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Studium interakce buněk s biomimetickým materiálem a jeho využití v biomedicíně
Sauerová, Pavla ; Hubálek Kalbáčová, Marie (advisor) ; Vandrovcová, Marta (referee) ; Jendelová, Pavla (referee)
Biomaterials are considered as very promising tools for regenerative medicine. They have compensatory or supporting function in organism and they are often developed to support specific conventional medical procedures. So-called biomimetic materials are developed to imitate natural environment of organism and to induce positive innate responses of organism. An essential part of biomaterial development is in vitro biological evaluation, which characterizes (often for the first time) the potential of developed material for its clinical application. This Ph.D. thesis deals with in vitro biological evaluation of three different biomimetic materials. In all three cases, the comprehensive evaluation was an integral part of the material development and optimization processes. Each material was in vitro characterised at the level of cell-material interactions with respect to its intended specific application.. In the first part, cell response to potential drug delivery system based on colloidal complexes of cationic surfactants with hyaluronic acid (HyA) was characterized. HyA protection ability and its limits were described; also the role of fetal bovine serum (FBS) in cell response to the stress stimuli was confirmed. Results considered surfactant-HyA complexes as promising system for drug delivery. In...
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Nanotechnology in the intensive care: Intravascular biocompatibility of carbon nanomaterials-effect of carbon nanotubes on blood platelets
Šemberová, Jana ; Straňák, Zbyněk (advisor) ; Dyr, Jan (referee) ; Hubálek Kalbáčová, Marie (referee)
Nanotechnology in the intensive care: Intravascular biocompatibility of carbon nanomaterials - effect of carbon nanotubes on blood platelets. EFFECT OF CARBON NANOTUBES ON BLOOD PLATELETS Carbon nanotubes (CNTs) are among the principal materials currently used in biomedical nanotechnologies. CNTs possess superior mechanical and chemical characteristics including enormous tensile strength, elasticity and conductivity. As a result they are very popular and attractive for use in various biomedical applications. Many of these applications may lead ultimately to contact of carbon nanomaterials and blood. Furthermore, CNTs may also be present intravascularly as a result of environmental or occupational exposure. Therefore, the investigation of the intravascular biocompatibility of CNTs is a critical safety issue. We studied the effects of structurally different purified CNT materials from different manufacturers on human platelets and compared their effects to amorphous carbon black nanoparticles (ACB), fullerene C60, fullerenol C60(OH)24 and NIST standard polystyrene nanobeads (PNBs). Using light transmission aggregometry of human platelet rich plasma, we found that various CNTs induce PLT aggregation and this occurs in a concentration dependent manner. In contrast to CNTs, ball-like shaped fullerene...
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Characterization of newly developed fluorescence probes in cellular systems
Kadlecová, Julie ; Hubálek Kalbáčová, Marie (advisor) ; Hendrych, Tomáš (referee)
Nanoparticles (NP) are currently a progressive area of scientific research. The possibility of synthesizing them according to the required parameters opens up possibilities for their wide use also in biomedicine. One example is a nanoparticle that can detect cellular processes, such as pH. We already know that the pH of healthy and cancer cells differs by the opposite gradient on the intracellular and extracellular side of the membrane. In this context, this work deals with the study of fluorescent silicon nanoparticles (SiNP) tested on a human keratinocyte cell line from a healthy donor (HaCaT) and from skin cancer donor (A431). Once found that even the highest concentrations of SiNP used are not cytotoxic, they can be further studied by fluorescence, confocal and super-resolution microscopy. In order to assess the pH detection properties of these SiNPs, a method for measuring intracellular pH with a fluorescent raciometric probe SNARF-1 using fluorescence spectroscopy and flow cytometry was introduced. Since the pH values of the intracellular environment are closely related to cellular metabolism, the metabolism of A431 and HaCaT cells was characterized and compared. To do this, methods for measuring analog glucose consumption (2-NBDG) and another new method for measuring real-time metabolism...
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Interaction of human immune cells with ultrasmall nanoparticles
Javorová, Pavlína ; Hubálek Kalbáčová, Marie (advisor) ; Krulová, Magdaléna (referee)
The application of nanoparticles in the field of theranostics requires knowledge of the specific interaction of nanoparticles with the immune system. One of the first cells with which nanoparticles interact when given to the body are cells of the mononuclear phagocytic system. The aim of this diploma thesis is to prepare an in vitro study that describes the effect of two types of gold and three types of silicon ultra-small nanoparticles on immune cells. Immune cells are presented in the form of primary PBMCs isolated from whole blood , and cells of monocytic cell line THP-1 in the form of monocytes and differentiated macrophages. During the experiments with primary cells, emphasis is placed on maintaining the concept of personalized protein corona. After characterization of the immune cells used, cells are subsequently stimulated with ultra-small nanoparticles and the influence of these nanoparticles on cell metabolism, viability, degree of differentiation and secretion of pro- and antiinflammatory cytokines is monitored. The outcome takes into account further use of the tested nanoparticles in the field of biomedicine. Key words: primary monocytes, cell lines, differentiation, macrophages, cytokines, cytotoxicity
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Interactions of cells with nanoparticles for bio-medical applications
Bělinová, Tereza ; Hubálek Kalbáčová, Marie (advisor) ; Benson, Veronika (referee) ; Topinka, Jan (referee)
In the past decades, nanoparticles have been viewed as a potentially powerful platform for various applications in biomedical sciences. The possible application of nanoparticles varies from drug delivery agents to novel imaging platforms and surely, some application potential still remains hidden. Thus, it is necessary to broadly study their in vitro behavior in order to assess the precise theranostic potential as well as to distinguish possible threats to human health. Even though nanoparticles are getting more and more attention in current research, still only a limited amount of information is available, especially regarding interactions of ultra-small (< 5 nm) nanoparticles with biological environment and cells. The aim of the work presented herein is to provide the reader with information concerning interactions of various ultra-small nanoparticles (silicon-based, gold, nanodiamonds) with biological environment and human cells. Dose- and time-dependent influence of the various nanoparticles on behavior of different human cells (osteoblasts, monocytes, keratinocytes, mesenchymal stem cells) was established under different conditions, stressing out the importance of protein corona (a layer of proteins originating from cultivation medium attached to nanoparticles). Biocompatibility of two...
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Dual effect of mesenchymal stem cells on cancer: its suppression or progression?
Matsakyanová, Victoria ; Hubálek Kalbáčová, Marie (advisor) ; Dibus, Michal (referee)
Mesenchymal stem cells (MSCs) are multipotent non-hematopoietic cells, capable of differentiation into osteogenic, adipogenic and chondrogenic cell types and can be isolated from bone marrow, adipose tissue or umbilical cord. Due to their differentiation, trophic and immunomodulatory properties, and also their ability to migrate spontaneously to the site of inflammation, damaged tissue or tumor microenvironment, MSCs bring a great potential to become not only a tool to support tissue repair and regeneration, but also for anticancer therapy. Their potential can be also supported by genetic manipulations, which may enhance their antitumor effect. However, in the context of tumor growth, the effect of MSCs in not so clear. While in some cases, MSCs play antitumorigenic role, in other cases they contribute to the development of tumor growth. This dual effect of MSCs on tumor growth is the result of many factors, which modulate the interaction between MSCs and tumor cells. This thesis summarizes the current knowledge of the dual effect of MSC on tumor cells and demonstrates the most promising factors that play the role in the dual effect of MSC. Key words: mesenchymal stem cells, cancer, tumor tropism, cancer suppression, cancer progression
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Targeting of Viral Nanoparticles to CD44 via Hyaluronic Acid
Hustedová, Anna ; Španielová, Hana (advisor) ; Hubálek Kalbáčová, Marie (referee)
Hyaluronic acid (HA) is widely studied as a targeting moiety to CD44 overexpressing cancer cells. Various types of nanoparticles (NPs) were modified by HA. Virus-like particles (VLPs) derived from mouse polyomavirus are an interesting class of NPs that can be modified by various targeting agents to increase their potential as gene or drug delivery vehicles for e.g. theragnostic purposes. HA has not been tested as a targeting moiety on VLPs, hence this was the focus of the current study. HA (~14 kDa) was attached to the VLPs via a bispecific Bodipy-derived fluorescent probe. To test the targeting potential of HA on comparable non-viral NPs, nanodiamonds were prepared in a similar manner. NPs functionalized with HA, together with Bodipy-labeled control variants, were tested on interactions with MDA-MB-231 cells overexpressing CD44. The NP-cell interaction via CD44 was assessed by a competitive cell-binding assay, where non-labeled HA competed for HA-binding sites at CD44 with the NPs. CD44 specific cell interactions were detected in studies with HA functionalized nanodiamonds, whereas VLP-HA* associated with cells in a less specific manner. Control VLPs with polyethylene glycol (PEG) did not interact with the cells. Results indicate that the HA targeting strategy for the VLPs requires optimization to...
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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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Cell-free scaffolds functionalized with bioactive compounds in bone tissue engineering
Lukášová, Věra ; Rampichová, Michala (advisor) ; Hubálek Kalbáčová, Marie (referee) ; Libusová, Lenka (referee)
There are good prospects for bone tissue engineering and therefore researcher is aimed towards the development of cell-free scaffolds. A cell-free scaffold serves as a temporal filling for critical size defects that do not heal spontaneously. Nevertheless, a suitable scaffold composition is yet to be discovered. Moreover, modifications of cell-free scaffolds with a drug delivery system activate the internal healing capacity. Platelets occur in the healing cascade as a natural source of growth factors (GFs), chemokines and cytokines. This autologous source of bioactive compounds enables the substitution of synthetic GFs. The aim of this thesis is to develop a bioactive cell-free scaffold with a drug delivery system supporting the physiological healing of bone defects. The centrifugal spinning method was used to produce nanofibrous poly-ε-caprolactone (PCL) scaffolds. PCL scaffolds were functionalized with different platelet concentrations. Bioactive compounds released from activated platelets were trapped within the formed fibrin net, enabling their gradual release. Improved metabolic activity, proliferation and alkaline phosphatase activity of MG-63 cells and human mesenchymal stem cells (hMSCs) were detected. The release of compounds lasted for two weeks and nearly reached the plateau phase,...
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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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