溶菌酶修饰的庆大霉素脂质体对抗细菌生物膜的研究

发布时间：2018-08-16 16:01

【摘要】：细菌生物膜引发的慢性感染是临床上棘手的问题之一。由于细菌生物膜对抗生素具有高度耐药性,而在缺乏新抗生素或者高效抗菌策略的前提下,只能加大现有抗生素的使用剂量、延长给药时间。长此以往,容易导致耐药性和耐药菌株的产生,同时,抗生素的毒副作用也会增加。因此,通过改良抗生素的现有给药方式,降低抗生素的使用剂量,提高作用效果,可以为细菌生物膜感染的治疗提供更好的解决办法。本文采用生物相容性好的带正电荷的溶菌酶修饰带负电荷的脂质体,提高了脂质体的稳定性,增强了对细菌生物膜的亲和力,提升了庆大霉素对生物膜的作用效果。具体研究内容如下:采用1,2-棕榈酰磷脂酰甘油(DPPG)和二棕榈酰磷脂酰胆碱(DPPC),通过挤压法制备了庆大霉素脂质体(LG),经磷钨酸钠沉淀法测定可知庆大霉素的包封率为0.6%。其次利用静电作用将带正电荷的溶菌酶吸附到负电荷的脂质体表面得到溶菌酶修饰的庆大霉素脂质体(LLG)。根据表面Zeta电位由LG的负值向LLG的正值转变可知,溶菌酶成功吸附到了脂质体表面。通过定时检测48 h内LG及LLG的粒径变化可知,溶菌酶的引入可以稳定脂质体。利用紫外分光光度计实时监测72 h内LG及LLG体外释放庆大霉素含量发现,LG在前48 h内,庆大霉素释放量超过了50%,而在72 h时70%以上的药物已经释放。相反地,在72 h时也仅有14%的药物从LLG中释放出来。由此也证明了溶菌酶吸附到庆大霉素脂质体表面可以有效改善脂质体的稳定性。通过体外构建铜绿假单胞杆菌(革兰氏阴性菌)和金黄色葡萄球菌(革兰氏阳性菌)生物膜,利用结晶紫染色法和MTT实验评价发现,与单独庆大霉素相比,LLG在破坏已形成的细菌生物膜和抑制细菌生物膜形成时更为有效。通过荧光显微镜及扫描电镜观察细菌生物膜发现,LLG不仅破坏了生物膜的形态及内部结构,也影响了单个细菌的形态结构。通过荧光定量罗丹明B与细菌生物膜的结合量,探讨了LLG破坏细菌生物膜的潜在机制,结果显示,LLG能更有效的吸附于负电荷的生物膜,进而定点释放药物,从而达到增强LLG的抗生物膜效果。综上所述,溶菌酶稳定脂质体的策略是可行的,LLG能够在一定程度上降低抗生素的使用剂量,减缓耐药菌株的形成,同时,也有助于减小抗生素的毒副作用。该策略为使用抗生素治疗细菌生物膜相关的顽固性感染提供了一种新的方案。
[Abstract]:Chronic infection caused by bacterial biofilm is one of the thorny problems in clinic. Because bacterial biofilms are highly resistant to antibiotics, the dosage of existing antibiotics can only be increased and the administration time can be prolonged under the premise of lack of new antibiotics or effective antibacterial strategies. In the long run, it is easy to cause drug resistance and resistant strains, and the toxic side effects of antibiotics will also increase. Therefore, by improving the current way of administration of antibiotics, reducing the dosage of antibiotics and increasing the effect of action, we can provide a better solution for the treatment of bacterial biofilm infection. In this paper, the biocompatible lysozyme with positive charge was used to modify the negatively charged liposome, which improved the stability of liposome, enhanced the affinity to bacterial biofilm, and enhanced the effect of gentamicin on biofilm. The main contents are as follows: the entrapment efficiency of gentamicin liposome (LG), was determined by sodium phosphotungstate precipitation method. The entrapment efficiency of gentamicin was 0.6 by extrusion method with 1 ~ 2-palmitylphosphatidylglycerol (DPPG) and dipalmitoylphosphatidylcholine (DPPC),). Secondly, the positive charge lysozyme was adsorbed on the surface of the negatively charged liposome by electrostatic action to obtain the lysozyme modified gentamicin liposome (LLG). According to the change of surface Zeta potential from negative value of LG to positive value of LLG, lysozyme was successfully adsorbed on the surface of liposome. The changes of LG and LLG particle size in 48 h were determined. The results showed that the introduction of lysozyme could stabilize liposomes. The release of gentamicin from LG and LLG in vitro was monitored by UV spectrophotometer in real time within 72 h. It was found that in the first 48 h, the release of gentamicin exceeded 50%, and more than 70% of the drugs had been released at 72 h. In contrast, only 14% of the drugs released from LLG at 72 h. It is also proved that lysozyme adsorbed on the surface of gentamicin liposomes can effectively improve the stability of liposomes. The biofilms of Pseudomonas aeruginosa (Gram-negative bacteria) and Staphylococcus aureus (Gram-positive bacteria) were constructed in vitro. Compared with gentamicin alone, LLG is more effective in destroying and inhibiting bacterial biofilm formation. Fluorescence microscope and scanning electron microscope (SEM) showed that LLG not only destroyed the morphology and internal structure of biofilm, but also affected the morphological structure of single bacteria. The potential mechanism of LLG destroying bacterial biofilm was discussed by fluorescence quantitative binding amount of Rhodamine B to bacterial biofilm. The results showed that LLG could be more effectively adsorbed on negative charge biofilm and then released drugs on site. In order to enhance the anti-biofilm effect of LLG. To sum up, the strategy of lysozyme stabilizing liposome is feasible. LLG can reduce the dosage of antibiotics to a certain extent, slow down the formation of drug-resistant strains, but also help to reduce the toxic side effects of antibiotics. This strategy provides a new strategy for the treatment of bacterial biofilm-related obstinate infections with antibiotics.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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