A real-time assay for cell-penetrating peptide-mediated delivery of molecular cargos

Schuyler B. Gentry; Scott J. Nowak; Xuelei Ni; Stephanie A. Hill; Lydia R. Wade; William R. Clark; Aidan P. Keelaghan; Daniel P. Morris; Jonathan L. McMurry

doi:10.1371/journal.pone.0254468

Peer Review History

Original SubmissionJune 22, 2021
9 Aug 2021 Decision Letter - Eugene A. Permyakov, Editor PONE-D-21-20501 A real-time assay for cell-penetrating peptide-mediated delivery of molecular cargos PLOS ONE Dear Dr. McMurry, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ============================== ACADEMIC EDITOR: Dear authors, Would you like to answer the reviewer's criticism and try to improve your manuscript according to his criticism, ============================== Please submit your revised manuscript by Sep 23 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. 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This does not alter our adherence to PLOS ONE policies on sharing data and materials.” We note that you received funding from a commercial source: “JLM and SJN” Please provide an amended Competing Interests Statement that explicitly states this commercial funder, along with any other relevant declarations relating to employment, consultancy, patents, products in development, marketed products, etc. Within this Competing Interests Statement, please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. Please include your amended Competing Interests Statement within your cover letter. We will change the online submission form on your behalf. 3. Please include captions for your Supporting Information files table at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. 4. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Major Comments: In the manuscript entitled “A real-time assay for cell-penetrating peptide-mediated delivery of molecular cargos”, the authors described a real-time method by integrating a cell containing flow chamber with a confocal microscope to allow for observation the cellular uptake kinetics of CPP-protein complex ab initio. By using this apparatus, it was found the CBS-MBP could be reversibly bonded with TAT-NMR-CaM and delivered into cells within seven minutes. The authors also performed a “distance analysis” which showed that CPP-adaptor-delivered cargo generated wider distribution throughout cells as compared to an analogous covalently-bound CPP-cargo, thus indicating the CPP-adaptor system could overcome the endosome entrapment and deliver the CBS-MBP into the cytosol. Though the apparatus-based assay showed advantages in providing a real-time analysis of the kinetic cellular uptake process, the superiority of the new method in comparison with the conventional static endpoint assay, however is less sufficiently presented. To this account, I’m afraid the current form of this manuscript is not sufficient for publication by the Plos one. And below are some major suggestions for the authors: # 1. CPPs are valuable tools capable of transporting various pharmaceutical molecules across cell membranes, but exact penetration mechanisms of CPPs and their conjugated cargos are still not clear. We also know little about the subsequent intercellular processes of the encapsulated CPP-cargo complex, such as endosome formation, escape, and redistribution to other subcellular organelles, which are critical for bioactivity of the therapeutic molecules. This work is noteworthy to provide a real-time analysis of the kinetic process of CPP mediated delivery which is important for us to study the transporting process of CPPs. However, as a new way of observing the cellular uptake process, the “real-time assay” should be compared with the conventional static endpoint assay in a number of ways, focusing on the key factors involved in the transportation and endosomal process such as cytoskeleton, tubulin, pH change, which could help us get a better understanding of this analytic tool. # 2. It is interesting that CPP-adaptor-delivered CBS-MBP once released from CPP-adaptor in the endosome, then the cargo could get out of the endosome and distribute into the cytosol by itself. To me it seems uncommon for a protein capable of endosome escape, so can other type of protein be excreted from endosome in the same way? #3. There are a number of mistakes with related to figures. 1) In the manuscript, line 274-275, it is saying “CaM with no CPP in complex with CBS-MBP moiety evinced even lower background that CBS-MBP alone (Fig 3C).” However no 3C image was found in Fig.3C. 2) Line 291: “a flow system installed on an incubated confocal stage was built (S1 Fig).” it refers to Fig S2, not S1. 3) Scale bar is missing in Fig.4 Reviewer #2: The manuscript entitled “A real-time assay for cell-penetrating peptide- mediated delivery of molecular cargos” by Gentry et al report the development of assay to monitor kinetics and mechanism of the delivery of cargos to the cytoplasm with the use on calmodulin in there studies. The CPP medicate delivery was found to be linear and fast (about 7 minutes). A major bottle neck of endosomal entrapment of CPP-cargos has been discussed and troubleshoted using various analytical data. Manuscript is well written with appropriate citations. ****** 6. 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PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0254468.r001
Revision 1
12 Aug 2021 Author Response August 10, 2021 Dr. Eugene A. Permyakov Academic Editor PLOS ONE RE: Manuscript PONE-D-21-20501 Dear Dr. Permyakov, Thank you for considering our manuscript, PONE-D-21-20501, for publication in PLOS ONE. We are appreciative of your and the reviewers’ efforts and honored that they found merit in our study and made constructive criticisms of the manuscript. We have made the suggested revisions, described in full below, and are proud to resubmit an improved, revised manuscript for further consideration. Our answers to editorial and reviewer points: Editor: 1) We have done our best to ensure that our manuscript meets the style requirements. 2) Competing Interests/Financial Disclosure. We are afraid there was a miscommunication in the disclosure. While two authors do have equity interest in New Echota Biotechnology, a start-up biotechnology company that has license to a patent on the CPP-adaptor technology, the study was not funded by any commercial source at all. Thus your statement that “We note you received funding from a commercial source: JLM and SJN” is in error. JLM and SJN are the initials of the authors, Jonathan L. McMurry and Scott J. Nowak, who have equity in the company. Both are employed by Kennesaw State University and were not supported or paid by the company in any way. The company has no funding and is mostly a dormant entity at the moment. We have amended the Competing Interests statement to eliminate the use of initials to avoid the miscommunication: I have read the journal's policy and the authors of this manuscript have the following competing interests: Authors McMurry and Nowak have equity interest in New Echota Biotechnology, a start-up company which has exclusive license to patent US10654894B2 on the CPP-adaptor technology and applications thereof. The other authors have no competing interests. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Please let us know if you need additional changes. 3) We have added captions for the Supporting Information files as requested. 4) We have eliminated the mention of data not shown, in one case providing a new figure in Supplemental Information and in the other referring to the paper with the data that was not shown. Reviewer #1: 1) Reviewer #1 notes in “Major Comments” that the superiority of the real-time assay to the static endpoint one is insufficiently presented. We have added a summary paragraph that more clearly describes the advantages of our method, lines 380-396. 2) The reviewer points out that the penetration mechanisms remain unclear (indeed they are a matter of great debate in the field) and implies that we should repeat the experiments “focusing on the key factors,” which include ”cytoskeleton, tubulin, pH change” and others. We completely agree that these and other factors are necessary to understanding the mechanisms and kinetics of CPP-mediated entry and delivery of cargo, but ask why is it we, of all the thousands of CPP studies are to be held to knowing the mechanisms that have for decades eluded understanding? We would instead plead that the study warrants publication now as it stand precisely so that other researchers with the resources to conduct those studies might incorporate our or similar assays in their work to better understand exactly these factors. We note that we did focus on some of the factors in our small molecule inhibitors experiments shown in Fig. 8 and the negative results we obtained strongly suggest that the mechanistic answers are likely very complex and will yet be a long time coming. We will continue to pursue them, but the scope of what the reviewer implies need be done is well beyond our ability to do at present and will require the efforts of the entire field. Best that they know about our assay now, we think. We hope the reviewer agrees. 3) Reviewer asks if other types of proteins can be excreted from the endosome. The brief answer is yes. Endosomal escape is recognized as the largest technical hurdle to be overcome in the development of CPP-based therapeutics. We direct him or her to our prior papers describing our system (references # 10 and 11, and our review on the subject, #6). Salerno et al. demonstrates delivery of three cargo proteins that vary in size and oligomerization. In Ngwa, et al., we delivered a model cargo protein to the nucleus, endoplasmic reticulum and lysosomes using appropriate signal peptides. The reviewer is absolutely correct that it is unclear how proteins escape the endosome, but we have shown that they do (i.e. how else would they wind up in the nucleus?). The distance analysis presented in the present study represents an advance in that we can now, at any moment in time during the course of a real-time assay, quantify how much better our CPP-adaptors deliver cargo than the standard linkage; it can be a tool to measure endosomal escape going forward. We have modified comments in the last paragraph to address this critique. 4) Figure mistakes: we have corrected them according to the reviewer’s comments. We also apologize as these are the sorts of errors we endeavor never to make, but somehow usually do. Thanks to the reviewer for catching them and sorry to have made doing so necessary. Reviewer #2: Reviewer #2 had no concerns that needed addressing in this letter. We also made a very few other minor edits where we caught typos or realized we could enhance clarity. We also neglected to put an umlaut in Göettingen in the materials and methods sections. The changes are marked on the track changes document. Again, we thank you and the reviewers for your efforts. They have resulted in a substantially improved manuscript we hope you will find worthy of publication. Sincerely, Jonathan McMurry, Ph.D. Corresponding Author Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0254468.r002
16 Aug 2021 Decision Letter - Eugene A. Permyakov, Editor A real-time assay for cell-penetrating peptide-mediated delivery of molecular cargos PONE-D-21-20501R1 Dear Dr. McMurry, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Eugene A. Permyakov, Ph.D., Dr.Sci. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0254468.r003
Formally Accepted
19 Aug 2021 Acceptance Letter - Eugene A. Permyakov, Editor PONE-D-21-20501R1 A real-time assay for cell-penetrating peptide-mediated delivery of molecular cargos Dear Dr. McMurry: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Prof. Eugene A. Permyakov Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0254468.r004

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