左氧氟沙星脂质体的肺靶向性研究
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摘要
目的:肺部感染是人类的跨世纪疾病,严重地危害着人类健康,是我国的常见病、多发病。虽然抗生素的开发与研究,为防治感染性疾病提供了有利条件。但由于抗生素的滥用,细菌多药耐药性的产生,降低了治疗肺部感染的效能,使治疗的成功率大为降低。此外,抗生素本身对肺部较低的靶向性及对其他部位一些副作用,也给肺部感染的治疗增加了难度。所以,研究和发展一种能够增强抗生素在肺部抗菌活性的药物传递系统是非常有必要的。
左氧氟沙星(Levofloxacin,LVFX)是第三代喹诺酮类药物,由于其抗菌谱广,抗菌力强,目前已成为临床治疗感染性疾病的主要药物。但是不良反应的发生、一些细菌对其产生的耐药性及广泛的分布性造成的在肺部位浓度低,在一定程度上也制约了其在临床上的应用。近年来,通过新的制剂手段如脂质体、纳米粒增加抗生素的活性,提高肺部治疗效能受到了广大科研工作者的青睐。
脂质体具有类似生物膜结构的双分子层膜,与细胞膜组成相似,即与细胞膜的有高度亲和性,通过和细胞膜发生融合作用,可增强细胞摄取,实现细胞内药物传输,能够提高生物利用度,抵抗细菌的抗药性,经静脉注射后能够实现靶向和缓释作用。通过控制脂质体的粒径可以实现被动靶向,粒径大于5μm的脂质体易被肺部的毛细血管截留,将药物包裹于大粒径的脂质体中可以实现抗生素在肺部的蓄积,提高其在肺部的抗菌活性。脂质体作为一种非常有前景的载体,在抗生素治疗肺部感染方面一直受到了科研工作者的关注。
基于上述特点我们构建了左氧氟沙星脂质体的给药系统,将左氧氟沙星包裹于大粒径脂质体中,将其被动靶向到肺,使其在肺部更好的发挥治疗效能,同时脂质体的包裹可使药物缓慢的释放,延长其半衰期,同时也降低左氧氟沙星在一些非靶部位的分布,有利于降低其副作用。由于左氧氟沙星为两亲性弱碱性药物,采用传统的被动载药法包封率低,而采用包封率高的硫酸铵梯度法来制备,又存在稳定性差、室温放置不稳定等缺陷,在试验中我们采用了硫酸铵梯度法制备了包封率高的脂质体,并采用PEG2000对脂质体进行包衣以期达到增加稳定性的目的。
方法与结果:采用紫外分光光度法测定左氧氟沙星的含量,辅料不干扰测定,专属性强,方法简便、灵敏,该法适合左氧氟沙星的测定。采用高速离心法对左氧氟沙星脂质体及包衣脂质体的包封率进行测定,可以将脂质体与未包封药物完全分离,回收率符合测定要求。
分别采用了薄膜分散法、逆相蒸发法、乙醇注入法及硫酸铵梯度法制备左氧氟沙星脂质体,以包封率为评价指标,在单因素考察基础上,采用正交试验设计优化脂质体的处方组成和制备工艺;将优化处方和工艺制备的脂质体,用PEG 2000包衣,考察PEG2000的加入方法及用量对稳定性的影响。结果表明:硫酸铵梯度法制备的左氧氟沙星脂质体包封率最高,优化的最佳处方组成为:磷脂与胆固醇之比为(8:1,g/g),药脂比为(1:8,g/g),硫酸铵的浓度为0.3mol/L;最佳制备工艺条件为:透析时间为18h,孵育温度为50℃,孵育时间为20min;将1%PEG2000水溶液和左氧氟沙星溶液同时加入空白脂质体中,50℃时孵育,稳定性最好。
采用电子透射显微镜观察脂质体的外观形态;激光散射粒度分析仪测定脂质体的粒径及其粒度分布;电势显微电泳仪测定脂质体表面Zeta电位;动态膜透析法测定脂质体的体外释药特性,以零级动力学方程、一级动力学方程、Higuchi方程和Weibull方程等数学模型拟合脂质体的体外释药动力学;冰箱4℃贮存,考察了两种脂质体的初步稳定性。结果表明:两种脂质体外观均呈椭圆形,大小均匀,未包衣左氧氟沙星脂质体指纹状结构明显,PEG包衣后的脂质体指纹状结构不明显,可能是PEG2000的加入覆盖了脂质体的外层,掩饰了脂质体的部分指纹;未包衣和包衣左氧氟沙星脂质体的平均粒径分别为(7.424±0.689)μm和(6.28±0.891)μm;Zeta电位分别为(+13.11±1.08)mV和(-12.88.±0.81)mV。两种脂质体的pH均在5.5-6.0之间,符合注射剂的要求;体外释放与药物溶液相比均表现了明显的缓释作用,均符合Weibull方程,其中PEG2000包衣脂质体的缓释作用更明显。初步稳定性试验结果表明,PEG2000脂质体的包衣对左氧氟沙星脂质体起到了明显的稳定作用。
采用HPLC法测定家兔血浆、小鼠血浆及组织中的左氧氟沙星的浓度,并进行了方法学考察。以左氧氟沙星注射液为对照,对左氧氟沙星脂质体及PEG包衣左氧氟沙星脂质体进行了药动学与组织分布动力学的研究;以相对摄取率(Re)、相对靶向效率(Te)、峰面积之比(Ce)等靶向性参数为指标,对脂质体的靶向性进行了评价。结果表明,在所选色谱条件下,内源性物质不干扰左氧氟沙星的测定,家兔血浆平均提取回收率大于90%,方法回收率在95%～105%之间,日内、日间精密度RSD小于4%,标准曲线方程A=40761 C+24093(r=0.9989),血药浓度在0.1～50μg/ml范围内线性关系良好;小鼠各组织器官平均提取回收率均大于90%,方法回收率在95%～105%之间,日内、日间精密度均小于4%,小鼠血浆、各组织线性范围为0.1～50μg/ml,线性关系良好,上述试验结果均满足生物样品测定要求。与注射液相比,两种脂质体家兔体内的药动学发生了明显变化,平均滞留时间(MRT)、消除半衰期(t_(1/2β),p＜0.05)、表观分布容积(V)均有所增加;PEG2000包衣脂质体t_(1/2β)较未包衣脂质体延长。靶向性评价结果表明,将左氧氟沙星制成脂质体后,肺靶向性大大提高,相反降低了心、肾中的药物浓度。PEG2000包衣脂质体与未包衣脂质体的体内分布没有显著性差异。
结论:本课题成功研制了具有肺靶向性的左氧氟沙星脂质体,所采用的制备工艺简便可行,重现性好,包封率较高,经PEG2000包衣后体外稳定性良好。将左氧氟沙星制备成脂质体后,明显改变了药物的体内配置,与注射剂相比,脂质体制剂使药物蓄积于肺部,增加了肺部的局部药物浓度,缓慢平稳释放药物,维持炎症部位长时间的有效药物浓度,并且可以减少非靶部位如心、肾、脑的蓄积,有效提高作用部位的药物浓度,降低非把部位的药物浓度,从而可提高疗效,降低副作用,减少用药次数,延长药物在作用部位时间,减轻病人痛苦,提高病人用药的顺应性和用药水平,具有良好的社会和经济效益。
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 (t_(1/2β)), 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 t_(1/2β). 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.
左氧氟沙星(Levofloxacin,LVFX)是第三代喹诺酮类药物,由于其抗菌谱广,抗菌力强,目前已成为临床治疗感染性疾病的主要药物。但是不良反应的发生、一些细菌对其产生的耐药性及广泛的分布性造成的在肺部位浓度低,在一定程度上也制约了其在临床上的应用。近年来,通过新的制剂手段如脂质体、纳米粒增加抗生素的活性,提高肺部治疗效能受到了广大科研工作者的青睐。
脂质体具有类似生物膜结构的双分子层膜,与细胞膜组成相似,即与细胞膜的有高度亲和性,通过和细胞膜发生融合作用,可增强细胞摄取,实现细胞内药物传输,能够提高生物利用度,抵抗细菌的抗药性,经静脉注射后能够实现靶向和缓释作用。通过控制脂质体的粒径可以实现被动靶向,粒径大于5μm的脂质体易被肺部的毛细血管截留,将药物包裹于大粒径的脂质体中可以实现抗生素在肺部的蓄积,提高其在肺部的抗菌活性。脂质体作为一种非常有前景的载体,在抗生素治疗肺部感染方面一直受到了科研工作者的关注。
基于上述特点我们构建了左氧氟沙星脂质体的给药系统,将左氧氟沙星包裹于大粒径脂质体中,将其被动靶向到肺,使其在肺部更好的发挥治疗效能,同时脂质体的包裹可使药物缓慢的释放,延长其半衰期,同时也降低左氧氟沙星在一些非靶部位的分布,有利于降低其副作用。由于左氧氟沙星为两亲性弱碱性药物,采用传统的被动载药法包封率低,而采用包封率高的硫酸铵梯度法来制备,又存在稳定性差、室温放置不稳定等缺陷,在试验中我们采用了硫酸铵梯度法制备了包封率高的脂质体,并采用PEG2000对脂质体进行包衣以期达到增加稳定性的目的。
方法与结果:采用紫外分光光度法测定左氧氟沙星的含量,辅料不干扰测定,专属性强,方法简便、灵敏,该法适合左氧氟沙星的测定。采用高速离心法对左氧氟沙星脂质体及包衣脂质体的包封率进行测定,可以将脂质体与未包封药物完全分离,回收率符合测定要求。
分别采用了薄膜分散法、逆相蒸发法、乙醇注入法及硫酸铵梯度法制备左氧氟沙星脂质体,以包封率为评价指标,在单因素考察基础上,采用正交试验设计优化脂质体的处方组成和制备工艺;将优化处方和工艺制备的脂质体,用PEG 2000包衣,考察PEG2000的加入方法及用量对稳定性的影响。结果表明:硫酸铵梯度法制备的左氧氟沙星脂质体包封率最高,优化的最佳处方组成为:磷脂与胆固醇之比为(8:1,g/g),药脂比为(1:8,g/g),硫酸铵的浓度为0.3mol/L;最佳制备工艺条件为:透析时间为18h,孵育温度为50℃,孵育时间为20min;将1%PEG2000水溶液和左氧氟沙星溶液同时加入空白脂质体中,50℃时孵育,稳定性最好。
采用电子透射显微镜观察脂质体的外观形态;激光散射粒度分析仪测定脂质体的粒径及其粒度分布;电势显微电泳仪测定脂质体表面Zeta电位;动态膜透析法测定脂质体的体外释药特性,以零级动力学方程、一级动力学方程、Higuchi方程和Weibull方程等数学模型拟合脂质体的体外释药动力学;冰箱4℃贮存,考察了两种脂质体的初步稳定性。结果表明:两种脂质体外观均呈椭圆形,大小均匀,未包衣左氧氟沙星脂质体指纹状结构明显,PEG包衣后的脂质体指纹状结构不明显,可能是PEG2000的加入覆盖了脂质体的外层,掩饰了脂质体的部分指纹;未包衣和包衣左氧氟沙星脂质体的平均粒径分别为(7.424±0.689)μm和(6.28±0.891)μm;Zeta电位分别为(+13.11±1.08)mV和(-12.88.±0.81)mV。两种脂质体的pH均在5.5-6.0之间,符合注射剂的要求;体外释放与药物溶液相比均表现了明显的缓释作用,均符合Weibull方程,其中PEG2000包衣脂质体的缓释作用更明显。初步稳定性试验结果表明,PEG2000脂质体的包衣对左氧氟沙星脂质体起到了明显的稳定作用。
采用HPLC法测定家兔血浆、小鼠血浆及组织中的左氧氟沙星的浓度,并进行了方法学考察。以左氧氟沙星注射液为对照,对左氧氟沙星脂质体及PEG包衣左氧氟沙星脂质体进行了药动学与组织分布动力学的研究;以相对摄取率(Re)、相对靶向效率(Te)、峰面积之比(Ce)等靶向性参数为指标,对脂质体的靶向性进行了评价。结果表明,在所选色谱条件下,内源性物质不干扰左氧氟沙星的测定,家兔血浆平均提取回收率大于90%,方法回收率在95%～105%之间,日内、日间精密度RSD小于4%,标准曲线方程A=40761 C+24093(r=0.9989),血药浓度在0.1～50μg/ml范围内线性关系良好;小鼠各组织器官平均提取回收率均大于90%,方法回收率在95%～105%之间,日内、日间精密度均小于4%,小鼠血浆、各组织线性范围为0.1～50μg/ml,线性关系良好,上述试验结果均满足生物样品测定要求。与注射液相比,两种脂质体家兔体内的药动学发生了明显变化,平均滞留时间(MRT)、消除半衰期(t_(1/2β),p＜0.05)、表观分布容积(V)均有所增加;PEG2000包衣脂质体t_(1/2β)较未包衣脂质体延长。靶向性评价结果表明,将左氧氟沙星制成脂质体后,肺靶向性大大提高,相反降低了心、肾中的药物浓度。PEG2000包衣脂质体与未包衣脂质体的体内分布没有显著性差异。
结论:本课题成功研制了具有肺靶向性的左氧氟沙星脂质体,所采用的制备工艺简便可行,重现性好,包封率较高,经PEG2000包衣后体外稳定性良好。将左氧氟沙星制备成脂质体后,明显改变了药物的体内配置,与注射剂相比,脂质体制剂使药物蓄积于肺部,增加了肺部的局部药物浓度,缓慢平稳释放药物,维持炎症部位长时间的有效药物浓度,并且可以减少非靶部位如心、肾、脑的蓄积,有效提高作用部位的药物浓度,降低非把部位的药物浓度,从而可提高疗效,降低副作用,减少用药次数,延长药物在作用部位时间,减轻病人痛苦,提高病人用药的顺应性和用药水平,具有良好的社会和经济效益。
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 (t_(1/2β)), 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 t_(1/2β). 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.
引文
[1]张淑慧,梁晋全,苏业璞.抗感染药物治疗学[M].石家庄:河北科技出版社,2001:341
[2]Kuo LC,Lai CC,Liao CH,et al..Multidrug-resistant Acinetobacter baumannii bacteraemia:clinical features antimicrobial therapy and outcome[J].Clinical Microbiology and Infection.2007,13(2):196-198.
[3]Davies T,Goering RV,Lovgren M,et al..Molecular epidemiological survey of penicillin-resistant Streptococcus pneumoniae from Asia,Europe,and North America[J].Diagn Microbiol Infect Dis.1999,34(1):7-12.
[4]Farrell DJ,Jenkins SG,Brown SD,et al..Emergence and spread of Streptococcus pneumoniae with erm(B) and mef(A) resistance[J].Emerg Infect Dis.2005,11(6):851-858.
[5]Pletz MW,McGee L,Jorgensen J,et al..2004.Levofloxacin-resistant invasive Streptococcus pneumoniae in the United States:evidence for clonal spread and the impact of conjugate pneumococcal vaccine[J].Antimicrob Agents Chemother.2004,48(9):3491-3497.
[6]Witte W,Cuny C,Klare I,et al..2008.Emergence and spread of antibiotic-resistant Gram-positive bacterial pathogens[J].International Journal of Medical Microbiology.2008,298(5-6):365-377.
[7]Fauci AS.Infectious diseases:considerations for the 21st century[J].Clin Infect Dis.2001,32(5):675-685.
[8]Li J,Rayner CR,Nation RL,Owen RJ,et al.Heteroresistance to colistin in multidrug-resistant Acinetobacter baumannii[J].Antimicrobial Agents and Chemotherapy.2006,50(9):2946-2950.
[9]Rodriguez-Hernadez MJ,Saugar J,Docobo-Perez F,et al..Studies on the antimicrobial activity of cecropin A-melittin hybrid peptides in colistin-resistant clinical isolates of Acinetobacter baumannii[J].Journal of Antimicrobial Chemotherapy.2006,58(1):95-100.
[10]Tewes F,Brillault J,Couet W,et al..Formulation of rifampicin-cyclodextrin complexes for lung nebulization[J].J Controlled Release.2008,129(2):93-99.
[11]杨藻寰.药理学和药物治疗学[M].北京:人民卫生出版社,2000:165-166
[12]干荣富.喹诺酮类药物发展趋势之探讨[J].世界临床药物.2007,28(9):570-576.
[13]刘双,杨京华.喹诺酮类药物研究进展[J].临床药物治疗杂志.2007,5(6):39-42.
[14]阿赛古丽,何烨.浅谈喹诺酮类抗生素[J].临床实践.2005,23(11):112
[15]苏红霞.喹诺酮类药物临床应用中应注意的问题[J].基层医学论坛.2007,11(10):927-928.
[16]张良海.我院127例左氧氟沙星注射液不良反应及用药监护[J].中国药房,2005,16(22):1732-1734
[17]李荣凌,张先洲,等.静脉输液临床应用分析[J].中国药房.2005,16(21):1637-1639
[18]高百春,肖春玲.左氧氟沙星不良反应[J].中国误诊学杂志志.2007,11(25):6191-6192.
[19]张志芬,张鹏.左氧氟沙星致精神症状1例[J].药物不良反应杂志,2006,8(3):223.
[20]缪秋琴.静滴喹诺酮类药物的注意事项及改进方法的探讨[J].基层医学论坛.2007,11(11):1036-1037.
[21]黄怡,刘忠令.常用抗菌药物耐药机制及对策[J].国外抗生素分册.1999,2(1):28-31.
[22]蒋红霞,曾振灵.细菌耐药机制与耐药性控制对策[J].动物医学进展.2001,22(4):4-7.
[23]Fresta M,Spadaro A,Cerniglia G,et al..Intracellular Accumulation of Ofloxacin-Loaded Liposomes in Human Synovial Fibroblasts.Antimicrobial Agents and Chemotherapy.1995,39(6):1372-1375.
[24]张灵芝.脂质体制备及其在生物医学中的应用[M].北京:北京医科大学中国协和医科大学联合出版社:1998:1.
[25]Bangham A.D.,Standish M.M.,Watkins J.C.,et al.Diffusion of univalent ions across the lamellae of swollen phospholipids.J Mol Biol.1965,13(1):238-252.
[26]Bangham A.D.Membrane models with phospholipids[J].Prog.Biophys.Mol.Biol.1968,18:29-95.
[27]Tyrrell DA,Heath TD,Colley CM,et al.New aspect of liposomes[J].Biochim Biophys Acta.1976,457(3-4):259-302
[28]Gregoridis G.,Liposomes in the therapy of lysosomal storage disease[J].Nature,1978,275(5682):695-696.
[29]Amselem S,Gabizon A,Barenholz Y.Optimization and upscaling of doxorubicin-containing liposomes for clinical use[J].J Pharm Sci.1990,79(12):1045-1052.
[30]Lian T,Ho RJ.Trends and developments in liposome drug delivery systems[J].J Pharm Sci,2001,90(6):667-680.
[31]平其能.现代药剂学[M].北京:中国医药科技出版社,1998:621.
[32]梁文权 生物药剂学与药物动力学[M].北京:人民卫生出版社,2005:110.
[33]Robillard NJ,Scarpa AL.Genetic and physiological characterization of ciprlevofloxacin resistance in Pseudomonas aeruginosa PAO[J].Antimicrob Agents Chemother.1988.32(4):535-539.
[34]Chamberland S,Bayer AS,Schollaardt T,Wong SA,Bryan LE.Characterization of mechanisms of quinolone resistance in Pseudomonas aeruginosa strains isolated in vitro and in vivo during experimental endocarditis[J].Antimicrob Agents Chemother.1989,33(5):624-634.
[35]孙维彤 张娜,李爱国等.托氟啶脂质体的研究[J].中国药学杂志2007,42(6):445-449.
[36]郑佳日失.脂质体主动载药及其研究进展[J].国外医学药学分册.2005,32(4):271-274.
[37]Cullis PR,Hope MJ,Bally MB,et al.Influence of pH gradients on the transbilayer transport of drugs,lipids,peptides and metal ions into large unilamellar vesicles[J].Biochim Biophys Acta,1997,1331(2):187-211.
[38]Nichols JW,Deamer DW.Catecholamine uptake and concentration by liposomes maintaining pH gradients[J].Biochim Biophys Acta.1976,455(1):269-271.
[39]Hwang SH,Maitani Y.Remote loading of diclofenac,insulin and fluorescein isothiocyanate labeled insulin into liposomes by pH and acetate gradient methods[J].Int J Pharm,1999,179(1):85-95.
[40]邓意辉,于彬,李焕秋.pH梯度法制备阿霉素脂质体[J].沈阳药科大学学报,1997,14(4):239-242.
[41]Eastman SJ,Hope MJ,Cullis PR.Transbilayer transport of phosphatidic acid in response to transmembrane pH gradients[J].Biochemistry.1991,30(7):1740-5
[42]环丙沙星脂质体的研究 沈阳药科大学硕士学位论文.
[43]Mayer LD,Tai LC,Bally MB,et al.Characterization of liposomal systems containing doxorubicin entrapped in response to pH gradients[J].Biochim Biophys Acta,1990,1025(2):143-151.
[44]Haran G,Cohen R,Bar LK,et al..Transmembrane ammonium sulfate gradients in liposomes produce efficient and stable entrapment of amphipathic weak bases Biochim Biophys Acta,1993,1151(1):201-215.
[45]Ishida T,Takanashi Y,Doi H,et al.Encapsulation of an antivasospastic drug,fasudil,intoliposomes,and in vitro stability of the fasudil-loadedliposomes.Int J Pharm,2002,232(1-2):59-67
[46]Harrigan PR,Wong KF,Redelmeier TE,et al..Accumulation of doxorubicin and other lipophilic amines into large unilamellar vesicles in response to transmembrane pH gradients[J].Biochim Biophys Aeta.1993,1149(2):329-38
[47]Blume G,Ceve G.Liposomes for the sustained drug release in vivo.Biochim Biophys Acta.1990 1029(1):91-7
[48]陆彬.药剂学[M].北京:中国医药科技出版社.2001:95
[49]Fielding RM,Lewis RO,Moon-McDermott L.Altered tissue distribution and elimination of amikacin encapsulated in unilamellar,low-clearance liposomes (MiKasome).Pharm Res.1998,15(11):1775-81.
[50]Wu NZ,Da D,Rudoll TL,et al Increased microvascular permeability contributes to preferential accumulation of Stealth liposomes in tumor tissue.Cancer Res.1993,53(16):3765-70.
[51]Gabizon A,Catane R,Uziely B,et al.Prolonged circulation time and enhanced accumulation in malignant exudates of doxorubicin encapsulated in polyethylene-glycol coated liposomes.Cancer Res.1994,54(4):987-92
[52]Mayer LD,Bally MB,Cullis PR.Uptake of adriamycin into large unilamellar vesicles in response to a pH gradient.Biochim Biophys Acta.1986,857(1):123-126.
[53]于波涛,张志荣,刘文胜。提高脂质体包封率的方法及其研究进展[J].中国医药工业杂志.2003,33(11):564-568.
[54]李维凤,牛晓峰,陈海英等。冻融法制备硝苯地平脂质体[J].西安交通大学学报医学版,2003,24(6):578-580.
[55]杜松,邓英杰,张威。主动载药法制备盐酸去氢骆驼蓬碱脂质体[J].中草药.2005,36(5):673-676.
[56]叶兆伟,承伟。脂质体包封率测定方法及影响因素[J].中国生物制品学杂志2007,20(10):789-782.
[57]邓意辉,于彬,李焕秋等。pH梯度法制备阿霉素脂质体[J].沈阳药科大学学报.1997,14(4):239-242.
[58]邓意辉,李焕秋,顾学裘。聚乙二醇单甲醚(2000)胆固醇琥珀酸酯包衣脂质体的研究[J].沈阳药科大学学报,2000,17(6):391-393.
[59]Furneri PM,Fresta M,Puglisi G,Tempera G.Ofloxacin-loaded liposomes:in vitro activity and drug accumulation in bacteria[J].Antimicrob Agents Chemother,2000.44(9),2458-2464.
[60]Clerc S,Barenholz Y..Loading of amphipathic weak acids into liposomes in response to transmembrane calcium acetate gradients[J].Biochim Biophys Acta.1995,1240(2):257-65.
[61]Arnhold J,Panasenko OM,Schiller J,et al.The action of hypochlorous acid on phosphatidylcholine liposomes in dependence on the content of double bonds. Stoichiometry and NMR analysis[J].Chem Phys Lipids,1995,78(1):55-64.
[62]Fomuso LB,Corredig M,Akoh CC.Metalcatalyzed oxidation of a structured lipid model emulsion[J].J Agric Food Chem,2002,50(24):7114-7119.
[63]Arifin DR,Palmer AF.Physical properties and stability mechanisms of poly (ethylene glycol) conjugated liposome encapsulated hemoglobin dispersions[J].Artif Cells Blood Substit Immobil Biotechnol,2005,33(2):137-162.
[64]蔡明志,王昆,黄复生.温度对冻干脂质体物理化学稳定性的影响研究[J].第三军医大学学报,2004,26(6):552-554.
[65]崔福德.药剂学[M].北京:人民卫生出版社,2004:256
[66]苏德森.物理药剂学(Ⅱ)(M).沈阳:沈阳药科大学,1998:11-19.
[67]Needham J,McIntosh TJ.,Lasic D.D..Repulse interactions and mechanical stability of polymer-grafted lipid membranes[J].Biochim.Biophys.A cta,1992(1),1108:40-48.
[68]Blume G.,Ceve G..Liposomes for the sustained drug release in vivo[J].Biochim Biophys.Acta,1990,1029:91-97
[69]苏德森.物理药剂学(M).沈阳,沈阳药科大学.1998:66-70.
[70]Cheng J,Zhu JB,Wen N,Xiong F..Stability and pharmacokinetic studies of o-palmitoylamylopectin anchored dipyridamole liposomes[J],Int J Pharm,2006,313(1-2):136-143.
[71]Cheng J,Wen N,Xiong F,Chen SJ,Zhu JB.Characterization,lung targeting profile and therapeutic efficiency of dipyridamole liposomes[J].J Drug Target,2006,14(10):717-724.
[72]王健松,朱家壁,吕瑞勤等.肺靶向阿奇霉素脂质体的制备及其在小鼠体内的分布[J].药学学报,2005,40(3):274-278.
[73]Wang JP,Maitani Y,Takayama K,et al.Pharmacokinetics and antitumor effect of doxorubicin carried by stealth and remote loading proliposome[J].Pharm Res,2000,17(7):782-787.
[2]Kuo LC,Lai CC,Liao CH,et al..Multidrug-resistant Acinetobacter baumannii bacteraemia:clinical features antimicrobial therapy and outcome[J].Clinical Microbiology and Infection.2007,13(2):196-198.
[3]Davies T,Goering RV,Lovgren M,et al..Molecular epidemiological survey of penicillin-resistant Streptococcus pneumoniae from Asia,Europe,and North America[J].Diagn Microbiol Infect Dis.1999,34(1):7-12.
[4]Farrell DJ,Jenkins SG,Brown SD,et al..Emergence and spread of Streptococcus pneumoniae with erm(B) and mef(A) resistance[J].Emerg Infect Dis.2005,11(6):851-858.
[5]Pletz MW,McGee L,Jorgensen J,et al..2004.Levofloxacin-resistant invasive Streptococcus pneumoniae in the United States:evidence for clonal spread and the impact of conjugate pneumococcal vaccine[J].Antimicrob Agents Chemother.2004,48(9):3491-3497.
[6]Witte W,Cuny C,Klare I,et al..2008.Emergence and spread of antibiotic-resistant Gram-positive bacterial pathogens[J].International Journal of Medical Microbiology.2008,298(5-6):365-377.
[7]Fauci AS.Infectious diseases:considerations for the 21st century[J].Clin Infect Dis.2001,32(5):675-685.
[8]Li J,Rayner CR,Nation RL,Owen RJ,et al.Heteroresistance to colistin in multidrug-resistant Acinetobacter baumannii[J].Antimicrobial Agents and Chemotherapy.2006,50(9):2946-2950.
[9]Rodriguez-Hernadez MJ,Saugar J,Docobo-Perez F,et al..Studies on the antimicrobial activity of cecropin A-melittin hybrid peptides in colistin-resistant clinical isolates of Acinetobacter baumannii[J].Journal of Antimicrobial Chemotherapy.2006,58(1):95-100.
[10]Tewes F,Brillault J,Couet W,et al..Formulation of rifampicin-cyclodextrin complexes for lung nebulization[J].J Controlled Release.2008,129(2):93-99.
[11]杨藻寰.药理学和药物治疗学[M].北京:人民卫生出版社,2000:165-166
[12]干荣富.喹诺酮类药物发展趋势之探讨[J].世界临床药物.2007,28(9):570-576.
[13]刘双,杨京华.喹诺酮类药物研究进展[J].临床药物治疗杂志.2007,5(6):39-42.
[14]阿赛古丽,何烨.浅谈喹诺酮类抗生素[J].临床实践.2005,23(11):112
[15]苏红霞.喹诺酮类药物临床应用中应注意的问题[J].基层医学论坛.2007,11(10):927-928.
[16]张良海.我院127例左氧氟沙星注射液不良反应及用药监护[J].中国药房,2005,16(22):1732-1734
[17]李荣凌,张先洲,等.静脉输液临床应用分析[J].中国药房.2005,16(21):1637-1639
[18]高百春,肖春玲.左氧氟沙星不良反应[J].中国误诊学杂志志.2007,11(25):6191-6192.
[19]张志芬,张鹏.左氧氟沙星致精神症状1例[J].药物不良反应杂志,2006,8(3):223.
[20]缪秋琴.静滴喹诺酮类药物的注意事项及改进方法的探讨[J].基层医学论坛.2007,11(11):1036-1037.
[21]黄怡,刘忠令.常用抗菌药物耐药机制及对策[J].国外抗生素分册.1999,2(1):28-31.
[22]蒋红霞,曾振灵.细菌耐药机制与耐药性控制对策[J].动物医学进展.2001,22(4):4-7.
[23]Fresta M,Spadaro A,Cerniglia G,et al..Intracellular Accumulation of Ofloxacin-Loaded Liposomes in Human Synovial Fibroblasts.Antimicrobial Agents and Chemotherapy.1995,39(6):1372-1375.
[24]张灵芝.脂质体制备及其在生物医学中的应用[M].北京:北京医科大学中国协和医科大学联合出版社:1998:1.
[25]Bangham A.D.,Standish M.M.,Watkins J.C.,et al.Diffusion of univalent ions across the lamellae of swollen phospholipids.J Mol Biol.1965,13(1):238-252.
[26]Bangham A.D.Membrane models with phospholipids[J].Prog.Biophys.Mol.Biol.1968,18:29-95.
[27]Tyrrell DA,Heath TD,Colley CM,et al.New aspect of liposomes[J].Biochim Biophys Acta.1976,457(3-4):259-302
[28]Gregoridis G.,Liposomes in the therapy of lysosomal storage disease[J].Nature,1978,275(5682):695-696.
[29]Amselem S,Gabizon A,Barenholz Y.Optimization and upscaling of doxorubicin-containing liposomes for clinical use[J].J Pharm Sci.1990,79(12):1045-1052.
[30]Lian T,Ho RJ.Trends and developments in liposome drug delivery systems[J].J Pharm Sci,2001,90(6):667-680.
[31]平其能.现代药剂学[M].北京:中国医药科技出版社,1998:621.
[32]梁文权 生物药剂学与药物动力学[M].北京:人民卫生出版社,2005:110.
[33]Robillard NJ,Scarpa AL.Genetic and physiological characterization of ciprlevofloxacin resistance in Pseudomonas aeruginosa PAO[J].Antimicrob Agents Chemother.1988.32(4):535-539.
[34]Chamberland S,Bayer AS,Schollaardt T,Wong SA,Bryan LE.Characterization of mechanisms of quinolone resistance in Pseudomonas aeruginosa strains isolated in vitro and in vivo during experimental endocarditis[J].Antimicrob Agents Chemother.1989,33(5):624-634.
[35]孙维彤 张娜,李爱国等.托氟啶脂质体的研究[J].中国药学杂志2007,42(6):445-449.
[36]郑佳日失.脂质体主动载药及其研究进展[J].国外医学药学分册.2005,32(4):271-274.
[37]Cullis PR,Hope MJ,Bally MB,et al.Influence of pH gradients on the transbilayer transport of drugs,lipids,peptides and metal ions into large unilamellar vesicles[J].Biochim Biophys Acta,1997,1331(2):187-211.
[38]Nichols JW,Deamer DW.Catecholamine uptake and concentration by liposomes maintaining pH gradients[J].Biochim Biophys Acta.1976,455(1):269-271.
[39]Hwang SH,Maitani Y.Remote loading of diclofenac,insulin and fluorescein isothiocyanate labeled insulin into liposomes by pH and acetate gradient methods[J].Int J Pharm,1999,179(1):85-95.
[40]邓意辉,于彬,李焕秋.pH梯度法制备阿霉素脂质体[J].沈阳药科大学学报,1997,14(4):239-242.
[41]Eastman SJ,Hope MJ,Cullis PR.Transbilayer transport of phosphatidic acid in response to transmembrane pH gradients[J].Biochemistry.1991,30(7):1740-5
[42]环丙沙星脂质体的研究 沈阳药科大学硕士学位论文.
[43]Mayer LD,Tai LC,Bally MB,et al.Characterization of liposomal systems containing doxorubicin entrapped in response to pH gradients[J].Biochim Biophys Acta,1990,1025(2):143-151.
[44]Haran G,Cohen R,Bar LK,et al..Transmembrane ammonium sulfate gradients in liposomes produce efficient and stable entrapment of amphipathic weak bases Biochim Biophys Acta,1993,1151(1):201-215.
[45]Ishida T,Takanashi Y,Doi H,et al.Encapsulation of an antivasospastic drug,fasudil,intoliposomes,and in vitro stability of the fasudil-loadedliposomes.Int J Pharm,2002,232(1-2):59-67
[46]Harrigan PR,Wong KF,Redelmeier TE,et al..Accumulation of doxorubicin and other lipophilic amines into large unilamellar vesicles in response to transmembrane pH gradients[J].Biochim Biophys Aeta.1993,1149(2):329-38
[47]Blume G,Ceve G.Liposomes for the sustained drug release in vivo.Biochim Biophys Acta.1990 1029(1):91-7
[48]陆彬.药剂学[M].北京:中国医药科技出版社.2001:95
[49]Fielding RM,Lewis RO,Moon-McDermott L.Altered tissue distribution and elimination of amikacin encapsulated in unilamellar,low-clearance liposomes (MiKasome).Pharm Res.1998,15(11):1775-81.
[50]Wu NZ,Da D,Rudoll TL,et al Increased microvascular permeability contributes to preferential accumulation of Stealth liposomes in tumor tissue.Cancer Res.1993,53(16):3765-70.
[51]Gabizon A,Catane R,Uziely B,et al.Prolonged circulation time and enhanced accumulation in malignant exudates of doxorubicin encapsulated in polyethylene-glycol coated liposomes.Cancer Res.1994,54(4):987-92
[52]Mayer LD,Bally MB,Cullis PR.Uptake of adriamycin into large unilamellar vesicles in response to a pH gradient.Biochim Biophys Acta.1986,857(1):123-126.
[53]于波涛,张志荣,刘文胜。提高脂质体包封率的方法及其研究进展[J].中国医药工业杂志.2003,33(11):564-568.
[54]李维凤,牛晓峰,陈海英等。冻融法制备硝苯地平脂质体[J].西安交通大学学报医学版,2003,24(6):578-580.
[55]杜松,邓英杰,张威。主动载药法制备盐酸去氢骆驼蓬碱脂质体[J].中草药.2005,36(5):673-676.
[56]叶兆伟,承伟。脂质体包封率测定方法及影响因素[J].中国生物制品学杂志2007,20(10):789-782.
[57]邓意辉,于彬,李焕秋等。pH梯度法制备阿霉素脂质体[J].沈阳药科大学学报.1997,14(4):239-242.
[58]邓意辉,李焕秋,顾学裘。聚乙二醇单甲醚(2000)胆固醇琥珀酸酯包衣脂质体的研究[J].沈阳药科大学学报,2000,17(6):391-393.
[59]Furneri PM,Fresta M,Puglisi G,Tempera G.Ofloxacin-loaded liposomes:in vitro activity and drug accumulation in bacteria[J].Antimicrob Agents Chemother,2000.44(9),2458-2464.
[60]Clerc S,Barenholz Y..Loading of amphipathic weak acids into liposomes in response to transmembrane calcium acetate gradients[J].Biochim Biophys Acta.1995,1240(2):257-65.
[61]Arnhold J,Panasenko OM,Schiller J,et al.The action of hypochlorous acid on phosphatidylcholine liposomes in dependence on the content of double bonds. Stoichiometry and NMR analysis[J].Chem Phys Lipids,1995,78(1):55-64.
[62]Fomuso LB,Corredig M,Akoh CC.Metalcatalyzed oxidation of a structured lipid model emulsion[J].J Agric Food Chem,2002,50(24):7114-7119.
[63]Arifin DR,Palmer AF.Physical properties and stability mechanisms of poly (ethylene glycol) conjugated liposome encapsulated hemoglobin dispersions[J].Artif Cells Blood Substit Immobil Biotechnol,2005,33(2):137-162.
[64]蔡明志,王昆,黄复生.温度对冻干脂质体物理化学稳定性的影响研究[J].第三军医大学学报,2004,26(6):552-554.
[65]崔福德.药剂学[M].北京:人民卫生出版社,2004:256
[66]苏德森.物理药剂学(Ⅱ)(M).沈阳:沈阳药科大学,1998:11-19.
[67]Needham J,McIntosh TJ.,Lasic D.D..Repulse interactions and mechanical stability of polymer-grafted lipid membranes[J].Biochim.Biophys.A cta,1992(1),1108:40-48.
[68]Blume G.,Ceve G..Liposomes for the sustained drug release in vivo[J].Biochim Biophys.Acta,1990,1029:91-97
[69]苏德森.物理药剂学(M).沈阳,沈阳药科大学.1998:66-70.
[70]Cheng J,Zhu JB,Wen N,Xiong F..Stability and pharmacokinetic studies of o-palmitoylamylopectin anchored dipyridamole liposomes[J],Int J Pharm,2006,313(1-2):136-143.
[71]Cheng J,Wen N,Xiong F,Chen SJ,Zhu JB.Characterization,lung targeting profile and therapeutic efficiency of dipyridamole liposomes[J].J Drug Target,2006,14(10):717-724.
[72]王健松,朱家壁,吕瑞勤等.肺靶向阿奇霉素脂质体的制备及其在小鼠体内的分布[J].药学学报,2005,40(3):274-278.
[73]Wang JP,Maitani Y,Takayama K,et al.Pharmacokinetics and antitumor effect of doxorubicin carried by stealth and remote loading proliposome[J].Pharm Res,2000,17(7):782-787.