The lack of specificity of traditional cytostatics and increasing resistance of cancer cells represent important challenges in cancer therapy. One of the characteristics of cancer cells is their intrinsic oxidative stress caused by higher metabolic activity, mitochondrial malfunction, and oncogene stimulation. This feature can be exploited in the pursuit of more selective cancer therapy, as there is increasing evidence that cancer cells are more sensitive to elevated concentrations of reactive oxygen species than normal cells. In this study, we demonstrate a new concept for cancer cell targeting by in situ production of radicals under physiological conditions. The biologically active radicals are produced in the milieu of cancer cells by enzymatic conversion from an inactive precursor, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt, by using miniature bioreactors represented by cell-sized microgels containing immobilized laccase. We utilize the pH-dependent activity of laccase to generate radicals only at a lower pH (5.7-6.1) that is characteristic of the tumor microenvironment. The composition of the microgels was optimized so as to allow sufficient substrate and radical diffusion, high enzyme activity, and stability under physiological conditions. The functionality of this system was evaluated on three cancer cell lines (HeLa, HT-29, and DLD1) and the cytotoxicity of in situ-produced radicals was successfully proven in all cases. These results demonstrate that cancer cell targeting by in situ-generated radicals using miniature enzymatic reactors may represent an alternative to traditional cytostatics. In particular, the pH-dependence of radical generation and their short-lived nature can ensure localized functionality in the tumor microenvironment and thereby reduce systemic side-effects.
Keywords: Spray-drying; cancer cell targeting; cytotoxicity; immobilization; laccase; microgel; microparticles; oxidative stress; pH-dependence; radicals.