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Biocompatible protein cages for encapsulation and internatization of small interfering RNA
Mokrý, Michal ; Balvan, Jan (referee) ; Heger, Zbyněk (advisor)
This thesis is focused on creation of apoferritin nanocarrier with encapsulated small interfering RNA marked with fluorescent dye. Main objectives are optimization of pH and amount of siRNA encapsulated into apoferritin cavity and physicochemical characteristics of created nanocarrier. First part deals with theoretical knowledge necessary for understanding concept of this thesis. Second part describes used methods and evaluated results. Created apoferritin nanocarriers were optimal in size with great hemocompatibility, but long-term stability didn’t meet our expectations.
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In silico design and validation of conotoxin-based peptides for neuroblastoma nanotherapy
Mokrý, Michal ; Gumulec, Jaromír (referee) ; Heger, Zbyněk (advisor)
Práca sa zaoberá in silico dizajnom a validáciou peptidov založených na konotoxíne - MrIA, izolovaného z morských slimákov druhu Conus marmoreus a možnosti využitia týchto peptidov v liečbe neuroblastómu pomocou cielenia norepinefrínového transportéru. Päť peptidov založených na tomto konotoxíne bolo simulovaných pomocou simulácii molekulárnej dynamiky, ich trajektórie boli analyzované pre zistenie vlastností týchto peptidov. Dva homologické modely ľudského norepinefrínového transportéru boli vytvorené pre analýzu väzobných vlastností peptidov založených na konotoxíne ku norepinefrínovému transportéru. Peptidy boli následne syntetizované a použité na pokrytie apoferitínových nanočastíc s elipticínom uväzneným vnútri apoferitínu. Vytvorené peptidy a nanočastice boli ďalej skúmané pre objasnenie ich fyzikálo-chemických vlastností. Interakcie a cytotoxicita boli skúmané aplokáciou nanočastíc na bunky neuroblastómu a epitelu. Z in silico a in vitro analýz vyšiel YKL-6 peptid ako najlepší kandidát na ďalší výskum.
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Study of expression of transferrin receptors (TfR1) and their utilization in nanomedicine
Krausová, Kateřina ; Fohlerová, Zdenka (referee) ; Heger,, Zbyněk (advisor)
Bachelor thesis deals with the expression of the transferrin receptor (TfR1) and its use in nanomedicine. During the last decade, nanotechnology emerged as one of the central milestones in connecting all scientific and technological disciplines. Nanomedicine already demonstrated efficacy not only in animal models of cancer but also in clinical practice. The theoretical part is not only aimed at cancer of the human population, but also at the possibilities of targeted drug delivery into the tumor tissue, which greatly reduces the otherwise serious side effects of conventional treatment – systemic toxicity. The practical part is focused on optimization for studying the expression of the transferrin receptor, a protein overexpressed by neoplastic cells aiming to enrich the higher metabolic needs of tumor cells. The optimal conditions were as follows: lysate of 50 000 cells applied with nonreducing nondenaturing buffer and the concentration of the primary antibody of 1.0 𝜇g/ml. Different levels of TfR1 expression were detected, depending on the type of tumor cells. The cell lines of neuroblastoma, prostate cancer (occurence in every 7th man) and breast cancer (occurence in every 8th woman) were selected for the next experiments. Via this transferrin receptor, apoferritin, which is a protein storaging iron ions in many organisms, can be internalized into cells. Artificially, the internal cavity of apoferritin may be used for encapsulation and transport of any molecules. In the case of this bachelor thesis, the apoferritin was used for delivery of doxorubicin. Doxorubicin has been used for cancer treatment for more than 30 years; however, its administered dose is limited by its high toxicity. This can be reduced by its encapsulation in a suitable vector for targeted transport to the tumor cells only. Apoferritin could serve as such suitable vector. In this thesis, the suitable usage of apoferritin as a nanocarrier for chemotherapeutic delivery was confirmed. Its molecule size of 10-12 nm allows it to employ the effect of increased permeability and retention. At the same time, this size makes it possible to avoid renal clearance. The properties of encapsulated doxorubicin are not affected by apoferritin, thus preserving its toxicity for cells with a high level of TfR1 expression (30% growth inhibition of these cells after 24 h of treatment).
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Preparation of mitotic inhibitors in the form of nanotransporters
Meskařová, Veronika ; Indra, Radek (advisor) ; Hýsková, Veronika (referee)
Cancer is one of the most widespread diseases that every third person will encounter it. It is a disease arising from the sudden growth and rapid division of own cells. Despite progress in treatment, an effectiveness is not sufficient, and cancer is the second most common cause of death. In addition, the treatment is associated with several side effects. Currently nanomedicine uses nanomaterials for transport drugs in cancer treatment. It is possible to deliver the drug to the target site, but also to reduce the systemic toxicity of the drug by binding the drug to a suitable nanotransporter. This diploma thesis deals with seeking the appropriate mass ratio of antitumor drugs, colchicine and docetaxel, and the nanotransporter apoferritin. The aim was to find suitable in vitro conditions for high drugs encapsulation into the nanotransporter. Drugs encapsulation took place under active and passive conditions. Active encapsulation was under way on opening and closing nanotransporter by changing pH environment in test tube. Drugs were dissolved in DMSO or water with addition of HCl to charge the drug molecules and improve the binding of the drug into nanotransporter. As part of passive encapsulation, drugs were incubated in surroundings of urea or bivalent metal ions. Overall, it can be concluded that...
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The study of properties of anticancer drugs ellipticine, etoposide and doxorubicin in the forms of nanocarriers
Lengálová, Alžběta ; Stiborová, Marie (advisor) ; Martínková, Markéta (referee)
Currently available anticancer therapies are inadequate and spur demand for improved technologies. Among others, the utilization of nanocarriers for anticancer drug delivery has shown great potential in cancer treatment. Nanocarriers can improve the therapeutic efficiency of the drugs with minimization of the undesirable side effects. To evaluate potential application of this technology, two forms of nanocarriers have been studied: multi-walled carbon nanotubes (MWCNTs) and apoferritin. The aim of this study was to determine, whether given cytostatics (ellipticine, etoposide and doxorubicin) are bound to these nanotransporters and how are they released from them, especially depending on pH. Since the pH of the tumor cells is lower than the pH of healthy cells it would be preferred that the drugs would release from nanocarriers at the lower pH while at the physiological pH the release of the drug would be eliminated. The results found show that ellipticine is actually released from its MWCNT- and apoferrtin-encapsulated form at acidic pH (5.0), while at pH 7.4 its interaction with nanocarriers is stable. Ellipticine released from MWCNT is activated by microsomal enzymes to reactive metabolites (13- hydroxyellipticine and 12-hydroxyellipticine) forming DNA adducts. The results indicate that both...
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In silico design and validation of conotoxin-based peptides for neuroblastoma nanotherapy
Mokrý, Michal ; Gumulec, Jaromír (referee) ; Heger, Zbyněk (advisor)
Práca sa zaoberá in silico dizajnom a validáciou peptidov založených na konotoxíne - MrIA, izolovaného z morských slimákov druhu Conus marmoreus a možnosti využitia týchto peptidov v liečbe neuroblastómu pomocou cielenia norepinefrínového transportéru. Päť peptidov založených na tomto konotoxíne bolo simulovaných pomocou simulácii molekulárnej dynamiky, ich trajektórie boli analyzované pre zistenie vlastností týchto peptidov. Dva homologické modely ľudského norepinefrínového transportéru boli vytvorené pre analýzu väzobných vlastností peptidov založených na konotoxíne ku norepinefrínovému transportéru. Peptidy boli následne syntetizované a použité na pokrytie apoferitínových nanočastíc s elipticínom uväzneným vnútri apoferitínu. Vytvorené peptidy a nanočastice boli ďalej skúmané pre objasnenie ich fyzikálo-chemických vlastností. Interakcie a cytotoxicita boli skúmané aplokáciou nanočastíc na bunky neuroblastómu a epitelu. Z in silico a in vitro analýz vyšiel YKL-6 peptid ako najlepší kandidát na ďalší výskum.
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The study of properties of anticancer drugs ellipticine, etoposide and doxorubicin in the forms of nanocarriers
Lengálová, Alžběta
Currently available anticancer therapies are inadequate and spur demand for improved technologies. Among others, the utilization of nanocarriers for anticancer drug delivery has shown great potential in cancer treatment. Nanocarriers can improve the therapeutic efficiency of the drugs with minimization of the undesirable side effects. To evaluate potential application of this technology, two forms of nanocarriers have been studied: multi-walled carbon nanotubes (MWCNTs) and apoferritin. The aim of this study was to determine, whether given cytostatics (ellipticine, etoposide and doxorubicin) are bound to these nanotransporters and how are they released from them, especially depending on pH. Since the pH of the tumor cells is lower than the pH of healthy cells it would be preferred that the drugs would release from nanocarriers at the lower pH while at the physiological pH the release of the drug would be eliminated. The results found show that ellipticine is actually released from its MWCNT- and apoferrtin-encapsulated form at acidic pH (5.0), while at pH 7.4 its interaction with nanocarriers is stable. Ellipticine released from MWCNT is activated by microsomal enzymes to reactive metabolites (13- hydroxyellipticine and 12-hydroxyellipticine) forming DNA adducts. The results indicate that both...
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Anticancer drugs in forms of nanoparticles and mechanisms potentiating their anticancer efficiency
Meskařová, Veronika ; Indra, Radek (advisor) ; Bělonožníková, Kateřina (referee)
Cancer has been one of the most common diseases of civilization for centuries. In the 18th century, some cancers were described and the first treatments were proposed. Currently, oncosurgery, chemotherapy, radiotherapy, immunotherapy and hormonal treatment are used to treat cancer. At the same time, efforts are being made to find new anticancer drugs that target tumor cells more selectively. Recently, nanomedicine has also started to be used. This bachelor thesis deals with minimizing the binding of the amount of cytostatic ellipticine to the surface of the nanotransporter apoferritin and achieving higher encapsulation efficiency. Two types of apoferritins at different weight ratios to ellipticine were studied. It has been found that by finding a suitable weight ratio of the two molecules, binding can be minimized, and encapsulation efficiency can be increased. When working with commercial apoferritin, there was a higher encapsulation and a lower binding of ellipticine to the surface at the weight ratio of 1:10. In contrast, when working with recombinant apoferritin, the encapsulation is higher and ellipticine binding are lower at the lower ratio of 1: 2,5. Key words: oncological diseases, anticancer drugs, nanomedicine, cytostatic, nanotransporter, ellipticine, apoferritin [IN CZECH]
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Study of tyrosin kinase inhibitor vandetanibe bound in apoferritin and liposomes
Jáklová, Kateřina ; Indra, Radek (advisor) ; Hýsková, Veronika (referee)
5 Abstract In this thesis the anticancer drug vandetanib was studied. Vandetanib is a tyrosine kinase inhibitor affecting signalling of vascular endothelial growth factor receptor (VEGFR), epidermal growth factor receptor (EGFR) or RET protooncogene (REarranged during Transfection). It is primarily used for the treatment of advanced tumors of the thyroid gland. Unfortunately, the usage of vandetanib in the cancer treatment is significantly limited by its toxicity and cardiotoxicity (one of the adverse effects is connected with long QT interval). One way, how to minimize these side effects, is binding a drug into a suitable transporter. Apoferritin and liposomes were used as a transport nanoparticles in this study. The aim of this thesis was to study the stability of the complex of nanoparticle apoferritin with vandetanib molecules (ApoVan) and to study the effect of pH on the release of inhibitor from the ApoVan form. Experiments have shown that ApoVan complex is relatively stable after its storage at 4 řC and - 20 řC for up to 8 weeks. Unfortunately after monitoring the effect of pH on the release of vandetanib from ApoVan, it was found that vandetanib is gradually released from its ApoVan form into the neutral environment at pH 7,4 as well as into the acidic environment at pH 6,5 and the way ApoVan is...
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Effect of cytochromes P450 on metabolism of anticancer drugs bound into apoferritin nanoparticle
Wilhelm, Marek ; Indra, Radek (advisor) ; Ptáčková, Renata (referee)
Tumour-related diseases are the second most common cause of death in the Czech Republic, right after cardiovascular diseases. Nanomedicine - a novel scientific discipline - shows captivating potential in anticancer treatment with help of so called nanotranporters - nanoparticles capable of transporting other molecules. Encapsulation of a cytostatic drug into a nanoparticle improves its pharmacokinetical and pharmacodynamical properties which helps to reduce adverse side effects on non-tumour healthy tissue. In the scope of this diploma thesis apoferritin - apo-form of ferritin - was studied, since this nanotransporter shows promise for clinical use in anticancer treatment. Effect of hepatic microsomes from premedicated and control rats on biotransformation of doxorubicin cytostatic (Dox) in free and apoferritin nanoparticle-bound forms was investigated at pH 7,4. Over the course of biotransformation two types of metabolites - M1 and M2 - were observed. Regardless of the employed inductor all studied microsomes have exhibited similar metabolism of free doxorubicin and its apoferritin encapsulated form (ApoDox). Our results also imply that doxorubicin can be metabolically processed by rat hepatic microsomes in both free and ApoDox form with similar efficiency. We have also studied biotransformation...
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