新型脂质体的可控制备及载药研究
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摘要
脂质体(liposomes)是一种新型药物载体,具有生物相容性好、可生物降解、无毒和无免疫原性等优点。且作为药物载体,可以提高药物治疗指数,降低药物毒副作用、降低药物剂量。但脂质体不太稳定,储存过程中易发生药物泄漏、脂质体聚集和破裂等,难以满足临床用药的要求。本课题正是基于对这一问题的考虑设计了可能具有较高稳定性和较高包封率的脂质体—新型脂质体,即在脂质体内部引入树枝状的骨架结构(dendrimer),使树枝状分支的末端链段掺入磷脂双分子层而形成类似脂质体的囊泡。而树枝状聚合物(dendrimer)的出现为这一研究设想提供了现实的可行性。为将树枝状聚合物末端有效掺入脂质双层,首先需聚合物末端疏水化修饰。本课题选择G3.5代的PAMAM dendrimer,将其末端羧基用DCC和NHS活化后与硬脂胺进行活泼酯反应,得到PAMAM dendrimer G3.5脂质小球。然后,采用薄膜超声法,以氢化豆磷脂、胆固醇以及PAMAM dendrimer G3.5脂质小球成功制备了新型脂质体。采用正交设计方法对最佳制备工艺进行实验,以氢化磷脂与胆固醇比例,氢化磷脂与PAMAM dendrimer G3.5脂质小球的比例,旋转蒸发温度,水化温度为4个因素,粒径为考察指标,筛选出新型脂质体最佳制备工艺。以鬼臼毒素为模型药物制备了载药新型脂质体,以透射电镜(TEM)观察形态,动态光散射(DLS)考察粒径分布,用葡聚糖凝胶柱分离法测定包封率,采用正交设计方法对最佳制备工艺进行实验,以氢化磷脂与胆固醇比例,氢化磷脂与PAMAM dendrimer G3.5脂质小球的比例,氢化磷脂与鬼臼毒素的比例,旋转蒸发温度为4个因素,包封率为考察指标,筛选出鬼臼毒素新型脂质体最佳制备工艺。并考察其对温度、乙醇、酸碱的耐受性,和体外经皮通透性的研究。还进行了鬼臼毒素新型脂质体的质量标准的考察。结果发现,优选出新型脂质体的制备工艺为氢化磷脂：胆固醇=20：3.26,氢化磷脂：PAMAM dendrimer G3.5脂质小球=20：0.5,氢旋转蒸发温度为55℃,水化温度为45℃,新型脂质体粒径均匀,平均粒径61.1nm；优选出鬼臼毒素新型脂质体的制备工艺为氢化磷脂：胆固醇=20：3.67,氢化磷脂：PAMAM dendrimerG3.5脂质小球=20：0.5,氢化磷脂：鬼臼毒素=20：1.5,旋转蒸发温度为45℃,测得该脂质体包封率为91.45%。稳定性试验证明鬼臼毒素新型脂质体能够耐受热压灭菌(115℃,67 kPa,30 min)、最终浓度为50%的乙醇、最终浓度为1 M的盐酸和最终浓度为0.1 M的盐酸；在体外经皮通透性研究中,达到了对药物释放的缓释作用；制备的鬼臼毒素新型脂质体在鬼臼毒素含量及分布的均一性方面均较为理想。综上所述,我们得知,和传统脂质体相比,新型脂质体提高了脂质体的包封率,稳定性,可以使装载的药物更好地发挥作用。
Liposomes are novel drug carriers, as they have good biocompatibility, biodegradability, innocuity, non-immunogenicity. As a drug carrier, they can elevate the therapeutic exponent, reduce their untoward effects, and decrease the dosage of the medicine. But liposomes are less stable. During its storage, the drug may be detrayed and liposomes may be aggregated, ruptured, and so on. These may lead to not meet the demand of clinical medication. Based on these problems, a novel drug carrier-liposomes-like with good stability and higher entrapment efficiency was designed by our studying team. Namely a dendritic framework structure was introduced into liposomes, which can make terminal chain segment of dendritic branch incorporate phospholipid bilayers and perform a vesicle similar to liposomes. The appearance of dendrimer provides realistic feasibility for the design. In order to introduce the dendritic framework structure into liposomes effectively, the terminal chain segment of dendrimer should be modified hydrophobically. PAMAM Dendrimer G3.5 was choosed, the carboxyls on terminal chain segment were activated with DCC and NHS, then the coupling reactionwere with stearylamine was formed via active ester. Finally the lipid globule of PAMAM dendrimer G3.5 were prepared successfully. We prepared the liposomes-like using hydrogenated soybean lecithin, cholesterol and the lipid globule of PAMAM dendrimer G3.5 by film dispersed method (TFDM) successfully. The optimization of the new type podophyllotoxin liposome preparation was performed based on the orthogonal experimental design. In this study, the size of the new type podophyllotoxin liposome were taken as the index and the influence of factors such as the proportion of hydrogenated phospholipid and Cholesterol, the proportion of hydrogenated phospholipid and PAMAM dendrimer G3.5 lipid globule, the temperature of rotary evaporation and the temperature of hydration. We prepared drug-loaded liposomes-like with Podophyllotoxin as a model drug. The shape of liposomes were observed by transmission electron microscopy (TEM), and the particles size was evaluated by dynamic scattering (DLS). The entrapment efficiency was determined using dextran gel column separation. The optimization of the new type podophyllotoxin liposome preparation was performed based on the orthogonal experimental design. In this study, the entrapment efficiency of the new type podophyllotoxin liposome were taken as the index and the influence of factors such as the proportion of hydrogenated phospholipid and Cholesterol, the proportion of hydrogenated phospholipid and PAMAM dendrimer G3.5 lipid globule, the proportion of hydrogenated phospholipid and podophyllotoxin and the temperature of rotary evaporation. Tolerance to temparature, ethanol and acid-base were studied, too. And vitro percutaneous permeability was studied. We also studied the Quality Standards inspection of the new type podophyllotoxin liposome. The results showed that the best formulation of the new type podophyllotoxin liposome were the proportion of hydrogenated phospholipid and Cholesterol was 20:3.26, the proportion of hydrogenated phospholipid and PAMAM dendrimer G3.5 lipid globule was 20: 0.5, the temperature of rotary evaporation was 55℃and the temperature of hydration was 45℃. And liposomes-like were successfully prepared with an average diameter of 61.1 run. The best formulation of the new type podophyllotoxin liposome were the proportion of hydrogenated phospholipid and Cholesterol was 20：3.67, the proportion of hydrogenated phospholipid and PAMAM dendrimer G3.5 lipid globule was 20:0.5, the proportion of hydrogenated phospholipid and podophyllotoxin was 20：1.5 and the temperature of rotary evaporation was 45℃. And the encapsulation efficiency was 91.45%. Curcumin liposomes-like were tolerant of steam sterilization (115℃、67kPa、30min),50% ethanol (final concentration),1M Hydrochloride (final concentration) and 0.1M NaOH (final concentration). In vitro percutaneous permeability study, it achieved the sustained-release effect of the drug release. In the podophyllotoxin content and distribution are more ideal in terms of uniformity. Conclusion demonstrated that liposomes-like have higher entrapment efficiency and better stability than traditional liposomes, and make the loaded drug play its role better.
Liposomes are novel drug carriers, as they have good biocompatibility, biodegradability, innocuity, non-immunogenicity. As a drug carrier, they can elevate the therapeutic exponent, reduce their untoward effects, and decrease the dosage of the medicine. But liposomes are less stable. During its storage, the drug may be detrayed and liposomes may be aggregated, ruptured, and so on. These may lead to not meet the demand of clinical medication. Based on these problems, a novel drug carrier-liposomes-like with good stability and higher entrapment efficiency was designed by our studying team. Namely a dendritic framework structure was introduced into liposomes, which can make terminal chain segment of dendritic branch incorporate phospholipid bilayers and perform a vesicle similar to liposomes. The appearance of dendrimer provides realistic feasibility for the design. In order to introduce the dendritic framework structure into liposomes effectively, the terminal chain segment of dendrimer should be modified hydrophobically. PAMAM Dendrimer G3.5 was choosed, the carboxyls on terminal chain segment were activated with DCC and NHS, then the coupling reactionwere with stearylamine was formed via active ester. Finally the lipid globule of PAMAM dendrimer G3.5 were prepared successfully. We prepared the liposomes-like using hydrogenated soybean lecithin, cholesterol and the lipid globule of PAMAM dendrimer G3.5 by film dispersed method (TFDM) successfully. The optimization of the new type podophyllotoxin liposome preparation was performed based on the orthogonal experimental design. In this study, the size of the new type podophyllotoxin liposome were taken as the index and the influence of factors such as the proportion of hydrogenated phospholipid and Cholesterol, the proportion of hydrogenated phospholipid and PAMAM dendrimer G3.5 lipid globule, the temperature of rotary evaporation and the temperature of hydration. We prepared drug-loaded liposomes-like with Podophyllotoxin as a model drug. The shape of liposomes were observed by transmission electron microscopy (TEM), and the particles size was evaluated by dynamic scattering (DLS). The entrapment efficiency was determined using dextran gel column separation. The optimization of the new type podophyllotoxin liposome preparation was performed based on the orthogonal experimental design. In this study, the entrapment efficiency of the new type podophyllotoxin liposome were taken as the index and the influence of factors such as the proportion of hydrogenated phospholipid and Cholesterol, the proportion of hydrogenated phospholipid and PAMAM dendrimer G3.5 lipid globule, the proportion of hydrogenated phospholipid and podophyllotoxin and the temperature of rotary evaporation. Tolerance to temparature, ethanol and acid-base were studied, too. And vitro percutaneous permeability was studied. We also studied the Quality Standards inspection of the new type podophyllotoxin liposome. The results showed that the best formulation of the new type podophyllotoxin liposome were the proportion of hydrogenated phospholipid and Cholesterol was 20:3.26, the proportion of hydrogenated phospholipid and PAMAM dendrimer G3.5 lipid globule was 20: 0.5, the temperature of rotary evaporation was 55℃and the temperature of hydration was 45℃. And liposomes-like were successfully prepared with an average diameter of 61.1 run. The best formulation of the new type podophyllotoxin liposome were the proportion of hydrogenated phospholipid and Cholesterol was 20：3.67, the proportion of hydrogenated phospholipid and PAMAM dendrimer G3.5 lipid globule was 20:0.5, the proportion of hydrogenated phospholipid and podophyllotoxin was 20：1.5 and the temperature of rotary evaporation was 45℃. And the encapsulation efficiency was 91.45%. Curcumin liposomes-like were tolerant of steam sterilization (115℃、67kPa、30min),50% ethanol (final concentration),1M Hydrochloride (final concentration) and 0.1M NaOH (final concentration). In vitro percutaneous permeability study, it achieved the sustained-release effect of the drug release. In the podophyllotoxin content and distribution are more ideal in terms of uniformity. Conclusion demonstrated that liposomes-like have higher entrapment efficiency and better stability than traditional liposomes, and make the loaded drug play its role better.
引文
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[3]Minko T, Pakunlu RI, Wang Y, Khandare JJ, Saad M. New generation of liposomal drugs for cancer. Anticancer Agents Med Chem,2006; 6(6):537～52
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