Cell-penetrating peptides: cytotoxicity, immunogenicity and application for tumor targeting
Date
2012-09-04
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Abstract
Viimastel aastatel, suur tähelepanu kasvajate ravis on pööratud mitmesugustele terapeutilise toimega ravimainete sisestamise strateegiatele, mis ühendavad endas nii ravimite spetsiifilist edastamist kui ka vähivastase kemoteraapiat. Rakku sisenevad peptiidid (RSPd või CPP-s, cell-penetrating peptides) osutunud väga tõhusateks vektoriteks selliste terapeutiliste ravimite sisseviimisel nagu proteiine ning nukleiinhappeid (plasmiidne DNA (pDNA) ja splaissingut korrigeerivaid oligonukleotiide (SKO)). RSPde kasutamine võib olla piiratud nende poolt põhjustatud kõrvaltoimete tõttu: tsütotoksilised efektid ning immunvastuse esilekutsumine.
Käesoleva töö peamiseks eesmärgiks oli analüüsida uute RSPde, PepFect-ide immunogeenset ja tsütotoskilist aktiivsust in vitro ja in vivo; uurida p53 analoogsete peptiidide tsütotoksilisust ning apoptootilist toimet rinnavähi rakumudelil ning välja töötada uus RSP, mis on võimeline sisenema ja spetsiifiliselt ära tundma peaajuvähi rakke gliobalstoomi (U87 MG) rakumudelil nii in vitro kui ka in vivo. Esimeses töös näidati, et uued RSPd PepFect-id, mis põhinevad peptiidi transportaan järjestusel, ei avaldanud tsütotoksilist ega immunogeenset toimet immuunsüsteemi rakkudele peptiidi kontsentratsioonidel 5 µM ja 10 µM in vitro tingimustes. Sarnane tulemus saadi ka immunokompetentsete hiirte süstimisel, kui kasutatud annused olid suuremad kui 5 mg/kg kohta. Teises töös disainiti ning sünteesiti lühikesi p53-analoogseid peptiide, mis lisaks efektiivsele rakku sisenemisele avaldasid ka apoptootilist toimet rinnavähi rakkudele in vitro (seerumivabas keskkonnas). Lühikesed p53-analoogsed peptiidid omavad head perspektiivi vähivastase strateegia arendamiseks.
Kolmandas töös kirjeldati uue transportvektori, gHoPe2, väljatöötamist, mis on võimeline spetsiifiliselt sihtmärkrakkudesse sisenema. Demonstreeriti ka selle potentsiaalseid kasutusvõimalusi ning toimet ainete transpordil vähirakkudesse.
Neljandas töös näidati, et mitteamfipaatse ja sünteetilise (RxR)4 peptiidi stearüülhappega N-terminaalse modifitseerimine tagab efektiivse ning mitte toksilise nukleiinhappete transporti rakkudesse.
Käesolev töö annab täiendava ülevaate vähispetsiifilistel RSPdel põhinevatest transportsüsteemidest, hinnates nende toksilisust, immunogeenset aktiivsust ning spetsiifilist transporti kasvajarakkudesse.
The delivery of therapeutic agents for the hard-to-treat tumors which comprise both targeting and anticancer chemotherapy strategies, have received much attention. Application of cell-penetrating peptides is one of very promising strategies for targeted drug delivery. However, similar issues as for any other drug delivery system: cytotoxicity and the tendency to induce innate immune response may limit their use. The current thesis is focused on the characterization of immunogenic and cytotoxic activities of cell-penetrating peptides PepFects, on the cytotoxic and apoptotic activities of newly designed cell-penetrating p53 analogues and on the development of a peptide-based glioma-targeted drug delivery vector. In Paper I, the cytotoxic and immunogenic activities of PepFect peptides and PepFect/nucleic acid complexes were analyzed in vitro and in vivo. The current study confirmed that PepFect peptides possess promising potential in nucleic acid delivery without evidence of undesired cytotoxicity and inflammation at the concentrations of 10 μM and 5 μM, respectively in vitro and at a dose of 5 mg/kg in vivo. In Paper II, we generated short p53-derived cell-penetrating protein analogues and analyzed their internalization efficacy, cytotoxic and apoptotic activities in the neuroblastoma and breast cancer cell lines. Even though the effect was observed under serum-free conditions, the novel protein analogues demonstrated selective apoptotic activity in the p53-non-active breast cancer cell line suggesting promising perspectives for a future anticancer strategy. In Paper III, a novel tumor-selective peptide-based drug delivery platform gHoPe2 has been developed demonstrating potential application of CPP-s for tumor-targeted delivery. In Paper IV, we showed that N-terminal modification of non-amphipathic (RxR)4 with stearic acid moiety resulted in efficient peptide vector for nucleic acids delivery in non-toxic manner. In conclusion, the current work provides evidence for the development of apoptotic protein-derived cell-penetrating peptides and tumor-targeted cell-penetrating peptide-based drug delivery vector with reduced toxicity and immunogenicity.
The delivery of therapeutic agents for the hard-to-treat tumors which comprise both targeting and anticancer chemotherapy strategies, have received much attention. Application of cell-penetrating peptides is one of very promising strategies for targeted drug delivery. However, similar issues as for any other drug delivery system: cytotoxicity and the tendency to induce innate immune response may limit their use. The current thesis is focused on the characterization of immunogenic and cytotoxic activities of cell-penetrating peptides PepFects, on the cytotoxic and apoptotic activities of newly designed cell-penetrating p53 analogues and on the development of a peptide-based glioma-targeted drug delivery vector. In Paper I, the cytotoxic and immunogenic activities of PepFect peptides and PepFect/nucleic acid complexes were analyzed in vitro and in vivo. The current study confirmed that PepFect peptides possess promising potential in nucleic acid delivery without evidence of undesired cytotoxicity and inflammation at the concentrations of 10 μM and 5 μM, respectively in vitro and at a dose of 5 mg/kg in vivo. In Paper II, we generated short p53-derived cell-penetrating protein analogues and analyzed their internalization efficacy, cytotoxic and apoptotic activities in the neuroblastoma and breast cancer cell lines. Even though the effect was observed under serum-free conditions, the novel protein analogues demonstrated selective apoptotic activity in the p53-non-active breast cancer cell line suggesting promising perspectives for a future anticancer strategy. In Paper III, a novel tumor-selective peptide-based drug delivery platform gHoPe2 has been developed demonstrating potential application of CPP-s for tumor-targeted delivery. In Paper IV, we showed that N-terminal modification of non-amphipathic (RxR)4 with stearic acid moiety resulted in efficient peptide vector for nucleic acids delivery in non-toxic manner. In conclusion, the current work provides evidence for the development of apoptotic protein-derived cell-penetrating peptides and tumor-targeted cell-penetrating peptide-based drug delivery vector with reduced toxicity and immunogenicity.
Description
Väitekirja elektrooniline versioon ei sisalda publikatsioone.
Keywords
bioloogiline transport, penetratsioon, peptiidid, tsütotoksilisus, immunogeneetika, vähirakud, plasmiidid, biological transport, penetration, peptides, cytotoxity, immunogenetics, cancer cells, plasmids