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Intravitreal Delivery of Corticosteroid Nanoparticles

Permanent Link: http://ufdc.ufl.edu/UFE0022065/00001

Material Information

Title: Intravitreal Delivery of Corticosteroid Nanoparticles
Physical Description: 1 online resource (109 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: choroidal, corticosteroids, intravitreal, nanoparticles, ocular, triamcinolone, vegf
Pharmacy -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Age-related macular degeneration and diabetic retinopathy occurring in the posterior region of the eye are the leading causes of blindness among the elderly. Treatments for these diseases currently include laser photocoagulation and photodynamic therapy. However, aforementioned methods help in eliminating only the existing neovascularization but do not treat the cause of the disease resulting in reoccurrence. Recently corticosteroids are being used to treat posterior ocular diseases due to their angiostatic and antipermeable properties. The rationale for the use of corticosteroids for these conditions is their ability to inhibit growth factors like vascular endothelial growth factor (VEGF). Drug delivery to the posterior segment of the eye is very challenging. One of the major concerns with intravitreal delivery is that repeated injections are disagreeable and lead to further complications. Sustained release systems including microspheres, liposomes and other implant devices offer an excellent alternative to multiple intravitreous injections. The development of drug delivery systems is becoming important in the treatment of vitreoretinal diseases not only to facilitate drug efficacy but also to attenuate the side effects. These systems can enhance the permeation of the drug, help in controlled release of the drug. Biodegradable poly(lactide-co-glycolide) corticosteroid nanoparticles were prepared and characterized for size, drug encapsulation and in vitro drug release. In order to obtain nanoparticles of desired size and drug loading, it is important to understand the effect of various formulation variables on particle characterization. Here we studied the effect of polymer, drug and surfactant concentration. Increasing the polymer concentration increased the size of the particles. While increase in surfactant concentration decreased the size of the particles and also drug loading. Cell culture studies were conducted to understand the effect of the corticosteroid nanoparticles on cell toxicity, cell uptake and VEGF secretion. Decrease in toxicity was seen with nanoparticles and use of soft steroid. The uptake of the particles was time and concentration dependent. Finally, we investigated the efficacy of intravitreal corticosteroid loaded nanoparticles on experimental choroidal neovascularization in a laser-induced mice model. The corticosteroid nanoparticles showed a significant decrease in neovascularization area compared to the control indicating that corticosteroid-loaded polymer nanoparticles can inhibit the development of experimental choroidal neovascularization.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Hochhaus, Guenther.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2008-11-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022065:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022065/00001

Material Information

Title: Intravitreal Delivery of Corticosteroid Nanoparticles
Physical Description: 1 online resource (109 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: choroidal, corticosteroids, intravitreal, nanoparticles, ocular, triamcinolone, vegf
Pharmacy -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Age-related macular degeneration and diabetic retinopathy occurring in the posterior region of the eye are the leading causes of blindness among the elderly. Treatments for these diseases currently include laser photocoagulation and photodynamic therapy. However, aforementioned methods help in eliminating only the existing neovascularization but do not treat the cause of the disease resulting in reoccurrence. Recently corticosteroids are being used to treat posterior ocular diseases due to their angiostatic and antipermeable properties. The rationale for the use of corticosteroids for these conditions is their ability to inhibit growth factors like vascular endothelial growth factor (VEGF). Drug delivery to the posterior segment of the eye is very challenging. One of the major concerns with intravitreal delivery is that repeated injections are disagreeable and lead to further complications. Sustained release systems including microspheres, liposomes and other implant devices offer an excellent alternative to multiple intravitreous injections. The development of drug delivery systems is becoming important in the treatment of vitreoretinal diseases not only to facilitate drug efficacy but also to attenuate the side effects. These systems can enhance the permeation of the drug, help in controlled release of the drug. Biodegradable poly(lactide-co-glycolide) corticosteroid nanoparticles were prepared and characterized for size, drug encapsulation and in vitro drug release. In order to obtain nanoparticles of desired size and drug loading, it is important to understand the effect of various formulation variables on particle characterization. Here we studied the effect of polymer, drug and surfactant concentration. Increasing the polymer concentration increased the size of the particles. While increase in surfactant concentration decreased the size of the particles and also drug loading. Cell culture studies were conducted to understand the effect of the corticosteroid nanoparticles on cell toxicity, cell uptake and VEGF secretion. Decrease in toxicity was seen with nanoparticles and use of soft steroid. The uptake of the particles was time and concentration dependent. Finally, we investigated the efficacy of intravitreal corticosteroid loaded nanoparticles on experimental choroidal neovascularization in a laser-induced mice model. The corticosteroid nanoparticles showed a significant decrease in neovascularization area compared to the control indicating that corticosteroid-loaded polymer nanoparticles can inhibit the development of experimental choroidal neovascularization.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Hochhaus, Guenther.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2008-11-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022065:00001


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1 INTRAVITREAL DELIVERY OF CORTICOSTEROID NANOPARTICLES By KEERTI MUDUNURI A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2008 Keerti Mudunuri

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3 To my parents and my husband

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4 ACKNOWLEDGMENTS I would like to express my grat itude to m y supervisor, Dr. Guenther Hochhaus, for giving me the opportunity to work with him for my Ph .D. I would like to thank him for his guidance, support and encouragement througho ut my Ph.D. I would also lik e to thank my advisor, Dr Shalesh Kaushal, for his valuable guidance and he lp especially with the animal experiments. I would also like thank Dr. Hartmut Derendorf a nd Dr. Jeffrey Hughes fo r being part of my Ph.D. supervisory committee. I w ould like to thank Dr. Veronika Butterweck for allowing me to use her laboratory facilities. I would also like to thank my fellow lab mates and graduate students for their valuable feedback and support. Finally, I would like to thank my parent s and my brother for their support and encouragement over all these years. None of th is work would have possible without them. I would also like to express my heartfelt grat itude to my husband Srinivas for his support, understanding and encouragement throughout this endeavor.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES.........................................................................................................................9 ABSTRACT...................................................................................................................................11 CHAP TER 1 INTRODUCTION..................................................................................................................13 Structure of the Eye........................................................................................................... .....13 Ocular Diseases......................................................................................................................14 Role of VEGF.........................................................................................................................15 Corticosteroids in Ocular Delivery.........................................................................................16 Routes of Drug Administration.............................................................................................. 17 Drug Delivery Systems...........................................................................................................19 Hypothesis..............................................................................................................................20 2 PREPARATION AND CHARACTERIZATION OF TRIAMCINOLONE ACETONIDE NANOPARTICLES ........................................................................................26 Introduction................................................................................................................... ..........26 Materials and Methods...........................................................................................................28 Nanoparticle Preparation.................................................................................................28 Nanoparticle Characterization......................................................................................... 28 Drug Encapsulation Efficiency........................................................................................ 29 In Vitro Release ...............................................................................................................29 Statistical Analysis.......................................................................................................... 30 Results.....................................................................................................................................30 Morphology.....................................................................................................................30 Effect of Polymer Poly (lactide-co-glycolide) on Size and Encapsulation ...................... 31 Effect of Triamcinolone Acetoni de on Size and E ncapsulation...................................... 31 Effect of Poly(vinyl alcohol ) on Size and Encapsulation ................................................31 In Vitro Release Studies ..................................................................................................32 Discussion...............................................................................................................................33 Conclusion..............................................................................................................................36 3 EFFECT OF TRIAMCINOLONE ACETO NIDE AND ITS NANOPARTICLES ON RETINAL PIGMENT EPITHELIAL CELLS....................................................................... 50 Introduction................................................................................................................... ..........50 Materials and Methods...........................................................................................................52

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6 Cell Culture................................................................................................................... ..52 Vascular Endothelial Grow th Factor Secretion ............................................................... 53 Cell Toxicity....................................................................................................................53 Cell Uptake.................................................................................................................... ..53 Statistical Analysis.......................................................................................................... 54 Results.....................................................................................................................................55 Vascular Endothelial Grow th Factor Secretion ............................................................... 55 Cell Toxicity....................................................................................................................55 Cell Uptake.................................................................................................................... ..56 Discussion...............................................................................................................................57 Conclusion..............................................................................................................................58 4 EFFECT OF INTRAVITREAL TRIAMCINOLONE ACETONIDE NANOPAR TICLES ON LASER INDUSED CHOROIDAL NEOVASCULARIZATION IN MICE.................................................................................. 67 Introduction................................................................................................................... ..........67 Materials and Methods...........................................................................................................69 Laser Induced Choroidal Neovascularization................................................................. 69 Treatment Administration............................................................................................... 69 Preparation of Flat Mounts.............................................................................................. 70 Statistical Analysis.......................................................................................................... 70 Results.....................................................................................................................................71 Discussion...............................................................................................................................71 Conclusion..............................................................................................................................74 5 LOTEPREDNOL ETABONATE NANOPARTICLES AND THEIR EFFECT ON LASER INDUCED CHOROIDAL NE OVASCULARIZATION IN MICE ......................... 79 Introduction................................................................................................................... ..........79 Materials and Methods...........................................................................................................80 Intraocular Pressure Measurements in Rabbits............................................................... 80 Preparation and Characterization of Loteprednol Etabonate Nanoparticles ................... 80 Effect of Loteprednol Etabonate on VEGF Secretion in ARPE-19 Cells ....................... 81 Cell Toxicity of Loteprednol Etabonat e and its N anoparticles on ARPE-19 Cells........ 81 Effect of Loteprednol Etabonate Nan oparticles on Laser Induced Choroidal Neovascularization in m ice.......................................................................................... 82 Statistical Analysis.......................................................................................................... 82 Results.....................................................................................................................................82 Intraocular Pressure Measurements in Rabbits............................................................... 82 Preparation and Characterization of Loteprednol Nanoparticles .................................... 82 Effect of Loteprednol on VEGF Secr etion in ARPE-19 Cells........................................ 83 Cell Toxicity of Loteprednol and its Nanoparticles on ARPE-19 Cells ......................... 83 Effect of Loteprednol Nanopartic les on Laser Induced Choroidal Neovascularization in Mice .........................................................................................83 Discussion...............................................................................................................................83 Conclusion..............................................................................................................................85

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7 6 CONCLUSIONS AND FUTURE WORK ............................................................................. 95 LIST OF REFERENCES...............................................................................................................97 BIOGRAPHICAL SKETCH.......................................................................................................109

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8 LIST OF TABLES Table page 1-1 Eye disease prevalence and projections in adults 40 y ears and older in the U.S ............... 21 1-2 Treatments being considered for AMD and DR................................................................ 21 1-3 Drug delivery systems for trea ting posterior ocular conditions .........................................22 2-1 Effect of polymer concentra tion on particle size and loading ........................................... 42 2-2 Effect of drug concentration on particle size and loading ................................................. 42 2-3 Effect of surfactant (PVA) concen tration on particle size and loading ............................. 42 2-4 Zero order and Higuchi equations of in vitro drug release showing the effect of polym er..............................................................................................................................43 2-5 The effect of drug on in vitro drug release shown by Zero order and Higuchi equations ............................................................................................................................44 2-6 The effect of surfactant on in vitro drug release shown by Ze ro order and Higuchi equations ............................................................................................................................45 2-7 Zero order and Higuchi equations of in vitro drug release showing the effect of TA, sm aller nanoparticles and larger nanoparticles..................................................................46 2-8 Relationship between various parameters to control the particle size and drug content for nanoparticles.................................................................................................................49 4-1 Treatment groups........................................................................................................... ....75 5-1 Size and encapsulation efficien cy of loteprednol nanoparticles ........................................88 5-2 Zero order and Higuchi equations of in vitro drug release showing the effect of polym er..............................................................................................................................88

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9 LIST OF FIGURES Figure page 1-1 Structure of the Eye....................................................................................................... ....23 1-2 Detailed structure of the retina...........................................................................................24 1-3 Routes of drug delivery to ta rget posterior region of eye .................................................. 25 2-1 Flow chart showing the nanopa rticle preparation process ................................................. 37 2-2 Scanning electron microscopy image of triam cinolone acetonide loaded 150 nm nanoparticles......................................................................................................................38 2-3 Scanning electron microscopy image of triam cinolone acetonide loaded 415 nm nanoparticles ................................................................................................................. ....39 2-4 Turbidity of micronized triamcinolon e aceton ide and triamcinolone acetonide nanoparticles prepared with 1:10 ratio............................................................................... 40 2-5 Particle size distribut ion with 172nm and 415 nm............................................................. 41 2-6 Effect of polymer concentration on in vitro cum ulative triamci nolone released from nanoparticles......................................................................................................................43 2-7 Effect of drug concentration on in v itro cumulative triamcinolone released from nanoparticles......................................................................................................................44 2-8 Effect of surfactant poly(vinyl alcohol) concentration on in v itro cumulative triamcinolone released from nanoparticles........................................................................45 2-9 Effect of size of the nan oparticles on in vitro cumulative triamcinol one released from nanoparticles......................................................................................................................46 2-10 Results of in vitro release studies extrapolated to 100% cumulative drug release............ 47 2-11 Scanning electron microscopy images taken during in vitro release studies ..................... 48 3-1 Effect ofTriamcinolone acetonide on VEGF secretion in ARPE-19 cells ......................... 60 3-2 Viability of ARPE-19 cells after 24 hours with m icroni zed triamcinolone acetonide......61 3-3 Viability of ARPE-19 cells after 24 hours with triam cinolone and triamcinolone nanoparticles......................................................................................................................62 3-4 Cell uptake of micronized triamcinolone com pared with its nanoparticles at 100 g/ml at 37 C at the end of 2 hours................................................................................... 63

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10 3-5 Effect of concentration on kinetics of nanoparticle uptake into ARPE-19 cells............... 64 3-6 Effect of time on kinetics of na noparticle uptake into ARPE-19 cells. ............................. 65 3-7 Effect of temperature on the kinetics of nanoparticle uptake into ARPE-19 cells. ........... 66 4-1 Choroidal flat mounts of the left and right eye .................................................................. 76 4-2 Representative images of laser induced choroidal neovascularization .............................. 77 4-3 Neovascularization area of four treatm ent groups.............................................................78 5-1 Intraocular pressure measurements (IOP ) for a period of 30 days in rabbits after intravitreal injection of tr iam cinolone and loteprednol..................................................... 87 5-2 In vitro release data of loteprednol etabonate and its nanoparticles .................................. 89 5-3 In vitro release studies of lote prednol nanoparticles extra polated to 100% cum ulative drug release........................................................................................................................90 5-4 Effect of loteprednol etabonate on VEGF secretion in ARPE-19 cells ............................. 91 5-5 Effect of loteprednol an d loteprednol nanoparticles on viability of ARPE19 Cells after 24 hours.....................................................................................................................92 5-6 Representative images of laser induced choroidal neovascularization .............................. 93 5-7 Neovascularization area of blank nanopa rticles, loteprednol nanoparticles and triam cinolone nanoparticles............................................................................................... 94

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11 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy INTRAVITREAL DELIVERY OF CORTICOSTEROID NANOPARTICLES By Keerti Mudunuri May 2008 Chair: Guenther Hochhaus Major: Pharmaceutical Sciences Age-related macular degeneration and diabet ic retinopathy occurri ng in the posterior region of the eye are the leading causes of b lindness among the elderly. Treatments for these diseases currently include laser photocoa gulation and photodynamic therapy. However, aforementioned methods help in eliminating only the existing neovasculari zation but do not treat the cause of the disease resulting in reoccurrence. Recently corticosteroids are being used to treat posterior ocular diseases due to their angiostatic and antipermeable properties. The rationale for the use of corticosteroids for these conditions is their ability to inhibi t growth factors like vascular endothelial growth factor (VEGF). Drug delivery to the posterior segment of the eye is very challenging. One of the major concerns with in travitreal delivery is th at repeated injections are disagreeable and lead to further compli cations. Sustained release systems including microspheres, liposomes and other implant devices offer an excellent alternative to multiple intravitreous injections. The development of drug delivery systems is becoming important in the treatment of vitreoretinal diseases not only to facilitate drug efficacy but also to attenuate the side effects. These systems can enhance the permeation of the drug, help in controlled release of the drug. Biodegradable poly(lactide-co-glycolide) cortic osteroid nanoparticles were prepared and

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12 characterized for size, drug encapsulation and in vitro drug release. In order to obtain nanoparticles of desired size and dr ug loading, it is important to unde rstand the effect of various formulation variables on particle characterization. Here we stud ied the effect of polymer, drug and surfactant concentration. Increasing the polymer concentrat ion increased the size of the particles. While increase in surfactant concentrat ion decreased the size of the particles and also drug loading. Cell culture studies were conducted to unders tand the effect of the corticosteroid nanoparticles on cell toxicity, cell uptake and VEGF secretion. Decrease in toxicity was seen with nanoparticles and use of soft steroid. The up take of the particles was time and concentration dependent. Finally, we investigated the efficacy of intrav itreal corticosteroid loaded nanoparticles on experimental choroidal neovascul arization in a laser-induced mice model. The corticosteroid nanoparticles showed a significant decrease in neovascularization area compared to the control indicating that corticos teroid-loaded polymer nanoparticles can inhibit the development of experimental choroida l neovascularization.

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13 CHAPTER 1 INTRODUCTION Structure of the Eye Eye is a complex sensory organ that has a distinct environment. Figure 1-1 shows the structure of the eye. The eye can be d ivided in to two main compartments the anterior segment and the posterior segment [1]. The anterior segmen t of the eye includes the cornea, iris, ciliary body, lens and consists of two flui d filled spaces: the anterior chamber and the posterior chamber that are filled with aqueous humor which is a thick watery substance be ing continually produced by the ciliary body and provides the nutrients to the surrounding struct ures. The posterior segment of the eye consists of three layers, sc lera, choroid and retina, surrounding the vitreous cavity, which is filled by the vitreous. Sclera is a dense, fibrous, viscoelastic connect ive tissue that forms the outer coat of the eye [2]. The vitreous humor is a transparent gel a nd is composed of hyaluronic acid and collagen [3, 4] in 98% water. The functions of the vitre ous include protecting the eye during mechanical trauma and providing adequate supp ort to the retina [5]. The retina, which is separated from the choroid by Burchs membrane, is the sensory inner coat of the posterior segment of the eye. One side of the retina contains the rod and cones an d is adjacent to the re tinal pigment epithelium (RPE) and the choroid; the other side of the reti na faces the vitreous. Figure1-2 shows the cross section of retina. Photor eceptor cells are located in the subret inal region near to the choroid. The macula is the center part of retina. Bloodretinal barrier (BRB) composed of the retinal vasculature and the retinal pigment epithelial cells separates the retina and the vitreo us from the systemic circulation and vitreous body. Two types of transmembrane proteins, occludin and the family of claudins, are responsible for the direct cell-to-cell attachment in the esta blishment of the tight j unction barrier [6, 7]. The

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14 choroid is vascular and pigmented tissue betwee n the retina and the sclera. The retinal pigment epithelium (RPE) is located betw een the choroid and the retina. Ocular Diseases Millions of people suffer from various sight threatening ocular conditions such as diabetic retinopathy, age related macular degeneration (A MD), cataract and glaucoma. Table 1-1 shows the statistics on number of people suffering from various ocular conditions in US. Among these diabetic retinopathy and age related macular dege neration occurring in the posterior segment of the eye are the leading causes of blindness in developed countries. Diabetic retinopathy is a complication of di abetes mellitus. It is characterized by the breakdown of blood-retinal barrier. Hyperglycemi a changes the formation of the blood-retinal barrier and also make retinal blood vessel become more permeable [1]. As the disease progresses neovascularization occurs due to lack of oxygen, forming frag ile blood vessels. These newly formed blood vessels cloud vision and bleed and damage the retina, resulting in increased vascular permeability leading to edema [8] and finally resulting in vascular proliferation [9]. Age-related macular degeneration (AMD) is a di sease associated with aging that gradually destroys sharp, central vi sion. AMD affects the macula, the part of the eye that allows us to see fine detail. Choroidal neovascular ization is associated with m acular degeneration. Choroidal (or subretinal) neovascularization (CNV) characteristic of macular de generation is a major cause of visual loss. Choroidal neovascularization originat es from choroidal vessels. It is accompanied by fluid and blood breaking through th e Bruchs membrane into the subretinal pigment epithelial space and/or into the subretinal space, resulting in irregular elevations of the surface of the retina. These diseases are mainly treated with la ser photocoagulation. La ser photocoagulation destroys neovascularization in age related macular degeneration and decreases vascular leakage

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15 and destroys new blood vessels in diabetic retin opathy. In this procedur e leaking blood vessels are burnt by the laser. Laser photoc oagulation does not help in restor ing the lost vision and has to be repeated several times in many patients. It can also lead to permanent damage of the retina and not effective for long term treatment. Ther efore, new therapeutic approaches are being sought for these conditions. Table 1-2 shows the various therapeuti c agents for retinal diseases. Recent preclinical studies have suggested that pharmacologic intervention or antiangiogenesis therapy may be useful to treat vari ous forms of ocular neov ascularization. Much of this work has focused on blocking vascular endothelial growth factor (V EGF) [10-13] which has been shown to play a major role in initiating these diseases. Role of Vascular Endothelial Growth Factor Vascular endothelial growth factor (VEGF) is a pot ent endo thelial cell mitogen and appears to be important in the development of ocular neovascu larization. Retinal cells forming the outer layer of BRB express VEGF [14]. It has been implicated in the onset and progress of diabetic retinopathy and choroidal neovascularization secondary to age related macular degeneration [15]. Studies in hum ans showed increased VEGF in aqueous humor and vitreous of patients with diabetic retinopathy [16-19]. Animal studies have shown th at injection of VEGF into the vitreous can cause a diab etic retinopathy like state [20]. Recent evidence also suggests a central role of VEGF in the development of choroidal neovascularization. Excised human choroidal neovascularization after experimental submacular surgery have shown elevated VEGF levels [21]. Vitreous VEGF levels were found to be significantly higher in patient s with age-related macular degeneration and choroidal neovascularization as compared to healthy controls [19]. VEGF also increases phosphorylation of tight junction proteins, such as occludin resulting in the breakdown of the blood retinal barrier

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16 [22, 23]. VEGF also plays a major role in inflammation by inducing intercellular adhesion molecule (ICAM-1) expression and leukocyte adhesion. The above information shows VEGF to be a good target for pharmace utical intervention. Recent studies showed that anti-VEGF therapy may inhibit breakdown of blood retinal barrier induced by diabetes in animals [24]. Several researchers showed that macromolecule s such as VEGF antibodies and aptamers to inhibit retinal vascular changes [25, 26] Studies have shown regr ession or prevention of neovascularization in retina [25, 27, 28] and choroid [26] in several animal models (primate [29] mouse [28] and rat [20] ) using anti-VEGF aptamers [25] and antibody fragments agent [26] But very little research with regard to lo w molecular weight compounds has been done. Corticosteroids are low molecular weight lipohilic compounds that are easier to administer and stable. They offer an alternative to the m acromolecules due to thei r anti angiogenic and antipermeable properties. Corticosteroids in Ocular Delivery Corticos teroids are traditionally used due to their anti inflammatory property, but recently they gained interest for treat ing posterior ocular conditions due to their angiostatic and antipermeable properties [30]. Corticosteroids show their effect by binding to the steroid receptors present in the cells. They then act by ei ther induction or repression of the target genes and inhibit inflammatory symptoms like edema and vascular permeability [31]. Posterior ocular diseases are charac terized by edema and neovascularization. Corticosteroids show their effect by action on various targets. Edema is mainly caused due to the breakdown of blood-retinal barrier and VEGF play s a critical role in causing the leakage. Corticosteroids act on VEGF directly by inhi biting VEGF secretion [32, 33] and also by countering the effects of VEGF, inhibiting cyt okine production, attenuating leukocyte adhesion.

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17 and inducing apoptosis [34]. Corticosteroids also affect the distribution of the tight junction proteins increasing their expressi on and also can reverse the phos phorylation of occludin (a tight junction protein) cau sed by VEGF [35]. Corticosteroids are also potent inhibitors of neovascularization. Corticosteroids act by inhibiting the basic fibrob last growth factor induced migra tion and tube formation [36], they further act by inhibiting extracellular matrix tu rn over by down regulation of metalloproteinase-2 production and down regulation of ICAM-1 expressi on [37] they also act by decreasing VEGF levels [32, 38] and decreasing the expression of major histocompatability complex-11 expression [39]. Routes of Drug Administration Traditional routes of adm ini stration of steroids are topi cal, systemic and periocular (including subconjunctival, sub-ten ons and retrobulbar), refer to Figure 1-3. The topical route using eye drops constitute approximately 97% of to tal formulations into the eye, but therapeutic concentrations are not achieved by this route mainly due to lacrim ation and presence of barriers. Although drug delivery to the posterior segment can be achieved by the systemic route, large doses are required to achieve th erapeutic concentration due to the presence of blood ocular barriers which can result in side effects. Peri ocular injections minimi ze most systemic side effects and eliminate the need for daily patient compliance, yet the blood ocular barrier can still impede from this route from atta ining therapeutic concentrations to its intended target tissue. Due to the poor accessibility, effective treatment of di seases related to the pos terior segment of the eye, the development of new delivery systems, r outes (intravitreal) and new corticosteroids will help to achieve high angiostatic and antipermeability concentrations and to reduce the adverse effects [30].

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18 Intravitreal injection provides the most direct approach in delivering drugs to the posterior segment, and therapeutic tissue drug levels can be achieved. Intravitreal delivery allows for sufficiently high local concentrations of corticos teroids to maximize their anti-inflammatory and angiostatic effects and attenuating adverse effect s. Intravitreal injec tion of triamcinolone acetonide (Kenalog-40) is routinely used in practi ce to treat diseases in the posterior region of the eye [40, 41]. The success of intr avitreal triamcinolone acetonide therapy has led to its use in a variety of diseases such as exudative macu lar degeneration and proliferative diabetic retinopathy (PDR) due to their potent antiangiogenic action. One of the most common adverse effects seen w ith corticosteroids is ocular hypertension [39, 41-43]. In one study, intravitreal injections of 25 mg of tr iamcinolone acetonide resulted in ocular hypertension in approxi mately 50% of eyes, commenci ng 1 to 2 months after the injection. Several large studies using 4 mg of triamcinolone s howed an elevated pressure incidence in 30% of patience. The factors for developing ocular hypertension are not yet understood [42]. Recent studies showed that cor ticosteroid treatment of human trabecular meshwork cells produced delaye d, progressive cellular and extr acellular glycoprotein induction [31]. Cataract is also a commonly seen complication caused by chronic use of corticosteroid treatments. Several mechanisms such as: form ation of covalent adducts with the steroid molecules with lysine residues of lens, lowe red ascorbic acid in the aqueous humor and metabolic changes like altered phospholipids meta bolism were shown to cause cataract. Because of their potential side effects a nov el approach to the use of cort icosteroids is highly desirable. This can be achieved by either developing soft dr ugs or by application of slow release systems. The development of soft drugs help s to reduce the risk of undesired side effects. The use of soft drugs is an effective way to prevent side effects as they metabolize at the s ite of action or at the

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19 site of application. Loteprednol etabonate is a site-active corticoste roid synthesized through structural modifications of prednisolone related compounds so that it will undergo a predictable transformation to an inactive metabolite [44]. Loteprednol etabonate was effective in the treatment of giant papillary conjunctivitis, seas onal allergic conjunctivitis, postoperative inflammation and uveitis. In a la rge double-blind study on corticos teroid responders loteprednol etabonate demonstrated less propens ity to cause clinically signif icant elevation in intraocular pressure when compared to prednisolone acetate [45]. Drug Delivery Systems Posterio r ocular diseases are chronic in nature and require multiple injections to maintain therapeutic concentrations, which ar e not only disagreeable but also increase the risk of cataract formation and retinal detachment with the fr equency of injection. Sustained drug delivery devices are an excellent way to avoid multiple intravitreal injection. The use of drug delivery systems (DDS) is an effective way of delivering drugs to the posterior region of the eye for an extended period. Table 1-3 shows the various drug delivery systems e ither clinically approved or under research for posterior ocular conditions. DDS can improve the permeation, help in prolonged release of the drug and also helps to attenuate the adverse effects. Recently implants of ganciclovir are being used, but surgery is re quired for placing the implant and its removal. Polymeric nanoparticles and microparticles offer an excellent way of sustained drug delivery [46, 47]. As smaller particles are better tolerated in the eye, we chose nanoparticles for drug delivery [46, 48]. Particles 200 nm or less are sh own to be localized in the RPE cells [46]. Nanoparticle formulation is one of the strategi es currently used to improve drug absorption across biological membranes [49]. In order to achieve sustai ned drug release and prolonged therapeutic effect using ophtha lmic drug-loaded nanoparticles, the entrapped drug must be released from the nanoparticles at an appropriate rate. If the re lease rate of the nanoparticle

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20 formulated drug is too fast it ma y fail to provide sustained exposure while too slow a release rate could prevent the drug from reaching a sufficien t concentration. In drug-loaded nanoparticles, the active molecules are confined within polym eric matrices by relatively strong noncovalent interactions such as ionic, hydrogen-bonding, hydrophobic, or dipole [50-52]. Biodegradable polymers such as poly(lactic-co-glycolic acid) and poly(lactic acid) ar e often used for the preparation of nanoparticles. They are aliphatic polyesters derived from glycolic acid and from lactic acid enantiomers. These polymers are bioc ompatible and can be synthesized in various molecular sizes allowing the encapsulation of sp ecifically adapted formulations. They degrade by hydrolysis of the ester linkages of polymer bac kbone and form lactic acid and glycolic acid and are eliminated from the body by Kr ebss cycle through normal excretion. Poly(lactide-co-glycolide) is widely used for surgical dressings fracture repairs and dental repairs. Several researchers have used poly(lactide-co-glycolide) for the preparation of DDS such as implants and nano/microspheres for controlled drug delivery. The degradation rate of these polymers can range from months to years depend ing on the molecular weight, conformation and copolymer composition [53]. Hypothesis We hypothesize that sustainedrelease corticosteroid na noparticles can im prove the therapeutic profiles of corticoste roids for posterior ocular diseas es. Our hypothesis will be tested by the following aims: Prepare and characterize corticosteroid nanopa rticles for: size, shape, drug encapsulation and in vitro release. Investigate the effect of steroids and th eir nanoparticles on VEGF expression, cellular uptake and cytotoxicity using ARPE-19 cells. Conduct in vivo study in mice and study the effect of co rticosteroid nanoparticles on laser induced choroidal neovascularization in mice.

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21 Table 1-1. Eye disease prevalence and projections in adults 40 years and older in the U.S Eye Disease Current Estimates (in millions) 2020 Projections (in millions) Advanced age-related macular degeneration (with associated vision loss) 1.8* 2.9 Glaucoma 2.2 3.3 Diabetic Retinopathy 4.1 7.2 Cataract 20.5 30.1 Table 1-2. Treatments being considered for AMD and DR Drug Class Drug Photodynamic therapy Visudyne [54] VEGF Inhibitors Anti-VEGF aptamer AntiVEGF antibodies Anti-VEGF antibody fragments VEGF-trap Pegaptanib [25] Bevacizumab [55] Ranizumab [55] VEGF-trap [56] PKCinhibitor Ruboxistaurin [55] Corticosteroids Flucinolone acetonide [57] Dexamethasone [58] Triamcinolone acetonide [59] Anacortave acetate [60]

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22 Table 1-3. Drug delivery systems for treating posterior ocular conditions Drug delivery system Drug Implants Ganciclovir [61-63] Flucinolonee acetonide [57, 64] Dexamethasone [65] Betamethasone phosphate [66, 67] Fluconazole [68] Triamcinolone [69] Cyclodextrins Dexamethasone [70, 71] Liposomes Cidofovir [72] Ionthophoresis Methotrexate [73] Dexamethasone [74] Nano/microparticles Budesonide [48] Cyclosporine [75] Peroxicam [76] PKC412 [56] Transdermal Prednisolone [77]

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23 Figure 1-1. Structure of the Eye

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24 Figure 1-2. Detailed structure of the retina

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25 Figure 1-3. Routes of drug delivery to target posterior region of eye

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26 CHAPTER 2 PREPARATION AND CHARACTERIZATI ON OF TRIAM CINOLONE ACETONIDE NANOPARTICLES Introduction Drug delivery to the posterior region of the eye rem ains a challenge. Drug delivery systems are becoming popular recently as a suitable way to sustain drug release. They have the advantage of delivering therapeutic agent to its site of action at an optimal rate. Slow release delivery systems can alter the drugs biopharmaceutics [78]. Th ey also can decrease toxic side effects due to slow drug release and also eliminate the inconvenience of re peated administration. Various drug delivery systems include: liposomes, implan ts, microparticles and nanoparticles. Several researchers showed the effectiveness of th ese systems for ocular drug delivery [79]. Recently there has been interest in the use of nanoparticle drug delivery to the posterior region of the eye. Nanoparticles are among the most widely studied colloidal systems over the past two decades. Nanoparticles are polyme ric particles, ranging from 10 nm to 1 m, in which the drug is dissolved, entrapped, en capsulated or adsorbed [49]. They have the advantage of easy administration by injection due to their small si ze. Unlike polymer implants, nanoparticles do not require surgical procedures for implantation and removal. Nanoparticles have been shown to be efficient as ocular drug delivery systems by prolonging the duration of the action of drugs. Bourges et al. examined intravitreal administer ed nanoparticles in rat ey es [46]. Poly(lactic-coglycolide) nanoparticles injected into the vitreous cavity of rats, were found within the vitreous cavity immediately after intravitre al injection. By 6 hours, the majo rity of the nanoparticles were found within the retinal pigmen t epithelium (RPE) and by 24 hours, a significant concentration of nanoparticles was observed in the cytoplasm of the RPE. With the rat model, intra cytoplasmic retinal cell concentrations of nanopa rticles remained elevated as far as 4 months after a single intravit real injection [46].

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27 Nanoparticles are made of natural or artifi cial polymers that are biocompatible. The various polymers used to fabr icate nanoparticles are polyacryl amide, polymethylmethacrylate, poly(lactide-co-glycolide) and E-caprolactone [79]. Biodegradab le nanoparticles formulated from poly(lactide-co-glycolide) and poly(vinyl alcohol) (PVA) pol ymers have been extensively investigated for various drug deli very applications. Poly(lactide-co-glycolide) is biocompatible, and more importantly, the degradation rates of polymer and the accompanying release of encapsulated drugs can be controlled by the pol ymers physicochemical properties, such as molecular weight, hydrophili city, and the ratio of lactide to glycolide [80]. Rate of drug release from controlled release fo rmulations depends on the characteristics of the particles, including particle size, size dist ribution, drug content, incorporation and surface morphology [81]. The particle size and drug conten t are particularly important characteristics that determine drug release. These characteristics depend on the specific formulation parameters [82]. To control these, it is im portant to study the effect of pr ocessing and materials parameter on particle size and drug content of the nanoparticles. Our goal wa s to prepare corticosteroid nanoparticles with biodegradable polymer poly(lactide-co-glyco lide) that can sustain drug release. In this study, we developed nanoparticles by an emulsion solvent evaporation technique using sonication. Initially an O/W emulsion was formed with the aqueous phase containing the emulsifier and organic phase in which the drug and the polymer are dissolved. The organic phase is then evaporated from the emulsion droplets resulting in the formation of nanoparticles which are collected through centrifugation. The formula tion parameters: amount of poly(lactide-coglycolide) polymer, triamcinolone acetonide drug and poly(vinyl alcohol) surfactant were varied to study the effect on size, encapsulation and drug release.

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28 Materials and Methods Materials: P oly(lactide-co-glycolide) (50:50) inherent viscosity of 0.68 dL/g was obtained from Lactel absorbable polymers (Pelham, AL ), polyvinyl alcohol (PVA) was obtained from Sigma Chemical Co. (St. Louis, MO), HPLC grade methylene chloride/dichloromethane acetonitrile were obtained from Fisher Scientific, dialysis membrane bags for in vitro release studies (molecular weight cut off 10,000; spec trum Laboratory (Rancho Dominguez, CA). Nanoparticle Preparation Nanoparticles were prepared by using solven t evaporation method [ 83]. The drug and the polym er were dissolved in organic phase, and th is solution was added to 10ml of 1% aqueous poly(vinyl alcohol) (PVA) solution. The resultan t mixture was sonicated at 40W for 1 minute with a probe sonicator (Sonics vibracell, Ne wtown, CT), to obtain an O/W emulsion. The O/W emulsion was then added to remaining 40ml of 1% aqueous PVA solution in a 50 ml conical flask. The contents were stirred at 250 rpm on a magnetic stirrer at room temperature overnight to evaporate the organic phase, allowing the form ation of turbid particulate suspension. The nanoparticles were separated by ultracentrifugation (B eckman Coulter, Inc.Fullerton, CA) at 35,000g for 1hour. The pellets were washed three tim es in double distilled water and freeze dried with Freezone 6 lyophilizer (Labconco Corpora tion, Kansas City, MO) to obtain lyophilized particles. [84]. Figure 2-1 shows th e nanoparticle preparation process. Nanoparticle Characterization The particle size of the cort icosteroid nanoparticles was de term ined with a NICOMP 380 ZLS (Particle Sizing Systems, Santa Barbara, CA). Sample of polymeric nanoparticles 5 mg were suspended in 5 ml of double distilled wate r, and the diluted suspension was subjected to particle size measurement. The morphology of the nanoparticles was analyzed by scanning electron microscope (SEM) JEOL JSM-6335F instrument (Major Analytical Instrument Center

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29 (MAIC), UF, Gainesville, FL). A small amount of the freeze dried sample was layered on the SEM stubs and coated with carbon in a high-vacuum evaporator. Th e coated samples were then observed for their surface morphology w ith the instrument set at 15 kV. Turbidity test was performed between the micronized triamcinolone acetonide and its nanoparticles by suspending them. Drug Encapsulation Efficiency Encapsulation efficiency of the particles wa s determ ined by weighing 5 mg of the freeze dried particles in a glass tube. Methylene chloride 2 ml was adde d and mixed thoroughly at room temperature. The resultant solution was eva porated to dryness under vacuum and the dried residue was reconstituted with acetonitrile wate r mixture (70:30). The reconstituted solution was vortexed for 1minute and centrifuged at 4000 rpm for 15minutes, and 100 l of the supernatant is injected into the HPLC column. The HPLC was pe rformed isocratically at ambient temperature, at a flow rate of 1 ml/min. The mobile phase comprised of acetonitrile and water (70:30 v/v). A Waters C18 column (4.6x150 mm) preceded by a gua rd column, and detection was accomplished using UV detection at a wave length of 254 nm. The calibrati on curve was obtained with standards from 1 to 50 g/ml and was linear (r2 > 0.99). The % encapsulation efficiency is cal culated using the following equation: 100 drug of amount l Theoretica drug of amount Actual efficiency tion %Encapsula 100 les nanopartic of amount Total les nanopartic in drug of Amount efficiency Loading % In Vitro Release In order to avoid the drawback of aggregation during freeze dr ying the release studies were performed with nanoparticles susp ensions immediately after their preparation. The concentration

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30 of the released drug was studied as a function of time. The re sults over 100 hour s are shown. Suspension of corticosteroid nanoparticles containing 300 g of the drug was transferred into floatable dialysis membrane unit (10,000 mol.wt), and the unit was placed in 50 ml centrifuge tube containing phosphate buffer solution (PBS, pH 7.4) at 37 C. Samples of 1 ml were collected at regular intervals and replaced by PBS. The cort icosteroid released was then analyzed using HPLC. Phosphate buffer solution containing the drug was directly injected into the HPLC column and acetonitrile: ammonium acetate buffer (50:50) was used as the mobile phase at a flow rate of 1 ml/min. Calibration curv e was obtained with standards from 0.5 g/ml to 20 g/ml and was linear ((r2 > 0.985). For release studies between micronized tria mcinolone, small nanoparticles of 145 nm and larger nanoparticles of 415 nm, in vitro release studies were conducted under sink conditions using 0.5% sodium dodecy l sulfate containing 650 g. of drug. Calibration curve was obtained with standards from 1 to 20 g/ml and was linear (r2 > 0.99). Statistical Analysis One way ANOVA was performed using GraphPad Prism 4 (GraphPad Software Inc., San Diego, CA) with p value less than 0.05 considered as significant. Results Morphology Figure 2-2 and Figure 2-3 show the SEM pictures of spherical nanopa rticles with smooth surface. Figure 2-4 shows turbidity of nanoparticles compared to micronized triamcinolone acetonide.

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31 Effect of Polymer Poly(lactide-co-gl ycolide) on Size and Encapsulation Effect of polymer poly(lactid e-co-glycolide) on nanoparticles characteristics is shown in Table 2-1. The polymer content was increased from 50 to 200 mg while keeping all the other processing conditions similar. Increasi ng the polymer concentration lead to gradual increase in the particle size from 115 to 200 nm. When the polymer amount was increased to 400 mg while keeping the ratio of drug to polym er 1:10, 67% of the particles showed mean concentration of 445 nm while 33% were 150 nm. Figure 2-5 shows th e distribution of the particles. Increasing the polymer concentration increases the viscosity of the organic phase and resisting droplet breakdown during sonication. Also increase in drug encapsulati on was seen with increase in polymer concentration in organic phase. The dr ug encapsulation efficiency increased from 48% to 65%. Increase in polymer increases particle si ze resulting in more drug being encapsulated. Effect of Triamcinolone Aceton ide on Size and Encapsulation The effect of drug concentration was studied by increasing triamci nolone acetonide from 5 to 15 mg while maintaining the polymer concen tration constant. Table 2-2 shows the observed results. The diameter of the particles remained independent of the amount of the drug present. As the drug concentration was increased from 5 to 15 mg there was a significant increase in the drug loading and encapsulation efficiency. Lower enca psulation was seen with 5 mg of drug. The lower encapsulation might be due to diffusion of the drug from the O/ W emulsion globule into the aqueous continuous phase resulting in lower encapsulation. Effect of Poly(vinyl alcohol) on Size and Encapsulation Emulsifier plays a critical role in the preparation of nanoparticles by emulsion solvent evaporation method. The amount of surfactant is crucial as it prevents the coalescence of the droplets and protects them. In order to study the effect of surfactant pol y(vinyl alcohol) on the nanoparticle properties, the concentration was va ried from 1 to 4% while keeping the other

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32 variables standard. The observed results are show n in Table 2-3. As the concentration increased from 1 to 4% there was a decrease in the size of the particles from 172 to 120 nm. Concentration of 4% emulsifier leads to smaller particles due stabilizing function of the surfactant, thus preventing the aggregation of particles. Increasing the PVA concentration decreased the drug encapsulation. This is mainly due to the decrease in particle size caused by increased stability. Decrease in particle size results in larger surface area which lead s to increased diffusion of the drug from the O/W emulsion into the conti nuous aqueous phase. Incr ease in encapsulation efficiency was observed when the aqueous phase was saturated with the drug, preventing the diffusion of the drug from O/W em ulsion into the aqueous phase. In Vitro Release Studies Release of corticosteroids from poly(lactid e-co-glycolide) nanoparticles into phosphatebuffered saline (PBS) was measured in vitro at 37C. Figure 2-6 to figure 2-9 show the cumulative release data of various formulations. A ll the formulations showed initial burst release of more than 10% on the first day which might be due to the presence of the drug on the surface of nanoparticles. A drug release of 50% wa s seen in 24, 34 and 96 hours for micronized triamcinolone acetonide, 145 and 415 nm nanopartic les respectively. The size of the particles plays a crucial role on the drug release. Larger particles showed slower release compared to smaller particles with similar formulation parame ters. Since smaller particles have larger surface area, the drug release is faster At the end of 192 hrs the cumu lative release for triamcinolone acetonide micronized, triamcinolone nanopart icles of 145 and 415 nm was 100, 92 and 72% respectively. Figure 2-8 shows the cumulative tr iamcinolone acetonide released from micronized triamcinolone acetonide and its na noparticles of different size. Unlike diffusion through the membrane of a reservoir system, where the rate of drug release is linear with time, matrix controlled di ffusion is linear with the square root of time for

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33 spherical structures, such as microspheres a nd nanospheres. In accordance with this our in vitro release data with all the formulations showed be tter fit with Higuchi equation compared to zero order. Tables 2-4 to 2-7 show the R2 values of the various form ulations calculated with zero order and Higuchi equations. Figure 2-9 shows the extrapolated in vitro data of micronized triamcinolone acetonide, nanoparticles of around 145 and 415 nm particles re spectively showing 100% drug release in 5, 7 and 16 days respectively. Figure 2-10 shows the SEM images taken during in vitro release studies over a 15 day period. The images show in itial swelling of the particles followed by pore formation and adhering. Discussion Drug delivery systems help in controlled delivery of therapeutic agents to its site of action and at optimal rate. An ideal c ontrolled-release formulation should release the entrapped drugs in a continuous manner over a desired time period. The results from the different studies show the potential of colloidal system s as ocular drug delivery syst ems for either hydrophobic or hydrophilic drugs [79]. Poly(lactide-co-glycolide) polymers are natura l biodegradable and biocompatible polymers that are most widely used. It is also approved for human use by the Food and Drug Administration. The nanoparticles formulation w ith a therapeutic agen t entrapped into the polymer matrix provides sustai ned drug release [49]. The lactide/glycolide polymers are cleaved by hydrolysis to form lactic acid and glycolic acid that are eliminated from the body through the Krebss cycle as carbon dioxide and water [85]. Typical emulsion solvent evaporation pr ocess was employed to produce polymeric nanoparticles and good reproducibility with si ze and encapsulation was observed. The nanoparticles were formed as a result of evapor ation of the organic solvent from the emulsion

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34 nanodroplets. The particle size and encapsulation efficiency are important parameters and can affect the biopharmaceutical propert ies of the nanoparticles. The si ze can also play an important role in endocytosis of na noparticles. The smaller the size of the particle, the better is the cellular uptake of particles [86]. The size of the particles was mainly affect ed by polymer concentration in the organic phase. Our results corroborate with those shown by other researchers that increase in resistance caused by larger amounts of polymer during the formation of emulsion results in larger nanoparticles [81]. The particle s prepared were unimodal belo w 200 mg of poly(lactide-coglycolide) when the polymer amount was increa sed to 400 mg, 67% of the particles were around 415 nm while 33% of them were 150 nm (figure 2-5). This formulation needs to be further optimized to prepare larg er particles with uniform size to prolong the dr ug release. This shows that the polymer concentration al ong with the sonication time and pow er need to be adjusted to obtain uniform particles of larger size. Previous studies have shown that factors that prevent the diffusion of the drug into the aqueous phase increase the encapsulation efficiency [87]. In our study, we observed that increase in encapsulation efficiency can be obtained by either increasing the dr ug concentration or by saturating the aqueous phase with the drug. Particle size and loading are important characteristics that can affect the release of the drug from the nanoparticles. In vitro release studies showed initia l burst followed by sustained drug release. Our in vitro release profiles are similar to those obtained by Feng et al. [88] who observed a 30 to 40% taxol release at the end of 2 weeks from nanospheres. Based on our drug release profiles and Higuchi plot s, it appears that the drug is entrapped in the polymer matrix.

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35 Kompella et al. showed cumulative release of 35 to 50% cumula tive budesonide release at the end of 2 weeks from nanoparticles followi ng an initial burst of 15 to 20% [89]. Our results showed a decrease in drug release wi th larger particles compared to the smaller particles. Smaller particles possess a larger surface area, which in turn can l ead to a faster release of the drug incorporated. Yoncheva et al. showed that the release properties of the nanoparticles and their size are interrelated [ 90]. Smaller nanoparticles also lead to a shorter average diffusion path of the matrix entrapped drug molecules. The release of the entrappe d drug from the polymer matrix has been found to occur through diffusion and degradation medi ated process. The release of the drug in the early stage is believed to occur mainly through diffusion in the polymer matrix while in the later phases the release occurs due to both diffusion and polym er degradation [91]. The change in surface morphology of nanoparticles with time following inc ubation in PBS is shown in figure 2-10. The particles showed roughness and pore formation follo wed by aggregation of the particles. Similar results were shown by Panyam et al. they also showed that the glass transition temperature (Tg) decreases with the decrease in polymer molecular weight resulti ng in more softer polymer prone to aggregation resulting in fusion of the particles [92]. Vishwanath et al. [93] showed that PLA and PLGA undergo deformation and aggregation owing to lowering of the polymer glass transition temperature below 37 C following its hydration in the buffer. The goal of this study was to understand the e ffect of various formulation parameters on nanoparticle properties and select a form ulation suitable for intravitreal delivery in vivo. Based on our results, the formulation with drug to polym er ratio of 1:10 with 1% PVA was selected as it gave us unimodal size particles with re producibility, good encapsulation efficiency and sustained drug release compared with micronized triamcinolone acet onide. It also showed less

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36 turbidity compared to micronized triamcinolone acetonide and as a result may provide better visibility after intravitreal delivery. As the volum e that can be injected into the vitreous is limited, higher polymer concentrations were not considered as the loadi ng efficiency decreases with increase in the amount of polymer. Conclusion Solvent evaporation method using sonication wa s successfully used in the preparation of triamcinolone acetonide nanoparticles. Particles produ ced were in the nanoparticle size range and unimodal, with good drug encapsulation. The various formulation factor s affecting the size, loading and release rate were studied. Table 2-8 shows the ef fect of various formulation parameters on particle characterization. In vitro release studies indicate sustained release of the drug following an initial burst. Nanoparticles can sustain drug release for a prolonged period preventing repeated intravitreal injections and hence can be us ed to treat posterior ocular conditions.

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37 Figure 2-1. Flow chart showing th e nanoparticle preparation process

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38 Figure 2-2. Scanning electron mi croscopy image of triamcinolone acetonide loaded 150 nm nanoparticles. Bar indicates 1 m

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39 Figure 2-3. Scanning electron micr oscopy image of triamcinolone acetonide loaded poly(lactideco-glycolide) 415 nm nanopartic les. Bar indicates 100 nm

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40 Figure 2-4. Turbidity of micronized triamci nolone acetonide and triamcinolone acetonide nanoparticles prepared with 1:10 ratio

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41 0 2 4 6 8 10 12 14 16 01002003004005006007008009001000 Diameter (nm)% channelA 0 1 2 3 4 5 6 7 8 9 01002003004005006007008009001000 Diameter (nm)% ChannelB Figure 2-5. Particle size dist ribution (A) 172 nm (B) 415 nm

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42 Table 2-1. Effect of polymer concentration on particle size and loading Formulation PVA Drug:Polymer Size (nm) Drug Loading (%wt/wt) EE% F1 1% 1:5 115 13* 12.7 0.9* 48.36 3.6* F2 1% 1:10 172 12 8.9 0.5 61.64 3.84 F3 1% 1:20 200 7 5.3 0.001* 65.51 2.30 F4** 1% 1:10 418* 9.02 0.17 78.8 *Statistically significant compared to F2 if P < 0.05. **400mg of polymer was used Table 2-2. Effect of drug concentr ation on particle size and loading Formulation Drug (mg) Drug:Polymer Size (nm) Drug Loading (%wt/wt) EE% F5 5 1:10 161 4 3.8 0.1* 20.19 0.1* F2 10 1:10 172 12 8.9 0.5 61.004 2.2 F6 15 1:10 153 4 10.5 1.1* 78.6 0.9* Statistically significant compared to F2 if P < 0.05 Table 2-3. Effect of surfactant (PVA) c oncentration on partic le size and loading Formulation PVA Drug:Polymer Size (nm) Drug Loading (%wt/wt) EE% F2 1% 1:10 172 12 8.9 0.5 61.0 2.2 F7 4% 1:10 120 30* 4.2 0.4* 20.0 3.0* F8** 4% 1:10 108 7.9 41.0 *Statistically significant compared to F2 if P < 0.05. ** shows 4% formulation where the aqueous phase was satu rated with the drug

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43 0 10 20 30 40 50 60 70 80 020406080100120140160180 Time (hrs)% Cumulative TA released 1:05 1:10 1:20 Figure 2-6. Effect of polymer concentration on in vitro cumulative triamcinolone released from nanoparticles. All the studies were perfor med at 37C. Data are expressed as the mean SD. Table 2-4. Zero order and Higuchi equations of in vitro drug release showing the effect of polymer Formulation Zero Order Higuchi Equation 1:5 R2 = 0.9818 Y=0.4305X + 5.0135 R2 = 0.9894 Y= 6.8924X +20.12 1:10 R2 = 0.9751 Y= 0.4341X + 8.1279 R2 = 0.9827 Y= 6.951X + 17.23 1:15 R2 = 0.9618 Y= 0.3902X + 24.416 R2 = 0.9881 Y= 6.305X +1.1349

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44 0 20 40 60 80 100 120 020406080100120140 Time (hrs)%CumulativeTA released 5mg 15mg 10mg Figure 2-7. Effect of drug concentration on in vitro cumulative triamcinolone released from nanoparticles. All the studies were perfor med at 37C. Data are expressed as the mean SD. Table 2-5. Zero order and Higuchi equations of in vitro drug release showing the effect of drug Formulation Zero Order Higuchi Equation 5 mg drug R2 = 0.9967 Y= 0.4247X + 63.901 R2 = 0.9973 Y= 6.7759X + 39.392 10 mg drug R2 = 0.9751 Y= 0.4341X + 8.128. R2 = 0.9827 Y= 6.951X + 17.230 15 mg drug R2 = 0.9551 Y=0.2434X + 31.584 R2 = 0.9705 Y= 3.8926X + 17.402

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45 0 10 20 30 40 50 60 70 80 90 050100150200 Time (hrs)% Cumulative TA released 1% 4% Figure 2-8. Effect of surfactant pol y(vinyl alcohol) concentration on in vitro cumulative triamcinolone released from nanoparticles. All the studies were performed at 37C. Data are expressed as the mean SD. Table 2-6. Zero order and Higuchi equations of in vitro drug release showing the effect of surfactant Formulation Zero Order Higuchi Equation 1% PVA R2 = 0.97510 Y= 0.4341X + 8.1279 R2 = 0.9827 Y= 6.951X + 17.23 4% PVA R2 = 0.09892 Y= 0.467X + 19.106 R2 = 1 Y= 7.4013X -. 6.7794

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46 0 20 40 60 80 100 120 0 50 100150200250 Time (hrs)% Cumulative TA released TA 145 nm 415 nm Figure 2-9. Effect of size of the nanoparticles on in vitro cumulative triamcinolone released from PLGA particles. All the studies were perfor med at 37C. Data are expressed as the mean SD. Table 2-7. Zero order and Higuchi equations of in vitro drug release showing the effect of micronized triamcinolone acetonide (TA), smaller nanoparticles (NP) and larger nanoparticles Formulation Zero Order Higuchi Equation TA R2 = 0.7395 Y= 0.4899X+ 34.699 R2 = 0.9007 Y= 8.116X+10.87 Smaller NP R2 = 0.8313 Y= 0.4592X +21.519. R2 = 0.9667 Y= 7.2552X + 1.2001 Larger NP R2 = 0.9042 Y=0.3184X+ 14.315 R2 = 0.9882 Y= 5.0435X +0.513

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47 0 20 40 60 80 100 120 0 5 10 15 20 Time (Days)% Cumulative TA released TA 145nm 415nm Figure 2-10. Results of in vitro release studies extrapolated to 100% cumulative drug release (A) micronized TA (B) smaller TA nanopa rticles (C) larger TA nanoparticles

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48 A B C D E Figure 2-11. Scanning electron mi croscopy images taken during in vitro release studies (A). day 0 (B) day 1 (C) day 5 (D) day 10 (E) day 15

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49 Table 2-8. Relationship between various parameters to control the partic le size and drug content for nanoparticles prepared by emulsification-solvent evaporation method Nanoparticle characteristics Effect of formulation parameters Effect on particle size Increase in polymer c oncentration in organic phase increased the size of the particle Decrease in surfactant concentration increased the size of the particles Increasing the energy during emulsifi cation decreased the size of the particle The drug concentration did not sh ow effect on particle size Effect on drug loading Increase in dr ug concentration increased drug loading Decrease in drug loading was seen with increase in surfactant concentration Increase in particle size increased drug loading Saturation of the aqueous phase w ith drug increased drug loading.

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50 CHAPTER 3 EFFECT OF TRIAMCINOLONE ACETONI DE AND ITS NANOPARTICLES ON RETINAL PIGMENT EPITHELIAL CELLS Introduction Retinal pigment epithelium is a major layer within the retina that separates the retina from the remaining posterior ocular tissue layers. Retinal pigment epithelial (RPE) cells express vascular endothelial growth factor (VEGF), whic h plays a critical role in the development of various posterior ocular conditions. VEGF, an angiogenic m itogen has been implicated in various vascular diseases in the eye including choroidal neovasculariza tion, macular edema and diabetic retinopathy. Elevated le vels of VEGF were reported in patients suffering from diabetic retinopathy and choroidal neovasc ularization [15]. Hence, agen ts that can inhibit VEGF secretion offer an attractive therapeutic modality. Most of the research has been on macromol ecules such as VEGF antibodies [94] and VEGF aptamers which have poor stability and permeability [17] Corticosteroids are gaining interest recently due to their angiogenic and antipermeable prope rties. These properties are in part due to their ability to inhibit VEGF secretio n. Previous studies show ed corticosteroids could inhibit VEGF secretion in airway and alveolar epithelial cells [95]. Intravitreal delivery is an effective route to de liver drugs to the posteri or region of the eye. It helps to overcome the various ocular barrie rs and the long diffusiona l distance required for attaining therapeutic con centration for posterior oc ular conditions. As repeat ed injections are not desirable, large doses of drug are needed. These hi gh concentrations may be toxic to ocular cells. Researchers have shown the toxicity of triamcinolone on ARPE-19 cells [96]. Retinal pigment epithelium plays a crucial role in maintaining the integrity of the outer retina blood barrier and supporting normal function of the retina. Since the retinal pigment epithelium is in close proximity to the vitreous space, separated from it only by the neuroretina, any damage to this

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51 monolayer poses the risk of its cytotoxicity and retin al dysfunction. Hence, particulate systems such as microparticles, nanoparticles and liposomes could be used for site-specific and sustained delivery of drugs [96-98]. Such delivery systems will likely modify the disposition of drugs at the site of RPE cells, sustain drug release, reduce toxicity and increase intracellular levels of the drugs in RPE. We hypothesized that nanoparticles can decrease cyto toxicity compared to the free drug and also sustain drug release. Cellular uptake is an important aspect of nanoparticle delivery. Because retinal pigment epithelial cells in vivo phagocytose particles [99], sustaine d high intracellular drug levels can likely be achieved by preparing pa rticulate drug delivery systems that can be injected into the vitreous. Previous studies demonstrate that uptake and retention of drug carriers like nanoparticles are affected by cellular processes such as endocytosis and e xocytosis. Studies have shown that nanoparticles formulat ed using polymers such as poly (lactide-co-glycolide), are taken up into cells through active pro cess such as endocytosis [100]. Although it is well-known that retinal pigment epithelial cells take up particles, the factors affecting the uptake are not well characterized [101, 102]. Understa nding the factors influencing pa rticle uptake would help in developing a suitable particulat e system for drug delivery to the retinal pigment epithelium. Human ARPE-19 cells have morphological an d functional properties similar to retinal pigment epithelial (RPE) cells in vivo [95]. They are capable of di fferentiating and proliferating similar to the RPE cells. In our studies we use ARPE-19 cells to show the effect of corticosteroids and their na noparticles on ocular cells. The overall objectives of thes e studies are as follows, determine the effect of triamcinolone aceton ide on VEGF secretion in ARPE-19 cells determine the toxicity of micronized triamci nolone acetonide compared to its nanoparticles on retinal pigment epithelial cells and

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52 determine the mechanism of uptake of triamc inolone acetonide nanopa rticles in ARPE-19 cells. Materials and Methods Materials: ARPE -19 cells, culture medium Dulbeccos modified Eagles medium Hams F-12 [DMEM-F12], fetal bovine serum (FBS), peni cillin-streptomycin, trypsin were obtained from Gibco (Carlsbad, CA). The cells were cu ltured either in cell culture flasks T-75 cm2 and the experiments were conducted in 96-well plates or 24 well plates obtained from Fisher (Pittsburgh, PA). Cell toxicity kit and cell ly sis reagent were obtained from Sigma (St. Louis, MO). VEGF ELISA kit was obtained from R&D systems (Mi nneapolis, MN). Pierce BCA protein assay kit (Rockford, IL) was used for pr otein analysis. Poly(lactide-co glycolide) was obtained from Lactel absorbable polymers (Pelham, AL). The HPLC grade methylene chloride and acetonitrile were obtained from Fisher Scientific. Cell Culture Human ARPE-19 cells were gr own in 1:1 (vol/vol) mixture of Dulbeccos modified Eagles and Hams F12 medium, co ntaining 10% fetal bovine serum, and antibiotic mixtures of 100 U/ml penicillin G and 100 g/ml streptomycin sulfate. The cells were cultured in 75 cm2 flasks. The cultures were maintained in a humidified 5% CO2 environment at 37C. Cells grown in a 75 cm2 T-flask are passed after they have reache d 80-90% of confluence at a split ratio of 1:2. The medium was changed every 2 days. For cell splitting, the old media in the flask is discarded and the cell la yer is briefly rinsed with PBS. Trypsin 5 ml is added and incubated for 10min. Later 5 ml of the media was added into the flask and the cell suspen sion is transferred into a centri fuge tube and spun at 125 g for 5 minutes. The supernatant is then discarded and cells are re-suspended in fresh growth media. Appropriate aliquots of cell su spension are transferred to new flasks and incubated at 37C.

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53 Vascular Endothelial Growth Factor Secretion Cells of ARPE-19 cultured in the tissue flask are trypsinised and seeded onto a 24 well plate and allowed to grow to confluence. The me dia was replaced every two days. On the day of the study media was replaced with 1% FBS media and allowed to remain in quiescence for 12 hours. After the quiescence period, the monolayer s are incubated with triamcinolone acetonide (1, 10 and 100 M). The culture media was collec ted at the end of 12 hours. The secreted VEGF in supernatants is quantified by ELISA met hod capable of detecti ng VEGF 165 and the cell protein content was assayed using the BCA protei n assay kit after lysing the cells and the VEGF secretion was normalized to total protein. Cell Toxicity Cells of ARPE-19 were seeded at a density of 10,000 cells/well and were allowed to attach overnight in 96-well plates. The cells were then e xposed to corticosteroids or their nanoparticles (0, 1, 10, 100 and1000 M). At the end of 24 hour incubation, the cells we re incubated with 25 l of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenylte trazolium bromide (MTT) solution (5 mg/ml in PBS) for 3 hours at 37C. Viable cells will reduce MTT to formazan. Finally 100 l of isopropanol was added to dissolve formazan crysta ls and the absorbance of this solution was measured at a wavelength of 540 nm using a mi cro-plate reader. The cell viability is then determined by the relative formazan formation after corticosteroid and the nanoparticles treatments compared to the control group. Cell Uptake Human ARPE-19 cells were seed ed in a 24-well plate at a de nsity of 50,000 cells/well and allowed to attach overnight. Cells were then trea ted with nanoparticle su spension or equivalent dose of triamcinolone acetonide solution in complete growth medium. To de termine the effect of dose of nanoparticles on uptake, ce lls were treated with various doses (50,100, 200 g/ml) of

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54 triamcinolone nanoparticles for 2 hours. To determin e the effect of time of treatment, cells were treated with constant dose (100 g /ml) of triamcinolone nanoparticles for varying periods of time (0.5, 1, 2 and 4 hours). To study the effect of te mperature on cellular upta ke of nanoparticles, cells were preincubated at 4 C for 1 hour and then treated with the nanoparticle suspension (100 g/ml) at 4 C for 2 hours. At the end of the treatment period, the cell monolayer was washed three times with ice cold PBS. Cells were then lysed using 150 l of cell lysis reagent. The pr otein content of the cell lysate was determined using the Pierce protein assay reagents. The cell lysates were then analyzed for corticosteroid content. Cell lysa tes were mixed with 300 l of methanol and incubated at 37 C for 6 hours on a shaker. The sample s were centrifuged at 13,000 rpm for 10 minutes. Supernatants were then analyzed fo r corticosteroids using HPLC. The drugs were separated with a C-18 column. The mobile phase for the assay consisted of acetonitrile and acetate buffer mixture (50:50 vol/vol). The flow rate was 1 ml/min. A standard plot was constructed for corticosteroids in cell lysate r eagent under identical conditions that was used for the sample preparation. Data was expressed as corticosteroid accumulation normalized to total cell protein. The calibration curve was pl otted with standards from 0.5 to 20 g/ml. (r2 > 0.99). Statistical Analysis All statistical analyses, one way ANOVA and t-test were performed using GraphPad Prism 4 (GraphPad Software Inc., San Diego, CA) with p value less than 0.05 considered as significant. The effect of VEGF secretion and effect of cellular uptake on time and concentration were analyzed using ANOVA. T-test was used to test the energy dependence on cellular uptake and the MTT assay results.

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55 Results Vascular Endothelial Growth Factor Secretion Effect of triamcinolone acet onide on VEGF secretion was studied using ARPE-19 cells. The cells were initially treated with 1, 10 and100 M concentrations of triamcinolone acetonide and the VEGF secretion at th e end of 12 hours was measured. Figure 3-1 shows the measured concentrations of VEGF secre tion. Compared with no treatm ent (control), triamcinolone acetonide at 1, 10, 100 M reduces the VEGF secretion by 60%, 51% and 54 %, respectively. Cell Toxicity Cell toxicity of corticosteroids and their nanoparticles was studied to determine their safety. Results are expressed as units of absorban ce of MTT at 540 nm. The absorbance readings under different treatments are furt her converted to relative percen t cell viability for comparative analysis. The percent viability in the presen ce of triamcinolone acetonide was calculated by dividing the absorbance reading of cells under different concentra tions of corticosteroids by the absorbance reading of cells under normal growth (assumed 100% viability) in th e absence of drugs. Experiments showed that triamcinolone acet onide causes a significant reduction in cell numbers as long as the cells had been exposed fo r more than 24 hours. Further, at 24 hours time point triamcinolone showed toxicity above 10 M. Figure 3-2 shows the e ffect of triamcinolone concentration on cell viability after 24 hours. The results show that the cell viability decreased from 100% for control to 74, 86 and 89% w ith 1000, 100, 10 M of free triamcinolone acetonide, respectively.

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56 Figure 3-3 shows the effect of triamcinolone, triamcinolone nanoparticles and blank nanoparticles on cell viability at 24 hour point at various levels of drug concentration. It was apparent that the cytotoxic effects of triamc inolone was concentrati on-dependent. Decrease in toxicity was seen with triamcinolone na noparticles compared to the free drug. Cell Uptake Cellular uptake of triamcinolone acetonide nanoparticles as nanoparticles or free drug was studied in ARPE-19 cells. Figur e 3-4 shows the cell uptake of free triamcinolone acetonide micronized compared with its nanoparticles. The results show that nanopa rticle formulation of triamcinolone acetonide has similar uptake compared to free drug. Kinetics of cellular accumulation of corticosteroid nanoparticles is studied by the effect of concentration, time and temperature on cellular accumulation. Figure 3-5 shows the effect of dose on cell uptake. At 50, 100 and 200 g/ml do se, cellular accumulation of 149, 352 and 748 g/mg protein was observed. This indicates an increase in uptake with increase in dose. Figure 3-6 shows the effect of time on cell uptake when the drug concentration was maintained at 100 g/ml. At 0.5, 1, 2 and 4 hours, the nanoparticles accumulation levels of 90, 305, 352 and 612 g/mg was observed. This indi cates an increase in uptake with time. Finally the energy dependence of nanoparticles uptake in cells was studied by comparing the uptake of cells by incubating at 37 C and 4 C. Figure 3-4 shows the observed results. Decreasing active processes in ce lls by incubating cells at 4 C decreased the nanoparticles uptake approximately 5times. Figure 3-7 shows the effect of temperature on the cell uptake of nanoparticles.

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57 Discussion Mechanism of action for angiostatic and antiper meable property of corticosteroids is not completely known. One of the reasons for their angiostatic and antiperme able properties might be due to their abil ity to inhibit VEGF secretion. VEGF is responsible for causing neovascularization and edema. The study was conducted in the presen ce of drug alone to determine the VEGF inhibitory property of triamcinolone acet onide. Our results show that triamcinolone acetonide was able to inhibit VEGF s ecretion in ARPE-19. This is consistent with the finding that corticosteroids reduce VEGF in cultured aortic vascular smooth muscle cells [103] and also can reduce VEGF mRNA and prot ein expression in cultured eosinophils [46]. Counter effects of corticosteroid s on VEGF have also been demons trated in a recent rabbit study, in which intravitreal VEGF injections cause d a time and dose-depe ndent breakdown of bloodretina and blood-aqueous barriers and led to va scular leakage. The br eakdowns were blocked both by dexamethasone and triamcinolone acetonide but not the nonsteroidal anti-inflammatory drug (NSAID) indomethacin. Retinal pigment epithelium is separated from the vitreous only by the presence of neuroretina. Therapeutic concentr ation to the posterior region of the eye can be achieved through intravitreal delivery. The drug de livered to the vitreous are at high dose and can be toxic. The cell toxicity study serves as a tool to demonstrate the sa fety of corticosteroids and their nanoparticles on cells in the posterior region of the eye. Narayana et al. showed that triamcinolone acetonide caused a significant decreas e in cell viability at concentrations of 100 and 200 g/ml at all the three time points of 2, 6 and 24 hours. Similar results were also seen by Yeung et al. from 0.01 to 1 mg/ml concentra tion [46, 104]. Drug delivery systems such as nanoparticles help in attenuating adverse effect s by sustaining the drug release without huge fluctuation in the peak concentr ation. A decrease in cell toxicity with nanoparticles compared

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58 with the free drug was seen. Th e use of nanoparticles is an effective way to overcome the adverse affects associated with triamcinolone ace tonide which is currently used in clinics. Though the exact mechanism of toxicity is not known, it has been reported that the glucocorticoid retinal cells cytotoxicity in vitro is mediated through alte rations of mitochondrial activity. In our study a slow increase in triamcinolone cellular uptake was seen with increase in concentration and time of incubati on of the cells with the nanoparticles. The uptake of drug into the cells could be either due to the slow release of the drug from the nanoparticles into the media, which is then taken up by the cells or due to the direct uptake of the nanoparticles by the ARPE19 cells, which then release the drug in the cells. The cells were washed three times with PBS to remove any free drug in the cells. Uptake of the na noparticles into the cells was considered as the nanoparticles used in our study were below 200 nm and nanoparticles of size below 200 nm have been reported to accumulate with in various ce lls including ARPE-19 cells [105]. Indicating that nanoparticles are taken up and concentrate within the cells. Decrease in cellular uptake wa s also seen at lower temper ature which indicates energy dependence of the cellular uptake of the particles. A lthough to confirm this we have not shown the decrease in uptake in the presence of metabo lic inhibitors, Panyam et.al showed that uptake of PLGA nanoparticles in muscle cells depends on concentration, time and energy. They showed that the uptake was inhibited in th e presence of metabolic inhibitors such as sodium azide [105]. Conclusion Our studies show that triamcinolone acetonide inhibits VEGF secretion in retinal pigment epithelial cells. Cell culture studies in retinal pi gment epithelial cells showed that triamcinolone acetonide loaded nanoparticles were less toxic compared to the micronized triamcinolone

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59 acetonide alone. Nanoparticles were internalized efficiently by AR PE -19 cells and the uptake of nanoparticles was dose and time dependent. Triamc inolone acetonide nanoparticles formulated from poly(lactide-co-glycolide) polymer offer a nontoxic and effi cient delivery system for the sustained intracellular delivery.

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60 0 20 40 60 80 100 120 140 ControlTA 1 MTA 10 MTA 100 MVEGF Secretion (% Control) Figure 3-1. Triamcinolone acetonid e inhibited secretion of VEGF in ARPE-19 cells. N=8.Data are expressed as the mean SD. *Significantly di fferent from the control at P < 0.05.

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61 50 60 70 80 90 100 110 120 Control1 M10 M100 M1 mM Cell viability (% Control) ** ** Figure 3-2. Viability of ARPE19 cells after 24 hours with micronized triamcinolone acetonide. Treatments as determined by MTT assay. Data as mean SD (N=8). *Significantly different from the control at P < 0.05, **Significant P < 0.01.

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62 60 65 70 75 80 85 90 95 100 105 110 115ControlBlank NPTA 1 MTA 10 MTA 100 MTANP 1 M TANP10 M TANP 100 M Cell viability (% Control) *** Figure 3-3. Viability of ARPE19 cells after 24 hours with tr iamcinolone and triamcinolone nanoparticles. Treatments as determined by MTT assay. Data as mean SD (N=8). *Significantly different from the contro l at P < 0.05, **Significant P < 0.01.

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63 Figure 3-4. Cell uptake of micr onized triamcinolone compared with its nanoparticles at 100 g/ml at 37 C at the end of 2 hours. No statisti cally significant difference was seen between the two groups. 0 100 200 300 400 500 600 700 TA NP TA TA accumulation (g/mg protein)

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64 Figure 3-5. Effect of concentration on kinetics of nanoparticle uptake into ARPE-19 cells. Data as mean SD (N=4). 0 100 200 300 400 500 600 700 800 900 50 g/ml 100 g/ml 200 g/mlTA accumulation (g/mg protein)

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65 Figure 3-6. Effect of time on ki netics of nanoparticle uptake into ARPE-19 cells. Data as mean SD (N=4). 0 100 200 300 400 500 600 700 800 30 min 1 hr 2 h r 4 h r TA accumulation (g/mg protein)

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66 Figure 3-7. Effect of temperatur e on the kinetics of nanoparticle uptake into ARPE-19 cells. Data as mean SD (N=4). **Significantly different at P < 0.01. 0 50 100 150 200 250 300 350 400 450 37C 4C TA accumulation (g/mg protein) **

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67 CHAPTER 4 EFFECT OF INTRAVITREAL TRIAMC INOLONE ACETONIDE NANOPARTICLES ON LASER I NDUSED CHOROIDAL NEOVASCULARIZATION IN MICE Introduction Age-related macular degeneration (AMD) is th e leading cause of blindness above the age of 55 in developed countries. However, the pa thogenesis of age-related macular degeneration remains unclear; 4 to 10 million Americans are estim ated to have some form of the disease. Severe loss of central vision fr equently occurs with the exuda tive (wet) form of age related macular degeneration, as a result of the formatio n of a pathological choroi dal neovascularization. Choroidal neovascularization contains abnormal blood vessels that leak fluid and blood. The leakage damages the structure and function of the retina especially at the macular region leading to loss of vision. Choroidal neova scularization is also generated in ocular histoplasmosis, high myopia, or some inflammatory diseases, after laser photocoagulation, an d ocular trauma. The main treatments currently in use or under development for exudativ e age related macular degeneration include photodynamic therapy [106], submacular surg ery, macular translocation [107], and transplantation [108] However, most treated pa tients do not show visual improvement, and the surgical procedures can ca use serious complications. The severe visual disability caused by this disease and the lack of an adequate treat ment for the disease has lead to the search for new therapeutic strategies. The recent resurgence in interest in choroida l neovascularization has been driven by the discovery of molecular mechanisms involved in this process and by the recognition that treatments targeting neovascularization will be a central strategy for treating the wet form of agerelated macular degeneration. As the factors involved in the pathogenesis of choroidal neovascularization (CNV) are be tter understood, pharmacologic th erapeutic agents are being explored. Antiangiogenic agents may be helpful in treating choroidal ne ovascularization without

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68 destruction of the retina. Agents such as vascul ar endothelial growth fact or (VEGF) inhibitors, matrix metalloproteinase inhibito rs, and glucocorticoids are being considered to treat choroidal neovascularization as they can ta rget the disease causing factors thus preventing reoccurrence. It may also prevent reoccurrence following laser treatment and might also be used as a prophylaxis[109]. Though VEGF inhib itors were shown to be effec tive in treating CNV, they are expensive and the systemic risks ar e yet to be investigated [110]. Inflammation and infiltration of inflammatory cells is associated with angiogenesis in biological systems [111]. Hist opathological examination of excised CNV specimens shows increase in inflammatory cells. Also a relative increas e in vascular endothe lial growth factor which has been implicated in angiogenesis to the amount of inflammatory cell in excised specimens with CNV was shown [112]. Corticoste roids have antiangiog enic, antifibrotic, and antipermeable properties and have demonstrated to markedly inhib it corneal and retinal neovascularization rodents and primates [113]. Recently, nonrandomized and randomized case series have described intravitreal triamcinolone acetonide monotherapy for the trea tment of choroidal neovascularization [114, 115]. In the majority of these reports, a short-term effect of improved retinal th ickness, decreased neovascular exudation, and, in some cases, improved visual acuity was noted. However, over the long term, intravitreal corticosteroid injection monotherapy was larg ely not beneficial. This might be due the lack of a sustained effect. Holecamp and associates tried to address this problem by using the sustai ned-release nonbiodegradable flucinolone acetonide implant [116]. The surgical placement of the nonerodable implan t carries potential surgical risks, including endophthalmitis, retinal tears or detachment, and vitreous hemorrhage [117].

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69 Since choroidal neovascularization is one of the most afflictive problems facing ophthalmology, new solutions are needed. We hypot hesized that nanoparticles of triamcinolone acetonide which are easy to administer via intravitr eal injection are a suitab le way to sustain drug delivery to overcome the progressive nature of ch oroidal neovascularization. In this study, we show that formation of a laser induced choroida l neovascularization can be prevented in a mouse model, by intravitr eal injections of cortic osteroid nanoparticles. Materials and Methods Laser Induced Choroidal Neovascularization Mice were anesthetized with pentobarbital sodi um (40 mg/kg) and pupils were dilated with compound tropicamide/phenylephrine. The 532 nm diode laser photocoagulation was delivered to the choroid of mice with 120 mW power for 100 msec duration, with a mean diameter of 100 m, three burns to each eye. Treatment groups in the study included (n=5) blank nanoparticles, triamcinolone acetonide nanoparticles delivered through intravitreal r oute, triamcinolone acetonide nanoparticles delivered through intraperitoneal route and triamcinolone acetonide phosphate solution given by intravit real route (Table 4-1). The laser-aiming beam was focused on the Bruchs membrane. The aim was to ruptur e the Bruchs membrane so the choroidal blood vessel would invade the subretinal space forming choroidal neovasculariza tion. The sure sign of the Bruchs membrane rupture by laser was the fo rmation of bubbling at the site of the laser application with or without hemorrhage. An intr avitreal administration wa s applied 2 days after photocoagulation. Treatment Administration Intravitreal injection of tr iamcinolone nanoparticles (1 l) containing 800 g/ml drug was performed in the right eye. The mice were anesthe tized with an intraperiton eal (i.p.) injection of sodium pentobarbital (4 0 mg/kg). Different treatments were injected into the vitreous under a

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70 dissecting microscope. A 27-gauge needle was first used to make an incision 0.5 mm posterior to the temporal limbus and the Hamilton needle wa s inserted through the in cision, approximately 1.5 mm deep, and angled toward the optic nerve un til the tip of needle was seen in the center of the vitreous. Intraperitoneal injection treatment group a dose of approximately 160 g of drug was given. Preparation of Flat Mounts Two weeks after lase r treatment, the size of choroidal neovas cularization lesions were evaluated. Mice used for the flat-mount technique were anes thetized and perfused with 3 ml of phosphatebuffered saline containing 50 mg /ml of fluorescein-labeled dextra n. The eyes were removed and fixed for 2 hour in 10% phosphate-buffered forma lin. The cornea and lens were removed and the entire retina was carefully dissected from the eyecup. Radial cuts (4 to 7, average 5) were made from the edge of the eyecup to the equator a nd the eyecup was flat-mount ed in aquamount with the sclera facing down and the choroid facing up. Figure 4-1 shows the flat mounts of the choroid. Flat mounts were examined by fluorescence microscopy. All images of neovascularization sites were obt ained at 40X objective with a Carl Zeiss Axioplan2 imaging channel. The neovascularization wa s quantified by image J software. ImageJ is a public domain, Java-based im age processing program developed at the National Institutes of Health. ImageJ can disp lay, edit, analyze, process images. Image J calculates area and pixel value statistics of user-defined selections. Statistical Analysis One way ANOVA analysis followed by Dunnette post test was perfor med using GraphPad Prism 4 (GraphPad Software Inc., San Diego, CA ) with p value less than 0.05 considered as significant.

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71 Results Figure 4-3 shows the neovascular ization area measured for vari ous treatments. The control group showed mean choroidal ne ovascularization area of 6002 2693 and 3025 2085 m2 in the left and the right eye respec tively. All the treatments were compared with the control group left eye. No statistically significant difference was seen with intravitreal delivery of triamcinolone acetonide phosphate solution. Intravitreal injection of triamcinolone acetonide nanoparticles had developed only 10% (614 71 m2) neovascularization in the treated eye. The contralateral eye also showed reduced neovascularization of only 21% (1296 57 m2) compared to the control. A significantly reduced chor oidal neovascularization was also seen with intraperitoneal administration of triamcinolone acetonide nanopart icles showing a mean area of neovascularization of 580 303 and 943 601 m2 in the left and the right eyes respectively. Figure 4-2 shows laser induced c horoidal neovasculariza tion images of the various treatments. Discussion The specific stimuli for choroidal neovasculariz ation are not fully understood. It generally occurs in the presence of a pro-inflammatory and pro-angiogenic environment. It is characterized by elevated levels of angiogenic factors. The appearance of choroidal neovascularization can cause vision loss and the current treatments have limited effectiveness. Recently there is great interest in identifying clinically relevant inhi bitors of ocular neovasc ularization and in the development of effective treatments targeting choroidal neovascularization. As the pathology of choroidal neovascularizati on is better understood, therapeutic agents that can treat the cause of choroidal neovascular ization are being considered. Growth factors and cell adhesion molecules that have been implicated in neovascularization include: intercellular adhesion molecular (ICA M-1), basic and acidic fibroblast growth factor (aFGF and bFGF), [118]

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72 and vascular endothelial growth factor (VEGF) [119]. Previous studies have demonstrated that VEGF is an angiogenic factor [36] that is upregulated in and localized to the CNV lesion suggesting that VEGF plays an important role in choroidal neovascul arization formation. Previous experiments have show n that sustained intravitreal delivery of VEGF in animal models can cause widespread retinal vascular dilation and compounds with anti-VEGF properties effectively suppress CNV. Although the principal e ffects of corticosteroids are the stabilization of the blood-retinal barrier, resorption of exudation and downregulation of inflammatory stimuli, they are also potent inhibitors of neovascularization [120]. A number of studies have show n that corticosteroids inhibited preretinal and subr etinal neovascularization; however, its mechanisms of antiangiogenesis remain unknown. Se veral researchers have suggest ed that corticosteroids can act on the neovascular cascade by processes such as directly decrea sing levels of VEGF, inhibiting bFGF-induced migration and tube formation in choroi dal microvascular endothelial cells, inhibiting extracellular matrix turnover by downregulati ng metalloproteinase-2 (MMP-2) production, downregulating inte rcellular adhesion molecule -1 (ICAM-1) expression, and reducing major histocompatibility co mplexII antigen expression [38]. In this study, we evaluated the effect of corticosteroid nanoparti cles on laser induced choroidal neovascularization in mice model. Neovascularization was stimulated in mice using high energy laser. High energy lase r causes a rupture of the Bruc hs membrane; thereafter, under the influence of various angiogeni c factors, an ingrowth of c horoidal vessels under the retinal pigment epithelium and then in to the subretinal space develo ps. Although, in this model, pathogenesis of the neovascularization is differe nt from age-related macular degeneration, the formation of CNV is believed to follow the simila r pattern and identical angiogenic factors such

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73 as VEGF [121] and bFGF [122] are expressed by the retinal pi gment epithelium and endothelial cells. Our observations showed that the formation of vascular tubes in CNV was blocked by triamcinolone acetonide nanoparticles in vivo in mice (Figure 4-3). C onsidering the reduced size of CNV in the treated group compared with the c ontrol group, it is likely that the proliferation and/or migration of vascular endo thelial cells are inhibited in co rticosteroid treated eyes. Given that VEGF production was found to be suppressed by triamcinolone acetonide treatment in our cell culture studies and by others in vivo [123], it is likely that angi ogenesis inhibitory action of triamcinolone is due to decrea se of VEGF directly or indi rectly. The effect of Kenalog (triamcinolone acetonide injectable suspension) which is curren tly used by ophthalmologists was studied by others in our group and only showed 15% neovascul arization compared to the control group (unpublished). This indicates that triamcinolone acetonide re tained its therapeutic effect when formulated as nanoparticles A significant decrease was seen in both the treated eye and the contralateral eye but the choroidal neovascularization is co mparatively less in the treated eye suggesting some targeted delivery of nanoparticles was achieved. Also choroidal neovascularization was d ecreased with intraperitoneal route of administration indicating the effect on the untreated eye might be due to the systemic entry of the drug from the treated eye. The targeted deliver y may be further improved by further decreasing the rate of drug release and determining the therapeutic concentration required to treat neovascularization. So far there is no info rmation regarding the therapeutic range of concentration of intraocular triamci nolone for antiproliferative effect.

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74 Our studies showed reduced choroidal neova scularization with triamcinolone acetonide nanoparticle formulation and with Kenalog, bot h of which release the drug slowly but no significant decrease was seen with triamcinolone acetonide phosphate solution. This implies that sustained release is essential for inhi bition of choroidal neovascularization. In this study, we demonstrated that intrav itreal triamcinolone acetonide poly(lactide-coglycolide) nanoparticles can inhibit the development of experimental choroidal neovascularization. It further supports previous animal studies that have indicated that corticosteroids can inhibit choroida l neovascularization formation [124]. Conclusions In conclusion, our results indicate a notable inhibitory effect of triamcinolone loaded nanoparticles on laser-induced c horoidal neovascularization in the mice model. However, the efficacy of this drug for the treatment of human CNV requires further investigation. We believe that triamcinolone acetonide loaded polymer na noparticles could be promising to inhibit human neovascularization. The use of corticosteroid nan oparticles will help to deliver effective dose to the posterior parts of the eye and prevent repeated intravitreal injection as they can sustain the drug release and can also ensure be tter visibility resulting in patient compliance. It should also be noted that this study, by its design, demonstrated inhibition or prev ention of choroidal neovascularization, but not regression of pre-ex isting neovascularization, which would be more relevant to humans.

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75 Table 4-1. Treatment groups Treatment Route Triamcinolone acetonide nanoparticles Intravitreal Triamcinolone acetonide nanoparticles Intraperitoneal Triamcinolone acetonide solution Intravitreal Blank nanoparticles Intravitreal

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76 Figure 4-1. Choroidal flat mount s of the left and right eye

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77 A B C D Figure 4-2. Representative images of laser i nduced choroidal neovascul arization (A) control group (B) Intravitreal injection triamcinolone solution (C) Intraperitoneal injection of triamcinolone nanoparticles (D) Intravitreal administ ration of triamcinolone nanoparticles

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78 Figure 4-3. Neovascularizati on area of four treatment gr oups. P< 0.05 was considered statistically significant *P < 0.05, **P < 0.01

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79 CHAPTER 5 LOTEPREDNOL ETABONATE NANOPARTICLES AND THEIR EFFECT ON LASER I NDUCED CHOROIDAL NEOVASCULARIZATION IN MICE Introduction Corticosteroids are the mainstay of therapy for inflammatory conditions. Traditionally used corticosteroids are associated w ith two clinically important ocul ar side effects: cataract and increased intraocular pressure [43]. Hence, there is a need for therapeutic agent with efficacy comparable to that of currently available cortic osteroids, but without th e adverse effects. The continuing development of ophthalmic steroids has resulted in compounds that have a low tendency to raise intraocular pressure (IOP). Loteprednol etabonate is a co rticosteroid which is the pr oduct of soft drug design (a compound that undergoes predictable metabolism to inactive metabolites) [125]. It has been developed as a topical treatment for ocular inflammation. Lotepr ednol etabonate is designed to be rapidly converted to inac tive and nontoxic metabolites, thus minimizing systemic adverse effects. Loteprednol has been approved by the Food and Drug Administration (FDA) as the active ingredient of three ophthalmic preparations (Lotemax, Alrex, Zylet) [125-127]. At present, it is the only corticosteroid a pproved by the FDA for use in all in flammatory and allergy-related ophthalmic disorders. The effect of loteprednol etabonate on neovasc ularization has not been studied. In this study, we investigate the effect of loteprednol on choroidal neova scularization in the posterior region of the eye. Loteprednol is very lipophilic in nature and if given in the present form will be cleared from the vitreous fast and may not show any therapeutic effect. Nanoparticle formulation of the drug will sustain the drug release and hence can be used to treat conditions in the posterior region of the eye without the adverse e ffects associated with other steroids. The goals of this study are:

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80 determine the effect of intrav itreal delivered loteprednol etab onate on intraocular pressure in rabbits determine the effect of loteprednol on VEGF secretion in ARPE-19 cells prepare and characterize lotepr ednol etabonate nanoparticles determine the effect of loteprednol and its nanoparticles on ARPE-19 cell viability finally to investigate th e effect of intravitreal delivered loteprednol nanoparticles on laser induced choroidal neovascularization Materials and Methods Intraocular Pressure Measurements in Rabbits Initially 2-3 drops of topical anesthetic was applied to the eye and the initial intraocular pressure (IOP) was measured with a pneumatonome ter, this will represent the preinjection IOP. Later the animals were anestheti zed with intramuscular injection of 35 mg/Kg of Ketamine and 5 mg/Kg of Xylazine. Steroid (100 l) was injected using tuberc ulin syringe with a 27-gauge needle into the vitreous cavity of the right eye. The needle was pointed toward the optic nerve to ensure that the lens was not in advertently nicked. As a control, the vehicle-only solution was injected in the other eyes at the same time. A cotton swab was placed immediately as the needle was withdrawn, to insure no reflux of steroid occurred. The eye was vigorously massaged to reduce the intraocular pressure because of increa sed volume resulting from the injection. This was done to reduce the IOP to a level within a few points of pr e-injection The IOP was then measured on 1, 5, 10, 15, 20 and 30 days. Preparation and Characterization of Loteprednol Etabonate Nanoparticles Materials and methods are simila r to that described in chap ter 2 for characterization of nanoparticles based on size, encapsulation efficiency and in vitro release. Briefly, the nanoparticles were prepared by solvent evaporat ion method and the prepar ed nanoparticles were characterized for size, shape, encapsulation and in vitro drug release. In vitro release studies

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81 were performed with 500 g of the drug in the dialysis bag, sink condition was maintained with 1% sodium dodecyl sulfate. The drug content in encapsulation studies was measured using HPLC. Acetonitrile and water (50:50 ) mobile phase was used with flow rate of 1 ml/min. A C-18 column was used and UV detection was accomp lished at 254 nm. Stock solution of 100 g/ml was prepared in acetonitrile. To determine the encapsulation efficiency the calibration curve was obtained from standards 1 to 50 g/ml (r2 > 0.99). In vitro drug release studies, acetonitrile: water: acetic acid (50:49.5:0.5 vol/vol) was used as the mobile phase. Calibration curv e was obtained from standards 1 to 30 g/ml in the release media (r2 > 0.99). Effect of Loteprednol Etabonate on VEGF Secretion in ARPE-19 Cells Materials and method used are similar to that described in chapter 3 for effect on VEGF in ARPE-19. Briefly, ARPE-19 cells were seeded onto a 24 well plate and allowed to grow to confluence. The monolayers are in cubated with loteprednol 10 M. The culture media is collected at the end of 12 hours. The secreted VEGF in supernatants is quantified by ELISA method capable of detecting VEGF 165 and the VE GF secretion was normalized to total protein assayed using BCA kit after lysing the cells. Cell Toxicity of Loteprednol Etabonate and its Nanoparticles on ARPE-19 Cells Materials and method used are similar to that described in chapter 3 for cell toxicity study ARPE-19 cells were seeded at a density of 10,000 cells/well and were allowed to attach overnight in 96-well plates. The cells were then exposed to loteprednol or its nanoparticles (1, 10 and 100 M). At the end of 24 hour incubation, th e cell viability was determined by MTT assay. The cell viability is then determ ined by the relative formazan formation after corticosteroid and the nanoparticles treatments compared to the control group.

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82 Effect of Loteprednol Etabonate Nanoparticles on Laser Induced Choroidal Neovascularization in mice Materials and methods are si milar to that described in chapter 4. The mice were anaesthetized and the pupils were dilated. The mice were lasered with 532-nm diode laser photocoagulation with 300 mW power for 200 ms ec duration, with a mean diameter of 50 m, with three lasers to each eye. Treatment groups included (n=5 ) blank nanoparticles, loteprednol nanoparticles and triamcinolone na noparticles delivered through intravitreal route in the right eye. After 2 weeks the mice were sacrificed and flat mounts we re prepared and the area of choroidal neovascularization was determined usi ng image J. Image J is a public domain, Javabased image processing program developed at the national institutes of health. Statistical Analysis All statistical analyses, T-test and ANOVA were performed using GraphPad Prism 4 (GraphPad Software Inc., San Diego, CA) with p value less than 0.05 considered as significant. Results Intraocular Pressure Measurements in Rabbits Mean intraocular pressu re (IOP) data of triamcinolone ace tonide and loteprednol etabonate (not nanoparticles) are presented in figure 5-1. Treatment-relate d differences in IOP were not apparent in our study. No other statistically significant differen ces were seen during the study. Mean IOP did not appear to be significantl y affected by intravitreal administration of triamcinolone or loteprednol. Preparation and Characterization of Loteprednol Nanoparticles The nanoparticles prepared were spherical and had a smooth surface. The size of the particles was 196 32 nm with good encapsulation efficiency of 72.56 1.75% (table 5-1). In vitro release studies showed sustai ned drug release with the nanopa rticles releasing the drug at

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83 slower rate compared to lotepr ednol etabonate alone (figure 5-2 ). Extrapolated results showed 100% drug release occurs in 25 days (figure 5-3). Effect of Loteprednol on VEGF Secretion in ARPE-19 Cells Figure 5-4 shows the effect of loteprednol on VEGF secretion. Loteprednol showed statistically significant decrea se of VEGF secretion at 10 M concentration. Compared to no treatment, the VEGF secretion was reduced by 52%. Cell Toxicity of Loteprednol and its Nanoparticles on ARPE-19 Cells The effect of loteprednol etabonate and its nanoparticles was studied on cell viability of ARPE-19 cells. Figure 5-5 shows that lotepre dnol and its nanoparticles did not show any significant decrease in ce ll viability across the enti re range from 1 to 100 M at the end of 24 hours. Effect of Loteprednol Nanoparticles on Las er Induced Choroidal Neovascularization in Mice Loteprednol nanoparticles were able to inhib it choroidal neovascularization. Compared to control group that showed a mean area of 2615 1954 m2 and 2151 1228 m2 in the left and right eye respectively, loteprednol nanoparticle s showed only 39% (1006 833 m2) and 28% (731 466 m2) choroidal neovascularization in the le ft and the right eye, respectively. Triamcinolone nanoparticles showed choroidal neovascularization of 379 217 and 180 140 m2 choroidal neovascularization compared to the control. Discussion Corticosteroids like triamcinolone acetonide can treat neovascularizati on but are associated with adverse effects. These adverse affects include elevati ons in intraocular pressure and the formation of, or acceleration of the development of, cataracts [42]. Elev ations in intraocular pressure are of particular con cern in patients who are already su ffering from elevated intraocular

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84 pressure, such as glaucoma patient s. Moreover, a risk exists that the use of corticosteroids in patients with normal intraocular pressure will cause elevation in pressure that result in damage to ocular tissue. Since therapy with corticosteroids is frequently long term, i.e., several days or more, a potential exists for signifi cant damage to ocular tissue as a result of prolonged elevations in intraocular pressure attributable to that therapy. Increased IOP is encountered with about 30% of patients using steroids [128], and is reported to develop about 2 months after administration of triamcinolone [129]. Cataract is reported in about up to 38% of patients receiv ing systemic corticos teroids [129], usually manifesting after 2 months to 1 year of expos ure. The absence of significant effects of intravitreal injection of triamcinolone on intraocu lar pressure in rabbits seen in our study might be due to the delayed onse t of intraocular pressure. Loteprednol etabonate has a lower propensity to induce elevation in intraocu lar pressure even when used in known steroid responders [130]. The pathophysiology behind steroids and raised IOP is at th e level of the trabecular meshwork whereby mucopolysaccharides from free-floating steroids bind to the ultrastructure of the trabecular meshwork and reduce the pore size and thus, reduce aqueous outflow. Steroids that typically increase intraocular pre ssure are the ones that have a ketone group in position 20 of the steroid skeleton. The ketone on th e C 20 of steroids is also belie ved to be responsible for the formation of Schiff bases between the steroid C 20 with nucleophilic grou ps such as -amino groups of lysine residues of proteins [131] resulting in cataract. Loteprednol etabonate appears to have an im proved safety profile compared to ketone corticosteroids, and may be more suitable than ketone corticosteroids for the treatment of conditions in which long-term therapy is nece ssary [132]. It is not completely known why loteprednol causes less intraocular [133] pressure increase than other co rticosteroids. One of

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85 reasons is that the predictable intraocular conversion of the drug to an inactive compound may reduce the amount of active corticoste roid in the trabecular meshwork. Nanoparticle formulation of loteprednol helps in sustaining the drug release and prevents the rapid elimination of the drug. The nanopa rticles had slower re lease compared to triamcinolone nanoparticles prepar ed with similar parameters. The difference in the release period of these two drugs might be due to the lip id solubility of the drugs. Lipophilic drugs can distribute more homogeneously in the matrix of polymer poly(lactide-co-glycolide), and can be released for a longer time. Our studies also showed that lo teprednol has anti-angiogenic eff ect as it was able to inhibit secretion of VEGF, a growth f actor that stimulated new blood vessel growth and hence can be used to treat neovascular conditions. Lotepr ednol nanoparticles reduced the occurrence of choroidal neovascularization in mice model. But the decrease was less compared with triamcinolone acetonide. The rate of drug release from the loteprednol nanoparticles might need to be optimized for higher release rate to show a better therapeutic effect. The decreased therapeutic effect might also be due to loteprednol being metabo lized within the nanoparticles by the various esterases in the eye. Conclusions Loteprednol etabonate inhibited VEGF secret ion responsible for neovascularization in human retinal pigment epithelia l cells. Nanoparticles formulat ed had better encapsulation and slower release than triamcinol one acetonide nanoparticles. Loteprednol or its nanoparticles did not cause toxicity in retinal pigment epithelia l cells and hence might be safer compared to triamcinolone acetonide for chronic use. Lotepr ednol nanoparticles were able to reduce laser induced choroidal neovascularization in mice. Although the formulation needs to be further

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86 optimized loteprednol nanoparticles might be a good option to treat neovascular conditions in the posterior eye.

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87 0 5 10 15 20 25 05101520253035 Time (Days)IOP (mm Hg) TA RIGHT TA LEFT LE LEFT LE RIGHT Figure 5-1. Intraocular pressure measurements (IOP) for a period of 30 days in rabbits after intravitreal injection of triamcinolone (T A) and loteprednol (LE). No statistically significant difference was seen.

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88 Table 5-1. Size and encapsulation effi ciency of lotepr ednol nanoparticles Formulation PVA Drug:Polymer Size (nm) Drug Loading (%wt/wt) EE% LE NP 1% 1:10 196 32 10.09 2.2 72.56 1.75 Table 5-2. Zero order and Higuchi equations of in vitro drug release showing the effect of polymer Formulation Zero Order Higuchi Equation LE R2 = 0.9709 Y= 0.3291X+ 1.2076 R2 = 0.9722 Y= 5.9567X+26.263 LE NP R2 = 0.9826 Y= 0.2148X +4.9107. R2 = 0.9922 Y= 4.2963X + 24.769

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89 0 10 20 30 40 50 60 70 0 50 100 150 200 Time (hrs) % Cumulative LE release LE LE NP Figure 5-2. In vitro release data of lotepre dnol etabonate and its nanop articles. All the studies were performed at 37C. Data are expressed as the mean SD of results in two experiments

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90 0 20 40 60 80 100 120 0200400600800 Time (hrs)% Cumulative LE released LENP Figure 5-3. In vitro release studies of loteprednol nanoparticles extrap olated to 100% cumulative drug release

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91 0 20 40 60 80 100 120 140 ControlLoteprednolVEGF Secretion (% Control)* Figure 5-4. Loteprednol eta bonate inhibited secretion of VEGF in ARPE-19 cells at concentration 10 M (N=8).Data are expressed as the mean SD. Significantly different from the control at P < 0.05

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92 60 70 80 90 100 110 120 130ControlLE 100 MLE 10 M LE 1 MLENP 100 M LENP 10 M LENP 1 M Cell viability (% control) Figure 5-5. Effect of loteprednol (LE) and loteprednol nanopa rticles (LE NP) on viability of ARPE-19 Cells after 24 hours. Treatm ents as determined by MTT assay. Data as mean SD (N=8). Significantly different from the control at P < 0.05.

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93 A B Figure 5-6. Representative images of laser i nduced choroidal neovascul arization (A) control group (B) loteprednol nanoparticles treated groups

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94 Figure 5-7. Neovascularizati on area of blank nanoparticles, loteprednol nanoparticles, triamcinolone nanoparticles in the left (untreated) and righ t (treated) eyes. Data as mean SD (N=5). Significantly different from the control at P < 0.05. *P < 0.05, **P < 0.01. 0 1000 2000 3000 4000 5000 LEFT RIGHT CONTROL LEFT RIGHT TRIAMCINOLONE LEFT RIGHT LOTEPREDNOL ** ** ** *Area m 2

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95 CHAPTER 6 CONCLUSIONS AND FUTURE WORK In this dissertation we addressed the use of nanoparticles of corticosteroids to treat conditions occurring in the posterior region of the eye, which are a m ajor cause of blindness among the elderly. Drug delivery to treat diseases like age-re lated macular degeneration and diabetic retinopathy is challenging. It is difficult to attain therap eutic concentration to treat them due to long diffusional distance and presence of ocular barriers. In travitreal delivery offers the most direct approach to deliver nanoparticles to the target area. Corticosteroid nanoparticles below 500 nm were successfully prepared using emulsion solvent evaporation technique. The nanoparticles of triamci nolone and loteprednol had good encapsulation efficiency and had longer drug re lease compared to the micronized drugs alone. Nanoparticles prepared were able to sustain the drug release as was shown in chapter 2 and hence can prevent repeated injections. The use of nanoparticle drug delivery syst em for triamcinolone acetonide showed a decrease in toxicity in ARPE-19 cells compared to the drug itself. Neither loteprednol nor its nanoparticles caused cell toxicity and hence are better suited for prolonged therapy. The cellular uptake studies showed that the nanoparticle uptake was time and dose dependent. Both triamcinolone acetonide and loteprednol were able to inhibit VEGF secretion responsible for neovascularization. Triamcinolone acetonide maintained its therapeu tic effect in the nanoparticle formulation inhibiting laser induced choroida l neovascularization in mice. Although some targeted delivery was achieved in the treated eye the formulati on has to be further optimized based on the therapeutic concentration required. Determining the systemic concentration of the drug and the

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96 understanding the transport of the drug to the contralateral eye will help to achieve better targeted delivery. In our study loteprednol nanopart icles showed less effect on choroidal neovascularization compared to triamcinolone nanoparticles. To show that the decreased effect is due slower release pharmacokinetic studies need to be conducted to determine the concentration of the drug in various tissues and the efficacy has to be shown over long term with no toxicity. In this study we showed that nontoxic corticosteroid nanopartic les pretreatment was able to prevent laser induced chor oidal neovascularization. The use of corticosteroid nanoparticles were able to deliver effective dose to the posterio r region of the eye and help prevent repeated intravitreal injection. Future st udies need to address the effect of corticosteroid nanoparticles on existing neovascular conditions.

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BIOGRAPHICAL SKETCH Keerti Mudunuri was born in Bhimavaram Andhra Pradesh, Indi a. She obtained her Bachelor of Pharmacy degree from the Vishnu College of Pharmacy, Andhra University, in 2003. She was admitted to the graduate program at the Department of Ph armaceutics, University of Florida in January 2004. She completed her Doctor of Philosophy in May 2008 under the supervision of Dr. Guenther Hochhaus.