Self-assembled lamellar complexes of siRNA with lipidic aminoglycoside derivatives promote efficient siRNA delivery and interference

Abstract

RNA interference requires efficient delivery of small doublestranded RNA molecules into the target cells and their subsequent incorporation into RNA-induced silencing complexes. Although current cationic lipids commonly used for DNA transfection have also been used for siRNA transfection, a clear need still exists for better siRNA delivery to improve the gene silencing efficiency. We synthesized a series of cationic lipids characterized by head groups bearing various aminoglycosides for specific interaction with RNA. siRNA complexation with such lipidic aminoglycoside derivatives exhibited three lipid/siRNA ratio-dependent domains of colloidal stability. Fluorescence and dynamic light-scattering experiments showed that cationic lipid/siRNA complexes were formed at lower charge ratios, exhibited a reduced zone of colloidal instability, and had smaller mean diameters compared with our previously described guanidinium-based cationic lipids. Cryo-transmission electron microscopy and x-ray-scattering experiments showed that, although the final in toto morphology of the lipid/siRNA complexes depended on the aminoglycoside type, there was a general supramolecular arrangement consisting of ordered lamellar domains with an even spacing of 67 Å. The most active cationic lipid/siRNA complexes for gene silencing were obtained with 4,5-disubstituted 2-deoxystreptamine aminoglycoside derivatives and were characterized by the siRNA being entrapped in small particles exhibiting lamellar microdomains corresponding to siRNA molecules sandwiched between the lipid bilayers. These results clearly show that lipidic aminoglycoside derivatives constitute a versatile class of siRNA nanocarriers allowing efficient gene silencing.gene silencing ͉ gene transfer vectors ͉ transfection R NAi has become widely used for knocking down the expression of a specific target gene by a posttranscriptional silencing mechanism and thereby it allows phenotypic analysis of gene function in cells (1, 2). Therapeutic approaches involving RNAi are also actively investigated (3, 4). To achieve gene silencing, sequence-specific double-stranded small interfering RNA (siRNA) molecules have to be delivered efficiently into the cytoplasm of cells (5, 6). Various methods have already been used for siRNA delivery, in particular, cationic lipids developed for plasmid DNA transfection (see ref. 7). These cationic lipids are composed of a hydrophobic moiety linked (by a spacer) to a cationic head group bearing either a quaternary ammonium [such as the lipids DOTMA {N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride} and DOTAP (1,2-dioleoyl-3-trimethylammonium-propane)], a polycation [such as the lipid DOGS (dioctadecylamidoglycylspermine) (8) and lipopolyamine RPR120535 (9)], or guanidinium groups [such as the lipid BGTC, bis(guanidinium)-tris(2-aminoethyl)amine-cholesterol (10, 11)]. A colipid such as dioleoyl phosphatidylethanolamine (DOPE) is usually combined with the cationic lipids to form liposomes. The electrostatic interactions between the plasmid D...

Cancer-Specific Transgene Expression Mediated by Systemic Injection of Nanoparticles

“…Finally, in zone C (i.e., for a charge ratio above 1.5), we observed colloidal stable, positively charged complexes with a mean diameter of 86 F 9 nm (n = 3). These results show that PPIG3/DNA nanoparticles followed the well-known three-stage model of colloidal stability described elsewhere (3,26,27,29). Next, we investigated the level of DNA condensation using fluorescence measurement after addition of ethidium bromide in all samples.…”

“…First, the colloidal stability of supramolecular assemblies formed between the dendrimers and plasmid DNA follows the well-known three-stage model of colloidal stability described with other synthetic vectors (3,4,26,27). This colloidal stability and the high positive charge of the particle are classic features of a nonviral gene delivery vector and might lead to the formation of monodispersed, nonaggregated particles, which could better diffuse and interact with cellular membranes and subsequently promote internalization via endocytosis or pynocytosis pathways (3,26,27,29,33). Second, at high charge ratio (e.g., ref.…”

“…Dynamic light scattering, fluorescence, and cryo-transmission electron microscopy (cryo-TEM) studies were done as previously described (26,27). PPIG3/DNA nanoparticles were prepared by combining increased concentration of PPIG3 dendrimer (0-3 Ag/AL) with a fixed-standardized concentration of DNA plasmid (10 Ag/mL) in 1 mL of 5% dextrose.…”

Cyclodextrin-scaffolded amphiphilic aminoglucoside clusters: self-assembling and gene delivery capabilities

“…A similar effect has been observed in cationic lipids furnished with aminoglycoside polar heads. 29 The presence of serum (10%) led to a generalized efficiency drop for both pGaCDs 2 and 4 when compared to paCD 1. Probably, the greater increase in size of the glycoCDplexes formed using 2 and 4 after interaction with serum proteins is at the origin of this observation.…”

“…Actually, the differential binding of aminoglycoside antibiotics to nucleic acids has already been exploited in the design of cationic lipidtype vectors. [29][30][31] Moreover, a number of glycosylated gene carriers have been shown to operate with transfection efficiencies that correlated with their glyco-dependent self-assembling capabilities in the presence of nucleic acids rather than with specific recognition events towards target protein receptors.…”