Encapsulation of plasmid DNA in stabilized plasmid-lipid particles composed of different cationic lipid concentration for optimal transfection activity

In previous work (Wheeler et al. (1999) Gene Therapy 6, 271-281) we have shown that plasmid DNA can be entrapped in "stabilized plasmid lipid particles" (SPLP) using low levels (5-10 mol%) of cationic lipid, the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), and a polyethyleneglycol (PEG) coating for stabilization. The PEG moieties are attached to a ceramide anchor containing an arachidoyl acyl group (PEG-CerC20). However, these SPLP exhibit low transfection potencies in vitro as compared to plasmid/cationic lipid complexes formed with liposomes composed of cationic and neutral lipid at a 1:1 lipid ratio. The objective of this study was to construct SPLPs with increased cationic lipid contents that result in maximum transfection levels. A phosphate buffer detergent dialysis technique is described resulting in formation of SPLP containing 7-42.5 mol% DODAC with reproducible encapsulation efficiency of up to 80%. An octanoyl acyl group was used as anchor for the PEG moiety (PEG-CerC8) permitting a quick exchange out of the SPLP to further optimize the in vitro and in vivo transfection. We have demonstrated that this technique can be used to encapsulate either linearized DNA or supercoiled plasmids ranging from 3-20 kb. The SPLP formed could be isolated from empty vesicles by sucrose density gradient centrifugation, and exhibited a narrow size distribution of approximately 75 +/- 6 nm as determined by cryo-electron microscopy. The high plasmid-to-lipid ratio observed corresponded to one plasmid per particle. The SPLP consist of a lipid bilayer surrounding the plasmid DNA as visualized by cryo-electron microscopy. SPLP containing a range of DODAC concentrations were tested for in vitro and in vivo transfection. In vitro, in COS-7 cells transfection reached a maximum after 48 h. The transfection efficiency increased when the DODAC concentration in the SPLP was decreased from 42.5 to 24 mol% DODAC. Decreasing the cationic lipid concentration improved transfection in part due to decreased toxicity. In vivo studies using an intraperitoneal B16 tumor model and intraperitoneal administration of SPLP showed maximum transfection activity for SPLP containing 24 mol% DODAC. Gene expression observed in tumor cells was increased by approximately one magnitude as compared to cationic lipid/DNA complexes. The SPLP were stable and upon storage at 4 degrees C no significant change in the transfection activity was observed over a one-year period. Thus this phosphate buffer detergent dialysis technique can be used to generate SPLP formulations containing a wide range of cationic lipid concentrations to determine optimal SPLP composition for high transfection activity and low toxicity.     

脂质体粒径的分光光度法检测

目的确立分光光度法判断脂质体相对大小,为评估脂质体物理稳定性提供一种简便、快捷的方法。方法以Rayleigh-Gans-Debye理论为基础,固定脂质体膜材的种类、比例及其在溶液中的总量,分别用乙醇注入法和高压乳匀法制备不同粒径的空间稳定脂质体,以电镜法和热力学光散射法测得的粒径作为标准,找出不同方法制得脂质体在436 nm波长处的吸光度A与粒径D的关系。结果单位磷脂浓度的脂质体溶液在436 nm处log(A_436nm/C_p)与脂质体粒径的对数logD呈线性相关(r²≥0.93,n=5)。结论脂质体溶液的吸光度能反映脂质体相对大小,可作为一种评定脂质体物理稳定性的方法。     

Liposomes encapsulating polymeric chitosan based vesicles--a vesicle in vesicle system for drug delivery

Drug delivery systems comprising vesicles prepared from one amphiphile encapsulating vesicles prepared from a second amphiphile have not been prepared previously due to a tendency of the bilayer components of the different vesicles to mix during preparation. Recently we have developed polymeric vesicles using the new polymer-palmitoyl glycol chitosan and cholesterol in a 2:1 weight ratio. These polymeric vesicles have now been encapsulated within egg phosphatidylcholine (egg PC), cholesterol (2:1 weight ratio) liposomes yielding a vesicle in vesicle system. The vesicle in vesicle system was visualised by freeze fracture electron microscopy. The mixing of the different bilayer components was studied by monitoring the excimer fluorescence of pyrene-labelled polymeric vesicles after their encapsulation within egg PC liposomes or hexadecyl diglycerol ether niosomes. A minimum degree of lipid mixing was observed with the polymeric vesicle-egg PC liposome system when compared to the polymeric vesicle-hexadecyl diglycerol ether niosome system. The polymeric vesicle-egg PC vesicle in vesicle system was shown to retard the release of encapsulated solutes. 28% of 5(6)-carboxyfluorescein (CF) encapsulated in the polymeric vesicle compartment of the vesicle in vesicle system was released after 4 h compared to the release of 62% of encapsulated CF from plain polymeric vesicles within the same time period.     

Pharmacokinetics of oligodeoxynucleotides encapsulated in liposomes: effect of lipid composition and preparation method

1. The effect of the method employed to prepare liposomes and their lipid composition were evaluated in terms of the encapsulation efficiency and pharmacokinetic features of two oligodeoxynucleotides of a 21 mer: the normal (N-Odn) and the phosphorothioate (S-Odn) oligodeoxynucleotide. 2. Liposomes were prepared by the classical method of multilamellar vesicles (MV) and by the dehydration-rehydration method (DR). Two lipid mixtures were used to prepare liposomes - the predominant lipid being phosphatidylcholine (PC) and sphingomyelin (SM) respectively. 3. The DR method for liposome preparation provided the highest encapsulation e ciency, regardless of liposome lipid composition and the type of oligodeoxynucleotide involved (N-Odn or S-Odn). 4. The pharmacokinetics of free and liposome encapsulated oligodeoxynucleotides was studied in mouse following i.v. administration. Liposome encapsulated oligodeoxynucleotides exhibited a significantly lower plasma clearance and longer half-life and residence time than free oligodeoxynucleotides. The method used to obtain the liposomes affected plasma clearance, which was lower for liposomes elaborated by the DR method than for liposomes prepared with the MV method. The use of S-Odn in place of N-Odn decreased the plasma clearance of oligodeoxynucleotide when administered encapsulated in liposomes, regardless of the lipid composition and method used to obtain the liposomes.     

Liposomes containing cationic dimethyl dioctadecyl ammonium bromide: formulation, quality control, and lipofection efficiency

This article describes a novel, simple, and relatively inexpensive method to prepare cationic liposomes using an ethanol injection/pressure extrusion method. The study also demonstrated that binding erythrosine dye to cationic liposomes results in a shift of the absorption maximum of the dye from 528 nm to 549 nm at pH 4.25, allowing quantification and visualization of these vesicles. In addition, a relatively simple Ficoll-based gradient centrifugation method for separation of lipoplexes from unbound molecules is presented. Laboratory-formulated dimethyl dioctadecyl ammonium bromide (DDAB) containing liposomes were just as efficient in complexing nucleic acids as commercially available types, and binding increased as the positive to neutral lipid ratio was increased. Transfection efficiency of the DDAB-containing liposomes increased as the ratio of cationic to neutral lipid was increased from 1:1 to 4:1 with either PtdChol or DOPE as the neutral lipid. A concomitant increase in cytotoxicity of CSU-SA1 cancer cells was noted as the ratio of positive to neutral lipid of the liposomes was increased. Nevertheless, our present study showed that the 2:1 liposome is a good choice since it delivers functional plasmids at a comparable rate to commercial liposome formulations, has similar toxicities to the less harmful commercial liposomes, and is at least 1000-fold more economical to prepare inhouse, a major factor to be considered in preclinical and clinical studies with these carriers.     

Pharmacokinetics of oligodeoxynucleotides encapsulated in liposomes: effect of lipid composition and preparation method

1. The effect of the method employed to prepare liposomes and their lipid composition were evaluated in terms of the encapsulation efficiency and pharmacokinetic features of two oligodeoxynucleotides of a 21 mer: the normal (N-Odn) and the phosphorothioate (S-Odn) oligodeoxynucleotide. 2. Liposomes were prepared by the classical method of multilamellar vesicles (MV) and by the dehydration-rehydration method (DR). Two lipid mixtures were used to prepare liposomes--the predominant lipid being phosphatidylcholine (PC) and sphingomyelin (SM) respectively. 3. The DR method for liposome preparation provided the highest encapsulation efficiency, regardless of liposome lipid composition and the type of oligodeoxynucleotide involved (N-Odn or S-Odn). 4. The pharmacokinetics of free and liposome encapsulated oligodeoxynucleotides was studied in mouse following i.v. administration. Liposome encapsulated oligodeoxynucleotides exhibited a significantly lower plasma clearance and longer half-life and residence time than free oligodeoxynucleotides. The method used to obtain the liposomes affected plasma clearance, which was lower for liposomes elaborated by the DR method than for liposomes prepared with the MV method. The use of S-Odn in place of N-Odn decreased the plasma clearance of oligodeoxynucleotide when administered encapsulated in liposomes, regardless of the lipid composition and method used to obtain the liposomes.     

Liposomes Containing Cationic Dimethyl Dioctadecyl Ammonium Bromide: Formulation,Quality Control,and Lipofection Efficiency

This article describes a novel, simple, and relatively inexpensive method to prepare cationic liposomes using an ethanol injection/pressure extrusion method. The study also demonstrated that binding erythrosine dye to cationic liposomes results in a shift of the absorption maximum of the dye from 528 nm to 549 nm at pH 4.25, allowing quantification and visualization of these vesicles. In addition, a relatively simple Ficoll-based gradient centrifugation method for separation of lipoplexes from unbound molecules is presented. Laboratory-formulated dimethyl dioctadecyl ammonium bromide (DDAB) containing liposomes were just as efficient in complexing nucleic acids as commercially available types, and binding increased as the positive to neutral lipid ratio was increased. Transfection efficiency of the DDAB-containing liposomes increased as the ratio of cationic to neutral lipid was increased from 1:1 to 4:1 with either PtdChol or DOPE as the neutral lipid. A concomitant increase in cytotoxicity of CSU-SA1 cancer cells was noted as the ratio of positive to neutral lipid of the liposomes was increased. Nevertheless, our present study showed that the 2:1 liposome is a good choice since it delivers functional plasmids at a comparable rate to commercial liposome formulations, has similar toxicities to the less harmful commercial liposomes, and is at least 1000-fold more economical to prepare inhouse, a major factor to be considered in preclinical and clinical studies with these carriers.     

Cytotoxicity evaluation of anionic nanoliposomes and nanolipoplexes prepared by the heating method without employing volatile solvents and detergents

Submicron lipid vesicles (nanoliposomes) are being used as carriers of bioactive compounds. In addition, complexes of nanoliposomes and nucleic acids (nanolipoplexes) are promising tools for the treatment of cancer, and viral and genetic disorders. Toxicity of some of these formulations, however, still remains a concern in their clinical utilisation. To address this problem, anionic liposomes were prepared by two different techniques, the conventional thin-film method, and the heating method (HM), in which no volatile organic solvent or detergent is used. An anionic nanolipoplex was constructed by incorporating plasmid DNA (pcDNA3.1/His B/lacZ) into the HM-nanoliposomes by the mediation of calcium. The toxicity of the nanoliposomes, with and without plasmid and Ca2+, was assessed using a human bronchial epithelial cell line (16HBE14o-) in the presence of serum. Cytotoxicity evaluations performed by two different assays (i.e. NRU and MTT) indicated that HM-nanoliposomes were completely non-toxic in the cell-line tested, whereas conventional liposomes revealed significant levels of toxicity. This may be due to the presence of trace amounts of chloroform and/or methanol applied during their preparation. Similar results were obtained for different sizes of lipid vesicles (prepared by 100 nm and 400 nm pore-size filters). In addition, it was observed that incorporation of DNA (15 microg/ 285 microg lipid) and Ca2+ (50 mM) to the nanoliposomes did not have any effect on their cytotoxicities. These findings indicate that the HM-liposomes have great potential as non-toxic delivery vehicles in human gene therapy and drug delivery applications while liposomes made using organic solvents should be used with caution.     

米诺地尔醇脂质体的制备

目的制备米诺地尔醇脂质体并评价其质量。方法采用乙醇注入法制备米诺地尔醇脂质体,以包封率为评价指标进行正交试验筛选出最佳的处方和工艺。采用HPLC测定主药含量、透析袋法测定醇脂质体的包封率。并对其粒径、电位、包封率等理化性质进行研究。结果各因素最佳的水平组合：药脂重量比为1∶10、胆固醇与大豆磷脂的重量比为1∶2、无水乙醇为处方量的30%。所制醇脂质体为乳黄色,平均粒径为1.103μm,Zeta电位为-3.69 mV,平均包封率为66.7%。结论米诺地尔醇脂质体的制备工艺简便可行,质量稳定可控,为开发新剂型奠定了实验基础。     

载基因阳离子脂质体的制备与包封率测定

目的:制备包封基因质粒的阳离子脂质体并考察其性质、测定包封率。方法:以DC-Chol和DOPE为材料,薄膜分散法制备阳离子脂质体,与可表达增强型绿色荧光蛋白的基因质粒结合并考察其转染性能,激光粒度分析仪测定阳离子脂质体和脂质复合物的粒径及Zeta电位;使用葡聚糖凝胶过滤法测定包封率,并对该法进行详细考察。结果:所制备的阳离子脂质体和脂质复合物的平均粒径分别为161.6和216.3 nm,Zeta电位分别为+22.2和+3.2 mV;基因质粒在0.1925～3.85μg.mL-1浓度范围内线性良好,精密度高,与葡聚糖无吸附作用,柱回收率高,测得脂质复合物的包封率为89.94%。结论:采用该处方和工艺可成功制备质量良好、能有效转染细胞的阳离子脂质体载体,葡聚糖凝胶过滤法可准确测定其包封率,该法快速、简便、有效。     

Boron-loaded liposomes in the treatment of hepatic metastases: preliminary investigation by autoradiography analysis

Boronophenylalanine (BPA)-loaded conventional and stabilized liposomes were prepared by the reversed phase evaporation method to treat liver metastases by boron neutron capture therapy. Conventional vesicles were composed of phosphatidylcholine and cholesterol, molar ratio 1:1. To obtain stealth liposomes, GM or PEG were included in the lipidic bilayer at a concentration of 6.67 or 5 mol%, respectively. Large unilamellar vesicles were formulated encapsulating BPA in the liposome aqueous compartment as a complex with fructose; BPA free base also was embedded into the lipidic bilayer. In vivo experiments were carried out after intravenous injection of liposome suspensions in BD-IX strain rats in which liver metastases had been induced. Alpha particle spectroscopy associated with histological analysis was performed to visualize boron spatial distribution in liver. Simultaneously, tissue boron concentrations were determined using inductively coupled plasma-mass spectroscopy. Results showed that PEG-modified liposomes accumulated boron in therapeutic concentrations (> 30 mug boron/g tissue) in metastatic tissue. The PEG-liposomes could be further explored in enhancing boron delivery to tumor cells.     

High Sensitivity Differential Scanning Calorimetry Study of DNA-Cationic Liposome Complexes

PURPOSE: To investigate plasmid DNA interactions with liposomes prepared from dimyristoylglyceroethylphosphocholine (EDMPC) and DOPE using high sensitivity differential scanning calorimetry (HSDSC). MATERIALS AND METHODS: Large unilamellar liposomes of EDMPC with DOPE (mol ratio 0-50%) were prepared. Plasmid DNA was added to give a final DNA/lipid (-/+) charge ratio of 0.5. Samples were placed into an HSDSC and cooled to 3 degrees C, held isothermally for 30 min and then the temperature was ramped to 120 degrees C at a rate of 1 degree C/min. RESULTS: On heating EDMPC liposomes, the main phase transition occurred at 21.2 degrees C, with a low temperature shoulder on the endothermic peak. At low DOPE concentrations the main phase transition temperatures and enthalpies of transition were lower than for pure EDMPC, with a peak corresponding to a pure EDMPC phase occurring at DOPE concentrations of 12-17 mol%. At 50 mol%, no main transition endotherm was observed. DNA solution produced two endothermic peaks with numerous 'satellite' peaks indicating thermal denaturation. DNA binding to EDMPC changed the shape of the thermogram, indicating alteration in lipid packing within the bilayer. DNA induced demixing in the bilayers of DOPE-containing liposomes. CONCLUSION: HSDSC provided information for characterizing liposome formulations and DNA interactions with such vesicles.     

Cationic Liposomes for Gene Transfection

Cationic liposomes spontaneously interact with the negatively charged DNA to form a stable complex that promotes the gene transfer to cells. The mode of formation and the size of cationic liposomes/DNA complexes were investigated using the atomic force microscopy (AFM). Also the most important physical–chemical factors involved in cationic liposome-mediated gene transfection, e.g. size and lipidic composition, were evaluated through the transfection of complexes with different liposomes/DNA molar ratio into three types of cultured cells. Cationic liposomes, composed of a neutral lipid (phosphatidilcoline), a cationic lipid dimethyldioctadecylammonium bromide (DDAB), a co-lipid 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) and a phospholipid derivative of polyethylene glycol (DSPE-mPEG) at different molar ratio, were mixed with a plasmid pCMVβ to form liposomes/DNA complexes. We have demonstrated that the complexes were made by complicated structures in which the liposomes tend to aggregate and the DNA is surrounded by lipidic material. In vitro transfection efficiency by liposomes/plasmid pCMVβ complexes was found to depend on the kind of lipid associated in the liposomes and the liposomes/DNA mixing ratio. The importance of associating DOPE in cationic liposomes was confirmed; this co-lipid is able to improve the ability of cationic liposomes to transfect cells but in addition, the AFM images and the EtBr fluorescence experiments have suggested that this lipid can also play an important role to facilitate the formation of stable liposomes, which efficaciously protect the DNA by nuclease digestion.     

Preparation of a TK/GCV administration system mediated by transferrin modified pro-cationic liposomes

Transferrin modified pro-cationic liposomes were prepared and used to investigate the effect of targeting therapeutic genes to human hepatoma carcinoma cells in vitro. The main lipid CHETA, cholest-5-en-3beta-yl[2-[[4-[(carboxymethyl)dithio]-1-iminobutyl]amino]ethyl] carbamate (C36H61N3O4S2), was synthesized and used to prepare pro-cationic liposomes. The thymidine kinase (TK) gene loaded pro-cationic liposomes were prepared by first mixing the plasmid DNA and protamine together, and then incubating the resulted polyplexes with blank pro-cationic liposomes preformed by the thin film dispersion-sonication method. Transferrin (Tf) was adsorbed on the surface of pro-cationic liposomes via electrostatic interactions to form transferrin modified pro-cationic liposomes. Cellular association was measured by fluorimetry at excitation and emission wavelengths of 490 and 520 nm, respectively. The viability of TK gene infected cells following administration of ganciclovir (GCV) was investigated by MTT assay. The transferrin modified TK gene pro-cationic liposomes had a mean diameter of 240 +/- 12 nm and zeta potential of -24.10 +/- 2.5 mV (n = 3). The transmission electron microscopy image indicated that most of the liposomes were relatively regular and spherical with a condensed core inside. Cell-associated fluorescence of Tf-liposomes and unmodified liposomes (without transferrin) was 7.8 x 10(6), and 3.2 x 10(6) per milligram protein, respectively. Compared to Lipofectamine 2000 (Invitrogen, USA) the pro-cationic liposomes and transferrin modified pro-cationic liposomes had less cytotoxicity to cells. The transduced TK gene HepG2 cells were more sensitive to GCV than the un-transduced TK gene ones and the human normal Chang liver cells were not affected by the TK/GCV system mediated by procationic liposomes.     

Cationic liposomes for gene transfection

Cationic liposomes spontaneously interact with the negatively charged DNA to form a stable complex that promotes the gene transfer to cells. The mode of formation and the size of cationic liposomes/DNA complexes were investigated using the atomic force microscopy (AFM). Also the most important physical-chemical factors involved in cationic liposome-mediated gene transfection, e.g. size and lipidic composition, were evaluated through the transfection of complexes with different liposomes/DNA molar ratio into three types of cultured cells. Cationic liposomes, composed of a neutral lipid (phosphatidilcoline), a cationic lipid dimethyldioctadecylammonium bromide (DDAB), a co-lipid 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) and a phospholipid derivative of polyethylene glycol (DSPE-mPEG) at different molar ratio, were mixed with a plasmid pCMVbeta to form liposomes/DNA complexes. We have demonstrated that the complexes were made by complicated structures in which the liposomes tend to aggregate and the DNA is surrounded by lipidic material. In vitro transfection efficiency by liposomes/plasmid pCMVbeta complexes was found to depend on the kind of lipid associated in the liposomes and the liposomes/DNA mixing ratio. The importance of associating DOPE in cationic liposomes was confirmed; this co-lipid is able to improve the ability of cationic liposomes to transfect cells but in addition, the AFM images and the EtBr fluorescence experiments have suggested that this lipid can also play an important role to facilitate the formation of stable liposomes, which efficaciously protect the DNA by nuclease digestion.     

Surfactant vesicle-mediated delivery of DNA vaccines via the subcutaneous route

Compared to naked DNA immunisation, entrapment of plasmid-based DNA vaccines into liposomes by the dehydration-rehydration method has shown to enhance both humoural and cell-mediated immune responses to encoded antigens administered by a variety of routes. In this paper we have compared the potency of lipid-based and non-ionic surfactant based vesicle carrier systems for DNA vaccines after subcutaneous immunisation. Plasmid pI.18Sfi/NP containing the nucleoprotein (NP) gene of A/Sichuan/2/87 (H3N2) influenza virus in the pI.18 expression vector was incorporated by the dehydration-rehydration method into various vesicle formulations. The DRV method, entailing mixing of small unilamellar vesicles (SUV) with DNA, followed by dehydration and rehydration, yielded high DNA vaccine incorporation values (85-97% of the DNA used) in all formulations. Studies on vesicle size revealed lipid-based systems formed cationic submicron size vesicles whilst constructs containing a non-ionic surfactant had significantly large z-average diameters (>1500 nm). Subcutaneous vesicle-mediated DNA immunisation employing two DRV(DNA) formulations as well as naked DNA revealed that humoural responses (immunoglobulin total IgG, and subclasses IgG1 and 1gG2a) engendered by the plasmid encoded nucleoprotein were substantially higher after dosing twice, 28 days apart with 10 microg DRV-entrapped DNA compared to naked DNA. Comparison between the lipid and non-ionic based vesicle formulations revealed no significant difference in stimulated antibody production. These results suggest that, not only can DNA be effectively entrapped within a range of lipid and non-ionic based vesicle formulations using the DRV method but that such DRV vesicles containing DNA may be a useful system for subcutaneous delivery of DNA vaccines.     

Dexamethasone Incorporating Liposomes: Effect of Lipid Composition on Drug Trapping Efficiency and Vesicle Stability

Effect of lipid composition on encapsulation and stability of dexamethasone (DXM) incorporating multilamellar vesicles (MLV) is studied. MLVs composed of phosphatidylcholine (PC) or distearoyl-glycero-PC (DSPC), with or without cholesterol (Chol), are prepared and the release of DXM during vesicle incubation in buffer or plasma proteins is evaluated. Incorporation of DXM is slightly higher in DSPC liposomes compared with PC, whereas the drug is displaced from liposomes, as the Chol content of liposome membranes increases. Plain lipid and Chol-containing liposomes lose similar fractions of vesicle-incorporated DXM during incubation in buffer or serum, whereas DXM release kinetics are similar (for each liposome type studied), implying that drug release is due mainly to dilution of liposome dispersions that leads to repartitioning of DXM.     

Dexamethasone incorporating liposomes: effect of lipid composition on drug trapping efficiency and vesicle stability

Effect of lipid composition on encapsulation and stability of dexamethasone (DXM) incorporating multilamellar vesicles (MLV) is studied. MLVs composed of phosphatidylcholine (PC) or distearoyl-glycero-PC (DSPC), with or without cholesterol (Chol), are prepared and the release of DXM during vesicle incubation in buffer or plasma proteins is evaluated. Incorporation of DXM is slightly higher in DSPC liposomes compared with PC, whereas the drug is displaced from liposomes, as the Chol content of liposome membranes increases. Plain lipid and Chol-containing liposomes lose similar fractions of vesicle-incorporated DXM during incubation in buffer or serum, whereas DXM release kinetics are similar (for each liposome type studied), implying that drug release is due mainly to dilution of liposome dispersions that leads to repartitioning of DXM.     

人参总皂苷脂质体的制备及质量评价

目的制备人参总皂苷(TSPG)脂质体,并进行质量评价。方法采用薄膜分散法、逆相蒸发法、乙醇注入法制备TSPG脂质体,以形态、包封率、载药量为指标筛选制备方法。结果薄膜分散法制备的TSPG脂质体外形规则、光滑、不黏连,包封率、载药量分别为38.70%、3.14%,较其他方法好。结论薄膜分散法制备的TSPG脂质体达到预期目标。     

Liposome-mediated DNA immunisation via the subcutaneous route

Compared to naked DNA immunisation, entrapment of plasmid-based DNA vaccines into liposomes by the dehydration-rehydration method has shown to enhance both humoural and cell-mediated immune responses to encoded antigens administered by a variety of routes. In this paper, we have investigated the application of liposome-entrapped DNA and their cationic lipid composition on such potency after subcutaneous immunisation. Plasmid pI.18Sfi/NP containing the nucleoprotein (NP) gene of A/Sichuan/2/87 (H3N2) influenza virus in the pI.18 expression vector was incorporated by the dehydration-rehydration method into liposomes composed of 16 micromol egg phosphatidylcholine (PC), 8 micromoles dioleoyl phosphatidylethanolamine (DOPE) or cholesterol (Chol) and either the cationic lipid 1,2-diodeoyl-3-(trimethylammonium) propane (DOTAP) or cholesteryl 3-N-(dimethyl amino ethyl) carbamate (DC-Chol). This method, entailing mixing of small unilamellar vesicles (SUV) with DNA, followed by dehydration and rehydration, yielded incorporation values of 90-94% of the DNA used. Mixing or rehydration of preformed cationic liposomes with 100 microg plasmid DNA also led to similarly high complexation values (92-94%). In an attempt to establish differences in the nature of DNA association with these various liposome preparations their physico-chemical characteristics were investigated. Studies on vesicle size, zeta potential and gel electrophoresis in the presence of the anion sodium dodecyl sulphate (SDS) indicate that, under the conditions employed, formulation of liposomal DNA by the dehydration-rehydration generated submicron size liposomes incorporating most of the DNA in a manner that prevents DNA displacement through anion competition. The bilayer composition of these dehydration-rehydration vesicles (DRV(DNA)) can also further influence these physico-chemical characteristics with the presence of DOPE within the liposome bilayer resulting in a reduced vesicle zeta potential. Subcutaneous liposome-mediated DNA immunisation employing two DRV(DNA) formulations as well as naked DNA revealed that humoural responses (immunoglobulin total IgG, and subclasses IgG1 and 1gG2a) engendered by the plasmid encoded NP were substantially higher after dosing twice, 28 days apart with 10 microg liposome-entrapped DNA compared to naked DNA. At all time points measured, mice immunised with naked DNA showed no greater immune response compared to the control, non-immunised group. In contrast, as early as day 49, responses were significantly higher in mice injected with DNA entrapped in DRV liposomes containing DOTAP compared to the control group and mice immunised with naked DNA. By day 56, all total IgG responses from mice immunised with both DRV formulations were significantly higher. Comparison between the DRV formulations revealed no significant difference in immune responses elicited except at day 114, where the humoural responses of the group injected with liposomal formulation containing DC-Chol dropped to significantly lower levels that those measured in mice which received the DOTAP formulation. Similar results were found when the IgG1 and IgG2a subclass responses were determined. These results suggest that, not only can DNA be effectively entrapped within liposomes using the DRV method but that such DRV liposomes containing DNA may be a useful system for subcutaneous delivery of DNA vaccines.