Samouspořádané polymerní systémy citlivé na vnější podněty pro biomedicínu

Stimuli-responsive self-assembled polymer nanoparticles are becoming increasingly more important tools in nanomedicine. In this thesis we studied two large sets of polymer samples designed to be capable of such self-assembly. Polymers in the first set, containing poly(2-methyl-2-oxazoline) or poly[N-(2-hydroxypropyl) methacrylamide] hydrophilic blocks and poly[N-(2,2-difluoroethyl)acrylamide] thermoresponsive block, were designed to act as 19F MRI contrast agents. Polymers in the second set were designed as drug delivery systems and were based on 2-methyl-2-oxazine for hydrophilic parts and 2-propyl-2-oxazoline for thermoresponsive or 2-butyl-2-oxazoline for hydrophobic parts. Both sets of copolymers were prepared with various ratios of monomers in their blocks and the second set was also prepared with gradient chain architecture. Properties of their self-assembled systems were studied in detail and compared with regard to their potential for biomedical applications. The primary used method of analysis was dynamic light scattering supported by a vast array of methods including static light scattering, small angle X-ray and neutron scattering, nuclear magnetic resonance and others. From the investigated copolymers the most promising candidates for biomedical applications were selected and highlighted.Samouspořádané polymerní systémy citlivé na vnější podněty se v současnosti stávají stále důležitějšími nástroji pro nanomedicínu. V této disertační práci jsme studovali dvě rozsáhlé sady polymerů schopných takovéhoto samouspořádání do nanočástic. Polymery z první sady, obsahující hydrofilní blok z poly(2-methyl-2-oxazolin)u nebo poly[N-(2- hydroxypropyl)methakrylamid]u a termoresponzivní blok z poly[N-(2,2- difluorethyl)akrylamid]u byly navrženy, aby sloužily jako kontrastní látky pro 19F MRI. Polymery z druhé sady byly navrženy jako nosiče léčiv a byly založeny na 2-methyl-2-oxazinu použitém pro hydrofilní části řetězců a 2-propyl-2-oxazolinu pro termoresponzivní nebo 2- butyl-2-oxazolinu pro hydrofobní části. V každé ze sad byly připraveny polymery s různým poměrem použitých monomerů a v druhé sadě byla kromě blokové použita i gradientová architektura řetězců. Vlastnosti jejich samouspořádaných systémů byly vyhodnoceny a porovnány s ohledem na jejich potenciál pro biomedicínské využití. Jako hlavní metoda analýzy byl využit dynamický rozptyl světla doplněný širokou škálou dodatečných metod včetně statického rozptylu světla, maloúhlového rozptylu rentgenového záření a neutronů, nukleární magnetické rezonance a dalších. Ze všech zkoumaných polymerů byly vybrány ty s největším potenciálem pro biomedicínské...Matematicko-fyzikální fakultaFaculty of Mathematics and Physic

The analysis of membrane potential recovery in yeast under CCCP-induced stress

The master's thesis is focused on the study of response of the intracellular pH of the yeast cells on various external environments, primarily in a relation to the protonophore carbonyl cyanide m-chlorophenylhydrazone, CCCP. To measure the intracellular pH of the yeast cells we used a genetically coded fluorescent probe the ratiometric pHluorin. Using the method of synchronously scanned fluorescent spectra we were able to measure the intracellular pH of the cells with high precision. As a part of these experiments we also studied the influence of ionic strength of the cell suspensions buffers on the surface potential as well as the influence of the mineral salt KCl on the depolarization of the yeast membranes and cytosolic acidification induced by the protonophore CCCP. We examined the changes of cytosolic pH as such but we also used the measured pH as an indicator of the processes and the state of environment outside the cell. One of the most notable outcomes of this thesis is a new method of monitoring the value of the surface potential of the yeast cells by measuring the titration curves of cytosolic acidification induced by the protonophore CCCP

The analysis of membrane potential recovery in yeast under CCCP-induced stress

Diplomová práce se zabývá studiem odezvy vnitrobuněčného pH kvasinkových buněk na různá vnější prostředí, především ve vztahu k protonoforu karbonylkyanidu m-chlorophenylhydrazonu, CCCP. K měření vnitrobuněčného pH kvasinkových buněk jsme využívali geneticky kódovanou fluorescenční sondu poměrový pHluorin. S využitím metody synchronně skenovaných fluorescenčních spekter jsme byli schopni měřit vnitrobuněčné pH buněk s vysokou přesností. Jako součást těchto experimentů jsme studovali také jak vliv iontové síly pufrů buněčných suspenzí na povrchový potenciál kvasinek, tak vliv anorganické soli KCl na depolarizaci membrán kvasinek a acidifikaci cytosolu vyvolanou protonoforem CCCP. Zkoumali jsme jednak přímo změny cytosolického pH a jednak jsme toto naměřené pH používali jako indikátor procesů a stavu prostředí mimo buňku. K nejvýznamnějším výsledkům této práce patří vyvinutí nové metody monitorování velikosti povrchového potenciálu kvasinek pomocí titračních křivek acidifikace cytosolu vyvolané protonoforem CCCP.The master's thesis is focused on the study of response of the intracellular pH of the yeast cells on various external environments, primarily in a relation to the protonophore carbonyl cyanide m-chlorophenylhydrazone, CCCP. To measure the intracellular pH of the yeast cells we used a genetically coded fluorescent probe the ratiometric pHluorin. Using the method of synchronously scanned fluorescent spectra we were able to measure the intracellular pH of the cells with high precision. As a part of these experiments we also studied the influence of ionic strength of the cell suspensions buffers on the surface potential as well as the influence of the mineral salt KCl on the depolarization of the yeast membranes and cytosolic acidification induced by the protonophore CCCP. We examined the changes of cytosolic pH as such but we also used the measured pH as an indicator of the processes and the state of environment outside the cell. One of the most notable outcomes of this thesis is a new method of monitoring the value of the surface potential of the yeast cells by measuring the titration curves of cytosolic acidification induced by the protonophore CCCP.Fyzikální ústav UKInstitute of Physics of Charles UniversityMatematicko-fyzikální fakultaFaculty of Mathematics and Physic

The analysis of membrane potential recovery in yeast under CCCP-induced stress

The master's thesis is focused on the study of response of the intracellular pH of the yeast cells on various external environments, primarily in a relation to the protonophore carbonyl cyanide m-chlorophenylhydrazone, CCCP. To measure the intracellular pH of the yeast cells we used a genetically coded fluorescent probe the ratiometric pHluorin. Using the method of synchronously scanned fluorescent spectra we were able to measure the intracellular pH of the cells with high precision. As a part of these experiments we also studied the influence of ionic strength of the cell suspensions buffers on the surface potential as well as the influence of the mineral salt KCl on the depolarization of the yeast membranes and cytosolic acidification induced by the protonophore CCCP. We examined the changes of cytosolic pH as such but we also used the measured pH as an indicator of the processes and the state of environment outside the cell. One of the most notable outcomes of this thesis is a new method of monitoring the value of the surface potential of the yeast cells by measuring the titration curves of cytosolic acidification induced by the protonophore CCCP

The analysis of membrane potential recovery in yeast under CCCP-induced stress

The master's thesis is focused on the study of response of the intracellular pH of the yeast cells on various external environments, primarily in a relation to the protonophore carbonyl cyanide m-chlorophenylhydrazone, CCCP. To measure the intracellular pH of the yeast cells we used a genetically coded fluorescent probe the ratiometric pHluorin. Using the method of synchronously scanned fluorescent spectra we were able to measure the intracellular pH of the cells with high precision. As a part of these experiments we also studied the influence of ionic strength of the cell suspensions buffers on the surface potential as well as the influence of the mineral salt KCl on the depolarization of the yeast membranes and cytosolic acidification induced by the protonophore CCCP. We examined the changes of cytosolic pH as such but we also used the measured pH as an indicator of the processes and the state of environment outside the cell. One of the most notable outcomes of this thesis is a new method of monitoring the value of the surface potential of the yeast cells by measuring the titration curves of cytosolic acidification induced by the protonophore CCCP

Response of plasma membrane potential and intracellular pH of yeast to changes in the concentration of extracellular K+

The bachelor thesis is focused on studying yeast cells and their response to various external conditions. Main focus was on the study of intracellular pH and membrane potential change under the condition of varying extracellular concentration of K+ ions. In particular we studied to what extent are the yeast cells able to compensate these changes. The ability of yeasts to resist the changes of external pH of the cell medium was studied in an experiment complementary to the measurements of intracellular pH. To measure the intracellular pH a genetically encoded fluorescent probe ratiometric pHluorin was used and to measure the changes of membrane potential a fluorescent probe diS-C3(3) was used. Also we successfully applied a method of synchronously scanned fluorescence to supress the cell autofluorescence

The analysis of membrane potential recovery in yeast under CCCP-induced stress

Diplomová práce se zabývá studiem odezvy vnitrobuněčného pH kvasinkových buněk na různá vnější prostředí, především ve vztahu k protonoforu karbonylkyanidu m-chlorophenylhydrazonu, CCCP. K měření vnitrobuněčného pH kvasinkových buněk jsme využívali geneticky kódovanou fluorescenční sondu poměrový pHluorin. S využitím metody synchronně skenovaných fluorescenčních spekter jsme byli schopni měřit vnitrobuněčné pH buněk s vysokou přesností. Jako součást těchto experimentů jsme studovali také jak vliv iontové síly pufrů buněčných suspenzí na povrchový potenciál kvasinek, tak vliv anorganické soli KCl na depolarizaci membrán kvasinek a acidifikaci cytosolu vyvolanou protonoforem CCCP. Zkoumali jsme jednak přímo změny cytosolického pH a jednak jsme toto naměřené pH používali jako indikátor procesů a stavu prostředí mimo buňku. K nejvýznamnějším výsledkům této práce patří vyvinutí nové metody monitorování velikosti povrchového potenciálu kvasinek pomocí titračních křivek acidifikace cytosolu vyvolané protonoforem CCCP.The master's thesis is focused on the study of response of the intracellular pH of the yeast cells on various external environments, primarily in a relation to the protonophore carbonyl cyanide m-chlorophenylhydrazone, CCCP. To measure the intracellular pH of the yeast cells we used a genetically coded fluorescent probe the ratiometric pHluorin. Using the method of synchronously scanned fluorescent spectra we were able to measure the intracellular pH of the cells with high precision. As a part of these experiments we also studied the influence of ionic strength of the cell suspensions buffers on the surface potential as well as the influence of the mineral salt KCl on the depolarization of the yeast membranes and cytosolic acidification induced by the protonophore CCCP. We examined the changes of cytosolic pH as such but we also used the measured pH as an indicator of the processes and the state of environment outside the cell. One of the most notable outcomes of this thesis is a new method of monitoring the value of the surface potential of the yeast cells by measuring the titration curves of cytosolic acidification induced by the protonophore CCCP.Katedra makromolekulární fyzikyDepartment of Macromolecular PhysicsFaculty of Mathematics and PhysicsMatematicko-fyzikální fakult

Response of plasma membrane potential and intracellular pH of yeast to changes in the concentration of extracellular K+

The bachelor thesis is focused on studying yeast cells and their response to various external conditions. Main focus was on the study of intracellular pH and membrane potential change under the condition of varying extracellular concentration of K+ ions. In particular we studied to what extent are the yeast cells able to compensate these changes. The ability of yeasts to resist the changes of external pH of the cell medium was studied in an experiment complementary to the measurements of intracellular pH. To measure the intracellular pH a genetically encoded fluorescent probe ratiometric pHluorin was used and to measure the changes of membrane potential a fluorescent probe diS-C3(3) was used. Also we successfully applied a method of synchronously scanned fluorescence to supress the cell autofluorescence

Self-assembled polymer systems responsive to external stimuli for biomedicine

Stimuli-responsive self-assembled polymer nanoparticles are becoming increasingly more important tools in nanomedicine. In this thesis we studied two large sets of polymer samples designed to be capable of such self-assembly. Polymers in the first set, containing poly(2-methyl-2-oxazoline) or poly[N-(2-hydroxypropyl) methacrylamide] hydrophilic blocks and poly[N-(2,2-difluoroethyl)acrylamide] thermoresponsive block, were designed to act as 19F MRI contrast agents. Polymers in the second set were designed as drug delivery systems and were based on 2-methyl-2-oxazine for hydrophilic parts and 2-propyl-2-oxazoline for thermoresponsive or 2-butyl-2-oxazoline for hydrophobic parts. Both sets of copolymers were prepared with various ratios of monomers in their blocks and the second set was also prepared with gradient chain architecture. Properties of their self-assembled systems were studied in detail and compared with regard to their potential for biomedical applications. The primary used method of analysis was dynamic light scattering supported by a vast array of methods including static light scattering, small angle X-ray and neutron scattering, nuclear magnetic resonance and others. From the investigated copolymers the most promising candidates for biomedical applications were selected and highlighted

Fluorine polymer probes for magnetic resonance imaging: quo vadis?

Over the last few years, the development and relevance of F-19 magnetic resonance imaging (MRI) for use in clinical practice has emerged. MRI using fluorinated probes enables the achievement of a specific signal with high contrast in MRI images. However, to ensure sufficient sensitivity of F-19 MRI, fluorine probes with a high content of chemically equivalent fluorine atoms are required. The majority of F-19 MRI agents are perfluorocarbon emulsions, which have a broad range of applications in molecular imaging, although the content of fluorine atoms in these molecules is limited. In this review, we focus mainly on polymer probes that allow higher fluorine content and represent versatile platforms with properties tailorable to a plethora of biomedical in vivo applications. We discuss the chemical development, up to the first imaging applications, of these promising fluorine probes, including injectable polymers that form depots that are intended for possible use in cancer therapy