Acid-sensitive polyethylene glycol conjugates of doxorubicin: preparation, in vitro efficacy and intracellular distribution

Coupling anticancer drugs to synthetic polymers is a promising approach of enhancing the antitumor efficacy and reducing the side-effects of these agents. Doxorubicin maleimide derivatives containing an amide or acid-sensitive hydrazone linker were therefore coupled to α-methoxy-poly(ethylene glycol)-thiopropionic acid amide (MW 20000 Da), α,ω-bis-thiopropionic acid amide poly(ethylene glycol) (MW 20000 Da) or α-tert-butoxy-poly(ethylene glycol)-thiopropionic acid amide (MW 70000 Da) and the resulting polyethylene glycol (PEG) conjugates isolated through size-exclusion chromatography. The polymer drug derivatives were designed as to release doxorubicin inside the tumor cell by acid-cleavage of the hydrazone bond after uptake of the conjugate by endocytosis. The acid-sensitive PEG conjugates containing the carboxylic hydrazone bonds exhibited in vitro activity against human BXF T24 bladder carcinoma and LXFL 529L lung cancer cells with IC70 values in the range 0.02–1.5μm (cell culture assay: propidium iodide fluorescence or colony forming assay). In contrast, PEG doxorubicin conjugates containing an amide bond between the drug and the polymer showed no in vitro activity. Fluorescence microscopy studies in LXFL 529 lung cancer cells revealed that free doxorubicin accumulates in the cell nucleus whereas doxorubicin of the acid-sensitive PEG doxorubicin conjugates is primarily localized in the cytoplasm. Nevertheless, the acid-sensitive PEG doxorubicin conjugates retain their ability to bind to calf thymus DNA as shown by fluorescence and visible spectroscopy studies. Results regarding the effect of an acid-sensitive PEG conjugate of molecular weight 20000 in the chorioallantoic membrane (CAM) assay indicate that this conjugate is significantly less embryotoxic than free doxorubicin although antiangiogenic effects were not observed.