Seznam abstraktů 2017: POSTEROVÁ SEKCE C

The topic revolving around the “origin of life” has been debated for several centuries and keeps doing so in our days. Among the various approaches chosen in the last decades, the one devised by Miller and Urey in their famous experiment in 1953 is certainly one of the most authoritative and universally accepted ones.[1] They followed the earlier observations by Oparin and Haldane, who postulated that the early atmosphere of the young Earth was mostly based on reducing gases like CH4, NH3, H2 together with water, and that this atmosphere could have generated the building blocks of organic life.[2] To prove this, Miller and Urey built a suitable reactor in which the proposed atmosphere of Oparin was reproduced, adding heating and electrical discharge as a source of energy. The result of this famous experiment was the “primordial soup”, in which several basic amino acids were found.
After this seminal experiment several other models were proposed, in which other gases, for example CO2 and H2S, were instead suggested as main constituents of the early atmosphere. More recent works indicate that hydrothermal vents, placed underwater or in the open atmosphere, in which local high temperatures are present, could be suitable points of origin for the early life on Earth.
In this work, we propose a pathway leading to the formation of the building blocks of the organic matter that evolved into the early life forms as based on the hydrothermal formation of formamide, which is in itself a strong candidate of abiogenesis emerged in recent works.[3] Formamide can lead to formation of nucleobases by a complex radical process. We propose, as a main difference from previous works, that the formation of formamide goes through a pathway involving first formic acid and then ammonium formate via the catalytic effect of metallic nanoparticles that could have been present in the early stages of the Earth.
REFERENCES: [1] S. L. Miller, Science 117, 1953, 528-9.; [2] A. I. Oparin, The origin of life, 1924.; [3] J. E. Šponer, J. Šponer, O. Nováková, V. Brabec, O. Šedo, Z. Zdráhal, G. Costanzo, S. Pino, R. Saladino, E. Di Mauro, Chem. Eur. J. 22, 2016, 3572-86.

Nanodiamond material possesses unique characteristics enabling its wide use in biomedical research and future clinical applications. We focus in detail on i) functionalized nanodiamond particles as transfection and monitoring agents of mammalian RNA and ii) boron doped nanocrystalline diamond (BDD) layers aiming for the development of advanced electrochemical sensors.
In order to develop a nanosensor enabling real-time monitoring, successful transfection, and effective intracellular release of antisense RNA we used fluorescent nanodiamond particles (FND). Plain FND were functionalized with short antisense RNA via a pH-sensitive polymer linker. Here we proved the sensor formation and stability as well as its uptake by specialized cancer cells and cytoplasmic release of RNA cargo that triggered degradation of specific intracellular target and subsequently reduced cell motility and apoptotic cell death.
The interaction of live cells with a nanodiamond layer is crucial for the development of biocompatible electrochemical sensors. Here we have tested the contact, attachment, and cellular response of fibroblasts to planar BDD deposited on silicon substrates as well as BDD deposited on a porous template formed by spin coating of SiO2 fibres. Such a surface retains the electrochemical sensitivity and offers great biocompatibility with mammalian cells as monitored by toxicity, proliferation, and cell cycle assays.
The research was financially supported by the Czech Ministry of Health (project 15-33094A) and the Czech Science Foundation (project 17-15319S).

This study was focused on interaction ZnO nanoparticles with protein. BSA (Bovine Serum Albumin) was chosen as a model protein to show how is the adsorption of protein successful on our prepared nanoparticles. Particles of ZnO were prepared by direct precipitation method from Zn(NO3)2 by addition of urea and further treatment. These particles were dispersed in water and characterised by distribution of size and compared with commercial ZnO. Average size of dispersed prepared particles was around 75 nm. Afterwards the zeta potential of particles was measured in different conditions (change of pH, concentration, ionic strength) and the isoelectric point was determined. These results were used for optimisation the appropriate conditions for adsorption of BSA on ZnO nanoparticles. The changes of zeta potential were measured during adsorption of BSA on ZnO nanoparticles. These results can help to understand the interaction of ZnO nanoparticles with protein which is important to know for use their antibacterial effect in application in medicine.

In the effort of exploring the environmentally friendly, easy and economical methodology, we report a mild and efficient one-pot protocol for the synthesis of biologically active tri substituted imidazole derivatives by a multi-component reaction involving 1, 2-diketone, aromatic aldehydes, NH4OAc and recyclable acid functionalized magnetically separable catalyst in ethanol. The functionalized magnetite nano particles were prepared by in situ co-precipitation method and their catalytic activity was investigated for the synthesis of 2,4,5- tri substituted imidazole derivatives .
The anticancer activity of synthesized imidazole derivatives was checked for HeLa cells.

Magnetic nanoparticles are widely studied as potential therapeutic vectors in number of conditions. One such potential application is in neurodegenerative diseases – Alzheimer’s & Parkinson’s disease and other diseases that have yet undescribed ethiopathology, but are known for production of aberant protein forms with β-amyloid structure. The ability to cross blood-brain barrier and the possibility of surface modification to get the nanoparticles into cells present unique opportunity to use high frequency electromagnetic hyperthermia to destroy such amyloids, which we investigated in our study. Starch covered 100 nm magnetite nanoparticles can be heated to hyperthermic temperatures by 3.5 MHz alternating electromagnetic field. Hen egg-white lysozyme amyloid fibrils are generally accepted as models for variety of amyloids. We prepared them in acidic conditions and added various micromolar amounts of magnetic nanoparticles, heated the mixture in electromagnetic field and evaluated the effect from absorbance peak shift from 490 nm to 540 nm in Congo red spectra, which is present when the dye binds with amyloid fibrils. Up to 40-45 µL of 25 mg/mL magnetic nanoparticles in 1 mL of hen egg-white amyloid fibrils did not have any considerable effect on the fibrils, but larger amounts than 50 µL exhibited ability to destroy the fibrils to such an extent that no red shift in Congo red spectra was observed.

Cationic gold nanorods (GNRs) have potential applications in medical diagnostics and therapy due to their unique optical and physical properties. Specifically their nanoscale size and positive surface charge allow them to enter the cells up to millions per cell. While scattering of light can be used for the particle imaging, the effective transformation of absorbed light into kinetic energy leads to the heating of the local environment and can be utilized for photothermal cancer therapy. However, despite their promise for biomedicine, little is known about the cellular pathways that facilitate the uptake of GNRs. Here we show the mechanism of cellular internalization and long-term fate of GNRs modified by (16-mercaptohexadecyl) trimethylammonium bromide (MTAB). The cellular uptake of MTAB-GNRs is a two-step process composed of fast passive adhesion of positively charged MTAB-GNRs on cell membrane by electrostatic interactions followed by slower active intracellular absorption. We identified sulfated proteoglycans carrying strong negative charge as MTAB-GNRs binding partners on the cell membrane. Transmembrane transport of individual MTAB-GNRs is provided via clathrin-mediated endocytosis while aggregated nanorods are engulfed via macropinocytosis. The MTAB-GNRs were distributed in cytoplasm inside membrane structures and end up in lysosomes. Finally, MTAB-GNRs were distributed into daughter cells during cell division, retained for long time in nondividing cells or exocytosed and reabsorbed by neighboring cells. These findings broaden our knowledge about cellular uptake of GNRs, a crucial prerequisite for future successful engineering of nanoparticles for medical applications.
ACKNOWLEDGEMENTS: This study was supported by The Grant Agency of the Czech Republic (GA16-13967S).

Graphene (two-dimensional carbon allotrope) with its basal planar structure and high surface area acts as a template for the delivery of biological molecules and drugs. However, direct covalent functionalization is limited by graphene’s low reactivity, stability and hydrophobicity. Here, we worked with a new type of recently developed graphene acid (GA) synthesized following our procedure [1], with a few modifications. In brief, cyanographene was synthesized from fluorinated graphite after reaction of fluorographene with NaCN and nucleophilic substitution of F atoms with -CN groups. Then, cyanographene was transformed to graphene acid, after acidic hydrolysis of the -CN moieties towards carboxylic groups. In the particular case, higher concentration of nitric acid was used (40-60 %), in order to simultaneously cleave the graphene sheets into smaller fragments usable in biomedicine. Complex cytotoxicity of GA studied by flow cytometry and optical microscopy confirmed its excellent biocompatibility. Moreover, after conjugation with cationic polymer polyethyleneimine (PEI), GA gains a highly positive charge and therefore has a strong ability to bind DNA. Here, we present GAPEI as a novel potential non-viral transfection agent, that is highly desired in gene therapy. Graphene acid was conjugated with polyethyleneimine (PEI) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry. Conjugation of PEI to GA was verified by X-ray photoelectron spectroscopy (XPS) and by measuring FTIR spectra. Size and Zeta potential measurements were also performed. After that, plasmid DNA pcDNA4_EGFP encoding green fluorescent protein was mixed with GAPEI in water and washed carefully by centrifugation. Binding of DNA was proven using UV spectroscopy and XPS. In conclusion, GA could be used as a new candidate for gene transfection and therapeutic application.
REFERENCES: [1] A. Bakandritsos, M. Pykal, P. Błoński, P. Jakubec, D.D. Chronopoulos, K. Poláková, V. Georgakilas, K. Čépe, O. Tomanec, V. Ranc, A.B. Bourlinos, R. Zbořil, M. Otyepka, Cyanographene and Graphene Acid: Emerging Derivatives Enabling High-Yield and Selective Functionalization of Graphene, ACS Nano. 11 (2017) 2982.

Aerogels based on a linear beta-1,3-glucan (Curdlan, CURD) together with polyethylene oxide (PEO) were prepared with different compositions via an environmentally friendly freeze drying process. The structural and thermal properties of the aerogels were studied using field emission scanning electron microscopy and thermogravimetric analysis, respectively. Volume shrinkage, mechanical performance, swelling, solubility and density were accessed. The effect of cellulose nanofibrils (CNF) on the mechanical properties and morphological structure of the prepared CURD-based aerogel was examined. The obtained results revealed that the as prepared PEO/CURD and PEO/CURD/CNF aerogels exhibited porous and layered morphologies. The PEO/CURD (40/60) aerogel reinforced with CNF (0.5 % w/v) displayed a significant improvement in the in-vitro release of a nonsteroidal anti-inflammatory drug (diclofenac sodium). Overall, it was found that the proposed aerogels are promising alternatives to non-biodegradable systems in current use in the biomedical domains.

In the presented work the simultaneous use of ultrasound in tracking and smart release was investigated. Hybrid microparticles based on an iron core coated by a polysaccharide were prepared and loaded with doxorubicin. The possibility to trigger and control the release of the payload was demonstrated by application of ultrasound cycles at different power and time length. Results clearly indicate a direct correlation between the intensity of the release with the two parameters. A preliminary test using Sus Domestic liver demonstrates the possibility to visualize the faith of the microparticles after injection at the target site by a linear array ultrasound transducer. The dual function of the ultrasound; to visualize the position of the microparticles and afterward trigger the release by changing the frequency was demonstrated offering a great perspective for a future biomedical application.

Magnetic particles play an important role in modern biomedical applications including targeted drug delivery, local embolization of blood veins, hyperthermia etc. Therefore, the development of more effective systems with high biocompatibility is of interest for many researchers. Nevertheless, these magnetic systems have to meet certain criteria necessary for in vivo applications. We have considered key requirements desired from such materials, and we have prepared a promising system based on core-shell particles via surface-initiated atom transfer polymerization (ATRP). The finest grade of the carbonyl iron particles was used as a suitable core, while the treatment of its surface in acidic environment ensured the presence of hydroxyl groups, which were further coupled with ethoxy groups of (3-Aminopropyl)triethoxysilane. After the functionalization, the immobilization of 2-Bromoisobutyryl bromide, which served as an initiator, was performed. Finally, the initiator-treated particles were grafted with poly(2-isopropenyl-2-oxazoline) (PIPOx) under ATRP conditions as the PIPOx has recently shown a great potential in biomedical applications. Thorough cleaning and washing procedures ensured high purity of the product. The reaction conversion, molar mass and dispersity of PIPOx grafts were investigated using nuclear magnetic resonance and gel permeation chromatography, respectively. The presence of grafted PIPOx was confirmed using Fourier-transform infrared, and energy-dispersive X-ray spectroscopies. The grafted PIPOx layer had negligible effect on particle magnetization as revealed via vibration-sample magnetometry. Synthesized core-shell structures may find utilization as a promising material for local embolization or may serve as a drug delivery system due to the presence of PIPOx bearing the active sites allowing the drug bonding.

It has been studied the changing of spectral characteristics of water-soluble 10 nm detonation nanodiamonds (DND) upon their complexation with a globular blood protein: bovine hemoglobin (Hb), human serum albumin (HSA) and rabbit immunoglobulin G (IgG) by fluorescence and absorption spectroscopy. The structure of the protein globule and its concentration in aqueous solution can significantly affect the complexation with DND (2·10-3 g/l) in the process of ultrasonic treatment (42 kHz). In all cases, it was observed when adding the DND the increasing of intensity of own luminescence of proteins (em = 330-346 nm) and bathochromic shift of protein luminescence bands. The magnitude of the shift of the maximum of protein luminescence bands at a protein concentration of 10-11 M was 5, 16, 18 nm, at the concentration 10-5 M - 7, 9, 2 nm for Hb, HSA and IgG, respectively. The aggregates’ sizes of DND was evaluated by AFM method in monocomponent film on the glass substrate. The results are of interest for investigations of the dynamic interaction of DND with biomacromolecules in vivo.

A new original technology of production of PHB hollow microspheres with addition of nanoparticular magnetite by carbon dioxide assisted nebulisation drying has been developed. Invertase enzyme (EC 3.2.1.26) was immobilised on the microparticles surface by a simple process exploiting hydrophobic interactions. The biocatalyst had very good activity and stability after repeated applications for sucrose hydrolysis to glucose and fructose. It can be used in effective biocatalytic production of inverted sugar syrup.

Platinum-based cytostatic represent a unique class of DNA-damaging antitumor agents and they are one the most frequently used drugs in oncology [1]. Problem is that some kind of cancer is resistant against this type of cytostatics. This resistance can be potentially caused by metalloproteins such as metallothioneins. MTs belong to the group of intracellular cystine-rich, metal-binding proteins and hold a number of functions in body [2]. One of them is detoxification of heavy metals. This ability of MTs can cause a decreased therapeutic effect of platinum-based cytostatics. In this work, the interaction between two isoforms of MTs (MT3 and MT2) and several types of platinum cytostatics (oxaliplatin, carboplatin and cisplatin) as well as platinum nanoparticles (size of 10 and 40 nm) was examined by fluorimetric analysis using a fluorescence zinc indicator (Fluozin-3). Both, stationary fluorescence spectrometry and capillary electrophoresis with laser-induced fluorescence detection (ex – 488 nm, em – 530 nm) was used in the study.
REFERENCES: [1] NEJDL L., KUDR J., BLAZKOVA I., CHUDOBOVA D., SKALICKOVA S., RUTTKAY-NEDECKY B., ADAM V., and KIZEK R., Mechanisms of Uptake and Interaction of Platinum Based Drugs in Eukaryotic Cells, in Platinum Metals in the Environment, F. Zereini and C.L.S. Wiseman, Editors. 2015, Springer: New York. p. 401-415.; [2] BUCHTELOVA H., DOSTALOVA S., MICHALEK P., KRIZKOVA S., STRMISKA V., KOPEL P., HYNEK D., RICHTERA L., RIDOSKOVA A., ADAM P., KYNICKY J., BRTNICKY M., HEGER Z., and ADAM V., Size-related cytotoxicological aspects of polyvinylpyrrolidone-capped platinum nanoparticles. Food and Chemical Toxicology, Vol. 105, No., 2017, pp. 337-346.

Cisplatin (cis-diamminedichloroplatinum II) is cytostatic currently used in the treatment of a various type of solid tumors, including cancers of the ovary, testis, bladder, head and neck, lung, cervix and endometrium. The health effects of platinum are strongly dependent upon the kind of bonds that are shaped and the exposure level and immunity of the person that is exposed. An in vivo interaction study was performed as a comparison of amino acid profiles in chicken embryo tissue before and after application of cisplatin and two platinum nanoparticles covered with polyvinylpyrrolidone. We found that cisplatin directly influences kidney amino acid representation even at low concentrations. In addition, we performed an interaction study that resulted in the determination of breaking points for each of analyzed amino acids. Proline, leucine, valine, serine, glutamine and isoleucine were determined as the most sensitive amino acids. Additionally, we compared amino acid profiles of liver and kidney before and after exposure to cisplatin and platinum nanoparticles. The amount of amino acids after interaction with cisplatin and platinum nanoparticles was significantly reduced (p ˂ 0.05). This fact points at an ability of cisplatin and Pt nanoparticles to induce changes in quantitative composition of amino acids in chicken tissues. Moreover, this confirms that the interactions between cisplatin, platinum nanoparticles and amino acids may act as another factor most likely responsible for adverse effects of cisplatin and platinum nanoparticles on chicken embryo tissues.

Paper tests are very cheap and fast diagnostic tools for self-testing in a wide range of health problems. Thus, the diagnostics of tumor development is of a great medical and socio-economic significance. Malignant prostate cancers are the most common malignancy in men. Procedures have been developed to diagnose this disease based on the monitoring of tumor marker molecules. Most commonly, PSA and fPSA are used. Although these tumor markers are significantly increased, the cancer at the biopsy is not always confirmed. Thus, the finding of other marker molecules is urgently needed. Recent studies have shown that changes in content of the amino acid sarcosine are associated with prostate tumors. Polyclonal chicken antibodies against sarcosine (AntiSar) were prepared. AntiSar antibodies were labeled with gold nanoparticles Au20 (size 20-30 nm, zeta potential - 38 mV) and CdTe QDs (emission 530 green QDs and 650 nm yellow QDs). Dot blot and flow lateral chromatography were used to develop a newly designed sarcosin detection procedure. The aggregation or fluorescence intensities were evaluated by newly designed software. Sarcosine was analyzed in a concentration range (0-150 μM) in PBS, arteficial urine and urine sample buffer. The calibration curves obtained in the concentration range (0-12 μM) were strictly linear (R2 = 0.99) with a relative error of 10%. Using the proposed methods sarcosine concentrations as low as 250-500 nM were analyzed. Moreover, the results obtained show a higher sensitivity (by 20-40%) of sarcosine detection using QDs in comparison with using gold nanoparticles.

Magnetically-responsive nanostructures represent very promising materials for drug delivery and diagnostic applications, especially in the field of cancer treatment. Such nanomaterials can be primarily self-assembled from superparamagnetic iron oxide nanoparticles (SPIONs) into SPION clusters, and then further to magnetic nanochains (MNCs) by applying magnetic assembly of its building blocks, SPION clusters. The main limitation of individual SPIONs, namely ineffective spatial guidance due to too small magnetic force acting on them, when exposed to magnetic field gradient, can be thus overcome. Magnetic SPION-based nanostructures are usually coated with a layer of porous silica which offers unique properties for drug loading and improves the biocompatibility of the nanosystem.
In the current study partially hollow SPION-based MNCs with the average length of about 300-500 nm were successfully prepared. The physical stability of obtained colloidal dispersions was systematically evaluated in two different physiologically acceptable media, namely HEPES and phosphate buffer, since it is a prerequisite for their application in biological environment. The zeta potential measurements revealed surface charge of the surface modified MNCs between -25 mV and -40 mV, in contrary to surface charge of unmodified MNCs which was between -3 mV and -10 mV, indicating better stability of the surface modified MNCs. The results revealed that partial dissolution of core nanoparticles in acidic conditions impair physical stability of the dispersion, since it decreased the surface charge of MNCs. Finally, partially hollow MNCs were loaded with a model poorly soluble drug via incubation of MNCs in ethanol drug solution. The preliminary experiments revealed very fast drug release from the novel magnetically-responsive drug delivery system (total drug release within the first 30 min), thus further optimization of MNCs structure and drug loading procedure is necessary to obtain controlled drug release.

Sarcosine is an amino acid that is formed by methylation of glycine and is present in trace amounts in the body. Increased sarcosine concentrations in blood plasma and urine are associated with sarcosinemia and some other diseases such as prostate cancer. In addition, sarcosine may be administered as a drug in some psychiatric disorders. Primarily, sarcosine in biological fluids is analyzed using liquid chromatography techniques, but this technique is not suitable for routine analysis. For this purpose, sarcosine detection using the nanomedicine approach was proposed. In the presented test, Au20 gold nanoparticles (size 20-30 nm, zeta potential -38 mV, absorption maximum at 529 nm), which exhibit pseudoperoxidase activity and convert a suitable substrate such as TMB, ABTS (1 mM) to a distinctive color product, were used. In the following experiments, the Au20 nanoparticle was linked to the chicken anti-sarcosine antibody. Subsequently, the ELISA method for the determination of sarcosine was prepared. Firstly, the construct for direct detection of sarcosine bound to the antibody was prepared by both the direct and the indirect method (dependencies were strictly linear, R2 = 0.996). The subsequent construct uses rabbit antibodies against the anti-sarcosine chicken antibody labelled with Au20 nanoparticle. The bound product is then monitored by the conversion of the TMB (655 nm) or ABTS (425 nm) substrate (the dependencies are strictly linear, R2 = 0.998). To further increase the sensitivity of the method (for more than 90% of the original signal), an aggregation agent was added to the used Au20 nanoparticles, which further increased adherence of gold nanoparticles to the antibodies. This procedure succeeded in achieving subnanomolar detection limits for the amino acid sarcosine.

Target therapy for oncologic diseases presents a big challenge for advance nanomedicine. In our work, we focused on multi-target approach development. Designed nanotransporter is based on polysaccharide chitosan which allows nanoparticles formation. These nanoparticles can bind metal ions, mainly zinc (moreover, zinc stabilize chitosan structure). We estimated, the zinc concentration was approximately 1 nmol/g of chitosan. In addition, chitosan nanoparticle (cage) irreversibly bind therapeutics which could be applied for targeted therapy of malignant tumours. Our designed chitosan structure (LMQ, 10 g) encapsulation efficiency for doxorubicin was 50%. The pH change (tested interval 8 - 5) caused 20% release of doxorubicin from the nanocage. The additional targeting of nanotransporter to the tumour matrix was mediated by PEG modification (10%). The specific interactions are oriented to cancer tissue due the fact the malignant cells highly express metallothionein (MT). The increased affinity of MT to zinc ions and the nanotransporter is preferentially bind to tumour regions with a high MT concentration. Our latest experimental results show the change of amino acid metabolism signalized by increasing the amino acid sarcosine amount. Therefore, the chitosan-based nanotransporter was modified by anti-sarcosine antibody. The functionality of designed nanotransporter was proved by ELISA with double detection of doxorubicin (fluorescence) and by peroxidase activity of ABTS substrate. In another system, magnetic separation and identification of individual components of the nanotransporter were used. The sarcosin binding activity was estimated around 50%.

Photodynamic therapy is a relatively new type of cancer treatment, or eventually it is used at the elimination of undesirable pathogenic microorganisms. Quantum dots (QDs) are semiconducting nanocrystals with a size of 2-20 nm and can be used in photodynamic therapy. Two types of CdTe QDs were prepared by microwave synthesis (500 W), typical absorption spectra had maxima λgreen = 554 nm, λ yellow = 580 nm. The determined size of the generated nanoparticles ranged between 5 - 10 nm. CdTe QDs were further studied by fluorescence analysis at excitation wavelength λ = 250 nm to obtain emission maxima (λem552) for green and (λem582) for yellow nanoparticles. Interaction study of CdTe QDs with bovine serum albumin (BSA) and with polyclonal chicken antibodies against sarcosine (AntiSar) was performed. BSA (100, 50, 25, 12.5, 6.3, 3.1, 1.6 and 0 μM) and AntiSar (20, 10, 5, 2.5, 1.3, 0.7, 0.3, and 0 g/L) were monitored with 1:1 addition of 50 μM QDsgreen or QDsyellow. The decrease in intensity of the normalized fluorescence signal in the presence of BSA or AntiSar was observed by 70-90%. The observed dependencies showed a linear trend (R2 = 0.9) with relative error of 9-12% calculated from 5 independent repetitions. In addition, it was possible to monitor the signal shift to shorter wavelengths at the highest applied BSA concentration by 4-16 nm. The obtained data suggest that the size of individual QDs will affect intensity of an interaction with biomolecule. Nanoconstructs should therefore be targeted according to these experimental data for their intended use. In further experiments, CdTe QDs modified with AntiSar will be used for targeted prostate cancer therapy using photodynamic effect.

This work is focused on the preparation and characterization of nanoparticles. Particles were prepared from negatively charged polymer (sodium hyaluronate) and oppositely charged polyaminoacid (polyarginine hydrochloride) in water and 0.15 M sodium chloride. Due to the composition of nanoparticles from biodegradable materials, particles may be attractive for medical applications. Particles were studied using dynamic light scattering and measuring of zeta potential.
The second part of this work is focused on study of electrostatic interactions between negatively charged hyaluronan chains and simple amino acids (lysine and arginine) in water. These interactions were studied by means of calorimetry and pH measurements.

The discovery of anti-carcinogenic drugs containing platinum (Pt), has led in the 1970s to synthesis new compounds containing Pt, which could provide better results in cancer therapy. Unfortunately most of these drugs have toxic side effects, therefore they are given in small doses with low concentration. One of these is cisplatin, which is widely used. However its antitumor activity is reduced by its side effects. A new form of cisplatin is liposomal cisplatin, which replaces the original one, reducing its toxicity and increasing efficiency. The aim of the study was to observe the effects of Pt cytostatics on cancer cell lines. Commercial cisplatin cytostatic was encapsulated into three types of liposomes (L8, L10 and L15) of a different chemical composition. Each of liposomes had different ability to close cytostatic. L8 differs from L10 by containing cholesterol. In despite of L10 and L8, L15 liposome contains a synthetic 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (C41H78NO8P) – DOPE, which is generally found in cell membranes. The size of the liposomes was characterized by Dynamic Light Scattering (DLS). The amount of Pt-drug in liposomes was verified by electrochemical determination. Their stability was observed by differential pulse voltammetry (DPV). The DPV is highly sensitive to platinum detection. These studies also demonstrated, that the cisplatin is stable in physiological solution for at least 24 hours, which making it a suitable medium storage for this drug. Finally, the encapsulated cisplatin was applied to cancer cell lines. Their viability was observed by MTT assay. The highest anti-carcinogenic activity was reported by liposomes L10.

Optical biosensors based on Surface Plasmon Resonance (SPR) imaging represent advanced optical label-free real-time biosensor technology. In these sensors, surface plasmons are excited by light waves by means of the attenuated total reflection (prism coupling) or diffraction on a metal grating (grating coupling). The strength of the coupling depends on the angle of incidence, grating period and refractive index of the dielectric medium in contact with the grating. However, the operating range (range of refractive index of the dielectric medium) of SPR imaging sensors utilizing conventional diffraction grating with a fixed period is rather limited.
In this work, we propose a special grating coupler, plasmonic gratings with linearly increasing period, and explore its ability to expand the operating range of SPR imaging sensors. The rigorous coupled wave theory was used for the analysis and design of the plasmonic grating with respect to normal light incidence of a wavelength of 785 nm. Plasmonic gratings were fabricated by the electron beam lithography and characterized by the scanning electron microscopy and atomic force microscopy. The potential of these gratings for SPR sensors was explored in a model refractometric experiment in which position of the dark band due to the excitation of surface plasmons was measured for different refractive indices (ranging from 1.329 to 1.347) of the medium in contact with grating. The experiments revealed that plasmonic gratings with a linearly increasing period provide SPR imaging sensors with a considerably larger operating range than those provided by grating with a fixed period.

The present work is focused on preparation of chitosan films, which were doped with silver nanoparticles (AgNPs). AgNPs were prepared and stabilized by reduction of silver nitrate with chitosan without adding reducing agents. It was tested whether the use of UV light could affect the AgNPs. UV light was used for 5 min: during preparation of chitosan solution and at the end of the process when the chitosan films were done. The presence of AgNPs was studied by X-ray photoelectron spectroscopy. Wettability and water absorption of the films were evaluated. Solid films were dissolved and then the solution was observed by the ultraviolet–visible spectroscopy and transmission electron microscopy. Concentration of AgNPs released into the solution during dissolution was studied by atomic absorption spectroscopy. The presence of AgNPs was confirmed both in the solid films and in the solutions by the above mentioned methods. The research was aiming on use of these films in medicine as a new type of wound dressing with antibacterial properties. These films could be used as a wound dressing, antimicrobial packaging material or as a long-term storage of AgNPs for various applications.

Extracellular matrix (ECM) forms essential part of tissue microenvironment. Compositional and structural properties of ECM modulate behaviour of cells, including differentiation, proliferation and replacement. Here we aimed at detailed characterization of the nanostructure of lung ECM and evaluation of its bioactivity on cells grown in vitro. Mouse lung tissue was decellularized using 0.2% sodium dodecyl sulphate, hypotonic solutions and DNase. Morphological analysis of the resulting ECM scaffolds was performed by means of transmission and scanning electron microscopy. The ECM scaffolds retained 3D architecture of the main lung anatomical regions: the alveolar region and the blood/airway network. The region-specific ECM nanotopology and organization of ECM macromolecules such as fibres of collagen, elastin, and fibrillin were characterized. The lung ECM scaffolds were also homogenized and applied as supplement of growth media on primary lung cells in vitro to test the bioactivity of lung ECM. We demonstrate that homogenzied ECM does not have negative impact on the prolifaration rate of primary lung cells. In conclusion we herein show in nanoscale the morphological characteristics of lung ECM and demonstrate that lung ECM is compatible with in vitro culture of lung cells. These findings add to better understanding of cells natural environment and can be valuable in applications of ECM for purposes of tissue engineering. This work was supported by grant COST CZ LD15144 awarded by the Ministry of Education, Youth and Sport of the Czech Republic and by MUNI/A/1369/2016.

Adsorption of antibodies to tularemia Francisella tularensis (F. name of the genus) on the surface of gold-plated glass chips and the subsequent reaction of antigen-antibody were studied by surface plasmon resonance (SPR) technique. It was found that these antibodies tend to polylayer adsorption on the gold surface. However, washing with a buffer solution leads to desorption of weakly associated molecules, and the monomolecular coating with the same parameters remains on the surface for all initial concentrations of antibody in the solution. The antibody monolayer characterized by surface protein concentration near 2.6 ng/mm2. This value corresponds to the area of about 100 nm2 per molecule of the immunoglobulin. This first adsorption layer adjacent to the surface irreversibly binds to it, which makes it possible to use such a system for subsequent diagnosis of tularemia microbes in solution. It was shown that rapid determination of the microbe in solution up to concentrations of 1.0 × 102 m.k / ml can be carried out using such biosensors using the SPR method. Based on the estimated area per molecule of the adsorbed antibody in the monolayer and the comparatively low signals of the antigen-antibody reaction, a conclusion was made about the lateral arrangement of the antibody molecules in the adsorption layer. The sensitivity of the proposed method can be improved by creating an oriented layer of antibody molecules, which promotes more effective interaction with the antigen.

This study is focused on the properties and behaviour of phase-separated hydrogels prepared by interaction of hyaluronan (Hya) with oppositely charged surfactants. Three representatives of surfactants, namely cetyltrimethylammonium bromide, chloride and p-toluensulfonate (CTAB, CTAC, CTAT), were selected for comparison. The method of rheology was used as a crucial one in this research. The fact that the system of Hya-CTAT forms the most rigid hydrogels has been proved. Higher molecular weight of polymer has also direct influence on the volume and stiffness of the hydrogels. The methods of preparation were compared as well. Mixing the stock solutions of entry components appeard to be the most suitable. The final results indicate that studied gels are to a certain extent competitive. Incorporation of hyaluronan into their structure is desirable with respect to its biological activity. This offers a potential usage of these substances in the field of medical applications.

Response of body to commonly used metal implants cannot be avoided. The long-term implant stability strongly depends on the surface properties (structural, mechanical, chemical) of the implant. Release of ions and wear particles from implants can be effectively prevented by robust, continuous, non-permeable and biocompatible coatings that adhere well to the bulk material. Diamond proved as excellent material supporting the adhesion, growth, viability, metabolic activity, and osteogenic differentiation of cells.
With view to practical medical applications we will show results of histology and micro-CT study after six and twelve months implantation of cortical screws in rabbit’s femur bone. These results are correlated with material characterization (morphology, composition, surface chemistry, hydrophilicity) of uncoated and diamond coated screws before and after in vivo tests. Self-tapping cortical screw HA 3.5x8 mm made of stainless steel and titanium were employed. For diamond deposition, we investigated two pre-treatment methods based on seeding by colloidal diamond nanoparticles in ultrasonic bath and immersion in a polymer composite with diamond nanoparticles. Application of a 150 nm a-Si, Cr, or WO3 interlayer was also evaluated. The diamond coating was then deposited by CVD in a low-pressure linear antenna microwave plasma system. Surfaces of the diamond-coated screw implants were further modified by oxygen plasma, a process expected to improve their hydrophilicity and adhesion of bone cells.
Histology of control implants (standard Ti alloy) after both 6 months and 1 year in rabbit femurs showed good osteointegration without any inflammatory reaction. Micro-CT examination after 1 year showed lamellar bone around the entire implant without any empty pockets. Diamond-coated screws examined after 6 months exhibited similarly good integration into the bone. Results with diamond-coated screws after 1 year are being analyzed. The study provides promising outlook for core material replacement (for avoiding Ti allergy) and for taking advantage of versatile and stable surface functionalization of diamond by diverse molecules (for faster osteointegration).
This study was supported by the Czech Health Research Council, Ministry of Health of the Czech Republic (grant No. 15-32497A).

Mesenchymal stem cells (MSCs) are multipotent cells present in the stromal fraction of many tissues. Their immunomodulatory properties and differentiation potential have been proven beneficial in number of autoimmune disease models. However a reliable in vitro approach to study MSCs differentiation in inflamed tissue and a clear insight into the involved signaling pathways may accelerate their application in vivo.
Fibrin nanoscaffolds have been developed for cartilage tissue engineering and provide a natural matrix that can mimic synovium microenvironment. Moreover fibrin hydrogels allow to recreate in vitro the same stiffness chondrocytes face in vivo, leading to improved chondrogenesis differentiation. Besides all these properties, fibrin can be completely degradated by nattokinase, allowing an easy single cell analysis.
The aim of this work is to investigate the role of NF-kB inflammatory pathway in MSCs chondrogenesis performed into fibrin nanoscaffolds. Thus we established NF-kB luciferase reporter line to monitor transcription factor activation. Combining degradable fibrin nanoscaffold technology to reporter line for monitoring transcription factors activity will provide a powerful platform to understand the role of inflammation in different stages of chondrogenesis.

Cell-based therapy of local tissue injuries or damages requires application of stem cells and inhibition of harmful inflammatory reaction which could impede the healing process. To increase the effectiveness of this therapy, a local administration of drugs can avoid their side effects associated with a systemic treatment. A local therapy requires suitable carriers, which can transfer the cells and drugs to the site of injury. As a promising carriers turned out nanofiber scaffolds prepared by electrospinning technology from various types of polymers. The main advantage of this technology is a possibility to define properties of nanofiber scaffolds, optimal for the growth and transfer of stem cells, and which could incorporate various types of immunosuppressive drugs. Here we describe the formation and use of nanofiber scaffolds prepared by needleless electrospinning technology from poly(L-lactic acid) (PLA) which are loaded with immunosuppressive drug cyclosporine A (CsA). We show that CsA-loaded nanofibers effectively and selectively inhibit proliferation of activated T-cells and suppress the production of T-cell cytokines in vitro. Simultaneously, these nanofiber scaffolds enable growth of mesenchymal stem cells (MSCs) and thus can serve as stem cell carriers. Moreover, using an experimental mouse model of skin transplantation, we showed that covering skin allografts with MSC-seeded and CsA-loaded nanofibers significantly inhibited the local production of proinflammatory cytokines IL-2, IL-17 and IFN-γ, and supports healing. Thus, nanofiber scaffolds seeded with stem cells and loaded with CsA can serve as carriers of cells and drugs for a local cell therapy and for simultaneous effective immunosuppression.

Advancement in organoid cultures from human pluripotent stem cells allows studying complexity of tissue specific signaling in vitro. This organ in a chip approach provide an important tool for translational research. Successful development of nanoparticles and nanostructures for clinical use has increased the need for more reliable experimental models beyond conventional cell culture.
Human induced pluripotent stem cells (iPSCs) can be differentiated to multiple cell types including tissue-like organoids. Here, we study the interaction of nanoparticles with cells in organoids and analyse their effects on various tissue organoids in vitro. Human tissue organoids are generated from iPSCs by growth factor induced differentiation method as well as by manipulation of various developmental signal pathways. The 3-dimentional structures of the bodies, with structural features similar to the appropriate human organ/tissue are obtained by embedding in a matrix. The organoids are injected with nano-sized particulate triggers such as glucans. The various tissue models are compared for their nanoparticle-induced stimulation by screening for inflammatory cytokines and release of danger associated molecular patters (DAMPs). Described novel model will allow studying human tissue interaction with nanoparticles which for example are used in drug delivery.

Oppositely charged polyelectrolytes and surfactants can form under specific conditions hydrogels containing micelle-like nanostructures. Due to its hydrophibic core, these structures are capable of solubilizing hydrophobic substances within hydrophilic soft solid matrix. Such hybrid materials can be of interest for medical applications. In this study, interactions between cationic dextrane and sodium dodecylsuplhate were studied and conditions of gel formation investigated. Using the techique of fluorescence probe, phase behavior was analyzed and region of the gel phase separation detected. Solubilization of hydrophobic dyes confirmed the formation of micellar domains. The gel formation was affected by ionic strength of gelling solutions.

Corneal damage is one of the most common causes of impaired vision or even blindness. When the injury is more extensive and the limbal region is involved, the natural regeneration of cornea is not sufficient. Such damage can lead to limbal stem cell deficiency (LSCD). Corneal transplantation is not sufficient in such cases. Nowadays the only option for LSCD treatment is transplantation of the limbal tissue or a transfer of limbal stem cells (LSCs) cultured from the healthy eye. In the case of bilateral LSCD the only option is allogenic transplantation of the limbus or cultivated LSCs with systemic administration of immunosuppressive drugs. Nevertheless the cell therapy is very promising tool for LSCD treatment. Transplantation of mesenchymal stem cells (MSCs) seeded on an appropriate scaffold turned out to be a suitable therapy of the LSCD. In our mouse model of LSCD we use nanofiber scaffold for MSC and LSC cultivation and for transplantation of these cells onto the chemically injured mouse eye. MSCs have immunosuppressive and immunomodulatory properties. We showed that MSCs have the ability to inhibit production of molecules associated with inflammation and restore epithelial regeneration in the damaged cornea. These inhibitory properties were confirmed in both in vitro and in vivo mouse model. Results thus showed beneficial effects of stem cell transplantation for murine corneal healing and for suppression of a local immune reaction which can impede the healing process.

The main aim of this work is the study of releasing of the solubilized substances from the phase separated hydrogels and to determine the solubilization capacity of these hydrogels. Hydrogels are colloidal materials which can be, under proper conditions, prepared by interaction between polymers and oppositely charged surfactants via electrostatic forces. Surfactants are able to form micellar structures suitable for solubilizing hydrophobic compounds in aqueous environment of the gel. Resulting materials (hydrogels) contain hydrophobic nanodomains which are possible to use in medicine, e.g. hydrophobic drugs for topical applications, drug delivery systems and so on.
For releasing tests were used fluorescein and acridine orange as fluorescent probes and hydrogels were monitored after 24, 48 and 72 hours from their preparation. For hydrogels monitoring was used fluorescence correlation spectroscopy because of the high sensitivity of this method. Hydrogels were prepared from hyaluronic acid and positively charges surfactant carbethopendecinium bromide.

Titanium and its alloys are widely used in orthopaedic and dental implants due to their biocompatibility and good mechanical properties. However, there have been problems with insufficient implants osteointegration. Formation of the nanostructures on the implants surface is a promising strategy to modulate cellular response such as adhesion and differentiation. The aim of this study was to investigate osteoblast – like cells (SaoS-2) adhesion, proliferation and differentiation on nanostructured titanium.
Nanotubes were fabricated using anodic oxidation of the commercially pure titanium (cpTi) on 10V and 20V. The cell response and adhesion of the SaoS 2 cells were studied in vitro on untreated and nanostructured cpTi. The activity of alkaline phosphatase (ALP) and expression of the osteogenic markers (collagen-I, osteocalcin) was measured using qRT-PCR. Collagen-I was also visualised using specific immunohistochemical staining followed by confocal microscopy.
No significant difference in cell proliferation among nanostructured or control cpTi was observed. Nevertheless, metabolic activity was significantly higher on nanostructured cpTi on day 1 and 10. ALP activity, which is an early marker of osteogenic differentiation, did not differ among samples. Synthesis of collagen-I and osteocalcin increased on nanostructured cpTi, while 20V showed the highest synthesis of both collagen-I and osteocalcin. Figures from SEM showed flatter and spreader cell morphology on nanotubes compared to smooth cpTi. The results indicate that the nanotubes present on the titanium surface are beneficial for adhesion and osteogenic differentiation of the Saos-2.
ACKNOWLEDGEMENTS: This study was supported by the Czech Science Foundation project No.16-14758S.

Nanofibrous matrices represent an attractive material for biomedical applications. The main attention has recently been focused on their application in wound healing and drug delivery. Both applications mentioned benefit from unique properties of nanofibres as safe 2D nanomaterial possessing features such as high porosity with small pore size which supports their application as a barrier system and also high specific surface increasing availability of the delivered drug. Another advantage to be mentioned is variability of the chemical composition allowing precise surface design and desired interactions between nanofibres and immobilised/ attached molecule.
The aim of this study was to develop a new nanofibrous material based on blend of two biocompatible and biodegradable polymers – poly-Ɛ-caprolactone (PCL) and polyethyleneimine (PEI). The electrospun matrices were evaluated in terms of morphology, chemical composition, functional groups availability and short term stability. Moreover, biocompatibility to the 3T3 fibroblastic cell line was verified. Binding capacity estimation was performed on a model molecule (BSA) in order to demonstrate binding capacity of the newly developed system.

Extracellular biosynthesis of nanoparticles have many important advantages such as well dispersed in aqueous solutions, low energy requirements, ecofriendly, non-toxic, low-costs and non-flocculate. This technique have shown significant promise as targeted drug delivery applications. In this investigation, for the first time, we examine the efficacy of targeted therapeutic delivery with t-PA and SK immobilized to biosynthesis of nanoparticles (CuNP) by using Streptococcus equi strains isolated from the horses of Iran and their ability to produce metallic nanoparticles. Also we compared them with their chemical synthesis. The S. equi was screened for its ability to produce MNPs. The different sizes and shapes (23 to 89 nm) are presented in the formation with good dispersion and high stability. Response Surface methodology was applied for the optimized production of biological CuNPs. The growth factors like pH, temperature and incubation time was changed. The optimum conditions to obtain CuNPs were found with the culture conditions of pH 7.5 in 120h at 35 ºC. To determine some of MNPs structural properties UV–vis absorption spectrophotometer, FTIR, XRD and SEM has characterized. The results provided some parameters may impact on the formation of biological MNPs. Lastly, these MNPs were conjugated with t-PA and SK, as a drug carrier. In addition, effective thrombolysis with magnet-guided SiO2-CuNPs-tPA-SK is demonstrated in rat embolism model where 18.59% of the regular t-PA dose and 15.78% of SK dose restored and 15–25 min reductions in blood clot lysis time were observed compared with runs with free t-PA and without magnet-guided and using the same drug dosage. The comparison between CuNPs with MNPs shows that thrombolysis had not been directed to the type of magnetic carrier under the magnetic guide.

The ability of cells to perceive the mechanics and nanostructure of the extracellular matrix (ECM) relies on the activity of a number of intracellular proteins, collectively defined as mechanosome.
By modifying the expression, the interaction or the localization of such proteins, the cell dynamically responds to modifications in ECM compliance and nanotopography by activating specific genetic programs. Among the intracellular proteins acting as mechanosensors, those composing the Focal Adhesions (FAs) and the effectors of Hippo pathway YAP/TAZ have been recently described as the main actors.
Here we show that both FAs and YAP/TAZ respond to cell area, to substrate mechanics and nanotopography by rearranging their structure or their localization, while they are not sensitive to cell polarity.
In particular, the assembly of FAs, measured by image analysis through vinculin spike formation at the periphery of the cell, and YAP/TAZ nuclear shuttling, are proven to be sensitive to substrate mechanics. Their activation by substrate stiffness favors cell spreading and increases cell tension propagation through cell cytoskeleton. The abolishment of the tension by pharmacological inhibitors of RhoA/ROCK or Myosin IIa pathways results in the inhibition of YAP/TAZ nuclear activity, the formation of FAs and the inability of the cell to spread and acquire a defined shape and size.
This effect is phenocopied by mimicking cell-cell interaction at single cell level, by activating the cadherin/catenin system in adherent cells.
Finally, by dynamically modifying substrate nanotopography, we demonstrate that both FAs and YAP/TAZ systems can be promptly controlled by ECM composition.

A simple procedure for synthesis of magnetic fluid stabilized by poly(methacrylic acid) was developed. This ferrofluid was used to prepare magnetically responsive chitosan composite material. Both ferrofluid and PMAA/chitosan microparticles were characterized in detail using optical microscopy, scanning electron microscopy, transmission electron microscopy, static and dynamic light scattering, small-angle neutron scattering, energy dispersive X-ray spectroscopy and Fourier transform infrared spectroscopy.
Magnetic PMAA/chitosan microparticles were employed as a magnetic carrier for immobilization of baker´s yeast; Saccharomyces cerevisiae cells were successfully incorporated into chitosan during its preparation.
PMAA/chitosan/yeast biocomposite was then tested as an adsorbent of selected organic dyes. The experimental data followed well the Langmuir adsorption isotherm model, and the maximum adsorption capacities were found to be 116.6 mg/g and 104.3 mg/g for crystal violet and safranin O, respectively. This biocompatible material was also utilized for sucrose hydrolysis; enzyme activity of one-day and one-month old biocatalyst remained unaltered.

The production of renewable, biobased and biodegradable bionanocomposites is dicussed in current research. The developement of such materials is mainly driven by rising concerns over environmental issues, sustainability and dependency on decreasing petroleum resources. The effect of blending, addition of heterogeneous nucleating agents and post-molding annealing on properties of poly(lactic acid) (PLA), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and lignin-coated cellulose nanocrystals (L-CNCs) bionanocomposites was investigated. Masterbatches were prepared by melt mixing using an internal mixer and further then were standard tensile test bars injection molded. The crystallization behaviour, morphology, impact strength and heat deflection temperature of neat and annealed PLA, PLA/PHBV blends and PLA/PHBV/L-CNC bionanocomposites were investigated. The results showed that both property modifiers (PHBV, L-CNC) and annealing conditons had significant influence on material properties.

Recent interest in industrial and medical usage of carbon nanomaterials arises the need for new testing protocols for characterizing their interaction with living systems. In this study we examined the anti-adhesive properties of differently terminated nanocrystalline diamond (NCD) layers deposited by plasma-enhanced CVD on glass or silicon against E. coli bacterium. We focused on the influence of cultivation medium in this process and compared bacterial adhesion to H-, O- and F-terminated NCD films in complex (LB) and mineral (M9) cultivation media that substantially differ in a content of organic molecules.
We found that hydrogen and fluorine terminated NCD layers were able to reduce bacterial adhesion by ca. 50%, however the effect was observed only in the mineral medium. In a complex LB medium we did not observe the reduction of bacterial adhesion. We assign this effect to the passivation of the H- or F-terminated NCD surfaces with organic molecules adsorbed from medium. For O-terminated NCD, no antiadhesive effect was observed, regardless on the cultivation medium.
We also found the properties of NCD layers to be independent of the base substrate, as the amount of biofilm was comparable for both NCD deposited on glass and on silicon. Our results show that nanocrystalline diamond surface is unfortunately not a universal antibacterial/antiadhesive coating.
ACKNOWLEDGEMENTS: This work was supported by the project GACR 15-01687S and SVV-2017-260426.

In the context of the development of nanotechnology in recent days, everyday applications of nanostructured noble metals, especially silver, are widespread in various industrial areas. However, their applications in cosmetics, household cleaners, textile industry, and first of all medicine, in which they are used for their unique antibacterial effects, may bring the risk connected with their potential cytotoxicity. Some of the toxicological studies indicate that the most cytotoxic form of nanostructured-metal materials are silver nanowires. For this reason, we investigated silver, as the most commonly used nanostructured noble metal, and palladium nanowires, as possibly safer alternative. Metal nanowires (NWs) were supported on polyethylene naphthalate (PEN), which is frequently used material for the preparation of thin and strong balloon catheters and other medical devices. The surface of metal/polymer composites was fully characterized by XPS, FIB-SEM and AFM. Thereafter, the biological testing of the samples was accomplished. Antibacterial properties of both, Ag and Pd NWs, were examined and compared between themselves using Gram-negative bacteria Escherichia coli and Gram-positive Staphylococcus epidermidis, which commonly cause hospital-acquired infections arising from long-term applications of catheters and other medical devices. Finally, the comparative cytotoxic study of both types of composites was carried out using mouse embryonic fibroblasts (L929). The biological testing served for i) the verification of significant antibacterial effects and cytotoxicity of Ag NWs referred in literature, and ii) the comparison of antibacterial and cytotoxic effects of Ag and Pd NWs to determine, whether Pd NWs are a suitable to substitute Ag in its medical applications.

Nanodiamonds (NDs) as well as non-thermal plasma are finding nowadays wider perspective for biomedical applications. Treatment of water for inhibiting bacterial growth is also of increasing importance. In this work we focus on the characterization of biocidal properties of water treated by non-thermal atmospheric plasma (i.e. so called plasma activated water - PAW) or PAW with nanodiamonds.
Employed nanodiamonds with average diameter of 5 nm in as-received form were produced by a detonation process. They are marked as H-ND due to their numerous C-H bonds and positive zeta potential. We activated water or aqueous colloidal solution of H-ND by corona discharge or transient spark, where the duration of activation was 1, 3 or 5 minutes. Antibacterial effects of PAW and H-ND in PAW were evaluated by comparison of number of CFU (colony forming units) of microorganism Escherichia coli, which were cultivated on Mueller-Hinton agar. We began the cultivation either immediately after mixing bacteria solution with examined solution or after cultivation for 5 h. Since there are only few data on synergistic antibacterial effects of nanodiamonds and plasma activation, we examined antibacterial effects of NDs and PAW separately and also their combined biocidal effects.
Our results show that the PAW alone indicates the most significant antibacterial effect for immediate exposure after atmospheric plasma activation (< 1 h cultivation) . In that case we could observe drop of CFUs compared to reference by 65% (corona discharge) or 55% (transient spark) for the longest activation period (5 minutes). Contrary to that, plasma activated solution with H-ND exhibits better antibacterial effect after the 5 h cultivation compared to the PAW alone. There was a drop of CFUs by 24% (corona discharge), which is slightly better than inactivated H-ND (20%), or by 67% (transient spark) for the longest activation period.. Bacteria growth-inhibition data are correlated with infrared spectroscopy of the nanodiamond showing modification of surface chemical groups. These and other factors that can affect antibacterial activity of water against the E. coli are discussed. The work has been supported by GACR 15-01687S.

Body implants are necessary part of contemporary medicine. Biocompatibility and life-time are one of the most frequently controlled parameters for material. One of the possible ways to ensure these requirements is to modify the surface of the implant with a thin layer. TiCN thin films with a differing composition were deposited on the surgical steel AISI 316 by cathodic arc evaporation of pure Ti with various different gas ratios of N2 and C2H2. The pres-sure in the vacuum chamber was 1.5 Pa and the deposition temperature was 400 °C during the deposition processes. Chemical stability, thickness of the layer and the layer adhesion to the substrate were studied, due to the possible use of those samples in the medical applica-tions. The deposition parameters affect physicochemical properties (the chemical composi-tion, roughness and surface energy) which have subsequently an impact on attachment of cells. Escherichia coli was used as the model bacterial strain. The importance was given to the observation of the willingness of the microorganisms to colonize individual samples.
This work presents the physicochemical properties of individual TiCN thin layers and also evaluates the relationship between surface properties and colonization of surface by bacte-rial cells.

Silver nanoparticles are widely used as effective antimicrobial agents in industry and medicine. The preparation method of «pure» silver hydrosol was developed. This hydrosol contains only silver NPs and stabilizing carbonate ions. It fully corresponds to the composition of natural freshwater. Preparation method consists in the photo-chemical reduction of silver ions by oxalate-ions in aerobic conditions under the action of low-pressure pulsed xenon lamp. The irradiation initiates Ag+ reduction with formation of metal nanoparticles and carbonate ions. 2nAg++nC2O42-+2nH2O=>Ag2n+2nH++2nHCO3-. The hydrosol was studied by spectrophotometry, dynamic light scattering, transmission and scanning electron microscopy. The hydrosol is aggregative stable for several weeks. It is free from toxic compounds that are usually employed as reducing agents or stabilizing additives and which may affect functioning of living organisms. The antibacterial properties of nanoparticles with an average size 22±2 nm were investigated on the Bacteria Escherichia coli. The influence of silver ions was also investigated for comparison. Experiments were carried out in the presence and in the absence of air. Different concentrations of silver NPs and silver ions have been inoculated into the Adkins medium with bacteria cells. After 5 days the cells’ number was counted and compared with control. The effect of cells division inhibition was observed. The parameters characterizing of inhibition were LD50 and LDmin. Suppressing of the activity of Escherichia coli has been found at lower concentrations of NPs under aerobic conditions in comparison with anaerobic conditions.
ACKNOWLEDGEMENTS: The work was funded by RFBR according to the research projects № 15-03-02068-a and 15-03-4854-a.

The aim of this study was to enhance the antimicrobial effect of natural extracts against a wide range of microorganisms. Extracts from various spices and herbs before and after encapsulation into liposomes were tested. Extracts were encapsulated individually or as mixtures, especially essential oils and various extracts from different parts of plants were used. The efficiency of encapsulation was determined by spectrophotometry. Also long-term stability of prepared liposome particles and amount of released component in model cosmetics conditions were monitored.
Liposomes were prepared from soybean lecithin using a sonication method. Size of prepared particles was determined by dynamic light scattering and colloid stability of particles was measured using a zeta potential. The antimicrobial activity was tested agains gram-positive (Micrococcus luteus) and gram-negative (Serratia marcescens) bacterial test strains. Also antifungal properties using Candida glabrata were studied. Antioxidant activity of prepared extract and particles was measured too.
It was confirmed that after encapsulation of plant extracts into liposomes significant enhancement of the antimicrobial effect has been achieved. Also during storage in cosmetic gel retain particles their antimicrobial and antioxidant activity. The prepared particles with encapsulated antimicrobial plants extract could thus be used as antimicrobial wound gel formulation.
ACKNOWLEDGEMENTS: This work was supported by project “Materials Research Centre – Sustainability and Development” No. LO1211 (Ministry of Education, Youth and Sport of the Czech Republic).

The compounds of boron and hydrogen, called boranes, have unigue and sometimes unusual properties that make them very stable and resistant. Boranes exist as 3D molecules, so-called clusters, that can be substituted to agents with wide application possibilities. For example, they have found their use in exploratory pharmacological research and may be monitored directly in living cells or organism by various imaging methods [1].
The main purpose of this project focused on the subsequent substitution of cobalt bisdicarba-closo-dodecaborane complexes with proven highly selective antimicrobial activity [2] by another functional group. Various molecular marker groups (e.g. iodine isotopes) with the possibility of their association with other, often used tags (e.g. biotin, fluorescent markers) were used for the secondary substitution of the active substances. The starting compounds were effectively substituted by nucleophilic substituentes on boron atoms or by electrophilic substitution of carbon cluster atoms in diverse reaction rates. The new substances were characterized by modern separation and structure identification analytical methods.
ACKNOWLEDGEMENTS: We would like to thank to project UJEP-SGS-172-07-05 for the financial support.
REFERENCES: [1] Gona K. B., Zaulet A., Gomez-Vallejo V., Teixidor F., Llop J., Vinas C.: Chem. Commun. 50 (2014).; [2] Kvasničková E., Masák J., Čejka J., Maťátková O., Šícha V.: J. Organomet. Chem. 827 (2017).

The synthesis process of composites based on graphene oxide is described and tested as a carrier of zinc oxide nanoparticles and subsequently tested as an antimicrobial agent for some bacterial strains (Staphylococcus aureus (S. aureus), methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli). Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), and confirmed the formation of this composite. The composite was tested by Colony-Forming Capability Test. Graphene oxide as well as zinc oxide exhibit antibacterial properties but the composite consisting of graphene oxide and zinc oxide shows stronger antimicrobial effect against pathogenic microorganisms. Stronger antimicrobial effect was found in S.aureus and MRSA as a G+ bacteria and E.coli as a G- one, due to different composition of cell wall.

Several tricyanofuran disperse dyes were synthesized under conventional and microwave heating. The dyes were prepared directly via Knoevenagel condensation in dry pyridine at room temperature employing acetic acid as catalyst. The reaction was performed between the active methyl-containing tricyanofuran moiety and tertiary amine-based arylaldehyde. The prepared tricyanofuran-based disperse colorants were dyed on polyester fabrics to afford satisfactory results. The susbtituents attached to the tertiary amine moiety influenced the absorption maxima. The fastness properties of the dyed polyester samples were measured. The biological activities of the synthesized dyestuffs against Gram-positive, Gram-negative pathogens were also evaluated.