Fig. 6. Synthesis of the region-confined PROTAC nanoparticle and its antitumor performance in MDA-MB-231 breast tumor model in vivo.

a General view of hypoxia-responsive and hypoxia-insensitive ARV771 derivates. b Representative profiles of HPLC analysis on ARV771-Nb and ARV771-Ph treated with different concentrations of Na₂S₂O₄. Western blot assay of BRD4 expression and its downstream protein of CDK4, CDK6 and p21 in MDA-MB-231 stem-like cells with (c) ARV771, (d) ARV771-Nb and (e) ARV771-Ph treatment for 24 h. f Number of the tumor spheroids (diameter > 50 μm) after the MDA-MB-231 cells with various treatments for 12 days (n = 5 independent experimental cell lines). Statistical analysis was performed by two-sided unpaired t-test. g DLS data and TEM image of PGDAT@N nanoparticle (scale bar = 50 μm). h Quantitative PCR detection of RNA expression post 24 h treatment with different formulations in MDA-MB-231 stem-like cells (n = 3 independent experimental cell lines). i Averaged and (j) individual tumor growth profiles of tumor-bearing mice treated with diverse formulations (insert: mice image at 27 days of experimental period, n = 6 mice). Two-sided unpaired t-test was used in the statistical analysis. k Survival curves of tumor-bearing mice (n = 6 mice). Statistical analysis was performed by two-sided unpaired log-rank (Mantel-Cox) test. l TUNEL staining of the tumor sections (scale bar = 100 μm). m IHC analysis of BRD4 expression in the tumor sections (scale bar = 100 μm). n Western blot assay of PROTAC nanoparticle induced intratumoral BRD4 degradation and differential expression of its downstream proteins. o Flow cytometry examination of CSCs percentage in the tumor tissue post different treatments (n = 3 mice). Statistical analysis was achieved by two-side unpaired t-test. All data are presented as mean ± SD.