Impaired closure of diabetic ulcers is a serious complication decreasing quality of patient's life. The cause is overexpression of metalloproteinases (MMPs) during reepithelialization. Inhibitors of metalloproteinases are low effective so far and their use leads to serious side effects. The proposed translational research is focused on preclinical development of a system for treatment of impaired closure of ulcers. It’s based on effective and safe gene therapy – RNA interference of selected MMP. The carrier, nanodiamonds – the carbon nanomaterial with the lowest known toxicity – bears polyethyleneimine which stabilizes the therapeutic siRNA and facilitates its transfection. The whole system is incorporated in nanofibers which serve for localized release of the therapeutic complex and maintenance of constant siRNA level for the time necessary for the closure of ulcer. The completion of project will enable to follow with preclinical trials in vivo using primates and possible translation of the therapeutic procedure to clinical outputs.

Špatné hojení ran je závažnou komplikací léčby diabetes zhoršující kvalitu života pacienta. Příčinou této poruchy je nadměrná exprese matrixových metaloproteinas (MMP) při reepitelizaci rány. Inhibitory MMP jsou zatím málo účinné a jejich použití vede k vážným vedlejším efektům. Předkládaný projekt spadá do oblasti translační medicíny a je zaměřený na preklinický vývoj systému pro léčbu špatně se hojících ran. Je založen na vysoce účinné a bezpečné genové terapii - RNA interferenci vybraných MMP. Jednotlivé stavební kameny systému již byly uchazeči funkčně vyzkoušeny a zde jsou synergicky využity. Nosičem jsou nanodiamanty – uhlíkový nanomateriál s nejnižší známou toxicitou – s navázaným polyethyleniminem, umožňujícím stabilizaci terapeutické siRNA a její transfekci do buněk. Celý systém bude inkorporován do nanovláken potřebných pro lokalizované uvolnění terapeutického komplexu a udržení stabilní hladiny siRNA po dobu nutnou k zahojení rány. Vyřešení projektu umožní pokračování in vivo zkoušek na primátech a potenciální translaci terapeutické procedury do klinického použití.

• MeSH
• hojení ran účinky léků   MeSH
• komplikace diabetu farmakoterapie   MeSH
• malá interferující RNA terapeutické užití   MeSH
• matrixové metaloproteinasy MeSH
• nanodiamanty terapeutické užití   MeSH
• nosiče léků MeSH
• terapie založená na RNAi MeSH
• translační biomedicínský výzkum MeSH
• uvolňování léčiv MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• farmacie a farmakologie
• genetika, lékařská genetika
• diabetologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

In the present work, we developed a novel needleless emulsion electrospinning technique that improves the production rate of the core/shell production process. The nanofibres are based on poly-ε-caprolactone (PCL) as a continuous phase combined with a droplet phase based on Pluronic F-68 (PF-68). The PCL-PF-68 nanofibres show a time-regulated release of active molecules. Needleless emulsion electrospinning was used to encapsulate a diverse set of compounds to the core phase [i.e. 5-(4,6-dichlorotriazinyl) aminofluorescein -PF-68, horseradish peroxidase, Tetramethylrhodamine-dextran, insulin growth factor-I, transforming growth factor-β and basic fibroblast growth factor]. In addition, the PF-68 facilitates the preservation of the bioactivity of delivered proteins. The system's potential was highlighted by an improvement in the metabolic activity and proliferation of mesenchymal stem cells. The developed system has the potential to deliver susceptible molecules in tissue-engineering applications.

• MeSH
• biokompatibilní materiály farmakologie   MeSH
• dextrany chemie   MeSH
• emulze chemie   MeSH
• jehly MeSH
• kolagen typ II metabolismus   MeSH
• křenová peroxidasa metabolismus   MeSH
• mezenchymální kmenové buňky cytologie   účinky léků   metabolismus   MeSH
• mezibuněčné signální peptidy a proteiny farmakologie   MeSH
• miniaturní prasata MeSH
• nanovlákna chemie   ultrastruktura   MeSH
• poloxamer chemie   MeSH
• polyestery chemie   MeSH
• prasata MeSH
• proteiny aplikace a dávkování   MeSH
• rhodaminy chemie   MeSH
• tkáňové inženýrství metody   MeSH
• tkáňové podpůrné struktury chemie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

MicroRNAs are short molecules of RNA regulating most cellular processes via the mechanism of RNA interference. Their dysregulation leads to a disease burden, making them important therapeutic targets. For the successful development of a therapeutic device, the uptake of a functionalized carrier by live cells and the sufficient release of effector therapeutic molecules are limiting factors. Here for the first time, the inhibition of oncogenic microRNA-21 in CT-26 colon cancer cells is achieved, using an advanced nanosystem consisting of fluorescent nanodiamond and antisense RNA. Stable nanocomplexes efficiently deliver antisense RNA into cell cytoplasm, encouraging further study of microRNA-21 function in target cells. Engaging the fluorescent nanoparticle enables monitoring of transfection and release of the antisense RNA load into cell cytoplasm. Importantly, the internalized antisense RNA effectively destroys target microRNA-21 in CT-26 cancer cells. The absence of oncogenic microRNA-21 liberates tumor suppressor genes Pdcd4 and Timp3 from silencing, and results in a decrease of cell invasion and migration, and in the induction of apoptotic cell death. This study uses a nanodiamond-based imaging and delivery system, and shows that the multidimensional performance of the presented device makes nanodiamond-based complexes promising therapeutic devices.

• MeSH
• lidé MeSH
• mikro RNA MeSH
• nádorové buněčné linie MeSH
• nanodiamanty * MeSH
• nekódující RNA MeSH
• proteiny regulující apoptózu MeSH
• RNA interference MeSH
• transfekce MeSH
• Check Tag
• lidé MeSH

Platelets are a popular source of native growth factors for tissue engineering applications. The aim of the study was to verify the use of platelet lysate as a fetal bovine serum (FBS) replacement for skin cell culture. The cytokine content of the platelet lysate was characterized using the Bio-Plex system. The cells (fibroblasts, melanocytes, and keratinocytes) were cultured on PCL nanofibrous scaffolds to mimic their natural microenvironment. The cytokine content of the platelet lysate was determined, and to the cells, a medium containing platelet lysate or platelet lysate in combination with FBS was added. The results showed that 7% (v/v) platelet lysate was sufficient to supplement 10% (v/v) FBS in the culture of fibroblasts and keratinocytes. The combination of platelet lysate and FBS had a rather inhibitory effect on fibroblasts, in contrary to keratinocytes, where the effect was synergic. Platelet lysate did not sufficiently promote proliferation in melanocytes; however, the combination of FBS and platelet lysate yielded a better outcome and resulted in bipolar morphology of the cultured melanocytes. The data indicated that platelet lysate improved cell proliferation and metabolic activity and may be used as an additive to the cell culture media.

• MeSH
• biomimetika metody   MeSH
• buněčná diferenciace MeSH
• buněčné kultury MeSH
• lidé MeSH
• nanovlákna chemie   MeSH
• trombocyty metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Glycan metabolism balance is critical for cell prosperity, and macromolecule glycosylation is essential for cell communication, signaling and survival. Thus, glycotherapy may be a potential cancer treatment. The aim of the present study was to determine whether combined synthetic glycoconjugates (GCs) induce changes in gene expression that alter the survival of colon cancer cells. The current study evaluated the effect of the GCs N‑acetyl‑D‑glucosamine modified polyamidoamine dendrimer and calix[4]arene scaffold on cancer cell proliferation, apoptosis, invasion and sensitivity to immune cell‑mediated killing. Using reverse transcription‑quantitative polymerase chain reaction, the expression of genes involved in the aforementioned processes was measured. It was determined that GCs reduce the expression of the glucosaminyltransferases Mgat3 and Mgat5 responsible for surface glycosylation and employed components of the Wnt signaling pathway Wnt2B and Wnt9B. In addition, the calix[4]arene‑based GC reduced cell colony formation; this was accompanied by the downregulation of the metalloproteinase Mmp3. By contrast, the dendrimer‑based GC affected the expression of the glucose transporter components Sglt1 and Egfr1. Therefore, to the best of our knowledge, the present study is the first to reveal that N‑acetyl‑D‑glucosamine‑dendrimer/calix[4]arene GCs alter mRNA expression in a comprehensive way, resulting in the reduced malignant phenotype of the colon cancer cell line HT‑29.

• MeSH
• apoptóza genetika   MeSH
• buňky HT-29 MeSH
• glukosa metabolismus   MeSH
• glykokonjugáty farmakologie   MeSH
• lidé MeSH
• molekuly buněčné adheze genetika   metabolismus   MeSH
• nádorové buněčné linie MeSH
• nádorové kmenové buňky účinky léků   metabolismus   MeSH
• nádory tračníku genetika   metabolismus   MeSH
• proliferace buněk MeSH
• proteiny usnadňující transport glukosy genetika   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• stanovení celkové genové exprese MeSH
• testy nádorových kmenových buněk MeSH
• transkriptom MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Efficient delivery of stabilized nucleic acids (NAs) into cells and release of the NA payload are crucial points in the transfection process. Here we report on the fabrication of a nanoscopic cellular delivery carrier that is additionally combined with a label-free intracellular sensor device, based on biocompatible fluorescent nanodiamond particles. The sensing function is engineered into nanodiamonds by using nitrogen-vacancy color centers, providing stable non-blinking luminescence. The device is used for monitoring NA transfection and the payload release in cells. The unpacking of NAs from a poly(ethyleneimine)-terminated nanodiamond surface is monitored using the color shift of nitrogen-vacancy centers in the diamond, which serve as a nanoscopic electric charge sensor. The proposed device innovates the strategies for NA imaging and delivery, by providing detection of the intracellular release of non-labeled NAs without affecting cellular processing of the NAs. Our system highlights the potential of nanodiamonds to act not merely as labels but also as non-toxic and non-photobleachable fluorescent biosensors reporting complex molecular events.

• MeSH
• buňky HT-29 MeSH
• DNA * MeSH
• lidé MeSH
• luminiscence MeSH
• myši inbrední DBA MeSH
• nanodiamanty * MeSH
• transfekce * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

OBJECTIVES: Faulty wound healing is a global healthcare problem. Chronic wounds are generally characterized by a reduction in availability of growth factors. New strategies are being developed to deliver growth factors more effectively. METHODS: In this study, we introduced electrospun scaffolds composed of polycaprolactone (PCL) nanofibers functionalized with adhered platelets, as a source of numerous growth factors. Three concentrations of platelets were immobilized to nanofibrous scaffolds by simple adhesion, and their influence on adhesion, proliferation and metabolic activity of seeded cells (murine fibroblasts, keratinocytes and melanocytes) was investigated. RESULTS: The data obtained indicated that presence of platelets significantly promoted cell spreading, proliferation and metabolic activity in all the skin-associated cell types. There were no significant differences among tested concentrations of platelets, thus even the lowest concentration sufficiently promoted proliferation of the seeded cells. CONCLUSIONS: Such complex stimulation is needed for improved healing of chronic wounds. However, the nanofibrous system can be used not only as a skin cover, but also in broader applications in regenerative medicine.

• MeSH
• buněčná adheze MeSH
• buněčné linie MeSH
• fibroblasty cytologie   metabolismus   MeSH
• hojení ran MeSH
• keratinocyty cytologie   metabolismus   MeSH
• melanocyty cytologie   metabolismus   MeSH
• myši MeSH
• nanovlákna chemie   ultrastruktura   MeSH
• polyestery chemie   MeSH
• proliferace buněk * MeSH
• tkáňové inženýrství MeSH
• tkáňové podpůrné struktury chemie   MeSH
• trombocyty cytologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

We show that fluorescent nanodiamonds (FNDs) are among the few types of nanosensors that enable direct optical reading of noncovalent molecular events. The unique sensing mechanism is based on switching between the negatively charged and neutral states of NV centers which is induced by the interaction of the FND surface with charged molecules.

• MeSH
• fluorescence MeSH
• molekulární sondy - techniky MeSH
• molekulární zobrazování MeSH
• nanodiamanty * chemie   MeSH
• nanostruktury MeSH