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Synthesis and characterization of potential drug delivery systems using nonionic surfactant "niosome"

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Title:
Synthesis and characterization of potential drug delivery systems using nonionic surfactant "niosome"
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English
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Leekumjorn, Sukit
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University of South Florida
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Subjects / Keywords:
fluorometer
gel exclusion chromatography
5(6)-carboxyfluorescein
encapsulation
sorbitan monoesters
Dissertations, Academic -- Chemical Engineering -- Masters -- USF   ( lcsh )
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government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Summary:
ABSTRACT: Niosomes are synthetic microscopic vesicles consisting of an aqueous core enclosed in a bilayer consisting of cholesterol and one or more nonionic surfactants. They are made of biocompatible, biodegradable, non-toxic, non-immunogenic and non-carcinogenic agents which form closed spherical structures (self assembly vesicles) upon hydration. With high resistance to hydrolytic degradation, niosomes are capable of entrapping many kinds of soluble drugs while exhibiting greater vesicle stability and longer shelf life. In this work, a potential drug delivery system has been designed, synthesized and characterized. For the synthesis of niosomes, a hydration process was developed with varying design parameters such as mass per batch, angle of evaporation, rotation speed of vacuum rotary evaporator and nitrogen flowrate to produce uniform thin film in 50 ml round bottom flask. The rehydration process was developed by varying the choice of solvents (H2O, phosphate buffer solution (PBS) and PBS/5(6)-carboxyfluorescein (CF) as a drug model) and hydrating temperature of below and above gel transition temperature. Lastly, a sonication process to produce unilamellar vesicles was partially optimized based on the particle distribution and the number of vesicles formed with sonication time. As a result of this process, unilamellar and multilamellar vesicles were formed with the combination of different nonionic surfactants (sorbitan monostearate-Span 60, sorbitan monopalmitate-Span40 and sorbitan monolaurate-Span20), cholesterol and an electrostatic stabilizer (dicetyl phosphate). The vesicles were examined using light scattering optical microscopy and UV microscopy. Optical sensing technology (Particle Sizing System) is used to determine the vesicles' size distribution. Gel exclusion chromatography (GEC) is discussed as a method to separate unencapsulated CF while retaining vesicle integrity. Particle Sizing System and luminescence spectrophotometer were used to determine CF encapsulation percentage and leakage. Result: Span 20, Span 40 and Span 60/Niosomes were made with mean particle size of 0.95-0.99 micro (mu)m. Typical concentrations of vesicle per ml/per mass of surfactant used were in the range of 1.46-1.79x10 8 . Typical encapsulation efficiencies were in the range of 48.8-62.9% for all three Span/Niosome systems. Niosomes were found to be stable for 9 days. The largest vesicles were observed with Span 60 with highest entrapment efficiency as compared to Span 20 and Span 40.
Thesis:
Thesis (M.S.Ch.E.)--University of South Florida, 2004.
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Includes bibliographical references.
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by Sukit Leekumjorn.
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Synthesis and Characterization of Poten tial Drug Delivery Systems using Nonionic Surfactant Niosome by Sukit Leekumjorn A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Chemical Engineering Department of Chemical Engineering College of Engineering University of South Florida Major Professor: Michael D. VanAuker, Ph.D. Venkat R. Bhethanabotla, Ph.D. Aydin K. Sunol, Ph.D. Date of Approval: March 24, 2004 Keywords: sorbitan monoesters, encapsulat ion, 5(6)-carboxyfluorescein, gel exclusion chromatography, fluorometer Copyright 2004, Sukit Leekumjorn

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DEDICATION To my family and my friends

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ACKNOWLEDGEMENTS I greatly acknowledge the guidance, advice and technica l support provided by Dr. Michael D. VanAuker who has b een my major professor during my stay at the University of South Florida. It was my sincerest honor to work under the mentorship of Dr. Aydin K. Sunol whose insights provided me with i nvaluable expertise and learning experiences. I also thank Dr. Venkat R. Bhethanabotla fo r providing technical support and guidance. Sincere thanks are due to Dr. Mark J. Jaro szeski and Dr. Bikash R. Pattnaik for their advice on fluorescein measurement, gel exclusion chromatography, and the synthesis technique, respectively. I thank Dr. Carl J. Biver for providing necessary equipment in the experimental setup. Also, I would lik e to acknowledge the c ontribution from my colleagues, Elizabeth Hood (for her contri bution in particle characterization and separation analysis); Ling Miao (for her work with 5(6) Carboxyfluorescein analysis); Sergio Guitierrez (for his advice on experi mental setup); Wilfredo Coln-Santiago and Naveed Aslam (for their assistance in th esis preparation). Special thanks to undergraduate research assi stance, Monica Gonzalez for her contribution in the experiment.

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i TABLE OF CONTENTS LIST OF TABLES .............................................................................................................iv LIST OF FIGURES ...........................................................................................................vi ABSTRACT .......................................................................................................................ix CHAPTER ONE: INTRODUCTION .................................................................................1 CHAPTER TWO: LITERATURE REVIEW .....................................................................3 2.1 Liposomes ............................................................................................................3 2.1.1 Phase Transition Temperature of Lipids .........................................................5 2.1.2 Stability/Surface Modification ........................................................................7 2.1.3 Cholesterol ......................................................................................................9 2.1.4 Liposome Preparation & Mechanisms Associated with Drug Encapsulation ..................................................................................................9 2.2 Niosomes ............................................................................................................10 2.2.1 General Characteristics of Nonionic Surfactant ...........................................11 2.3 Summary ............................................................................................................13 CHAPTER THREE: DESIGN CONCEPT ......................................................................14 3.1 Material and Equipment Selection .....................................................................14 3.2 Encapsulation Technique ...................................................................................17 3.3 Separation Technique .........................................................................................19 3.4 Particle Characterization ....................................................................................21 CHAPTER FOUR: PROCEDURES AND EXPERIMENT PROTOCOLS ....................23 4.1 Procedures ..........................................................................................................23 4.1.1 Buffer Solution Preparation ..........................................................................23 4.1.2 Niosome Stock Solution ...............................................................................23 4.1.3 5(6)-Carboxyfluorescein (5 mM) in PBS Stock Solution .............................24 4.1.4 Gel Chromatography Column Preparation ...................................................25 4.1.5 Dehydration of Stock Niosome .....................................................................25 4.1.6 Rehydration of Thin Film .............................................................................26 4.1.7 Gel Exclusion Chromatography Separation ..................................................26 4.1.8 UV Microscope .............................................................................................27 4.1.9 Encapsulated Drug Measurement .................................................................27 4.1.10 5(6)-Carboxyfluorescein Standardize Curve ................................................28 4.1.11 Particle Sizing System 780 (PSS 780) ..........................................................28

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ii 4.2 Experimental Protocols ......................................................................................29 4.2.1 Determination of Mass per Batch .................................................................29 4.2.2 Determination of Angle of Eva poration and Speed of Rotation ...................30 4.2.3 Determination of Nitrogen Flow Rate during the Dehydration Process .......31 4.2.4 Effect of Different Hydrating Solvents .........................................................31 4.2.5 Effect of Different Hydrating Temperatures .................................................31 4.2.6 Effect of Particle Formation during Sonication ............................................32 4.3 Quantitative Measurements used in Niosome Characterization ........................33 4.3.1 Effect of Niosome Vesicles in PBS-CF Solution over Time ........................33 4.3.2 Separation Pattern Using Gel Exclusion Chromatography ...........................34 4.3.3 Leakage Studies and Encapsulation Measurement .......................................34 CHAPTER FIVE: RESULTS AND DISCUSSION .........................................................37 5.1 Particle Formation ..............................................................................................37 5.1.1 Determination of Mass per Batch .................................................................38 5.1.2 Determination of Angle of Eva poration and Speed of Rotation ...................39 5.1.3 Determination of Nitrogen Flow Rate during the Dehydration Process .......40 5.1.4 Effect of Different Hydrating Solvents .........................................................41 5.1.5 Effect of Different Hydrating Temperatures .................................................48 5.1.6 Effect of Particle Formation during Sonication ............................................54 5.2 5(6)-Carboxyfluorescein ....................................................................................61 5.2.1 5(6)-Carboxyfluoresce in Standard Curve .....................................................61 5.3 Quantitative Measurements used in Niosome Characterization ........................65 5.3.1 Effect of Niosome Vesicles in PBS-CF Solution over Time ........................66 5.3.2 Separation Pattern Using Gel Exclusion Chromatography ...........................67 5.3.3 UV Microscopic Observation .......................................................................76 5.3.4 Leakage Studies ............................................................................................76 CHAPTER SIX: CONCLUSIONS ...................................................................................81 6.1 Future Work: Using Niosomes in Cardiovascular Therapy ...............................82 6.1.1 Statins ............................................................................................................83 6.1.2 ACE-Inhibition/Angiotensin II Receptor Blockade ......................................84 6.1.3 PPARAgonist ...........................................................................................84 6.2 Future Work: Background on Targ eting Atherosclerotic Plaque ......................85 6.2.1 Mechanisms of leukocyte Adhesion (Clu es for Targeting Atherosclerotic Plaque) ..........................................................................................................85 6.3 Summary ............................................................................................................88 REFERENCES ..................................................................................................................89 APPENDICES.................................................................................................................100 Appendix A Comparison between niosomes produced at 40C and 60C with the increase in particles size distribution that are greater ~0.58 m...............................................101

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iii Appendix B Comparison between niosomes produced at Day 0, Day 1 and Day 14 with the increase in particles size distri bution that is greater ~2 m....................................104 Appendix C Sample calculation of PSS ASCII-file from PSS: total volume calculation with Span60/Niosomes after gel exclusion column...........................................................106

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iv LIST OF TABLES Table 1: The effect of transition te mperature vs. acyl composition (hydrocarbon chain length) .......................................................................................................5 Table 2: Chemical speci fications of Sp20, Sp40, S p60, cholesterol, dicetyl phosphate and CF .............................................................................................12 Table 3: Phase transition te mperature of Sp20, Sp40, and Sp60 ...................................12 Table 4: List of materi al used in the experiment ............................................................14 Table 5: List of equipm ent used in the experiment ........................................................15 Table 6: Gel exclusion packing materials and their specifications ................................19 Table 7: Recommended volume of sample per column volume ....................................20 Table 8: The range of values of each vari able tested for manipulated parameters in making niosomes .............................................................................................29 Table 9: Molar ratio of Span/Niosome stock solution (weighted mass in Table 10/MW in Table 2) ...........................................................................................38 Table 10: Stock solution of Span/Niosome prepared for dehydration process ................38 Table 11: Observation of the thin film from different injected volumes of stock solutions ...........................................................................................................39 Table 12: Observation of the thin film from different angles and rotation speeds ..........40 Table 13: The effect of nitrogen fl ow rate on the dehydrated thin film ...........................41 Table 14: 3 PSS runs of S p60/Niosomes hydrating in H 2 0@ 40C .................................42 Table 15: 3 PSS runs of Sp60/Ni osomes hydrating in PBS@ 40C ................................44 Table 16: 3 PSS runs of Sp60/Ni osomes hydrating in PBS-CF@ 40C ..........................46 Table 17: 3 PSS runs of Sp60/Ni osomes hydrating in PBS-CF@ 60C ..........................48 Table 18: 3 PSS runs of Sp40/Ni osomes hydrating in PBS-CF@ 60C ..........................52

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v Table 19: 3 PSS runs of Sp20/Ni osomes hydrating in PBS-CF@ 60C ..........................52 Table 20: Sp60/Niosomes particle count s and size distribution from 0-4 minutes sonication time .................................................................................................54 Table 21: Sp20/Niosomes particle count s and size distribution from 0-4 minutes sonication time .................................................................................................56 Table 22: Sp40/Niosomes particle count s and size distribution from 0-4 minutes sonication time .................................................................................................57 Table 23: CF intensities with respect to known concentrations .......................................62 Table 24: Comparison of Sp60/ Niosomes particle counts a nd mean size of particles 0-14 days ..........................................................................................................66 Table 25: GEC separation of unencapsu lated CF from 0.25 ml injection of Sp60/Niosomes ................................................................................................68 Table 26: GEC separation of unencapsu lated CF from 1.00 ml injection of Sp60/Niosomes ................................................................................................69 Table 27: GEC separation of unencapsulated CF from 0.10 ml injection of Sp60/Niosomes................................................................................................69 Table 28: Particle size distribution of Sp60/Niosomes sample collected after GEC.......71 Table 29: Comparison of counts per ml and background intensity vs. cumulative volume of sample .............................................................................................73 Table 30: 2 PSS runs of Sp20/Niosomes after GEC (5-10 ml cumulative volume and 0.5 ml injection) ...............................................................................................74 Table 31: 2 PSS runs of Sp40/Niosomes after GEC (5-10 ml cumulative volume and 0.5 ml injection) ...............................................................................................74 Table 32: 2 PSS runs of Sp60/Niosomes after GEC (5-10 ml cumulative volume and 0.5 ml injection) ...............................................................................................74 Table 33: Leakage study of Sp20, Sp40 a nd Sp60/Niosomes from day 0 to day 9 .........78 Table 34: Summary of Sp20, Sp40 and S p60/Niosomes CF entrapment percentage ......80 Table 35: Summary of optimized para meters in making Span/niosome system .............81

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LIST OF FIGURES Figure 1: Chemical structure of Phosphatidyl-choline (PC) ................................................3 Figure 2: Possible formation of liposome vesicles when hydrating in aqueous solution ....4 Figure 3: PC immerse in water to form bilayer. Elongation and several vesicles were observed .............................................................................................................4 Figure 4: The effect of transition temperature vs. acyl composition ...................................6 Figure 5: Modified liposomes using phosphatidylethanolamine substitution .....................8 Figure 6: Preparation of liposome using dehydration/rehydration technique ....................10 Figure 7: Critical packing parameter of surfactants ...........................................................11 Figure 8: Bilayer membrane structure ...............................................................................11 Figure 9: Chemical structure of Sp60 (A), Sp40 (B) and Sp20 (C) ...................................16 Figure 10: Chemical structure of dicetyl phosphate ..........................................................16 Figure 11: Chemical structure of cholesterol .....................................................................17 Figure 12: Proposed structure of niosome vesicle (a) in 2D (A=span, B=cholesterol, C=dicetyl phosphate), in 3D (b) ......................................................................17 Figure 13: Chemical structure of 5(6)-carboxyfluorescein ................................................18 Figure 14: Brightness vs. quenching, self quenching properties of common fluorescent agents .............................................................................................18 Figure 15: Schematic drawing of gel exclusion chromatography (GEC) separation ........19 Figure 16: Econo-Pace 10DG column specification ..........................................................20 Figure 17: Hemocytometer specification: square sized (0.05 mm) ...................................21 Figure 18: PSS auto-diluter scheme ...................................................................................22 vi Figure 19: Microscopic observation of niosome vesicles at (A) 67.1400 (B) 67.6400 ........................................................................................................37

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vii Figure 20: Run #1, Span60/Niosomes hydrating in H 2 0 ....................................................43 Figure 21: Run #1, Span60/Niosomes hydrating in PBS ...................................................45 Figure 22: Run #1, Span60/Niosomes hydrating in PBS-CF ............................................47 Figure 23: Run #1, Run#2 and Run#3, Span60/Niosomes hydrating in PBS-CF .............49 Figure 24: Sp60/Niosomes in PBS 60C (A), (B) and (C) and (D) at different magnifications ..................................................................................................50 Figure 25: Comparison of Sp60/Niosomes pa rticle size distribution at 40C and 60C ...51 Figure 26: Comparison of Sp 20, Sp40 and Sp60/Niosomes using PBS-CF .....................53 Figure 27: PSS results, Sp60/Niosomes pa rticle distribution from 0-4 minutes sonication time .................................................................................................55 Figure 28: PSS results, Sp20/Niosomes pa rticle distribution from 0-4 minutes sonication time .................................................................................................56 Figure 29: PSS results, Sp40/Niosomes pa rticle distribution from 0-4 minutes sonication time .................................................................................................57 Figure 30: PSS total particle counts of Sp20, Sp40 and Sp60/Niosomes from 0-4 minutes sonication ...........................................................................................59 Figure 31: PSS average particle distribution of Sp20, Sp40 and Sp60/Niosomes from 0-4 minutes sonication .....................................................................................60 Figure 32: Sp60/Niosomes observation A) before sonication and B) after sonication ......60 Figure 33: Standard curve of floresce nce intensity vs. concentration of CF .....................63 Figure 34: Standard curve of florescence inte nsity vs. concentration of CF (linear fit) ....64 Figure 35: Comparison of CF stock so lution batch #1 and batch #2 based on concentration and intensity ..............................................................................65 Figure 36: Sp60/Niosomes particle size dist ribution 14 days afte r rehydrating process ...67 Figure 37: GEC counts per ml vs. cumulative volume collected ......................................70 Figure 38: GEC, mean diameter vs. cumulative volume ...................................................72 Figure 39: Comparison of counts per ml and background intensity vs. cumulative volume of sample .............................................................................................73

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viii Figure 40: Comparison of Run#1 from S p20, Sp40 and Sp60/Niosomes formation after GEC .........................................................................................................75 Figure 41: Observation of Sp60/Niosomes unde r A) regular light scattering mode and B) UV mode .....................................................................................................76 Figure 42: Stability of Sp20, Sp40 and Sp60/Niosomes, day 0 to day 9 ...........................77 Figure 43: Average intensity increased for Sp20, Sp40 and Sp60/Niosomes from day 0 to day 9 ..........................................................................................................79

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ix SYNTHESIS AND CHARACTERIZATION OF POTENTIAL DRUG DELIVERY SYSTEMS USING NONIONIC SURFACTANT NIOSOME Sukit Leekumjorn ABSTRACT Niosomes are synthetic microscopic vesi cles consisting of an aqueous core enclosed in a bilayer consisting of choleste rol and one or more nonionic surfactants. They are made of biocompatible, biodegr adable, non-toxic, nonimmunogenic and noncarcinogenic agents which form closed spherica l structures (self a ssembly vesicles) upon hydration. With high resistance to hydrolyt ic degradation, niosomes are capable of entrapping many kinds of soluble drugs while exhibiting greater vesicle stability and longer shelf life. In this work, a potential drug delivery sy stem has been designed, synthesized and characterized. For the synthesis of niosomes, a hydration process was developed with varying design parameters such as mass per ba tch, angle of evaporation, rotation speed of vacuum rotary evaporator and nitrogen flowra te to produce uniform thin film in 50 ml round bottom flask. The rehydration process was developed by varying the choice of solvents (H 2 O, phosphate buffer solution (PBS) and PBS/5(6)-carboxyfluorescein (CF) as a drug model) and hydrating temperature of be low and above gel transition temperature. Lastly, a sonication process to produce unilame llar vesicles was partially optimized based on the particle distribution and the number of vesicles formed with sonication time.

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As a result of this process, unilamellar and multilamellar vesicles were formed with the combination of different nonionic surfactants (sorbitan monostearate-Span 60, sorbitan monopalmitate-Span40 and sorbitan monolaurate-Span20), cholesterol and an electrostatic stabilizer (dicetyl phosphate). The vesicles were examined using light scattering optical microscopy and UV microscopy. Optical sensing technology (Particle Sizing System) is used to determine the vesicles size distribution. Gel exclusion chromatography (GEC) is discussed as a method to separate unencapsulated CF while retaining vesicle integrity. Particle Sizing System and luminescence spectrophotometer were used to determine CF encapsulation percentage and leakage. Result: Span 20, Span 40 and Span 60/Niosomes were made with mean particle size of 0.95-0.99 m. Typical concentrations of vesicle per ml/per mass of surfactant used were in the range of Typical encapsulation efficiencies were in the range of 48.8-62.9% for all three Span/Niosome systems. Niosomes were found to be stable for 9 days. The largest vesicles were observed with Span 60 with highest entrapment efficiency as compared to Span 20 and Span 40. 81079.146.1 x

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1 CHAPTER ONE: INTRODUCTION Development of new drugs is difficult, expensive and rather time consuming in the process involving p reclinical testing, i nvestigational new drug application (IND), clinical trials, phase I, II, & III, new drug application (NDA) and FDA approval. Improving safety and efficacy of existing drugs has been attempted using different methods such as individualizing drug th erapy, dose titration and therapeutic drug monitoring and, most importantly, delivering dr ugs at controlled rate s at targeted sites [] 1 Drug delivery systems could provide extended ci rculating half-lives so that less drug is required for therapeutic effectiv eness (for a longer period of time) relieving the patient of side effects caused by non-specific tissu e uptake and provide protection against enzymatic degradation [] 2 Recently discovered biological drugs often require drug delivery systems due to several side effects of the active ingredients to minimize the number of injections required during the course of therapy. Today, lipid and nonionic surfactant based drug delivery systems have drawn much attention from researchers as potential carriers of various bi oactive molecules that could be used for therapeutic applications. Several commercial liposome/niosome-based drugs have already been marketed with a great success. For example, liposomes and niosomes have been used to encapsulate colchicines [] 3 estradiol [] 4 tretinoin [ ] 5 6 dithranol [ ] 7 8 enoxacin [] 9 for applications such as an ticancer, anti-tubercular, antileishmanial, anti-inflammatory, hormonal drugs and oral vaccines [ , , , ] 10 11 12 13 14 15 16 17

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2 Based on these successes and the advantag es of niosomes over liposomes, the objective of this research was to determin e an appropriate synthesis technique for a niosome drug carrier system, to characterize the resultant particles and to encapsulate 5(6)-carboxyfluoresce in (CF) to demonstrate encapsulation efficiency, and leakage/release over time.

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CHAPTER TWO: LITERATURE REVIEW The focus of this literature review is on liposomes and niosomes as drug delivery systems (synthesis technique, targeting surface modification, biocompatibility and their delivery applications). 2.1 Liposomes A liposome is a microscopic vesicle consisting of an aqueous core enclosed in one or more phospholipid layers, used to convey vaccines, drugs, enzymes, or other substances to target cells or organs [] 18 Liposomes are bilayered structures made of amphipathic (both hydrophobic and hydrophilic) phospholipids/cholesterol that spontaneously form closed structures when hydrated in aqueous solutions (Figure 1, Figure 2 and Figure 3). 3 Hydrophilic head group Hydrophobic tails Si g ma-Aldrich Catalo g Figure 1: Chemical structure of Phosphatidyl-choline (PC)

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4 Figure 2: Possible formation of liposome vesicles when hydrating in aqueous solution www.avantili p ids.com www.avantili p ids.com 0.05 mm Figure 3: PC immerse in water to form bilayer. Elongation and several vesicles were observed In Figure 2, depending on the number of bilayers, liposomes are classified as multilamellar (MLV), large unilamellar (LUVs) or small unilamellar (SUVs) and range in size from 0.025~20 m in diameter. The size and morphology of liposomes are regulated

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5 by the method of preparation and composition. Mainly, factors that contribute to the overall vesicle formation are phase transition temperature, stability and cholesterol. 2.1.1 Phase Transition Temperature of Lipids The phase transition temperature is define d as the temperature required to induce a change in the lipid physica l state from the ordered gel pha se (hydrocarbon chains are fully extended and closely packed) to the disordered liquid crystalline phase (hydrocarbon chains are random ly oriented and fluid) [] 19 There are several factors which directly affect the phase tr ansition temperature; however, the length of the hydrocarbon chain provides a major contribu tion to the overall transition temperature (Table 1, Figure 4). It is noted that PC=phosphatidyl choline, PE=phosphatidylethanolamine, PS=Phosphatidylserine, PA=phosphatidic acid and PG=phosphatidyglycerol. Table 1: The effect of transition temperature vs. acyl composition (hydrocarbon chain length) Phase Transition Temperature (C) Acyl Composition avantilipids.com PC PE PS PA PG 12 -1 29 17 31 -3 13 14 14 23 50 35 50 23 15 33 16 41 63 54 67 41 17 48 18 55 74 68 75 55 19 60 20 66 83 21 72 22 75 23 79 24 80

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12141618202224020406080 Phase Transition Temperature (C)Fatty Acyl Composition PC Phosphatidylcholine PE Phosphatidylethanolamine PS Phosphatidylserine PA Phosphatidic Acid PG Phosphatidylglycerol www.avantili p ids.com Figure 4: The effect of transition temperature vs. acyl composition As the hydrocarbon chain length increases, molecular (van der Waals) interactions become stronger, requiring more energy to disrupt the ordered packing; thus the phase transition temperature increases (Figure 4). The stability of the final structure depends on the type of emulsion which is created (oil in water or water in oil). The larger the hydrophobic chains, the greater the intermolecular force on the hydrophilic part of the molecule. This forms a structure which extends the hydrophilic end (head) into the water substrate while the hydrophobic chains (tails) are separated from this substrate by the 6

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7 intrabilayer spaces of the sphere In general, high transition lipids provide a stable (nonleaky) delivery system [ ] 20 2.1.2 Stability/Surface Modification Lipid compounds that are easily oxidized (unsaturated) tend to have a shorter shelf life. Stability issues due to hydrolyti c degradation are a general problem with lipid products. Aqueous formulations of drug pr oducts tend to be less stable since the presence of excess or bulk water leads to rapid hydrolytic degradation in lipid preparations [ , ] 21 22 23 This hydrolysis is dependant on several factors including pH [21] temperature [21, 23] buffer species [23] ionic strength, acyl ch ain length and headgroup [22] and the state of aggregation [22] Nevertheless, there have been successes in delivering various drugs and therapeutic genes in both animal and human trials [ , , , ] 24 25 26 27 28 29 30 In light of the short half-l ife of conventional liposomes several researchers have introduced a protective coat by designing liposomes that are non-reactive or polymorphous [ , ] 31 32 33 Phospholipids with a polyethylene glycol (PEG ) coating help prevent liposomes from sticking to each ot her and to blood cells or vascular walls [] 34 This coating also reduces th e uptake of liposomal vesicles in the liver and extends circulation time [31] Furthermore, by incorporating targ eting ligands on the surface of the liposomes, it was possible to direct these vesicles to certain organs using the PEGlycation technique [ , ] 35 36 37 38 Branched amino acids have also been incorporated on the surface of liposomal vesicles in order to stabilize th e vesicle and to deliver to targeted sites [39, 40] Today, liposomes are used in drug delivery systems [ , ] 41 42 43 For example, liposomal systems integrated with recepto r protein conjugates are being studied for

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cardiovascular treatments targeting activated platelets [] 44 Acoustic liposomes have been created for echocardiographic enhancement of pathological components of atherosclerotic lesions [44] Modified liposome has been shown to attach to early atheroma in animal model when targeted to vascular cell adhesion molecule type 1 (VCAM-1), or intercellular adhesion molecule-1 (ICAM-1) on atherosclerotic lesion [44, ] 45 46 Furthermore, covalently bonded monoclonal antibodies to small unilamellar liposomes has been accomplished using dipalmitoyl lecithin and cholesterol and variable quantities of phosphatidylethanolamine substituted with the heterobifunctional cross-linking reagent N-hydroxysuccinimidyl 3-(2-pyridyldithio) propionate (SPDP) [] 47 Figure 5 represents the molecular structure of heterobifunctional cross-linking reagent and modified liposome. A) N-hydroxysuccinimidyl 3-(2-pyridyldithio) propionate B) Dipalmitoylphosphatidylethanolamine 3-(2-pyridyldithiopropionate) Figure 5: Modified liposomes using phosphatidylethanolamine substitution 8 Once the modified liposomes were formed, they were then reacted and incubated with antibody derivatized with the same reagent at 5 to 20 fold molar excess and it was found

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9 that more than 40% of antibody could be reproducibly bound to liposomes without the loss of encapsulat ed carboxyfluorescein [47] By knowing the fact that several types of antibodies covalently bind to modify liposomes and the availability of anti-ICAM-1 and anti-VCAM-1, exploring the usage of these antibodies may improve drug delivery systems specifically targeti ng atherosclerotic plaque. 2.1.3 Cholesterol Cholesterol (C 27 H 45 OH) is a cell membrane consti tuent found in most animal systems. It modulates membrane fluidity, elasticity, and permeability by closing the gaps created by imperfect packing of other lipid species when proteins are embedded in the membrane [] 48 Furthermore, cholesterol enables the formation of vesicles, reduces aggregation and provides greater stability [] 49 Based on the size and orientation of cholesterol molecule during vesi cle formation, the best molar ra tio of lipid to cholesterol was reported to be one to one [] 50 2.1.4 Liposome Preparation & Mechanisms Associated with Drug Encapsulation There are two major techniques for ma king liposomes: dehydration-rehydration and reverse phase synthesis. In this work, the dehydrati on-rehydration method was used (see Figure 6).

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www.avantili p ids.com Figure 6: Preparation of liposome using dehydration/rehydration technique After the thin film is prepared, the aqueous phase is introduced. Upon agitation, liposomes form multilamellar (MLV), large unilamellar vesicles (LUVs) or small unilamellar (SUVs) upon rehydration (Section 2.1). While in the assembly mode, there are two primary mechanisms such as encapsulation (formation of liposomes passively entrapped water soluble drug in the interlamellar spaces) and partitioning (formation of liposomes passively entrapped organic soluble drug in the intrabilayer spaces). 2.2 Niosomes Because of a liposomes instability, alternative nonionic surfactants have been investigated [20] Using similar techniques, nonionic surfactant vesicles or niosomes have been synthesized [50] These formulations use alternative materials to phospholipids such as Span 60, Span 40 and Span 20 (Table 1), which are inexpensive and widely available permitted food additives [50 51 , , ] 52 53 54 55 56 For example, niosomes has been used to encapsulate colchicines [3] estradiol [4] tretinoin [5,6] dithranol [7,8] enoxacin [9] 10

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and for application such as anticancer, anti-tubercular, anti-leishmanial, anti-inflammatory, hormonal drugs and oral vaccine [10, 11, 12, 13, 14, 15, 16, 17] 2.2.1 General Characteristics of Nonionic Surfactant The ability of nonionic surfactant to form bilayer vesicles instead of micelles is dependant on the hydrophilic-lipophilic balance values (HLB) of the surfactant, the chemical structure of the components and the critical packing parameter [] 57 The relationship between the structure of the surfactant including size of hydrophilic head group, and length of lipophilic alkyl chain in the ability to form vesicles is described in Figure 7. Figure 7: Critical packing parameter of surfactants [57] The general form of a single bilayer vesicle is shown in Figure 8. 11 Figure 8: Bilayer membrane structure

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12 Nonionic surfactants investigated in this res earch are shown in Table 2. For HLB values greater than 6, cholesterol must be added to the surfactant in order for a bilayer vesicle to form [52] For lower HLB values, cholesterol is ad ded to make vesicles more stable as described earlier in Section 2.1.3. Also, th e addition of cholesterol enables more hydrophobic surfactants to form vesicles, suppr esses the tendency of the surfactants to form aggregates, and lends greater stability to the bilayer membranes by raising the gel liquid transition temperature of the vesicle [53] Below are the phase transition temperatures of nonionic surfactants (Table 3). Table 2: Chemical specifications of Sp20, Sp40, Sp60, cholesterol, dicetyl phosphate and CF Chemical Specification Description MW (g/mol) MW Formula HLB [54] Sorbitan monolaurate (Span20) Clear viscous liquid 346.5 C 18 H 34 O 6 8.6 Sorbitan monopalmitate (Span40) Yellowish powder 402.6 C 22 H 42 O 6 6.7 Sorbitan monostearate (Span60) White powder 430.6 C 24 H 46 O 6 4.7 Cholesterol White powder 386.7 C 27 H 46 O N/A Dicetyl phosphate White powder 546.9 C 32 H 67 O 4 P N/A 5(6)-carboxyfluorescein Yellowish powder 376.0 C 21 H 12 O 7 N/A Table 3: Phase transition temp erature of Sp20, Sp40, and Sp60 Nonionic Surfactant Acyl composition Gel Transition Temperature Sorbitan monolaurate (Span20) C9 Liquid at room temperature Sorbitan monopalmitate (Span40) C13 46-47C Sorbitan monostearate (Span60) C15 56-58C The transition temperatures of surfactants increased 46-47C to 56-58C as the hydrocarbon length is increased (C9-C15). This stabilit y decreases leakage of the vesicles and stabilizes against osmotic gradients [53]

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13 2.3 Summary Above all, liposomes and niosomes have been successfully used to entrap several types of water soluble drugs. Niosome system s have been shown to be more chemically stable, commercially less expensive, and less cumbersome in handling, production and storage than liposomes [50]

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14 CHAPTER THREE: DESIGN CONCEPT As a first step, optimization of the process design parameters for making niosomes was prepared. This included pa rameters such as mass per batch, angle of evaporation, dehydration nitrogen flowrate hydrating solvents, hydrating temperature and sonication time. 3.1 Material and Equipment Selection All materials and equipments used in the experiments are listed below (Table 4 and Table 5). Table 4: List of material used in the experiment Sigma Aldrich Cooperation Sorbitan Monostearate, Span 60 (S-7010) Sorbitan Monopalmitate, Span 40 (S-6885) Sorbitan Monolaurate, Span 20 (S-6635) Cholesterol; 5-cholesten-3 -ol (C-8503) Dicetyl Phosphate, Dihexadecyl phosphate (D-2631) Phosphate Buffered Saline, pH 7.4 (P-3813) Sodium Hydroxide (S-0899) Sephadex G-50 (G-50-80) Fisher Scientific Chloroform, 99%A.C.S. HPLC grade (C606-1) Triton X-100 (BP151) Biotium, Inc. Hayward, California 5-(and-6)-Carboxyfluorescein, catalog #51013

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15 Table 5: List of equipment used in the experiment Mass balance: Denver Instrument Company A-250 Buchi-Vaccum Controller V-800 Buchi-Rotavapor R-200 Buchi-Heating Bath B-490 Rotary evaporator: Buchi Vac V-500 Laboratory Supplies CO., INC Model G1125PIG 115 Volts, 50-60 Hz, 3.5 Amps. Sonicator: Output 80 KC, 80 Watts Particle Sizing Systems (PSS), Model 770A Accusizer TM Autodiluter Pat Particle sizing: Partial Particle Sizer TM Fluorescence Spectrometer: Perkin Elmer, LS-3B Cooling System: PolyScience Refrigeration Circulation Model 90 Olympus BH-2, 4x, 10x, 20x Dplan 40x Splan NFK 1.67 x LD125 Microscope: NFK 6.7 x LD125 Lecia Type 090-135.002 II/02 UV Microscope: LEP-LTD ARC Lamp 50W, HBO-AC, 6V max, 35W Sony CCD Color Video Camera Video Camera: Model SSC-C370 Glassware: 50 ml Pyrex Mexico, 24/40 neck size, No. 4320A 1-10 L 10-100 L 100-1000 L Eppendorf Research Pipette: 1000-5000 L Container Wrap: PARAFILMR M Laboratory film Gel Exclusion Column: EconoPac 10DG column, Biorad Laboratories, Inc Water Filter System: Waterwise Model A30D/W3616 Figure 9 shows the chemical structur e of sorbitan monostearate (Span 60), sorbitan monopalmitate (Span 40) and sorbit an monolaurate (Span 20). Their physical properties were reported in Table 2.

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Sigma-Aldrich Catalog Figure 9: Chemical structure of Sp60 (A), Sp40 (B) and Sp20 (C) Span 20, Span 40 and Span 60 are widely available food additives and the main ingredient in some cosmetic applications. They are characterized as amphipathic (hydrophobic head and hydrophilic tail) molecules that can spontaneously form closed structures when hydrated in aqueous solutions. Dicetyl phosphate is also used in to prevent aggregation between vesicles and provide greater stability. Chemical structure of dicetyl phosphate is shown in Figure 10. Sigma-Aldrich Catalog Figure 10: Chemical structure of dicetyl phosphate Cholesterol, 5-cholesten-3-ol (Figure 11) is used in combination with nonionic surfactant. 16

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Sigma-Aldrich Catalog Figure 11: Chemical structure of cholesterol The structure of the resultant niosome is shown in Figure 12. 17 a) 2-dimension b) 3-dimension andrew.cmu.edu/~ j amesv/ Research-Lab.html Figure 12: Proposed structure of niosome vesicle (a) in 2D (A=span, B=cholesterol, C=dicetyl phosphate), in 3D (b) 3.2 Encapsulation Technique Dehydration/rehydration technique is used to form niosomes. During rehydration, solution containing 5(6)-carboxyfluorescein (Figure 13), as a drug model, in sodium hydroxide (NaOH) and phosphate buffered saline (PBS) is used.

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Biotium Catalog Figure 13: Chemical structure of 5(6)-carboxyfluorescein Premade CF solutions, in the range of 5 mM, exhibit self-quenching (higher concentration of CF molecules with a bright fluorescein color decrease the intensity reading of the fluorescent from fluorometer) and can be entrapped inside niosomes, exhibiting low intensity fluorescence. Brightness vs. number of molecules of several types of dyes is shown in Figure 14 where self-quenching occur at higher concentration. Amersham Biosciences Figure 14: Brightness vs. quenching, self quenching properties of common fluorescent agents However, when niosomes are broken down with Triton X-100 (nonionic detergent used to solubilize membranes), the changes in the intensity can be used to estimate the amount of CF entrapped inside the niosomes. 18

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3.3 Separation Technique Excess CF remaining in the hydrating solution was separated from the vesicles by gel exclusion chromatography (GEC). The gel matrix consists of spherical beads with pores of a specific size distribution. As small molecules diffuse into the gel pores, their flow through the column is retarded, while large molecules bypass the pores and are rapidly eluted. Sephadex G-50 was used as a packing material in the column (Table 6), to separate niosomes from other molecules such as unencapsulated CF, cholesterol molecules, small micelles, and fragments of surfactants (Figure 15). Table 6: Gel exclusion packing materials and their specifications Gel Type (Sigma) Fractionation Range, Globular Proteins (Da) Approx. Bed Volume (ml swelled per gram dry Approx. Dry Bead Diameter (m) Approx. Void Volume (% of total bed volume) G-10 700 23 40-120 10-30 G-15 1500 2.53.5 40-120 10-30 G-25 10005000 46 50150 20-40 G-50 1500000 9 5050 20-40 Figure 15: Schematic drawing of gel exclusion chromatography (GEC) separation 19

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Resolution of separation depends on particle size, pore size, flow rate, column dimensions, and sample volume. In general, the highest resolution is obtained with low flow rates, long/narrow columns (25-50 ml column volume, Figure 16), small-particle-size gels (Sephadex G50), small sample volumes and large differences in molecular weights (molecular weight of CF as compare to niosomes). EconoPac 10DG column Bed volume 20 ml Total column volume 30 ml Packing buffer PBS Flow rate 1.1-1.5 ml/min Column material Polypropylene Frit material Polypropylene Desalting gel Porous bead pH 2-10 pH range Operating temperature 2-45 C www.bio-rad.com Figure 16: Econo-Pace 10DG column specification Table 7: Recommended volume of sample per column volume Column Volume (www.bio-rad.com) Volume of Sample 100-200 ml 0.5 ml 50-100 ml 0.25 ml 25-50 ml 0.125 ml 20

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3.4 Particle Characterization Particle characterization is accomplished using light scattering optical microscopy, UV microscopy, luminescence spectroscopy (fluorometry) and light scattering and light obscuration techniques. Both UV and light scattering optical microscope provide a magnification range from 4004 plus eye piece magnification. The light microscope also gives an estimation of particle diameter using a hemocytometer (Figure 17). The UV microscope provides a dark background image with a trace of fluorescence entrapped inside the niosome. 10 0.05 mm Figure 17: Hemocytometer specification: square sized (0.05 mm) As described earlier, the excitation and emission wavelength of CF are I excitation and I emission are 492/514 nm respectively. This property can be used in fluorometry to detect CF, and the intensity of the signal is proportional to intensity reading over diluted ranges. By measuring the intensity of fluorescence and calibrating against sets of standard solutions, fluorometer can be use to detect CF in extremely dilute solutions. 21

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Particle sizing of the vesicle dispersion can be achieved by the Particle Sizing System 780 (PSS). This technology utilizes forward light scattering and light obscuration technique equipped with auto-dilution scheme (Figure 18). Particle Sizing System Figure 18: PSS auto-diluter scheme Scattered light is collected onto a photodiode to produce an electrical pulse, the voltage which is proportional to the light intensity detected from each particle and for each particle size. The photodiode pulses are amplified and this electrical signal measured by a counter. In general, PSS-780 has the ability to count and size particles in the range of 0.5-500 m. The operation of the particle sizer and counter depends, primarily, on the low concentration value of the particles in solution. Autodilution in the PSS-780 reduces concentration to eliminate the possibility that multiple particles pass through the detecting chamber. Higher concentration solutions exhibit higher probabilities of false readings because more than one particle might pass between the photodiode array detector and the light scattering emitter. By requiring very low concentrations the possibility of light extinction due to the passage of more than one particle is reduced. 22

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23 CHAPTER FOUR: PROCEDURES AND EXPERIMENT PROTOCOLS 4.1 Procedures The basic procedures listed below were de termined based on literature surveys. Design parameters were then varied to optimize the procedure (Section 4.2) 4.1.1 Buffer Solution Preparation 1) For 10x solution, open one phosphate buffered saline package (Sigma, P3813), place the powder into 1 L containe r and fill up the bottle with 100 ml of H 2 O (equivalent ultrapure water, Waterwise Model A30D/W3616). 2) For 1x solution, open one phosphate buffere d saline package (Sigma, P-3813), place the powder into 1 L container and fill up the bottle with 1.0 L of H 2 O. 4.1.2 Niosome Stock Solution 1) For Span 60/cholesterol/DCP stock solution: weigh out 0.1600 g of dicetyl phosphate (Sigma, D-2631), 0.8800 g of cholesterol (Sigma, C-8503) and 1.0000 g of Span 60 (Sigma, S-7010) in th e mass balance and place it in into a clean, dry 50 ml or 100 ml screw type bot tle. These values were precalculated to have a molar ratio of 7.5/7.5/1 of Span60, cholesterol and DCP respectively [ ] 58 59 Pipette 100.0 ml chloroform into the bottle. Close the bottle tightly and place it in a warm water bath, at 40C, to ensure complete dissolution.

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24 2) For Span 40/cholesterol/DCP stock solution: weigh out 0.1600 g of dicetyl phosphate (Sigma, D-2631), 0.8800 g of cholesterol (Sigma, C-8503) and 0.9360 g of Span 60 (Sigma, S-6885) in th e mass balance and place it in into a clean, dry 50 ml or 100 ml screw type bot tle. These values were precalculated to have a molar ratio of 7.5/7.5/1 of Span 40, cholesterol and DCP respectively [58, 59] Pipette 100.0 ml chloroform (Fishersci, C606-1) into the bottle. Close the bottle tightly and place it in a warm water bath, at 40C, for 10 minutes to ensure complete dissolution. 3) For Span 20/cholesterol/DCP stock solution: weigh out 0.1600 g of dicetyl phosphate (Sigma, D-2631), 0.8800 g of cholesterol (Sigma, C-8503) and 0.8060 g of Span 20 (Sigma, S-6635) in th e mass balance and place it in into a clean, dry 50 ml or 100 ml screw type bot tle. These values were precalculated to have a molar ratio of 7.5/7.5/1 of Span 20, cholesterol and DCP respectively [58, 59] Pipette 100.0 ml chloroform (Fishersci, C606-1) into the bottle. Close the bottle tightly and place it in a warm water bath, at 40C, for 10 minutes to ensure complete dissolution. 4.1.3 5(6)-Carboxyfluorescein (5 mM) in PBS Stock Solution 1) Pipette 4.0 ml of H 2 0 in to the 100 mg CF container (Biotium, 51013) and place the solution into a 60 ml plastic container (milky yellow should be observed). 2) Pipette 11.0 ml of NaOH (0.05 M, pH 13) into the container at 0.5 ml interval of injection (clear orange solution should be observed).

PAGE 38

25 3) Pipette 32.9 ml H 2 O into the container (clear green-orange should be observed). The value was precalculate d to achieve 5 mM CF stock solution. 4) Wrap the container with the aluminum fo il and keep it in the refrigerator for further analysis. 5) Calibration using fluorometer is required for the successive batch of CF stock solution to maintain the correct intensity readings. 4.1.4 Gel Chromatography Column Preparation 1) Weight out 5.0000 g of Sephadex G-50 (Sigma, G-50-80) in to a 500 ml beaker. Fill up the beaker with PBS to twice the original volume of Sephadex G-50. Allow 3 hours for a complete absorbsion of Sephadex G-50. 2) Pipette Sephadex G-50 solution in to a gel chromatography column slowly. There should be no air bubbles present during packing process. 3) Allow Sephadex G-50 to settle down gravitationally, and repack the column again until desired column height. 4.1.5 Dehydration of Stock Niosome 1) Clean 50 ml round bottom flask with detergent and dry completely. 2) Pipette 1.0 ml of stock soluti on into the round bottom flask. 3) Pipette 3.0 ml of chloroform into the flask and allow completely mixing for 12 minutes. 4) After 1-2 minutes, allow nitrogen to flow in at the rate of 3.0 L/min.

PAGE 39

26 5) The rotation of the evaporator should be slow and the level of the chloroform should remain undisturbed during this pr ocess. The film should be smooth and slightly cloudy. 6) Allow the film to completely dry for 15 min, turn on the vacuum rotary evaporator and dry for another 15min while reducing pressure from 1013 mmbar to approximately less than 100 mmbar. 7) Turn off the vacuum and quickly allow nitrogen gas to remove oxygen inside the flask and close the flask with wrap paper. 4.1.6 Rehydration of Thin Film 1) Turn on the vacuum evaporator hot bath (60C) and remove the wrap paper. 2) Pipette 3.0 ml of CF stock solution into the round bottom flask, place the flask back on to the evaporator and lower the flask into the water bath. 3) Allow hydration for at least 2 hours. 4) Place the flask in the sonicator bath and allow sonication for approximately 2 minutes. 5) Place the solution in a closed lid cont ainer and wrap the container with aluminum foil. 4.1.7 Gel Exclusion Chromatography Separation 1) Pipette 0.1 ml of the rehydration sample and place th at into the top of the column and let the sample settle down for 1 minute.

PAGE 40

27 2) Allow the sample to flow through the co lumn. Note: there should be clear color separation. 3) Collect the sample and wrap each bot tle with aluminum foil for further analysis. 4.1.8 UV Microscope 1) Turn on the power supply to both the microscope and UV power generator. 2) Place a drop on glass slide and place it on the platform of the microscope. 3) Adjust the microscope to normal m ode (visible light) and adjust the magnification. 4) Switch mode form normal mode to UV mode. 4.1.9 Encapsulated Drug Measurement 1) Turn on the power supply to Perkin Elme r, LS-3B and adjust the excitation to and emission to . 2) Pipette 1.0 ml PBS solution into an empt y cuvette and place it in the sample holder on the front of fluorometer. 3) Close the lid, take initial reading and tare the reading us ing zero button. 4) Add 500 L of PBS solution follow by 500 L of the sample. 5) Place the cuvette back, close the lid and record the intensity. 6) Remove the cuvette and pipette 20 L of Triton X-100 into the solution. 7) Allow complete mixing of the solu tion using vortex mixer and place the cuvette back to take the second measurement.

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28 4.1.10 5(6)-Carboxyfluorescein Standardize Curve 1) Turn on the power supply to Perkin Elme r, LS-3B and adjust the excitation to and emission to . 2) Pipette 1.0 ml PBS solution into an em pty cuvette and place it in the sample holder on the front of fluorometer. 3) Close the lid, take initial reading and tare the reading us ing zero button. 4) Add 500 L of PBS solution follow by 5 L of the sample obtained from CF stock solution. 5) Place the cuvette back, close the lid and record the intensity. 6) Remove the cuvette and pipette 100 L of PBS into the solution. 7) Place the cuvette back, close the lid and record the intensity. 8) Remove the cuvette and pipette anothe r 100 L of PBS in to the solution. 9) Repeat the same procedure until 2.5 ml of sample in the cuvette. 10) Repeat this experiment for two more tim es and take the average values of the results. 11) Construct a standardize curve based on the known concentration with respect to their intensities. 4.1.11 Particle Sizing System 780 (PSS 780) 1) Turn on the power supply to Particle Sizing System and initiate the PSS program on the connected computer. 2) Drain and fill the round bottom flask. The standard sample volume of H 2 0 inside this vessel was set to 35 ml.

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29 3) Flush the system until the particle count per ml is less than 20 and stop flush. 4) Pipette 10 L of the solution obtained from gel exclusion chromatography into the vessel and closes the cap. 5) Start the sample counting by click go one the PSS program and save the results into a created software folder. 6) Repeat the same procedure with another sample. 4.2 Experimental Protocols In this study, the following parameters were manipulated (Table 8). Table 8: The range of values of each variable tested for manipulat ed parameters in making niosomes Manipulated Parameters Span20 Span 40 Span 60 Mass per Batch 0.0185-0.0738 gram 0.0198-0.0790 gram 0.0204-0.0816 gram Angle of Dehydration 30-60 degree Rotation Speed on Rotary Evaporator 1-3 revolution/sec Nitrogen Dehydration Flowrate 2-5 L/min Hydrating Solvent H2O, PBS, PBS-CF Hydrating Temperature 40C and 60C Sonication Time 0-4 min 4.2.1 Determination of Mass per Batch 1) Clean four 50 ml of round bottom flasks with detergent and dry completely. 2) Pipette 1.0 ml of Span 60/cholesterol/DCP stock solution into the first flask and 3.0 ml of chloroform. 3) Repeat the procedures listed in Section 4.1.5; observe and record the characteristic of the film. 4) Repeat the procedures described previ ously and pipette the second flask with 2.0 ml of Span 60/choleste rol/DCP stock solution a nd 2.0 ml of chloroform.

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30 5) Repeat the procedures described previous ly and pipette the third flask with 3.0 ml of Span 60/cholesterol/DCP stoc k solution and 1.0 ml of chloroform. 6) Repeat the procedures described previously and pipette the fourth flask with 4.0 ml of Span 60/choleste rol/DCP stock solution. 4.2.2 Determination of Angle of Evaporation and Speed of Rotation 1) Clean twelve 50 ml of round bottom flasks with detergent and dry completely. 2) Pipette 1.0 ml of Span 60 stock soluti on in twelve flasks and 3.0 ml of chloroform. 3) Repeat the procedures listed in Section 4.1.5, however, the an gle of the four flasks are as follow 30, 45, 52.7 and 60 respectively and the rotation speed of 1-1.5 rev/sec. 4) Observe and record the characteristic of the thin film. 5) Repeat the same procedures again, however, the rotation speed of the four flasks increase to 1.5-2 rev/sec. 6) Observe and record the characteristic of the thin film. 7) Repeat the same procedures again, however, the rotation speed of the four flasks increase to 2-3 rev/sec. 8) Observe and record the characteristic of the thin film.

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31 4.2.3 Determination of Nitrogen Flow Rate during the Dehydration Process 1) Clean four 50 ml of round bottom flasks with detergent and dry completely. 2) Pipette 1.0 ml of Span 60/cholesterol/DCP stock solution in all four flasks and 3.0 ml of chloroform. 3) Repeat the procedures listed in Se ction 4.1.5, however, the nitrogen flowrate during the dehydration process increase from 2-5 L/min (1 L/min increment). 4) Observe and record the characteristic of the thin film. 4.2.4 Effect of Different Hydrating Solvents 1) Clean nine 50 ml of round bottom flasks with detergent and dry completely. 2) Pipette 1.0 ml of Span 60/cholesterol/DCP stock solution all nine flasks and 3.0 ml of chloroform. 3) Repeat the procedures listed in Sec tion 4.1.5 and Section 4.1.6 with the first three flasks, however, the choi ce of hydrating solvent are H 2 O, PBS, PBS-CF. 4) Run the three samples in the PSS-780 w ith 10 L injection and record the output data prior so nication process. 5) Repeat the procedures for two more times. 4.2.5 Effect of Different Hydrating Temperatures 1) Clean six 50 ml of round bottom flasks with detergent and dry completely. 2) Pipette 1.0 ml of Span 60 stock soluti on in all six flasks and 3.0 ml of chloroform.

PAGE 45

32 3) Repeat the procedures listed in Se ction 4.1.5 and Section 4.1.6 with PBS-CF solution, however, the hydrating temp erature is set at 40C and 60C. 4) Run the three samples in the PSS-780 with 10 L injection and record the output data prior so nication process. 5) Repeat the procedures for two more time. 4.2.6 Effect of Particle Formation during Sonication 1) Clean three 50 ml of round bottom flasks with detergent and dry completely. 2) Pipette 1.0 ml of Span 60 stock solu tion the first flasks and 3.0 ml of chloroform. 3) Pipette 1.0 ml of Span 40 stock solu tion the first flasks and 3.0 ml of chloroform. 4) Pipette 1.0 ml of Span 20 stock solu tion the first flasks and 3.0 ml of chloroform. 5) Repeat the procedures listed in Se ction 4.1.5 and Section 4.1.6 with PBS-CF on all three flasks. 6) Run the three samples in the PSS-780 w ith 10 L injection and record the output data prior so nication process. 7) Run the three samples in the PSS-780 with 10 L injection after sonicate for 1 minute and record the output data. 8) Run the three samples again in the PSS-780 with 10 L injection after sonicate for another 1 minute (2 minutes in total) and reco rd the output data.

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33 9) Run the three samples in the PSS-780 with 10 L injection after sonicate for another one 1 minute (3 minutes in to tal) and record the output data. 10) Run the three samples in the PSS-780 with 10 L injection after sonicate for another one 1 minute (4 minutes in to tal) and record the output data. 4.3 Quantitative Measurements used in Niosome Characterization After optimizing parameters, measuremen ts based on light scattering optical microscopy, UV microscopy and fluorometer were made. 4.3.1 Effect of Niosome Vesicles in PBS-CF Solution over Time 1) Clean a 50 ml of round bottom flask w ith detergent and dry completely. 2) Pipette 1.0 ml of Span 60 stock solu tion in the flasks and 3.0 ml of chloroform. 3) Repeat the procedures listed in Se ction 4.1.5 and Section 4.1.6 with PBS-CF on all three flasks according to th e optimized parameters previously determined. 4) Run the three samples in the PSS-780 w ith 10 L injection and record the output data. 5) Keep the three sample solutions in clos e lid container at 4C (refrigerator). 6) After one week, run the three samples in the PSS-780 with 10 L injection and record the output data. 7) Again, keep the three sample soluti ons in close lid container at 4C (refrigerator).

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34 8) After one week (total 2 weeks), run the three samples in the PSS-780 with 10 L injection and record the output data. 4.3.2 Separation Pattern Using Gel Exclusion Chromatography 1) Pipette 1.0 ml, 0.25 ml or 0.10 ml of the Span60/Niosomes sample after rehydration into the top of the column. 2) The column should not be a llowed to dry at all time. 3) Allow the sample to flow through the column. There should be clear color separation based on observation. 4) Collect sample into separate bottles a nd wrap each bottle w ith aluminum foil. 5) Run each sample collected with PSS-780. 4.3.3 Leakage Studies and Encapsulation Measurement 1) The sample of Span60/Niosomes, Sp an40/Niosomes and Span20/Niosomes collected after gel exclusion chromatogr aphy (previously described in Section 4.3.2) are kept in the dark at room temperature for the next 9 days. 2) During this period, these samples are test ed for the leakage of CF from the niosome vesicles based on the intens ity reading from fl uorometer (0.5 ml injection to the fluorometer+0.5 ml of H 2 0). 3) Add 20 L of Triton X-100 and record the changes in the intensity. 4) To calculate the entrapment efficiency, the intensity measurements of Span20/Niosomes, Span 40/Niosomes and Span60/Niosomes produced in Day 0 (before and after breaking niosomes using Triton X-100) were

PAGE 48

recorded and the difference in intensity can be used to calculate the amount of mole of CF in the solution. With the interior volume previously calculated based on the result of PSS-780, the final concentration can be calculated. 5) The entrapment efficiency was calculated based on the following equations: CountedParticlemcmmmmDiametermlorcmVolumeInterior363100102)(34)( Equation 1 Equation 1, both diameter and particle counted can be obtained from PSS-780 ASCII file (Sp60/Niosome in appendix C). Note, the particle counted has to be multiplied by the dilution factor. Based on the sample collected between 5-10 ml (cumulative volume) after GEC, the average values of the changes in intensities (Table 33, Day #0) were calculated from Equation 2. testedsampleIinitialIfinalIntensityAverage# Equation 2 Moreover, the number of moles of CF in the niosomes was calculated using Equation 3 based on the average intensity obtained in equation #2 and the proportionality constant between the intensity and concentration LmoleIntensity81060.3 obtained from CF standardized curve (Figure 34). 35

PAGE 49

)(0.110001060.3)(8testedvolumetotalmlmlLLmoleIntensityensityAverageIntmoleentrapCF Equation 3 Furthermore, to obtain the number of mole of CF entrapped, the total volume tested in the cuvette for the fluorometer (0.5 ml CF sample plus 0.5 ml of PBS) was multiplied to the calculated concentration (Equation 3). Finally, to make a comparison the initial mole of CF present inside the niosomes, taking the initial concentration of CF (5 mM) and multiplied to the interior volume (previously calculated in Equation 1) of the niosomes in a solution assuming the initial concentration was 5 mM (Equation 4). VolumeInteriormlLLmolemoleentrapCFInitial1000105)(3 Equation 4 36

PAGE 50

CHAPTER FIVE: RESULTS AND DISCUSSION 5.1 Particle Formation Essentially, there are several factors which influence particle formation including total mass per batch, equipment setup, and dehydration using nitrogen gas and reduced pressure, hydrating solvent, hydrating temperature and sonication time. Using techniques described in Section 4, niosome vesicles were formed using 0.0204 gram per batch (7.95:7.78:1 molar ratio of span60, cholesterol and dicetyl phosphate respectively), 45 angle of evaporation, 1-1.5 rev/sec dehydrating rotation speed, 3 L/min of nitrogen flowrate, 40C hydrating temperature and 2 minute sonication time. Figure 19 shows a microscopic image of spherical vesicles. Large vesicles with about 10 m in diameter were observed. Their presence confirms the success of the first step of the process. 37 A) 10 m B) 50 m Figure 19: Microscopic observation of niosome vesicles at (A) 67.1400 (B) 67.6400

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38 5.1.1 Determination of Mass per Batch Using the dehydration and rehydration technique, the synthesis of nonionic vesicles was achieved using stock solutions of Span60/cholesterol/dicetyl phosphate, Span40/cholesterol/dicetyl phosphate and Sp an20/cholesterol/dicet yl phosphate (Table 9). Table 9: Molar ratio of Span/Nio some stock solution (weighted ma ss in Table 10/MW in Table 2) Stock Solution Span (mole) Cholesterol (mole) DCP (mole) Sp60/Chol/DCP 7.94 7.78 1.00 Sp40/Chol/DCP 7.95 7.78 1.00 Sp20/Chol/DCP 7.95 7.78 1.00 For each batch during dehydration process, there were limits on the amount of Span/cholesterol/dicetyl phosphate that coul d be placed into a 50 ml round bottom flask to produce a uniform thin film before rehydr ation process. Base d on the molar ratio given from the literature reports of 7.5:7. 5:1 of span, cholesterol (chol) and dicetyl phosphate (DCP) respectively [32, 33] the stock solutions were made as follows. Table 10: Stock solution of Span/Niosome prepared for dehydration process Stock Solution Span (g) Chol (g) DCP (g) Total Mass (g) Sp60/Chol/DCP 1.0000 0.8800 0.1600 2.0400 Sp40/Chol/DCP 0.9360 0.8800 0.1600 1.9760 Sp20/Chol/DCP 0.8060 0.8800 0.1600 1.8460 Since 100 ml of chloroform was used to prep are the stock solution (Table 10), series of experiments were carried out to determin e the optimal value of mass per batch of dehydration. Based on the observation of the thin film inside 50 ml round bottom flask, the optimal mass per batch was 0.0204 grams for Sp60/Niosome, 0.01976 grams for Sp40/Niosome and 0.01846 grams for Sp20/Niosome (Table 11).

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39 Table 11: Observation of the thin film from different injected volumes of stock solutions Sp#/Chol/DCP Angle of Evaporation ~52.7 Rotation Speed (rev/sec) ~1-1.5 Nitrogen Flowrate (L/min) 3 Stock Solution Volume of Stock Solution (ml) Volume of Chloroform (ml) Total Volume/Batch (ml) Observation After Dehydration Mass Per batch (gram) Sp60/Chol/DCP 1.0 3.0 4.0 Smooth thin film 0.0204 Sp40/Chol/DCP 1.0 3.0 4.0 Smooth thin film 0.0198 Sp20/Chol/DCP 1.0 3.0 4.0 Smooth thin film 0.0185 Sp60/Chol/DCP 2.0 2.0 4.0 Aggregation formed 0.0408 Sp40/Chol/DCP 2.0 2.0 4.0 Aggregation formed 0.0395 Sp20/Chol/DCP 2.0 2.0 4.0 Aggregation formed 0.0369 Sp60/Chol/DCP 3.0 1.0 4.0 Aggregation formed 0.0612 Sp40/Chol/DCP 3.0 1.0 4.0 Aggregation formed 0.0593 Sp20/Chol/DCP 3.0 1.0 4.0 Aggregation formed 0.0554 Sp60/Chol/DCP 4.0 0 4.0 Aggregation formed 0.0816 Sp40/Chol/DCP 4.0 0 4.0 Aggregation formed 0.079 Sp20/Chol/DCP 4.0 0 4.0 Aggregation formed 0.0738 Consistent with the literature reports [32, 33] the total mass of the process per batch should not exceed 0.0250 g per 4.0 ml of total volume of a solution in 50 ml round bottom flask. For experiments that exceed 0.0250 g, aggregat es usually form at the bottom of the flask after dehydration process. 5.1.2 Determination of Angle of Evaporation and Speed of Rotation During dehydration process, the position of the round bottom flask and the rotation speed of the rotary evaporator were also important factors in producing a smooth

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40 uniform thin film. A series of experime nts was conducted to determine the angle of dehydration by adjusting the rotary evaporator A smooth thin film of Sp60/Niosomes was produced for the angle less than 52.7 with the rotation speed of 1-1.5 rev/sec (Table 12). Table 12: Observation of the thin film from different angles and rotation speeds Sp60/Chol/DCP Mass Per Batch (gram) 0.0204 Nitrogen Flowrate (L/min) 3 Speed ~30 ~45 ~52.7 ~60 1-1.5 rev/sec Smooth thin film, not uniform surface Smooth thin film, not uniform surface Smooth thin film, uniform surface Aggregation form 1.5-2 rev/sec Smooth thin film, not uniform surface Aggregation form Aggregation form Aggregation form 2-3 rev/sec Aggregation form Aggregation form Aggregation form Aggregation form For experiments that exceed 52.7, aggregations usually formed at the bottom of the flask after dehydration process. As a result, the angle of evaporat ion was set at 52.7 and 1-1.5 rev/sec throughout the experiments. 5.1.3 Determination of Nitrogen Flow Rate during the Dehydration Process There were two steps to dehydrating the chloroform, a volatile organic solvent used to dissolve a mixture of Span/choleste rol/dicetyl phosphate. First, a stream of nitrogen gas was passed though the mixt ure containing Span/cholesterol/dicetyl phosphate in a round bottom flask wh ile rotating in the evaporator at a slow flowrate of 3 L/min (inert gas which was used to prevent oxidation of the thin film). Table 13 shows the effect of nitrogen flow rate on the dehydrated thin film after chloroform evaporated slowly upon nitrogen gas contact.

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41 Table 13: The effect of nitrogen flow rate on the dehydrated thin film Stock Solution Mass Per batch (gram) Angle of Evaporation Rotation Speed (rev/sec) Nitrogen Flowrate (L/min) Observation Sp60/Chol/DCP 0.0204 52.7 1-1.5 2 Smooth thin film Sp60/Chol/DCP 0.0204 52.7 1-1.5 3 Smooth thin film Sp60/Chol/DCP 0.0204 52.7 1-1.5 4 Aggregations formed Sp60/Chol/DCP 0.0204 52.7 1-1.5 5 Aggregations formed At higher flowrates, non-uniform thin film or aggregation was usually observed at the end of each run. Once a uniform thin film was established using nitrogen gas, a vacuum rotary evaporator pulled a vacuum from 1013 mmbar to approximately less than 100 mmbar in approximately 15 min to ensure a complete removal of chloroform. 5.1.4 Effect of Different Hydrating Solvents With an appropriate setup and operating pa rameters, a series of experiments was conducted using a stock solution of Span/choleste rol/dicetyl phosphate, prepared earlier. After dehydrating of Span60/c holesterol/dicetyl phosphate, H 2 0, PBS and CF/PBS were used as a hydrating solvent. Using 4.0 ml H 2 0 as a hydrating solvent and hydrating temperature of 40C for 1 hour with 2 minutes sonication ti me, niosomes distributions are determined by Particle Sizing System 780 (Table 14) and micros copic observation of Run #1 (Figure 20).

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42 Table 14: 3 PSS runs of Sp60/Niosomes hydrating in H 2 0@ 40C Caption: 3 runs on sp60/Niosome in H2O, 10 L PSS injection, 40C Hydrating Temperature. Sample Run# Run 1 Run 2 Run 3 Total Particles Sized 291208 303125 299425 Total Particles in Sample 3034386 3070656 2985267 Dilution Factor 10.42 10.13 9.97 Mean (m) 0.76 0.75 0.77 Mode (m) 0.58 0.58 0.58 Median (m) 0.62 0.62 0.62 Average Total Particles in the Sample 3030103 Standard Deviation 42855 Average Mean Particles Sized (m) 0.76 According to Table 14, Total Particles Sized coupled with the Dilution Factor provided the Total Particles in Sample. By taking the result from the 3 runs, Average Total Particles in the Sample was 3,030,103 and the Standard Deviation was 42,855. Notice that the total particle counts of S p60/Niosome lie within one standard deviation (3,030,103,855). The Average Mean Partic les Sized was determined to be 0.76 m.

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123020000400006000080000100000120000140000160000180000200000 Total Count (particles per injection)Diameter (um) 50 m Figure 20: Run #1, Span60/Niosomes hydrating in H 2 0 Note that particle sizing and quantitative analysis of the vesicle dispersion was done by optical light scattering and extinction techniques using a PSS-780. The instrument counts and sizes particles by a combination of light scattering and light extinction technologies, but has a lower detection limit of 0.5 m. Since all of the calculation was accomplished based on particle that are larger than 0.5 m, there is a small error associated with mean particle sizing, counts, counts per ml and the calculated interior volume of niosome vesicles. In other words, the mean particle size would be smaller if PSS-780 detected 43

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44 particles below 0.5 m, count and count pe r ml would be higher. Presently, the calculated interior volume of niosomes would be underestimated. Similarly, using 4.0 ml PBS as a hydrating solvent and hydrating temperature of 40C for 1 hour with 2 minutes sonication time, niosomes distributions are determined by Particle Sizing System 780 (Table 15) and microscopic observation (Figure 21). Table 15: 3 PSS runs of Sp60/Niosomes hydrating in PBS@ 40C Caption: 3 runs on sp60/Niosome in PBS, 10 L PSS injection, 40C Hydrating Temperature. Sample Run# Run 1 Run 2 Run 3 Total Particles Sized 316500 302112 319325 Total Particles in Sample 3206145 3084564 3247535 Dilution Factor 10.13 10.21 10.17 Mean ( m) 0.76 0.76 0.75 Mode ( m) 0.58 0.58 0.58 Median ( m) 0.62 0.62 0.62 Average Total Particles in the Sample 3179415 Standard Deviation 84709 Average Mean Particles Sized ( m) 0.76 According to Table 15, the Average Tota l Particles in the Sample was 3,179,415 and the Standard Deviation was 84,709. No tice that the total particle counts of Sp60/Niosome lie within one standard devi ation (3,179,415,709), si milar to the result obtained previously using H 2 O as hydrating solvent. The Average Mean Particles Sized was also determined to be 0.76 m.

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123020000400006000080000100000120000140000160000180000200000 Total Count (particles per injection)Diameter (um) 50 m Figure 21: Run #1, Span60/Niosomes hydrating in PBS Moreover, using 4.0 ml PBS-CF as a hydrating solvent and hydrating temperature of 40C for 1 hour with 2 minutes sonication time, niosomes distributions are determined by Particle Sizing System 780 (Table 16) and microscopic observation (Figure 22). 45

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46 Table 16: 3 PSS runs of Sp60/Niosomes hydrating in PBS-CF@ 40C Caption: 3 runs on sp60/Niosome in PBS-CF, 10 L PSS injection, 40C Hydrating Temperature. Sample Run# Run 1 Run 2 Run 3 Total Particles Sized 308518 303102 320114 Total Particles in Sample 3776260 3603883 3700518 Dilution Factor 12.24 11.89 11.56 Mean ( m) 0.75 0.76 0.74 Mode ( m) 0.58 0.58 0.58 Median ( m) 0.64 0.64 0.64 Average Total Particles in the Sample 3693554 Standard Deviation 86399 Average Mean Particles Sized ( m) 0.75 Again, according to Table 16, the Average Total Particles in the Sample was 3,693,554 and the Standard Deviation was 86,399. Notice that the total particle counts of Sp60/Niosome lie within one standard devi ation (3,693,554,399), si milar to the result obtained previously using H 2 O and PBS as hydrating solven t. The Average Mean Particles Sized was also determined to be 0.75 m.

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123020000400006000080000100000120000140000160000180000200000 Total Count (particles per injection)Diameter (um) 50 m Figure 22: Run #1, Span60/Niosomes hydrating in PBS-CF Based on the results obtained, niosomes formed using solvents such as H 2 O, PBS or PBS-CF yielded similar niosome mean size distribution (0.75-0.76 m). The total particles detected were in the range of particles per 10 L sample. However, there are aggregates formed as a result of low hydrating temperature of 40C which took place at a temperature below the gel to liquid crystal transition temperature of the surfactant (suggested temperature of 60C) 66107.3100.3 [5] Overall, niosomes can form using H 2 O, PBS or PBS-CF without significant change in particle size and size distribution. For the following experiments, PBS or PBS-CF will be used as a hydrating solvent. 47

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48 5.1.5 Effect of Different Hydrating Temperatures At a higher hydrating temperat ure above gel-liquid transi tion temperate, niosomes are formed with small amount of aggregations. Three expe riments were performed using Span 60 stock solution, 4 ml of PBS for hydr ation, and 60C hydrating temperature for 1 hour and 2 minutes sonication. Results fr om Particle Sizing System 780 (Table 17, Figure 23) and microscopic observa tions are shown in Figure 24. Table 17: 3 PSS runs of Sp60/Niosomes hydrating in PBS-CF@ 60C Caption: 3 runs on sp60/Niosome in PBS-CF, 10 L PSS injection, 60C Hydrating Temperature. Sample Run# Run 1 Run 2 Run 3 Total Particles Sized 298294 293063 314825 Total Particles in Sample 3385637 3050786 3714935 Dilution Factor 11.35 10.41 11.8 Mean ( m) 1.15 1.16 1.14 Mode ( m) 0.58 0.58 0.58 Median ( m) 0.85 0.85 0.85 Average Total Particles in the Sample 3383786 Standard Deviation 332078 Average Mean Particles Sized ( m) 1.15 Based on Table 17, the Average Total Par ticles in the Sample was 3,383,786 and the Standard Deviation is 332,078. Notice that the total particle counts of Sp60/Niosome lie within one standard deviation (3,383, 786,078); similar to the result obtained previously using PBS-CF as hydrating solven t at 40C. However, the Average Mean Particles Sized has increased from 0.75 m to 1.18 m.

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49 246810 0200040006000800010000 Count (particles)Diameter (um) Run#1 PBS-CF,60C hydrating Temperature Run#2 PBS-CF,60C hydrating Temperature Run#3 PBS-CF,60C hydrating Temperature 0.500.751.001.251.501.752.00050001000015000200002500030000350004000045000 Total Count (particles per injection)Diameter (um) Figure 23: Run #1, Run#2 and Run#3, Span60/Niosomes hydrating in PBS-CF

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50 A) 400x1.67 magnification B) 400x1.67 magnification 50 m 50 m C) 400x1.67 magnification 10 m 50 m D) 400x6.67 magnification Figure 24: Sp60/Niosomes in PBS 60C (A), (B) and (C) and (D) at different magnifications In all three experiments, niosomes are formed using PBS at 60C hydrating temperature yielded a larger mean size distribution (1.16-1.19 m), median (0.85 m), and mode (0.58 m) then niosomes produced at 40C. Consistently, three experiments produced very similar particle size distribution (Figure 23) and the total particles detected were in the range of particles per 10 L sample. Taking PSS-780 results of Run#1 (60C hydrating temperature using PBS-CF) and compared it to Run#1 (40C hydrating temperature using PBS-CF), the change in particle distribution can be observed (Figure 25). 66101.3109.2

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12345020000400006000080000100000120000140000160000180000 Total Count (particle per injection)Diameter (um) Run #1:PBS-CF, 40C hydrating Temperature Run #1:PBS-CF, 60C hydrating Temperature. Figure 25: Comparison of Sp60/Niosomes particle size distribution at 40C and 60C Based on Figure 25, hydrating temperature played an important role in successfully producing niosomes with a higher yield (43% increase in particles counted that are greater 0.58 m compared to niosomes produced at 40C, Appendix A) than those produced below gel-liquid transition temperature. For different types of surfactant used in this process, a series of experiment were carried out using stock solution of Span 20, Span 40 and Span 60. Using dehydration/rehydration method described earlier, niosomes was produced using PBS-CF as a hydrating solvent, 60C hydrating temperature for 1 hour and 2 minute sonication time. Niosomes distributions are determined by Particle Sizing System 780 (Table 18, Table 19). 51

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52 Table 18: 3 PSS runs of Sp40/Niosomes hydrating in PBS-CF@ 60C Caption: 3 runs on sp40/Niosome in PBS-CF solution, 10 L PSS injection Sample Run# Run 1 Run 2 Run 3 Total Particles Sized 302748 323115 314456 Total Particles in Sample 3184909 3344240 2867839 Dilution Factor 10.52 10.35 9.12 Mean (m) 1.04 1.07 1.05 Mode (m) 0.58 0.58 0.58 Median (m) 0.85 0.85 0.85 Average Total Particles in the Sample 3132329 Standard Deviation 242514 Average Mean Particles Sized (m) 1.05 According to Table 18, the result from the 3 runs showed that the Average Total Particles in the Sample was 3,132,329 and the Standard Devi ation was 242,517. Notice that the total particle counts of Sp60/Niosome lie within one standard deviation (3,132,329,517). The Average Mean Particles Sized was also determined to be 1.05 m. Table 19: 3 PSS runs of Sp20/Niosomes hydrating in PBS-CF@ 60C Caption: 3 runs on sp20/Niosome in PBS-CF solution, 10 L PSS injection Sample Run# Run 1 Run 2 Run 3 Total Particles Sized 294893 294582 313483 Total Particles in Sample 2842769 3196215 3605055 Dilution Factor 9.64 10.85 11.5 Mean (m) 0.83 0.87 0.84 Mode (m) 0.58 0.58 0.58 Median (m) 0.85 0.85 0.85 Average Total Particles in the Sample 3214679 Standard Deviation 381478 Average Mean Particles Sized (m) 0.85 According to Table 19, the result from the 3 runs showed that the Average Total Particles in the Sample was 3,214,679 and the Standard Devi ation was 381,478. Notice that the total particle counts of Sp60/Niosome lie within one standard deviation (3,214,679,478). The Average Mean Particles Sized was also determined to be 0.85 m.

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Furthermore, taking PSS-780 results of Run#1 of Sp60/Niosomes, Run#1 of Sp40/Niosomes and Run#1 of Sp20/Niosomes and plotted on the same graph, the change in particle distribution can be observed (Figure 26). 0.500.751.001.251.501.752.00020000400006000080000100000120000140000160000180000 Total Count (particles per injection)Diameter (um) Sp20 Run#1: PBS-CF, 60C Sp40 Run#1: PBS-CF, 60C Sp60 Run#1: PBS-CF, 60C 0.550.600.650.700.75020000400006000080000100000120000140000160000180000 Total Count (particles per injection)Diameter (um) 0.751.001.251.501.752.0002000400060008000100001200014000160001800020000220002400026000 Total Count (particles per injection)Diameter (um) Figure 26: Comparison of Sp 20, Sp40 and Sp60/Niosomes using PBS-CF Based on Table 17, Table 18, Table 19, Span60/Niosomes have the largest Average Total Particles of 3,383,786 particles per 10L injection and the Average Mean Particle Sized of 1.18 m as compared to Span40/Niosome (3,132,329 counts per 10 L, 1.05 m) and Span20/Niosome (3,214,679 counts per 10 L, 0.85 m). For Span60/Niosomes, there are greater numbers of particles are formed in the range of 1-2 m. Span 20/Niosome and Span 53

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54 40/Niosome formed smaller particle s are formed closer to or le ss than 1 m. Overall, Span 60/Niosome demonstrated a good candidate for drug delivery applications. 5.1.6 Effect of Particle Formation during Sonication Sonication time also played an important role in the formation of niosomes. A series of experiments was conducted to determin e the effects of sonication on mean size distribution and total population after sonication. First, Span 60/Niosomes were produced using techniques described earlier using PBS-CF as hydrating solvent. After rehydrating process, 10 L of sample was injected to Par ticle Sizing System 780 to record the particle distribution at time zero. The rest of the sample was then placed on the sonicator and, for every minute, 10 L of sample was drawn out to record the particle distribution. Below are results Span60/Niosomes formed 0-4 min sonication (Table 20, Figure 27). Table 20: Sp60/Niosomes particle counts and size distribution from 0-4 minutes sonication time Caption: 0-4 min sonication sp60/Niosome in PBS-CF solution, 10 L PSS injection PSS Sample Sonication Time 0 min 1 min 2 min 3 min 4 min Total Particles Sized 256938 309428 353337 328783 313347 Total Particles in Sample 1264135 2803418 3731239 3501539 2976797 Dilution Factor 4.92 9.06 10.56 10.65 9.50 Mean (m) 1.21 1.17 1.13 1.00 0.95 Mode (m) 0.58 0.58 0.58 0.58 0.58 Median (m) 0.77 0.74 0.70 0.68 0.67

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0.500.751.001.251.501.752.00020000400006000080000100000120000140000160000 55 Total Count (particles per injection)Diameter (um) Sp60 0minsonicate Sp60 1minsonicate Sp60 2minsonicate Sp60 3minsonicate Sp60 4minsonicate 0.550.600.650.700.75020000400006000080000100000120000140000160000 Total Count (particles per injection)Diameter (um)0.751.001.251.501.752.000500010000150002000025000 Total Count (particles per injection)Diameter (um) Figure 27: PSS results, Sp60/Niosomes particle distribution from 0-4 minutes sonication time According to Figure 27, at time zero, Span60/Niosomes had the largest particle distribution where particle are formed in multilamellar structure. After every minute of sonication, particle distribution below approximately 1 m was shifting upward and particle distribution greater than 1 m was shifting downward as a result of small lamella formation. Having observed a well defined particle formation pattern where particle distribution shift to the left (small lamella formation) with respect to sonication time, similar experiments were conducted with Span20/Niosomes and Span40/Niosome. Below were results 0-4 minutes sonication (Table 21, Figure 28, Table 22, Figure 29).

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Table 21: Sp20/Niosomes particle counts and size distribution from 0-4 minutes sonication time Caption: 0-4 min sonication sp20/Niosome in PBS-CF solution, 10 L PSS injection PSS Sample Sonication Time 0 min 1 min 2 min 3 min 4 min Total Particles Sized 272578 296068 294893 298637 273703 Total Particles in Sample 999875 2304347 2843782 3186408 2552591 Dilution Factor 3.67 7.78 9.64 10.67 9.33 Mean (m) 1.11 0.90 0.83 0.82 0.79 Mode (m) 0.58 0.58 0.58 0.58 0.58 Median (m) 0.76 0.70 0.68 0.67 0.66 0.500.751.001.251.501.752.00020000400006000080000100000120000140000160000180000 Total Count (particles per injection)Diameter (um) Sp20 0minsonicate Sp20 1minsonicate Sp20 2minsonicate Sp20 3minsonicate Sp20 4minsonicate 0.550.600.650.700.75020000400006000080000100000120000140000160000180000 Total Count (particles per injection)Diameter (um) 0.751.001.251.501.752.000500010000150002000025000 Total Count (particles per injection)Diameter (um) Figure 28: PSS results, Sp20/Niosomes particle distribution from 0-4 minutes sonication time 56

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Table 22: Sp40/Niosomes particle counts and size distribution from 0-4 minutes sonication time Caption: 0-4 min sonication sp40/Niosome in PBS-CF solution, 10 L PSS injection PSS Sample Sonication Time 0 min 1 min 2 min 3 min 4 min Total Particles Sized 314758 302748 324115 313710 303525 Total Particles in Sample 762212 2272110 3410857 3700323 3084138 Dilution Factor 2.42 7.50 10.52 11.80 10.16 Mean (m) 1.17 1.11 1.04 0.97 0.97 Mode (m) 0.58 0.58 0.58 0.58 0.58 Median (m) 0.86 0.72 0.72 0.70 0.70 0.500.751.001.251.501.752.00020000400006000080000100000120000140000160000 Total Count (particles per injection)Diameter (um) Sp40 0minsonicate Sp40 1minsonicate Sp40 2minsonicate Sp40 3minsonicate Sp40 4minsonicate 0.550.600.650.700.75020000400006000080000100000120000140000160000 Total Count (particles per injection)Diameter (um) 0.751.001.251.501.752.000500010000150002000025000 Total Count (particles per injection)Diameter (um) Figure 29: PSS results, Sp40/Niosomes particle distribution from 0-4 minutes sonication time According to Figure 28 and Figure 29, similar to experiment perform using Span60/Niosomes, Span20/Niosomes and Span40/Niosomes had the largest particle distribution where particles were formed in multi-lamellar structure at time zero. After 57

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58 every minute of sonication, particle distri bution below approximately 1 m was shifting upward and particle distribution greater than 1 m was shifting downwar d as a result of small-lamellar formation. According to Figure 30, the total counts of Span20/Niosomes, Span40/Niosomes and Span60/Niosomes increased as samples we re sonicated from 0 to approximately 2-3 minutes. However, for sonication times greater than 3 minutes, particle counts decreased as a result of particles that are formed less than 0.5 m (below the detection limit of PSS780). For optimization of niosome production, a 2 minute sonication time was set for all experiments. The particle average diameter also decreased with sonication time as a result of small-lamellar formation (Figure 31). For Span20/Niosomes, Span40/Niosomes and Span60/Niosomes, particles average diameter decreased from 1.11-0.79 m, 1.17-0.97 m and 1.21-0.95 m respectively (Figure 31).

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594 012360000080000010000001200000140000016000001800000200000022000002400000260000028000003000000320000034000003600000380000040000004200000 Total Counts (particles)Sonication Time (min) Sp20/Niosome Sp40/Niosome Sp60/Niosome Figure 30: PSS total particle counts of Sp20, Sp40 and Sp60/Niosomes from 0-4 minutes sonication

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604 01230.750.800.850.900.951.001.051.101.151.201.251.30 Average Diameter (um)Sonication Time (min) Sp20/Niosome Sp40/Niosome Sp60/Niosome Figure 31: PSS average particle distribution of Sp20, Sp40 and Sp60/Niosomes from 0-4 minutes sonication Figure 32 demonstrated the size reduction of prepared Span 60/Niosomes after 2 minutes sonication based on microscopic observation with hemocytometer slice for a clear comparison. A) 50 m 50 m B) Figure 32: Sp60/Niosomes observation A) before sonication and B) after sonication

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61 5.2 5(6)-Carboxyfluorescein A calibration curve relating intensity to concentration was established to determine entrapment efficiency of CF inside niosomes. 5.2.1 5(6)-Carboxyfluorescein Standard Curve During rehydration, solution containing CF and sodium hydroxide (NaOH) in PBS is used as a drug model. In general, a direct measurement of quantities of CF encapsulated and leaked out of niosomes can be calculated using standardized curve measurement. Briefly, a series of known CF concentrations were prepared from the CF stock solution. The intensity reading for each concentration using the fluorometer is shown in Table 23, Figure 33 and Figure 34.

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62 Table 23: CF intensities with respect to known concentrations Stock Solution Concentration (mol/L) Volume of Stock Solution Injection (ml) Volume of PBS injection (ml) Total Volume (ml) Average Intensity Concentration (mol/L) 5.00E-03 1 0 1 2.3 5.00E-03 5.00E-04 1 0 1 47.5 5.00E-04 5.00E-05 1 0 1 835.5 5.00E-05 5.00E-06 1 0 1 556.3 5.00E-06 5.00E-07 0.9 0.1 1 176.3 4.50E-07 5.00E-07 0.9 0.2 1.1 159.7 4.09E-07 5.00E-07 0.9 0.3 1.2 140.6 3.75E-07 5.00E-07 0.9 0.4 1.3 119.4 3.46E-07 5.00E-07 0.9 0.5 1.4 108 3.21E-07 5.00E-07 0.9 0.6 1.5 100.3 3.00E-07 5.00E-07 0.9 0.7 1.6 93.4 2.81E-07 5.00E-07 0.9 0.8 1.7 88 2.65E-07 5.00E-07 0.9 0.9 1.8 82.8 2.50E-07 5.00E-07 0.9 1 1.9 78.6 2.37E-07 5.00E-07 0.9 1.1 2 74.5 2.25E-07 5.00E-07 0.9 1.2 2.1 70.7 2.14E-07 5.00E-07 0.9 1.3 2.2 67.1 2.05E-07 5.00E-07 0.9 1.4 2.3 64.3 1.96E-07 5.00E-07 0.9 1.5 2.4 61.7 1.88E-07 5.00E-07 0.9 1.6 2.5 59.2 1.80E-07 5.00E-07 0.9 1.7 2.6 56.8 1.73E-07 5.00E-07 0.9 1.8 2.7 54.8 1.67E-07 5.00E-07 0.3 0.7 1 62.8 1.50E-07 5.00E-07 0.3 0.8 1.1 55.3 1.36E-07 5.00E-07 0.3 0.9 1.2 47.1 1.25E-07 5.00E-07 0.3 1 1.3 42.1 1.15E-07 5.00E-07 0.3 1.1 1.4 39 1.07E-07 5.00E-07 0.3 1.2 1.5 36.2 1.00E-07 5.00E-07 0.3 1.3 1.6 34 9.38E-08 5.00E-07 0.3 1.4 1.7 31.9 8.82E-08 5.00E-07 0.3 1.5 1.8 30.2 8.33E-08 5.00E-07 0.3 1.6 1.9 28.6 7.89E-08 5.00E-07 0.3 1.7 2 27.1 7.50E-08 5.00E-07 0.3 1.8 2.1 25.8 7.14E-08 5.00E-07 0.3 1.9 2.2 24.5 6.82E-08 5.00E-07 0.3 2 2.3 23.4 6.52E-08 5.00E-07 0.3 2.1 2.4 22.5 6.25E-08 5.00E-07 0.3 2.2 2.5 21.6 6.00E-08 5.00E-07 0.3 2.3 2.6 20.7 5.77E-08 5.00E-08 0.9 0.1 1 18.8 4.50E-08 5.00E-08 0.9 0.2 1.1 16.4 4.09E-08 5.00E-08 0.9 0.3 1.2 14 3.75E-08 5.00E-08 0.9 0.4 1.3 12.6 3.46E-08 5.00E-08 0.9 0.5 1.4 11.6 3.21E-08 5.00E-08 0.9 0.6 1.5 10.8 3.00E-08 5.00E-08 0.9 0.7 1.6 10.2 2.81E-08 5.00E-08 0.9 0.8 1.7 9.6 2.65E-08 5.00E-08 0.9 0.9 1.8 9 2.50E-08 5.00E-08 0.9 1 1.9 8.5 2.37E-08 5.00E-08 0.9 1.1 2 8.1 2.25E-08 5.00E-08 0.9 1.2 2.1 7.7 2.14E-08 5.00E-08 0.9 1.3 2.2 7.4 2.05E-08 5.00E-08 0.9 1.4 2.3 7 1.96E-08 5.00E-08 0.3 0.7 1 6.4 1.50E-08 5.00E-08 0.3 0.8 1.1 5.5 1.36E-08 5.00E-08 0.3 0.9 1.2 4.7 1.25E-08 5.00E-08 0.3 1 1.3 4.2 1.15E-08 5.00E-08 0.3 1.1 1.4 3.8 1.07E-08 5.00E-08 0.3 1.2 1.5 3.6 1.00E-08 5.00E-08 0.3 1.3 1.6 3.4 9.38E-09 5.00E-08 0.3 1.4 1.7 3.1 8.82E-09 5.00E-08 0.3 1.5 1.8 3 8.33E-09 5.00E-08 0.3 1.6 1.9 2.8 7.89E-09 5.00E-08 0.3 1.7 2 2.7 7.50E-09 5.00E-08 0.3 1.8 2.1 2.5 7.14E-09 5.00E-08 0.3 1.9 2.2 2.4 6.82E-09 5.00E-08 0.3 2 2.3 2.3 6.52E-09 5.00E-08 0.3 2.1 2.4 2.2 6.25E-09 5.00E-08 0.3 2.2 2.5 2.1 6.00E-09

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1E-81E-71E-61E-51E-41E-30100200300400500600700800900 IntensityConcentration (mol/l) Figure 33: Standard curve of florescence intensity vs. concentration of CF According to Figure 34, a linear relationship between intensity and CF concentration was observed in a low concentration range of mol/l. However, at a higher concentration of CF, the relative intensity decreased as a result of CFs self quenching described earlier. Since a linear relationship was observed at lower end of the CF concentration, fitting the data points was accomplished using a trend-line program (Figure 34 and Equation 5). 71050 (mole/L) ionconcentratC andintensity I where1060.38CI Equation 5 63

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Therefore, dilution of encapsulated CF with Span/Niosomes before proceeding to the measurement of CF using fluorometer was necessary to prevent a misleading reading from self quenching phenomena. 0.03.0x10-86.0x10-89.0x10-81.2x10-71.5x10-71.8x10-70102030405060 I=3.60E8xCR2=.994IntensityCF Concentration (mole/L) Measured Intensity Linear Fit of CF Measured Intensity Figure 34: Standard curve of florescence intensity vs. concentration of CF (linear fit) Furthermore, to determine the variability between CF batches (stock solution), the intensities readings of CF stock solution were recorded and compared between batch #1 and batch #2. Batch #2 was made and tested for their consistency using the same procedures of that batch #1 (Figure 35). 64

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1E-51E-4450475500525550575600625650675700725750775800825850875900925 IntensityCF Concentration (mole/L) Batch #1 Batch #2 Figure 35: Comparison of CF stock solution batch #1 and batch #2 based on concentration and intensity Notice that the concentration and their intensity of the two stock solutions are in the range of % of intensity values obtained in batch #2 using batch #1 as a standard solution. As a result, new stock solution was used in the experiment without creating a new standard curve. 5.3 Quantitative Measurements used in Niosome Characterization With an appropriate setup (mass per batch, angle of evaporation, rotation speed and nitrogen flowrate during dehydration process) and operating parameters (types of 65

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66 hydrating solvents, hydrating temp erature and sonication time), a series of experiments were conducted using PBS-CF rehydrating solution of Span20/Niosomes, Span40/Niosomes and Span60/Niosomes. Qu antitative measurements based on light scattering optical microscopy, UV micr oscopy, fluorometer and PSS-780 were conducted. First, the stability of niosome vesicles in PBS-CF solution over time is observed using PSS-780. Second, separating of unencapsulated CF using GEC is characterized using PSS-780 and fluorometer. Th irdly, after GEC, CF entrapped inside niosomes is observed under UV microscope. La stly, the amount of CF entrapped inside niosomes can be determined using fluoromet ry and previously obtained standard CF curve (Section 5.2). 5.3.1 Effect of Niosome Vesicles in PBS-CF Solution over Time To determine their stability, Span60/ Niosome batch using PBS-CF hydrating solvent with 2 minutes sonication were kept se parately for 2 week period. The stability of the vesicles over time is demonstrated in Figure 36. Dispersions of Span60/CF niosomes were monitored for 2 weeks for cha nges in particle size distribution and mean particle size using Particle Sizing System 780 (Table 24 and Figure 36). Table 24: Comparison of Sp60/Niosomes particle counts and mean size of particles 0-14 days Caption: span 60 Day 0-Day 14 10 L injection PBS-CF Day 0 Day 1 Day 14 Total Particles Sized 312943 314825 325961 Total Particles in Sample 3311120 3714429 3650685 Dilution Factor 10.58 11.8 14.27 Fluid Volume Sampled (ml) 60 60 60 Mean (m) 1.15 1.19 1.24 Mode (m) 0.58 0.58 0.58 Median (m) 0.85 0.85 0.85

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123456789100500010000150002000025000300003500040000 67 Span60/Niosomes Day 0 Span60/Niosomes Day 1 Span60/Niosomes Day 14Count (particles)Diameter (um) 234567891005001000150020002500300035004000 Count (particles)Diameter (um) 1-10 m scale Figure 36: Sp60/Niosomes particle size distribution 14 days after rehydrating process The increase in particle counts in the range of greater than 2 m for Day 1 after rehydrating process as compared to Day 0 was computed to be 9% and 29% for Day 14 (Appendix B). The shift in mean particle size (Table 24) may be due to aggregation. 5.3.2 Separation Pattern Using Gel Exclusion Chromatography Next, Span20, Span40 or Span60/Niosomes solutions were placed on top of the gel exclusion chromatography described earlier to separate out the unencapsulated dye.

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68 Investigations of how this column operates for Span60/Niosomes at different sample size of 0.1 ml, 0.25 ml and 1.0 ml were perf ormed (Table 25, Table 26 and Table 27). The data in the first and second column in Table 25, Table 26 and Table 27 represents the volume of sample collected and cumulative volume of sample. The third column shows the volume injection of collect ed sample into PSS-780. The data in the forth and the fifth column (Count and Count/ml) was results obtained from PSS-780. Finally, the last column was calculated ba sed on the product of the Counts/ml (PSS780) and Volume of Sample collected to obtain the number of particles in the sample. Furthermore, the sum of all of the particles in the last column was compared to the initial count. Percent recoveries of the 3 experi ments were reported (Table 25, Table 26 and Table 27). Table 25: GEC separation of unencapsulated CF from 0.25 ml injection of Sp60/Niosomes Span60/Niosomes 0.25 ml injection Volume of Sample Collected (ml) Cumulative volume (ml) PSS volume Inject (ml) Count (PSS) Count/ml (PSS) Total Count 3 3 0.5 134947 269894 809682 1 4 0.5 302707 605414 605414 1 5 0.5 5015185 10030370 10030370 1 6 0.25 4361015 17444060 17444060 1 7 0.25 2880461 11521844 11521844 1 8 0.25 1664320 6657280 6657280 1 9 0.25 1238906 4955624 4955624 1 10 0.25 911021 3644084 3644084 1 11 0.25 751149 3004596 3004596 5 16 0.5 103618 207236 1036180 10 26 0.5 75156 150312 1503120 Final Count 61212254 Initial Count 76284375 % of Recovery ~80%

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69 Table 26: GEC separation of unencapsulated CF from 1.00 ml injection of Sp60/Niosomes Span60/Niosomes 1.00 ml injection Volume of Sample Collected (ml) Cumulative volume (ml) PSS volume Inject (ml) Count (PSS) Count/ml (PSS) Total Count 3 3 0.5 189916 379832 1139496 1 4 0.5 191779 383558 383558 1 5 0.1 91992 919920 919920 1 6 0.01 488153 48815300 48815300 1 7 0.01 549349 54934900 54934900 1 8 0.01 446928 44692800 44692800 1 9 0.01 307263 30726300 30726300 1 10 0.01 223464 22346400 22346400 1 11 0.01 130354 13035400 13035400 5 16 0.01 34913 3491300 17456500 10 26 0.01 5363 536300 5363000 Final Count 239813574 Initial Count 321476353 % of Recovery ~75% Table 27: GEC separation of unencapsulated CF from 0.10 ml injection of Sp60/Niosomes Span60/Niosomes 0.10 ml injection Volume of Sample Collected (ml) Cumulative volume (ml) PSS volume Inject (ml) Count (PSS) Count/ml (PSS) Total Count 3 3 0.5 153279 306558 919673 1 4 0.5 281163 562325 562325 1 5 0.5 4221766 8443531 8443531 1 6 0.5 3528291 7056582 7056582 1 7 0.5 1926008 3852015 3852015 1 8 0.5 1432339 2864678 2864678 1 9 0.5 938226 1876452 1876452 1 10 0.5 762494 1524988 1524988 1 11 0.5 598329 1196658 1196658 5 16 0.5 57164 114327 571636 10 26 0.5 19562 39125 391248 Final Count 29259786 Initial Count 33976389 % of Recovery ~86%

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Notice that the initial and final counts were decreased in all three cases (76,284,375 to 61,212,254 counts in 0.25 ml injection, 321,476,353 to 239,813,574 counts in 1.00ml injection and 33,976,389 to 29,259,786 counts in 0.10ml injection). It was estimated that 80% recovery was obtained in the 0.25 ml injection, 75% recovery was obtained in the 1.00 ml injection and 86% recovery was obtained in the 0.10 ml injection. As a result, 0.10 ml of sample injection was set for all gel exclusion chromatography separation processes. Based on result obtained from Table 25, Table 26 and Table 27, the separation pattern can be observed through the particle counts per volume collected (Figure 37). 24681012141618202224260500000010000000150000002000000025000000300000003500000040000000450000005000000055000000 Counts per mlCumulative Volume Collected 0.10ml injection GEC 0.25ml injection GEC 1.00ml injection GEC Figure 37: GEC counts per ml vs. cumulative volume collected 70

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71 Based on Table 25, Table 26 and Table 27 and Figure 37, most of the particles exited the gel exclusion chromatography column betw een 4-26 ml volume collected with the maximum amount of Span60/Niosomes ar ound 6ml sample collection for 0.25 ml injection, 7 ml sample collection for 1.00 ml injection and 5ml sample collection for 0.10 ml injection. Overall, for the following gel exclusion chromatography separation experiment, the samples were collected at 5-10 ml cumulative volume where the highest particle counts were observed. Furthermore, based on the collected particle mean diameter of Span60/Niosomes, highest particle distribution around 5-15 cu mulative volume were observed in both 0.25 ml sample injection and 0.10 ml sample inject ion (Table 28 and Figure 38). For 1.00 ml sample injection, niosomes did not exit the column at a particular distribution (only increased in mean particle diameter). Table 28: Particle size distribution of Sp60/Niosomes sample collected after GEC GEC Results (PSS-780): Mean Diameter for various Injection of Span60/Niosomes Volume of Sample Collected (ml) Cumulative volume (ml) 1.00ml Injection ( m) 0.25ml Injection ( m) 0.10ml Injection ( m) 3 3 0.76 0.90 0.91 1 4 0.77 0.91 0.91 1 5 0.77 0.91 0.92 1 6 0.93 0.94 0.91 1 7 0.94 0.93 0.94 1 8 0.93 0.94 0.94 1 9 0.94 0.96 0.94 1 10 0.95 0.95 0.96 1 11 0.96 0.97 0.98 5 16 1.05 0.95 0.97 10 26 1.09 0.94 0.93 Notice that the particle mean diameters determined by PSS-780 are less than 1 m in both 0.25 ml sample injection and 0.10 ml sample injection (Table 28). This is because,

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during the separation process, diluting media provide trace of small impurities that resulting in the shift in particle distribution to a lower range. 0510152025300.60.70.80.91.01.11.2 0510152025300.60.70.80.91.01.11.2 0510152025300.60.70.80.91.01.11.2 Mean Diameter (um)Cumulative Volume Collected (ml) 0.1ml injectionMean Diameter (um)Cumulative Volume Collected (ml) 1.00ml injectionMean Diameter (um)Cumulative Volume Collected (ml) 0.25ml injection 1 standard deviation error bar Figure 38: GEC, mean diameter vs. cumulative volume By taking the result from PSS-780 obtained previously for 0.1 ml injection GEC, similar relationships between particle counts per ml and CF intensity measurements using a fluorometer were observed (Table 29 and Figure 39). 72

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Table 29: Comparison of counts per ml and background intensity vs. cumulative volume of sample Cumulative volume (ml) Count/ml (PSS) Recorded Intensity Average Intensity 3 306558 0.0 0.0 0.0 0.0 4 562325 0.1 0.1 0.1 0.1 5 8443530 7.6 7.7 7.8 7.7 6 7056580 7.6 7.7 7.6 7.6 7 3852020 6.2 6.1 6.3 6.2 8 2864680 4.8 4.8 5.0 4.9 9 1876450 3.1 3.5 3.3 3.3 10 1524990 1.5 1.7 1.3 1.5 11 1196660 0.7 0.8 0.7 0.7 16 114327 0.1 0.1 0.1 0.1 26 39125 0.1 0.0 0.0 0.0 2468101214161820222426280246810 Relative IntensityCumulative Volume Collected (ml) Rel. intensity, 0.1ml injection GEC2468101214161820222426280100000020000003000000400000050000006000000700000080000009000000 Count per mlCumulative Volume Collected (ml) Count/ml, 0.1ml injection GEC Figure 39: Comparison of counts per ml and background intensity vs. cumulative volume of sample 73

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74 Notice that particle counts and CF intensit y had a highest peak at 5 ml collection and highest distribution between 5-10 ml of samp le collection. Based on collected sample, 0.5 ml of Span20/Niosomes, Span40/Niosomes and Span60/Ni osomes (0.1 ml injection into gel exclusion chromatography separation) were injected to PSS-780 and result was observed (Table 30, Table 31, Table 32 and Figure 40). Table 30: 2 PSS runs of Sp20/Niosomes after GEC (5-10 ml cumulative volume and 0.5 ml injection) Caption: 2 runs on sp20/Niosome Draw #2 after GEC Sample Run# Run 1 (0.5 ml injection) Run 2 (0.5 ml injection) Total Particles Sized 287422 266019 Total Particles in Sample 1655168 1920591 Dilution Factor 5.76 7.22 Mean (m) 0.94 0.95 Mode (m) 0.58 0.58 Median (m) 0.66 0.67 Table 31: 2 PSS runs of Sp40/Niosomes after GEC (5-10 ml cumulative volume and 0.5 ml injection) Caption: 2 runs on sp40/Niosome Draw #2 after GEC Sample Run# Run 1 (0.5 ml injection) Run 2 (0.5 ml injection) Total Particles Sized 292319 297572 Total Particles in Sample 1446979 1234924 Dilution Factor 4.95 4.15 Mean (m) 0.95 1.02 Mode (m) 0.58 0.58 Median (m) 0.71 0.74 Table 32: 2 PSS runs of Sp60/Niosomes after GEC (5-10 ml cumulative volume and 0.5 ml injection) Caption: 2 runs on sp60/Niosome Draw #2 after GEC Sample Run# Run 1 (0.5 ml injection) Run 2 (0.5 ml injection) Total Particles Sized 282137 279057 Total Particles in Sample 1690219 2434632 Dilution Factor 5.99 8.72 Mean (m) 0.99 0.98 Mode (m) 0.58 0.58 Median (m) 0.76 0.74

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0.500.751.001.251.501.752.00010002000300040005000600070008000900010000110001200013000 Count (particles)Diameter (um) Span20 5-10ml sample 0.5ml injection Span40 5-10ml sample 0.5ml injection Span60 5-10ml sample 0.5ml injection Figure 40: Comparison of Run#1 from Sp20, Sp40 and Sp60/Niosomes formation after GEC Based on Figure 40, there are similarities between the plots behaviors obtained prior to gel exclusion chromatography separation as described earlier (Figure 26). Furthermore, particles size distribution was obtained in the range of 0.94-0.99 m with the particles count in the range of counts per 0.5 ml or approximately vesicle counts per ml/per mass of surfactant (Table 11, Table 30, Table 31 and Table 32). 66103.2108.1 81079.146.1 75

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5.3.3 UV Microscopic Observation Following the previous process, samples collected between 5-10 ml after running through gel exclusion chromatography separation, were examined under the light/UV microscope to demonstrate that niosomes have encapsulated CF. A small droplet of collected sample was placed on a slide under the microscope and observed at 67.1400 magnification. Presence of CF inside Span60/Nisomes was observed in Figure 41. A) B) Figure 41: Observation of Sp60/Niosomes under A) regular light scattering mode and B) UV mode Span60/Nisosomes were observed under regular light scattering mode (A). Switching off to the UV mode, bright fluorescent green dots were observed (B). Based on this observation, it was clear that Span60/Niosomes encapsulated CF inside the vesicle membrane and maintained their structural integrity inside a solution. 5.3.4 Leakage Studies Sample of Span20/Niosomes, Span40/Niosomes and Span60/Niosomes collected between 5-10 ml after gel exclusion chromatography separation were kept for 9 days in 76

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the dark for leakage analysis. Starting from day zero, 0.5 ml of a sample and 0.5 ml of PBS was placed in the cuvette to take an initial reading from fluorometer. Then, 20 L of Triton X-100 was added into the sample to disrupt the membrane to obtain the final reading. For the following days, the same procedures were repeated to determine the leakage from niosomes (Figure 42 and Table 33). 0123456789912151821 0123456789912151821 0123456789912151821 0123456789912151821 0123456789912151821 0123456789912151821 Sp60/CF final reading Sp40/CF initial reading Sp20/CF initial readingIntensityDays Sp60/CF initial readingIntensityDays Sp40/CF final readingIntensityDays Sp20/CF final reading 1 standard deviation error bar Figure 42: Stability of Sp20, Sp40 and Sp60/Niosomes, day 0 to day 9 77

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78 Table 33: Leakage study of Sp20, Sp40 and Sp60/Niosomes from day 0 to day 9 Membrane permeability of various Niosomes, Day # 0-9 using CF intensity measurement Span 20 Span 40 Span 60 Day Initial Intensity Final Intensity Initial Intensity Final Intensity Initial Intensity Final Intensity 15.20 22.90 12.50 16.70 9.70 14.80 15.40 22.30 13.20 17.20 8.20 13.20 0 15.80 22.80 14.80 19.10 8.20 13.10 Average 15.47 22.67 13.50 17.67 8.70 13.70 Span 20 Span 40 Span 60 Day Initial Intensity Final Intensity Initial Intensity Final Intensity Initial Intensity Final Intensity 15.40 21.40 13.70 17.50 11.10 15.70 15.00 21.10 14.40 18.70 11.10 16.40 1 15.70 21.60 15.20 19.30 9.90 14.80 Average 15.37 21.37 14.43 18.50 10.70 15.63 Span 20 Span 40 Span 60 Day Initial Intensity Final Intensity Initial Intensity Final Intensity Initial Intensity Final Intensity 15.20 21.70 12.20 16.60 9.40 14.00 15.80 22.70 11.60 15.30 9.60 14.70 2 16.00 22.90 12.30 16.20 9.50 14.30 Average 15.67 22.43 12.03 16.03 9.50 14.33 Span 20 Span 40 Span 60 Day Initial Intensity Final Intensity Initial Intensity Final Intensity Initial Intensity Final Intensity 15.10 21.10 12.70 17.30 8.80 13.60 14.20 20.40 11.80 15.70 9.10 14.10 3 14.60 20.70 12.00 16.60 9.60 14.90 Average 14.63 20.73 12.17 16.53 9.17 14.20 Span 20 Span 40 Span 60 Day Initial Intensity Final Intensity Initial Intensity Final Intensity Initial Intensity Final Intensity 13.50 21.10 10.90 15.50 7.80 12.10 13.70 20.40 10.80 15.60 7.80 12.10 7 13.90 20.70 10.20 14.40 8.80 13.30 Average 13.70 20.73 10.63 15.17 8.13 12.50 Span 20 Span 40 Span 60 Day Initial Intensity Final Intensity Initial Intensity Final Intensity Initial Intensity Final Intensity 13.70 19.60 10.60 13.40 8.20 13.50 13.20 19.20 10.70 13.60 8.50 13.70 9 13.50 19.40 10.50 13.20 8.40 13.60 Average 13.47 19.40 10.60 13.40 8.37 13.60

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In Figure 42, the initial value represents the background fluorescence of the sample, and the final intensity of the fluorescence signal after the vesicles were disrupted using Triton X-100. Apparent fluctuation in the results may have been caused by inherent variability of injection volumes ( by volume for pipette injections at 10 L). The overall trends of these data indicated that the vesicles show very little leakage over 9 days period. %5 Based on the average intensity increased (Figure 43) after adding Triton X-100 and the result obtained from the ASCII file output from Particle Sizing System 780, entrapment efficiency was calculated based on the Equation 1, Equation 2, Equation 3 and Equation 4. 012345678902468 012345678902468 012345678902468 IntensityDays Span60/NiosomesIntensityDays Span40/NiosomesIntensityDays Span20/Niosomes 1 standard deviation error bar Figure 43: Average intensity increased for Sp20, Sp40 and Sp60/Niosomes from day 0 to day 9 79

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80 The result of the calculation is shown below: Table 34: Summary of Sp20 Sp40 and Sp60/Niosomes CF entrapment percentage Surfactant Interior volume (cm3) Average Intensity (Day #0) CF entrap (mole) Actual CF (mole) Concentration (mol/l) Percent Entrapment Sp20 8.23E-06 7.2 2.01E-11 4.11E-11 2.44E-03 48.8% Sp40 4.71E-06 4.2 1.16E-11 2.36E-11 2.47E-03 49.3% Sp60 4.43E-06 5.0 1.39E-11 2.21E-11 3.15E-03 62.9% The concentration of CF entrapped in niosom es was calculated to be 48.8-62.9% of the original 5 mM. The concentration of CF insi de niosomes was estimated to be 2.44-3.15 mM for Span 60, Span40 and Span20. Consistent w ith literatures [34, 31, ] 60 the increased size of the lipophilic alkyl chai n of surfactants (Span 60, Span40 and Span20) has shown to produce larger vesicles which would expl ain the increasing entrapment capability of the vesicles.

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81 CHAPTER SIX: CONCLUSIONS The present study has partia lly optimized synthesis technique for niosome as a drug carrier system and characterized its ability to encapsulate drug model 5(6)carboxyfluorescein (CF) and leakage studies. Vesicles were formed by the combination of surfactants (Span 20, Span40 and Span60) cholesterol and dicet yl phosphate using appropriate setup and operating parameters. This included optimization parameters such as mass per batch, angle of evaporation, dehydration nitrogen flowrate, hydrating solvents, hydrating temperature and sonication time (Table 35). Table 35: Summary of optimized parame ters in making Span/niosome system Flask Size Molar Ratio Mass Per Batch Angle of Evaporation Nitrogen Flowrate Hydrating Solvent Hydrating Temperature. Sonication Time Sp60 50ml 7.94:7.78:1.0 0.02040 ~52.7degree ~1-1.5 rev/sec PBS/PBS-CF 60C 2 min Sp40 50ml 7.94:7.78:1.1 0.01976 ~52.7degree ~1-1.5 rev/sec PBS/PBS-CF 60C 2 min Sp20 50ml 7.94:7.78:1.2 0.01846 ~52.7degree ~1-1.5 rev/sec PBS/PBS-CF 60C 2 min After gel exclusion chromatogr aphy separation (0.1 ml injection and 5-10 ml cumulative volume collected after running the sample), niosome vesicles were observed under both light microscope and UV microscope to demonstrate CF entrapment. They are stable in solution over time (approximately 9 days) and exhibit low leakage over the time. Vesicle size was dependent on choice of surfactant where Span 60 surfactant has shown to produce the largest particle size distribution (0.99 m). The highest entrapment percentage based on calculated particle volume using Particle Sizing System 780 and CF

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measurement from fluorometer was obtained from Span60/Nisomes (62.9%) follow by Span40/Niosome (49.3%) and Span20/Niosome (48.8%). Typical concentrations of vesicle per ml/per mass of surfactant used were in the range of Based on the choice of surfactants (Span 20, Span 40 or Span 60), the increase in size of alkyl chain in making niosomes (increase in HLB values of Span 20, Span 40 and Span 60 respectively according to Table 2) has shown to produce larger vesicles which would explain the increase in entrapment capability. 81079.146.1 6.1 Future Work: Using Niosomes in Cardiovascular Therapy In this present study, CF is used specifically for this model and has been entrapped by niosome vesicles. For future study, repeated investigation is necessary when using a chosen drug, and depending on the molecular structure, as well as the physical and chemical properties of the drugs and their respective synthesis techniques, characterization protocols and quantitative measurements may be necessary. In reference to atherosclerotic plaque, it has become apparent that inflammation plays a key role in the plaque development [60, , ] 61 62 63 64 It has also been recognized that pentameric protein in liver, fibrinogen, leukocyte counts, inflammatory cytokines, adhesion molecules and makers of leukocyte activation are involved in the atherosclerosis process [61, 62, 63] Many researchers have developed and tested the effectiveness of cardiovascular drug such as statins, ACE-Inhibition/Angiotensin II receptor blockade and peroxisome proliferator activated receptors (PPAR-) to reduce inflammatory biomarker in atherosclerosis [] 65 Some of these are clinically available, others are available for experimentation. Although these drugs have shown promising 82

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83 results, drug delivery systems may increase the effectiveness by delivering active ingredients, minimized the side effects by inhibiting uptake by the immune system and lowering toxicity to unaffected tissues. 6.1.1 Statins Clinically, inhibition of this pathway by statins, potent inhi bitors of 3-hydroxy-3methyl-CoA (HMG-CoA) reductase, has been shown to reduce plas ma levels of LDL cholesterol and several clinical trials with this group of drugs have demonstrated a remarked reduction in monocyteendothelial interaction in at herosclerosis during plaque progression [ , ] 66 67 68 69 Several datas also suggested that the benefits of drug interventions such as statin therapy is the most effective among those with elevated inflammatory markers and medi ators such as CRP, cytokines, cellular adhesion molecule levels, platelet activation fact ors of vascular inflammation [ , , , , , ] 70 71 72 73 74 75 76 77 78 79 80 Other studies have shown that lipid-lowering therapy using statin-derivative therapy changes plaque morphology and leads to coronary plaque stabilization [ , ] 81 82 83 Above all are beneficial results of this class of drug, however, recent focus on the effects of statins that are more frequently observed at higher doses and in combination with other lipid lowering drugs may need careful considera tion in individuals at risk of adverse drug reactions based on genetic variability of an individual and the analysis of genetic variants of drug therapy [ ] 84 85

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84 6.1.2 ACE-Inhibition/Angiotensin II Receptor Blockade Angiotensin converting enzyme (ACE) inhi bitors has been sh own to reduce the development of atherosclerosis in hyperchol esterolemic animals ac ross a wide range of species and has proven to be effective in reducing the surface area of the lesions [ , ] 86 87 88 Although the mechanism for these effects has not been well defined, it was assumed that both Angiotensin II suppression and interfer ence with the breakdown of bradykinin, nanopeptides which help increase vascular permeability, dilate blood vessels, contract non-vascular smooth muscle and may cause pain are involved. In one study, by blocking the degradation of bradykinin, ACE inhibitors potentiate the abili ty of bradykinin to reduce blood pressure and stimulate the releas e of tissue-type plasminogen activator from the vasculature which resulte d in removing the blockage [] 89 Another study in a genetically hypercholesterolemic rabbit mode l showed that suppression of the reninangiotensin system by ACE inhibition causes comparab le inhibition of aortic atherosclerosis and mild reduction of blood pressure [] 90 Finally, synthesized or nonsulfhydryl ACE inhibitor, enalap ril, injected into cholestero l-fed rabbits has been shown to significantly reduce the percen t plaque areas in the aortas [] 91 6.1.3 PPARAgonist Several animal studies have shown that fibrate treatment for ligands, peroxisome proliferator-activated receptor (PPAR), leads to a reduction of the coronary events associated with factors related to atheroscle rosis. One study has s hown that the effective treatment with the PPARagonist on the development of atherosclerotic lesions in apolipoprotein (apo) E-deficient mice resulted in the reduction of aortic cholesterol

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85 content [] 92 Other studies have shown that PPAR(Wy 14643) partially inhibit ox-LDL and cytokine such as TNFand reduce transforming growth factor (TGF), highly expressed in the lesions, whic h suggested beneficial effect s on the vascular wall of atherosclerotic plaque [ , , ] 93 94 95 96 97 98 Finally, the influence of PPARactivators on intracellular cholesterol homeostasis was investigated and found to decrease the cholesterol ester (CE):free cholesterol (FC) ratio as a result of blocking TNFinduced CE formation [ , ] 99 100 101 Above all, PPARhas shown a positive result in animal experimentation, however, this particular drug has not been proven effective in some experiments such as severely aggravates hypercholesterolemia and accelerates the development of atherosclerosis in mice lacking apolipoprotein E [] 102 6.2 Future Work: Background on Targeting Atherosclerotic Plaque The following subsection backgrounds provide important clues of how atherosclerotic plaque developed. In ot her words, the chemokines that are normally associated with the biological processes l eading to the plaque formation may open a way to specifically target these niosome ve sicles, where progressing plaques occur. 6.2.1 Mechanisms of leukocyte Adhesion (C lues for Targeting Atherosclerotic Plaque) High levels of LDL have been shown to acce lerate atherosclerosi s in experimental animals and in humans. Introducing LDL to endothelial cells has a significant impact on the plaque growth and nu mber of monocyte adhesion [ ] 103 104 This effect was described as an oxidative process or the release of oxygen free radicals in th e arterial lining to

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86 oxidized LDL, triggering atherosclerosis. As a result, excess oxidized LDL (ox-LDL) transfers into endothelial cell membrane, serv ing as a basis for cholesterol build up on the arterial wall and atherosclerotic plaque. Mo reover, due to excess ox-LDL, the viscosity of the plasma increased to create disturbed flow and increased shear stress which could explain the preference of leukocyte adhesion particularly at the vessel orifices and bifurcations. Although, it shoul d be noted that specific plaq ue adhesion prefers the lower shear stress neighborhood over the higher sh ear stress neighborhood, in the region in which the flow exhibits a change in stre ss rate. Furthermore, ox-LDL was found to promote adhesion effect of leukocytes to th e endothelium as a re sult of chemotactic activities based on oxidative process [] 105 and oxidized LDL exert direct chemotactic effects on monocytes [] 106 in a common feature in early atherogenesis [] 107 In vivo, studies have shown that signif icant amounts of ox-LDL were present in human plasma and atherosclerotic lesi ons, inducing mRNA for the human monocyte chemoattraction protein-12 (MCP-1) which ulti mately increases the monocytes adhesion to endothelial cells [] 108 The details of these studies showed that ox-LDL stimulates the rolling, tethering and adhering of circulating leukocytes to the endot helium in arterioles and venules and to aortic endothelium [107] For example, leukocyte adhesion in response to ox-LDL was associated with increased permeability of fluorescent stained macromolecules across endothelium in a rat mesenteric model [] 109 Beside chemotactic signals generated by the dysfunction of the endothelium, incorporation of ox-LDL into monocyte/macrophage lipid bilaye r by phagocytosis process enhanced the adhesivity due to physical changes in the membrane [ ] 110 111 Several researcher s have found expression such as leukocyte function-associated antig en 1 (LFA-1), Mac-1 and p150,95, adhesion

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87 receptors belong to the 2-integrin family using several types of monoclonal antibodies. For example, ox-LDL induced leukocyte adhesi on to venular and ar teriolar endothelium was shown to be prevented significantly by pr etreating with the monoclonal antibody to the CD11b subunit of Mac-1 [ , ] 112 113 114 115 This observation showed that anti-Mac-1 inhibited adhesion of all leukocytes and s uggested the role of monocyte adhesion, activation and releas e of mediators to recruitmen t of more leukocytes, including lymphocytes to endothelium. Moreover, self adhesions of circulating leukocytes further increase the size of atherosclerotic plaque c ould be considered as the contribution of the chemotactic attraction, adhesion and emigration. Many studies have shown that vascular cell adhesion molecule type 1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), Pselectin and E-selectin on atherosclerotic lesion [ ] 116 117 These expressions promoted th e adhesion of both leukocytes and lymphocytes through binding receptor of the 1 and 2-integrin family. Rolling, tethering and adhering of circ ulating leukocytes to the e ndothelium in arterioles and venules and to aortic endothelium were observed as a result of 1 and 2-integrin family receptors. For example, immunohistochemical staining in aortas of normal chow-fed wild-type mice and rabbits, VCAM -1 and ICAM-1 were expressed by endothelial cells in regions predisposed to atherosclerotic lesi on formation predominantly by endothelium on early and by intimal cells in more advanced lesions [112] The overall process of cellular adhesion molecules may take several hours in vivo and can be summarized into three steps. First, P-selectin and E-selectin on the endothelium and L-selectin on the surface of leukocytes slowed down leukocytes as they passed through the vessel and roll on the su rface of the endothelial before it became

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88 completely stopped. Secondly, the slowed leukocytes have enough time to respond to chemokines signaling molecules on the endothe lial surface, and th en engaged in the cellular adhesion molecules us ing leukocytes integrins ( 1 and 2) to cause firm adhesion and initiate migration. 6.3 Summary Based on the following reviews, using thes e structural dynamic carriers such as niosomes, the chemokines (Anti-ICAM-1 and Anti-VCAM-1) which played an important role in biological processes leading to the plaque formation may open a way to specifically target these niosome vesicles, where progressing plaques occur. Following the attachment and release of cardiovascular dr ugs, it is believed that this process may help create an anatomical and physiologi cal barrier to plaque growth (plaque stabilization) with little or no further treatments.

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89 REFERENCES 1 Gieringer D H, The safety and efficacy of new drug approval. Cato Journal. 1985;5:177. 2 Chen Y, Jia Z, Schaper A, Kristiansen M, Smith P, Wombacher R, Wendorff J H, Grie A. Hydrolytic and Enzymatic Degradation of Liquid-Crystalline Aromatic/Aliphatic Copolyesters Biomacromolecules, 2004;5:11 3 Hao Y, Zhao F, Li N, Yang Y, Li K. Studies on a high encapsulation of colchicine by a niosome system. International J ournal of Pharmaceutics 244 (2002) 73. 4 Fang J Y, Yu S Y, Wu P C, Huang Y B, Tsai Y H. In vitro skin permeation of estradiol from various proniosome form ulations. International Journal of Pharmaceutics. 2001;215:91. 5 Manconi M, Sinico C, Valenti D, L oy G, Fadda A M. Niosomes as carriers for tretinoin. I. Preparation and properties. International Journal of Pharmaceutics. 2002;234:237. 6 Manconi M, Valenti D, Sinico C, Lai F, Loy G, Fadda A M. Niosomes as carriers for tretinoin II. Influence of vesicular incorporation on tretinoin photostability. International Journal of Pharmaceutics. 2003;260:261. 7 Touitou E, Junginger H E, Weiner N D, Nagai T, Mezei M. Liposomes as carriers for topical and transdermal delivery. Journal of Pharmaceutical Sciences. 1994;83:1189. 8 Agarwal R, Katare O P, Vyas S P. Preparation and in vitro evaluation of liposomal/niosomal delivery systems for antipsoriatic drug dithranol. International Journal of Pharmaceutics. 2001;228:43. 9 Fang J Y, Hong C T, Chiu W T, Wang Y Y. Effect of liposomes and niosomes on skin permeation of enoxaci n. International Journal of Pharmaceutics. 2001;219:61 72. 10 Udupa N, Chandraprakash K S, Umadevi P, Pillai G K. Form ulation and evaluation of methotrexate niosomes. Drug De velopment and Industrial Pharmacy. 1993;19:1331.

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90 11 Parthasarathi G, Udupa N, Umadev i P, Pillai G K. Niosome-encapsulated vincristine sulfate: improved anticancer ac tivity with reduced toxicity in mice. Journal of Drug Targeting. 1994;2:173. 12 Uchegbu I F, Double J A, Turton J A, Fl orence A T. Distribution, metabolism and tumoricidal activity of doxorubicin administ ered in sorbitan monostearate (Span 60) niosomes in the mouse. Pharmaceutical Research. 1995;12:1019. 13 Jain C P, Vyas S P. Preparation a nd characterization of niosomes containing rifampicin for lung targeting. Journa l of Microencapsulation. 1995;12:401. 14 Williams D M, Carter K C, Baillie A J. Visceral leishmaniasis in the BALB/c mouse: a comparison of the in vivo activ ity of five nonionic surfactant vesicle preparations of sodium stibogluconate Journal of Drug Targeting. 1995;3:1. 15 Arunothayanun P, Turton J A, Uchegbu I F, Florence A T. Preparation and In Vitro/In Vivo Evaluation of Luteinizing Hormone Releasing Hormone (LHRH)Loaded Polyhedral and Spherical/Tubular Niosomes. Journal of Pharmaceutical Sciences. 1999;88:34. 16 Rentel C O, Bouwstra J A, Naisbett B, Junginger H E. Niosomes as a novel peroral vaccine delivery system. International Journal of Pharmaceutics. 1999;186:161. 17 Ruckmani K, Jayakar B, Ghosal S K. Nonionic surfactant vesicles (niosomes) of cytarabine hydrochloride for effective treatment of leukemias: encapsulation, storage, and in vitro release. Drug Development and Industrial Pharmacy. 2000;26:217. 18 Webster Medical Dictionary. 19 Small D M. Handbook of Lipid Research: Th e Physical Chemistry of Lipids, From Alkanes to Phospholipids, Vol. 4, Plenum Press, New York, 1986. 20 Lasic D D, Papahadjopoulos D. Lipos omes and biopolymers in drug and gene delivery. Current Opinion in Solid State & Materials Science 1996;1:392. 21 Frrkjaer S, Hjorth E L, Wrrts O. Stabili ty and storage of liposomes, in Optimization of Drug Delivery. Bundgaard H, Bagger Ha nsen A, Kofod H, Eds. Munksgaard, Copenhagen. 1982:384. 22 Kensil C R, Dennis E A. Alkaline hydrol ysis of phospholipids in model membranes and the dependence on their state of a ggregation. Biochemistry. 1981;20:6079. 23 Grit M, de Smidt J H, Struijke A, Crommelin D J A. Hydrolysis of phosphatidylcholine in aqueous liposome dispersions. Int. J. Pharm. 1989;50:1.

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91 24 Ostro M J, Cullis P R. Use of liposomes as injectable-drug delivery systems. Am J Hosp Pharm. 1989;46:1576. 25 Gupta C M. Liposome-based targeted dr ug delivery in malaria and leishmania infections. Journal of Para sitic Diseases. 1996; 20:67. 26 Park J W. Liposome-based drug delivery in breast cancer treatment. Breast Cancer Res. 2002;4:95. 27 Gal S, Pinchuk I, LichtenbergD. Peroxidation of liposomal palmitoyllinoleoylphosphatidylcholine (PLPC), effects of surface charge on the oxidizability and on the potency of antioxidants. Chemistr y and Physics of Lipids. 2003;126:95. 28 Wong J P, Yanga H, Blasettib K L, Schnella G, Conleyb J, Schofield L N. Liposome delivery of ciprofloxacin against intracellular Francisella tularensis infection. Journal of Cont rolled Release. 2003;92:265. 29 Igarashi A, Konno H, Tanaka T, Nakamura S, Sadzuka Y, Hirano T, Fujise Y. Liposomal photofrin enhances therapeutic efficacy of photodynamic therapy against the human gastric cancer. Toxi cology Letters. 2003;145:133. 30 Li W, Watarai S, Iwasaki T, Kodama H. Suppression of Salmonella enterica serovar Enteritidis excretion by intraocular vaccina tion with fimbriae proteins incorporated in liposomes. Developmental and Comparative Immunology. 2004;28:29. 31 Moghimi S M, Szebeni J. Stealth liposomes and long circulating nanoparticles:critical issues in pharmac okinetics, opsonization and protein-binding properties. Progress in Lipid Research. 2003;42:463. 32 Managit C, Kawakami S, Nishikawa M, Yamashita F, Hashida M Targeted and sustained drug delivery using PEGylated ga lactosylated liposomes. International Journal of Pharmaceutics. 2003;266:77. 33 Silvander M, Bergstrand N, Edwards K. Linkage identity is a major factor in determining the effect of PEGylated surfactants on permeability of phosphatidylcholine liposomes. Chemistr y and Physics of Lipids. 2003;126:77. 34 Wichmann C, Naumann P T,Spangenberg O,Konrad M, Mayer F, Hoppert M. Liposomes for microcompartmentation of en zymes and their influence on catalytic activity. Biochemical and Biophysical Research Communi cations. 2003;310:1104 1110.

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92 35 Saul J M, Annapragada A, Natarajan J V, Bellamkonda R V. Controlled targeting of liposomal doxorubicin via the folate receptor in vitro. Journal of Controlled Release. 2003;92:49. 36 Schiffelers R M, Koning G A,Hagen T L M, Fens M H A M, Schraa A J, Janssen A P C A, Kok R J, Molema G, Storm G. Anti-tumor efficacy of tumor vasculaturetargeted liposomal doxorubicin. Journa l of Controlled Release. 2003;91:115. 37 Jain S, Mishra V, Singh P, Dubey P K, Saraf D K, Vyas S P. RGD-anchored magnetic liposomes for monocytes/neu trophils-mediated brain targeting. International Journal of Pharmaceutics. 2003;261:43. 38 Chiua G N C, Marcel B B, Mayer L D. Targeting of antibody conjugated, phosphatidylserine-containing liposomes to vascular cell adhesion molecule 1 for controlled thrombogenesis. Biochimica et Biophysica Acta. 2003;1613:115. 39 Szab R, Hudecz F, Reig F. Interfacia l interactions between poly[L-lysine]-based branched polypeptides and phospholipid m odel membranes. Journal of Colloid and Interface Science. 2003;267:18. 40 Seonga H, Choia W M, Kima J C, Thompsonb D H, Park K. Preparation of liposomes with glucose binding sites: li posomes containing di-branched amino acid derivatives. Biomat erials. 2003;24:4487. 41 Tianshun L, Rodney H J Y. Trends and developments in liposome drug delivery systems. Journal of Pharmaceutical Sciences. 2001;90:66780. 42 Naruya T, Ryuichi M, Jitsuo H, Toshio O. Novel molecular therapeutic approach to cardiovascular disease based on hepato cyte growth factor. Journal of Atherosclerosis and Thrombosis. 2000;7:1. 43 Vinod L, Cunxian S, Robert L J. Nanopart icle drug delivery system for restenosis. Advanced Drug Delivery Reviews. 1997;24:63. 44 Lestini B J, Sagnella S M, Xu Z, Shive M S, Richter N J, Jayaseharan Case A J, Kottke-Marchant K, Anderson J M, Marc hant R E. Surface modification of liposomes for selective cell targeting in cardiovascular drug de livery. Journal of Controlled Release. 2002;78:235. 45 Poston R N, Haskard D O, Coucher J R, Gall N P, Johnson-Teidy R R. Expression of intercellular adhesion molecule-1 in atherosclerotic plaques. Am J Pathol. 1992;140:665. 46 Printseva O Y, Peclo M, Gown A M. Vari ous Cell Types in human atherosclerotic lesions express ICAM-1. Am J Pathol 1992;140:889.

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93 47 Barbet J, Machy P, Leserman L D. Monoclonal antibody covalently coupled to liposomes: specific targeting to cells J Supramol Struct Cell Biochem. 1981;16:243. 48 Van Ly H. Effect of Short-Chain Alcohols on Elasticity, Stability, and Phase Transition of Lipid Membranes. Univer sity of California, Davis. 2003:1. 49 Bouwstra J A, van Hal D A, Hofland H E J, et al. Pr eparation and characterization of nonionic surfactant vesicles. Colloids and Surfaces A: Physiochemical and Engineering Aspects 1997; 123-124: 71-80. 50 Uchegbu I F, Florence A T. Non-Ionic Surf actant vesicles (Niosomes): Physical and Pharmaceutical Chemistry. Advances in Colloid and Interface Science. 1995;58:1 55. 51 Yoshioka T, Sternberg B, Florence A T. Preparation of vesicles (niosomes) of sorbitan monoesters (Span 20, 40, 60 and 80) and a sorbitan trimester (Span 85). Int J Pharm. 1994;105:1-6. 52 Lawrence M J. The formation, characteri zation and stability of non-ionic surfactant vesicles. STP Pharma Sciences. 1996;1:49. 53 Bouwstra J A, van Hal D A, Hofland H E. Preparation and characterization of nonionic surfactant vesicles. Colloids a nd Surfaces A: Physiochemical and Engineering Aspects. 1997;123-124:71. 54 Manosroi A, Wongtrakul P, Manosroi J, Sakai H, Sugawara F, Yuasa M, Abe M. Characterization of vesicles prepared with various non-i onic surfactants mixed with cholesterol. Colloids and Surf aces B: Biointerfaces. 2003;30:129. 55 Nasseri B, Florence A T. Microtubules fo rmed by capillary extrusion and fusion of surfactant vesicles. International Journal of Pharmaceutics. 2003;266:9198. 56 Nasseri B, Florence A T. A vesicular shuttl e: transport of a vesi cle within a flexible microtube. Journal of Controlled Release. 2003;92:233. 57 Uchegbu, IF, Vyas SP. Non-ionic surfactan t vesicles (Niosomes) in drug delivery. Int J of Pharm. 1998;172:33-70. 58 Oommen E, Tiwari S, Udupa N, Kama th R. Niosome Entrapped b-Cyclodextrin Methotrexate Complex as a Drug Delivery System. Indian Journal of Pharmacology. 1999;31:279-284. 59 Shahiwala A, Misra A. Studies in Topical Application of Niosomally Entrapped Nimesulide. J Pharm Pharmaceut Sci. 2002;5:220-225.

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94 60 Jean-Francois A, Pierre G, Barbara G S, Eric D; Francis B. Usefulness of experimental models to understand the va scular effects of estrogens. Medicine Sciences. 2003;19:1226. 61 Fabrizio T, Filippo C, Achille D, Fran cesco V, Anna G S, Luigi C, Pier G A. Unstable angina and elevated C-reac tive protein levels predict enhanced vasoreactivity of the culprit lesion. Circul ation. 2001;104:1471. 62 Tomai F, Crea F, Gaspardone A, Versaci F, Ghini A S, Chiariello L, Gioffre P A. Unstable angina and elevated c-reactive protein levels predict enhanced vasoreactivity of the culprit lesion. Circul ation. 2001;104:1471. 63 Enrique G. Link between intracellular pathogens and cardiovascular diseases. Clinical Microbiology and In fection. 1998;4:S33S36. 64 Libby P. The interface of atherosclero sis and thrombosis: basic mechanisms. Vascular Medicine 1998;3:225. 65 Jeffrey A L, John C F, Benjamin H D, Joseph M B. Cardiovascular pharmacogenomics: Current status, future prospects. Journal of Cardiovascular Pharmacology and Therapeutics. 2003;8:71. 66 Weber C, Erl W, Weber K S C, Webe r P C. HMG-CoA Reductase Inhibitors Decrease CD11b Expression and CD11b-Dependent Adhesion of Monocytes to Endothelium and Reduce Increased Adhesiveness of Monocytes Isolated From Patients With Hypercholesterolemia. JACC. 1997;30:1212. 67 Rajamannan N M, Subramaniam M, Spri ngett M, Sebo T C, Niekrasz M, McConnell J P, Singh R J, Stone N J, Bonow R O, Spelsberg T C. Atorvastatin Inhibits Hypercholesterole mia-Induced Cellular Prolif eration and Bone Matrix Production in the Rabbit Aortic Valve. Circulation. 2002;105:2660. 68 Yoshida M. Potential role of statin s in inflammation and atherosclerosis. Atheroscler Thromb. 2003;10(3):140. 69 Davidson M H. Newer pharmaceutical agents to treat lipid disorders. Curr Cardiol Rep. 2003;5:463. 70 Ridker P M, Rifai N, Pfeffer M A, Sack s F, Braunwald E, Investigators Long-Term Effects of Pravastatin on Plasma Concentra tion of C-reactive Prot ein. Circulation. 1999;100:230. 71 Ridker P M, Hennekens C H, Buring J E, Rifai N. C-Reactiv e Protein and Other Markers of Inflammation in the Prediction of Cardiovascular Disease in Women. NEJM 2000;342:836.

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95 72 Wagner A H, Kohler T, Ruckschloss U, Just I, Hecker M. Improvement of Nitric OxideDependent Vasodilatation by HMG-CoA Reductase Inhibitors Through Attenuation of Endothelial Superoxide Ani on Formation. Arterioscler Thromb Vasc Biol. 2000;20:61. 73 Albert M A, Danielson E, Rifai N, Ridker P M. Effect of Statin Therapy on CReactive Protein Leve ls. JAMA 2001;286:64. 74 Saw J, Steinhubl S R, Berger P B, Ke reiakes D J, Serebruany V L, Brennan D, Topol E J. Lack of Adverse ClopidogrelA torvastatin Clinical Interaction From Secondary Analysis of a Randomized, Placebo-Controlled Clopidogrel Trial. Circulation. 2003;108:921. 75 Pate G E, Tahir M N, Murphy R T, Foley J B. Anti-inflammatory Effects of Statins in Patients with Aortic Stenosis. J Cardiovasc Pharmacol Ther. 2003;8:201. 76 Hognestad A, Endresen K, Wergeland R, Stokke O, Geiran O, Holm T, Simonsen S, Kjekshus J K, Andreassen A K. Plasma C-Reactive Protein as a Marker of Cardiac Allograft Vasculopathy in Heart Transplant Recipients. J Am Coll Cardiol. 2003;42:477. 77 Blake G J. Inflammatory biomarkers of the patient with myocardial insufficiency. Curr Opin Crit Care. 2003;9:369. 78 Nawawi H, Osman N S, Yusoff K, Khalid B A. Reduction in serum levels of adhesion molecules, interleukin-6 and Cr eative protein following short-term lowdose atorvastatin treatment in patient s with non-familial hypercholesterolemia. Horm Metab Res. 2003;35:479. 79 Meyers C D, Tannock L R, Wight T N, Chait A. Statin-exposed vascular smooth muscle cells secrete proteoglycans with decreased binding affinity for LDL. 2003;M300252JLR200:1. 80 Halouia M, Meilhaca O, Jandrot-Perrusb M, Mi chel J B. Atorvastatin limits the proinflammatory response of rat aortic smoot h muscle cells to thrombin. European Journal of Pharmacology. 2003;474:175. 81 Bellosta S, Via D, Canavesi M, Pfister P, Fumagalli R, Paoletti R, Bernini F. HMGCoA Reductase Inhibitors Reduce MMP-9 Secretion by Macrophages. Arterioscler Thromb Vasc Biol. 1998;18:1671. 82 Bellamy M F, Pellikka P A, Klarich K Y, Tajik J, Enriquez-Sarano M. Association of Cholesterol Levels, Hydroxymethylglut aryl Coenzyme-A Reductase Inhibitor Treatment, and Progression of Aortic St enosis in the Community. J Am Coll Cardiol. 2002;40:1723.

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96 83 Takano M, Mizuno K, Yokoyama S, Seimiya K, Ishibashi F, Okamatsu K, Uemura R. Changes in Coronary Plaque Color and Morphology by Lipid-Lowering Therapy With Atorvastatin: Serial Evaluation by Co ronary Angioscopy. J Am Coll Cardiol. 2003;42:680. 84 Frolkis J P, Pearce G L, Nambi V, Minor S, Sprecher D L. Statins Do Not Meet Expectations for Lowering Low-Density Lipoprotein Cholesterol Levels When Used in Clinical Practice. Am J Med. 2002;113:625. 85 Schmitz G, Drobnik W. Pharmacogenomi cs and pharmacogenetics of cholesterollowering therapy. Clin Chem Lab Med. 2003;41:581. 86 Kim J A, Berliner J A, Nadler J L. Angiotensin II Increases Monocyte Binding to Endothelial Cells. Biochemical and Biophysical Research communications. 1996;226:862. 87 Hernndez-Presa M, Bustos C, Ortego M, Tuon J, Renedo G, Ruiz-Ortega M, Egido J. Angiotensin-Converting Enzyme Inhibition Prevents Arterial Nuclear FactorB Activation, Monocyte Chemoa ttractant Protein1 Expression, and Macrophage Infiltration in a Rabbit Model of Early Accelerated. Atherosclerosis Circulation. 1997;95:1532-1541. 88 Strawn W B, Chappell M C, Dean R H, Kivlighn S, Ferrario C M. Inhibition of Early Atherogenesis by Losartan in Monkeys With Diet-Induced Hypercholesterolemia Circ ulation. 2000;101:1586. 89 Vaughan D. Pharmacology of ACE inhibito rs versus AT1 blockers. Can J Cardiol. 2000;E:36EE. 90 Greenwood J P, Scott E M, Mary D A S G, Stoker J B. Effect of Chronic Moxonidine Therapy on Peripheral Sympathetic Activity in Essential Hypertension. AJH. 1999;12:28AA. 91 Schuh J R, Blehm D J, Frierdich G E, McMahon E G, Blaine E H. Differential effects of renin-angiotensin system blockade on atherogenesis in cholesterol-fed rabbits. J Clin Invest. 1993;91:1453. 92 Duez H, Chao Y S, Hernandez M, Torpie r G, Poulain P, Mundt S, Mallat Z, Teissier E, Burton C A, Tedgui A, Fruchart J C, Fievet C, Wright S D, Staels B. Reduction of Atherosclerosis by the Peroxi some Proliferatoractivated Receptor Agonist Fenofibrate in Mice. The Journal of Biological Chemistry. 2002;277:48051.

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97 93 Takano H, Nagai T, Asakawa M, Toyozaki T, Oka T, Komuro I, Saito T, Masuda Y. Peroxisome ProliferatorActivated Recep tor Activators Inhi bit LipopolysaccharideInduced Tumor Necrosis Factor-a Expre ssion in Neonatal Rat Cardiac Myocytes. Circ Res. 2000;87:596. 94 Martin-Nizard F, Furman C, Delerive P, Kandoussi A, Fruchart J C, Staels B, Duriez P. Peroxisome proliferator-activated receptor activators inhibit oxidized low density lipoprotein-induced endothelin-1 secr etion in endothelial cells. J Cardiovasc Pharmacol. 2002;40:822. 95 Kintscher U, Lyon C, Wakino S, Br uemmer D, Feng X, Goetze S, Graf K, Moustakas A, Staels B, Fleck E, Hsueh W A, Law R E. PPAR_ Inhibits TGF-_ Induced _5 Integrin Transcription in Vascular Smooth Mu scle Cells by Interacting With Smad4. Circ Res. 2002;91:e35e44. 96 Beltowski J, Wojcicka G, Jamroz A. Pe roxisome proliferator-activated receptors (PPAR) in pathophysiology of the circul atory system and prospective use of agonists of these receptors in ther apy. Postepy Hig Med Dosw. 2003;57:199. 97 Chen Y E, Fu M, Zhang J, Zhu X, Li n Y, Akinbami M A, Song Q. Peroxisome proliferator-activated receptors and the cardiovascular system. Vitam Horm. 2003;66:157. 98 Puddu P, Puddu G M, Muscari A. P eroxisome proliferator-activat ed receptors: are they involved in atherosclerosis progressi on? International Jour nal of Cardiology. 2003;90:133. 99 Chinetti G, Lestavel S, Fruchart J C, Clavey V, Staels B. Peroxisome ProliferatorActivated Receptor Reduces Cholesterol Esterifica tion in Macrophages. Circ Res. 2003;92:212. 100 Sridhar G R. Peroxisome proliferator-act ivated receptors as molecular targets for drug therapy. J Assoc Physicians India. 2003;51:49. 101 Ziouzenkova O, Asatryan L, Sahady D, Or asanu G, Perrey S, Cutak B, Hassell T, Akiyama TE, Berger JP, Sevanian A, Pl utzky J. Dual roles for lipolysis and oxidation in peroxisome proliferation-activator receptor responses to electronegative low density lipopr otein. J Biol Chem. 2003;278:39874. 102 Fu T, Kashireddy P, Borensztajni J. Th e peroxisome-proliferat or-activated receptor agonist ciprofibrate severely aggrav ates hypercholesterolaemia and accelerates the development of atheroscle rosis in mice lacking apoli poprotein E. Biochem. J. 2003;373:941.

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98 103 Alderson L M, Endemann G, Lindsey S, Pronczuk A, Hoover R L, Hayes K C. LDL enhances monocyte adhesion to endot helial cell in vitro. Am J Pathol. 1986;123:334. 104 Pritchard K A, Schwarz S M, Medow M S, Stemerman M B. Effect of low-density lipoprotein on endothelial cell membrane flui dity and mononuclear cell attachment. Am J physiol. 1991;260:c43c49. 105 Turner S R, Campbell J A, Lynn W S, Polymorphonuclear leukocyte chemotaxis toward oxidized lipid components of cell membranes. J Exp Med. 1975;141:1437 1441. 106 Quinn M T, Parthasarathy S, Fong L G, Steinberg D. Oxidatively modified low density lipoproteins: a potential role in recruitment and retention of monocyte/macrophages during atherogene sis. Proc Natl Acad Sci. 1987;84:2995 2998. 107 Lehr H A, Becker M, Marklund S L, Hubner C, Arfors K E, Kohlschutter A, Messmer K. Superoxide-dependent stimulation of leukocyte adhesion by oxidatively modified LDL in vivo. Arterioscler. Thromb. 1992;12:824. 108 Liao F, Berliner J A, Mehrabian M, Navab M, Demer L L, Lusis A J, Fogelman A M. Minimally modified low density lipoprot ein is biologically active in vivo in mice. J Clin Invest. 1991;87:2253. 109 Liao L, Asako H, Kurose I, Auerbach B, Newton R S, Granger D N. Oxidized lipoproteins elicit leukocyte-endothelial cell adhesion in mesenteric venules. Abstract FASEB Meeting, 1993. 110 Rogers K A, Hoover R L, Castellot J J, Robinson J M, Karovsky M J. Dietary cholesterol-induced changes in macrophage characteristics. Am J patho. 1986;125:284. 111 Lichtenstein I H, Zaleski E M, MacGregor R R. Neutrophil dysfunction in the rabbit model of spur cell anem ia. J Leukoc Biol. 1987;42:156. 112 Lehr H A, Krober M, Hubner C, Vajkoczy P, Menger M D, Nolte D, Kohlschutter A, Messmer K. Stimulation of leukocy te/endothelium interaction by oxidized lowdensity lipoprotein in hairless mice: involvement of CD11b/CD18 adhesion receptor complex. 1993;68:388. 113 Carlos T M, Harlan J M. Leukocyte-Endothelial Adhesion Molecules. Blood. 1994;84:2068.

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99 114 Johnston B, Issekutz T B, Kubes P. The 4-Integrin Supports Leukocyte Rolling and Adhesion in Chronically Inflamed Post capillary Venules In Vivo. J Exp Med. 1996;183:1995. 115 Crutchfield K L, Shinde V R, Campbell C J, Parkos C A, Allport J R, Goetz D J. CD11b/CD18-coated microspheres attach to E-selectin under flow. J Leukoc Biol. 2000;67:196. 116 Poston R N, Haskard D O, Coucher J R, Gall N P, Johnson-Teidy R R. Expression of intercellular adhesion molecule-1 in atherosclerotic plaques. Am J Pathol. 1992;140:665. 117 Printseva O Y, Peclo M, Gown A M. Vari ous Cell Types in human atherosclerotic lesions express ICAM-1. Am J Pathol 1992;140:889.

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100 APPENDICES

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101 Appendix A Comparison between niosomes produced at 40 C and 60C with the increase in particles size distribution that are gr eater ~0.58 m. Calculati ng result are shown below: Mean Particle Distribution Niosomes 40C Niosomes 60C Difference (60C-40C) 0.57609 174019 78839 0 0.58392 119138 94428 0 0.59185 110076 91666 0 0.59989 102447 60827 0 0.60804 101525 86875 0 0.6163 47054 55844 8790 0.62467 91757 81697 0 0.63315 41366 59046 17680 0.64175 86775 57535 0 0.65047 93398 53708 0 0.65931 44149 53709 9560 0.66826 81009 56379 0 0.67734 75568 56608 0 0.68654 38917 53997 15080 0.69586 79076 26286 0 0.70532 73335 53205 0 0.7149 34218 27748 0 0.72461 62799 51709 0 0.73445 60556 24116 0 0.74443 59915 47955 0 0.75454 26422 22932 0 0.76479 48871 48171 0 0.77517 44545 23725 0 0.7857 44416 44166 0 0.79638 39963 20223 0 0.80719 17792 40912 23120 0.81816 33671 20661 0 0.82927 32513 41053 8540 0.84054 31253 20313 0 0.85195 27474 36894 9420 0.86352 24281 36321 12040 0.87525 24190 38390 14200 0.88714 23915 18525 0 0.89919 20650 34800 14150 0.91141 18782 32732 13950 0.92379 29037 33687 4650 0.93633 17043 35103 18060 0.94905 14989 30959 15970 0.96194 13660 41660 28000 0.97501 12979 27449 14470 0.98825 17148 26148 9000 1.00168 10066 24036 13970 1.01528 15027 36137 21110 1.02907 9335 23545 14210 1.04305 12674 33724 21050 1.05722 12797 31937 19140 1.07158 11476 21106 9630 1.08614 9575 28895 19320 1.10089 6478 26438 19960 1.11584 8919 26729 17810 1.131 7989 24879 16890 1.14636 7578 23038 15460 1.16193 6837 21447 14610 1.17772 9036 20976 11940 1.19371 6305 27455 21150 1.20993 4948 18448 13500 1.22636 5179 18309 13130 1.24302 4806 24006 19200 1.2599 6095 16015 9920 1.27702 4522 21392 16870 1.29436 3901 20681 16780 1.31194 5245 18205 12960

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102 Appendix A (continued) 1.32976 3396 17276 13880 1.34783 4663 17003 12340 1.36613 3832 16162 12330 1.38469 4901 15121 10220 1.4035 3343 13823 10480 1.42256 4153 16973 12820 1.44189 3854 12684 8830 1.46147 2751 12051 9300 1.48132 3360 14770 11410 1.50144 3395 13205 9810 1.52184 2850 10110 7260 1.54251 2722 11882 9160 1.56346 2347 12107 9760 1.5847 2283 10493 8210 1.60622 2669 10159 7490 1.62804 2480 10220 7740 1.65015 2129 9159 7030 1.67257 2098 8538 6440 1.69529 2366 8136 5770 1.71832 2447 8347 5900 1.74166 1665 9215 7550 1.76531 2134 7594 5460 1.78929 2117 8847 6730 1.81359 1506 8506 7000 1.83823 1695 7915 6220 1.8632 1888 6178 4290 1.88851 1829 7499 5670 1.91416 1339 6899 5560 1.94016 1398 6448 5050 1.96651 1437 6797 5360 1.99322 1696 6876 5180 2.0203 1415 7535 6120 2.04774 738 5388 4650 2.07555 849 5589 4740 2.10375 696 5296 4600 2.13232 775 6235 5460 2.16129 722 5022 4300 2.19064 501 5711 5210 2.2204 625 4725 4100 2.25056 496 5446 4950 2.28113 573 4813 4240 2.31211 882 5122 4240 2.34352 651 5421 4770 2.37535 533 4283 3750 2.40762 613 4833 4220 2.44032 444 4934 4490 2.47347 671 4841 4170 2.50706 460 4820 4360 2.54112 525 4525 4000 2.57563 490 4310 3820 2.61062 412 4682 4270 2.64608 567 3957 3390 2.68202 513 4023 3510 2.71845 499 4209 3710 2.75538 480 3930 3450 2.7928 469 4439 3970 2.83074 328 4058 3730 2.86919 466 3746 3280 2.90816 487 4027 3540 2.94766 455 3435 2980 2.9877 384 3884 3500 3.02828 317 3757 3440 3.06942 526 3966 3440 3.11111 455 3825 3370 3.15337 508 3338 2830 3.1962 459 3829 3370 3.23961 379 3599 3220 3.28362 488 3668 3180 3.32822 407 2887 2480 3.37343 366 3426 3060

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103 Appendix A (continued) 3.41925 385 3445 3060 3.46569 458 3208 2750 3.51277 389 3009 2620 3.56048 256 3136 2880 3.60885 365 3345 2980 3.65787 372 2902 2530 3.70755 251 2821 2570 3.75791 365 2775 2410 3.80896 246 3206 2960 3.86069 413 2743 2330 3.91313 322 2742 2420 3.96629 361 2491 2130 4.02016 353 2743 2390 4.07477 273 2453 2180 4.13012 304 2854 2550 4.18622 311 2571 2260 4.24308 290 2590 2300 4.30071 245 2295 2050 4.35913 350 2470 2120 4.41834 252 2082 1830 4.47835 187 2237 2050 4.53918 273 2013 1740 4.60084 409 1899 1490 4.66333 190 2070 1880 4.72668 200 2110 1910 4.79088 212 2342 2130 4.85595 277 1777 1500 4.92191 153 2143 1990 4.98877 339 1949 1610 5.05653 270 1880 1610 Total Particle (40C) 2420917 Particle Increased 1046680 Percent Increased 43%

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104 Appendix B Comparison between niosomes produced at Day 0, Day 1 and Day 14 with the increase in particles size distribution that is greater ~2 m. Calculating results are shown below: Mean Particle Distribution Niosomes (Day 0) Niosomes (Day 1) Niosomes (Day 14) Difference (Day1Day 0) Difference (Day14-Day 0) 2.0203 6730 6340 6910 0 180 2.04774 5550 6250 5620 700 70 2.07555 5270 6200 5880 930 610 2.10375 5270 7050 5320 1780 50 2.13232 6200 6010 6370 0 170 2.16129 4690 6690 4900 2000 210 2.19064 5650 5760 5770 110 120 2.2204 4720 6150 4700 1430 0 2.25056 5620 5550 5530 0 0 2.28113 4460 5540 4540 1080 80 2.31211 5320 5590 5270 270 0 2.34352 5280 4800 4810 0 0 2.37535 4330 5480 4500 1150 170 2.40762 5380 4860 4550 0 0 2.44032 4780 5490 4810 710 30 2.47347 4690 5020 4570 330 0 2.50706 4210 4870 4950 660 740 2.54112 4690 4770 4030 80 0 2.57563 4310 4710 4190 400 0 2.61062 4350 4610 4490 260 140 2.64608 3900 4600 4130 700 230 2.68202 3950 4290 3920 340 0 2.71845 3870 3960 4120 90 250 2.75538 3880 4280 4030 400 150 2.7928 3790 3760 4120 0 330 2.83074 3500 3900 3700 400 200 2.86919 3610 4120 3850 510 240 2.90816 4050 3340 4600 0 550 2.94766 3400 4050 3300 650 0 2.9877 3930 3460 4190 0 260 3.02828 3440 3530 3730 90 290 3.06942 4040 3670 4260 0 220 3.11111 4120 3070 4030 0 0 3.15337 3170 3250 3590 80 420 3.1962 3540 3080 4050 0 510 3.23961 3670 3080 3870 0 200 3.28362 3560 2600 4220 0 660 3.32822 3010 3150 3270 140 260 3.37343 3400 2950 4140 0 740 3.41925 3170 3080 3780 0 610 3.46569 3280 3120 3840 0 560 3.51277 3320 3320 3860 0 540 3.56048 3750 2730 4210 0 460 3.60885 3170 2770 3880 0 710 3.65787 3180 2860 3900 0 720 3.70755 3110 2810 3350 0 240 3.75791 2750 2970 3610 220 860 3.80896 3030 2600 4390 0 1360 3.86069 2610 2670 3590 60 980 3.91313 3160 2390 4350 0 1190 3.96629 2560 2620 3790 60 1230 4.02016 2910 2350 4440 0 1530 4.07477 2470 2480 4010 10 1540 4.13012 2850 2250 4280 0 1430 4.18622 2390 1960 4730 0 2340 4.24308 2470 2270 3730 0 1260 4.30071 2680 1980 4040 0 1360 4.35913 2590 2080 4080 0 1490 4.41834 2430 2090 3740 0 1310 4.47835 2460 1790 4030 0 1570 4.53918 1870 2070 3260 200 1390

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105 Appendix B (continued) 4.60084 2050 1850 4220 0 2170 4.66333 2160 1880 3510 0 1350 4.72668 1940 2140 3640 200 1700 4.79088 2510 1810 3980 0 1470 4.85595 1990 1900 3260 0 1270 4.92191 2010 2110 3210 100 1200 4.98877 2120 1610 3800 0 1680 5.05653 1880 1960 3270 80 1390 5.12522 1770 1460 3640 0 1870 5.19483 1980 1640 2940 0 960 5.26539 1800 1640 3230 0 1430 5.33691 1560 1460 3270 0 1710 5.40941 1440 1560 3020 120 1580 5.48288 1470 1470 2840 0 1370 5.55736 1360 1410 3040 50 1680 5.63284 1430 1460 2330 30 900 5.70936 1560 1350 2340 0 780 5.78691 1240 1260 2170 20 930 5.86551 1230 1360 1970 130 740 5.94518 1170 1310 2160 140 990 6.02594 990 1290 1760 300 770 6.10779 960 890 1820 0 860 6.19075 870 1010 1550 140 680 6.27484 990 950 1810 0 820 6.36007 920 1110 1660 190 740 6.44646 730 920 1390 190 660 6.53403 640 880 1270 240 630 6.62278 710 980 1670 270 960 6.71274 600 850 1330 250 730 6.80392 470 960 1210 490 740 6.89634 570 940 1300 370 730 6.99001 500 820 1180 320 680 7.08496 420 910 1020 490 600 7.18119 560 720 1050 160 490 7.27873 430 750 1040 320 610 7.3776 410 810 1010 400 600 7.47781 420 730 1080 310 660 7.57939 430 720 950 290 520 7.68234 370 720 850 350 480 7.78669 280 750 860 470 580 7.89246 280 600 880 320 600 7.99966 340 520 900 180 560 8.10832 300 370 770 70 470 8.21846 270 530 810 260 540 8.33009 390 490 820 100 430 8.44324 280 470 800 190 520 8.55792 260 440 630 180 370 8.67417 320 590 800 270 480 8.79199 240 430 720 190 480 8.91141 250 390 740 140 490 9.03246 170 430 530 260 360 9.15515 280 320 700 40 420 9.2795 170 350 540 180 370 9.40555 270 430 510 160 240 9.5333 170 370 690 200 520 9.6628 190 350 590 160 400 9.79405 230 340 660 110 430 9.92708 120 240 490 120 370 10.06192 130 0 490 0 360 10.19859 230 0 0 0 0 Total Particle (Day 0) 285370 Particle Increased 25390 81880 Percent Increased 9% 29%

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106 Appendix C Sample calculation of PSS ASCII-file from PSS: total volume calculation with Span60/Niosomes after gel exclusion column. Below is the equation for calculating the total interior volume of niosomes from 0.56~20 m. PSS-780 ASCII files for Span20/Niosomes after GEC (0.5 ml injection) Particle Sizing Systems, Inc. Santa Barbara, Calif., USA Model 780 AccuSizer Caption: span20 cf 1st draw 5 ml--0.5 ml inj File Name = 711s2ge2.001 Time Date = 7/11/2003 11:44 Sensor Model: LE400-0.5 SUM S/N: 8910 Cal. File: 0008910s.sns Elapsed Time of Data Collection = 60 Sec. Background File = NONE Total # Part. Sized (>=Thres. 0.53 um ) = 266019 Calculated Total No. of Particles in Sample = 1920591 Dilution Factor = 7.22 Fluid Volume Sampled = 60 ml No. of Channels = 512 NUM-WT Mean = 0.95 um Mode = 0.58 um Median = 0.67 VOL-WT Mean = 8.27 um Mode = 12.66 um Median = 8.1 Summary of Detailed Distribution, Weightings Diameter # Part. Cum Num Num % Vol % Cum Num % Vol Num % # Part. Interior Volume (microns) Sized >=Diam. >=D iam. >=Diam. w/dilution ml 0.53851 8158 266019 3.067 0.059 100 100 59064 4.8295E-09 0.54583 8787 257861 3.303 0.066 96.933 99.941 63618 5.41689E-09 0.55324 5994 249074 2.253 0.047 93.63 99.875 43397 3.84764E-09 0.56076 9729 243080 3.657 0.079 91.377 99.829 70438 6.50334E-09 0.56837 10314 233351 3.877 0.087 87.72 99.75 74673 7.1789E-09 0.57609 10122 223037 3.805 0.089 83.843 99.662 73283 7.33626E-09 0.58392 11370 212915 4.274 0.104 80.038 99.573 82319 8.58139E-09 0.59185 10632 201545 3.997 0.102 75.763 99.469 76976 8.35578E-09 0.59989 6732 190913 2.531 0.067 71.767 99.367 48740 5.5093E-09 0.60804 9679 184181 3.638 0.1 69.236 99.3 70076 8.24829E-09 0.6163 5906 174502 2.22 0.064 65.598 99.2 42759 5.24092E-09

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107 Appendix C (continued) 0.62467 8562 168596 3.219 0.096 63.377 99.136 61989 7.9116E-09 0.63315 5866 160034 2.205 0.069 60.159 99.04 42470 5.64416E-09 0.64175 5676 154168 2.134 0.069 57.954 98.971 41094 5.68692E-09 0.65047 5251 148492 1.974 0.067 55.82 98.902 38017 5.47849E-09 0.65931 5110 143241 1.921 0.067 53.846 98.836 36996 5.55171E-09 0.66826 5329 138131 2.003 0.073 51.925 98.768 38582 6.02864E-09 0.67734 5127 132802 1.927 0.073 49.922 98.695 37119 6.03977E-09 0.68654 4624 127675 1.738 0.069 47.995 98.621 33478 5.67221E-09 0.69586 2256 123051 0.848 0.035 46.256 98.552 16333 2.88165E-09 0.70532 4453 120795 1.674 0.072 45.408 98.517 32240 5.92309E-09 0.7149 2371 116342 0.891 0.04 43.734 98.445 17166 3.28401E-09 0.72461 4331 113971 1.628 0.076 42.843 98.406 31356 6.24652E-09 0.73445 1979 109640 0.744 0.036 41.215 98.33 14328 2.97214E-09 0.74443 3671 107661 1.38 0.07 40.471 98.293 26578 5.74107E-09 0.75454 1821 103990 0.685 0.036 39.091 98.224 13184 2.96547E-09 0.76479 3737 102169 1.405 0.077 38.407 98.188 27056 6.33705E-09 0.77517 1720 98432 0.647 0.037 37.002 98.111 12453 3.03708E-09 0.7857 3379 96712 1.27 0.076 36.355 98.074 24464 6.21292E-09 0.79638 1470 93333 0.553 0.034 35.085 97.998 10643 2.81459E-09 0.80719 2997 91863 1.127 0.073 34.533 97.964 21698 5.97518E-09 0.81816 1642 88866 0.617 0.041 33.406 97.891 11888 3.40898E-09 0.82927 2987 87224 1.123 0.079 32.789 97.85 21626 6.45744E-09 0.84054 1324 84237 0.498 0.036 31.666 97.771 9586 2.98058E-09 0.85195 2532 82913 0.952 0.072 31.168 97.735 18332 5.93531E-09 0.86352 2414 80381 0.907 0.072 30.216 97.663 17477 5.89239E-09 0.87525 2480 77967 0.932 0.077 29.309 97.591 17955 6.30355E-09 0.88714 1258 75487 0.473 0.04 28.377 97.515 9108 3.32962E-09 0.89919 2235 74229 0.84 0.075 27.904 97.474 16181 6.15984E-09 0.91141 2109 71994 0.793 0.074 27.063 97.399 15269 6.05278E-09 0.92379 1977 69885 0.743 0.072 26.271 97.326 14313 5.90831E-09 0.93633 2086 67908 0.784 0.079 25.528 97.254 15103 6.4914E-09 0.94905 1882 65822 0.707 0.074 24.743 97.175 13626 6.09851E-09 0.96194 2500 63940 0.94 0.103 24.036 97.101 18100 8.4357E-09 0.97501 1598 61440 0.601 0.068 23.096 96.998 11570 5.61489E-09 0.98825 1483 59842 0.557 0.066 22.495 96.93 10737 5.42599E-09 1.00168 1385 58359 0.521 0.064 21.938 96.864 10027 5.27684E-09 1.01528 2071 56974 0.779 0.1 21.417 96.8 14994 8.21627E-09 1.02907 1322 54903 0.497 0.066 20.639 96.7 9571 5.46139E-09 1.04305 1841 53581 0.692 0.096 20.142 96.633 13329 7.91966E-09 1.05722 1688 51740 0.635 0.092 19.45 96.537 12221 7.56146E-09 1.07158 1135 50052 0.427 0.064 18.815 96.445 8217 5.29428E-09 1.08614 1592 48917 0.598 0.094 18.389 96.381 11526 7.73282E-09 1.10089 1410 47325 0.53 0.087 17.79 96.287 10208 7.13162E-09 1.11584 1407 45915 0.529 0.09 17.26 96.2 10187 7.41032E-09 1.131 1306 44508 0.491 0.087 16.731 96.11 9455 7.16256E-09 1.14636 1287 43202 0.484 0.089 16.24 96.023 9318 7.34986E-09 1.16193 1184 41915 0.445 0.086 15.756 95.934 8572 7.04091E-09 1.17772 1094 40731 0.411 0.082 15.311 95.848 7921 6.77455E-09 1.19371 1497 39637 0.563 0.117 14.9 95.766 10838 9.65285E-09 1.20993 990 38140 0.372 0.081 14.337 95.648 7168 6.64742E-09 1.22636 974 37150 0.366 0.083 13.965 95.568 7052 6.81005E-09 1.24302 1207 36176 0.454 0.107 13.599 95.485 8739 8.78777E-09 1.2599 843 34969 0.317 0.078 13.145 95.378 6103 6.39106E-09 1.27702 1097 34126 0.412 0.105 12.828 95.3 7942 8.66038E-09 1.29436 1024 33029 0.385 0.102 12.416 95.195 7414 8.41787E-09 1.31194 958 32005 0.36 0.1 12.031 95.093 6936 8.20058E-09 1.32976 944 31047 0.355 0.102 11.671 94.993 6835 8.41451E-09 1.34783 835 30103 0.314 0.094 11.316 94.891 6045 7.75049E-09 1.36613 754 29268 0.283 0.089 11.002 94.796 5459 7.2876E-09 1.38469 778 28514 0.292 0.095 10.719 94.708 5633 7.83023E-09 1.4035 766 27736 0.288 0.098 10.426 94.613 5546 8.02792E-09 1.42256 863 26970 0.324 0.115 10.138 94.515 6248 9.41802E-09 1.44189 658 26107 0.247 0.091 9.814 94.4 4764 7.47755E-09 1.46147 602 25449 0.226 0.087 9.567 94.31 4358 7.12366E-09 1.48132 751 24847 0.282 0.113 9.34 94.223 5437 9.25388E-09 1.50144 680 24096 0.256 0.106 9.058 94.11 4923 8.72509E-09 1.52184 514 23416 0.193 0.083 8.802 94.004 3721 6.86764E-09 1.54251 606 22902 0.228 0.103 8.609 93.921 4387 8.43129E-09 1.56346 625 22296 0.235 0.11 8.381 93.818 4525 9.05477E-09 1.5847 615 21671 0.231 0.113 8.146 93.708 4453 9.27798E-09

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108 Appendix C (continued) 1.60622 525 21056 0.197 0.1 7.915 93.595 3801 8.2473E-09 1.62804 502 20531 0.189 0.1 7.718 93.495 3634 8.21176E-09 1.65015 463 20029 0.174 0.096 7.529 93.395 3352 7.88658E-09 1.67257 470 19566 0.177 0.101 7.355 93.299 3403 8.33658E-09 1.69529 442 19096 0.166 0.099 7.178 93.198 3200 8.16378E-09 1.71832 429 18654 0.161 0.1 7.012 93.099 3106 8.251E-09 1.74166 485 18225 0.182 0.118 6.851 92.999 3511 9.71335E-09 1.76531 385 17740 0.145 0.098 6.669 92.88 2787 8.02899E-09 1.78929 425 17355 0.16 0.112 6.524 92.783 3077 9.22929E-09 1.81359 425 16930 0.16 0.117 6.364 92.671 3077 9.61044E-09 1.83823 452 16505 0.17 0.129 6.204 92.554 3272 1.06433E-08 1.8632 318 16053 0.12 0.095 6.035 92.424 2302 7.79727E-09 1.88851 376 15735 0.141 0.117 5.915 92.33 2722 9.60026E-09 1.91416 358 15359 0.135 0.116 5.774 92.213 2592 9.5182E-09 1.94016 363 15001 0.136 0.122 5.639 92.097 2628 1.00498E-08 1.96651 324 14638 0.122 0.114 5.503 91.975 2346 9.34051E-09 1.99322 313 14314 0.118 0.114 5.381 91.861 2266 9.39609E-09 2.0203 334 14001 0.126 0.127 5.263 91.747 2418 1.04407E-08 2.04774 274 13667 0.103 0.108 5.138 91.62 1984 8.91892E-09 2.07555 304 13393 0.114 0.125 5.035 91.512 2201 1.03041E-08 2.10375 275 13089 0.103 0.118 4.92 91.387 1991 9.70627E-09 2.13232 327 12814 0.123 0.146 4.817 91.269 2367 1.20183E-08 2.16129 254 12487 0.095 0.118 4.694 91.122 1839 9.72098E-09 2.19064 313 12233 0.118 0.152 4.599 91.004 2266 1.24737E-08 2.2204 235 11920 0.088 0.119 4.481 90.853 1701 9.75211E-09 2.25056 259 11685 0.097 0.136 4.393 90.734 1875 1.1192E-08 2.28113 236 11426 0.089 0.129 4.295 90.598 1709 1.06194E-08 2.31211 238 11190 0.089 0.136 4.206 90.469 1723 1.11517E-08 2.34352 226 10952 0.085 0.134 4.117 90.333 1636 1.10269E-08 2.37535 215 10726 0.081 0.133 4.032 90.199 1557 1.09234E-08 2.40762 212 10511 0.08 0.136 3.951 90.066 1535 1.1216E-08 2.44032 220 10299 0.083 0.147 3.872 89.93 1593 1.21199E-08 2.47347 203 10079 0.076 0.142 3.789 89.783 1470 1.16454E-08 2.50706 211 9876 0.079 0.153 3.713 89.641 1528 1.26042E-08 2.54112 185 9665 0.07 0.14 3.633 89.488 1339 1.15076E-08 2.57563 184 9480 0.069 0.145 3.564 89.348 1332 1.19181E-08 2.61062 188 9296 0.071 0.154 3.495 89.203 1361 1.26802E-08 2.64608 164 9108 0.062 0.14 3.424 89.049 1187 1.15183E-08 2.68202 181 8944 0.068 0.161 3.362 88.909 1310 1.32374E-08 2.71845 180 8763 0.068 0.167 3.294 88.748 1303 1.3708E-08 2.75538 177 8583 0.067 0.171 3.226 88.581 1281 1.40364E-08 2.7928 167 8406 0.063 0.168 3.16 88.411 1209 1.37903E-08 2.83074 173 8239 0.065 0.181 3.097 88.243 1253 1.48759E-08 2.86919 164 8066 0.062 0.179 3.032 88.062 1187 1.46845E-08 2.90816 158 7902 0.059 0.179 2.97 87.884 1144 1.47316E-08 2.94766 136 7744 0.051 0.161 2.911 87.704 985 1.32041E-08 2.9877 176 7608 0.066 0.216 2.86 87.544 1274 1.77935E-08 3.02828 141 7432 0.053 0.18 2.794 87.328 1021 1.48438E-08 3.06942 166 7291 0.062 0.221 2.741 87.147 1202 1.81976E-08 3.11111 145 7125 0.055 0.201 2.678 86.926 1050 1.6552E-08 3.15337 125 6980 0.047 0.181 2.624 86.725 905 1.48584E-08 3.1962 140 6855 0.053 0.211 2.577 86.544 1014 1.73287E-08 3.23961 152 6715 0.057 0.238 2.524 86.333 1100 1.95911E-08 3.28362 147 6563 0.055 0.24 2.467 86.095 1064 1.97294E-08 3.32822 140 6416 0.053 0.238 2.412 85.855 1014 1.9566E-08 3.37343 131 6276 0.049 0.232 2.359 85.617 948 1.90644E-08 3.41925 143 6145 0.054 0.263 2.31 85.386 1035 2.16703E-08 3.46569 124 6002 0.047 0.238 2.256 85.122 898 1.95672E-08 3.51277 128 5878 0.048 0.256 2.21 84.884 927 2.10328E-08 3.56048 138 5750 0.052 0.287 2.162 84.629 999 2.36125E-08 3.60885 128 5612 0.048 0.277 2.11 84.341 927 2.28062E-08 3.65787 117 5484 0.044 0.264 2.062 84.064 847 2.17074E-08 3.70755 114 5367 0.043 0.268 2.018 83.8 825 2.20243E-08 3.75791 102 5253 0.038 0.249 1.975 83.533 738 2.052E-08 3.80896 120 5151 0.045 0.306 1.936 83.283 869 2.51384E-08 3.86069 128 5031 0.048 0.339 1.891 82.977 927 2.79217E-08 3.91313 109 4903 0.041 0.301 1.843 82.638 789 2.47592E-08 3.96629 105 4794 0.039 0.302 1.802 82.337 760 2.48359E-08 4.02016 100 4689 0.038 0.299 1.763 82.035 724 2.46302E-08 4.07477 103 4589 0.039 0.321 1.725 81.736 746 2.6417E-08

PAGE 122

109 Appendix C (continued) 4.13012 110 4486 0.041 0.357 1.686 81.414 796 2.93777E-08 4.18622 113 4376 0.042 0.382 1.645 81.057 818 3.14255E-08 4.24308 83 4263 0.031 0.292 1.603 80.675 601 2.40358E-08 4.30071 102 4180 0.038 0.374 1.571 80.383 738 3.0758E-08 4.35913 105 4078 0.039 0.401 1.533 80.009 760 3.29705E-08 4.41834 101 3973 0.038 0.402 1.494 79.608 731 3.30245E-08 4.47835 84 3872 0.032 0.348 1.456 79.207 608 2.86003E-08 4.53918 90 3788 0.034 0.388 1.424 78.859 652 3.19089E-08 4.60084 100 3698 0.038 0.449 1.39 78.471 724 3.69189E-08 4.66333 97 3598 0.036 0.453 1.353 78.022 702 3.72904E-08 4.72668 89 3501 0.033 0.433 1.316 77.569 644 3.56284E-08 4.79088 100 3412 0.038 0.507 1.283 77.136 724 4.16853E-08 4.85595 92 3312 0.035 0.486 1.245 76.629 666 3.99344E-08 4.92191 87 3220 0.033 0.478 1.21 76.143 630 3.9324E-08 4.98877 113 3133 0.042 0.647 1.178 75.665 818 5.31858E-08 5.05653 84 3020 0.032 0.501 1.135 75.019 608 4.11694E-08 5.12522 104 2936 0.039 0.645 1.104 74.518 753 5.30772E-08 5.19483 99 2832 0.037 0.64 1.065 73.873 717 5.26122E-08 5.26539 88 2733 0.033 0.592 1.027 73.233 637 4.8698E-08 5.33691 89 2645 0.033 0.624 0.994 72.641 644 5.12858E-08 5.40941 97 2556 0.036 0.708 0.961 72.018 702 5.82048E-08 5.48288 99 2459 0.037 0.752 0.924 71.31 717 6.18584E-08 5.55736 76 2360 0.029 0.601 0.887 70.558 550 4.94489E-08 5.63284 83 2284 0.031 0.684 0.859 69.957 601 5.62338E-08 5.70936 69 2201 0.026 0.592 0.827 69.273 500 4.86798E-08 5.78691 76 2132 0.029 0.679 0.801 68.681 550 5.5833E-08 5.86551 69 2056 0.026 0.642 0.773 68.002 500 5.27842E-08 5.94518 86 1987 0.032 0.833 0.747 67.361 623 6.85063E-08 6.02594 71 1901 0.027 0.716 0.715 66.528 514 5.88939E-08 6.10779 59 1830 0.022 0.62 0.688 65.812 427 5.09614E-08 6.19075 50 1771 0.019 0.547 0.666 65.192 362 4.49715E-08 6.27484 70 1721 0.026 0.797 0.647 64.645 507 6.55606E-08 6.36007 61 1651 0.023 0.723 0.621 63.848 442 5.94912E-08 6.44646 46 1590 0.017 0.568 0.598 63.125 333 4.67153E-08 6.53403 46 1544 0.017 0.591 0.58 62.557 333 4.8645E-08 6.62278 53 1498 0.02 0.71 0.563 61.966 384 5.83625E-08 6.71274 45 1445 0.017 0.627 0.543 61.256 326 5.15999E-08 6.80392 55 1400 0.021 0.798 0.526 60.629 398 6.56716E-08 6.89634 51 1345 0.019 0.771 0.506 59.83 369 6.34108E-08 6.99001 45 1294 0.017 0.708 0.486 59.059 326 5.82617E-08 7.08496 46 1249 0.017 0.754 0.47 58.351 333 6.20165E-08 7.18119 48 1203 0.018 0.819 0.452 57.597 348 6.73857E-08 7.27873 30 1155 0.011 0.533 0.434 56.778 217 4.38556E-08 7.3776 38 1125 0.014 0.703 0.423 56.245 275 5.7845E-08 7.47781 48 1087 0.018 0.925 0.409 55.541 348 7.60855E-08 7.57939 36 1039 0.014 0.722 0.391 54.616 261 5.94213E-08 7.68234 39 1003 0.015 0.815 0.377 53.894 282 6.7032E-08 7.78669 36 964 0.014 0.783 0.362 53.079 261 6.44315E-08 7.89246 37 928 0.014 0.838 0.349 52.296 268 6.89566E-08 7.99966 27 891 0.01 0.637 0.335 51.457 195 5.23981E-08 8.10832 38 864 0.014 0.934 0.325 50.82 275 7.67915E-08 8.21846 38 826 0.014 0.972 0.311 49.887 275 7.99636E-08 8.33009 35 788 0.013 0.932 0.296 48.914 253 7.66928E-08 8.44324 32 753 0.012 0.888 0.283 47.982 232 7.30154E-08 8.55792 39 721 0.015 1.127 0.271 47.094 282 9.2663E-08 8.67417 22 682 0.008 0.662 0.256 45.968 159 5.44307E-08 8.79199 31 660 0.012 0.971 0.248 45.306 224 7.98657E-08 8.91141 20 629 0.008 0.652 0.236 44.335 145 5.36545E-08 9.03246 24 609 0.009 0.815 0.229 43.683 174 6.7045E-08 9.15515 25 585 0.009 0.884 0.22 42.868 181 7.27233E-08 9.2795 23 560 0.009 0.847 0.211 41.983 167 6.96689E-08 9.40555 25 537 0.009 0.959 0.202 41.136 181 7.88551E-08 9.5333 25 512 0.009 0.998 0.192 40.178 181 8.21121E-08 9.6628 24 487 0.009 0.998 0.183 39.179 174 8.20838E-08 9.79405 20 463 0.008 0.866 0.174 38.181 145 7.12286E-08 9.92708 30 443 0.011 1.353 0.167 37.316 217 1.11256E-07 10.06192 21 413 0.008 0.986 0.155 35.963 152 8.10959E-08 10.19859 18 392 0.007 0.88 0.147 34.977 130 7.23819E-08 10.33712 23 374 0.009 1.171 0.141 34.097 167 9.63083E-08 10.47753 20 351 0.008 1.06 0.132 32.926 145 8.72055E-08

PAGE 123

110 Appendix C (continued) 10.61985 18 331 0.007 0.994 0.124 31.866 130 8.17269E-08 10.7641 14 313 0.005 0.805 0.118 30.872 101 6.61909E-08 10.91031 15 299 0.006 0.898 0.112 30.067 109 7.38482E-08 11.05851 18 284 0.007 1.122 0.107 29.17 130 9.22783E-08 11.20872 13 266 0.005 0.844 0.1 28.048 94 6.93983E-08 11.36097 14 253 0.005 0.946 0.095 27.204 101 7.78236E-08 11.51528 14 239 0.005 0.985 0.09 26.258 101 8.1038E-08 11.6717 12 225 0.005 0.879 0.085 25.273 87 7.23304E-08 11.83024 15 213 0.006 1.145 0.08 24.393 109 9.41476E-08 11.99093 15 198 0.006 1.192 0.074 23.249 109 9.80363E-08 12.1538 15 183 0.006 1.241 0.069 22.057 109 1.02086E-07 12.31889 12 168 0.005 1.034 0.063 20.816 87 8.50419E-08 12.48622 21 156 0.008 1.884 0.059 19.782 152 1.54971E-07 12.65582 21 135 0.008 1.962 0.051 17.898 152 1.61372E-07 12.82773 4 114 0.002 0.389 0.043 15.936 29 3.20071E-08 13.00197 9 110 0.003 0.912 0.041 15.547 65 7.49907E-08 13.17857 14 101 0.005 1.477 0.038 14.635 101 1.2147E-07 13.35758 11 87 0.004 1.208 0.033 13.158 80 9.93833E-08 13.53902 13 76 0.005 1.487 0.029 11.95 94 1.22304E-07 13.72292 5 63 0.002 0.596 0.024 10.463 36 4.89832E-08 13.90932 3 58 0.001 0.372 0.022 9.867 22 3.06039E-08 14.09825 9 55 0.003 1.162 0.021 9.495 65 9.56038E-08 14.28975 7 46 0.003 0.941 0.017 8.333 51 7.743E-08 14.48385 4 39 0.002 0.56 0.015 7.392 29 4.60733E-08 14.68059 4 35 0.002 0.583 0.013 6.831 29 4.79764E-08 14.87999 4 31 0.002 0.607 0.012 6.248 29 4.9958E-08 15.08211 0 27 0 0 0.01 5.641 0 0 15.28697 2 27 0.001 0.329 0.01 5.641 14 2.70852E-08 15.49462 3 25 0.001 0.514 0.009 5.311 22 4.23059E-08 15.70508 4 22 0.002 0.714 0.008 4.797 29 5.87378E-08 15.91841 3 18 0.001 0.558 0.007 4.083 22 4.5873E-08 16.13463 4 15 0.002 0.774 0.006 3.525 29 6.36904E-08 16.35379 1 11 0 0.202 0.004 2.751 7 1.65803E-08 16.57593 0 10 0 0 0.004 2.549 0 0 16.80108 2 10 0.001 0.437 0.004 2.549 14 3.59566E-08 17.02929 2 8 0.001 0.455 0.003 2.112 14 3.74418E-08 17.2606 3 6 0.001 0.711 0.002 1.657 22 5.84825E-08 17.49505 0 3 0 0 0.001 0.946 0 0 17.73269 0 3 0 0 0.001 0.946 0 0 17.97356 0 3 0 0 0.001 0.946 0 0 18.21769 0 3 0 0 0.001 0.946 0 0 18.46515 1 3 0 0.29 0.001 0.946 7 2.38669E-08 18.71596 0 2 0 0 0.001 0.656 0 0 18.97018 1 2 0 0.315 0.001 0.656 7 2.58792E-08 19.22786 0 1 0 0 0 0.341 0 0 19.48903 1 1 0 0.341 0 0.341 7 2.80613E-08 Total Interior Volume 8.22522E-06


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Leekumjorn, Sukit.
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Synthesis and characterization of potential drug delivery systems using nonionic surfactant "niosome"
h [electronic resource] /
by Sukit Leekumjorn.
260
[Tampa, Fla.] :
University of South Florida,
2004.
502
Thesis (M.S.Ch.E.)--University of South Florida, 2004.
504
Includes bibliographical references.
516
Text (Electronic thesis) in PDF format.
538
System requirements: World Wide Web browser and PDF reader.
Mode of access: World Wide Web.
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Title from PDF of title page.
Document formatted into pages; contains 123 pages.
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ABSTRACT: Niosomes are synthetic microscopic vesicles consisting of an aqueous core enclosed in a bilayer consisting of cholesterol and one or more nonionic surfactants. They are made of biocompatible, biodegradable, non-toxic, non-immunogenic and non-carcinogenic agents which form closed spherical structures (self assembly vesicles) upon hydration. With high resistance to hydrolytic degradation, niosomes are capable of entrapping many kinds of soluble drugs while exhibiting greater vesicle stability and longer shelf life. In this work, a potential drug delivery system has been designed, synthesized and characterized. For the synthesis of niosomes, a hydration process was developed with varying design parameters such as mass per batch, angle of evaporation, rotation speed of vacuum rotary evaporator and nitrogen flowrate to produce uniform thin film in 50 ml round bottom flask. The rehydration process was developed by varying the choice of solvents (H2O, phosphate buffer solution (PBS) and PBS/5(6)-carboxyfluorescein (CF) as a drug model) and hydrating temperature of below and above gel transition temperature. Lastly, a sonication process to produce unilamellar vesicles was partially optimized based on the particle distribution and the number of vesicles formed with sonication time. As a result of this process, unilamellar and multilamellar vesicles were formed with the combination of different nonionic surfactants (sorbitan monostearate-Span 60, sorbitan monopalmitate-Span40 and sorbitan monolaurate-Span20), cholesterol and an electrostatic stabilizer (dicetyl phosphate). The vesicles were examined using light scattering optical microscopy and UV microscopy. Optical sensing technology (Particle Sizing System) is used to determine the vesicles' size distribution. Gel exclusion chromatography (GEC) is discussed as a method to separate unencapsulated CF while retaining vesicle integrity. Particle Sizing System and luminescence spectrophotometer were used to determine CF encapsulation percentage and leakage. Result: Span 20, Span 40 and Span 60/Niosomes were made with mean particle size of 0.95-0.99 micro (mu)m. Typical concentrations of vesicle per ml/per mass of surfactant used were in the range of 1.46-1.79x10 8 Typical encapsulation efficiencies were in the range of 48.8-62.9% for all three Span/Niosome systems. Niosomes were found to be stable for 9 days. The largest vesicles were observed with Span 60 with highest entrapment efficiency as compared to Span 20 and Span 40.
590
Adviser: VanAuker, Michael D.
653
fluorometer.
gel exclusion chromatography.
5(6)-carboxyfluorescein.
encapsulation.
sorbitan monoesters.
690
Dissertations, Academic
z USF
x Chemical Engineering
Masters.
773
t USF Electronic Theses and Dissertations.
4 856
u http://digital.lib.usf.edu/?e14.272