Biophysical Bulletin

Influence of redox cyclers on thiol oxidation in the presence of nanoparticles

Wed, 06 Aug 2025 08:32:04 +0000

Background: An increase in metabolic rate interconnected with oxidative imbalance are major features of tumor process. Higher ROS (reactive oxygen species) levels make tumor cells more sensitive to oxidative stress compared to normal cells. Therefore, generating additional ROS can lead to cancer cell death. Redox cycling is a crucial process responsible for the production of ROS by various clinical and experimental anticancer agents. Among these compounds are quinones and ascorbic acid, which exhibits a synergistic antitumor effect. Elevated glutathione levels and glutathione-dependent antioxidant enzymes play a key role in protecting cancer cells from intracellular oxidative stress. Nanoparticles with glutathione depletion properties can act as smart chemodynamic agents, disrupting the cellular antioxidant defense system. In this work, inorganic nanoparticles based on rare earth elements are used as catalytic amplifiers of one- electron transfer with the formation of organic and oxygen radicals in the redox cycles of ascorbic acid and vitamin K3.

Objectives: The thiol oxidation was studied in the presence of nanoparticles in combination with redox cyclers.

Materials and methods: As an indicator of the pro-oxidant efficiency of nanoparticles (CeO2 (2 nm, 20 μg/ml) or GdYVO4:Eu³⁺ (2 nm, 20 μg/ml)) combined with organic compounds (ascorbic acid (100 or 200 µM) and vitamin K3 (4 μM)) changes in the level of thiols (glutathione (200 μM), L-cysteine (200 μM) or dithiothreitol (500 μM)) in the model system were used.

Results: It was shown that GdYVO4:Eu³⁺ and CeO2 nanoparticles enhances oxidation of thiols under an influence of the redox active molecule as well as their combination. The efficiency of bare nanoceria as well as in redox cyclers combinations was higher compared to respective orthovanadate nanoparticles combinations (including time dynamics) that was especially pronounced in the dithiothreitol oxidation system.

Conclusions: The data obtained indicate the ability of nanoceria to significantly enhance the oxidation of thiols induced by redox cyclers revealing the perspective of this approach in solving the problem of increased thiol level in tumor cells.

Effects of cryoprotective agents on osmotic tolerance limit of testicular interstitial cells

Wed, 06 Aug 2025 08:35:12 +0000

Background: Cryopreservation is a multistep process, which includes stages affecting biological material mechanically, osmotically and toxically. The use of cryopreservation of biological materials is cost-effective and affording long-term storage at cryogenic temperatures. It also guarantees the stability of the genetic component of cells and reduced contamination of the biological material.

Objectives: The objective of the research is to evaluate the effects of cryoprotective agents (CPAs) (dimethyl sulfoxide (DMSO), dextran (D40), hydroxyethyl starch, polyethylene glycols (PEG1500 and PEG400), and fetal bovine serum) and their combinations on the interconnection between the osmotic tolerance of testicular interstitial cells (ICs) and cryoprotection.

Materials and Methods: The osmotic tolerance limit (OTL) of ICs and the toxic effect of the CPA were investigated in the phosphate buffer saline based media of different osmolarities: isosmotic (300 mOsm), hypo-osmotic (225 mOsm), hyperosmotic (600 mOsm). Similar osmotic conditions can develop during cryopreservation of cells in the temperature interval from +4 to -30 °C.

Results: The indicators of cell survival after incubation in the media differed depending on osmolarities of incubation media. They were compared with the indicators obtained after cooling ICs to -30 °C followed by warming and CPA removal. We have shown that the non-toxic additive D40 increased the OTL of ICs in hypo-osmotic medium and decreased negative effects of DMSO on the cells. These effects were accompanied by high indicators of ICs survival obtained after cooling ICs to -30 °C with 100 mg/ml D40 and 0.7 M DMSO.

Conclusions: These results unveil the mechanisms of cryoprotection of 0.7DMSO+D40 and partially explain the superiority of 0.7DMSO+D40 media shown in our previous works compared with other investigated media. Understanding the mechanisms of cryodamage and cryoprotection of 0.7DMSO+D40 paves a way toward the development of new serum-/xeno-free cryoprotective compositions and improvement of cryopreservation protocols for cell suspensions that include many types of cells. Further studies are required to reveal the effects of DMSO on membranes and intracellular metabolic processes.

Dose effects of ultraviolet and terahertz laser radiation on the plasma membrane of erythrocytes

Wed, 06 Aug 2025 08:38:07 +0000

Background: In modern medicine, various technical means and developments are actively used in invasive intravascular (IVL) and non-invasive transcutaneous blood irradiation methods, including low-intensity laser radiation (LLLR) of various ranges. Despite the positive clinical results of such exposure, the physical and molecular mechanisms remain incompletely understood. Ultraviolet (UV) and terahertz (THz) ranges of electromagnetic radiation are classified as biogenic; therefore, establishing their effects at the level of blood cells will allow them to be recommended for use in medical and biotechnological practice after the development of appropriate exposure methods.

The objective was to study the biogenic activity of ultraviolet (UV) and terahertz (THz) ranges of LLLR on the structural and functional state of red blood cells of rats in vitro.

Materials and methods: Using methods of microelectrophoresis, spectrophotometry and chemical erythrograms, the following was studied: zeta potential of red blood cells; the content of the primary products of lipid peroxidation in the red blood cells (fatty acid conjugates of membrane of red blood cells phospholipids — diene (DK), triene (TK), and tetraene (TTK), oxodiene (ODK); resistance of erythrocytes to the action of an acid hemolytic after previous laser irradiation in the UV and THz ranges. Laser irradiation of the samples in the UV range was carried out by a nitrogen pulsed laser (λ = 0.337 μm), with the average radiation power 5 mW; irradiation in the THz range with a continuous CO₂ laser (λ = 118.8 μm), the average radiation power is 20 mW. When the samples were irradiated, the density of radiation power W did not exceed 7 W/m². The exposure dose under nitrogen laser irradiation was D_1UV=0.109 J/cm², D_2UV=0.327 J/cm², D_3UV=0.546 J/cm². Under CO₂ laser irradiation, the exposure dose was D_1THz=0.624 J/cm², D_2THz=1.871 J/cm², D_3THz=3.119 J/cm². The thickness of irradiated red blood cell suspension layer was 1 mm.

Results: The effect of low-dose LLLR UV and THz radiation causes physicochemical changes in the plasma membrane of erythrocytes. A nonlinear dose-dependent decrease in the electrokinetic potential of the cell surface and an increase in the hemolytic sensitivity of erythrocytes against the background of activation of lipid peroxidation processes in erythrocyte membranes have been established.

Conclusions: Under the influence of LLLR, the free-radical peroxidation of lipids of erythrocyte membranes is stimulated. Pronounced biogenic activity of UV of LLLR was detected at the level of erythrocyte membranes. The obtained data can be used to expand the spectrum of application of LLLR of the UV range in IVL techniques.

Optical and electron microscopy imaging of model circulating tumour cells

Wed, 06 Aug 2025 08:40:36 +0000

Background: Microscopy is a key tool in biophysical research for visualizing living cells' morphology, structure, and dynamic processes. Depending on the specific research goals, various optical and electron microscopy techniques can be applied, each offering unique benefits. Determining the structural features of the cytoskeleton, nucleus, and membrane of circulating tumor cells in the model Lewis lung carcinoma (LLC) is an important biophysical and biological task, as it will allow identifying targets for antitumor and antimetastatic therapy, as well as investigating the mechanisms of action of antitumor drugs. However, comprehensive multimodal imaging of such cells — particularly under non-adherent conditions — remains limited in the literature due to the difficulty of working with such models.

Objectives: This study aimed to visualize Lewis lung carcinoma (LLC) cells cultured under de-adhesive conditions using various microscopy techniques for their characterization and to examine the cancer cells' structure and functionality. The goal was to evaluate each method's individual capabilities and combined strengths in revealing cellular morphology, internal structure, and nanoparticle interactions.

Materials and Methods: LLC cells were obtained from the National Bank of Cell Lines and Tumor Strains of the IEPOR (NAS of Ukraine) and cultured in RPMI-1640 medium under conditions of de-adhesive growth. Imaging was performed using inverted optical microscopy (Euromex Oxion), fluorescence and confocal microscopy (Carl Zeiss LSM 510) with F-actin (Alexa Fluor 488-phalloidin) and nuclear (Hoechst 33342) staining, and label-free Coherent Anti-Stokes Raman Scattering (CARS) microscopy (Leica TCS SP8). Scanning electron microscopy (TESCAN MIRA3 LMU) was used for high-resolution surface imaging. 2D-MoS₂ nanoparticles, as well as 2D-MoS₂ and doxorubicin simultaneously, were applied to investigate nanoparticle-mediated labeling and cellular uptake.

Results: A comparative analysis of multiple imaging modalities — including optical, fluorescence, confocal, CARS, and SEM — was applied to Lewis lung carcinoma (LLC) cells. Fluorescence microscopy with specific fluorophores made it possible to analyze the size and properties of actin fibers and showed that nuclei occupy most of the deadhesive cells. Electron microscopy revealed numerous filopodia on the cell surface. CARS showed the presence of lipid droplets in the cells.

Conclusions: Each microscopy method provided complementary insights into cell morphology, cytoskeletal organization, lipid content, and surface ultrastructure. Nanoparticles demonstrated high utility as dual imaging and therapeutic agents. This work represents the first detailed SEM study of non-adherent LLC cells and highlights the potential of integrated multimodal imaging for studying circulating tumor cell models.

Anticoronavirus activity of C60 fullerene: in silico and in vitro screening

Wed, 06 Aug 2025 08:42:27 +0000

Background: The search for potential therapeutic agents against the most common coronaviruses, which can pose a threat to human and animal life, is an urgent issue of modern biomedicine.

Objective of the work was to evaluate in silico the ability of C₆₀ fullerene to interact with the membrane protein ACE2, thereby preventing the formation of the "coronavirus-ACE2" complex and its further penetration into the host cell, as well as the effectiveness of the anticoronavirus action of these carbon nanoparticles in in vitro systems.

Methods: The Protein Data Bank was used to study the structural organization of the human ACE2 membrane protein. The CHARMM-GUI and SwissParam web resources were used to construct the membrane and C₆₀ fullerene, respectively. Potential binding pockets for C₆₀ fullerene in the ACE2 structure were determined using the Caver software package. The system molecular docking algorithm (sdock+) was used to study the interaction between C₆₀ fullerene and ACE2. Molecular dynamics (MD) calculations were performed using the Gromacs 2020 software package. Cytotoxicological and virological methods were used in in vitro experiments. Statistical processing of experimental results was carried out using the Statistica 13.3 program.

Results: It was found three potential binding sites between the groove of the peptidase domain of the ACE2 protein and C₆₀ fullerene. According to the results of molecular docking and MD, it follows that C₆₀ fullerene forms two stable complexes with the ACE2 protein, thus blocking its potential interaction with coronaviruses. According to the results of in vitro studies, it follows that C₆₀fullerenes at a maximum allowable concentration of 37.5 μg/ml act on the coronaviruses of swine (α-coronavirus) and cattle (β-coronavirus) at the early stage of replication (1 h) in sensitive cellular systems, significantly reducing their infectious activity by 2.00 TCID₅₀/ml and ≥2,28 TCID₅₀/ml, respectively.

Conclusions: C₆₀ fullerene has been shown to form two stable complexes with the membrane protein ACE2, thereby inhibiting its functional activity and blocking potential interaction with coronaviruses. It was established that the C₆₀ fullerenes show antiviral activity against coronaviruses of two groups at the initial stage of infection when interacting with sensitive host cells.

C60 fullerene promotes recovering rat musculus gastrocnemius contracting activity after neurogenic atrophy

Wed, 06 Aug 2025 08:44:22 +0000

Background: Recovery of functional activity of skeletal muscles after neurogenic atrophy is a complex and long-term process. Therefore, the search for effective therapeutic means for its acceleration is an extremely urgent task in modern biomedicine. Skeletal muscle atrophy is characterized by a reduction in muscle mass, increased fatigue, and decreased contractile strength, driven by oxidative stress, inflammation, and activation of catabolic reactions. Denervation triggers complex signaling cascades involving Ca²⁺ and transcription factors that promote protein degradation and impair myocyte function. Mitochondrial dysfunction, mitophagy, and disrupted oxidative phosphorylation exacerbate degenerative changes in muscle fibers. In addition to muscle loss, surrounding tissues undergo remodeling, including fibrosis and devascularization, which further hinder regeneration. Oxidative stress is a key initiator of muscle atrophy, and current therapeutic strategies focus on its mitigation through antioxidants, stem cells, and extracellular vesicles.

Objective of the work was to study of the C₆₀ fullerenes with antioxidant properties on the mechanokinetics of rat m. gastrocnemius contraction for 30 days after neurogenic atrophy caused by sciatic nerve damage.

Methods: When analyzing the miotic response using tensometry, such mechanokinetic parameters as the level of maximum muscle contraction force, the time to reach its maximum contraction force and the time of holding the maximum force of m. gastrocnemius contraction were evaluated. Additionally, histological studies of m. gastrocnemius were performed.

Results: It was shown that in injured animals that received daily oral administration of C₆₀ fullerene aqueous solution (C₆₀FAS) at a dose of 1 mg/kg throughout the experiment, there was the positive dynamics in the studied mechanokinetic parameters of m. gastrocnemius contraction at the level of 29-39±3% compared to the group of animals that did not use C₆₀FAS, which was confirmed by histological analysis of these muscles on the 30^th day after the initiation of sciatic nerve damage.

Conclusions: The results obtained indicate the prospects of using C₆₀FAS to recovering the functional activity of skeletal muscles after neurogenic atrophy.