A nuclease-mimetic platinum nanozyme induces concurrent DNA platination and oxidative cleavage to overcome cancer drug resistance

Abstract

Platinum (Pt) resistance in cancer almost inevitably occurs during clinical Pt-based chemotherapy. The spontaneous nucleotide-excision repair of cancer cells is a representative process that leads to Pt resistance, which involves the local DNA bending to facilitate the recruitment of nucleotide-excision repair proteins and subsequent elimination of Pt-DNA adducts. By exploiting the structural vulnerability of this process, we herein report a nuclease-mimetic Pt nanozyme that can target cancer cell nuclei and induce concurrent DNA platination and oxidative cleavage to overcome Pt drug resistance. We show that the Pt nanozyme, unlike cisplatin and conventional Pt nanoparticles, specifically induces the nanozyme-catalyzed cleavage of the formed Pt-DNA adducts by generating in situ reactive oxygen species, which impairs the damage recognition factors-induced DNA bending prerequisite for nucleotide-excision repair. The recruitment of downstream effectors of nucleotide-excision repair to DNA lesion sites, including xeroderma pigmentosum groups A and F, is disrupted by the Pt nanozyme in cisplatin-resistant cancer cells, allowing excessive accumulation of the Pt-DNA adducts for highly efficient cancer therapy. Our study highlights the potential benefits of applying enzymatic activities to the use of the Pt nanomedicines, providing a paradigm shift in DNA damaging chemotherapy.

Fig. 1. Schematic illustration of NMPNs-mediated concurrent DNA platination and oxidative cleavage to overcome Pt resistance of cancer.

The outermost mPEG_5K-AC-CA of NMPNs can be selectively dissociated in tumour acidic microenvironment due to the hydrolysis of the β-thiopropionate group, thus exposing TAT peptides to promote the endocytosis, endosomal escape, and nuclear transportation. NMPNs can efficiently release Pt ions to induce Pt-DNA adducts in the nucleus of cisplatin-resistant tumour cells. Meanwhile, due to the highly active OXD and POD-like activities, NMPNs generate in situ reactive oxygen species (ROS) to induce the oxidative DNA cleavage near the Pt-DNA binding sites, which prohibits further DNA bending and thus destroying the DNA conformation required for NER. Intriguingly, the recruitment of NER effectors (XPA and XPF) to the DNA lesion sites has been disrupted. Consequently, the NMPNs induced and tailored Pt-DNA adducts can efficiently accumulate in the tumour cells without NER-mediated repairing, thus inducing the apoptosis of cisplatin-resistant tumour cells, as well as exerting a potent anti-tumour effect in vivo.

Fig. 2. Designed synthesis and characterization of NMPNs with high performance catalytic activity.

a Schematic diagram of the synthesis of NMPNs. b Representative TEM image of PtNCs in chloroform. Scale bar: 100 nm; Insert: high-resolution TEM image of PtNCs. Scale bar: 2 nm. n = 3 independent experiments. c TEM image of NMPNs in water. Scale bar: 100 nm. n = 3 independent experiments. d The analysis of shedding behaviour of mPEG_5K-AC-CA from the surface of NMPNs at pH 6.5 and 7.4. Rhodamine B isothiocyanate (RITC) was conjugated to the terminal of mPEG_5K-AC-CA to obtain RITC-mPEG_5K-AC-CA, and the fluorescence intensity of RITC-mPEG_5K-AC-CA fragments released from NTNPs surface was analyzed. n = 3 independent experiments. Statistical significance was analyzed by two-tailed Student’s t-test. e XPS spectra of NMPNs, confirming the co-existence of Pt⁰ (B.E. at 71.7 and 75.3 eV), Pt²⁺ (B.E. at 72.3 and 76.3 eV), and Pt⁴⁺ (B.E. at 73.1 and 77.9 eV) on the surface of NMPNs. f ESR spectra of NMPNs by using BMPO as spin-trapping agents for detecting •O₂⁻. g ESR spectra of NMPNs by using DMPO as spin-trapping agents for detecting •OH. h DFT calculation of catalytic reaction on metallic Pt of PtNCs. i DFT calculation of catalytic reaction on oxidized Pt of PtNCs. j, k The proposed mechanism of NMPNs to catalyze H₂O₂ and O₂ into •OH and •O₂⁻. Source data are provided as a Source Data file.

Fig. 3. pH-dependent cell nucleus-targeting of NMPNs.

a CLSM images of intracellular localization of FITC-labelled NMPNs and FITC-labelled PNPs after incubated for 6 h under different pH conditions. Scale bar: 20 μm. Arrows indicate NMPNs in the cytoplasm after the escape from endosomes in cisplatin-resistant Huh7 cells. b CLSM images of intracellular localization of FITC-labelled NMPNs and FITC-labelled PNPs after incubated for 12 h under different pH conditions. Scale bar: 40 μm. Asterisks indicate NMPNs in the nucleus of cisplatin-resistant Huh7 cells. c Quantitative analysis of the colocalization between lysotracker and FITC-labelled NMPNs or PNPs. d Quantitative analysis of the colocalization between DAPI and FITC-labelled NMPNs or PNPs. e Bio-TEM images of cells after incubation for 12 h with NMPNs under different pH conditions. Arrowheads indicate NMPNs accumulated in the nucleus of Huh7 cells. Black asterisks indicate NMPNs in nucleus. Scale bar: 1 μm. f Schematic diagram of the NMPNs to target the tumour cell nucleus. All data are presented as means ± SEM, n = 3 independent experiments. Statistical significance was analyzed by one-way ANOVA with multiple comparisons test (c, d). Source data are provided as a Source Data file.

Fig. 4. NMPNs induce Pt-DNA adducts formation and oxidative cleavage of DNA strand.

a The release of Pt ions from NMPNs at different time points under neutral conditions after the discharge of mPEG_5K-AC-CA. b The agarose gel electrophoresis of DNA treated with different concentrations of NMPNs. c Schematic illustration of Pt ion binding to DNA backbone. d Schematic illustration of ROS coordinating with Pt ion and causing oxidative DNA cleavage. e Comparison of hydrolysis and desorption process of complete DNA double-stranded fragments (upper) and DNA double-stranded fragments attacked by ROS (lower). The hydrolysis energy of DNA strand fragments attacked by ROS is −4.66 eV, lower than that of the complete double-stranded DNA (3.00 eV). Besides, the desorption energy of the unpaired base strand in ROS-attacked DNA is 3.05 eV, which is also lower than that of the bases with pairs in complete double-stranded DNA (9.22 eV). All data are presented as means ± SEM, n = 3 independent experiments. Source data are provided as a Source Data file.

Fig. 5. NMPNs circumvent NER pathway by concurrent of DNA platination and oxidative cleavage.

a The level of Pt-DNA adducts in the nucleus of cisplatin-resistant Huh7 cells after treatment with NMPNs at pH 6.5. Scale bar: 40 μm. Scale bar: 40 μm. b ROS levels in the nucleus of cisplatin-resistant Huh7 cells after treatment with PNPs or NMPNs at pH 6.5. Scale bar: 40 μm. c Immunofluorescence of the γ-H2AX in cisplatin-resistant Huh7 cells after different treatments at pH 6.5. Scale bar: 40 μm. d Immunofluorescence of the XPA and Pt-DNA adducts in cisplatin-resistant Huh7 cells after different treatments at pH 6.5. Scale bar: 40 μm. e Quantitative analysis of the colocalization between XPA and Pt-DNA adducts. f Immunofluorescence of the XPF and Pt-DNA adducts in cisplatin-resistant Huh7 cells after different treatments at pH 6.5. Scale bar: 40 μm. g Quantitative analysis of the colocalization between XPF and Pt-DNA adducts. h Western blot analysis of XPF expression in the nucleus of cisplatin-resistant Huh7 cells after different treatments. i Quantitative analysis of XPF expression in cisplatin-resistant Huh7 cells after different treatments at pH 6.5. j The schematic illustration of the mechanism underlying NMPNs to induce DNA platination and oxidative cleavage to combat Pt resistance in tumour cells. In comparison to Pt compounds and PNPs that cannot effectively generate ROS in the nucleus, NMPNs can readily accumulate in the nucleus by acidity-induced exposure of TAT peptides, and induce in situ ROS generation to induce DNA oxidative cleavage, thus destroying the DNA conformation required for NER. It hampers the recruitment of XPA and XPF and thus inhibiting NER pathway. All data are presented as means ± SEM, n = 3 independent experiments. Statistical significance was analyzed by one-way ANOVA with multiple comparisons test. Source data are provided as a Source Data file.

Fig. 6. NMPNs promote cisplatin-resistant tumour cell apoptosis by inducing Pt-DNA adducts without NER repairing.

a Immunofluorescence of the Pt-DNA adducts in cisplatin-resistant Huh7 cells after different treatments at pH 6.5. Scale bar: 40 μm. b Immunofluorescence of the Pt-DNA adducts in cisplatin-resistant Huh7 cells after treatments with NMPNs or NMPNs+NAC at different time points. Scale bar: 40 μm. c Quantitative analysis of Pt-DNA adducts in cisplatin-resistant Huh7 cells after treatments with NMPNs or NMPNs+NAC at different time points. n = 6 independent experiments. d Quantitative analysis of DNA damage of cisplatin-resistant Huh7 cells after treatment with cisplatin or NMPNs. Cisplatin: n = 46; NMPNs: n = 44. e Flow cytometry analysis of cell apoptosis after different treatments at pH 6.5. f and corresponding quantitative results. n = 5 independent experiments. g The inhibition effect of cisplatin and NMPNs on cisplatin-resistant Huh7 cells growth at different incubation times. h The cell viabilities of cisplatin-resistant Huh7 cells or siXPA-transfected cisplatin-resistant Huh7 cells after treatment with NMPNs, PNPs or cisplatin. n = 4 independent experiments. All the data are presented as means ± SEM, Statistical significance was analyzed by one-way ANOVA with multiple comparisons test. Source data are provided as a Source Data file.

Fig. 7. NMPNs suppress tumour in vivo and improve overall therapeutic outcome.

a Schematic illustration of the establishment of orthotopic liver tumour mice model and treatment process. b Representative bioluminescence (BLI) images of each group after different treatments. c Quantitative BLI signals of tumours after different treatments. n = 5 mice. Statistical significances were analyzed by two-tailed unpaired Student’s t-test. d Representative images of liver collected from each treatment group on day 31. Black arrows indicate the tumour tissues. e Representative TUNEL and DAPI staining images of tumour tissues. Scale bar: 200 μm. f, g Western blot analysis and quantitative analysis of γ-H2AX, XPA and XPF in the nucleus of tumour cells after different treatments. n = 3 independent experiments. h The body weight changes of mice with different treatments. n = 5 mice. i The survival curves of orthotopic liver tumour mice after different treatments. n = 9 mice. j The schematic illustration of NMPNs to induce Pt-DNA adduct formation and oxidative cleavage for inhibiting the recruitment of XPA and XPF, which nullifies NER pathway and thus suppresses the tumour growth in vivo. All data are presented as means ± SEM. Statistical significances were analyzed by one-way ANOVA with multiple comparisons test (g, h) and the log-rank (Mantel–Cox) test (i). Source data are provided as a Source Data file.