A.E. Lugovtsov

The influence of nanodiamond on the oxygenation states and micro rheological properties of human red blood cells in vitro

Abstract. Nanodiamond has been proven to be biocompatible and proposed for various biomedical applications. Recently, nanometer-sized diamonds have been demonstrated as an effective Raman/fluorescence probe for bio-labeling, as well as, for drug delivery. Bio-labeling/drug delivery can be extended to the human blood system, provided one understands the interaction between nanodiamonds and the blood system. Here, the interaction of nanodiamonds (5 and 100 nm) with human red blood cells (RBC) in vitro is discussed. Measurements have been facilitated using Raman spectroscopy, laser scanning fluorescence spectroscopy, and laser diffractometry (ektacytometry). Data on cell viability and hemolytic analysis are also presented. Results indicate that the nanodiamonds in the studied condition do not cause hemolysis, and the cell viability is not affected. Importantly, the oxygenation/deoxygenation process was not found to be altered when nanodiamonds interacted with the RBC. However, the nanodiamond can affect some RBC properties such as deformability and aggregation in a concentration dependent manner. These results suggest that the nanodiamond can be used as an effective bio-labeling and drug delivery tool in ambient conditions, without complicating the blood’s physiological conditions. However, controlling the blood properties including deformability of RBCs and rheological properties of blood is necessary during treatment.

Nanodiamonds for Medical Applications: Interaction with Blood in Vitro and in Vivo

Nanodiamonds (ND) have emerged to be a widely-discussed nanomaterial for their applications in biological studies and for medical diagnostics and treatment. The potentials have been successfully demonstrated in cellular and tissue models in vitro. For medical applications, further in vivo studies on various applications become important. One of the most challenging possibilities of ND biomedical application is controllable drug delivery and tracing. That usually assumes ND interaction with the blood system. In this work, we study ND interaction with rat blood and analyze how the ND surface modification and coating can optimize the ND interaction with the blood. It was found that adsorption of a low concentration of ND does not affect the oxygenation state of red blood cells (RBC). The obtained in vivo results are compared to the results of in vitro studies of nanodiamond interaction with rat and human blood and blood components, such as red blood cells and blood plasma. An in vivo animal model shows ND injected in blood attach to the RBC membrane and circulate with blood for more than 30 min; and ND do not stimulate an immune response by measurement of proinflammatory cytokine TNF-α with ND injected into mice via the caudal vein. The results further confirm nanodiamonds’ safety in organisms, as well as the possibility of their application without complicating the blood’s physiological conditions.

Study of laser beam scattering by inhomogeneous ensemble of red blood cells in a shear flow

Laser ektacytometry is a technique widely used for measuring the deformability of red blood cells (erythrocytes) in blood samples in vitro. In ektacytometer, a flow of highly diluted suspension of erythrocytes in variable shear stress conditions is illuminated with a laser beam to obtain a diffraction pattern. The diffraction pattern provides information about the shapes (shear-induced elongations) of the cells under investigation. This paper is dedicated to developing the technique of laser ektacytometry so that it would enable one to measure the distribution of the erythrocytes in deformability. We discuss the problem of calibration of laser ektacytometer and test a novel data processing algorithm allowing to determine the parameters of the distribution of erythrocytes deformability. Experimentally, we examined 12 specimens of blood of rats under the action of 4 shear stresses. Analysis of the data shows that in conditions of a limited range of digitizing the diffraction patterns, the measurement errors for the mean deformability, deformability scatter and the skewness of erythrocytes distribution in deformability by our method are respectively 15%, 20% and 20%.

Red blood cells in laser beam field: calculations of light scattering

The main goal of this work is to develop a fast tool for calculation of light scattered by single large optically soft spheroidal particles. In particular, such particles mimic biological cells (e.g., red blood cells (RBC)). Methodology of calculation of light scattering by arbitrarily oriented optically soft spheroidal particles in Ray-Wave Approximation (RWA) is presented. We show that RWA permits to quickly and accurately calculate the angular distributions of the intensities of light scattered from particles mimicking RBC.

Reduced erythrocyte deformability in Krushinsky-Molodkina rats with acute hemorrhagic stroke

Acute hemorrhagic stroke in Krushinsky-Molodkina rats was used to assess the ability of erythrocytes to change their shape in a shear flow. Membrane rigidity and internal viscosity of erythrocytes were measured by laser diffractometry (i.e., obtaining a diffraction pattern from a thin layer of an erythrocyte suspension in a shear flow followed by computer processing of the image). The results testify to reduced deformability of erythrocytes under hemorrhagic stroke.

Optical study of the dynamics and deformation of erythrocytes in the flow

Optical study of the dynamics and deformation of erythrocytes in flow was performed by two methods. Cells were trapped and manipulated with laser tweezers. Laser tweezers allow observing the deformation of a single cell or a cell aggregate under various experimental conditions, such as at rest or in flow, and also studying the aggregation of erythrocytes, which is an important process that affects blood rheology. Another method — ektacytometry 3— is based on obtaining information about the microrheological parameters of erythrocytes from diffraction patterns of laser radiation on a diluted suspension of cells in a flow and at rest. This method makes it possible to determine the average shear strain of a whole ensemble that includes many thousands of particles from changes in the diffraction pattern depending on the shear stress applied to the cells. The forces of interaction between two erythrocytes in an aggregate are measured and the cell deformation parameters are determined. The data on erythrocyte deformation obtained by the two methods agree well with each other.

Semax increases erythrocyte deformability in the shearing blood stream in intact rats and rats with cerebral ischemia.

208 Cerebral ischemia changes the rheological proper� ties of the blood, including the erythrocyte deform� ability (ED) (1). Its decrease may additionally aggra� vate brain microcirculation and worsen the pathologi� cal process. The synthetic drug Semax (ACTH4-7 - ProGlyPro), which has no hormonal properties, demonstrates neuroprotective effects during cerebral ischemia (2, 3). One of the mechanisms of the protec� tive action of Semax against brain ischemia is probably related to its influence on ED and, respectively, on the blood microcirculation. In this study, we examined in vitro the effects of Semax on ED in intact rats and rats with cerebral ischemia. Male Wistar rats weighing 210 ± 5 g were used for the study. In the first series of experiments, we studied the effects of Semax on ED in intact rats (n = 8). In the second series of experiments, we studied the effects of Semax on ED in rats with cerebral ischemia induced by simultaneous occlusion of the common carotid arteries performed under ether anesthesia (n = 8). For this purpose, the blood was sampled prior to and 90 min after the surgery. The blood was sampled from the jugular vein into test tubes containing 7% EDTA. The blood samples were divided into aliquots, which were mixed with a Semax solution with final concen� trations from 2 × 10 -9 to 2 × 10 -5 M or with a 0.9% NaCl solution, and incubated for 1 h. Then, these samples were diluted with a solution of polyethylenox� ide (MM = 5 × 10 6 ) at a ratio of 1 : 300 in order to get a suspension with a viscosity of 13 cP. We measured the ED in the shearing blood stream using a laser rota� tional diffractometer (7). The shear rate in the stream of erythrocyte suspension in the rheological gap of the diffractometer was changed by steps in the range from 13.8 to 1550 s

Analysis of laser beam scattering by an ensemble of particles modeling red blood cells in ektacytometer

Using a simple theoretical model, we have obtained approximate relations between the characteristics of particles, modeling red blood cells, and the parameters of the diffraction pattern, produced by a laser beam diffracted in the ektacytometer. We have estimated, in particular, the effect of the particles size dispersion on the diffraction pattern visibility. The estimate shows, that relation of light intensities in the first minimum and the first maximum in the diffraction pattern is a parameter, which is rather sensitive to the particles size dispersion.

Light scattering by biological spheroidal particles in geometric optics approximation

The main goal of this work is the fast analysis of light scattered by a large optically soft spheroidal particle (size parameter ka > 20). In particular, these particles mimic single cells (e.g., red blood cells (RBC)) and their aggregates. Methodology of calculation of light scattering by arbitrarily oriented optically soft spheroidal particles in geometric optics approximation is presented. We show that the geometric optics approximation permits to quickly calculate the angular distributions of the intensities and phases of light scattered from optically soft particles, such as erythrocytes.

Laser diffraction analysis of shear deformability of human and rat erythrocytes in norm and ischemia

Ischemic diseases of people and animals are accompanied with deterioration of microrheologic properties of their blood, in particular, with impairing red blood cells (RBC) deformability. In this work, the analysis of human and rat RBC deformability in norm and ischemia was performed by means of the laser diffractometry - a modern technique allowing for measuring the flexibility of RBC, which determines the blood flow parameters in vessels. Human RBC were obtained from the blood of healthy individuals and from patients suffering from ischemic diseases. Human RBC deformability from both groups of individuals was measured. Rat RBC were obtained from a control group of animals and from a group with experimentally induced ischemia (EII). This animal model is frequently used for studying the response of an organism to ischemia. The effect of Semax, a medication that is frequently used for therapeutic treatments of human brain diseases in clinical practice, on RBC deformability was studied with its application in vitro and in vivo. It is shown that in human ischemic patients, the deformability of RBC was lower than that from healthy individuals. Both in vivo and in vitro applied semax positively influences the impaired deformability properties of RBC of ischemic rats.