A biomimetic nanoreactor for synergistic chemiexcited photodynamic therapy and starvation therapy against tumor metastasis

Photodynamic therapy (PDT) is ineffective against deeply seated metastatic tumors due to poor penetration of the excitation light. Herein, we developed a biomimetic nanoreactor (bio-NR) to achieve synergistic chemiexcited photodynamic-starvation therapy against tumor metastasis. Photosensitizers on the hollow mesoporous silica nanoparticles (HMSNs) are excited by chemical energy in situ of the deep metastatic tumor to generate singlet oxygen (1O2) for PDT, and glucose oxidase (GOx) catalyzes glucose into hydrogen peroxide (H2O2). Remarkably, this process not only blocks the nutrient supply for starvation therapy but also provides H2O2 to synergistically enhance PDT. Cancer cell membrane coating endows the nanoparticle with biological properties of homologous adhesion and immune escape. Thus, bio-NRs can effectively convert the glucose into 1O2 in metastatic tumors. The excellent therapeutic effects of bio-NRs in vitro and in vivo indicate their great potential for cancer metastasis therapy.

prolonged survival of the mice. The effect of the biomimetic nanoreactor seems to be remarkable and its concept is interesting. However, results and methods were not appropriately described. There are apparent mistakes which remarkably decreased the reliability of data as stated below.
Specific comments. 1. Fig.5 (e). Legends indicated that red line shows the control group. However, all mice survived in this group. How all mice in control group could survive? If this is a simple mistake, it remarkably decreases the reliability of results in this paper.
2. Fig.5 (C). This graph may show the percentage of mice with metastasis. If so, the mice inoculated with B16F10 which is known to highly metastatic melanoma cell lines developed lung metastasis in only 60% of mice without therapy. These results are inconsistent with previous reports showing high metastatic potential of this cell line.
3. In ln 179, authors stated that BalB/C nude mice were used in this experiment. Why nude mice were used for experiments with B16F10 murine melanoma cell line? On the other hand, authors stated in the Methods section (ln425) that BalB/C mice were used. Authors should carefully explain what type of mice were used for each experiment.
4. How authors determined the dose of biomimetic nanoreactor for in vivo experiments? Authors should state the reason why 40mg/kg biomimetic nanoreactor was used.
5. Ln 225-226. The mean of the sentence "In contrast, many aggressive metastasis can be seen throughout the lung." is unclear.

Answers to the questions and changes of the manuscript
Reviewer #1 (Remarks to the Author): The presented work describes a CRET-based biomimetic nanoreactor (bio-NR) to perform synergistic photodynamic-starvation therapy against tumor metastases by converting glucose into 1 O 2 in cancer cells. Although this work is interesting, some of the results are contradictory and unconvincing. In addition, some of the following points should also be critically taken into account.
1. The author mentioned that "After amino modification, the surface area and average pore size of HMSNs were decreased from 417.12 m2/g to 147.17 m2/g and 11.4 nm to 8.4 nm, respectively" in the manuscript (page 5 lines 6-9). This data decreased too sharp only with simple amination process. More data should be presented.

Answer:
We thank the reviewer very much. The amination reagent APTES is a silylation reagent, which will react with SiO 2 scaffold and form thin SiO 2 shell on the surface. This process will directly change the pore size, which has a great influence on the surface area of mesoporous materials. And some previous researches based on mesoporous silica reported the similar results (Angew. Chem. Int. Ed., 2010, 49, 7281-7283;Chem. Mater., 2012, 24, 3895-3905). Fig. 4d, liver and kidney in the mice treated with HMSNs-Ce6@C exhibited a brighter signal than lung with metastatic tumors.

From in vivo images of
However, Si content in liver and kidney actually lower than that in lung with metastatic tumors (Fig. S6). Moreover, where does the tumor treatment group come from? The mouse model was not lung-bearing metastatic tumors? Please explain it.

Answer:
We thank the reviewer very much. We are sorry for the misunderstanding. The mouse model was lung-bearing metastatic tumors.
Because the metastatic tumors were separated from the normal lung tissues before Si content measurement by ICP-AES and the mass of metastatic tumors was small, the results showed the calculated ID %/g of Si content in metastatic tumor was higher than that in liver and kidney 4. According to Fig. S5, "The data showed that PDT or starvation therapy alone resulted in higher cell viability (62.0% and 73.8%, respectively) than the combination of therapies" should be corrected as "The data showed that PDT or starvation therapy alone resulted in higher cell viability (73.8% and 62.0%, respectively) than the combination of therapies". At first, the viability of B16F10 cells caused by PDT just reduced to 26.2% after 24 h incubation, this result was not convinced. Moreover, the viability of B16F10 cells treated with starvation therapy has no difference with combination of therapies (in anaerobic conditions) and calculated around 62% (Fig. S5). It means PDT had no effect under anaerobic conditions; this result was too theoretical to be believed.

Answer:
We thank the reviewer for the valuable comment. The manuscript has been revised and now the data is consistent with the experiments. Because the intracellular H 2 O 2 concentration was very low (less than 0.1 µM) and it is a key reactant for chemical energy generation, the efficiency of chemiluminescence resonance energy transfer based PDT is much lower than that under light irradiation. In addition, as O 2 is a key factor for both PDT and glucose catalysis in starvation therapy, the anaerobic conditions (1 % O 2 ) will also severely limit the effect of starvation therapy. We also conducted MTT assay to explain it. The results showed that the cell viabilities of starvation therapy alone and PDT alone under anaerobic conditions were 83.7% and 89.6%, respectively ( Supplementary   Fig. S6). Therefore, PDT under anaerobic conditions can also have effect on cell death. Fig. 2 and Fig. 4 were confusing: "Hydrodynamic size distributions (f) and zeta potentials (g) of the nanoparticles." should be corrected as "Hydrodynamic size distributions (h) and zeta potentials (i) of the nanoparticles." "ELISA analysis of IL-6 (f) and IL-12 (g) after the mice were injected with HMSNs-GOx-Ce6@CPPO-PFC@C or
We have corrected them and check the manuscript carefully.
6. In Fig. 5F and Fig. S8, the scale bars should be added.

Answer:
We thank the reviewer very much for the valuable comment. The scale bars have been added in Fig. 5F and Fig. S8.
7. CRET should be provided full name when it was first used.

Answer:
We thank the reviewer for bringing this point to our notice. The full name "chemiluminescence resonance energy transfer" has been added in the place where CRET was first used.
8. In the page 5 lines 9-11 and page 6 lines 8-9, the sentence "The amino content of HMSNs-NH2 was calculated to be 1.12 μmol/mg by TGA" was repeated twice in this manuscript, please delete one of them.

Answer:
We thank the reviewer. We have deleted the sentence in the page 6 lines 8-9.
9. Statistical analysis of the Fig. S5 and S6 should be provided.

Answer:
We thank the reviewer for the valuable comment. The statistical analysis of cell viability and Si content in different organs was added in the revised supplementary information.
10. Authors need to carefully check the language of the paper. There are many obvious errors in the text, such as "Disscussion" should be corrected as "Discussion"; In the page 6 lines 7-8, " 1-12.9±1.2 mV for bio-NRs" should be corrected as "-12.9±1.2 mV for bio-NRs". Please check it.

Answer:
We thank the reviewer for pointing out the mistakes. We have revised the manuscript according to the reviewer's comments and checked the manuscript carefully.
Reviewer #2 (Remarks to the Author): Authors developed biomimetic nanoreactors which achieve PDT with no light excitation. Furthermore, coating of the nanoreactor with cancer cell membrane increased accumulation to lung by decreasing its clearance in vivo. In the lung metastasis model with B16-F10 melanoma cells, treatment with the biomimetic nanoreactor decreased production of lung metastasis and prolonged survival of the mice. The effect of the biomimetic nanoreactor seems to be remarkable and its concept is interesting. However, results and methods were not appropriately described.
There are apparent mistakes which remarkably decreased the reliability of data as stated below.
Specific comments.
1. Fig.5 (e). Legends indicated that red line shows the control group. However, all mice survived in this group. How all mice in control group could survive? If this is a simple mistake, it remarkably decreases the reliability of results in this paper.

Answer:
We thank the reviewer very much for pointing out the mistake. We made an obvious mistake that the control group and HMSNs-GOx-Ce6@CPPO-PFC/O 2 @C group were really reverse. We sincerely apologize for our carelessness. We have corrected the mistake in the revised manuscript and checked the manuscript carefully.

Response to Reviewer #2
Comment 2. Authors replied that they showed "Mass percentage of metastases" (Mass percentage of metastases = weight of metastatic tumors/weight of lung. This description is also very unclear, because the methods in detail for measurement of lung and lung metastases for Fig 5(c) was not stated in the Methods section. The red bar in Fig 5 (c) probably shows the mass percentage of metastasis in control group and it is about 60%. However, this reviewer can not understand how authors could measure the weight of metastatic tumors and lung separately. It is also very unclear whether "weight of lung" means weight of total lung with metastases or weight of normal lung.

Answer:
We thank for the referee very much and sorry for the misunderstanding. The metastatic tumors were separated from normal lung tissue by cutting the black B16-F10 metastatic tumors from the total lung tissue to the maximum extent.
Subsequently, the mass of metastatic tumors and normal lung tissue was measured using an analytical balance. And the mass percentage of metastases was calculated according to the weight of metastatic tumors / weight of normal lung. The experimental details have been added in the "In vivo therapeutic effect of the bio-NRs" section of Method. In addition, "the weight of lung" means "the weight of normal lung", which has been changed in the revised manuscript for better understanding.