Myricetin Nanofibers as Amorphous Delivery System

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Author,

The manuscript presents a relevant formulation approach and includes a broad set of experimental data. However, several important issues related to experimental design, statistical description, analytical validation, and data interpretation need further clarification. In its current form, these points limit the clarity and reliability of the conclusions. Therefore, major revision is required, and the following comments are provided to help improve the overall quality of the manuscript.

1. Despite the use of a Box–Behnken design, none of the investigated electrospinning parameters reached statistical significance with respect to myricetin solubility. Therefore, the applied design appears to function primarily as a parameter screening rather than a true optimization, and this limitation should be clearly acknowledged by the authors.
2. The manuscript does not include a dedicated statistical analysis section. The authors should clearly specify the statistical methods used, including the applied tests, software, significance criteria, number of replicates, and explicitly indicate which statistical test was applied to each experimental dataset to ensure transparency and reproducibility.
3. The reported myricetin loading and encapsulation efficiencies exceeding 100% raise methodological concerns and require clearer justification. Although the authors attribute this outcome to the presence of localized myricetin agglomerates observed by SEM, such values suggest potential sample heterogeneity or analytical overestimation. The authors should clarify how these results were calculated and interpreted and explicitly discuss the limitations of using these values for quantitative comparison.
4. The amorphous nature of myricetin is inferred mainly from XRPD results; however, the lack of complementary thermal analysis limits the strength of this conclusion. This limitation should be explicitly acknowledged.
5. The in vitro dissolution data are presented without release kinetic analysis (zero, first order, Higuchi etc.). The authors should either justify the absence of kinetic modeling or, if feasible, evaluate the release mechanism using appropriate kinetic models.
6. The in vitro dissolution profiles are presented without error bars, which limits the assessment of data variability and reproducibility. The authors should include appropriate measures of variability (e.g., standard deviation or standard error) to support the reliability of the dissolution data.
7. Although the analytical method is referenced from the literature, reliance on previously published methods alone is not sufficient to ensure quantitative reliability in a formulation-specific context. Since each formulation matrix may affect analytical performance, method specificity should be demonstrated, including chromatographic evidence showing the absence of interference from formulation components, together with basic validation parameters.

Sincerely,

Author Response

Dear Reviewer,

The authors sincerely thank them for their time in reviewing our article and providing valuable comments. Based on these comments, changes were made to the manuscript to improve its quality. These changes were implemented via "track changes."

The manuscript presents a relevant formulation approach and includes a broad set of experimental data. However, several important issues related to experimental design, statistical description, analytical validation, and data interpretation need further clarification. In its current form, these points limit the clarity and reliability of the conclusions. Therefore, major revision is required, and the following comments are provided to help improve the overall quality of the manuscript.

1. Despite the use of a Box–Behnken design, none of the investigated electrospinning parameters reached statistical significance with respect to myricetin solubility. Therefore, the applied design appears to function primarily as a parameter screening rather than a true optimization, and this limitation should be clearly acknowledged by the authors.

Thank you for your valuable comment. Based on the comments from reviewers 1 and 3, the authors have made appropriate corrections to the manuscript to ensure the content is not misleading. The Box–Behnken design did not reveal statistically significant effects of the studied variables on the analyzed response parameters, as confirmed by the Pareto charts (p > 0.05). Therefore, no statistically optimized formulation could be identified within the tested design space.Consequently, BB5 was selected for further studies based on a practical performance criterion rather than statistical optimization. Specifically, this formulation demonstrated the highest MYR concentration after 24 h and maintained a stable supersaturated state without a noticeable decline in concentration over time. Since long-term supersaturation is considered a key parameter for improving bioavailability, this behavior was regarded as the most relevant factor for subsequent investigations.

Accordingly, the term “optimal formulation” has been revised to “selected formulation for further studies” to more accurately reflect the selection rationale.

 

2. The manuscript does not include a dedicated statistical analysis section. The authors should clearly specify the statistical methods used, including the applied tests, software, significance criteria, number of replicates, and explicitly indicate which statistical test was applied to each experimental dataset to ensure transparency and reproducibility.

Thank you for your valuable comment. We added dedicated statistical analysis section.

 

3. The reported myricetin loading and encapsulation efficiencies exceeding 100% raise methodological concerns and require clearer justification. Although the authors attribute this outcome to the presence of localized myricetin agglomerates observed by SEM, such values suggest potential sample heterogeneity or analytical overestimation. The authors should clarify how these results were calculated and interpreted and explicitly discuss the limitations of using these values for quantitative comparison.

4. The amorphous nature of myricetin is inferred mainly from XRPD results; however, the lack of complementary thermal analysis limits the strength of this conclusion. This limitation should be explicitly acknowledged.

 

The authors introduced a section on DSC analysis in the manuscript and discussed the obtained results in detail.

 

5. The in vitro dissolution data are presented without release kinetic analysis (zero, first order, Higuchi etc.). The authors should either justify the absence of kinetic modeling or, if feasible, evaluate the release mechanism using appropriate kinetic models.

 

We thank the Reviewer for this comment. The dissolution experiments in the present study were conducted under non-sink conditions and were designed to evaluate apparent solubility and supersaturation behavior rather than classical drug release kinetics. Therefore, the obtained profiles represent concentration–time curves characterized by rapid supersaturation followed by partial concentration decline, rather than diffusion-controlled release from a matrix. Under such conditions, traditional kinetic models (e.g., Higuchi, Korsmeyer-Peppas, or first-order release models) are not fully applicable. Instead, the dissolution behavior was analyzed using concentration-based descriptors, including Cmax, Tmax, and supersaturation maintenance. These parameters more appropriately describe the performance of supersaturating systems.

We have clarified this point in the revised manuscript.

 

6. The in vitro dissolution profiles are presented without error bars, which limits the assessment of data variability and reproducibility. The authors should include appropriate measures of variability (e.g., standard deviation or standard error) to support the reliability of the dissolution data.

 

We corrected it.

 

7. Although the analytical method is referenced from the literature, reliance on previously published methods alone is not sufficient to ensure quantitative reliability in a formulation-specific context. Since each formulation matrix may affect analytical performance, method specificity should be demonstrated, including chromatographic evidence showing the absence of interference from formulation components, together with basic validation parameters.

We added HPLC results.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled Myricetin Nanofibers as an Amorphous Delivery System: Optimization by Box Behnken Design reports on the fabrication of electrospun polymeric nanofibers loaded with myricetin using a Box Behnken experimental design. The study includes an extensive physicochemical characterization and presents a substantial amount of experimental data, which could potentially make it suitable for publication. However, in its current form, I am reluctant to recommend the manuscript for publication. This is primarily due to limited novelty and concerns regarding the design and justification of the dissolution study.

Points and remarks:

• The manuscript does not include in vitro cell viability or cytocompatibility studies, which significantly limits the biological and pharmaceutical relevance of the proposed myricetin-loaded nanofiber delivery system.
• The novelty of the work is not sufficiently demonstrated. The formulation strategy and reported outcomes appear largely similar to previously published studies on myricetin nanofibers (e.g., https://doi.org/10.3390/pharmaceutics15030906 ) The authors should clearly delineate the advances of the present work beyond existing literature.
• The choice of dissolution medium and experimental conditions lacks adequate justification in terms of physiological relevance. The authors expect to clarify how the selected dissolution conditions relate to the intended route of administration (e.g., oral versus topical delivery).
• The manuscript would benefit from a systematic revision of its structure to improve clarity and logical flow.
• Several language issues and typographical errors are present and should be addressed. Additionally, the authors are encouraged to discuss the challenges associated with generating such nanofibrous structures and to highlight their advantages compared to existing preparation techniques.

The MS should address the points outlined above, which would significantly strengthen the manuscript and help bring it closer to the threshold for reconsideration in Pharmaceuticals.

END

Author Response

Dear Reviewer,

The authors sincerely thank them for their time in reviewing our article and providing valuable comments. Based on these comments, changes were made to the manuscript to improve its quality. These changes were implemented via "track changes."

Points and remarks:

• The manuscript does not include in vitro cell viability or cytocompatibility studies, which significantly limits the biological and pharmaceutical relevance of the proposed myricetin-loaded nanofiber delivery system.

 

We thank the Reviewer for this valuable comment. We agree that cytocompatibility and in vitro biological evaluation are essential for further development of the system. However, such studies were beyond the scope of the present formulation-oriented investigation and will be addressed in future research. It is also worth noting that both MYR (for example https://doi.org/10.1016/j.fct.2015.06.016) and PVP are well-documented compounds with established safety profiles in pharmaceutical and biomedical applications, which supports the feasibility of further biological evaluation of the developed system.

 

• The novelty of the work is not sufficiently demonstrated. The formulation strategy and reported outcomes appear largely similar to previously published studies on myricetin nanofibers (e.g., https://doi.org/10.3390/pharmaceutics15030906 ) The authors should clearly delineate the advances of the present work beyond existing literature.

 

Thank you for your valuable comment. The authors expanded on this discussion in their paper. The presented nanofiber preparation strategy and physicochemical characterization differ significantly from previously published work. For example, earlier studies did not examine the effect of electrospinning parameters on the MYR dissolution profile as thoroughly.

 

• The manuscript would benefit from a systematic revision of its structure to improve clarity and logical flow.

 

Corrected it.

 

• Several language issues and typographical errors are present and should be addressed. Additionally, the authors are encouraged to discuss the challenges associated with generating such nanofibrous structures and to highlight their advantages compared to existing preparation techniques.

We sincerely thank you for your valuable comments. The authors have made changes to the manuscript and discussed the advantages of electrospinning.

The MS should address the points outlined above, which would significantly strengthen the manuscript and help bring it closer to the threshold for reconsideration in Pharmaceuticals.

We sincerely thank the Reviewer for their constructive and detailed comments. All points raised have been carefully addressed in the revised version of the manuscript. We believe that these revisions have significantly strengthened the scientific clarity and methodological consistency of the manuscript. We respectfully hope that the revised version now meets the standards for reconsideration.

Reviewer 3 Report

Comments and Suggestions for Authors

1. Although a Box–Behnken design was applied to evaluate the effects of electrospinning parameters (applied voltage, needle-to-collector distance, and flow rate) on myricetin solubility, none of the investigated factors or their quadratic terms were statistically significant at either T1 (15 min) or T2 (1 h) (p > 0.05). Despite this, the manuscript repeatedly refers to “optimization” in the title, objectives, and conclusions. This represents a clear inconsistency between the experimental results and the central claim of the study.

2. The choice of BB5 is primarily based on 24 h apparent solubility data (T3) provided in the Supplementary Materials, while T3 was not defined as a response variable in the Box–Behnken model nor included in the statistical analysis. If long-term supersaturation is the decisive criterion, it should be explicitly defined as a response variable and incorporated into the DOE. Otherwise, the term “optimal formulation” should be revised.

3. The reported apparent solubility values in the mg/mL range are several orders of magnitude higher than the intrinsic aqueous solubility of crystalline myricetin. It is therefore unclear whether the measured concentrations reflect true molecular solubility or are influenced by transient supersaturation, colloidal/nanoparticulate species, or polymer-assisted solubilization. Filtration through a 0.22 µm membrane does not fully exclude colloidal fractions, and this limitation should be addressed and discussed more explicitly.

4. Despite being prepared using the same electrospinning parameters, BB5-A, BB5-B, and BB5-C fibers exhibit pronounced differences in morphology, agglomerate formation, and measured myricetin content. These variations raise concerns regarding process robustness and solution homogeneity and weaken the claim of an optimized and reproducible formulation.

5. Encapsulation efficiencies of 119–128% are physically implausible and most likely arise from heterogeneous drug distribution, agglomeration of myricetin, or sampling artefacts. The use of the term “encapsulation efficiency” is therefore inappropriate in this context and should be replaced with more suitable parameters such as drug content or drug loading variability, accompanied by proper statistical treatment.

6. While XRPD confirms amorphization of myricetin within the PVP30 nanofibers, no thermal analysis (e.g., DSC) or short-term physical stability studies are provided. Given the inherent recrystallization risk of amorphous systems, stability data are essential to substantiate the proposed amorphous delivery system.

7. The DPPH and CUPRAC assays were conducted without the inclusion of a positive reference standard (e.g., Trolox or ascorbic acid), and the number of replicates and statistical comparisons are insufficiently reported. In addition, the lack of measurable antioxidant activity for crystalline myricetin may be influenced by methodological constraints and should be interpreted more cautiously.

8. Although HPLC–DAD is extensively used for quantitative determination of myricetin throughout the study, no validation data (calibration curve, linearity range, correlation coefficient, LOD/LOQ, precision, or system suitability parameters) are reported. Without these data, the reliability of the reported concentration values cannot be fully assessed.

9. The manuscript states only the supplier of myricetin without providing information on product code, batch/lot number, purity grade, certificate of analysis (CoA), or impurity profile. Commercial myricetin is typically not 100% pure, and assuming complete purity may lead to systematic errors in quantitative analyses, including solubility, loading, and antioxidant activity measurements. These details should be clearly reported and discussed.

10. The study is based exclusively on in vitro solubility, dissolution, and antioxidant activity data. Claims related to improved bioavailability should therefore be phrased more cautiously and limited to potential implications unless supported by in vivo evidence.

Comments on the Quality of English Language

The manuscript is generally understandable; however, the English language requires professional editing, particularly to improve sentence structure, reduce redundancy, and enhance clarity in the Results and Discussion sections.

Author Response

Dear Reviewer,

The authors sincerely thank them for their time in reviewing our article and providing valuable comments. Based on these comments, changes were made to the manuscript to improve its quality. These changes were implemented via "track changes."

1. Although a Box–Behnken design was applied to evaluate the effects of electrospinning parameters (applied voltage, needle-to-collector distance, and flow rate) on myricetin solubility, none of the investigated factors or their quadratic terms were statistically significant at either T1 (15 min) or T2 (1 h) (p > 0.05). Despite this, the manuscript repeatedly refers to “optimization” in the title, objectives, and conclusions. This represents a clear inconsistency between the experimental results and the central claim of the study.
2. The choice of BB5 is primarily based on 24 h apparent solubility data (T3) provided in the Supplementary Materials, while T3 was not defined as a response variable in the Box–Behnken model nor included in the statistical analysis. If long-term supersaturation is the decisive criterion, it should be explicitly defined as a response variable and incorporated into the DOE. Otherwise, the term “optimal formulation” should be revised.

Thank you for your valuable comment. Based on the comments from reviewers 1 and 3, the authors have made appropriate corrections to the manuscript to ensure the content is not misleading. The Box–Behnken design did not reveal statistically significant effects of the studied variables on the analyzed response parameters, as confirmed by the Pareto charts (p > 0.05). Therefore, no statistically optimized formulation could be identified within the tested design space.Consequently, BB5 was selected for further studies based on a practical performance criterion rather than statistical optimization. Specifically, this formulation demonstrated the highest MYR concentration after 24 h and maintained a stable supersaturated state without a noticeable decline in concentration over time. Since long-term supersaturation is considered a key parameter for improving bioavailability, this behavior was regarded as the most relevant factor for subsequent investigations.

Accordingly, the term “optimal formulation” has been revised to “selected formulation for further studies” to more accurately reflect the selection rationale.

3. The reported apparent solubility values in the mg/mL range are several orders of magnitude higher than the intrinsic aqueous solubility of crystalline myricetin. It is therefore unclear whether the measured concentrations reflect true molecular solubility or are influenced by transient supersaturation, colloidal/nanoparticulate species, or polymer-assisted solubilization. Filtration through a 0.22 µm membrane does not fully exclude colloidal fractions, and this limitation should be addressed and discussed more explicitly.

The authors re-examined the protocols and spreadsheets. Appropriate sections of the manuscript were revised.

4. Despite being prepared using the same electrospinning parameters, BB5-A, BB5-B, and BB5-C fibers exhibit pronounced differences in morphology, agglomerate formation, and measured myricetin content. These variations raise concerns regarding process robustness and solution homogeneity and weaken the claim of an optimized and reproducible formulation.

The authors discuss the obtained results in the "Results and discussion" section.

5. Encapsulation efficiencies of 119–128% are physically implausible and most likely arise from heterogeneous drug distribution, agglomeration of myricetin, or sampling artefacts. The use of the term “encapsulation efficiency” is therefore inappropriate in this context and should be replaced with more suitable parameters such as drug content or drug loading variability, accompanied by proper statistical treatment.

The authors re-examined the protocols and spreadsheets. Appropriate sections of the manuscript were revised.

6. While XRPD confirms amorphization of myricetin within the PVP30 nanofibers, no thermal analysis (e.g., DSC) or short-term physical stability studies are provided. Given the inherent recrystallization risk of amorphous systems, stability data are essential to substantiate the proposed amorphous delivery system.

The authors introduced a section on DSC analysis and stability study in the manuscript and discussed the obtained results in detail.

7. The DPPH and CUPRAC assays were conducted without the inclusion of a positive reference standard (e.g., Trolox or ascorbic acid), and the number of replicates and statistical comparisons are insufficiently reported. In addition, the lack of measurable antioxidant activity for crystalline myricetin may be influenced by methodological constraints and should be interpreted more cautiously.

In the revised version of the manuscript, the authors included the results for ascorbic acid and improved the discussion of the results.

8. Although HPLC–DAD is extensively used for quantitative determination of myricetin throughout the study, no validation data (calibration curve, linearity range, correlation coefficient, LOD/LOQ, precision, or system suitability parameters) are reported. Without these data, the reliability of the reported concentration values cannot be fully assessed.

The authors supplemented the missing information in the "Materials and Methods" section.

9. The manuscript states only the supplier of myricetin without providing information on product code, batch/lot number, purity grade, certificate of analysis (CoA), or impurity profile. Commercial myricetin is typically not 100% pure, and assuming complete purity may lead to systematic errors in quantitative analyses, including solubility, loading, and antioxidant activity measurements. These details should be clearly reported and discussed.

Thank you for your valuable comment. The authors have provided the missing information.

10. The study is based exclusively on in vitro solubility, dissolution, and antioxidant activity data. Claims related to improved bioavailability should therefore be phrased more cautiously and limited to potential implications unless supported by in vivo evidence.

We thank the Reviewer for this important comment. We fully agree that conclusions regarding improved bioavailability should be drawn cautiously in the absence of in vivo data. In the revised manuscript, all statements referring to bioavailability have been carefully rephrased and limited to potential implications only. The discussion now emphasizes enhancement of apparent solubility and supersaturation performance, while any reference to bioavailability is described as a possible outcome requiring further in vivo validation. Additionally, the limitation related to the lack of in vivo studies has been explicitly acknowledged in the revised version of the manuscript.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Author,

The revised manuscript has been carefully evaluated, and the authors have satisfactorily addressed the previously raised concerns. The clarification regarding the Box–Behnken design, the inclusion of complementary DSC analysis, and the improvements in statistical reporting and data presentation have significantly strengthened the scientific clarity and reliability of the study. In its current form, the manuscript is scientifically sound and suitable for publication.

Best regards.

Author Response

Dear Reviewer,
We sincerely thank you for your careful re-evaluation of our revised manuscript and for your positive assessment. We greatly appreciate your constructive comments and are pleased that the revisions have strengthened the manuscript. Thank you for your time and valuable feedback.

Best Regards

Reviewer 2 Report

Comments and Suggestions for Authors

The suggestions were accordingly followed, and the manuscript can be accepted.

Comments on the Quality of English Language

it will be great to work a bit on the English. 

Author Response

Dear Reviewer,
Thank you very much for your positive evaluation of our manuscript and for recommending its acceptance. We sincerely appreciate your constructive feedback. In response to your suggestion, we carefully revise the manuscript once more to further improve the English language and ensure clarity and fluency throughout the text.

Thank you again for your valuable comments.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript has been substantially improved and most major concerns have been addressed. However, before final acceptance, the following clarifications are still required:

1. It should be clearly and explicitly stated in the manuscript that the reported “apparent solubility” values do not necessarily represent true molecular solubility. Given the possibility of supersaturation, polymer-assisted solubilization, and/or colloidal species formation, this distinction should be clearly discussed to avoid potential misinterpretation of the data.

2. To better demonstrate process robustness and reproducibility, the relative standard deviation (RSD%) values for drug content and/or solubility measurements (particularly for BB5-A, BB5-B, and BB5-C batches) should be presented in a clear tabulated format. This will strengthen the reliability of the reported results.

3. The term “selected formulation” should be used consistently throughout the manuscript. Please ensure that terms such as “optimal formulation” or similar expressions are fully removed to maintain conceptual consistency with the revised study design.

4. The limitations paragraph should be further strengthened. In particular, it should explicitly acknowledge the constraints related to in vitro-only evaluation, potential artefacts in apparent solubility measurements, and the absence of in vivo validation.

Addressing these points will further improve clarity, transparency, and scientific rigor.

Author Response

We sincerely thank the Reviewer for the thorough evaluation of our manuscript and for the constructive comments provided. Below, we address each comment point by point.

Reviewer’s comment (1):
It should be clearly and explicitly stated in the manuscript that the reported “apparent solubility” values do not necessarily represent true molecular solubility. Given the possibility of supersaturation, polymer-assisted solubilization, and/or colloidal species formation, this distinction should be clearly discussed to avoid potential misinterpretation of the data.

Response (1):
We sincerely thank the Reviewer for this important and insightful comment. We fully agree that the distinction between apparent solubility and true thermodynamic molecular solubility should be clearly stated to avoid potential misinterpretation.

Accordingly, we have revised the manuscript to explicitly clarify this issue. A definition of “apparent solubility” and its distinction from true molecular solubility has been added in Section 2.2. Furthermore, in Section 2.4, we have expanded the discussion to emphasize that the reported concentration values may reflect a metastable supersaturated state and may include contributions from polymer-associated species and/or colloidal drug-rich aggregates formed under non-equilibrium conditions.

These revisions clearly define the terminology and ensure proper interpretation of the reported data.

 

Reviewer’s comment (2):

To better demonstrate process robustness and reproducibility, the relative standard deviation (RSD%) values for drug content and/or solubility measurements (particularly for BB5-A, BB5-B, and BB5-C batches) should be presented in a clear tabulated format. This will strengthen the reliability of the reported results.

Response (2):

We sincerely thank the Reviewer for this valuable suggestion. In response, Tables 1 and 2 have been revised to explicitly include the measurement errors (expressed as mean ± SD), thereby improving the transparency and reliability of the reported drug content and solubility data. Additionally, a new Supplementary Table (Table S2) has been introduced in the Supplementary Materials, presenting the detailed results of the In Vitro Dissolution Test for BB5-A, BB5-B, and BB5-C, including the corresponding variability data.

 

Reviewer’s comment (3):

The term “selected formulation” should be used consistently throughout the manuscript. Please ensure that terms such as “optimal formulation” or similar expressions are fully removed to maintain conceptual consistency with the revised study design.

Response (3):

We sincerely thank the Reviewer for this important comment. The manuscript has been carefully revised. All expressions suggesting optimization (e.g., “optimal formulation,” “most favorable,” “best-performing,” or similar wording) have been removed or rephrased to maintain full conceptual consistency with the revised study design. The terminology has been unified in manuscript to avoid any implication of statistical optimization.

 

Reviewer’s comment (4):

The limitations paragraph should be further strengthened. In particular, it should explicitly acknowledge the constraints related to in vitro-only evaluation, potential artefacts in apparent solubility measurements, and the absence of in vivo validation.

Response (4):

We thank the Reviewer for this important and constructive suggestion. In revised version of manuscript the Authors have added a new section (Section 2.7 Limitations of the Study), in which the key limitations of the present work are explicitly discussed.