Solunulkoisten vesikkeleiden sisäänottomekanismien erottaminen elinaikaerotteisella fluoresenssimikroskopialla : Elinaikaerotteisesta fluoresenssimikroskopiasta saatavan tiedon analysointimenetelmien vertailua

Solunulkoiset vesikkelit ovat potentiaalisia lääkeaineenkantajia, diagnostisia välineitä sekä terapeuttisia tuotteita niiden biologisten funktioidensa vuoksi, mutta niiden toimintaa soluissa ei vielä tunnetta hyvin. Leimaamalla solunulkoiset vesikkelit fluoresoivalla merkkiaineella niiden kulkua soluihin ja solujen sisällä voidaan tutkia elinaikaerotteisella fluoresenssimikroskopialla, jolla saadaan selville sekä merkkiaineen fluoresenssin intensiteetti sekä elinaika jokaisessa kuvan pikselissä. Tässä diplomityössä eturauhasen syöpäsoluista eristetyt solunulkoiset vesikkelit leimattiin roottori-BODIPY-merkkiaineella, jonka fluoresenssin elinaika muuttuu ympäristön viskositeetin muuttuessa. Käytetyt solunulkoiset vesikkelit on nimetty eksosomeiksi ja mikrovesikkeleiksi, ja ne on erotettu toisistaan eri laskeutumisnopeuksien ja nostetiheyksien perusteella differentiaalisella ultrasentrifugoinnilla. Merkkiaineella leimattujen ja ultrasuodatuksella puhdistettujen vesikkelien kulkua eturauhasen syöpäsoluihin tutkittiin elinaikaeroteisella fluoresenssimikroskopialla. Vaikkakin aikaisemmissa tutkimuksissa oli huomattu, että eksosomit ja mikrovesikkelit kulkeutuisivat soluissa eri paikkoihin, ei tässä tutkimuksessa päädytty tällaiseen lopputulokseen. Huomattiin kuitenkin, että käytetyllä roottori-BODIPY-merkkiaineella voitiin seurata solunulkoisten vesikkeleiden luonnollista kulkeutumista solussa. Tässä diplomityössä havaittiin, että käytetty merkkiaine muodostaa aggregaatteja vesiliuoksissa, ja että aggregaattien fluoresenssi havaitaan pidemmillä aallonpituuksilla kuin monomeerisen merkkiaineen. Elinaikaerotteisesta fluoresenssimikroskopiasta saatua tietoa käsiteltiin eri tavoin, jotta saatiin selville parhaimmat analysointimenetelmät tulevaisuudessa tehtäviä kokeita varten. Kun fluoresenssin vaimenemiskuvaajiin tehtiin kahta erilaista sovitusta, saatiin elinaikakomponenteille suurin piirtein samansuuruiset arvot. Elinaikahistogrammeihin taas sovitettiin kahta Gaussin käyrää, jolloin saatiin tietoa elinajan lisäksi myös elinaikakomponenttien populaatioiden suuruuksista ja vaihteluista seurannan aikana. Histogrammeista saadut elinajat kuitenkin poikkesivat jonkin verran fluoresenssin vaimenemiskuvaajiin tehtyjen sovitusten elinajoista, joten se ei ole yhtä luotettava tapa tarkkaan elinajan määritykseen.Extracellular vesicles are potential diagnostic and therapeutic vehicles and drug delivery systems because of their biological functions. However, their actions inside the cells are not well known. By labelling extracellular vesicles with fluorescent labels their uptake and trafficking in cells can be monitored with fluorescence lifetime imaging microscopy, which gives the information about intensity and lifetime of the fluorescence signal in every pixel of the image. In this master’s thesis, extracellular vesicles separated from the growth medium of prostate cancer cells were labelled with a fluorescent label (rotor-BODIPY) which fluorescence lifetime changes when viscosity of the environment changes. Used extracellular vesicles are divided into exosomes and microvesicles according to their sedimentation rate and buoyant density by differential ultracentrifugation. The trafficking of extracellular vesicles in prostate cancer cells was followed with fluorescence lifetime imaging microscopy. There are previous studies were the fluorescence signal of the label is different depending on the used delivery system. In this thesis, this phenomenon was not observed. However, rotor-BODIPY allowed us to follow the natural trafficking of extracellular vesicles inside the cells. It was noticed, that used label formed aggregates in aqueous environments, and the fluorescence of aggregates shifted to longer wavelengths than the fluorescence of monomeric label. Different analysis methods were used to obtain the best method for future studies. Fluorescence decay curves were analyzed in two different ways, that both gave same values for the fluorescence lifetime components. Two Gaussian curves were fitted to the lifetime histograms of fluorescence lifetime imaging microscopy images, and among the lifetime data, also knowledge of each lifetime component’s population size and change during measurements were obtained. The fluorescence lifetime data gotten from the lifetime histograms differed from the data that was obtained from the fluorescence decay curves, so it is not as reliable way to estimate the values of the fluorescence lifetime components

Addressing challenges in the removal of unbound dye from passively labelled extracellular vesicles

Correction: 10.1039/d1na90120fStudies of extracellular vesicles (EVs), their trafficking and characterization often employ fluorescent labelling. Unfortunately, little attention has been paid thus far to a thorough evaluation of the purification of EVs after labelling, although the presence of an unbound dye may severely compromise the results or even lead to wrong conclusions on EV functionality. Here, we systematically studied five dyes for passive EV labelling and meticulously compared five typical purification methods: ultracentrifugation (UC), ultracentrifugation with discontinuous density gradient (UCG), ultrafiltration (UF), size exclusion chromatography (SEC), and anion exchange chromatography (AEC). A general methodology for evaluation of EV purification efficiency after the labelling was developed and tested to select the purification methods for the chosen dyes. Firstly, we found that some methods initially lead to high EV losses even in the absence of the dye. Secondly, the suitable purification method needs to be found for each particular dye and depends on the physical and chemical properties of the dye. Thirdly, we demonstrated that the developed parameter E-rp (relative purification efficiency) is a useful tool for the pre-screening of the suitable dye-purification method combinations. Additionally, it was also shown that the labelled EVs properly purified from the unbound dye may show significantly reduced contrast and visibility in the target application, e.g. in the live cell fluorescence lifetime imaging.Peer reviewe

Assembly of Bleomycin Saccharide-Decorated Spherical Nucleic Acids

Glyco-decorated spherical nucleic acids (SNAs) may be attractive delivery vehicles, emphasizing the sugar-specific effect on the outer sphere of the construct and at the same time hiding unfavorable distribution properties of the loaded oligonucleotides. As examples of such nanoparticles, tripodal sugar constituents of bleomycin were synthesized and conjugated with a fluorescence-labeled antisense oligonucleotide (AONARV7). Successive copper(I)-catalyzed azide-alkyne and strain-promoted alkyne-nitrone cycloadditions (SPANC) were utilized for the synthesis. Then, the glyco-AONARV7 conjugates were hybridized with complementary strands of a C60-based molecular spherical nucleic acid (i.e., a hybridization-mediated carrier). The formation and stability of these assembled glyco-decorated SNAs were evaluated by polyacrylamide gel electrophoresis (PAGE), UV melting profile analysis, and time-resolved fluorescence spectroscopy. Association constants were extracted from time-resolved fluorescence data. Preliminary cellular uptake experiments of the glyco-AONARV7 conjugates (120 nM solutions) and of the corresponding glyco-decorated SNAs (10 nM solutions) with human prostate cancer cells (PC3) showed an efficient uptake in each case. A marked variation in intracellular distribution was observed.</p

Solunulkoisten vesikkeleiden sisäänottomekanismien erottaminen elinaikaerotteisella fluoresenssimikroskopialla : Elinaikaerotteisesta fluoresenssimikroskopiasta saatavan tiedon analysointimenetelmien vertailua

Solunulkoiset vesikkelit ovat potentiaalisia lääkeaineenkantajia, diagnostisia välineitä sekä terapeuttisia tuotteita niiden biologisten funktioidensa vuoksi, mutta niiden toimintaa soluissa ei vielä tunnetta hyvin. Leimaamalla solunulkoiset vesikkelit fluoresoivalla merkkiaineella niiden kulkua soluihin ja solujen sisällä voidaan tutkia elinaikaerotteisella fluoresenssimikroskopialla, jolla saadaan selville sekä merkkiaineen fluoresenssin intensiteetti sekä elinaika jokaisessa kuvan pikselissä. Tässä diplomityössä eturauhasen syöpäsoluista eristetyt solunulkoiset vesikkelit leimattiin roottori-BODIPY-merkkiaineella, jonka fluoresenssin elinaika muuttuu ympäristön viskositeetin muuttuessa. Käytetyt solunulkoiset vesikkelit on nimetty eksosomeiksi ja mikrovesikkeleiksi, ja ne on erotettu toisistaan eri laskeutumisnopeuksien ja nostetiheyksien perusteella differentiaalisella ultrasentrifugoinnilla. Merkkiaineella leimattujen ja ultrasuodatuksella puhdistettujen vesikkelien kulkua eturauhasen syöpäsoluihin tutkittiin elinaikaeroteisella fluoresenssimikroskopialla. Vaikkakin aikaisemmissa tutkimuksissa oli huomattu, että eksosomit ja mikrovesikkelit kulkeutuisivat soluissa eri paikkoihin, ei tässä tutkimuksessa päädytty tällaiseen lopputulokseen. Huomattiin kuitenkin, että käytetyllä roottori-BODIPY-merkkiaineella voitiin seurata solunulkoisten vesikkeleiden luonnollista kulkeutumista solussa. Tässä diplomityössä havaittiin, että käytetty merkkiaine muodostaa aggregaatteja vesiliuoksissa, ja että aggregaattien fluoresenssi havaitaan pidemmillä aallonpituuksilla kuin monomeerisen merkkiaineen. Elinaikaerotteisesta fluoresenssimikroskopiasta saatua tietoa käsiteltiin eri tavoin, jotta saatiin selville parhaimmat analysointimenetelmät tulevaisuudessa tehtäviä kokeita varten. Kun fluoresenssin vaimenemiskuvaajiin tehtiin kahta erilaista sovitusta, saatiin elinaikakomponenteille suurin piirtein samansuuruiset arvot. Elinaikahistogrammeihin taas sovitettiin kahta Gaussin käyrää, jolloin saatiin tietoa elinajan lisäksi myös elinaikakomponenttien populaatioiden suuruuksista ja vaihteluista seurannan aikana. Histogrammeista saadut elinajat kuitenkin poikkesivat jonkin verran fluoresenssin vaimenemiskuvaajiin tehtyjen sovitusten elinajoista, joten se ei ole yhtä luotettava tapa tarkkaan elinajan määritykseen.Extracellular vesicles are potential diagnostic and therapeutic vehicles and drug delivery systems because of their biological functions. However, their actions inside the cells are not well known. By labelling extracellular vesicles with fluorescent labels their uptake and trafficking in cells can be monitored with fluorescence lifetime imaging microscopy, which gives the information about intensity and lifetime of the fluorescence signal in every pixel of the image. In this master’s thesis, extracellular vesicles separated from the growth medium of prostate cancer cells were labelled with a fluorescent label (rotor-BODIPY) which fluorescence lifetime changes when viscosity of the environment changes. Used extracellular vesicles are divided into exosomes and microvesicles according to their sedimentation rate and buoyant density by differential ultracentrifugation. The trafficking of extracellular vesicles in prostate cancer cells was followed with fluorescence lifetime imaging microscopy. There are previous studies were the fluorescence signal of the label is different depending on the used delivery system. In this thesis, this phenomenon was not observed. However, rotor-BODIPY allowed us to follow the natural trafficking of extracellular vesicles inside the cells. It was noticed, that used label formed aggregates in aqueous environments, and the fluorescence of aggregates shifted to longer wavelengths than the fluorescence of monomeric label. Different analysis methods were used to obtain the best method for future studies. Fluorescence decay curves were analyzed in two different ways, that both gave same values for the fluorescence lifetime components. Two Gaussian curves were fitted to the lifetime histograms of fluorescence lifetime imaging microscopy images, and among the lifetime data, also knowledge of each lifetime component’s population size and change during measurements were obtained. The fluorescence lifetime data gotten from the lifetime histograms differed from the data that was obtained from the fluorescence decay curves, so it is not as reliable way to estimate the values of the fluorescence lifetime components

Assembly of Bleomycin Saccharide-Decorated Spherical Nucleic Acids

Glyco-decorated spherical nucleic acids (SNAs) may be attractive delivery vehicles, emphasizing the sugar-specific effect on the outer sphere of the construct and at the same time hiding unfavorable distribution properties of the loaded oligonucleotides. As examples of such nanoparticles, tripodal sugar constituents of bleomycin were synthesized and conjugated with a fluorescence-labeled antisense oligonucleotide (AONARV7). Successive copper(I)-catalyzed azide-alkyne and strain-promoted alkyne-nitrone cycloadditions (SPANC) were utilized for the synthesis. Then, the glyco-AONARV7 conjugates were hybridized with complementary strands of a C60-based molecular spherical nucleic acid (i.e., a hybridization-mediated carrier). The formation and stability of these assembled glyco-decorated SNAs were evaluated by polyacrylamide gel electrophoresis (PAGE), UV melting profile analysis, and time-resolved fluorescence spectroscopy. Association constants were extracted from time-resolved fluorescence data. Preliminary cellular uptake experiments of the glyco-AONARV7 conjugates (120 nM solutions) and of the corresponding glycodecorated SNAs (10 nM solutions) with human prostate cancer cells (PC3) showed an efficient uptake in each case. A marked variation in intracellular distribution was observed.Peer reviewe