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Preparation and characterization of nanodiamonds modified with biocompatible polymers
Kvaková, Klaudia ; Skoumalová, Petra (oponent) ; Cígler, Petr (vedoucí práce)
Nanodiamonds (NDs, 250 nm) were oxidized, separated by size and the polyglycerol layer was polymerized on the ND's surface. The polymerization was achieved by acid catalyzed ring opening multi-branching polymerization of glycidol with the hydroxyl and carboxylic groups formed, during oxidation on the NDs surface. The polyglycerol layer increases the stability and biocompatibility of the NDs. After the coating, a terminal propargyl group was introduced by glycidyl propargyl ether addition. The presence of the alkyne group enables further modification using copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC-click). Employing CuAAC click reaction, a mannose (with azide linker) was attached to the NDs surface. Thus prepared NPs were characterised from the physicochemical point of view. The size distribution was measured by dynamic light scattering (DLS), and it was further confirmed by transmission electron microscopy (TEM). Inertness towards biological conditions was evaluated (stability tests in 1xPBS, 10xPBS and 100 % FBS) and zeta potential was measured. Next, fluorescent nanodiamonds (FNDs, 250 nm) with attached D-mannose were prepared. According to the thermogravimetry results, DLS measurement and TEM images, it was determined, that NDs are covered by thin polyglycerol layer. Presence of glycidol coating was further confirmed by stability tests. Presence of attached D-mannose was supported by Fourier-transform infrared spectroscopy (FTIR). Prepared polyglycerol coated NDs are well suited for in vitro and in vivo testing. That was proven by their stability in high salt concentration media such as phosphate buffer saline (PBS) and low protein binding properties in fetal bovine serum (FBS).
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Fluorescent Nanoprobes Emitting in Near-Infrared Region for Biomedical Applications
Kvaková, Klaudia ; Cígler, Petr (vedoucí práce) ; Bogdanowicz, Robert (oponent) ; Poláková, Kateřina (oponent)
Hlavním cílem této práce je podrobný výzkum fluorescenčních nanosond emitujících v blízké infračervené oblasti a jejich využití v biomedicíně. Jsou studovány zejména fluorescenční nanodiamanty (FND) a zlaté nanoklastry (AuNC). Nejprve byl zkoumán potenciál FND pro in vivo vizualizaci sentinelových lymfatických uzlin v myším modelu. Tato studie ukázala, že FND modifikované manózou vykazují vyšší internalizaci makrofágy a lepší retenci v sentinelových lymfatických uzlinách v porovnání s výchozími FND. Kromě využití v bio-zobrazování byly FND použity i pro přesné měření teploty v buňkách s difrakčním rozlišením. V této práci byly FND použity jako sondy pro lokální měření změn teploty v důsledku aktivity hipokampálních neuronů. Dále byl zkoumán potenciál AuNC, jakožto možných fluorescenčních značek pro značení a zobrazování buněk. Jednoduchou syntetickou cestou byly připraveny stabilní AuNC. Dalšími modifikacemi pomocí různých ligandů bylo dosaženo zvýšení intenzity fotoluminiscence a prodloužení doby života fotoluminiscence. Práce dále popisuje reverzibilní světelně a tepelně indukované účinky na fotoluminiscenční vlastnosti syntetizovaných AuNC. Bylo též prokázáno zesílení fotoluminiscence AuNC pomocí použití konjugace s plazmonickým systémem - zlatými nanotyčemi. Nakonec byla dosažena internalizace...
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Preparation and characterization of nanodiamonds modified with biocompatible polymers
Kvaková, Klaudia ; Skoumalová, Petra (oponent) ; Cígler, Petr (vedoucí práce)
Nanodiamonds (NDs, 250 nm) were oxidized, separated by size and the polyglycerol layer was polymerized on the ND's surface. The polymerization was achieved by acid catalyzed ring opening multi-branching polymerization of glycidol with the hydroxyl and carboxylic groups formed, during oxidation on the NDs surface. The polyglycerol layer increases the stability and biocompatibility of the NDs. After the coating, a terminal propargyl group was introduced by glycidyl propargyl ether addition. The presence of the alkyne group enables further modification using copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC-click). Employing CuAAC click reaction, a mannose (with azide linker) was attached to the NDs surface. Thus prepared NPs were characterised from the physicochemical point of view. The size distribution was measured by dynamic light scattering (DLS), and it was further confirmed by transmission electron microscopy (TEM). Inertness towards biological conditions was evaluated (stability tests in 1xPBS, 10xPBS and 100 % FBS) and zeta potential was measured. Next, fluorescent nanodiamonds (FNDs, 250 nm) with attached D-mannose were prepared. According to the thermogravimetry results, DLS measurement and TEM images, it was determined, that NDs are covered by thin polyglycerol layer. Presence of glycidol coating was further confirmed by stability tests. Presence of attached D-mannose was supported by Fourier-transform infrared spectroscopy (FTIR). Prepared polyglycerol coated NDs are well suited for in vitro and in vivo testing. That was proven by their stability in high salt concentration media such as phosphate buffer saline (PBS) and low protein binding properties in fetal bovine serum (FBS).
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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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Fluorescent nanodiamonds: The new platform for construction of chemo- and biosensors
Cígler, Petr ; Ledvina, Miroslav ; Tvrdoňová, Monika ; Řezáčová, V. ; Nesládek, M. ; Kratochvílová, Irena ; Fendrych, František ; Štursa, Jan ; Kučka, Jan ; Ráliš, Jan
Over the past few years, fluorescent nanodiamonds (FNDs) have been recognized as potential fluorophores for use in bioimaging, owing to their unique and attractive chemical and particularly spectral properties. The source material for their preparation, synthetic nanodiamond (ND), is non-expensive and commercially available. Notably, NDs have the highest biocompatibility of all carbon nanomaterials and their surface can be chemically modified by various techniques. FNDs are capable of fluorescing with almost quantitative quantum yields from point defects of crystall lattice - nitrogen-vacancy (NV) centers. In particular, the long-wavelength emission, high brightness, no photobleaching, no photoblinking, and an exceptional resistance to chemical degradation make them almost ideal core for development of fluorescent bioimaging probes.
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