基于多巴胺过表达调节的分子印迹-固相生物催化型给药系统的构建及评价

The abuse of amphetamine-type stimulants (ATS) in our country is becoming more and more serious, triggering serious social problems and bringing heavy medical burdens. This study is aimed at the core neurological mechanism of the regular drug use stage in ATS addiction. It is suggested that a drug delivery system, which is capable of specifically adsorbing and metabolizing over-expression dopamine (DA) in the synaptic cleft, should be constructed and can reverse the ATS craving. In this study, membrane-bound catechol-O-methyltransferase (MB-COMT), which has highly metabolic activity for DA, is extracted from liver and brain tissue. Making use of molecular imprinting and enzyme immobilization technology, the molecular imprinting-solid phase biocatalytic drug delivery system is synthesized by utilizing DA molecular imprinting microspheres as the solid phase carriers and introducing with 6-carbon spacer arms and MB-COMT. The selective adsorption property and specific enzyme metabolic activity are optimized in vitro. In the methamphetamine self-administration model and methamphetamine-induced conditioned place preference model, the behavioral and neurochemical influences of the drug delivery system on the methamphetamine addiction are evaluated. This drug delivery system is biodegradable and has special molecular recognition and metabolic effect for DA, which is expected to achieve a highly specific and effective removal of DA that is not reabsorbed by neurons. The study provides a research basis for the design of ATS addictive therapeutic agents.

我国苯丙胺类兴奋剂（ATS）的滥用形式日趋严重，引发严重社会问题、带来沉重医疗负担。本研究针对ATS成瘾过程中规律性用药阶段的核心神经学机制，拟构建给药系统能够特异性吸附并代谢突触间隙过量表达的多巴胺（DA），逆转ATS渴求。在本研究中，从肝和脑中提取纯化对DA具有高效代谢活性的膜结合型儿茶酚-O-甲基转移酶（MB-COMT），利用分子印迹技术和酶固定化技术，以DA分子印迹微球作为固相载体，引入6-碳间隔臂和MB-COMT合成分子印迹-固相生物催化型给药系统，体外优化其对DA的选择性吸附性能和专属性酶代谢活性，并在小鼠甲基苯丙胺静脉自身给药模型和甲基苯丙胺诱导的条件性位置偏爱模型中，对成瘾小鼠的行为学和神经化学影响进行系统评价。该系统可生物降解，对DA具有专属性分子识别和代谢作用，预期能够高度专一、有效的清除未被神经元重吸收的DA，为ATS成瘾治疗剂的设计提供研究基础。

本文首次利用仿生溶胶-凝胶法和降解酶固定化技术来制备分子印迹-固相生物催化型给药系统（MAO-MIXPs），制备成功的MAO-MIXPs可高选择性识别、吸附并降解多巴胺，有望用于多巴胺的表达调节。仿生溶胶-凝胶法首先是DA和PEI聚合，而后通过非共价键氢键的结合而与TMOS自组装偶联，经洗脱模板分子留下“记忆腔”，然后将DA降解酶MAO固定化在MIXPs。MAO-MIXPs因为洗脱模板分子后留下的DA印迹位点以及MAO可对DA降解，可降低表达DA。DA吸附实验是在人工脑脊液下进行的，即通过模拟脑中DA过表达环境并为体内研究提供了理论基础。吸附机理实验研究验证该聚合物更倾向于均一单层吸附，为今后MAO-MIXPs剂型的制备提供了理论基础。进一步，在研究中选择静脉注射作为给药系统的给药方式，口服冰片作为渗透增强剂，给药系统与冰片的共给药提高了入脑的靶向效率。当冰片增加BBB的渗透性时，载药系统可以直接穿过BBB进入脑实质，然后绕过星形胶质细胞和内皮细胞的末端之间的连接，有效地吸收到神经元中，实现大脑皮层和海马中的神经元靶向，使该载体脑内特定脑区非侵入式递送和神经元靶向成为可能。