Scavenger receptors as a target for nucleic acid delivery with peptide vectors
Failid
Kuupäev
2017-03-16
Autorid
Ajakirja pealkiri
Ajakirja ISSN
Köite pealkiri
Kirjastaja
Abstrakt
Geeniekspressiooni reguleerimine rakkudesse suunatud nukeiinhapete ja sünteetiliste oligonukleotiidide (ON) abil omab suurt potentsiaali geeniteraapias mitmete haiguste ravil. Suure molekulmassi ning negatiivse laengu tõttu ei ole ON-id võimelised iseseisvalt rakke ümbritsevat plasmamembraani läbima ning rakusisese sihtmärgini jõudma. Selleks, et tagada bioaktiivsete molekulide pääsemine rakkudesse ning jõudmine sihtmärgini, on kasutusele võetud mitmeid transportsüsteeme. Üheks perspektiivseimaks võimaluseks on nende transport rakku sisenevate peptiidide (RSP) abil. RSP-d on lühikesed, enamasti positiivselt laetud ja/või amfipaatsed peptiidid, mida iseloomustab võime läbida rakkude plasmamembraani ning rakkudesse suunata erineva omadustega molekule. Tänu positiivsele laengule moodustavad RSP-d negatiivset laengut kandva ON-ga spontaanseid komplekse elektrostaatiliste interaktsioonide vahendusel, ning sellisel kujul on ON-d võimelised efektiivselt rakkudesse sisenema.
Vaatamata sellele, et RSP-sid on intensiivselt uuritud enam kui 20 aastat, pole veel teada nende täpsed rakkudesse sisenemise mehhanismid. Varem arvati, et positiivselt laetud RSP-ON komplekside rakkudesse jõudmise eelduseks on seondumine negatiivselt laetud rakupinna komponentidega, mis indutseerib edasise sisenemise rakku erinevate endotsütoosi mehhanismide vahendusel. Hiljuti on aga näidatud, et vastupidiselt eeldatule, moodustavad RSP-d ON-ga hoopis negatiivset laengut omavaid komplekse ning sellised molekulid vajavad sisenemiseks retseptoreid rakkude pinnal. Spetsiifiliste rakupinna retseptorite tuvastamine ning nende mehhanismide täpsem uurimine aitab paremini mõista ON-de transporti rakkudesse nii RSP-de kui ka muude transportsüsteemide vahendusel.
Käesoleva doktoritöö eesmärgiks oli uurida püüdurretseptorite toimimise mehhanisme RSP-ON komplekside omastamisel keskkonnast, sealhulgas analüüsida retseptorite suunamist plasmamembraanile, uurida nende rakkudesse sisenemise mehhanisme pärast RSP-ON komplekside sidumist ha järgnevat liikumist rakkude sees.
Nucleic acids and synthetic oligonucleotides (ONs) are promising tools for regulation of gene functions and for the treatment of various diseases. However, the plasma membrane that surrounds cells provides an impermeable barrier for such big and negatively charged molecules. In order to facilitate the intracellular delivery of ONs and gain their access to target sites, various transfection methods have been developed. Among these, the cell-penetrating peptides (CPPs) have gained increased popularity. CPPs are short, usually cationic and/or amphipathic peptides that can efficiently cross the barrier of the cell plasma membrane. Due to the positive charge, CPPs form complexes with negatively charged ONs via electrostatic and hydrophobic interactions, and thereby facilitate cellular uptake of ONs. Despite extensive research in the field of CPPs, the cell entry mechanisms of CPP-ON complexes into cells are still largely unknown. Until quite recently it has been thought that positively charged CPP-ON complexes associate with negatively charged cell surface molecules, which induce the uptake of the complexes through endocytic mechanisms. Very recently, contrarily to expectation we found the nanocomplexes of CPP and ON to be negatively charged. Induction of internalization of such complexes into cells through simple electrostatic interactions with cell membrane is unlikely, and probably it occurs in a receptor-dependent manner. Identifying the specific receptors involved, and understanding the exact mechanisms of how CPPs and their complexes with ONs reveal their activity inside cells are highly essential in order to harness such complexes for medical purposes. The aim of this study was to examine the mechanisms of scavenger receptors at recognition of extracellular ONs and their complexes with CPPs. In more detail we describe the recruitment of the receptors to the plasma membrane by CPP-ON nanocomplexes, subsequent cell entry mechanisms and intracellular destination of the nanocomplexes.
Nucleic acids and synthetic oligonucleotides (ONs) are promising tools for regulation of gene functions and for the treatment of various diseases. However, the plasma membrane that surrounds cells provides an impermeable barrier for such big and negatively charged molecules. In order to facilitate the intracellular delivery of ONs and gain their access to target sites, various transfection methods have been developed. Among these, the cell-penetrating peptides (CPPs) have gained increased popularity. CPPs are short, usually cationic and/or amphipathic peptides that can efficiently cross the barrier of the cell plasma membrane. Due to the positive charge, CPPs form complexes with negatively charged ONs via electrostatic and hydrophobic interactions, and thereby facilitate cellular uptake of ONs. Despite extensive research in the field of CPPs, the cell entry mechanisms of CPP-ON complexes into cells are still largely unknown. Until quite recently it has been thought that positively charged CPP-ON complexes associate with negatively charged cell surface molecules, which induce the uptake of the complexes through endocytic mechanisms. Very recently, contrarily to expectation we found the nanocomplexes of CPP and ON to be negatively charged. Induction of internalization of such complexes into cells through simple electrostatic interactions with cell membrane is unlikely, and probably it occurs in a receptor-dependent manner. Identifying the specific receptors involved, and understanding the exact mechanisms of how CPPs and their complexes with ONs reveal their activity inside cells are highly essential in order to harness such complexes for medical purposes. The aim of this study was to examine the mechanisms of scavenger receptors at recognition of extracellular ONs and their complexes with CPPs. In more detail we describe the recruitment of the receptors to the plasma membrane by CPP-ON nanocomplexes, subsequent cell entry mechanisms and intracellular destination of the nanocomplexes.
Kirjeldus
Väitekirja elektrooniline versioon ei sisalda publikatsioone.
Märksõnad
retseptorid, rakku sisenevad peptiidid, nukleiinhapped, oligonukleotiidid, rakud, rakumembraan, transportvalgud, bioloogiline transport, receptors, cell-penetrating peptides, nucleic acids, oligonucleotides, cells (biology), cell membrane, transport proteins, biological transport