Production and Properties of Quercetin-Loaded Liposomes and Their Influence on the Properties of Galactomannan-Based Films

Round 1

Reviewer 1 Report

The authors studied galactomannan films obtained from Cassia grandis seeds. The reported properties are quite macroscopic. Some figures are to be redrawn. The paper remains on a descriptive level.

The paper is clearly more oriented towards food industry (use of seeds are primary material) or medical applications (drug delivery, pharmaceutical applications, study of exposure to the skin). The authors fail to justify the submission to the applied nano journal. The submission to Applied Nano is a misfit in  my opnion.

I strongly recommend the authors to seek for a better-suited journal to disseminate their work in order to reach the adapted readership.

Author Response

Dearest Reviewer, we are very grateful for your understanding about the relevance of the manuscript. We modified the archive trying to accomplish your requirements. Please see the new “Introduction” and the “Conclusions” topics, which we think that fit the scope of the prestigious Applied Nano Journal.  

Author Response File: Author Response.docx

Reviewer 2 Report

Comments for author File: Comments.pdf

Author Response

RESPONSE TO REVIEWER 2 Authors have reported the development of quercetin-loaded liposome-embedded galactomannan films and their physicochemical characterization using various analytical techniques like DLS, SEM, FTIR, XRD, etc. The present study does not report any application. This article needs significant improvement before it can be considered for publication in the Applied Nano journal.  Comments: 1. The introduction has too basic information, and the knowledge gap needs to be established better. - Reply:

Dearest reviewer, we are very grateful for your valuable comments, which highly improved the work. We recognized our fault; because of this, we added more than 10 new references to the revised manuscript. We hope that the marked changes are now satisfactory and allow the manuscript improvement.

 

2. Lecithin is a mixture of mainly phosphatidylcholine and phosphatidylethanolamine. Both the phospholipids are zwitterionic at physiological pH. Then why the Zeta potential is showing ~ -45 mV value?- Reply:Dearest reviewer, thank you for your constructive comment. Considering that lecithin is a mixture of phospholipids, we believe that the negatively-charged groups of phospholipids (pKa values of anionic phosphatidic groups are typically around 1.5; Ogawa, Decker, & McClements, 2004) directly influence values of zeta potential. This behavior was also reported by Gibis, Vogt, and Weiss (2012; Encapsulation of polyphenolic grape seed extract in polymer-coated liposomes) for 1% (w/v) lecithin liposomes, which had negative surface charges regardless of pH. Additionally, the magnitude of surface charges decreased from -21.8 at pH 3 to -57.7 at pH 11. More recently, Talón et al., (2019; Encapsulation of eugenol by spray-drying using whey protein isolate or lecithin: Release kinetics, antioxidant and antimicrobial properties) also reported a zeta potential value of -45.7 mV for liposomes based on lecithin and maldodextrin. This charge was inverted when chitosan was incorporated, leading to a zeta potential of +61.5 mV. 3. The method section needs to clarify the antioxidant activity assay. Was quercetinreleased from QT-LL for this study? If so, then the quercetin shall behave like a free quercetin. Therefore, why is there a difference in antioxidant activity between quercetin and QT-LL? - Reply:Dearest reviewer, thank you for your constructive comment. Greater antioxidant activities of free molecules were also observed, for example, by Talón et al. (2019) working with the encapsulation of eugenol by spray-drying in lecithin and maltodextrin matrices. Pure eugenol showed the best antioxidant activity when compared to emulsions based on lecithin as wall material, with or without oleic acid as carrier agent, and chitosan as coating. It is important to mention that the ethanol injection method used here is faster and simpler than other methods reported by the scientific literature: magnetic stirring (<1000 rpm) and approximately 20 minutes are able to produce liposomes with small particle sizes, good monodispersed properties, without the need for sonication, extrusion or homogenization by Rotor Stator. The lower antioxidant activities of QT-LLs than free-quercetin could be due to the partial oxidation of the compound during the process. 4. Please verify the loading capacity (%) calculation. For example, the theoretical loading capacity for 100 μg/mL of quercetin is = (100/25000) x 100 % = 0,4 %. However, it is reported as ~ 5 % in Figure 2B. Please clarify.- Reply:Dearest reviewer, we recognize our fault. The equations were corrected. Please consider the new formula:

EE (%) = (Wtotal - Wfree)/Wtotal x 100,

LC (%) = (Wtotal – Wfree)/Wvehicle) x 100,

 5. Figure 3C, finding any difference among film morphology without or with QT-LL is challenging. Also, authors are requested to include improved images to support their claims. The presence of QT-LL in the films shall be imaged in high resolution. - Reply:Dearest reviewer, thank you for your constructive comment. The figure was update as requested. Please see now SEM images with 10000x magnification. 6. The release kinetics of QT-LL and films + QT-LL must be provided to check if the QTLL immobilization in films has any effect on drug release. - Reply:

Dearest reviewer, thank you for your constructive comment. In this study, we produced quercetin-loaded liposomes immobilized on galactomannan-based films aiming at the development of a matrix valuable for Medicine and Biology. We recognize our fault on the absence of the release kinetics of QT-LL and films + QT-LL; however, it is now one of the objectives of our research group and should be submitted to related journals in a few months. We strongly hope that the changes made in this version allowed the manuscript improvement, and are now satisfactory for you.

 7. Authors shall present a skin permeation study of QT-LL films to support the enhanced skin permeability hypothesis. This way, authors will have at least one application for their present system.- Reply:

Dearest reviewer, thank you for your constructive comment. In this study, we produced quercetin-loaded liposomes immobilized on galactomannan-based films aiming at the development of a matrix valuable for Medicine and Biology. The applicability of this matrix, especially in the nanopharmacology field, is now one of the objectives of our research group and should be submitted to related journals in a few months. We strongly hope that the changes made in this version allowed the manuscript improvement, and are now satisfactory for you.

 

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript needs to be improved (see attached file).

Comments for author File: Comments.pdf

Author Response

RESPONSE TO REVIEWER 3 On the manuscript «Effect of quercetin-loaded liposomes on the physicochemical properties of galactomannan-based films obtained from Cassia grandis seeds»by Priscilla Barbosa Sales de Albuquerque, Marthyna Pessoa de Souza, Ana Isabel Bourbon, Miguel Cerqueira, Lorenzo Pastrana, Paula Jauregi, José A. Teixeira, Maria das Graças Carneiro-da-Cunha. This manuscript is devoted to the design of nanomaterials based on biologically active components such as galactomannan, quercetin and lecithin. Lecithin liposomes loaded with quercetin were obtained and some of their physicochemical properties were studied in the work. In addition, galactomannan-based films with the inclusion of lecithin liposomes were obtained. The resulting film materials were comprehensively studied. In general, the presented liposomes and films, taking into account the natural origin of their initial reagents, are very promising for the design of various, including personalized dosage forms. The studies are relevant and confirmed by modern methods of analysis. There are, however, a number of comments and questions about the manuscript. 1. Since a significant part of the manuscript is devoted to the preparation and study of the properties of quercetin-loaded lecithin liposomes, I recommend revising the title of it. I would propose the following title: “Production and properties of quercetin-loaded liposomes and their influence on the properties of galactomannan-based films”.- Reply: Dearest reviewer, the title was modified accordingly your comment. 2. Literature-patent search shows the presence of a huge number of publications on the synthesis of lecithin liposomes, including the method of ethanol injection. Below are some of them.1. Ueno, M., Ohkuma, A., Kikuchi, H., Ohsawa, S., & Shibusawa, K. (1985). The preparation of single bilayer liposome by injection method and the effect of some additives on the size distribution. Yakugaku Zasshi - Journal of the Pharmaceutical Society of Japan, 105(3), 224-231.2. Imura, T., Otake, K., Hashimoto, S., Gotoh, T., Yuasa, M., Yokoyama, S., ... & Abe, M. (2003). Preparation and physicochemical properties of various soybean lecithin liposomes using supercritical reverse phase evaporation method. Colloids and Surfaces B: Biointerfaces, 27(2-3), 133-140. https://doi.org/10.1016/S0927-7765(02)00048-63. Suk, V. R. E., & Misran, M. (2017). Preparation, characterization and physicochemical properties of DOPE-PEG2000 stabilized oleic acid-soy lecithin liposomes (POLL). Colloids and Surfaces A: Physicochemical and Engineering Aspects, 513, 267-273. https://doi.org/10.1016/j.colsurfa.2016.10.0534. Kanda, H., Katsube, T., & Goto, M. (2021). Preparation of liposomes from soy lecithin using liquefied dimethyl ether. Foods, 10(8), 1789. https://doi.org/10.3390%2Ffoods10081789 Does the proposed method for obtaining lecithin liposomes differ from those presented in the above works? It would be good to supplement the manuscript with a comparative description of the obtained quercetin-loaded liposomes and similar vesicles presented in other authors’ publications.- Reply:

Dearest reviewer, we are very grateful for your valuable comments, which highly improved the work. We recognized our fault; because of this, we added more than 10 new references to the revised manuscript, including those mentioned by the referee. We hope that the marked changes are now satisfactory and allow the manuscript improvement.

 3. It is desirable to supplement Section 3.1 with electron microscopy data (SEM/TEM/AFM) for visualization of finished quercetin-loaded lecithin liposomes.- Reply:Dearest reviewer, thank you for your constructive comment. A SEM image of the QT-LL 75 was added to Figure 3. This was the only possible modification at this moment, with a few days to accomplish all of the corrections on the manuscript. We strongly hope that the changes made in this version allowed the manuscript improvement, and are now satisfactory for you. 4. When analyzing films using scanning electron microscopy, the morphology of the film surface is undoubtedly important. It is also desirable to provide an EDX analysis of the zones of morphological inhomogeneities and uniformities of films. It would be nice to see the film in cross section. This would make it possible to fix the state of liposomes and the multilayer nature of the film, if any.- Reply:Dearest reviewer, thank you for your constructive comment. The figure was update; please see now SEM images with 10000x magnification. This was the only possible modification at this moment, with a few days to accomplish all of the corrections on the manuscript. We strongly hope that the changes made in this version allowed the manuscript improvement, and are now satisfactory for you.  5. It is necessary to provide X-ray diffractometry patterns and calculate the degree of crystallinity (χ, %) of the films.- Reply:Dearest reviewer, we are grateful for your constructive comment. Please see the degree of crystallinity of the films on section 3.4.3 of the revised manuscript. Considering that, in the present form, out manuscript has 3 figures and 5 tables, it is a consense of all of the authors that the addition of the drffractometry patterns of the films could be harmful for the publication process. In our point of view, this was the best possible organization. We strongly hope that the changes made in this version allowed the manuscript improvement, and are now satisfactory for you. 6. It is necessary to give the numerical values of the contact angles of wetting θ°of the surface of the films.- Reply:Dearest reviewer, we are grateful for your constructive comment. Please see table 5 [Values of contact angle for (A) the control film and the films (B-F) with different concentrations of QT-LL 75 (values expressed as average ± standard deviation] on the revised manuscript. 7. The conclusions are not successful and are inconsistent with the purpose of the work. This section needs to be updated and revised. The title of the manuscript, objectives and conclusions should be consistent with each other.- Reply:

Dearest reviewer, we are grateful for your constructive comment. We modified the title and the conclusions of the manuscript trying to accomplish your requirements. We hope that now you observe the consistency in our work.

   

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors have made relevant changes to their article which can now be published. The suitability for the journal is quite well introduced in the introduction.

Author Response

RESPONSE TO REVIEWER 1 

The authors have made relevant changes to their article which can now be published. The suitability for the journal is quite well introduced in the introduction.

- Reply:

Dearest Reviewer, we are very grateful for your understanding about the relevance of the manuscript. Thank you for your important opinion about the acceptance of the manuscript.  

Author Response File: Author Response.docx

Reviewer 2 Report

The authors have addressed all comments to improve the manuscript. This article can now be considered for publication. 

Author Response

RESPONSE TO REVIEWER 2 The authors have addressed all comments to improve the manuscript. This article can now be considered for publication.

- Reply: Dearest Reviewer, we are very grateful for your understanding of the relevance of the manuscript. Thank you for your important opinion about the acceptance of the manuscript.  

  

 

Author Response File: Author Response.docx

Reviewer 3 Report

Attached review

Comments for author File: Comments.pdf

Author Response

RESPONSE TO REVIEWER 3 on the manuscript «Effect of quercetin-loaded liposomes on the physicochemical properties of galactomannan-based films obtained from Cassia grandis seeds»by: Priscilla Barbosa Sales de Albuquerque*, Marthyna Pessoa de Souza, Ana Isabel Bourbon, Miguel Cerqueira, Lorenzo Pastrana, Paula Jauregi, José A. Teixeira, Maria das Graças Carneiro-da-Cunha. It should be noted that the authors have responded to a number of my comments and made appropriate corrections. However, some questions remain open. The authors need to answer them and make the appropriate changes:  1. Does their proposed method for obtaining lecithin liposomes differ from those presented in the literature? Please supplement your manuscript with a comparative description of the obtained quercetin-loaded liposomes and similar vesicles presented in the publications of other authors; - Reply:Dearest reviewer, thank you for your constructive comment. We added the requested information at the end of setio 3. Resultds and discussion, just above the topic 4. Conclusions. 2. When analyzing films using scanning electron microscopy, the morphology of the film surface is undoubtedly important. It is also desirable to provide an EDX analysis of the zones of morphological inhomogeneities and uniformities of the film. It would be good to see the film in cross section. This would make it possible to fix the state of liposomes and the multilayer nature of the film, if any. - Reply:Dearest reviewer, thank you for your constructive comment. Unfortunately, it was not possible to provide an EDX analysis of the morphological shape of the films because we had a few days to accomplish all of the corrections on the manuscript. We recognize the importance of your requirement and strongly hope that the other changes made in this second version allowed the manuscript improvement. 3. It is necessary to provide X-ray patterns. If the number of figures in a manuscript is limited, please place the X-ray patterns in supplementary materials to the manuscript. - Reply:Dearest reviewer, thank you for your constructive comment. A DRX image was added as Figure 5. 4. The authors present data on the crystallinity degree of their films. It is not clear how they calculated it. Usually the degree of crystallinity is measured in percents.- Reply:Dearest reviewer, thank you for your constructive comment. The degree of crystallinity (%), or crystallinity index, was calculated by considering the total diffracted area and the area under the crystallinity peaks; after integration and correction of the data for absorption, the ratio of the crystalline area to the total area was obtained and considered as the crystallinity index (%). Please verify this information in the revised manuscript version 2. 

 

Author Response File: Author Response.docx

Round 3

Reviewer 3 Report

The authors corrected the manuscript taking into account all the comments of the reviewer. The manuscript may be published in Applied Nano.