聚(2-乙基丙烯酸)脂肪酰胺衍生物构建的酸敏脂质体

本实验合成了系列聚(2-乙基丙烯酸)长链脂肪酰胺衍生物，并采用高分子插入法制备了聚(2-乙基丙烯酸)酸敏高分子脂质体。应用荧光指示剂、粒径仪、荧光显微镜及细胞实验，系统研究了高分子修饰和脂肪胺的链长对高分子衍生物嵌入脂质体的效率和质量的影响。结果表明，高分子插入法可以制备聚(2-乙基丙烯酸)酸敏高分子脂质体。(1) 高分子嵌入量与高分子脂肪胺的链长无关，但与高分子修饰度相关。(2) 高分子嵌入量与起始的高分子-脂质体比例成正比。(3) 在酸性条件下聚(2-乙基丙烯酸)脂质体可产生显著的脂质体融合及释药行为。(4) 聚(2-乙基丙烯酸)脂质体在细胞内呈现出良好的酸敏诱导释药特性。实验证明这种方法制备的脂质体具有良好的酸敏释药性能，并且制备方法简便，可控性好，实用性强。     

聚(2-乙基丙烯酸)脂质体的制体及其热敏性研究

采用插入法以脂肪酰胺修饰的聚(2-乙基丙烯酸)衍生物构建热敏递药的高分子脂质体。用荧光分析法，借助荧光分光光度仪和粒径仪系统地研究了高分子脂质体的热敏特性。结果发现，采用脂肪胺修饰的聚(2-乙基丙烯酸)制备的脂质体具有明显的热敏释药特性，其释药特性与插入的高分子结构有关，还与制备脂质体的磷脂组成有关，同时采用聚(2-乙基丙烯酸)制备的脂质体还具有显著的酸敏释药特性。以聚(2-乙基丙烯酸)为热敏诱导介质制备的脂质体在体外实验中呈现出良好的热敏释药特性，且制剂制备方法简便、可靠。     

聚(2-乙基丙烯酸)脂质体的制体及其热敏性研究

采用插入法以脂肪酰胺修饰的聚(2-乙基丙烯酸)衍生物构建热敏递药的高分子脂质体.用荧光分析法,借助荧光分光光度仪和粒径仪系统地研究了高分子脂质体的热敏特性.结果发现,采用脂肪胺修饰的聚(2-乙基丙烯酸)制备的脂质体具有明显的热敏释药特性,其释药特性与插入的高分子结构有关,还与制备脂质体的磷脂组成有关,同时采用聚(2-乙基丙烯酸)制备的脂质体还具有显著的酸敏释药特性.以聚(2-乙基丙烯酸)为热敏诱导介质制备的脂质体在体外实验中呈现出良好的热敏释药特性,且制剂制备方法简便、可靠.     

荧光素作为酸敏指示剂评价酸敏材料和脂质体酸敏性的方法研究

目的:建立一种简单而快速的评价酸敏材料和脂质体酸敏性的实验分析方法。方法:利用脂质体包裹的荧光素在高浓度环境的自屏蔽作用,以及从脂质体中释放后自屏蔽消失的特性,通过检测试样的荧光发射光信号,来评价酸敏物质诱发细胞膜融合通透性能的强弱和脂质体的酸敏释药性能。结果:选用不同磷脂组分制备的脂质体可以成功的包裹高浓度荧光素,被包裹的荧光素稳定并且能屏蔽自己的发射光;在酸性条件,酸敏物质破坏脂质体膜,荧光素可以从脂质体中释放出来。酸敏材料的酸敏能力、溶液酸度、脂质体磷脂组成都影响荧光素从脂质体中释放的能力和速度;酸度影响荧光素的荧光强度,但在 pH 6.5～10.0不受影响。结论:利用脂质体包裹高浓度荧光素的自我屏蔽性,建立一种简单而快速的实验方法,用来评价酸敏物质诱发细胞膜融合通透性能的强弱以及评价脂质体的酸敏释药性能。     

酸敏脂质体的制备及其生物学活性

目的　对不同方法制备酸敏脂质体进行包封率测定及形态学观察 ,探讨酸敏脂质体包裹反义寡核苷酸的生物学效应。方法　测定四种不同方法制备包裹1 2 5I IL 8脂质体的包封率 ;在超高倍显微分析仪下观察形态结构。用包裹细胞磷脂酶A2 (cPLA2 )反义寡核苷酸的酸敏脂质体转染U937细胞 ,提取细胞RNA ,采用反转录 聚合酶链反应 (RT PCR) ;免疫印迹 (Westernblot)法检测cPLA2 蛋白的表达。结果　反相蒸发法与钙融合法联合制备的酸敏脂质体具有良好地包封率和形态结构 ,经酸敏脂质体包裹cPLA2 反义寡脱氧核苷酸可明显抑制cPLA2基因及蛋白的表达。结论　制备所得的酸敏脂质体可良好的发挥其生物学效应     

聚乙二醇POPA磷脂衍生物构建的酶敏脂质体

以磷酰胺键将聚乙二醇高分子MePEG2000-NH₂与磷脂POPA连接在一起, 合成聚乙二醇磷脂衍生物, 以聚乙二醇磷脂衍生物为主要膜材构建酸敏脂质体。采用荧光分析法系统研究了聚乙二醇磷脂衍生物脂质体在酸性条件下对荧光染料的释药特性。以聚乙二醇磷脂衍生物构建的酸敏脂质体,在pH 6.5～7.5时稳定,其稳定性与制备脂质体的磷脂种类及胆固醇含量密切相关,在pH 5.0时发生显著的荧光泄漏,泄漏率与环境酸性的强度及处于酸性的时间呈正相关。聚乙二醇磷脂衍生物构建的脂质体具有开发成酸敏释药脂质体的前景。
     

甲氧基聚乙二醇-磷脂酰乙醇胺的制备及其对脂质体的稳定作用

采用两步反应法制备脂质体空间稳定膜材料甲氧基聚乙二醇-磷脂酰乙醇胺(MPEG—EPE)，并用以制备空间稳定脂质体(SLs)，同时制备不含MPEG—EPE的传统脂质体(CLs)，比较两者放置过程中粒径变化、加乙醇后浊度变化、包封钙黄绿素后在冻干-再水化过程中的荧光泄漏程度及其与人血浆蛋白的吸附指标，评价MPEG2000-EPE对脂质体的稳定作用。结果表明，试验前后SLs的粒径及浊度变化不大，荧光泄漏程度和人血浆蛋白吸附量也远小于CLs，提示其对脂质体有良好的稳定作用。     

唑来膦酸阳离子脂质体的制备及其体外特性表征

目的制备唑来膦酸阳离子脂质体,并对其体外特性进行表征。方法采用薄膜分散法制备唑来膦酸阳离子脂质体,以包封率、载药量、平均粒径、Zeta电位为评价指标,对处方及工艺进行单因素考察,并对其体外特性进行表征。结果确定处方工艺为DPPC与DC-Chol比例为3∶1,PBS水化体积10 m L,旋转蒸发时间60 min,超声均化5 min,制备得到的阳离子脂质体的平均粒径、聚分散指数、包封率、载药量和Zeta电位分别为(106.76±1.94)nm,0.262±0.027,(38.54±0.99)%,(3.42±0.27)%,+(42.37±2.60)m V,唑来膦酸阳离子脂质体体外释药具有缓释靶向特性,药物释放曲线符合Weibull方程模型。结论采用薄膜分散法制备的唑来膦酸阳离子脂质体具有较高的稳定性,为其药动学和药效学研究奠定了基础。     

利福平脂质体温敏型原位凝胶的制备及其体外释药特征研究

目的 制备利福平脂质体温敏型原位凝胶,并对其体外性质进行研究。方法 采用薄膜分散法制备利福平脂质体,并对利福平脂质体进行表征研究;以泊洛沙姆188、泊洛沙姆407为凝胶基质,制备利福平脂质体温敏型原位凝胶;以无膜溶出模型研究温敏型原位凝胶体外累积溶蚀率;采用透析袋法分析药物体外释放情况。结果 制备的利福平脂质体平均粒径、聚分散指数、Zeta电位、包封率和载药量分别为(149.0±5.67)nm、0.275±0.056、-(29.8±1.59)mv、(79.6±2.67)%,(18.6±0.25)%;利福平脂质体温敏型原位凝胶的胶凝温度为(34.3±0.6)℃。体外溶蚀曲线和体外释药曲线均符合零级动力学特征。结论 利福平脂质体温敏型原位凝胶的制备工艺简单易行,体外释放显示其有很好的缓释作用。     

盐酸环丙沙星-聚氰基丙烯酸正丁酯纳米溶胶的制备

目的:制备盐酸环丙沙星-聚氰基丙烯酸正丁酯纳米溶胶,并对其处方因素进行考察。方法:以氰基丙烯酸正丁酯为载体材料,采用乳化聚合法制备盐酸环丙沙星-聚氰基丙烯酸正丁酯纳米溶胶,以粒径、外观为主要指标,用单因素方法优化处方,得到二步法制备纳米溶胶的条件和工艺。结果:制备的纳米溶胶的平均粒径为(82.6±15)nm,包封率为61.9%,载药量为34.2%(g/g),平均Zeta电位为(-25.1±9.92)mV。结论:本实验制备的盐酸环丙沙星-聚氰基丙烯酸正丁酯纳米溶胶的粒径小,分布窄,稳定性好。     

寡核苷酸聚赖氨酸修饰物的脂质体制备及其对HepG2细胞的细胞毒活性研究

目的研究反义寡核苷酸的聚赖氨酸修饰物对脂质体包封率的影响及对HepG2细胞活性的初步测定。方法利用反义药物的聚赖氨酸修饰物,采用薄膜分散法制备反义寡核苷酸及其聚赖氨酸修饰物的脂质体;紫外分光光度法测定包封率的差异并考察对HepG2细胞细胞毒活性的影响。结果高密度正电荷的聚赖氨酸与带负电荷的反义寡核苷酸偶联后,脂质体的载药量大大增加,并且细胞的摄入量增加,对HepG2细胞生长具有抑制作用。结论聚赖氨酸修饰物增加了反义寡核苷酸的脂质体包封率,有效诱导人肝癌细胞的凋亡作用。     

Use of the Gamma-Ray Perturbed Angular Correlation (PAC) Technique for Monitoring Liposomal Phospholipid Bilayer Integrity

A membrane labeling method based on the principle of gamma-ray perturbed angular correction (PAC) was developed to monitor the structural integrity of liposomal membranes. The reporter group was ¹¹¹In(III) complexed with the lipophilic diethylenetriaminepentaacetic acid (DTPA) derivative of dipalmitoylphosphatidylethanolamine (DPPE) embedded in the phospholipid bilayers of small unilamellar liposomes. Using this method, complete chemical digestion of the constituent phospholipids in these DTPA-conjugated liposomes by phospholipase A₂ or phospholipase C in the presence of Ca^{2 +} was found not to be followed by an immediate disruption of the liposomal membrane. Compared with other methods, the method developed permits the continuous noninvasive monitoring of the micro-environment of the lipid bilayer at the molecular level. It may potentially be applicable to evaluate liposomal fusion, screen for penetration enhancers under development for enhancement in mucosal drug penetration, and monitor liposomal degradation within the living animal.Karl J. Hwang: Deceased.     

Boosting 5-ALA-based photodynamic therapy by a liposomal nanomedicine through intracellular iron ion regulation

5-Aminolevulinic acid (5-ALA) has been approved for clinical photodynamic therapy (PDT) due to its negligible photosensitive toxicity. However, the curative effect of 5-ALA is restricted by intracellular biotransformation inactivation of 5-ALA and potential DNA repair of tumor cells. Inspired by the crucial function of iron ions in 5-ALA transformation and DNA repair, a liposomal nanomedicine (MFLs@5-ALA/DFO) with intracellular iron ion regulation property was developed for boosting the PDT of 5-ALA, which was prepared by co-encapsulating 5-ALA and DFO (deferoxamine, a special iron chelator) into the membrane fusion liposomes (MFLs). MFLs@5-ALA/DFO showed an improved pharmaceutical behavior and rapidly fused with tumor cell membrane for 5-ALA and DFO co-delivery. MFLs@5-ALA/DFO could efficiently reduce iron ion, thus blocking the biotransformation of photosensitive protoporphyrin IX (PpIX) to heme, realizing significant accumulation of photosensitivity. Meanwhile, the activity of DNA repair enzyme was also inhibited with the reduction of iron ion, resulting in the aggravated DNA damage in tumor cells. Our findings showed MFLs@5-ALA/DFO had potential to be applied for enhanced PDT of 5-ALA.KEY WORDS: 5-Aminolevulinic acid, Biotransformation interference, Iron ion regulation, DNA repair inhibition, ALKBH2, Membrane fusion liposomes, Photodynamic therapy, Drug delivery     

Interactions Between Aminoglycosides and Phospholipids Using Liposomes: A Possible Mechanism of Nephrotoxicity

Interactions of aminoglycosides with phospholipids were estimated by the increase in turbidity of liposomes consisting of various phospholipids. The turbidity of liposomes containing negatively charged phospholipids was increased by gentamicin, the highest increase in turbidity being observed for phosphatidylinositol-4,5-diphosphate-containing liposomes. The extent of turbidity was dependent on the concentration of acidic phospholipid in the liposomal membrane as well as the number of amino groups of the aminoglycosides. The release of glucose from glucose-entrapped liposomes depended on the concentration of gentamicin. The turbidity of liposomes containing lipids extracted from rat renal cortex was also increased by aminoglycosides depending on the number of amino groups. From electron microscopic observations, the increase in turbidity of liposome suspensions was caused by liposome fusion.     

Isolation of bovine serum albumin fragment P-9 and P-9-mediated fusion of small unilamellar vesicles

Fusion peptide P-9 was isolated from bovine serum albumin by controlled pepsin degradation in the presence of caprylic acid, followed by Sephadex G-75 gel filtration and ion-exchange chromatography of CM-cellulose. By this procedure, P-9 could be strictly separated from peptic fragment P-Phe, which has a molecular weight close to that of P-9. P-Phe has no fusogenic activity. The addition of P-9 to phosphatidylcholine (PC) liposomes containing cholesterol (Chol) gave rise to an increase of absorption intensity at around pH 4.0. The increase of turbidity by P-9 addition did not decrease with increasing pH, indicating P-9-mediated fusion of PC liposomes. The extent of the fusion of PC liposomes was strongly dependent on the PC chain length and temperature. The membrane fluidity close to the polar head groups of the fatty acyl chains of PC affected markedly the extent of P-9-mediated liposome fusion. However, there was no correlation between membrane fluidity near the hydrophobic end of the fatty acyl chains and the extent of liposome fusion. The rate of liposome fusion was dependent on both lipid composition and PC chain length. These results suggest that a contact or an interaction of P-9 with liposomal membrane occurs in the rigid regions. The character of the membrane-water interface region in the liposome controls a triggering effect for P-9-mediated fusion.     

Poly(styrene-co-maleic acid)-based pH-sensitive liposomes mediate cytosolic delivery of drugs for enhanced cancer chemotherapy

pH-responsive polymers render liposomes pH-sensitive and facilitate the intracellular release of encapsulated payload by fusing with endovascular membranes under mildly acidic conditions found inside cellular endosomes. The present study reports the use of high-molecular weight poly(styrene-co-maleic acid) (SMA), which exhibits conformational transition from a charged extended structure to an uncharged globule below its pK(1) value, to confer pH-sensitive property to liposomes. The changes in the co-polymer chain conformation resulted in destabilization of the liposomes at mildly acidic pH due to vesicle fusion and/or channel formation within the membrane bilayer, and ultimately led to the release of the encapsulated cargo. The vesicles preserved their pH-sensitivity and stability in serum unlike other polymer-based liposomes and exhibited no hemolytic activity at physiological pH. The lysis of RBCs at endosomal pH due to SMA-based liposome-induced alterations in the bilayer organization leading to spherocyte formation indicated the potential of these vesicles to mediate cytosolic delivery of bio-active molecules through endosome destabilization. The SMA-loaded liposomes exhibiting excellent cytocompatibility, efficiently delivered chemotherapeutic agent 5-Fluorouracil (5-FU) within colon cancer cells HT-29 in comparison to neat liposomes. This caused increased cellular-availability of the drug, which resulted in enhanced apoptosis and highlighted the clinical potential of SMA-based vesicles.