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Extracellular Matrix-Derived Nanoparticles for Imaging and Immunomodulation

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Extracellular Matrix-Derived Nanoparticles for Imaging and Immunomodulation EXTRACELLULAR MATRIX-DERIVED NANOPARTICLES FOR IMAGING AND IMMUNOMODULATION BY: JOHN KRILL A THESIS SUBMITTED TO JOHNS HOPKINS UNIVERSITY IN CONFORMITY WITH THE REQUIREMENTS FOR THE DEGREE OF MASTER IN SCIENCE AND ENGINEERING BALTIMORE, MARYLAND MAY, 2016 © 2016 JOHN KRILL ALL RIGHTS RESERVED ABSTRACT The extracellular matrix (ECM) is a complex component of tissue that includes collagens, glycoproteins, proteoglycans, and elastic fibers. These proteins serve both as a structural foundation upon which cells organize and communicate, and to inherently direct many physiological phenomena within cells, such as migration, proliferation, and differentiation. Due to these unique properties of ECM, laboratory generated matrix derived from the decellularization of native tissue, has become a preferred scaffolding material for use in tissue engineering and regenerative medicine. ECM particulate forms have been of increasing interest, as they provide several advantages over macro-scale patches. Particles enable minimally invasive delivery alternatives, including injections. In addition, by reducing ECM particle size to the nanoscale, ECM can be directly taken up by cells, such as macrophages, through phagocytosis or other engulfment methods. This internalization of ECM may lead to enhanced potency of directing cell behavior or differentiation versus simple material contact. This is highly relevant in the development of immune therapies that aim to modulate the host immune response for more positive outcomes in cancer treatment, implant integration, and vaccines. This thesis has two major parts. First, the methodology and characterization of successfully produced ECM nanoparticles derived from several porcine tissue sources are provided. This encompasses the tissue decellularization process and nanoparticle generation procedure. The second part focuses on modification of ECM nanoparticles with a variety of molecules, including fluorescent dyes, polyethylene glycol and functional peptides to alter their properties. Preliminary data regarding the ability of non-modified ECM nanoparticles to influence macrophage polarization in vitro is offered, with changes in cytokine expression suggesting immunomodulatory effects. Overall, ECM nanoparticles are a promising biomaterial for medical imaging, cancer research and immunology, and thus deserve further exploration. Thesis Readers: Dr. Jennifer H. Elisseeff, Dr. Kevin Yarema, and Dr. Hai-Quan Mao ii SPECIAL THANKS TO: Dr. Matthew Wolf Tony Wang Christopher Anderson And the rest of the Elisseeff Lab for their assistance and guidance throughout this entire project, as well as members from the Green and Yarema Labs who facilitated many of these studies. iii TABLE OF CONTENTS List of Tables ............................................................................................................................... v List of Figures ........................................................................................................................... vi 1. Introduction 1.1 Overview of ECM ......................................................................................................................................... 1 1.2 ECM as a Biomaterial in Tissue Repair ............................................................................................... 4 1.3 ECM in Immunology ................................................................................................................................... 8 1.4 ECM Particulates: Unlocking New Applications ........................................................................... 12 2. Decellularization Process 2.1 Tissue Decellularization Methodology ............................................................................................. 14 2.2 ECM Characterization .............................................................................................................................. 15 3. ECM Nanoparticle Preparation 3.1 Cryomilling to Generate Micron-Scale Powder ............................................................................ 17 3.2 Processing of ECM Powder Into Nanoparticles ............................................................................ 20 3.3 ECM Nanoparticle Characterization .................................................................................................. 22 3.4 ECM Nanoparticle Cytotoxicity Studies ........................................................................................... 25 4. ECM Nanoparticle Modification and Functionalization 4.1 Fluorescent Marker Conjugation ........................................................................................................ 29 4.2 PEGylation .................................................................................................................................................... 39 5. Effect of ECM Nanoparticles on Macrophage Polarization 5.1 Intrinsic ability of ECM Nanoparticles to Influence Macrophage Polarization ............... 44 5.2 Functionalization of ECM Nanoparticles for Immune Therapy: Future Works .............. 47 6. Conclusion ............................................................................................................................ 50 References ................................................................................................................................ 51 Curriculum Vitae .................................................................................................................... 55 iv LIST OF TABLES Table 1: List of common ECM components, their functions and where they can be found within the body ................................................................................................................................... 3 Table 2: Examples of commercially available scaffolds composed of ECM ................................. 8 Table 3: Abbreviated list of associated factors for M1 and M2 macrophage phenotypes ............. 11 Table 4: Overview of PAA/Triton decellularization procedure ..................................................... 14 Table 5: Overview of SDS Decell Procedure ................................................................................ 15 Table 6: Settings used for SPEX sampleprep 6870 freezer/mill .................................................... 18 Table 7: Emulsiflex operating pressures for ECM nanoparticle generation .................................. 21 Table 8: List of NHS dyes successfully conjugated to ECM nanoparticles................................... 38 v LIST OF FIGURES Figure 1: H&E comparison of decellularized ECM batches with native tissue counterparts ........ 16 Figure 2: Masson’s trichrome comparison of decellularized ECM batches with native tissue counterparts ........................................................................................................................... 17 Figure 3: Size comparisons of multiple cryomilled ECM batches ................................................ 18 Figure 4: SEM images highlighting morphology differences between ECM powders ................. 20 Figure 5: Representative size profile for ECM nanoparticle batches ............................................ 23 Figure 6: Cumulative Z-averages and PDIs of ECM nanoparticle batches ................................... 23 Figure 7: Effects of filtering on ECM nanoparticle batches .......................................................... 24 Figure 8: Cell counts for polystyrene beads added to hASCs ....................................................... 26 Figure 9: Cell counts for lower concentrations of polystyrene beads added to hASCs ................. 27 Figure 10: hASC viability images under addition of various nanoparticles .................................. 28 Figure 11: Cell counts for hASC viability images under addition of various nanoparticles ......... 29 Figure 12: NHS-ester chemistry overview..................................................................................... 30 Figure 13: Fluorescence microscope image of ECM particles ...................................................... 31 Figure 14: Centrifuge method to wash synthetic particles ............................................................. 32 Figure 15: Sizing data of ECM nanoparticle batch before and after centrifugation ...................... 33 Figure 16: Overview of centrifuge filter unit method to wash nanoparticles. ............................... 33 Figure 17: Size profile changes of FITC-ECM nanoparticles after centrifuge washing ................ 34 Figure 18: Flow-through and fluorescence data of free dye and FITC-ECM nanoparticle batches ............................................................................................................................................... 34 Figure 19: Comparison of FITC-NHS, fluorescein and glycine quenched ECM nanoparticle batches ................................................................................................................................... 35 vi Figure 20: Addition of FITC-tagged ECM nanoparticles to bone marrow-derived macrophages 37 Figure 21: Signal-to-noise ratios of several dyes conjugated to ECM nanoparticles .................... 38 Figure 22: Distribution of Licor-tagged ECM nanoparticles introduced by tail vein injection ..... 39 Figure 23: Size profiles of ECM nanoparticle batches conjugated with NHS-PEG ...................... 40 Figure 24: Zeta potentials of ECM nanoparticle batches conjugated with NHS-PEG . ................ 41 Figure
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