Nanocomposites of Terbium Sulfide Nanoparticles with a Chitosan Capping Agent for Antibacterial Applications

Round 1

Reviewer 1 Report (Previous Reviewer 1)

I consider that this manuscript is acceptable for publication in J. Comp. Sci. after being corrected the following  minor changes.

1.- Pag. 3, line 109: 4,000 rpm should be changed by 4000 rpm. Same in line 112.

2.- Figure 1 overlapes text in line 206.

After those minor modifiations, I consider that this manuscript is suitable for publication in J. Comp. Sci.

Author Response

Authors Response to Reviewer comments on MS ID: jcs-2107054

 

Reviewer #1

I consider that this manuscript is acceptable for publication in J. Comp. Sci. after being corrected the following  minor changes.

1.- Pag. 3, line 109: 4,000 rpm should be changed by 4000 rpm. Same in line 112.

Response: The rpm values for lines 109 and 112 were harmonized.

2.- Figure 1 overlapes text in line 206.

Response: The overlap of text for Fig. 1 was addressed.

 

After those minor modifiations, I consider that this manuscript is suitable for publication in J. Comp. Sci.

Summary: We acknowledge reviewer #1 for the constructive comments.  The manuscript has been further checked for language, clarity, and syntax throughout to meet the high standards of the Journal of Composites Science.

Author Response File: Author Response.docx

Reviewer 2 Report (Previous Reviewer 2)

The paper entitled "Nanocomposites of Terbium Sulfide Nanoparticles with a Chitosan Capping Agent for Antibacterial Applications" described the Te2S3 Chitosan hybrids for antibacterial applications. This topic is interesting, I think it will attract many readers. The experimental data supported the conclusions to some extent. But I think the authors should address the following question:

1. Can you explain why you fixed the pH values between 9-11. How about other pH values, like pH > 11?

2. It is better to add TEM images of the CS-Tb2S3 hybrid to confirm the core-shell structure.

3. What is the weight percentage of Tb2S3 in the CS-Tb2S3 hybrids?

4. It is better to list a table to compare the inhibition zone of antibacterial of different CS-Tb2S3 hybrids.

Author Response

Authors Response to Reviewer comments on MS ID: jcs-2107054

 

Reviewer #2

The paper entitled "Nanocomposites of Terbium Sulfide Nanoparticles with a Chitosan Capping Agent for Antibacterial Applications" described the Te2S3 Chitosan hybrids for antibacterial applications. This topic is interesting, I think it will attract many readers. The experimental data supported the conclusions to some extent. But I think the authors should address the following question:

1. Can you explain why you fixed the pH values between 9-11. How about other pH values, like pH > 11?

Response: The pH was set at three different conditions (pH 9, 10, and 11) during the synthesis of the chitosan capped Tb₂S₃ particles. We thank the reviewer for this comment. In the one-pot synthesis method, NPs are normally formed in alkali environment, though in some cases, the reactions can undergo at ambient pH. The reason is to ensure the formation of metal sulfide salts, which are expected to agglomerate and form NPs, and to avoid the formation of hydrogen sulfide. On the other hand, the pH of the solution should be lower than 12 (or even lower). As for the case of the alkali environments, the metal ions have a tendency to form hydroxide salts. In this study, the pH was 9 to 11, where the most favourable composite was obtained at pH 10, based on the antimicrobial results (cf. Fig. 5).

 

2. It is better to add TEM images of the CS-Tb2S3 hybrid to confirm the core-shell structure.

Response: We agree with the reviewer the TEM images would be the preferred technique to illustrate the core-shell morphology. Due to the inability to obtain access to TEM instrumentation, the results presented relied on SEM and other complementary methods. Additional SEM images were obtained in another independent sample run for chitosan capped Tb₂S₃ nanoparticles (cf. Fig. S2, Supplementary Material). Several images were obtained in different sample regions (1, 2, and 3) at greater magnification versus the original results presented in Fig. 4 in the manuscript. With reference to Fig. S2 (Supplementary Material), the surface of the sample reveals a number of pseudo-spherical structures in the 10-30 nm size regime, according to the various sample regions (1, 2, and 3). There is evidence of dispersed nanoparticles and aggregated structures on the sample surface, where the light colored regions reveal emission of secondary electrons, attributed to the Tb₂S₃ which resides closer to the sample interface. The variation of light and dark colored regions is related to thinner vs. thicker chitosan shells surrounding the Tb₂S₃ nanoparticles, respectively. This is in accordance with the use of chromium films for contrast enhancement of SEM images (https://doi.org/10.1111/j.1365-2818.1991.tb03095.x), along with evidence of Tb species (ca. 0-4 wt.%) based on the EDX analysis of regions 1-3 of Fig. S2 (results not shown) that concur with previous results listed in Table 3.

Taken together with the other complementary results (DLS particle size analysis, zeta-potential, Raman, XPS, and TGA results, the updated SEM results (cf. Fig. S2)) provide strong experimental support for the core-shell morphology of such chitosan capped Tb₂S₃ nanoparticle materials that is illustrated in Scheme 1.  

 

3. What is the weight percentage of Tb2S3 in the CS-Tb2S3 hybrids?

Response: Based on the SEM-EDX analyses listed in Table 3, the Tb₂S₃ content in the hybrid particles varies across a range (3.05 to 4.45 wt.%) for particles prepared at variable pH conditions, as outline in Table 3, corresponding to the SEM images presented in Fig. 4.

 

4. It is better to list a table to compare the inhibition zone of antibacterial of different CS-Tb2S3 hybrids.

Response:

For the case of CS-Tb₂S₃ hybrids, the materials prepared at pH 10 showed the most favourable antibacterial results, as illustrated for dispersed nanoparticle hybrids in Fig. 5 (cf. dashed circle).  To verify the role of terbium in these composites, the antibacterial properties were evaluated against E. coli and S. aureus, as illustrated in Fig. S3 (b,c). The antibacterial activity of metal nitrate salts were compared for terbium, zinc, and europium against a positive control (tetracycline), along with chitosan. The results indicate an overall greater antibacterial property for the metal salts versus negligible activity for chitosan, where the activity of terbium/europium nitrate compares favourably to that of the positive control (tetracycline) for both bacterial strains, whereas zinc nitrate shows the highest activity overall among the metal salts.

Based on the variable steric effects of the chitosan capping agent, the attenuated antibacterial properties for the hybrid nanoparticles in Fig. 5 can be understood. Taken together with the results presented in Fig. S3 (b,c), the antibacterial properties of terbium sulfide capped nanoparticles is further supported. Nevertheless, there is a need to further explore the optimization of the antibacterial properties of this system in future work. The antibacterial properties of metal sulfide, metal oxide materials display peroxidase-like enzyme activity, which has been outlined in a recent review (https://doi.org/10.3390/antibiotics11030390). However, we contend that the thickness of the capping agent and accessibility of the metal sulfide interface may require further optimization in order to enhance the electron transfer mechanism to effect enhanced antimicrobial activity for this system.

Summary: We acknowledge reviewer #2 for the insightful and constructive comments.  The manuscript has been further checked for language, clarity, and syntax throughout to meet the high standards of the Journal of Composites Science.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report (Previous Reviewer 1)

This new version is suitable for publication in its actual form.

Reviewer 2 Report (Previous Reviewer 2)

The authors have addressed my comments. I recommend it to publish in this journal.

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

 This manuscript entitled "Nanocomposites of terbium sulfide quantum dots with chotosan capping agent for antibacterial applications"  is focused on the eventual application of these compounds for fighting certain infections produced by staphylococcus  aureus in commercial contact lens.

In this regards, and in spite of the interest for finding new new compounds with  antibacterial activity, their conclusions are very poor, supported only on the well known chitosan antibacterial activity,  showing in any case, a  worsen activity than tetracycline.

For all the said above, I regret to recommend the rejection of this manuscript in its actual form.

Author Response

Authors’ Response to Reviewer Comments on MS ID: jcs-2027284

 

Reviewer #1

 

 This manuscript entitled "Nanocomposites of terbium sulfide quantum dots with chotosan capping agent for antibacterial applications"  is focused on the eventual application of these compounds for fighting certain infections produced by staphylococcus  aureus in commercial contact lens.

In this regards, and in spite of the interest for finding new new compounds with  antibacterial activity, their conclusions are very poor, supported only on the well known chitosan antibacterial activity,  showing in any case, a  worsen activity than tetracycline.

For all the said above, I regret to recommend the rejection of this manuscript in its actual form.

 

Response:

We thank the reviewer the insightful and constructive comments. We understand that the manuscript is quite specific on the synthesis of chitosan-encapsulated Tb₂S₃ nanoparticles (NPs) and their antibacterial activity properties against the S. aureus bacterial strain. An emphasis of this work is explorative in nature rather than definitive. This relates to the sparse use of rare earth metal elements (REEs) to form NPs, despite the fact that REEs have many interesting physical and chemical properties that are also useful for advanced technology applications. However, a key problem is the selection of proper capping agent, where many attempts that employ REE-based NPs reveal their highly hygroscopic nature. Therefore, in this study we have used chitosan to passivate the metallic atoms on the NPs surface. In turn, the use of chitosan offers interesting properties due to its antibacterial activity and biocompatibility (doi: 10.3390/molecules26237136). In this study, chitosan-encapsulated Tb₂S₃ NPs were tested against the S. aureus bacterial strain. Whereas the antibacterial activity of the chitosan-encapsulated Tb₂S₃ NPs is lower than tetracycline (which is a standard antibiotic), it should be understood that the chitosan-encapsulated Tb₂S₃ NPs may not efficiently facilitate any electron transfer from the Tb₂S₃ to the chitosan matrix. In general, electron transfer processes are one of the important recognized mechanisms of action for the antimicrobial activity of nanoparticles (doi: 10.1016/s0748-5514(86)80040-x). Nevertheless, the formation of chitosan-encapsulated Tb₂S₃ NPs evidenced that the biopolymer is useful to passivate various metallic atoms, including REEs, on the NPs surface. Since chitosan is also rich with hydroxyl and amino groups, which cap the NPs, the biopolymer can also bind with auxiliary organic pharmaceutical compounds. Thus, the chitosan-encapsulated Tb₂S₃ QDs might be used as multifunctional drug carrier systems with multifunctional properties in future research and development efforts (doi: 10.3390/molecules26237136). 

 

Yan D, Li Y, Liu Y, Li N, Zhang X, Yan C. Antimicrobial Properties of Chitosan and Chitosan Derivatives in the Treatment of Enteric Infections. Molecules. 2021, 26(23), 7136. doi: 10.3390/molecules26237136.

Ames JR, Ryan MD, Kovacic P. Mechanism of antibacterial action: electron transfer and oxy radicals. J Free Radic Biol Med. 1986, 2(5-6), 377-91. doi: 10.1016/s0748-5514(86)80040-x.

In summary, authors wish to acknowledge the insightful and constructive comments provided by reviewer #1. The authors feel that the feedback provided has improved the quality of this contribution through the inclusion of additional results and editing. The revised manuscript was comprehensively edited for language, syntax and clarity throughout to meet the high standards of this journal.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript entitled “Nanocomposites of Terbium Sulfide Quantum Dots with a Chitosan Capping Agent for Antibacterial Applications” described the synthesis of TeS2 and the antibacterial application of TeS2/chitosan composites. The topic is interesting, but the experimental data cannot well support the conclusions. I think this paper should add more experiment and resubmit it later. The detail comments are as follows:

1.       It is better to add the TEM image of the synthesized TeS2 quantum dots to confirm its dimension.

2.       Can you explain why you fixed the pH values between 9-11. Have you synthesized the TeS2 in acid environment or have you try strong alkali environment?

3.       The authors claimed that there are some interaction between TeS2 and chitosan. Can you confirm the conclusion by different characterization?

4.       Figure 3 and Figure 4 are wrong. “SEM images of quantum dots that prepared at different pH”. The scale bar of these SEM images is 2 micrometer. It cannot observe the quantum dots at such low magnification.

5.       Chitosan is well known antibacterial material. I cannot confirm the antibacterial function of TeSe quantum dots. More experiment should be added.

6.       The inhibition zone is not clear in Fig6-8.

7.       More antibacterial mechanism should be explained. In addition, how about other properties of the composite films?

 

Author Response

Authors’ Response to Reviewer Comments on MS ID: jcs-2027284

 

 

Reviewer #2

The manuscript entitled “Nanocomposites of Terbium Sulfide Quantum Dots with a Chitosan Capping Agent for Antibacterial Applications” described the synthesis of TeS2 and the antibacterial application of TeS2/chitosan composites. The topic is interesting, but the experimental data cannot well support the conclusions. I think this paper should add more experiment and resubmit it later. The detail comments are as follows:

1. It is better to add the TEM image of the synthesized TeS2 quantum dots to confirm its dimension.

Response:

We agree with the reviewer’s comment. In this study, we could only access SEM to acquire images of chitosan-encapsulated Tb₂S₃ nanoparticles (NPs). In part, this is due mainly to the immiscible nature of this kind of NPs in any solvent. Actually, upon drying, chitosan-encapsulated Tb₂S₃ NPs forms thin film, rather than agglomeration of small particles, making more difficult for them to be dispersed in different solvents. With this problem in mind, we have analyzed the formation of the chitosan-encapsulated Tb₂S₃ NPs based on TGA, Raman spectroscopy, XPS, light scattering, SEM, EDX, and XRD analyses. Among these methods, XRD and SEM provides the most direct support for the nanoscale size range of the terbium sulfide particles. The DLS results and zeta-potential support that the terbium sulfide is complexed within the chitosan matrix, in agreement with the TGA, XPS, and Raman results. The particle size estimates of chitosan and the chitosan-encapsulated Tb₂S₃ NPs provide support of the formation of chitosan composites that contain Tb₂S₃ NPs, as described in the revised manuscript.

 

2. Can you explain why you fixed the pH values between 9-11. Have you synthesized the TeS2 in acid environment or have you try strong alkali environment?

Response:

We thank the reviewer for this comment. In the one-pot synthesis method, NPs is normally formed in alkali environment, though in some cases the reactions can undergo at ambient pH. The reason is to ensure the formation of metal sulfide salts, which are expected to agglomerate and form NPs, and to avoid the formation of hydrogen sulfide. On the other hand, pH of the solution should be lower than 12 (or even lower), as for the case of the alkali environments for the metal ions that have a tendency to form hydroxide salts. Herein, the pH was 9 to 11, where the most favourable composite was obtained at pH 10.

3. The authors claimed that there are some interaction between TeS2 and chitosan. Can you confirm the conclusion by different characterization?

Response:

We thank the reviewer for their insightful comment. Interaction between capping agent and metal on the QDs surface could be demonstrated using several techniques. The simplest technique is vibrational spectroscopy, such as infrared or Raman spectral analysis. The IR spectra shown in Fig. are dominated by the chitosan spectral signatures due to the greater mole fraction of chitosan over Tb(III) [38: 1 mole ratio of chitosan monomer: Tb(III)], which limits detection of the metal sulfide fraction, along with spectral overlap. We have obtained Raman spectra of chitosan and that for the CS-Tb₂S₃ nanocomposite, as shown in the figure below. There is evidence spectral intensity variation in the following regions: 2900 cm^-1, 1200-1500 cm^-1, 800-1200 cm^-1, and 300-600 cm^-1. While the wavenumber values do not show significant change, the spectral intensities are more pronounced, which suggests that the metal sulfide nanoparticles interact with the surface functional groups of chitosan (-OH, -NH₂) through various donor-acceptor interactions. Evidence for the potential chelation of the –COH groups of chitosan with terbium sulfide finds support in the XPS results (cf. Fig. S5 & S13, Supplementary Material). The IR results of Lim et al. reveal that the broad band at 420–811 cm^-1 related to CdS vibrations which showed prominent peaks at 601 and 720 cm^-1 , where the arrangement of the biopolymer chains of chitin was generally unchanged upon encapsulation CdS nanoparticles by chitin. A more detailed analysis of the Raman spectrum was precluded due to the large fluorescence background emission from the CS-Tb₂S₃ nanocomposite sample. Finally, the XPS spectrum of the CS-Tb₂S₃ nanocomposite was obtained by mounting the sample onto carbon tape prior to analysis. The XPS results provide further support for the incorporation of terbium sulfide within the matrix of chitosan as the capping agent.  Thus, Fig. 2 (now Scheme 1) was revised and the caption highlights that the mode of interactions are meant to be illustrative rather than specific in nature.

4. Figure 3 and Figure 4 are wrong. “SEM images of quantum dots that prepared at different pH”. The scale bar of these SEM images is 2 micrometer. It cannot observe the quantum dots at such low magnification.

Response:

We agree with the reviewer’s comment. As indicated in Q#2, we could not acquire any TEM images due to lack of access to such facilities. Therefore, SEM was used to characterize the surface morphology of the chitosan fraction of the nanocomposite that serves to encapsulate the Tb₂S₃ nanoparticles. While SEM cannot resolve the nanoscale NPs, the SEM features, it can support the role of a layered composite between chitosan and the Tb₂S₃ nanoparticles. Additional evidence for the composite formation was obtained from TGA, Raman, XPS, DLS, and zeta-potential results, conceptually illustrated in Scheme 1.

5. Chitosan is well known antibacterial material. I cannot confirm the antibacterial function of TeSe quantum dots. More experiment should be added.

Response:

We agreed with the reviewer’s comment. While the antibacterial efficacy of chitosan is known, its role is considered secondary in nature or questionable relative to terbium sulfide. Various studies reveal that it was found to have no antibacterial activities against Gram positive and Gram negative bacteria. The research studies show that the antibacterial activity of chitosan were performed at elevated concentration (ca. mg/mL range) which could be prepared at slightly acidic conditions. In this case, we have evaluated carefully the antibacterial activity of chitosan against Gram positive and Gram negative bacteria, and we found that the minimum inhibition concentration (MIC) was near 1 mg/mL. Note that the composite was prepared under alkaline conditions at pH values above the pK_a of chitosan to yield a solid phase nanocomposite, which may impede electron transfer processes that attenuate the production of reactive oxygen species. It should be noted that the antibacterial properties of metal sulfide, metal oxide materials display peroxidase-like enzyme activity, which has been outlined in a recent review (https://doi.org/10.3390/antibiotics11030390). However, we contend that the thickness of the capping agent and accessibility of the metal sulfide interface may require further optimization in order to enhance the electron transfer mechanism to effect enhanced antimicrobial activity for this system.

6. The inhibition zone is not clear in Fig6-8.

Response:

We agreed with the reviewer’s comment. We have increased the resolution of the images shown in Fig. 6-8.

7. More antibacterial mechanism should be explained. In addition, how about other properties of the composite films?

Response:

We understand that the mechanism of action of antibacterial activity is still an open question but several established mechanisms were outlined in the introduction with appropriate leading citations. Based on literature survey and our experience, we believe that the antibacterial activity should depend on the electron transfer process and the polarity of compounds, which could interact and destroy the lipid membrane bacterial structure, leading to the cell death. In some cases, small nanoparticles can directly penetrate the cell wall and cause cell death. In this study, we infer that the antibacterial properties of nanoparticles is affected by capping substances, along with the nanoparticle size, and the surface accessibility of the metal sulfide nanoparticle. Chitosan has the capability to facilitate electron transfer, mainly at slightly acidic conditions, where protonation of the amine group occurs. The use of chitosan offers stabilization of the metal nanoparticles as the capping agent but it may also introduce steric effects that influence the surface accessibility between the bacteria and metal sulfide interface. These factors will be further explored as part of future planned studies.  

In summary, authors wish to acknowledge the insightful and constructive comments provided by reviewer #2. The authors feel that the feedback provided has served to improve the quality of this contribution. The revised manuscript was further edited by including extra experimental results. As well, comprehensive editing for language, syntax and clarity was made throughout to meet the high standards of this journal.

Author Response File: Author Response.docx

Reviewer 3 Report

The study from "Nanocomposites of Terbium Sulfide Quantum Dots with a Chitosan 2 Capping Agent for Antibacterial Applications" shows an interesting concept for creating an antibacterial layer by the use of a simple method using chitosan coated Tb2S3 Quantum dots. 

However, the paper has some serious shortcomings that cannot be ignored and therefore I recommend the rejection of the paper in the current form. 

A first and general problem of the article is that the section on the results does not have any description of the data and how it was treated and interpreted. It is just a collection of figures and tables with a too short caption.

1.) The authors present "quantum dots", however they do not show them. The only things that are shown are SEM images that show some porous structure. They should have used TEM to show the particles. If there would be quantum dots inside the structure the resolution to see and analyse them would not be sufficient. Why did they not show how pristine Quantum dots look like? Instead they used XRD which is fine for assesing the crystaline structure of the QDs, but not the size. If so please cite the method, how this is done. Also it is not clear how they made the image analysis, which particles structures were counted? 

2.) The loading of "QDs" to the contact lense was analysed by UV absorption at ca. 210 nm, and not by their fluorescence properties. The authors cite Zhang et al. 2021 for reference , but Zhang et al. investigated Tb complexes and not "Tb2S3" and I see no evidence that the photonic properties of these materials are similar. Also analysing the fluorescence of the QDs would have been more appropiate, but maybe also insufficient. In addition, also the contact lenses, which are made of some type of plastic could absorb and the differences may have also influenced the result.

3.) The material suggested to be used with a product that is in contact with human tissue (the eye), but there is no discussion about the toxicity of the material (Tb2S3), only about chitosan. In addition, the athors should discuss, if there is any risk originating from "Nanosize". There is a huge discussion about the safety of nanometrials (e.g. doi: 10.1021/acs.chemrestox.9b00519). 

4.) The authors should add to the introduction also alternatives for bactericidic agents, that are used today and why they believe that the Tb2S3 QDs are better suited. 

Also the most important paragraph (L281-295) is not very well writen and it was not clear, if the method did work or not. 

 

Author Response

Authors’ Response to Reviewer Comments on MS ID: jcs-2027284

 

 

Reviewer #3

The study from "Nanocomposites of Terbium Sulfide Quantum Dots with a Chitosan 2 Capping Agent for Antibacterial Applications" shows an interesting concept for creating an antibacterial layer by the use of a simple method using chitosan coated Tb2S3 Quantum dots. 

However, the paper has some serious shortcomings that cannot be ignored and therefore I recommend the rejection of the paper in the current form. 

A first and general problem of the article is that the section on the results does not have any description of the data and how it was treated and interpreted. It is just a collection of figures and tables with a too short caption.

• The authors present "quantum dots", however they do not show them. The only things that are shown are SEM images that show some porous structure. They should have used TEM to show the particles. If there would be quantum dots inside the structure the resolution to see and analyse them would not be sufficient. Why did they not show how pristine Quantum dots look like? Instead they used XRD which is fine for assesing the crystaline structure of the QDs, but not the size. If so please cite the method, how this is done. Also it is not clear how they made the image analysis, which particles structures were counted? 

Response:

Due to the inability to obtain access to TEM facilities, the results presented are reliant on SEM and other complementary methods. For example, DLS particle size analysis, zeta-potential, Raman, XPS, and TGA results were included to support the characterization of the nanoparticles and the formation of nanocomposites between chitosan and teribium sulfide. For the case of SEM results, Image J software was used to analyze the size of the terbium sulfide domains at higher magnification, where various regions of the sample (n=75) were averaged to obtain the particle size estimates in the revised manuscript. The data was reported as a histogram to determine the particle size distribution formed using “Origin” software to yield the histogram, where the average nanoparticle sizes for  nanoparticles at pH 9, 10 and 11 are 57.8 nm; 74.9 nm and 114.6 nm.

We agree with the reviewer concerning the limitations of XRD for analysis of the nanoparticle size due to the bias toward the crystalline domains of the nanoparticles. The restricted access to TEM led us to examine particle size using dynamic light scattering as a complementary tool to further address the reviewer concern.

Differences in measurement results using FESEM and XRD are due to differences in the respective measurement methods and the ability to measure all fractions of terbium sulfide (amorphous and crystalline domains). The Scherrer equation uses XRD line width analysis to estimate the actual crystal grain size since the order of X-ray waves can be diffracted by the distance between atoms in the crystalline domains. Meanwhile, the measurement uses FESEM because it uses the scanning electron principle only to reveal the surface morphology so that the visible particles are not restricted to the crystal grains solely.

 

• The loading of "QDs" to the contact lense was analysed by UV absorption at ca. 210 nm, and not by their fluorescence properties. The authors cite Zhang et al. 2021 for reference , but Zhang et al. investigated Tb complexes and not "Tb2S3" and I see no evidence that the photonic properties of these materials are similar. Also analysing the fluorescence of the QDs would have been more appropiate, but maybe also insufficient. In addition, also the contact lenses, which are made of some type of plastic could absorb and the differences may have also influenced the result.

Response: Raman spectral analysis of the nanoparticles (NPs) was attempted; however, a relatively high fluorescence background was noted, where the spectral features are dominated by the capping agent. However, evidence of Raman spectral intensity changes are noted which may suggest interaction of the polar functional groups (-NH, -OH) of chitosan with the terbium sulfide, which is further supported by XPS results.  The high background fluorescence emission precluded a detailed analysis of the vibrational spectra. The study of uncapped nanoparticles presents a challenge in terms of controlling the nanoparticle size during synthesis and potential aggregation effects. The major component of the nanocomposite is chitosan (~95%) which presents challenges for study of the vibrational spectra (IR and Raman) of the materials since this is approaching the limits of detection for sensitivity. For the case of IR results for composites containing CdS with chitin, the loading levels were twice higher than that reported by Lim et al. (2021) than the loading levels used herein.  The results obtained from light scattering, zeta potential, XPS and TGA for the nanoparticle systems indicate that Tb₂S₃ are dispersed within the chitosan matrix, along with their nanoparticle size range.

 

• The material suggested to be used with a product that is in contact with human tissue (the eye), but there is no discussion about the toxicity of the material (Tb2S3), only about chitosan. In addition, the athors should discuss, if there is any risk originating from "Nanosize". There is a huge discussion about the safety of nanomatrials (e.g. doi: 10.1021/acs.chemrestox.9b00519).

 

 

Response:

Thank you to the reviewer for this insightful comment. Based on the other research using terbium (Tb) compound, the toxicity of the terbium compound such as in the Tb(NO₃)₃(OH₂)₃.(18C6) compound is mild. The cytotoxicity and genotoxicity studies indicated that the Tb(NO₃)₃(OH₂)₃.(18C6) complex and its Tb(NO₃)₃.6H₂O salt have excellent antiamoebic activity with very low IC50 values are 7 and 2.6 μg/mL, respectively, with significant decrease (p < 0.05) in Acanthamoeba viability when the concentration was increased from 0 to 30 μg/mL. The mode of cell death in Acanthamoeba cells following treatment with the Tb complex was apoptosis (Sarawat et al., 2019; Dengler et al, 2010; Xinming et al,, 2008). This is in contrast to the Tb(NO₃)₃.6H₂O salt treated Acanthamoeba, which exhibited necrotic type cells. The percentage of DNA damage following treatment with all the compounds at the IC25 values showed high percentage of type 1 with the % nuclei damage are 14.15 ± 2.4; 46.00 ± 4.2; and 36.36 ± 2.4, respectively for untreated, treated with Tb complex and Tb salt (Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 153 (2016) 714–721).

Sarawat, S., Stapleton, F., Willco, M. & Roy, M. (2019). Quantum dots in Ophthalmology: A literature review. Current Eye Research.

Dengler, M., Saatchi, K., Dailey, J. P., Matsubara, J., Mikelberg, F. S., Hafeli, U. O., Zborowski, M. (2010). Targeted Delivery of Magnetic Cobalt Nanoparticles to the Eye Following Systemic Administration.

Xinming, L., Yingde, C., Lloyd, A. W., Mikhalovsky, S. V., Sandeman, S. R., Howel, C. A. & Liewen, L. (2008). Polymeric hydrogels for novel contact lens-based ophthalmic drug delivery systems: A review. Contact Lens and Anterior Eye, 31(2), 57-64.

 

Since the physicochemical and biological properties of nanomaterials vary with the size, composition, morphology of the particles. The structure-function relationships can be altered according the size, shape, surface chemical properties, where these factors are known to impact the biological safety and toxicology, especially for clinical applications. Thus, further attention and efforts should be directed at such nanoparticle systems in future work. Further details on the toxicity and clinical applications of related metal sulfides are outlined in a recent review (https://doi.org/10.3390/antibiotics11030390). Herein, this work contributes to the field by demonstrating the potential utility of terbium sulfide nanoparticles as potential antibacterial agents, where the efficacy of such systems is anticipated to have additional promise through further optimization of these systems.

 

 

• The authors should add to the introduction also alternatives for bactericidic agents, that are used today and why they believe that the Tb2S3 QDs are better suited. 

Response:

As an antibacterial strategy, the ultimate goal of nanomaterials such as nanoparticle systems relate to their in vivo application and clinical transformation. Various antimicrobial agents act by interfering with (1) cell wall synthesis, (2) plasma membrane integrity, (3) nucleic acid synthesis, (4) ribosomal function, and (5) folate synthesis.

By comparison with conventional antipbiotics, nanomaterials have biocatalytic properties such as peroxidase-like activity are reported to disrupt biofilms but lack a stabilizing coating that is required for clinical applications. Iron–oxide nanoparticles coated with dextran reveal (ACS Nano 2019, 13,4960–4971) strong peroxidase-like activity at acidic pH values that target biofilms with high specificity, and prevented severe caries without impacting surrounding oral tissues in vivo.

Recently, Yin et al. (ACS Nano 2016, 10, 11000–11011) reported that PEG functionalized molybdenum sulfide nanoflowers (PEG-MoS2 NFs) with peroxidase-like catalytic activity and high photothermal

conversion efficiency in the near-infrared (NIR) region combined the catalysis with

PTT to provide rapid and efficacious antibacterial effects.

Notwithstanding current potential issues of toxicity, there is a need to carry out further studies of new biocompatible nanomaterials that possess antibacterial capabilities since such systems represent a new class of antibiotics alternatives (https://doi.org/10.3390/antibiotics11030390). Due to the unique structure-properties of these systems, these materials will serve to address urgent global issues related to antibiotic resistance of conventional antibiotics.

 

 

Also the most important paragraph (L281-295) is not very well writen and it was not clear, if the method did work or not. 

Response:

The suggested paragraph and section was rewritten as noted in the markup version of the revised manuscript to address the reviewer comments.

 In summary, authors wish to acknowledge the insightful and constructive comments provided by reviewer #3. The authors feel that this feedback has served to improve the quality of this contribution. The revised manuscript was further edited for language, syntax and clarity throughout to meet the high standards of this journal.

Author Response File: Author Response.docx