随着细菌对各种一线抗生素耐药性的产生，开发无毒高效的抗菌药物，辅助制定多层面的抗菌策略成为当务之急。本文通过自组装制备了由小檗碱（berberine，BBR）和绿原酸（chlorogenic acid，CGA）组成的超分子中药纳米复合物（berberine and chlorogenic acid nanocomposition，BBR/CGA），并对其体内外抑制金黄色葡萄球菌（Staphylococcus aureus，S. aureus）和耐多药金黄色葡萄球菌（multidrug-resistant Staphylococcus aureus，MRSA）性能进行了研究。

1 超分子中药纳米复合物BBR/CGA的制备表征及性能研究

本章试验采用透析沉淀法制备了超分子中药纳米复合物BBR/CGA并对其物理、化学及生物性能进行了研究。研究首先利用分子动力学模拟了BBR和CGA单体的自组装过程；其次以平均水合粒径、多分散指数（polydispersity，PDI）为评价指标筛选用于合成BBR/CGA的最佳物料配比；随后通过紫外可见吸收光谱（ultraviolet visible absorption spectrum，UV-vis）、荧光发射光谱、傅里叶变换红外光谱（fourier transform infrared spectroscopy，FT-IR）、透射电镜（transmission electron microscope，TEM）、Zeta电位、电喷雾离子化-高分辨质谱（electrospray ionization-high resolution mass spectrometric，ESI-HRMS）等技术对BBR/CGA进行表征；进一步采用UV-vis研究了BBR/CGA的药物体外释放性能；利用CCK-8法评估不同浓度BBR/CGA对小鼠乳腺上皮细胞毒性。结果显示：分子动力学模拟预测BBR和CGA单体在50 ns时可发生自组装现象；在摩尔比为1:1时，制备的BBR/CGA平均水合粒径和PDI最小，分别为358.6±2.4 nm和0.28±0.01；各种表征数据也证实了BBR/CGA的成功制备；BBR/CGA在24 h内有53±0.78%的BBR释放，稳定性好、缓释效果优良；在0-12 μM浓度范围内，BBR/CGA对小鼠乳腺上皮细胞抑制率小于5%，有良好的生物相容性。以上结果表明：试验成功制备了具有优良性能的超分子中药纳米复合物BBR/CGA，有较好的生物安全性。

2 超分子中药纳米复合物BBR/CGA体外抑菌作用的研究

本章试验以S. aureus和MRSA为研究对象，探究了超分子中药纳米复合物BBR/CGA的体外抑菌性能。研究通过微量肉汤稀释法、琼脂平板涂覆法和动态抗菌法测定了BBR/CGA对S. aureus及MRSA的抑菌效果；采用经典结晶紫染色法、银染法评价了BBR/CGA抑制生物被膜形成的性能及MTT法、吖啶橙染色法评价其破坏生物被膜的性能；利用碘化丙啶（propidium iodide，PI）染色法结合流式细胞术分析BBR/CGA对细菌质膜通透性的影响；进一步利用RT-PCR技术研究BBR/CGA对细菌生物被膜形成、细胞质膜形成、营养摄取、核苷酸代谢等基因表达的影响；通过生长曲线法探究BBR/CGA在恢复MRSA对β-内酰胺类抗生素敏感性中的作用。试验结果表明：当浓度达到1.5 μM时，BBR/CGA对S. aureus和MRSA的抑制效果最佳，显著高于BBR、CGA单体和苯唑西林、氨苄西林两种抗生素（P<0.05）；与BBR、CGA单体相比，BBR/CGA不仅能显著抑制S. aureus和MRSA生物被膜形成（P<0.05），还可有效破坏成熟的生物被膜且清除率分别达到了75.73±2.55%和66.60±0.05%；与MRSA组相比，BBR/CGA破坏S. aureus细菌质膜能力更强（高出约1.6倍）；亚抑菌浓度的BBR/CGA不仅能显著下调与生物被膜形成相关基因如agrA、hlaC、cidA、icaA的表达水平，还能显著下调与病原菌质膜形成、营养摄取等功能密切相关的相关基因如hrtA、hrtB、rpmG3、deoD、secE等的表达水平（P＜0.05）；亚抑菌浓度的BBR/CGA与氨苄青霉素联合有明显的协同抑制作用，可以重新恢复氨苄青霉素对MRSA的抑菌能力，这可能与BBR/CGA显著下调MRSA耐药相关基因erm、mecA、pbpB的表达水平有关（P＜0.05）。综上所述，BBR/CGA不仅具有良好的抑菌性能，还在消除MRSA的β-内酰胺耐药性方面发挥重要作用。

3 超分子中药纳米复合物BBR/CGA的安全性评价及体内抑菌研究

本章试验以伤口分别感染S. aureus和MRSA的小鼠为研究对象，探究了超分子中药纳米复合物BBR/CGA的体内抗菌性能。研究首先通过体外红细胞溶血试验和体内安全性试验评估了BBR/CGA的生物相容性；其次成功构建S. aureus和MRSA感染小鼠皮肤伤口模型，观察BBR/CGA改善创面愈合的效果并利用H&E染色法评估了伤口愈合过程；对皮肤伤口组织载菌量计数验证了BBR/CGA的体内抗菌效果；最后利用全自动血常规分析仪对小鼠全血进行血常规分析。试验结果表明：BBR/CGA在0-12 μM浓度范围内无明显溶血现象；与对照组相比，BBR/CGA处理组对小鼠心、肝、脾和肾四个重要的器官无明显伤害，表明BBR/CGA具有良好的生物相容性且在体内无明显毒副作用。与PBS处理组相比，BBR/CGA在加速创面修复和皮肤再生方面具有显著优势，能加快伤口炎症反应期的消退并促进皮肤创面愈合，创面组织菌落计数（colony forming units，CFU）显著低于PBS处理组（P<0.05）。研究结果证实，在更为复杂的体内病理条件下，BBR/CGA仍具有良好的生物相容性和抗菌活性，其治疗感染伤口愈合速度快、效果好。

Along with bacteria resistant to various first-line antibiotics, developing nontoxic, efficient antibiotics and formulating multidimensional strategies to combat bacterial infections are urgent. In this paper, supramolecular Chinese medicine berberine and chlorogenic acid nanocompositions (BBR/CGA) were prepared by the self-assembly of Chinese herb medicine monomer berberine (BBR) and chlorogenic acid (CGA), subsequently the inhibitory effect BBR/CGA against Staphylococcus aureus (S. aureus) and multidrug-resistant Staphylococcus aureus (MRSA) in vitro and in vivo were studied.

1 Preparation, characterization and properties study of supramolecular traditional Chinese medicine nanocomposites BBR/CGA

In this chapter, the supramolecular traditional Chinese medicine nanocomposites BBR/CGA were prepared by dialysis precipitation method, and its physical, chemical and biological properties were studied. First, molecular dynamics simulation was used to understand the self-assembly process of BBR and CGA monomers. The preparation ratio of BBR/CGA was optimized by taking particle size and polydispersity (PDI) as evaluation indexes. Then, BBR/CGA were characterized by ultraviolet visible absorption spectrum (UV-vis), fluorescence emission spectrum, fourier transform infrared spectroscopy (FT-IR), transmission electron microscope (TEM), zeta potential, and electrospray ionization-high resolution mass spectrometry (ESI-HRMS). At last, the drug release properties of BBR/CGA in vitro were studied by UV-vis and the cytotoxicity of BBR/CGA on mouse mammary epithelial cells was evaluated by CCK-8 method. Molecular dynamics simulation result predicted that BBR and CGA monomers would self-assemble at 50 ns. When the molar ratio was 1:1, the particle size and PDI of BBR/CGA were the best, which were 358.6±2.4 nm and 0.28±0.01, respectively. Various characterization data also confirmed the successful preparation of BBR/CGA and only 53±0.78% of BBR was released from BBR/CGA at 24 h, suggesting that BBR/CGA maintained good stability and excellent sustained-release effect. BBR/CGA had no negative effects on mouse mammary epithelial cells in the range of 0-12 μM. The above results showed that the supramolecular traditional Chinese medicine nanocomposites BBR/CGA with excellent properties had been successfully prepared.

2 Study on antibacterial effect of supramolecular traditional Chinese medicine nanocomposites BBR/CGA in vitro

In order to explore the antibacterial properties of supramolecular traditional Chinese medicine nanocomposites BBR/CGA, S. aureus and MRSA were used as the research objects. Firstly, the antibacterial effect of BBR/CGA on S. aureus and MRSA was determined by micro broth dilution, agar plate coating and dynamic antibacterial method. Secondly, the classical crystal violet staining and silver staining method were used to evaluate the inhibition performance of BBR/CGA on biofilm formation, and MTT and acridine orange staining method were used to evaluate its destruction performance of biofilm. The effect of BBR/CGA on bacterial plasma membrane permeability was analyzed by propidium iodide (PI) staining combined with flow cytometry. Further, RT-PCR was used to study the effects of BBR/CGA on gene expression such as biofilm formation, bacterial plasma membrane formation, nutrient uptake and nucleotide metabolism. Finally, the role of BBR/CGA in restoring the sensitivity of MRSA to β-lactam antibiotics was studied by growth curve method, and its potential drug resistance regulation mechanism was explored. The results showed that when the concentration reached 1.5 μM, BBR/CGA had the best inhibitory effect on S. aureus and MRSA, which was higher than that of BBR, CGA, oxacillin and ampicillin (P<0.05). Compared with BBR and CGA monomers, BBR/CGA could not only significantly inhibit the biofilm formation of S. aureus and MRSA (P<0.05), but also destroy the biofilm more effectively, and the clearance rates were 75.73±2.55% and 66.60±0.05%, respectively. Compared with MRSA group, BBR/CGA had stronger ability to destroy the plasma membrane of S. aureus (about 1.6 times higher). Meanwhile, BBR/CGA with sub-inhibitory concentration can not only significantly down regulate the expression level of genes related to biofilm formation, such as agrA, hlaC, cidA, icaA , but also significantly down regulate the expression level of genes closely related to pathogen plasma membrane formation and nutrient uptake, such as hrtA, hrtB, rpmG3, deoD, secE (P<0.05). In addition, the combination of BBR/CGA with sub-inhibitory concentration and ampicillin had obvious synergistic inhibitory effect, which can restore the antibacterial ability of ampicillin against MRSA. It may be related to BBR/CGA significantly down regulating the expression levels of MRSA resistance related genes erm, mecA, pbpB (P<0.05). To sum up, BBR/CGA not only had good anti-biofilm and antibacterial properties, but also played an important role in abrogation of β-lactam resistance of MRSA.

3 Study on the safety evaluation and in vivo antibacterial activity of supramolecular traditional Chinese medicine nanocomposites BBR/CGA 

In order to explore the antibacterial properties of supramolecular traditional Chinese medicine nanocomposites BBR/CGA in vivo, mice with wounds infected with S. aureus and MRSA were used as the research objects. Firstly, the biocompatibility of BBR/CGA was clarified by in vitro hemolysis and in vivo safety evaluation test. Then, the mice skin wound models infected with S. aureus and MRSA were successfully constructed, and the effect of BBR/CGA on wound healing was observed and evaluated by H&E staining. Subsequently, the bacterial load of skin wound tissue was counted to verify the antibacterial effect of BBR/CGA in vivo. Finally, the whole blood of mice was analyzed by automatic blood routine analyzer. The results showed that BBR/CGA had no obvious hemolysis in the concentration range of 0-12 μM. Compared with the control group, BBR/CGA treatment did not cause significant damage to four important organs, heart, liver, spleen and kidney, indicating that BBR/CGA had good biocompatibility and no side effects in vivo. Compared with PBS treatment group, BBR/CGA had significant advantages in accelerating wound repair and skin regeneration, which can accelerate the regression of wound inflammatory response period and promote skin wound healing. Meanwhile, the colony forming units (CFU) of wound tissue treated with BBR/CGA was significantly lower than that treated with PBS (P<0.05), suggesting that the rapid healing speed and short cycle of infected wound treated with BBR/CGA may be due to its antibacterial effect. Obviously, even under more complex in vivo pathological conditions, BBR/CGA still has good antibacterial activity and good biocompatibility.