Faculty Bibliography

A method has been defined to interfacially photopolymerize poly(ethylene glycol) diacrylates (PEG diacrylates) to form a crosslinked hydrogel membrane upon the surfaces of porcine islets of Langerhans to serve as an immune barrier for allo- and xenotransplantation. A sensitivity study of six key parameters in the interfacial photopolymerization process was performed to aid in determination of the optimal encapsulation conditions, leading to the most uniform hydrogel membranes and viable islets. The key parameters included the concentrations of the components of the initiation scheme, namely eosin Y, triethanolamine, and 1-vinyl 2-pyrrolidinone. Other parameters investigated included the duration and flux of laser irradiation and the PEG diacrylate molecular weight. Each parameter was doubled and halved from the standard conditions used in the encapsulation process while holding all the remaining parameters at the standard conditions. The effects of changing each parameter on islet viability, encapsulation efficiency, and gel thickness were quantified. Islet viability was sensitive to the duration of laser illumination, viability significantly increasing as the duration was reduced. Encapsulation efficiency was sensitive to the concentrations of eosin Y, triethanolamine, and 1-vinyl 2-pyrrolidinone, to the laser flux, and to the PEG diacrylate molecular weight. Increasing the concentration of eosin Y significantly improved the encapsulation efficiency, while decreasing the concentration of 1-vinyl 2-pyrrolidinone and increasing the concentration of triethanolamine had the greatest effects in significantly reducing the encapsulation efficiency. Gel thickness was sensitive to the concentrations of triethanolamine and 1-vinyl 2-pyrrolidinone, to the duration of laser illumination, and to the PEG diacrylate molecular weight. Increasing the PEG diacrylate molecular weight significantly increased the gel thickness, while decreasing the concentration of 1-vinyl 2-pyrrolidinone and increasing the concentration of triethanolamine had the greatest effects in significantly reducing the gel thickness. From this sensitivity study, conditions were determined to encapsulate porcine islets, resulting in greater than 90% islet viability and greater than 90% encapsulation efficiency.

BACKGROUND:We present the synthesis, characterization and initial structure-function analysis of a new class of bioactive agent that allows the application of techniques from colloid science to biological surfaces. Stable colloidal suspensions can be generated by immobilizing a dense brush of soluble polymer at the colloidal surface, creating a zone protected against the adhesion of approaching particles, a phenomenon termed polymeric steric stabilization. This is often accomplished for aqueous colloidal dispersions using adsorbing block copolymers. We demonstrate that water-soluble block copolymers can be designed to adsorb onto heterogeneous biological surfaces and block cell-cell and cell-surface adhesion, using polymer compositions and architectures that are quite different from surfactants used for stabilizing nonbiological colloidal dispersions. RESULTS:Comb copolymers were synthesized having polycationic backbones (poly-L-lysine, PLL), serving as the anchor for binding to the net negatively charged biological surfaces, grafted with water-soluble polynonionic chains (polyethylene glycol, PEG), to block biological recognition, producing PLL-graft-PEG copolymers. Specific copolymers were found to sterically stabilize red blood cells from lectin-induced hemagglutination and fibroblasts from adhesion to fibronectin-coated surfaces. The polymer design principles, which appear to be unique for adsorption to heterogeneous biological surfaces, require the use of very high molecular weight comb copolymers, perhaps because anionic sites are non-uniformly distributed on biological surfaces, and the ability of larger copolymers to span between highly anionic sites. CONCLUSIONS:Water-soluble copolymers were produced that can block recognition at biological surfaces, on the basis of nonspecific physicochemical phenomena rather than specific biochemical interactions.

Photopolymerized crosslinked networks of poly(ethylene glycol; PEG) diacrylate (MW 8000) were derivitized throughout their bulk with Arg-Gly-Asp (RGD)-containing peptide sequences. Incorporation was achieved by functionalizing the amine terminus of the peptide with an acrylate moiety, thereby enabling the adhesion peptide to copolymerize rapidly with the PEG diacrylate upon photoinitiation. PEG diacrylate hydrogels derivitized with RGD peptide at surface concentrations ranging from 0.001 to 1 pmol/cm2 were studied in vitro for their ability to promote spreading of human foreskin fibroblasts over 24 h. Hydrogels not derivitized with peptides were poor substrates for adhesion, permitting spreading of only 5% of the seeded cells. When immobilized with no spacer arm, both RGD and RDG (inactive control) supported spreading of approximately 50% and approximately 15% of cells at 1 and 0.1 pmol/cm2 surface concentrations respectively; lower concentrations did not promote spreading. When a MW 3400 PEG spacer arm was incorporated between the hydrogel and the peptide linkage, incorporation of 1 pmol/cm2 RGD promoted 70% spreading whereas RDG at the same concentration did not promote spreading. In addition, when cells were seeded in serum-free medium, only RGD peptides incorporated with a spacer arm were able to promote spreading. Thus peptide incorporated into PEG 8000 diacrylate hydrogels without a spacer arm nonspecifically mediated cell spreading whereas incorporation via a MW 3400 PEG spacer arm was required to permit cell spreading to be specifically mediated.

Poly(tetrafluoroethylene) (PTFE) films were surface-modified by employing a reaction solution of benzophenone and sodium hydride in anhydrous dimethylformamide at a temperature of 150 degrees C for 12 h. Electron spectroscopy for chemical analysis (ESCA) showed defluorination, oxygen incorporation, and extensive unsaturation within the treated PTFE surfaces. The suitably of these reduced PTFE films as substrates for graft polymerization was initially assessed via photograft polymerization of the sodium salt of styrenesulfonic acid (SS-Na), which permitted unequivocal surface analysis by the introduction of a new atom, as well as poly(ethylene glycol) monoacrylate (PEG-Ac). All photograpt polymerization was performed employing ultraviolet irradiation with 2,2-dimethoxy-2-phenylacetophenone as an initiator. Photograft polymerization of SS-Na was verified by further reduction of fluorine atomic content and the appearance of new sulfur and sodium atomic peaks on ESCA survey spectra, and that of PEG-Ac was verified by further reduction of fluorine atomic content and increase of atomic percent ratio of O/C from ESCA survey spectra as well as appearance of a new ester peak on high resolution ESCA C 1s spectra. Dynamic water contact angles on reduced and PEG-Ac photograft polymerized films were measured and showed that the PTFE film surface became more hydrophilic after reduction (from 120 to 89 deg) and the reduced film became more hydrophilic after photograft polymerization with PEG-Ac (from 89 to 36 deg). Modification of the complete surface of expanded PTFE (ePTFE), i.e. of the lumenal, outside and pore surfaces, was performed by employing the reaction described above, except at 105 degrees C for 1 day, followed by photograft polymerization of PEG-Ac. ESCA was performed on the superficial surfaces (i.e. the lumen and exterior) as well as on cross-sections of the ePTFE to permit analysis of the pore surfaces. This analysis showed that both the initial surface reduction and subsequent photograft polymerization were successful as indicated from F/C and O/C atomic percent ratios from ESCA survey spectra, from overall peaks shapes of high resolution ESCA C 1s spectra and from generation of new ester peaks on high resolution ESCA C 1s spectra of ePTFE graft polymerized with PEG-Ac, which demonstrated an O/C atomic percent ratio close to that of PEG-Ac homopolymer. Low voltage scanning electron microscopy confirmed minimal morphological damage to the ePTFE microstructure after reduction and graft polymerization. The approach explored thus provides a means for modulation of biological interactions at ePTFE surfaces with only minimal modification of material morphology, with some surface texture appearing on a length scale of 50-100 nm.

Novel lactide-based poly(ethylene glycol) (PEG) polymer networks (GL9-PEGs) were prepared by UV copolymerization of a glycerol-lactide triacrylate (GL9-Ac) with PEG monoacrylate (PEG-Ac) to use as scaffolds in tissue engineering, and the surface properties and biocompatibility of these networks were investigated as a function of PEG molecular weight and content. Analysis by ATR-FTIR and ESCA revealed that PEG was incorporated well within the GL9-PEG polymer networks and was enriched at the surfaces. From the results of SEM, AFM, and contact angle analyses, GL9-PEG networks showed relatively rough and irregular surfaces compared to GL9 network, but the mobile PEG chains coupled at their termini were readily exposed toward the aqueous environment when contacting water such that the surfaces became smoother and more hydrophilic. This reorientation and increase in hydrophilicity were more extensive with increasing PEG molecular weight and content. As compared to GL9 network lacking PEG, protein adsorption as well as platelet and S. epidermidis adhesion to GL9-PEG networks were significantly reduced as the molecular weight and content of PEG was increased, indicating that GL9-PEG networks are more biocompatible than the GL9 network due to PEG's passivity. Based on the physical and biological characterization reported, the GL9-PEG materials would appear to be interesting candidates as matrices for tissue engineering.

Functional porcine islets, free of known pathogens, can serve as a source of insulin producing cells for the treatment of experimentally induced insulin dependent Diabetes Mellitus. Porcine islets can be conformally coated (microencapsulated) with a covalently linked, stable permselective membrane while maintaining islet viability and function. The PEG conformal coating is immunoprotective in a discordant xenograft animal model (porcine islets to rat).