Laboratory of Radionuclides and Labeled Compounds

Chaschin I.S., Perepelkin E.I., Sinolits M.A., Badun G.A., Chernysheva M.G., Ivanova N.M., Vasilev V.G., Kizas O.A., Anuchina N.M., Khugaev G.A., Britikov D.V., Bakuleva N.P.

The patterns of formation of chitosan nanoparticles doped with vancomycin and coatings based on them in carbonate solutions have been investigated for the first time in this study. Using a technique of radioactive indicators, it was found that at a CO

Bunyaev V.A., Sinolits A.V., Badun G.A.

Zhidkova E.M., Tilova L.R., Fetisov T.I., Kirsanov K.I., Kulikov E.P., Enikeev A.D., Budunova I.V., Badun G.A., Chernysheva M.G., Shirinian V.Z., Yakubovskaya M.G., Lesovaya E.A.

Glucocorticoids (GCs) are widely used for treating hematological malignancies despite their multiple adverse effects. The biological response to GCs relies on glucocorticoid receptor (GR) transrepression (TR) that mediates the anticancer effects and transactivation (TA) associated with the side effects. Selective GR agonists (SEGRAs) preferentially activating GR TR could offer greater benefits in cancer treatment. One of the well-characterized SEGRAs, 2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium-chloride (CpdA), exhibited anticancer activity; however, its translational potential is limited due to chemical instability. To overcome this limitation, we obtained CpdA derivatives, CpdA-01–CpdA-08, employing two synthetic strategies and studied their anti-tumor activity: 4-(1-hydroxy-2-(piperidin-1-yl)ethyl)phenol or CpdA-03 demonstrated superior GR affinity and stability compared to CpdA. In lymphoma Granta and leukemia CEM cell lines, CpdA-03 ligand exhibited typical SEGRA properties, inducing GR TR without triggering GR TA. CpdA-03 effects on cell viability, growth, and apoptosis were similar to the reference GR ligand, dexamethasone (Dex), and the source compound CpdA. In vivo testing of CpdA-03 activity against lymphoma on the transplantable P388 murine lymphoma model showed that CpdA-03 reduced tumor volume threefold, outperforming Dex and CpdA. In conclusion, in this work, we introduce a novel SEGRA CpdA-03 as a promising agent for lymphoma treatment with fewer side effects.

Zhidkova E.M., Tilova L.R., Fetisov T.I., Kirsanov K.I., Kulikov E.P., Enikeev A.D., Budunova I.V., Badun G.A., Chernysheva M.G., Shirinian V.Z., Yakubovskaya M.G., Lesovaya E.A.

Glucocorticoids (GCs) are widely used in blood cancer treatment despite their multiple adverse effects. Biological response to GCs is mediated by glucocorticoid receptor (GR) by transactivation (TA), associated with side effects, and transrepression (TR), mediating anticancer effects. Selective GR agonists (SEGRAs) preferentially activating GR TR could be better option for cancer treatment. One of well-characterized SEGRAs is 2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium-chloride (CpdA), demonstrated anticancer activity in vitro and in vivo, however its translational potential is limited due to chemical instability. CpdA derivatives CpdA-01-08 were designed by two synthetic strategies. Derivative CpdA-03 demonstrated superior GR affinity and stability compared to CpdA. Analysis of CpdA-03 ligand properties in lymphoma Granta and leukemia CEM cell lines revealed its SEGRA profile: it induced GR TR without triggering GR TA, as evidenced by changes in expression of GR target genes. Effects of CpdA-03 on cell viability, growth, and apoptosis were similar to reference GR ligand, dexamethasone (Dex), and the proto-type compound CpdA. In vivo testing of CpdA-03 anti-lymphoma activity in transplantable P388 murine lymphoma model showed that CpdA-03 reduced tumor volume threefold, outperforming Dex and CpdA. In conclusion, this work introduces CpdA-03 as promising new SEGRA with potential for further development as anti-lymphoma drug with fewer side effects.

Bunyaev V.A., Chernysheva M.G., Badun G.A.

Complexes of carbon nanotubes (CNTs) with bovine serum albumin (BSA) and chitosan are synthesized via adsorption from aqueous solutions. Using tritium-labeled compounds, it is shown that the amount of biopolymers bound to CNTs is proportional to their concentration in solution. It is shown that the resulting complexes are stable; they do not degrade after removing the adsorbate solution and washing with water. CNTs with the studied substances are modified sequentially. It is found that the order of sequential modification does not affect the composition of the complex, which can be attributed to the absence of competition for binding sites. It is shown that the synthesized complexes have high stability of sedimentation in aqueous media. The electrokinetic potential of the CNT–chitosan complex reaches a value of +36 mV at a modifying agent concentration of 8 mg/mL. Upon the sequential modification of CNTs with BSA first and then with chitosan, the electrokinetic potential of the complex falls to +20 mV. The electrokinetic potential of the CNT–BSA complex changes from −10 to −18 mV upon raising the concentration of protein in the solution from 0.2 to 5 mg/mL. The sequential modification of CNTs with chitosan first and then with BSA produces complexes with low electrokinetic potentials. Measurements of the angle of contact show that modifying CNTs with chitosan and BSA raises the hydrophilicity of the coatings deposited on glass.

Chernysheva M.G., Shen T., Badun G.A., Mikheev I.V., Chaschin I.S., Tsygankov Y.M., Britikov D.V., Hugaev G.A., Bakuleva N.P.

Coatings with xenogenic materials, made of detonation nanodiamonds, provide additional strength and increase elasticity. A functionally developed surface of nanodiamonds makes it possible to apply antibiotics. Previous experiments show the stability of such coatings; however, studies on stability in the bloodstream and calcification of the material in natural conditions have yet to be conducted. Tritium-labeled nanodiamonds (negative and positive) were obtained by the tritium activation method and used to develop coatings for a pork aorta to analyze their stability in a pig’s bloodstream using a radiotracer technique. A chitosan layer was applied from a solution of carbonic acid under high-pressure conditions to prevent calcification. The obtained materials were used to prepare a porcine conduit, which was surgically stitched inside the pig’s aorta for four months. The aorta samples, including nanodiamond-coated and control samples, were analyzed for nanodiamond content and calcium, using the radiotracer and ICP-AES methods. A histological analysis of the materials was also performed. The obtained coatings illustrate a high in vivo stability and low levels of calcification for all types of nanodiamonds. Even though we did not use additional antibiotics in this case, the development of infection was not observed for negatively charged nanodiamonds, opening up prospects for their use in developing coatings.

Badun G.A., Severin A.V., Zaitseva E.A.

[3H]Hyaluronic acid with a molecular weight of 2.37 MDa and a specific radioactivity of 35 GBq/g was obtained using the tritium thermal activation method. Tritium labeled hyaluronic acid (HA) with a molecular weight of 2.37, 0.20, and 0.10 MDa were used to study their adsorption on hydroxyapatite (HAP) in two textural modifications: aqueous suspension and powder. Differences in adsorption kinetics and adsorption isotherms were observed due to variations in the interaction between polysaccharide molecules and the sorbent forms under consideration. The adsorption isotherms of HA on HAP were proven linear. It is shown that strong HA–HAP complexes are formed, and no desorption of HA into water and 0.9% NaCl solution were detected in 2 days.

Badun G.A., Bunyaev V.A., Chernysheva M.G.

Erratum

Badun G.A., Bunyaev V.A., Chernysheva M.G.

Abstract The possibility of tritium introduction into graphene oxide (GO) by tritium thermal activation method was demonstrated. It was established that, in order to produce the highest possible specific radioactivity, thin films of GO with a thickness of 5.6 mg/m2 must be treated with tritium atoms. The experiment conducted at 77 K showed a number of advantages. GO was processed with tritium atoms, the resulting specific activity of [3H]GO reached 2.6 Ci/mg in term of the weight of the initial GO (0.7 Ci/mg after removal of the labile tritium). Specific energy release of [3H]GO with this specific activity is 22.3 W/kg, which is quite sufficient for its application as a component of a nuclear battery.

Chernysheva M.G., Badun G.A., Popov A.G., Chashchin I.S., Anuchina N.M., Panchenko A.V.

Creation of biocompatible coatings for xenogenic materials that can be used to manufacture prosthetic heart valves is an urgent and, unfortunately, still unsolved problem. It is necessary to obtain a biomaterial that would comply with the mechanical characteristics of a human valve and possess antimicrobial properties, which are of critical importance during the first postsurgical days. Biocompatible coatings can be used for this purpose, and it has turned out that detonation nanodiamonds are suitable for their preparation. The developed functional surface of nanodiamonds allows them to adsorb antibiotics; nanodiamonds are nontoxic and do not cause additional calcification. In this study, we have proposed to prepare a composite coating composed of nanodiamonds, lysozyme, and miramistin as broad-spectrum antimicrobial agents. The use of tritium-labeled nanodiamonds has made it possible to study the distribution of nanodiamond–lysozyme complexes after intravenous administration to mice and showed that the majority of the material remains at the place of injection. It has been shown that nanodiamond–lysozyme–miramistin composites exhibit strong antimicrobial activity, while the nanodiamond–miramistin complex shows no toxicity with respect to Staphylococcus aureus. Thus, the nanodiamond–lysozyme–miramistin composite can be used to create coatings for materials of prosthetic heart valves.

Kangina O.A., Chernysheva M.G., Badun G.A., Lishai A.V., Tsygankova N.G., Savitskaya T.A., Grinshpan D.D.

This work is devoted to studying the adsorption of sodium dodecyl sulfate (SDS) and sodium N-lauryl sarcosinate (NLS) on activated carbons produced from hydrolytic lignin using laboratory and pilot setups. Commercially available activated carbons produced by “Sorbent” Ltd. and SKT-3 are used for comparison. The surface of the activated carbons is modified (hydrophilized) by cellulose sulfoacetate (CSA) adsorption from aqueous solutions. Amounts of surfactants adsorbed on the activated carbon surface are determined from their radioactivity using tritium-labeled compounds. It has been found that the maximum adsorption of anionic surfactants is higher on activated carbons obtained from lignin. Adsorption values in the saturation region are 1.7 and 1.5 mmol/g for SDS and NLS on activated carbons obtained from lignin and 0.8 mmol/g for both surfactants adsorbed on commercial carbons. It has been shown that the preliminary application of CSA does not affect the maximum adsorption of NLS at its high concentrations; however, it reduces its adsorption in the initial region of the isotherm. Adsorption of CSA and its ability to be retained on the surface of the activated carbons is also studied. Tritium-labeled CSA has been used to show that the isotherm of its adsorption on activated carbon obtained from lignin is S-shaped beginning from a concentration of 0.5 g/L and reaches a maximum value of 25 mg/g. In the presence of NLS, the polymer is desorbed from the carbon surface by more than 50%.

Chernysheva M.G., Shen T., Chaschin I.S., Badun G.A., Vasil'ev V.G., Mikheev I.V., Bakuleva N.P.

Multicomponent coatings of collagen-rich matrices of bovine pericardium were prepared from nanodiamonds and antibiotics, namely, amikacin, levofloxacin and chitosan. Preservation of the multilayers that consist of nanodiamonds, antimicrobial agents and chitosan on the material surface and preventing the calcification of the material were revealed.

Skrabkova H.S., Chernysheva M.G., Baygildiev T.M., Shnitko A.V., Kasperovich A.V., Egorova T.B., Badun G.A., Arutyunyan A.M., Ksenofontov A.L., Rodin I.A.

Lysozyme complexes with amikacin and levofloxacin were studied by spectroscopy approaches as well as using a tritium probe. Tritium was used as a labeling agent to trace labeled compound concentration in a system of two immiscible liquids and in the atomic form to determine the possible position of the binding site. Co-adsorption of protein and drug at the liquid-liquid interface was analyzed by scintillation phase method that allowed us to directly determine the amount of protein and drug in the mixed adsorption layer. Also, tensiometric measuring of the interfacial tension was used for calculation of binding parameters accordingly to Fainerman model. The treatment of complexes with atomic tritium followed by trypsinolysis and analysis of tritium distribution in the lysozyme peptides reveals the binding sites, binding energies in which were analyzed using molecular docking. Formation of complexes with amikacin and levofloxacin preserves secondar structure of protein. However, the formation of complex with amikacin leads to the almost total loss of the enzymatic activity of lysozyme and the redshift of the maximum on the lysozyme fluorescence band. A slight decrease in the distribution coefficient of lysozyme in the presence of amikacin assumes that the complex has higher hydrophilicity in comparison to lysozyme without additives. The most favorable for binding were the positions of the active centers that included amino acids Asp52 and Glu35, as well as in the vicinity of peptide His15-Arg21, with the participation of amino acids Tyr20, Arg14. In the case of levofloxacin, the formation of lysozyme-ligand complex in aqueous solution is possible without changing the microenvironment of the active center of the protein. Binding of levofloxacin to the active center of the enzyme was the most favorable, but Asp52 and Glu35 that are responsible for the enzymatic activity of lysozyme, were not affected.

Ksenofontov A.L., Baratova L.A., Semenyuk P.I., Fedorova N.V., Badun G.A.

Coat proteins (CP) of the potato virus A virions (PVA) contain partially disordered N-terminal domains, which are necessary for performing vital functions of the virus. Comparative analysis of the structures of coat proteins (CPs) in the intact PVA virions and in the virus particles lacking N-terminal 32 amino acids (PVAΔ32) was carried out in this work based on the tritium planigraphy data. Using atomic-resolution structure of the potato virus Y potyvirus (PVY) protein, which is a homolog of the CP PVA, the available CP surfaces in the PVY virion were calculated and the areas of intersubunit/interhelix contacts were determined. For this purpose, the approach of Lee and Richards [Lee, B., and Richards, F. M. (1971) J. Mol. Biol., 55, 379-400] was used. Comparison of incorporation profiles of the tritium label in the intact and trypsin-degraded PVA∆32 revealed position of the ΔN-peptide shielding the surface domain (a.a. 66-73, 141-146) and the interhelix zone (a.a. 161-175) of the PVA CP. Presence of the channels/cavities was found in the virion, which turned out to be partially permeable to tritium atoms. Upon removal of the ∆N-peptide, decrease in the label incorporation within the virion (a.a. 184-200) was also observed, indicating possible structural transition leading to the virion compactization. Based on the obtained data, we can conclude that part of the surface ∆N-peptide is inserted between the coils of the virion helix thus increasing the helix pitch and providing greater flexibility of the virion, which is important for intercellular transport of the viruses in the plants.

Shen Т., Chernysheva M.G., Badun G.A.

Procedure of tritium labeling vancomycin synthesis using tritium thermal activation method was developed. The influence of target mass on the specific and total radioactivity was revealed. [3H]Vancomycin was used for studying its equilibrium adsorption on nanodiamonds as well as its amount that tightly bonded to surface and did not remove with water. It was found that vancomycin is adsorbed from aqueous solution via strong bonding to nanodiamonds and is not washed out with water. Application of 0.028 M phosphate buffer (pH 6.7 and 2.7) increased the equilibrium vancomycin adsorption about one and a half times, while vancomycin amount in the adsorption complex with nanodiamonds after washing with water was significantly reduced. Such behavior of vancomycin is due to the presence of phosphate-ions that contribute to vancomycin adsorption but are washed out with water. Molecular mechanics simulation allows the suggestion that the formation of multiple hydrogen bonds is required for generation of a durable adsorption complex of vancomycin with nanodiamonds.