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Pegylation of niosomes

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Title:
Pegylation of niosomes
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English
Creator:
Elliott, John
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University of South Florida
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Tampa, Fla
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Subjects / Keywords:
Targeted drug delivery
Polyethylene glycol
Nanoparticle
Liposome
Inflammation
Dissertations, Academic -- Chemical Engineering -- Doctoral -- USF   ( lcsh )
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non-fiction   ( marcgt )

Notes

Abstract:
ABSTRACT: The research presented in this dissertation describes the creation and characterization of a novel antibody-vesicle conjugate modified with polyethylene glycol (PEG) that possesses enhanced binding to and uptake by inflammation-activated endothelial cells with improved storage stability and longer shelf-life and potential reduction in immunogenic potential compared to previous designs. Targeted drug delivery provides an effective means of delivering therapeutic concentrations of a drug to the site or organ of action. The drug is delivered using a niosome, a vesicle with an aqueous core and a bilayer membrane composed of non-ionic surfactants and cholesterol. Antibodies that recognize specific cell antigens are attached to the niosome to complete the targeting molecule, an immuno-niosome (IN). When functionalized PEG, a water soluble, biologically inert polymer, is attached to proteins, it can protect the protein, increasing its half-life in vivo. The immuno-niosome synthesis process is modified to include PEG incorporation into the niosome membrane, a process known as PEGylation. Since the vasculature connects the entire body, immuno-niosomes targeted to endothelial cells were used. When endothelial cells are activated during disease, stress and injury, certain receptors are expressed and upregulated. One such receptor, CD44, is upregulated in response to vascular inflammation associated with atherosclerosis. The research hypothesis is that the addition of polyethylene glycol to the drug delivery vesicle (immuno-niosome) using cyanuric chloride linking chemistry will improve colloidal stability and binding performance of the PEGylated immuno-niosomes to endothelial cell surface receptors expressed during an inflammatory response. The research presented in this dissertation provides the following evidence to support this hypothesis: -Construction and characterization of a modified drug delivery vesicle using non-ionic surfactants conjugated with PEG and functionalized with antibodies against endothelial cell surface receptors (PEGylated immuno-niosomes) improves the colloidal stability over previously designed vesicles. -Binding of PEGylated CD44-IN to activated endothelial cells is improved over previously designed vesicles. -Binding of PEGylated CD44-IN to activated endothelial cells under physiological conditions (flow) is demonstrated. -Uptake of PEGylated immuno-niosomes by activated or injured endothelial cells is demonstrated using confocal microscopy.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2010.
Bibliography:
Includes bibliographical references.
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by John Elliott.
General Note:
Title from PDF of title page.
General Note:
Document formatted into pages; contains X pages.
General Note:
Includes vita.

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ABSTRACT: The research presented in this dissertation describes the creation and characterization of a novel antibody-vesicle conjugate modified with polyethylene glycol (PEG) that possesses enhanced binding to and uptake by inflammation-activated endothelial cells with improved storage stability and longer shelf-life and potential reduction in immunogenic potential compared to previous designs. Targeted drug delivery provides an effective means of delivering therapeutic concentrations of a drug to the site or organ of action. The drug is delivered using a niosome, a vesicle with an aqueous core and a bilayer membrane composed of non-ionic surfactants and cholesterol. Antibodies that recognize specific cell antigens are attached to the niosome to complete the targeting molecule, an immuno-niosome (IN). When functionalized PEG, a water soluble, biologically inert polymer, is attached to proteins, it can protect the protein, increasing its half-life in vivo. The immuno-niosome synthesis process is modified to include PEG incorporation into the niosome membrane, a process known as PEGylation. Since the vasculature connects the entire body, immuno-niosomes targeted to endothelial cells were used. When endothelial cells are activated during disease, stress and injury, certain receptors are expressed and upregulated. One such receptor, CD44, is upregulated in response to vascular inflammation associated with atherosclerosis. The research hypothesis is that the addition of polyethylene glycol to the drug delivery vesicle (immuno-niosome) using cyanuric chloride linking chemistry will improve colloidal stability and binding performance of the PEGylated immuno-niosomes to endothelial cell surface receptors expressed during an inflammatory response. The research presented in this dissertation provides the following evidence to support this hypothesis: -Construction and characterization of a modified drug delivery vesicle using non-ionic surfactants conjugated with PEG and functionalized with antibodies against endothelial cell surface receptors (PEGylated immuno-niosomes) improves the colloidal stability over previously designed vesicles. -Binding of PEGylated CD44-IN to activated endothelial cells is improved over previously designed vesicles. -Binding of PEGylated CD44-IN to activated endothelial cells under physiological conditions (flow) is demonstrated. -Uptake of PEGylated immuno-niosomes by activated or injured endothelial cells is demonstrated using confocal microscopy.
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PEGylation of Niosomes by John A. Elliott A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemical and Biomedical Engineering College of Engineering University of South Florida Co Major Professor: Michael VanA uker Ph.D. Co Major Professor: Mark Jaroszeski Ph.D. Joel Strom, M.D. Nathan Gallant, Ph.D. Ryan Toomey, Ph.D. Karl Muffly, Ph.D. John Wolan, Ph. D. Date of Approval: November 16 20 09 Keywords: targeted drug delivery, polyethylene glycol, nanoparticle, liposome, inflammation Copyright 2010 John Elliott

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Dedication I would like to dedicate this document to my wife Jill and my two children Tyler and Claire as well as all those who have inspired me in the past and those who I hope to encourage making a positive impact on the scientific community.

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Acknowledgments I would like to acknowledge Dr. Bre nda Flam and Dr. Elizabeth Hood for being great mentors and for all the encouragement and help I have received from them over the course of my research A special acknowledgement is in order for all of my committee member s and especially, Dr s VanAuker, Muffly and Strom for their guidance and support during this process. I would also like to acknowledg e the faculty and staff at USF H ealth for the use of their f acilities and all of the technical support they have provided

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i Table of Contents List of Tables ................................ ................................ ................................ .................... i ii List of Figures ................................ ................................ ................................ .................... iv Abstract ................................ ................................ ................................ ........................... viii 1 Project Background ................................ ................................ ................................ .. 1 1.1.0 Drug Delivery Introduction ................................ ................................ ......... 4 1.1.1 Basics of Pharmacokinetics ................................ ............................. 4 1.1.2 Compartment Models ................................ ................................ ....... 6 1. 2 0 Introduction to the Cardiovascular System ................................ .................. 8 1. 2 1 Blood ................................ ................................ ................................ 8 1. 2 2 Heart ................................ ................................ ............................... 11 1.2.3 Circulation ................................ ................................ ...................... 12 1.3.0 Cell Introduction ................................ ................................ ........................ 1 3 1.3.1 Endothelial Cells ................................ ................................ ............ 1 4 1.3.2 Endothelial Cell Mass Transport ................................ ................... 1 6 1.3.3 Endocytosis ................................ ................................ .................... 19 1.4.0 Cell Surface R eceptors ................................ ................................ ............... 20 1.4.1 Proteins ................................ ................................ .......................... 24 1.4.2 Antibodies ................................ ................................ ...................... 24 1.4.2.1 Immunoglobulin G ................................ ........................ 2 6 1.4.2.2 Monoclonal Antibodies ................................ ................. 2 8 1.4. 3 CD44 Receptor ................................ ................................ ............... 2 9 1.4.4 Intercellular Adhesion Molecule ( ICAM ) ................................ ..... 30 1.4.5 Vascular Cellular Adhesion Molecule (VCAM) ........................... 31 1.5.0 Cytokines and Inflammation ................................ ................................ ...... 32 1.5.1 Tumor Necrosis Factor Alpha ................................ ........................ 33 1.5.2 Interleukin 1 ................................ ................................ ................... 34 1.6.0 Cardiovascular Disease ................................ ................................ .............. 34 2. Construction and Characterization of Modified Vesicle Design ........................... 3 7 2.1.0 Amphiphiles ................................ ................................ ............................... 3 7 2.1.1 Non I onic Surfactants ................................ ................................ .... 3 9 2.1.2 Cholesterol ................................ ................................ ..................... 40 2.2.0 Polyethylene G lycol ................................ ................................ ................... 4 1 2.3.0 Molecule Synthesis ................................ ................................ .................... 42 2.3.1 Funct ionalized Non Ionic Surfactant ................................ ............. 43 2. 3.2 Functionalized Cholesterol ................................ ............................ 4 6 2.4.0 Vesicle Synthesis ................................ ................................ ....................... 4 7

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ii 2.4.1 Thin Film Formation and Hydration ................................ .............. 4 8 2.4.2 Vesicle Extrusion ................................ ................................ ........... 4 8 2.4.3 Size Exclusion Chromatography Introduction ................................ ................................ .................... 4 9 2.4.3.1 SEC Volumetric Theory ................................ .............. 50 2.4.3.2 SEC Methods ................................ ............................... 51 2 .4.4 Antibody Conjugation ................................ ................................ .... 5 3 2.5.0 Vesicle Metrology ................................ ................................ ...................... 5 4 2.5.1 Thin Layer Chromatography Introduction ................................ ..... 5 4 2.5.1.1 Thin Layer Chromatography Methods ......................... 55 2.5.1.2 Thin Layer Chromatography Results ........................... 57 2.5.2 Fluorescent Microscopy ................................ ................................ 58 2.5.3 Flow Cytometr y ................................ ................................ ............. 6 1 2.5.4 Transmission Electron Microscopy ................................ ............... 65 2.5.5 Dynamic Light Scattering ................................ .............................. 69 3. Evaluation of Receptor Conjugated Vesicles ................................ ........................ 73 3.1.0 Cell Culture ................................ ................................ ................................ 7 3 3. 2 0 Fixed Cell Binding ................................ ................................ ..................... 7 4 3.3.0 Live Cell Binding ................................ ................................ ....................... 77 3.3.1 Multiw ell Plate Experiments ................................ .......................... 77 3.3. 2 Confocal Laser Scanning Microscopy ................................ ........... 80 3.3.3 Tr ansmission Electron Microscopy ................................ ............... 89 3.4.0 pH Sensitive Dye Study ................................ ................................ ............. 92 3.5.0 Vesicle Binding in Flow ................................ ................................ ............ 9 5 4. Enhancement of the Modified Vesicle Designs for Targeted Delivery in Vivo ................................ ................................ ................................ .... 97 4.1.0 Multi Well Plate Imaging ................................ ................................ ........... 97 4.2.0 Phantom Mouse Imaging ................................ ................................ ........... 99 5. Discussion and Future Direction ................................ ................................ .......... 103 5.1.0 Vesicle Design and Manufacturing ................................ .......................... 103 5.2.0 Experimental Conclusions ................................ ................................ ....... 107 5.3.0 Future Directions ................................ ................................ ..................... 109 References ................................ ................................ ................................ ....................... 111 Bibliography ................................ ................................ ................................ .................... 125 Appendices ................................ ................................ ................................ ...................... 127 Appendix A : Transmission Electron Microscopy Notes ................................ ... 128 About T he Author ................................ ................................ ................................ .. End P age

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iii List of Tables Table 1.0 Project A ims ................................ ................................ ................................ 3 Table 1.1 Physical P roperties of B lood ................................ ................................ ........ 9 Table 1. 2 Blood Cell T ypes ................................ ................................ ....................... 10 Table 1. 3 Endothelial Transport Equations [36] ................................ ........................ 1 7 Table 1.4 Integral Membrane Protein Types ................................ ............................. 21 Table 1. 5 Common C ell Adhesion Molecules (CAMs) ................................ ............. 22 Table 1. 6 Human I mmunoglobulin T ypes ................................ ................................ 26 Table 2.0 Immuno niosome Materials ................................ ................................ ....... 4 3 Table 2.1 TLC Materials ................................ ................................ ............................ 5 5 Table 2.2 5% PEG Sample Event Population ................................ ............................ 63 Ta ble 2.3 5% PEG Sample Median Distribution ................................ ....................... 63 Table 2.4 10% Functionalized Cholesterol Median Population ................................ 64 Table 2.5 Transmission Electron Microscopy Materials ................................ ........... 65 Table 3.0 Static Binding Results ................................ ................................ ................ 77 Table 3.1 Injury Binding Results ................................ ................................ ............... 83

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iv List of Figures Figure 1.0 PEGylated Immuno niosome ................................ ................................ ....... 3 Figure 1.1 Simple Compartmental Model ................................ ................................ ..... 7 Figure 1. 2 Endothelial Cell on Aortic V alve (arterial) ................................ ................ 15 Figure 1. 3 Immunoglobulin G (IgG) ................................ ................................ ........... 2 8 Figure 1. 4 CD44 Genomic Sequence and Receptor Structure [82,83] ....................... 2 9 Figure 1.5 ICAM 1 ................................ ................................ ................................ ...... 31 Figure 1.6 VCAM 1 ................................ ................................ ................................ .... 32 Figure 2.0 H ydrodynamic Shell of a Surfactant M olecule ................................ .......... 38 Figure 2.1 Molecules : Span 60 and T ween 61 ................................ ........................... 40 Figure 2 .2 Cholesterol ................................ ................................ ................................ 40 Figure 2 3 Polyethylene G lycol (PEG) ................................ ................................ ........ 4 1 Figure 2.4 PEGylated Surfactant S ynthesis ................................ ................................ 4 5 Figure 2.5 Functionalized Cholesterol Synthesis ................................ ........................ 4 7 Figure 2.6 Size Exclusion Chromatography ................................ ................................ 50 Figure 2.7 Size Exclusion Chromatography Elution Profile ................................ ....... 5 1 Figure 2.8 Elution Profile f rom 2 ml Sample Size ................................ ..................... 5 2 Figure 2.9 Elution Profile f rom 500 ul Sample Size ................................ ................... 5 3 Figure 2.10 Antibody Conjugation ................................ ................................ ................ 5 4 Figure 2.11 TLC Image Analysis ................................ ................................ .................. 5 7 Figure 2.12 Atorvastatin Standards ................................ ................................ ............... 5 8

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v Figure 2.13 Jablonski Diagram ................................ ................................ ..................... 5 9 Figure 2.14 PEGylated FITC Conjugated Immuno niosomes ................................ ...... 60 Figure 2.15 FITC Conjugated Immuno niosomes Using Functionalized Cholesterol ................................ ................................ ................................ 60 Figure 2.16 5% PEG Sample Forward Scatter Distribution ................................ .......... 62 Figure 2.17 5% PEG Sample Particle Size Distribution ................................ ............... 62 Figure 2.18 5% PEG Sample Dynamic Light Scattering Data ................................ ...... 63 Figure 2. 19 10% Functionalized Cholesterol Forward Scatter Distribution ................. 64 Figure 2.20 10% Functionalized Cholesterol Sample Dynamic Light Scattering Data ................................ ................................ ................................ ............ 65 Figure 2. 21 TEM of Bulk Solution ................................ ................................ ............... 67 Figure 2. 22 TEM of Extruded 5% PEGylated Niosome s ................................ ............. 68 Figure 2. 23 TEM of 5% PEGylated FeO Loaded Niosomes ................................ ........ 68 Figure 2.24 Dynamic Light Scattering ................................ ................................ .......... 69 Figure 2.25 Ni osome Sample Correlation Data ................................ ............................ 71 Figure 2. 26 Data from Stability Comparison Study on Day90 ................................ ..... 72 Figure 3.0 Fixe d Cell Binding: 10% Tween Fluorescein loaded IN (CD44, KM201) ................................ ................................ .................... 7 6 Figure 3.1 Fixed Cell Binding: 5% PEG Fluorescein loaded IN (CD44, KM201) ................................ ................................ .................... 76 Figure 3.2 Nonspecific Binding Results ................................ ................................ ...... 79 Figure 3.3 Live Binding Performances ................................ ................................ ....... 80 Figure 3.4 One H our S cratch I njury ICAM ( 200X M agnification ) ............................ 82 Figure 3.5 Graph of Fluorescent Intensity f rom Region of Interest 1 ......................... 82 Figure 3.6 Injury Model Region of Interest ( 200X M agnification ) ............................ 83

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vi Figure 3. 7 (A) 4 Hour PEGylated CD44 Immuno niosomes, (B) Control, 4 Hour PEGylate d CD44 Immuno niosomes, (630X M agnification) .......... 85 Figure 3.8 (A) 3 Hour PEGylated CD44 Immuno niosomes, (B) Control, 3 Hour PEGylated CD44 Immuno n iosomes, (630X M agnification) .......... 85 Figure 3.9 (A) 2 Hour PEGylated CD44 Immuno niosomes, (B) Control, 2 Hour PEGy lated CD44 Immuno n iosomes, (630X M agnification) .......... 86 Figure 3.10 (A) 1 Hour PEGylated CD44 Immuno niosomes, (B) Control, 1 Hour PEGylated CD44 Immuno niosomes, (630X M agnification) .......... 86 Figure 3.11 (A D) Z stack of the 1 4 Hour PEGylated CD44 Immuno niosomes, (630X M agnificati on) ................................ ............................... 87 Figure 3. 12 (A) 4 Hour PEGylated VCAM 1 Immuno niosomes, (B) Control, 4 Hour PEGylated VCAM 1 Immuno niosomes, (630X M agnification) ................................ ................................ ................ 88 Figure 3. 13 (A) 4 Hour ICAM Cholesterol Immuno niosomes, (B) Control, 4 Hour ICAM Cholesterol Immuno niosomes ( 630X M agnification) ......... 89 Figure 3. 14 Gold L oaded, Internalized Immuno niosomes ................................ .......... 91 Figure 3. 15 Gold Loaded Immuno niosome Bound to Cell Membrane ....................... 92 Figure 3. 16 Fluorescence Spe ctra of 1 M S NARF 4 in PBS, pH 5.45 to 7.2 ............. 93 Figure 3. 17 Spectrum of SNARF 4 Load ed Span 60 Niosomes at pH 7.2 ................... 94 Figure 3. 18 Spectrum of Cholesterol ................................ ................................ ............ 95 Figure 3. 19 Flow Cell ................................ ................................ ................................ .... 95 Figure 3. 20 PEGylated CD44 C onjugated I mmuno niosomes B ound to E ndothelial C ells U nder F low C onditions (400x M agnification) .............. 96 Figure 4.0 IRDye 800CW Bulk Solutions ................................ ................................ .. 97 Figure 4.1 IRDye 800CW Reference Samples ................................ ............................ 98 Figure 4.2 4nmol IRDye 800CW Loaded 5% PEGylated N iosomes .......................... 99 Figure 4.3 IRDye 800CW Pipette Standards for Phantom Mouse ............................ 100

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vii Figure 4. 4 Minimum Detection Limits with Epi illumination ................................ .. 101 Fig ure 4. 5 Phantom Mouse Imaged Under Trans illumination ................................ 102

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viii PEGylation of Niosomes John A. Elliott ABSTRACT The research presented in this dissertation describes the creation and characterization of a novel antibody vesicle conjugate modified with polyethylene glycol (PEG) that possesses enhanced binding to and uptake by inflammation activated endothelial cells with improved storage stability and longer shelf life and potential reduction in immunogenic potential compared to previous designs Targeted drug delivery provides an effective means of delivering therapeutic concentrations of a drug to the site or organ of action. The drug is delivered using a niosome, a vesicle with an aqueous core and a bilayer membrane composed of non ionic surfactants and cholesterol. Antibodies that recognize specific cell antigens are attached to the niosome to complete the targetin g molecule, an immuno niosome (IN). When func tionalized PEG a water soluble, biologically inert polymer, is attached to proteins, it can protect the protein, increasing its half life in vivo. The immuno niosome synthesis process is modified to include PEG incorporation into the niosome membrane, a process known as PEGylation. Since the vasculature connects the entire body, immuno niosomes targeted to endothelial cells were used. When endothelial cells are activated during disease, stress and injury, certa in receptors are expressed and upregulated. One such receptor, CD44, is upregulated in response to vascular inflammation associated with atherosclerosis.

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ix The research hypothesis is that the addition of polyethylene glycol to the drug delivery vesicle (imm uno niosome) using cyanuric chloride linking chemistry will improve colloidal stability and binding performance of the PEGylated immuno niosomes to endothelial cell surface receptors expressed during an inflammatory response. The research presented in this dissertation provides the following evidence to support this hypothesis: Construction and characterization of a modified drug delivery vesicle using non ionic surfactants conjugated with PEG and functionalized with antibodies against endothelial cell surface receptors (PEGylated immuno niosomes) improves the colloidal stability over previously designed vesicles. Binding of PEGylated CD44 IN to activated endothelial cells is improved over previously designed vesicles. Binding of PEGylated CD44 IN to act ivated endothelial cells under physiological conditions (flow) is demonstrated. Uptake of PEGylated immuno niosomes by activated or injured endothelial cells is demonstrated using confocal microscopy.

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1 1 Project Background Generally, the most effective and least toxic method of delivery of a therapeutic agent is targeting directly to the site of action. Therefore, methods to enhance and improve the design of targeted delivery vesicles are increasingly important to avoid the deleterious health and drug dilution effects associated with systemic administration. In 1965 Bangham et al., described the liposome, a self assembly bilayer phospholipid vesicle composed of the cell membrane materials [1 3] They determined that ions encapsulated within the liposomes could diffuse out, similar to the process of particle diffusion across a cell or biological membrane. Then, in the 1970's, Gregoriadis determined that liposomes could be used to encapsul ate proteins, chemotherapeutic drugs and other therapeutic agents [4] Also, in the 1970's, scientists in the cosmetics industry developed niosomes, bilayer membrane vesicles composed of non ionic surfactants [5, 6] Since the phospholipids in liposomes are prone to oxidation, niosomes offer better stab ility and shelf life [7] Next, with the advent of monoclonal antibody production in 1975 by Schwaber and Cohen [8] targeting specific sites located on cellular targets became feasible. Additionally, the graft ing of small molecular weight drug molecules to polymer chains, e.g. polyethylene glycol (PEG), was reported to alter the immunological properties of the molecule [9] and also reduce renal excretion of the molecule [10, 11] Therefore, to improve the delivery vesicle (niosome) circulation time and provide colloidal stability, a biodegradable polymer chain made from polyethylene glycol is attached to the niosome surface, a process known as PEGylation. The hydrophilic PEG

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2 chain attracts water molecules to the vesicle surface and this hydration inhibits plasma protein attachment. Allen et al. and others have shown that PEGylation inhibits reticuloendothelial system uptake and provides a site for antibody conjugation that is exten ded away from the vesicle surface [12 15] During the vesicle production process, the functionalized surfactant, PEG, is incorporated into the membrane of the niosome. Antibodies targeted to a specific cell surface receptor are conjugated to the terminal end of the PEG chain, creating the immuno niosome [16, 17] In many disease states, e.g. trauma, atherosclerosis, infection and inflammation, specific receptors are expressed on the endothelial cell surface [18 19] For example, when exposed to cytokines and abnormal shear stress, endothelial cells become activated, initiate an inflammatory response and express cellular adhesion molecules [20] Cellular adhesion molecules are transmembrane glycoproteins involved in leukocyte recruitment, adhesion and t ransmigration, thus making them potential targets for targeted delivery vesicles [20 23] In particular, CD44, a smooth muscle and endothelial cell surface receptor, is involved in inflammation medi ated processes, making it an attractive therapeutic target [24] CD44 plays a critical role in inflammation associated with atherosclerosis, the buildup of plaque causing hardening of the arteries and progress ion to heart disease Blocking antigen binding to the ac tivated cell surface CD44 can reduce evidence of inflammation [18] CD44 is also a potential target for solid tumor cells th at express the cell surface receptor and in some metastatic cancers where elevated levels of soluble CD44 can be found in the circulation [25 28] PEGylated immuno niosomes are antibody conjugated targeted delivery vesicles made from PEGylated non ionic surfactants shown in figure 1 .0

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3 Figure 1.0 PEGylated Immuno niosome The research hypothesis is that the addition of polyethylene glycol to the targeted delivery vesicle (niosome) using cyanuric chloride linking chemistry will improve the colloidal stability and binding performance of the PEGylated immuno niosomes to endothelial cell surface receptors expressed during an inflammatory response. Through successful completion of the proposed aims in table 1.0, we pr ovide a stable targeted delivery vesicle, the PEGylated immuno niosome, capable of delivering therapeutic agents to specific cellular sites with high specificity. Table 1.0 Project A ims Aim 1 Construct and characterize a modified vesicle design usin g non ionic surfactants conjugated with PEG and functionalized with antibodies against endothelial cell surface receptors (PEGylated immuno niosomes). Aim 2 Compare and observe binding of PEGylated immuno niosomes to endothelial cell surface inflammatory recepto rs Aim 3 Develop vesicle designs and techniques for in vivo delivery and imaging of the PEGylated immuno niosomes Cholesterol Span 60 Tween 61 PEGylated Tween61 Antibody

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4 1.1.0 Drug Delivery Introduction Design of a targeted drug delivery molecule requires knowledge of biology, transport phenomena and pharmacokinetics. Integrating the knowledge derived from these fields provides the foundation for modern research and development of most drug delivery vesicles and conjugates. Therapeutic agents can be delivered by various routes, e.g. oral, ocular, rect al, bronchial, trans dermal and systemic Most of the methods use the cardiovascular system for distribution and removal of the agent once it has entered the body. The cardiovascular system provides the driving force and infrastructure for the distribution of the therapeutic agent throughout the body. Depending on the location of the targeted site, d istribution is carried out by diffusive and convective forces. When the blood is used to transport material through the body the point of first contact for the targeting vesicle is the membrane of the endot helial cell surface. 1.1.1 Basics of Pharmacokinetics Pharmacokinetics is the study of how the body distributes, absorbs, metabolizes, and clears a drug after administration while pharmac odynamics describes the biochemical and physiological effects of the drug on the body The basic overview of the process of pharmacokinetics is described by the acronym LADME which represents Liberation (L), Absorption (A), D istribution (D), Metabolism (M) and Excretion (E). These processes are not discreet as some can take place simultaneously For example, s imple compartment models can be used to estimate serum levels in order to prevent the occurrence of nephrotoxicity. Inputs and o utputs are the t wo types of operation described by LADME Liberation and absorption describe input processes where liberation is the release of the

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5 drug from its original form and absorption is the movement of the drug from its original site of administration into the blood stream D istribution metabolism and excretion are output operations Distribution is where the drug diffuses or travels from the intravascular space into the tissue Metabolism is where the drug is broken down or converted into forms that are easy to elim inate. Excretion is the elimination of the original compound or its metabolites from the body through renal hepatic or pulmonary routes. In the design of drug delivery molecules, t he first thing to consider is how the drug is to be administered or introdu ced into the body D rugs can be delivered by site specific methods such as ocular dermal and bronchial or by broader distribution methods of delivery such as oral and intravenous routes The dermal methods are based on adsorption through the skin by a pat ch that releases the drug over time T he drug is absorbed into the blood and distributed, e.g. nicotine and nitroglycerine patches. Oral methods of delivery use pills containing simple starch or gelatin binders to control release rates in the gastrointestinal tract (GI), with the la t ter providing a slower release rate in the stomach and intestine A more sophisticated oral delivery method uses an osmotic pump across a polymer shell so the drug can be delivered for a longer time The drawback to oral delivery methods is the ha r sh environment of the GI tract and the low ab sorption rates of some drugs from the GI tract into the blood. T he benefit is greater compliance since typically the drugs can be taken by the patient without aid from a medical professional and without the pain or complications that can be associated with intravenous delivery methods. The route chosen for this study is the intravenous route since the entire dosage of the administered drug reaches the circulatory system wher e it i s

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6 then distributed throughout the body prior to clearance by hepatic renal and proteolytic enzyme routes. 1.1.2 Compartment Model s When the drug enters the blood stream the c entral volume (Vc) describes the volume of blood in the vessels This includes any tissues that are highly perfused with blood and where the drug may distribute. The peripheral volume (Vp) describes blood in the tissues outside of the central volume. Together Vc and Vp make up the volume of distribution (Vd), which is the total flui d volume the drug is dissolved in [29] Together the terms Vc, Vp and Vd are used to approximate the amount of drug in the body, peak serum levels and the clearance of the drug from the body. The clearance (Cl) of the drug from the blood is another import ant term that describes the behavior of the drug in vivo. The rate of drug clearance is described by a constant calle d the elimination coefficient ( k el ), which describes the fra ction o f drug eliminated per unit time. The constant is patient specific becaus e it is based on the capacity and functionality of specific organs that can be impacted by disease and the administered drug treatment. The clearance calculation is shown in equation 1.0 Cl = k el *Vd Equation 1.0 The simplest way of characterizing a drug in the body is to use first order kinetics and a singl e compartment model. In reality, first order kinetics hold s up for most drugs while the si ngle compartment model work s if serum samples are collected after the drug moves into the tissues. The one compartment model is used for drugs that perfuse rapidly while the two compartment model takes into account the distribution phase. When graphing the two models the one compartment model will yield a straight line on a

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7 logarithm ic y axis. The two compartment model yields a biphasic line on the same graph, indicating the distribution phase. Some drugs when administered by intravenous infusion over a time course will f ollow a three compartment model consisting of distribution, el imination and tissue release. In encapsulated drug delivery design, the tissue release of the drug can be coordinated with the time release from the vesicle s This is a beneficial attribute of encapsulated delivery methods since drug plasma concentrations are maintained for long time periods without the initial high drug plasma concentration s associated with non encapsulated delivery techniques The body can be modeled as a set of compartments connected in parallel and / or series with the blood plasma as the conduit between the organs, as shown in figure 1. 1 Figure 1.1 Simple Compartment al Model With rate constants representing the coefficients for drugs entering and leaving a particular compartment (k12, k21) the liver and kidneys act as a sink (k20) where the drug is metabolized and/ or extracted from the plasma through hepatic and renal clearance. This multiple compartment model is still incomplete because t here are intercellular and ex tracellular space interactions and int r a cellular compartment interactions within each organ. M ore advanced modeling is being investigated in order to

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8 incorporate physiological data from experiments [30] Another method used to characterize the drug in the body is to take into account the complex design of the vascular bed and to i mpleme nt fractal analysis [31, 32] 1. 2.0 Introduction to the Cardiovascular System The major components of the cardiovascula r system are the heart, vessels and blood circulated throughout the body. The entire vascular s ystem is lined with endothelial cells providing a blood contact surface Therefore the cells play an active role in the cardiovascular system. Th e system is considered a closed loop because it is a sealed system where all the fluid is maintained and circu lated within an enclosed body It should be noted that a water cycle exists where fluids are absorbed through the GI tract replenish ing the large water content that leaves the system through renal excretion, sweat and respiratory function. The system provides transport of vital nutrients and flu ids to all the organs in the human body making it an attractive avenue for drug delivery schemes. Because the operation of the cardiovascular system is essential for life, it is also a target for the rapeutic intervention and treatment. 1. 2 1 Blood The fundamental role of blood is maintenance and homeostasis. It is a fluid composed of a multitude of components ranging from cells to proteins and trace minerals. Blood density is very close to pure wat er as shown in table 1.1 105 0 kg/m 3 compared t o 1000 kg/m 3

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9 Table 1.1 Physical P roperties of B lood Property Values Density 10 5 0 7 [ kg/m 3 ] @ 37 C Viscosity at shear rate 3.26 0.43 [mPa*s] at shear rate of 100 [1/s] 4.37 0.60 [mPa*s] at shear rate of 50 [1/s] 5.46 0.84 [mPa*s] at shear rate of 1 [1/s] pH 7.4 0.05 [H + ] Osmotic colloidal pressure 284 [mm H 2 O] The cellular component of blood can be divided into three main groups with sub types as shown in table 1. 2 The t h ree main cell types are e r y throcytes or red blood cells (RBC), leukocytes or white blood cells (WBC) and thrombocytes or platelets. Whole blood contains all the cellular and fluid components. Plasma contains the whole blood soluble factors only, with t he cells separated out by centrifugation. Once whole blood has clotted the remaining liquid component is serum. It is similar to plasma except that it lacks the clotting factors. The plasma makes up approximately 55% of the blood by volume and carri es nutrients, proteins and trace minerals and acts as a pH buffer The plasma is composed primarily of water and contains numerous low molecular weight organic and inorganic materials and about 7% protein, e.g. albumin fibrinogen and four major groups of globulins.

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10 Table 1.2 Blood C ell T ypes Type Sub Type Function Erythrocytes Red Blood Cells Carry oxygen to tissue s in the body Leukocytes Neutrophil Bacterial and fungal targeting Eosinophil Parasite targeting and allergy mechanisms Basophil Contains histamine and heparin, involved in specific inflammatory and alle rg ic processes Lymphocytes (Natural Killer cell) (B cell and T cell) N K cells remove tumors and viral infected cells by cytokine induced apoptosis. B and T cells are frontline agents in the adaptive immune system. Monocyte Replenish macrophages and fight infection Thrombocytes Platelets Re gulation in blood clotting and inflammation A Newtonian fluid is d escribed as a fluid that is non compressible T he viscosity is a function of temperature and pressure only and is described by Newton s Law of Viscosity where the shear stress is a function of the velocity profile multiplied by a constant of proportionality as shown in equation 1.1 Equation 1.1 Blood can be approximated as a Newtonian fluid when circulating in large vessels. H owever, when the vessel radius approaches that of capillaries the red blood cells and plasma separate into two phases and blood can no l onger be considered a Newtonian fluid The blood flow is described as smooth ( laminar ) or turbulent depending on the

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11 fluid path. In smooth flow, there are no disruptions in the axial direction and the cross sectional vel ocity pattern s are described as p lug or p arabolic In plug flow the cross section al velocity is constant creating high shear rates that act on the vessel wall. In plug flow it is assumed there is no boundary layer at the wall surface When the velocity profile is parabolic the fluid in the cent er of the vessel has the maximum velocity and the fluid at the wall has the slowest velocity. The parabolic profile creates conditions that can impede particles from reaching the vessel wall. This condition was first described by Segre and Silberberg in 1962 The phenomenon causes particles that are near the wall to mig rate toward the axial position and particles that are in th e axial position migrate toward the wall. When blood is initially ejected from the heart, it travels through th e large arteries in a plug profile which becomes parabolic as the cycle continues T his effect is less pronounced as the blood travels further away from the heart into smaller vessels (not including the capillaries) Turbulent flow occur s at bifurcations a nd in pathological conditions such as atherosclerosis where hardening of the arteries and plaque formation disrupt the normal blood flow. Fluid flow can be described by the velocity profile, pressure drop and the wall shear stress where the difference in p ressure is the driving force causing fluid flow Blood is circulated based on the principle that a fluid flows from an area of high er pressure to lower pressure analogous to heat flow The ventricle s in the heart contract and force the blood out creating the driving force so the blood will move from an area of higher to lower pressure. 1. 2.2 Heart The human heart is a four chamber pump driven by and composed of myocardial muscle tissue and it provides the driv ing force for blood circulation thr ough the entire

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12 system. The myocardial muscle tissue is surrounded by the pericardium, a tough inelastic multi layered tissue. T he inner walls of the myoc ardium are lined with endocardial tissue composed of endothelia l cells covering trabeculae. The left an d right coronary arteries are positioned on the outer surface of the muscle tissue and feed blood to the myocardial muscle To control the direction of blood flow there are four valves two valves that are actuated by papillary muscle s and two cuspid valves that are passive and are operated by the pressure differential across the valve. 1. 2 3 Circulation The blood is returned from the body to the right atrium through two veins, the superior and inferior vena cava. B lood flows from the right atrium through a tricuspid valve into the right ventricle and is pumped through the right and left pulmonary arteries to the lungs through a semi lunar valve. In the lungs a gas exchange occurs between the air in the alveolar sac s and the lung capill aries After this exchange process the blood is now ready to deliver oxygen to the various tissues in the body. The oxygenated blood is returned to the heart through the right and left pulmonary veins to the left atrium. The blood traverses across the mit ral valve into the left ventricle where it is ejected across the aortic valve into the aortic arch and the descending aorta From the major arteries the vessels continue to branch out into smaller arteries transitioning from the arterioles to the smallest group of vessels called capillaries. The arterioles are responsible for the peripheral resistance in the cardiovascular system; this resistance is directly related to arterial pressure from the restriction of blood flowing from the arteries through the arterioles The capillaries contain the largest total cross s ectional surface area in the body and are present in every tissue They contain a single layer of endothelial cells

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13 surrounded by basal lamina Cap illaries are the pathwa y for the delivery and exchange of oxygen carbon dioxide and critical nutrients to the surrounding tissues in the body making them a focus for targeted drug delivery techniques. The fluid that carries nutrients from the capillaries is col lected in the lymphatic duct s. The lymphatic system is responsible for removing fluids from the tissue interstitial spaces around the body and returning those fluids to the blood stream The blood is returned to the heart by a complementary set of v essels called veins which completes the closed loop of the cardiovascular system. 1. 3.0 Cell Introduction Cells are contained within the tissues of all living creatures. Cells can be divided into two distinct types, eukaryotes, cells with a nucleus and well dev eloped intracellular organelles and prokaryotes, cells without a discrete nuclear membrane The basic structure of a cell consists of a fluid core surrounded by a bi layer lipid membrane T he internal space of eukaryotic cells the cytoplasm, is a f luid core with membrane bound organelles and a nucleus. The cytoplasm contains many small and large molecules and the cell transports material into the cytoplasm and between organelles using small membrane bound vesicles The nucleus contains the informati on the cell need s to function and replicate. Energy for cellular processes is generated in the mitochondri on. M itochondria produce adenosine triphosphate (ATP) that is used as an activated energy carrier in biosynthesis reactions and is also responsi ble for cellular functions such as the cell cycle and growth. The nucleus is surrounded by a double layer membrane with pores that allow the transfer of material. Ribosomes within the cytosol are sites of protein synthesis where messenger RNA transcribed in th e nucleus is translated into protein on

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14 the ribosomes. An additional multifunctional cytoplasmic organelle is the endoplasmic reticulum (ER). The rough endoplasmic reticulum (RER) is coated with ribosome s The formed proteins can be further modified on the surface of the RER There is another part of the ER called the smooth endoplasmic reticulum (SER) that is responsible for specialized functions like lipid and steroid production. The proteins formed at the free ribosomes, RER and SER are transported to an organelle called the golgi apparatus. The golgi appa ratus functions primarily as a cellular post office where the material is sorted packaged and sent to the proper destination within the cell Lysosomes import and recycle materi als using digestive enzym es Lysosomes have an internal pH of 4.8. Material s transported into the cell by endocytotic vacuoles are typically shuttled to an acidic vacuole called an endosome. The e ndosome sorts recyclable materials to the plasma membrane or delivers them to the lysosome. This transport and recycling feature is important in developing drug delivery strategies because therapeutic agents can be degraded by the acidic conditions. 1.3.1 Endothelial Cells The vascula ture is lined with a single layer of endothel ia l cells providing the blood contact surface in the heart, arter ies, veins and lymphatic system The endothelial cell is a subtype of the epithelium which lines human body cavities and tissue structures. One feature of the endothelial cell type is that it expresses the E cadhe r in ad hesion molecule on its surface. There are dif f erences between arterial and venous endothelial cells and it has been shown that the genetic coding determines the cell phenotype [33] In fact dif ferent phenotypes are seen in the five different types of endothelia l cells found in the heart (endocardial, coronary, arterial capillary and lymphatic) [34] There are

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15 orga nizational/orientation differences between the endothelial cells (arterial versus ventricular) covering the aortic valve. The c ells are aligned in different patterns to compensate for the physiological stress es they encounter. A transmission electron micro scope image of an endothelial cell on an aortic valve is shown in figure 1. 2 Figure 1.2 Endothelial C ell on A ortic V alve ( arterial ) Variations in p ermeability of the endothelial cell layer in large arteries and capillaries are caused by the underlying structural components of the different vessels. The large arteries transport blood and the capillaries function to deliver vital molecules and fluids to the tissue intracellular space s The main arteries allow passage of oxyg en and small molecul es from the blood through the endothelial cells into the arterial substructure to maintain the smooth muscle cells. The lymphatic system is a low flow

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16 system that maintains intracellular fluids and has the potential to become a target for developing anti inflammatory treatments [35] There are also interactions between tumor cells and the lymphatic sy stem making it a focus for anti cancer treatment strategies. 1.3.2 Endothelial Cell Mass Transport For intravenous targeted drug delivery, the endothelial cell surface is the first point of contact, whether the surface of the cell is the intended site of therapy the drug is to be transported into the cell or the drug needs to cross the endothelial cell barrier to reach the underlying tissue s T he select ion of the cell surface receptor i s matched to the delivery vesicle using knowledge of the receptor s temporal expression and binding affinity The targeting strategy dictates t he size and composition of the delivery vesicle to provide the behavior required in the area of interest since it may need to overcome convective forces due to blood flow momentum in great vessels or shear forces in smaller vessels. Depending on the application of the therapeutic agent the mass transport interactions with the endothelium can be cha racterized as: a reactive surface, a reactive wall or a semi permeable membrane shown in table 1. 3 [36]

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17 Table 1. 3 Endothelial Transport Equations [36] Process Equation Important Condition s Reactive Surface C s /C b =1/(1+ Da r /Sh) Equation 1. 2 Da r << Sh Da r >> Sh Semi Permeable Membrane C s /C b =1/(1+ Da c /Sh), Equation 1. 3 Da c >> Sh Reactive Wall P s,A /P b,A =1 Da w /Sh E quation 1. 4 Da r << Sh Da r Sh As a reactive surface Michaelis Menten kinetics (MMK) are used to model the interactions at the surface. MMK describe biological reactions involving enzymes and assume the reaction is irreversible and that the product does not bin d or occupy the enzyme. The Damkohler number (Da) is a ratio rel ating the reaction rate to the mass transport rate and the Sherwood number (Sh) is used to relate the convective and diffusive forces. W hen the surface concentration is equal to the concentration found in the blood stream reactive surface e quation 1.2 is reaction limited. When the Damkohler number is much greater than the Sherwood number ( Da r >> Sh) the reactants are present at the wall in concentrat ions high enough or in excess for the reaction Th e diffusive and convective forces that bring the reactan ts from the blood stream to the cell wall do not influence the reaction. When the endothelium is described as a semi permeable membrane for small molecules e quation 1.3 shows that when the Damkoh l er number is much larger than the Sherwood number the system is t ransport limite d or fluid phase limited In larger vessels

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18 this means it will be more difficult for large molecules and p articles to move to the endothelial surface In contrast, smooth flow patterns will allow smaller particles to diffuse to the endothelial surface more quickly This illustrates how a vesicle that is attached to the endothelial surface at a site of therapeutic interest can take advantage of this process In the capill ary vessels there is a phenomenon called shear thinning that takes place. This occurs because the diameter of the red blood cell is similar to that of the vessel. The red blood cells separate from the plasma and travel down the center of the vessel an d the plasma is pushed toward the wall surface creating a n on Newtonian liquid with two distinct phases. The effect of the plasma being located near the wall creates a fluid phase that the delivery vesicle will occupy when in close proximity to the binding site on the vessel wall. At the reactive wall there is con sumption or a sink on the mass balance. The act of oxygen transport from the blood stream to the vessel wall is an important process that is required to maintain homeostasis of the vessel. Equation 1. 4 shows that when the Damkohler number is much smaller than the Sherwood number the reaction is limited by the reaction rates associated with the wall and transport phenomena does not play an important role. As the Damkohler and Sherwood number s approach one another the reaction is limited by transport of the reactant to the wall and convective and diffusive forces become important. This has been used to explain endothelial dysfunction in coronary arteries leading to a leaky vascular wall and allowing the influx of low density lipoproteins a primary step in atherosclerotic plaque formation.

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19 1. 3 3 Endocytosis Endocytosis is t he uptake of nutrients, fluids, large and small molecules and particles in cells There are t wo main pathways for endocytosis phagocytosis and pinocytosis. P hagocytosis is described as a process where the cell engulfs large particle s (>250nm) by folding its membrane around the particle P inocytosis is a process where a cell uptakes liquids and small molecules (<150nm) inside p its that are formed in the cell membrane and that pinch off forming vacuoles that carry the contents to various intracellular locations [37] Phagocytosis is a process that specialized cells in the immune system use to remove pathogens and debris and is the process that mac rophages use to complete housekeep ing duties throughout the body. Phagocytosis is a major pathway for specialized cells like macrophages but is rare in endothelia l cells in sit u. Ho w ever Muro et al. repo r ted that endothelial cells can internalize large p articles up to 5 um in size [22] Considering the rarity of phagocytosis in endothelia l cells in vivo it has not been a focus of most targeted delivery schemes However, since macrophage activity is a pro cess associated with tissue inj ury and inflammation the macrophage is an attractive target for therapeutic treatment [38] Pinocytosis and receptor mediated pathways are also used to deliver a payload to the site of interest. Pi nocytosis is a non specific uptake of extracellular fluids and small molecules and can be used to deliver a reservoir to the cell surface The reservoir contents are eluted into the extracellular fluid and diffusion becomes t he driving force for delivery. Uptake pathways are described by four main groups: macropinocytosis, clathrin media ted endocytosis (CME), caveolae mediated endo cytosi s (CavME), and clathrin caveolae independent internalization [39, 40] CME

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20 and CavME both provide non specific pathways for the uptake of adsorbed molecules through invaginat ions found on the cell membrane The caveolae are small 60 80 nm plasma membrane invaginations used for endocytosis, e xocytosis, transcytosis and the internal transport of macromolecules [41] They do not have a large volume an d are typically lined with receptors such as gp60, an albumin specific receptor The CavME pathway has been well studied and provides a promising route for delivery to the endosome, caveosome and the cell nucleus [42] The selection of these different delivery routes is based on interactions o f the surface lipid rafts, inte grins and proteins. Caveolae are involved in the transcytotic and exocytotic process es which are directed by secondary protein interactions. 1. 4 0 Cell Surface R eceptors Targeted drug delivery can be used to target cell types base d on size charge and binding schemes using cell surface receptors and receptors found within the pits formed during pinocytosis Ce lls continually express protein and carbohydrate receptors for ligands both externally on their membrane surface and internally for a variety of functions. The receptors that are expressed vary between cell type s and some of them may only be up regulated during certain stages of disease or injury such a s receptors for inflammation Cell surface receptors may be either integral or peripheral ly attached to the membrane Peripheral receptors are composed of proteins that are temporarily attached to the cell membrane and function in a r egulatory manner. They are not a s common as the integral types Peripheral and integral membrane receptors are required cellular homeostasis. The functions of the receptors vary from adhesion, signaling and communicati on to the extracellular matrix and other cells as well as t riggering cellular

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21 processes such as apoptosis, endocytosis and mitosis. Transmembrane receptors are integral in nature and their protein structures penetrate through the membrane into the cytoplasm for intra cellular signaling The integral membrane proteins are typically made from alpha helices and beta sheets Integral proteins can be grouped into six categories based on how the structure interact s with the lipid membrane of the cell ( table 1.4 [14] ) Table 1.4 Integral Membrane Protein Types Type Description I One helix structure with terminal amino group on the outside and carboxyl group on the inside of the cell membrane. II One helix structure with terminal carboxyl group on the outside and amino group on the inside of the cell membrane. III Multiple helix structure penetrating the cell membrane using a single polypeptide IV Multiple helix structure formed by different polypeptides creating a channel through the membrane V Proteins that are attached to the lipid membrane by covalently linking to lipid molecules without passing through the membrane VI Single polypeptide structure that passes through the cell membrane and has a type V lipid anchor. The delivery vesicle encounters the cell membrane surface receptor and this is the first target for most delivery schemes. A secondary interaction with peripheral receptors could provide additional targeting benefit s Inflammatory receptors are cell surface

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22 receptors that are up regulated by the cell during cond itions of disease, injury and infection. Inflammatory receptors include the cell ular adhesion molecules (CAMs) which can be expressed on the cell surface intracellularly or within the extracell ular matrices of tissues. The CAM family can be grouped into f ive groups based on protein type. These are: immunoglobulins selectins addressins, integri ns and cadherins. Of these five, three have be en used for targeting the arterial endothelium The CAM groups, the immunoglobulin s selectins and cadhe rins, provide potential binding sites for therapeutic particle delivery (table 1.5 ) Table 1.5 Common Cell Adhesion Molecules (CAM s ) Type Name Function IgG ICAM 1 In t erCellular Adhesion M olecule 1 is expressed by leukocytes and endothelial cells and is responsible for leukocyte extravasation into tissues from the blood stream during an inflammatory response [43] IgG CD44 Is involved in cell to cell interactions and is expressed by endothelial cells during inflammation, atherosclerosis, rheumatoid arthritis and cancer [44 46] IgG V CAM 1 Vascular Cell Adhesion M olecule 1 is expressed by activated endothelium during inflammation. It is e xpressed by large and small vessels and is associated with a therosclerosis. Some melanomas use it to attach to the endothelial cell surface [47 49]

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23 Table 1.5 (Continued) IgG PECAM Platelet Endothelial Cell Adhesion M ole cule is found on endothelial cells, plat e lets, macrophages, neutrophil s and Kupffer cells. It plays an important signaling role in thrombosis and between macrop hage cells and dying neutrophils [50, 51] Selectin E selectin A low affinity c ell adhesion molecule expressed only by endothelial cells during inflammation It allow s leukocytes to bind in a reversible manner known as leukocyte rolling [52, 53] Selectin P selectin A cell adhesion molecule similar to E selectin but expressed very quickly as it is stored in endothelial cells pr eformed and does not need to be synthesized [54, 55] Selectin L sel ectin A cell adhesion molecule that is associated with lymphatic endothe lial cells. It functions like E selectin and P selectin and is used for leukocyte recruitment [56 58] Cadherin VE cadherin VE cadherin is an intracellular adhesion molecule that is responsible for the integrity of the junc tion s between endothelial cells [59, 60] Cadherin T cadherin H cadherin T cadherin is highly expressed i n the heart and aorta and is up regulated in atherosclerotic lesions and tumor penetrating blood vessels [61 63] H cadherin is associated with cancer and tumor developmen t [64 67]

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24 1.4.1 Proteins Proteins are responsible for most of the functions that take pla ce in a cell. They are the most complex molecules found with regard to their structure and functio n They are comprised of a combination of amino acids that have side chains that give them distinct chemical functions. Polypeptides are formed when t he amino acid molecules are linked together by a covalent peptide bond forming linear polymers with functional side chains Some proteins have a negative or positive charge some are uncharged or polar and some are hydrophilic. The noncovalent interactions betwee n the side chains cause the linear polypeptide sequence to take shape. Hydrogen bonds, ionic bonds and van der Waals attractio ns are responsible for secondary structure of the molecule. The peptides will shift to a confo rmation with the lowest energy level. This is determined by temperature pH and the solvent. The t wo major structures that are commonly formed are the alpha helix and beta pleated sheet. eta pleated sheet structures resemble ribbons with polypeptide chains running in the same direction in a parallel structure or an altern ating direction forming an anti parallel sheet structure. Proteins form alpha helix structures to cross the cell membrane. 1.4.2 Antibodies T he immunoglobulin superfamily (IgSF ) is a large group of c ell surface and soluble proteins made from amino acids that contain an immunoglobulin (Ig) domain The Ig domain s contain two layers of pleated sheets and three to four anti parallel polypeptide chains T he Ig structure anti parallel beta sheets are char acterized by the conserved disulfide bond that links the two sheets together. All the proteins with these immunoglobulin doma ins belong to the IgSF (t he structures are involved with cell

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25 recognition, binding, adhesion and receptors for antigens and cytokin e s ). Antibodies belong to this superfamily and bind to their specific antigens with high affinity and selectivity [68] These antibodies are used to target inflammatory receptors in the described studies There are five different classes or isotypes of human antibodies shown in table 1. 6 They contain polypeptide structures grouped into regions called chains with constant regions that are conserved and variable regions that form the binding sites for specific antigens. All five classes have the same fundamental four chain structure made from a pair of identical heavy chains and a pair of identical light chains held together as a tetramer with disulfide bonds In 1959 Porter showed that the antibody could be cleaved using the proteo lytic enzyme papain into three major structures that compose 90% of the original molecule (figure 1.3) He noted that two of the structures retained the ability to bind with antigen and had nearly identical chemical properties. He also noted the third fragment had very different chemical properties and did not bind with antigen. This was the first clue that imm unoglobulin s composed of the three groups he found after the papain digest. The two identical structures are called fragment antigen binding arms (Fab) and the third region i s called fragment crystalline (Fc) [69] ( figure 1. 3 ). The five classes of antibodies each have distinct heavy chains which are used for classifica tion as listed in table 1.6.

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26 Table 1.6 Human Immunoglobulin Types Type Heavy Chain Description IgA Dimer that is found in mucous and breast milk provides protection from pathogen colonization [70] Can survive conditions in the gastrointestin al tract [71] IgD Monomer that is located on the surface of B cells and acts as a n antigen receptor [72] IgE Monomer that binds its Fc region to leukocytes and binds to allergens to trigger the release of histamine. [73] It also initiates a n immune response to parasitic worms [74] [75] IgG Monomer and the immunoglobulin that is responsible fo r the secondary immune response. Comprises 75% of the immunoglobulin found in serum [76] IgM Pentamer immunoglobulin that is the largest and first found in the blood or in monomer form on the cell membrane [77] 1.4.2.1 Immunoglobulin G The IgG family of antibodies comprises 75 % of the antibodies found in human plasma The four major classes of human IgG are IgG(1), IgG(2), IgG(3) and IgG(4) with IgG(1) being the most abundant of the four classes [78] Immunoglobulin contains glyco proteins produced by specialized lymphocyte cells The se specialized lymphocytes

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27 originate from hematopoietic stem cell s and terminally differentiate into plasma cells an d memory B cells. The genetic code that is developed during this maturation process is based on four genes, t he variable (V), diversity (D), joining (J) and constant (C) gene. The cell line that produces IgG has the following lin e age The stem cell s differ entiate into p ro B cells p re B cells, and then immature B cells A t this stage the cells are located in the bone marrow and they are antigen independent. The cells then migrate into the spleen, tonsils and other peripheral lymphoid tissue s and some become mature B cells, and collect in areas that are rich in helper T ce lls. T hese specialized areas in the tissues are known as germinal centers. When the mature B cell is activated by a helper T cell it divide s and differentiate s into a centeroblast by a process cal led somatic hyper mutation After somatic hypermutation the precursor cell is called a centrocyte prior to becoming a plasma cell or a memory B cell. During B cell maturation the genetic code that is responsible for the makeup of the vari able binding region is determined through a process called VDJ recombination The variable chain has three domains that are encoded by the V, D and J genes. During the VDJ recombination process base pair changes are introduced for both heavy and light ch ain variable regions. In this way, the immune system is able to make a diverse group of antibodies that are able to recognize multiple antigen types

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28 Figure 1.3 Immunoglobulin G (IgG) 1.4.2.2 Monoclonal Antibodies The advent of methods of monoclonal antibod y production in the was made possible by Paul bullet concept The technique was first described in 1973 by Schwaber and Cohen and they reported being able to produce human mouse antibodies by fusing mouse myeloma and human peripheral lymphocytes creating h ybridomas [8, 79] There has been controversy over this paper and Kohler and Milstein in 1975 reported production of monoclonal antibodies by fusing mouse melanoma cells with spleen cells from an immunized mouse [80] They were recognized as the first group to produce antibodies using the hybridoma technique

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29 1.4. 3 CD44 Receptor CD 44 shown in figure 1. 4 is a cell surface receptor that remains expressed on the cell surface or that can be shed or released into circulation by enzymatic cleavage, becoming soluble CD44 [27] CD44 is involved in many functions including cel l to cell interactions with leukocytes and endothelial cells and remodeling of the extracellular matrix during cell to substrate interactions in embryonic development [81] There are typically two forms of CD44, the standard and the variant form. A sin gle gene with 20 exons encodes CD44, and the standard form is the most prevalent consisting of exons 1 5, 16 18 and 20. Exons 6 15 are called variant exons v1 v10. T he variant exons are spliced in different combinations in the stem portion of the receptor [82] Figure 1.4 CD44 Genomic Sequence and R eceptor Structure [82, 83]

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30 CD44 is a transmembrane glycoprotein in the cellular adhesion molecule family and is the major receptor for hyaluronan [84] CD44 is expressed by many cell types throughout the body including the keratinocytes [85] and red blood cells [86] T he CD44 receptor expressed by both endothelial and smooth muscle cells is involved in inflammation mediated processes, making it a n attractive therapeutic target [24] It has been shown to play a critical role in inflammation associated with atherosclerosis, and blocking binding to the CD44 receptor can reduce evidence of inflammation [18] CD44 is also e xpressed on various cancer cell surface s and has been implicated as a marker for cancer when found in high levels in its soluble form in the circulation [25, 28, 44] 1.4.4 Intercellular Adhesion M olecule ( ICAM ) Intercellular adhesion molecule (ICAM) is found in three forms ( ICAM 1, ICAM 2 and ICAM 3 ) and is part of the immunoglobulin superfamily It is expressed by cytokine activated vascul ar endothelial cells macrophages fibroblasts smooth mu s cle cells, keratinocytes and leukocytes. The normal function of ICAM is to provide binding between endothel ial cells and leukocytes during injury and infection by binding to leukocyte function association antigen (LFA) or macrophage 1 antigen (Mac 1) ICAM is also a receptor for human rhinoviruses and malaria infected erythrocytes [87] ICAM 1, 2 and 3 are type I transmembrane glycoprotein s that are all involved in the LFA immune response [88] ICAM 1 was first discovered in 1986 [89] followed by the discovery of ICAM 2 in 1989 [90] and fina l ly ICAM 3 in 1992. ICAM 3 is expressed by lymphoid but not endothelial cells [91] The ICAM 1 and ICAM 2 receptor s are normally express ed in low concentrations on endothelial cells but are highly upregulated after exposure to the pro

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31 inflammatory cytokines tumor necrosis factor al pha ( TNF ) and interleukin 1 ( IL 1 ) making them an attractive binding target for drug therapy ICAM 1 is encoded by human chromosome 19. It has a total of seven domains. F ive are in the extracellular space with D1 and D3 as active binding sites [92] as shown in figure 1.5 Figure 1.5 ICAM 1 1.4.5 Vascular Cellular Adhesion M olecule (VCAM) Vascular cellular adhesion molecule (VCAM 1) is a mem ber of the immunoglobulin super family and is encoded by human chromosome 1 It is expressed only by cytokine activated endothelial cells in large and small vessels and like ICAM 1 it bin ds leukocytes via the leukocyte function association antigen (LFA) [93] It differs from CD44 and ICAM since it has two bind ing sites in domains 1 and 4 for very late antigen (VLA 4) expressed by T and B lymphocytes monocytes, basophils and eosinophils [94] (f igure 1.6). Metastatic melanoma cells bind to VCAM expressed on

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32 endothelial cells [95] It has also been shown that the adhesion molecule facilitates migration of leukocytes into the lymp h nodes [96] VCAM is upregulated at sites where arterial atherosclerotic plaques develop during the progression of this chronic disease VCAM expression is associated with the recruitment of monocytes and macrophages that infiltrate arterial tissue and venous grafts making it an attractive drug delivery tar get [97, 98] Figure 1.6 VCAM 1 1.5.0 Cyt okines and Inflammation Cytokines are extracellular signaling molecules that are required for cell cell communication T hey share several properties, which include: secreted briefly and are self lim ited, can be produ ced by multiple cell types, act on diffe rent cells types, action can be redundant and can influence the production and action of other cytokines. Cytokine chemical signaling can be grouped into autocrine, paracrine and endocrine

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33 action. A utocrines are chemical agents that influence or act on the cell from which it was secreted P aracrine action is where a cytokine acts on a different type of cell than the one that secreted it in t he same area of a tissue. E ndocrine action is where a cytokine secreted in the blood stre am is distributed into tissues by blood and extracellular fluids. Cytokines are classified into three major grou ps that describe their function: interleukins, lymphokines and chemokines. Interleukins were first classified as molecules that were produced by a population of blood leukocytes and that interact ed with other leukocyte populations [99] H owever they are produced by other cell types and many newly discovered cytokines are designated with the interl e ukin nomenclature to prevent naming ambiguity [100] Lymphokines are a class of cytokines that are produced by activated T lymph ocyt es in response to specific immunity [101] C hemokines are pro inflammatory cytokines released by cells and are used to signal or attract other cells to a site of injury or infection The name is derived from chemota c tic cytoki nes Two specific cytokines t umor necrosis factor alpha and interleukin 1, are further described in section s 1.5.1 and 1.5.2, because of their interactions in vascular tissues and one is used later in experiments to invoke a desired reaction from cultured endothelial cells. 1.5.1 Tumor Necrosis Factor Alpha Since i nflammation is a cellular response to injury stress or infection cells will up regulate specific receptors in response to those stresses. The inflammatory response is defined by the recruit ment of leukocytes to the a ff ected tissue and a shift in the fluid exchange balance through the arterial wall [102, 103] Tumor necrosis factor alpha (TNF ) and interleu kin 1 (IL 1 ) are both mediators of inflammation. The process of inflammation was elucidated by Menkin in 1959 when

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34 he described a mediator he called necrosin as being endogenous in nature. In 1975 the mediator TNF It behaved l ike necrosin, previously described by Menkin [104] TNF is highly conserved among species [105] and its function is concentration dependent. At low concentrations it is a pro inflammatory cytokine and causes the up regulation of endothelial cell receptors and a ctivation and up regulation of macrophages and leukocytes. At higher concentrations it cau ses cell apoptosis and has anti tumor ogenic properties. [106] 1.5.2 Interleukin 1 Interleukin 1 (IL 1) was one of the first cytokines described by scientist s like Menkin and Beeson in the 1940s as an endogenou s pyro gen because of its ability to induce fever when purified samples were administe red to normal animals [107] The entire interleukin g roup contains over 30 members and IL 1 is a family of pro inflammatory molecules containing 11 members all encoded by separate genes [100] Both IL 1 alpha (IL 1 ) and beta (IL 1 ), are pro inflam m atory molecules in their mature or active forms. The molecules are encoded by a 7 exon gene on chromosome 2 of the human genome and are produced as precursor peptides The precursors are proteolytically cleaved to become the mature or active forms 1. 6.0 Cardiovascular Disease Cardiovascular disease is the number one killer of adults in the United States and according to the American Heart Association 864,480 Americans died in 2005 from some form of cardiovascular disease. Cancer was responsible for 559,312 deaths and accidents for 117,809 deaths Coronary heart disease caused one in five deaths in the United States in 2004 [108] Coronary heart disease is caused by a therosclerosis a

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35 pathological disease causing narrowing of the coronary arteries due to the buildup of oxidized lipids, macrophages and smooth muscle cells in the vessel wall. This process is characterized by an inflammatory response perpetuated by endothelial dysfunction [109] followed by the accumulation of lipids [110] recruitment of leukocytes [111] and activation of smooth muscle cells [112] which eventually form mature plaque s This pathological process occurs over the course of several decades and can be described in six stages. The first stage has been shown to start in as early a s the first decade of human life [113 115] The start of lesion development occurs in areas of endothelial dysfunction. The endothelial cells become permeable and ex press adhesion molecules on their surface In stage I lesions some of the macrophage s develop into isolated foam cells after ingesting oxidized low density lipoproteins In s tage II lesions the lipids accumulate within the smooth muscle cells the macrophage foam cells accumulate and both cell types form distinct layers that are seen grossly as fatty streaks The accumulation of macrophages stimulates an inflammatory response in the tissue causing increased production of cytokines. Stage III lesions contain the same cells and cholesterol esters as seen in stage I and II lesions with the addition of extracellular lipid droplets. This accumulation of extracellular material is the link between early and advanced stages of atherosclerosis. Stages I, II and III can occur during the first three decades of life respectively [116] Advanced stages of atherosclerosis are described by atheromas, lesions that disrupt the intimal structure. Stage IV lesions contain a lipid core caused by the accumulation of extracellular lipids in a distinct region This core disrupts the normal intima and tissue remodeling is initiated by invasive macrophages and migrating smooth muscle cells Stage V lesions can develop

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36 into three different forms. Type V(a) have multiple layers or cores separated by thick fibrous tissue. Type V(b) lesions have a calcified lipid core, causing sever e changes in the elastic properties of the intima. Type V(c) is used to describe lesions with small or absent lipid cores but with extensive fibrous tissue. Stage VI lesions are stage V and IV lesions that have further modifications. Type VI(a) lesions have surface disruptions, type VI(b) lesions have either hematoma or hemorrhage associated with the lesion and type VI(c) lesions have thrombotic deposits. The se different types of surface modification are sometimes called complicated lesions [117]

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37 2 Construction and Characterization of Modified Vesicle Design Vesicles for targeted drug delivery can be made from several different types of materials such as natural or synthetic surfactants polymers, peptides, minerals and metals Vesicles fabricated from semiconductors and single wall nanotubes have also been used as drug carriers [118] They are very stable and have long circulation times but are not biodegradable and present possible long term health risks. Membrane vesicle designs using mole cules that self organize when place d into aqueous solutions and form non covalent structures provide a design that has been used in nature This is the design chosen for this study and the construction is described in the following sections 2.1.0 Amphiphiles Auto assembly vesicles and cell membr anes are formed from molecules called amphiphiles. These are molecules that have two different domains with opposite behaviors towards aqueous environments. Amphiphiles have one distinct region that attracts water molecules ( hydrophilic) while the other region repel s water molecules (hydrophobic) T his property makes them a surface active agent also known as a surfactant. Amphiphiles can be made from natural and synthetic surfact ants as well as peptides and co block polymers. The molecule properties can be further described by their tendency to dissolve in a n aqueous liquid (hydro philic ity ) or a fatty liquid ( lip ophilic ity ) known as the hydrophilic lip ophilic balance (HLB). The HLB is commonly described on a dimensionless scale where 0 is very lip ophilic and 20 is very hydrophilic.

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38 There are two major classes of surfactants non ionic and ionic The ionic surfactants can be cationic (positively charged) anionic (negatively charged) or zwitterionic ( both positive and negative domains in the head group ) that can be positive or nega tive depending on the pH of the solvent The ionic and non ionic surfactants have hydrophobic tail sections composed of long hydrocarbon chains that are attached to hydrophilic head sections with a large hydrodynamic radius co mpared to the tail section as shown in figure 2.0 The difference in the hydrodynamic radius of the head and tail section is responsible for the formation of the non covalent structures made from the aggregating surfactant molecules. Figure 2.0 Hydrodynamic S hell of a S urfactant M olecule On e equation describing this basic behavior of the molecule is the non dimensional critical packing parameter (CPP) Equation 2 .0

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39 In equation 2.0, V is the volume of the molecule, a is the effective head group area and l is the hydrophobic tail chain length. Amphiphiles that have a CPP value below 0 .5 will form micelle structures. C ylindrical micelles will form when the CPP is in the range of 0.3 to 0.5. W hen the CPP range is between 0.5 and 1 amphiphiles will form both vesicles and flexible bi layers Therefore, this is the target range for the surfactants used in this study When the CPP range is equal to 1 then lamella and planar structures are formed. Inverse micelle s are formed w hen value of the calculated CPP is greater than 1 2. 1.1 Non Ionic Surfactants Non i onic surfactants are synthetic amphiphiles that are commonly used as dete rgent emulsifiers. Th e two surfactants used in this study are sorbitan monostearate (Span 60) and polyethylene sorbitan monostearate (Tween 61) B oth contain fatty acid tails composed of approximately equal amounts of stearic acid ( CH 3 (CH2) 16 COOH ) and palmitic acid ( CH 3 (CH2) 1 4 COOH ) Three hydroxyl groups attached to the head groups makes the molecules hydrophilic The Tween 61 molecule has three oligomeric chains of polyoxyethylene used to tether the hydroxyl groups to the head of the molecule as shown in figure 2. 1

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40 Figure 2 .1 Molecules: Span 60 and Tween 61 2. 1 2 Cholesterol C holesterol is a steroid which exhibits a slight amphiphil i c tendency due to the hydroxyl group at the head of the ring structure as shown in figure 2. 2 Cholesterol with an HLB factor of 2.96 is used to stabilize the bilayer membrane by adding rigidity due to the planar configuration of the molecule. S teroid molecules that are closely related to cholesterol, such as cholestanol, sitosterol and stigmasterol can be used to tailor the leakage and stability of the membrane [119] Figure 2.2 Cholesterol

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41 2.2 .0 Poly ethylene G lycol Polyethylene glycol (PEG) is a hydrophilic polymer made from repeating units of ethylene oxide and is shown in figure 2. 3 The polymer is also named polyethylene oxide (PEO) and polyoxyethylene ( POE). PEG is commonly used to refer to polymers with m olecular weights below 20,000 Da while polymers with molecular weights greater than 20,000 Da are commonly referred to as PEO POE is used to describe all weights of the polymer Figure 2.3 Polyethylene G lycol (PEG) The polymer behavior is based on temperature, solvent, molecular weight and the concentration of the polymer. When t he polymer is synthesized it may have a distribution of molecular weights. S eparation techniques are used to sort the polymer chains on a size basis The polydispers i ty index (PDI) is a measure of how similar the molecules are to one another in size and weight (e quation 2.3 ) It can be calculated by the ratio in the number average molar mass ( e quation 2.1 ) an d the weigh t average molar mass (e quation 2.2 ). = ( n i M i / M i ) Equation 2.1 = ( w i M i / w i ) Equation 2.2 PDI = / Equation 2.3 PEG has been used in a variety of drug delivery appli cations. Conjugation of PEG to peptides and proteins is used modify their behavior in the body Since PEG is highly water soluble it can be used to modify proteins and drugs that are not very soluble in aqu eous solutions [120] PEG conjugation can b e used to control the membrane

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42 permeability of small molecules used as chemotherapeutic agents [121] It is also used to modify protein and peptide circulation time while mainta ining bioavailability and slowing enzymatic degradation [122 125] PEG can also be conjugated or adsorbed onto the surface of small particles or vesicles. The addition of the polymer to the su rface helps to slow the attachment of serum proteins that tag the particles for removal [126] This approach has been used in liposome production to hide them from phagocytes creating stealth liposomes [12, 15, 127 135] Another benefit of PEG conjugation is steric stabilization of the particle by physicall y separating the vesicle membrane s from one another thereby preventing vesicle fusion [136] The conformation of the grafted PEG chains is dependent on the distance between surface attachment points (D) and chain length of the polymer (R). The re are three primary conformations : pancake (D>>R) mushroom (D>R) and brush with transition phases between them. For a PEG chain of 2000 Da (PEG 2000), t he pancake conformation is typical when th e PEG surface concentrations are 1.3 mol %. Mushroom conformation is found when PEG surface concen trations are at 4.5 mol%. At higher PEG concentrations of 9 mol% the polymer interaction forces the chains to extend into the brush conformation [137] In lipos omes it has been shown that PEG 2000 with a 5 mol% surface concentration extends blood circulation times compared to liposomes without the PEG chains. For the immuno niosome vesicle design, a bifunctional PEG 2000 was used 2.3.0 Molecule Synthesis Vesicles used as d rug carriers can be constructed loaded and functionalized using a variety of methods and chemistries Drug loaded niosomes that can be functionalized

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43 after construction by using a linker molecule activated by adjustment to an alkaline pH provide a functionalization method that is simple and nondestructive to the encapsulated therapeutic agent. The surfactant is modified to form a functionalized surfactant molecule that can be use d to create the niosome vesicles. Table 2.0 Immuno n iosome Materials Material description Catalog number Manufacturer Tween 61 P 3065 Sigma Aldrich Span 60 85546 Fluka Omega Hydroxyl alpha amino PEG, MW 2000 Da HO PEG2000 NH2 JenKem Technology Co., Ltd. Cholesterol 362794 5G Sigma Aldrich N,N diisopropylethylamine, redistilled 387649 100ML Sigma Aldrich Chloroform AC61028 1000 Acros Organics Dicetyl phosphate 101546 MP Biomedicals, LLC Cyanuric chloride C95501 Sigma Aldrich 2. 3 .1 Functionalized Non Ionic Surfactant Polyoxyethyle ne sorbitan monostearate (Tween 61) is a non ionic surfactant with a fatty acid hydrophobic tail structure composed primarily of stearic acid and a hydrophilic head group with three short polyoxyethylene chains. The linker molecule cyanuric chloride ( CC ) is covalently attached to the surfactant through nucleophilic substitution to the hydroxyl group on the terminal end of one of the polyoxyet hylene chain s Tween 61 dissolved in dry chloroform and CC in a 0.8 molar ra tio of [CC:Tween

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44 61] in the presence of a 3 fold molar ratio of N,N d iisopropylethylamine ( DIPEA ) are mixed together T o for m the functionalized surfactant Tween C C, t he mixture is placed in a 50 ml round bottom flask attached to a sealed rotary evaporator under a nitrogen atmosphere for 36 hours Next, a n intermediate molecule is created by covalently binding the amine group on the bi functional PEG molecule to the previously functionalized Tween CC surfactant in a 0.9 molar ratio [PEG:Tween CC] in the presence of 2 fold m olar ratio of DIPEA. To functionalize this intermediate molecule, CC is covalently bound to the terminal hydroxyl group on the PEG chain in a 0.8 molar ratio [CC:Tween CC PEG ] in the presence of 2 fold molar DIPEA for 36 hours to form Tween CC PEG CC. The reaction steps are shown in figure 2.4.

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45 Figure 2. 4 PEGylated Surfactant Synthesis

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46 2. 3.2 Functionalized Cholesterol In addition to the functionalized PEGylated molecule described in section 2.3.1 a second functionalized molecule was synthesized to provide options for targeted dru g delivery Cholesterol is a fat soluble molecule that can be incorporated in vesicles and micelles made from non ionic surfactants. Using functionalized micelles lip ophilic and hydrophobic agents that w ould normally destabilize a bi layer membrane can be delivered to a specific target. The cyanuric chloride (CC) functionalized cholesterol provides a site for antibody conjugation that is in close proximity to the vesicle surface thereby enhancing the fusi b ility of the vesicle with the endothelial cell outer membrane. This occurs when the vesicle is less than 2nm from the cell surface and provides an additional delivery strategy The synthesis was carr ied out as previously described. T he cholesterol dissolv ed in dry chloroform was reacted for 36 hours with CC in a 1:0.9 molar ratio in the presence of DIPEA under a nitrogen atmosphere. The CC replaces the hydroxyl head group on the cholesterol molecule form ing the molecule cholesterol CC as shown in figure 2. 5

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47 F i gure 2.5 Functionalized Cholesterol Synthesis 2.4.0 Vesicle Synthesis Vesicle formation is a multi step process driven by the reduction of the amphiphile surfactant molecule energy state that forms ordered non covalent structures as descri bed in section 2.1 0. Membrane formation is a process that requires a temperature dependent phase chang e and mechanical shear forces. The materials used are a combination of surfactant functionalized surfactant cholesterol and an electrostatic agent. The ratios of materials are derived from previous studies where a 1:1:0.01 molar ratio of cholesterol surfactant and an electrostatic agent respectively, provide a stab le membrane structure. Here, dicetyl phosphate (DCP) is used as the electrostatic agent since it provide s a slight negative charge which helps the fo rmed vesicle s repe l one an other in solution.

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48 2.4.1 Thin Film Formation and Hydration The first step in vesicle formation is the production of lamella that are formed from the materials in rou nd bottom glass flasks on a rotary evaporator Functionalized and non functionalized non ionic surfactants cholesterol and dicetyl phosphate are dissolved in chloroform The mixture is reduced under a nitrogen atmosphere at low pressures. When the solution is reduced to a thin film the pressure is reduced to 50 bar for 2 hours and then purged with nitrogen under atmospheric pressure at 4 L/min to remove trace chloroform from the film. To form vesicles the thin film s are hydrat ed using an aqueous solution containing a hydrophilic drug or dye T he aqueous solution is placed in a round bottom flask on a sealed rotary evaporator that has been purged with nitrogen. The temperature is raised above the gel crystalline phase using a ho t water bath and a shear force is applied by rotation of the flask on a rotary evaporator to separate the lamella into sheets to form a c ombination of multilayer and bi layer vesicles in solution. 2.4.2 Vesicle Extrusion To control the vesicle size distrib ution a barrel extruder (LIPEX Northern Lipids Inc.) and polycarbonate membranes are used. The membranes are commercially available with uniform pore sizes ranging from 1um down to 50 nm allowing control of the desired extruded vesicle size. The extrud er is surrounded by a water jacket in order to maintain the extrusion temperature above the gel crystalline phase. The raw solution of multilayer vesicles (MLVs) and bi layer vesicles (BLVs) is loaded into the extruder and pushed across the polycarbonate m embranes using high pressure nitrogen gas Multiple passes are performed to create uniform vesicle dispersion s in the bulk solution.

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49 2.4.3 Size Exclusion Chromatography Introduction Size exclusion chromatography (SEC) is a method used to separate molecule s from a solution based on their size Other types of SEC include: gel filtration chromatography (GFC) aqueous system and gel permeation chromatography (GPC) non aqueous system used for polymer separation. Both types of SEC use a non adsorptive stationary phase to eliminate any reactive process of the sample with the stationary phase The separation process is driven by two primary phenomena, diffusion and exclusion. This is accomplished by placing the sample in a column with a porous packing material stationary media The material allow s small molecules to diffuse within the stationary packing material while the larger molecules are excluded by size and flow freely around the packing material. This difference in traveling time or rete ntion time allows for sample separation as shown in figure 2.6

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50 Figure 2.6 Size Exclusion Chromatography 2.4.3 .1 SEC Volumetric Theory The separation process is based on the variation in hydrodynamic sizes of the sample molecules to be processed and their interaction with the packing material. The porosity of the packing material and the packing geometry create the volumetric parameters that are used to describe how the sample elutes from the column. Equation 2.4 This volumetric distribution is described by equation 2.4, where K av is the average volumetric distribution coefficient, Ve is the elution volume, Vt is the total volume or the sum of the outer (Vo) and the inner volume (Vi) of th e packing material, sephadex. Vi

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51 is the inner volume of the packing material and is equal to Vt Vo. Ve Vo is the retention volume and if the packing material particles do not retain the solute (sample), then the value of Kav =0. A g raphical depiction is sho wn in figure 2.7 Figure 2.7 Size Exclusion Chromatography Elution Profile 2. 4.3 .2 SEC Methods In fluorescent dye loaded niosome production the formed vesicles must be separated from the free ( unencapsulated ) dye The bulk sample containing dye loaded niosomes and free dye is injected in a column that is packed with small porous spheres made from cross linked dextran (Sephadex) To prevent the vesicles from collapsing or swelling, th e aqueous solvent phase used is pho sphate buffere d saline solution with the same osmotic strength as the dye solvent. A n Ak ta Prime (GE H ealthcare) chromatography system equipped with a pH probe, conductivity probe, pressure sensor

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52 mercury lamp and UV filter set is used for the separation The column is a 70 cm model XK 16 and is used for separating large bulk samples up to 2 ml however we have found that sample sizes of 500 ul avoid fouling in the entrance, causing back mixing of the sample Figure 2.8 is the elution profile of a 2 ml sample where the peak spread from where the sample clogged the packing material as it entered the column. Figure 2.8 Elution Profile f rom 2 ml Sample Size Figure 2.9 is the elution profile from a 500 ul sample that was injected into the column. The narrow peak sho ws that the sample was able to pass through the entrance without fouling the packing material to the point that causes back mixing of the sample.

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53 Figure 2. 9 Elution Profile f rom 500 ul Sample Size 2.4.4 Antibody Conjugation Immuno niosomes are created when anti bodies are conjugated to the exterior surface of the niosomes. This procedure is conducted at room temperature (25 C) on a n orbi tal shaker table for 16 hours. Fir st, t he conjugation is initiated by adjusting the pH of the niosome solution to 8.8 as described by Bendas and Hood [16, 17] At pH 8.8 the chloride groups on the cyanuric chloride molecule are activated and thus a covalent bond is formed via nucleophilic substitution with one of the disulfide groups on the Fc region of the IgG as shown in figure 2.10

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54 Figure 2. 10 Antibody Conjugation After antibody conjugation the solution is loaded in a disposa ble PD 10 chromatography column packed with sephadex G 25. The immuno niosome fraction is eluted with one column volume of 0.01M phosphate buffered saline The pH of the sample is verified and adjusted if necessary. The excess unconjugated antibodies can be recovered by subsequent column elution. 2.5.0 Vesicle Metrology To determine vesicle size stability and dispersion properties a variety of me thods were used to gather data on the niosomes after synthesis The methods include chromatography, microscopy flow cytometry and dynamic light scattering techniques. 2. 5 .1 Thin Layer Chromatography Introduction Thin layer chromatography (TLC) provides an economical method of sample analysis without the need for expensive equipment and materials typically used in high performa nce liquid chromatography (HPLC) A lthough HPLC is more sensitive and can detect trace concentrations it is recommended as a follow up procedure to TLC TLC is also recommended for develop ment and optimization of the solvent system used for the

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55 separation The flat plates allow for visual inspection of the separation Samples that have components that interfere with the separation or are at concentrations below the limit of detection will need to be pre processed by separation or concentration respectively prior to using TLC. Separation can be completed using a simple solvent partitioning procedure ( F olch method [138] ) ion exchange columns [139] or solid phase extraction columns [140] The TLC separation process is based on a mobile phase composed of a combination of solvents that carry the sample up the plate by convective forces and a stationary phase acting as the adsorbent and attracting the sample to the matrix by hydrogen bonds and dipole interactions The solvent system contains polar and nonpolar solvents creating an organic/ inorganic partition on the plate T he sample is separated into its components as the mobile phase moves up the plate 2.5.1.1 Thin Layer Chromatography Methods Table 2.1 TLC Materials Item Description Manufacture r TLC Plates 20 x 20, Silica Gel 60 250um Flu orescent Indicator. Whatman Catalog No: 4861 820 TLC Tank Glass 25 cm x 25 cm Tank Fisher Micro Pipettes 1 Lambda (1 ul) Drummond # 1 000 0010 Micro Pipettes 5 Lambda (5 ul) Drummond # 1 000 0050 Atorvastatin Atorvastatin Calcium Pfizer Chloroform Solvent Fisher

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56 Table 2.1 (C ontinued ) Item Description Manufacture r Diethyl Ether Solvent Fisher Hexane Solvent Fisher Acetic Acid Solvent Fisher Methanol Solvent Fisher UV Lamp 254nm Ultra violet hand held lamp Cole Parmer Camera 35mm SLR digital EOS 20D Cannon For t he TLC separation, 20x20 cm glass plates coated with a thin layer of silica (stationary phase) impregnated with a fluorescent indicator were used When the TLC plate is exposed to ultraviolet light, the separated components block the fluorescent signal as indicated by a dark spot on the plate If the component of interest do es not fluoresce or absorb, a stain can be applied to the plate which will react with or stain the separated component so it can be imaged. At orvastatin calcium was encapsulated in a previously designed immuno niosom e. TLC analysis was used to determine the actual concentration of atorvastatin calcium within the immuno niosomes. Some of t he samples were processed prior to TLC separation to conce ntrate and separate components. Standards were applied directly to the plate using disposable glass capillary tubes (microcap ). Two solvent systems were chosen. T he first separation was completed in chloroform (a proton donor ), methanol (polar) and 4% acet ic acid in a [45:55:4] volumetric ratio T he second development was completed using hexane (as the diluent) diethyl ether (proton acceptor) and acetic acid in a [80:20:1.5] volumetric ratio The methanol and chloroform form a part ition on the

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57 plate based on their polar ity The hexane and ether are used to separate lipids and cholesterol. T he acetic acid is used as a solvent modifier t o reduce tailing of the sample during separation when lipids are present 2.5.1.2 Thin Layer Chromatography Results The plates are analyzed using a n open software routine in MATlab, that determines the spot density from an image (shown in Figure 2. 11 ) taken with a 35mm SLR digital camera [141] The plate is illuminated by a mounted 2 54 nm lamp. The focal distance of the camera, position of the lamp and position of the sample spot are held constant. Figure 2.11 TLC Image Analysis The c alibration curve in Figure 2.1 2 was generated using known concentrations of pure atorvastatin dissolved in methanol. The standards were directly spotted onto the TLC plate and then developed. The linearity of the standards (R 2 =0.9898) show s the sample

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58 follows the Beer Lambert law [142] Standards and unknowns are included on each TLC plate to allow for comparison bet ween plates To determine the actual encapsulation of atorvastatin used to treat apolipoprotein E knockout mice immuno niosome samples were dehydrated on a vacuum concentrator and reconstituted using methanol. The atorva statin dissolves freely in methanol while cholesterol and Span 60 (niosome components) do not Samples were rehydrated in 1/10 th the volume of the original sample in order to concentrate the drug Additionally, standards were separated on the same plate to provide a concentration reference. It was determined that the concentration of atorvastatin encapsulated in the immuno niosomes was 1.68E 05 m g/ml, well below the expected concentration of 0.15 m g/ml Figure 2.12 Atorvastatin Standards 2. 5.2 Fluorescent Microscopy Fluorescent microscopy is used to confirm specific binding of the immuno niosomes to cell surface receptors. The antibody conjugation to the immuno niosomes is also imaged using this technique Materials may exhibit fluorescent beha vior when they y = 1E+09x + 12.63 R = 0.9898 0 5 10 15 20 25 1E 10 2.1E 09 4.1E 09 6.1E 09 8.1E 09 Optical Density Moles of atorvastatin Atorvastatin Standards AV Standards Linear (AV Standards)

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59 have been excited by a beam of light. Fluorescence was first described by Stokes when he noticed that some minerals emit ted visible light when an invisible source light was used to illuminate them [143] Th is process can be described in three steps using a simple Jablonski diagram ( Figure 2.1 3 ). Figure 2.13 Jablonski Diagram First, the energy state of the molecule is increased by the absorption of light from a source. The second step is the release of a photon from the molecule after it has been excited by an external source The third step is the energy level drop to the normal state after the release of a photon. This energy difference between the two states is called the and was described by Alexander Jablonski in 1935 [144] Fluorescence microscopy takes advantage of the abi lity to separate a specific spectrum of light that is emitted from a molecule by using optical filters. To confirm antibody conjugation to the functionalized surfactant large vesicles (>1um) were produced with 5% functionalized PEG2000 to create the PEGyl ated immuno niosomes Immuno niosomes conjugated with flu orescein isothiocyanate (FITC) labled antibodies are shown by the arrow in f igure 2.14 ( 4 0 0X magnification )

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60 Figure 2.14 PEGylated FITC Conjugated Immuno niosomes Another type of functionalized molecule was synthesized t o provide alternative conjugation strategies that are described in section 5. 3 .0 This was accomplished using cholesterol and functionalizing the molecule with the cyanuric chloride linker chemistry Confirmation of the functionalized cholesterol was accomplished by imaging large vesicles (>1um) that were conjugated with fluorescentl y tagged antibodies. Figure 2.15 is a black and white image taken of the fluorescent tagged immuno niosomes made with 10% functionalized cholesterol (200x magnification, bright spots indicate fluorescent tagged immuno niosomes). Figure 2.15 FITC Conjugated Immuno n iosomes Using Functionalized Cholesterol 10 0um

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61 2.5.3 Flow Cytomet ry Flow c ytometry is a tech nique used to examine cells and microscopic particles in suspension. For the experiments shown below, an LSR II flow cytometer (Becton Dicki n son) was used. Cells or particles are either tagged with a fluorescent labeled antibody or a fluorescent dye can be encapsulated in the particles. The particles are suspended and move in a flowing stream through a laser path. Median fluorescent intensity and forward and side scatter from the dye and part icle is detected using a photo multiplier tube. Forward scatter dat a is used to indicate cell volume and side scatter data is used to examine the complexity of internal structures of the cell or particle. Flow cytometry is commonly used to count fluorescently labeled cells in a mixed population. In this study it was used to confirm antibody conjugation and provide a map of the population distribution of the vesicles, to confirm data collected using dynamic light scattering instrumentation. I mmuno niosome samples were conjugated with Fluorescein isothiocyanate ( FITC ) labeled goat anti rat IgG antibodies Figures 2.16 and 2.1 7 and tables 2. 2 and 2. 3 show forward and side scatter data along with median fluorescence intensity for the two populations from a sample of 5% PEGylated immuno niosomes that were hydrated with ph osphate buffered saline and conjugated with FITC labeled IgG. Figure 2.1 8 is dynamic light scattering data for the same sample illustrating the two different size vesicle populations. Previously the immuno niosomes size was controlled using extrusion techn iques and size exclusion chromatography. By combining these size and purification techniques 99.7% of the population was the same size.

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62 Figure 2. 16 5% PEG Sample Forward Scatter Distribution Figure 2. 17 5% PEG Sample Particle Size Distribution

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63 Fi gure 2.18 5% PEG Sample Dynamic Light Scattering Data Table 2. 2 5% PEG Sample Event Population Table 2. 3 5% PEG Sample Median Distribution Fluorescent dye loaded niosomes were prepared using 10% funct ionalized cholesterol To maintain large vesicle sizes, the niosomes were not extruded. However they were vortexed to create large vesicles and then separated from the free dye using P1 P2

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64 size exclusion chromatography. The different sized fractions were mixed t ogether. Forward scatter data illustrating the three different population sizes is shown in figure 2.1 9 and table 2. 4 The three different population sizes are also shown using dynamic light scattering as shown in figure 2. 20 Table 2. 4 10% Functionalized Cholesterol Median Population Figure 2. 19 10% Functionalized Cholesterol F orward Scatter Distribution

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65 Figure 2. 20 10% Functionalized Cholesterol Sample Dynamic Light Scattering Data 2.5.4 Transmission Electron Microscopy Transmission electron microscopy (TEM) is used to provide sample images with high resolution and magnification that can not be achieved with light microcopy. This is accomplished in a device that shoots a focused beam of electrons through the sample T he signal can be detected using an electronic camera or the resulting beam can be viewed on a phosphorus screen. A beam of high energy electrons emanate from an electron gun that is passed through a very thin section of mater ial to produce images. The electron beam must be under vacuum to prevent unwanted molecular interactions Table 2.5 Transmission Electron Microscopy Materials Item Description Manufacture 2% glutaraldehyde Fixative made from 50% stock Fis her 2% Osmium Tetroxide Fixative and contrast stain EMS P1 P2 P3

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66 Table 2.5 (continued) 2% Uranyl Acetate Stain EMS Acetone Solvent Fisher Ethanol Anhydrous agent Fisher Propylene Oxide S olvent EMS Embed 812 Resin EMS Araldite 502 Resin EMS Dodecenyl Succinic Anhydride Anhydride EMS 2,4,6 tridimethylamino methyl phenol (DMP 30) Accelerator EMS Lead Nitrate Stain EMS Sodium Citrate Stain EMS 1N Sodium Hydroxide Reagent Fisher 20nm Gold Particles Electron dense marker EMS 5nm Iron Oxide particle Magnetic particle Oceannanotech Copper Grids Sample holder TEM EMS Formvar Coated Grids Sample holder TEM EMS A transmission electron micrograph of the solution containing 5% PEGylated surfactants hydrated with 0.01M phosphate buffered saline is shown in figure 2.21 T he solution was stained with uranyl acetate, placed on a formvar coated carbon grid and imaged. The arrows indicate multi layer membranes from the vesicles in the bulk solution.

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67 Figure 2. 21 TEM of Bulk Solution A transmission electron micrograph of niosomes prepared with 5% PEGylated surfactant and hydrated with 0.01M phosphate buffered saline is shown in f igure 2. The vesicles were treated with osmium tetroxide to stabilize the vesicle membr ane through chemical fixation. The vesicle su spension was placed on a formvar coated carbon grid dried and imaged.

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68 Figure 2. 22 TEM of Extruded 5% PEGylated Niosomes A transmission electron micrograph of 5% PEGylated surfactant niosomes containing 5nm paramagnetic FeO particles in 0.01M phosphate buffered saline is shown in figure 2.23. The vesicle suspension was placed on a formvar coated carbon grid dried and imaged. Figure 2. 23 TEM of 5% PEGylate d FeO Loaded Niosomes

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69 2.5.5 Dynamic Light Scattering Dynamic light scattering (DLS) is also known as photon correlation spectroscopy (PCS) or quasi elastic light scattering (QLS ). DLS uses the physics of small particle behavior to correlate data from light scatte re d by the particles in solution [145, 146] The sample is placed into a c uvet te and a monochr om at ic light source such as a laser is passed through the sample ( f igure 2. 24 ). The scattered light that has collided with the particles in the sample is collected and the data is analyzed. Figure 2. 24 Dynamic Light Scattering The theory of dynamic light scattering is based on Brownian motion caused by thermal energy causing random motion of a particle in solution F or spherical particles the relationship of the radius and the diffusion coefficient ( e quation 2.5 ) is given by the Stokes Ein stein equation [147] w here the diffusion coefficient (D) is equal to the mobility of the particle ( p ) multiplied by Boltz B ) constant and the absolute

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70 temperature (T). The mobility (e quation 2.6 ) of a particle is the ratio of the pa rticle s terminal drift velocity (V p ) to an applied force (F) (e quation 2.7 ) relates the energy of the microscopic particle to the energy level of the macroscopic surroundings. (N A ). Applying Stokes law for frictional force (F d ) ( e quation 2.8 ) (e quation 2 .9 ) Using Stok es law the Einstein equation becomes a relationship of the apparent diffusion coefficient and the radius of the particle ( e quation 2.10 ) that can be rearranged to yield the apparent radius of a particle when the diffusion coefficient is known ( e quation 2.11 ) [148] D = p k B T Equation 2.5 p = V p / F Equation 2.6 k B = R / N A = 1.38 E 23 [ JK 1 ] Equation 2.7 F d = 6 RV Equation 2.8 = 6 R Equation 2.9 D = ( k B T) / ( 6 R ) [m 2 s 1 ] Equation 2.10 R App = ( k B T) / ( 6 D App ) Equation 2.11 In dynamic light scattering data analysis, t h e light scattered from the sample is used to determine the time it takes for the particle to move from its original starting position The correlation function is used to relate the change in time and the difference of the current position of the particle compared to its original position. At low concentrations, the correlation function can relate the size of the particle to the tempera ture and the viscosity of the fluid A graph of the correlation function for a

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71 niosome sample is shown in figure 2.25 On the left side near time =0 the correlation of the position of the particle to its original position is equal to one meaning the star ting position of the particle is directly related t o its current position. As time increases, this relation ship is eventually lost and there is no correlation of the current position of the particle to where it originated. The rate of decay is used to calc ulate the radius of the particles Figure 2. 25 Niosome Sample Correlation Data Niosome s containing Span 60 and 10% Tween or Span 60 and 5% P EG, were placed in sealed cuvettes and measured every 7 days over a 90 day period. Care was taken not to disturb the samples during the time course and particle sedimentation was noted in the non PEGylated niosome samples. The results from these data suggest the PEG had a positive effect in maintaining vesicle suspension and size dispers ion, shown in figure 2. 26

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72 Figure 2. 26 Data from Stability Comparison Study on Day90 5% PEG 10% Tween

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73 3 Evaluation of Receptor Conjugated Vesicles Binding studies were conducted t o investigate immuno niosome interact ion s with endothelial cells Several methods were used to gather data to e nsure the results were not biased by the detection technique. Most of the techniques were based on an encapsulated fluorescent dye to detect the cell bound immuno niosomes. Gold particle s were encapsulated for transmission electron microscopy experiments. 3.1.0 Cell Culture Primary bovine aortic endothelia l cells (BAEC) and endothelial basal media (EB M 2) were purchased from Lonza. The cells were plated at a n average density of 6.7 x10 5 ce lls/ cm 2 and grown in T 25 culture flasks (C ostar) using EBM 2 supplemented with 10% heat inactivated calf serum and 100 units/ml penicillin and 100 ug/ml streptomycin in a humidified 5% CO 2 /95% air incubator at 37 C. When the cells reached 85% confluency aliquots were frozen back in a 50:50 (V:V) mixture of supplemented EBM 2 and DMSO supplemented cryo preservative (P rofreeze Cambrex Bio Science ) Endothelial c ells used in binding experiments were allowed to proliferate in tissue culture flasks to a cell viability of 98% prior to high density plating and were used from passage 2 to passage 5. T he cells were rinsed three times with 0.01M phosphate buffered saline solution (PBS) and then were detached from the flask bottom using 4% trypsin/EDTA in 0.01M PBS for 2 minutes. The cells were col lected and resuspended in serum supplemented EBM 2 to neutralize the trypsin. The suspension was centrifuged at 900 Gs to form a cell pellet The cell pellet was resuspended in fresh supplemented EBM

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74 2 and plated onto growing surfaces of choice, i.e. flasks, glass slides, glass cover slips or 96 and 24 multi well plates. The glass slides and cover slips were cleaned with 100% ethanol (ETOH) and dried prior to steam sterilization. For some experiments, the glass su rfaces were treated with sterile 1M NaOH for 30 minutes and rinsed with 0.1M PBS three times to improve cell adhesion. 3. 2 .0 Fixed Cell Binding Fixed endothelial cells under static conditions were used t o determine the binding efficiency of the PEGylated i mmuno niosomes ( INs ) to endothelial cells. For receptor specific binding t he cells were treated with the cytokine tumor necrosis factor alpha ( TNF ) to invoke an inflammatory response in the endothelial cells for expression of the targeted cell surface receptors. This was accomplished by treating the cells with 20 ng/ml TNF 2 for 24 hours before the experiment. After incubation with TNF o prevent internalization or turnover of the expressed receptors over long time courses The cells were fixed with a solution made from 2% glutaraldehyde in 0.01M PBS to maintain the osmotic balance in the sample. The fixation causes cross linking between protein molecules G lutar aldehyde was used for the fixation because it is a larger molecule than formaldehyde and is able to bridge greater distances. For some experiments t he commercial preservative Histochoice was used since it has less auto fluorescence signal than glutaraldehy de. After fixation the cells were rinsed three times with 0.01M PBS. Some PBS was left on the cells after t he last ri nse to maintain sample moisture. It was removed just prior to immuno niosome sample application

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75 The niosome concentration is indicated by the total surfactant content of the sample. Black plastic 96 well culture plates with clear bottom s were used for the experiments using fluorescent dye to reduce signal cross talk between wells BAEC were plated and grown as previously des cribed in section 3.1.0. The cells were treated with TNF eated with a 2% solution of normal goat serum in 0.01 M PBS to block nonspecific binding. The cells were then incubated with fluorescein loaded immuno niosomes that wer e conjugated with CD44 clone KM201. The PEGylated immuno niosomes were compared to the previous immuno niosome design to determine the effect of the PEG tether used for antibody conjugation. The endothelial cells were incubated for 5, 10, 15, 30, 60 and 1 20 minutes with 1.5, 3, and 6 mM of 10% Tween immuno niosomes or 5% PEGylated immuno niosomes at 37 C Following incubation, each cell well was rinsed three times with 0.01M PBS to remove un bound immuno niosomes The plate was imaged on a Typhoon 9410 flat bed fluorescent scanner (GE Healthcare) using the 488nm laser to excite the fluorescent dye. Data was collected at 528nm with a s etting of 600volts on the photo multiplier tube a pixel size of 100 micron s and a focal plane of 3mm to account for the di stance from the bottom of the plate to the imaging surface. The cell density was determined by treating the cells with a solution of 20ng/ml propidium iodide and using excitation /emission of 535/620nm on the Typhoon The raw data was analyzed with Quantity One software For each well the fluorescent volume was calculated by taki ng 5 mm regions of interest at 5 different locations. The error bars in figures 3.0 and 3.1, indicate the standard deviation for the measurement. The result s from th e vesicle compar ison study showed an increase in the binding performance of the 5% PEGylated INs over the previous 10% Tween IN design

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76 This was most evident with the high er vesicle concentration and long er incubation times. The results of the vesicle comparison study are shown in figures 3.0 and 3.1 and the data is summarized (percent difference in binding) in table 3.0 Figure 3.0 Fixed Cell Binding: 10% Tween Fluorescein loaded IN (CD44, KM201) Figure 3.1 Fixed Cell Binding: 5% PEG Fluorescein loaded IN (CD44, KM201) 9.50E+05 1.05E+06 1.15E+06 1.25E+06 1.35E+06 1.45E+06 1.55E+06 5 10 15 30 60 120 Volume Time [minutes] 10% Tween Static Binding Results 10% Tween [6mM] 10% Tween [3mM] 10% Tween [1.5mM] 9.50E+05 1.05E+06 1.15E+06 1.25E+06 1.35E+06 1.45E+06 1.55E+06 5 10 15 30 60 120 Volume Time [minutes] 5% PEG Static Binding Results 5% PEG [6mM] 5% PEG [3mM] 5% PEG [1.5mM]

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77 Table 3.0 Static Binding Results 3. 3 .0 Live Cell Binding We sought to determine how the immuno niosomes interact ed with live cells and to confirm BAEC uptake of the immuno niosomes using CD44 as the target receptor A study c omparing the CD44 receptor to other receptors was conducted using carboxyrhodami ne 110 fluorescent dye loaded immuno niosomes conjugated with CD44 I CAM 1 or V CAM 1 antibodies C onfocal m icroscopy was used to confirm uptake using the CD44 receptor and to examine binding patterns between I CAM 1 and CD44 INs binding to the BAEC 3.3.1 M ultiwell Plate E xperiments Two different e xperiments were conducted using live cells grown in multiwell plates to provide IN and active cell surface receptor binding data For both experiments t he cells were grown and plated as previously described in section 3.1.0. In t he first experiment cells were plated in black plastic 96 well tissue culture plates with clear Time [minutes] 6mM % Diff. 3mM % Diff. 1.5mM % Diff. 5 1.21 0.84 2.12 10 1.16 1.82 1.73 15 3.11 0.44 4.63 30 1.93 2.10 6.37 60 10.76 8.03 7.96 120 17.00 9.25 12.82

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78 bottoms and grown until they were 85% confluent. This experiment was designed to determine if antibodies that target different receptors hav e similar nonspecific binding properties To determine nonspecific binding, the cells were not treated with cytokines prior to incubation with the immuno niosomes. The immuno niosomes were conjugated with I CAM 1, V CAM 1 or the CD44 antibody A control vesi cle was not conjugated with antibodies. A 3mM immuno niosome solution in M edium (DMEM) was used for the experiment. Cells were incubated with immuno niosomes or control vesicles for 0.5, 1.0, 2.0 and 4.0 hours in a humidified 5% C O 2 /95% air incubator at 37 C. Each cell well was rinsed three times with 0.01M phosphate buffered saline (PBS) to remove unbound vesicles. The plate was imaged on a Typhoon 9410 flat bed fluorescent scanner (GE Healthcare) using the 488nm laser to excite t he fluorescent dye. Data was collected at 528nm with a se tting of 600volts on the photo multiplier tube, a pixel size of 100 microns and a focal plane of 3mm The cell density was determined by treating the cells with a solution of 20ng/ml propidium iodide and imaging using an excitation/emission of 535/620nm on the Typhoon. The results of the e xperiment indicate that the non specific binding is antigen independent as shown in figure 3.2

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79 Figure 3.2 Nonspecific Bind ing Results The second experiment was designed to compare the binding performance of the different receptor targeted immuno niosomes to live cells. The cells were plated in black plastic 24 well tissue culture plates with clear bottoms and grown until they were 85% co nfluent. Control cells were not treated while the other cells were treated with 20ng/ml TNF Then all the cells were treated with a 2% solution of normal goat serum in 0.01M PBS to reduce nonspecific binding prior to incubation with the immu no niosomes The immuno niosomes were conjugated with I CAM 1, V CAM 1 or the CD44 antibody A control vesicl e was not conjugated with antibodies. A 1.5 mM immuno niosome solution in DMEM was used in the experiment. Cells and antibody conjugated immuno niosom es or control vesicles were incubated for 1, 2 and 4 hours in a humidified 5% CO 2 /95% air incubator at 37 C. Each cell well was rinsed three times with 0.01M phosphate buffered saline (PBS) to remove unbound vesicles. The plate was imaged on a Typhoon 9410 flat bed fluorescent scanner (GE Healthcare) using the 488nm laser to excite the fluorescent dye. Data was collected as previously described The cell density R = 0.9748 0.00E+00 5.00E+05 1.00E+06 1.50E+06 2.00E+06 2.50E+06 3.00E+06 3.50E+06 4.00E+06 4.50E+06 0 1 2 3 4 5 Volume Time [] hours Nonspecific Binding Results No CAM CD54, I CAM CD106, V CAM CD44 Linear (CD54, I CAM)

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80 was determined by treating the cells with a solution of 20ng/ml propidium iodide and imaging usi ng an excitation/emission of 535/620nm on the Typhoon. The results of the experiment indicate that by four hours post immuno niosome binding, the three antigens have similar binding to the endothelial cells as shown in figure 3.3 Figure 3.3 Live Binding Performances 3.3. 2 Confocal Laser Scanning M icroscopy Con focal microscopy was used to determine binding characteristics of the immuno niosomes ( INs ) to activated bovine aortic endothelial cells ( BAECs ) Confocal microscopy uses multiple focal plane s, called the z stack to reconstruct images. Each plane is scanned with a focused beam of laser light. This is accomplished one line at a time over the surface from one end of the image to the other. The software that reconstructs the images uses a setting called maximum projection. This setting uses the maximum average intensity from each plane to reconstruct the compiled image. This method is an improvement over standard fluorescent microscopy since data from the out of focus plane is not used, providing better image contrast and resolution. 5.00E+07 1.00E+08 1.50E+08 2.00E+08 2.50E+08 3.00E+08 0 1 2 3 4 5 Volume Hours Live Binding Performance ICAM Vcam CD44

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81 Two types of experiments were conducted using confocal microscopy. The cells were grown as previously described in section 3.1.0 and plated in 8 well glass tissue culture chamber slides (Lab Tek II, Fisher Scientific) In t he first experiment t he cells were activated using an injury model T he injury was created by scratching the cells in each well in a n a 200ul pipet tip 24 hours prior to incubation with the INs. The INs were loaded with 1mM carboxy rhodamine 110 dye since it is more photo stable than fluorescein based dyes The INs were conjugated with either I CAM 1 or CD44 antibodies and inc ubated with the injured cells f o r one hour. The cells were rinsed three times with 0.01M phosphate buffered sa line (PBS) solution before fixation with H istochoice fixative to preserve the cells and bound vesicles. The slides were cover slipped with a number 1.5 glass cover and mounting media containing 4',6 diamidino 2 phenylindole ( DAPI ) stain to identify the cell nucleus. DAPI is a stain that w hen bound to double stranded DNA fluo resces approximately 20 times g reater than DAPI alone. An i mage of the cells and ICAM 1 INs from the injury model is shown in figure 3.4, with arrows pointing a t the injury border. T he fluorescent intensity of across the injury (ROI 1) is shown in f igure 3.5

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82 Figure3.4 One H our S cratch I njury I CAM ( 200X M agnification ) Figure 3 .5 Graph of Fluorescent Intensity f rom Region of Interest 1 The injury model was used to compare the binding of ICAM 1 and CD44 antibodies against the receptors expressed by the i njured endothelial cells. Figure 3.6 is an image of

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83 how the regions of interest were located; t he results are shown in table 3. 1 (fluores cence standard deviation) Figure 3. 6 Injury Model Region of Interest ( 20 0X M agnification ) Table 3.1 Injury Binding Results Pixel Density CD44 I CAM 1 Injured ROI 1052828 (+/ 993.21) 383184 (+/ 325.96) Non Injured ROI 633041 (+/ 543.01) 179019 (+/ 168.65) Difference 419787 204165 Uptake of the antibody conjugated immuno niosomes into the activated endothelial cells was confirmed in the second set of experiments using confocal microscopy techniques. The INs were loaded with 1mM Carboxyr hodamine 110

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84 fluorescent dye, extruded across 200nm polycarbonate membranes and purified using size exclusion chromatography. The dye loaded niosomes had an average size of 168nm based on dynamic light scattering data. Th e INs were conjugated with CD44, IC AM 1 or VCAM 1 antibodies. The cells were grown as previously described in section 3.1.0 and plated in 8 well glass tissue culture chamber slides and grown until they were 85% confluent. The cells were treated with 20 ng/ml TNF treated with a 2% solution of normal goat serum in 0.01M PBS for one hour to reduce nonspecific binding prior to incubation with the immuno niosomes The cells were incubated with a 3mM solution of immuno niosomes in medium (DMEM) for 1, 2, 3 and 4 hours. The cells were rinsed three times with 0.01M PBS before fixation with H istochoice fixative to preserve the cells and bound vesicles. A 5 ug/ml solution of w heat germ agglutinin conjugate d to Alexa Fluor 633 (WGA Alexa Fluor 633 Invitrogen ) in 0.01M PBS was used to stain the cell membrane The slides were cover slipped with a number 1.5 glass cover slip and mounting media containing 4',6 diamidino 2 phenylindole ( DAPI ) nucleic acid stain to id entify the cell nucleus. In figures 3.7 through 3.1 3 the carboxyrhodamine 110 fluorescent dye is shown in green. Figures tagged with an (A) indicate TNF activated endothelial cells and figures tagged with a (B) indicate control endothelial cells that we re not treated with TNF Internalized dye within the cell is indicated by the arrow in figure 3.7(A). Endothelial cells with no injury or TNF treatment se r ved as the control as shown in f igure 3.7(B).

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85 Figure 3.7 (A) 4 Hour PEGylated CD44 Immuno n iosomes (B) Control, 4 Hour PEG ylated CD44 Immuno niosomes ( 630X M agnification) Figure 3.8 (A) 3 Hour PEGylated CD44 Immuno niosomes, (B) Control, 3 Hour PEG ylated CD44 Immuno niosomes (630X M agnification) A B A B

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86 Figure 3.9 (A) 2 Hour PEGylated CD44 Immuno niosomes (B) Control, 2 Hour PEGylated CD44 Immuno niosomes, (630X M agnification) Figure 3.10 (A) 1 Hour PEGylated CD44 Immuno niosomes, (B) Control, 1 Hour PEG ylated CD44 Immuno niosomes (630X M agnification) Figures 3.8 (A and B) shows endothelial cells that were incubated for 3 hours with PEGylated CD44 conjugated immuno niosomes The a rrows point at immuno niosomes that have formed clusters on lipid rafts on the cellular membrane as they are being internalized. Figures 3.9 (A and B) show endothelial cells that were incubated for 2 hours with PEGylated CD44 conjugated immuno niosomes Th e clusters of immuno niosomes have formed, but they are not as distinct as the clusters in the 3 and 4 hour incubations. This suggests that the endocytotic pathway involves lipid rafts and clustering of cell surface receptors. Figures 3.10 (A and B) show e ndothelial cells that were A B A B

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87 incubated for 1 hour with PEGylated CD44 conjugated immuno niosomes. The immuno niosomes are bound to the cell surface but have not formed clusters as seen with longer incubation times. Internalization is confirmed by the Z stack images shown in figure 3.11 (A D) showing the internalized dye in the 1 4 hour treated samples (A) from figures 3.7 3.10. Figure 3.11 (A D) Z stack of the 1 4 Hour PEGylated CD44 Immuno niosomes, (630X M agnification)

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88 To compare immuno niosome b inding to endothelial cells using a different antigen PEGylated niosomes were conjugated with VCAM 1. Endothelial cells incubated for four hours with PEGylated VCAM 1 conjugated immuno niosomes are shown in figures 3.1 2 (A and B) The arrows point at immu no niosomes that have formed cluste rs on lipid rafts on the cell membrane, similar to PEGylated immuno niosomes that were conjugated with CD44. Figure 3.1 2 (A) 4 Hour PEGylated VCAM 1 Immuno niosomes, (B) Control, 4 Hour PEGylated VCAM 1 Immuno niosomes, (630X M agnification) Confocal microscopy was also used to image immuno niosomes made from functionalized cholesterol. The steroid was functionalized with cyanuric chloride as previously described in section 2.3.2 and immuno niosomes were made with 10% functionalized cholestero l loaded with 1mM carboxyrhodamine 110 and conjugated with ICAM 1 antibodies. The cell s were grown and plated in 8 well glass tissue culture chamber slides. They were treated with 20 ng/ml TNF prior to incubation with immuno nio somes as previously described. Figure 3.1 3 (A) and (B) are images of e ndothelial cells incubated for 4 hours with immuno niosomes made A B

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89 with 10% functionalized cholesterol. The vesicles appear to have formed dense clusters on the endothelial cell surface. Figure 3.1 3 (A) 4 Hour ICAM Cholesterol Immuno niosomes (B) Control, 4 Hour ICAM Cholesterol Immuno niosomes (630X M agnification) 3.3. 3 Transmission E lectron M icroscopy Transmission e lectron microscopy ( TEM ) was used to image immuno niosomes loaded wit h albumin coated 20nm gold nano particles. G old is an electron dense material that provides a strong signal when viewed with TEM. The immuno niosomes were conjugated with antibodies that target the CD44 cell surface receptor. Endothelial cells were grown in T 25 culture flasks as previously described in section 3.1.0. The cells were treated with 20 ng/ml TNF supplemented EBM 2 for 24 hours. The cells were fixed with 2% glutaraldehyde in 0.01M phosphate buffered saline (PBS) for 15 min utes. The cells were washed three times with 0.01M PBS to remove excess glutaraldehyde. The cells were treated with 2% osmium tetroxide (OsO 4 a heavy metal compound that is used as a fixative and a stain ) for 30 minutes and rinsed with deionized water thr ee times to remove excess osmium and salts. The cells were removed from the flasks using a cell scraper and were recovered by centrifugation at 900g to form a cell pellet. Since epoxide A B

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90 resins are not miscible with water the samples must be dehydrated. T o protect the cells during this process, the dehydration is done slowly and great car e is taken to e nsure the samples do no t dry completely between steps by ensuring the sample is always covered in solution. Samples are dehydrate d for 2 minutes each step in 20 % (v/v) 30 %, 50%, 70%, 90% ethanol at 4 C followed by 100% ethanol at room temperature and 100% ethanol at 4C Prepared resin ( Appendix A note 1 ) is diluted with solvent in a 1:1 (v/v) ratio The cell pellets are immersed in this solution for one h our and agitated to ensure adequate infiltration of the cells. The cells are spun at 900g for 5 minutes to form a pellet and the excess resin is removed and replaced with pure resin at room temperature for one hour This step is repeated one additional tim e To embed the samples, t he cell pellets are then placed in labeled casting trays with pure resin and heated to 60 C for 24 48 hours to polymerize the resin. Thick sections of the embedded cells are cut and stained with semeithin stain and viewed using light microscopy to inspect the samples. Then thin sections, 50 70um are cut from the embedded samples and floated on prepared mesh grids ( Appendix A note 2) and placed sample side up for drying in p etri dishes lined with filter paper. Uranyl acetate (U A) staining of the thin sections is perfo rmed by prepping a disposable 4 well petri dish with a section of dental wax and adding a drop of prepared UA stain ( Appendix A note 3) corresponding to each grid to be stained Grids with samples are placed sample side down on the UA stain for 10 minutes. The samples are washed with d e ionized water in a series of three beakers The grids are a gitate d by moving the sample vertically in each beaker. T he washed grids are placed on the filter paper and dried before lea d staining. Lead staining is performed on dry grids as foll ows. The p etri dish is prepared as

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91 previously described. Sodium hydroxide ( NaOH ) pellets (to remove CO 2 ) are placed in the chamber with the dental wax P repared lead citrate stain is added ( Appendix A note 4). The grids are floated sample side down on the drops of lead stain for 1 3 minutes. T he grids are washed as before in a series of three beakers filled with deionized water and allowed to dry sampl e side up on filter paper A transmissio n electron micrograph taken at 30kv of gold loaded, internalized PEGylated CD44 conjugated immuno niosome is shown in f igure 3. 1 4. The arrow points to gold particles that have been internalized inside the endothelial cell. The arrow in f igure 3. 1 5 points t o a gold loaded PEGylated CD44 conjugated immuno niosome bound to the cell membrane. Because gold is not metabolized sections of tissue samples from the liver and spleen (from in vivo experiments) would provide information on how the immuno niosomes interact with the organs. Figure 3.1 4 G ol d L oaded Internalized Immuno niosomes

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92 Figure 3. 1 5 Gold Loaded Immuno niosome Bound to Cell Membrane 3. 4 .0 pH Sensitive Dye Study When the endothelial cell internalizes the targeted delivery vesicle by the process of endocytosis the process can be tracked by using a fluorescent agent that shifts spectrum with changing pH conditions. A family of pH sensitive dyes, seminaphthorhodafluor carboxylic acid (SNARF ) can be used to track such environmental pH changes. [149 156, 157 ] It was hypothesized that encapsulation of the pH sensitive dye could be used to track the different steps associated with vesicle internalizat ion [158 161] It is well understood that cellular internalization pathways utilize vacuoles that employ a pH change. This intracellular change creates a condition that causes conformational changes in proteins, thus enabling the sortin g and recycling of cellular receptors to the cell surface. Endocytosis is a multistep process that can involve mult iple transport vacuoles such as early endosomes, late endosomes and lysosomes [162] This process can be monitored by using an indicator to determine the time course of the pH shift in the micro environment. Carboxy SNARF 4 was selected for the study

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93 based on reports of SNARF 1 interacting with the liposome membrane [163] Samples of SNARF 4 dye were placed in a steady state spectrofluorometer ( ISS PC1) to gather spectral data from the dye a t different pH values. The stock solution of dye was diluted to a 1 M concentration in 0.01M phosphate buffered saline solution and t he pH was adjusted using 1N hydrochloric acid. The samples were excited at 488nm and the spectra of the 1 M SNARF 4 dye with a pH ranging from 5.45 to 7.2 were recorded as shown in figure 3. 1 6 Figure 3. 1 6 Fluorescence Spectra of 1 M SNARF 4 in PBS, pH 5.45 to 7.2 There was a strong pH dependent shift in the SNARF 4 dye spectra It shifted from 650 nm at a pH of 7.2 to 580 nm at pH 5.4. This data suggested the dye could indicate the intracellular pH changes in the different cellular compartme nts. Therefore 1 M SNARF 4 dye was encapsulated in niosomes made from Span 60 chol esterol and dicetyl phosphate. The samples were then placed into the spectrofluorometer and the spectra at various emission wavelengths were recorded. When the SNARF 4 loaded niosomes at a pH of 7.2 were excited at 488nm, the spectrum showed a peak at 520 nm 0 20000 40000 60000 80000 100000 120000 140000 160000 180000 200000 500 520 540 560 580 600 620 640 660 680 700 720 740 760 Fluorescence Intensity Emission (nm) 1uM SNARF 4 in PBS 7.2pH 7.03pH 6.79pH 6.61pH 6.28pH 6.01pH 5.79pH 5.45pH Excitation 488 nm

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94 ( f igure 3. 16 ). This result was unexpected, therefore the experiment was repeated using new samples of niosomes loaded with SNARF 4 dye. The results of t he two experiments confirmed the spectra of the SNARF 4 loaded niosomes had a peak at 520 nm when excited at 488 nm at a pH of 7.2, as shown in figure 3. 1 7 Figure 3. 1 7 Spectr um of SNARF 4 Lo aded Span 60 Niosomes at pH 7.2 The spectra of the individual components of the niosomes were recorded to determine the source of the 520 nm emission peak Cholesterol was found to fluoresce with a strong signal when placed in 0.01M pho sphate buffered saline solution, the same concentrat ion was to make niosomes at a pH of 7.2 (figure 3.1 8 ) The ring stru cture of cholesterol shown in figure 2.2 is thought to be the source of the signal. 0 100000 200000 300000 400000 500000 600000 700000 800000 450 550 650 750 Fluorescence Intensity Emission (nm) SNARF 4 Loaded Span 60 Niosomes SNARF 4 Loaded Span 60 Niosomes Excitation = 488 nm

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95 Figure 3. 1 8 Spectrum of Cholesterol 3. 5 .0 Vesicle B inding in F low In preparation for in vivo s tudies, the confirmation of the ability of PEGylated CD44 conjugated immuno niosomes to bind to endothelial cells in flow was confirmed usin g a parallel plate flow chamber (CytoDyne La Jolla, C A .) The chamber was attached to a syringe pump as shown in figure 3. 1 9 Figure 3. 1 9 Flow Cell E ndothelial cells were cultured as previo usly described in section 3.1.0 Cells were plated at a density of 2x10 3 total cells on 75 mm x 38 mm glass slides that were steam sterilized 0 50000 100000 150000 200000 250000 300000 350000 400000 450 550 650 750 Fluorescence Intisity Emission (nm) Cholesterol in 0.01M PBS Cholesterol in 0.01M PBS Excitation = 488 nm

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96 and t reated with 1M sodium hydroxide. The cells were grown to 80% confluency before treatment with 20 ng/ml TNF for 24 hours. The cells were fixed with a solution made from 2% glutaraldehyde in 0.01M phosphate buffered saline (PBS) and rinsed to remove excess glutaraldehyde A 60 ml syringe was loaded with 0.01M PBS and the system was primed with 10 ml PBS to remove any air. The slide was placed on the flooded chamber then vacuum was applied to hold the slide in place. Before starting the pump, a 2ml bolus inj ection of a 3mM solution of PEGylated CD44 conjugated immuno niosomes loaded with 5mM 5(6)carboxy fluorescein was injected upstream of the chamber The n the pump wa s started and 50 ml of PBS was eluted at a flow rate (Q) of 1 4 ml/min through the chamber. Us ing equation 3.0 the calculated endothelial cell d ynes/cm 2 u sing a viscosity 9x10 3 dyn/s*cm 2 channel height (h) of 2.2x10 2 cm and a channel width (b) of 2.5 cm [164] 2 ) Equation 3.0 Fluorescence microscopy was used to verify immuno niosome binding in flow to endothelial cells placed in the para l lel plate flow chamber. T he samples were post stained with diamidino 2 phenylindole, dihydrochloride (DAPI) to determin e the location of the cell nucleus. The a rrow s in figure 3. 20 point at bound vesicles on the endothelial cells. Figure 3. 20 PEGylated CD44 C onjugated I mmuno niosomes B ound to E ndothelial C ells U nder F low C onditions (400x M agnification)

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97 4 Enhancement of the Modified Vesicle Designs for Targeted Delivery in Vivo To determine the bio distribution of the immuno niosomes in vivo it is necessary to use a fluorescent dye where there is minimal imaging interference from living tissues, e.g. hemoglobin, collagen, cholesterol and water. A near infrared dye with a maximum emission spectrum of 800 nm was used (IRDye 800CW c arboxylate, LiCor Biosciences) 4.1.0 Multi Well Plate Imaging Free and encapsulated IRDye 800CW was imaged using an in vivo imaging system ( Xenogen IVIS Spectrum Caliper Life Sciences). Both epi and trans illumination methods were used to measure fluore scence intensity at excitation/emission 745/800 nm Figure 4.0 shows a 96 well plate with two concentrations of IRDye 800CW (A 1 3 ) 1nmol, (B 1 3 ) 4 nmol and (C 1 3 ) 0.01M phosphate buffered saline solution (PBS) The minimum IRDye 800CW concentration detecta ble using epi illuminat ion with a 5 second exposure time was 1 nmol Figure 4.0 IRDye 800CW Bulk Solutions In the next experiment, 0.01M PBS was encapsulated in 5% PEG an d 10% Tween niosomes (f igure 4.1 A1 3 and B1 3 respectively) IRDye 800CW was encapsulated in

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98 5% PEG niosomes in 1 nmol (Lane D 1 3 ) and 4 nmol (Lane C 1 3 ) concentrations Three different volumes of the samples 100 ul ( column 3) 200 ul ( column 2) and 300 ul ( column 1 ) were imaged using epi illumination (5 second exposure, F2 aperture, small binning) and the image is shown in figure 4.2. It was determined that the PBS loaded niosomes did not have a signal that would interfere with the dye, and that 4 nmol IRDye 800CW loaded niosomes were required for imaging at these settin gs. Figure 4.1 IR Dye 800CW Reference Samples Based on these results niosomes were loaded with 4 nmol IRDye 800CW, extruded across 200 nm polycarbonate films and purified using size exclusion chromatography

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99 (SEC) as described in Section 2.4.3. Three volumes of 4 nmol IRDye 800CW loaded niosomes, 300, 200, 100 ul (Row A, B, C respectively in Figure 4. 2 ) were imaged to confirm previous results The results shown in Figure 4. 2 show that the method is repeatable and that extruded, SEC purified niosom es loaded with 4 nmol IRDye800CW can be imaged using epi illumination on the Xenogen IVIS Spectrum Figure 4. 2 4nmol IRDye 800CW Loaded 5% PEGylated N iosomes 4.2.0 Phantom Mouse Imaging To determine preliminary concentrations of fluorescent dye a nd immuno niosomes for in vivo studies, a synthetic mouse called the phantom mouse was used to image samples of niosomes loaded with the IR Dye 800CW fluorescent dye. IVIS

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100 Spectrum generated epi illumination images of pipettes loaded with free and encapsulated 4, 8, and 12 fmol IR800CW dye (1,2 and 3ul of 4 n M dye) as shown in figure 4.3. Figure 4.3 IRDye 800CW Pipette Standards for Phantom Mouse

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101 Figure 4.4 Minimum Detection Limits with Epi illumination To determine the limit of detection for the IRDye800CW loaded niosomes, the phantom mouse was imaged in the ventral position with a 12 fmol IRDye 800CW loaded niosome sample for 1 second with F1 aperture, high lamp, binning set at level 4 with epi illumina tion as shown in Fig ure 4.4 For comparison the phantom mouse was placed in the dorsal position and the sam e sample was imaged using trans i llumination, shown in figure 4.5 The sample emitted a signal of 2.6E9 photons/sec/cm 2 when imaged for 5 seconds F2 aperture, high lamp, binning at level 4. Results indicate the niosome and dye concentrations are adequate for pilot in vivo imaging studies

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102 Figure 4.5 Phantom Mouse Imaged Under Trans illumination

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103 5 Discussion and Future Direction 5.1 .0 Vesicle Design and Manufacturing Many vesicle designs have been developed to transport therapeutic agents to specific sites in the body. While no one design provides a solution for every application, techniques using bilayer membranes to entrap therapeu tic agents have proven successful. Polymers provide vesicle stability, but the harsh conditions used for the polymerization process can be detrimental to the agent being entrapped. Amphiphiles made from peptides are easily metabolized in the body making th em biocompatible. However, because the molecules can be broken down so easily, the vesicle life span is low, and spontaneous release of the entrapped drug can cause toxic dosage levels. In the niosome production, the use of synthetic non ionic surfactants provides an economical and physiological advantage compared to traditional phospholipids used in liposome production. Because phospholipids have the tendency to oxidize over time, great care must be taken to ensure the product remains safe for use. Synthet ic surfactants also have an advantage because surfactants derived from natural sources require extra processing to ensure there are no pathological contaminants. The 200 nm size chosen for the niosome design provides an internal volume that can contain a substantial dosage of the therapeutic agent, yet is small enough to evade opsonization (coating of the vesicles by plasma proteins) and recognition by the reticulo endothelial system. The 200 nm size is large enough to eliminate the non specific uptake of the vesicles through the process of cellular pinocytosis. The 200 nm size also

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104 allows the vesicle to remain in suspension for an extended period of time. The additi on of polyethylene glycol to the niosome (PEGylation) provides a mechanical boundary between vesicles. This steric stabilization further enhances the stability of the niosome suspension. The PEG chains also reduce the attachment of plasma proteins to the n iosome membrane surface. This is a very important feature that has been shown by other groups to increase the circulation time of the vesicle in vivo. Targeting specific endothelial cell surface receptors allows for intravenous delivery of the vesicles. T his route delivers all of the vesicles to the blood where they will encounter the endothelial cells lining the vessel walls. CD44, an inflammatory receptor expressed by activated endothelial cells after cytokine exposure or physical injury, is the targeted receptor. The CD44 receptor is also present on many cancer cells and provides a method of delivering a therapeutic vesicle directly to the tumor. In addition to targeting CD44, ICAM 1 and VCAM 1, other inflammatory receptors expressed by endothelial cells were targeted, and bindi ng of PEGylated immuno niosomes (IN) with the three different receptors types was compared. We investigated nonspecific binding and the ability of the PEGylated immuno niosomes to bind fixed and live endothelial cells. We showed by confocal microscopy that PEGylated immuno niosomes can be internalized by the endothelial cell when they bind to the CD44 receptor (figure 3.11) This suggests that the targeting scheme is capable of delivering therapeutics to specific cells. Two methods were used to stimulate the upregulation of inflammatory cell surface receptors. One method was the exposure of the cell to the pro inflammatory cytokine, tumor necrosis factor alpha (TNF been used in vitro to mimic the natural process of inflammation. An injury model was

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105 used to investigate the capacity of the PEGylated immuno niosomes to target cells that h a ve experienced physical trauma. The concentration of atorvastatin encapsulated in immuno niosomes was d etermined using thin layer chromatography (TLC). It was found that two solvent systems were required to separate all the immuno niosome components from the atoravastatin. The atorvastatin loaded immuno niosomes were pre processed prior to TLC separation to remove the surfactant and to concentrate the sample. Using known atorvastatin concentrations, a standard curve was created. We determined that this TLC separation method gave us the ability to accurately determine concentration as indicated by the linear correlation coefficient (R 2 ) of 0.9898 between the known and unknown atorvastatin concentrations. In the previous immuno niosome design developed in our lab by Hood et al., cyanuric chloride was used to link the Tween 61 and CD44 antibody used to target t he niosome to the endothelial cells. In the work reported here, polyethylene glycol with cyanuric chloride linking chemistry was added to this previous design, creating the PEGylated immuno niosome (figure 1.0) The linker chemistry was used to attach and functionalize the PEG to the head group of the Tween 61 surfactant. The size and amount of PEG used was based on previous liposome design s [12 15] and the results from the fixed cell binding experiments (figures 3.0 and 3.1) which showed improved bindi ng of PEGylated CD44 IN as compared with CD44 IN to the activated endothelial cells. The current PEGylated INs contain PEG surfactant on both the interior and exterior of the vesicle. The immuno niosome design can be further modified by incubating niosomes

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106 with PEGylated micelles to created PEGylated immuno niosomes with the PEG only on the immuno niosome outer surface. Therapeutic agent or dye entrapment occurs during the thin film hydration process. This process time was extended from one to four hours, keeping other conditions constant, to provide a bulk solution that was more homogenous than previously obtained. Use of a more homogenous bulk solution lessened the previously seen fouling of the polycarbonate membranes used for vesicle extrusion. With a b ulk solution hydration time of one hour, the membranes in the extruder needed to be replaced after four passes. When the hydration time of the bulk solution was extended to two hours, eight ten passes through the extruder could be accomplished before the m embranes fouled. When the hydration time of the bulk solution was extended to four hours, the number of passes before the membranes fouled approached 20. The high number of passes translates to better size control of the vesicles and lower numbers of vesic les becoming entrapped in the extruder memb ranes ( less waste ) After bulk solution extrusion, the encapsulated and free therapeutic agent or dye was separated using size exclusion chromatography This type of separation results in some sample loss and sam ple dilution. Under theoretical conditions, separation of vesicles based on size is based on the assumption that there are no entrance or exit effects from the column. However, empirically we showed that when the sample was loaded on the column, the intera cti on of the bulk solution and the entrance region caused back mixing (figure 2.8) The sample volume loaded on the column was reduced from 2 ml to 500 l (figure 2.9) The reduction in sample volume loaded resulted in symmetric elution profiles where the vesicle size correlated with the elution fraction. To determine PEG

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107 incorporation efficiency in the niosomes, the bulk solution was spiked with free PEG. The spiked sample was loaded and eluted through the column The resulting elution profile showed an ex tra peak that was not present in samples that were not spiked with free PEG. These results indicated that the PEG modified surfactant was incorporated in the PEGylated niosome (results not shown) A difference in PEGylated niosome versus niosome size dist ribution was determined using dynamic light scattering. The addition of PEG to a vesicle can alter the niosome solution viscosity and change its light absorbance properties which will affect data collected using dynamic light scattering. To minimize the ef fects of the PEG on the PEGylated niosome size distribution measurements, the samples were diluted with 0.01 M phosphate buffered saline, to maintain osmolarity and prevent PEGylated niosome rupture. The large dilution of the PEGylated niosome sample cause d the sample viscosity to approach pure solvent (phosphate buffered saline). The size distribution changed by only 4 nm, indicating that the PEG did not affect PEGylated niosome size distribution and that the original measurements were valid. The PEGylated niosome size distribution was also measured using transmission electron microscopy (figure s 2.17 and 2.18 ) and f low cytometry (figure s 2.11 and 2.12 tables 2.0 and 2.1 ). The PEGylated niosome size distribution supports the original measurements using dynamic light scattering (figure 2.13) 5.2 .0 Experiment al Conclusions The binding efficiency of the PEGylated and non PEGylated immuno niosomes to tumor necrosis factor alpha (TNF activated endothelial cells was compared. The endothelial cells were pl at ed in black/clear bottom multiwe ll culture plates. 5,6

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108 Carboxyfluorescein was encapsulated in the PEGylated CD44 IN and CD44 IN to visualize binding to the endothelial cells. To prevent the endothelial cell surface receptors from being internalized or re cycled over the two hour experimental time course, the cells were fixed prior to incubation with the PEGylated CD44 IN or CD44 IN. Binding of the PEGylated CD44 IN to activated endothelial cells was increased 17% over binding of the CD44 IN t o activated en dothelial cells (figures 3.0, 3.1, t able 3.0). These data demonstrate that PEGylation of the immuno niosomes improves specific binding to activated endothelial cells. Next, experiments were conducted to determine the non specific binding of the PEGylated immuno niosomes to live endothelial cells. Fluorescent dye was encapsulated in the PEGylated CD44 IN, VCAM 1 IN and ICAM 1 IN to visualize binding. Non specific binding of the PEGylated CD44 IN, VCAM 1 IN and ICAM 1 IN to unactivated endothelial cells was measured. It was determined that non specific binding was linear over the four hour time course (Figure 3.2). These data demonstrate that non specific binding of the PEGylated IN to the unactivated endothelial cells was not antibody dependent. Next, experi ments were conducted to determine the interaction of the PEGylated immuno niosomes with live endothelial cells. Fluorescent dye was encapsulated in the PEGylated CD44 IN, VCAM 1 IN and ICAM 1 IN to visualize binding. The endothelial cells were activated wi th TNF to induce an inflammatory response. Specific binding of the PEGylated CD44 IN, VCAM 1 IN and ICAM 1 IN to the activated endothelial cells was measured. It was determined that all three PEGylat ed IN had similar binding to the activated endothelial

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109 cells over the four hour time course (Figure 3.3). These data suggest that the use of antibodies on the PEGylated IN that target CD44 is as effective as those that target VCAM 1 or ICAM 1. Next, exper iments were conducted to confirm uptake of the PEGylated immuno niosomes by activated endothelial cells. Fluorescent dye was encapsulated in the PEGylated CD44 IN, VCAM 1 IN and ICAM 1 IN to visualize binding and uptake to activated endothelial cells using a confocal microscopy The images captured provided confirmation that the immuno niosome payload was internalized by the endothelial cells (Figures 3.7 3.13 ) This was a major accomplishment that had not been seen before using the vesicle design and antib ody combination. Next, experiments were conducted to determine the interaction of the PEGylated immuno niosomes with physically injured endothelial cells using confocal microscopy The cells were injured 24 hours prior to incubation with 5,6 c arboxyrhodam ine loaded immuno niosomes that were conjugated with CD44 or ICAM antibodies. The results indicated the immuno niosomes can target endothelial cells that have sustained trauma from a physical injury (Figures 3. 4 3.5 and 3.6, Table 3.1 ) This is another im portant finding and the results suggest that injuries sustained from battle field wounds can be targeted by therapeutic immuno niosomes. This also means that the immuno niosome could deliver therapeutic agents to venous grafts after coronary surgery. 5.3 .0 Future Directions E xperiments designed to determine the performance of the PEGylated immuno niosome s in vivo have been a pproved and funded through the S ignatu re Interdisciplinary Program In Cardiovascular R esearch (SIPCVR), a pilot grant program funded by USF

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110 Health. The proposed experiment s will assist us in determining the bio distribution of the CD44, ICAM or VCAM conjugated immuno niosomes in apolipoprotein E k nockout (apoE / ) mice and wild type control mice (C57BL/6). The data will also give insig ht to the pharmacokinetics (described in section 1.1.1) of the PEGylated immuno niosomes in vivo The experimental design is to target sites of vascular inflammation caused by atherosclerosis in the apo E / mice The mice will be injected with fluorescen t dye loaded immuno niosomes fluorescent dye loaded niosomes without antibodies or free dye. Near infrared fluorescent dye, IRDye 800cw will be used so t he animals can be imaged live using the Xenogen IVIS Spectrum (described in section 4 .2.0 ). The organs will be imaged whole and then sectioned to give greater detail of the interactions and binding of the immuno niosomes with organs and cells The next experiments will be based on the information gained from the pilot study. To improve endothelial cell b inding in large arteries modification to the immuno niosome design will be made using the functionalized cholesterol (described in section 2.3.2) and a biotinylated PEG to conjugate both antibodies and sel ectins, respectively. The se modification s will provide a design that will allow the immuno niosome to attach with low affinity to the vessel wall via selectin binding and then roll to a si t e of inflammation where the antibody can bind with high affinity, mimicking leukocyte binding

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125 Bibliography 1. Rene P. Schwarzenbach, P.M.G., Dieter M. Imboden, ENVIRONMENTAL ORGANIC CHEMISTRY Second ed. 2003, Hoboken, New Jersey: John Wiley and Sons, Inc. 1313. 2. C.G. Caro, T.J.P., R.C. Schroter, W.A. Seed, The MECHANICS of the CIRCULATION 1978, Walton Street, Oxford OX2 6DP: Oxford University Press. 527. 3. Saltzman, W.M., Drug Delivery ed. K.E. Gubbins. 2001, 198 Madison Avenue, New York, New York 10016: Oxford University Press. 372. 4. B. Bonavida, G.R.G., H. Kirchner, L.J. Old, TUMOR NECROSIS FACTOR/CACHECTIN and RELATED CYTOKINES 1988, P.O. Box CH 4009 Basel, Switzerland: S. Karger AG. 276. 5. D.L. Nelson, M.M.C., Lehininger PRINCIPLES OF BIOCHEMISTRY Fourth ed. 2005, 41 Madison Avenue, New York, New York 10010: W.H. Freeman and C ompany. 6. M.P Lefranc, G.L., THE IMMUNOGLOBULIN FactsBook 2001, 525 B street, Suite 1900, San Diego, California 92101 4495: Academic Press. 457. 7. Cazes, J., Handbook of Thin Layer Chromatography Second ed, ed. B.F. J. Sherma. Vol. 55. 1996, 270 Madi son Avenue, New York, New York 10016: Marcel Dekker, Inc. 1104. 8. Hamley, I.H., INTRODUCTION TO SOFT MATTER 2000, Baffins Lane, Chichester, West Sussex PO19 UD, England: John Wiley and Sons. 342. 9. J.H. Fuhrop, J.K., MEMBRANES and MOLECULAR ASSEMBLIES : THE SYNKINETIC APPROACH Monographs in Supramolecular Chemistry, ed. F. J.F. Stoddart. Vol. 5. 1994, Thomas Graham House, Science Park, Milton Road, Cambridge CB4 4WF, UK: The Royal Society of Chemistry. 227. 10. Huglin, M.B., LIGHT SCATTERING FROM POLY MER SOLUTIONS Physical Chemistry, ed. M.B. Huglin. Vol. 27. 1972, 111 Fifth Avenue, New York, New York 10003: Academic Press Inc. 885. 11. D.A. Lauffenburger, J.J.L., RECEPTORS 1996, 198 Madison Avenue, New York, New York 10016: Oxford University Press, Inc. 365.

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127 Appendices

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128 Appendix A : Transmission Electron Microscopy Notes note 1: Embedding resin is made by combining 13.75 grams of Embed 812, 8.5 grams Araldite502 and stirring thoroughly without introducing bubbles. Then the resin is activated by stirring in as before 27.5 grams of DDSA and 0.7 0.9 grams of DMP 30. note 2: The grids are prepared by sonicating them in technical grade acetone for one minute followed by reagent grade acetone for one minute and finally in 95% ethanol. Decant ethanol and invert petri dish over filer paper until grids fall from the dish and have completely dried. note 3: To make 50 ml of UA stain heat 50 ml DiH2O to 60 degrees Celsius. Under fume hood add 0.5 grams of UA (CAUTION)to the hea ted DiH2O. Allow to cool and do not agitate before use. note 4: Simple lead citrate is prepared by adding 0.01 0.04 grams of lead citrate to 10ml of freshly boiled and cooled H2O in a screw top vial. Then add 0.1ml of 10N NaOH and shake until lead dissolve s.

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About The Author John Elliott received a n AA in Computer Science from Saint Petersburg College in 2002. He continued his education and received a B.S. and M.S. in Chemical Engineering from the University of South Florida, in 2006. He then enrolled in the PhD program and has worked as a member of a multidisciplinary team working on a project Department of Defense. He has presented his work at several conferences during his undergraduate and graduate studies. He prese nted at the 3rd and 4th Annual USF College of Engineering Undergraduate Research Symposium, 2005 and 2006. He has presented at the 16th and 19th USF Health Research Day, 2006 and 2009. He presented his graduate work at the Central Society for Clinical Rese arch and Midwestern Section American Federation for Medical Research (CSCR/MWAFMR) Joint Meeting, 2009. He also presented at the Association of American Physicians and the American Society for Clinical Investigation, American Physicians Scientists Associat ion (ASCI/AAP), (APSA) 2009, and at the 25th Southern Biom edical Conference, 2009. He plans to continue his research in targeted drug delivery after graduation.