Synthesis and evaluation of protein-based biopolymer in production of silver nanoparticles as bioactive compound versus carbohydrates-based biopolymers

This overall process deals with evaluating the performance of silver nanoparticles, synthesized from sodium caseinate (SC) as green biological active agent, in comparison with widely produced from carboxymethyl cellulose, other carbohydrates (oxidized nanocellulose fibres (OC) and starch (St)). The TGA, FTIR and TEM, as well as its antimicrobial activities toward pathogenic Gram-positive and Gram-negative bacteria in addition to the yeast strain Candida albicans NRRL Y-477 were examined. In addition, with regard to their anti-tumour activity, the evaluation was studied via many cancer cell lines against RPE1 (normal retina cell line). The results revealed that the SC–Ag(I) and CMC–Ag(I) complexes were formed in six- and five-membered chelate rings, respectively, as nanoparticles, while linear chelation structure was formed in case of OC–Ag(I) and St–Ag(I) complexes. The complexation of SC with Ag(I) ions was recommended as promising stable and antimicrobial agent, with lower free Ag(I) ions and particle size than other Ag-complexes. Moreover, it provided anti-tumour activity of most tested cell lines (in vitro), with the following sequence HCT116 > PC3 > HePG 2 > MCF-7 > A549 with IC50 and IC90 values of 25.8 and 54.73 µg ml−1, 45.1 and 66.7 µg ml−1, 64.3 and 110.7 µgml−1, 71.4 and 114.8 µgml−1 and 80.1 and 127.7 µgml−1, respectively. The promising effect of SC–Ag complex was also clear from its selective index versus RPE1 (normal retina cell line).


Recommendation? Major revision is needed (please make suggestions in comments)
Comments to the Author(s) his work focuses on protein-based and carbohydrate-based biopolymer in production of silver nanoparticles, evaluating the antimicrobial activities and anti-tumor activities. The novelty of this study relies on the comparison between protein-based-Ag nanoparticles and carbohydrate-based-Ag nanoparticles. Overall this manuscript needs drastic revision before acceptance for publication. 1) When discussing the electiveness of protein-based biopolymer in production of silver nanoparticles, how to evaluate the effectiveness while there's not enough data to support it, such as yield and load of AgNPs? 2) Fig.2 shows the possibilities of Ag-complex formation vs biopolymer type, I'm afraid it's not proper to display the structure of biopolymers in your way without long polymer chain.
3) The connection mode between AgNPs and polymers isn't clear though FTIR assay can prove the bind with little difference. 4) In the assay of MIC, all samples were dissolved in DMSO. There's no doubt causing protein denaturation, making the results implausible. 5) The paper is riddled with grammatical or spelling mistakes, needing drastic revision. For example, FTIR-Spectra is spelt as FTRR-Spectra, biopolymers is spelt as biopoltmers and there's no dot in line 12 between 2106 cm-1 and These bands.

Decision letter (RSOS-200928.R0)
We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.

Dear Professor Basta:
Title: Electiveness of protein-based biopolymer in production of silver nanoparticles as high efficient bioactive compound versus Carbohydrates-based biopol Manuscript ID: RSOS-200928 Thank you for your submission to Royal Society Open Science. The chemistry content of Royal Society Open Science is published in collaboration with the Royal Society of Chemistry.
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Introduction: Start from here "Many years ago, silver regards as the unique metal of valuable uses in treating and preventing the diseases. Its complexes were applied as biocide and/or biostatic [21]; while its salts succeeded in treating some infections, appearing resistance to the modern antibiotics and drops of silver nitrate are placed in newborn`s eyes at birth for the prevision of contracting gonorrhea from mother [22]. Synthesizing of green silver nanoparticles gained attention by many researchers due to its biological activity, as a result of its ability to anchor the microorganism cell wall, followed by penetrating, changing the cell structure that can lead to cell [23,24]. " and introduction part much be enriched by mechanistic approach of biological function of silver nano-particles.
Author may be improve introduction part in the light of following references--Antibacterial silver nanomaterials synthesis from Obliteration of bacterial growth and biofilm through ROS generation by facilely synthesized green silver nanoparticles. PloS one 12 (8) Comments to the Author(s) his work focuses on protein-based and carbohydrate-based biopolymer in production of silver nanoparticles, evaluating the antimicrobial activities and anti-tumor activities. The novelty of this study relies on the comparison between protein-based-Ag nanoparticles and carbohydrate-based-Ag nanoparticles. Overall this manuscript needs drastic revision before acceptance for publication. 1) When discussing the electiveness of protein-based biopolymer in production of silver nanoparticles, how to evaluate the effectiveness while there's not enough data to support it, such as yield and load of AgNPs? 2) Fig.2 shows the possibilities of Ag-complex formation vs biopolymer type, I'm afraid it's not proper to display the structure of biopolymers in your way without long polymer chain.
3) The connection mode between AgNPs and polymers isn't clear though FTIR assay can prove the bind with little difference. 4) In the assay of MIC, all samples were dissolved in DMSO. There's no doubt causing protein denaturation, making the results implausible. 5) The paper is riddled with grammatical or spelling mistakes, needing drastic revision. For example, FTIR-Spectra is spelt as FTRR-Spectra, biopolymers is spelt as biopoltmers and there's no dot in line 12 between 2106 cm-1 and These bands.

RSOS-200928.R1 (Revision)
Review form: Reviewer 2 Is the manuscript scientifically sound in its present form? Yes

Are the interpretations and conclusions justified by the results? Yes
Is the language acceptable? Yes

Recommendation?
Accept as is

Comments to the Author(s)
The authors have carefully addressed the concerns of reviewers.

Decision letter (RSOS-200928.R1)
We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.
Dear Professor Basta: Title: Synthesis and evaluation of protein-based biopolymer in production of silver nanoparticles as bioactive compound versus carbohydrates-based biopolymer Manuscript ID: RSOS-200928.R1 It is a pleasure to accept your manuscript in its current form for publication in Royal Society Open Science. The chemistry content of Royal Society Open Science is published in collaboration with the Royal Society of Chemistry.
The comments of the reviewer(s) who reviewed your manuscript are included at the end of this email.
Thank you for your fine contribution.

Dear Editor Prof. Anthony Stace and the Asso. Editor Dr Ya-Wen Wang.
First, we hope you are keeping well at this difficult period, too. May our LORD GOD safe you and your dears.
We would like to present my deep thanks for efforts made by you and reviewers to improve my submitted Ms with Ref. # (Ms ID: RSOS-200928). All the reviewers' remarks are considered in revised version, and the changes made in text are highlighted in grey color. Following it our Answers to Reviewer's remarks, and we hope this revised version will meet with approval.
Reviewer 1 * Manuscript "Electiveness of protein-based biopolymer in production of silver nanoparticles as high efficient bioactive compound versus Carbohydratesbased biopolymers" is looking valuable but acceptable after revision Answer Many thanks for your kind decision, which will give us the chance for extending our publication in this estimated Journal * Title: Dear author kindly revise title and make more simple and eye catchy for readers Answer Thanks for your kind advice, the title was changed to " Synthesis and evaluation of proteinbased biopolymer in production of silver nanoparticles as bioactive compound versus carbohydrates-based biopolymers".
* Abstract: is too lengthy and silver material related information missing, kindly update with release kinetics and size and stability data.

Answer
This section was modified and the # of words it reduced to 222, , with thanks * Introduction Start from here "Many years ago, silver regards as the unique metal of valuable uses in treating and preventing the diseases. Its complexes were applied as biocide and/or biostatic [21]; while its salts succeeded in treating some infections, appearing resistance to the modern antibiotics and drops of silver nitrate are placed in newborn`s eyes at birth for the prevision of contracting gonorrhea from mother [22]. Synthesizing of green silver nanoparticles gained attention by many researchers due to its biological activity, as a result of its ability to anchor the microorganism cell wall, followed by penetrating, changing the cell structure that can lead to cell [23,24]. " and introduction part much be enriched by mechanistic approach of biological function of silver nano-particles.
Author may be improve introduction part in the light of following references- Antibacterial silver nanomaterials synthesis from Obliteration of bacterial growth and biofilm through ROS generation by synthesized green silver nanoparticles. PloS one 12 (8), e0181363  Mesoflavibacter zeaxanthinifaciens and targeting biofilm formation. Frontiers in pharmacology 10, 801.  Antimicrobial and anticancer activities of silver nanoparticles synthesized from the root hair extract of Phoenix dactylifera. Materials Science and Engineering: C 89, 429-443 Answer We added your valuable suggested References. Could you please check the highlighted 2 nd paragraph in revised version, with thanks.
The 2 nd paragraph of Introduction section of original article was deleted, and based on your advice we modified the 3 rd paragraph and become to be 2 nd . Based on this modification the References numbers were changed, and added the following References.
Dr. Please permit me to keep the 1st paragraph as it summarized the applications of biopolymers and their complexes with metal ions, with many thanks.. -Reviewer 2 This work focuses on protein-based and carbohydrate-based biopolymer in production of silver nanoparticles, evaluating the antimicrobial activities and anti-tumor activities. The novelty of this study relies on the comparison between protein-based-Ag nanoparticles and carbohydrate-based-Ag nanoparticles.
Overall this manuscript needs drastic revision before acceptance for publication.
1) When discussing the electiveness of protein-based biopolymer in production of silver nanoparticles, how to evaluate the effectiveness while there's not enough data to support it, such as yield and load of AgNPs? Answer Thank you for your advice; also as response to the 1 st Reviewer we changed the title to become "" Synthesis and evaluation of protein-based biopolymer in production of silver nanoparticles as bioactive compound versus carbohydrates-based biopolymers". I hope it convenient with your inquiry.
2) Fig.2 shows the possibilities of Ag-complex formation vs biopolymer type, I'm afraid it's not proper to display the structure of biopolymers in your way without long polymer chain. Answer Thank you, We modified the proposed structures of biopolemer-based AgNPs in Fig. 2, as follow; I hope it will be convenient 3) The connection mode between AgNPs and polymers isn't clear though FTIR assay can prove the bind with little difference. Answer Thanks for this valuable comment, but I ask you to check the highlighted paragraphs related to FT-IR spectra  Under sub title 3.1.1, lines # 10-18, as we reported all changes accompanied the complexation of SC (as example) with Ag(I) ions, from shifting the bands related to NH and COO groups and the increase of band intensity at wave-number range related to M-O (Ag-O bond formation). Moreover, we realized the increase in Ag(I) content which was noticed in Table 1, with the proposed structure (presence more chelating gps.; NH and COO groups chelating sides),  In case of CMC-AgNPs (in the same sub-title, 2nd paragraph),we proposed the chelating side with Ag(I) is via COO groups and not OH, based on the position of OH stretching vibration of OH band on complexation is slightly shifted to higher wavenumber (from 3425 to 3428 cm -1 ); while the bands related to C=O of COOare decreased with disappearance of band related to C-O (1419 cm -1 ). This observation is evidence the OH groups not included the chelation, but the chelation sides are via COOand ether linkage of 1ry alcohol, with formation five member ring chelation geometry (Fig. 2).  Also, these proposed structures were emphasized from the data of TGA measurements [please check the following paragraph which was reported in text of original version [highlighted paragraph in grey color, page # 9-10, started from the last line in page 9] [The possible structure of Sc-Ag complex with 6-membered ring is provided higher thermal stability than the case of five-member ring as the case of CMC-Ag complexes. In other words, and based on the greatest affinity of SC to chelate Ag(I) ions than CMC, as clear from the Ag contents (Table 1) included SC-Ag ( 16.9 mM) and SCMC-Ag complexes(5.0 mM). This leads to the formation of more SC-Ag chelated bonds, which need higher energy for decomposition. The data of thermal stability or activation energy of degradation is the net of bond formation (coordinated and ionic bonds) and weakness of hydrogen bonds due to chelating of Ag (I) ions with COOand NH2 groups, as observed from data of MHBS (Table 1)].

4)
In the assay of MIC, all samples were dissolved in DMSO. There's no doubt causing protein denaturation, making the results implausible. Answer Thank you for this comment, but we would like to clear, this test was carried out according to Ref. [36]. The use of DMSO (2% in Aqueous solution), in MIC, because it was the well known organic solvent that do not exhibit any inhibition on the pathogenic microorganism growth, beside the tested samples were found soluble in DMSO, but not soluble in other organic solvents. Following some published articles on using DMSO-antimicrobial test of AgNPs 5) The paper is riddled with grammatical or spelling mistakes, needing drastic revision. For example, FTIR-Spectra is spelt as FTRR-Spectra, biopolymers is spelt as biopoltmers and there's no dot in line 12 between 2106 cm-1 and These bands. Answer I'm extremely sorry about it. We checked the article and made the corrections about typo and language. All the changes are highlighted in grey color, once again thanks for your efforts and time.
Finally, I hope our response to Reviewers comments and the revised version are convenient for publication.