Green Synthesis of a Novel Silver Nanoparticle Conjugated with Thelypteris glandulosolanosa (Raqui-Raqui): Preliminary Characterization and Anticancer Activity

Round 1

Reviewer 1 Report

1-      DLS provides information about the polydispersity of NPs too. The authors should be discuss polydispersity index (PdI). Please interpret the polydispersity index when it is equal to 0.4.

2-      Usually, NPs sizes measured by SEM or STEM are smaller than the hydrodynamic size of NPs (by DLS). Please the authors discuss that what is the reason difference between the size of nanoparticles measured by STEM and nanoparticles measured by DLS?

3-      I can't believe that you claim the synthesis of AgNPs without using XRD. Do you know that XRD is necessary, and without this analysis, you can never be told about the synthesized AgNPs?

4-      Nowadays, gold nanoparticles (AuNPs) have mainly portion in green medicine, especially in cancer therapy. The authors have to explain why silver nanoparticles were preferred to AuNPs for this study

5-      Nowadays, plant-based nanomaterials have the main portion in green medicine. The author did not discuss recent ref on applying plants in nanomedicine. In this context, the authors should be cited the most recent ref, such as DOI: 10.1016/j.scp.2022.100606

6-      In figure 3, STEM images of AgNPs-RR don’t have any scale bar. Please use original STEM micrographs.

7-      In the introduction, the authors need to discuss the potential of nanoparticles firstly to treat different cancers broadly speaking and next discuss the application of silver nanoparticles to treat cancers. In this context, the following paper must be cited and discussed (DOI: 10.3390/nano12071102; DOI: 10.1016/j.molstruc.2020.127889; DOI: 10.3390/pr8040430; DOI: 10.1016/j.mtcomm.2020.101511)

8-      Detoxifying nanoparticles from a patient's body is a critical issue in nano-based therapy. It is recommended to explain the detoxification and filtration of idle AgNPs from the body of the treated cancer patients in the discussion. In this context, the following paper must be cited and discussed (DOI: 10.1016/j.mehy.2013.01.035)

9-      The conclusion needs to be rewritten and improved

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript describes the green synthesis of silver nanoparticles using aqueous extract of Thelypteris glandulosolanosa (Raqui-Raqui), which implies the novelty of the paper. The elaborated nanoparticles were subjected to UV-Vis, DLS, STEM analysis and cytotoxicity assay. The manuscript reads well, specific comments and suggestions are listed below:

1) The part in the introduction regarding the conducted  study (lines 96-120) is recommended to be presented briefer. Please double check the used terms and abbreviations (lines 101/106).

2) The influence of the different studied concentrations aqueous extracts of Thelypteris glandulosolanosa (Raqui-Raqui) on the formation and characteristics of AgNPs is recommended to be discussed. Please specify the obtained sizes, morphology, etc. to which concentration of reducing agent are related. The same goes for the conducted cytotoxicity assay.

3) Please add the letters “A”, “B”, “C” to the images on Figure 3.

Author Response

Please see the attachment

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Authors addressed all comments completely.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The paper deals with the fabrication of silver nanoparticles using Thelypteris glandulosolanosa extract as reducing agents and with analysis of the dose-dependent effects of AgNPs exposure in vitro. Novelty of the manuscript is major issue in this work. There are many thousands and tens of thousands of articles are available related to biogenic synthesis of silver nanoparticles using plants. Unfortunately the scientific value of the manuscript is minimal too. The nanoparticles have not been sufficiently characterized. The authors used only UV-vis-spectroscopy and SEM. However, other techniques to characterize the surface of AgNPs-RR, which is extremely important for describing their anticancer properties (IR-spectroscopy, NMR, XPS and others) were not used. Cell toxicity of AgNPs-RR insufficiently described. It is also necessary to provide data on the anti-cancer activity of the Thelypteris glandulosolanosa extract itself without Ag. So, the article has serious flaws and additional experiments needed. I thus cannot recommend this paper for publication. My additional comments and questions:

1. 1. UV-visible spectra analysis of AgNPs and AgNPs-RR : Any information about the synthesis and study of AgNPs is missing in the manuscript.
2. It is necessary to provide data on the output of Ag Nps.
3. The authors give an average particle size by SEM image analysis. This technique is not reliable because the diameter may be distorted. TEM image analysis is more commonly used because in this case the observed diameter is the true diameter. It is necessary to provide TEM images of Ag Nps.
4. The bar lines on the figure 3 are completely misreadable.
5. Please, check the significant figures in the numbers along all the research.
6. DLS analysis of AgNPs-RR does not take into account particle agglomeration.

Reviewer 2 Report

The paper is extremely interesting and describes the green process of nanoparticle synthesis in the fight against cancer. Unfortunately, the paper has certain shortcomings, two key shortcomings, which the authors must clarify, whether the process of nanoparticle synthesis is taken from the literature or they designed it and from the formulation aspect a very important issue. The authors obtained completely different particle sizes by all methods, which is unfortunately unacceptable, and especially their explanation in one sentence. And the difference in particle size is up to 50 times. This will certainly influence the behavior of these modern, exceptional therapeutic carriers in the in vivo system.

1. The authors do not state the correct distribution of nanoparticle size in the abstract. The fact is that systems with a unimodal particle size distribution were formulated and about 60% of the nanoparticles had a size of about 39 nm and slightly less than 40% a particle size of about 260 nm. This is evident from Figure 2. This is indicated by the relatively high value of PdI. This can be a realistic representation of particle size, and perhaps unfortunately a consequence of inadequate sample preparation. It is usual to prepare the samples before the measurement, and then store them for 24/48 hours in order to stabilize the system. Because there is a certain instability here, even three factions, I don't know what the consequence is. Of course, this must be corrected in the abstract, and you can only repeat the measurement for safety, although you have put a reference with this procedure.
2. Too many abbreviations in the abstract, try to introduce them, give full names first and then abbreviations.
3. I find lines 103 and 104 redundant.
4. In the introduction, I am a little missing what other types of carriers were used for this purpose, one paragraph. What are the challenges in formulation? Somehow you skipped that part of me.
5. Part of the materials is missing (2.1.).
6. Chapter 2.1. make it clear what is new and what you may have already taken from some other silver nanoparticle synthesis processes.
7. For part 3.2.look at the work below which I suggest, to try to explain the causes of system instability, as well as sample preparation, it is true that these are not the same systems, nor exactly the same type of carrier.But the essence of the problem of your work is the measurement at the time of making, bimodal, even trimodal particle size distribution.There is a potential possibility that the system is unstable for a long time, we do not have a study of thermodynamic stability, or stability over time.It can potentially happen that there is a "leak" of content and thus your nanoparticles, which only at that moment of creation have a debatable particle size, who knows what happens with time.I sincerely hope that this paper will help you discuss the results.Because two factions are represented to you in a very high degree, unfortunately.

Krstić M, Ražić S, Đekić Lj, Dobričić V, Momčilović M, Vasiljević D, Ibrić S. Application of a mixture experimental design in the optimization of the formulation of solid self-emulsifying drug delivery systems containing carbamazepine. Latin American Journal of Pharmacy. 2015; 34 (5): 885-894.

8. 3.3. Please reinforce the explanation for the particle size measured by the two methods, the difference is 50 times the particle size. You do not need to agree with any other work, but to make your results compatible. Even though you have completely taken over the procedure from that paper, that is what I asked you to state in 3.1. My sincere advice, follow the procedure from the paper I gave you in chapter 3.2 , measure the particle size after 0, 24, 48h, use highly purified water (or at least water for injections) for dispersing, see if you get a better fit . It’s only half an hour of work and two days of waiting, but I think everything will be far clearer. It is a very significant result on cell cultures, but consider what will happen if your system is absolutely unstable and you apply it in vivo, unfortunately it will not reach the tumor tissue. Or at least try to reinforce the explanation for the carrier itself in Chapter 3.2. and in 3.3. whence so many differences.
9. Increase slightly 3.4. and conclusion. This is a work of a completely formulation type, and there is a lot of ambiguity. Is the procedure yours or taken over. Huge differences in particle size by two methods.