免疫脂质体作为靶向给药系统的研究进展

免疫脂质体是用抗体或其片段修饰的脂质体,能与靶细胞表面抗原或受体结合,从而对靶细胞具有分子水平上的识别能力。与游离药物、非特异抗体脂质体、单独单抗等相比,免疫脂质体有更好的选择性和更强的杀伤活性。在动物体内,免疫脂质体可使药物特异性分布在病灶部位,从而增强药物疗效、减轻不良反应,并且表面聚乙二醇化还增强了体内的循环时间。本文综述了用于修饰的不同种类抗体、抗体与免疫脂质体偶联方式,并总结了免疫脂质体在抗肿瘤药、基因治疗、活体成像技术以及在传染病、自身免疫和神经退行性疾病治疗方面的应用。     

八聚精氨酸修饰的载紫杉醇脂质体的制备工艺研究

目的 研究八聚精氨酸(R8)修饰的载紫杉醇脂质体的制备工艺.方法 采用有机相反应法,将R8修饰到脂质体表面,并以薄膜分散-探头超声法制备载紫杉醇脂质体,以细胞摄取、粒径、PDI、包封率为主要指标对磷脂/胆固醇、药脂比、水化液种类、R8密度、DSPE-PEG2000密度进行筛选,进一步优化处方.结果 最优处方为磷脂-胆固醇2∶1、药脂比1∶40、水化液为PBS(pH6.5)、R8密度5％、DSPE-PEG2000密度3％,所制脂质体的粒径为156 ±2 nm,PDI＝ 0.25,包封率为80.87％±8.9％.结论 成功制备了八聚精氨酸(R8)修饰的载紫杉醇脂质体.     

新型阿霉素热敏脂质体的研制

目的:探讨以二棕榈酰磷脂酰胆碱(DPPC)和单棕榈酰磷脂酰胆碱(MPPC)为原料制备的温度敏感阿霉素脂质体的制备工艺和理化性质.方法:采用膜过滤挤压法和pH梯度法制备阿霉素脂质体,以紫外分光光度法检测样本中阿霉素含量,计算阿霉素热敏脂质体在不同温度下的药物释放特性,并对其包裹率、粒径,pH值进行研究.结果:阿霉素热敏脂质体在39℃下迅速释放,前20 s内释放药物50%,42℃下药物释放达到60%以上.包封率99.5%,平均粒径90.8 nm,pH值为6.5.结论:制备的热敏感阿霉素脂质体优于常规脂质体,具有良好的温度控释特性.     

载基因脂质体用于治疗肿瘤的最新研究进展

基因传递是基因治疗中极具挑战性的领域,尤其是当全身给药时,生理学和生物学屏障是需要考虑的主要障碍。脂质体作为基因载体在治疗肿瘤中拥有独特优势,近几年备受关注。研究者们对脂质体的新制备材料、制备工艺、修饰方法等进行了广泛的研究,以期进一步提高其靶向性,降低其毒副作用。除了阳离子脂质体、长循环脂质体、pH敏感脂质体、免疫脂质体以及受体介导的脂质体,本文重点强调了新型的细胞穿膜肽修饰的脂质体在治疗肿瘤方面的最新研究进展。     

脂质体面临的聚乙二醇“窘境”及其解决方法

聚乙二醇(polyethylene glycol,PEG)因其优越的性能被广泛应用于脂质体等纳米载体表面修饰,但随着研究的深入,PEG化脂质体也产生了相应的负面影响,如PEG链抑制靶细胞对脂质体的摄取、妨碍p H敏感脂质体细胞内的"核内体逃逸"以及对同一动物体内重复注射PEG化脂质体诱发的加速血液清除(accelerated blood clearance,ABC)现象等,这些称为PEG"窘境"(PEG dilemma)。PEG"窘境"为携载抗肿瘤药物的PEG化脂质体的靶向递送、包封基因和蛋白类药物的PEG化p H敏感脂质体发生有效的细胞内释放以及需要重复注射的PEG化脂质体都带来了严峻的挑战,因此解决PEG"窘境"刻不容缓。本文对PEG"窘境"的定义、分类和几种克服PEG"窘境"的方法进行了综述。     

葡萄糖受体靶向的钆喷酸葡胺长循环脂质体的肿瘤细胞靶向研究

目的：制备葡萄糖受体靶向的钆喷酸葡胺（GdDTPA）长循环脂质体，并研究其对高表达葡萄糖受体肿瘤细胞的靶向性。方法：合成新型含葡萄糖表面活性剂（N-棕榈酰葡萄糖胺），利用逆向蒸发法制备Gd—DTPA脂质体、PEG修饰Gd—DTPA脂质体、葡萄糖修饰Gd-DTPA脂质体以及葡萄糖PEG修饰GdDTPA脂质体。将4种脂质体分别与前列腺癌细胞一起培养，用MRI检测前列腺癌细胞摄取的Gd-DTPA浓度。结果：成功合成新型含葡萄糖表面活性荆（N棕榈酰葡萄糖胺），以及制备4种Gd-DTPA脂质体。前列腺癌细胞对4种脂质体都有摄取，每种脂质体MRI检测的SBTIW信号值分别为Gd-DTPA脂质体362、PEG修饰Gd-DTPA脂质体299、葡萄糖修饰GdDTPA脂质体397、葡萄糖PEG修饰GdDTPA脂质体377。结论：葡萄糖的修饰有利于脂质体对肿瘤细胞的靶向。     

吐温80对9-硝基喜树碱脂质体药物动力学性质的影响

目的:考察吐温80对9-硝基喜树碱(9-NC)脂质体体内药物动力学以及内酯型/羧酸盐型平衡的影响。方法:采用薄膜法制备9-NC脂质体以及吐温80修饰的9-NC脂质体;12只大鼠随机分为两组,按1.5 mg.kg-1剂量分别给予9-NC普通脂质体和吐温80修饰的脂质体,于不同时间点取血,处理后测定9-NC内酯型浓度和总浓度(内酯/羧酸盐)。采用统计矩模型利用3P97程序计算药物动力学参数。结果:采用表面活性剂吐温80进行修饰后,9-NC内酯型和总浓度的AUC分别提高了1.47倍和1.65倍,内酯型和总浓度的清除率CL和表观分布容积Vss显著下降(P<0.01)。此外,总浓度的MRT以及t1/2延长(P<0.05)。结论:吐温80修饰使得9-NC内酯型比例有所下降,但没有显著性差异,9-NC吐温修饰对9-NC脂质体具有一定的长循环效果。     

不同表面活性剂对伊文思蓝脂质体体内外性质的影响

目的　寻找与二硬脂酰磷脂酰乙醇胺 聚乙二醇 (DSPE PEG)功能相似的表面活性剂 ,以增加脂质体的稳定性 ,改善其体内分布 ,提高靶向性。方法　制备伊文思蓝 (EB)脂质体 ,考察胆固醇与磷脂的比例对伊文思蓝脂质体包封率的影响 ;比较用DSPE PEG、吐温 80 (Tween 80 )和苄泽 3 5 (Brij 3 5 )修饰后的EB脂质体的包封率和在大鼠体内组织分布状况的变化。结果　EB脂质体的包封率最高为 2 5 3 0 %。用DSPE PEG、Tween 80和Brij 3 5修饰后使EB脂质体的包封率略有下降 ,但差别不显著 ;体内分布实验结果显示 :修饰后的脂质体在肝、脾和肾中EB的浓度均有不同程度的降低 ,脑中EB的浓度有所提高 ,而且以Tween 80修饰组最显著。结论　DSPE PEG、Tween 80和Brij 3 5对EB脂质体的包封率影响较小。Brij 3 5对EB脂质体的作用与DSPE PEG相似 ,能提高脂质体逃避网状内皮系统吞噬的能力 ;Tween 80主要能增加EB脂质体在大鼠脑组织中的分布 ,为脑靶向脂质体的研究提供了有益信息     

脂质体作为抗肿瘤药物载体的应用研究

目前，应用脂质体作为抗肿瘤药物载体已成为趋势。脂质体可明显提高抗肿瘤药物的靶向性，延长药物的作用时间，降低药物毒性。作者对国内外普通脂质体和修饰脂质体（包括长循环脂质体、免疫脂质体、温度敏感脂质体和pH敏感脂质体）的相关文献进行了综述。结果表明，脂质体是抗肿瘤药物的理想载体，在肿瘤治疗中有着广阔的应用前景。     

乳糖化-去甲斑蝥素磷脂复合物及其pH敏感型脂质体的制备

目的:合成乳糖化-去甲斑蝥素磷脂复合物,并制备其pH敏感型脂质体。方法:将乳糖化-去甲斑蝥素与磷脂聚合成药物磷脂复合物,并采用FT-IR、DSC和1H-NMR对其进行表征。逆向蒸发法制备药物磷脂复合物脂质体;利用羧甲基壳聚糖与脂质体表面的静电吸附作用,使羧甲基壳聚糖吸附在脂质体表面,制备乳糖化-去甲斑蝥素磷脂复合物pH敏感型脂质体;考察了药物与磷脂的复合率,磷脂复合物脂质体的包封率,粒径大小和分布,以及体外释药特性。结果:药物磷脂复合率为(97.2±2.01)%,磷脂复合物脂质体的平均包封率为(70.00±1.30)%,平均粒径为(47.18±4.16)nm,粒径跨距为(0.70±0.07),电镜显示其形态圆整,体外释药符合Weibull方程。结论:乳糖化-去甲斑蝥素磷脂复合率高,制成的pH敏感型脂质体性质稳定,且具有缓释特性。     

Long-circulating, pH-sensitive liposomes sterically stabilized by copolymers bearing short blocks of lipid-mimetic units

A major hurdle towards in vivo utilization of pH-sensitive liposomes is their prompt sequestration by reticuloendothelial system and hence short circulation time. Prolonged circulation of liposomes is usually achieved by incorporation of pegylated lipids, which have been frequently reported to deteriorate the acid-triggered release. In this study we evaluate the ability of four novel nonionic copolymers, bearing short blocks of lipid-mimetic units to provide steric stabilization of DOPE:CHEMs liposomes. The vesicles were prepared using the lipid film hydration method and extrusion, yielding liposomes of 120–160 nm in size. Their pH-sensitivity was monitored via the release of encapsulated calcein. The incorporation of the block copolymers at concentration up to 10 mol% did not deteriorate the pH-sensitivity of the liposomes. A selected formulation was tested for stability in presence of 25% human plasma and proved to significantly outclass the plain DOPE:CHEMs liposomes. The ability of calcein-loaded liposomes to deliver their cargo inside EJ cells was investigated using fluorescent microscopy and the results show that the surface-modified vesicles are as effective to ensure intracellular delivery as plain liposomes. The pharmacokinetics and organ distribution of a selected formulation, containing a copolymer bearing four lipid anchors was investigated in comparison to plain liposomes and PEG (2000)–DSPE stabilized liposomes. The juxtaposition of the blood clearance curves and the calculated pharmacokinetic parameters show that the block copolymer confers superior longevity in vivo. The block copolymers utilized in this study can be consider as promising sterically stabilizing agents for pH-sensitive liposomes.     

On the formulation of pH-sensitive liposomes with long circulation times

Strategies used to enhance liposome-mediated drug delivery in vivo include the enhancement of stability and circulation time in the bloodstream, targeting to specific tissues or cells, and facilitation of intracytoplasmic delivery. pH-sensitive liposomes have been developed to mediate the introduction of highly hydrophilic molecules or macromolecules into the cytoplasm. These liposomes destabilize under acidic conditions found in the endocytotic pathway, and usually contain phosphatidylethanolamine (PE) and titratable stabilizing amphiphiles. Formulations without PE have also been developed. Encapsulated compounds are thought to be transported into the cytoplasm through destabilization of or fusion with the endosome membrane. Incorporation of a low mole percentage of poly(ethylene glycol) (PEG)-conjugated lipids into pH-sensitive liposomes confers prolonged circulation times to these liposomes, which are otherwise cleared rapidly. While the incorporation of PEG-lipids reduces the pH-dependent release of encapsulated fluorescent markers in vitro, it does not hinder the cytoplasmic delivery of the markers per cell-associated liposome. This suggests that intracellular delivery is not dictated simply by the destabilization of the liposomes. Antibodies or ligands to cell surface receptors can be coupled to pH-sensitive or sterically stabilized pH-sensitive liposomes for targeting. pH-sensitive liposomes have been used to deliver anticancer drugs, antibiotics, antisense oligonucleotides, ribozymes, plasmids, proteins and peptides to cells in culture or in vivo.     

In vitro and in vivo evaluation of sanguinarine liposomes prepared by a remote loading method with three different ammonium salts

Sanguinarine liposomes were prepared by a remote loading method using three different ammonium salts. A series of studies, including in vitro release, in vitro and in vivo anti-tumor effects and pharmacokinetics in rats, were conducted. The three liposomes showed pH-sensitive release characteristics in vitro, but there were obvious variations in their release profiles. Among the three liposomes, the liposomes made using ammonium citrate and phosphate possessed better anti-tumor activity in vitro and in vivo, compared with the liposome using ammonium sulfate. Pharmacokinetics test results in rats indicated that sanguinarine liposomes have notably elevated AUC (P<0.05) and markedly lower CL (P<0.05) compared with the solution, but there were no obvious differences between the three liposomes. The present study may be useful for better understanding and better choice of a suitable ammonium salt for the remote loading method.     

pH-sensitive liposomes--principle and application in cancer therapy

The purpose of this review is to provide an insight into the different aspects of pH-sensitive liposomes. The review consists of 6 parts: the first introduces different types of medications made in liposomal drug delivery to overcome several drawbacks; the second elaborates the development of pH-sensitive liposomes; the third explains diverse mechanisms associated with the endocytosis and the cytosolic delivery of the drugs through pH-sensitive liposomes; the fourth describes the role and importance of pH-sensitive lipid dioleoylphosphatidylethanolamine (DOPE) and research carried on it; the fifth explains successful strategies used so far using the mechanism of pH sensitivity for fusogenic activity; the final part is a compilation of research that has played a significant role in emphasizing the success of pH-sensitive liposomes as an efficient drug delivery system in the treatment of malignant tumours. pH-Sensitive liposomes have been extensively studied in recent years as an amicable alternative to conventional liposomes in effectively targeting and accumulating anti-cancer drugs in tumours. This research suggests that pH-sensitive liposomes are more efficient in delivering anti-cancer drugs than conventional and long-circulating liposomes due to their fusogenic property. Research focused on the clinical and therapeutic side of pH-sensitive liposomes would enable their commercial utility in cancer treatment.     

Steric stabilization of liposomes by pH-responsive N-isopropylacrylamide copolymer

The aim of this study was to characterize a pH-sensitive liposome formulation bearing a terminally alkylated N-isopropylacrylamide (NIPAM) copolymer with regard to its pH responsiveness, surface properties, and pharmacokinetics. The interacting forces between two lipid bilayers bearing the anchored NIPAM copolymer were measured with a surface force apparatus. The pH-triggered content release was evaluated in buffer before and after incubation in human serum. The pharmacokinetics was determined in rats following the intravenous injection of 67Ga-loaded liposomes with or without the polymer coating. The force measurements between lipid bilayers showed that NIPAM copolymers provide a steric barrier that was dependent on pH. The pH-sensitive liposomes maintained their pH sensitivity after incubation in serum. In vivo, the polymer-coated liposomes exhibited a prolonged circulation time in rats, with an area under the blood concentration-time curve that is 1.6-fold higher than the control formulation. This study showed that liposomes can be rendered pH sensitive by anchoring a terminally alkylated NIPAM copolymer at their surface. At neutral pH, the polymer provides a steric barrier that increases the liposome circulation time in vivo.     

Estrogen-anchored pH-sensitive liposomes as nanomodule designed for site-specific delivery of doxorubicin in breast cancer therapy

The present investigation reports the development of nanoengineered estrogen receptor (ER) targeted pH-sensitive liposome for the site-specific intracellular delivery of doxorubicin (DOX) for breast cancer therapy. Estrone, a bioligand, was anchored on the surface of pH-sensitive liposome for drug targeting to ERs. The estrone-anchored pH-sensitive liposomes (ES-pH-sensitive-SL) showed fusogenic potential at acidic pH (5.5). In vitro cytotoxicity studies carried out on ER-positive MCF-7 breast carcinoma cells revealed that ES-pH-sensitive-SL formulation was more cytotoxic than non-pH-sensitive targeted liposomes (ES-SL). The flow cytometry analysis confirmed significant enhanced uptake (p < 0.05) of ES-pH-sensitive-SL by MCF-7 cells. Intracellular delivery and nuclear localization of the DOX was confirmed by fluorescence microscopy. The mechanism for higher cytotoxicity shown by estrone-anchored pH-sensitive liposomal-DOX was elucidated using reactive oxygen species (ROS) determination. The in vivo biodistribution studies and antitumor activities of formulations were evaluated on tumor bearing female Balb/c mice followed by intravenous administration. The ES-pH-sensitive-SL efficiently suppressed the breast tumor growth in comparison to both ES-SL and free DOX. Serum enzyme activities such as LDH and CPK levels were assayed for the evaluation of DOX induced cardiotoxicity. The ES-pH-sensitive-SL accelerated the intracellular trafficking of encapsulated DOX, thus increasing the therapeutic efficacy. The findings support that estrone-anchored pH-sensitive liposomes could be one of the promising nanocarriers for the targeted intracellular delivery of anticancer agents to breast cancer with reduced systemic side effects.     

Efficiency of Cytoplasmic Delivery by pH-Sensitive Liposomes to Cells in Culture

The intracellular processing of pH-sensitive liposomes composed of cholesterylhemisuccinate (CHEMS) and dioleoylphosphatidylethanolamine (DOPE) by eukaryotic cell lines has been compared to non-pH-sensitive liposomes made of CHEMS and dioleoylphosphatidylcholine (DOPC). The pH-sensitive liposomes can deliver encapsulated fluorescent molecules [calcein, fluoresceinated dextran, fluoresceinated polypeptide, and diphtheria toxin A chain (DTA)] into the cytoplasm. Cytoplasmic delivery can be blocked in the presence of ammonium chloride or EDTA, indicating that the process requires a low-pH environment and the presence of divalent cations. Inhibition of cellular protein synthesis by DTA delivery from the pH-sensitive liposome is orders of magnitude greater than from the non-pH-sensitive liposome composition. The delivery of DTA into the cytoplasm by pH-sensitive liposomes is at least 0.01% of cell-associated liposomal DTA. There is no significant difference in the degradation rate of bovine serum albumin (BSA) or the rate of acidification of pH-sensitive dye, 8-hydroxy-l,3,6-pyrene-trisulfonate (HPTS), when delivered to cells in pH-sensitive and non-pH-sensitive liposomes. Thus the efficiency of cytoplasmic delivery is less than 10% of the cell-associated liposome contents, which is the smallest difference that can be detected by these two assays. Based upon the various assays used to measure liposome content disposition in the cell, we conclude that the efficiency of cytoplasmic delivery by the CHEMS/DOPE liposomes is greater than 0.01% and less than 10% of the cell-associated liposomal contents.     

pH-sensitive, serum-stable and long-circulating liposomes as a new drug delivery system

The lack of stability in blood and the short blood circulation time of pH-sensitive liposomes are major drawbacks for their application in-vivo. To develop pH-sensitive, serum-stable and long-circulating liposomes as drug delivery systems, the impact of polyethylene glycol-derived phosphatidylethanolamine (DSPE-PEG) on the properties of pH-sensitive liposomes was investigated. pH-sensitive liposomes were prepared with dioleoylphosphatidylethanolamine (DOPE) and oleic acid (DOPE/oleic acid liposome) or DOPE and 1,2-dipalmitoylsuccinylglycerol (DOPE/DPSG liposome). The inclusion of DSPE-PEG enhanced the serum stability of both DOPE/oleic acid and DOPE/DPSG liposomes, but also shifted the pH-response curve of pH-sensitive liposomes to more acidic regions and reduced the maximum leakage percentage. The impact of DSPE-PEG, however, was much lower in the DOPE/DPSG liposomes than in the DOPE/oleic acid liposomes. In tumour tissue homogenates, where the pH is lower than normal healthy tissues, the pH-sensitive DOPE/DPSG liposomes released the entrapped markers rapidly, in comparison with pH-insensitive dipalmitoylphosphatidylcholine/cholesterol/DSPE-PEG liposomes. Moreover, the release rate was not affected by the content of DSPE-PEG. The blood circulation time of methotrexate incorporated in DOPE/UDPSG liposomes was significantly prolonged with increasing content of DSPE-PEG. Taken together, the liposomes composed of DOPE, DPSG and DSPE-PEG (up to 5%) were pH sensitive, plasma stable and had a long circulation time in the blood. The complete destabilization of the liposomes at tumour tissues suggests that the liposomes might be useful for the targeted delivery of drugs such as anticancer agents.     

Delivery of Plasmid DNA into Mammalian Cell Lines Using pH-Sensitive Liposomes: Comparison with Cationic Liposomes

We compare the transfection efficiency of plasmid DNA encoding either luciferase or (-galactosidase encapsulated in pH-sensitive liposomes or non-pH-sensitive liposomes or DNA complexed with cationic liposomes composed of dioleoyloxypropyl-trimethylammonium:dioleoylphosphatidyl-ethanolamine (1:1, w/w) (Lipofectin) and delivered into various mammalian cell lines. Cationic liposomes mediate the highest transient transfection level in all cell-lines examined. pH-sensitive liposomes, composed of cholestryl hemisuccinate and dioleoylphosphatidylethanolamine at a 2:1 molar ratio, mediate gene transfer with efficiencies that are 1 to 30% of that obtained with cationic liposomes, while non-pH-sensitive liposome compositions do not induce any detectable transfection. Cationic liposomes mediate a more rapid uptake of plasmid DNA, to about an eightfold greater level than that obtained with pH-sensitive liposomes. The higher uptake of DNA mediated by Lipofectin accounts for part of its high transfection efficiency. Treatment of cells with chloroquine, ammonium chloride, or monensin decreases (threefold) transfection using pH-sensitive liposomes and either has no effect on or enhances cationic liposome-mediated transfection. Therefore plasma membrane fusion is not the only mechanism available to cationic liposomes; in certain cell lines DNA delivery via endocytosis is a possible parallel pathway and could augment the superior transfection efficiency observed with cationic liposomes.     

A review of mechanistic insight and application of pH-sensitive liposomes in drug delivery

Abstract

The pH-sensitive liposomes have been extensively used as an alternative to conventional liposomes in effective intracellular delivery of therapeutics/antigen/DNA/diagnostics to various compartments of the target cell. Such liposomes are destabilized under acidic conditions of the endocytotic pathway as they usually contain pH-sensitive lipid components. Therefore, the encapsulated content is delivered into the intracellular bio-environment through destabilization or its fusion with the endosomal membrane. The therapeutic efficacy of pH-sensitive liposomes enables them as biomaterial with commercial utility especially in cancer treatment. In addition, targeting ligands including antibodies can be anchored on the surface of pH-sensitive liposomes to target specific cell surface receptors/antigen present on tumor cells. These vesicles have also been widely explored for antigen delivery and serve as immunological adjuvant to enhance the immune response to antigens. The present review deals with recent research updates on application of pH-sensitive liposomes in chemotherapy/diagnostics/antigen/gene delivery etc.