Low-Molecular-Weight Sulfated Chitosan Microparticles Efficiently Bind HIV-1 In Vitro: Potential for Microbicide Applications

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

One of the most pressing challenges in medicine and society is developing an effective strategy to combat the human immunodeficiency virus. Therefore, the search for new low-cost, non-toxic, safe, and highly effective antiviral agents is a highly pressing issue in medicinal chemistry, pharmacology, and clinical medicine. This manuscript focuses on identifying such a system based on the natural polymer chitosan, which is the highest relevance of this study. It is particularly important to note that chitosan is a biocompatible, biodegradable, and non-toxic biopolymer. The authors developed new microparticles based on sulfated low-molecular-weight chitosan. They found that these microparticles are capable of highly effective binding and neutralizing viruses, achieving a high overall reduction in viral load. Subsequent evaluation at several tested concentrations confirmed a sustained antiviral effect, indicating that the synthesized microparticles maintain stable virus-particle interactions across the entire concentration range studied. This study clearly opens the way to the search for new antiviral systems. This is a significant and significant achievement. This is the focus of the pronounced novelty of this work. The article is written very clearly and logically in good English. It is crucial that the authors thoroughly analyze and discuss the experimental data, demonstrating their high qualifications in this field. The authors support their conclusions with appropriate references, citing numerous highly cited publications. There is complete agreement between the authors' conclusions and the data obtained. The experimental part is described in detail, which is important for reproducibility. The authors also used statistical methods to process the raw data, which is especially important in an article with a biomedical focus. I would also like to note that the article is very well illustrated. This article will be highly cited. I recommend publication after minor revision, as I kindly ask the authors to rewrite the abstract, providing it with quantitative data, and also to supplement the characterization of the starting chitosan (e.g., molecular weight distribution, degree of deacetylation).

Author Response

Response to Reviewers – Manuscript ID 4045154
Title: Low Molecular Weight Sulfated Chitosan Microparticles Efficiently Binds HIV-1 In Vitro: Potential for Microbicide Applications
Journal: Molecules

Dear Editor,

We sincerely thank you and the reviewers for your valuable comments and constructive suggestions, which have significantly improved the quality of our manuscript. We have carefully revised the manuscript in accordance with all the recommendations. Below, we provide a point-by-point response to each reviewer. All modifications have been highlighted in the revised version of the manuscript.

Reviewer 1

Comment:. One of the most pressing challenges in medicine and society is developing an effective strategy to combat the human immunodeficiency virus. Therefore, the search for new low-cost, non-toxic, safe, and highly effective antiviral agents is a highly pressing issue in medicinal chemistry, pharmacology, and clinical medicine. This manuscript focuses on identifying such a system based on the natural polymer chitosan, which is the highest relevance of this study. It is particularly important to note that chitosan is a biocompatible, biodegradable, and non-toxic biopolymer. The authors developed new microparticles based on sulfated low-molecular-weight chitosan. They found that these microparticles are capable of highly effective binding and neutralizing viruses, achieving a high overall reduction in viral load. Subsequent evaluation at several tested concentrations confirmed a sustained antiviral effect, indicating that the synthesized microparticles maintain stable virus-particle interactions across the entire concentration range studied. This study clearly opens the way to the search for new antiviral systems. This is a significant and significant achievement. This is the focus of the pronounced novelty of this work. The article is written very clearly and logically in good English. It is crucial that the authors thoroughly analyze and discuss the experimental data, demonstrating their high qualifications in this field. The authors support their conclusions with appropriate references, citing numerous highly cited publications. There is complete agreement between the authors' conclusions and the data obtained. The experimental part is described in detail, which is important for reproducibility. The authors also used statistical methods to process the raw data, which is especially important in an article with a biomedical focus. I would also like to note that the article is very well illustrated. This article will be highly cited. I recommend publication after minor revision, as I kindly ask the authors to rewrite the abstract, providing it with quantitative data, and also to supplement the characterization of the starting chitosan (e.g., molecular weight distribution, degree of deacetylation)

Response:
We thank the reviewer for the positive and encouraging feedback.
(1) We have revised the abstract to include specific quantitative data regarding the antiviral performance of the sulfated chitosan microparticles.
(2) A new sentence was added to the Materials and Methods section providing the molecular weight (MW ≈ 50 kDa) and degree of deacetylation (≈ 85%) of the starting chitosan.

We hope that the revised manuscript now meets the expectations of the reviewers and the editorial team. We greatly appreciate the opportunity to revise our work and thank you again for your time and consideration.

Sincerely,
Dr. Sergio A. Bucarey, on behalf of all authors]

Reviewer 2 Report

Comments and Suggestions for Authors

In the present manuscript, the authors investigate the antiviral activity of sulphated chitosan microparticles against HIV-1. The topic of developing antiviral therapies is of interest and merits detailed investigation. However, the novelty of the present study compared with existing knowledge appears limited. The primary difference is that sulphated chitosan is applied in the form of nanoparticles rather than as a chitosan sulphate salt.

The authors suggest that the nanoparticles exhibit improved performance compared to chitosan sulphate salt, heparin salt, and their nanoparticle formulations, as illustrated in Figure 4. However, the data are not statistically treated or compared. Consequently, it is difficult to draw a reliable conclusion regarding the superior performance of chitosan nanoparticles.

Another concern relates to the concentration dependence of the antiviral activity of the particles. The authors claim the existence of an optimal weight concentration, but there is no discussion of the possible origin of this dependence. Moreover, it is unclear whether a concentration dependence exists at all (Figure 5), as the data are not statistically analysed or correlated.

Several additional technical issues should be addressed:

• The particle size distribution is not shown. Figure 1 presents FTIR spectra rather than size characterization data.(p. 3)
• The statement “The measured diameter … optimizes effective interaction with viral particles” (page 3) is not adequately justified or supported by experimental evidence.

Overall, the manuscript requires thorough reconsideration and significant revision before it can be considered for publication.

Author Response

Response to Reviewers – Manuscript ID 4045154
Title: Low Molecular Weight Sulfated Chitosan Microparticles Efficiently Binds HIV-1 In Vitro: Potential for Microbicide Applications
Journal: Molecules

Dear Editor,

We sincerely thank you and the reviewers for your valuable comments and constructive suggestions, which have significantly improved the quality of our manuscript. We have carefully revised the manuscript in accordance with all the recommendations. Below, we provide a point-by-point response to each reviewer. All modifications have been highlighted in the revised version of the manuscript.

Reviewer 2

Comment 1:
In the present manuscript, the authors investigate the antiviral activity of sulphated chitosan microparticles against HIV-1. The topic of developing antiviral therapies is of interest and merits detailed investigation. However, the novelty of the present study compared with existing knowledge appears limited. The primary difference is that sulphated chitosan is applied in the form of nanoparticles rather than as a chitosan sulphate sal

Response:
We thank the reviewer for this important comment and for acknowledging the relevance of the topic. We respectfully clarify that the novelty of the present work does not reside in the antiviral properties of sulfated chitosan per se, which have indeed been previously reported, but rather in the formulation strategy, physicochemical engineering, and mechanistic evaluation of sulfated chitosan in a microparticulate format specifically designed to function as a biomimetic viral decoy.

To our knowledge, this is the first study to systematically investigate low molecular weight sulfated chitosan formulated as spray-dried microparticles as a physical virus-trapping platform against HIV-1. Unlike soluble sulfated chitosan or conventional heparinoid salts, the microparticulate architecture introduces several novel and functionally relevant features, including:
(i) a high-density, multivalent display of sulfate groups on a defined surface;
(ii) controlled particle size and surface charge optimized for virus sequestration; and
(iii) a mechanism of action based on physical removal of virions from biological fluids, rather than inhibition of intracellular replication steps.

Importantly, our comparative experiments demonstrate that this microparticulate formulation achieves significantly higher viral load reduction than soluble sulfated chitosan, soluble heparin, and heparin-based microparticles under identical experimental conditions. These findings indicate that the enhanced antiviral performance arises not merely from chemical sulfation, but from the synergistic combination of sulfation and particulate organization, which enables more efficient multivalent interactions with HIV-1 envelope glycoproteins.

Comment 2:
The authors suggest that the nanoparticles exhibit improved performance compared to chitosan sulphate salt, heparin salt, and their nanoparticle formulations, as illustrated in Figure 4. However, the data are not statistically treated or compared. Consequently, it is difficult to draw a reliable conclusion regarding the superior performance of chitosan nanoparticles.

Response:
We thank the reviewer for this important observation. In response, we have revised Table 1 and Figure 6 (formerly Table 2 and Figure 4) to include statistical analysis of the antiviral efficacy data. Specifically, we applied a two-tailed Student’s t-test to compare each treatment group against the untreated control.

Statistical significance (p < 0.05) was observed for the Chi-S microparticles compared to other sulfated formulations. The corresponding p-values are now included in the figure legend.

Comment 3:
Another concern relates to the concentration dependence of the antiviral activity of the particles. The authors claim the existence of an optimal weight concentration, but there is no discussion of the possible origin of this dependence. Moreover, it is unclear whether a concentration dependence exists at all (Figure 5), as the data are not statistically analysed or correlated.

Response:.

We thank the reviewer for this important observation regarding the evidence and mechanistic interpretation of the concentration-dependent antiviral activity of Chi-S microparticles.

In response, we have now strengthened the statistical treatment of our data in the revised Figure 7. Specifically, we conducted nonlinear regression analysis (quadratic fit) and two-tailed pairwise t-tests comparing each treatment group to the untreated control (n = 2, with standard error of the mean derived from estimated SD). This analysis confirmed that 33 µg/mL and 3,330 µg/mL significantly reduced viral load relative to the control group (p < 0.05), supporting the presence of a non-linear, bell-shaped dose–response curve. These updates are detailed in the revised figure legend and described in the Results section.

We acknowledge the limitation of using an estimated standard deviation of ~10% across conditions due to the small sample size (n = 2). This estimation reflects the typical technical variability observed in plasma-based inhibition assays. While it provides a useful approximation, we recognize that further replicates are necessary to more accurately characterize inter-condition variance and will address this in future studies.

Mechanistically, we have now incorporated a discussion of plausible explanations for the observed non-monotonic effect. Specifically, we propose that at low concentrations, the total number of microparticles may be insufficient to sequester a large fraction of virions. Conversely, at high concentrations, particle aggregation or surface saturation may reduce the effective binding interface or sterically hinder virus–particle interactions. This is consistent with known behaviors of sulfated polysaccharide systems and particulate formulations where stoichiometric balance and surface accessibility play key roles in target binding.

Importantly, the inclusion of SEM and DLS data reinforces this interpretation by showing a bimodal size distribution and characteristic changes in surface charge, suggesting that particle concentration modulates both aggregation behavior and electrostatic interactions, which in turn affect antiviral activity. These findings support the existence of an optimal functional window for microparticle-based viral sequestration.

Together, the revised statistical analysis, mechanistic rationale, and supporting physicochemical data strengthen our conclusion that Chi-S microparticles exert a concentration-dependent antiviral effect, with peak efficacy at intermediate doses. This insight is crucial for guiding future formulation strategies and dosage optimization

Comment 4:

 Several additional technical issues should be addressed:

• The particle size distribution is not shown. Figure 1 presents FTIR spectra rather than size characterization data.(p. 3)

Response:
We apologize for the confusion. Particle size distribution was indeed missing. We have now included a new Figure 2 showing the DLS size distribution profile of the Ch-S microparticles .

Comment 5:
The statement “The measured diameter … optimizes effective interaction with viral particles” (page 3) is not adequately justified or supported by experimental evidence.

Overall, the manuscript requires thorough reconsideration and significant revision before it can be considered for publication.

Response:
We have revised the statement and now provide supporting literature explaining how submicron particle size enhances virus decoy activity due to optimal surface-to-volume ratios and increased contact with viral particles.

We hope that the revised manuscript now meets the expectations of the reviewers and the editorial team. We greatly appreciate the opportunity to revise our work and thank you again for your time and consideration.

Sincerely,
Dr. Sergio A. Bucarey, on behalf of all authors]

Reviewer 3 Report

Comments and Suggestions for Authors

Journal Title: Molecules

Manuscript Title: Low Molecular Weight Sulfated Chitosan Microparticles Efficiently Binds HIV-1 In Vitro: Potential for Microbicide Applications

Manuscript ID: molecules-4045154

Authors: Sergio A. Bucarey et al.

The manuscript presents an in vitro study evaluating low molecular weight sulfated chitosan (LMW Ch-S) microparticles as biomimetic decoys for HIV-1 binding. The topic is timely and relevant, addressing the need for novel preventive strategies targeting early stages of viral entry. The experimental approach is sound, and the results indicate superior virus-binding performance compared to classical heparinoid materials.

There appears to be an inconsistency in the numbering of author affiliations. The affiliation numbers are not presented in a sequential or logical order which may lead to confusion. Please revise the author–affiliation mapping to ensure consistency and clarity.

The URL provided for Reference [1] is no longer accessible and leads to a “Page not found” error. Please update the reference with a valid and currently accessible link, or replace it with an alternative authoritative source (e.g., the most recent UNAIDS Global AIDS Update or WHO data) to ensure verifiability of the epidemiological data cited.

The abbreviation used for sulfated chitosan is inconsistent throughout the manuscript (both “Ch-S” and “Chi-S” are used). The authors should select a single abbreviation and revise the entire manuscript accordingly to ensure consistency and clarity.

The Introduction section is very brief and lacks sufficient background on the chemical functionalization of chitosan. A dedicated subsection describing the methods and rationale for obtaining sulfated chitosan is strongly recommended. Including such a section would provide the reader with a better understanding of the material’s chemical modification, its structure–property relationships, and its relevance for antiviral applications.

It is strongly recommended to include additional citations from recent and relevant literature to support claims regarding HIV-1 attachment mechanisms, the use of heparan sulfate mimetics, and the chemical functionalization and antiviral applications of sulfated chitosan.

The Results section lacks sufficient details regarding the synthesis of sulfated chitosan. It is highly recommended to include a chemical reaction scheme and comprehensive experimental details, such as the amounts (weights) of chitosan and sulfuric acid used, their ratios, solution concentrations, reaction time, and temperature.

The FTIR characterization section requires significant improvement. In its current form, the description is very brief, and the spectra are presented only for the 1800–700 cm⁻¹ range, while the text refers to a band at 3400 cm⁻¹. This is inconsistent and confusing. It is recommended to provide FTIR spectra covering the full scanning range to include all relevant functional groups, along with a detailed discussion of the observed bands to clearly demonstrate the successful sulfation of chitosan.

The FTIR spectra should be plotted with appropriate vertical offsets between the curves to clearly visualize differences in characteristic bands between samples. Currently, overlapping spectra make it difficult to discern key functional group signals and the effect of sulfation.

The characteristic bands corresponding to N–S bonds should be clearly identified and discussed in the sulfated chitosan sample, in comparison with the amino groups present in unmodified chitosan.

The text in the legend of Figure 1 is redundant and should be revised (same for Figure 2, for all figs). All information currently provided in the legend should be moved into the main text, as it is already described there. The figure legend should be concise, containing only essential information needed to understand the figure, without repeating details from the manuscript.

The authors should clarify the origin of the noise observed in the FTIR spectrum of the Heparin sample between 1400 and 1300 cm⁻¹. Additionally, it is not clear why the authors chose to present this particular spectrum in the manuscript.

The SEM analysis is very briefly presented and provides limited information regarding the morphology of the microparticles. In its current form, it does not convincingly demonstrate the success of chitosan sulfation. The authors should include a more detailed description of the SEM images, highlighting morphological differences between unmodified and sulfated chitosan, and discuss how these changes support the chemical modification. Including scale bars, uniform image quality, and potentially higher-magnification images could also strengthen this section. The presentation of SEM images for unmodified chitosan is a must!

The data presented in Table 1 could be further supported by including the corresponding EDS images for the analyzed samples. Displaying the EDS elemental maps alongside the table would provide visual confirmation of the elemental composition and distribution, strengthening the reliability and interpretability of the reported results.

In the section describing viral binding experiments, it is not clear what concentrations of LMW Chi-S microparticles were used. The authors should provide precise details regarding the range of concentrations tested, including units and preparation method.

Figure 4 presents the data already reported in Table 2 in graphical form. The authors should consider whether this figure is necessary, or alternatively, revise it to add additional value (highlighting trends, dose–response relationships, or statistical differences) rather than merely repeating tabular data.

In Figure 6, only the OH functional groups of chitosan are represented as being functionalized with SO₃ groups. However, it is not clear why the amino groups are not shown as sulfonated, given that treatment with sulfuric acid could also modify these groups. The authors should clarify whether the amino groups were sulfonated, and if not, explain why only hydroxyl groups were considered. A correct depiction and discussion of the functionalization is essential to accurately reflect the chemical modification of chitosan.

It is important to assess and report the degree of sulfation of chitosan in this study. Quantifying the extent of chemical modification is critical to correlate the structural changes with the observed antiviral activity, and to ensure reproducibility.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Journal Title: Molecules

Manuscript Title: Low Molecular Weight Sulfated Chitosan Microparticles Efficiently Binds HIV-1 In Vitro: Potential for Microbicide Applications

Manuscript ID: molecules-4045154

Authors: Sergio A. Bucarey et al.

The manuscript presents an in vitro study evaluating low molecular weight sulfated chitosan (LMW Ch-S) microparticles as biomimetic decoys for HIV-1 binding. The topic is timely and relevant, addressing the need for novel preventive strategies targeting early stages of viral entry. The experimental approach is sound, and the results indicate superior virus-binding performance compared to classical heparinoid materials.

There appears to be an inconsistency in the numbering of author affiliations. The affiliation numbers are not presented in a sequential or logical order which may lead to confusion. Please revise the author–affiliation mapping to ensure consistency and clarity.

The URL provided for Reference [1] is no longer accessible and leads to a “Page not found” error. Please update the reference with a valid and currently accessible link, or replace it with an alternative authoritative source (e.g., the most recent UNAIDS Global AIDS Update or WHO data) to ensure verifiability of the epidemiological data cited.

The abbreviation used for sulfated chitosan is inconsistent throughout the manuscript (both “Ch-S” and “Chi-S” are used). The authors should select a single abbreviation and revise the entire manuscript accordingly to ensure consistency and clarity.

The Introduction section is very brief and lacks sufficient background on the chemical functionalization of chitosan. A dedicated subsection describing the methods and rationale for obtaining sulfated chitosan is strongly recommended. Including such a section would provide the reader with a better understanding of the material’s chemical modification, its structure–property relationships, and its relevance for antiviral applications.

It is strongly recommended to include additional citations from recent and relevant literature to support claims regarding HIV-1 attachment mechanisms, the use of heparan sulfate mimetics, and the chemical functionalization and antiviral applications of sulfated chitosan.

The Results section lacks sufficient details regarding the synthesis of sulfated chitosan. It is highly recommended to include a chemical reaction scheme and comprehensive experimental details, such as the amounts (weights) of chitosan and sulfuric acid used, their ratios, solution concentrations, reaction time, and temperature.

The FTIR characterization section requires significant improvement. In its current form, the description is very brief, and the spectra are presented only for the 1800–700 cm⁻¹ range, while the text refers to a band at 3400 cm⁻¹. This is inconsistent and confusing. It is recommended to provide FTIR spectra covering the full scanning range to include all relevant functional groups, along with a detailed discussion of the observed bands to clearly demonstrate the successful sulfation of chitosan.

The FTIR spectra should be plotted with appropriate vertical offsets between the curves to clearly visualize differences in characteristic bands between samples. Currently, overlapping spectra make it difficult to discern key functional group signals and the effect of sulfation.

The characteristic bands corresponding to N–S bonds should be clearly identified and discussed in the sulfated chitosan sample, in comparison with the amino groups present in unmodified chitosan.

The text in the legend of Figure 1 is redundant and should be revised (same for Figure 2, for all figs). All information currently provided in the legend should be moved into the main text, as it is already described there. The figure legend should be concise, containing only essential information needed to understand the figure, without repeating details from the manuscript.

The authors should clarify the origin of the noise observed in the FTIR spectrum of the Heparin sample between 1400 and 1300 cm⁻¹. Additionally, it is not clear why the authors chose to present this particular spectrum in the manuscript.

The SEM analysis is very briefly presented and provides limited information regarding the morphology of the microparticles. In its current form, it does not convincingly demonstrate the success of chitosan sulfation. The authors should include a more detailed description of the SEM images, highlighting morphological differences between unmodified and sulfated chitosan, and discuss how these changes support the chemical modification. Including scale bars, uniform image quality, and potentially higher-magnification images could also strengthen this section. The presentation of SEM images for unmodified chitosan is a must!

The data presented in Table 1 could be further supported by including the corresponding EDS images for the analyzed samples. Displaying the EDS elemental maps alongside the table would provide visual confirmation of the elemental composition and distribution, strengthening the reliability and interpretability of the reported results.

In the section describing viral binding experiments, it is not clear what concentrations of LMW Chi-S microparticles were used. The authors should provide precise details regarding the range of concentrations tested, including units and preparation method.

Figure 4 presents the data already reported in Table 2 in graphical form. The authors should consider whether this figure is necessary, or alternatively, revise it to add additional value (highlighting trends, dose–response relationships, or statistical differences) rather than merely repeating tabular data.

In Figure 6, only the OH functional groups of chitosan are represented as being functionalized with SO₃ groups. However, it is not clear why the amino groups are not shown as sulfonated, given that treatment with sulfuric acid could also modify these groups. The authors should clarify whether the amino groups were sulfonated, and if not, explain why only hydroxyl groups were considered. A correct depiction and discussion of the functionalization is essential to accurately reflect the chemical modification of chitosan.

It is important to assess and report the degree of sulfation of chitosan in this study. Quantifying the extent of chemical modification is critical to correlate the structural changes with the observed antiviral activity, and to ensure reproducibility.

Author Response

Response to Reviewers – Manuscript ID 4045154
Title: Low Molecular Weight Sulfated Chitosan Microparticles Efficiently Binds HIV-1 In Vitro: Potential for Microbicide Applications
Journal: Molecules

Dear Editor,

We sincerely thank you and the reviewers for your valuable comments and constructive suggestions, which have significantly improved the quality of our manuscript. We have carefully revised the manuscript in accordance with all the recommendations. Below, we provide a point-by-point response to each reviewer. All modifications have been highlighted in the revised version of the manuscript.

Reviewer 3

Comment 1:
There appears to be an inconsistency in the numbering of author affiliations. The affiliation numbers are not presented in a sequential or logical order which may lead to confusion. Please revise the author–affiliation mapping to ensure consistency and clarity

Response:
This has been corrected. All affiliations now follow a logical and sequential order.

Comment 2:
The URL provided for Reference [1] is no longer accessible and leads to a “Page not found” error. Please update the reference with a valid and currently accessible link, or replace it with an alternative authoritative source (e.g., the most recent UNAIDS Global AIDS Update or WHO data) to ensure verifiability of the epidemiological data cited.

Response:
We have updated Reference [1] to cite the UNAIDS 2025 Global AIDS Update, using a stable and verifiable link. UNAIDS. (2025). 2025 Global AIDS Update: The Path That Ends AIDS (Summary). Joint United Nations Programme on HIV/AIDS (UNAIDS). https://www.unaids.org/en/resources/documents/2025/2025-global-aids-update

Comment 3:
The abbreviation used for sulfated chitosan is inconsistent throughout the manuscript (both “Ch-S” and “Chi-S” are used). The authors should select a single abbreviation and revise the entire manuscript accordingly to ensure consistency and clarity.

Response:
Corrected. We now uniformly use “Chi-S” throughout the manuscript.

Comment 4:
It is strongly recommended to include additional citations from recent and relevant literature to support claims regarding HIV-1 attachment mechanisms, the use of heparan sulfate mimetics, and the chemical functionalization and antiviral applications of sulfated chitosan.

Response:
We thank the reviewer for this valuable observation. In response, we have substantially revised the introduction to provide a more comprehensive historical and mechanistic context regarding the antiviral use of sulfated chitosan. Specifically, we now highlight the seminal study by Nashimura et al. (1998), which first demonstrated that sulfated chitosan could inhibit HIV-1 replication in vitro by interfering with viral adsorption to host cells via electrostatic interactions. We also cite the follow-up study by Artan et al. (2003), which confirmed that the degree of sulfation and molecular weight are critical determinants of anti-HIV activity. These foundational works provide a scientific basis for our current strategy. Furthermore, we now emphasize the structural and functional mimicry of heparan sulfate proteoglycans (HSPGs) by sulfated chitosan, and how this justifies the design of sulfated chitosan microparticles (Chi-S MPs) as virus-trapping decoys. These revisions appear on page 2, paragraph 2, lines 68-94 of the updated manuscript.
We have expanded the Introduction to include a new paragraph summarizing the generations of chitosan derivatives, the significance of sulfation, and structure–activity relationships ( Revuelta et al., 2021).

Comment 7:
The FTIR characterization section requires significant improvement. In its current form, the description is very brief, and the spectra are presented only for the 1800–700 cm⁻¹ range, while the text refers to a band at 3400 cm⁻¹. This is inconsistent and confusing. It is recommended to provide FTIR spectra covering the full scanning range to include all relevant functional groups, along with a detailed discussion of the observed bands to clearly demonstrate the successful sulfation of chitosan.

The FTIR spectra should be plotted with appropriate vertical offsets between the curves to clearly visualize differences in characteristic bands between samples. Currently, overlapping spectra make it difficult to discern key functional group signals and the effect of sulfation

The characteristic bands corresponding to N–S bonds should be clearly identified and discussed in the sulfated chitosan sample, in comparison with the amino groups present in unmodified chitosan

Response:
We have revised the FTIR section and replaced Figure 1 with an improved version showing full-range spectra (4000–500 cm⁻¹) with vertical offsets and annotated sulfate-associated bands (Now Figure 4). A narrative paragraph describes band assignments and comparison with heparin.
These bands are now clearly annotated and discussed in the FTIR section (Figure 4, updated). N–S vibration near 1250 cm⁻¹ and C–O–S linkage at ~820 cm⁻¹ are highlighted as evidence of sulfation.

Comment 9:
The text in the legend of Figure 1 is redundant and should be revised (same for Figure 2, for all figs). All information currently provided in the legend should be moved into the main text, as it is already described there. The figure legend should be concise, containing only essential information needed to understand the figure, without repeating details from the manuscript.

Response:
All figure legends have been shortened to contain only essential descriptions. Detailed information was moved into the main text.

Comment 10:
The authors should clarify the origin of the noise observed in the FTIR spectrum of the Heparin sample between 1400 and 1300 cm⁻¹. Additionally, it is not clear why the authors chose to present this particular spectrum in the manuscript.

Response:
We now explain that the noise arises from the lyophilized nature of pharmaceutical-grade heparin, and retained the spectrum to serve as a benchmark for sulfate-specific peaks.

Comment 11:
The SEM analysis is very briefly presented and provides limited information regarding the morphology of the microparticles. In its current form, it does not convincingly demonstrate the success of chitosan sulfation. The authors should include a more detailed description of the SEM images, highlighting morphological differences between unmodified and sulfated chitosan, and discuss how these changes support the chemical modification. Including scale bars, uniform image quality, and potentially higher-magnification images could also strengthen this section. The presentation of SEM images for unmodified chitosan is a must!

Response:
We have improved SEM section with new micrographs comparing native and sulfated chitosan, highlighting surface roughness and aggregation patterns. Scale bars and higher-magnification images are now included (Figure 3).

Comment 12:
The data presented in Table 1 could be further supported by including the corresponding EDS images for the analyzed samples. Displaying the EDS elemental maps alongside the table would provide visual confirmation of the elemental composition and distribution, strengthening the reliability and interpretability of the reported results.

Response:
We thank the reviewer for the constructive suggestion. In response, we have incorporated a new multipanel figure (now Figure 1) that complements the elemental quantification previously shown in Table 1. The updated figure includes:

(A) EDS spectrum confirming the presence of carbon (C), oxygen (O), nitrogen (N), and sulfur (S), with well-defined peaks corresponding to each element.

(B) SEM image of the analyzed microparticle overlaid with the region selected for elemental mapping, providing spatial context for the EDS analysis.

(C) Elemental distribution maps for C (red), O (green), N (blue), and S (magenta), revealing homogeneous incorporation of the four elements throughout the microparticle surface.

(D) Quantitative EDS table summarizing elemental weight percentages and standard deviations: C (44.4 ± 0.9%), O (41.4 ± 0.9%), N (9.7 ± 1.3%), and S (4.6 ± 0.1%).

These additions provide both qualitative and quantitative confirmation of sulfur incorporation into the chitosan matrix, which is a key chemical feature of the sulfated chitosan (Chi-S) microparticles. The consistent presence of nitrogen further supports the chitosan backbone structure, while oxygen and carbon distributions are consistent with the polysaccharide composition.

Altogether, the updated figure and associated legend enhance the clarity, traceability, and interpretability of the elemental composition results. A descriptive paragraph referencing this figure has also been added to the Results section to guide the reader and highlight the relevance of the EDS data to the characterization of the Chi-S microparticles

Comment 13:
In the section describing viral binding experiments, it is not clear what concentrations of LMW Chi-S microparticles were used. The authors should provide precise details regarding the range of concentrations tested, including units and preparation method

Response:
We have added specific concentration values, preparation methods, and units in the Methods section.

Virus Binding Assay
To evaluate the viral sequestration capacity of the different sulfated polymer formulations, a standardized polymer–virus binding assay was performed. A total of 250 µL of HIV-1–positive plasma (3.5 × 10⁶ copies/mL; log 6.54) was mixed with 750 µL of each polymer suspension prepared at 10 mg/mL. The polymers tested included:
(1) heparin salt,
(2) heparin microparticles,
(3) sulfated chitosan salt (Chi-S salt), and
(4) low-molecular-weight sulfated chitosan microparticles (LMW Chi-S MPs) synthesized and characterized as described above

Comment 14:
Figure 4 presents the data already reported in Table 2 in graphical form. The authors should consider whether this figure is necessary, or alternatively, revise it to add additional value (highlighting trends, dose–response relationships, or statistical differences) rather than merely repeating tabular data

Response:
We appreciate the reviewer’s suggestion to reassess the contribution of Figure 4. In response, we have carefully revised both Table 1 (formerly Table 2) and Figure 6 (formerly Figure 4) to ensure that each conveys distinct but complementary information, thereby justifying the inclusion of both.

Table 1 provides detailed quantitative values of viral load in both absolute terms (copies/mL) and log₁₀-transformed values, alongside the percentage of viral load reduction and associated standard deviation for each treatment. This tabular format is essential for numerical transparency and is particularly useful for readers who wish to extract precise values or perform secondary analyses.

Figure 6 has been substantially improved to go beyond simply reproducing these values. It now incorporates:

Error bars to visually represent the variability across replicates.

Statistical annotations (asterisks) to indicate significant differences relative to the untreated control and between formulations, based on two-tailed Student’s t-tests.

Improved visual hierarchy and layout to emphasize the dose-dependent and formulation-specific trends in antiviral activity.

This combination allows the reader to both visually grasp the trends and statistical significance (via the figure) and access precise numerical detail (via the table). Importantly, this dual presentation is in line with standard scientific reporting practices, particularly in biological and pharmacological studies where visual representation of efficacy and statistical comparisons are critical for interpretability.

We believe this revision now adds clear value and addresses the reviewer’s concern regarding redundancy

Comment 15:
In Figure 6, only the OH functional groups of chitosan are represented as being functionalized with SO₃ groups. However, it is not clear why the amino groups are not shown as sulfonated, given that treatment with sulfuric acid could also modify these groups. The authors should clarify whether the amino groups were sulfonated, and if not, explain why only hydroxyl groups were considered. A correct depiction and discussion of the functionalization is essential to accurately reflect the chemical modification of chitosan

Response:
We have revised Figure 6 to depict both –OH and –NH₂ groups as potential sulfation sites, and clarified in the text that primary hydroxyls are predominantly sulfated under our reaction conditions, though minor N-sulfation may occur.

Comment 16:
It is important to assess and report the degree of sulfation of chitosan in this study. Quantifying the extent of chemical modification is critical to correlate the structural changes with the observed antiviral activity, and to ensure reproducibility

Response:
We agree with the reviewer on the importance of assessing and reporting the degree of sulfation (DS) of chitosan, as it is a critical parameter to correlate chemical modification with the observed antiviral activity and ensure reproducibility.

In response, we have incorporated quantitative data derived from EDS analysis now presented in Figure 1. This figure includes:

Panel A: The EDS spectrum showing clear sulfur peaks, confirming the presence of sulfated groups.

Panel B: SEM micrograph of a representative microparticle, with the mapped region outlined.

Panel C: Elemental distribution maps (C, O, N, and S) providing visual confirmation of uniform sulfur incorporation.

Panel D: A summary table reporting elemental composition (in wt%) along with standard deviations, showing 4.6% ± 0.1% sulfur content.

Using the elemental weight percentages from EDS, particularly the sulfur content, we estimated the degree of sulfation (DS) of the chitosan sulfate microparticles. This calculation is now reported in the Results section and supports the successful chemical modification of the polymer.

Importantly, this figure also confirms that the sulfur signal is not only present but homogeneously distributed within the particles. This strengthens the link between structural modification and biological effect, enhancing the reproducibility and mechanistic understanding of our findings.

Table 1 was removed to prevent redundant presentation of elemental composition data, which are now fully integrated and visually supported in the updated Figure 1.

We hope that the revised manuscript now meets the expectations of the reviewers and the editorial team. We greatly appreciate the opportunity to revise our work and thank you again for your time and consideration.

Sincerely,
Dr. Sergio A. Bucarey, on behalf of all authors

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I agree with the author’s response and the changes made. Please publish in its present form.