layer is composed of the inner segments and outer segments of rods and cones. There are roughly 6 million cones and more than 100 million rods within the retina36.

The outermost retinal layer is called retinal pigment epithelium (RPE). It contributes to blood- retinal barrier (BRB) in conjunction with the endothelium of the retinal vessels and has many functions including electrolyte transport and secretion of growth factors and cytokines (Fig. 1-2). The RPE cells intermingle with outer segments of rod and cone cells. RPE cells play an important role in the support and maintenance of both photoreceptor cells and underlying capillary endothelium37.

Figure 1. 2 Structure of the retina38

Choroid: choroid is mainly a vascular structure inundated with numerous blood vessels that bathes outer portion of retina. It has numerous unique properties: It has many huge membrane-lined pores, which may act as an instrument of lymphatic drainage, and it is also able to alter volume, hence changing amount of choroid thickness39. It is also composed of non-vascular smooth muscle cells, primarily at the back of fovea. Contraction of these muscle cells leads to thinning of choroid, leading to opposition of the thickening action caused by expanding lacunae. Choroid also contains intrinsic neurons, mainly at the back of central retina. These neurons are responsible for controlling the muscles and may also modulate flow of blood in the choroid40.

The choroid performs many functions: Its blood vessels are responsible for the outer retina; blockage of oxygen supply from choroid to retina may be a reason for development of AMD41. The choroidal blood flow may also be responsible for temperature control in retina. Also due to its vascular actions, the choroid is composed of secretory cells, mostly responsible for control of vascularization as well as scleral growth. Lastly, drastic modulation in thickness of choroid controls motion of backward and forward movement of retina causing photoreceptors cells to be in the plane of focus42.

Vitreous Humor: The vitreous humor occupies a major part of the eyeball. It consists of a gel like fluid with clear consistency. It is usually present in region at the back of lens and in anterior portion of the retina at the posterior portion of the ocular system43-44. Since eyes are responsible for processing visual information, vitreous humor must be transparent enough for light rays to conveniently penetrate through it. Most of this humor is composed of aquatic component, and a small percentage of collagen, salt, and sugar45.

Vitreous humor lacks mobility, has no blood supply and it is not actively created or replenished which is a stark contrast to the aqueous humor. If any external object gets entry into vitreous humor, it will remain suspended there till it gets removed by surgery. These may be a clump of cellular debris, or may be blood, are known as floaters, which, if not taken care of, may negatively affect a person’s vision46. As one grows old, viscosity of this fluid decreases. This may cause posterior vitreous detachment. This is a condition in which vitreous is removed from retina. It is common in geriatric 7 population over seventy years of age. Problems of vitreous humor may also cause retina to detach eventually which may require surgical intervention. If proper care is not taken, retinal detachment may cause permanent blindness47.

1.2 Ocular Drug Delivery Barriers

There are many barriers that limit the delivery of drug such as blood retinal barrier (BRB), nasolacrimal drainage, blinking reflex, epithelial tight junction, efflux pumps and tear turnover (Fig. 1-

3)48-50.

Figure 1. 3 Barriers for ocular drug delivery51

1.2.1 Epithelial Tight Junction (ZO)

The corneal epithelium is the initial barrier to absorption of therapeutics when delivered via topical administration. It has several stratifications and is composed of a basal layer of cells which are columnar in shape, two to three layers of wing cells and finally one or two squamous cellular layers in 8 the outer periphery. The outer cellular layer is surrounded by a region of intercellular tight junctions also known as zonula occludens (ZO). The ZO function as barriers that prevent therapeutics from permeating via paracellular route. The ZO consists of anastomotic strands that is responsible for prevention of therapeutic absorption via paracellular route. There are four protein associated with tight junctions. These are ZO-1, cingulin, ZO-2, and occluden52. Occluden is the most significant of all the four tight junction proteins as far as functional importance is concerned. Concentration of calcium in extracellular and intracellular region of ZO affects the permeability of drugs. If ZO membrane integrity is broken or calcium ions present in extracellular region are chelated out via artificial agents like EDTA, permeation of therapeutic molecules are enhanced throughout ZO. Pores present in epithelial region of cornea are negatively charged at physiological pH. This causes negatively charged agents to move slowly as compared to positively charged agents. Also, concentration of calcium in the cellular layer levels as well as actin filaments that are present as component of cytoskeleton act as significant parameter in retaining ZO consistency53.

1.2.2 Reflex Blinking

A normal eyedropper can transfer approximately 25–56 µL of topical formulation with average volume being around 39 µL. But an eye can hold 30 µL in a temporary manner. The remaining volume is wasted due to nasolacrimal drainage or reflex blinking (5–7 blinks/min). This lowers amount of available therapeutics significantly54.

1.2.3 Metabolism in Ocular Tissues

Therapeutics that consists of aromatic hydrocarbons are subjected to metabolism and degradation in pigmented epithelium and ciliary body the eye. They are converted to their corresponding epoxides and phenols and are subsequently even more degraded by other enzymes present in ocular fluids and tissues55. Hayakawa et al. showed that less than optimum absorption of therapeutics of peptide origin and insulin is a function of large-scale degradation while undergoing permeation process through conjunctival region56. This observation was made in albino rabbits, an animal model that can mimic human eye. Schoenwald et al. showed clearance of therapeutic molecules 9 via aqueous humor turnover is significantly lesser in contrast to other pathways that are involved with the clearance process. This indicates most of the administered therapeutics get eliminated by metabolic pathways57.

1.2.4 Tear Turnover

A major barrier to topical delivery for ocular diseases is tear turnover. After a drug is administered via the topical route, it leads to an enhancement in amount of cul-de-sac. This causes reflex blinking which leads to greater secretion in tear, finally causing fast depletion in therapeutics from precorneal area58. Loss of therapeutic solution continues via tear turnover and nasolacrimal drainage till tear volume of the conjunctiva cul-de-sac is in normal range (7–9 µL) again59.

1.2.5 Nasolacrimal Drainage

As elucidated above, most of applied therapeutic concentration gets removed due to tear turnover or nasolacrimal drainage. Approximately 95% of applied therapeutic dose gets removed via conjunctival as well as nasolacrimal duct. The lacrimal drainage system in human adults acts as an intermediary for flow of tear from eye to nasal cavity60. The pathway of this flow is composed of the following components: puncta, canaliculi, lacrimal sac, and nasolacrimal duct. In a Histological section, it is observed that walls of lacrimal sac and nasolacrimal duct are highly perfused with blood vessels61. This creates an ideal environment for systemic absorption of therapeutic absorption.

Following topical application of drugs, eye drop solution mixes with lacrimal fluid in the first phase.

The time of contact available for drug with ocular tissues is approximately 1–2 min. This extremely limited timeframe is due to constant production of lacrimal fluid62. Almost half of applied drug volume flows into upper canaliculus. The remaining drug volume passes into lower canaliculus of lacrimal sac.

Flow of therapeutic drug solution continues into nasolacrimal duct, following which drainage in nasal region occurs. A few important parameters that control concentration of therapeutics that are applied topically are i) volume of applied therapeutic solution ii) reflex blinking and iii) age of patient. If volume of instilled drug is high, it will mostly be drained into nose from the nasolacrimal sac, and if volume is low, elimination occurs rapidly via lacrimal sac63. 10

1.2.6 Efflux Pumps

Efflux proteins are present either on apical or basolateral side of cell membrane. These proteins are responsible for restriction or enhancement of absorption of therapeutic molecules, depending on their polar location. The ATP-binding cassette, popularly referred to as ABC proteins, consists of super family of proteins64. These proteins are encoded by an MDR1 gene, and these proteins causes efflux of numerous substrates from cell membrane and cytoplasm into extracellular environment. There are mainly two major classes of efflux proteins that cause drug resistance: (a) P-glycoprotein, which prevents entry of amphipathic molecules (b) multidrug resistant protein (MRP), responsible for extruding out organic anions and conjugated compounds65.

P-glycoprotein 1 (P-gp), also known as MDR1 or ABCB1, consists of a ~170 kDa ATP dependent efflux pump. It is usually situated on apical surface of polarized cells. It causes depletion of therapeutics in multidrug-resistant cells. P-gp has been observed to be expressed on numerous ocular cells and tissues. These include ocular conjunctival epithelial cells, ciliary non-pigmented epithelium, human cornea, iris and ciliary muscle cells, and retinal capillary endothelial cells etc. Expression mRNA transcript of P-gp has also been observed in human cornea. Constable et al. investigated expression of P-gp in three human RPE derived cell lines- ARPE19, D407, and h1RPE. It was observed

D407 cells express P-gp in a significant amount and may be used for in vitro drug transport studies66.

MRP is a ~190 kDa membrane-bound efflux protein that is encoded by ABCC1 gene. It is usually found on basal side of polar cells. It functions as an organic anionic transporter. It is responsible for transport of glutathione, cysteinyl leukotrienes, glucuronides, sulfate conjugates and bile salts. It has been observed that mRNA transcript of MRP is expressed in the human corneal epithelium67-68.

Like P-gp, MRP is also a family of proteins and has many isoforms. It has been demonstrated that

MRP5 expression is significantly more compared to MDR1, MRP1–MRP4, MRP6, and BCRP in ocular tissues69. Chen et al. studied localization of efflux protein expression in numerous ocular tissues.

It was observed that human cornea efflux transporters like MRP1–4, MRP6 were situated in basal side of corneal epithelium, while MRP7 and MDR1 were present in almost all sides of corneal epithelium70. 11

In contrast to corneal epithelium, in human conjunctiva, MRP2–4, MRP6, MDR1, and BCRP were present in basal side while MRP1, MRP7 were observed to be present in all sides of conjunctival epithelium. In human iris ciliary body, MRP1–2, MDR1 were observed in stromal cells.

1.3 Ocular Drug Delivery Routes

Therapeutics delivered to eye can be divided into three groups; topical (drops, emulsions, suspensions, ointment and gels), systemic (oral and I.V), and intraocular implants/injections (Fig. 1-4).

Figure 1. 4 Ocular and Systemic Pharmacokinetic Models for Drug Discovery and

Development

1.3.1 Systemic Route

One of the major drawbacks of systemic delivery of ocular therapeutics is that blood–aqueous barrier (BAB) and blood–retinal barrier (BRB) act as significant barriers for anterior and posterior segment ocular delivery of therapeutics, respectively. BAB is composed of two discontinuous cell layers. These two layers are situated in the front part of the eye and are composed of endothelial cells of the iris/ciliary blood vessels and the ciliary non-pigmented epithelial cells. These two cellular structures induce expression of tight junctional protein complexes that creates barrier to entrance of 12 solutes71. Blood–retinal barrier (BRB) prevents successful entry of drugs from circulatory system to posterior ocular segment. BRB consists of two kinds of cellular layers. These are retinal capillary endothelial cells and retinal pigment epithelium cells (RPE). These two layers are referred to as the inner BRB and outer BRB, respectively. RPE, situated in-between neural retina and choroid, consists of a single layer of cells performing highly specialized function. RPE helps in biochemical and metabolic activities by allowing selective transportation of biomolecules in-between photoreceptor cells and choriocapillary layer. Also, it is responsible for maintenance of vision by taking up of and transformation of retinoids72. However, ZO of RPE is efficient in prevention of permeation into intercellular space. Following delivery of therapeutics using oral or intravenous route, drugs permeate with ease into choroid as it is highly perfused with blood vessels in contrast to retinal capillaries.

Organization of choriocapillary are fenestrated in nature causing fast equilibration of therapeutics in the circulatory system with non-perfused portion of choroid. Nevertheless, outer BRB which is composed of RPE prevents subsequent penetration of therapeutics from choroid into retina. Although it is advisable to dose therapeutics to retina using systemic route, its successful deployment is difficult due to presence of BRB, which tightly controls entry of therapeutics from systemic circulation to retina73.

1.3.2 Topical Route

Topical delivery of ocular therapeutics are in most cases administered as drops and are used for treatment of diseases related to anterior ocular segment. Majority of topically administered therapeutics act at various strata of cornea, conjunctiva, sclera, and similar tissue segment of anterior ocular segment like iris and ciliary body74. Once topical drugs are applied, precorneal factors and anatomical barriers exert adverse effects on bioavailability of topically applied therapeutics. Precorneal factors that are involved in this adverse event are drainage of applied solution, blinking of eye, tear film, turnover of tear, and externally affected lacrimation. Lacrimation constituent as well as volume of tear film determines health of ocular surface and forms initial resistant barrier to drug permeation as it has significant turnover rate. Gel forming mucin proteins in tear film provides protection as it forms 13 a hydrophilic layer which removes xenobiotic and ocular insults by moving over the glycocalyx75.

Human tear volume is approximately 7 μl, and ocular cul-de-sac may temporarily hold nearly 30 μl of topically administered ocular drop. Nevertheless, tear film exhibits fast restoration time of 2–3 min, which causes washout of topically applied dosage form within a rapid time frame of 15–30 s following administration76.

Apart from tear film and tear turnover, different layers of cornea, conjunctiva, and sclera function as an important determinants in effective drug permeation. Cornea consists of most anterior component of the ocular system. It functions as mechanical barrier that prevents entry of xenobiotics into eye and provides a degree of protection to ocular tissues. It may be classified into a) epithelium b) stroma c) endothelium. Each strata of cornea has its unique polarity with associated barrier that prevents permeability of therapeutic molecules77. The first layer of epithelium is hydrophobic and consists of lipid molecules. It provides substantial barrier to drug permeability of hydrophilic therapeutic molecules that are topically administered. Also, superficial epithelium of cornea is connected to each another via desmosomes and is ribbon-like tight junction proteins that surround this structure. These tight junctional proteins reduce permeability of therapeutics via paracellular route52.

The second layer of cornea, stroma provides 90% of the thickness that cornea occupies, and consists of an extracellular matrix which is comprised of collagen fibrils that are arranged in a laminar manner. Stroma has high water content and is a major barrier to absorption of hydrophobic therapeutic molecules. Finally, endothelium is the final layer of cornea comprised of hexagonal-shaped cells arranged in a single layer. Hence corneal layers, mainly the first two layers of epithelium as well as stroma, act as principal barriers to topical ocular drug administration78.

1.3.3 Intravitreal Injection (IVT) and Implants

In the last 30 years, intravitreal route of drug administration via injection has been popularized.

Numerous therapeutic platforms are being synthesized only for delivery via intravitreal route. It is now considered routine in treatment of many eye diseases. IVT delivers therapeutic entities such as solution, suspension, or depot formulations directly injected into the vitreous humor through the pars plana. IVT 14 is a highly efficient method for delivering drugs into the posterior segment. It is widely used for diabetic macular edema and AMD79. One of the major advantages of IVT injection includes targeted delivery of therapeutics that overrides all the involved drawbacks. Disadvantages with IVT injection involves peri-injection pain, intraocular and subconjunctival bleeding, increased intra ocular pressure, leaky wound, and finally toxic reaction on surface of eye due to use of cleanser prior to injection. Infectious endophthalmitis is a major side effect of IVT injection. Other major complication associated with IVT injection are retinal detachment, increase in intraocular pressure, retinal detachment, and macular infarction (in case of intravitreal gentamycin administration)80. Table 1-1 is the summary of current biologics used for age-related macular degeneration (AMD)

Table 1. 1 List of current biologics used in AMD

TRADE GENERIC SUMMARY/FDA-APPROVAL DATE MWT REF NAME NAME

Beovu® Brolucizumab Humanized single chain Antibody 26 k 81 fragment/ FDA approved for wet AMD Da on October 8, 2019

Lucentis® Ranibizumab Humanized Anti-VEGF Fab fragment / 48 k 82 FDA approved for wet AMD on June Da 30, 2006 VEGFR 1/2 - Fc fusion protein, FDA Eylea® Aflibercept approved for wet AMD on November 115 k 83 21, 2011 Da

Avastin® Bevacizumab Full antibody VEGF inhibitor / Off 149 k 84-85 label for AMD Da

Macugen® Pegaptanib Single strand of nucleic acid anti-VEGF 50 86 aptamer/ Off label for AMD kDa

Currently there are three long-acting corticosteroid implants which are FDA approved for

AMD: the fluocinolone acetonide implants Retisert® and Iluvien® and the dexamethasone drug delivery system Ozurdex®. They offer an alternative approach in treatment of AMD87. Their advantage over treatment with biologics is that they provide a long-term control of inflammation and macular edema with a less frequency of injections. Side effects like cataract and glaucoma are common with 15 implants, therefore, careful patient selection and close monitoring is essential. The development of new devices is a future challenge in order to improve drug delivery systems (Table 1-2) 88.

Table 1. 2 FDA approved intravitreal implants for AMD

TRADE NAME GENERIC NAME SUMMARY/FDA-APPROVAL DATE REF

Retisert® Fluocinolone 0.59 mg is implanted into the posterior 89 acetonide segment of the affected eye / April 08, 2005

Iluvien® Fluocinolone 0.19 mg is indicated for the treatment of 90 acetonide diabetic macular edema (DME) / September 26, 2014 0.7 mg (700 mcg) dexamethasone in the Ozurdex® Dexamethasone intravitreal implant / June 6, 2009 91

1.3.4 Periocular Injection

Generally, steroids are administered via periocular route. This route is usually adopted in case of vitritis and CME. This route is also used for treatment of posterior or intermediate uveitis to prevent toxic effects involved with systemic immunosuppressive agents. These injections may be beneficial for delivery of local immunosuppressive agents to affected eye of patients suffering from assymetric disease who have already been prescribed systemic immunosuppressant92. In anterior uveitis, this route of administration may be utilized for treatment of CME as it can create a localized depot of corticosteroid if patient is noncompliant.

Periocular steroid therapy is usually considered if visual acuity has been reduced to between

20/40 and 20/60. It can be functionally durable for a period of up to 3 months, but it may not start exerting its functional efficacy prior to 6 weeks following injection. This may not be useful for patients with severe vitritis or CME, who will require faster onset of action. Also, few patients of vitritis and

CME may exhibit resistant to repeated periocular steroidal injections. Development of elevated intraocular pressure (IOP) and cataract are some of the side effects associated with periocular injection93.

1.3.5 Sub-conjunctival Injection

Subconjunctival injections may be administered either below eyeball or below conjunctiva that lines the eyelid. Subconjunctival route for injecting therapeutics is usually opted to treat disorders in cornea, sclera, anterior uvea and vitreous layer. A major advantage of sub-conjunctival injection is it allows therapeutics to avoid epithelial layer, a principal obstacle that prevents drug entry as discussed previously. Major systemic exposure to therapeutics takes place due to this route of drug delivery.

Therapeutic concentration of drug may be achieved in both anterior and posterior vitreous segment of the eye via subconjunctival injection94. This leads to sub-conjunctival route of administration being a preferred route of drug delivery for anti-infective drugs. It is possible to use subconjunctival space to administer drugs to posterior ocular tissues in therapeutic concentrations by utilizing drugs that undergo slow dissolution alone or together with semisolid foundations. Retinal concentrations of Celecoxib are observed after a subconjunctival suspension depot is administered. This shows it may be used in disorders related to blood vessel leaks of posterior ocular segments95.

1.4 Age-Related Macular Degeneration (AMD)

1.4.1 AMD Background

The human eye is one of the most sensitive body organs, which performs crucial functions and helps to see things clearly . Indeed, anything which tampers with an individual’s vision causes a lot of stress and discomfort to the person. Age-related macular degeneration (AMD) is one of the most common causes of irreversible damage of the macular region in elderly population. Despite intensive clinical research, the pathogenesis is not yet comprehensively delineated. However, a complex interaction of genetics and environmental factors, aging process, oxidative stress, chronic changes in in the macular region may contribute to the onset and final picture of the disease. AMD is one of numerous eye conditions, which is common with people as they get older96. AMD refers to a situation, in which macula, an integral part of retina deteriorates. Macula plays a fundamental role in the vision process by enabling a sharper view of objects looked at from a distance. This chapter covers essential information regarding AMD, such as data about disease, types of AMD, pathophysiology of the 17 condition, available therapy methods for the disease, drawbacks of anti-VEGF therapy, and finally, use of nanotechnology in AMD. Clinically, there are two types of AMD: dry and wet AMD. The dry form

(geographic atrophy) is characterized by degeneration of retinal pigmented epithelial cells (RPE) and photoreceptor sensitive cells97. The wet (exudative form) is associated with choroidal neovascularization (CNV) beneath macular region with retinal bleeding which result in sudden loss of central vision. Drusen which is made up of fatty acids, lipids and some cell-fragments are common feature for both AMD types98.

1.4.2 AMD Pathogenesis

There are four major risk factors for development of AMD: age, ethnicity, genetics and environmental factors. Environmental factors include cigarette smoking, fat rich food, and UV exposure. Aging, cigarette smoking and UV exposure share the ability for more reactive oxygen species

(ROS) generation and promotion of oxidative stress99. During cellular metabolism humans produce

ROS from oxygen (O2). ROS levels in the retina are regulated to continue the process of cellular homeostasis. Oxidative stress is defined as a condition in which ROS levels are higher than the capacity of antioxidant system in the cells. This will result in irreversible damage to RPE and photoreceptors sensitive cells100

The exact cause of AMD is still to be determined, but combination of genetics and environmental factors may play a crucial role for the development of the disease. AMD causes drastic changes in the normal functioning of the macular and the entire retina part of the eye. Several physiological changes occur depending on the type of AMD that an individual is suffering from. As such, while focusing on dry AMD first, it is worth mentioning that the illness results in changes in the color of the retinal pigment epithelium. This is a crucial pigment, which is always darker in color and helps in nourishing and ensuring that photo-receptors work well .Therefore, there is a need to pinpoint that by distorting the color of the pigment, photo-receptors, which depend on this pigment, may not function well; as such, an individual would not adopt to both light and darkness101. Fig. 1-5 shows an illustration of the pathogenesis of AMD. 18

Accumulation of drusen interferes with functioning of macula because the cells are cut off from access to nutrients. Macular cells need those nutrients to enhance visual acuity. It is worth admitting that pile-up of waste materials such as fatty lipids and proteins from the retina also leads to the creation of abnormal shape or lining underneath this part of the eye, which limits it from wholly trapping light and translating it into signals that can then help in visualization of the given image.

Retina has a thin layer to allow passage of view, but the wastes interfere with its original shape, making it hard for it to perform its roles correctly102.

Wet AMD leads to abnormal development of cells underneath the retina. Leakage of blood cells causes more harm to macula as it makes the part to swell and lose its shape, causing its epithelial lining to detach from the retina103. Therefore, changes to macula not only lead to shifts in the physical structure of the eye part, but they also alter how the eye functions. Absorbed fluids from leaking blood vessels limit macula from working correctly. Wet AMD condition may worsen due to the disorder’s severity and rapid destruction of retina’s structures. People who suffer from this type of AMD are most definitely bound to blindness, a condition, which deprives a person a chance to see again or to conduct basic things in life as was earlier performed.

Figure 1. 5 Schematic representation of the pathogenesis of AMD104

1.4.3 AMD Statistics

Approximately 9 million individuals are affected with AMD in the United States (U.S) alone, with a global prevalence of 170 million in 2018105. Aging is the greatest risk factor; therefore, the prevalence of AMD in the U.S is anticipated to increase to 22 million by the year 2050, while the global prevalence may increase to 288 million by the year 2040105. The number of people suffering from AMD is expected to rise tremendously with the rapid increase in global population106. AMD accounts for approximately 8% of all the blindness across the world107. Such huge numbers are worrying, while medical reports and research studies indicate that people should expect more AMD complications in the coming years107. This alone shows magnitude of this disease and how it adversely affects the entire human population. AMD is painless, but the damage it causes to the eye is severe. The cure for this disease is not yet established. Many studies have shown that Americans who have an AMD condition are most likely to live in a depressing life situation, since by losing their visions, they automatically become dependent on others to help them108. The researchers state that currently, over 1.5 million

Americans are living distressed lives because they can no longer do things the way they did before the disease. As per the scholars’ analysis, recent research has not been detailed enough to analyze how such conditions can be prevented or reduced in society109.

Fisher et al110. also mention that with limited information on a proper treatment of this disease, several people will have to suffer before such inventions come true. A research study conducted by the scientists among the Hispanic Americans indicates that AMD is one of the leading causes of visual impairment among the community members110. As such, the authors mention that thorough research needs to be done to find solutions to this problem. They suggest that over 50% of the population living with this condition become frustrated with life, while others become depressed when they remember that their visions will never be the same again due to the irreversible nature of the disease110.

1.4.4 Dry AMD

There are generally two primary types of AMD disease. The first type of illnesses is called dry

AMD. In this kind of ailment, waste materials deposited by retina accumulate beneath it. The wastes are known as drusen, which are always yellow, and as the wastes pile up, they block movement of essential nutrients to retina98. Once the retina has been cut off from nutrients, the cells located in macula begin to lack vital nutrients needed to bring sharp and stray visions. As such, cells will die, leaving eye with no functional macula. Bartlett111 et al. stipulate that the worst part of this disease is that it is always noticed in its later stage; this is due to it is painlessness, and hence, the attention of an individual may not be drawn to the progress of the disease. The pathogenesis of early AMD is characterized by thickening of Bruch membrane (BrM) due to lipid and protein accumulation that lead to formation of sub-RPE deposits that occur as discrete accumulations, called drusen, which can be hard or soft, or as continuous accumulations.

Drusen comes in two forms, that is, the hard drusen and the soft one. The soft ones are irregular in shape and take the yellow form, while the hard ones take the round shapes. These deposits line up beneath retina, they interfere with an individual’s visual acuity112. Dry AMD may start with distortion of vision from one eye, and in its advanced stage, the disease may affect both eyes, where it would be difficult for an individual to perform necessary activities such reading or seeing people. People with dry AMD may see a straight line as a bent one. This means that for such people, the ability to view directly and sharply has been distorted, what is called visual acuity. People with dry AMD may also require very bright light whenever they read a book or look at an object. The reasons for this, is that the reduced clarity in vision due to the pile-up of drusen makes it difficult to read in the dark or places with a limited supply of light103. As such, an individual would want to be exposed to areas with high light intensity so that they can at least have a view of an object. Yonekawa et al. say103 that when such complications intensify in an individual, they may proceed on to a level that a person loses the sight completely. The scholars state that another symptom of this disorder is the reduced clarity in vision where an individual may still observe an image, but the given image may come out as a blurry object103. 21

A large percentage of people, nearly over 80%, experiencing AMD suffer from this type of illness. The disorder is common among people of older age where it leads to increased dependency for movement, reading, or driving113.

1.4.5 Wet AMD

This is the second type of disease, which is most severe compared to the other kind. It is termed as wet AMD because it involves abnormal leak of blood from vessels to the macula114. Mostly, it originates from dry AMD, which mainly leads to blockage of the retina with the waste materials.

Ertekin et al.114 explain that what first happens is abnormal growth of blood vessels beneath retina, and with their proximity to the macula, they may leak blood to sensitive part of retina. Due to leakage, the macula may swell and, in turn, take a completely different shape, hence resulting in a change in visual capability. Ertekin et al. state that at least 20% of people having AMD face wet type, which is more severe than any other kind of the disorder114. Wet AMD occurs when new abnormal blood vessels develop under the retina in a process called choroidal neovascularization (abnormal new vessel formation). Localized macular edema or hemorrhage may elevate an area of the macula or cause a localized retinal pigment epithelial detachment.

It is crucial for people of older ages to go for frequent eye exams to determine the status of their eyes. Ertekin et al114. mention that just like dry AMD distorts the visual acuity, wet AMD does it the same way. Wet AMD is always considered advanced stage of the disorder where permanent loss in eyesight is a definite outcome of the condition. Patients with this kind of disease are most likely to see black spots anytime they try to view an object114. These spots form in the middle of their vision field, and when the entire macula is covered by blood, they lose total sight.

Patients with the form of wet AMD are most likely to see straight lines as bent or irregular.

Ertekin et al115. mention that such kinds of views result from the distortion of macular shape where the retina element loses its smooth lining. The scholars state that at the moment, thousands of Americans who are old have reported this kind of disorder. The researchers, however, advise that such people should receive proper care, and that family members should always be by their sides to offer help in all 22 aspects of life. The elderly needs maximum attention of people who live with them, and if they do not have family members to stay with them, then there is a need to employ people who can offer them support in their old age. Such support may reduce any chances of stress or depressions among patients.

1.4.6 Therapies for AMD

1.4.6.1 Nutritional Therapy

The first type of therapy available for AMD is nutritional therapy. This treatment refers to process of administering nutrients, which contain high antioxidant content. The role of this kind of cure is to help in replenishing macular cells in early stages of dry AMD116. As such, during early stages of illness, cells of the macula will definitely lack nutrients due to accumulation of wastes117. As more waste increases, the condition of macula worsens as more cells in retina are deprived of essential nutrients to carry out crucial activities. All physiological processes rely on nutrients absorbed by cells.

As dry AMD condition deteriorates, more nutrient administration may be prescribed to help in boosting physiological processes of the cell. It is worth mentioning that it reaches a level when continued pumping of nutrients may not be helpful, since even if amount of nutrients is increased, the blockage of cells by drusen may limit cells from getting access to required amount of nutrients for them to function correctly117.

It is, therefore, always recommended for people of older age to visit eye clinic regularly so that any changes in their eyesight may be noticed quickly and at an early stage for appropriate actions to be taken117. The number of older people visiting eye clinics in America is still lower than expected, and according to Tisdale et al117., this happens due to numerous factors. The first factor is lack of awareness, which limits people from attending eye clinics on time. The scholars mention that there is a need to increase awareness among elderly so that many people can understand the benefits of reporting to opticians during the early stages of AMD conditions. As stated by Tisdale et al117., nutritional therapy is not a permanent cure for AMD illness. The scholars suggest that there is a need for more research to help in finding solutions to the illness. According to them, the government should set aside more funds to assist in researching the possible solutions how to treat dry AMD. According to the scientists, 23 projecting an increase in the number of people who will most likely suffer from the condition is not enough. They mention that as researchers and governmental organizations spend more time in forecasting increases in AMD among the American population and entirely across the globe, there is a need for relevant organizational departments to conduct thorough research, which may help in coming up with the long-lasting solutions to the AMD problem117.

1.4.6.2 Anti-VEGF Therapy

This is a common form of therapy currently used to manage wet AMD situations. The process involves administering intravitreal injection of anti-VEGF therapies such as ranibizumab, bevacizumab and aflibercept. The biologics prevent the development of more blood vessels underneath the retina.

One of the primary causes of wet AMD is leakage of abnormal blood vessels, which is always formed as the condition worsens118. These blood vessels are weaker than normal blood vessels, and as such, they tend to leak, unlike healthy blood vessels, which have strong walls to cushion any pressure. The abnormal growth of blood vessels may also cover a large portion of the retina, hence squeezing available space for essential elements such as macula119.

Patel et al.120 assert that injecting anti-VEGF can, however, lead to adverse effects when the body reacts unfavorably to injection. More so, some investigators mention that such doses should be administered by experienced eye specialists who understand the process well. They also add that entire process is very expensive, and hence, it cannot be afforded by everyone in the society118. The researchers, however, say that there are several success reports obtained after injections of the drug into eye. Administering these drugs has reportedly reduced chances of permanent blindness among people by 50%118. According to the researchers, this latest invention in the treatment of we AMD is of great importance, as many people have recorded huge benefits from its use.

1.4.7 Limitations of Anti-VEGF Therapy

Even though the use of anti-VEGF has proved to be effective due to many success reports, there are several drawbacks linked to the therapy. One shortcoming of this therapy is that it may lead to severe eye pain, which may last for many days. Human eye is one of the most sensitive organs, 24 which, when injected, may suffer from excessive stress and pain. , Usui-Ouchi and Friedlander118 mention that as patients are taken through this kind of therapy, there is a need to advise them on the potential adverse effects, such as pain in the eye.

The next limitation is that the anti-VEGF treatment may lead to complete changes in vision of an individual. Though there are many successful reports for this drug injection, the scholars add that challenges that generally arise from such factors are always more significant and can lead to severe health implications. Anti-VEGF therapy may cause retinal detachment, thus leading to serious ocular complications coupled with little to no interpretation of received light80.

Other limitations are that this kind of therapy is expensive and may lead to permanent vision loss. There is a high possibility for the eye to react with the drug injected; hence, there is a risk of the reduction of the vitreous quantity80. Vitreous is a crucial fluid component of the eye, which helps in maintaining the shape of the organ. When the amount of this fluid reduces, an individual may suffer from various challenges, such as the loss in the form of the eye and increased dryness beneath the retina, a condition, which may make more eye cells die. It is evident that despite the mentioned benefits of anti-VEGF therapy, many perils accompany it. Therefore, it is high time for the scientific community to develop alternative therapy which will be safer and painless, which may reduce the challenges that people have to go through in their old ages. It is, however, crucial to mention that there has been tremendous progress in the diagnosis of this condition. Various health agencies need to work together so that long-lasting solutions for this eye condition can be identified.

1.4.8 Intravitreal Implants

Intravitreal implants are generally used for administration of steroidal medications. They are a fairly novel mode of treatment for ocular disorders in general and macular edema in particular. They are also used for treatment of retinal vein occlusion, uveitis, and also in some cases diabetic retinopathy.

The steroidal medication is enclosed in a small capsule-like structure, which is subsequently injected into eye121.

The steroidal medication is released in a sustained manner for a few months. In general, there are two kinds of widely used implants, Ozurdex® – an implant of dexamethasone and Retisert®-an implant of fluocinolone acetonide. Ozurdex® is a popular and widely implant; Retisert® is deployed in some cases. Intravitreal steroidal implants are widely deployed to treat leakage (macular edema) associated with retinal vein occlusion, uveitis etc. Implants of fluocinolone acetonide may be useful as it provides for drug release for up to nine months and no repeat dosage is required but it has risk of severe side effects such as cataract. Intravitreal implants are not only useful for treatment of posterior uveitis patients, the steroidal therapeutic causes reduction of inflammation as well122.

One month following implantation, drugs begin exerting pharmacological effects, with subsequent improvement in vision for most patients. To begin with, mostly there is a temporary improvement in visual acuity, with a required subsequent injection after a month. There is a reduction of macular edema, with an observed maximum reduction of edema within two weeks, but the efficacy begins to disappear after three months. The injection of implant causes IOP to increase. Hence people suffering from glaucoma suffer from higher risk of side effects123. A significant portion of patients receiving this implant develops serious ocular infection124. Symptoms of infection are numerous and may include - blurred vision, pain, photophobia, redness, floaters, lid swelling, discharge, and there is a 1% risk of a retinal tear after this injection. Another major side effect associated with implants is the possibility of retinal tear. It manifests as sudden flashes of light or floaters in the months subsequent to injections125.

1.5 The Use of Nanotechnology in AMD

Even though the retina is an accessible part of the eye, permanent treatment of AMD has proven to be extremely difficult. Delivery of drugs into the retina poses a serious challenge to doctors due to anatomical complications126. As such, scholars state that use of nanoparticles can be of great help in ensuring efficient delivery of drugs into the retina. They mention that instead of using big objects, which may lead to severe eye problems, there is a need to employ use of nanoparticles to distribute the drugs to intended sections of retina. 26

Nanoparticles can also carry a both hydrophobic and hydrophilic drugs, even though the sizes of the particles are microscopic3. According to Patel et al.126, this is a crucial way of getting rid of unsafe ocular injections. Another benefit of the nano-technology implementation is that it involves drops of liquid drugs, which can spread to a vast region of eye, hence reaching various parts of the eye, which would not have been achieved through injection. The authors mention that injections solely focus on one region of the eye, and although a professional doctor should conduct an injection into the eye, various structures still may be destroyed126. To minimize such problems, use of nanotechnology seems to be promising.

It is worth concluding that AMD is one of the leading causes of vision loss among older people.

At the moment, there is no cure for the two types of this illness. There is a need for the government to fund more research, which will focus on study of this disease and ways for its treatment. It is a common disease among the elderly, and as such, there is a necessity to support these people both emotionally and physically in all their daily activities.

Method of drug delivery can have a huge impact on its activity and potency for treatment of different diseases. Various delivery systems are currently under development to overcome delivery obstacles such as to increase drug solubility, to prevent harmful side effects, and to slow down the degradation pattern of a particular drug. Nanoparticulate systems such as nanoparticles, nanomicelles, and liposomes have gained a lot of interest in pharmaceutical industry specifically in the drug delivery field. Nanotechnology is defined as use of materials which are less than 100 nm size127.

Nanotechnology has been used in diverse fields such as engineering, electronics, and physics; however, other aspects for nanotechnology in pharmaceutics include drug delivery systems which are being explored due to their massive impact in improving solubility, bioavailability and targetability for many drugs and many diseases. Nanotechnology and nanoscience have emerged rapidly in the last decade. It has opened a new era in the development of materials, especially those for medical applications.

Nanomaterials have many different size, shapes and compositions. The traditional forms of nanomaterials include polymeric nanomicelles, liposomes, quantum dots, nanoparticles and theranostic 27 microparticles. Nanomaterials have a potential role for improving ocular treatments128. The anterior segment of the eye can be easily reached with hydrophilic drugs. On the contrary, drug delivery to back of the eye is considered a challenge due to many reasons such as clearance mechanisms, ocular barriers and static barriers which prevent the access of the hydrophobic compounds129. So, different nanoparticulate systems in the following sections will be presented.

1.5.1 Nanoparticles

Nanoparticles have been extensively studied as a drug-carrier in ophthalmic preparation for ocular diseases. They can offer significant advantages over the conventional drug delivery such as the high drug pay load, and the ability to control drug release for long periods of time. Nanoparticles are defined as a colloidal solid particle with a diameter of 1 to 1000 nm. To enhance solubility of drugs, they can be entrapped inside nanoparticles or they can be adsorbed or attached to the surface.

Nanoparticles are generally prepared from biodegradable or non-biodegradable materials such as natural or synthetic polymers, golds, metals, and lipids130. Therefore, different types of nanoparticles have been studied for the diagnosis and the treatment of ocular diseases. There are different approaches for how nanoparticles are prepared. Selection of suitable method of preparation depends on different factors such as the physicochemical properties of the polymer and drug that will be entrapped.

Polymeric nanoparticles are prepared as the following:

1.5.1.1 Emulsion-Solvent Evaporation Method

This type of preparation is the most frequent one and it involves two steps. The first step is to emulsify polymeric organic solution in aqueous solution. The second step is to remove the organic phase using rotavap or Vacuum. Then, nanoparticles will be washed with water several times to remove unentrapped drug and followed by lyophilization. The size of nanoparticles here can be controlled by different factors such as type of dispersing agent, stirring time, and viscosity of the drug, polymers, organic phase and aqueous phase. The major drawback for this technique is the limitation of loading hydrophilic compounds131.

1.5.1.2 Double Emulsion and Evaporation Method

Double emulsions also called “emulsions of emulsions” and have the potential for encapsulating both hydrophilic and hydrophobic drugs. This method has a major advantage compared to emulsion-solvent evaporation method which cannot encapsulate hydrophilic compounds. The preparation process involves addition of aqueous drug solution to organic polymer solution under vigorous stirring to form water in oil (w/o) emulsion. The resultant emulsion is usually added to into second aqueous solution to form w/o/w emulsion. Then, the organic phase solvent will be removed with evaporator and the particles will be washed with water several times followed by lyophilization.

Variation in nanoparticle size is common in this method which is considered as a drawback of this technique132-133.

1.5.1.3 Salting-Out Method

Salting out method is based on separation of water-miscible solvent from aqueous phase through salting out agent. Magnesium chloride, magnesium acetate and calcium chloride are commonly used salting out agents. Their role is to prevent the miscibility of organic phase in aqueous phase which results in emulsion formation. The advantage of salting out method is to avoid use of organic solvents completely which may be toxic to human cells. Briefly, both drug and polymer are dissolved in organic solvent miscible in water. The homogenous solution is added to aqueous solution of salting out agent and stabilizer under vigorous stirring. To reverse effect of salting out agent, emulsion is diluted in excess water which will lead to precipitation of the polymer. Therefore, drug will be encapsulated inside the polymeric matrix which results in formation of nanoparticles134.

1.5.1.4 Nanoprecipitation Method

Nanoprecipitation is a simple method used for encapsulation of both hydrophilic and hydrophobic drugs inside core of nanoparticles. The formation of nanoparticles here is based on the precipitation process135. Typically, both drug and polymer must be dissolved in the first system (the solvent), but not in the second (non-solvent). The major drawback of such technique is wide range of the particle size and it requires filtration which might affect overall yield. 29

1.5.2 Microparticles

Microparticles are defined to be any particulate matter with a diameter ranging from 1-1000 micrometers, regardless of the internal or external architecture. Microparticles may be broadly classified as a) microspheres which are microparticles spherical in nature, and b) micro-capsules are microparticles with a core inside a layer composed of components different from center. The core may have any physical state.

Despite this general categorization, numerous investigators tend to use these two terms in an interchangeable manner that may create confusion. Generally, it is considered that a microsphere consists of a smooth blend of polymer and therapeutics, while microcapsules comprised of one single layer of therapeutics, at least, if not more. As the category and subcategory of therapeutic inside microcapsules become increasingly reduced, microcapsules transform into microparticles136.

Usually, it is considered a microcapsule when size ranges between 50 nm to 2 mm with a core component. Strictly considered, microspheres are spheres with a vacant interior. Microparticles have widespread applications in the domain of drug delivery platforms. Microscopic examinations through scanning electron microscopy (SEM) and transmission electron microscopy (TEM) have demonstrated intricate architecture of microcapsules, which has been revealed to be complicated and varied. It may possess a single core or may maintain several layers of the center. Covering the core several concentric layers of coating may exist. Microparticles may clump together in a different shapes as well as sizes and may also develop new outer coating walls. Ideal microcapsules are created by having a liquid core or creating microcapsules as a dispersed solution before it is solidified. Although micro-structure of each membrane and interior may be detected by SEM of surfaces or sections, their physical quality, involving consistency, tortuosity, and crystallinity, is difficult to characterize quantitatively in microcapsules137.

However, some progress has been made, and efforts are on to calculate permeability and porosity from unharnessed information, dimensions, densities, and core/wall ratios. The impact of size and form distribution has been studied recently. Microcapsules are finally distributed in numerous 30 dosage forms, like exhausting gelatin capsules, which can be enteric-coated, soft gelatin capsules, or suspensions in liquids, all of which permit dispersion of individual microcapsules upon release.

Microcapsules may still be of enough interest due to relative ease in design and formulation and also due to the benefits of microparticulate delivery systems. The latter factor is due to sustained release from every individual microcapsule, and they also provide more significant uniformity and dependability. A further advantage over other dosage forms includes safety in case of a burst or defective release in dosage forms. It has also been suggested that in case of microparticles138, several particulate systems are spread over sufficient area of gastro-intestinal tract, that will lead to (a) reduced therapeutic dose with low concentrations and thereby result in low toxicity and irritability profile, and

(b) smaller variation in rate of absorption and time of transit. A broad form of coating materials is out there for microencapsulation. Some patented innovative coating polymers have conjointly been developed for a few specialized applications, notably among bio-adhesives and muco-adhesive.

However, several older coating materials are satisfactory for deployment within the alimentary canal139.

Selection of suitable coating material from an extended list of candidates depends on the following criteria: a) particular dosage forms or product needs, like stabilization, reduced volatility, unharness characteristics, and environmental conditions; b) coating material that may justify financial investment; and c) microencapsulation methodology that is best suited to accomplish the coated product objectives. The selection of suitable coating material decides physical and chemical properties of resultant microcapsules/ microspheres. While choosing a candidate coating material, parameters that need to be considered are: stabilization, reduced volatility, release characteristics, environmental conditions, etc. Chemical constituents of the coating material should be able to form bonds that are cohesive with core materials. They have to be chemically compatible, non-reactive with core material, and supply specified coating properties like strength, flexibility, solidness, optical properties, and stability140.

Generally, hydrophilic polymers, hydrophobic polymers, or a mixture of each are used for the microencapsulation method. A variety of coating materials have been used successfully; samples of 31 these include: gelatin, polyvinyl alcohol, alkyl radical polyose, cellulose ester phthalate, and vinyl resin maleic compound. The film thickness varies significantly based on the area of inner architecture to be coated and other physical characteristics of the system140. The micro-capsules might encompass one particle or clusters of particles. Once isolation from the liquid phase is complete, the dosage form is supposed to be in the form of a free-flowing powder. The powder is appropriate for formulation as compressed tablets, exhausting gelatin capsules, suspensions, and alternative dose forms.

1.5.3 Core Materials

The core material is the material over which coating is applied. Core material could also be in the form of solids or droplets of liquids and dispersions. The composition of core material will vary and therefore provide definite flexibility and allow effectual style and development of specified microcapsule properties. A substance could also be microencapsulated for a variety of reasons.

Examples might include the protection of volatile compounds from their surrounding environment, safe and convenient handling of the materials that are otherwise toxic, taste masking. This means that for controlled release properties means challenging to handle liquids as solids; it allows the advantage of the ease of preparation of free flow powders.

1.5.4 Mechanism and Mechanics of Drug Release

1.5.4.1 Diffusion

Diffusion is the most common mechanism whereby the dissolution fluid penetrates the coating, dissolves the core and forms opening channels or pores. Thus, the general release depends on, (a) the rate at which dissolution fluid penetrates the wall of microcapsules, (b) the rate at which drug dissolves within the dissolution fluid, and (c) the rate at which dissolved drug is released and dispersed from the surface141. The mechanics of such drug release obeys Higuchi’s equation as below:

1/2 Q = [D/J (2A - ε CS) CS t]

Where, Q is that the number of drug-free per unit space of exposed surface in time t; D is the diffusion constant of the matter within the solution; A is the total amount of drug per unit volume; ε is the consistency of the wall of microcapsule; J is the crookedness of the capillary system in the wall. CS 32 represents the drug solubility in the matrix. J is the tortuosity of the capillary system in the wall. The above equation can be simplified to Q = vt where, v is the apparent release rate.

1.5.4.2 Dissolution

The dissolution rate of the chemical coat determines discharge rate of drug from microcapsule once the coat is soluble within the dissolution fluid. The thickness of the coat and its solubility within the fluid dissolution influence the discharge rate142.

1.5.4.3 Osmosis

The chemical coat of microcapsule acts as a semi leaky membrane. Osmosis occurs with the creation of a force per unit area differential between the internal and external environment of the microcapsule and drives drug release from microcapsule through tiny pores within the coat142.

1.5.4.3 Erosion

Erosion of coat due to pH and/or catal143ytic chemical reaction causes drug release from microparticles fabricated using coating components like monostearate, bee’s wax, and stearyl alcohol144.

1.5.4.4 Applications of Microcapsules and Microspheres

a) Sustained release dosage forms. The microencapsulated drug will be administered, as microencapsulation is probably most helpful for the preparation of tablets, capsules or canal dose forms143.

b) Microencapsulation may be used to prepare enteric-coated dosage forms, so the drug is absorbed explicitly from the bowel instead of being metabolized in the stomach143.

c) Due to its coating component, it masks the bitterness of drugs143.

d) Microencapsulation allows formulation of hydrophobic drugs into solid dosage forms. This addresses issues inherent in manufacturing tablets, like granulations and direct compression to tablets.

e) Microparticles also protect therapeutics from environmental hazards like humidity, light, heat, etc143.

f) Microencapsulation leads to a decrease in the volatility of therapeutics. Volatile therapeutics may get extended shelf life due to microencapsulation143.

g) Microencapsulation also reduces the potential danger of handling of poisonous or noxious substances. The toxicity occurred thanks to the administration of fumigants, herbicides, pesticides, and pesticides that are reduced by microencapsulation143.

h) Many therapeutics are microencapsulated to lower internal organ irritation143.

1.5.6 Nanomicelles

Delivery of medication at therapeutic concentrations to specific targeted site of action with a negligible adverse reaction to ocular tissues not affected by the disease is an active area of investigation for ocular scientists with no breakthrough reported. The intricate architecture of ocular tissues acts as a significant barrier to the targeted delivery of therapeutics and medication reaching its site of action.

Also, there is existence of both static and dynamic barriers in the eye. These include epithelial layer of the cornea, which is hydrophobic, a hydrophobic stromal layer of sclera and cornea, lymphatic system of the conjunctiva, blood vessels of the choroids, and finally the significant blood-retinal barrier

(BRB)145.

Topical route of therapeutic delivery via eye drops is a widely used approach that is preferable to other routes, is convenient, and comparatively better patient compliance- thus mainly adopted for the treatment of ophthalmic disorders146. Even though topical delivery of therapeutics is the preferred route when it comes to ophthalmic dosage form design, achieving drug concentration in therapeutic range is a significant challenge as the bioavailability of the drug is low following topical delivery via eye drop. For posterior ocular disorders, intravitreal injections are usually deployed. However, as mentioned previously, intravitreal injections have significant drawbacks that cause poor patient compliance, lead to severe damage to tissues, allow pathogenic infection and other severe side effects.

Hence an effective topical dosage form that leads to effective drug delivery to both anterior and posterior ocular disorders is an absolute necessity. The disorders that need to be addressed include dry eye syndrome, glaucoma, AMD, DR, and congenital retinal disorders. Ocular disorders exert a 34 considerable cost on healthcare. Hence many therapeutics are developed to address this segment.

However, many such effective therapeutics do not achieve success due to the non-availability of optimized drug delivery platforms. The majority of therapeutics that are developed for treatment of ocular disorders are lipophilic. Hence there is a need for the development of aqueous-based drug delivery platforms that will be able to deliver a therapeutic concentration of drugs in diseased ocular tissues. To address these issues, nanotechnology must make rapid strides to create smart drug-delivery platforms like nanomicelles for ocular delivery147.

Nanomicelles may be defined as nanosized drug delivery platforms that are self-assembled

(they generally have a with particle size within a variety of 10 to 100 nm) colloidal dispersions with a hydrophobic core and hydrophilic shell148. Currently, nanomicelles are deployed as drug delivery platforms to solubilize lipophilic drugs. Hydrophobicity is taken into account as a severe limiting factor for formulating clear aqueous solutions with concentrations sufficient to achieve therapeutic levels in ocular tissues.

Nanomicelles solubilize hydrophobic drugs by entrapping them within a mixed micellar hydrophobic core with a corona composed of hydrophilic chains extending outwards, leading to a transparent aqueous formulation. Nanomicelles function as excellent pharmaceutical carriers due to their ability to stop or minimize drug degradation, lower adverse side effects, and improved drug permeation through ocular epithelia with minimal or no irritation, ultimately resulting in enhanced ocular bioavailability148. Mitra et al. pursued a scientifically relevant investigation on nanomicellar formulation. A distinct, previously unexplored nanomicellar formulation of voclosporin, which happens to be an inhibitor of calcineurin149, was developed for treatment of dry eye syndrome. The following mixed micellar formulation fabricated from two non-ionic surfactants- vitamin E TPGS and octoxynol-40, in a predefined ratio150. The resultant micellar formulation had the following properties: a) relatively small size (in nanometer) b) this small size causes homogeneous nature of formulation c) transparent and clear in appearance. A major success of this mixed micellar formulation was that it was capable of encapsulating a comparatively high dose of therapeutics149. This is a considerable success 35 considering the high amount of drug that was able to be loaded in this particular formulation. Almost

0.2% of voclosporin was loaded onto the formulation despite being hydrophobic. Apart from the fact that a high dose of local delivery of voclosporin, these nanomicellar formulations exhibited prolonged stability compared to the emulsion. When delivered topically, therapeutic concentration was observed in retina and choroid in the posterior ocular tissues. Surprisingly no detectable amount of drug was observed in lens or vitreous. This pattern of drug distribution is efficacious in terms of avoiding debilitating adverse events like the elevation of IOP or the formation of cataracts. These adverse effects often cause non-compliance of patients and discontinuation of therapy. Hence, these formulations developed by Mitra et al 146. may be applied topically that will lead to enhanced drug uptake by ocular tissues and patient compliance.

Drug loaded nanomicelles may possess a few disadvantages. A prominent drawback of deploying nanomicellar formulation as a drug delivery platform is that it is not possible to formulate hydrophilic therapeutics and macromolecular drugs like protein and peptide-based therapeutics as well as enzymes151. Also, oral delivery of few nanomicellar formulations with polymeric composition may lead to irritability in the gastric mucosa. Finally, drug-loaded nanomicellar constructs get diluted rapidly when it enters the systemic circulation following delivery via intravenous injection. This may cause rapid drug release before the onset of pharmacological action, also referred to as burst release152.

Finally, nanomicelles have the advantage of having a small size, reduced toxic response, enhanced solubilization of lipophilic drugs, and enhanced bioavailability146. The nanomicellar drug- delivery platform allows for the development of a topically applied dosage form to deliver lipophilic drugs. Also, this formulation strategy has been observed to be patient compliant and leads to a successful drug delivery strategy to posterior ocular tissues in case of disorders AMD, DR, DME, etc.

As recent in 2018, the FDA has approved Cequa®, which is essentially nanomicellar formulation of distinctive cyclosporine-A without any additive. Cequa® has been approved for the treatment of the dry eye-related disorder. Due to its lipophilic nature, cyclosporine-A is usually formulated as an oil-

36 based emulsion with all its associated disadvantages. Hence there was an unmet need that was fulfilled by this aqueous nanomicellar formulation.

In conclusion, nanomicelles possess the benefits of exhibiting low toxicity, increasing the solubility of hydrophobic drugs, and achieving therapeutic concentrations. The nanomicellar drug- delivery platform appears to be a possible pharmaceutical carrier for the topical administration of hydrophobic drugs. Moreover, this technology often leads to improved patient compliance and should enable non-invasive drug delivery to back-of-the-eye disorders like age-related degeneration, diabetic retinopathy, diabetic macular edema, and posterior uveitis. Recently in 2018, the first FDA-approved nanomicellar formulation named Cequa®, a unique and first-in-class preservative-free cyclosporine-A

(CsA) nanomicellar topical formulation for the treatment of dry eye disease153. As it is highly lipophilic,

CsA is famous as an oil-based emulsion, with all the disadvantages involved with an emulsion-based formulation. There is an unmet need for the development of an aqueous and clear formulation of CsA for a safe and better drug product. In this regard, a novel, clear, aqueous nanomicellar solution of CsA was developed, which has the potential to deliver therapeutic concentrations of CsA with minimal discomfort to patients, as shown in Fig 1-6. It is anticipated that many nanomicellar formulations will likely be approved sooner by FDA for different ocular diseases. Substantial progress still must be made to realize sustained drug release from the micelles relative to other larger particulate systems like nanoparticles, microparticles, and liposomes.

Figure 1. 6 Cequa (cyclosporine nanomicellar solution) approved by the FDA153

1.5.7 Liposomes

Liposomes, as drug delivery platforms, have been investigated extensively, more than any other dosage forms, as they may be fabricated into different forms154. Membranes prepared from the bilayer structure of phospholipids can form spherical structures containing internal hydrophilic core. This architecture is obtained via introduction of phospholipids into an aqueous phase, and these structural forms are referred to as liposomes155; Nearly forty years ago, Bangham et al. defined liposomes to be as spherical shaped vesicles of small size which is composed of reagents like non-toxic surfactants, cholesterols, phospholipids, etc. they may even be fabricated out of membrane proteins154. This group of investigators also reported that liposomes might also be used as drug delivery platforms, with therapeutic compounds contained in the core of liposomes. Liposomes are also capable of incorporating and acting as delivery vehicles for both hydrophobic and hydrophilic drugs. Constituents of liposome- like lipids are amphipathic with hydrophobic and hydrophilic aspects. Liposomes may consist of either single or multiple bilayers of lipid molecules, which are created by lipophilic and hydrophilic reactions occurring in the aqueous phase154. The aqueous phase repels the lipophilic aspect of liposomes, causing liposomes to self-assemble. Also, Phosphatidylcholine (PC) and Dipalmitoyl PC are commonly used to prepare liposome generation. Two major advantages of liposomes, in utilization as drug delivery platforms, are that they are biocompatible and biodegradable. This is a function of the lipidic component of the liposomes. Various kinds of lipids and amphiphilic compounds may be used to fabricate liposomes156.

1.5.7.1 Liposome Preparation

Fabrication of liposomes includes few techniques that may involve mechanical methodology, use of organic solvents, or phase separation of detergent from phospholipid detergent mixture. In

38 liposome fabrication, nature and quantity of phospholipid and the electrochemical aspects of the aqueous phase are important parameters that affect the final architecture of liposome156.

Multilamellar liposome vesicles fabrication: Preparation of multilamellar liposome is the most uncomplicated technique among all techniques practiced for preparing liposomes157. In this method, first stage of preparation involves use of an organic phase to dissolve lipid. The mixture thus formed is completely dried. A common practice is the use of a mixture of lipids like egg lecithin, cholesterol, and phosphatidylglycerol in a molar ratio of 0.9:1.0:0.1158. The organic phase that is commonly used includes chloroform or a combination of chloroform/methanol typically in a ratio of

2:1 in the first step, each of the lipid constituents is dissolved in the organic phase separately. In the second step, mixing occurs in required ratio with different solubilized lipids to ensure uniform lipid distribution in the resultant mixture. Following this step, nitrogen stream is applied to the mixture in a tube. Also, to remove any traces of organic solvent, the film of lipid is dried in an evacuated chamber for at least 4-6 hours.

Unilamellar vesicles fabrication: The unilamellar vesicle is a popular choice for liposomes preparation159. Its architecture provides room for a robust distribution of entrapped therapeutic agents within internal aqueous phase. There are a few techniques to prepare these liposomes. These include ultrasonication, extrusion through polycarbonate filters, freeze-thawing, ethanol injection, detergent method, and preparation of sterile large unilamellar vesicles. Bhatia et al160. used a mixture of many small unilamellar vesicles (SUVs) populations to obtain a final GUV (Giant unilamellar vesicle) of uniform property.

Giant Unilamellar Liposomes Preparation: There exist numerous techniques for the fabrication of giant liposomes supported utilizing only water, non-electrolyte, or zwitterions161. In this methodology, an enhanced attraction exists between membranes due to the existence of ions, which create a total charged environment. This creates a scenario in which membrane sheets cannot move apart from each other during the preparation phase in which rehydration and swelling are taking place.

Recently, it has been demonstrated that buffers of physiological strength may be used to prepare giant 39 liposomes162. There are numerous techniques that may be deployed to prepare such formulations use of electro-formation, osmotic shock, etc. Also, Karamdad et al162. developed a novel method that deployed a microfluidic approach to prepare GUV that was characterized in a mechanically for loading and encapsulation efficiency of hydrophilic drugs. Drugs of hydrophilic nature, when encapsulated in GUV, causes hydration of mixed-phase of lipid and hydrophilic therapeutics162. This way, therapeutics may permeate into core of the formulation, and the remainder of excipients remain external to the platform.

The available excipient may lower drug entrapment, and hence there needs to be implemented a mechanism to remove these from the liposomal formulation. In order to perform this purification of drug and unused excipients, gel filtration chromatography and dialysis may be used. For macromolecular therapeutics, sequential dehydration followed by rehydration may be used161.

Encapsulation of Hydrophobic Drugs: Liposome formulation possesses a lipid bilayer composed of a phospholipid, which may be the interface where hydrophobic drugs may be entrapped.

Hydrophobic therapeutics like verteporfin (Visudyne®), drug mobility may be slowed down towards externally oriented hydrophilic phase and internal hydrophilic environment of liposome. Hence encapsulation of therapeutics may be performed through a solubilization step in which the therapeutic is dissolved in organic phase and phospholipid component of a liposome.

CHAPTER 2. INTRODUCTION

2.1 Overview

Eyes are sensitive and incredibly complex organs responsible for visual system. They control vision, obtain and process visual information. They also perform few light dependent activities that are independent of vision163. Eyes are responsible for detection of light, converting it into electrochemical signals which are transduced to brain via neurons. In this manner body maintains a relationship with environment164. There are different ocular disorders that affect our vision. These disorders represent a significant burden on healthcare worldwide and in the US165. This thesis work focuses on diseases affecting posterior segment of the eye. Age-related macular degeneration (AMD), macular edema

(ME), glaucoma, diabetic macular edema (DME), proliferative vitreoretinopathy (PVR), cytomegalovirus retinitis (CMV), non-infectious uveitis, endophthalmitis and diabetic vitreoretinopathies are few of the posterior ocular disorders which may cause loss of vision if timely medical intervention does not occur166. Of all the posterior ocular disorders mentioned above, AMD will be a major focus of this work. There is currently no effective treatment available to completely cure AMD167. Anti-VEGF therapy is currently considered treatment of choice. However, it is invasive in nature and does not offer substantial long-term relief to AMD patients168. Other treatment options include thermal laser photocoagulation, and surgery. None of these therapeutic approaches are safe and effective to be considered treatment of choice169. Hence there is a real need for development of an alternative, safe, effective and non-invasive therapeutic approach. This dissertation work addresses this problem via development of nanomicellar formulation of FDA approved polymers incorporating therapeutics whose efficacy has been clinically established.

2.2 Statement of the Problem and Hypothesis

Approximately 11 million individuals with age over 40 years are currently suffering from AMD in the US and global prevalence of approximately170 million170. Inflammation in macular region of the eye coupled with enhanced VEGF expression and upregulation of pro-inflammatory cytokines, are 41 major factors causing choroidal neovascularization (CNV) involved in AMD171. Existing therapeutic approaches like steroid implants are invasive in nature and do not affect enhanced VEGF level and pro- inflammatory cytokines172. Also, oxidative stress may have a huge impact on the pathogenesis and the progression of AMD173. Anti-VEGF therapy are administered as intravitreal injection monthly leading to some serious complications like retinal detachment, uveitis, subconjunctival hemorrhage etc. This leads to poor patient compliance80. Hence there is an immediate requirement for development of better strategies for AMD treatment which is less invasive with lowered side effects and high patient compliance. Not only there is requirement for better drug delivery strategies, there is also an urgent need for alternative therapeutic agents. This research proposal aims to address these issues. We aim to develop a topical eye drop of CXB, CUR and RSV loaded nanomicelles for delivery to back of the eye, especially to the retinal region. Proposed size of nanomicelles (around 20 nm) will allow them to permeate effectively through the aqueous channels/pores of the sclera. The hydrophilic corona will help to prevent washout of drugs into systemic circulation by conjunctival/choroidal blood vessels and lymphatics. Physicians would be able to prescribe this formulation for repeated administration, since the topical route is a non-invasive and patient compliant route.

It is hypothesized that the proposed work which aims to develop nanomicellar formulation fabricated from a combination of HCO-40 and OC-40 polymers will be utilized to entrap CUR and

RSV leading to development of a safe, clear and stable formulation for efficient, non-invasive topical delivery to the eye for AMD treatment. Also, biotin conjugated nanomicelles loaded with CXB will be developed that is preferentially taken up by retinal and conjunctival cells through SMVT transporters, which are overexpressed retinal cells174. Utilization of targeted approach is hypothesized to enhance uptake of CXB by retinal cells.

2.2.1 Rationale for Using Statistical Design of Experiments (DOE)

Design of Experiment (DOE) is a systematic method to determine the relationship between factors affecting a process and the output of that process. DOE helps scientists learn the pattern of interactions among independent factors and their effects on dependent factors. This method can reduce 42 a number of experiments for optimization. This process saves a lot of time and cost. Recently, design of experiment (DOE) has been widely utilized to optimize formulation. It is a structured powerful approach for conducting experiments to identify and explain the influence of individual factors and their interactions on response variables. In this project, DOE is used to identify the effect of two different polymers that may enhance the entrapment efficiency and loading efficiency, the interaction among factors (polymer-drug and polymer-polymer) may enhance or lower drug loading in nanomicelles. Based on information from the model, we will optimize and modify nanomicelle components to achieve higher aqueous solubility for steroids.

2.2.2 Rational for Using Curcumin (CUR), Celecoxib (CXB), and Resveratrol (RSV)

The first drug proposed to be deployed in this research proposal is CXB which is a selective non-steroidal anti-inflammatory drug (NSAID) exerting its pharmacological effect by selectively affecting cyclooxygenase-2 (COX-2). It is widely used for treatment of pain and inflammatory disorders associated with spondylitis and arthritis. Also, COX-2 is also implicated in production of

VEGF175. It has been widely reported that inflammatory pathways are majorly responsible for development of AMD176. The use of COX-2 inhibitor is an intelligent strategy that may potentially offer enhanced therapeutic relief in AMD treatment through relieving associated inflammation and reducing

VEGF release and inhibiting pro-inflammatory cytokines secretion from macrophages177.

Resveratrol (RSV), (3,5,4’-trihydroxy-trans-stilbene), is a polyphenolic compound which is originally derived from peanuts, grapes, red wines and blueberries178. RSV gained so much attention because of its antioxidant, anti-inflammatory and anticancer effects179. Moreover, some studies have shown the ability of RSV to reduce VEGF levels in vitro and in vivo, hence reducing the incidence of angiogenesis formation and choroidal neovascularization (CNV) in AMD patients180-181. However,

RSV possesses poor aqueous solubility (< 50 µg/ml) and low bioavailability (< 1%) which limits its pharmacological activity and biological effect182 . Therefore, there is an urgent need to develop a delivery system that can enhance the solubility and stability of RSV.

Another drug that may potentially create a paradigm shift in AMD treatment is Curcumin, which is a biologically active component extracted from turmeric. Curcumin (CUR) has a variety of important pharmacologically relevant applications as it may act as an antioxidant, anti-inflammatory agent, and may also exhibit anti-proliferative effects183-184. This makes it an attractive therapeutic agent potentially able to be an effective therapy for useful for AMD. It has also been shown to have protective effects by preventing oxidative stress from causing damage to RPE cells. This may prevent early onset AMD185. Therefore, targeted interventions to reduce oxidative stress to RPE cells might be a useful therapy for AMD186.

2.3 Objectives

2.3.1 Optimization of Curcumin (CUR) Nanomicellar Formulation

In this proposed work two amphiphilic polymers will be selected namely: Hydrogenated castor oil 40 (HCO-40) and Octoxynol-40 (OC-40). Both polymers are FDA approved and considered to be safe for ophthalmic preparations. The objective is to develop and optimize aqueous CUR loaded nanomicellar formulation. The ratio of HCO-40 and OC-40 will be optimized to achieve highest entrapments of CUR nanomicelles with high drug loading. The formulations will be optimized using

JMP (version 8.0, SAS Institute Inc., Cary, NC, USA) experimental design software where HCO-40,

OC-40 and CUR will be considered as independent variables. On the other hand, drug loading (%DL), entrapment efficiency (%EE), size, PDI, precipitation will be considered as dependent variables. The validity of the overall model will be checked with checkpoint analysis. The optimized formulation will be tested on human corneal epithelial cells (HCEC) and human retinal pigmented epithelial cells

(D407). The experiments will be designed as follows:

❖ To develop and validate bioanalytical quantitative RP-HPLC method for CXB.

❖ To use of full factorial design of experiment (DOE), JMP 9.0 experimental design software, to

prepare aqueous CUR loaded nanomicelles using various ratios of HCO-40 and OC-40.

❖ To characterize the optimized formulation regarding average micellar size, polydispersity index

(PDI), critical micellar concentration (CMC), Zeta potential, morphological analysis, viscosity, 44

appearance, entrapment efficiency (%EE), drug loading efficiency (%DL), and in vitro drug

release kinetics.

❖ To characterize the optimized formulation with 1H-NMR, FT-IR and XRD instruments.

❖ To perform cytotoxicity studies and cell viability assays: LDH and MTT assays

❖ To measure the reduction of VEGF level in D407 and HCEC cell lines using VEGF-A ELISA

assay.

❖ To assess the antioxidant activity of CUR-NMF on retinal cells using H2O2 as a model of

oxidative stress.

2.3.2 Optimization of Celecoxib (CXB) Nanomicellar Formulation

In this proposed work Hydrogenated castor oil 40(HCO-40) and Octoxynol-40(OC-40) will be utilized like the previous section. The objective of this work is to develop and optimize aqueous CUR loaded nanomicellar formulation. The ratio of HCO-40 and OC-40 will be optimized to achieve stable nanomicelles with high drug loading. The formulations will be optimized using JMP 9 experimental design software where HCO-40, OC-40 and drug will be considered as independent variables. On the other hand, drug loading, entrapment efficiency, size, PDI, critical micellar concentration (CMC), and light transmittance will be considered as dependent variables. The optimized formulation will be tested on human corneal epithelial cells (HCEC) and human retinal pigmented epithelial cells (D407). The experiments will be designed as follows:

❖ Biotin was conjugated to octoxynol-40 polymer (OC-40) to form an ester bond with help of

EDC/DMAP chemistry.

❖ 1H-NMR and FT-IR were used for analyzing the product formation.

❖ DLS was used to assess the size, PDI and Zeta potential of the optimized formulation.

❖ RP-HPLC was used for measuring entrapment efficiency (%EE), drug loading (%DL) and drug

release kinetics from biotinylated CXB nanomicelles and the naked drug.

❖ TEM was used to analyze the morphology of the formulation.

❖ MTS and LDH assays were be performed to analyze the safety of our formulation on retinal

pigmented epithelial cells (D407), human corneal epithelial cells (HCEC) and choroidal cells

(CCL20.2).

❖ VEGF-A ELISA was performed to determine VEGF release from D407 (retinal epithelial) cells

upon exposure to our formulation.

❖ Pro-inflammatory cytokines such TNF-훼 and IL-1β release from macrophage cells (RAW 264.7)

were determined with ELISA before and after treatment.

❖ LC-MS/MS and confocal microscopy were used to determine the uptake of biotinylated

nanomicelles loaded CXB on retinal cells.

2.3.3 Preparation of Resveratrol (RSV) Nanomicellar Formulation

In this work, similar to the previous two sections, Hydrogenated castor oil 40(HCO-40) and

Octoxynol-40(OC-40) were selected. The objective of this work is to enhance the solubility of RSV.

The ratio of HCO-40 and OC-40 will be optimized to achieve stable nanomicelles with high drug loading. The formulations will be optimized using JMP 9 experimental design software where HCO-

40, OC-40 and drug will be considered as independent variables. On the other hand, drug loading, entrapment efficiency, size, PDI, critical micellar concentration (CMC), and light transmittance will be considered as dependent variables. The optimized formulation will be tested on human corneal epithelial cells (HCEC) and human retinal pigmented epithelial cells (D407). The experiments will be designed as follows:

❖ To develop and validate bioanalytical quantitative HPLC method for RSV.

❖ To characterize the optimized formulation regarding average micellar size, PDI, CMC, Zeta

potential, morphological analysis, viscosity, appearance, entrapment efficiency, loading efficiency,

In vitro drug release.

❖ To perform cell cytotoxicity studies and cell viability assays: LDH and MTT assays

❖ To measure the reduction of VEGF level in D407 and HCEC cell lines using VEGF-A ELISA

assay VEGF expression by quantitative RT-PCR. 46

❖ To perform cell uptake studies using confocal microscopy

❖ To assess the antioxidant activity of RSV nanomicelles on retinal cells using 500 µM tertiary

butyl hydrogen peroxide solution (tBHPS) as a model of oxidative stress.

CHAPTER 3. SELF-ASSEMBLING TOPICAL NANOMICELLAR FORMULATION TO

IMPROVE CURCUMIN ABSORPTION ACROSS OCULAR TISSUES

3.1 Rationale

Age-related macular degeneration (AMD) is one of the leading cause of lower visual acuity in the US and globally 187-189. Approximately 11 million individuals aged ≥ 40 years are currently affected with AMD in the US with a global prevalence of 170 million 187. Since age is the most significant risk factor, incidence of AMD is growing and expected to reach 22 million in the US by 2040. The global prevalence is also expected to be 228 million by 2040 187-188. AMD selectively affects the macular region of the retina, which is comprisedof millions of photoreceptor cells. Depending on the severity of symptoms, AMD is characterized into early, intermediate and advanced stages 190. The term “dry

AMD” refers broadly to the early or intermediate stage 191. Dry AMD is characterized by drusen, yellow deposits underneath the macula 192. The term “wet AMD” refers to the advanced neovascular stage of the disease representing rapid vision loss. Wet AMD is caused by VEGF-stimulated abnormal proliferation of blood vessels in the retina. The neovascular tissue associated with exudative AMD is also characterized as choroidal neovascularization (CNV). These newly formed blood vessels are fragile and prone to leaking which leads to irreversible macular damage 191. Although wet AMD comprises a small portion of total AMD, it accounts for the majority of blindness from AMD 193.

While there is no current treatment for dry AMD, various treatment strategies have been developed for wet AMD. The most effective treatments consist of anti-VEGF agents which inhibit angiogenesis. These drugs include ranibizumab, bevacizumab, aflibercept, and pegaptanib. The doses are injected directly into the posterior segment of the eye by intravitreal route 194-197. Despite their effectiveness, these treatment regimens require a monthly intravitreal injection and are associated with higher rates of side effects such as retinal detachments, subconjunctival hemorrhage, and uveitis ultimately leading to poor patient compliance 191, 198. Although Anti-VEGF therapy improves visual acquity, it does not provide retinal cells protection from oxidative stress leading to retinal degeneration.

Thus, antioxidant minerals and vitamins such as vitamin E, vitamin C, zinc and beta carotene can reduce 48 the risk of vision loss from AMD 199. Other experimental treatments include retinal pigment epithelial

(RPE) transplants 192 and implantable telescope lenses 200. However, these procedures carry much higher safety risks than anti-VEGF therapies, rendering this therapy less than ideal for AMD treatment

192, 200.

Ocular diseases of the posterior segment include age-related macular degeneration (AMD), diabetic retinopathy (DR) and diabetic macular edema (DME). Delivering drugs to the posterior segment of the eye is considered to be challenging due to presence of tight junctions of the blood- retinal barrier (BRB). Therefore, development of an alternative and less-invasive drug delivery platform is pivotal. In such cases, an aqueous nanomicellar formulation utilizing amphiphilic polymers with high compliance can deliver drugs for the back of the eye. Use of nanomicelles holds several advantages such as improved drug solubility and uptake, noninvasive delivery of hydrophobic drugs, and improved patient compliance due to a nonirritating clear aqueous solution 201-203. Despite their small size (less than 20 nm in diameter), nanomicelles follow conjunctival/scleral pathway rather than uveo/scleral pathway after topical administration and as a result, IOP change and cataract formation are minimized 203.

Although the pathophysiology of AMD is not delineated yet, oxidative stress may play an essential role in its development. Oxygen is necessary for the retina to maintain its integrity and function. However, high oxygen tension on retina caused by light or reactive oxygen species (ROS) may result in irreversible damage to the RPE, which is linked to AMD188. Recently we have selected

H2O2 as a model of oxidative stress and then examined the antioxidant activity of our formulation.

Curcumin with log p-value of 3.2 posseeses several pharmacological activities including antioxidant, anti-inflammatory, and antiVEGF effects 204. Curcumin has been shown to protect RPE cells against oxidative stress, in turn, preventing the onset of AMD 186, 205. Therefore, interventions of targeted oxidative stress reduction in RPE cells might be a beneficial therapy for AMD. Moreover, some studies have shown the ability of curcumin to reduce VEGF level in vivo, hence reduce

49 angiogenesis formation 206. However, curcumin possesses a poor water solubility and low bioavailability.

In the present study, the objective is to develop and optimize nanomicellar formulations of curcumin with full factorial design of experiments (DOE). JMP software analysis was performed to acquire highest entrapment efficiency (EE), low size, drug loading (DL) and polydispersity index(PDI) without any precipitation. Two FDA- approved amphiphilic polymers, namely hydrogenated castor oil-

40 (HCO-40) and octoxynol-40 (OC-40), were applied as an effective method for preparing nanomicellar formulations of curcumin (CUR-NMF). This preparation can be delivered as eye drops to the retina. The optimal formulation is characterized for size, PDI, EE, DL, precipitation, and in-vitro drug release. Furthermore, H2O2 was used as a model of oxidative stress to investigate the antioxidant avticvity of CUR-NMF on D407 cells. Moreover anti-VEGF activity is also carried out and assessed with ELISA. Therfore, the antioxidant and anti-VEGF activities of CUR-NMF were conducted on retinal pigmented epithelial cells(D407) where H2O2 used as a model of oxidative stress and oxidative stress induced VEGF release.

Research group of Dr. Mitra has succeeded in preparing cyclosporine nanomicelles, Cequa®, utilizing same polymers, and the formulation was recently approved by the FDA for dry eye disease153.

It has been hypothesized that such a nanomicellar formulation has potential to deliver curcumin as eye drops to retina. The optimal nanomicellar formulation has been characterized for size, PDI, EE, DL, precipitation, and in-vitro drug release. Furthermore, H2O2 was utilized as a model of oxidative stress to investigate the antioxidant activity of CUR-NMF on retinal (D407) cells.

Additionally, anti-VEGF activity was assessed using ELISA. The novelty and significance of the work come from addressing the etiology of both types of AMD, dry and wet, oxidative stress, and high VEGF level (angiogenesis) in one single formulation which is CUR-NMF. Fig 3-1 shows a schematic representation of curcumin nanomicellar formulation (CUR-NMF)

Figure 3. 1 Schematic representation of curcumin nanomicellar formulation (CUR-NMF)

3.2 Material and Methods

Curcumin, in curcuminoid form (curcumin, demethoxycurcumin, bisdemethoxycurcumin)

+98%, and H2O2 was purchased from Sigma Aldrich, MO, USA. Hydrogenated Castor Oil 40

(HCO40) was purchased from the Peboc division of Eastman Company, UK. Octoxynol-40 (Igepal

CA-897) was purchased from Rhodio Inc., NJ, USA. HPLC grade acetonitrile was procured from

Fisher Scientific, NH, USA. Cell Titer 96® Aqueous nonradioactive cell proliferation assay (MTS) and lactate dehydrogenase (LDH) assay kits were purchased from Promega Corporation, WI, USA, and Takara Bio Inc., Japan, respectively. VEGF-A ELISA kit was purchased from Thermo Fisher

Scientific, MA, USA. D407 cell line was a generous gift from Dr. Araki-Sasaki (Kinki Central

Hospital, Japan).

3.3 Methods

3.3.1 HPLC Analysis

Reversed-phase HPLC (RP-HPLC) method was applied to analyze samples with Shimadzu

LC pump (Waters Corporation, Milford, MA), Alcott autosampler (model 718 AL), Shimadzu

UV/Vis detector (SPD-20AV), and Phenomenex C18 column (Spherisorb 250 × 4.60 mm, five μm).

The mobile phase was composed of acetonitrile and water (60:40 v/v) with a ﬂow rate set at 0.5 mL/min and a UV detector at 425 nm. Curcumin-calibration curve (1 to 125 μg/mL) was constructed by injecting 30 μL curcumin-ethanol solution into the column.

3.3.2 Experimental Design

The effects of formulation parameters were analyzed using a 23 full factorial experimental designs with three center points. The analysis was performed in JMP software (version 8.0, SAS

Institute Inc., Cary, NC, USA). A total of 11 experimental runs were conducted by varying the weight percent of three independent variables, including Curcumin (X1) HCO40 (X2) and OC-40 (X3) (Table

3-1). The dependent variables, namely, CUR-NMF size (Y1), polydispersity index (PDI) (Y2), entrapment efficiency (Y3), loading efficiency (Y4), and precipitation (Y5) were further evaluated based on the statistical design of experiments (DOE) (Table 3-2). The optimal CUR-NMF was obtained by maximizing the desirability function and its acceptability. The validity of the overall model was evaluated with a checkpoint analysis (Table 3-3). The independent and dependent variables, as well as their coded values, are presented in Table 3-1. In Table 3-1 -1, +1, 0, and -1 represent the upper, middle, and lowest values, respectively, for a given independent variable.

Table 3. 1 Independent variables with coded and uncoded levels.

Coded design Uncoded design

Formulation code

X1 X2 X3 X1 X2 X3 (CUR wt%) (HCO-40 wt%) (OC-40 wt%)

F1 + - + 0.2 0.5 2

F2 (3x) 0 0 0 0.1 2.5 1

F3 - + + 0.05 4 2

F4 + + - 0.2 4 0.1

F5 - - - 0.05 0.5 0.1

F6 + + + 0.2 4 2

F7 + - + 0.05 0.5 2

F8 + - - 0.2 0.5 0.1

F9 - + - 0.05 4 0.1

Table 3. 2 Summary of the independent factors and recorded dependent variables responses Formulation CUR HCO- OC- Size % %DL PDI %precipitation Code wt% 40 wt% 40 (nm) EE wt% F1 0.2 0.5 2 13.38 48 3.56 0.102 40

F2 (3X) 0.1 2.5 1 17.87 72 2 0.227 NA

F3 0.05 4 2 17.39 70 0.58 0.188 NA

F4 0.2 4 0.1 22.5 80 3.49 0.301 NA

F5 0.05 0.5 0.1 19.32 64 4.92 0.178 60

F6 0.2 4 2 16.35 78 0.981 0.18 NA

F7 0.05 0.5 2 25.5 73 1.43 0.655 30

F8 0.2 0.5 0.1 14.79 17 4.25 0.055 25

F9 0.05 4 0.1 21.01 73 0.88 0.177 NA

Table 3. 3 Checkpoint analysis. Responses Run Predicted value Measured value P value (mean ± SD) (mean ± SD) Student’s t test 0.05 Size F12 19.90 ± 2.11 18.93 ± 0.025 0.4853

F13 18.18 ± 2.12 18 ± 0.05 0.0939

F14 18.50 ± 0.94 18.64 ± 0.12 0.8810

PDI F12 0.178 ± 0.09 0.176 ± 0.002 0.7107

F13 0.170 ± 0.09 0.185 ± 0.05 0.6693

F14 0.230 ± 0.04 0.202 ± 0.03 0.0926

%EE F12 70.96 ± 15.9 53.27 ± 0.57 0.788

F13 68.51± 15.8 82.63 ± 0.51 0.2234

F14 64 ± 7 70.69 ± 0.91 0.4754

%LD F12 2.79 ± 1.54 3.55 ± 0.015 0.8336

F13 3.722 ± 1.54 3.86 ± 0.026 0.2514

F14 2.54 ± 0.68 2.73 ± 0.112 0.7352

% precipitation F12 26.04 ± 30 NA 0.8164

F13 8.125 ± 30.99 NA 0.1146

F14 15.10 ± 13.69 NA 0.7819

3.3.3 Preparation of Curcumin Nanomicellar Formulation (CUR-NMF)

CUR-NMF was prepared following the solvent evaporation method207. Briefly, predetermined amounts of curcumin, OC-40, and HCO-40 were dissolved separately in ethanol. Consequently, a homogenous solution was obtained by additions of OC-40 to HCO-40. Then, the calculated amount of 55 curcumin solution was added to the mixture in a dropwise pattern. The organic solvent (ethanol) was evaporated with a highspeed vacuum evaporator (Genevac Technologies VC3000D speed vacuum,

USA) over 9 hours to obtain a thin film. The resultant solid was rehydrated with deionized (DI) water.

An equal volume of 2x phosphate buffer (pH 6.85) was added to optimize the formulation pH. The solution was sonicated for 10 minutes and then filtered through a 0.22 µm membrane filter to remove the un-entrapped drug or other foreign substances. Then, povidone K-90 was used as a viscosity enhancer.

3.3.4 Size, Polydispersity Index, and Surface Charge

The mean hydrodynamic nanomicellar size, distribution, PDI, and surface potential of CUR-

NMF were measured by dynamic light scattering (DLS) (Brookhaven Instruments Corporation, TX,

USA). DLS is a non-destructive technique to measure the molecule/ particle size at the sub-micron level at a laser wavelength of 659.0 nm and a temperature of 25°C. A sample volume of 500μL was utilized for size, distribution, and PDI studies, and a 1000 μL volume was utilized for potential surface measurement. The average values of three micellar diameter measurements for 12 runs were considered for all samples.

3.3.5 Viscosity

Viscosity was determined with Ostwald-Cannon-Fenske viscometer. The relative viscosity of

CUR-NMF was compared with the viscosity of the water. Briefly, one end of the viscometer was filled with either 5 ml CUR-NMF or 5 ml water with high care to avoid the formation of air bubbles. The micellar formulations, as well as water, were aspirated from the other end. The time required for the formulations and water to flow down under gravity was measured. The density was also measured. All measurements were performed in triplicate and expressed as mean ±SD.

Viscosity (CUR-NMF) = (Density (CUR-NMF) × time (CUR-NMF) × Viscosity (water)) /

(Density (water) × time (water))

Viscosity (water) = 0.89 centipoise (Cp) at 25°C with a Density (water) = 1 g/ml

3.3.6 Dilution Study

The effect of dilution on the formulation was studied by rapidly dissolving CUR-NMF samples up to 100 times in STF (simulated tear fluid) and measuring for size and PDI with DLS instrument (Table 3-4).

Table 3. 4 Dilution effect on CUR-NMF size and PDI.

Dilution factor Size (nm) (mean± SD) PDI (mean± SD)

0 17.01±0.06 0.123±0.004

20 19.99±0.28 0.200±0.012

40 18.54±0.10 0.115±0.008

100 18.51±0.28 0.177±0.005

3.3.7 Entrapment and Loading Efficiency

The entrapment efficiency is the percentage of drug-loaded with respect to the amount of initial drug. In this study, the total amount of entrapped drug in the formulation was determined by reverse phase-HPLC following published protocol207. One milliliter of each CUR-NMF sample was collected into 1.5-mL microcentrifuge tubes and centrifuged at 10,000 rpm for 10 min at 4°C to remove unincorporated curcumin. Approximately 0.5 mL of supernatant was carefully collected into a fresh tube and lyophilized to obtain a solid pellet. This pellet was resuspended in 0.5 mL of dichloromethane that resulted in a clear solution. Due to the addition of dichloromethane, curcumin (CUR) was released in the surrounding organic environment and underwent reversal of hydrophobic and hydrophilic segments to form reversed nanomicelles. The solution was evaporated under a speed vac to obtain a solid pellet. It was diluted with HPLC mobile phase, and the amount of drug present in the diluted sample was determined. The amounts of curcumin in the core of nanomicelles were calculated by 57 subtracting the total amount of unentrapped drug. The percent entrapment and loading efficiency of curcumin were calculated according to equations 3-1 and 3-2, respectively.

mass of curcumin in nanomicelles∗100 Percent Drug Entrapped (%EE) = (3- mass of curcumin added in the formulation

mass of curcumin in nanomicelles∗100 1) Loading Efficiency(%DL ) = (3-2) mass of curcumin added+mass of polymers added

A qualitative analysis was conducted with FT-IR and proton nuclear magnetic resonance (1H

NMR) to determine the presence of free curcumin in solution. Studies were performed for curcumin alone, blank nanomicelles and CUR-NMF

3.3.8 Critical Micelle Concentration (CMC) Determination

The critical micellar concentration was determined with a blend of HCO-40 and OC-40 using iodine as a probe. Iodine solution was prepared by maintaining a 1:2 ratio of I2 and KI, respectively.

The absorbance of hydrophobic iodine in the hydrophobic core of nanomicelles was determined by adding each of the diluted polymer solutions. With the help of a microplate reader (DDX 880; Beckman

Coulter, Inc., Jersey City, NJ), the Iodine entrapment was measured at 460nm.

3.3.9 Transmission Electron Microscopy (TEM)

TEM was used to assess the morphology and the shape of the CUR-NMF. Briefly, around 50

μL of CUR-NMF was placed on a carbon-coated copper grid. Samples were negatively stained by phosphotungstic acid and dried entirely before taking TEM images.

3.3.10 Cytotoxicity Studies

The toxicity of CUR-NMF was evaluated by performing lactate dehydrogenase (LDH) and

MTT assays on human D407 cells. Cell viability assay was performed to determine toxicity of CUR-

NMF on D407 cells according to the manufacturer’s protocol. Briefly, 10,000-12,000 cells/well were plated into 96-well plates and allowed to grow for 24h. Then 100 μL of blank and different dilutions of

0.05% CUR NMF (1, 5, 10, 20, and 40 μM) were dissolved in serum-free media added to each well.

All cells were exposed to formulations for 24 hr. Cell culture medium and 10% Triton X-100 served as negative and positive controls, respectively. Cell viability percentage was calculated based on negative

(medium) control (100%). Premixed WST-1 cell proliferation reagent was added according to the manufacturer’s protocol (Clontech, Mountain View, CA). Cell proliferation assay is based on the enzymatic cleavage of the tetrazolium salt (WST-1). A water-soluble formazan dye is detected by absorbance at 450 nm with a microtiter plate reader (Analyst GT, Molecular Devices, Sunnyvale, CA).

The amount of formazan formed is directly proportional to the number of viable cells.

3.3.11 Lactate Dehydrogenase Assay (LDH)

If any membrane damage occurs due to CUR-NMF exposure, then lactate dehydrogenase activity will increase in the culture media. The LDH assay was performed to assess the plasma membrane damage on D407 cells with Takara LDH cytotoxicity detection kit (Takara Bio, INC.,

Mountain View, CA). D407 cells were grown on 96-well plates at a density of 10,000 cells/well. After

48 hours of growth, D407 cells in each well were added to 100 μL of serum-free cell culture medium.

One hundred μL of blank and different concentrations of CUR-NMF (1, 5, 10, 20, and 40 μM) were added the in serum-free culture media. The cells were incubated for two h at 37°C. In this experiment, cell culture media served as negative controls. After incubation, the plate was centrifuged at 250 × g for 10 min in a dark condition. One hundred μL of supernatant from each well was transferred to an optically clear 96-well flat bottom plate. Then a solution containing equal volumes of catalyst and dye solution (prepared following manufacturer’s protocol) was added to each well. The mixture was incubated for 15 min at room temperature in the dark and then measured with a plate reader at 490 nm absorbance. A rise in a reading indicates higher LDH release in the culture medium, which directly correlates with the amount of formazan produced. Therefore, the amount of formazan produced is proportional to the number of plasma membrane-damaged cells. Cytotoxicity was calculated according to the equation (3-3)

(Observed experimental value− cell culture medium value)∗100 %Cytotoxicity = (3-3) Triton X−100− cell culture medium value

3.3.12 Antioxidant Activity Using ELISA

The antioxidant activity of CUR-NMF was assessed on D407 cells using H2O2 as a model of oxidative stress. Retinal cells exposed to oxidative stress enhance VEGF expression and release, leading to angiogenesis186, 208. Therefore, the amount of VEGF release was measured as a marker of antioxidant activity.

3.3.13 ELISA (Enzyme-linked immunosorbent assay)

VEGF-A ELISA estimated the amount of VEGF released by D407 cells post-exposure to 25,

50, and 100 µM of H2O2. Briefly, a pre-coated 96-well-microplate with anti-human VEGFA antibody was utilized for this purpose. Then a 50 µl of either samples or standards were added to all wells and mixed gently and incubated for 2 hours. Then it was washed thoroughly with a wash buffer to remove the unbound antigen. Subsequently, a 100 µl of secondary detecting antibody coupled to streptavidin-

HRP was added and incubated for 1 hr. After a 3X5min thorough wash, the TMB substrate was added, which result in a colorimetric signal because of the reaction with HRP. Lastly, UV-spectrophotometer was used to quantify the signal and the OD net =OD 450 – OD550208

3.3.14 In-Vitro Drug Release

The curcumin-release from nanomicelles was determined by in vitro release study in PBST

(8mM Na2HPO4, 150mM NaCl, 2mM KH2PO4, 3mM KCl, pH 7.4 and 0.5% Tween-80). Then, 1000

µl of CUR-NMF was added in a dialysis bag with a MWCO of 1000 Da. Both ends of the bag were tied and then immersed in 5 ml of PBST at 37C. At predetermined time points, the entire buffer was changed with a fresh PBST. The amount of curcumin released from CUR-NMF was assessed by the

RP-HPLC method. The release of curcumin was measured in triplicate, and the data were plotted as mean ±SD.

Statistical Analysis

GraphPad® Prism 7 and JMP software were used for statistical analysis. Data were summarized as mean ± SD. One-way ANOVA was used to determine the significance between control and different groups. Results with a p-value of < 0.05 were considered to be 60 significant. Statistical signiﬁcance was deﬁned as p < 0.05. Using the Tukey post-hoc test produced the same results as using the Bonferroni post-hoc test.

3.4 Results and Discussion

3.4.1 Experimental Design

The most influential factors in CUR-NMF formulation were identified by applying a full factorial experimental design in JMP software. All dependent variables were recorded and analyzed.

The formulations were characterized for size, PDI, EE, DL, and precipitation. Both HCO-40 and OC-

40 are water-soluble; therefore, any precipitation in a formulation of CUR-NMF will be unentrapped drug. Curcuminoids are not water-soluble and readily precipitated out in aqueous mixtures when not encapsulated. The percent of precipitation was determined using equation 3-4:

푊푒𝑖푔ℎ푡표푓푐푢푟푐푢푚𝑖푛푝푟푒푐𝑖푝𝑖푡푎푡푒 % Precipitation = × 100 (3-4) 퐼푛𝑖푡푎푙푡표푡푎푙푤푒𝑖푔ℎ푡표푓푐푢푟푐푢푚𝑖푛

Curcumin precipitate was isolated by centrifuging the mixture in an Eppendorf tube and separating the supernatant. The precipitate was further dried and weighed on a microbalance.

The prediction models for each dependent variable were initially evaluated using the coefficient of determination (R2), ANOVA, and lack-of-fit tests, and the results are presented in Table 3-3. It also summarizes the overall response obtained for each dependent variable along with their average and standard deviation. According to software, an acceptable model should produce a p-value in ANOVA analysis ≤ 0.05, and it is a coefficient of determination 0.70 ≤ R2. In the current study, all the dependent variables have R2 values higher than 0.70, indicating strong correlations between the independent and dependent variables. Furthermore, all p values for the ANOVA test are ≤ 0.05, indicative of a statistically significant model fit at a 95% confidence level. Also, p values for the lack-of-fit test are higher than 0.05, indicating no lack-of-fit for all the models. Thus, all the models for predicting the dependent variables are acceptable.

Fig. 3-2 A and B are a summary of the Pareto charts showing a summary of the effect of all independent variables on the dependent variables. Critical, independent variables that significantly affect any given response exert an effect size higher than the critical t value (tcrit= 2.776, df= 4, α=

0.05) indicated by a vertical blue line. As illustrated in the chart, the interactions of HCO-40 and CUR significantly increase CUR-NMF size while the interaction of OC-40 and HCO-40, as well as the interaction of CUR and OC40 significantly, reduces micelle particle size. It has also been observed that

OC-40 and HCO40 do not significantly alter nanomicelles particle size. In addition, OC-40 and the interaction factor CUR and OC-40 tend to expand nanomicelles polydispersity significantly.

Interactions of CUR and OC-40, as well as the interaction factor of HCO40 and OC-40, significantly decrease CUR-NMF polydispersity. Nonetheless, HCO-40 did not significantly affect the PDI. When considering the %EE, both HCO-40 and the interaction factors between CUR and HCO-40 significantly increased drug entrapment into the nanomicelles. However, CUR alone significantly decreases the entrapment efficiency.

Figure 3. 2 (A) Pareto charts summarizes the effect of independent variables and their interaction on CUR-NMF size, PDI, %EE, %LD and % precipitation, respectively. Bar graphs extend

64 past the tcrit cut off (blue vertical line) indicating statistical significance (p<0.05) and (B) Surface profile for all the 5 responses.

Other parameters such as CUR and OC-40 interaction, as well as OC-40, did not significantly affect %EE. Furthermore, %LD was only significantly affected (decreased) by increasing HCO-40 concentrations. It was also observed that % precipitation significantly lower with higher HCO-40 concentrations. None of the remaining independent variables affected the % precipitation. The CUR-

NMF prepared with the lowest HCO-40 concentration (0.05 wt%) always led to the formation of a precipitate during storage.

The overall effects of the independent variables and their interactions on the dependent variables are expressed by the following equations:

Y1= 18.53 – 2.025X1 – 0.625X3 + 0.5325X2 – 1.265X1X3 + 2.1375X1X2 – 1.8175X3X2

(3-5)

Y2= 0.2288 – 0.07X1 + 0.05175X3 – 0.018X2 – 0.07025X1X3 + 0.099X1X2 – 0.07925X3X2

(3-6)

Y3= 64.55 – 8.25X1 + 4.5X3 – 11.25X2 + 3X1X3 + 9.75X1X2 – 5.5X3X2

(3-7)

Y4= 2.5 + 0.735X1 – 0.6975X3 – 0.8535X2 + 0.25X1X3 + 0.37X1X2 + 0.3475 X3X2

(3-8)

Y5= 14.167 – 3.17X1 – 1.895X3 – 19.3075X2 + 5.6425X1X3 + 3.08X1X2 + 1.855X3X2

(3-9)

(X1= Drug wt%, X2=HCO-40 wt% and X3= OC-40 wt%)

Fig. 3-3 summarizes the prediction profiler as well as the desirability plot for the overall model.

The profiler allows a prediction of the dependent variables as a change with the independent factors. In this study, the goal (desirability) is to minimize nanomicelles size (Y1), PDI (Y2) and precipitation

(Y5) increase drug encapsulation efficiency (Y3) and loading efficiency (Y4).

Figure 3. 3 Prediction profiler and desirability plot showing the effects of independent variables on CUR-NMF size, PDI, %EE, %LD and precipitation.

Based on these criteria, the optimal CUR-NMF attained with experimental conditions of lowest

CUR concentration (0.05 wt%), lowest OC-40 concentration (0.1 wt%) and medium HCO-40 concentration (2.5 wt%). This condition yielded a maximum desirability value of 0.582.

Checkpoint Analysis

The resulting model, as well as the optimal formulation, were validated in a checkpoint analysis. It was conducted using a predicted optimal formulation F12 (X1 =0.2, X2 =2.5, X3=0.1) and two additional random formulations F13 (X1 =0.05, X2 = 2.5, X3= 0.1) and F14 (X1 =0.1, X2 =2.5,

X3=0.1). Each formulation was prepared in triplicates (n=3), and the dependent variables recorded and analyzed by ANOVA in comparison to the predicted values from the model. Table 3-3 summarizes the 66 predicted and measured values for all three checkpoint analysis formulations. No significant differences were observed between predicted and measured values, as indicated by the probability values obtained by ANOVA (P>0.05). Therefore, the model appears to be valid and could accurately predict the CUR-

NMF size, PDI, %EE, %LD, and precipitation. Among different formulations prepared, F13 exhibited the highest entrapment efficiency with the optimal percent of drug loading. Therefore, formulation F13 was used for further experiments.

3.4.2 Osmolarity and pH

Normal human tear pH varies from 6.5 to 7.6209. Therefore, CUR-NMF pH was adjusted to

6.85 with phosphate buffer. Osmolality is also a very crucial factor to be considered, especially for ophthalmic preparations. The osmolarity of CUR-NMF was shown as 312 mOsm/kg, which is similar to tear film osmolality.

3.4.3 Size, PDI and Surface Charge

CUR-NMF size and polydispersity indices were determined for all the prepared formulations.

In Fig. (3-4 A), results show a size range from 14 to 26 nm for CUR-NMF. Polydispersity indices were less than 0.6. CUR-NMF did not show any significant difference in size. We hypothesize that the size of CUR-NMF can traverse across aqueous sclera channels/ pores that range between 20-80 nm. These nanomicelles display a narrow and unimodal particle size distribution. Since these nanomicelles are in the same size range as membrane receptors, proteins, and other bio-molecules, we hypothesize that these nanocarriers can bind and traverse across cellular barriers. The surface charge of CUR-NMF is slightly negative (Fig. 3-4 B).

3.4.4Entrapment and Loading Efficiency

Curcumin entrapment and loading into the mixed nanomicelles were determined using an RP-

HPLC method. CUR-NMF drug entrapment and loading efficiencies are summarized in Table 3-2.

Among the formulations prepared and including checkpoint analysis, F13 was the optimum formulation with the highest entrapment efficiency and optimal drug loading. All the formulations were evaluated for appearance, viscosity, osmolality, size, PDI, and surface charge. 67

3.4.5 Viscosity

The viscosity of the formulation is one of the essential factors that need to be addressed, especially for ophthalmic preparations. The mean residence time and drainage of ophthalmic preparation depend on the viscosity of the instilled solution. Upon the addition of povidone K-90 to the formulation, the viscosity of the optimized CUR-NMF formulation was 1.90±0.032 cP (mean ± SD)

(n=3), at RT (25°C) which is similar to the viscosity of water, 0.89 cP. All formulation viscosities were less than 4.4 cP, indicating that the drainage rate may not be affected unless reaching the critical value210.

3.4.6 Dilution Effect

Tear dilution and protective mechanisms in the human eye prevent external materials from reaching inside the eye. Dilution effect study is vital because ocular barriers may cause premature release of the drug before reaching the back of the eye. Therefore, the dilution experiment was implemented to check the stability of the nanomicellar formulation upon serial dilutions. The dilution effect on CUR-NMF was studied through the rapid mixing of CUR-NMF formulation in stimulated tear fluid (STF) up to 100 times. No significant change was seen in the size and PDI of the nanomicelles after analyzing the samples in DLS. The size and PDI before dilution were found to be 17.01nm and

0.123, respectively. After 20, 40, and100 times dilution, the size, and PDI ranged between 18 to 20 nm

0.123 to 0.200, respectively (Table 3-4). Moreover, nanomicellar formulations were stable to the dilution effects.

3.4.7 Critical Micellar Concentration (CMC)

The ocular static and dynamic barriers are most challenging to overcome in order to deliver therapeutic drug concentrations to the back of the eye. Generally, 20% of a drug is available for absorption after tear dilution. Tear holding capability for the precorneal pocket is about 10 µl without overflow, and the turnover rate is 0.7 µl/min approximately75. Therefore, to avoid any disruption of micellar absorption, a very low CMC value is pivotal for topical delivery. Low CMC value maintains the stability of nanomicelles in the blood circulation. Iodine in the core of polymers ensured 68 sustainability by generating I3- to I2 from the potassium iodide solution. This experiment was conducted in triplicate. The CMC value of the blend of HCO-40 and OC-40 was found to be 0.01 (Fig.

3-4 C), indicating that the present formulation is highly stable and capable of resisting the tear dilution effect.

1 5 0

1 0 0

5 0

0 5 0 1 0 0 1 5 0

Figure 3. 4 (A) Size distribution of the nanomicelles, (B) Zeta potential for CUR-NMF and (C) CMC:

Plot of ultraviolet absorbance of I2 versus concentration of HCO-40/OC-40 nanomicelles (2.5:0.1) in water. CMC was calculated by corresponding polymer concentration at which a sharp increase in absorbance was observed.

3.4.81HNMR, FT-IR and XRD Characterizations

1HNMR spectra were recorded on a Varian 400 MHz spectrometer (Varian, USA) in deuterated water (D2O) or deuterated DMSO (DMSO-d6). HNMR spectral analysis suggested that the resonance peaks corresponding to curcuminoid present only in DMSO-d6. No peaks were observed in CUR-NMF in D2O (Fig. 3-5 A). These results proved that all curcuminoid molecules are effectively entrapped inside the hydrophobic core of the nanomicelles. There is no peak difference between blank nanomicelles and CUR-NMF, which proves that all curcumin molecules are entirely entrapped inside the core of the nanomicelles. FT-IR was conducted in three different groups: blank micelle, CUR-NMF, and curcumin alone (Fig. 3-5 B). In pure curcumin, C=C band stretch was noticed at 1630 cm1, and C-

H bending peaks were noticed at 1500 cm1. On the contrary, in blank micelle and CUR-NMF, we see the broad peak of OH from 3550 to 3200 cm1. Curcumin peaks were not shown in CUR-NMF, which means all curcumin molecules are effectively entrapped inside the hydrophobic core of the nanomicelles. XRD data of the polymers (HCO-40 and OC-40) and Curcumin are shown in (Fig. 3-5

C) 71

Figure 3. 5 (A) Qualitative 1HNMR studies. 1HNMR spectrum for (i) blank nanomicelles,

(ii) CUR-NMF and (iii) curcumin, (B) (i) Qualitative FT-IR spectrum for CUR-NMF, (ii) blank nanomicelles and (iii) curcumin alone, (C) XRD spectrums of HCO-40, OC-40 and curcumin alone.

3.4.9 Transmission Electron Microscopy (TEM)

Curcumin entrapped nanomicelles are clearly shown in spherical and round shapes. The size range of nanomicelles is from 14 to 26 nm which is consistent with sizes data obtained from DLS

(Fig. 3-6).

Figure 3. 6 TEM micrograph of CUR-NMF suggests as spherical shape.

3.4.10 Cytotoxicity Studies

The LDH and cell proliferation assay were performed under 24h of exposure to determine the long-exposure cytotoxic effects of the CUR-NMF. It was assumed that this incubation period would be sufficient to evaluate any toxicity211. Viable cells of D407 for blank nanomicelles, 1 µM, 5 µM, and 10

µM CUR-NMF showed consistency with that of the negative control (culture medium) (Fig. 3. 7 A).

Triton X-100 served as a positive control, which reduced the percent cell viability to ̴ 20 % for D407 cells. LDH assay was performed with D407 cells. The amount of LDH released in culture media directly

75 correlates with membrane damage and cytotoxicity. As expected, Triton X-100 caused more serious membrane damage. After exposure to blank nanomicelle and CUR-NMF (1 µM, 5 µM, and 10 µM), the percent cytotoxicity of D407 over 24hour appeared to be similar to the negative control (Fig. 3-7

B). However, 20µM and 40µM CUR-NMF showed slightly higher LDH release indicating cell membrane damage. This observation suggests that cell membrane damage by CUR-NMF is dose- dependent. If the dose of CUR-NMF is kept between 1-10 µM concentrations, the formulations can be safely for in-vitro model.

Figure 3. 7 Cell cytotoxicity assays: (A) MTT (B) LDH conducted on D407 following 24h of

CUR-NMF exposure. Triton-X 100 served as a positive control and culture medium as a negative control.

3.4.11 Cell Viability of D407 Cells Under Oxidative Stress by H2O2

Different concentrations (25, 50, and 100 µM) of hydrogen peroxide (H2O2) were mixed with culture medium and then exposed to D407 cells for 24h. Cell viability was verified with MTT assay following manufacturer protocol. Cell viability was dependent on H2O2 concentration. One hundred

µM concentration of H2O2 showed the highest cell death, with around 26% of cell viability (Fig. 3-8

A).

3.4.12 Protective-Effect of CUR-NMF on D407 Cells against H2O2-Induced Oxidative Stress

D407 cells were grown in a 96-well plate for 24 h. Cells were arranged in three groups and pretreated with blank nanomicelles, 10 µM curcumin, and 10 µM CUR-NMF for four h. Then all the formulations were removed, and cells were rinsed 3X5min with PBS. Since 100 µM hydrogen peroxide caused maximum cell death (Fig. 3-8 A), the same concentration was administered to study the

77 protective effect of CUR-NMF. A culture medium containing 100µM hydrogen peroxide was added to each well and incubated for another 24 h. Fig. 3-8 B shows that blank nanomicelles did not protect the cells from oxidative stress, and only 25% of cells survived. Curcumin (10 µM) alone also demonstrated almost similar results with a little increase in cell viability. However, 10µM CUR-NMF produced the highest cell viability ~ (60%) and thus provided the highest level of protection against oxidative stress- induced by H2O2. The failure of curcumin alone in protecting the cells against oxidative stress is possibly due to its low solubility and stability issues.

When curcumin alone was incubated with serum-free media, around 90% of it degraded within

30 minutes, which is consistent with our results212. Secondly, curcumin is highly hydrophobic and insoluble in an aqueous medium. Without nanomicellar formulation, curcumin is usually dissolved in

DMSO with dilutions in serum-free media. Therefore, there might be some possibility of microscopic precipitation of curcumin, which in turn reduces its antioxidant activity. In contrast, CUR-NMF is highly soluble and stable in the aqueous medium, leading to a higher protection of retinal cells in response to oxidative stress, and consequently, higher cell viability. A

Figure 3. 8 (A) MTT assay onD407 cell line after exposing various H2O2 concentration for 24h.

Data were expressed as mean ± SD (n=5), a p-value of <0.005 was considered to be statistically significant from the control and represented by *** and (B) Cells were pretreated for 4 hours with blank nanomicelles, 10 µM curcumin, 10 µM CUR-NMF, then all formulations were removed, rinsed with

PBS, followed by the addition of 100 µM of H2O2. N=5, data are expressed as mean ±SD. A p-value of < 0.005 considers statistically significant from control and represented by ***.

3.4.13 Oxidative Stress-Induced VEGF Secretion

D407 cells were exposed to 25 µM, 50 µM, and 100 µM of H2O2 for 24hour in the presence or absence of 10 µM CUR-NMF. D407 cells without any treatment were considered as a negative control.

Then the culture medium was collected and centrifuged to analyze the concentration of VEGF by

ELISA. The VEGF standard concentration varied from 31.25 to 2000 pg/ml, with an approximate of

R2= 0.9938. The concentration of VEGF in D407 retinal cells, without any treatment (control), was

(574 ± 45 pg/ml) with n=4 (mean ±SD) after 24hour of incubation. When cells were exposed to 25µM,

79 and 50 µM of H2O2 for 24hour, an increased amount of VEGF secretion was observed. However,

VEGF secretion was dramatically reduced in the presence of 100 µM H2O2, possibly due to the reduced number of cell survival. Post-exposure to 25 µM H2O2 and 50 µM H2O2, the amount of VEGF secretion was increased to ~1988 and 1415 pg/ml, respectively. On the contrary, when the D407 cells were exposed to oxidative stress by H2O2 in the presence of 10 µM CUR-NMF, a concentration-dependent decrease in VEGF secretion was observed. These results indicate higher anti-VEGF activity of 10 µM

CUR-NMF in the culture media (Fig. 3-9). Therefore, our CUR-NMF formulation is suitable to inhibit

VEGF production in response to oxidative stress in this model. The massive reduction in the VEGF level by CUR-NMF is possibly due to the inhibition of cascade of various biochemical pathways, including VEGF, COX-2, and IL-2, by curcumin213. Further studies need to be performed to investigate all the molecular pathways involved in VEGF signaling that can be inhibited by CUR-NMF.

3.4.14 In-Vitro Drug Release

To investigate the release of curcumin from the nanomicellar formulation, we performed the release study in PBST (Phosphate Buffer Solution + Tween 80) at a physiological pH 7.4, 37C, under sink condition. Cumulative percent of curcumin release from nanomicelles was very slow, i.e., ~85% of the drug was released over one month without any burst effect (Fig.3-10). Such a controlled release of curcumin suggests that the formulation will sustain the release under physiological conditions, which may help to achieve therapeutic concentrations of curcumin in the posterior segment of the eye with less frequent doses. Moreover, the sustained curcumin release can help in counteracting the retinal oxidative stress and provide long-term protection of retinal cells.

The slow release may be attributed to the lipophilic nature of curcumin. Also, the strong hydrophobic interactions between the hydrophobic portions of the amphiphilic polymers and 81 curcumin may also slow the release rate. Our results suggest that nanomicelles as a delivery system are capable of providing a sustained release of curcumin.

Figure 3. 10 In vitro drug release in PBST (pH 7.4), at 37°C under sink conditions. Data are expressed in mean ± SD where n=3.

3.5 Conclusion

The study demonstrates the preparation, optimization, and characterization of the natural curcumin-loaded nanomicellar formulation (CUR-MNF) for topical ocular delivery. A clear aqueous

CUR-NMF was prepared using amphiphilic polymers (HCO-40 and OC-40) with the solvent evaporation technique. A full factorial design was utilized with JMP software to optimize formulation size, PDI, entrapment efficiency, drug loading, and precipitation. Cell cytotoxicity assays showed that

5-10 µM dose of CUR-NMF is safe for topical application. The CUR-MNF formulation has shown a significant protective effect against H2O2 induced oxidative stress in D407 cells as well as the anti-

VEGF effect. The hydrophobic interactions between polymers and curcumin allowed the sustained release of curcumin over 1 month, indicating its potential in providing long-term protection of retinal cells against oxidative stress. These promising findings indicate that CUR-NMF may allow topical delivery of therapeutic concentrations of curcumin for the treatment of both types of AMD through its antioxidant, anti-inflammatory, and anti-VEGF actions.

CONFLICTS OF INTEREST

The authors declare that they have no conflict of interest.

ACKNOWLEDGEMENTS

The authors would like to thank Abrar Alnafaisah, and Dr. Nathan Oyler (UMKC, Chemistry department) for helping with FT-IR instrumentation and spectrum.

CHAPTER 4. SELF-ASSEMBLED BIOTINYLATED NANOMICELLES OF

CELECOXIB FOR BACK OF THE EYE DISEASES

4.1 Rationale

Age related macular degeneration (AMD) is a prevalent cause of blindness affecting approximately 11 million Americans and the number is expected to be doubled by the end of 2050 214.

Recent ophthalmic investigations suggest that AMD is a low grade inflammatory disease which is characterized by increased vascular endothelial growth factor (VEGF) release and expression, increased pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) production and action, and lastly some degree of oxidative stress 215. Currently anti-VEGF therapies such as ranibizumab, bevacizumab, pegaptanib and aflibercept are considered the corner stone for AMD management. These biologics are administered as intravitreal injection every 4-6 weeks based on the severity of the symptoms, disease progression and neovascular leakage 187, 216-218. However, high cost, retinal detachment and patient non-compliance render such approach to be unsuitable option 219. Moreover, anti-VEGF therapy does not address other factors involved in the pathogenesis of the diseases such as high production of pro-inflammatory cytokines 220-221.

Poor aqueous solubility and specificity have been a major problem for the ocular administration of BCS II class drugs, such as celecoxib. It is important to find an effective approach to enhance the solubility and the specificity of the drug to retinal cells of the eye. Celecoxib (CXB), a cyclooxygenase-

2 (COX-2) inhibitor, possesses anti-inflammatory, anti-VEGF and anti-proliferative activities, which can offer a potential therapeutic approach for AMD 95, 222. CXB is very hydrophobic with aqueous solubility of only 3.2 µg/mL at room temperature and requires a delivery system in order to reach back of the eye segment 223. Several delivery systems have been studied to deliver CXB to the back of the eye including nanoparticles and microparticles as intravitreal injections 224-225. However, such delivery systems lack patient compliance and are difficult to apply especially for elder people80. Nanomicelles are self-assembling colloidal materials with hydrophobic core and hydrophilic shell. Topical

84 administration in the form of nanomicelles offer advantages over the intravitreal route due to easy administration, smaller nanomicellar size and provide better patient compliance 153.

Transporter targeted drug delivery is a promising strategy to enhance the cellular internalizations and the specificity of nanoconstructs 226. A previous study has shown high expression of sodium dependent multivitamin transporter (SMVT) in retinal pigmented epithelial cells (D407) and human corneal epithelial cells (HCEC) 227. SMVT is transmembrane protein which is responsible for translocating vitamins, biotin and other factors to retinal cells. Therefore, we postulated that utilizing biotin as a targeting moiety may enhance cellular accumulation of biotinylated nanomicelles in retinal cells, and consequently enhance retinal bioavailability.

The purpose of this study was to design, characterize and evaluate self-assembled biotinylated,

CXB loaded nanomicelles and optimize the formulation based on entrapment efficiency, drug loading, size and polydispersity index (PDI). We have demonstrated the significant diminished release of

VEGF-A from D407, TNF-α and IL-6 by RAW (macrophages) cells in response to biotinylated nanomicelles loaded CXB. We conducted both MTT and LDH assays on pigmented retinal epithelial cells (D407), human corneal epithelial cells (HCEC) and human conjunctival cells (CCL 20.2) to indicate the safety and tolerability of CXB nanomicelles for in vitro model. Fig. 4-1 shows a schematic representation of self-assembled biotinylated nanomicelles loaded with CXB.

Figure 4. 1 Schematic drawing image of self-assembled biotinylated nanomicelles loaded with CXB

4.2 Materials

Celecoxib, shown in Fig 4-2, was obtained from Fischer Scientific with > 99% purity.

Hydrogenated Castor Oil 40 (HCO-40) was purchased from the Peboc division of Eastman Company,

UK. Octoxynol-40 (IgepalCA-897) was obtained from Rhodio Inc., NJ, USA. HPLC grade acetonitrile, methanol, and formic acid were procured from Fisher Scientific, USA. DMAP and water-soluble EDC were purchased from Fisher Scientific, USA. Cell-Titer 96® AQueous nonradioactive cell proliferation assay (MTS) kit and lactate dehydrogenase (LDH) assay kit was obtained from Promega Corp and

Takara Bio Inc., respectively. RAW 264.7 and CCL 20.2 cells were procured from the American Type

Culture Collection (ATCC, Manassas, VA). Dr. Richard Hunt (University of South Carolina,

Columbia, SC, USA) kindly provided D407 cells. HCEC is SV-40 virus transfected human

86 immortalized corneal cells; this cell line was a generous gift from Dr. Araki-Sasaki (Kinki Central

Hospital, Japan). VEGF-A, TNF-α, IL-Iβ, and IL-6 ELISA kits were received from eBioscience.

Figure 4. 2 Chemical structure of CXB (celecoxib)

4.2.1 Cell Culture

D407, CCL 20.2, and RAW 264.7 cell lines were cultured first in T75 flasks in Dulbecco’s

Modified Eagle Medium (DMEM) that contains high l-glutamine and glucose concentrations, 1% nonessential amino acids, and 10% FBS (heat-inactivated). 100 IU/mL streptomycin and 100 IU/ mL penicillin were added to DMEM to protect cells from bacteria. The pH of the medium was adjusted to

7.4, similar to human blood pH. Cells were incubated at 37°C in an atmosphere containing 5% CO2 and 90% relative humidity. The media was replaced every alternate day until cells reached 90% confluence.

4.3 Methods

4.3.1 Experimental Design

The central composite design (CCD) is more useful methodology for modelling various technological processes in the case of small number of preformed experiments compared with “one variable at a time” approach228. The effects of formulation parameters were analyzed using a CCD with three center points. The analysis was performed in JMP software (version 8.0, SAS Institute Inc., Cary,

NC, USA). A total of 16 experimental runs were conducted by varying the weight percent of three independent variables, including Celecoxib (X1) HCO40 (X2) and OC-40 (X3) (Table 4-1). The dependent variables, namely, size (Y1), entrapment efficiency (Y2), and loading efficiency (Y3) were

87 further evaluated based on the statistical design of experiments (DOE) (Figure 4. 3). The optimal CXB nanomicelles was obtained by maximizing the desirability function and its acceptability. The independent and dependent variables, as well as their coded values, are presented in Fig 4-3. -1, +1, 0, and -1 represent the upper, middle, and lowest values, respectively, for a given independent variable.

Figure 4. 3 Independent variables with coded, uncoded levels and their responses.

4.3.2 Chemical Synthesis

Octoxynol 40 (OC-40) is an amphiphilic polymer which has a free terminal hydroxyl (OH) group. On the other hand, biotin has a free carboxylic (COOH) group. Therefore, with the help of

EDC/DMAP chemistry, an ester bond has been established to form a product called biotinylated OC-

40227. The molar ratio of OC-40 to biotin was 1:15. The product was purified with a dialysis bag

(MWCO 1000 Da) against DI water for 2 days to remove unreacted biotin, EDC, DMAP and other impurities followed by lyophilization. The lyophilized powder was analyzed with 1H-NMR and FT-IR and used for further experiments.

4.3.3 Characterization of The Synthesized Biotin Conjugates with 1H-NMRand FT-IR

1H-NMR 400 MHz was used to analyze biotin, OC-40, and biotinylated OC-40 conjugates.

Briefly, 3 mg of OC-40 and biotinylated OC-40 were dissolved in D2O. On the other hand, the same

88 amount of biotin was dissolved in deuterated DMSO (d-DMSO) since it has low solubility in D2O. All samples were lyophilized and scanned for FT-IR.

4.3.4 Preparation of 0.1% CXB Loaded Biotinylated Nanomicelles

Biotinylated nanomicellar formulations loaded with 0.1% CXB were prepared following solvent evaporation and film rehydration technique as described previously 229. Briefly, predetermined amount of CXB was accurately weighed and dissolved in ethanol. Then, HCO-40 and biotinylated OC-

40 were weighed out and dissolved separately in ethanol. The ethanolic solution of HCO-40 and OC-

40 were mixed which is the resultant solution. Next, 0.1% of CXB ethanolic solution was added dropwise to the resultant solution. The organic phase (ethanol) was evaporated using high speed vacuum (Genevac) for 8 hours cycle to obtain a thin film. The resultant thin film was rehydrated with

DDI water, and the volume was built up with PBS containing Povidone K 90 (BASF SE,

Ludweigshafen, Germany). Povidone K 90 was used as a viscosity enhancer to increase the residence time of the formulation at action site. The solution was then vortexed for 10 minutes. Lastly, all the formulations were filtered with 0.22 µm filter membrane to remove unentrapped CXB aggregates and other foreign particulates.

4.3.5 Size, Polydispersity Index (PDI), Zeta Potential and Surface Morphology

Size, PDI and zeta potential of CXB nanomicelles were obtained with Zeta Sizer (Zetasizer

Nano ZS, Malvern Instruments Ltd, Worcestershire, UK) which employs dynamic light scattering technique for particle size measurement at room temperature (RT) (Fig. 4). Briefly, 1 ml of CXB nanomicellar solution (1 mg/mL) was placed into a glass cuvette. The samples were measured at a scattering angle of 173 º and 25 ºC. Data were collected in triplicate measurements as mean ± standard deviation (SD). The morphology of the nanomicelles was analyzed with transmission electron microscopy (TEM) as a shown in Fig. 5.

4.3.6 Entrapment Efficiency and Drug Loading

The total amount of CXB entrapped inside the core of the nanomicelles was determined by reversed-phase HPLC (RP-HPLC). One milliliter of each formulation in the Eppendorf tube was 89 centrifuged at 10,000 rpm at 4 °C for 10 minutes. Five hundred microliter of the supernatant was precisely collected from the Eppendorf tube and transferred to a new vial followed by lyophilization to obtain thin film. Five hundred microliter of the dichloromethane was added to reverse the micelle and release all the CXB in the surrounding organic phase. The solution mixture was evaporated using speed vacuum to obtain solid pellets of reversed micelles. The pellets were then dissolved in the mobile phase

(80% acetonitrile/20% water with 0.1% formic acid), and the amount of the drug was determined with

RP-HPLC. The method was applied to analyze samples with Shimadzu LC pump (Waters Corporation,

Milford, MA), Alcott autosampler (model 718 AL), Shimadzu UV/Vis detector (SPD-20AV), and

Phenomenex C18 column (Spherisorb 250 × 4.60 mm, five μm). The mobile phase was composed of acetonitrile and water (80:20, v/v) with 0.1% formic acid with the ﬂow rate set at 0.5 mL/min and UV detector at 254 nm. Appropriately 1 mg/mL CXB (stock) solution, which was diluted to use as internal phase. Calibration curve was constructed by injecting 30 µL of serially diluted CXB (500 µg/mL) in the mobile phase showed the highest linearity with R2 = 0.9998 for a range of 1 to 500 µg/mL concentration range. The percent of the entrapment efficiency and drug loading was calculated according to the following equations and summarized in Table 4-1.

Table 4. 1 Size, loading and entrapment efficiency of CXB nanomicelles at varying wt. % ratio of polymers Formulation CXB wt% Composition of (HC0-40: DL (%) EE (%) Size(nm) biotinylated OC-40)(wt. %) 29.4 ± 25.33 ± F1 0.1 0.5: 1 1.93 ± 0.03 2.76 0.23 99.3 ± 14.70 ± F2 0.1 2.5: 1 3.67 ± 0.06 1.56 0.07 35.5 ± 28.95 ± F3 0.1 0.5: 0.01 2.10 ± 0.09 3.12 0.43

89.1 ± 16.62 ± F4 0.1 2.5: 0.01 3.56 ± 0.12 2.11 0.09

푎푚표푢푛푡표푓퐶푋퐵푞푢푎푞푛푡𝑖푓𝑖푒푑𝑖푛푛푎푛표푚𝑖푐푒푙푙푒푠 퐸푛푡푟푎푝푚푒푛푡푒푓푓푖푐푖푒푛푐푦(%퐸퐸) = × 100 … … … 푎푚표푢푛푡표푓퐶푋퐵푎푑푑푒푑𝑖푛푡ℎ푒푛푎푛표푚𝑖푐푒푙푙푒푠

(4-1)

푎푚표푢푛푡표푓퐶푋퐵푞푢푎푞푛푡𝑖푓𝑖푒푑𝑖푛푛푎푛표푚𝑖푐푒푙푙푒푠 퐿표푎푑푖푛푔푒푓푓푖푐푖푒푛푐푦(%퐷퐿) = × 100 … … … 푎푚표푢푛푡표푓퐶푋퐵푎푑푑푒푑+푎푚표푢푛푡표푓푝표푙푦푚푒푟푠푢푠푒푑

(4-2)

4.3.7 Osmolality and pH

Since ocular fluids are isotonic, the topical ophthalmic preparations should be isotonic as well.

The formulation was adjusted with 0.9 % NaCl to normalize the osmolarity using Osmometer Vapro

5520. The pH of the formulation was adjusted with phosphate buffer to match lacrimal fluid pH which is 7.4.

4.3.8 Clarity Examination

The light transmittance percentage (%T) (n=4) was measured at different wavelength range with a UV-Vis spectrophotometer. DI water served as a negative control, and all measurement was recorded in triplicate.

4.3.9 Viscosity

Relative Viscosity of biotinylated nanomicelles loaded CXB was determined with Ostwald-

Cannon-Fenske viscometer, where the viscosity of the formulation was compared to the viscosity of water. Povidone K 90 was added to all the formulations to increase the viscosity and hence enhance the mean residence time of the formulation at the site of the action. Briefly, one end of the viscometer was filled with either 5 mL CXB nanomicelles or 5 mL DI water with extreme care to avoid air bubbles forming. The time required for the formulations and water to flow down under gravity was determined.

The density of the formulation, as well as DI water, were also measured. All obtained data were collected in triplicate and expressed as mean ± SD. Table 4-2 summarized the physical properties of the blank and biotinylated nanomicelles, including viscosity. Relative viscosity was obtained according to the following equation:

Viscosity (CXB nanomicelles) = (Density (CXB nanomicelles) × time (CXB nanomicelles) ×

Viscosity (water)) / (Density (water) × time (water)) … … … Eq. (4-3)

Where Viscosity (water) = 0.89 centipoise (Cp) at 25°C with a Density (water) = 1 g/mL.

Table 4. 2 Physical properties of blank nanomicelles and CXB loaded biotinylated nanomicelles (F2)

Formulation Size PDI Zeta %EE %DL Viscosity(cP) potential (mV) Blank 0.146 ± 17.3 ± 0.21 0.94 ± 0.20 NA NA 1.98 ± 0.06 nanomicelles 0.012

CXB loaded 0.154 ± 3.67 ± biotinylated 14.7 ± 0.07 0.22 ± 0.13 99.33 ± 1.56 1.92 ± 0.09 0.018 0.06 nanomicelles

4.3.10 In-Vitro Cytotoxicity (MTT assay)

Cytotoxicity of naked CXB and CXB-loaded biotinylated nanomicelles were evaluated by

MTT (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and lactate dehydrogenase (LDH) assays on human corneal epithelial cells (HCEC), retinal pigmented epithelial cells (D407) and choroidal endothelial cells (CCL20.2) according to the manufacturer’s protocol. Briefly, 10,000 cells/well in 100 µL of DMEM solution containing 10% FBS were seeded in 96-well plates. Cells were cultured and allowed to attach for 24 hours at 37 °C in an environment containing 5% CO2 and 90% relative humidity. Then, all formulations were prepared in serum-free DMEM medium and filtered with a sterile 0.22 µm filter membrane under laminar flow inside the hood. After that, DMEM media was removed, and cells were rinsed with DPBS twice.

Then, cells were exposed to different micromolar concentrations of CXB and biotinylated nanomicelles loaded with CXB for 24 h at 37°C. Serum-free medium served as negative controls. After

24 hours of incubation, 10 µL of MTT stock solution (5 mg/mL) was added to all the wells. Cell viability percentage was calculated based on the absorbance (Abs.) at 490 nm wavelength of samples, negative and positive control. The amount of formazan product is directly proportional to the number of viable cells. Cell viability was expressed according to the following equation:

Cell viability (%) = (Abs. of the sample – Abs. of serum-free medium)/ (Abs. of Triton X 10%

– Abs. of serum free medium) * 100 … … … Eq. (4-4)

4.3.11 Lactate Dehydrogenase Assay (LDH)

Lacated dehydrogenase (LDH) is an enzyme that is released to the surround tissues only if there is cellular damage 230. Therefore, if any membrane damage occurs to ocular cells due to CXB nanomicelles exposure, then LDH release will increase in the cell culture media. The LDH assay was performed to address the plasma membrane damage on D407, HCEC, and CCL20.2 cells with Takara

LDH cytotoxicity detection kit (Takara Bio, Inc., Mountain View, CA). Formulations were added to serum-free culture media and filtered with a 0.22 µm sterile nylon filter. All cells were grown on 96- well plates at a density of 4 cells/well. After 24 hours, 100 μL of blank and approximately 30 µM 93 concentration of CXB nanomicelles were added to the cells. In this experiment, serum-free cell culture medium and Triton X-10% served as a negative and positive control, respectively. After incubation, the plate was centrifuged at 250 ×g for 10 min in the dark, and 100 µL supernatant from each well was transferred to an optically clear 96-well flat bottom plate. Then a solution containing equal volumes of catalyst and dye solution was prepared according to the manufacturer’s protocol and added to each well. The mixture was incubated for 15 min at room temperature in the dark, and then absorbance was measured with a plate reader at 490 nm. A rise in a reading indicates higher LDH release in the culture medium, which directly correlates with the amount of formazan produced. Therefore, the amount of formazan produced is proportional to the number of plasma membrane damaged cells. Cytotoxicity was calculated according to the equation (5-5)

% Cytotoxicity = (Observed experimental value − cell culture medium value) ∗100 Triton

X−100 – cell culture medium value) … … … Eq. (4-5)

4.3.12 Enzyme-Linked Immunosorbent Assay (ELISA)

4.3.12.1 VEGF-A

VEGF is a signaling protein produced and released from different tissues in the human body, which plays a crucial role in angiogenesis development, the formation of new blood vessels231. The role of VEGF in wet AMD development has been studied extensively 232-234. Therefore, the anti-VEGF activity of CXB nanomicelles was assessed with VEGF-A ELISA. Briefly, D407 cells were grown on

96-well plate at a density of 20,000 cells/well and treated with different µM concentrations (1 - 30 µM) of CXB nanomicelles for 24 h and incubated at 37 °C in an environment containing 5% CO2 and 90% relative humidity. Cell-free supernatants were carefully collected and analyzed with a VEGF-A ELISA kit following the manufacture’s protocol (Promega).

4.3.12.2 Pro-inflammatory Cytokines Analysis (TNF-α and IL-6)

Recent studies have shown that TNF-α, IL-6, and ILβ cytokines are involved in inflammation

235-236. To investigate the effect of blank and CXB nanomicelles on cytokines release from lipopolysaccharides (LPS) treated group, macrophages cells were grown in 96 well plates at a cell 94 density of 5000 cells/well for 24 h. We noticed that when the cell counts were less than 5, the basal

TNF-α and IL-6 were not detected by ELISA, most possibly because the released cytokines levels were below the lower limit of detection by these ELISA assays. This experiment was composed of five different groups: serum-free medium, control (serum-free medium with cells), 1 µg/mL LPS, 1 µg/mL

LPS + 30 µM blank nanomicelles and 1 µg/mL LPS + 30 µM CXB nanomicelles. Basal TNF-α and

IL-6 secretion from RAW cells were very low, 30, and 5 pg/mL, respectively. Therefore, 1 µg/mL LPS was used to stimulate TNF-α and IL-6 release from macrophages cells. Cells were incubated with 30

µM blank and CXB nanomicelles for 8 hours, then all the formulations were removed, and cells were incubated with LPS for 24 hours at 37 °C, 5% CO2 incubator. Supernatants were collected carefully, and the concentration of TNF-α and IL-6 cytokines were analyzed with an ELISA kit following the manufacturer’s protocols (eBioscience).

4.3.13 Drug Release Study

CXB has a very low aqueous solubility of 3.3 µg/mL. A fixed volume of (300 µg/300 µL) of naked CXB and CXB loaded nanomicelles was transferred to membrane tubing (MWCO 2000 Da,

Spectrum Laboratories, CA, USA). Samples were immersed in 5 ml of PBST (PBS with 3 % v/v Tween

-80) in shaking water bath at 50 rpm and 37°C to maintain sink condition. At each time interval, the entire 5 ml medium was taken out and replaced with a fresh 5 ml PBST to maintain sink condition. The reason for adding 3% v/v Tween-80 is to solubilize all the released CXB, which showed a saturation solubility of 977 ± 4 µg/mL. The amount of CXB released was determined with RP-HPLC.

4.3.14 LC-MS/MS Analytical Method Development

To analyze CXB, LCMS/MS method was developed. An AB Sciex 3200 Q-Trap Spectrometer

(Foster City, CA) coupled to a Shimadzu UFLC system (Columbia, MD) was employed with electrospray ionization (ESI) in negative mode with Analyst v.1.4.2 software. A stock solution of 100

µM of celecoxib was prepared in acetonitrile and diluted to 10 µM in 50% water + 50% acetonitrile with 0.1% formic acid, which was then infused into a mass spectrometer to optimize different compound dependent parameters (Table 4-3) using Analyst quantitative optimization wizard. 95

Chlorzoxazone was selected as an internal standard (IS). Initially, various compound dependent parameters were similarly optimized using a 10 µM Chlorzoxazone stock solution in 50% water + 50% acetonitrile with 0.1% formic acid. The most intense fragments (Q3) for both celecoxib and chlorzoxazone were selected for the final method. Source dependent parameters were optimized using flow injection analysis (FIA) operation of Analyst quantitative optimization wizard using the same 10

µM standard. Chromatographic detection was performed on a Phenomenex C18 column (100 ×4.6 mm,

5 μm) with a flow rate of 0.6 mL/min at ambient temperature. The optimized gradient was 30% C (0-1 min), 30-100% C (1-8 min), 100% C (8-9 min), 100-30% C (9-10 min) and 30% C (10-15 min) where solvent A is water with 0.1% formic acid and solvent C is acetonitrile with 0.1% formic acid.

Table 4. 3 Mass spectrometric parameters for the most intense MS/MS transition of celecoxib and chlorzoxazone in negative ESI mode detection a.

Compound Parent Fragment Declustering Entrance Collision Collision Collision Ion Ion (Q3) Potential Potential Cell Energy Cell Exit (Q1) (V) (V) Entrance (V) Potential Potential (V) (V)

Celecoxib 380.2 316.2 -95 -9.5 -18 -32 -4

Chlorzoxazone 168.1 132.1 -50 -11.5 -10 -28 -2 aGlobal method parameters were: source temperature 300 ºC, curtain gas (CUR) 20, ion spray voltage (IS) -4500 V, Gas flow (GS1 and GS2) 50 (arbitrary units), collision associated dissociation (CAD) High.

4.4.15 Linearity, Sensitivity, Matrix effect, Recovery and Cell Uptake Study

Standard 80 µM celecoxib with 10 µM chlorzoxazone was serially diluted in a step of 2 for 11 steps in water-acetonitrile (50:50, v/v) with 0.1% formic acid containing 10 µM chlorzoxazone as

96 internal standard and analyzed by the developed method in triplicate to construct the standard curve.

Limit of detection (LOD) and lower limit of quantification (LLOQ) was calculated using (3.3×Standard deviation of the lowest concentration)/slope of the standard curve and (10×Standard deviation of the lowest concentration)/slope of the standard curve (Table 4-4). The matrix effect was analyzed using retinal cellular extract to calculate the recovery of celecoxib. Firstly, matrix was prepared and checked for proper dilution with an acceptable recovery237. Briefly, 1.25×105 retinal cells/mL were grown to confluence and washed 3 times with 1 mL DPBS. Each time, after the addition of DPBS, the cells were left on ice for 5 minutes, and then the supernatants were discarded. Finally, 1 mL of cold DPBS was added, and the plate was kept at -80 °C overnight. The next day, cells were extracted with 3 mL acetonitrile with 10 µM internal standard (chlorzoxazone), mixed well on ice and transferred to 15 mL falcon tube. The extract was then centrifuged at 5000 ×g for 30 minutes at 4 °C. The supernatant was collected on a fresh, chilled falcon tube. To the well-containing cells, 2 mL of cold acetonitrile was added for a second extraction, mixed well and combined with pellets obtained from the 1st centrifugation, and re-suspended well. The cellular suspension was then centrifuged at 5000 ×g for 15 mins at 4°C. The supernatant was collected and combined with the 1st supernatant. To this 2 mL of cold water was added, freeze-dried under vacuum, and reconstituted in 200 µL water-acetonitrile

(50:50, v/v) with 0.1% formic acid to prepare the matrix. Matrix was serially diluted and spiked with an intermediate concentration of celecoxib (10 µM) to determine the exact condition allowing the detection of celecoxib without any interferences.

Similarly, the matrix effect was also assessed by filtering the acetonitrile-water extract. Three concentrations (low, intermediate, and high) were then selected to check the matrix effect and calculate the recovery. Matrix was spiked with 1, 10, and 80 µM of CXB. Similarly, three concentrations of CXB were prepared in water-acetonitrile (50:50, v/v) with 0.1% formic acid. These samples were analyzed by the developed method. Matrix effect was calculated by subtracting 1 from the ratio of area unit of celecoxib in the matrix to water and then multiplying by 100. Recovery was calculated by taking the ratio of the area unit of celecoxib in the matrix to that in standard solvent and then multiplying with 97

100 (Table 4-4). For the cell uptake study, HCEC were seeded at 12 well plates at a density of 50,000 cells/well in total 24 ml of DMEM and maintained until they reach confluence. Then, cells were treated with biotinylated nanomicelles (targeted), non-targeted nanomicelles (NT), targeted nanomicelles + 1 mM biotin, and celecoxib alone. Cells were lysed following the protocol mentioned above at 2h, 6h and 24 h intervals. The samples were prepared using the protocol described above for analysis by LC-

MS/MS using the validated analytical method to evaluate cellular uptake of celecoxib from nanomicelles in concerted efforts with confocal microscopy.

Table 4. 4 Recovery of celecoxib in retinal extract at three different concentration.

Concentration (µM) Recovery ± SEa

1 129.1 ± 3.9

10 122.5 ± 6.9

80 117.1 ± 4.1 aSE = Standard error

4.4.16 Cell Uptake Study by Confocal Microscopy

To validate cellular internalization and co-localization of the nanomicelles, in-vitro uptake studies were carried out on CCL20.2 cells and assessed with confocal laser scanning microscopy

(CLSM). The native solubility of coumarin-6, hydrophobic fluorescent dye, in water is very low, similar to CXB. Therefore, coumarin-6 was used as a fluorescent probe for cellular uptake studies. The various treatment groups were as follows; (i) control groups that contained serum-free media, (ii) coumarin-6 loaded non-targeted nanomicelles, (iii) coumarin-6 loaded biotin targeted nanomicelles and (iv) 1 mM biotin followed by addition of biotin-targeted nanomicelles (T). CCL 20.2 were seeded at 8- well

98 chamber slide at a density of 30,000 cells/well in total 200 µL of DMEM in each well. Then, cells were treated with various treatment groups. All formulations were in DPBS at a concentration of 50 µg/ml.

The cells were incubated for one hour with formulations, washed twice with cold DPBS to remove the excess formulations, and fixed with 4% cold buffered paraformaldehyde for 20 min. The cells were rinsed 3x5min with cold PBS and store at 4 °C for further analysis. The excitation wavelength of coumarin-6 is 490 nm.

Statistical analysis

At least three replicates experiments were performed, and results were expressed as mean ±

SD. Student’s t-test or one-way ANOVA were employed to determine statistical significance among groups where p < 0.05 indicated statistical significance in experiments. Statistical signiﬁcance was deﬁned as p < 0.05. Using the Tukey post-hoc test produced the same results as using the Bonferroni post-hoc test.

4.5 Results and Discussion

4.5.1 Experimental Design

The most influencing factors in CXB nanomicelles formulation were identified by applying a central composite design (CCD) in JMP software. CCD method is used to determine the number of experiments to be assessed for the optimization of the independent variable and responses. The minimum, intermediate, and maximum values of each variable are labeled as -1, 0, and +1. This is illustrated in fig 4. 3. All dependent variables were recorded and analyzed. The formulations were characterized for size, EE, and DL. Both HCO-40 and biotinylated OC-40 are water-soluble; therefore, any precipitation in a formulation of CXB nanomicelles will be the unentrapped drug. CXB is not water-soluble and readily precipitated out in aqueous mixtures when not encapsulated.

The prediction models for each dependent variable were initially evaluated using the coefficient of determination (R2), ANOVA, and lack-of-fit tests. It also summarizes the overall response obtained for each dependent variable along with their average and standard deviation. According to software, an

99 acceptable model should produce a p-value in ANOVA analysis ≤ 0.05, and it is a coefficient of determination 0.70 ≤ R2.

In the current study, all the dependent variables have R2 values higher than 0.70, indicating strong correlations between the independent and dependent variables. Furthermore, all p values for the

ANOVA test are ≤ 0.05, indicative of a statistically significant model fit at a 95% confidence level.

Also, p values for the lack-of-fit test are higher than 0.05, indicating no lack-of-fit for all the models.

Thus, all the models for predicting the dependent variables are acceptable.

Fig. 4-4 A, B and Care a summary of the Pareto charts showing a summary of the effect of all independent variables on the dependent variables. Critical, independent variables that significantly affect any given response exert an effect size higher than the critical t value (tcrit= 2.776, df= 4, α= 0.05) indicated by a vertical blue line. As illustrated in the chart, it is clear the biotinylated OC-40 has significantly reduces nanomicellar size. When considering the %EE, both HCO-40 and the interaction factors between CXB and HCO-40 significantly increased drug entrapment into the nanomicelles.

However, CXB alone significantly decreases the entrapment efficiency. Other parameters such as biotinylated OC-40 interaction with drug, as well as biotinylated OC-40, did not significantly affect

%EE. Regarding the %DL, both drug wt% and HCO-40 significantly increased the drug loading.

Furthermore, %LD was only significantly affected (decreased) by increasing HCO-40 concentrations.

Figure 4-5 summarizes the prediction profiler as well as the desirability plot for the overall model. The profiler allows a prediction of the dependent variables as a change with the independent factors. In this study, the goal (desirability) is to minimize nanomicelle size (Y1), increase drug encapsulation efficiency (Y2) and loading efficiency (Y3). Based on these criteria, the optimal CXB nanomicelles attained with experimental conditions of intermediate CXB concentration (0.1 wt%), lowest OC-40 concentration (0.1 wt%) and medium HCO-40 concentration (2.5 wt%). This condition yielded a maximum desirability value of 0.625.

100

101

Figure 4. 4 Pareto charts and surface profile for all responses summarizes the effect of independent variables and their interaction on CXB nanomicelles size (A), %EE (B), and %LD (C), respectively. Bar graphs extend past the tcrit cut off (blue vertical line) indicating statistical significance (p<0.05)

Figure 4. 5 Prediction profiler and desirability plot showing the effect of independent variables on biotinylated CXB nanomicelles size, %EE and %LD

102

4.5.2 Chemical Synthesis

The biotinylated-oc-40 conjugate was achieved by activating the free carboxylic acid of biotin with EDC in one beaker, and DMAP was added to OC-40 to activate the free terminal OH on the other beaker. The two solutions were mixed, and the reaction was continued for 24 hours at RT to establish an ester bond. Then, the product was transferred in a dialysis bag of 1000 Da MWCO and dialyzed against water for two days to remove the unreacted biotin, EDC, DMAP, and other foreign particulates.

The product was then lyophilized and characterized for further experiments. Fig.4. 6 shows a chemical synthesis scheme of biotinylated OC-40.

Figure 4. 6 Chemical synthesis schemes of biotinylated OC-40

4.5.3 1H-NMR and FT-IR

1H-NMR spectra of the biotinylated OC-40 conjugate and OC-40 were obtained on a 400

MHz NMR spectrometer with samples dissolved in deuterium oxide (D2O) at a concentration of 2 mg/mL. Biotin alone was dissolved in d-DMSO at the same concentration. All data were plotted on one graph for a more straightforward analysis (Fig. 4-7) The most common featured protons of biotin analysis in 1H-NMR are carboxylic group protons, and it can be seen in the downfield at 12 ppm, as shown in Fig 4-7. Also, the two duplets at 4 and 4.1 ppm, which represent the proton of the adjacent carbons (b, b̴ ) below the NH group [27]. For OC-40 H-NMR spectral interpretation, a broad peak is visible at 3.5 ppm, representing the (O-CH2-CH2)40 portion of the molecule. The product 1H-NMR analysis revealed two peaks at 4.2 and 4.3 ppm, which represent the presence of biotin and one characteristic peak at 3.5 ppm, which is coming from OC-40. Moreover, the absence of a carboxylic proton peak in the product proved that the conjugation or ester formation occurred at the terminal

103

COOH of biotin. The FT-IR results are shown in Fig. 4-8. In the finished product, biotinylated OC-

40, the ester bond can be clearly observed at 1760 cm-1.

NONAME00 0.45

0.40

0.35

0.30

0.25

0.20

0.15

0.10 Normalized Intensity Normalized 0.05 a 0

-0.05 b -0.10

-0.15 c

14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 Chemical Shift (ppm) Figure 4. 7 1H-NMR spectrums of (a) OC-40 in D2O, (b) biotin in d-DMSO and (c) biotinylated OC-40 in D2O

104

105

C C

Figure 4. 8 FT-IR spectrums of (a) OC-40 in D2O, (b) biotin in d-DMSO and (c) biotinylated

OC-40 in D2O Preparation of the 0.1% CXB loaded biotinylated nanomicelles

Biotinylated nanomicelles loaded CXB was prepared by a solvent evaporation method as described in the method section229. Using this technique, we were able to achieve a large encapsulation efficiency with 99% of CXB entrapped inside the hydrophobic core of the nanomicelles.

4.5.4 Size, Polydispersity Index and Zeta Potential

The observed hydrodynamic diameter of biotinylated CXB loaded nanomicelles was at the range of 14 nm, whereas the blank one at the range of 17 nm. The significant difference in mean diameter size between CXB and blank nanomicelles is attributed to the hydrophobic interactions between the drug and the hydrophobic portions of the polymers resulting in further size reduction for

CXB nanomicelles. On the contrary, blank nanomicelles have no drug hence less hydrophobic interactions in the core of the micelle leading to less size reduction. Both CXB and blank nanomicelles are monodisperse, as shown in Fig. 4-9 with a PDI of less than 0.2. The surface charge of the formulations was slightly negative. High cationic charge formulations lead to systemic toxicity, whereas high anionic charge creates difficulties due to repulsion between cell membrane and micelles with similar anionic charges238.

106

107

Figure 4. 9 (A) Size distribution, (B) intensity and (C) Zeta potential of biotinylated nanomicelles loaded CXB with DLS

4.5.5 Entrapment Efficiency and Drug Loading

Entrapment and drug loading are essential physicochemical properties of polymeric nanomicelles. Various methods have been used to quantify the concentration of drugs of interest inside the nanomicelles such as dialysis, gel filtration, ultrafiltration, and RP-HPLC. RP-HPLC is more sensitive than the other techniques; hence it has been utilized for determining drug loading and entrapment239. Different wt% ratio of polymers exhibited a range of entrapment and drug loading, as shown in Table 4-1. Among different formulations, F2 exhibited the highest entrapment efficiency and drug loading. Therefore, F2 was characterized further for size, PDI, zeta potential and morphology with

TEM (Fig.4-9).

108

Figure 4. 10 TEM figure for CXB nanomicelles showing round smooth morphology

4.5.6 Osmolality and pH

The lacrimal fluid is known to be isotonic to blood. Therefore, to optimize the tonicity of the formulation, a 0.9% w/w NaCl was added to all the formulations to adjust physiological tear osmolarity of 289 mOsm/L. The pH of the lacrimal fluids in the eye is typically about 7.4, and it has been shown that these fluids contain numerous buffer solutions such as carbonic acid and weak organic acids, which help to neutralize the tear. Therefore, the pH of all formulations was optimized with PBS (pH 7.4) to mimic the lacrimal fluid environment.

4.5.7 Clarity Examination

UV-Vis spectrophotometer was used to perform the clarity testing of nanomicelles at 400 nm wavelength. As shown in Fig. 4-11, the percentage of light transmittance of the biotinylated nanomicelles loaded with CXB was similar to DI water which means hardly any visible foreign 109 particles in CXB biotin targeted nanomicellar formulation. Therefore, this nanomicellar solution is clear and suitable for ophthalmic delivery.

DI water Nanomicelles

Figure 4. 11 Clarity test for CXB nanomicelles and DI water

4.5.8 Viscosity

Prolonging contact time with the cornea and different eye tissues may be obtained by enhancing the formulation’s viscosity. Therefore, povidone K-90 was added to all formulations as a viscosity enhancer, which will enhance the mean residence time and the intact time of the formulation for ocular drug delivery, as shown in Table 4-2. The viscosity of the formulation after adding PBS was similar to water (0.9 cP); however, it has increased to 1.98 ± 0.06cP and 1.92 ± 0.09cP for blank and CXB nanomicelles respectively after povidone K-90 addition. Recent studies have shown that maximum ocular tissue penetration by ophthalmic solutions occurs when the viscosity falls into the range of 15 to 150 mPa240.

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4.5.9 Drug Release Study

Determining the drug release profile of hydrophobic compounds from nanomicelles is a challenging task and requires a fine adjustment. Before performing drug release studies, a suitable dissolution media for hydrophobic compounds such as CXB should be evaluated based on saturation solubility. There are many approaches to enhance the solubility of hydrophobic compounds in aqueous dissolution media such as pH alteration, surfactants, and to increase the volume of the dissolution media. Therefore, 3% v/v Tween-80 (surfactant) was added to the dissolution media to enhance the solubility of the naked and released CXB in PBS. To determine the release pattern of naked CXB and

CXB polymeric nanomicelles, we performed release experiments in PBST at 37 ºC, as shown in Fig.

4-12. Nearly all the CXB has been released from the nanomicelles over one month without any burst effect. The slow-release profile of CXB from nanomicelles can be attributed to the hydrophobic nature of CXB, hydrophobic-hydrophobic interactions between CXB, and the hydrophobic portion of the polymers. Such a controlled release of CXB indicates that the formulation will sustain the release under physiological conditions, which will help to achieve therapeutic concentrations of CXB in the posterior segment of the eye with less frequent doses. Our results suggest that nanomicelles as a delivery system can provide a sustained release of CXB.

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Figure 4. 12 Drug release study of naked CXB and CXB loaded nanomicelles carried out on

PBST (pH 7.4) where n=3.

4.5.10 In-Vitro Cytotoxicity: MTT and LDH Assays

Cell proliferation assay, MTT assay was used to test the percent of cell viability after 24 hours of exposure to CXB alone and biotinylated nanomicelles loaded CXB in D407, HCEC, and CCL20.2 cell lines. As shown in Fig.4-13 (A, B and C) as we increase the dose of CXB nanomicelles to 100 µM, the cells were safe, well tolerated, and suitable for topical eye drops. On contrary, 100 µM CXB alone exhibited a significant toxicity on different eye cells. This can be attributed to the slow-release of the drug from the nanomicelles. LDH is an enzyme released from cells as a response to cellular damage by exogenous stimuli240. LDH assay (Fig.4-13 D) did not indicate any differences between control cells and biotinylated nanomicelles loaded CXB exposed cells indicating no toxicity by 24 h of exposure.

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113

114

C Conjunctival cells

150 CXB nanomicelles

% CXB alone

y 100

l l 50

e ✱ C

M M M M M µ µ µ µ µ 0 1 1 0 0 . 1 0 0 1

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Figure 4. 13 Cell viability assay MTT on (A) D407, (B) HCEC and (C) Conjunctival cells respectively, after 24 h of exposure to different concentration of CXB nanomicelles and CXB alone.

(D) LDH release from of D407, CCL20.2 and HCEC cells after 24hr of exposure to blank and CXB loaded biotinylated nanomicelles. (Paired t-test)

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4.5.11 ELISA

4.5.11.1 VEGF-A Release from D407

VEGF-A plays a crucial role in the angiogenesis or the formation of new blood vessels [21].

The newly formed blood vessels are abnormal, leaky, and fenestrated; hence the blood comes out very easily, resulting in wet AMD. Through inhibition of VEGF-A release, CXB nanomicelles may provide a solution for wet AMD. As depicted in Fig. 4-14 A, CXB nanomicelles were able to reduce VEGF-A release from retinal cells in a dose-dependent manner. Celecoxib at a concentration of 30 µM (around

11.43 µg/mL) inhibited 50% of VEGF-A secretion after 24 h exposure proving the anti-VEGF-A activity of CXB nanomicelles. These CXB nanomicelles are capable of sustained drug release for a prolonged period and capable of producing more inhibition of VEGF-A release. Therefore, CXB nanomicelles may provide a solution for wet AMD by inhibition of VEGF-A release.

4.5.11.2 Pro-inflammatory Cytokines TNF-α and IL-6

TNF-α and IL-6 are pro-inflammatory cytokines that are released from macrophages resulting in ongoing inflammation241. The experiment was designed to examine whether CXB nanomicelles can aid in reducing the inflammatory cytokines released from RAW 264.7 (macrophages) cells in the presence of LPS or not. Treatment of RAW 264.7 cells with LPS alone results in a significant increase in cytokine production as compared to the control group. Surprisingly, the level of TNF-α and IL-6 were significantly decreased compared to the LPS group after addition of CXB nanomicelles Fig. 4-14

B and Fig. 4-14C, respectively. Our results corroborate with previous studies that CXB is capable of relieving arthritis through the reduction of inflammation242.

Interestingly, blank nanomicelles that had no drug exhibited anti-TNF-α and anti-IL-1β activities, and this can be attributed to the presence of ricinoleic acid in HCO-40, the primary fatty acid found in castor oil, which is known for its anti-inflammatory activity243. CXB nanomicelles revealed a very significant reduction in inflammatory cytokines compared to blank nanomicelles, which may be due to additive activities of CXB and ricinoleic acid present in HCO-40 polymer. Further studies needed for screening different components in HCO-40, which might have potential anti-inflammatory activity. 117

A B

Figure 4. 14 ELISA analysis of (A) VEGF-A release from retinal cells D407 after exposure to different conc. of CXB nanomicelles; (B) TNF-α release in LPS-stimulated RAW cells after exposure to blank and 30 µM CXB nanomicelles; (C) IL-6 release in LPS-stimulated RAW cells after exposure to blank and CXB nanomicelles. The values are mean ± SD of triplicate. *p < 0.05, **p < 0.01 of blank vs biotinylated nanomicelles loaded CXB. Cells were exposed to treatment and control groups for 24 h.

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4.5.12 LC-MS/MS Chromatogram of Celecoxib with Internal Standard

Well resolved peaks for celecoxib and chlorzoxazone (IS) were obtained in multiple reaction monitoring (MRM) scan mode with reverse phase chromatography using C18 column and standard formic acid (pH 2.0) solvent system (Fig. 4-15 A & B). Average retention time for celecoxib and chlorzoxazone (IS) was 7.81 min and 4.92 min respectively with a standard error of 0.001 calculated from injection of 40 samples during constructing standard curve.

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Figure 4. 15 (A) Standard curve and linearity characteristics of celecoxib; (B) LC-MS/MS chromatogram of 10 µM celecoxib and 10 µM chlorzoxazone (IS).

4.5.13 LC-MS/MS Method Validation

Limit of detection (LOD) and lower limit of quantification (LLOQ) for celecoxib were calculated as 0.24 µM and 0.73 µM respectively from the standard curve (R2=0.9973). The area under the curve with increasing concentration showed excellent linearity in the concentration range of 0-80

µM showing the dynamic range of the developed method. Matrix effect and recovery were calculated using the formula described in the method section. Filtering the cell extract helped significantly with minimizing the matrix effect and maximizing recovery. Unfiltered matrix showed a matrix effect (%ME

± SE) of -18.6 ± 6.6 compared to filtering, which reduced the matrix effect (%ME ± SE) to -2.8 ± 1.8

26. A recovery study was done following this filtering of the matrix approach. For all three concentrations, recovery of celecoxib was in the range of 117.1-129.1% with an average standard error of 5.0% 120

4.5.14 Celecoxib Uptake Study into Retinal Cells from Nanomicelles

Drug uptake study using LC-MS/MS revealed that there is a significant difference between non-targeted nanomicelles and targeted nanomicelles, with a slight elevation (2 folds) in celecoxib uptake with time in the latter group (Fig. 4-16). Celecoxib uptake into cells from non- targeted and targeted nanomicelles were 7.1% and 16%, respectively, of the loaded dose. Biotinylated nanomicelles with 1 mM unlabeled biotin showed significantly reduced uptake (3.1%) of celecoxib. Since excess one mM biotin competitively blocked the SMVT transporter receptor, hence less biotinylated nanomicelles were taken up by retinal cells. On the other hand, the control group exhibited lower uptake at all-time intervals. A possible explanation for this is that the drug is not soluble in serum-free medium, and therefore, less drug was taken up by retinal cells.

Figure 4. 16 Celecoxib uptake study on HCEC cell line at 2, 6 and 24 h

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4.5.15 Determination of Micellar Uptake by Confocal Microscopy

Fig. 4-17 illustrates high internalization of biotinylated nanomicelles by conjunctival cells over

24 hours of treatment. Biotinylated nanomicelles utilize the SMVT transporter therefore, once SMVT transporter reaches saturation with excess biotin around 1 mM, the uptake of the biotinylated nanomicelles was very minimal. This data is consistent with LC-MS/MS data that uptake of CXB was substantially reduced when biotinylated nanomicelles loaded CXB was administered to retinal cells in the presence of 1 mM unlabeled biotin. The uptake of targeted nanomicelles are high and significantly reduced when cells were incubated with excess biotin which saturates SMVT transporter. Therefore, this strongly supports the hypothesis that biotinylated nanomicelles utilize SMVT transporter for translocation

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Figure 4. 17 Confocal localizations on conjunctival cells where Control = retinal cells with

PBS; NT = non-targeted nanomicelles loaded coumarin as a hydrophobic fluorescent dye; T= biotinylated (targeted) nanomicelles; and T + B= biotinylated (targeted) nanomicelles+ 1 mM biotin.

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4.6 Conclusion

CXB loaded biotinylated nanomicelles were synthesized following solvent evaporation thin- film rehydration technique. Previous studies227 showed an expression of SMVT, a biotin transporter in retinal cells; hence biotin was applied as a targeting moiety and conjugated with OC-40 with the help of EDC/DMAP chemistry. The ratio of 2.5: 0.1, HCO-40: biotinylated OC-40, was used with the entrapment efficiency and drug loading of 99.3% and 3.67%, respectively. 1H-NMR and FT-IR were used to analyze the purified product. Cell viability and cytotoxicity assays carried out in D407, HCEC, and CCL20.2 revealed that CXB nanomicelles have low in vitro toxicity potential and have potential for ophthalmic preparation. The nanomicelles were uniform and spherical, as shown by their TEM images. The significant reduction of VEGF-A secretion from D407 cells after exposure to CXB nanomicelles proved the anti-VEGF activity of CXB nanomicelles. Furthermore, the addition of CXB nanomicelles diminished LPS induced TNF-α and IL-6 release from macrophages cells (RAW 264.7), proving the anti-inflammatory activity. Cellular internalization study was performed with LC-MS/MS showed high cellular uptake of biotinylated nanomicelles loaded CXB compared to non-biotinylated one, which was confirmed by confocal microscopy data showing higher uptake of biotinylated nanomicelles. This study suggests that biotinylated nanomicelles loaded CXB can be a promising delivery system for managing AMD.

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CHAPTER 5. RESVERATROL NANOMICELLES AS A POTENTIAL TRATEMENT

FOR AGE RELATED MACULAR DEGENERATION

5.1 Rationale

Oxidative stress is associated with different eye diseases such as cataract, proliferative vitreoretinopathy, diabetic macular edema (DME), and age-related macular degeneration (AMD)244-245.

AMD is a neurodegenerative disease that affects the macula, which the most sensitive part of the retina.

AMD is the leading cause of blindness in the US and western countries, while 10% of the population over 60 are suffering from its consequences105. Retinal pigmented epithelial cells (RPE) is located between photoreceptor sensitive cells and choriocapillaris. RPE plays a vital role by controlling the exchange of the nutrients, oxygen and waste products between choroidal capillaries and photoreceptor sensitive cells. Hence RPE are subjected to oxidative stress as a result of high metabolic rate, high oxygen consumption, and high exposure to UV light246. Oxidative stress results in irreversible degeneration of RPE cells, which plays a crucial role in the pathogenesis of AMD.

Traditionally there are two types of AMD: dry and wet. The most common form of AMD is dry, which accounts for 80-90% of all AMD cases, and is characterized by the presence of deposits beneath retina called drusen. These drusen are made up of lipids, fatty acids, and proteins. The second type of AMD called wet AMD, which accounts for 10-15% of all AMD cases. The hallmark feature of wet AMD is the presence of new blood vessels as a result of the process of angiogenesis, the formation of new blood vessels. These new blood vessels are fragile, abnormal, leaky, and fenestrated, and hence sub-retinal bleeding is the primary feature of wet AMD247.

Currently, there is no treatment available for dry AMD. On the other hand, anti-VEGF biologics are considered as the cornerstone for the treatment of wet AMD. These biologics include ranibizumab, bevacizumab and aflibercept all given to the patients as a monthly intravitreal injection.

Although these biologics improve visual acuity, nearly in half of the patients, high cost, high patient non-compliance, and retinal detachments are still major problems for such delivery248-249. It has been hypothesized that antioxidant agents may protect cells from oxidative stress in the retina by 125 counteracting the free radicals that are produced in the process of light absorption. Higher dietary levels of antioxidant vitamins and minerals may reduce the risk of progression of age-related macular degeneration (AMD)250. Many studies have shown that taking antioxidant agents slows down the progression of dry AMD to late-stage AMD. Oxidative stress and inflammation play an essential role in the pathogenesis of AMD, which lead to permanent vison loss if left untreated.

Some herbals are known for their antioxidant, and anti-inﬂammatory actions, may have a remarkable effect in the prevention and treatment of AMD. Among several other plant-derived products, there is huge interest in the therapeutic effects of resveratrol on the posterior ocular diseases251-252.

Resveratrol (RSV), (3,5,41-trihydroxy-trans-stilbene), is a polyphenolic compound that is originally derived from peanuts, grapes, red wines and blueberries. RSV has gained so much of attention because of its antioxidant, anti-inflammatory and anti-cancer effect. Moreover, some studies have shown the ability of RSV to reduce VEGF levels in vitro and vivo, hence reducing the incidence of angiogenesis and choroidal neovascularization (CNV) in AMD patients253-254. Most of the beneficial effects of RSV are associated with its antioxidant activity. However, RSV possesses poor aqueous solubility (<50 µg/ml) and low bioavailability (< 1%), which limits its pharmacological activity and biological effect255. Therefore, there is an urgent need to develop a delivery system that can enhance the solubility and stability of RSV.

HCO-40 (Hydrogenated castor oil 40) and OC-40 (Octoxynol 40) are amphiphilic polymers that have been approved by the FDA as vehicles for ocular drug delivery. In the present study, we developed self-assembled nanomicellar formulations of RSV utilizing HCO-40 and OC-40 polymers.

Currently, there is no efficient treatment for AMD and delivery systems that enhance the uptake of the antioxidants might be important for the prevention of the disease. Our laboratory has succeeded in preparing cyclosporine nanomicelles, Cequa®, utilizing the same polymers and the formulation was recently approved by the FDA for dry eye disease153. We believe such a nanomicellar formulation has

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the potential to deliver RSV as eye drops for drug delivery to the retina. Fig 5-1 showed a schematic

representation of the RSV nanomicelles and the chemical structure of RSV.

Figure 5. 1 (A) Resveratrol (RSV) structure and (B) schematic representation of RSV nanomicelles

5.2 Materials

Resveratrol (RSV) was purchased from T.C.I. (Tokyo Chemical Industry) > 99% purity.

Hydrogenated Castor Oil 40 (HCO40) was purchased from Peboc division of Eastman Company, UK.

Octoxynol-40 (Igepal CA-897) was purchased from Rhodio Inc., NJ, USA. Tertiary-butyl hydrogen

peroxide solution (tBHPS) was purchased from Sigma Aldrich, MO, USA. HPLC grade acetonitrile

was procured from Fisher Scientific, NH, USA. Cell Titer 96® Aqueous nonradioactive cell

proliferation assay (MTT) and lactate dehydrogenase (LDH) assay kits were purchased from Promega

Corporation, WI, USA and Takara Bio Inc., Japan, respectively. VEGF-A ELISA kit was purchased

from Thermo Fisher Scientific, MA, USA. D407 cell line was a generous gift from Dr. Araki-Sasaki

(Kinki Central Hospital, Japan). Tertiary butyl hydrogen peroxide solution (tBHPS) was purchased

from Sigma-Aldrich, MO, USA.

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5.3 Methods

5.3.1 Cell Culture

The human retinal pigment epithelial cell line (D407) was obtained from Dr. Araki-Sasaki, Japan. The cells were maintained in DMEM supplemented with 10% fetal bovine serum, 1mM sodium pyruvate,

100U/mL penicillin, 100 mg/mL streptomycin, and 2.5 mg/mL amphotericin B, at 37°C, 5 % CO2, and

95% relative humidity. The cells were seeded in 96-well plates (10,000 cells/well). After reaching 80% conﬂuence, the growth medium was removed and replaced by a medium containing resveratrol.

Resveratrol was dissolved in ethanol and diluted to the appropriate concentration (25, 50, and 100mM) in the cell culture medium. The concentration of ethanol did not exceed 0.01% concentration in the culture medium. Control cells received the same concentration of ethanol in the culture medium. To ensure reproducibility, assays were carried out between passage no. 25 and passage no. 50

5.3.2 HPLC Analysis

Reversed-phase HPLC (RP-HPLC) method was applied to analyze samples with Shimadzu LC pump (Waters Corporation, Milford, MA), Alcott autosampler (model 718 AL), Shimadzu UV/Vis detector (SPD-20AV), and Phenomenex C18 column (Spherisorb 250 × 4.60 mm, 5 μm). The mobile phase was composed of acetonitrile and water (60:40 v/v) with a ﬂow rate set at 0.5 mL/min and UV detector at 305 nm. RSV-calibration curve (1 to 250 μg/mL) was constructed by injecting 30 μL RSV- ethanol solution into the column.

5.3.3 Preparation of RSV Nanomicelles

RSV nanomicelles were prepared following the solvent evaporation method. Briefly, predetermined amounts of RSV, OC-40, and HCO-40 were dissolved separately in ethanol.

Consequently, a homogenous solution was obtained by additions of OC-40 to HCO-40. Then, the calculated amount of RSV solution was added to the mixture in a dropwise pattern. The organic solvent

(ethanol) was evaporated with a highspeed vacuum evaporator (Genevac Technologies VC3000D speed vacuum, USA) over 9 hours to obtain a thin film. The resultant solid was rehydrated with deionized (DI) water. An equal volume of 2x phosphate buffer (pH 6.85) was added to optimize the 128 formulation pH. The solution was sonicated for 10 minutes and then filtered through a 0.22 µm membrane filter to remove the un-entrapped drug or other foreign substances. Then, povidone K-90 was used as a viscosity enhancer.

5.3.4 Entrapment and Loading Efficiency

The entrapment efficiency is the percentage of drug-loaded concerning the amount of initial drug. In this study, the total amount of entrapped drug in the formulation was determined by reverse phase-HPLC following published protocol13. 1 ml of each RSV nanomicelles sample was collected into 2 mL Eppendorf tubes and centrifuged at 10,000 rpm for 10 min at 4°C to remove unincorporated

RSV. Approximately 0.5 mL of supernatant was carefully collected into a fresh tube and lyophilized to obtain a solid pellet. This pellet was resuspended in 0.5 mL of dichloromethane (DCM) that resulted in a clear solution. Due to the addition of DCM, RSV was released in the surrounding organic environment underwent reversal of hydrophobic and hydrophilic segments to form reversed nanomicelles. The solution was evaporated under a speed vac to obtain a solid pellet. It was diluted with HPLC mobile phase, and the amount of drug present in the diluted sample was determined. The amounts of RSV in the core of nanomicelles were calculated by subtracting the total amount of unentrapped drug. The percent entrapment and loading efficiency of curcumin were calculated according to equations 5-1 and

5-2 respectively.

mass of resveratrol in nanomicelles∗100 Percent Drug Entrapped(%EE) = (5-1) mass of resveratrol added in the formulation

mass of resveratrol in nanomicelles∗100 Loading Efficiency(%DL) = (5-2) mass of resveratrol added+mass of polymers added

5.3.5 Size, Polydispersity Index (PDI), and Surface Charge

The mean hydrodynamic nanomicellar size, distribution, PDI, and surface potential of RSV nanomicelles were measured by dynamic light scattering (DLS) (Brookhaven Instruments Corporation,

TX, USA). DLS is a non-destructive technique to measure the molecule/ particle size at the sub-micron level at a laser wavelength of 659.0 nm and a temperature of 25°C. A sample volume of 1000 μL was

129 utilized for size, distribution, polydispersity index, and surface charge. The average values of three micellar diameter measurements for 12 runs were considered for all samples.

5.3.6 Clarity Test

The percentage transmittance of light through samples (N=4) was measured at the different wavelength range from 400nm to 600 nm with a UV-Vis spectrometer (Model: Biomate-3, Thermo

Spectronic, Waltham, MA). Percent of light transmission was recorded. Distilled deionized water served as blank. All measurements were performed in triplicate. One of the primary objectives of this study was to prepare a clear aqueous solution of RSV. Optical clarity/appearance refers to 90% or higher transmission of light of 400 nm wavelength in a 1.0 cm path. Micelle size, typically smaller than the smallest wavelength of visible light radiation (about 350 nm), denotes clarity of the solution. In general, light scattering occurs when any particle interferes with the visible light wavelengths. Due to the tiny size of the nanomicelles, light is not scattered, which denotes a transparent/clear aqueous solution. Ophthalmic compositions of the present RSV formulations are substantially transparent, with an absorption unit below 0.05 measured at 400 nm. The optical clarity of the formulation was compared with distilled deionized water as blank. This study indicates that all the RSV nanomicelles are similar to water with no particulate matter present.

5.3.7 Osmolality and pH

Osmolality is an essential attribute for the topical eye drop formulation. It was measured using the Wescor Vapor Pressure Osmometer Vapro 5520 to follow the manufacturer manual. Briefly, 10 µl of RSV nanomicelles was loaded in the center of the sample disc, and immediately the instrument measure and show osmolality value. The pH of the RSV nanomicelles was adjusted similar to the tear pH ~ 6.85 with phosphate buffer.

5.3.8 Dilution Effect

The dilution effect of our RSV nanomicelles was studied by diluting the sample from 0 to 200 times with simulated tear fluid (STF, composition: NaCl 0.68 g, NaHCO3 0.22 g, CaCl2⋅2H2O 0.008 g,

KCl 0.14 g, and distilled deionized water to 100 mL). Diluted RSV nanomicelles were measured for 130 size characterization following an earlier described protocol. Briefly, the RSV nanomicelles were diluted with an appropriate volume of STF according to dilution factor, and NMF size and PDI were recorded from the DLS analyzer.

5.3.9 Viscosity

Viscosity was determined with Ostwald-Cannon-Fenske viscometer. The relative thickness of

RSV nanomicelles was compared with the viscosity of the water. Briefly, one end of the viscometer was filled with either 5 ml RSV nanomicelles or 5 ml water with high care to avoid the formation of air bubbles. The micellar formulations, as well as liquid, were aspirated from the other end. The time required for the formulations and water to flow down under gravity was measured. The density was also measured. All measurements were performed in triplicate and expressed as mean ±SD.

Viscosity (RSV nanomicelles) = (Density (RSV nanomicelles) × time (RSV nanomicelles) × Viscosity (water)) /

(Density (water) × time (water))

Viscosity (water) = 0.89 centipoise (Cp) at 25°C with a Density (water) = 1 g/ml

5.3.10 Solubility Studies in Distilled Deionized Water (DDI)

Aqueous solubility studies of naked RSV and RSV nanomicelles were carried out according to a previously published protocol from our laboratory. Briefly, saturated solutions of RSV and RSV nanomicelles were freshly prepared in DDI in siliconized tubes and placed in a shaker bath for 24h at

RT. At the end of 24h, tubes were centrifuged for 10min at 10,000 rpm to separate the undissolved drug. Supernatants were carefully separated and filtered through 0.45 µm membrane filter (Nalgene syringe filter). Samples were further diluted appropriately and analyzed by HPLC

5.3.111H-NMR and FT-IR

A qualitative analysis was conducted with FT-IR and proton nuclear magnetic resonance (1H

NMR) to determine the presence of free RSV in solution. Studies were performed for RSV alone, blank nanomicelles and RSV nanomicelles

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5.3.12 Powder X-Ray Diffraction (XRD)

XRD analysis was performed for RSV, freeze-dried blank nanomicelles, and freeze-dried RSV nanomicelles. A Rigaku MiniFlex powder automated X-ray diffractometer (Rigaku, The Woodland,

Texas, USA) was utilized for the analysis at RT. Cu Kα radiation (λ=1.5418Å) at 30 kv and 15mA was utilized. The diffraction angle covered from 2ϴ 4.0° to 45.0°, and a step of 0.05° with 3 sec/step were applied. The diffraction patterns were processed using Jade 8 (Materials Data, Inc., Livermore, CA).

5.3.13 Transmission Electron Microscopy (TEM)

TEM was used to assess the morphology and the shape of the RSV nanomicelles. Briefly, around 50 μL of RSV nanomicelles was placed on a carbon-coated copper grid. Samples were negatively stained by phosphotungstic acid and dried completely before taking TEM images.

5.3.14 MTT Assay

The toxicity of RSV nanomicelles was evaluated by performing lactate dehydrogenase (LDH) and MTT assays on human D407 cells. Cell viability assay was performed to determine the toxicity of

RSV nanomicelles on D407 cells according to the manufacturer’s protocol. Briefly, 10,000-12,000 cells/well were placed into 96-well plates and allowed to grow for 24h. Then different concentrations of RSV nanomicelles were prepared, dissolved in serum free media and added to each well. For the naked RSV since it has very low aqueous solubility, it has been dissolved first in DMSO and then serial dilutions were made to match the concentration of RSV in nanomicelles. All cells were exposed to formulations for 24 hr. Cell culture medium and 10% Triton X-100 served as negative and positive controls, respectively. Cell viability percentage was calculated based on negative (medium) control

(100%). Premixed WST-1 cell proliferation reagent was added according to manufacturer’s protocol

(Clontech, Mountain View, CA). Cell proliferation assay is based on the enzymatic cleavage of the tetrazolium salt (WST-1). A water-soluble formazan dye is detected by absorbance at 450 nm with a microtiter plate reader (Analyst GT, Molecular Devices, Sunnyvale, CA). The amount of formazan formed is directly proportional to the number of viable cells.

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5.3.15 Lactate Dehydrogenase Assay (LDH)

The LDH assay was performed to assess the plasma membrane damage on D407 cells with

Takara LDH cytotoxicity detection kit (Takara Bio, INC., Mountain View, CA). D407 cells were grown on 96-well plates at a density of 10,000 cells/well. After 48 hours of growth, D407 cells in each well were added to 100 μL of serum-free cell culture medium. Different micromolar concentration of RSV nanomicelles and RSV alone were added in the in serum-free culture media. The cells were incubated for 2 h at 37°C. In this experiment, cell culture media served as negative controls. After incubation, the plate was centrifuged at 250 × g for 10 min in a dark condition. One hundred μLof supernatant from each well was transferred to an optically clear 96-well flat bottom plate. Then a solution containing equal volumes of catalyst and dye solution (prepared following manufacturer’s protocol) was added to each well. The mixture was incubated for 15 min at room temperature in the dark and then measured with a plate reader at 490 nm absorbance. A rise in a reading indicates higher LDH release in the culture medium, which directly correlates with the amount of formazan produced. Therefore, the amount of formazan produced is proportional to the number of plasma membrane-damaged cells. Cytotoxicity was calculated according to the equation (5-3)

(Observed experimental value− cell culture medium value)∗100 %Cytotoxicity= (5-3) Triton X−100− cell culture medium value

5.3.16 Antioxidant Activity of RSV Nanomicelles on D407 Cells

Oxidative stress plays an important role of the pathogenesis and the progression of AMD100.

Therefore, antioxidant activity testing is very crucial for our formulation. To assess the antioxidant activity in vitro, exogenous chemical compound tBHPS was used as a model of oxidative stress. Briefly, retinal cells (D407) were grown in 96 well plate at a density of 10,000 cells per well. Then Effect of

RSV nanomicelles, tBHPS, and their combined treatment on the viability and membrane integrity of human retinal pigment epithelial cells were assessed. Cells were treated with RSV nanomicelles for 4 hrs and then they were rinsed and washed with DPBS for 3 time.

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5.3.16 Development and Optimization of a LC-MS/MS Method for Quantification of

Resveratrol

Liquid chromatography tandem mass spectrometry analysis was performed on an AB Sciex

3200 QTrap Spectrometer (Foster City, CA) coupled to a Shimadzu UFLC system (Coumbia, MD) using electrospray ionization (ESI) in negative mode with Analyst v.1.4.2 software. A stock solution of resveratrol at a concentration of 1 mg/mL was prepared in 100% acetonitrile and diluted to 10 µg/mL in 30% 10 mM ammonium acetate + 50% acetonitrile which was then infused into mass spectrometer to optimize different compound dependent parameters (Table 1) were optimized using Analyst quantitative optimization wizard. Chlorzoxazone was selected as an internal standard and various compound dependent-parameters were similarly optimized using a 10 µg/mL in 30% 10 mM ammonium acetate + 50% acetonitrile. Specifications for optimizing various compound dependent parameters were set as investigation of four most intense product ion peaks and build final method based on the best three, minimum m/z of product ion of 50, exclude product ions with m/z ± 5 amu and a threshold area intensity of 0 cps. Various source dependent parameters were optimized using flow injection analysis (FIA) operation of Analyst quantitative optimization wizard using same 10 µg/mL standard solution of resveratrol and chlorzoxazone. Chromatographic detection was performed on a

Kromasil 3.0 × 100 mm C18 column with a flow rate of 0.3 mL/min at ambient temperature. The optimized gradient was 30% B (0-1 min), 30-50%B (1-4 min), 50-100% B (4-10 min), 100-30% B (10-

11 min) and 30% B (11-15 min) where solvent A is 10 mM ammonium acetate (pH 6.5) and solvent B is 30% A + 70% acetonitrile.

5.3.17 Statistical Analysis

GraphPad® Prism 8 software was used for statistical analysis. Data was summarized as mean

± SD. One-way ANOVA was used to determine significant between control and different groups.

Results with p-value of < 0.05 were significant. Bonferonni post hoc test was used for multiple comparisons with pretreatment conditions and insults tBHPS as a variable. Statistical significance was denied as p < 0.05. 134

5.4 Results and Discussion

5.4.1 Entrapment Efficiency and Drug Loading

RSV entrapment and loading into the mixed nanomicelles were determined using an RP-

HPLC method. RSV drug entrapment and loading efficiencies are summarized in Table 5-1.

Table 5. 1 Physicochemical properties of blank and RSV nanomicelles.

Zeta Formulation Size PDI potential(mV) %EE %DL Viscosity(cP)

Blank 16.3 ± 0.132 ± nanomicelles 0.44 0.019 1.12 ± 0.20 NA NA 2.02 ± 0.09

RSV 18.9 ± 0.111 ± 96.33 ± 4.90 ± nanomicelles 0.07 0.280 1.22 ± 0.13 1.56 0.08 2.10 ± 0.02

5.4.2 Size, Polydispersity Index (PDI), and Surface Charge

RSV nanomicelles size and polydispersity indices were determined for all the prepared formulations. In Fig. 5. 2, results show a size range from 14 to 26 nm for RSV nanomicelles.

Polydispersity indices were less than 0.6. RSV nanomicelles did not show any significant difference in size. We hypothesize that the size of RSV nanomicelles can traverse across aqueous sclera channels/ pores that range between 20-80 nm. These nanomicelles display a narrow and unimodal particle size distribution. Since these nanomicelles are in the same size range as membrane receptors, proteins, and other biomolecules, we hypothesize that these nanocarriers can bind and traverse across cellular barriers. The surface charge of RSV nanomicelles is slightly negative.

135

Figure 5. 2 Average RT nanomicellar size and PDI using dynamic light scattering (DLS)

5.4.3 Clarity Testing

An important subject in this study was to prepare an aqueous clear solution of RSV. Optical clarity/appearance refers to 90% or greater transmission of light of 400 nm wavelength in a 1.0 cm path. nanomicellar size, typically smaller than the smallest wavelength of a visible light radiation

(about 350 nm), denotes clarity of the solution. Regularly, light scattering is a process where particles of the drug of interest interferes with the visible light wavelengths. Due to extremely small size of the nanomicelles light is not scattered, which express a transparent/clear aqueous solution. Ophthalmic compositions of the present RSV nanomicelles are substantially clear. The optical clarity of the formulation was compared with distilled deionized water as blank. This study indicates that all the RSV nanomicelles are similar to water with no particulate matter present. Clarity testing is shown in Fig 5-

136

DI water RSV

nanomicelles

Figure 5. 3 Clarity testing for RSV nanomicelles and DI water

5.4.4 Osmolality and pH

pH was adjusted to human tear pH, around 7.4. Osmolality is also a very crucial factor to be considered especially for ophthalmic preparations. Osmolality of RSV nanomicelles was shown as 327 mOsm/kg which is similar to tear film osmolality (Table 5-2).

Table 5. 2 Osmolality and pH of the Blank and RSV nanomicelles

Osmolality Formulation HCO-40 wt % OC-40 wt % (mmol/kg) pH

Blank nanomicelles 2.5 0.1 325 6.8

RSV nanomicelles 2.5 0.1 375 6.8

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5.4.5 Dilution Effect

Tear dilution and protective mechanisms in human eye prevent external materials to reach inside the eye. Dilution effect study is vital because ocular barriers may cause premature release of the drug before reaching the back of the eye. Therefore, the dilution experiment was implemented to check the stability of the nanomicellar formulation upon serial dilutions. Dilution effect on RSV nanomicelles was studied through the rapid mixing of RSV nanomicelles formulation in stimulated tear fluid (STF) up to 100 times. No significant change was seen in the size and PDI of the nanomicelles after analyzing the samples in DLS. The size and PDI before dilution were found to be 18.72 nm and 0.158 respectively.

After 20, 40 and100 times dilution, the size and PDI ranged between 18 to 25 nm 0.158 to 0.211 respectively (Table 5-3). Moreover, nanomicellar formulations were stable to the dilution effects.

Table 5. 3 Dilution effect on RSV nanomicelles size and PDI.

Dilution factor Size (nm) (mean± SD) PDI (mean± SD)

0 18.72 ± 0.06 0.158 ± 0.009

20 20.88 ± 0.77 0.170 ± 0.011

40 19.21 ± 0.23 0.169 ± 0.006

100 25.18 ± 0.44 0.211 ± 0.003

5.4.6 Viscosity

The viscosity of the formulation is one of the essential factors that need to be addressed especially for ophthalmic preparations. In the preparation of the ophthalmic solutions a suitable thickening agent is frequently added to increase the viscosity. Although they reduce the surface tension

138 significantly, their primary benefit is to increase the ocular contact time, thereby decreasing the drainage rate and increasing drug bioavailability. Different techniques are being used to measure the viscosity of the formulation of interest such as capillary viscometer methods, rotational rhometer methods, and rolling ball viscometer methods. The mean residence time and drainage of ophthalmic preparation depend on the viscosity of the instilled solution. Upon addition of povidone K-90 to the formulation, the viscosity of the optimized RSV formulation was 2.10 ±0.088cP (mean ± SD) (n=3), at

RT (25°C) which is similar to the viscosity of water, 0.89 cP. All formulation viscosities were less than

4.4 cP, indicating that the drainage rate may not be affected unless reaching the critical value.

5.4.7 Solubility Studies in Distilled Deionized Water (DDI)

Aqueous solubility values of RSV and RSV nanomicelles were found to be 93 ± 17 and 2000

± 82 µg/mL. The solubility of RSV nanomicelles were 20 times higher than RSV alone. Saturation solubility is shown in Fig 5-4.

Figure 5. 4 Saturation solubility of resveratrol and resveratrol nanomicelles in water

139

5.4.8 1HNMR, FT-IR and XRD Characterizations

1H-NMR spectra were recorded on a Varian 400 MHz spectrometer (Varian, USA) in

deuterated water (D2O) or deuterated DMSO (DMSO-d6). H-NMR spectral analysis suggested that the

resonance peaks corresponding to RSV present only in (DMSO-d6). No peaks of RSV are available in

RSV nanomicelles in D2O (Fig. 5 A). These results proved that all RSV molecules are effectively

entrapped inside the hydrophobic core of the nanomicelles. There is no peak difference between blank

nanomicelles and RSV nanomicelles which proves that all resveratrol molecules are entirely entrapped

inside the core of the nanomicelles. FT-IR was conducted in three different groups: blank micelle, RSV

nanomicelles and RSV alone (Fig. 5 B). In pure powder of resveratrol, three characteristic intense bands

at 1383.85, 1586.53 and 1606.21 cm-1 correspond to C–O stretching, C–C olefinic stretching and C–C

aromatic double-bond stretching. On the contrary, in blank micelles and RSV nanomicelles we see the

broad peak of OH from 3550 to 3200 cm1. Resveratrol peaks were not shown in RSV nanomicelles

which means all resveratrol molecules are effectively entrapped inside the hydrophobic core of the

nanomicelles. XRD data of the polymers (HCO-40 and OC-40) and resveratrol are shown in (Fig. 5 C).

RSVND2O A 0.065 0.060

0.055

0.050

0.045 0.040

0.035

0.030

Normalized Intensity Normalized 0.025

0.020

0.015

0.010 0.005

14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 Chemical Shift (ppm)

140

0.065 BND2O B 0.060

0.055

0.050

0.045

0.040

0.035

0.030

Normalized Intensity Normalized 0.025

0.020 0.015

0.010

0.005

14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 Chemical Shift (ppm)

1 Figure 5. 5 H-NMR spectrums of (A) RSV and (B) Blank nanomicelles in D2O

141

Figure 5. 6 FT-IR spectra of (A) Blank nanomicelles, (B) Resveratrol, and (C) Resveratrol nanomicelles

142

iii

Figure 5. 7 XRD analysis of (i) RSV nanomicelles, (ii) RSV and (iii) Blank nanomicelles

5.4.9 Transmission Electron Microscopy (TEM)

Resveratrol entrapped nanomicelles are clearly shown in spherical and round shapes. The size range of nanomicelles is from 14 to 26 nm which is consistent with sizes data obtained from DLS

(Fig. 6).

5.4.10 Cytotoxicity Studies

The LDH and cell proliferation assay were performed under 24h of exposure to determine the long- cytotoxic effects of the resveratrol nanomicelles. It was assumed that this incubation period would be enough to evaluate any toxicity. Retinal pigmented epithelial cells (D407) were treated with different micromolar concentrations of both RSV alone and RSV nanomicelles. RSV alone is highly

143 hydrophobic, therefore, DMSO was used to solubilize it and then serial dilutions were made with serum free medium. On the other hand, RSV nanomicelles are already water soluble and they were added to serum free medium to make the proposed concentrations. As shown in Figure 5.6 RSV nanomicelles were not toxic even at higher concentrations like 200 µM. This is can be attributed to the slow release kinetics of RSV from the hydrophobic core of the nanomicelles. On contrary, 200 µM RSV alone was causing more damage to D407 cells. 10% Triton X-100 served as a positive control which reduced the percent cell viability to ̴ 10 % for D407 cells. LDH assay was performed to D407 cells. The amount of

LDH released in culture media directly correlates with membrane damage and cytotoxicity. Triton X-

100 caused higher membrane damage. After exposing to blank nanomicelles and RSV nanomicelles, the percent cytotoxicity of D407 over 24 hr appeared to be similar with negative control (Fig. 5.).

Figure 5. 8 Cell viability assays conducted on D407 cells after 24 of exposure to different concentration of RSV alone and RSV nanomicelles (Paired t-test)

144

Figure 5. 9 LDH assay conducted on D407 after 24 hr of exposure to different concentration of RSV alone and RSV nanomicelles. (Paired t-test)

5.4.11 Protective Effect of RSV Nanomicelles on D407 Cells Against tBHPS Induced Oxidative

Stress

The retinal pigmented epithelial cells (RPE) are vulnerable to a normal high level of oxidative stress, as a result of the high metabolic rate, and high oxygen consumption. Oxidative damages to the

RPE have been suggested to be involved in the pathogenesis of AMD. Few studies deal with resveratrol effects on the prevention of eye diseases or on the protection of eye cells. Oxidative stress is involved in the pathogenesis and the progression of AMD. The retina’s higher susceptibility to oxidative imbalance and increased vulnerability to oxidative stress is likely due to less effective antioxidant systems and higher oxygen demand. After replicating the experiments, we found some variations in cell viability, which attributed to the stability problem of H2O2. Therefore, tertiary butyl hydrogen peroxide solution (tBHPS) was used as extracellularly applied chemicals inducing oxidative stress. In

96 well plates, D407 cells were treated with 100 µM of tBHPS for 4 hrs. Cells were arranged in two

145 groups and pretreated with RSV nanomicelles, and resveratrol for 4 h. Then all the formulations were removed, and cells were rinsed 3X5min with PBS. Since 100 µM tBHPS caused maximum effect, the same concentration was administered to study the protective effect of RSV nanomicelles. However,

RSV nanomicelles produced the highest cell viability ~ (69%) and thus provided the highest level of protection against oxidative stress-induced by tBHPS. Resveratrol is highly hydrophobic and insoluble in an aqueous medium. Without nanomicellar formulation, resveratrol is usually dissolved in DMSO with dilutions in serum-free media. Therefore, there might be some possibility of little precipitation of resveratrol, which in turn reduces its antioxidant activity. In contrast, RSV nanomicelles are highly soluble and stable in the aqueous medium, leading to a higher protection of retinal cells in response to oxidative stress, and consequently, higher cell viability. Treatment with 100 µM tBHPS for 1h decreased the cell viability to 9.3% (P<0.001). Resveratrol treatment alone did not cause cytotoxic effects or a significant extent of cell death. However, resveratrol pretreatment showed a statistically significant protective effect against tBHPS induced oxidative stress (P<0.05) by increasing the cell viability to 69%. Treatment with resveratrol alone did not result in a significant modification of LDH activity, the absorbance values being lower than the controls but not statistically significant.

146

Figure 5. 10 Antioxidant Activity testing using tBHPS (500 µM) as a model of oxidative stress.

Retinal cells were treated with tBHPS showed a significant decrease (p < 0.001) in viability compared to control (untreated cells). D407 cells pretreated with RSV nanomicelles and then tBHPS treated showed a significant increase (p < 0.05) in viability compared to tBHPS treated. All experiments were performed in triplicate and values were expressed as mean ± SD. Post hoc test (Bonferroni was used to compare tBHPS vs treated.

147

5.4.12 Development and Optimization of a LC-MS/MS Method for Quantification of

Resveratrol

Liquid chromatography tandem mass spectrometry analysis was performed on an AB Sciex

3200 QTrap Spectrometer (Foster City, CA) coupled to a Shimadzu UFLC system (Coumbia, MD) using electrospray ionization (ESI) in negative mode with Analyst v.1.4.2 software. A stock solution of resveratrol at a concentration of 1 mg/mL was prepared in 100% acetonitrile and diluted to 10 µg/mL in 30% 10 mM ammonium acetate + 50% acetonitrile which was then infused into mass spectrometer to optimize different compound dependent parameters (Table 5-4) were optimized using Analyst quantitative optimization wizard. Chlorzoxazone was selected as an internal standard and various compound dependent-parameters were similarly optimized using a 10 µg/mL in 30% 10 mM ammonium acetate + 50% acetonitrile. Specifications for optimizing various compound dependent parameters were set as investigation of four most intense product ion peaks and build final method based on the best three, minimum m/z of product ion of 50, exclude product ions with m/z ± 5 amu and a threshold area intensity of 0 cps. Various source dependent parameters were optimized using flow injection analysis (FIA) operation of Analyst quantitative optimization wizard using same 10 µg/mL standard solution of resveratrol and chlorzoxazone. Chromatographic detection was performed on a

Kromasil 3.0 × 100 mm C18 column with a flow rate of 0.3 mL/min at ambient temperature. The optimized gradient was 30% B (0-1 min), 30-50%B (1-4 min), 50-100% B (4-10 min), 100-30% B (10-

11 min) and 30% B (11-15 min) where solvent A is 10 mM ammonium acetate (pH 6.5) and solvent B is 30% A + 70% acetonitrile.

148

Table 5. 4 Mass spectrometric parameters for the most intense MS/MS transition of Resveratrol and Chlorzoxazone in negative ESI mode detectiona.

Collision Collision Parent Declustering Entrance Cell Collision Fragment Cell Exit Compound Ion Potential Potential Entrance Energy Ion (Q3) Potential (Q1) (V) (V) Potential (V) (V) (V) Resveratrol 227.1 143.1 -85 -10 -10 -28 -2

Chlorzoxazone 168.1 132.1 -50 -11.5 -10 -28 -2 aGlobal method parameters were: source temperature 300 ºC, curtain gas (CUR) 20, ion spray voltage (IS)-4500 V, Gas flow (GS1 and GS2) 50 (arbitrary units), collision associated dissociation (CAD) High.

5.4.13 Linearity and Sensitivity Study

Standard 100 µg/mL resveratrol with 1 µg/mL chlorzoxazone was serially diluted in a step of

2 for 13 steps in 10 mM ammonium acetate (pH 6.5): acetonitrile (50:50, v/v) containing 1µg/mL

chlorzoxazone as internal standard (IS) and analyzed by the developed method in triplicate to construct

the standard curve (Figs 5-11, 5-12, 5-13)237. Limit of detection (LOD) and lower limit of quantification

(LLOQ) was calculated using (3.3×Standard deviation of the lowest concentration)/slope of the

standard curve and (10×Standard deviation of the lowest concentration)/slope of the standard curve.

149

150

Figure 5. 11 LC-MS/TOF of the RSV

151

7.0E+06

6.0E+06 y = 104118x + 117905 R² = 0.9913 5.0E+06

4.0E+06

3.0E+06 Area (cps) Area 2.0E+06

1.0E+06

0.0E+00 0 10 20 30 40 50 60 Concentration (µg/mL)

Figure 5. 12 Standard curve and linearity characteristics of Resveratrol

152

1.2E+05 Resveratrol 7.06 Chlorzoxazone (IS)

1.0E+05 ---

8.0E+04

Intensity (cps) Intensity 6.0E+04

--- 4.73

4.0E+04

2.0E+04

0.0E+00 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 --- Time (min) ---

Figure 5. 13 LC-MS/MS chromatogram of 10 µg/mL resveratrol and 1 µg/mL chlorzoxazone

(IS)

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5.4.14 Uptake study of RSV alone and RSV nanomicelles

D407 cells were grown in T-25 Flask. For the cell uptake study, D407 were seeded at 12 well plates at a density of 50,000 cells/well in total 24 ml of DMEM and maintained until they reach confluence. Then, cells were treated with RSV nanomicelles, RSV alone in medium, and RSV alone in

DMSO. Cells were lysed following the protocol mentioned above at 2h, 6h and 24 h interval. At each time point, cells were washed 3 times with DPBS (3 × 5 minutes). The samples were prepared using the protocol described above for analysis by LC-MS/MS using the validated analytical method to evaluate cellular uptake of RSV from nanomicelles. Drug uptake study using LC-MS/MS revealed that the uptake of RSV nanomicelles is time dependent. The nanomicelles have sustained the drug release from the core of it. The uptake of RSV alone in medium was very minute, because the drug has a very poor solubility in DMEM medium which is less than 3 µg/mL. On the contrary, RSV alone in DMSO has the highest uptake which is due to high solubility of the drug in DMSO, and the high permeability of the lipophilic drug across the cell membrane which is a lipid bilayer in nature. However, DMSO can not be given to ocular tissues due to its profound toxicity.

150

Control (RSV in )

L medium)

m / g RSV nanomicelles  100

(g/mL) in medium

0 0

1 RSV (g/mL) in DMSO

(

f o

k a

p U 0 2 hr 6 hr 24 hr

Time-dependent uptake

154

Figure 5. 14 Resveratrol uptake study on D407 cell line at 2, 6 and 24 h

5.5 Discussion

ROS play a major role in cellular damage. Like other human cells, retinal pigmented epithelial cells (RPE) utilizes antioxidants enzymes as a mechanism to counteract ROS action. Various antioxidant enzymes act as a primary or secondary antioxidant defense of RPE cells such as catalase, Cu, Zn, and glutathione S-transferase. Moreover, RPE contains natural antioxidants components such as to- copherol, ascorbic acid, and lutein. Supplementation with antioxidants represents a strategy to counteract the deleterious effects of ROS. Many studies have shown the ability of resveratrol (RSV) to protect cells against hydrogen peroxide-induced oxidative stress, and pretreatment with RSV promoted cell survival. RSV is known for its antioxidant, anti-inflammatory, anti-VEGF, and anticancer effect. How- ever, low aqueous solubility, less than 50 µg/mL at room temperature, and poor stability limit its clinical and biological impact. Ocular drug delivery with no side effects after topical application is a challenging approach. Ocular barriers like static and dynamic represent a challenge by impeding drug transport across the cell membrane and different ocular tissues. After topical application, a large portion of the drug will be lost due to excessive tear production and drainage through the nasolacrimal duct. Moreo- ver, sub-optimal physicochemical properties of drugs will retard aqueous solubility, thereby impeding drug delivery. In such a scenario, clear aqueous nanomicellar formulation appears to be highly promising.

Nanomicelles are colloidal constructs composed of amphiphilic polymers with two different portions, a small hydrophobic head, and a long hydrophilic tail. The hydrophobic core of the nanomicelles inter- acts with hydrophobic drugs, whereas the hydrophilic tail helps to surround with water and enhance solubility. Hence, nanomicelles can encapsulate hydrophobic drugs easily. In our study, an aqueous clear nanomicellar RSV formulation was successfully prepared following a novel solvent evaporation technique. The addition of RSV (0.1 wt%) to the amphiphilic polymers, namely HCO-40 and OC-40, was successfully encapsulated inside the hydrophobic core of the nanomicelles. The prepared aqueous

155 nanomicellar formulation needs to be maintained in the physiological range for osmolality and pH. In general, to adjust tonicity, agents such as glycerol, boric acid, or sodium chloride are added. The hy- pertonic or hypotonic aqueous solution may cause irritation to the eye tissue. The pH of the eye varies from 6.5 to 7.6. Therefore, osmolality and pH were maintained at ~ 300 mOsm/Kg and ~ 6.85, respectively. RSV nanomicelles produced much higher drug entrapment (> 95%) and loading efficiencies

(4.90%). RSV nanomicelles showed a small particle size ranging between 15 to 70 nm. This data is also in agreement with real-time TEM images. The viscosity of RSV nanomicelles was similar to that of water 0.9 centipoise (cP). Topical application results in immediate loss due to tear drainage, spill on the cheeks, or through lacrimal drainage. Therefore, it is recommended to use a viscosity enhancer to increase the intact time between the formulation and different ocular tissues. In our study, povidone K-

90 was used because it has a bioadhesive and viscosity enhancer properties. The dual behavior of povidone K-90, viscosity enhancer, and bioadhesive property, may increase the intact time of the formulation with precorneal pocket and hence the bioavailability of the drug on the eye. The average flow rate of the tears in humans is 1.2 µL/min. Therefore, to assess the stability of our RSV nanomicelles in terms of size and PDI, serial dilutions of the formulation were made on simulated tear fluid (STF) up to 100 dilutions. The dilution of RSV nanomicelles caused a slight increase in the average size. In addition,

PDI was slightly increased upon the dilution in 100x. Dilution of the nanomicelles up to 100x produced a negligible effect on the size and demonstrated high stability of the nanomicelles upon dilutions. There- fore, we expect the RSV nanomicelles to behave similarly in vivo and exhibit high stability for future in vivo studies. RSV ha a poor aqueous solubility of less than 50 µg/mL at room temperature. There is a possibility that a small amount/concentration of RSV, due to its aqueous solubility, may be present in the outer aqueous solution of nanomicelles. We conducted qualitative proton nuclear magnetic resonance (1H-NMR) and Fourier-transform infrared spectroscopy (FT-IR) studies that may determine the presence of free/unentrapped RSV molecules in a solution. Formulations were prepared in different

1 media such as CDCl3 and D2O. H NMR spectroscopy studies were conducted for RSV, the mixture of

HCO-40/OC40 in D2O, and RSV nanomicelles in D2O. Excessive addition of RSV above its aqueous 156 solubility results in drug precipitation. In nanomicelles, we did not observe any RSV precipitation indicating drug entrapment inside the hydrophobic core of the nanomicelles, at a level higher than its aqueous solubility. On the other hand, results show that in CDCl3, the resonance peaks corresponding to RSV and nanomicelles are present, as evidenced by the spectra. However, in D2O peaks corresponding to nanomicelles are only detected, whereas peaks for RSV were not evident. These results indicate that RSV is entrapped in the inner hydrophobic core of nanomicelles in D2O. Suspended nanomicelles when dried and resuspended in CDCl3, resonance peaks corresponding to RSV were evident, and the spectra were similar. These results indicate that the nanomicelles entrapped RSV in the inner core, and when subjected to organic solvent as the outer environment, nanomicelles reversed and released RSV.

1 Therefore, H-NMR identified peaks corresponding to RSV in CDCl3. However, in an aqueous solvent

(D2O), nanomicelles entrapped RSV inside the core, and the nuclear magnetic resonance signals are lost. The absence of drug peak in H-NMR and FT-IR confirm the drug encapsulation. In the present study we used micromolar concentrations of RSV nanomicelles and compare it with naked RSV alone in protecting the cells from the damage of tertiary butyl hydroperoxide solution (tBHPS) according to previous studies regarding the antioxidant potential of resveratrol in various cultured cells. The results of our study indicate that resveratrol protects D407 RPE cells tBHPS induced cytotoxicity, as evident from the viability (MTT) and cytotoxicity (LDH leakage). tBHPS is an external chemical compound that induces oxidative stress in vitro. In this study, we induced an acute, sub-lethal oxidative stress applying 100 µM tBHPS to the culture medium, in non-adapted RPE cells. At this concentration, we observed a severe decrease in cell viability, a change in cell morphology (rounding), but not a severe change of cell density. Shamsi et al256. reported that treatment of RPE cells with 100mM hydrogen peroxide for 5h determined signiﬁcant changes in cell morphology and a cytopathic effect, whereas, for 500mM hydrogen peroxide, they observed a 32% decrease of RPE cells’ viability. We showed that

RSV nanomicelles pretreatment did not have cytotoxic effects in the absence of tBHPS, but that it partially reversed the cytotoxic effects of tBHPS in cells exposed to oxidative stress. Resveratrol was proved to have an intrinsic antioxidant capacity due to the redox properties of its hydroxyl groups and 157 the delocalization of electrons. The hydroxyl group in the 4¢ position and the trans-conﬁguration of resveratrol are structural determinants for the antioxidant activity of resveratrol257. Resveratrol was found to be an effective scavenger of hydroxyl, superoxide, and metal-induced radicals and to protect against lipid peroxidation and DNA damage caused by ROS in a cell model. The antioxidant potential of resveratrol was demonstrated in the D407 cultured cell model by protecting retinal cells against oxidative-stress induced tBHPS. Resveratrol is efﬁciently absorbed and metabolized very rapidly, mainly into sulfo-conjugates and glucuronides, which are eliminated by urine. Consequently, the plasma concentration of free resveratrol is very low, even after high dosage oral administrations. How- ever, due to its lipophilic nature, resveratrol may be bound to the cellular fraction in blood or in lipophilic tissues, which lead to the underestimation of its concentration. Despite a large number of studies, the distribution of resveratrol in tissues and cells, including the eye, remains to be determined. Several studies on resveratrol pharmacokinetics and safety in humans showed that it is well-tolerated, and no marked toxicity was reported178. The highest dose was 5g of resveratrol as a single intake, and it did not cause serious adverse effects. Currently, several strategies are developed to improve resveratrol bioavailability. RPE is continuously subjected to the aggressive attack of ROS, resulting in either phag- ocytosis or metabolic processes. It is considered that oxidative stress at the RPE level is one of the major causes of AMD. This study demonstrates that resveratrol can contribute to the protection of RPE cells by enhancing antioxidant enzyme activities. Taking into account the lack of toxicity, studies on human subjects are necessary to see if a resveratrol-rich diet can have a preventing effect on AMD.

158

5.6 Conclusion

The study demonstrates the preparation, optimization, and characterization of the RSV nanomicelles for topical ocular delivery. A clear aqueous RSV nanomicelles was prepared using amphiphilic polymers (HCO-40 and OC-40) with solvent evaporation technique. Cell cytotoxicity assays showed that 0.5 to 0.2% RSV nanomicelles is safe for topical application. The RSV nanomicelles have shown a significant protective effect against tBHPS induced oxidative stress in D407 cells. These promising findings indicate that RSV nanomicelles may allow topical delivery of therapeutic concentrations of resveratrol for the treatment of both types of AMD through its antioxidant, anti- inflammatory, and anti-VEGF action. RSV nanomicelles may provide a potential for prevention and treatment of AMD.

159

CHAPTER 6. SUMMARY AND RECOMMENDATIONS

6.1 Summary

Topical formulation administration is considered as the most convenient route for the ocular disease patients. Although this route is viable for the treatment of the anterior segment of the eye, it still considered as a major challenge for posterior eye segment diseases such diabetic macular edema (DME) and age -related macular degeneration (AMD). The complex structure of the eye, static and dynamic barriers limit the penetration of hydrophobic therapeutic. Static barriers include choroid, sclera, conjunctival epithelium, Bruch’s membrane and corneal epithelium work along with dynamic barriers such as lacrimation, lymphatic drainage and efflux pumps to prevent the entry foreign substance to ocular tissues. Polymeric nanomicelles as such is a delivery system which have been studied to enhance the solubility of hydrophobic drugs and target different eye tissues. The small size of the nanomicelles, less than 20 nm, allow them to transverse through aqueous channel pores (30-100nm) located in trans- scleral pathway and deliver the drugs to retina. Curcumin, celecoxib and resveratrol have shown anti- inflammatory, anti-VEGF and anti-proliferated activity on different retinal cells and has been widely used in many cancers. The hydrophobicity nature of the previous compounds, low bioavailability and low aqueous solubility limit their clinical use. Therefore, there is an urgent need to enhance the solubility and subsequent the bioavailability of them through nanomicelles. Polymeric nanomicelles is an ideal drug delivery system which can improve aqueous solubility of curcumin, celecoxib and resveratrol by dissolving it in the hydrophobic core of the nanomicelles. Polymeric nanomicelles can be made through simple straightforward technique called solvent evaporation film rehydration technique.

In chapter 3, the objective was to develop a clear aqueous nanomicellar formulation (NMF) of curcumin (CUR-NMF) with a combination of nonionic surfactant hydrogenated castor oil 40 (HCO-

40) and octoxynol-40 (Oc-40). In order to delineate the effects of drug-polymer interactions on entrapment efficiency (EE), loading efficiency (LE), size, PDI and precipitation, a design of experiment

(DOE) was performed to optimize the formulation. In this study, full factorial design has been used 160 with curcumin wt %, HCO-40 and OC-40 as independent variables. All formulations were prepared following solvent evaporation and film rehydration method, characterized with size, polydispersity, shape, morphology, EE, LE and CMC. A specific blend of 0.05 wt % of curcumin, and HCO-40 and

Oc-40 at a particular wt% ratio (2.5:1) produced highest drug EE, LE, and smallest size, PDI and no precipitation. Solubility of curcumin in NMF improved 32 folds relative to normal aqueous solubility.

Qualitative 1H-NMR and FT-IR studies revealed the absence of free drug in the outer aqueous NMF medium. Moreover, 5-10 µM of CUR-NMF appeared to be highly stable and well tolerated on human retinal pigment epithelial cells (D407 cells). The drug release studies showed a sustained drug release over nearly a month. Moreover, CUR-NMF has shown a protective effect of retinal cells against H2O2- induced oxidative stress. In addition, VEGF-A ELISA study on retinal pigmented epithelial cells

(D407) suggested that the formulation has reduced the vascular endothelial growth factor release

(VEGF). Overall, this study suggests that 5-10 µM CUR-NMF potentially could be used for human topical ocular drop application.

In chapter 4, the objective was to develop self-assembled biotinylated nanomicelles loaded celecoxib

(CXB) with a combination of nonionic surfactant hydrogenated castor oil 40 (HCO-40) and biotinylated octoxynol-40 (OC-40). Transporter mediated drug delivery may enhance the localization of the drug inside the cell. Previous data from our lab have shown high expression of sodium dependent multivitamin transporter (SMVT) in retinal pigmented epithelial cells (D407) and human corneal epithelial cells (HCEC). SMVT is known for transporting vitamins, and other essential factors such as biotin. Therefore, biotin was used as a targeting moiety. The conjugation of biotin with Octooxynol-40

(OC-40) was carried out with the help of EDC/DMAP chemistry. The product was dialyzed against water for 2 days to remove the unreacted biotin, EDC and DMAP. The purified product was lyophilized for further analysis. 1HNMR and FT-IR data confirmed that the product was formed. Then the nanomicelles were prepared following solvent evaporation thin film rehydration technique. Wt% ratio of (HCO-40: biotinylated Oc-40) (2.5:1) has shown the highest entrapment efficiency (% EE) and drug loading (% DL) with around 99% and 3.76%, respectively. Moreover, biotinylated nanomicelles loaded 161

CXB appeared very stable with dilution factors up to 200 times and well tolerated on human corneal epithelial cells (HCEC) and human retinal pigment epithelial cells (D407 cells), and human conjunctival cells. Biotinylated nanomicelles loaded CXB have shown a sustained drug release over one month in PBST medium. In addition, VEGF-A ELISA analysis revealed the ability of CXB nanomicelles to reduce VEGF release from retinal cells in dose dependent manner. The capability of our nanomicelles to reduce LPS-induced TNF-α and IL-6 cytokines from macrophages were observed with ELISA testing. The cellular uptake studies were tested utilizing confocal microscopy and LC-

MS/MS and data suggested that biotinylated nanomicelles have more cellular internalization 2 folds more than non-targeted nanomicelles. When SMVT transporter was blocked with 1 mM excess biotin, the uptake of biotinylated nanomicelles was significantly reduced suggesting that biotinylated nanomicelles utilize SMVT transporter. Overall this study suggests that self-assembled biotinylated nanomicelles potentially could be used for human topical ocular drop application. All the hydrophobic drugs are successfully encapsulated inside the hydrophobic core of the nanomicelles. The solubility of these hydrophobic drugs has been enhanced.

In chapter 5, Resveratrol (RSV), polyphenolic stilbene, is originally derived from peanuts, grapes, red wines and blueberries. RSV has been shown to have antioxidant, anti-inflammatory and anti- proliferative effect which can help in AMD. However, poor aqueous solubility, low bioavailability limits its clinical use in ocular diseases. RSV nanomicelles were prepared through solvent evaporation thin film rehydration technique by utilizing HCO-40 and OC-40 as amphiphilic polymers. The size,

PDI, Zeta potential of RSV nanomicelles was determined with DLS. Entrapment efficiency and loading were determined with RP-HPLC. 1H-NMR and FT-IR were used for qualitative analysis of the formulation. MTT and LDH were performed to assess the cell viability and toxicity, respectively. tBHPS was used as a model of oxidative stress and the antioxidant activity of the RSV nanomicelles and RSV alone was determined.

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6.2 Recommendations

A few recommendations can be made to move further with this work based on the results obtained from the above studies. For the first research project which is about CUR-NMF, testing the permeation of the nanomicelles across different eye tissues on ex vivo model (bovine eye) will give more detailed about the pharmacokinetics profile of the formulation. In vivo studies on rabbit eye also give more detailed about the retention and the concentration of the drug across different eye tissues such as retina, choroid, Bruch’s membrane and macula.

For the Second work, since the OC-40 polymer has a free OH group, it can be conjugated to peptides or folic acid which is known to be expressed in retinal cells. Therefore, the cellular internalization and the uptake of the transporter targeted nanomicelles will be higher compared to non-targeted one.

Conjugation of peptide and/or folate molecule to drug and simultaneous loading into NMF should be undertaken in order to come up with the best possible formulation.

For the third work, it is known that oxidative damage might play a role in the development of AMD.

The retina is particularly susceptible to oxidative damage due its high oxygen consumption and demand. Therefore, developing in vivo mouse models of oxidative stress particularly on retina is great idea.

163

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VITA

Meshal Alshamrani was born on 19th October 1989, in Albaha City, Saudi Arabia.

He completed his Pharm. D from King Abdul-Aziz University in 2013, Jeddah, Saudi Arabia.

After completion of his Pharm. D, he worked for Jizan University as a teaching assistant.

He joined the Interdisciplinary Ph.D. program at UMKC in October 2015 to pursue a Ph.D. degree in Pharmaceutical Science and Chemistry. He is an active member of American

Association of Pharmaceutical Scientists (AAPS). He completed his doctoral studies in April

2020 under the guidance of Dr. Melchert. He has authored/co-authored some reviewed publications and has presented his work in a number of international and national conferences such as AAPS and PGSRM.

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