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Nanofiber dressing consisting of antisense rna-functionalized nanodiamonds for therapy of non-healing wounds in diabetic individuals
Neuhoferová, Eva ; Petráková, V. ; Vocetková, K. ; Kindermann, Marek ; Křivohlavá, Romana ; Benson, Veronika
Non-healing wounds are serious complication in diabetic patients and represent an attractive challenge for development of suitable carrier system possessing constant and localized release of therapeutic biomolecule into the wound without any undesired side effects. Given the fact that these non-healing wounds are result of impaired balance in metalloproteinases synthesized by immune cells residing the wounds, gene therapy offering knock down of such enzymes is of great interest. \nHere we challenged a development of functional and biocompatible wound dressing enabling controlled release of trackable carrier loaded with therapeutic siRNA. Our dressing consists of scaffold from degradable polymer nanofibers enriched with fluorescent nanodiamond particles (FND). We have previously shown the nanodiamond particles are great carriers for antisense RNAs. Their advantages represent high biocompatibility, stable luminescence giving us the possibility to track the carrier system in the wound, and effective release of antisense RNA in the wound. Embedding of nanodiamond-siRNA systems into nanofiber scaffold enables continuous release of siRNA and maintaining the stable siRNA concentration in the wound site resulting in a promotion of wound healing. \nWe developed FND-siRNA complexes specific to MMP-9 that efficiently inhibit the expression of target MMP-9 mRNA. The complexes were embedded into core/shell nanofibers from PVA and PCL, visualized by confocal microscopy, and characterized by electron microscopy. Real-time PCR was used to assess the silencing effect of siRNA that has been delivered to target murine fibroblasts by FND released from nanofiber dressing. Nanofiber system with embedded FNDs was applied on wounds in diabetic animal models to evaluate its suitability regarding short and long term toxicity, efficacy, and handling in vivo. \n
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Nanodiamonds as an innovative system for intracellular delivery of mirna-34a inprostatic cancer therapy
Bitti, G. ; Abate, M. ; Neuhoferová, Eva ; Kindermann, Marek ; Petráková, V. ; Boccellino, M. ; Quagliuolo, L. ; Filová, Eva ; Benson, Veronika ; Caraglia, M. ; Amler, Evžen
The microRNA(miRNA)-34a is an important regulator of tumor suppression. It controls the expression of several target proteins involved in cell cycle, differentiation and apoptosis, and antagonizes processes that are necessary for basic cancer cell viability as well as cancer stemness, metastasis, and chemoresistance. It is downregulated in numerous cancer types, including prostatic cancer, and inhibits malignant growth by repressing genes involved in various oncogenic signaling pathways. Given the anti-oncogenic activity of miR-34a, here we proved the substantial benefits of a new therapeutic concept based on nanotechnology delivery of miRNA mimics. In order to monitor the miRNA-34a replacement, we used a fluorescent nanodiamond particles (FND) system with linked miRNA-34a mimic, which was delivered to PC3 and DU145 prostatic cancer cell lines. We used functionalized nanodiamonds coated with polyethylenimine to transfer miRNA-34a into PC3 and DU145 prostatic cancer cell lines and we measured the zeta-potential of these complexes before using them for in vitro experiments. A replacement of miRNA-34 was observed by monitoring levels of miRNA-34 via real-time PCR. Moreover, our in vitro experiments demonstrated that miRNA-34a replacement, using this FND delivery system, decreased viability and induced apoptosis in prostatic cancer cell lines. Our findings suggest the replacement of oncosuppressor miRNA-34a provides an effective strategy for cancer therapy and the FND-based delivery systems seems to be an excellent strategy for a safe and effective targeting of the tumor.
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UNICELLULAR ALGA CHLORELLA AS A FEED SUPPLEMENT, CURRENT EXPERIENCE AND FUTURE PERSPECTIVES
Kotrbáček, V. ; Doucha, Jiří ; Kuchařová, V. ; Doubek, J.
n experiments with farm animals we studied the effect of Chlorella biomass as a feed supplement. In laying hens we found that diet supplemented with 1 % (i.e. 10 g/kg) of dry disintegrated heterotrophically grown Chlorella biomass significantly increased content of carotenoids in egg yolks of 8 experimental animals already after the first week of supplementation (P<0,01). Compared to 8 control hens these differences remained significantly higher until the end of the eight-week experiment. The average carotenoid concentration was 17,3 mu g per g of yolk in control group and 25,3 mu g per g of yolk in experimental animals (P<0,01). Autotrophically grown Chlorella was supplemented to the 30 gilts one month prior the expected first heat in a dose of 1,5 g per animal per day. These sows entered heat earlier and the number of pregnancy was higher in comparison to the 30 control animals. Other studies observed digestibility of selenium organically bound in Se-enriched Chlorella. The level of selenium in blood, colostrum and also tissues of new born piglets was determined. Group of 8 sows was supplemented with Se-enriched alga during the pregnancy and 3 days after parturition at a dose of 0,3 mg of selenium per 1 kg of feed. The same amount of selenium in the form of sodium selenite was administrated to the control group of 8 individuals. Organic form of selenium significantly increased concentration of Se in blood of sows and colostrum (P<0,05). Piglets born to supplemented sows had also significantly higher level of Se in heart, muscles and liver (P<0.01). The use of fresh Chlorella biomass produced outdoors on a dairy farm was experimentally tested. Location near biogas station enables utilization of flue gas as a source of carbon for algal photosynthesis. The liquid fresh biomass applied to dairy cows during the pregnancy at a dose of 15 g dry mass per cow increased the dry matter content in colostrum from 17,5 g per 100 ml of colostrum in control group to 20,5 g per 100 ml of colostrum in supplemented cows (P<0,05). This pilot experiment demonstrated possibilities of cheaper Chlorella production and its use in ruminant nutrition.
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