基于载药微胶囊构筑的多孔复合水凝胶和电纺纤维的制备及其蛋白质控释应用

The controlled release drug delivery system for protein or peptides are receiving increasing attenation in order to solve the problem of both an initial burst of the drug and a limited release time.In this project, we design to frabrication two types biodegradable and controlled release systems for protein by the combination of layer-by-layer assembly (LBL), click chemistry and reactive electrospinning.The protein is coprecipited in CaCO3 particles followed by multilayer assembly using by furan-functioned chtisan (CS-g-PEG-furan) and maleimide-funcationed hyaluronic acid (HA-Mal).Cs-g-PEG-furan and HA-Mal are assembled via electrostatics and are subsequently covalently stabilized using Diels-Alder click. After removal the CaCO3 template, the microcapusles loaded protein with furan or maleimide groups (clickable microcapusle) on the surface will be obtained. The microcapusle with controlled shell structure, drug loading and release rate will be obtained by adjusting the thickness, permeability and bidegradation rate of the shell. Then, the porous nanocomposite hydrogel is synthesized via in situ Diels-Alder click using the cilickable microcapusles as crosslinkers.The release rate, duration of release, and magnitude of the drug burst can all be tuned by adjusting the affinity of each of the microcapusle and bulk hydrogel for the target drug as well as the cross-link density of the bulk hydrogel phase, which regulates both drug diffusion and hydrogel degradation. The porous nanocomposite hydrogel for long-term, time-controlled release of protein should be obtained. The relationship between the structure parmeters, degradation rate between the nanocomption hydrogel and the release kinetics of protein from the hydrogel should be estiblished. The nanocomposite hydrogel fibers will be frabrcated using the clickable microcapusles as crosslinkers via the dual-syring reactive electrospinning. The release kinetics will be studied in detail for protein relesed from the nanocomposite hydrogel fibers.

以实现蛋白质药物长效、时控型释放并保持其生物活性为出发点，构建载药量和释药速率可控的蛋白质药物控释体系是生物医用高分子材料的研究热点之一。本项目旨在结合逐层自组装法（LBL）、点击反应和静电纺丝技术制备两类新型可生物降解的蛋白质控释体系：以透明质酸和壳聚糖作为构筑材料，利用LBL与点击反应相结合制备表面携带可点击基团的原位载药微胶囊，掌握控制微胶囊结构和释药特性的关键因素，获得囊壁结构、载药量和释药速率可控的可点击微胶囊，并以此为交联剂，利用原位点击反应制备多孔纳米复合水凝胶，通过调控微胶囊和水凝胶基体的结构参数、降解速率，实现复合水凝胶对蛋白质的高装载、长效安全、时控型释放，建立复合水凝胶结构参数、降解速率与蛋白质释放动力学间的经验关系；在此基础上利用反应性静电纺丝技术制备载药的电纺复合水凝胶纤维，实现对蛋白质的控制释放，掌握并深化关键技术，以拓展纳米复合水凝胶在生物医药领域的应用。

本项目设计合成了几类刺激响应型的微胶囊、微凝胶、纳米复合水凝胶和纳米复合水凝胶纤维，并研究了其药物控释特性。以巯基化透明质酸（HA-SH）和巯基化壳聚糖（CS-SH）为原料，结合层层自组装（LBL）和辣根过氧化物酶（HRP）酶促交联反应制备了二硫键交联的原位装载蛋白质的透明质酸/壳聚糖微胶囊。壳交联可有效地减少溶核过程中药物的暴释，调控巯基取代度可有效地调控壳层交联密度进而调控蛋白质药物的释放速率。以HRP/酪胺酶促交联HA-SH中巯基制备了二硫键交联的HA水凝胶，该体系具备凝胶化可控以及还原降解可控的双重优点。通过单独改变HRP和酪胺的浓度可调控凝胶化速率，可在20~50 min内实现快速凝胶化。结合反相乳液法制备了尺寸可控的二硫键交联的高载药量HA微凝胶，将其原位包埋于HA凝胶中制备了纳米复合水凝胶，以FITC-Dextran和盐酸阿霉素作为模型药物，通过调控凝胶前体浓度、水凝胶和微凝胶交联密度和模型药物的流体动力学直径均能有效降低突释效应。这种二硫键交联的药物载体在还原性介质下能够快速降解，实现还原响应的药物控释。将装载有布洛芬的pH和温度响应的壳层渗透性可调的聚（甲基丙烯酸-co-二乙烯基苯）-g-聚（N-异丙基丙烯酰胺）（P(MAA-co-DVB)-g-PNIPAm）微胶囊和牛血清蛋白（BSA）均匀分散于酪胺和巯基改性的透明质酸（HA-Tyr-SH）水溶液中， 通过HRP/H2O2酶促交联，制备了包埋载药微胶囊和BSA的HA纳米复合水凝胶。调控H2O2、HRP和HA-Tyr-SH浓度，能有效地调节水凝胶的凝胶化时间和交联密度，凝胶化时间从30 s到15 min。可同时实现对装载的布洛芬和BSA的控制释放，通过改变环境温度、pH和透明质酸酶的浓度可有效地调控药物释放动力学。结合静电纺丝技术和HRP酶促交联反应成功地制备了包埋载药微胶囊的纳米复合水凝胶纤维，纤维直径约为300 nm。本项目设计开发的蛋白质药物纳米复合水凝胶体系具有凝胶化速率快、可生物降解、高载药量、结构和药物释放速率可控等优点，可为新型蛋白质药物控释载体设计和开发提供创新思路和实验基础。