一种基于天然 α-螺旋肽自组装的新型靶向抗菌纳米的设计及构建

This study will firstly prepare a fusion peptide of Ts-linker-EDED-IFCM. Ts (S-thanatin) is an active peptide of specific bactericidal property, and IFCM is a short peptide derived from the α-helical consensus motif of the intermediate filament protein which constitutes cytoskeleton together with the microtubule and microfilament. In the recombinant Ts-linker-EDED-IFCM, IFCM is fused after the C-terminal of Ts with a flexible GGGS linker, and a negatively-charged region composed of 4 acidic amino-acid (EDED) is inserted between the linker and IFCM. After trigged with the positively-charged antibiotic of minocycline (Mc) with benzene rings structure, Ts-linker-EDED-IFCM will self-assemble into a spherical nanoparticle which is characterized of bacterial specific targeting potential , pH-sensitive release, double bactericidal mechanism, and synchronous killing both drug-susceptible and -resistant bacteria. The prepared nanoparticles will be characterized, and the acute- and subacute- toxicity has to be investigated. The in vitro antimicrobial potential could be evaluated by MICs and MBCs against standard strains and clinic isolates. The in vivo antimicrobial potential could be evaluated with a septic mouse model caused by different bacteria including drug-susceptible and -resistant bacteria. The in vivo structure-dosage-effect relationship and differences between drug-susceptible and -resistant bacteria or between Gram+ and Gram- bacteria will be studied as well. In conclusion, we will explore the feasibility to construct a novel self-assembly nanoparticle against bacteria based on the α-helical peptide. This research will provide theoretical basis and practical evidences for pre-clinic research and other nanoparticles preparation using chemicals or antibiotics beside the model drug minocycline used in this study.

本项目拟以细胞骨架成分-中间丝状蛋白α-螺旋保守基序肽（IFCM）为材料，N端融合具有细菌靶向及杀菌作用的生物活性肽S-thanatin （Ts），中间插入柔性连接片段（GGGS）及4个酸性氨基酸（EDED）组成的负电荷载药区，构成靶向融合肽Ts-linker-EDED-IFCM；用具有多苯环结构的阳离子抗生素米诺环素（Mc）激发，使之自组装形成一种具有细菌靶向、pH敏感释放、双重杀菌机制、耐药与非耐药菌同步杀灭的新型抗菌纳米；对其进行表征及急性/亚急性毒性试验研究；用标准菌株及临床分离多重耐药菌进行体外杀菌试验；建立革兰氏阳性和阴性标准菌株及耐药菌腹膜炎败血症模型，考察靶向载药纳米在体内对耐药和非耐药菌的作用效果，以获得结构-剂量-效应变化的多点数据，为靶向纳米抗菌药物的临床前研究打下基础；同时，也为除模式药物以外的其它种类抗生素或化合物自组装纳米的构建提供了理论和实验依据。

细菌的耐药性在全球范围内正以惊人的速度增加，寻找并设计克服多重耐药机制的新型化合物成为当前的研究热点。本项目合成了抗菌肽TS，发现其具有广泛的体内外抑菌活性，特别是对革兰氏阴性菌的抑菌活性最强。进一步深入研究了其抗菌机制，发现没有溶血活性的TS通过取代二价阳离子和竞争性结合脂多糖，破坏了细菌外层细胞膜的完整性。此外，抗菌肽TS通过与细菌DNA相互作用，从内部分解细菌，从而抑制和杀死大肠杆菌。抗菌肽TS的多重抗菌机制可能不易诱导细菌耐药，提示其在未来可作为抗细菌感染的抗菌药物使用。本研究进一步设计合成了系列小肽，包括Ts -linker -EDED-IFCM、PTS-linker-MT、P1c、IFP、P60、P51、P41、P45等小肽，用不同的药物激发，发现可激发不同肽的自组装，初步发现了组装疏水药物和亲水药物α-螺旋肽的结构特征，即必须具有同源二聚体或三聚体的性质。利用人源α-螺旋肽自组装构建了一种联合多种治疗为一体的自组装多肽纳米，并评估了其治疗效果。本项目的实施为对于促进新肽的开发利用，临床微生物感染的治疗，支持新抗生素的开发具有重要意义。同时，本项目也为靶向纳米抗菌药物的临床前研究打下基础；为除模式药物以外的其它种类抗生素或化合物自组装纳米的构建提供了理论和实验依据。