Influence of the Polymer and Solvent Variables on the Nanoencapsulation of the Flavonoid Quercetin: Preliminary Study Based on Eudragit^® Polymers

Round 1

Reviewer 1 Report

Comments are in the attached file

Comments for author File: Comments.docx

Minor errors should be check

Author Response

Dear reviewer, we appreciate the review of our work and all your comments; we look forward to answering all your questions. The English language and typos were also reviewed.

The article “Influence of the Polymer & Solvent Variables on the Nanoencapsulation of the Flavonoid Quercetin: Preliminary Study 3 Based on Eudragit® Polymers” is well written and have significance in the domain.

Reply.- We appreciate your kind comment. Also, as suggested by other reviewers we will improve the images.

However, there is a questions regarding Characterization of the Nanoencapsulation.

• Why the characterization of Nanoencapsulation not determined by XRD, UV, spectrometer and FTIR？

Reply.- Thank you for your observation.

Since the main objective of this research is to develop a simple methodology to encapsulate quercetin and to determine how the main variables are affected by the technique used, in our work, we used the methodology employed by Pacheco et al. (2022, Doi: 10.15517/rev.biol.trop..v70i1.48746), where the percentage of encapsulation was determined by performing a calibration curve of the analyte to determine the concentration of quercetin in the supernatants after ultracentrifugation. This basic methodology is effective because it does not require very sophisticated equipment, is reproducible, and provides a quantification of the analyte under study (https://doi.org/10.1016/j.jcis.2022.03.144).

Reviewer 2 Report

My comments are as follow:

·         In Figure 1, I suggest to improve the quality of the images.

·         In lines 79 and 81, you repeated the same “Therefore” connector.  I suggest to modify some of them.

·         In lines 351 and 352, you stated that “All four polymers and Qr showed no significant (p ≤ 0.001) cytotoxicity”. In this way, statistically “no significant” is when a “p-value is higher than 0.05 (p > 0.05)”.

·         In all the Tables and numbers of your manuscript, I suggest to work with two decimals for numbers with one integer number (i.e., Table 3, 3.71 it is OK), with one decimal for numbers with two integer numbers (i.e, in Table 3, round to 80.7 instead of 80.69), and without decimals for numbers with three or more integer numbers (i.e, in Table 3, round to 152 instead of 151.68).  I suggest to do the same with all the numbers and tables in your manuscript.

·         In Figure 5, I suggest to apply a two-way ANOVA to evaluate the effect of  different concentrations of the polymers (from 100 to 1,000 μg/mL) between each methacrylate Eudragit polymers (EPO, E100, L100, & L100-55) and Quercetin (Qr) (i.e., you can compare statistically differences for a concentration of 100 μg/mL between EPO, E100, L100, L100-55 and Qr, then for 200 μg/mL, and so on).

·         In your manuscript, sometime you indicate “significant for p ≤ 0.05”; then you indicate “not significant for p ≥ 0.05”. Consequently, what happen if in statistical result of a research we obtain exactly a p = 0.05? is significant? Is not significant?

Author Response

Dear reviewer, we appreciate the review of our work and all your comments; we look forward to answering all your questions. The changes made are marked with the change control tool. The English language was also reviewed.

In Figure 1, I suggest to improve the quality of the images.

Reply.- Figures were improved; hopefully, the images will be more understandable to readers.

In lines 79 and 81, you repeated the same “Therefore” connector.  I suggest to modify some of them.

Reply.- The connectors were changed, as well as the paper was revised to avoid this error.

In lines 351 and 352, you stated that “All four polymers and Qr showed no significant (p ≤ 0.001) cytotoxicity”. In this way, statistically “no significant” is when a “p-value is higher than 0.05 (p > 0.05)”.

Reply.- Thanks for the observation.  In the hemolytic activity assay the results revealed that four polymers and Qr at different concentrations showed high significant difference compared to the Positive Control, which consists of Distilled Water and causes total hemolysis of the red cells (p < 0.001 when compared to positive control).

We apologize for the confusion in the interpretation of the results, we accept that it was our typing error.

In all the Tables and numbers of your manuscript, I suggest to work with two decimals for numbers with one integer number (i.e., Table 3, 3.71 it is OK), with one decimal for numbers with two integer numbers (i.e, in Table 3, round to 80.7 instead of 80.69), and without decimals for numbers with three or more integer numbers (i.e, in Table 3, round to 152 instead of 151.68).  I suggest to do the same with all the numbers and tables in your manuscript.

Reply.- Thank you for your suggestion, your comment was taken into account, and the corresponding changes were made.

In Figure 5, I suggest to apply a two-way ANOVA to evaluate the effect of  different concentrations of the polymers (from 100 to 1,000 μg/mL) between each methacrylate Eudragit polymers (EPO, E100, L100, & L100-55) and Quercetin (Qr) (i.e., you can compare statistically differences for a concentration of 100 μg/mL between EPO, E100, L100, L100-55 and Qr, then for 200 μg/mL, and so on).

En la figura 5, sugiero aplicar un ANOVA de dos vías para evaluar el efecto de las diferentes concentraciones de los polímeros (de 100 a 1.000 μg/mL) entre cada uno de los polímeros de metacrilato Eudragit (EPO, E100, L100 y L100-55) y la quercetina (Qr) (es decir, se pueden comparar estadísticamente las diferencias para una concentración de 100 μg/mL entre EPO, E100, L100, L100-55 y Qr, luego para 200 μg/mL, y así sucesivamente).

Reply.- Thanks for the observation, analyses were performed between the same concentrations between treatments, which can be seen in Table 4.

In your manuscript, sometime you indicate “significant for p ≤ 0.05”; then you indicate “not significant for p ≥ 0.05”. Consequently, what happen if in statistical result of a research we obtain exactly a p = 0.05? is significant? Is not significant?

Reply.- As in question 3, it was a typo; the changes have been made. However, to answer your question. The p-value is a statistical measure that indicates the probability of obtaining a result equal to or more extreme than the observed result, assuming the null hypothesis is true. In general, if the p-value is less than or equal to a predefined significance level, such as 0.05 (also known as the alpha level), the results are statistically significant (PMID: 20445438). This means that there is a 5% probability that the observed results are due to chance or random chance and not due to a real relationship or effect.

In summary, if p equals 0.05, the results are considered to be significant at a 5% significance level. We hope we have satisfactorily answered the question.

Reviewer 3 Report

1.      Figure 1, 2, 3 resolution are too low, please consider providing higher resolution images.

2.       Any explanation why increases the volume of OP and AP causing the decrease of the size?

3.       Similarly, please explain why increase the volume of PA, the size decreases first and then increase again.

4.       I think the current work is too preliminary, I would recommend the author add some toxicity studies of the PNPs, and may provide an potential application of the PNPs.

English is fine. 

Author Response

Dear reviewer, we appreciate the review of our work and all your comments; we look forward to answering all your questions. The changes made are marked with the change control tool. The English language was also reviewed.

Comments and Suggestions for Authors

1. Figure 1, 2, 3 resolution are too low, please consider providing higher resolution images.

Reply.- Figures were improved; hopefully, the images will be more understandable to readers. In addition, as supplementary material, a table has been included with the results corresponding to Figure 2 for each of the formulations evaluated at different concentrations.

2. Any explanation why increases the volume of OP and AP causing the decrease of the size?

Reply.- The successful formulation of stable and efficient nanocarriers requires the preparation of homogeneous (monodisperse) populations of nanocarriers of a given size. However, it is difficult to control the particle size distribution without considering the composition of the nanocarriers and the nature of the solvents and co-solvents used during their preparation (PMID: 29783687).

Among the various precipitation techniques, liquid-antisolvent addition nanoprecipitation has been the most widely reported. In this process, a solvent (can be any organic solvent such as acetone, ethanol, or methanol), preferably in a water-miscible solvent (in which the drug has a suitable solubility), is mixed with an antisolvent (water, in most cases) by applying mixing forces. For it has been observed that the addition of some external factors (such as ultrasonic waves or high revolutions in the mixture) usually results in a smaller particle size or a narrower particle size distribution (PSD) (PMID: 18082385, PMID: 23333709).

The smaller particle size obtained by these methods is the result of faster mixing to affect the nucleation stage or by arresting particle growth (PMID: 23333709). Thus, we assume that the decrease in size is given first by sonication to dissolve the polymers in the corresponding solvent but mainly on the application of mixing forces. In addition, the use of surfactants has been reported to help decrease the average particle size in nanoformulations (PMID: 30678132) such as Qr-PNPs.

3. Similarly, please explain why increase the volume of PA, the size decreases first and then increase again.

Reply.- As in the previous question, the use of high mixing forces helps in particle decrease, however when the masses of polymer were increased under the same condition, there was a gradual increase in size; this is due to the degree of supersaturation, which is the ratio between the concentration of the solute in that solution at that particular condition (PMID: 23333709). Therefore, the solubility profile of the solute in different solvent/antisolvent combinations at different temperatures must be known beforehand to calculate the degree of supersaturation; (in our study, it was at room temperature).

Any condition that increases the concentration of the solute or drug in solution or reduces the solubility of the drug (increase in antisolvent, decrease in temperature) ultimately increases the degree of supersaturation. Thus, if the degree of supersaturation is low and reached slowly (in case of poor mixing), the nucleation rate is slow, and the system takes longer, which favors particle growth over nucleation (PMID: 23333709).

However, high supersaturation also increases the mass transfer gradient and can increase particle size. Thus, the effect of supersaturation on final particle size depends solely on how supersaturation is achieved. This can be modified by altering some parameters of the antisolvent phase (volume, temperature) and/or of the solvent phase (volume, concentration) (PMID: 12404417).

We hope we have answered your questions. The related information has been added to the discussion section.

4. I think the current work is too preliminary, I would recommend the author add some toxicity studies of the PNPs, and may provide an potential application of the PNPs.

Reply.- Thank you for your feedback. The objective of the present work was the development of an easy, fast methodology that does not require sophisticated equipment for the encapsulation of water-insoluble molecules such as the flavonoid quercetin. This is to be able to administer either quercetin or other metabolites. It was decided to use Eudragit polymers because they are safe and widely used by the pharmaceutical industry. Since, as you comment, it is necessary to perform more toxicity tests in normal cells such as Vero cells or PBMC, we evaluated the formulations in human erythrocytes as a toxicity test and to prove that they have potential for future research (which is a future perspective) in in vivo models as well as the evaluation of other biological activities such as antioxidant, microbiological and against tumor cells.

Round 2

Reviewer 3 Report

All the comments are addressed. 

English is fine