POSTER SESSION C

The present work deals with preparation and subsequent modification of aligned polycaprolactone microfibers made by new semiautomatic drawing technique. This advanced scaffold for tissue engineering combines 3D architecture, high surface to volume ratio, alterable conductivity which closely imitates native extracellular matrix and actively modulating cell functions. A new synthetic approach has been chosen for derivatization of aligned polymer fibers, which lack any surface functional groups for immobilization of biomolecules, grow factors or specific amino acid sequences. To overcome this drawback the thin conductive polypyrrol layer was applied on the polycaprolactone fibers by chemical oxidation from the solution. Thus, in the one simple synthetic step we can modify original fibers with various functional groups presented in the beta-position of the pyrrol. Our future goal is to functionalize microfibrous surface with cyclodextrine units using highly efficient copper free Click reaction. Finally we gain an aligned microfibrous scaffold covered by immobilized cyclodextrine macrocycles that allow direct complexation of proteins during cell cultivation. These nonbonding interactions are relatively weak but the cumulative effect makes them efficient in the complexation of various neutral or ionic molecules. Therefore, better environment for cell adhesion and proliferation is being provided.

Over the last decade, electrospun nanofibres were approved to be an interesting material for many applications thanks to a number of unique properties including enormous specific surface area, high porosity and small pore size. Demands on individual properties and their range vary according to the application field. Materials applied in regenerative medicine, drug and cell delivery are supposed to meet a number of specific requirements. Besides the very basic demands on biocompatibility and non-toxicity, more specific requirements depend on certain application, action period and site. For many applications, degradation period and kinetics play a crucial role. The systems for confluent cell layer delivery may require a bioactive nanostructured surface enabling improved cell adhesion and proliferation combined to optimal degradation kinetics. Silica nanofibres prepared by sol-gel method were confirmed to be biocompatible, non-toxic and biodegradable easily. Their applicability in cell delivery and bioactive coatings effects were confirmed. This study claims to reveal effect of the bioactive coating effect on degradation kinetics and mechanism of silica nanofibres degradation in vitro. The degradation characteristics of uncoated and coated silica nanofibres were tested under certain conditions (37°C, 5% CO2). Progress of degradation over time was investigated using electron microscopy (SEM), mass spectrometry and particle size analysis.
ACKNOWLEDGEMENTS:
Research reported in this paper was supported in part by the Project OP VaVpI (CZ.1.05/2.1.00/01.0005), the National Programme for Sustainability I (LO1201), and the SGS project (21066/115) at the TUL.

Hyaluronan as negatively charged polyelectrolyte can interact with positively charged surfactant micelles via electrostatic interactions to form core-shell like nanoparticles. These aggregates can solubilize hydrophobic active substances and therefore are potential carriers in drug delivery applications. This research was conducted with the aim to prepare nanoparticles consisting of hyaluronan and cationic micelles and evaluate strength of electrostatic interactions between components.
Interactions of hyaluronan and surfactants in aqueous solution were investigated using turbidimetry method and stability of nanoparticles was studied using dialysis technique. Turbidimetric titration was chosen as indicator of the loss of intensity of transmitted light due to the scattering effect of particles associated from hyaluronan and surfactant in it.
Results of turbidimetry revealed that aggregates formation depends on hyaluronan concentration while surfactant concentration (above critical micelle concentration) affects interaction insignificantly. On the basis of turbidimetric results a system for dialysis experiments was selected. Dialysis experiments showed that a part of surfactant molecules is bound to hyaluronan chain and a rest of molecules diffuse to water through dialysis membrane and after a lapse of time the concentrations of the retentate and permeate were balanced. The system can be suitable for the preparation of targeted carriers of biologically active substances.

This paper deals with the fluorescence study of microviscosity changes of hydrogel composed by hyaluronan and cationic surfactant cetyltrimethylammonium bromide during fortnight-ageing. The steady state anisotropy values obtained in the experiments were used to estimate the microviscosity of the corresponding hydrogel regions. Two different fluorescent probes were used, perylene for hydrophobic domains and rhodamine 6G for hydrophilic region. It is observed that the microviscosity in the hydrophobic domains of hydrogel is roughly constant, indicating that these domains does not undergo significant structural changes during ageing. However, the microviscosity of hydrophilic parts gradually decreased, which indicates structural changes in the nearby area of rhodamine molecules.

Nanofibers are valued for their ultra-high specific surface areas and have been found potentially useful in many biomedical applications. Modification or functionalization of nanofibers is necessary in order to engineer specific features that will help maximize their end-use performance. A spectrum of bioactive molecules, including antibacterial agents, anti-cancer drugs, enzymes, proteins, can be incorporated into/onto nanofibers via different approaches1.
We have focused on enzyme immobilization providing many advantages over use of its soluble form: enhanced enzyme activity, reusability, simplified processing, better storage properties and pH stability2.
Here we describe the immobilization of trypsin (EC 3.4.21.4) on silica nanofibers. After silanisation incorporating 72 ± 12.6 µg NH2 per mg of sheets, trypsin was bound using combinatory chemistry of EDAC and sulfo-NHS. The amount (215 ± 9.8 µg protein/cm2) and activity (24.8 ± 2.7 I.U./cm2) of immobilized enzyme as well as storage and operational stability was measured. The proteolytic activity of immobilized trypsin measured immediately after immobilization and during one week was high and stable. Prepared bio-functional nanofiber material can find the application in wound healing process (enzyme debridement).
ACKNOWLEDGEMENTS:
The research was supported by the National Programme for Sustainability I (LO1201) at the Technical University of Liberec and project SGS 2015 (21066/115).
REFERENCES:
[1] Zhong, J Textile Sci Eng 2012, 2:7; [2] Bílková Z. et al. J. Chrom. B 770 (2002) 177-181.

Electrospun nanofibers with their nanoscale structure and the ability to mimic native extracellular matrix (ECM) harbor a potential for biotechnology, biomedicine, and tissue engineering. Inorganic nanofibers, such as silica-based nanofibers, have attracted attention for their physical and chemical stability, surface functionalities and hydrophilic nature.
In this study, we tested electrospun silica-based nanofibers as a scaffold for human bone marrow mesenchymal stem cells (hMSC), cells promising for regenerative medicine. Biodegradable SiO2 nanofibers prepared by sol-gel method and needleless electospinning were tested for cytotoxicity and capability to support cell adhesion.
For cytotoxicity testing, the nanofibers were incubated for 24 or 72 hours in phosphate-buffered saline (PBS) or cell culture media supplemented with serum. The eluate was then applied to hMSC for either 24 hours or 4 days and cytotoxicity and real-time toxicity were evaluated using MTT assay and xCELLigence device, respectively. While eluates from PBS-treated nanofibers showed no impact on the cells, eluates from nanofibers treated with serum-containing media had a negative impact on cell metabolic activity and cell morphology.
The adhesion of hMSC to nanofibers was studied using confocal microscopy. The nanofibers supported the adhesion, however cell survival was limited. Coating the nanofibers with collagen strongly limited the negative impacts.
In conclusion, silica-based nanofibers supported the adhesion of hMSC, however they were not sufficient for extended cultivation and the eluates exhibited negative impact on the viability of the cells. Additional tests will be necessary to clarify the interactions of hMSC with silica nanofibers and to optimize conditions for long-term cell survival.
ACKNOWLEDGEMENTS:
This research was supported by Centre for Nanomaterials, Advanced Technologies and Innovation (CZ.1.05/2.1.00/01.0005), the NPU project (LO1201) and VaVpI MŠMT Pre-Seed project (CZ.1.05/3.1.00/14.0295), UNCE c. 204022.

A new nanofibrous membrane with interesting chemical and strength properties was prepared through the interaction between polyamide nanofibres and oak bark extract, which is rich in the content of hydrolysable tannins.
The aim of this study was to verify the bond strength of tannins and polyamide nanofibres depending on the different pH of the aqueous medium.

This study is focused on development of novel nanofibrous material with immobilized Ag ions. These nanofibers have a great potential for biomedical applications, specifically for antimicrobial wound dressing. The hybrid silica/PVA nanofibers were prepared by electrospinning and thermally stabilized. Consequently, Ag ions were immobilized onto the surface of the nanofibers. It was proved that amount of immobilized Ag ions depends on immobilizing process time. As SEM analysis shown, Ag ions were immobilized onto single fibers in continuous density. According to preliminary antibacterial in-vitro test, the hybrid nanofibers with Ag ions show significant antibacterial activity.

Nanofibers biofunctionalized by enzymes is materials of interest in the biomedical field due to their potential application in wound healing. This work describes the production and characterisation of electrospun chitosan nanofibers biofunctionalized by microbial collagenase. The morphology and microstructure of the chitosan nanofibers prepared by Nanospider technology were examined using a scanning electron microscopy (SEM). Amino groups of chitosan nanofiber were connected with the carboxylic acid groups of collagenase using EDAC and sulfo-NHS methodology. Collagenase enzymatic activity was evaluated by peptidic substrate (Pz-peptide, 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg) in order to estimate the ability of this biomaterial to be used as an enzymatic debriding wound dressing. Characterisation of collagenase chitosan nanofibers in term of apparent Michaelis-Menten constants (KM(app) and vmax) measurements revealed higher enzyme to substrate affinity in comparison with soluble form of collagenase. The resulting novel collagenase chitosan nanofibers showed excellent operational stability and long shelf life for at least 6 weeks. Therefore, the novel collagenase chitosan nanofibers are expected to be a potential scaffold for wound healing applications, as e.g. enzymatic debridement.

A novel core-shell hybrid nanomaterial based on surface active maghemite nanoparticles (SAMNs) and chromate Cr2O72- was developed. The SAMN@Cr2O7 complex was characterized by in field (5 T) Mössbauer spectroscopy at 5 K, which showed the appearance of Fe(II) atoms at the interface between nanoparticles and chromium. Moreover, high-angle annular dark-field imaging (HAADF) and electron energy loss spectroscopy (EELS) for elemental mapping in a scanning transmission electron microscope (STEM) in parallel with energy-dispersive X-ray spectroscopy analysis, mapped chromium distribution on SAMN surface mirroring iron(III) binding sites distribution. Furthermore, electrochemical techniques showed lower charge transfer resistances, higher capacitive current, better electrochemical performances, as well as more reversible electrochemical behavior with respect to bare SAMNs. Chromium shell improved electrocatalytic properties of SAMNs toward hydrogen peroxide electroreduction. Furthermore, an enzyme, namely, bovine serum amino oxidase (BSAO), was immobilized on the surface of SAMN@Cr2O7, leading to an biologically active bio-nano-conjugate for polyamine oxidation (SAMN@Cr2O7-BSAO), which was exploited for the development of a new reagentless electrochemical biosensor for polyamine detection. The system successfully distinguished tumor tissues from healthy liver by polyamine content in human liver extracts from biopsies.

In this paper we computationally investigate a Nanobiosensors behavior based on field effect transistor (FET). The nanoFET sensor comprises Source, Drain, Channel, Insulator layer and aqueous medium that contain ions and target molecules and receptor that cover in channel that absorption target molecules. When receptors absorb target molecules, conductance of channel change. Changing in conductance is factor that we understand absorption occur. Simulations are tools that help us to predicted behavior of sensors. There are kind of computational tools such as quantum and classic methods. One of classic method is scaling-law. In this method the sensor is assumed to be a circuitry network that contains nodes covering sensory receptors. To investigate the sensor response, a number of target molecules are randomly adsorbed by the receptors. Randomization here is translated into the concentration of target molecules in the sensor. In this paper we study the impact of aspect ratio of the sensing channel upon the sensitivity of the sensor. We assume sensing channel is plenary with a length and width that respectively define the number of nodes in length and width of network. It is firstly assumed that all of nodes contain receptors and normalized conductance between each node (∆) is 1. Then receptors are randomly covered with target molecules. Two kinds of sensor behavior are there studied as the first being depletion where the carriers are run out of the region while the second being accumulation where the carrier density increases there. In this study we conclusion that in depletion sensitivity isn`t function of width of sensors but is function of value of conductance between each node. This kind of sensor appropriate for low concentration of target molecules. Accumulation model appropriate for high concentration of target molecules and at this model in long length and wide width, by increasing length, there is not a major shift in sensitivity and increasing in value of conductance lead to increasing of sensitivity.

The aim of the work is to compare the biogenic silica nanoparticles which have been isolated from rice husks with synthetically produced silica brand Cab-O-Sil LM-150. The comparison is based on an evaluation of the two systems from the point of view of chemical composition, particle size and structure, ability to form clusters of particles and interactions with selected bacterial systems. Methods used for comparison were: SEM, EDX, TEM, FTIR, ICPOES and bacteriological tests. Husks are standardly contaminated by accompanying ions that are important for plant growth, but undesirable for obtaining a quality product. Rice husk used for the silica isolation were purified by boiling in 10% HCl for 2 hours. Rice husks were then washed with distilled water until pH 7. The dried and purified husks were burned in an oven at 650oC of temperature rise 10oC/min during 2 hours. The obtained product has a chemical composition analogous to the synthetic product. Biogenic particles reach a size of about 20 nm, about 5 nm synthetic. Both materials have an amorphous structure. Interaction with bacterial systems was performed with Gram-negative bacteria Escherichia coli W3110 strain and Gram-positive bacteria Bacillus cereus. In both cases analogous behaviour was observed depending on the concentration of nanoparticles and reducing the growth rate of cultures of 8% for E. coli and 5% for B. cereus resulted nanoparticles at the concentration of 150 mg/ litter of medium.

Biocompatible and bactericidal coatings were deposited onto different titanium based implant materials. For this the plasma chemical oxidation (PCO) process as well as the atmospheric pressure plasma chemical vapour deposition (APCVD) was used successively. With the PCO technique about 10 µm thick oxide films with high amounts of amorphous calcium phosphate and a defined morphology structure can be deposited onto the titanium implant substrates. It can be shown that the morphological structure and the film composition have a positive influence on the adhesion and the growth of human cells, tested with HaCaT keratinocytes and therefore are suited to improve the biocompatibility compared to pure metals or alloys. With the APCVD thin composite SiOx films can be deposited on the modified titanium substrates. Silver, copper or zinc was used as bactericidal agent, the APCVD film thickness is in the range of less than 150 nm. It could be shown that the bactericidal efficiency of these composite coatings depends slightly on the amount of agent in the films, the agent itself and the used bacteria strains with their specific cell structure. For both of the shown agents Ag and Cu a therapeutic range could be determined, without cytotoxic but bactericidal properties. Beside the description of the coating processes and test methods diverse film properties like structural images as well as the bactericidal and cytotoxic behaviour of the coatings will be presented.

In this study were prepared and compared surfaces of pure polyethylene and polyethylene filled with organo-modified vermiculite. Both polymers were prepared from industrial polyethylene powders. The polyethylene composite were filled with 3% of additive. The additive was vermiculite with chlorhexidine diacetate (CA). CA is used as antibacterial agent in dentist medicine. The infection is major problem of implant surgery. Smoothening of polyethylene surface can result in difficult adherence of bacteria and viruses. Combination of smooth surface with antibacterial effect should to very promising material for implant medicine. Two processes were compared – heat treatment and chemical modification (boiling in hexane). Pure polyethylene shows deep depression and sharp tips formed by preparation process. The heating method creates finer surface and more flat surfaces without sharp tips. On the other hand boiling in hexane create surface with fine roughness resulting in dissolution of polyethylene.
ACKNOWLEDGEMENTS:
This work was supported by the “Nanocomposite materials based on polymers and modified clay minerals with special effects” project (No. SP2015/59) and “IT4 Innovations Centre of Excellence project” (No.CZ1.05/1.1.00/02.0070)

Titanium and its alloys are widely used as hard tissue implants for their high biocompatibility and suitable physical properties. Along with other surface treatment methods, anodization technique is known to enhance osseointegration. The aim of this study is to evaluate the adhesion, growth and osteogenic differentiation of human osteoblast like SAOS-2 cells on Ti 6Al 4V samples anodized in electrolytes composed of a stable volume of KOH (336.48 g/l) and variable volumes of liquid glass (124.47 g/l, 84,12 g/l and 37.38 g/l; samples s5, s6 and s7, respectively). Non anodized Ti 6Al 4V samples and cell culture polystyrene (PS) served as control materials.
On days 2 and 4 after seeding, the cell number did not differ significantly among the tested samples. However, on day 7, the cell number on s6 samples reached the lowest values, which could be attributed to a non homogeneous TiO2 film on s6 samples formed during anodization. Nevertheless, the osteogenic differentiation, estimated by the intensity of fluorescence of collagen I in SAOS 2 cells grown in a differentiation medium, was the highest on s6 samples. On s5 samples, coated with homogeneous TiO2 films, both cell numbers and intensity of fluorescence of collagen I was relatively high. The bone matrix mineralization, evaluated by Alizarin Red staining, was the highest on s5 samples in standard culture medium, and similar on all tested samples in differentiation medium. Thus, the surface modification of s5 samples could be considered the most suitable for application in bone implants.
ACKNOWLEDGEMENTS:
Supported by TACR (TA04011214) and the Ministry of Health of the CR (15 32497A).

Adipose-derived stem cells (ASCs) are of a great importance for bone tissue engineering, and the adipose tissue is a significant source of the stem cells thanks to its abundance [1]. We compared ASCs obtained by liposuction under two pressures (-200 mmHg and -700 mmHg). The number of cells isolated under -700 mmHg (ASC-H) was higher than under -200 mmHg (ASC-L). Five days after seeding, ASCs-H reached a population density of ~63 000 cells/cm2, while ASCs-L only ~40 000 cells/cm2. The adhesion and growth of ASCs were studied on Ti-6Al-4V samples either unmodified (K, Ra=0.28) or modified by shot blasting and tarnishing (A, Ra=0.2), vibratory finishing (B, Ra=0.1) and vibratory finishing, shot blasting and polishing (C, Ra=0.08). All modified samples were more wettable than the K samples. On day 1, the largest cell spreading areas were found on samples A and K (1060-1310 μm2), and the smallest on B (730-820 μm2). At the same time, these areas were larger in ASCs-H than in ASCs-L, and also the growth was faster in ASCs-H. On day 3, ASCs-H reached the highest population density on samples B. On day 7, the numbers of ASCs-H evened out on all modified samples and were higher than on non-modified samples. ASCs-H seems to be more appropriate for faster colonization of bone implants than ASCs-L, and all studied modifications of Ti-6Al-4V enhanced the cell growth.
ACKNOWLEDGEMENTS:
Supported by the TACR (TA04011214) and the Ministry of Health (15-33018A).
REFERENCES:
[1] Zhang Y et al.: Scientific World Journal 2012: 793823, 2012.

SPRi (Surface Plasmon Resonance Imaging) biosensors are used for detection in real time with advantage of processing large number parallel measurements. Our goal is fast detection of low concentrations proteins. A low concentration testing of proteins in urea or blood serum is needed for clinical tests.
This article shows the problems of measuring proteins in real samples of body fluids. The original biochip for detecting albumin (HSA - Human serum albumin) was implemented and tested both by standard tests, and by the patient samples.

In recent years, microfluidics has shown considerable promise for improving diagnostics and biology research. Certain properties of microfluidic technologies, such as rapid sample processing and the precise control of fluids in an assay, have made them attractive candidates to replace traditional experimental approaches [1]. Here, an example of such application is presented: ELISA that uses magnetic particles as the solid phase (MELISA) implemented into a microfluidic device. The microfluidic Rhombic chamber chip (120 μl chamber volume, hydrophilized) in ChipGenie® edition-P holder (microfluidic ChipShop, Jena, D) with strong integrated magnets enabling to fix the magnetic particles in reaction chamber and arrange them as a fluidized bed was employed. The behaviour and colloidal stability of 11 various types of superparamagnetic particles varying in composition and diameter (0.5–100 µm) were compared. Parameters as optimal amount of magnetic particles inside the chamber, rate of aggregation, clogging the chip channels and stability of magnetic cloud inside the chip were evaluated. The microparticles with the best parameters were then coated with specific antigen (chymotrypsin) and traditional MELISA with such bioactive carrier and variously diluted porcine serum was performed in microplate arrangement and simultaneously in microfluidic chip. Only the final step, measurement of sample absorbance was performed off-line in microplate spectrophotometer. Our results repeatedly confirmed that MELISA can be easily adopted for microfluidic version drawing all benefits associated with the miniaturization.

Graphene and its derivatives found their usage in many fields of science due to the exceptional properties. In this work we focus on the theoretical description of the adsorption of three biochemical molecules (ascorbic acid, dopamine and nicotinamide adenine dinucleotide) on the surfaces of graphene and its fluorinated derivatives with various degree of fluorine coverage. Non-local correlation density functional that approximately accounts for dispersion interactions was used for prediction of adsorption geometries and adsorption energies of molecules on considered surfaces. Studied systems were compared and the most suitable surface for each molecule was chosen as recommendation for possible usage in construction of electrochemical sensors.

Nowadays, growing demand of more effective antimicrobial agents is observed in many areas involving food processing and packaging, water cleaning, hygiene or medicine. Therefore, except finding new ones, various ways of improvements of existing agents are searched. One of the most promising ways is the modification of administration (i.e. pure solution, incorporation into composite material, etc.) especially by the docking on suitable matrix because this may help to achieve the controlled release and, therefore, prolonged and more environmentally friendly activity. Wide range of possible matrices varying in effectivity, cost as well as stability, may be used. Natural minerals being a low-cost environmentally stable materials represent reasonable choice.
This work is focused on exploration of capability of zero charged clay minerals kaolinite and pyrophyllite to carry antibacterial chlorhexidine and antifungal nystatin, respectively. Investigation of host-guest interaction was based on force field molecular dynamics which was performed on model surfaces in periodic boundary conditions. Comparison of mean binding energies was used in selection of appropriate matrix and improvement of antibacterial activity was estimated on the basis of available experimental data. Further investigation included calculation of mean square displacements of antimicrobial agents in order to estimate their diffusion over the surface and thus make an estimation of adsorption rate. Finally, differences of guest’s shape were observed on the basis of structural characteristic - radius of gyration.

Bacteria attached to solid surfaces and forming bacterial biofilms present serious complications both in medicine and industry. Developing of anti-adhesive coatings is therefore of great importance nowadays. Nanocrystalline diamond (NCD) is one of the promising materials because of its favourable mechanical and chemical properties. It was also reported to be highly compatible with mammalian tissue cells. However, the knowledge on interactions of this material with bacteria is still rather scarce.
The NCD thin films used in this study were grown by chemical vapour deposition method on silica fused glass substrates. To achieve different wetting properties, the NCD were terminated by hydrogen or oxygen plasma. The NCD films were then characterized by atomic force microscopy, scanning electron microscopy, Raman spectroscopy, X-ray photoelectron spectroscopy and contact angle measurement. AFM measurements confirmed differences in surface roughness between uncoated and NCD coated glass. Oxidized NCD films were hydrophilic compared to hydrogenated ones.
In this contribution, we report slightly higher attachment of gram-negative model bacterium Escherichia coli to NCD surface compared to uncoated glass. Bacteria adhered more to hydrogenated - i.e. more hydrophobic - NCD surface. For attachment experiments, continuous cultivation in commercially available CDC Bioreactor was used. We also present the effect of three different traditional sterilizing methods (autoclaving, dry heat, ultra-violet light) on the wetting properties of NCD films.
ACKNOWLEDGEMENTS:
This work was supported by the GACR project 15-01687S.

The aim of the presented paper was the investigation of the barrier properties of Diamond Like Carbon (DLC) thin films used for biomedical applications. The DLC films were prepared by the Plasma Enhanced Chemical Vapour Deposition (PECVD) from methane. Stainless steel AISI type 316L was used as a substrate. Samples were placed on the excitation RF electrode. The self-bias ranged from -700V to -800V and the deposition time varied from 1 min to 15 min. The working gas methane pressure during process was 20 Pa. Morphology of obtained DLC layers was characterized by AFM and SEM microscopy and the coating thickness by calotest method. The surface chemical composition was identified by EDS analysis. The coated samples as well as the pure substrate were tested for leaching of allergenic nickel into environment. Physiological solution was used as a model matter for this purpose. The content of nickel in the solution was monitored using ICP-OES analysis. The obtained results proved that the barrier effect was achieved even in case of 50 nm thick DLC film.

Use of supercritical fluid (SCF) is fast developing and promising way of submicron particles and nanoparticles preparation. The methods of preparation are relatively cheap and allow many modifications, which give a great potential to using supercritical fluids. CO2 is the most used SCF because of low critical points and non-toxic properties.One of possible methods is rapid expansion of supercritical fluid (RESS). This process is consisted of the saturation of the supercritical fluid with solid substrate followed by the depressurization of the solution through a heated nozzle into a low pressure chamber.
This research introduces results from preparation of submicron particles and nanoparticles of caffeine particles by supercritical CO2 using supercritical rapid expansion of supercritical solution method. Commercial system Spe-ed SFE-4 (Applied Separations, Inc.), which is primarily designed for extractions of organic compounds from solid matrices, was used. The aim of the work was to determine effect of supercritical conditions - pressure and especially temperature on the particle size. The aim was to prepare caffeine submicron particles and nanoparticles in the narowest particle size distribution. The products were characterized with using SEM and particle size distribution analyses.
ACKNOWLEDGEMENTS:
This research has been funded by the Ministry of Education, Youth and Sports of Czech Republic project SP 2015/56 and the European Regional Development Fund in the IT4Innovations Centre of Excellence (CZ.1.05/1.1.00/02.0070).

A number of materials studied in materials research and in bio-applications (polymers, glass, silicates) have a wide range of interesting properties, but their use is often limited to certain applications due to inappropriate surface properties. Particularly for bio-applications are very important surface properties like chemistry, surface charge, morphology and porosity of the materials studied. Therefore, we focus on the possibility of modification of the surface properties so that they are suitable for pre-planned applications. We use a wide range of analytical methods for characterization of the samples before and after modifications. The aim of work is to modify the surface and surface properties of different substrates. Make it suitable for bio-applications, namely to: be useful for adhesion and proliferation of cells, or to exhibit antibacterial effects.
We have modified surfaces of polymers and glass by chemical methods, and we studied the resultant changes of surface properties of the modified substrates. The surfaces of solid substrates were firstly pre-treated by piranha solutions. The activated surfaces were grafted with selected vicinal compounds and then with some boron cluster compounds - highly fluorescent boron hydride cluster anti-B18H22 or its thiolated derivative 4,4’-(HS)2-anti-B18H20.
ACKNOWLEDGEMENTS:
This work was supported by the internal grant of the J. E. Purkinje University in Ústí nad Labem in the Czech Republic (SGA project 5322215001401).

Magnetic nanoparticles (MNP) of magnetite become widespread in biomedical researches and practice during the last decade. Coating of MNP with gold shell reduces their aggregation in colloid solution, decreases their toxicity for living organisms and facilitates functionalization.
Herein, we present the synthesis, purification, functionalization processes and characterization of magnetite-gold nanoparticles with target properties for biomedical application. Magnetite nanoparticles were obtained by co-precipitation and then covered with gold shell by reduction of HAuCl4 with Na citrate or NH2OH*HCl. Obtained nanoparticles were functionalized with sulphur-containing ligands (mercapto-PEG-acid, lipoic acid etc.). As an enzyme for modification a chymotrypsin was chosen.
Magnetite and magnetite-gold nanoparticles were characterized by TEM, XRD, VSM. The formation of «core-shell» structure was proved by HRTEM combined with EDX-analysis. Transverse relaxation rate (R2) was determined to be 250-300 mM-1s-1 for magnetite and 150-300 mM-1s-1 for magnetite-gold that exceeds R2 of commercial MRI-contrasting agents. The effect of alternating magnetic field on catalytic properties of chymotrypsin immobilized on magnetite nanoparticles, notably the slowdown of catalyzed reaction at the level of 30-35 % was found. The most probable reason for it is the change of active centers topology on the enzyme surface as a result of its deformation.
Thereby, obtained systems have promising application in MRI as contrasting agents and also in targeted drug delivery for remote control of enzyme catalytic activity.
ACKNOWLEDGEMENTS:
This work was supported by grants from Ministry of Education and Science of the Russian Federation (grants 11.G34.000 and K1-2014-022), and from Russian Science Foundation (RSF grant 14-13-00731)

We study the surface properties and cell-substrate interactions of amine functionalized carbon nanoparticles (CNPs) grafted on biopolymer film. Poly-L-lactic acid, in form of polymer film, was treated in an inert argon plasma discharge and, subsequently, grafted with functionalized carbon nanoparticles. Selected samples were thermally stressed during or following the grafting procedure. The surface properties were studied using multiple methods (goniometry, X-ray photoelectron spectroscopy). Cell-substrate interactions were determined in vitro by studying adhesion, proliferation and viability of vascular smooth muscle cells from the aorta of a rat. Cell-substrate interactions on pristine and modified substrates were compared to standard tissue culture polystyrene. Our results show that CNPs affect surface morphology and wettability and therefore adhesion, proliferation and viability of cultured cells.

Magnetic nanoparticles offer several advantages for isolation of biological materials. Since they are broadly used in diagnostic immunoassays and in various reactions involving enzymes, proteins, and DNA, this study considers the interaction of four surface modified magnetic microparticles with selected DNA fragments. The magnetic microparticle precursors are prepared by reduction of from iron(III)nitrate and sodium borohydride in ammonia solution. Different surface modifications, named as MAN 37, MAN 127, MAN 158 and MAN 164 were obtained by targeted chemical treatments. The zeta potential and particle size measurements were performed in a zetasizer MALVERN instrument. All considered magnetic microparticles are characterized by positive zeta-potentials, while the DNA fragments in solution show an approx. zeta-potential of -0.22 mV. All the particles were polycharged. MAN 37 and MAN 127 reveal the highest initial zeta potentials. Upon exposure to DNA, MAN 37 and MAN 127 particles lowered their zeta-potential near zero, meanwhile due to the DNA binding, in the case of MAN 164 the zeta potential decreases until -1.25 mV. All particles considered are classified as monodisperse systems. MAN 37 showed the smallest sizes of approx. 0.5 μm and almost no difference in size toward DNA exposure. MEN 158 distinguished for their initial high size of approx. 3.75 μm compared to MAN 127 and MAN 158, possessing sizes of approx. 3 μm each. MAN 164 particles exhibit the biggest change in contact to DNA, from approx. 3.0 μm up to 5 μm when exposed to DNA fragments. All results were confirmed by quantitative real-time PCR.

This work involves the synthesis of magnetite nanoparticles (NPs) functionalized with lauric acid (LA), oleic acid (OA) and lysine (Lys). The synthesis was carried out using a chemical route of co-precipitation. This route allows the production of NPs functionalized using a basic infrastructure, low cost of production. After synthesis of functionalized NPs these were characterized with the techniques: X-ray diffraction (XRD), transmission electron microscopy (TEM), Mössbauer spectroscopy (MS), vibrating sample magnetometry (VSM), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR) and thermogravimetry analysis (TGA). Of the analysis made by the different techniques we concluded that functionalized NPs are of very good quality. Of the VSM measurements we have that the magnetic saturation, for NPs coated with AL, is 60 emu/g. The XRD and TEM measurements show that the NPs have an average size between 9 and 11 nm with spinel crystal structure with lattice parameter of 8.37 Å. XPS measures determined that iron atoms has a valence of +3 and +2, with a total ratio of iron atoms Fe3+:Fe2+ of 2:1. Of the FTIR measurements we show that AL and AO molecules are chemically bound to the surface of the NPs. By TGA measures we calculate the number of functionalized molecules. In the case of NPs coated with AL and AO were 1974 and 1486, respectively.

During the past few years there has been a remarkable growth of research activities exploring properties and biomedical applications of nano-sized materials. Size, shape, size distribution, morphology, surface functionalization and stability of nanomaterials have an influence on resulting biological and other effects of nanomaterials. Due to the unnecessary overuse of antibiotics in the second half of the 20^th century, mankind must face the incidence increase of resistant and/or multiresistant bacterial strains, which may significantly decrease the efficiency of current medical treatment. Nanoparticles or nanomaterials could potentially represent the new medicines because no acquired bacterial resistance after application of nanomaterials has been observed yet. ZnO nanoparticles(NPs) were synthesized by the reaction between zinc chloride and sodium hydroxide. Stevensite/ZnO nanocomposite (SEZN) was prepared by addition of the aqueous mixture of zinc chloride and sodium hydroxide solutions into the aqueous suspension of stevensite. The modification of the standard microdilution method was used for the evaluation of the antibacterial activity of ZnO NPs and composite stevensite/ZnO.
ACKNOWLEDGEMENTS:
The study was created on the Faculty of Metallurgy and Materials Engineering in the Project No. LO1203 "Regional Materials Science and Technology Centre - Feasibility Program" funded by Ministry of Education, Youth and Sports of the Czech Republic and supported by projects reg. No. SP2015/54 SP2015/60 (Ministry of Education, Youth and Sports of the Czech Republic).

One of the most promising type of hybrid nanolayers prepared by sol-gel method are antimicrobial nanolayers based on TMSPM (3-(Trimethoxysilyl)propyl methacrylate) containing ionically bonded cations of silver, copper and zinc. Antimicrobial layer with long-term activity may be applied to the surfaces of almost all materials (metals, glass, ceramics, fabrics, plastics except Teflon). Similar type of nanolayers can be potentially used for different applications, such as organic agent immobilization (drugs, enzymes, etc.) and inert or conversely active interlayer.
The final antimicrobial nanolayer shows significant antimicrobial (against Staphylococcus aureus, MRSA, Escherichia coli, Proteus vulgaris, Acinetobacter baumanii, P. aeruginosa and other pathogenic bacterial strains), antiviral (HIV, H1N1, Dengue, HSV1, hepatitis viruses) and antimycotic (Candida albicans, Candida glabrata) activity. The basic function - keeping the microbial purity of the spaces is active in normal fluorescent light as well as in the dark. When treated surfaces are irradiated with UV-A radiation (sunlight, UV-A fluorescent lamp), the antimicrobial activity increases significantly. Elimination of almost all bacteria and viruses on the surface is achieved in a few tens of minutes.
The technology is applicable in medical institutions (operating theaters, intensive care units, transplant and neonatal units, offices, waiting rooms, hospices etc.), in the social sphere (schools, cafeterias, senior homes and other types of social institutions) or food (food processing plants, warehouses and stores). The long-term activity is also demonstrated by the preservation of antimicrobial activity on the fabrics after 50 washing cycles.
Developed antibacterial surface treatment technology is patented in the Czech Republic as well as abroad.

Formation of specific non-covalent complexes among nucleic acids and proteins seems to be essential for regulation of cellular processes and biochemical pathways. On the molecular level, the interactions between DNA and proteins are playing crucial role in the control of DNA replication, transcription, translation, and also repair of damaged DNA. Thus, detailed characterization of DNA-protein interactions improves the knowledge about abnormal cells and provides a better understanding of tumor growth , its prevention and medical treatment.
The interaction between ssDNA and the ssDNA-binding protein SSB was analyzed using atomic force microscopy (NanoWizzard3) providing images of the formed complexes on mica. Furthermore, this was complemented by determination of binding forces using atomic force spectroscopy with SSB-modified cantilever tip. The interaction was also characterized using the surface plasmon resonance based real-time bioanalysis (Biacore) providing reference data on kinetics of the interaction. In all cases, the direct label-free approach was conveniently employed and the disturbing labelling was not required.

Biometrology is currently rapidly evolving part of metrology. The need of accurate and standardized measurements is urgent in various fields of biology, especially in laboratory diagnostic in healthcare and in the pharmaceutical industry. Many of the metrological institutes worldwide are trying to achieve the similar results through pilot studies and key comparisons focused on metrology of nucleic acids and proteins, however the variability of living world is still a few steps ahead this attempts.

The presented study was focused on preparation, characterization and application of liposomes as transport systems for active components. Liposomes were prepared from mixture of egg/soy lecithin and cholesterol by sonication and thin layer evaporation methods. As active components complex natural sources of caffeine and fruit extracts were used individually as well as in mixtures.
The efficiency of encapsulation was determined by HPLC/PDA (individual phenolics, caffeine, vitamin C) and by spectrophotometry (phenolics). Long-term stability of particles and amount of released component in model/real foods, in model cosmetic conditions and in a model physiological environment were monitored too. Size of prepared liposomes was determined by dynamic light scattering. Stability of the particles was measured using a zeta potential. All types of prepared liposomes exhibited good stability. Liposomes showed a very good long-term stability mainly in water conditions with neutral pH, too. Liposomes with co-encapsulated active components exhibited relatively high encapsulation efficiency and better stability than liposomes with one type of active component.
Liposome as delivery systems for caffeine enables enhancing of active component permeation through the skin and controlled release. Particles also maintained their integrity during passage through the gastrointestinal tract until they reached their target destination. Prepared particles with encapsulated caffeine as well as other tested antioxidants and vitamins could be used to modern cosmetics products and also to food and beverage industry as active components of energy drinks and food supplements.
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).