Objective: To investigate the in vitro eradicative effect of self-assembled azithromycin/rhamnolipid nanoparticles (AZI-RHL NPs) on P seudomonas aeruginosa ( P. aeruginosa) biofilm.
Methods: AZI-RHL NPs were prepared and characterized. The minimum inhibitory concentration (MIC) of AZI-RHL NPs on planktonic P. aeruginosa was measured by the broth microdilution method. The eradicative effect of AZI-RHL NPs on P. aeruginosa biofilm was evaluated via crystal violet staining and SYTO 9/PI live/dead staining. Fluorescence labeling was used to measure the eradicative effect of NPs on extracellular polymeric substances (EPS). In addition, crystal violet staining was performed to evaluate the inhibitory effect of AZI-RHL NPs on the adhesion of P. aeruginosa on human bronchial epithelial BEAS-2B cells. To investigate the ability of AZI-RHL NPs to penetrate mucus, the interaction between NPs and mucin was measured via particle size changes after co-incubation with mucin solution.
Results: The AZI-RHL NPs had a particle size of about 121 nm and were negatively charged on the surface, displaying a high encapsulation efficiency and a high drug loading capacity of 96.72% and 45.08% for AZI, respectively and 99.38% and 53.07% for RHL, respectively. The MIC of AZI-RHL NPs on planktonic P. aeruginosa was half of that of using AZI alone. AZI-RHL NPs displayed the capacity to effectively destroy the biofilm structure and remove the proteins and polysaccharides in EPS, eradicating biofilms in addition to reducing the survival rate of bacteria in the biofilm. AZI-RHL NPs were shown to have inhibited P. aeruginosa adhesion on BEAS-2B cells and prevented the residual bacteria from forming a new biofilm. There was no significant change in the particle size of NPs after co-incubation with mucin solution, indicating a weak interaction between NPs and mucin, and suggesting that NPs could penetrate the mucus and reach the P. aeruginosa infection sites.
Conclusion: AZI-RHL NPs were able to effectively enhance the removal of P. aeruginosa biofilm through a four-step strategy of biofilm eradication, including penetrating the mucus, disintegrating the biofilm structure, killing the bacteria dispersed from biofilm, and preventing the adhesion of residual bacteria. We hope that this study will provide a replicable common strategy for the treatment of refractory infections caused by P. aeruginosa and other types of biofilms.
目的: 探索阿奇霉素/鼠李糖脂自组装纳米粒（AZI-RHL NPs）在体外对铜绿假单胞菌（Pseudomonas aeruginosa, P. aeruginosa）生物被膜的清除作用。
方法: 制备并表征AZI-RHL NPs。采用微量肉汤稀释法测定AZI-RHL NPs对游离P. aeruginosa的最低抑菌浓度（MIC）。采用结晶紫染色法和SYTO 9/PI死活菌染色法评价AZI-RHL NPs对生物被膜的清除作用。采用荧光标记法探究NPs对生物被膜胞外聚合物（EPS）的清除作用。通过结晶紫染色法观察NPs对P. aeruginosa在人正常肺上皮细胞（BEAS-2B）黏附中的抑制作用。通过测定NPs与黏蛋白溶液孵育后的粒径变化考察其与黏蛋白的相互作用，以推测其黏液穿透能力。
结果: AZI-RHL NPs的粒径约为（121.4±6.0） nm，表面带负电，具有高包封率高载药量，对AZI的包封率为96.72%，载药量为45.08%，对RHL的包封率为99.38%，载药量为53.07%。AZI-RHL NPs对游离P. aeruginosa的MIC是AZI组的一半。AZI-RHL NPs可有效破坏生物被膜的结构，去除EPS中的蛋白质和多糖，在清除生物被膜的同时降低膜内细菌存活率，并抑制P. aeruginosa在BEAS-2B细胞上的黏附，避免残余细菌形成新的生物被膜。与黏蛋白溶液共孵育后NPs粒径无明显变化，提示NPs与黏蛋白相互作用较弱，推测其可穿透黏液到达P. aeruginosa感染部位。
结论: AZI-RHL NPs通过“穿透黏液-破坏生物被膜-杀死膜内细菌-抑制残余细菌再黏附”的“除膜四部曲”策略，有效提高了P. aeruginosa生物被膜的清除，为治疗包括P. aeruginosa在内的由生物被膜引起的顽固性感染提供了一种可复制的通用型策略。
Keywords: Antibiofilm; Azithromycin; Pseudomonas aeruginosa; Rhamnolipid; Self-assembled nanoparticles.
Copyright© by Editorial Board of Journal of Sichuan University (Medical Sciences).