Natalia A. Mazurkova

High-performance method for specific effect on nucleic acids in cells using TiO2~DNA nanocomposites

Nanoparticles are used to solve the current drug delivery problem. We present a high-performance method for efficient and selective action on nucleic acid target in cells using unique TiO2·PL-DNA nanocomposites (polylysine-containing DNA fragments noncovalently immobilized onto TiO2 nanoparticles capable of transferring DNA). These nanocomposites were used for inhibition of human influenza A (H3N2) virus replication in infected MDCK cells. They showed a low toxicity (TC50 ≈ 1800 μg/ml) and a high antiviral activity (>99.9% inhibition of the virus replication). The specificity factor (antisense effect) appeared to depend on the delivery system of DNA fragments. This factor for nanocomposites is ten-times higher than for DNA in the presence of lipofectamine. IC50 for nanocomposites was estimated to be 1.5 μg/ml (30 nM for DNA), so its selectivity index was calculated as ~1200. Thus, the proposed nanocomposites are prospective for therapeutic application.

Efficient inhibition of human influenza a virus by oligonucleotides electrostatically fixed on polylysine-containing TiO2 nanoparticles

Antiviral activity of the TiO2·PL·DNA nanobiocomposites was studied on the MDCK cell culture infected with influenza A virus (subtype H3N2). DNA fragments in the nanocomposites are electrostatically bound to titanium dioxide nanoparticles precovered with polylysine. It was shown that TiO2·PL·DNA(v3′) nanocomposite bearing the DNA(v3′) fragment targeted to the 3′-end of the noncoding region of segment 5 of viral RNA specifically inhibited the virus reproduction with the efficiency of 99.8% and 99.9% (i.e., by factors of ∼400 and 1000, respectively) at a low concentration of DNA(v3′) in nanocomposite (0.1 and 0.2 μM, respectively). The TiO2·PL·DNA(r) nanocomposite containing an oligonucleotide noncomplementary to viral RNA or oligonucleotide DNA(v3′) unbound to the nanoparticles show very low antiviral activity (inhibition by factors of ∼3.5 and 1.3, respectively).

[Composites of Peptide Nucleic Acids with Titanium Dioxide Nanoparticles. IV+. Antiviral Activity of Nanocomposites Containing DNA/PNA Duplexes].

Antiviral activity of TiO2 · PL · DNA/PNA nanobiocomposites was studied on the MDCK cell culture infected with influenza A virus (H3N2 subtype). A PNA fragment in nanocomposites was electrostatically bound in the form of a DNA/PNA heteroduplex to titanium dioxide nanoparticles precovered with polylysine (TiO2 · PL). It was shown that TiO2 · PL · DNA1/PNA1 nanobiocomposite bearing the PNA1 fragment targeted to the 3′-end of the noncoding region of segment 5 of viral RNA specifically inhibited the virus reproduction with an efficiency of 99.8%. The 50% cytotoxic concentration (TC50) and 50% effective inhibitory concentration (IC50) of the TiO2 · PL · DNA1/PNA1 nanocomposite were evaluated to be more than 1200 μg/mL and less than 3 μg/mL, respectively. Based on these data, the selectivity index (SI) for TiO2 · PL · DNA1/PNA1 nanobiocomposite defined as the TC50/IC50 ratio, was calculated to be more than 400. Thus, TiO2 · PL · DNA/PNA nanobiocomposites were shown to not only penetrate through cell membranes, but exhibit a high specific antisense activity without toxic effects on the living cells.

High antiviral effect of TiO2·PL–DNA nanocomposites targeted to conservative regions of (−)RNA and (+)RNA of influenza A virus in cell culture

Summary Background: The development of new antiviral drugs based on nucleic acids is under scrutiny. An important problem in this aspect is to find the most vulnerable conservative regions in the viral genome as targets for the action of these agents. Another challenge is the development of an efficient system for their delivery into cells. To solve this problem, we proposed a TiO2·PL–DNA nanocomposite consisting of titanium dioxide nanoparticles and polylysine (PL)-containing oligonucleotides. Results: The TiO2·PL–DNA nanocomposites bearing the DNA fragments targeted to different conservative regions of (−)RNA and (+)RNA of segment 5 of influenza A virus (IAV) were studied for their antiviral activity in MDCK cells infected with the H1N1, H5N1, and H3N2 virus subtypes. Within the negative strand of each of the studied strains, the efficiency of DNA fragments increased in the direction of its 3’-end. Thus, the DNA fragment aimed at the 3’-noncoding region of (−)RNA was the most efficient and inhibited the reproduction of different IAV subtypes by 3–4 orders of magnitude. Although to a lesser extent, the DNA fragments targeted at the AUG region of (+)RNA and the corresponding region of (−)RNA were also active. For all studied viral subtypes, the nanocomposites bearing the DNA fragments targeted to (−)RNA appeared to be more efficient than those containing fragments aimed at the corresponding (+)RNA regions. Conclusion: The proposed TiO2·PL–DNA nanocomposites can be successfully used for highly efficient and site-specific inhibition of influenza A virus of different subtypes. Some patterns of localization of the most vulnerable regions in IAV segment 5 for the action of DNA-based drugs were found. The (−)RNA strand of IAV segment 5 appeared to be more sensitive as compared to (+)RNA.

Efficient inhibition of influenza A viral replication in cells by deoxyribozymes delivered by nanocomposites

Nucleic-acid-based drugs are a promising class of novel therapeutics; however, their use in medicine is widely limited because of insufficient delivery into cells. This article proposes a new delivery strategy of nucleic acid fragments into cells as components of TiO2-based nanocomposites. For the first time, unmodified Dz molecules were non-covalently immobilized on TiO2 nanoparticles precovered with polylysine (TiO2•PL) with the formation of (TiO2•PL)•Dz nanocomposites. DNAzymes in the proposed nanocomposites were shown to retain their ability to cleave the RNA target in a cell-free system with the same selectivity as unbound Dz molecules. It was shown by confocal laser microscopy that the fluorescein-labelled (TiO2•PL)•DzFlu nanocomposites penetrate into eukaryotic cells, where DzFlu is internalized in the cytoplasm and predominantly in nuclei. Delivery of deoxyribozymes into cells in the proposed nanocomposites permits very efficient interactions with RNA targets inside cells. This was demonstrated by an example of inhibition of H5N1 influenza A virus replication (inhibition by a factor of ca. 3000). This effect was one order of magnitude higher than with using lipofectamine as the transfection agent. The proposed (TiO2•PL)•Dz nanocomposites demonstrated high antiviral activity and are thus potent as nucleic-acid-based drugs.

Non-agglomerated silicon–organic nanoparticles and their nanocomplexes with oligonucleotides: synthesis and properties

The development of efficient and convenient systems for the delivery of nucleic-acid-based drugs into cells is an urgent task. А promising approach is the use of various nanoparticles. Silica nanoparticles can be used as vehicles to deliver nucleic acid fragments into cells. In this work, we developed a method for the synthesis of silicon–organic (Si–NH2) non-agglomerated nanoparticles by the hydrolysis of aminopropyltriethoxysilane (APTES). The resulting product forms a clear solution containing nanoparticles in the form of low molecular weight polymer chains with [─Si(OH)(C3H6NH2)O─] monomer units. Oligonucleotides (ODN) were conjugated to the prepared Si–NH2 nanoparticles using the electrostatic interaction between positively charged amino groups of nanoparticles and negatively charged internucleotide phosphate groups in oligonucleotides. The Si–NH2 nanoparticles and Si–NH2·ODN nanocomplexes were characterized by transmission electron microscopy, atomic force microscopy and IR and electron spectroscopy. The size and zeta potential values of the prepared nanoparticles and nanocomplexes were evaluated. Oligonucleotides in Si–NH2·ODN complexes retain their ability to form complementary duplexes. The Si–NH2 Flu nanoparticles and Si–NH2·ODNFlu nanocomplexes were shown by fluorescence microscopy to penetrate into human cells. The Si–NH2 Flu nanoparticles predominantly accumulated in the cytoplasm whereas ODNFlu complexes were predominantly detected in the cellular nuclei. The Si–NH2·ODN nanocomplexes demonstrated a high antisense activity against the influenza A virus in a cell culture at a concentration that was lower than their 50% toxic concentration by three orders of magnitude.

Nitraria schoberi L. hairy root culture as a source of compounds with antiviral activity against influenza virus subtypes А(H5N1) and А(H3N2)

The hairy root culture of halophyte Nitraria schoberi was obtained by transforming seedlings’ primary leaves with a wild strain of Agrobacterium rhizogenes 15834 SWISS (transformation frequency was 78.7%). The roots’ transgenic status was confirmed by PCR analysis using rolB-, rolC-, virC-, virD1-specific primers. There was a significant increase in the biosynthesis of flavonoids, hydroxycinnamic acids, pectins, protopectins and saponins in hairy roots as compared to intact plant roots. The сatechin content in ethanol extracts of transformed roots’ exceeded this factor 3.8 times in plant roots, but the compound composition of catechin nature in the latter was more diverse. The high antiviral activity of N. schoberi hairy root ethanol extracts against influenza virus subtypes A(H5N1), A(H3N2) was revealed, which manifested itself in the effective inhibition of infectivity both in vitro—in MDCK cell culture—and in vivo increasing survival of mice infected with them.