Purification of extracellular vesicles by immunoaffinity methods and their characterization

[L. Filipović]

90 str.

Hemijske nauke - Biohemija / Chemistry - Biochemistry

Nauka i medicina obiluju najfascinantnim istorijskim otkrićima. Mnoga od njih, značajna za čovečanstvo, dogodila su se slučajnim opažanjem. Ekstracelularne vezikule (EVs) su otkrivene u studijama koje su u to vreme imale sasvim drugi cilj. Istraživanja u poljima hematologije, ćelijske biologije, virusologije dovela su do otkrića malih struktura u cirkulaciji, koje nisu pripadale dotad poznatim kategorijama. Nakon njihovog otkrivanja (1930. godine), prošlo je mnogo vremena dok šira naučna zajednica nije prihvatila i proširila potpuno novi koncept razumevanja međućelijske komunikacije. Zbog toga je ova oblast dugo ostala neistražena, a tek u poslednjih 30 godina doživljava svoj procvat. Danas je poznato da su to lipidne strukture oslobođene iz ćelija, koje između ostalog imaju ključnu ulogu u održanju homeostaze organizma. One omogućavaju komunikaciju između ćelija, prenoseći informacije poput proteina, RNK i DNK molekula. Otkriće da sve ćelije mogu prenositi informacije putem EVs značajno je povećalo njihov potencijal za primenu u medicini i kliničkoj praksi. Međutim, jedan od glavnih problema rada sa EVs je njihovo izolovanje iz heterogenih izvora. Dosadašnje metode omogućavaju izolovanje EVs, ali takvi preparati sadrže brojne kontaminacije ne-vezikularne prirode. Cilj ove naučne disertacije bio je razvoj nove, brze, jeftine, reproducibilne metode za izolovanje EVs iz različitih bioloških uzoraka. U prvoj fazi korišćen je čvrsti nosač za imobilizaciju nanoantitela specifičnih za prepoznavanje EVs. Selektovana antitela su proizvedena rekombinantno u E. coli sistemu, uz visok prinos od 3-5 mg/L medijuma, čime su značajno smanjeni troškovi proizvodnje sistema za izolovanje EVs. Imobilizacijom nanoantitela na nosaču razvijena je imunoafinitetna hromatografija za izolovanje EVs, koja je testirana na nekoliko bioloških izvora. Karakterizacija EVs obavljena je kombinacijom više različitih biohemijskih i instrumentalnih metoda. U završnom delu disertacije, praćeno je preuzimanje ovako izolovanih vezikula obojenih lipofilnom bojom (OilRed).

Science and medicine are abundant with some of the most fascinating historical discoveries. Many of these, significant for humanity, were made through accidental observations. Extracellular vesicles (EVs) were discovered in studies that originally had entirely different objectives. Research in the fields of haematology, cell biology, and virology led to the discovery of small structures in circulation that did not belong to previously known categories. After their discovery (in the 1930s), it took a long time for the broader scientific community to accept and expand upon this completely new concept of intercellular communication. As a result, this field remained unexplored for a long time and has only experienced significant growth in the last 30 years. Today, it is known that EVs are lipid structures released from cells, which, in addition to numerous roles, play a key part in maintaining the homeostasis of the organism. They enable communication between cells, transferring information such as proteins, RNA, and DNA molecules. The discovery that all cells can transfer information through EVs has significantly increased their potential for applications in medicine and clinical practice. However, one of the main challenges in working with EVs is their isolation from heterogeneous sources. Current methods allow for the isolation of EVs, but such preparations often contain numerous non-vesicular contaminants. The aim of this scientific dissertation was to develop a new, fast, cost-effective, and reproducible method for isolating EVs from various biological samples. In the first phase, a solid support was used for the immobilization of nanoantibodies specific for EV recognition. The selected antibodies were recombinantly produced in the E. coli system, with a high yield of 3–5 mg/L of medium, significantly reducing production costs of the EVs purification system. By immobilizing nanoantibodies onto the support, immunoaffinity chromatography was developed for the isolation of EVs, which was tested on several biological sources. The characterization of EVs was performed using a combination of various biochemical and instrumental methods. In the final part of the dissertation, uptake of the vesicles isolated using nanobody-based system was observed after staining EVs with a lipophilic dye (OilRed).

ekstracelularne vezikule, nanoantitela, polimetakrilatni nosač, imunoafinitetno prečišćavanje, mikroskopija, protočna citometrija, Hek293, Skbr3, plazma, urin

extracellular vesicles, nanobodies, polymethacrylate carrier, immunoaffinity purification, microscopy, flow cytometry, Hek293, Skbr3, plasma, urine

577.112(043.3)

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