| Objective With the increasing prevalence of diabetes, to avoid the current clinical "exogenous insulin therapy" with frequent injection, blood glucose monitoring and repeated dose adjustment. This study focused on synthesizing injectable glucose sensitive biological materials encapsulated insulinoma cells to mimick bioartificial pancreas, to achieve the regulation of insulin secretion according to blood glucose level.Method Based on ammonium persulfate(APS) and tetramethylethylenediamine(TMEDA) redox system, 3-aminophenyl boronic acid(AAPBA) and N-isopropyl acrylamide(NIPAM) were crosslinked by dextran-maleic anhydride(Dex-Ma) to turn into hydrogels rapidly at 37?. By adjusting the feed ratios between AAPBA and NIPAM, dosages of crosslinking agent and concentrations of redox system, we synthesized of temperature sensitive hydrogels with a liquid state under low temperature and a solid state at 37?. The structure of the materials was analyzed by infrared spectrum(FT-IR). Scanning electron microscope(SEM) was used to detect the morphology of the materials. The swelling properties, rheological properties and in vitro insulin release behavior of the materials were also analyzed. The cytotoxicity of the materials was detected by MTT assay. Based on the above characterization, we selected an optimum feeding system with good biocompatibility and the best property, to encapsulate a beta cell line INS-1 832/13 secreted insulin. SEM was used to detect the morphology of the materials encapsulated cells. Fluorescence microscope was used to detect the morphology and cell viability of the encapsulated cells. ELISA method was used to detect the insulin secretion of cells encapsulated in materials stimulated by different concentrations of glucose.Result By adjusting the feed ratios of AAPBA and NIPAM, the mass ratios between AAPBA and NIPAM of 2:68, 4:66 and 6:64 were selected, with Dex-Ma of 1 mg/m L, a fixed initiator concentration of 20 mmol/L. They had good temperature sensitivity with a liquid state under low temperature and a solid state at 37?. FT-IR suggested that the material was synthesized. SEM revealed that the three materials were porous with a diameter between several micrometers and tens of micrometers, and the pores were enlarged with the increase of AAPBA. Swelling ratios of the materials increased to the maximum(2 ~ 4 times) rapidly in the first 10 ~ 30 minutes. The swelling properties of the materials decreased with the increase of AAPBA. Rheological measurement showed that the storage modulus(G') and loss modulus(G'') increased with the increase of temperature. A2N68, A4N66 and A6N64 had a phase transition temperature of 25?, 18? and 14?, respectively. Shear thinning test indicated that the viscosity decreased with increasing shear stress. In vitro insulin release assay indicated that the cumulative release increased gradually in 12 h, and tended to slow that. The results of MTT showed that the biocompatibility of the three materials was good. Materials with minimal cytotoxicity were selected to encapsulate INS-1 832/13 cells. SEM suggested a good combination of cells and materials. Acridine orange/ethidium bromide(AO/EB) double fluorescence staining showed that the cells grew well in hydrogels and turned into a cell ball over time. ELISA assay indicated that the insulin secretion of cells encapsulated in the materials changed according to the glucose level, and the content of AAPBA in materials had an effect on glucose sensing.Conclusion In this study, we had synthesized hydrogels with both temperature sensitivity and glucose sensitivity. They were injectable attributed to the characteristics of reversible phase transition between low temperature and 37?. INS-1 832/13 cells encapsulated in the hydrogels grew in clusters gradually, and had an insulin secretion pattern according to the glucose concentration. It achieved to mimic the microenvironment of cells highly, and might provide theoretical basis for the application of injectable hydrogels in islet transplantation. |
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