Nina G. Morozova

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.

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. 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%).

Synthesis of bivalent neogalactolipids via modified Staudinger reaction

The synthesis of bivalent galactose-containing neutral lipids for the development of a targeted gene delivery system to the hepatocytes has been described. For introducing galactosyl ligands into the lipid molecule, a convenient approach based on modified Staudinger reaction was used.

Synthesis and transfection activity of novel galactosylated polycationic lipid

In this study, we synthesized a new galactosylated cationic lipid and investigated its biological activity. The structure of lipid combines both spermine residue for DNA compaction and galactose moiety for the improvement of aggregation behavior of lipoplexes. Lipid was low toxic for different mammalian cells, and was able both to compact plasmid DNA and to mediate cellular accumulation of various nucleic acids (ODN, pDNA and siRNA) exhibiting biological activity (transgene expression, gene silencing).

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.

Synthesis of mannose-containing neoglycolipids as a component of targeted delivery system for transfer of nucleic acids into antigen-presenting cells

Two approaches to the preparation of mannose-containing neoglycolipid, designed for development of nonviral targeted delivery system for transfer of nucleic acids into antigen-presenting cells, were described. They differed from each other in the binding of the targeting ligand and the lipid anchor, which was effected either through the formation of an amide bond or via the chemoselective binding agent 3,4-diethoxycyclobut-3-ene-1,2-dione.

A Novel Disulfide-Containing Polycationic Amphiphile: 1,28-Di[(cholest-5-en-3β-yl)disulfanyl]-4,25-dioxo-3,8,12,17,21,26-hexaazaoctacosane Tetrahydrochloride

The absence of highly effective delivery systems is a major challenge for gene therapy. Our work was aimed at the development of novel cationic liposomes possessing high transfection efficiency. For this purpose, a novel disulfide polycationic amphiphile 2S4 was synthesized. Cationic liposomes based on 2S4 and a helper lipid DOPE were formed by the thin film hydration method and exhibited effective plasmid DNA delivery into the HEK293 cells, with a transfection activity superior to that of the commercial agent Lipofectamine® 2000. Our results suggest that the polycationic amphiphile 2S4 is a promising candidate for in vitro nucleic acid delivery.

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

Graphical abstract Figure. No Caption available. Abstract 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‐3&bgr;‐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 ± 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.

Novel bivalent spermine-based neutral neogalactolipids for modular gene delivery systems

New bivalent spermine-based neutral neogalactolipids have been synthesized to develop effective modular gene delivery systems (MGDS) targeting hepatocyte asialoglycoprotein receptors. MGDS composed of new neogalactolipids, cationic gemini-amphiphile, and DOPE were agglutinated in the presence of lectin RCA120 and provided efficient delivery of fluorescently-labeled oligodeoxyribonucleotides into HepG2 cells.