Modulation of a membrane lipid lamellar-nonlamellar phase transition by cationic lipids: A measure for transfection efficiency

Synthetic cationic lipids can be used as DNA carriers and are regarded to be the most promising non-viral gene carriers. For this investigation, six novel phosphatidylcholine (PC) cationic derivatives with various hydrophobic moieties were synthesized and their transfection efficiencies for human umbilical artery endothelial cells (HUAEC) were determined. Three compounds with relatively short, myristoleoyl or myristelaidoyl 14:1 chains exhibited very high activity, exceeding by ∼ 10 times that of the reference cationic derivative dioleoyl ethylPC (EDOPC). Noteworthy, cationic lipids with 14:1 hydrocarbon chains have not been tested as DNA carriers in transfection assays previously. The other three lipids, which contained oleoyl 18:1 and longer chains, exhibited moderate to weak transfection activity. Transfection efficiency was found to correlate strongly with the effect of the cationic lipids on the lamellar-to-inverted hexagonal, L_α → H_II, phase conversion in dipalmitoleoyl phosphatidylethanolamine dispersions (DPoPE). X-ray diffraction on binary DPoPE/cationic lipid mixtures showed that the superior transfection agents eliminated the direct L_α → H_II phase transition and promoted formation of an inverted cubic phase between the L_α and H_II phases. In contrast, moderate and weak transfection agents retained the direct L_α → H_II transition but shifted to higher temperatures than that of pure DPoPE, and induced cubic phase formation at a later stage. On the basis of current models of lipid membrane fusion, promotion of a cubic phase by the high-efficiency agents may be considered as an indication that their high transfection activity results from enhanced lipoplex fusion with cellular membranes. The distinct, well-expressed correlation established between transfection efficiency of a cationic lipid and the way it modulates nonlamellar phase formation of a membrane lipid could be useful as a criterion to assess the quality of lipid carriers and for rational design of new and superior nucleotide delivery agents.     

Bilayer Mixing,Fusion, and Lysis Following the Interaction of Populations of Cationic and Anionic Phospholipid Bilayer Vesicles

Cationic, O-alkylphosphatidylcholines, recently developed as DNA transfection agents, form bilayers indistinguishable from those of natural phospholipids and undergo fusion with anionic bilayers. Membrane merging (lipid mixing), contents release, and contents mixing between populations of positive vesicles containing O-ethylphosphatidylcholine (EDOPC) and negative vesicles containing dioleolylphosphatidylglycerol (DOPG) have been determined with standard fluorometric vesicle-population assays. Surface-charge densities were varied from zero to full charge. All interactions depended critically on surface-charge density, as expected from the adhesion-condensation mechanism. Membrane mixing ranged from zero to 100%, with significant mixing (>10 <70%) occurring between cationic vesicles that were fully charged and anionic vesicles that had fractional surface charges as low as 0.1. Such mixing with membranes as weakly charged as cell membranes should be relevant to transfection with cationic lipids. Unexpectedly, lipid mixing was higher at high than at low ionic strength when one lipid dispersion was prepared from EDOPC plus DOPG (in different proportions), especially when the other vesicles were of EDOPC; this may somehow be a consequence of the ability of the former mixture to assume non-lamellar phases. Leakage of aqueous contents was also a strong function of charge, with fully charged vesicles releasing essentially all of their contents less than 1 min after mixing. EDOPC was more active in this regard than was DOPG, which probably reflects stronger intermolecular interactions of DOPG. Fusion, as measured by contents mixing, exhibited maximal values of 10% at intermediate surface charge. Reduced fusion at higher charge is attributed to multiple vesicle interactions leading to rupture. The existence of previously published data on individual interactions of vesicles of the same composition made it possible for the first time to compare pairwise with population interactions, confirming the likelihood of population studies to overestimate rupture and hemifusion and underestimate true vesicle fusion.     

Lipoplex formulation of superior efficacy exhibits high surface activity and fusogenicity, and readily releases DNA

Lipoplexes containing a mixture of cationic phospholipids dioleoylethylphosphatidylcholine (EDOPC) and dilauroylethylphosphatidylcholine (EDLPC) are known to be far more efficient agents in transfection of cultured primary endothelial cells than are lipoplexes containing either lipid alone. The large magnitude of the synergy permits comparison of the physical and physico-chemical properties of lipoplexes that have very different transfection efficiencies, but minor chemical differences. Here we report that the superior transfection efficiency of the EDLPC/EDOPC lipoplexes correlates with higher surface activity, higher affinity to interact and mix with negatively charged membrane-mimicking liposomes, and with considerably more efficient DNA release relative to the EDOPC lipoplexes. Observations on cultured cells agree with the results obtained with model systems; confocal microscopy of transfected human umbilical artery endothelial cells (HUAEC) demonstrated more extensive DNA release into the cytoplasm and nucleoplasm for the EDLPC/EDOPC lipoplexes than for EDOPC lipoplexes; electron microscopy of cells fixed and embedded directly on the culture dish revealed contact of EDLPC/EDOPC lipoplexes with various cellular membranes, including those of the endoplasmic reticulum, mitochondria and nucleus. The sequence of events outlining efficient lipofection is discussed based on the presented data.     

Lipoplex formulation of superior efficacy exhibits high surface activity and fusogenicity, and readily releases DNA

Lipoplexes containing a mixture of cationic phospholipids dioleoylethylphosphatidylcholine (EDOPC) and dilauroylethylphosphatidylcholine (EDLPC) are known to be far more efficient agents in transfection of cultured primary endothelial cells than are lipoplexes containing either lipid alone. The large magnitude of the synergy permits comparison of the physical and physico-chemical properties of lipoplexes that have very different transfection efficiencies, but minor chemical differences. Here we report that the superior transfection efficiency of the EDLPC/EDOPC lipoplexes correlates with higher surface activity, higher affinity to interact and mix with negatively charged membrane-mimicking liposomes, and with considerably more efficient DNA release relative to the EDOPC lipoplexes. Observations on cultured cells agree with the results obtained with model systems; confocal microscopy of transfected human umbilical artery endothelial cells (HUAEC) demonstrated more extensive DNA release into the cytoplasm and nucleoplasm for the EDLPC/EDOPC lipoplexes than for EDOPC lipoplexes; electron microscopy of cells fixed and embedded directly on the culture dish revealed contact of EDLPC/EDOPC lipoplexes with various cellular membranes, including those of the endoplasmic reticulum, mitochondria and nucleus. The sequence of events outlining efficient lipofection is discussed based on the presented data.     

Dendritic cationic lipids with highly charged headgroups for efficient gene delivery

Gene therapy is expected to lead to powerful new approaches for curing many diseases, a potential that is currently explored in worldwide clinical trials. Nonviral DNA delivery systems are desirable to overcome the inherent problems of viral vectors, but their current efficiency requires improvement and the understanding of their mechanism of action is incomplete. We have synthesized new multivalent cationic lipids with highly charged dendritic headgroups to probe the structure-transfection efficiency relationships of cationic liposome (CL)-DNA complexes, a prevalent nonviral vector. The lipid headgroups are constructed from ornithine cores and ornithine or carboxyspermine endgroups. The dendritic lipids were prepared on a gram scale, using a synthetic scheme that permits facile variation of the lipid building blocks headgroup, spacer, and hydrophobic moiety. They carry four to sixteen positive charges in their headgroups. Complexes of DNA with mixtures of the dendritic lipids and neutral 1,2-dioleoyl-sn-glycero phosphatidylcholine (DOPC) exhibit novel structures at high contents of the highly charged lipids, while the well-known lamellar phase is formed at high contents of DOPC. DNA complexes of the new dendritic lipids efficiently transfect mammalian cells in culture without cytotoxicity and, in contrast to lamellar complexes, maintain high transfection efficiency over a broad range of composition.     

Synthesis and evaluation of vitamin D-based cationic lipids for gene delivery in vitro

A new panel of steroidal cationic lipids has been synthesized for gene delivery. Using commercially available vitamin D2 (calciferol) or vitamin D3 (cholecalciferol) as hydrophobic motifs and a variety of cationic head groups as binding sites for negatively charged phosphate groups in DNA, we demonstrated that the transfection activity of the synthetic vitamin D-based cationic lipids 1d, 2d formulated with dioleoylphosphatidylethanolamine (DOPE) as a co-lipid is comparable to that of 3-(-[N-N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol). These synthetic lipids are effective in transfecting a variety of cell lines. These results suggest that vitamin D-based cationic lipids are useful transfection reagents for in vitro gene transfer studies.     

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.     

Synthesis and gene transfer activities of novel serum compatible cholesterol-based gemini lipids possessing oxyethylene-type spacers

Four novel cholesterol-based gemini cationic lipids differing in the length of oxyethylene-type spacers [-CH2-(CH2-O-CH2)n-CH2-] between each ammonium headgroup have been synthesized. These formed stable suspensions in aqueous media. Cationic liposomes were prepared from each of these lipids individually and as mixtures of cationic lipid and DOPE. These were used as nonviral gene delivery agents. All the cholesterol-based gemini lipids induced better transfection activity than their monomeric counterpart. Inclusion of DOPE in co-liposomal formulation of the cationic gemini lipid potentiates their gene transfer activity significantly. A major characteristic feature of these oxyethylene spacer based cholesterol gemini lipids was that serum does not inhibit the transfection activity of these gemini lipids, whereas the transfection activity of their monomeric counterpart decreased drastically in the presence of serum. One of the cholesterol-based gemini lipids 2a possessing a -CH2-CH2-O-CH2-CH2- spacer showed the highest transfection activity.     

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.     

Characterisation of three novel cationic lipids as liposomal complexes with DNA

Cationic lipids (CLs) are being increasingly exploited as transfection vectors for the delivery of DNA into eukaryotic cells. To obtain further insight to the complex formation and interactions between cationic liposomes and DNA, we characterised three novel cationic lipids, viz. bis[2-(11-phenoxyundecanoate)ethyl]-dimethylammonium bromide, N-hexadecyl-N-?10-[O-(4-acetoxy)-phenylundecanoate]ethyl?- dimethylammonium bromide, and bis[2-(11-butyloxyundecanoate)ethyl]dimethylammonium bromide. These lipids bear the same charged headgroup yet have different hydrophobic parts. Accordingly, we may anticipate their electrostatic interactions with DNA to be similar while differing in both thermal phase behaviour and physicochemical properties of their complexes with DNA. In keeping with the above all three lipids formed complexes with DNA as evidenced by light scattering, fluorescence spectroscopy and Langmuir film balance. Differential scanning calorimetry revealed very different phase behaviours for the binary mixtures of the three CLs with dimyristoylphosphatidylcholine and also provided evidence for DNA-induced lipid phase separation. These data were confirmed by compression isotherms and fluorescence microscopy of monolayers residing on an aqueous buffer, recorded both in the presence and absence of DNA. Importantly, binding to cationic liposomes appears to prevent thermal denaturation of DNA upon heating of the complexes. Likewise, renaturation of heat-treated DNA complexed with the cationic liposomes appears to be abolished as well.     

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.     

Novel imidazole-functionalized cyclen cationic lipids: Synthesis and application as non-viral gene vectors

A series of novel 1,4,7,10-tetraazacyclododecanes (cyclen)-based cationic lipids bearing histidine imidazole group 10a–10e were synthesized. These amphiphilic molecules have different hydrophobic tails (long chain, cholesterol or α-tocopherol) and various type of linking groups (ether, carbamate or ester). These molecules were used as non-viral gene delivery vectors, and their structure–activity relationships were investigated. As expected, the imidazole group could largely improve the buffering capabilities comparing to cyclen. The liposomes formed from 10 and dioleoylphosphatidyl ethanolamine (DOPE) could bind and condense plasmid DNA into nanoparticles with proper size and zeta-potentials. Comparing with Lipofectamine 2000, the formed lipoplexes gave lower transfected cells proportion, but higher fluorescence intensity, indicating their good intracellular delivering ability. Furthermore, results indicate that transfection efficiency of the cationic lipids is influenced by not only the hydrophobic tails but also the linking group. The cyclen-based cationic lipid with α-tocopherol hydrophobic tail and an ester linkage could give the highest transfection efficiency in the presence of serum.     

Basis for selectivity of cationic antimicrobial peptides for bacterial versus mammalian membranes

Novel cationic antimicrobial peptides typified by structures such as KKKKKKAAXAAWAAXAA-NH2, where X = Phe/Trp, and several of their analogues display high activity against a variety of bacteria but exhibit no hemolytic activity even at high dose levels in mammalian erythrocytes. To elucidate their mechanism of action and source of selectivity for bacterial membranes, phospholipid mixtures mimicking the compositions of natural bacterial membranes (containing anionic lipids) and mammalian membranes (containing zwitterionic lipids + cholesterol) were challenged with the peptides. We found that peptides readily inserted into bacterial lipid mixtures, although no insertion was detected in model "mammalian" membranes. The depth of peptide insertion into model bacterial membranes was estimated by Trp fluorescence quenching using doxyl groups variably positioned along the phospholipid acyl chains. Peptide antimicrobial activity generally increased with increasing depth of peptide insertion. The overall results, in conjunction with molecular modeling, support an initial electrostatic interaction step in which bacterial membranes attract and bind peptide dimers onto the bacterial surface, followed by the "sinking" of the hydrophobic core segment to a peptide sequence-dependent depth of approximately 2.5-8 A into the membrane, largely parallel to the membrane surface. Antimicrobial activity was likely enhanced by the fact that the peptide sequences contain AXXXA sequence motifs, which promote their dimerization, and possibly higher oligomerization, as assessed by SDS-polyacrylamide gel analysis and fluorescence resonance energy transfer experiments. The high selectivity of these peptides for nonmammalian membranes, combined with their activity toward a wide spectrum of Gram-negative and Gram-positive bacteria and yeast, while retaining water solubility, represent significant advantages of this class of peptides.     

Membrane perturbation and the mechanism of lipid-mediated transfer of DNA into cells

Mixtures of cationic lipids and unsaturated phosphatidylethanolamine are used extensively for the intracellular delivery of plasmids and antisense oligodeoxynucleotides (ODN) in vitro. However, the mechanism by which cytoplasmic delivery of these large molecules is achieved remains unclear. The common hypothesis is that phosphatidylethanolamine promotes fusion of lipid/DNA particles with endosomal membranes, but this is inconsistent with several reports that have failed to correlate the fusogenic activity of a wide variety of lipid/DNA particles, measured by lipid mixing techniques, with their transfection activity. To address this issue further we have conducted a detailed analysis of the lipid mixing and DNA transfer activity of two, physically similar but functionally different, lipid/DNA particles composed of equimolar dioleyldimethylammonium chloride (DODAC) and dioleoylphosphatidylethanolamine (DOPE) or dioleoylphosphatidylcholine (DOPC). In combination with DODAC both phospholipids form almost identical lipid/DNA particles, they are endocytosed by cells to the same extent and each undergoes equivalent lipid mixing with cell membranes after uptake. Despite this, DNA transfer is 10- to 100-fold more extensive for lipid/DNA particles containing DOPE. We conclude that lipid mixing between lipid-based delivery systems and endosomal membranes must occur for DNA transfer to occur. However, the potency of different lipid/DNA particles correlates better with the ability of the exogenous lipid to disrupt membrane integrity.     

Factors governing the assembly of cationic phospholipid-DNA complexes

The interaction of DNA with a novel cationic phospholipid transfection reagent, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC), was investigated by monitoring thermal effects, particle size, vesicle rupture, and lipid mixing. By isothermal titration calorimetry, the heat of interaction between large unilamellar EDOPC vesicles and plasmid DNA was endothermic at both physiological and low ionic strength, although the heat absorbed was slightly larger at the higher ionic strength. The energetic driving force for DNA-EDOPC association is thus an increase in entropy, presumably due to release of counterions and water. The estimated minimum entropy gain per released counterion was 1.4 cal/mole- degrees K (about 0.7 kT), consistent with previous theoretical predictions. All experimental approaches revealed significant differences in the DNA-lipid particle, depending upon whether complexes were formed by the addition of DNA to lipid or vice versa. When EDOPC vesicles were titrated with DNA at physiological ionic strength, particle size increased, vesicles ruptured, and membrane lipids became mixed as the amount of DNA was added up to a 1.6:1 (+:-) charge ratio. This charge ratio also corresponded to the calorimetric end point. In contrast, when lipid was added to DNA, vesicles remained separate and intact until a charge ratio of 1:1 (+:-) was exceeded. Under such conditions, the calorimetric end point was 3:1 (+:-). Thus it is clear that fundamental differences in DNA-cationic lipid complexes exist, depending upon their mode of formation. A model is proposed to explain the major differences between these two situations. Significant effects of ionic strength were observed; these are rationalized in terms of the model. The implications of the analysis are that considerable control can be exerted over the structure of the complex by exploiting vectorial preparation methods and manipulating ionic strength.     

Cationic carbosilane dendrimers-lipid membrane interactions

The aim of this work was to study interactions between cationic carbosilane dendrimers (CBS) and lipid bilayers or monolayers. Two kinds of second generation carbosilane dendrimers were used: NN16 with Si-O bonds and BDBR0011 with Si-C bonds. The results show that cationic carbosilane dendrimers interact both with liposomes and lipid monolayers. Interactions were stronger for negatively charged membranes and high concentration of dendrimers. In liposomes interactions were studied by measuring fluorescence anisotropy changes of fluorescent labels incorporated into the bilayer. An increase in fluorescence anisotropy was observed for both fluorescent probes when dendrimers were added to lipids that means the decreased membrane fluidity. Both the hydrophobic and hydrophilic parts of liposome bilayers became more rigid. This may be due to dendrimers' incorporation into liposome bilayer. For higher concentrations of both dendrimers precipitation occurred in negatively charged liposomes. NN16 dendrimer interacted stronger with hydrophilic part of bilayers whereas BDBR0011 greatly modified the hydrophobic area. Monolayers method brought similar results. Both dendrimers influenced lipid monolayers and changed surface pressure. For negatively charged lipids the monitored parameter changed stronger than for uncharged DMPC lipids. Moreover, NN16 dendrimer interacted stronger than the BDBR0011.     

DNA release from lipoplexes by anionic lipids: correlation with lipid mesomorphism, interfacial curvature, and membrane fusion

DNA release from lipoplexes is an essential step during lipofection and is probably a result of charge neutralization by cellular anionic lipids. As a model system to test this possibility, fluorescence resonance energy transfer between DNA and lipid covalently labeled with Cy3 and BODIPY, respectively, was used to monitor the release of DNA from lipid surfaces induced by anionic liposomes. The separation of DNA from lipid measured this way was considerably slower and less complete than that estimated with noncovalently labeled DNA, and depends on the lipid composition of both lipoplexes and anionic liposomes. This result was confirmed by centrifugal separation of released DNA and lipid. X-ray diffraction revealed a clear correlation of the DNA release capacity of the anionic lipids with the interfacial curvature of the mesomorphic structures developed when the anionic and cationic liposomes were mixed. DNA release also correlated with the rate of fusion of anionic liposomes with lipoplexes. It is concluded that the tendency to fuse and the phase preference of the mixed lipid membranes are key factors for the rate and extent of DNA release. The approach presented emphasizes the importance of the lipid composition of both lipoplexes and target membranes and suggests optimal transfection may be obtained by tailoring lipoplex composition to the lipid composition of target cells.     

Oxazole yellow homodimer YOYO-1-labeled DNA: a fluorescent complex that can be used to assess structural changes in DNA following formation and cellular delivery of cationic lipid DNA complexes.

To improve transfection efficiency following delivery of plasmid expression vectors using lipid-based carriers, it is crucial to define structural characteristics of the lipid/DNA complexes that optimize transgene expression. Due to its strong affinity for DNA and high quantum yield, the fluorescent DNA intercalator YOYO-1 was used as a tool to assess changes in DNA that occur following lipid binding and cell delivery. In this study, the stability of the dye/DNA complex following binding of poly-L-lysine or monocationic lipids is characterized. More than 98% of the fluorescence measured for a defined DNA/YOYO-1 complex was lost when DNA was condensed using poly-L-lysine. This loss in fluorescence could be attributed to displacement of bound dye. In contrast, more than 30% of the fluorescence of the dye-labeled DNA was retained after formation of cationic lipid/DNA complexes. Significantly, the results illustrate differences in structural changes cationic lipids and PLL exert on plasmid DNA. The fluorescent lipid/DNA complex was used to assess DNA delivery to murine B16/BL6 cells in vitro. An assay relying on fluorescence resonance energy transfer between bound YOYO-1 and propidium iodide was used to distinguish between DNA attached to the cell surface and internalized DNA.     

Photoenhancement of transfection efficiency using novel cationic lipids having a photocleavable spacer

New cationic lipids having an o-nitrobenzyl moiety as a photocleavable spacer between its hydrophilic and hydrophobic region were synthesized. To improve the efficiency of transfection with lipoplexes, after transfecting the cationic lipid aggregate/DNA complex, photoirradiation was performed. Photochemical decomposition of lipids would not only make the vector's membrane unstable to facilitate the fusion with endocytic vesicles, but also promote dissociation of cationic lipid-DNA complex, thus aiding the escape of DNA from the endocytic vesicles. Using a luciferase gene as a model, we show that UV irradiation of photoresponsive lipoplex-treated COS-1 cells induces a substantial increase in the efficiency of transfection. Herein, we show a novel photoresponsive gene delivery system.     

Mixtures of cationic lipid O-ethylphosphatidylcholine with membrane lipids and DNA: phase diagrams

Ethylphosphatidylcholines are positively charged membrane lipid derivatives, which effectively transfect DNA into cells and are metabolized by the cells. For this reason, they are promising nonviral transfection agents. With the aim of revealing the kinds of lipid phases that may arise when lipoplexes interact with cellular lipids during DNA transfection, temperature-composition phase diagrams of mixtures of the O-ethyldipalmitoylphosphatidylcholine with representatives of the major lipid classes (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, cholesterol) were constructed. Phase boundaries were determined using differential scanning calorimetry and synchrotron x-ray diffraction. The effects of ionic strength and of DNA presence were examined. A large variety of polymorphic and mesomorphic structures were observed. Surprisingly, marked enhancement of the affinity for nonlamellar phases was observed in mixtures with phosphatidylethanolamine and cholesterol as well as with phosphatidylglycerol (previously reported). Because of the potential relevance to transfection, it is noteworthy that such phases form at close to physiological conditions, and in the presence of DNA. All four mixtures exhibit a tendency to molecular clustering in the gel phase, presumably due to the specific interdigitated molecular arrangement of the O-ethyldipalmitoylphosphatidylcholine gel bilayers. It is evident that a remarkably broad array of lipid phases could arise in transfected cells and that these could have significant effects on transfection efficiency. The data may be particularly useful for selecting possible "helper" lipids in the lipoplex formulations, and in searches for correlations between lipoplex structure and transfection activity.