生理环境pH响应高分子/蛋白复合制剂的活性控制和安全性问题研究

Surface modification of therapeutic proteins by polymers, such as PEGylation, is one of the most commonly used methodologies for improving the proteins' stability, solubility and protecting the proteins from enzyme degradation, which has gained prolongation of circulation duration of the proteins in vivo. However, a major drawback of covalent attachment of polymer chains is to always cause the loss of biological activity of the proteins due to the polymers' non-specific shelding effect as well as the permanent change of protein structure. In this project, we aim to develop a new strategy for "traceless" reversible modification of proteins, by the application of dynamic covalent chemistry. Urokinase plasminogen activator (uPA) is selected as model protein and will be reacted with benzaldhyde functionalized polymers to form a benzoic-imine linkge.The low-pH labile ability of the linkage will endow the resultant conjugates to exhibit higher bioactivity under very weak acidic conditions as in the cerebral ischemic area of stroke patients (pH 6-6.5). Conjugates with vary kinds of architectures, in particular polymer-protein conjugates and protein-crosslinking nanogels, will be fabricated to demonstrate the influence of microstructure of conjugate on its perperties, and thus to help the design of protein formulation with controlled bioactivity. Another objective of this project is to clarify the prossible safety issues raised by the dynamic covalent conjugation, such as the pharmacological conern of re-attachment of free benzaldhyde functionalized polymers with serum proteins after the hydrolysis of the conjugates in vivo. Therefore we will carefuly check the reactivity of -CHO containing polymers to various kinds of proteins and intend to select out those having a conformation/aggregate structure leading to low protein affinity in aqueous media. The construction of such polymer chains on protein surface will be gained via either in situ polymerizaiton, or by the synthesis of crosslinked protein nanogels with amphiphilic block copolymers. We hope the above routes would be able to diminish the non-specific protein conjugation of the freed polymer chains by hiding of their aldhyde groups inside coils or in the core of the formed aggregates once the conjugates hydrolized. The pharamcokinetics, biodistribution and transpotation mechanism of benzaldhyde functionalized polymers as well as uPA conjugates will be evaluated in vitro and in vivo, in order to screen optimized formulations for further trials.

高分子修饰对提高蛋白药物的抗降解能力、延长体内循环时间有着重要作用，但往往导致蛋白活性下降。本项目提出通过构筑高分子/蛋白动态共价复合结构进行蛋白可逆修饰的思路，以血栓治疗蛋白尿激酶为模型，由苯甲醛官能化高分子与蛋白表面氨基反应形成苯甲酰亚胺动态共价键的路径制备蛋白复合制剂，利用动态共价键在正常生理环境下稳定、在血栓半暗带等弱酸性微环境中(pH 6-6.8)加速水解的特性实现蛋白的pH值响应释放，提高制剂在病灶区的活性。本项目将制备包括高分子/蛋白复合物、蛋白交联结构（纳米凝胶）等不同体系，考察微结构对复合物表观活性及其pH响应性的影响。此外，还将重点对高分子/蛋白动态共价复合体系可能带来的安全性问题进行研究，以期为所合成的蛋白制剂和其它亚胺基动态共价聚合物的生物医学应用提供实验基础，为解决高分子/蛋白复合工作中的稳定性与生物活性矛盾提供可借鉴方法。

通过本项目研究，我们掌握了通过苯甲酰亚胺动态共价键制备高分子/蛋白复合结构的基本方法和规律；确认了通过动态共价键制备的高分子/蛋白复合物的安全性；以尿激酶为模型，设计、制备了不同结构、形态以及对血栓周围乏氧组织生理pH值具有一定响应能力的高分子/尿激酶复合体系，使其水解动力学在一定时间和pH值范围内可调，继而通过微观结构和pH响应能力控制其表观生物活性。在对上述高分子/蛋白复合结构的表征中，我们发现，以溶栓蛋白为交联点、具有三维交联结构的聚乙二醇/尿激酶纳米凝胶对类似血栓周围半暗带的弱酸性pH值微环境有着较好的响应性，在该pH值下，高分子/蛋白复合体系较在正常生理pH值下具有更高的活性，因此能够对血栓周围组织形成被动靶向作用；而由动态共价键构筑的核壳结构高分子中空微凝胶在上述pH值的变化范围内具有较高的稳定性，但同时对超声场具有响应性，可由外场介导溶栓蛋白释放和溶栓过程。据此，我们研究了高分子/尿激酶复合制剂的溶栓特点，发现上述纳米、亚微米聚集结构的复合制剂能够在大血管阻塞的情况下，在血栓周围半暗带结构中实现控制释放，溶解微血管和侧枝循环中的微血栓，从而帮助远端缺血脑组织建立微循环，改善预后。上述工作结果不仅为蛋白的高分子修饰提供了新方法，也为提高尿激酶制剂时间窗外的血管再通率提供了借鉴，为急性缺血性卒中的治疗提供了新思路。