Effect of titanium dioxide nanoparticles on human red blood cells microrheologic properties: in vitro studies by laser techniques
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. For the time being there is no sufficient evidence confirming that certain NP when administered into blood flow will not negatively influence the blood circulation, spontaneous aggregation and forced disaggregation parameters of blood cells and therefore the patient’s health . 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.
The aim of this work is to evaluate the dependence of RBC microrheologic properties on the concentration of titanium dioxide NP incubated with whole blood and obtain the conditions, at which these particles are safe in microrheologic terms for administering into the blood flow.
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 ultrasonic sonicator (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 blood clotting. Then, solutions of disaggregated NPs were incubated with whole blood for 1 hour. All experiments were performed within 3 hours after blood sampling.
In this work we used the laser aggregometer “Rheoscan” , which applies the method of laser aggregometry  to study blood aggregation properties. Rheoscan allows for measuring the aggregation kinetics of RBC – the time-dependence of the intensity of forward light scattered by a layer of whole blood during the spontaneous aggregation of RBC. By analyzing the obtained curve 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 – the time that characterizes the rate of cell aggregation; amplitude (AMP) – the characteristic of the cells ability to deform in flow conditions; critical shear stress (CSS) – the hydrodynamic strength of the aggregates.
The results obtained with the Rutile RODI TiO2 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 TiO2 at a concentration of 8 μg/ml compared to the control sample, and it decreases by (20.9 ± 7.1)% in a sample with a concentration of 400 μg/ml. Also there is a general tendency to a decrease in the RBC hydrodynamic strength with increasing TiO2 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 TiO2 at a 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 affect the microrheologic properties of whole blood.
To conclude, our experiments demonstrate that after surpassing the concentration of 4 µg/ml a dramatic impairment of aggregation parameters take place.
This work was supported by Russian Science Foundation grant №20-45-08004.
 Health Effects of Nanoparticles. The IRSST report “Nanoparticles: Current knowledge about occupational health and safety risks and prevention measures”, IRSST, Quebec, Canada, 2006.
 Shin S., Hou J. X., Suh J. S., Singh M. Validation and application of a microfluidic ektacytometer (RheoScan-D) in measuring erythrocyte deformability, Clinical Hemorheology and Microcirculation, 37(4):319-328, 2007.
 Lugovtsov A. E. et al. Optical assessment of alterations of microrheologic and microcirculation parameters in cardiovascular diseases, Biomedical Optics Express, 10(8):3974–3986, 2019.
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Faculty of Physics, Lomonosov Moscow State University, Russia
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