Seznam abstraktů 2022: POSTEROVÁ SEKCE C

Neurodegenerative disease (ND) is characterised by the gradual loss of nerve cells, which is associated with severe psychological and neurological symptoms. However, treatment options for NO are still insufficient. Dysregulated autophagy has been shown to play a key role in the pathogenesis of NO. Because the exact mechanism and individual signaling pathways of autophagy in NO are not well understood, studying the mechanism of autophagy thus represents a major challenge and hope for effective treatment of NO.
The aim of this work is the controlled induction, accurate detection and description of the molecular mechanism of autophagy in healthy and neurodegenerative cells using 2D materials (GO, rGO and GA), Raman spectroscopy and supporting methods of confocal microscopy and molecular biology. In particular, Raman spectroscopy will be used for the first time ever to study autophagy at the molecular level. This study opens the door to potential therapeutic strategies to combat neurodegenerative diseases through the regulation of autophagy using 2D graphene materials, and its results will clearly expand the knowledge of the mechanism of autophagy and contribute to new treatment options for NO.

Point of care testing (POCT) represents important part of the diagnostics and subsequent treatment of patients. Diagnostic tests performed from the comfort of the patient's home with a smartphone readout enable diagnosis without the laboratory equipment and the presence of a specialized laboratory trained personnel. The tests are designed to be simple and thus allow the patient to do them themselves. They also have the potential for testing in medical facilities due to their small dimensions and lower economic demands than other classical diagnostic methods based on the PCR reaction. The importance of POC approaches increases during the pandemies, like in case of SARS-CoV-2.
In the present work, a microfluidic device, which uses RNA isolated by magnetic nanoparticles with
a specifically modified surface, is developed. Magnetic nanoparticles represent a fast tool for obtaining the RNA of high quality, compatibility with other downstream RNA detection platforms and the possibility of miniaturizing into a chip. For RNA analysis, we have developed a sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay, which utilizes a fluorescence readout with sensitivity and specificity comparable to the RT-qPCR. Its advantage lies primarily in the isothermal profile of the reaction and a shorter time of the whole analysis. The fluorescent detector could be replaced by an electrochemical sensor, which could provide even higher sensitivity of the detection. The difference between both types of detection has been preliminary studied in this work.

Chondroitin sulfate (CS) is one of the main classes of glycosaminoglycans that are present in the extracellular matrix and play a unique role in modulating cellular functions. Due to its biocompatibility and biodegradability, it is considered a promising biomimetic material for drug delivery systems (DDS). In this work, hydrophobically modified CS with two different degrees of substitution via covalent attachment of oleyl groups (CS-OL) were synthesized. The behavior of the obtained polymers in PBS solution was studied using the fluorescence method (determination of the critical aggregation concentration), dynamic light scattering (size and zeta potential of the particles), and cryogenic transmission electron microscopy (morphology of the structures). Subsequently, the possibility of developing drug carriers using CS-OL was tested. Curcumin, a natural polyphenol, was selected as a model hydrophobic drug. The effect of curcumin on the size and morphology of CS-OL structures was investigated. In addition, the ability to accumulate the drug was evaluated by determining the drug loading capacity (DLC) and encapsulation efficiency (EE) parameters for the systems. Drug release studies were carried out to obtain information on the curcumin release profile from the nanostructures. Finally, the stability of curcumin-loaded CS-OL particles was determined over time and under physiological conditions. Our research showed promising results for the potential application of hydrophobically modified CS as curcumin nanocarriers. ACKNOWLEDGEMENTS: The authors thank to the National Science Centre Poland for the financial support (grant nr 2019/35/B/ST5/02147).

The contribution deals with the current topic of hydrogels. These are materials that have long been attracting attention in a wide range of fields - from agriculture to medicine. Depending on the type of hydrogel prepared, different medical applications are also considered. In this study, we focus on the use of chitosan hydrogels in the field of wound dressings. Chitosan stands out for its biocompatibility [1], relatively low cost and the possibility of biodegradation. In this application, they should serve both as wound covers that prevent further mechanical tissue damage and additionally prevent pathogens from penetrating the wound.
In the study, various chitosan hydrogels (different molecular weight, degree of deacetylation, concentrations, etc.) were prepared and their mechanical properties were studied. The possibility of incorporating drugs, commonly used for the treatment of more or less serious wounds (anti-inflammatories and antibiotics), was also studied for successful and fastest possible treatment. Subsequently, the impact on the internal structure of the prepared gels and the diffusion properties of these drugs were studied. The ability to release drugs into the wound over time was determined. The determination is carried out by LC-MS by release into physiological solution at 37ºC after 6, 12, 24, 48 and 72 hours.
ACKNOWLEDGEMENTS: The work has been supported by the University Specific Research, project No. CEITEC VUT/FCH-J-22-7916; Faculty of Chemistry, BUT. REFERENCES: [1] RODRIGUES, Susana, Marita DIONÍSIO, Carmen LÓPEZ a Ana GRENHA. Biocompatibility of Chitosan Carriers with Application in Drug Delivery. Journal of Functional Biomaterials. 2012, 3(3), 615-641. ISSN 2079-4983. Doi:10.31661/gmj.v0i0.1362.

Confocal Raman Microscopy (CRM) is a powerful technique capable of identifying internalized nanoconjugates in live cells without the use of fluorescent labels. Recently emerged family of photothermally active 2D nanoparticles, such as molybdenum oxides MoOx and MXenes, are able to absorb infrared radiation, due to the existence of localized surface plasmons, thereby generating heat and inducing cell death. By attaching an active targeting agent to their surfaces, we create nanoconjugates with selective binding properties. Monoclonal antibody M75 specifically target carbonic anhydrase IX (CA IX) located on the cancer cells surface. CA IX is hypoxia-inducible enzyme involved in the maintenance of pH homeostasis of tumor cells facilitating their survival. Inorganic 2D nanomaterials based bioconjugates are relatively successful in in vitro targeted drug delivery studies. However, to increase the positive outcomes of clinical trials, further research is needed involving advanced imaging techniques that mimic the cells natural environment and functions. Here we show that with the help of label free live cell CRM, we are able to trace the internalization of MoOx M75 and MXene M75 conjugates to specific cell regions in CA IX expressing cells, while comparing different conjugation strategies involving polyethylene glycol and/or aminophosphonic acids linkers. Photothermal therapy mediated via nanomaterials is a promising technique to eradicate cancer due to its spatially highly specific and minimally invasive nature. CRM study of live cells is able to provide spatially resolved biochemical information from living cells and internalized nanoconjugates, providing feedback on the conjugation process. ACKNOWLEDGEMENTS: Supported by APVV-20-0485, VEGA 2/0117/22 and VEGA 2/0029/20.

Specific targeting of tumor cells with high efficiency is still an open question in the field of Theranostics, especially in cancer research. One of the most direct solutions is to use biomarkers specific to a given tumor type. In our research, we are focusing on the targeting of tumor associated carbonic anhydrase IX. As CAIX expression is induced by hypoxia, it enables the targeting of a wide range of solid tumors characterized by development of hypoxic areas. We are proposing a non-conventional method, single molecule force spectroscopy, to evaluate the affinity of CAIX specific antibody (M75) in different cell types. We have tested SMFS with Bruker Multimode8 AFM complemented with liquid cell on a well described antigen-antibody bond. We developed our in-house software (in Labview) for the evaluation of the antigen-antibody bond dissociation and tested it on avidin-biotin bond. The AFM tips were functionalized with biotin, and avidin layer was prepared on Mica substrate. The results of the rupture events were in accordance with published data, and our software was an effective tool in assorting the force curves.

Nanofibrous scaffolds are materials used in medical applications for tissue replacement. It would be a remarkable achievement to fabricate nanofibrous scaffolds directly on a wound (in situ deposition) instead of surgical implantation. Nanofibrous materials are produced mainly by electrospinning. Portable electrospinning devices provide nanofibrous production in vitro using high voltage, which can be dangerous for the human organism. Electrospinning appears as an unsuitable method for in situ deposition of nanofibrous scaffolds. Air-jet spinning (or solution blow spinning) is a method of nanofibrous production using compressed air or gas and seems relatively safe for applying nanofibers directly to wounds. In this work, air-jet spinning of polycaprolactone, poly(L-lactic acid), poly(vinyl alcohol) and poly(ethylene oxide) was performed utilising a commercial airbrush for in situ application of skin wound dressings. The average fibre diameter of air-jet spun fibres was assessed using a scanning electron microscope. Cytotoxicity of fibrous mats and DNA quantification were investigated. Chemical changes in the structure of polymers in fibrous mats were examined by Fourier-transform infrared spectroscopy. The degree of crystallinity was set via differential scanning calorimetry. Finally, the enzymatically catalysed degradation of biodegradable fibrous materials was observed.

Our study is devoted to the development of the nanocomposite gels for biomedical application (e.g., for local treatment of cancer diseases, as an injectable therapeutic scaffold for bone and tissue regeneration). The nanocomposite gels are based on a water-soluble, biocompatible polysaccharide gels (alginic & hyaluronic acids) with embedded complex nanoparticles (NPs) (ultrasmall iron oxide, silver, hydroxyapatite NPs) (hereinafter referred to as gelNPs). Coupling of these types of NPs allowed us to obtain nanomaterials with the combined properties of its components: magnetic, optical, and bioactive [1,2]. Thus, iron oxide NPs provide magnetic properties and can enhance contrast at magnetic resonance imaging (as T2 contrast agent). Calcium phosphate NPs serve as a source of Ca2+ and (PO4)3− ions needed for bone regeneration. Silver NPs are known to reveal bioactive (e.g., antimicrobial, antiviral, antifungal, anticancer) properties and ability to enhance optical signal (e.g., for phototherapy) [3]. Complex NPs were produced using mushroom extracts (Amanita muscaria, Sparassis crispa). Latest research showed that polysaccharides extracted from A. muscaria exhibit significant antitumor activity and are able to reduce the neurogenic pain [4]. S. crispa was reported to exhibit antitumor, antimicrobial, and antifungal properties [5]. The antitumor effect of S. crispa is usually related to fungal glucans. Antimicrobial and antifungal activity of S. crispa is related to active low molecular compounds (e.g., sparassol). A. muscaria and S. crispa extracts were used as a capping agent during the synthesis of NPs; its compounds formed surface layer on the NPs. Surface layer from A. muscaria and S. crispa compounds provided the stability of gelNPs (no precipitation of NPs for several months) and, additionally, biologically active properties. One of the main focuses of this study is the investigation of microstructure of the gelNPs. Our aim was to answer the question: “How would gel microstructure behave in contact with tissue?”. To answer the question, porous materials (pumice and polyurethane sponge) and biological tissue (humerus bones, muscle tissue) were used as a substrate for gelNPs exposition. The substrates with gelNPs were kept in refrigerator (4 °C, 24 h) in humid atmosphere, whereupon examined using cryo-SEM technique. Our results showed that the gelNPs microstructure has an ability to self-reassemble, mimicking the substrate morphology. In particular, exposition on pumice caused reassembly of the nanocomposite gel into a 10-times smaller scale structure (element size decreased from 10 to ≤1 µm). Exposition on a section of humerus bone also induced the gelNPs microstructure transformation: it became spongy with pore size ≤3 µm. Exposition on muscular tissue also forces gelNPs microstructure transformation. Herein, the microstructure scale also decreased: the pore diameter became 1−4 µm (for the tissue across the fibrils) and 3−6 µm (for the tissue along the fibrils). Interestingly, that not each porous substrate caused reassembly of the gel: polyurethane sponge, highly hydrophilic and porous material, did not induce reorganization of microstructure, it only disturbed it. Our results confirm that gelNPs has an ability to self-reassemble, mimicking the substrate morphology. In living organism, it would mimic morphology of surrounding tissue, accelerating formation of extracellular matrix and tissue regeneration.
REFERENCES: [1] O. Ivashchenko, B. Peplińska, L. Przysiecka, E. Coy, M. Jarek, K. Chybczyńska and S. Jurga, ACS Appl. Mater. Interfaces, 2020, 12, 7840-7853.; [2] O. Ivashchenko, Ł. Przysiecka, B. Peplińska, M. Jarek, E. Coy and S. Jurga, Sci. Rep., 2018, 8, 13260.; [3] S. P. Deshmukh, S. M. Patil, S. B. Mullani and S. D. Delekar, Mater. Sci. Eng. C, 2019, 97, 954-965.; [4] T. Kiho, M. Katsurawaga, K. Nagai, S. Ukai and M. Haga, Carbohydr. Res., 1992, 224, 237-243.; [5] L. Thi Nhu Ngoc, Y.-K. Oh, Y.-J. Lee and Y.-C. Lee, Int. J. Mol. Sci., 2018, 19, 1487.

Exosomes (EXs) derived from the plasmatic membranes of cells are important indicators of physiological and pathological processes of the organism as well as individual tissues and cells. EXs can occur in various of body fluids and are significant for transport of informatory and regulatory molecules such as protein, lipids microRNAs or they can function as vectors in pathogenesis. Because of that EXs are considered an additional mechanism for intercellular communication and can fundamentally influence the development of metastases in cancer or the progression of degenerative changes, modulation of immune responses. At the same time, EXs are widely studied for their ability to transport embedded regulatory molecules or drugs and can be considered as an alternative to traditional nanoparticle approaches in drug delivery systems. EXs can cross the blood-brain barrier and are therefore interesting transporters for central nervous system (CNS) therapies. The production and composition of EXs depending on therapeutic approaches is still a little studied area that can provide essential information about intercellular communication during therapy. An unknown topic is also the modulation of EXs after interaction with nanomaterials, the transfer of nanomaterials through EXs, and also the possibility of transporting EXs with nanomaterials to the CNS across the blood-brain barrier. Given the widespread field of nanomedicine and the possibilities that nanomaterials offer, such as targeted drug delivery, minimizing the side effects of drugs themselves, or optimizing drug circulation in the body, the transport of nanomaterials via EXs is an important experimental goal [1-3].
REFERENCES: [1] SAINT-POL, Julien, Fabien GOSSELET, Sophie DUBAN-DEWEER, Gwënaël POTTIEZ a Yannis KARAMANOS. Targeting and Crossing the Blood-Brain Barrier with Extracellular Vesicles. Cells [online]. 2020, 9(4), 1-13. ISSN 20734409. Dostupné z: doi:10.3390/cells9040851; [2] THÉRY, Clotilde, Sebastian AMIGORENA, Graça RAPOSO a Aled CLAYTON. Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids. Current Protocols in Cell Biology [online]. 2006, 30(1), 1-29. ISSN 1934-2500. Dostupné z: doi:10.1002/0471143030.cb0322s30; [3] DRUZHKOVA, T. A. a A. A. YAKOVLEV. Exosome Drug Delivery through the Blood-Brain Barrier: Experimental Approaches and Potential Applications. Neurochemical Journal [online]. 2018, 12(3), 195-204. ISSN 1819-7124. Dostupné z: doi:10.1134/s1819712418030030

This contribution is focused on the encapsulation of erlotinib to liposomes with different properties which are suitable for inhalation drug delivery. Erlotinib is a hydrophobic drug used for the biological treatment of non-small cell lung cancer (NSCLC) in the case of overexpression of epidermal growth factor receptors. The current form of this drug is a film-coated tablet (Tarceva®) for oral administration. Accumulation of erlotinib is not that selective and causes side effects such as skin rash and toxic effects on pulmonary, hepatic and renal systems [1-2]. The liposomal form of erlotinib is a perspective solution for increasing the concentration of erlotinib in tumors and thus reducing its side effects. Moreover, in combination with inhalation as a local type of administration, the efficiency of NSCLC treatment by this drug could be significantly increased.
In this study, the encapsulation efficiency (EE) of erlotinib in liposomes with different types of stabilization (electrostatic, steric and membrane) was estimated. The EE was significantly increased by electrostatic stabilization. The change of physicochemical properties of liposomes after the encapsulation of erlotinib was also studied and it was found that only a few liposomes formula alternated their properties. Liposomes with the highest EE were nebulized by air-jet and vibrating-mesh nebulizers to estimate leakage of the encapsulated drug during the nebulization process. ACKNOWLEDGEMENTS: This work was supported by the University Specific Research, project No. FCH/FSI-J-22-7859. REFERENCES: [1] X. Zhou et al., Drug Des. Devel. Ther., 2017, 12, 1-8.; [2] Ö. Çoban and Z. Değim, Turkish J. Pharm. Sci., 2018.

Tissue engineering and regenerative medicine is an ever-growing multidisciplinary field. Along with the development of tissue carriers, methods for testing the biocompatibility of these materials are also being developed. This work deals with research, development, and optimization of the preparation of a fiber system intended for monitoring the direct interaction of the 3T3 mouse fibroblast cell line with polymeric materials in real-time (live cell imaging). The fabricated fiber model (grid) was seeded with the 3T3 mouse fibroblast cell line and the interaction between the materials and the cells were monitored for 3 days by optical microscope with an incubation chamber. The temperature in the chamber was 37 °C. Every 15 minutes was taken a picture of cells on the fibers. The advantage of this method is the absence of other chemicals like chemicals for visualization cells (fluorescence staining) or chemicals for the measure of cell viability because cells which have adhered to fibers are visible in transmitted light. The results of monitoring the cell interaction with the fiber model (grid) were compared with the testing of cell adhesion and proliferation on compatible fiber layers of biodegradable polyesters produced by DC spinning. On layers were tested cell viability and morphology of cells by fluorescence and scanning electron microscope.

Exosomes are small membrane nanovesicles naturally produced by various cell types, including mammalian, fungal and plant cells. For their specific properties, such as the presence of plasma membrane on their surface, size (usually 30 - 150 nm) and the possibility to transfer various molecules, such as small RNAs or proteins, they are able to enter recipient cells where they can participate in modulation of cell physiological processes.
Although it was not clear for a long time whether plants release exosomes, today we know that it is so. It was shown, plant exosome-like nanovesicles (PELNs) are able to enter cells of different species, including human, where they can modulate pathophysiological processes. PELNs were proven to have therapeutic effects in human diseases, such as cancer, inflammatory or neurodegenerative diseases. Due to their ideal native structure and properties, exosomes appear to be suitable drug delivery vehicles, as they are able to encapsulate the drug, cross the plasma membrane and deliver the drug into the target site without eliciting an immune response in the recipient organism.
Our goal was to determine whether tobacco exosomes are able to enter various cell types, indicating they could be used for drug delivery into human cells. To obtain best results, we compared different plant materials for PELNs isolation, including new sources - tobacco callus and suspension cultures, as well as different methods of exosome isolation. We evaluated biophysical and biochemical properties of plant vesicles, as well as differences between isolates. We show tobacco-derived PELNs successfully enter tobacco and mesenchymal cell lines.

This work is focused on the solubilization of vitamin A and vitamin E into catanionic vesicles, which were formed from hexadecyltrimtehylammonium-dodecyl sulphate (HTMA-DS) ion pair amphiphiles, positively charged double-chained surfactant dioctadecyldimethylammonium chloride (DODAC) and cholesterol. Due to their spherical structure with hydrophilic and hydrophobic domains, some hydrophilic as well as hydrophobic substances can be encapsulated into these domains. Catanionic vesicles may be used as an alternative to liposomes, e.g., such as drug delivery system. In this work, the encapsulation efficiency of two hydrophobic vitamins was monitored using HPLC with a UV-VIS detector. The encapsulation efficiency of vitamin A was determined to 87 % and for vitamin E to 69 % by HPLC measurement. Higher encapsulation values for vitamin A may be due to the smaller molecule of vitamin, which resulted in easier encapsulation into vesicles. And also due to the lower sensitivity of vitamin A to light degradation. In addition, the change in size and stability of vesicles after encapsulation of vitamins by dynamic and electrophoretic light-scattering was also measured. Vesicles without vitamins had the smallest size (around 90 nm). With the addition of vitamin A, the size changed only slightly, but with vitamin E, the size of vesicles increased by up to 37 nm. Despite this, vesicles with/without vitamins were stable, the zeta potential was in the range 60-75 mV. However, during 20 days of measurement, there was a decrease in both size and zeta potential, which is due to the high instability of vitamins in light.

Hydrogels are a very interesting and sought material due to their properties and possible applications in several sectors, namely pharmacy, medicine, agriculture, and many others. For several possible applications, their biocompatibility and biodegradability are essential. The most suitable representatives for these conditions are physically crosslinked hydrogels. Which, they, however, lack especially in terms of mechanical properties. For this reason, such hydrogels are often modified. One possibility of modification is the addition of amphiphilic substances that self-organize inside the hydrogel and modify its properties. Rheology appears to be a suitable method for characterizing mechanical properties, however, measuring and evaluating the results obtained while measuring the hydrogel samples is quite difficult. This work tries to describe various tests on these modified hydrogel materials and their evaluation for appropriate and correct descriptions of hydrogel materials.
The basic test that will tell us a lot about hydrogels is the amplitude deformation test, which will provide us with information about the linear viscoelastic region, the cross-over point, and information needed to set up other tests. The next test is the frequency test, which provides us with information to calculate the network density and mesh size. Relaxation tests are very important, i.e. three interval thixotropy and creep tests. They provide us with information about the regeneration of the hydrogels after stress. Together, these tests will help us characterize the hydrogel material and also be able to recognize the differences between modified hydrogels and hydrogels without modifications.

Agarose is a biocompatible polysaccharide naturally occurring in algae, which in an aqueous environment at proper conditions can form a hydrogel structures. In them, agarose chains are crosslinked via hydrogen bonds and final structure reports a viscoelastic behaviour with storage modulus exceeding loss modulus in the linear viscoelastic region (LVR). Although agarose chains are considered almost uncharged, a small amount of sulphate groups can be present in these molecules.
In proposed work, an influence of various surfactants (cationic, anionic, non-ionic) on the rheological behaviour of agarose hydrogels was studied. Whereas both anionic and non-ionic surfactants at different concentrations did not affect the rheological behaviour, cationic surfactants reported a significant impact both on the absolute values of storage and loss moduli as well as the scope of LVR in broad range of surfactant concentration (0.1 to 200 mM). With decreasing cationic surfactant concentration (cetyltrimethylammonium bromid), crossover point shifted to larger values of strains from 0.8 ± 0.2 % for the reference sample to 37.0 ± 18.3 % for sample with 0.2 mM CTAB solution. To confirm electrostatic interactions of negatively charged sulphate groups with cationic surfactants, measurements with different ionic strength were conducted, since high ionic strength can disrupt these interactions. With increasing sodium chloride content, the crossover point shift to higher strain values was less significant.
It may be assumed, that even a small amount of sulphate groups on the agarose backbone can have a huge impact on the rheology behaviour of agarose gels when interacting with oppositely charged surfactants.

In the past decades, hydrogels have been attracting extensive attention due to their ability to meet diverse needs of biomedical, environmental and personal care application. Moreover these materials can effectively mimic the interfacial, chemical, mechanical, structural and biological functions of native tissues. The application of bulk hydrogel constructs with uniform physical and chemical properties, however, does not fully reflect the in vivo-like situation, since tissues usually display various types of gradients. [1] Thus, there is a growing interest in developing approaches for the design and fabrication of gradient hydrogels that demonstrate direction-dependent properties (pore size, microstructure, stiffness etc.) and could thoroughly mimic
or simulate the particular structures. Despite the already existing wide range of preparation methods of gradient hydrogels, characterization techniques, that could unambiguously confirm the existence of gradient structure, are still being pursued. This work introduces some
of existing preparation and characterization techniques, intended for selected gradient materials,
as well as the main factors affecting the spatial distribution of the final gradient.
REFERENCE: [1] Sauty B. et.al. Enabling Technologies for Obtaining Desired Stiffness Gradients in GelMA Hydrogels Constructs. Macromol. Chem. Phys. 2022, 223(2). ISSN 1022-1352. doi:10.1002/macp.202100326

Protein-nanoparticle conjugates represent a class of hybrid nanomaterials which are object of constant increasing interest due to the opportunity of developing new biological properties, which may significantly differ from parent macromolecules. Herein, a new ternary hybrid was self-assembled by a simple wet reaction, combining a copper-containing enzyme (Bovine Serum Amino Oxidase, BSAO) and magnetic nanoparticles (surface active maghemite nanoparticles, SAMNs), pre-coated with a tannic acid (TA) shell as docking surface for the immobilization of the enzyme. The as-obtained nanobioconjugate (SAMN@TA@BSAO) maintained the enzyme’s catalytic activity toward polyamines oxidation. Noteworthy, the optimum pH shifted from 7.2 for the soluble enzyme to pH 5.0 upon immobilization onto the nanoparticles. The influence of pH on the secondary structure of the functionalized BSAO was assessed by circular dichroism and FTIR spectroscopies. Our findings showed a different pH-related behaviour of the secondary structure of the immobilized enzyme in comparison to the native protein. The former was sensitive to the medium conditions, responding to the increase of acidity with an enhancement of β-sheet to α-helix ratio, whereas the latter was apparently insensitive to pH variations. Aiming at a new solution for cancer treatment, the SAMN@TA@BSAO-catalysed oxidation of polyamines can be ideally exploited for the in situ production of cytotoxic substances (aldehydes and hydrogen peroxide) in tumour tissues, which actually are acidic and amines-rich environments.

Bacteria resistance to antibiotics emerged attention of researchers. New substances with antibacterial activity are constantly being developed. In the last decade, a group of nanostructured antibacterial materials called "nanoantibiotics" has been widely studied. Especially, selenium nanoparticles attracted considerable attention due to their unique antibacterial properties as well as anticancer. Moreover, elemental selenium is an essential trace element important for several major metabolic pathways. Besides SeNPs' excellent biogenic properties, the aggregation and sedimentation stability are one of the significant limitations of their application.
In the present study, two reduction agents were used for SeNPs synthesis, sodium borohydride or ascorbic acid. Also, the influence of several stabilizing agents was investigated, namely PDDA, PEI, sodium oleate, quercetin, and gelatine. The particle size of formatted SeNPs ranges around 100 nm, as revealed by dynamic light scattering and transmission electron microscopy measurements. Most prepared dispersions maintained aggregation and sedimentation stability for more than six months. SeNPs dispersions performed strong antibacterial effects, especially against Gram‑positive bacteria. SeNPs exhibit significant antibacterial efficacy, especially to Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus strains at concentrations as low as 1 mg/l, that has not been published elsewhere. SeNPs synthesized using sodium borohydride showed low cytotoxicity (EC50 > 100 mg/l). Interestingly, SeNPs reduced by ascorbic acid showed much higher cytotoxicity (EC50 6.8 mg/l) against NIH/3T3 cell line.

The nanoporous systems represent a promising tool for biosensing. The ordered nanopores surfaces has been studied for the detection of the affinity interactions (antigen-antibody, aptamer-protein, DNA hybridization) and were able to visualize a direct detection event. From techniques, that create the solid-state nanopores, some require sophisticated equipment and complex facilities (e.g. focused ion beam etching). Simpler way to form nanopores is by anodic oxidation of aluminium, which is frequently used. Another way is creation by packing spherical nanoparticles in a dense planar arrangement. The nanopores are formed as interspatial space between nanoparticles. Thanks to wide variety of material, shape, size, and modification of the surface, the nanoporous system can be prepared according to the needs and create specific design for applications. In this work the polystyrene nanoparticles with diameters 40 and 80 nm were used for formation of nanopores matrix on the electrode surface. They are uniform, nonconductive and it is possible to modified them with diverse functional groups. The diameter of nanoparticles modulated the pore size. The increasement of surface area could provide higher capacity for immobilization of biorecognition molecules. Here, the nanopore-based enzymatic biosensor for the detection of analytes was developed and characterized using electrochemical methods in presence of redox mediator. The biosensor promotes basic knowledge of nanopores sensors and have potential in point of care devices.

The worldwide SARS-CoV-2 pandemic triggered an urgent need for effective sensing and therapeutic solutions to detect, prevent, and treat viral infections efficiently. However, a great number of infectious as well as civilization diseases may be efficiently diagnosed upon detection of specific molecular biomarkers (proteins, nucleic acids, proteins, lipids metabolites, and others). The emergence of the electrochemical methods in this field, and the rapid growth of electrochemical reported biosensors, were possible as these offer very low detection limits, rapid detection duration, and ease of performing the test without additional training. On the other hand, the primary concern in modern electrochemical biosensing platforms lies in their structural complexity, functionalization complexity, as well as usually low selectivity allowing them to be used in particular for screening purposes.
When it comes to impedimetric biosensors, the most commonly used identification approach is the variation of charge transfer resistance upon specific analyte adsorption. However, the detection may also base on other factors, such as adsorption layer capacitance or even disturbance in frequency dispersion of capacitance. Recently, multisinusoidal monitoring with Dynamic Electrochemical Impedance Spectroscopy (DEIS) was proposed by our group for simultaneous tracking of the impedimetric characteristic upon alteration of the polarization conditions. As a result, we obtain the unique fingerprint of the electric parameter changes, dependent on macromolecular charge, size, hydrophilicity, and others. Our approach, combining potentiodynamic DEIS measurement with extended statistical studies, was used for human-specific DEF-B1 aptasensor from saliva and, more recently, allowed us to study and detect subtle changes in the electric properties responsible for the susceptibility of various cellular receptors to SARS-CoV-2. The proposed approach neglects some of the reproducibility issues induced by non-specific adsorption and fouling and reveals the measurement conditions that offer the highest variation of studied parameters upon specific analyte adsorption.

Serotonin is an important neurotransmitter regulating behavioural functions e.g. appetite and feeding, circadian rhythm, or aggression. It is also a very important molecule on the brain-gut axis. Its imbalance is correlated with some psychiatric disorders such as depression, anorexia nervosa, and some anxiety disorders.
In this work, a novel method of surface-enhanced Raman spectroscopy (SERS) detection of serotonin is being invented. Magnetic nanocomposite was chosen as the signal enhancing platform as it allows simple analyte separation and its preconcentration, and it does not cause significant interference in the Raman spectra. The magnetic nanocomposite is composed of a magnetite core, polymeric layer, and immobilized silver nanoparticles. Different polymers were tested, such as chitosan or cellulose. It is not further functionalized with a serotonin selector; this approach relies purely on physical adsorption.
The influence of the used polymer, pH of the solution, excitation laser wavelength, addition of chloride ions, and more were tested to find the best set of conditions for serotonin detection. There are just a few SERS methods capable of detecting serotonin at physiological concentrations. This method aims at future detection of serotonin and dopamine simultaneously from mice brain tissue, which could be advantageous in clinical research of neurotransmitter imbalance.

Controlled release of active compounds has been investigated in current medical research and there were developed many approaches, how to control it. For instance, complexation of active compound, encapsulation in matrix, or release from gels. However, there are still opportunities to improve its mechanisms.
This contribution presents potentially promising method of controlled release, which is based on two modifications of standard mechanisms: i. release from nanostructured layered material, ii. release forced by capillarity. That mechanism allows us to design systems with gradual release of the active substance.
The release of indigotin dye was performed from materials where layers of non-woven chitosan fabric alternate with gelatine layers. Active compound was incorporated into each layer. Gradual wetting of layers is caused by capillarity effect. The controlled release can be observed visually, e.g. by optical spectroscopy. In the future, the colour compound will be substituted by medical active substances, such as antibiotics etc.
The final systems will be composed of natural and biocompatible materials, such as polysaccharides and proteins. The active compound will be in synergy with the chitosan matrix. Chitosan is a unique natural polymer with positive charge on the surface, it exhibits mucoadhesive and antibacterial properties. Gelatine was chosen because it is a protein with a negative charge on its surface, which forms a polyionic mixture with positively charged chitosan.
Such material will improve the current practice in the field of medical materials. It will allow us to design patches treated with an active substance that could improve the therapeutic effect.

Nanofibrous materials are an essential part of research in the field of tissue engineering because their structure resembles the native environment for cells. Research in this area is focusing to improve the in vitro environment for cells even more, for example by using some external stimulus. The stimulus can be electrical stimulation of cells. The literature confirms that electrical stimulation has a positive effect on cell proliferation. It would lead to more realistic imitation of the native conditions in the human organism in vitro. Many current publications focus on the development of a suitable nanofibrous scaffold to provide appropriate cell support during electrical stimulation. Those scaffolds should be biocompatible and conductive. In this work, polycaprolactone nanofibrous scaffolds with incorporated graphene were developed using AC electrospinning. To increase the conductivity, the materials were pressed. Materials were characterized (by scanning electron microscope, fiber diameters and wettability were measured). The 3T3 mouse fibroblast cell line was cultured on the materials for 14 days. Using fluorescence microscopy and scanning electron microscopy, the number of cells and their morphology were evaluated. Nanofibrous materials with incorporated graphene have been evaluated as cytocompatible. ACKNOWLEDGEMENTS: This work was supported by the Student Grant Competition of the Technical University of Liberec under the project No. SGS-2022-6050.

Chitosan is used in a wide range of industries - from agricultural and biotechnological to medical applications. In medicine, chitosan films are often used as wound covers, especially due to their low cost, ease of preparation, mechanical properties, high biocompatibility, easy application and strong adhesion of the film to the wound, while fully adapting to the shape and relief of the wound. Equally important is the film's ability to permeate water vapour and gases, while keeping the wound clean and remaining impermeable to bacteria and other microorganisms. Nevertheless, the possibility of incorporating drugs (e.g. anti-inflammatory drugs and antibiotics) is also important due to their subsequent gradual release into the wound area. The objective of the wound cover application is to accelerate the wound healing process thanks to the mechanical coverage of the wound, but also due to the effects of drugs on potential pathogens that could enter the wound.
This project aimed to prepare different chitosan-based films (various molecular weights, degrees of deacetylation and concentrations of chitosan were used). Subsequently, the mechanical properties of these chitosan films were studied and the possibility of incorporating different medicines and the rate of release of these drugs from the chitosan film by diffusion was investigated. The prepared films were placed in physiological solution at a constant temperature of 37 °C and subsequently sampled at 6; 12; 24; 48 and 72 hours. The experiments were done in triplicate and collected samples were subsequently analyzed by LC-ESI-MS/MS.

Poly(ether-ether-ketone) (PEEK) was introduced as spinal implant artificial bone material in the 1990s because its elastic modulus is closer to that of human cortical bone. However, its biocompatibility needs to be further improved. One of the promising substances that may enhance the biocompatibility of PEEK is strontium (Sr). It is known that Sr has a dual effect of stimulating osteoblast function and inhibiting osteoclast function and can be used for osteoporosis treatment. In this study, the deposition of strontium oxide (SrOx) was conducted by atomic layer deposition (ALD) and plasma-enhanced ALD on PEEK samples. The advantage of these processes is the atomic-level control of the film growth that enables conformal coating and nanometric scale mixing with other materials. However, the deposition temperature of SrOx film had to be limited to maximum 250 °C due to PEEK temperature sensitivity. First, the deposition was optimized on a silicon substrate. Since the ALD process depends significantly on the surface chemistry, it turned out that the deposition on pure PEEK was challenging. Therefore, PEEK was coated with TiO2 ALD or magnetron-sputtered Ti films before the SrOx ALD. The deposited films were analyzed by X-ray photoelectron spectroscopy and atomic force and scanning electron microscopies. The oxide film thicknesses were determined by ellipsometry or reflectometry.

Biodegradable nanofiber materials are widely used for biomedical applications such as tissue engineering. These materials are characterized by submicron fibers diameter, small pore size, and large specific surface area (surface to volume ratio), and this structure is very similar to the natural extracellular matrix and is a favorable environment for the growth of eukaryotic cells. These days research focused on other modifications of nanofiber structures for upgrades these structures or to create specific properties e.g. antimicrobial character. Fibers’ morphology and structure are able modified during their preparation, a suitable method for that is electrospinning technology and post-process modification. Electrospun materials with nanofiber backbone decorated with protruding periodic pattern of polymer crystals are termed nanofiber shish-kebabs. The shish-kebab structure can be formed by post-process recrystallization in suitable liquids (dilute polymer solution, partial solvent, or a mixture of solvent and non-solvent, solvent or partial solvent, or dilute polymer solution evaporation. The crystal period can be controlled to be a few hundreds of nanometres. Partial enzyme-catalyzed degradation of electrospun nanofibers is another method to prepare a similar structure. In this case, amorphous parts of polymer nanofibers are preferentially degraded and the remaining macromolecules form new structures mainly crystal structures resembling nanofiber shish-kebab. The aim of this study was preparation, description, and characterization of mentioned structure.

There is an ongoing interest in the production of antibacterial textiles, i.e. materials capable to inhibit the growth of microbes or completely eliminate them. In this study, we investigate the possibility to produce such materials by means of low-pressure DC magnetron sputtering of silver onto textile substrates (non-woven mask, PEEK and PP mesh). It is shown that this simple and straightforward technique allows for the effective coating of the textiles with a thin Ag layer and assures high antibacterial action of fabricated materials connected with the release of bactericidal Ag+ ions from the coating in contact with water. Furthermore, the combined biological tests and atomic absorption measurements revealed that the amount of released silver and thus also antibacterial action of metalized textiles may be tailored simply by the Ag deposition time.

Nanofibrous materials for medical applications are currently produced mainly by electrospinning. There is a whole range of modifications of electrospinning technology leading to different arrangements of fibers, their shape, and especially surface changes. Morphology change is an essential factor for biomimetic mimicry of the intercellular mass. Mastering the possibilities of structural changes in nano and microfiber materials is important for the repeatability of production and predicting the resulting properties of tissue carriers or wound covers.
Among widely used biodegradable polymer materials are aliphatic polyesters: polycaprolactone (PCL) or a copolymer of caprolactone and lactic acid (PLCL). Many different modifications of electrospinning can be used to change the fiber structure and, therefore, to set optimal, especially morphological, material effects for application, testing, or treatment with these means. Structural or shape changes include not only fiber diameters, their distribution, but also fiber orientation, their interconnection, their smooth or rough surface, and also the significant arrangement of the internal networks of polymer chains represented, for example, by the degree of crystallinity. The process and material parameters of the production of nanofibrous materials must still be studied to ensure the resulting properties of the materials lead to predictable effects on cell adhesion, viability, proliferation, hemocompatibility, or biodegradability.
A general understanding of the effect of five different solvent systems and especially the effect of air humidity (from 20 to 50% RH) during the needleless electrospinning process (NanospiderTM) on the formation of nanofibrous materials from biodegradable polyesters (PCL, PLCL) is evaluated concerning the actual spinning process and the resulting morphology assessed by SEM. The internal chemical structure or arrangement evaluated using especially DSC, FTIR and GPC is necessary to ensure the reproducibility of the production of materials and thus their application results. A detailed description and study of the production of biomaterials is the first step and a necessary basis for subsequent biological evaluations.

Titanium layers doped with biogenic elements attract much attention due to their ability to enhance the biological integration of implant materials. However, the addition of biogenic elements to the coating/layer changes its composition, structure, properties and surface morphology, which affects their application possibilities. Therefore, the research and surface characterization of such layers is needed. In the present work, the mixed titanium layers with calcium and phosphorus have been synthetized by a dual electron beam evaporation method. The effect of doping in these mixed-titanium layers on their surface morphology and mechanical properties have been investigated. Atomic force microscopy was used for morphological characterization and nanoindentation was used to measure the mechanical properties (indentation hardness and reduced modulus). The surface layers are characterized in connection with tuning the deposition conditions and increasing dopant content.

This experimental study aims to assess the effect of selected filler types on the longitudinal thermal expansion of composite systems. The studied issue concerns the dimensional stability or durability of white dental fillings, composite systems of photo polymerizing polymer with particulate fillers of micrometer, and nanometer dimensions, mainly based on silica and zirconia. ChS Epoxy 455 epoxy resin cured with P11 hardener was used as the matrix of the composite systems on which the effect of selected filler types was evaluated. The fillers were particulate and fibrous materials of different chemical compositions, sizes, and combinations. Synthetic silica nanoparticles, cellulose fibers, hybrid fillers derived from modified rice husk, and cellulose nanocrystals were used as fillers. The longitudinal thermal expansion of the prepared composite systems was evaluated in the temperature range of 30 ° to 60 °C, which corresponds to the temperatures to which dental fillings are usually exposed. The selected fillers and the fracture surfaces of samples were analyzed on a scanning electron microscope, and the longitudinal thermal expansion was measured on a thermomechanical analyzer. Based on the dimensions and chemical compositions of fillers used, the quality of the interfacial interface, and the arrangement of the fillers in the polymer matrix, the contribution of these reinforcing elements to the potential influence of the thermal expansion coefficient was evaluated. Given the results obtained, the following experiments will focus on the study of material combinations of particulate and fibrous fillers, the evaluation of the influence of the environment on materials, and machinability.

Over the last years, drug delivery and wound management have undergone a significant evolution as novel nano-based dosage forms and wound dressing pads for active wound management of contaminated and slow-to-heal wounds have been developed. Among all these nanomaterials, nanofibres have attracted attention as potential matrix for drug delivery and wound healing due to their unique properties leading to barrier effect and increased drug bioavailability. In addition to these properties held by nanofibres in general, the chemical nature is a crucial parameter, possibly able to modulate wound healing process through molecules released into the wound site. From this point of view, silica nanofibres represent a very promising material for this application, combining traditional properties of nanofibres and unique properties given by their inorganic nature - such as release of orthosilicic acid during degradation. Another indisputable advantage of silica nanofibres is their easy-to-modify surface available for functionalization and drug conjugation. This parameter may play a significant role due to diversity of bacterial strains related to the contaminated wound development. As one of the outcomes of current progress in novel wound dressings development is need for personalized medicine approach and variability drugs available for conjugation.
In this work, we present results of silica nanofibres-based wound dressing initial screening as potential robust system for antibiotics delivery into the wound site. Interactions of silica nanofibres and five model antibiotics with low water solubility were evaluated in terms of drug quantity, obtained antimicrobial activity and biocompatibility in vitro.