دانشگاه: University of the Sciences in Philadelphia
: 8.84 MB
Abstract: Doxorubicin (DOX) is one of the most potent anthracycline antibiotics used for treatment of multiple tumor types including breast cancer treatment. However, its efficacy is limited by fatal toxicities associated with DOX therapy causing damage to healthy tissues and organs. In this project, a high molecular weight gelatin-doxorubicin (GDOX) conjugate was synthesized and purified. Gelatin was linked to DOX in this conjugation via glygly spacer and an acid labile hydrazone bond. GDOX was characterized for molecular weight distribution of gelatin by size exclusion chromatography. Drug load on the conjugate was measured by UV-visible spectroscopy. DOX release from conjugate was measured at three different pH: 4.8, 6.5 and 7.4. A minimal DOX release of 9% ± 3 % was observed at systemic pH (7.4) and maximum release of 49 ± 1.8 % was observed at tumor microenvironment at pH 4.8. GDOX was then evaluated in an in vitro model consisting of two breast cancer cell lines: MCF7 cells and a triple negative breast cancer (TNBC) MDA-MB-231 cells for intracellular trafficking with emphasis on lysosomal targeting. Cell uptake and localization of an equivalent concentration of 10 µM DOX in GDOX was determined with fluorescence microscopy in these cells and fluorescence quantification was performed with Fiji software. Whole cell fluorescence showed localization of GDOX in both cell lines with higher conjugate accumulation in MDA-MB-231 cells. Lysosomal membrane permeabilization (LMP) was studied using a fluorescent labelled dextran. After 24 h, GDOX triggered 100% LMP in TNBC cells but surprisingly no LMP was observed in MCF7 cells. Finally, to evaluate GDOX induced DNA damage in these cells, TUNEL assay was performed which labels DNA strands breaks using a fluorescent tag. Images were captured on a fluorescent microscope and evaluated for percent apoptotic cell count. A similar extent of apoptosis was seen in both the cell lines and was comparable to free DOX. Overall, these results suggest that conjugate has potential of inducing toxicity to TNBC cells via lysosomal pathway which may induce apoptosis and thus has a potential for treatment of TNBC tumors. However, MCF7 cell studied results indicates a different pathway of toxicity in inducing apoptosis.
Keywords: Lysosomal targeting,Cell damage,Breast Cancer,In vitro,Doxorubicin ,Antibiotics,Tumor treatment, DNA damage