曲妥珠单抗修饰的阿霉素免疫脂质体的制备及体外性质研究

目的:制备表面键合曲妥珠单抗(trastuzumab,TMAB)的阿霉素免疫脂质(Doxorubicin-loadedimmunoliposome,DOX-IML),并对其体外性质进行研究。方法:将磷脂酰胆碱、胆固醇、阿霉素、DSPE-MPEG2000以一定比例混合,采用薄膜超声分散法制备阿霉素脂质体,将聚乙二醇衍生物(1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[succinimidyl(polyethylene glycol)-3400]、DSPE-PEG3400-NHS)连接到TMAB;再与阿霉素脂质体连接得到DOX-IML。研究不同浓度的TMAB对DOX-IML入胞能力及细胞毒性的影响;测定免疫脂质体的包封率、载药率、粒径、电荷及稳定性等性质;动态透析法模拟体外释药特性,激光共聚焦观察免疫脂质体对AU565细胞抗体介导的入胞作用;MTT法研究DOX-IML抑制肿瘤细胞的生长。结果:成功制备了表面键合TMAB的阿霉素免疫脂质体,配体载入率分别是53%、75.5%、84%;每毫克DOX-IML中抗体的含量分别是37、83、108μg·mg-1;阿霉素的包封率为76.85%、载药量为8.03%;粒径131.8nm;表面电荷-27mV。抗体含量83μg·mg-1的DOX-IML组的细胞存活率最低,细胞内荧光强度最高,且该免疫脂质体稳定性良好,具有一定缓释作用。DOX-IML具有较强的特异性靶向作用,其入胞能力和细胞毒性均高于阿霉素脂质体。结论:DOX-IML具有较强的特异性靶向作用,其入胞能力和细胞毒性均高于阿霉素脂质体,抗体含量适中时其入胞能力和细胞毒性最强。     

盐酸米托蒽醌聚乙二醇化脂质体的制备及包封率考察

目的:制备盐酸米托蒽醌聚乙二醇化(PEG化)脂质体,建立包封率测定方法.方法:采用乙醇注入结合高压均质法制备空白PEG化脂质体;以铵根离子梯度法进行主动载药制备盐酸米托蒽醌PEG化脂质体;采用G-25葡聚糖凝胶色谱分离脂质体和游离药物;使用紫外-可见分光光度法测定脂质体的包封率.结果:空白脂质体平均粒径为88.7nm,载药后粒径为95.3nm;在所建立色谱条件下,脂质体与游离米托蒽醌分离良好;盐酸米托蒽醌在0.5～10μg·ml-1范围内线性关系良好(R 2=0.9997),精密度高;脂质体的平均包封率大于96％.结论:乙醇注入-高压均质法结合铵根离子主动载药法适用于制备盐酸米托蒽醌PEG化脂质体;所建立分析方法简单快捷、准确可靠,可用于盐酸米托蒽醌长循环脂质体包封率的测定.     

人表皮细胞生长因子脂质体制备及促大鼠烫伤修复研究

目的:制备重组人表皮细胞生长因子(rhEGF)脂质体,并考察其促大鼠烫伤创面愈合的作用.方法:采用pH梯度法制备rhEGF脂质体;超滤-离心法分离rhEGF脂质体混悬液中的游离rhEGF,ELISA法测定rhEGF含量,计算脂质体包封率;采用透射电镜观察脂质体的外观形态;采用纳米粒度及Zeta电位分析仪分别测定脂质体的粒径和Zeta电位;以大鼠烫伤模型观察给药后各试验组创面愈合过程中的形态、愈合时间和愈合率的变化.结果:制备的rhEGF脂质体包封率为57.7±1.1％;脂质体形状较为规则,呈完整圆球形或椭圆形的单室囊泡;脂质体粒度分布均匀,呈正态分布,平均粒径为63.7 nm;脂质体的Zeta电位为+9.2mV,带正电荷;rhEGF脂质体高、中剂量组能显著性促进大鼠烫伤创面愈合,使创面愈合时间明显提前,低剂量组促烫伤修复效应不明显.结论:pH梯度法制备的rhEGF脂质体包封率较高,rhEGF脂质体对大鼠烫伤创面的愈合有明显促进作用.     

盐酸洛拉曲克脂质体的制备及其质量考察

目的:制备盐酸洛拉曲克脂质体并考察其理化特性。方法:采用薄膜挤压-硫酸铵梯度法制备盐酸洛拉曲克脂质体,透射电镜及激光粒度分析仪分别观察和检测其粒径大小及分布,通过紫外分光光度法测定包封率及评估体外释药试验。结果:制备的盐酸洛拉曲克脂质体包封率达83.6%±2.37%,粒径103.5±26nm且分布均匀。24h体外释放实验结果提示约有66.5%的盐酸洛拉曲克从脂质体释放出来。结论:新制备的盐酸洛拉曲克脂质体粒径大小均匀,包封率尚有提高空间,具有体外缓慢释药的特性。     

万古霉素脂质体的制备及质量考察

目的:制备万古霉素脂质体并考察其质量.方法:采用薄膜分散冻干法制备万古霉素脂质体,透射电镜观察其粒径分布,通过紫外分光光度法测定包封率和体外释要特性.结果:透射电镜结果显示脂质体粒径大小均匀,测得平均包封率为79.23%±4.13%,体外释放度实验结果提示,振荡24h后,约有65%的万古霉素药物从脂质体释放出来,体外抑菌试验显示,6周后仍然有抑菌圈的形成.结论:薄膜分散冻干法适于制备万古霉素脂质体,该制剂稳定性好,粒径大小均匀,包封率高,具有体外缓慢释药的特性.     

盐酸洛拉曲克脂质体的制备及其质量考察

目的：制备盐酸洛拉曲克脂质体并考察其理化特性。方法：采用薄膜挤压-硫酸铵梯度法制备盐酸洛拉曲克脂质体,透射电镜及激光粒度分析仪分别观察和检测其粒径大小及分布,通过紫外分光光度法测定包封率及评估体外释药试验。结果：制备的盐酸洛拉曲克脂质体包封率达83.6%±2.37%,粒径103.5±26nm且分布均匀。24h体外释放实验结果提示约有66.5%的盐酸洛拉曲克从脂质体释放出来。结论：新制备的盐酸洛拉曲克脂质体粒径大小均匀,包封率尚有提高空间,具有体外缓慢释药的特性。     

酸敏阿霉素脂质纳米粒的制备及其体外抗肿瘤活性研究

目的:本研究旨在制备具有被动靶向和酸敏特性的脂质混合纳米粒,以期提高阿霉素(doxorubicin,DOX)的靶向递药效率,降低DOX的毒副作用,提高抗肿瘤活性。方法:采用微乳法制备磷酸钙纳米粒核,薄膜分散法制备脂质混合纳米粒,硫酸铵梯度法包封DOX。采用透射电镜观察外观形态,用zeta电位及纳米粒度分析仪测定纳米粒的粒径及zeta电位,透析法评价阿霉素脂质纳米粒体外释药特征。用MTT方法研究阿霉素脂质混合纳米粒对A549细胞的细胞毒性。采用流式细胞仪和激光共聚焦显微镜观察A549细胞对阿霉素脂质纳米粒的摄取。结果:体外释药结果显示阿霉素脂质纳米粒具有酸敏特性。流式结果说明A549细胞对阿霉素脂质纳米粒的摄取具有明显的时间依赖性,激光共聚焦显示阿霉素脂质纳米粒能将阿霉素递送至细胞核中。结论:阿霉素脂质体对A549细胞有明显的细胞毒性,为进一步进行体内实验提供了基础。     

正交试验法优选PP1脂质体的制备工艺

目的:制备PP1脂质体,筛选最优处方。方法:用薄膜水化法制备PP1脂质体,以高效液相色谱法(HPLC)测定PP1脂质体的包封率,以包封率为主要指标,选取药脂比、胆固醇磷脂比、温度和水化时间为因素,用正交试验筛选最优配方。结果:用薄膜水法制备的PP1脂质体的最优处方的组成为:药脂比为1:10,胆固醇与二棕榈酰磷脂酰胆碱(DPPC)比为1:8,温度为40℃,水化时间为3 min。平均包封率为(63.27±3.32)%。PP1脂质体的平均粒径为(157.73±9.74)nm,Zeta电位为(-4.74±0.44)m V。结论:用薄膜水化法制备出的PP1脂质体包封率高,形态和粒径均匀,重现性好,为研究其在眼部的缓释作用奠定了基础。     

依托泊苷长循环热敏前体脂质体的制备与表征

目的研究依托泊苷长循环热敏前体脂质体的制备并对其性质进行考察。方法采用薄膜分散法制备依托泊苷长循环热敏脂质体,再用冷冻干燥技术制备依托泊苷长循环热敏前体脂质体。利用zeta电势测定仪、高效液相色谱等技术对该脂质体的粒径、电位、包封率、载药量、稳定性、释放度等进行系统的研究。结果依托泊苷长循环热敏前体脂质体水合后形成依托泊苷长循环热敏脂质体,粒径均值为(108.6±3.6)nm,Zeta电位的均值为(-12.2±1.8)mV,包封率可达96.2%;该脂质体在相变温度42℃下药物释放达到95%以上。结论依托泊苷长循环热敏前体脂质体的制备工艺稳定,载药量大,包封率高。含量及其包封率测定方法简单、快速、准确。本实验可为依托泊苷静脉注射用热敏脂质体新制剂的开发提供研究基础。     

杨梅苷脂质体的制备研究

目的：制备杨梅苷脂质体。方法：采用逆相蒸发法制备杨梅苷脂质体。用冷冻离心法分离脂质体和游离药物,用高效液相色谱法测定药物含量并计算包封率。采用激光粒度仪测定平均粒径。结果：杨梅苷脂质体制备的最佳处方和工艺为：卵磷脂：杨梅6：1,卵磷脂：胆固醇2：1,有机相：水相4：1,磷酸盐缓冲溶液的pH值为6.86,浓度为0.005 mol.L-1;超声时间为5分钟。结论：最佳条件下制备的杨梅苷脂质体包封率较高,粒径分布好,质量稳定。     

The effect of doxorubicin incorporated into a phospholipid delivery nanosystem on HepG2 cells proteome

A phospholipid drug delivery nanosystem with particle size up to 30 nm elaborated at the Orekhovich Institute of Biomedical Chemistry (Russian Academy of Medical Sciences) has been used earlier for incorporation of doxorubicin (Doxolip). This system demonstrated higher antitumor effect in vivo as compared with free doxorubicin. In this study the effect of this nanosystem containing doxorubicin on HepG2 cell proteome has been investigated. Cells were incubated in a medium containing phospholipid nanoparticles (0.5 μg/mL doxorubicin, 10 μg/mL phosphatidylcholine). After incubation for 48 h their survival represented 10% as compared with untreated cells. Cell proteins were analyzed by quantitative two-dimensional gel electrophoresis followed by identification of differentially expressed proteins with MALDI-TOF mass spectrometry. The phospholipid transport nanosystem itself insignificantly influenced the cell proteome thus confirming previous data on its safety. Doxorubicin, as both free substance and Doxolip (i.e., included into phospholipid nanoparticles) induced changes in expression of 28 proteins. Among these proteins only four of them demonstrated different in response to the effect of the free drug substance and Doxolip. Doxolip exhibited a more pronounced effect on expression of certain proteins; the latter indirectly implies increased penetration of the drug substance (included into nanoparticles) into the tumor cells. Increased antitumor activity of doxorubicin included into phospholipid nanoparticles may be associated with more active increase of specific protein expression.     

Interaction of liposomes bearing a lipophilic doxorubicin prodrug with tumor cells

When used as nanosized carriers, liposomes enable targeted delivery and decrease systemic toxicity of antitumor agents significantly. However, slow unloading of liposomes inside cells diminishes the treatment efficiency. The problem could be overcome by the adoption of lipophilic prodrugs tailored for incorporation into lipid bilayer of liposomes. We prepared liposomes of egg yolk phosphatidylcholine and yeast phosphatidylinositol bearing a diglyceride conjugate of an antitumor antibiotic doxorubicin (a lipophilic prodrug, DOX-DG) in the membrane to study how these formulations interact with tumor cells. We also prepared liposomes of rigid bilayer-forming lipids, such as a mixture of dipalmitoylphosphatidylcholine and cholesterol, bearing DOX in the inner water volume, both pegylated (with polyethylene glycol (PEG) chains exposed to water phase) and non-pegylated. Efficiency of binding of free and liposomal doxorubicin with tumor cells was evaluated in vitro using spectrofluorimetry of cell extracts and flow cytometry. Intracellular traffic of the formulations was investigated by confocal microscopy; co-localization of DOX fluorescence with organelle trackers was estimated. All liposomal formulations of DOX were shown to distribute to organelles retarding its transport to nucleus. Intracellular distribution of liposomal DOX depended on liposome structure and pegylation. We conclude that the most probable mechanism of the lipophilic prodrug penetration into a cell is liposome-mediated endosomal pathway.     

The Use of Transmembrane pH Gradient-Driven Drug Encapsulation in the Pharmacodynamic Evaluation of Liposomal Doxorubicin

Abstract

The toxicity and efficacy properties of doxorubicin entrapped inside liposomes are sensitive to the physical characteristics of the vesicle carrier system. Studies addressing such relationships must use preparation procedures with the ability to independently vary vesicle size, lipid composition and drug to lipid ratio while maintaining high trapping efficiencies. The transmembrane pH gradient-driven encapsulation technique allows such liposomal doxorubicin formulations to be prepared. Pharmacokinetic, toxicology and antitumour studies with these systems have revealed several important relationships between liposome physical properties and biological activity. The acute toxicity of liposomal doxorubicin is related primarily to the ability of the liposomes to retain doxorubicin after administration. Including cholesterol and increasing the degree of acyl chain saturation of the phospholipid component in the liposomes significantly decreases drug leakage in the blood, reduces cardiac tissue accumulation of doxorubicin and results in increased LD₅₀ values. In contrast, the efficacy of liposomal doxorubicin is most influenced by liposome size. Specifically, liposomes with a diameter of approximately 100 nm or less exhibit enhanced circulation lifetimes and antitumour activity. While these relationships appear to be rather straightforward, there exist anomalies which suggest that a more thorough evaluation of liposomal doxorubicin pharmacokinetics may be required in order to fully understand its mechanism of action. A key feature in this regard is the ability to differentiate between non-encapsulated and liposome encapsulated doxorubicin pools in the circulation as well as in tumours and normal tissues. This represents a major challenge that must be addressed if significant advances in the design of more effective liposomal doxorubicin formulations are to be achieved.     

Engineering liposomes of leaf extract of seabuckthorn (SBT) by supercritical carbon dioxide (SCCO2)-mediated process

Seabuckthorn (SBT; Hipphophae rhamnoides) leaf extract obtained by supercritical carbon dioxide (SCCO(2)) using ethanol as an entrainer, containing mainly flavanoids as bioactive principles with antioxidant and antibacterial properties, was used for the preparation of liposomes. Liposomes are promising drug carriers with sustained release because they can enhance the membrane penetration of drugs, deliver the entrapped drugs across cell membranes, and improve extract stability and bioavailability. The aim of the present study was to compare the two different methods of liposome production: the Bangham thin-film method and SCCO(2) gas antisolvent method (SCCO(2) GAS) for the incorporation of SBT leaf extract in terms of particle size, morphology, encapsulation efficiency, antioxidant activity, and thermal stability. Liposomes obtained with the thin-film method were multilamellar vesicles with average particle size (3,740 nm), encapsulation efficiency (14.60%), and particle-size range (1.57-6.0 μm), respectively. On the other hand, liposomes by the SCCO(2) GAS method were nanosized (930 nm) with an improved encapsulation efficiency (28.42%) and narrow range of size distribution (0.48-1.07 μm), respectively. Further, the antioxidant activity of leaf extract of SBT was determined by the 2 diphenyl-1-picrylhydrazyl method and expressed as Trolox equivalents as well as of the intercalated extract in liposomes. The oxidative stability of SBT encapsulated in liposomes was again estimated using differential scanning calorimetry (DSC). Thermal-oxidative decomposition of the samples (i.e., pure liposomes and encapsulated extracts) and the modification of the main transition temperature for the lipid mixture and the splitting of the calorimetric peak in the presence of the antioxidants were also studied by DSC. After encapsulation in liposomes, antioxidant activity proved to be higher than those of the same extracts in pure form.     

Rate of biodistribution of STEALTH® liposomes to tumor and skin: influence of liposome diameter and implications for toxicity and therapeutic activity

The influence of diameter on the pharmacokinetic and biodistribution of STEALTH® liposomes into the tumor (4T1 murine mammary carcinoma) and cutaneous tissues (skin and paws) of mice was studied to ascertain the time course of liposome accumulation and to determine if a preferential accumulation of liposomes into tumor over skin or paws could be achieved by altering liposome size. These tissues were chosen as the dose-limiting toxicity for Caelyx™/Doxil® in humans is palmar-plantar erythrodysesthesia, a cutaneous toxicity. We examined liposomes of four diameters: 82, 101, 154, or 241 nm. Liposomes with the three smallest diameters showed similar accumulation profiles that were significantly higher than the largest liposomes in all three tissues of interest. We were unable to achieve a preferential accumulation of liposomes into tumor over skin or paws based on size alone, as evidenced by the tumor to skin and tumor to paw ratios. However, there were differences in the time courses of liposome accumulation in these three tissues. Liposome levels plateaued in tumors and paws within 24 h, whereas skin levels plateaued between 24 and 48 h. The therapeutic activity of liposomal doxorubicin of three diameters (100, 157, and 255 nm) was tested in the same model. All formulations delayed tumor growth, with liposomes of 100 or 157 nm being equally efficacious and superior to liposomes of 255 nm.     

Rate of biodistribution of STEALTH liposomes to tumor and skin: influence of liposome diameter and implications for toxicity and therapeutic activity

The influence of diameter on the pharmacokinetic and biodistribution of STEALTH liposomes into the tumor (4T1 murine mammary carcinoma) and cutaneous tissues (skin and paws) of mice was studied to ascertain the time course of liposome accumulation and to determine if a preferential accumulation of liposomes into tumor over skin or paws could be achieved by altering liposome size. These tissues were chosen as the dose-limiting toxicity for Caelyx/Doxil in humans is palmar-plantar erythrodysesthesia, a cutaneous toxicity. We examined liposomes of four diameters: 82, 101, 154, or 241 nm. Liposomes with the three smallest diameters showed similar accumulation profiles that were significantly higher than the largest liposomes in all three tissues of interest. We were unable to achieve a preferential accumulation of liposomes into tumor over skin or paws based on size alone, as evidenced by the tumor to skin and tumor to paw ratios. However, there were differences in the time courses of liposome accumulation in these three tissues. Liposome levels plateaued in tumors and paws within 24 h, whereas skin levels plateaued between 24 and 48 h. The therapeutic activity of liposomal doxorubicin of three diameters (100, 157, and 255 nm) was tested in the same model. All formulations delayed tumor growth, with liposomes of 100 or 157 nm being equally efficacious and superior to liposomes of 255 nm.     

Intrahepatic Distribution of Long-Circulating Liposomes Containing Polyethylene Glycol) Distearoyl Phosphatidylethanolamine

Abstract

We investigated the intrahepatic distribution in rats of liposomes of 85 or 130 nm diameter, which were sterically stabilized with a polyethylene glycol) derivative of phosphatidylethanolamine (PEG-PE) so as to increase their circulation time in blood. Various times after intravenous injection of radiolabeled ([³H-]cholesterylether) liposomes, parenchymal and non-parenchymal cells of the liver were isolated and their radioactivity content was determined. Control liposomes of 85 nm without PEG-PE distributed in an approximately 80:20 ratio to hepatocytes (H) and macrophages (M), respectively; the 130-nm control liposomes showed a 50:50 H/M distribution. Incorporation of PEG-PE reduced the rate of total liver uptake about 4-fold for liposomes of either size and shifted the H/M ratio to 60:40 for the smaller vesicles and to 40:60 for the larger ones. For both liposome sizes, PEG-PE apparently causes a shift in intrahepatic distribution in favor of the macrophages. It is concluded that PEG-PE has a stronger inhibitory effect on liposome uptake by hepatocytes than on uptake by macrophages. Attempts to shift liposome uptake more in favor of hepatocytes, by incorporation of lactosylceramide, failed. This compound, although causing an increase in hepatic uptake, particularly for the 130-nm liposomes, shifted the H/M ratio further towards the macrophages. We conclude that the galactose moiety of the glycolipid is sufficiently exposed on the surface of (PEG-PE)-containing liposomes to allow interaction with the galactose-binding lectin at the surface of the liver macrophage and that the extent of exposure is dependent on vesicle size.     

The Use of Liposomes as Carriers of Lipophilic Anthracycline Antibiotics

Abstract

Tumor drug resistance and lack of tumor selectivity are the two main limitations of current systemic anticancer therapy. Liposomes have been shown to decrease certain doxorubicin (Dox)-related toxicities. By modifying liposome size and composition, the tumor localization of liposome entrapped drugs can be greatly enhanced. Through extensive structure-activity studies aimed at identifying anthracycline antibiotics which combine an enhanced affinity for lipid membranes and an ability to overcome multidrug resistance (MDR), we have identified Annamycin (Ann), which is ideally suited for entrapment in liposomes of different size and composition and has shown remarkable in vivo antitumor activity in different tumor models that display natural or acquired resistance to Dox. The unprecedented liposome formulation flexibility offered by Ann is expected to facilitate current efforts aimed at developing pharmaceutically acceptable liposomal-Ann formulations with optimal tumor targeting properties.