Synthesis of Titanium Nitride Nanoparticles by Pulsed Laser Ablation in Different Aqueous and Organic Solutions.

Owing to a strong photothermal response in the near-IR spectral range and very low toxicity, titanium nitride (TiN) nanoparticles (NPs) synthesized by pulsed laser ablation in liquids (PLAL) present a novel appealing object for photo-induced therapy of cancer, but the properties of these NPs still require detailed investigation. Here, we have elaborated methods of femtosecond laser ablation from the TiN target in a variety of liquid solutions, including acetonitrile, dimethylformamide, acetone, water, and H2O2, to synthesize TiN NPs and clarify the effect of liquid type on the composition and properties of the formed NPs. The ablation in all solvents led to the formation of spherical NPs with a mean size depending on the liquid type, while the composition of the NPs ranged from partly oxidized TiN to almost pure TiO2, which conditioned variations of plasmonic peak in the region of relative tissue transparency (670-700 nm). The degree of NP oxidation depended on the solvent, with much stronger oxidation for NPs prepared in aqueous solutions (especially in H2O2), while the ablation in organic solvents resulted in a partial formation of titanium carbides as by-products. The obtained results contribute to better understanding of the processes in reactive PLAL and can be used to design TiN NPs with desired properties for biomedical applications.

Laser-synthesized TiN nanoparticles for biomedical applications: Evaluation of safety, biodistribution and pharmacokinetics.

Having plasmonic absorption within the biological transparency window, titanium nitride (TiN) nanoparticles (NPs) can potentially outperform gold counterparts in phototheranostic applications, but characteristics of available TiN NPs are still far from required parameters. Recently emerged laser-ablative synthesis opens up opportunities to match these parameters as it makes possible the production of ultrapure low size-dispersed spherical TiN NPs, capable of generating a strong phototherapy effect under 750-800 nm excitation. This study presents the first assessment of toxicity, biodistribution and pharmacokinetics of laser-synthesized TiN NPs. Tests in vitro using 8 cell lines from different tissues evidenced safety of both as-synthesized and PEG-coated NPs (TiN-PEG NPs). After systemic administration in mice, they mainly accumulated in liver and spleen, but did not cause any sign of toxicity or organ damage up to concentration of 6 mg kg-1, which was confirmed by the invariability of blood biochemical parameters, weight and hemotoxicity examination. The NPs demonstrated efficient passive accumulation in EMT6/P mammary tumor, while concentration of TiN-PEG NPs was 2.2-fold higher due to "stealth" effect yielding 7-times longer circulation in blood. The obtained results evidence high safety of laser-synthesized TiN NPs for biological systems, which promises a major advancement of phototheranostic modalities on their basis.

Heating and convection associated with alkaline electrophoresis.

Alkaline version of the single-cell gel electrophoresis (Comet assay) is widely used in toxicological, environmental, and monitoring studies to assess the DNA damage levels in individual cells. The change in the temperature of the electrophoretic solution is one of the reasons leading to interlaboratory variation of Comet assay results. In this work, changes of surface temperature of the solution during electrophoresis were studied using technique of real-time thermal imaging. It has been found that the electrophoresis is accompanied by nonuniform temperature rise in different areas of the electrophoretic chamber. The maximum of heating was observed in the central region of the chamber, where temperature increased by an average of 7°C. The minimum temperature rise in other parts of the chamber was about 5°C. After removing the solution, the temperature on the surface of slides was higher than that on the surface of the solution. We believe that (1) nonuniform heating of the electrophoretic solution and convection could be the reasons responsible for the variability of results both in inter- and intralaboratory studies; (2) the spatial distribution of heating of the solution depends on the size and configuration of the electrophoretic chambers used.

DNA damage in blood leukocytes from mice irradiated with accelerated carbon ions with an energy of 450 MeV/nucleon.

PURPOSE: The objective of the study was to estimate the DNA damage in blood leukocytes at long terms after irradiation of mice with carbon ions (450 MeV/nucleon) both before and in the Bragg peak. MATERIALS AND METHODS: White outbred SHK male mice were exposed to whole-body irradiation with carbon ions at doses of 0.1-2 Gy in the spread-out Bragg peak and at a dose of 6 Gy before and in the Bragg peak. At different times after irradiation (1-75 days), whole blood was collected from the tail of each mouse and analyzed by the comet assay. Mice X-irradiated in the same dose range served as a positive control. The level of the expression of mRNA of CDKN1A, APEX1, BBC3, TXN2, and ß-ACT genes in bone marrow cells was determined in animals irradiated with carbon ions at doses of 0.1-2 Gy using the real-time PCR. RESULTS: It was found that, 24 h after 12C-irradiation, a dose-dependent (0.1-2 Gy) increase in the DNA damage of leukocytes occurred, which was accompanied by a decrease in their concentration and an increase in the expression of the CDKN1A and BBC3 genes in bone marrow cells. The expression of the APEX1 and TXN2 genes did not change. In mice 12C-irradiated at a dose of 6 Gy before and in the Bragg peak, the level of DNA damage changed as follows: by day 3, it increased; by day 23 it returned to the control level; by day 30, it increased again; and by day 75, it fell to the control level on irradiation before the Bragg peak and was significantly higher (p < .05) than in the control after irradiation in the Bragg peak. CONCLUSIONS: The dynamics of changes in the level of DNA damage in leucocytes of 12C-irradiated mice within 30 days is similar to that in mice exposed to sublethal doses of X-radiation. The retention of the high level of DNA damage by day 75 after 12C-irradiation in the Bragg peak indicates a significant injury of cells from different cell pools of the blood system. The high level of DNA damage may be related not only to complex DNA injuries but also to chronic oxidative stress.

Some causes of inter-laboratory variation in the results of comet assay.

We performed an inter-laboratory study to determine the variation of comet assay results and to identify its possible reasons. An exchange of slides between Labs in different stages of the comet assay protocol was performed. Because identical slides, durations of alkali treatment and electrophoresis, and similar electric field strengths (2.0 V/cm and 2.14 V/cm) were used, we concluded that the observed inter-laboratory difference in the results is directly associated with the electrophoresis step. In Lab 1, mouse bone marrow cells were exposed to methyl methanesulfonate at concentrations of 10, 25 and 50 µM for 3 h at 37 °C. In Lab 2, cells the same as in Lab 1 were immobilized in LMA on slides and exposed to X-rays at doses of 3-8 Gy. We found that the transportation of slides after lysis or electrophoresis step, as well as different dyes used for scoring did not produce any significant effect on the results. No substantial difference in the data was also revealed when various software packages were used for image analysis. The temperature of the alkaline solution was shown to increase during electrophoresis and, besides, the temperature heterogeneity of the solution took place in the area of the platform, with a maximum in the middle of the chamber. The temperature heterogeneity could affect the rate of conversion of alkali labile sites into single stranded breaks. Thus, it was clearly indicated that real temperature variations during the alkali treatment and electrophoresis were an essential factor in the variability of the results between our Labs.

The quality and fertility of sperm collected from European common frog (Rana temporaria) carcasses refrigerated for up to 7 days.

There is a catastrophic decrease in the biodiversity of amphibians coupled with the loss of genetic variation. The perpetuation of amphibian biodiversity demands a multifaceted approach, including the use of reproduction technologies (RTs), to enable efficient reproduction in captivity and to prevent the loss of genetic variation. Reproduction technologies for the storage of amphibian sperm for days to weeks, when refrigerated at 4°C, or for millennia when cryopreserved have recently undergone rapid development. Sperm from amphibians may be obtained through excision and maceration of testes; however, this is sometimes not possible with rare or endangered species. Alternate methods of obtaining sperm are through hormonal induction, or as spermatozoa from the carcasses of recently dead amphibians. The use of sperm from carcasses of recently dead amphibians is particularly valuable when sampled from genetically important founders in conservation breeding programs, or where catastrophic mortality is occurring in natural population. Sperm harvested over a period of 7 days from the testes of European common frog (Rana temporaria) carcasses stored in a refrigerator were assessed for percentage and progressive motility, cell membrane integrity, nuclear DNA fragmentation, and fertilizing ability. In addition, the survival of resulting embryos to hatch was recorded. Results indicated that some sperm of R. temporaria remain motile and fertile when harvested from frog carcasses refrigerated up to 7 days post-mortem, and resulting embryos can develop to hatch.

DNA-binding proteins of mammalian mitochondria.

Acid-soluble proteins were isolated from liver and spleen mitochondria and their ability to form complexes with DNA was investigated. According to electrophoresis data, acid-soluble proteins include about 20 polypeptides ranging in the molecular mass from 10 to 120 kDa. It was found that acid-soluble proteins form stable DNA-protein complexes at a physiological NaCl concentration. Different polypeptides possess different degrees of DNA affinity. There is no significant difference between DNA-binding proteins of mitochondria from liver and those from spleen as to their ability to form complexes with mtDNA and nDNA. In the presence of 5 microg of DNA most polypeptides were bound to DNA, and further increase in DNA amount affected little the binding of proteins to DNA. There was no distinct difference in DNA-protein complex formation of liver mitochondrial acid-soluble proteins with nDNA or mtDNA. Also, it was detected that with these mitochondrial acid-soluble proteins, proteases that specifically cleave these proteins are associated. It was shown for the first time that these proteases are activated by DNA. DNA-binding proteins including DNA-activated mitochondrial proteases are likely to participate in the regulation of the structural organization and functional activity of mitochondrial DNA.