DOTAP cationic liposomes prefer relaxed over supercoiled plasmids

Cationic liposomes and DNA interact electrostatically to form complexes called lipoplexes. The amounts of unbound (free) DNA in a mixture of cationic liposomes and DNA at different cationic lipid:DNA molar ratios can be used to describe DNA binding isotherms; these provide a measure of the binding efficiency of DNA to different cationic lipid formulations at various medium conditions. In order to quantify the ratio between the various forms of naked DNA and supercoiled, relaxed and single-stranded DNA, and the ratio between cationic lipid bound and unbound DNA of various forms we developed a simple, sensitive quantitative assay using agarose gel electrophoresis, followed by staining with the fluorescent cyanine DNA dyes SYBR Green I or SYBR Gold. This assay was compared with that based on the use of ethidium bromide (the most commonly used nucleic acid stain). Unlike ethidium bromide, SYBR Green I DNA sensitivity and concentration-dependent fluorescence intensity were identical for supercoiled and nicked-relaxed forms. DNA detection by SYBR Green I in solution is approximately 40-fold more sensitive than by ethidium bromide for double-stranded DNA and approximately 10-fold for single-stranded DNA, and in agarose gel it is 16-fold more sensitive for double-stranded DNA compared with ethidium bromide. SYBR Gold performs similarly to SYBR Green I. This study shows that: (a) there is no significant difference in DNA binding isotherms to the monocationic DOTAP (DOTAP/DOPE) liposomes and to the polycationic DOSPA (DOSPA/DOPE) liposomes, even when four DOSPA positive charges are involved in the electrostatic interaction with DNA; (b) the helper lipids affect DNA binding, as DOTAP/DOPE liposomes bind more DNA than DOTAP/cholesterol; (c) in the process of lipoplex formation, when the DNA is a mixture of two forms, supercoiled and nicked-relaxed (open circular), there is a preference for the binding to the cationic liposomes of plasmid DNA in the nicked-relaxed over the supercoiled form. This preference is much more pronounced when the cationic liposome formulation is based on the monocationic lipid DOTAP than on the polycationic lipid DOSPA. The preference of DOTAP formulations to bind to the relaxed DNA plasmid suggests that the binding of supercoiled DNA is weaker and easier to dissociate from the complex.     

Factors affecting DNA binding and stability of association to cationic liposomes

Lipoplexes are complexes formed between cationic liposomes (L(+)) and polyanionic nucleic acids (P(-)). They are commonly used in vitro and in vivo as a nucleic acid delivery system. Our study aims are to investigate how DOTAP-based cationic liposomes, which vary in their helper lipid (cholesterol or DOPE) and in media of different ionic strengths affect the degree, mode of association and degree of condensation of pDNA. This was determined by ultracentrifugation and gel electrophoresis, methods based on different physical principles. In addition, the degree of pDNA condensation was also determined using the ethidium bromide (EtBr) intercalation assay. The results suggest that for cationic lipid compositions (DOTAP/DOPE and DOTAP/cholesterol), 1.5 M NaCl, but not 0.15 M NaCl, both prevent lipoplex formation and/or induce partial dissociation between lipid and DNA of preformed lipoplexes. The higher the salt concentration the greater is the similarity of DNA condensation (monitored by EtBr intercalation) between lipoplex DNA and free DNA. As determined by ultracentrifugation and agarose gel electrophoresis, 30-90% of the DNA is uncondensed. SDS below its critical micellar concentration (CMC) induced "de-condensation" of DNA without its physical release (assessed by ultracentrifugation) for both DOTAP/DOPE and DOTAP/cholesterol lipoplexes. As was assessed by agarose gel electrophoresis SDS induced release of 50-60% of DNA from the DOTAP/cholesterol lipoplex but not from the DOTAP/DOPE lipoplex. This study shows that there are conditions under which DNA is still physically associated with the cationic lipids but undergoes unwinding to become less condensed. We also proved that the helper lipid affects level and strength of the L(+) and DNA(-) electrostatic association; these interactions are weaker for DOTAP/cholesterol than for DOTAP/DOPE, despite the fact that the positive charge and surface pH of DOTAP/cholesterol and DOTAP/DOPE are similar.     

Interplay in lipoplexes between type of pDNA promoter and lipid composition determines transfection efficiency of human growth hormone in NIH3T3 cells in culture.

This study was aimed to investigate if and to what extent there is an interplay between lipoplex physicochemical properties and plasmid promoter type affecting transfection efficiency in vitro. To reduce the number of variables only one cell type (NIH3T3 cells), one gene (human growth hormone), one cationic lipid (DOTAP) in a plasmid >85% in supercoiled form, and the same medium conditions were used. The variables of the physicochemical properties included presence and type of helper lipid (DOPE, DOPC, or cholesterol, all in 1:1 mole ratio with DOTAP), size and lamellarity of the liposomes used for lipoplex preparation (large unilamellar vesicles, LUV, versus multilamellar vesicles, MLV), and DNA(-)/cationic lipid(+) charge ratio, all containing the same human growth hormone but differing in their promoter enhancer region. Two of the promoters were of viral origin: (a) SV40 promoter (simian virus early promoter) and (b) CMV promoter (cytomegalovirus early promoter); two were of mammalian cell origin: (c) PABP promoter (human poly(A)-binding protein promoter) and (d) S16 promoter (mouse ribosomal protein (rp) S16 promoter). Transfection studies showed that, irrespective of promoter type, large (> or =500 nm) MLV were superior to approximately 100 nm LUV; the extent of superiority was dependent on liposome lipid composition (larger for 100% DOTAP and DOTAP/DOPE than for DOTAP/DOPC and DOTAP/cholesterol). The optimal DNA(-)/DOTAP(+) charge ratio for all types of lipoplexes used was 0.2 or 0.5 (namely, when the lipoplexes were positively charged). Scoring the six best lipoplex formulations (out of 128 studied) revealed the following order: pCMV (DOTAP/DOPE) > pSV (DOTAP/DOPE)=pCMV(DOTAP/cholesterol)=pS16 (100% DOTAP)=pS16 DOTAP/DOPE > pCMV (DOTAP/DOPC). The lack of trivial consistency in the transfection efficiency score, the pattern of transfection efficiency, and statistical analysis of the data suggest that there is cross-talk between promoter type and lipoplex lipid composition, which may be related to the way the promoter is associated with the lipids.     

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.     

Use of a quaternary ammonium detergent in liposome mediated DNA transfection of mouse L-cells

Sonicated liposomes composed of dioleoylphosphatidylethanolamine (DOPE) and a quaternary ammonium detergent (dodecyl-, tetradecyl-, or cetyl-trimethylammonium bromide) mediates functional transfer of pSV2 CAT plasmid DNA to mouse L929 fibroblasts. Successful transfection was determined by assaying for chloramphenicol acetyltransferase activity in cell lysates collected 40 h after exposure to the lipid-DNA complexes. Liposomes prepared with the quaternary ammonium detergents were less toxic than the free detergents at the same concentrations and were more efficient in their delivery of the plasmid DNA to the cells. Analysis of the three detergents in combination with the lipid showed that cetyltrimethylammonium bromide was least toxic to the cells. This detergent, at a minimal concentration of 20 mol% in DOPE, allowed for stable liposome preparations and efficient transfection. Optimal efficiency of transfection occurred with 30 micrograms of DNA. Further increases in the DNA concentration caused a decrease in the transfection efficiency, perhaps due to charge repulsions between the liposomes now saturated with negatively charged DNA and the negatively charged cell surface. The transfection activity of the liposome was limited by its cytotoxicity at high liposome concentrations. These results are compared with that of the Lipofectin, another positively charged liposome preparation which is commercially available. Although the overall transfection activity of the liposome containing the quaternary ammonium detergent is somewhat lower than that of the Lipofectin, it may serve as an inexpensive and convenient alternative.     

Morphology of Cationic Liposome/DNA Complexes in Relation to Their Chemical Composition

Abstract

Electron microscopy is used to show the morphology of liposome/DNA complexes as related to their cationic component, the molar ratio of the helper lipid (usually DOPE¹), the nature of the DNA-component, as well as the composition of the media. Liposomes made of monovalent cationic amphiphiles adhere and fuse during interaction with negatively charged DNA thereby complexing the DNA. The size of the resulting complexes is depending upon charge neutralization and is smallest at a slightly positive net charge. At molar ratios of DOPE, to the cationic component of ≥ 1.5, hexagonal lipid tubules are formed, especially in media containing high salt concentrations, and even in the control lipid mixture, not interacting with any DNA or oligonucleotide. Complexes, made of plasmid-DNA, monovalent cationic amphiphiles, and DOPE at a lower molar ratio, show additionally to the semifused or fused liposomes a new structure, called spaghetti-like structure, representing a bilayer-coated, supercoiled DNA. Single-strand and short oligonucleotides seem not to form such structures during interaction with monovalent cationic liposomes. Neither fusion nor spaghetti formation is observed during interaction of DNA with liposomes made of polyvalent cationic amphiphiles. In general, small complexes consisting of some few semifused liposomes bearing the self-encapsulated nucleic acid and additionally the spaghetti-like structure, free or connected with these complexes, seem to be candidates for the transfectionactive structure rather than large extended H_II¹-lipid arrangements.     

Liposome-entrapped plasmid DNA: characterisation studies

Plasmid DNA pRc/CMV HBS (5.6 kb) (100 microg) encoding the S (small) region of hepatitis B surface antigen was incorporated by the dehydration-rehydration method into liposomes composed of 16 micromol egg phosphatidylcholine (PC), 8 micromol dioleoylphosphatidylcholine (DOPE) and 1, 2-diodeoyl-3-(trimethylammonium)propane (DOTAP) (cationic liposomes) or phosphatidylglycerol (anionic liposomes) in a variety of molar ratios. The method, entailing mixing of small unilamellar vesicles (SUV) with the DNA, followed by dehydration and rehydration, yielded incorporation values of 95-97 and 48-54% of the DNA used, respectively. Mixing of preformed cationic liposomes with 100 microg plasmid DNA also led to high complexation values of 73-97%. As expected, the association of DNA with preformed anionic liposomes was low (9%). Further work with cationic PC/DOPE/DOTAP liposomes attempted to establish differences in the nature of DNA association with the vesicles after complexation and the constructs generated by the process of dehydration/rehydration. Several lines of evidence obtained from studies on vesicle size and zeta-potential, fluorescent microscopy and gel electrophoresis in the presence of the anion sodium dodecyl sulphate (SDS) indicate that, under the conditions employed, interaction of DNA with preformed cationic SUV as above, or with cationic SUV made of DOPE and DOTAP (1:1 molar ratio; ESCORT Transfection Reagent), leads to the formation of large complexes with externally bound DNA. For instance, such DNA is accessible to and can be dissociated by competing anionic SDS molecules. However, dehydration of the DNA-SUV complexes and subsequent rehydration, generates submicron size liposomes incorporating most of the DNA in a fashion that prevents DNA displacement through anion competition. It is suggested that, in this case, DNA is entrapped within the aqueous compartments, in between bilayers, presumably bound to the cationic charges.     

Adeno-associated virus mediated gene transfer into primary rat brain neuronal and glial cultures: enhancement with the pH-sensitive surfactant dodecyl 2-(1'-imidazolyl) propionate

This study evaluated the effects of a novel, pH-sensitive surfactant, dodecyl 2-(1'-imidazolyl) propionate (DIP), on cationic lipid mediated transfection in primary rat brain neuronal and glial cultures. The cationic lipid complex DOTAP/DOPE (1, 2-dioleoyl-3-trimethylammonium propionate and dioleoyl phosphatidylethanolamine, respectively) was added over a range of concentrations (0-120 microg/ml) with DNA concentration kept constant (1.6 microg/ml). The neuron-specific enolase (NSE) and cytomegalovirus (CMV) promoters were found to drive green fluorescent protein (GFP) expression in neuron-enriched and glial cultures, respectively, using adeno-associated virus (AAV) derived constructs. NSE-driven GFP expression was not observed in glial cultures. Addition of DOTAP/DOPE increased transfection efficiency over a wide range of lipid concentrations (5-50 microg/ml) keeping DNA concentration constant (1.6 microg/ml). Addition of DIP to the lipid/DNA complex increased maximum transfection efficiencies in glial and neuronal cultures 2-3-fold. Transfection efficiencies were at their maximum with a similar total lipid concentration (50 microg/ml) in both cell-types in the presence of DIP. Neuronal cultures were more sensitive than glia to the toxic actions of DOTAP/DOPE, with or without DIP. These results indicate that AAV-mediated gene-transfer to neurons and glia can be facilitated by addition of a pH-sensitive surfactant to cationic liposome/DNA complexes and that endosomal escape could be a limiting factor in transgene expression.     

Transfection property of a new cholesterol-based cationic lipid containing tri-2-hydroxyethylamine as gene delivery vehicle

A novel cholesterol-based cationic lipid containing a tri-2- hydroxyethylamine head group and ether linker (Chol- THEA) was synthesized and examined as a potent gene delivery vehicle. In the preparation of cationic liposome, the addition of DOPE as helper lipid significantly increased the transfection efficiency. To find the optimum transfection efficiency, we screened various weight ratios of DOPE and liposome/DNA (N/P). The best transfection efficiency was found at the Chol-THEA:DOPE weight ratio of 1:1 and N/P weight ratio of 10~15. Most of the plasmid DNA was retarded by this liposome at the optimum N/P weight ratio of 10. The transfection efficiency of Chol-THEA liposome was compared with DOTAP, Lipofectamine, and DMRIE-C using the luciferase assay and GFP expression. Chol-THEA liposome with low toxicity had better or similar potency of gene delivery compared with commercial liposomes in COS-7, Huh-7, and MCF-7 cells. Therefore, Chol-THEA could be a useful non-viral vector for gene delivery.     

Isothermal titration calorimetric analysis of the interaction between cationic lipids and plasmid DNA

The effects of buffer and ionic strength upon the enthalpy of binding between plasmid DNA and a variety of cationic lipids used to enhance cellular transfection were studied using isothermal titration calorimetry at 25.0 degrees C and pH 7.4. The cationic lipids DOTAP (1,2-dioleoyl-3-trimethyl ammonium propane), DDAB (dimethyl dioctadecyl ammonium bromide), DOTAP:cholesterol (1:1), and DDAB:cholesterol (1:1) bound endothermally to plasmid DNA with a negligible proton exchange with buffer. In contrast, DOTAP: DOPE (L-alpha-dioleoyl phosphatidyl ethanolamine) (1:1) and DDAB:DOPE (1:1) liposomes displayed a negative enthalpy and a significant uptake of protons upon binding to plasmid DNA at neutral pH. These findings are most easily explained by a change in the apparent pKa of the amino group of DOPE upon binding. Complexes formed by reverse addition methods (DNA into lipid) produced different thermograms, sizes, zeta potentials, and aggregation behavior, suggesting that structurally different complexes were formed in each titration direction. Titrations performed in both directions in the presence of increasing ionic strength revealed a progressive decrease in the heat of binding and an increase in the lipid to DNA charge ratio at which aggregation occurred. The unfavorable binding enthalpy for the cationic lipids alone and with cholesterol implies an entropy-driven interaction, while the negative enthalpies observed with DOPE-containing lipid mixtures suggest an additional contribution from changes in protonation of DOPE.     

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.     

Zeta potential of transfection complexes formed in serum-free medium can predict in vitro gene transfer efficiency of transfection reagent

We have tested the zeta potential (zeta, the surface charge density) of transfection complexes formed in serum-free medium as a rapid and reliable technique for screening transfection efficiency of a new reagent or formulation. The complexes of CAT plasmid DNA (1 microgram) and DC-chol/DOPE liposomes (3-20 nmol) were largely negatively charged (zeta=-15 to -21 mV), which became neutral or positive as 0.5 microgram or a higher amount of poly-L-lysine (PLL, MW 29300 or MW 204000) was added (-3.16+/-3.47 to +6.04+/-2.23 mV). However, the complexes of CAT plasmid DNA (1 microgram) and PLL MW 29300 (0.5 microgram or higher) were neutral or positively charged (-3.22+/-2.3 to +6.55+/-0.64 mV), which remained the same as 6.6 nmol of the liposomes was added. The complexes formed between two positively charged compounds, PLL MW 29300 (0.5 microgram) and the liposomes (3-20 nmol), were as closely positively charged as DNA/PLL or DNA/liposomes/PLL complexes (+3.31+/-0.41 to 7.16+/-1.0 mV). These results indicate that PLL determined the overall charge of the DNA/liposome/PLL ternary complexes. The complexes formed with histone (0.75 microgram or higher) were also positively charged, whose transfection activity was as high as PLL MW 29300. However, the complexes formed with protamine or PLL MW 2400 remained negatively charged. These observations are in good agreement with the transfection activity of the formulation containing each polycationic polymer. The presence of PLL MW 29300 did not change the hydrodynamic diameter of DNA/liposome/PLL complexes (d(H)=275-312 nm). The complexes made of different sizes of PLL (MW 2400 and 204000) also did not significantly change their size. This suggests that DNA condensation may not be critical. Therefore, zeta of the transfection complex can predict the transfection efficiency of a new formulation or reagent.     

Efficient gene transfer by transferrin lipoplexes in the presence of serum

Cationic lipids are being used increasingly as reagents for gene delivery both in vitro and in vivo. One of the limitations to the application of cationic lipid-DNA complexes (lipoplexes) in vivo is the inhibition of gene delivery by serum. In this study, we have shown that transferrin (Tf)-lipoplexes, which had transferrin adsorbed at their surface via electrostatic interactions, are much more effective than plain lipoplexes in transfecting cells in the presence of relatively high concentrations (up to 60%) of fetal bovine serum (FBS). Serum even enhanced transfection by Tf-lipoplexes composed of 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP)/dioleoylphosphatidylethanolamine (DOPE)/pCMVLacZ at high lipid/DNA (+/-) charge ratios, and inhibited lipofection for those with low charge ratios when they were added to the cells immediately after the preparation of complexes. The effect of serum on lipofection was dose-dependent. Preincubation of the complexes at 20 degrees C for 6 h led to serum resistance, even for the negatively charged transferrin-lipoplexes. A similar tendency was observed for DOTAP/cholesterol and DOTAP/DOPE/cholesterol liposomes. The percentage of cells transfected, measured by beta-galactosidase expression, also increased with the serum concentration. Cell viability was not affected significantly when the cells were incubated with the complexes for 4 h at 37 degrees C, followed by a 48-h incubation. Our findings extend the scope of previous studies where transferrin-lipoplexes were used to introduce DNA into cells, rendering these complexes and their future derivatives potential alternatives to viral vectors for gene delivery in vivo.     

Hydration of lipoplexes commonly used in gene delivery: follow-up by laurdan fluorescence changes and quantification by differential scanning calorimetry.

Lipoplexes, which are formed spontaneously between cationic liposomes and negatively charged nucleic acids, are commonly used for gene and oligonucleotide delivery in vitro and in vivo. Being assemblies, lipoplexes can be characterized by various physicochemical parameters, including size distribution, shape, physical state (lamellar, hexagonal type II and/or other phases), sign and magnitude of electrical surface potential, and level of hydration at the lipid-DNA interface. Only after all these variables will be characterized for lipoplexes with a broad spectrum of lipid compositions and DNA/cationic lipid (L(+)) mole (or charge) ratios can their relevance to transfection efficiency be understood. Of all these physicochemical parameters, hydration is the most neglected, and therefore the focus of this study. Cationic liposomes composed of DOTAP without and with helper lipids (DOPC, DOPE, or cholesterol) or of DC-Chol/DOPE were complexed with pDNA (S16 human growth hormone) at various DNA(-)/L(+) charge ratios (0.1-3.2). (DOTAP=N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride; DC-Chol=(3beta-[N-(N',N'-dimethylaminoethane)-carbamoyl]-cholester ol; DOPC=1, 2-dioleoyl-sn-glycero-3-phosphocholine; DOPE=1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine). The hydration levels of the different cationic liposomes and the DNA separately are compared with the hydration levels of the lipoplexes. Two independent approaches were applied to study hydration. First, we used a semi-quantitative approach of determining changes in the 'generalized polarization' (GP) of laurdan (6-dodecanoyl-2-dimethylaminonaphthalene). This method was recently used extensively and successfully to characterize changes of hydration at lipid-water interfaces. Laurdan excitation GP at 340 nm (GP(340)DOTAP. The GP(340) of lipoplexes of all lipid compositions (except those based on DC-Chol/DOPE) was higher than the GP(340) of the cationic liposomes alone and increased with increasing DNA(-)/L(+) charge ratio, reaching a plateau at a charge ratio of 1. 0, suggesting an increase in dehydration at the lipid-water interface with increasing DNA(-)/L(+) charge ratio. Confirmation was obtained from the second method, differential scanning calorimetry (DSC). DOTAP/DOPE lipoplexes with charge ratio 0.44 had 16.5% dehydration and with charge ratio 1.5, 46.4% dehydration. For DOTAP/Chol lipoplexes with these charge ratios, there was 17.9% and 49% dehydration, respectively. These data are in good agreement with the laurdan data described above. They suggest that the dehydration occurs during lipoplex formation and that this is a prerequisite for the intimate contact between cationic lipids and DNA.     

A multi-step lipid mixing assay to model structural changes in cationic lipoplexes used for in vitro transfection.

Formation of liposome/polynucleotide complexes (lipoplexes) involves electrostatic interactions, which induce changes in liposome structure. The ability of these complexes to transfer DNA into cells is dependent on the physicochemical attributes of the complexes, therefore characterization of binding-induced changes in liposomes is critical for the development of lipid-based DNA delivery systems. To clarify the apparent lack of correlation between membrane fusion and in vitro transfection previously observed, we performed a multi-step lipid mixing assay to model the sequential steps involved in transfection. The roles of anion charge density, charge ratio and presence of salt on lipid mixing and liposome aggregation were investigated. The resonance-energy transfer method was used to monitor lipid mixing as cationic liposomes (DODAC/DOPE and DODAC/DOPC; 1:1 mole ratio) were combined with plasmid, oligonucleotides or Na(2)HPO(4). Cryo-transmission electron microscopy was performed to assess morphology. As plasmid or oligonucleotide concentration increased, lipid mixing and aggregation increased, but with Na(2)HPO(4) only aggregation occurred. NaCl (150 mM) reduced the extent of lipid mixing. Transfection studies suggest that the presence of salt during complexation had minimal effects on in vitro transfection. These data give new information about the effects of polynucleotide binding to cationic liposomes, illustrating the complicated nature of anion induced changes in liposome morphology and membrane behavior.     

Using disaccharides to enhance in vitro and in vivo transgene expression mediated by a lipid-based gene delivery system

BACKGROUND: Lipid-based vectors have been widely applied to in vivo and in vitro gene delivery. Disaccharides can effectively stabilize lipid membranes. This study examined whether disaccharides could enhance the transgene expression mediated by lipid-based vectors. METHODS: Different disaccharides were incorporated into the vectors prepared with DOTAP/protamine/DNA (LPD) or with DNA/cationic liposomes containing DOTAP, DOTAP/Chol, DOTAP/DOPE, or DC-Chol/DOPE. The levels of transgene expression and internalized plasmid of CHO cells were represented by the percentages of GFP-positive cells and the fluorescence intensity of ethidium-monoazide covalently labeled plasmid, respectively. The vectors containing either cellobiose or trehalose were also intravenously injected into mouse tail vein to investigate the potentials of in vivo applications. RESULTS: For enhancing the transgene expression, cellobiose was found to be effective for all the vectors whereas maltose decreased the effectiveness of DOTAP/Chol liposomes and LPD. For the internalization of plasmid, most disaccharides were able to increase the cellular delivery of DOTAP, DOTAP/Chol, and DOTAP/DOPE liposomes, but caused decreases in the cellular entry of DC-Chol/DOPE liposomes. An approximately linear correlation between the internalized plasmid and the transgene expression was observed for all the treatments in this study. When the vectors were administered to mouse by intravenous injection, 10-fold and 3-fold increases in the luciferase expression of lung were observed for DOTAP liposomes containing 330 mM cellobiose and trehalose, respectively. CONCLUSIONS: This study showed that using trehalose and cellobiose with a lipid-based delivery system provides a straightforward approach to effectively enhance both in vitro and in vivo transgene expression.     

Biophysical and lipofection studies of DOTAP analogs

In order to investigate the relationship between lipid structure and liposome-mediated gene transfer, we have studied biophysical parameters and transfection properties of monocationic DOTAP analogs, systematically modified in their non-polar hydrocarbon chains. Stability, size and (by means of anisotropy profiles) membrane fluidity of liposomes and lipoplexes were determined, and lipofection efficiency was tested in a luciferase reporter gene assay. DOTAP analogs were used as single components or combined with a helper lipid, either DOPE or cholesterol. Stability of liposomes was a precondition for formation of temporarily stable lipoplexes. Addition of DOPE or cholesterol improved liposome and lipoplex stability. Transfection efficiencies of lipoplexes based on pure DOTAP analogs could be correlated with stability data and membrane fluidity at transfection temperature. Inclusion of DOPE led to rather uniform transfection and anisotropy profiles, corresponding to lipoplex stability. Cholesterol-containing lipoplexes were generally stable, showing high transfection efficiency at low relative fluidity. Our results demonstrate that the efficiency of gene transfer mediated by monocationic lipids is greatly influenced by lipoplex biophysics due to lipid composition. The measurement of fluorescence anisotropy is an appropriate method to characterize membrane fluidity within a defined system of liposomes or lipoplexes and may be helpful to elucidate structure-activity relationships.     

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.     

Interactions between pH-sensitive liposomes and model membranes

The structure and dynamics of two different pH-sensitive liposome systems were investigated by means of cryo-transmission electron microscopy and different photophysical techniques. Both systems consisted of dioleoylphosphatidylethanolamine (DOPE) and contained either oleic acid (OA) or a novel acid-labile polyethylene glycol-conjugated lipid (DHCho-MPEG5000) as stabiliser. Proton induced leakage, lipid mixing and structural changes were studied in the absence and presence of EPC liposomes, as well as in the presence of liposomes designed to model the endosome membrane. Neither DHCho-MPEG5000- nor OA-stabilised liposomes showed any tendency for fusion with pure EPC liposomes or endosome-like liposomes composed of EPC/DOPE/SM/Cho (40/20/6/34 mol.%). Our investigations showed, however, that incorporation of lipids from the pH-sensitive liposomes into the endosome membrane may lead to increased permeability and formation of non-lamellar structures. Taken together the results suggest that the observed ability of DOPE-containing liposomes to mediate cytoplasmic delivery of hydrophilic molecules cannot be explained by a mechanism based on a direct, and non-leaky, fusion between the liposome and endosome membranes. A mechanism involving destabilisation of the endosome membrane due to incorporation of DOPE, seems more plausible.     

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.