NickFects – novel cell-penetrating peptides. Design and uptake mechanism
Date
2013-10-01
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Abstract
Vajadus uute ja spetsiifilisemate ravimite järgi, kui hetkel kasutatavad madalmolekulaarsed ravimid, kasvab pidevalt. Nukleiinhapped ja nende analoogid omavad väga suurt potentsiaali paljude raskete haiguste raviks. Oma füsiko-keemilistest omadustest tingituna ei ole nukleiinhapete molekulid võimelised iseseisvalt rakku sisenema ja vajavad terapeutilise toimekohani jõudmiseks transportvektorite abi. Viirustel põhinevad vektorid on väga efektiivsed, kuid nende kasutamisel võivad ilmneda mitmed kõrvaltoimed nagu immunoloogiline reakstioon või transporditava DNA insertsioon valesse genoomi piirkonda. Sellest tulenevalt keskendutakse üha enam mitteviiruslike vektorite arendamisele.
Rakku sisenevad peptiidid (RSPd) on üks grupp mitteviiruslikke vektoreid, mida on laialdaselt kasutatud nukleiinhapete rakku viimiseks nii in vitro kui ka in vivo tingimustes. RSPd on lühikesed, 5–30 aminohappe jäägist koosnevad katioonsed ja/või amfipaatsed peptiidid, mis on võimelised sisenema rakku ja transportima sinna ka endaga seotud lastmolekuli. Lastmolekuli sidumiseks on peamiselt kaks moodust: kovalentne, kus lastmolekul seotakse RSPga kovalentse sideme kaudu ja mitte-kovalentne, kus kasutatakse elektrostaatilisi ja hüdrofoobseid interaktsioone. Antud töös kasutati mitte-kovalentset sidumise strateegiat, kus lastmolekulidest ja RSPdest moodustati nanopartiklid. Kuigi mitte-kovalentne sidumisstrateegia on palju lihtsam ja odavam kui kovalentne strateegia, on selle peamiseks puuduseks moodustunud nanopartiklite iseloomustamise keerukus. Seetõttu pöörati antud töös erilist tähelepanu RSP-nukleiinhapete komplekside omadustele. Moodustunud kompleksid sisenevad rakku enamasti endotsütoosi teel, millest tulenevalton suur tõenäosus jääda kinni endotsütootilistesse vesiikulitesse. See omakorda vähendab oluliselt lastmolekulide bioloogilist aktiivsust, sest lastmolekulide sihtmärgid asuvad kas rakutuumas või tsütoplasmas. RSPde kui transportvektorite kasutamisel on väga oluline tagada RSP/lastmolekuli komplekside vabanemine endotsütootilistest vesiikulitest ning lastmolekuli jõudmine oma sihtmärgini rakus.
Käesoleva töö peamiseks eesmärgiks oli disainida uued efektiivsed peptiidsed vektorid, NickFectid (NF), erinevate nukleiinhapete transpordiks rakku. Uute vektorite disainil lähtuti stearüleeritud TP10 peptiidist. Disaini eesmärgiks oli parandada peptiidse vektori võimet moodustada komplekse lastmolekuliga, suurendada komplekside sisenemist rakkudesse ja vabanemist endostütootilistest vesiikulitest.
Esimeses töös kirjeldame NF1 ja NF2. Disanil aluseks võetud peptiidi järjestusse lisame fosforüülrühma ja hüdrofoobsuse muutmiseks asendame isoleutsiin treoniiniga. Nimetatud modifikatsioonid parandavad olulisest RSP võimet transportida rakkudesse splaissingut-korrigeerivaid oligonukleotiide. Teises töös modifitseerime TP10 koostisosi, mastoparaani ja galaniini fraktsioone siduvat ühenduslüli ning disainime „kõvera“ peptiid NF51. Saadud peptiid transpordib rakkudesse nii plasmiidset DNAd, splaissingut-korrigeerivaid oligonukleotiide kui ka väikseid interfeeruvaid RNAsid. Lisaks näitame, et NF51 on kasutatav transfetsiooni reagendina valkude tootmises, antud juhul kasutades QMCF tehnoloogiat. Kolmandas töös uurime kahe erineva RSP, NF1 ja NF51 omadusi. Näitame, et kuigi käesolevad peptiidid omavad erinevat keemilist struktuuri on nende võime transportida rakku plasmiidset DNAd sarnane. Võrdleme nende peptiidide ja plasmiidse DNA komplekside omadusi, interaktsiooni rakupinnaga, rakku sisenemiseks kasutatavaid endotsütoosi radasid ning rakusisest liikumist.
Kokkuvõttes on antud töös disainitud peptiidid, NickFectid, on vähe toksilised, sobivad nukleiinhapete transpordiks ja omavad head perspektiivi nii geeniteraapias kui valkude tootmises.
Nucleic acids and their analogues are highly potential candidates to be utilized for the treatment of various devastating diseases. The clinical potential of these biomolecules remains restricted so far because of their poor stability in the presence of serum and low uptake into the cells resulting from the high molecular weight, negative charge and hydrophilic nature of the nucleic acids. Therefore, the development of macromolecule-based drugs is dependent on the progress and improvement of carrier molecules that can facilitate their transfection and protect the cargo from degradation. CPPs are relatively short peptides, 5–30 amino acids in length, with the ability to gain access to the cell interior via energy-dependent and/or independent mechanisms, and facilitate intracellular delivery of associated cargo molecules to intracellular targets. This thesis focuses on the design and characterization of a new family of CPPs, named NickFect. The peptides are designed to deliver various types of bio-active cargos, including plasmid DNA, splice-correcting oligonucleotides and small interfering RNAs, using non-covalent nanoparticle formation approach. In order to enhance the nanoparticle formation activity, uptake efficacy and endosomolytic properties, we insert different modifications or make amino acid substitutions to the backbone of the parental peptide, stearyl-TP10. For instance, addition of phosphoryl-group yielded NF1 and NF2, highly efficient peptide-based transfection reagents for the intracellular delivery of splice-correcting oligonucleotides. Another radical modification, insertion of a kink, resulted in NF51 that proved to deliver nucleic acids to the targets both in the nucleus and cytoplasm. Additionally, we demonstrate the applicability of NF51 in protein production system. Furthermore, we unravel the uptake mechanism of two NickFect family members, NF1 and NF51, related to the biological activity of transfected plasmid DNA. Taken together, our results demonstrate that the performed chemical modifications in NickFects enhanced the activity of these peptides for delivering nucleic acids. Studies of the uptake mechanism gave us valuable information on how to enhance the bioavailability of different genetic materials non-covalently linked to NickFects for further in vivo applications and for designing more efficient carrier vectors and achieving bioavailability of the macromolecule-based drugs. Conclusively, NickFects have remarkable potential for the delivery of nucleic acids both in vitro and in vivo.
Nucleic acids and their analogues are highly potential candidates to be utilized for the treatment of various devastating diseases. The clinical potential of these biomolecules remains restricted so far because of their poor stability in the presence of serum and low uptake into the cells resulting from the high molecular weight, negative charge and hydrophilic nature of the nucleic acids. Therefore, the development of macromolecule-based drugs is dependent on the progress and improvement of carrier molecules that can facilitate their transfection and protect the cargo from degradation. CPPs are relatively short peptides, 5–30 amino acids in length, with the ability to gain access to the cell interior via energy-dependent and/or independent mechanisms, and facilitate intracellular delivery of associated cargo molecules to intracellular targets. This thesis focuses on the design and characterization of a new family of CPPs, named NickFect. The peptides are designed to deliver various types of bio-active cargos, including plasmid DNA, splice-correcting oligonucleotides and small interfering RNAs, using non-covalent nanoparticle formation approach. In order to enhance the nanoparticle formation activity, uptake efficacy and endosomolytic properties, we insert different modifications or make amino acid substitutions to the backbone of the parental peptide, stearyl-TP10. For instance, addition of phosphoryl-group yielded NF1 and NF2, highly efficient peptide-based transfection reagents for the intracellular delivery of splice-correcting oligonucleotides. Another radical modification, insertion of a kink, resulted in NF51 that proved to deliver nucleic acids to the targets both in the nucleus and cytoplasm. Additionally, we demonstrate the applicability of NF51 in protein production system. Furthermore, we unravel the uptake mechanism of two NickFect family members, NF1 and NF51, related to the biological activity of transfected plasmid DNA. Taken together, our results demonstrate that the performed chemical modifications in NickFects enhanced the activity of these peptides for delivering nucleic acids. Studies of the uptake mechanism gave us valuable information on how to enhance the bioavailability of different genetic materials non-covalently linked to NickFects for further in vivo applications and for designing more efficient carrier vectors and achieving bioavailability of the macromolecule-based drugs. Conclusively, NickFects have remarkable potential for the delivery of nucleic acids both in vitro and in vivo.
Description
Väitekirja elektrooniline versioon ei sisalda publikatsioone.
Keywords
nukleiinhapped, peptiidid, penetratsioon, farmatseutiline keemia, ravimidisain, nucleic acids, peptides, penetration, pharmaceutical chemistry, drug design