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From gene delivery to gene silencing: plasmid DNA-transfecting cationic lipid 1,3-dimyristoylamidopropane-2-[bis(2-dimethylaminoethane)] carbamate efficiently promotes small interfering RNA-induced RNA interference.

ABSTRACT: The cationic lipid 1,3-dimyristoylamidopropane-2-[bis(2-dimethylaminoethane)] carbamate (1,3lb2) was applied as a delivery system for small interfering RNA (siRNA) to inhibit the production of vascular endothelial growth factor (VEGF) in vitro in human prostate carcinoma cell line PC-3. VEGF protein silencing peaked at 94% when cationic lipid-nucleic acid complexes (lipoplexes) were formulated at a nitrogen:phosphorothioate ratio (N:P) of 2 with a dose concentration of 53.7 nM, and the performance of these lipoplexes was not impeded by serum. Knockdown efficiency was maintained for at least 72 h, and an IC(50) of 12 nM lasted for 48 h. Only 20% of the total siRNA became cell-associated at this N:P, at a rate of 25 ng/h. Lipoplexes of the optimal formulation were relatively monodisperse, having an average diameter of 634 nm and a zeta potential of -21.3 mV. Formation of the 1,3lb2-siRNA complex reached 94% at an N:P of 2 and was positively cooperative; the binding constant was calculated in the range of 10(5) M(-1), and a Hill coefficient of 3 was determined. 1,3lb2 was found to be a nontoxic and potent carrier of siRNA that binds to the nucleic acid effectively and whose lipoplexes promote long-lasting inhibition, have high biological activity at low N:Ps, and are functional in the presence of serum.

SUBMITTER: Spelios M

PROVIDER: S-EPMC2905058 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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From gene delivery to gene silencing: plasmid DNA-transfecting cationic lipid 1,3-dimyristoylamidopropane-2-[bis(2-dimethylaminoethane)] carbamate efficiently promotes small interfering RNA-induced RNA interference.

Spelios Michael M Kearns Molinda M Savva Michalakis M

Biochemistry 20100701 27

The cationic lipid 1,3-dimyristoylamidopropane-2-[bis(2-dimethylaminoethane)] carbamate (1,3lb2) was applied as a delivery system for small interfering RNA (siRNA) to inhibit the production of vascular endothelial growth factor (VEGF) in vitro in human prostate carcinoma cell line PC-3. VEGF protein silencing peaked at 94% when cationic lipid-nucleic acid complexes (lipoplexes) were formulated at a nitrogen:phosphorothioate ratio (N:P) of 2 with a dose concentration of 53.7 nM, and the performan ...[more]

PMID: 20515078

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Project description:Type 1 diabetes and thyroid disease are T cell-dependent autoimmune endocrinopathies. The standard substitutive administration of the deficient hormones does not halt the autoimmune process; therefore, development of immunotherapies aiming to preserve the residual hormonal cells, is of crucial importance. PTPN22 C1858T mutation encoding for the R620W lymphoid tyrosine phosphatase variant, plays a potential pathophysiological role in autoimmunity. The PTPN22 encoded protein Lyp is a negative regulator of T cell antigen receptor signaling; R620W variant, leading to a gain of function with paradoxical reduced T cell activation, may represent a valid therapeutic target. We aimed to develop novel wild type PTPN22 short interfering RNA duplexes (siRNA) and optimize their delivery into Jurkat T cells and PBMC by using liposomal carriers. Conformational stability, size and polydispersion of siRNA in lipoplexes was measured by CD spectroscopy and DLS. Lipoplexes internalization and toxicity evaluation was assessed by confocal microscopy and flow cytometry analysis. Their effect on Lyp expression was evaluated by means of Western Blot and confocal microscopy. Functional assays through engagement of TCR signaling were established to evaluate biological consequences of down-modulation. Both Jurkat T cells and PBMC were efficiently transfected by stable custom lipoplexes. Jurkat T cell morphology and proliferation was not affected. Lipoplexes incorporation was visualized in CD3+ but also in CD3- peripheral blood immunotypes without signs of toxicity, damage or apoptosis. Efficacy in affecting Lyp protein expression was demonstrated in both transfected Jurkat T cells and PBMC. Moreover, impairment of Lyp inhibitory activity was revealed by increase of IL-2 secretion in culture supernatants of PBMC following anti-CD3/CD28 T cell receptor-driven stimulation. The results of our study open the pathway to future trials for the treatment of autoimmune diseases based on the selective inhibition of variant PTPN22 allele using lipoplexes of siRNA antisense oligomers.

Dataset Information

From gene delivery to gene silencing: plasmid DNA-transfecting cationic lipid 1,3-dimyristoylamidopropane-2-[bis(2-dimethylaminoethane)] carbamate efficiently promotes small interfering RNA-induced RNA interference.

Publications

From gene delivery to gene silencing: plasmid DNA-transfecting cationic lipid 1,3-dimyristoylamidopropane-2-[bis(2-dimethylaminoethane)] carbamate efficiently promotes small interfering RNA-induced RNA interference.

Similar Datasets

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