Utilizing TPGS for Optimizing Quercetin Nanoemulsion for Colon Cancer Cells Inhibition

Round 1

Reviewer 1 Report

In this study, the authors adopted a self-assembly technique to prepare the nanoemulsion (NE) by using the chosen oils according to quercetin (QR) maximum solubility and TPGS as a surfactant. They aimed to develop and optimize QR-loaded NE to overcome the QR drawbacks and improve QR release rate and antitumor efficacy. In the authors’ opinion, their work has significance in presenting a way to strengthen the QR’s anticancer effect and prove the safety of the QR-NE. However, the experimental data shown in this paper are not sufficient to draw a final conclusion, and the experiment itself is not reasonable and has obvious flaws. Therefore, this work needs major revisions before publication in Colloids and interfaces.

There are several questions as follow:

1.       In line 249 and line 260, authors gave two equations according to the Figure 2 and Figure 3. It’s confusing that none of the results shown in Table 1 match the two equation! Please explain this question!

2.       Please tell us the purpose for presenting the equations in line 249 and line 260!

3.       Please add the scale bar in Figure 4B!

4.       The pictures in Figure 2 and 3 are not clear enough.

5.       The authors foucus their work on the improved QR release rate. It would be better to introduce the relative background in the introduction or discussion! And please give the reasons why the authors used the group of QR in the drug release assays! I could not understand why the free drug need be released! How could the free drug be released from the free drug? Or please redisign a reasonable and suitable solution to support your argument.

 

6.       Please define the abbreviation at first mention and use it consistently thereafter, e.g. TPGS in line 17.

Author Response

Dear / Editor-in-Chief, Colloids and Interfaces Journal

Dear/ Respected reviewers

Thank you for giving us the opportunity to submit a revised draft of our manuscript titled [Utilizing TPGS for Optimizing Quercetin Nanoemulsion for Colon Cancer Cells Inhibition], manuscript ID: colloids-1859055 to Colloids and Interfaces.  I and my co-authors appreciate the time and effort that you and the reviewers have dedicated to providing your valuable and positive feedback on our manuscript. We are grateful to the reviewers for their insightful comments on our paper. We tried as much as possible to respond to most of the enquiries and suggestions provided by the respected reviewers. All changes were made through Microsoft word track changes. Here is our point-by-point response to the reviewers’ comments and concerns followed by references to some responses.

Accept our regards.

Reviewer # 1
Comment	Response
1.            In line 249 and line 260, authors gave two equations according to the Figure 2 and Figure 3. It’s confusing that none of the results shown in Table 1 match the two equations! Please explain this question!	The equations were calculated using NCSS 2020 software. For more precision, the results were re-evaluated using Design Expert 7.0.0 Stat Ease.Inc. software and more explanation was added to the discussion.
2.            Please tell us the purpose for presenting the equations in line 249 and line 260!	The equations were added to indicate the effect of each factor on the evaluated parameters (PS and EE). The represented coded sign could indicate the effect of each factor.
3.            Please add the scale bar in Figure 4B!	We have added the scale bar as suggested. Here is how the figure look when we put the scale on some selected particles.
4.            The pictures in Figure 2 and 3 are not clear enough.	New clear figures were added.
5.            The authors focus their work on the improved QR release rate. It would be better to introduce the relative background in the introduction or discussion! And please give the reasons why the authors used the group of QR in the drug release assays! I could not understand why the free drug need be released! How could the free drug be released from the free drug? Or please redesign a reasonable and suitable solution to support your argument.	We have modified the manuscript as suggested. The nano drug particle size in the nanoemulsion formulations facilitates the drug release rate which will be higher than free drug, besides the smaller particle size increases the drug solubility. The presence of surfactants such as TPGS in NE formulations could enhance the QR release rate via increasing QR solubility. TPGS could as a SAA improve the drug wettability in contact with water via the adsorption effect on a larger surface area of nano particles and rapid drug partitioning into diluted dissolution medium, mainly from small droplets. We have taken many studies as a reference with similar design, and many have concluded results in agreement with our results, which showed that NE formulations usually result in enhanced hydrophobic drugs release rate due to the effect of oil and interfacial film barriers.
6.            Please define the abbreviation at first mention and use it consistently thereafter, e.g. TPGS in line 17.	Thank you for the comment. The text has been modified as suggested.

 

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript entitled "Utilizing TPGS for Optimizing Quercetin Nanoemulsion for 2 Colon Cancer Cells Inhibition" described the formulation of quercetin nanoemulsion using TPGS as a surfactant. The nanoemulsion was characterized and tested in two cancer cell lines. The tissue of kidney, liver, and spleen of animals receiving the NE were observed for toxicity. There are some comments required to be addressed below.

1. Please review the examples of nanoemulsion use for cancer therapy in the Introduction.

2. In the introduction, please describe the novelty of this work.

3. Why was absorbance of quercetin measured at 258 nm not 380 nm?

4. Please include zeta potential values to demonstrate the charge of NE.

5. For NE, which is different from course emulsion, the colloidal stability is required to confirm the quality of the product. The authors are recommended to include size, PDI and zeta potential of NE after storage.

6. Please give a rationale of using 10%alcohol water as a release medium with reference.

7. Please specify MTT concentration in the method of cell viability test.

8. Line 278: It is not correct to say that the optimal PDI value is more than 0.9. It should be less than 0.3. 

9. Line 286: It might not be able to conclude that the nanoemulsion has a uniform size from TEM. This result is contrast to PDI values obtained from dynamic light scanning technique.

10. Please discuss more on the correlation of quercetin released from NE and the cytotoxicity results and compare with quercetin alone.

11. Please include details of animal study such as how animals took the NE etc.

Author Response

Dear / Editor-in-Chief, Colloids and Interfaces Journal

Dear/ Respected reviewers

Thank you for giving us the opportunity to submit a revised draft of our manuscript titled [Utilizing TPGS for Optimizing Quercetin Nanoemulsion for Colon Cancer Cells Inhibition], manuscript ID: colloids-1859055 to Colloids and Interfaces.  I and my co-authors appreciate the time and effort that you and the reviewers have dedicated to providing your valuable and positive feedback on our manuscript. We are grateful to the reviewers for their insightful comments on our paper. We tried as much as possible to respond to most of the enquiries and suggestions provided by the respected reviewers. All changes were made through Microsoft word track changes. Here is our point-by-point response to the reviewers’ comments and concerns followed by references to some responses.

Accept our regards.

 

 

Reviewer # 2
Comment	Response
1.     Please review the examples of nanoemulsion use for cancer therapy in the Introduction.	We have addressed the reviewer’s comment as suggested.
2.     In the introduction, please describe the novelty of this work.	“Quercetin (QR) is a polyphenolic lipophilic compound that was chosen due to its colorectal anticancer activity. Nanoparticles could improve cancer therapy via tumor targeting by utilizing D-tocopheryl polyethylene glycol succinate (Vitamin-E TPGS) TPGS as a surfactant in nanoemulsion preparation which is considered an efficient drug delivery system for enhancing lipophilic antineoplastic agents. Thus, this study is aimed to develop and optimize QR-loaded nanoemulsion (NE) using TPGS as a surfactant to enhance the QR antitumor activity.” The text has been mentioned in the abstract to indicate the importance of utilizing new formulation methods for enhancing the nanoemulsion utilizing different types of oils.
3.     Why was absorbance of quercetin measured at 258 nm not 380 nm?	The absorbance of quercetin used in this study was 370nm not 258nm. That was selected according to manufacturer of the product. We modified that in the manuscript.
4.     Please include zeta potential values to demonstrate the charge of NE.	The zeta potential of selected NE was done, and it was -21 as shown in the picture below.
5.     For NE, which is different from course emulsion, the colloidal stability is required to confirm the quality of the product. The authors are recommended to include size, PDI and zeta potential of NE after storage.	The PS was measured for selected F-5 Nanoemulsion as shown in the picture below at days (1, 30, 60) and it was very close to what have been found in the first day after NE formulation.
6.     Please give a rationale of using 10% alcohol / water as a release medium with reference.	Quercetin is a bioflavonoid compound with low water solubility[1]. Alcohol was added to the media to provide a sink condition in which the solubility of QR was different in nano-formulation[2] .
7.     Please specify MTT concentration in the method of cell viability test.	Thanks for the reviewer for the suggestion. We have included the concentrations in the method section as suggested.  “MTT assay was used to evaluate the viability of HCT-116 and HT-29 colon cancer cell lines following 48h of treatment with quercetin at 5, 10, 20, 50, and 100 µM.“
8.     Line 278: It is not correct to say that the optimal PDI value is more than 0.9. It should be less than 0.3.	The PDI data was deleted as usually using dynamic light scanning technique does not give accurate results about the particle size compared to laser method (zeta size method). Additionally, PDI numerical value is ranged from 0.0 to 1.0 with no certain specific number for each formulation. FDA’s “does not mention the criteria for an acceptable PDI [3].
9.     Line 286: It might not be able to conclude that the nanoemulsion has a uniform size from TEM. This result is contrast to PDI values obtained from dynamic light scanning technique.	The PS was measured for selected F-5 Nanoemulsion as shown in the TEM picture below and it was very close to what have been found that nanoemulsion has particle size range from 9-56nm. We said it was uniform according to the results obtained from zetasizer.
10.  Please discuss more on the correlation of quercetin released from NE and the cytotoxicity results and compare with quercetin alone.	It was rephrased (line 387-390).
11.  Please include details of animal study such as how animals took the NE etc.	We have added and modified section 2.9 as required. “The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Animal Care and Use Committee at Taif University, Taif, Saudi Arabia. The protocol number is (42-0112).”

 

References

1. Van Hecke, E. and M. Benali, Solid dispersions of quercetin-PEG matrices: Miscibility prediction, preparation and characterization. Food Bioscience, 2022. 49: p. 101868.
2. Sun, M., et al., Development of nanosuspension formulation for oral delivery of quercetin. Journal of biomedical nanotechnology, 2010. 6(4): p. 325-332.
3. Danaei, M., et al., Impact of particle size and polydispersity index on the clinical applications of lipidic nanocarrier systems. Pharmaceutics, 2018. 10(2): p. 57.

 

Author Response File: Author Response.docx

Reviewer 3 Report

The current study has a relevant topic and is within the aim and scope of the journal. It is of interest to the readers of this journal. The manuscript is clearly written in professional, unambiguous language throughout. QR-loaded nanoemulsion using TPGS as a surfactant is prepared. Results from in vitro experiments show that QR-loaded nanoemulsion outperformed free QR in killing cancer cells. In vivo studies suggest QR-loaded nanoemulsion did not induce any adverse effects in rats. The weakness, however, is the conclusion drawn from the data reported, abnormal release profile reported, and missing description of in vivo study.

 

My biggest concern is the missing description of rat treatment. For example, how the QR-NE was administered. Only the histological analysis was mentioned. Additionally, the statement of the experiments conducted in this study are approved by the Animal Experiment Ethical Committee is missing.

 

The next important item is the conclusion (line 335, 382-383). MTT assay is a cell proliferation assay and is used to assess cytotoxicity. Data from the MTT assay does not support the conclusion that cellular uptake efficiency is improved. Please remove this statement or provide further justification.

 

Thirdly, the PDI value reported does not support the statement that QR-NE has a narrow and homogenous particle size distribution (lines 222-224). A PDI of 0.3 and below is considered to be a homogenous population. The particle size distribution of QR-NE in this study is polydispersed.

 

The release profile of QR in figure 5 is contrary to what we commonly observe. The entrapped cargo would show a slower release than its free counterpart. However, figure 5 shows the opposite. Please provide the raw data of the release study for review.

 

Lastly, in figure 1(a), the QR solubility in different oils is compared. However, whether there is a significant difference among groups is not mentioned. This is important since figure 1(a) justifies the reason why oleic oil is selected. If there is indeed a significant difference, please add an indication such as “*” in the graph to reflect the statistical significance.

 

The minor comments are listed below.

 

Please add a scale bar in figure 4(b) to help readers of the journal assess how large the particles in the figure are.

 

Throughout the manuscript, IC50 values of QR, blank nanoemulsion, and QR-loaded nanoemulsion from in vitro study are not mentioned. Please provide this data set to support the conclusion that QR-loaded nanoemulsion has better anti-cancer properties. (line 333).

Author Response

Dear / Editor-in-Chief, Colloids and Interfaces Journal

Dear/ Respected reviewers

Thank you for giving us the opportunity to submit a revised draft of our manuscript titled [Utilizing TPGS for Optimizing Quercetin Nanoemulsion for Colon Cancer Cells Inhibition], manuscript ID: colloids-1859055 to Colloids and Interfaces.  I and my co-authors appreciate the time and effort that you and the reviewers have dedicated to providing your valuable and positive feedback on our manuscript. We are grateful to the reviewers for their insightful comments on our paper. We tried as much as possible to respond to most of the enquiries and suggestions provided by the respected reviewers. All changes were made through Microsoft word track changes. Here is our point-by-point response to the reviewers’ comments and concerns followed by references to some responses.

Accept our regards.

 

Reviewer # 3
Comment	Response
My biggest concern is the missing description of rat treatment. For example, how the QR-NE was administered. Only the histological analysis was mentioned. Additionally, the statement of the experiments conducted in this study are approved by the Animal Experiment Ethical Committee is missing	We have added and modified section 2.9 as required. “The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Animal Care and Use Committee at Taif University, Taif, Saudi Arabia. The protocol number is (42-0112).”
The next important item is the conclusion (line 335, 382-383). MTT assay is a cell proliferation assay and is used to assess cytotoxicity. Data from the MTT assay does not support the conclusion that cellular uptake efficiency is improved. Please remove this statement or provide further justification.	The statement has been deleted as the reviewer suggested and it has been modified all over the manuscript.
Thirdly, the PDI value reported does not support the statement that QR-NE has a narrow and homogenous particle size distribution (lines 222-224). A PDI of 0.3 and below is considered to be a homogenous population. The particle size distribution of QR-NE in this study is polydispersed.	We agreed with the reviewer’s comment. The PDI data was deleted as usually using dynamic light scanning technique does not give accurate results about the particle size compared to laser method (zeta size method). Additionally, PDI numerical value is ranged from 0.0 to 1.0 with no certain specific number for each formulation. FDA’s “does not mention the criteria for an acceptable PDI [1].
The release profile of QR in figure 5 is contrary to what we commonly observe. The entrapped cargo would show a slower release than its free counterpart. However, figure 5 shows the opposite. Please provide the raw data of the release study for review.	The nano drug particle size in the nanoemulsion formulations facilitates the drug release rate which will be higher than free drug, besides the smaller particle size increases the drug solubility. The presence of surfactants such as TPGS in NE formulations could enhance the QR release rate via increasing QR solubility. TPGS could as a SAA improve the drug wettability in contact with water via the adsorption effect on a larger surface area of nano particles and rapid drug partitioning into diluted dissolution medium, mainly from small droplets[2]. Many studies have concluded results in agreement with our results, which showed that NE formulations usually result in enhanced hydrophobic drugs release rate due to the effect of oil and interfacial film barriers [2, 3].
Lastly, in figure 1(a), the QR solubility in different oils is compared. However, whether there is a significant difference among groups is not mentioned. This is important since figure 1(a) justifies the reason why oleic oil is selected. If there is indeed a significant difference, please add an indication such as “*” in the graph to reflect the statistical significance..	It was rephrased (lines 276-278).
The minor comments are listed below.   Please add a scale bar in figure 4(b) to help readers of the journal assess how large the particles in the figure are.	We have added the scale bar as suggested.
Throughout the manuscript, IC50 values of QR, blank nanoemulsion, and QR-loaded nanoemulsion from in vitro study are not mentioned. Please provide this data set to support the conclusion that QR-loaded nanoemulsion has better anti-cancer properties. (line 333).	We have added IC50 values in the manuscript as suggested.

References

1. Danaei, M., et al., Impact of particle size and polydispersity index on the clinical applications of lipidic nanocarrier systems. Pharmaceutics, 2018. 10(2): p. 57.
2. Buyukozturk, F., J.C. Benneyan, and R.L. Carrier, Impact of emulsion-based drug delivery systems on intestinal permeability and drug release kinetics. Journal of controlled release, 2010. 142(1): p. 22-30.
3. Md, S., et al., Formulation design, statistical optimization, and in vitro evaluation of a naringenin nanoemulsion to enhance apoptotic activity in A549 lung cancer cells. Pharmaceuticals, 2020. 13(7): p. 152.

 

Round 2

Reviewer 1 Report

The authors have improved their quality of manuscript in the revision version.  In my opinion, their work could be accepted in Journal of "Colloids and Interfaces".

Reviewer 2 Report

The manuscript is now acceptable for publication.

Reviewer 3 Report

The comments from the first round of review have been addressed. Details on the rat treatment have been added to the manuscript including the animal care guideline. Using MTT assay as a measurement for cellular uptake has been removed from the manuscript. The release profile has been clarified as well. In terms of minor comments, the scale bar and IC50 value have been provided.