新型靶向双功能聚酰胺-胺树枝状抗菌分子的设计合成及其抗菌作用研究

The emergence and wide spread of multidrug resistant gram negative (G-) bacteria in the world has become a serious threat to human beings. Development of new antibiotic drugs, especially those that do not induce antibiotic resistance among bacteria, is a new trend for novel antibiotic agent research. Our previous results showed that low generation amino-terminated poly(amidoamine) dendrimers (PAMAM) had strong bactericidal effects against multidrug resistant G- bacteria strains, and had unique advantages of novel structures without inducing resistance. However, PAMAM exhibited cytotoxicity to eukaryotic cells at higher concentration and the cytotoxicity was positively correlated with the number of surface amino group of PAMAM. Therefore, one of the most critical issues for optimizing the antibacterial property of PAMAM is to improve the selectivity of the antibacterial effects of PAMAM and reduce the cytotoxicity of PAMAM. In this project, we plan to design and synthesis novel PAMAM dendrimers by conjugating with carboxylated LED209, which could specifically bind with QseC receptor of G- bacteria and significantly inhibit the expression of virulence genes of G- pathogens. The synthesized PAMAM dendrimers possess bifunctional properties which can target G- bacteria and inhibit bacteria virulence. This research includes synthesis of a series of PAMAM dendrimers modified with LED209 at different ratios by covalent bond, evaluation of their targeting effects, bacterial virulence, the antibacterial activity in vitro and in vivo, and exploration of the underlying antibacterial mechanisms. This project aims to clarify the design rules and parameters for targeting antimicrobial agents based on PAMAM dendrimers and reveal their antibacterial effects and mechanisms, which will provide a new train of thought and tactics for the research of novel antimicrobial agents.

多重耐药革兰氏阴性（G-）菌的不断出现和蔓延，已成为严重威胁人类健康的难题，研制不易诱导细菌耐药的新型抗菌药物是未来的研究方向。我们前期研究发现，氨基末端聚酰胺-胺树枝状分子（PAMAM）能有效杀灭多种多重耐药G-菌，且具有不易诱导耐药的突出优点。但该分子在高浓度时对真核细胞呈现一定毒性，且毒性大小与其末端氨基数目呈正比。因此如何降低PAMAM的细胞毒性，提高其对细菌作用的选择性，是将其优化为理想抗菌分子的关键问题。本课题拟采用共价偶联的设计思路，将能特异性识别G-菌群体感受器QseC的配体LED209与PAMAM末端氨基偶联，合成出细胞毒性低、细菌选择性高的抗菌新分子，使其具备靶向识别G-菌和抑制细菌毒力的双重作用；并对该分子展开细菌靶向、细菌毒力、细胞毒性、体内外抗菌活性及作用机理等方面研究，旨在阐明靶向双功能树枝状抗菌分子的设计规律、抗菌作用及机理，为新型抗感染药物的研究探索新路径。

背景：多重耐药革兰氏阴性菌（MDR-GNB）的不断出现和蔓延，已成为严重威胁人类健康的难题。我们前期研究发现，氨基末端聚酰胺-胺树枝状分子（PAMAM）能有效杀灭多种MDR-GNB，且具有不易诱导耐药的突出优点。但该分子在高浓度时对真核细胞呈现一定毒性，且毒性大小与其末端氨基数目呈正比。因此如何降低PAMAM的细胞毒性，提高其对细菌作用的选择性，是将其优化为理想抗菌分子的关键问题。内容：本课题采用共价偶联的设计思路，将能特异性识别GNB群体感受器QseC的配体LED209与PAMAM末端氨基偶联，合成出细胞毒性低、细菌选择性高的抗菌新分子，使其具备靶向识别GNB和抑制细菌毒力的双重作用；并对该分子展开细菌靶向、细菌毒力、细胞毒性、体内外抗菌活性及作用机理等方面的研究。结果：将间位LED209羧基衍生物与不同代数PAMAM（G2、G3和G4）末端氨基以不同比例（n=2、4、8）共价连接，成功设计合成出九种新型分子（LPDs）；MTT结果发现，末端氨基修饰后PAMAM细胞毒性显著降低，修饰比例越高，毒性越低；最低抑菌浓度（MIC）结果显示，末端氨基修饰后，PAMAM对不同种属GNB的MIC值升高，修饰比例越高，MIC值升高越显著；综合筛选结果优选G3 LPD （n=2）作为后续研究对象。形态学表征显示，G3 PAMAM 和G3 LPD 在水溶液中粒径分别为20 nm和5 nm，Zeta电位分别为−3.48 ± 0.78和11.51 ± 3.19 mV，提示G3 LPD分散性和稳定性均增加。PCR结果显示，G3 LPD（n=2）可显著抑制EHEC致病基因在mRNA水平的表达，与对照组对比有统计学差异。靶向验证结果提示，与G3 PAMAM-FITC相比，G3 LPD-FITC与细菌EHEC具有更高的选择性；耐药实验显示，不容易对G3 LPD产生耐药。动物实验结果发现用G3 LPD预处理5 h 的S. typhimurium感染组小鼠，生存时间长于未处理和G3 PAMAM处理组。透射电镜显示，与对照组相比，EHEC与G3 PAMAM或 G3 LPD共孵育300 min后，EHEC细胞质浓缩，胞膜上空泡形成。结论：G3 LPD（n=2）可选择性识别GNB，通过抑制QseC受体降低细菌毒力的同时，保留相当的抗菌活性，是一个毒性小且不易诱导耐药的双功能靶向抗菌候选分子。

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