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Nanotransporters for theranostics
Dostálová, Simona ; Adam,, Vojtěch (referee) ; Kizek, René (advisor)
Master thesis deals with the use of bacteriophage as a theranostic drug nanocarrier. The term theranostics is used in recent years for systems that allow connecting of diagnostics, targeted drug delivery and monitoring of patient’s response to administered treatment in a single modality. These systems are very suitable especially with heterogeneous diseases, such as cancer. Nowadays, the treatment of cancer has often severe side effects to the patient’s body, which lowers his capability to fight the disease. Theoretical part of this work is focused on the properties of viral capsids, proteins and inorganic materials as drug nanocarriers. In practical part of this work, different methods for cultivation of bacteriophage are compared, both in liquid and solid medium and with different concentrations of the maltose, trough whose receptors bacteriophage is able to enter the host cell. Optimal was cultivation with 0.2% maltose in solid medium. Practical part is focused mainly on the use of bacteriophage as a nanocarrier for cytotoxic drug doxorubicin. Bacteriophage was able to encapsulate all applied concentrations of doxorubicin (0; 12.5; 25; 50; 100 and 200 g/ml), which was proved using fluorescent detection. Different times of encapsulation (2; 4; 8 and 12 hours) were studied. Optimal time was 2 hours. Encapsulation properties of bacteriophage were compared to apoferritin. Bacteriophage was able to encapsulate 4× higher concentrations of doxorubicin and its release during rinsing with water was 10× lower compared to apoferritin. This work concludes that bacteriophage is a very suitable platform for targeted drug delivery in theranostics.
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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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Cílený transport bioaktivních molekul pomocí ferritinových nanoklecí
Rogotovskaya, Alexandra
Abstract This bachelor's thesis deals with the topic of targeted transport of bioactive molecules using ferritin nanocages. Nanoparticles are widely tested for use in contemporary medicine, not only for their use in cancer management. The use of these materials is preferred due to their ability to decrease unwanted and toxic effects of conventional chemotherapy. The use of nanoparticles ensures targeted transport, which results in reduction of the toxic effect of drugs. Among the most promising nanomedicinal vehicles belong ferritins. Ferritin is a globular protein and the main natural intracellular iron storage molecule in almost all living organisms. Ferritin specifically recognizes transferrin receptor 1 (TfR1), which is usually highly expressed on various types of tumor cells. Ferritin exhibits unique ability of reversible self-assembly. Furthermore, the surface of ferritins can be chemically or genetically modified to bear variety of cancer targeting ligands. In addition, due to its origin, ferritins are biocompatible and do not induce unwanted immune reactions. Thus, ferritins are promising delivery vehicles for targeted cancer therapy.
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Optimalizace přípravy lipozomálních nosičů pro cílený transport terapeutických nukleových kyselin
Maráková, Ester
This bachelor thesis titled 'Optimization of the preparation of liposomal carriers for targeted transport of therapeutic nucleic acids' deals with the use of liposomal nanocarriers and the mechanism of RNA interference by means of siRNA in targeted therapy. The aim of this work was to optimize the preparation of complex of liposomes, polyethylene glycol and siRNA for further research, and to write an expert treatise of the issue. In the experimental part of this work, cationic liposomes were prepared from a mixture of lipids in a molar ratio of 20:50:10:20 (DODAG, DOPE, cholesterol, CPA·HCl), into which a control siRNA was encapsulated after their extrusion. Further, two types of polyethylene glycol, PEG2000 and PEG2120, were attached to the liposomal surface. The experimental part of this work consisted of three main experiments: monitoring the effect of encapsulated siRNA amount, monitoring the effect of PEGylation rate and a stability study of liposomes stored for 3 months. At the end of this work, selected samples of liposomes were imaged by scanning electron microscope. In each experiment, size, PDI and ζ potential were measured using dynamic light scattering. In the experiment, where the effect of volume of encapsulated siRNA was studied, we observed a slight increase in size with increasing amount of encapsulated siRNA. Based on results in the second-mentioned experiment, liposomes with the PEGylation rate of 5% containing PEG2000 and the PEGylation rate of 2% containing PEG2120 were selected for the stability study as well as phosphate-buffered saline (PBS) (the effect of PEGylation rate was also compared with samples prepared in 20 mM HEPES + 135 mM NaCl buffer). In a long-term storage study, we were able to demonstrate the potential of liposomes to be used as stable carriers for therapeutics. However, given that the structures of these complexes appeared to be disintegrated after 28 days, further optimization steps, like use of fast protein liquid chromatography (FPLC) or use of lipids with PEG already incorporated in their structure are needed in order to reduce the error rate.
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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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Testování inhibitorů GTPas pro studium endocytózy bionanomateriálů
Gráfová, Karolína
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.
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Využití funkčních potravin v terapii nádorových onemocnění
Vašíčková, Kateřina
This diploma thesis with the name „Use of Functional Food in Cancer Therapy“ is focused on the study of the possibility of using nutraceuticals of functional food in cancer therapy. Literary part of this work deals with the topic of functional food and their positive effects on human body, especially in prevention and treatment of various type of diseases. One of the main topic is cancer and its treatment options. These days, research using nutraceuticals as a supplementary treatment or monotherapy modality of cancer is often the subject of research. In this work, the effect of nutraceutical curcumin, whose antitumor effects were tested in vitro in both monotherapy and supplementary breast cancer treatment, were investigated. As a supplementary treatment, curcumin was tested in synergy with the cytostatic doxorubicin. Biologically active molecules have been encapsulated into a cavity of protein nanocarrier apoferritin, which has been promoted to support molecule´s therapeutic effects while protecting non-transformed breast cells. The encapsulation efficiency was about 68–72 % for all applied molecules. Stability of encapsulated curcumin and doxorubicin complexes stored in aqueous medium at 4 °C and 20 °C for 48 hours was determined by monitoring the change in optical properties, the size and surface ζ-potential of the nanoparticles or premature drug release from the nanotransporter. In addition, a colloidal stability test have been confirmed the increased protection of biologically active molecules by the process of direct encapsulation. In solutions simulating the internal environment of the organism (blood plasma, cytoplasm, endosomes), the amount of the released molecules from the apoferritin cavity was monitored at 37 °C for 48 hours. The colloidal stability test determined the blood plasma behavior for 48 hours. The toxicity of the tested complexes against the tumor cell lines MDA-MB-231 and MDA-MB-468 was determined by several cell experiments. The HBL-100 cell line was consider as a control non-transformed cells. The research of this diploma thesis determined the oxidative stress of cell lines induced by prepared com-plexes and the rate of internalization of these formulations into cells. Biocompatibility was determined by monitoring protein corona formation on the surface of nanoparticles, activation of C3 complement component, genotoxic effects on healthy breast cell lines and hemolysis of erythrocytes in human blood.
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Nanoparticle forms of anticancer drugs and the mechanisms influencing their efficiency
Urbanová, Tereza ; Stiborová, Marie (advisor) ; Hýsková, Veronika (referee)
Currently, cancer is one of the major diseases of civilization. The disadvantage of conventional chemotherapy, which began in the 1940s, is its non-specific effect, so the cytostatics are toxic to healthy cells. However, if the cytostatic is inserted into a nanotransporter, it increases its specific efficacy and reduces the negative side effects. One of the possible nanotransporters is protein called apoferritin (a protein component of ferritin, an iron-carrying protein) that contains light and heavy subunits differing in their function in iron uptake. In this bachelor thesis, the ability of apoferritin to encapsulate two cytostatics (ellipticine and doxorubicin), depending on its origin and the proportion of light and heavy apoferritin subunits, was studied.
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Functionalized hybrid polymer structures for biomedical applications
Rabyk, Mariia ; Štěpánek, Petr (advisor) ; Sedláková, Zdeňka (referee) ; Kotek, Jan (referee)
This doctoral thesis is dedicated to the synthesis and characterization of novel functionalized hybrid structures for biomedical purposes. Systems reported in this work can be subdivided into the two main groups: natural-based materials and synthetic amphiphilic block copolymers. Both groups were studied as perspective theranostic agents for medical applications. In the first group, natural polysaccharides glycogen and mannan were selected as starting materials for preparation of novel nanoconjugates that possess ability for multimodal detection in vivo. Because grafting of natural macromolecules with synthetic polymers generally slows down the biodegradation rate, both polysaccharides were modified in two different ways to form nanoprobes with or without poly(2-methyl-2-oxazoline)s chains. The prepared nanoconjugates were functionalized with N-hydroxysuccinimide-activated fluorescence and magnetic resonance imaging labels. The resulting materials were tested both in vitro and in vivo and were shown to be completely biocompatible, biodegradable and exhibit some extra benefits in terms of their practical usage in biomedicine. Glycogen was functionalized with allyl and propargyl groups with following freeze-drying from aqueous solutions to form nano- and microfibrous materials. The presence of both...
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Responsive and non-responsive soft matter nanomedicines for biomedical applications
Jäger, Eliézer ; Štěpánek, Petr (advisor) ; Sedláková, Zdeňka (referee) ; Poučková, Pavla (referee)
The thesis outlines possible medical applications of soft matter assemblies as nanotechnology based systems as well as their potential in the emerging field of nanomedicine. Nanomedicine can be defined as the investigation area encompassing the design of diagnostics and therapeutics at the nanoscale, including nanobots, nanobiosensors, nanoparticles and other nanodevices, for the remediation, prevention and diagnosis of a variety of illnesses. The ultimate goal of nanomedicine is to improve patient quality-of-life. Because nanomedicine includes the rational design of an enormous number of nanotechnology-based products focused on miscellaneous diseases, a variety of nanomaterials can be employed. Therefore, the thesis is driven by a focus on recent advances in the manufacture of soft matter-based nanomedicines specifically designed to improve cancer diagnostics and chemotherapy efficacy. It will in particular highlight liposomes, polymer-drug conjugates, drug- loaded block copolymer micelles and biodegradable polymeric nanoparticles, emphasizing the current investigations and potential novel approaches towards overcoming the remaining challenges in the field as well as a brief overview of formulations that are in clinical trials and marketed products. Based on vehicle-related and...
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