Preparation and characterization of extracellular vesicles for cancer therapy

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http://urn.fi/URN:ISBN:978-951-51-6007-2
Title: Preparation and characterization of extracellular vesicles for cancer therapy
Author: Saari, Heikki
Contributor: University of Helsinki, Faculty of Pharmacy, Farmaseuttisten biotieteiden osasto
Doctoral Programme in Biomedicin
Publisher: Helsingin yliopisto
Date: 2020-05-14
URI: http://urn.fi/URN:ISBN:978-951-51-6007-2
http://hdl.handle.net/10138/313983
Thesis level: Doctoral dissertation (article-based)
Abstract: Drug delivery aims to optimize the systemic distribution of administered drugs to reach their target tissue in an effective and specific manner. This approach is arising also in the field of cancer therapy, since traditional small molecule chemotherapeutics can cause severe side effects and more sophisticated biomolecular therapeutics cannot always reach the target cells on their own. For these purposes, therapeutics can be packed into carriers that improve their pharmacokinetics by providing a suitable, protected environment for the cargo to travel in the body, armed with targeting molecules that guide them to the site of interest. Synthetic drug carriers include liposomes, porous silica and different polymer particles that can be modified to contain necessary surface structures for targeted delivery and improved biocompatibility. While they have shown much promise in research settings, their success in cancer treatment has been limited to only a handful of commercially available formulations, some of which have presented liver toxicity as a result of continuous administration. Additionally, targeting cancer cells is difficult because cancer is a very individual disease, and heterogeneity exists even within tumors, so finding any universal cancer-specific markers to target is extremely challenging. Nature has adopted a similar approach for delivering molecules containing biologically functional cargo from cell to cell. These naturally occurring carriers are called extracellular vesicles (EVs), in contrast to intracellular vesicles that mediate cargo trafficking inside of the cells. EVs are 50 – 1000 nm in diameter, consisting of a lipid bilayer with embedded membrane proteins that encloses water-soluble biological cargo within. Their exact composition and cargo vary from cell to cell and depending on the condition of the cells that produce them. Additionally, cells have multiple pathways for EV secretion and production. For these reasons EVs comprise a very heterogeneous population of vesicles in composition and they also have multiple biological functions that have not been completely clarified yet. EVs have been found in practically all body fluids and organisms that have been studied, taking part in the normal and pathological functions of the organism. For example, procoagulant EVs are found in blood and saliva that participate in hemostasis, while EVs secreted by cancer cells promote the survival, growth and metastasis of the tumor. They are able to affect cells via membrane interactions and by delivering functional cargo into their recipient cells. It has been shown that EVs are even able to target cells selectively by binding to specific cellular receptors, enabling targeted cargo delivery. Given their natural cargo delivery properties, EVs present remarkable potential for drug delivery applications. As cancer cells also use EVs to communicate with each other, their EVs reflect the same heterogeneity that exists within the cells themselves, and may provide useful insights for cancer-targeted drug delivery. However, the internalization mechanisms of drug-carrying EVs and especially the fate of the drugs they carry is not well understood. In this thesis, the use of EVs was assessed for the delivery of chemotherapeutic drug paclitaxel and an oncolytic adenovirus that represent small molecular and biological chemotherapeutics, with cancer cell-derived EVs as the model carrier. The studies presented here focus on the preparation, characterization, intracellular tracking and effectiveness of these EVs for cancer therapy. The obtained results provide novel methods and knowledge for the future development of EV-based therapeutics for the treatment of cancer. First, EVs were loaded with paclitaxel by incubation, which enhanced the cytotoxic effect of the drug, changing its internalization from passive diffusion to endocytosis. A novel approach using fluorescence lifetime microscopy was introduced for tracking the release of paclitaxel from the EVs inside of the cells, identifying distinct patterns of subcellular drug release in individual cells and its intracellular kinetics. This method was able to show details about the drug release mediated by EVs that could not be observed with conventional fluorescence microscopy. Additionally, the relationship between EVs and adenoviruses was explored, revealing a previously undocumented pathway of spreading adenoviral infection via EVs. This EV-mediated infective delivery of the viral genome occurred separately from the classical pathway of adenoviral life cycle, and produced infective particles resembling EVs more than virions. EVs can in theory enhance the pharmacokinetics of oncolytic viruses by hiding them from the immune system and providing them alternative pathways for cell targeting and internalization, however it was found that infective EVs do react with adenovirus neutralizing antibodies. Taken together, these results suggest that EVs can act as versatile carriers of therapeutic cargo for cancer treatment, ranging from small molecule chemotherapeutics to oncolytic viruses, though they require further development. New methods are reported constantly for preparing and studying therapeutic EVs with new, innovative approaches that will help in the future treatments of cancer and other diseases.Solunulkoiset vesikkelit (EV, Extracellular Vesicle) toimivat solujen välisinä viestinviejinä ja omaavat siksi huomattavia sovellutusmahdollisuuksia lääkkeenkuljetuksessa. Koska syöpäsolut myös käyttävät EV:itä kommunikoidakseen toistensa kanssa, niiden EV:t voivat tarjota hyödynnettävissä olevaa tietoa syöpään kohdennettavaan lääkkeenkuljetukseen. Lääkeaineita kantavien EV:iden sisäänottomekanismeja ja erityisesti lääkeaineiden kohtaloa ei kuitenkaan tunneta tarkasti. Tässä väitöskirjatyössä selvitettiin EV:iden käyttöä kemoterapiassa käytetyn paklitakselin ja onkolyyttisen adenoviruksen kuljetuttajina, jotka edustavat pienmolekulaarista ja biologista kemoterapiaa, käyttäen syöpäsolujen EV:itä mallikantajina. Tutkimuksissa keskityttiin näiden EV:iden valmistukseen, karakterisointiin, solunsisäiseen kulkeutumiseen ja toiminnallisuuteen syövän hoidossa. Saadut tulokset tarjoavat uusia menetelmiä ja tuntemusta tulevaisuuden EV-pohjaisten syöpähoitojen kehittämiseen. Ensin, EV:t lastattiin paklitakselilla inkuboimalla niitä yhdessä, mikä tehosti lääkeaineen sytotoksista vaikutusta, ohjaten sen sisäänottoa passiivisesta diffuusiosta endosytoosiin. Lisäksi esiteltiin uudenlaista lähestymistapaa paklitakselin EV:istä vapautumisen seuraamiseksi solujen sisällä fluoresenssin elinaikamikroskopialla, minkä avulla tunnistettiin lääkeaineen solunsisäisen vapautumisen mekanismeja yksittäisissä soluissa ja sen solunsisäistä kinetiikkaa. Lisäksi selvitettiin EV:iden ja adenovirusten välistä suhdetta, josta havaittiin aiemmin tunnistamaton reitti adenovirusinfektion levittämiseksi EV:illä. Tämä EV-välitteinen infektiivinen virusgenomin kuljetus ilmeni itsenäisenä adenoviruksen klassisesta elinkaaresta, infektiivisten partikkelien muistuttaen enemmän EV:itä kuin virioneja. Nämä tulokset viittaavat siihen, että EV:t voivat toimia monipuolisina kantajina syövän hoidossa käytettävälle terapeuttiselle lastille pienmolekyyleistä onkolyyttisiin viruksiin, tosin niiden käyttö edellyttää lisää kehitystyötä. Uusia menetelmiä esitetään jatkuvasti terapeuttisten EV:iden valmistamiseen ja tutkimukseen uusin, innovatiivisin lähestymistavoin, jotka tulevat edistämään tulevaisuudessa syövän ja muiden sairauksien hoitoa.
Subject: biofarmasia
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