制备抗菌肽LL37修饰的金纳米颗粒及其体外转染和抗菌实验研究  

Preparation of Gold Nanoparticles Modified by Antimicrobial Peptide LL37 and Its in Vitro Transfection and Antibacterial Experiments

作　　者：王淞 陈宏鑫 徐凯 吴娟娟 吴高贤 杨斌 WANG Song;CHEN Hongxin;XU Kai;WU J uanjuan;WU Gaorian;YANG Bin(Department of Burn and Plastic Surgery,General Hospital of Central Theater Command,Wuhan Hubei 430070,China)

机构地区：[1]中部战区总医院烧伤整形科,湖北武汉430070 [2]中部战区总医院保健科,湖北武汉430070

出　　处：《联勤军事医学》2023年第9期725-730,共6页Military Medicine of Joint Logistics

基　　金：国家自然科学基金青年项目(82102340);中部战区总医院博士后项目(BSH022)。

摘　　要：目的 制备抗菌肽LL37修饰的金纳米颗粒(gold nanoparticles, AuNPs)(AuNPs@LL37),并研究其理化性质的表征参数、体外转染效率及其抗菌能力。方法 通过化学还原法和配体交换法制备AuNPs@LL37,紫外分光光度仪检测吸收光谱;纳米粒度仪测定其水合粒径、表面电位(Zeta电位);透射电镜观察其形态特征;超敏型二喹啉甲酸(micro bicinchoninic acid assay, micro BCA)蛋白定量试剂盒测量其表面固定的多肽量;荧光猝灭排除实验检测AuNPs@LL37与质粒DNA的结合情况;细胞计数试剂盒(cell counting kit 8,CCK-8)实验检测AuNPs@LL37的细胞毒性;体外转染实验检测AuNPs@LL37的转染能力;体外抗菌实验检测AuNPs@LL37的抗菌能力。结果 制备得到带正电荷[Zeta电位为(35.40±1.20)mV、直径为(7.10±1.60)nm],表面固定LL37多肽的AuNPs@LL37。当AuNPs@LL37∶pDNA=5∶1时,AuNPs@LL37/pDNA Zeta电位达到正电荷饱和;荧光猝灭排除实验表明,AuNPs@LL37与DNA有较强的结合能力;体外转染实验表明,AuNPs@LL37能够介导增强型绿色荧光蛋白质粒-血管内皮生长因子165(plasmid enhanced green fluorescent protein-vascular endothelial growth factor 165,pEGFP-VEGF165)转染细胞并在细胞中表达绿色荧光蛋白,其转染率可达(89.52±7.36)%;体外抗菌实验表明,AuNPs@LL37和AuNPs@LL37/pDNA均具有杀菌作用。结论 AuNPs@LL37是一种新型的功能化纳米颗粒,既能够作为基因载体又具有抗菌能力。Objective To prepare gold nanoparticles modified by antimicrobial peptide LL37(AuNPs@LL37), and study the characterization parameters of their physicochemical properties, in vitro transfection efficiency and antibacterial ability. Methods AuNPs@LL37 was prepared by chemical reduction method and ligand exchange method, the absorption spectrum was detected by ultraviolet spectrophotometer;the hydrated particle size and surface potential(Zeta potential) were measured by nanoparticle particle size analyzer;the morphological characteristics were observed by transmission electron microscopy;the amount of peptides fixed on the surface was measured by micro bicinchoninic acid assay(micro BCA) protein quantification kit;fluorescence quenching exclusion experiments were performed to detect the combination of AuNPs@LL37 and plasmid DNA;the cytotoxicity of AuNPs@LL37 was detected by cell counting kit 8(CCK-8) assay;the transfection ability of AuNPs@LL37 was detected by transfection experiment in vitro;the antibacterial ability of AuNPs@LL37 was detected by in vitro antibacterial assay. Results The positive charge [Zeta potential was(35.40±1.20) mV, diameter was(7.10±1.60)nm] and LL37 polypeptide was fixed on the surface of AuNPs@LL37. When AuNPs@LL37∶pDNA=5∶1, AuNPs@LL37/pDNA Zeta potential reached positive charge saturation, in vitro transfection experiments showed that the nanoparticles could mediate the transfection of plasmid enhanced green fluorescent protein-vascular endothelial growth factor 165(pEGFP-VEGF165) and express a green fluorescent protein in cells, with the transfection rate reaching(89.52±7.36)%;in vitro antibacterial experiments showed that, AuNPs@LL37 and AuNPs@LL37/pDNA both had bactericidal effects. Conclusion AuNPs@LL37 is a new type of functional nanoparticle, which can be used as a gene carrier and has an antibacterial ability.

关 键 词：抗菌肽LL37 金纳米颗粒 细胞转染 抗菌能力 

分 类 号：R945[医药卫生—微生物与生化药学] R96[医药卫生—药剂学]