Towards understanding the colloidal stability and detection of extracellular vesicles
Date
2023-02-13
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Abstract
Ekstratsellulaarsed vesiikulid (EVd) on nanoosakesed, mida eraldavad stressiseisundis olevad rakud . Nende nanoosakeste (NP) interaktsioonides erinevate bioloogiliste süsteemidega ja mängivad olulist rolli osakeste suurus ja pinnalaeng. EVd moodustavad kolloidlahuseid ning tänu nende pinnal olevatele lipiididele ning pinnavalkudele on nad tavaliselt negatiivselt laetud. EVde uurimisel ja nende eesmärgipärasel kasutamisel on vajalik nende struktuuri ja funktsionaalsuse säilimine ning agregeerumise vältimine. Selleks on vajalik iseloomustada EVde füsikokeemilisi omadusi ning leida tingimused, mille juures säilib nii kolloidlahuste stabiilsus kui ka osakeste bioloogiline aktiivsus. Standardne metoodika selle uurimiseks seni puudub.
Käesoleva doktoritöö eesmärgiks oligi uurida EVsid sisaldavate kolloilahuste stabiilsust mõjutavad tegureid, ning rakendada fluorestsentseeruvate nanoosakeste jälgimise analüüsi (fNTA) membraanset päritolu ekstratsellulaarsete vesiikulite määramiseks. EVde pindlaengut (Z-potentsiaali) mõjutavatest teguritest uuriti puhverlahuse kontsentratsiooni, detergendi, ioonse jõu ning lahuse pH mõju. Leiti, et EVde Z-potentsiaali (ZP) negatiivne väärtus väheneb puhverlahuse kontsentratsiooni suurenemisel. Samas EVde ZP negatiivne väärtus lahuse pH väärtuse suurenedes kasvab. See on seotud EVde vaheliste elektrostaatiliste tõukejõudude suurenemisega, mis suurendab ka kolloidlahuse stabiilsust. Võrreldes tervete inimeste ning psoriaasihaigete vereseerumist eraldatud EVde füüsiko-keemilisi omadusi, olulisi erinevusi ZPde väärtustes ega osakeste suurusjaotustes ei täheldatud. Töös uuriti ka lipofiilse membraanvärvi CellMask™ Green (CMG) mõju erinevatel meetoditel eraldatud EVde füüsikalistele omadustele. Uurimise all olid nii inimese kooriokartsinoomi rakkudest kui veiste follikulaarvedelikust ja seemnevedelikust pärinevate EVd. fNTA analüüsil selgus, et CMG kontsentratsiooni suurendamine mõjutas fluorestseeruvate nanoosakeste mõõdetavat suurust. Samas sõltus osakeste keskmine suurus ka EVde puhastamiseks kasutatud metoodikast. Läbiviidud uuringud näitavad EVde iseloomustamiseks kasutatavate mõõtmismetoodikate standardiseerimise vajalikkust.
Nanoparticle (NP) size is an important aspect of cellular uptake by different interactions with biological systems. Aside from their size, NPs also possess surface charge enabling their interaction with their surroundings. EVs are membrane-bound NPs secreted by most cell types under stress. Naturally, EVs exist as colloidal suspensions when resuspended in media and carry a net negative charge due to the presence of lipids, membrane and glycosylated proteins on their surface. It is crucial to maintain EV physical characteristics during storage and surface manipulation, and overcome the issue of EV aggregation for EVs applications. The colloidal stability of EVs is one of the crucial parameters for understanding their fates in different conditions. However, currently there is no a standard methodology to detect and quantify EVs or to assess the colloidal stability of EVs. The aim of this study was to determine the colloidal stability of EVs in different conditions and study the applicability of fluorescence NTA (fNTA) for the detection of membranous EVs. The ZP of EVs was influenced by the buffer concentration, detergent, ionic strength and pH. The ZP value of EVs shifts towards less negative values along the increase of buffer concentration. ZP of EVs also shifted to more negative values as pH increased, favoring electrostatic repulsions between EV particles and increasing their colloidal stability. We also assessed the physical characteristics of blood serum EVs derived from healthy and psoriasis patients, but no difference in particle size distribution and ZP values was found. Finally, we showed the effect of membrane dye CellMask™ Green (CMG) labeling on the physical characteristics of JAr EVs isolated using different methods, as well as EVs derived from biological fluids (e.g. bovine follicular fluid and seminal plasma). fNTA analysis revealed that the increase in CMG concentration altered the particle size of fluorescent nanoparticles. Our results also showed the mean particle size of fluorescent and total NPs of JAr EVs were affected by the EV purification method. Biologically derived EVs showed a greater variation CMG labelling than cell culture-derived EVs. Our studies highlight the importance of using standardized ZP measurements and the potential of exploiting fNTA for the detection of membrane dye-labelled EVs.
Nanoparticle (NP) size is an important aspect of cellular uptake by different interactions with biological systems. Aside from their size, NPs also possess surface charge enabling their interaction with their surroundings. EVs are membrane-bound NPs secreted by most cell types under stress. Naturally, EVs exist as colloidal suspensions when resuspended in media and carry a net negative charge due to the presence of lipids, membrane and glycosylated proteins on their surface. It is crucial to maintain EV physical characteristics during storage and surface manipulation, and overcome the issue of EV aggregation for EVs applications. The colloidal stability of EVs is one of the crucial parameters for understanding their fates in different conditions. However, currently there is no a standard methodology to detect and quantify EVs or to assess the colloidal stability of EVs. The aim of this study was to determine the colloidal stability of EVs in different conditions and study the applicability of fluorescence NTA (fNTA) for the detection of membranous EVs. The ZP of EVs was influenced by the buffer concentration, detergent, ionic strength and pH. The ZP value of EVs shifts towards less negative values along the increase of buffer concentration. ZP of EVs also shifted to more negative values as pH increased, favoring electrostatic repulsions between EV particles and increasing their colloidal stability. We also assessed the physical characteristics of blood serum EVs derived from healthy and psoriasis patients, but no difference in particle size distribution and ZP values was found. Finally, we showed the effect of membrane dye CellMask™ Green (CMG) labeling on the physical characteristics of JAr EVs isolated using different methods, as well as EVs derived from biological fluids (e.g. bovine follicular fluid and seminal plasma). fNTA analysis revealed that the increase in CMG concentration altered the particle size of fluorescent nanoparticles. Our results also showed the mean particle size of fluorescent and total NPs of JAr EVs were affected by the EV purification method. Biologically derived EVs showed a greater variation CMG labelling than cell culture-derived EVs. Our studies highlight the importance of using standardized ZP measurements and the potential of exploiting fNTA for the detection of membrane dye-labelled EVs.
Description
Väitekirja elektrooniline versioon ei sisalda publikatsioone
Keywords
extracellular vesicles, colloids, stability, research methods, biochemical markers, psoriasis