Comparative study of cytotoxicity of ferromagnetic nanoparticles and magnetitecontaining polyelectrolyte microcapsules

The cytotoxicity of magnetite nanoparticles (MNP) stabilized with citrate acidand polyelectrolyte multilayer microcapsules containing these particles in the shell is analyzed. Microcapsules were prepared by co-precipitation of iron (II) and (III) chlorides. Polyelectrolyte microcapsules synthesized by the layer-by-layer method from biodegradable polymers polyarginine and dextran sulfate. Cytotoxicity of the synthesized objects was studied on the L929 cells culture and human leucocytes. It was also investigated the phagocytic activity of leukocytes for the MNP and magnetite containing polyelectrolyte microcapsules (MCPM). A set of tests (MTT assay, neutral red uptake assay, lactate dehydrogenase release assay) was used to study the cytotoxicity in vitro. All the tests have shown that the magnetic nanoparticles have a greater cytotoxicity in comparison with microcapsules containing an equivalent amount of magnetite. In contrast to the mouse fibroblast culture, human leukocytes were more resistant to the toxic effects of magnetite. At the concentrations used in our studies no significant reduction in the viability of leukocytes has been registered. Both MNP and MCPM undergo phagocytosis, however, the phagocytic activity of leukocytes for these particles was lower than for the standard objects (latex microparticles). Introduction Polyelectrolyte microcapsules (PM) are a promising system for packaging and magneticaly driven delivery of verious substances. Microcapsules are commonly synthesized by layer-by-layer (LBL) technology when oppositely charged polyelectrolytes are applied in layers on a core of inorganic material which can contain different substances[1]. Then the core is dissolved by an acid and the substance remains in the PM. Inclusion of different functional groups into the core or shell of capsules allows to control the delivery and release of the drug.A method for the synthesis of PM from biocompatible and biodegradable polymers (polyarginine and dextran-sulphate) with nanoparticles of magnetite incorporated into the shell was proposed in paper [2]. It has been shown in in-vitro International Symposium Physics, Engineering and Technologies for Bio-Medicine IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 784 (2017) 012038 doi:10.1088/1742-6596/784/1/012038 International Conference on Recent Trends in Physics 2016 (ICRTP2016) IOP Publishing Journal of Physics: Conference Series 755 (2016) 011001 doi:10.1088/1742-6596/755/1/011001 Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Published under licence by IOP Publishing Ltd 1 experimentsthat these capsules are more actively phagocytosed by cells [2].andcan act as intracellular anchors that allow manipulating cells by an external magnetic field [3]. However, there are several problems that limit the use of this technology. One of the main problems is the toxicity of magnetic nanoparticles. Iron is not a foreign substance to the body being a part of its major systems (hemoglobin, cytochrome p450, etc.). There are several systems involved in assimilation, transportation and excretion of iron in the human body. However, magnetically driven drug delivery requires parenteral administration of rather large amount of iron which can lead to some side effects [4]. In particular, the ionized iron can activate lipid peroxidation and be toxic for cell membranes [5]. However, the isolation of iron from contact with the blood cells and endothelium can decrease the cytotoxicity [6]. In the present paper, the cytotoxicity of magnetite nanoparticles (MNP), and magnetite containing polyelectrolyte microcapsules (MCPM) with these nanoparticles included in the shell was studied. 1. Materials and methods: 1.1. Reagents Iron chloride (III) (FeCl3 • 6H2O) (99.99%, Sigma-Aldrich), Iron chloride (II) (FeCl2 • 4H2O) (99.99%, Sigma-Aldrich), Ammonium hydroxide 25% (99.8% Vekton, Russia) Citric acid (99.99%, Sigma-Aldrich), Sodium ethylenediaminetetraacetate (99.9% Vekton) Sodium carbonate (99.9% Vekton) Calcium chloride (99.9% Vekton) Sodium dextran sulfate (DS) (M ~ 100 kDa, Sigma-Aldrich), Polyarginine hydrochloride (PA) (M ~ 15-70 kDa, Sigma-Aldrich), Sodium chloride (99.9% Vekton) Hydrochloric acid 38% (99.8% Vekton). Dulbecco’s Modified Eagles Media (DMEM),Biolot (Russia). Fetal Bovine Serum (FBS), Biolot (Russia). Isopropanol (99.9% Vekton) Neutral red, Sigma-Aldrich. MTT (3[4,5dimethylthiazol-2-yl-2,5-difeniltetrazolium bromide), Sigma-Aldrich. LDH Cytotoxicity kit, (The Thermo Scientific, USA). The latex particles (PanEco, USA). Trypan blue, Sigma Aldrich. All reagents were used without further purification. 1.2. Synthesis of ferromagnetic nanoparticles Magnetite nanoparticles were prepared by coprecipitation of iron (II) and (III) chlorides in a molar ratio of 1:2 [Volodkin] using citric acid as a stabilizer. Iron chlorides (2.35g of FeCl3•6H2O and 0.86 g of FeCl2•4H2O) were dissolved in 40 mL of deionized water. The obtained solution was poured into three-necked flask and heated to 80°C under nitrogen. Then ammonium hydroxide (5 ml of 25% solution) were slowly added to a heated solution with vigorous stirring and kept for 15 minutes. Then citric acid (4 ml of25% solution) was added to the particles colloidand kept stirring for 1 hour at 90°C. The magnetite particles were purified by dialysis(cellulose membrane Q1210-55 F3 ("Orange scientific", Belgium) with 12-14 kDa pore, the eluent 0.15 MNaCl, the volume ratio suspension/eluent equal 1/1500, the treatment time 24 hours). Then the purified colloid was centrifuged at 16 000 min -1 for 5 minutes to remove large particles. 1.3. Synthesis of magnetite containing polyelectrolyte microcapsules International Symposium Physics, Engineering and Technologies for Bio-Medicine IOP Publishing IOP Conf. Series: Journal of Physics: Conf. Series 784 (2017) 012038 doi:10.1088/1742-6596/784/1/012038