A Study On Lysozyme Associated Liposomal Gentamicin Against Bacterial Biofilm

The chronic infection related to biofilm is one of the most difficult problems in clinic.As the bacterial biofilm is highly resistant to antibiotics,and in the absence of new antibiotics or efficient antibacterial strategy,only increase the dose of existing antibiotics,prolong the administration time can solve the problem.Over time,it is easy to cause the emergence of drug resistance and resistant strains,while the side effects of antibiotics will increase.Therefore,there is an urgent need for effective solutions to bacterial biofilm by improving the existing mode of administration,reducing the dose,improving the effect of antibiotics.In this study,we use the biocompatible and positive lysozyme to modify negative liposomes.This strategy could improve the stability of liposomes and enhance the affinity to bacterial biofilm,further improve the therapeutic effect of gentamicin.Specific studies as follows:The liposomal gentamicin(LG)were prepared by extrusion method using 1,2-dipalmitoyl-sn-glycero-3-phosphor-(1'-rac-glycerol)(DPPG)and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine(DPPC).The encapsulation efficiency of gentamicin determined by sodium phosphotungstate precipitation method was 0.6%.To prepare lysozyme associated liposomal gentamicin(LLG),we use the positively charged lysozyme adsorbing to the negative liposomal surface through electrostatic interaction.According to the change of the Zeta potential of the surface from the negative value of LG to the positive value of LLG,the lysozyme was successfully adsorbed to the surface of the liposome.It can be proved that the introduction of lysozyme can stabilize the liposome by regular detection of the LG and LLG hydrodynamic size in deionized water within 48 hours.We detected the release of gentamicin from LG and LLG within 72 hours by UV spectrophotometer,found that LG released more than 50% gentamicin in the first 48 hours and over 70% at 72 hours,on the contrary,only 14% of the drugs were released from the LLG at 72 hours.It has also been shown that lysozyme adsorption to liposomal gentamicin can improve the stability of liposomes.The LLG was more effective at damaging established biofilms and inhibiting biofilm formation of pathogens including Pseudomonas aeruginosa(Gram-positive bacteria)and Staphylococcus aureus(Gram-negative bacteria)than gentamicin alone proved by crystal violet staining and MTT assay.Observing the bacterial biofilm by fluorescence microscopy and scanning electron microscopy,we found that LLG could not only destroy the morphology and internal structure of biofilms,but also affected the morphological structure of individual bacteria.We quantified the fluorescence intensity between Rhodamine B and bacterial biofilm,explored the underlying mechanism by which LLG disrupted bacterial biofilm above,found that LLG could be more effectively adsorbed on the negative biofilm,then released the drug,accordingly enhanced the anti-biofilm effect.In summary,the strategy using lysozyme to stabilize liposome is workable.LLG could reduce the dosage and side effects of antibiotics and slow down the formation of resistant strains,which provides a new approach for the antibiotics to treat intractable infections associated with bacterial biofilm.