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Study of Cellular Toxicity of Representative Nanoparticles in Tissue Cultures.
Filipová, Marcela ; Holada, Karel (advisor) ; Benson, Veronika (referee) ; Hubálek Kalbáčová, Marie (referee)
Safety concerns arising from cytotoxic behavior of nanoparticles (NPs) in complex biological environment remain the main problem limiting NPs application in biomedicine. In this study, we have investigated cytotoxicity of NPs with different composition, shape and size, namely SiO2 NPs (SiNPs, 7-14 nm), superparamagnetic iron oxide NPs (SPIONs, 8 nm) and carboxylated multiwalled carbon nanotubes (CNTCOOHs, diameter: 60-100 nm, length: 1-2 μm). Cytotoxicity was evaluated with newly designed screening assay capable to simultaneously assess activity of cell dehydrogenases, activity of lactate dehydrogenase (LDH) released from cells into environment and number of intact cell nuclei and apoptotic bodies in human umbilical vein endothelial cell (HUVEC) culture growing in the very same well of the 96-well plate. Aforementioned attributes were subsequently utilized to obtain information about cell viability and necrotic and apoptotic aspects of cell death. Results from this "three-in-one" cell death screening (CDS) assay showed that SiNPs and CNTCOOHs evoked pronounced cytotoxic effect demonstrated as decrease of cell viability and development of apoptotic bodies formation. In contrast to this, SPIONs induced only mild cytotoxicity. Moreover, SiNPs impaired cell membrane leading to increased LDH release...
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The Cell-Nanomaterial Interactions and Their Application in Biomedicine
Vrabcová, Lucie ; Hubálek Kalbáčová, Marie (advisor) ; Pešta, Martin (referee) ; Benson, Veronika (referee)
Nanomaterials entered the biomedicine already at the beginning of the millennium and they still bring new and unique advances and possibilities for treatment, diagnosis and regeneration, thus they continue to facilitate the development of personalized medicine. Interactions at nanoscale allow until then unconceivable opportunities to influence processes on molecular level. The completely new perspectives of nanomaterial applications jointly raise attention concerning health and environmental safety issues. Although a lot of novel biomedical applications of nanomaterials have emerged, the vast majority is still found to be at the stage of a concept. The consistent basic in vitro research of elemental interactions of nanomaterial with biological environment should represent an essential part of its development. The concern of this thesis was to describe the cytocompatibility and interactions of two types of nanomaterials with different human cells. First, ultra-fine grain titanium was tested for prospective use in implant development. We confirmed its positive effect mainly on the growth of osteoblasts and recommended the further pre-clinical trials of this material in a form of a bone or dental implant. Second, several types of ultra-small (˂ 5 nm) nanoparticles of different origin (silicon, gold...
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Effect of nanodiamond particles on neuronal tissue
Šimková, Veronika ; Benson, Veronika
High-Pressure High-Temperature nanodiamonds are considered to be highly biocompatible due to\ntheir low acute toxicity, which makes them suitable for biomedical applications. However, next to interaction with the cell membrane, nanoscale allows particles to penetrate the plasma membrane and even accumulate in the cell cytoplasm. Recently it brought up questions regarding sustainable nanoparticle application in vivo and potential toxicity hazards. The neuronal tissue consists of neurons and glial cells, and it is separated by a bloodbrain barrier from the peripheral blood system. Neurons are nerve cells forming a complex network of synapses that carry signals in the brain. The neurons are nourished and protected by glial cells. This project aims to find out whether nanodiamonds pass the blood-brain barrier when applied intravenously as a drug carrier and whether they directly interact with the neuronal tissue.
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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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Biocompatibility of diamond-based platforms with neuronal cells.
Gottfriedová, Kristýna ; Benson, Veronika (advisor) ; Hubálek Kalbáčová, Marie (referee)
5 Abstract Microelectrode arrays represent therapeutic approach to neurodegenerative diseases treatment. The development of electrode platforms is rather challenging due to the direct interaction of the material with neuronal tissue. Nanodiamond is one of the researched materials because of its biochemical properties: biocompatibility with many cell types, chemical inertness, high wear and corrosion resistance. During nanodiamond platforms development, biological research focuses on biocompatibility of used material with primary neurons and the evaluation of their adherence to the nanodiamond that is important for recording of electrical activity of neurons. Both, the biocompatibility as well as the adherence depend on the used nanodiamond, manufacturing, roughness, and surface modification. The amount of boron dopant enabling electric conductivity of nanodiamond is also important. In my thesis, I have summarized research on biocompatibility and adherence of neural cells on behalf of above mentioned parameters. I discuss here the variability of experimental results in order to surface modification and cultured cells type. According to the research reports, it seems that neuronal cells prosper well and prefer to adhere to platforms covered with molecules of extracellular matrix or at least poly-lysine...
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Targeted biocompatible nanoparticles for therapy and cancer diagnostics.
Neburková, Jitka ; Cígler, Petr (advisor) ; Benson, Veronika (referee) ; Schirhagl, Romana (referee)
Nanoparticles (NPs) have considerable potential in targeted medicine. NPs can merge various functions and serve as labels for imaging or as nanocarriers in therapy. Modification of NPs with targeting ligands can lead to highly specific interactions with targeted cancer cells. However, the efficacy of targeting depends on the ratio between specific and non-specific interactions of a NP with the cell. Non-specific interactions of NPs are unrelated to targeted receptors and need to be eliminated in order to decrease background noise during imaging and adverse effect of drugs on healthy tissues. In this thesis, surface modifications of NPs were explored mainly on biocompatible carbon NPs called nanodiamonds (NDs), which have exceptional fluorescent properties such as long fluorescence lifetime, no photobleaching and photoblinking and sensitivity of their fluorescence to electric and magnetic field. Main issues addressed in this thesis are low colloidal stability of NDs in buffers and media, their non-specific interactions with proteins and cells and limited approaches for ND surface modifications. These issues were solved by coating NDs with a layer of biocompatible, hydrophilic, and electroneutral poly(ethylene glycol) or poly[N-(2- hydroxypropyl) methacrylamide] polymers. Optimized polymer coating...
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Biocompatibility of diamond-based platforms with neuronal cells.
Gottfriedová, Kristýna ; Benson, Veronika (advisor) ; Hubálek Kalbáčová, Marie (referee)
Existuje mnoho typů platforem, které jako povrch, bezprostředně interagující s buňkami, využívají diamant. Hlavní rozdíly jsou v typu použitých diamantů. Jedná se o nanodiamanty a ultrananokrystalické diamanty, které mohou být různě strukturované a také dopované o atomy boru. Ačkoli by pravděpodobně bylo hezké, kdyby na platformách nemusela být žádná povrchová úprava. Podle výsledků výzkumů to nevypadá jako lehký úkol, výzkum v tomto směru probíhá, ale zatím bez významných úspěchů. Nejlepších výsledků se při kultivaci buněk na platformách dosahuje, když je povrch potažen alespoň částečkami molekul extracelulární matrix. Jako o trošku méně účinné se jeví využití molekul, které na základě elektrostatických sil navodí adhezi buňky. Jedná se například o molekulu lysinu. Dalším krokem ve výzkumu je ověření, zda povrchy, které se v první fázi zkoumání jevily jako biokompatibilní, budou i po dopování stále optimální pro růst buněk. Z níže uvedených výzkumů zatím vyplývá, že například dopování nanodiamantu borem nemá významný vliv na prosperitu buněk. Klíčová slova nanodiamant, nervová buňka, biokompatibilita, mikroelektrodové pole
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The study of signaling pathways that modulate multidrug resistance
Dvořák, Pavel ; Souček, Pavel (advisor) ; Daum, Ondřej (referee) ; Benson, Veronika (referee)
The study of signaling pathways that modulate multidrug resistance The theme of cancer cell resistance to anti-cancer drugs including the common mechanisms of resistance development and the theory of cancer stem cells was introduced in the Introduction to the doctoral thesis. The theoretical part was focused more deeply on the two topics - the role of ATP-binding cassette (ABC) transport proteins and chromosomal abnormalities in the development of cancer chemoresistance. The possible therapeutic potential for the treatment of cancer was stressed for both topics. The Results were composed of the commentaries on the five published works, which the author of the thesis conducted as the main author. The first work brought the evidence supporting the hypothesis of the existence of ABC gene expression profiles (signatures), which are common to multiple types of tumors and are associated with significant clinical consequences. These general ABC gene expression profiles could possibly form a new hallmark of cancer. The second work studied more closely a group of acute myeloid leukemia patients, who did not achieve complete cytogenetic remission after two attempts to maintain remission of the malignant disease. The new entity, consisting of patients with the translocation t(2;11)(p21;q23) without the...
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Histone deacetylase inhibitors in plasma cell leukemia treatment: effect of the bone marrow microenvironment
Burianová, Ilona ; Stöckbauer, Petr (advisor) ; Pour, Luděk (referee) ; Benson, Veronika (referee)
Multiple myeloma and its aggressive variant, plasma cell leukemia, are still considered to be incurable diseases despite the progressive treatment approaches comprising novel drugs. This can be attributed to the presence of the bone marrow microenvironment which plays an important role in drug resistance of myeloma cells. Hematopoietic cell lines derived from hematologic malignancies are suitable models for the study of etiopathogenesis of these malignant diseases and for testing new potential drugs. Establishment of these cell lines is still considered to be coincidental and rare event. The first part of the thesis is focused on establishment and characterization of the cell line UHKT-944 derived from a patient with primary plasma cell leukemia, and on completion of characterization of the cell line UHKT-893 derived from a patient with multiple myeloma. Additional analysis of UHKT-893 cell line were performed including sequence analysis of IgVH gene rearrangements and cytogenetic analysis which contributed to more detailed characterization of this cell line. During cultivation of UHKT-944 cells, we monitored the cell growth and confirmed dependence on interleukin-6 (IL-6). Immunophenotype analysis revealed the presence of surface markers characteristic of malignant plasma cells. UHKT-944 cells...
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