ИСТИНА

Last achievements in nanoparticle engineering shed the light on developing a unique way of diagnostics and therapy of different diseases in medicine [1]. In most cases the administration of nanoparticles into human organism is supposed to be performed via blood flow by means of intravenous injection. In order to apply the nanoparticles successfully one needs to be sure of their biocompatibility and nontoxicity. In particular, it is important to know how they interact with blood components [2,3]. Silicon nanoparticles (Si NPs) have attracted broad attention due to their excellent optical and mechanical properties, in particular, strong fluorescence and photostability. Another main advantage of Si NPs over other nanomaterials, e.g., iron nanoparticles or quantum dots, is their low toxicity. Many organic compounds in human body include silicon, so after biodegradation Si NPs can be bound with other natural minerals or harmlessly eliminated via renal clearance [4]. Therefore, these materials are proposed for use as potential vehicles for biomedical imaging and real-time diagnosis [5,6,7]. The main aims of this work are: 1) determination of the effect of Si NPs on the intrinsic microrheologic property of red blood cells (RBC) – their spontaneous aggregation; 2) analysis of changes in the absorption and fluorescence spectra of RBC after incubation of the whole blood sample with Si NPs. We performed our experiments with fresh blood samples drawn from a cubital vein of a healthy volunteer. Two types of Si NPs with average sizes of 5 and 100 nm were used. To fulfill the first task we used two light scattering techniques to conduct the measurements: laser aggregometry based on diffuse light scattering from whole blood samples and optical tweezers. In the first case, we obtained the parameters characterizing the ability of RBC to spontaneously aggregate (average aggregation time and aggregation index - the percent of the cells that have aggregated during first 10 seconds after the start of the aggregation process) from the experimental dependences of the intensity of light scattered from large ensembles of cells incubated with Si NPs. In the second case, in order to analyze the cells microrheologic properties (aggregation time and interaction forces) single RBC were trapped and manipulated by optical tweezers without mechanical contact with the cells. The following task was the determination of Si NPs influence on optical properties of RBC. In this experiment, spectral instruments photometer and fluorometer were based in one spectrometer (FluoroMax®-4, Japan) were used to obtain the absorption and fluorescence spectra of RBC incubated with Si NPs (average size 5 nm). To confirm the adsorption of nanoparticles on the cells’ membrane, the whole blood after incubation with Si NPs at concentrations ranging from 100 to 1000 μg / ml during 1 hour was centrifuged and the RBC were separated from the plasma. Absorption spectra were obtained, which were compared with those of the original sample of whole blood. The experiments carried out by laser aggregometry using whole blood and Si NPs with average size 100 nm showed an increase in the RBC aggregation time after their incubation with Si NPs while the aggregation index decreased by 40 ± 8 % from the norm (see Fig. 1). For example, the characteristic time of RBC aggregates formation increased by 50 ± 6 % at the particle concentration of 100 μg/ml in comparison with the case without incubation with nanoparticles. The experiments with optical tweezers also showed an increase in the time of aggregation of individual RBC. The forces of RBC interaction during their aggregation and disaggregation were found not to change. Both of the applied methods showed compatible results. Thus, we conclude that Si NPs actually affect the microrheologic properties of blood decreasing the ability of RBC to spontaneously aggregate. This influence is dependent on the size and concentration on the NPs and on the duration of the RBC incubation with the NPs. Figure 1: Aggregation index of blood after 60 min incubation with NPs versus Si NPs concentration ranging from 0 to 100 µg/ml. Figure 2: Fluorescence spectra of whole blood (black) and RBC after incubation of whole blood with Si NPs (red). The obtained spectra of blood incubated with Si NPs (Fig. 2) confirms the fact of adsorption of these NPs on the RBC membrane. The obtained differences in the spectra allow for speculating on a possibility of applying the Si NPs as fluorescence nanoagents for diagnostic purposes in medicine. Acknowledgment: The authors acknowledge the financial support provided to this study by RSF grant № 20-45-08004. REFERENCES [1] A. Jiang, B Song , X. Ji, F. Peng, H. Wang, Y. Su, and Y. He, “Doxorubicin-loaded silicon nanoparticles impregnated into red blood cells featuring bright fluorescence, strong photostability, and lengthened blood residency,” Nano Res., vol. 11, no. 4, pp. 2285–2294, 2018. [2] Y. Zhao, X. Sun, G. Zhang, B. G. Trewyn, I. I. Slowing, and V. S. Y. Lin, “Interaction of mesoporous silica nanoparticles with human red blood cell membranes: Size and surface effects,” ACS Nano, vol. 5, no. 2, pp. 1366–1375, 2011. [3] Y Lin, L. Tsai, E. Perevedentseva, H. Chang, C. Lin, D. Sun, A. E. Lugovtsov, A. V. Priezzhev, J. Mona, C. Cheng, “The influence of nanodiamond on the oxygenation states and micro rheological properties of human red blood cells ,” J. Biomed. Opt., vol. 17, no. 10, p. 101512, 2012 [4]F. Peng, Y. Su, Y. Zhong, C. Fan, S. T. Lee, and Y. 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