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5MEDICAL SCIENCES
ADHESION PROPERTIES OF HYDROGEL FILED WITH FERRO NANOPARTICLES
Derbyshev G.S.,
Ural State Medical University, Ekaterinburg, Russian Federation
Melekhin V. V.,
Ural State Medical University, Ekaterinburg, Russian Federation Institute of Medical Cell Technologies, Ekaterinburg, Russian Federation
Baldanshirieva A.D.,
Ural State Medical University, Ekaterinburg, Russian Federation
Sichkar D.A.,
Ural State Medical University, Ekaterinburg, Russian Federation
Kostyukova S.V.,
Ural State Medical University, Ekaterinburg, Russian Federation Institute of Medical Cell Technologies, Ekaterinburg, Russian Federation
Makeev O.G.
Ural State Medical University, Ekaterinburg, Russian Federation Institute of Medical Cell Technologies, Ekaterinburg, Russian Federation
Abstract
The article presents the data of analysis of polymeric gel with magnetic nanoparticles of iron oxide, statistical analysis of cell adhesion on hydrogel.
Keywords: hydrogel, nanoparticles, adhesion
Introduction
Developing regenerative medicine involves the development of means for the targeted delivery of active substances as well as cells directly to the damaged area of the body. The means of targeted delivery are well known, of which liposomes [7], antibodies [5] and nanoparticles [6] are considered the most studied. However, the effectiveness of targeted delivery depends on very many factors, which leads to a more or less even distribution of therapeutic agents in the body.
Therefore, the means of directional delivery are constantly being improved. Ferrogels, polymeric biodegradable gels with magnetic nanoparticles, which are considered to be a promising direction in this field of research, can be referred to such improved means. On the one hand, magnetic nanoparticles themselves are used in medicine, for example, for MRI diagnostics, drug delivery with controlled release, modeling of local hyperthermia, etc. [6]. On the other hand, a composite of hydrogel and magnetic particles opens up prospects in the development of magnetically controlled biocompatible materials for the needs of biomedicine. A feature of ferrogel can be considered ease of use - gel in the form of micro particles, administered intravenously, will accumulate in the selected area when it is exposed to a sufficient magnetic field strength created by a magnet. It does not require specific agents that deliver the active substance or cells to specific targets. Under the influence of a magnetic field, a ferrogel can in a controlled manner deliver drugs or cells, squeezing them out, contracting like a sponge [4]. The initiation of the mechanism of the release of drugs can also be triggered by a high-frequency magnetic field, in which metal particles will be heated [8]. In addition, the gel prevents the oxidation of metal particles with the subsequent local toxicity of oxides, which makes the use of ferrogel among the promising means for targeted delivery.
The purpose of the study is to analyze the adhesion properties of a ferrogel based on a polyacrylamide hydrogel with various concentrations of iron oxide nano-particles.
Materials and methods
The study was conducted on a human dermal fi-broblast cell culture (5th passage) obtained from a clinically healthy donor for 40 years based on prior informed consent. Primary culture was obtained by enzymatic disaggregation of tissue using collagenase (Sigma Aldrich, USA).
The substrate for planting cells was a polyacryla-mide gel, which is a hydrated three-dimensional network of polyacrylamide filaments.
The samples studied included 6 groups: the first group - gel without iron oxide nanoparticles (0%), in the second group the concentration of iron oxide nanoparticles was 0.25%, in the third - 0.5%, in the fourth -0.75%, in the fifth - 1%, the sixth group - the control (cell passage on the culture plastic of the corresponding area).
To assess the adhesion properties of a polymeric material, an adhesion index was determined. To this end, cells were planted on a 96-well plate (Orange Scientific, Belgium), the bottom part of the wells of which was previously coated with a gel with different concentrations of iron oxide nanoparticles. The number of replications in each group is 8. The seeding concentration is 4 • 103 cells per well. The culture medium is DMEM / Ham F-12 (Sigma Aldrich, USA) with 10% fetal bovine serum (Sigma Aldrich, USA). After passage, cell cultures were incubated at 37 ° C, 5% CO2 and 95% humidity in a Sanyo 18AIC incubator (Japan) for 6 hours. At the end of this time, cultures were washed out of non-adherent cells with a solution of DPBS (Sigma Aldrich, USA). The adhered cells were examined microscopically using a ZOE microscope (Bio-Rad,
USA), photo documented and removed from the surface with a 0.25% trypsin-Versen solution. Cells were counted on an automatic cell counter (Scepter, USA). The adhesion index, expressed as a percentage, was determined as the ratio of the number of adherent cells to the seed concentration multiplied by 100.
Statistical data processing was performed using the RStudio program (Version 0.99.903 - © 2009-2016 RStudio, Inc.). To compare the groups, the non-parametric Mann-Whitney test was used. The relationship of the variables was estimated by the Spearman correlation coefficient.
Results and discussion
Microscopic examination of the cells adhered to the gel surface revealed morphological differences. Thus, when using a gel without the inclusion of iron oxide nanoparticles, the adherent cells remained predominantly shocked, and with an increase in the concentration of nanoparticles, an increase in the proportion of cells of the normal spindle shape was recorded.
Table 1.
The average values of adhesion for the studied groups and the comparison of the experimental groups with the control by the non-parametric criterion of Mann-Whitney (in the calculations, confidence inter-
A comparatively low adhesion index was obtained on a gel without nanoparticles, averaging 82.8% (Table 1). With an increase in the concentration of nanoparticles, an increase in the adhesion properties of the material under study was observed (Fig. 1). Moreover, with an iron oxide concentration of 1%, the adhesion index reached 98.6%, which is not statistically different from the control group (99.1%) with cell adhesion on culture plastic (Mann-Whitney test, w = 19, p = 0, 19 - Table 1).
The interrelation of the adhesion index with the concentration of iron oxide nanoparticles (from 0 to 1%) in the polymeric material was also quantified. The non-parametric Spearman coefficient demonstrated a high positive correlation between these variables and amounted to 0.705 in this case (s = 2694.4; p = 7.681e-
Group Average U-criterion (w, p)
Control 99.102245 -
Gel - 0% Fe2O3 82.789375 0, 0.0001554
Gel - 0.25% Fe2O3 91.12498571 0, 0.0003108
Gel - 0.5% Fe2O3 93.97357113 12, 0.03792
Gel - 0.75% Fe2O3 95.95826286 5, 0.005905
Gel - 1% Fe2O3 98.57999145 19, 0.1949
It is important that the absence of a pronounced cytotoxic effect may indicate that the nanoparticles, fixed in the crystal lattice of the gel, are not capable in free form to enter the culture medium and have a damaging effect on the cells.
Thus, the results obtained indicate differences in the adhesive properties of the gel, depending on the concentration of iron oxide nanoparticles. Probably, the observed effects are due to the fact that the presence of iron oxide nanoparticles in the gel structure changes the conformation of the gel surface and, thus, improves the adhesion of fibroblasts to the polymeric material. Probably, the magnetic moment created by particles in the gel also plays an important role. Regardless of which of these factors or their combinations influence the result obtained, an increase in the concentration of nanoparti-cles in the gel leads to an increase in cell adhesion to ferrogels. In general, the obtained results are in good agreement with the data of other researchers. Thus, weak cell adhesion to polyacrylamide gel was previously established [2], however, it was shown that loading the hydrogel with Fe2O3 nanoparticles promotes adhesion and proliferation of osteoblast cells and chon-drocytes [3], other authors noted similar results with different cell types [1].
Conclusion:
1. High adhesion properties to the cells of the fi-broblastic differen give reason to characterize the synthesized material as promising for further study.
2. Cell adhesion to ferrogel is the higher, the greater the concentration of iron oxide nanoparticles in it.
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SUBSTANTIATION OF CLINICAL DIAGNOSIS BY THE APPLICATION OF GENETIC
TRANSFORMATION MARKER
Desyatova M.A.,
Ural State Medical University, Ekaterinburg, Russian Federation
(Junior research scientist)
Korotkov A.V.,
Ural State Medical University, Ekaterinburg, Russian Federation
(Junior research scientist) Institute of Medical Cell Technologies, Ekaterinburg, Russian Federation
Kostyukova S.V.,
Ural State Medical University, Ekaterinburg, Russian Federation
(Junior research scientist) Institute of Medical Cell Technologies, Ekaterinburg, Russian Federation
Makeev O.G.
Ural State Medical University, Ekaterinburg, Russian Federation
(Junior research scientist) Institute of Medical Cell Technologies, Ekaterinburg, Russian Federation
Abstract
In this article, the application principle of the polymorphic marker TP53 had been considered and discussed as a universal methodic for assessment of the disruption of the stability of the genetic apparatus in patients with a previously established fact of oncological disease.
Keywords: Genomic instability, DNA, Single nucleotide polymorphism (SNPs), mutation.
Introduction
Recently, there is a need to detect the presence of mutations leading to malignant transformation of the cell. Understanding genetic variations can provide the ability to track cancer diseases, most of which are caused by carcinogenesis, and as a result, obtain information about the mechanisms leading to changes in the internal integrity of the genetic apparatus, as well as identify critical factors affecting the occurrence of cellular transformations. This may be an advantage when
creating a preventive strategy and a preliminary assessment of the predisposition to mutation. At this stage, we are at the introductory stage of DNA diagnostics, as a tool for analyzing a single nucleotide polymorphism. Replacing a single base can lead to some changes in the genome. However, the vast majority of such substitutions may not cause changes in the expression and functioning of the gene, but manifest themselves in the characteristics of the phenotype. In this study, it is relevant
