两栖类皮肤抗菌肽chensinin-1b自组装纳米结构的构建、体内稳定性及抗菌作用机制研究

Antimicrobial peptide chensinin-1b, a peptide with a random coil conformation derived from amphibian skin secretions, shows broad-spectrum antimicrobial activities against pathogenic microbes and remains benign to mammalian cells. The development of peptide drug based on chensinin-1b is limited by its poor stability and short circulation time in vivo. Based on previous research results, the self-assembly approach is considered to be effective to improve the stability and therapeutic efficacy of the peptide in vivo. In this study, to built self-assemblies of antimicrobial peptide chensinin-1b, the sequence and structure of chensinin-1b are re-constructured and optimized from two aspects: the conjugation of aliphatic acid with different lengths or other bulky hydrophobic group, such as Ala, Leu, Val and etc, to C- and N-terminus of chensinin-1b to built peptide amphiphiles; and/or the intrinsic sequence mutation or modification of chensinin-1b, which result the amphiphilic pattern and secondary structure content of chensinin-1b are changed. The modulated self-assembly process to form stable nanostructure of the antimicrobial peptide chensinin-1b with the random coil conformation is investigated. The antimicrobial activity, serum stability, and the ability to control the release of peptide drug of self-assemblies are examined. Meanwhile, the relationship between peptides nanostructure, bioactivity and stability are also determined. The structure and morphology of bacterial cell membrane affected by the peptide nanostructure are examined, and subsequently, the bactericidal way, target and action mode on chensinin-1b nanostructure are also studied by the changes of cytoplasmic membrane, membrane receptor and etc. In addition, the experimental mouse model infected by pathogenic microbes is built to examine the stability and therapeutic efficacy of chensinin-1b nanostructure in vivo. In this proposal, we hope to demonstrate the self-assembly approach applied to antimicrobial peptide chensinin-1b for the development of carrier-free drug delivery system and present an additional stimulus to consideration of antimicrobial peptides with special conformation as new therapeutic agents.

两栖类皮肤抗菌肽chensinin-1b具有良好的广谱抗菌活性，在膜环境下呈现任意伸展结构，其在体内易于被蛋白酶水解以及循环半衰期短影响了其作为抗菌药物的临床使用。本研究通过利用疏水性基团修饰chensinin-1b的N端或C端构建类表面活性剂分子或优化并改造chensinin-1b的内在序列，使其具有不同的两亲性模式，进而构建chensinin-1b自组装体以期解决抗菌肽的体内稳定性问题并控制多肽药物的体内释放。研究此类非经典结构抗菌肽自组装形成稳定纳米结构的条件，考察其抗菌活性并研究肽纳米结构、活性和稳定性这三者之间的关系。从自组装纳米颗粒对细菌细胞膜结构、形态等方面的影响系统研究此类纳米颗粒杀死细菌的主要途径、作用靶点和机制；构建病原微生物感染的小鼠模型，考察其体内稳定性及杀菌活性，为开发此类两栖类皮肤抗菌肽纳米结构作为新型无载体抗菌药物的可行性提供理论依据。

两栖类皮肤抗菌肽chensinin-1b显示了较好的广谱抗菌活性，但易被蛋白酶降解以及循环半衰期短影响了其临床使用。本研究中，我们对chensinin-1b类似物抗菌活性及机制研究的工作基础上，对此类肽的抗癌活性及机制、质粒转染性能和靶向递送性能进行了研究。疏水性强的脂肽PA-C1b作用于MCF-7细胞比chensinin-1b表现出更强的抗癌活性，差异在于chensinin-1b通过破坏细胞膜的方式杀死癌细胞；PA-C1b不破坏细胞膜，通过诱导细胞凋亡杀死癌细胞。PA-C1b由于聚集状态发生变化其在蛋白酶存在下的稳定性得到了较大的提升。抗癌机制研究表明chensinin-1b及其相关脂肽具有穿膜肽的功能，因此我们构建了抗菌肽/DNA复合物，使其能够转染治疗基因到癌细胞内表达后杀死癌细胞，并协同利用肽自身杀死癌细胞。为了考察抗菌肽的转染性能，选择chensinin-1b及其相关脂肽作为质粒载体，以空载体eGFP质粒作为报告基因，转染实验结果表明chensinin-1b及其系列脂肽与质粒eGFP的质量比在1:8时显示了较好的转染性能，且转染能力遵循PA-C1b>LA-C1b>OA-C1b>chensinin-1b这一顺序。利用p53基因作为治疗基因，PA-C1b作为载体，实验结果表明该基因能够被转染到肿瘤细胞内经表达后可实现杀死肿瘤细胞的目的，并发现在荷瘤小鼠模型中PA-C1b/eGFP复合物在肿瘤部位的转染效率要明显高于单独质粒转染的情况。进一步我们构建基于多孔纳米材料的靶向递送系统 MSN@Cy7-PA-C1b@FA-GO 用于抗菌肽PA-C1b的靶向递送，包括抗菌肽在多孔材料中的吸附，利用氧化石墨烯对吸附后含PA-C1b的纳米材料进行封堵，利用叶酸修饰材料表面构建靶向性。结果表明MCF-7细胞能够摄取MSN@Cy7-PA-C1b@FA-GO材料并能够在近红外照射后释放出Cy7-PA-C1b，且均分布于细胞质和细胞核中。在荷瘤小鼠模型中，腹腔注射MSN@Cy7-PA-C1b@FA-GO后在不同的时间点检测Cy7-PA-C1b荧光信号发现，随着时间的增加，抗菌肽可靶向于肿瘤部位并发生聚集，发挥抗癌作用。基于对chensinin-1b序列的设计改造产生多条活性序列，改变其聚集状态，考察相关生物活性，为后续两栖类皮肤抗菌肽作为新型抗生素和抗癌药物提供理论基础。