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。}     

联合包载阿霉素和siRNA阳离子脂质体的制备及体外评价

目的制备联合包载阿霉素和siRNA阳离子脂质体并进行体外评价。方法采用薄膜分散法制备载阿霉素阳离子脂质体(doxorubicin cationic liposomes,DCL),与siRNA静电复合得联合载药阳离子脂质体(liposome complexes,lipoplexes);动态激光散射法测定lipoplexes的粒径和zeta电位;透射电镜观察lipoplexes形态;超滤离心法测定lipoplexes中阿霉素和siRNA的包封率;琼脂糖凝胶阻滞实验考察lipoplexes中siRNA的结合能力;荧光显微镜观察MCF-7/ADR细胞对siRNA的摄取情况。结果所制备的lipoplexes外形圆整、分散均匀,当(2,3-二油氧基丙基)三甲基氯化铵与siRNA质量比为20时,其平均粒径为(125.7±2.7)nm,zeta电位为((45.8±1.5)m V,阿霉素包封率为(52.3±2.6)%,siRNA包封率为(88.1±1.8)%,且lipoplexes中siRNA可以紧密结合。与游离siRNA相比,lipoplexes可显著增加MCF-7/ADR细胞对siRNA的摄取。结论联合包载阿霉素和siRNA阳离子脂质体体外性质良好,能增加MCF-7/ADR细胞对siRNA的摄取,可用于小分子化疗药物和siRNA的共输送。     

RGD类似物修饰的阿霉素隐形脂质体的制备及体外细胞结合试验

目的研究用精氨酸-甘氨酸-天冬氨酸(RGD)类似物(RGDm)修饰隐形脂质体(SL)，以增加抗癌药物在肿瘤部位积蓄的同时，增加抗癌药物向肿瘤细胞内的传递。方法合成RGDm，将其通过PEG链与二硬脂酰磷脂酰乙醇胺(DSPE)连接形成导向化合物DSPE-PEG-RGDm，在此基础上制备RGDm修饰的隐形脂质体(RGDm-SL)，阿霉素(DOX)作为模型药物通过硫酸铵梯度法装载。体外实验中，用pH探针(BCECF-AM)标记黑色素瘤细胞，通过细胞黏附试验考察导向化合物与肿瘤细胞的黏附情况；通过流式细胞实验和激光共聚焦显微实验考察肿瘤细胞对SL包封的阿霉素(SL-DOX)及RGDm-SL包封的阿霉素(RGDm-SL-DOX)的结合或摄取情况。结果与DSPE-PEG相比，黑色素瘤细胞与导向化合物DSPE-PEG-RGDm的黏附显著增加，过量游离RGDm的加入能抑制其黏附；与SL-DOX相比，RGDm-SL-DOX与黑色素瘤细胞共同孵育后，细胞对阿霉素的结合及摄取均显著增加。结论RGDm修饰的隐形脂质体可作为肿瘤靶向的载体通过受体介导的方式促进抗肿瘤药物向肿瘤细胞内的传递。     

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。     

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。     

麦胚凝集素修饰的长春瑞滨阳离子脂质体的处方优选及细胞毒性试验

目的:制备麦胚凝集素(WGA)修饰的长春瑞滨(VRB)阳离子脂质体(WGA-VRB阳离子脂质体),优化处方并进行细胞毒性试验。方法:以磷脂、胆固醇为辅料,以3β-[N-(N′-N′-二甲基氨基乙烷)-氨基甲酰基]胆固醇盐酸盐(DC-Chol)为阳离子材料,以二硬脂酰磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000)为长循环链,采用薄膜分散法及硫酸铵梯度法制备WGA-VRB阳离子脂质体。以包封率为指标,采用星点设计-响应面法优化处方中DC-Chol、胆固醇和VRB用量。测定所制VRB脂质体和WGA-VRB阳离子脂质体中VRB含量,比较二者和空白阳离子脂质体作用于人乳腺癌细胞MCF-7和人非小细胞肺癌细胞A549后对细胞存活率的影响。结果:5 m L WGA-VRB阳离子脂质体的最优处方为磷脂22 mg、胆固醇12 mg、DC-Chol 8 mg、VRB 0.5 mg,其包封率为(92.24±1.21)%(n=3),与预测值的相对误差为5.3%。VRB脂质体和WGA-VRB阳离子脂质体中VRB含量分别为(96.01±3.26)、(93.39±1.59)μg/m L(n=3);与空白阳离子脂质体和VRB脂质体比较,WGA-VRB阳离子脂质体可明显降低MCF-7、A549细胞的存活率(P<0.05)。结论:成功制得WGA-VRB阳离子脂质体,其对MCF-7、A549细胞存活的抑制作用强于VRB脂质体。     

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。     

整合素配体RGD修饰的阳离子脂质体作为siRNA输送载体的体外研究

本研究构建了能够靶向肿瘤新生血管的RGD修饰阳离子脂质体（RGD-Lipo），作为靶向耐药相关基因MDR1的siRNA输送载体并评价其相关的药剂学性质。该脂质体与siRNA形成的复合物粒径控制在200nm以内，并且对其中所包载的siRNA具有一定的保护作用。体外实验结果表明，经RGD修饰的脂质体（RGD-Lipo）细胞粘附能力显著增强，并可增加细胞内siRNA的转染效果。与利用前插法进行靶向修饰相比，利用后插法进行RGD修饰可有效地改善siRNA的溶酶体释放效率。细胞毒试验结果表明，后插法制备的pRGD-Lipo-siRNA能够有效逆转人卵巢癌SKV03／A细胞的耐药性，并增加阿霉素药物在细胞内的蓄积。体外研究结果证实，使用pRGD-Lipo-siRNA有利于提高耐药细胞对化疗药阿霉素的敏感度，并将有可能应用于临床耐药肿瘤的治疗。     

主动包载维替泊芬阳离子脂质体用于新生血管和肿瘤靶向光动力治疗

本研究设计了一种主动稳定包载维替泊芬(verteporfin, BPD)的新生血管及肿瘤靶向阳离子脂质体(cationic liposome with drug active-loaded in lumen, CLL),并对其体内外理化性质进行探究。采用醋酸钙梯度法对光敏剂BPD进行主动包载,并通过后插法加入阳离子脂质(2,3-二油酰基-丙基)-三甲胺[(2,3-dioleoy-loxy-propyl)-trimethylammonium, DOTAP]成功制备CLL。体外表征结果显示, CLL粒径约100 nm,电位在28 mV左右,体外稳定性较被动载药制剂显著增强。此外,包载于脂质体内腔的BPD通过分子间荧光共振能量转移(homo-FRET)可在递送过程中关闭光敏效应,使光毒性降低。细胞摄取及毒性实验结果证明,正电荷靶向可显著提高血管内皮细胞及肿瘤细胞中药物摄取量, CLL光动力药效显著增强。体内实验结果显示,相比于被动载药制剂, CLL血浆清除速率降低且在肿瘤中特异性蓄积增强。离体组织定量结果显示, CLL在正常组织中分布少,有利于提高其体内安全性。动物实验均按照北京大学医学部实...     

肺靶向阿奇霉素脂质体的制备及其在小鼠体内的分布

目的研究肺靶向阿奇霉素阳离子脂质体的制备方法并考察其在小鼠体内的分布。方法利用旋转薄膜-冻融法制备肺靶向阿奇霉素脂质体。用高效液相色谱法测定给药后小鼠体内各组织中的药物浓度。结果制得的脂质体平均粒径为6.582 μm，表面电荷为+19.5 mV，包封率大于75%，稳定性好。药物体外释药符合Higuchi方程。小鼠尾静脉给药后，阳离子脂质体主要被肺摄取，在肺部的滞留时间明显延长，AUC值约为阿奇霉素溶液的8.4倍。结论采用薄膜-冻融法，添加十八胺可制得具有较高包封率及稳定性的阿奇霉素阳离子脂质体，在小鼠肺部的分布优于注射液，能达到肺靶向目的。     

Cellular association and cytotoxicity of doxorubicin-loaded immunoliposomes targeted via Fab' fragments of an anti-CD74 antibody

The purpose of our research was to evaluate in vitro therapeutic efficacy of doxorubicin (DXR)-loaded immunoliposomes with Fab' fragments of the anti-CD74 antibody LL1 attached to the surface. LL1 is well suited for targeting purposes because it is internalized very fast by B-lymphoma cells. However, at in vivo application whole antibodies show fast clearance in circulation. Taking this fact into consideration, this study was initiated to elucidate the prospects of using Fab' fragments of LL1 in stead of the whole antibody for future targeting in vivo of DXR-loaded liposomes. The Fab' fragments were covalently attached to the surface of sterically stabilized liposomes by use of a PEG-based heterobifunctinal coupling agent. LL1 Fab' conjugated sterically stabilized DXR liposomes showed approximately six times faster accumulation of the drug in Raji human B-lymphoma cells than nontargeted liposomes. In vitro cytotoxicity, quantitated by a tetrazolium assay, against Raji cells gave IC(50) values of 0.13, 0.45, and 0.11 microM for DXR-loaded immunoliposomes, DXR-loaded liposomes and free drug, respectively. The results from this study suggest that DXR-loaded immunoliposomes targeted with Fab' fragments from the anti-CD74 antibody LL1 could be a useful system for future in vivo experiments.     

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.     

Specific binding of sterically stabilized anti-B-cell immunoliposomes and cytotoxicity of entrapped doxorubicin

Administration of doxorubicin (DXR) formulated in sterically stabilized liposomes, (SL) containing engrafted poly(ethylene glycol)-modified phosphatidylethanolamine (PEG-PE) on their surface, has been shown to increase the therapeutic index of the drug. A further improvement could be achieved through targeting of liposome-entrapped drug selectively to cancer cells. This paper describes the conjugation of the anti-B-cell lymphoma monoclonal antibody LL2 to the surface of DXR-loaded liposomes by use of a PEG-based heterobifunctional coupling agent. Competitive-binding ELISA of the resulting immunoliposomes (SIL) against the monoclonal anti-idiotype antibody, WN, indicated preserved immunological activity. The pH-sensitive probe, HPTS was used to study the binding of liposomes with target cells. The results showed a 3.8-fold increased cellular association of SIL compared to that of SL and an apparent internalization of SIL into low pH compartments. Addition of an excess of unconjugated free LL2 displaced about 72% of the HPTS-SIL association with cells. Experiments with 125I-labeled free and SIL-bound LL2 showed approximately 50% degradation for both preparations. In vitro MTT cytotoxicity tests against neoplastic B cells gave IC(50) values of 1.6, 2.9 and 0.35 microM for DXR-SIL, DXR-SL and free DXR, respectively. Leakage of drug from the liposomes apparently reduced the specificity of the cytotoxic action of DXR-SIL.     

Direct measurement of the extravasation of polyethyleneglycol-coated liposomes into solid tumor tissue by in vivo fluorescence microscopy

The extravasation of liposomes of different sizes into solid tumors after i.v. injection was visualized by in vivo fluorescence microscopy in mouse neuroblastoma C-1300-bearing mice. Liposomes composed of distearoylphosphatidylcholine/cholesterol (1/1 molar ratio) and 6 mol% distearoylphosphatidylethanolamine derivative of polyethyleneglycol (PEG) were prepared. The PEG-coated liposomes were fluorescently labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) as a liposome marker or with doxorubicin (DXR) as an aqueous-phase marker. Liposomes with an average diameter of 100–200 nm showed the greatest tumor accumulation. With time after injection of DiI-labeled liposomes, the tumor interstitial fluorescence intensity increased. Most fluorescent spots were located outside and around the vessel wall, indicating extravasation of intact liposomes. The perivascular distribution was heterogeneous. We also obtained the same fluorescence localization pattern with DXR released from extravasated liposomes after injection of DXR-encapsulated liposomes. No fluorescence from extravasated liposomes was detected in normal s.c. tissue; the fluorescent spots were observed only in the vessel wall. Our results indicate that small-size long-circulating liposomes are able to traverse the endothelium of blood vessels in tumors and extravasate into interstitial spaces. Moreover, encapsulated drug was released from extravasated liposomes in the tumor.     

Gelatin-coated magnetic iron oxide nanoparticles as carrier system: drug loading and in vitro drug release study

Magnetic iron oxide nanoparticles (IOPs) were coated with gelatin A and B and drug-loading efficiency was investigated using doxorubicin (DXR) as a model drug to evaluate their potential as a carrier system for magnetic drug targeting. Drug loading to coated IOPs was done using adsorption as well as desolvation/cross-linking techniques to understand their role. Drug loading by adsorption technique was done by incubating mixture of coated IOPs and drug in various conditions of pH, DXR-to-coated IOPs ratio, gelatin types and IOPs amounts. Drug loading by desolvation/cross-linking technique was done by adding acetone and glutaraldehyde (GTA) to the mixture of coated IOPs and DXR. The results indicated involvement of electrostatic interaction during loading of DXR-to-coated IOPs. Compared to adsorption technique, desolvation/cross-linking technique improved the efficiency of drug loading regardless of type of gelatin used for the coating. The DXR-loaded particles showed pH responsive drug release leading to accelerate release of drug at pH 4 compared to pH 7.4.     

Comparative study on preparative methods of DC-Chol/DOPE liposomes and formulation optimization by determining encapsulation efficiency

Three most commonly used preparative methods, dry-film, reverse phase evaporation and ethanol injection were employed to prepare cationic liposomes composed of DC-Chol and DOPE, respectively. The resulting samples were contrasted through morphology observation, particle size and zeta potential analysis. Sephadex filtration method with high selectivity was developed to determine the encapsulation efficiency of plasmid DNA-loaded cationic vectors, on this basis, cationic liposomes formulation was further optimized by applying Box Behnken design with encapsulation efficiency as evaluation index. The results showed that liposomes prepared by dry-film method were of best quality and stability, moreover, the optimum formulation of cationic liposomes and optimal value of each influencing factors were quantitatively obtained, measured value was highly consistent with predicted results. These findings preliminarily clarified the effect of preparative methods on performance of cationic liposome, as well as formulation factors on encapsulation efficiency, and will provide important methodological reference for further study of liposomes carriers for gene delivery.     

Vascular targeting of doxorubicin using cationic liposomes

Tumor vessel has been recognized as an important target for anticancer therapy. Cationic liposomes have been shown to selectively target tumor endothelial cells, thus can potentially be used as a carrier for chemotherapy agents. In this study, cationic liposomes containing 20 mol% cationic lipid dimethyl dioctadecyl ammonium bromide (DDAB) and loaded with doxorubicin (DOX) were prepared and characterized. The cationic liposomal DOX showed 10.8 and 9.1 times greater cytotoxicity than control PEGylated liposomal DOX in KB oral carcinoma and L1210 murine lymphocytic leukemia cells, and 7.7- and 6.8-fold greater cytotoxicity compared to control neutral non-PEGylated liposomal DOX, repectively, in these two cell lines. Although cationic liposomal DOX had higher tumor accumulation at 30 min after intravenous administration compared to control liposomes (p<0.05), DOX uptake of these liposomes at 24h post-injection was similar to that of PEGylated liposomal DOX (p>0.05) and approximately twice the levels of the free drug and non-PEGylated liposomes. In a murine tumor model generated using L1210 cells, increased survival rate was obtained with cationic liposomal DOX treatment compared to free DOX (p<0.01), neutral liposome control (p<0.01), as well as PEGylated liposomes (p<0.05). In conclusion, the cationic liposomal DOX formulation produced superior in vitro cytotoxicity and in vivo antitumor activity, and warrants further investigation.     

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.