Biomaterials 19 (1998) 1287 ��� 1294 Characterization of permeability and network structure of interfacially photopolymerized poly(ethylene glycol) diacrylate hydrogels Gregory M. Cruise!,",1, David S. Scharp#, Jeffrey A. Hubbell!,* !Division of Chemistry and Chemical Engineering, California Institute of Technology, Mail Stop 210-41, 391 S. Holliston Avenue, Pasadena, CA 91125, USA "Department of Chemical Engineering, University of Texas at Austin, Austin, TX 78712, USA #Neocrin Company, 31 Technology Drive, Suite 100, Irvine, CA 92618, USA Received 7 July 1997 accepted 25 January 1998 Abstract Hydrogel membranes formed by interfacially photopolymerizing poly(ethylene glycol) (PEG) diacrylate precursor solution were prepared from PEG diacrylate of molecular weights (MW) ranging from 2000 (2K) to 20 000 (20K) with concentrations ranging from 10% to 30% w/w. The effects of PEG diacrylate MW and concentration in the membrane precursor solution upon the diffusivities of vitamin B 12 , myoglobin, ovalbumin, albumin, and IgG were determined. Regardless of the concentration of the PEG diacrylate in the precursor solution, hydrogels prepared with PEG 2K, 4K, and 8K diacrylate were impermeable to proteins with a size equal to or larger than myoglobin (22 kDa), while hydrogels prepared with PEG 20K diacrylate were impermeable to proteins with a size equal to or larger than ovalbumin (45 kDa). Similarities between hydrogels formed from PEG 2K, 4K, and 8K diacrylates were also seen in calculations of the molecular weight between crosslinks and the mesh size, with values in the range of 150���750 g/mol and 15���35 A, respectively, depending on PEG diacrylate concentration. In contrast, hydrogels formed from PEG 20K diacrylate had molecular weight between crosslinks ranging from 1150 to 2000 g/mol and mesh sizes ranging from 45���70 A, with larger values being observed in membranes polymerized from more dilute PEG diacrylate precursor. ( 1998 Published by Elsevier Science Ltd. All rights reserved Keywords: Poly(ethylene glycol) Hydrogel Diffusion Proteins 1. Introduction Transplantation of islets of Langerhans shows promise for the treatment of diabetes. Encapsulation of islet allo- and xenografts in a semipermeable membrane is one type of immunomodulation that has been explored to prevent rejection. Such an encapsulation membrane should allow for diffusion of dissolved gases, nutrients, wastes, glucose (the primary secretogogue for insulin), and insulin���all compounds that have relatively low mo- lecular weights. However, the membrane should be im- permeable to the components of the immune system, certainly including complement protein C1q and perhaps also immunoglobulins���compounds that have much * Corresponding author. Institute for Biomedical Engineering, Swiss Federal Institute of Technology, and University of Zu ��rich, Mousson- strasse 18, CH-8044 Zu ��rich, Switzerland. 1 Present address: Cohesion Technologies, 2500 Faber Place, Palo Alto, CA 94303, USA. larger molecular weights���as well as all cells of the im- mune system. The diffusion of proteins through various types of membranes has been studied, including poly (vinyl alco- hol) [1], poly(N-isopropylacrylamide) [2], hyaluronic acid esters [3] and collagen [4] membranes. The network structures of radiation-crosslinked linear [5] and star [6] PEG, materials with similar structures to those studied herein, have been characterized. These studies showed the potential of manufacturing a permselective mem- brane to potentially prevent rejection of transplanted tissues. A technique to interfacially photopolymerize PEG dia- crylate directly upon the surfaces of individual islets of Langerhans to serve as permselective barriers has been reported [7]. A photoinitiator (eosin Y, a xanthine dye [8]) is adsorbed to the islet surface from a saline solution, and the non-adsorbed photoinitiator is removed from the islet suspension by washing. The photoinitiator-stained islets are then suspended in an aqueous solution of PEG diacrylate with the other components of the initiation 0142-9612/98/$19.00 ( 1998 Published by Elsevier Science Ltd. All rights reserved. PII S 0 1 4 2 - 9 6 1 2 ( 9 8 ) 0 0 0 2 5 - 8

scheme, namely triethanolamine, and a polymerization accelerator, 1-vinyl 2-pyrrolidinone (the solution of all components is referred to herein as the precursor solution). Irradiation of this suspension results in poly- merization of the precursor solution only where all the components of the initiation scheme meet, namely at the islet surfaces, with the precursor solution not near an islet surface remaining unpolymerized and liquid. Be- cause the PEG macromer is a diacrylate, polymerization results in a conformal, crosslinked hydrogel comprised of nodes of oligoacrylate connected by linear PEG esterified thereto. Porcine islets encapsulated as described above in conformal PEG diacrylate hydrogel membranes have been shown to be functional in vitro, using static glu- cose stimulation and perifusion cultures, and in vivo, by maintenance of normoglycemia in streptozotocin- induced diabetic athymic mice [9]. To aid in selection of the molecular weight and concentration of the PEG diacrylate in the membrane precursor solution that results in potentially xenoprotective membranes, we studied the effect of these two encapsulation parameters on the diffusivity of biological molecules through the membrane, the average molecular weight between crosslinks, and the mesh size of the hydrogel network. A model system was developed to be able to perform measurements of permeation and hydrogel character- istics. Membranes formed by interfacial photopolymeri- zation upon an islet surface are not amenable to permeation measurements using a two-chamber diffu- sion cell, and membrane swelling is difficult to character- ize upon an islet, which itself contains a large amount of water. To enable placement of interfacially polymerized PEG diacrylate hydrogel membranes in a diffusion cell, photopolymerization of PEG diacrylate was performed upon a poly (vinylidiene fluoride) microporous filter, which took the place of the islet as the substrate for interfacial photopolymerization of the PEG diacrylate precursor. 2. Materials and methods 2.1. PEG diacrylate synthesis All solvents used in the synthesis were reagent grade or better and the reactants were used as received. Fifty grams of PEG diol (Union Carbide) with a molecular weight of either 1350 (2K), 3350 (4K), 8000 (8K) or 20,000 (20K) were dissolved in 750 ml of benzene (Fisher) and water was removed by azeotropically distilling 250 ml of benzene. Triethylamine (Aldrich), in four fold molar ex- cess based on PEG diol end groups, was added to the PEG solution at room temperature. Acryloyl chloride (Aldrich), in four fold molar excess based on PEG diol end groups, was added dropwise to the PEG solution to form acrylate diesters of PEG. The mixture was stirred overnight at 35��C under argon. The insoluble triethylamine salts formed during the reaction were re- moved by filtration and the PEG diacrylate product was precipitated by the addition of 1.4 l of diethyl ether (Fisher) chilled to 4��C. The PEG diacrylate precipitate was collected on a fritted funnel, redissolved in 100 ml of benzene, and reprecipitated with 1.4 l of chilled diethyl ether twice more. The polymer was dried 24 h in a vac- uum oven at 35��C. PEG diacrylates were analyzed using nuclear magnetic resonance (NMR) spectroscopy and gel permeation chromatography (GPC). The degree of substitution of the PEG terminal alcohol for acrylate was determined using the NMR spectrum of PEG diacrylates and the method of Dust et al. [10]. Briefly, the method compares the ratio of the integration from the PEG backbone ( \ 3.5 ppm) and the acrylate peaks ( \ 5.8���6.4 ppm) to the known PEG weight average molecular weight. The extent of acrylation substitution was calculated using the follow- ing formula: % acrylation"MPEG molecular weightN/ M(integral of PEG backbone)/[(integral of acrylates)/ 6]/4]44N. 2.2. Preparation of hydrogels PEG diacrylate precursor solutions were prepared in phenol-free M199 cell culture medium (Gibco) with 225 mM triethanolamine (Aldrich) and 37 mM 1-vinyl 2- pyrrolidinone (Aldrich), and were adjusted to pH 8 with 6 M HCl (VWR). The precursor solution was filter-steril- ized using a 0.2 lm syringe filter (Corning). PEG diacrylate hydrogels were prepared on hy- drophilic poly (vinylidiene fluoride) (PVF) filters with a pore size of 0.22 lm (Millipore). The PVF filters were stained in deionized and distilled water saturated with ethyl eosin (Aldrich) for 18 h. The stained filters were removed from the ethyl eosin solution and placed in a 500 ml sterile filter flask (Corning) with 200 ml of de- ionized and distilled water. The PVF filters were seated on top of the filter flask filter and the entrapped ethyl eosin was washed into the filter flask with the filtrate, leaving only the stained PVF filter. The stained PVF filter was placed in a 60 mm petri dish (Corning) and 5 ml of PEG diacrylate precursor solution was added. The air bubbles were removed and the stained filter was illuminated with an argon ion laser for 2 min at a flux of 145 mW cm~2. After illumina- tion, the filter-supported hydrogel membrane was re- moved from the PEG diacrylate solution and placed in HEPES-buffered saline for at least 12 h to permit equilibration of the hydrogel and the diffusion of any free PEG diacrylate entrained in the hydrogel membrane. For each of the experimental conditions, five replicates were analyzed. 1288 G.M. Cruise et al. / Biomaterials 19 (1998) 1287���1294