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Surface functionalization of the biological gold nanoparticles for micro-rna targeting
Pourali, Parastoo ; Benada, Oldřich ; Benson, Veronika
Among non-viral gene carriers with low toxicity and high transfection efficiency, the use of gold nanoparticles (AuNPs) is of particular interest due to their biocompatibility and special properties. This is the first time we attempted to functionalize the surface of the biological AuNPs in order to conjugate them with antimiR-135b through electrostatic interactions and knockdown the microRNA-135b gene expression inside the cells. A fungal strain, Fusarium oxysporum, was cultured in Sabouraud Dextrose Broth (SDB), centrifuged, and the mycelium-free supernatant was challenged with 1 mmol final concentration of HAuCl4.3H2O and incubated for 24 h at 37°C in a shake flask. AuNPs were characterized by visible spectrophotometry, Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), Energy-Dispersive X-ray spectroscopy (EDS), and a zetasizer. The washed and sterilized AuNPs were used for cytotoxicity and conjugation assays. First transferrin (Tf) and then polyethylenimine (PEI) were used to functionalize and change the surface charge of the AuNPs and then antimiR-135b was conjugated to the AuNPs trough electrostatic interactions. Their association was confirmed by visible spectrophotometry and electrophoresis. Confocal microscopy was used to investigate the internalization of the AuNPs-antimiR-135b complex. The results proved the formation of AuNPs with a maximum absorption peak at 528 nm, round and oval shapes (15-20 nm), and average zeta potential of -21.02 mV. The AuNPs-antimiR-135b showed delayed electrophoresis unlike antimiR-135b or AuNPs alone. Functionalized AuNPs did not cause any toxicity in cell culture and confocal microscopy showed successful transfection of AuNPs-antimiR-135b into the vast majority of 4T1 cells. We concluded that the biological AuNPs were non-toxic and they could carry antimiR-135b to enable gene silencing
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Mineralised polylactide and polycaprolactone soft foams with hierarchical micro-macro porous structure for tissue engineering
Beran, M. ; Berková, E. ; Musílková, Jana ; Sedlář, Antonín ; Slepička, P. ; Fajstavr, D.
The purpose of the study was development of soft foams from resorbable polymers with unique micro-macro porous interconnected hierarchical structure specially designed as scaffold for engineering of soft tissues. The foams have been prepared by freeze-drying of solutions of polylactide (PLA) and polycaprolactone (PCL) in 1,4-dioxane. The foams prepared by freeze-drying had interconnected porous aerogel characteristics. The hierarchical structure with bimodal micro-macro pore size distribution were obtained after addition of sucrose or NaCl porogens with defined crystal size distributions to the solutions before freeze-drying and leaching the porogen crystals from the freeze-dried foams with demineralised water. Polyethyleneimine was chemically conjugated to the alkali-treated foams followed by conjugation of citric acid using carbodiimide chemistry. Finally, they were mineralised by immersing and incubating in a simulated body fluid with ionic concentration similar to that of human blood plasma, to obtain tissue engineering scaffolds. To verify their biocompatibility, the scaffolds were seeded with adipose-derived stem cells (ASC) and sarcoma osteogenic-2 (SaOs-2) human osteoblast-like cells. Morphology of the cells attached to the scaffolds was evaluated and their viability was verified by a metabolic test. Biocompatibility and usability of the scaffolds was successfully verified by incubation with adipose-derived stem cells and SaOs-2 human osteosarcoma cell line. Mineralised scaffolds are more suitable growth supports for both the cell types than unmineralized collagen scaffolds. The scaffolds have been specially designed for engineering of soft tissues, but they can be used in other categories of tissue engineering, too.
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The co-culture of keratinocytes and fibroblasts on a multi-layered polyester nanofibrous membrane enriched with platelet lysate
Blanquer, Andreu ; Filová, Elena ; Jenčová, V. ; Brož, Antonín ; Kuželová Košťáková, E. ; Lisnenko, M. ; Procházková, R. ; Bačáková, Lucie
The prevalence of chronic wounds is increasing due to the population ageing and specific illnesses like diabetes mellitus and vascular diseases. Nanofibrous membranes fabricated using synthetic polymers are promising materials to enhance skin wound healing. PCL and PVA membranes are being studied to be used as scaffolds for skin tissue engineering and hydrogels for controlled drug delivery, respectively. The present study considers the development of a multi-layered membrane made of PCL and PVA loaded with platelet lysate (PL). PCL nanofibers allowed cell adhesion and growth, whereas PVA acted as a hydrogel that releases the bioactive compounds of platelet lysate. The cytocompatibility of the membranes containing PL and without it was demonstrated on two cell types involved in wound healing, i.e. keratinocytes and fibroblasts. Both cell types were able to adhere and proliferate on the membranes. In addition, the membrane containing PL enhanced the proliferation of fibroblasts. A co-culture study was also performed by seeding each cell type on one side of the membrane. The cells were co-cultured for 7 days and the results showed that PL increased the proliferation of cells achieving a monolayer of keratinocytes or fibroblasts on each side of the membrane. Thus, the beneficial effect of PCL-PVA+PL membranes on monocultures and co-cultures of skin cells was demonstrated, and these membranes can be considered potential scaffolds for treatment of chronic wounds.
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Differentiation of mesenchymal stem cells on fibrin assemblies supported by immobilized growth factors FGF2 and VEGF
Musílková, Jana ; Filová, Elena ; Kaplan, Ondřej ; Bačáková, Lucie
Bioartificial heart valves and vascular grafts prepared from decellularized tissues could be recellularized with bone marrow-derived mesenchymal stem cells (MSCs) that are able to differentiate into both smooth muscle cells and endothelial cells. MSCs differentiation is facilitated by sustained release of growth factors. In our study assemblies based on fibrin, fibrin with heparin, fibrin with adsorbed or covalently-immobilized vascular endothelial growth factor A165 (VEGF) or basic fibroblast growth factor (FGF-2) via binding to heparin attached to fibrin have been prepared and were evaluated for their stimulation of MSCs differentiation. We estimated the mRNA expression of endothelial marker CD31 (PECAM1), smooth muscle marker α-actin (ACTA2), osteoblast markers osteocalcin (BGLAP) and alkaline phosphatase (ALP). The gene expression was estimated using RT-PCR on days 1, 7 and 21 after seeding. The cell morphology and viability was evaluated by LIVE/DEAD staining. VEGF, both adsorbed and covalently bound, increased significantly the expression of smooth muscle marker α-actin. The mRNA expression of ACTA2 on day 7 and 21 raised more than 200 times in comparison to control samples (undifferentiated cells before seeding). The ACTA2 gene expression significantly exceeded the expression of all other evaluated genes at all time intervals. Moreover, on day 21, the late smooth muscle marker desmin (DES) was steeply rising in cells cultivated on assemblies containing heparin and covalently bound VEGF. The expression of osteocalcin was minimal. We conclude that fibrin assembly containing covalently bound VEGF is the most convenient for MSCs differentiation towards smooth muscle cells.
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Composite coatings containing nanometric constituents prepared by plasma spraying with liquid feedstocks
Mušálek, Radek ; Tesař, Tomáš ; Medřický, Jan ; Čížek, Jan ; Lukáč, František
Plasma spraying of liquid feedstocks allows deposition of materials with novel microstructures. Due to the combination of a liquid carrier and ultrafine powders (in case of suspensions) or in-situ formation of the final material in the plasma jet (in case of solutions), preparation of ultrafine composites is possible, typically in a form of coatings on the substrates. Moreover, both suspension and solution spraying routes can be easily combined, either together or even with conventional plasma spraying of coarse dry powders, thus merging benefits of “nanometric” and “micrometric” constituents in the final composite microstructure. As a consequence, liquid feedstock plasma spraying represents an industrially-relevant deposition process with wide variability, providing coatings with properties tailored for various applications ranging from compact wear-resistant layers, porous thermal barrier coatings, bio-compatible medical coatings, catalyst carriers, fuel-cells, etc. The paper introduces several examples of such ultrafine coatings recently deposited at IPP CAS using hybrid water-stabilized plasma torch.
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A sampler of diffraction and refraction optically variable image elements
Horáček, Miroslav ; Krátký, Stanislav ; Matějka, Milan ; Chlumská, Jana ; Meluzín, Petr ; Pirunčík, J. ; Aubrecht, I. ; Kotrlý, M. ; Kolařík, Vladimír
Diffraction and refraction optically variable image elements are basic building blocks of planar structures for advanced security of documents and valuables. A sampler formed by an array of 36 diffraction structures binary, tertiary, quaternary and blazed gratings (period range 400 nm 20,000 nm) represents a cross-section throughout technological steps mastering, galvanic replication and embossing. Electron-beam writing technology with Gaussian beam and electron energy of 100 keV, with very small forward scattering of high energy electrons and with the possibilities to create a linear grating with the minimal period of 100 nm, was used to create the master. An important advantage of high-resolution electron-beam lithography is its substantial flexibility in combining possible planar structures with significantly different parameters, such as very dense and relatively shallow structures together with deep structures (approx. 10 microns) with precise shapes (micro-lenses or Fresnel structures). For protection of documents and valuables, interesting results are induced with planar optical structures consisting of non-periodic arrangements, which are characterized by high robustness to counterfeiting and imitation. While the origination process is available for grating period down to 100 nm, the mass replication technology appears to be a bottleneck of the entire technological process. Measurement of topology and profiles of the structures by atomic forces microscope and documenting the quality of technological process of the three steps of replication of planar optically variable elements was performed for all 36 structure types of sampler.
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General overview of GaN devices and transport properties of AlGaN/GaN HEMT structures - impact of dislocation density and improved design
Hulicius, Eduard ; Hájek, František ; Hospodková, Alice ; Hubík, Pavel ; Gedeonová, Zuzana ; Hubáček, Tomáš ; Pangrác, Jiří ; Kuldová, Karla
GaN-based nanostructures are used for many present semiconductor devices. The main topics are structures for blue LEDs and LDs, but there are also other interesting and important GaN devices namely for power electronics, scintillators and detectors as well as High Electron Mobility Transistors (HEMT). Reduction of dislocation density considerably increases electron mobility in 2DEG. All presented results support our expectation that a suitably designed AlGaN back barrier can help to prevent this phenomenon.
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Emergence of dark ZnO nanorods by hydrogen plasma treatment
Remeš, Zdeněk ; Buryi, Maksym ; Sharma, Dhananjay K. ; Artemenko, Anna ; Mičová, J. ; Rezek, B. ; Poruba, A. ; Hsu, H.S. ; Potocký, Štěpán ; Babin, Vladimir
We employed a custom-built inductively coupled plasma (ICP) 13.56 MHz reactor with up to 300 W RF discharge power. Hydrothermally grown ZnO nanorods were exposed to the ICP plasma with a mixture of hydrogen and argon for up to 30 min, followed in-situ by plasma oxidation. Plasma properties were monitored by optical emission spectroscopy (OES) and by measuring the self-bias potential of the stainless steel sample holder separated from the ground by a blocking capacitor. The exciton-related UV photoluminescence of ZnO nanorods and optical absorption increases significantly after the plasma treatment. We attribute it to the complex changes of ZnO surface electronic states that also give rise to its black color visually.
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Degradation of polycaprolactone electrospun materials - methods of analysis
Havlíčková, K. ; Řezanka, M. ; Kobera, Libor ; Kuželová Košťáková, E. ; Lukáš, D. ; Jenčová, V.
Biodegradable electrospun materials are widely used for medical application. Polycaprolatone is polymer suitable for electrospinning technology and is very often used to create nanofibrous scaffolds for tissue engineering. The time to disintegration or biodegradation of such materials is very important here. However, testing is not entirely easy. It is not possible to create exactly body-like conditions in vitro. Moreover, it is not easy to find suitable analytical methods that would show exactly what happens in the nanofibrous polycaprolactone electrospun samples at certain stages of degradation, ie how the internal structure of decaying nanofibers changes. This paper describes the traditional use of methods for testing polycaprolatone nanofibers by enzymatically catalysed degradation. Morphological changes are studied using scanning electron microscope images. However, it also offers a non-traditional analysis of polycaprolactone electrospun materials using the ssNMR method.
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