Electrospinning repellents in polyvinyl alcohol-nanofibres for obtaining mosquito-repelling fabrics

Recently, the use of repellents for preventing the transmission of mosquito-borne diseases is getting increasingly more attention. However, most of the current repellents are volatile in nature and must be frequently re-applied as their efficacy is only limited to a short period of time. Therefore, a slow release and abrasion-resistant mechanism is needed for prolonging the protection time of the repellents. The focus of this study is on the direct micro-encapsulation of repellents from an emulsion and integration of already encapsulated repellents into nanofibres via electrospinning. Different repellents were electrospun in polyvinyl alcohol (PVA) nanofibrous structures, namely p-menthane-3,8-diol micro-capsules, permethrin, chilli and catnip oil. The repellents were successfully incorporated in the nanofibres and the tensile properties of the resulting samples did not have a significant change. This means that the newly created textiles were identical to current PVA nanofibrous textiles with the added benefit of being mosquito repellent. Principally, all incorporated repellents in the nanofibrous structures showed a significantly reduced number of mosquito landings compared to the control. Consequently, the currently described method resulted in a new and very effective repelling textile material that can be used in the prevention against mosquito-associated diseases.

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Comments to Author:
Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) Ciera and coworkers present an interesting paper on the efficacy of repellent-treated fabrics. Unfortunately, the authors don't seem familiar with the current literature and make many unsubstantiated claims in the introduction and discussion. Some mayor revisions would be required before this paper could be published. Introduction: Page 2, Line 13: permethrin is a pyrethroid class insecticide with repellent properties. It is different from the other repellents that can be sprayed on peoples skins. Line 14-16 The citations used to show that 'synthetic repellents' are toxic to humans are not valid. There is little in the literature to suggest that for example DEET is more toxic to humans like for example PMD. The authors should take a critical look at the literature and rewrite their introduction.
Line 19-20 Better say that there is a large market for 'alternative repellents'. Line 6-42 Many of the citations in the introduction (like for example Science Daily) are not acceptable and need to be replaced with scientific paper citations.

Materials -should be Materials & Methods
Page 5 Line 27 -please describe the species of mosquitoes used. Line 51 the bioassay is not well described. Please add a scheme and photo so the reader can understand how this assay works. Page 6 Line 6 -Provide details of the solution from Mukabana and add citation.
Discussion: Many of the citations are simply missing Page 16 line 48 the authors contrast synthetic repellents with biological repellents. This is unacceptable language. Line 52 none of the repellents tested here are novel. Line 53 Permethrin-treated clothing is already on the market. Page 17 Line 10 Is there any proof that the textiles actually slowly release the repellents? If not these claims must be removed.

Reviewer: 2
Comments to the Author(s) Comments to the editor This paper reports micro-encapsulation of biological mosquito repellents into nanofibers through electrospinning for controlled slow release. This reviewer finds the paper to have interesting technical contents and is likely to attract a wider readership and therefore recommends its publications after some minor revisions. Comments to the author Why were the mosquito nanofabrics not tested in the field because this could have given a clear perspective of the application of these innovative textiles? Generally the author has mixed up present and past tense-this need to be revised There seems to be some grammatical errors that need to be corrected There is a grammatical error in the abstract-last sentence of paragraph one For example page 2 line26-28 Page 7 line 44 -replace "is" and "smaller" with "was" and "less", respectively. Page 10 line 18 -replace "is" with "was" Page 11 line 49 "shows " should be "showed" same case for page 11 line 53

RSOS-182139.R1 (Revision)
Review form: Reviewer 2 Is the manuscript scientifically sound in its present form? Yes

Comments to the Author(s)
The authors of this work have made satisfactory corrections and the current reviewer recommends the publication of this manuscript as is.

Do you have any ethical concerns with this paper? No
Have you any concerns about statistical analyses in this paper? I do not feel qualified to assess the statistics

Comments to the Author(s)
The manuscript reports a preparation of mosquito repelling nanofibrous fabric by electrospinning repellents-containing polymer solution. It is interesting and of practically useful of the functionalized material by a facile technique. During the revision, most of the comments are well replied. However, this reviewer would suggest a minor revision with several additional questions: 1) Since the used mosquito repellents are volatile that will be easily evaporated during electrospinning, so is it possible to verify the exact amount of repellents remaining in the nanofiber after electrospinning? 2) PAV is a water-soluble polymer, as claimed by the authors it is environmental; however, just because it is water-soluble it would be easily dissolved for the resulting PVA nanofibers since applying environment is moist/wet/high humidity, and for human skin, it also might be sweating. So, how to address this issue? 3) SEM image of nanofiber containing different amount of repellents should be provided. 4) The size of the microcapsules is 5 ±1 μm, how does such a big size particles being encapsulated in nanofibers with the diameter of about several hundreds nanometers? 5) In the experiment section, it is written that the PVA solution was prepared under stirring at room temperature (21 ±1°C), but as I know the PVA could usually be dissolved under heating (~90°C). Can you answer this question? 6) What is the thickness of the nanofiber mat used for the mechanical test (both the directly measured and calculated ones)?

Review form: Reviewer 4
Is the manuscript scientifically sound in its present form? Yes

Recommendation?
Accept as is

Comments to the Author(s)
The authors have addressed all the comments appropriately from the reviewers and the work of using electrospun fibers loaded with repellents to repel mosquito is very interesting and can be useful.

04-Jul-2019
Dear Dr Ciera: On behalf of the Editors, I am pleased to inform you that your Manuscript RSOS-182139.R1 entitled "Electrospinning repellents in polyvinyl alcohol-nanofibers for obtaining mosquito repelling fabrics" has been accepted for publication in Royal Society Open Science subject to minor revision in accordance with the referee suggestions. Please find the referees' comments at the end of this email.
The reviewers and Subject Editor have recommended publication, but also suggest some minor revisions to your manuscript. Therefore, I invite you to respond to the comments and revise your manuscript.
• Ethics statement If your study uses humans or animals please include details of the ethical approval received, including the name of the committee that granted approval. For human studies please also detail whether informed consent was obtained. For field studies on animals please include details of all permissions, licences and/or approvals granted to carry out the fieldwork.
• Data accessibility It is a condition of publication that all supporting data are made available either as supplementary information or preferably in a suitable permanent repository. The data accessibility section should state where the article's supporting data can be accessed. This section should also include details, where possible of where to access other relevant research materials such as statistical tools, protocols, software etc can be accessed. If the data has been deposited in an external repository this section should list the database, accession number and link to the DOI for all data from the article that has been made publicly available. Data sets that have been deposited in an external repository and have a DOI should also be appropriately cited in the manuscript and included in the reference list.
If you wish to submit your supporting data or code to Dryad (http://datadryad.org/), or modify your current submission to dryad, please use the following link: http://datadryad.org/submit?journalID=RSOS&manu=RSOS-182139.R1 • Competing interests Please declare any financial or non-financial competing interests, or state that you have no competing interests.
• Authors' contributions All submissions, other than those with a single author, must include an Authors' Contributions section which individually lists the specific contribution of each author. The list of Authors should meet all of the following criteria; 1) substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; 2) drafting the article or revising it critically for important intellectual content; and 3) final approval of the version to be published.
All contributors who do not meet all of these criteria should be included in the acknowledgements.
We suggest the following format: AB carried out the molecular lab work, participated in data analysis, carried out sequence alignments, participated in the design of the study and drafted the manuscript; CD carried out the statistical analyses; EF collected field data; GH conceived of the study, designed the study, coordinated the study and helped draft the manuscript. All authors gave final approval for publication.
• Acknowledgements Please acknowledge anyone who contributed to the study but did not meet the authorship criteria.
• Funding statement Please list the source of funding for each author.
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Reviewer comments to Author: Reviewer: 2 Comments to the Author(s) The authors of this work have made satisfactory corrections and the current reviewer recommends the publication of this manuscript as is.

Reviewer: 3
Comments to the Author(s) The manuscript reports a preparation of mosquito repelling nanofibrous fabric by electrospinning repellents-containing polymer solution. It is interesting and of practically useful of the functionalized material by a facile technique. During the revision, most of the comments are well replied. However, this reviewer would suggest a minor revision with several additional questions: 1) Since the used mosquito repellents are volatile that will be easily evaporated during electrospinning, so is it possible to verify the exact amount of repellents remaining in the nanofiber after electrospinning? 2) PAV is a water-soluble polymer, as claimed by the authors it is environmental; however, just because it is water-soluble it would be easily dissolved for the resulting PVA nanofibers since applying environment is moist/wet/high humidity, and for human skin, it also might be sweating. So, how to address this issue? 3) SEM image of nanofiber containing different amount of repellents should be provided. 4) The size of the microcapsules is 5 ±1 μm, how does such a big size particles being encapsulated in nanofibers with the diameter of about several hundreds nanometers? 5) In the experiment section, it is written that the PVA solution was prepared under stirring at room temperature (21 ±1°C), but as I know the PVA could usually be dissolved under heating (~90°C). Can you answer this question? 6) What is the thickness of the nanofiber mat used for the mechanical test (both the directly measured and calculated ones)?
Reviewer: 4 Comments to the Author(s) The authors have addressed all the comments appropriately from the reviewers and the work of using electrospun fibers loaded with repellents to repel mosquito is very interesting and can be useful.

24-Jul-2019
Dear Dr Ciera, I am pleased to inform you that your manuscript entitled "Electrospinning repellents in polyvinyl alcohol-nanofibers for obtaining mosquito repelling fabrics" is now accepted for publication in Royal Society Open Science.
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RE: Manuscript ID RSOS-182139 revision:
Thank you for allowing us to resubmit a revised version of our manuscript entitled, "Electrospinning repellents in polyvinyl alcohol-nanofibers for obtaining mosquito repelling fabrics" for publication consideration in your Journal (Royal Society Open Science).
We appreciate the reviewer comments, which we have carefully considered. An outline of the reviewers' comments and our changes and/or rebuttal is described below. Our responses to the reviewer's comments appear in italic. Thank you for your continued assistance throughout this process.

Introduction:
Page 2, Line 13: permethrin is a pyrethroid class insecticide with repellent properties. It is different from the other repellents that can be sprayed on peoples skins.
We didn't indicate that permethrin is the same with other repellents that can be applied on the skin. We just mentioned it as an example of a synthetic repellent. Please see the paragraph below: The most effective and most commonly used insect repellents for preventing mosquito bites present on the market today are synthetic in nature which include DEET (N, N-diethyl-3methylbenzamide), permethrin and Picaridin [2,4,5]. However, some of these synthetic repellents are associated with some harmful and toxic effects, some resulting in allergic reactions and damage to the nervous system [6,7]. Moreover, some mosquitoes have become resistant to the current synthetic repellents and environmental concerns have prevented their effective use [3,5] Line 14-16 The citations used to show that 'synthetic repellents' are toxic to humans are not valid. There is little in the literature to suggest that for example DEET is more toxic to humans like for example PMD. The authors should take a critical look at the literature and rewrite their introduction.
We were not comparing the toxicity of the different repellents. We just mentioned that some synthetic repellents can be harmful. Please see below However, some of these synthetic repellents are associated with some harmful and toxic effects, some resulting in allergic reactions and damage to the nervous system [6,7]. Moreover, some

The description is long and will make the manuscript too long instead we have added the citation. Please below
The strips were then impregnated with attractive compounds by bringing them into an Eppendorf tube containing 1 mL of solution as described in [21]. Thereafter, the strips were stored overnight in a refrigerator at 4°C and later hung up for half an hour under a fume hood to allow excess fluids to leak out. Finally, the strips were immediately used or packed in aluminium foil and stored at 4°C in a refrigerator until they were used.

Corrected. The missing citations were added
Page 16 line 48 the authors contrast synthetic repellents with biological repellents. This is unacceptable language.
Line 52 none of the repellents tested here are novel.

Our resulting Nano fabric is what we are referring to as novel not the repellents. Please see below
These results clearly indicate that the novel textile materials with incorporated biological repellents can be used in the protection against mosquitoes. Recently, biological repellents are considered as a tool in vector control preventing mosquito house entry [32,42,43], providing a promising application for our novel repelling textiles in push-pull system [44]. In light of these potential applications, future studies should focus on the durability of the material, such as their washing durability and the longevity of the repellency efficacy.
To conclude, mosquito repellents were successfully spun into PVA nanofibers with electrospinning to obtain a possible slow release mosquito repellent textile. Two techniques were used, namely electrospinning of microcapsules with incorporated repellent and direct emulsion electrospinning of the repellents. These methods both proved very successful in developing new and very effective mosquito repelling textile material that can be used in the prevention against mosquito associated diseases. Whereas, incorporation of microcapsules in the nanofibers gives long term functionality it still needs active breakage of the microcapsules to release the functional additive. Here we only tested PMD, but it is obvious that these microcapsules can contain many different repellents and even mixtures, making this an attractive, quick method for developing a variety of repelling textiles. On the other hand, emulsion electrospining encapsulates the functional additives in a one-step process into nanofibers making it a simple and cheap technique. Moreover, because the encapsulating wall is so thin, it may be possible that the additives are continuously released slowly and it is therefore needless to break the nano-capsules to release the additive. However, this possible continuous release may result in short term use. Both methods show great potential in creating novel and very efficient repelling textiles, not only on a lab-scale but also for industrial-scale productions. We would like to thank you for your email and your continuous effort to improve our paper. We have read and answered the comments of the reviewers in great detail. As reviewer 2 and 4 did not have any more questions/remarks we only could answer the remarks of reviewer 3 who had some valid points: An outline of the reviewers' comments and our changes and/or rebuttal is described below. Our responses to the reviewer's comments appear in italic. Thank you for your continued assistance throughout this process. 1) Since the used mosquito repellents are volatile that will be easily evaporated during electrospinning, so is it possible to verify the exact amount of repellents remaining in the nanofiber after electrospinning? This is a valid point and some of the repellent will evaporate during the electrospinning process, however we think this loss will be very minimal during the process for the following reasons: -The repellent is in emulsion in PVA and is oil like, therefore water from the PVA solution will still be evaporated first and in greater amounts than the repellent, although it is possible that some small quantities of the repellent could evaporate. -We still see beads in the fibres compared to the blank meaning that the repellent did get included in the PVA nanofibrils ( Figure 2) -We showed that the repellents are present in the fibrous mats with infrared which is very clear ( Figure 5) -We can show a repellent effect on mosquitos of our fibrous mats which is a macroscopic effect of a nanofibrous mat, meaning that the repellent that is integrated into the electrospun mats must be significantly high ( Figure 6) So in general, we are convinced that the repellent is integrated in the electrospun mats and in large quantities. In the future, we of course will quantify these amounts but it is extremely hard to also show this. As for now there is no straight forward technique to do this. So yes, this will be done in the future but is not part of the current paper.
2) PVA is a water-soluble polymer, as claimed by the authors it is environmental; however, just because it is water-soluble it would be easily dissolved for the resulting PVA nanofibers since applying environment is moist/wet/high humidity, and for human skin, it also might be sweating. So, how to address this issue?