Huang Hui Yu, Shaw James, Yip Christopher, Wu Xiao Yu
Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada.
Pharm Res. 2008 May;25(5):1150-7. doi: 10.1007/s11095-007-9507-x. Epub 2007 Dec 11.
Polymeric membranes containing pH-sensitive nano-hydrogels and glucose oxidase were found to exhibit glucose-responsive insulin release. To verify that this glucose-responsiveness stemmed from the decrease in the internal pH of the membranes, we determined the spatial and temporal pH profiles inside the composite membranes in situ for the first time.
A pH-sensitive fluorescent dye and an inert internal reference was incorporated in the membranes consisting of poly(N-isopropylacrylamide-co-methacrylic acid) nanoparticles in a polymer matrix, with or without glucose oxidase and catalase. The fluorescence intensity versus time was measured by laser scanning confocal microscopy. The intensity ratios of the two fluorescent dyes were used to determine the internal pH profiles of the membranes in buffer solutions of various pH or glucose levels.
The internal pH was found to be lower than the external pH of buffer solutions. The pH decreased with an increase in glucose concentration, incubation time and the distance towards the center of the membranes due to the relative rates of glucose oxidation and solute diffusion.
The results provided direct experimental evidence of acidic internal pH that inversely related to external glucose concentration in an external medium of constant neutral pH.
含有pH敏感纳米水凝胶和葡萄糖氧化酶的聚合物膜表现出葡萄糖响应性胰岛素释放。为了验证这种葡萄糖响应性源于膜内部pH值的降低，我们首次原位测定了复合膜内部的时空pH分布。
将一种pH敏感荧光染料和一种惰性内部参比物掺入由聚（N-异丙基丙烯酰胺-共-甲基丙烯酸）纳米颗粒在聚合物基质中组成的膜中，该膜含有或不含有葡萄糖氧化酶和过氧化氢酶。通过激光扫描共聚焦显微镜测量荧光强度随时间的变化。两种荧光染料的强度比用于确定膜在不同pH值或葡萄糖水平的缓冲溶液中的内部pH分布。
发现膜内部pH低于缓冲溶液的外部pH。由于葡萄糖氧化和溶质扩散的相对速率，pH值随着葡萄糖浓度、孵育时间以及朝向膜中心距离的增加而降低。
结果提供了直接的实验证据，表明在恒定中性pH的外部介质中，内部酸性pH与外部葡萄糖浓度呈负相关。
