Improved anticancer efficacy of doxorubicin mediated by human-derived cell-penetrating peptide dNP2

Although cell penetrating peptides (CPPs) have been extensively studied as an approach to deliver anti-cancer drugs into the tumor cells for the last 30 years, no FDA-approved CPP-based drugs are available, implying that the existing CPPs may have less efficiency in human or have side effects such as toxicity. Herein, we established a tumor targeting drug delivery system by attaching a human-derived cell-penetrating peptide dNP2 (CKIKKVKKKGRKKIKKVKKKGRK) to N-(2-hydroxypropyl)-methacrylamide (HPMA) copolymer doxorubicin conjugates. Firstly, in vitro cytotoxicity of free dNP2 peptide and dNP2-modified blank HPMA copolymer were examined. A classic CPP-R8 (CRRRRRRRR) was chosen for comparison and the results showed that 200 μM free R8 reduced cell viability to 68.4% but dNP2 did not induce any toxicity at the same concentration. After conjugation to HPMA copolymer, a similar trend was also observed which indicated the excellent biocompatibility of dNP2. Next, effect of dNP2 modification on cellular uptake, DNA damage, apoptosis and anticancer activity of HPMA copolymer doxorubicin conjugates were evaluated. It was excited that dNP2 modified HPMA copolymer (P-(dNP2)-DOX) not only had a higher uptake by HeLa cell compared with non-modified copolymer (P-DOX) but resulted in an enhanced drug distribution in nuclei. Furthermore, P-(dNP2)-DOX exhibited greater DNA damage ability (10.5 folds higher than P-DOX) in comet assay and induced more apoptosis cells (46.0%). P-(dNP2)-DOX also showed a stronger cell cytotoxicity (3-fold to P-DOX) as well as in 3D tumor spheroid assay (inhibition rate 78%). All these results suggested that the human-derived cell-penetrating peptide dNP2 could facilitate tumor nuclear-accumulation of anti-cancer drugs and improve anticancer efficacy. More importantly, dNP2 has less toxicity compared with classic CPP-R8 thus shows the potential for the clinic cancer therapy.