Abstract
Elaboration of non-viral vehicles for delivery of therapeutic nucleic acids, in particular siRNA, into a cell is an actively growing field. Gold nanoparticles (AuNPs) occupy a noticeable place in these studies, and various nanoconstructions containing AuNPs are reported. We aimed our work to the rational design of AuNPs-based siRNA delivery vehicle with enhanced transfection efficiency. We optimized the obtaining of non-covalent siRNAs-AuNPs cores: ionic strength, temperature and reaction time were determined. Formation of cores was confirmed using gel electrophoresis. Stable associates were prepared, and then enveloped into a lipid layer composed of phosphatidylcholine, phosphatidylethanolamine and novel pH-sensitive lipidoid. The constructions were modified with [Str-(RL)4G-NH2] peptide (the resulting construction). All intermediate and resulting nanoconstructions were analyzed by dynamic light scattering (DLS) and transmission electron microscopy (TEM) to control their physico-chemical properties. To examine the biological effect of the delivery vehicle, green fluorescent protein (GFP)-expressing human embryonic kidney (HEK) Phoenix cells were incubated with the resulting construction containing anti-GFP siRNA, with the siRNA effect being studied by flow cytometry and confocal microscopy. Transfection of the cells with the resulting construction reduced the GFP fluorescence as efficiently as Lipofectamin 3000. Thus, siRNA vehicle based on non-covalently bound siRNA-AuNP core and enveloped into a lipid layer provides efficient delivery of siRNA into a cell followed by specific gene silencing.
Highlights
Elaboration of non-viral vehicles for delivery of therapeutic nucleic acids (TNA) into a cell is one of the actively growing fields of gene therapy
SiRNA vehicle based on non-covalently bound siRNA-AuNP core and enveloped into a lipid layer provides efficient delivery of siRNA into a cell followed by specific gene silencing
Lipid-AuNPs nanoconstructions were used to deliver various nucleic acids into a cell: plasmids [1,2,3], antisense oligonucleotides [4] and siRNA. siRNA has been attached onto the outer surface of a construction [5]
Summary
Elaboration of non-viral vehicles for delivery of therapeutic nucleic acids (TNA) into a cell is one of the actively growing fields of gene therapy. We suppose that the use of pre-prepared AuNPs provides the ability to control the attachment of siRNA onto AuNPs surface, and, respectively, the preparation of the nanoconstruction core. We applied non-covalent attachment of siRNA onto the AuNPs surface and have shown the stability of such construction. We show how it is possible to optimize preparation of non-covalent siRNA-Au associates, in particular, how to increase a number of siRNA molecules on one AuNPs. Lipid-based nanoconstructions for TNAs delivery are considered the most promising delivery vehicles due to the lipids’ ability to fuse with cell membranes and vast room for the modification of lipid layers. We believe the approach to use non-covalently assembled siRNA-Au core to be promising, since it permits siRNA to be released from the core in the functional form [9], which is crucial for gene silencing [12,13], and could optimize amount of siRNA molecules delivered into a target cell
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