反义寡核苷酸脂质体复合物性质对细胞摄入行为的影响

目的研究影响反义寡核苷酸脂质体复合物的性质和细胞摄取的因素。方法逆相蒸发法制备3种不同的空白脂质体,与反义寡核苷酸混合得到复合物,显微镜观察其形态,琼脂糖电泳分析载药量,流式细胞仪测定阳性细胞百分率和平均荧光强度。结果高电荷密度的脂质体和低离子强度介质可使复合物发生凝聚,载药量和细胞摄入量依赖于空白脂质体和药物的比例以及脂质体膜表面的电荷密度。结论阳离子脂质体可以提高载药量和细胞的摄入,其程度与复合物比例、脂质体膜表面电荷密度等有关。     

Effect of ultrasonication on the stability of oligonucleotides adsorbed on nanoparticles and liposomes

In the present study, oligonucleotides were adsorbed onto the surface of cationic liposomes and nanoparticles at different ratios. As a result, the surface charges of the colloidal carriers were decreased with increasing oligonucleotide concentration. At a certain oligonucleotide concentration, complete charge neutralization led to the aggregation of the carrier systems. Further increasing oligonucleotide concentrations reversed the surface charge of liposomes and nanoparticles to a negative one. Ultrasonication was investigated as a possible means for the homogenization of the formed aggregates. However, the use of ultrasonication led to a time-dependent damage of oligonucleotides adsorbed onto AH-Chol liposomes and MMAEMC-nanoparticles, as well as of unbound oligonucleotides. Nearly 60% of the oligonucleotides adsorbed to MMAEMCnanoparticles and 65% of ODNs adsorbed to the liposomes were degraded by the effect of cavitation produced by ultrasonication within 10min. In contrast, the oligonucleotides were protected from degradation when DEAE-stabilized PHCA-nanoparticles were employed as ODN carriers. More than 80% of the oligonucleotides entangled in thesurface matrix of these nanoparticles remained intact.     

Effect of ultrasonication on the stability of oligonucleotides adsorbed on nanoparticles and liposomes.

In the present study, oligonucleotides were adsorbed onto the surface of cationic liposomes and nanoparticles at different ratios. As a result, the surface charges of the colloidal carriers were decreased with increasing oligonucleotide concentration. At a certain oligonucleotide concentration, complete charge neutralization led to the aggregation of the carrier systems. Further increasing oligonucleotide concentrations reversed the surface charge of liposomes and nanoparticles to a negative one. Ultrasonication was investigated as a possible means for the homogenization of the formed aggregates. However, the use of ultrasonication led to a time-dependent damage of oligonucleotides adsorbed onto AH-Chol liposomes and MMAEMC-nanoparticles, as well as of unbound oligonucleotides. Nearly 60% of the oligonucleotides adsorbed to MMAEMC-nanoparticles and 65% of ODNs adsorbed to the liposomes were degraded by the effect of cavitation produced by ultrasonication within 10 min. In contrast, the oligonucleotides were protected from degradation when DEAE-stabilized PHCA-nanoparticles were employed as ODN carriers. More than 80% of the oligonucleotides entangled in the surface matrix of these nanoparticles remained intact.     

Physicochemical properties of liposomes incorporating hydrochlorothiazide and chlorothiazide

In an attempt to study the effect of hydrophobic drugs on liposome properties, multilamellar liposomes (MLV) consisting of phosphatidylcholine (PC) and incorporating chlorothiazide (CT) or hydrochlorothiazide (HCT), were prepared and characterized. Liposome size, surface charge, stability (in buffer, plasma and sodium cholate) and calcium-induced aggregation were studied for drug-incorporating liposomes and empty liposomes for comparison. Results show that drug incorporation affects liposome size, z-potential and stability in presence of buffer and plasma proteins. Indeed, drug-incorporating liposomes are slightly larger and have a negative surface charge, which increases with the amount of drug incorporated in the lipid membrane. The membrane integrity of drug incorporating liposomes (in absence and presence of plasma proteins) is significantly higher when compared with that of empty liposomes (for both drugs studied). On the contrary, vesicle membrane integrity in presence of sodium cholate and calcium induced vesicle aggregation, are not affected by drug incorporation. Leakage of thiazides from liposomes was demonstrated to be induced by dilution. Low amounts of thiazides (around 10-15%) are released when lipid concentration is over 0.1 mM, while further dilution increased drug leakage exponentially. Concluding, results demonstrate that the presence of HCT or CT in liposome membranes has a significant effect on main vesicle properties, which are known to influence vesicle targeting ability. Thereby, it is very interesting to continue studies in this respect, especially with more lipophilic drugs.     

Novel cationic liposome formulation for the delivery of an oligonucleotide decoy to NF-kappaB into activated macrophages.

Nuclear factor-kappaB (NF-kappaB) is involved in several pathological processes, such as inflammation. Pro-inflammatory genes expression can be down-regulated by using an oligonucleotide (ODN) decoy to NF-kappaB. Cationic liposomes are largely used to improve ODN uptake into cells, although a higher transfection efficiency and a lower toxicity are required to use them in therapy. In this work, we investigated the potential of a novel liposome formulation, based on the recently synthesised cationic lipid (2,3-didodecyloxypropyl) (2-hydroxyethyl) dimethylammonium bromide (DE), as the delivery system for a double stranded ODN decoy to NF-kappaB. Liposomes composed of DE or DE mixed with 1,2-dioleyl-sn-glycero-3-phosphoethanolamine or cholesterol as helper lipids were complexed with ODN at different +/- charge ratios. In vitro uptake and the effect of ODN, naked or complexed with DE-containing liposomes, were evaluated in lipopolysaccharide-stimulated RAW 264.7 macrophages. The use of helper lipids increased liposome physical stability up to 1 year at 4 degrees C. ODN complexed with DE/cholesterol liposomes, at the +/- charge ratio of 8, showed a limited cytotoxicity and the highest inhibition of nitrite production, inducible nitric oxide synthase protein expression and NF-kappaB/DNA binding activity. Confocal microscopy confirmed a high ODN cell uptake obtained with DE/cholesterol liposomes at the highest +/- charge ratio.     

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。     

Differential Adhesion of Normal and Inflamed Rat Colonic Mucosa by Charged Liposomes

PURPOSE: To study the adhesion properties of charged liposomes to the healthy and inflamed (colitis-induced) rat intestinal epithelium. METHODS: Neutral, positively charged, and negatively charged liposomes were prepared and tagged. The cationic or anionic liposomes contained increasing amounts (13, 22, or 36 mol%) of either the cationic lipid dimethyl-dioctadecylammoniumbromide (DODAB) or the anionic lipid 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DSPG). Colitis was induced in rats by DNBS. Adhesion of the various types of liposomes was assessed in rat colon sacs. The effects of charge type, charge density (mol%), liposome size, and incubation time on the adhesion of the liposomes were compared in the inflamed and healthy epithelial tissues. RESULTS: Three times as many cationic liposomes adhered to the healthy colonic mucosa than neutral or anionic liposomes. However, anionic liposome adherence to the inflamed colonic mucosa was 2-fold that of either neutral or cationic liposomes (a finding that was verified by charged-dyes studies). Adherence was directly correlated with charge density. An inverse correlation was identified between cationic liposome size and healthy tissue adherence in short incubation periods. The adherence of cationic liposomes, which was also found to be time-dependent, decreased in healthy mucosa in the presence of high concentrations of aqueous Mg2+ rinse. CONCLUSIONS: Anionic liposomes could be useful for the topical delivery of anti-inflammatory drugs in inflammatory bowel disease therapy.     

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.     

Comparative Study of Separation of Non-encapsulated Drug from Unilamellar Liposomes by Various Methods

The purpose of this study was to compare the various methods available to separate non-encapsulated drug from large unilamellar liposomes (LUV). Multilamellar liposomes (MLV) were prepared by thin film hydration using distearoylphosphatidylcholine:cholesterol (2:1 molar ratio). MLVs were passed through a 0.2-μm polycarbonate membrane using an extruder to prepare LUVs. Particle size of liposome preparations was characterized using a submicron particle-size analyser. The non-encapsulated drug was separated by: filtering through Centrifree tubes; passing through gel (Sepharose-4B and Sephadex G-25M); passing through minicolumn; ficoll density gradient; protamine aggregation; or dialysis. The dialysis method was found to be unsuitable for separation of non-encapsulated drug due to equilibration of encapsulated drug as the free drug was dialyzed. The upper limit for lipid concentration was 5 mg mL^?1 using the Centrifree method. Separation using gel chromatography led to dilution of liposome preparation. Minicolumn and density gradient techniques did not lead to sample dilution, however the minicolumn method was tedious. The time required for separation of liposomes by protamine aggregation was longer for neutral liposomes. Thus it was concluded that the Centrifree was the fastest method to estimate encapsulation; the density gradient method was ideal to separate non-encapsulated drug; and protamine aggregation was the least expensive method to estimate encapsulation efficiency.     

Liposomes as drug carrier systems. Preparation, classification and therapeutic advantages of liposomes]

Bangham et al. (1965) created first the concept of the liposome as a microparticulate lipoidal vesicle separated from its aqueous environment by one or more lipid bilayers. Later Gregoriadis and Ryman (1972) suggested to use liposomes as drug carrier systems. Nowadays liposomes are under extensive investigation for improving the delivery of therapeutic agents, enzymes, vaccines and genetic materials. Liposomes offer an excellent opportunity to selective targeting of drugs which is expected to optimize the pharmacokinetical parameters, the pharmacological effect and to reduce the toxicity of the encapsulated drugs. To understand the system it is important to know the basic properties of these lipoidal vesicles. Our aim was to focus on the lipid composition and the method of liposome preparation what determine the liposomal membrane fluidity, permeability, vesicle size, charge density, steric hindrance and stability of the liposomes as principle factors those influence the fate of liposomes, their interactions with the blood components and other tissues after systemic administration or local use.     

Modeling cytoplasmic release of encapsulated oligonucleotides from cationic liposomes

Transfection activity of antisense oligodeoxynucleotides (ODN)-loaded cationic liposomes is mainly restricted by uptake and ODN release into cytoplasm, which is difficult to evaluate in cell culture studies. Well-designed models of cellular membranes, aim of the present study, might facilitate investigation of such processes. In this investigation, a phosphorothioate ODN was actively encapsulated in a DODAP-containing cationic liposome by ethanol injection with 73% efficiency. ODN release was determined by fluorescence dequenching of FITC-ODN upon incubation of liposomes with early endosomal (EE), late endosomal (LE) and plasma membranes (PM) models. LE provided the highest release (up to 76%) in a temperature-dependent manner. Release by EE (<16%), total PM (<11%) and PM external layer ( approximately 0) were not temperature sensitive. These differences are attributed to lipid charge, chain mobility, critical packing parameter and cholesterol content of the models. Intracellular distribution of FITC-ODN, determined by fluorescence microscopy and flowcytometry in the presence and absence of sodium azide, confirmed that liposomes were internalized mainly via endocytosis; hence inability of our PL models to simulate such active processes. Instead, release of ODN from endosomes into cytoplasm was pH-sensitive and in good agreement with model membrane studies in terms of amount and mechanism.     

槐定碱阳离子脂质体的制备及体外抗肿瘤活性的研究

目的制备槐定碱阳离子脂质体,并探讨其对肿瘤细胞的抑制作用。方法采用主动载药法制备槐定碱阳离子脂质体,并对其进行表征研究,采用MTS方法考察槐定碱阳离子脂质体对3种肿瘤细胞的抑制作用。结果制备得到的槐定碱阳离子脂质体呈类圆形,表面光滑,其平均粒径和聚分散指数分别为242.2 nm和0.180,表面电荷为+32.5 m V,其包封率和载药量分别为88.62%和5.97%。槐定碱阳离子脂质体对3种肿瘤细胞的IC50值均明显高于槐定碱,而空白阳离子脂质体对细胞并无明显的抑制作用。结论采用阳离子脂质体作为槐定碱的载体有利于将药物透过细胞膜,提高抗肿瘤作用,值得进行深入的系统研究。     

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.     

A new vaginal delivery system of amphotericin B: a dispersion of cationic liposomes in a thermosensitive gel

Amphotericin B (AmB) is used in the treatment of fungal infections; however, its clinical use is limited by its toxic side effects. In this study, AmB-loaded cationic liposome gels were formulated with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and cholesterol (CH) at a molar ratio of DOPE:DOTAP:CH?=?4:5:1 in thermosensitive gel composed of poloxamer 407 (P407) and poloxamer 188 (P188). To enhance the solubility of AmB, 6 mol% of distearoyl phosphatidyl ethanolamine–polyethylene glycol was added prior to encapsulation of the drug into liposomes. Scanning electron microscopy was used to observe the AmB encapsulated cationic liposome gels. In vitro release, stability and cytotoxicity of AmB in cationic liposome gels were evaluated. The particle size and zeta potential of AmB-loaded liposomes were in the range of 400–500?nm and 40–60 mV, respectively. The thermosensitive gel at the ratio of P407:P188?=?15:15 (w/w) gelled at 37°C, approximating body temperature. Encapsulation efficiency of AmB was ～50–60%, which was influenced by the ratio of AmB to lipid. Moreover, AmB-loaded cationic liposome gels were more stable and less toxic than free AmB. From these results, cationic liposome gel formulations may be useful for vaginal delivery of AmB.     

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.     

In vivo gene transfection via intravitreal injection of cationic liposome/plasmid DNA complexes in rabbits

To optimize the in vivo ocular transfection efficiency of plasmid DNA (pDNA)/cationic liposome complexes, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)/dioleoylphosphatidylethanolamine (DOPE) (1:1 molar ratio) liposomes and DOTMA/cholesterol (Chol) (1:1 molar ratio) liposomes were prepared with varying amounts of pDNA. pDNA/cationic liposome complexes were intravitreally injected (100 microL) in rabbits, and luciferase activity in the cornea, aqueous humor, iris-ciliary body, lens, vitreous body, and retina was measured. Transfection efficiency of pDNA alone did not change with pDNA ranging from 40 to 85 mg. In contrast, transfection efficiency of pDNA complexed with DOTMA/Chol liposomes significantly increased with the amount of pDNA ranging from 40 to 85 microg (P < 0.05). pDNA complexed with DOTMA/DOPE liposomes could not be prepared with pDNA greater than 60 microg. Among these experiments, pDNA (85 microg) complexed with DOTMA/Chol liposomes (pDNA:cationic liposome charge ratio (- : +) = 1.0:2.0) showed the highest transfection efficiency in the ocular tissue and its transfection-mediated luciferase activity peaked at 3 days. Among the ocular tissues, the highest gene expression was observed in the aqueous humor.     

Physicochemical characterization and skin permeation of liposome formulations containing clindamycin phosphate

This study was undertaken to evaluate the physicochemical properties and skin permeation of liposome formulations containing clindamycin phosphate (CP), especially when charge was imparted to the liposome. Five different liposome formulations were prepared using Phospholipon 85G (PL) and cholesterol (CH) by conventional lipid film hydration technique. Molar ratio of CH to PL was varied in the range of 0.16–1.0. Charged liposomes were prepared in the same way with addition of 1,2-dioleoyl-3-trimethylammonium-propane chloride salt (DOTAP) and 1,2-dimyristoyl-sn-glycero-3-phosphate monosodium salt (DMPA) as charge carrier lipid for cationic or anionic charge of the liposome, respectively. Fresh full-thickness mice skin was taken and used for skin permeation study using Keshary-Chien diffusion cell with 1.77 cm² diffusion area at 37°C. All liposome formulations prepared showed homogeneous size distribution with mean particle size of about 1 μm or less. Among the five liposome formulations prepared, formulation with the molar ratio of 0.5 showed the best result in the physicochemical properties such as polydispersity index, entrapment efficiency, size evolution, and ability of the liposome to retain CP as of entrapped in the vesicles. Charge-impartation of the formulation with cationic charge carrier lipid resulted in additional benefit in terms of inhibition of size evolution, the ability of the liposome to retain CP in the vesicles, and skin permeation. Steady state flux of the drug through the mice skin in the cationic liposome vesicles was 0.75 ± 0.01 μg/cm²h while that in the control (dissolved into mixed alcohol solution) was 0.17 μg/cm²h. One half molar ratio of CH to PL was optimal in terms of physicochemical properties of the liposome formulation containing CP, and incorporation of cationic charge carrier lipid appeared to provide additional benefits for the stability of the liposome formulation and skin permeation of the drug.     

Effect of cationic charge on receptor-mediated transfection using mannosylated cationic liposome/plasmid DNA complexes following the intravenous administration in mice

The purpose of this study was to evaluate the effect of cationic charge of complexes after intravenous administration of cholesten-5-yloxy-N-[4-[(1-imino-2-D-thiomannosyl-ethyl)amino]butyl]formamide (Man-C4-Chol) containing cationic liposomes/pDNA complexes in mice. Transfection efficiency after intravenous administration of complex at a charge ratio (- : +) of 1.0:2.3 and/or 1.0:3.1 in liver and spleen expressing a mannose receptor on the cell surface were higher than those in lung. When complexes were formed at a charge ratio (- : +) of 1.0:4.7, on the other hand, transfection efficiency in the lung was highest, suggesting a non-specific interaction. Although asialoglycoprotein receptors are expressed on hepatocytes, a liver-selective gene transfection was not achieved by the intravenous administration of pDNA complexed with cholesten-5-yloxy-N-[4-[(1-imino-2-D-thiogalactosyl-ethyl)-amino]butyl]formamide (Gal-C4-Chol)/DOPE liposomes at a charge ratio (- : +) of 1.0 : 2.3. This information supports the design of pDNA/ligands-grafted cationic liposome complexes for cell-specific gene delivery after intravenous administration.     

A novel cationic cholesterol derivative,its formulation into liposomes,and the efficient transfection of the transformed human cell lines HepG2 and HeLa

A novel cationic cholesterol derivative, 3 β[N-(N',N',N'-trimethylaminopropane)-carbamoyl] cholesterol iodide (Chol-Q), has been formulated with equimolar amounts of dioleoyl phosphatidylethanolamine (DOPE) into stable unilamellar liposomes up to 100 nm in size for DNA delivery into mammalian cells. When compared with similarly constituted liposomes containing the tertiary analogue 3 β[N-(N',N'-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T) in a band shift assay, liposomes displayed similar DNA binding affinities and appeared to afford complete protection to plasmid DNA against serum nuclease catalysed degradation at liposome:DNA ratios (w/w) of 2.5:1, 5:1, and 10:1 in incubation mixtures containing 5% fetal bovine serum at 37 C for 90 min. Chol-Q liposomes were, however, markedly less toxic to cells in culture over a wide range of concentrations with cells numbering 76% of untreated controls at 37.5 μg/mL complete medium in the human hepatocellular carcinoma line HepG2 and 75% at 30 μg/mL in cervical carcinoma HeLa cells. At these levels of Chol-T liposomes, cell numbers were 37% and 15%, respectively. Gene transfer experiments with pSV2CAT and pRSVCAT plasmids in HepG2 cells showed maximum efficiency at a Chol-Q liposome:DNA ratio of 5:1 (w/w) and at a Chol-T liposome:DNA ratio of 10:1. In HeLa cells, both liposome preparations performed best at a ratio of 2.5:1. Differences in transfection efficiencies over the liposome range of 5-20 μg/ mL were rather less pronounced with Chol-Q lipoplexes suggesting a greater versatility of this system.     

Biotin-directed assembly of targeted modular lipoplexes and their transfection of human hepatoma cells in vitro

The asialoglycoprotein receptor, which is abundantly and near exclusively expressed on hepatocytes, has received much attention in the design of non-viral hepatotropic DNA delivery systems. Thus, asialoglycoproteins and hexopyranosyl ligands have been coupled to DNA-binding cationic polymers and liposomes in the assembly of complexes intended for uptake by liver parenchymal cells. The aim of the study was to construct a hepatocyte-targeted multimodular liposome-based transfecting complex, in which the biotin-streptavidin interaction provides the cohesive force between the ligand asialorosomucoid and the liposome bilayer, and to evaluate its transfection capabilities in the hepatocyte-derived human transformed cell line HepG2. Dibiotinylated asialoorosomucoid was attached to cationic liposomes constructed from 3β[N-(N’,N’-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T):dioleoylphosphatidylethanolamine:biotinylcholesterylformylhydrazide (MSB1) (48:50:2 mole ratio) through streptavidin interposition. Liposome-pGL3 DNA interactions were studied by gel band shift and ethidium displacement assays. The cytotoxicity of assemblies was evaluated in the HepG2 cell line and transfection capabilities determined by measuring the activity of the transgene luciferase. Binding assays showed that all DNA was liposome associated at a DNA (negative):liposome (positive) charge ratio of 1:1. Accommodation of a streptavidin dibiotinylated asialoorosomucoid assembly was achieved at a DNA:liposome:streptavidin dibiotinylated asialoorosomucoid ratio of 1:4:9 (weight basis). Complexes showed optimal transfection activity at this ratio, which was reduced 10-fold by the presence of the competing ligand asialofetuin. The streptavidin-biotin interaction has been applied for the first time to the assembly of hepatocyte-targeted lipoplexes that display asialoorosomucoid and that are well tolerated by a human hepatoma cell line in which transfection is demonstrably achieved by receptor mediation. Favorable size and charge ratio characteristics suggest that this system may be suitable for in vivo application.