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.

Small interfering RNA (siRNA) is the most important tool for the manipulation of mRNA expression and needs protection from intracellular nucleases when delivered into the cell. In this work, we examined the effects of siRNA modification with the phosphoryl guanidine (PG) group, which, as shown earlier, makes oligodeoxynucleotides resistant to snake venom phosphodiesterase. We obtained a set of siRNAs containing combined modifications PG/2′-O-methyl (2′-OMe) or PG/2′-fluoro (2′-F); biophysical and biochemical properties were characterized for each duplex. We used the UV-melting approach to estimate the thermostability of the duplexes and RNAse A degradation assays to determine their stability. The ability to induce silencing was tested in cultured cells stably expressing green fluorescent protein. The introduction of the PG group as a rule decreased the thermodynamic stability of siRNA. At the same time, the siRNAs carrying PG groups showed increased resistance to RNase A. A gene silencing experiment indicated that the PG-modified siRNA retained its activity if the modifications were introduced into the passenger strand.

Use of multicellular spheroids in studies of nanoparticles (NPs) has increased in the last decade, however details of NPs interaction with spheroids are poorly known. We synthesized AuNPs (12.0 ± 0.1 nm in diameter, transmission electron microscopy (TEM data) and covered them with bovine serum albumin (BSA) and polyethyleneimine (PEI). Values of hydrodynamic diameter were 17.4 ± 0.4; 35.9 ± 0.5 and ±125.9 ± 2.8 nm for AuNPs, AuBSA-NPs and AuPEI-NPs, and Z-potential (net charge) values were −33.6 ± 2.0; −35.7 ± 1.8 and 39.9 ± 1.3 mV, respectively. Spheroids of human hepatocarcinoma (HepG2) and human embryo kidney (HEK293) cells (Corning ® spheroid microplates CLS4515-5EA), and monolayers of these cell lines were incubated with all NPs for 15 min–4 h, and fixed in 4% paraformaldehyde solution. Samples were examined using transmission and scanning electron microscopy. HepG2 and HEK2893 spheroids showed tissue-specific features and contacted with culture medium by basal plasma membrane of the cells. HepG2 cells both in monolayer and spheroids did not uptake of the AuNPs, while AuBSA-NPs and AuPEI-NPs readily penetrated these cells. All studied NPs penetrated HEK293 cells in both monolayer and spheroids. Thus, two different cell cultures maintained a type of the interaction with NPs in monolayer and spheroid forms, which not depended on NPs Z-potential and size.

Fluorophore (FD) labeling is widely used for detection and quantification of various compounds bound to nanocarriers. The systems, composed of gold nanoparticles (GNPs) and oligonucleotides (ONs) labeled with FDs, have wide applications. Our work was aimed at a systemic study of how FD structure (in composition of ON-FDs) influenced the efficiency of their non-covalent associates’ formation with GNPs (ON-FD/GNPs). We examined ONs of different length and nucleotide composition, and corresponding ON-FDs (FDs from a series of xanthene, polymethine dyes; dyes based on polycyclic aromatic hydrocarbons). Methods: fluorometry, dynamic light scattering, high performance liquid chromatography, gel electrophoresis, molecular modeling and methods of thermodynamic and statistical analysis. We observed significant, differing several times, changes in surface density and Langmuir constant values of ON-FDs vs. ONs, evidence for the critical significance of FD nature for binding of ON-FDs with GNPs. Surface density of ON-FD/GNPs; hydrophobicity and total charge of ON or ON-FD; and charge and surface area of FDs were revealed as key factors determining affinity (Langmuir constant) of ON or ON-FDs for GNPs. These factors compose a specific set, which makes possible the highly reliable prediction of efficiency of ONs and ON-FDs binding with GNPs. The principal possibility of creating an algorithm for predictive calculation of efficiency of ONs and GNPs interaction was demonstrated. We proposed a hypothetical model that described the mechanism of contact interaction between negatively charged nano-objects, such as citrate-stabilized GNPs, and ONs or ON-FDs.

Understanding the patterns of noncovalent adsorption of double-stranded nucleic acids (dsDNA) on gold nanoparticles (GNPs) was the aim of this study. It was found that the high-affinity motifs in DNA can and do act as an “anchor” for the fixation of the whole molecule on the GNP (up to 98 ± 2 single-stranded (ss)DNA molecules per particle with diameter of 13 ± 2 nm). At the same time, the involvement of an “anchor” in the intramolecular DNA interaction can negatively affect the efficiency of the formation of ss(ds)DNA–GNP structures. It has been shown that the interaction of GNP with DNA duplexes is accompanied by their dissociation and competitive adsorption of ssDNAs on GNP, wherein the crucial factor of DNA adsorption efficiency is the intrinsic affinity of ssDNA to GNP. We propose a detailed scheme for the interaction of dsDNA with GNPs, which should be taken into account in studies of this type. Researchers focused on this field should accept the complicated nature of such objects and take into account the many competing processes, including the processes of adsorption and desorption of DNA on gold as well as the formation of secondary structures by individual DNA strands.