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.     

Immunostimulation of dendritic cells by cationic liposomes

A nano-aggregate liposome-polycation-DNA (LPD), composed of a cationic lipid, protamine and plasmid DNA was found to effectively deliver a human papillomavirus (HPV)-E7 epitope antigen to the antigen presenting cells of the immune system, eliciting enhanced anti-tumor immune responses in mouse models of cervical carcinoma. Both the cationic liposome and plasmid DNA were essential for the full immunostimulation activity of LPD. Interestingly, cationic liposomes alone could stimulate the antigen presenting dendritic cells (DC) leading to the expression of co-stimulatory molecules, CD80 and CD86. However, cationic lipids could not stimulate DC for the expression of pro-inflammatory cytokines. Moreover, they were unable to enhance the expression of NF-κB, suggesting that dendritic cells stimulation by cationic lipids is signaled through an NF-κB independent mechanism. DC stimulation was specific to cationic lipids, the zwitterionic and anionic lipids showed little or no activity. The ability of different cationic lipids to stimulate the expression of co-stimulatory molecules on DC varied significantly. In general, the cationic lipids bearing ethyl phosphocholine head groups were better stimulants than their trimethylammonium counterparts. In case of the cationic lipids bearing trimethyl ammonium head groups, the ones bearing unsaturated or shorter saturated hydrophobic chains exhibited enhanced immunostimulatory activity. The LPS-induced TNF-α expression by dendritic cells was inhibited by active cationic lipids but not the inactive ones, suggesting the possible involvement of lipopolysaccharide binding protein (LBP) in cationic lipid mediated DC stimulation. Based on the structure-specific activation of dendritic cells by cationic lipids, a model for the immunostimulation of DC by such lipids is proposed.     

Immunostimulation of dendritic cells by cationic liposomes

A nano-aggregate liposome-polycation-DNA (LPD), composed of a cationic lipid, protamine and plasmid DNA was found to effectively deliver a human papillomavirus (HPV)-E7 epitope antigen to the antigen presenting cells of the immune system, eliciting enhanced anti-tumor immune responses in mouse models of cervical carcinoma. Both the cationic liposome and plasmid DNA were essential for the full immunostimulation activity of LPD. Interestingly, cationic liposomes alone could stimulate the antigen presenting dendritic cells (DC) leading to the expression of co-stimulatory molecules, CD80 and CD86. However, cationic lipids could not stimulate DC for the expression of pro-inflammatory cytokines. Moreover, they were unable to enhance the expression of NF-kappaB, suggesting that dendritic cells stimulation by cationic lipids is signaled through an NF-kappaB independent mechanism. DC stimulation was specific to cationic lipids, the zwitterionic and anionic lipids showed little or no activity. The ability of different cationic lipids to stimulate the expression of co-stimulatory molecules on DC varied significantly. In general, the cationic lipids bearing ethyl phosphocholine head groups were better stimulants than their trimethylammonium counterparts. In case of the cationic lipids bearing trimethyl ammonium head groups, the ones bearing unsaturated or shorter saturated hydrophobic chains exhibited enhanced immunostimulatory activity. The LPS-induced TNF-alpha expression by dendritic cells was inhibited by active cationic lipids but not the inactive ones, suggesting the possible involvement of lipopolysaccharide binding protein (LBP) in cationic lipid mediated DC stimulation. Based on the structure-specific activation of dendritic cells by cationic lipids, a model for the immunostimulation of DC by such lipids is proposed.     

Synthesis and properties of novel tetraalkyl cationic lipids

The synthesis, physical properties, and transfection potencies of two representives of a new class of divalent, tetraalkyl cationic lipids is described. These cationic lipids are dimers of N,N-Dioleyl-N,N-dimethylammonium chloride (DODAC) joined by a hydrocarbon tether three or six carbons in length (TODMAC3 and TODMAC6, respectively). It is shown that TODMAC6 can display improved transfection properties in comparison to DODAC when formulated into plasmid DNA-cationic lipid complexes. These improved transfection potencies are observed at cationic lipid to DNA charge ratios of two or higher. It is also shown that TODMAC6 exhibits equivalent or improved ability (as compared to DODAC) to induce nonbilayer structure in mixtures with anionic lipid. This is consistent with the hypothesis that the ability of cationic lipids to induce nonbilayer structures when mixed with anionic lipids is correlated to their transfection potency. Complexes containing TODMAC3 on the other hand exhibit lower transfection potencies than achieved with DODAC, behavior that is consistent with steric effects limiting the formation of ion pairs with anionic lipids. It is concluded that TODMAC6 exhibits potential as a transfection agent for in vitro and in vivo use and that the design of cationic lipids according to their ability to induce nonbilayer structure provides a useful guide for synthesis of new cationic lipids.     

Cationic lipids and cationic ligands induce DNA helix denaturation: detection of single stranded regions by KMnO4 probing

Cationic lipids and cationic polymers are widely used in gene delivery. Using 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) as a cationic lipid, we have investigated the stability of the DNA in DOTAP:DNA complexes by probing with potassium permanganate (KMnO4). Interestingly, thymidines followed by a purine showed higher susceptibility to cationic ligand-mediated melting. Similar studies performed with other water-soluble cationic ligands such as polylysine, protamine sulfate and polyethyleneimine also demonstrated melting of the DNA but with variations. Small cations such as spermine and spermidine and a cationic detergent, cetyl trimethylammonium bromide, also rendered the DNA susceptible to modification by KMnO4. The data presented here provide direct proof for melting of DNA upon interaction with cationic lipids. Structural changes subsequent to binding of cationic lipids/ligands to DNA may lead to instability and formation of DNA bubbles in double-stranded DNA.     

Transfection activity of binary mixtures of cationic o-substituted phosphatidylcholine derivatives: the hydrophobic core strongly modulates physical properties and DNA delivery efficacy

A combination of two cationic lipid derivatives having the same headgroup but tails of different chain lengths has been shown to have considerably different transfection activity than do the separate molecules. Such findings point to the importance of investigating the hydrophobic portions of cationic amphiphiles. Hence, we have synthesized a variety of cationic phosphatidylcholines with unusual hydrophobic moieties and have evaluated their transfection activity and that of their mixtures with the original molecule of this class, dioleoyl-O-ethylphosphatidylcholine (EDOPC). Four distinct relationships between transfection activity and composition of the mixture (plotted as percent of the new compound added to EDOPC) were found, namely: with a maximum or minimum; with a proportional change; or with essentially no change. Relevant physical properties of the lipoplexes were also examined; specifically, membrane fusion (by fluorescence resonance energy transfer between cationic and anionic lipids) and DNA unbinding (measured as accessibility of DNA to ethidium bromide by electrophoresis and by fluorescence resonance energy transfer between DNA and cationic lipid), both after the addition of negatively charged membrane lipids. Fusibility increased with increasing content of second cationic lipid, regardless of the transfection pattern. However, the extent of DNA unbinding after addition of negatively charged membrane lipids did correlate with extent of transfection. The phase behavior of cationic lipids per se as well as that of their mixtures with membrane lipids revealed structural differences that may account for and support the hypothesis that a membrane lipid-triggered, lamellar-->nonlamellar phase transition that facilitates DNA release is critical to efficient transfection by cationic lipids.     

Induction of genuine autophagy by cationic lipids in mammalian cells

Autophagy is a cellular stress response that results in the activation of a lysosomal degradation pathway. In this report, we showed that cationic lipids, a common-used class of transfection reagents, induced genuine autophagy in mammalian cells. Extensive LC3 dot formation was observed by treatment with cationic lipids (with or without DNA), but not neutral lipids, in a HeLa cell line stably expressing GFP-LC3 (HeLa-LC3). Further proofs for autophagy were obtained by the co-localization of the LC3 dots with lysosome-specific staining patterns, observation of LC3-I to LC3-II form conversion and appearance of autophagic vacuoles under TEM. The autophagic flux assay with bafilomycin A1 and degradation of p62/SQSTM1 suggested that the autophagy induced by cationic lipids was primarily due to increased formation of autophagosomes and not decreased turnover. Moreover, cationic lipids induced autophagy in an mTOR-independent manner.     

Cell Biological and Biophysical Aspects of Lipid-mediated Gene Delivery

Cationic lipids are conceptually and methodologically simple tools to deliver nucleic acids into the cells. Strategies based on cationic lipids are viable alternatives to viral vectors and are becoming increasingly popular owing to their minimal toxicity. The first-generation cationic lipids were built around the quaternary nitrogen primarily for binding and condensing DNA. A large number of lipids with variations in the hydrophobic and hydrophilic region were generated with excellent transfection efficiencies in vitro. These cationic lipids had reduced efficiencies when tested for gene delivery in vivo. Efforts in the last decade delineated the cell biological basis of the cationic lipid gene delivery to a significant detail. The application of techniques such as small angle X-ray spectroscopy (SAXS) and fluorescence microscopy, helped in linking the physical properties of lipid:DNA complex (lipoplex) with its intracellular fate. This biological knowledge has been incorporated in the design of the second-generation cationic lipids. Lipid-peptide conjugates (peptoids) are effective strategies to overcome the various cellular barriers along with the lipoplex formulations methodologies. In this context, cationic lipid-mediated gene delivery is considerably benefited by the methodologies of liposome-mediated drug delivery. Lipid mediated gene delivery has an intrinsic advantage of being a biomimetic platform on which considerable variations could be built to develop efficient in vivo gene delivery protocols.     

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.     

Synthesis of novel cholesterol-based cationic lipids for gene delivery

The new cholesterol-based cationic lipids B, C, and D with an ether linked spacer were synthesized by using aminopropyl chain extension with acrylonitrile. The cholesterol-based cationic lipid A with carbamoyl linkage were also synthesized in order to compare the difference in transfection efficiency of the two linkage types. To this end, GFP expression of these cationic lipids was confirmed respectively.     

Nature as a source of inspiration for cationic lipid synthesis

Synthetic gene delivery systems represent an attractive alternative to viral vectors for DNA transfection. Cationic lipids are one of the most widely used non-viral vectors for the delivery of DNA into cultured cells and are easily synthesized, leading to a large variety of well-characterized molecules. This review discusses strategies for the design of efficient cationic lipids that overcome the critical barriers of in vitro transfection. A particular focus is placed on natural hydrophilic headgroups and lipophilic tails that have been used to synthesize biocompatible and non-toxic cationic lipids. We also present chemical features that have been investigated to enhance the transfection efficiency of cationic lipids by promoting the escape of lipoplexes from the endosomal compartment and DNA release from DNA-liposome complexes. Transfection efficiency studies using these strategies are likely to improve the understanding of the mechanism of cationic lipid-mediated gene delivery and to help the rational design of novel cationic lipids.     

Cationic lipid-mediated nucleic acid delivery: beyond being cationic

Realization of the potential of nucleic acids as drugs is intricately linked to their in vivo delivery. Cationic lipids demonstrated tremendous potential as safe, efficient and scalable in vitro carriers of nucleic acids. For in vivo delivery of nucleic acids, the extant two component liposomal preparations consisting of cationic lipids and nucleic acids have been largely found to be insufficient. Being a soft matter, liposomes readily respond to many physiological variables leading to complex component and morphological changes, thus confounding the efforts in a priori identification of a “competent” formulation. In the recent past many chemical moieties that provide advantage in facing the challenges of barriers in vivo, were incorporated into cationic lipids to improve the transfection efficiency. The cationic lipids, essential for DNA condensation and protection, definitely require additional components to be efficient in vivo. In addition, formulations of cationic lipid carriers with non-lipidic components, mainly peptides, have demonstrated success in in vivo transfection. The present review describes some recent successes of in vivo nucleic acid delivery by cationic lipids.     

Cationic liposomal lipids: from gene carriers to cell signaling

Cationic lipids are positively charged amphiphilic molecules which, for most of them, form positively charged liposomes, sometimes in combination with a neutral helper lipid. Such liposomes are mainly used as efficient DNA, RNA or protein carriers for gene therapy or immunization trials. Over the past decade, significant progress has been made in the understanding of the cellular pathways and mechanisms involved in lipoplex-mediated gene transfection but the interaction of cationic lipids with cell components and the consequences of such an interaction on cell physiology remains poorly described. The data reported in the present review provide evidence that cationic lipids are not just carriers for molecular delivery into cells but do modify cellular pathways and stimulate immune or anti-inflammatory responses. Considering the wide number of cationic lipids currently available and the variety of cellular components that could be involved, it is likely that only a few cationic lipid-dependent functions have been identified so far.     

Cationic lipids activate cellular cascades. Which receptors are involved?

Cationic lipids have been extensively used as carriers of biologically active molecules (nucleic acids, peptides and proteins) into cells. Recent data provided evidence that cationic lipids are not just inert transporters but do activate specific cellular cascades. This review illustrates these activating properties with a few examples. Cell activation raises the question of which receptors are involved. Some cationic lipids seem to satisfy specific structural requirements of Toll-like receptors (TLR4) as they activate TLR4-dependent pathways. However, cationic lipids display a large structural diversity and it is likely that they are also recognized by receptors with a broader specificity. Alternatives are proposed and discussed to explain this broad specificity.     

Synthesis and biological activity of carbamate-linked cationic lipids for gene delivery in vitro

We have introduced a convenient synthesis method for carbamate-linked cationic lipids. Two cationic lipids N-[1-(2,3-didodecylcarbamoyloxy)propyl]-N,N,N-trimethylammonium iodide (DDCTMA) and N-[1-(2,3-didodecyl carbamoyloxy)propyl]-N-ethyl-N,N-dimethylammonium iodide (DDCEDMA), with identical length of hydrocarbon chains, alternative quaternary ammonium heads, carbamate linkages between hydrocarbon chains and quaternary ammonium heads, were synthesized for liposome-mediated gene delivery. Liposomes composed of DDCEDMA and DOPE in 1:1 ratio exhibited a lower zeta potential as compared to those made of pure DDCEDMA alone, which influences their DNA-binding ability. pGFP-N2 plasmid was transferred by cationic liposomes formed from the above cationic lipids into Hela and Hep-2 cells, and the transfection efficiency of some of cationic liposomes was superior or parallel to that of two commercial transfection agents, Lipofectamine2000 and DOTAP. Combined with the results of the agarose gel electrophoresis and transfection experiment, the DNA-binding ability of cationic lipids was too strong to release DNA from complex in the transfection, which could lead to relative low transfection efficiency and high cytotoxicity.     

Tunable pH-sensitive liposomes composed of mixtures of cationic and anionic lipids

The pH-dependent fusion properties of large unilamellar vesicles (LUVs) composed of binary mixtures of anionic and cationic lipids have been investigated. It is shown that stable LUVs can be prepared from the ionizable anionic lipid cholesteryl hemisuccinate (CHEMS) and the permanently charged cationic lipid N,N-dioleoyl-N, N-dimethylammonium chloride (DODAC) at neutral pH values and that these LUVs undergo fusion as the pH is reduced. The critical pH at which fusion was observed (pH(f)) was dependent on the cationic lipid-to-anionic lipid ratio. LUVs prepared from DODAC/CHEMS mixtures at molar ratios of 0 to 0.85 resulted in vesicles with pH(f) values that ranged from pH 4.0 to 6.7, respectively. This behavior is consistent with a model in which fusion occurs at pH values such that the DODAC/CHEMS LUV surface charge is zero. Related behavior was observed for LUVs composed of the ionizable cationic lipid 3alpha-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol hydrochloride (DC-Chol) and the acidic lipid dioleoylphosphatidic acid (DOPA). Freeze-fracture and (31)P NMR evidence is presented which indicates that pH-dependent fusion results from a preference of mixtures of cationic and anionic lipid for "inverted" nonbilayer lipid phases under conditions where the surface charge is zero. It is concluded that tunable pH-sensitive LUVs composed of cationic and anionic lipids may be of utility for drug delivery applications. It is also suggested that the ability of cationic lipids to adopt inverted nonbilayer structures in combination with anionic lipids may be related to the ability of cationic lipids to facilitate the intracellular delivery of macromolecules.     

Gene transfer efficacies of novel cationic amphiphiles with alanine, beta-alanine, and serine headgroups: a structure-activity investigation

Herein, we report on the relative in vitro efficacies of nine novel non-glycerol based cationic amphiphiles with increasing hydrophobic tails and the amino acids serine, alanine and beta-alanine as the headgroup functionalities (lipids 1-9, Scheme 1) in transfecting multiple cultured cells including CHO, COS-1, MCF-7, and HepG2. The gene transfer efficiencies of lipids 1-9 were evaluated using the reporter gene assays in all the four cell lines and the whole cell histochemical X-gal staining assays in representative CHO cells. In CHO, HepG2, and MCF-7 cells, cationic lipids with alanine (4-6) and beta-alanine (7-9) headgroups were found to be remarkably more transfection efficient than their serine headgroup counterparts (1-3). Most notably, in CHO, HepG2, and MCF-7 cells, in combination with cholesterol as auxiliary lipid, the transfection efficiencies of the cationic lipids with alanine and beta-alanine headgroups and myristyl and palmityl tails (lipids 4, 5, 7 and 8) were significantly higher (2-3-fold) than that of LipofectAmine-2000, a widely used commercially available liposomal tranfection vectors. Surprisingly, in COS-1 cells, although cationic lipids with beta-alanine headgroups (7-9) were strikingly transfection efficient (3-4-fold more efficacious than LipofectAmine-2000), the gene transfer properties of both their structural isomers (4-6) and their serine headgroup counterparts (1-3) were adversely affected. In summary, the present structure-activity investigation demonstrate that high gene delivery efficacies of cationic amphiphiles containing alanine or beta-alanine headgroups can get seriously compromised by substituting the alanine or beta-alanine with serine presumably due to the enhanced sensitivity of DNA associated with such serine-head-containing cationic lipids.     

Surface charge markedly attenuates the nonlamellar phase-forming propensities of lipid bilayer membranes: calorimetric and (31)P-nuclear magnetic resonance studies of mixtures of cationic, anionic, and zwitterionic lipids

The lamellar/nonlamellar phase preferences of lipid model membranes composed of mixtures of several cationic lipids with various zwitterionic and anionic phospholipids were examined by a combination of differential scanning calorimetry and (31)P NMR spectroscopy. All of the cationic lipids utilized in this study form only lamellar phases in isolation. Mixtures of these cationic lipids with zwitterionic strongly lamellar phase-preferring lipids such as phosphatidylcholine form only the lamellar liquid-crystalline phase even at high temperatures, as expected. Moreover, mixtures of these cationic lipids with strongly nonlamellar phase-preferring zwitterionic lipids such as phosphatidylethanolamine exhibit a markedly reduced propensity to form inverted nonlamellar phases, again as expected. However, when mixed with anionic lipids such as phosphatidylserine, phosphatidylglycerol, cardiolipin, or phosphatidic acid, a marked enhancement of nonlamellar phase-forming propensity occurs, despite the fact both components of the mixture are nominally lamellar phase-preferring. An examination of the lamellar/nonlamellar phase transition temperatures and the nature of the nonlamellar phases formed, as a function of temperature and of the composition of the mixture, indicates that the propensity to form inverted nonlamellar phases is maximal in mixtures where the mean surface charge of the membrane surface approaches neutrality and decreases markedly with increases in the density of positive or negative charge at the membrane surface. Moreover, the onset temperatures of the reversed hexagonal phase rise more steeply than do those of the inverted cubic phase as the ratio of cationic and anionic lipids is varied, suggesting that the formation of inverted hexagonal phases is more sensitive to this surface charge effect. These results indicate that surface charge per se is a significant and effective modulator of the lamellar/nonlamellar phase preferences of membrane lipids and that charged group interactions at membrane surfaces may have a major role in regulating this particular membrane property.     

Carbamate-linked cationic lipids for gene delivery

Series of cationic lipids 1a-p, with variable length of hydrocarbon chains, alternative quaternary ammonium heads, carbamate linkages between hydrocarbon chains and quaternary ammonium heads, as well as different anion combined with them, were synthesized for liposome-mediated gene delivery. Two plasmid DNAs, pGL3-control and pGFP-N2, were transferred by cationic liposomes formed from the above cationic lipids into five mammalian cell lines, and the transfection efficiency of some of the cationic liposomes was superior or parallel to that of two commercial transfection agents, Lipofectamine2000 and Sofast.