CEA-Functionalized Gold Nanoparticles for Oral Prophylaxis: An In Vivo Evaluation of Safety, Biodistribution, and Cytokine Expression in Healthy Mice

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The author evaluated the immunogenicity, biodistribution and therapeutic effect of a CEA-AuNP in a mouse model of colorectal cancer through histological, hyperspectral and ELISA based cytokine analysis, providing theoretical support for further research on CEA-AuNP nanostructures as oral immunotherapy drugs for cancer prevention. However, this paper should be further improved by a major modification.

Specific comments:

1. The quality of the pictures in the text is poor. It is recommended that they be made clearer to improve the readability of the article.
2. The sizes of the pictures in Figure 2A are inconsistent.
3. Line 189 and Line 193 etc. To indicate the location of the abnormal phenomenon, it is recommended that relevant arrows are added to the tissue section.
4. Line 213 and 244, the font used for the legend content is inconsistent with that used for other legends.
5. Line 243, it is important to establish the reason why only the blank group, 5 mg and 50 mg were detected in the hyperspectral dark-field microscope, while images of other concentrations were not provided.
6. Neither Figure 4 nor Figure 5 indicates the magnification or scale.
7. Line 265-278, the font format differs from that used in other parts.
8. Line 280, Figure 7 does not mark the horizontal coordinate groups of IL-1β.
9. Figures 6 and 7 do not have a P value.
10. The references are not in a uniform format. For example, see reference 16 on line 448.

Author Response

We sincerely thank the reviewer for their thorough and constructive feedback on our manuscript. We appreciate the insightful suggestions, which have helped us significantly improve the quality and clarity of our work. Below, we provide detailed responses to each comment, along with the corresponding revisions made to the manuscript.

 

Comment 1: The quality of the pictures in the text is poor. It is recommended that they be made clearer to improve the readability of the article.

Response 1: Thank you for this observation. All figures have been replaced with higher resolution images to enhance visual clarity. We have ensured that the updated figures are optimized for print and digital formats.

 

Comment 2: The sizes of the pictures in Figure 2A are inconsistent.

Response 2: We have reformatted Figure 2A to ensure that all subfigures are of uniform size and layout. This improves consistency and presentation quality. We also rearranged the figures and are now stratified by organ and nanoparticle dose, which we hope significantly enhances interpretability.

 

Comment 3: Line 189 and Line 193 – To indicate the location of the abnormal phenomenon, it is recommended that relevant arrows are added to the tissue section.

Response 3: We have added arrows and markers in the relevant images to clearly indicate the locations of the observed abnormal phenomena. Corresponding figure legends have also been updated for clarity.

 

Comment 4: Line 213 and 244 – The font used for the legend content is inconsistent with that used for other legends.

Response 4: The font in all figure legends has been standardized throughout the manuscript to maintain consistency in formatting.

 

Comment 5: Line 243 – It is important to establish the reason why only the blank group, 5 mg, and 50 mg were detected in the hyperspectral dark-field microscope, while images of other concentrations were not provided.

Response: We appreciate the reviewer’s observation regarding the selection of dose groups represented in the hyperspectral imaging figures. In our in vivo study design, animals were administered CEA-AuNP formulations at escalating doses (5, 10, 20, and 50 mg/kg/day), along with a control group receiving PBS. For hyperspectral analysis, tissue samples were collected from all groups. However, only the control, 5 mg, and 50 mg groups were chosen for detailed image presentation in the manuscript for the following reasons:

• Representative Spectrum Visualization: The 5 mg and 50 mg/kg/day groups were selected to illustrate low-dose and high-dose accumulation patterns, which demonstrated a clear and dose-dependent contrast in nanoparticle distribution as seen via hyperspectral dark-field microscopy. The control group served as a necessary baseline to confirm the absence of nanoparticle-associated reflectance.
• Signal Intensity Thresholds: In intermediate-dose groups (10 mg and 20 mg/kg/day), spectral signals were detected but were subtle and spatially inconsistent, making them less suitable for clear visual representation. Quantitative spectral data confirmed the dose-dependent trend, but these images did not add further illustrative value beyond what was shown in the 5 mg and 50 mg examples.
• Figure Space and Readability Constraints: To maintain clarity and conciseness in visual presentation, we prioritized the clearest and most contrasting results. Including all five dose groups in figures would have required significantly more space and potentially compromised the readability and interpretability of the data.
• Clarification Added: This rationale has now been clearly stated in the revised manuscript under the Results section and in the figure legends for Figures 4 and 5.

We thank the reviewer for highlighting this point and believe the clarification strengthens the transparency and rationale of our data presentation.

 

Comment 6: Neither Figure 4 nor Figure 5 indicates the magnification or scale.

Response 6: Scale bars and magnification details have been added to Figures 4 and 5. Figure legends have also been revised to include this information.

 

Comment 7: Line 265–278 – The font format differs from that used in other parts.

Response 7: We have corrected the font formatting in this section to match the rest of the manuscript.

 

Comment 8: Line 280 – Figure 7 does not mark the horizontal coordinate groups of IL-1β.

Response 8: We have updated the figure (Figure 11 in the revised manuscript) to include proper labels on the horizontal axis for IL-1β. This revision improves clarity and interpretability of the data.

 

Comment 9: Figures 6 and 7 do not have a P value.

Response 9: We thank the reviewer for this valuable observation. The comment refers to Figures 10 and 11 in the revised manuscript, which depict cytokine levels in spleen and liver tissue, respectively. These results were obtained using ELISA kits and are presented as absolute mean values.

However, we would like to clarify that these cytokine determinations were performed in single measurements (n = 1 per animal, 6 animals per group), and raw replicate data were not recorded at the time of analysis. As such, statistical significance testing (e.g., ANOVA, t-tests) was not feasible due to the absence of replicate values per sample.

To address this, we have clearly stated in the Materials and Methods and Figure Legends that cytokine results represent descriptive, qualitative data only, intended to indicate expression trends rather than support statistical comparisons. This limitation is also acknowledged in the Discussion section, and future studies are planned to include full quantitative analyses with replicates and statistical validation.

We appreciate the reviewer’s attention to data robustness and have made corresponding clarifications throughout the manuscript.

 

Comment 10: The references are not in a uniform format. For example, see reference 16 on line 448.

Response 10: The reference list has been thoroughly revised to ensure consistency in formatting, in accordance with the journal’s guidelines.

 

We thank the reviewer once again for their valuable comments, which have contributed to strengthening the manuscript. We hope the revisions meet the expectations and are now suitable for publication.

Reviewer 2 Report

Comments and Suggestions for Authors

Gold nanoparticles (AuNPs) represent a promising carrier system for immunotherapy and an innovative platform for nanovaccines in immunoprophylaxis, owing to their inherent capacity, shared with other nanoparticles, to accumulate preferentially in immune system compartments. The topic holds significant relevance in oncology and vaccinology, the article under review exhibits substantial methodological and presentational deficiencies.

The title “CEA-Functionalized Gold Nanoparticles for Oral Immunoprophylaxis: In Vivo Evaluation of Safety, Biodistribution, and Immunogenicity in a Murine Colorectal Cancer Model” is incongruent with the described methodology. The study reportedly employed ordinary laboratory mice (Murine Colorectal Cancer Model) without clarifying whether a genetically engineered, chemically induced, or xenograft-based colorectal cancer model was utilized.

Nanoparticle Synthesis and Characterization: The synthesis protocol for AuNPs (e.g., citrate reduction, seed-mediated growth) is unspecified.

No physicochemical characterization data (e.g., dynamic light scattering for hydrodynamic diameter, zeta potential, TEM/SEM imaging) are provided to confirm nanoparticle size, morphology, or surface charge.

The CEA conjugation methodology (e.g., EDC/NHS coupling, thiol-gold bonding) lacks description, as do details about the CEA protein source (commercial vendor? recombinant production?) and its final concentration.

In Vivo Experimental Design: Critical animal model parameters—including mouse strain (BALB/c? C57BL/6?), age (weeks), and weight range—are omitted, preventing reproducibility.

The rationale for selecting a 30-day administration regimen remains unexplained, with no references to pharmacokinetic studies or prior dose-optimization experiments.

Analytical and Statistical Deficiencies: Hyperspectral dark-field microscopy protocols lack specimen preparation details (e.g., tissue fixation, sectioning thickness, staining methods).

Statistical analysis is inadequately described: the software (GraphPad Prism? SPSS?) and tests (ANOVA? t-test?) are unspecified, and Figures 6–7 omit error bars, measures of central tendency (mean/median), and dispersion metrics (SD/IQR). Significance thresholds (e.g., p < 0.05) and post-hoc comparisons are absent.

Control Group and Histopathological Findings: Section 3.2 fails to include control group histology, compromising the assessment of nanoparticle-induced toxicity. The figures would benefit from a side-by-side presentation of experimental and control organ sections, stratified by nanoparticle concentration.

Concentration Rationale: The use of AuNP concentrations requires justification. Citations to prior biodistribution studies or toxicity profiles in similar models are needed to dose selection.

Author Response

Comment 1: The title “CEA-Functionalized Gold Nanoparticles for Oral Immunoprophylaxis: In Vivo Evaluation of Safety, Biodistribution, and Immunogenicity in a Murine Colorectal Cancer Model” is incongruent with the described methodology. The study reportedly employed ordinary laboratory 1mice (Murine Colorectal Cancer Model) without clarifying whether a genetically engineered, chemically induced, or xenograft-based colorectal cancer model was utilized.

Response 1: We appreciate the reviewer’s observation and fully acknowledge the inconsistency between the original title and the actual experimental design. The study was conducted in healthy BALB/c mice without induction of a colorectal cancer model. The goal was to evaluate the immunoprophylactic potential of CEA-functionalized gold nanoparticles, not their therapeutic efficacy against established tumors.

To address this, we have revised the title to accurately reflect the methodology and scope of the study. The new title is:

“CEA-Functionalized Gold Nanoparticles for Oral Immunoprophylaxis: In Vivo Evaluation of Safety, Biodistribution, and Cytokine Expresion in Healthy Mice”

This revised title clarifies that the evaluation was conducted in a non-tumor-bearing model and focuses on safety, biodistribution, and immunological responses, which are essential precursors to future efficacy studies in disease models.

We thank the reviewer for prompting this important clarification, which has improved the accuracy and transparency of our manuscript.

 

Comment 2: The synthesis protocol for AuNPs (e.g., citrate reduction, seed-mediated growth) is unspecified.

No physicochemical characterization data (e.g., dynamic light scattering for hydrodynamic diameter, zeta potential, TEM/SEM imaging) are provided.

The CEA conjugation methodology (e.g., EDC/NHS coupling, thiol-gold bonding) lacks description, as do details about the CEA protein source (commercial vendor? recombinant production?) and its final concentration.

Response 2: We thank the reviewer for these important observations. We have revised the Materials and Methods section to include a full description of the synthesis procedure (Turkevich method), characterization (UV-VIS, DLS, AFM), and CEA conjugation (DTT-reduction and thiol-gold interaction). The peptide source (MyBiosource) and purification method are also specified. These additions enhance reproducibility and scientific rigor.

 

Comment 3: In Vivo Experimental Design: Critical animal model parameters—including mouse strain (BALB/c? C57BL/6?), age (weeks), and weight range—are omitted, preventing reproducibility.

Response 3: We thank the reviewer for pointing out this omission. The in vivo experiments were conducted on female BALB/c mice, aged 6–8 weeks, with an initial weight range of 18–22 grams. These details have now been explicitly added to the Materials and Methods section under the “Study Design for In Vivo Experiments” subsection to ensure transparency and reproducibility.

 

Comment 4: The rationale for selecting a 30-day administration regimen remains unexplained, with no references to pharmacokinetic studies or prior dose-optimization experiments.

Response 4:We thank the reviewer for highlighting this omission. The 30-day administration period was selected based on commonly accepted protocols for subacute toxicity assessment, as outlined in OECD Guideline 407 for repeated dose 28-day oral toxicity study in rodents. Although no prior pharmacokinetic or dose-optimization studies were available for our specific nanomaterial formulation, the selected dosing duration aimed to capture potential cumulative effects associated with repeated exposure. This rationale has now been added to the “Study Design for In Vivo Experiments” subsection in the revised manuscript, along with an appropriate reference to support this standardized approach.

 

Comment 5: Analytical and Statistical Deficiencies: Hyperspectral dark-field microscopy protocols lack specimen preparation details (e.g., tissue fixation, sectioning thickness, staining methods).

Response 5: We appreciate the reviewer’s observation regarding the need for additional methodological details. In response, we have revised the “Tissue Collection and Processing” section to specify that all tissues analyzed by hyperspectral dark-field microscopy were formalin-fixed (10% neutral buffered formalin), paraffin-embedded, and sectioned at 5 µm thickness. To preserve the intrinsic optical properties of gold nanoparticles, no histological stains were applied to these sections. These clarifications have been added to enhance transparency and reproducibility of the imaging protocol.

 

Comment 6: Statistical analysis is inadequately described: the software (GraphPad Prism? SPSS?) and tests (ANOVA? t-test?) are unspecified, and Figures 6–7 omit error bars, measures of central tendency (mean/median), and dispersion metrics (SD/IQR). Significance thresholds (e.g., p < 0.05) and post-hoc comparisons are absent.

Response 6: We thank the reviewer for this detailed and constructive observation. We acknowledge the omission of statistical parameters in Figures 10 and 11 (previously Figures 6–7). As clarified in the revised Materials and Methods section, cytokine determinations were performed on pooled tissue lysates with a single measurement per analyte. While six animals were included in each group, logistical constraints at the time of analysis led to the use of pooled homogenates from selected animals, and individual replicate data were not preserved.

As a result, the reported values represent pooled means without available standard deviation or replicate counts. Therefore, statistical tests (e.g., ANOVA or t-tests), error bars, or significance thresholds could not be applied. We have revised the figure captions and relevant text in both the Materials and Methods and Results sections to emphasize that these cytokine results are presented as part of a qualitative, descriptive analysis only and do not support statistical inference.

We appreciate the reviewer’s emphasis on statistical rigor and have incorporated this limitation transparently in the manuscript.

 

Comment 7: Control Group and Histopathological Findings: Section 3.2 fails to include control group histology, compromising the assessment of nanoparticle-induced toxicity. The figures would benefit from a side-by-side presentation of experimental and control organ sections, stratified by nanoparticle concentration.

Response 7: We thank the reviewer for this important observation. At the time of experimentation, histological examination of control animals (administered PBS only) was performed, but representative micrographs were not archived for long-term documentation, as no pathological changes were observed and tissues appeared histologically normal. Therefore, control group images were not included in the original manuscript.

We fully agree that the inclusion of control histology images would have enhanced the comparative value of the findings. The low-dose group (10 mg/kg/day) did not exhibit histological changes and can partially serve as a reference, In the revised histological presentation, representative micrographs are displayed side by side for each organ, across different nanoparticle doses, to facilitate direct visual comparison.

 We acknowledge this limitation and explicitly state it in the revised Discussion section, and we are committed to including control histology documentation in future studies for more comprehensive toxicity assessment. We appreciate the reviewer’s suggestion and have revised the text accordingly to reflect this consideration.

 

Comment 8: Concentration Rationale: The use of AuNP concentrations requires justification. Citations to prior biodistribution studies or toxicity profiles in similar models are needed to support dose selection.

Response 8: We thank the reviewer for this valuable observation. The selection of gold nanoparticle (AuNP) concentrations (5–50 mg/kg/day) was based on a combination of prior in vitro cytocompatibility testing (data accepted for publication by the same authors in Vaccines Journal) and reference to previous in vivo studies demonstrating acceptable safety margins for orally administered AuNPs in murine models. Specifically, comparable oral or systemic exposure levels have been reported in studies evaluating nanoparticle biodistribution, toxicity, and immune modulation [References added: Almeida et al., 2014; Ferrando et al., 2020; Dykman et al., 2012]. These references have now been included in the revised manuscript to support dose rationale. We have also clarified this rationale in the “Study Design for In Vivo Experiments” subsection.

Reviewer 3 Report

Comments and Suggestions for Authors

The article is devoted to the study of the effects of gold nanoparticles functionalized with CEA. Despite the topical and interesting subject, significant shortcomings were found in the text of the article

 

1. The introduction contains the phrase - "Building on prior in vitro validation of antigen loading, cytocompatibility, and macrophage uptake....", but there is no reference and the reviewer was able to find only unpublished data from this group of authors (https://www.preprints.org/manuscript/202505.0830/v1). It is not correct to refer to the preprint.

 

 

2. Nanoparticles are not characterized, the text contains a reference to unpublished data. It is recommended to make accompanying materials with characteristics of nanoparticles. In addition to assessing the particle size, it is also important to describe in detail the functionalization procedure. Which CEA was used? How was the fact of cross-linking of protein and nanoparticles confirmed? Does the species specificity of CEA matter?

 

3. Study Design for In Vivo Experiments - the title of the article mentions a colorectal cancer model, but there is no reason to think so. How did the authors induce inflammation? Did they use azoxymethane (AOM)/dextran sulfate sodium? (https://doi.org/10.7717/peerj.16159). Justify why the period of 30 days was chosen.

 

4. Describe how tissue samples were prepared for Hyperspectral Dark-Field Microscopy.
5. Biochemical and Immunological Analysis - ELISA kits (Elab Biosciences - please specify country). Describe how organ homogenates were prepared. Why do you use "biochemical" when you only assessed cytokines? Perhaps it would be better to write "cytokine levels"?

 

6.Add a new point to the text of the article - statistical data processing.

7.Macroscopic Findings - your data allow you to provide full statistics on the mass of the spleen in the text. It would be better if you presented these results in a table and indicated the mean and error of the mean, you will be able to mathematically confirm splenomegaly.

 

 

8. Histopathological Findings - the data should be presented more correctly. First of all, the control group data is not provided. You can group the photos by organs, presenting the control photo in one line, then the nanoparticles by increasing concentration. In this way, it will be possible to trace the effect of the nanoparticles.
9. Hyperspectral Dark-Field Microscopy - «Quantitative analysis demonstrated a dose-dependent relationship between signal intensity»- what do you mean? If this is a count of dots on a photo, then why is this data not presented? For example, these photos can be processed in ImageJ and get numerical data that can be compared mathematically.

 

10. 6 and Fig. 7. - it is necessary to design the histograms differently. There is no need to label each column. However, it is necessary to indicate what it is - the average, the median? Also indicate the standard deviation or interquartile range on the histograms, conduct statistical analysis using appropriate non-parametric criteria.

 

Comments on the Quality of English Language The authors use terms incorrectly. The title mentions "Murine Colorectal Cancer Model" but then does not explain what the authors mean. The title includes "Immunogenicity" but it was not studied.

 

Author Response

Comments and Suggestions for Authors

The article is devoted to the study of the effects of gold nanoparticles functionalized with CEA. Despite the topical and interesting subject, significant shortcomings were found in the text of the article

 

Comment 1: The introduction contains the phrase - "Building on prior in vitro validation of antigen loading, cytocompatibility, and macrophage uptake....", but there is no reference and the reviewer was able to find only unpublished data from this group of authors (https://www.preprints.org/manuscript/202505.0830/v1). It is not correct to refer to the preprint.

Response 1: We thank the reviewer for this important observation. At the time of initial submission, the referenced data were indeed available only as a preprint. However, we are pleased to report that this study has since been formally accepted for publication in the peer-reviewed journal Vaccines. Accordingly, we have updated the manuscript to reflect the final citation (now listed as reference 11) and removed any mention of preprints. This ensures the reference now corresponds to a peer-reviewed source, improving the rigor and transparency of the introduction. 

 

Comment 2: Nanoparticles are not characterized, the text contains a reference to unpublished data. It is recommended to make accompanying materials with characteristics of nanoparticles. In addition to assessing the particle size, it is also important to describe in detail the functionalization procedure. Which CEA was used? How was the fact of cross-linking of protein and nanoparticles confirmed? Does the species specificity of CEA matter?

Response 2: We thank the reviewer for this important observation. The current version of the manuscript has been substantially revised to address these concerns:

1. Nanoparticle Synthesis and Characterization:

The synthesis of AuNPs via the Turkevich method is now detailed in Section 2. “Materials and Methods.” We provide characterization data including UV-Vis spectroscopy (plasmon peak at 522 nm), dynamic light scattering (hydrodynamic size ~20 nm before and ~80 nm after functionalization), and atomic force microscopy (AFM), confirming size, morphology, and colloidal stability.

2. Functionalization Procedure and CEA Source:

The CEA conjugation protocol is described using thiol-gold chemistry. Specifically, commercially sourced CEA peptide (MyBiosource.com) was reduced with DTT to expose thiol groups, then incubated with AuNPs. The conjugation was confirmed by a redshift and broadening in the UV-Vis spectrum, hydrodynamic size increase via DLS, and changes in AFM spectral fingerprints.

3. Evidence of Protein–Nanoparticle Cross-linking:

As noted above, the redshift in UV-Vis spectra and DLS size increase post-conjugation provide biophysical evidence of successful surface modification. These findings are consistent with literature standards (see references [13–16, 36]).

4. Species Specificity of CEA:

The functionalized CEA peptide sequence is derived from a conserved region of the human CEA protein. While the murine immune system may respond differently to human CEA, this was intentional: the construct simulates a heterologous immunogen, aligning with current nanovaccine design strategies where cross-species antigen presentation promotes robust immune priming. We have clarified this rationale in the revised Discussion section.

5. Reference to Unpublished Data:

The previously mentioned preprint has now been formally cited in the References section [11] and identified as non-peer-reviewed preliminary data. We acknowledge the reviewer’s concern and clarify that the conclusions drawn in this manuscript are based solely on the in vivo study, with the preprint serving to contextualize prior construct validation.

We hope these clarifications and methodological expansions fully address the reviewer’s concerns.

 

Comment 3: Study Design for In Vivo Experiments - the title of the article mentions a colorectal cancer model, but there is no reason to think so. How did the authors induce inflammation? Did they use azoxymethane (AOM)/dextran sulfate sodium? (https://doi.org/10.7717/peerj.16159). Justify why the period of 30 days was chosen.

Response 3: We thank the reviewer for drawing attention to this important issue. We acknowledge that the original title inaccurately referred to a “colorectal cancer model,” which could lead to misinterpretation, as no tumor induction or inflammation was performed in this study.

To clarify the experimental model, this study employed healthy female BALB/c mice without the use of any inflammatory or carcinogenic agents. Neither AOM/DSS nor any genetic or chemical tumor induction method was applied. The model was intended to evaluate systemic and local immune responses, as well as the safety and biodistribution of orally administered CEA-functionalized gold nanoparticles, in the context of prophylactic nanovaccine assessment.

The manuscript title has been revised to eliminate reference to a colorectal cancer model. It now reads:

“CEA-Functionalized Gold Nanoparticles for Oral Immunoprophylaxis: In Vivo Assessment of Biodistribution, Safety, and Immune Activation in Healthy Mice.”

All corresponding mentions of a cancer model have been removed or corrected throughout the manuscript text for consistency and accuracy.

No Inflammation Was Induced. As noted, this study did not use AOM/DSS or other inflammation-inducing agents. The experimental setup aimed to assess immune activation under physiological conditions, mimicking a preclinical preventive (rather than therapeutic) setting for mucosal immunomodulation.

Justification for the 30-Day Administration Period: the 30-day exposure regimen was selected based on the following:

• OECD Test Guideline 407 for subacute oral toxicity testing of chemicals and nanomaterials;
• Literature demonstrating that biodistribution and immune priming after oral nanoparticle administration require repeated dosing over weeks to capture cumulative effects;
• Relevance to vaccine development, where multi-dose schedules over similar time frames are common to ensure antigen processing and memory response formation.

These clarifications have been added to Section 2, “Study Design for In Vivo Experiments,” to enhance reproducibility and contextual justification.

 

Comment 4: Describe how tissue samples were prepared for Hyperspectral Dark-Field Microscopy.

Response 4: We thank the reviewer for this helpful observation. We have now added a dedicated description of tissue preparation for Hyperspectral Dark-Field Microscopy (HSDM) in the “Materials and Methods” section. Specifically, all tissue samples were fixed in 10% neutral-buffered formalin, paraffin-embedded, and sectioned at 5 μm using a microtome. Sections intended for HSDM were left unstained to preserve the native optical properties of gold nanoparticles and mounted on clean glass slides with DPX mounting medium. These steps have now been clearly specified in the revised text.

 

Comment 5: Biochemical and Immunological Analysis - ELISA kits (Elab Biosciences - please specify country). Describe how organ homogenates were prepared. Why do you use "biochemical" when you only assessed cytokines? Perhaps it would be better to write "cytokine levels”?

Response 5: We appreciate the reviewer’s detailed feedback.  The country of the ELISA kit supplier has now been added: Elabscience Biotechnology Inc., USA.

A full description of organ homogenate preparation has been included. Briefly, tissues were weighed, mechanically homogenized in PBS with protease inhibitors, and centrifuged to collect the supernatant.

We agree that the term “biochemical” may be misleading in this context, as the analysis was limited to cytokine profiling. Accordingly, we have revised the section title and text throughout the manuscript to refer specifically to “Cytokine Analysis” or “Cytokine Levels.”

 

Comment 6: Add a new point to the text of the article - statistical data processing.

Response 6: We thank the reviewer for this suggestion. As described in our previous responses, statistical analysis was not feasible for all parameters in this study. Specifically, cytokine levels were determined from pooled organ samples with no replicate measurements per sample; thus, results are presented descriptively without statistical inference. These details are now explicitly stated in the revised “Materials and Methods” and “Results” sections for clarity.

 

Comment 7: Macroscopic Findings - your data allow you to provide full statistics on the mass of the spleen in the text. It would be better if you presented these results in a table and indicated the mean and error of the mean, you will be able to mathematically confirm splenomegaly.

Response 7: We thank the reviewer for this valuable suggestion. The full in vivo study included five experimental groups; however, detailed macroscopic and histopathological evaluation (including spleen measurements) was performed only for the control group and the high-dose group (50 mg/kg/day), as per the approved experimental protocol (Authorization No. 287/19.01.2022, Cluj County Veterinary Authority). Due to this limitation in dataset completeness, group-wise statistical analysis (e.g., means, standard deviations, or ANOVA) could not be performed.

Instead, we have descriptively reported the spleen lengths observed in these two groups to highlight the observed difference. Specifically, the spleen length reached 31.25 mm in the high-dose group, compared to 20.78 mm in the control group, suggesting marked splenomegaly potentially related to nanoparticle exposure. We have revised the Results section to transparently present this observation while clearly stating that statistical confirmation was not feasible.

In future studies, we aim to include systematic organ measurement across all groups to enable full quantitative and statistical interpretation.

 

Comment 8: Histopathological Findings - the data should be presented more correctly. First of all, the control group data is not provided. You can group the photos by organs, presenting the control photo in one line, then the nanoparticles by increasing concentration. In this way, it will be possible to trace the effect of the nanoparticles.

Response 8: We appreciate the reviewer’s suggestion regarding improved visual presentation of histopathological findings. We fully agree that side-by-side comparison of control and treated tissues, stratified by organ and nanoparticle dose, would significantly enhance interpretability. Unfortunately, due to technical limitations during initial tissue processing and archiving, representative control images of certain organs were not successfully retained or imaged under the same optical conditions. Therefore, we are unable to include control histology photos in the current version.

However, we have revised the figure layout to group experimental images by organ and dose, and we now provide enhanced descriptive histological comparisons to the control group, based on observations made at the time of analysis. Where relevant, we reference normal histoarchitecture to clearly distinguish nanoparticle-induced changes. We also acknowledge this limitation in the revised manuscript under the “Discusions” subsection and intend to address this rigorously in future work through systematic archiving of matched control images.

 

Comment 9: Hyperspectral Dark-Field Microscopy - «Quantitative analysis demonstrated a dose-dependent relationship between signal intensity»- what do you mean? If this is a count of dots on a photo, then why is this data not presented? For example, these photos can be processed in ImageJ and get numerical data that can be compared mathematically.

Response 9: We thank the reviewer for this excellent observation. In the original manuscript, the phrase “quantitative analysis” referred to a qualitative visual estimate of nanoparticle reflectance signals observed across dose groups. We recognize that this wording was misleading and have now revised the phrasing to clarify the semi-quantitative nature of the assessment.

While we did not conduct automated pixel or particle quantification using ImageJ or similar software, we agree this would significantly enhance the rigor and reproducibility of our results. We are currently in the process of validating an image analysis pipeline using hyperspectral spectral library mapping and ImageJ particle count thresholds.

To maintain transparency, we have updated the manuscript to clarify that signal comparisons were visual/semi-quantitative, note the absence of numerical reflectance quantification and acknowledge this as a limitation and opportunity for future work.

 

Comment 10 : Fig. 6 and Fig. 7. - it is necessary to design the histograms differently. There is no need to label each column. However, it is necessary to indicate what it is - the average, the median? Also indicate the standard deviation or interquartile range on the histograms, conduct statistical analysis using appropriate non-parametric criteria.

 Response 10: We thank the reviewer for this valuable observation. In the revised manuscript, Figures 6 and 7 now appear as Figures 10 and 11, and the accompanying legends have been updated to clarify the data presentation.

 The cytokine measurements were performed on pooled tissue lysates per group, meaning each data point represents a single pooled measurement from multiple animals, without replicate values. As such, the values shown are absolute mean values derived from pooled samples, and no statistical variation (e.g., standard deviation, interquartile range) or inferential testing could be applied.

 To avoid any confusion, we have revised the Materials and Methods (Cytokine Analysis subsection), Results, and figure legends to clearly indicate the descriptive nature of these data and the absence of statistical analysis. We agree that in future studies, cytokine analysis should be performed on individual animal samples to allow proper statistical evaluation, and we plan to implement this in subsequent experiments.

 

Reviewer Comments on the Quality of English Language Comments on the Quality of English Language:

The authors use terms incorrectly. The title mentions "Murine Colorectal Cancer Model" but then does not explain what the authors mean. The title includes "Immunogenicity" but it was not studied.

Response: We thank the reviewer for this accurate and constructive feedback. We acknowledge that the original title may have overstated the scope of the study:

• The term “Murine Colorectal Cancer Model” was inappropriate, as no cancer-inducing protocol (e.g., AOM/DSS, xenografts, or transgenics) was employed. All experiments were conducted in healthy BALB/c mice, and the focus was on evaluating prophylactic safety and biodistribution, not disease modulation.
• The inclusion of “Immunogenicity” in the title was also too strong, as the study only evaluated cytokine expression profiles as preliminary indicators of immune modulation. It did not assess antigen-specific responses, antibody production, or T-cell activation typically associated with immunogenicity studies.

To address this, we have revised the title as follows:

“CEA-Functionalized Gold Nanoparticles for Oral Prophylaxis: In Vivo Evaluation of Safety, Biodistribution, and Cytokine Expression in Healthy Mice”

The updated title more accurately reflects the study design and avoids misinterpretation.

 

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed most of the previous concerns, and the manuscript has been improved. However, minor revisions are still required to further enhance the quality and presentation of the study.
Specific comments:

1. The image resolution remains suboptimal, exhibiting noticeable blurring upon magnification.
2. The annotation arrows lack visual prominence and present an unrefined stylistic appearance.
3. Several images retain black borders, creating formatting inconsistencies with other graphical elements. (e.g., Figure 2A, 3B)

Author Response

Comment 1: The image resolution remains suboptimal, exhibiting noticeable blurring upon magnification.

Response 1: Thank you for pointing this out. We have replaced all figures with higher-resolution versions, ensuring improved clarity and sharpness at standard and magnified view.

 

Comment 2: The annotation arrows lack visual prominence and present an unrefined stylistic appearance.

Response 2: All annotation arrows have been redesigned with improved color contrast, consistent thickness, and clean vector styling to enhance visual clarity and presentation quality.

 

Comment 3: Several images retain black borders, creating formatting inconsistencies with other graphical elements (e.g., Figure 2A, 3B).

Response 3: The figures have been revised to remove black borders. We reviewed all image panels to ensure consistent formatting and alignment throughout.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for addressing the comments and incorporating the suggested corrections. After the comments were addressed, the points of concern were resolved and the manuscript became clearer. However, several issues remain that warrant further attention:

2.5 Cytokine Quantification
Pooling samples from multiple animals into a single composite sample eliminates the ability to assess biological variability within each group. Consequently, the effective sample size is reduced to one per group per organ, which is insufficient for conducting statistical analyses or making meaningful comparisons. With only a single measurement per group and no technical or biological replicates, it is impossible to estimate variability, calculate confidence intervals, or perform hypothesis testing. This significantly undermines the scientific rigor and interpretability of the data.
Presenting pooled data as absolute values without accompanying statistical analysis is appropriate only for preliminary or exploratory purposes. While such data may illustrate general trends, they cannot substantiate claims regarding differences between groups or treatment effects.

3.1 Macroscopic Findings
The absence of data from intermediate dose groups limits the ability to characterize the dose-response relationship comprehensively. Without statistical analysis, it is not possible to determine whether the observed differences are statistically significant.

3.2 Histopathological Findings
Using representative images from the low-dose group as a baseline for comparison, without including control group images, may lead readers to incorrectly assume equivalence between the low-dose and control groups. This approach is unconventional and may compromise the clarity and interpretability of the findings. Inclusion of control images alongside low-dose images is recommended to provide a clear visual baseline.

Table 2. Summary of Cytokine Profile Changes
In the absence of statistical analysis, the table should include only descriptive summaries of the results. Statements regarding statistical significance or dose-dependent effects should be omitted unless supported by appropriate statistical testing.

Author Response

Comment 1: 2.5 Cytokine Quantification
Pooling samples from multiple animals into a single composite sample eliminates the ability to assess biological variability within each group. Consequently, the effective sample size is reduced to one per group per organ, which is insufficient for conducting statistical analyses or making meaningful comparisons. With only a single measurement per group and no technical or biological replicates, it is impossible to estimate variability, calculate confidence intervals, or perform hypothesis testing. This significantly undermines the scientific rigor and interpretability of the data.
Presenting pooled data as absolute values without accompanying statistical analysis is appropriate only for preliminary or exploratory purposes. While such data may illustrate general trends, they cannot substantiate claims regarding differences between groups or treatment effects.

Response 1: We thank the reviewer for highlighting this limitation. Indeed, pooling organ samples from multiple animals per group resulted in a single composite lysate, precluding the calculation of biological variability or statistical comparisons. While we acknowledge the limitation of pooled samples, this approach was chosen to increase sensitivity in detecting low-abundance cytokines and minimize inter-individual variability at the exploratory stage. The data were not subjected to statistical testing and are intended solely to provide a qualitative overview of cytokine trends, which will guide the design of future replicated studies. As such, we acknowledge that the cytokine data are purely descriptive and exploratory in nature. To address this, we have revised the relevant section to explicitly state that the results are intended only to illustrate general trends in cytokine modulation, and we have refrained from making any claims regarding statistically significant differences between treatment groups. Future studies will incorporate both biological and technical replicates to enable rigorous statistical evaluation of immune responses.

Comment 2: 3.1 Macroscopic Findings
The absence of data from intermediate dose groups limits the ability to characterize the dose-response relationship comprehensively. Without statistical analysis, it is not possible to determine whether the observed differences are statistically significant.

Response 2: We thank the reviewer for this observation. We acknowledge that the absence of intermediate dosing groups limits our ability to fully explore and interpret a dose–response relationship. Additionally, we agree that, in the absence of statistical analysis, it is not possible to determine whether the observed macroscopic differences are statistically significant. However, we would like to clarify that the current experiment was designed as a preliminary, exploratory investigation, primarily aimed at identifying potential gross morphological effects at the highest exposure level. The observations were intended to serve as qualitative indicators that could guide the development of future, more comprehensive studies.

To address the reviewer’s concern, we have revised Section 3.1 of the manuscript to explicitly acknowledge the descriptive nature of the macroscopic findings and to state that these should not be interpreted as evidence of dose-dependence or statistical significance. We have also clarified the exploratory context of the study and emphasized the need for further investigation with additional dose groups and statistical rigor.

 

Comment 3: 3.2 Histopathological Findings
Using representative images from the low-dose group as a baseline for comparison, without including control group images, may lead readers to incorrectly assume equivalence between the low-dose and control groups. This approach is unconventional and may compromise the clarity and interpretability of the findings. Inclusion of control images alongside low-dose images is recommended to provide a clear visual baseline.

Response 3: We thank the reviewer for this valuable observation. During the histopathological evaluation, all findings were assessed in comparison to established normal histological architecture for each organ, and deviations—whether present or absent—were carefully recorded. While representative images from the low-dose group were included to illustrate the absence of pathological changes, the evaluation itself was conducted with reference to known normal histological standards, not under the assumption that the low-dose group was equivalent to controls. However, we recognize that the absence of visual control images in the manuscript may reduce clarity for the reader. In response, we have revised the manuscript to clarify that the histological interpretation was based on comparisons to standard normal histology. 

 

Comment 4: Table 2. Summary of Cytokine Profile Changes
In the absence of statistical analysis, the table should include only descriptive summaries of the results. Statements regarding statistical significance or dose-dependent effects should be omitted unless supported by appropriate statistical testing.

Response 4: We thank the reviewer for this important and constructive comment. We fully agree that no claims regarding statistical significance or dose-dependence can be made. In response, we have revised Table 2 to include only descriptive summaries of observed cytokine trends in the spleen and liver. The updated table now presents qualitative observations alongside cautious interpretations of the immune response, without suggesting statistical comparisons. All language implying significance or dose-responsiveness has been removed. Additionally, a clarifying note has been added to the table legend indicating that the interpretations are exploratory in nature.

Reviewer 3 Report

Comments and Suggestions for Authors

To the Authors,

The reviewer thanks the authors for their thorough work with the article’s data and for addressing the questions raised. The authors have significantly improved the manuscript, as well as the way the data are presented. In addition, the title of the article now accurately reflects its content. The abstract is also consistent with the article’s content.

Nevertheless, a couple of unresolved issues remain.

First, in section 2.5, it is stated that “Each cytokine was measured once per pooled group lysate (n = 1 per group per organ).” The reviewer has never previously encountered such an experimental design. Each group (control and experimental) included 6 animals, but for the assessment of cytokine levels, material from several animals was pooled. Please explain the rationale behind this experimental design and whether there are any precedents for it in the scientific literature.

How do you explain the fact that the sterility of the nanoparticles used was not taken into account?

Figures 10 and 11 – If you have decided to indicate cytokine levels, please remove the decimal values. The axes also need to be labeled. What does “prot-FICAT” mean? Under the figure, only the figure title and note should remain. The description of the results is better placed in the main text.

Table 2 – I am not sure that in this case you can use the terms “significant” and “dose-dependent,” as they imply a mathematical basis. In this situation, it is only an increase or decrease.

Line 167 – Please specify how many animals were included in the pool (n=6?).

Author Response

Comment 1: First, in section 2.5, it is stated that “Each cytokine was measured once per pooled group lysate (n = 1 per group per organ).” The reviewer has never previously encountered such an experimental design. Each group (control and experimental) included 6 animals, but for the assessment of cytokine levels, material from several animals was pooled. Please explain the rationale behind this experimental design and whether there are any precedents for it in the scientific literature.

Response 1: We thank the reviewer for this important and thoughtful observation. Indeed, as indicated in Section 2.5, cytokine analysis was performed on pooled lysates from each group, resulting in a single measurement per group per organ. This approach was chosen due to the limited availability of sample material per animal and the relatively high volume required for reliable quantification using the multiplex assay platform. In this context, pooling was a practical compromise to allow simultaneous measurement of multiple cytokines across all groups. 

We acknowledge the limitation that this design does not permit statistical inference or assessment of inter-individual biological variability. Our intention was to use these data for descriptive and exploratory purposes—to identify general trends in cytokine modulation between treatment groups and organs. To avoid overstating these findings, we have ensured that the revised text, figure legends, and tables clearly indicate that the data are presented without statistical analysis and should be interpreted accordingly. Regarding precedents, while individual-level cytokine quantification is certainly the gold standard, pooling has been reported in exploratory or screening studies where technical or resource constraints exist: [Broadhurst DI. Kell DB. Statistical strategies for avoiding false discoveries in metabolomics and related experiments. Metabolomics, 2006;2:171-196]. We have now added a brief justification to this approach in the revised manuscript to clarify this for readers.

 

Comment 2: How do you explain the fact that the sterility of the nanoparticles used was not taken into account?

Response 2: The nanoparticles used in our study were administered via oral gavage, a route that does not require strict sterility, as the gastrointestinal tract naturally encounters a high microbial load. Nevertheless, to minimize the risk of introducing potential contaminants or confounding variables (e.g., endotoxins), nanoparticle suspensions were prepared using sterile materials and solutions. We acknowledge that this detail was not clearly stated in the original manuscript and have now included a clarification to reflect the precautions taken, while also noting that full sterility is not a requirement for oral administration routes.

Comment 3: Figures 10 and 11 – If you have decided to indicate cytokine levels, please remove the decimal values. The axes also need to be labeled. What does “prot-FICAT” mean? Under the figure, only the figure title and note should remain. The description of the results is better placed in the main text.

Response 3: We thank the reviewer for these detailed observations regarding Figures 10 and 11. In response:

• Decimal values have been removed from the indicated cytokine levels for visual clarity and consistency, as these measurements are qualitative and not statistically analyzed.
• Axes have been labeled appropriately to clearly indicate the cytokines analyzed and their relative expression.
• The label “prot-FICAT” has been clarified to avoid confusion protein - Liver
• The figure captions have been revised to include only the title and essential explanatory note, in accordance with journal formatting guidelines. The descriptive summary of the cytokine findings has been moved to the main Results section to improve clarity and manuscript structure. 

 

Comment 4: Table 2 – I am not sure that in this case you can use the terms “significant” and “dose-dependent,” as they imply a mathematical basis. In this situation, it is only an increase or decrease.

Response 4: We thank the reviewer for this important observation. We fully agree that the use of terms such as “significant” or “dose-dependent” implies a statistical basis, which is not applicable in our current experimental design due to the use of pooled samples (n = 1 per group per organ) and the absence of statistical testing.

In response, we have carefully revised Table 2 and the associated text to use only qualitative and descriptive language (e.g., “elevated,” “mild increase,” “no apparent change”). All references to statistical significance or dose dependence have been removed to avoid misinterpretation. A clarifying note has also been added to the table to indicate that the data are intended for exploratory purposes only.

 

Comment 5: Line 167 – Please specify how many animals were included in the pool (n=6?).

Response 5: We thank the reviewer for this helpful suggestion. Yes, each pooled sample included tissue from six animals per group. We have now clarified this point in the text to avoid ambiguity.