Photothermal Bacterial Clearance Using Gold Nanoshells Grown on Chitosan Nanoparticles Dielectric Templates

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Summary

This research article is about the development of gold nanoshells, grown on thiolated chitosan nanoparticles, as an approach for creating antibacterial solutions/surfaces in the context of antibiotic resistance. Results showed that chitosan nanoparticles gelation and surface modification processes to add a thiol functional group were characterized. Optimal synthesis condition resulted in nanoparticles of ~215 nm in diameter and a Zeta potential of ~+16 mV. Optimal chitosan nanoparticles where further used as core to grow a gold shell where thiol groups acted as nucleation sites. Then the laser-mediated photothermal effect of the gold nanoshells was determined and modeled. Finally, the gold nanoshells were used in combination with laser irradiation on E. coli and S. aureus bacteria. Data showed that nanoshells alone had a strong effect on E. coli viability. The bactericidal effect was further amplified by photothermal effect. Conversely, gold nanoshells did not impact significantly the viability of S. aureus, which was explained by the thick peptidoglycan layer of the gram + bacteria. The article concludes that gold nanoshells show favorable photothermal properties with potential antibacterial applications but required further optimization, especially for gram+ bacteria.

General appreciation

The article is well-written overall. The experimental approach is adequate but needs more detail in the Materials and Methods section (see specific comments below). The results are convincing but would benefit, in some case, from statistical analyses (see specific comments below). While the experimental work is rigorous, the novelty of the study also needs to be further discussed. For instance, the idea of thiolated chitosan as a core template for gold nanoshells is not new (see PMID: 31521288). In addition, the concept of using photothermal treatment with gold NPs, nanoshells, nanorods, etc. to treat bacterial infection or to be used as antibacterial applications has been already demonstrated in many other studies with, in some case, higher antibacterial efficiency for S. aureus (e.g. PMID: 25834427, PMID: 35500384, PMID: 31337206, https://doi.org/10.1021/acsanm.5c00324). Hence, the authors are strongly encouraged to discuss the novelty of their work compared to other similar studies. Additionally, it is unclear why the authors chose to irradiate a liquid solution rather than a surface coated with their gold nanoshells, as performed in many similar studies. Testing on a coated surface would have been more convincing and easier to control (e.g. energy density, surface, etc.), as the phototreatment of a liquid solution can be influenced by the container's thickness and material, the duration and surface area of the irradiation, and the absorption of energy by the surrounding liquid. The experimental setup described in this study lacks clarity on how the irradiation was performed and the anticipated penetration depth of the irradiation within the liquid solution containing gold nanoshells and bacteria.

Specific comments

Introduction

• Lines 55-56: replace “no effective action is taken” with “no effective actions are taken”.
• Line 88: This is the first time the acronym GNSs is introduced in the main text. Please, define properly GNSs and precise the difference with GNPs.

Results

• Figure 1: FTIR-ATR spectra recorded for TCS is not really convincing in showing the thiol functional group (2548 cm^-1). Authors are encouraged to confirm their observations with another experimental approach such as a thiol quantification kit.
• Figure and Table captions should also include the number of replicates performed.
• Table 1: Typically, zeta potential values below 30 indicate incipient instability, where NPs are unstable and can aggregate. Please discuss this point. It would also be relevant to present a representative DLS size distribution curve for the optimal TCNPs.
• Line 153: replace “similarly” with “similar”.
• Section 2.3: This section should be discussed more with relevant and similar studies (see PMIDs listed in the “General appreciation” section of the review report).
• Section 2.3 (lines 223-224): The authors should discuss more the effect of non-irradiated TCNP@AU treatment on E. coli and compare, with statistical evidence, the impact of photothermal treatment following irradiation. The TCNP@AU alone seems to affect greatly the bacteria survival without the need for irradiation treatment.
• Figure 5: Please provide statistical data between experimental conditions. Also, data should be reported in CFU/mL instead of percentage of viability.

Materials and Methods

• In the materials section, please provide the necessary information for the following chemicals and materials used in the study:  NHS, DMF, EDAC, dialysis membranes, bacteria culture, etc.)
• Line 232: Please precise the percentage of deacetylation for the chitosan used in this study.
• Please cite the reference appropriately (see lines 238 and 270)
• Please introduce properly all the acronyms of chemicals (NHS, 3-MPA, DMF, EDAC, etc.)
• Section 3.1.1: There is no need for a subsection here. I strongly encouraged the authors to merge this paragraph with the paragraph above.
• Section 3.2 (line 265): Since centrifuge RPM is not informative without specifying the rotor's size and shape, the author should report the centrifugal force in RCF (× g) instead,
• Section 3.3 (line 282): Please precise the exclusion size and the dialysis condition.
• Section 3.4.1: Please precise how the zeta potential was measured (e.g. sample condition, solvent composition, etc.)
• Section 3.5: Please precise how the samples were prepared for ΔT profile acquisition. A figure or a schematic presenting the experimental setup and conditions would greatly help to better understand how data were obtained and how that information could be transposed to the assays with bacteria solutions.
• Section 4.5: This section would also benefit more detail information. A figure or a schematicpresenting the experimental setup and conditions would greatly help to better understand how the photothermal experiments were conducted.
• Section 4.5 (line 338): please justify the rational behind the irradiation duration chosen.
• Section 4.5 (line 338-339): Please precise the energy density (e.g. J/cm²) that was used for irradiating the bacterial solutions.

Author Response

The Authors sincerely appreciate all suggestions and recommendations provided by the Reviewers to enhance the manuscript quality. We have addressed majority of recommendations as detailed below:

Reviewer #1

Specific comments

Introduction

• Lines 55-56: replace “no effective action is taken” with “no effective actions are taken”.

Thank you for your advice. The changes was made as recommended.

• Line 88: This is the first time the acronym GNSs is introduced in the main text. Please, define properly GNSs and precise the difference with GNPs.

Thank you for this valuable observation. We have now clearly defined the acronym was gold nanoshell (GNS).

Results

• Figure 1: FTIR-ATR spectra recorded for TCS is not really convincing in showing the thiol functional group (2548 cm^-1). Authors are encouraged to confirm their observations with another experimental approach such as a thiol quantification kit.

We appreciate the suggestion to quantify thiol groups using a specific kit. Unfortunately, we could not implement this experimental approach due to logistical constraints, as the Ellman’s reagent necessary for such analysis would require approximately two month to obtain, exceeding the manuscript submission timeline. To addres your concern, we further analyzed and emphatized the amide II band in FTIR spectra, providing stronger evidence of the successful chemical modification of chitosan. The manuscript now includes:

The black line corresponds to the FTIR spectrum of native CS, which shows characteristic stretching bands centered at 3299 cm^-1, attributed to -OH and -NH₂ groups. The stretching vibrations of -CH₂- o in the pyranose ring and -CH₃ of acetyl groups of CS appeared at the range of 2950-2780 cm-¹ 10.1002/app.46459. The peak at 1661 cm^-1 corresponds to C=O stretching vibration of amide I, while  the peak at 1578 cm^-1 result from the overlapping of -NH in amide II and -NH₂ groups 10.1002/app.46459 https://doi.org/10.3389/fsufs.2022.881434  https://doi.org/10.1002/fsn3.1486. Additional bands at 1420 cm^-1 and 1058 cm^-1are correspond to the asymmetric bending vibrations of -CH₂- and stretching C-O-C of pyranose rings, respectively. Peaks at 1318 cm^-1 and 1025 cm^-1 are related to C-N stretching (amide III) and the symmetric C-O-C stretching [14], [15]. In the spectrum of TCs (blue line), increased peak intensities for amide I and amide II, -CH₂-, C=O, and amide III indicate successful chemical modification [16]. Additionally, a weak shoulder at 2548 cm^-1 confirms the presence of thiol (-SH) group [12], corroborating the successful thiolation.

• Figure and Table captions should also include the number of replicates performed.

Thank you for your advice. The number of replicates is now specified accordingly.

• Table 1: Typically, zeta potential values below 30 indicate incipient instability, where NPs are unstable and can aggregate. Please discuss this point. It would also be relevant to present a representative DLS size distribution curve for the optimal TCNPs.

We agree whit this observation and have expanded the discussion of zeta potential. The manuscript now includes the following clarification:

Interestingly, despite the relatively low zeta potential values (+14 mV) obtained for the TCNPs under optimal formulation, the nanosystem exhibited substantial colloidal stability. Literature indicates that colloidal stability can be inferred from its zeta potential: values between |30|-|20| mV suggest stability due sufficient electrostatic repulsion between nanoparticles; values between |20|-|10| mV indicate a metastable system; and values below |10| mV typically signify an unstable colloidal system prone to aggregation.  

• Line 153: replace “similarly” with “similar”.

Thank you for your suggestion. This correction has been made.

• Section 2.3: This section should be discussed more with relevant and similar studies (see PMIDs listed in the “General appreciation” section of the review report).

We greatly appreciate the bibliography and suggestion provided. A detailes comparative discussion with relevant studies has been incorporated at the end of section 2.3, discussing the photothermal effect of TCNP@Au against gram positive and Gram-negative bacteria

These results are consistent with the antibacterial activity reported for various photothermal nanosystems, such as spherical gold nanoparticles (SGNP) and gold nano rods (GNR) and GNSs. For instance, Millenbaugh et. al. evaluated the photothermal effect of SGNP, both conjugated and non-conjugated with antibodies specific to S. aureus. Interestingly, bacteria survival remained relatively high (75%) when exposed to laser irradiation in the presence of non-functionalized SGNPs, whereas antibody conjugated SGNPs exhibited a significantly stronger antibacterial effect, reducing bacterial survival to 36 % (doi 10.2147/IJN.S76150). An innovative antibacterial photothermal platform was reported by Uusitalo et. al., who immobilized GNRs on the surface of titanium and glass substrates. To evaluate the photothermal effect on the viability of S. aureus and E. coli, the GNR-substrates were placed in direct contact with bacterial cultures on solid agar culture and irradiated with NIR laser at varying irradiance levels (0-20 W/cm²). Both the GNP-titanium substrate and GNR-glass substrate significantly reduced bacterial viability. Specifically, the GNP-titanium substrate decreased S. aureus viability to 18 CFU/cm² at 10 W/cm², while the GNR-glass substrate reduced the viability of both E. coli and S. aureus to nearly undetectable levels at 20 W/cm². The enhanced activity observed in Uusitalo’s study is due to the considerably higher irradiation power applied (doi 10.1021/acsanm.5c00324). In other study, Ma et. al. reported that GNRs were capable to eliminating approximately ~99% of E. coli and 88% of  S. aureus under NIR irradiation. This high antibacterial efficacy was attributed to the strong photothermal conversion (η>60%) and the positively charged double layer of hydroxide coating GNR, which can promotes electrostatic interaction with the bacteria cell wall (10.1021/acsami.9b10373). Manivasagan et. al. investigated thiolated chitosan-wrapped gold nanoshells for their photothermal antibacterial effect against S. aureus. They report a negligible bacterial viability when cells were treated with 115 μg/mL of chitosan-wrapped GNSs and subjected to NIR irradiation for just 5 min (doi: 10.1016/j.carbpol.2019.115228). The enhanced antibacterial performance in their study may be attributed to the use of silica nanoparticles as templates, which enable the formation of continuous gold shell layer capable of generating temperatures exceeding 55 °C upon irradiation.

 

• Section 2.3 (lines 223-224): The authors should discuss more the effect of non-irradiated TCNP@AU treatment on E. coli and compare, with statistical evidence, the impact of photothermal treatment following irradiation. The TCNP@AU alone seems to affect greatly the bacteria survival without the need for irradiation treatment.

Thank you for your recommendation. We have included the requested statistical analysis:

Figure 5b shows that E. coli viability was significantly affected by the presence of TCNP@Au, with or without irradiation. A repeated measures one-way ANOVA followed by Tukey’s post hoc test revealed a significant reduction in viability compared to control conditions (p = 0.0250 for non-irradiated TCNP@Au and p = 0.0166 for irradiated TCNP@Au). Moreover, irradiation of TCNP@Au led to a statistically significant enhancement in the bactericidal effect compared to the non-irradiated condition (p = 0.0106). These findings confirm the intrinsic antibacterial activity of TCNP@Au and demonstrate the potentiation of its bactericidal properties via photothermal activation. The same statistical analysis was performed for S. aureus, but no statistically significant differences were observed between conditions (p > 0.05), supporting the notion that S. aureus exhibits greater tolerance to photothermal treatment under the tested parameters. The heightened susceptibility of E. coli to photothermal treatment can be attributed to the structural differences between Gram-negative and Gram-positive bacteria. Unlike S. aureus, E. coli possesses a lipid bilayer with a thin peptidoglycan layer, making it more vulnerable to thermal damage induced by TCNP@Au.

• Figure 5: Please provide statistical data between experimental conditions. Also, data should be reported in CFU/mL instead of percentage of viability.

Thank you for your valuable suggestion. Figure 5 has been modified accordingly.

Materials and Methods

• In the materials section, please provide the necessary information for the following chemicals and materials used in the study: NHS, DMF, EDAC, dialysis membranes, bacteria culture, etc.)

Thank you for your advice. The required information has been  specified clearly.

• Line 232: Please precise the percentage of deacetylation for the chitosan used in this study.

Thank you for your advice. We attended this observation.

• Please cite the reference appropriately (see lines 238 and 270)

Thank you. Citations have been revised and properly formatted.

• Please introduce properly all the acronyms of chemicals (NHS, 3-MPA, DMF, EDAC, etc.)

Thank you for your advice. Acronyms have been defined upon first mention.

• Section 3.1.1: There is no need for a subsection here. I strongly encouraged the authors to merge this paragraph with the paragraph above.

This revision has been completed as recommended

• Section 3.2 (line 265): Since centrifuge RPM is not informative without specifying the rotor's size and shape, the author should report the centrifugal force in RCF (× g) instead,

The centrifugal force in RFC (x g) has been included in the manuscript.

• Section 3.3 (line 282): Please precise the exclusion size and the dialysis condition.

Thank you for your advice. The dialysis membrane details have been added.

• Section 3.4.1: Please precise how the zeta potential was measured (e.g. sample condition, solvent composition, etc.)

Thank you for your recommendation. Detailed measurement conditions have neem described in Materials and Methos We include the following section in Material and Methods.

3.4.1. Nanoparticles hydrodynamic size (D_H) and zeta potential (ζP)

The hydrodynamic diameter (D_H) and zeta potential (ζP) of the nanoparticles were determined using a Zetasizer Nano ZS (Malvern instruments, UK) equipped with a 633 nm red laser (He-Ne, 4mW). For dynamic light scattering (DLS), 0.6 mL of each sample was placed into disposable square cuvette. For the zeta potential measurements 1.2 mL of each sample was loaded in a folded capillary (U-shape) cell. All measurements were performed in triplicate at a constant temperature of 25 °C. Results are reported as mean ± standard deviation.

• Section 3.5: Please precise how the samples were prepared for ΔT profile acquisition. A figure or a schematic presenting the experimental setup and conditions would greatly help to better understand how data were obtained and how that information could be transposed to the assays with bacteria solutions.

Thank you for your suggestion. Relevant figure illustrating the experimental setups have been included.

 

 

Set up of the photothermal measurement assay.

• Section 4.5: This section would also benefit from more detail information. A figure or a schematic presenting the experimental setup and conditions would greatly help to better understand how the photothermal experiments were conducted.

Thank you for your suggestion. Relevant figure illustrating the experimental setups have been included.

 

 

Scheme of the photothermal effect measurements on the viability of bacteria by microdilution method. 

• Section 4.5 (line 338): please justify the rational behind the irradiation duration chosen,

Section 4.5 is a number mistake; the correct section is 3.6.

The irradiation duration of 15 minutes was chosen based on precious experimental assays, where CFU analysis indicated reduced viability after this period. Section 4.5 (line 338-339): Please precise the energy density (e.g. J/cm²) that was used for irradiating the bacterial solutions.

The energy density used (4.76 W/cm²) has been indicated in the text.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Reviewer's Comments

In this research article titled "Photothermal Bacterial Clearance Using Gold Nanoshells Grown on Chitosan Nanoparticles Dielectric Templates," the authors have developed gold nanoshells (GNSs) that are deposited onto the surface of chitosan nanoparticles to serve as a photothermal agent for treating bacterial infections.

Some of the comments below may help improve the article.

Comments

1. “In the second step, a chitosan (CS) solution (0.02% w/v) was prepared using HCl solution (0.1 M).”

Can the authors confirm whether they used 0.2% w/v of chitosan during the synthesis?

2. What was the concentration of TCNP@Au in the test tube in section 4.5 of the method section? It would be great if the author could add a schematic of the setup.
3. How much was the temperature increase of TCNP@Au without converting it into ΔT?
4. It would be interesting to see the agar plates (pictures) of TCNP@Au exposed to NIR light so we can easily distinguish between the grown colonies of E. coli and S aureus in your treated groups.
5. What was the source of the NIR laser? Please provide its details.

Author Response

We sincerely appreciate all suggestions and recommendations provided by the reviewers to enhance the manuscript quality. We have addressed all additional reviewer comments in detail as follows:

Comments

1. “In the second step, a chitosan (CS) solution (0.02% w/v) was prepared using HCl solution (0.1 M).”

Can the authors confirm whether they used 0.2% w/v of chitosan during the synthesis?

Thank you for this observation. We made a significant error in stating the concentration incorrectly. The actual concentration used was 2 % w/v. This has been corrected in the manuscript.

1. What was the concentration of TCNP@Au in the test tube in section 4.5 of the method section? It would be great if the author could add a schematic of the setup.

Thank you for highlighting this issue. We made an error in the numbering of sections in the initial version; this should correctly refer to section 3.6, and this numbering has been corrected in the current manuscript. Regarding your question, we regret not having precisely recorded the concentration of TCNP@Au used during the photothermal viability assay. However, we can provide an approximate concentration in terms of Au³⁺, assuming complete gold-shell formation from the initial gold addition. This, the TCNP@Au concentration, expressed as equivalent of Au³⁺ is approximately 10.97 mg/mL. This information, however, was not included in the main text.

A schematic setup illustrating the photothermal assay has been incorporated into the manuscript.

 

 

Set up of the photothermal measurement assay.

1. How much was the temperature increase of TCNP@Au without converting it into ΔT?

The minimum temperature (room temperature) recorded was approximately 20 °C, and the maximum temperature reached during the irradiation process was around 26 °C. Despite the seemingly modest temperature increase, the photothermal effect substantially impacted on the viability of E. coli bacteria but did not significantly affect S. aureus. Similar temperature profiles have previously been reported for gold nanoshells, gold nanospheres, gold nanostars (urquis-like shape), and gold nanorods. It is important to know that the extent of heating strongly depends on the concentration of gold nanoparticles, as well as the laser power.

https://doi.org/10.1016/j.heliyon.2025.e42738 https://doi.org/10.1021/acs.jpcc.9b03122 https://doi.org/10.1002/lsm.22072

1. It would be interesting to see the agar plates (pictures) of TCNP@Au exposed to NIR light so we can easily distinguish between the grown colonies of E. coli and S aureus in your treated groups.

Thank you for your suggestion. Images of agar plates from the bacterial cultures following treatments have been included in the relevant section of the manuscript.

1. What was the source of the NIR laser? Please provide its details.

The laser source was a diode module, and detailed specifications have been added to the manuscript text.

We thank the reviewers again for their thorough and constructive feedback.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The revised manuscript addresses most of the reviewer’s comments. However, there are still some minor elements that require attention:

• In lines 268, 278, 283 and 287: cited references are not properly formatted. Some references are cited as DOI ID instead of [##]. 
• In Figure 5C caption, please provide more information regarding the dilution of bacteria samples that were plated and the incubation conditions.
• Line 342: The dialysis exclusion size has still not been provided in the “Materials and Methods” section.

Author Response

The Authors sincerely appreciate all suggestions and recommendations provided by the Reviewer to enhance the manuscript quality. We apologize for the error in the initial round, as we mistakenly attached an incorrect draft, corresponding to one of the intermediate versions in which recommendations were progressively addressed. This draft contrasts with the final document, which incorporated all recommended corrections. We now attach the appropriate revised document. Additionally, we have carefully addressed all new suggestions provided by the Reviewer in this second round of revision, as detailed in the attached file.

The sumitted ZIP file includes the following documents:

manuscript.v3.docx: final version of manuscritp.

manuscript.v3B.docx: final version with changes highlited in yellow for reference.

raw_data: this file, as requested by the Assistant Editor, contains the raw data for UV-Vis spectroscopy, FTIR, and AFM analysis.

Author Response File: Author Response.pdf