Preparation of Glucose-Responsive Phenylboronic Acid-Containing Polymers and Their Application to Artificial Pancreas

研究了一种对葡萄糖浓度敏感的新型“智能”聚合物体系，作为化学调节胰岛素释放系统的候选材料。苯硼酸能与顺式二醇类物质形成可逆结合。利用苯硼酸聚合物作为关键材料，研究了葡萄糖酸修饰胰岛素（G-Ins）与葡萄糖分子交换反应的葡萄糖反应性胰岛素释放系统。用正面色谱法在pH 8.5下评价了各种含二醇分子与PBA凝胶珠[聚(3-甲基丙烯酰胺苯硼酸-co-丙烯酰胺-co-N，N'-亚甲基双-（丙烯酰胺）]的络合作用。含二醇分子的结构特征强烈影响其与PBA凝胶珠的结合。特别是，开链单糖的缔合常数（约9.5X10X^2至5.1X10X^3 l/mol）高于葡萄糖（约6.3X10X^2 l/mol）。合成G-Ins，并将其结合到PBA凝胶柱上。然后，研究了pH为8.5时不同葡萄糖浓度下G-Ins的释放曲线。G-Ins的释放浓度随葡萄糖浓度的变化呈脉动性变化。葡萄糖浓度与释放的G-lns峰高呈线性关系，并且释放响应对葡萄糖浓度的变化没有滞后时间。为了在生理pH 7.4下增强PBA与葡萄糖的结合，在PBA[聚(3-甲基丙烯酰胺苯硼酸-co-N，N-二甲氨基丙烯酰胺-co-丙烯酰胺-N，N'-亚甲基双（丙烯酰胺）]的聚合物链中加入了一种胺成分。采用批量法测定与葡萄糖配合的PBA基团数量与总PBA基团数量的比例。与不含胺组分合成的PBA共聚物相比，该比例随着胺含量和缓冲液pH的增加而增加。这些结果证实，邻近胺（未质子化）与PBA的相互作用加强了与葡萄糖的结合，特别是在pH 7.4及以上。采用液相色谱法评价了pH 7.4条件下含胺PBA凝胶珠中G-Ins的释放特性。与PBA凝胶相比，含胺PBA凝胶在缓冲冲洗下柱中G-Ins的下降速度较慢。从含胺的PBA凝胶中释放的G-Ins的量远远大于PBA凝胶，在生理ph下对葡萄糖的添加做出反应。长期释放研究的结果表明，含胺的PBA凝胶对葡萄糖的挑战有超过120小时的反应能力。此外，G-Ins的释放量与葡萄糖浓度呈线性关系。这些结果证明了胰岛素反应系统在生理条件下实现胰岛素释放以响应葡萄糖的可行性。少

A new "Intelligent" polymer system sensitive to glucose concentration have been studied as a candidate material for chemically regulated insulin release system. Phenylboronic acid is capable to form reversible binding with cis-diol substances. Glucose responsive insulin release system has been studied with utilization of phenylboronic acid polymers for the key material to exchange reaction between gluconic acid modified insulin (G-Ins) and glucose molecule. CompIexation of various diol-containing molecules with PBA gel beads [poly (3-methacrylamidophenylboronic acid-co-acrylamide-co-N,N'-methylenebis- (acrylamide)] was evaluated using frontal chromatography at pH 8.5. The structural features of the diol-containing molecules strongly influenced their binding to PBA gel beads. In particular, open-chain mono saccharides demonstrated higher association constants (ca. 9.5X10X^2 to 5.1X10X^3 l/mol) than glucose (ca. 6.3X10X^2 l/mol). G-Ins was synthesized, and was bound onto PBA gel column. … More Then, the G-Ins release profile in response to varying glucose concentrations was investigated at pH 8.5. The released concentration of G-Ins from the polymer was pulsatile in response to the repeated stepwise concentration changes of glucose. Other remarkable results are that the linearity between glucose concentration and peak height of released G-lns, and that the release response have no lag time to change of glucose concentration. To enhance binding between PBA and glucose under physiological pH 7.4, an amine component was incorporated into the polymer chain along with PBA [poly (3-methacrylamidophenylboronic acid-co-N,N-dimethylaminopropylacrylamide-co-acrylamide-co-N,N'-methylenebis (acrylamide)]. The proportion of the amount of PBA groups complexed with glucose versus total amount of PBA groups was determined by batch method. Compared to PBA copolymer synthesized without the amine component, the proportion increased as a function of the amine content and the pH of the buffer. These results confirm that the interaction of neighboring amines (unprotonated) with PBA strengthens the binding with glucose, especially at pH 7.4 and above. Release property of G-Ins from the amine-containing PBA gel beads at pH 7.4 was evaluated using a liquid chromatography system. Amine-containing PBA gel showed less rapid decrease of G-Ins in the column under buffer-rinse over PBA gel. The released amount of G-Ins from amine-containing PBA gel is much greater than for the PBA gel, responding to the addition of glucose at physiological pH.The results from long-term release studies demonstrate that the amine-containing PBA gel has the capability to respond to glucose challenge over 120 hours. Furthermore, a linearity of released G-Ins was observed with glucose concentration. These results demonstrate the feasibility of an insulin-responsive system to achieve insulin release in response to glucose under physiological conditions. Less