MAGNETICALLY SENSITIVE NANOCOMPOSITES AND MAGNETIC LIQUIDS BASED ON MAGNETITE, GEMCITABINE, AND ANTIBODY HER2

1 Chuiko Institute of Surface Chemistry of National Academy of Sciences of Ukraine 17 General Naumov Str., Kyiv, 03164, Ukraine, E-mail: phorbyk@ukr.net 2 Ivan Franko Zhytomyr State University 40 V. Berdychivska Str., Zhytomyr, 10008, Ukraine 3 R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology of National Academy of Sciences of Ukraine 45 Vasylkivska Str., Kyiv, 03022, Ukraine


INTRODUCTION
At the beginning of the 21st century, the works in the field of creation of medicobiological and biotechnological nanomachines and "nanoclinics" have got a priority [1][2][3][4]. In these works [5,6], the ideas have been developed for creation of multilevel magnetically sensitive nanocomposites (NC) with a hierarchical nanostructure, which [7,8] have a complex of functions characteristic for biomedical nanorobot: recognition of microbiological objects in biological environments; targeted delivery of medicines to specific cells and organs, and deposit; complex therapy with chemo-, immune, neutron capture radiation, photodynamic, hyperthermic methods and realtime diagnostics; adsorption of cellular decomposition remnants, ions of heavy metals, viral particles, and their removal from the body through an external magnetic field. It should be noted that for today, the concept of magnetically sensitive NC with multilevel hierarchical nanoscale architecture and functions of nanorobots was experimentally substantiated and confirmed through chemical construction of core-shell type nanostructures with complex multilevel hierarchical structure, their combined research and comprehensive check of the capability to perform these functions effectively [9][10][11][12][13][14][15][16][17][18][19].
For in vitro and in vivo systems, the benefits of using of magnetic liquid (ML) compared to the traditional use of cisplatin (CP) have been proven [20]. It has been found that the biggest cytotoxic activity of the nanocomposite, indicated in the MCF-7/CP resistance line, is related to the most active accumulation of ferrimagnetic nanoparticles in cells due to the high level of transferrin receptors and the disturbance of the antioxidant protection system of resistant cells. It has been shown that a magnetic liquid is capable to cause more pronounced cytomorphological changes and genotoxic effects in the cells of the resistance line compared to cells of the sensitive line. Thus, it has been found that ML on the basis of magnetite and cisplatin is characterized by the capability to redox regulation of cells with phenotype of medicine resistance, which testifies the prospect of its use for pathogenetically grounded targeted therapy of malignant tumors.
It has been experimentally shown that the use of ML containing NC on the basis of magnetite and CP, on the general and biochemical parameters of blood does not make more toxic effects on the body, compared with the official antitumor medicine cisplatin, in according doses.
In papers [16,[21][22][23] the results are presented concerning researches into NC made on the basis of nanosized Fe 3 O 4 with modified (by hydroxyapatite, aminopropylsiloxane, polyacrylamide, etc.) surface, conjugated with CP and antibody (AB) CD95. In vitro NC were characterized by recognition of MCF-7 human breast cancer cells, determined activity of CP, CD95, and synergism of their combined activity with Fe 3 O 4 , which resulted in vitro to the death of cells in an amount that exceeded the effect of control samples of CP and CD95 in 1.4-2.7 times as dependent on the composition of the NC.
Taking all this into consideration, it is an actual task from the scientific and practical point of view to clarify the possibility of using of the above mentioned approaches to create new effective antitumor medicines based on ML, containing magnetically sensitive NC, other relevant chemotherapeutic medicines and antibodies [24][25][26][27][28][29][30]. Solving of this task can expand the functionality of NCs, improve their specificity, promote the creation of new multifunctional antitumor medicines for targeted delivery and local therapy, and provide new data concerning the interaction of oncological drugs with tumor cells.
Therefore, the purpose of this work is to synthesize new nanocomposites and magnetic liquids based on magnetite, gemcitabine and HER2 antibody, to investigate their properties and bioactivity in relation to HCC hepatic cells of HepG2 line.

EXPERIMENTAL PART AND DISCUSSION
In this work, gemcitabine -(2-deoxy-2', 2) difluorocytidine monochloride) is used as a drug of the chemotherapeutic mechanism of action. It is a cytotoxic drug, an antimetabolite from the pyrimidine antagonists group. GC belongs to the List of the main medicines of the World Health Organization and the most effective and safe medicines needed in the healthcare system. It is characterized by considerable antitumor activity for the list of solid tumors (non-small cell lung cancer, pancreatic, bladder, breast and ovarian cancer), satisfactory endurance and the capability to combine successfully with other antitumor medicines. It is used for treatment of cholangiocarcinoma and other types of biliary cancer. The general side effects include bone marrow suppression, liver and kidney problems, nausea, fever, rash, shortness of breath and hair loss [31][32][33][34]. Therefore, for today, the possibility is actively studied to use GC in composition of magnetically sensitive NC and ML on their basis to create multi-functional antitumor medicines for targeted delivery and local therapy [35][36][37][38].
In this work as an AB, HER2 (Neu, ErbB-2, CD340) was chosen. It is a membrane protein, a tyrosine proteinkinase of the EGFR / ErbB epidermal growth factor receptor family encoded by the human gene ERBB2. Amplification of the HER2 gene plays an important role in the pathogenesis and progression of certain aggressive types of cancer [39][40][41]. HER2 is an important biomarker and therapeutic target of the disease, associated with aggressiveness of the tumor and unfavorable prognosis. It is known that HER2 AB is considered to be one of the optimal for treatment of such diseases as gastrointestinal tract cancer, in particular in the presence of liver metastasis [42]. Therefore, for in vitro researches, we have chosen HER2 AB in combination with GC in composition of ML based on magnetite. Hepatocellular carcinoma (HCC) is the most common primary malignant form of liver cancer, the result of malignant transformation of hepatocytes, a severe, high mortality disease. Every year in the world about 600 thousand cases of this disease are diagnosed. In order to test the antitumor activity of the synthesized ML in vitro, the cells of the HCC human liver of HepG2 line were selected in this work.

METHODS OF RESEARCH
Adsorption of GC on the surface of nanosized magnetite was carried out from aqueous solutions of different concentration. Gemcitabine TEVA (Pharmachemie BV, the Netherlands) was used for research.
Adsorption capacity of samples A (mg/g), extraction extent U (%), and separation factor E (mL/g) were determined by techniques described in [13,14].
The amount of adsorbed substance on the surface of magnetite was determined by measuring the concentration of GC in contact solutions before and after adsorption using a spectrophotometer Spectrometer Lambda 35 UV/Vis Perkin Elmer Instruments.
Specific surface area (S sp ) of the samples was determined by nitrogen thermal desorption by means of a KELVIN 1042 device of "COSTECH Instruments" firm. The size of NP was appreciated by formula D BET = 6/(ρS BET ), where ρ is density of a particle of NC, S BET is a value of specific surface area calculated by the polymolecular adsorption theory of Brunauer, Emmett and Teller (BЕТ).
The hysteresis loops of the magnetic moment of the samples were measured using a laboratory vibration magnetometer of Foner type at the room temperature. The description of the installation and the method of measurement are set out in [43]. To prevent interactions, the demagnetized nanoparticles were distributed in the paraffin matrix with a volume concentration  The cells under research were cultured in a complete nutrient medium DMEM with 10 % FTS and 40 μg/mL of gentamicin in plastic dishes in a humidified atmosphere at 5 % СО 2 and 37 °С. The medium changes and transitions of the cells were carried out according to the standard method [44]. For the experiments we used cells that were in the exponential phase of growth. After 24-48 h after the last transition, the cells were planted for cultivation at a concentration of 1-1.5×10 4 cells /well of 96-well cell plate. After 24 h, various amounts of experimental substances were added to the respective wells according to the study scheme and their cytotoxic activity was determined.
The vitality of cells in the experiment was evaluated by means of colorimetric method by colouring cells with crystalline violet. Adhesive cells are separated from the substrate after death. This property is used in order to determine the number of living cells after their treatment with the investigational agents. One of the ways to determine attached (living) cells is to dye them with crystalline violet dye which binds to proteins and DNA. The results were evaluated using a multi-spectrophotometer (wave length is 540 nm). The percentage of viable cells was calculated according to the formula: The cytotoxic and antiproliferative activity was determined using IC50 indicator.

SYNTHESIS AND PROPERTIES OF MAGNETITE
The synthesis of nanodisperse magnetite was carried out according to the method [7], by coprecipitation of iron salts according to the reaction: The synthesized ensembles of Fe 3 O 4 NP were characterized by dimensions of 3-23 nm. The average size of the NP (d 0 ) depended on the synthesis conditions and was 6-13 nm, the size distribution could be technologically managed. The specific surface area (S sp ) of synthesized magnetite, dependent on the average particle size, was S sp = 90-180 m 2 /g. In this work we used specimens for which S sp = 110±1 % m 2 /g. The value of mean diameter of the NP Fe 3 O 4 , calculated from the results of studies of X-ray diffractograms using the Scherrer formula, D XRD was 10.5 nm. Functional OH groups with concentration of 2.2 mmol/g were detected in the study of infrared spectra of magnetite surface [7,14].
The synthesized magnetite is characterized by satisfactory magnetic characteristics: the coercivity H c = 55.0±2.5 % Oe, the specific magnetization of saturation σ s =56.2±2.5 % Gs ·cm 3 /g, relative residual magnetization M r /M s = 0.2±2.5 %. Such characteristics are important for medical and biological applications [7,44], for example, while transporting the medicine through blood vessels of small diameter, in which the embolization and aggregation of particles are extremely undesirable.
In [13,14] it has been established that the synthesized magnetite with the given properties is characterized by superparamagnetism of nanoparticles and is in a completely singledomain state. It is known that superparamagnetism is a form of magnetism, which is manifested in ferro-and ferrimagnetic particles.
If such particles are of sufficiently small size, they are converted into a singledomain state, that is, they become homogeneously magnetized through the entire volume at any values and directions of the field H. To the peculiarities of the singledomain state of these particles, one can also refer the existence of domains not only in alloys and compounds in the solid state, but also in liquid media (suspensions and colloids).
Adsorption of gemcitabine on a surface of nanosized Fe 3 O 4 . Adsorption of GC on the surface of magnetite Fe 3 O 4 was carried out in a medium of saline solution in the range of concentrations C 0 = 0.02-0.67 mg/mL (m = 0.03 g, V = 5 mL, pH = 3.0) during 2 h in static mode at room temperature. The pH of GC solution medium in 0.9 % NaCl was given by 0.1 N HCl.
From the results of researches into the dependence of adsorption activity on pH, the maximal values of А (mg/g), U (%) and E (mL/g) for Fe 3 O 4 are observed in the acid medium at рН = 3. According to the results of spectroscopic researches, at the value of рН = 3, up to 70 % of GC is in protonated form, which corresponds to the medicinal form of gemcitabine hydrochloride.
The isotherm of adsorption of GC on the surface of Fe 3 O 4 , and the results of its mathematical processing are given in Fig. 1.
For quantitative description of equilibrium processes in the investigated range of concentrations the Langmuir, Freundlich, and BET [45] models were used. Linearized equations of these models are used to determine the values of the constants (for the Langmuir model R 2 = 0.849, the Freundlich -0.996, BЕТ -0.948), included in the isotherm equation.
In our experiments, there has been a deviation in the adsorption character from the Langmuir scheme, which may be caused by the presence of several types of binding centers, characterized by different values of equilibrium constants (energy heterogeneity) and/or the effects of a compatible adsorption. Therefore, we have used the Freundlich model ( Fig. 1 а,  Table 1), that describs the isotherm of adsorption on a heterogeneous surface: А = К F С р 1/n, in the linearized form lnA = lnK F + 1/n lnС.
The experimental values of the adsorption capacity А were ~ 37.2 mg/g, the extraction extent U = 33.13 %, the separation coefficient E = 82.58 mL/g at С 0 = 0.67 mg/mL (m = 0.03 g, V = 5 mL, pH = 3.0). The results of the studies indicate that nanosized magnetite may be promising for the producing of magnetically sensitive adsorption materials for medical purposes, for example, for detoxification of an organism after GC treatment.   Fig. 2 (σ is specific magnetization, H is magnetic field strength). Table 2 shows the magnetic properties of magnetite and NC with adsorbed gemcitabine derived from experimental hysteresis loops.
In the table: H c , Oe -coercive force; σ s , emu/g -specific magnetization of saturation of NC; σ r , emu/g -residual specific magnetization of NC; σ r /σ s -relative residual magnetization; α Fe3O4 calccalculated mass concentration of Fe 3 O 4 in NC, %.   PS was used as the dispersion medium of samples of magnetic liquids for research. It should be noted, that the use of distilled water as dispersion medium, does not change the magnetic properties of colloidal systems substantially.
Nanosized magnetite was used as a dispersed phase, in the singledomain state, or related NC Fe 3 O 4 @GC.
The mass of sodium oleate m Ol.Na used to stabilize the surface of the NP and NC in the composition of the ML was calculated taking into account the concentration of hydroxyl groups on the surface of magnetite. The calculation was made using the formula: It is known that PEG interferes with adsorption interactions of liquid components with protein molecules [45], which is important for medical applications of magnetic liquids. Additional modification with PEG-2000 was carried out in a dynamic mode using a shaker, the amount of polymer was 10-15 % of the weight of the bulk of NP Fe 3 O 4 , or NC [13,14]. Fig. 4 shows the hysteresis loops of aqueous ML Fe 3 O 4 @Ol.Na/PEG+PS (1) and Fe 3 O 4 @GC/Ol.Na/PEG+PS (2). The thickness of the layer of GC is ~ 2.3 nm. The thickness of the layer of the stabilizer ~ 3 nm, and the volumes of water in the ML are the same.
As known, taking into account the distribution of nanocomposite particles by volumes p(V), the magnetization curve of ML (Fig. 5) v -volume of a NC particle, s M -magnetization of the saturation of the core, v c -volume of the core, , χ -magnetic susceptibility of the shell, v s -shell volume. The main contribution to the magnetization of ML is introduced by the superparamagnetism of the cores and the paramagnetism of the shells of the сore@shell particles. Compared to superparamagnetism of cores in the fields H = 0-10 kOe, in most cases the paramagnetism of the shells (the second term in (1)) can be neglected.
The modification of ML Fe 3 O 4 @GC/Ol.Na/PEG+PS with AB HER2 (receiving ML Fe 3 O 4 @GC/Ol.Na/PEG/AB HER2+PS) was performed dynamically with use of a shaker. The concentration of GC and AB HER2 in such ML is determined by the therapeutic necessity. In the starting ML, the concentration of GC and AB HER2 has been 1.25 mg/mL and 3.75 μg/mL, respectively, which allows one providing the necessary dosages of the medicine in the experimental samples by diluting the initial ML. In addition, such a liquid is characterized by satisfactory rheological properties and sedimentation resistance. In particular: 1. IC50 for ML was 0.155 mg/mL (control 1).

Investigation of influence of experimental samples on viability of HepG2 hepatocellular carcinoma cells of human liver in vitro.
In samples with a ML concentration more than 0.19 mg/mL, the number of living cells is not determined, which is, likely, due to high optical density of samples. 2. The HER2 AB in monoapplication (control 3) in the studied concentrations does not affect the viability or proliferation of hepatocellular carcinoma of human liver cells of the HepG2 line, as its effect does not decrease the cell viability and does not differ practically from the influence of PS (control 5). 3. The cultivation of HepG2 cells at the same time in the presence of ML and AB HER2 at concentrations less than 0.05 mg/mL and 0.013 μg/mL, respectively, did not practically affect the viability of cells of liver carcinoma. However, the complex application of ML and HER2 at concentrations of 0.1 mg/mL and 0.025 μg/mL, respectively, reduced the number of viable cells of the said line to ~ 85.9 %. 4. IC50 for GC was 0.002 mg/mL (control 2).
The influence of GC in monoapplication at the concentration of 0.008 mg/mL left ~ 78 % of cells in a viable state. The use of GC in this concentration in combination with ML (0.1 mg/mL) showed a synergistic effect and increased the effectiveness of cytostatic drug up to ~ 10 % (the number of alive cells was ~ 68 %). 5. The combined application of GC and AB HER2 (control 4) at concentrations of 0.008 mg/mL and 0.025 μg/mL, respectively, also showed a synergistic effect, that resulted in a decrease of number of viable cells to ~ 65 %. 6. The use of a composite system consisting of GC and HER2 at concentrations of 0.008 mg/mL and 0.025 μg/mL, respectively, and ML (0.1 mg/mL with respect to Fe 3 O 4 ) results in a reduction of the number of viable cells of HepG2 liver carcinoma to ~ 55 %, that indicates a significant synergistic effect of the said components. The revealed synergistic cytotoxic/cytostatic effect can be explained by the high biological activity of the complex Fe 3 O 4 -GC-HER2 with the integrated ligand due to recognition of HepG2 tumor cell receptors and pharmacological correction of endogenous iron exchange, which is ensured by the use of iron-containing ML, GC, and HER2 antibodies.
Indeed, in mechanisms of implementation of the apoptosis program due to formation of the medicinal effect of NC, violations of the exchange of endogenous iron in tumor cells play an essential role [47]. The said violations cause an increased need of iron for cells, which is satisfied by the accumulation of a significant number of Fe 3 O 4 nanoparticles with ML. The high level of "free iron" in the form of accumulated Fe 3 O 4 and acidic medium in the cells, causes accelerated formation of iron ions and active forms of oxygen (Fenton reaction), which in its turn, leads to oxidative stress of cells and apoptosis. Thereby an increase occurs also in the effectiveness of both GC and HER2 AB. As an example we can take the increase up to ~ 10 % in the action of GC at the concentration of 0.008 mg/mL in the composition of ML and the appearance of cytotoxic action of the ML+ HER2 complex at a level of ~ 10 % at the concentration of HER2 of 0.025 μg/mL, compared to the absence of activity of AB in these doses in monoapplication.
Thus, the combined effect of ML, GC, and HER2 on the HepG2 cells significantly exceeds their effects in monouse at the same concentrations, which results in synergistic effect.
So, in vitro, an example of the effect of a new magnetocarried colloidal system, containing magnetite, antitumor component GC and HER2 antibody, on HCC human liver cells of HepG2 line shows the possibility to achieve a cytotoxic effect at substantially lower concentrations of chemo-and immunotherapeutic medicines and to create conditions for reducing of toxico-allergic reactions of the organism as a whole. In addition, the revealed experimental data indicate that the investigated MLs can be promising for use in the method of targeted delivery and local therapy of oncological diseases.

CONCLUSION
The processes of adsorption of GC on the surface of nanosized singledomain magnetite (Fe 3 O 4 ) have been studied. In the experiment, the value of the adsorption capacity A reached The ML were synthesized based on magnetite and PS, stabilized with Ol.Na and PEG, containing GC and HER2 AB (Fe 3 O 4 @GC/Ol.Na/PEG + PS). The properties of ML were studied as well as cytotoxic/cytostatic activity in relation to HCC of human liver of HepG2 line.
The obtained data can be useful for predicting of the nanoscale architecture of magnetically sensitive NC and ML in the production of medicines on their basis.
The synergistic nature of the effect of GC/Fe 3 O 4 /HER2 complex on HepG2 cells was revealed. It has been shown that HER2 AB alone does not affect HepG2 cell viability/ proliferation in investigated concentrations.
GC suppressed cell proliferation of liver carcinoma, the IC50 value was 0.002 mg/mL in vitro. The use of ML in combination with GC can increase the cytotoxic activity of the composite up to 8-10 %. The ML + GC + HER2 complexes caused a synergistic effect and increased the cytotoxic activity, compared with GC in monouse, to 18-20 %, while GC content reduced to 0.008 mg/mL.
The results of the studies indicate that the use of ML on the basis of magnetite, gemcitabine and antibody increases the effectiveness of the antitumor medicines with a significant reduction in their dose and, respectively, the toxico-allergic reactions of the body, and nanosized magnetite may be promising for the manufacture of magnetically sensitive adsorption materials for medical purposes, for example, detoxification of an organism after GC therapy.