Gene Transfection in High Serum Levels: Case Studies with New Cholesterol Based Cationic Gemini Lipids

Abstract

BackgroundSix new cationic gemini lipids based on cholesterol possessing different positional combinations of hydroxyethyl (-CH2CH2OH) and oligo-oxyethylene -(CH2CH2O)n- moieties were synthesized. For comparison the corresponding monomeric lipid was also prepared. Each new cationic lipid was found to form stable, clear suspensions in aqueous media.Methodology/Principal FindingsTo understand the nature of the individual lipid aggregates, we have studied the aggregation properties using transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential measurements and X-ray diffraction (XRD). We studied the lipid/DNA complex (lipoplex) formation and the release of the DNA from such lipoplexes using ethidium bromide. These gemini lipids in presence of a helper lipid, 1, 2-dioleoyl phophatidyl ethanol amine (DOPE) showed significant enhancements in the gene transfection compared to several commercially available transfection agents. Cholesterol based gemini having -CH2-CH2-OH groups at the head and one oxyethylene spacer was found to be the most effective lipid, which showed transfection activity even in presence of high serum levels (50%) greater than Effectene, one of the potent commercially available transfecting agents. Most of these geminis protected plasmid DNA remarkably against DNase I in serum, although the degree of stability was found to vary with their structural features.Conclusions/Significance-OH groups present on the cationic headgroups in combination with oxyethylene linkers on cholesterol based geminis, gave an optimized combination of new genera of gemini lipids possessing high transfection efficiency even in presence of very high percentage of serum. This property makes them preferential transfection reagents for possible in vivo studies.

Factors influencing the transfection efficiency and cellular uptake mechanisms of Pluronic P123-modified polypropyleneimine/pDNA polyplexes in multidrug resistant breast cancer cells

“…(3) FBS played pivotal role in the transportation of polyplexes which eventually results in effective transfection. It indicated that P123-PPI/pDNA polyplexes had significant serum dependent transfection activity, which was consistent with the results obtained from the other report [40]. The transfection efficiency of P123-PPI/pDNA polyplexes showed a 'bell'-shaped serum concentration dependence behavior when FBS percentage increased from 5 to 50% (Fig.…”

Recent progress in gene therapy to deliver nucleic acids with multivalent cationic vectors

“…Other groups have reported the electrostatic interaction when the GCL contains an oligo-oxyethylene spacer [93,115], and/or other headgroups as imidazolium or pyridinium [45, 57-59, 75, 99, 116]. Looking for more biocompatible vectors, GCLs that incorporate aminoacid rests, such as GCL-lysine 12 based and GCL-arginine based [60,62,64], GCL-cholesterol based [117][118][119], and GCL-cyclen-based [120] have been also investigated. Lipoplexes containing a MVCL with dendritic-type headgroup have been also analyzed [67,121,122].…”

“…TEM and/or cryo-TEM studies have reported many micrographs to characterize the size, shape, and morphology of a wide variety of lipoplexes as: (a) pDNA with GCLs [45,54,57,62,64,75,93,115,117,118,120], MVCLs [63], cationic polymers [107,133,[169][170][171] or polycations [40,84,100,136]; and (b) siRNA with cationic polymers [90,104]. Fig.…”

“…As a result of that, the transfection capability [75]. Recent studies reporting TE of lipoplexes and polyplexes have been investigated: (i) divalent GCLs, as cholesterol-based with alkyl or oligooxyethylene spacers [118,119,121], aromatic-based with alkyl or oligo-oxyethylene spacers [46,93], SS14 linkers [66], ammonium with alkyl or oligo-oxyethylene spacers [54,71,93,179], imidazolium or pyridinium derivatives with alkyl-or oligooxyethylene spacers [45,59,75], aminoacid-based [62,64] or gemini-like amphiphilic peptides [150], cyclen-based CLs [120,180], and archael tetraether-based lipids [138];…”

“…The results confirm that all these optimized formulations did not report any toxicity to the cell lines studied, the cell viability after transfection being considerably better than the commercial control Lipo2000. The cell viability/cytotoxicity of several complexes between nucleic acids and a wide variety of recent synthesized MVCVs has been analyzed: (i) GCLs as cholesterol-based with alkyl or oligo-oxyethylene spacers [118,119,121,206,207], aromatic-based with alkyl or oligo-oxyethylene spacers [46], ammonium derivatives [54,71,93] imidazolium derivatives [45,75,99] and pyridinium derivatives [57] with alkyl type spacers, aminoacid-based [64,208], SS14 linkers [66], gemini-like amphiphilic peptides [150] or cyclen-based CLs [120,180]; (ii) several MVCLs [67,72,122,181,182]; (iii) cationic polymer-derivatives from PEI [124,127,183,209], PAMAM [140], PDMAEMA [123,133], chitosan [188], polyphosphoramidates [189], polyhydroxyalkanoates [92], polyamines [107,187] or polyaminoacids [127,190]; (iv)…”