Sustainable Coated Nanostructures Based on Alginate and Electrospun Collagen Loaded with Antimicrobial Agents

Round 1

Reviewer 1 Report

In the manuscript “Sustainable Coated Nanostructures based on Alginate and Electrospun Collagen Loaded with Antimicrobial Agents”, the author reported sustainable coated nanostructures containing commercial antimicrobial agents by mixing them with collagen glue extracted from rabbit skin waste and deposited as electrospun nanofibers onto sodium alginate support. The paper fit the aims and scope of Coatings. The paper seems to be acceptable but, i it requires some modifications. Additionally, several questions should be answered by the authors in detail.

Introduction:

There are so many reports on the electrospun nanofibers with bactericidal activity. How about the novelty of the manuscript? You should explain why it is interesting to do the experiments you describe and especially what is new compared to these published papers. Rabbit skin can be considered as waste or by-products from the meat industry or leather industry. In my opinion, the reuse of waste or by-products in food industry deserves to be emphasized, which can increase economic value and environmental benefits. Therefore, the incipit has to be supported with proper suitable literature references, in order to evidence the need to reuse agro-food waste by-products, including”Reutilization of Food Waste: One-Step Extration, Purification and Characterization of Ovalbumin from Salted Egg White by Aqueous Two-Phase Flotation. Foods, 2019, 8(8): 286. doi:10.3390/foods8080286”, ”Effective separation of prolyl endopeptidase from Aspergillus Niger by aqueous two phase system and its characterization and application. International Journal of Biological Macromolecules 2020,169: 384-395. doi: 10.1016/j.ijbiomac.2020.12.120”, ”Direct Separation and Purification of α-Lactalbumin from Cow Milk Whey by Aqueous Two-phase Flotation of Thermo-sensitive Polymer/Phosphate, Journal of The Science of Food and Agriculture, 2020, doi: 10.1002/jsfa.11055”

Research on protein-based functional materials, especially antibacterial or antioxidant materials, and protein-based nanofiber materials should be mentioned.

1. Fabrication and Characterization of a Microemulsion Stabilized by Integrated Phosvitin and Gallic Acid. Journal of Agricultural and Food Chemistry 2020, 68, 5437-5447. doi 10.1021/acs.jafc.0c00945
2. Preparation and Characterization of Coating Based on Protein Nanofibers and Polyphenol and Application for Salted Duck Egg Yolks. Foods 2020, 9, 449. doi:10.3390/foods9040449

it is strongly suggested to indicate at the end of the Introduction section the main employed characterisation techniques in order to achieve their purpose.

Materials and Methods

The Materials and Methods section is well organized and described in details, but some details need to be improved.

Alg was defined as alginate film in abstract (line 18) but it was defined to be Sodium alginate in line 103. It should be consistent. TEM in line 212 should be defined.

In vitro (line 178) should be in italic.

Line 125-126: Was the Col prepared into two type of solution with different solvent?

Results

The author should explain why a relatively wide distribution range of the fiber diameter was obtained.

In Figure 4, the concerned characteristic peaks might be marked on the FTIR graphs to make it clear.

The statistical analyses must be performed for the replicated experiments using proper methods (for example; ANOVA) and the results must be discussed properly. For the replicated data, it must be also mentioned that whether the results were given as “average ± standard deviation” or not.

Author Response

We would like to sincerely thank you for evaluating our manuscript. We found your comments and suggestions very constructive and we accordingly revised the manuscript. All changes are highlighted in the revised manuscript.

1. Introduction. Thank you for your opinion and remarks about Introduction.

Lines 77-78 were completed with „In a circular economy concept, the use of resources within closed-loop systems represents an approach about the integration of the well-being of the environment with economic activities [33].  For example, the reuse of agro-food waste by-products such as salted egg whites [34], Aspergillus niger prolyl endopeptidase (An-PEP) from fermentation broth [35], or α‐Lactalbumin from cow milk whey  [36] is beneficial for the reduction of environmental pollution and increase economic value simultaneously. Developing of protein-based functional materials derived from food industry [37,38], which possess especially antioxidant activity and protein-based nanofiber materials for wound healing [39] with potential to killing the bacteria has also been reported. Rabbit skin can be considered as waste or by-products resulted from the meat industry [40,41] or leather industry [42]. In this context, the choice of biomaterials with proper bioactive properties for the production of wound healing is a good strategy.”

The novelty of this paper consists in the use of indigenous natural resources based on collagen from rabbit skin glue, in a circular economy, for production of the antimicrobial biohybrid nanofibers wound dressing. The coated nanostructures revealed uniform nanofibers with a relative wide distribution range of the fiber diameter and high surface roughnesses as compared. From the tested coated nanostructures, only those with chitosan and Ag425K passed the cell proliferation test. The Alg-Col-ZnONPs nanostructure did not pass the proliferation test but it showed clear inhibition zones against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 and decreased with at least 4 logarithmic units in CFU/mL values compared to the growth control.

We highlighted the main employed characterization techniques at the end of the Introduction section: ”In this paper, the morphological (Scanning Electron Microscopy coupled with the Energy Dispersive X-ray - SEM/EDS, and Atomic Force Microscopy - AFM), structural (Attenuated Total Reflectance Fourier-Transform Infrared spectroscopy - ATR-FT-IR) and biological properties of the coated nanostructures were investigated’’.

2. Materials and Methods.

Alg is a code for the sodium alginate film. So we correct this code in the abstract (line 22).

We defined TEM as Transmission Electron Microscopy (line 132).

In all manuscript the word “in vitro” is written in italic.

Indeed, first the collagen rabbit glue was dissolved in distilled water, then, 40% acetic acid was added to obtain a homogeneous dispersion.

3. Results.

Based on our previous experience with nanofibers production, we think that the prepared solutions for electrospinning as well as selection of the electrospinning parameters contributed to the obtaining of a relatively wide distribution range of the fiber diameter. Also these measurement results are due to the nanometric dimension of commercial antimicrobial agents, as well as electrical conductivity of the solutions (Table 1).

 Lines 256-259 were modified accordingly.

The Figure 4 was corrected according your requirement.

Significant differences between the means of triplicate experiments and the control were determined by using one-way ANOVA statistical analysis (significance difference was noted as *for p < 0.05, and **for p < 0.01) for biological tests, since the mean values ± the standard deviations (SD) were expressed for electrical conductivity and pH measurements (Table 1).

Reviewer 2 Report

In this sudy by Matei et al., the authors analyzed how electrospun fibers filled with nanoparticles exhibit antimicrobial properties. The report fits the scope of Coatings journals and the results themself are quite interesting. However, there are some issues, which should be addressed before it can be accepted. Please find the suggestions below:
1) " Subsequently, the dispersions were sonicated for 30 min in order to remove air bubbles." (Lines 132-133). It is commonly known that sonicaton can cause aeration due to excessive cavitation in the solution. Therefore, it casts doubt if this technique is best for removal of air bubbles. Please comment on this issue and provide sonication parameters in the revised version of the manuscript.
2) Table 1 - how was the electrical characterization done to obtain the conductivity? Was it conducted using a 2- or 4-probe method? How was the thickness of the material evaluated.
3) A serious concern is the lack of statistics at some places which puts in question if these particular results can be interpreted. For instance, the values of electrical conductivity reported in Table 1 should be accompanied by the uncertainty values. pH values reported a row below should also have standard deviations reported.
4) SEM acceleration voltage is not given.
5) How did the authors ensure that the sputtering with gold did not thicken the fibers? At some point later in text the fiber dimensions are discussed.
6) SEM images in Fig. 1 should have professional scale bar markers.
7) Why the corresponding SEM images in Fig. 2 are not of the same magnification? Besides, raw data from these plots should be reused to produce proper plots. It is not recommended to copy-paste native plots from EDS software.
8) Overall, data presentation is of rather low quality. Most of the figures do not have the same formatting and they overflow from one page to the other which is confusing and makes it hard to follow the narration. It is advised to make these images arranged and formatted better paying attention that they should not span several pages.
9) It would be good to show TEM micrographs of nanoparticles used for the study to prove that indeed these were nanoparticles. They play an important fole in the behavior of the developed material, so it is important to elucidate their structure.

10) Transmittance of some samples in Fig. 4 extends beyond 100% which is obviously impossible. Please explain it. 

Author Response

We would like to sincerely thank you for evaluating our manuscript. We found your comments and suggestions very constructive and we accordingly revised the manuscript. All changes are highlighted in the revised manuscript.

1) Indeed, sonication sometimes destroy the polymer structure. As alternatives, centrifuge and vacuum are recommended. In this manuscript, sonication parameters (i.e. 50 W sonication intensity, 24 kHz frequency, at 30 °C for 30 min) are provided (line 145).

2) We think there is a misunderstanding of the electrical conductivity reported in Table 1. We did not measure the electrical conductivity on the films, but on prepared solutions with collagen and commercial antimicrobial agents, before to obtain electrospun nanofibers.

3) Table 1 was completed with the standard deviations for electrical conductivity and pH measurements.

4) Line 168: 5 kV was the SEM acceleration voltage.

5) All the prepared samples before SEM analysis were sputtered with gold, the coating having 5 nm thicknesses. We think that this gold ultrathin coating did not influence the thickness of nanofibers. However a conducting path must be established for microscopic examination.

6) Figure 1 was corrected with a professional scale bar.

7) Thank you for your comment. In this paper, we used 20000X the magnification for all coated samples before crosslinking and 10000X the magnification for all crosslinked coated samples in order to view the dimension of nanofibers. All EDS native plots were replaced with the proper plots (Figure 2).

8) In this revised form, Figures 1, 2 and 3 were formatted as not to overflow from one page to another.

9) Thank you for your comment about the dimension of nanoparticles. We provide you a short data about the characteristic of nanoparticles from manufacturers and our try to detect their structure.

Ag425K acquired from TiPE Nanotechnology in life, Shanghai, China. The producer, TiPe Solution declares the particle size dimension of 6-8 nm (https://www.tipe.com.cn/ag-series/). According to our measurements the particle size diameter is under 25 nm, both determinations performed by CryoTEM and Zetasizer Nano ZS (Malvern). Below we present some unpublished results related to the measurement of average particle size of Ag425K using CryoTEM equipment coupled with EDX detector (FEI Tecnai F20 G² TWIN Crio-TEM and X-MaxN 80T detector) and Zetasizer Nano ZS (Malvern).

 Figure A and B about the composition of Ag425K by EDX (STEM mode), TEM images for particle size measurement; and Average particle size measured by DLS of 23.7 nm was uploaded as a separate document. 

ZnO nanoparticle dispersion supplied by Sigma-Aldrich, Darmstadt, Germany, has the average particle size, declared through technical specification (https://www.sigmaaldrich.com/Graphics/COfAInfo/SigmaSAPQM/SPEC/72/721077/721077-BULK_______ALDRICH__.pdf), below 100 nm by TEM and below 40 nm by APS (aerodynamic particle sizer). According to the literature the particle analysis showed particle  sizes under 35 nm by DLS and of 13.2-16.5 nm by TEM (Rossi T., Penfold T.J., Rittmann-Frank M.H., Reinhard M., Rittmann J., Borca C.N., Grolimund D., Milne C.J., Chergui M., Characterisating the Structure and Defects Concetration of ZnO Nanoparticles in a Colloidal Solution, The Journal of Physical Chemistry, 118, 19422-19430, 2014).

10) The transmittance exceeded beyoung 100% due to the baseline correction. In this manuscript, Figure 4(a) was replaced with another containing the principal characteristic peaks of samples, according to observation of another reviewer.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Thank you for the corrections. There are two more issues, but they can be handled at the proof stage:
1) There is a mistake in Fig. 1, two panels are named "e" and there is a typo.
2) One can see lots of unexpected space e.g. on Page 7. Please tidy up the article.

Author Response

Thank you for your comment. We found your comments and suggestions very constructive and we accordingly revised the manuscript.