抗菌肽的控制释放及稳定性研究

Antimicrobial peptides constitute a very important component of the innate immune system. They are consisted of amino acid residues from 6 to 100 and have amphiphilic structure. Antimicrobial peptides have a good prospect because they have broad antimicrobial activity, which have a significant role in the killing in multi-drug resistant bacteria, and they are not easy to produce drug resistance. But the clinical application of antimicrobial peptides is also facing some problems such as poor stability, susceptible to protease degradation and production. In the project, several new amphiphilic materials are designed to load antimicrobial peptides, and the peptide loading in linear materials is compared with dobby materials. The microsphere loading antimicrobial peptides is prepared with emulsion method (O/W or W1/O/W2) and Layer-by-Laye (LBL) assembly, and the structure and particle size of microsphere are studied with SEM and laser light scattering. The duration of antimicrobial peptides sustained release formulation is adjusted by changing the structure, composition and molecular weight of carrier material. The stability of antimicrobial peptides is studied by in vitro release and in vivo release. This project will provide a new way to solve the stability and sustained release of antimicrobial peptides. And the success of the project will broaden the scope of application of antimicrobial peptides in clinical medicine, which has the large theoretical and practical significance.

抗菌肽是生物内在免疫的重要组成部分，一般由6-100个氨基酸残基组成，具有两亲性结构。抗菌肽具有广谱抗菌活性，尤其是其对多药物耐受的细菌具有明显的杀灭作用，且不易产生耐药性，具有良好的应用前景。但抗菌肽的临床应用也面临着一些问题如稳定性差、易被蛋白酶降解、量产困难等。项目通过设计几个新型的两亲性新材料来对抗菌肽进行负载研究，并对比线性材料及多臂材料对多肽负载量的影响。分别用乳液法(O/W或W1/O/W2)及Layer-by-layer(LbL)自组装方法制备微球负载抗菌肽，并利用SEM、激光光散射等手段研究微球的结构和粒径。通过改变载体材料的结构、组成、分子量等来调节抗菌肽缓释剂型的持续作用时间，通过体外释放及动物体内的释放来研究抗菌肽的稳定性。本项目的研究将为解决抗菌肽的稳定性及持续释放提供一个新的途径，项目的成功将拓宽抗菌肽在临床医学上的应用范围，具有较大的理论及实际意义。

抗菌肽具有广谱的抗菌活性、较低的抑菌浓度而且具有不易引发细菌耐药性的独特杀菌机制等优良特性，被认为是最有可能代替抗生素的候选药物分子之一。为了解决抗菌肽体内半衰期短、稳定性差的问题，不同的可生物降解高分子材料被用来制备载抗菌肽微球，并对其进行控制释放研究。.用具有良好生物相容性和可生物降解的聚乳酸基材料制备了不同分子量、不同嵌段和比例的mPEG–PLGA、四臂PEG–PLGA 、poly(LA-co-MA) 和4-arm PEG-PMA材料，通过复乳溶剂挥发法（W/O/W）对抗菌肽BF-30进行包埋，制备得到不同的载药微球。通过扫描电子显微镜（SEM）来观察微球的形貌，微球具有完整的球形和光滑的表面。通过萃取法，用高效液相色谱法（HPLC）测定poly(LA-co-MA) 和4-arm PEG-PMA微球的包埋率为92.47 ± 1.21%、81.26±1.41%。对载抗菌肽微球进行了体外释放和降解实验研究，结果表明，载BF-30的微球能持续稳定释放25天，并且释放过程中无明显的突释效应。在持续降解50天内，微球的形貌发生改变，降解介质的 pH、微球的分子量(Mn)和重量损失也都发生改变。为了研究分析微球释放液中多肽的稳定性，结合HPLC、圆儿色谱（CD）以及抑菌实验进行分析，结果表明释放液中多肽的极性和二级结构得到了保持，其仍具有生物活性。为了检测微球的生物效应，对载抗菌肽微球进行了溶血性实验和细胞毒性实验，结果表明，在一定的微球浓度范围内，微球能达到对细胞无毒性且不引起溶血效应的要求。释放肽和天然肽，以释放介质PBS为空白对照组，对沙门氏菌、志贺氏菌和金黄色葡萄球菌的抑菌圈实验证明释放肽没有失去抗菌活性。项目的研究开展为解决抗菌肽的稳定性及持续释放提供一个新的途径，拓宽了抗菌肽在临床医学上的应用范围，具有较大的理论及实际意义。