Abstract 4663: Lysosomal targeting from a gelatin-doxorubicin conjugate produces evidence of both nuclear and lysosomal cytotoxcity pathways in mcf-7 breast cancer cells
Authors: Mohammed Alvi, Bayan Eshmawi, Rachel Nicoletto, Hyun Kate Kim, Christopher Cammarata, Clyde Ofner
Journal: Cancer Research (2018)

Abstract Background: Intra-lysosomal targeting has been investigated for the treatment of cancer but details of the cytotoxicity pathways remain unclear. A high molecular weight gelatin - doxorubicin conjugate (GDox) has been synthesized to target acidic lysosomes for intra-lysosomal drug release. The purpose of this investigation was to explore GDox feasibility for drug delivery and to make a preliminary determination of a cytotoxic pathway. Methods: GDox was synthesized in formamide using the carbodiimide, EDC. Drug load was determined spectroscopically, drug release at pH 4.8 was determined by HPLC, and molecular weight and its reduction in FBS was determined by HPSEC. Cellular localization was determined for free Dox and GDox at 10 µM or its equivalent Dox concentration using fluorescence microscopy with appropriate subcellular stains. Three to four independent trials (n=3-4) were examined with 200 to 700 cells per time point. Cytotoxicity was determing by the MTT procedure (n=3). Viabilities, growth inhibition profiles and their IC50 values were calculated with GraphPad Prism 7. The nucleus content of free Dox or Dox released from GDox after incubation was determined by UHPLC using fluorescent detection (n=3-4). Results: A typical GDox contained 5.6% w/w Dox, had a molecular weight of ~ 160 kDa, and released 58+2.3% Dox at pH 4.8 representing lysosomal pH. After incubation in FBS at 37°C for 24 hr the low molecular weight species increased a relatively small 16%. Fluorescent images with LysoTracker Green confirmed GDox localization to the lysosome which was detectable by 2 hr but substantial in swollen lysosomes by 24 and 48 hrs. No released Dox was observed in the nucleus, but a slight red nucleus haze at later times suggested this possibility. Cell debri from GDox was not observed from free Dox. As expected, free Dox rapidly accumulated in the nucleus by 2 hr. Viability determinations at 0.1, 1, and 10 µM for 2 to 48 hr showed no effect from either agent before 24 hr. By 48 hr at 10 µM, the viability of Dox (30+12%) and GDox (36+13%) were indistinguishable (p=0.68). However, by 72 hr, IC50 values varied 10-fold at 0.094 and 0.96 µM, respectively. The nucleus drug content after incubation at 10 µM for 2 to 48 hr was 0.27+0.07 to 1.39+0.6 µg/106 cells (p&amp;lt0.01) for free Dox, and 0.014+0.01 to 0.11+0.06 µg/106 cells (p&amp;lt0.01) for released Dox. Conclusions: Taken together these results suggest good feasibility of GDox lysosomal targeting and Dox release in MCF7 breast cancer cells. Of note, after 48 hr incubation GDox produced the same cytotoxicity as the free drug but with 13-fold less Dox in the nucleus. This striking nucleus difference and the substantial lysosomal GDox content suggest a lysosomal pathway of GDox cytotoxicity in addition to a nuclear pathway. Support: NIH/NCI R15CA135421 and the Agnes Varis Trust for Women's Leadership and Health. Citation Format: Mohammed Alvi, Bayan Eshmawi, Rachel Nicoletto, Hyun Kate Kim, Christopher Cammarata, Clyde Ofner. Lysosomal targeting from a gelatin-doxorubicin conjugate produces evidence of both nuclear and lysosomal cytotoxcity pathways in mcf-7 breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4663.