Open-source hardware- and software-based cryomicroscopy system for investigation of phase transitions in cryobiological research.

The development of inexpensive equipment adapted for the study of a specific biological object is very important for cryobiology. In the presented work, we have proposed a simple system for microscopy utilising open-source platform Arduino. Testing this system showed that it had sufficient sensitivity to determine the physical processes occurring in a cryopreserved sample such as intra- and extracellular water crystallisation and salt eutectic. Utilising this system, we investigated the mechanisms of cryoprotection and cryodamage of testis interstitial cells (ICs) in cryoprotective media, which included cryoprotective agents such as dimethyl sulphoxide (Me2 SO), as well as foetal bovine serum or polymers (dextran, hydroxyethyl starch and polyethylene glycol). It was shown that a serum-/xeno-free medium that included 0.7 M Me2 SO and 100 mg/mL dextran was able to reduce intracellular water crystallisation in cells, change the structure of extracellular ice, and reduce salt eutectic and recrystallisation. All these effects correlated with better IC survival after cryopreservation in the medium. This medium is potentially less toxic as it has lower concentrations of Me2 SO compared to serum-containing media developed for cryopreservation of testicular cells. This would pave a way for the creation of nontoxic serum-free compositions that does not require removal before use of cryopreserved living cells for laboratory practice or in clinics.

Oxidative stress-modifying effects of TiO2nanoparticles with varying content of Ti3+(Ti2+) ions.

Nanoparticles (NPs) with reactive oxygen species (ROS)-regulating ability have recently attracted great attention as promising agents for nanomedicine. In the present study, we have analyzed the effects of TiO2defect structure related to the presence of stoichiometric (Ti4+) and non-stoichiometric (Ti3+and Ti2+) titanium ions in the crystal lattice and TiO2NPs aggregation ability on H2O2- and tert-butyl hydroperoxide (tBOOH)-induced ROS production in L929 cells. Synthesized TiO2-A, TiO2-B, and TiO2-C NPs with varying Ti3+(Ti2+) content were characterized by x-ray powder diffraction, transmission electron microscopy, small-angle x-ray scattering, x-ray photoelectron spectroscopy, and optical spectroscopy methods. Given the role of ROS-mediated toxicity for metal oxide NPs, L929 cell viability and changes in the intracellular ROS levels in H2O2- and tBOOH-treated L929 cells incubated with TiO2NPs have been evaluated. Our research shows that both the amount of non-stoichiometric Ti3+and Ti2+ions in the crystal lattice of TiO2NPs and NPs aggregative behavior affect their catalytic activity, in particular, H2O2decomposition and, consequently, the efficiency of aggravating H2O2- and tBOOH-induced oxidative damage to L929 cells. TiO2-A NPs reveal the strongest H2O2decomposition activity aligning with their less pronounced additional effects on H2O2-treated L929 cells due to the highest amount of Ti3+(Ti2+) ions. TiO2-C NPs with smaller amounts of Ti3+ions and a tendency to aggregate in water solutions show lower antioxidant activity and, consequently, some elevation of the level of ROS in H2O2/tBOOH-treated L929 cells. Our findings suggest that synthesized TiO2NPs capable of enhancing ROS generation at concentrations non-toxic for normal cells, which should be further investigated to assess their possible application in nanomedicine as ROS-regulating pharmaceutical agents.

Antibacterial and antioxidant activity of gold and silver nanoparticles in dextran-polyacrylamide copolymers.

Search for new antimicrobial agents is of great significance due to the issue of antimicrobial resistance, which nowadays has become more important than many diseases. The aim of this study was to evaluate the toxicity and biological effects of a dextran-graft-polyacrylamide (D-PAA) polymer-nanocarrier with/without silver or gold nanoparticles (AgNPs/D-PAA and AuNPs/D-PAA, respectively) to analyze their potential to replace or supplement conventional antibiotic therapy. The toxicity of nanocomplexes against eukaryotic cells was assessed on primary dermal fibroblasts using scratch, micronucleus and proliferation assays. DPPH (2,2-diphenyl-1-picrylhydrazylradical) assay was used to evaluate the antioxidant capacity of D-PAA, AgNPs/D-PAA and AuNPs/D-PAA. DNA cleavage, antimicrobial and biofilm inhibition effects of nanocomplexes were investigated. Nanocomplexes were found to be of moderate toxicity against fibroblasts with no genotoxicity observed. AgNPs/D-PAA reduced motility and proliferation at lower concentrations compared with the other studied nanomaterials. AgNPs/D-PAA and AuNPs/D-PAA showed radical scavenging capacities in a dose-dependent manner. The antimicrobial activity of AgNPs/D-PAA against various bacteria was found to be much higher compared to D-PAA and AuNPs/D-PAA, especially against E. hirae, E. faecalis and S. aureus, respectively. D-PAA, AgNPs/D-PAA and AuNPs/D-PAA showed DNA-cleaving and biofilm inhibitory activity, while AgNPs/D-PAA displayed the highest anti-biofilm activity. AgNPs/D-PAA and AuNPs/D-PAA were characterized by good antimicrobial activity. According to the findings of the study, AgNPs/D-PAA and AuNPs/D-PAA can be evaluated as alternatives for the preparation of new antimicrobial agents, the fight against biofilms, sterilization and disinfection processes. Our findings confirm the versatility of nanosystems based on dextran-polyacrylamide polymers and indicate that AgNPs/D-PAA and AuNPs/D-PAA can be evaluated as alternatives for the preparation of novel antimicrobial agents.

Hemocompatibility of dextran-graft-polyacrylamide/zinc oxide nanosystems: hemolysis or eryptosis?

Aim. In this study, blood compatibility of ZnO nanoparticles-polymer nanocomplex (D-PAA/ZnONPs(SO42-)) synthesizedin situinto dextran-graft-polyacrylamide (D-PAA) using zinc sulphate as a precursor was tested using hemolysis, osmotic fragility and eryptosis assays.Materials and methods. Dose-dependent ability to induce eryptosis was assessed following 24 h incubation at concentrations of 0-800 mg l-1analyzing hallmarks of eryptosis (cell shrinkage and phosphatidylserine externalization), as well as reactive oxygen species generation. Hemolysis was detected spectrophotometrically based on hemoglobin release following exposure to the D-PAA/ZnONPs(SO42-) nanocomplex. Osmotic fragility test (OFT) involved detection of hemolysis of red blood cells exposed to 0.2% saline solution following incubation with the D-PAA/ZnONPs(SO42-) nanocomplex. Additional incubation of the nanocomplex in the presence or absence of either ascorbic acid or EGTA was used to reveal the implication of oxidative stress- or Ca2+-mediated mechanisms in D-PAA/ZnONPs(SO42-) nanocomplex-induced erythrotoxicity.Results. Hemocompatibility assessment of the D-PAA/ZnONPs(SO42-) nanocomplex revealed that it induced hemolysis and reduced resistance of erythrocytes to osmotic stress at concentrations of above 400 and 200 mg l-1, respectively. Oxidative stress- or Ca2+-mediated mechanisms were not involved in D-PAA/ZnONPs(SO42-) nanocomplex-induced hemolysis. Strikingly, the D-PAA/ZnONPs(SO42-) nanocomplex did not promote cell membrane scrambling, cell shrinkage and oxidative stress in red blood cells following the direct exposure for 24 h. Thus, the D-PAA/ZnONPs(SO42-) nanocomplex did not induce eryptosisin vitro. Eryptosis is generally considered to occur earlier than hemolysis in response to stress in order to prevent hemolytic cell death. Counterintuitively, our data suggest that hemolysis can be triggered by nanomaterials prior to eryptosis indicating that eryptosis and hemolysis assays should be used in combination for testing blood compatibility of nanomaterials.Conclusions. The D-PAA/ZnONPs(SO42-) nanocomplex has a good hemocompatibility profile at low concentrations. Hemocompatibility testing in nanotoxicology should include both eryptosis and hemolysis assays.

Antimicrobial Effects of Nanostructured Rare-Earth-Based Orthovanadates.

The search for novel antimicrobial agents is of huge importance. Nanomaterials can come to the rescue in this case. The aim of this study was to assess the cytotoxicity and antimicrobial effects of rare-earth-based orthovanadate nanoparticles. The cytotoxicity against host cells and antimicrobial activity of LaVO4:Eu3+ and GdVO4:Eu3+ nanoparticles were analyzed. Effects of nanomaterials on fibroblasts were assessed by MTT, neutral red uptake and scratch assays. The antimicrobial effects were evaluated by the micro-dilution method estimating the minimum inhibitory concentration (MIC) of nanoparticles against various strains of microorganisms, DNA cleavage and biofilm inhibition. GdVO4:Eu3+ nanoparticles were found to be less toxic against eukaryotic cells compared with LaVO4:Eu3+. Both nanoparticles exhibited antimicrobial activity and the highest MIC values were 64 mg/L for E. hirae, E. faecalis and S. aureus shown by GdVO4:Eu3+ nanoparticles. Nanoparticles demonstrated good DNA cleavage activity and induction of double-strand breaks in supercoiled plasmid DNA even at the lowest concentrations used. Both nanoparticles showed the biofilm inhibition activity against S. aureus at 500 mg/L and reduced the microbial cell viability. Taken the results of host toxicity and antimicrobial activity studies, it can be assumed that GdVO4:Eu3+ nanoparticles are more promising antibacterial agents compared with LaVO4:Eu3+ nanoparticles.

Effect of zinc oxide micro- and nanoparticles on cytotoxicity, antimicrobial activity and mechanical properties of apatite-polymer osteoplastic material.

This work is devoted to the comparison of the physical and biological properties of synthesized osteoplastic composites with an experimentally determined content (375 µg/g) of the micro (ZnOMPs) and nano (ZnONPs) particles, immobilized in Hydroxyapatite-Alginate-Chitosan matrix (HA-Alg-CS). ZnONPs show pronounced antimicrobial activity against E.coli ATCC 25922 and S. aureus ATCC 25923, while ZnOMPs only in the CS presence. Composites containing ZnONPs/MPs do not have a toxic effect on bone-forming cells - osteoblasts, preserving their ability to biomineralization. ZnOMPs and ZnONPs to varying degrees, but significantly affect composites' swelling, porosity, shape stability, and prolong vitamin D3 release for 120h, compared to Control. Composites do not demonstrate unwanted "burst release." ZnONPs/MPs increase Youngs' modulus of the HA-Alg matrix, namely 348 â†’ 419 MPa (ZnOMPs), 348 â†’ 646 MPa (ZnONPs), and weaken the plastic (irreversible) deformations. The compressive strength of HA-Alg and HA-Alg/CS matrixes containing ZnONPs (178 MPa and 251 MPa, respectively) is in the range of values for native cortical bone (170-193 MPa). Biocompatibility and lack of toxic effect give both composites a perspective for osteoplastic application, but composites doped with ZnONPs are more attractive.

Marine Polysaccharides Carrageenans Enhance Eryptosis and Alter Lipid Order of Cell Membranes in Erythrocytes.

Aim In the current study, hemocompatibility of three major commercially available types of carrageenans (ι, κ and λ) was investigated focusing on eryptosis. MATERIALS AND METHODS: Carrageenans of ι-, κ- and λ-types were incubated with washed erythrocytes (hematocrit 0.4%) at 0-1-5-10 g/L for either 24 h or 48 h. Incubation was followed by flow cytometry-based quantitative analysis of eryptosis parameters, including cell volume, cell membrane scrambling and reactive oxygen species (ROS) production, lipid peroxidation markers and confocal microscopy-based evaluation of intracellular Ca2+ levels, assessment of lipid order in cell membranes and the glutathione antioxidant system. Confocal microscopy was used to assess carrageenan cellular internalization using rhodamine B isothiocyanate-conjugated carrageenans. RESULTS: All three types of carrageenans were found to trigger eryptosis. Pro-eryptotic properties were type-dependent and λ-carrageenan had the strongest impact inducing phosphatidylserine membrane asymmetry, changes in cell volume, Ca2+ signaling and oxidative stress characterized by ROS overproduction, activation of lipid peroxidation and severe glutathione system depletion. Eryptosis induction by carrageenans does not require their uptake by erythrocytes. Changes in physicochemical properties of cell membrane were also type-dependent. No carrageenan-induced generation of superoxide and hydroxyl radicals was observed in cell-free milieu. CONCLUSIONS: Our findings suggest that ι-, κ- and λ-types trigger eryptosis in a type-dependent manner and indicate that carrageenans can be further investigated as potential eryptosis-regulating therapeutic agents.

Assessing the Cytotoxicity of TiO2-x Nanoparticles with a Different Ti3+(Ti2+)/Ti4+ Ratio.

Titanium dioxide (TiO2) nanoparticles are promising biomedical agents characterized by good biocompatibility. In this study, we explored the cytotoxicity of TiO2-x nanoparticles with a different Ti3+(Ti2+)/Ti4+ ratio and analyzed the efficiency of eryptosis indices as a tool in nanotoxicology. Two types of TiO2-x nanoparticles (NPs) were synthesized by the hydrolysis of titanium alkoxide varying the nitric acid content in the hydrolysis mixture. Transmission electron microscopy (TEM) images show that 1-TiO2-x and 2-TiO2-x NPs are 5 nm in size, whereas X-ray photoelectron spectroscopy (XPS) reveals different Ti3+ (Ti2+)/Ti4+ ratios in the crystal lattices of synthesized NPs. 1-TiO2-x nanoparticles contained 54% Ti4+, 38% Ti3+, and 8% Ti2+, while the relative amount of Ti4+ and Ti3+ in the crystal lattice of 2-TiO2-x nanoparticles was 63% and 37%, respectively. Cell viability and cell motility induced by TiO2-x nanoparticles were investigated on primary fibroblast cultures. Eryptosis modulation by the nanoparticles along with cell death mechanisms was studied on rat erythrocytes. We report that both TiO2-x nanoparticles do not decrease the viability of fibroblasts simultaneously stimulating cell migration. Data from in vitro studies on erythrocytes indicate that TiO2-x nanoparticles trigger eryptosis via ROS- (1-TiO2-x) and Ca2+-mediated mechanisms (both TiO2-x nanoparticles) suggesting that evaluation of eryptosis parameters is a more sensitive nanotoxicological approach for TiO2-x nanoparticles than cultured fibroblast assays. TiO2-x nanoparticles are characterized by low toxicity against fibroblasts, but they induce eryptosis, which is shown to be a promising tool for nanotoxicity screening. The Ti3+ (Ti2+)/Ti4+ ratio at least partly determines the cytotoxicity mechanisms for TiO2-x nanoparticles.

Combined Dextran-Graft-Polyacrylamide/Zinc Oxide Nanocarrier for Effective Anticancer Therapy in vitro.

Introduction: Cancer chemotherapy faces two major challenges - high toxicity of active substances and tumor resistance to drugs. Low toxic nanocarriers in combination with anticancer agents can significantly increase the effectiveness of therapy. Modern advances in nanotechnology make it easy to create materials with the necessary physical and chemical properties. Methods: Two hybrid nanosystems of dextran-polyacrylamide/ zinc oxide nanoparticles (D-PAA/ZnO NPs) were synthesized in aqueous solution with zinc sulphate (D-PAA/ZnO NPs (SO42-)) and zinc acetate (D-PAA/ZnO NPs (-OAc)). The light absorption, fluorescence, dynamic light scattering and transmission electron microscopy for nanocomposite characterization were used. MTT, neutral red uptake and scratch assays were selected as fibroblasts cytotoxicity assays. Cytotoxicity was tested in vitro for normal fibroblasts, MAEC, prostate (LNCaP, PC-3, DU-145) and breast (MDA-MB-231, MCF-7) cancer cells lines. Immunocytochemical methods were used for detection of Ki-67, p53, Bcl-2, Bax, e-cadherin, N-cadherin and CD44 expression. Acridine orange was used to detect morphological changes in cells. Results: The radius of ZnO NPs (SO42-) was 1.5 nm and ZnO NPs (-OAc) was 2 nm. The nanosystems were low-toxic to fibroblasts, MAEC. Cells in the last stages of apoptosis with the formation of apoptotic bodies were detected for all investigated cancer cell lines. Proapoptotic proteins expression in cancer cells indicates an apoptotic death. Increased expression of E-cadherin and N-cadherin was registered for cancer cells line LNCaP, PC-3, DU-145 and MCF-7 after 48 h incubation with D-PAA/ZnO NPs (SO42-). Conclusion: The nanosystems were low-toxic to fibroblasts, MAEC. The D-PAA/ZnO NPs nanosystem synthesized using zinc sulphate demonstrates high cytotoxicity due to destruction of various types of cancer cells in vitro and potentially increases adhesion between cells. Thus, our findings indicate the selective cytotoxicity of D-PAA/ZnO NPs against cancer cells and can be potentially used for cancer treatment.

Cytotoxicity of Hybrid Noble Metal-Polymer Composites.

The aim of the present research was to assess the cytotoxicity of gold and silver nanoparticles synthesized into dextran-graft-polyacrylamide (D-PAA) polymer nanocarrier, which were used as a basis for further preparation of multicomponent nanocomposites revealed high efficacy for antitumor therapy. The evaluation of the influence of Me-polymer systems on the viability and metabolic activity of fibroblasts and eryptosis elucidating the mechanisms of the proeryptotic effects has been done in the current research. The nanocomposites investigated in this study did not reduce the survival of fibroblasts even at the highest used concentration. Our findings suggest that hybrid Ag/D-PAA composite activated eryptosis via ROS- and Ca2+-mediated pathways at the low concentration, in contrast to other studied materials. Thus, the cytotoxicity of Ag/D-PAA composite against erythrocytes was more pronounced compared with D-PAA and hybrid Au/polymer composite. Eryptosis is a more sensitive tool for assessing the biocompatibility of nanomaterials compared with fibroblast viability assays.