Cationic liposomes for gene delivery: from biophysics to biological applications

The use of an efficient carrier for nucleic acid-based medicines is considered to be a determinant factor for the successful application of gene therapy. The drawbacks associated with the use of viral vectors, namely those related with safety problems, have prompted investigators to develop alternative methods for gene delivery, cationic lipid-based systems being the most representative. Despite extensive research in the last decade on the use of cationic liposomes as gene transfer vectors and the development of elegant strategies to enhance their biological activity, these systems are still far from being viable alternatives to the use of viral vectors in gene therapy. In this review considerations are made regarding the structure-activity relationships of cationic liposome/DNA complexes and the key formulation parameters influencing the features of lipoplexes are presented and discussed in terms of their effect on biological activity. Particular emphasis is given to the interaction of the lipoplexes with serum components as well as to novel strategies developed to circumvent difficulties that may emerge upon iv administration of the complexes. Finally, since the ability of the lipoplexes to be stored while preserving their transfection activity is a crucial issue for the repeated use of such carriers, approaches reported on the improvement of their physical stability are also reviewed.     

In vivo comparative study of lipid/DNA complexes with different in vitro serum stability: effects on biodistribution and tumor accumulation

To evaluate the in vivo biodistribution and expression of DOTAP-Chol/DNA complexes (lipoplexes) with different in vitro serum stability, quantitative real-time PCR, in vitro luciferase expression and whole body luminescence imaging were used. In general, less tissue biodistribution, lower luciferase expression and whole body luminescence were observed for DOTAP:Chol (mol/mol 1:4)/DNA lipoplexes which had higher in vitro serum stability as compared to DOTAP:Chol (mol/mol 1:1)/DNA lipoplexes. Plasmid DNA biodistribution and expression were mainly confined to the lungs, and the results suggest that in vitro serum stability may serve as a predictor of transfection in the lung. No correlation between plasmid DNA tissue biodistribution and gene expression was observed by simultaneous determination of the level of plasmid DNA tissue biodistribution and gene expression. While high doses of the formulation possessing increased in vitro serum stability did exhibit reduced entrapment in the lung, no corresponding increase in the plasmid levels of other tissues was observed. However, this formulation did show increased accumulation in tumors that was not further enhanced by PEGylation.     

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.     

Anionic liposomal delivery system for DNA transfection

The present study investigates the use of novel anionic lipoplexes composed of physiological components for plasmid DNA delivery into mammalian cells in vitro. Liposomes were prepared from mixtures of endogenously occurring anionic and zwitterionic lipids, 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DOPG) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), respectively, at a molar ratio of 17:83 (DOPG:DOPE). Anionic lipoplexes were formed by complexation between anionic liposomes and plasmid DNA molecules encoding green fluorescence protein (GFP) using Ca2+ ions. Transfection and toxicity were evaluated in CHO-K1 cells using flow cytometry and propidium iodide staining, respectively. Controls included Ca2+-DNA complexes (without lipids), anionic liposomes (no Ca2+), and a cationic liposomal formulation. Efficient delivery of plasmid DNA and subsequent GFP expression was achieved using anionic lipoplexes. Transfection efficiency increased with Ca2+ concentration up to 14 mM Ca2+, where transfection efficiency was 7-fold higher than in untreated cells, with minimum toxicity. Further increase in Ca2+ decreased transfection. Transfection efficiency of anionic lipoplexes was similar to that of cationic liposomes (lipofectAmine), whereas their toxicity was significantly lower. Ca2+-DNA complexes exhibited minimal and irregular transfection with relatively high cytotoxicity. A model was developed to explain the basis of anionic lipoplex uptake and transfection efficacy. Effective transfection is explained on the formation of nonbilayer hexagonal lipid phases. Efficient and relatively safe DNA transfection using anionic lipoplexes makes them an appealing alternative to be explored for gene delivery.     

Cationic liposomes for gene delivery

Cationic liposome–DNA complexes (lipoplexes) constitute a potentially viable alternative to viral vectors for the delivery of therapeutic genes. This review will focus on various parameters governing lipoplex biological activity, from their mode of formation to invivo behaviour. Particular emphasis is given to the mechanism of interaction of lipoplexes with cells, in an attempt to dissect the different barriers that need to be surpassed for efficient gene expression to occur. Aspects related to new trends in the formulation of lipid-based gene delivery systems aiming at overcoming some of their limitations will be covered. Finally, examples illustrating the potential of ca-tionic liposomes in clinical applications will be provided.     

In vivo characteristics of cationic liposomes as delivery vectors for gene therapy

After a decade of clinical trials, gene therapy seems to have found its place between excessive ambitions and feasible aims, with encouraging results obtained in recent years. Intracellular delivery of genetic material is the key step in gene therapy. Optimization of delivery vectors is of major importance for turning gene therapy into a successful therapeutic method. Nonviral gene delivery relies mainly on the complexes formed from cationic liposomes (or cationic polymers) and DNA, i.e., lipoplexes (or polyplexes). Many lipoplex formulations have been studied, but in vivo activity is generally low compared to that of viral systems. This review gives a concise overview of studies on the application of cationic liposomes in vivo in animal models of diseases and in clinical studies. The transfection efficiency, the pharmacokinetic and pharmacodynamic properties of the lipid-DNA complexes, and potentially relevant applications for cationic liposomes are discussed. Furthermore, the toxicity of, and the induction of an inflammatory response in association with the administration of lipoplexes are described. Increasing understanding of lipoplex behavior and gene transfer capacities in vivo offers new possibilities to enhance their efficiency and paves the path to more extensive clinical applications in the future.     

New synthetic glycolipids for targeted gene transfer: synthesis, formulation in lipoplexes and specific interaction with lectin

Nonviral gene delivery systems are a promising approach for gene therapy applications, despite their low in vivo gene transfer efficiency. One approach to enhance this efficiency is to incorporate targeting elements into cationic lipid/DNA complexes (lipoplexes). Ligand-containing lipoplexes have to retain their efficiency while exposing accessible ligand on their surface. Physicochemical properties (particle size, surface charge, and efficacy of DNA complexation) of the lipoplexes largely determine their gene transfer efficiency. We synthesized glycolipids with various galactosylated head ligand and incorporated them into lipoplexes. We showed that incorporation of up to 33% mol of glycolipid did not change the physicochemical properties of lipoplexes. Some of our glycolipids yielded lipoplexes whose galactosyl heads were well exposed on the surface as demonstrated by a strong interaction with Ricinus communis agglutinin. Glycolipid-containing lipoplexes gave an efficient gene transfer on hepatocytes, although no ligand-targeted transfection could be observed.     

Anionic pH-sensitive pegylated lipoplexes to deliver DNA to tumors

Anionic pegylated lipoplexes have been prepared from the combined formulation of cationic lipoplexes and pegylated anionic liposomes. To this end, two original (bis- and tetra-) carboxylated cholesterol derivatives have been synthesised. Titration of the particle surface charge was realised to determine the ratio between anionic and cationic lipids that would give pH-sensitive complexes. This ratio has been optimised to form particles sensitive to pH change in the range 5.5-6.5. Compaction of DNA into these newly formed anionic complexes was checked by DNA accessibility to picogreen and DNA electrophoresis on an agarose gel. Gene expression of the formulated gene was similar for the cationic formulation taken as a control and the anionic formulations prepared. The pH-sensitive properties of these formulations was shown in vitro using bafilomycin, a vacuolar H(+)ATPase inhibitor. The efficiency of the new formulations to deliver DNA to the tumor was compared with cholesteryl hemisuccinate (CHEMS) formulations. The tetracarboxylated compound gave the most efficient formulations for tumor delivery in vivo.     

New Synthetic Glycolipids for Targeted Gene Transfer: Synthesis,Formulation in Lipoplexes and Specific Interaction with Lectin

Nonviral gene delivery systems are a promising approach for gene therapy applications, despite their low in vivo gene transfer efficiency. One approach to enhance this efficiency is to incorporate targeting elements into cationic lipid/DNA complexes (lipoplexes). Ligand-containing lipoplexes have to retain their efficiency while exposing accessible ligand on their surface. Physicochemical properties (particle size, surface charge, and efficacy of DNA complexation) of the lipoplexes largely determine their gene transfer efficiency. We synthesized glycolipids with various galactosylated head ligand and incorporated them into lipoplexes. We showed that incorporation of up to 33% mol of glycolipid did not change the physicochemical properties of lipoplexes. Some of our glycolipids yielded lipoplexes whose galactosyl heads were well exposed on the surface as demonstrated by a strong interaction with Ricinus communis agglutinin. Glycolipid-containing lipoplexes gave an efficient gene transfer on hepatocytes, although no ligand-targeted transfection could be observed.     

Cationic liposomes for gene delivery

Cationic liposome-DNA complexes (lipoplexes) constitute a potentially viable alternative to viral vectors for the delivery of therapeutic genes. This review will focus on various parameters governing lipoplex biological activity, from their mode of formation to in vivo behaviour. Particular emphasis is given to the mechanism of interaction of lipoplexes with cells, in an attempt to dissect the different barriers that need to be surpassed for efficient gene expression to occur. Aspects related to new trends in the formulation of lipid-based gene delivery systems aiming at overcoming some of their limitations will be covered. Finally, examples illustrating the potential of cationic liposomes in clinical applications will be provided.     

Increased resistance of DNA lipoplexes to protein binding in vitro by surface-modification with a multivalent hydrophilic polymer

The potential of cationic liposomes as DNA delivery vehicles for gene therapy is significantly limited by their instability upon systemic administration. Their strong positive charge induces non-specific binding of serum proteins and subsequent clearance from the circulation. This work investigates the ability of the multivalent reactive copolymer of poly[N-(2-hydroxypropopyl) methacrylamide], pHPMA (MA-GG-ONp) to shield lipoplexes from non-specific protein binding. The polymer was found to react with cationic liposome-DNA complexes (lipoplexes) in both an electrostatic and covalent manner to form an external polymer coat. Polymer coating resulted in an increase in lipoplex diameter (by up to 100 nm) that was proportional to the amount of polymer used, with a concomitant reduction in surface charge from strongly positive to neutral (from 30 to 0 mV). Polymer-coated lipoplexes exhibited increased stability to protein binding compared to untreated liposomes and reduced non-specific uptake into cells in vitro.     

Cell-specific delivery of genes with glycosylated carriers.

Cationic liposomes and polymers have been accepted as effective non-viral vectors for gene delivery with low immunogenicity unlike viral vectors. However, the lack of organ or cell specificity sometimes hampers their application and the development of a cell-specific targeting technology for them attracts great interest in gene therapy. In this review, the potential of cell-specific delivery of genes with glycosylated liposomes or polymers is discussed. Galactosylated liposomes and poly(amino acids) are selectively taken up by the asialoglycoprotein receptor-positive liver parenchymal cells in vitro and in vivo after intravenous injection. DNA-galactosylated cationic liposome complexes show higher DNA uptake and gene expression in the liver parenchymal cells in vitro than DNA complexes with bare cationic liposomes. In the in vitro gene transfer experiment, galactosylated liposome complexes are more efficient than DNA-galactosylated poly(amino acids) complexes but they have some difficulties in their biodistribution control. On the other hand, introduction of mannose residues to carriers resulted in specific delivery of genes to non-parenchymal liver cells. These results suggest advantages of these glycosylated carriers in cell-specific targeted delivery of genes.     

Serum-resistant lipopolyplexes for gene delivery to liver tumour cells.

In this study, an efficient non-viral gene transfer system has been developed by employing polyethylenimine (PEI 800, 25 and 22kDa) and DOTAP and cholesterol (Chol) as lipids (lipopolyplex), at three different lipid/DNA molar ratios (2/1, 5/1 and 17/1) by using five different protocols of formulation. Condensation assays revealed that PEI of 800, 25 and 22kDa were very effective in condensing plasmid DNA, leading to a complete condensation at N/P ratios above 4. Addition of DOTAP/Chol liposomes did not further condense DNA. Increasing the molar ratio lipid/DNA in the complex resulted in higher positive values of the zeta-potential, while the particle size increased in some protocols, but not in others. High molecular weight PEI (800kDa) used in the formulation of lipopolyplexes lead to a bigger particle size, compared to that obtained with smaller PEI species, whether branched (25kDa) or linear (22kDa). These vectors were also highly effective in protecting DNA from attack by DNAse I. Transfection activity was maximal by using protocols 3 and 4 and a lipid/DNA molar ratio of 17/1. These complexes showed high efficiency in gene delivery of DNA to liver cancer cells, even in the presence of high concentration of serum (60% FBS). On the other hand, complexes formed with linear PEI (22kDa) were more effective than lipopolyplexes containing branched PEI (800 or 25kDa). The complexes resulted to be much more efficient than conventional lipoplexes (cationic lipid and DNA) and polyplexes (cationic polymer and DNA). The same behaviour was observed for complexes prepared in the presence of the therapeutic gene pCMVIL-12. Toxicity assays revealed a viability higher than 80% in all cases, independently of the protocol, molar ratio (lipid/DNA), molecular weight and type of PEI.     

Efficient and safe delivery of siRNA using anionic lipids: Formulation optimization studies

Novel formulations based on physiologically occurring anionic lipids have been designed to achieve safe and efficient siRNA delivery. Anionic liposomes (DOPG/DOPE) were complexed with siRNA using calcium ion bridges to prepare anionic lipoplexes. Various formulation parameters (liposome composition, lipid and calcium concentration) were evaluated and optimized to achieve efficient silencing and high cell viability in breast cancer cells. The optimal anionic lipoplexes composed of 1μg/mL lipid (40:60 (DOPG/DOPE m/m)), 2.4mM calcium and 10nM siRNA, showed maximum silencing (～70% knockdown) without being cytotoxic. These lipoplexes also showed stability and high efficiency in the presence of serum. Additionally, optimal anionic lipoplexes showed efficient intracellular uptake and endosomal escape. Characterization studies indicated the optimal anionic formulations were 324.2±19.6nm with a surface charge of (-22.9±0.1)mV and 98.5±1.4% encapsulation efficiency. Control cationic lipoplexes (Lipofectamine 2000) showed silencing comparable to the anionic lipoplexes but were highly cytotoxic as indicated by IC50 values (cationic - 22.9μg/mL, compared to anionic - greater than 10(7)μg/mL). Calcium-siRNA complexes (without liposomes) showed low efficiency (～50% silencing), and highly variable results. The optimized anionic formulations may offer a safer alternative to conventional cationic based systems for efficient in vitro as well as in vivo delivery of therapeutic siRNAs.     

Efficient serum-resistant lipopolyplexes targeted to the folate receptor

In this work, we have developed and evaluated a new targeted lipopolyplex (LPP), by combining polyethylenimine (PEI), 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP)/Chol liposomes, the plasmids pCMVLuc/pCMVIL-12, and the ligand folic acid (FA), able to transfect HeLa and B16-F10 cells in the presence of very high concentration of serum (60% FBS). These complexes (Fol-LPP) have a net positive surface charge. The combination of folic acid with lipopolyplexes also enhanced significantly the transfection activity of the therapeutic gene interleukin-12 (IL-12), without any significant cytotoxicity. The specificity of the folate receptor (FR)-mediated gene transfer was corroborated by employing a folate receptor deficient cell line (HepG2). This formulation improved gene delivery showed by conventional lipoplexes or polyplexes resulting an efficient, simple, and nontoxic method for gene delivery of therapeutic genes in vitro and very probably in vivo.     

Preparation and gene delivery of alkaline amino acids-based cationic liposomes

Cationic lipids 1, 2, and 3, based on hydrophobic cholesterol linked to L-lysine, L-histidine or L-arginine, respectively, were designed and tested as gene delivery vectors. Physicochemical and biological properties of all liposomes and lipoplexes were evaluated, including lipid-DNA interactions, size, morphology, zeta potential, acid-base buffering capability, protection of DNA from DNase I digestion, and cytotoxity. The efficiency of luciferase gene transfection of lipoplexes 1-3 was compared with that of commercial dioleoyl-trimethylammonium propane (DOTAP) and polyethyleneimine (PEI) in 293T cells and HepG2 cells with or without poly(ethylene glycol) PEG stabilizer. The complexation and protection of DNA of liposome 3 was the strongest among the three liposomes. The efficiency of gene transfection of liposomes 1-3 was two-to threefold higher than that of PEI and/or DOTAP in 293T cells. Liposomes 1 and 3 in PEG as stabilizer showed sixfold higher transfection efficiency than that of PEI and/or DOTAP, whereas liposome 2 showed very low transfection efficiency. In HepG2 cells, the transfection efficiency of all the cationic liposomes was much lower than that of DOTAP. In conclusion, lipids 1-3 were efficient and non-toxic gene vectors; the headgroup of cationic lipids and the stabilizer of liposome formulation had an important influence on gene transfection.     

Aerosolization of lipoplexes using AERx® pulmonary delivery system

The lung represents an attractive target for delivering gene therapy to achieve local and potentially systemic delivery of gene products. The objective of this study was to evaluate the feasibility of the AERx Pulmonary Delivery System for delivering nonviral gene therapy formulations to the lung. We found that “naked” DNA undergoes degradation following aerosolization through the AERx nozzle system. However, DNA formulated with a molar excess of cationic lipids (lipoplexes) showed no loss of integrity. In addition, the lipoplexes showed no significant change in particle size, zeta (ζ) potential, or degree of complexation following extrusion. The data suggest that complexation with cationic lipids had a protective effect on the formulation following extrusion. In addition, there was no significant change in the potency of the formulation as determined by a transfection study in A-549 cells in culture. We also found that DNA formulations prepared in lactose were aerosolized poorly. Significant improvements in aerosolization efficiency were seen when electrolytes such as NaCl were added to the formulation. In conclusion, the data suggest that delivery of lipoplexes using the AERx Pulmonary Delivery System may be a viable approach for pulmonary gene therapy.     

Aerosolization of lipoplexes using AERx Pulmonary Delivery System

The lung represents an attractive target for delivering gene therapy to achieve local and potentially systemic delivery of gene products. The objective of this study was to evaluate the feasibility of the AERx Pulmonary Delivery System for delivering nonviral gene therapy formulations to the lung. We found that "naked" DNA undergoes degradation following aerosolization through the AERx nozzle system. However, DNA formulated with a molar excess of cationic lipids (lipoplexes) showed no loss of integrity. In addition, the lipoplexes showed no significant change in particle size, zeta (zeta) potential, or degree of complexation following extrusion. The data suggest that complexation with cationic lipids had a protective effect on the formulation following extrusion. In addition, there was no significant change in the potency of the formulation as determined by a transfection study in A-549 cells in culture. We also found that DNA formulations prepared in lactose were aerosolized poorly. Significant improvements in aerosolization efficiency were seen when electrolytes such as NaCl were added to the formulation. In conclusion, the data suggest that delivery of lipoplexes using the AERx Pulmonary Delivery System may be a viable approach for pulmonary gene therapy.     

阳性脂质体介导基因转染及其研究进展*

基因治疗是一种很有前景的治疗模式,而阳性脂质体介导的基因转染是目前基因治疗的研究热点之一。现综述近5年来有关阳性脂质体的文献,介绍了阳性脂质体的基本组成,并从生物学、理化性质及制剂学等几个方面介绍了影响阳性脂质体/DNA复合物转染效率的主要因素,最后从新的阳性脂质成分及阳性脂质体或阳性脂质体/DNA复合物的表面修饰等方面介绍了近年来有关改善阳性脂质体介导基因转染的研究进展。     

Effects of lipid composition and preparation conditions on physical-chemical properties, technological parameters and in vitro biological activity of gemcitabine-loaded liposomes

The effects of lipid composition and preparation conditions on the physicochemical and technological properties of gemcitabine-loaded liposomes, as well as the in vitro anti-tumoral activity of various liposome formulations were investigated. Three liposome formulations were investigated: DPPC/Chol/Oleic acid (8:3:1 molar ratio, liposomes A), DPPC/Chol/DPPS (6:3:1 molar ratio, liposomes B) and DPPC/Chol/DSPE-MPEG (6:3:1 molar ratio, liposomes C). Multilamellar liposomes were prepared by using the TLE, FAT and DRV methods, while small unilamellar liposomes were obtained by extrusion through polycarbonate filters. Light scattering techniques were used to characterize liposome formulations. Loading capacity and release profiles of gemcitabine from various liposome formulations were also investigated. Caco-2 cells were used to evaluate in vitro the antitumoral activity of gemcitabine-loaded liposomes with respect to the free drug and also the intracellular drug uptake. Preparation methods and liposome lipid composition influenced both physicochemical parameters and drug delivery features. Liposomes with a size ranging from 200 nm to 7 microm were obtained. The gemcitabine entrapment was higher than that expected probably due to an interaction with the liposome lipid components. The following decreasing loading capacity order was observed: liposome B>liposome C>liposome A. Gemcitabine release from various liposome formulations is modulated by two different processes, i.e. desorption from and permeation through liposomal bilayers. MTT assay showed a greater cytotoxic effect of gemcitabine-loaded liposomes with respect to the free drug. The following decreasing anticancer activity order was observed between the various liposome formulations: liposome C>liposome A>liposome B. The increased anticancer activity is correlated to the ability of the colloidal carrier to increase the intracellular drug uptake. Due to the encouraging results and to the high liposome modularity various applications of potential therapeutic relevance can be envisaged for liposomes.