Chitosan-olive oil microparticles for phenylethyl isothiocyanate delivery: Optimal formulation

Phenylethyl isothiocyanate (PEITC), a chemopreventive compound, is highly reactive due to its considerably electrophilic nature. Furthermore, it is hydrophobic and has low stability, bioavailability and bioaccessibility. This restricts its use in biomedical and nutraceutical or food applications. Thus, the encapsulation of this agent has the function of overcoming these limitations, promoting its solubility in water, and stabilizing it, preserving its bioactivity. So, polymeric microparticles were developed using chitosan-olive oil-PEITC systems. For this, an optimisation process (factors: olive oil: chitosan ratio and PEITC: chitosan ratio) was implemented through a 3-level factorial experimental design. The responses were: the particle size, zeta-potential, polydisperse index, and entrapment efficiency. The optimal formulation was further characterised by FTIR and biocompatibility in Caco-2 cells. Optimal conditions were olive oil: chitosan and PEITC: chitosan ratios of 1.46 and 0.25, respectively. These microparticles had a size of 629 nm, a zeta-potential of 32.3 mV, a polydispersity index of 0.329, and entrapment efficiency of 98.49%. We found that the inclusion process affected the optical behaviour of the PEITC, as well as the microparticles themselves and their interaction with the medium. Furthermore, the microparticles did not show cytotoxicity within the therapeutic values of PEITC. Thus, PEITC was microencapsulated with characteristics suitable for potential biomedical, nutraceutical and food applications.

The reviewer is correct, and this was always considered as can be seen in the design itself when we specified in line 139 "(…) the process began with the addition of 4 mL of CS (…)", which means that the concentration of chitosan was a constant in all experiments. As perhaps it was not entirely clear, now we highlight it in the text.

Comment 3: It is not clear why olive oil was incorporated in formulation.
Lines 64-66 say "Besides, PEITC was already stabilised with vegetable oils, such as olive oil, once vegetable oil protects non-polar ITCs from decomposition or volatilisation." This may have gone very unnoticed in the text, which is why it is now reinforced between lines 94-96 of the introduction, in which the objectives of the work are highlighted.

Comment 4:
To determine the significant variable, ANOVA tablet must be included. It is not possible for the reader to conclude if the factor is significant without ANOVA table. Line 453-547 mention the factors are significant; however, it is not possible to confirm without ANOVA table.
As mentioned in line 323 all the data and statistics of the experimental design are available in the S1 Appendix. Comment 5: All particle size graphs don't have 100% intensity; Y-axis should be explained in the manuscript. In addition, dilution factor used for PSD is not mentioned in the paper.
The intensity distribution is naturally weighted according to the scattering intensity of each particle fraction or family, therefore 100% is obtained by integrating the area under the curve. According to the reviewer's request, we add a few exclamatory words in the methodology. For more information you can consult the link: https://www.materials-talks.com/blog/2017/01/23/intensity-volume-number-which-size-is-correct/ To carry out the measurements of size and zeta-potential there was no dilution, it was done directly on the formed system.

Comment 6: Preparation method for microparticles is not incorporated in the manuscript.
We regret not agreeing with the reviewer, but that is not correct, Section "2.2. Microparticle production" specifies the entire preparation of the microparticles.

Comment 7:
The formulation composition is not shown in the table.
The formulation is what is optimised, that is why it is well reported, and even in Table 5 the optimal composition is shown. Thank you very much for noticing the formatting error, it is fixed in the new version.

2.
Page 10, Line 214-216: The tested concentrations for different treatments were different. Please explain and make it clear in these lines.
We appreciate the comment, it is clarified in the new version.

Please introduce design of experiments in the introduction section.
Thank you very much for the comment, in the new version we mention the experimental design on line 112 of the introduction.

4.
Page 20, Section 4: In-vitro biocompatibility studies: Two kinds of fluorescence measurements have been described in the methods section. Please clearly distinguish and elaborate on the fluorescence measurements at excitation wavelength of 560 nm and an emission of 590 nm; the fluorescence intensities at 485 nm. Only % metabolic inhibition was represented in the results and figures.
Thank you very much for noticing the error. It was really a typing error, because we use the same formula for XTT protocol (which is cheaper, but PrestoBlue is more sensitive) which absorbs at 485 nm.

5.
Authors should clearly state the optimal formulation chosen for all the characterization studies carried out.
The optimal formulation was specified in Table 5. In line with the reviewer's request, a clarification was added on line 411.

6.
Please include the importance of central composite design in the discussion by citing any previous works.
We appreciate the comment, unfortunately in this case we do not agree. The factorial design, at least in this work, is hardly a statistical tool, commonly used in all engineering for formulations. That is why we think that mentioning its importance in the discussion is like talking about any of the other analytical methodologies used, that would dilute the focus on the interpretation of the results.

7.
It is also recommended to cite any reports referring to the safe use of microparticle components especially highlighting the dosage.
We appreciate the recommendation, and note that this is already mentioned in the introduction.

8.
English needs to be thoroughly checked and can be improved at many places in the manuscript.
Thank you very much for the comment, the article was completely revised.