DOTAP/DOPE and DC-Chol/DOPE lipoplexes for gene delivery: zeta potential measurements and electron spin resonance spectra

Non-viral vectors represent an important alternative in gene delivery. Among these vectors, cationic liposomes are widely studied, because of their ability to form stable complexes with DNA fragments (lipoplexes). In the present work, we report on the characterization by electron spin resonance (ESR) spectroscopy and zeta potential measurements of cationic liposomes and of their complexes with oligonucleotides. Liposomes were made with a zwitterionic lipid, DOPE, and a cationic lipid, either DOTAP or DC-Chol. Oligonucleotides were the 20-base single strand polyA, the 20-base single strand polyT, and the corresponding double strand dsAT. The zeta potential as a function of the oligonucleotide/lipid⁺ ratio gave an S-shaped titration curve. Well-defined surface potential changes took place upon charge compensation between the cationic lipid heads and the phosphate groups on the oligonucleotides. The inversion point depended on the specific system under study. The bilayer properties and the changes that occurred with the incorporation of DNA fragments were also monitored by ESR spectroscopy of appropriately tailored spin probes. For all the systems investigated, the ESR spectra showed that no major alteration took place after lipoplex formation and molecular packing remained substantially unchanged. Both zeta potential and ESR measurements were in favor of an external mode of packing of the lipoplexes.     

DOTAP/DOPE and DC-Chol/DOPE lipoplexes for gene delivery studied by circular dichroism and other biophysical techniques

Cationic liposomes give rise to stable complexes with DNA molecules (lipoplexes) that are of great interest for gene delivery applications. In particular, liposomes made up by a cationic lipid (DOTAP or DC-Chol) and a zwitterionic lipid (DOPE), produce stable adducts with single and double-stranded DNA oligonucleotides. Formation of these lipoplexes has been further addressed here by circular dichroism spectroscopy (CD) and by other independent biophysical methods. Titration of DNA oligonucleotides with cationic liposomes resulted into significant modifications of their circular dichroic bands. Such spectral modifications were ascribed to progressive DNA condensation and loss of native conformation, as a consequence of the electrostatic interactions taking place between the phosphate groups of DNA and the positively charged head groups of cationic lipids. In all cases, the loss of the CD feature characteristic of the native DNA conformation closely matched the inflection point of Zeta potential profiles. The resulting adducts showed peculiar and non-canonical CD spectra, while exhibiting appreciable stability at physiological pH.     

DNA alters the bilayer structure of cationic lipid diC14-amidine: A spin label study

Cationic lipids-DNA complexes (lipoplexes) have been used for delivery of nucleic acids into cells in vitro and in vivo. Despite the fact that, over the last decade, significant progress in the understanding of the cellular pathways and mechanisms involved in lipoplexes-mediated gene transfection have been achieved, a convincing relationship between the structure of lipoplexes and their in vivo and in vitro transfection activity is still missing. How does DNA affect the lipid packing and what are the consequences for transfection efficiency is the point we want to address here. We investigated the bilayer organization in cationic liposomes by electron spin resonance (ESR). Phospholipids spin labeled at the 5th and 16th carbon atoms were incorporated into the DNA/diC14-amidine complex. Our data demonstrate that electrostatic interactions involved in the formation of DNA-cationic lipid complex modify the packing of the cationic lipid membrane. DNA rigidifies the amidine fluid bilayer and fluidizes the amidine rigid bilayer just below the gel-fluid transition temperature. These effects were not observed with single nucleotides and are clearly related to the repetitive charged motif present in the DNA chain and not to a charge-charge interaction. These modifications of the initial lipid packing of the cationic lipid may reorient its cellular pathway towards different routes. A better knowledge of the cationic lipid packing before and after interaction with DNA may therefore contribute to the design of lipoplexes capable to reach specific cellular targets.     

Effect of the preparation procedure on the structural properties of oligonucleotide/cationic liposome complexes (lipoplexes) studied by electron spin resonance and Zeta potential

Lipoplexes with different surface charge were prepared from a short oligonucleotide (20 mer, dsAT) inserted into liposomes of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phospho-ethanolamine (DOPE). The starting liposomes were prepared by two different procedures, i.e. progressive dsAT addition starting from plain liposomes (titration) and direct mixing of dsAT with pure liposomes (point to point preparation). Lipoplexes were characterized from a molecular point of view by Electron Spin Resonance (ESR) of a cationic spin probe and by Nuclear Magnetic Resonance. Structural and surface features were analysed by Zeta potential (zeta) measurements and Cryo-TEM micrographs. The complete set of results allowed to demonstrate that: i) the interactions between dsAT and cationic lipids were strong and occurred at the liposome surface; ii) the overall shape and physicochemical properties of liposomes did not change when short nucleic acid fragments were added before surface charge neutralization; iii) the bilayer structure of the lipids in lipoplexes was substantially preserved at all charge ratios; iv) the physical status of lipoplexes with electrical charge far from neutrality did not depend on the preparation method.     

Thermodynamic aspects and biological profile of CDAN/DOPE and DC-Chol/DOPE lipoplexes

The DNA complexation and condensation properties of two established cationic liposome formulations, CDAN/DOPE (50:50, m/m; Trojene) and DC-Chol/DOPE (60:40, m/m), were investigated by using a combination of isothermal titration calorimetry (ITC), circular dichroism (CD), photon correlation spectroscopy (PCS), and turbidity assays. Plasmid DNA (7528 bp) was titrated with extruded liposomes (90 +/- 15 nm) and a thermodynamic profile established. ITC data revealed that the two liposome formulations differ substantially in their DNA complexation characteristics. Equilibrium dissociation constants for CDAN/DOPE (K(d) = 19 +/- 3 microM) and DC-Chol/DOPE liposomes (K(d) = 2 +/- 0.5 microM) were obtained by fitting the experimental data in a one-site binding model. Both CDAN/DOPE and DC-Chol/DOPE binding events take place with a negative binding enthalpy (DeltaH degrees = -0.5 and -1.7 kcal/mol, respectively) and increasing system entropy (TDeltaS = 6 +/- 0.3 and 6.2 +/- 0.3 kcal/mol, respectively). Interestingly, CDAN/DOPE liposomes undergo substantial rehydration and protonation prior to complexation with pDNA, which is observed as two discrete exothermic signals during titration. No such biphasic effects are seen with respect to the binding between DC-Chol/DOPE and pDNA that appears to be otherwise instantaneous with no rehydration effects. The rehydration and protonation characteristics of CDAN/DOPE liposomes in comparison with those of DC-Chol/DOPE cationic liposomes are confirmed by ITC; CDAN/DOPE liposomes have strongly exothermic dilution characteristics and DC-Chol/DOPE liposomes only mildly endothermic characteristics. Furthermore, analysis of cationic liposome-pDNA binding by CD spectroscopy reveals that CDAN/DOPE-pDNA lipoplexes are more structurally fluid than DC-Chol/DOPE-pDNA lipoplexes. CDAN/DOPE liposomes induced considerable fluctuation in the DNA structure for at least 60 min, whereas liposomes obtained from DC-Chol/DOPE lack the same effect on the DNA structure. Turbidity studies show that DC-Chol/DOPE lipoplexes exhibit greater resistance to serum than CDAN/DOPE lipoplexes, which showed substantial precipitation after incubation for 100 min with serum. Transfection studies on HeLa and Panc-1 cells reveal that CDAN/DOPE lipoplexes are superior in efficacy to DC-Chol/DOPE lipoplexes. CDAN/DOPE liposomes tend to transfect best in normal growth medium (including 10% serum and antibiotics), whereas DC-Chol/DOPE lipoplexes transfect best under serum free transfection conditions.     

Interaction of cationic liposomes and their DNA complexes with monocytic leukemia cells

Cationic liposomes complexed with DNA have been used extensively as non-viral vectors for the intracellular delivery of reporter or therapeutic genes in culture and in vivo. We examined the relationship between the characteristics of the lipoplexes, their mode of interaction with monocytic THP-1 cells and their ability to transfect these cells. We determined the size and zeta potential of cationic liposomes (composed of 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and its mixtures with neutral lipids), and lipoplexes at different (+/-) charge ratios. As the (+/-) charge ratio of the lipoplexes decreased to (1/1), a significant reduction in zeta potential and an increase in size was observed. The increase in size resulted from fusion between liposomes promoted by DNA, as demonstrated by a lipid mixing assay, and from aggregation of the complexes. Interaction of liposomes and lipoplexes with THP-1 cells was assessed by monitoring lipid mixing ('fusion') as well as binding and cell association. While no lipid mixing was observed with the 1/2 (+/-) lipid/DNA complexes, lipoplexes with higher (+/-) charge ratios underwent significant fusion in conjunction with extensive cell binding. Liposome binding to cells was dependent on the positive charge of the liposomes, and their fusion could be modulated by the co-lipid. DOTAP/phosphatidylethanolamine (1:1) liposomes fused with THP-1 cells, unlike DOTAP/phosphatidylcholine (1:1) liposomes, although both liposome types bound to the cells to a similar extent. The use of inhibitors of endocytosis indicated that fusion of the cationic liposomes with cells occurred mainly at the plasma membrane level. The presence of serum increased the size of the cationic liposomes, but not that of the lipoplexes. Low concentrations of serum (3%) completely inhibited the fusion of cationic liposomes with cells, while inhibiting binding by only 20%. Our results suggest that binding of cationic liposomes and lipoplexes to cells is governed primarily by electrostatic interactions, whereas their fusion is regulated by the lipid composition and sterically favorable interactions with cell surface molecules. In addition our results indicate no correlation between fusion of the lipoplexes with the plasma membrane and the levels of transfection.     

Gene delivery mediated by cationic liposomes: from biophysical aspects to enhancement of transfection

Cationic liposomes complexed with DNA have been used extensively as non-viral vectors for the intracellular delivery of reporter or therapeutic genes in culture and in vivo. However, the relationship between the features of the lipid-DNA complexes (`lipoplexes') and their mode of interaction with cells, the efficiency of gene transfer and gene expression remain to be clarified. To gain insights into these aspects, the size and zeta potential of cationic liposomes (composed of 1,2-dioleoyl-3- (trimethylammonium) propane (DOTAP) and its mixture with phosphatidylethanolamine (PE)), and their complexes with DNA at different (+/-) charge ratios were determined. A lipid mixing assay was used to assess the interaction of liposomes and lipoplexes with monocytic leukaemia cells. The use of inhibitors of endocytosis indicated that fusion of the cationic liposomes with cells occurred mainly at the plasma membrane level. However, very limited transfection of these cells was achieved using the above complexes. It is possible that the topology of the cationic liposome-DNA complexes does not allow the entry of DNA into cells through a fusion process at the plasma membrane. In an attempt to enhance transfection mediated by lipoplexes composed of DOTAP and its equimolar mixture with dioleoylphosphatidylethanolamine (DOPE) two different strategies were explored: (i) association of a targeting ligand (transferrin) to the complexes to promote their internalization, presumably by receptor-mediated endocytosis; and (ii) association of synthetic fusogenic peptides (GALA or the influenza haemagglutinin Nterminal peptide HA-2) to the complexes to promote endosomal destabilization and release of the genetic material into the cytoplasm. These strategies were effective in enhancing transfection in a large variety of cells, including epithelial and lymphoid cell lines, as well as human macrophages, especially with the use of optimized lipid/ DNA (+/-) charge ratios. Besides leading to high levels of transfection, the ternary complexes of cationic liposomes, DNA, and protein or peptide, have the advantages of being active in the presence of serum and being non-toxic. Moreover, such ternary complexes present a net negative charge and, thus, are likely to alleviate the problems associated with the use of highly positively charged complexes in vivo, such as avid complexation with serum proteins. Overall, the results indicate that these complexes, and their future derivatives, may constitute viable alternatives to viral vectors for gene delivery in vivo.     

Gene delivery mediated by cationic liposomes: from biophysical aspects to enhancement of transfection.

Cationic liposomes complexed with DNA have been used extensively as non-viral vectors for the intracellular delivery of reporter or therapeutic genes in culture and in vivo. However, the relationship between the features of the lipid-DNA complexes ('lipoplexes') and their mode of interaction with cells, the efficiency of gene transfer and gene expression remain to be clarified. To gain insights into these aspects, the size and zeta potential of cationic liposomes (composed of 1,2-dioleoyl-3- (trimethylammonium) propane (DOTAP) and its mixture with phosphatidylethanolamine (PE)), and their complexes with DNA at different (+/-) charge ratios were determined. A lipid mixing assay was used to assess the interaction of liposomes and lipoplexes with monocytic leukaemia cells. The use of inhibitors of endocytosis indicated that fusion of the cationic liposomes with cells occurred mainly at the plasma membrane level. However, very limited transfection of these cells was achieved using the above complexes. It is possible that the topology of the cationic liposome-DNA complexes does not allow the entry of DNA into cells through a fusion process at the plasma membrane. In an attempt to enhance transfection mediated by lipoplexes composed of DOTAP and its equimolar mixture with dioleoylphosphatidylethanolamine (DOPE) two different strategies were explored: (i) association of a targeting ligand (transferrin) to the complexes to promote their internalization, presumably by receptor-mediated endocytosis; and (ii) association of synthetic fusogenic peptides (GALA or the influenza haemagglutinin N-terminal peptide HA-2) to the complexes to promote endosomal destabilization and release of the genetic material into the cytoplasm. These strategies were effective in enhancing transfection in a large variety of cells, including epithelial and lymphoid cell lines, as well as human macrophages, especially with the use of optimized lipid/DNA (+/-) charge ratios. Besides leading to high levels of transfection, the ternary complexes of cationic liposomes, DNA, and protein or peptide, have the advantages of being active in the presence of serum and being non-toxic. Moreover, such ternary complexes present a net negative charge and, thus, are likely to alleviate the problems associated with the use of highly positively charged complexes in vivo, such as avid complexation with serum proteins. Overall, the results indicate that these complexes, and their future derivatives, may constitute viable alternatives to viral vectors for gene delivery in vivo.     

Physico-chemical characterization and transfection efficacy of cationic liposomes containing the pEGFP plasmid

Cationic liposomes-DNA complexes (lipoplexes) are largely used in gene delivery. Deciphering specific chemical and physical properties of lipoplexes is a necessary step to unravel the mechanisms underlying transfection and to improve transfection efficacy in each experimental model. In the present paper we investigated the physico-chemical features of lipoplexes containing a plasmid encoding for the GFP protein, in order to correlate these results with transfection efficacy. Cationic unilamellar vesicles (mean diameter 100 nm) were prepared, from the cationic DC-Chol lipid and the zwitterionic phospholipid DOPE. The two components of the liposome bilayer were used at molar ratio close to unity. ESR spectra were recorded and zeta potential zeta was measured on liposomes complexed with the plasmid. One of the main points of interest in this paper resided in the fact that both kinds of measurements were carried out in the same conditions (i.e. lipid concentration, medium composition, and pH) employed for cell transfection experiments. Transfection was performed on CHO cells; the percentage of fluorescent cells was evaluated and compared with the above physico-chemical features. It emerged that the composition and pH of the medium, the lipoplex/cell ratio, as well as the amount of lipoplex added to the cell culture were critical parameters for transfection efficacy. Finally, lipoplex surface charge played a fundamental role to achieve a high transfection level.     

Dependence of the cellular internalization and transfection efficiency on the structure and physicochemical properties of cationic detergent/DNA/liposomes

BACKGROUND: Control of the structure and physicochemical properties of DNA complexed with nonviral vectors is essential for efficient biodistribution and gene delivery to cells. Cationic liposomes interact with DNA giving transfection competent but large and heterogeneous aggregates. On the other hand, cationic detergents condense DNA into small homogeneous but reversible complexes inefficient for transfection. METHODS: In order to combine the favorable features of both vectors, ternary complexes were prepared by adding cationic liposomes to plasmid DNA condensed by cationic detergents. The structure and physicochemical properties of these complexes were investigated by electron microscopy, quasi-elastic light scattering, gel electrophoresis and fluorescence techniques. These data were then correlated with the transfection efficiency and intracellular trafficking of the ternary complexes determined by luciferase gene expression and confocal microscopy, respectively. RESULTS: The ternary complexes were found to form small, homogeneous, globular, stable and positively charged particles with a highly dense and packed lamellar internal structure differing from the multilamellar structure (L(alpha)(C)) of the corresponding lipoplexes. In the presence of serum, the ternary complexes were more efficiently internalized into cells, less toxic and showed 20-fold higher transfection efficiency than lipoplexes. CONCLUSIONS: This study showed that small, monodisperse and highly stable complexes could be obtained by precompaction of DNA with cetyltrimethylammonium bromide, followed by addition of cationic lipids. The higher efficiency of the ternary complexes with respect to their corresponding lipoplexes was related to their internal structure which prevents their dissociation by serum proteins and allows efficient internalization in the target cells.     

Physicochemical characterization and purification of cationic lipoplexes.

Cationic lipid-nucleic acid complexes (lipoplexes) consisting of dioleoyltrimethylammoniumpropane (DOTAP) liposomes and plasmid DNA were prepared at various charge ratios (cationic group to nucleotide phosphate), and the excess component was separated from the lipoplex. We measured the stoichiometry of the lipoplex, noted its colloidal properties, and observed its morphology and structure by electron microscopy. The colloidal properties of the lipoplexes were principally determined by the cationic lipid/DNA charge ratio and were independent of the lipid composition. In lipoplexes, the lipid membranes as observed in freeze-fracture electron microscopy were deformed into high-radius-of-curvature features whose characteristics depended on the lipid composition. Lipoplexes prepared at a threefold or greater excess of either DOTAP or DNA could be resolved into complexes with a defined stoichiometry and the excess component by sedimentation to equilibrium on sucrose gradients. The separated, positively charged complex retained high transfection activity and had reduced toxicity. The negatively charged lipoplex showed increased transfection activity compared to the starting mixture. In cryoelectron micrographs the positively charged complex was spherical and contained a condensed but indistinct interior structure. In contrast, the separated negatively charged lipoplexes had a prominent internal 5.9 +/- 0.1-nm periodic feature with material projecting as spikes from the spherical structure into the solution. It is likely that these two lipoplexes represent structures with different lipid and DNA packing.     

Physico-Chemical Characteristics of Lipoplexes Influence Cell Uptake Mechanisms and Transfection Efficacy

Background

Formulation of DNA/cationic lipid complexes (lipoplexes) designed for nucleic acid delivery mostly results in positively charged particles which are thought to enter cells by endocytosis. We recently developed a lipoplex formulation called Neutraplex that allows preparation of both cationic and anionic stable complexes with similar lipid content and ultrastructure.

Methodology/Principal Findings

To assess whether the global net charge could influence cell uptake and activity of the transported oligonucleotides (ON), we prepared lipoplexes with positive and negative charges and compared: (i) their physicochemical properties by zeta potential analysis and dynamic light scattering, (ii) their cell uptake by fluorescence microscopy and flow cytometry, and (iii) the biological activity of the transported ON using a splicing correction assay. We show that positively or negatively charged lipoplexes enter cells cells using both temperature-dependent and -independent uptake mechanisms. Specifically, positively charged lipoplexes predominantly use a temperature-dependent transport when cells are incubated OptiMEM medium. Anionic lipoplexes favour an energy-independent transport and show higher ON activity than cationic lipoplexes in presence of serum. However, lipoplexes with high positive global net charge and OptiMEM medium give the highest uptake and ON activity levels.

Conclusions

These findings suggest that, in addition to endocytosis, lipoplexes may enter cell via a temperature-independent mechanism, which could be mediated by lipid mixing. Such characteristics might arise from the specific lipoplex ultrastructure and should be taken into consideration when developing lipoplexes designed for in vivo or ex vivo nucleic acid transfer.     

Effect of "helper lipid" on lipoplex electrostatics

Lipoplexes, which are complexes between cationic liposomes (L+) and nucleic acids, are commonly used as a nucleic acid delivery system in vitro and in vivo. This study aimed to better characterize cationic liposome and lipoplex electrostatics, which seems to play a major role in the formation and the performance of lipoplexes in vitro and in vivo. We characterized lipoplexes based on two commonly used monocationic lipids, DOTAP and DMRIE, and one polycationic lipid, DOSPA--each with and without helper lipid (cholesterol or DOPE). Electrical surface potential (Psi0) and surface pH were determined using several surface pH-sensitive fluorophores attached either to a one-chain lipid (4-heptadecyl hydroxycoumarin (C17HC)) or to the primary amino group of the two-chain lipids (1,2-dioleyl-sn-glycero-3-phosphoethanolamine-N-carboxyfluorescein (CFPE) and 1,2-dioleyl-sn-glycero-3-phosphoethanolamine-N-7-hydroxycoumarin) (HC-DOPE). Zeta potentials of the DOTAP-based cationic liposomes and lipoplexes were compared with Psi0 determined using C17HC. The location and relatively low sensitivity of fluorescein to pH changes explains why CFPE is the least efficient in quantifying the differences between the various cationic liposomes and lipoplexes used in this study. The fact that, for all cationic liposomes studied, those containing DOPE as helper lipid have the least positive Psi0 indicates neutralization of the cationic charge by the negatively-charged phosphodiester of the DOPE. Zeta potential is much less positively charged than Psi0 determined by C17HC. The electrostatics affects size changes that occurred to the cationic liposomes upon lipoplex formation. The largest size increase (based on static light scattering measurements) for all formulations occurred at DNA-/L+ charge ratios 0.5-1. Comparing the use of the one-chain C17HC and the two-chain HC-DOPE for monitoring lipoplex electrostatics reveals that both are suitable, as long as there is no serum (or other lipidic assemblies) present in the medium; in the latter case, only the two-chain HC-DOPE gives reliable results. Increasing NaCl concentrations decrease surface potential. Neutralization by DNA is reduced in a NaCl-concentration-dependent manner.     

Polyethylenimine of various molecular weights as adjuvant for transfection mediated by cationic liposomes

Over the last years significant progress has been made in non-viral gene delivery mediated by cationic liposomes. However, the results obtained are still far from being satisfactory regarding transfection efficiency, particularly when compared to that achieved using viral vectors. We have previously demonstrated that association of transferrin with cationic liposomes significantly improves transfection in a large variety of cells, both in vitro and in vivo. In this work, several strategies have been explored in order to further improve transfection mediated by transferrin-associated lipoplexes. To this regard, the effect on transfection of pre-condensation of DNA with polyethylenimine of low MWs (2.7, 2.0 and 0.8 KDa) at various N/P ratios, lipid composition, cationic lipid/DNA (+/-) charge ratio and the presence of a surfactant in the lipoplexes was investigated. Two different modes for preparing the liposomes were tested and the extent of cell association of their complexes with DNA as well as their capacity to protect the carried DNA were evaluated. Our results show that complexes generated from cationic liposomes prepared by the ethanol injection method in which the carried DNA was pre-condensed with low MW polyethylenimine are highly efficient in mediating transfection. The differential modulating effect observed upon association of transferrin to various liposome formulations on transfection mediated by the polyethylenimine-complexes suggests that these complexes enter into the cells through different pathways (involving clathrin versus caveolin), most likely by taking advantage of their intrinsic biophysical properties to escape from the endosome to the cytosol.     

Effect of lipid composition on the structure and theoretical phase diagrams of DC-Chol/DOPE-DNA lipoplexes

Lipoplexes constituted by calf-thymus DNA (CT-DNA) and mixed cationic liposomes consisting of varying proportions of the cationic lipid 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol) and the zwitterionic lipid, 1,2-dioleoyl-sn-glycero-3-phosphoetanolamine (DOPE) have been analyzed by means of electrophoretic mobility, SAXS, and fluorescence anisotropy experiments, as well as by theoretically calculated phase diagrams. Both experimental and theoretical studies have been run at several liposome and lipoplex compositions, defined in terms of cationic lipid molar fraction, α, and either the mass or charge ratios of the lipoplex, respectively. The experimental electrochemical results indicate that DC-Chol/DOPE liposomes, with a mean hydrodynamic diameter of around (120 ± 10) nm, compact and condense DNA fragments at their cationic surfaces by means of a strong entropically driven electrostatic interaction. Furthermore, the positive charges of cationic liposomes are compensated by the negative charges of DNA phosphate groups at the isoneutrality L/D ratio, (L/D)(?), which decreases with the cationic lipid content of the mixed liposome, for a given DNA concentration. This inversion of sign process has been also studied by means of the phase diagrams calculated with the theoretical model, which confirms all the experimental results. SAXS diffractograms, run at several lipoplex compositions, reveal that, irrespectively of the lipoplex charge ratio, DC-Chol/DOPE-DNA lipoplexes show a lamellar structure, L(α), when the cationic lipid content on the mixed liposomes α ≥ 0.4, while for a lower content (α = 0.2) the lipoplexes show an inverted hexagonal structure, H(II), usually related with improved cell transfection efficiency. A similar conclusion is reached from fluorescence anisotropy results, which indicate that the fluidity on liposome and lipoplexes membrane, also related with better transfection results, increases as long as the cationic lipid content decreases.     

Cationic liposome-mediated gene delivery: biophysical study and mechanism of internalization

To identify factors affecting cationic liposome-mediated gene delivery efficiency, we studied the relationship between the biophysical characteristics of liposome/DNA complexes (lipoplexes) at different (+/-) charge ratios, their structures as monitored by atomic force microscopy (AFM), and their mechanism(s) of internalization into the cells. Significant changes were observed in the particle size and zeta potential of liposomes and their structures assessed by AFM upon addition of DNA, which depended on (+/-) charge ratios. AFM images showed that lipoplexes were formed from extensively fused and apparently homogeneous lipid particles encapsulating DNA. Lipoplexes were found to internalize the cells through the endocytosis pathway. Lipoplex-cell fusion was found to occur mainly at the plasma membrane level; however, this lipoplex-cell membrane fusion was found to be essential for the uptake of the large particles. A new perspective for the internalization of large lipoplex particles into cytoplasm is discussed.     

Lipid composition of cationic nanoliposomes implicate on transfection efficiency

Abstract

Cationic liposome (CL)-DNA complexes (lipoplexes) have appeared as leading nonviral gene carriers in worldwide gene therapy clinical trials. Arriving at therapeutic dosages requires the full understanding of the mechanism of transfection. However, using CLs to deliver therapeutic nucleic acids and drugs to target organs have some problems, including low transfection efficiency. The aim of this study was developing novel CLs containing four neutral lipids; cholesterol, 1,2-dioleoyl phosphatidylethanolamine, distearoylphosphatidylcholine and dipalmitoylphosphatidylcholine as a helper lipid and dimethyl dioctadecyl ammonium bromide as a cationic lipid to increase transfection efficiency. We have investigated the correlation between number of lipid composition and transfection efficiency. The morphology, size and zeta potential of liposomes and lipoplexes were measured and lipoplexes formation was monitored by gel retardation assay. Transfection efficiency was assessed using firefly luciferase reporter assay. It was found that transfection efficiency markedly depended on liposome to plasmid DNA (pDNA) weight ratio, lipid composition and efficiency of pDNA entrapment. High transfection efficiency of plasmid by four component lipoplexes was achieved. Moreover, lipoplexes showed lower transfection efficiency and less cytotoxicity compared to Lipofectamine?. These results suggest that lipid composition of nanoliposomes is an important factor in control of their physical properties and also yield of transfection.     

Human serum albumin enhances DNA transfection by lipoplexes and confers resistance to inhibition by serum

Cationic liposome-DNA complexes ('lipoplexes') are used as gene delivery vehicles and may overcome some of the limitations of viral vectors for gene therapy applications. The interaction of highly positively charged lipoplexes with biological macromolecules in blood and tissues is one of the drawbacks of this system. We examined whether coating cationic liposomes with human serum albumin (HSA) could generate complexes that maintained transfection activity. The association of HSA with liposomes composed of 1, 2-dioleoyl-3-(trimethylammonium) propane and dioleoylphosphatidylethanolamine, and subsequent complexation with the plasmid pCMVluc greatly increased luciferase expression in epithelial and lymphocytic cell lines above that obtained with plain lipoplexes. The percentage of cells transfected also increased by an order of magnitude. The zeta potential of the ternary complexes was lower than that of the lipoplexes. Transfection activity by HSA-lipoplexes was not inhibited by up to 30% serum. The combined use of HSA and a pH-sensitive peptide resulted in significant gene expression in human primary macrophages. HSA-lipoplexes mediated significantly higher gene expression than plain lipoplexes or naked DNA in the lungs and spleen of mice. Our results indicate that negatively charged HSA-lipoplexes can facilitate efficient transfection of cultured cells, and that they may overcome some of the problems associated with the use of highly positively charged complexes for gene delivery in vivo.     

Spectroscopic characterization of DMPC/DOTAP cationic liposomes and their interactions with DNA and drugs

Gene and synthetic drug-delivery vectors have been developed and characterized to treat several genetic diseases and cancers. Our study aims at characterizing cationic liposomes containing the zwitterionic phospholipid DMPC and the cationic lipid DOTAP as well as their interactions with two types of DNA and a new class of antineoplastic agents derived from arylchloroethylureas (CEU). Results obtained using FTIR spectroscopy as well as ³¹P and ²H NMR indicate that DMPC and DOTAP form cationic liposomes in a highly disordered fluid phase at a molar ratio of 1:1. In addition, the FTIR results indicate that the presence of DNA or CEUs within the liposomes does not significantly affect the conformational order of both the DMPC and DOTAP acyl chains. Our results therefore provide a detailed characterization of complexes between cationic liposomes and both DNA and drugs and indicate that these complexes are stable and fluid assemblies.     

Structure and internal organization of overcharged cationic-lipid/peptide/DNA self-assembly complexes

The combination of cationic lipids with cationic peptides and DNA vectors can produce synergistic effects in gene delivery to eukaryotic cells. Binary complexes of cationic lipids with DNA are well-studied whereas little information is available about the structure of the ternary lipid/peptide/DNA (LPD) complexes and mechanisms defining DNA protection and delivery. Here we use synchrotron small angle X-ray scattering and dynamic light scattering zeta-potential measurements to determine structure and the net charge of supramolecular aggregates of complexes in mixtures of plasmid DNA, cationic liposomes formed from DOTAP, plus a linear cationic ε-oligolysine with the pendant α-amino acids Leu-Tyr-Arg (LYR), ε-(LYR)K10. These ternary complexes display multilamellar structures with relatively constant separation between DOTAP bilayers, accommodating a hydrated monolayer of parallel DNA rods. The DNA-DNA distance in the complexes varies as a function of the net positive to negative (lipid+peptide)/DNA charge ratio. An explanation for the observed dependence of DNA-DNA distance on charge ratio was proposed based on general polyelectrolyte properties of non-stoichiometric polycation-DNA mixtures.