Metal Nanozymes: New Horizons in Cellular Homeostasis Regulation

Round 1

Reviewer 1 Report

The finding that nanozymes possess mimic oxidoreductases (e.g., peroxidase, oxidase, superoxide dismutase (SOD), and catalase), hydrolases (e.g., phosphatase and phosphatase), and lyases such as carbonic anhydrase, which broke the traditional concept that inorganic materials were bio-inert, and stimulated researchers’ extensive interest in exploring other new enzyme-like nanomaterials. This review discusses the use of metal nanozymes in the regulation of cellular homeostasis and related biomedical applications, which is a very interesting and fascinating topic, and will provide ideas for the development of nanozymes,will attract researchers’attention and effort to bio-inspired nanozymes. However, there are some minor questions that need to be solved for improving the quality of the manuscript.  

1. Your title is focus on the metal nanozymes, therefore metal nanozymes also is needed to as a key word but not nanozyme in abstract.
2. The general classification of nanozymes requires a brief introduction in the part of “A short history of nanozymes”, for example, nanozymes can be roughly divided into three categories according to material types: metal-based nanozymes (e.g.,gold, platinum, and cobalt), metal oxides or metal sulfidenanozymes (e.g., ferric oxide, cerium dioxide, vanadiumpentoxide, and iron sulfide), and carbon-based nanozymes, (e.g., fullerenes, graphene, and carbon dots),or as your idea. In addition, metal nanozymes that you raised include metal-based nanozymes and metal oxides or metal sulfidenanozymes?
3. O2 ° -,need to revised as O₂^-, but not circle in line 109.
4. It is better to list the comparison of enzyme-like activity of different metal nanozymes in a table(such as KM (mmol L^-1) Vmax (M s^-1),which will help readers to understand which design of metal nanozymes have a strong enzyme-like
5. In the part of biomedical applications, the order needs to be tweaked. I commended that “Metal nanozymes as Enzyme Inhibitors”is first, biosensing or diagnosis is second, and the following is neurodegenerative diseases, cancer, and pathogens, which belong to disease 
6. In the part of fighting pathogens, vaccines is also an ideal strategy against pathogens, it is better to referencea paper that “Mucosal Vaccination for Influenza Protection Enhanced by Catalytic Immune-Adjuvant. Advanced Science. 2020;7(18):2000771. doi: 10.1002/advs.202000771.”, which provides an antiviral alternative for designing nasal vaccines based on IONzyme, as a catalytic immune-adjuvant, to combat influenza infection, which also is an ideal vaccine design strategy against COVID19.
7. In conclusions, more details about the bottleneck problem of metal nanoenzyme recently and related solution strategy or future development direction need to be further introduced.  

Author Response

Reviewer 1 - answers

Dear Reviewer,

Thank you for the evaluation of our manuscript and all the valuable comments that allowed us to improve our paper. Below you will find a point-by-point response to all your comments.

The finding that nanozymes possess mimic oxidoreductases (e.g., peroxidase, oxidase, superoxide dismutase (SOD), and catalase), hydrolases (e.g., phosphatase and phosphatase), and lyases such as carbonic anhydrase, which broke the traditional concept that inorganic materials were bio-inert, and stimulated researchers’ extensive interest in exploring other new enzyme-like nanomaterials. This review discusses the use of metal nanozymes in the regulation of cellular homeostasis and related biomedical applications, which is a very interesting and fascinating topic, and will provide ideas for the development of nanozymes,will attract researchers’attention and effort to bio-inspired nanozymes. However, there are some minor questions that need to be solved for improving the quality of the manuscript.  

1. Your title is focus on the metal nanozymes, therefore metal nanozymes also is needed to as a key word but not nanozyme in abstract.

 

Thank you, done

 

 

1. The general classification of nanozymes requires a brief introduction in the part of “A short history of nanozymes”, for example, nanozymes can be roughly divided into three categories according to material types: metal-based nanozymes (e.g.,gold, platinum, and cobalt), metal oxides or metal sulfidenanozymes (e.g., ferric oxide, cerium dioxide, vanadiumpentoxide, and iron sulfide), and carbon-based nanozymes, (e.g., fullerenes, graphene, and carbon dots),or as your idea. In addition, metal nanozymes that you raised include metal-based nanozymes and metal oxides or metal sulfidenanozymes?

 

The following fragment was added to the line 76:

Nanozymes can be divided into three categories according to material types: metal-based nanozymes (e.g.,gold, platinum, and cobalt), metal oxides or metal sulfidenanozymes (e.g., ferric oxide, cerium dioxide, vanadiumpentoxide, and iron sulfide), and carbon-based nanozymes, (e.g., fullerenes, graphene, and carbon dots). Herein, according to the keynote of the special edition, we will focus on the metal- and metal oxide-based nanozymes.

 

1. O2 ° -,need to revised as O₂^-, but not circle in line 109.

 

Thank you, done

 

1. It is better to list the comparison of enzyme-like activity of different metal nanozymes in a table(such as KM (mmol L^-1) Vmax (M s^-1),which will help readers to understand which design of metal nanozymes have a strong enzyme-like

 

Thank you very much for this suggestion, the following fragment has been added in line 81:

The catalytic reaction of nanozymes is like that of natural enzymes, which conforms to the kinetic curve of Michaelis-Menten equation. Kinetics analysis performer by researchers [1–5],  indicated that nanozymes showed typical Michaelis−Menten behavior toward H₂O₂ , TMB (3, 3, 5, 5-tetramethylbenzidine) glucose and other substrates. The selected examples of the K_m (Michaelis-Menten constant) values and V_max (maximum rate) of the reaction for certain substrates are shown in Table 1 (see also [1]).

Table 1: Comparison of the Km and Vmax of selected nanozymes.

Nanozymes	substrate	Km (mM)	Vmax (M x s-1)	Ref.
V2O5	TMB H2O2	0.738 0.232	1.85 x 10-5 1.29 x 10-5	[3]
Fe3O4 MNPs	TMB H2O2	0.434 154	10.00 x 10-8 9.78 x 10-8	[3]
MOF(Co/2Fe0	TMB H2O2	0.25 4.22	0.38 x 10-7 0.49 x 10-7	[2]
AuNPs	glucose	0.41	0.10 x 1--6	[4]
ZnO	starch	0.022	1.81 x 10-6	[5]
GOx-Fe0@EM-A	H2O2	29.59	3.81 x 10-8	[6]

 

TMB - 3, 3, 5, 5-tetramethylbenzidine

K_m – Michaelis-Menten constant

V_max – maximum rate

 

1. Stasyuk, N.; Smutok, O.; Demkiv, O.; Prokopiv, T.; Gayda, G.; Nisnevitch, M.; Gonchar, M. Synthesis, Catalytic Properties and Application in Biosensorics of Nanozymes and Electronanocatalysts: A Review. Sensors 2020, 20, 4509, doi:10.3390/s20164509.
2. Yang, H.; Yang, R.; Zhang, P.; Qin, Y.; Chen, T.; Ye, F. A Bimetallic (Co/2Fe) Metal-Organic Framework with Oxidase and Peroxidase Mimicking Activity for Colorimetric Detection of Hydrogen Peroxide. Microchim Acta 2017, 184, 4629–4635, doi:10.1007/s00604-017-2509-4.
3. Sun, J.; Li, C.; Qi, Y.; Guo, S.; Liang, X. Optimizing Colorimetric Assay Based on V2O5 Nanozymes for Sensitive Detection of H2O2 and Glucose. Sensors 2016, 16, 584, doi:10.3390/s16040584.
4. Fan, L.; Lou, D.; Wu, H.; Zhang, X.; Zhu, Y.; Gu, N.; Zhang, Y. A Novel AuNP‐Based Glucose Oxidase Mimic with Enhanced Activity and Selectivity Constructed by Molecular Imprinting and O ₂ ‐Containing Nanoemulsion Embedding. Adv. Mater. Interfaces 2018, 5, 1801070, doi:10.1002/admi.201801070.
5. Khade, B.S.; Gawali, P.G.; Waghmare, M.M.; Dongre, P. Adsorption of α-Amylase and Starch on Porous Zinc Oxide Nanosheet: Biophysical Study. Food Biophysics 2021, 16, 280–291, doi:10.1007/s11483-021-09669-9.
6. Liu, W.; Ruan, M.-L.; Liu, L.; Ji, X.; Ma, Y.; Yuan, P.; Tang, G.; Lin, H.; Dai, J.; Xue, W. Self-Activated in Vivo Therapeutic Cascade of Erythrocyte Membrane-Cloaked Iron-Mineralized Enzymes. Theranostics 2020, 10, 2201–2214, doi:10.7150/thno.39621.

 

 

 

 

 

1. In the part of biomedical applications, the order needs to be tweaked. I commended that “Metal nanozymes as Enzyme Inhibitors”is first, biosensing or diagnosis is second, and the following is neurodegenerative diseases, cancer, and pathogens, which belong to disease 

 

Done, according to the Reviewer 1 and 2 comment

 

 

 

1. In the part of fighting pathogens, vaccines is also an ideal strategy against pathogens, it is better to referencea paper that “Mucosal Vaccination for Influenza Protection Enhanced by Catalytic Immune-Adjuvant. Advanced Science. 2020;7(18):2000771. doi: 10.1002/advs.202000771.”, which provides an antiviral alternative for designing nasal vaccines based on IONzyme, as a catalytic immune-adjuvant, to combat influenza infection, which also is an ideal vaccine design strategy against COVID19.

 

A fragment concerning this novel research along with the figure has been added:

IONs can be also used to develop new catalytic mucosal vaccine adjuvants by exploiting its POD-like activity in acidic lysosomes to direct DC maturation.

Mucosal vaccination to protect against influenza enhanced by a catalytic ION-based immune adjuvant has recently been elaborated by Gao et al. [266]. who designed a new mucosal vaccine based on the combination of chitosan (CS) functionalized iron oxide nanozyme (CS-IONzyme) and H1N1 whole inactivated virus (WIV). First, CS-IONzyme was synthesized by a solvothermic method in which CS in the reaction system acted as both a ligand and a surface functionalization factor. CS was shown to cause a positive surface charge of IONzyme increasing potential antigen delivery. In a mouse model, CS-IONzyme was shown to have good biosafety. When mice were immunized intranasally with CS-IONzyme and H1N1 WIV complexes, the amount of the CS-IONzyme-H1N1 WIV complex was about 30-fold increased on the nasal mucosa compared to the H1N1 WIV only formulation after 15 minutes of incubation. The complex adhered stably to the mucosa surface. Furthermore, in in vivo study, CS-IONzyme facilitated H1N1 WIV to enhance CCL20-driven submucosal dendritic cell (DC) recruitment and transepithelial dendrite (TED) formation for viral uptake via the toll-like receptor(TLR) 2/4-dependent pathway. Moreover, IONzyme catalyzed a reactive oxygen species (ROS)-dependent DC maturation, which further enhanced the migration of H1N1 WIV-loaded DCs into the draining lymph nodes for antigen presentation. In result, CS-IONzyme-based nasal vaccine triggered an 8.9-fold increase of IgA-mucosal adaptive immunity in mice, providing a 100% protection against influenza, while only a 30% protection by H1N1 WIV alone (Figure 13).

 

A figure (Figure 13 has been added.

 

 

1. In conclusions, more details about the bottleneck problem of metal nanoenzyme recently and related solution strategy or future development direction need to be further introduced.  

 

Conclusions have been modified.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Dear Authors,

This article is a thorough review of nanozymes, covering aspects such as the types of nanomaterials, their mode of action, and biomedical applications. Each section is well structured, and each topic is clearly addressed using examples from the literature, which has also been properly cited. Nevertheless, careful revision of the manuscript should be considered, and a list of specific (minor) comments is suggested.

1) In general, the manuscript is well structured. Nonetheless, I would suggest that the subheading “5. Nanozymes in the fighting of neurodegenerative diseases” should be described after section 6 and 7, as the use of nanozymes as enzyme inhibitors and in biosensing could have broader applications, and then the following topics on neurodegeneration, cancer, and pathogens can be considered as more targeted applications.

2) Regarding the figures, please revise the typos in figure 1 (“biosenising”) and in figure 4 (“mimmicking”). The quality of figure 2 should be improved and the axis titles should also be included. Also, on page 5, line 182, it should be figure 3, not figure 2.

3) In table 1, please consider moving the references for a later column for better readability (page 14).

4) The manuscript should be checked for the use of abbreviations. When an abbreviation is first used, it should come after the full name and then always be given in the abbreviated form. For example, on page 4, line 153, the names of these enzymes were previously abbreviated. Throughout the manuscript, the terminology of the nanozymes alternates between full name and abbreviation. In addition, there are many abbreviations without the full name, making it difficult to read. For example, line 224 (POD), line 502 (G-CeO₂NPs), line 713 (GSPs), line 764 (PET, SPECT), line 649 (nano-ZIFs), etc. Please revise.

5) In the sentence starting at line 636, do the authors mean that the non-fluorescent substrate is converted to a fluorescent-active product? Please revise the sentence if it should mean substrate or product.

6) In section 9, the spelling of bacterial names must be in italics and after the first mention in the text, the genus name can be abbreviated. Please revise.

7) The italic spelling of in vitro and in vivo should be consistent throughout the manuscript, which is not the case.

8) Finally, the notation of symbols and units in equation 1 and equation 3 has some typos (e.g., missing subscripts and superscripts), and there are other typos and split sentences throughout the text that should be carefully revised.

Author Response

Reviewer 2 - answers

Dear Reviewer,

Thank you for the evaluation of our manuscript and all the valuable comments that allowed us to improve our paper. Below you will find a point-by-point response to all your comments.

This article is a thorough review of nanozymes, covering aspects such as the types of nanomaterials, their mode of action, and biomedical applications. Each section is well structured, and each topic is clearly addressed using examples from the literature, which has also been properly cited. Nevertheless, careful revision of the manuscript should be considered, and a list of specific (minor) comments is suggested.

1) In general, the manuscript is well structured. Nonetheless, I would suggest that the subheading “5. Nanozymes in the fighting of neurodegenerative diseases” should be described after section 6 and 7, as the use of nanozymes as enzyme inhibitors and in biosensing could have broader applications, and then the following topics on neurodegeneration, cancer, and pathogens can be considered as more targeted applications.

Done, according to the Reviewer 1 and 2 comment.

 

2) Regarding the figures, please revise the typos in figure 1 (“biosenising”) and in figure 4 (“mimmicking”). The quality of figure 2 should be improved and the axis titles should also be included. Also, on page 5, line 182, it should be figure 3, not figure 2.

This has been amended, thank you.

 

3) In table 1, please consider moving the references for a later column for better readability (page 14).

 

Done.

4) The manuscript should be checked for the use of abbreviations. When an abbreviation is first used, it should come after the full name and then always be given in the abbreviated form. For example, on page 4, line 153, the names of these enzymes were previously abbreviated. Throughout the manuscript, the terminology of the nanozymes alternates between full name and abbreviation. In addition, there are many abbreviations without the full name, making it difficult to read. For example, line 224 (POD), line 502 (G-CeO₂NPs), line 713 (GSPs), line 764 (PET, SPECT), line 649 (nano-ZIFs), etc. Please revise.

The abbreviations have been carefuly checked and amended throughout the text. Full names were given for the first use and then abbreviations were used.

 

5) In the sentence starting at line 636, do the authors mean that the non-fluorescent substrate is converted to a fluorescent-active product? Please revise the sentence if it should mean substrate or product.

This has been rephrased, as follows:

In the pH range 4−7, HRP/H₂O₂ oxidized ampliflu to nonfluorescent resazurin. In contrast, at pH 6-8 nanoceria oxidized ampliflu to the intermediate oxidation fluorescent product resorufin, while at or below pH 5.0, nanoceria also yielded the terminal oxidized nonfluorescent product resazurin. Thus, the ability of nanoceria to oxidize ampliflu to a stable fluorescent product in the pH range 6−8 was used to develop a sensitive cell-based ELISA at neutral pH without the use of H₂O₂.

6) In section 9, the spelling of bacterial names must be in italics and after the first mention in the text, the genus name can be abbreviated. Please revise.

This has been done, thank you.

7) The italic spelling of in vitro and in vivo should be consistent throughout the manuscript, which is not the case.

Amended.

8) Finally, the notation of symbols and units in equation 1 and equation 3 has some typos (e.g., missing subscripts and superscripts), and there are other typos and split sentences throughout the text that should be carefully revised.

This has been amended, thank you.

 

Author Response File: Author Response.docx

Reviewer 3 Report

This review paper systematically described the development and prospects of metal or metal oxide nanozymes. The authors not only proposed the efficient improving methods for the design of new nanozymes, but summarized the mechanisms of pro-oxidative and antioxidant action of nanozymes，especially of the nanozymes based on metal or metal oxide. At last, it exhaustively listed the extensive application of metal nanozymes such as in fighting of neurodegenerative diseases、biosensing as well as cancer treatment etc. It will be of broad interest to its readership in the highly interdisciplinary research areas. I recommend this paper for publication in Applied Sciences after a minor revision. Detailed comments are as follows:

1. Some specific writing format is not reasonable (Page 3,line 109, “O2 °” ,the expression of free radicals should be “O₂^·- ”), please check and revise in full paper.
2. More papers can be cited to discuss the antioxidant activity of cerium oxide nanozymes (such as Molecules 2021, 26(12), 3747 https://doi.org/10.3390/molecules26123747 )
3. Coherence before and after some sentences is not good and the English presentation in introduction section needs to be polished.

Author Response

Reviewer 3 - answers

Dear Reviewer,

Thank you for the evaluation of our manuscript and all the valuable comments that allowed us to improve our paper. Below you will find a point-by-point response to all your comments.

This review paper systematically described the development and prospects of metal or metal oxide nanozymes. The authors not only proposed the efficient improving methods for the design of new nanozymes, but summarized the mechanisms of pro-oxidative and antioxidant action of nanozymes，especially of the nanozymes based on metal or metal oxide. At last, it exhaustively listed the extensive application of metal nanozymes such as in fighting of neurodegenerative diseases、biosensing as well as cancer treatment etc. It will be of broad interest to its readership in the highly interdisciplinary research areas. I recommend this paper for publication in Applied Sciences after a minor revision. Detailed comments are as follows:

1. Some specific writing format is not reasonable (Page 3,line 109, “O2 °” ,the expression of free radicals should be “O₂^·- ”), please check and revise in full paper.

 

Thank you, this has been corrected. Editorial amendments have been introduced throughout the paper.

 

1. More papers can be cited to discuss the antioxidant activity of cerium oxide nanozymes (such as Molecules 2021, 26(12), 3747 https://doi.org/10.3390/molecules26123747 )

 

Thank you for this remark, widening the point of view on the nanozyme potential activity depending on the applied system. we added this report to the review:

 

The cerium oxide nanoparticle (nanoceria) was shown to posses high peroxidase activity [252,253]. Provided that high peroxidase activity is more conducive to promoting the production of ROS, the CeO₂-H2O2 system might be regarded generally a good solution for bacterial disinfection. However, as shown and discussed by Bing et al. [254] CeO₂ nanoparticles show multiple enzyme-like activities depending on their shapes and sizes [1,2]. Beside the high peroxidase activity, CeO2 also have high superoxide dismutase and antioxidant activities, and depending on the conditions could as well be used as ROS scavengers. In the system investigated by Bing et al. [254] the peroxidase activity of CeO₂ did not contribute to the antibacterial effect, instead, the ROS scavenging capacity of CeO₂ could protect the bacteria from being killed by H₂O₂.

 

1. Coherence before and after some sentences is not good and the English presentation in introduction section needs to be polished.

The introduction section has been improved.

 

 

Author Response File: Author Response.docx