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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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Modifikace ferritinových nanoklecí cílícími modalitami
Kurcová, Jana
In last two decades, application of protein nanoparticles has emerged as an excellent candidate in a field of biomedical research, especially as a tool for targeted drug delivery. Nanocarriers can ensure specific drug delivery into the damaged cells without the risk of premature release of the drug hence damage to normal cells. It is possible to modify the surface of nanocarriers with targeting modalities and use active targeting to improve cell specificity and internalization of nanocarriers. Therefore, this thesis is focused on specific surface modifications of nanocarriers with respect to active targeted drug delivery. Herein, a new method of covalent binding of the hexameric HWR (HWRGWV) peptide to the surface of fluorescently labeled apoferritin cages (APO-Cy7) using a zero-length crosslinker N,N’ dicyclohexylcarbodiimide (DCC), is presented. HWR peptide can specifically bind Fc region of antibodies enabling its application for site directed conjugation with various targeting antibodies. The optimal ratio of HWR to APO for maximum reaction effect was 1.26 mg HWR to 1 mg APO-Cy7. Furthermore, the stability of as-prepared fluorescent nanocarrier was confirmed after the binding with peptide. Fluorescence of nanocarrier increased in the emission maximum (λ = 773 nm) from 1943 ± 209 a.u. to 2594 ± 62 a.u. due to the transfer of electrons from the tryptophan residues of the peptide to Cy7. The size of the nanocarrier was 12.71 ± 0.49 nm with a surface ζ-potential of -14.37 ± 3.15 mV. The correct binding orientation of the peptide was confirmed using polyclonal IgG antibodies. After incubation and following purification, the presence of antibodies was proven spectrophotometrically, via SDS-PAGE and by changing the size of the nanocarrier. Thus, the presented work demonstrates a versatile platform suitable for a surface modification with targeting antibodies for utilization in active anticancer nanomedicine.
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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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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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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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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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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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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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