Encapsulation of Cannabidiol in Chitosan-Stabilized Argan Oil Nanoemulsion as a Potential Dermal Drug Delivery System for Psoriasis Treatment

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript presents a comprehensive study on the development of chitosan-stabilized CBD nanoemulsions for psoriasis treatment. While the research addresses a relevant topic and employs a multifaceted characterization approach, the quality of the presentation and the depth of scientific rigor require significant improvement. The following are critical concerns that must be addressed before the manuscript can be considered suitable for publication.

1. The introduction fails to state a clear, testable hypothesis. The objectives, while implied, are not concisely listed. A dedicated "Aim of the Study" section is needed to guide the reader and frame the experimental design.
2. The methods for determining the Degree of Deacetylation (DDA) and Molecular Weight (Mw) of chitosan are omitted (Page 4). This is a critical oversight as these parameters govern chitosan's solubility, charge density, and biological activity. The source and specifications of Tocobiol C (mentioned on Page 4) are also missing.
3. Methodological Omissions and Ambiguities:

The method for ensuring the complete dissolution of CBD crystals in the lipid phase is not described.

The sonication parameters for nanoemulsion preparation are provided, but the probe type and immersion depth are not specified, affecting reproducibility.

The "low-energy method" for nanoemulsion formation is referenced but not adequately described or cited.

4. The manuscript consistently violates fundamental conventions for reporting mean ± standard deviation (SD). According to standard practice (e.g., APA, many scientific journals), the SD should determine the precision of the mean. The mean should be rounded to the last decimal place of the SD, and the SD itself should typically be reported with one or two significant figures (with two often used when the first digit is 1). The manuscript fails to adhere to this. For example, the value -26.47 ± 7.67 mV should be presented as -26 ± 8 mV.
5. Figures 5 and 6 (Pages 13-15) suffer from poor temporal resolution. The critical period between 8 and 24 hours is represented by only a single endpoint measurement at 24h. This makes it impossible to accurately model release kinetics or identify potential plateau phases during this long interval.
6. The severe in vivotoxicity of chitosan-coated formulations in  elegans (Page 17-18) starkly contrasts with the in vitro non-cytotoxicity in HaCaT cells. The discussion attributes this to the complexity of a whole organism but fails to provide a mechanistic hypothesis. Furthermore, the potential role of residual acetic acid (pH 4.3-4.9) in causing toxicity is mentioned as an afterthought but not investigated or controlled for, representing a major experimental flaw.
7. The claim of "strong correlation" with zero-order kinetics (Page 14, R² = 98-99%) for release across a dialysis membrane is misleading. Such high R² values are common for cumulative release data and do not uniquely prove a zero-order mechanism. The use of more discriminative models (e.g., Korsmeyer-Peppas) is necessary to elucidate the true release mechanism (Fickian diffusion, swelling, etc.).
8. The 3-month stability study (Page 11-12) monitors physical and colloidal parameters but does not include a chemical assay for CBD degradation (e.g., HPLC quantification of remaining CBD). A decrease in pH in argan oil formulations suggests possible hydrolysis, but its impact on CBD integrity is not assessed.
9. The discussion section often reiterates results without providing deeper insight. For example, the speculation that CBD "moderated the structural rigidity imparted by chitosan" (Page 11) is not supported by any direct structural analysis (e.g., microscopy, FTIR).
10. The text alternates between "zeta potential," "ζ-potential," and the symbol "ζ" without consistency. The abbreviation "MS" is used once (Page 8, "oil-to-MS ratio") instead of the previously defined "SM" (Surfactant Mixture).
11. The conclusion fails to synthesize the key, albeit contradictory, findings. It does not explicitly reconcile the promising in vitroperformance with the alarming in vivo toxicity, nor does it propose a clear path for the "further optimization" it mentions.
12. The ex vivopermeation study uses pure CBD in argan oil as a control but does not include a control with chitosan solution alone (without nanoemulsion) to decouple its penetration-enhancing effect from the nanoemulsion system itself.
13. The method states worms were "categorized as either alive or dead" after 24 hours (Page 7). The specific, operational criteria for determining death (e.g., lack of movement in response to tactile stimulus) are not provided, which is essential for reproducibility in lethality assays.
14. The study fixes the CBD concentration at 0.1% w/w without providing a pharmacological justification or reference to effective doses from prior in vitroor pre-clinical studies for psoriasis models.

Comments on the Quality of English Language

The manuscript is difficult to read, and the poor expression obscures the scientific content. It requires thorough revision by a native English speaker with expertise in pharmaceutical sciences or a professional academic editing service. Correcting the language is not a superficial task but is integral to ensuring the research is communicated accurately and effectively. This issue alone warrants a decision of "Major Revision."

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors, After reading your MS, I really appreciate your idea.

Overall, the paper has a clear practical goal: help cannabidiol (CBD) work better on skin by putting it into very tiny oil droplets (“nanoemulsions”) made with argan oil and then testing whether adding a chitosan coating improves stability and skin delivery. That story comes through well in the abstract and early sections, and the experiments you chose (droplet size/charge, viscosity, storage stability, release through a membrane, permeation through pig skin, and basic safety checks) are logical for a first “formulation and characterization” study.

From a non-expert reader’s view, the biggest barrier is that many key terms are introduced as if everyone already knows them. For example, “PDI” and “zeta potential” are important, but a simple one-sentence explanation would help a lot: PDI is basically “how even the droplet sizes are,” and zeta potential is “how strongly droplets repel each other electrically, which helps stop clumping.” When these are explained in plain language right where they first appear, the reader can follow why changes matter.

 

In methods, some details feel a bit hard to reproduce because the “final formulation” is not always crystal clear. You explain the oil/surfactant ratios tested and the HLB target, and then you explain that chitosan is dissolved in 1% acetic acid and mixed 1:1 with the nanoemulsion, followed by sonication. It would be helpful if you state in one place (one short paragraph) the exact final composition of the chosen “best” formulation(s): final % oil, final % surfactants, final % water, final % chitosan, final % CBD, and whether the acetic acid remains in the final product or is neutralized/adjusted.

 

In results, when you discuss “enhanced stability,” the storage data actually show that the chitosan-containing systems grow more in droplet size and become less uniform over time (PDI rising), which reads like a loss of stability compared with the non-chitosan versions. So the wording should be softened and made more honest: chitosan improves some desirable properties (positive charge, viscosity, likely adhesion), but it may also make long-term physical stability more challenging unless further optimized (pH adjustment, chitosan type/molecular weight, concentration, etc.).

 

The release and skin permeation sections are interesting, but the practical “so what?” needs to be stated more plainly. The two nanoemulsions show fairly similar release and permeation overall, and even when cumulative permeation differs, the steady-state flux is described as not statistically different.

 

The safety section is the most important part to handle carefully, because it can change how readers interpret the whole study. In keratinocyte cells (HaCaT), the formulations look reasonably compatible (mostly “weak cytotoxicity” or better), and you even suggest the CBD + chitosan combination looks the most biocompatible in that test. But in the whole-organism assay (C. elegans), the chitosan-containing formulations appear severely toxic, while DMSO and non-chitosan nanoemulsions are well tolerated.  I think, this sounds alarming: “safe in cells, but lethal in a living organism.” You do offer reasonable possible explanations (positive charge interactions, formulation form, residual acetic acid/pH effects), but you should make the implications clearer: this doesn’t automatically mean it’s unsafe for human skin, but it does mean the chitosan formulation needs additional safety work before anyone can claim it is ready for topical use.

The “psoriasis treatment” angle is promising, but right now it reads more like “a delivery platform that might be useful for psoriasis,” not a demonstrated psoriasis therapy. The paper does not show anti-psoriatic efficacy in a psoriasis model (cell inflammatory markers, psoriatic skin equivalents, or animal psoriasis-like models).

 

Finally, I suggest the simplest improvement is to rewrite a few key sentences in the Introduction and Results so that every technical measurement is immediately tied to an everyday meaning (e.g., “smaller droplets usually spread better and can help penetration,” “more uniform droplets usually mean fewer stability problems,” “higher viscosity can help it stay on the skin longer”).

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

This paper talks about the development of CBD-loaded argan oil nano emulsions (NE) stabilized with chitosan and presents an extensive evaluation of drug release behavior using in vitro release studies and ex vivo skin permeation studies. The authors also conducted comprehensive toxicity assessment including both in vitro and in vivo testing. While the study provides a strong methodological introduction to NE-based topical delivery of CBD, the observed in vivo toxicity associated with chitosan-containing formulations indicates that further formulation optimization and mechanistic investigation are required before translational relevance can be established.

I do not have major comments regarding the experimental design or interpretation of results. As a minor suggestion:

• Including a schematic illustrating the NE preparation process would improve clarity for readers.
• Incorporation of representative live/dead assay imaging could strengthen the toxicity assessment, as visual data would complement the extensive quantitative graphs and help convey the biological outcomes more intuitively.
• Including representative microscopy images of the NE would improve clarity and strengthen the overall characterization.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed the majority of my concerns. Three minor issues require attention prior to acceptance:

1. Temporal resolution in release/permeation studies (Comment 5): While the authors appropriately reframed their kinetic analysis to focus on the initial linear phase, the experimental design limitation between 8–24 h remains unaddressed. Please add a brief statement acknowledging that the absence of intermediate timepoints during this interval precludes definitive identification of potential plateau phases or secondary release mechanisms.
2. Justification of CBD concentration (Comment 14): The response states that 0.1% w/w is "supported by prior pre-clinical and clinical studies," but no specific references are provided in the manuscript. Please either cite 2–3 key publications that employed similar topical CBD concentrations for inflammatory skin conditions or revise the statement to reflect that this concentration represents an empirically selected starting point based on preliminary solubility and formulation feasibility studies.
3. Chitosan-only control for permeation (Comment 12): The explanation regarding CBD hydrophobicity is noted. However, to strengthen mechanistic interpretation, please add a single sentence in the Discussion acknowledging that the relative contribution of chitosan versus the nanoemulsion matrix to enhanced permeation cannot be fully decoupled without a chitosan-in-oil control or hydrophilic model drug experiments, and that this represents a limitation for mechanistic attribution.

Comments on the Quality of English Language

The manuscript has been improved compared to the initial submission, but several language issues remain that require attention prior to publication. Some sentences exhibit awkward syntax or imprecise word choice that detracts from the scientific message.

Author Response

For research article

 

 

Response to Reviewer 1 Comments
1. Summary
Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted in the re-submitted files.
2. Questions for General Evaluation	Reviewer’s Evaluation	Response and Revisions
Does the introduction provide sufficient background and include all relevant references?	Yes	_
Is the research design appropriate?	Can be improved	We have addressed two key points to clarify and strengthen the design justification. First, we have now provided specific references to support the selection of the 0.1% w/w CBD concentration. Second, while the hydrophobic nature of CBD prevented the inclusion of a simple aqueous chitosan control, we have now explicitly acknowledged this as a limitation for mechanistic interpretation.
Are the methods adequately described?	Yes	_
Are the results clearly presented?	Can be improved	We have revised the manuscript to improve the clarity of the results presentation. Specifically, we have added a statement acknowledging the limitation of the sampling interval in the in vitro release study (Section 3.5) to ensure the results are not over-interpreted.
Are the conclusions supported by the results?	Yes	_
Are all figures and tables clear and well-presented?	Yes	_
3. Point-by-point response to Comments and Suggestions for Authors
Comments 1: Temporal resolution in release/permeation studies (Comment 5): While the authors appropriately reframed their kinetic analysis to focus on the initial linear phase, the experimental design limitation between 8–24 h remains unaddressed. Please add a brief statement acknowledging that the absence of intermediate timepoints during this interval precludes definitive identification of potential plateau phases or secondary release mechanisms.
Response 1: Thank you for this valuable feedback. We agree with the reviewer that the lack of sampling timepoints between 8 and 24 hours limits a comprehensive interpretation of the release profile during that interval. We have added a brief statement to the manuscript acknowledging this limitation (Results section, Page 16).   “It is important to note that due to the absence of sampling timepoints between 8 and 24 hours, the current data cannot definitively confirm whether a plateau phase or a secondary release mechanism occurs during this interval.”
Comments 2: Justification of CBD concentration (Comment 14): The response states that 0.1% w/w is "supported by prior pre-clinical and clinical studies," but no specific references are provided in the manuscript. Please either cite 2–3 key publications that employed similar topical CBD concentrations for inflammatory skin conditions or revise the statement to reflect that this concentration represents an empirically selected starting point based on preliminary solubility and formulation feasibility studies.
Response 2: Thank you for highlighting the need for clearer justification regarding the selected CBD concentration. We agree that the manuscript should provide explicit support for the chosen formulation parameters. In response to your comment, we have revised the manuscript to include specific references that support the use of 0.1% w/w CBD for topical applications targeting inflammatory skin conditions. The following key publications have now been cited: -          A study by Kim et al. (2024) investigating CBD effects on imiquimod-induced psoriasis in mice, which demonstrated that 0.1% CBD significantly reduced PASI scores, epidermal thickness, and inflammatory markers. -          A clinical study by Umpreecha et al. (2023) evaluating topical 0.1% CBD for recurrent aphthous ulcers (RAU), which reported reduced ulcer size and accelerated healing without adverse effects.   (Results section, Page 9).   “The 0.1% w/w CBD concentration employed in this study was selected based on prior evidence demonstrating its therapeutic efficacy in inflammatory skin conditions. Preclinical studies have shown that topical 0.1% CBD significantly reduces psoriasis-like inflammation, epidermal thickness, and hyperproliferation in murine models [16]. Furthermore, clinical application of 0.1% CBD has been reported to accelerate healing and reduce lesion size in inflammatory mucosal conditions without adverse effects [17], supporting its suitability for dermal delivery in the present formulation study.”
Comments 3: Chitosan-only control for permeation (Comment 12): The explanation regarding CBD hydrophobicity is noted. However, to strengthen mechanistic interpretation, please add a single sentence in the Discussion acknowledging that the relative contribution of chitosan versus the nanoemulsion matrix to enhanced permeation cannot be fully decoupled without a chitosan-in-oil control or hydrophilic model drug experiments, and that this represents a limitation for mechanistic attribution.
Response 3: Thank you for this constructive comment. In response to your suggestion, we have added a single sentence in the Discussion section explicitly acknowledging this limitation. The sentence has been placed after our discussion of chitosan's proposed effects, to ensure transparency regarding the mechanistic attribution.     (Results section, Page 17).   “This interpretation is subject to the limitation that the relative contribution of chitosan versus the nanoemulsion matrix to enhanced permeation cannot be fully decoupled without comparative studies using a hydrophilic model drug.”
4. Response to Comments on the Quality of English Language
Point 1: The manuscript has been improved compared to the initial submission, but several language issues remain that require attention prior to publication. Some sentences exhibit awkward syntax or imprecise word choice that detracts from the scientific message.
Response 1: Thank you for this feedback. We have carefully reviewed the manuscript and addressed the language issues. The text has been edited to improve sentence structure, correct imprecise word choices, and enhance overall clarity.

 

Reviewer 2 Report

Comments and Suggestions for Authors

The authors revised well.

Author Response

For research article

 

 

Response to Reviewer 2 Comments
1. Summary
Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted in the re-submitted files.
2. Questions for General Evaluation	Reviewer’s Evaluation	Response and Revisions
Does the introduction provide sufficient background and include all relevant references?	Yes	_
Are all the cited references relevant to the research?	Yes	_
Is the research design appropriate?	Can be improved	We have addressed two key points to clarify and strengthen the design justification. First, we have now provided specific references to support the selection of the 0.1% w/w CBD concentration. Second, while the hydrophobic nature of CBD prevented the inclusion of a simple aqueous chitosan control, we have now explicitly acknowledged this as a limitation for mechanistic interpretation.
Are the methods adequately described?	Can be improved	We have ensured that the rationale for choosing 0.1% w/w CBD is now explicitly supported by cited literature within the manuscript, strengthening the description of our methodological choices.
Are the results clearly presented?	Can be improved	We have revised the manuscript to improve the clarity of the results presentation.
Are the conclusions supported by the results?	Can be improved	To ensure our conclusions are appropriately supported, we have added a caveat regarding the in vitro release data, explicitly stating that the absence of intermediate timepoints precludes definitive conclusions about plateau phases or secondary mechanisms. In the ex vivo section, we have nuanced our conclusions about chitosan's role by adding a limitation statement, ensuring the claims do not overreach the data.
Are all figures and tables clear and well-presented?	Can be improved	We have reviewed all figures and tables to ensure they are clear and well-presented. No specific issues were raised by the reviewer regarding the figures themselves, but we have verified that all axes are correctly labeled, legends are clear, and the resolution is suitable for publication.
3. Point-by-point response to Comments and Suggestions for Authors
Comments 1: The authors revised well.
Response 1: Thank you for your contribution to the quality of our work.
4. Response to Comments on the Quality of English Language
Point 1: The English is fine and does not require any improvement.
Response 1: We thank the reviewer for their comment.