Citation
Fabrication, Characterization, and Co-Localization of Drug-Loaded Plga Microparticles on Microarray Platforms

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Title:
Fabrication, Characterization, and Co-Localization of Drug-Loaded Plga Microparticles on Microarray Platforms
Creator:
Agrawal, Nikunj Kumar
Place of Publication:
[Gainesville, Fla.]
Florida
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University of Florida
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english
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1 online resource (100 p.)

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Biomedical Engineering
Committee Chair:
KESELOWSKY,BENJAMIN G
Committee Co-Chair:
RINALDI,CARLOS
Committee Members:
BRUSKO,TODD MICHAEL
Graduation Date:
8/9/2014

Subjects

Subjects / Keywords:
Antigens ( jstor )
Cells ( jstor )
Cytokines ( jstor )
Dendritic cells ( jstor )
Encapsulation ( jstor )
Immunology ( jstor )
In vitro fertilization ( jstor )
Polymers ( jstor )
Solvents ( jstor )
Type 1 diabetes mellitus ( jstor )
Biomedical Engineering -- Dissertations, Academic -- UF
diabetes -- microarrays -- microparticles -- plga
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Biomedical Engineering thesis, M.S.

Notes

Abstract:
Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease occurring in the pancreatic islets and accounts for diabetes in children and adolescents. T1DM is associated with pathogenic action of self -reactive effector T cells (Teffs) on the beta cells. However, several recent studies have shown that the disease pathology is also attributed to a second T cell subset, known as regulatory T Cells (Tregs), which plays a critical role in the development of T1DM. Newer therapeutic approaches try to maintain the homeostatic balance between Teff and Treg cells in patient affected with T1DM. Of particular interest to our research group is DC based immunotherapy approach where we aim to utilize the tolerogenic potential of DCs as therapeutic agents for autoimmune diseases and transplant rejection. A number of biological and pharmaceutical agents, including IL-10, thymic stromal lymphopoietin (TSLP), and transforming growth factor (TGF-beta) have been reported to promote DCs with capacity to instigate CD25+ T cell proliferation and differentiation from CD25- T cells. Therefore, our lab is interested in investigating the induction of tolerogenic DCs via use of several drugs of interest (Aspirin, Curcumin, Epigallocatechin gallate, Menadione, Ergosterol, TGF Beta) which will help in increasing the levels of tolerogenic signaling, thereby inducing production of Tregs. One way to facilitate the investigation of small molecules and their potential in up regulating the tolerogenic DC subset is by synthesizing drug loaded biodegradable microparticles and investigating them in a high throughput manner. Screening of immune cell response towards these microparticle based vaccines on a microarray platform is therefore of interest to our group. Microarray technology has emerged as a valuable tool in biological sciences, particularly for high-throughput applications. Furthermore, it helps in economical savings by using smaller volume of expensive reagents, high throughput analysis of data, portability and waste reduction as compared to conventional biological assay that use traditional 12/24/96/384 multi-well plates. This further leads to the long envisioned reality of personalized medicine where better treatment can be offered to individuals based on their responding behavior rather than the name of the disease they are suffering from. ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (M.S.)--University of Florida, 2014.
Local:
Adviser: KESELOWSKY,BENJAMIN G.
Local:
Co-adviser: RINALDI,CARLOS.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2015-08-31
Statement of Responsibility:
by Nikunj Kumar Agrawal.

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Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Embargo Date:
8/31/2015
Classification:
LD1780 2014 ( lcc )

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1 FABRICATION, CHARACTERIZATION, AND CO LOCALIZATION OF DRUG LOADED PLGA MICROPARTIC LES ON MICROARRAY PLATFORMS By NIKUNJ K. AGRAWAL A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2014

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2 © 2014 Nikunj K . Agrawal

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3 To Grandpa Om

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4 ACKNOWLEDGMENTS This work would not have been possible without the help, support and encouragement of several people. Firstly, I would especially like to express my sincerest gratitude to my graduate advisor Dr. Benjamin Keselowsky. Under his mentoring, guidance, positive and inspiring advice, I have matured as a research engineer. Thanks to his guidance, I feel I have become a stronger, more independent student and I will have a better approach to my future research projects. I would like to express my gratitude to my su pervisory committee, Dr. Carlos Rinaldi and Dr. Todd Brusko for having accepted to examine this work and all their useful suggestions. Their diverse scientific backgrounds afforded me the exceptional opportunity of expert advice. Thank you for your guidanc e and constructive suggestions. I thank Matt Carstens for being a mentor and guiding me throughout the project, helping me setting up my experiments, reviewing my results and for being so patient. I would also like to express my appreciation to my other c olleagues in the laboratory for their understanding and support during my studies. Thank you Jamal Lewis, Evelyn Bracho, Josh Stewart, Robert Gresham, Sufi Mohsin, Lawrence Fernando, Lirong Yang and Azedeh. To my family for always being a driving force fo r me and I would like to express my deepest gratitude to my parents, Ajai and Sushma and Grandma Saroj. I acknowledge the love, support and unremitting encouragement I received from Uncle Satendra, Aunt Barb, Aunt Karen, Aunt Lynn, Uncle Ravi, Aunt Sangeet a and Uncle Sandeep. I thank Alice, Abhijit, Immanuel, Niraj, Rahul, Ankit, Chris, Antonietta for their without their support.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF FIGURES ................................ ................................ ................................ .......... 8 LIST OF ABBREVIATION S ................................ ................................ ........................... 10 ABSTRACT ................................ ................................ ................................ ................... 13 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 15 Background ................................ ................................ ................................ ............. 15 Type 1 Diabetes Mellitus ................................ ................................ .................. 15 Dendritic Cells ................................ ................................ ................................ .. 17 Tolerogenic Dendritic Cells ................................ ................................ ............... 19 Regulatory T Cells ................................ ................................ ............................ 22 Specific Aims ................................ ................................ ................................ .......... 25 Specific Aim 1: Fabrication and Optimization of Drug Loaded Biodegradable PLGA Microparticles ................................ ................................ ..................... 26 Specific Aim 2: Particle Characterization: Evaluation of Encapsulation Efficiency and In Vitro Release Profile of Drug Loaded PLGA Microparticles ................................ ................................ ................................ 26 Specific Aim 3: Development of Microarray and Microwell Platforms with Co localization of Drug Loaded MPs for Investigation of Dendritic Cell Responses in High Throughput ................................ ................................ ..... 27 2 FABRICATION AND OPTIMIZATION OF DRUG LOADED BIODEGRADABLE PLGA MICROPARTICLES ................................ ................................ ..................... 29 Background ................................ ................................ ................................ ............. 29 Biodegradability and Biocompatibility ................................ ............................... 29 Poly (Lactic Co Glycolic) Acid (PLGA) ................................ ............................. 30 Fabrication Techniques ................................ ................................ .................... 32 Solvent evaporation method: ................................ ................................ ..... 33 Phase separation (coacervation) ................................ ............................... 34 Spray drying: ................................ ................................ .............................. 35 Experimental P rocedure ................................ ................................ ......................... 35 Preparation of 1 2 µm Drug Loaded PLGA Microparticles ............................... 35 Preparation of 10 µm Rhodamine Dye Loaded PLGA Microparticles ............... 36 Preparation of 1 2 µm LPS Microparticles ................................ ........................ 37 Particle Size Measurements ................................ ................................ ............. 38 Re sults ................................ ................................ ................................ .................... 39

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6 3 PARTICLE CHARACTERIZATION EVALUATION OF ENCAPSULATION EFFICIENCY AND IN VITRO RELEASE PROFILE OF DRUG LOADED MICROPA RTICLES ................................ ................................ ................................ 41 Background ................................ ................................ ................................ ............. 41 Aspirin ................................ ................................ ................................ .............. 41 Curcumin ................................ ................................ ................................ .......... 43 EGCG ................................ ................................ ................................ ............... 45 Vitamin D3 ................................ ................................ ................................ ........ 47 Ergosterol ................................ ................................ ................................ ......... 49 Menadione ................................ ................................ ................................ ........ 50 Compounds of Future Interest ................................ ................................ .......... 51 TGF beta ................................ ................................ ................................ .... 51 AhR compounds ................................ ................................ ........................ 53 PDE inhibitors ................................ ................................ ............................ 54 Drug Release Behavior ................................ ................................ .................... 55 Experimental Procedure ................................ ................................ ......................... 56 Characterization of Microparticles ................................ ................................ .... 56 Efficiency of drug encapsulation ................................ ................................ 56 Degradation of microparticles ................................ ................................ .... 57 Results ................................ ................................ ................................ .................... 58 4 DEVELOPMENT OF MICROARRAY AND MICROWELL PLATFORMS WITH CO LOCALIZATION OF DRUG LOADED MPs FOR INVESTIGATION OF DENDRITIC CELL RESPONSES IN HIGH THROUGHPUT ................................ .. 69 Background ................................ ................................ ................................ ............. 69 Microarrays ................................ ................................ ................................ ....... 69 Non Fouling Surfaces ................................ ................................ ....................... 70 Polydimethylsiloxane (PDMS) ................................ ................................ .......... 71 Microwells ................................ ................................ ................................ ......... 72 Miniaturization Technique ................................ ................................ ................. 74 Experimental Procedure ................................ ................................ ......................... 75 Particle Array Fabrication ................................ ................................ ................. 75 Preparation of PDMS Microwells ................................ ................................ ...... 78 Cell Seeding ................................ ................................ ................................ ..... 79 Staining and Ima ge Analysis ................................ ................................ ............ 8 0 Results ................................ ................................ ................................ .................... 80 LIST OF REFERENCES ................................ ................................ ............................... 86 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 100

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7 LIST OF TABLES Table page 3 1 Mass factor, encapsulation efficiency and 4 weeks release of PLGA microparticles loaded with curcumin. ................................ ................................ .. 59 3 2 Mass factor, encapsulation efficiency and 4 weeks release of PLGA microparticles loaded with aspirin. ................................ ................................ ...... 61 3 3 Mass factor, encapsulation efficiency and 4 weeks release of PLGA microparticles loaded with EGCG. ................................ ................................ ...... 63 3 4 Mass factor, encapsulation efficiency and 4 weeks release of PLGA microparticles loaded with menadione. ................................ ............................... 65 3 5 Mass factor, encapsulation efficiency and 4 weeks release of PLGA microparticles loaded with ergosterol. ................................ ................................ 67 3 6 Mass factor, encapsulation efficiency and 4 weeks release of PLGA microparticles loaded with immuno modulatory agents. ................................ ..... 68

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8 LIST OF FIGURES Figure page 1 1 Dendri ................................ ......................... 20 2 1 Molecular structure of PLGA molecule. ................................ .............................. 30 2 2 Particle size distribution of 1 2 µm PLGA microparticles. ................................ ... 39 2 3 Particle size distribution of 10 µm PLGA microparticles. ................................ .... 40 3 1 Molecular structure of aspirin ................................ ................................ .............. 41 3 2 Molecular structure of curcumin ................................ ................................ .......... 43 3 3 Molecular structure of EGC G ................................ ................................ .............. 45 3 4 Molecular structure of 1,25 dihydroxyvitamin D3 ................................ ................ 48 3 5 Molecular structure of ergosterol ................................ ................................ ........ 49 3 6 Molecular structure of menadione. ................................ ................................ ..... 50 3 7 Curcumin encapsulation standard curve. ................................ ........................... 58 3 8 Curcumin release standard curve. ................................ ................................ ...... 58 3 9 In vitro release profile of curcumin. ................................ ................................ ..... 59 3 10 Aspirin encapsulation standard curve. ................................ ................................ 60 3 11 Aspirin release standard curve. ................................ ................................ .......... 60 3 12 In vitro release profile of aspirin. ................................ ................................ ......... 61 3 13 EGCG encapsulation standard curve. ................................ ................................ 62 3 14 EGCG release standard curve. ................................ ................................ ........... 62 3 15 In vitro release profile of EGCG. ................................ ................................ ......... 63 3 16 Menadione encapsulation standard curve. ................................ ......................... 64 3 17 Menadione release standard curve. ................................ ................................ ... 64 3 18 In vitro release profile of menadio ne. ................................ ................................ .. 65 3 19 Ergosterol encapsulation standard curve. ................................ .......................... 66

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9 3 20 Ergosterol release standard curve. ................................ ................................ ..... 66 3 21 In vitro release profile of ergosterol. ................................ ................................ ... 67 4 1 Molecular structure of a poly (dimethylsiloxane). ................................ ................ 71 4 2 Microparticle/dendritic cell (MP/DC) array fabrication. ................................ ........ 77 4 3 Geometric representation of the silicon mold. ................................ ..................... 78 4 4 Microarray and microwell platforms. ................................ ................................ ... 79 4 5 Quantitation of 10µm microparticle printing on microarray chip. ......................... 80 4 6 Linear fit slope for 10µm microparticles printed on microarray chip. .................. 81 4 7 Quantitation of 1 2µm microparticle printing on microarray chip. ....................... 81 4 8 Linear fit slope for 1 2µm microparticles printed on microarray chip. ................. 82 4 9 Quantita tion of 10µm microparticle printing on microarray well. ......................... 82 4 10 Linear fit slope for 10µm microparticles printed on microarray well. ................... 83 4 11 Quantitation of 1 2µm microparticle printing on microarray well. ........................ 83 4 12 Linear fit slope for 1 2µm microparticles printed on microarray well. .................. 84 4 13 Fluorescence images of serial 1:2 dilutions of rhodamine dye Loaded PLGA MPs printed on a microarray ................................ ................................ ............... 84 4 14 Fluorescence micrograph of co localized Fadu cells and microparticles. ........... 85

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10 LIST OF ABBREVIATIONS AhR Aryl hydrocarbon r eceptor APC Antigen presenting c ells BMDC Bone m arrow derived dendritic cell cAMP C yclic adenosine monophosphate CERB cAMP response element binding protein Cdc Conventional or classical dendritic c ell COX Cyclo oxygenase CTLA Cytotoxic T lymphocyte a ntigen CurcDC Curcumin treated dendritic c ells DC Dendritic c ells DCM Dichloromethane DI Deionized DIM 3, 3 diindolylmethane DNA Deoxyribonucleic acid EAE Experimental autoimmune e ncephalomyelitis EGCG Epigallocatechin gallate FasL Fas l igand FoxP3 For khead winged h elix p rotein 3 GDF Growth differentiation factors GM CSF Granulocyte macrophage colony stimulating factor HIV Human immunodeficiency v irus Hu Mo DC Human monocyte derived dendritic c ells I3C I ndole 3 carbinol iDC Immature dendritic c ells

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11 IDO Indoleamine 2, 3 dioxygenase IL Interleukin ILT Immunoglobulin like transcript iNKT I nvariant natural killer T itDC Induced tolerogenic dendritic c ells iTreg Induced T regulatory cells JDRF Juvenile diabetes research foundation LOC Lab on a chip LPS Lipopolysaccharide MatDC Mature dendritic c ells MC Methylene c hloride MP Microparticle MHC Major histocompatibility c omplex NF kB Nuclear factor kappa b eta NOD Non obese diabetic NSAID Non steroidal anti inflammatory d rug ntDC Natural tolerogenic dendritic c ells nTreg natural T regulatory cells PCL polycarprolactone pDC Plasmacytoid dendritic c ell PDEi Phophodiesterases inhibitor PDL Programmed death l igand PDMS Poly dimethylsiloxane PGA Poly glycolic acid

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12 PGE 2 Prostagland in E2 PKA P rotein kinase A PLA Poly lactic acid PLGA Poly lactic co glycolic acid PVA Polyvinyl alcohol RA Retinoic a cid RNA Ribonucleic acid ROS Reactive oxygen s pecies T1DM Type 1 diabetes m ellitus TCDD 2, 3, 7, 8 Tetrachlorodibenzo pdioxin TCR T cell receptor tDCs Tolerogenic dendritic c ells Teff Effector T cells Transforming growth factor b eta TH T helper TLR T lymphocyte r eceptor Tumor necrosis f TSLP Thymic stromal l ymphopoietin Treg Regulatory T cells TRAIL TNF related apoptosis inducing ligand

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13 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science FABRICATION, CHARACTERIZATION, AND CO LOCALIZATION OF DRUG LOADED PLGA MICROPARTICLES ON MICROARRAY PLATFORMS By Nikunj K. Agrawal August 2014 Chair: Benjamin G. Keselowsky Major: Biomedical Engineering Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease occurring in the pancreatic islets and accounts for diabetes in children and adolescents. T1DM is associated with pathogenic action of self However, several recent studies have shown that the disease pathology is also attributed to a second T cell subset, known as regulatory T Cells (Tregs), which plays a critical role in the development of T1DM. Newer therapeutic approaches try to maintain the homeostatic balance between Teff and Treg cells in patient s affected with T1DM. Of particul ar interest to our research group is a DC based immunotherapy approach where we aim to utilize the tolerogenic potential of DCs as therapeutic agents for autoimmune diseases and transplant rejection. A number of biological and pharmaceutical agents, inc luding IL 10, thymic stromal lymphopoietin (TSLP), and transforming growth factor (TGF reported to promote DCs with capacity to instigate CD25+ T cell proliferation and differentiation from CD25 T cells. Therefore, our lab is interested in in vestigating the induction of tolerogenic DCs via use of several drugs of interest (Aspirin, Curcumin,

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14 Epigallocatechin gallate, Menadione, Ergosterol, TGF Beta) which will help in increasing the levels of tolerogenic signaling, thereby inducing production of Tregs. One way to facilitate the investigation of small molecules and their potential in up regulating the tolerogenic DC subset is by synthesizing drug loaded biodegradable microparticles and investigating them in a high throughput manner. Screening o f immune cell response towards these micropar ticle based vaccines on a micro array platform is therefore of interest to our group. Microarray technology has emerged as a valuable tool in biological sciences, particularly for high throughput applications. F urthermore, it helps in economical savings by using smaller volume of expensive reagents, high throughput analysis of data, portability and waste reduction as compared to conventional biological assay that use traditional 12/24/96/384 multi well plate s. Th is further leads to the long envisioned reality of personalized med icine where better treatment can be offered to individuals based on their responding behavior rather than the name of the disease they are suffering from .

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15 CHAPTER 1 INTRODUCTION Background Type 1 Diabetes M ellitus Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease that results from T cell mediated destruction of insulin producing beta cells in islets of pancreas [ 1 ] . This leads to hypo insulinemia, and severely altered glucose homeostasis. Individuals become hyperglycemic due to loss in critical produce insulin, a hormone that helps the body move the glucose (i.e., sugar) contained mass is unable to match insulin deman ds of the body [ 2 ] . The common symptoms are polyuria (frequent urination), polydipsia (increased thirst), polyphagia (increased hunger), and weight loss. It is also known as insulin dependent diabetes and juvenile diabetes. As per the Juvenile diabetes research foundation (JDRF), each year, more than 15,000 children and 15,000 adults approximately 80 people per day are diagnosed with T1DM in the U.S. alone. The prevalence of T1DM in Americans under age 2 0 rose by 23 percent between 2001 and 2009. T1DM accounts for $14.9 billion in healthcare costs in the U.S. alone each year. The socioeconomic impact of T1DM, in terms of personal, reduced productivity and related health care cost is huge. For instance, ch ildren faced with a lifetime of glucose monitoring and insulin injections have approximately twice the healthcare cost than children without T1DM [ 3 5 ] . T1DM is associated with pathogenic action of self reactive effector T cells

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16 is also attributed to a second T cell subset, known as regulatory T Cells (Tregs), which plays a critical role in the development of T1DM [ 6 ] . Under normal circumstances the Tregs counterbalance the auto reactive action of Teff, thus maintaining immune homeostasis. The important reason for the development of T1DM is the imbalance in the activity of Teff and Treg cells . In response to antigen presentation and co stimulation by Antigen presenting cells (APC) , CD4+ T lymphocytes in the local pancreatic lymph nodes proliferate and differentiate into auto reactive CD4+ effector T cells (Teffs). Within the pancreatic islets , these activated Teffs release a host of cytokines including interferon ( IFN ) and interleukin 2 ( IL 2 ) , resulting in recruitment of cytotoxic macrophages and CD8+ T lymphocytes [ 7 ] . Cytotoxic inflammatory cells ultimately infiltrate and destroy the isle t M patients require life long insulin supply and are at high risk for heart disease, strokes, ki dney failure, blindness, etc. which decreases the life expectancy and contributes to early mortality for these patients. Type 1 d iabetes is different from T2D M in the way that it can only be treated by taking insulin delivered either via multiple syringe injections subcutaneously (under the skin) or through an insulin pump. Traditionall y, life long insulin replacement via several delivery methods such as sub cutaneous, nasal and oral etc. have been used to manage glucose levels in type 1 diabetes mellitus patients [ 8 10 ] . However, inconvenience associated with continuous injection of insulin to control blood glucose levels, excessive cost of care and physiologic complications like obesity and hypogl ycemia has led to the need for the development of innovative new therapies for

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17 T1DM within the past 3 decades [ 8 , 11 ] . Some of the newer approaches to maintain the homeostatic balance between Teff and Treg cells in patient s affected with T1DM include generation of immunological tolerance with the help of transplantation of insulin producing organ (pancreas) and cells [ 10 , 12 ] . However there are several associated risks such as limited donor availability and long term immuno suppressive drug treatment [ 10 , 13 ] . Currently, there are no means to prevent the events that initiate the destruction of o there is no therapeutic approach to control the stages of T1DM. This residual m ass presents a potential therapeutic opportunity to reverse T1DM by focusing on blocking Teff activity during early period of TIDM and inducing the formation of Treg cells. ets of pancreas seems to be an attrac tive strategy to treat T1DM going forward. Of particular interest to our research group is a DC based immunotherapy approach. In this personalized medical procedure, blood derived monocytes are extracted from patients, manipulated ex vivo to produce antige n specific, tolerogenic DCs which are then replaced into the prevent autoimmune disease [ 14 ] . In accordance with this perspective, there has been emergence of therapies that increase the activity of Tregs and restore the balance between the Treg and Teff in order t o maintain immunological peace. Dendritic C ells Dendritic cells (DCs) are professional antigen presenting cells which are a subset of leukocytes and have mostly been studied as potent stimulators of adaptive immunity

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18 [ 15 ] . DCs were discovered by Steinman and Cohn in 1973 and ever since their discovery they have become increasingly recognized for their role as ke y regulator of innate and adaptive immunity [ 16 ] . Their purpose is to acquire, process and present antigen to T cells and induce the activation and differentiation of naïve T cells into effector and memory cells [ 15 ] . DCs significantly contribute to central and T cell peripheral tolerance. Pattern recognition receptors like Toll like receptors (TLRs) present on DCs helps in recognizing pathogen. Once recognized, DCs migrate to T cell areas of lymphoid organ to present pathogen derived antigen to antigen specific T cells [ 17 ] . Activated DCs up regulate co stimulatory molecul es and produce cytokines that d rive T cell priming and differentiat ion. However In the absence of activation, antigen presentation by steady state DCs might lead to T cell unresponsiveness and might promote tolerance [ 17 ] . of auto antigens that initiate and drive di sease progression. islet cell derived antigens can be captured by DCs and presented to auto reactive T cells after the migration of DCs to the pancreatic lymph nodes, thereby in islet cell derived antigens to T cells in the pancreatic lymph nodes, but why they induce active immune responses rather than tolerance in response to self antigens is not yet clea rly understood [ 17 ] . DCs are exquisitely adept at acquiring, processing and presenting antigens to T cells. They also adjust the context (and hence the outcome) of antigen presentation in

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19 response to a plethora of environmental inputs that signal the occurrence of pathogens or tissue damage. Such signals generally boost DC maturation, which promotes their migration from peripheral tissues into and within secondary lymphoid organs and their capacity to induce and regulate effector T cell responses [ 16 ] . Tolerogenic Dendritic C ells The importance of Tregs in controlling the immune response and ensuring immunological peace has been investigated thoroughly, though there is much more to learn of this critical cell type. Failure of Tre gs has resulted in the development of a host of autoimmune diseases [ 18 ] . By contrast, cellular therapy of adoptive transfer of Tregs has shown efficacy in many of these disorders [ 19 ] . It is therefore important to understand the role of DCs in Treg activation and differentiation for the development of therapeutic strategies in several disease settings. The race is now to utilize the tolerogenic potential o f DCs as therapeutic agents for autoimmune diseases and transplant rejection. DCs can prevent, inhibit or modulate T cell mediated effector responses through a variety of mechanisms ranging from the production of pleiotropic anti inflammatory factors that exert broadly attenuating effects, deletion of self reactive T cells mediated by thymic DCs (central tolerance), and T cell anergy induction and generation of Treg cells for peripheral tolerance [ 20 , 21 ] . Tolerogenic DCs include immature, maturation resistant or alternatively activated DCs th at express surface MHC class I and class II molecules, have a low co stimulatory to inhibitory signal ratio and have an impaired ability to synthesize T helper 1 (TH1) cell driving cytokines (such as interleukin 12p70). Various anti inflammatory and immuno suppressive agents potentiate or confer tolerogenicity on DCs (in vitro or in vivo).

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20 Naïve T cell s require three concomitant inputs to differentiate into full fledged effector cells (Teffs): signal 1 is the antigenic stimulus provided by MHC molecules dis playing a cognate peptide; signal 2 is provided by costimulatory molecules; and signal 3 is provided by cytokines produced by DCs or other microenvironmental sources [ 22 ] . As mentioned above, iDCs are typically tolerogenic [ 23 ] , so the maturation status or rather, the absence of maturation provides a hint for the tolerogenic capacity of DCs. Classic immunobiology describes the basis of T cell anergy to be the lack of concomitant costimulation or c ytokines (signal 2 and 3). Figure 1 1 Dendritic cell and T cell interaction . (Source: https://www.google.com/#q=dendritic+cells+and+T+cells+interaction ) In addition t o anergy, peripheral tolerance is maintained by induction of antigen [ 24 , 25 ] . It has been antigens . Depletion of this subset of T cells has been shown to accelerate and induce autoimmunity in various animal models [ 26 28 ] . Suppression of effector T cells is

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21 accomplished by the ability of Tregs to impair antigen presentation by mature DCs. Thus, the crucial step in Treg mediated immu no suppression is generation of Tregs which is initiated by dendritic cells of definite phenotypes [ 24 ] . In addition to the fact that immature tDCs present little or no signals 2 and 3, they can receive tolerance entiation in response to maturation stimuli. These signals can be mimicked in vitro to induce tDCs under tissue culture conditions. Thus, we can differentiate between tDCs that arise naturally from hematopoietic precursors (natural tDCs or ntDCs) and tDCs that have received instructive signals that may cement or modulate their tolerogenic phenotype (induced tDCs or itDCs) [ 16 ] . A number of biological and pharmace utical agents, including IL 10 and transforming growth factor (TGF to instigate CD 25+ T cell proliferation and differentiation from CD25 T cells [ 29 , 30 ] . For example, incubation of murine splenic or bone marrow derived DCs (BMDCs), or of human monocyte derived DCs (huMoDC) or rat BMDC with IL 10 alone or in combination with other cytokines confers a certain capacity to induce suppressive lymphocytes, including CD4+CD25+, CD8+ etc. [ 16 ] . Signaling through the IL 10 receptor (IL10R) maintains iDCs in their immature state even in the presence of maturation signals [ 31 , 32 ] Tregs and oth er sources in many tissues, also has profound effects on DCs in vitro. Laouar et al have neuropathology associated with E AE [ 33 ]

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22 The maturation state of these DCs is also important and it has been demonstrated that Treg inducible DCs exhibit eith er immature or semi mature profiles. Phenotypic characteristics of tolerogenic DCs include: increase d expression of surface markers programmed death ligand (PDL) 1 and PDL 2, immunoglobulin like transcript (ILT) 3 and ILT 4; and reduced MHC II and co stimu latory molecules (CD80 and CD86). Functionally, tolerogenic DCs show increased production of anti inflammatory cytokines (IL 10) and indoleamine 2, 3 dioxygenase (IDO). Tolerogenic DCs, therefore, offer a number of immunosuppressive modalities with poten tial for autoimmune protection. Administration of biological agents like cytokines (IL 10, TGF ), vitamin D3 or pharmacological drugs such as aspirin, curcumin, menadione, EGCG, ergosterol and rapamycin may be therapeutically beneficial. These factors hav e been shown to induce DCs, capable of generating CD25+, Foxp3+ T cells (Tregs) and further halt or reverse autoimmunity in animal models [ 34 ] . Regulatory T C ells In the late 1970s and early 1980s, the studies on neonatally thymectomized mice revealed the existence of T cell population which when suppressed caused multi organ autoimmunity in the mice [ 35 ] . Seminal study by Sakaguchi renewed the concept of the inhibitory capability of T cells playing an indispensable role in maintaining self toleran ce. It was shown that the mice receiving an adoptive transfer of T cells devoid of CD4+CD25+ cells developed multi organ system autoimmunity [ 36 ] . This set of T cells is now known as regulatory T cells (Tregs), responsib le to maintain immunological peace by keeping a check on auto reactive T cells, thereby preventing undesirable immune responses, such as autoimmune diabetes T1DM [ 37 ] . Thus, the knowledge that

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23 CD4+ Tregs has an important and central role in maintaining self tolerance has led to prospects that these cells have great potential as a cell based treatment in order to restore self tolerance and to treat autoimmune diseases. As such, Tregs may be able to target T cell responses and perhaps antigen specific responses without broadly immunosuppressive effects. Studies have shown that there are peripheral tolerance. nTregs, suc h as CD3+CD4+CD25+Foxp3+ cells are developed in the thymus and iTregs are differentiated in the periphery under certain conditions [ 38 ] . In general, it has been considered that weak T cell receptor (TCR) stimulation [ 39 , 40 ] or TCR stimulation under tolerogenic conditions, such as with immature DCs, promotes induction of iTregs [ 29 ] . Because of the limited phenotypic characterization, iTregs that arise physiologically are often indistinguishable from nTregs. Functionally, iTregs also share many characteristics with nTregs [ 41 ] and have been shown to inhibi t Tns proliferation in vitro and in vivo. They can also inhibit the differentiation of other Th cell subsets through the production of IL 10 and TGF such as IFN production [ 42 , 43 ] cell antigen pulsed immature DCs have also been shown to protect pre diabetic NOD recipients from developing diabetes, p robably through the in vivo induction of Tregs [ 12 ] . These findings confirm that iTregs can control t he autoimmune response in T1D. All Treg cell population has shown to express CD25, the high affinity receptor for IL transcription factor Foxp3 [ 44 46 ] . Compelling evidence suggest that loss of control of

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24 the autoimmune response by FoxP3+ Tregs is a major contributing factor in the development in T1DM. Foxp3 absence in mice and humans results in fatal diseases like scurfy and IPEX syndrome respectively, highlighting the importance of Tregs in self tolerance. Interestingly, the most common endocrinopathy encountered in IPEX is T1DM, suggesting that Tregs may be central to the suppression of cir specific T cells, and such pre effector T cells may be in relative abundance compared to other self reactive T cells [ 47 49 ] . Naïve T lymphocytes and T lymphocytes subsets such as Th1, Th2, Th17 and Tregs possess a great deal of plasticity in their differentiation, mostly depending on the local microenvironment. Studies from several groups have shown that differentiation of T cells into CD4+ iTregs is favored by an environment rich in factors lik e transforming growth factor (TGF 10 [ 50 , 51 ] . Immune suppressive function of Teff cells and expansion of iTregs has been shown to increase by the active secretion of TGF inflammatory cytokine IL 10 [ 52 55 ] . iTregs suppress Teff activity through multiple mechanisms, it is known that Tregs can specifically suppress Teff cells via the secretion of the above mentioned inhibitory cytokines (TGF 10) [ 56 58 ] . Tregs interplay with Teff cells to achieve balance between the pro inflammatory and anti inflammatory responses thereby maintaining immune homeostasis. Neither T lymphocyte subset is capable of acting in isolation . Tregs and Teff cell subtypes both compete for same self antig en with same T cell receptor (TCR). They both become active and proliferate in response to antigen. Although Treg cells possess higher affinity for self antigen and are less reliant on co stimulation as compared to Teffs, which could be the mechanism that causes Treg generation in non inflammatory sites, thereby

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25 promoting self tolerance [ 59 62 ] . Conversely, Teff gets activated and proliferate more rapidly in pro inflammatory states thus promoting Teff/Treg balance towards pathogenesis. The outcome of tolerance or pathogenesis is determined by local immune microenvironment and agents such as antigen and cytokines, influencing the critical balance between Teff and Treg cells. Thus for a therapy to be effective, strategies sh cell specific Teff pathogenic activity thereby trying to prevent and reverse T1DM. From the above mentioned studies, it can be determined that Tregs exhibit decreased functionality wit h respect to Teff cell suppression in T1DM as observed in human and NOD mice studies. Reduced Treg function is a result of imbalance in the Treg/Teff response which causes failure to maintain self tolerance, resulting in pro inflammatory over anti inflamma tory response. Specific Aims This research focuses on the engineering of biomaterial cell interactions, and targeted controlled release of immune modulating factors in order to direct immune cell function. It involves synthesis and characterization of drug load ed PLGA microparticles of various sizes, calculating their encapsulation efficiency, and analyzing release behavior for controlled drug delivery. Further, it involves fabrication o f microarray and microwell platforms, co localization and imaging of pro tolerogenic factors loaded micropart icles onto microarray platforms for investigation of dendritic cell responses in a high throughput manner.

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26 Specific Aim 1: Fabrication and O ptimization of Drug Loaded B i odegradable PLGA M icroparticle s Induction of immune tolerance for the treatment of autoimmune diseases, type 1 diabetes for instance, is one of the most concerning and talked about issues in modern vaccine technology development. Biodegradable, microparticle based vaccines could be us ed to deliver antigens and other immuno modulatory agents to a targeted phagocytic cell population, dendritic cells (DC) in this case. DCs are the most efficient antigen presenting cells (APC) and one of the major regulators of the immune system. They acti vely process and present antigen, thus providing either antigen specific tolerance or autoimmunity. Several drug delivery vehicles have been investigated and of these, MPs fabricated using PLGA are most widely used because of its approval from U.S. Food a nd Drug Administration (FDA) for biodegradable surgical sutures and drug delivery products. PLGA MPs of appropriate size are phagocytosed efficiently by DCs providing direct delivery of antigens and other immuno modulatory factors such as adjuvants for im mune recognition. Moreover, these vaccine microparticles can be engineered by surface modifications to modulate activation, uptake and direct them to target a specific subset of antigen presenting cells, dendritic cells. This approach of developing drug l oaded MPs will help in addressing issues related to manufacturing, storage and shipping as encapsulation of vaccine inside biomaterial provides stability and improved shelf life. Specific Aim 2: Particle Characterization: E v aluation of E ncapsulation Effic iency and In Vitro Release Profile o f Drug Loaded PLGA M icroparticles I t is important to monitor the effects of drugs on the numerous immuno modulatory pathways which they can affect . Therefore it is important to determine the encapsulation efficiency of d rug loaded microparticles . Knowledge of encapsulation

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27 efficiency will help in determining the accurate dosing regimen. This can be achieved by co localizing appropriate number of drug loaded microparticle onto microarray platform via contact printing techn ique . It further ensures cost benefits via proper utilization and less wastage of expensive drugs. A critical characteristic in drug delivery application is the control led delivery of small molecules and other agents in a reproducible, time dependent fashi on . The release kinetics is directly controlled by the various processes under which PLGA undergoes degradation. This degradation of PLGA copolymer is a collection of processes including bulk diffusion, bulk erosion, surface diffusion, and surface erosion . Therefore, we focus on targeting intracellular delive ry of pro tolerogenic factors to DCs using poly (lactic co glycolicacid) (PLGA) degradable microparticles. As part of its anti inflammatory effects, these factors will hinde r antigen presentation by DCs . It is therefore important to obtain drug release kinetics of various drug loaded into PLGA MPs. Specific A im 3: Development of Microarray and Microwell P latforms with Co localization of Drug Loaded MPs for Investigation of Dendritic Cell Responses in High T hroughput The ability to perform high throughput screening of biological agents on specific cell populations is highly desired in drug discovery efforts. Fabri cation of microarrays leverages the use of standard contact printing mini arraying equipment in order to achieve localization of particles on isolated islands while providing background non adhesive surfaces to prevent off island cell migration. This can b e achieved by robotically printing amine terminated silane islands onto glass substrates and backfilling with poly ethylene glycol (PEG) based non adhesive background onto a clean glass. Microwell devices made of polydimethylsiloxane ( PDMS ) have increasing ly become popular as they consume fewer reagents due to their lower requisite sample volumes.

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28 One of the major advantages of miniaturized platforms is the ability to perform high throughput analysis on small cell population. Furthermore, it also helps in m inimizing the use of expensive biological reagents which adds to cost effectiveness of this technique.

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29 CHAPTER 2 FABRICATION AND OPTIMIZATION OF DRUG LOADED BIODEGRADABLE PLGA MICROPARTICLES Background Biodegradability a nd Biocompatibility The biodegrada bility or biocompatibility of a material is an essential property for pharmaceutical applications. Biodegradability is defined as a material whose components are degraded into harmless components which are either metabolized or excreted. Biocompatibility i s defined by a material whose components should be physiologically tolerable and should not cause an adverse local or systemic response after administration. There has been significant increase in the number of such materials used in various biomedical app lications over the last decade. The most common use is in controlled delivery of various drugs to a desired site which can be broadly classified as synthetic biodegradable polymers mainl y consisting of hydrophobic mate rials like PLGA, polyanhydrides, and n aturally occurring polymers such as complex sugar and inorganics. Biodegradable materials could be either natural or synthetic in origin. These can further be degraded either enzymatically or non enzymatically or both in order to produce toxicologically sa fe, biocompatible by products which are finally eliminated via normal metabolic pathways. Polymers based on a C C backbone tend to resist degradation, whereas heteroatom containing polymer backbones allow biodegradability [ 63 ] . Thus biodegradability can be engineered into polymers by the careful addition of chemical linkages such as amide linkages, anhydride, ester etc. One of the important requirement for polymer to be used in medical devices and targeted drug release applications is to be non toxic, biocompatible and capable of

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30 controlled degradation in response to biological conditions [ 63 ] . Biocompatibility is often critically influenced by the chemical nature of the degradation products rather than the polymer itself. Polymers based on polylactide (PLA), polyglycolide (PGA), and polycarprolactone (PCL) have been extensively employed as bio materials. Degradation products of these polymers yield the corresponding hydroxyl acids, making them safe for in vivo use. Poly (Lactic Co G lycolic) A cid ( PLGA ) PLGA or poly (lactic co glycolic) acid is a copolymer which owing to its biocompatibility an d biodegradability is used in host of Food and Drug Administration (FDA) approved therapeutic applications [ 64 , 65 ] . PLGA microparticles are among the favorites in the current biomaterials research as drug delivery systems, mainly for their controlled and targeted drug deliver y. Polyester PLGA is a copolymer of poly lactic acid (PLA) and poly glycolic acid (PGA). PLGA is generally an acronym for poly D,L lactic coglycolic acid where D and L lactic acid forms are in equal ratio. Different forms of PLGA can be obtained dependin g on the ratio of lactide to glycolide used for the polymerization. It can be dissolved by large range of common solvents like methylene chloride, ethyl acetate , tetrahydrofuran, acetone etc. Figure 2 1. Molecular structure of PLGA molecule . (Source: http://polyscitech.com/PLGA/PLGA.php )

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31 PLGA degrades by hydrolysis of its ester linkages in the presence of water. It has been observed that the time required for the degradation depends on the monomer ratio used in the polymerization. The higher the content of glycolide units, the lower the time required for degradation. The reason for success for PLGA as biodegradable polymer is it undergoing hydrolysis in the body to produce original monomers lactic acid a nd glycolic acid. These two monomers under normal physiological conditions are by products of various metabolic pathways in the body. Since the body effectively deals with these two monomers, there is a minimal systemic toxicity associated with PLGA applic ation in drug delivery techniques. Additionally the possibility of tailor the polymer degradation time by changing the ratio of two monomers also make PLGA common choice for biomedical and drug delivery applications [ 66 , 67 ] . Additionally drug release profiles ranging from days to months can be obtained by modifying parameters like polymer molecular weight, particles size, preparation conditions etc. The initial burst of drug release occurs via diffusion of surface and the controlled release is contributed by both polymer erosion and drug diffusion [ 68 , 69 ] . PLGA can be fabricated into nanoparticles and microparticles of multiple geometries ranging from 1µm 30µm depending upon the requirements using solvent evaporation technique . PLGA polymers are physically strong and highly biocompatible and have been extensively studied as delivery vehicles for drugs, proteins and various other macromolecules such as DNA, RNA and peptides [ 65 , 70 , 71 ] . These properties make PLGA an excellent carrier of vaccines using encapsulated antigen such as proteins or peptides along with immuno modulatory agents such as adjuvants. Addit ionally, PLGA based MPs can be surface modified which can be used to modulate,

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32 uptake, activation and targeting a particular subset of antigen presenting cell, the dendritic cell [ 72 74 ] . Targeted delivery of factors to DCs is of particular interest as DCs play a pivotal role in the activation and maintenance of suppressive networks within the immune system [ 75 ] . Thus PLG A particulate systems have been identified as a valuable immuno therapeutic tool and in past two decades poly lactic co glycolic acid (PLGA) has been among the most attractive polymeric candidates used to fabricate devices for drug delivery and tissue engi neering applications. In particular, PLGA has been extensively studied for the development of devices for controlled delivery of small molecule drugs, proteins and other macromolecules in commercial use and in research. Fabrication Techniques Poly lactic co glycolic acid (PLGA) has proved to be among the most attractive polymeric candidates used to fabricate devices for drug delivery and tissue engineering applications. In addition to its biocompatibility, drug compatibility, suitable biodegradation kineti cs , mechanical properties, and most importantly being approved by FDA for therapeutic use, PLGA can be easily processed and fabricated in various forms and sizes. Particle shape, in addition to size, is becoming increasingly recognized as important in the design of drug carriers for in vivo use. It has been shown that high aqueous phase viscosity; basic aqueous phase pH and hydrophilic polymer side chains and end groups are all conditions that favor the formation of spheroidal particles. There are various fabrication techniques to form the PLGA controlled drug delivery devices [ 76 ] . There have been several method s described for the fabrication of drug loaded PLGA microparticle. Few of these methods are as follows:

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33 Solvent evaporation m ethod: Microencapsulation by solvent evaporation technique is widely used in pharmaceutical industries. It facilitates a controlle d release of a drug, which has many clinical benefits such as: drug targeting to specific locations resulting in a higher efficiency, reducing of dosing frequency and more convenience and acceptance for patients [ 77 , 78 ] . The obtained drug loaded polymer can degrade and re lease the encapsulated drug slowly with a specific release profile. Water insoluble polymers are used as encapsulation matrix using this technique. Biodegradable polymer PLGA (poly(lactic co glycolic acid)) is frequently used as encapsulation material. Mic roencapsulation using solvent evaporation technique can be used in different ways. Hydrophobicity or hydrophilicity of the drug decides the choice of technique to be used for efficient drug encapsulation. Single emulsion technique : This is the simplest met hod and other methods are derived from this technique. Oil in water emulsification process is an example of single emulsion technique. This method is frequently used for hydrophobic (insoluble or poorly water soluble) drugs such as steroids. Under this tec hnique single phase solution is first prepared by dissolving appropriate amount of polymer in volatile organic solvent for instance Dichloromethane (DCM). The drug is later added to the solution to produce dispersion in the solution. The solution obtained is later emulsified in large volume of water in the presence of an emulsifier such as polyvinyl alcohol (PVA). This is done in appropriate temperature with continuous stirring. The organic solvent is then allowed to evaporate by transferring the solution t o a large quantity of water with surfactant. The resulting solid microspheres are then washed and dried (flash frozen in liquid nitrogen) under appropriate conditions to obtain final drug loaded microparticles [ 79 , 80 ] .

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34 Double emulsio n technique : The above mentioned technique is not suitable when dealing with hydrophilic drugs. This is due to the reason that hydrophilic drug may not be soluble in organic solvent and the drug will diffuse into the continuous phase during emulsion, leadi ng to the loss of drug. For these reasons water in oil in water emulsion technique is preferred to encapsulate hydrophilic drugs for instance proteins, peptides and vaccines. In this method polymer is dissolved in organic solvent like DCM or chloroform an d appropriate amount of drug dissolved in aqueous phase (DI water) is later added to organic phase with continuous stirring to give water in oil emulsion. Later this water in oil emulsion is added to PVA aqueous solution and further emulsified. The organic solvent is then allowed to evaporate and resulting microparticles are washed and dried under appropriate conditions to yield drug loaded microparticles [ 81 , 82 ] . For the same drug, the drug encapsulation efficiency may vary depending on the method used [ 83 ] . The physical properties of obtained micr ospheres are strongly dependent on the nature of materials and also on the parameters during the manufacturing of microspheres [ 84 ] . Phase separation (c oacervation) This technique is used to prepare drug loaded microparticles by using liquid liquid phase separation method. This process gives rise to two liquid phases. One of these phases includes polymer contai ning coacervate phase and other is supernatant phase depleted in polymer. Thus there are three main steps involved in this technique; phase separation of the coating polymer solution, adsorption of the coacervate around the drug particles and finally the q uenching of the microparticles. The morphology and size of the microparticles depends upon the parameters like quenching time, concentration of the polymer, quenching temperature composition of the solvent [ 85 ,

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35 86 ] . Finally microparticles are obtained by washing, sieving, filtration, centrifugation and freeze drying. Spray d rying: Phase separation methods are not suitable for mass production since it tends to produce agglomerated particles and require removal of large quantit ies of the organic phase from the microparticles. Under spray drying technique drug loaded microparticles are prepared by spraying solid in oil dispersion or water in oil emulsion in a stream of heated air. Solubility of drug in water decides the nature of solvent to be used in the process. This technique is rapid, convenient and has few processing parameters, which makes it preferable for large scale processing. This technique has shown to encapsulate all kinds of drugs, proteins and peptides into micropar ticles without significant loss in their biological activity. Experimental Procedure Preparation of 1 2 µm Drug Loaded PLGA M icroparticles A 50:50 (PLA/PGA M/M %) polymer composition of poly (D , L lactide co glycolide) (Purac Biomaterials, Netherlands) w as used to generate microparticles. Poly vinyl alcohol (PVA) (MW ~ 15,000 g/mol) was purchased from MP Biomedicals (Santa Ana, CA, USA) and was used as an emulsion stabilizer. ddH2O was used as the aqueous phase to form the emulsions while methylene chlori de (Fisher Scientific, NJ, USA) was used as an organic solvent to dissolve PLGA polymer. Microparticles were formed using standard oil in water solvent evaporation technique [ 87 ] . Briefly, the PLGA polymer was dissolved in methylene chloride at 5% concentration. Known amount of fluorescent dye like rhodamine, a red fluorescent dye (RHOD) (Sigma Aldrich) and 9 anthracenecarboxylic acid, blue fluorescent dye (ACA)

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36 (Sigma A ldrich) and several drugs like aspirin (Acros Organics), curcumin (Sigma Aldrich), EGCG (Sigma Aldrich), menadione, ergosterol (Sigma Aldrich) etc. were added to the solution to produce dispersion in the solution. This solution was later added to 50 ml of 5% PVA solution in ddH2O and was homogenized using tissue miser homogenizer (Dremel, Wisconsin, USA) at 25,000 rpm for 2 minutes to form the emulsion. Then, the emulsion was added to 500 ml of 1% PVA solution. The particles thus formed were agitated using a magnetic stirrer (Fisher Scientific, NJ, USA) for 24 h to evaporate residual methylene chloride. The remaining solution was centrifuged at 10,000g for 10 min to collect MPs which were subsequently washed three times with ddH2O. The water was aspirated fr om the centrifuged MPs, which were then flash frozen in liquid nitrogen and kept under vacuum overnight. The MPs were stored at 20 ° C until used. Preparat ion of 10 µm Rhodamine Dye L oaded PLGA Microp articles A 50:50 (PLA/PGA M/M %) polymer composition o f poly (D,L lactide co glycolide) (PLGA) was used to generate microparticles. Poly vinyl alcohol (PVA) (MW ~ 15,000 g/mol) was purchased from MP Biomedicals (Santa Ana, CA, USA) and was used as an emulsion stabilizer. ddH2O was used as the aqueous phase t o form the emulsions while methylene chloride (Fisher Scientific, NJ, USA) was used as an organic solvent to dissolve PLGA polymer. Microparticles were formed using standard oil in water solvent evaporation technique [ 87 ] . Briefly, the known amount of PLGA polymer was dissolved in methylene chloride at 15% concentration with the help of vortex and sonication. After PLGA is completely dissolved known amount of fluorescent dye rhodamine, a red fluorescent dye (RHOD) (Sigma Aldrich) was added to the solution to produce dispersion in the solution. This

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37 solution was later added to 50 ml of 5% PVA solution in ddH2O and was homogenized using tissue miser homogenizer (Dremel, Wisc onsin, USA) at 18,000 rpm for 2 minutes to form th e emulsion. Then, the emulsion was added to 5 00 ml of 1% PVA solution. The particles thus formed were agitated using a magnetic stirrer (Fisher Scientific, NJ, USA) for 24 h to evaporate residual methylene chloride. The remaining solution was centrifuged at 10,000g for 10 min to collect MPs which wer e subsequently washed three times with ddH2O. The water was aspirated from the centrifuged MPs, which were then flash frozen in liquid nitrogen and kept under vacuum overnight. The MPs were stored at 20C until used. Preparation of 1 2 µm LPS Micro partic les LPS is positive control for dendritic cells as they help in stimulating DCs differentiation. A 50:50 (PLA/PGA M/M %) polymer composition of poly(D,L lactide co glycolide) (PLGA) was used to generate microparticles. Poly vinyl alcohol (PVA) (MW ~ 15,000 g /mol) was purchased from MP Biomedicals (Santa Ana, CA, USA) and was used as an emulsion stabilizer. ddH2O was used as the aqueous phase to form the emulsions while methylene chloride (Fisher Scientific, NJ, USA) was used as an organic solvent to dissolve PLGA polymer. Microparticles were formed using standard water oil water solvent evaporation technique [ 87 ] . Known amount of PLGA polymer was dissolved in methylene chlo ride at 5% concentration with the help of vortex and sonication. After PLGA is completely dissolved 200 µl of LPS solution was emulsified with 2 ml of 5% PLGA solution using a tissue miser homogenizer (Dremel, Wisconsin, USA) at 25,000 rpm for 2 minutes to form a primary emulsion. The primary emulsion was added to 10 mL of 5% PVA solution in ddH2O and the homogenizing was continued at 25,000 rpm for 2 minutes to form the

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38 secondary emulsion. Then, the secondary emulsion was added to 100 ml of 1% PVA solution . The particles thus formed were agitated using a magnetic stirrer (Fisher Scientific, NJ, USA) for 24 h to evaporate residual methylene chloride. The remaining solution was centrifuged at 10,000g for 10 min to collect MPs which were subsequently washed th ree times with ddH2O. The water was aspirated from the centrifuged MPs, which were then flash frozen in liquid nitrogen and kept under vacuum overnight. The MPs were stored at 20 ° C until used. Particle Size M easurements Particle size was characterized by dynamic laser diffraction (Beckman Coulter, Brea, CA). A total of 10 mg of particles were re suspended in 10 mL of de ionized (DI) water via sonication in a sonicating bath for 2 min (Branson 2510, Paragon Electronics, FL). The sample solution was run th rough the instrument and the size was verified through measuring the angular variation in intensity of light scattered as a laser beam passes through a dispersed particulate sample.

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39 Resu lts Figure 2 2 . Particle size distribution of 1 2 µm PLGA microparticles. A size distribution curve of PLGA microparticles quantified via laser diffraction technique using Beckman coulter counter (based on volume estimation). The average size of particles is 1.612

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40 Figur e 2 3 . Particle size distribution of 10 µm PLGA microparticles. A size distribution curve of PLGA microparticles quantified via laser diffraction technique using Beckman coulter counter (based on volume estimation). The average size of particles is 10.18 m with standard deviation of 9.187

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41 CHAPTER 3 PARTICLE CHARACTERIZATION EVALUATION OF ENCAPSULATION EFFICIENCY AND IN VITRO RELEASE PROFILE OF DRUG LOADED MICROPARTICLES Background Aspirin Acetylsalicyclic acid, commonly known as Aspirin is a salicylate drug. It is used for variety of medical purposes such as antipyretics to reduce fever, as analgesic to relieve minor pain, as anti platelet and as an anti inflammatory medication. It is commo nly used to control the symptoms of arthritis and rheumatism and is also regarded as a life saver in variety of atherosclerotic cardiovascular complications. Salicylic acid is the main metabolite of aspirin and is an integral part of human and animal metab olism. Figure 3 1. Molecular structure of aspirin (Source: https://www.google.com/#q=aspirin+structure ) Aspirin is a member of the non steroidal anti inflammatory agent (NSAID) class of dr ugs. NSAIDs mediate their effect by inhibition of prostaglandin and thromboxane synthesis by the enzyme cylclo oxygenase (COX). The two distinct enzymatic sites on COX are involved in the conversion of arachidonic acid to prostaglandins and thromboxanes, which mediate various events such as vasodilation and inflammatory associated events [ 88 ] . Aspirin d iffers from other NSAIDS in the mechanism of action

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42 since it is the only NSAID that does so in an irreversible manner, by acetylation of the enzyme [ 89 ] . Since aspirin is a well known drug which inhibits cyclooxygenase activity, the correlation between cytokine regulation and cyclooxygenase inhibition by aspirin was investigated by Ho et al [ 90 ] . In peripheral tissues, prostaglandin E2 (PGE 2) causes stimulatory effect on iDCs, causing the DC maturation. In addition PGE 2 also mediates IL 12 production and affects other chemokine receptor expression and also causes apoptosis of antigen presenting cells (APCs). Since prostanoids such as PGE 2 has been shown to mediate DC maturation [ 91 93 ] , aspirin thus shows it effects in inhibiting human DC maturation and stimulatory function [ 90 , 94 , 95 ] . It was determined that while aspirin promotes initial DC development and functional activity, it later stalls DC development at an immature state [ 95 ] . In vitro effects of aspirin treated murine bone marrow derived DC has shown to decrease the surface expression of cell receptors like, MHC II, CD 40, CD80, and CD 86. In further paper by Ho et al. [ 90 ] , the effect of aspirin was determined on LPS stimulated DC to express co stimulation and maturation markers. The study demonstrated that aspirin inhibits the maturation process of human DC encountering pathogen LPS. The effects of aspirin response were shown to be rapid with decreased LPS induced IL 12 and IL 10 in human DCs after just 3 hours of aspirin exposure [ 90 ] . There has been considerable amount of data presented in past couple of years that clearly suggests that aspirin media tes immuno modulation by several mechanisms. Its ability to induce tolerogenic DCs (tDCs) and to mediate T regulatory (Treg) cells production has been of particular interest in the field of induction of immunological self -

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43 tolerance. It has been shown that aspirin can inhibit autoimmune response by actively increasing CD4+ CD25+FOXP3+ Treg cells and by mediating tolerogenic DCs response which induce hypo responsiveness in naïve T cells. Curcumin Curcumin is the principal curcuminoid of the popular South Asi an spice turmeric, which is a member of the ginger family. Curcumin is a polyphenolic compound isolated from the rhizome of the plant Curcuma longa (turmeric) that has traditionally been used for pain and wound healing. Turmeric has a long history of use i n Ayurvedic medicine for the treatment of inflammatory disorders. The curcuminoids are natural phenols that are responsible for the yellow color of turmeric. It has a long history of medical use for anti inflammation in India and Southeast Asia. Curcumin is safe, non toxic and demonstrates anti inflammatory [ 96 ] anti oxidant [ 97 , 98 ] , anti microbial [ 99 101 ] and anti proliferative [ 102 ] activity. Figure 3 2. Molecular structure of curcumin (Source: https://www.google.com/#q=cu rcumin+structure ) Earlier studies have shown that dietary curcumin inhibits blood sugar levels in diabetic patients and its animal models [ 103 , 104 ] cells are susceptible to cells against re active oxygen species (ROS) mediated damage by enhancing antioxidants and reduces hyperglycemia in chemically induced diabetes [ 105 ] . Cu rcumin treatment

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44 also inhibits diabetes associated complications such as renal lesion, wound healing, and cataracts in human patients and animal models [ 106 109 ] Curcumin is a lipophilic agent that is nearly insoluble in water yet quite stable in the acidic pH of the stomach. Further more, it has been prized since ancient times for its various pharmacological benefits associated with its antioxidant and anti inflammatory properties. Curcumin was also found to suppress several inflammatory cytokines such as tumor necrosis factor alpha ( TNF a), interleukins (IL 1, 6, and 8) [ 110 ] , and cyclooxygenase 2 (COX 2). It can generate Treg s and these Treg s are a ble to function in vivo. Recent studies have shown that curcumin helps in suppressing various autoimmune disorders like multiple sclerosis, rheumatoid arthritis, psoriasis, and inflammatory bowel disease in human or animal models. Although the beneficial e ffects of nutraceuticals are traditionally achieved through dietary consumption at low levels for long periods of time, the use of purified active compounds such as curcumin at higher doses for therapeutic purposes needs to be carefully monitored. Rogers et al. investigated the immunomodulatory effects of curcumin on human monocyte derived (hu mo DC) and murine DC. Hu mo DC when exposed to curcumin show impaired ability to undergo phenotypic and functional maturation following exposure to LPS, a potent T l ymphocyte r eceptor (TLR) agonist. They found that CurcDC demonstrates minimal CD83 expression, downregulated expression of CD80 and CD86 and reduction in major histocompatibility complex MHC II and CD40 expression compared to matDC. In addition Curc DC also displayed decreased Interleukin IL 12 mRNA and protein expression. It was shown that curcumin directs DC

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45 differentiation towards a tolerogenic phenotype that results in forkhead winged helix protein 3 (FoxP3+) Tregs in vitro and in vivo [ 111 113 ] In a further study by Cong et al. [ 114 ] showed that CurcDC induced differentiation of Treg s resembling Treg s in the intestine, including both CD4+ CD25+ Fox p3+ Treg and IL 10 producing cells. Such Treg induction required IL RA (Retinoic Acid metabolite of dietary component Vitamin A) produced by curcumin modulated DC. Thus curcumin educates DC to tolerogenic phenotype, including the ability to differentiate naive T cells into intestine protective Treg s . These effects are a plausible mechanism for curcumin mediated anti inflammatory function. EGCG Epigallocatechin gallate (EGCG), also known as epigallocatechin 3 gallate, is the ester of epigallocatechin and gallic acid, and is a type of catechin. It is the most abundant catechin in tea accounti ng for more than 40% of polyphenols in green tea [ 115 ] , and is a potent antioxidant that may have therapeutic applications in the treatment of many disorders, including anti proliferative and cancer chem opreventive activities [ 116 , 117 ] . EGCG also induced apoptosis of human monocytes, another possible mechanism of its anti inflammatory and immunosu ppressive effects [ 118 ] . Figure 3 3. Molecular structure of EGCG (Source: https://www.google.com/#q=EGCG+structure )

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46 It is found in green tea, but not in black tea; during black tea production, the catechins are converte d to theaflavins and thearubigins. One of the proposed health benefits of consuming green tea is its protective effect on autoimmune diseases. T cells, particularly CD4(+) T helper (Th) cells, play a key role in mediating many aspects of autoimmune disease s. Upon antigenic stimulation, naive CD4(+) T cells proliferate and differentiate into different effector subsets [ 119 ] . Th1 and Th17 cells are the pro inflammatory subsets of Th cells respon sible for inducing autoimmunity whereas regulatory T cells (Treg) have an antagonistic effect. Green tea and its active ingredient, epigallocatechin 3 gallate (EGCG), have been shown to improve symptoms and reduce the pathology in some animal models of aut oimmune diseases. It has been observed that EGCG inhibited CD4(+) T cell expansion in response to either polyclonal or antigen specific stimulation. EGCG impedes Th1 and Th17 differentiation and prevents IL 6 induced inhibition on Treg development. In a study conducted by Yoneyama et al. effect of EGCG on human monocyte derived DCs (MODCs) and, consequently, on the T cell mediated immune response was investigated. The group studied the induction of apoptosis, and the detailed phenotypic and functional cha nges of MODCs, generated by culture of peripheral blood monocytes in the presence of GM CSF , induced by EGCG. They found that the EGCG induced apoptosis and affected the phenotype of the developing DCs. Exposure of monocytes to EGCG together with GM CSF an d IL 4 inhibited their differentiation to DCs and, in addition, induced apoptosis of DC precursors and immature DCs. The expression of CD83, CD80, CD11c and MHC class II, which are surface markers and co stimulatory molecules essential for antigen presenta tion by DCs were down -

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47 regulated by EGCG. Moreover, EGCG blocked the terminal maturation process of already differentiated DCs. Several studies have identified IL 10 as the major cytokine that can prevent both differentiation of DCs from monocytes and their maturation by blocking release of IL 12 [ 120 ] . IL 10 also inhibits expression of co stimulatory molecules and consequently causes inhibition of TH1 responses [ 121 ] . On LPS stimulation, human MODCs cultured with EGCG produced high amounts of IL 10, dose dependently of EGCG. Therefore IL 10 prod uction by MODCs may be a possible mechanism of the inhibitory effect of EGCG on DC maturation. Effects of EGCG on human MODCs include inhibition of their differentiation, terminal maturation and antigen presenting function. EGCG dose dependently induced ap optosis of DC precursors. Thus EGCG is a potential immunosuppressive agent by inducing apoptosis of human MODCs and exerting strong inhibitory effects on the differentiation of and the antigen presentation by DCs, effects possibly mediated by increased pro duction of IL 10. Therefore, EGCG treatment seems to be an effective alternative to generate tolerogenic DCs, which may be useful in the treatment of allergic and autoimmune disorders as well as allograft rejection. Vitamin D3 The role of 1,25 dihydroxyvit amin D3 (1,25(OH)2D3) in T1DM has been recently widely recognized as an immunomodulatory agent on pancreatic and immune cells. 1,25(OH)2D3 deficient mice were at higher risk of developing DM, even more aggressively when deficiency is present early in life [ 122 ] . Early administration of 1,25(OH)2D3 performs dual action on the pancreatic beta cell s and on immune cells by protecting against or reducing the severity of pancreatic insulitis [ 123 ] .

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48 Figure 3 4 : Molecular structure of 1,25 dihydroxyvitamin D3 (Source: https://www.google.com/#q=1%2C25dihydroxyvita min+D3+(1%2C25 (OH)2D3) ) 1,25(OH)2D3 has shown to decrease the expression of proinflammatory cytokine Il 6 also acts as a direct stimulator of Th17 cells. This further leads to decreased T cell prolifera tion and differentiation, increased Tregs production and arrest of autoimmune process [ 124 ] . Additionally 1,25(OH)2D3 decreases MHC class I expression which causes reduced vulnerability of islet cells to cytotox ic T lymphocytes [ 125 ] . In addition to its above mentioned activity at pancreati c islets, 1,25(OH)2D3 also inhibits differentiation and maturation of DCs and promotes their apoptosis, thereby preventing their transformation into APCs which is the first step in the initiation of autoimmune response [ 126 ] . 1,25(OH)2D3 immunomodulatory effects have shown significant protection against pancreatic insulitis in animal studies [ 123 125 ] . In humans, retrospective analysis and observational studies demonstrated high prevalence of 25 OH D deficiency in patients with T1DM [ 127 ] and suggested a contributory role in the pathogenesis of T1DM.

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49 Ergosterol Ergosterol is the major component of the fungal cell membrane. It is a white crystalline organic solid of the molecular formula C28H44O belonging to the steroid family. It acts as a bioregulator of membrane fluidity, asymmetry, and integrity [ 128 ] . It is the predominant sterol component in the plasma membrane of fungi (e.g, Saccha romyces and other yeasts and Claviceps purpurea ) and is named for ergot , a common name for the members of the fungal genus Claviceps from which it was first isolated. It serves the same function that cholesterol serves in animal cells in the structure and functions. Ergosterol does not occur in plant or animal cells. Figure 3 5 : Molecular structure of ergosterol (Source: https://www.google.com/#q=Ergosterol+structure ) Ergosterol is a biological precursor (a provitamin ) to vitamin D2 . Ergosterol is converted by ultraviolet irradiation into ergocalciferol, or vitamin D2, a nutritional factor that promotes proper bone development in humans and other mammals. For this reason, when yeast ( ) and fungi (mushrooms), are exposed to UV light, significant amounts of vitamin D are produced. Because ergosterol is present in cell membranes of fungi, yet absent in those of animals, it is a useful target for antifungal drugs [ 129 ] . Immunomod ulatory functions of ergosterol can be observed by its role in suppressing T cell proliferation. Some studies have shown ergosterol may have antitumor properties [ 130 , 131 ]

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50 Menadione Menadione is a synthetic chemical compound sometimes used as a nutritional supplement because of its vitamin K activity. Primary known function of vitamin K is to maintain healthy blood clotting and prevent exces sive bleeding and hemorrhage, it also plays important role in normal bone calcification for carbohydrate storage in the body. It is an analog of 1, 4 naphthoquinone with a methyl group in the 2 position [ 132 ] . Figure 3 6 : Molecular structure of menadione. (Source: https://www.google.com/#q=menadione+structure ) Menadione is a fat soluble vitamin precursor that is converted into menaquinone in the liver. Vitamin K1 and K2 are the naturally occurring types of vitamin K. The former, which is also known as phylloquinone, is synthesized by plants and can be found in such foods as spinach, broccoli, lettuce, and soybeans. The latter, sometimes alternatively referred to as menaquinone, is primarily produced by bacteria in the anterior part of the gut and the intestines. Vitamin K3, on the other hand, is one of the many manmade ver sions of vitamin K. Also called menadione, this yellowish, synthetic crystalline substance is converted into the active form of the K2 vitamin inside of the animal body. Immunomodulatory functions of menadione can be observed by its role in suppressing T c ell proliferation. It has been found to inhibit the production of proinflammatory cytokines like TNF 6 and IL 4 [ 133 ] , thereby attenuat ing the T cell

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51 mediated activity by inhibiting the proliferative response of T cells and increasing the number of FoxP3+ Tregs cells. Compounds of Future I nterest TGF beta Transforming growth factor beta (TGF secreted by immune and other non hematopoietic cells. It controls cellular differentiation, proliferation, and several other critical functions like cellular matura tion, embryonal development, wound healing, immune regulation etc. in most cells. It helps by keeping a check on immune system by acting as a potent immune suppressor through the inhibition of proliferation, differentiation, and activation and effector fun ction of immune cells thus maintaining immune homeostasis. The TGF growth factor beta superfamily which consists of more than 40 members . There are three TGF 2, and TGF expressed in different tissues but have similar functions [ 134 ] . It has been studied that to differentiate into an effector T cell, the naïve T cells require t hree signals; TCR stimulation, co stimulation and cytokines. Th1 cells are induced by IL 12 and Th2 cells are induced by IL 4. TGF known to inhibit the expression of surface receptors of pro inflammatory cytokines like IL 12 and IL 4 thereby inhibitin g the differentiation of T cells [ 135 , 136 ] . Furthermore, studies have shown TGF homeostasis, and function of iTregs in the periphery. In fact Treg cell number has been shown to significantly decreased in the abse nce of TGF / ) [ 55 ] . It has been demonstrated in mice [ 53 ] and later in humans [ 137 , 138 ] that TGF P3

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52 expre ssion in TCR stimulated T cells. This helps in maintain immune peace in two ways; the generated cells are not only unresponsive to TCR stimulation, but can also produce TGF 10. Both TGF 10 are potent suppressors of proliferation a nd cytokine production in vitro. Additionally, TGF induction of Foxp3 expression. It has been shown that CD4+ T cells which are deficient in TGF either in vivo or in vitro [ 139 , 140 ] . Moreover, the systemic increa se in TGF increase Foxp3+ Treg cell count in mice [ 141 , 142 ] . Therefore, therapies that increase TGF destruction. A transient pulse of TGF hase of diabetes is sufficient to inhibit disease onset by promoting the expansion of intra islet CD4+CD25+FOXP3+ T cell pool [ 143 ] . Furthermore, TGF promote tolerogenic DCs and induces the differentiation of DC with low expression of MHC II, low level of CD1d, and co stimulatory molecules including CD80, CD83, and CD86. These results in DCs with an immature phenotype and are known to have tolerogenic properties. TGF system. It acts on CD4, CD8 T cells etc. Its deficiency can lead to severe autoimmune dysregulation and the development of autoimmune diseases. Thus TGF essential regulator of autoimmune diseases. It has shown to delay the onset and reduce the severity of autoimmune diseases [ 144 , 145 ] .

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53 AhR compounds Aryl hydrocarbon receptor (AhR) also known as dioxin receptor, is a ligand activated transcription factor that serves as a receptor for various environmental t oxins. More recently it was found that AhR ligation can also regulate T cell differentiation, specifically through activation of Foxp3+ regulatory T cells (Tregs) and downregulation of the proinflammatory Th17 cells. The importance of the AhR in immunologi cal processes can be illustrated by expression of this receptor on a majority of immune cell types. In addition, AhR signaling pathways have been reported to influence a number of genes responsible for mediating inflammation and other immune responses [ 146 ] . The AhR gene has been found in various cell types present in mammals, amphibians, reptiles, and birds [ 147 ] and the fact that the AhR gene has been so highly conserved provides evidence of the fundamental importance of AhR in biological systems. Studies revealed AhR to be a key regulator in the metabolism of foreign chemicals, commonly referred to as xeno biotics. Number of AhR ligands, both synthetic and natural, has been discovered and these ligands, typically components of environmental pollutants, include the halogenated aromatic hydrocarbons such as polychlorinated dibenzo p dioxins, dibenzofurans, an d biphenyls as well as polycyclic aromatic hydrocarbons such as benzo(a)pyrene, anthracene, and 3 methylcholanthrene. 2, 3, 7, 8 Tetrachlorodibenzo pdioxin (TCDD), a halogenated aromatic hydrocarbon, is one of the most immunotoxic and immunosuppressive AhR ligands [ 148 ] . In addition to these synthetic AhR ligands, number of natural ligands has also been discovered. Most of natural AhR ligands are introduced into biological systems by oral consumption of foods and herbal medicines, as in the cases of flavonoids, stilbenes , carotenoids, and indoles [ 148 ] .

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54 The functions of AhR in T cells depend on the specific ligand bound to the receptor . For instance, binding of TCDD to AhR suppresses experimental autoimmune encephalomyelitis (EAE) by promoting the development of Foxp3+ Treg cells, whereas 6 formylindolo[3,2 b] carbazole enhances EAE by inducing the differentiation of IL 17 producing T cells [ 149 ] . In macrophages and dendritic cells ( DCs), AhR is anti inflammatory. In response to LPS, Ahr deficient macrophages show increased production of pro inflammatory cytokines, such as IL 6 and TNF deficient DCs produce less of the anti inflammatory cytokine IL 10 [ 149 ] . In fact, due to their di verse properties and inconsequential side effects, resveratrol (class of stilbenes) [ 150 ] , Indole compounds, such as indole 3 carbinol (I3C) and its chief metabolite 3,3` diindolylmethane (DIM) and other dietary products such as carotenoids and curcumin have been found to interact with the AhR are currently being used in clinical trials to treat diabetes. PDE inhibitors Various cellular pathways and inflammatory responses are mediated by cyclic adenosine monophosphate (cAMP), an essential intracellular second messenger made up of phosphodiester bonds. The role of cAMP in cell signaling and homeostasis was established, and regulation of this pathway by PDE inhibitors arose as a field of considera ble interest [ 151 ] . Phophodiesterases inhibitors (PDEis) act by inhibiti ng the catabolism of cyclic nucleotides, cAMP and cGMP, which are ubiquitously expressed in cells of the immune system. Phosphodiesterases degrade the cyclic nucleotides, cGMP and cAMP to AMP, respectively. Their critical role in int racellular signalling has designated cAMP and cGMP as potential new therapeutic targets. PDEis act by

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55 prolonging the action of cyclic nucleotides inside the cells. There are 11 PDEs discovered till date. PDEis can be non specific like theophylline or speci fic like rolipram for PDE4 and sildenafil, tadalafil or vardenafil for PDE5. PDE inhibition results in the accumulation of the intracellular second messenger cAMP, downstream activation of protein kinase A (PKA), and subsequent phosphorylation of the tran scription factor cAMP response element binding protein (CREB). Activation of this pathway modulates gene transcription of numerous cytokines, proinflammatory and destructive pro perties [ 152 ] . Drug Release B ehavior It has been sho wn in both in vivo and in vitro experiments for various types of drug loaded PLGA microparticles that PLGA copolymer undergoes degradation by hydrolysis or biodegradation through cleavage of its backbone ester linkages into oligomers and later monomers [ 153 , 154 ] . This degradation of PLGA copolymer is collection of processes like bulk diffusion, bulk erosion, surface diffusion and surface erosion. The rate of degradation for PLGA copolymers is dependent on the polymer composition, molecular weight of the polymer a nd the degree of crystallinity of the polymer. Effect of polymer composition The molar ratio of the lactic and glycolic acids in the polymer chain is the most important factor to determine the rate of degradation. It has been shown that weight loss of po lymer is accelerated with increase in glycolic acid percentage [ 155 , 156 ] . For instance PLGA 50:50 (PLA/PGA) shows faster degradation than PLGA 65:35. This effect is due to preferential degradation of glycolic acid due to higher hydrop hi l l icity.

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56 Effect of Molecular weight Polymers with higher molec ular weight have longer chains which in turn require more time to degrade compared to smaller polymer chains. Thus polymers with high molecular weight exhibits lower degradation rates [ 157 ] . Effect of size and shape of polymer matrix The ratio of surface area to volume is another important factor determin ing the degradation of polymer. Polymers with higher surface area ratio results in higher degradation of polymer matrix . Effect of Crystallinity Properties such as crystallinity and glass transition temperature are affected by copolymer composition and have indirect effect on degradation rate of polymer. The release of drug from degrading PLGA polymer has been shown to exhibit patterns like Initial burst and slow progressive release [ 68 , 153 , 154 ] . Initial burst happens when drug in contact with the medium, on the surface of the polymer is released as a function of solubility and penetration of water into polymer matrix. Progressive release happens when water inside the matrix hydrolyzes the polyme r creating a passage for drug to be released by the process of diffusion and erosion till entire polymer dissolves. Experimental Procedure Characterization of M icroparticles Efficiency of drug encapsulation A known weight of MPs loaded with drug was disso lved in DMSO. The efficiency of encapsulated drug in the MPs was then quantified by measuring the absorbance at specific wavelength by spectrophotometer (Nanodrop Technologies Inc., DE, USA) and comparing absorbance to a standard curve made from known conc entration of drug in DMSO. The standard curve was made by dissolving known weight of drug in DMSO

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57 and serially diluting 1:1 with DMSO 12 times (n=3). Blank PLGA particles were then added to each dilution to account for PLGA absorbance. Absorbance was measu red using a Nano Drop (ND 1000) spectrophotometer at specific wavelength. Degradation of microparticles A known weight of MPs loaded with drug was dissolved in PBS with 2 % Tween 20 (v/v) and incubated at 37°C with rocking for 30 days (n=3). Samples were taken at day 0, 1, 2, 4, 7, 21, and 28 by centrifuging at 10,000 rpm for 10 min to separate the drug loaded microparticles from solution of PBS with 2 % Tween 20 (v/v). PBS solution with 2 % Tween 20 (v/v) containing the released drug was then carefully pi petted out and saved. Equal amount of PBS with 2 % Tween 20 (v/v) was then replaced and kept in the incubator at 37°C. The concentration of released drug was then quantified by measuring the absorbance at specific wavelength by spectrophotometer (Nanodrop Technologies Inc., DE, USA) and comparing absorbance to a standard curve made from known concentration of drug in PBS with 2 % Tween 20 (v/v). The standard curve was made by dissolving known weight of drug in PBS with 2% Tween 20 (v/v) and serially dilutin g 1:1 with PBS with 2% Tween 20 (v/v) 12 times (n=3). Blank PLGA particles were then added to each dilution to account for PLGA absorbance if any. Absorbance was measured using a Nano Drop (ND 1000) spectrophotometer at specific wavelength.

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58 Results F igure 3 7 . Curcumin encapsulation standard curve. The standard curve was made by dissolving 10 mg of curcumin in 1 ml of DMSO and serially diluting 1:1 with DMSO 12 times (n=3). 5 mg of blank particles were then added to each dilution. Absorbance was measu red using a Nano Drop (ND 1000) spectrophotometer at 435 nm. Linear fit slopes were obtained with parameters shown. Figure 3 8 . Curcumin release standard curve. The standard curve was made by dissolving 10 mg of curcumin in 1 ml of PBS with 2% Tween 20 (v/v) and serially diluting 1:1 with PBS with 2% Tween 20 (v/v) 12 times (n=3). 5 mg of blank particles were then added to each dilution. Absorbance was measured using a Nano Drop (ND 1000) spectrophotometer at 435 nm. Linear fit slopes were obtained with parameters shown. y = 0.016x + 0.0197 R² = 0.9986 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0.0 20.0 40.0 60.0 80.0 100.0 Absorbance @ 435 NM Concentration (µg /ml ) Curcumin encapsulation standard curve y = 0.0047x + 0.0198 R² = 0.9972 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 0.0 50.0 100.0 150.0 200.0 Absorbance @ 435 NM Concentration (µg /ml) Curcumin release standard curve

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59 Figure 3 9 . In vitro release profile of curcumin. 5 mg of curcumin particles were dissolved in 1 ml of PBS with 2 % Tween 20 (v/v) and incubated at 37°C with rocking for 30 days. Table 3 1. Mass factor, encapsulation efficiency and 4 weeks release of PLGA microparticles loaded with curcumin. 0.0% 10.0% 20.0% 30.0% 40.0% 50.0% 60.0% 70.0% 80.0% 90.0% 100.0% 0 5 10 15 20 25 30 Cumulative release % Days Curcumin release profile

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60 Figure 3 10 . Aspirin encapsulation standard curve. The standard curve was made by dissolving 10 mg of aspirin in 1 ml of PBS with 2% Tween 20 (v/v) and serially diluting 1:1 with DMSO 12 times (n=3). 5 mg of blank particles were then added to each dilution. Absorban ce was measured using a Nano Drop (ND 1000) spectrophotometer at 275 nm. Linear fit slopes were obtained with parameters shown. Figure 3 11 . Aspirin release standard curve. The standard curve was made by dissolving 10 mg of aspirin in 1 ml of PBS with 2 % Tween 20 (v/v) and serially diluting 1:1 with PBS with 2% Tween 20 (v/v) 12 times (n=3). 5 mg of blank particles were then added to each dilution. Absorbance was measured using a Nano Drop (ND 1000) spectrophotometer at 275 nm. Linear fit slopes were obt ained with parameters shown. y = 0.0006x + 0.0267 R² = 0.9982 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 200 400 600 800 1000 1200 1400 Absorbance @ 275 NM Concentration (µg / ml) Aspirin encapsulation standard curve y = 0.0002x + 0.0223 R² = 0.9954 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2 0 100 200 300 400 500 600 700 Absorbance @ 275 NM Concentration (µg / ml) Aspirin release standard curve

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61 Figure 3 12 . In vitro release profile of aspirin. 5 mg of aspirin particles were dissolved in 1 ml of PBS with 2 % Tween 20 (v/v) and incubated at 37°C with rocking for 30 days. Table 3 2 . Mass factor, encapsulation effici ency and 4 weeks release of PLGA microparticles loaded with aspirin. 0.0% 20.0% 40.0% 60.0% 80.0% 100.0% 120.0% 0 5 10 15 20 25 30 Cumulative release % Days Aspirin release profile

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62 Figure 3 13 . EGCG encapsulation standard curve. The standard curve was made by dissolving 10 mg of vitamin E in 1 ml of DMSO and serially diluting 1:1 with DMSO 12 times (n=3). 5 mg of blank particles were then added to each dilution. Absorbance was measured using a Na no Drop (ND 1000) spectrophotometer at 278 nm. Linear fit slopes were obtained with parameters shown. Figure 3 14 . EGCG release standard curve. The standard curve was made by dissolving 10 mg of vitamin E in 1 ml of PBS with 2% Tween 20 (v/v) and seriall y diluting 1:1 with PBS with 2% Tween 20 (v/v) 12 times (n=3). 5 mg of blank particles were then added to each dilution. Absorbance was measured using a Nano Drop (ND 1000) spectrophotometer at 278 nm. Linear fit slopes were obtained with parameters shown. y = 0.0021x + 0.1398 R² = 0.9959 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 0.00 100.00 200.00 300.00 400.00 500.00 600.00 700.00 Absorbance @ 278 NM Concentration (µg / ml) EGCG encapsulation standard curve y = 0.0019x + 0.0289 R² = 0.9997 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.0 50.0 100.0 150.0 200.0 250.0 300.0 350.0 Absorbance @ 278 NM Concentration (µg/ ml) EGCG release standard curve

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63 Figure 3 15 . In vi tro release profile of EGCG. 5 mg of EGCG particles were dissolved in 1ml of PBS with 2 % Tween 20 (v/v) and incubated at 37°C with rocking for 30 days. Table 3 3 . Mass factor, encapsulation efficiency and 4 weeks release of PLGA mi cr oparticles loaded with EGCG . 0.0% 20.0% 40.0% 60.0% 80.0% 100.0% 120.0% 0 5 10 15 20 25 30 Cumulative release % Days EGCG release profile

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64 Figure 3 16 . Menadione encapsulation standard curve. The standard curve was made by dissolving 10 mg of menadione in 1 ml of DMSO and serially diluting 1:1 with DMSO 12 times (n=3). 5 mg of blank particles were then added to each dilution. Absorbance was measured using a Nano Drop (ND 1000) spectrophotometer at 334 nm. Linear fit slopes were obtained with parameters shown. Figure 3 17 . Menadione release standard curve. The standard curve was made by dissolving 10 mg of menadione in 1 ml of PBS with 2 % Tween 20 (v/v) and serially diluting 1:1 with PBS with 2 % Tween 20 (v/v) 12 times (n=3). 5 mg of blank particles were then added to each dilution. Absorbance was measured using a Nano Drop (ND 1000) spectrophotometer at 334 nm. Linear fit slopes were obtained with parameters shown. y = 0.0015x + 0.0185 R² = 0.9995 0 0.2 0.4 0.6 0.8 1 0 100 200 300 400 500 600 700 Absorbance @ 334 NM Concentration (µg / ml) Menadione encapsulation standard curve y = 0.0012x + 0.0085 R² = 0.998 0 0.05 0.1 0.15 0.2 0.25 0.0 50.0 100.0 150.0 200.0 Absorbance @ 334 NM Concentration (µg / ml) Menadione release standard curve

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65 Figur e 3 18 . In vitro release profile of menadione. 5 mg of menadione particles were dissolved in 1 ml of PBS with 2 % Tween 20 (v/v) and incubated at 37°C with rocking for 30 days. Table 3 4. Mass factor, encapsulation efficiency and 4 weeks release of PLGA microparticles loaded with menadione. 0.0% 5.0% 10.0% 15.0% 20.0% 25.0% 30.0% 35.0% 40.0% 45.0% 50.0% 0 5 10 15 20 25 30 Cumulative release % Days Menadione release profile

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66 Figure 3 19 . Ergosterol encapsulation standard curve. The standard curve was made by dissolving 10 mg of ergosterol in 1 ml of DMSO and serially diluting 1:1 with DMSO 12 times (n=3). 5 mg of blank particles were then added to each dilution. Absorbance was measur ed using a Nano Drop (ND 1000) spectrophotometer at 284 nm. Linear fit slopes were obtained with parameters shown. Figure 3 20 . Ergosterol release standard curve. The standard curve was made by dissolving 10 mg of ergosterol in 1 ml of PBS with 2 % Twee n 20 (v/v) and serially diluting 1:1 with PBS with 2 % Tween 20 (v/v) 12 times (n=3). 5 mg of blank particles were then added to each dilution. Absorbance was measured using a Nano Drop (ND 1000) spectrophotometer at 284 nm. Linear fit slopes were obtained with parameters shown. y = 0.0012x + 0.0229 R² = 0.9978 0 0.1 0.2 0.3 0.4 0.5 0.0 50.0 100.0 150.0 200.0 250.0 300.0 350.0 Absorbance @ 284 NM Concentration (µg /ml ) Ergosterol encapsulation standard curve y = 5E 05x + 0.0232 R² = 0.9878 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0 500 1000 1500 2000 2500 3000 Absorbance @ 284 NM Concentration (µg / ml) Ergosterol release standard curve

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67 Figure 3 21 . In vitro release profile of ergosterol. 5 mg of ergosterol particles were dissolved in 1 ml of PBS with 2 % Tween 20 (v/v) and incubated at 37°C with rocking for 30 days. Table 3 5 . Mass factor, encapsulation efficiency and 4 weeks release of PLGA microparticles loaded with ergosterol. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 0 5 10 15 20 25 30 Cumulative mass release Days Ergosterol release profile

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68 Table 3 6. Mass factor, encapsulation efficiency and 4 weeks release of PLGA microparticles loaded with immuno modulatory agents. * Total mass release

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69 CHAPTER 4 DEVELOPMENT OF MICROARRAY AND MICROWELL PLATFORMS WITH CO LOCALIZATION OF DRUG LOADED MPs FOR INVESTIGATION OF DENDRITIC CELL RESPONSES IN HIGH THROUGHPUT Background Microarrays Traditionally, biological assays require th e use of conventional 12/24/96/384 well plates which require high volumes of expensive biological reagents and cells. This multi well plate format suffers from other limitations such as wastage while washing out chemicals, high t hroughput screening of data . An alternative to this approach lies in more promising microarray technology. Microarray technology has emerged as a valuable tool in biological sciences, particularly for high throughput applications. Micro arrays are multiplex lab on a chip devices whic h are 2D arrays on a solid substrate, commonly glass slide or silicon that assays large amount of biological material using high throughput analysis [ 158 ] . Ability to design parallel experiments helps in generating large amount of data in a short amount of time. Several types of m icro arrays exist including DNA, prot eins and ce llular microarrays. Early micro arrays consisted of DNA microarrays, which were oligonucleotides immobilized on the solid support. The cellular components of interest are then extracted, labeled, and hybridized to their immobilized complimentary capture probes where they are measured and quantified. Following these developments protein microarrays came into existence, which are similar in design with an exception of oligonucleotides being replaced with cell lyses solutions or biological samples su ch as serum. Also of note, the capture molecule which is attached to the substrate can vary. The most commonly used molecule is a monoclonal antibody; however other proteins,

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70 aptamers and enzymes can be used. For detection, proteins are typically attached with a fluorophore that can be quantified with standard fluorescence detection techniques. Following the evolution of protein microarray technology, the desire to investigate living cells rather than simply cell lysates has resulted in the development of cellular microarrays. Early cellular microarrays would arrange capture probes and detector probes in such a way so that the single cell would be isolated on the array. The detector probes, which are molecules that bind soluble factors secreted by cells, al low functional analysis of the bound cells to be performed. This technology allowed for the ability to molecularly delineate the characteristics of individual cells from heterogeneous population helping to identify the cellular behavior in healthy and dise ase states. More recently, microarray technology has expanded to include patterning of biodegradable polymers embedded with small molecules. This approach employed robotically printing PLGA impregnated with drugs of interest in an arrayed fashion. Non Fo uling S urfaces Fouling is the accumulation of undesirable or unwanted material on solid surfaces to the detriment of function. The fouling material can consist of either living organisms (bio fouling) or a non living substance (inorganic or organic). It is usually distinguished from other surface growth phenomena in that it occurs on a surface of a component, system or plant performing a defined and useful function, and that the fouling process impedes or interferes with this function. Generally, materials placed in a biological environment will experience the adsorption of proteins and cells on their surface. It has been observed that typically surfaces that bind proteins will generally bind cells. In order to deal with such processes, surface modification strategies have been investigated to reduce the

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71 adsorption of proteins and thus to biomaterials [ 159 ] . The most studied way among all is the use of polyethylene glycol (PEG) ( H (O CH2 CH2)n OH). Surfaces coated with the polymer poly (ethylene glycol) have been shown to exhibit a particularly low degree of prot ein adsorption, achieving a more than hundred fold lower reduction in the amount of adsorbed protein. Polydimethylsiloxane (PDMS) Polydimethylsiloxane (PDMS) falls under the category of polymeric organo silicon compounds that are commonly referred to as s ilicones. It is made of silicon, carbon, hydrogen and oxygen. PDMS is the most widely used silicon based organic polymer, is used in wide range of biomedical applications. In the last decade laboratory applications have moved from using of glass and silic on structures towards polymers because of their ease of manufacturing, moderate cost and increased portability. Microwell devices made of PDMS have increasingly become popular as they consume fewer reagents and can work with lower sample volumes. PDMS belo ngs to group of siloxanes. It consists of an inorganic slioxane group and side group of methyl. The chemical formula for PDMS is CH3 [Si(CH3)2O]nSi(CH3)3, where n is the number of repeating monomer [SiO(CH3)2] units. PDMS consists of a flexible (Si O) back bone and a repeating (Si(CH3)2O) unit. The number of the repeating (Si(CH3)2O) units generally defines the molecular weight, and consequently many of the viscoelastic properties of the material. Figure 4 1. Molecular structure of a poly (dimethylsiloxane) . C ombines both organic and inorganic groups. (Source: https://www.google.com/#q=PDMS+structure )

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72 From a biomedical point of view, PDMS is non toxic, inert, highly hydrophobic, non fluorescent, non flammable and optically clear translucent polymer that does not bio and good transparency [ 16 0 ] . It has wide range of applications from medical devices and implants through catheters to soft contact lenses, mold rel ease agents, waterproofing etc. In the last few years its use has become popular for fabricating or replicating structure using sof t lithography technique. This application has made it popular in the development of DNA microarrays, PCR, capillary electrophoresis, micro wells etc . [ 161 , 162 ] . For laboratory applications, researchers prefer to use PDMS Sylgard® 184 (Dow Corning Corporation) which can be fabricated using two part heat curable kit. In most cases, the pre polymer is crosslinked with the curing agent in 10:1 ratio (weight/weight). Although by changing the specific ratios o f pre polymer and curing agent PDMS of different properties can be obtained. The elasticity of PDMS is directly related to the amount of crosslinking. More crosslinking leads to less elasticity. As a result of its several favorable properties, there has b een increasing use of PDMS for lab on chip (LOC) devices. Aggressive research in this field is likely to result in many more uses of PDMS in analytical application. Microwells The use of miniaturized platforms has successfully increased our capabilities to understand the fundamental biological mechanisms which were previously masked by traditional laboratory systems. Many complex microbiological and immunological phenomenon such as cell migration, cell proliferation and differentiation, drug release,

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73 cell t o cell communication etc. can now be studied and understood in a microenvironment resembling the in vivo condition. Techniques such as micro electrochemical systems (MEMS) and sensors are commercially available which aid in understanding such complex biolo gical mechanisms but the cost associated with such commercial techniques and difficulties in designing and manufacturing these micoplatforms in laboratories make these technological advancements unattainable for many such applications. We introduce a simp le PDMS based microwell platform fabricate d with the help of soft lithography technique. The advances in high throughput microwell platforms greatly facilitated drug discovery and cell culture research [ 163 ] . The microwell plate designed is useful for all types of cell culture studies in cluding high resolution microsc opy and automated imaging. This microwell platform is easy to fabricate and can be used for routine laboratory settings. The microwell array can be directly put into an incubator to allow the maintenance of desired cell growth with optimum temperature, humidity, air pressure, oxygen and CO2 concentration. These platforms can be useful for resear chers for rare cell populations and expensive reagents. The silicon mold for developing microwell platform was fabricated with the help of electron beam lithography technique. The major advantage of this technique is the ability to draw custom patterns with high resolution of up to 10 nm. The technique is used to get desired custom shapes by scanning a focused beam of electrons on a surface it causes selective removal of either exposed or no n exposed regions of resist by changing its solubility and immersing it in a solvent.

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74 Miniaturization T echnique research and there are several reasons behind why this is nec essary and beneficial when it comes to understanding how single cell line or even individual cell respond to drug targeting and cellular behavior. First, the size compatibility between molecules of interest and micron sized tools has given a way to micro a nd nano technologies to move into the field of life sciences. Second, given the scenario to run several different tests on a limited sample would require a miniaturized technique where low sample volume could turn out to be sufficient in carrying out analy sis. Third, it helps in experiment parallelization where several different experiments can be run side by side in a limited space and results can be compared. Furthermore, the technique helps in economical savings by using smaller volume of expensive reage nts, high throughput analysis of data, portability and waste reduction [ 164 ] . The c onventional tools such as 12/24/96/384 well plates are relatively big than the molecules under study and also require larger volumes of expensive reagents. Attempts have been made worldwide for developing a strategy to study single cell line behavior. Miniaturized techniques are very often design to study the behavior of single cell line or individual cells. The information received from such analysis can aid in understanding the role of particular cell in pr ocessing complex inputs and the molecular machinery of the cell. For instance, outcome of the culture of million cells in response to a drug is an average response for all the cells in the culture. It has been shown that such responses could be misleading [ 165 ] . This problem on the other hand can be resolved with the help of and provides information about drug response, proliferation, cell division etc. This

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75 further leads to one of the long envisioned and now approaching reality of personalized medicine or per sonalized healthcare where better treatment can be offered to individuals based on their responding behavior rather than the name of the disease they are suffering from. More recently, micro wells technique has emerged as a common method to isolate cells by mechanically separating them by p hysical boundaries instead of separating them by creating chemical barrier on a chip via PEG based pluronic F 127 adsorbed onto CH3 alkanethiol monolayer to provide non adhesive background against silane layer. Microwell d evice s can be designed in several different ways depending on the necessity. This can be achieved by changing parameters like size, shape, number of wells or even elastic properties of the polymer. Considering the applications such as imaging, it is import ant to have a transparent material with good optical properties. One such material which is now commonly used is the biologically inert PDMS [179]. Today, commercially available arrays are available which provide application specific, miniaturized assays. Miniaturization techniques are an expanding field of research and have great potential in a field of single cell line analysis. Microwell and microarrays have successfully been able to acquire data for statistically significant results. Experimental Proce dure Particle Array F abrication Glass coverslips (22x22 mm 2 , Fisher scientific) were cleaned in an oxygen plasma etcher (Terra Universal, CA, USA) for 6 min. An array of (3 Aminopropyl) trimethoxysilane (NH2 terminated silane) (Sigma Aldrich) as obtained f rom the manufacturer was diluted in DI water. The silane solution was printed on clean

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76 coverslips using a Calligrapher Miniarrayer (BioR ad) contact printer, with 360 µ m diameter solid metal pin. The printed coverslips were then coated with 200 Å of titanium (Ti 99.995% pure) followed by 200 Å of gold (Au 99.999% pure) (Williams Advanced Materials, IL, USA). Gold coated coverslips were then sonicated in 70% ethanol in DI water for 15 min to remove gold coating from over the printed islands exposing NH2 terminated silane arrayed spots, while leaving the gold coating intact around the islands for further processing. The coverslip was then washed with DI water without letting it dry, to ensure the lifted gold was removed. The coverslips were dried by blowing nitrogen gas on the coverslip. The coverslips were then incubated with 0.01 M, methyl terminated alkanethiol (CH3(CH2)11SH, Sigma Aldrich) for 1 h followed by washing wit h 200 proof ethyl alcohol (Fisher Scientific). Substrates were then incubated in 10% pluronic F 127 (BASF Corporation, USA) in DI water, for 4 h to render the surface around the islands cell resistant. The coverslip was washed with DI water and re suspende d MPs of PLGA were over printed on the exposed islands. Microparticles were printe d using a pin of 360 µ m diameter. The MPs on the islands were allowed to dry completely by incubating them at room temperature under vacuum for 30 min. The coverslip was then rinsed with DI water. Micrographs were obtained using MosaiX module of Axiovision software (CarlZeiss). The fluorescently labeled MPs were counted To design a printed array of a dilut ion of number of single fluorescent dye encapsulated MPs, we re suspended the desired MPs in 1 mL of DI water via combination of vortex mixing and sonication. The MP concentration was verified by taking micrographs of fluorescent MPs on a hemocytometer and utilizing Automatic

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77 object Measurement Program, Axiovision software. A serial 1:1 dilution of MPs in DI water was generated, with a starting concentration of 680 X 10^6/mL. Figure 4 2. Microparticle/dendritic cell (MP/DC) array fabrication. (A) Surfac e engineering MP array: (1) Amine (NH2 ) terminated silane is printed on O2 plasma cleaned glass in an array. (2) Titanium and gold are deposited on glass coverslip having NH2 terminated silane spots. (3) The coverslip is sonicated to remove gold deposited on the NH2 terminated silane spots. (4) Methyl (CH3 ) terminated alkanethiol is assembled on gold. (5) Pluronic F 127 is adsorbed preferentially on the hydrophobic CH3 terminated alkanethiol. (B) Cross section of a single spot in a MP array illustrating physisorbed MPs on NH2 terminated spots with a polyethylene glycol based non adhesive background surface chemistry (not to scale). (C) Dendritic cells are seeded on MP arrays, selectively adherent to NH2 terminated spots providing co localized DCs/MPs arra ys. Large numbers of DC targeting MP formulations can be tested in parallel for modulation of DC function [ 166 ]

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78 These suspensions were added into separate wells in a 384 well plate which was used as the source plate to over spot MPs ont o the arrayed NH2 terminated substrate. Fluorescence micrographs of the entire array were obtained and the number of MPs on each island counted using Axiovision software. Preparation of P DMS M icrowells PDMS was fabricated using Sylgard® 184 silicone elast omer (Dow Corning Corporation). A known amount of elastomer was mixed with a curing agent (10:1) and mixed vigorously. After mixing, the entrapped air was removed by desiccation. After desiccation, the mixture was poured over a silicon mold. The diameter a nd the height of the well on the mold were 400 µm and 200 µm respectively. The center to center distance between two wells on mold was 600 µm. A . B . Figure 4 3. Geometric representation of the silicon mold. A ) Top view of the mold. B ) Side view of the mold. In order to prevent PDMS from sticking to the silicon mold, salinization of the mold was performed by exposing the mold to the vapor of trichloro(1H, 1H, 2H, 2H perfluoro octyl)silane (Sigma Aldrich) in a vacuum chamber for 0.5 h. The thickness

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79 was contr olled using supporters and spacers of appropriate dimensions. After the PDMS was poured over the mold, it was put in an oven at 75°C for 24 hours to cure. Following, the cured polymer was peeled away from the mold and cut into desired shape. Figur e 4 4 . Microarray and microwell platform s. A) Titanium and gold deposited microarray with amine terminated islands B) PDMS based microwell platform. Cell S eeding Fadu cells were maintained in dulbecco's modified eagle medium (DMEM) supplemented with 10% fe tal bovine serum (Thermo Scientific, Waltham, MA), 1% penicillin G and 1% streptomycin (Thermo Scientific). The cells were cultured at 37ºC in a humidified incubator containing 5% CO2.Following microarray fabrication, 100,000 Fadu cells were seeded over ea ch array in 3 ml serum free media and allowed to incubate on a rocking plate at room temperature until cell attachment to the MPs occurred, with minimal attachment to background, typically 10 15 min. Microarrays were gently washed in PBS, placed in a 35 mm petri dish with complete media, and placed in an incubator for 24 72 h.

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80 Staining and Image A nalysis Rhodamine loaded microarrays w as fixed in 4% paraformaldehyde, and placed in PBS with Hoechst dye 34580 (Invitrogen, USA) for 30 min. Array was then moun ted with DAPI mounting dye and imaged using an Axiovert 200M microscope (Carl Zeiss, Oberkochen, Germany). Result s Figure 4 5 . Quantitation of 10 µm microparticle printing on microarray chip . Rhodamine encapsulated microparticles were printed over silane treated glass coverslips. Surface adsorbed microparticles were quantified by fluorescence image analysis. 0 200 400 600 800 1000 1200 5.6 11.1 22.2 44.4 88.8 177.6 355.2 710.4 Average # of particles Particle concentration in source plate ( x 10^6 / ml ) 10um particle printing microarray

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81 Figure 4 6 . Linear fit slope for 10 µm m icroparticles printed on microarray chip . Av erage delivered microparticles values with standard deviations were plotted as a function of source p late concentration. Linear fit slopes were obtained with parameters shown. Figure 4 7 . Quantitation of 1 2 µm microparticle printing on micro array chip . Rhodamine encapsulated microparticles were printed over silane treated glass coverslips. Surface adsorbed microparticles were quantified by fluorescence image analysis. y = 2.9458x 3.9951 R² = 0.9958 0 100 200 300 400 500 600 700 0 50 100 150 200 Average # of particles Particle concentration in source plate ( x 10^6 / ml) 10um particle printing microarray 0 100 200 300 400 500 600 700 800 900 1000 5.3 10.6 21.3 42.5 85.0 170.1 340.1 680.2 Average # of particles Particle concentration in source plate ( x 10^6 / ml) 1 2µm particle printing microarray

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82 Figure 4 8 . Linear fit slope for 1 2 µm m icroparticles printed on microarray chip . A verage delivered microparticles values with standard deviations were plotted as a function of source p late concentration. Linear fit slopes were obtained with parameters shown. Figure 4 9 . Quantitation of 10 µm microparticle printing on micro array well . Rhodamine encapsulated microparticles were printed over silane treated glass coverslips. Surface adsorbed microparticles were quantified by fluorescence image analysis. y = 1.7387x + 19.168 R² = 0.9935 0 100 200 300 400 500 600 700 0 50 100 150 200 250 300 350 400 Average # of particles Particle concentration in source plate ( x 10^6 / ml) 1 2µm particle printing microarray 0 10 20 30 40 50 60 70 80 8.05 16.10 32.20 64.40 128.80 257.60 515.20 Average # of particles Particle concentration in source plate ( x 10^6 / ml) 10µm particle printing microwell

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83 Figure 4 10 . Linear fit slope for 10 µm m icroparticles printed on microarray well . Average delivered microparticles values with standard deviations were plotted as a function of source p late concentration. Linear fit slopes were obtained with parameters shown. Figure 4 11 . Quantitation of 1 2 µm microparticle printing on micro array well . Rhodamine encapsulated microparticles were printed over si lane treated glass coverslips. S urface adsorbed microparticles were quantified by fluorescence image analysis y = 0.0669x + 28.992 R² = 0.9564 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 0 100 200 300 400 500 600 Average # of particles Particle concentration in source plate ( X 10^6/ml ) 10 um particle printing microwell 0 20 40 60 80 100 120 140 4.175 8.35 16.7 33.4 66.8 133.6 267.2 534.4 Average # of particles Particle concentration in source plate ( X 10^6 / ml) 1 2µm particle printing microwell

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84 Figure 4 12 . Linear fit slope for 1 2 µm m icroparticles printed on microarray well . Average delivered microparticles values with standard deviations were plotted as a function of source p late concentration. Linear fit slopes were obtained with parameters shown. Figure 4 13 . Fluorescence images of serial 1:2 dilutions of rhodamine dye Loaded PLGA MPs printed on a microarray . D ilution 1 (10m g/ml) 710*10^6 particles/ml. y = 0.1826x + 23.482 R² = 0.957 0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 0 100 200 300 400 500 600 Average # of particles Particle concentration in source plate ( X 10^6 /ml) 1 2µm particle printing microwell Decreasing source plate particle concentration

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85 Figure 4 14 . Fluorescence micrograph of co localized Fadu cells and microparticles. Fadu cells were seeded onto array of printed rhodamine encapsulated MPs. Fluorescence micrograph overlay of nuclei staining in blue, and MPs shown in red.

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86 LIST OF REFERENCES [1] Haller MJ, Atkinson MA, Schatz D. Type 1 diabetes mellitus: etiology , presentation, and management. Pediatric clinics of North America. 2005;52:1553 78. [2] Kabelitz D, Geissler EK, Soria B, Schroeder IS, Fandrich F, Chatenoud L. Toward cell based therapy of type I diabetes. Trends in immunology. 2008;29:68 74. [3] Permutt MA, Wasson J, Cox N. Genetic epidemiology of diabetes. The Journal of clinical investigation. 2005;115:1431 9. [4] Goudy KS, Tisch R. Immunotherapy for the prevention and treatment of type 1 diabetes. International reviews of immunology. 2005;24:307 26. [ 5] Zipris D. Epidemiology of type 1 diabetes and what animal models teach us about the role of viruses in disease mechanisms. Clinical immunology. 2009;131:11 23. [6] Bluestone JA, Tang Q, Sedwick CE. T regulatory cells in autoimmune diabetes: past challen ges, future prospects. Journal of clinical immunology. 2008;28:677 84. [7] Lehuen A, Diana J, Zaccone P, Cooke A. Immune cell crosstalk in type 1 diabetes. Nature reviews Immunology. 2010;10:501 13. [8] Raman VS, Heptulla RA. New potential adjuncts to trea tment of children with type 1 diabetes mellitus. Pediatric research. 2009;65:370 4. [9] Sherr J, Tamborlane WV. Past, present, and future of insulin pump therapy: better shot at diabetes control. The Mount Sinai journal of medicine, New York. 2008;75:352 6 1. [10] Ichii H, Ricordi C. Current status of islet cell transplantation. Journal of hepato biliary pancreatic surgery. 2009;16:101 12. [11] Danne T, Becker D. Paediatric diabetes: achieving practical, effective insulin therapy in type 1 and type 2 diabete s. Acta paediatrica. 2007;96:1560 70. [12] Lo J, Peng RH, Barker T, Xia CQ, Clare Salzler MJ. Peptide pulsed immature dendritic cells reduce response to beta cell target antigens and protect NOD recipients from type I diabetes. Annals of the New York Acade my of Sciences. 2006;1079:153 6. [13] Guo T, Hebrok M. Stem cells to pancreatic beta cells: new sources for diabetes cell therapy. Endocrine reviews. 2009;30:214 27. [14] Clare Salzler MJ, Brooks J, Chai A, Van Herle K, Anderson C. Prevention of diabetes i n nonobese diabetic mice by dendritic cell transfer. The Journal of clinical investigation. 1992;90:741 8.

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87 [15] Guerder S, Joncker N, Mahiddine K, Serre L. Dendritic cells in tolerance and autoimmune diabetes. Current opinion in immunology. 2013;25:670 5. [16] Maldonado RA, von Andrian UH. How tolerogenic dendritic cells induce regulatory T cells. Advances in immunology. 2010;108:111 65. [17] Ganguly D, Haak S, Sisirak V, Reizis B. The role of dendritic cells in autoimmunity. Nature reviews Immunology. 2013 ;13:566 77. [18] Cabrera SM, Rigby MR, Mirmira RG. Targeting regulatory T cells in the treatment of type 1 diabetes mellitus. Current molecular medicine. 2012;12:1261 72. [19] Roncarolo MG, Battaglia M. Regulatory T cell immunotherapy for tolerance to self antigens and alloantigens in humans. Nature reviews Immunology. 2007;7:585 98. [20] Steptoe RJ, Thomson AW. Dendritic cells and tolerance induction. Clinical and experimental immunology. 1996;105:397 402. [21] Morelli AE, Hackstein H, Thomson AW. Potentia l of tolerogenic dendritic cells for transplantation. Seminars in immunology. 2001;13:323 35. [22] Cronin SJ, Penninger JM. From T cell activation signals to signaling control of anti cancer immunity. Immunological reviews. 2007;220:151 68. [23] Steinman R M, Hawiger D, Nussenzweig MC. Tolerogenic dendritic cells. Annual review of immunology. 2003;21:685 711. [24] Yamazaki S, Inaba K, Tarbell KV, Steinman RM. Dendritic cells expand antigen specific Foxp3+ CD25+ CD4+ regulatory T cells including suppressors o f alloreactivity. Immunological reviews. 2006;212:314 29. [25] Tarbell KV, Yamazaki S, Steinman RM. The interactions of dendritic cells with antigen specific, regulatory T cells that suppress autoimmunity. Seminars in immunology. 2006;18:93 102. [26] Urban BC, Willcox N, Roberts DJ. A role for CD36 in the regulation of dendritic cell function. Proceedings of the National Academy of Sciences of the United States of America. 2001;98:8750 5. [27] Verbovetski I, Bychkov H, Trahtemberg U, Shapira I, Hareuveni M, Ben Tal O, et al. Opsonization of apoptotic cells by autologous iC3b facilitates clearance by immature dendritic cells, down regulates DR and CD86, and up regulates CC chemokine receptor 7. The Journal of experimental medicine. 2002;196:1553 61. [28] Mahn ke K, Johnson TS, Ring S, Enk AH. Tolerogenic dendritic cells and regulatory T cells: a two way relationship. Journal of dermatological science. 2007;46:159 67.

PAGE 88

88 [29] Luo X, Tarbell KV, Yang H, Pothoven K, Bailey SL, Ding R, et al. Dendritic cells with TGF beta1 differentiate naive CD4+CD25 T cells into islet protective Foxp3+ regulatory T cells. Proceedings of the National Academy of Sciences of the United States of America. 2007;104:2821 6. [30] Yang H, Cheng EY, Sharma VK, Lagman M, Chang C, Song P, et a l. Dendritic cells with TGF beta1 and IL 2 differentiate naive CD4+ T cells into alloantigen specific and allograft protective Foxp3+ regulatory T cells. Transplantation. 2012;93:580 8. [31] Lang R, Patel D, Morris JJ, Rutschman RL, Murray PJ. Shaping gene expression in activated and resting primary macrophages by IL 10. Journal of immunology. 2002;169:2253 63. [32] Moore KW, de Waal Malefyt R, Coffman RL, O'Garra A. Interleukin 10 and the interleukin 10 receptor. Annual review of immunology. 2001;19:683 76 5. [33] Laouar Y, Town T, Jeng D, Tran E, Wan Y, Kuchroo VK, et al. TGF beta signaling in dendritic cells is a prerequisite for the control of autoimmune encephalomyelitis. Proceedings of the National Academy of Sciences of the United States of America. 20 08;105:10865 70. [34] Morelli AE, Thomson AW. Dendritic cells: regulators of alloimmunity and opportunities for tolerance induction. Immunological reviews. 2003;196:125 46. [35] Kojima A, Prehn RT. Genetic susceptibility to post thymectomy autoimmune disea ses in mice. Immunogenetics. 1981;14:15 27. [36] Sakaguchi S, Fukuma K, Kuribayashi K, Masuda T. Organ specific autoimmune diseases induced in mice by elimination of T cell subset. I. Evidence for the active participation of T cells in natural self toleran ce; deficit of a T cell subset as a possible cause of autoimmune disease. The Journal of experimental medicine. 1985;161:72 87. [37] Tang Q, Bluestone JA. The Foxp3+ regulatory T cell: a jack of all trades, master of regulation. Nature immunology. 2008;9:2 39 44. [38] Bluestone JA, Abbas AK. Natural versus adaptive regulatory T cells. Nature reviews Immunology. 2003;3:253 7. [39] Bresson D, Togher L, Rodrigo E, Chen Y, Bluestone JA, Herold KC, et al. Anti CD3 and nasal proinsulin combination therapy enhances remission from recent onset autoimmune diabetes by inducing Tregs. The Journal of clinical investigation. 2006;116:1371 81. [40] Kretschmer K, Heng TS, von Boehmer H. De novo production of antigen specific suppressor cells in vivo. Nature protocols. 2006; 1:653 61. [41] Vignali DA, Collison LW, Workman CJ. How regulatory T cells work. Nature reviews Immunology. 2008;8:523 32.

PAGE 89

89 [42] Weiner HL. Induction and mechanism of action of transforming growth factor beta secreting Th3 regulatory cells. Immunological re views. 2001;182:207 14. [43] Barrat FJ, Cua DJ, Boonstra A, Richards DF, Crain C, Savelkoul HF, et al. In vitro generation of interleukin 10 producing regulatory CD4(+) T cells is induced by immunosuppressive drugs and inhibited by T helper type 1 (Th1) a nd Th2 inducing cytokines. The Journal of experimental medicine. 2002;195:603 16. [44] Fontenot JD, Rasmussen JP, Gavin MA, Rudensky AY. A function for interleukin 2 in Foxp3 expressing regulatory T cells. Nature immunology. 2005;6:1142 51. [45] Hori S, No mura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science. 2003;299:1057 61. [46] Khattri R, Cox T, Yasayko SA, Ramsdell F. An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nature immunology. 200 3;4:337 42. [47] Bennett CL, Christie J, Ramsdell F, Brunkow ME, Ferguson PJ, Whitesell L, et al. The immune dysregulation, polyendocrinopathy, enteropathy, X linked syndrome (IPEX) is caused by mutations of FOXP3. Nature genetics. 2001;27:20 1. [48] Gambi neri E, Torgerson TR, Ochs HD. Immune dysregulation, polyendocrinopathy, enteropathy, and X linked inheritance (IPEX), a syndrome of systemic autoimmunity caused by mutations of FOXP3, a critical regulator of T cell homeostasis. Current opinion in rheumato logy. 2003;15:430 5. [49] Ochs HD, Ziegler SF, Torgerson TR. FOXP3 acts as a rheostat of the immune response. Immunological reviews. 2005;203:156 64. [50] Josefowicz SZ, Rudensky A. Control of regulatory T cell lineage commitment and maintenance. Immunity. 2009;30:616 25. [51] Curotto de Lafaille MA, Lafaille JJ. Natural and adaptive foxp3+ regulatory T cells: more of the same or a division of labor? Immunity. 2009;30:626 35. [52] Groux H, O'Garra A, Bigler M, Rouleau M, Antonenko S, de Vries JE, et al. A C D4+ T cell subset inhibits antigen specific T cell responses and prevents colitis. Nature. 1997;389:737 42. [53] Chen W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, et al. Conversion of peripheral CD4+CD25 naive T cells to CD4+CD25+ regulatory T cells by TGF beta induction of transcription factor Foxp3. The Journal of experimental medicine. 2003;198:1875 86. [54] Li MO, Sanjabi S, Flavell RA. Transforming growth factor beta controls development, homeostasis, and tolerance of T cells by regulatory T cell de pendent and independent mechanisms. Immunity. 2006;25:455 71.

PAGE 90

90 [55] Marie JC, Letterio JJ, Gavin M, Rudensky AY. TGF beta1 maintains suppressor function and Foxp3 expression in CD4+CD25+ regulatory T cells. The Journal of experimental medicine. 2005;201:10 61 7. [56] Asseman C, Mauze S, Leach MW, Coffman RL, Powrie F. An essential role for interleukin 10 in the function of regulatory T cells that inhibit intestinal inflammation. The Journal of experimental medicine. 1999;190:995 1004. [57] Fahlen L, Read S, Gorelik L, Hurst SD, Coffman RL, Flavell RA, et al. T cells that cannot respond to TGF beta escape control by CD4(+)CD25(+) regulatory T cells. The Journal of experimental medicine. 2005;201:737 46. [58] Nakamura K, Kitani A, Strober W. Cell contact depend ent immunosuppression by CD4(+)CD25(+) regulatory T cells is mediated by cell surface bound transforming growth factor beta. The Journal of experimental medicine. 2001;194:629 44. [59] Coombes JL, Siddiqui KR, Arancibia Carcamo CV, Hall J, Sun CM, Belkaid Y, et al. A functionally specialized population of mucosal CD103+ DCs induces Foxp3+ regulatory T cells via a TGF beta and retinoic acid dependent mechanism. The Journal of experimental medicine. 2007;204:1757 64. [60] Hsieh CS, Liang Y, Tyznik AJ, Self SG , Liggitt D, Rudensky AY. Recognition of the peripheral self by naturally arising CD25+ CD4+ T cell receptors. Immunity. 2004;21:267 77. [61] Jordan MS, Boesteanu A, Reed AJ, Petrone AL, Holenbeck AE, Lerman MA, et al. Thymic selection of CD4+CD25+ regulat ory T cells induced by an agonist self peptide. Nature immunology. 2001;2:301 6. [62] Rigby MR, Trexler AM, Pearson TC, Larsen CP. CD28/CD154 blockade prevents autoimmune diabetes by inducing nondeletional tolerance after effector t cell inhibition and reg ulatory T cell expansion. Diabetes. 2008;57:2672 83. [63] Peter SJ, Miller MJ, Yasko AW, Yaszemski MJ, Mikos AG. Polymer concepts in tissue engineering. Journal of biomedical materials research. 1998;43:422 7. [64] Tamber H, Johansen P, Merkle HP, Gander B . Formulation aspects of biodegradable polymeric microspheres for antigen delivery. Advanced drug delivery reviews. 2005;57:357 76. [65] Jain RA. The manufacturing techniques of various drug loaded biodegradable poly(lactide co glycolide) (PLGA) devices. B iomaterials. 2000;21:2475 90. [66] Okada H, Toguchi H. Biodegradable microspheres in drug delivery. Critical reviews in therapeutic drug carrier systems. 1995;12:1 99.

PAGE 91

91 [67] Jiang W, Gupta RK, Deshpande MC, Schwendeman SP. Biodegradable poly(lactic co glyco lic acid) microparticles for injectable delivery of vaccine antigens. Advanced drug delivery reviews. 2005;57:391 410. [68] Faisant N, Siepmann J, Benoit JP. PLGA based microparticles: elucidation of mechanisms and a new, simple mathematical model quantify ing drug release. European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences. 2002;15:355 66. [69] Yeo Y, Park K. Control of encapsulation efficiency and initial burst in polymeric microparticle sy stems. Archives of pharmacal research. 2004;27:1 12. [70] Bouissou C, Rouse JJ, Price R, van der Walle CF. The influence of surfactant on PLGA microsphere glass transition and water sorption: remodeling the surface morphology to attenuate the burst release . Pharmaceutical research. 2006;23:1295 305. [71] Ruhe PQ, Hedberg EL, Padron NT, Spauwen PH, Jansen JA, Mikos AG. rhBMP 2 release from injectable poly(DL lactic co glycolic acid)/calcium phosphate cement composites. The Journal of bone and joint surgery A merican volume. 2003;85 A Suppl 3:75 81. [72] Kempf M, Mandal B, Jilek S, Thiele L, Voros J, Textor M, et al. Improved stimulation of human dendritic cells by receptor engagement with surface modified microparticles. Journal of drug targeting. 2003;11:11 8 . [73] Faraasen S, Voros J, Csucs G, Textor M, Merkle HP, Walter E. Ligand specific targeting of microspheres to phagocytes by surface modification with poly(L lysine) grafted poly(ethylene glycol) conjugate. Pharmaceutical research. 2003;20:237 46. [74] B onifaz L, Bonnyay D, Mahnke K, Rivera M, Nussenzweig MC, Steinman RM. Efficient targeting of protein antigen to the dendritic cell receptor DEC 205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and p eripheral CD8+ T cell tolerance. The Journal of experimental medicine. 2002;196:1627 38. [75] Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature. 1998;392:245 52. [76] Shive MS, Anderson JM. Biodegradation and biocompatibility o f PLA and PLGA microspheres. Advanced drug delivery reviews. 1997;28:5 24. [77] Berkland C, King M, Cox A, Kim K, Pack DW. Precise control of PLG microsphere size provides enhanced control of drug release rate. Journal of controlled release : official jour nal of the Controlled Release Society. 2002;82:137 47. [78] Freiberg S, Zhu XX. Polymer microspheres for controlled drug release. International journal of pharmaceutics. 2004;282:1 18.

PAGE 92

92 [79] King TW, Patrick CW, Jr. Development and in vitro characterization of vascular endothelial growth factor (VEGF) loaded poly(DL lactic co glycolic acid)/poly(ethylene glycol) microspheres using a solid encapsulation/single emulsion/solvent extraction technique. Journal of biomedical materials research. 2000;51:383 90. [80 ] Rosca ID, Watari F, Uo M. Microparticle formation and its mechanism in single and double emulsion solvent evaporation. Journal of controlled release : official journal of the Controlled Release Society. 2004;99:271 80. [81] Chaisri W, Hennink WE, Okonogi S. Preparation and characterization of cephalexin loaded PLGA microspheres. Current drug delivery. 2009;6:69 75. [82] Mao S, Xu J, Cai C, Germershaus O, Schaper A, Kissel T. Effect of WOW process parameters on morphology and burst release of FITC dextran loaded PLGA microspheres. International journal of pharmaceutics. 2007;334:137 48. [83] Herrmann J, Bodmeier R. Biodegradable, somatostatin acetate containing microspheres prepared by various aqueous and non aqueous solvent evaporation methods. European jo urnal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik eV. 1998;45:75 82. [84] McGinity JW, O'Donnell PB. Preparation of microspheres by the solvent evaporation technique. Advanced drug d elivery reviews. 1997;28:25 42. [85] Hua FJ, Kim GE, Lee JD, Son YK, Lee DS. Macroporous poly(L lactide) scaffold 1. Preparation of a macroporous scaffold by liquid -liquid phase separation of a PLLA -dioxane -water system. Journal of biomedical materials research. 2002;63:161 7. [86] Graham PD, Brodbeck KJ, McHugh AJ. Phase inversion dynamics of PLGA solutions related to drug delivery. Journal of controlled release : official journal of the Controlled Release Society. 1999;58:233 45. [87] Newman KD, Samuel J, Kwon G. Ovalbumin peptide encapsulated in poly(d,l lactic co glycolic acid) microspheres is capable of inducing a T helper type 1 immune response. Journal of controlled release : official journal of the Controlled Release Society. 1998;54:49 59. [88] M arnett LJ, Kalgutkar AS. Cyclooxygenase 2 inhibitors: discovery, selectivity and the future. Trends in pharmacological sciences. 1999;20:465 9. [89] Smith WL, Meade EA, DeWitt DL. Pharmacology of prostaglandin endoperoxide synthase isozymes 1 and 2. Annal s of the New York Academy of Sciences. 1994;714:136 42. [90] Ho LJ, Chang DM, Shiau HY, Chen CH, Hsieh TY, Hsu YL, et al. Aspirin differentially regulates endotoxin induced IL 12 and TNF alpha production in human dendritic cells. Scandinavian journal of rh eumatology. 2001;30:346 52.

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93 [91] Jonuleit H, Kuhn U, Muller G, Steinbrink K, Paragnik L, Schmitt E, et al. Pro inflammatory cytokines and prostaglandins induce maturation of potent immunostimulatory dendritic cells under fetal calf serum free conditions. E uropean journal of immunology. 1997;27:3135 42. [92] Rieser C, Bock G, Klocker H, Bartsch G, Thurnher M. Prostaglandin E2 and tumor necrosis factor alpha cooperate to activate human dendritic cells: synergistic activation of interleukin 12 production. The Journal of experimental medicine. 1997;186:1603 8. [93] Whittaker DS, Bahjat KS, Moldawer LL, Clare Salzler MJ. Autoregulation of human monocyte derived dendritic cell maturation and IL 12 production by cyclooxygenase 2 mediated prostanoid production. Jour nal of immunology. 2000;165:4298 304. [94] Matasic R, Dietz AB, Vuk Pavlovic S. Cyclooxygenase independent inhibition of dendritic cell maturation by aspirin. Immunology. 2000;101:53 60. [95] Hackstein H, Morelli AE, Larregina AT, Ganster RW, Papworth GD, Logar AJ, et al. Aspirin inhibits in vitro maturation and in vivo immunostimulatory function of murine myeloid dendritic cells. Journal of immunology. 2001;166:7053 62. [96] Yadav VS, Mishra KP, Singh DP, Mehrotra S, Singh VK. Immunomodulatory effects of c urcumin. Immunopharmacology and immunotoxicology. 2005;27:485 97. [97] Balasubramanyam M, Koteswari AA, Kumar RS, Monickaraj SF, Maheswari JU, Mohan V. Curcumin induced inhibition of cellular reactive oxygen species generation: novel therapeutic implicatio ns. Journal of biosciences. 2003;28:715 21. [98] Ruby AJ, Kuttan G, Babu KD, Rajasekharan KN, Kuttan R. Anti tumour and antioxidant activity of natural curcuminoids. Cancer letters. 1995;94:79 83. [99] Apisariyakul A, Vanittanakom N, Buddhasukh D. Antifung al activity of turmeric oil extracted from Curcuma longa (Zingiberaceae). Journal of ethnopharmacology. 1995;49:163 9. [100] Mazumder A, Raghavan K, Weinstein J, Kohn KW, Pommier Y. Inhibition of human immunodeficiency virus type 1 integrase by curcumin. B iochemical pharmacology. 1995;49:1165 70. [101] Negi PS, Jayaprakasha GK, Jagan Mohan Rao L, Sakariah KK. Antibacterial activity of turmeric oil: a byproduct from curcumin manufacture. Journal of agricultural and food chemistry. 1999;47:4297 300. [102] Gar g AK, Buchholz TA, Aggarwal BB. Chemosensitization and radiosensitization of tumors by plant polyphenols. Antioxidants & redox signaling. 2005;7:1630 47. [103] Srinivasan M. Effect of curcumin on blood sugar as seen in a diabetic subject. Indian journal of medical sciences. 1972;26:269 70.

PAGE 94

94 [104] Babu PS, Srinivasan K. Influence of dietary curcumin and cholesterol on the progression of experimentally induced diabetes in albino rat. Molecular and cellular biochemistry. 1995;152:13 21. [105] Srivivasan A, Meno n VP, Periaswamy V, Rajasekaran KN. Protection of pancreatic beta cell by the potential antioxidant bis o hydroxycinnamoyl methane, analogue of natural curcuminoid in experimental diabetes. Journal of pharmacy & pharmaceutical sciences : a publication of t he Canadian Society for Pharmaceutical Sciences, Societe canadienne des sciences pharmaceutiques. 2003;6:327 33. [106] Sidhu GS, Mani H, Gaddipati JP, Singh AK, Seth P, Banaudha KK, et al. Curcumin enhances wound healing in streptozotocin induced diabetic rats and genetically diabetic mice. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society. 1999;7:362 74. [107] Suresh Babu P, Srinivasan K. Amelioration of renal lesions associated with diabetes by dietary curcumin in streptozotocin diabetic rats. Molecular and cellular biochemistry. 1998;181:87 96. [108] Kumar PA, Suryanarayana P, Reddy PY, Reddy GB. Modulation of alpha crystallin chaperone activity in diabetic rat lens by curcumin. Mole cular vision. 2005;11:561 8. [109] Suryanarayana P, Saraswat M, Mrudula T, Krishna TP, Krishnaswamy K, Reddy GB. Curcumin and turmeric delay streptozotocin induced diabetic cataract in rats. Investigative ophthalmology & visual science. 2005;46:2092 9. [11 0] Shirley SA, Montpetit AJ, Lockey RF, Mohapatra SS. Curcumin prevents human dendritic cell response to immune stimulants. Biochemical and biophysical research communications. 2008;374:431 6. [111] Feng G, Gao W, Strom TB, Oukka M, Francis RS, Wood KJ, et al. Exogenous IFN gamma ex vivo shapes the alloreactive T cell repertoire by inhibition of Th17 responses and generation of functional Foxp3+ regulatory T cells. European journal of immunology. 2008;38:2512 27. [112] Feng G, Wood KJ, Bushell A. Interferon gamma conditioning ex vivo generates CD25+CD62L+Foxp3+ regulatory T cells that prevent allograft rejection: potential avenues for cellular therapy. Transplantation. 2008;86:578 89. [113] Wang Z, Hong J, Sun W, Xu G, Li N, Chen X, et al. Role of IFN gamma in induction of Foxp3 and conversion of CD4+ CD25 T cells to CD4+ Tregs. The Journal of clinical investigation. 2006;116:2434 41. [114] Cong Y, Wang L, Konrad A, Schoeb T, Elson CO. Curcumin induces the tolerogenic dendritic cell that promotes differentia tion of intestine protective regulatory T cells. European journal of immunology. 2009;39:3134 46.

PAGE 95

95 [115] Nakagawa K, Miyazawa T. Chemiluminescence high performance liquid chromatographic determination of tea catechin, ( ) epigallocatechin 3 gallate, at picomole levels in rat and human plasma. Analytical biochemistry. 1997;248:41 9. [116] Lambert JD, Yang CS. Cancer chemopreventive activity and bioavailability of tea and tea polyphenols. Mutation research. 2003;523 524:201 8. [117] Cao Y, Cao R. Angiogene sis inhibited by drinking tea. Nature. 1999;398:381. [118] Kawai K, Tsuno NH, Kitayama J, Okaji Y, Yazawa K, Asakage M, et al. Epigallocatechin gallate induces apoptosis of monocytes. The Journal of allergy and clinical immunology. 2005;115:186 91. [119] W u D, Wang J, Pae M, Meydani SN. Green tea EGCG, T cells, and T cell mediated autoimmune diseases. Molecular aspects of medicine. 2012;33:107 18. [120] Xia CQ, Kao KJ. Suppression of interleukin 12 production through endogenously secreted interleukin 10 in activated dendritic cells: involvement of activation of extracellular signal regulated protein kinase. Scandinavian journal of immunology. 2003;58:23 32. [121] De Smedt T, Van Mechelen M, De Becker G, Urbain J, Leo O, Moser M. Effect of interleukin 10 on d endritic cell maturation and function. European journal of immunology. 1997;27:1229 35. [122] Zella JB, McCary LC, DeLuca HF. Oral administration of 1,25 dihydroxyvitamin D3 completely protects NOD mice from insulin dependent diabetes mellitus. Archives of biochemistry and biophysics. 2003;417:77 80. [123] Mathieu C, Laureys J, Sobis H, Vandeputte M, Waer M, Bouillon R. 1,25 Dihydroxyvitamin D3 prevents insulitis in NOD mice. Diabetes. 1992;41:1491 5. [124] Gregori S, Giarratana N, Smiroldo S, Uskokovic M, Adorini L. A 1alpha,25 dihydroxyvitamin D(3) analog enhances regulatory T cells and arrests autoimmune diabetes in NOD mice. Diabetes. 2002;51:1367 74. [125] Riachy R, Vandewalle B, Belaich S, Kerr Conte J, Gmyr V, Zerimech F, et al. Beneficial effect of 1 ,25 dihydroxyvitamin D3 on cytokine treated human pancreatic islets. The Journal of endocrinology. 2001;169:161 8. [126] Arnson Y, Amital H, Shoenfeld Y. Vitamin D and autoimmunity: new aetiological and therapeutic considerations. Annals of the rheumatic d iseases. 2007;66:1137 42. [127] Janner M, Ballinari P, Mullis PE, Fluck CE. High prevalence of vitamin D deficiency in children and adolescents with type 1 diabetes. Swiss medical weekly. 2010;140:w13091.

PAGE 96

96 [128] Geber A, Hitchcock CA, Swartz JE, Pullen FS, Marsden KE, Kwon Chung KJ, et al. Deletion of the Candida glabrata ERG3 and ERG11 genes: effect on cell viability, cell growth, sterol composition, and antifungal susceptibility. Antimicrobial agents and chemotherapy. 1995;39:2708 17. [129] Kathiravan MK, Salake AB, Chothe AS, Dudhe PB, Watode RP, Mukta MS, et al. The biology and chemistry of antifungal agents: a review. Bioorganic & medicinal chemistry. 2012;20:5678 98. [130] Takaku T, Kimura Y, Okuda H. Isolation of an antitumor compound from Agaricus bla zei Murill and its mechanism of action. The Journal of nutrition. 2001;131:1409 13. [131] Yazawa Y, Yokota M, Sugiyama K. Antitumor promoting effect of an active component of Polyporus, ergosterol and related compounds on rat urinary bladder carcinogenesis in a short term test with concanavalin A. Biological & pharmaceutical bulletin. 2000;23:1298 302. [132] Castro FA, Mariani D, Panek AD, Eleutherio EC, Pereira MD. Cytotoxicity mechanism of two naphthoquinones (menadione and plumbagin) in Saccharomyces cer evisiae. PloS one. 2008;3:e3999. [133] Hatanaka H, Ishizawa H, Nakamura Y, Tadokoro H, Tanaka S, Onda K, et al. Effects of vitamin K and K on proliferation, cytokine production, and regulatory T cell frequency in human peripheral blood mononuclear cells. L ife sciences. 2014. [134] Govinden R, Bhoola KD. Genealogy, expression, and cellular function of transforming growth factor beta. Pharmacology & therapeutics. 2003;98:257 65. [135] Gorelik L, Constant S, Flavell RA. Mechanism of transforming growth factor beta induced inhibition of T helper type 1 differentiation. The Journal of experimental medicine. 2002;195:1499 505. [136] Gorham JD, Guler ML, Fenoglio D, Gubler U, Murphy KM. Low dose TGF beta attenuates IL 12 responsiveness in murine Th cells. Journal o f immunology. 1998;161:1664 70. [137] Fantini MC, Becker C, Monteleone G, Pallone F, Galle PR, Neurath MF. Cutting edge: TGF beta induces a regulatory phenotype in CD4+CD25 T cells through Foxp3 induction and down regulation of Smad7. Journal of immunolog y. 2004;172:5149 53. [138] Rao PE, Petrone AL, Ponath PD. Differentiation and expansion of T cells with regulatory function from human peripheral lymphocytes by stimulation in the presence of TGF {beta}. Journal of immunology. 2005;174:1446 55. [139] Apost olou I, Verginis P, Kretschmer K, Polansky J, Huhn J, von Boehmer H. Peripherally induced Treg: mode, stability, and role in specific tolerance. Journal of clinical immunology. 2008;28:619 24.

PAGE 97

97 [140] Liu Y, Zhang P, Li J, Kulkarni AB, Perruche S, Chen W. A critical function for TGF beta signaling in the development of natural CD4+CD25+Foxp3+ regulatory T cells. Nature immunology. 2008;9:632 40. [141] Luo X, Yang H, Kim IS, Saint Hilaire F, Thomas DA, De BP, et al. Systemic transforming growth factor beta1 ge ne therapy induces Foxp3+ regulatory cells, restores self tolerance, and facilitates regeneration of beta cell function in overtly diabetic nonobese diabetic mice. Transplantation. 2005;79:1091 6. [142] Perruche S, Zhang P, Liu Y, Saas P, Bluestone JA, Che n W. CD3 specific antibody induced immune tolerance involves transforming growth factor beta from phagocytes digesting apoptotic T cells. Nature medicine. 2008;14:528 35. [143] Peng Y, Laouar Y, Li MO, Green EA, Flavell RA. TGF beta regulates in vivo expansion of Foxp3 expressing CD4+CD25+ regulatory T cells responsible for protection against diabetes. Proceedings of the National Academy of Sciences of the United States of America. 2004;101:4572 7. [144] Kuruvilla AP, Shah R, Hochwald GM, Liggitt HD, P alladino MA, Thorbecke GJ. Protective effect of transforming growth factor beta 1 on experimental autoimmune diseases in mice. Proceedings of the National Academy of Sciences of the United States of America. 1991;88:2918 21. [145] Racke MK, Dhib Jalbut S, Cannella B, Albert PS, Raine CS, McFarlin DE. Prevention and treatment of chronic relapsing experimental allergic encephalomyelitis by transforming growth factor beta 1. Journal of immunology. 1991;146:3012 7. [146] Sun YV, Boverhof DR, Burgoon LD, Fielden MR, Zacharewski TR. Comparative analysis of dioxin response elements in human, mouse and rat genomic sequences. Nucleic acids research. 2004;32:4512 23. [147] Hahn ME, Karchner SI, Shapiro MA, Perera SA. Molecular evolution of two vertebrate aryl hydrocar bon (dioxin) receptors (AHR1 and AHR2) and the PAS family. Proceedings of the National Academy of Sciences of the United States of America. 1997;94:13743 8. [148] Busbee PB, Rouse M, Nagarkatti M, Nagarkatti PS. Use of natural AhR ligands as potential ther apeutic modalities against inflammatory disorders. Nutrition reviews. 2013;71:353 69. [149] Nguyen NT, Hanieh H, Nakahama T, Kishimoto T. The roles of aryl hydrocarbon receptor in immune responses. International immunology. 2013;25:335 43. [150] Brasnyo P, Molnar GA, Mohas M, Marko L, Laczy B, Cseh J, et al. Resveratrol improves insulin sensitivity, reduces oxidative stress and activates the Akt pathway in type 2 diabetic patients. The British journal of nutrition. 2011;106:383 9.

PAGE 98

98 [151] Henney CS, Lichtenst ein LM. The role of cyclic AMP in the cytolytic activity of lymphocytes. Journal of immunology. 1971;107:610 2. [152] Spina D. PDE4 inhibitors: current status. British journal of pharmacology. 2008;155:308 15. [153] Ramchandani M, Robinson D. In vitro and in vivo release of ciprofloxacin from PLGA 50:50 implants. Journal of controlled release : official journal of the Controlled Release Society. 1998;54:167 75. [154] Amann LC, Gandal MJ, Lin R, Liang Y, Siegel SJ. In vitro in vivo correlations of scalable P LGA risperidone implants for the treatment of schizophrenia. Pharmaceutical research. 2010;27:1730 7. [155] Lu L, Peter SJ, Lyman MD, Lai HL, Leite SM, Tamada JA, et al. In vitro and in vivo degradation of porous poly(DL lactic co glycolic acid) foams. Bio materials. 2000;21:1837 45. [156] Lu L, Garcia CA, Mikos AG. In vitro degradation of thin poly(DL lactic co glycolic acid) films. Journal of biomedical materials research. 1999;46:236 44. [157] Park TG. Degradation of poly(lactic co glycolic acid) microsph eres: effect of copolymer composition. Biomaterials. 1995;16:1123 30. [158] Broadhead ML, Clark JC, Dass CR, Choong PF. Microarray: an instrument for cancer surgeons of the future? ANZ journal of surgery. 2010;80:531 6. [159] Hoffman AS. Non fouling surfac e technologies. Journal of biomaterials science Polymer edition. 1999;10:1011 4. [160] McDonald JC, Duffy DC, Anderson JR, Chiu DT, Wu H, Schueller OJ, et al. Fabrication of microfluidic systems in poly(dimethylsiloxane). Electrophoresis. 2000;21:27 40. [1 61] Moorcroft MJ, Meuleman WR, Latham SG, Nicholls TJ, Egeland RD, Southern EM. In situ oligonucleotide synthesis on poly(dimethylsiloxane): a flexible substrate for microarray fabrication. Nucleic acids research. 2005;33:e75. [162] Effenhauser CS, Bruin G J, Paulus A, Ehrat M. Integrated capillary electrophoresis on flexible silicone microdevices: analysis of DNA restriction fragments and detection of single DNA molecules on microchips. Analytical chemistry. 1997;69:3451 7. [163] Kim T, Cho YH. A pumpless c ell culture chip with the constant medium perfusion rate maintained by balanced droplet dispensing. Lab on a chip. 2011;11:1825 30. [164] Friedman M, Lindstrom S, Ekerljung L, Andersson Svahn H, Carlsson J, Brismar H, et al. Engineering and characterizatio n of a bispecific HER2 x EGFR binding affibody molecule. Biotechnology and applied biochemistry. 2009;54:121 31.

PAGE 99

99 [165] Di Carlo D, Lee LP. Dynamic single cell analysis for quantitative biology. Analytical chemistry. 2006;78:7918 25. [166] Acharya AP, Clare Salzler MJ, Keselowsky BG. A high throughput microparticle microarray platform for dendritic cell targeting vaccines. Biomaterials. 2009;30:4168 77.

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100 BIOGRAPHICAL SKETCH Nikunj K. Agrawal was born in 1987, in Rampur, India. He completed his secondary education in 2005 from Rampur and received a Certificate of Merit from tificates of merit state that he was among the top 0.1% successful cand idates across the country in high school and senior secondary examinations. He also represented his school in various extracurricular biotechnology in 2012 from Amity University, India where he stood top of his class for junior a nd senior year with a GPA of 9.92 /10 and 9.54/10 respectively. He also received scholarships for all four years of my under graduation, an honor that only a few can have. During his under graduation Nikunj attempt ed to expand the horizon of his knowledge through various r esearch and industrial projects and worked on several projects both at the University and at other reputed government and corporate laboratories. To further pursue his interest in biomedical research he came to University of Florida for a MS degree in fall 2012 and started working under Dr. Benjamin s research focus e s on engineering of biomaterial cell interactions, and targeted controlled release of immune modulating factors in order to direct immune cell fun ct ion. It involves fabrication, characterization , co localization and imaging of drug loaded PLGA microparticles of various sizes onto microarray platforms. Additionally, he developed novel PDMS based microwell platform s for high throughput screening of de ndritic cell response s. plans to continue his education further to a Ph.D. program.



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Microparticlesurfacemodi cationstargetingdendriticcellsfornon-activating applicationsJamalS.Lewis,ToralD.Zaveri,CharlesP.CrooksII,BenjaminG.Keselowsky*JCraytonPruittFamilyDepartmentofBiomedicalEngineering,UniversityofFlorida,POBox116131,Gainesville,FL32611-6131,USAarticleinfoArticlehistory: Received10May2012 Accepted22June2012 Availableonline12July2012 Keywords: Microparticle Dendriticcell Phagocytosis Non-activating Microsphere VaccineabstractMicroparticulatesystemsfordeliveryoftherapeuticstoDCsforimmunotherapyhavegainedattention recently.However,reportsaddressingtheoptimizationofDC-targetingmicroparticledeliverysystems arelimited,particularlyforcaseswherethegoalistodeliverpayloadtoDCsinanon-activatingfashion. Here,weinvestigatetargetingDCsusingpoly(dlactide-co-glycolide)microparticles(MPs)inanonstimulatorymannerandassessef cacy invitro and invivo .Wemodi edMPsbysurfaceimmobilizing DCreceptortargetingmolecules e antibodies(anti-CD11c,anti-DEC-205)orpeptides(P-D2,RGD),where anti-CD11cantibody,P-D2andRGDpeptidestargetintegrinsandanti-DEC-205antibodytargetsthe c-typelectinreceptorDEC-205.Ourresultsdemonstratethemodi edMPsareneithertoxicnoractivating,andDCuptakeofMPs invitro isimprovedbytheanti-DEC-205antibody,theanti-CD11cantibody andtheP-D2peptidemodi cations.TheP-D2peptideMPmodi cationsigni cantlyimprovedDC antigenpresentation invitro bothatimmediateanddelayedtimepoints.Notably,MPfunctionalization withP-D2peptideandanti-CD11cantibodyincreasedtherateandextentofMPtranslocation invivo by DCsandMFs,withtheP-D2peptidemodi edMPsdemonstratingthehighesttranslocation.Thiswork informsthedesignofnon-activatingpolymericmicroparticulateapplicationssuchasvaccinesfor autoimmunediseases. 2012ElsevierLtd.Allrightsreserved.1.Introduction Poly(lactic-co-glycolicacid)(PLGA)particulatesystemshave gainedwidespreadattentionasaviableoptionfordeliveryof vaccinesinthelastquartercentury [1] .PLGAparticlesatthemicron andsub-micronsizesarecontemporaryimmuno-therapytools beingusedtodeliverantigen [2,3] ,adjuvant [4] ,DNA [5 e 7] and pharmacologicaldrugs [8,9] .PLGAasabiomaterialhasbeen extensivelycharacterizedandhasbeenshowntodemonstrate qualitiessuchasbiocompatibilityandbiodegradability [1,10] . Additionally,PLGAparticulatesystemsoffercontrolofsizeand shapeofthedeliverysystem,hydrophobicity,loadingandrelease kineticsofawiderangeofbiomolecules,modulationofimmunogenicity,antigenprocessingandpresentation.Furthermore,PLGA particulatematterprovidecapabilityforsurfacefunctionalization [11] .ThesequalitiescombinedmakePLGAmicroparticulate systemsidealforvaccinedeliverytoantigenpresentingcells(APCs) includingdendriticcells(DCs). Firstdiscoveredin1973bythe Steinman group,itisnowwell understoodthatDCsaredirectlyinvolvedininitiationandmodulationofTcellandBcellimmunity [12] .DendriticCellsarethemost ef cientantigenpresentingcellsduetotheirexceptionalabilityto uptake,processandpresentantigen [13 e 15] .Morerecently,ithas beenrecognizedthatDCsplayacriticalroleincentraltoleranceand maintenanceofperipheraltolerance.Theimplicationisthat throughDCs,thedirectionandmagnitudeofimmuneresponsecan bemanipulated.Therefore,DCspresentatherapeutictargetfor modulationofautoimmunediseasesandtransplantrejection [16] . TheversatilityofDCstoguideimmuneresponsesisattributedto itslineageand,maturationstate [12] .Immaturedendriticcells (iDCs)circulatethroughoutthebodyandareableto ‘ scavenge’ pathogens,foreignmaterials,andapoptoticornecroticcells.They areequippedwithawidearrayofendocyticandphagocyticsurface receptorsthatrecognizeahostofmoleculesincludingproteins, lipids,sugars,glycoproteins,glycolipidsandoligonucleotides [17,18] .Notably,thereceptortypeengagedduringphagocytosisby DCsdirectssubsequentchangeinmaturation [18] .Researchers havesoughttoexploitthesetraitsbyincorporatingtargeting moleculessuchaspathogen-associatedmolecularpatterns,and antibodiesagainstsurfacereceptorsinconjunctionwithproteins, polymericparticlesandotherdrugcarriers [19 e 21] .These * Correspondingauthor.Tel.: 13522735878;fax: 13523929791. E-mailaddresses: bgk@u .edu , bkeselowsky@bme.u .edu (B.G.Keselowsky). Contentslistsavailableat SciVerseScienceDirectBiomaterialsjournalhomepage:www.elsevi er.com/locate/biomaterials 0142-9612/$ e seefrontmatter 2012ElsevierLtd.Allrightsreserved. http://dx.doi.org/10.1016/j.biomaterials.2012.06.049 Biomaterials33(2012)7221 e 7232

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approachesareintendedtoaugmentdruguptakebyDCsaswellas bolsteradjuvantactivityforincreasedimmunogenicity [20,22 e 24] . However,therearenumerousapplicationsinwhichtargeting factorstoDCsinanon-stimulatingcontextisperceivedtobe desirablesuchasmicroparticle(MP)-basedvaccinescorrecting T1D [25] .Fornon-stimulatoryapplications,DCreceptorsthatdo nottriggerimmuno-stimulatorypathways,orthataretoleranceinducing,areappropriate. Theendocyticreceptor,DEC-205(CD205)representsonesuch potentialcandidatefornon-activationDC-targeting.DEC-205isan integralmembraneproteinhighlyexpressedonthesurfaceofDCs foundinlymphoidareascriticalforimmunityandtolerance [26] .It isamemberoftheC-typelectinfamilywhichbindscarbohydrates andmediatesendocytosis [26] .Considerableefforthasgone towardstargetingDCsviaDEC-205antibodiesandsingle-chain fragmentvariables(scFv) [21,23,27] . Bonifaz etal.showedthat proteinstargetedtothisreceptorimproveantigenpresentationby 100-fold [21] .Further,DEC-205targetinghasbeenlinkedwithDC abilitytoinducetolerance invitro aswellasinanimalmodels [28,29] .Therefore,iDCscanpossiblybeprimedalongatolerogenic pathwaythroughtargetingoftheDEC-205receptor.Theimplicationsofthiscannotbeoverstatedifthegoalisthedevelopmentof aDC-targetingMPvaccineforautoimmunediseases.Another surfacereceptorabundantlypresentonDCswhichprovides arationalchoiceforDCtargetingistheCD11csurfacemolecule.The CD11c/CD18proteinispartofthefamilyofb2integrinsexpressed exclusivelybyleukocytesparticularlymyeloidDCs [13,30] .TargetingofDCsviatheCD11cantibodieshasbeenshowntoenhance humoralresponsesinmice [13,30,31] .Inadditiontotheuseof antibodies,DC-speci ctargetingthroughtheCD11csurface receptormayalsobeeffectedthroughtheuseofreceptor-binding peptides.TheP-D2peptideisderivedfromtheIg-likedomain2 ofintercellularadhesionmolecule4(ICAM-4) [32] .Allfour membersoftheb2integrinfamilyhaveastrongbindingaf nityfor ICAM-4whichhasbeenshowntobeinvolvedinerythrophagocytosis e aprocessthoughttobeinvolvedinselfrecognitionandimmunehomeostasis [33,34] .Whilewearenotawareof anyworkwhichdirectlyprovidesevidencethatP-D2peptidecould enhanceDCphagocytosis,blockingstudiesby Ihanus etal.highlight thehighaf nitythatP-D2peptidehasforCD11c,andmotivate investigationforuseinMPtargeting [32] .Theuseoftargeting peptideshasthebene tthat,unlikewholeantibodies,itwon ’ tbind toin ammation-causingFcreceptorsandarelessexpensiveto producethanantibodies [35] . ThegoalofcouplingDCtargetingligandstothesurfaceofMPsis toprovideincreasedpayloadofdrug/biological/antigendelivered, therebyimprovingresponseandreducingthenumberofadministrationsrequired.TheDCtargetingligandsinvestigatedheremay functioninanon-activatingmanner,whichcouldproveusefulfor applicationssuchasimmunotherapiestocorrectautoimmune diseasesorpromotetransplanttolerance.2.Methodsandmaterials 2.1.Preparationof uorescentmicroparticles A50:50polymercompositionofpoly(dlactide-co-glycolide)(PLGA;average molecularweight w 44,0000g/mol)inmethylenechloride(Lactel,AL,USA)was usedtogenerateMPs.Poly-vinylalcohol(PVA;molecularweight w 100,000g/mol) waspurchasedfromFisherScienti c(NJ,USA)andwasusedasanemulsionstabilizer.Distilledwater(DiH2O)wasusedastheaqueousphasetoformtheemulsions whilemethylenechloride(FisherScienti c,NJ,USA)wasusedastheorganicsolvent todissolvePLGApolymer.Microparticleswereformedusingastandardoil-water solventevaporationtechnique. Brie y,100mgofPLGApolymerwasdissolvedinmethylenechlorideat5%w/v ratio.Fluorescentdye(eitherrhodamine-6g[Sigma e Aldrich,MO,USA],1,10-dioctadecyl-3,3,30,30-tetramethylindodicarbocyanine,4-chlorobenzenesulfonatesalt [DiD;Invitrogen,Karlsruhe,Germany]orAnthracenecarboxylicacid[ANC;Fluka, Buchs,Switzerland])wasdirectlyloadedinto2mLofthe5%PLGAinmethylene chloridesolutionandemulsi edat35,000rpmfor180susingatissue-miser homogenizer(FisherScienti c,NJ,USA)toformaprimaryemulsion.Theprimary emulsionwasaddedto2mLof5%PVAsolutioninDiH2Oandthehomogenizingwas continuedat19,500rpmfor60s.Thiswasaddedto30mLof0.5%PVAsolution.The particlesthusformedwereagitatedusingamagneticstirrer(FisherScienti c,NJ, USA)for24htoevaporateresidualmethylenechloride.Theremainingsolutionwas centrifugedat10,000 gfor10mintocollectMPswhichweresubsequently washedthreetimeswithDiH2O.ThewaterwasaspiratedfromthecentrifugedMPs, whichwerethen ash-frozeninliquidnitrogenandkeptundervacuumindryice overnight.TheMPswerestoredat 20Cuntilused.Reagentswhosevendorswere notspeci edwerepurchasedfromSigma e Aldrich. 2.2.Cross-linkingofligandstomicroparticles LigandsweretetheredtothesurfaceofPLGAMPsbyconjugationofunbound availableaminegroupsinligandstofreecarboxylterminalsofthepolymerusing carbodiimidechemistry.ThefreecarboxylgroupsonPLGAMPswereactivatedwith a1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride(EDC)(Acros Organics,Belgium)and N -hydroxysulfosuccinimide(NHS)(PierceBiotechnology,IL, USA)solutionfor15minwhileagitatingat50rpm.Theligand(e.g.Anti-mouseCD11c mAb[cloneHL3,IgG1,l2(BDPharmingen)],Anti-mouseDEC-205mAb[clone 205yekta,IgG2a(Ebioscience)])wasintroducedtothesuspensionwhichwasshaken vigorouslyfor16h.Afterincubation,theparticleswerecentrifugedat10,000rpmfor 10minandwashedtwicewithphosphatebufferedsaline(PBS)(Hyclone,UT,USA) andresuspendedforuse.Thepeptides,P-D2[aminoacidsequence:GGVTLTYQFAAGPRDK]andRGD[aminoacidsequence:GGGRGDSPCGGDK]wereproducedby theUniversityofFloridaICBRpeptidesynthesiscorefacility.Forquanti cation purposes,a uorescenttag(either5-carboxytetramethylrhodamine[5-TAMRA]or 7-amino-4-methylcoumarin[MCA])wasaddedtothecarboxylterminalofthese peptides,leavingtheaminoterminusfreetotargetforcrosslinkingtoMPs.The concentrationoftheligandsusedduringtheconjugationstepwasbasedon10:1mol ratiooftheligandtotheamountofPLGApolymerpresent. Asacontrolgroupforsomeexperiments,polyethyleneglycolmoietieswere adsorbedontothesurfaceofMPsbyincubatingaknownmassofPLGAMPsina10% PluronicF127(BASF)solutionfor16hundergentleagitation.Thisgroupwasclassi edasthePEGMPgroup. 2.3.Quanti cationofcross-linkedligands Themethodusedtoquantifyconjugatedligandlevelswasdependentonligand type.Forpeptides,eachPLGAMPbatch(1mgdrymass)conjugatedwith uorescently-taggedpolypeptidewassuspendedin100mLof6mg/mLbovine pancreatictrypsin(MPBiomedicalsLLC,Solon,OH)tocleavethe uorescenttag portionofthepolypeptidederivativefromthesurfaceofthePLGAMPs.Standards containingTAMRA-RGDandtrypsininsolutionwereincubatedsimultaneously.The suspensionswerecentrifugedtopelletMPsand75mLofsupernatantweretransferredtoblack, at-bottom,halfarea,polystyrene96wellplates(CorningInc., Corning,NY)and uorescencequanti edusingaSpectraMaxM5platereader (MolecularDevices,LLC). AntibodiestetheredontothesurfaceofPLGAMPswerequanti edusingadot blotprocedure.Antibody-conjugatedMPsincubatedwith6mg/mLtrypsinovernight.Sampleswerethenboiledin2%SDSfor15min,cooledandspottedonto aPVDFmembrane(BioRad).Standardsconsistingofserialdilutionsofthetrypsinizedantibodyin2%SDSwerealsospottedontothePVDFmembrane.Thespotted membraneswereallowedtodryfor3hatroomtemperatureandblockedovernight at4Cusing20mLofBlotto(5%milkcasein,0.2%Tween-20,0.02%sodiumazidein PBS).Blockingsolutionwasthendecantedoffand20mLoftheantibodysolution (1:10,000dilutionsofalkalinephosphatase-taggedantibodyraisedagainstthe tetheredantibody)wasincubatedwiththemembraneatroomtemperaturefor1h withgentleshaking.Theantibodysolutionwasthenremovedandthree20mL washeswithTBS-tween(20mMTrispH7.6,0.8%NaCl,0.1%Tween-20indeionized water)wereperformedtoremoveunboundantibody.Themembranewasthen washedwith20mLofPBStoremoveresidualTBS-tweensolution.Thebottomof alight-shieldedcontainerwascoveredwith1mLofanalkalinephosphatase colorimetricsubstrate(ECF substrateforWesternblotting,GEHealthcare)andthe membranewaslaidfacedownontothesubstrate.Themembranewasincubatedat roomtemperatureinthesubstratefor25minthenrinsedwithDIwater.AMolecular DynamicsStorm850Imagerwasusedtodetermineimage uorescence(480/ 520nmexcitation/emission)whichwasanalyzedwithAxioVisionsoftware. Densitometricmeansweretakenbyrecordingaveragepixelbrightnessinthespot andwerebackgroundcorrected.Antibodylevelswerecomparedtoproteinantibody standardsforquanti cation. 2.4.Sizedistributionandz-potentialofligand-modi edmicroparticles TheMPsizedistributionwasmeasuredbyaMicrotracNanotracDynamicLight ScatteringParticleAnalyzer(Microtrac,Montgomery,PA).Thezetapotentialof ligand-conjugatedMPswasdeterminedusingaBrookhavenZetaPluszetaJ.S.Lewisetal./Biomaterials33(2012)7221 e 7232 7222

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potentialanalyzer(BrookhavenInstrumentsCorp.,NY,USA).Foreachexperimentalcoatingandcontrol,threesampleswereanalyzedatroomtemperaturein distilledwater. 2.5.Dendriticcellandmacrophagecellculture DendriticCellsandMFsweregeneratedfrombonemarrowobtainedfrom8to 12weekold,female,C57BL6/jandNon-ObeseDiabetic(NOD)miceinaccordance withguidelinesapprovedbyUniversityofFloridausingamodi ed10dayprotocol. ForDCculture,micewereeuthanizedbyCO2asphyxiationfollowedbycervical dislocationandtibiasandfemurswereharvestedforisolatingmarrowcells.The marrowcellswereobtainedby ushingtheshaftoftheboneswitha25gneedle usingRPMImedium(MPBiomedicals,OH,USA)containing1%fetalbovineserum (Lonza,Walkersville,MD)and1%penicillin-streptomycin(Hyclone)andmixedto makeahomogenoussuspension.Thesuspensionwasthenstrainedusing70mm cellstrainers(BectonDickinson,NJ,USA)andcellswerecollectedat1300rpmfor 7min.Theredbloodcells(RBCs)wereremovedbylysingwithACKlysisbuffer (Lonza,Walkersville,MD)followedbycentrifugationat1500rpmfor5minto recoverleukocytes.Leukocyteswerethenre-suspendedinDMEM/F-12withL-glutamine(Cellgro,Herndon,VA),10%fetalbovineserum,1%sodiumpyruvate (Lonza,Walkersville,MD),1%non-essentialaminoacids(Lonza,Walkersville,MD), 1%penicillin-streptomycin(Hyclone)and20ng/mLGM-CSF(R&Dsystems,MN, USA)(DCmedia)andplateontissueculture asksfor2dinordertoremove adherentcells.At2dthe oatingcellsweretransferredtolowattachmentplates andculturedinfreshDCmediaforexpansionofDCprecursorcells.At7d,cells weretransferredtotissuecultureplatestoallowforDCadhesionandproliferation. At10d,theywereliftedfromtissuecultureplatesandused.MFswereobtained similarlyusingMFmediasupplementedwith20ng/mLG-CSF(Millipore,MA, USA) [36] . 2.6.Internalizationofmicroparticles PhagocytosisofMPsbyDCsandMFswascon rmedbyconfocallaserscanning microscopy(OlympusDSU-IX81SpinningDisc,MA,USA).2 105cellsperwellwere culturedinLabTek(Nunc,Roskilde,Denmark)eight-wellchamberglassslidesone daypriortobeingfedsurface-modi edrhodamine-loadedMPsata10:1ratio(MPscells).Afterincubationat37Cfor1h,un-phagocytosedparticleswereremovedby washingthreetimeswith1 PBSand xedusing4%paraformaldehydeatroom temperaturefor10min.Themorphologyofthecellwaselucidatedbystainingwith OregonGreen 488phalloidin(Invitrogen,Karlsruhe,Germany)whilethenucleus washighlightedbyHoechst33342nucleicacidstain(Invitrogen,Karlsruhe, Germany). 2.7.Dendriticcellactivationandcytokineanalysis DCmaturationwasquanti edbymeasuringcellsurfacemarkerlevelsusing owcytometry.Brie y,DCswereliftedbyincubatingwith5mMNa2EDTAsolution in1MPBSsolutionat37Cfor20min.Dendriticcellswerethenwashedwith1% fetalbovineseruminPBSandincubatedwithantibodiesraisedagainstCD16/CD32 (FcgIII/IIReceptor)(clone2.4G2,IgG2b,k),(BDPharmingen,CA,USA)for40minat 4CinordertoblockFcgreceptors.Cellswerethenwashedandstainedwith antibodiesagainstCD80(clone16-10A1,IgG2,k),CD86(cloneGL1,IgG2a,k),MHCII (cloneM5/114.15.2,IgG2b,k),andCD11c(cloneHL3,IgG1,l2)(BDPharmingen)for 40minat4C.Speciesspeci cisotypeswereusedascontrols.Dataacquisitionwas performedusing(FACScalibur,BectonDickinson,NJ,USA) owcytometryandthe geometric uorescentintensitiesdetermined.Morethan10,000eventswere acquiredforeachsampleanddataanalysiswasperformedusingFCSExpressversion 3(DeNovoSoftware,LosAngeles,CA). Cellculturesupernatantswerecollectedafter24hofcellculturewithvarious surface-modi edMPs,centrifugedtoremoveanycelldebrisandstoredat 20C untilanalysis.TheIL-12cytokinesubunit,IL-12p40,andIL-10cytokineproduction wasanalyzedusingsandwichenzyme-linkedimmunosorbantassay(ELISA)kits (BectonDickinson,NJ,USA)accordingtomanufacturer ’ sdirections. 2.8.Cellviability Toexaminethepotentialforcytotoxicityofthesurfacemodi edPLGAMPs, alactatedehydrogenasekit(Roche,Mannheim,Germany)wasusedpermanufacturerinstructions.Inbrief,1 106cellswereplatedin6welltissuecultureplates. Afterbeingexposedtothevarioussurfacemodi edPLGAMPsfor24h,the conditionedmediawerecollectedandcentrifugedtoremovedebrisandthe supernatantswerestoredat 20Cuntilfurthermeasurement.Dendriticcells werelysedbyadding10%TritonX-100tomediafor1h,andthelysateswere collectedasa ‘ deadcell ’ control.Anadditionalcontrolwasusedasthe ‘ livecell ’ controlconsistingofuntreatedDCs.LactateDehydrogenaseactivityinsupernatants andcontrolsweredeterminedbyanincubationwiththechromogenicsubstrateat 37Cfor1h,andtheopticaldensityvaluewasdetectedat490nmbyamicroplate reader(MRXII,Dynextechnologies,VA,USA).Therelativecytotoxicityofeach treatmentwascalculatedby ndingthedifferencebetweenthesampleandthe deadcellcontrol,normalizedtothedifferencebetweenthe ‘ livecell ’ and ‘ deadcell ’ controls. 2.9.Singlecellandmixedculturepreferentialphagocytosisstudies MicroparticlephagocytosisbyDCsandMFsinsuspensionwasinvestigated. DendriticcellsorMFswereincubatedinaDiDsolution(1mM)for1htotracklive cells.FollowingawashingtoremovefreeDiD,cells(1 105cellsin0.2mL)were co-culturedwithvarioussurface-modi edrhodamine-loadedMPsfor1hwhile beinggentlyagitatedat50rpmat37Cina2mMdextrosesolution.Microparticles outnumberedcellsbya10:1ratio.Flowcytometricanalysiswasthencarriedoutto determinethemean uorescentintensityofcellsintherhodaminechannelas ameasureofphagocyticactivity.Controlsincludingcell-onlyandMP-only suspensionswerealsoassessedby owcytometry. Asimilarmethodwasusedtostudypreferentialphagocytosisinamixedculture ofDCsandMFs.Cellswerepre-labeledwitheithercalcein(Invitrogen) e DCsor DiD e MFs,countedandsubsequentlymixedina1:1ratioina2mMdextrose solution.ExperimentalandcontrolANC-loadedMPswereaddedtocellsina10:1 ratio.Thesesuspensionswereagitatedat50rpmfor1hat37Cand owcytometric analysistodeterminethemean uorescentintensityofcellsintheANCchannel afterincubationasameasureofMPuptakelevels.Theappropriatecontrols includingcell-onlyandMP-onlysuspensionswerealsoassessedby owcytometry. InordertoblockFcreceptorsfrombindingtheFcportionofsurfaceimmobilizedantibodies,FcBlock(anti-CD16/32,clone2.4G2)wasaddedtothe suspensionsthatcontainedMPsmodi edwithantibodies.Allexperimentswere repeatedusingthreeseparatepreparationswiththreereplicateseachtimeforeach ligand-modi edMPgroup. 2.10.Antigenpresentationbydendriticcellsafterphagocytosisofsurface-modi ed microparticles NODDCs(2.5 104/well)wereco-incubatedwithsurfacemodi ed,1040-55 mimetope-loadedMPs,aswellastherelevantcontroltreatments,ina96welltissue cultureplatefor1hin2mMdextroseat37C.MPsoutnumberedNODDCsby100:1. Afterthoroughlywashingawayallun-phagocytosedandunboundMPs,NODBDC2.5CD4 T-cells(1.25 105/well)wereaddedtoeachwellandincubatedat 37Cfor3d.Bromodeoxyuridine(BrdU)(kitfromBecktonDickinson)wasaddedto thecultureforthelast4h.T-cellswerethenimmuno uorescentlystainedforBrdU accordingtomanufacturer ’ sspeci cations.Flowcytometrywasthenusedto quantifyTcellproliferationforthedifferenttreatments. InordertodeterminehowprolongedantigenpresentationwasimpactedbyMP surfacemodi cation,MP-treatedDCsafterwashingwereleftinDCculturemediafor 4d.Subsequenttothisperiod,NOD-BDC2.5CD4 Tcellswereaddedfora3day mixedlymphocytecoupling(MLC)followedbyBrdUinoculationandstainingfor quanti cationofproliferation. 2.11.Uptakeandtranslocationofmicroparticlestolymphnodes Thisexperimentwasdesignedtodeterminethetargetingef cacyofourligandmodi edMPsinan invivo environment.Female,6weekoldC57Bl/6micewere dividedinto4experimentalgroups(basedonMPsurfaceligandtype)andthree controlgroups( n 4foreachgroup).Eachanimalfromtheexperimentalgroups receivedasubcutaneousinjectionintothefootpadofbothhindlimbs,containing 0.5mgofligand-conjugatedDiD-loadedMPssuspendedin50mLPBS.Theanimals inthecontrolgroupswere:unmodi edDiD-loadedMPs,50mmdiameterDiDloadedMPs(whicharetoolargetobephagocytosed),andunmodi edunloaded MPs.The50mmdiameterDiD-loadedMPwasincludedasacontroltolocally releaseDiDstainingcellsattheinjectionsitewithoutbeingtakenupinorderto determineifthiseffectcontributedtothenumberofDiD-positivecellstranslocated totheLN. At24,48and72hpost-injection,miceweresacri cedbyCO2asphyxiationand cervicaldislocation,andthedrainingpopliteallymphnodesfromeachhindlimb harvestedandpreparedintosinglecellsuspensions.Cellswerethenpreppedfor owcytometrybyincubationat4Cfor0.5hinacocktailofantibodiesspeci cfor DCs(anti-CD11c,cloneHL3),MFs(anti-F4/80,cloneA3-1)andTcells(anti-CD3, clone17A2).AllantibodieswerefromBDBiosciencesPharmingen(CA,USA)except anti-F4/80whichwasfromAbDSerotec(Oxford,UK).Appropriateisotypeswere usedforeachantibodyspeciesasnegativecontrols. Followingstaining,multi-color owcytometrywasusedtodeterminethe fractionofeachcelltypestainedwiththeDiDdyeasameasureoftheextentparticle uptakeandtranslocationbyDCsandMFs. 2.12.Signi cancetesting StatisticalanalyseswereperformedusinggenerallinearnestedmodelANOVA. BasedontheoverallANOVA p value( p 0.05),aposthocassessmentusingTukey testwassubsequentlyperformedtomakepair-wisecomparisons.Differenceswere consideredsigni cantwhen p 0.05usingSystat(Version12,SystatSoftware,Inc., SanJose,CA).J.S.Lewisetal./Biomaterials33(2012)7221 e 7232 7223

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3.Results 3.1.Microparticlecharacterization Wecharacterizedsurface-modi edMPs,determiningsize distribution,ligandsurfaceloadingandzetapotentials.PLGAMPs werepreparedviaadoubleemulsionsolventevaporationtechnique.FabricatedMPsweredeterminedtohaveanaveragediameterof w 1mmviadynamiclightscatteringtesting,calculatedby volumewitharepresentativeplotshownin Fig.1 A.Conjugationof ligandstotheMPsurfacedidnotalterthesizedistributionofthe particles(datanotshown).Ligandsweretetheredtothesurfaceof PLGAMPs.CarboxylgroupspresentattheMPsurfacewereactivatedbyEDC/NHSandcoupledtoprimaryaminegroupsonthe decoratingmolecules(exceptPEG,whichwassurface-adsorbed). Surfacemodi cationofMPsbyPEG,DEC-205andCD11cantibodies,P-D2andRGDpeptideswascharacterizedbyzetapotentialanalysis.Unmodi edPLGAMPsdemonstratedanegativezeta potential( w 46mV).SurfaceconjugationwithCD11cantibody furtherreducedthezetapotentialtoapproximately 59mV. ConverselycoatingwithPEGmaskedthenegativesurfacecharge to 20mV.Theseobservationsareconsistentwithsimilarpublishedstudies [37] .ConjugationofAnti-DEC-205,P-D2andRGD peptidesonlyslightlyincreasethesurfacecharge( w 37 to 42mV).Weveri edantibodyconjugationbyenzymatic cleavingoftheproteins.Cleavedantibodieswerespottedonto PDVFmembranesandquanti edbydotblotanalysis.Peptide conjugationwasalsovalidatedbyenzymaticcleavageof uorescently-taggedpeptidesfromtheparticlesurfacefollowedby detectionusingaplatereader.ThesurfaceloadingoftheCD11cAb, DEC-205Ab,P-D2peptideandRGDpeptidewererevealedtobe substantialat109ng/mg,88ng/mg,4359ng/mgand19ng/mg PLGArespectivelyastabulatedin Fig.1 B.Itwasalsodetermined, thatthesurfacedensityofallligandswassubstantiallyincreasedby crosslinking.ThesurfacedensityoftheCD11cAb,DEC-205Ab,P-D2 peptideandRGDpeptide,intheabsenceofthecrosslinker,was determinedtobe77ng/mg,60ng/mg,3373ng/mgand11ng/mg PLGArespectively. 3.2.Internalizationofmicroparticles Confocallaserscanningmicroscopywasusedtocon rmthe internalizationofMPsbyDCsandMFs.Themorphologyofthecell waselicitedbystaining lamentousactinwhilethenucleuswas highlightedbyanucleicacidstainandrhodamine-loadedMPs wereusedtoshowparticleengulfment. Fig.2 isarepresentative confocalmicroscopicimageoftheDCcytoskeleton(green) surroundingtherhodamine-loadedMP(orange)inboththex e y andx e zplanes,con rmingMPinternalization. 3.3.Dendriticcellmaturationandviability ToassesswhetherourMPsurfacemodi cationswererelevant fornon-activatingapplications,weexaminedtheireffectonDC maturation.Speci cally,weinvestigatedwhetherornotthe variousligand-graftedMPsincreasedexpressionoftheDCmaturationmarkers,CD80,CD86andMHC-IIonbonemarrow-derived iDCs. ThelevelsofexpressionofCD80,CD86andMHCIIonDCscoculturedwithanyofthesurface-graftedMPswerefoundtobe notstatistically( p -value > 0.05)differenttothatofiDCsand representativedotplotsareshown( Fig.3 ).Incontrast,DCschallengedwithLPSasapositivecontrol,showsigni cantlyhigher expressionlevelsofCD80,CD86andMHCII.Theseresultssuggest theseligand-graftedMPmodi cationsdonotactivateDCs. Furthermore,wedemonstratedthatthereisnoDCtoxicityassociatedwithMPco-incubation( p -value > 0.05)( Fig.4 ). Surface Ligand Zeta Potential (mV) Ligand Surface Loading (ng/ mg PLGA) Hamster Anti-mouse CD11c -59 3 109 11 Rat Anti-mouse DEC-205 -42 3 88 13 P-D2 Peptide -37 1 4359 613 RGD Peptide -42 2 19 4 PEG (10% Pluronic) -20 3 Unmodified -46 3 A B Microparticle Diameter (nm) 010002000300040005000 0 5 10 15 20 25 30 D Av = 1150 nm % Volume Fig.1. Poly(dlactide-co-glycolide)microparticle(MP)characterization e (A)Microparticlesizewasdeterminedtobe1.15mmusingDLS.(B)PLGAMPssurface-modi edwith variousligandsandPEGwerecharacterizedbyzetapotentialmeasurementand uorescentquanti cationtechniquestocon rmsurfaceloading.Zetapotentialdatashown representthemean standarderrorfor vereadingsofeachsample.Theligandsurfaceloadingdatashownrepresentmean standarddeviationforthreereplicatesofeach sample. J.S.Lewisetal./Biomaterials33(2012)7221 e 7232 7224

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3.4.Microparticlephagocytosisbydendriticcellsandmacrophages Inordertoinvestigatethe invitro uptakeofMPssurface modi edwithligandsbyDCsandMFs,weincubatedcellswith uorescently-labeledMPsinsuspension,serum-free.FcBlock (anti-CD16/32,clone2.4G2)wasincludedtotheantibody-modi ed MPgroupstoblockrecognitionbyFcreceptorsinordertodrive uptakebythetargetedreceptors.ThepercentofcellsthatphagocytosedMPswasnotaffectedbyMPtreatment,andranged between94%and97%forDCs,and91% e 96%forMFs. Cellularuptakelevelsweredeterminedbasedonthemean intensityofMP uorescenceassociatedwithcells.Thelevelof phagocytosisofMPsbyDCswasalteredbasedonthesurface modi cationoftheMP( Fig.5 A).Allsurfacemodi cations,withthe exceptionofthePEGandRGDmodi edMPs,demonstratedthe capacitytosigni cantlyincreaseMPuptakebyDCscomparedto theunmodi edMP.Theantibodygroups,anti-CD11candanti-DEC205bothsigni cantlyimprovedDCuptakeby w 50%comparedto unmodi edMP.TheP-D2peptideenhancedDCphagocytosisthe most,doublingthenumberofMPsphagocytosedoverthe unmodi edMPs. ModulationofMFuptakeofsurface-modi edMPswasalso investigated( Fig.5 B).MPssurface-conjugatedwiththeP-D2 peptidesubstantiallyincreasedphagocytosisby w 40%overuptake levelsforunmodi edMPs.Allothersurfacemodi cationsyielded similarlevelsofMPuptakeastheunmodi ed. Because invivo ,MFsandDCscancompeteforMPuptake,we wereinterestedindeterminingiftheseligandsdemonstratetargetingspeci cityforeitherDCsorMFs.Usingamixedcultureof DCsandMFs(1:1ratio),weinvestigateduptakewhenthesecells haveequalaccesstoMPs( Fig.6 ).Wefoundthatunderthese conditions,DCstakeupmoreMPsthanMFsregardlessofthe surfacemodi cation,andtheextentofMPuptakeisin uencedby ligandsurfacemodi cation.Similartrends,basedonthetypeof surfacemodi cation,seenforuptakeinbothsinglecellsuspension uptakesystemswereobservedforuptakeinthemixedculture competitiveuptakesystem.Anti-CD11candanti-DEC-205antibodiesandP-D2peptidesigni cantlyimproveduptakeforboth DCsandMFswhencomparedtouptakeofunmodi edMPs. Moreover,differencesinuptakespeci citywereobserved,suggestingthatcomparedtoothertreatments,theanti-DEC-205 antibodyMPmodi cationprovidedbothhighlevelsofuptake andspeci cityofDCtargeting.Incontrast,whileMPssurfacemodi edwithanti-CD11cantibodyandP-D2demonstratedhigh levelsofuptake,DCselectivitywaslowinthis invitro model. 3.5.ImprovedantigenpresentationbyDCsafterphagocytosisof surface-modi edMPs Inordertodetermineef cacyofDCantigenpresentation followingphagocytosisofantigen-loadedMPs,NOD-derivedDCs cellswereallowedtotakeupligand-graftedMPsencapsulatingthe peptideantigen,1040-55mimetope,thensubsequentlyincubated withNOD-BDC2.5T-cells.TheNOD-BDC2.5mouseisengineered withT-cellreceptorsthatarespeci callyengagedbythe1040-55 mimetope.Uponbindingthe1040-55peptide(e.g.whenpresented onAPCs),T-cellsarestimulatedtoproliferate.Inourexperimental setup,MPsloadedwith1040-55peptidewereincubatedwithDCs for1h,unboundMPsremoved,andthenDCswereeitherimmediatelyco-culturedwithBDC2.5T-cells,orwereculturedfor4d beforetheadditionofBDC2.5T-cells.Bycomparingthe4ddelayed experimenttothenodelaytest,theabilityforMP-loadingto provideprolongedantigenpresentationbyDCstoTcellswas investigated( Fig.7 ). Regardingtheearlyantigenpresentationoftheno-delay experiment,theP-D2-modi edMPswastheonlygroupthat signi cantlyimprovedT-cellresponseabovethelevelobservedfor unmodi ed,1040-55peptide-loadedMPs( Fig.7 A).Thepositive control,asolubleboluswiththeequivalentamountofpeptide encapsulatedintheMPswasincluded.Thiscontrolprovokedthe largestTcellresponse,over2-foldhigherthantheP-D2modi cation,whileunloadedMPsshowedminimalTcellproliferation. Investigatingprolongedantigenpresentationbytheinclusionof a4ddelay,MPsurfacemodi cationwasfoundtoimpactthe response.Theincubationdelayresultedinadramaticdeclineinthe responseofT-cellstothesolubleantigenicpeptidecontrol.MostMP modi cationsmaintainedalow-magnitudeTcellresponsesimilar tothatseenwithouttheincubationdelay.OnlytheP-D2peptideconjugatedMPloadedwithantigensigni cantlyincreasedproliferation( Fig.7 B).Theunloaded,unmodi edMPgroupwasincluded asacontroltodemonstratethatTcellproliferationwasindeedtothe encapsulationof1040-55peptideinotherMPmodi cations. 3.6.InVivouptakeandtranslocationofsurface-modi ed microparticlestolymphnode Forapplicationstargetingphagocytes invivo ,itiscriticalto investigatetheeffectofMPsurfacemodi cationsusingan invivo model.Wethereforeusedamousefootpadinjectionmodelto determinetherateandextentofsurface-modi edMPtranslocation fromtheinjectionsitetoproximallymphnodes,viauptakefrom DCsandMFs.DiD-loadedMPsweresurface-modi edandinjected intothefootpadofmice.Subsequently,proximaldraininglymph nodeswereexcisedandanalyzedforcell-MPco-localization.The percentofMPcellsharvestedfromdraininglymphnodeswas usedasameasureofuptakeandtranslocationfromtheinjection site.DCsandMFsweredesignatedbystainingpositiveforeither CD11corF4/80,respectively.At24hafterinjection,nomodi cation showeddistinctioninimprovingtraf ckingtothedraininglymph node( Fig.8 A).However,by48h,lymphnodeharvestedDCs recoveredfrommiceinjectedwithanti-CD11c-andP-D2-modi ed MPsdisplayedamarkedincreaseinthepercentofMPDCsto4.8% MPand6.2%MPrespectivelycomparedto2%MPforunmodiedMPs.AsimilartrendwasobservedforMFsatthis48htime Fig.2. Internalizationofsurface-modi edMPsbydendriticcells(DCs)isdemonstratedviaconfocalmicroscopy.DCswereincubatedrhodamine-loadedsurfacemodi edMPs(orange)for1h, xedandstainedfortheactincytoskeleton(green)and thenucleus(blue).Thisimageshowsthex yopticalsectionaswellasx z projection,showingengulfmentoftheMPs.(Forinterpretationofthereferencesto colourinthis gurelegend,thereaderisreferredtothewebversionofthisarticle.) J.S.Lewisetal./Biomaterials33(2012)7221 e 7232 7225

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Surface Ligand/ Condition CD80 v. CD86CD80 v. MHC II FITC Anti-CD86 100101102103104 100101102103104 1.16% 2.30% 70.86% 25.68% PE Anti-MHC II 100101102103104 100101102103104 1.34% 2.13% 81.00% 15.54% FITC Anti-CD86 100101102103104 100101102103104 50.21% 32.35% 17.09% 0.35% PE Anti-MHC II 100101102103104 100101102103104 15.46% 67.10% 12.60% 4.84% FITC Anti-CD86 100101102103104 100101102103104 1.33% 1.18% 80.45% 17.04% PE Anti-MHC II 100101102103104 100101102103104 1.37% 1.89% 88.86% 7.88% FITC Anti-CD86 100101102103104 100101102103104 1.62% 1.78% 76.78% 19.83% PE Anti-MHC II 100101102103104 100101102103104 1.25% 2.15% 88.15% 8.45% iDC (neg. control) +LPS (pos. control) CD11c Ab -MP Dec 205 Ab -MP Fig.3. Immaturedendriticcellphenotypeismaintainedevenafterexposuretoligand-conjugatedMPmodi cations.Dendriticcellswereincubatedwithvarioussurface-modi ed MPsfor24hat37C.MPsoutnumberedcellsbya10:1ratio.CellswerethenextensivelywashedtoremoveunboundMPsandstainedwith uorescently-taggedanti-CD80,CD86, MHCIIandCD11cantibodies.FlowcytometricassessmentrevealedthelevelsofexpressionforthesemoleculesonDCs.ImmatureDCs(iDCs)and LPSgroupsareincludedas negativeandpositivecontrols,respectively.Representativeplotsfromthreeseparateexperimentsareshown. J.S.Lewisetal./Biomaterials33(2012)7221 e 7232 7226

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point,butwithsigni canceonlyfortheCD11cantibodymodi cation,whichincreasedthenumberofcellswithMPsto2%MPcomparedtothe0.5%MPseenforunmodi edMPs.After72hthe P-D2modi edparticlessubstantiallyimprovedtraf ckingto draininglymphnodesforDCs(4.4%MP)andMFs(2.3%MP)cells comparedtothecontrols(1.8%MPand0.8%MP,respectively). Unexpectedly,theisotypeantibodyforCD11calsoimprovedthe numberofMPtraf ckedDCsat72himplicatingtheroleofDCsFc receptorsatthistimepoint.Thisillustratesthein uencethechoice ofantibodyspeciesandIgGtypehavewhenusingwholeantibodies for invivo targeting. InadditiontoelucidatingtheextentofMPtraf cking,thistime seriesstudyalsocontextualizestheimpactofMPsurfacemodi cationonthekineticsofMPtraf cking.Thesedatastronglysuggest FITC Anti-CD86100101102103104 100101102103104 1.18% 1.45% 82.60% 14.77% PE Anti-MHC II100101102103104 100101102103104 0.59% 2.04% 88.98% 8.39% FITC Anti-CD86100101102103104 100101102103104 1.27% 1.93% 76.90% 19.90% PE Anti-MHC II100101102103104 100101102103104 1.17% 2.03% 86.63% 10.17% FITC Anti-CD86100101102103104 100101102103104 1.44% 2.31% 75.53% 20.72% PE Anti-MHC II100101102103104 100101102103104 1.02% 2.73% 84.39% 11.87% P-D2-MP RGD-MP PEG-MP UnmodifiedMP Fig.3. ( continued ). J.S.Lewisetal./Biomaterials33(2012)7221 e 7232 7227

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thattherateofuptakeandtranslocationofMPsisgreatlysuperior whenMPsaresurface-modi ed,primarilywiththeP-D2peptide andtoalesserextenttheCD11cantibody,forDCs( Fig.8 B)andMFs ( Fig.8 C)overa3dayperiod.TheCD11cantibody-graftedMPshows improvementforbothDCsandMFswhichpeaksat48hbutthen dropstocontrollevelsat72h.TheP-D2MPalsoshowsimproved uptakeandtraf ckingforDCswhichpeaksat48hbutthendrops tocontrollevelsat72h.Incontrast,forMFs,theP-D2MPmaintainsincreasinglevelsthroughouttheexperimentaltimeperiod.In general,these invivo resultscorrelatewellwiththe invitro results, exceptthatthemodi cationwiththeanti-DEC-205antibodydid nottranslatetouptakeandtraf ckinglevelsashighasindicated invitro . This invivo studyalsoillustratesdifferencesinthekineticsof uptakeandtranslocationbetweenDCsandMF.Itisevidentthatfor allmodi cations,themaximumnumberofMPCD11ccells occursat48h.ForF4/80cells,thereisnopeakforthedurationof thestudyforanyofthemodi cationswiththeexceptionofP-D2graftedMPsinthestudytimeperiod.These ndingscorroborate reportsofslowerlymphnodehomingofMFscomparedtoDCs [38] . 4.Discussion PLGAparticulatesystemshavebeenidenti edasavaluable immuno-therapeutictool.Particlesatthemicronandsub-micron levelallowfortargeteddeliveryofaplethoraofimmunomodulatingagentstocriticalelementsoftheimmunesystem [1,2] . TargeteddeliveryoffactorstoDCsisofparticularinterestasDCsplay apivotalroleintheactivationandmaintenanceofsuppressive networkswithintheimmunesystem [12] .EffortsatcontrollingDC polarityformitigationofautoimmunedisorders,suchastype1 diabetes(T1D)haveintensi edinthepastdecade [25] .WhileMFs aremoreabundantthanDCsandcompeteforuptakeofparticulate matter,MFsarelessef cientatantigenpresentationandtherefore maybeconsideredtobelesspotentatmanipulationoftheimmune system [13,39] .Notably,researchershavebeguntoincorporatePLGA MPsasacellimmuno-modulatorysystemintoDCimmunotherapy [9,11] .Phillipsandassociates [40] demonstratedpreventionofT1D inNODmicebypolymericmicrospheresloadedwithanti-sense Macrophage Microparticle Phagocytosis Microparticle Surface Ligand CD11c Ab MP DEC-205 Ab MP P-D2 MP RGD MP Unmodified MP PEG MP Microparticle Uptake (RFU) 0 100 200 300 400 500 600 700 * Dendritic Cell Microparticle Phagocytosis Microparticle Surface Ligand CD11c Ab MP DEC-205 Ab MP P-D2 MP RGD MP Unmodified MP PEG MP Microparticle Uptake (RFU) 0 200 400 600 800 1000 1200 * * * AB Fig.5. Microparticle(MP)phagocytosisby(A)dendriticcells(DCs)and(B)macrophagesMFsismodulatedbyMPsurfaceligand.Cellswereculturedwithsurface-modi ed rhodamine-loadedMPsfor1hwithagitationat37C.Microparticlesoutnumberedcellsbya10:1ratio.Flowcytometricanalysiswascarriedouttodeterminethemean uorescentintensityofcells(intherhodaminechannel)afterincubationasameasureofMPuptake.Pair-wisesigni cantdifferencefromcelluptakeofunmodi edPLGAmicroparticles isdenotedbythe*symbol(p value < 0.05). CD11c Ab MP DEC-205 Ab MP P-D2 MP RGD MP Unmodified MP PEG MP iDC 0 20 40 60 80 100 120 Viability (%)Microparticle Type Fig.4. Co-cultureofsurface-modi edMPswithdendriticcells(DCs)hasnegligible effectsoncellviability.Dendriticcellswereincubatedwithvarioussurface-modi ed MPsfor24hat37C.Microparticlesoutnumberedcellsbya10:1ratio.Cellsupernatantswerecollectedafter24hforacytotoxicityassay.Thepercentageviabilityfor eachspecimenwascalculatedbasedonlive/deadcellcontrols.Nodifferencesbetween groupswerefoundbyANOVA(p > 0.1). J.S.Lewisetal./Biomaterials33(2012)7221 e 7232 7228

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oligonucleotidesthatpassivelytargetedDCsbyvirtueofbeing micronrangeparticulate,andwhichweredemonstratedtomanipulatetheimmuno-regulatoryfunctionofDCs.Othershaveshown thatactivetargetingofDCsusingsurface-modi edparticulate systemsimprovesDCuptakeandtraf cking,andescalatingdownstreamimmuno-stimulatoryresponses,primarilyforcancervaccine applications [41] . ActivetargetingofMPsisaccomplishedbysurfaceligationof ligandsthatspeci callybindmoleculesonthesurfaceofDCs.Prior workhasdemonstratedef cacioustargetingofMPstoDCreceptors suchastoll-likereceptors,CD40,avb3andavb5integrins [42] . However,bindingthesereceptorscanresultinDCactivation.For example,itiswellestablishedthattoll-likereceptorsandCD40 receptorarestimulatoryinnature [23] .Additionally,wehave recentlyshownthatDCadhesiontovariousadhesivesubstrates,as wellasRGDpeptidesurfacedensitygradientsthroughtheavintegrinsarealsoactivating [43e 45].Similarly,othershaveshown thattargetingMPstoavintegrinsresultsinDCactivation [42] . Giventhestimulatorypathwaysinducedbythesereceptors,other candidateswhichareeithernon-in ammatoryorpro-tolerogenic areofinterestfornon-activatingapplications.Ligandsthattarget DCsurfacereceptorssuchastheintegrinCD11c/CD18andtheDEC205receptorhavebeenidenti edaspotentialcandidatesforthis goal.Wewerethereforeinterestedintheuseofantibodiesagainst CD11candDEC-205aswellastheCD11c-bindingpeptide,P-D2 peptideasligandsinthisclass.BothCD11candDEC-205antibodies havebeenpreviouslycoupledtoantigen-loadedliposomesfor targetingofDCstoenhanceprotectionfromtumorinmicechallengedwithmalignantmelanomacells [23,46] . Bonifaz etal. [47] and Birkholz etal. [48] conjugatedDEC-205monoclonalantibody Mixed Culture Microparticle Phagocytosis CD11c Ab MP DEC-205 Ab MP P-D2 MP RGD MP Unmodified MP PEG MP 200 400 600 800 1000 1200 1400 1600 1800 DC MAC Microparticle Uptake (RFU)Microparticle Type * ** Fig.6. Microparticle(MP)uptakeinamixedculture(DCsandMFs)isin uencedby MPsurfacemodi cation.AnequalnumberofDCsandMFswereco-culturedwith varioussurface-modi edAMC-loadedMPsfor1hat37Cwhilebeinggentlyagitated. Microparticlesoutnumberedcellsbya10:1ratio.Flowcytometricanalysiswascarried outtodeterminethemean uorescentintensity(intheAMCchannel)ofDCsandMFs afterincubationasameasureofMPuptakelevel.Pair-wisesigni cantdifferenceofMP uptakebetweenDCsandMFsisdenotedbythe*symbol(p value < 0.05). Early Antigen Presentation Microparticle/ Treatment CD11c Ab MP Dec 205 Ab MP P-D2 MP RGD MP PEG MP 1040-loaded Unmodified MP Empty Unmodified MP Sol 1040 Proliferation Index 0 2 4 6 8 10 Prolonged Antigen Presentation Microparticle/ Treatment CD11c Ab MP DEC-205 Ab MP P-D2 MP RGD MP PEG MP 1040-loaded Unmodified MP Empty Unmodified MP Sol 1040 Proliferation Index 0 1 2 3 4 * *B*A Fig.7. (A)P-D2peptidesurface-modi edPLGAMPsloadedwithantigenicpeptideleadstoincreasedantigenpresentationand(B)improvedprolongedantigenpresentation.NonobesediabeticmouseDCswereculturedwitheitherMPsloadedwith1040-55mimitopeorsolublepeptide(asacontrol,atanequalmasstothatloadedintheMPs)ata100:1MP toDCratiofor1h,followedbywashingtoremoveunboundMPs.Subsequently,freshly-isolatedBDC2.5CD4 Tcellswereaddedtoculturewells(eitherimmediately(early)orafter 4d(prolonged))andco-cultureda3-daymixedlymphocytereaction.T-cellproliferationwasthenmeasuredusingaBrdUproliferationassayasameasureoffunctionalantigen presentation.Datashownrepresentthemeanproliferationindices standarderror(n 3).Pair-wisesigni cantdifferencefromtheunmodi edPLGAMPgroup(byANOVAand Tukeysigni cancetest)isdenotedbythe*symbol(p value < 0.05). J.S.Lewisetal./Biomaterials33(2012)7221 e 7232 7229

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andanti-DEC-205singlechainfragmentvariabledirectlytoantigenicproteinsfortargetingtoDCstoimproveantigen-speci c immunestimulation.Morerecently,Fahmyandassociatesreportedgraftingofanti-DEC-205toPLGAnanoparticlesanditseffecton DCuptakeandfunction.Theirstudydemonstratedthatsurface graftedDEC-205nanoparticlesinduceIL-10expressionbutareonly modestlyeffectiveinimprovingnanoparticleinternalizationby DCs [49] .Asthepopularityofparticulatesystemsascontemporary medicaldrugdeliverydevicesincreases,theneedforparticle optimizationarises(includingactivetargeting,releasekinetics, proteinrepulsion,etc).Thisstudybeginstoaddresstheseparameters,principallyforapplicationsrequiringtargetingtophagocytes whilemaintaininganon-activatedstate.Anotherconsiderationis thatMPmodi cationwithtargetingpeptidesinsteadofantibodies isaparticularlyattractivecandidateasitwillnottargetFcreceptors andhasalowerproductioncost [50] . Topromotephagocytosis,wefabricatedourparticleswith arangeof0.5 e 2.5mminsizewithanaveragediameterof1.15mm andconfocalmicroscopycon rmedthattheMPswerereadily takenupbyDCs.Belowthismicronsizerange,particlescanbe takenupbypinocytosiswhichisnotlimitedtoantigenpresenting cells.Inthisregard,nanoparticlesarethereforenotasselectiveas microparticlesforphagocytes [51,52] .Well-establishedEDC/NHS chemistrywasusedtoligateligandstothesurfacesofPLGAMPs. Veri cationofconjugationwasaccomplishedbypeptideand proteinquantitativemethods,andcorroboratedbyzetapotential measurement.Theseresultsshowthatasubstantialamountofeach moleculeisimmobilizedonthesurfaceoftheMP,atlevels comparabletothosepreviouslyreported [42,53] .Differencesin uptakeobservedbetweenthedifferentsurfacemodi cationsof MPscanbeattributedtofactorsaffectingtheactivityofthe immobilizedligandincludingthequantityofimmobilizedligand, CF4/80 + Cells Time (Days) 0.51.01.52.02.53.03.5% MP+ Cells 0.0 0.5 1.0 1.5 2.0 2.5 3.0 CD11c Ab-MP CD11c ISO Ab-MP DEC-205 Ab-MP DEC-205 ISO Ab-MP P-D2-MP Unmodified-MP B Cell Type (Timepoint) CD11c+ (24h) F4/80+ ( 24h) CD11c+ (48h) F4/80+ (48h) CD11c+ (72h) F4/80+ (72h) % MP+ Cells 0 1 2 3 4 5 6 7 CD11c Ab-MP CD11c ISO Ab-MP DEC-205 Ab-MP DEC-205 ISO Ab-MP P-D2-MP Unmodified-MP * * * * * * CD11c + Cells Time (Days) 0.51.01.52.02.53.03.5% MP+ Cells 0 1 2 3 4 5 6 7 CD11c Ab-MP CD11c ISO Ab-MP DEC-205 Ab-MP DEC-205 ISO Ab-MP P-D2-MP Unmodified-MP A Fig.8. Phagocytosisandtraf ckingofmicroparticles(MPs)bydendriticcells(DCs)andmacrophages(MFs)isenhancedthroughMPsurfacemodi cation.Female,6weekold C57Bl/6miceweregivenfootpadinjectionscontaining0.5mgofligand-conjugatedDiD-MPsalongwithcontrolMPs.Atdifferenttimepoints(24h,48h,72h)thedraininglymph nodeswererecovered,processedandstainedforCD11c(DC)andF4/80(MF)surfacemarkersfollowedby owcytometryanalysis.ThepercentageofeachcelltypeMP (DiD channel)wasdeterminedandanalyzedbyANOVA.MicroparticleuptakeforbothDCsandMFsissummarizedforallMPgroups(A),aswellasdetailedseparatelyforDCs(B)and MFs(C).The*symboldenotespair-wisecomparisonsusingtheTukeytestshowingsigni cantdifferenceincomparisontounmodi edMPs(p < 0.05). J.S.Lewisetal./Biomaterials33(2012)7221 e 7232 7230

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thespeci creceptortargeted,thesiteofligandbindingtothe receptor(i.e.,doesbindingactivate/de-activatethereceptor)and theeffectivebindingaf nityoftheimmobilizedligand [39,42,54] . Thesefactorscanexplainwhymodi cationsinourhandsmayact differentlyfrompriorreports,andtheirpotentialin uenceoverour resultsisdiscussedfurtherbelow. Invitro testingtodeterminethestimulatorycapacityofMP modi cationsshowedthereisnosigni cantchangeinthe expressionofDCactivationmarkersinvestigatedwithanyofthe surface-modi edMPs.Further,neitherthemodi edorunmodi ed MPsshowedanytoxiceffects.Theseoutcomesareimportant considerationsfordesigningDC-targetingMPsforapplications suchasvaccinesforameliorationofautoimmunedisorders. Wealsodeterminedthatsecretionofcytokines,IL-12andIL-10, doesnotvarysigni cantlyforDCsexposedtoanyoftheMP modi cations(datanotshown).WithregardtoDEC-205MPs,this resultisseeminglycontrarytoworkbytheFahmygroupwhich describedanincreaseinDCsecretionofIL-10uponDEC-205 nanoparticleexposure [49] .Thisdisparitymaybeexplainedby thefactthattheirreportedsurfacedensitywas50-foldgreaterthan thedensitymeasuredonourDEC-205MP,thesizeoftheirparticle wasmuchsmallerat200 e 250nm,andtheDEC-205presentation ontheirparticlewasdifferent,consistingofpalmitate e avidin moleculesincorporatedintothesurfaceoftheparticlebinding abiotinylatedDEC-205antibody. TheeffectofsurfacefunctionalizationonearlyuptakeofMPsby DCsandMFswasinitiallyevaluated invitro .TheCD11candDEC205antibody-modi edandP-D2peptidesurfacefunctionalized MPsprovidedsigni cantenhancementofDCphagocytosiswhile CD11candP-D2surfacegraftingaugmentedMFuptakeofMPs. RGDpeptidegraftingfailedtoincreaseeitherDCorMFphagocytosisofMPs.Peptidequanti cationmethodsrevealedthatthe surfacedensityoftheRGDpeptidewas19ng/mgwhichequatesto approximately11,500fmol/cm2. Petrie etal. [55] reportedthatan RGDsurfacedensityofapproximately2,000fmol/cm2onsolid substratespromotedmurineadhesion.Therefore,itwasexpected ourRGD-modi edMPsshouldencourageMPphagocytosis.Given thatourRGDmodi cationdidnotimproveuptakemaysuggestthat peptidepresentationinourformulationwassub-optimal.Alternatively,increasingMPphagocytosismaysimplyrequirehigher thresholdlevelsofRGDsurfacedensitythanlevelsrequiredonlyfor adhesion,asCorreandassociates [50] reportedthatmicrospheres functionalizedwith20-foldhigherRGDsurfacedensityin uences MFphagocytosis.Whilstligandquantityandpresentationare limitingfactors,differencesinDCandMFlevelsofexpressionand activationofthetargetedreceptorsalsoin uenceDCandMFuptake [56,57] .Notably,thesereceptorlevelsandactivationstates cancertainlydifferbetween invitro and invivo scenarios. Particulate-basedantigendeliveredtoAPCscanserveasan intracellularantigendepotprovidingprolongedantigenpresentationduetosustainedrelease [58] .WeinvestigatedMPsurface modi cationtoincreaseintracellularstoresofantigenthereby increasingprolongedpresentationofantigen.Wedeterminedthat ataninitialtimepoint,ligand-directedMPuptakein uences antigenpresentationsigni cantlyfortheP-D2peptideligandand toalesserextentandsigni cancelevel( p < 0.09)fortheDEC-205 antibodymodi edMPgroup.Further,wedemonstratedthat microparticulatesystemsallowforprolonged,continuouspresentationofantigen,andthatligandsurfacemodi cation,particularly withP-D2peptide,increasesthelevelofthisprotractedpresentation.Thisisimportant,becausetakingintoaccounttimeconsiderationsaftersubcutaneousinjectionforAPCantigeninterception, uptakeandtranslocation,antigenadministeredinsolubleformcan bedilutedanddegradedbeforeeffectivefunctionalinteractionby APCscanoccurinTcellrichregions [2] .Thisresultindicates controlledreleasepackagingofantigenintargetedPLGAMPscan overcomethisissue. The invitro studiesprovidedawell-controlledplatformto identifythebestcandidatesforDCuptake.Additionally,these resultscanguideapplicationsutilizing invitro / exvivo manipulation ofAPCs,whichcanbeenhancedusingpolymericmicrospheres [17,58] .However,applicationsaimedat invivo targetingofMPsto DCsrequireuseof invivo experimentalsystemstodetermineef cacy.Basedonthelow invitro uptakelevels,weeliminatedtheRGD modi edgroupandcompared invivo uptakeandtranslocationto draininglymphnodeaftersubcutaneousinjectionofourbestMP modi cations.TheP-D2peptide-conjugatedMPsinparticular,and toalesserextent,theanti-CD11c-conjugatedMPswereeffectivein increasing invivo traf ckingofMPstodraininglymphnodesbyboth DCsandMFsat2dand3d,respectively.Notably,inconsistentwith our invitro result,theanti-DEC-205-conjugatedMPsfailedtoalter MPtraf ckingbyDCs invivo .Thismaybeexplainedbydifferences inDCreceptors(expressionlevelsandactivationstates)presentin the invitro and invivo conditions [30] .Forinstance, Jefford etal. demonstratedthatperipheralbloodDCsgenerated invivo differed from invitro monocyte-derivedDCsinphenotype,migratory capacityandT-cellstimulation [59] .Furthermore,DCsubpopulations invivo showphenotypicdiversity [60,61] .WhileSteinmanand colleagues [21] reportedthatantigen-coupledDEC-205antibody injectedintravenouslysuccessfullytargetedlymphoidDCs,itis possiblethattheDCspresentatthesubcutaneousinjectionsitehave alowerexpressionofDEC-205 [60] .Consequently,subcutaneously injected-MPsmodi edwiththeanti-DEC-205antibodyfornonactivatingDC-targetingmayproveineffective. 5.Conclusion Inthisstudy,weinvestigatedstrategiestoachieveactivetargetingofMPstoDCsinanon-activatingmodeandassessedthe ef cacyofeachoftheseapproaches invitro aswellas invivo . Moreover,wehavedemonstratedthatligationoftheP-D2peptide (andtoalesserextent,theanti-CD11candanti-DEC-205antibodies)tothesurfaceofPLGAMPsisapromising,newapproachfor activeDCtargeting.Ingeneral,conjugationoftheseligandstoPLGA MPswerenon-stimulatingtoDCs,enhancedDCMPuptake, improvedantigenpresentationlevelanddurationbyDCsandalso boosted invivo traf ckingofMPstothedraininglymphnode.The knowledgegarneredfromthisstudyisinstructiveforthedesignof non-activatingpolymericmicroparticulateapplicationssuchas vaccinesforauto-immunedisease. Acknowledgement ThisworkwassupportedinpartbytheNationalInstitutesof Health(R01DK091658,andR56DK091658),andbyanInnovative GrantfromtheJuvenileDiabetesResearchFoundation. References[1]JiangW,GuptaRK,DeshpandeMC,SchwendemanSP.Biodegradable poly(lactic-co-glycolicacid)microparticlesforinjectabledeliveryofvaccine antigens.AdvDrugDelivRev2005;57(3):391 e 410. [2]OhaganDT,RahmanD,McgeeJP,JefferyH,DaviesMC,WilliamsP,etal. Biodegradablemicroparticlesascontrolledreleaseantigendeliverysystems. Immunology1991;73(2):239 e 42. [3]JaganathanKS,VyasSP.Strongsystemicandmucosalimmuneresponsesto surface-modi edPLGAmicrospherescontainingrecombinantHepatitisB antigenadministeredintranasally.Vaccine2006;24(19):4201 e 11. [4]RajapaksaTE,LoDD.Microencapsulationofvaccineantigensandadjuvants formucosaltargeting.CurImmunolRev2010;6(1):29 e 37. [5]PerezC,SanchezA,PutnamD,TingD,LangerR,AlonsoMJ.Poly(lacticacid)poly(ethyleneglycol)nanoparticlesasnewcarriersforthedeliveryofplasmid DNA.JControlRelease2001;75(1e 2):211 e 24.J.S.Lewisetal./Biomaterials33(2012)7221 e 7232 7231

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[6]Tinsley-BownAM,FretwellR,DowsettAB,DavisSL,FarrarGH.Formulationof poly(D,L-lactic-co-glycolicacid)microparticlesforrapidplasmidDNA delivery.JControlRelease2000;66(2e 3):229 e 41. [7]WalterE,MoellingK,PavlovicJ,MerkleHP.MicroencapsulationofDNAusing poly(DL-lactide-co-glycolide):stabilityissuesandreleasecharacteristics. JControlRelease1999;61(3):361 e 74. [8]MohammadiG,ValizadehH,Barzegar-JalaliM,Lot pourF,AdibkiaK, MilaniM,etal.Developmentofazithromycin-PLGAnanoparticles:physicochemicalcharacterizationandantibacterialeffectagainst Salmonellatyphi . ColloidsSurfB2010;80(1):34 e 9. [9]AcharyaAP,Clare-SalzlerMJ,KeselowskyBG.Ahigh-throughputmicroparticlemicroarrayplatformfordendriticcell-targetingvaccines.Biomaterials 2009;30(25):4168 e 77. [10]JohansenP,MenY,MerkleHP,GanderB.RevisitingPLA/PLGAmicrospheres: ananalysisoftheirpotentialinparenteralvaccination.EurJPharmBiopharm 2000Jul3;50(1):129 e 46. [11]Waeckerle-MenY,GroettrupM.PLGAmicrospheresforimprovedantigen deliverytodendriticcellsascellularvaccines.AdvDrugDelivRev2005;57(3): 475 e 82. [12]BanchereauJ,SteinmanRM.Dendriticcellsandthecontrolofimmunity. Nature1998;392(6673):245 e 52. [13]BanchereauJ,BriereF,CauxC,DavoustJ,LebecqueS,LiuYT,etal.Immunobiologyofdendriticcells.AnnuRevImmunol2000;18:767. [14]MatzingerP.Tolerance,danger,andtheextendedfamily.AnnuRevImmunol 1994;12:991 e 1045. [15]ShortmanK,LiuYJ.Mouseandhumandendriticcellsubtypes.NatRev Immunol2002;2(3):151 e 61. [16]SteinmanRM,HawigerD,NussenzweigMC.Tolerogenicdendriticcells.Annu RevImmunol2003;21:685 e 711. [17]TackenPJ,deVriesI,TorensmaR,FigdorCG.Dendritic-cellimmunotherapy: fromexvivoloadingtoinvivotargeting.NatRevImmunol2007;7(10): 790 e 802. [18]GogolakP,RethiB,HajasG,RajnavolgyiE.Targetingdendriticcellsfor primingcellularimmuneresponses.JMolRecognit2003;16(5):299 e 317. [19]WattendorfU,CoullerezG,VorosJ,TextorM,MerkleHP.Mannose-based molecularpatternsonstealthmicrospheresforreceptor-speci ctargetingof humanantigen-presentingcells.Langmuir2008;24(20):11790 e 802. [20]YangL,YangH,RideoutK,ChoT,IlJooK,ZieglerL,etal.Engineeredlentivectortargetingofdendriticcellsforinvivoimmunization.NatBiotechnol 2008;26(3):326 e 34. [21]BonifazL,BonnyayD,MahnkeK,RiveraM,NussenzweigMC,SteinmanRM. Ef cienttargetingofproteinantigentothedendriticcellreceptorDEC-205in thesteadystateleadstoantigenpresentationonmajorhistocompatibility complexclassIproductsandperipheralCD8( )Tcelltolerance.JExpMed 2002;196(12):1627 e 38. [22]SwChoe,AcharyaAP,KeselowskyBG,SorgBS.Intravitalmicroscopyimaging ofmacrophagelocalizationtoimmunogenicparticlesandco-localizedtissue oxygensaturation.ActaBiomater2010;6(9):3491 e 8. [23]CheongC,ChoiJH,VitaleL,HeLZ,TrumpfhellerC,BozzaccoL,etal.Improved cellularandhumoralimmuneresponsesinvivofollowingtargetingofHIVgag todendriticcellswithinhumananti-humanDEC205monoclonalantibody. Blood2010;116(19):3828 e 38. [24]NchindaG,KuroiwaJ,OksM,TrumpfhellerC,ParkCG,HuangY,etal.The ef cacyofDNAvaccinationisenhancedinmicebytargetingtheencoded proteintodendriticcells.JClinInvest2008;118(4):1427 e 36. [25]KeselowskyBG,XiaCQ,Clare-SalzlerM.Multifunctionaldendriticcelltargetingpolymericmicroparticles:engineeringnewvaccinesfortype1 diabetes.HumVaccines2011;7(1):37 e 44. [26]JiangWP,SwiggardWJ,Heu erC,PengM,MirzaA,SteinmanRM,etal.The receptorDec-205expressedbydendriticcellsandthymicepithelial-cellsis involvedinantigen-processing.Nature1995;375(6527):151 e 5. [27]WangB,KuroiwaJM,HeLZ,CharalambousA,KelerT,SteinmanRM.The humancancerantigenmesothelinismoreef cientlypresentedtothemouse immunesystemwhentargetedtotheDEC-205/CD205receptorondendritic cells.AnnNYAcadSci2009;1174:6 e 17. [28]SternJN,KeskinDB,KatoZ,WaldnerH,SchallenbergS,AndersonA,etal. Promotingtolerancetoproteolipidprotein-inducedexperimentalautoimmuneencephalomyelitisthroughtargetingdendriticcells.PNatlAcadSci 2010;107(40):17280 e 5. [29]YamazakiS,DudziakD,HeidkampGF,FioreseC,BonitoAJ,InabaK,etal. CD8( )CD205( )splenicdendriticcellsarespecializedtoinduceFoxp3( ) regulatoryTcells.JImmunol2008;181(10):6923 e 33. [30]AmmonC,MeyerSP,Schwarz scherL,KrauseSW,AndreesenR,KreutzM. Comparativeanalysisofintegrinexpressiononmonocyte-derivedmacrophagesandmonocyte-deriveddendriticcells.Immunology2000;100(3): 364 e 9. [31]BerryJD,LiceaA,PopkovM,CortezX,FullerR,EliaM,etal.Rapidmonoclonal antibodygenerationviadendriticcelltargetinginvivo.HybridHybridomics 2003;22(1):23 e 31. [32]IhanusE,UotilaLM,ToivanenA,VarisM,GahmbergCG.Red-cellICAM-4is aligandforthemonocyte/macrophageintegrinCD11c/CD18:characterization ofthebindingsitesonICAM-4.Blood2007;109(2):802 e 10. [33]BaillyP,TonttiE,HermandP,CartronJP,GahmbergCG.TheredcellLWblood groupproteinisanintercellularadhesionmoleculewhichbindstoCD11/ CD18leukocyteintegrins.EurJImmunol1995;25(12):3316 e 20. [34]IhanusE,UotilaL,ToivanenA,StefanidakisM,BaillyP,CartronJP,etal. CharacterizationofICAM-4bindingtotheIdomainsoftheCD11a/CD18and CD11b/CD18leukocyteintegrins.EurJBiochem2003;270(8):1710 e 23. [35]MesserschmidtSK,MusyanovychA,AltvaterM,ScheurichP,P zenmaierK, LandfesterK,etal.Targetedlipid-coatednanoparticles:deliveryoftumor necrosisfactor-functionalizedparticlestotumorcells.JControlRelease2009; 137(1):69 e 77. [36]ZaveriTD,DolgovaNV,ChuBH,LeeJ,WongJ,LeleTP,etal.Contributionsof surfacetopographyandcytotoxicitytothemacrophageresponsetozincoxide nanorods.Biomaterials2010;31(11):2999 e 3007. [37]LiYP,PeiYY,ZhangXY,GuZH,ZhouZH,YuanWF,etal.PEGylatedPLGA nanoparticlesasproteincarriers:synthesis,preparationandbiodistributionin rats.JControlRelease2001;71(2):203 e 11. [38]KiamaSG,CochandL,KarlssonL,NicodLP,GehrP.Evaluationofphagocytic activityinhumanmonocyte-deriveddendriticcells.JAerosolMed:JIntSoc AerosolsMed2001;14(3):289 e 99. [39]ThieleL,MerkleHP,WalterE.Phagocytosisandphagosomalfateofsurfacemodi edmicroparticlesindendriticcellsandmacrophages.PharmRes 2003;20(2):221 e 8. [40]PhillipsB,NylanderK,HarnahaJ,MachenJ,LakomyR,StycheA,etal. Amicrosphere-basedvaccinepreventsandreversesnew-onsetautoimmune diabetes.Diabetes2008;57(6):1544 e 55. [41]HamdyS,HaddadiA,HungRW,LavasanifarA.Targetingdendriticcellswith nano-particulatePLGAcancervaccineformulations.AdvDrugDelivRev2011; 63(10 e 11):943 e 55. [42]KempfM,MandalB,JilekS,ThieleL,VorosJ,TextorM,etal.Improved stimulationofhumandendriticcellsbyreceptorengagementwithsurfacemodi ed microparticles.JDrugTarget2003;11(1):11 e 8. [43]AcharyaAP,DolgovaNV,XiaCQ,Clare-SalzlerMJ,KeselowskyBG.Adhesive substratesmodulatetheactivationandstimulatorycapacityofnon-obese diabeticmouse-deriveddendriticcells.ActaBiomater2011;7(1):180 e 92. [44]AcharyaAP,DolgovaNV,MooreNM,XiaCQ,Clare-SalzlerMJ,BeckerML,etal. ThemodulationofdendriticcellintegrinbindingandactivationbyRGDpeptidedensitygradientsubstrates.Biomaterials2010;31(29):7444 e 54. [45]AcharyaAP,DolgovaNV,Clare-SalzlerMJ,KeselowskyBG.Adhesive substrate-modulationofadaptiveimmuneresponses.Biomaterials2008; 29(36):4736 e 50. [46]FahamA,AltinJG.Ag-bearingliposomesengraftedwithpeptidesthatinteract withCD11c/CD18inducepotentAg-speci candantitumorimmunity.IntJ Cancer2011;129(6):1391 e 403. [47]BonifazLC,BonnyayDP,CharalambousA,DargusteDI,FujiiSI,SoaresH,etal. InvivotargetingofantigenstomaturingdendriticcellsviatheDEC-205 receptorimprovesTcellvaccination.JExpMed2004;199(6):815 e 24. [48]BirkholzK,SchwenkertM,KellnerC,GrossS,FeyG,Schuler-ThurnerB,etal. TargetingofDEC-205onhumandendriticcellsresultsinef cientMHCclass II-restrictedantigenpresentation.Blood2010;116(13):2277 e 85. [49]BandyopadhyayA,FineRL,DementoS,BockenstedtLK,FahmyTM.The impactofnanoparticleliganddensityondendritic-celltargetedvaccines. Biomaterials2011;32(11):3094 e 105. [50]BrandhonneurN,ChevanneF,VieV,FrischB,PrimaultR,LePotierMF,etal. Speci candnon-speci cphagocytosisofligand-graftedPLGAmicrospheres bymacrophages.EurJPharmSci2009;36(4e 5):474 e 85. [51]SinghM,ChakrapaniA,O ’ HaganD.Nanoparticlesandmicroparticlesas vaccine-deliverysystems.ExpertRevVaccines2007;6(5):797 e 808. [52]MailenderV,LandfesterK.Interactionofnanoparticleswithcells.Biomacromolecules2009;10(9):2379 e 400. [53]TanH,HuangD,LaoL,GaoC.RGDmodi edPLGA/gelatinmicrospheresas microcarriersforchondrocytedelivery.JBiomedMaterResB2009;91B(1): 228 e 38. [54]LinSX,MalletWG,HuangAY,Max eldFR.Endocytosedcation-independent mannose6-phosphatereceptortraf csviatheendocyticrecyclingcompartmentenroutetothetrans-golginetworkandasubpopulationoflateendosomes.MolBiolCell2004;15(2):721 e 33. [55]PetrieTA,CapadonaJR,ReyesCD,GarciaAJ.Integrinspeci cityandenhanced cellularactivitiesassociatedwithsurfacespresentingarecombinant bronectinfragmentcomparedtoRGDsupports.Biomaterials2006;27(31): 5459 e 70. [56]KouPM,BabenseeJE.Macrophageanddendriticcellphenotypicdiversityin thecontextofbiomaterials.JBiomedMaterResA2011;96(1):239 e 60. [57]HashimotoD,MillerJ,MeradM.Dendriticcellandmacrophageheterogeneity invivo.Immunity2011;35(3):323 e 35. [58]FredriksenBrN,GripJ.PLGA/PLAmicro-andnanoparticleformulationsserve asantigendepotsandinduceelevatedhumoralresponsesafterimmunization ofAtlanticsalmon( SalmosalarL .).Vaccine2012;30(3):656 e 67. [59] JeffordM,SchnurrM,ToyT,MastermanKA,ShinA,BeecroftT,etal.FunctionalcomparisonofDCsgeneratedinvivowithFlt3ligandorinvitrofrom bloodmonocytes:differentialregulationoffunctionbyspeci cclassesof physiologicstimuli.Blood2003;102(5):1753 e 63. [60]InabaK,SwiggardWJ,InabaM,MeltzerJ,MirzaA,SasagawaT,etal.Tissue distributionoftheDec-205proteinthatisdetectedbythemonoclonalantibodyNldc-145.1.Expressionondendriticcellsandothersubsetsof mouseleukocytes.CellImmunol1995;163(1):148 e 56. [61]GuoM,GongS,MaricS,MisulovinZ,PackM,MahnkeK,etal.Amonoclonal antibodytotheDEC-205endocytosisreceptoronhumandendriticcells.Hum Immunol2000;61(8):729 e 38.J.S.Lewisetal./Biomaterials33(2012)7221 e 7232 7232