Abstract: 1st Year: Create an alginate-based biomimic ECM (extracellular matrix) to enhance cell-matrix interaction and prolong drug release
Surface modification of biomaterials with cell-recognition sites can enhance interaction between cells and matrix materials. Cell adhesion ligands such as arginine-glycine-asparagine (RGD) sequences can be immobilized on alginate to promote cell anchorage[1]. Alginate (Pronova, NovaMatrix) will be chemically modified with GRGDY (Yao-Hung Biotech. Inc.) peptides utilizing aqueous carbodiimide chemistry to enhance cell-matrix interaction[2]. 1-ethyl-(dimethylaminopropyl) carbodiimide (EDC), a water-soluble carbodiimide will be used to form amide linkages between amine containing molecules and the carboxylate moieties on the alginate polymer backbone[3]. The chemistry will be optimized with regard to the carbodiimde: uronic acid ratio, pH, and NaCl molarity by the previous studies. The aerodynamic encapsulation device will be used to form the modified alginate microspheres by tuning the parameter such as air pressure, nozzle size and flow rate. The structure,properties,and standard drug release kinetics of alginate-based microspheres will be characterized by XRD, FTIR, SEM, the dynamic rheological and swelling ratio behaviors will also be performed[4].
2nd year: Characterize the endothelial progenitor cell (EPC) behavior with peptide-modified alginate
Human peripheral blood mononuclear cells from healthy adults will be isolated by density gradient centrifugation under 400g for 30min. (HISTOPAQUE, Sigma). The mononuclear cells will be cultured in endothelial cell growth medium (Cambrex). After 24 hrs, the non-adherent cells will be collected and continued to be cultured for 1 week. The ex vivo expanded cells will adapt endothelial-like characteristics and will be considered as endothelial progenitor cells (EPCs)[5]. In vitro cell adhesion will be performed by seeding the EPCs on the alginate surface at a density of 25000 cells/cm2 in a 24 well plate. After 4hr, the wells will be rinsed with fresh medium at 4hr to remove unattached cells from the surfaces. Alginate disks will be rinsed and fixed with 4% formaldehyde for 10 minutes at 16hr and cells will be visualized under inverted microscope. In vitro cell proliferation will be determined by performing the MTS assay (Promega) on a 96 well plate at 3,5,7,10 days after culture[6].
3rd year: Synthesize the multifunctional microspheres and in vivo evaluation of cocktail therapy in treating murine Peripheral Arterial Disease (PAD)
Aptamer selected EPCs, therapeutic reagent (i.e. pro-angiogenic DNA aptamer) loaded in mesoporous nanoparticles will be encapsulated in the modified alginate microspheres. The microspheres will be cultured up to 10days. To determine the viability of encapsulated cells, the microspheres will be depolymerized by soaking in depolymerization solution containing 100mM sodium citrate (Fisher Scientific), 10mM MOPS(Sigma) and 27mM NaCl for 30min at 37℃. The solution will be centrifuged at 1200rpm for 10min. The cell pellet will then be resuspended in medium and cell density/ viability was determined by trypan blue staining.
In the in vivo study, administration of different treatments group: saline control, cocktails containing microspheres, cells only, therapeutic reagent only (n=10 for each group. N=40) will be carried out in the mice hindlimb ischemia model by direct injection. The blood flow will be evaluated with Laser Doppler Blood Perfusion (LDBP) method at day 0, 7, 14,and 28 days . The animal will be sacrificed at 28 days post treatment and cryo-sectioned. Histology will be evaluated by H&E stain and blood vessel cells will be immunostained. The vessel density will be quantified and human cell marker will also be detected for exogenous cell retention and integration into the region of interests[7-9].
References
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