"Nanobiotechnology for health-care"

Investigating on the interaction between nanodiamonds and hemoglobin in vitro by spectrophotometric analysis
Batyuk L.V.1, Kizilova N.N.2, Muraveinik O.A.3 
1Department of Medical and Biological Physics and Medical Information Science, Kharkiv National Medical University, 4 Nauky Ave., Kharkiv, 61022, Ukraine.
E-mail: liliya-batyuk@ukr.net

2 Warsaw University of Technology, Institute of Aviation and Applied Mechanics, Poland; PoreLab, Department of Chemistry, Norwegian University of Science and Technology, Norway.
E-mail: n.kizilova@gmail.com

3Kharkiv City Clinical Hospital No 7, Kharkiv, Ukraine
E-mail: muraveinik-oksana@ukr.net

One of the conditions for the practical application of nanodiamonds in medicine is the need for a preliminary study of the effects of these nanoparticles on various components of human blood. We have previously identified certain changes in the cellular and biochemical composition of blood under the influence of ultradisperse nanodiamonds (NDs) of detonation synthesis [1-2]. In particular, an important role of NDs in the regulation of quantitative imbalance of erythrocyte membrane proteins in patients with acute ischemic stroke was found [3]. One of the most important mechanisms for increasing the structural stability of erythrocyte membranes is their interaction with the main protein of the cell, namely with hemoglobin (Hb). 
This work is devoted to the study of the features of binding of Hb with NDs in the experiment in vitro. A suspension of erythrocytes from the blood of cancer patients who underwent radiation therapy, namely, patients with stomach cancer (n = 8) and breast cancer (n = 11), was used as a model. As a control, a suspension of erythrocytes was used, which was obtained from the blood of healthy donors (n = 10). Hb was obtained according to the standard method. In experiments were performed using an aqueous suspension of NDs, with an initial concentration of nanodiamonds of 0.1–0.3% by weight. For studies, the suspension was diluted to 0.045–0.035% to a final concentration of 0.9% NaCl, depending on the setting of the experiments. The stock suspensions of NDs with various concentrations were prepared using 0.01 M PBS (pH 7.0), which were sonicated for 2 h. Dispersions of nanoparticles at various concentrations were added to the Hb suspension and kept for a specified time. The concentrations of hydroxy and methemoglobin were calculated based on the equations for the spectrophotometric analysis of mixtures of Hb [4]. The absorbance of these solutions was measured at 522 nm (the isobestic point for HbO2 and MetHb). Calculations of Fe(III) content and the degree of hemoglobin denaturation was calculated on the basis of absorbance values at 630 nm as the relation between absorbance of the examined solution and absorbance of the solution that contains 100% HbO2 (0% Fe(III)) and absorbance of the solution totally converted into MetHb (100% Fe(III)) [4]. The kinetics of spontaneous Hb oxidation was considered at a temperature of 43 °C and fixed pH values ​​in the range of 5–8 according to the method [5]. The results were statistically processed using the MATLAB program. Differences between two independent samples were determined using the Mann-Whitney nonparametric test. It is known that the level of Hb affects tumor-induced neoangiogenesis, which can cause tumor growth and metastasis. It was found that, in the presence of NDs, a decrease in the kinetics of spontaneous oxidation of Hb is observed in comparison with the control, which may be due to the more rapid adsorption of Hb on the NDs surface due to hydrophobic forces. Binding to NDs can lead to partial unfolding and exposure of hydrophobic Hb residues, which leads to intermolecular interactions between the unfolded Hb and NDs surfaces; this system can be thermodynamically stable. The curve shapes indicate a large difference between the exhibit hemolysis Hb as a function of incubation time suspensions with various concentration of NDs for healthy donors and oncologic patients. The time course analysis may provide further materials for underlying mechanisms interaction between NDs and geme protein.

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