谷甾醇葡萄糖苷和卞泽双重修饰肝实质细胞靶向阳性脂质体介导的基因转染和抗乙肝病毒作用

目的研究载乙型肝炎病毒(HBV)反义寡核苷酸的双重表面修饰肝实质细胞靶向阳性脂质体的基因转染，抗乙肝病毒作用和其介导基因转染的机制。方法以3β-［N-(N′,N′-二甲氨基乙基)-氨甲酰基］胆固醇(DC-Chol)和二棕榈酰磷脂酰胆碱(DPPC)为脂材，分别以谷甾醇葡萄糖苷(sito-G)和卞泽(Brij 35)为膜表面修饰成分，制备载HBV反义寡核苷酸的阳性脂质体。采用大鼠原代肝实质细胞和人肝癌细胞HepG 2.2.15，通过流式细胞分析、荧光显微镜观察和酶联免疫吸附试验(ELISA)，考察脂质体对基因转染的促进作用及其病毒抑制作用；通过评价渥曼青霉素、尼日利亚菌素以及无涎胎球蛋白对其病毒抑制作用的影响，探讨其转染机制。结果以sito-G和Brij 35对脂质体进行双重表面修饰，显著提高了脂质体的转染率和病毒抑制作用；荧光显微镜下观察到较强转染，反义寡核苷酸的胞内分布以在细胞核中为主；渥曼青霉素、尼日利亚菌素和无涎胎球蛋白均不同程度地降低了载反义寡核苷酸脂质体的病毒抑制作用。结论Brij 35和sito-G双重修饰阳性脂质体显示出较高的基因转染效率和显著的病毒抑制作用，其基因转染过程以内吞和膜融合为主，并表现出肝实质细胞表面去唾液酸糖蛋白受体 (ASGPR)的靶向选择性。     

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。     

转铁蛋白修饰的载基因前阳离子脂质体制备及其表达研究

本文制备、优化转铁蛋白修饰的前阳离子脂质体，并研究其相关性质。通过薄膜分散膜挤压法制备空白前阳离子脂质体；以鱼精蛋白缩合质粒DNA与空白前阳离子脂质体作用形成载基因前阳离子脂质体(PLPD)；转铁蛋白(transferrin，Tf)再与PLPD作用形成转铁蛋白修饰的载基因前阳离子脂质体(Tf-PLPD)；中心组合设计优化制备工艺；以lacZ为报告基因转染人肝癌细胞株HepG2；测定形态、粒径、电位和转染效率。结果显示，PLPD形态近似于球体，平均粒径为(228.9±8.0) nm，多分散指数为0.122±0.020(n=3)；zeta电位为(-25.08±2.50) mV(n=3)，转染效率(12.18±3.80) mU·mg^-1(protein)。Tf-PLPD平均粒径为(240±12) nm，多分散指数为0.150±0.030(n=3)；zeta电位为(-24.10±2.50) mV(n=3)；转染效率(24.26±2.60) mU·mg^-1(protein)是裸质粒的20倍；实验结果也表明血清的存在不影响PLPD和Tf-PLPD的转染效率；PLPD和Tf-PLPD小于阳离子脂质体LPD对人肝癌细胞HepG2，SMMC7721和张氏正常肝细胞3种细胞株的毒性。由此可见，转铁蛋白修饰的前阳离子脂质体作为基因转运的非病毒载体具有良好的应用前景。     

大豆糖苷修饰阳离子脂质体的体外肝细胞靶向性

目的研究肝靶向物质大豆糖苷(soybean-derived sterylglucoside,SG)的加入对阳离子脂质体肝细胞靶向性的影响。方法以荧光素钠(FS)为模型药物，采用HepG₂ 2.2.15细胞模型和SD雄性大鼠，检测SG，SG/Brij-35(卞泽-35)和SG/PEG-DSPE(polyethylene glycol-distearoylphosphatidylethanolamine)修饰的阳离子脂质体的物理化学性质，在细胞培养水平和离体肝脏水平考察阳离子脂质体的转染和肝细胞选择性。结果未修饰以及SG，SG/Brij-35和SG/PEG-DSPE修饰的FS阳离子脂质体在中性溶液中的包封率分别为91.74%，88.46%，89.70%和83.12%，粒径分别为124.4，113.7，110.8和93.0 nm，空白脂质体在溶液中表面电荷为正。细胞培养和肝脏灌流结果说明，阳离子脂质体的转染率显著高于中性脂质体，SG单独修饰后的阳离子脂质体的细胞转染率较未修饰有显著提高，SG/Brij-35修饰的阳离子脂质体则表现出肝实质细胞选择性。结论阳离子脂质体可以促进FS进入肝脏细胞，具有较高的肝细胞摄取率，而SG/Brij-35的修饰可以提高脂质体的肝细胞选择性。     

齐多夫定棕榈酸酯半乳糖化脂质体在小鼠体内的分布

肝实质细胞作为人类免疫缺陷病毒的贮库，与病毒不断地向外周循环扩散有关。本文制备了含自制［(2-乳糖酰胺基)乙胺基］甲酸胆固醇酯(［(2-lactoylamido) ethylamino］ formic acid cholesterol ester， CH-ED-LA)为“肝自导向”辅料的齐多夫定棕榈酸酯(azidothymidine palmitate， AZTP)半乳糖化脂质体(GalLs)， 并对齐多夫定棕榈酸酯半乳糖化脂质体在小鼠体内的分布进行了研究。采用乙醇注入-超声分散法制备了以下4种脂质体， 脂质构成分别为：大豆磷脂(SPC)/胆固醇(CH)/CH-ED-LA(80∶10∶10, 10% GalLs)， SPC/CH/CH-ED-LA(80∶15∶5, 5% GalLs)， SPC/CH/CH-ED-LA(80∶17∶3, 3% GalLs)和SPC/CH(80∶20, CL)， 测得各脂质体的包封率均大于95%，平均粒径均小于100 nm； CH-ED-LA的加入量对AZTP脂质体的膜电位和药物含量均无影响。小鼠尾静脉注射齐多夫定溶液剂15.85 mg·kg^-1、 齐多夫定棕榈酸酯普通脂质体及半乳糖化脂质体各30 mg·kg^-1， HPLC法测定药物在小鼠体内各组织中的分布。各脂质体组的药物半衰期较溶液组均有显著增加(P<0.05)；与齐多夫定溶液剂相比，齐多夫定棕榈酸酯普通脂质体和CH-ED-LA分别占脂质总量3%， 5%， 10%(mol/mol)的半乳糖化脂质体的药物肝靶向摄取率(r_e)分别为1.32和1.48， 2.13和1.50。结果表明含新糖脂CH-ED-LA的脂质体可显著改善齐多夫定棕榈酸酯的肝靶向性，可望成为一种有用的靶向肝的药物传递载体。     

脂质体肝实质细胞靶向性研究

目的 脂质体经配体修饰后介导受体细胞,以达靶向给药。方法 肝实质细胞上无唾液酸糖蛋白受体(ASGP-R)的配体无唾液酸胎球蛋白(AF)修饰脂质体得AF-SSL。将ASGP-R重组于脂质双分子膜(BLM)得ASGP-R-BLM,与AF-SSL相互作用,监测BLM膜电参数变化,确定受体-配体间识别反应;用3H标记方法测AF-SSL与肝实质细胞在体内外靶向性;将AF-SSL包抗癌药阿霉素(ADM),考察其抗癌药效。结果ASGP-R-BLM稳定时间随AF-SSL加入量增加而缩短,表现出明显的量效关系,证明在ASGP-R-BLM与AF-SSL之间有特异识别反应;体外AF-SSL与肝实质细胞结合率明显高于无AF修饰脂质体SSL(在第10和90 min时P<0.05,在第30和60 min时P<0.01);抗肝癌疗效,AF-SSL组大鼠存活期显著长于无AF修饰组,且对心、肾、肺的毒副作用小。结论配体修饰脂质体达到主动靶向对应受体细胞是可行的,本文为脂质体细胞水平靶向给药提供依据。     

茉莉酸甲酯诱导人神经母细胞瘤细胞株BE(2)-C凋亡作用机制

探讨茉莉酸甲酯对人神经母细胞瘤细胞株BE(2)-C的生长抑制作用及其对凋亡抑制蛋白家族成员XIAP和survivin表达的影响。1～2 mmol·L^-1茉莉酸甲酯处理BE(2)-C细胞6～24 h后， MTT法检测瘤细胞生长活性， 克隆形成实验检测细胞增殖能力， PI单染流式细胞术检测细胞周期， AO/EB荧光染色法、 Hoechst 33258荧光染色法、 Annexin V-FITC/PI双染流式细胞术检测细胞凋亡， 半定量逆转录聚合酶链反应(RT-PCR)、 实时荧光定量逆转录聚合酶链反应(real-time RT-PCR)检测细胞周期素D1(cyclin D1)、 X连锁凋亡抑制蛋白(X-linked inhibitor of apoptosis protein， XIAP)及survivin表达。结果表明， 各浓度茉莉酸甲酯作用后， BE(2)-C细胞呈时间、 浓度依赖性生长抑制， 1， 1.5和2 mmol·L^-1茉莉酸甲酯作用24 h后， 细胞生长抑制率为20.6%～85.5%(P<0.01)， IC₅₀为1.35 mmol·L^-1； S期细胞比率增高， 部分瘤细胞发生典型的凋亡形态学改变， 早期凋亡细胞(FITC⁺ PI^-)分别占13.51%、 17.32%和24.59%(均P<0.01)，细胞死亡率分别为29.36%、 54.73%和75.52%(均P<0.01)； XIAP和survivin表达分别下调18.5%～68.9%和22.4%～48.7%(均P<0.05)， 而cyclin D1表达无显著变化(P>0.05)。可见， 茉莉酸甲酯能通过诱导细胞周期阻滞和凋亡抑制人神经母细胞瘤细胞株BE(2)-C的生长活性， 下调XIAP和survivin基因表达可能是其作用机制之一。     

以叶酸受体为靶向的阳离子脂质体的制备与性质考察

为了研制一种能通过叶酸受体途径靶向肿瘤细胞的叶酸受体靶向脂质体，将叶酸(folate，folic acid，F)、 聚乙二醇二胺(polyoxyethylene-bis-amine，NH₂-PEG-NH₂)、 琥珀酸酐(succinic anhydride，SUC)和二硬脂酰磷脂酰乙醇胺(distearoylphosphatidylethanolamine，DSPE)按序共价连接， 并使用薄层色谱和飞行时间质谱确证合成产物为叶酸-聚乙二醇-二硬脂酰磷脂酰乙醇胺(folate-polyethyleneglycol-distearoylphosphatidylethanolamine，F-PEG-DSPE)。膜材选用二棕榈酰磷脂酰胆碱(dipalmitoylphosphatidylcholine，DPPC)， 3β-［N-(N′,N′-二甲基胺乙基)胺基甲酰基］胆固醇(3β-［N(N′,N′-dimethylaminoethane) carbamoyl］ cholesterol，DC-Chol)和F-PEG-DSPE，以10∶10∶0.75(摩尔比)的配比，以荧光素标记的阴离子葡聚糖(dextran fluorescein anionic，DFA)为模型，用薄膜分散法制备含DFA的叶酸受体靶向脂质体，其包封率较高(>55%)、稳定性好，平均粒径为144 nm，体外释放慢。MTT法考察其对细胞的毒性结果表明该阳离子脂质体具有一定的细胞毒性，在低浓度时(0.012 5～0.1 μmol·L^{-1)脂质体的细胞毒性与DC-chol浓度成正比。流式细胞技术检测KB细胞和HepG2细胞对DFA脂质体的摄取，结果表明叶酸受体靶向的长循环阳离子脂质体能提高细胞对脂质体的摄取。该研究为进一步研究叶酸受体靶向阳离子脂质体在肿瘤基因治疗中的应用提供了理论基础。}     

丹参水溶性成分拮抗脂多糖诱导HTR8/SVneo滋养层细胞凋亡的作用机制

目的 探讨丹参水溶性成分拮抗脂多糖(lipopolysaccharide,LPS)诱导HTR8/SVneo细胞凋亡的相关作用机制。方法 以不同浓度(0,100,200,400,800,1 600 ng·mL^-1)LPS处理HTR8/SVneo细胞,采用实时定量聚合酶链式反应验证HTR 8/SVneo细胞炎症模型的建立;细胞活力检测试剂盒检测各组细胞活力;划痕试验评估各组细胞向损伤区域迁移能力;流式细胞术检测各组细胞凋亡率并分析各组细胞线粒体膜电位。结果 200 ng·mL^-1 LPS干预后HTR8/SVneo细胞的NLRP3炎症小体指标及炎症因子表达量增高(P<0.05)。与LPS组相比,丹酚酸B(0.08 μmol·L^-1)、丹酚酸C(0.4 μmol·L^-1)、紫草酸(0.08 μmol·L^-1)干预后HTR8/SVneo细胞活性明显升高(P<0.01)。与LPS组比较,丹参水溶物质组凋亡率显著降低(P<0.05或P<0.01),线粒体膜电位水平显著升高。结论 丹参水溶物质能够提高LPS干预后HTR8/SVneo细胞活力,改善细胞迁移能力,提高细胞线粒体膜电位,进而抑制细胞凋亡。     

n,n′-二乙酰-L-胱氨酸对半乳糖胺联用脂多糖引起小鼠免疫性肝衰竭的作用

目的研究n,n′-二乙酰-L-胱氨酸(DiNAC)对免疫性肝衰竭的治疗作用。方法观察DiNAC对Balb/C小鼠由半乳糖胺联用脂多糖引起免疫性肝衰竭的作用。半乳糖胺/脂多糖攻击6 h后，小鼠血清ALT，AST和外周血T细胞亚群分别用全自动生化仪、流式细胞仪测定，并用光镜观察肝组织病理切片，统计半乳糖胺/脂多糖攻击24 h后的小鼠存活率。结果给肝衰竭小鼠ip DiNAC(50，200，800 mg·kg^-1)，能明显阻止小鼠血清ALT和AST活力增高，使肝组织损害减轻及提高小鼠存活率，并呈剂量依赖关系；DiNAC能增强免疫性肝衰竭小鼠外周血CD4⁺,CD8⁺,Th1和Th2 T淋巴细胞的增殖分化。结论DiNAC对免疫性肝衰竭动物有明显的治疗作用，这一作用与其免疫调节有关。     

Characteristics and biodistribution of soybean sterylglucoside and polyethylene glycol-modified cationic liposomes and their complexes with antisense oligodeoxynucleotide

A novel cationic liposome modified with soybean sterylglucoside (SG) and polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE) as a carrier of antisense oligodeoxynucleotide (ODN) for hepatitis B virus (HBV) therapy was constructed. Characteristics of the cationic liposomes modified with SG and PEG (SG/PEG-CL) and their complexes with 15-mer phosphorothioate ODN (SG/PEG-CL-ODN complex) were investigated by incorporation efficiency, morphology, electrophoresis, zeta potentials, and size analysis. Antisense activity of the liposomes and ODN complexes was determined as hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in HepG₂ 2.2.15 cells by ELISA. Their tissue and intrahepatic distribution were evaluated following intravenous injection in mice. The complexes gained high incorporation efficiency and intact vesicular structure with mean size at ∼200 nm. The SG/PEG-CL-ODN complexes enhanced the inhibition of both HBsAg and HBeAg expression in the cultured HepG₂ 2.2.15 cells relative to free ODN. The uptake of SG/PEG-CL and nonmodified cationic liposomes (CL) was primarily by liver, spleen, and lung. Furthermore, the concentration of SG/PEG-CL was significant higher than that of CL in hepatoctyes at 0.5 hr postinjection. The biodistribution of SG/DSPE-CL-ODN complex compare with free ODN showed that liposomes enhanced the accumulation of ODN in the liver and spleen, while decreasing its blood concentration. SG/PEG-CL-mediated ODN transfer to the liver is an effective gene delivery method for cell-specific targeting, which has a potential for gene therapy of HBV infections. SG and PEG-modified cationic liposomes have proven to be an alternative carrier for hepatocyte-selective drug targeting.     

Characteristics and Biodistribution of Soybean Sterylglucoside and Polyethylene Glycol-Modified Cationic Liposomes and Their Complexes with Antisense Oligodeoxynucleotide

A novel cationic liposome modified with soybean sterylglucoside (SG) and polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE) as a carrier of antisense oligodeoxynucleotide (ODN) for hepatitis B virus (HBV) therapy was constructed. Characteristics of the cationic liposomes modified with SG and PEG (SG/PEG-CL) and their complexes with 15-mer phosphorothioate ODN (SG/PEG-CL-ODN complex) were investigated by incorporation efficiency, morphology, electrophoresis, zeta potentials, and size analysis. Antisense activity of the liposomes and ODN complexes was determined as hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in HepG₂ 2.2.15 cells by ELISA. Their tissue and intrahepatic distribution were evaluated following intravenous injection in mice. The complexes gained high incorporation efficiency and intact vesicular structure with mean size at ～200 nm. The SG/PEG-CL-ODN complexes enhanced the inhibition of both HBsAg and HBeAg expression in the cultured HepG₂ 2.2.15 cells relative to free ODN. The uptake of SG/PEG-CL and nonmodified cationic liposomes (CL) was primarily by liver, spleen, and lung. Furthermore, the concentration of SG/PEG-CL was significant higher than that of CL in hepatoctyes at 0.5 hr postinjection. The biodistribution of SG/DSPE-CL-ODN complex compare with free ODN showed that liposomes enhanced the accumulation of ODN in the liver and spleen, while decreasing its blood concentration. SG/PEG-CL–mediated ODN transfer to the liver is an effective gene delivery method for cell-specific targeting, which has a potential for gene therapy of HBV infections. SG and PEG-modified cationic liposomes have proven to be an alternative carrier for hepatocyte-selective drug targeting.     

Cationic liposome (DC-Chol/DOPE=1:2) and a modified ethanol injection method to prepare liposomes, increased gene expression

Cationic liposomes composed of 3beta-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and dioleoylphosphatidylethanolamine (DOPE) (DC-Chol/DOPE liposome, molar ratio, 1:1 or 3:2) prepared by the dry-film method have been often used as non-viral gene delivery vectors. The formulation and preparation of DC-Chol/DOPE liposomes, as well as the formation of their lipoplexes were investigated in an attempt to improve transfection efficiency in vitro. A more efficient transfection in medium with serum was achieved using DC-Chol/DOPE liposomes (molar ratio, 1:2) than those (3:2), and preparation method by a modified ethanol injection than the dry-film. The most efficient DC-Chol/DOPE liposome for gene transfer was molar ratio (1:2) and prepared by a modified ethanol injection method. The enhanced transfection might be related to an increase in the release of DNA in the cytoplasm by the large lipoplex during incubation in optiMEM, not to an increased cellular association with the lipoplex. The use of a modified ethanol injection method might enhance the role of DOPE that is aid in destabilization of the plasma membrane and/or endosome. These findings suggested that cationic liposomes rich in DOPE prepared by a modified ethanol injection method will help to improve the efficacy of liposome vector systems for gene delivery.     

阳离子脂质材料和聚乙二醇对脂质体细胞转染率及膜流动性的影响阳离子脂质材料和聚乙二醇对脂质体细胞转染率及膜流动性的影响

目的制备包封荧光素钠（FS）的脂质体，考察阳离子脂质材料（DC-chol）和聚乙二醇（PEG）对脂质体包封率、细胞转染率及膜流动性的影响。方法以FS作为模型物质，制备并分离脂质体，测定脂质体包封率；通过观察荧光光谱的变化考察FS与脂质体膜之间的相互作用；以HepG₂ 2.2.15为细胞模型观察脂质体对FS细胞转染率的影响；通过荧光偏振技术考察阳离子脂质材料和PEG对脂质体膜流动性的影响。结果阳离子脂质材料和PEG能提高脂质体包封率（0.64％～86.57％）、细胞转染率（2.18％～48.46％）及脂质体膜流动性，PEG分子质量的增大有利于包封率、转染率的提高，并增加脂质体膜的流动性。结论在脂质体处方中加入阳离子脂质材料和高分子量的PEG有利于提高包封率、细胞转染率及增加脂质体膜的流动性。     

DC-Chol/DOPE cationic liposomes: A comparative study of the influence factors on plasmid pDNA and siRNA gene delivery

Cationic liposomes (CLs) composed of 3β-[N-(N′,N′-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) and dioleoylphosphatidylethanolamine (DOPE) (DC-Chol/DOPE liposomes) have been classified as one of the most efficient gene delivery systems. Our study aims to examine the effect of the molar ratio of DC-Chol/DOPE, PEGylation and serum on the pDNA (plasmid pDNA) and siRNA (small interfering RNA) transfection of DC-Chol/DOPE liposomes. The results showed that the most efficient DC-Chol/DOPE liposomes for pDNA or siRNA delivery were at a 1:2 or 1:1 molar ratio of DC-Chol/DOPE, respectively. The transfection efficiency of DC-Chol/DOPE liposomes increased along with increased weight ratio of DC-Chol/siRNA. However, the pDNA transfection efficiency decreased along with increased weight ratio of DC-Chol/pDNA from 3/1. As expected, PEGylation decreased siRNA and pDNA transfection efficiency of DC-Chol/DOPE liposomes. In PEGylated DC-Chol/DOPE liposomes, increased weight ratio of DC-Chol/pDNA from 3/1 did not lead to higher pDNA transfection efficiency, whereas increased weight ratio of DC-Chol/siRNA resulted in increased siRNA transfection efficiency. Furthermore, the serum did not significantly inhibit the pDNA and siRNA transfection efficiency of DC-Chol/DOPE liposomes. In conclusion, our results elucidated the influence factors of DC-Chol/DOPE liposome transfection and would reveal that siRNA and pDNA transfection mechanisms were different in DC-Chol/DOPE liposomes.     

Effect of Pluronic on cellular uptake of cationic liposomes- mediated antisense oligonucleotides

Cationic liposomes modified by different Pluronic block copolymers were prepared. The influence of Pluronic on the cellular uptake of antisense oligonucleotides (ODN) based on cationic 3beta[N-(N', N'-dimethylaminoethan)-carbamoyl] cholesterol (DC-Chol) liposomes was studied by flow cytometric analysis. It showed that DC-Chol liposomes containing Pluronic gave 1.7-2.3 times higher capacity of cellular uptake of ODN, despite the diminution of ODN loading efficiency. The level of improvement by Pluronic is related to the hydrophobic propylene oxide (PO) units Pluronic contains as well as the lipophile/hydrophile value of the molecule. This preliminary study indicated that modifying liposomes with another excipient is a useful pharmaceutical technique to improve ODN delivery.     

响应面法优化花生凝集素修饰长春花碱隐形阳离子脂质体处方

目的:采用响应面法优化花生凝集素(PNA)修饰长春花碱隐形阳离子脂质体的处方。方法:采用硫酸铵梯度法制备花生凝集素修饰长春花碱隐形阳离子脂质体;以半数抑制浓度IC₅₀为指标,考察卵磷脂与3β-[N-(N',N'-二甲基胺乙基)胺基甲酰胺基]胆固醇(EPC/DC-Chol)摩尔比、卵磷脂与聚乙二醇-二硬脂酰磷脂酰乙醇胺(EPC/ PEG₂₀₀₀-DSPE)摩尔比、花生凝集素(PNA)质量百分比3个因素对考察指标的影响,并对各个因素进行二项式拟合,通过响应面Box-Behnken设计优选最佳处方。结果:优选出的处方为EPC/DC-Chol 摩尔比为1.5:1、EPC/ PEG_2000-DSPE摩尔比20:1、PNA质量百分比为0.1%。按照优化后的处方所制备脂质体的Zeta电位稳定、粒径分布均匀。结论:该方法应用简便、预测性好,制备的PNA修饰长春花碱隐形阳离子脂质体符合设计要求。