Characterization of cell-penetrating peptide/nucleic acid nanocomplexes and their cell-entry mechanisms
Kuupäev
2016-06-09
Autorid
Ajakirja pealkiri
Ajakirja ISSN
Köite pealkiri
Kirjastaja
Abstrakt
Kõrge spetsiiflisuse ja madala kõrvalmõjude tekkeriski tõttu peetakse nukleiinhappeid väga suure ravipotentsiaaliga molekulideks. Bioloogilise aktiivsuse saavutamiseks on vajalik nukleiinhapete sisenemine rakkudesse ning jõudmine sihtkohta kas rakutuumas või tsütoplasmas. Suure molekulmassi ja negatiivse laengu tõttu pole aga nukleiinhapped võimelised ise rakke ümbritsevat plasmamembraani läbima. Seetõttu on arendatud mitmesuguseid meetodeid, mille abil nukleiinhappeid efektiivsemalt rakkudesse suunata, ning üheks neist on rakku sisenevate peptiidide (RSP; i.k. cell-penetrating peptide, CPP) kasutamine. RSP-d on lühikesed, enamasti katioonsed ja/või amfipaatsed aminohappelised järjestused, mis on võimelised läbima plasmamembraani ning transportima rakkudesse erinevaid bioaktiivseid molekule.
Selleks, et RSPd saaksid nukleiinhappeid rakkudesse transportida, on vajalik peptiidse vektori sidumine lastmolekuliga. Lihtsaim võimalus on RSP siduda lastmolekuliga kompleksi moodustamise teel, millega saadakse RSP/nukleiinhappe nanopartiklid, mida hoiavad koos laengulised ja hüdrofoobsed interaktsioonid. RSP/nukleiinhappe nanokompleksid sisenevad rakkudesse efektiivselt ning tagavad kõrge lastmolekuli bioaktiivsuse, samas ei ole antud süsteem veel jõudnud kliiniliste katsete või kasutuseni. Üheks põhjuseks siinjuures on tekkivate nanokomplekside omaduste (nt suurus, kuju, laeng) komplitseeritud kirjeldamine. Teine probleem, mis takistab mittekovalentse strateegia rakendamist, on vähene arusaam nanokomplekside rakku sisenemise mehhanismidest ja rakusisesest suunamisest. Nii RSP/nukleiinhappe komplekside omaduste selgitamine kui nende rakku sisenemise mehhanismide ja rakusisese suunamise mõistmine on aga äärmiselt vajalikud, et parendada transportpeptiidide omadusi ja tagada lastmolekulide võimalikult kõrge aktiivsus nende kasutuselevõtuks biomeditsiinis, vältides sealjuures soovimatuid kõrvalmõjusid.
Antud töös kirjeldasime hiljuti väljatöötatud PepFect ja NickFect tüüpi peptiidide ja nukleiinhapete vaheliste nanokomplekside suurust, kuju ja laengut. Lisaks sellele identifitseerisime uuritud RSP/nukleiinhappe komplekside peamised rakku sisenemise mehhanismid ning analüüsisime rakku viidud nukleiinhappe paiknemist ja selle muutumist ajas.
Nucleic acids are highly promising candidates for the treatment of various diseases. In order to achieve biological functionality, nucleic acids need to be internalized by cells and reach their action site in cytoplasm or nucleus. However, due to the large size and negative charge, naked nucleic acids are not capable of traversing the plasma membrane of cells. A wide variety of delivery vectors have been designed to facilitate the cellular uptake of nucleic acids. One class of such vectors are cell-penetrating peptides (CPPs), short sequences of 5-40 amino acid residues, which are capable of gaining access to the interior of cells, and importantly, mediate the internalization of coupled cargo molecules. The simplest way to couple CPPs to nucleic acids is to mix the peptide and cargo. The co-incubation of CPPs and nucleic acids leads to the formation of nanocomplexes due to electrostatic and hydrophobic interactions between the peptide and cargo. Co-incubation strategy has been shown to yield high bioactivities of cargos in numerous studies. However, this approach has not reached clinical trials yet. One reason behind this is the complicated physicochemical characterization of the forming nanocomplexes. However, in order to be considered for implementation in biomedicine the properties of CPP/nucleic acid complexes such as size, morphology and charge need to be characterized in detail. Another bottleneck which impedes the implementation of non-covalent strategy for nucleic acid delivery is the poor knowledge of the cellular uptake mechanisms and intracellular trafficking of CPP/nucleic acid nanocomplexes. However, detailed characterization of the cell internalization pathways and cellular trafficking of CPP/cargo complexes are essential for avoiding undesired side effects and refining their properties in order to yield higher activities of delivered cargo. The main objectives of the current thesis were to characterize the physicochemical properties of CPP/nucleic acids nanocomplexes formed by PepFect and NickFect type carrier peptides, and to examine the cellular uptake mechanisms and intracellular trafficking of nanocomplexes and nucleic acids.
Nucleic acids are highly promising candidates for the treatment of various diseases. In order to achieve biological functionality, nucleic acids need to be internalized by cells and reach their action site in cytoplasm or nucleus. However, due to the large size and negative charge, naked nucleic acids are not capable of traversing the plasma membrane of cells. A wide variety of delivery vectors have been designed to facilitate the cellular uptake of nucleic acids. One class of such vectors are cell-penetrating peptides (CPPs), short sequences of 5-40 amino acid residues, which are capable of gaining access to the interior of cells, and importantly, mediate the internalization of coupled cargo molecules. The simplest way to couple CPPs to nucleic acids is to mix the peptide and cargo. The co-incubation of CPPs and nucleic acids leads to the formation of nanocomplexes due to electrostatic and hydrophobic interactions between the peptide and cargo. Co-incubation strategy has been shown to yield high bioactivities of cargos in numerous studies. However, this approach has not reached clinical trials yet. One reason behind this is the complicated physicochemical characterization of the forming nanocomplexes. However, in order to be considered for implementation in biomedicine the properties of CPP/nucleic acid complexes such as size, morphology and charge need to be characterized in detail. Another bottleneck which impedes the implementation of non-covalent strategy for nucleic acid delivery is the poor knowledge of the cellular uptake mechanisms and intracellular trafficking of CPP/nucleic acid nanocomplexes. However, detailed characterization of the cell internalization pathways and cellular trafficking of CPP/cargo complexes are essential for avoiding undesired side effects and refining their properties in order to yield higher activities of delivered cargo. The main objectives of the current thesis were to characterize the physicochemical properties of CPP/nucleic acids nanocomplexes formed by PepFect and NickFect type carrier peptides, and to examine the cellular uptake mechanisms and intracellular trafficking of nanocomplexes and nucleic acids.
Kirjeldus
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Märksõnad
rakku sisenevad peptiidid, nukleiinhapped, nanokompleksid, bioloogiline transport, endotsütoos, cell-penetrating peptides, nucleic acids, nanocomplexes, biological transport, endocytosis