ANTITUMOR VECTOR SYSTEMS BASED ON BIOACTIVE LECTIN OF BACILLUS SUBTILIS ІМВ B-7724

С ombination of properties of lectins and magnetically sensitive iron-containing nanocomposites (NC) for use in oncology is actual and promising from scientific and applied point of view. The aim of the research is to synthesize and to study new iron-containing NC and magnetic fluids containing bioactive bacterial lectin, promising for use /C NC. The dependence of the adsorption capacity on time was studied. A magnetic fluid (MF) based on single-domain Fe 3 O 4 , containing lectin was synthesized and investigated. Immobilization of lectin on MF particles was carried out in a dynamic mode at room temperature for 3 hours. The concentration of lectin in the composition of MF was 0.2 mg/mL. MF with immobilized lectin was further modified with PEG-2000. The synthesis of Fe 3 O 4 /ol.Na/lectin/PEG (ol.Na – sodium oleate) vector system was carried out in a dynamic mode for 3 hours. Modification of the surface of nanoparticles with polyethylene glycol was performed in order to increase the stability of the magnetic fluid, reducing the aggregation of particles. To determine the effect of experimental samples on the viability of MCF-7 cells in vitro, the following samples were prepared: Fe 3 O 4 /ol.Na/PEG (MF), C Fe3O4 = 3 mg/mL; cytotoxic lectin of B. subtilis IMB B-7724 (CL), C CL = 0.2 mg/mL; nanobiocomposite (NBC). Nanobiocomposite based on MF and bacterial lectin was found to have a synergistic cytotoxic effect on MCF-7 human breast cancer cells, causing up to 40 % cell death. The IC 50 values for the nanobiocomposite and lectin in relation to MCF-7 cells were 100 and 125 μ g/mL, respectively. The results of research show that the combination of properties of lectins and magnetically sensitive iron-containing NC for use in oncology is a promising direction in creating new effective antitumor vector systems for targeted drug delivery and combined local therapy of cancer. The use of natural components in vector systems is a way to minimize the side effects on the body and improve compatibility with other antitumor remedies.

Сombination of properties of lectins and magnetically sensitive iron-containing nanocomposites (NC) for use in oncology is actual and promising from scientific and applied point of view. The aim of the research is to synthesize and to study new iron-containing NC and magnetic fluids containing bioactive bacterial lectin, promising for use as prototypes of new effective antitumor vector systems for targeted drug delivery and combined local therapy of cancer with minimized side effects on the body and improved compatibility with other remedies.
To create vector systems, nanodisperse magnetite was synthesized by the Elmore reaction. The synthesis of aluminum-containing coating on the surface of Fe 3

O 4 was carried out by double chemical modification with aluminum isopropylate. The obtained Fe 3 O 4 /Al 2 O 3 NC was impregnated with sucrose solutions. Carbonization of the carbohydrate shell of NC was carried out in argon (500 °C). As a result, Fe 3 O 4 /Al 2 O 3 /C NC was obtained.
The magnetic properties of nanostructures were measured using a laboratory vibration magnetometer of Foner type at room temperature. Adsorption immobilization of lectin was performed in 0. 9  The results of research show that the combination of properties of lectins and magnetically sensitive ironcontaining NC for use in oncology is a promising direction in creating new effective antitumor vector systems for targeted drug delivery and combined local therapy of cancer. The use of natural components in vector systems is a way to minimize the side effects on the body and improve compatibility with other antitumor remedies.

INTRODUCTION
As confirmed by many well-known oncologists in the world, biotherapy will become one of the foundations of cancer treatment in the XXI century. According to modern ideas, biotherapy of cancer is carried out using methods that activate the body's antitumor defenses by the immune system, as well as affect the factors and mechanisms that control the processes of angiogenesis and apoptosis. Biotherapy means are a wide range of different factors, including antitumor vaccines, sera, modern biotechnology products using monoclonal antibodies, cytokinins, cellular factors, regulators of genome activity and other molecular processes of cell life [1].
In recent years, researchers have growing interest in immunotherapy as one of the main methods of biotherapy for patients with cancer. In 2018, the Nobel Prize in medicine was awarded to James Allison (USA) and Tasuku Hondze (Japan) for a revolutionary technique of immunotherapy of cancer using T-cells [2].
It is known that the use of monoclonal antibodies in oncological practice is a method of passive immunotherapy [3,4]. Clinical data suggest that passive immunotherapy is certainly effective and comparable to chemotherapy, but its toxicity is much lower. The combined use of these methods is still widely used, as it significantly expands the possibilities of treatment with modern drugs and helps to increase their effectiveness.
In our works [5,[6][7][8], the results have been shown of researches into magnetosensitive nanocomposites (NC) made on the basis of nanoscale one-domain magnetite (Fe 3 O 4 ) with a modified surface (by hydroxyapatite, aminopropylsiloxane, polyacrylamide, etc.), conjugated with a chemotherapeutic drugcisplatin (CP), and antibody (AB) CD95. In vitro NC were characterized by the recognition of MCF-7 human breast cancer cells, showed the activity of CP, CD95, and the synergism of their joint action with Fe 3 O 4 , which led in vitro to cell death in the quantity that exceeded the action of control samples CP and CD95 in 1.4-2.7 times, as dependent on the composition of NC.
In [9,10] we synthesized magnetic fluid (MF) based on magnetite, gemcitabine (GC) and HER2 AB, studied their properties and bioactivity of the created colloidal systems against hepatocellular carcinoma (HCC) cells HepG2 of human liver. HER2 (Neu, ErbB-2, CD340) is a membrane protein, tyrosine protein kinase of the EGFR/ErbB epidermal growth factor receptor family, which is encoded by the human ERBB2 gene. Amplification of the HER2 gene plays an important role in the pathogenesis and progression of certain aggressive types of cancer [11][12][13]. HER2 is an important biomarker and therapeutic target of the disease, it is associated with tumor aggressiveness and unfavorable prognosis. It has been found that using MF based on physiologic saline and single-domain Fe 3 O 4 in the complex with GC and HER2 AB, the antitumor activity of the composite can be increased in vitro by 8-10 %. MF+GC+HER2 complexes caused the synergistic effect and the increase in cytotoxic activity, compared to GC in monotherapy, up to 18-20 %, with decrease in GC contents to 0.008 mg/mL.
The revealed synergistic cytotoxic/cytostatic effect was explained by high biological activity of the complex with integrated ligand MF-GC-HER2 due to recognition of receptors of HepG2 tumor cells and pharmacological correction of endogenous iron metabolism provided by the use of HER2 and GC in the composition of iron-containing MF. Thus, it has been shown that using magnetic fluid based on magnetite, gemcitabine and HER2 antibody, the effectiveness of cytotoxic/cytostatic action of antitumor drugs increases with a significant reduction in their dose and, accordingly, toxicoallergic reactions of the body.
The said data led to the formulation of scientifically-practical issues related to this work, as an interdisciplinary direction on the border of nanotechnology, surface chemistry and physics, biology and medicine, based on the use of natural components in the composition of iron-containing bioactive NC and magnetic fluids [14][15][16] in creating effective vector systems for antitumor therapy with minimized side effects on the human body and improved compatibility with other drugs. Such natural components, which have unique properties, significant and not yet realized potential opportunities for practical use, include, in particular, lectins [17][18][19][20][21].
Lectins are known to be a group of substances of protein nature (proteins and glycoproteins) of non-immune origin which have a capability to reversely and selectively bind carbohydrates and carbohydrate determinants of biopolymers without altering their covalent structure. They are substances of primary synthesis and are present in all kingdoms, types and classes of living organisms [17][18][19][20]. Lectins are capable to highly specific bind carbohydrate moieties on cellular surface and participate in cell recognition. For example, some pathogens use lectins to attach to the cells of an affected organism.
Many lectins were found to have cytotoxic properties, due to which they are considered promising for use in antitumor therapy. Their antitumor activity is carried out by various mechanisms, including apoptosis, autophagy and inhibition of tumor growth [18]. However, at present, lectins are more common used in medical research and in vitro experiments than in clinical practice. However, due to their involvement in cell recognition, more and more researchers are turning to the investigation of possibility to use lectins for the treatment of cancer. In this aspect, in our opinion, the combination of properties of lectins and magnetically sensitive iron-containing NC [14][15][16] is also interesting and promising from a scientific and applied point of view for use in oncology [22][23][24][25][26][27][28].
Therefore, the purpose of research in this work was the synthesis and study of new ironcontaining NC and magnetic fluids containing bioactive bacterial lectin, promising for use as prototypes of new effective antitumor vector systems for targeted drug delivery and complex local therapy of cancer with minimized side effects on the body and improved compatibility with other drugs.

MATERIALS AND METHODS OF RESEARCH
Samples of magnetite and magnetite/carbon NC were used for research. In the composition of NC, carbon was used due to the known unique adsorption properties and biocompatibility.
Synthesis of nanosized magnetite and its main characteristics. Nanodisperse magnetite was synthesized by the Elmore reaction: Fe 2+ +2Fe 3+ +8NH 4 OHFe 3 O 4 +4H 2 O+8NH 4 + . The ensemble of Fe 3 O 4 nanoparticles (NP) was characterized by sizes of 3-23 nm, with their average size (d 0 ) ~ 11 nm, as determined by the Scherrer formula, and the specific surface area (S sp ) was 105 m 2 /g. Magnetite NP were in a completely single-domain state, coercive force H c = 81.0 Oe, saturation magnetization σ s = 55.6 Gs•cm 3 /g. In the study of IR spectra of magnetite surface, functional groups OH were revealed, the concentration of which was 2.2 mmol/g. The synthesis and properties of magnetite have been described in more detail in [16].

Synthesis of Fe 3 O 4 /Al 2 O 3 /C NC.
To protect magnetite from oxidation under the influence of the temperature (500 °C) required to obtain a carbon shell, the surface of magnetite was modified with a protective layer of alumina.
The synthesis of aluminum-containing coating on the surface of Fe 3 O 4 was carried out by two-fold chemical modification with aluminum isopropylate. To remove isopropyl radicals from the surface of the synthesized NC, the modified samples were calcined in argon stream at the temperature of 500 °C for 2 hours. As a result of the polycondensation reaction, the surface of magnetite acquires an amphoteric character due to Al-OH groups [22]. Magnetic properties. Hysteresis loops of the magnetic moment of the samples were measured using a laboratory vibrating magnetometer of the Foner type at room temperature. The description of an installation and the measurement technique have been described in [19,20]. Demagnetized NP were distributed in paraffin matrix with a volume concentration ~ 0.05 to prevent interaction. For comparison, we used materials with a known value of the specific saturation magnetization (σ s ): a tested sample of nickel and magnetite NP (98 %) manufactured by Nanostructured & Amorphous Materials Inc., USA. In relation to the reference sample, the measurement error σ s did not exceed 2.5 %.
Adsorption immobilization of lectin.  [17]. The obtained MF was characterized by optimal values of the sizes of stabilized nanoparticles, saturation magnetization, sedimentation stability, dynamic viscosity and density [28,29]. Immobilization of lectin on the particles of MF was carried out in a dynamic mode at room temperature for 3 hours. In the composition of Fe 3 O 4 /ol.Na MF, the concentration of lectin was 0.2 mg/mL.
The obtained magnetic fluid with adsorbed lectin was further modified with PEG-2000. In the colloidal solution of Fe 3 O 4 /ol.Na/lectin, the portion of PEG was added at the rate of 15 % (mass) of the polymer by weight of the portion of magnetite. The synthesis of Fe 3 O 4 /ol.Na/lectin/PEG vector system was carried out in a dynamic mode for 3 hours.
Modification of the surface of nanoparticles with polyethylene glycol was performed in order to increase the stability of the magnetic fluid, reduce particle aggregation [28,29]. In addition, when using a magnetic fluid in a biological environment, the presence of polymer chains effectively prevents the adsorption of biomolecules onto the particles of MF, in particular, their agglutination by components that are part of the blood.  Cell cultivation. MCF-7 cells were cultured in complete DMEM medium with 10 % fetal calf serum (FCS) and 40 μg/mL of gentamicin in plastic ware for cell culture in humidified atmosphere at 5 % CO 2 and 37 °C. Changing of medium and reseeding of cells were performed according to the standard technique [26]. Cells were reseeded after reaching 80-90 % of the monolayer with the help of Versene solution. In the experiments, we used the cells being in the exponential growth phase.

Determination of cell viability by colorimetric method.
In 24 hours after reseeding, MCF-7 cells were removed from the substrate with the aid of Versene solution, and the number of cells in suspension was assessed using trypan blue in Goryaev chamber. The number of cells has been determined by the formula: X = (a/80)×10 6 , where X is the number of cells in 1 mL; a -the number of cells counted diagonally in 5 large squares of the chamber.
To assess the effect of the experimental drugs on the viability and proliferation of human breast cancer (BC) cells, the cells were seeded in wells of a 96-well plate in DMEM nutrient medium with 10 % FCS at the concentration of 1×10 4 /well. Cells were cultured in a humidified atmosphere at 5 % CO 2  Physiologic saline was used as a negative control. Cells were cultured in a humidified atmosphere at 5 % CO 2 and 37 o C for another 72 hours.
Calculation of results. After incubation, the results (number of living cells) were calculated visually (direct microscopy method) and by colorimetric method, staining living cells with crystal violet [30]. The nutrient medium was removed from the plates, and 50 μL of a solution of crystal violet (5 mg of dye in 1 mL of 70 % methyl alcohol) was added into each well. In 10 min, the dye was washed three times with running water. Further, the plate was dried at room temperature, and the dye was eluted with 96 % ethyl alcohol (100 μL/well) for 10 min using a mini-shaker PSU-2T at 300 rpm. The results were recorded using a multiwell spectrophotometer with a vertical beam path at the excitation wavelength of 540 nm. The number of living cells (X) in each well of the plate was expressed as a percentage, calculated by the formula: where A 0 is the average value of the optical density in the wells of the negative control, A 1 is the average value of the optical density in the well of the experimental group.
Using human BC cell models in vitro, IC 50 for test preparations was determined with the help of regression analysis.
Calculations of the mean value of the experimental indices (M) and the arithmetic mean error (m) were performed using the software package Excel 2016. To assess the significance levels of the difference in the mean values between the groups, Student's t-test was used. The calculations were performed using the software package STATISTICA 6.0. The difference was noted at a level of p < 0.05.

RESULTS AND DISCUSSION
The measured hysteresis loops of magnetite particles and composites are shown in Fig. 1. Table 1  and Fe 3 O 4 /Al 2 O 3 /C NC dependent on time was carried out in static mode at pH = 7, the volume of the solution was 5 mL, the weight of the adsorbent was 0.03 g, the lectin concentration was 0.116, 0.221 and 0.375 mg/mL (Fig. 2, a, b).
The high rate of sorption in the first 30 min is characteristic for the rapid movement of sorbate molecules to a sorbent surface and may indicate the rate of chemical binding to the active centers of the sorbent. The equilibrium between the solution and the sorbent for concentrations of 0.116 and 0.221 mg/mL is reached in 90 min.  Note: *) error ± 2.5 %, H c , Oe -coercive force, σ s , emu/g -specific saturation magnetization (its value is obtained by extrapolation of the experimental curve σ(H -1 ) to the y-axis), σ H = 8 kOe , emu/g is the specific magnetization in the field of 8 kOe, σ r , emu/g is the residual specific magnetization, σ r /σ s is the relative residual magnetization, σ s NC /σ s Fe3O4 is the mass fraction of magnetite in NC The extraction extent of lectin R (%) was 12-38 % for magnetite and 46-67 % for Fe 3 O 4 /Al 2 O 3 /C NC. Adsorption saturation of the surface was observed.

Study of the effect of nanobiocomposite on the viability of MCF-7 human BC cells.
Visualization of the effect of the studied preparations on the number of living human BC cells is shown in Fig. 3.
The effect of different concentrations of cytotoxic lectin of B. subtilis IMB B-7724, MF, NBC preparations on the viability of human BC cells was studied in vitro. The results are given in Table 2.  To assess the cytotoxic effect of the studied preparations on human BC cell cultures, we used the value of IC 50 (cytotoxicity index) -the concentration of half-maximal inhibition, the concentration of drug required for 50 % inhibition of the test response (number of alive tumor cells) in vitro.
On the base of results presented in Table 2, using the method of regression analysis, the values of IC 50 were calculated for the test preparations on human BC cell models (Table 3). When realizing comparative analysis of the cytotoxic activity of NBC to the experimental compounds in mono-use on human BC cells, a synergistic effect of the nanobiocomposite and the complex of preparations on MCF-7 cells was revealed ( Table 3). The treatment of tumor cells with NBC or the complex of preparations led to a statistically reliable decrease in the value of IC 50 for lectin by 57 %, and for MF, the value of IC 50 decreased by 64 %, compared to mono-use.
The said data show that the combination of properties of lectins and magnetically sensitive iron-containing NC for use in oncology is a promising direction for creation of new effective antitumor vector systems for targeted drug delivery and complex local therapy of cancer with minimized side effects on the body and improved compatibility with other remedies. The results of research show that the combination of properties of lectins and magnetically sensitive iron-containing NC for use in oncology is a promising direction for creation of new effective antitumor vector systems for targeted drug delivery and complex local therapy of cancer. The use of natural components in vector systems is a way to minimize the side effects on the body and improve compatibility with other antitumor remedies.