| Objective: A great many people in the world are suffering from pulmonary infection causing various pathogens and might lose their lives if the treatment could not be given promptly and effectively. And antibiotics have been playing significant roles in treatment of infection. However, with abuse of various antibiotics, mutidrug-resistance aggravates the incidence of failure treatment. In addition, low targeting efficiency to lung of antibiotics usually results in an abortive treatment of pulmonary infection. Hence, there is a great deal of need to study and develop novel delivery systems to improve the antibacterial activity of antibiotics in lung.Levofloxacin is described as a third-generation quinolone and a front-line drug in curing various inflammations such as pulmonary, urinary, intestinal infection due to its extensive distribution. However the facts that its extensive distribution usually induces neurologic and hematological side effects, low targeting efficiency and the occurrence of resistance limit the use of levofloxacin in the treatment of pulmonary inflammation clinically to some extent. And in the past few decades, in order to improve the therapeutic efficiency of antibiotic in the treatment of pulmonary inflammation some measures such as nanoparticles and liposomes are given attention greatly.Liposomes are possible carriers for controlled drug delivery and targeting by the intravenous route. As with most drug carriers, liposomes have been extensively used in an attempt to improve the selective delivery and the therapeutic index of antimicrobial agents. In the event of a failure treatment, antibiotic- susceptibility of the infectious organisms often reduces, which results in that the antibiotic levels that can be achieved at the site of infection are too low for an efficient bactericidal action. In these cases, access to the target site especially inflammation site is a major determinant of antibacterial activity. So increase accumulation of antibiotics in lung using liposomes could enhance its antibacterial activity and further improve the therapy efficient for curing pulmonary infection. Surprisingly, targeting to different body sites can be realized by modulating liposome size in the intravenous route. Liposomes with size larger than 5μm could be trapped passively by the vascular network of the lung. Consequently, increased therapeutic efficiency of pulmonary inflammation could be gained using encapsulation of drug in liposomes. And liposomes have been attracting considerable attention as one of the most promising drug carriers for antibiotics to treatment pulmonary infection.Based on the information above, levofloxacin-loaded liposomes are developed. In this study levofloxacin loaded in large sized liposomes possessing passive lung targeting efficiency would deliver the drug to lung and can release the drug slowly, which can improve the therepeutic efficiency in the treatment of pulmonary inflammation and also can decrease the side effects of non-target tissues. Besides in order to improve the stability of levofloxacin-loaded liposomes, PEG2000 is used to coat the liposomes.Methods and results: UV spectrophotometry method was established to determine the levofloxacin concentration in vitro which is easy, convenient and correct. High speed centrifugation was applied to separate the free drug and liposomes which performed good accuracy and accorded with the requirement of separation.Levofloxacin-loaded liposomes were prepared by passive methods and remote loading method which finally were chosen to encapsulate levofloxacin into liposomes. The optimal formulation was obtained by orthogonal experimental design studies, with the entrapment efficiency as evaluation index. The optimized procedure was as follows: the dialysis time was 18h, incubation temperature was 55℃, incubation time was 20min, and the optimized prescription was as follows: lecithin/cholesterol (8:1, g/g), drug/lipids (1:8, g/g) and concentration of ammonium sulfate (mol/L) (0.3mol/L) according to the analytical results using Orthogonality Experiment Assistant version 3.1 (Sharetop Software Studio). PEG2000 is used to coating levofloxacin-loaded liposomes and in our study the quantity of PEG2000 and the adding method were investigated. The results showed that the stability was the best when 1 % PEG2000 was incubated with the liposomes and levofloxacin solutin.The optimal formulations of levofloxacin formulated in liposomes and PEG coating liposomes with appropriate drug encapsulation percentage and homogenous particle size distribution were selected to investigate the physiochemical properties in vitro. The properties of LVFX-liposomes and PEG coating LVFX-liposomes such as diameter and size distribution were observed by transmission electric microscope (TEM) and laser dispersive analyzing apparatus for granularity, and Zeta potential was measured by micro-electrophoresis apparatus. In vitro releases of LVFX-liposomes and PEG coating LVFX-liposomes were performed by dialysis method with levofloxacin solution as a control. The results showed: the formulated liposomes were found to be relatively uniform in size (7.424±0.689μm) with a positive zeta potential (+13.1 1±1.08mV). The range of drug entrapment efficiency was 82.19%-86.23%. But it presents low stability and to improve its stability, we use PEG2000 to coat the prepared levofloxacin-loaded liposomes. After being coated, the liposomes with EE of (79.33±2.07)% performed better stability than the original liposomes and its zeta potatial changed to negative (-12.88.±0. 81mV), which can be explained by the success of PEG2000 coating . pH of levofloxacin- loaded lipsomes and coating liposomes were in the range of 5.5-6.0 in accordance with the requirement of injections. In vitro drug release of levofloxacin-loaded liposomes and PEG coating liposomes were both monitored for up to 3 days, and the release behavior were both in accordance with Weibull-equation. PEG coating liposomes performed slower release than non-coating liposomes. Besides PEG coating liposomes performed better stabilitythan non-coating lipsomes.HPLC method was established to investigate levofloxacin concentrations of plasma and tissue in rabbits and mice and the results showed that endogenous substances did not interfere with levofloxacin determination in the conditions of selected chromatographic. The extraction recovery and method recovery of drug in blood of rabbits were larger than 90% and between 95%-105%, and the RSDs of intra-day and inter-day were less than 4%. There was a good linearity of the drug concentration within the range of 0.1-50μg/ml and the linear equation was A =40761 C +24093 (r = 0.9989) . The extraction recovery and method recovery of drug in tissues of mice was larger than 90% and between 95%-105%, and the RSDs of intra-day and inter-day were less than 4%. There was a good linearity of the drug concentration in the plasma and the tissues of mice within the range of 0.1-50μg/ml. The results above could satisfy the need of analysis for biological detection. The optimal formulations of levofloxacin formulated in liposome and PEG-coating liposomes with appropriate drug encapsulation percentage and homogenous particle size distribution were selected to investigate pharmacokinetic, biodistribution and lung targeting efficiency after intravenous administeration utilizing rabbit and mice as animal models compared with levofloxacin injection. The levofloxacin-loaded liposomes and PEG-coating liposomes both exhibited a longer elimination half life (t1/2β), MRT and VL in vivo compared with levofloxacin solution after intravenous injection to New Zealand rabbits. The encapsulation of levofloxacin in the two types liposomes also changed its biodistibution in mice after intraveneous injection in caudal vein. And compared with non-coating liposomes, the coating liposomes performed a t1/2β. Two types of liposomal levofloxacin performed excellent lung targeting efficiency with AUC, Te and Re of lung all showing obvious elevation. Besides, liposomal formulations presented accumulative activity in spleen and liver. Conversely the biodistribution of liposomal formulation in non-RES sites such as kidney, brain, heart and plasma decreased with descending Ce compared with levofloxacin injection, which potentially resulted in the reduction of side effects of free drug. And there were no obvious difference to compare coating liposomes with non coating liposomes.Conclusion: In this study, levofloxacin was successfully encapsulated into liposomes for application of injection. Levofloaxacin-loaded liposomes had a higher entrapment efficiency using remote loading method with simple, feasible preparation technology, and good reproducibility. Coating liposomes with PEG2000 can improve the stability of liposomes greatly in vitro. Compared with the control group, the distribution of drugs significantly changed in the group of levofloxacin-liposomes and coating liposomes. Levofloxacin encapsulated in liposomes could improve drug accumulate in lung, extende retention time of drug in lung, enhance local levofloxacin concentration in the lung and improve the therapeutic efficiency. In addition, the side effects in non-target tissues could be avoided and patients' compliance and the medication effect could be improved accompany by good social and valuable economic benefits. Levofloxacin-loaded liposomes were promising passive targeting to lung for pulmonary infection treatment and through PEG coating the stability of liposomes was greatly improved which accomplish our original goals.
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