Optimizing cell-penetrating peptide-based nanoparticles for delivery of nucleic acid therapeutics
Date
2024-07-09
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Abstract
Geenide avaldumise mõjutamine, manustades erinerva toimemehhanismiga nukleiinhappelisi (NH) ravimeid, on leidnud üha enam rakendust meditsiinis nii kaasasündinud kui omandatud haiguste raviks. Paraku ei ole sellised terapeutilised molekulid organismis piisavalt stabiilsed ning suure molekulmassi ja negatiivse laengu tõttu ei suuda need efektiivselt läbida bioloogilisi barjääre. NH efektiivseks toimetamiseks märklaudrakkudesse on tänapäeval välja töötatud mitmeid kandursüsteeme. Üheks tõhusaks kandursüsteemiks on rakkudesse penetreeruvad peptiidid (RPP). Need on kuni 30 aminohappe pikkused peptiidid, mis on võimelised läbima rakumembraane ja koos endaga transportima rakkudesse lastmolekule. RPP-d on valdavalt positiivselt laetud ning seostudes negatiivselt laetud NH-ga moodustavad nad elektrostaatiliste ja hüdrofoobsete interaktsioonide tulemusena nanoosakesi.
Käesolevas töös uurisime tegureid, mis pärsivad RPP/NH komplekside tõhusust märklaudgeenide mõjutamisel, ning viise, kuidas vastavat efektiivsust suurendada. Mudelpeptiidina kasutasime peptiidi PepFect14 (PF14), mille laeng on +5 ning mis on modifitseeritud stearüülhappega. Doktoriprojekti esimeses etapis analüüsisime valgukrooni teket PF14/NH osakeste pinnale. Valgukroon on valkude kiht, mis moodustub verre või muudesse biovedelikesse sattuvate osakeste pinnale ning võib põhjustada osakeste agregatsiooni, suurendada toksilisust ja suunata organismist eemaldamisele. Näitasime, et osakestele moodustuva valgukrooni koostis sõltub kasutatud seerumist ning valgukroon mõjutab oluliselt osakeste transfektsiooni ja efektiivsust kultuuris kasvatatavates rakkudes. Seejärel uurisime kas ja kuidas mõjutab Ca²⁺ ja Mg²⁺ ioonide lisamine RPP/NH osakeste omadusi ja efektiivsust. Näitasime, et mõlemad ioonid, eriti Ca²⁺, suurendavad RPP/NH osakeste efektiivsust rakukultuuris mitmekordselt, tõenäoliselt hõlbustades NH vabanemist endosoomidest. Järgnevalt rakendasime PF14 ka mRNA transfekteerimiseks rakkudesse. Selleks moodustasime mRNA-st ja PF14-st nanoosakesed ja varieerisime abiainete lisamisega nende koostist eesmärgiga suurendada osakeste efektiivsust. Näitasime, et PF14 transpordib mRNA efektiivset püsiliinide rakkudesse, aga mõnevõrra vähem tõhusalt inimese primaarsetesse rakkudesse koekultuuris ja kõrvakoesse hiires.
Nucleic acids (NAs) are therapeutics with high potential, enabling the treatment of hereditary and acquired diseases. However, delivering NAs into target cells is extremely challenging. The large size and highly negative charge of NAs result in poor serum stability and hinder their ability to penetrate cellular membranes effectively. Furthermore, if cellular internalization occurs, most NA molecules are degraded by lysosomal enzymes after becoming entrapped in acidic endosomes. To tackle these issues, various NA delivery systems have been developed. Among these, cell-penetrating peptides (CPPs) are highly promising candidates. These up to 30 amino acid-long peptides exhibit an inherent ability to cross biological membranes, carrying cargo molecules into cells. Positively charged CPPs readily form non-covalent complexes with NAs based on electrostatic interactions, and the forming nanoparticles have been shown to effectively deliver various NAs in vitro and in vivo. The current thesis's focus was to understand better the factors determining the efficiency of CPP-based NA delivery and explore methods for improvement. As a model CPP, we used PepFect14 (PF14), an amphipathic stearylated CPP with a charge of +5. Firstly, we analyzed the protein corona of CPP/NA complexes – a layer of proteins forming around nanoparticles once exposed to a serum-containing environment, e.g., the bloodstream. We showed that the protein corona composition is highly dependent on the serum used and affects the nanoparticles' properties, cellular association, and resulting biological effect. Secondly, we investigated the impact of added Ca²⁺ and Mg²⁺ ions on the properties and functionality of the PF14/NA nanoparticles. We showed that these ions, especially Ca²⁺, greatly enhance the biological effect of NAs delivered by PF14, most likely facilitating the endosomal escape of the cargo. Finally, we investigated the ability of PF14 to transfect messenger RNA into cell lines, primary cells, and in vivo. We supplemented the nanoparticles with different excipients to achieve high mRNA transfection efficiency in cultured cells and in vivo. In summary, our findings provide novel insights into the challenges associated with developing CPP/NA nanoparticles for clinical use and open potential avenues for enhancing their efficiency.
Nucleic acids (NAs) are therapeutics with high potential, enabling the treatment of hereditary and acquired diseases. However, delivering NAs into target cells is extremely challenging. The large size and highly negative charge of NAs result in poor serum stability and hinder their ability to penetrate cellular membranes effectively. Furthermore, if cellular internalization occurs, most NA molecules are degraded by lysosomal enzymes after becoming entrapped in acidic endosomes. To tackle these issues, various NA delivery systems have been developed. Among these, cell-penetrating peptides (CPPs) are highly promising candidates. These up to 30 amino acid-long peptides exhibit an inherent ability to cross biological membranes, carrying cargo molecules into cells. Positively charged CPPs readily form non-covalent complexes with NAs based on electrostatic interactions, and the forming nanoparticles have been shown to effectively deliver various NAs in vitro and in vivo. The current thesis's focus was to understand better the factors determining the efficiency of CPP-based NA delivery and explore methods for improvement. As a model CPP, we used PepFect14 (PF14), an amphipathic stearylated CPP with a charge of +5. Firstly, we analyzed the protein corona of CPP/NA complexes – a layer of proteins forming around nanoparticles once exposed to a serum-containing environment, e.g., the bloodstream. We showed that the protein corona composition is highly dependent on the serum used and affects the nanoparticles' properties, cellular association, and resulting biological effect. Secondly, we investigated the impact of added Ca²⁺ and Mg²⁺ ions on the properties and functionality of the PF14/NA nanoparticles. We showed that these ions, especially Ca²⁺, greatly enhance the biological effect of NAs delivered by PF14, most likely facilitating the endosomal escape of the cargo. Finally, we investigated the ability of PF14 to transfect messenger RNA into cell lines, primary cells, and in vivo. We supplemented the nanoparticles with different excipients to achieve high mRNA transfection efficiency in cultured cells and in vivo. In summary, our findings provide novel insights into the challenges associated with developing CPP/NA nanoparticles for clinical use and open potential avenues for enhancing their efficiency.
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