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。     

酸敏高分子诱导的脂质体膜融合性能研究

本文研究了具有酸敏特性的高分子聚(2-乙基丙烯酸)诱导的脂质体-脂质体及脂质体-细胞膜之间的融合特性。采用脂肪胺修饰的聚(2-乙基丙烯酸)制备酸敏高分子脂质体。用荧光共振能量转移法定量测定脂质体间的融合程度，采用粒径仪研究脂质体在发生膜融合时的粒径变化。结果显示，聚(2-乙基丙烯酸)酸敏高分子脂质体有很强的酸敏融合性能。在酸性条件下高分子酸敏脂质体之间可以相互融合，其程度与脂质体表面酸敏高分子聚(2-乙基丙烯酸)的分子质量成正比；高分子酸敏脂质体的酸敏融合性与构成其磷脂膜的磷脂种类有关，另外脂质体膜中加入胆固醇可降低高分子酸敏脂质体的膜融合性能；高分子酸敏脂质体与血红细胞膜影融合；酸敏高分子诱导的脂质体融合会引起脂质体膜通透性的增加。聚(2-乙基丙烯酸)诱导的酸敏高分子脂质体的膜融合特性说明其可用于制备酸敏控释脂质体。     

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。
     

反义寡核苷酸阳离子脂质体的制备和体外细胞摄取研究反义寡核苷酸阳离子脂质体的制备和体外细胞摄取研究

目的制备高效促进细胞摄取反义寡核苷酸(ASON)和保护ASON的脂质体。方法以3β［n-(n′,n′-二甲氨基乙基)氨甲酰基-胆固醇(DC-Chol)为类脂成分制备阳离子脂质体(以下简称DC-Chol脂质体),与ASON混合得到载药脂质体,测定载药率。用琼脂糖凝胶电泳分析载药脂质体的结构特点;流式细胞仪检测不同条件下细胞摄取荧光标记ASON的情况;变性聚丙烯酰胺电泳考察DC-Chol脂质体对ASON的保护作用。结果载药率与DC-Chol脂质体和药物的+/-电荷比有关,当+/-电荷比大于2时,载药率达90%以上;琼脂糖凝胶电泳显示ASON同时存在于DC-Chol脂质体的周围和包裹于其内部的两种形式;流式细胞仪测定结果表明,DC-Chol脂质体可明显增加细胞对ASON的摄取,阳性细胞染色率和胞内平均荧光强度均较对照组有明显增加,增加程度主要取决于+/-电荷比例,血清可降低细胞的摄取;变性聚丙烯酰胺电泳证实DC-Chol脂质体具有保护ASON的作用。结论DC-Chol脂质体具有显著增加细胞摄取ASON和保护ASON的作用,有望成为反义类药物的高效传递系统。     

K237修饰的紫杉醇热敏脂质体的制备、处方优化和体外释放度研究

目的：以紫杉醇（PTX）为模型药物,构建K237修饰的热敏脂质体（K237-PTX-TSL）,系统研究K237-PTX-TSL的制备工艺、理化性质,处方优化和体外释放特性。方法：采用NH2末端PEG化技术合成靶向磷脂材料DSPE-PEG-K237,采用薄膜分散法制备K237修饰的紫杉醇热敏脂质体（K237-PTX-TSL）,HPLC法测量药物的包封率和载药量;采用马尔文激光粒度仪测定K237-PTX-TSL的粒径及粒径分布和Zeta电位;利用差示扫描量热法（DSC）测量相变温度（Tm）;采用透析袋法测量相变温度下的释药规律并拟合释放曲线。结果：优化的处方为：DPPC：DSPG：MSPC：DSPE-PEG-NHS=9：1：1：1,药脂比为1/20,磷脂浓度为5.0%,DSPE-PEG-K237占处方磷脂总量为1%。制备得到的K237-PTX-TSL包封率为（94.23±0.76）%;粒测得K237-PTX-TSL粒径为（88.3±4.7） nm,电荷为-4.5 mv,PDI值为0.13±0.01;K237-PTX-TSL的相变温度为40.805℃,K237-PTX-TSL在42℃时的体外释放最优拟合为一级动力学模型,方程为In（100-Q）=-0.063 8t+4.713 0（r=0.994 4）。42℃时20 min内紫杉醇累计释放度为72.45%,60 min的累计释放度为98.84%。结论：K237修饰的热敏脂质体载药量和包封率较高、粒径较小,热敏释药性质良好,1 h内药物基本释放完全。     

奥沙利铂长循环热敏脂质体及其药效学研究

目的制备奥沙利铂长循环热敏脂质体(oxaliplatinlong-circulating-thermosensitive liposomes,OLTL),并考察其性质及抑瘤作用。方法采用薄膜分散法制备OLTL;透析法考察OLTL在37℃和42℃下的体外释药;免疫抑制法建立荷Lewis肺癌小鼠模型,以瘤重为指标考察OLTL的抑瘤作用。结果带有蓝色乳光的OLTL粒径在100～130 nm之间,包封率大于90%;42℃条件下,OLTL释药率达90%以上;OLTL组抑瘤率为70.79%。结论 OLTL具有良好的热敏释药特性,可明显提高奥沙利铂(oxaliplatin)的抑瘤作用。     

脒修饰的包载荧光探针香豆素-6脂质体的制备

目的研究3,5-二-十五烷氧基苯甲脒(DBH)修饰的香豆素-6脂质体的制备工艺,并初步考察该脂质体的体外释放性能和肾小球靶向性。方法首先合成DBH。再以其为配基,胆固醇、大豆磷脂为载体材料,采用薄膜分散-超声法制备脒修饰的载香豆素-6荧光探针脂质体,考察其粒径分布、Zeta电位、包封率及体外累积释药率。结果脒基修饰的香豆素-6脂质体形态圆整,粒径分布为120.7±2.4 nm,Zeta电位为12.6±1.6 m V,包封率为99.7%±1.8%,体外的48 h累积释药量小于2%。结论脒基修饰的香豆素-6脂质体的制备工艺简便易操作,包封率高,性质稳定。     

月桂酸二乙醇酰胺修饰利福喷丁脂质体的制备、性质及肺部给药

目的制备表面活性剂修饰利福喷丁(RIF)脂质体，进行该脂质体水化性能、载药量、释药速度和肺部给药研究。方法采用薄膜超声法制备利福喷丁脂质体，比较月桂酸二乙醇酰胺(LDEA)，Tween 80和azone修饰利福喷丁脂质体的形态、包封率、释药速度和离体猪肺膜透过性，通过纤支镜进行肺部给药研究。结果RIF-LDEA脂质体粒径在15～50 nm，包封率为83.0%，表观透膜系数Kp为44.29；LD₅₀为675 mg·kg^-1。结论LDEA修饰使利福喷丁脂质体的载药量增加1倍、释药速度的可调性强及安全性好。经纤支镜介导灌注给药治疗肺内膜结核的效果显著。     

紫杉醇长循环热敏前体脂质体的制备与表征

目的:研究紫杉醇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紫杉醇长循环热敏脂质体,再用冷冻干燥技术制备紫杉醇长循环热敏前体脂质体;采用激光粒度仪考察粒径和Zeta电位;采用高效液相色谱法研究其含量与包封率;并考察脂质体的体外释药特性.结果:紫杉醇长循环热敏前体脂质体水合后形成紫杉醇长循环热敏脂质体,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:紫杉醇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,具有良好的热敏性;含量及其包封率测定方法简单、快速、准确.本实验可为紫杉醇静脉注射用新制剂的开发提供研究基础.     

Development of lysolipid-based thermosensitive liposomes for delivery of high molecular weight proteins

The purpose of the present investigation was to determine the feasibility of using lysolipid-based thermal sensitive liposomes (145 nm) for high molecular weight molecule delivery. Fluorescein isothiocyanate conjugate-albumin was used as a model drug (MW 66 kDa). Thermal sensitive liposomes, which encapsulated the protein were prepared using a passive encapsulation methodology involving freeze-thawing cycles followed by extrusion. In vitro release studies at various temperatures indicated rapid release behaviour of the encapsulated protein at 42 and 44.5 °C but a good stability at 37.5 °C. The current findings suggest that lysolipid-based thermal sensitive liposomes can be used to deliver high molecular weight molecules.     

Taguchi正交法优化奥沙利铂长循环脂质体的制备

目的通过优化奥沙利铂(L-OHP)长循环脂质体的制备工艺,以提高其包封率(entrapment efficiency,EE%)并达到缓释效果。方法采用Taguchi正交法L9(34)(TOA),以包封率作为考察指标并采用超滤法检测包封率。考察了L-OHP脂质体的粒径、zeta电位、形态以及体外释放行为。结果药物与脂质间的比例(W/W),胆固醇与磷脂酰胆碱的比值(M/M)和超声时间是影响最大的三个因素(P<0.05)。此外,优化后的奥沙利铂脂质体经mPEG修饰后与未修饰的奥沙利铂脂质体相比较,具有更好的物理化学特性,其粒径为(204±1.1)nm,包封率高达25.40%±2.5%。修饰后的脂质体在透射电子显微镜下呈现球形结构,具有较大的内部空间,脂质体表面可见白色mPEG层状结构。经mPEG修饰的奥沙利铂脂质体相比于未修饰的脂质体,具有更低的体外释放速率,具有一定缓释能力。修饰与未修饰的脂质体以及奥沙利铂溶液均符合一级释放模型,拟合系数为0.990 2。结论经Taguchi正交法优化后的奥沙利铂长循环脂质体可作为一种新型的肿瘤靶向药物传递系统。     

非特异靶向荷正电高分子磷脂脂质体的构建和体外评价

采用一种新设计的末端带有枝状结构寡赖氨酸聚乙二醇高分子磷脂为主要功能性辅料, 依照常规薄膜-水合和后续高分子插入两种方法制备了系列外表面带正电荷的高分子脂质体。激光衍射粒径仪研究证明, 常规脂质体制备方法和后续高分子插入都可以制备纳米、粒径均匀、外表面携带不同密度正电荷的高分子磷脂脂质体, 正电荷密度不同并不影响脂质体的粒径和分布。后续高分子插入过程不影响脂质体的载药工艺, 不干扰脂质体的载药量, 不引起脂质体的形态和粒径变化, 也不诱发脂质体内部包裹的药物早期泄漏。体外细胞学实验证明这种荷正电高分子脂质体局部电荷密度高, 对细胞有显著非特异性靶向作用。     

阿霉素的不同盐型对其脂质体体外药物泄漏和体内长循环的影响

目的探讨阿霉素不同盐型对脂质体体内外稳定性的影响。方法以薄膜分散-挤压法制备含有不同缓冲对的空白脂质体，用pH梯度和化学梯度法包载阿霉素，对其在脂质体内状态进行观察，测定了阿霉素脂质体理化性质；用透析法检测阿霉素脂质体在不同介质中的药物泄漏；用HPLC法研究不同盐型阿霉素脂质体在大鼠体内药代动力学行为。结果甘氨酸盐缓冲液、柠檬酸盐缓冲液和硫酸铵溶液作内水相制得的空白脂质体的平均粒径分别为(103±8)，(102±12)和(97±8) nm，zeta电位分别为(-21.3±0.5)，(-21.7±0.4)和(-20.9±0.7) mV，对阿霉素的包封率分别为47.8%，96.7%和98.6%。甘氨酸盐制得的脂质体体外泄漏最快，硫酸铵制得的脂质体泄漏最慢；甘氨酸盐缓冲液、柠檬酸盐缓冲液和硫酸铵溶液作内水相制得的阿霉素脂质体大鼠体内平均滞留时间分别为12.13，23.31和29.79 h。结论阿霉素在脂质体内水相中不同盐型影响其脂质体的体内外稳定性，以硫酸铵为内水相制得的阿霉素脂质体最稳定，其稳定次序与内水相中酸的强度有关，酸性越弱其脂质体稳定性越高。     

Enhanced Delivery and Antitumor Activity of Doxorubicin Using Long-Circulating Thermosensitive Liposomes Containing Amphipathic Polyethylene Glycol in Combination with Local Hyperthermia

Enhanced delivery of doxorubicin (DXR) to a solid tumor subjected to local hyperthermia was achieved by using long-circulating, thermosensitive liposomes (TSL) composed of dipalmitoyl phosphatidylcholine (DPPC)/distearoyl phosphatidylcholine (DSPC) (9:1, m/m) and 3 mol% amphipathic polyethylene glycol (PEG) in colon 26-bearing mice. Inclusion of 3 mol% of distearoyl phosphatidylethanolamine derivatives of PEG (DSPE-PEG, amphipathic PEG) with a mean molecular weight of 1000 or 5000 in DPPC/DSPC liposomes resulted in decreased reticuloendothelial system (RES) uptake and a concomitant prolongation of circulation time, affording sustained increased blood levels of the liposomes. Concomitantly, DXR levels in blood were also kept high over a long period. The presence of amphipathic PEG did not interfere with the encapsulation of DXR by the pH gradient method (>90% trapping efficiency) or with the temperature-dependent drug release from the liposomes. The optimal size of these liposomes was 180 – 200 nm in mean diameter for thermosensitive drug release and prolonged circulation time. The DXR levels in the tumor after injection of long-circulating TSL (DXR-PEG1000TSL or DXR-PEG5000TSL, at a dose of 5 mg DXR/ kg) with local hyperthermia were much higher than after treatment with DXR-TSL lacking PEG or with free DXR, reaching 7.0 – 8.5 DXR µg/g tumor (approximately 2 times or 6 times higher than that of DXR-TSL or free DXR, respectively). Furthermore, the combination of DXR-PEGTSL and hyperthermia effectively retarded tumor growth and increased survival time. Our results indicate that the combination of drug-loaded, long-circulating, thermosensitive liposomes with local hyperthermia at the tumor site could be clinically useful for delivering a wide range of chemotherapeutic agents in the treatment of solid tumors.     

靶向肿瘤新生血管的阿霉素阳离子脂质体的体外研究

本研究采用3β-［N-(N′,N′-二甲基胺乙基)胺基甲酰胺基］胆固醇(DC-Chol)和二棕榈酰磷脂酰胆碱为脂材制备了各种DC-Chol含量不同的阿霉素阳离子脂质体，考察了阿霉素阳离子脂质体的体外性质，同时以大鼠主动脉内皮细胞为模型，考察它们对不同阳离子脂质体的摄取情况，并采用静脉注射FITC-Dextran(M_r 500 000)标记体内肿瘤新生血管，为体内靶向肿瘤血管提供依据。结果表明阿霉素阳离子脂质体包封率均在90%以上，粒径在100~200 nm。随着DC-Chol含量的增加，zeta电位升高，但PEG的加入会降低zeta电位。DC-Chol含量的增加会增大阿霉素的释放量，同时也促进脂质体被内皮细胞的摄取，加快摄取速度。因此在进行体内靶向肿瘤血管考察时应充分关注这些体外实验结果。FITC-Dextran标记法可以显影体内新生血管，为体内肿瘤血管靶向实验提供直观的观察方法。     

甘草次酸阳离子脂质体的制备及稳定性考察

目的:制备甘草次酸阳离子脂质体,并研究其稳定性。方法:用乙醇注入法制备甘草次酸阳离子脂质体。考察其粒径、包封率、过氧化值、在血浆中的稳定性和放样稳定性等性质。结果:所得脂质体的粒径小而均匀,呈球形和类球形,包封率为(91.6±1.2)%;离心加速试验结果显示脂质体的稳定性参数KE值较小,脂质体在血浆中释放缓慢,在4℃下放置6个月,其外观、包封率、粒径等各项指标无明显改变。结论:制得的甘草次酸脂质体包封率较高,稳定性良好。     

Enteral Absorption of Insulin in Rats from Mucoadhesive Chitosan-Coated Liposomes

Purpose. The mucoadhesiveness of polymer-coated liposomes was evaluated to develop a novel drug carrier system for oral administration of poorly absorbed drugs such as peptide drugs. Methods. Multilamellar liposomes consisting of dipalmitoylphosphatidylcholine (DPPC) and dicetyl phosphate (DCP) (DPPC:DCP = 8:2 in molar ratio) were coated with chitosan (CS), polyvinyl alcohol having a long alkyl chain (PVA-R) and poly (acrylic acid) bearing a cholesteryl group. The adhesiveness of the resultant polymer-coated liposomes to the rat intestine was measured in vitro by a particle counting method with a Coulter counter. The CS-coated liposomes containing insulin were administered to normal rats and the blood glucose level was monitored. Results. The existence of polymer layers on the surface of liposomes was confirmed by measuring the zeta potential of liposomes. The CS-coated liposomes showed the highest mucoadhesiveness and the degree of adhesion was dependent on the amount of CS on the surface of the liposomes. The blood glucose level of rats was found to be significantly decreased after administration of the CS-coated liposomes containing insulin. The lowered glucose level was maintained for more than 12h after administration of the liposomal insulin, which suggested mucoadhesion of the CS-coated liposomes in the intestinal tract of the rats.     

Thermo-responsive magnetic liposomes for hyperthermia-triggered local drug delivery

We prepared and characterised thermo-responsive magnetic liposomes, which were designed to combine features of magnetic targeting and thermo-responsive control release for hyperthermia-triggered local drug delivery. The particle size and zeta-potential of the thermo-responsive magnetic ammonium bicarbonate (MagABC) liposomes were about 210?nm and ?14?mV, respectively. The MagABC liposomes showed encapsulation efficiencies of about 15% and 82% for magnetic nanoparticles (mean crystallite size 12?nm) and doxorubicin (DOX), respectively. The morphology of the MagABC liposomes was visualised using transmission electron microscope (TEM). The MagABC liposomes showed desired thermo-responsive release. The MagABC liposomes, when physically targeted to tumour cells in culture by a permanent magnetic field yielded a substantial increase in intracellular accumulation of DOX as compared to non-magnetic ammonium bicarbonate (ABC) liposomes. This resulted in a parallel increase in cytotoxicity for DOX loaded MagABC liposomes over DOX loaded ABC liposomes in tumour cells.