The purpose of this study was to control the fabrication of new labile supramolecular assemblies by formulating associations of DNA molecules with inorganic layered double hydroxides (LDHs). The results show that LDH/DNA hybrids synthesized by a coprecipitation route involving the in situ formation of LDHs around DNA molecules acting as templates were characterized by a lamellar organization, with DNA molecules sandwiched between hydroxide layers, exhibiting a regular spacing of 1.96 nm. Our results indicate that labile complexes resulting from the association of nucleic acids and inorganic materials can be obtained not only by anion exchange but also by a direct self-assembly route.
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...
We reported that amphiphilic block copolymers hold promise as nonviral vectors for the delivery of plasmid DNA, ranging from 4.7 to 6.2 kb, to healthy muscle for the production of local or secreted proteins. To evaluate the efficiency of these vectors to deliver large plasmid DNA molecules to pathological muscles, plasmid DNAs of various lengths were complexed with Lutrol or poloxamine 304 and injected intramuscularly into dystrophic muscles. Lutrol-DNA and poloxamine 304-DNA complexes promoted gene transfer into muscles of the naturally occurring mouse model for DMD (mdx) in a dose- and plasmid DNA size-dependent manner. For small plasmid DNAs encoding reporter genes, this improvement over naked DNA was smaller in mdx than in the wild-type control strain. By contrast, Lutrol enabled us to deliver the large plasmid (16.1 kb) encoding the rod-deleted dystrophin in mdx mouse muscle, whereas the same amount of naked DNA did not lead to dystrophin expression, under the same experimental conditions. Lutrol-treated mdx mice showed the production of dystrophin in large numbers of muscle fibers. More importantly, we also found that expressing dystrophin with Lutrol led to restoration of the dystrophin-associated protein complex. Thus, we conclude that block copolymers constitute a novel class of vectors for the delivery of large plasmid DNA not only to healthy muscles but also to pathological muscle tissues.
Various pulmonary disorders, including cystic fibrosis, are potentially amenable to a treatment modality in which a therapeutic gene is directly delivered to the lung. Current gene delivery systems, either viral or nonviral, need further improvement in terms of efficiency and safety. We reported that nonionic amphiphilic block copolymers hold promise as nonviral gene delivery systems for transfection of muscular tissues. To evaluate the efficiency of these vectors in the lung, intratracheal instillation or aerosolization of reporter genes complexed with Lutrol or PE6400 was performed. Lutrol-DNA and, to a lesser extent, PE6400-DNA complexes promoted efficient gene transfection into mouse airways in a dose-dependent manner. This improvement over naked DNA was observed irrespective of the reporter gene. Lutrol enabled us to deliver significantly higher DNA amounts than current nonviral vectors, with even greater increases in gene expression and without the formation of colloidally unstable complexes. Time course studies showed that Lutrol-DNA complexes permitted prolonged gene expression for up to 5 days whereas with poly(ethylenimine) (PEI)-DNA polyplexes, expression peaked on days 1-2 postinstillation, was strongly reduced by day 5, and reached background levels on day 7. Aerosolized delivery of Lutrol-DNA complexes, a less invasive approach to deliver genes to the lung, gave 5- to 15-fold higher reporter gene expression compared with PEI-DNA polyplexes administered via the same delivery route. After intratracheal instillation of Lutrol-DNA complexes, histochemical staining for beta-galactosidase expression showed the presence of large blue areas. Histopathological analysis showed that Lutrol alone did not elicit inflammation, and that the inflammatory response after intratracheal instillation of Lutrol-DNA complexes was reversible and was observed only with the highest amounts of DNA. We also found that Lutrol can efficiently deliver genes to the airways of cystic fibrosis mice. Thus, we conclude that Lutrol is a highly promising vector for gene delivery to the lung.
We hypothesized that a nonviral gene delivery of the hyperpolarization-activated HCN2 channel combined with the beta(2)-adrenergic receptor (ADRB2) would generate a functional pacemaker in a mouse model of complete atrioventricular block (CAVB) induced by radiofrequency ablation of the His bundle. Plasmids encoding HCN2 and ADRB2 mixed with tetronic 304, a poloxamine block copolymer, were injected in the left ventricular free wall (HCN2-ADRB2 mice). Sham mice received a noncoding plasmid. CAVB was induced 5 days later. Ventricular escape rhythms in HCN2-ADRB2 mice were significantly faster than in sham mice at day 15 after ablation and later. In HCN2-ADRB2 mice, QRS complexes were larger than in sham mice and characterized by abnormal axes. Immunostaining of GFP-HCN2 fusion protein showed an expression of HCN2 channel in left ventricular myocardium for at least 45 days after injection. In the mouse, CAVB induces progressive hypertrophy and heart failure leading to 50% mortality after 110 days. HCN2-ADRB2 mice survived 3 weeks longer than sham mice. Finally, beta-adrenergic input increased ventricular escape rhythms significantly more in HCN2-ADRB2 mice than in sham mice. In conclusion, nonviral gene transfer can produce a functional cardiac biological pacemaker regulated by sympathetic input, which improves life expectancy in a mouse model of CAVB.
Intramuscular injection of plasmid DNA formulated with PE6400 provides an efficient and simple method for secretion and production of non-muscle proteins.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.