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

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.


4-Figure 4:
A-In panel B, why is the DCF fluorescence so localized to one pole of the nucleus? B-In panel C, in their direct testing of the mechanism that oxidation is important following DNA platination, the authors show that NAC co-treatment reduces gH2AX formation, but the key controls showing equivalent NMPNs uptake in the presence of NAC are missing, and the presence of equivalent amounts of the Pt-DNA adduct are also missing. This Pt-DNA missing data, which is shown in panel F suggests that the NAC treatment has major effects on DNA platination itself, not just oxidative cleavage, and makes it impossible to use the results to validate the direct importance of the ROS-dependent cleavage effects. All of the data needs to be quantified.
C-What effect does NAC have on the PNP effects? D-In panels D and E, the authors show primary data for XPA, which they do not quantify, and quantified data for XPF, which they do not show primary data for. Please show IF images for all of the relevant XP proteins and quantify all of the data for both NMPNs and PNPs. E-In panel I, how reproducible is the effect? How many times was the experiment done? Are the differences statistically significant? F-In panel J, how exactly is viability being measured? What is CCK-8 solution?
5-This is a critical point -what direct proof do the authors have that lack of NER is the primary mechanism responsible for the enhanced cytotoxicity? To make this claim, the authors need to compare the effects of the NMPNs on isogenic cells that are NER-proficient versus NER-deficient, such as XP knock-outs, and show that the cytotoxicity, compared to PNPs is only enhanced in NERproficient cells. Figure 5: A-Images in Panel e are so dim that they are impossible to see, so the authors claims cannot be verified.

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B-In panel f, please show gH2AX immunofluorescence, not just a Western blot 7-The models in Figure 4K and 5J are not adequately supported by the data, as I mention in comment #5.
In summary, the authors clearly appear to have created a novel nanoparticle with potential utility, but the mechanism that underlies the cellular effects is not adequately validated. I think the results are important even if the NER-focused mechanism is wrong, but additional experimental data is required.
1. The authors did a comprehensive work on clarifying that the NMPNs-generated ROS can inhibit the recruitment of NER-related proteins by disrupting the required DNA structure. However, to fully understand the roles of NMPNs in the suppression of cisplatin-resistant cells in vitro, a control group of the combination of NAC and NMPNs is needed.
Response: Thank you for your valuable comment. According to your suggestions, we analyzed the viability of cisplatin-resistant Huh7 cells after treatment with saline, NMPNs, NAC, or NMPNs+NAC. The results show that NMPNs treatment can dramatically suppress the growth of cisplatin-resistant Huh7 cells. However, the inhibitory effect of NMPNs on cisplatin-resistant Huh7 cell growth can be significantly compromised in the presence of a ROS scavenger NAC. Consistently, the cotreatment of NMPNs and NAC can suppress DNA double stranded breaks and decrease the accumulation of Pt-DNA adducts (Fig. 3c,d). These results demonstrate that ROS generated by NMPNs for DNA structure disruption is indispensable to the inhibitory effects of NMPNs on cisplatin-resistant cells.
Our modification to the manuscript: The results were added as Supplementary Fig.   2 23 in the revised supporting information. In addition, the following sentences and methods were added on pages 12 and 30-31 in the revised manuscript, respectively.
• Figure S23 Supplementary Figure 23. The inhibition effect of saline, NMPNs, NAC, or NMPNs+NAC on cisplatin-resistant Huh7 cells growth. n = 5 independent experiments, data are presented as means ± S.E.M. Source data are provided as a Source Data file.
• Page 12 "Compared with cisplatin, NMPNs cause more severe DNA damage (Fig. 5d), effectively inducing apoptosis to inhibit the proliferation of cisplatin-resistant Huh7 cells ( Fig. 5e-g). However, the inhibitory effect of NMPNs on cisplatin-resistant Huh7 cell growth is significantly compromised in the presence of NAC (Supplementary Fig. 23)." • Page 30-31 Method In vitro cytotoxicity. Cisplatin-resistant Huh7 cells were seeded into 96-well plates (1 × 10 4 cells/well) for 24 h and treated with cisplatin, PNPs or NMPNs with a concentration of 20 μg/mL for 6 h at pH 6.5. Then, the incubation medium containing cisplatin, PNPs or NMPNs were removed and replaced with fresh medium. After 12, 18 and 24 h, the cells were incubated with CCK-8 solution for each well and incubated for another 3 h, the absorbance of each well was measured at 450 nm by a microplate reader.
Cisplatin-resistant Huh7 cells were seeded into 96-well plates (1 × 10 4 cells/well) for 24 h and treated with NMPNs (20 μg/mL) in the presence or absence of NAC for 6 h at pH 6.5. Then, the cells were incubated with CCK-8 solution for each well and 3 incubated for another 3 h, the absorbance of each well was measured at 450 nm by a microplate reader.
L02 cells were seeded into 96-well plates (1 × 10 4 cells/well) for 24 h and treated with NMPNs (20 μg/mL) for 6 h at pH 7.4. Then, the cells were incubated with CCK-8 solution for each well and incubated for another 3 h, the absorbance of each well was measured at 450 nm by a microplate reader.
2. NMPNs are expected to act at tumor area, it is necessary to provide the information about the concentration of NMPNs in tumor tissues. Also, did these NMPNs show unwanted toxicity to normal cells and tissues around tumors in the liver?
Response: Thank you for your kind suggestion. Accordingly, the biodistribution of NMPNs was studied by using inductively coupled plasma mass spectrometry (ICP-MS). The results show that NMPNs can effectively accumulate in tumour tissue. In addition, the effect of NMPNs has been evaluated on normal liver cell (e.g., L02 cell lines). The results demonstrated that NMPNs exert no inhibitory effect on the L02 cells growth under neutral conditions. Consistently, immunohistochemical analysis and TUNEL staining images demonstrated that NMPNs showed no obvious toxicity to normal liver tissues around tumours. These results indicate that NMPNs can effectively accumulate in tumour tissues and suppress tumour growth, and exert no evident toxicity to normal cells and tissues around tumours in the liver, which is likely due to cancer cell nucleus targeting capability of NMPNs as demonstrated in the manuscript.
Our modification to the manuscript: The results were added as Supplementary Figs. 25,26,29,30 in the revised supporting information. In addition, the following sentences and methods were added on pages 12-13, 30-31, and 35-36 in the revised manuscript, respectively.
• Figure S25 Supplementary • Figure S29 Supplementary Figure 29. Representative hematoxylin and eosin (H&E) staining images of normal tissues around tumour in the liver from saline or NMPNs-treated mice. n = 3 independent mouse livers. Scale bar: 500 μm.

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• Figure S30 Supplementary Figure 30. Representative TUNEL staining images of normal tissues around tumour in the liver from saline or NMPNs-treated mice. n = 3 independent mouse livers. Scale bar: 100 μm.
• Page 12 "Moreover, NMPMs show no obvious inhibitory effect on the normal cell growth, such as L02 cell, under neutral conditions ( Supplementary Fig. 25)." • Page 13 "NMPNs can efficiently accumulate in tumour tissues, likely due to their capability to expose TAT peptides under acidic tumour microenvironment ( Supplementary Fig. 26)". "Moreover, NMPNs show no obvious toxicity to normal tissues around the tumour in the liver and other major organs, suggesting the excellent biosafety of NMPNs ( Fig. 6h and Supplementary Fig. 29-31)." • Page 30-31 Method In vitro cytotoxicity. Cisplatin-resistant Huh7 cells were seeded into 96-well plates (1 × 10 4 cells/well) for 24 h and treated with cisplatin, PNPs or NMPNs with a concentration of 20 μg/mL for 6 h at pH 6.5. Then, the incubation medium containing cisplatin, PNPs or NMPNs were removed and replaced with fresh medium. After 12, 18 and 24 h, the cells were incubated with CCK-8 solution for each well and incubated for another 3 h, the absorbance of each well was measured at 450 nm by a microplate reader. 6 Cisplatin-resistant Huh7 cells were seeded into 96-well plates (1 × 10 4 cells/well) for 24 h and treated with NMPNs (20 μg/mL) in the presence or absence of NAC for 6 h at pH 6.5. Then, the cells were incubated with CCK-8 solution for each well and incubated for another 3 h, the absorbance of each well was measured at 450 nm by a microplate reader.
L02 cells were seeded into 96-well plates (1 × 10 4 cells/well) for 24 h and treated with NMPNs (20 μg/mL) for 6 h at pH 7.4. Then, the cells were incubated with CCK-8 solution for each well and incubated for another 3 h, the absorbance of each well was measured at 450 nm by a microplate reader.
• Page 35-36 Biodistribution of NMPNs via ICP-MS analysis. Mice were intraperitoneally administered with NMPNs three times a week (2.5 mg/kg body weight) for two weeks. Then, the mice were sacrificed and the hearts, livers, spleens, lungs, kidneys, and tumours were harvested. The quantifiable amounts of hearts, livers, spleens, lungs, kidneys, and tumours were mixed with aqua regia and incubated at 60℃ for 72 h. After dilution and filtration, the Pt concentrations were quantified by ICP-MS analysis.
• Page 36 Hematoxylin and eosin (H&E) and TUNEL staining. Mice were treated with saline, cisplatin, PNPs or NMPNs (2.5 mg/kg body weight) three times a week, respectively. After 14 days, livers, spleens, lungs, kidneys, and tumour tissues were harvested, and fixed in 10% formalin, embedded in paraffin, sectioned, and then stained by H&E or TUNEL for further analysis.
3. Considering that ROS can inhibit tumors insensitive to Pt by disrupting the NER pathway, it is necessary to assess the ROS level in tumor tissues after the treatment of NMPNs. More discussion is also needed.  Fig. 3 and 4 of the manuscript.

Response
Our modification to the manuscript: The TEM results were added as Supplementary  Fig. 3, and the DLS size and zeta potential of PtNC@PEG has been added into previous Supplementary Figs. 5 and 6 in the revised supporting information. In addition, the following sentences were added on page 6 in the revised manuscript.
• Page 6 "PtNCs were further grafted with the heterobifunctional SH-PEG2K-COOH to provide the carboxyl groups for 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)mediated coupling with the amino groups of the TAT peptides and then shielded with pH-responsible mPEG5K-AC-CA (Supplementary Figs. 2,3)." "The successful modification of TAT peptide is verified by Fourier transform infrared (FT-IR) spectra ( Supplementary Fig. 3), corresponding with the increased zeta potential and size relative to PtNC@PEG ( Supplementary Fig. 5, 6)." 5. The colloidal stability of NMPNs should be evaluated by analyzing their timedependent size and surface charge.
Response: Thank you very much for your valuable comments. Per your suggestion, the time-dependent sizes and zeta potentials of NMPNs were detected using a Zetasizer Nano ZS90 to confirm their colloidal stability. No obvious size or zeta potential changes were observed over a week, which demonstrates the high colloidal stability of NMPNs.
Our modification to the manuscript: The results were added as Supplementary Fig.  7 in the revised supporting information. In addition, the following sentences were added on page 6 in the revised manuscript.
• Figure S7 Supplementary Figure 7. a, b, The sizes (a) and zeta potentials (b) of NMPNs over a week. n = 3 independent experiments, data are presented as means ± S.E.M. Source data are provided as a Source Data file.
• Page 6 "NMPNs are well-dispersed in water ( Fig. 1c) with the high colloidal stability ( Supplementary Fig. 7)" 6. In addition to the results regarding the endosomal escape and nucleus targeting of NMPNs, the description of the cellular uptake pathway of NMPNs are required.

Response: Thank you very much for your valuable comments. To investigate how
NMPNs enter Huh7 cells, we quantified the cellular uptake of RITC-labelled NMPNs after adding methyl-b-cyclodextrin (MβCD, an inhibitor of caveolin-mediated endocytosis), chlorpromazine (an inhibitor of clathrin-mediated endocytosis) or amiloride (an inhibitor of macropinocytosis) <Ref. Colloid. Surfaces B 2018, 161, 10-17;Nat. Commun. 2016, 7, 11284>. In  Our modification to the manuscript: The results were added as Supplementary Fig.  15 in the revised supporting information. In addition, the following sentences and methods were added on pages 8-9 and 29 in the revised manuscript, respectively.

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• Figure S15 Supplementary Figure 15. Flow cytometry analysis of internalization of NMPNs into HuH7 cells after pre-treatment with serum-free medium (vehicle), amiloride (an inhibitor of macropinocytosis), chlorpromazine (an inhibitor of clathrin-mediated endocytosis), or methyl-b-cyclodextrin (MβCD, an inhibitor of caveolin-mediated endocytosis). n = 3 independent experiments, data are presented as means ± S.E.M. Source data are provided as a Source Data file.
• Page 8-9 "Moreover, we further studied the cellular endocytosis mechanism of NMPNs in Huh7 cells. In accordance with previously reported TAT peptide-modified nanoparticles 47 , the cellular uptake of NMPNs can be inhibited when Huh7 cells were pre-incubated with amiloride, chlorpromazine, and MβCD, respectively, indicating that NMPNs can be internalized into Huh7 cells through multiple pathways, among which the MβCDmediated endocytosis plays a leading role in the cellular endocytosis of NMPNs ( Supplementary Fig. 15)." • Page 29 Method Endocytic pathway of NMPNs. To study the cellular uptake mechanisms of NMPNs by cisplatin-resistant Huh7 cells, three specific endocytic inhibitors were used: (1) chlorpromazine, an inhibitor to probe clathrin-mediated endocytosis; (2) amiloride, an inhibitor of micropinocytosis; (3) methyl-β-cyclodextrin (MβCD), an inhibitor of caveolin-mediated endocytosis. Specifically, cisplatin-resistant Huh7 cells were preincubated in serum-free RPMI DMEM medium with chlorpromazine (40 μM, 30 min), amiloride (100 μM, 30 min) or MβCD (5 mM, 30 min), respectively. The medium was then changed to fresh serum-free medium containing the inhibitors plus NMPNs (20 μg/mL) and further incubated for 6 h at 37℃. The cells were collected for quantitative analysis.

Reference
7. Please unify the font size in the Figure 5.
Response: Thank you for your kind suggestion. We have unified the font size in the Fig.  5 (Now is Fig.6 in the revised manuscript).
Our modification to the manuscript: We have unified the font size in Fig. 5 (Now is Fig. 6) in the revised manuscript.

Reviewer #2 -nanoparticles and cancer (Remarks to the Author):
Platinum (Pt) resistance compromises the clinical usage of Platinum-based chemotherapeutics. In this manuscript, by exploiting the structural vulnerability of nucleotide excision repair (NER) process and the mechanism of Platinum (Pt) resistance, the authors developed a nuclease-mimetic Pt nanozyme (NMPNs) which can target to the cancer cell nucleus and induce concurrent DNA platination and oxidative cleavage to overcome Pt drug resistance. When NMPNs accumulated in tumor tissue, the acid-responsive layer on the surface was detached and the nucleus-targeting peptide TAT was exposed for the intracellular nucleus targeted delivery. The existence of Pt 0 , Pt 2+ , and Pt 4+ on the surface endowed NMPNs with potent oxidase and peroxidaselike activities to generate ROS for the cleavage of double-stranded DNA. Besides, NMPNs can efficiently suppress the recruitment of NER associated factors to inhibit NER process, thus leading to the excessive formation of Pt-DNA adducts and dramatically inducing the apoptosis of Pt-resistant tumor cells. Overall, this is a promising strategy to promote the clinical application of Platinum-based chemotherapeutics. The major conclusions are fully supported by the experimental data. I would suggest its publication on Nature communication after addressing the following comments.
Response: Thank you very much for your encouraging comments. Based on your kind suggestions, we have made point-to-point responses and modified the manuscript.
1. The quality of Figure 2b is not high enough to distinguish lysosome and nanoparticle signals. The authors can supply the separated single fluorescence channels or improve the resolution.
Response: Thank you very much for your suggestion. Per your suggestion, we have provided the CLSM images with high quality and corresponding quantitative result in the revised manuscript.
Our modification to the manuscript: We have added the CLSM images with higher quality and the corresponding quantitative result as Fig. 2a and c in the revised manuscript, respectively. In addition, the following sentences were added on page 8 in the revised manuscript.   μm. f, Schematic diagram of the NMPNs to target the tumour cell nucleus. All the data are presented as means ± S.E.M., n = 3 independent experiments. Source data are provided as a Source Data file.
• Page 8 "Under acidic conditions, the FITC signals diffuse across the cytoplasm of the cells, indicating the efficient cellular uptake and endosomal escape of NMPNs. In stark contrast, FITC signals of NMPNs are mostly confined in endosomes in a neutral environment. Moreover, FITC labelled PNPs are trapped in endosomes both in acidic and neutral environments (Fig. 2a,c)." 2. The Figure 4a should also contain the PNPs group in the main text.
Response: Thank you very much for your kind suggestion. Actually, we have provided the capacity of PNPs to induce Pt-DNA adducts in Fig. 5a. Compared with NMPNs treatment, PNPs cannot significantly enhance the production of Pt-DNA adducts in the nucleus of cisplatin-resistant cells, likely result from the lack of nucleus-targeting capability.
•  3. Please provide the quantitative analysis of Figure 4i.

Response: Thank you for your valuable suggestion. Per your suggestion, we have added the quantitative results of flow cytometry analysis of cell apoptosis after different treatments at pH 6.5.
Our modification to the manuscript: The results were added as Fig. 5f in the revised manuscript. In addition, the following sentences were added on page 12 in the revised manuscript.

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• Figure 5 experiments. Statistical significance was analyzed by one-way ANOVA with multiple comparisons test. Source data are provided as a Source Data file.
• Page 12 "Compared with cisplatin, NMPNs cause more severe DNA damage (Fig. 5d), effectively inducing apoptosis to inhibit the proliferation of cisplatin-resistant Huh7 cells ( Fig. 5e-g)." 4. Please describe how the experiment in Figure 3b was carried out in detail.

Response: Thank you for your kind suggestion. Per your suggestion, we have added the detailed description of experimental method.
Our modification to the manuscript: The method of the experiment in Fig. 3b were added on page 27 in the revised manuscript. •

Page 27
Method Study of DNA cleavage after NMPNs treatment. NMPNs were treated with PBS (pH 6.5) for 6 h. Then, PBS (pH 6.5) was removed by ultrafiltration and the NMPNs were collected. 300 ng of plasmid DNA (pet-32a+) were incubated with different amounts of the obtained NMPNs (2.5, 5, 10, 20 g/L) in PBS (pH 7.4) at 25℃ for 12 h. These samples were mixed with 0.20 volume of the desired 6× loading buffer and slowly added into the 1% agarose gel slots in an electrophoresis tank containing 1× TBE electrophoresis buffer respectively, which were then applied with a voltage of 90 V until the DNA samples migrate a sufficient distance through the gel. Lastly, the gel was stained for 30 min by using SYBR dyes at room temperature and an image of the gel was captured under UV transillumination.
5. The stability of NMPNs in physiological condition should be evaluated.
Response: Thank you very much for your valuable comments. Per your suggestion, the size and zeta potential of NMPNs in PBS and DMEM were detected using a Zetasizer Nano ZS90. No obvious size or zeta potential changes was observed over a week, which demonstrates their high colloidal stability.

Our modification to the manuscript:
The results were added as Supplementary Fig.  7 in the revised supporting information. In addition, the following sentences were added on page 6 in the revised manuscript.

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• Figure  6. There is another important aspect from the future translational aspect: how was the production capacity for making this new type of nanoparticle? Is this line of production capable for integration with the industrial scale production?

Reviewer #3 -DNA repair, cancer and chemotherapy. (Remarks to the Author):
Review of Li et al., "A nuclease-mimetic platinum nanozyme induces concurrent DNA platination and oxidative cleavage to overcome drug resistance" In this manuscript the authors construct nuclease-mimetic Pt-nanoparticles designed to induce concurrent DNA platination and oxidative cleavage of the platinated lesion to prevent recognition and repair by nucleotide excision repair, with the intent of utilizing these particles for cancer therapy. The idea is clever and worth pursuing. However, many of the claims in the paper are based on correlation, key controls are missing, and the data is over-interpreted. The relative importance of NER in suppressing the cytotoxicity response to platinum treatments, compared to other repair pathways such as HR and the Fanconi pathway, remain subjects of active controversy. The authors do not definitively prove that the enhanced effects of their NMPNs are truly the result of NER disruption by the oxidative damage of the particle, and other parts of their data suggest that the oxidative effects may be more important for inducing other types of damage or modulating the platinum-induced damage in a manner that is independent of any effects on NER. Several of the Figure captions are so brief that it is difficult to know what exactly is being shown, and many parts of the work have not been quantified.

Response: Thank you very much for your encouraging comments. Based on your kind suggestions, we have made point-to-point responses and modified the manuscript. We believe that your comments have significantly improved the quality of our manuscript.
Major comments: 1-The cellular images shown in Figure 2B and C are so poorly illuminated that it is not possible to actually see what is claimed in the Results section about pH-dependent nuclear uptake, on page 8-9, lines 166-184, hence the reviewer is unable to determine if the claims are substantiated by the data.

Response: Thank you very much for your comments. Per your suggestion, we provide the CLSM images with high quality to clarify the endosomal escape and nucleustargeting capacity of NMPNs in cisplatin-resistant Huh7 cells at different pH
conditions. In addition, the corresponding quantitative analyses have been added. As shown in Fig. 2b Our modification to the manuscript: We have provided CLSM images with high quality in Fig. 2a (previous Fig. 2b) and Fig. 2b (previous Fig. 2c) and the corresponding quantitative analyses (Fig. 2c,d). In addition, the following sentences were added on page 8 in the revised manuscript.   μm. f, Schematic diagram of the NMPNs to target the tumour cell nucleus. All the data are presented as means ± S.E.M., n = 3 independent experiments. Source data are provided as a Source Data file.
• Page 8 "Under acidic conditions, the FITC signals diffuse across the cytoplasm of the cells, indicating the efficient cellular uptake and endosomal escape of NMPNs. In stark contrast, FITC signals of NMPNs are mostly confined in endosomes in a neutral environment. Moreover, FITC-labelled PNPs are trapped in endosomes both in acidic and neutral environments (Fig. 2a,c). These results indicate that the acid-induced exposure of TAT peptides can facilitate the endosomal escape of NMPNs 45 , contributing to the efficient nucleus targeting. Indeed, in an acidic microenvironment, NMPNs can efficiently accumulate in the cell nucleus (Fig. 2b,d), which is beneficial from the surface TAT peptides that can promote the entry into the nucleus via the importin α/β pathway 46 . In contrast, PNPs can barely reach the nucleus, regardless of the pH conditions (Fig. 2b,d)." Figure 2D and Supplemental Figure 12 that we are actually seeing Pt nanoparticles in the nucleus would be better if supported by some type of independent analysis directly showing the presence of platinum. Perhaps some type of EXAFS study or nuclear isolation and flame spectrometry could be done. Alternatively, the authors could use their anti-Pt:DNA adduct antibody as in Figure 4A. None of this data is quantified, which would also help make the findings more convincing. Fig. 14). Moreover, to fully study the capability of NMPNs to target the tumour cell nucleus, we further analyzed the amount of Pt-DNA adducts after treatment with NMPNs or PNPs. The result demonstrated the enhanced accumulation of Pt-DNA adducts in the nucleus after NMPNs treatment as compared to that of PNPs treatment under the acidic conditions ( Supplementary Fig. 17 a,b). These results can clearly show the accumulation of NMPNs in the nucleus of Huh7 cells under acidic conditions is more than that under neutral conditions, demonstrating the capability of NMPNs to target the nucleus of cisplatin-resistant Huh7 cells.

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Our modification to the manuscript: The results were added as Supplementary Fig.  14 and 17 in the revised supporting information. In addition, the following sentences and method were added on pages 8-9 and 30 in the revised manuscript, respectively.
• Figure S14 Supplementary Figure 14. The concentrations of Pt ions in the nucleus of cisplatinresistant Huh7 cells after treatment with NMPNS or PNPs under different pH conditions. n = 3 independent experiments, data are presented as means ± S.E.M. Source data are provided as a Source Data file.
• Figure S17 Supplementary Figure 17. a, Immunofluorescence images of the Pt-DNA adducts in cisplatin-resistant Huh7 cells after different treatments at pH 6.5. Scale bar: 40 μm. b, Quantification analysis of Pt-DNA adducts in cisplatin-resistant Huh7 cells after different treatments at pH 6.5. n = 3 independent experiments, data are presented as means ± S.E.M. Source data are provided as a Source Data file.

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• Page 8 "The Bio-TEM images and inductively coupled plasma mass spectrometry (ICP-MS) analysis of Pt ions in the nucleus also show the accumulation of NMPNs in the nucleus of Huh7 cells under acidic conditions ( Fig. 2d and Supplementary Fig. 13, 14)."

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Page 9 "These results demonstrate that, different from PNPs, NMPNs implement the pHresponsive discharge of the "protective shield" under acidic conditions to exposure TAT peptides for facilitating the endosomal escape and subsequent nucleus targeting ( Fig.  2f and Supplementary Fig. 16), which can readily initiate the formation Pt-DNA adducts ( Supplementary Fig. 17a,b)." • Page 30

Method The concentration of NMPNs in the nucleus of cisplatin-resistant Huh7 cells.
Cisplatin-resistant Huh7 cells were treated with NMPNs or PNPs (20 μg/mL) for 24 h at pH 7.4 or pH 6.5, respectively. The cell nucleus was extracted and then mixed with aqua regia and incubated at 60℃ for 72 h. After dilution and filtration, the Pt concentrations were quantified by ICP-MS analysis. Figure 3A.

3-Figure 3: A-Please describe what is actually being measured in
Response: Thanks for your comment. In Fig. 3a Fig. 18).
Finally, we have also measured the concentrations of Pt ions in the nuclei of Huh7 cells after treatment with NMPNs by using ICP-MS. The result shows that the Pt ion in the nuclei reaches up to ~1.1 μg/10 7 cells (~0.22 g/L) ( Supplementary Fig. 14), which is comparable to the concentration of NMPNs (0.25 g/L) that can induce DNA cleavage in cell-free experiment, demonstrating that NMPNs can efficiently target and accumulate in the nucleus of cisplatin-resistant Huh7 cells to induce Pt-DNA adducts and DNA cleavage.
Our modification to the manuscript: The results were added as Fig. 3b and Supplementary Fig. 14 and 18 in the revised manuscript. In addition, the following sentences and method were added on pages 8-9, 27-28, and 30 in the revised manuscript, respectively.

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• Figure S14 Supplementary Figure   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 the data are presented as means ± S.E.M., n = 3 independent experiments. Source data are provided as a Source Data file.
• Page 8 "The Bio-TEM images as well as inductively coupled plasma mass spectrometry (ICP-MS) analysis of Pt ions in the nucleus also show the accumulation of NMPNs in the nucleus of Huh7 cells under acidic conditions ( Fig. 2e and Supplementary Fig. 13, 14)."

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Page 27 Method Study of DNA cleavage after NMPNs treatment. NMPNs were treated with PBS (pH 6.5) for 6 h. Then, PBS (pH 6.5) was removed by ultrafiltration and the NMPNs were collected. 300 ng of plasmid DNA (pet-32a+) were incubated with different amounts of the obtained NMPNs (2.5, 5, 10, 20 g/L) in PBS (pH 7.4) at 25℃ for 12 h. These samples were mixed with 0.20 volume of the desired 6× loading buffer and slowly added into the 1% agarose gel slots in an electrophoresis tank containing 1× TBE electrophoresis buffer respectively, which were then applied with a voltage of 90 V until the DNA samples migrate a sufficient distance through the gel. Lastly, the gel was stained for 30 min by using SYBR dyes at room temperature and an image of the gel was captured under UV transillumination.
Study of kinetic of NMPNs-induced DNA cleavage. 300 ng of Plasmid DNA were incubated with NMPNs with different concentrations (0.42, 0.84, 1.68, 3.36 μM) in PBS (pH 7.4) for 3, 9, 12 h, respectively. Then, the integrity of DNA was investigated by using gel electrophoresis. The band intensities were quantified using Image J software (version 1.8.0) to calculate the percentage of cleaved DNA. The percentage of cleaved DNA vs. time followed pseudo-first-order kinetic profiles. Then, GraphPad Prism Software Version 8.0 was used for the non-linear curve fitting.  (Figs. 1f,g and 3a) on their effects on DNA (Fig. 3b and Fig. 4a,c). It has been reported that Pt nanoparticles can release Pt ions (Fig. 3a), which can react with phosphate groups of DNA and form Pt-DNA adducts (Fig. 4a) Fig 3c), which is similar to the intermediate products of cisplatin verified in the experiment <Ref. J. Med. Chem. 2007, 50, 2601-2604Phys. Chem. Chem. Phys., 2014, 16, 19290-19297;Ref. Comput. Theor. Chem., 2016, 1094. The sugar radical generated by DNA binding to Pt ions is highly reactive and easily oxidized, causing the P-O bond breaks and DNA cleavages <Ref. Chem. Rev., 2010, 110, 1018 Considering the capability of NMPNs to generate ROS (Fig.  1f,g), in Fig 3d, (Fig. 3b), which is consistent with the results of previous literature <Ref. Proc. Natl. Acad. Sci. USA, 2001, 98, 8241-8246>, and ultimately (Supplementary Fig. 19).

Our modification to the manuscript:
The results were added as Fig. 3b and Supplementary Fig. 19 in the revised manuscript. In addition, the following sentences and method were added on pages 10 and 28 in the revised manuscript, respectively.  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 the data are presented as means ± S.E.M., n = 3 independent experiments. Source data are provided as a Source Data file.

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• Figure S19 Supplementary Figure 19. The analysis of Pt-DNA binding sites in the DNA extracted from cisplatin-resistant Huh7 cells after treatment with NMPNs. The blue fluorescence and the green fluorescence correspond to DNA fibers and Pt-DNA binding sites, respectively. Arrows indicate the DNA fragmentations. Asterisks indicate the Pt-DNA binding sites at the end of DNA fragmentations. n = 3 independent experiments. Scale bar: 100 μm.
• Page 10 "In line with the DFT results, we found that NMPNs can induce the formation of Pt-DNA adducts and DNA breakage in the cisplatin-resistant Huh7 cells (Fig. 4a,c). Moreover, we extracted the DNA fragmentations from NMPNs-treated cisplatinresistant Huh7 cells and analyzed the location of Pt-DNA binding in the DNA fragmentations. Intriguingly, the result shows that the Pt-DNA binding sites mainly localize at the end of DNA fragmentation (Supplementary Fig. 19)."

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Page 28 Method The analysis of locations of Pt-DNA binding in the DNA. Cisplatin-resistant Huh7 cells were cultured in 6-well plates with 2 mL culture medium. NMPNs (20 μg/mL, pH 6.5) were added and incubated for 24 h, cells were subsequently collected and quickly resuspended in 500-600 μL of ice-cold PBS. Then, 2.5 μL of cell resuspension was spotted onto a pre-cleaned glass slide and mixed with 7.5 μL of spreading buffer (0.5% SDS in 200 mM Tris-HCl (pH 7.4), 50 mM EDTA). After 10 min, the slides were tilted to 15° to spread DNA fibers along the length. Then, air-dry the DNA spreads and fixed 34 in 3:1 methanol/acetic acid for 20 min at -20℃. After washing with PBS three times, slides were blocked with 1% BSA in PBS for 30 min at room temperature and incubated with anti-cisplatin modified DNA antibodies (GeneTex, GTX17412, dilution 1:100) to detect Pt-DNA adduct. After 1 h of incubation, slides were washed with PBS three times and stained with Alexan Fluor 488-conjugated AffiniPure Rabbit anti-Rat IgG (H+L) (Boster, BA1129, dilution 1:200) for 2 h at room temperature in the dark. After washing with PBS three times, slides were incubated with DAPI solution for 15 min in the dark. Finally, the cells were detected by using CLSM. D-The ultimate claim that these nanoparticles are causing ssDNA hydrolysis and a double strand break near the Pt site needs to be definitively demonstrated experimentally. (Fig. 3b). (Fig. 4c) (Fig. 5a). In light of these results, we extracted DNA fragmentations from the nucleus of cisplatin-resistant Huh7 cells after NMPNs treatment, and analyzed the Pt-DNA binding sites by using confocal laser scanning microscopy. The result shows that the green signals corresponding to the Pt-DNA binding mainly localize at the end of DNA fibers (Supplementary Fig. 19). These results demonstrate that NMPNs can induce the double strand break of DNA near the Pt-DNA binding sites.

Our modification to the manuscript:
The results were added as Supplementary Fig.  19 in the revised manuscript. In addition, the following sentences and method were added on pages 10 and 28 in the revised manuscript, respectively.  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 the data are presented as means ± S.E.M., n = 3 independent experiments. Source data are provided as a Source Data file.    experiments. Statistical significance was analyzed by one-way ANOVA with multiple comparisons test. Source data are provided as a Source Data file.
• Figure S19 Supplementary Figure 19. The analysis of Pt-DNA binding sites in the DNA extracted from cisplatin-resistant Huh7 cells after treatment with NMPNs. The blue fluorescence and the green fluorescence correspond to DNA fibers and Pt-DNA binding sites, respectively. Arrows indicate the DNA fragmentations. Asterisks indicate the Pt-DNA binding sites at the end of DNA fragmentations. n = 3 independent experiments. Scale bar: 100 μm.
• Page 10 "In line with the DFT results, we found that NMPNs can induce the formation of Pt-DNA adducts and DNA breakage in the cisplatin-resistant Huh7 cells (Fig. 4a,c). Moreover, we extracted the DNA fragmentations from NMPNs-treated cisplatinresistant Huh7 cells and analyzed the location of Pt-DNA binding in the DNA fragmentations. Intriguingly, the result shows that the Pt-DNA binding sites mainly localize at the end of DNA fragmentation (Supplementary Fig. 19)."

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Page 28 Method The analysis of locations of Pt-DNA binding in the DNA. Cisplatin-resistant Huh7 cells were cultured in 6-well plates with 2 mL culture medium. NMPNs (20 μg/mL, pH 6.5) were added and incubated for 24 h, cells were subsequently collected and quickly resuspended in 500-600 μL of ice-cold PBS. Then, 2.5 μL of cell resuspension was spotted onto a pre-cleaned glass slide and mixed with 7.5 μL of spreading buffer (0.5% SDS in 200 mM Tris-HCl (pH 7.4), 50 mM EDTA). After 10 min, the slides were tilted to 15° to spread DNA fibers along the length. Then, air-dry the DNA spreads and fixed in 3:1 methanol/acetic acid for 20 min at -20℃. After washing with PBS three times, slides were blocked with 1% BSA in PBS for 30 min at room temperature and incubated with anti-cisplatin modified DNA antibodies (GeneTex, GTX17412, dilution 1:100) to detect Pt-DNA adduct. After 1 h of incubation, slides were washed with PBS three times and stained with Alexan Fluor 488-conjugated AffiniPure Rabbit anti-Rat IgG (H+L) (Boster, BA1129, dilution 1:200) for 2 h at room temperature in the dark. After washing with PBS three times, slides were incubated with DAPI solution for 15 min in the dark. Finally, the cells were detected by using CLSM.
E-When is this double strand break actually being formed? Isn't it more likely to form during S-phase, at which time both HR and NHEJ are likely to outcompete NER as repair mechanisms?
Response: Thank you for your valuable comments. As the reviewer mentioned, both HR and NHEJ are likely to outcompete NER as DSBs repair mechanisms during Sphase <Molecular Cell, 2012, 47, 320-329>. However, in this study, NMPNs can release Pt ions that interact with DNA to form Pt-DNA adducts, which is cell cycle phase nonspecific <Ref. Brit. J. Cancer, 2007, 96, 231-240;Nanomedicine, 2010, 5, 51-64>. Moreover, the accumulated NMPNs in the nucleus can efficiently generate ROS, which can induce the break of DNA strains around the Pt-DNA binding site, which can lead to the hydrolysis of the other DNA strain, ultimately resulting in the DSBs (Fig. 4b). Therefore, the DSB induced by NMPNs can be formed in any phase of cell cycles. Notably, the repair of DSBs in S phase mediated by HR and NHEJ can be hindered by the Pt ions that bind to the end of DNA via steric effect ( Supplementary  Fig. 19) <Ref. Mol. Cancer Res., 2005, 3, 277-285;DNA and Chromosomes, 2012, 287, 24263-24272>. Notably, NER is the dominant way to remove the Pt-DNA adducts <Ref. Nature, 2009Nature, , 461, 1071Nature, -1078Sci. Rep., 2017, 7, 11785>. Consequently,

the NMPNs induced the formation of Pt-DNA adducts and the subsequent oxidative cleavage of Pt-DNA adducts can impair the structure basis required for NER, thus inhibiting the DNA repair and suppressing the growth of cisplatin-resistant cancer cells.
Our modification to the manuscript: The results were added as Supplementary Fig.  19 in the revised manuscript. In addition, the following method were added on page 28 in the revised manuscript.  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 the data are presented as means ± S.E.M., 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.
• Figure S19 Supplementary Figure 19. The analysis of Pt-DNA binding sites in the DNA extracted 43 from cisplatin-resistant Huh7 cells after treatment with NMPNs. The blue fluorescence and the green fluorescence correspond to DNA fibers and Pt-DNA binding sites, respectively. Arrows indicate the DNA fragmentations. Asterisks indicate the Pt-DNA binding sites at the end of DNA fragmentations. n = 3 independent experiments. Scale bar: 100 μm. •

Page 28 Method
The analysis of locations of Pt-DNA binding in the DNA. Cisplatin-resistant Huh7 cells were cultured in 6-well plates with 2 mL culture medium. NMPNs (20 μg/mL, pH 6.5) were added and incubated for 24 h, cells were subsequently collected and quickly resuspended in 500-600 μL of ice-cold PBS. Then, 2.5 μL of cell resuspension was spotted onto a pre-cleaned glass slide and mixed with 7.5 μL of spreading buffer (0.5% SDS in 200 mM Tris-HCl (pH 7.4), 50 mM EDTA). After 10 min, the slides were tilted to 15° to spread DNA fibers along the length. Then, air-dry the DNA spreads and fixed in 3:1 methanol/acetic acid for 20 min at -20℃. After washing with PBS three times, slides were blocked with 1% BSA in PBS for 30 min at room temperature and incubated with anti-cisplatin modified DNA antibodies (GeneTex, GTX17412, dilution 1:100) to detect Pt-DNA adduct. After 1 h of incubation, slides were washed with PBS three times and stained with Alexan Fluor 488-conjugated AffiniPure Rabbit anti-Rat IgG (H+L) (Boster, BA1129, dilution 1:200) for 2 h at room temperature in the dark. After washing with PBS three times, slides were incubated with DAPI solution for 15 min in the dark. Finally, the cells were detected by using CLSM. Figure 4: A-In panel B, why is the DCF fluorescence so localized to one pole of the nucleus?

4-
Response: Thank you for your comments. Previous studies reported that the lysosome location itself tends to be distributed on one side of the cell nucleus <Ref. Chem. Sci., 2020, 11, 596;Sensor. Actuat. B-Chem., 2020, 15, 128302;Sensor. Actuat. B: Chem., 2021, 15, 130397;Adv. Funct. Mater., 2020, 30, 1909999>. Once NMPNs escape from the lysosomes, they also tend to enter the nucleus on the side of the nucleus that is adjacent to the lysosome via the importin α/β pathway, and accumulate at one pole of the nucleus (Fig. 2b). Consequently, NMPNs generate ROS in situ and the higher level of ROS (as indicated by the DCF fluorescence signals) was observed at one pole of the nucleus (Fig. 4b).
Our modification to the manuscript: The following sentences were added on page 10 in the revised manuscript.

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Page 10 "As mentioned above, NMPNs accumulated in the nucleus can effectively induce the formation of Pt-DNA adducts by releasing Pt ions (Fig. 4a), and facilitate ROS generation in the nucleus (Fig. 4b) for inducing intensive oxidative cleavage of DNA strands (Fig. 4c), mimicking a biomimetic nuclease." B-In panel C, in their direct testing of the mechanism that oxidation is important following DNA platination, the authors show that NAC co-treatment reduces gH2AX formation, but the key controls showing equivalent NMPNs uptake in the presence of NAC are missing, and the presence of equivalent amounts of the Pt-DNA adduct are also missing. This Pt-DNA missing data, which is shown in panel F suggests that the NAC treatment has major effects on DNA platination itself, not just oxidative cleavage, and makes it impossible to use the results to validate the direct importance of the ROSdependent cleavage effects. All of the data needs to be quantified. Fig. 5b,c, there is no significant difference in the amount of Pt-DNA adducts between NMPNs-treated and NMPNs+NAC-treated cisplatin-resistant Huh7 cells at 6 h and 12 h, indicating that NAC shows no effects on the priming the formation of Pt-DNA adducts. This is because NAC shows no effect on the cellular uptake of NMPNs, which enables the comparable release of Pt ions. However, 18 h or 24 h later, we found the remarkable enhancement of Pt-DNA adducts in NMPNs-treated cisplatin-resistant Huh7 cells as compared to that of NMPNs+NAC-treated cisplatin-resistant Huh7 cells. It is understood that NMPNs can simultaneously release Pt ions to form Pt-DNA adducts and generate ROS to induce the cleavage of Pt-DNA adducts near the Pt-DNA binding sites, which can impair the DNA structure required for NER and thus inhibiting the NER-mediated DNA repairing. In contrast, the cotreatment with NAC can scavenge ROS generated by NMPNs, avoiding the oxidative cleavage of Pt-DNA adducts, which enables the NER-mediated DNA repairing. These results demonstrate that NMPNs can behave as a biomimetic nuclease to rupture the very structure of DNA required for NER by generating in situ ROS, which can induce DNA oxidative cleavage and facilitate the accumulation of Pt-DNA adducts.

Response: Thank you for your valuable suggestions. Per your suggestion, we studied the NMPNs uptake in the presence or absence of NAC. The result shows no obvious difference in the intracellular concentration of Pt ions after treatment with NMPNs or NMPNs+NAC, demonstrating that NAC exerts no effects on the cellular uptake of NMPNs. Additionally, we assessed the amount of Pt-DNA adducts in cisplatin-resistant Huh7 cells after treatments with NMPNS or NMPNS+NAC at different time points. As shown in
Our modification to the manuscript: The results were added as Supplementary Fig.  22 and Fig. 5b,c in the revised supporting information. In addition, the following sentences and methods were added on pages 11-12 and 30 in the revised manuscript, 45 respectively.
• Figure S22 Supplementary Figure   experiments. Statistical significance was analyzed by one-way ANOVA with multiple comparisons test. Source data are provided as a Source Data file.
• Page 11 "The cotreatment with ROS scavenger NAC shows no interference with the cellular uptake of NMPNs ( Supplementary Fig. 22)."

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Page 11-12 "To fully understand the capability of NMPNs to facilitate the buildup of Pt-DNA adducts, we further investigated the Pt-DNA adducts formation and accumulation in cisplatin-resistant Huh7 cells after incubation with NMPNs in the presence or absence of NAC. After 6-hour or 12-hour incubation, we found that both NMPNs and NMPNs+NAC treatment can initiate the formation of Pt-DNA adducts by releasing Pt ions in the cisplatin-resistant Huh7 cells. However, after 18-hour or 24-hour incubation, only NMPNs can facilitate the accumulation of Pt-DNA adducts, demonstrating that NMPNs can not only release Pt ions to induce the formation of Pt-DNA adducts, but also effectively generate in situ ROS to destroy the DNA conformation required for NER 26 , promoting the accumulation of cytotoxic Pt-DNA adducts. (Fig. 5b,c)." • Page 30 Method The concentration of NMPNs in cisplatin-resistant Huh7 cells in the presence or absence of NAC. Cisplatin-resistant Huh7 cells were treated with NMPNs (20 μg/mL) in the presence or absence of NAC for 24 h at pH 6.5, respectively. Then the cells were mixed with aqua regia and incubated at 60℃ for 72 h. After dilution and filtration, the Pt concentrations were quantified by ICP-MS analysis.
C-What effect does NAC have on the PNP effects?

Response: Thank you for your kind comment. Based on your suggestion, we studied the PNPs effect on the induction of DNA damage in cisplatin-resistant Huh7 cells in the presence or absence of NAC. The representative images of each group and corresponding quantitative analysis demonstrate that the cotreatment with NAC can compromise the effect of PNPs on the DNA damage induction in cisplatin-resistant Huh7 cells.
Our modification to the manuscript: The results were added as Supplementary Fig.  20 in the revised supporting information. In addition, the following sentences were added on pages 10 and 31-32 in the revised manuscript, respectively.

48
• Figure S20 Supplementary Figure 20. a, Immunofluorescence images of the γ-H2AX in cisplatinresistant Huh7 cells after treatments with PNPs with or without NAC at pH 6.5. Scale bar: 40 μm. b, Quantification analysis of Pt-DNA adducts in cisplatin-resistant Huh7 cells after treatments with PNPs with or without NAC at pH 6.5. n = 4 independent experiments, data are presented as means ± S.E.M. Source data are provided as a Source Data file.
• Page 10 "The cotreatment with NAC can compromise the effect of NMPNs and PNPs on the induction of oxidative cleavage of DNA ( Fig. 4c and Supplementary Fig. 20)." • Page 31-32 Method Immunofluorescence detection of γ-H2AX. The cisplatin-resistant Huh7 cells were treated with cisplatin, NMPNs, NMPNs+NAC, PNPs or PNPs+NAC for 24 h at pH 6.5. Then, cells were fixed by 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.5% Triton X-100 for another 20 min, and then blocked with 5% bovine serum albumin in PBS for 1 h at room temperature. After which, the cells were incubated with primary antibodies at room temperature for 1 h: anti-γ-H2AX (dilution 1:100, ab81299) from Abcam (Cambridge, UK). Then, the cells were incubated with Alexa Fluor 488-conjugated goat anti rabbit IgG (H+L) from Boster Biological Technology Co., Ltd. for 1 h at room temperature. In addition, the DAPI solution was added for 15 min in the dark for nucleus staining. Finally, the cells were detected by using CLSM.
D-In panels D and E, the authors show primary data for XPA, which they do not quantify, and quantified data for XPF, which they do not show primary data for. Please 49 show IF images for all of the relevant XP proteins and quantify all of the data for both NMPNs and PNPs.
Response: Thank you for your valuable comments. Per your suggestions, we have added the quantitative results for the IF images of XPA. In addition, the image of Western blot results and their corresponding quantitative analysis are also presented in Fig. 5h,   • Page 10-11 "Intriguingly, as compared to PNPs, nearly no XPA colocalizes with Pt-DNA adducts after NMPNs treatment, indicating the recruitment of XPA is suppressed (Fig. 4d,e and Supplementary Fig. 21a,b). XPF, a pivotal NRE factor for making incisions near DNA lesion sites 52 , is also significantly downregulated after the treatment with NMPNs as indicated by the decreased red fluorescence signals corresponding to XPF (Fig. 4f,g) and XPF protein expression level in the nucleus of cisplatin-resistant Huh7 cells (Fig. 4h,i). In comparison, PNPs and cisplatin show no impact on the NER process (Fig. 4h,i and Supplementary Fig. 21c,d)."

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Page 33 Method Immunofluorescence detection of XPA or XPF and anti-cisplatin modified DNA. The Huh7 cells were treated with PNPs, NMPNs or NMPNs plus NAC for 24 h at pH 6.5. The control group was set for no treatment. Then, cells were fixed by 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.5% Triton X-100 for another 20 min, and then blocked with 5% bovine serum albumin in PBS for 1 h at room temperature. After which, the cells were incubated with primary antibodies at room temperature for 1 h: anti-XPA (1:100, AF5336) from Beyotime Biotechnology (Shanghai, China) or anti-XPF (1:100, OM201253) from Omnimabs (California, USA) or anti-cisplatin modified DNA (dilution 1:100, GTX17412) from GeneTex (California, USA). Then, the cells were incubated with Alexan Fluor 488-conjugated rabbit anti-rat IgG (H+L) or Alexa Fluor 488-conjugated goat anti rabbit IgG (H+L) or Alexa Fluor 555-conjugated goat anti mouse IgG (H+L) from Boster Biological Technology Co., Ltd. for 1 h at room temperature. In addition, the DAPI solution was added for 15 min in the dark for nucleus staining. Finally, the cells were detected by using CLSM. E-In panel I, how reproducible is the effect? How many times was the experiment done? Are the differences statistically significant?
Response: Thank you for your valuable suggestion. The flow cytometry analysis of cell apoptosis was independently repeated three times, all showing that NMPNs can effectively induce the apoptosis of cisplatin-resistant Huh7 cells. In addition, we have added the quantitative results to clarify the statistical significance.
Our modification to the manuscript: The results were added as Fig. 5f in the revised manuscript. In addition, the following sentences were added on pages 12, 23, and 29 in the revised manuscript, respectively.  experiments. Statistical significance was analyzed by one-way ANOVA with multiple comparisons test. Source data are provided as a Source Data file.
• Page 12 "Compared with cisplatin, NMPNs cause more severe DNA damage (Fig. 5d), effectively inducing apoptosis to inhibit the proliferation of cisplatin-resistant Huh7 cells (Fig. 5e-g)" F-In panel J, how exactly is viability being measured? What is CCK-8 solution?
Response: Thank you for your comments. We have clarified the CCK-8 measuring method of cell viability in the "Methods" section. The CCK-8 solution is a solution that contains WST-8, which is purchased from Shenzhen Sunview Technology Co., Ltd. In the presence of an electron-coupled reagent, WST-8 is reduced by some dehydrogenases within the mitochondria to produce the orange-yellow formazan.

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Page 30-31 In vitro cytotoxicity. Cisplatin-resistant Huh7 cells were seeded into 96-well plates (1 × 10 4 cells/well) for 24 h and treated with cisplatin, PNPs or NMPNs with a concentration of 20 μg/mL for 6 h at pH 6.5. Then, the incubation medium containing cisplatin, PNPs or NMPNs were removed and replaced with fresh medium. After 6, 12 and 18 h, the cells were incubated with cell counting kit-8 (CCK-8) solution for each well and incubated for another 3 h, the absorbance of each well was measured at 450 nm by a microplate reader. Cisplatin-resistant Huh7 cells were seeded into 96-well plates (1 × 10 4 cells/well) for 24 h and treated with NMPNs (20 μg/mL) in the presence or absence of NAC for 6 h at pH 6.5. Then, the cells were incubated with CCK-8 solution for each well and incubated for another 3 h, the absorbance of each well was measured at 450 nm by a microplate reader.
L02 cells were seeded into 96-well plates (1 × 10 4 cells/well) for 24 h and treated with NMPNs (20 μg/mL) for 6 h at pH 7.4. Then, the cells were incubated with CCK-8 solution for each well and incubated for another 3 h, the absorbance of each well was measured at 450 nm by a microplate reader.
5-This is a critical point -what direct proof do the authors have that lack of NER is the primary mechanism responsible for the enhanced cytotoxicity? To make this claim, the authors need to compare the effects of the NMPNs on isogenic cells that are NERproficient versus NER-deficient, such as XP knock-outs, and show that the cytotoxicity, compared to PNPs is only enhanced in NER-proficient cells.
Response: Thank you for your valuable comment. We carried out XPA siRNA transfection in cisplatin-resistant Huh7 cells by using two double-stranded siRNA sequences of XPA-Homo: (1) sense (5'-3')-GACCUGUUAUGGAAUUUGATT, antisense (5'-3')-UCAAAUUCCAUAACAGGUCTT; (2) sense (5'-3')-GGAGACGAUUGUUCAUCAATT, anti-sense (5'-3')-UUGAUGAACAAUCGUCUCCTT, resulting in the XPA knock-out ( Supplementary  Fig. 24 Our modification to the manuscript: The results were added as Fig. 5h in the revised supporting information. In addition, the following sentences and methods were added on pages 12 and 34-35 in the revised manuscript, respectively.   • Figure S24 Supplementary Figure 24. Western blot analysis of XPA expression in cisplatinresistant Huh7 cells, siNC-transfected cisplatin-resistant Huh7 cells and siXPAtransfected cisplatin-resistant Huh7 cells, respectively. Source data are provided as a Source Data file. • Page 12 "To further investigate the role of NER in NMPNs-mediated therapeutic effect on cisplatin-resistant Huh7 cells, we checked the cytotoxicity of NMPNs, PNPs, or cisplatin to cisplatin-resistant Huh 7 cells (which are proficient in NER) and siXPAtransfected cisplatin-resistant Huh 7 cells (which are deficient in NER) ( Supplementary  Fig. 24). The results show that both PNPs and cisplatin exert enhanced cytotoxicity against NER-deficient Huh7 cells as compared to that of NER-proficient cisplatinresistant Huh7 cells, indicating that NER deficiency can sensitize cisplatin-resistant Huh7 cells to PNPs and cisplatin. However, there is no obvious difference between the cytotoxicity of NMPNs against NER-proficient and NER-deficient Huh7 cells. In NERdeficient Huh7 cells, the XPA expression level is too low to be recruited to the Pt-DNA adducts for repairing 54 . While in NER-proficient Huh7 cells that express a high level of XPA, NMPNs can inhibit the recruitment of XPA to the Pt-DNA adducts by destroying the NER-required DNA bending structure (Fig. 5h)."

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Page 34-35 Method XPA knockdown by small interfering RNA (siRNA). To silence the gene expression of XPA, XPA-specific siRNA (siXPA) and control siRNA (siNC) were obtained from Genepharma (Shanghai, China). Cisplatin-resistant Huh7 cells were seeded into 6-well plates (2 × 10 5 cells per well) overnight and then transfected with siRNA (siXPA or siNC) mixed with Lipofectamine 2000 transfection reagent, according to the recommended protocols by the manufacturer. The medium (Opti-MEM) was replaced at 6 h post-transfection. After 24 h, the cells were treated with cisplatin, PNPs, and NMPNs.

Response:
Thank you for your comment and we're sorry to make you confused. We have provided representative images with high quality of each group in the revised manuscript.
Our modification to the manuscript: The TUNEL (green fluorescence) and DAPI (blue fluorescence) staining images with high quality were added as Fig. 5e (Now is Fig. 6e) in the revised manuscript. 60 • Figure 6 Fig. 6 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 biologically independent animals. d, Representative images of liver collected from each treatment group on day 31. Black arrows indicate the tumour tissues. e, Representative TUNEL (green fluorescence) and DAPI (blue fluorescence) 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 tumours cells after different treatments. n = 3 independent experiments. h, The body weight changes of mice with different treatments. n = 5 biologically independent animals. i, The survival curves of orthotopic liver tumour mice after different treatments. n = 9 biologically independent animals. j, The schematic illustration of NMPNs to induce Pt-DNA adduct formation and oxidative cleavage for inhibiting the recruitment of XPA and XPF, which 61 nullifies NER pathway and thus suppresses the tumour growth in vivo. All the data are presented as means ± S.E.M. Statistical significance was analyzed by one-way ANOVA with multiple comparisons test (g,h) or two-tailed multiple t-tests with Bonferroni-Dunn correction (c). Source data are provided as a Source Data file.
B-In panel f, please show gH2AX immunofluorescence, not just a Western blot Response: Thank you for your kind comment. Based on your suggestion, γ-H2AX immunofluorescence is performed and representative images are shown in Supplementary Fig. 27, whose results are consistent with Western blot analysis.
Our modification to the manuscript: The results were added as Supplementary Fig.  27 in the revised supporting information. In addition, the following sentences and methods were added on pages 13 and 36 in the revised manuscript, respectively.
• Page 13 "Western blot analysis of tumour tissue reveals that NMPNs can decrease the expression of XPA and XPF in the nucleus to inhibit the DNA repair mediated by NER, thus inducing more severe DNA damage (Fig. 6f,g). Consistently, the higher signal of green fluorescence corresponding to γ-H2AX is observed in the tumour tissues of mice treated with NMPNs as compared with that of mice treated with cisplatin or PNPs ( Supplementary Fig. 27)." 62 • Page 36 Method Immunofluorescence detection of γ-H2AX of tumour tissue. Mice were treated with saline, cisplatin, PNPs or NMPNs (2.5 mg/kg body weight) three times a week. After 14 days, tumour tissues were collected for further analysis of γ-H2AX expression by immunohistochemical staining. 7-The models in Figure 4K and 5J are not adequately supported by the data, as I mention in comment #5.
Response: Thank you for your valuable comment. We carried out XPA siRNA transfection in cisplatin-resistant Huh7 cells by using two double-stranded siRNA sequences of XPA-Homo: (1) sense (5'-3')-GACCUGUUAUGGAAUUUGATT, antisense (5'-3')-UCAAAUUCCAUAACAGGUCTT; (2) sense (5'-3')-GGAGACGAUUGUUCAUCAATT, anti-sense (5'-3')-UUGAUGAACAAUCGUCUCCTT, resulting in the XPA knock-out ( Supplementary  Fig. 24). To investigate whether NER plays a primary role in the enhanced cytotoxicity of NMPNs against cisplatin-resistant Huh7 cells, we compared the inhibitory effect of NMPNs, PNPs, and cisplatin on the growth of cisplatin-resistant Huh7 cells. These results show that both PNPs and cisplatin exert the enhanced cytotoxicity against NERdeficient Huh7 cells as compared to that against NER-proficient cisplatin-resistant Huh7 cells, indicating that NER deficiency can sensitize cisplatin-resistant Huh7 cells to PNPs and cisplatin. However, there is no obvious difference in the cytotoxicity of NMPNs against NER-proficient and NER-deficient Huh7 cells. In NER-deficient Huh7 cells, the XPA expression level is too low to be recruited to the Pt-DNA adducts for repairing <Ref. Nat. Commun., 2020, 11, 4124>. Similarly, in NER-proficient Huh7 cells that expresses high level of XPA, NMPNs can inhibit the recruitment of XPA to the Pt-DNA adducts by destroying the NER-required DNA bending structure. Moreover, we extracted DNA fragmentations from the nucleus of cisplatin-resistant Huh7 cells after NMPNs treatment, and analyzed the Pt-DNA binding sites by using confocal laser scanning microscopy. The result shows that the green signals corresponding to the Pt-DNA binding mainly localize at the end of DNA fibers (Supplementary Fig. 19). These results demonstrate that the enhanced cytotoxicity of NMPNs to cisplatin-resistant Huh7 cells is highly dependent on their capacity to impair the NER pathway by inducing oxidative cleavage of Pt-DNA adducts.
Our modification to the manuscript: The results were added as Supplementary Fig.  19, 24 and Fig. 5h in the revised manuscript. In addition, the following sentences and methods were added on pages 10, 12, 28, and 34-35