Intratumoral synthesis of nano-metalchelate for tumor catalytic therapy by ligand field-enhanced coordination

The iron gall ink-triggered chemical corrosion of hand-written documents is a big threat to Western cultural heritages, which was demonstrated to result from the iron gall (GA-Fe) chelate-promoted reactive oxygen species generation. Such a phenomenon has inspired us to apply the pro-oxidative mechanism of GA-Fe to anticancer therapy. In this work, we construct a composite cancer nanomedicine by loading gallate into a Fe-engineered mesoporous silica nanocarrier, which can degrade in acidic tumor to release the doped Fe3+ and the loaded gallate, forming GA-Fe nanocomplex in situ. The nanocomplex with a highly reductive ligand field can promote oxygen reduction reactions generating hydrogen peroxide. Moreover, the resultant two-electron oxidation form of GA-Fe is an excellent Fenton-like agent that can catalyze hydrogen peroxide decomposition into hydroxyl radical, finally triggering severe oxidative damage to tumors. Such a therapeutic approach by intratumoral synthesis of GA-Fe nano-metalchelate may be instructive to future anticancer researches.

The level of experimental detail was sufficient for a researcher to reproduce the work.
This paper provides novel method to cancer tumour treatment and i feel the work will be of interest to researchers and clinicians working in the oncology field. I also feel the comprehensive materials synthesis and characterisation will be of much interest to a wide materials research community.
Reviewer #2: Remarks to the Author: In this study, the authors prepared a nanosystem for cancer therapy by loading gallate into a Feengineered hollow silica nanocarrier. Intracellularly released Fe3+ and gallate can trigger the coordination reactions with each other to form GA-Fe nanocomplex in situ, leading to oxygen reduction reaction for generating hydrogen peroxide. In addition, GA-Fe as a Fenton-like agent can catalyze hydrogen peroxide into hydroxyl radical to provide oxidative damage to tumors. The manuscript can be considered for publication after addressing the following major issues: 1. Since both Fe3+ and gallate are small in size, what is the advantage of choosing hollow silica over mesoporous silica? 2. The authors demonstrated the formation of GA-Fe structure in solution. How to directly prove the formation of the GA-Fe nanocomplex delivered by hollow silica nanocarrier intracellularly, considering that intracellular microenvironment is more complicated? 3. In addition, how to directly prove that the GA-Fe nanocomplex promotes two steps of sequential one-electron ORRs generating hydrogen peroxide intracellularly? 4. There are intrinsic Fe3+ presence in cells. Would such Fe3+ participate in GA-Fe nanocomplex formation intracellularly? 5. From Figure 7d, the tumors still show a growth trend after treating with FHPG for 14 days. The FHPG nanosystem with such complicated therapeutic pathway (delivery, dissociation, GA-Fe formation, and catalysis) seems not efficient in terms of cancer therapy when compared with other nanomedicine. 6. Key liver function index and blood chemistry metrics before and after treating with the nanosystem in vivo should be measured to further support the biosafety.
Remarks to the Author: In this manuscript, a nanomedicine for cancer therapy by loading gallate in a Fe-engineered hollow MSN functionalized with PEG was constructed. In situ, a nano-dimensional hexacoordinated GA-Fe complex was formed to promote the two-electron reduction of O2 into H2O2 and further promote the generation of highly oxidizing •OH. The overall study is complete. This paper is recommended for publication after addressing following issues: 1. The water-soluble drug gallate is loaded in mesoporous silica without blocking the hole. Does the drug leak through the blood circulation? Will this leakage cause side effects? 2. What is the specific concentration of the drug and the Fe iron content in FHPG? 3.The drug release experiment in response to the acidity should be provided. 4. In Figure 7a, the blood circulation half-time of FHPG was calculated to be 1.75 h. Without appropriate reference value, what can "1.75h" represent? 5. In Figure S26, Supporting Information, the safety of FHPG on mouse HC11 cells was confirmed and the authors ascribe the low toxicity of nanoparticles in the HC11 cells to the neutral environment in normal cells was not conductive to Fe release. However, is there any possibility of iron ions could be endocytosed and then degraded in the acidic lysosome? 6. In Figure 4h, does the "Fe 2p1/2" mean the zero-valent iron peaks?
Response to reviewer I.

Comments from reviewer I:
The authors describe the synthesis of a GA/Fe Fe-engineered mesoporous silica nanocarrier for the treatment of cancer. The authors provide comprehensive materials characterization using multiple experiential approaches.
They draw upon a well know chemical reaction that is attributed to the breakdown of written works. Their approach to use the underlying chemistry responsible for this to treat cancer tumours is very novel. The authors provide clear and scientifically sound discussions and conclusions based on their experimental data at all stage of materials development and cellular evaluation.
Please provide some clarification in the text regarding the possible dangers of the mesoporous material degrading at neutral pH. Whilst this degradation is shown to be slow, what is the likely hood that the nanocarrier could breakdown outside the tumour environment and cause damage to other organs. The research does not use a tumour targeted approach and relies on the vascular system to deliver the material to the tumour, therefore off target effects would be likely.
Many of the figures contain data with error bars. Are these standard errors, standard deviations or something else? Also there was no statistics provided on the significance of data variation.
The level of experimental detail was sufficient for a researcher to reproduce the work.
This paper provides novel method to cancer tumour treatment and I feel the work will be of interest to researchers and clinicians working in the oncology field. I also feel the comprehensive materials synthesis and characterization will be of much interest to a wide materials research community.
Response: Thank you very much for the positive comment and kind recommendation. According to your suggestion, the possible danger of nanomedicine at neutral pH has been provided in the revised manuscript (Page 31).
All the error bars in this work are standard deviations, and the related statements have also been Response to reviewer II.

Comments from reviewer II:
In this study, the authors prepared a nanosystem for cancer therapy by loading gallate into a Fe-engineered hollow silica nanocarrier. Intracellularly released Fe 3+ and gallate can trigger the coordination reactions with each other to form GA-Fe nanocomplex in situ, leading to oxygen reduction reaction for generating hydrogen peroxide. In addition, GA-Fe as a Fenton-like agent can catalyze hydrogen peroxide into hydroxyl radical to provide oxidative damage to tumors.
The manuscript can be considered for publication after addressing the following major issues: Response: Thank you very much for the kind question. We cannot totally exclude the possibility of the reaction between endogenous intracellular Fe 3+ and the delivered gallate.

Response
However, as the concentration of endogenous Fe 3+ is relatively low (3.5-230 μM according to the literature: Free Radical Biol. Med. 2013, 65, 143-149), the generation of GA-Fe nanocomplex mainly rely on the delivered Fe 3+ engineered in the nanocarrier. Figure 7d, the tumors still show a growth trend after treating with FHPG for 14 days.

From
The FHPG nanosystem with such complicated therapeutic pathway (delivery, dissociation, GA-Fe formation, and catalysis) seems not efficient in terms of cancer therapy when compared with other nanomedicine.
Response: Thank you very much for pointing out this issue. The tumor therapeutic efficiency relies not only on the intrinsic properties of nanomedicines, but also on other specific experiment factors such as injected dose and tumor subtype. Therefore, it may be not easy and appropriate to directly compare the therapeutic efficacies of different nanomedicines reported in different sources. We expect that the therapeutic efficacy of FHPG can be further improved substantially during its future anticancer applications, based on the current relatively high antitumor efficacy that significantly slows down tumor growth. Fortunately, thanks to the negligible toxicity of the nanomedicine compared to the conventional highly toxic chemodrugs, the dosages of the nanomedicine can be largely elevated, which is expected to show much better anti-tumor performance In this study, distinct enhancement of therapeutic efficacy can be observed after the combination of nanocarrier and gallate in one single nanosystem, providing a feasible strategy to significantly promote oxidative damage of tumor, by the well-designed sequential reactions favoring the generation of large amount of •OH. We hope that such a design of nanomedicine can inspire future works in this field.
6. Key liver function index and blood chemistry metrics before and after treating with the nanosystem in vivo should be measured to further support the biosafety.
Response: Thank you very much for the kind suggestion. The key hepatic function index and blood chemistry metrics before and after treating with the nanosystem have been provided in the supporting information (control group and FHPG group in Figure S35 of revised supporting information), which demonstrate the satisfactory biocompatibility of the nanomedicine.
Response to reviewer III.

Comments from reviewer III:
In this manuscript, a nanomedicine for cancer therapy by loading gallate in a Fe-engineered hollow MSN functionalized with PEG was constructed. In situ, a nano-dimensional hexacoordinated GA-Fe complex was formed to promote the two-electron reduction of O 2 into H 2 O 2 and further promote the generation of highly oxidizing •OH. The overall study is complete. This paper is recommended for publication after addressing following issues: Response: Thank you very much for the positive comment and kind recommendation. Please find the following detailed responses to your suggestions.
1. The water-soluble drug gallate is loaded in mesoporous silica without blocking the hole.
Does the drug leak through the blood circulation? Will this leakage cause side effects?
Response: Thank you very much for the kind questions. We cannot exclude the possibility of potential gallate leakage through the blood circulation. In fact, it also an unsolved issue encountered by all the currently-published non-blocked drug delivery systems. However, as the gallate is mainly loaded in the inner cavity of the hollow nanocarrier, while the shell of the nanocarrier is relatively thick, it can be inferred that the leakage of gallate is not significant.
Based on our experimental results such as hematological and histological evaluations ( Figure   S35-37 in the revised supporting information), this minor leakage will not trigger distinct side effect, which may be attributed to the intrinsic good biocompatibility of gallate.
2. What is the specific concentration of the drug and the Fe iron content in FHPG?
Response: Thank you very much for the constructive question. The mass percentages of Fe and gallate in FHPG are 5.707 wt% and 16.817 wt%, respectively, for keeping the stoichiometric ratio of Fe to gallate to be 1:1. We have further supplemented the data in the revised manuscript according to your question (Page 34).
3. The drug release experiment in response to the acidity should be provided.
Response: Thank you very much for the kind suggestion. In fact, we have also ever tried to directly investigate the release kinetics of gallate in acidic environment. However, as the released gallate will react immediately with the co-released Fe 3+ to form GA-Fe nanocomplex, which is hard to be separated from the nanomedicine precipitate after centrifugation, therefore we are sorry to say, it is indeed hard to directly quantify the concentration of released gallate in the solution.
4. In Figure 7a, the blood circulation half-time of FHPG was calculated to be 1.75 h. Without appropriate reference value, what can "1.75h" represent?
Response: Thank you very much for pointing out this issue. The blood circulation half-time of FHPG is roughly equivalent to that of PEGylated pristine MSN (1.85 h) according to our previous report on the pharmacokinetics of spherical MSNs (small 2011, 7, 271-280).
According to your question, we have revised the related discussion and cited the literature in the revised manuscript (Page 29, Ref 42).