Spacer structure and hydrophobicity influences transfection activity of novel polycationic gemini amphiphiles

Three novel polycationic gemini amphiphiles with different spacers were developed and evaluated in terms of their physiochemical properties and transfection efficiencies. Cationic liposomes formed by these amphiphiles and the helper lipid DOPE were able to successfully condense DNA, as shown by gel mobility shift and ethidium bromide intercalation assays. Transfection activity of the liposomes was superior to Lipofectamine^® 2000 and was dependent on spacer structure, hydrophobicity, and nucleic acid type (pDNA or siRNA). We demonstrated that the cationic liposomes 2X6/DOPE and 2X7/DOPE are potential non-toxic vehicles for gene delivery.     

[12]aneN3-based lipid with naphthalimide moiety for enhanced gene transfection efficiency

Three cationic lipids derived from [12]aneN₃ modified with naphthalimide (1a), oleic acid (1b) and octadecylamine (1c) were designed and synthesized. In vitro transfection showed that all these liposomes can deliver plasmid DNA into the tested cell lines. Among these liposomes, 1a gave the best transfection efficiency (TE) in A549 cells, which was higher than that of lipofectamine 2000. More importantly, the TE of 1a was dramatically increased in the presence of 10% serum. These results suggested that 1a might be a promising non-viral gene vector, and also give further insight for developing novel high performance gene delivery agents.     

Novel nonviral vectors for gene delivery: Synthesis and applications

We have synthesized novel cationiclipids for gene delivery bearing an ester bond betweenthe lipid moiety and the polyamine head. We have foundthat an intramolecular rearrangement occurs duringpurification of one of the products. The rearrangementled to a cyclic lipopolyamine which was active for DNAgene transfer. The formation ofcyclization products depends on the spacer foundbetween the lipid and the polyamine. The introduction ofarginine in the linker position prevents the formation ofcyclic products. Linear as well as cyclicanalogues showed high-efficiency gene transfer whentested in vitro for luciferase gene expressionas compared to dioctadecylamidoglycyl spermineor lipofectamine and also in vivo in the Lewislung carcinoma model. The introduction of arginine in thelinker position promoted increased transfectionactivity, demonstrating that a diversity of linkers,such as short peptides or glycosides, can beintroduced into cationic lipids for targeted gene transfer.     

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.     

Synthesis of cationic cardiolipin analogues

An approach was developed to synthesize a new class of cationic cardiolipin analogues containing two quaternary ammonium groups with tetra alkyl groups retaining "glycerol" moiety, the central core of the molecule. Cationic cardiolipin analogues were modified via introduction of either two or four oxyethylene groups to enhance the solubility in polar solvents. These newly synthesized cationic cardiolipin analogues can be applied to a broad range of drug delivery systems such as transfection reagents.     

Transfection of "naked" siRNA results in endosomal uptake and metabolic impairment in cultured neurons

RNA interference is rapidly becoming a powerful tool for gene silencing in mammalian cells. Introduction of siRNA into primary cells, however, remains one of the major difficulties of this novel technique. Using cationic lipid-based transfection reagents satisfactory transfection results are observed in cell lines, but low transfection efficiency and cytotoxicity limit applications in primary cells, especially primary neurons. The application of "naked" siRNA has been previously used successfully in nematodes and mammals in vivo. We therefore evaluated the effects of non-cationic-lipid-based siRNA application to primary hippocampal neuron cultures. "Naked" siRNA was bound to the cell surface and was taken up into endosomes. No significant silencing effect of endogenous or reporter genes was observed, rather application of "naked" siRNA was accompanied by a moderate downregulation of metabolic activity in culture. We postulate that endosomal degradation of "naked" siRNA in neurons prevents the induction of significant RNAi-mediated mRNA-downregulation and is accompanied by a global impairment of the cell metabolism. Transfection methods circumventing the endosomal pathway therefore might prove useful for siRNA transduction of primary neurons.     

Synthesis of diamine maleyl chitosans, and in vitro transfection studies

Three novel diamine-modified chitosan derivatives were synthesized from N-maleyl chitosan via Michael addition reaction with 1,2-diaminoethane, 1,4-diaminobutane, and 1,6-diaminohexane, respectively. These chitosan derivatives exhibited well binding ability of condensing plasmid DNA to form complexes with size ranging from 150 to 500 nm when the chitosan derivative/DNA weight ratios were above 10. The complexes observed by scanning electron microscopy (SEM) exhibited a compact and spherical morphology. The cytotoxicity of the chitosan derivatives presented a dependence on their side-chain structures. The gene transfection experiments were evaluated in 293 T and HeLa cells. The data obtained demonstrated that the gene transfection efficiencies of these chitosan derivatives were better than that of chitosan, suggesting these chitosan derivatives as potential gene vectors in vitro.     

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.     

Synthesis and delivery activity of new cationic cholesteryl glucosides

Cholesterol amphiphiles containing positively charged groups (pyridinium, N-methylimidazolium, N-methylmorpholinium, and N-methylpiperidinium) linked via β-glucosyl spacer were prepared by alkylation of the corresponding bases with 6-О-mesyl-β-d-cholesteryl glucopyranoside. IC₅₀ values were in the range 20-35 μM for the series of compounds and liposomal formulations with DOPE (1:1) were significantly less toxic. The liposomal formulations provided the accumulation of FITC-labeled oligonucleotide in cells, and the efficiency of this process was comparable to that of Lipofectamine® 2000. Cationic liposomes were able to deliver siRNA into the cells, and the liposomal formulation 7d/DOPE provided the most pronounced down-regulation of EGFP expression both in the presence and in the absence of serum (up to 30%).     

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.     

Polycations. 17. Synthesis and properties of polycationic derivatives of carbohydrates

In our continuing investigation of polycationic salts for purposes of antimicrobial action, ion-channel blocking, and construction of ionic liquids, we have prepared several series of polycationic salts derived from carbohydrate precursors. These salts are currently being investigated for optimal efficacy as antibacterials and antifungals, as well as for other applications. The syntheses of such series of salts are described here along with preliminary antibacterial testing results and a discussion of their properties indicating their potential utility for several purposes.     

Gene delivery mediated by cationic liposomes: from biophysical aspects to enhancement of transfection

Cationic liposomes complexed with DNA have been used extensively as non-viral vectors for the intracellular delivery of reporter or therapeutic genes in culture and in vivo. However, the relationship between the features of the lipid-DNA complexes (`lipoplexes') and their mode of interaction with cells, the efficiency of gene transfer and gene expression remain to be clarified. To gain insights into these aspects, the size and zeta potential of cationic liposomes (composed of 1,2-dioleoyl-3- (trimethylammonium) propane (DOTAP) and its mixture with phosphatidylethanolamine (PE)), and their complexes with DNA at different (+/-) charge ratios were determined. A lipid mixing assay was used to assess the interaction of liposomes and lipoplexes with monocytic leukaemia cells. The use of inhibitors of endocytosis indicated that fusion of the cationic liposomes with cells occurred mainly at the plasma membrane level. However, very limited transfection of these cells was achieved using the above complexes. It is possible that the topology of the cationic liposome-DNA complexes does not allow the entry of DNA into cells through a fusion process at the plasma membrane. In an attempt to enhance transfection mediated by lipoplexes composed of DOTAP and its equimolar mixture with dioleoylphosphatidylethanolamine (DOPE) two different strategies were explored: (i) association of a targeting ligand (transferrin) to the complexes to promote their internalization, presumably by receptor-mediated endocytosis; and (ii) association of synthetic fusogenic peptides (GALA or the influenza haemagglutinin Nterminal peptide HA-2) to the complexes to promote endosomal destabilization and release of the genetic material into the cytoplasm. These strategies were effective in enhancing transfection in a large variety of cells, including epithelial and lymphoid cell lines, as well as human macrophages, especially with the use of optimized lipid/ DNA (+/-) charge ratios. Besides leading to high levels of transfection, the ternary complexes of cationic liposomes, DNA, and protein or peptide, have the advantages of being active in the presence of serum and being non-toxic. Moreover, such ternary complexes present a net negative charge and, thus, are likely to alleviate the problems associated with the use of highly positively charged complexes in vivo, such as avid complexation with serum proteins. Overall, the results indicate that these complexes, and their future derivatives, may constitute viable alternatives to viral vectors for gene delivery in vivo.     

Calcium enhances the transfection potency of stabilized plasmid-lipid particles

Previous work from this laboratory has shown that plasmid DNA can be encapsulated in small (70-nm-diameter) stabilized plasmid-lipid particles (SPLP) that consist of a single plasmid encapsulated within a bilayer lipid vesicle. SPLP preferentially transfect tumor tissue following intravenous administration. Although the levels of transgene expression in vivo are greater for SPLP than can be achieved with naked DNA or complexes, they are lower than may be required for therapeutic benefit. In the present work we examine whether Ca2+ can enhance the transfection potency of SPLP. It is shown that Ca2+ can enhance SPLP transfection potency in bovine hamster kidney cells by 60- to 100-fold when treated in serum containing medium and an additional 60-fold when serum is absent for the initial 10 min of the transfection period. When cells are treated with SPLP in the presence of Ca2+, there is a fivefold increase in intact plasmid in the cell. It is also shown that this Ca2+ effect involves the formation of calcium phosphate precipitates; however, these precipitates are not directly associated with the SPLP plasmid DNA. The ability of calcium phosphate to facilitate delivery of other macromolecules without direct association is also demonstrated by the release of large-molecular-weight dextrans from endosomal/lysosomal compartments in the presence of calcium phosphate. Finally, it is shown that, unlike naked DNA, SPLP transfection potency in the presence of calcium phosphate is not affected by nuclease activity.     

Lipophosphonate/lipophosphoramidates: a family of synthetic vectors efficient for gene delivery

Lipophophoramidates constitute a class of synthetic vectors which were especially designed for gene delivery. In this family of compounds, the phosphorus functional group links two lipid chains to a spacer ended by a polar headgroup. Such vectors, which can readily be obtained, offer an alternative to the numerous examples of glycerolipid-based vectors that have been more exhaustively studied. Since the pioneering work describing this series of synthetic vectors, several chemical modifications have been proposed with the aim of correlating the molecular structure with the gene transfection efficacy. It has indeed been observed that some modifications which may be considered as minor at first glance, actually have important consequences on both the transfection efficacy and cytotoxic side effects. We herein discuss the modification of the structure of lipophosphoramidates, in particular of their lipidic part and of the nature of the cationic polar head which may be constituted by a trimethylammonium, trimethylphosphonium or trimethylarsonium motif. We also report that, as well as the in vitro transfection efficacy which governs the selection of the most promising vectors for in vivo studies, other aspects related to the synthetic pathway must be also considered for the development of new synthetic vectors (such as modularity of the synthesis, scaling-up).     

Natural lipid extracts and biomembrane-mimicking lipid compositions are disposed to form nonlamellar phases, and they release DNA from lipoplexes most efficiently

A viewpoint now emerging is that a critical factor in lipid-mediated transfection (lipofection) is the structural evolution of lipoplexes upon interacting and mixing with cellular lipids. Here we report our finding that lipid mixtures mimicking biomembrane lipid compositions are superior to pure anionic liposomes in their ability to release DNA from lipoplexes (cationic lipid/DNA complexes), even though they have a much lower negative charge density (and thus lower capacity to neutralize the positive charge of the lipoplex lipids). Flow fluorometry revealed that the portion of DNA released after a 30-min incubation of the cationic O-ethylphosphatidylcholine lipoplexes with the anionic phosphatidylserine or phosphatidylglycerol was 19% and 37%, respectively, whereas a mixture mimicking biomembranes (MM: phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine /cholesterol 45:20:20:15 w/w) and polar lipid extract from bovine liver released 62% and 74%, respectively, of the DNA content. A possible reason for this superior power in releasing DNA by the natural lipid mixtures was suggested by structural experiments: while pure anionic lipids typically form lamellae, the natural lipid mixtures exhibited a surprising predilection to form nonlamellar phases. Thus, the MM mixture arranged into lamellar arrays at physiological temperature, but began to convert to the hexagonal phase at a slightly higher temperature, ∼ 40-45 °C. A propensity to form nonlamellar phases (hexagonal, cubic, micellar) at close to physiological temperatures was also found with the lipid extracts from natural tissues (from bovine liver, brain, and heart). This result reveals that electrostatic interactions are only one of the factors involved in lipid-mediated DNA delivery. The tendency of lipid bilayers to form nonlamellar phases has been described in terms of bilayer “frustration” which imposes a nonzero intrinsic curvature of the two opposing monolayers. Because the stored curvature elastic energy in a “frustrated” bilayer seems to be comparable to the binding energy between cationic lipid and DNA, the balance between these two energies could play a significant role in the lipoplex-membrane interactions and DNA release energetics.     

Highly efficient gene transfer into hepatocyte-like HepaRG cells: New means for drug metabolism and toxicity studies

HepaRG progenitor cells are capable of differentiating into hepatocyte-like cells that express a large set of liver-specific functions. These cells, however, only express small amounts of an important cytochrome P450, the CYP2E1, which limits their use for toxicological studies of drugs metabolized by this pathway. Our aim was to establish an efficient transfection protocol to increase CYP2E1 expression in HepaRG cells. Transfection protocols of the green fluorescent protein (GFP) reporter gene were evaluated using electroporation and cationic lipids belonging to the lipophosphonates, lipophosphoramidates and lipids derived from glycine betaine. Following optimization of the charge ratios, plasmid DNA and formulations with neutral co-lipids, the lipophosphoramidate compounds KLN47 and BSV10, allowed expression of the GFP in ∼50% of adherent progenitor HepaRG cells, while electroporation targeted GFP expression in ∼85% of both progenitor and differentiated cells in suspension. Transient enforced expression of active CYP2E1 was also achieved in progenitors and/or differentiated HepaRG cells using the electroporation and the lipophosphoramidate compound BSV10. Importantly, in electroporated cells, CYP2E1 expression level was correlated with a significant increase in CYP2E1-specific enzymatic activity, which opens new perspectives for this CYP-dependent drug metabolism and toxicity studies using HepaRG cells.     

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.     

Synthesis,anticancer activity and cytotoxicity of galactosylated epothilone B

Epothilones are the 16-membered macrolide compounds, exhibit microtubule-promoting activity, have the same anti-tumor mechanism as paclitaxel, and are expected to be the ideal substitutes for paclitaxel. However, natural epothilone compounds have been found to have disadvantages such as high toxicity in vivo, poor selectivity to tumor cells, and susceptibility to drug resistance. Herein, epothilone B was synthesized by fermentation, and it was galactosylated by chemical method. The toxicity in vitro of epothilone B and its galactosylated derivative was investigated by the MTT method. The anticancer activity evaluation in vitro was performed using a method similar to the antibody-directed enzyme-prodrug therapy (ADEPT) method. It indicated that the ratio of cytotoxicity between the free epothilone B and the galactosylated epothilone B was about 150. This would lay the foundation for the targeted treatment of cancer with epothilone glycosides.