Igor Dmitrievich Zlotnikov

Arginase is an enzyme of medical significance that plays a crucial role in research on macrophages, in the field of oncology, and serves as a biomarker in various areas. However, the current methods for assessing its activity rely on indirect, time-consuming colorimetric assays, which cannot provide real-time kinetic data in complex media. This is a critical aspect in biomedical research. To address this issue, we have developed two novel spectroscopic methods without the use of reagents, based on circular dichroism (CD) spectroscopy and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. The approach based on CD proved to be particularly effective in capturing the complete progress curve of arginine hydrolysis within a single measurement, with high sensitivity, enabling detection limits as low as 0.026 U/mL. Nonetheless, the use of CD is primarily restricted to homogeneous and optically transparent solutions. In contrast, ATR-FTIR allows for real-time monitoring of specific bands within the substrate and product (arginine and ornithine, respectively) in complex and non-transparent media such as cell cultures, without the need for sample preparation. This method enables us to measure arginase activity in a living culture of Lewis lung carcinoma cells (LLC1), where the liposomal form of arginase demonstrates a remarkable 6.4-fold increase in specific activity compared to the native enzyme.

Fluoroquinolones (ciprofloxacin and levofloxacin) are broad-spectrum antibiotics, but their use is limited by low solubility, toxicity, and the risk of resistance. The use of cyclodextrins and polymeric carriers is a promising approach to overcome these limitations. In this work, we systematically studied the complexation of ciprofloxacin (CF) with β-cyclodextrin derivatives — 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), methyl-β-cyclodextrin (M-β-CD), and sulfobutyl ether β-cyclodextrin (SBE-β-CD) — using IR and UV spectroscopy, equilibrium dialysis, and solubility assays. The most stable complex is formed with HP-β-CD (Kdis = 1.7 × 10−4 M), which increases the solubility of CF by 20% (pH 4.0) and 64% (pH 7.4) and provides sustained release. Based on these data, two types of polymeric complexes of levofloxacin (LF) with enterosgel, and silica gel/HP-β-CD were developed for aquaculture. Influence of dihydroquercetin on physicochemical parametres of complexes has been studied. The complexes showed high loading efficiency (85–95%) and sorption capacity up to 1.0 mg/mg. The solubility of LF complexes was reduced by 6 times compared to the free drug, and release was prolonged up to 3 days. The synthesized complexes antimicrobial activity against E. coli and B. subtilis was higher than that of the commercial drug "Antibak". The developed complexes represent a promising environmentally safe strategy for aquaculture.

Arginine-deprivation therapy exploits the arginine auxotrophy of numerous tumor types, positioning arginase (EC 3.5.3.1) as a next-generation antitumor biocatalyst. Robust continuous activity assays are essential to drive the rational design of new drug formulations and support clinical diagnostics, where arginase serves as a disease biomarker and an indicator of macrophage polarization states. However, current evaluation relies on indirect, stop-and-develop colorimetric assays that cannot resolve real-time kinetics in complex, turbid media. Here, we developed two label-free, real-time kinetic assays that monitor the native substrate and product directly: far-UV absorbance and circular dichroism (CD) at 205 nm, and FTIR tracking the guanidinium (~1630–1675 cm–1) and carboxylate/amine bands of L-arginine and L-ornithine. These methods eliminate the need for coupled enzymes and chromogenic reagents. Both approaches resolved the full progress curve of arginine hydrolysis in a single run and returned Michaelis-Menten parameters consistent with literature values, featuring detection limits down to 0.025 U/mL. To demonstrate our methodology in complex microheterogeneous media, we evaluated a liposomal arginase formulation (lipo-ARG), a relevant delivery system for targeting hepatocellular carcinoma. Kinetic testing revealed that encapsulation enhances the enzyme's operational stability, increasing its functional persistence by up to 200-fold under stress. To elucidate this stabilization mechanism, we analyzed thermograms, conformational dynamics, and protein structures. CD spectroscopy and thermodynamic analysis demonstrated strong structural protection: while the free enzyme completely unfolds and aggregates under acute thermal stress, the liposomal formulation retains a significant portion of its intact secondary structure even at 80 °C. The lipid bilayer acts as a rigid molecular scaffold, raising the unfolding enthalpy from 156 to 466 kJ/mol and preventing bulk aggregation. Structural locking correlates with the sustained residual catalytic activity of the liposomal enzyme. The developed continuous assays successfully validate liposome encapsulation as an effective strategy for creating robust enzyme formulations for oncotherapy.

A series of molecularly imprinted polymers (MIPs) based on hydroxypropyl-β-cyclodextrin (HPCD) crosslinked with 1,6-hexamethylene diisocyanate (HMD) or toluene diisocyanate (TDI), as well as hybrid chitosan–HPCD polymers crosslinked with genipin, were synthesized using levofloxacin and fluorescein as template molecules. The structure and spatial organization of the obtained materials were characterized by FTIR spectroscopy, FTIR microscopy mapping, and ζ-potential measurements. The influence of pH, crosslinker content, and template structure on sorption performance was investigated. All MIPs exhibited maximum sorption at pH 3.0. The highest sorption capacity toward levofloxacin was achieved for the LV–Chit–HPCD–Genipin_MIP (74.8 mg/g), whereas the fluorescein-imprinted FL–HPCD–TDI_MIP (1:1) demonstrated the highest sorption capacity (135.5 mg/g) and selectivity coefficient (84.1). Dynamic column experiments confirmed efficient analyte extraction, reducing the analyte concentration by more than 90% after ten loading cycles. All synthesized MIPs exhibited excellent regenerability, with less than 3% loss of sorption efficiency after ten consecutive sorption–desorption cycles. The applicability of the developed sorbents to real matrices was demonstrated using milk and blood plasma samples after minimal sample preparation. Fluorescein extraction efficiencies reached 97.6% and 93.6% for milk and plasma, respectively. The obtained results demonstrate that HPCD-based MIPs combine high sorption capacity, exceptional selectivity, operational stability, and applicability to complex biological matrices, making them promising materials for selective sample preparation, analyte preconcentration, and controlled drug delivery systems.

A series of molecularly imprinted polymers (MIPs) based on hydroxypropyl-β-cyclodextrin (HPCD) crosslinked with 1,6-hexamethylene diisocyanate (HMD) or toluene diisocyanate (TDI), as well as hybrid chitosan–HPCD polymers crosslinked with genipin, were synthesized using levofloxacin and fluorescein as template molecules. The structure and spatial organization of the obtained materials were characterized by FTIR spectroscopy, FTIR microscopy mapping, and ζ-potential measurements. The influence of pH, crosslinker content, and template structure on sorption performance was investigated. All MIPs exhibited maximum sorption at pH 3.0. The highest sorption capacity toward levofloxacin was achieved for the Chit–HPCD–G polymer (74.8 mg/g), whereas the fluorescein-imprinted HPCD–TDI (1:1) MIP demonstrated the highest sorption capacity (135.5 mg/g) and selectivity coefficient (84.1). Dynamic column experiments con-firmed efficient analyte extraction, reducing the analyte concentration by more than 90% after ten loading cycles. All synthesized MIPs exhibited excellent regenerability, with less than 3% loss of sorption efficiency after ten consecutive sorption–desorption cycles. The applicability of the developed sorbents to real matrices was demonstrated using milk and blood plasma samples after minimal sample preparation. Fluorescein extraction efficiencies reached 97.6% and 93.6% for milk and plasma, respectively. The obtained results demonstrate that HPCD-based MIPs combine high sorption capacity, exceptional selectivity, operational stability, and applicability to complex biological matrices, making them promising materials for selective sample preparation, analyte pre-concentration, and controlled drug delivery systems.

The secondary immunomodulatory effects of conventional therapeutics, such as antibiotics and cytostatics, are frequently overlooked despite their significant clinical implications. Building on our previous findings that drugs like paclitaxel and doxorubicin heavily influence macrophage polarization—potentially driving metastasis or inflammation—this study systematically evaluates the secondary immune-modulating actions of standard drugs and natural adjuvants. Using patient-derived bronchoalveolar lavage (BAL) fluid (ex vivo alveolar macrophages), we developed an analytical platform using synthetic carbohydrate-functionalized fluorescent ligands targeting key receptors (CD206, CD209, CD280, CD301). Integrating ligand-binding profiles with Linear Discriminant Analysis (LDA) yielded quantitative immune-state vectors capable of differentiating favorable and unfavorable prognostic signatures and imbalanced immune states. Pro-filing samples across heterogeneous respiratory conditions revealed highly con-text-dependent responses. While some treatments synergistically corrected unfavorable imbalanced profiles, others provoked dysregulation. Notably, in pneumonia or bronchitis with an asthma-prone M2-dominant profile, specific antibiotic regimens are critical; doxycycline, for instance, may exacerbate patient deterioration by further driving M2a polarization. Crucially, we identified that natural adjuvants (e.g., curcumin, coumarins, polyphenols) exhibit potent properties capable of correcting these adverse secondary drug effects. Ultimately, this profiling platform highlights the necessity of evaluating patient-specific secondary drug effects, offering a functional blueprint for precision immunotherapy and the rational design of adjuvant-enhanced treatments.

The secondary immunomodulatory effects of conventional therapeutics, such as antibiotics and cytostatics, are frequently overlooked despite their significant clinical implications. Building on our previous findings that drugs like paclitaxel and doxorubicin heavily influence macrophage polarization—potentially driving metastasis or inflammation—this study systematically evaluates the secondary immune-modulating actions of standard drugs and natural adjuvants. Using patient-derived bronchoalveolar lavage (BAL) fluid (ex vivo alveolar macrophages), we developed an analytical platform using synthetic carbohydrate-functionalized fluorescent ligands targeting key receptors (CD206, CD209, CD280, CD301). Integrating ligand-binding profiles with Linear Discriminant Analysis (LDA) yielded quantitative immune-state vectors capable of differentiating between favorable prognostic signatures and imbalanced immune states. Profiling samples across heterogeneous respiratory conditions revealed highly context-dependent responses. While some treatments synergistically corrected imbalanced profiles, others provoked dysregulation. Notably, in pneumonia or bronchitis with an asthma-prone M2-dominant profile, specific antibiotic regimens are critical; doxycycline, for instance, may exacerbate patient deterioration by further driving M2a polarization. Crucially, we identified that natural adjuvants (e.g., curcumin, coumarins, polyphenols) exhibit potent properties capable of correcting these adverse secondary drug effects. Ultimately, this profiling platform highlights the necessity of evaluating patient-specific secondary drug effects, offering a functional blueprint for precision immunotherapy and the rational design of adjuvant-enhanced treatments.

The balance of alveolar macrophage (AM) polarization is severely disrupted in chronic inflammatory diseases like bronchiectasis, where a persistent pro-inflammatory (M1) phenotype perpetuates inflammation. To address this, we developed a high-throughput platform using a series of synthetic glycoligands (L1-L5) on a polyethyleneimine (PEI) scaffold. These ligands, which have varying affinities for macrophage lectin-like receptors, were used for phenotypic “fingerprinting” of AM subpopulations from pediatric bronchiectasis patients and a healthy control. Analysis of bronchoalveolar lavage fluid (BALF) revealed a pathogenic, M1-dominant profile (55% M1) in patients, confirming a state of chronic inflammation, which starkly contrasted with the quiescent, M0-dominant profile in the healthy control. We then leveraged this platform for targeted immunomodulation, using a drug-ligand conjugate to steer the dysregulated macrophage population toward a healthy state. The most potent conjugate, Dox-L5, dramatically suppressed the pathogenic M1 population (from 55% to 16%). This M1 suppression was accompanied by a significant shift toward the M2a (tissue-repair) phenotype and the emergence of a quiescent M0-like population, effectively remodeling the AM profile. This work validates a glycan-based platform for both diagnosing and correcting pathological macrophage imbalances. Our targeted approach offers a precise strategy to resolve chronic inflammation in bronchiectasis by suppressing M1 macrophages and promoting a pro-resolving M0/M2 phenotype, thereby restoring lung homeostasis.

Background.The research laboratory Progressive biotechnologies in aquaculture of the Saratov State University of Genetics, Biotechnology and Engineering named after N.I. Vavilov studied the effect of complexes of ß-cyclodextrin on the healing of skin wounds in hybrid sturgeon fish. The total microbial count of sturgeon cut wounds was determined using the culture method and the composition of the microflora of the fish colon using the qPCR method under the influence of the ß-cyclodextrin complex. This complex was ß- cyclodextrin with levofloxacin included in a chitosan shell. The complex was adsorbed on the surface of the feed that the sturgeons received daily. Changes in the total microbial count demonstrate the nature of the inflammatory process and the effectiveness of the use of therapeutic drugs. The study of the colon microbiota also demonstrates the result of using these complexes in fish. It was found that the use of antibacterial complexes of ß-cyclodextrins with chitosan on sturgeons leads to a significant decrease in the total microbial count of the wound surface. The highest antimicrobial activity was established for the chitosan-ß-cyclodextrin complex with 20 and 15% levofloxacin content. The presence of bacteroides, eubacteria, clostridia, peptostreptocci, enterobacteria, lactobacilli and staphylococci were determined in the composition of the normal flora. At the same time, by the end of the experiment, the microbiome of the large intestine of fish is characterized by the restoration of the number of lactic acid bacteria. The research results can be used in aquaculture in the process of fish cultivation in the treatment of injuries to the outer coverings received during transportation and sorting. Purpose.Reproduction and cultivation of fish in aquaculture is inevitably associated with trauma to the skin of fish, infection with saprophytic, opportunistic and pathogenic microorganisms during transportation, sorting, dense planting, and transition to new feed. All these factors reduce the immune status of the fish organism and lead to the occurrence of various diseases that affect all metabolic processes. Currently, antibiotics of various groups with a wide spectrum of action are use to combat infectious diseases, as well as for prevention purposes. Due to the specific lifestyle of aquatic organisms, antibiotics are introduced into the fish organism in aquaculture using medicinal baths, injections, or orally with feed. Materials and methods.The effect of the chitosan-ß-cyclodextrin complex on the fish organism under aquarium conditions was studied. This complex was synthesized and provided by the Department of Chemical Enzymology of the Lomonosov Moscow State University. The studied complex is a light yellow powder, slowly soluble in water due to the content of chitosan and cyclodextrin and added to fish feed. For the experiment, 5 groups of sturgeons with wounds in the form of dorsal cuts of the skin 2 cm long and 0.5 cm deep were formed using the pair-analogue method. Before the experiment, the fish were fed with compound feed with a peroxide value of 24.68±2.22 for 10 days to form a model digestive disorder and intestinal dysbiosis. The fish received feed with the preparation daily (3 times a day). Individuals of the 1st and 2nd control groups did not receive the studied complex, in addition, individuals of the 2nd control group continued to receive low-quality feed during the experiment. The experimental groups received high-quality feed with the complex in different dosages of levofloxacin (the first - a complex with 20%, the second - with 15% and the third - with 10% of the antibiotic, respectively) for 7 days. Results.The study of sturgeon wound microflora showed that the studied chitosan-ß-cyclodextrin complex with an antibiotic reduces the TMC of cut wounds. In individuals of the control groups (K1 and K2), the inflammatory process in the wounds continued until the end of the experiment (day 8). On the 8th day, the greatest suppressive effect on the microflora of fish wounds was exerted by complexes with 15 and 20% antibiotic content: in both groups, the decrease in TMC relative to day 1 was 1000 times, below K1 by 1000 times, and K2 by 10000 times. The results of assessing the diversity of sturgeon large intestine microflora indicate that the control group of fish is characterized by normal microflora: bacteroides, eubacteria, clostridia, peptostreptocci, enterobacteria, lactobacilli and staphylococci. In the experimental groups a decrease in the number of lactobacilli, enterobacteria, fusobacteria, eubacteria and clostridia genomes was recorded. Lactate-utilizing bacteria were not detected. Gradual recovery of the fish organism is confirmed by the presence of lactobacilli on the 14th day in the same quantity as before injury and the use of the complex, as well as the absence of mycoplasmas, streptococci and Candida fungi. Conclusion.A study of wound microflora using the culture method showed that chitosan-ß-cyclodextrin complexes with levofloxacin, used in the treatment of sturgeons, have a significant antimicrobial effect on the number of microorganisms compared to the control groups, with the best effect by the end of the experiment being provided by complexes with 15 and 20% levofloxacin. A study of wound microflora using the culture method showed that chitosan-ß-cyclodextrin complexeswith levofloxacin, used in the treatment of sturgeons, have a significant antimicrobial effect on the number of microorganisms compared to the control groups, with the best effect by the end of the experiment being provided by complexes with 15 and 20% levofloxacin. Molecular genetic research using the PCR method in real time determined the presence of bacteroids, eubacteria, clostridia, peptostreptocci, enterobacteria, lactobacilli and staphylococci in the composition of the normal flora. In the 14 days the microflora of the...

The balance of alveolar macrophage (AM) polarization is severely disrupted in chronic in-flammatory diseases like bronchiectasis, where a persistent pro-inflammatory (M1) phe-notype perpetuates inflammation. To address this, we developed a high-throughput plat-form using a series of synthetic glycoligands (L1-L5) on a polyethyleneimine (PEI) scaf-fold. These ligands, which have varying affinities for macrophage lectin-like receptors, were used for phenotypic "fingerprinting" of AM subpopulations from pediatric bronchi-ectasis patients and a healthy control. Analysis of bronchoalveolar lavage fluid (BALF) revealed a pathogenic, M1-dominant profile (55% M1) in patients, confirming a state of chronic inflammation, which starkly contrasted with the quiescent, M0-dominant profile in the healthy control. We then leveraged this platform for targeted immunomodulation, using a drug-ligand conjugate to steer the dysregulated macrophage population toward a healthy state. The most potent conjugate, Dox-L5, dramatically suppressed the pathogenic M1 population (from 55% to 16%). This M1 suppression was accompanied by a signifi-cant shift toward the M2a (tissue-repair) phenotype and the emergence of a quiescent M0-like population, effectively remodeling the AM profile. This work validates a gly-can-based platform for both diagnosing and correcting pathological macrophage imbal-ances. Our targeted approach offers a precise strategy to resolve chronic inflammation in bronchiectasis by suppressing M1 macrophages and promoting a pro-resolving M0/M2 phenotype, thereby restoring lung homeostasis.