Introduction. The polypeptide produced by monocyte-macrophage cells and known as tumor necrosis factor (TNF), acts not only as the cytotoxicity effector mediator ensuring destruction of cancer target cells, but also participates in the regulation of various physiological and pathological processes in the body. Research of the biological activity of TNF, especially the mechanisms of its implementation is important for the elaboration of indications and methods for its clinical application not only in the treatment of patients with malignant tumors, but also in other areas of clinical medicine, and for creation of new drugs that affect its production in the body [1].
Modifications of the substance of the target recombinant protein usually lead to improving the biological properties, increase activity and reduce the toxicity of TNF [2]. In our previous studies, it was shown that combining the nanoparticles of cerium dioxide (CND) with interferon [3] or vaccines [4] ensures the growth of their biological activity. One of the possible mechanisms for implementation of the above-mentioned effect involves the nano-biocomposite formation. Based on the preliminary data, we have explored the process of interaction CND with TNF.
Materials and methods. The interaction of CND with TNF and the formation of nano-biocomposite lead to an increase in peptide hydrodynamic diameter (HD). HD was monitored via dynamic light scattering (DLS) method using the two angle particles and molecular size analyzer Zetasizer Nano ZS (Malvern, UK). As a model object, the Human Recombinant Tumor Necrosis Factor-α (rhTNF-α) was used (Promega Corporation Part# 9PIG524). The rhTNF-α concentration was 3.8•104 U/ml (1µg/ml). The CND (size ~5.8 [5,65-5,85] nm, ζ-potential ~ +12 mV) was synthesized by O.S. Ivanova (IGIC RAS) as an aqueous 0.01 M sol [5]. CND was added to the rhTNF-α in the weight ratio of 1:3 and analyzed ex tempore. Interaction of rhTNF-α with CND-sol was analyzed in dynamics. The measurements of the HD were carried out for 17 hours at intervals of 3-4 minutes. All measurements were performed at a constant temperature (25°C) and neutral media (pH=7.2). The obtained data were analyzed using methods of mathematical statistics and were presented as the median and interquartile range.
Results and discussion. The initial HD of rhTNF-α was ~0.62 [0.56-0.71] µm. Adding CND to rhTNF-α caused an increase of HD. Four hours after introduction of CND, the HD of solute was twofold increased. By the tenth hour, the size of the objects was ~1.57 [1.46-1.74] µm. In this time interval, the growth of the H|D of the particles was almost linear. In the range of 10 to 17 hours of observation, the CND-rhTNF-α nano-biocomposites size was stabilized. By the end of his observations, the HD was ~1.69 [1.66-1.74] µm. Thus, the modification of rhTNF-α by CND leads to the formation of stable nano-biocomposites having increased HD. Our preliminary in vitro study [6] showed that such nano-biocomposite of CND-modified rhTNF-α is more active than the pristine rhTNF-α (probably, due to adjuvant-mimic properties of CDN). The findings are the rationale for the creation of new highly efficient nanocomposite drugs.
References.
1) Gough DB, Winstanley FP et al. Regulation of tumor necrosis factor production in healthy humans and in patients with cancer. Arch Surg. – 1992; 127(6):713-717.
2) Pat. RF № 2,386,447 Anticancer drug based on nanoparticles bearing recombinant human tumor necrosis factor alpha. Masycheva VI, Lebedev LR, Danilenko ED. – Publ. 20.04.2010.
3) Zholobak N.M., Krivokchatska L.D., Shcherbakov A.B., et al. The influence of nanoceria dioxide colloid solution on the activity of viferon. Conference “Nanotechnology in oncology – 2010” – Moscow, 30.10.2010; P.44-48.
4) Zholobak N.M., Mironenko A.P., Shcherbakov A.B., et al. Cerium dioxide nanoparticles increase immunogenicity of the influenza vaccine. Antiviral Research 2016; 127; P.1-9.
5) Ivanov V.K., Polezhaeva O.S., Shcherbakov A.B., et al. Microwavehydrothermal synthesis of stable nanocrystalline ceria sols for biomedical uses. Russian Journal of Inorganic Chemistry, 2010; 55:1-5
6) Shydlovska O.A, Kharchenko E, Zholobak N.M., et al. Nanoparticles of cerium dioxide increase activity of recombinant tumor necrosis factor-alpha. XIV International Scientific Conference of Students, PhD Students & Young Scientists: Shevchenkivska vesna: Biology – 2016; P.23–24.
