Preparation Of Celastrol Cationic Liposomes And Its Antibacterial Effect Against MRSA

Staphylococcus aureus(S.aureu)is an important pathogenic bacterium in animal husbandry.It not only poses a great threat to animal health,but also may be transmitted from animals to humans.Methicillin resistant Staphylococcus aureus(MRSA)is a drug-resistant strain of S.aureus.Compared with sensitive strains,MRSA infection increases the mortality of sick animals to more than half,causing great economic losses to the breeding industry.Therefore,the development of effective drugs for MRSA infectious diseases is of great significance.Celastrol is one of the active ingredients of the traditional Chinese medicine Leigonin,which has good antibacterial effects.However,as a class IV drug in the biopharmaceutics classification system(BCS),the intrinsic low solubility and poor permeability of celastrol significantly limited clinical translation.In recent years,nanotechnology-based drug delivery systems have shown great potential in improving overall efficacy and safety.Among them,cationic liposomes,which can not only improve the solubility and permeability of drugs,but also to promote their uptake by bacteria,have attracted much attention in the field of formulation science.Thus,in this study,celastrol cationic liposomes were prepared by thin-film hydration method and characterized.Then the in vitro and in vivo antibacterial effects of celastrol cationic liposomes against MRSA were explored and the antibacterial mechanisms were further elucidated.The obtained results were as follows:(1)By examining the particle size,polydispersity index,Zeta potential and encapsulation rate of the liposomes,we determined the prescription and the process conditions of thin film hydration method for the celastrol cationic liposomes and successfully prepared.The prescriptions were as follows: Stearamine addition was 5 mg,cholesterol to phospholipid ratio was 1:6,drug input was 8mg,Tween 80 addition was 20 μL,hydration volume was 20 m L.The process conditions of film hydration method are that hydration time was 90 min,hydration temperature was 55 ℃,and probe ultrasonication time was 5 min.(2)The celastrol cationic liposomes were characterized and evaluated for safety using transmission electron microscopy,laser particle size measurement and UV spectrophotometer,with the following results.The particle size of liposomes was 109.4 nm,the zeta potential was26.11±0.22 m V,the morphology was irregular spherical structure,and the encapsulation efficiency and drug loading capacity were 81.58±1.09% and 9.71±0.22%,respectively.In addition,the obtained celastrol cationic liposomes are stable,safe and showed a sustained-release behavior.(3)The antibacterial tests in vitro and in vivo showed that compared with celastrol,celastrol cationic liposomes reduced the minimum inhibitory concentration(MIC)of MRSA in vitro,inhibited the time growth curve of MRSA more obviously,and significantly interfered with the formation of MRSA biofilm;In addition,compared with celastrol,celastrol cationic liposomes showed enhanced therapeutic effect in vivo,showing significant reduction of the inflammatory symptoms and the number of colonies in the subcutaneous cyst in mice.(4)The mechanisms of antibacterial action of celastrol cationic liposomes were elucidated using microspectrophotometer,scanning electron microscope and enzyme marker.The results were as follows: celastrol cationic liposomes significantly enhance the permeability of MRSA cell membrane,resulting in DNA and protein leakage and cell membrane damage.The leakage of alkaline phosphatase(ALP)leads to the destruction of cell wall,followed by the destruction of cell wall.At the same time,it can significantly affect the metabolism of MRSA and reduce the content of ATP in the bacteria.Compared with ordinary liposomes,the binding and uptake of MRSA and cationic liposomes were significantly enhanced.In conclusion,this study successfully prepared stable,safe and slow-release celastrol cationic liposomes,which enhanced the anti-MRSA effect of rhodopsin by targeting MRSA,improving the uptake of the drug by MRSA,enhancing cell membrane permeability,disrupting cell wall integrity,and blocking the antibacterial mechanism of metabolism.This study provides a new research and development strategy for traditional Chinese medicine preparation in the treatment of MRSA infection.