Design of new polymer cell- penetrating peptide conjugates for improvment of drug tumor penetration

Introduction

One of the greatest challenges for tumor targeted drug delivery systems is to achieve deep and efficient penetration to all regions of the tumor while minimizing internalization into healthy tissues. Using CPPs is one way to overcome this challenge. Cell penetrating peptides (CPPs) are short cationic peptides usually rich in Arginine and Lysine amino acid residues, which give the peptides a positive charge that allows them to easily penetrate the membranes of different cells ^[1]. CPPs have been conjugated to a various variety of macromolecular carriers in order to facilitate their transport inside the cell ^[2]. However the lack of cell specificity is the main drawback for their clinical use ^[3]. In this study different CPP sequences [namely Octaarginine (R8), TAT and Penetratin (Pen)] were conjugated to the water-soluble N-(2hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (DOX) conjugates in order to improve intratumor penetration. The comparable cell penetration activity and cytotoxicity was studied in 4T1 breast cancer and PC3 prostate cancer cell lines.

Results and Discussion

HPMA Copolymer-CPP conjugates, with or without a DOX, namely P-(R8)-FITC, P-(TAT)-FITC, P-(PEN)-FITC, and P-(R8)-DOX-FITC, P-(TAT)-DOX-FITC, P-(PEN)-DOX-FITC were synthesized by radical precipitation copolymerization, and their cell penetration activity was tested in 4T1 and PC3 cells. The cell penetration activity of the targeted HPMA copolymer bearing R8 and TAT peptides was significantly higher in both PC3 and 4T1 cancer cell lines, relative to HPMA copolymer bearing Pen peptide which showed less penetration activity. The reference copolymer, which has no CPP conjugated, showed minimal cell penetration activity. The results of the cytotoxicity assay were used to calculate the IC50 relative to control of non-treated cells. P-(TAT)-FITC was not toxic towards the cells. P-(TAT)-DOX-FITC demonstrated a dose dependent cytotoxicity towards the cells and was ~ 10-times less toxic towards when compared to free DOX, suggesting a different mechanism of cell entry.

Refrences

[1] Heitz, Frederic, May Catherine Morris, and Gilles Divita. "Twenty years of cell‐penetrating peptides: from molecular mechanisms to therapeutics." British journal of pharmacology157.2 (2009): 195-206.

[2] Koren, Erez, and Vladimir P. Torchilin. "Cell-penetrating peptides: breaking through to the other side." Trends in molecular medicine 18.7 (2012): 385-393.

[3] Vivès, Eric, Julien Schmidt, and André Pèlegrin. "Cell-penetrating and cell-targeting peptides in drug delivery." Biochimica et Biophysica Acta (BBA)-Reviews on Cancer1786.2 (2008): 126-138.