Studies On A Retro-inverso D-SP5 Peptide Modified Polymeric Micelles For Tumor Targeting Drug Delivery

The application of tumor targeting ligands for the treatment of cancer holds the promise of enhanced efficacy and reduced toxicity. In the recent years, several peptides that are capable to target tumor blood vessels have been discovered using in vivo phage display technology. L-SP5 (L (SVSVGMKPSPRP)), first identified in 2007 as SP5-52, was a natural peptide (L-peptide) that could recognize tumor neovasculature but not normal blood vessels. However, the tumor targeting efficacy of L-SP5 was highly limited due to the protease sensitivity during the process of serum circulation.In this article, to improve its in vivo stability and potentially strengthened binding bioactivity, the retro-inverso version of L-SP5, D-SP5 was designed and synthesized according to the retro-inverso theory. The main content of this study includes several parts:First, peptides and their derivatives were synthesized and the serum stability of L-SP5 and D-SP5 were investigated and compared. Second, tumor targeting capability of D-SP5 was studies both qualitatively and quantitatively. Third, D-SP5-PEG-DSPE was synthesized and determined, and D-SP5-conjugated polymeric micelles were prepared and characterized. Fourth, Dox-loaded micelles were evaluated in-vitro and in-vivo for antitumor effect.Peptides D-SP5 and parental L-SP5 are shown to compete for the same target sites of a yet unknown cellular target and possess a dual-targeting bioactivity for both activated endothelial cells (HUVEC) and several tumor cell lines. Cellular uptake experiments showed superior in vitro targeting abilities of D-SP5 compared with L-SP5, such as enhanced internalization into stimulated HUVEC or KB, U87, and SGC tumor cells. A radioligand receptor binding assay revealed a higher cell affinity of D-SP5 in all tested cell lines, with Kd values for D-SP5 about 2-fold lower than for L-SP5.125I-D-SP5 bound to stimulated HUVEC, KB, with Kd of 131 and 588 pmol/L, while L-SP5 bound to the cells with Kd of 232 and 1250 pmol/L. Maximum binding capacity (Bmax) of D-SP5 to the stimulated-HUVEC, KB were 61 and 243 fmol per 106 cells and those of L-SP5 were 24 and 160 fmol per 106 cells. Fluorescein-labeled D-SP5 upon intravenous administration displayed strong association with tumor endothelium. D-SP5-conjugated PEG-DSPE micelles displayed enhanced tumor homing (evidenced by near infrared in vivo imaging). When loaded with Doxorubicin, D-SP5 micelles could markedly suppress tumor growth with higher efficacy than L-SP5 micelles both in vitro and in vivo in KB tumor xenografts. In summary, the data demonstrate that D-SP5 displays higher binding affinities towards tumor endothelium as well as tumor cells and enhanced tumor targeting capability in vitro and in vivo.