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Preparation and characterisation of mRNA/DNA transfection vectors
Horák, Tomáš ; Chmelíková, Larisa (referee) ; Skopalík, Josef (advisor)
This diploma thesis deals with genetic engineering, especially the transfection of DNA into MSCs (Mesenchymal stromal cells) and dendritic cells. Both lipoplexes and metal magnetic nanoparticles were tested to introduce the vector into cells. The research was focused on finding more efficient methods of transfection. According to analysis on MADLS and gel electrophoresis, aspects playing an important role in conjugation and subsequent transfection were found. Conjugation occurs after only 4 minutes, as evidenced by an increase in zeta potential, but to achieve full conjugation it is necessary to incubate the sample for 20 minutes. Incomplete conjugation to iron nanoparticles resulted in strong carrier-carrier interactions, which formed an unwanted conglomerates. Encapsulation into liposomes with cationic surface treatment was without complications. The success rate of GFP-labeled protein expression after transfection by these methods was calculated to be 95%, resp. 91%. This result is due to low cytotoxicity. However, commercial tested kits on dendritic cells had a success rate below 5% with high cytotoxicity.
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Preparation and characterisation of mRNA/DNA transfection vectors
Horák, Tomáš ; Chmelíková, Larisa (referee) ; Skopalík, Josef (advisor)
This diploma thesis deals with genetic engineering, especially the transfection of DNA into MSCs (Mesenchymal stromal cells) and dendritic cells. Both lipoplexes and metal magnetic nanoparticles were tested to introduce the vector into cells. The research was focused on finding more efficient methods of transfection. According to analysis on MADLS and gel electrophoresis, aspects playing an important role in conjugation and subsequent transfection were found. Conjugation occurs after only 4 minutes, as evidenced by an increase in zeta potential, but to achieve full conjugation it is necessary to incubate the sample for 20 minutes. Incomplete conjugation to iron nanoparticles resulted in strong carrier-carrier interactions, which formed an unwanted conglomerates. Encapsulation into liposomes with cationic surface treatment was without complications. The success rate of GFP-labeled protein expression after transfection by these methods was calculated to be 95%, resp. 91%. This result is due to low cytotoxicity. However, commercial tested kits on dendritic cells had a success rate below 5% with high cytotoxicity.
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Preparation and characterization of superparamagnetic inorganic/polymer particles for biomedical application
Zasońska, Beata Anna ; Horák, Daniel (advisor) ; Šafařík, Ivo (referee) ; Sysel, Petr (referee)
Superparamagnetic -Fe2O3 nanoparticles were synthesized by coprecipitation of ferric and ferrous salts with a base. Resulting nanoparticles were coated with shells, such as poly(N,N- dimethylacrylamide) (PDMAAm), neat and functionalized silica (SiO2 and SiO2-NH2), and polyaniline (PANI). PDMAAm shell was introduced by modification of iron oxide nanoparticle surface with an initiator and N,N-dimethylacrylamide was polymerized producing -Fe2O3&PDMAAm core-shell particles. In case of SiO2-NH2 shell, tetramethyl orthosilicate was used to yield -Fe2O3&SiO2 nanoparticles, which were subsequently modified by (3-aminopropyl)triethoxysilane to prepare γ-Fe2O3&SiO2-NH2 particles. Oxidation of aniline hydrochloride with ammonium persulfate in an aqueous solution of poly(N-vinylpyrrolidone) in the presence of iron oxides produced -Fe2O3&PANI nanoparticles. Finally, the last type of the particles was based on thionin-modified poly(carboxymethyl methacrylate) (PCMMA&Th). The particles were characterized by techniques, such as scanning and transmission electron microscopy (SEM and TEM) and dynamic light scattering (DLS) to determine the particle morphology and hydrodynamic diameter. The presence of the functional groups, chemical composition, and the iron content were investigated by Fourier-transform...
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