All chemicals were of analytical grade. Na₂HPO₄, disodium phosphate, NaH₂PO₄, sodium phosphate monobasic monohydrate, KH₂PO₄, monopotassium phosphate, FITC CoA, Concanavalin A-Fluorescein Isothiocyanate from Canavalia ensiformis (Jack Bean) type IV, FITC WGA, Wheat Germ Agglutinin- Fluorescein Isothiocyanate from Triticum vulgare and NaCl were purchased from Sigma-Aldrich (St Louis, MO, United States). DAPI, 4´, 6-Diamidino-2´-phenylindole dihydrochloride (10236276001) was purchased from Roche Diagnostics GmbH, Mannheim, Germany. Phenylmethanesulfonyl fluoride (PMSF) was purchased from Fluka Chemie, AG. KCl was purchased from Riedel-de-Haën AG, D-3016 Seelze 1. Glycerol (1, 2, 3-Propanetriol, Glycerin) was obtained from NBCo Biochemicals (Division of ICN Biochemicals, Inc., Cleveland, Ohio 44128).

Venom blood samples are taken from healthy human subjects and β-thalassemia patients using an EDTA-anticoagulant vacutainer study. The present study complied with the ethical regulations and legislation in Europe and Bulgaria. The experiment was performed in compliance with the WMA Declaration of Helsinki and the Ethical Principles for Medical Research Involving Human Subjects (https://www.wma.net>wma-declaration-of-helsinki-ethical-principles-for-medical-research-involving-human-subjects/09/06/2022). Written informed consent was obtained from all subjects recruited for this study. Ethics Committee of Sofia University, “St. Kliment Ohridski”, Approval Codes: RD-04-91, 25 February 2022, and RD-04-68, 2 February 2023.

Blood samples were collected from the following groups according to the principles of the World Medical Declaration of Helsinki (https:// www.wma.net/>wma-declaration-of-helsinki-ethical-principles-for-medical-research-involving-human-subjects/09/06/2022):

1. Nine healthy subjects with normal hematological values and indices;

Blood samples were centrifuged (1,000 × g, 5 min, 4°C). Plasma and buffy coat were discarded, and the erythrocytes were washed three times under the same conditions of centrifugation with two volumes of phosphate buffered saline (PBS), pH 7.4 (PBS: 10.1 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 136.9 mM NaCl, 0.2 mM KCl) at 4°C. After centrifugation of the blood samples, the erythrocyte ghosts were prepared using the method described by Burton et al. (Burton et al., 1981; Yoshimine et al., 1992) with modifications. The erythrocyte ghosts were performed by hemolysis and washing the erythrocytes (500 µL) in 15 mL of 5 mM sodium phosphate buffer (Na₂HPO₄/NaH₂PO₄, pH 8.0) at 4°C for 20 min. This is followed by the centrifugation in the same solution at 17500 × g (Janetzki 24, Engelsdorf (Bez. Leipzig), Germany) for 30 min. The pellet was washed with 17500 × g (Janetzki Engelsdorf (Bez. Leipzig), Germany) in 15 mL 2.5 mM sodium phosphate buffer, pH 7.75 for 30 min at 4°C. The pellet was washed twice in 15 mL of 1.25 mM sodium phosphate buffer, pH 7.6 for 30 min at 4°C. The final pellet of erythrocyte membranes (erythrocyte ghosts) was suspended in 100 µL of cold Sorensen’s phosphate buffer (1/15 M Na₂HPO₄/NaH₂PO₄, pH 7.4), 0.7 mM PMSF at 4°C.

The erythrocyte membranes were stored at 4°C for no more than 4 h before sample preparation for microelectrophoretic measurements. Erythrocyte ghosts were stored at −20°C in the presence of 20% glycerol before sample preparation for the fluorescence microscopy experiments. The protein concentration was measured as previously described (Lowry et al., 1951).

Electrophoretic mobility (EPM) measurements were performed using particle electrophoresis (microscopic (visual) microelectrophoresis) with an OPTON Cytopherometer (Feintechnik Ges., Germany). Electrophoretic migration is measured in a rectangular chamber and platinum electrodes at a constant electric field of 5 mA and 25°C. The movement of the cells over a known distance (16 μm) was timed for forward and backward (reversed field) runs. The erythrocytes were observed under a light microscope connected to a Sony video camera at a magnification of 2000 ×. The cells were observed on a JVC monitor (Victor Company of Japan, Ltd., Yokohama, Japan). Erythrocytes of approximately equal size were measured. The results are expressed using the EPM ( u per 10^–8 m² V⁻¹s⁻¹ ± standard deviation for each sample). The standard deviation of the electrophoretic mobility u was 2%–5%. The electrical conductance and viscosity of different media, including erythrocytes, were measured using a CyberScan PC 510 (Eutech Instruments, Singapore) pH/conductivity meter and a Rheo (Germany) viscometer, respectively. Values represent the mean of three independent measurements (54–96 erythrocytes) with three replicates each. The zeta potential (ζ) is calculated from the electrophoretic mobility, u, using the Helmholtz-Smoluchowski equation (Delgado et al., 2007): ζ = η u ε r ε 0 , (1) where ζ   e l e c t r o k i n e t i c , z e t a   p o t e n t a i l is in units of mV; ε r = 78.5   a t   25 ℃ is the relative dielectric permittivity of the aqueous phase; ε 0 = 8.8542 × 10 − 12   F m − 1 is the permittivity of free space, and η = 0.001393   P a . s is the viscosity of the phosphate buffered saline (PBS), pH 7.4 at 25°C as in McLaughlin, (1977).

The electrostatic potential in the aqueous phase of the membrane surface at x = 0 and surface charge density ( σ is given by: 136.6   x   σ √ C = sin ⁡ h z Ψ o 51.38 , (2) where Ψ o is in mV (McLaughlin, 1977). The surface electrical charge is expressed as Cm⁻² (in Coulombs per meter squared; The Coulomb is defined as the quantity of electricity transported in one second by a current of one ampere). The value of surface charge density is calculated using the assumption that ζ ≅ Ψ o (McLaughlin, 1977).

The electrostatic Coulombic part of the electrical work done in charging up the surface of the erythrocyte membrane can be calculated. The location of charged groups plays a role in determining the types of interactions in which they participate, and the contributions they make to the electrostatic free energy of the SMF-treated erythrocyte cells in norm and pathology (McLaughlin, 1977; Cevc, 1990).

In the linear regime of Gouy-Chapman approximation the dominant contribution to the electrostatic free energy comes from the internal energy, which is proportional to the volume of the integral of the square of the electric field (Cevc, 1990): G e l e c s = G G o u y = − ∫ 0 Ψ σ d Ψ 0 (3)

The integral in Eq. 3 is due to the electrical work done in moving ions from the bulk to a surface with a final net surface charge density σ e l , electrostatic surface potential Ψ 0 , where electrostatic potential Ψ ( σ , σ p = 0 ), and free energy G e l e c .

The Coulombic free energy of most membranes is only small, below 2 kJmol⁻¹, owing to the interfacial structure effects (Cevc, 1990). The electrostatic free energy per 1 cm² of the double electrical layer near the outer surface of the erythrocyte membrane was calculated: G G o u y = –   8 N A k T λ cosh y 0 2   –   1 , (4) where σ = 2 N A D k T π ½ sinh y 0 2 , y 0 = Z e Ψ 0 k T , e = 1.6021 × 10 − 19 C is the elementary electronic charge, Z is the ion valence, k = 1.3805 × 10 − 2 J K − 1 is the Boltzmann constant, N A = 6.022 × 10 23 m o l − 1 , is the Avogadro constant, T = 298 .   15 ° K is the absolute temperature.

The Debye-Hückel length by the general definition in all electrostatic theory: λ = 2 e 2 N A n 0 Z i 2 ε k T 1 2 = 2 e 2 N A c Z i 2 1000 ε k T 1 2 ≅ 0.328 · 10 − 10 c Z 2   m − 1 , (5) where the summation goes over all ionic species and the factor 10 3 appears because the bulk ion concentration is normally given in moles per liter; n 0 i = concentration of the n_oi(x) species of the i^th - ion at definite point x with potential E(x) at 25°C; Z i = electrolyte valence of the i^th ion; c = electrolyte ionic strength = bulk concentration of the i^th species, ideally for x → ∞ , but practically just far enough away from the surface charge region.

In vitro fluorescence microscopy was performed using a BX50-FLA (Olympus, Tokyo, Japan). Fluorescein isothiocyanate (FITC)-labeled lectin (CoA) and wheat germ agglutinin (WGA) were used. Concanavalin A FITC labeled from C. ensiformis (Jack bean) Type IV, FITC content 3.6 mol/mol lectin (Mol.Wt of lectin approx. 102,000) has an affinity for terminal α−D−mannosyl and α−D−glycosyl residues. CoA-FITC inhibitory carbohydrates are α−Methylmannoside and α−Methylglucoside. Lectin from Triticum vulgaris (Wheat Germ) fluorescein isothiocyanate labeled contains 2 mg protein containing 2.2 mol FITC per mole lectin (Mol. Wt of lectin approx. 36,000). The secondary structure of the protein (FITC-WGA) is rich in strands and contains carbohydrate-binding sites on its surface. It is used for the fluorescence detection of glycoproteins containing β (1 β 4)−N-acetyl-D-glucosamine. WGA is not blood-specific but has an affinity for N−acetyl−β−D−glucosamine oligomers. WGA does not contain protein-bound carbohydrates. FITC-Con A (MW 102,000 Da) or FITC-WGA (MW 36,000 Da) labeling was dissolved in distilled water at 2 mg/mL as a stock solution in dark tubes. The solution was diluted with PBS (pH 7.4–20 µg/mL) before use. Erythrocyte membranes were then transferred to the staining solution. Erythrocyte ghosts were exposed to SMF (2.0 T) for 15 min at 25°C. After 60 min incubation with the staining solution, erythrocyte ghosts (2 × 10⁶ cells/mL) were washed three times with PBS (15 min, 12,000 × g) and examined under an epifluorescence microscope (BX50-FLA, Olympus, Tokyo). Nuclear counterstain DAPI (4´,6-Diamidino-2´-phenylindole dihydrochloride; 5 mg/mL stock solution; Excitation at 358 nm/Emission at 461 nm) was used to assess gross cell morphology (Klinge et al., 2022). DAPI staining allowed multiple uses of cells eliminating the need for duplicate samples (Tarnowski et al., 1991).

The intensity of light scattering (LS) at 90° and λ = 480 nm from the erythrocyte suspension is registered using a laboratory-made apparatus using Specol 10 Spectrophotometer (Carl Zeiss, Germany) and titration attachment type Ti. Cut-off filters (red glass filter with λ ≥ 640 nm and blue-green glass filter with λ ≤ 600 nm) are used. The reaction medium used in the LS experiment contained PBS (pH 7.4). The scattering level at an angle of 90^o represents the level of cell aggregation.

Malondialdehyde (MDA) content was measured according to (Halliwell and Gutteridge, 1999) with slight modifications. The erythrocyte suspensions (500 μL erythrocytes in PBS, pH 7.4, 2 mM NaN₃, Ht = 20%) are homogenized in 400 μL of 28% trichloroacetic acid (TCA) and centrifuged at 12,500 × g for 15 s. After centrifugation, 1 mL of the supernatant was mixed with 500 μL of 1% thiobarbituric acid (TBA) in 20% NaOH, and the mixture was incubated in boiling water for 15 min. The reaction was terminated by cooling the samples in an ice bath. In the TBA test, MDA reacts to generate a colored product. In the acidic solution, the product absorbed light at λ = 532 nm. The MDA content was estimated using an extinction coefficient of 154 mmol L⁻¹ cm⁻¹. Lipid peroxidation in erythrocyte membranes is determined by the production of thiobarbituric acid reactive substances (TBARS) and expressed in mol L^-1 (Halliwell and Gutteridge, 1999).

Electrophoretic mobility, zeta potential, and surface electrical charge data are expressed in u x 10^–8, m²V⁻¹s⁻¹, ζ, mV, and σ, Cm⁻², respectively. The light scattering levels were expressed as arbitrary units. All electrokinetic and electrostatic free energy data are reported as the mean ± SD. The results were averaged from three independent experiments, each with three replicates. Statistical differences between means were determined using Analysis of Variance (ANOVA). One-way analysis of variance was performed using the Holm-Sidak method to compare the significance of the treatments taking p ≤ 0.05 as significant, p ≤ 0.01 as highly significant, and p ≤ 0.001 as extremely significant. Statistical analyses were performed using Minitab.v.17.

Erythrocytes from healthy subjects and β-thalassemia patients were used as model systems to evaluate the effect of SMF on membrane surface charge. The SMF treatment was applied for 15 min. The EPM of erythrocytes after exposure of norm and β-thalassemia samples to SMF (0.1–2.0 T) is shown in (Figures 2A, B). There was a decrease in the EPM values of healthy subjects after SMF exposure (0.5 T) compared to those of non-treated cells (p = 0.038). The zeta potential of erythrocytes from healthy subjects (ζ = −29.9 mV) exposed to SMF (0.5 T) and to (2.0 T) was significantly decreased in comparison with the values of the electrokinetic potential of non-treated erythrocytes (ζ varied from −25.9 mV under SMF (0.5 T) exposure of up to −26.5 mV at the SMF (2.0 T) treatment) (Figure 3A). There was a significant increase in the electrokinetic potential of erythrocyte membranes from healthy subjects upon exposure to SMF (0.1 T, p < 0.001; 1.0 T, p = 0.007, and 1.5 T, p < 0.001) (Figure 3A). Figure 2A shows the increase in EPM (p = 0.014) and net surface charges of erythrocyte membranes from healthy subjects (Figure 4A) after SMF (1.5 T) treatment.

No statistically significant changes were observed in the electrophoretic mobility of β-thalassemia erythrocytes under the influence of applied SMF intensities over the entire range of exposures used (Figure 2B). SMF exposure (0.1 T; 2.0 T) led to a significant reduction in the electrokinetic potential of β-thalassemia erythrocytes (Figure 3B, p < 0.001). A decrease in ζ under SMF (1.0 T, 1.5 T) exposure was observed (Figure 3B, p = 0.011) compared to the control values of non-SMF-treated β-thalassemia erythrocytes (ζ = −23.8 mV).

However, there was a significant difference in the surface electrical charge of erythrocyte membranes from healthy subjects and β-thalassemia patients (p < 0.05) (Figures 4A, B). There was a reduction in the σ of non-treated erythrocytes in norm after SMF (0.5 T) exposure (σ = −0.0273 Cm⁻²) and SMF (2.0 T) (σ = −0.0281 Cm⁻²) compared to the surface charge of non-treated erythrocytes, called controls (σ = −0.0320 Cm⁻²) (p < 0.05) (Figure 4A). An increase of σ values of erythrocytes in norm after SMF (0.1 T; 1.5 T) treatment (p < 0.05) is observed.

A decrease in the negative electrical charges on the outer surface of β-thalassemia membranes was observed after SMF (0.1–2.0 T) treatment (p < 0.05) (Figure 4B). Net surface charge varies from σ of control samples without SMF exposure (σ = −0.0232 Cm⁻²) to σ = −0.0232 Cm⁻² for SMF (0.1 T) treatment or σ = −0.0208 C m^-2 at SMF (2.0 T) (p < 0.05). A slight decrease in negative electrical charges on the outer surface of β-thalassemia membranes at SMF (0.5 and 1.5 T) was observed (p < 0.05) (Figure 4B).

Our research shows that the action of 0.1 and 0.5 T SMF causes an increase in the negative values ​​of the electrostatic free energy (G_{electrostatic}), which corresponds to spontaneous processes as a result of the impact of the magnetic field on healthy erythrocytes (p < 0.05, Figure 5A), in contrast to the strong reduction of G_{electrostatic} at doses of 0.5 T. Lower negative values ​​of the electrostatic free energy of beta-thalassemia erythrocytes after exposure to SMF were calculated compared to the observed values ​​of G_{electrostatic} from healthy cells (p < 0.05, Figure 5B). The presence of spontaneous chemical processes in beta-thalassemia erythrocytes after exposure to 0.1 T and 2.0 T SMF is shown in Figure 5B (p < 0.05).

Shape changes are accompanied by a significant lowering of the electrostatic energy and arise in the surface area of the cell (Jadhao et al., 2014). Normally, erythrocytes have the shape of a double-concave disk, which is also visible in the microelectrophoretic microscopic measurements of the electric field of the cells. Erythrocytes treated under the norm with 0.1 T SMF are characterized by the appearance of small vesicles, and these particles are assumed to have detached from the cell membrane with the formation of vesicles according to the thermodynamic principle of maintaining maximum stability of the particle. Aggregated cells of smaller sizes resembling “cluster” aggregates are also observed (Figure 5A). SMF (0.5 T) leads to a decrease in the stability of erythrocytes in norm as a result of the decreased electrokinetic potential of the cells. Strong aggregation was observed because of the reduced zeta potential, with cells present in a larger area encompassing several cells. SMF (1.0 T) leads to the fragmentation of the erythrocyte membrane and a change in cell morphology, the serrated ends of which are well expressed on the outer surface of cells under physiological conditions. SMF (1.5 T) causes the appearance of smaller erythrocyte vesicles and the aggregation of at least two smaller cells compared to an erythrocyte in norm. We assumed that these small vesicles detached from the erythrocyte membrane because of the applied impact with the (1.5 T) SMF. Treatment of erythrocytes with SMF (2.0 T) led to the appearance of an increased number of aggregates with electrokinetic potentials close to those of healthy erythrocytes not exposed to SMF (2.0 T). There is also a fraction of erythrocytes in the norm with a smaller volume that sediments slowly when moving in an electric field and has a significantly lower zeta potential because of the lower value of the surface electric charge when treated with (2.0 T) SMF.

The β-thalassemia erythrocytes are characterized by a target-like cell shape and are larger than healthy erythrocytes. The membrane surfaces around the center of the cell (i.e., the center of the target) were slightly protruding and not convex, as was the case when the erythrocytes were exposed to certain SMF intensities (Figure 5B). When exposed to β-thalassemia erythrocytes with (0.1 T) SMF, swollen cells with protruding ends of the membrane above the main plane of the erythrocyte cell are observed. There was a change in the morphology of erythrocytes treated with (0.1 T) SMF, which were elongated along the main plane, where erythrocytes with protrusions on their surfaces were observed. SMF (0.5 T) exposure changed the erythrocyte shape. A change in the morphology of the β-thalassemia erythrocytes and reorientation and oscillation of the cells during their movement in the electric field was observed in EPM measurements after the physical impact. The target-like cells are large and swollen at their ends. Erythrocytes may have an elongated shape compared to β-thalassemia control erythrocytes, with a reduced cell target area. Treatment of β-thalassemia erythrocyte membranes with an SMF (1.0 T) caused a change in erythrocyte morphology. The cells have a smaller concave portion and highly convex membrane surfaces. A jagged outer cell envelope is also observed. SMF (1.5 T) also changed the shape of β-thalassemia erythrocytes, with protrusions above the plane of the outer membrane of the cell. Thus, changes in the morphology of β-thalassemia cells were caused by protrusions above the membrane surface. Erythrocytes isolated from patients with β-thalassemia treated with 1.5 T SMF move in the electric field by changing the direction of their movement from horizontal (in the direction of the opposite pole of the negatively charged cell) to the vertical position that appeared upright in their movement (occupying a perpendicular position relative to the plane of the electrophoretic chamber). β-thalassemia erythrocytes, after being treated to (2.0 T) SMF, have a changed cell morphology. Swollen “target-like cells” have been recorded, which appear swollen around the erythrocyte cavity with a smaller area. The shape change of β-thalassemia erythrocytes possesses protrusions above the membrane, protruding swollen ends of the plasma membrane of the cell, and cells with larger sizes of the “target-like” β-thalassemia erythrocytes.

The level of light scattering (LS) at a 90° angle with respect to the incident beam direction has been used to monitor erythrocyte aggregation after SMF exposure (Doltchinkova et al., 2006).

The exposure of erythrocytes from healthy subjects to SMF is shown in Figure 6A. The LS of erythrocytes was reduced following the treatment with SMF (0.5 T). The intensities of the SMF (0.1 T, 1.0–2.0 T) significantly enhanced the LS value (p < 0.05). There was a significant decrease in the LS of β-thalassemia erythrocytes in SMF (1.5 T and 2.0 T) (p < 0.05) in comparison to that of control erythrocytes in pathology without SMF treatment (Figure 6B).

The extent of lipid peroxidation (LP) was estimated by analyzing thiobarbituric acid reactive substances (TBARS) according to the method described by Halliwell and Gutteridge (1999) with modifications.

Magnetic fields are one of the most common environmental factors that influence living systems by increasing the lifespan of free radicals. Magnetic fields influence the kinetics of reactions with radical pair intermediates (McLauchlan and Steiner, 1991; Grissom, 1995). We have studied: i) whether SMF (0.1–2.0 T) induces lipid peroxidation stress in erythrocytes in normal and pathological conditions and ii) compared the lipid peroxidation status of erythrocytes in the presence of 50 mM H₂O₂ (second positive control), where the maximal LP changes of erythrocytes in normal conditions are observed.

The content of MDA, a marker of lipid peroxidation stress, was examined in SMF (2.0 T)-pre-exposed erythrocytes (Figures 7A, B). In an isotonic solution, the lipid peroxidation rate of erythrocytes under normal conditions increased upon adding 50 mM H₂O₂ (Figure 7A). The SMF exposure promotes the lipid peroxidation of erythrocytes from healthy subjects at doses of SMF (0.5–2.0 T), compared to the control SMF-non-treated cells (Figure 7A). SMF (1.0 T) treatment of β-thalassemia erythrocytes enhanced secondary LP products in the cells (p < 0.05).

Erythrocyte ghosts prepared by lysis and washed with hypotonic phosphate buffer had the same lipid content as intact red blood cells. Figure 8A shows the changes in FITC-lectin labeling in the epifluorescence (expressed as the amount of FITC-lectin labeling of the cells) in erythrocyte membranes (erythrocyte ghosts) before and after SMF (2.0 T) treatment.

Healthy erythrocyte membranes treated with FITC- concanavalin A (FITC-CoA) and FITC-wheat germ agglutinin (FITC-WGA) are presented with intense fluorescence due to lectins binding to the membrane (Figure 8A). Weaker fluorescence resulting from lectin-labeling of treated erythrocyte membranes from healthy subjects was observed after exposure to SMF (2.0 T) (Figure 8A) (p < 0.05). The binding of FITC-CoA to thalassemia erythrocyte membranes was reduced compared to that in non-treated ghosts in pathology without SMF (2.0 T).

Fluorescent microscopy images of FITC-CoA and FITC-WGA labeling of erythrocyte membranes after SMF (2.0 T) treatment in normal and β-thalassemia are presented in Figure 8B. The erythrocyte model systems can be further utilized to obtain information on protein-membrane interactions under SMF (2.0 T) exposure.