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Antibody targeting of non ionic surfactant vesicles to vascular inflammation

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Title:
Antibody targeting of non ionic surfactant vesicles to vascular inflammation
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English
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Hood, Elizabeth D
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University of South Florida
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Tampa, Fla.
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Subjects / Keywords:
Drug delivery
Niosomes
Vesicles
Immunotargeting
Atherosclerosis
Dissertations, Academic -- Chemical Engineering -- Doctoral -- USF   ( lcsh )
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Summary:
ABSTRACT: Cardiovascular disease (CVD) and particularly atherosclerosis is a leading cause of morbidity in the developed world. Atherosclerosis and the rupture of vulnerable atherosclerotic plaque cause 70% of deaths from CVD. The progression of atherosclerosis has been identified as a pathological inflammatory process. Targeting atherosclerotic drug therapies to inflammatory markers has emerged as an important and growing research area. The adhesion molecule CD44 has been implicated in the onset and build-up of atherosclerotic lesions throughout the course of development. The research in this dissertation is aimed at targeting anti-inflammatory therapy to activated vascular endothelium with directed with an anti-CD44 antibody, IM7, conjugated to a non ionic surfactant vesicle (niosome) drug carrier. The IM7 conjugated immunoniosome has been shown to bind to endothelial and synovial lining cells in vitro.The preliminary research is involved with the development of the drug delivery vesicle, and the antibody linkage chemistry, along with an analysis of vesicle characteristics and stability. A novel linking chemistry using polyoxyethylene sorbitan monostearate and cyanuric chloride allows antibodies to be conjugated to vesicle surface polymer groups without prior derivatization. Subsequent research tested the resulting 'immunoniosome's' ability to bind to target antigens with selectivity and specificity. Bovine aortic endothelial cells activated with cytokines provide a model of inflammation. Analysis of binding was done through fluorescent and scanning electron microscopy. In vivo uptake of vesicles at sites of inflammation is size dependent. In order to overcome this barrier to uptake, niosome suspensions were thermally extruded to create uniform 200 nm vesicles.Further analysis of the efficacy of the system looked at live cell uptake of the immunoniosomes measured by confocal and transmission electron microscopy. Preparation for in vivo murine studies required that the antibody component was modified to counteract the immune response. Finally, the conjugation of antibody fragments to niosomes and the binding and uptake of the vesicles in a live endothelial cell model is evaluated. A viable drug delivery particle showing binding and cellular uptake capabilities in inflammatory cells was produced by this research using a novel surfactant-antibody linker.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2007.
Bibliography:
Includes bibliographical references.
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Statement of Responsibility:
by Elizabeth D. Hood.
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Title from PDF of title page.
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Includes vita.
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Document formatted into pages; contains 186 pages.

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ABSTRACT: Cardiovascular disease (CVD) and particularly atherosclerosis is a leading cause of morbidity in the developed world. Atherosclerosis and the rupture of vulnerable atherosclerotic plaque cause 70% of deaths from CVD. The progression of atherosclerosis has been identified as a pathological inflammatory process. Targeting atherosclerotic drug therapies to inflammatory markers has emerged as an important and growing research area. The adhesion molecule CD44 has been implicated in the onset and build-up of atherosclerotic lesions throughout the course of development. The research in this dissertation is aimed at targeting anti-inflammatory therapy to activated vascular endothelium with directed with an anti-CD44 antibody, IM7, conjugated to a non ionic surfactant vesicle (niosome) drug carrier. The IM7 conjugated immunoniosome has been shown to bind to endothelial and synovial lining cells in vitro.The preliminary research is involved with the development of the drug delivery vesicle, and the antibody linkage chemistry, along with an analysis of vesicle characteristics and stability. A novel linking chemistry using polyoxyethylene sorbitan monostearate and cyanuric chloride allows antibodies to be conjugated to vesicle surface polymer groups without prior derivatization. Subsequent research tested the resulting 'immunoniosome's' ability to bind to target antigens with selectivity and specificity. Bovine aortic endothelial cells activated with cytokines provide a model of inflammation. Analysis of binding was done through fluorescent and scanning electron microscopy. In vivo uptake of vesicles at sites of inflammation is size dependent. In order to overcome this barrier to uptake, niosome suspensions were thermally extruded to create uniform 200 nm vesicles.Further analysis of the efficacy of the system looked at live cell uptake of the immunoniosomes measured by confocal and transmission electron microscopy. Preparation for in vivo murine studies required that the antibody component was modified to counteract the immune response. Finally, the conjugation of antibody fragments to niosomes and the binding and uptake of the vesicles in a live endothelial cell model is evaluated. A viable drug delivery particle showing binding and cellular uptake capabilities in inflammatory cells was produced by this research using a novel surfactant-antibody linker.
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Antibody Targeting of Non Ionic Surfact ant Vesicles to Vascular Inflammation by Elizabeth D. Hood A dissertation submitted in the partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemical Engineering College of Engineering University of South Florida Major Professor: Michael D. VanAuker, Ph.D. Joel A. Strom, M.D. Karl Muffly, Ph.D. Mark Jaroszeski, Ph.D. Stanley Kranc, Ph.D. Date of Approval: November 7, 2007 Keywords: drug delivery, niosomes, vesi cles, immunotargeting, atherosclerosis Copyright 2007, Elizabeth D. Hood

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Dedication I dedicate this document to my sons Aaron and Lucas, and to my parents, Alan and Mary, who were always allies to the common enemy in the middle. This work was only possible with the lo ve, support, and patience of my family. Thank you all.

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Acknowledgments I would like to acknowledge the me mbers of my committee for their mentorship and support. Dr. Michael Va nAuker and Dr. Strom both challenged and motivated me to present this resear ch as it developed over the years in conferences and symposia which provided me with a wide range of experience and knowledge greater t han if I had not ventured out of the lab. Their mentorship gave me the opportunity to present our work at national and international conferences in San Francisco, San Diego New York City, and New Orleans. Thanks to Dr Mark Jaroszeski for being an excellent instructor in the classroom and in the laboratory. His ex perience and guidance with metrology, cell culture and microscopy are very much appreciated. I want to especially thank Dr Karl Mu ffly for help with microscopy and cell culture. His endless patience and open doo r made the research not just possible but enjoyable. I would also like to extend my gratitude to the patience and instruction generously given by Ed Hall er, USF Health Pathology Department and manager of the Microscopy Core Fa cility, for endless hours of assistance and guidance in microscopy and tissue and lipid fixation techniques. Thanks to John Elliot for the Matlab™ im age analysis program and TEM images. Research is impossible without funding; I gratefully acknowledge funding from the University of South Florida New Researc hers Award, the Florida Chapter of the Arthritis Foundation for a summer research fellowship, and the Department of Defense US Army Medical Research Acquisition Activity Grant No.W81XWH-05-1-0585, all of whic h made this work possible.

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Note to Reader: The original of this document contains color that is necessary for understanding the data. The or iginal dissertation is on file with the University of South Florida library in Tampa, Florida.

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i Table of Contents List of T ables .......................................................................................................vii List of Fi gures..................................................................................................... viii List of Abbr eviations ............................................................................................xii ABSTRACT.........................................................................................................xv 1. Prelude ..........................................................................................................1 2. Literature Ex amination...................................................................................4 2.1. Overview................................................................................................4 2.2. Cardiovascular Disease and Atheroscl erosis.........................................5 2.2.1. Implications of Card iovascular Di sease..........................................5 2.2.2. Atherosclerosis Developm ent.........................................................6 2.2.3. Atherosclerosis and Inflamma tion...................................................9 2.3. Adhesion Mole cules.............................................................................11 2.3.1. Adhesion Molecu le CD 44..............................................................13 2.4. Drug Target ing.....................................................................................14 2.4.1. Vesicular Drug Delivery.................................................................16 2.4.1.1. Liposom es..............................................................................16 2.4.1.2. Niosomal Drug Delivery.........................................................19 2.5. Background and Current Practices in Antibody Mediated Drug Targeting .............................................................................................................20

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ii 2.6. Cardiovascular Antibody M ediated Imaging and Therapy....................23 2.6.1. Imaging and Therapy ....................................................................23 2.6.2. Current Research in Vesicle Mediated Drug Delivery in Inflammation and Cardio vascular Dis ease..................................................25 3. Project Descr iption ......................................................................................29 3.1. Development and Testing of an Immunoni osome................................30 3.2. Binding and Uptake of Vesi cles............................................................32 4. Vesicle Deve lopment ...................................................................................34 4.1. Immunoniosome Synthesis Project Descr iption ...................................34 4.2. Material and Methods ...........................................................................35 4.2.1. Introducti on...................................................................................35 4.2.1.1. Gel-Liquid Crystal Trans ition Temper ature............................38 4.2.2. Material s.......................................................................................38 4.2.2.1. Chemical s..............................................................................38 4.2.2.2. Vesicle Characterization Materials and Methods...................39 4.2.2.3. Vesicle Purification Ma terials and Methods............................43 4.2.2.3.1. Gel Exclusion Ch romatogr aphy.........................................44 4.2.2.3.2. Fluorescence In tensity.......................................................47 4.2.2.3.3. UV Absorb ance..................................................................48 4.2.2.3.4. Ultras ound......................................................................... 49 4.2.2.4. Equipm ent..............................................................................50 4.2.3. Methods........................................................................................51 4.2.3.1. Vesicle Sy nthesis ...................................................................51

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iii 4.2.3.1.1. Thin Film Hydrat ion Techni ques........................................51 4.2.3.1.2. GEC Purifi cation................................................................54 4.2.3.1.3. UV Absorb ance..................................................................54 4.2.3.1.4. Niosomes of Differing Su rfactant Components..................55 4.2.3.1.5. Increased Lipid C oncentrati ons.........................................56 4.2.3.2. Development of Tween 61-Span 60 Nio some........................56 4.2.3.2.1. Tween-Span Mixed Formulati ons......................................57 4.2.3.3. Functionalization of Tween 61 with Cyanuric Chloride...........58 4.2.3.4. Attachment and Verification of Antibody Conjugation............59 4.2.3.5. Synovial Lining Cell Culture Me thods....................................61 4.2.3.6. Immunoniosome Synovial Lining Cell Incubation Methods....61 4.2.3.7. Proof of Concept Cell Bind ing................................................62 4.2.3.8. Statistical Methods.................................................................62 4.2.4. Experimental Designs ...................................................................64 4.2.4.1. Vesicle Formulati on Assessm ent...........................................64 4.2.4.1.1. Sorbitan Ester Formulati ons..............................................64 4.2.4.1.2. Tween-Span US Exposure Stability Study.........................64 4.2.4.2. Polyoxyethylene Sorbitan Monos tearate-Cyanuric Chloride Linker Chem istry......................................................................................65 4.3. Results.................................................................................................66 4.3.1. Sorbitan Ester Vesi cle Formula tions.............................................66 4.3.1.1. Results of the Varied Sorb itan Ester Formu lations................66 4.3.1.2. Results of Ultrasound Ex posure on Nio somes.......................69

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iv 4.3.2. Results of Surfac tant Blen ding ......................................................71 4.3.3. Results of Linking Ch emistry Deve lopment ...................................73 4.3.4. Binding of Immunoniosomes to Synovial Lini ng Cells...................76 4.4. Discussio n............................................................................................78 5. Fixed Endothelial Cell and Immun oniosome Bindin g Studies......................81 5.1. Backgroun d..........................................................................................81 5.1.1. Endothelial Cells Antibody-T argeted Drug Delivery......................81 5.2. Materials and Methods .........................................................................82 5.2.1. Materials and C hemicals...............................................................82 5.2.2. Methods........................................................................................82 5.2.2.1. Endothelial Cell Culture.........................................................82 5.2.2.1.1. Gelatin Coating Procedure.................................................84 5.2.2.2. Cell Fixation...........................................................................84 5.2.2.3. Immunohistochemical Staining..............................................85 5.2.3. Incubation Ex periments.................................................................86 5.2.3.1. Design of Ex periment s...........................................................86 5.2.4. Description of Methods..............................................................88 5.2.4.1. Incubation of Niosomes wit h Endothelia l Cell s.......................88 5.2.4.2. Fluorescent Microscopy Im aging of Inc ubated Ce lls..............88 5.2.4.3. Image Anal ysis.......................................................................88 5.2.4.4. Scanning Electron Microsc opy...............................................91 5.3. Results.................................................................................................93 5.3.1. Endothelial Cell-Imm unoniosome Images.....................................93

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v 5.3.1.1. Fluorescent Mi crograp hs........................................................93 5.3.1.2. Scanning Electron Mi crograph Im ages..................................94 5.4. Discussio n............................................................................................96 6. Live Endothelial Ce ll Uptake Studies ...........................................................98 6.1. Backgroun d..........................................................................................98 6.1.1. Live Endothelial Cells in Targeted Drug Delivery..........................98 6.1.2. Dynamic Light Scatteri ng..............................................................99 6.1.3. Monoclonal Antibody Fragments.................................................101 6.2. Materials and Methods .......................................................................103 6.2.1. Materials and C hemicals.............................................................103 6.2.2. Methods ......................................................................................104 6.2.2.1. Extrusion of Niosomes .........................................................104 6.2.2.2. Dynamic Light Scatteri ng Measurem ents............................107 6.2.2.3. Antibody Fragment ation.......................................................110 6.2.2.4. Endothelial Cell Cu lture and Fixation Techniques................110 6.2.2.5. Fixation Techniques for C onfocal Micr oscopy......................111 6.2.2.6. Fixation Techniques for Transmission Electron Microscopy111 6.2.3. Confocal Micr oscopy ...................................................................112 6.3. Experimental Designs ........................................................................114 6.4. Results...............................................................................................115 6.4.1. Extrusion Re sults........................................................................115 6.4.2. TEM............................................................................................120 6.5. Conclusion s........................................................................................121

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vi 7. Conclusions and C ontributi ons..................................................................123 7.1. Introducti on........................................................................................123 7.2. Contributio ns......................................................................................124 7.3. Future Work.......................................................................................125 Referenc es.......................................................................................................128 Appendice s.......................................................................................................146 Appendix A. Particle Sizing Syst ems Data and Softw are Output.................147 Appendix B. Akta Prime Chro matography Data Output...............................155 Appendix C. Fluoresc ent Plate Reader........................................................ 158 Appendix D. Matlab ™ Image Anal ysis.........................................................162 Appendix E. Confocal Image Processi ng Data ............................................171 About the Aut hor.....................................................................................E nd Page

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vii List of Tables Table 4.1. Equipment for Vesi cle Development and Synthes is.........................50 Table 4.2 Surfactant Stru ctures and Pr operties ..................................................53 Table 4.3 Low Concentration Ex ample of Vesicl e Component s........................56 Table 4.4 Masses of Vesicle Component s for Varied Tween 61 Surfactant Percentages Ratios per F ilm..............................................................................57 Table 5.1 Experimental Variables of BAEC-IN Bindi ng Experim ents..................87 Table 6.1 Equipment for Uptake Studi es..........................................................104 Table 6.2 Confoc al Setti ngs..............................................................................113 Table 7.1 Future Work: In Vivo Atherosclerotic Mouse St udy..........................126 Table A.1 Data Exported Fr om PSD ASC II File................................................148

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viii List of Figures Figure 2.1 The Stages of At herosclerotic Plaque.................................................7 Figure 2.2 The Phases of Atheroscler osis............................................................8 Figure 2.3 Vesicular Inte ractions wit h Cells ........................................................18 Figure 3.1 Interdependence of Elements of the Drug Deliv ery System.............29 Figure 3.2 Immunoniosome Membrane Stru cture..............................................32 Figure 4.1 Critical Packing Pa rameter and Bilaye r Membrane..........................36 Figure 4.2 Autodilution Scheme of Particle Sizi ng System.................................40 Figure 4.3 Particle Size Distribution Plot of a Mix of Three Sizes of Polystyrene Latex Stan dards.................................................................................................41 Figure 4.4 Stabili ty of CF....................................................................................42 Figure 4.5 Calibrati on Curve of CF.....................................................................43 Figure 4.6 Calibration Curve with a Wide Range of C oncentrati ons..................43 Figure 4.7 Sephadex G 50 Hydrated Beads. ......................................................45 Figure 4.8 Chromatogram of Span 60 Nio somes of Varied PBS Concentrations ...........................................................................................................................46 Figure 4.9 Release of Dy e from Nios omes.........................................................48 Figure 4.10 Intrinsic UV Ab sorbance of Niosome s.............................................55 Figure 4.11 Tween 61 Cyanuric Ch loride Linking Mechanism............................59 Figure 4.12 Antibody Conjugat ion......................................................................60 Figure 4.13 Encapsulation of CF by Surfac tant Ty pe.........................................66

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ix Figure 4.14 Formation of Niosomes afte r Hydration by Surfactant Type............67 Figure 4.15 The Effect of Sonication Time on Partic le Size and Counts............68 Figure 4.16 Sonica tion Effects............................................................................68 Figure 4.17 PSD Soni cation Effe cts...................................................................69 Figure 4.18 PSDs with Res pect to US Exposure................................................70 Figure 4.19 The Effect of US Ex posure Time on Re lease of CF........................70 Figure 4.20 The Effect of MI on Rel eased CF in a Tw een 61 Niosome..............71 Figure 4.21 The Effect of MI on Particle Retention in the Span 60 Niosomes....71 Figure 4.22 The Retention of CF in Tween 61-Span 60 Niosome s....................72 Figure 4.23 Increased UV Si gnal with Increased Lipid Concentration in Tw-CCCF Niosome s. .................................................................................................73 Figure 4.24 Elution of IN wit h Fluorescent Antibodie s........................................74 Figure 4.25 Fluorescent Mi crograph of IN with Fluor escent Antibodies..............75 Figure 4.26 Stability of the 10% Tw-CC-CR Post GE C Niosome ........................76 Figure 4.27 Experimental and Control Images of Sy novial Lining Cells.............77 Figure 4.28 Synovia l Lining Ce lls.......................................................................78 Figure 5.1 Matlab Program Importing and Cropping...........................................89 Figure 5.2 Matlab Progr am Image So rting..........................................................90 Figure 5.3 Matlab Progr am Image A nalysis. .......................................................90 Figure 5.4 Matlab Program Fluoresc ent Nuclei and IN Overlay..........................91 Figure 5.5 Immunohistochemic al Stain fo r CD44. ..............................................93 Figure 5.6 Fluorescent and Contra st Overlay of BAECs and IN s.......................93 Figure 5.7 Scanning Electron Micrographs of BAECs. ......................................94

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x Figure 5.8 Binding Densit y with Respect to Time and Concentration................ 95 Figure 5.9 Binding Dens ity with Respect to An tibody Concent ration..................95 Figure 6.1 IgG Antibod y Structur e....................................................................103 Figure 6.2 Extruder Assembly .........................................................................105 Figure 6.3 Nitrogen Deliv ery to Ex truder.........................................................106 Figure 6.4 Particle Size Distributi on of 60 nm and 220 nm Standards..............109 Figure 6.5 Correlation Function Curve of the Standards. .................................109 Figure 6.6 Leica Confocal Softw are Image Acquisiti on Window. .....................113 Figure 6.7 Spatial Setting for Scan Mode Imaging. .........................................114 Figure 6.8 Elution of Extrusions 0-10 for a 10% TW-CC-CF Hydration Sample. .........................................................................................................................116 Figure 6.9 Dynamic Light Scatteri ng Data of Extr uded Sample s......................116 Figure 6.10 Confocal BAECs 20 Minute Inc ubation. ........................................117 Figure 6.11 Confocal BAECs I Hour Inc ubation. ...............................................118 Figure 6.12 Confocal BAECs 2 Hour Inc ubation...............................................118 Figure 6.13 Confocal BAECs Cont rol..............................................................119 Figure 6.14 Confocal Cross Secti ons..............................................................119 Figure 6.15 Fab-IN BAEC Confocal Image.......................................................120 Figure 6.16 TEM of Au-INs ..............................................................................120 Figure A.1 Particle Size Dis tribution of Standards ............................................147 Figure A.2 Sample Num ber Calculat ion...........................................................148 Figure B.1 Akta Prime Elution Chro matogram Showing UV Absorbance and Conductivi ty......................................................................................................155

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xi Figure B.2 Akta Prim e Method No tes...............................................................156 Figure B.3 Akta Prime Chromatogr am Showing All Measures .........................157 Figure C.1 Fluorescent Plate R eader: Protocol De finition................................158 Figure C.2 Fluorescent Plate Reader: Defined Plate Reader Geometry..........159 Figure C.3 Fluorescent Plate Reader: De fine Individual Well Measurement Types .........................................................................................................................160 Figure C.4 Fluorescent Plate R eader: Stored Standar d Curve.........................160 Figure C.5 Fluorescent Plate Reader: Fl uorescence Intensit y by Wells...........161 Figure C.6 Fluorescent Plate Reader: Intens ity Data Exported to Microsoft Excel .........................................................................................................................161

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xii List of Abbreviations Alexa Fluor ™ 488 AF American Heart Association AHA Bovine aortic endothelial cell BAEC Bovine serum albumin BSA Carboxyfluorescein CF Carboxyrhodamine CR Cellular adhesion molecules CAMs Cyanuric chloride CC Deionized DI Deoxyribonucleic acid DNA Dicetyl phosphate DCP Diisopropylethylamine DIPEA Dulbecco’s Modified Eagl e Medium DMEM Dynamic light scattering DLS Endothelial cells ECs Extracellular matrix ECM Fluorescein isothiocyanate FITC Gel exclusion chromatography GEC Hank’s Balanced Saline HBS

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xiii Hydrophilic-lipophilic balance HLB Immunohistochemical IHC Institutional Animal Care and Use Committee IACUC Intracellular adhesion molecule-1 ICAM-1 Immunoglobulin IgG Immunoliposome IL Immunoniosome IN Interleukin-1 IL-1 Leica Confocal Software LCS Molar M Monoclonal antibody mAb Particle size distribution PSD Particle Sizing Systems PSS Phosphate buffered saline PBS Photomultiplier tube PMT Platelet endothelial adhesi on molecule-1 PECAM-1 Polyethylene glycol PEG Polyethylene oxide PEO Polyoxyethylene sorbitan monos tearate Tween 61 Reticuloendothelial system RES Scanning electron microscopy SEM Sorbitan monostearate Span 60 Sorbitan monolaurate Span 40

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xiv Sorbitan monopalminate Span 20 Standard error of the mean SEM Synovial lining cells (synoviocytes) SLs Tissue factor TF Tissue necrosis factor alpha TNFTransmission electron microscopy TEM Ultrasound US Ultraviolet UV University of South Florida USF Vascular adhesion molecule-1 VCAM-1

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xv Antibody Targeting of Non Ionic Surfact ant Vesicles to Vascular Inflammation Elizabeth D. Hood ABSTRACT Cardiovascular disease (CVD) and particularly atherosclerosis is a leading cause of morbidity in the developed worl d. Atherosclerosis and the rupture of vulnerable atherosclerot ic plaque cause 70% of deaths from CVD. The progression of atherosclerosis has been id entified as a pathological inflammatory process. Targeting athero sclerotic drug therapies to inflammatory markers has emerged as an important and growing rese arch area. The adhesion molecule CD44 has been implicated in the onset and build-up of atherosclerotic lesions throughout the course of devel opment. The research in this dissertation is aimed at targeting anti-inflammatory therapy to activated vascular endothelium with directed with an anti-CD44 antib ody, IM7, conjugated to a non ionic surfactant vesicle (niosome) drug carrier. Th e IM7 conjugated immunoniosome has been shown to bind to endothelia l and synovial lining cells in vitro The preliminary research is involv ed with the development of the drug delivery vesicle, and the antibody linkage chemistry, along with an analysis of vesicle characteristics and stability A novel linking chemistry using polyoxyethylene sorbitan monostearate and cyanuric chloride allows antibodies to be conjugated to vesicle surface polym er groups without prior derivatization.

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xvi Subsequent research tested the resulting ‘immunoniosome’s’ ability to bind to target antigens with selectivity and spec ificity. Bovine aortic endothelial cells activated with cytokines provide a model of inflammation. Analysis of binding was done through fluorescent and sc anning electron microscopy. In vivo uptake of vesicles at sites of inflammation is si ze dependent. In order to overcome this barrier to uptake, niosome suspensions were thermally extruded to create uniform 200 nm vesicles. Further analysis of the effi cacy of the system looked at live cell uptake of the immunonios omes measured by confocal and transmission electron microscopy. Preparation for in vivo murine studies required that the antibody component wa s modified to counteract the immune response. Finally, the conjugation of antibody fragments to niosomes and the binding and uptake of the vesicles in a live endothelial cell model is evaluated. A viable drug delivery particle showing binding and cellul ar uptake capabilities in inflammatory cells was produced by this research using a novel surfactantantibody linker.

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1 1. Prelude Immunotherapy, the use of antibodies to treat disease, is of great interest in the fields of chronic infectious dis ease, cancer, cardiovascular, and arthritis research, among others. In 1796, Edwa rd Jenner used a cowpox virus to develop a vaccine against small pox. He discovered that deliberate infection with the virus brought on a mild state of the disease and a subsequent immunity to it1. It was Paul Erlich at the beginning of t he 20th century who originally discovered antibodies and described the role they play in humoral immunity. He proposed the ‘magic bullet’ concept of using anti bodies to send therapeutic agents to target cells2. He also imaged that “a carrier by which to bring therapeutically active groups to the organ in ques tion” would be advantageous3. Not until the development of monoclona l antibody (mAb) production could his ‘magic bullet’ concept be realized. In the 1970s the B cell melanoma was identified as producing a single type of antibody. Georges Jean Franz Khler and Csar Milstein invented the process to produce monclonal antibodies in 1975 for which they won a Nobel Prize in Physiology or Medicine in 19844. Gregory Winter developed techniques to humanize monoc lonal antibodies for therapeutic uses5. In 1965 Dr. Alec Bangham published ‘Diffusion Of Univalent Ions Across Lamellae Of Swollen Phospho lipids’ which is the seminal work of liposome6, and therefore, vesicular drug delivery. Mo re than forty years later strategic drug delivery using phospholipid or other bila yer model structures continues to be pursued for myriad applications such as vaccinations7, gene delivery8,

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2 thrombolysis9, topical applications10, tumor targeting 11, vascular targeting12, transdermal13, and ophthalmic treatments10,14 among many others. The combination of immunotherapy and ve sicular drug delivery originated with Paul Erlich at t he beginning of the 20th century. Almost eighty years later in 1981, Torchilin and Klibanov reviewed methods for immobilizing proteins on the surfaces of liposomes15. Immunotargeting of drugs using an antibody vector bound to a drug carrier has continued to be dev eloped in the fields of cancer and tumor therapy16-27, and cardiovascular research28-34. The use of immunotargeted drug delivery to effect treatment and blo ck progression of damaging inflammatory processes has not been as widely pursued as cancer and tumor targeting, most likely due to the more highly toxic effe cts that anticancer drugs have on healthy tissues. However, the prevalence of cardiovascular disease and especially atherosclerosis35, the challenges of restenosis of arteries after angioplasty, the build up of plaques on st ent implants, and other e ffects of inflammatory processes in cardiovascular disease, as well as the questionable systemic side effects produced by cardiovascular drugs36,37 provide motivation and opportunities for a more su ccinct therapeutic approach. Current research into innovative anti -atherosclerotic treatment includes identifying and manipulating the inflamma tory mechanisms consistent with the progression of the disease from initia l fatty streaks to fibrous plaques to vulnerable complex lesions, uptake me chanisms of macrophage derived foam cells38, and the identification of the regulat ory factors controlling inflammatory response in endothelial cells and uptake of lipoproteins39,40.

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3 The hypothesis of this research is that an antibody conjugated non ionic surfactant vesicle could be developed and targeted to inflammation with the potential to provide therapeutic benefit by targeting specific inflamed tissues. In order to test this hypothesis the following aims were pursued: Aim 1. Develop and characterize a targeted drug delivery system using non ionic surfactant vesicles conjugated to monoclonal antibodies specific to an inflammatory target antigen. Aim 2. Quantify and optimize the immunoniosome-antigen binding in fixed culture aortic endothelial cells with respect to concentrations of both vesicle concentration and antibody density using fluorescence microscopy and computer image analysis. Aim 3. Observe, describe, and quantify cellular uptake of immunoniosomes in live cells using confocal and electron microscopy. Aim 4. Once the drug delivery system is sufficiently described, develop a protocol for the in vivo evaluation of the adherence and effect of the immuniosomes encapsulating atorvastat in on plaque development in the ApoE knockout mouse model. The successful completion of these aims would produce a well described drug delivery system ready for in vivo testing in an atherosclerotic model.

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4 2. Literature Examination 2.1. Overview Developing a drug delivery system provokes inquiry into several different disciplines of research. The original i dea was to develop a targeting system that would treat atherosclerosis, specifically the vulnerable plaque of atherosclerosis. Investigations into the recent developments and conclusions about atherosclerosis revealed that the disease, like numerous other chronic pathologies such as arthritis, lupus, and chronic obstructive pulmonary disease, is an inflammatory disorder. Systemic implications and common mechanisms in the build up of inflammatory disease ar e demonstrated by the high incidence of atherosclerosis in rheumatoid arthritis pat ients without traditional risk factors for the disease41. The onset and characteristic phases of atherosclerosis are described, as well as the mechanisms of inflammation and the disease progression. Literature reviews of athero sclerosis lead to investigation of other inflammatory pathologies as a means to identify potential treat ment strategies. Furthermore, the mechanisms of di sease onset and progression provide targeting strategies; cellular adhesion mole cules are mediators of progression of atherosclerosis, among other inflammatory diseases, and are reviewed generally, and the candidate adhesion molecule, CD44 specifically. Identifying the appropriate drug delivery vehicle provoked research into the large body of work representing drug targeting generally, vesicular drug targeting, and liposomal and niosomal drug delivery specifically Drug targeting may be directed passively or actively; acti ve targeting is usually mediated with a

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5 vector molecule, and some of the methods and applications of actively targeted drug delivery are reviewed. Finally, a re view of targeted im aging and therapy in cardiovascular medicine is reviewed. 2.2. Cardiovascular Disease and Atherosclerosis 2.2.1. Implications of Cardiovascular Disease The American Heart Association (AHA ) lists prevalence among Americans of having one or more types of cardiovascu lar disease (CVD) at 37.1% for 2004. Over 36% of all deaths of Americans in 2004 were attributed to CVD35. Of the different types of CVD, coronary heart disease and its underlying cause, atherosclerosis, is the lar gest single cause of death42. Atherosclerosis is a type of arteriosclerosis, which describes t he thickening and hardening of the arteries generally. Atherosclerosis is the build up of lipids, cholesterol, calcium, cellular waste, and fibrin and the proliferation of smooth muscle and inflammatory cells within the sub endothelial space of medium to large arteries43, specifically including the aorta, carotid, coronary, and peripheral arteries44. This evolving accumulation of plaque causes narrowing of the arteries reducing the lumen and the area available for blood flow, and decreases flexibility of vessels to absorb the pressures created by blood flow. Stenos is is defined as “the constriction or narrowing of a passage or orifice”45. Atherosclerotic plaques are prone to erosion and/or rupture which produces a release of core lipid and infla mmatory contents into the blood stream. As the coagulation factors in the blood contact the infla mmatory tissue factor (TF) expressed by the macrophages and the sm ooth muscle cells, along with other

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6 expressed materials, a blood clot or thrombus forms in the lumen of the blood vessel42. A thrombus is the product of t he coagulation cascade of the blood; the platelets activated by thrombin form a plug immediately and are further enforced as fibrin fibers are formed by the cascade of coagulation factors46. This process is necessary for hemostasis, to reduce blood loss in a damaged blood vessel, for example, however the formation of occl usive thrombi as a result of plaque rupture is the major instigat ing factor of coronary events47. Additionally it has been shown that the plaque’s co mposition is a greater pr edictor to susceptibility to rupture than size or degree of stenosis42. In fact, many plaques prone to rupture do not appear to be severely stenotic in a coronary angiogram48-51. 2.2.2. Atheroscler osis Development Traditionally, atherosclerosis was considered to be a passive lipid processing disorder with plaques gradually evolving over time to eventual occlusion of the blood vessel. In this view the blood vessel itself is considered to be an ‘inert tube’, not a dynamic structure interacting wit h blood and extracellular matrix elements and cells, and proteins, wh ich are seen in the normal artery but also actively contribute to the progression of the disease. Plaques may form as either stable, or unstable, vulnerable. Stable plaques are characterized by a thick fibrous cap, a small core of lipid materials and an unchanged lumen area, wher eas vulnerable plaques are characterized by a thin fibrous cap covering a large lipid core containing inflammatory cells. Atherosclerosis is classified in stages assigning levels of severity to plaques as they develop as seen in Figure 2.144. The initial stage, Phase 1, is

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7 the development of the fatty streak and proliferation of monocytes; the endothelium may be unaffected at this stage and no symptoms are evident52 as the vasculature compensates by ‘remodel ing’ and maintaining the size of the lumen53. Figure 2.1 The Stages of Atheroscl erotic Plaque Development. From 47. Figure 2.2 shows an outline of t he stages describing the disease progression as defined by atherosclerot ic lesion development and architecture47. At the first stage the small lesions are of three types of increasing complexity; type I consists of lipid-filled foam cells derived from macrophages. Type II lesions contain smooth muscle cells and lipids from the extracellular matrix along with fatty foam cells, and in type III lesions the smooth muscle cells are immersed in connective tissue, fibrils and lipids54. Phase 2 is described as advanced, with pre-stenotic le sions with potential to rupt ure, and progresses into either Phase 3 or 4. Plaques at Phas e 2 are either type IV, or type Va; the

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8 former characterized by a large lipid vo lume and smaller fibrous cap, and the latter having a more extensive cap47. Phase 3 lesions are type VI developed from type IV or Va lesions that hav e ruptured or eroded and form non occlusive thrombi. Phase 4, as shown in Figure 2.2, is the alternate path from Phase 2, and has severe characteristic symptoms from type VI lesions. Occlusions may be constant or periodic and pr esent clinically as acute coronary syndrome. Because a third of occlusive thrombi come from non stenotic pl aque disruption the syndrome may not be obvious until ther e is severe sudden ischemia or a myocardial infarction (MI)47. Finally, in Phase 5 lesions are type Vb or Vc and are either, respectively, calcified or fibrous and may both cause angina (chest pain due to constricted oxygen flow) or ischemia44. Figure 2.2 The Phases of Atherosclerosis. From 55 To further complicate an intric ate process, the progression of atherosclerotic plaques vulnerable to rupture and thrombus formation and their

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9 triggering events are not homogenous across di fferent areas of the vasculature. Plaque disruption is a critical trigger for acute events in coronary arteries, less so in the carotid, and in the peripheral ar teries thrombogenicity (the propensity to form thrombi) is more crucial42. 2.2.3. Atherosclerosis and Inflammation Inflammatory processes play a role in vascular disease, rheumatoid and osteoarthritis, chronic obstructive pulm onary disease, and inflammatory bowel disease, lupus, among others. The inflammatory proc ess is characterized by accumulation of inflammatory cells leukocytes, and macrophages that perpetuate the process and contri bute to tissue destruction. Once understood to be a disorder of lipid accumulation and cholesterol metabolism, atherosclerosis is now described as a “chronic inflammatory disease of the arterial system”56; inflammatory factors are implicated in every phase of plaque development. Endothelial cells contacting flowing blood mediate interactions of underlying vessel tissue with blood cells and components. In normally functioning endothelium, which forms a monolayer of cells on a basement membrane that cover the intima l layer, expression of inflammatoryresponse cellular adhesion molecules (CAM s) and the subsequent attachment of circulating leukocytes, is minimal42. There is general agreement that atherosclerosis begins with an inciting injury to the endothelium provoking an inflammatory immune response57. When normal homeostasis is not restored, endothelial cell dysfunction continues as the disease progresses58. It is the expression of adhes ion molecules and the influx

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10 of leukocytes that perpetuate the dis ease throughout the process. Vascular adhesion molecule-1 (VCAM-1) has been shown to play a major role in leukocyte recruitment, binding to monocytes and T lymphocytes59. Further description on the role and mechanisms of adhesion molecu les in the inflammatory process will be discussed in Section 2.3. A mono cyte crossing the endothelium and entering the tunica intima becomes a tissue macrophage, which then takes up lipids and lipoproteins, in particular oxidized low density lipoprotein (LDL) in the plaque and further transforms into a foam cell60. The build up of foam cells is a characteristic of plaques. The foam cells produce proinflammatory cytokines which further provoke immune response in the plaque an d promote reactive oxygen species61. Eventually the foam cells accumulate in t he center of the lesion, die, and form a necrotic core within the plaque. Death of th e foam cells is attributed to either apoptosis (programmed cell deat h) or the toxic effect s of oxidized lipoprotein uptake62. The products of the inflammatory cells within the plaques disrupt the stability of the fibrous cap covering the plaque. Macrophages and smooth muscle cells release proteinases, such as collagenase and elastase, which break down the structural proteins collagen and elastin. The degradation of elastin also disrupts the ability of cells to move though the plaque61.

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11 2.3. Adhesion Molecules Cellular adhesion molecules (CAMs) mediate blood -e ndothelial cell interactions common to all segments of the vasculature under physiological or pathological conditions 62. They are glycoproteins which have cytoplasmic, transmembrane and extracellular domains63. CAMs are expressed by nearly every cell type55, and are characterized by strong ligand binding. They participate in cell to cell and cell to matrix interactions and in some cases also signalling, migration, motility, gene transcription and differentiation64. As was mentioned previously, a hallm ark of endothelial dysfunction at the initiation of atherosclerosis is the persi stent adherence of circulating leukocytes and their subsequent uptake into the tunica intima65. The inflammatory cells are recruited by CAMs. CAMs expressed by the endothelium include intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), and platelet-endothelial cell adhesion molecule-1 (PECAM-1), integrins, and selectins66. Once the adhesion molecules bind with monocytes, macrophages, lymphocytes or platelet s, those bound elements becom e activated themselves, and release proinflammatory cytokines, membrane receptors, and a myriad of enzymes, including the interleukins, tissue necrosis factor alpha (TNF), interferon, and numerous others. Cytokines have been shown in increase the expression of adhesion mo lecules including TNFand interleukin-1 IL-1 ) and increase the binding of l eukocytes to the endothelium67. The expression of inflammatory proteins creates a positive feedback loop by further inciting uptake

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12 of inflammatory cells, promoting aggregat ion of oxidized LDL on the endothelial surface and thus causing injury to the endothelium, and excreting further inflammatory mediators68. Since VCAM-1 is at the forefront of monocyte recr uitment in lesion formation69 it has been identified as a potential t herapeutic target and has been widely studied70. In knock out gene studies with mouse models the elimination of VCAM-1 but not ICAM-1 was show n to reduce plaque formation in atherosclerotic mice71. In vitro studies with monocytes co-cultured with endothelial cells showed that adhesion wa s reduced when cells were treated with antibodies raised against VCAM-1 and E-selectin, but not P-selectin61. Levels of expressed VCAM-1 are used as a measur ement in the efficacy of drug therapy72. Adhesion molecule P-selectin is expr essed on activated endothelial cells and platelets. Targeting P-selectin demonstrated therapeutic efficacy in an atherosclerotic mouse model which are cross bred with a P-selectin null mouse model. Reduction in plaque formation and proliferation of leukocytes was observed. However, the presence of so luble P-selectin in the bloodstream complicates the strategy73. Depending on the therapeutic strategy, these adhesion molecules could provide drug targeting candidates. There have been numerous studies using anti-VCAM74, anti-ICAM-175, and the anti-selectins76 antibodies as targeting vectors which show great potential in vitro however, there has been very little translation of these studies in vivo over the last decade. Collaboration with arthritis researchers70 led to the investigation of another adhesion molecule

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13 candidate, CD4477, ligand to hyaluronan (HA), a polysaccharide a major component in the extracellular matr ix (ECM) of mamma lian cells. The interactions of CD44 and HA have been implicated in cancer, autoimmune diseases, and inflammatory processes78. 2.3.1. Adhesion Molecule CD44 CD44 is a family of adhesion molecules that are characterized by their function, and mainly described as hyalurona n receptors. There are 10 standard isoforms and 10 variant isoforms of CD44, and the most populous is the CD44s isoform. The variation among the forms is found in ECM domain of the structure and accounts for the isoforms variation in function78. CD44 isoform CD44-v6 is highly expressed in the smooth muscle ce lls of the intima and media of injured arteries78, CD44 isoforms including CD44-v6 are expressed on the endothelium suggesting regulatory function of growth factors28 CD44 v10 is expressed by aortic endothelial cells28. Isoforms CD44-v3 and CD44-v6 where shown to be expressed on plaque microvessel, wher eas CD44H and CD44v6 both express on endothelial cells after exposure to IL-1 and TNF. Cd44H, CD44-v5, CD44-v6, CD44-v7/8 isoforms are all expres sed on macrophages and are all highly regulated by the cytokines74. CD44 and HA were shown to mediate leukocyte endothelial cell adhesion, previously thought to be the sole domain of the selectin family79. These studies taken all together indicate that CD44 isoforms are implicated in the pathogenic inflammatory process of atherosclerosis and that their role is highly complex and regulated.

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14 Interruption of the inflammatory pr ocess has been studied using CD44 blocked by antibody IM7 (anti-CD44)80. Expression of CD44 and its variants was augmented when exposed to pro-infl ammatory cytokines within human atheroma, implicating CD44 expressi on with the pathogenesis of arterial diseases74. CD44 was further implicated in t he progression of atherosclerosis. Hyaluronan was shown, in a low molecu lar weight form, to stimulate vascular cellular adhesion molecule (VCAM-1) and proliferation of smooth muscle cells (SMC), whereas high molecu lar weight forms of HA inhibit SMC proliferation81. Atherosclerotic prone ApoE -deficient mice bred with CD44-null mice showed a 50-70% reduction in aortic lesions compared to CD44 heterozygous and wild type mice 82. These results suggest that CD44 promotes atherosclerosis by both mediating inflammatory cell recruitment to atherosclerotic lesions and by altering smooth muscle function 81. 2.4. Drug Targeting Drug targeting is a strategy aiming at the delivery of a compound to a particular tissue of the body. Drugs can be delivered singly or in large amounts by using drug carriers. Drug carriers are substances that facilitate timecontrolled delivery, organ-specific targeting, protection, prolonged in vivo function, and decrease of toxicity of drugs to unspecified tissues. Drug targeting may allow for increased permeability of membrane barriers, allowing for molecular movement between tissues. I deally, drug targeting would provide a high local concentration of drug at the si te of disease and a concentration below levels of toxicity in healthy tissues81. Examples of drug carriers are myriad,

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15 including but not limited to, liposomes, micelles, polymeric vesicles, and nanoparticles6. Normal administration of drugs or therapeutic agents does not allow for concentrated accumulation of drug at diseased sites due to an essentially uniform distribution of drug throughout t he body. In order to adequately treat affected sites using traditional systemic administration high doses of drug must be delivered. This not only increases cost s, but also can creat e toxic side effects as normal tissues and organs are needl essly exposed to pharmaceuticals 83. Encapsulation of drugs for passive targeting, either by liposome 81, niosome 68, or polymeric 81 media has shown increased retent ion time, decreased therapeutic dose, and reduced toxicity to unspecified tissues. Targeting schemes include direct appl ication into the affected organ or tissue, passive application to tissues th rough leaky vasculature-tumors, infarcts, or inflammation. Another scheme is ph ysical targeting, which can be based on manipulating an abnormal pH or temperature at the ta rget by using pH or temperature sensitive drug carriers. Magnetic target ing of drugs to affected areas within the body can be achieved us ing paramagnetic carriers attached to magnetic drugs and then directed by an external magnetic field84. Active drug targeting is generally descr ibed as the use of a vect or molecule with a high specific affinity toward the affected tissues bound to a drug or drug carrier 21. Active drug targeting using a monoclonal ant ibody vector is the approach of this research.

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16 2.4.1. Vesicular Drug Delivery Among drug carriers listed previously vesicular drug carriers provide advantages over individual single molecule carriers. They not only allow greater payload of drug to be delivered, but also provide isolation of the drug from the system overall and thereby provide pr otection immune responses. Vesicle bilayers mimic biological membranes which enhance absorption of encapsulated drugs across cell membranes and into tissues. Vesicular drug delivery allows protection of the encapsulated dr ugs from enzymatic degradation85, prolonged circulation time, and therefore a reduced rate of release of drug into the bloodstream86, and the shielding of an immunogenic drug from recognition by the reticuloendothelial system (RES)85. 2.4.1.1. Liposomes Vesicles made from organically-d erived amphiphilic (having both hydrophilic and hydrophobic moieties) phosp holipids, like those that comprise cell membranes, are the most prevalent and widely researched drug delivery particle87. These liposomes, or “fat bodies” from the Greek, were first observed by researchers in the 19th century using lecithin in blood clotting studies. In 1911, Otto Lehmann published a r epresentation of micrograph of what he called ‘artificial cells’ which resembled a di spersion of multi-lamellar liposomes 88. Even though work with lecithin dispersions c ontinued the properties and potential applications of these dispersions were not described until Dr. Alec Bangham and his colleagues published their seminal work 89. They described how these particles, formed from the hy dration of lipid thin film s, retained some of the

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17 hydrating solution that they formed in within their core creating a ‘permeability membrane’. Researchers used the self a ssembly vesicles to study biological membranes and other applications, and thes e pursuits eventually led them to drug delivery. Liposome applications that developed thereafter, both commercially and within academia, are ma ny and varied including multiple scientific disciplines; medicine, immunol ogy, diagnostics, cosmetics, ecology, cleansing and the food industry 90. Promising laboratory re search did not always translate to commercially viable scale-up within the medical realm, however, and repeatability and stability problems c aused early liposome based start-up pharmaceutical companies to fail91. Liposomes’ interaction with cells has been studied not just for drug targeting purposes but also in devel oping further understanding of cell-cell interactions. Liposomes’ interaction with cells can be characterized in four ways shown in Figure 2.3. First, the liposom e may exchange material, either lipids or proteins, with cell membranes. Secondl y, liposomes may bind or adsorb with cells, and once bound may be internaliz ed through either endocytosis or phagocytosis. Alternately, the bound lip osome may instead fuse with the membrane. Whichever of these possibi lities arise is dependent on the size, charge, and makeup of the vesicle92.

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18 Figure 2.3 Vesicular Interactions with Cells. (Adapted from Lasic 81) The problem of early and rapid elimination from the bloodstream of the first liposome formulations was solved by the development of the ‘Stealth’ or ‘sterically stabilized’ liposomes93. The addition of small amounts of a hydrophilic polymer, frequently polyethylene glycol (PEG), extended the half life of a ‘classic’ liposome from a few minut es to several hours94. Researchers have developed several synthesis techniques to enhance de sired characterist ics. Along with varying methods of synthesis, vesicular drug delivery also varies in physical makeup. Multiple vesicle layers have been shown to enhance release rates significantly when compared to free drug and uni-lamellar vesicle delivery95. Development of commercially availabl e vesicle applications, passive and active, have been pioneered by cancer research96. One multiple-application technology that is being developed co mmercially is called DepoFoam™ and is made by the Swiss company SkyePharma. They produce foams of spherical

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19 lipid-bilayer aqueous chambers, or multivescular liposomes that are used to encapsulate drugs, vaccines, DNA, peptides or large particle bio-therapeutics which may be administered locally or systemically97. 2.4.1.2. Niosomal Drug Delivery Niosomes are analogues of liposom es synthesized from synthetic surfactants rather than or ganically derived phospholipids. They are also self assembly vesicles usually composed of a surfactant, cholesterol and a steric stabilizer, such as dicetyl phosphate98. Niosomes behave similarly to liposomes in vivo by prolonging circulation time of the encapsulated drug and altering chemical distribution within the body 99,100. However, niosomes have advantages over liposomes as drug carriers, includ ing chemical stability, lower cost, easier storage and handling, and a reduced like lihood of becoming toxic through oxidation 8. Like liposomes, niosomal encapsula tion reduces toxicity of drugs to untargeted tissues in many different app lications and therapies. Niosomal drug delivery has been studied using vari ous methods of administration 7 including intramuscular 10, intravenous 101, peroral, 102and transdermal 103. Niosomes can be used to solubilize insoluble agents86. Nebulized surfactants entrapping alltrans-retinoic acid were delivered as an inhaled aerosol reduci ng the drug toxicity and altering the pharmacokinetics 84. In addition, as drug delivery vesicles, niosomes have been shown to enhance abs orption of some drugs across cell membranes 26, to localize in targeted organs 17 and tissues 84,104, and to elude the RES 105. Cellular uptake of niosomes can be via endocytosis 106; however

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20 they have been shown to bind and fuse with cell plasma membranes via cellular receptors when vesicle surface charge is sufficiently negative 26. Niosome entrapment of methotre xate administered orally and intravenously in a mouse model was show n to increase residence time in the body and decrease clearance27. Tumor targeting of ni osomal formulations of doxorubicin26, vincristine sulfate105, cisplatin107, camptothecin24, shown increased residence times and anti-tumor efficacy compared to free drug. Recent examples of niosomal dr ug delivery research are varied and include topical vaccination generally24 and of DNA108,106 and treatment of acne109, alternate insulin delivery methods2, oral immunizations110, transdermal anti-inflammatories111, and ophthalmic applications112, among others. 2.5. Background and Current Practices in Antibody Mediated Drug Targeting The discovery of the nature of ant ibodies and the development and refinement of monoclonal antibody producti on had a revolutionary effect of the treatment of infectious disease and has provoked the pursuit of numerous targeting strategies in cancer and cardiovascular medicine113. Anticancer therapy, and especiall y tumor targeting, has been the forerunner in the development of liposomal 28, niosomal 32, and other drug delivery systems32 and is the most widely pursued application of antibody mediated targeted drug delivery 32. Early clinically approved and commercially available nanoscale (200 nm or sma ller) systems for untargeted drug delivery were developed for cancer treatment and include DOXIL, a liposomal encapsulation of doxorubicin (1995), and Ambisome, liposomal amphotericin

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21 (1990)32. Innovations in vesicle-antibody li nking schemes were also driven by cancer researchers. In order to incr ease circulation time and decrease the rapid clearance of classic liposomes from circ ulation, polymer groups (usually PEG groups) were added to liposomes surfaces32. These liposomes showed favorable passive targeting to tumors and leaky vasculature. The addition of site specific targeting vectors, monoclona l antibodies and fragments most prevalent among them, provided active targeting capabilities to improve therapeutic capacity and decrease non specific tissue interactions. When antibodies were coupled to the vesicle surface of a PEG coated liposome both the ability of the antibody to bind to the surface of the liposome was hindered, as well as its capacity to attach to the targeted antigen site68. The PEG groups that inhibited the desired qualities of an immunoliposom e provided an attractive site for antibody attachment and increased anti body-antigen site recognition versus attachment of antibodies at the vesicle surface34. Liposomal immunotargeting has been us ed extensively for cancer and cardiovascular applications. Antibody-ves icle conjugation chemistries are varied but there are similar physical configurations of linkers that result in increased efficacy of antigen binding when the liga nd is attached distal to the vesicle surface. This increases rotational freedom of the targeting moiety and decreases hindrance by the bulky polyethelyne glyco l (PEG) groups at the surface of a ‘stealthy’ liposome114. Attachment of ligand distal to the vesicle on a PEG terminus was found to have increased bind ing to target cells compared to attachment on the surface31. Development of a polyethylene end group on a

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22 phospholipid molecule functionalized with cyanuric chloride allowed for attachment of antibodies without pr ior derivatization of antibodies 115. Improved tumor targeting was shown using niosom es with PEG-glucose conjugates using a paramagnetic agent encapsulant28. Echogenic liposomes conjugated with targeting antibodies show in vitro pr omise for simultaneous imaging capabilities and targeted therapeutics 116. Differing antibody-polymer linkage schemes were developed, usually by creating a functionalized PEG group coupled to a phospholipid molecule (‘linker lipid’92,95,117-126) and incorporated into the vesicle membrane for subsequent antibody or targeting vector coupli ng. These schemes included differing formulations of thioether bonds coupling an antibody to the terminal end of a PEG group, either by activating a ma leimide group attached to a thiolized antibody 7,8,91,99,100,102,127-131, or hydrazide group reac ted with an aldehyde group on an oxidized antibody10,99,132-135, among others. An end-group functionalized PEG linkage was developed that did not requi re activation or functionalizing of the antibody105. A cyanuric chloride molecule to was added to the head group of a membrane component phospholipid linking a PEG molecule to that, and then another cyanuric chloride molecule to t he terminal end of the PEG which would then be coupled to an antibody via nucleophilic substitution115,136-138. Although the group subs equently found unfavorable in vivo blood circulation times of their i mmunoliposome (IL) formulation19,28,29,139, subsequent studies have identified the Fc region of the whole antibodies used as the culprit in provoking an immune response, and prom oting rapid clearance of ILs.

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23 Researchers have demonstrated this wit h liposomes conjugated with anti-HER2 monoclonal antibody fragments (the protein HER2 is a member of the epidermal growth factor family and is over-expresse d in breast cancer and implicated in its pathogenesis) 105. The anti-HER2 IL-were shown to have identical blood residence time as compared to non-tar geted sterically stabilized liposomes in vivo and further showed no increased clearance with subsequent administrations. Incubation with cells that over-expressed HER2 had a 700 fold increase in cellular uptake of dr ug compared to non-targeted liposome140. Active targeting of niosomes was shown using gl ucose targeting with the inclusion of a glucose-palmitoyl glyco l chitosan conjugate in a sorbitan monostearate niosome141. Improved tumor targeting wa s shown using niosomes with PEGglucose conjugates using a paramagnetic agent encapsulant142. To our knowledge there is no literat ure on any other group studying antibody targeting of niosomes. 2.6. Cardiovascular Antibo dy Mediated Imaging and Therapy 2.6.1. Imaging and Therapy Diagnostic medical imaging is a prevalent and non invasive technology widely used in obstetric and cardio vascular disciplines, among others. Innovations in technology have transfo rmed ultrasound (US) images from grainy and poorly-resolved to sharp digital imaging115. The non invasive nature of US tissue penetration makes it an attractive therapeutic strategy. Active therapeutic targets within the cardiovascular system in clude atherosclerotic plaques, or other

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24 areas of damage within the vascular walls and bed, infarcts, thrombi and blood elements115. US targeted imaging 88 and imaging driven therapy of thrombi has been widely pursued143. Contrast agents have been studied not just for the ability to enhance ultrasound (US) imaging bu t also for thrombolytic potential created by the very high localized pre ssures created when US exposed contrast agents burst driving their therapeut ic load into target thrombi88. Ultrasound imaging of a liposomal combination of echogenic contrast agent and drug delivery vehicle has shown also the abili ty to guide therapy and disrupt thrombi 88,144. Additionally contrast agents, or ‘microbubbles’, have been studied as potential gene delivery vector s by exploiting the explos ive cavitation of US exposure driven bursting to pr opel plasmid DNA into vessels88. Detection of expressed adhesion molecu les as antibody directed contrast agents bind locally can be used to identif y inflammation and early detection of cardiovascular pathologies115. Atherosclerotic anima l models demonstrate the facility of targeted MRI imaging of th rombus producing ruptured plaque to potentially guide therapy136. Atherosclerotic plaques can also be imaged using targeted US contrast techniques acce ssing either expre ssed inflammatory adhesion molecules on the endot helial surface or activated bound leukocytes through contrast agent antigen receptors136. The potential to direct therapy through imaging and simultaneously control drug delivery and release could address se veral challenges of inflammatory pathologies as greater under standing of the complex chemical and molecular

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25 interactions of inflammatory proce sses drive plaque bui ldup, instability and vessel restenosis115. 2.6.2. Current Research in Ve sicle Mediated Drug Delivery in Inflammation and Cardiovascular Disease. Reviews of cardiovascular target ed drug delivery cover the myriad applications pursued in targeting pharma ceuticals to cardiovascular disease145. Thrombus treatments and imaging have bee n targeted with and wi thout antibody or other protein mediati on, and through magnetically driven thrombolytics. Atherosclerotic lesions, circulating blood cells and elements, and endothelial cells have all been addressed as therapeutic target s. In treatment of lung disease, aerosols have been developed to accumulate drug delivery in the lungs92, as have antibody-mediated delivery to antigens in the lungs146, to malignant lung disease145, and immuno-liposomes directed to the pulmonary endothelium30,147149. The mechanisms and techniques of successful targeted and untargeted accumulation of drug delivery vesicles in the pulmonary endothelium are relevant to developing other vascular inflammation strategies. Imaging of pathogenesis in the heart th rough targeting has been studied post myocardioal infaction and in myocardi tis. Liposomal accumulation has been shown in ischemic heart tissue 150. Liposomal cardiovascular targeting has been reviewed in detail specifically32. While limited to intravascular tissues only, in vitro and in vivo studies of liposomes and immuno liposome as a drug carriers to many antigens were addressed151. Among the topics reviewed was the targeting of vessel wall injury. Multiple pathol ogies, atherosclerosis, and coronary

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26 thrombosis among them, are initiated by vessel injuries which promote platelet activation and binding. Early detec tion of the disrupted endothelium through targeting of expressed antigens provid ing a ‘signal’ has been studied. This included an in vitro examination of targeting extrac ellular matrix antigens where collagen gaps provide binding sites for liposomes conjugated with antibodies to type 1 collagen, and similarly proven against laminin and fibronectin also. These studies showed good affinity, specificity, and selectivity. Also the liposomal antibody-antigen binding mimicked nature: cell binding studies showed that liposomes conjugated with ant ibodies provided a dissociation constant (describes the affinity between ligand and protein) on the order of 10-9 M, which corresponds to physiological antigen to free antibody binding constants151. Human endothelial cells incubated with immunoliposomes conjugated with cell surface antibodies ( anti-T antigen A25) were shown to bind at 4 C and endocytose 30% of bound ILs at 37 F. Liposomes and ILs were seen to accumulate in an ischemic heart provid ing imaging and therapeutic targeting potentials to damaged myocardium. Posi tively charged untargeted liposomes were observed to accumulate in experimen tally produced myocardial tissues as early as the 1970s; further studies confi rmed further the propensity for liposomes to accumulate in depolarized myocytes and in ischemic tissues in general151. These findings provided the groundwork for liposome passive targeting to ischemic tissue and infarcted heart tissue. Active targeting of anti-myosin conjugated ILs to myocardial tissue was studied in vivo and showed good accumulation in the ischemic myocardial tissue.

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27 This effect is limited in traditional liposom es when access is restricted because of lack of blood supply and vesicles are e liminated by the RES before accumulation occurs. As with other traditional li posomes, surface alterations by the incorporation of PEG gr oups along with antibodies provided targeting and prolonged circulation time showed greater penetration152. Another consequence of ischemia is t he formation of lesions of the cell membrane. Loss of membrane integrit y is further exacerbated upon reperfusion leading to cell death as cell contents ar e exposed and reperfusion injury ensues. The capacity to target and seal lesions created in hypoxic in vitro conditions using cardiocytes with immunoliposomes (ILS) shown to be effective, greatly increasing post hypoxic cell viability afte r IL incubation. Furthermore, hypoxic and IL treated cells were shown to grow normally in culture after the event109. Immuno-liposomes targeted to interc ellular adhesion molecule 1 (ICAM-1) were shown to bind to and be internalized by epithelial cells in vitro demonstrating their potential of the anti-ICAM antibody bear ing vesicles to deliver an anti-inflammatory drugs to si tes of increased ICAM expression 153. With an aim to combat restenosis following cor onary angioplasty, liposomes conjugated with peptide sequences were created to tar get to glycoprotein IIb-IIIa receptors on activated platelets34. Platelet aggregat ion and deposition is implicated in the pathogenesis of restenosis through depos ition of growth factors and inflammatory mediators. To increase ci rculation time they modified the vesicle surfaces with an oligodextran polymer, analogous to the use of PEG groups to elude the RES 152. The effect of stress by reactive oxygen species on

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28 endothelial cells is implic ated in cardiovascular diseases making antioxidant drugs therapeutic candidates. Antiox idant enzymes such as superoxide dismutase and catalase make good therapeutic candidates but are rapidly eliminated from the bloodstream, inhibi ting their efficacy. Muzykantov’s group30 showed that by encapsulating the enzymes which they coupled to PEGs, the elimination was greatly reduced and the efficacy and bioavailability of the enzymes were increased. Binding to expressed antigens in the endothelium in vivo was shown by targeting angiotensin converting enzyme (ACE) and adhesion molecules (ICAM-1 and PECAM-1). Vascu lar accumulation, endothelial uptake, and increased antioxidant pr otection were all shown with this targeting scheme136. ICAM-1, but not PECAM-1 wa s shown to be susceptible to pathological stimulus, exhibiting incr eased expression and uptake in and by endothelial cells. Anti-ICAM1 target ed immunoliposomes of 100-300 nm diameters showed specificit y of uptake compared to non specific antibodies or large vesicle targeting115. The aim of the literature review was to not only establish a foundation of knowledge in the different disciplines involv ed in creating a targeted drug delivery system but also to identify the most appropriate carrier system, antigen target, mode of delivery and mec hanism of uptake.

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29 3. Project Description The objective of this project wa s to develop and test a drug delivery system that could be targeted to vulnerab le atherosclerotic plaque. Figure 3.1 shows the interdependent relationship of the physical elem ents needed to meet this objective. In order to design the system effectively, the a ffinity of the drug delivery vehicle must be established for both the drug of in terest and the drug delivery vector that will be targeting the bi ological site. The targeting vector is chosen not only for its ability to seek and deliver the drug delivery system to the site of interest but also to help facilitat e the uptake of the drug on site. Obviously the efficacy of the drug deliv ered must be appropriate to treat the affected site. Figure 3.1 Interdependence of Element s of the Drug De livery System. The drug delivery vehicle that was chosen for the system is a non ionic surfactant vesicle, or niosom e. The vesicle’s stability, versatility, biocompatibility, and cost made the choice compelling. Surfactant components available are numerous, allowing versatility in formulati on strategies to not only manipulate

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30 stability136 (the ability to retain the encapsulated component) but also to allow the potential to incorporate various linkers to the vesicle surface105. The biological target provides the therapeutic conduit, and therefore should be specific to the disease pathogenesis and if possible, imp licit in it. The development of atherosclerosis and the morphological ch anges in plaque along the progression of the disease provide varied targeting strategies105. The inflammatory processes implicated in each progressi ve step of plaque development and the shift in stability of the plaques have associ ated biomarkers which provide not only a targeting potential for in situ drug delivery, but also a mechanism to interrupt disease progression. Adhesion molecule s are mediators in the process of inflammation and make logical therapeutic targets70. 3.1. Development and Test ing of an Immunoniosome The use of an antibody-conjugated non ionic surfactant vesicle or ‘immunoniosome’ to target vascular in flammation is an original concept. Niosome research has been largely in the realm of cancer154, immunization155, and topical therapies156. The process of vesicle development included evaluating the formation and stability of niosom es composed of sorbitan monoester components that had been well described the literature and whose surfactants were commercially available71. Liposome literature describes the widespread use of polyethylene glycol (PEG) groups on the surface of liposomes, originally incorporated to elude the immune system, as a desirable site for targeting vector attachment. The addition of PEG may be accomplished by adding the polymers after vesicle formation to linkers attached to phospholipids accessible on the

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31 liposome surface or by creating phos pholipid PEG compound molecules and incorporating those as the vesicles form157. A similar polymer group was incorporated in one of our sorbitan este r surfactants but t he stability of the polyoxyethylene sorbitan monostearate (T ween 61) niosome was not favorable. This led to investigating blending t he surfactant components to evaluate the potential to introduce a linking site while maintaining the desired properties of stability and entrapment. Liposome lit erature reviews described multiple schemes linking proteins to vesicles141. Cyanuric chloride has been used in the past to adhere proteins to surfaces and was described as a linker in immunoliposomes12,28,32,158. From these ideas I developed the linking chemistry of the polyoxyethylene sorbitan monos tearate functionalized with cyanuric chloride in order to provide a protein binding site on the surface of a formed vesicle. This is a novel process and a non-provisional patent application has been filed and published 159. The resulting immunonios ome (IN), see Figure 3.2, was tested in a fixed synovial lining cell model for specificity, selectivity and binding. Adhesion molecule CD44 was id entified as a target antigen implicated in the progression of atherosclerosis160, so the binding studies were done with anti-CD44 as the targeting vector. Bovine aortic endothelial cells were used to test the ability of the niosomes to binding to inflammatory cells specif ically expressing target antigens. The binding of fluorescent INs to cells was measured using a customized MatlabTM program (see Appendix D), which relates the binding of immunoniosomes to the cells relative to experimental variables.

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32 Figure 3.2 Immunoniosome Membrane Structure. 3.2. Binding and Uptake of Vesicles Uptake of INs by cells was investigat ed. Prior to the live cell incubation studies, changes in the synthesis process were necessary. Reduction of niosome size by extrusion created niosom es of 200 nm, a size demonstrated as preferential in uptake by atherosclerotic lesions148. Additionally, in some studies antibodies were fragmented prior to c onjugation and incubation with cells to evaluate the ability of the linkers to attach Fab fragments. Studies showing unfavorable uptake of whole antibody immunoliposomes implicated the conserved Fc region of the antibody107,161, so in preparation for in vivo studies, uptake of Fab conjugat ed immunoniosomes, as well as the uptake of whole antibody targeted vesicles by cells was tested using confocal and TEM microscopy.

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33 Finally, in order to test the efficacy of the system in vivo a protocol for a study using an atherosclerotic mouse model and treated with atorvastatin containing immunoniosomes compared to free drug treatment was developed and approved by the USF Institutional An imal Care and Use Committee (IACUC) and the US Army Medical Research and Material Command Animal Care and use Review Office.

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34 4. Vesicle Development 4.1. Immunoniosome Synthesis Project Descripti on The development of the immunoniosom e took place in four distinct phases. In the first phas e, the general formulation of the vesicle had to be identified, synthesized, measured, and test ed for viability. Niosome literature and availability of components favored using surfactants from the sorbitan ester family, both for stability, encaps ulation capacity, and economy 30. Vesicles of different compositions of those so rbitan esters, and their encapsulation and retention capacities were evaluated using fluorescent dye as a drug model. Chromatography, particle sizing, fluorescence intensity, and microscopy were employed to purify and measure vesicle formulations162-164. Vesicles were stressed with diagnostic levels of ultras ound to evaluate membrane stability. Also of interest was the potential to create a ‘tuneable’ ta rgeted drug delivery vesicle that would not only be site spec ific but would have controlled release upon activation with US exposure. The nex t phase was a refinement of the first, and looked at the potential of blending surfactant components in quantities that retained desired stability properties while providing potential linker candidates. Once the blended formulation was estab lished, the next phase involved the development and evaluation of the linker ch emistry. Finally, once the ability of the surfactant linker to bind a monoclonal antibody to the surface of a niosome was established the last phase test ed the ability of the resulting

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35 ‘immunoniosome’ to bind to a target ant igen on a fixed cell using an activated fixed synoviocyte as a cell model of inflammation. 4.2. Material and Methods 4.2.1. Introduction The ability of lipids to form bilayer vesicles instead of micelles is dependent on the hydrophilic-lipophilic balance (HLB) value of the surfactant, the chemical structure of the components and the critical packi ng parameter (CCP) which is the relationship between the struct ure of the surfactant including size of hydrophilic head group, and lengt h of lipophilic alkyl chain (Figure 4.1). The formula is 0a l v CCPc where lc = the length of the alkyl chain, v = the volume of the hydrophobic chain volume, and a0= area of the hydrophilic head group115. The HLB value can measured experimentally for each surfactant by reversed phase thin layer chromatography165. The value represents a relative proportion of the hydrophobic and hydrophobic groups comprising the molecule and provides a guide for evaluating potential ve sicle formation. Generally it has been reported that HLB for sorbit an esters are between 4.0-8.0115. For of an HLB value of greater than ~6, cholesterol must be added to the surfactant in order for a vesicle to form166. The general form of a single bilayer vesicle is shown in the left of Figure 4.1.

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36 Figure 4.1 Critical Packing Paramete r and Bilayer Membrane. Left: Schematic for calculating the critical packing par ameter of an amphiphi le (Adapted from Uchegbu 1998115.) Right: Bilayer Membrane Structure Niosomes are similar to liposomes in structure and methods of synthesis. The fundamental component of both is an amphiphile, a molecule containing both a hydrophobic and hydrophilic moiety, and the structure of the resulting vesicle is formed by a single or multiple bilayer membrane enclosing an aqueous core. There is a wide array of co mmercially available, inexpensive, biocompatible surfactants that enable s pecified structural design of vesicles167. Niosomes can form from many of these surfactants, and are commonly made by the combination of a single alkyl chain nonionic surfactant and cholesterol, in addition to an ionic electros tatic stabilizer, such as dicetyl phosphate. The addition of cholesterol enables more hy drophobic surfactants to form vesicles and suppresses the tendency of the surf actants to flocculate or form aggregates167. The addition of cholesterol has been shown to lend greater stability to the bilayer membrane by raisi ng the gel liquid transit ion temperature of the vesicle. This stability decreases leakage of the vesicles and stabilizes against osmotic gradients168.

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37 The formation of single or multiple bilayer (lamellar) vesicles upon hydration is a result of the amphiphilic surfactant’s (surface active agent’s) interaction with the aqueous solution caus ed by the high interfacial tension between the water and the hydrophobic alkyl chains. This tension causes the hydrocarbon regions to associate simu ltaneously as the hy drophilic head groups orient towards the water; the actions of each opposing force culminate in the formation of the vesicle assembly 168. The thermodynamics of formation dictated by Gibbs free energy ( GH S 0, at a given temperature) states that in order for the self assembly to go forward there must be sufficient enthalpy ( H) to overcome the negative entropy of the system (S) and the reduction in free energy (G). The van der Waals attrac tions, the hydrophobic-hydrophilic interactions, hydrogen bonding, and electr ostatic interaction contribute the necessary enthalpy of formation. In order for the associ ation of the molecules to proceed there must be an energy gradi ent between their associated versus isolated states, and Israelachvili stated that self assembly will proceed as long as the interaction free energy per associated monomer 0 N, ( where N is the number of associated monomers) is greater that the mean interacti on free energy of the isolated monomers; 0 1 0 N. This is true as long as 0 N decreases with N, with 0 N reaching a limiting value 168. The energy gradient driven interactions of the molecules described underlie the HL B dependence of vesicle formation mentioned earlier as related to the shape and size of the hydrophilic head groups, and the lengths of the hydrophobic alkyl tails that dictate the HLB balance169.

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38 4.2.1.1. Gel-Liquid Crystal Transition Temperature Formation of lamellar vesicles is dependent on not only the components themselves, but also on temperature. The bilayer structures have different phases in which they exist. The gel st ate is the most ordered, and the liquid crystal state is less ordered. Within the gel phase the alkyl tails of the amphiphiles are crystall ized and can not diffuse. In the liquid crystal state there is lateral diffusion of the bilayer co mponents. The liquid crystal phase of a system always exists above the gel phase. In order for niosomal vesicles to form the hydration must be set above the gelliquid crystal transit ion temperature of the surfactant167,169. The gel phase transition te mperature of Span 60 is 57 C, and therefore the hydration and extrus ion temperatures are set to 60 C167. 4.2.2. Materials 4.2.2.1. Chemicals Niosome preparations and surfactant derivatization and treatments were made from sorbitan monos tearate (Span 60), sorbit an monopalminate (Span 40), sorbitan monolaurate (Span 20), polyoxyet helene sorbitan monostearate (Tween 61), cholesterol, and dicetyl phosphate ( DCP), diisopropylethylamine (DIPEA), cyanuric chloride (CC), Triton-X 100, whic h all came from Sigma Chemical, St. Louis, MO. Surfactant proper ties are listed in Table 4.2 in Section 4.2.3.1. Fluorescent dyes, 5(6) carboxyfluoresce in (CF) and 5(6) carboxyrhodamine (CR) were obtained from Biotium, Hayward, CA. Phosphate buffered saline (PBS), and Sephadex G50, Histochoice tissue fi xative, Hank’s Balanced Saline (HBS), Dulbecco’s Modified Eagle Medium (DMEM), Bovine serum albumin (BSA) and

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39 goat serum were obtained from Fisher Scientific, Suwannee GA. Collegenase P was obtained from Roche Appl ied Science, Indianapolis IN. The Alexa Fluor 488 protein fluorescent labeling kit ca me from Molecular Probes (A20181). 4.2.2.2. Vesicle Characte rization Materials and Methods Particle analysis included light scatte ring, single particle optical sensing technology, optical microscopy, chromatography techniques and fluorescence measurements. Fluorescence c hanges were measured to evaluate encapsulation by the vesicles and leakage over time. The mean particle size and distribution of formed vesicles were det ermined using an Accusizer 780A optical particle analyzer from Particle Sizing Systems. The algorithm behind the PSS technology combines single-particle light scattering and light extinction technologies. Light scattering is used for particles less than 1 m in diameter, with a lower limit of 0.5 m, and light extinction is used for particle greater than 1.0 m. Small volumes (0.005 0.1 ml, depending on concentration) of niosome suspensions are added to the diluting chamber and diluted and cycled through the through the sensor until the concentration of particles is such that they pass through individually, eliminating ‘coincidenc e’ of particles. As the particles pass through a uniformly illuminated ‘optical parti cle sensor’ they obscure part of the sensor signal which produces an output pul se from a 35mW infrared diode. The pulses represent these discrete particl es whose magnitude corresponds to a particle diameter. Figure 4.2 shows a sc hematic diagram of the auto-dilutor system. The accumulation of the pulses, interpreted by a signal processor, produces a particle size and concentration distribution plot ( PSD). Figure 4.3

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40 shows a PSD plot of a polystyrene standard c ontaining a mixture of particles with three different diameters, 0.7, 1.0, and 5 m. The known sizes and concentrations of the standar ds allowed for calibration and verification of the instrument. Figure 4.2 Autodilution Scheme of Part icle Sizing System. (PSS User manual) The vesicle suspensions were also characterized by observing the changes of their chromatography elution pr ofiles details of which are described in the GEC purification below. Essentially, the constituents of a vesicle suspension are separated by size as they elut e through a packed bed matrix, and those constituents can be measured using UV abs orbance or fluorescence intensity. By comparing changes in elution chromat ograms the relative stability of the formed vesicles can be monitored.

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41 Figure 4.3 Particle Size Distribution Plot of a Mix of Three Sizes of Polystyrene Latex Standards. Fluorescent dyes were used to study entrapment and retention in the niosomes because of the sensitivity and pr ecision of fluorescent measurements. We were able to observe incremental changes as small as tenths of nano-moles of dyes in suspension. The sensitivity of carboxyfluorescein dye to changes in fluorescence intensity with exposure to light was tested by exposing varied concentrations to as much as five day’s exposure to ambient light. The results shown in Figure 4.4 indicate the dye’s resistance to decay despite exposure, reassuring us about any potential conf ounding effects of short term light exposure on signal changes. Nonethele ss, dye solutions were always stored away from light in 4 C.

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42 CF Fluorescence Intensity Decay with Exposure to light versus Time 0.0 20.0 40.0 60.0 80.0 100.0 120.0 140.0 160.0 5.00E-062.50E-061.00E-065.00E-071.00E-07Concentration (mol/L)Intensity t=0 days t=2 days t=5 days Figure 4.4 Stability of CF. The De cay of Varied Concentration of Carboxyfluorescein. At low concentrations fluorescenc e intensity is proportional to dye concentration so encapsulation of dye c an be calibrated by use of a standard curve, as shown in Figure 4.5. At hi gher concentrations the relationship is not linear and measured intensity will decreas e with increased concentration due to self quenching as shown in Figure 4.6. Entrapment of dye by niosomes was measured by first disrupting the sus pended niosomes with Triton X 100, a nonfluorescing detergent, and then measuring th e fluorescence intensity of the dye released by the vesicles into the suspending buffer solution70. Light and fluorescence microscopy were also us ed to verify vesicle formation and disruption.

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43 CF Concentration versus Fluorescence Intensity Calibration Curvey = 3.0698E+08x R2 = 0.9994 0 20 40 60 80 100 120 140 160 0.00E+001.00E-072.00E-073.00E -074.00E-075.00E-076.00E-07Concentration (mol/L)Intensity Figure 4.5 Calibration Curve of CF. Car boxyfluorescein Molar Concentration vs. Fluorescence Intensity CF Concentration vs Fluorescence Intensity0 100 200 300 400 500 600 700 800 900 0.0000000010.000000010.00000010.0000010.000010.00010.001Concentration (mol/L)Intensity Figure 4.6 Calibration Curve with a Wide Range of Concentrations. Fluorescence Intensity with Respect to a Wide Range of Concentrations of Carboxyfluorescein Dye. 4.2.2.3. Vesicle Purification Materials and Methods Niosome literature describes several methods of separating formed vesicles from un-encapsulated hydrating solutions and unformed lipids33. Among

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44 them are gel exclusion chromatogr aphy, dialysis, centrifugation, and ultracentrifugation. Gel exclusion ch romatography was chosen as an appropriate bench top method for this research. Ge l exclusion chromatography (GEC) is also referred to as size exclusion chromatography, and pr ovides relatively rapid separation of formed vesicles from unf ormed lipids and unencapsulated dye (or drug). Dialysis is time consuming and only eliminates the free dye and not the unformed lipids. Centrifugation provides only partial separation of unformed lipids, created vesicle disruption, and has limited utility depending on vesicle density. Ultracentrifugation was expensiv e and largely unavailable for our routine experimentation. 4.2.2.3.1. Gel Exclusion Chromatography The theory behind GEC is straightforwar d. Gel filtration media is packed into a column to form a packed bed. The medium is a porous matrix of spherical particles, such as Sephadex G50, that are chemically and physically stable and non reactive. Micrographs of hy drated Sephadex G50 beads at two magnifications are shown in Figure 4.7. The packed bed is equilibrated by flushing with a buffer solution (the mob ile phase) such as phosphate buffered saline (PBS). The liquid inside the matrix is referred to as the stationary phase. The void volume refers to the volume of the column not taken up by the matrix. Once a suspended sample is added to the gel matrix, buffer and sample move through the column. Larger particles do not enter the pores of the gel matrix; they move through the column at the same rate as the buffer, and elute within the first column volume. Particles small enough to be caught up in the gel matrix

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45 pores elute later. The separation process takes place as one total column volume of buffer passes through the gel filtration medium. Gel filtration is an isocratic chromatography technique, meaning that s eparation is achieved without the use of any kind of gradient. Figure 4.7 Sephadex G50 Hydrated Beads. Left: 100x magnification, right: 200x. According to the theory the particl es or molecules will be separated according to the Stokes radius of the par ticle. The equation for the Stokes radius isas M where M is molecular weight, a is the Stokes radius, and s is the sedimentation coefficient, ) 1 ( 60 N, where 0 is the viscosity of the solvent, is the partial specific volume of the particle, is the density of the solvent, and N is Avogadro’s number. Vesicle size c an be calculated using the relationship between the volumes of the system; 0 0V V V V kt e d where V0 is the void volume, Ve is the elution volume, Vi is the pore volume, and Vt is the total volume, kd is then the volume fraction of t he stationary phase that is available to an eluting species170.

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46With GEC, hydrated samples were not only separated and purified, but also the sample’s stability and entrapment over time could be evaluated by studying the elution profile of the purified samples as they changed over time. The plot in Figure 4.8 shows an over lay of two elution profiles, or chromatograms, of two niosome sus pensions of differing encapsulated PBS concentrations. The abscissa is volume eluted and the ordinate represents the UV absorbance (fluorescence is also meas ured) of the eluting volume. The first peak is the signal from the niosomes which are eluted in the void volume, and the second from the unencapsulated dye. As a suspension of vesicles deteriorates over time, the first peak decreases and the dye released increases the second peak. Theoretically, a newly purified stable suspension would have no second peak. From the lack of a first peak signal in the 0.1M we discerned that vesicles were not viable at the hi gher salt concentration due to the osmotic gradient with the eluting buffer. Figure 4.8 Chromatogram of Span 60 Nio somes of Varied PBS Concentrations.

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474.2.2.3.2. Fluorescence Intensity Fluorescent dyes are used as dr ug models commonly in research because of the high sensitivity of fluore scence spectroscopy, which can be as much as two orders of magnit ude more sensitive than absorbance spectroscopy82. Fluorescence spectroscopy uses changes in vibrational energy levels of the molecule or substanc e being measured to assess levels of fluorescence. The sample is excited by exposing it to a known quantity of energy. By absorbing the energy in the fo rm of a photon of lig ht at a particular wavelength the substance changes from a ground electronic state (low energy) to a high frequency state (higher energy). Collisions between highly vibrating molecules cause loss and emission of ener gy as they drop back down from the excited high energy state to ground state and emit photons18,25. Instrumental analysis of the energy absor bed and emitted at particu lar filter wavelengths provides fluorescence intensity values that can be used to interpret sample concentrations when compared to a standard curve ( e.g. Figure 4.5.) Initial fluorescence measurements were taken with a single measurement fluorescence spectrophotometer, and later measurements were taken using a fluorescent plate reader which allows for multiple readings to be taken at once. Figure 4.9 shows the effect on vesicle disruption and subsequ ent release of dye from the vesicles on the concentration of dye in the measuri ng container (cuvette or plate well). The vessel on the left shows the dye conc entration consistent within and outside the formed vesicles after hydration, while the middle shows the vesicles suspended in PBS with dye contained within them, and finally, the right vessel

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48indicates the release of the dye from the vesicles into the suspending PBS. The dye released from the disrupted vesicles creates the change in signal of the fluorescence (FL) intensity. Figure 4.9 Release of Dye from Niosomes. 4.2.2.3.3. UV Absorbance UV absorbance of materials is comm only used to measure concentration in solutions of proteins and nucleic ac ids. The theory is straightforward; materials which absorb ultraviolet light (wavelengths from 200-400 nm in the near UV range171) have properties which obey the Beer-Lambert Law. The law is expressed in different formulas shown below. The transmittance of light (T) through the material is a ratio of t he intensity of the incident light (I0) and the intensity of the light after passing through it (I1). lc AI I T 10 101 0, where 1 0 10log I I A and k 4 so that lc A A is absorbance at a particular wavelength l is the path length in cm c is concentration in M

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49k is the extinction coeffi cient for the material is the wavelength of the light So for a material with a known extinction coefficient, at a particular wavelength, and a known path length, molar absorbance is proportional to concentration148,172,173. 4.2.2.3.4. Ultrasound Energy can be added to order or to disrupt a system; in our niosome development experiments we used ultr asound for both of those purposes. Sound is the phenomenon we experience per ceiving the propagatio n of pressure waves through air or water. Mechanical vibrations create vibrating pressure waves transferring energy from the source to the pressure waves and to anything the waves contact. Audible (or acoustic) sound frequencies range from 20 Hz to 20KHz. Ultrasound waves are above 20KHz and outside the range of human hearing141. Exposure of a biological membrane to ultrasound causes sonoporation which is the temporary, non-destructive perforation of the cell membrane. This transient state enhances permeability of therapeutic agents into cells and tissues115. Similarly, the bilayer membrane structure of a self assembly vesicle exhibits a temporary permeability with exposure to acoustic forces174. Our suspensions of formed niosomes were exposed to ultrasound in a bath sonicator to reduce lamellarity and size which is a common practice in vesicle synthesis175. Experiments were conducted exposing different formulations of niosomes to diagnostic US frequencies to test the potential of US mediated drug release in vivo Intensity levels of US exposure vary for different

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50medical applications. Powe r levels greater than 10 W/cm2 are used in surgical applications; therapeutic US ranges from 0.5-3 W/cm2, and diagnostic US ranges from 0.1m W/cm2 to 0.5 W/cm2, 176. 4.2.2.4. Equipment Table 4.1. Equipment for Vesicle Development and Synthesis. Equipment Manufacturer Model Mass balance Denver Instrument Company A-250 Rotary evaporator Buchi Laboratory, Switzerland Vaccuum Controller V-800 Buchi-Rotavapor R-200 Buchi-Heating Bath B-490 Buchi VacR V-500 Bath sonicator Laboratory Supplies CO., INC Model G1125PIG Particle sizing Particle Sizing Systems Accusizer 780ATM Fluorescence spectrophotometer Perkin Elmer LS-3B Fluorescence Plate Reader Biotek Flx800 UV Microscope Lecia Type 090-135.002 Inverted Fluorescence Microscope Olympus IX71 Chromatography column GE Healthcare (Amersham Biosciences) Superdex HiLoad XK 16/60 Chromatography system GE Healthcare (Amersham Biosciences) KTAprime Vortex Mixer Fisher Scientific Touch Mixer 231 pH meter Echocardiography machine Acuson Aspen

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514.2.3. Methods 4.2.3.1. Vesicle Synthesis 4.2.3.1.1. Thin Film Hydration Techniques Traditional thin film hydration tec hniques were first described in the 1960s6. In niosome synthesis this method involves dissolving surfactants, cholesterol and, often, an electrostatic st abilizer (such as dicetyl phosphate) in a non polar solvent, such as chloroform, and then evaporating the solvent and forming a thin film of the dried chemic als on a vessel. Once all solvent is removed, usually by employing a vacuum or nitrogen gas, t he addition of an aqueous solution hydrates the thin film. The hydrophobic/hydrophilic interactions of the amphiphilic molecules when exposed to the aqueous environment cause the self-assembly of lipid bilayer vesi cles. As the hydrophobic tails orient themselves together, they shield them selves from exposure to the water molecules, and the hydrophilic head gr oups line up together inwardly and outwardly exposed to the aqueous solution. Figure 4.1 (left) earlier in the text shows a drawing of the orient ation of the amphiphiles. Initially several different formulations were investigated. While different sorbitan ester surfactants were assessed, a consistent ratio of surfactant to cholesterol to dicetyl phosphate was used throughout. The stability of vesicles made using an equimolar ratio of surfactant to cholesterol with the addition of 13% dicetyl phosphate had already been well described177,178. A stable vesicle is defined as one which maintains a consistent size and retains a constant level of

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52encapsulant178. The first ‘generation’ of niosomes had lipid concentrations approximately 10 mM. The concentration refers to the total lipid concentration in the niosome suspension after GEC. It is calculated from the initial concentration of lipids used to make thin films and taking into account the dilution of the suspensions during GEC, and the process loss of vesicle as measured by particle counts. As a general rule the dilu tion from post hydration to post GEC is 10x and the retention of formed vesicles is approximately 75%. The lipids were dissolved in chlo roform and aliquoted into 50 ml round bottomed flasks. Early films were creat ed by blowing a constant stream of nitrogen gas at 5L/min. Later it was det ermined that residual chloroform could be further reduced using a vacuum and so the vacuum pump of the rotary evaporator was employed. Once the films dried they were hydrated by adding either 0.01 M PBS, 5.0 mM CF, or 1.02.0 mM CR rotating for 1-2 hours in a 60C water bath. At regular intervals during hy dration, the solutions were agitated on a vortex mixer. Once complete, the solution was sonicated for 5-30 minutes in a bath sonicator at 80 KHz and 80 watts. Resi dual chloroform was measured by mass balance. After 24 hours, the chloroform remaining was measured to be less than 0.35% of original solvent by mass before hydration. Table 4.2 shows the structures and descripti ons of the surfactants investigated in our research. The sorbitan monoesters ar e a family of alkyl esters of identical head groups and varying alkyl chain leng th. Polyoxyethylene (PEO) sorbitan monostearate (Tween 61) has the same st ructure as sorbitan monostearate, as the names suggest, but includes multiple PE O polymer chains on its head group.

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53The sorbitan monoester (Span) family wa s investigated because of its well documented facility to form vesicles and it s biocompatibility; they are widely used in food, pharmaceutical and cosmetic industries93. Table 4.2 Surfactant Structures and Properties.

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544.2.3.1.2. GEC Purification Once the hydration process is completed, 2 ml of 60 C sample is injected into the sample loop of the chroma tography system. A pr eprogrammed elution method is loaded from a PC. The sample is injected by the system into the column from the sample loop through t he injection port as 0.01M PBS is added continuously at 1.0 ml/min. As the el ution progresses, t he system provides information about temperature, pressure conductivity, and UV absorbance at 280 nm. The elution method is split into phases where the eluted materials are either captured or sent to waste; in the first phase the sample is introduced to the column. Then the system resets the baselin e UV reading to 0. For the first third of the void volume the eluted material is discarded, after which the eluted material containing the separated niosomes is captured in 2 ml fractions. Finally, once the void volume has passed through the column, the eluted material is again sent to waste, and the free dye is flushed from the column. The maximum total volume of the XK 16/60 column is 124 ml In order to flus h completely after a separation, the buffer is run for 2 total column volu mes or 240 ml, although the bed packing volume is usually about 100 ml, plus or minus 3-5%. 4.2.3.1.3. UV Absorbance The Akta prime system incorporated a UV sensor and automatically generated chromatograms for each separati on or assessment elution that was run. Although UV is not as sensitive to changes in fluorescent signals as fluorescence microscopy is, the curves provided useful run-by-run comparisons

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55of different formulations. Unfortunately, the results did not prove as easily quantifiable as those of the fluorescenc e reading because both the niosomes and the fluorescent dye contained within t hem contributed to t he signal at the niosome elution peak as seen in Figure 4. 10. The absorbance of niosomes at 280 nm which contain no absorbing solution can be explained by the presence of double bonds and ring groups in the surfactants. UV Absorbance vs Elution Time 0 100 200 300 400 500 600 051015202530354045 Time (min)mAu Tw CC CF Tw CC PBS Figure 4.10 Intrinsic UV Absorbance of Niosomes. Tw-CC-CF represents a niosome encapsulating CF dye, and Tw-CC-PBS represents a niosome encapsulating PBS. The Tw-CC-CF curve has a second peak due to the absorbance of free dye. PBS does not absorb UV at 280 nm. 4.2.3.1.4. Niosomes of Diffe ring Surfactant Components Niosomes made from Span 20, S pan 40, Span 60, or Tween 61 as the surfactant component in 1:1:0.105 molar ratio with cholesterol and DCP were made as described encapsulating a 5mM so lution of CF. Table 4.3 shows the masses of lipid components used. The encapsulation of the dye was measured by disrupting the vesicles with Triton X 100 in a de-ionized water (DI) solution.

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56Concentrations of entrapped dye were found by comparing the disrupted niosome fluorescence intensity to a st andard curve. Standard curves were created for each type of dye and new dye solu tion. Particle sized distributions were run for each of the formulations. Table 4.3 Low Concentration Example of Vesicle Components. Surfactant mass(g) Mass (g) Total mols Sp20 SP40 Sp60 TW61 cholesterol DCP 0.148 0.0437 0.00635 2.37E-04 0.01 0.088 0.016 2.80E-04 0.094 0.088 0.016 4.90E-04 0.081 0.088 0.016 4.91E-04 4.2.3.1.5. Increased Lipid Concentrations In order to increase the dye entrapment capacity of the niosomes, the lipid concentrations were increased 7.5-10 fold, maintaining the original molar ratios of the components. The GEC met hods had to be adapted to the more concentrated hydrated solution. 4.2.3.2. Development of Tween 61-Span 60 Niosome In order to conjugate a ta rgeting moiety to a niosome, a linking agent had to be conceived of that would either be attached or inserted into the niosomes after they are formed, or to be in corporated within t he membrane during formation. The latter approach would not require an additional process step or potentially affect vesicle stability. T he surfactant Tween 61 shown in the right side of Figure 4.12 (also seen in Table 4.2) is nearly identical in structure to Span 60 except for the additional incorporat ion of polyethylene branches on the hydrophilic head group. The polyethylene ox ide (PEO) groups on the polar head

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57of Tween 61 surfactant potentially could be exploited as a linker for antibody conjugation. This linkage is analogous to antibody coupling on the distal end of PEG groups added to immunoliposomes 90,179. 4.2.3.2.1. Tween-Span Mixed Formulations Based on the results of the stability studies blends of the surfactant component of the vesicles were examin ed. A combination of Span 60 and Tween 61 was selected with the aim of retain ing the desirable qualities of Span 60 stability and retention, while maintain ing the option to use the PEO polymer groups from the Tween 61 on the membrane surface for antibody attachment. We looked at combinations of small amounts of Tween 61 (0-10%) incorporated with Span 60 while maintaining the 1:1:0.1 molar ratio of surfactant to cholesterol to dicetyl phosphate. Table 4.4 shows ma sses of components used to make films of the varied combinations of surfactant s. For each formulation the retention of dye over time of the vesicles was co mpared to the solely Span 60 surfactant composition. Table 4.4 Masses of Vesicle Component s for Varied Tween 61 Surfactant Percentages Ratios per Film. % Tween Tween 61 (g) Span 60 (g) Cholesterol (g) DCP(g) 1.00% 0.000890.088260.10132 0.00752 3.25% 0.00280.084170.10333 0.00767 5.50% 0.004670.080270.10525 0.00781 7.75% 0.006430.076550.10707 0.00795 10.00% 0.008110.073000.10882 0.00808

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584.2.3.3. Functionalization of Tween 61 with Cyanuric Chloride Tween 61 was functionalized prior to ni osome synthesis by activation of the hydroxyl groups on the ends of t he PEO chains. In the presence of diisopropyl ethyl amine (DIPEA), Tween 61 and cyanuric chloride were incubated in a nitrogen environment. The overall me chanism is shown in Figure 4.11. The cyanuric chloride undergoes nucleophilic substitution binding to the terminal hydroxyl group of a PEO chain on the Tween 61 molecule. The molar ratio of Tween:CC:DIPEA was 1:0.8:2 90,180 and a 0.2 g/ml solution was made by combining 1g Tween 61, 0.124 g CC, 0.274 ml DIPEA, and 5 ml chloroform. The Tween 61 and chloroform were combined in a round bottom flask. Cyanuric chloride was added and the DIPEA was withdrawn from a sealed flask using a long sharp metal syringe tip and added directly into the mixture. The flask was rotated in a nitrogen environment for 36 hour s. The excess solution was stored and remained stable at -4C for several m onths. The resulting functionalized Tween-CC solution was added to the surfact ants and lipids in chloroform prior to forming a thin film.

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59 Figure 4.11 Tween 61 Cyanuric Chloride Linking Mechanism. 4.2.3.4. Attachment and Veri fication of Antibody Conjugation The GEC purified niosome solutions were adjusted to pH 8.8 by titrating 1 l at a time of 1 M sodium hydroxide drop-wise while monitoring the pH. IgG monoclonal antibodies (either fluorescent ly tagged Alexa-488 (AF)-IgGs or anti CD44 IM7) were incubated with the nios omes at a concentr ation of and gently shaken for 16 hours in the dar k. At pH 8.8 the anti body binds to the cyanuric chloride linker distal to the vesicle surf ace shown in Figure 4.12. Linking of antibodies to either of the available chloride groups has equivalent potential energy, however linking a subsequent antibody to the second available chloride group would require much greater energy of activation, and is not favored when other linkers on other molecules are available105. After antibody conjugation the immunoniosome (IN) solution pH is re stored to 7.4 using 0.1 M PBS. Concentration of antibodies incubated was 5 g protein /ml niosomes which is equivalent to 2.78 g protein/ mol lipid. Concentration of total lipids in the post GEC niosome solution is 1.8 mM, found by calculation from the original hydration

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60concentration of 0.0144 M accounting for th e 6 times dilution factor and particle retention efficiency during GEC. Viab le binding groups of Tween-CC linkage are 52% of the Tween component by calculation. This value is based on the reaction molar ratio (1:0.8 of Tween:CC) and a pub lished binding efficiency (65%) of the reaction of CC with 1.2-Dipalmitoylsn -glycero-3-phosphoethanolamine polyethylene glycol (DPPE-PEG)24. The overall molar percentage of Tween in the total lipid concentration of the nios omes was 4.76%, making the Tween-CC linker 2.47% of tota l lipids, or 0.045 mol Tween-CC/ml niosomes. This provides 2.68 1016 binding sites/ml niosomes. Antibody incubation of 5 g antibodies/ml of niosomes relates to a ratio of gr eater than 1300:1 Tween-CC binding sites to antibodies. In order to verify the efficacy of the linker chemistry and establish the ability to bind antibodies to the surf ace of the vesicles, antibodies were fluorescently tagged and incubated with non fluorescing niosomes for conjugation. Fluorescent tagging of antibodies was done as described in the protocol from a commercially ava ilable kit from Molecular Probes. Figure 4.12 Anti body Conjugation.

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614.2.3.5. Synovial Lini ng Cell Culture Methods Primary cell cultures of bovine syn oviocytes were used in niosome incubation experiments. These cells we re provided grown and already fixed by a collaborator’s laboratory. The following description of the synoviocyte primary culture comes from t he resulting publication70. The synovial membranes were harvested from the metacarpal phalangeal joints of 3 month old bovines. After washing with phosphate buffered saline ( PBS) the tissues were dispersed in 0.1% collagenase P in 4% bovine serum al bumin (BSA). Isolated cells were washed with PBS suspended in Dulbecco’s DMEM containing 10% fetal calf serum at a concentration of 105 cells per ml. The cells were plated in Labtek 8 well micro-slides. Cells were allow ed to attach overni ght. The media was removed, and the cells washed with PBS and fixed for 2 hours in Histochoice. A subset of fixed cells was i mmuno-stained with IM7 using standard immunohistochemical techniques. 4.2.3.6. Immunoniosome Synovial Lining Cell Incubation Methods The fixed cell layers were pre-in cubated in 0.01 M PBS with 2% goat serum with or without solubl e IM7 antibody for 1 hour at room temperature prior to incubation with immunoniosomes. Ce lls were rinsed with PBS and incubated for 1 hour at 37 C with fluorescent niosomes with or without antibody conjugation. The cells were well rinsed to remove unbound niosomes and examined by fluorescent microscopy. Im aging of cells, cell nuclei, and INs was done with an Olympus 1X71 inverted fluore scent microscope fitted with DAPI and FITC (fluorescein isothiocyanate) filter s allowing imaging of the green fluorescent

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62niosomes and the blue stai ned cell nuclei. For a given image, a phase contrast picture was captured, and t hen the light and filters were changed to fluorescent mode to image the cell nuclei using the DAPI filter, and the FITC filter to image the fluorescent niosomes bound to the cells. These images were captured and combined using DP-BWR image analysis software to overlay them. 4.2.3.7. Proof of Concept Cell Binding To establish proof-of-concept cell binding, bovine synovial membrane lining (SL) cells were plated at 1x 103 cells/mm2 on glass multi-well slides and maintained in DMEM with 20% fetal bovi ne serum, until cells reached near 90% confluence. The cell layers were was hed and fixed for 24 hours in Histochoice fixative and rewashed Hanks Buffered Sali ne (HBS). The fixed cell layers were pre-incubated in PBS with 2% goat serum wi th or without soluble IM7 antibody for 1 hour at room temperat ure to prevent non specific binding. Niosomes used in SL cell studies encapsulated 5( 6) carboxyrhodamine 110 (CR), a photobleaching resistant fluorescent dye. (P hotobleaching is a loss of fluorescence intensity due to exposure to intense ligh t.) Cells were rinsed and incubated for 1 hour at 37 C with fluorescent niosomes or fluorescent niosomes derivatized with IM7 (purified IgG). The cells were well rinsed to remove unbound niosomes and were examined by fluorescent microscopy. 4.2.3.8. Statistical Methods Statistical differences in experimen tal values were calculated using standard error of the mean (SEM) of at least three repeated measures. The

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63formula for SEM is the ratio of the standar d deviation of a data set divided by the square root of the number of repeated measures, n. The standard deviation of a set of measured values is defined as the square root of the variance. The variance is the summed deviations of t he measured values from the mean of those values. If xi is an observation among n observations and x is the mean of all the observations (the sum of all m easured values divided by the number of values measured) then the variance is: 1 ) (1 2 2 n x x sn i i and the standard deviation is therefore; 1 ) ( .1 2 n x x Dev Stn i i. The standard error of the mean is n Dev St SEM . The SEM is represented graphically as y-axis error bars in the graphs in this document. P-values are used as another statistical method included in some of the reported data. In hypothesis testing resulti ng data are evaluated as being different than the mean of the measured values wit hin a range of values. A p-value is defined as the probability of a measure to be different t han the mean of the measured values. If a p-value of <0.001 is repor ted then there is less than 0. 1% chance that there is no difference in the means of the measur ed value versus the comparison value, usually a control 181.

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644.2.4. Experimental Designs 4.2.4.1. Vesicle Formulation Assessment 4.2.4.1.1. Sorbitan Ester Formulations Entrapment efficiency and membrane pe rmeability of varied formulations of niosomes were evaluated. Size r eduction through sonication over time was assessed in each formulation. Dye encapsulation capacity was assessed in each formulation. Additionally the effect of sonication on vesicle size and count was evaluated in the different Span compos itions. The ability to accurately evaluate the entrapment of dye by niosom es depends on the ability to completely disrupt them. 4.2.4.1.2. Tween-Span US Exposure Stability Study Based on the results of the studies done on the different surfactant candidates, niosome made from either Tween or Span 60 as the surfactant component were evaluated for stability. The different vesicle formulations were exposed to diagnostic ultrasound at 0, 5, 10 minutes using a transducer and an Accuson Aspen echocardiography machine. Machine settings were kept constant throughout the experiment wit h the frequency set to 3.5 MHz, the dynamic range set to 70dB, and the initia l gain set to 10 dB. During the experiment the mechanical index of the instru ment was varied. Mechanical index (MI) is the ratio of the acoustic pressure over the square root of frequency. Therefore max MI corresponds to a ma ximum acoustic pressure at a given

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65frequency. The formula is f P MI where P is pressure in MPa, and f is frequency in MHz. The spatial pe ak temporal average was 46 mW/cm2, and the total absolute power was 65 mW. Fluore scence readings of the niosomes and suspending fluid were obtained at 5 mi nute increments over 15 minutes of ultrasound exposure. All vesicle charac terization measurements were repeated three times at each 5 minute time increment. 4.2.4.2. Polyoxyethylene Sorbitan Monostearate-Cyanuric Chloride Linker Chemistry The Tween-CC linkers were prepared as described and tested by first attaching the fluorescently tagged IgG ant ibodies to non fluorescing niosomes and assessing the vesicle morphology and fluorescence of the resulting suspension, by GEC elution of the vesicl e suspension. To further verify the antibody binding to the niosomes, t he post GEC purified AF-immunoniosomes were examined with an Olympus 1X71 in verted fluorescent microscope. Approximately 50 l of immunoniosome suspension was pipetted onto a glass microscope slide and viewed at 10 and 40x using a FITC filter to verify the presence of fluorescent spherical particl es. Fluorescent images where captured using the Olympus DP-BSW software.

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664.3. Results 4.3.1. Sorbitan Ester Vesicle Formulations 4.3.1.1. Results of the Vari ed Sorbitan Ester Formulations The varied formulations were evaluat ed for entrapment by fluorescent measurements and particle sizing. The entrapment of dye by each formulation is shown in Figure 4.13. The Tween 61 niosome shows the greatest encapsulation of dye, followed by Span 60. Entrapment decreases with decreasing alkyl chain length. Figur es 4.14 show that Span 60 forms the greatest number of vesicles after hydrat ion and retains the greatest amount of dye after GEC. Figure 4.13 Encapsulation of CF by Surfactant Type.

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67 Vesicle Formation0.00E+00 1.25E+08 2.50E+08 3.75E+08 5.00E+08 Span 20Span 40Span 60Tween 61Surfactant TypeNumber of Niosomes/ml Figure 4.14 Formation of Niosomes afte r Hydration by Surfactant Type. In order to reduce vesicl e size and lamellar number (number of bilayers) bath sonication over a range of times was tested in the Span 20 and Span 40 niosomes after hydration and before GEC. The data in Figure 4.15 show that vesicle size (number weight mean) decr eases and vesicle count increases over sonication time to an asymptotic value. The effect of sonicati on on the different surfactants is not identical however. T he concentration of par ticles greater than 0.5 m (the detection limit of particle size analyzer) of the Span 20 vesicles increases and reaches an asymptotic lim it with sonication time while the concentration of the Span 40 particles increase to a greater degree and fluctuated more greatly. With both su rfactants the mean particle diameter decreases with sonication then levels out. This is to be expected since sonication of vesicles reduces the number of bilayers in the vesicles and disrupts aggregates182 and is directly seen in the micrograph shown in Figure 4.16 which shows a light microscopy image of a PBS containing niosome sample on a hemocytometer slide before and after soni cation which was used to reduce and unify particle size and lamellarity 183.

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68Figure 4.17 shows the effect on sonica tion over the entire vesicle size distribution. The curves show the particl e size distribution of the unsonicated versus sonicated vesicles of Span 60 ni osomes. The size distribution clearly shifts downward after sonica tion. Based on the results of the Span 20 and Span 40 niosomes it is assumed that a portion of the population of the Span 60 niosomes that are not seen in the post sonication distribution have dropped below the detection limit of the PSS. Span 20 and 40: Mean Vesicle Size and Concentration with Sonication time0 0.5 1 1.5 2 050100150200250300 time (sec)Mean Particle Size (um)0.00E+00 2.00E+08 4.00E+08 6.00E+08 8.00E+08Particle Counts/ml Span 40: Mean Particle Size Span 20: Mean Particle Size Span 40: Particles/ml Span 20: Particles/ml Figure 4.15 The Effect of Sonicati on Time on Particle Size and Counts. Figure 4.16 Sonication Effects. The E ffect of Sonication on Vesicle Size Distribution in Span 60 Niosomes.

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69 Figure 4.17 PSD Sonication Effects. The graph shows the shift in Particle Size Distribution in a bath sonicated Span 60 nisome sample. The blue curve is the size distribution of the sample before s onication, and the red curve represent the distribution after sonication. 4.3.1.2. Results of Ultra sound Exposure on Niosomes Ultrasound exposure experiments we re conducted on niosomes whose surfactant component was either Tween 61 or Span 60, based on the results of the earlier sorbitan ester experiments, es tablishing the two as the candidate surfactants. Vesicle suspens ions of the different formul ations were monitored by particle size distribution and fluorescenc e intensity measurements. The graphs shown in Figure 4.18 represent the particle size distributions (PSD) of the control and experimental samples over the duration of the experiment. On the left the curves represent the PSDs of the Span 60 niosomes before (blue) and after (red) US exposure. The right side of Figure 4.18 shows the same thing for the Tween 61 niosomes. It is clear by the closeness of the curves in each that the disruptive effect of US on the formed particles wa s minimal. The particle counts are expressed in numbers per ml of sample.

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70 Figure 4.18 PSDs with Respect to US Ex posure. Left: Span 60 niosomes; right; Tween 61 niosomes. For each blue is prior to US exposure, and red is after. In Figure 4.19 the effect of US exposur e time is seen to be significantly greater in the Tween 61 niosomes than the Span 60 niosomes. Figure 4.20 indicates that release of dye is shown to increase with increased mechanical index in Tween 61 niosomes, whereas the st ability of the vesicles in the Span 60 with MI as shown in Figure 4.21. Taken all together, the stability of the Span 60 niosome and its ability to retain encaps ulated materials is demonstrated to be greater than that of the Tween 61. Ho wever, for controlled rapid release a Tween 61 vesicle may be desirable. % Increase in Free CF in Tween 61 and Span 60 Niosome Suspensions vs US Exposure Time at MI=1.6 0% 5% 10% 15% 0510 Time (min) Tween 61 Span 60 Figure 4.19 The Effect of US Exposur e Time on Release of CF. Error bars represent SEM of n>=3.

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71 % Increase in Free CF in Tween 61 Niosome Suspension vs Mechanical Index for t=10 min0% 5% 10% 15% 00.61.21.8 MI Figure 4.20 The Effect of MI on Released CF in a Tween 61 Niosome. Error bars represent SEM of n>=3. Span 60 Vesicle Counts vs Mechanical Index75% 88% 100% 00.511.52 MI% .0 represents the control sample not MI=0 Figure 4.21 The Effect of MI on Particle Retention in the Span 60 Niosomes. Error bars represent SEM of n>=3. 4.3.2. Results of Surfactant Blending Since the formulation of niosomes wit h Tween 61 as the sole surfactant component were seen to be less stabl e than the Span 60 niosomes both statically, and when stressed, the effect of the inclusion of a ra nge of small molar percentages of Tween 61 in a Span 60 ni osome on vesicle entrapment capacity and membrane stability was evaluated. This was measured by retention of

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72entrapped dye over time at 4C in a PBS suspension. Although Tween 61 niosomes are reported to have a greater entr apment capacity 184 (which was confirmed by the data shown in Figure 4. 13), the observation was that niosomes whose surfactant component was entirel y composed of Tween 61 lost three times more encapsulated dye relative to niosome formulations whose surfactant component was purely Span 60 under static conditions holding cholesterol and DCP molar ratios constant as shown in t he tabulated values in the right side of Figure 4.22. On the left si de of the figure the data indica te that the inclusion of up to 10% by mole of Tween 61 had no signif icant effect on the retention of dye over the time examined. Statistical differences were judged by evaluating the overlap of standard error of the mean graphi cally. Also, in order to increase the entrapment capacity of the vesicles, and therefore increase potential drug entrapment, the overall mass of lipids used was increased. The results of the increase can be seen in the overlay ch romatogram shown in Figure 4.23. 0.75 0.8 0.85 0.9 0.95 1 05101520 Time (days)Nt/N0 1% Tween 3.25% Tween 5.5% Tween 10.0% Tween %Tween % CF Retained 0 88.0% 1 85.9% 3.25 84.8% 5.5 89.1% 10 88.0% 100 62.2% Figure 4.22 The Retention of CF in Tween 61-Span 60 Niosomes. Left: graphical depiction of the inclusion of 1-10% Tween. Right: Over all retention of dye in various formulations in 15 or more days.

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73 Figure 4.23 Increased UV signal with Increased Lipid Concentration in Tw-CCCF Niosomes. 4.3.3. Results of Linki ng Chemistry Development The verification of the linking chemis try was tested in two ways; first was the assessment of the el ution of PBS containing ni osomes conjugated to Alexafluor tagged antibodies. After inc ubation with the antibodies the niosomes were purified using GEC which should have resulted in a suspension of non fluorescing niosomes conjugated to fluor escent antibodies. That purified suspension was eluted through a GEC co lumn and measured by UV absorbance at a wavelength of 280 nm. Figure 4.24 shows a signal in the niosome elution volume. There is also a very slight si gnal in the antibody el ution volume (before 120 ml) which show a few free AF anti bodies; the protein and the bound dye would both contribute to the signal at that wavelength. This indicates that there are negligib le numbers of free antibodies in the solution and demonstrates successful atta chment of Alexa Fluor tagged IgGs to PBS containing niosomes.

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74Secondly, the niosome suspensi on was imaged using fluorescence microscopy as seen in Figure 4.25. Further confirmation of the conjugation of AF antibodies was evident when fluoresc ent spheres were evident under a fluorescent microscope us ing a FITC filter. The presence of distinct and discrete spherical fluorescent particles confi rms the conjugation of the fluorescent antibodies to the non fluorescing niosomes a nd the efficacy of the linker to attach the antibodies to the surfac e of the niosome membrane. The discrete fluorescent spheres appear to be of the same size and re lative size distribution of niosomes. These two independent measures indicate successful antibody conjugation of the AF antibodies to non fluorescing niosomes. Figure 4.24 Elution of IN with Fluorescent Antibodies.

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75 Figure 4.25 Fluorescent micrograph of IN with Fluorescent Antibodies. The effect of the Tween-CC linker on the 10% Tween 61 niosome was evaluated over 22 days. Figure 4.26 shows an overlay of four separate elutions of the same sample of post-GEC 10%Tween 61 niosomes. Due to slight differences in column packing and sus pension injection the chromatograms do not line up perfectly, but do indicate very little variation in the suspension over time.

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76 Stability of 10%Tw-CC-CR Niosome Suspensions 0 5 10 15 20 25 30 35 051015202530 Time (min)mAU Tw-CC-CR t=0 days Tw-CC-CR t=8 days Tw-CC-CR t=18 days Tw-CC-CR t=22 days Figure 4.26 Stability of the 10%Tw-CC-CR Post GEC Niosome. 4.3.4. Binding of Immunoniosom es to Synovial Lining Cells The binding of the immunoniosomes to the target anti gen in a fixed cell model was confirmed as follows. The fluorescent and light micrographs shown in Figure 4.27 demonstrate the specificity and selectivity of immunoniosome binding to target antigens. The upper micrograph figures of cells (A, C, and E) correspond exactly to the fluorescent mi crographs below them (B, D, and F). Parts A and B of Figure 4.27 correspond to the cells incubated with IM7 tagged niosomes and show binding of the imm uno-niosomes evident by the bright spherical shapes attached at cell proc esses and cell membranes. Whereas the cells pre-incubated with fr ee IM7, shown in C and D, do not show the small spherical attachments due to blocking of the targeted binding sites. This demonstrates the targeting selectivity of antibody antigen bindi ng. In parts E and F, cells have been incubated with unconjuga ted niosomes. Additionally, the absence of binding in the cells incubat ed with untagged niosomes, parts E and F,

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77demonstrates the specificity. In all of the fluorescence images some autofluorescence of cells is evident, but clear ly distinct from t he brighter point-like images of the fluorescent niosomes. In the left hand side of Figure 4.28, IHC staining techniques show the CD44 expres sion at the cell processes and at the cell membranes as indicated by the red arrows. Correspondingly, binding of green fluorescent INs in t he right hand side of Figure 4.28 is seen at cell processes and membranes. Figure 4.27 Experimental and Control Images of Synovial Lining Cells. The upper slides (A), (C), and (E) show c ontrast micrographs and the lower slides (B), (D), and (F) are the corresponding fluorescent micrographs of those above them captured using a FITC filter. (A) and (B) show SL cells incubated with IM7tagged niosomes containing 1 mM CR dy e. (C) and (D) are SL cells preincubated with free IM7. (E) and (F ) are cells incubated with untargeted niosomes (no IM7).

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78 Figure 4.28 Synovial Lining Cells. Le ft: IHC stained for CD 44 with nuclear counterstaining. Arrows indicate CD 44 expressed on cell processes. (40 x magnification Olympus BH 2) 4.4. Discussion The results of the experiments to this point confirm the capacity to develop monoclonal antibody conjugated niosomes ta rgeted to specific cell receptors. The investigation of member s of sorbitan ester family as the candidate surfactant in our vesicle revealed that Span 60 had the best combination of entrapment capacity, stability and retention versus the others studied. However, since the polyoxyethylene polymers on the Tween 61 head group extend distal to the surface of the formed nios ome they would provide ideal linking moieties for antibody conjugation. Blending the surfactants in order to retain the desired properties of each surfactant was conc eived and the results showed that the inclusion of as much as 10% of Tween 61 within a niosome composed of Span 60 in the surfactant component maintai ned the desirable stability and retention properties of the ‘purely’ Span 60 ni osome. Sorbitan monostearate based niosomes can be functionalized through inclusion of a cyanuric chloride

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79derivatized polyoxyethylene monostear ate to conjugate monoclonal IgG antibodies to the vesicle surf aces without requiring derivatiz ation of the antibody. The coupling of IgG antibodies to formed and purified vesicles was verified. The resulting ‘immunoniosome’ was s hown to bind to target antigens in fixed cells. In the fixed SL cell m odel targeting shows high selectivity and specificity demonstrated by the lack of IN binding to the target antigen when blocked with free antibodies specific to the antigen, and also the inability of untargeted (non antibody conjugat ed) niosomes to bind to cells. The results of the fixed cell SL binding studies demonstrat ed that further investigations were warranted to investigate binding and t hen uptake by inflamed endothelial cells in vitro and in vivo The implications for therapeutic treatment of inflammatory diseases is significant not only in the capacity to target chemical therapy to affected tissues but also to block rec eptors of inflammatory pathways and to interrupt the perpetuating effect of the process 178. Also, since the attachment of antibodies is independent of the type and generic to any IgG antibody, the system’s therapeutic targeting is flexible and may include more than one targeting vector if desired. In order to pursue live cell uptake experiments the size and particle distribution of INs would have to be addre ssed. Size reduction protocols using extrusion, and monitored by sub-micron particle sizing, would modify and verify vesicles of size conducive to cellular uptake177. Additionally, measurement of optimal antibody coating dens ity would need to be pursued. By using activated fixed aortic endothelial cells (an appropriate model fo r vascular inflammatory

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80drug uptake studies) optimizations of the drug targeting system could be conducted prior to pursuing live cell uptake studies. In liposome applications, as few as tens of antibodies or antibody fragments 185 conjugated per vesicle have shown binding and cellular uptake, necessi tating the optimization of our immunoconjugation.

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81 5. Fixed Endothelial Cell and I mmunoniosome Binding Studies 5.1. Background 5.1.1. Endothelial Cells Anti body-Targeted Drug Delivery Endothelial cells are a type of epithelia l cell; they are single-layer simple squamous epithelial cells which line blood vessels and ar e therefore the mediating layer between the bl ood stream and the tissue t hat they line. Varying targeting drug delivery strategies hav e been studied using activated endothelial cells expressing adhesion molecules. Di fferent targeting schemes have been reviewed and/or studied including ca rdiovascular liposome targeting, nanomedicine, pulmonary targeting of li posomes with antioxidants and antithrombotics, and enzymatic therapeutics, also using polymeric nanocarriers. Vesicular or nanocarrier cardiovascular tar geting strategies frequently include an endothelial cell model with a target adhesion molecule and the corresponding antigen vector. In the studies descri bed in this section 10%Tween-CC niosomes conjugated with an anti-CD44 ant ibody were targeted to bovine aortic endothelial cells (BAECs). The aim was to verify and quantify bindi ng of the INs to activated BAECS under differing experimental variables such as incubation time, concentration of lipid and antibody coating. Thr oughout these BAEC fixed cell binding experiments the niosome abbreviation IN refe rs to the formulation defined in Part 4, the blended surfactant niosome with the Tween 61-cyanuric chloride linker,

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82containing the photobleaching resistant dy e, carboxyrhodami ne (CR), conjugated to the anti-CD44 antibody, IM7. 5.2. Materials and Methods 5.2.1. Materials and Chemicals Included in this list are the materials and chemicals new to this phase of the project that were not included in Sect ion 4.2.2.1. Ca mbrex endothelial cell basal media (EBM), bovine calf serum, penicillin-streptomycin, 0.05% (w/v) Trypsin-EDTA solution, 99% HEPES so dium salt, and 100% ethanol all came from Fisher Scientific, as did all th e pipettes, flasks, filters, syringes, and centrifuge tubes. The secondary antibod ies used for IHC came from Sigma Chemical as did the Tris buffer, and sodium acetate. Cryo-preserved endothelial cells (BAEC) came from Clonetics, Inc. The IHC ABC staining kit (Cat# PK 7200) came from Vector labs, as did the diamino benzidine (DAB) substrate kit (SK4100), the diam idino-2-phenylindole ( DAPI) nuclear mounting stain, and the hard set mounting media. The potassium ferricyanide and uranyl acetate were a kind gift from Ed Haller from the Pathology Department of USF Health. Osmium tetroxide and gelatin came from USF Health Core facilities. 5.2.2. Methods 5.2.2.1. Endothe lial Cell Culture Endothelial cells were grown using cl assical sterile techniques in 25cm2 sized cell culture flasks in endothelial bas al media supplemented with 10% fetal calf serum, 1% penicillin-streptomycin and 1% non essential amino acids. Cryo-

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83preserved cells were started on fla sks that had been coated with collagen to encourage adhesion. Cell growth media wa s changed every 2-3 days. The cells were cultured to a confluent monolayer and then sub-cultured into 8 well microslides for incubation with INs and subsequent imaging. Cells were sub-cultured by releasing their adherence from flasks with the enzyme trypsin, which is a serine protease of the pancreas whic h breaks down pept ide bonds of the carboxyl groups of amino acids arginine and lysine. The commercially available, purified form of trypsin is used commonly in cell culture to release cells from culture vessel surfaces. To release the cells from the flasks, first the growth media was removed by pipette, and then the cells were rinsed several times with 37 C 0.01 M PBS to eliminate the anti-tr ypsin agents present in the endothe lial media. Next 2 ml of 0.05% trypsin was added to the flask and incubated at 37 C for 5-10 minutes. Release of cells from the surface was monitored using a phase contrast microscope. Once the cells were rel eased, the vessel wall was washed further with PBS, and the suspended cells were centri fuged for 3-4 minutes at 3000 rpm. The trypsin and PBS were removed by pipette from the resulting pellet of cells which were re-suspended in growth media and 400 l aliquots were added to the individual wells of the micro-slides. The cells grew to near confluence in 1-2 days. A day prior to incubation with INs, fresh media suppl emented with 7 ng/ml TNF(an inflammatory cytokine whose role in adhesion molecule ex pression is described in Section 2.3) was added to the culture to activate t he cells and induce the expression of

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84adhesion molecule CD44. Prior to inc ubation with INs the cells were fixed overnight with either Histochoice, for IHC staining, or wit h paraformadehyde, for fluorescence imaging. 5.2.2.1.1. Gelatin Coating Procedure Coating of culture vessels with either collagen, gelatin or fibronectin helps encourage primary or cryo-pre served endothelial cells to proliferate in initial culturing. A 2% by weight solution of solid gelatin was prepared in pyrogen free deionized (DI) water for flask coat ing. The DI was heated to 80C and the gelatin was dissolved in the water. The dissolved gelatin and water were kept at the increased temperature for 10 minutes and then the solution was cooled to about 30 C and filtered with a 0.22 m syringe filter. If the solution is allowed to cool too much then filtration becomes ve ry difficult. Then 1.5-2.0 ml of the solution was added to 25 cm2 culture flasks and incubated at 37 C for 4 hours or longer. The excess solution was drawn off with a pipette and discarded while working under the sterile hood. Unus ed gelatin solution can be stored at 4 C for later use. 5.2.2.2. Cell Fixation Fixation of cells stabilizes the struct ure and allows solvents to penetrate the plasma membrane. Paraformaldehy de (PF), which does not fix by cross linking, has been identified as being the bes t preservative of cell structures. Because PF does not induce autofluorescence it is also a preferred fixative in

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85fluorescent microscopy applications. Typi cally concentrations of 2-4% in buffer solution are reported for cell fixation. 5.2.2.3. Immunohistochemical Staining Immunohistochemistry (IHC) is used to demonstrate the presence and location of antigens in cells or tiss ues. There are several factors which determine how well the antigen will be detec ted, chief among them the density and dispersity of the antigens presented on the cells. The fixative type can have an effect on antibody-antigen binding if t he cells structure is greatly changed in the fixation process. Detection is also impaired due to the antigen signal versus non specific background binding. This c an be offset by using a blocking buffer, usually a non specific protein or serum of the antibody host. The specificity of the antibody used is also important. Monoclonal antibodies give the most specific binding and lowest background, especially when used after a binding buffer treatment. A subset of the experiment al cells which had been TNFactivated but not incubated with INs underwent IHC staini ng to verify the expression of the target antigen, CD44. The commercially available kit from Vector Labs that was used employs an immunoperoxidase proced ure with avidin-biotin binding. Avadin is a 68 kDa glycoprotein that has a very high affinity (1015/M) to bind to the smaller biotin molecule. This affinity makes the binding essentially irreversible, and is 106 times greater than any antibodyantigen association. The kit is purchased specific to the cell ty pe (e.g. mouse IgG) and primary antibody employed, and the primary antibody is s pecific to the target antigen to be

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86detected. The general idea is that the primary antibody will adhere to the antigen that you wish to identify, and a secondary biotinylated antibody will adhere to the primary antibody. The biotin groups on the secondary antibody provide binding sites for the avidin biotinylated en zyme complex containing horseradish peroxidase. Cell nuclei were counters tained with diaminobenz idine (DAB) which provided an orientation of t he expressed antigen relative to the rest of the cell. The staining procedure was carried out a ccording to the instructions of the manufacturer. 5.2.3. Incubation Experiments 5.2.3.1. Design of Experiments The aim of the experiments was to assess the ability to bind the 10%Tween-CC-CR niosome conjugated to IM7 with activated endothelial cells, and to examine the effect of experimental and design variables on binding. Variables examined, shown in Table 5.1, were incubation time, lipid concentration, and antibody coating densit y. Each variable was given an individual eight well microslide. Whenever PBS is mentioned the concentration was 0.01M unless otherwise indicated.

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87Table 5.1 Experimental Variables of BAEC-IN Bindi ng Experiments. Concentration of IgG (ug/ml) Lipid Conc. (mM) Incubation Time (hours) 5.00 1.0125 mM 4 0.50 5.0625 mM 2 0.05 10.125 mM 1 Control niosomes (non INs) 10.125 mM Control Abs (pre IM7 post IN) 10.125 mM After incubation the cells were imaged with fluorescence microscopy to ascertain niosome binding relative to cell binding density by measuring the fluorescence intensity of the green bound niosomes and the blue DAPI stained cell nuclei. A custom made MatlabTM analysis program described in Section 5.2.4.3 allowed for the analysis of t he fluorescent images and quantify the fluorescence intensity of binding relative to cell density. The first set of experiments were done at an ant ibody concentration of 5.0 g/ml antibody, and then the other antibody densities were ex amined with optimal variables found in the time and lipid concentration studies. In order to verify bi nding independent of the fl uorescent techniques a subset of IN incubated BAECs were separ ately fixed and coated for imaging by scanning electron microscopy (SEM).

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885.2.4. Description of Methods 5.2.4.1. Incubation of Nioso mes with Endothelial Cells Cells grown in 8 well micro-slides were fixed with 4% paraformaldehyde diluted in 0.01 M PBS at 4 C overnight. Prior to incubation the fixative was rinsed from cells three times with PBS. The cells were pre-incubated with 2% goat serum in PBS for 1 hour at 37 C to prevent non specific protein interactions and then were rinsed three times with PBS. The niosomes were added to the cells and incubated for the designated time. After the IN-cell incubation the cells were rinsed three times with PBS and a drop of DAPI nuclear stain was added to each well. After staining, the wells were removed from the s lide and the mounting media was added drop-wise to the slide and a cover slip was placed over the cells and was left to dry before imaging. 5.2.4.2. Fluorescent Microsco py Imaging of Incubated Cells Fluorescent and phase contrast imaging of cells, cell nuclei, and INs was done as described in Section 4.3.2.6. Each treat ment variable was imaged multiple times (3-5) in multiple wells (2-4) at 20x and 40 x magnification. 5.2.4.3. Image Analysis Overlays of fluorescent and phase contrast images were created as described in Section 4.2.3.6 in order to visualize binding. In order to quantify binding the images were analyzed usi ng a customized image analysis Matlab™ (Mathworks) program. Example output and compiled data from the program is included in Appendix D Essentially a graphic user interface was developed in

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89which the user first is pr ompted to import the fluoresc ent image of the nuclei as shown in Figure 5.1 left. The image incl udes a scale bar, and the program uses it for calibration. The right side of Fi gure 5.1 demonstrates the next step as the user is prompted to crop the image (if desired), before setting a threshold value of image intensity. Setting the threshol d allows the user to compensate for variability in image clarity and intensity and can be an iterative process with the desired outcome being the identification of the individual nuclei and the removal of background noise, as shown by the image in Figure 5.2. Once the threshold is established, the nuclei are individually id entified and counted, shown in the right side of Figure 5.2, by running Process 1. Process 1 measures the area of each identified nuclei and creates minimum, maximum, and average values for the group, and then, using those values, it eval uates whether each one is a single or greater count (Figure 5.3) and then sums the group. Figure 5.1 Matlab Program Importing and Cropping. Left: Importing of the fluorescent nuclei image. Right: The original image is cropped for analysis.

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90 Figure 5.2 Matlab Program Image Sorting. Left: Setting the threshold in process 1. Right: The identified objec ts are sorted and counted. Figure 5.3 Matlab Program Image Analysis. The fluorescent image of the niosomes which correspond to the same field as the previously imported nuclei image was imported (Figure 5.4 left). The same process of setting a threshold wa s repeated. Process 2 sorts the niosome images. Once the overall process was finished the program has calculated the number of cells, the total area of cell nuclei and the ce ll density, the niosome count, and created an overlay image of the two processed fluorescent images as seen in Figure 5.4 right. The discret e counting of niosomes was amended to

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91calculating fluorescent area of the niosomes instead, reasons for which will be discussed in greater detail in the re sults section, Section 5.3.1. Figure 5.4 Matlab Program Fluorescent Nu clei and IN Overlay. Left: Importing the IN fluorescent images. Righ t: Overlay of processed images 5.2.4.4. Scanning Electron Microscopy Scanning electron microscopy was used to verify the binding of niosomes to cells independent of t he fluorescent measures, and to assess the patterns of surface binding of niosomes to endothelial cells. The cells were grown on square glass cover slips to allow for fixation and metal coating prior to SEM imaging. Before incubation with INs, the BAECs were rinsed with 37 C PBS several times to remove serum proteins, and then were fixed with 2.5% glutaraldehyde in PBS overnight at 4 C. The fixed cells were rinsed three times with PBS (cells may be stored at 4 C in PBS at this point, if requir ed). Cells were then incubated with niosomes at 37 C for the designated times and rinsed with PBS three times again afterwards. A 1.5% potassium ferri cyanide-1% osmium tetroxide solution

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92was prepared under a fume hood in DI and added to the cells to incubate in the dark for 1 hour1. Once the osmium fixation step was comp leted, the cells were rinsed three times with a 0.1 M sodium acetate buffer. The lipids of the niosome membranes were fixed with the addition of a 1.5% urany l acetate solution in 0.1 M Tris buffer for an hour. Before the dehy dration began, the fixative was rinsed three times with sodium acetate buffer. Dehydr ation was done in a graded series for 5 minutes per treatment starting with 30% ethanol, then 70%, 95%, and 100% twice. Once the cells were dehydrat ed, they were substituted with a 1:1 preparation of hexamethyldisilazane (H MDS) and 100% ethanol for five minutes followed by two five minute treatments of pure HMDS. The coverslips of fixed cells and niosomes were dried under the fume hood. After this fixation process they were mounted on stubs and sputter coat ed by Ed Haller in the USF Health core facilities lab. The SEM imaging was done with Betty Loorman of the Biological Electron Microscope Facility of the Biology Department at USF on a JOEL JSM 35 scanning electron microscope. 1 Extreme care must be taken worki ng with these strong fixatives which can rapidly fix mucous membranes at low concentrations, and all contact with liquid and fumes must be avoided. Dispose of chemicals in a dedicated OsO4 container.

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935.3. Results 5.3.1. Endothelial Ce ll-Immunoniosome Images 5.3.1.1. Fluorescent Micrographs Figure 5.5 shows the IHC stained BAEC s with nuclear counterstain after activation with TNF-. White arrows indicate ex pressed CD44 on cell surfaces and processes. Correspondingly, the two images in Figure 5.6 shows the green fluorescent niosomes attached to the ce ll processes and surf aces at different magnifications. Both images represent INs conjugated to 5g/ml IM7 antibodies and incubated for 2 hours prio r to imaging. Figure 5.5 Immunohistochem ical stain for CD44. Figure 5.6 Fluorescent and Contrast Overlay of BAECs and INs. Left: 40x magnification. Right 20x.

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945.3.1.2. Scanning Electron Micrograph Images The left side of the SEM image shown in Figure 5.7 shows fixed cells incubated with INs, and the right side is a c ontrol of the same group of cells that were not incubated with INs. The vesicl es seen on the cell surface of the left image are spherical and appear to be approx imately the same size as the post GEC niosomes. The comparison was m ade by matching the vesicles to the 1 m white line above the 10 m scale line as shown. Figure 5.7 Scanning Electron Micrographs of BAECs. Left: INs bound to cell surfaces. Right: Control. The graph in the left side of Figure 5.8 shows the effect of lipid concentration on relative binding density at different incubation times. The graph in the right side of Figure 5.8 displays the same data relative to incubation time at the different lipid concentrations, and t he control data shown there reflect the

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95incubation of non targeted niosomes ( without conjugated anti bodies) at the maximum lipid concentration. Figure 5.9 shows the affect of antibody concentration on the relative binding of INs to fixed BAECs. In all graphs the error bars represent the standard error of the mean with n>=3. Figure 5.8 Binding Densit y with Respect to Time and Concentration. Left: Binding density with respect to lipid conc entration at different incubation times. Right: Binding with respect to time at di fferent lipid concentrations as compared to controls. All p<0.01 as compared to controls. Figure 5.9 Binding Densit y with Respect to Antibody Concentration. Graph shows relative binding density at 1 and 2 hour incubation times. Cell-Niosome Binding Density vs Concentration0 5 10 15 20 25 30 35 40 0246810 Concentration of lipids (mM)Relative Binding Density 1 hour incubation 2 hour incubation 4 hour incubation Binding Density vs. Incubation Time0 5 10 15 20 25 30 35 40 124 Time (hr)Relative Binding Density Conc= 1.0125 mM Conc= 5.0625 mM Conc= 10.125 mM Controls Conc=10.125 mM

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965.4. Discussion The aim of the experiments was to es tablish definitivel y that the drug targeting system would bind to fixed endothe lial cells at the target antigen. The IHC staining of Figure 5.5 indicates that CD44 was expressed on the fixed bovine endothelial cells at the cell processes and surface. The two fluorescent micrograph-overlay images shown in Figure 5.6 indicate binding of the vesicles in those regions. Furthermore, examinati on of the SEM images in Figure 5.7 shows that vesicles of the right size and shape were observed bound to cell surfaces and that similarly shaped features were not observed in the control image. The binding of the vesicles to cell surfac es is observed in both cases to be nonhomogeneous, and the accumulation of multip le vesicles concentrated in small areas as compared to relatively sparse binding in other areas led us to believe that discrete counting of bound INs by fluorescent measures would be inaccurate. The original MatlabTM routine created a ratio of discrete counts of cell nuclei to discrete counts of niosomes. Once the SEM data revealed that niosomes may be bound closely together in groups making individual counting unlikely, the analysis was reconfigured to measure fluorescence intensity by area of binding and the data are shown to reflect binding area relative to controls. The graph in Figure 5.8 left show s that the binding of INs at 1hour incubation time is independent of concentrati on of lipids. The binding intensity at 1 hour corresponded exactly to what was obs erved in the synovia l lining studies. At increased incubation times the binding increased at the 5 mM lipid concentration, but little increased binding was observed in either an increase in

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97incubation time or in an increase in lipid concentration. The graph of Figure 5.8 right shows the same data with the controls included to cl arify the effect of time on binding density with respect to each of the lipid concentrations. The control data at each incubation time represent non-immunoniosomes incubated at the maximum lipid concentration. Immunoniosome binding relative to different coatings of antibody concentrations is shown in Figure 5.9. With increasing anti body concentrations, increased relative binding was observ ed, although at the lower antibody concentrations there appeared to be no variat ion in binding at 4 hours. Binding of INs at 1 hour was not studied based on the previous data. Ad ditional testing of binding relative to concentrati ons of antibodies between 1-5 g/ml may yield an optimum point. In conclusion, the results shown indi cate that INs bind to activated fixed endothelial cells specifically and in a non homogeneous pattern. The binding relative to time and lipid concentration in creased to a limiting value but antibody concentration did not for the concentrations studied. For the treatments studied, 5 mM at 2 hours appears to be optimum in binding density while conserving material. Further studies will be conduc ted to investigate uptake up by live endothelial cells in vitro to prepare for in vivo animal studies. Section 6 describes the uptake of IM7 conj ugated immunoniosomes in live cells.

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98 6. Live Endothelial Cell Uptake Studies 6.1. Background 6.1.1. Live Endothelial Cells in Targeted Drug Delivery Live endothelial cells have been used as a cell model for testing drug targeting efficacy for many applications including immunoliposomes targeting inflammation and for delivering anti-thrombolytics, and echogenic immunoliposomes for imaging and targeting. The cells have been used in flow experiments for tumor ta rgeting, using antibody fragments, and to address targeting of quiescent versus proliferati ng cells. Uptake of antibody targeted drug delivery vesicles is highly dependent on the target antigen, as previously described (Section 2.3). In order to further investigate the IM7 conjugated immunoniosome as an effective treatment a gainst inflammation the uptake of the vesicles into cells needed to be verified using live cells. Additionally, since accumulation, permeability, and uptake of vesicle by cells has been well established as being size dependent the si ze of the vesicles would need to be reduced. A study involving liposomes s uggests that the preferential size for uptake by atherosclerotic lesions is 200 nm. Size reduction of niosomes has been reviewed115 and includes probe sonication, extrusion through polycarbonate filters, micro-fluidization, and high pre ssure homogenization. Extrusion gave the best size control while maintaining co mparable levels of encapsulation.

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99Microscopic methods used for investi gating IN uptake by activated BAECs were confocal microscopy and transmissi on electron microscopy (TEM). With confocal microscopy fluorescent images ar e captured in multiple x-y planes in a z direction. The result provides thr ee-dimensional information about cellular uptake of a fluorescent vesicle. TE M provides very high magnification and resolution because the energy source from which the image is formed is a beam of electrons rather than light, which has a resulting wavelength (in a vacuum and at a small angle) 3 or ders of magnitude lower than that of light. 6.1.2. Dynamic Light Scattering Dynamic Light Scattering (DLS), also referred to as photon correlation spectroscopy, is used widely to measure the size and distribution of dispersed particles by measuring the changes in laser light projected through the particles. The random movement of small particles in a suspension is called Brownian motion, and is caused as particles collide with molecules from their surroundings and diffuse randomly. When polychromatic li ght hits small suspended particles, the light is scattered in many directions; th is is referred to as Raleigh scattering. However, when monochromatic light is shined into a solution of particles undergoing Brownian motion, the light hitting the moving particles undergoes a Doppler shift when changing wavelength, which can be measured. The change in wavelength is related to the size of the particle that the light hit. Particles undergoing Brownian motion trav el at different speeds related to their size; smaller particles move faster than larger ones. This relationship is

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100defined by the Stokes-Einstein equation. First, the Einstein relation describes diffusion of particles: T k DB p ; where D is the diffusion constant, p is the mobility of the particles, kB is Boltzmann’s constant (a physical constant which relates energy and temperature;1.38 x 10 -23 J/K) and T is the temperat ure in K. Under conditions of low Reynolds number (the rati o of inertial to viscous forces) the mobility is related inversely to the drag coefficient, where under Stokes Law, spherical particles of a radius r: r 6 ; is the viscosity of the medi a in which the particle is dispersed. Therefore taken together, the Stokes -Einstein equation becomes r T k DB6 Diffusion coefficient units are in m2/s The hydrodynamic radius is then found by: D T k rB H6 Through these relations, given viscosity and absorption of suspending media, and the temperature of syst em, the DLS system algorithms measure particle size and/or molecular weight of a suspension through the use of a digital correlator. The correlator measures how cl osely two signals are over a period of time. Correlation is a statistical tec hnique for reducing noise and finding a real signal within it; it measures the degree by which a signal is not random when it would appear to be. T he second order equation is:

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101 2 0 0) ( ) ( ) ( ) ( t I t I t I G where the function relates the product of the intensity at t0 by the intensity at t0 increased by some value as a ratio to the square of the intensity overall squared. 6.1.3. Monoclonal Antibody Fragments Another important consi deration in effective tar geted drug delivery is the persistence of the system wit hin the bloodstream. Vesicle encapsulation of drugs has been shown to increase circulation time, improve bio-distribution, and prolong therapeutic plasma levels in vivo versus free drug administrations. If the drug delivery vesicle or targeting vector provokes an immune response within the body and subsequent rapid clearance from the blood stream there may not be sufficient time or payload of drug deliver ed to be efficacious. Concern about the rapid and increasing clearance of immunoliposomes in vivo with subsequent intravenous injections was solved when antibody fragments were substituted for whole antibodies. While antibodies have been discussed in earlier sections, a description of the physical structure was not given in detail. An antibody is a glycoprotein of molecular weight 150 kDa which is made up of two general types of polypeptide chains, two heavy chains and two light chai ns. The light chains are about 25 kDa each, and each heavy chain is about 50 kDa. There are five subcategorizes of antibodies, IgA, IgE, IgD, IgM, and IgG. These differ by their heavy chains types and structural makeup. The chains are held together by a series of disulfide bonds. Figure 6.1 shows the general structur e of an antibody with respect to the

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102different chains. An IgG, which is the s ubgroup of interest for this research, is composed of either of two types of light chains, or and the 1 heavy chains, which designate the subgroup. The antigen binding (variable) region is indicated at the to p of Figure 6.1, and is specific to the exact antigen to which the antibody binds. The regions below are conserved, and consistent by antibody type. The area between them is referred to as the hinge region. Over all above the hinge is referred to as the F(ab’)2, and below the Fc region. The immune response to the immunoliposomes was provoked by t he Fc region of the antibody and was negated when Fab fragments were used. As Figure 6.1 indicates, fragmentation of antibodies can include a single or a c oupled Fab antigen binding group, in fact many more configurations and combi nations have been studi ed and engineered for use in biopharmaceuticals, biosensors and diagnostics.

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103 Figure 6.1 IgG Antibody Stru cture. (Source: Invitrogen) Fragmentation of antibodies is done using proteolysis reactions (degradation of protein by enzymes) using enzymes such as papain, or pepsin or ficin, and then subsequent purification st eps are taken to separate Fc segments and non-fragmented whole antibodies fr om the desired Fab or F(ab’)2 fragments. 6.2. Materials and Methods 6.2.1. Materials and Chemicals Included in this list are the material s, chemicals, and equipment new to this phase of the project that were not included in the previous sections. Polycarbonate filters and polystyrene latex particle standards where obtained from Sigma Chemical. The antibody fragmentation kit (Immunopure IgG1 Fab

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104and F(ab’)2 44880) was purchased from Pierce Bi otechnology. Propidium iodide nuclear stain (H-1300) was obt ained from Vector Labs. The gold particles were a gift from Ed Haller, USF Health Pa thology. Embed media and components for TEM (and assistance) were provided, kindly, by Dr Karl Muffly. Table 6.1 Equipment for Uptake Studies. Equipment ManufacturerModel Lipid Extruder and Assembly Northern Lipids Thermobarrel 10 ml Water bath Fisher Isotemp Open-Bath Circulator Submicron Dynamic Light Scattering Instrument Malvern Nano-S Confocal Microscope Leica SP3 6.2.2. Methods 6.2.2.1. Extrusion of Niosomes Prior to extrusion the niosomes we re prepared through the hydration step and then frozen overnight. Freezing and thawing the vesicle preparations prompts hydration of the lip ids and equal distribution of encapsulated materials. The niosomes were extruded using a bench top stainless steel 10 ml Thermobarrel LipexTM Extruder. Figure 6.2 shows t he extruder and indicates the functional parts. In order to set up t he extrusion process the extruder must be disassembled so that the filter package c an be put in place. Two polycarbonate filters were stacked on top of a mesh disk and a support disk within the cavity of the filter support base and secured under an o-ring. A drop or two of de-ionized

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105(DI) water was added between each filter while stacking to ensure good seating. The correct alignment of t he filters is crucial to t he extrusion process; poor alignment increases extrusion time and may necessitate changing filters before the extrusion sequence is complete. The barrel of the extruder was replaced and secured with wing nuts. Figure 6.2 Extruder Assembly. (Source: Northern Lipids Extruder Handbook) The vesicle suspension was push ed through the polycarbonate filters using high pressure nitrogen gas (N2) supplied through high pressure lines. The direct pressure into the system from the N2 tank was controlled through a valve on the delivery tubing which is coupled to the gas inlet.

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106 Figure 6.3 Nitrogen Delivery to Extruder. (Adapted from Northern Lipids Extruder Handbook.) The water jacket of the extruder was supplied 60 C DI water by a heated water bath which was circulated by a perista ltic pump. Once the circulating water equilibrated the extruder tem perature, the filter asse mbly was conditioned by expressing 8-10 ml of heated PBS through it. The pressure of the PBS delivery was relatively low; setting the pressu re to 200 psig, and rapidly opening and closing the valve was suffici ent to expel the liquid. Once the filters are conditioned, the heated niosome suspension was pipetted into the sample inlet. Control of the niosome suspens ion temperature is important because extrusion must be done above the gel-to-liquid crystalline phase transition temperature of the surfactant component (or in the case of phospholipids, the component with the saturated alkyl tail) as described in Section 4.2.1.1. The te mperature of the sample adde d to the inlet was allowed to equilibrate for a moment before starting the extrusion (this is repeated at each successive step). The valve was opened to allow the pressure to push the niosome suspension through the filters. The extruded sample was expressed

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107from the outlet tube once t he extrusion process was underway. If sample was not expressed within a few seconds, the pressure was increased until the solution began to flow from the tube. The maximum pressure set by the manufacturer was 800 psig. Once t he extruded sample was captured, the system pressure was vented by opening the pressure relief valve, and the sample was returned to the inlet. This process was repeated ten times and loss from the process was usually less t han 10%. After extrusion, the niosome sample was ready for the GEC process. 6.2.2.2. Dynamic Light Scattering Measurements Dynamic light scattering was used to measure the size of the extruded and purified vesicle suspensions using a Malvern Instruments Nanosizer S. Since the PSS particle analyzer (Section 4. 2) had a lower detec tion limit of 0.5 m diameter particles, the new instrument was required to monitor the size and stability of the extruded vesicles. The Malvern system provides size, molecular weight, and dispersity data using dynam ic light scattering measurements with autocorrelation function. The data results in a poly dispersity index which is a measure of the width of the particle size distribution peak of the sample. The instrument allowed measurements of post GEC particles suspended in PBS. Operating parameters were programmed for measuring sa mples specifically in a standard operating procedure (SOP) in the instrument software (DTS). The material properties, refrac tive index and UV absorbance of the material to be measured and the dispersant are input. Refractive index was measured on a refractometer, and the UV absorbance of the niosome suspension at red laser

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108wavelength (633 nm) was measured using a UV spectrophotometer. These values are used to allow the conversion of the intensity distribution to a volume or number distribution. Outputs from individual measur ements include intensity and volume particle size distributions, cumulants and distribution fit curves, intensity and volume statistics, and the correlation data. Calibration of the system was done with polystyrene latex standards. Figure 6.4 shows the intensity PSD of a 60 nm and 220 nm mixture of polyst yrene latex particle standards, and the Figure 6.5 shows the correlation function curve. T he PSS system (as described in Section 4.2.2.2) provided actual counts of particles, due to the single particle sensing measurement algorithm, whereas the Malver n instrument provides a distribution relative to a measured populat ion-the area under the curve of the size distribution of the Nanosizer data is always 100%, and the area under the curve of the PSS size distribution is the number of particles measured and counted by the instrument. Due to the difference in the underlying physics of these particle characterization tools, their data did not ali gn well. Despite clai ms to the contrary by the manufacturer, dispersions of vesicles between 700-1000 nm did not measure accurately or repeatedly in the Malv ern instrument, at any dilution. This would probably be due to the size limitation of Brownian motion.

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109 Figure 6.4 Particle Size Distri bution of 60 nm and 220 nm Standards. Figure 6.5 Correlation Func tion Curve of the Standards.

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1106.2.2.3. Antibody Fragmentation Antibodies were fragmented using a comme rcially available kit from Pierce according to the manufacturer’s instru ctions. While the components of the columns and buffers are proprietary, ess entially the process is the pH dependent digestion of the hinge protei ns of the antibody by the thiol protease enzyme ficin (MW 25 kDa) in the presence of the amino acid cysteine. At a concentration of 1 mM cysteine, ficin will produce F(ab’)2 fragments, and at a concentration of 10 mM cysteine, ficin will produce Fab fragm ents. The digestion reaction takes place in a column and the resulting fragment s are eluted out in different fractions using different buffers. The UV absorpt ion of the fractions was measured and a balance of proteins found that the initial yield was 70%. The protein concentrations were measured us ing extinction coefficients of Fab= 7.5 x 104, and IgG= 22.1 x 104 using Beers Law (see Section 4.2). 6.2.2.4. Endothelia l Cell Culture and Fixation Techniques Cells were cultured and activated as prev iously described in section 5.2. For confocal studies the cells were gr own on 8 well micro-slides, for TEM the cells were grown on cover slips to allow for embedding into the resin. Prior to incubation with live cells, the INs we re filtered for sterility using a 0.2 m syringe filter to eliminate po ssible contaminants. For the TEM studies a dilute solution of 15 nm spherical gold particles was encapsulated in 10% Tw-CC niosomes wh ich were extruded, purified with GEC, and conjugated to IM7 antibodi es in the usual manner. The gold particles were

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111used in the niosomes to provide a strong signal during the TEM imaging. Prior to IN incubation, the cells were well rinsed with warmed PBS. 6.2.2.5. Fixation Techniques for Confocal Microscopy Fluorescent IN suspensions were diluted 1:1 in serum free endothelial media (to avoid unwanted protein interactions ) and incubated with live bovine aortic endothelial cells (BAECs) at 37 C for varied times. Afte r incubation the vesiclemedia mixture was discarded and the cells were rinsed three times with room temperature PBS and then were fixed wit h 1% paraformaldehyde in PBS for 15 minutes. After fixation, the cells were PBS rinsed again and incubated with 0.1% Triton X 100 solution for three minutes, to encourage membrane permeability for the nuclear stain, propidium iodide, wh ich was left to penetrate for 30 minutes. Once the staining was complete, the cells were rinsed again, and the wells were removed. A few drops of hard setti ng mounting solution were added to each slide and a glass coverslip was placed over the cells and allowed to harden overnight. 6.2.2.6. Fixation Techniques for Transmission Electron Microscopy Incubation of cells with INs with t he gold containing INs was done as described for the confocal studies. Once the incubation was complete the cells were rinsed three times with room tem perature PBS. Cells were fixed with a 2.5% solution of glutaraldehyde for 15 minutes at room temperature. Fixation of cells for TEM was similar to that of the SEM procedure. A 1.5% potassium ferricyanide/1% osmium tetroxide soluti on in DI water was added to the rinsed

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112cells and left to fix for an hour in the dark. The cells we re rinsed three times for 10 minutes each in 0.1M sodium acetate buffer. The lipid membranes were stabilized by fixing in a 1.5% solution of ur anyl acetate in a 0.1 Tris buffer, pH 6.3 for 30 minutes and then were rinsed three times with DI water. Once the final fixation step was comple te the cells went through a series of dehydration steps. A se ries of ethanol in water was added as follows for 5 minutes each, 30%, 70%, 95%, and 100% ethanol for 15 minutes. After the alcohol series, the cells were cleared with 100% propylene oxide for 15 minute, and then a mixture of 100% propylene oxide mixed 1:1 with the embedding media. The embedding media was mixed toge ther per the instructions from the EMS Technical Data Sheet for Aral dite 502/EMBED-812 Embedding media as follows: Embed-812 -13.75 g, Araldite 502 -8 .5 g, DDSA -27.5 g, DMP-30-0.9 g. The cells were embedded in the 1:1 mixt ure and allowed to dry overnight, and then was capsulated in pure embedding media. The embedded cells were sectioned with a diamond knife and fixed onto copper grids stained with 8% uranyl acetate and lead citrate. 6.2.3. Confocal Microscopy The confocal images were captur ed with a Leica SP2 using 40x oil objectives with FITC and rhodam ine filters. The argon lase r (blue light for green labels) provided wavelengths 458-514 nm and the helium neon laser (red light for far red labels) provided light at a wa velength of 633 nm. The Leica confocal software (LCS) was used for imaging and for post-image capture processing.

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113Once the fluorescently stained cell nuc lei images were visually confirmed and focused by viewing through the micr oscope eyepiece, the image capture software was set up. The two photomulti plier (PMT) gains and offsets were set for clarity and reduction of background noi se, where possible. Figure 6.6 shows the image acquisition screen wher e the settings are adjusted in the software. Table 6.2 shows the PMT gain and offset and z position settings for the samples examined. Figure 6.6 Leica Confocal Software Image Acquisition Window. Table 6.2 Confocal Settings. PMT 1 PMT 2 Z/Y Position Sample Gain Offset Gain Offset (microns) Control 632 -11%616-7%-42 20min-dim 520 -15%464-4%-49 20min-brt 549 -14%568-14%-28 1hour-run1 862.3 -5.10%847-3.60%-29 1hour-run2 862.3 -5.10%847-3.60%-29

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114Once the settings are established the image capture stack has to be defined prior to going into scan mode. Figure 6. 7 shows the schematic for setting the z/y position in the LCS software. By scro lling through the image using the z position the bottom and top positions were set to mark the start and end of the captured stack. Figure 6.7 Spatial Setting for Scan Mode Imaging. 6.3. Experimental Designs Extrusion experiments were evaluated by taking aliquots of sample at each extrusion pass. These samples were examined for size and entrapment of dye by GEC elution and DLS. The GEC eluti on profiles were created by fluorescent measurements of sequentially eluted fracti ons. Particle size distribution was measured by DLS to see the effect of extrusion passes on vesicle formation and dispersity.

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115For the confocal uptake studies with whole antibodies conjugated to fluorescent immuniosomes incubation times examined were 20 mins, 1 hour, 2 hours, and a control of 2 hours at 4 C to check for non specific binding. Confocal images of INs conjugated to IM7 Fab fragments were m easured at 1 hour. TEM incubation time was 1 hour. All antibody or fragment coating was at a done at concentrations of 5 g/ml. 6.4. Results 6.4.1. Extrusion Results In Figure 6.8 the fluorescence chromat ograms show the GEC elution of each sample. The first peak represents the fluorescent signal fr om the niosomes (partially quenched due to concentrati on) and the second peak represents the unencapsulated free dye. Figur e 6.9 shows the particle si ze distribution of the niosome fractions captured during GEC.

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116 Figure 6.8 Elution of Extrusions 0-10 for a 10% TW-CC-CF Hydration Sample. Figure 6.9 Dynamic Light Scatte ring Data of Extruded Samples. The cell nuclei are stained red in the c onfocal images, and the INs are green. All images shown are at the maximum ar ea of cell nuclei. Figure 6.10 shows the 20 min incubation. The z value represent s the depth within t he stack that the

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117image represents. Figure 6.11 shows the 1 hour incubation, and Figure 6.12 is the 2 hour incubation. The control cells are shown in Figure 6.13. These figures represent incubations wit h whole antibody bound INs. The LCS software provided a post imaging function called slice that allowed for a cross section of xz or y-z plane to be viewed. The narro w images shown in Figure 6.14 are individual cross sections for each incubat ion slide already shown. The final confocal image is seen in Figure 6.15 r epresenting the incubation of BAECs with fragment conjugated immunoniosomes. Figure 6.10 Confocal BAECs 20 Minut e Incubation. Image at -7.001 mm (Range of stack: 0,-12.537 mm)

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118 Figure 6.11 Confocal BAECs I Hour Inc ubation. Image at -11.56 mm (Range of stack: 0,-20.00 mm) Figure 6.12 Confocal BAECs 2 Hour Inc ubation. Image at -11.07 mm (Range of stack: 0, 20.03 mm)

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119 Figure 6.13 Confocal BAECs Control. Incubation at 4 C for 2 hrs. Image at 4.88 mm (Range of stack: 11.07,0 mm) Figure 6.14 Confocal Cross Sections.

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120 Figure 6.15 Fab-IN BAEC Confocal Image. 6.4.2. TEM Figure 6.16 shows the TEM image a singl e endothelial cell c aptured from the incubation of INs containing gold particles with BAECs. The arrows indicate the niosomes bound to the surfac e of the endothelial cell. Figure 6.16 TEM of Au-INs.

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1216.5. Conclusions The extrusion studies indicated that eight passes through the system is preferable for these vesicles versus the suggested ten passes used in the synthesis of liposomes. Each successi ve extrusion pass up until the eight showed an increase in the formed nios ome signal of Figure 6.8, and correspondingly showed a taller and sharper peak in the dynamic light scattering data of Figure 6.9. In each data set, reduction of niosome formation and peak was seen in the tenth pass sample. The confocal images suggest uptake of immunoniosomes by live endothelial cells. Penetration of IM7-niosomes continues between 20 min and 1 hour, but does not appear to increase appreciatively thereafter. The cr oss section images raise the possibility that INs are attach ing to the junctions between the cells, although there are niosomes visible in so me images near nuclei. Controls show no binding at 4 C. Absolute verification of location of the vesicles either within or on the surface of the cell remains to be done. Membrane stai ning was attempted to clarify the imaging, but since the nat ure of the vesicle membrane is analogous to that of a cell membrane, cross staining did not allo w for the desired distinction of IN position using that technique. An independent measure is needed to confirm uptake such as radio labeling of vesicle components or encapsulated material. The IN-Fab incubation showed very spar se binding. This may be due to disruption of the antigen c ouple of the fragment dissoci ating when exposed to the binding pH. Using the F(ab’)2 fragments instead of the single Fab’s may provide

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122binding closer to that of the whole antibodies and should better preserve the antigen binding sites. As it stands although the potential for fragment use is demonstrated, the fragment bindi ng requires optimization. The TEM image shows adhesion to the surf ace of cell by the gold INs, none were observed within the cell, however a very small sample of cells were identified in the media, and an improvement of the cell embedding technique may allow for clearer data.

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123 7. Conclusions and Contributions 7.1. Introduction The research that this document represents created a new drug delivery system with a novel linking chemistry ta rgeted to treat a very significant pathological condition, atherosclerosis. T he long term goal of this research was to create, test, and optimize a drug deliver y carrier with a tar geting vector that could be used to treat the vulnerable pl aque of atheroscleros is and test it experimentally in vivo in an atherosclerotic animal model. The fixed cell binding results of this research strongly dem onstrate and ability of the immunoniosome to bind to a tar get antigen and to do so selectively and specifically. The uptake results do not def initively demonstrate the exact fate of the IM7 conjugated niosomes when bound to inflammatory endothelial cells. There has been sufficient demonstration withi n the body of liposome literature to establish that the correct antigen-anti body couple will enable the uptake of antibody bearing vesicles into the endothelium through endocytosis. Further studies evaluating uptake of immunoniosom es by endothelial cells will need to be pursued. Regardless of uptake, the binding of niosomes bearing drug payload has the potential for therapeutic effect by virtue of localization and antigen binding site blocking. If a drug bearing particle is localized to a pathological site, in situ drug

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124release will provide higher concentrati on of drug at the des ired location than a systemic delivery would. Also, a critical fa ctor in the progression of inflammatory diseases, and the build up of athero sclerotic plaque, is the destructive proliferation of inflammatory cells at the sites of chronic inflammation. By blocking the binding of inflammatory cells and the cell uptake sites, their accumulation within the tissues would be mitigated. W hether the targeted vesicles are taken up by the cells through endocytosis, or remain on the surface of the inflammatory site, the potential for treatment remains. The immunoniosome system was evaluat ed in this research for use in targeting inflammatory processes; it would not necessarily be limited to that application. The system is capable of encapsulating a solution of either hydrophilic or hydrophobic dr ug, and of binding to any IgG and, except where limited by size or surface hindrances, any protein. With these capacities, many different applications of the system are pos sible and remain to be pursued. The creation of the Tween 61-cyanur ic chloride linker provides a highly reactive binding moiety which can undergo nucleophi lic substitution reactions easily and without prior derivatization of the binding species. T he potential of the system to be used for other applications is significant. 7.2. Contributions The contributions of this research to the field of targeted drug delivery include the synthesis and characterization of a novel formulation of an antibody targeted non ionic surfactant vesicle drug carrier. There is no information in the literature describing niosomes composed of a bl end of surfactants as a tool to

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125simultaneously maintain required physi cal properties while creating linking moieties. The creation of an antibody linker by the functionalization of a polyoxyethylene sorbitan monostearate molecu le is novel, as is the creation of the polyoxyethylene sorbit an monostearate-cyanuric chloride linker molecule. There is no reported use of non ionic surf actant vesicles designed for active targeting with monoclonal antibodies. 7.3. Future Work Future work in the vesicle characteri zation of this research includes the further investigation of uptake of IN s by activated endothelial cells, and a refinement of the antibody fragment binding process. Entrapment of an HMG CoA reductase inhibitor drug, atorvastatin, will be pursued prior to the onset of the in vivo animal study. The protocol for the study has already been developed and granted approval by the USF IACUC and the US Army Medical Research and Material Command Animal Care and us e Review Office. Table 7.1 outlines the experimental design of the research. The atherosclerotic animal model that will be used in the experiments is a knock out mouse which lacks the ligand ap olipoprotein E (apoE) which promotes lipoprotein clearance. The apoEdeficient mice are bred using homologous recombination in embryonic stem ce lls to be susceptible to developing atherosclerosis. The animals will be fed a high fat di et to encourage the formation and progression of atherosclerotic lesions and plaque. After the 8-10 week feeding

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126period, the treatment phase lasts 6 weeks. Treatments of ex perimental groups are outlined in Table 7.1. Table 7.1 Future Work: In Vivo Atherosclerotic Mouse Study Treatment Group (n=6 for all) Treatment Test low 1. Surfactant vesicle coated with antibody fragments and containing drug atorvastatin (test substance-low drug concentration) Test high 2. Surfactant vesicle coated with antibody fragments and containing atorvastatin (test substance-high drug concentration) Control targeted non drug 3. Su rfactant vesicle coated with antibody fragments and containing buffered saline solution Control non targeted with drug 4. Surfactant vesicle without antibody containing atorvastatin Control free drug low 5. Fr ee drug atorvastatin low concentration (control) Control free drug high 6. Fr ee drug atorvastatin high concentration (control) Control injection 7. Buffered saline (control) Treatments will be administered by tail vein injection, and weekly blood samples will be taken by submandibular bl eeding to assess blood cholesterol, triglycerides, and atherosclerot ic markers (e.g. C-reactive protein). Weekly body weight will also be measured. At the end of the treatm ent period, the mouse aortas will be evaluated for differences in plaque progression, the bio-di stribution of the at orvastatin drug in the animal’s organs will be assessed, and the lipid blood levels will be studied. The expectation from this study is that the targeted drug tr eatment group will show a reduction of plaque progression relative to the free drug groups and

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127controls. Additionally the study may dem onstrate the extrahepatic effects of the statin on the build up of plaques, since statin treatment does not affect the production of cholesterol in t he liver in the mouse model.

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146 Appendices

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147Appendix A. Particle Sizing S ystems Data and Software Output The images below are Particle Sizi ng Systems Accusizer software output from a 3 size polystyrene latex standar d for size and count calibration of instrument. The axes scales can be set, and the y axis can display units of number of particles counted (# Part.) or numbers of particles counted per ml of sample injected for analysis (# Part./ml as shown). For a given injection volume the cumulative number of counts can be f ound in the volume fraction calculation menu in the display menu. To obtain the number of particles per ml divide the calculated number of particles listed in the volume fraction calculation by sample volume injected. For example in the sa mple below 0.025 ml was injected. The calculated # is 147011. The tota l number per ml is 5.88 x 106 particles per ml. Figure A.1 Particle Size Distribution of Standards

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148Appendix A. (Continued) Figure A.2 Sample Number Calculation : Table A.1 Data Exported From PSD ASCII File Particle Sizing Systems, Inc. Santa Barbara, Calif., USA Model 780 AccuSizer Caption: standard MML 0.7, 1. 5 um peaks 25 uL injection File Name = 3_24_03.003 Sensor Model: LE400-0.5 SUM S/N: 8910Cal. File: 0008910s.sns Elapsed Time of Data Collection = 60Sec. Background File = NONE Total # Part. Sized (>=Thres. 0.52 um ) = 101593 Calculated Total No. of Particles in Sample = 150625 Dilution Factor = 1.48 Fluid Volume Sampled = 60ml No. of Channel s = 512 NUM-WT Mean = 0.91um Mode = 0.73um Median = 0.73 VOL-WT Mean = 7.93um Mode = 5.34um Median = 5.32 Summary of Detailed Distribution, Weightings Diameter # Part. Cum Num Num % Vol % Cum Num % Vol Num % (microns) Sized >=Diam. >=Diam. >=Diam. 0.52418 28810159 3 0.2830.007100 100 0.5313 32810130 5 0.3230.00999.717 99.993 0.53851 31210097 7 0.3070.00999.394 99.984

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149Appendix A. (Continued) 0.55324 40010031 3 0.3940.01298.74 99.965 0.56076 368999130.3620.01298.346 99.953 0.56837 389995450.3830.01397.984 99.941 0.57609 818991560.8050.02897.601 99.928 0.58392 799983380.7860.02896.796 99.901 0.59185 715975390.7040.02696.01 99.872 0.59989 643968240.6330.02595.306 99.846 0.60804 631961810.6210.02594.673 99.821 0.6163 265955500.2610.01194.052 99.796 0.62467 571952850.5620.02593.791 99.784 0.63315 285947140.2810.01393.229 99.76 0.64175 701944290.690.03392.948 99.747 0.65047 1084937281.0670.05392.258 99.714 0.65931 709926440.6980.03691.191 99.66 0.66826 1647919351.6210.08890.493 99.624 0.67734 2336902882.2990.1388.872 99.536 0.68654 1655879521.6290.09686.573 99.407 0.69586 5436862975.3510.32784.944 99.311 0.70532 7096808616.9850.44579.593 98.984 0.7149 3987737653.9240.2672.608 98.539 0.72461 8772697788.6340.59668.684 98.279 0.73445 9730610069.5770.68860.049 97.683 0.74443 8951512768.8110.65950.472 96.995 0.75454 3857423253.7970.29641.661 96.336 0.76479 6126384686.030.48937.865 96.04 0.77517 4295323424.2280.35731.835 95.55 0.7857 2792280472.7480.24227.607 95.193 0.79638 1624252551.5990.14624.859 94.951 0.80719 509236310.5010.04823.26 94.805 0.81816 680231220.6690.06622.759 94.757 0.82927 447224420.440.04622.09 94.691 0.84054 331219950.3260.03521.65 94.645 0.85195 250216640.2460.02821.324 94.61 0.86352 202214140.1990.02321.078 94.582 0.87525 181212120.1780.02220.879 94.559 0.88714 225210310.2210.02820.701 94.538 0.89919 180208060.1770.02320.48 94.51 0.91141 181206260.1780.02420.303 94.486 0.92379 354204450.3480.0520.124 94.462 0.93633 267200910.2630.03919.776 94.412 0.94905 197198240.1940.0319.513 94.373 0.96194 190196270.1870.0319.319 94.343 0.97501 196194370.1930.03219.132 94.312 0.98825 313192410.3080.05418.939 94.28 1.00168 254189280.250.04618.631 94.226 1.01528 794186740.7820.14818.381 94.18 1.02907 784178800.7720.15317.6 94.032 1.04305 1684170961.6580.34116.828 93.88 1.05722 2565154122.5250.54115.17 93.538 1.07158 2938128472.8920.64612.646 92.997 1.08614 261099092.5690.5979.754 92.352 1.10089 131072991.2890.3127.185 91.754 1.11584 125959891.2390.3125.895 91.442

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150Appendix A. (Continued) 1.131 67047300.6590.1734.656 91.13 1.14636 32840600.3230.0883.996 90.957 1.16193 13437320.1320.0383.673 90.869 1.17772 10635980.1040.0313.542 90.831 1.19371 6034920.0590.0183.437 90.8 1.20993 4734320.0460.0153.378 90.782 1.22636 3133850.0310.013.332 90.767 1.24302 3933540.0380.0133.301 90.757 1.2599 3733150.0360.0133.263 90.744 1.27702 2232780.0220.0083.227 90.73 1.29436 1732560.0170.0073.205 90.722 1.31194 3432390.0330.0143.188 90.716 1.32976 3232050.0310.0133.155 90.702 1.34783 1931730.0190.0083.123 90.688 1.36613 2831540.0280.0133.105 90.68 1.38469 2331260.0230.0113.077 90.667 1.4035 1431030.0140.0073.054 90.656 1.42256 2230890.0220.0113.041 90.65 1.44189 1330670.0130.0073.019 90.638 1.46147 1330540.0130.0073.006 90.631 1.48132 1430410.0140.0082.993 90.624 1.50144 1030270.010.0062.98 90.616 1.52184 1530170.0150.0092.97 90.61 1.54251 830020.0080.0052.955 90.6 1.56346 1129940.0110.0082.947 90.595 1.5847 1429830.0140.012.936 90.588 1.60622 1629690.0160.0122.922 90.578 1.62804 629530.0060.0052.907 90.566 1.65015 729470.0070.0062.901 90.561 1.67257 1029400.010.0082.894 90.556 1.69529 1429300.0140.0122.884 90.547 1.71832 629160.0060.0052.87 90.535 1.74166 929100.0090.0082.864 90.53 1.76531 929010.0090.0092.855 90.521 1.78929 1328920.0130.0132.847 90.512 1.81359 828790.0080.0092.834 90.499 1.83823 328710.0030.0032.826 90.491 1.8632 528680.0050.0062.823 90.487 1.88851 728630.0070.0082.818 90.481 1.91416 628560.0060.0082.811 90.473 1.94016 328500.0030.0042.805 90.465 1.96651 1028470.010.0142.802 90.462 1.99322 528370.0050.0072.793 90.448 2.0203 128320.0010.0012.788 90.441 2.04774 328310.0030.0052.787 90.439 2.07555 328280.0030.0052.784 90.435 2.10375 328250.0030.0052.781 90.43 2.13232 328220.0030.0052.778 90.425 2.16129 328190.0030.0052.775 90.42 2.19064 328160.0030.0062.772 90.414 2.2204 928130.0090.0182.769 90.409 2.25056 428040.0040.0082.76 90.391 2.28113 428000.0040.0082.756 90.383 2.31211 427960.0040.0092.752 90.375

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151Appendix A. (Continued) 2.34352 327920.0030.0072.748 90.366 2.37535 427890.0040.012.745 90.359 2.40762 127850.0010.0022.741 90.349 2.44032 327840.0030.0082.74 90.347 2.47347 427810.0040.0112.737 90.339 2.50706 327770.0030.0082.733 90.328 2.54112 227740.0020.0062.73 90.32 2.57563 227720.0020.0062.729 90.314 2.61062 327700.0030.012.727 90.308 2.64608 327670.0030.012.724 90.298 2.68202 02764002.721 90.288 2.71845 327640.0030.0112.721 90.288 2.75538 227610.0020.0072.718 90.278 2.7928 227590.0020.0082.716 90.27 2.83074 02757002.714 90.262 2.86919 427570.0040.0172.714 90.262 2.90816 127530.0010.0042.71 90.245 2.94766 327520.0030.0142.709 90.241 2.9877 327490.0030.0142.706 90.227 3.02828 527460.0050.0252.703 90.213 3.06942 127410.0010.0052.698 90.188 3.11111 327400.0030.0162.697 90.183 3.15337 02737002.694 90.167 3.1962 227370.0020.0122.694 90.167 3.23961 127350.0010.0062.692 90.155 3.28362 127340.0010.0062.691 90.149 3.32822 02733002.69 90.143 3.37343 327330.0030.0212.69 90.143 3.41925 02730002.687 90.122 3.46569 02730002.687 90.122 3.51277 02730002.687 90.122 3.56048 02730002.687 90.122 3.60885 127300.0010.0082.687 90.122 3.65787 227290.0020.0172.686 90.114 3.70755 02727002.684 90.097 3.75791 127270.0010.0092.684 90.097 3.80896 127260.0010.012.683 90.087 3.86069 327250.0030.0312.682 90.077 3.91313 02722002.679 90.046 3.96629 227220.0020.0222.679 90.046 4.02016 527200.0050.0582.677 90.024 4.07477 327150.0030.0362.672 89.966 4.13012 227120.0020.0252.669 89.93 4.18622 227100.0020.0262.667 89.905 4.24308 127080.0010.0142.666 89.878 4.30071 427070.0040.0572.665 89.865 4.35913 227030.0020.032.661 89.808 4.41834 627010.0060.0922.659 89.778 4.47835 126950.0010.0162.653 89.686 4.53918 626940.0060.12.652 89.67 4.60084 626880.0060.1042.646 89.57 4.66333 426820.0040.0722.64 89.465 4.72668 826780.0080.1512.636 89.393 4.79088 2626700.0260.5112.628 89.242

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152Appendix A. (Continued) 4.85595 2526440.0250.5112.603 88.732 4.92191 2126190.0210.4472.578 88.22 4.98877 3925980.0380.8652.557 87.773 5.05653 5825590.0571.3392.519 86.909 5.12522 10425010.1022.52.462 85.57 5.19483 22223970.2195.5572.359 83.07 5.26539 46821750.46112.1992.141 77.512 5.33691 77217070.7620.9551.68 65.313 5.40941 5679350.55816.0260.92 44.359 5.48288 2063680.2036.0630.362 28.333 5.55736 471620.0461.440.159 22.27 5.63284 191150.0190.6060.113 20.83 5.70936 9960.0090.2990.094 20.223 5.78691 8870.0080.2770.086 19.924 5.86551 2790.0020.0720.078 19.647 5.94518 7770.0070.2630.076 19.575 6.02594 4700.0040.1560.069 19.313 6.10779 3660.0030.1220.065 19.156 6.19075 2630.0020.0850.062 19.034 6.27484 8610.0080.3530.06 18.95 6.36007 1530.0010.0460.052 18.597 6.44646 2520.0020.0960.051 18.551 6.53403 3500.0030.1490.049 18.455 6.62278 6470.0060.3110.046 18.306 6.71274 3410.0030.1620.04 17.994 6.80392 038000.037 17.832 6.89634 1380.0010.0590.037 17.832 6.99001 1370.0010.0610.036 17.774 7.08496 036000.035 17.713 7.18119 1360.0010.0660.035 17.713 7.27873 035000.034 17.647 7.3776 035000.034 17.647 7.47781 2350.0020.1490.034 17.647 7.57939 3330.0030.2330.032 17.497 7.68234 4300.0040.3240.03 17.264 7.78669 2260.0020.1690.026 16.94 7.89246 4240.0040.3510.024 16.772 7.99966 020000.02 16.42 8.10832 020000.02 16.42 8.21846 020000.02 16.42 8.33009 020000.02 16.42 8.44324 020000.02 16.42 8.55792 1200.0010.1120.02 16.42 8.67417 019000.019 16.309 8.79199 019000.019 16.309 8.91141 019000.019 16.309 9.03246 019000.019 16.309 9.15515 2190.0020.2740.019 16.309 9.2795 1170.0010.1430.017 16.034 9.40555 016000.016 15.892 9.5333 016000.016 15.892 9.6628 1160.0010.1610.016 15.892 9.79405 1150.0010.1680.015 15.731

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153Appendix A. (Continued) 9.92708 014000.014 15.563 10.06192 014000.014 15.563 10.19859 014000.014 15.563 10.33712 014000.014 15.563 10.47753 1140.0010.2050.014 15.563 10.61985 013000.013 15.358 10.7641 013000.013 15.358 10.91031 1130.0010.2320.013 15.358 11.05851 012000.012 15.126 11.20872 012000.012 15.126 11.36097 1120.0010.2620.012 15.126 11.51528 011000.011 14.864 11.6717 011000.011 14.864 11.83024 011000.011 14.864 11.99093 011000.011 14.864 12.1538 011000.011 14.864 12.31889 1110.0010.3340.011 14.864 12.48622 010000.01 14.53 12.65582 010000.01 14.53 12.82773 010000.01 14.53 13.00197 010000.01 14.53 13.17857 1100.0010.4090.01 14.53 13.35758 190.0010.4260.009 14.121 13.53902 08000.008 13.696 13.72292 08000.008 13.696 13.90932 08000.008 13.696 14.09825 08000.008 13.696 14.28975 180.0010.5210.008 13.696 14.48385 07000.007 13.175 14.68059 07000.007 13.175 14.87999 07000.007 13.175 15.08211 07000.007 13.175 15.28697 07000.007 13.175 15.49462 07000.007 13.175 15.70508 07000.007 13.175 15.91841 170.0010.720.007 13.175 16.13463 160.0010.750.006 12.454 16.35379 05000.005 11.704 16.57593 05000.005 11.704 16.80108 05000.005 11.704 17.02929 05000.005 11.704 17.2606 05000.005 11.704 17.49505 05000.005 11.704 17.73269 05000.005 11.704 17.97356 150.0011.0370.005 11.704 18.21769 04000.004 10.668 18.46515 140.0011.1240.004 10.668 18.71596 03000.003 9.543 18.97018 130.0011.2190.003 9.543 19.22786 02000.002 8.324 19.48903 02000.002 8.324 19.75375 120.0011.3760.002 8.324 20.02207 01000.001 6.948 20.29403 01000.001 6.948

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154Appendix A. (Continued) 20.56969 01000.001 6.948 20.84909 01000.001 6.948 21.13229 01000.001 6.948 21.41933 01000.001 6.948 21.71027 01000.001 6.948 22.00517 01000.001 6.948 22.30407 01000.001 6.948 22.60702 01000.001 6.948 22.9141 01000.001 6.948 23.22534 01000.001 6.948 23.54082 01000.001 6.948 23.86058 01000.001 6.948 24.18468 01000.001 6.948 24.51318 01000.001 6.948 24.84615 01000.001 6.948 25.18363 01000.001 6.948 25.52571 01000.001 6.948 25.87242 01000.001 6.948 26.22385 01000.001 6.948 26.58006 01000.001 6.948 26.9411 01000.001 6.948 27.30704 01000.001 6.948 27.67796 01000.001 6.948 28.05391 01000.001 6.948 28.43497 01000.001 6.948 28.82121 01000.001 6.948 29.21269 01000.001 6.948 29.60949 01000.001 6.948 30.01168 01000.001 6.948 30.41933 01000.001 6.948 30.83252 01000.001 6.948 31.25132 01000.001 6.948 31.67581 01000.001 6.948 32.10607 01000.001 6.948 32.54217 01000.001 6.948 32.9842 01000.001 6.948 33.43222 01000.001 6.948 33.88634 110.0016.9480.001 6.948 34.34662 00000 0 34.81316 00000 0 35.28603 00000 0 35.76532 00000 0 36.25113 00000 0 36.74353 00000 0

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155Appendix B. Akta Prime Ch romatography Data Output Generated reports for each sample run show user designated data, method, run and evaluation notes. Figure B.1 Akta Prime Elution Chro matogram Showing UV Absorbance and Conductivity

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156Appendix B. (Continued) Figure B.2 Akta Prime Method Notes

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157Appendix B. (Continued) Each programmed method run created an overlay of all experimental curves generated. The data for each curv e represented in the overlay can be individually exported as an ASCII file. Figure B.3 Akta Prime Chromat ogram Showing All Measures

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158Appendix C. Fluore scent Plate Reader The BioTek Instruments fluorescent pl ate reader uses KC Junior software for creating an operating procedure and tr anslating the intensity data into concentration data based on a designated calibration curve. Below are the steps for setting up a 96 well plate and the resulting data available for export to Microsoft Excel. C.1 Defined Protocol Figure C.1 Fluorescent Plate Reader: Protocol Definition

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159 Appendix C. (Continued) Figure C.2 Fluorescent Plate Reader : Defined Plate Reader Geometry

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160Appendix C. (Continued) Figure C.3 Fluorescent Plate Reader: De fine Individual Well Measurement Types C.2 Stored Standard Curve Example Figure C.4 Fluorescent Plate Reader: Stored Standard Curve

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161Appendix C. (Continued) Measured Intensity Figure C.5 Fluorescent Plate Reader: Fluorescence Intensity by Wells C.3 Data Exported to Excel Figure C.6 Fluorescent Plate Reader: Intensity Data Exported to Microsoft Excel

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162Appendix D. Matlab™ Image Analysis The Matlab program which produced th is example output was constructed by John Elliott, M.S. ChE while working in the Chemical E ngineering Department at USF in 2006-2007. The overall proce ss of the image analysis and the images produced was described in Sect ion 5.2.4.3. Below is the data output from the Matlab Command Window. These examples were run with 20x magnification images files from BAECs incubated with INs for 2 hours at 37 C. Command Window Output caldata = 6x1 struct array with fields: Area Centroid BoundingBox X_Cal = 0.4669 handles = figure1: 153.0455 pushbutton7: 7.0457 pushbutton6: 6.0457 listbox1: 5.0457 pushbutton5: 4.0457 pushbutton4: 3.0457 pushbutton3: 2.0457 pushbutton2: 1.0457 pushbutton1: 0.0457

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163Appendix D. (Continued) togglebutton1: 159.0455 axes3: 154.0455 output: 153.0455 fil ename: 'W1_20x_D1.tif' root_file: 'C:\Program Files\MATLAB71\work\images\10-05-06\' banner: [1024x1360x3 uint8] Cal: 0.2180 h = 1 h = 2 celldata = 76x1 struct array with fields: Area Centroid BoundingBox max_area = 1243 cell_mean = 104.2558 max_area2 = 476 h = 3 filename2 = W1_20x_F1a.tif root_file2 =

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164Appendix D. (Continued) C:\Program Files\MATLAB71\work\images\10-05-06\ filename2 = W1_20x_F1.tif root_file2 = C:\Program Files\MATLAB71\work\images\10-05-06\ h = 4 celldata22 = 1699x1 struct array with fields: Area Centroid BoundingBox handles = figure1: 153.0455 pushbutton7: 7.0457 pushbutton6: 6.0457 listbox1: 5.0457 pushbutton5: 4.0457 pushbutton4: 3.0457 pushbutton3: 2.0457 pushbutton2: 1.0457 pushbutton1: 0.0457 togglebutton1: 159.0455 axes3: 154.0455 output: 153.0455 fil ename: 'W1_20x_D1.tif' root_file: 'C:\Program Files\MATLAB71\work\images\10-05-06\' banner: [1024x1360x3 uint8] Cal: 0.2180 myX: [1 1360] myY: [1 1024] su b_A: [571x571x3 uint8]

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165Appendix D. (Continued) rect_userD: [10. 0968 248.8353 570.2515 570.2515] sub_B: [571x571 uint8] sub_BB: [571x571 uint8] su b_C: [571x571 logical] l abeled: [571x571 double] numObjects: 76 RGB_labe l: [571x571x3 uint8] numObjects2: 42 red_set: [5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 6 4 11 14] ful_ set: [5 0 0 0 0 1 13 29 14 7 4 1 0 0 0 0 0 0 0 2] fatcell: [1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 2 2 1 1 2] A2: [1024x1360x3 uint8] s ub_A2: [571x571x3 uint8] RGB_label 2: [571x571x3 uint8] nio_ful_set: [1630 41 13 6 2 1 0 0 2 1 0 0 1 0 0 0 0 1 0 1] nio_red_set: [1630 41 13 6 2 1 0 0 2 1 0 0 1 0 0 0 0 1 0 1] handles = figure1: 153.0455 pushbutton7: 7.0457 pushbutton6: 6.0457 listbox1: 5.0457 pushbutton5: 4.0457 pushbutton4: 3.0457 pushbutton3: 2.0457 pushbutton2: 1.0457 pushbutton1: 0.0457 togglebutton1: 159.0455 axes3: 154.0455 output: 153.0455 f ilename: 'W1_20x_D1.tif' root_file: 'C:\Program Files\MATLAB71\work\images\10-05-06\' banner: [1024x1360x3 uint8] Cal: 0.2180 myX: [1 1360] myY: [1 1024] sub_A: [571x571x3 uint8] re ct_userD: [10.0968 248.8353 570.2515 570.2515] sub_B: [571x571 uint8] sub_BB: [571x571 uint8] sub_C: [571x571 logical] labeled: [571x571 double] numObjects: 76

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166Appendix D. (Continued) RGB_label: [ 571x571x3 uint8] numObjects2: 42 red_set: [5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 6 4 11 14] ful_ set: [5 0 0 0 0 1 13 29 14 7 4 1 0 0 0 0 0 0 0 2] fatcell: [1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 2 2 1 1 2] A2: [1024x1360x3 uint8] su b_A2: [571x571x3 uint8] RGB_labe l2: [571x571x3 uint8] nio_ful_set: [1630 41 13 6 2 1 0 0 2 1 0 0 1 0 0 0 0 1 0 1] nio_red_set: [1630 41 13 6 2 1 0 0 2 1 0 0 1 0 0 0 0 1 0 1] final pic: [571x571x3 uint8] cellTotalCount: 80 handles = figure1: 153.0455 pushbutton7: 7.0457 pushbutton6: 6.0457 listbox1: 5.0457 pushbutton5: 4.0457 pushbutton4: 3.0457 pushbutton3: 2.0457 pushbutton2: 1.0457 pushbutton1: 0.0457 togglebutton1: 159.0455 axes3: 154.0455 output: 153.0455 f ilename: 'W1_20x_D1.tif' root_file: 'C:\Program Files\MATLAB71\work\images\10-05-06\' banner: [1024x1360x3 uint8] Cal: 0.2180 myX: [1 1360] myY: [1 1024] sub_A: [571x571x3 uint8] re ct_userD: [10.0968 248.8353 570.2515 570.2515] sub_B: [571x571 uint8] sub_BB: [571x571 uint8] sub_C: [571x571 logical] labeled: [571x571 double] numObjects: 76 RGB_l abel: [571x571x3 uint8] numObjects2: 42 red_set: [5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 6 4 11 14] ful_ set: [5 0 0 0 0 1 13 29 14 7 4 1 0 0 0 0 0 0 0 2]

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167Appendix D. (Continued) fatcell: [1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 2 2 1 1 2] A2: [1024x1360x3 uint8] su b_A2: [571x571x3 uint8] RGB_labe l2: [571x571x3 uint8] nio_ful_set: [1630 41 13 6 2 1 0 0 2 1 0 0 1 0 0 0 0 1 0 1] nio_red_set: [1630 41 13 6 2 1 0 0 2 1 0 0 1 0 0 0 0 1 0 1] final pic: [571x571x3 uint8] cellTotalCount: 80 totalArea: 7.0877e+004 handles = figure1: 153.0455 pushbutton7: 7.0457 pushbutton6: 6.0457 listbox1: 5.0457 pushbutton5: 4.0457 pushbutton4: 3.0457 pushbutton3: 2.0457 pushbutton2: 1.0457 pushbutton1: 0.0457 togglebutton1: 159.0455 axes3: 154.0455 output: 153.0455 f ilename: 'W1_20x_D1.tif' root_file: 'C:\Program Files\MATLAB71\work\images\10-05-06\' banner: [1024x1360x3 uint8] Cal: 0.2180 myX: [1 1360] myY: [1 1024] sub_A: [571x571x3 uint8] re ct_userD: [10.0968 248.8353 570.2515 570.2515] sub_B: [571x571 uint8] sub_BB: [571x571 uint8] sub_C: [571x571 logical] labeled: [571x571 double] numObjects: 76 RGB_l abel: [571x571x3 uint8] numObjects2: 42 red_set: [5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 6 4 11 14] ful_ set: [5 0 0 0 0 1 13 29 14 7 4 1 0 0 0 0 0 0 0 2] fatcell: [1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 2 2 1 1 2] A2: [1024x1360x3 uint8] su b_A2: [571x571x3 uint8]

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168Appendix D. (Continued) RGB_label2: [571x571x3 uint8] nio_ful_set: [1630 41 13 6 2 1 0 0 2 1 0 0 1 0 0 0 0 1 0 1] nio_red_set: [1630 41 13 6 2 1 0 0 2 1 0 0 1 0 0 0 0 1 0 1] final pic: [571x571x3 uint8] cellTotalCount: 80 totalArea: 7.0877e+004 cell_density: 0.0011 handles = figure1: 153.0455 pushbutton7: 7.0457 pushbutton6: 6.0457 listbox1: 5.0457 pushbutton5: 4.0457 pushbutton4: 3.0457 pushbutton3: 2.0457 pushbutton2: 1.0457 pushbutton1: 0.0457 togglebutton1: 159.0455 axes3: 154.0455 output: 153.0455 f ilename: 'W1_20x_D1.tif' root_file: 'C:\Program Files\MATLAB71\work\images\10-05-06\' banner: [1024x1360x3 uint8] Cal: 0.2180 myX: [1 1360] myY: [1 1024] sub_A: [571x571x3 uint8] re ct_userD: [10.0968 248.8353 570.2515 570.2515] sub_B: [571x571 uint8] sub_BB: [571x571 uint8] sub_C: [571x571 logical] labeled: [571x571 double] numObjects: 76 RGB_l abel: [571x571x3 uint8] numObjects2: 42 red_set: [5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 6 4 11 14] ful_ set: [5 0 0 0 0 1 13 29 14 7 4 1 0 0 0 0 0 0 0 2] fatcell: [1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 2 2 1 1 2] A2: [1024x1360x3 uint8] su b_A2: [571x571x3 uint8] RGB_labe l2: [571x571x3 uint8]

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169Appendix D. (Continued) nio_ful_set: [1630 41 13 6 2 1 0 0 2 1 0 0 1 0 0 0 0 1 0 1] nio_red_set: [1630 41 13 6 2 1 0 0 2 1 0 0 1 0 0 0 0 1 0 1] final pic: [571x571x3 uint8] cellTotalCount: 80 totalArea: 7.0877e+004 cell_density: 0.0011 nioTotalCount: 1699 handles = figure1: 153.0455 pushbutton7: 7.0457 pushbutton6: 6.0457 listbox1: 5.0457 pushbutton5: 4.0457 pushbutton4: 3.0457 pushbutton3: 2.0457 pushbutton2: 1.0457 pushbutton1: 0.0457 togglebutton1: 159.0455 axes3: 154.0455 output: 153.0455 f ilename: 'W1_20x_D1.tif' root_file: 'C:\Program Files\MATLAB71\work\images\10-05-06\' banner: [1024x1360x3 uint8] Cal: 0.2180 myX: [1 1360] myY: [1 1024] sub_A: [571x571x3 uint8] re ct_userD: [10.0968 248.8353 570.2515 570.2515] sub_B: [571x571 uint8] sub_BB: [571x571 uint8] sub_C: [571x571 logical] labeled: [571x571 double] numObjects: 76 RGB_l abel: [571x571x3 uint8] numObjects2: 42 red_set: [5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 6 4 11 14] ful_ set: [5 0 0 0 0 1 13 29 14 7 4 1 0 0 0 0 0 0 0 2] fatcell: [1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 2 2 1 1 2] A2: [1024x1360x3 uint8] su b_A2: [571x571x3 uint8] RGB_labe l2: [571x571x3 uint8] nio_ful_set: [1630 41 13 6 2 1 0 0 2 1 0 0 1 0 0 0 0 1 0 1]

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170Appendix D. (Continued) nio_red_set: [1630 41 13 6 2 1 0 0 2 1 0 0 1 0 0 0 0 1 0 1] final pic: [571x571x3 uint8] cellTotalCount: 80 totalArea: 7.0877e+004 cell_density: 0.0011 nioTotalCount: 1699

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171Appendix E. Confocal Image Processing Data The following is a text file generated by the Leica Confocal Software which details the operating parameters and the st acking instructions for combining the fluorescent images. Leica Microsystems Heidelberg GmbH This file is intended for read-only pur poses changes here will not affect the images. Date: Tuesday, June 19, 2007 Time: 16:05 File Version: 26000000 EXPERIMENT INFORMATION Number of Images: 3 Type: Se ries with 'tif'-files DIMENSION DESCRIPTION #0 Pixel Size in Byte: 1 Resolution in Bit: 8 Max Value: 255.0000000000 Min Value: 0.000000e+000 Label: I Number of Dimensions: 4 Dimension_0: 120 Logical Size: 512 Physical Length: 1.365967e-004 m Physical Origin: 0.000000e+000 m Dimension_1: 121 Logical Size: 512 Physical Length: 1.365967e-004 m Physical Origin: 0.000000e+000 m Dimension_2: 6815843 Logical Size: 2 Physical Length: 0.000000e+000 Physical Origin: 0.000000e+000 Dimension_3: 122 Logical Size: 1 Physical Length: 0.000000e+000 Physical Origin: 0.000000e+000 Series Name: Series038

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172Appendix E. (Continued) Description: HARDWARE PARAMETER #0 AOTF (458) 49.328449 AOTF (476) 0.000000 AOTF (488) 50.671551 AOTF (514) 0.000000 AOTF (561) 50.012210 AOTF (633) 0.000000 AOTF (458) 0.000000 AOTF (476) 0.000000 AOTF (488) 0.000000 AOTF (514) 0.000000 AOTF (561) 0.000000 AOTF (633) 0.000000 PMT 1 Active Active PMT 1 (Offs.) -21.600000 PMT 1 (HV) 631.887456 PMT 2 Active Active PMT 2 (Offs.) -13.600000 PMT 2 (HV) 616.060961 PMT 3 Inactive Inactive PMT Trans Inactive Inactive Beam Expander Beam Exp 6 Beam Exp 6 Excitation Beam Splitter FW DD 488/568 DD 488/568 External Detection FW Mirror Mirror Hardware Type No. 2.000000 Scan Field Rotation -0.038943 Rotation Direction 1 X Scan Actuator Active Active X Scan Actuator (Gain) 2.745308 X Scan Actuator (Offs.) 0.000000 Y Scan Actuator Active Active Y Scan Actuator (Gain) 2.745308 Y Scan Actuator (Offs.) 0.000000 Z Scan Actuator Inactive Inactive Z Scan Actuator (POS) -0.000048 Scan Speed 200.000000 Phase 10.546875 Y-Phase 0.122100 SP Mirror 1 (left) 500.000000 SP Mirror 1 (right) 550.000000 SP Mirror 1 (stain) FITC FITC SP Mirror 2 (left) 570.000000

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173Appendix E. (Continued) SP Mirror 2 (right) 700.000000 SP Mirror 2 (stain) TRITC TRITC SP Mirror 3 (left) 750.000000 SP Mirror 3 (right) 850.000000 SP Mirror 3 (stain) None None Objective HCX PL APO CS 40.0x 1.25 OIL UV HCX PL APO CS 40.0x1.25 OIL UV Order number (Obj.) 506179 Numerical aperture (Obj.) 1.250000 SCANNER INFORMATION #0 RoiScan 0 IsSequential 0 ChaserUVShutter 0 ChaserVisibleShutter 0 MPShutter 0 UVShutter 0 VisibleShutter 1 ScanMode xyz Active Pinhole [m] 0.000081 Pinhole [airy] 0.998666 Size-Width [m] 136.596680 Size-Height [m] 136.596680 Size-Depth 0.000000 StepSize [m] 0.040703 Voxel-Width [m] 0.266790 Voxel-Height [m] 0.266790 Voxel-Depth 0.000000 Zoom 2.745308 Scan-Direction 1 Y-Scan-Direction 1 SequentialMode 0 Frame-Accumulation 1 Frame-Average 1 Line-Average 1 Resolution 8 Channels 2 Format-Width 512 Format-Height 512 Sections 1 TIME INFORMATION #0 Stamped Dimension: 2 Stamp_0: 2007: 06:19,15:38:12:546

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174Appendix E. (Continued) Stamp_1: 2007: 06:19,15:38:12:546 LUT DESCRIPTION #0 LUT_0 Name: Green Inverted (1=yes / 0=no): 0 LUT_1 Name: Red Inverted (1=yes / 0=no): 0 SEQUENTIAL INFORMATION #0 Sequence Count: 0 SERIES INFORMATION #0 Number of Series: 3 IMAGES INFORMATION #0 Number of Images: 2 Image Width: 512 Iamge Length: 512 Bits per Sample: 8 Samples per Pixel: 1 ********************************** ***** NEXT IMAGE ********************* ************ DIMENSION DESCRIPTION #1 Pixel Size in Byte: 1 Resolution in Bit: 8 Max Value: 255.0000000000 Min Value: 0.000000e+000 Label: I Number of Dimensions: 4 Dimension_0: 120 Logical Size: 512 Physical Length: 1.365967e-004 m Physical Origin: 0.000000e+000 m Dimension_1: 121 Logical Size: 512 Physical Length: 1.365967e-004 m Physical Origin: 0.000000e+000 m Dimension_2: 6815843 Logical Size: 2 Physical Length: 0.000000e+000 Physical Origin: 0.000000e+000 Dimension_3: 122

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175Appendix E. (Continued) Logical Size: 3 Physical Length: -3.256268e-007 m Physical Origin: -3.661267e-005 m Series Name: Series045 Description: HARDWARE PARAMETER #1 AOTF (458) 49.328449 AOTF (476) 0.000000 AOTF (488) 50.671551 AOTF (514) 0.000000 AOTF (561) 50.012210 AOTF (633) 0.000000 AOTF (458) 0.000000 AOTF (476) 0.000000 AOTF (488) 0.000000 AOTF (514) 0.000000 AOTF (561) 0.000000 AOTF (633) 0.000000 PMT 1 Active Active PMT 1 (Offs.) -21.600000 PMT 1 (HV) 631.887456 PMT 2 Active Active PMT 2 (Offs.) -13.600000 PMT 2 (HV) 616.060961 PMT 3 Inactive Inactive PMT Trans Inactive Inactive Beam Expander Beam Exp 6 Beam Exp 6 Excitation Beam Splitter FW DD 488/568 DD 488/568 External Detection FW Mirror Mirror Hardware Type No. 2.000000 Scan Field Rotation -0.038943 Rotation Direction 1 X Scan Actuator Active Active X Scan Actuator (Gain) 2.745308 X Scan Actuator (Offs.) 0.000000 Y Scan Actuator Active Active Y Scan Actuator (Gain) 2.745308 Y Scan Actuator (Offs.) 0.000000 Z Scan Actuator Inactive Inactive Z Scan Actuator (POS) -0.000043 Scan Speed 200.000000 Phase 10.546875 Y-Phase 0.122100

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176Appendix E. (Continued) SP Mirror 1 (left) 500.000000 SP Mirror 1 (right) 550.000000 SP Mirror 1 (stain) FITC FITC SP Mirror 2 (left) 570.000000 SP Mirror 2 (right) 700.000000 SP Mirror 2 (stain) TRITC TRITC SP Mirror 3 (left) 750.000000 SP Mirror 3 (right) 850.000000 SP Mirror 3 (stain) None None Objective HCX PL APO CS 40.0x 1.25 OIL UV HCX PL APO CS 40.0x1.25 OIL UV Order number (Obj.) 506179 Numerical aperture (Obj.) 1.250000 SCANNER INFORMATION #1 RoiScan 0 IsSequential 0 ChaserUVShutter 0 ChaserVisibleShutter 0 MPShutter 0 UVShutter 0 VisibleShutter 1 ScanMode xyz Active Pinhole [m] 0.000081 Pinhole [airy] 0.998666 Size-Width [m] 136.596680 Size-Height [m] 136.596680 Size-Depth [m] 0.325627 StepSize [m] 0.162813 Voxel-Width [m] 0.266790 Voxel-Height [m] 0.266790 Voxel-Depth [m] 0.162813 Zoom 2.745308 Scan-Direction 1 Y-Scan-Direction 1 SequentialMode 0 Frame-Accumulation 1 Frame-Average 1 Line-Average 1 Resolution 8 Channels 2 Format-Width 512 Format-Height 512 Sections 84

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177Appendix E. (Continued) TIME INFORMATION #1 Stamped Dimension: 2 Stamp_0: 2007: 06:19,15:45:23:593 Stamp_1: 2007: 06:19,15:45:23:593 Stamp_2: 2007: 06:19,15:45:26:843 Stamp_3: 2007: 06:19,15:45:26:843 Stamp_4: 2007: 06:19,15:45:30:93 Stamp_5: 2007: 06:19,15:45:30:93 LUT DESCRIPTION #1 LUT_0 Name: Green Inverted (1=yes / 0=no): 0 LUT_1 Name: Red Inverted (1=yes / 0=no): 0 SEQUENTIAL INFORMATION #1 Sequence Count: 0 IMAGES INFORMATION #1 Number of Images: 6 Image Width: 512 Iamge Length: 512 Bits per Sample: 8 Samples per Pixel: 1 ********************************** ***** NEXT IMAGE ********************* ************ DIMENSION DESCRIPTION #2 Pixel Size in Byte: 1 Resolution in Bit: 8 Max Value: 255.0000000000 Min Value: 0.000000e+000 Label: I Number of Dimensions: 4 Dimension_0: 120 Logical Size: 512 Physical Length: 1.372375e-004 m Physical Origin: 0.000000e+000 m Dimension_1: 121 Logical Size: 512 Physical Length: 1.372375e-004 m Physical Origin: 0.000000e+000 m Dimension_2: 6815843

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178Appendix E. (Continued) Logical Size: 2 Physical Length: 0.000000e+000 Physical Origin: 0.000000e+000 Dimension_3: 122 Logical Size: 125 Physical Length: -2.018886e-005 m Physical Origin: -3.815939e-005 m Series Name: Series049 Description: HARDWARE PARAMETER #2 AOTF (458) 49.328449 AOTF (476) 0.000000 AOTF (488) 50.671551 AOTF (514) 0.000000 AOTF (561) 50.012210 AOTF (633) 0.000000 AOTF (458) 0.000000 AOTF (476) 0.000000 AOTF (488) 0.000000 AOTF (514) 0.000000 AOTF (561) 0.000000 AOTF (633) 0.000000 PMT 1 Active Active PMT 1 (Offs.) -21.600000 PMT 1 (HV) 631.887456 PMT 2 Active Active PMT 2 (Offs.) -13.600000 PMT 2 (HV) 616.060961 PMT 3 Inactive Inactive PMT Trans Inactive Inactive Beam Expander Beam Exp 6 Beam Exp 6 Excitation Beam Splitter FW DD 488/568 DD 488/568 External Detection FW Mirror Mirror Hardware Type No. 2.000000 Scan Field Rotation -0.038943 Rotation Direction 1 X Scan Actuator Active Active X Scan Actuator (Gain) 2.732488 X Scan Actuator (Offs.) 0.000000 Y Scan Actuator Active Active Y Scan Actuator (Gain) 2.732488 Y Scan Actuator (Offs.) 0.000000 Z Scan Actuator Inactive Inactive

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179Appendix E. (Continued) Z Scan Actuator (POS) -0.000048 Scan Speed 200.000000 Phase 10.546875 Y-Phase 0.122100 SP Mirror 1 (left) 500.000000 SP Mirror 1 (right) 550.000000 SP Mirror 1 (stain) FITC FITC SP Mirror 2 (left) 570.000000 SP Mirror 2 (right) 700.000000 SP Mirror 2 (stain) TRITC TRITC SP Mirror 3 (left) 750.000000 SP Mirror 3 (right) 850.000000 SP Mirror 3 (stain) None None Objective HCX PL APO CS 40.0x 1.25 OIL UV HCX PL APO CS 40.0x1.25 OIL UV Order number (Obj.) 506179 Numerical aperture (Obj.) 1.250000 SCANNER INFORMATION #2 RoiScan 0 IsSequential 0 ChaserUVShutter 0 ChaserVisibleShutter 0 MPShutter 0 UVShutter 0 VisibleShutter 1 ScanMode xyz Active Pinhole [m] 0.000081 Pinhole [airy] 0.998666 Size-Width [m] 137.237549 Size-Height [m] 137.237549 Size-Depth [m] -20.188864 StepSize [m] 0.162813 Voxel-Width [m] 0.268042 Voxel-Height [m] 0.268042 Voxel-Depth [m] 0.162813 Zoom 2.732488 Scan-Direction 1 Y-Scan-Direction 1 SequentialMode 0 Frame-Accumulation 1 Frame-Average 1 Line-Average 1 Resolution 8

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180Appendix E. (Continued) Channels 2 Format-Width 512 Format-Height 512 Sections 125 TIME INFORMATION #2 Stamped Dimension: 2 Stamp_0: 2007: 06:19,15:54:21:453 Stamp_1: 2007: 06:19,15:54:21:453 Stamp_2: 2007: 06:19,15:54:24:703 Stamp_3: 2007: 06:19,15:54:24:703 Stamp_4: 2007: 06:19,15:54:27:953 Stamp_5: 2007: 06:19,15:54:27:953 Stamp_6: 2007: 06:19,15:54:31:203 Stamp_7: 2007: 06:19,15:54:31:203 Stamp_8: 2007: 06:19,15:54:34:437 Stamp_9: 2007: 06:19,15:54:34:437 Stamp_10: 2007: 06:19,15:54:37:687 Stamp_11: 2007: 06:19,15:54:37:687 Stamp_12: 2007: 06:19,15:54:40:937 Stamp_13: 2007: 06:19,15:54:40:937 Stamp_14: 2007: 06:19,15:54:44:187 Stamp_15: 2007: 06:19,15:54:44:187 Stamp_16: 2007: 06:19,15:54:47:437 Stamp_17: 2007: 06:19,15:54:47:437 Stamp_18: 2007: 06:19,15:54:50:687 Stamp_19: 2007: 06:19,15:54:50:687 Stamp_20: 2007: 06:19,15:54:53:937 Stamp_21: 2007: 06:19,15:54:53:937 Stamp_22: 2007: 06:19,15:54:57:187 Stamp_23: 2007: 06:19,15:54:57:187 Stamp_24: 2007: 06:19,15:55:00:437 Stamp_25: 2007: 06:19,15:55:00:437 Stamp_26: 2007: 06:19,15:55:03:687 Stamp_27: 2007: 06:19,15:55:03:687 Stamp_28: 2007: 06:19,15:55:06:937 Stamp_29: 2007: 06:19,15:55:06:937 Stamp_30: 2007: 06:19,15:55:10:187 Stamp_31: 2007: 06:19,15:55:10:187 Stamp_32: 2007: 06:19,15:55:13:437 Stamp_33: 2007: 06:19,15:55:13:437 Stamp_34: 2007: 06:19,15:55:16:687 Stamp_35: 2007: 06:19,15:55:16:687 Stamp_36: 2007: 06:19,15:55:19:937

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181Appendix E. (Continued) Stamp_37: 2007: 06:19,15:55:19:937 Stamp_38: 2007: 06:19,15:55:23:187 Stamp_39: 2007: 06:19,15:55:23:187 Stamp_40: 2007: 06:19,15:55:26:437 Stamp_41: 2007: 06:19,15:55:26:437 Stamp_42: 2007: 06:19,15:55:29:687 Stamp_43: 2007: 06:19,15:55:29:687 Stamp_44: 2007: 06:19,15:55:32:937 Stamp_45: 2007: 06:19,15:55:32:937 Stamp_46: 2007: 06:19,15:55:36:187 Stamp_47: 2007: 06:19,15:55:36:187 Stamp_48: 2007: 06:19,15:55:39:437 Stamp_49: 2007: 06:19,15:55:39:437 Stamp_50: 2007: 06:19,15:55:42:687 Stamp_51: 2007: 06:19,15:55:42:687 Stamp_52: 2007: 06:19,15:55:45:937 Stamp_53: 2007: 06:19,15:55:45:937 Stamp_54: 2007: 06:19,15:55:49:187 Stamp_55: 2007: 06:19,15:55:49:187 Stamp_56: 2007: 06:19,15:55:52:437 Stamp_57: 2007: 06:19,15:55:52:437 Stamp_58: 2007: 06:19,15:55:55:687 Stamp_59: 2007: 06:19,15:55:55:687 Stamp_60: 2007: 06:19,15:55:58:937 Stamp_61: 2007: 06:19,15:55:58:937 Stamp_62: 2007: 06:19,15:56:02:187 Stamp_63: 2007: 06:19,15:56:02:187 Stamp_64: 2007: 06:19,15:56:05:437 Stamp_65: 2007: 06:19,15:56:05:437 Stamp_66: 2007: 06:19,15:56:08:687 Stamp_67: 2007: 06:19,15:56:08:687 Stamp_68: 2007: 06:19,15:56:11:937 Stamp_69: 2007: 06:19,15:56:11:937 Stamp_70: 2007: 06:19,15:56:15:187 Stamp_71: 2007: 06:19,15:56:15:187 Stamp_72: 2007: 06:19,15:56:18:437 Stamp_73: 2007: 06:19,15:56:18:437 Stamp_74: 2007: 06:19,15:56:21:687 Stamp_75: 2007: 06:19,15:56:21:687 Stamp_76: 2007: 06:19,15:56:24:937 Stamp_77: 2007: 06:19,15:56:24:937 Stamp_78: 2007: 06:19,15:56:28:187 Stamp_79: 2007: 06:19,15:56:28:187 Stamp_80: 2007: 06:19,15:56:31:437

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182Appendix E. (Continued) Stamp_81: 2007: 06:19,15:56:31:437 Stamp_82: 2007: 06:19,15:56:34:687 Stamp_83: 2007: 06:19,15:56:34:687 Stamp_84: 2007: 06:19,15:56:37:937 Stamp_85: 2007: 06:19,15:56:37:937 Stamp_86: 2007: 06:19,15:56:41:187 Stamp_87: 2007: 06:19,15:56:41:187 Stamp_88: 2007: 06:19,15:56:44:437 Stamp_89: 2007: 06:19,15:56:44:437 Stamp_90: 2007: 06:19,15:56:47:687 Stamp_91: 2007: 06:19,15:56:47:687 Stamp_92: 2007: 06:19,15:56:50:937 Stamp_93: 2007: 06:19,15:56:50:937 Stamp_94: 2007: 06:19,15:56:54:187 Stamp_95: 2007: 06:19,15:56:54:187 Stamp_96: 2007: 06:19,15:56:57:437 Stamp_97: 2007: 06:19,15:56:57:437 Stamp_98: 2007: 06:19,15:57:00:687 Stamp_99: 2007: 06:19,15:57:00:687 Stamp_100: 2007: 06:19,15:57:03:937 Stamp_101: 2007: 06:19,15:57:03:937 Stamp_102: 2007: 06:19,15:57:07:187 Stamp_103: 2007: 06:19,15:57:07:187 Stamp_104: 2007: 06:19,15:57:10:437 Stamp_105: 2007: 06:19,15:57:10:437 Stamp_106: 2007: 06:19,15:57:13:687 Stamp_107: 2007: 06:19,15:57:13:687 Stamp_108: 2007: 06:19,15:57:16:937 Stamp_109: 2007: 06:19,15:57:16:937 Stamp_110: 2007: 06:19,15:57:20:187 Stamp_111: 2007: 06:19,15:57:20:187 Stamp_112: 2007: 06:19,15:57:23:437 Stamp_113: 2007: 06:19,15:57:23:437 Stamp_114: 2007: 06:19,15:57:26:687 Stamp_115: 2007: 06:19,15:57:26:687 Stamp_116: 2007: 06:19,15:57:29:937 Stamp_117: 2007: 06:19,15:57:29:937 Stamp_118: 2007: 06:19,15:57:33:187 Stamp_119: 2007: 06:19,15:57:33:187 Stamp_120: 2007: 06:19,15:57:36:437 Stamp_121: 2007: 06:19,15:57:36:437 Stamp_122: 2007: 06:19,15:57:39:687 Stamp_123: 2007: 06:19,15:57:39:687 Stamp_124: 2007: 06:19,15:57:42:937

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183Appendix E. (Continued) Stamp_125: 2007: 06:19,15:57:42:937 Stamp_126: 2007: 06:19,15:57:46:187 Stamp_127: 2007: 06:19,15:57:46:187 Stamp_128: 2007: 06:19,15:57:49:437 Stamp_129: 2007: 06:19,15:57:49:437 Stamp_130: 2007: 06:19,15:57:52:687 Stamp_131: 2007: 06:19,15:57:52:687 Stamp_132: 2007: 06:19,15:57:55:921 Stamp_133: 2007: 06:19,15:57:55:921 Stamp_134: 2007: 06:19,15:57:59:171 Stamp_135: 2007: 06:19,15:57:59:171 Stamp_136: 2007: 06:19,15:58:02:421 Stamp_137: 2007: 06:19,15:58:02:421 Stamp_138: 2007: 06:19,15:58:05:671 Stamp_139: 2007: 06:19,15:58:05:671 Stamp_140: 2007: 06:19,15:58:08:921 Stamp_141: 2007: 06:19,15:58:08:921 Stamp_142: 2007: 06:19,15:58:12:171 Stamp_143: 2007: 06:19,15:58:12:171 Stamp_144: 2007: 06:19,15:58:15:421 Stamp_145: 2007: 06:19,15:58:15:421 Stamp_146: 2007: 06:19,15:58:18:671 Stamp_147: 2007: 06:19,15:58:18:671 Stamp_148: 2007: 06:19,15:58:21:921 Stamp_149: 2007: 06:19,15:58:21:921 Stamp_150: 2007: 06:19,15:58:25:171 Stamp_151: 2007: 06:19,15:58:25:171 Stamp_152: 2007: 06:19,15:58:28:421 Stamp_153: 2007: 06:19,15:58:28:421 Stamp_154: 2007: 06:19,15:58:31:671 Stamp_155: 2007: 06:19,15:58:31:671 Stamp_156: 2007: 06:19,15:58:34:921 Stamp_157: 2007: 06:19,15:58:34:921 Stamp_158: 2007: 06:19,15:58:38:171 Stamp_159: 2007: 06:19,15:58:38:171 Stamp_160: 2007: 06:19,15:58:41:421 Stamp_161: 2007: 06:19,15:58:41:421 Stamp_162: 2007: 06:19,15:58:44:671 Stamp_163: 2007: 06:19,15:58:44:671 Stamp_164: 2007: 06:19,15:58:47:921 Stamp_165: 2007: 06:19,15:58:47:921 Stamp_166: 2007: 06:19,15:58:51:171 Stamp_167: 2007: 06:19,15:58:51:171 Stamp_168: 2007: 06:19,15:58:54:421

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184Appendix E. (Continued) Stamp_169: 2007: 06:19,15:58:54:421 Stamp_170: 2007: 06:19,15:58:57:671 Stamp_171: 2007: 06:19,15:58:57:671 Stamp_172: 2007: 06:19,15:59:00:921 Stamp_173: 2007: 06:19,15:59:00:921 Stamp_174: 2007: 06:19,15:59:04:171 Stamp_175: 2007: 06:19,15:59:04:171 Stamp_176: 2007: 06:19,15:59:07:421 Stamp_177: 2007: 06:19,15:59:07:421 Stamp_178: 2007: 06:19,15:59:10:671 Stamp_179: 2007: 06:19,15:59:10:671 Stamp_180: 2007: 06:19,15:59:13:921 Stamp_181: 2007: 06:19,15:59:13:921 Stamp_182: 2007: 06:19,15:59:17:171 Stamp_183: 2007: 06:19,15:59:17:171 Stamp_184: 2007: 06:19,15:59:20:421 Stamp_185: 2007: 06:19,15:59:20:421 Stamp_186: 2007: 06:19,15:59:23:671 Stamp_187: 2007: 06:19,15:59:23:671 Stamp_188: 2007: 06:19,15:59:26:921 Stamp_189: 2007: 06:19,15:59:26:921 Stamp_190: 2007: 06:19,15:59:30:171 Stamp_191: 2007: 06:19,15:59:30:171 Stamp_192: 2007: 06:19,15:59:33:421 Stamp_193: 2007: 06:19,15:59:33:421 Stamp_194: 2007: 06:19,15:59:36:671 Stamp_195: 2007: 06:19,15:59:36:671 Stamp_196: 2007: 06:19,15:59:39:921 Stamp_197: 2007: 06:19,15:59:39:921 Stamp_198: 2007: 06:19,15:59:43:171 Stamp_199: 2007: 06:19,15:59:43:171 Stamp_200: 2007: 06:19,15:59:46:421 Stamp_201: 2007: 06:19,15:59:46:421 Stamp_202: 2007: 06:19,15:59:49:671 Stamp_203: 2007: 06:19,15:59:49:671 Stamp_204: 2007: 06:19,15:59:52:921 Stamp_205: 2007: 06:19,15:59:52:921 Stamp_206: 2007: 06:19,15:59:56:171 Stamp_207: 2007: 06:19,15:59:56:171 Stamp_208: 2007: 06:19,15:59:59:421 Stamp_209: 2007: 06:19,15:59:59:421 Stamp_210: 2007: 06:19,16:00:02:671 Stamp_211: 2007: 06:19,16:00:02:671 Stamp_212: 2007: 06:19,16:00:05:921

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185Appendix E. (Continued) Stamp_213: 2007: 06:19,16:00:05:921 Stamp_214: 2007: 06:19,16:00:09:171 Stamp_215: 2007: 06:19,16:00:09:171 Stamp_216: 2007: 06:19,16:00:12:421 Stamp_217: 2007: 06:19,16:00:12:421 Stamp_218: 2007: 06:19,16:00:15:671 Stamp_219: 2007: 06:19,16:00:15:671 Stamp_220: 2007: 06:19,16:00:18:921 Stamp_221: 2007: 06:19,16:00:18:921 Stamp_222: 2007: 06:19,16:00:22:171 Stamp_223: 2007: 06:19,16:00:22:171 Stamp_224: 2007: 06:19,16:00:25:421 Stamp_225: 2007: 06:19,16:00:25:421 Stamp_226: 2007: 06:19,16:00:28:671 Stamp_227: 2007: 06:19,16:00:28:671 Stamp_228: 2007: 06:19,16:00:31:921 Stamp_229: 2007: 06:19,16:00:31:921 Stamp_230: 2007: 06:19,16:00:35:171 Stamp_231: 2007: 06:19,16:00:35:171 Stamp_232: 2007: 06:19,16:00:38:421 Stamp_233: 2007: 06:19,16:00:38:421 Stamp_234: 2007: 06:19,16:00:41:671 Stamp_235: 2007: 06:19,16:00:41:671 Stamp_236: 2007: 06:19,16:00:44:921 Stamp_237: 2007: 06:19,16:00:44:921 Stamp_238: 2007: 06:19,16:00:48:171 Stamp_239: 2007: 06:19,16:00:48:171 Stamp_240: 2007: 06:19,16:00:51:421 Stamp_241: 2007: 06:19,16:00:51:421 Stamp_242: 2007: 06:19,16:00:54:671 Stamp_243: 2007: 06:19,16:00:54:671 Stamp_244: 2007: 06:19,16:00:57:921 Stamp_245: 2007: 06:19,16:00:57:921 Stamp_246: 2007: 06:19,16:01:01:171 Stamp_247: 2007: 06:19,16:01:01:171 Stamp_248: 2007: 06:19,16:01:04:421 Stamp_249: 2007: 06:19,16:01:04:421 LUT DESCRIPTION #2 LUT_0 Name: Green Inverted (1=yes / 0=no): 0 LUT_1 Name: Red

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186Appendix E. (Continued) Inverted (1=yes / 0=no): 0 SEQUENTIAL INFORMATION #2 Sequence Count: 0 IMAGES INFORMATION #2 Number of Images: 250 Image Width: 512 Iamge Length: 512 Bits per Sample: 8 Samples per Pixel: 1

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About the Author Elizabeth Hood received a B.S. in Chemical Engineering, magna cum laude, from USF in 2000. In 2002 she returned to USF to pursue a PhD in Biomedical Engineerin g and has presented and publis hed proceedings and abstracts at several national and inter national meetings in cluding AIChE 2003 and 2006 Annual Meetings, American Soci ety of Echocardiography’s 15th Annual Scientific Sessions 2004, and t he 26th Annual Inter national Conference of the IEEE Engineerin g in Medicine and Biology Society 2004. Also, Drug Delivery and Translational Research Symposium in 2006 at the Polytechnic University in Brooklyn, NY, the presti gious American College of Cardiology Annual Scientific Session in New Or leans in 2007, and several USF symposia. She is a contributor in three pat ent disclosure applications, and the primary inventor in one. Elizabeth rece ived the Peter Brown Fellowship 20042005, the Central Florida Arthritis Fellowship Summer 2005, and a Southern Section-American Federation for Medica l Research grants in both 2006 and 2007. Elizabeth received an award for re cognition of excellence at the USF Health Research Day 2006. Her first peer-reviewed journal article entitled ‘Immuno-targeting of Noni onic Surfactant Vesicles to Inflammation’ was published in 2007 in the International Journal of Pharmaceutics. After graduating as one of the first to receive a Ph.D. in Biomedical Engineering from USF Elizabet h will be pursuing a National Institute of Health National Research Service Award post docto ral fellowship at the University of Pennsylvania’s Institute for Environmental Medicine.