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.     

The synthesis of cholesterol-based cationic lipids with trimethylamine head and the effect of spacer structures on transfection efficiency

Five cholesterol-based cationic lipids were newly synthesized based on cholest-5-en-3β-oxyethane-N,N,N-trimethylammonium bromide (Chol-ETA) structure where the cholesterol backbone is linked to cationic head via various lengths of ether-linked carbon spacer. The transfection efficiency of these compounds was increased in order of three (Chol-PRO) < four (Chol-BTA) < two (Chol-ETA) methylene unit in their spacer, and was decreased by an addition of isomethyl group to Chol-PRO spacer. In case of the presence of multiple bonds in the spacer, it required the more cationic lipids in liposome formulation than single bond in the spacer to present similar transfection efficiency.     

In vitro delivery of curcumin with cholesterol-based cationic liposomes

A new cholesterol-based cationic lipid was synthesized; liposomes prepared on its basis were evaluated as drug delivery vehicles for curcumin. Free and liposome-encapsulated curcumin cytotoxicity against HeLa, A549, HepG2, K562 and 1301 cell lines was assessed. Liposomal curcumin with ED₅₀ values ranging from 2.5–10 μM exhibited 2–8 times higher cytotoxicity than free curcumin. The synthetic cholesterol-based cationic lipid also enhanced cellular uptake of curcumin into tested cells. Cationic liposome alone showed low cytotoxicity at high doses with ED₅₀ values of 90–210 μM.     

Solid phase synthesis of novel asymmetric hydrophilic head cholesterol-based cationic lipids with potential DNA delivery

Twenty-four asymmetric divalent head group cholesterol-based cationic lipids were designed and synthesized by parallel solid phase chemistry. These asymmetric head groups composed of amino functionality together with trimethylamino, di(2-hydroxyethyl)amino or guanidinyl groups. Spacers between cationic heads and linker were both equal and unequal in length. These lipids were subjected to evaluation for DNA binding affinities by gel retardation assay and were screened for their transfection efficiency on HEK293 cells. Cationic lipids with equal chain length exhibited high transfection efficiency when polar part contained asymmetric polar heads. In contrast, lipids with unequal chain length exhibited high transfection efficiency when polar part contained symmetric heads. According to the optimal formulation, seven lipids exhibited higher transfection efficiency than the commercially available transfection agents, Effectene?, DOTAP and DC-Chol, to deliver DNA into PC3 human prostate adenocarcinoma cells. 3β-[N-(N′-Guanidinyl)-2′-aminoethyl)-N-(2-aminoethyl)carbamoyl] cholesterol (5) bearing amino and guanidinyl polar heads exhibited highest transfection efficiency with minimal toxicity. The morphology of active liposomes was observed by transmission electron microscopy (TEM) and size of liposomes were around 200–700 nm.     

Anchoring and bola cationic amphiphiles for nucleotide delivery. Effects of orientation and extension of hydrophobic regions.

Novel cationic amphiphiles, based on lithocholic acid derivatives with two structural motifs, anchoring lipids and bola lipids, were designed and synthesized. Both bear extended hydrophobic space-filling substituents. A significant effect of the orientation and extension of hydrophobic regions around the ether linkage at the 3-position was found on the efficiency of DNA delivery.     

Physicochemical characteristics associated with transfection of cationic cholesterol-based gene delivery vectors in the presence of DOPE

The physicochemical properties of a novel series of cholesterol-based cationic lipids in the presence of DOPE were studied by various techniques in an effort to correlate cationic lipid structure with transfection efficacy. It was found that while DOPE improves the β-gal activity of the active AC and MC derivatives, the overall zeta potential of the particles, pDNA complexation and condensation is not improved. This is in stark contrast with the tertiary amine derivative DC whose dispersion properties were improved and its monolayer surface potential is restored at high molecular surface density in the presence of DOPE. Overall the transfection activity mediated by DC and the quaternary ammonium TC derivative was greatly improved in the presence of DOPE and is attributed to decreased cytotoxicity, improved fusogenicity and cellular association.     

Cellular pathway of plasmids vectorized by cholesterol-based cationic liposomes.

We investigated by transmission electron microscopy the cellular route in tumor MCF7 cells of DNA labeled with digoxigenin, carried by cationic liposomes (Lip+) prepared from TMAEC-Chol [3 beta(N-(N',N',N'-trimethylaminoethane)-carbamoyl)cholesterol iodide] and TEAPC-Chol [3 beta(N-(N',N',N'-triethylaminopropane)-carbamoyl)cholesterol iodide], two cholesterol-based cationic lipids containing a quaternary ammonium. In a previous work we showed the pathway of cationic lipid/plasmid complexes from the beginning of endocytosis until their entry into the perinuclear area. Beyond this limit, unlabeled exogenous plasmids cannot be distinguished with nuclear DNA. This work dealt with the cellular fate of cationic liposome-vectorized plasmids labeled with digoxigenin using an immunogold procedure. Early after the beginning of transfection (30 min, 1 hr, 5 hr), gold particles were observed only in the cytoplasm and in endosome-like vesicles, whereas after 24 hr gold particles were densely present in the nucleus. These results demonstrate the nuclear localization of plasmids vectorized by the cationic liposomes used. The results are discussed in comparison with transfection efficiency measurements.     

Synthesis and structure-activity relationships of a series of increasingly hydrophobic cationic steroid lipofection reagents

The use of cholesterol-based cationic lipids and the ability of glucocorticoids to reduce local inflammatory response to lipoplexes motivated an investigation of structure-activity relationships for cationic steroids. A one-step synthetic scheme using iminothiolane was developed to link spermine to the 21-OH position of steroids via an amidine linkage. Five steroids (cortisol, dexamethasone, corticosterone, 11-deoxycortisol, and 11-deoxycorticosterone) with increasing hydrophobicity of the parent steroid (Log P(ster) from 1.51 to 3.01) were conjugated with spermine, formulated with dioleoylphosphatidylethanolamine (DOPE) at DOPE : steroid mole ratios (R) of R = 0.5 to 2, and then complexed with 1 microg enhanced green fluorescent protein (EGFP) plasmid DNA at charge ratios (CR) = 2 to 24 amines per phosphate (0.5 to 6 steroids per phosphate). The resulting 105 different formulations of the cationic steroid series were used to lipofect bovine aortic endothelial cells. Transgene expression data at either 24 or 48 h post-lipofection for all formulations was collapsed onto master curves when plotted against a single empirical dimensionless parameter, the lipofection index (LI) = CR (Log P(liposome))(Log P(ster)/|DeltaLog P|) [R/(R + 1)] where DeltaLog P = Log P(DOPE)- Log P(ster) and Log P(liposome) is a mole-weighted average of the DOPE/cationic steroid liposome hydrophobicity. For 7 < LI < 29, the EGFP expression at 24 or 48 h post-lipofection increased linearly with LI (EGFP approximately 0 for LI < 7), but did not increase further for LI > 29, thus providing a predictive design rule based on Log P of the hydrophobic moiety of the cationic steroid lipid.     

Effect of the headgroup variation on the gene transfer properties of cholesterol based cationic lipids possessing ether linkage

Eight cholesterol based cationic lipids differing in the headgroup have been synthesized based on the ether linkage between the cationic headgroup and the cholesterol backbone. All the lipids formed stable suspensions in water. Transfection efficacies were examined in the absence and presence of serum using their optimized liposomal (lipid:DOPE) formulations. Our results showed that the transfection activities depend on the nature of the headgroup. Lipid bearing 4-N,N'-dimethylaminopyridine (DMAP) as headgroup showed the maximum transfection efficacy in the presence of serum. Importantly, the optimized formulation for this cationic lipid does not require DOPE, which is being used by most commercially available formulations. Cytotoxicity studies showed that the introduction of the positive charge decreases the cell viability of the cationic lipid formulations. Gel electrophoresis and Ethidium bromide exclusion assay revealed the different DNA binding abilities of formulations depending upon the headgroup of the cholesteryl lipid.     

Cationic oxyethylene lipids. Synthesis, aggregation, and transfection properties

Four cationic lipids (1-4) with oligo-oxyethylene units at the linkage region between the pseudoglyceryl backbone and the hydrocarbon chains have been synthesized. Two of these lipids (1 and 2) have an equal number of (CH(2)CH(2)O)(n)() units attached to both C-1 and C-2 positions of the pseudoglyceryl backbone, making their linkage regions similar, while the other two (3 and 4) are unsymmetrical in terms of the number of oxyethylene units in the linkage. Synthesis of lipids 1 and 2 involved the coupling of benzyl glycerol with the corresponding tosylates as a key step. Each of these lipids formed membranous aggregates when dispersed in water and exhibited clear thermotropic phase transitions typical of vesicular assemblies. The lipids 1-4 exhibited enhanced biological activities as gene transfer agents compared to their non-oxyethylene diether analogue, DHTMA. Transfection experiments using aqueous suspensions of these lipids and also their mixtures with cholesterol or dioleoyl phosphatidyl ethanolamine (DOPE) were performed on HeLa cells. The best transfection activity was demonstrated by unsymmetrical lipid 3, which had two oxyethylene units only at the C-1 position of the pseudoglycerylbackbone.     

Advantage of the ether linkage between the positive charge and the cholesteryl skeleton in cholesterol-based amphiphiles as vectors for gene delivery

Twelve novel cationic cholesterol derivatives with different linkage types between the cationic headgroup and the cholesteryl backbone have been developed. These have been tested for their efficacies as gene transfer agents as mixtures with dioleoyl phosphatidylethanolamine (DOPE). A pronounced improvement in transfection efficiency was observed when the cationic center was linked to the steroid backbone using an ether type bond. Among these, cholest-5-en-3b-oxyethane-N,N,N-trimethylammonium bromide (2a) and cholest-5-en-3b-oxyethane-N,N-dimethyl-N-2-hydroxyethylammonium bromide (3d) showed transfection efficiencies considerably greater than commercially available reagents such as Lipofectin or Lipofectamine. To achieve transfection, 3d did not require DOPE. Increasing hydration at the headgroup level for both ester- and ether-linked amphiphiles resulted in progressive loss of transfection efficiency. Transfection efficiency was also greatly reduced when a 'disorder'-inducing chain like an oleyl (cis-9-octadecenyl) segment was added to these cholesteryl amphiphiles. Importantly, the transfection ability of 2a with DOPE in the presence of serum was significantly greater than for a commercially available reagent, Lipofectamine. This suggests that these novel cholesterol-based amphiphiles might prove promising in applications involving liposome-mediated gene transfection. This investigation demonstrates the importance of structural features at the molecular level for the design of cholesterol-based gene delivery reagents that would aid the development of newer, more efficient formulations based on this class of molecules.     

Effect of the headgroup variation on the gene transfer properties of cholesterol based cationic lipids possessing ether linkage

Eight cholesterol based cationic lipids differing in the headgroup have been synthesized based on the ether linkage between the cationic headgroup and the cholesterol backbone. All the lipids formed stable suspensions in water. Transfection efficacies were examined in the absence and presence of serum using their optimized liposomal (lipid:DOPE) formulations. Our results showed that the transfection activities depend on the nature of the headgroup. Lipid bearing 4-N,N′-dimethylaminopyridine (DMAP) as headgroup showed the maximum transfection efficacy in the presence of serum. Importantly, the optimized formulation for this cationic lipid does not require DOPE, which is being used by most commercially available formulations. Cytotoxicity studies showed that the introduction of the positive charge decreases the cell viability of the cationic lipid formulations. Gel electrophoresis and Ethidium bromide exclusion assay revealed the different DNA binding abilities of formulations depending upon the headgroup of the cholesteryl lipid.     

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.     

Synthesis and Transfection Efficiencies of Divalent Ammonium Headgroup Cationic Lipids with Different Hydrophobic Tails

Russian Journal of Bioorganic Chemistry - Cationic lipids with amide or carbamate linker and divalent cationic headgroups were synthesized for a transfection study. Sixteen cationic lipids with...     

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.     

Gene-transferring efficiencies of novel diamino cationic lipids with varied hydrocarbon chains

Utilizing three biocompatible components, a series of novel cationic lipids has been chemically synthesized and tested for their gene-transferring capabilities in 293 transformed kidney cells and B16BL6 mouse melanoma cells. The synthesized cationic lipids consisting of a core of lysine and aspartic acid with hydrocarbon chains of varied length were assigned the acronyms DLKD (O,O'-dilauryl N-lysylaspartate), DMKD (O,O'-dimyristyl N-lysylaspartate), DPKD (O,O'-dipalmityl N-lysylaspartate), and DSKD (O,O'-distearyl N-lysylaspartate). The gene-transferring capabilities of these cationic lipids were found to be dependent on the hydrocarbon chain length. Under similar experimental conditions, the order of gene transfection efficiency was DMKD > DLKD > DPKD > DSKD. Addition of cholesterol or dioleoyl phosphatidylethanolamine (DOPE) as a colipid did not change this order. Colipid addition affected the transfection efficiency positively or negatively depending on the length of the cationic lipid acyl chain. On the whole, the length of the hydrophobic carbon chain was a major factor governing the gene-transferring capabilities of this series of cationic lipids. The observed differences in transfection efficiency may be due to differing binding affinities to DNA molecules as well as differences in the surface charge potential of the liposome-DNA complexes (lipoplexes) in the aqueous environment.     

Synthesis of diether-linked cationic lipids for gene delivery.

Quaternary ammonium lipids 1b-d, with diether linkages between hydrocarbon chains and butane or hexane backbone, were synthesized for cationic liposome-mediated gene delivery. The synthetic strategy of using C-4 or C-6 synthon permits the achievement of the variation of the hydrophobic domain as well as changes of space between the quaternary ammonium head and the hydrophobic domain in the diether-linked cationic lipids.     

New cationic lipids for gene transfer with high efficiency and low toxicity: T-shape cholesterol ester derivatives

New degradable cationic ester lipids with 'T-shape' configurations were synthesized and tested for gene delivery carrier. Their transfection efficiency and toxicity were compared with commercially available cationic lipids, DOTMA, DOSPA, and DC-Chol. They showed efficient transfection activity and almost no toxicity on mammalian cell lines. Their ester bond degradation was monitored by (1)H NMR.     

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.     

Synthesis and in vitro evaluation of glutamide-containing cationic lipids for gene delivery

A novel series of cationic amphiphiles based on dialkyl glutamides with cationic pyridinium head group were synthesized as potential gene delivery agents. Four cationic lipids with glutamide as linker and varying chain lengths were tested for their transfection efficiency in three cell lines. The DNA-lipid complexes were characterized for their ability to bind to DNA, protection from nuclease digestion, size, zeta-potential, and toxicity. All four lipids demonstrated efficient transfection in MCF-7, COS, and HeLa cells, and the reporter gene expression was much higher with DOPE as the helper lipid in the formulation when compared to cholesterol. Among these 14-carbon lipids, lipid 2 has shown the highest transfection efficiency, complete protection of DNA from nuclease digestion, and low toxicity. Interestingly, lipid 2 has also shown remarkable enhancement in transfection in the presence of serum.