Synthesis of Nanoscale TiO2 and Study of the Effect of Their Crystal Structure on Single Cell Response

To study the effect of nanoscale titanium dioxide (TiO2) on cell responses, we synthesized four modifications of the TiO2 (amorphous, anatase, brookite, and rutile) capable of keeping their physicochemical characteristics in a cell culture medium. The modifications of nanoscale TiO2 were obtained by hydrolysis of TiCl4 and Ti(i-OC3H7)4 (TIP) upon variation of the synthesis conditions; their textural, morphological, structural, and dispersion characteristics were examined by a set of physicochemical methods: XRD, BET, SAXS, DLS, AFM, SEM, and HR-TEM. The effect of synthesis conditions (nature of precursor, pH, temperature, and addition of a complexing agent) on the structural-dispersion properties of TiO2 nanoparticles was studied. The hydrolysis methods providing the preparation of amorphous, anatase, brookite, and rutile modifications of TiO2 nanoparticles 3–5 nm in size were selected. Examination of different forms of TiO2 nanoparticles interaction with MDCK cells by transmission electron microscopy of ultrathin sections revealed different cell responses after treatment with different crystalline modifications and amorphous form of TiO2. The obtained results allowed us to conclude that direct contact of the nanoparticles with cell plasma membrane is the primary and critical step of their interaction and defines a subsequent response of the cell

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

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

Investigation of the Antiviral Activity of Experimental Samples Obtained from the Grass and Roots of Alchemilla vulgaris L. Against Vaccinia Virus and Ectromelia Virus

Introduction. The abolition of smallpox vaccination after its elimination in 1980 led to a decrease in the immunocompromised immunity in humans. Zoonotic monkeypox, camelpox, buffalopox and cowpox viruses that are close to the variola virus also pose a danger to humans. In Russia today there are no effective and safe medicines for the prevention and treatment of smallpox and other orthopoxvirus infections in humans and animals. The Lady's mantle (Alchemilla vulgaris) is a promising source for the development of new antiviral drugs. Previous studies have found that Alchemilla vulgaris shows activity against influenza virus and herpes simplex virus.Aim. The aim of this work was to study the chemical composition and antiviral activity of extracts from the roots and the grass of Alchemilla vulgaris against orthopoxviruses.Materials and methods. Qualitative analysis of the samples was performed by high performance liquid chromatography. Quantitative analysis was performed using a complex of spectrophotometric methods. To determine the toxicity and antiviral activity of experimental samples from Alchemilla vulgaris in vitro, a transplantable Vero cell culture was used. Antiviral activity of the obtained preparations was evaluated by reducing the infectivity (titer) of orthopoxviruses in the monolayer of Vero cells infected with orthopoxviruses in the presence of preparations with different concentrations relative to the cell culture infected with orthopoxviruses without the preparations.Results and discussion. It was shown that the experimental sample from Alchemilla vulgaris obtained by the method of ethyl acetate extraction from the roots of the studied plant and purified with chloroform contains mainly catechins and leucoanthocyanins (70 %). In parallel, extract from the raw mass of the grass of the plant purified with chloroform and ethanol contains the amount of flavonoids (71 %). Wherein the content of flavonoids in unpurified ethanol extracts from the roots and the grass of Alchemilla vulgaris composed 5 % and 6 %, respectively. It was revealed that purified preparations obtained from Alchemilla vulgaris roots when using ethyl acetate and ethanol as extractants showed antiviral activity against vaccinia virus and ectromelia virus, as well as preparations obtained from grass by ethanol extraction.Conclusion. Thus, purified ethyl acetate extracts from the roots and ethanol extracts from the wet grass mass of Alchemilla vulgaris exhibit antiviral activity against orthopoxviruses in vitro

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–NH2Flu nanoparticles and Si–NH2·ODNFlu nanocomplexes were shown by fluorescence microscopy to penetrate into human cells. The Si–NH2Flu 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

Safety and Pharmacokinetics of the Substance of the Anti-Smallpox Drug NIOCH-14 after Oral Administration to Laboratory Animals

Background: Since most of the modern human population has no anti-smallpox immunity, it is extremely important to develop and implement effective drugs for the treatment of smallpox and other orthopoxvirus infections. The objective of this study is to determine the main characteristics of the chemical substance NIOCH-14 and its safety and bioavailability in the body of laboratory animals. Methods: The safety of NIOCH-14 upon single- or multiple-dose intragastric administration was assessed according to its effect on the main hematological and pathomorphological parameters of laboratory mice and rats. In order to evaluate the pharmacokinetic parameters of NIOCH-14 administered orally, a concentration of ST-246, the active metabolite of NIOCH-14, in mouse blood and organs was determined by tandem mass spectrometry and liquid chromatography. Results: The intragastric administration of NIOCH-14 at a dose of 5 g/kg body weight caused neither death nor signs of intoxication in mice. The intragastric administration of NIOCH-14 to mice and rats at doses of 50 and 150 µg/g body weight either as a single dose or once daily during 30 days did not cause animal death or critical changes in hematological parameters and the microstructure of internal organs. The tissue availability of NIOCH-14 administered orally to the mice at a dose of 50 µg/g body weight, which was calculated according to concentrations of its active metabolite ST-246 for the lungs, liver, kidney, brain, and spleen, was 100, 69.6, 63.3, 26.8 and 20.3%, respectively. The absolute bioavailability of the NIOCH-14 administered orally to mice at a dose of 50 µg/g body weight was 22.8%. Conclusion: Along with the previously determined efficacy against orthopoxviruses, including the smallpox virus, the substance NIOCH-14 was shown to be safe and bioavailable in laboratory animal experiments