MOSCOW STATE UNIVERSITY Saratov Fall Meeting 2020 Effect

MOSCOW STATE UNIVERSITY Saratov Fall Meeting 2020 Effect of titanium dioxide nanoparticles on human red blood cells microrheologic properties: in vitro studies by laser techniques Neznanov 1*, Kadanova 1, A. I. I. M. A. E. Lugovtsov 1, 2, A. V. Priezzhev 1, 2, A. P. Popov 3 1 Department of Physics of M. V. Lomonosov Moscow State University, Moscow, Russia Laser Center of M. V. Lomonosov Moscow State University, Moscow, Russia 3 VTT Technical Research Centre of Finland, Oulu, Finland *E-mail: neznanov. ai@mail. ru 2 International

Introduction Nanoparticles (NP) have strong potential for diagnostic and therapeutic applications in medicine. Regarding the administration of NP into human organism via intravenous injection as most probable, detailed study of their interaction with blood components and possible toxicity is required. The key properties that determine blood flow are the mechanical and aggregation properties of red blood cells (RBC), which are the major constituents of the blood. The image of the sample smear aggregates of RBC at 60 × zoom. For the time being there is no sufficient evidence confirming that certain NP when administered into the blood flow will not negatively influence the blood circulation, spontaneous aggregation and forced disaggregation parameters of blood cells and therefore the patient’s health [1]. So it is vital to assess the interaction of NP with blood cells in in vitro conditions before conducting the in vivo experiments and pursuing clinical applications. [1] Health Effects of Nanoparticles. The IRSST report “Nanoparticles: Current knowledge about occupational health and safety risks and prevention measures”, IRSST, Quebec, Canada, 2006. 2

Introduction The aim of this work is to evaluate the dependence of RBC microrheologic properties (especially, aggregation properties) on the concentration of titanium dioxide NP incubated with whole blood and obtain the conditions, at which these particles are safe in the microrheologic terms for administering the into the blood flow. Image of 250 nm Ti. O 2 NP localizations on the RBC incubated with whole blood at a concentration of 100 mcg / ml in vitro [2] Avsievich T. , Popov A. , Bykov A. & Meglinski I. Mutual interaction of red blood cells influenced by nanoparticles. Scientific Reports 9(5147), 2018. 3

Laser aggregometry technique for evaluating RBC microrheologic properties The laser aggregometer “Rheoscan” [3] allows for measuring the aggregation/dissagregation kinetics of RBC – the time-dependence of the intensity of forward/backward light scattered by a layer of whole blood during the spontaneous aggregation and forced dissagregation of RBC. By analyzing the obtained curves we can calculate the following microrheologic parameters: • Aggregation index (AI) – the ratio of the cells that aggregated during the first 10 seconds of the spontaneous aggregation • Characteristic aggregation time (T 1/2) – the time that characterizes the rate of cell aggregation Microcuvette with a rotatable magnetic bar, illuminating laser and • Amplitude (AMP) – the characteristic of forward scattered light detecting Process of AMP, T 1/2 and AMP measuring. the cells ability to deform in flow unit. conditions; 4

Laser aggregometry technique for evaluating RBC microrheologic properties • Critical shear stress (CSS) – the minimum shear stress that must be applied to the flow of aggregates in order to break down them. Cuvette for measuring CSS, illuminating laser and backward scattered light detecting unit. Process of CSS measuring. 5

Sample preparation Dry NP were dissolved in phosphate buffered saline (PBS) at the concentrations of 4 - 400 μg/ml. Next, the resulting solutions were placed for 5 minutes into an ultrasonicator (CODYSON CD-4800) in order to destroy the NP aggregates. Blood samples were drawn from healthy donors between the age of 20 - 25 years and were stabilized with EDTA to prevent the blood clotting. All experiments were performed within 3 hours after blood sampling. Then, solutions of disaggregated NPs were incubated with whole blood for an 1 hour. 6

Results Dependence of aggregation time of RBC on Ti. O 2 concentration after 1 hour incubation RBC with NP. Dependence of aggregation index of RBC on Ti. O 2 concentration after 1 hour incubation of RBC with NP. 7

Results Dependence of AMP on Ti. O 2 concentration after 1 hour incubation of RBC with NP. Dependence of CSS of RBC aggregates on Ti. O 2 concentration after 1 hour incubation of RBC with NP. 8

Results The results obtained with the Rutile RODI Ti. O 2 particles show that the number of cells aggregated during the fixed time (typically 10 sec) is highly dependent on the NP concentration. The aggregation rate of RBC increases by (14. 2 ± 4. 8)% in a whole blood sample incubated with Ti. O 2 at the concentration of 8 μg/ml compared to the control sample, and it decreases by (20. 9 ± 7. 1)% in a sample with the concentration of 400 μg/ml. The characteristic aggregation time T 1/2 reduces by (24. 2 ± 9. 4)% in the sample with the concentration of 8 μg / ml, and it increases by (35. 1 ± 7. 5)% in the sample with the concentration of 400 μg / ml compared to the control sample. Also there is a general tendency to a decrease in the RBC hydrodynamic strength with increasing Ti. O 2 NP concentration. The AMP parameter, that characterize the ability RBC to deform, falling by (17. 1 ± 4. 7)% in a whole blood sample incubated with Ti. O 2 at the concentration of 400 μg/ml relatively not incubated with NP sample. It means that the safe concentration range is up to 4 μg/ml in terms of the effect of these NP on the microrheologic properties of whole human blood. Above this concentration, NPs strongly affect the microrheologic properties of the whole blood. 9

Conclusions To conclude, our experiments demonstrate that after surpassing the concentration of 4 µg/ml a dramatic impairment of aggregation parameters take place. Ti. O 2 is a proaggregant at low concentrations of the NP (0 - 8 μg / ml) and it is an antiaggregant above the concentration of 100 μg / ml. This work is supported by Russian Science Foundation (Grant № 20 -45 -08004). 10

Thank you for attention!