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Self-Assembled Polymer Systems Based on Poly[(N-2,2-difluoroethyl)acrylamide] as Diagnostic and Theranostic Tracers for 19F Magnetic Resonance Imaging
Kolouchová, Kristýna ; Hrubý, Martin (advisor) ; Matějíček, Pavel (referee) ; Poučková, Pavla (referee)
In this doctoral thesis we describe a series of stimuli-responsive polymers which could be used as diagnostic tools or as smart drug delivery systems with simultaneous diagnosis (theranostics). All hereby mentioned polymers are thermoresponsive copolymers of (N-2,2-difluoroethyl)acrylamide exhibiting lower critical solution temperature in aqueous milieu. This means that they are water-soluble at low temperature while when heated above a certain temperature, they self-assemble into nano- or macro- sized assemblies. Because of the high concentration of fluorine atoms, all these polymers could be used as 19 F MRI tracers. We designed multiple different thermoresponsive, thermo- and pH-responsive, thermo-and reactive-oxygen-species-responsive polymers that could find numerous discussed applications in human medicine. We investigated their physico-chemical properties with 1 H and 19 F nuclear magnetic resonance (NMR), size exclusion chromatography (SEC), elemental analysis, dynamic light scattering (DLS), static light scattering (SLS), small angle neutron scattering (SANS), small-angle X-ray scattering (SAXS), and turbidimetry. After the physicochemical optimization of the parameters for the selected applications, their biocompatibility was tested in vitro. Several promising polymers were tested in vivo...
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Lipid-based nanoparticles for the delivery of naphthoquinone derivatives for the treatment of psoriasis
Ulrichová, Tereza ; Hrubý, Martin (advisor) ; Poučková, Pavla (referee)
Psoriasis is an autoimmune chronic skin disease with a global prevalence of around 2-4%. Pharmacological treatment currently serves mainly to suppress the symptoms of the disease. However, many drugs have major side effects and therapy must often be discontinued. On the other hand, advances in understanding the pathophysiology of psoriasis have pointed out several molecular targets to be considered for future treatment. Traditional Chinese medicine has been using herbal extracts containing various types of 1,4-naphthoquinones for psoriasis treatment. These naphthoquinones have attracted much attention from the pharmaceutical industry for their structural diversity and wide range of pharmacological effects. Colored 1,4-naphthoquinones shikonin, juglon and their derivatives have already been tested on a numerous cell lines and animal models. The problem is their limited solubility in water, thermolability and susceptibility to oxidation and polymerization. Topical application is complicated by their strong binding to the upper layer of the skin, which causes skin discoloration that is difficult to wash off and reduces their penetration into the deeper layers of the skin, where their therapeutic effect is needed. To solve these problems, we developed within this thesis colorless prodrugs based on...
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Antitumour efficacy of gels containing liposomes with hydroxy-aluminium phthalocyanine in photodynamic therapy
Kobrle, Pavel ; Poučková, Pavla (advisor) ; Štukavec, Jan (referee)
The targets of the diploma thesis presented here were to establish the antitumour efficacy of a new liposomal product with hydroxy-aluminium phthalocyanine (PC) in photodynamic therapy (PDT) of malignant tumours and determine the optimum time interval between the topical application of the product and PDT (tumour exposure to light). Effects were examined of the PC concentration on the PDT efficacy in a colon carcinoma cell line (SW-620), mammary carcinoma cell line (MDA-MB-231) and human neuroblastoma cell line (UKF NB-3). Hydroxy-aluminium phthalocyanine was employed as a photosensitizer. Outbred athymic female nude CD-1 mice already bearing the above mentioned xenotransplanted tumours were used in the experiments. The area comprising the tumour was exposed to light from a xenon lamp (600-700 nm, 80 J/cm2 ). The results were statistically evaluated, summarized in tables and plotted in graphs. In the diploma work, the liposomal product containing phthalocyanine (9-18 mg/ml) was shown to have considerable antitumour effects in the types of tumours chosen. The optimum time interval for the PDT is of 10 min after the product topical application. Key words: photodynamic therapy; hydroxy-aluminium phthalocyanine; liposomal drug delivery
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A study on the activity of α-tomatine and newly synthetized derivatives of benzo[c]fluorene
Suchá, Lenka ; Tomšík, Pavel (advisor) ; Pejchal, Jaroslav (referee) ; Poučková, Pavla (referee)
Many of anticancer drugs are of natural origin. Plants from the Solanaceae family contain various steroidal glycoalkaloids that are toxic to cancer cells. The aim of the first part of this work was to evaluate the potential anticancer effect of α-tomatine - a steroidal glycoalkaloid from Solanaceae plant family. The second part of this work was focused on the anticancer research of newly synthetized derivatives of benzo[c]fluorene. Benfluron, one of the derivatives, was reported 30 years ago to have anticancer effects in vitro and in vivo almost. Tomatine inhibited the proliferation and viability of MCF-7 cells. In this cancer cell line treated with tomatine, no DNA damage, no changes in p53 and p21WAF1/CIP1 protein levels, neither the induction of apoptosis was demonstrated. However, the decrease in ATP levels was found. New synthetized derivatives of benfluron and dimefluron caused higher cytotoxic effect in the different cancer cell lines compared with their original compounds. On the other hand, these derivatives did not caused higher tumour growth inhibition when compared with their original compounds in in vivo studies. Benfluron and hydrazone of dimeflurone prolonged the survival of mice with inoculated solid form of Ehrlich tumour. In this work, we demontrated that tomatine did not induce apoptosis...
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Mitochondria as a target of anticancer therapy.
Dvořák, Aleš ; Ježek, Petr (advisor) ; Poučková, Pavla (referee) ; Vecka, Marek (referee)
Mitochondrial isocitrate dehydrogenase 2 (IDH2) catalyzes reductive carboxylation (RC, reverse Krebs cycle pathway) and 2HG synthesis (2HG) - metabolite of which many scientists are interested. 2HG may be concurrently synthetized in cytosol by IDH1. RC is involved in anabolic reactions necessary for cell proliferation - produces citrate, fatty acid precursor - especially in hypoxia. IDH2 and IDH1 are not the only enzymes that are involved in 2HG synthesis. Recently, several enzymes, which participate in 2HG production, have been discovered. 2HG is useful in cancer diagnostics due to its overproduction by transformed cells. Moreover, 2HG may cause epigenetic changes via inhibition of 2-oxoglutarate dependent dioxygenase. In this work, the importance of RC and 2HG synthesis in cancer and healthy cells was investigated by gas chromatography with mass spectrometry detection as well as IDH2 influence. We found that IDH2 significantly participates in reverse RC and 2HG synthesis in breast cancer cell lines and uses glutaminolysis as a supplementary anaplerotic pathway. RC is increased by hypoxia, inhibition of respiration, and decreased by activation of respiration or hypocapnia. We confirmed 2HG synthesis and RC in healthy cells (fibroblasts, breast epithelial cells etc.) as well as in cancer cells....
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Autoflurescence spectroscopy of colorectal carcinoma
Ducháč, Vítězslav ; Horák, Ladislav (advisor) ; Hoch, Jiří (referee) ; Poučková, Pavla (referee)
The submitted dissertation addresses the issue of autofluorescence diagnostics of colorectal carcinoma. The following objectives were set: to verify the possibilities of exploiting the autofluorescence properties of normal colon tissue and tissue changed by tumour to differentiate them and to verify the possibility of using an experimentally created portable measuring set to carry out an "optical biopsy" during the colorectal carcinoma operation. The project solution was divided into three phases: Phase I focused on experimental verification of the fluorescence characteristics of normal colorectal tissue and tissue changed by tumour with the use of argon and a helium-neon laser. The obtained values confirmed the possibility to differentiate not only in terms of quality (change in the spectral curve) but quantity as well (change in intensity of the autofluorescence emission at selected wavelengths). When excited by the He-Ne laser, an approx. 30 a.u. contribution was observed practically throughout the measured interval, the maximum being between 670 - 700 nm, which corresponds to the fluorescence spectre of porphyrins. Normal tissue did not show hardly any activity. When the Ar laser was used, intensity discriminant values ranged only in individual units. Phase II was devoted to verification of the...
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Contribution to radiodiagnostics and to treatment of chosen pathological lesions of femur in childhood and in adults
Horák, Martin ; Poučková, Pavla (advisor) ; Marek, Josef (referee) ; Vítek, František (referee)
7 Abstract (AJ) Introduction Radiology examination using specialized modern imaging methods, including CT and MRI, is essential in the diagnosis of congenital and acquired diseases of the musculoskeletal system. The first part of the dissertation deals with certain congenital defects of the short femur, known in the literature as proximal femoral focal deficiency (PFFD). This part summarizes our experience with the radiological findings in the preoperative and postoperative period, with the main attention to the vascular supply to the affected area. The second part of the presentation deals with some aspects of autologous chondrocyte transplantation fixed at two different carriers implanted into post-traumatic articular cartilage defects of the distal femur. Radiological findings are evaluated in the relation to the histopathological findings. Objectives The first part of the study after the distribution of patients with PFFD by current commonly used radiographic classification sets the objective in the extent of scans of the hip joints to specify diagnosis PFFD in each patient and to evaluate in detail changes in the area of disability, especially a course of blood vessels. The evaluation of the radiation burden of repeated X-ray measurements was done with respect to the age of the patients. Tissue samples...
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Self-Assembled Polymer Systems Based on Poly[(N-2,2-difluoroethyl)acrylamide] as Diagnostic and Theranostic Tracers for 19F Magnetic Resonance Imaging
Kolouchová, Kristýna ; Hrubý, Martin (advisor) ; Matějíček, Pavel (referee) ; Poučková, Pavla (referee)
In this doctoral thesis we describe a series of stimuli-responsive polymers which could be used as diagnostic tools or as smart drug delivery systems with simultaneous diagnosis (theranostics). All hereby mentioned polymers are thermoresponsive copolymers of (N-2,2-difluoroethyl)acrylamide exhibiting lower critical solution temperature in aqueous milieu. This means that they are water-soluble at low temperature while when heated above a certain temperature, they self-assemble into nano- or macro- sized assemblies. Because of the high concentration of fluorine atoms, all these polymers could be used as 19 F MRI tracers. We designed multiple different thermoresponsive, thermo- and pH-responsive, thermo-and reactive-oxygen-species-responsive polymers that could find numerous discussed applications in human medicine. We investigated their physico-chemical properties with 1 H and 19 F nuclear magnetic resonance (NMR), size exclusion chromatography (SEC), elemental analysis, dynamic light scattering (DLS), static light scattering (SLS), small angle neutron scattering (SANS), small-angle X-ray scattering (SAXS), and turbidimetry. After the physicochemical optimization of the parameters for the selected applications, their biocompatibility was tested in vitro. Several promising polymers were tested in vivo...
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Novel hybrid polysaccharide-based polymers for biomedicine
Loukotová, Lenka ; Hrubý, Martin (advisor) ; Sedláková, Zdeňka (referee) ; Poučková, Pavla (referee)
1 Abstract This doctoral thesis is focused on the synthesis and characterization of novel hybrid polysaccharide-based polymers applicable for biomedicine, specifically for a conceptually new bimodal cancer treatment - immunoradiotherapy. For this purpose, polysaccharides β-glucan from Auricularia auricula-judae and κ-carrageenan from Kappaphycus alvarezii, exhibiting immunostimulatory and anticancer activities, were chosen to be grafted with thermoresponsive poly(2-isopropyl-2-oxazoline-co-2-butyl-2-oxazoline)s (POXs) (with different graft lengths and grafting densities) that induced a lower critical solution temperature of the final polymers. The thermoresponsive behavior of resulting polymers was studied with temperature-dependent light scattering methods, fluorescence measurements and also nuclear magnetic spectroscopy to select a polymer material with the most suitable properties for the intended application, aiming at a polymer depot formation after the injection of a polymer solution into the body. The chosen polymer, β-glucan-graft-POX with graft length of 2500 Da, was then modified to bear 1,4,7,10-tetraazacyclododecane-1,4,7,10- tetraacetic acid and a fluorescent dye Dyomics-615 at the graft ends and tested first in vitro to investigate its immunostimulatory properties and also the cellular uptake....
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