Vol 50, No 6 (2024)

This review presents an analysis of literature, including our own work, on various aspects of using RBC as an in vitro model in the comprehensive evaluation of antioxidant activity of a wide range of natural and synthetic compounds, their mixtures, and plant extracts. The existing practice of using human, laboratory, and domestic animal red blood cells is examined. The characteristics of the most commonly used initiators of oxidative stress in such studies, 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) and H₂O₂, as well as the mechanisms underlying the development of the hemolytic process are discussed. A critical analysis of methodological approaches to assessing the level of hemolysis is provided. The review further discusses the evaluation of erythrocyte survival under oxidative stress conditions and the ability of the tested compounds to act as membrane protectors. The text considers the criteria for a comprehensive assessment of erythrocytes, facilitating the study of cellular and molecular mechanisms underlying antioxidant activity of a wide range of substances on a model of oxidative hemolysis of erythrocytes. Traditional methods include assessment of the intensity of membrane lipid peroxidation (LPO) processes through measurement of concentration of products that react with 2-thiobarbituric acid, a s well assessment of relative content of oxidized forms of hemoglobin in erythrocytes. The use of modern fluorescent methods is another promising approach. In particular, the fluorescence of heme degradation products, the decrease in intensity of which can indicate the presence of antioxidant activity in the compounds under investigation, is a sensitive marker of oxidative stress in erythrocytes. Another prominent fluorescent method is the assessment of the level of oxidative stress by measuring the intracellular concentration of ROS in erythrocytes. Analysis of our own and literature data allows us to recommend the method of oxidative hemolysis of erythrocytes as the method to screen newly developed compounds in order to select the most interesting candidates for further in-depth studies. It is appropriate for establishing the structure-activity relationship and developing a strategy for the targeted synthesis of new biologically active compounds combining high hemocompatibility and antioxidant activity, promising for biomedical applications.

Vitamin B₁₂ is a vital biologically active compound for human and is involved in a wide range of metabolic processes. The widespread vitamin B₁₂ deficiency and vitamin’s low penetrating ability into cells determine the urgency of delivery systems development for the design of formulations with improved biopharmaceutical properties. This work provides a brief discussion of the main chemical and biochemical properties of the vitamin B₁₂, as well as considers oral, injectable and transdermal multicomponent dosage forms of vitamin B₁₂ that are aimed at solving the issue. Moreover, the literature analysis of the prospects of using vitamin B₁₂ as an auxiliary component for both passive and active delivery of other drug molecules, for example, peptide nucleic acids and antitumor drugs, is presented. The review describes in detail the types of proposed delivery systems for biologically active compounds, in which vitamin B₁₂ is one of the components.

A new approach to the automated synthesis of N-unsubstituted phosphoramidate oligodeoxyribonucleotides (P-NH₂) based on an optimized solid-phase phosphoramidite protocol using the Staudinger reaction has been proposed. The rapid and efficient oxidation of model P^(III)-containing phosphite triethers by the organic azide (9H-fluoren-9-yl)methylcarbonylazide (FmocN₃) to the corresponding phosphamides –(OPO(OR)(NFmoc))–, where R is a residue of nucleoside or alkyl nature, has been demonstrated. Removal of the alkaline-labile fluorenyl group from the modified internucleoside linkage allows the production of electroneutral, under physiological conditions of pH ~7, N-unsubstituted phosphoramidate (–(OPO(O)(NH₂))– or (P-NH₂)) residues in the oligonucleotide chain instead of the classical negatively charged phosphodiester (–(OPO(O)(O)(O¯))–) or (P-O)) residues. In optimizing the synthetic protocol, it has been demonstrated that to improve the efficiency of P-NH₂-oligonucleotide synthesis, it is necessary to include an additional Fmoc-group cleavage step in the automatic synthesis protocol after each oxidation step of the growing oligomer chain via the Staudinger reaction. An almost complete absence of dependence of the P-NH₂-oligonucleotide yield on both the localization of the P-NH₂-strand in the chain and the type of dinucleotide fragment being modified was shown. A set of mono- and bis-modified octadeoxyribonucleotides was obtained, and a detailed study of the thermal stability of complementary DNA/DNA complexes under different buffer conditions was performed. It was shown that under high ionic strength conditions (1 M NaCl, pH 7.2), the introduction of a single P-NH₂ strand reduced the thermostability of the DNA complex by an average of 1.3°C. When the ionic strength of the solution decreases, the destabilizing effect of the P-NH₂-modification decreases significantly, which further confirms the electroneutral status of the introduced phosphoramidate linkage. Thus, we have developed a protocol for the preparation of partially modified oligonucleotide derivatives bearing uncharged but isostructured to native P-O-strands – phosphoramidate residues P-NH₂.

Many regulatory neuropeptides contain a large amount of proline residues. The unique proline peptide bond conformation protects these peptides from enzymatic degradation; therefore enzymes cleaving the proline peptide bonds in neuropeptides are of particular interest. The abnormal activity of serine peptidases that cleave peptides at the carboxyl group of proline residues prolyl endopeptidase (PEP) and dipeptidyl peptidase IV (DPP-IV) were observed in patients with anxiety disorders. PEP is involved in the maturation and degradation of neuropeptides and peptide hormones, it also is associated with the regulation of blood pressure and various disorders of the central nervous system. DPP-IV is involved in many physiological processes, in particular in glucose homeostasis in type II diabetes and immunity. When studying the metabolism of the N-acyl derivative of the aminoacyl-2-cyanopyrrolidine PEP inhibitor a decreasing in the activity of DPP-IV at the initial time was detected. This was an unexpected effect observed for inhibitors of the general formula X-Y-2-S-cyanopyrrolidine, where X represents the N-protective group and Y represents the amino acid (any besides glycine and proline). Molecular dynamics simulations of inhibitor complexes with proteases revealed the possibility of PEP inhibitors binding in the DPP-IV active site with hydrogen bonds and hydrophobic interactions that allow linkage of the nitrile group with the catalytic serine residue in the DPP-IV active site. The present study opens the prospect of creating new pharmacologically active ligands of PEP and DPP-IV.

Despite a large number of works focused on the search for the mechanisms of formation of IgE-producing B cells, the question of the relative contribution of germinal centers and extrafollicular foci B cells in this process still remains controversial. Of particular interest is the study of the mechanisms of stimulation of the allergic immune response under the influence of air pollutants. The aim of the work was to study the connection between the adjuvant effect of benzo(a)pyrene (BaP) on the production of specific IgE in a novel low-dose allergy model with changes in the subpopulation composition of B-cells in the tissue of the immunization site and secondary lymphoid organs. Antigen without any stimuli was administrated to one group of BALB/c mice for 9 weeks in a low (0.3 μg) dose. BaP was administrated to another group of mice along with antigens at a dose of 4 ng. B-cell subpopulations were analyzed by flow cytometry. BaP significantly stimulated the production of allergen-specific IgG₁ at early (3 weeks) time point, and allergen-specific IgE at late (9 weeks) time point. The aeropollutant increased the content of CD19⁺CD38^–CD95⁺B220⁺ germinal center B-cells with the phenotype and their precursors (CD19⁺CD38⁺CD95⁺B220⁺) with the phenotype in the spleen at early and late time points, but not in the lungs or regional lymph nodes. Under its influence, the content of CD19⁺CD38^–CD95⁺B220^– and CD19⁺CD38⁺CD95⁺B220⁺ extrafollicular plasmablasts in the spleen at an early time point and in lung tissue at a later time point also increases. In the spleen, BaP increased the content of CD138⁺CD19^–B220⁺ and CD138⁺CD19^–B220^– mature plasma cells, and in regional lymph nodes the content of CD138⁺CD19⁺B220^– immature plasma cells at a later time point. The adjuvant effect of BaP on the production of specific IgE was largely associated with stimulation of the formation of germinal centers in the spleen and with extrafollicular activation of B cells in lung tissue.

Stability and monodispersity are important properties of nanoparticles and nanocomposites that ensure the reliability of their application in biological systems and the reproducibility of results. The preparation of non-agglomerated oligonucleotide-containing nanocomposites based on anatase titanium dioxide nanoparticles (Ans~ODN) is the aim of this work. The immobilization of oligodeoxynucleotides on TiO₂ nanoparticles has been studied by the dynamic light scattering and transmission electron microscopy. The antiviral activity of the synthesized samples has been performed on VERO cells infected with herpes simplex virus of the first type. The effect of NaCl on the agglomeration of nanoparticles and nanocomposites in aqueous solutions has been studied. The presence of NaCl leads to agglomeration of nanoparticles and nanocomposites. It has been shown that nanocomposites are formed in an aqueous solution in the absence of NaCl. A comparison of the biological activity of nanocomposites prepared in water and saline solution has been carried out with an example of inhibition of replication of the herpes simplex virus of the first type in the cell culture. The studied nanocomposite, regardless of the preparation method (in water or 0.9% NaCl), inhibited virus replication by 4.5 orders of magnitude when used 1 day after preparation. After 10 days of storage, the activity of the sample prepared in saline solution was two orders of magnitude lower than that of the active sample prepared in water. We have developed the method for the preparation of non-agglomerated oligonucleotide-containing nanocomposites based on anatase nanoparticles and demonstrated their potential use for the study of their biological activity. Unlike nanocomposites prepared in the presence of salt, which lose their efficacy during storage, nanocomposites that are not prone to agglomeration can be obtained in water for future use.