Study On Targeting Levofloxacin Liposome Against Drug-resistant Bacteria And Its Preparation

Objective:On the basis of preliminary studies on antimicrobial peptides and nano-liposome technology, prepare levofloxacin liposome with the effect of targeting drug-resistant bacteria by modifying its surface with Ts, optimize the preparation processes and prescriptions, and study its antibacterial activity in vitro.Method:1. Use UV spectrophotometry to detect the content of levofloxacin, determine the detection wavelength, and conduct methodological study. Use dialysis method to test the encapsulation efficiency of targeting levofloxacin liposome and measured its recovery rate; 2, Take the encapsulation efficiency as evaluation indicator, pH gradient method and ammonium sulfate gradient method were used to prepare levofloxacin liposome and screen the liposome preparation method. According to single factor optimization method, select the best preparation process, and use orthogonal test design to optimize the levofloxacin liposome preparation method. Then modified the liposome with targeting Ts and prepared targeting levofloxacin liposome. Study the physicochemical properties and stability of targeting levofloxacin liposome prepared under optimal conditions; 3. Use Laser Scanning Confocal Microscope to study the targeting effect of levofloxacin liposome labeled with coumarin to standard Klebsiella pneumoniae strains. Test the MIC of free levofloxacin, levofloxacin combined with Ts, non-targeting and targeting levofloxacin liposome to drug-resistant strains and analyze synergistic effect. By testing the time-kill activity to standard Klebsiella pneumoniae strain and drug uptake test, evaluate in vitro antibacterial activity of levofloxacin liposomes. Use crystal violet staining and scanning electron microscopy to study the influence of targeting levofloxacin liposome to biofilm growth.Results:Use UV spectrophotometry to test the content of levofloxacin in liposome, maximum absorption at 290nm wavelength, without interference of minor ingredient. The method is simple and sensitive with high specificity. Use dialysis method to test the liposome encapsulation efficiency, which is convenient and can effectively separate the liposome from free drugs with high recovery rate. To prepare levofloxacin liposome, ammonium sulfate gradient method is better than pH gradient method. The optimal preparation conditions of ammonium sulfate gradient method are:0.2 mol/L ammonium sulfate, dialysis for 24h to form ammonium sulfate gradient, drug incubation at 60℃ for 20min; the best prescription is:30 mg/mL phospholipid, phospholipid and cholesterol mass ratio of 4:1, drug and lipid ratio of 1:10. Using the best preparation process and prescription to prepare three groups levofloxacin liposome in parallel average encapsulation rate of 75.26+1.35%, coupling ratio of Ts was 68.43±1.45%, average particle size of 152.5±3.2 nm, polydispersity index (PDI) was 0.149±0.02, no difference compared with non-targeting liposomes, zeta potential +20.68±1.72 mV, good process reproducibility, good stability within a month. MIC of targeting liposome was lower than other formulations, FIC≤0.5, indicating synergistic effect; maximum bacteria inhibition during the same time and the time-kill activity is remarkable; accumulation of levofloxacin was maximum in bacterial cells; inhibition effect of targeting liposome is stronger than Ts.Conclusion:This experiment developed the targeting levofloxacin liposome successfully with ammonium sulfate gradient method. Its preparation process is simple with high encapsulation efficiency, good reproducibility and stability. Compared with free drug and non-targeting liposomal formulation, targeting levofloxacin liposomes have a stronger antibacterial activity to drug-resistant bacteria, which can improve the curative efficacy and reduce the drug dose, providing theoretical basis for its clinical development and application.