Metalothionein (MT) is a polypeptide of molecular weight in the range of 6-10 kDa. MT typically contains 60 to 68 amino acid residues. MT is characterized by its unique content of metal ions as well as its sulfur content. Higher MT levels were observed in proliferating cells. This fact demonstrates the importance of MT in the process of cellular regulation (relationship to cancer).
The most widely used drug for patients with breast cancer metastases is an anthracycline antitumor antibiotic doxorubicin (DOX). However, the clinical use of DOX is limited by dose-related heart muscle damage (cardiomyopathy), more prevalent with increasing cumulative doses. For this reason, creation of novel pharmaceutical formulations based on using alternative methods as nanocarriers for targeted drug delivery to tumour cells is a crucial task in modern pharmacology. The aim of this work was to design a nanotechnological construct. The construct is designed as two separate nanotransporters. The nanotransporter (A) is formed by an antibody-modified AgNPs particle and a carbon nanotube with encapsulated DOX (AgNPs / Ab1 / MWCNT / DOX / ODN1). The nanotransporter (B) is engineered with SPION particle modified with antibody and with bound MT (SPION / Ab2 / MT / ODN2). Construct AgNPs/Ab1/MWCNT/DOX/ODN1-SPION/Ab2/MT/ODN2 is formed using an oligonucleotide anchor. Individual parts of the nanotransporter were studied using appropriate methods. The presence of MT was monitored electrochemically by Brdicka method in connection with the transfer technique (AdTSV). Characteristic MT signals RS2CO (-1.15 V), Cat1 (-1.25 V), Cat2 (-1.45 V), Cat3 (-1.75 V) were observed at accumulation time of 120s. SDS PAGE confirmed the presence of MT on SPION nanoparticles at sizes 7 to 15 kDa. The DOX signal was fluorometrically monitored (Em 590 nm, Ex 490 nm). AgNPs sizes ranged from 15 - 20 nm, and the SPION nanoparticles ranged from 20 - 50 nm. Additionally, used AgNPs nanoparticles exhibited significant antiproliferative activity (growth inhibition by 20 - 40 %) on a model culture S. cerevisiae. Created nanoconstruct A showed growth inhibition for S. cerevisiae by more than 50%. The nanoconstruct after these various analysis shows a high potential as an anticancer drug and may be an innovative way how to deal with the breast cancer in a targeted therapy
Given the miRNA-34 family role as tumor suppressor in cancer, it was showed that using miRNA-34a as a new anticancer agent has a great therapeutic potential. Unfortunately, biopharmaceutical issues, rapid degradation in biological fluids, poor uptake into cells and not specific distribution into the body, prevent the use of wild-type non-coding RNA by systemic administration. To overcome these limits, in our previous study we developed a new therapeutic concept based on nanotechnology delivery of miRNA-34a into prostate cancer using fluorescent nanodiamond particles coated with polyethylenimine. In the current study, in order to achieve a more conservative, stable and controlled release of miRNA-34a, we designed a system based on nanodimaonds-miRNA34a complexes encapsulated into nanofibers. We successfully encapsulate nanodiamonds into fibers of polycaprolactione, a biodegradable polymer with a slow degradation rate and a high cellular adhesion, using an electropsinning process. We cultveted prostate cancer cells (PC3 and DU145) on nanofibers scaffolds and we analysed the presence of nanodiamonds in cells using confocal microscopy. Moreover, in these prostate cancer cell lines we observed a replacement of miRNA-34a via real-time PCR and a decreased viability testing the matabolic activity. Our preliminar results suggest a more increased and stable mid-term replacement of oncosuppressor miRNA-34a in prostate cancer when the nanodiamonds-miRNA complexes are delivered encapsulating them in nanofibers. Therefore we provided an effective strategy for cancer therapy ready to be tested for in vivo experiments. ACKNOWLEDGEMENTS: The project was supported by the Ministry of Education, Youth and Sports of the Czech Republic under NPU: LO1309 and LO1508.
SERS has become an attractive analytical tool for recognition of various biomolecules [Guselnikova et al., 2017, doi.org/10.1016/j.colsurfa.2016.12.040]. In this work, we created a universal platform, based on surface modified gold multibranched nanoparticles (AuMs), SPP coupling with localized surface plasmon (LSP), and utilization of artificial neural network for analysis and estimation of SERS spectra, measured on the „normal“ and „tumor“ cells cultivation media. At the first stage, we used the AuMs which have an extremely high number of plasmonic hot spots per single nanoparticle and provide excellent SERS signal intensification [Kalachyova et al., 2017, doi.org/10.1039/C7CP01828B]. The surface of AuMs was decorated with charged organic functional moieties to entrap (bio)molecules from targeted solution [Guselnikova et al., 2018, doi.org/10.1016/j.snb.2018.03.016]. Chemical and morphological structures of the prepared nanoparticles were examined with different techniques such as SEM-EDX and Raman spectroscopy. The decorated AuMs were placed in the cultivation media of melanoma and fibroblast lines in vitro. After interaction with cultivation media the AuMs were drop-deposited on the periodical gold grating, to achieve SPP-LSP coupling and even more enhance the SERS signal from entrapped (bio)molecules [Kalachyova et al., 2017, doi.org/10.1039/C7CP01828B]. Raman spectra were collected from several kinds of melanoma cells lines and further analyzed using ANN with the aim to create the express, unpretentious and reliable methods of melanoma recognition at the early stages of tumor development. We truly believe that our application has a promising practical application for reducing the time and complexity of (bio) analyzing in medical laboratories.
ACKNOWLEDGEMENTS: This work was supported by the Grant Agency of Health Ministry 15-33459A.
Sarcosine oxidase (SOX) is an enzyme, that catalyzes the oxidative demethylation of sarcosine with the glycine as a product and is physiologically active in human body and other mammals. However prostate cancer cells have a high expression of sarcosine. In this study the superparamagnetic iron oxide nanoparticles (SPIONs) were prepared and their surface was modified with gold nanoparticles (AuNPs). These AuNPs were modified with chitosan (CS) and SOX. Obtained AuNPs were characterized by physicochemical methods, such as dynamic light scattering or spectrophotometry, where the pseudo-peroxidase activity of the AuNPs was used. Hydrogen peroxide decomposes because of the pseudo-peroxidase activity with the appearance of a blue coloration of 3,3’,5,5’-tetramethylbenzidine (TMB). For the analysis of AuNPs enzymatic activity the SDS-PAGE with silver staining has been used. Gels (7.5%) were prepared using acrylamide stock solution 30% (m / V) with bisacrylamide 1% (m/V). Separating gel contained: acrylamide 7.5% (m / V), bisacrylamide 0.5% (m/V), 0.4 M Tris/HCl, 0.1% (m/V) sodium dodecyl sulfate (SDS), pH 8.8. Stacking gel contained: 4.5% acrylamide (m/V), 0.15% bisacrylamide (m/V), 0.1% SDS (m / V), 0.1M Tris/HCl, pH 6.8. Nanoconstructs were diluted 2:1 with a loading buffer (PLB Max). Each well contained 15 µl of the diluted solutions. Electrophoretic measuring conditions were: 120 V, 1.5 hours in a running buffer (24mM Tris, 0.2M glycine and 3mM SDS). After measurement the gel was stained with silver, scanned and evaluated by Colortest in the laboratory system Qinslab. The SPIONs or AuNPs cannot be detected themselves alone using SDS-PAGE, therefore this method served as a confirmation, that all parts of the nanoconstruct are connected and we are able to analyze them and use them for other research or possible diagnostic purposes.
Histamine, biologically active amine, is normally present in a body and it is involved in a local regulation of physiological processes. It occurs in food as a product of microbial decarboxylation of amino acid histidine, and consumption of histamine-rich food can lead to intoxication. Hence, its identification, quantification, and awareness of this foodborne toxin are important for food safety as it can serve as an indicator of food spoilage. This study presents a synthesis of various γ-Fe2O3 nanoparticles, which differ in surface functionalization, application of particles for histamine detection and isolation, and subsequent reaction of desorbed histamine with ninhydrin for final ion exchange chromatography quantification. The aim of this study was to develop easy to use, cost-effective, and error-free procedure for quantification of histamine. The applicability in natural complex samples and the analytical parameters of this sensor have been performed in the quantification of histamine in freshwater fish.
In the last years, the nanosized noble metal (gold, silver) became increasingly in the focus of interest in the sensor and medical researches thanks to their unique properties. Because of their outstanding electrical, magnetic and optical properties, they can be the basis of potential biosensors and the intense interest is directed towards the feasible role of gold nanoparticles in medical diagnostics and human therapies [1]. In our research group, we have been paying close attention to the development of biocompatible syntheses of plasmonic Au nanoparticles (NPs) and fluorescence Au nanoclusters (NCs) as well as the characterization of the produced gold/biomolecule hybrid systems: study the relationship between the structure and their optical properties and investigation of their potential utilizations. These different nanostructures can be synthesized depending on the molar ratio of gold precursor and the ligand [2]. Small ligand excess results the formation of plasmonic NPs with diameter larger than 2 nm. In contrast, at high biomolecule excess sub-nanometer sized clusters are formed (d < 2nm). The ultra-small Au NCs show unique physical and chemical properties such as well-defined molecular structure, discrete electronic transitions and characteristic photoluminescence. If the gold NCs consist of only a few-atoms (blue-emitting NCs) the presence of the emission peak depends on the number of metal atoms in the clusters. Furthermore, if the size of the NCs reaches the few nm (red-emitting NCs) both the oxidation state of the surface metal atoms (for example Au0 or AuI state) and the surface ligand effect influence the wavelength of the emission maximum. The luminescent few nanometer Au NPs (the size more than 2 nm) show plasmonic feature, because the collective oscillation of the free electron is occurred. It has to mention that, the plasmonic Au NPs can show characteristic short-time fluorescence, which is depend on the surface roughness or the grain size effect.
The proteins are the well-known reducing and stabilizing agents during the “green” synthesis methods. The production of plasmonic and fluorescent gold-base nanohybrid systems was successful by using the γ-globulin as reducing and stabilizing agent. The several nm sized γG-Au NCs exhibit red emission at 645 nm and show high kinetic stability at physiological conditions. Thus, they may be suitable for selective detection of L-kynurenine (Kyn), which is the one of the intermediate of the physiologically important kynurenine pathway. The detection limit (LOD) of 15 μM and the dynamic range of 15-100 μM were obtained. Beyond the definition of the analytical parameters (KSV, k0, kq) the nature as well as the proposed mechanism of the quenching process was also suggested based on the determinative thermodynamic parameters (ΔG, ΔH°, ΔS°) of the interaction.
ACKNOWLEDGEMENTS: The research was supported by National Research, Development and Innovation Office-NKFIH through project K116323 and GINOP-2.3.2-15-2016-00038.
REFERENCES: [1] D. Ungor, E. Csapó, B. Kismárton, Á. Juhász, I. Dékány, Nucleotide-directed syntheses of gold nanohibrid systems with structure-dependent optical features: Selective fluorescence sensing of Fe3+ ions, Colloids and Surfaces B: Biointerfaces 155, 135-144 (2017), doi: 10.1016/j.colsurfb.2017.04.013; [2] V. Hornok, E. Csapó, N. Varga, D. Ungor, D. Sebők, L. Janovák, G. Laczkó, I. Dékány, Controlled syntheses and structural characterization of plasmonic and red-emitting gold/lysozyme nanohybrid dispersions, Colloid and polymer science 294, 49-58 (2016), doi: 10,1007/s00396-0,15-3781-7.
Infectious diseases are known to be a constant threat to human health also in the beginning of the 21st century. The rapid growth of the human population has led to the agricultural intensification, which has caused an increase in resistant bacterial species in the environment. The aim of the project was to design and verify a theranostic tool combining the diagnostic and therapeutic parts for targeting the bacterial cells. The construct was composed of: A) silver nanoparticles prepared by green synthesis (AgNPsGS); B) antibiotic encapsulated in apoferritin (APO); C) modified magnetic gold nanoparticles (AuNPs). The effect of theranostic nanotransporter on prokaryotic cell culture was model-monitored by altering their growth characteristics (OD monitoring). During the project, 20 various types of AgNPsGS were prepared. The best antibacterial effect (85% growth inhibition of S. aureus) with the minimal inhibitory concentration (MIC50) of 0.4 μg/mL was observed by AgNPsGS4 (Thymus serpyllum L.). SPIONs (20 mg/mL, magnetic AuNPs) were modified with antibody (1 mg/mL IgY) and oxidized graphene sheets (1 mg/mL). The EDC/NHS polymer (2,2 mM EDC, 4 mM NHS, 3 h, 37 °C) was used for binding. The apoferritin (SPIONs/Au/APO) was attached to the SPION/AuNPs. In the SPIONs/Au/APO cavity (12 nm), the antibiotic doxorubicin (2 μM DOXO, the DOXO encapsulation efficiency was around 13% of the applied concentration) was model-sealed. The created SPIONs/Au/AgNPsGS4/APO/DOXO construct was applied to the S. aureus model culture. In the presence of 70 μg of SPIONs/Au/AgNPsGS4/APO/DOXO, a dramatic growth inhibition of S. aureus (95% growth inhibition and 13 mm wide inhibition zone on agar medium) was observed. The biological effect of the nanotransporter lies in ROS (reactive oxygen species) formation, DNA destruction and cell membrane damage.
Breast cancer is the most commonly diagnosed cancer in women. Circulating tumor DNA (ctDNA) is related to breast cancer development and thus, screening of tumors using ctDNA blood detection is highly sensitive and may significantly improve early-stage breast tumor diagnosis. Recently, a nanomaterial-assisted PCR using gold nanoparticles (AuNPs) has been applied to dramatically improve the specificity and sensitivity of PCR, achieving better gene detection. In this study, we aimed to optimize AuNP-assisted PCR, using a new AuNPs-ODN-ThiC3 nanocomposite. Synthesized AuNPs-ODN-ThiC3 nanocomposites, obtained after 60 min of interaction between AuNPs and ODN-ThiC3, were characterized by differential UV/VIS spectroscopy (AuNPs 35 mg/mL), AuNP peroxidase-like activity (0.5 mM TMB, 60 min, 30 cycles), PAGE 25% gel electrophoresis with silver staining visualization (V = 120 mV, I = 150 mA, P = 20 W) and adsorptive stripping voltammetry (HMDE, from start 0 to end -1.75 V, time of accumulation 240 s). AuNP-ODN-ThiC3-assisted PCR was performed using Elizyme HS Robust Polymerase (5 U/µL), denaturation: 95°C/15 s, annealing: 59°C/15 s, extension: 72°C/15 s, 35 cycles. Analysis of AuNPs modification was achieved with ODN-ThiC3 (in different concentrations) and, therefore, new AuNPs-ODN-ThiC3 nanocomposites were obtained. We observed an increased absorbance signal of AuNPs at 527 nm, with the increase of ODN-ThiC3 concentration (at 260 nm) in the AuNPs-ODN-ThiC3 mixture (y = -0.0397 + 0.0734x, r = 0.99). Mixing ODNs-ThiC3 with AuNPs increased the peroxidase-like activity of AuNPs by 17%. LS voltammograms of cytosine and adenine (CA) reduction (the average potential at -1.34 V) showed a linear dependence (r = 0.99) between peak current and concentration of ODN-ThiC3 in the AuNPs-ODN-ThiC3. A linear dependence (r = 0.99) between bands density and ODN-ThiC3 concentration was also achieved. Analysis of PCR protocols: AuNPs-ODN-ThiC3-assisted PCR (AuNPs 814 µg/mL, ODN-ThiC3 4 µg/mL) showed a better yield (140% of conventional PCR and 120% of AuNP-assisted PCR). Conventional PCR can be optimized by integrating ODN-ThiC3 on the AuNP surface during PCR. This improvement represents an innovative PCR method for breast cancer detection.
Ferrous nanoparticles have a great potential as a theranostic tool for diverse medical applications. Our tests have shown, that stem cells which adopt nanograms of ferrous magnetic nanoparticles display sensitivity to magnetic field.
The first set of experiments was focused to attraction of the cells in static water solution. Human and rat mesenchymal stem cells were labelled by fluorescence markers and placed in physiological solution in a sample chamber of a microscope. An external magnetic field of 50 - 300 mT of a magnet or electromagnet was applied. The magnetic field resulted in slow motion of non-adherent cells as visualized by an optical recording system used for precise ultrafast recording of their trajectories.
The second set of experiments was focused on a study of magnetic capture of cells under flow conditions (flow 0.02 - 0.1 cm/s) mimicking real value of blood flow in human capillaries in vivo. When the cells were injected to a linear fluidic system (transparent microtubes), trajectories of cells were modified from linear to parabolic by the magnet or electromagnet. When the force exerted on the cells by the magnetic field overcame the particle hydrodynamic drag force in specific time interval, the cells were able to reach the microtube wall. Further tests quantified limits of magnetic forces which were needed for long time cells adherence on the surface of microtubes with fluidic flow. The parameters depended on the type of surface of microtubes (inorganic plastic versus an organic endothelial layer mimicking a real capillary).
The easy and fast detection of drug content and concentration levels is demanded in biological research as well as in clinical practice. Therefore, label-free platforms based on electrical detection are intensively developed. Nanomaterials and structures are often introduced to provide a fast and sensitive response. Here we study on microscopic level how nanodiamonds and gold nanoparticles interact with drug molecules. We use experimental multifunctional molecule for treatment of Alzheimer disease [J. Med. Chem. 57 (2014) 8576]. The study is performed directly on interdigitated impedance sensor, thus, it is directly comparable with electronic sensor response.
The sensors were made of interdigitated electrodes (IDT, Pt or Au electrode, 15-50 um gap, overall size 5 × 6 mm2) coated by nanodiamonds. Suspensions of detonation nanodiamond particles (hydrogenated H-DND and air-annealed O-DND) were prepared by ultrasonication and centrifugation in water and then drop casted (10 ul) on the active sensor area. Both types of sensors were also further combined with Au colloidal nanoparticles (AuNP, size 20 nm) for providing nanoscale binding sites (spacing 100 nm). The sensors were then functionalized in MTAB (myristyltrimethylammonium bromide) and finally exposed to 1-EN-142 drug in DMSO (10% solution) and rinsed by water and methanol to remove all excess residues. The morphological differences of the prepared sensors were studied by scanning electron microscopy (SEM). Elementary EDX spectra for chlorine, oxygen and gold were measured on a Phenom microscope at 15 kV. Atomic force microscopy (Bruker ICON, CF4 treated Multi75AL cantilevers) was employed to measure local tip-surface adhesion forces and surface topography. Characteristic sensor area of 1x1 um2 was examined at a scan rate of 0.1 Hz and resolution of 512x512 pixels.
The obtained SEM, EDX, and AFM adhesion data show that the drug binds to all nanoparticle materials (H-DND, O-DND, AuNP) be their standalone or combined in a composite, although it binds in different configurations as indicated by different nanoscale adhesion. The effect of drug on nanoscale adhesion is the most evident on the composite H-DND / AuNP sensor, where adhesion increased on AuNP by 4.5 nN (that is by 316%) and on H-DND by 2.5 nN (that is 332%) after the application of drug molecules. Similarly pronounced yet opposite change of adhesion was observed on O-DND based sensors. Contrary, on AuNP-only sensor, adhesion to AuNP increased only by 0.4 nN (that is by 25%). The difference is most likely related to different AuNP charge when interacting with different substrates [J. Nanopart. Res. 16 (2014) 2364]. This is supported by the observation that the presence of H-DND also increased the density of deposited Au nanoparticles compared to other substrates. We discuss implications of these microscopic data for electronic biosensor performance. The work has been supported by the GACR project 17-19968S.
Extracts from naturally occurred sources, such as plants or microorganisms, have been recently tested for their interesting properties. Some kind of plants and microbial cells might be producing bioactive compounds that can be used in food or cosmetic industry. Hydrophobic or/and hydrophilic substances isolated from plants or microbial cells are effectively used to reduce negative influences in human body. Isolated extracts are encapsulated into nanoparticles to increase extracts stability, delay their release and to protect them from degradation due to external conditions. The food and cosmetic industries expect increasingly complex properties from food ingredients, which often would not be able to be achieved without encapsulation.
The aim of this work is to encapsulate microbial compounds, some plant extracts and also model hydrophilic and hydrophobic substances. Chosen components were encapsulated into liposomes.
Particle size and distributions of liposomes were analyzed by dynamic light scattering and their stability by using zeta potential. Spectrophotometric methods were used to measure encapsulation efficiency and to characterize the chosen plant extracts by its amount of total polyphenols, flavonoids and antioxidant activity.
Chosen samples were analyzed for their antimicrobial activity and cytotoxicity. Serratia marcescens, Escherichia coli as gram-negative bacterias, Micrococcus luteus as gram-positive bacteria and fungal strain (Candida glabrata) were used to test antimicrobial activity. Human keratinocytes and mouse melanoma cells were used to cytotoxicity determination.
It has been proved that encapsulation techniques help slowly release the active compounds from plant extracts and transport them to targeted location. Antimicrobial effect was also seen before and after encapsulation into liposomes. Moreover, none of the extracts showed evidence of cytotoxicity.
ACKNOWLEDGEMENTS: This work was also supported by the project “Materials Research Centre at FCH BUT—Sustainability and Development” no. LO1211 of the Ministry of Education, Youth and Sports of the Czech Republic.
At present, great attention is given to silver nanoparticles (AgNPs), which thanks to its unique properties, such as good electric conductivity, photoelectrochemical activity and antimicrobial activity are widely used. Green synthesis of nanoparticles uses biological molecules from living organisms. Biological extracts may contain molecules which exhibit significant antibacterial, antiviral and cytotoxic effects. The aim of this work was to study the diverse AgNPs synthesized from 10 different types of plant extracts. Extracts from dried plants (0.5 g/25 mL 18 MΩ water) were prepared at 70 ° C by heating for 20 minutes. After filtration, the leachates were mixed in the 1:1 ratio with 0.1 M AgNO3 and allowed to stir at room temperature for 18 hours. They were then mixed in the 1:1 ratio with methanol, shaken for 5 minutes on the rotary mixer, and centrifuged at 12,000 g for 30 minutes. After removing the supernatant, the pellet was dried at 60°C for 24 hours. After weighing, the purified AgNPs were dissolved in 18 MΩ of water. AgNPs were yellow, orange and brown. The extraction efficiency was monitored by organic solvents (methanol, ethanol, acetone, propanol). The yields of AgNPs ranged from 15 to 5%, and the most suitable solvent was methanol with an average AgNPs yield of about 10%. AgNPs were characterized spectrally (spectral maxima were in the range of 300-500 nm) by determining the zeta potential and the size of nanoparticles (30-80 nm). Furthermore, antioxidant activity was monitored using ABTS (670 nm), DPPH (517 nm), and FRAP (595 nm) methods. Two methods (ABTS and DPPH) based on the elimination of synthetic radicals and the FRAP method based on the reduction of iron complexes were used to monitor the antioxidant activity. Antioxidation assays were evaluated using calibration curve equations in which the standard was gallic acid. The results for the ABTS and DPPH methods were also expressed as percentage of inhibition of the radicals and in the FRAP method as percentage of reduction activity. The results were calculated using the ABTS method in the range (25.9 - -84.9%), in the DPPH method (19.2-86.6%) and in the FRAP method (8.5 --93.2%). Most AgNPs prepared by green synthesis showed significant antioxidant activity.
Reactive oxygen species (ROS) are important signalling molecules that play crucial role in cellular metabolism. The most prominent is hydrogen peroxide (H2O2), which is under physiological conditions enzymatically converted into water and oxygen. Under pathological conditions, the cell is unable to control this process, which results in oxidative stress leading to damage of cellular structures. Such produced H2O2 is present at a nanomolar concentration and currently there is a lack of suitable electrochemical sensors for its sensitive detection.
This work is focused on a preparation of H2O2 sensor using modification of screen printed electrodes (SPE) with 2D-nanomaterial quantum dots, such as MoS2, MoSe2, WS2 and WSe2 from the family of transition metal dichalkogenides. 2D-nanomaterial quantum dots were produced by liquid phase exfoliation. This technique requires very low amounts of source material (0.1 - 1 g). Nanoparticle shape and size was characterized using atomic force microscopy. Modification of SPE was done by drop casting of the suspension of nanomaterial on a working electrode. To determine the effect of individual 2D-materials, cyclic voltammetry was performed in the environment of fresh solutions of ferro-ferricyanide, phosphate buffer saline and hydrogen peroxide. Data obtained from individual cyclic voltamograms were evaluated and compared with non-modified electrodes. 2D-material based sensors represent a vast potential for employment in next-generation devices due to the unique structural and electronic properties. The high surface area to volume ratio enables the development of sensors and biosensors with potentially exceptional sensitivity.
Composite films based on furcellaran (FUR) and carbon quantum dots (CQDs) has been obtained by casting method. The effects of CQDs films as nanofiller on optical, physical, mechanical, microstructural, and antibacterial properties of furcellaran films were evaluated. The synthesized composites were characterized using Scanning Electron Microscopy (SEM), Fourier-transform infrared spectroscopy (FT-IR) and ultraviolet-visible spectroscopy (UV-Vis). The results from scanning electron microscopy (SEM) confirmed that the composite films have homogeneous and compact structure, with no fractures or cracks. Tensile strength (TS) and elongation at break (EAB) values increased with the increase of CQDs concentration. The UV-Vis results confirmed that composite films exhibited enhancement of photosensitivity. The FUR/CQDs composite films have high potential to be used as antimicrobial films in many applications.
Prostate cancer is one of the most common cancers among men and can in its later stages cause serious medical problems. Due to the limited suitability of current diagnostic biochemical markers, new markers for the detection of prostate cancer are highly demanded. Determination of Prostate-specific antigen (PSA) is widely used for a prostate cancer screening and increases cancer detection by 81% over the use of the digital rectal examination alone (1). PSA can be found in a free form (freePSA) and bonded to proteins. Nowadays performed analysis of the total amount of PSA is limited by its relatively low specificity when serum concentrations are moderately elevated (4.0 to 10.0 ng/mL), and it was shown 75% of consecutively performed biopsies were negative and thus unnecessary (2). Previous reports demonstrated a direct correlation between a percentage of freePSA and prostate volume as well as an indirect correlation between the percentage of freePSA and unfavorable prostate cancer pathology, particularly among men with an elevated PSA (3).
Here, we developed a new multiplex analytical methodology for a determination of PSA and freePSA in a single analytical run. The method is based on magnetic Fe3O4@Ag nanocomposite covalently functionalized using respective AntiPSA antibodies. The nanocomposite allows isolating the PSA from the sample (human plasma) by an application of the external magnetic field. Following spectral analysis using magnetically assisted surface enhanced Raman spectroscopy MA-SERS analysis allows to distinguish between a signal of freePSA and bonded PSA and determine their levels with a LOD = 5 ng.mL.
ACKNOWLEDGEMENTS: The authors gratefully acknowledge support from projects NPU LO1305 of the Ministry of Education, Youth and Sports of the Czech Republic, and project IGA_PrF_2018_021.
REFERENCES: [1] Shahab A. A., Soebadi D. M., Djatisoesanto W., Hardjowijoto S., Soetojo S. and Hakim L. Prostate Int. 1 (2013); 23-30.; [2] Grossklaus D. J., Smith J. A., Shappell S. B., Coffey Ch. S., Chang S. S. and Cookson M. S. Urologic Oncology 7 (2002); 195-198.; [3] Kuriyama M., Uno H., Watanabe H., Yamanaka H., Saito Y. and Shida K. Jpn. J. Clin. Oncol. 29 (1999); 617-622.
Superparamagnetic iron oxides (SPIO) nanoparticles (NPs) are used as negative contrast agents in magnetic resonance imaging (MRI). Moreover, thanks to the amazing magnetic behavior of SPIO particles they can act also as magnetic drug delivery nanocarriers which is highly desirable and challenging field in today’s personalized medicine1. However, controlled synthesis of highly stable, biocompatible, reproducible SPIO NPs in mild conditions is still very rare which is demonstrated by the fact that many commercial contrast agents have been withdrawn from the market due to the problems with effective and reproducible synthesis. Here, we introduce a facile and controlled synthesis of Gd-doped SPIO NPs. We have found that different amount of Gadolinium (5, 10 and 20%) replacing the Fe mols in the beginning of the synthesis influence the final physico-chemical properties of the later products. With increasing amount of Gd ions, the higher amount of organic versus inorganic content is calculated (from TGA analysis) which is mirrored in decreasing of saturation magnetization (from 78Am2/kg for SPIO without Gd to 15Am2/Kg for 20% Gd-doped SPIO). All prepared samples are stable in PBS and biocompatible till high concentrations (500 ug/ml). Only 20% Gd-SPIO reveal IC 50 at 250 ug/ml. Moreover, 10% Gd-SPIO show the most stable properties also in a high ionic strength (1 M of NaCl). This sample was used for mesenchymal stem cells (MSC) labeling. After 24 hours, the robust internalization of Gd-SPIO into the MSC was confirmed by fluorescence optical microscopy and by strong negative signal of MSC in clinical MRI.
Polyelectrolyte-surfactant mixtures are progressive systems studied for their potential use in numerous applications in daily life including delivery systems for pharmaceutics. Mixing polyelectrolytes with oppositely charged surfactant ions in aqueous media typically leads to a strong association providing a simple route for constructing ordered materials with a great scientific and application potential. In this study, the combination of dextran with SDS surfactant was used for the preparation of two types of hydrogels. The diffusion of Nile red and Atto dyes from their solutions in SDS or NaCl into the hydrogels was monitored in time. The time development of the diffusion flows was used for the determination of basic transport characteristics of hydrogels. The diffusion of the probe dyes in prepared hydrogels was compared and discussed.
Hydrogels are ubiquitous materials with three dimensional polymer network in which water is trapped due to its surface tension effect. Recently, there has been an increasing interest in hydrogels, since they can be used in a broad field of applications, e.g., human health and cosmetics (contact lenses, wound healing dressings, and artificial replacement tissues), drug delivery systems, bioengineering etc.
The aim of our work was to assess the distribution and size of pores of agarose hydrogel in dependence on its concentration. Four concentrations were prepared (0.5 %, 1.0 %, 2.0 % and 4.0 % of dry weight content) and a number of methods - both direct and indirect - were executed on dried (rapidly/gradually frozen and subsequently lyophilized) and wet (original) samples in order to measure the pore sizes within the hydrogel structure: mercury porosimetry and SEM (scanning electron microscopy) for dried samples and cryo SEM and turbidimetry for wet samples.
Reasonable results were obtained for the wet samples as they were closer to their native state they are usually used for applications in; the structure of dried samples, especially the gradually frozen ones, was disrupted to some extent as a result of water crystal growth during the freezing procedure. Cryo SEM and turbidimetry provided comparable results of pore diameters, however, turbidimetry was not sufficient enough for assessing differences between individual concentrations, whereas cryo SEM method allowed to compare pore diameters dependant on the concentrations, moreover, it showed more detailed and realistic structure.
ACKNOWLEDGEMENTS: The research was supported by CSF (17-15451S), MEYS CR (LO1212), its infrastructure by MEYS CR and EC (CZ.1.05/2.1.00/01.0017).
Polyelectrolytes are able to interact with oppositely charged surfactants via electrostatic forces. In these highly interacting systems a strong electrostatic attraction between the individual components occurs. After exceeding the critical aggregation concentration (CAC) that is significantly smaller than the critical micellization concentration (CMC) of the surfactant in solution, the aggregates begin to form.
The main aim of this study was to describe the initial formation of the interaction in polyelectrolyte-surfactant system and to identify the state, which precedes the formation of the hydrogel. For this study a cationic polysaccharide diethylaminoethyl-dextran hydrochlorid (DEAED) and anionic surfactant sodium dodecyl sulfate (SDS) were used. The formation of the interactions between DEAED and SDS was studied by the light scattering based techniques using Zetasizer Nano ZS (Malvern Instruments), high resolution ultrasonic spectroscopy (HR-US, Ultrasonic scientific) and densitometry (DSA 5000M, Anton Paar). Moreover, the thermodynamic parameters of aggregation process were tested by micro-calorimetric titrations using a modular microcalorimeter TAM III (TA Instruments).
The aim of this study was to examine the properties and behaviour of phase-separated hydrogels. These can be prepared by interaction of specific biopolymer (sodium hyaluronate - Hya) with oppositely charged surfactant (carbethopendecinium bromide - Septonex). Using the method of rheology, the effect of pH and addition of multivalent ions to the mixture of Hya-Septonex, was studied. Samples underwent both oscillatory and flow measurements. Generally, pH suppressed the elasticity of Hya-Septonex hydrogels by decreasing both the elastic and viscous moduli in comparison to standard pH-uncontrolled samples. Molecular weight of polymer has also direct influence in presence of pH influence. Higher molecular weight of hyaluronan caused the elastic modulus to be higher than viscous even in case of lower pH. However, using the pH around or below the pKa of Hya (pH=3.5) caused that the most of the COO- groups of Hya were not dissociated, thus the gels were not formed. On the other hand, higher pH (pH=9) resulted in the most rigid hydrogels. The influence of multivalent ion environment on the rheological properties was also studied. Three representatives, Ca2+, Mg2+ and Fe3+, were selected for comparison. There was no significant impact on elastic and viscous moduli nor the flow behavior, compared to standard NaCl environment, with exception of Fe3+ ions. These samples evinced the highest values of elastic modulus leading to stiff, almost unmeasurable, hydrogels. The results of this work bring us closer to understand the properties and behaviour of these types of systems and their potential utilization in medical applications.
The presented work was focused on the study of interactions of azo dyes (model diffusion probes) with cationic biopolymer chitosan. The interactions were realized via diffusion processes in hydrogel media based on thermoreversible agarose. The main aim was study of influence of pH of solution on the diffusion process. Interactions of used dyes are based on electrostatic character. The aminogroup of chitosan interacts with the functional group of chosen anionic dyes (sulfonic group) and thus affects the process of diffusion. For this purpose were chosen organic anionic dyes Chicago sky blue 6B (C.I. 24 410), Sirius red (C.I. 35 780) and Reactive blue 49 (C.I. 621 526). Used diffusion technique was based on monitoring of the time progression of diffusion profile by UV-VIS spectrophotometry. The presented work follows the previous experiments and shows comprehensive view of the reactivity of chitosan and its behavior in different systems. Unsteady diffusion in cuvettes appears to be an universal method for the study of reactivity of biopolymers and for the study of transport processes in hydrogel media.
Surface-enhanced Raman spectroscopy (SERS), stemming from the gigantic light energy focusing near the plasmon-active surface, provides the possibility to reveal the presence of targeted analytes under extremely dilute conditions [1,2]. At present, SERS sensors have been making significant progress regarding practical applications in the analytical and bioanalytical fields [3,4]. The main delaying factors in SERS development are related to the poor signal reproducibility. Proposed work is aimed at the achievement of highly-reproducible SERS results. The high sensitivity of SERS has been achieved through the immobilization of sharp-edges bimetallic specific nanoparticles - gold/platinum nanourchins (Au@PtNPs), on the gold grating surface, through the 1,4-benzendithiol (BDT) or 4-sulfobenzenediazonium tosylate (ADT-SH). Gold grating, able to support the surface plasmon polariton (SPP) excitation and propagation, was created on the polymer substrate using the excimer laser modification with further metal deposition. Bimetallic nanoparticles, able to efficiently excite the localized plasmon resonance (LSP), were synthesized using the wet chemical approach. Immobilization of Au@PtNPs on the gold grating allows to achieve SPP-LSP coupling and significantly SERS enhancement, with the maintaining of signal reproducibility, provided by the periodical surface. More detailed experiments show that in the case of ADT-SH, the better Au@PtNPs grafting density and SERS enhancement factor were achieved. Moreover, the produced structure allows SERS measurements with a portable Raman spectrophotometer.
ACKNOWLEDGEMENTS: This work was supported by the Grant Agency of Health Ministry of Czech Republic under the project 15-33459A
REFERENCES: [1] Kalachyova, Y., Mares, D., Jerabek, V., Ulbrich, P., Lapcak, L., Svorcik, V., Lyutakov, O., 2017. Ultrasensitive and reproducible SERS platform of coupled Ag grating with multibranched Au nanoparticles. Phys. Chem. Chem. Phys. 19, 14761-14769. https://doi.org/10.1039/C7CP01828B; [2] Guselnikova, O., Kalachyova, Y., Hrobonova, K., Trusova, M., Barek, J., Postnikov, P., Svorcik, V., Lyutakov, O., 2018. SERS platform for detection of lipids and disease markers prepared using modification of plasmonic-active gold gratings by lipophilic moieties. Sensors and Actuators B: Chemical 265, 182-192. https://doi.org/10.1016/j.snb.2018.03.016; [3] Kalachyova, Y., Erzina, M., Postnikov, P., Svorcik, V., Lyutakov, O., 2018. Flexible SERS substrate for portable Raman analysis of biosamples. Applied Surface Science 458, 95-99. https://doi.org/10.1016/j.apsusc.2018.07.073; [4] Guselnikova, O., Postnikov, P., Erzina, M., Kalachyova, Y., Švorčík, V., Lyutakov, O., 2017. Pretreatment-free selective and reproducible SERS-based detection of heavy metal ions on DTPA functionalized plasmonic platform. Sensors and Actuators B: Chemical 253, 830-838. https://doi.org/10.1016/j.snb.2017.07.018.
A sensitive detection of microorganisms plays an important role in prevention and identification of infectious and deadly diseases. However, conventional methods of bacterial detection are time-consuming, laborious or expensive. A promising way to overcome these problems offers the strategy of molecularly imprinted polymers (MIPs), which use molecular templates to create selective binding sites in cross-linked polymers. By this approach, it is possible to prepare recognition materials for tailored not only according to the target analyte but also according to the detection method available. In this work, the novel sensitive method for Staphylococcus aureus (SA) isolation and detection was investigated. This method utilized the non-covalent molecular imprinting technique employing dopamine as a functional monomer. A layer of molecularly imprinted polymer was created on a surface of magnetic particles (MPs). MPs were chosen due to the ability to pre-concentrate bacteria from large sample volumes and thereby allow for the extremely sensitive detection.
Phytoestrogens are assumed to play an important role in the prevention of tumor or heart disease, and osteoporosis. Soy food is the most significant source of these compounds present as acetylglucosides, malonylglucosides and/or methyl ethers. The most commonly used methods for phytotestrogen determination include chromatographic or electrophoretic separation techniques.
In this work, the benefits of coupling of capillary electrophoresis with molecularly imprinted polymers for analysis of phytoestrogens are shown. Polydopamin imprinted layer formed on the surface of magnetic particles enables efficient extraction/isolation of model compounds (genistein and biochani A) from milk sample and subsequent microcolumn separation with absorbance detection enables to distinguish nonspecifically bound interferents.
Sulfur nanoparticles (S NPs) were prepared by a quick precipitation method in the absence and presence of tetraoctylammonium bromide (TOAB) that was used as a stabilizer to control the nanoparticles size. Scanning electron microscope (SEM), transmission electron microscope (TEM) and X-Ray Diffraction (XRD) were used to characterize the obtained S NPs.
The average size of S NPs was in the range 2.4 - 8.9 nm, with spherical like shape. The NPs size was controlled by changing the preparation parameters: temperatures, different acid concentrations, different acid types and the presence of TOAB stabilizer.
The anticancer activity of the prepared S NPs has been tested on various types of cancer cell clones including leukemia, kidney and colon cancers.The desired S NPs revealed a high promising activity and selectivity toward killing kidney cancer Cells.
Broth micro-dilution method was applied to deter-mine antibacterial activity of S-NPs against Staphy-lococcus aureus, Escherichia coli and Pseudomonas aeruginosa reference strains. S-NPs exhibited antimi-crobial activity (MIC = 5.47µg/ml) against Staphylococcus aureus strain (Gram-positive bacte-rium). On the other hand, no antimicrobial activity was detected against Gram-negative bacteria isolates (Escherichia coli and Pseudomonas aeruginosa) at 0.68 to 800 µg/ml.
PDMS microchamber and microchannels were evaluated in many studies for specific in vitro cultivations. Modern microlitography of PDMS brings new wide posibilities how to prepare transparent planar blocks with micro-channels or isotropic system of linear lamellas. On the other side, the planar nanofiber sheets are another modern entities, which can display well cell adhesion and can be used as semi-transparent in vitro culture substrate.
The PDMS planar blocks with isotropic parallel lamellas (triangle shape, wide 3 microns, distance 4, 10 and 20 microns) were prepared by method of silicon/S1813 microlitography. The final PDMS surface was modified by collagen I solution or fibronectin coating solution. The final topography mimicks of extracellular matrix. Second type of in vitro culture plates, the polycaprolactone nanofiber sheets, was prepared by method of electrospining. The surface was modified also by the fibronectin coating.
The neonatal myocytes and HL-1 cardiac cells were seeded on these two types of plates. The cell adherence, cell viability and final orientation of cells after 24 hours were statistically evaluated. The PDMS plate (with 10 micron distance of lamellas and fibronectine coating) has shown the best ratio of adherent/nonadherent neonatal myocytes and viability of neonatal myocytes (48h). More than 75% of neonatal myocytes adopt parallel orientation with micro-lamellas. Similar ratio of adherent cells and isotropicity were detected also for HL-1 cells. Isotropic in vitro cultures of myocytes should be used as modern model of heart tissue (especiallly optical tracking of excitation) or platfrom for transplantable "cell sheets" generation.
Electrospinning method was used to prepare polycaprolactone/gelatin fibers using polymer solutions of five different gelatin and polycaprolactone ratios dissolved in acetic acid. These materials were then modified with plasma treatment in air at atmospheric pressure. All samples of modified and unmodified nanofiber materials were studied using methods of scanning electron microscopy (SEM), infrared spectroscopy (IR) and energy dispersive x-ray spectroscopy (EDX). Samples underwent a test of hydrolytic stability. According to these results a series of materials were selected for toxicity testing using mesenchymal stem cells (MSC). Results were analyzed using live/dead assay and MTT assay. Acknowledgement: This research has been supported by the project LO1411 (NPU I) funded by Ministry of Education Youth and Sports of Czech Republic.
The work deals with the functionalization of nanofibrous structures for tissue engineering. The purpose of this functionalization is to increase the useful properties of these structures - for example, cell viability or hydrophilicity. Substrates used for functionalization were planar nanofibrous structures from biodegradable polyesters. Bioactive molecules in the form of growth factors based on specific recombinants or human platelet derivatives were used for functionalization. The binding of growth factors will be solved by polyphenolytic coatings. The effectiveness of functionalization was monitored by immunological methods and biological in-vitro tests.
In this work, silica dioxide nanofibers (SiNFs) were prepared by sol-gel method followed by electrospinning to be further functionalized by silylation. To improve the chemical modification, plasma treatment of various parameters was applied and the effect on the surface properties before and after plasma activation was studied. The increase of the amine groups available on the surface after the combined physico-chemical modification, together with the improved hydrophilicity of the so-modified inorganic nanomats, were demonstrated. Finaly, the optimal chosen parameters of the plasma activation were applied and biocompatibility as well as time stability of the physical modification were proven.
Plasma-polymerized organosilicon coatings have been playing an important role in many research studies due to their wide range of applications. These materials can be used in industry as transparent wear resistant layers, protective hard a-Si:C:H or SiO2-like films, corrosion protection coatings, barrier films etc. Plasma-polymerization using organosilicon precursors has been investigated lately for development of unique thin film materials for bioapplications as well, including surface coating of surgical and dental implants to improve their biocompatibility. In the present work, low pressure RF capacitively coupled discharge was used to deposit thin solid films suitable for medical application from mixture of trimethylsilyl acetate (TMSA) and oxygen. Since resistance to liquid environment is the critical parameter that determines usability of the material for bioapplications, the deposited TMSA plasma polymers were exposed to water for 48 hours. The degree of stability varied with deposition parameters as well as other properties of organosilicon plasma polymers. This study summarizes changes of chemical structure and mechanical properties of resulting coatings in dependence on the ratio of TMSA and oxygen flow rates during the deposition process. Results of water stability tests included in present study proved that it is possible to prevent delamination of the coating during exposition to the aqueous environment by appropriate choice of discharge parameters.
In this work function and application of titanium dioxide as a filler in a composite system PCL/ TiO2 was examined. Titanium dioxide was applied by ultrasonic device on already electrospun polymer scaffold treated and non-treated with NaOH.
A procedure for the surface modification of electrospun poly(ε-caprolaktone) (PCL) fibrous scaffold was made to enhance the interaction of the surface with TiO2 particles. The surface modification was performed using NaOH treatment for the formation of carboxyl groups on the fiber surfaces.
Contact angle was measured by goniometer machine to prove change from hydrophobic to hydrophilic polymer surface. After that, SEM photographies were taken. Mechanical properties were tested through differential scanning calorimetry. Moreover, weight of titanium dioxide was measured by termogravimetric analysis.
Nowadays, broad spectrum of materials, including ceramics, metals and polymers, are frequently used in biological applications. Likewise, polymers are advantageously used as matrices for composite materials containing metal nanostructures, in particular, due to their long-term stability and excellent workability. For the preparation of metal nanostructures/polymer composites with improved biological properties (biocompatibility and antibacterial properties), one can (i) incorporate nanostructures into the volume of polymeric matrix or (ii) prepare thin metal coating (nanolayer) on the surface of biocompatible polymer. In this study, we prepared silver and palladium nanolayers (NLs) on the surface of biocompatible polyethylene naphthalate by means of cathode sputtering. Such prepared NLs were transformed into nanoislands (NIs) by low temperature post-deposition annealing to determine the differences between biological properties of these two metal nanostructures. Secondary objective of this study was to compare biological properties of commonly used Ag and novel nanostructures based on Pd. It was found that Pd nanostructures exhibited significantly higher antibacterial effects than Ag ones. Surprisingly, in case of both examined metal composites, increase of the surface roughness by the formation of NIs unfortunately did not lead to improved biological properties.
Polycaprolactone is known as biocompatible material for long time and it is considered as possible material for bioscaffolds. Since natural structure of hard tissues (bones) and most of soft tissues (skin, neural tissue) is fibrous, there are attempts to mimic such a structure.
Polycaprolactone (PCL) nanofibers with diameter of approximately 180 nm were prepared by electrospinning method. For better biocompatibility and bioactivity, a composite fibrous structure was prepared. Hydroxyapatite nanoparticles were synthetized via precipitation reaction and further hydrothermal treatment. The ceramic particles were added to modified polycaprolactone precursor and the solution was electrospun.
The prepared fibers and as well as dried original precursor were in-vitro tested for cytotoxicity by direct contact test. Mouse fibroblast from L929 line were used for cell cultivation. It was found out that fibrous structure and presence of ceramic particles have a positive influence on cell activity and proliferation - the growth rate of the cells was significantly higher compared to bulk polymer precursor.
Platelets are effective source of growth factors and other bioactive molecules which influence cellular fate. They can be combined with an appropriate scaffold where the mechanical properties of scaffold and bioactive properties of platelets modulate cells behavior in the site of the injury. This composite scaffold is a promising tool in tissue engineering applications.
The aim of this study was to compare different concentrations of platelets adhered on PCL nanofibers and evaluate their effect on mouse 3T3 fibroblasts.
Three concentrations of the platelets adhered on nanofibers were evaluated in cells. The concentration of selected mediators was measured. 3T3 fibroblasts were tested in vitro to observe the difference in cellular response to PCL nanofibers and platelets. Cell metabolic activity and proliferation were evaluated. To visualize the cells, the confocal microscopy and SEM was used.
Platelets in maximum concentration significantly stimulate the viability when its compared with control. Proliferation of fibroblasts was significantly higher than in the control group cultured in FBS. Cells were well- spread on the scaffolds with adhered platelets, whereas on plain scaffolds, the cells created clusters. The effect of platelets was dose-dependent. It is obvious, that the combination of bioactive substances and PLC nanofibres significantly stimulates cells growth and metabolic activity of the tested cell type.
ACKNOWLEDGEMENTS: This study has been supported by the Ministry of Education, Youth, and Sports of the Czech Republic, Research Program NPU I:LO1508, Prague Competitiveness CZ.2.16/3.1.00/24006 and Research Program NPU I:LO1508, Prague Competitiveness CZ.2.16/3.1.00/21528, Internal Grant Agency of the Ministry of Health of the Czech Republic No 17-32285A, Ministry of Industry and Trade within TRIO program grant no FV30311
In case of long-term in vivo implant placement the release of ions and wear particles from implants to the body must be limited or blocked. Therefore, to prevent the bone implants failure, the diamond film has been proposed as chemically stable, non-permeable and biocompatible protective coating. The histology and micro-CT study after up to one-year implantation of diamond coated cortical screws showed good osteointegration without any inflammatory reaction. Nevertheless, to investigate the long-term stability of diamond coated screw implants the characterization of its surface after implantation needs to be realized as well.
The self-tapping cortical screw HA 3.5×8 mm implants made of stainless steel and titanium alloy were covered by the thin diamond film in low-temperature low-pressure linear antenna microwave plasma system. Before diamond deposition, selected screws were coated with amorphous silicon interlayer to improve diamond adhesion. The diamond coated screws were implanted in rabbit femur bones for 6 and 12 months. Then the screws were gently extracted from bone tissue and washed by phosphate buffered saline (PBS) and distilled water. The extracted screws were analyzed by optical microscopy, scanning electron microscopy (SEM) and Raman micro-spectroscopy measurements on places exposed to different stress.
The SEM analysis detected microscopically localized delamination of the diamond coating from the screw and presence of non-diamond material on the surface of the implant. The optical images and corresponding Raman spectroscopy maps confirmed partial delamination of diamond from the screw surface. The adhesion of the diamond coating to strained parts of screws may thus not be sufficient for such hard tissue implants, and there is a possible risk of contamination by the material residues in bones. The biological tests also showed good osteointegration. The impact on possible applications in bone implants is discussed.
ACKNOWLEDGEMENTS: This study was supported by the Czech Health Research Council, Ministry of Health of the Czech Republic (grant No. 15-32497A). The financial support of the program for International Mobility of Researchers (CZ.02.2.69/0.0/0.0/16_027/0008465) is appreciated.
Clay minerals are well known to mankind for thousands of years and they have found their place in many applications - ceramics, construction or cosmetics and pharmacy. Clays have layered structure with unique feature to take up ions or organic molecules into the interlayer space. This modified structure acquires new physical and chemical properties and can serve as a selective sorbent or a drug carrier. The electrospinning process enables incorporation of clays and modified clays into polymeric nanofibrous structure. Prepared electrospun materials could be used in many fields - from biomedicine to electrical engineering.
The aim of this study was to prepare self-supporting homogenous nanofibrous layers with a presence of pristine clay minerals and clay minerals containing antibacterial agent chlorhexidine acetate in their interlayer space.
One clayy and two polymers were used in this study. Vermiculite was used as obtained with size fraction < 40 μm. Chlorhexidine/vermiculite was prepared through the intercalation process. Nanofibers were made of hydrophobic polymers, polyurethane (PU) and polycaprolactone (PCL), to gain water stable and durable layers. Polymer solutions for electrospinning contained 2, 5 and 8 wt. % (according to the total weight of the solution) of clay or chlorhexidine/clay. These suspensions were homogenized and immediately spun using 4SPIN LAB. Prepared self-supporting homogenous fibrous layers were further analysed.
Morphology was characterized using scanning electron microscopy (SEM) and presence of clay minerals in the layer was confirmed by EDX mapping. From SEM images, diameter of the fibres and size of clay particles were evaluated.
Wound healing is a complex process which, in some cases, might be disrupted and slowed down by ongoing infection and inflammation. Over the course of time, these processes, if not treated, may lead to life-threatening states. According to the actual wound treatment trends, attention of researchers is dedicated to in situ drug application and number of novel nanoparticulate dosage forms has been developed. Solid lipid nanoparticles (SLN) belonging to semisolid dosage forms group represent an interesting dosage form easy to produce and offering very good encapsulation efficiency for variety of compounds. In this study, we present gentamicin doped SLN for contaminated wound treatment characteristic by low melting temperature enabling sustained drug release at the body conditions over 72 hours. SLN formed by emulsification technique were evaluated particle size distribution, melting point, antibacterial activity against model strains E. Coli and S. Gallinarum and cytocompatibility to Hacat keratinocytes in vitro.
Zinc phosphate particles were prepared by reaction of zinc nitrate with hydrogen phosphate, diphosphate and triphosphate, and characterized by Dynamic Light scattering (DLS) and Scanning Electron Microscopy (SEM). Antibacterial effect of ZnO nanoparticles and zinc phosphates ones were compared. Bacterial strains of (Escherichia coli NCTC 13216, Staphylococcus aureus NCTC 8511, and Methicillin-resistant S. aureus CCM 7110 were used for the testing. Oxidative stress was determined the GSH/GSSG ratio by using High-pressure liquid chromatography with twelve-channel CoulArray electrochemical detector (HPLC-ED). It has been demonstrated the zinc phosphate particles shown better antibacterial properties and smaller toxicity, than the conventional ZnO.
The synthesis of TiO2 nanoparticles (NPs) under various temperature treatments was described and TiO2 NPs was subsequently tested as an antibacterial agent. Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, Dynamic light scattering (DLS) were used to confirm structure of TiO2 NPs and detect differences between individual batches treated with different temperature. Antibacterial properties were tested by Time kill assay on three different bacterial strains: (Staphylococcus aureus (S. aureus), methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli). TiO2 NPs as photocatalyst was incubated with bacterial cells under ambient light. Changes in temperature treatment can affect diameter size and crystal structure of TiO2 NPs as well as its antibacterial properties.
TiO2 is widely used as a material interacting with biomolecules due to its biocompatibility, stability or selectivity (R. Kupcik et al., ACS Applied Materials & Interfaces, 2017). Electrostatic interactions with phosphate groups in phosphopeptides with TiO2 has been widely employed prior to their enrichment (K. Engholm-Keller et al., Proteomics, 2013). Other important biomolecules containing phosphate moieties interacting with TiO2 are nucleic acids. For isolation of biomolecules, an affinity chromatography is widely preferred due to high clearance of impurities, however current materials and techniques have several drawbacks, such as insufficient selectivity. The aim of our work is to demonstrate the unique characteristics of TiO2 nanotubes decorated by Fe3O4 nanoparticles (TiO2NTs@Fe3O4NPs) that provide both higher selectivity for phosphopetides (as compared to commercial particles) and capability to isolate nucleic acids.
For the both applications, cell lysates were used as an initial sample. Phosphopeptides were enriched after tryptic digestion of proteins in the lysate and analyzed using MALDI-MS and nLC-ESI-MS/MS. Nucleic acids were pre-isolated from the lysate, spiked with defined oligonucleotides and analyzed with gel electrophoresis before and after TiO2NTs@Fe3O4NPs isolation. As compared to commonly used TiO2 microspheres, TiO2NTs@Fe3O4NPs showed an enhanced selectivity for phosphopeptides and presence of non-phosphorylated peptides in the eluate was significantly reduced. It was also possible to isolate nucleic acids which means not only their interaction but also their elution. Finally, the material shows magnetic properties applicable for simple and quantitative separation from complex liquid samples.
ACKNOWLEDGEMENTS: This work was supported by OP RDE project with acronym NANOBIO, reg. n. CZ.02.1.01/0.0/0.0/17_048/0007421.
The wear in the pair of friction of the composite elements has a significant effect on the operational life of the hip joint endoprostheses. The structure of the surface layers of the contacting elements plays a key role in the wear process. To improve the tribological characteristics of metal endoprostheses, reinforcing nanostructured coatings are used. In this case, titanium is used as the metal base material, and titanium nitride (TiN) is used as the coating. The structure of the coating is determined by its application modes. Promising is the application of assisted powder coating nitriding method. In pre-clinical studies, computer simulation is used to predict the mechanical behavior of endoprostheses. In the case of a numerical study of endoprostheses of hip joint with coatings, continual methods account for structural features by the homogenization method, i.e. features of the structure are taken into account implicitly and are reflected in the integral characteristics of materials. However, this approach may give an incomplete pattern of the endoprostheses failure. In this paper, we represent a numerical model of the friction pair «head - acetabulum cup» of endoprostheses on the basis of the movable cellular automata method. The main advantage of this method in solving such problems is the possibility of explicit consideration of material discontinuities and fracture processes. The influence of the structure of the coatings on the mechanical behavior of the friction pair of the endoprosthesis is studied.
ACKNOWLEDGEMENTS: The reported study was funded by RFBR according to the research project № 18-38-00323.
The undesirable adhesion of bacteria, cells or proteins to solid surfaces of the medical implants often leads to infection at the implant-tissue interface and causes the severe health problems. So-called anti-biofouling surfaces avoid such non-specific adsorption and thus prevent the biofilm formation. Poly(2 oxazolines) (POx) are a class of synthetic polymers, which among other features (e.g. biocompatibility, water solubility, synthetic versatility) possess anti-biofouling properties and accordingly, they are very promising for this purpose. In our contribution, we investigate the effect of cold atmospheric pressure plasma generated by diffuse coplanar surface barrier discharge (DCSBD) on thin POx-based films. Our main objective is to adjust the appropriate conditions of the plasma treatment to achieve the cross-linking of the POx layer, while the anti-biofouling properties stay maintained. For this purpose, an aqueous solution of the defined statistical copolymer based on POx containing 5 mol% of double bonds in the polymer backbone was deposited in three different concentrations (2, 5 and 10 wt.%) on plasma activated silicon wafers by spin-coating. Achieved POx layers were exposed to cold DCSBD plasma and chemical, and morphological changes were characterized by means of X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), Fourier-transform infrared spectroscopy (FTIR) and profilometry. POx coatings are usually prepared by challenging multi-step grafting; therefore, our new approach would be faster, easier, and economically friendly. Moreover, the cross-linking of prearranged polymers based on POx by plasma treatment, which were not investigated until now represents the new way for the preparation of anti-biofouling surfaces.
Aberrant neuronal growth and inter-neuronal communication underlie the prefrontal cortical (PFC) pathology of many psychiatric diseases (PDs) such as schizophrenia. Current treatments are limited to conventional pharmacotherapy strategies which lead to various side effects.1
Potential therapeutic targets include promoting neuronal communication by enhancing neuronal arborisation, synaptic formation and neural transmission in the central nervous system (CNS). Electrical stimulation is a modern treatment method which can includes electroconvulsive therapy,2 deep-brain stimulation (DBS), epidural stimulation. Stewart et al reported the interaction of human neural stem cells (hNSCs) against electrical stimulation using conducting polymeric films, polypyrrole (PPy). 2-3 Electrical stimulation of PPy(DBS) induced hNSCs to predominantly b-III Tubulin (Tuj1) expressing neurons, with lower induction of glial fibrillary acidic protein (GFAP) expressing glial cells. 4 Organic conductors are an exciting class of materials in which are utilized in microelectrodes due to having excellent mechanical stability, electrical conductivity, biocompatibility, flexibility and ability to fabricate and scale up. 5 Therefore, their integration with biocompatible polymers shows extensive potentials to develop micro electordes that are capable of electrically stimulating of cells with respect to fulfill other requirements (e.g. biocompatibility, flexibility, ability to fabricate).
This work presents the fabrication of three-dimensional flexible organic conductors composed of reduced liquid crystalline graphene oxide (rLCGO) and conducting polymers which hosts neural cells proliferation and development as well as performing the electrical field.
In animal nutrition, the nanotechnology is mostly used for the preparation of nanominerals, especially trace minerals, due to their low bioavailability. These nanominerals also showed advantageous effects at lower doses compared to conventional minerals. In addition, nanominerals can improve digestive efficacy, immunity and overall performance in livestock. In that case, we suggested and synthesized four types of zinc phosphate-based nanoparticles (ZnNPs), which achieved a significant antibacterial activity. After successful in vitro testing, we realized also in vivo study. We investigated the effect of zinc nanoparticles on rats after oral administration. Among others, the influence on total aerobic bacteria and coliforms in rat feces at day 10 or day 30 of treatment was monitored. In general, it has been observed a decrease of total aerobic and coliform bacteria population compared to untreated control group.
Antibacterial nanoparticles have been used and tested over last years due to their unique properties and excessive need for bacteriostatic products in medical care, food industry, clothing etc. Silver nanoparticles, leading group of these nanoparticles in terms of production, were proven to poses not only bacteriostatic but bactericidal character, and also have shown number of negative biological effects and cytotoxicity. Such reasons have been driving power to search for alternative concepts. One of the proposed ways to overcome the negative effects of silver NPs might be composite production combining their antibacterial nature with a convenient biocompatible material. In this study, possibilities of such core-shell nanoparticles were explored. Silver-shielded silica nanoparticles were produced by two-step chemical synthesis, characterized and their antimicrobial activity and biocompatibility were compared to commercial silver NPs (<100nm) used as a standard.
The present work focuses on the preparation and characterization of polysaccharide films based on methylcellulose. Films were doped with silver nanoparticles (AgNPs). AgNPs were prepared and stabilized by reduction of silver nitrate. Polyethylene glycol and glycerol have been used as additives for better material properties of methylcellulose films. Properties of these films were analyzed with a number of analytical techniques. Wettability and water absorption of films were evaluated. Concentration of AgNPs released into the solution during dissolution was studied by atomic absorption spectroscopy. The presence of AgNPs in solutions and solid films was observed by ultraviolet-visible spectroscopy. Solid films were dissolved at different temperatures and release of AgNPs from the films was observed during the dissolution time by ultraviolet-visible spectroscopy. The presence of AgNPs was confirmed both in the solid films and in the solutions by the above mentioned methods. It has been found that additives have changed material properties. Our research was aimed at the use of these films in medicine as a new type of wound dressing with antibacterial properties or as a drug delivery system.
This is a first report for the synthesis of silver nanoparticles using branch extract of Betula pendula - one of the most common trees in Europe, China and southwest Asia. The present study is dealing with ecofriendly green synthesis of silver nanoparticles (AgNPs) using the aqueous extract of branches from B. pendula tree. The reaction mixture contained only the extract (10 or 25% of the total volume of the reaction mixture) from the branches and the source of silver ion without the addition of any catalyst, surfactant or template. The bio-synthesis was carried out at room temperature for 24 hours. The described method is perfectly green, simple, coast-effective and eco-friendly. Reducing substance from birch branches are purely natural and can be obtained simply without damaging the tree. The AgNPs were characterized by UV-vis., DLS and TEM. Surface plasmon spectra for AgNPs are centered at 430-436 nm. The synthesized AgNPs were found to be spherical in shape with size 22.7 ± 6.9 nm and 25.2 ± 7.5 nm, respectively for a reaction mixture containing 10 or 25% of the extract. Biomolecules from branches acted as a reducing agent as well as stabilizer. Common green synthesis uses "green" parts of plants, especially leaves or fruits. It should be noted that in countries with alternating seasons, leaves are not available throughout the year. Branches, on the other hand, are available all year round and their composition is constant regardless of the season.
ACKNOWLEDGEMENTS: The authors gratefully acknowledge the support provided by project LO1305 of the Ministry of Education, Youth and Sports of the Czech Republic.
The biotransformation of silver ions (Ag+) to atomic silver is a green nanotechnology, that meets the need of eco-friendly methods for synthesis of various nanostructures.
In the present study, an extracellular biosynthesis of silver nanoparticles (AgNPs) from AgNO3 solution by using the cell-free extract of Trichoderma reesei biomass was performed. The influence of the medium components, where the biomass of T. reesei was obtained, the initial concentration of AgNO3 solution and the time for Ag+ reduction to AgNPs was studied.
The process of AgNPs formation was monitored by UV-Vis absorption spectroscopy. The isolated nanoparticles were characterized by Transmission Electron Microscopy (TEM) and Inductively-Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES).
In the UV-vis absorption spectra well-defined band at 380 nm was observed, indicating the presence of AgNPs. TEM images of prepared NPs in the reaction mixture with different AgNO3 concentrations, showed almost monodispersed nanoparticles, with spherical shape and size in the range 2-10 nm. The IPC analysis revealed that the AgNPs concentration varies depending on the initial AgNO3 concentration and the different growth media in which the biomass of T. reesei was obtained. Based on the ICP quantitative data the degree of biotransformation of Ag+ to Ag0 at various experimental conditions has been evaluated.
In conclusion, it is worth to mention that the major advantages of extracellular biosynthesis of AgNPs by reduction with a cell-free extract of Trichoderma reesei biomass are the small size (3-5 nm) of the silver particles, their narrow size distribution and limited amount of chemical reagents used.