Positively charged cyclodextrins as effective molecular transporters of active phosphorylated forms of gemcitabine into cancer cells

Violeta Rodriguez-Ruiz,Yannis G Lazarou,Giuseppina Salzano,Maria Lampropoulou,Konstantina Yannakopoulou,Valentina Agostoni,Andrey Maksimenko,Patrick Couvreur,Ruxandra Gref

doi:10.1038/s41598-017-08727-y

Abstract

Positively charged cyclodextrins (PCCDs) are molecular carriers of particular interest for their ability to readily enter into cancer cells. Of main interest, guanidino- and aminoalkyl- PCCDs can be conveniently synthesized and form stable and strong inclusion complexes with various active molecules bearing phosphate groups. We have addressed here the challenge to deliver into cancer cells phosphorylated gemcitabine drugs well known for their instability and inability to permeate cell membranes. NMR data corroborated by semiempirical theoretical calculations have shown that aminoalkyl-CDs form sufficiently stable complexes with both mono- and tri-phosphate forms of gemcitabine by simple mixing of the compounds in aqueous solution at physiological pH. Confocal microscopy and radioactivity counting experiments revealed that the developed systems enabled phosphorylated gemcitabine to penetrate efficiently into aggressive human breast cancer cells (MCF7), eventually leading to a substantial reduction of IC50 values. Moreover, compared to free drugs, phosphorylated metabolites of gemcitabine encapsulated in PCCDs displayed improved in vitro activities also on the aggressive human cancer cells CCRF-CEM Ara-C/8 C, a nucleoside transport-deficient T leukemia cell line. The current study offers the proof-of-principle that phosphorylated nucleoside drugs could be efficiently transported by PCCDs into cancer cells.

Highlights

Nucleoside analogue prodrugs encompass a range of antiviral and anticancer agents
DFdCMP is phosphorylated to the diphosphate[4] and to the active triphosphate form[5] which competes with the natural substrates for incorporation into DNA resulting in inhibition of nucleic acid synthesis and enzymes of nucleotide metabolism[6]
® intracellular concentration of the model triphosphorylated hydrophilic BODIPY TR ATP into cancer cells. These findings suggest that the positively charged cyclodextrins (PCCDs) host 1 can promote efficient complexation with phosphorylated drugs, enabling the entire complexes to cross the cell membranes and efficiently deliver their cargo inside the cells

Summary

Introduction

Nucleoside analogue prodrugs encompass a range of antiviral and anticancer agents. Among them, the cytidine analogue gemcitabine (2,2′-difluorodeoxycytidine, dFdC) (Fig. 1a) is a first line drug used to treat various solid tumors including non-small-cell lung cancer and pancreatic cancer[1]. Various strategies aimed at increasing the stability and efficiency of active forms of dFdC have been investigated, including their incorporation in colloidal delivery systems as well as their direct conjugation to lipophilic molecules[9,10,11]. As an alternative to colloidal nanoparticles, we propose here the use of molecular carriers as delivery systems to transport active phosphorylated dFdC inside cancer cells. A pKa of 3.6 for the protonated amino group of dFdC has been reported, meaning that at physiological pH dFdC is mostly unprotonated[17] In light of these considerations, cell-penetrating PCCD derivatives including per-[6-(2-aminoalkylamino) -6-deoxy]-βCD (hosts 1, 2) and per-[6-guanidino-6-deoxy]-βCD (host 3) (Fig. 1b), which are mostly protonated in neutral aqueous solutions (pKa values of host 1 are 6.4 and 9.5; pKa values of guanidino-terminated derivative are 7.8 and 11.012) were selected in this study

Methods

Results

Conclusion