The adsorption of oligonucleotides on citrate-coated gold nanoparticles (AuNPs) is studied under conditions "right after the synthesis", i.e., in a weak citrate solution at a pH value close to neutral (5.8 ± 0.2). We found that short-term elevation of reaction temperature under these conditions provides fast and strong adsorption of oligonucleotides on the surface of AuNPs. The affinity of oligonucleotides to AuNPs depends on the length of the oligonucleotide and its nucleotide composition. The shortest oligonucleotide in this study, T, is the most affine, having the equilibrium binding constant K = 0.10 ± 0.04 nM and the highest surface density-up to 200 molecules per one particle. Olygothymidylates are at least as affine to AuNPs as oligoadenylates, while oligocytidilates show the lowest affinity. We also studied the interaction of resulting DNA/AuNPs with a series of low- and high-molecular thiols, which provide a variety of operations with adsorbed oligonucleotides: displacement (complete or partial) and encapsulation in a secondary shell. These experiments imitate someway the conditions in a living cell or serum, and show that DNA/AuNPs obtained by this method can be applied in a number of bionanotechnological applications, including delivery of nucleic acid therapeutics and theranostics.

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.

The ability of gold nanoparticles to bind different substances has resulted in the high interest of researchers determining their usage as a promising carrier of various biological substances including nucleic acids (NAs) for therapeutic applications. Most publications report covalent binding (conjugation) of an NA to spherical AuNPs via the Au-S bond. In this work, we obtained non-covalent associates of different ssDNA, ssRNA and siRNAs with spherical gold nanoparticles (AuNPs) and examined their physico-chemical properties and stability in media mimicking intracellular space (bacterial 'cytosol') and cell culture media (10% FBS in DMEM). The 'cytosol' was obtained from E. coli and possessed nuclease activity. For the first time, we used the phosphoryl guanidine (dimethylimidazolidin-2-imine, Dmi) group for modification of 3'-ends to enhance the stability of ssRNAs and siRNAs against nuclease destruction. Trying to evaluate the material balance, we analyzed the whole nucleotide species obtained after incubation of NA-AuNPs associates in 'cytosol' and FBS and evaluated the degree of NAs destruction, a share of full-size NAs remained on the surface of the AuNPs and in the solution. Native ss- and siRNAs, both free and in composition of non-covalent associates with AuNPs, were less resistant to degrading factors than ssDNA. The introduction of two Dmi-groups into the ssDNA increased its stability in 'cytosol' three times within 2.5 h. Dmi-modified siRNAs in non-covalent associates with AuNPs were two times more stable than unmodified siRNA within 4 h. We showed that non-covalent siRNA-AuNPs associates serve as a kind of storage for full-size NAs and thereby prolong their presence in nuclease-active media. Our study showed that non-covalent binding of siRNAs with a surface of AuNPs provides desorption of both strands, which is necessary for siRNA functioning in living cells, and could be considered as an important way to construct siRNA and ssDNA delivery systems based on AuNPs.