(W0930-01-01) Targeting TNBC Tumor Using Hypoxia-Responsive Nanoparticles containing Synergistic Drug Combinations

Purpose: Triple-Negative Breast Cancer (TNBC) is an aggressive malignancy without the expressions of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Common treatment strategies include surgery, chemotherapy, radiation, and hormonal therapy. However, the effectiveness of chemotherapy is often limited by the harmful side effects. In addition, solid tumors in TNBC develop hypoxia, leading to aggressiveness, increased metastasis, and become resistant to chemotherapy. Napabucasin (NAPA), a STAT-3 inhibitor, is undergoing clinical trials as an adjuvant therapy for solid tumors. In these studies, we have developed hypoxia-responsive nanoparticles containing NAPA that would decrease the stemness and increase the effectiveness of the anticancer drug doxorubicin (DOX) in killing TNBC cells under hypoxia.

Methods: We used MDA-MB-231 cells and performed a synergy experiment with NAPA and Dox under normoxia (21% oxygen) and hypoxia (0.2% oxygen) to determine the effect of the drugs. We performed immunofluorescence (IF) assays using MDA-MB-231 and patient-derived (PDX) TNBC cells under both normoxia and hypoxia to detect the neuropilin-1 (NRP-1) receptor expression. Using hypoxia-responsive iRGD peptide, we developed PEG-Azo-PLA nanoparticles encapsulating NAPA and DOX. Furthermore, we characterized the developed nanoparticles for their drug release pattern, toxicity, and effectiveness using MDA-MB 231 and patient-derived TNBC cells

Results: We observed a prominent synergistic effect of NAPA and DOX in regulating the MDA-MB-231 cell proliferation in both normoxia and hypoxia. Interestingly, the synergistic effect of NAPA and DOX was higher in hypoxic conditions than in normoxia. Further, we observed a significant increase in NRP-1 expression on MDA-MB-231 and PDX TNBC cells in hypoxia. The developed nanoparticles had a diameter of 130 ± 15 nm and a NAPA encapsulation efficacy of 25%. The DOX-encapsulated nanoparticles had a diameter of 210 ± 20 nm with an encapsulation efficacy of 60%. We tested these nanoparticles on MDA-MB-231 cells and found an increased release of encapsulated NAPA and DOX compared to non-encapsulated counterparts. Additionally, we found that cancer cell viability was significantly reduced when treated with NAPA and DOX nanoparticles together in hypoxic conditions compared to normoxic conditions.

Conclusion: The combination of NAPA and Dox showed promising synergy in regulating the proliferation of MD-MB-231 cells. The NRP-1 targeted hypoxia-responsive polymersomes showed effective delivery of NAPA and Dox in the hypoxic regions of TNBC cells. Delivering the synergistic drug to TNBC during hypoxia can reduce cell growth and metastasis, suppress stemness markers, and mitigate chemotherapy-associated side effects.

Acknowledgements: DaCCoTA (NIGMS, U54GM128729) , Confocal fluorescence microscope: Pharmaceutical Sciences Department, purchased with DaCCoTA funds.​​, NIH (NIGMS) 2R01 GM114080​

Figure 1: Schematic showing the study hypothesis