阳离子膜融合脂质体介导反义寡核苷酸在Hela细胞中的转染实验研究

目的研究阳离子膜融合脂质体(CFL)介导反义寡核苷酸(ASON)的细胞转染效率及影响因素。方法 逆相蒸发法制备3种不同阳离子含量的脂质体(CL),在CL上引入仙台病毒形成CFL,将制得的阳离子膜融合脂质体与反义寡核苷酸混合得到复合物,考察形态学及载药量,用MTT法考察该载体的细胞毒性,流式细胞仪测定阳性细胞百分率和平均荧光强度。结果制得的CFL形态均匀,粒径为(168±65) nm。载药量随着磷脂/ASON(+/-)电荷比增加而增加。CFL细胞毒性明显低于相同电荷比的CL,细胞转染效率是随阳离子含量、磷脂/ASON(+/-)电荷比增加而增加,血清和低温均对CFL的细胞转染有影响。结论阳离子膜融合脂质体作为载体在低电荷比条件下可降低细胞毒性并可提高细胞转染效率,可作为该ASON的给药系统而进一步研究。     

阴离子脂质体－阳离子脂质体复合物介导的基因转染

目的评价阴离子脂质体-阳离子脂质体复合物介导质粒转移至HepG2肝癌细胞中及其毒副作用。方法制备携载表达绿色荧光蛋白质粒的阳离子脂质体,与阴离子脂质体形成复合物。测定脂质体复合物的zeta电位,凝胶阻滞实验考察质粒包封情况,流式细胞仪测量各阴离子脂质体-阳离子脂质体复合物的转染效率,MTT法检测细胞毒性。结果复合物能完全包裹质粒,其zeta电位低于阳离子脂质体zeta电位;脂质体复合物介导的转染效率略低于阳离子脂质体,其细胞生存率高于阳离子脂质体。结论阴离子脂质体-阳离子脂质体复合物在降低细胞毒性的同时,可实现对HepG2细胞较高的转染效率。     

反义寡核苷酸脂质体复合物性质对细胞摄入行为的影响

目的研究影响反义寡核苷酸脂质体复合物的性质和细胞摄取的因素。方法逆相蒸发法制备3种不同的空白脂质体,与反义寡核苷酸混合得到复合物,显微镜观察其形态,琼脂糖电泳分析载药量,流式细胞仪测定阳性细胞百分率和平均荧光强度。结果高电荷密度的脂质体和低离子强度介质可使复合物发生凝聚,载药量和细胞摄入量依赖于空白脂质体和药物的比例以及脂质体膜表面的电荷密度。结论阳离子脂质体可以提高载药量和细胞的摄入,其程度与复合物比例、脂质体膜表面电荷密度等有关。     

载多柔比星二氧化钛纳米粒的制备及体外评价

目的制备载多柔比星（doxorubicin,DOX）的二氧化钛（Ti02）纳米粒,并考察其体外释放百分率及细胞毒性。方法通过水热法合成DOX的Ti02纳米粒,采用透射电镜及X-射线衍射仪对其进行表征,紫外可见分光光度法测定载药量及体外释放,采用MTT法分析其对MCF-7细胞和Hela细胞的细胞毒性。结果所制备的纳米粒分散均匀。外观呈梭状,长度约为200nm,在水中的载药量达10．85％,体外释放具有pH敏感性,空白纳米粒细胞毒性较低,载药纳米粒的细胞毒性与游离多柔比星相当。结论所制备的TiO2纳米粒具有较高的载药量及pH敏感的体外释放性能,可作为DOX的载体。     

脂质体介导C-erbB-2反义寡核苷酸转染小鼠乳腺癌TM40D细胞的效率及毒性

目的 探讨阳离子脂质体为载体的反义寡核苷酸（ODNs）转染小鼠乳腺癌TM40D细胞的最佳转染效率及对细胞的毒性作用。方法 分别将有／无脂质体介导的反义ODNs转染小鼠乳腺癌TM40D细胞,应用流式细胞仪观察不同时间的反义ODNs的转染效率,荧光显微镜观察反义ODNs在细胞内的分布,全自动生化分析仪检测转染培养上清液的乳酸脱氢酶（LDH）浓度。结果 无脂质体介导的反义ODNs转染细胞效率随着时间的延长增加,6h达到63．00％。脂质体介导的反义ODNs转染细胞4h时转染效率达到高峰为72．23％。细胞内荧光强度与细胞转染效率相关。无脂质体介导的FAM标记的反义ODNs分布在细胞浆,脂质体介导的反义ODNs在细胞浆及细胞核内均有分布。各组LDH浓度无统计学差异。结论 脂质体介导组反义ODNs转染效率高于无脂质体组,同时转染高峰提前出现。脂质体促进反义ODNs进入细胞核内。短时间内阳离子脂质体及反义ODNS对细胞的毒性不明显。     

多西他赛他莫昔芬复方脂质体逆转乳腺癌细胞耐药性的研究

目的 在体外考察多西他赛(Docetaxel, DTX)他莫昔芬(Tamoxifen, TMX)复方脂质体对乳腺癌细胞耐药性的逆转作用。方法 在体外,利用乳腺肿瘤细胞MCF-7和耐药乳腺肿瘤细胞MCF-7/ADR,通过MTU实验和中效原理评价多西他赛和他莫昔芬不同浓度时的协同作用。通过薄膜分散法制备具有协同作用的多西他赛他莫昔芬复方脂质体。在体外通过MTT实验考察复方脂质体细胞毒性和逆转肿瘤耐药作用。结果 在MCF-7细胞和MCF-7/ADR细胞,TMX-DUX混合物(M∶M=7∶1)的中效原理联合指数值都小于1,具有协同作用。对MCF-7细胞,游离药物DTX在浓度0.001 nM到50 nM有细胞毒性,游离TMX没有细胞毒性,DTX-TMX复方脂质体具有细胞毒性。对耐药MCF-7/ADR细胞,游离药物DTX在浓度0.001 nM到50 nM没有细胞毒性,游离TMX没有细胞毒性,DTX-TMX复方脂质体具有细胞毒性,并且细胞毒性强度大于对MCF-7细胞。结论 DTX-TMX复方脂质体能够逆转肿瘤耐药性。     

卡铂前体脂质体的制备及安全性的初步评价

目的：制备卡铂前体脂质体,并对用药安全性进行初步评价.方法：采用薄膜挤压法制备卡铂脂质体,加入冻干支持剂冷冻干燥后得到卡铂前体脂质体.对豚鼠全身用药的过敏性、家兔全身用药的血管刺激性以及溶血性进行考察.结果：制备所得的卡铂前体脂质体水合后的包封率为72.0%,载药量为24.0%,平均粒径为125.1 nm.卡铂前体脂质体不引起豚鼠过敏反应,不引起家兔溶血和红细胞凝集反应,静脉注射对家兔血管无刺激性.结论：制备所得的卡铂前体脂质体有较高的包封率和载药量,水合后粒径均匀,形态圆整,且具有较好的用药安全性.     

阳离子脂质体介导肽核酸转染K562细胞的研究

目的 优化阳离子脂质体介导肽核酸(PNA)转染K562细胞的条件.方法 以阳离子脂质体lipofectamine 2000为载体,将标记有FITC荧光基团的PNA转染K562细胞,在荧光显微镜下观察并计算其转染效率,采用Cell Counting Kit(CCK-8)检测其细胞毒性.应用正交试验筛选出最佳的PNA和脂质体的用量及比例,并优化稀释用培养基血清浓度,以获得最优的转染效果.结果 以2.5× 105/mL～3×105/mL的细胞密度接种,100 μL/孔的培养基中加入PNA 6.25 pmol,PNA与脂质体的体积比为1∶3.5,稀释用培养基未加胎牛血清时,细胞转染效率和细胞毒性最佳,转染效率为87.2％,细胞存活率(RGR)为94.1％.结论 PNA可通过阳离子脂质体转染进入K562细胞,优化条件后得到的细胞转染效率和细胞存活率能够满足基因表达研究的实验要求.     

硫酸铵梯度法制备莫西沙星脂质体的工艺研究

目的 制备具有较高包封率和载药量且体外放置稳定的莫西沙星脂质体。方法 采用硫酸铵梯度法制备包载莫西沙星的脂质体,以粒径及其分布和包封率、载药量为指标对处方和工艺进行优化。结果 最佳制备条件为：空白脂质体中硫酸铵质量浓度70 mg/mL、磷脂质量浓度50 mg/mL、脂质体粒径120 nm左右、透析时间5 h、载药时药脂比2∶3、孵育温度40 ℃、孵育时间30 min。制备得到的莫西沙星脂质体粒径为（143.00±3.98）nm,包封率为（74.56±3.21）%,载药量为（26.39±1.88）%。结论 硫酸铵梯度法制备的莫西沙星脂质体包封率较高,粒径均一,室温放置稳定性良好。     

载山柰酚长循环靶向脂质体的处方筛选及抗卵巢癌疗效评估

目的:筛选载山柰酚长循环靶向脂质体(c-Lipo/KPF)的最佳处方工艺，并初步研究其抗卵巢癌疗效和机制。方法:以综合评分为指标，用正交试验法筛选c-Lipo/KPF的最优处方；用透射电镜、激光粒度仪、动态透析法、可见分光光度法等检测c-Lipo/KPF的形态、粒径、体外释药行为、血清稳定性和血液相容性；用MTT法、荧光显微镜和流式细胞术等检测空白脂质体的细胞相容性、c-Lipo/KPF对卵巢癌细胞的靶向效率和细胞毒性；用活性氧(ROS)检测试剂盒、凋亡检测试剂盒、周期检测试剂盒考察c-Lipo/KPF对卵巢癌细胞内ROS水平、凋亡和周期分布的影响。结果:c-Lipo/KPF的最佳处方工艺为磷脂、胆固醇、药物的摩尔比为80∶5∶4,超声时间为8 min;其粒径、Zeta电位、包封率(EE)和载药量(DL)分别为129 nm,-29 mV,95.2%和1.7%;溶血率低于5%;和普通脂质体(Lipo/KPF)相比，其血清稳定性、对A2780细胞的靶向效率和细胞毒性、升高细胞内ROS水平、诱导细胞G₂/M期阻滞和凋亡的能力均显著提高。结论:按最佳处方工艺制备的c-Li...     

Surface-engineered liposomes for dual-drug delivery targeting strategy against methicillin-resistant Staphylococcus aureus (MRSA)

This study focused on the encapsulation of vancomycin (VAN) into liposomes coated with a red blood cell membrane with a targeting ligand, daptomycin–polyethylene glycol–1,2-distearoyl-sn-glycero-3-phosphoethanolamine, formed by conjugation of DAPT and N-hydroxysuccinimidyl-polyethylene glycol-1,2-distearoyl-sn-glycero-3-phosphoethanolamine. This formulation is capable of providing controlled and targeted drug delivery to the bacterial cytoplasm. We performed MALDI-TOF, NMR and FTIR analyses to confirm the conjugation of the targeting ligand via the formation of amide bonds. Approximately 45% of VAN could be loaded into the aqueous cores, whereas 90% DAPT was detected using UV–vis spectrophotometry. In comparison to free drugs, the formulations controlled the release of drugs for > 72 h. Additionally, as demonstrated using CLSM and flow cytometry, the resulting formulation was capable of evading detection by macrophage cells. In comparison to free drugs, red blood cell membrane–DAPT–VAN liposomes, DAPT liposomes, and VAN liposomes reduced the MIC and significantly increased bacterial permeability, resulting in > 80% bacterial death within 4 h. Cytotoxicity tests were performed in vitro and in vivo on mammalian cells, in addition to hemolytic activity tests in human erythrocytes, wherein drugs loaded into the liposomes and RBCDVL exhibited low toxicity. Thus, the findings of this study provide insight about a dual antibiotic targeting strategy that utilizes liposomes and red blood cell membranes to deliver targeted drugs against MRSA.     

荧光偏振法用于止血剂与脂质体及血液红细胞主-客体分子间作用机制的研究

以磷脂脂质体模拟血液细胞,用1,6-二苯基已三烯(DPH)为荧光探针,荧光偏振法研究了安络血、止血芳酸、维生素K₁和6-氨基已酸4种止血剂作为客体分子与脂质体及血液红细胞两类主体分子互相作用而形成的超分子化合物。利用偏振度(P)与微粘度(η)的定量关系,计算微粘度的变化,并以微粘度的变化推测止血剂与红细胞的作用机制。结果表明,止血剂与脂质体或血红细胞的相互作用力主要是超分子作用力。不同血凝机制的止血剂与红细胞作用方式也不尽相同。本文还对止血剂与红细胞的结合方式进行了探讨。     

Improved antitumor efficacy and reduced toxicity of liposomes containing bufadienolides

Long-circulating liposomes are used extensively nowadays for enhancing the therapeutic effect and reducing the toxicity of anticancer drugs. In this paper, a traditional Chinese medicine, toad venom, which has long been used in the clinic for tumor therapy with unpleasant side effects, was incorporated into poloxamer modified liposomes to increase its antitumor effect and reduce its toxicity. Our preparation of bufadienolides liposomes had a particle size of around 70 nm and an entrapment efficiency of about 87.6%. Lyophilized liposomes well retained their appearance, particle size and encapsulation efficiency for 3 months. The in vitro release results verified the sustained release properties of the bufadienolides liposomes. The concentration of bufadienolides in modified liposomes that caused 50% cell killing was much lower than that of free drug for both Lovo cells and NCI-H157 cells. Compared to the bufadienolides solution and the unmodified liposomes, the bufadienolides liposomes significantly prolonged the retention time and increased the area under the curve in vivo. The antitumor efficiency of the bufadienolides liposomes against mice bearing H22 liver cancer cells and Lewis pulmonary cancer cells were 2.15 and 2.96, respectively, times that of a bufadienolides solution at the same toxicity. The safety test results demonstrated that the bufadienolides liposomes had an LD₅₀ that was 3.5 times the LD₅₀ of bufadienolides solution and caused no allergen-related or blood vessel irritation effects. All these results proved that poloxamer modified bufadienolides liposomes have improved antitumor efficacy and safety.     

THE EFFECT OF ADRIAMYCIN AGAINST A LIVER METASTATIC MODEL BY ENCAPSULATION IN LIPOSOMES

Antitumour activities of liposomes containing adriamycin (L-ADM) and their distribution process into tumour cells were analysed. The lipid composition of the liposomes was dimyristoylphosphatidylglycerol (DMPG)/egg phosphatidylcholine/cholesterol/adriamycin (ADM) in a molar ratio of 11·4 : 2 : 12 : 1·3. Liver-metastasizing murine tumour models, M5076 and L5178Y-ML, were used. In vivo antitumour effect against these tumour models was assessed from increase in life span (ILS). The survival prolongation effect of L-ADM in mice with liver failure caused by M5076 was significantly higher than that of F-ADM. In contrast, significant enhancement of the effects by encapsulation in liposomes was not observed in L5178Y-ML-bearing mice. In vitro cytostatic activities of L-ADM against M5076 cells as well as against other tumour cell lines were lower than those of F-ADM. The in vitro kinetic study of the distribution of L-ADM to the tumour cells revealed that ADM in L-ADM was taken up into the tumour cells mainly after it was released from the liposomes rather than taken up as the liposomal form. Among the cell lines tested, M5076 cells had the highest phagocytic activity and therefore the highest uptake activity of ADM during incubation with L-ADM. These findings suggest that the augmented antitumour activity of L-ADM in M5076-bearing mice was the result of phagocytosis of L-ADM by M5076 cells as well as the reduction of toxicity, prolonged retention of ADM in systemic circulation, and liver accumulation of ADM after administration of L-ADM.     

Increased intracellular targeting to airway cells using octaarginine-coated liposomes: in vitro assessment of their suitability for inhalation

Delivery of macromolecular drugs to airway cells after inhalation can be limited by rapid clearance, in vivo degradation, and poor intracellular targeting. Liposome carriers offer an effective method of improving drug stability, but conventional liposomes have limited intracellular targeting capacity and are cleared rapidly by the lungs. Further modification is required to improve liposome-cell interaction and intracellular targeting. Therefore, we proposed conjugating three arginine-rich membrane translocating peptides, namely, HIV-TAT, Antennapedia, and octaarginine, to neutral liposomes as a biocompatible alternative to cationic lipids for intracellular delivery of macromolecules to airway cells. Conjugation did not significantly affect liposome stability, and each system was nebulized to produce aerosols of mean aerodynamic diameter < 1.5 microm. The peptides caused a significant (p < 0.05) increase in liposome-airway cell association compared to untagged liposomes and to DOTAP liposomes. Up to 30% of the peptide-conjugated liposomes added were bound and internalized (via a temperature-dependent, endocytic process) after just 2 h. The novel carriers all delivered encapsulated dextrans rapidly and efficiently to the cytoplasm of Calu-3 cells. Once internalized by the cells, the modified carriers localize for the most part in the cytoplasm with only a small amount of nuclear localization. These peptide-conjugated liposomes were significantly (p < 0.05) less toxic than DOTAP liposomes with octaarginine-coated liposomes the least toxic. These systems, particularly octaarginine-coated liposomes, offer many advantages for drug delivery to airway epithelial cells including increased stability, improved cell binding, and cell uptake with an improved toxicity profile.     

双配体修饰的阿霉素脂质体靶向于脑胶质瘤的体外研究

目的筛选和优化转铁蛋白、叶酸共同修饰的阿霉素脂质体的处方及制备工艺,以期得到具有良好的脑胶质瘤靶向治疗作用的给药系统。方法采用薄膜分散和硫酸铵梯度法制备阿霉素脂质体。将叶酸连接至二硬脂酸磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000-NH2)得到DSPE-PEG2000-Folic,考察不同磷脂种类、药脂比、水化介质和载药时间,对脂质体粒径、包封率和稳定性的影响,确定脂质体的处方工艺。以大鼠的脑毛细血管内皮细胞(bEnd3)和星形胶质细胞组成体外血脑屏障(blood-brain barrier,BBB),并结合大鼠胶质瘤C6细胞,构建体外模拟胶质瘤靶向治疗的复合BBB模型。考察阿霉素脂质体在bEnd3细胞中的摄取机制和透过BBB的转运速率及对C6细胞的毒性。结果确定了DSPC作为主要磷脂组分,并以120 mmol.L 1的硫酸铵作为水化介质,药脂比为1∶1 5,载药时间选择60 min,成功制备了高包封率和稳定性的双配体脂质体。其在bEnd3细胞中摄取远大于普通脂质体(P<0.05),摄取过程受网格蛋白和小窝内陷介导的细胞内吞作用,并受转铁蛋白和叶酸的影响;同时其在BBB模型中的药物透过速率、及其进一步透过BBB后对下层C6细胞的毒性,均显著高于其他脂质体组。结论转铁蛋白和叶酸共同修饰的阿霉素脂质体具有较好的体外脑胶质瘤靶向治疗作用。     

Evaluation of cell tolerability of a series of lipoamino acids using biological membranes and a biomembrane model

The interaction of a series of amphiphilic 2-alkyl aminoacids (lipoamino acids, LAAs) with different cell cultures and biomembrane models was investigated. LAAs can be useful promoieties to modify the physico-chemical properties of many drugs, and in particular their lipophilicity. Tests were performed in vitro on mammalian cells (murine astrocytes) and human red blood cells (haemolysis), and in vivo on rabbit eye as alternative models to assess the tolerability or the potential damaging effects of these compounds on different biological systems. The mode of interaction of LAAs with pure phospholipid multilamellar liposomes, taken as a biomembrane model, was also analysed by differential scanning calorimetry experiments. Different tolerability/toxicity patterns were obtained in the various models; in particular, the most lipophilic terms of the series, methyl 2-aminohexadecanoate (LAA16), displayed haemolytic activity and toxicity for mouse astrocyte cultures. A specific assay confirmed that LAA16 acted at level of cell membranes, while neither any damaging effects on nucleus or apoptotic induction were observed. The shorter-chain LAAs and the tetradecyl homologue (LAA14) showed the best compatibility with the various cell models.     

Multivariate toxicity screening of liposomal formulations on a human buccal cell line

The influence of various formulation factors on the in vitro cellular toxicity of liposomes on human buccal cells (TR146), were studied by using the concept of statistical experimental design and multivariate evaluation. The factors investigated were the type of main phospholipid (egg-PC, DMPC, DPPC), lipid concentration, the type of charge, liposome size, and amount and nature of the charged component (diacyl-PA, diacyl-PG, diacyl-PS, stearylamine (SA), diacyl-TAP) in the liposomes. Both full factorial design and D-optimal designs were created. Several significant main factors and interactions were revealed. Positively charged liposomes were shown to be toxic. The toxicity of negatively charged liposomes was relatively low. Diacyl-TAP was less toxic than SA, and DPPC was less toxic than DMPC. Low level of positively charged component was favourable and essential when using egg-PC as the main lipid. The amount of negatively charged component, the liposome size, and the total lipid concentration did not affect the toxicity within the experimental room. DPPC appeared to be a good candidate when formulating both positively and negatively charged liposomes with low cellular toxicity. The concept of statistical experimental design and multivariate evaluation was shown to be a useful approach in cell toxicity screening studies.     

M cells prefer archaeosomes: an in vitro/in vivo snapshot upon oral gavage in rats

The archaeolipids (lipids extracted from archaebacterias) are non saponificable molecules that form self sealed mono or bilayers (archaeosomes-ARC). Different to liposomes with bilayers made of conventional glycerophospholipids, the bilayer of ARC posses a higher structural resistance to physico chemical and enzymatic degradation and surface hydrophobicity. In this work we have compared the binding capacity of ARC exclusively made of archaeols containing a minor fraction of sulphoglycophospholipids, with that of liposomes in gel phase on M-like cells in vitro. The biodistribution of the radiopharmaceutical (99m)Tc-DTPA loaded in ARC vs that of liposomes upon oral administration to Wistar rats was also determined. The fluorescence of M-like cells upon 1 and 2h incubation with ARC loaded with the hydrophobic dye Rhodamine-PE (Rh-PE) and the hydrophilic dye pyranine (HPTS) dissolved in the aqueous space, was 4 folds higher than upon incubation with equally labeled liposomes. Besides, 15% of Rh-PE and 13 % of HPTS from ARC and not from liposomes, were found in the bottom wells, a place that is equivalent to the basolateral pocket from M cells. This fact suggested the occurrence of transcytosis of ARC. Finally, 4 h upon oral administration, ARC were responsible for the 22.3 % (3.5 folds higher than liposomes) shuttling of (99m)Tc-DTPA to the blood circulation. This important amount of radioactive marker in blood could be a consequence of an extensive uptake of ARC by M cells in vivo, probably favored by their surface hydrophobicity. Taken together, these results suggested that ARC, proven their adjuvant capacity when administered by parenteral route and high biocompatibility, could be a suitable new type of nanoparticulate material that could be used as adjuvants by the oral route.