Effects of Microencapsulated Ferulic Acid or Its Prodrug Methyl Ferulate on Neuroinflammation Induced by Muramyl Dipeptide
, Anna Bianchi 1,†, Barbara Pavan 2,*, Paola Tedeschi 1, Valentina Albanese 3, Luca Ferraro 4, Federico Spizzo 5, Lucia Del Bianco 5 and Alessandro Dalpiaz 1
Round 1
Reviewer 1 Report
The manuscript by Botti et al. describes the use of stearic acid-based microparticles as carriers of a methylated ferrulic acid as a drug precursor. The Authors described in great detail the process of synthesis of the target drug formulation (taking into account all steps) as well as provided the detailed characterization of the final product. Particularly noteworthy is the comprehensive analysis of the distribution and interaction of the prodrug and its active form with the carrier matrix, carried out using various techniques (DSC, XRD). In addition, the stability and kinetics of the pro-drug hydrolysis under storage and in physiological conditions have been studied in depth, which is of great importance from the point of view of target applications. The manuscript undoubtedly carries considerable scientific novelty and remains within the scope of IJERPH journal. I have some comments on technical issues and the way the results are described. The manuscript is very long and at some moments quite disorganized, with numerous digressions. The discussion of results and conclusions at times do not correspond directly with the results presented. Nevertheless, after revision, this work can be considered for publication.
Please find my detailed comments below:
1) The manuscript is very long - consider moving less important information (such as detailed descriptions of characterization procedures) to Supplementary Materials
2) Sometimes it is difficult to separate the results and their critical analysis from the far-reaching perspective drawn from them by the Authors. This makes the manuscript at times difficult for the reader. The end of the Results section (p. 22) is practically a summary and conclusions mixed with numerous future prospects, so that the article loses its clarity - it is difficult to really grasp what, according to the Authors, directly follows from the results of the experiments, and what is the authors' interpretation.
3) The source and explanation of the abbreviation describing trifluoroacetic acid (TFA) is not provided in the Materials section.
4) Line 149: the notation "1-2" is misleading and may suggest that this is a mixture of compounds. I recommend a more explicit notation or suggestion that these are syntheses carried out in parallel
5) Line 212: why do the Authors use the concentration in scientific notation (5-10-2M), while in other cases prefixes are used - I recommend standardization.
6) Subsection 2.6 - more experimental details describing incubation conditions during kinetics analysis is needed (whether closed vessel/air access, humidity control, etc.).
7) Subsection 2.13 - the description of the immunoenzymatic assay is poorly described. Please describe the procedure in more detail or indicate the specific detection kit that was used.
8) Are the authors confident that sample preparation for SEM analysis does not disrupt sample morphology?
9) On what basis do the Authors make the assumption that a 15-minute incubation in hot methanol leads to full dissolution of SLMs? Has this been verified?
10) Section 3.1 in Results is more like Introduction, as it has little correspondence with the experimental results described next.
11) Table 1. Please explain the term "Drug loading (%)" in Figure caption for better clarity.
12) Labels on the axes in Fig. 6 and 7 (XRD data) are blurry and the font is small. I will ask you to improve the quality of the graphs.
Author Response
Reviewer - The manuscript by Botti et al. describes the use of stearic acid-based microparticles as carriers of a methylated ferrulic acid as a drug precursor. The Authors described in great detail the process of synthesis of the target drug formulation (taking into account all steps) as well as provided the detailed characterization of the final product. Particularly noteworthy is the comprehensive analysis of the distribution and interaction of the prodrug and its active form with the carrier matrix, carried out using various techniques (DSC, XRD). In addition, the stability and kinetics of the pro-drug hydrolysis under storage and in physiological conditions have been studied in depth, which is of great importance from the point of view of target applications. The manuscript undoubtedly carries considerable scientific novelty and remains within the scope of IJERPH journal. I have some comments on technical issues and the way the results are described. The manuscript is very long and at some moments quite disorganized, with numerous digressions. The discussion of results and conclusions at times do not correspond directly with the results presented. Nevertheless, after revision, this work can be considered for publication.
Authors - We thank the reviewer for appreciating our manuscript and his/her work aimed to improve its quality. The manuscript was modified according to all the comments.
Please find my detailed comments below:
- Reviewer - The manuscript is very long - consider moving less important information (such as detailed descriptions of characterization procedures) to Supplementary Materials.
Authors – We thank the reviewer for this remark that we have carefully taken into account. Accordingly, the data of chromatography and NMR referred to FER-Me and Caf-Me were moved to Supplementary Materials. Moreover, all information about chromatographic precision, calibration method and accuracy of HPLC measurements were moved to Supplementary Materials
2) Reviewer - Sometimes it is difficult to separate the results and their critical analysis from the far-reaching perspective drawn from them by the Authors. This makes the manuscript at times difficult for the reader. The end of the Results section (p. 22) is practically a summary and conclusions mixed with numerous future prospects, so that the article loses its clarity - it is difficult to really grasp what, according to the Authors, directly follows from the results of the experiments, and what is the authors' interpretation.
Authors – This criticism raised by the reviewer has been carefully considered by the authors. Therefore, the end of the results section (page 22 of the original manuscript) was erased and the sentences about the future prospects were simplified and moved to the “Conclusions” section which in the revised manuscript was named “Conclusions and Future Perspectives”.
3) Reviewer - The source and explanation of the abbreviation describing trifluoroacetic acid (TFA) is not provided in the Materials section.
Authors – The information indicated by the reviewer was inserted in the revised manuscript
4) Reviewer - Line 149: the notation "1-2" is misleading and may suggest that this is a mixture of compounds. I recommend a more explicit notation or suggestion that these are syntheses carried out in parallel.
Authors - The manuscript was modified as recommended by the reviewer.
5) Reviewer - Line 212: why do the Authors use the concentration in scientific notation (5-10-2M), while in other cases prefixes are used - I recommend standardization.
Authors - 5×10-2 M was substituted with 50 mM
6) Reviewer - Subsection 2.6 - more experimental details describing incubation conditions during kinetics analysis is needed (whether closed vessel/air access, humidity control, etc.).
Authors - Further experimental data were added in subsections 2.6 and 2.19.
7) Reviewer - Subsection 2.13 - the description of the immunoenzymatic assay is poorly described. Please describe the procedure in more detail or indicate the specific detection kit that was used.
Authors – As requested by the reviewer, the procedure was described in more detail in the revised manuscript.
8) Reviewer - Are the authors confident that sample preparation for SEM analysis does not disrupt sample morphology?
Authors -The point raised by the reviewer is appropriate. We would remark that the SEM micrographs reported in Figure 5 suggest that the samples are constituted by aggregates of microparticulate systems, whose spherical shape appears more evident for the samples based on tristearin. The gold coating is proposed as a consolidate procedure for SEM analysis of micro and nano-particulate systems based on both polymeric and solid lipid matrixes. We have previously adopted this procedure for the SEM analysis of microparticles based on tristearin or stearic acid evidencing that their conformation can depend on the type of loaded drug (refs 27,28,54).
9) Reviewer - On what basis do the Authors make the assumption that a 15-minute incubation in hot methanol leads to full dissolution of SLMs? Has this been verified?
Authors - We have adopted the same procedure as previusly reported (reference 28 of the revised manuscript). We have taken into account that 80° C allow to melt the lipid phases, allowing to facilitate their dissolution in methanol and induce the complete relase of the drugs. We have verified that at these conditions the complete release of loaded compounds is obtained within 5 min. Reference 28 was quoted in section 2.16.
10) Reviewer - Section 3.1 in Results is more like Introduction, as it has little correspondence with the experimental results described next.
Authors – We thank the reviewer for this remark. Accordingly, section 3.1 was strongly reduced and focalized to the synthesis of Caf-Me and Fer-Me.
11) Reviewer - Table 1. Please explain the term "Drug loading (%)" in Figure caption for better clarity.
Authors – The explanation of the term “Drug loading (%) was inserted as a note of Table 1
12) Reviewer - Labels on the axes in Fig. 6 and 7 (XRD data) are blurry and the font is small. I will ask you to improve the quality of the graphs.
Authors – Labels on the axes of XRD figures were improved.
Reviewer 2 Report
The comments for authors are appended below:
11. Remove full stop (.) at the end of the title.
22. Authors used several times ‘we’ in the manuscript. Suggest removing and rephrase the statements (eg Line 374, 377, 490).
33. Line 174: …as a control…
44. Line 176: …as a reference….
55. Line 317: Remove “by omitting the” and rephrase as “without the drugs”.
66. Line 363: Justify the reason for using a high stirring speed (100 rpm) in dissolution and release studies and include the same explanation in Results and Discussion. Have authors studied the effect of stirring speed at low or medium on release of Fer and Fer-Me prior to using a high speed? If yes, it is good to add the data into the manuscript.
77. Line 366: Suggest to add a statement “The collected samples were quantified for Fer& Fer-Me using the developed HPLC method” after …to maintain sink conditions….
88. Line 553: The mentioned regression value is for “r or r2”? Suggested to recheck.
99. Line 564, Table 1: Fer & Fer-Me encapsulations were high with stearic acid compared to Tristearin. Discuss the possible reasons in the results and discussion section.
Author Response
- Reviewer - Remove full stop (.) at the end of the title.
Authors – The full stop (.) at the end of the title is removed in the revised manuscript and Supplementary Materials
- Reviewer - Authors used several times ‘we’ in the manuscript. Suggest removing and rephrase the statements (eg Line 374, 377, 490).
Authors – All the statements with the term “we” were rephrased as suggested by the reviewer.
- Reviewer - Line 174: …as a control…
Authors – The sentence was corrected as indicated by the reviewer.
- Reviewer - Line 176: …as a reference….
Authors – The sentence was corrected as indicated by the reviewer.
- Reviewer -Line 317: Remove “by omitting the” and rephrase as “without the drugs”.
Authors – The sentence was rephrased as indicated by the reviewer.
- 66. Reviewer - Line 363: Justify the reason for using a high stirring speed (100 rpm) in dissolution and release studies and include the same explanation in Results and Discussion. Have authors studied the effect of stirring speed at low or medium on release of Fer and Fer-Me prior to using a high speed? If yes, it is good to add the data into the manuscript.
Authors - The reviewer raises an interesting point. The authors are sorry, but the effects of different stirring speeds on dissolution or release of Fer or Fer-Me were not performed. The 100 rpm were adopted in accordance with previous studies performed for release studies from solid lipid microparticles (Refs. 27, 28, 54).
- Reviewer - Line 366: Suggest to add a statement “The collected samples were quantified for Fer& Fer-Me using the developed HPLC method” after …to maintain sink conditions….
Authors – We thank the reviewer for this suggestion. The statement was added in the revised manuscript.
- Reviewer - Line 553: The mentioned regression value is for “r or r2”? Suggested to recheck.
Authors – We thank the reviewer for the remark. All mentioned regression values reported in the manuscript are “r”.
- Reviewer - Line 564, Table 1: Fer & Fer-Me encapsulations were high with stearic acid compared to Tristearin. Discuss the possible reasons in the results and discussion section.
Authors – As requested by the reviewer, the possible reasons of higher loading of drugs in stearic acid than in tristearin microparticles have been discussed in the results and discussion section.
