MMP-2-triggered, mitochondria-targeted PROTAC-PDT therapy of breast cancer and brain metastases inhibition

Abstract

Proteolytic targeting chimera (PROTAC) technology is a protein-blocking technique and induces antitumor effects, with potential advantages. However, its effect is limited by insufficient distribution and accumulation in tumors. Herein, a transformable nanomedicine (dBET6@CFMPD) with mitochondrial targeting capacity is designed and constructed to combine PROTAC with photodynamic therapy (PDT). In this work, we demonstrate that dBET6@CFMPD exhibits great biodistribution and retention, and can induce potent antitumor response to suppress primary and metastatic tumors, becoming a nanomedicine with potential in cancer combination therapy.

Fig. 1. Preparation and characterization of nanomedicines.

a Schematic illustration of the composition of dBET6@CFMPD and its therapeutic effect on primary breast cancer and brain metastases. a Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. The DLS results and TEM images of b dBET6@CPD, c dBET6@CFPD, and d dBET6@CFMPD (scale bar = 200 nm). The molecular mass changes of CFPD (e) and CFMPD (f) after incubation with MMP-2 for 4 and 24 h were detected by MALDI-TOF-MS analyses. MALDI-TOF-MS spectra of e, f are included in Supplementary Figs. 10 and 11. g The TEM images of dBET6@CFMPD after incubation with MMP-2 (scale bar = 2 μm). The TEM images and enlarged images of cells treated with CFPD (h) and CFMPD (i). Red arrows and blue arrows indicate spherical nanoparticles and nanofibers, respectively (scale bar = 500 nm). Source data are provided as a Source Data file.

Fig. 2. Cellular uptake, retention, and mitochondria targeting.

a, b Cellular uptake assay of different formulations by fluorescence microscope (scale bar = 100 μm). c, d Cellular uptake assay of different formulations by flow cytometry (n = 3 independent cell lines). e Confocal images of 4T1 MSCs incubated with free Ce6, CPD, CFPD, and CFMPD for 12 h (scale bar = 100 μm). f Quantitative analyses of tumor spheroid sections at 100 μm. g Mitochondrial localization analyses of different formulations in 4T1 cells (scale bar = 20 μm). h TEM image of cells treated with CFMPD (scale bar = 1 μm). Red arrows indicate mitochondrial cristae. All data are presented as mean ± SD. Two-tailed Student’s t test and One-way analysis of variance (ANOVA) with a Tukey post hoc test were used for the statistical comparison between the two groups and among multiple groups, respectively. A significant difference was considered when the p value was <0.05. Source data are provided as a Source Data file.

Fig. 3. BRD4 inhibition and antitumor effect in vitro.

a WB analyses of BRD4 inhibition and EMT downregulation on 4T1 cells. The G1–G6 represent control, dBET6&Ce6 + L, CFMPD + L, dBET6@CPD + L, dBET6@CFPD + L, and dBET6@CFMPD + L, respectively. b PD-L1 expression of 4T1 cells treated with different formulations. c Detection of intracellular ROS generation by flow cytometry (n = 3 independent experimental cell lines). MTT assay after incubating 4T1 cells with parent nanoparticles (d) and BET6-loaded nanoparticles (e). n = 3 independent cell lines. f Apoptosis assay of 4T1 cells treated with various formulations. g Statistical results of apoptosis analysis (n = 3 independent cell lines). h Live/dead double staining of 4T1 cells incubated with different preparations (scale bar = 100 μm). All data are presented as mean ± SD. Two-tailed Student’s t test and One-way analysis of variance (ANOVA) with a Tukey post hoc test were used for the statistical comparison between the two groups and among multiple groups, respectively. A significant difference was considered when the p value was <0.05. Source data are provided as a Source Data file.

Fig. 4. Evaluation of ICD, macrophage polarization, and anti-metastasis effect.

a, b CRT exposure on the 4T1 cell surface was detected by confocal microscope and flow cytometry (scale bar = 50 μm, n = 3 independent cell lines). c, d Extracellular HMGB-1 and ATP levels of tumor cells after incubation with different formulations (n = 3 independent cell lines). e–g Secreted IL-10, TGF-β, and IFN-γ by RAW264.7 cells after treatment with different formulations (n = 3 independent cell lines). h, i Flow cytometry analysis of the abundance of CD86⁺ and CD206⁺ RAW264.7 cells after incubation with various formulations (n = 3 independent cell lines). j–l Microscopy images of wound healing assay, migration assay, and invasion analysis of 4T1 cells treated with different formulations (scale bar = 100 μm). All data are presented as mean ± SD. Two-tailed Student’s t test and One-way analysis of variance (ANOVA) with a Tukey post hoc test were used for the statistical comparison between the two groups and among multiple groups, respectively. A significant difference was considered when the p value was < 0.05. Source data are provided as a Source Data file.

Fig. 5. In vivo targeting efficiency and retention effect of nanomedicines.

a In vivo imaging of mice at 1, 2, 4, 8, 12, 24 h post intravenous injection. b Ex vivo imaging of tumors. c Semi-quantification of major organs and tumors at 24 h post intravenous injection (n = 4 mice). d In vivo fluorescent imaging of mice at 5 min, 4, 8, 12, 24, 36, and 48 h post intra-tumoral injection. e Ex vivo imaging of tumors at 48 h. f Semi-quantification of tumors (n = 4 mice). g Fluorescent distribution of frozen sections of tumors (scale bar = 100 μm). All data are presented as mean ± SD. Two-tailed Student’s t test and One-way analysis of variance (ANOVA) with a Tukey post hoc test were used for the statistical comparison between the two groups and among multiple groups, respectively. A significant difference was considered when the p value was < 0.05. Source data are provided as a Source Data file.

Fig. 6. Evaluation of brain-targeting ability.

a Schematic representation of the in vitro construction process for blood-brain barrier (BBB) and the traversal of BBB. Figure 1a Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. b Fluorescence intensity of the supernatant liquid in the lower chamber after the introduction of NPs for 4 h (n = 3 independent experimental units). c Fluorescence intensity of nanoparticles taken up by 4T1 cells in the lower chamber (n = 3 independent cell lines). d In vivo imaging of mice bearing with brain-metastatic tumor at 1, 2, 4, 8 h post intravenous injection. e, f Ex vivo imaging of major organs and brains at 8 h post intravenous injection. g, h Semi-quantification of major organs and brains (n = 4 mice). All data are presented as mean ± SD. Two-tailed Student’s t test and One-way analysis of variance (ANOVA) with a Tukey post hoc test were used for the statistical comparison between the two groups and among multiple groups, respectively. A significant difference was considered when the p value was < 0.05. Source data are provided as a Source Data file.

Fig. 7. In vivo antitumor and anti-metastasis effects.

a Tumor volume of 4T1 tumor-bearing mice recording every 2 days (n = 6 mice). b Tumor weights of 4T1 breast cancer models (n = 6 mice). c Body weight of mice administrated with different formulations (n = 6 mice). d Ki67 and TUNEL staining assay of sectioned tumors (scale bar = 2 mm). e, f Ex vivo imaging and statistical results of lung metastases (n = 5 mice). g HE staining images of lung tissues (scale bar = 5 mm in HE images, scale bar = 100 μm in enlarged images). h, i Weights and ex vivo image of recurrent tumors (n = 5 mice). The G1–G8 represent Control, dBET6, Ce6 + L, CFMPD + L, dBET6@CFMPD, dBET6@CPD + L, BET6@CFPD + L, and BET6@CFMPD + L, respectively. All data are presented as mean ± SD. Two-tailed Student’s t test and One-way analysis of variance (ANOVA) with a Tukey post hoc test were used for the statistical comparison between the two groups and among multiple groups, respectively. A significant difference was considered when the p value was < 0.05. Source data are provided as a Source Data file.

Fig. 8. Inhibition of primary and brain-metastatic tumors.

a Bioluminescence imaging of mice bearing with brain-metastatic tumors. b Semi-quantification results of bioluminescence imaging on the 8th, 16th, and 24th post-tumor inoculation (n = 10 mice). c Tumor volume of primary tumors (n = 10 mice). d, e The ex vivo image and weights of primary tumors (n = 10 mice). The G1–G4 represent Control, CFMPD + L, BET6@CFPD + L, and BET6@CFMPD + L, respectively. f Percent survival analyses of mice bearing with brain-metastatic and primary tumors (n = 10 mice). And survival study was analyzed through Two-sided log-rank (Mantel–Cox) test. g H&E staining of brain tissues (scale bar = 2 mm). All data are presented as mean ± SD. Two-tailed Student’s t test and One-way analysis of variance (ANOVA) with a Tukey post hoc test were used for the statistical comparison between the two groups and among multiple groups, respectively. A significant difference was considered when the p value was < 0.05. Source data are provided as a Source Data file.

Fig. 9. In vivo antitumor immune response.

a Representative immunofluorescent images of tumor sections to detect the CRT and HMGB1 levels (scale bar = 50 μm). b–d Percentages of CD80⁺ CD86⁺ DCs, CD8⁺ T cells, and CD4⁺ T cells in LNs. Quantification data of CD8⁺ T cells (e), central memory T cells (Tcm, CD44⁺CD62L⁺) (f), and effector memory T cells (Tem, CD44⁺CD62L⁻) (g) in spleens. Abundances of IFN-γ⁺ CD8⁺ T cells (h), CD4⁺ T cells (i), and T_reg cells (j) in tumors. k Percentages of PD-L1⁺ cells in tumors after different treatments. Flow cytometry analyses of M1 (l) and M2 (m) cells in tumors. Flow cytometry measurements of CD8⁺ T cells (n) and CD4⁺ T cells (o) in the DLNs of mice bearing with primary and brain metastatic tumors. Percentages of M1 (p) and M2 (q) cells in the brains after different treatments. r Representative immunofluorescent images of the brain to observe CD86⁺ and CD206⁺ cells (scale bar = 1 mm). Green and red fluorescence signals indicate CD86 and CD206, respectively. n = 4 mice per group for all the studies. All data are presented as mean ± SD. Two-tailed Student’s t test and One-way analysis of variance (ANOVA) with a Tukey post hoc test were used for the statistical comparison between the two groups and among multiple groups, respectively. A significant difference was considered when the p value was < 0.05. Source data are provided as a Source Data file.

Fig. 10. Anti-metastasis effect and antitumor immune response on E0771-bearing mice.

a Bioluminescence imaging of mice bearing with brain-metastatic tumors. b Semi-quantification results of bioluminescence imaging on the 8th, 16th, and 24th post-tumor inoculation. c, d The ex vivo image and weights of primary tumors. e–g Percentages of CD80⁺ CD86⁺ DCs, CD8⁺ T cells, and CD4⁺ T cells in LNs. h–k Abundances of CD80⁺ CD86⁺ DCs, CD8⁺ T cells, CD4⁺ T cells, and Tcm cells in spleens. l–o Abundances of CD8⁺ T cells, PD-L1⁺ cells, T_reg cells, and M1 cells in tumors. n = 9 mice per group for primary tumor growth and bioluminescence imaging studies in (a, b, d). n = 5 mice per group for other studies. All data are presented as mean ± SD. Two-tailed Student’s t test and One-way analysis of variance (ANOVA) with a Tukey post hoc test were used for the statistical comparison between the two groups and among multiple groups, respectively. A significant difference was considered when the p value was < 0.05. Source data are provided as a Source Data file.