Transfection with fluorinated lipoplexes based on fluorinated analogues of DOTMA,DMRIE and DPPES

Fluorinated double-chain (poly)cationic lipids (one or both of these chains being ended by a highly fluorinated tail) which are close analogues of DOTMA, DMRIE or DPPES were designed as synthetic vectors for gene delivery. For N/P ratios (N=number of amine functions of the lipid; P=number of DNA phosphates) from 0.8 to 5, these fluorinated cationic lipids condensed DNA, with or without the use of DOPE, to form fluorinated lipoplexes. No specific cell toxicity was evidenced for these new fluorinated lipoplexes. The efficiency of some of the fluorinated lipoplexes to transfect lung epithelial A549 cells was comparable to that of the first generation of fluorinated lipoplexes made from fluorinated analogues of DOGS (Transfectam) [Bioconjug. Chem. 12 (2001) 114]. These results, combined with the higher in vivo transfection potential found for fluorinated lipoplexes than for conventional lipoplexes or PEI polyplexes [J. Gene Med. 3 (2001) 109], confirm that fluorinated lipoplexes are very promising gene transfer systems.     

Transfection with fluorinated lipoplexes based on fluorinated analogues of DOTMA, DMRIE and DPPES

Fluorinated double-chain (poly)cationic lipids (one or both of these chains being ended by a highly fluorinated tail) which are close analogues of DOTMA, DMRIE or DPPES were designed as synthetic vectors for gene delivery. For N/P ratios (N=number of amine functions of the lipid; P=number of DNA phosphates) from 0.8 to 5, these fluorinated cationic lipids condensed DNA, with or without the use of DOPE, to form fluorinated lipoplexes. No specific cell toxicity was evidenced for these new fluorinated lipoplexes. The efficiency of some of the fluorinated lipoplexes to transfect lung epithelial A549 cells was comparable to that of the first generation of fluorinated lipoplexes made from fluorinated analogues of DOGS (Transfectam) [Bioconjug. Chem. 12 (2001) 114]. These results, combined with the higher in vivo transfection potential found for fluorinated lipoplexes than for conventional lipoplexes or PEI polyplexes [J. Gene Med. 3 (2001) 109], confirm that fluorinated lipoplexes are very promising gene transfer systems.     

In vitro cationic lipid-mediated gene delivery with fluorinated glycerophosphoethanolamine helper lipids.

There is a need for the development of nonviral gene transfer systems with improved and original properties. "Fluorinated" lipoplexes are such candidates, as supported by the remarkably higher in vitro and in vivo transfection potency found for such fluorinated lipoplexes as compared with conventional ones or even with PEI-based polyplexes (Boussif, O., Gaucheron, J., Boulanger, C., Santaella, C., Kolbe, H. V. J., Vierling, P. (2001) Enhanced in vitro and in vivo cationic lipid-mediated gene delivery with a fluorinated glycerophosphoethanolamine helper lipid. J. Gene Med. 3, 109-114). Here, we describe the synthesis of fluorinated glycerophosphoethanolamines (F-PEs), close analogues of dioleoylphosphatidylethanolamine (DOPE), and report on their lipid helper properties vs that of DOPE, as in vitro gene transfer components of fluorinated lipoplexes based on pcTG90, DOGS (Transfectam), or DOTAP. To evaluate the contribution of the F-PEs to in vitro lipoplex-mediated gene transfer, we examined the effect of including the F-PEs in lipoplexes formulated with these cationic lipids (CL) for various CL:DOPE:F-PE molar ratios [1:(1 - x):x with x = 0, 0.5 and 1; 1:(2 - y):y with y = 0, 1, 1.5, and 2], and various N/P ratios (from 10 to 0.8, N = number of CL amines, P = number of DNA phosphates). Irrespective of the F-PE chemical structure, of the colipid F-PE:DOPE composition, and of the N/P ratio, comparable transfection levels to those of their respective control DOPE lipoplexes were most frequently obtained when using one of the F-PEs as colipid of DOGS, pcTG90, or DOTAP in place of part of or of all DOPE. However, a large proportion of DOGS-based lipoplexes were found to display a higher transfection efficiency when formulated with the F-PEs rather than with DOPE alone while the opposite tendency was evidenced for the DOTAP-based lipoplexes. The present work indicates that "fluorinated" lipoplexes formulated with fluorinated helper lipids and conventional cationic lipids are very attractive candidates for gene delivery. It confirms further that lipophobicity and restricted miscibility of the lipoplex lipids with the endogenous lipids does not preclude efficient gene transfer and expression. Their transfection potency is rather attributable to their unique lipophobic and hydrophobic character (resulting from the formulation of DNA with fluorinated lipids), thus preventing to some extent DNA from interactions with lipophilic and hydrophilic biocompounds, and from degradation.     

Efficient lipid-mediated transfection of DNA into primary rat hepatocytes

Cationic lipids are an effective means for transfecting nucleic acids into a variety of cell types. Very few of these lipids, however, have been reported to be effective with primary cells. We report on the efficacy of several commercially available cationic lipid reagents to transfect plasmid DNA into primary rat hepatocytes in culture. The reagents tested in this study include TransfectAce, LipofectAmine, Lipofectin, N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammoniumchloride (DOTMA), (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate (DOTAP), and cetyltrimethyl-ammonium bromide/dioleoylphosphatidylethanol-amine (CTAB/DOPE). Electron micrographic (EM) studies indicate that similar size Lipofectin and DOTAP vesicles contain DNA-like material internally and that these vesicles attach to the cell membrane. DOTAP vesicles are multilamellar, appear as clusters, and have a high DNA-to-lipid ratio. Lipofectin vesicles appear to attach to the cell surface as individual vesicles. The EM observations are consistent with current theories on the mechanism of transfection by cationic lipids. While Lipofectin has proven to be effective in transfection studies of primary cells in culture, we have found DOTAP to be a viable alternative. DOTAP yields transfection rates in hepatocytes comparable to DOTMA and Lipofectin, however, at lower concentrations of reagent and at considerably less cost. Optimal conditions for transfecting 5 μg of plasmid DNA with DOTAP were achieved by utilizing multilamellar (vortexed) vesicles at a concentration of 15 μg DOTAP per 2 ml media in 60-mm plates for 2 h transfection time. In this study, DOTAP has proven to be economical, easy to prepare, and very effective in transfecting DNA into primary rat hepatocytes.     

Real-time Observation of Lipoplex Formation and Interaction with Anionic Bilayer Vesicles

A novel development has allowed for the direct observation of single, pairwise interactions of linear DNA with cationic vesicles and of DNA-cationic lipid complexes with anionic vesicles. A new cationic phospholipid derivative, l,2-dioleoyl-sn-glycero-3-ethylphosphocholine, was used to prepare giant bilayer vesicles and to form DNA-cationic lipid complexes (lipoplexes). The cationic vesicles were electrophoretically maneuvered into contact with DNA, and similarly, complexes were brought into contact with anionic phospholipid vesicles composed of dioleoylphosphatidylglycerol (DOPG; 100%), DOPG/dioleoylphosphatidylethanolamine (DOPE; 1:1) or DOPG/dioleoylphosphatidylcholine (DOPC; 1:1). Video fluorescence microscopy revealed that upon contact with phospholipid anionic vesicles, lipoplexes exhibited four different types of behavior: adhesion, vesicle rupture, membrane perforation (manifested as vesicle shrinkage and/or content loss), and expansion of DNA (which was always concomitant with membrane perforation.) In one instance, the lipoplex was injected into the target vesicle just prior to DNA expansion. In all other instances, the DNA expanded over the outer surface of the vesicle, and expansion was faster, the larger the area of vesicle over which it expanded. Given the likelihood of incorporation of cellular anionic lipids into lipoplexes, the expansion of the DNA could be important in DNA release during cell transfection. Upon contact with naked DNA, giant cationic vesicles usually ruptured and condensed the DNA into a small particle. Contact of cationic vesicles that were partially coated with DNA usually caused the DNA to wrap around the vesicle, leading to vesicle rupture, vesicle fusion (with other attached vesicles or lipid aggregates), or simply cessation of movement. These behaviors clearly indicated that both DNA and vesicles could be partly or fully covered by the other, thus modifying surface charges, which, among others, allowed adhesion of DNA-coated vesicles with uncoated vesicles and of lipid-coated DNA with uncoated DNA.     

Structural and fusogenic properties of cationic liposomes in the presence of plasmid DNA.

The structural and fusogenic properties of large unilamellar vesicles (LUVs) composed of the cationic lipid N-[2,3-(dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and 1,2-dioleoyl-3-phosphatidylethanotamine (DOPE) have been examined in the presence of pCMV5 plasmid and correlated with transfection potency. It is shown, employing lipid mixing fusion assays, that pCMV5 plasmid strongly promotes fusion between DOTMA/DOPE (1:1) LUVs and DOTMA/1,2-dioleoyl-3-phosphatidylcholine (DOTMA/DOPC) (1:1) LUVs such that at a cationic lipid-to-DNA charge ratio of 3.0, approximately 80% fusion is observed. The anions citrate and chloride can also trigger fusion, but at much higher concentrations. Freeze-fracture electron microscopy studies demonstrate the tendency of cationic vesicles to form clusters at low pCMV5 content, whereas macroscopic fused aggregates can be observed at higher plasmid levels. 31P NMR studies of the fused DNA-DOTMA/DOPE (1:1) complexes obtained at high plasmid levels (charge ratio 1.0) reveal narrow "isotropic" 31P NMR resonances, whereas the corresponding DOPC containing systems exhibit much broader "bilayer" 31P NMR spectra. In agreement with previous studies, the transfection potency of the DOPE-containing systems is dramatically higher than for the DOPC-containing complexes, indicating a correlation between transfection potential and the motional properties of endogenous lipids. Interestingly, it was found that the complexes could be separated by centrifugation into a pellet fraction, which exhibits superior transfection potencies, and a supernatant fraction. Again, the pellet fraction in the DOPE-containing system exhibits a significantly narrower 31P NMR resonance than the corresponding DOPC-containing system. It is suggested that the 31P NMR characteristics of complexes exhibiting higher transfection potencies are consistent with the presence of nonbilayer lipid structures, which may play a direct role in the fusion or membrane destabilization events vital to transfection.     

Synthesis, in vitro transfection activity and physicochemical characterization of novel N,N'-diacyl-1,2-diaminopropyl-3-carbamoyl-(dimethylaminoethane) amphiphilic derivatives

A novel series of N,N'-diacyl-1,2-diaminopropyl-3-carbamoyl-(dimethylaminoethane) cationic derivatives was synthesized and screened for in vitro transfection activity at different charge ratios in the presence and absence of the helper lipids DOPE and cholesterol. Physicochemical properties of lipid-DNA complexes were studied by gel electrophoresis, fluorescence spectroscopy and dynamic light scattering. The interfacial properties of the lipids in isolation were studied using the Langmuir film balance technique at 23 degrees C. It was found that only lipoplexes formulated with the dioleoyl derivative, 1,2lmt[5], mediated significant in vitro transfection activity. Optimum activity was obtained with 1,2lmt[5]/DOPE mixture at a +/-charge ratio of 2. In agreement with the transfection results, 1,2lmt[5] was the only lipid found to complex and retard DNA migration as verified by gel electrophoresis. Despite the efficient complexation, no significant condensation of plasmid DNA was observed as indicated by fluorescence spectroscopy measurements. Monolayer studies showed that the dioleoyl derivative 1,2lmt[5] was the only lipid that existed in an all liquid-expanded state with a collapse area and collapse pressure of 59.5 A2 and 38.7 mN/m, respectively. This lipid was also found to have the highest elasticity with a compressibility modulus at monolayer collapse of 80.4 mN/m. In conclusion, increased acyl chain fluidity and high molecular elasticity of cationic lipids were found to correlate with improved transfection activity.     

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.     

In vitro lipofection with novel asymmetric series of 1,2-dialkoylamidopropane-based cytofectins containing single symmetric bis-(2-dimethylaminoethane) polar headgroups

Novel N,N'-diacyl-1,2-diaminopropyl-3-carbamoyl[bis-(2-dimethylaminoethane)] bivalent cationic lipids were synthesized and evaluated for in vitro transfection activity against a murine melanoma cell line. In the absence of the helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), only the dioleoyl derivative 22 (1,2lb5) elicited transfection activity. The transfection activity of this lipid was reduced when formulated with DOPE. Contrary to that, the dimyristoyl derivative 19 (1,2lb2) mediated no activity when used alone but induced the highest levels of marker gene expression in the presence of DOPE. In an effort to correlate the transfection activity with cationic lipid structures, the physicochemical properties of cationic lipids in isolation and of lipoplexes were studied with surface tensiometry, photon correlation spectroscopy, gel electrophoresis mobility shift assay, and fluorescence techniques. In regard to the lipoplex properties, gel electrophoresis mobility shift assay and EtBr exclusion fluorescence assay revealed that the 1,2lb5 was the only lipid to associate and condense plasmid DNA, respectively. Photon correlation spectroscopy analysis found that 1,2lb5/DNA complexes were of relatively small size compared to all other lipoplexes. With respect to the properties of isolated lipids, Langmuir monolayer studies and fluorescence anisotropy on cationic lipid dispersions verified high two-plane elasticity and increased fluidity of the transfection competent dioleoyl derivative 1,2lb5, respectively. The results indicate that high transfection activity is mediated by cationic lipids characterized by an expanded mean molecular area, high molecular elasticity, and increased fluidity.     

Evaluation of cationic assemblies constructed with amino acid based lipids for plasmid DNA delivery

We synthesized cationic lipids bearing lysine, histidine, or arginine as a cationic headgroup for use in gene transfer studies. The cationic assemblies formed from lysine- or arginine-type lipids gave unilamellar vesicles (approximately 100 nm diameter), whereas the morphology of the histidine-type lipids was tube-like. The competences of the cationic assemblies were sufficient to form lipoplexes, and the resulting lipoplexes were evaluated in terms of gene expression efficiencies with COS-7 cells. The lysine- or arginine-type lipids exhibited higher gene expression efficiencies than that of Lipofectamine2000, a conventional transgenic reagent, indicating that stable lipoplexes could be prepared between spherical cationic assemblies and plasmid DNA. The gene expression efficiency in relation to the cationic headgroup of the lipids was as follows: lysine > or = arginine > histidine. In addition, gene expression efficiency was enhanced by decreasing the length of the alkyl chain of the hydrophobic moiety. Unlike Lipofectamine2000, no reduction in transfection efficiency in the presence of fetal bovine serum was observed for the lipoplexes formed using synthetic cationic lipids. Moreover, the synthetic cationic lipids revealed remarkably low cytotoxicity compared with Lipofectamine2000. In conclusion, cationic assemblies formed from 1,5-ditetradecyl-N-lysyl-L-glutamate or 1,5-ditetradecyl-N-arginyl-L-glutamate can be used as an effective plasmid DNA delivery system.     

Transfection with fluorinated lipoplexes based on new fluorinated cationic lipids and in the presence of a bile salt surfactant

The synthesis of two fluorinated cationic lipids, which are analogues of frequently used synthetic gene carrier agents (including the cationic 2,3-dioleoyloxy-N-[2-(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium (DOSPA) component of the commercially available liposomal Lipofectamine), and the disintegration and DNA accessibility (evaluated by the ethidium bromide (BET) intercalation assay) as well as the in vitro transfection efficacy of cationic lipoplexes formulated with these new lipids in conjunction with conventional or fluorinated helper lipids, in the absence or presence of sodium taurocholate (STC), a powerful anionic bile salt detergent, is reported. A higher stability, with respect to the STC lytic activity and DNA accessibility, of the fluorinated cationic lipoplexes as compared with their respective lipofectamine-based ones was demonstrated. Indeed, while the Lipofectamine lipoplexes were fully disintegrated at a [STC]/[lipid] molar ratio of 2000, only 40-60% of the DNA intercalation sites of the lipoplexes based on the fluorinated analogue of DOSPA were accessible to ethidium bromide. A higher transfection potential in the presence of STC was further found for the lipoplexes formulated with the fluorinated analogue of DOSPA as compared with the Lipofectamine preparation. For a STC concentration of 7.5 mM, lipofection mediated with these fluorinated lipoplexes was significantly higher (nearly 30- to 50-fold, p < 0.05) than with the Lipofectamine ones. These results confirm the remarkable transfection potential of fluorinated lipoplexes.     

Analysis and optimization of the cationic lipid component of a lipid/peptide vector formulation for enhanced transfection in vitro and in vivo

We have previously described a lipopolyplex formulation comprising a mixture of a cationic peptide with an integrin-targeting motif (K16GACRRETAWACG) and Lipofectin, a liposome consisting of DOTMA and DOPE in a 1:1 ratio. The high transfection efficiency of the mixture involved a synergistic interaction between the lipid/peptide components. The aim of this study was to substitute the lipid component of the lipopolyplex to optimize transfection further and to seek information on the structure-activity relationship of the lipids in the lipopolyplex. Symmetrical cationic lipids with diether linkages that varied in alkyl chain length were formulated into liposomes and then incorporated into a lipopolyplex by mixing with an integrin-targeting peptide and plasmid DNA. Luciferase transfections were performed of airway epithelial cells and fibroblasts in vitro and murine lung airways in vivo. The biophysical properties of lipid structures and liposome formulations and their potential effects on bilayer membrane fluidity were determined by differential scanning calorimetry and calcein-release assays. Shortening the alkyl tail from C18 to C16 or C14 enhanced lipopolyplex and lipoplex transfection in vitro but with differing effects. The addition of DOPE enhanced transfection when formulated into liposomes with saturated lipids but was more variable in its effects with unsaturated lipids. A substantial improvement in transfection efficacy was seen in murine lung transfection with unsaturated lipids with 16 carbon alkyl tails. The optimal liposome components of lipopolyplex and lipoplex vary and represent a likely compromise between their differing structural and functional requirements for complex formation and endosomal membrane destabilization.     

Molecular shape of the cationic lipid controls the structure of cationic lipid/dioleylphosphatidylethanolamine-DNA complexes and the efficiency of gene delivery

Pyridinium amphiphiles, abbreviated as SAINT, are highly efficient vectors for delivery of DNA into cells. Within a group of structurally related compounds that differ in transfection capacity, we have investigated the role of the shape and structure of the pyridinium molecule on the stability of bilayers formed from a given SAINT and dioleoylphosphatidylethanolamine (DOPE) and on the polymorphism of SAINT/DOPE-DNA complexes. Using electron microscopy and small angle x-ray scattering, a relationship was established between the structure, stability, and morphology of the lipoplexes and their transfection efficiency. The structure with the lowest ratio of the cross-sectional area occupied by polar over hydrophobic domains (SAINT-2) formed the most unstable bilayers when mixed with DOPE and tended to convert into the hexagonal structure. In SAINT-2-containing lipoplexes, a hexagonal topology was apparent, provided that DOPE was present and complex assembly occurred in 150 mm NaCl. If not, a lamellar phase was obtained, as for lipoplexes prepared from geometrically more balanced SAINT structures. The hexagonal topology strongly promotes transfection efficiency, whereas a strongly reduced activity is seen for complexes displaying the lamellar topology. We conclude that in the DOPE-containing complexes the molecular shape and the nonbilayer preferences of the cationic lipid control the topology of the lipoplex and thereby the transfection efficiency.     

Synthesis of novel cationic lipids having polyamidoamine dendrons and their transfection activity

We designed a novel type of cationic lipid, lipids with a cationic polar group in the polyamidoamine dendron, because these dendron-bearing lipids are expected to form complexes with plasmid DNA and achieve efficient transfection of cells by synergy of endosome buffering and membrane fusion with the endosome, both of which are useful for the promotion of the transfer of plasmid DNA from endosome to cytosol. Four kinds of lipids with polyamidoamine dendrons of first to fourth generations, DL-G1, DL-G2, DL-G3, and DL-G4, were synthesized. The lipid with a dendron of a higher generation exhibited greater ability to form lipoplexes with plasmid DNA, as estimated by agarose gel electrophoresis. While the DL-G1 lipoplex did not transfect CV1 cells, the lipoplexes containing the DL-G2, DL-G3, or DL-G4 could induce transfection of the cells, and their activity was elevated with increasing generation of the dendron. Addition of dioleoylphosphatidylethanolamine (DOPE), which is known to increase fusion ability of a lipid membrane, into the lipoplexes greatly enhanced their transfection activity. In addition, the comparison with DC-Chol-containing lipoplex, which is widely used as a nonviral vector, showed that the DL-G3-DOPE lipoplex exhibits more efficient transfections. These findings imply that these dendron-bearing lipids may form the basis for a novel family of cationic lipids for efficient gene delivery.     

TACN-based cationic lipids with amino acid backbone and double tails: Materials for non-viral gene delivery

Cationic lipids have become an efficient type of non-viral vectors for gene delivery. In this Letter, four cationic lipids containing 1,4,7-triazacyclononane (TACN) headgroup, glutamic/aspartic acid backbone and dioleyl tails were designed and synthesized. The TACN headgroup gives these lipids excellent pH buffering capacities, which were higher than branched 25 kDa PEI. Cationic liposomes prepared from these lipids and DOPE showed good DNA affinity, and full DNA condensation was found at N/P ratio of 3 via agarose gel electrophoresis. The lipoplexes were characterized by dynamic light scattering (DLS) assay, which gave proper particle sizes and zeta-potentials for transfection. In vitro gene transfection results in two cell lines reveal that TAN (with aspartic acid and amide bond in the structure) shows the best transfection efficiency, which is close to commercially available transfection agent Lipofectamine 2000.     

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.     

trans-2-Aminocyclohexanol-based amphiphiles as highly efficient helper lipids for gene delivery by lipoplexes

Lipidic amphiphiles equipped with the trans-2-aminocyclohexanol (TACH) moiety are promising pH-sensitive conformational switches (“flipids”) that can trigger a lipid bilayer perturbation in response to increased acidity. Because pH-sensitivity was shown to improve the efficiency of several gene delivery systems, we expected that such flipids could significantly enhance the gene transfection by lipoplexes. Thus a series of novel lipids with various TACH-based head groups and hydrocarbon tails were designed, prepared and incorporated into lipoplexes that contain the cationic lipid 1,2-dioleoyl-3-trimethylammonio-propane (DOTAP) and plasmid DNA encoding a luciferase gene. B16F1 and HeLa cells were transfected with such lipoplexes in both serum-free and serum-containing media. The lipoplexes consisting of TACH-lipids exhibited up to two orders of magnitude better transfection efficiency and yet similar toxicity compared to the ones with the conventional helper lipids 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol. Thus, the TACH-lipids can be used as novel helper lipids for efficient gene transfection with low cytotoxicity.     

In vitro lipofection with novel series of symmetric 1,3-dialkoylamidopropane-based cationic surfactants containing single primary and tertiary amine polar head groups

A novel series of symmetric double-chained primary and tertiary 1,3-dialkoylamido monovalent cationic lipids were synthesized and evaluated for their transfection activities. In the absence of the helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), only the primary and tertiary dioleoyl derivatives 1,3lmp5 and 1,3lmt5, respectively elicited transfection activity. This is a striking difference between symmetrical 1,2-diacyl glycerol-based monovalent cationic lipids that always found both dioleoyl and dimyristoyl analogues being efficient transfection reagents. In the presence of helper lipid, all cationic derivatives induced marker gene expression, except the dilauroyl analogues 1,3lmp1 and 1,3lmt1 that elicited no transfection activity. Combining electrophoretic mobility data of the lipoplexes at different charge ratios with transfection activity suggested two requirements for high transfection activity with monovalent double-chained cationic lipids, that is, binding/association of the lipid to the plasmid DNA and membrane fusion properties of the lipid layers surrounding the DNA.     

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.     

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.