Diploma thesis entitled“Testing of GTPase inhibitors for the study of endocytosis of bionanomaterials” is summarizing the importance of bionanotransporters with a special emphasis on apoferritin, characterizes mechanisms of cellular internalization driven by GTPases and also their inhibitors. The following experimental part shows efficacy of apoferritin fluorescence labelling concluding that Cy2 NHS ester would be suitable for following analyses. Afterwards, expression of TfR1 and SCARA5 receptors was deter-mined and toxicity of dynamine inhibitor dynasore was tested. Internalization inhibition of Cy2 labelled apoferritin by dynasore was tested on five cell lines derived from breast tissue (HBL-100, T-47D, MCF-7, MDA-MB-231 and MDA-MB-468). The same cell lines were used for testing of internalization inhibition of Cy2 APO using dynasore, sodium azide or sucrose. The obtained results confirm internalization inhibition by dynasore and also by dynasore in combination with the other two inhibitors. At the same time, complexity of endocytic mechanisms is emphasized, highlighting need for further testing.

abstract: Sensitivity is a fundamental challenge for in vivo molecular magnetic resonance imaging (MRI). Here, I improve the sensitivity of metal nanoparticle contrast agents by strategically incorporating pure and doped metal oxides in the nanoparticle core, forming a soluble, monodisperse, contrast agent with adjustable T2 or T1 relaxivity (r2 or r1). I first developed a simplified technique to incorporate iron oxides in apoferritin to form "magnetoferritin" for nM-level detection with T2- and T2* weighting. I then explored whether the crystal could be chemically modified to form a particle with high r1. I first adsorbed Mn2+ ions to metal binding sites in the apoferritin pores. The strategic placement of metal ions near sites of water exchange and within the crystal oxide enhance r1, suggesting a mechanism for increasing relaxivity in porous nanoparticle agents. However, the Mn2+ addition was only possible when the particle was simultaneously filled with an iron oxide, resulting in a particle with a high r1 but also a high r2 and making them undetectable with conventional T1-weighting techniques. To solve this problem and decrease the particle r2 for more sensitive detection, I chemically doped the nanoparticles with tungsten to form a disordered W-Fe oxide composite in the apoferritin core. This configuration formed a particle with a r1 of 4,870mM-1s-1 and r2 of 9,076mM-1s-1. These relaxivities allowed the detection of concentrations ranging from 20nM - 400nM in vivo, both passively injected and targeted to the kidney glomerulus. I further developed an MRI acquisition technique to distinguish particles based on r2/r1, and show that three nanoparticles of similar size can be distinguished in vitro and in vivo with MRI. This work forms the basis for a new, highly flexible inorganic approach to design nanoparticle contrast agents for molecular MRI. / Dissertation/Thesis / Ph.D. Bioengineering 2012

The diploma´s thesis “Antitumor activity and targeted transport of rutin” deals with the effect of flavonoid rutin on tumor and non-tumor cells. The thesis is divided into the theoretical part and practical part. The theoretical part deals with rutin itself, tumor diseases and nanocarrier apoferritin. The practical part has three main parts. The first one deals with the properties of the rutin itself, its stability for storage and stability in solutions simulating distinct physiological environments. Furthermore, toxicity of rutin for tumor and non-tumor cells and the effect on the expression of proteins involved in the malignant potential of tumor cells were investigated. The second part deals with encapsulation of rutin into apoferritin (to form aporutin), characterization of this complex by means of its stability and toxicity for tumor and non-tumor cells. The third and last part focuses on a combined therapy in terms, of the synergistic action of rutin/aporutin together with doxorubicin on tumor and non-tumor cells was investigated.

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...

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...

Theme of this diploma thesis is usage of electrochemical techniques for study of apoferritin, which is artificially synthetized protein derived from ferritin. Apoferritin has empty cavity which can be used for completion of broad amount of substances. Encapsulation of anticancer drugs can decrease their unwanted side effects such as strong toxicity and therefore increase it’s effectiveness at the tumor site. Substances chosen in this diploma thesis were standard platinum and platinum drugs cisplatin and it’s analogy oxaliplatin. Measurement was performed by electrochemical method differential pulse voltammetry by which was determined encapsulation capacity of apoferritin for chosen anticancer drugs. For determination size and zeta potential of nanoparticles was used colloid analyzator ZetaSizer Nano.

Protein crystallization is significant in both biotechnology and biomedical applications. In biotechnology, crystallization is essential for determining the structure of both native and synthesized therapeutically important proteins. It can also be used as a final purification step and as a stable form for protein storage. With regard to biomedical systems, protein crystallization appears to be involved in the development and manifestation of certain human diseases. In particular, there exists evidence that L-rich ferritin crystals are involved in Hereditary Hyperferritinemia Cataract Syndrome (HHCS). In the current research a microbatch crystallization apparatus has been introduced that enables (1) multiple batch crystallization experiments at various temperatures and solution conditions in parallel and (2) quantitative monitoring of crystal growth without disturbing the progress of an experiment for observation. The primary application of the apparatus is, but not limited to, screening of protein crystallization conditions, although the system can also be used for other macromolecular and small-molecule crystallization experiments. Multiwell microbatch experiments demonstrated the dependence of apoferritin crystal growth kinetics and final crystal size on temperature and cadmium concentration. Although the solubility of apoferritin might be independent of temperature, the results of this study show that the crystal growth kinetics are affected by temperature, profoundly under some conditions. For apoferritin under near physiological conditions the solution thermodynamics in the form of the second virial coefficient have proofed to be a valuable predictor for the crystallization outcome. Furthermore, the significance of the elevated level of some divalent cations in cataractous lenses has been studied both in dilute solutions and under crystallization conditions and cadmium seems to be sole menace in apoferritin condensation.

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...

Crystallization of proteins in the human body can lead to the development of diseases such as sickle cell anemia and cataract. Understanding protein crystallization can give insight into such diseases. Furthermore, protein crystallization is necessary for protein structure resolution. This is important since resolution of protein structure is the first step towards establishing structure/function relations, and possibly towards performing specific structural modifications that may change the function in desirable directions. Another important application of protein crystallization is in downstream processing in the pharmaceutical industry where it is used for separation and as a final purification step. The present study increases knowledge of interactions between protein molecules during crystallization and hence the crystallization process. Crystallization of proteins in the human body can lead to the development of diseases such as sickle cell anemia and cataract. Understanding the processes involved in protein crystallization can help us gain a better understanding of such diseases. Crystallization of human gamma D-crystallin (HGD) and apoferritin, two proteins found in the lens, was studied in relation to cataract formation. Crystallization of both proteins was studied in the presence of divalent cations which are found at elevated concentrations in cataractous lenses. Results indicate that the divalent cations studied enhance crystallization of these proteins. A thermodynamic property, the osmotic second virial coefficient, was measured in protein solutions and its value was correlated with the occurrence of crystallization. It was found that the second virial coefficient successfully predicted crystallization of both proteins. A new method was developed for indirect measurement of the second virial coefficient using dynamic light scattering. This new method is more robust and efficient than the traditional static light scattering method. Finally the ability of solubilizers to prevent crystallization in HGD solutions was studied. A commercial solubilizer, NDSB-201, was found to increase the energy barrier to nucleation. Although this did not prevent crystallization, it resulted in fewer and smaller crystals being obtained. The naturally occurring alpha A-crystallin was a superior solubilizer to NDSB-201, as it suppressed aggregation and prevented crystallization of HGD under conditions for which NDSB-201 did not. The findings in the present study provide insight into the processes by which protein crystallization occurs and hence into diseases associated with protein crystallization. The findings in the present study provide insight into the processes by which protein crystallization occurs. Using the second virial coefficient to assess whether a protein will crystallize out of solution, approaches for retardation and prevention of protein crystallization, and implications for future research, are discussed.

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.