PEGylated Elesclomol@Cu(Ⅱ)-based Metal‒organic framework with effective nanozyme performance and cuproptosis induction efficacy for enhanced PD-L1-based immunotherapy

Abstract

Nanozymes constitute a promising treatment strategy for antitumor therapy. However, the catalytic function of metal‒organic framework (MOF)-based nanozymes during cuproptosis remains unclear. In this study, a Cu(Ⅱ)-based MOF nanocomposite loaded with the copper ionophore elesclomol and surface modified with polyethylene glycol polymer (PEG) was developed (ES@Cu(Ⅱ)-MOF) for effective cuproptosis induction. The peroxidase (POD)-like activity of ES@Cu(Ⅱ)-MOF generated an abundance of hydroxyl radicals (•OH) via a Fenton-like reaction, and its glutathione peroxidase (GSH-Px)-like activity converted Cu²⁺ into more toxic Cu⁺ ions to efficiently consume endogenous GSH. Notably, the rapid accumulation of Cu⁺ and ES in tumor cells induced the aggregation of lipoylated dihydrolipoamide S-acetyltransferase (DLAT) and the downregulation of Fe‒S cluster proteins, ultimately leading to cuproptosis. ES@Cu(Ⅱ)-MOF exhibited extraordinary cytotoxicity against breast cancer cells in vitro and significantly suppressed 4T1 breast tumor growth in vivo. Moreover, ES@Cu(Ⅱ)-MOF induced immunogenic cell death (ICD) to increase the antitumor immune response. Furthermore, combining ES@Cu(Ⅱ)-MOF with an anti-programmed cell death-ligand 1 (PD-L1) antibody converted the immunosuppressive tumor microenvironment to an immunogenic microenvironment, thus effectively inhibiting breast tumor growth. Overall, this work provides an innovative approach utilizing nanozymes to facilitate cuproptosis for cancer treatment, which potentially enhances the effectiveness of immune checkpoint inhibitor-based immunotherapy.

Keywords: Anti-PD-L1 antibody; Breast cancer; Cu(Ⅱ)-MOF; Cuproptosis; Immunotherapy; Nanozyme.

Graphical abstract

Fig. 1

Schematic illustration of the synthesis of ES@Cu(Ⅱ)-MOF nanozymes and the mechanism of ES@Cu(Ⅱ)-MOF-induced cuproptosis for enhancing combined immunotherapy.

Fig. 2

Characteristics of the ES@Cu(Ⅱ)-MOF NPs. A) Schematic illustration of the synthesis of the ES@Cu(Ⅱ)-MOF NPs. B) SEM images of the Cu(Ⅱ)-MOF NPs and ES@Cu(Ⅱ)-MOF NPs. Scale bar = 50 nm. C) Elemental mapping (Cu, C, N, O, and S) of the Cu(Ⅱ)-MOF NPs and ES@Cu(Ⅱ)-MOF NPs. Scale bar = 100 nm. D) Hydrodynamic sizes of the Cu(Ⅱ)-MOF NPs (average size = 152.7 nm, polydispersity index = 0.13) and ES@Cu(Ⅱ)-MOF NPs (average size = 171.9 nm, polydispersity index = 0.18). E) Zeta potentials of the Cu(Ⅱ)-MOF NPs, ES, and ES@Cu(Ⅱ)-MOF NPs (n = 3). F) UV–vis absorption spectra of the Cu(Ⅱ)-MOF NPs, ES, and ES@Cu(Ⅱ)-MOF NPs. G) FTIR spectra of the Cu(Ⅱ)-MOF NPs, ES, and ES@Cu(Ⅱ)-MOF NPs. H) XRD patterns of the Cu(Ⅱ)-MOF NPs and ES@Cu(Ⅱ)-MOF NPs. I) XPS analysis of the Cu(Ⅱ)-MOF NPs and ES@Cu(Ⅱ)-MOF NPs. The chemical formula of ES is C₁₉H₂₀N₄O₂S₂. The data are presented as the means ± SDs.

Fig. 3

The nanozyme activity of the ES@Cu(Ⅱ)-MOF NPs. A) Schematic illustration of the nanozyme activity of the ES@Cu(Ⅱ)-MOF NPs. B, C) Cumulative release of Cu and ES from the ES@Cu(Ⅱ)-MOF NPs in PBS at pH values of 4.5, 5.5, and 7.5, respectively (n = 3). D, E) Analyses of the POD-like activities of ES@Cu(Ⅱ)-MOF NPs treated with various concentrations of ES@Cu(Ⅱ)-MOF NPs and reaction times using TMB as a substrate. F) •OH generation detected by ESR spectroscopy. DMPO was used as a spin trap for •OH. G) Michaelis‒Menten kinetic analysis of ES@Cu(Ⅱ)-MOF NPs with H₂O₂ as a substrate. H, I) Analyses of the GSH-Px-like activities of ES@Cu(Ⅱ)-MOF NPs treated with various concentrations of ES@Cu(Ⅱ)-MOF NPs and reaction times using DTNB as a substrate. J) Michaelis‒Menten kinetic analysis for ES@Cu(Ⅱ)-MOF NPs with GSH as a substrate. The data are presented as the means ± SDs; ∗∗p < 0.01; ∗∗∗p < 0.001.

Fig. 4

Effects of ES@Cu(Ⅱ)-MOF NPs on cuproptosis induction. A) CLSM imaging analysis of the cellular uptake of FITC@Cu(Ⅱ)-MOF NPs (green) in 4T1 cells after 8 h of incubation. The cell nuclei were stained with Hoechst 33342 (blue), and the lysosomes were stained with LysoTracker (pink). Scale bar: 25 μm. The fluorescence intensities of the FITC@Cu(Ⅱ)-MOF (green) and lysosome (pink) signals across the regions indicated with white arrows were analyzed and shown in line plots (Fig. S7). B) ICP‒MS analysis of the intracellular Cu content in 4T1 cells after 8 h of incubation (n = 3). C) CLSM images of the intracellular ROS level (green) in 4T1 cells following the indicated treatments. The cells were stained with Dil (red). Scale bar: 50 μm. D) CLSM images of DLAT foci (green) in 4T1 cells after the indicated treatment. The cell nuclei were stained with DAPI (blue), and the mitochondria were stained with MitoTracker (red). Scale bars: 10 μm. The fluorescence intensities of DLAT foci (green) and mitochondrial (red) signals across the regions indicated with white arrows were analyzed and shown in line plots (Fig. S10). E) Quantification of DLAT foci in 4T1 cells after the indicated treatment (n = 3). F) Western blot analysis of the expression of Fe‒S cluster proteins (FDX1 and DLD) in 4T1 cells after different treatments. The intensity of each protein band was quantified via NIH ImageJ software. G) Schematic illustration of the mechanism of ES@Cu(Ⅱ)-MOF nanozyme-induced cuproptosis. 4T1 cells treated with CuCl₂ + ES were used as the positive control [3]. The data are presented as the means ± SDs; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Fig. 5

The antitumor effect of ES@Cu(Ⅱ)-MOF-induced cuproptosis in vitro. A-D) Relative viabilities of 4T1, MDA-MB-231, MDA-MB-468, and BEND3 cells following 24 h of incubation with a gradient concentration of ES@Cu(Ⅱ)-MOF NPs (n = 3). E) Morphological injury of 4T1 cells after different treatments. Scale bar: 50 μm. F) CLSM images of 4T1 cells stained with calcein-AM (green, live cells) and PI (red, dead cells) after different treatments. Scale bar: 50 μm. G) Flow cytometric analysis of the degree of necrosis in 4T1 cells following various treatments. H) Quantification of Annexin V-FITC⁺/PI⁺ 4T1 cells after the indicated treatment (n = 3). I) Colony formation ability of 4T1 cells after different treatments. J) Quantification of the number of colonies formed by 4T1 cells after the indicated treatment (n = 3). 4T1 cells treated with CuCl₂ + ES were used as the positive control [3]. The data are presented as the means ± SDs; ∗∗p < 0.01; ∗∗∗p < 0.001.

Fig. 6

ICD triggered by ES@Cu(Ⅱ)-MOF NPs in vitro. A) CLSM images of the released HMGB-1 (green) in 4T1 cells after the indicated treatment. The cell nuclei were stained with DAPI (blue). Scale bars: 50 μm. B) CLSM images of exposed CRT (green) after the indicated treatment. The cell nuclei were stained with DAPI (blue). Scale bars: 50 μm. C) Released LDH levels in 4T1 cells after the indicated treatments (n = 3). D) Intracellular ATP levels in 4T1 cells after the indicated treatments (n = 3). E) Schematic illustration of the transwell experiment. The conditioned medium following the indicated treatments was added to the lower chamber, and RAW264.7 macrophages were cultured in the upper chamber. F) Chemotaxis of RAW264.7 macrophages stimulated with the indicated conditioned medium. Scale bars: 200 μm. 4T1 cells treated with CuCl₂ + ES were used as the positive control [3]. The data are presented as the means ± SDs; ∗∗∗p < 0.001.

Fig. 7

The antitumor effect of ES@Cu(Ⅱ)-MOF NPs combined with an Anti-PD-L1 antibody in vivo. A) IVIS imaging of subcutaneous tumor-bearing mice after intravenous injection of IR820 or IR820@Cu(Ⅱ)-MOF NPs. B) IVIS imaging of mouse tumors and major organs (heart, lung, kidney, liver, and spleen) collected at 48 h postinjection. C) Treatment schedule for the antitumor effect of ES@Cu(Ⅱ)-MOF + Anti-PD-L1 combination therapy in vivo. D-F) Photographs of tumors (D), growth curves (E), and tumor weights (F) of tumor tissues collected from 4T1 tumor-bearing mice after different treatments (n = 5). H) H&E staining and Ki-67 (nuclear positive) and FDX1 (cytoplasmic positive) IHC images of tumor tissues collected from 4T1 tumor-bearing mice following various treatments. Scale bars: 25 μm. The data are presented as the means ± SDs; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Fig. 8

ES@Cu(Ⅱ)-MOF + Anti-PD-L1 combination therapy activated the antitumor immune response in vivo. A) T-SNE analyses of the distribution of TILs in tumor tissues collected from 4T1 tumor-bearing mice following various treatments. B) Flow cytometric analysis of CD3⁺ T lymphocytes in tumor tissues from 4T1 tumor-bearing mice following various treatments. C) Flow cytometric analysis of CD3⁺CD8⁺ CTLs in tumor tissues from 4T1 tumor-bearing mice following the indicated treatments. D) Flow cytometric analysis of CD3^-B220⁺ B cells in tumor tissues from 4T1 tumor-bearing mice following the indicated treatments. E-G) Quantification of the proportions of CD3⁺, CD3⁺CD8⁺, and CD3^-B220⁺ cells in 4T1 tumor-bearing mice following the indicated treatments (n = 4). H-J) Serum IFN-γ, TNF-α, and IL-6 levels in 4T1 tumor-bearing mice following the indicated treatments (n = 5). The data are presented as the means ± SDs; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.