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.

Design, synthesis and transfection efficiency of a novel redox-sensitive polycationic amphiphile.

A novel redox-sensitive polycationic amphiphile (2S3) with disulphide linkers for nucleic acid delivery was developed. Cationic liposomes formed by 2S3 and the helper lipid DOPE demonstrated effective DNA delivery into HEK293 cells with a maximal transfection activity that is superior than both nonredox-sensitive cationic liposomes and Lipofectamine® 2000 at an N/P ratio of 6/1. Redox-sensitivity was tested by experiments with extracellular glutathione which shown the ability of disulphide linker degradation. Our results suggest that polycationic amphiphile 2S3 is a promising candidate for nucleic acid delivery.

Structure-transfection activity relationships in a series of novel cationic lipids with heterocyclic head-groups.

Cationic liposomes are promising candidates for the delivery of various therapeutic nucleic acids. Here, we report a convenient synthesis of carbamate-type cationic lipids with various hydrophobic domains (tetradecanol, dialkylglycerol, cholesterol) and positively charged head-groups (pyridinium, N-methylimidazolium, N-methylmorpholinium) and data on the structure-transfection activity relationships. It was found that single-chain lipids possess high surface activity, which correlates with high cytotoxicity due to their ability to disrupt the cellular membrane by combined hydrophobic and electrostatic interactions. Liposomes containing these lipids also display high cytotoxicity with respect to all cell lines. Irrespective of chemical structures, all cationic lipids form liposomes with similar sizes and surface potentials. The characteristics of complexes composed of cationic liposomes and nucleic acids depend mostly on the type of nucleic acid and P/N ratios. In the case of oligodeoxyribonucleotide delivery, the transfection activity depends on the type of cationic head-group regardless of the type of hydrophobic domain: all types of cationic liposomes mediate efficient oligonucleotide transfer into 80-90% of the eukaryotic cells, and liposomes based on lipids with N-methylmorpholinium cationic head-group display the highest transfection activity. In the case of plasmid DNA and siRNA, the type of hydrophobic domain determines the transfection activity: liposomes composed of cholesterol-based lipids were the most efficient in DNA transfer, while liposomes containing glycerol-based lipids exhibited reasonable activity in siRNA delivery under serum-free conditions.

Targeted delivery of nucleic acids into xenograft tumors mediated by novel folate-equipped liposomes.

Folate receptors (FR) are cellular markers highly expressed in various cancer cells. Here, we report on the synthesis of a novel folate-containing lipoconjugate (FC) built of 1,2-di-O-ditetradecyl-rac-glycerol and folic acid connected via a PEG spacer, and the evaluation of the FC as a targeting component of liposomal formulations for nucleic acid (NA) delivery into FR expressing tumor cells. FR-targeting liposomes, based on polycationic lipid 1,26-bis(cholest-5-en-3ß-yloxycarbonylamino)-7,11,16,20-tetraazahexacosan tetrahydrochloride (2X3), lipid helper dioleoylphosphatidylethanolamine (DOPE) and novel FC, formed small compact particles in solution with diameters of 60 ±â€¯22 nm, and were not toxic to cells. Complexes of NAs with the liposomes were prepared at various nitrogen to phosphate ratios (N/P) to optimize liposome/cell interactions. We showed that FR-mediated delivery of different nucleic acids mediated by 2X3-DOPE/FC liposomes occurs in vitro at low N/P (1/1 and 2/1); under these conditions FC-containing liposomes display 3-4-fold higher transfection efficiency in comparison with conventional formulation. Lipoplexes formed at N/P 1/1 by targeted liposomes and cargo (Cy7-labeled siRNA targeting MDR1 mRNA) in vivo efficiently accumulate in tumor (∼15-18% of total amount), and kidneys (71%), and were retained there for more than 24 h, causing efficient downregulation of p-glycoprotein expression (to 40% of control) in tumors. Thus, FC containing liposomes provide effective targeted delivery of nucleic acids into tumor cells in vitro and in xenograft tumors in vivo.

Multicomponent mannose-containing liposomes efficiently deliver RNA in murine immature dendritic cells and provide productive anti-tumour response in murine melanoma model.

Here we demonstrate the ability of mannosylated liposomes (ML) targeted to mannose receptors (MR) to perform the targeted delivery of model plasmid DNA encoding EGFP and total tumour RNA into murine bone-marrow-derived dendritic cells (DCs) and enhance the efficiency of anti-tumour response triggered by these DCs in murine melanoma model. ML consist of cationic lipid 2X3 (1,26-Bis(cholest-5-en-3ß-yloxycarbonylamino)-7,11,16,20-tetraazahexacosan tetrahydrochloride), helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), and 2.5, 5 or 10% mol. of novel mannosylated lipoconjugates. In the structure of lipoconjugates D-mannose was attached to ditetradecylglycerol residue via succinyl (lipoconjugate 1) or diethylsquarate (lipoconjugate 2) linker groups. ML spontaneously form complexes with plasmid DNA and RNA due to electrostatic interaction between positively charged lipid amino group and negatively charged phosphate of nucleic acids. ML demonstrated the benefit in transfection efficiency (TE) of pDNA into DC progenitors and immature DCs in comparison with the control liposomes at low N/P (nitrogen to phosphate) ratios (1/1 and 2/1) but not at high N/P ratios where the TE was comparable with control liposomes. Moreover, ML at low N/P were more effective in RNA delivery into immature DCs in comparison with DC progenitors. At high N/P ratios liposomal formulations containing 5 and 10% mol. of mannosylated lipoconjugate 2 with diethylsquarate linker were the most effective (up to 50% of transfected cells). DCs transfected ex vivo with ML/melanoma B16 RNA complexes after i.v. injection into mice caused five- to six-fold inhibition of melanoma lung metastasis number. Moreover, the i.v. injection of ML/melanoma B16 RNA complexes into mice induced generation of the melanoma B16-specific cytotoxic T-lymphocytes, which were two-fold more efficient in B16 cell killing than those from control liposome group.

Novel cationic liposomes provide highly efficient delivery of DNA and RNA into dendritic cell progenitors and their immature offsets.

Here we report on the application of cationic liposomes formed by new cationic lipids and the lipid-helper DOPE (dioleoylphosphatidylethanolamine) for the transfection of plasmid DNA and mRNA into dendritic cells (DCs) progenitors and immature DCs of bone-marrow origin in vitro and the use of these DCs to induce the suppression of B16 melanoma metastases in vivo. The cationic lipids contain one (X2, S1, S2 and S3) or two (2X3) cholesterol residues or long-chain hydrocarbon substituent (2D3) linked with spermine. Data show that liposomes 2X3-DOPE, 2D3-DOPE, X2-DOPE and S2-DOPE display high transfection efficiency in respect to DNA (30-47% of DC progenitors and up to 57% of immature DC were transfected) and RNA (up to 57% of cells were transfected). The studied lipids exhibited an efficiency of DNA and RNA delivery in DCs several times higher in comparison with Lipofectamine 2000. Observed ex vivo the higher transfection efficiencies of DCs with mRNAs encoding of a set of tumor-associated antigens provided by cationic liposomes 2X3-DOPE and 2X2-DOPE corresponded to a 3-5 fold suppression of metastasis number in a model of murine B16 melanoma in vivo. The injection into mice of these pulsed DCs resulted in a slight pro-inflammatory response which was balanced by the positive effect of the antitumor cytokine production induced by the DCs. The obtained data show that the novel spermine-based polycationic lipids can be applied in the preparation of antitumor DC-based vaccine.

Novel cholesterol spermine conjugates provide efficient cellular delivery of plasmid DNA and small interfering RNA.

New polycationic lipids corresponding to the two different classes of amphiphiles ("head-tail" and "gemini") were designed and used as components of non-viral gene delivery systems. The hydrophobic domain of lipids is based on the cholesterol residue and the hydrophilic one--on the naturally occurring polyamine--spermine. Ester and carbamate linker groups as well as oligomethylene spacers of various lengths were used to connect cholesterol and spermine motifs in order to estimate the structure-activity relationships of novel polycationic lipids and to determine an effective and safe transfectant suitable for the delivery of different nucleic acids. The cationic liposomes composed of the synthesized polycationic lipids and DOPE provided delivery of FITC-labeled oligonucleotide, plasmid DNA and siRNA into HEK293 cells with an efficiency significantly higher than that of Lipofectamine 2000. We found that the transfection activity of polycationic lipids is influenced by a linker type, a spacer length and the amount of cholesterol residues. The lipid containing two cholesterol units, carbamate linker and spacer of six methylene groups demonstrated the best in vitro transfection results among other analogues tested and was defined as a promising candidate for further transfection studies to be hold in vivo.

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 ß-glucosyl spacer were prepared by alkylation of the corresponding bases with 6-О-mesyl-ß-D-cholesteryl glucopyranoside. IC(50) values were in the range 20-35µM 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%).

Novel cholesterol-based cationic lipids for gene delivery.

Gene therapy based on gene delivery is a promising strategy for the treatment of human disease. Here we present data on structure/biological activity of new biodegradable cholesterol-based cationic lipids with various heterocyclic cationic head groups and linker types. Enhanced accumulation of nucleic acids in the cells mediated by the lipids was demonstrated by fluorescent microscopy and flow cytometry. Light scattering and atomic force microscopy were used to find structure/transfection activity correlations for the lipids. We found that the ability of the lipids to stimulate intracellular accumulation of the oligodeoxyribonucleotides and plasmid DNA correlates well with their ability to form in solution lipid/NA complexes of sizes that do not exceed 100 nm. Screening of the lipids revealed the most promising transfection agents both in terms of low toxicity and efficient delivery: cholesterol-based lipids with positively charged pyridine and methyl imidazole head groups and either the ester or carbamate linker.