Synthesis and Characterization of Quercetin–Iron Complex Nanoparticles for Overcoming Drug Resistance

Round 1

Reviewer 1 Report

In this manuscript, Bao et al. synthesized the Quercetin-Iron nanoparticles. The nanoparticles increased the stability and solubility compared to free quercetin. The preliminary cellular testing suggested that the Q-Fe nanoparticles could effectively inhibit the P-gp efflux pump. These NPs have great potential in overcoming pharmacokinetic limitations of free Q, and open possibility for future applications.

The experiments are rationally designed, and the manuscript is well written. It was recommended to accept after minor revision.

Minor questions:

1. In Figure 2D: The degree sign in the figure need to be corrected.

2. In Figure 5A: The Q-Fe ratios should be 1:5, 1:4, and 1:3 in the Table.

Author Response

Comments 1: In Figure 2D: The degree sign in the figure need to be corrected.

Responses: Thanks! The degree signs have been corrected!

 

Comments 2: In Figure 5A: The Q-Fe ratios should be 1:5, 1:4, and 1:3 in the Table.

Responses: Thanks! We have fixed the table ratios and made it in consistent sequence with the figure caption.

Reviewer 2 Report

The authors present a study where they tested different rates of a combination of Fe and Quercetin as well as different pH, solutions, and temperatures to obtain the best conditions to obtain the Q-Fe complex that improves the characteristics of solubility, and concentration, and present fewer quercetin breakdown products.   The authors concluded that the ratio to form the best Q-Fe complex was 2:1 and that the solubility is better when dissolved in DI water or 150mM NaCl.   However, the title does not reflect the work presented because most of the experiments are aimed at finding the best conditions for the complex, and only at the end of the paper they showed the results of an experiment carried out with BEC cells trying to demonstrate its effect on drug resistance and propose it as an anticancer treatment. They did not indicate what type of cells the BECs are, and a significant result is not observed, so it is necessary to carry out additional experiments testing different culture conditions (dose, incubation time) and measure more parameters to corroborate if the complex presents better anticancer effects than quercetin itself. I recommend that this paper be published in a technical journal or that additional experiments be included to demonstrate the title.

Author Response

Responses: (1) To reflect the significant amount work on characterization, we have changed the
tittle to “Synthesis and Characterization of Quercetin–Iron Complex Nanoparticles for
Overcoming Drug Resistance”.
(2) Detailed description of iBECs was included in the experimental methods. In brief,
it is human induced pluripotent stem-cell (iPSC) derived brain-like endothelial cells
(iBECs).
(3) We have included cytotoxicity studies and P-gp expression immunostaining on
MDA-MB 231 br, a variant of triple negative and highly metastatic breast cancer cells
line and iBECs. Please note that the P-gp expression is prevalent in drug-resistant
231 cell lines, which takes several month to develop from a regular cancer cell line by
selection. Therefore, we used iBECs that have high level expression of P-gp as a
model system. We have illustrated the P-gp expression in Figure 7.

Reviewer 3 Report

(1) In figure 1, what is the pH of samples without pH adjustment? The pH should be measured and provided. 

     Line 186, the text "no changes in absorption" should be checked, since the intensity was different between no pH adjustment and pH 7.

(2) Lines 251-252, the typical peaks of Q were not observed in Figure 2 is possibly due to the degradation of Q, how to exclude the possibility?

(3) Fe3+ is a strong oxidant, while quercetin is an antioxidant. Why did the reduction-oxidation reaction not between them? How to differentiate the Fe3+ from Fe2+ in UV-Vis and IR spectra? HPLC or MS is suggested to check the original structure of quercetin in Q-FeNPs.

(4) The percentage of Fe and quercetin in nanoparticles should be measured and given.

 

Author Response

Comments: (1) In figure 1, what is the pH of samples without pH adjustment? The pH should be
measured and provided.
Responses: The pH of the solution without pH adjustment was 6.4, which was added in the text
as follows: “The Q methanol solution without pH adjustment was around 6.4, after .. .”
Line 186, the text "no changes in absorption" should be checked, since the intensity was different
between no pH adjustment and pH 7.
Responses: The texts were rewritten to better reflect the plots as follows: “The Q methanol
solution without pH adjustment was around 6.4, after adjusting the solution pH to 7
with 100 mM NaOH, no changes in absorption peak position and relative peak
intensity between the two bands were observed after 2 hours at room temperature,
where the intensity difference was likely due to the baseline variation.”

Comments: (2) Lines 251-252, the typical peaks of Q were not observed in Figure 2 is possibly
due to the degradation of Q, how to exclude the possibility?
Responses: The absorption peak of the main degradation products are around 330 nm as shown in
Figure 1B. The lack of this peak and even no should peak in the UV-vis absorption
plots suggested the absence of degradation products.
Comments: (3) Fe3+ is a strong oxidant, while quercetin is an antioxidant. Why did the
reduction-oxidation reaction not between them? How to differentiate the Fe3+ from Fe2+ in UVVis
and IR spectra? HPLC or MS is suggested to check the original structure of quercetin in QFeNPs.
Responses: The coordination between Q-Fe has been well documented (Ref. 26, 29, 30, 47);
however, it is difficult to completely rule out the formation of Fe2+ during the process. Because
of the nanoparticle formation, HPLC or MS is not doable on these samples. For our future studies,
we plan to perform some XPS analysis to differentiate Fe2+ versus Fe3+ in the sample or ICP
quantification.
Comments: (4) The percentage of Fe and quercetin in nanoparticles should be measured and
given.
Responses: Yes! We have measured the percentage of Fe and quercetin by TGA, and detailed
analysis was shown in Table 1.

Reviewer 4 Report

A comprehensive report on the generation of FeQNPs, in different conditions, determining their stability nd solubility. Well organized, well written and interesting for a wide group of readers. 

Authors should have provided more biological data (antioxidant effect, effect on apoptosis etc). At least in part these  studies should be planned and discussed in the discussion section.

Author Response

Thanks for the suggestions! We have included the following additional data in the
manuscript:
(1) We have performed DPPH assay to evaluate the antioxidant activity of FeQNPs
with various Q-Fe ratios (new Figure 6).
(2) We have included P-gp expression immunostaining data on MDA-MB 231 br, a
variant of triple negative and highly metastatic cancer cells line and iBECs (new
Figure 7).
(3) We have also included cytotoxicity studies of FeQNPs as a new Figure S6

Reviewer 5 Report

It was a manuscript about the synthesis and evaluation of quercetin-iron nanocomplex for overcoming the drug resistance ability of cancer cells. Here are some comments on this study that should be considered before publication:

1.       Please improve the quality of the abstract.

2.       For chemical subheading you should write them as a sentence. Materials that were bought from the same company should be mentioned in one sentence. Moreover, you need to add the city and country of the companies that the materials were bought from them.

3.       TEM analysis should be written as a separate subheading. Moreover, please write the brand name and manufacturer country of the used instruments.

4.       Section 2.6 should be mixed with section 2.5.

5.       Please rewrite section 2.7.

6.       You didn’t mention anything about the fabrication of formulation in different temperatures in the method part.

7.       Please add XRD to section 2.5. Moreover, please do the XRD test for the prepared sample before increasing the temperature. The XRD result shown in figure S5 is not good as well. Please do it again.

8.       “… suggested an increase of water coordination with NP formation …” please rewrite.

9.       There are grammatical mistakes in the text. Please check and correct.

10.   Please improve the quality of the conclusion.

11.   Please do cytotoxicity tests (on both cancer and normal cell lines) to check the effectiveness of your proposed component.

 

 

Author Response

Comments: 1. Please improve the quality of the abstract.

Responses: Abstract was edited!

Comments: 2. For chemical subheading you should write them as a sentence. Materials that were bought from the same company should be mentioned in one sentence. Moreover, you need to add the city and country of the companies that the materials were bought from them.

Responses: Corrected!

Comments 3: TEM analysis should be written as a separate subheading. Moreover, please write the brand name and manufacturer country of the used instruments.

Responses: Related information was included.

Comments 4: Section 2.6 should be mixed with section 2.5.

Responses: Combined!

Comments 5 : Please rewrite section 2.7.

Responses: Section 2.7 was edited!

Comments: 6. You didn’t mention anything about the fabrication of formulation in different temperatures in the method part.

Responses: Experimental procedure for NP formation at different temperatures was added!

Comments: 7. Please add XRD to section 2.5. Moreover, please do the XRD test for the prepared sample before increasing the temperature. The XRD result shown in figure S5 is not good as well. Please do it again.

Responses: Corresponding description of XRD pattern collection was added to the characterization section (now section 2.6). As prepared samples are Q-Fe complex NPs, which did not show any signal. We have redone the XRD in Figure S5.

Comments: 8. “… suggested an increase of water coordination with NP formation …” please rewrite.

Responses: Sentence was edited.

Comments: 9. There are grammatical mistakes in the text. Please check and correct.

Responses: Double checked! Thanks!

Comments: 10. Please improve the quality of the conclusion.

Responses: Conclusion was edited!

Comments: 11. Please do cytotoxicity tests (on both cancer and normal cell lines) to check the effectiveness of your proposed component.

Responses: included cytotoxicity studies of FeQNPs as a new Figure S6.

Round 2

Reviewer 2 Report

The authors have satisfactorily responded to all the recommendations, so I agree to accept the paper for publication.

Reviewer 5 Report

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