Quantitative cytotoxicity analysis of antibacterial Janus nanoparticles in immune and cancer cells

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However, concerns about their cytotoxicity, particularly their effects on mammalian cells, must be thoroughly addressed to ensure therapeutic safety. Amphiphilic Janus NPs, which have segregated hydrophobic and polycationic ligands on two hemispheres, have previously been shown to exhibit potent antibacterial activity. Methods In this study, we evaluated the cytotoxicity of amphiphilic Janus NPs in immune and cancer cell lines. Cytotoxicity assays were performed to assess the effects of Janus NPs on cell viability and membrane integrity, with a particular focus on how internalization of the nanoparticles influenced cellular responses. Results The results revealed that both immune and cancer cells exhibited negligible cytotoxic effects when exposed to Janus NPs. However, phagocytic immune cells demonstrated greater susceptibility to membrane damage and viability loss, suggesting that internalization plays a significant role in nanoparticle-induced cytotoxicity. Conclusions Amphiphilic Janus NPs show great potential as highly effective antibacterial agents with minimal cytotoxicity. While immune cells may be more vulnerable to nanoparticle-induced damage due to their internalization capacity, these findings support the further investigation of Janus NPs for clinical applications. " } { "@context": "http://schema.org", "@type": "BreadcrumbList", "itemListElement": [ { "@type": "ListItem", "position": "1", "item": { "@id": "https://f1000research.com/", "name": "Home" } }, { "@type": "ListItem", "position": "2", "item": { "@id": "https://f1000research.com/browse/articles", "name": "Browse" } }, { "@type": "ListItem", "position": "3", "item": { "@id": "https://f1000research.com/articles/13-1340", "name": "Quantitative cytotoxicity analysis of antibacterial Janus nanoparticles..." } } ] } Home Browse Quantitative cytotoxicity analysis of antibacterial Janus nanoparticles... ALL Metrics - Views Downloads Get PDF Get XML Cite How to cite this article Johnson B, Peck M, Richman H et al. Quantitative cytotoxicity analysis of antibacterial Janus nanoparticles in immune and cancer cells [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2024, 13 :1340 ( https://doi.org/10.12688/f1000research.156592.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Brief Report Quantitative cytotoxicity analysis of antibacterial Janus nanoparticles in immune and cancer cells [version 1; peer review: 1 approved with reservations, 1 not approved] Benjamin Johnson 1 , Matthew Peck 1 , Hunter Richman 1 , Swagata Bhattacharyya 1 , Yan Yu https://orcid.org/0000-0001-6496-5045 1 Benjamin Johnson 1 , Matthew Peck 1 , [...] Hunter Richman 1 , Swagata Bhattacharyya 1 , Yan Yu https://orcid.org/0000-0001-6496-5045 1 PUBLISHED 08 Nov 2024 Author details Author details 1 Department of Chemistry, Indiana University, Bloomington, IN, 47405, USA Benjamin Johnson Roles: Data Curation, Formal Analysis, Writing – Original Draft Preparation Matthew Peck Roles: Data Curation, Formal Analysis, Writing – Original Draft Preparation Hunter Richman Roles: Data Curation, Formal Analysis, Writing – Original Draft Preparation Swagata Bhattacharyya Roles: Data Curation, Formal Analysis, Writing – Original Draft Preparation Yan Yu Roles: Conceptualization, Project Administration, Supervision, Writing – Review & Editing OPEN PEER REVIEW DETAILS REVIEWER STATUS Abstract Background Nanoparticles (NPs) hold promise as alternatives to antibiotics in the fight against multi-drug-resistant bacteria. However, concerns about their cytotoxicity, particularly their effects on mammalian cells, must be thoroughly addressed to ensure therapeutic safety. Amphiphilic Janus NPs, which have segregated hydrophobic and polycationic ligands on two hemispheres, have previously been shown to exhibit potent antibacterial activity. Methods In this study, we evaluated the cytotoxicity of amphiphilic Janus NPs in immune and cancer cell lines. Cytotoxicity assays were performed to assess the effects of Janus NPs on cell viability and membrane integrity, with a particular focus on how internalization of the nanoparticles influenced cellular responses. Results The results revealed that both immune and cancer cells exhibited negligible cytotoxic effects when exposed to Janus NPs. However, phagocytic immune cells demonstrated greater susceptibility to membrane damage and viability loss, suggesting that internalization plays a significant role in nanoparticle-induced cytotoxicity. Conclusions Amphiphilic Janus NPs show great potential as highly effective antibacterial agents with minimal cytotoxicity. While immune cells may be more vulnerable to nanoparticle-induced damage due to their internalization capacity, these findings support the further investigation of Janus NPs for clinical applications. READ ALL READ LESS Keywords Janus nanoparticles, cell cytotoxicity, anti-bacterial nanomaterials, cell internalization, cell viability Corresponding Author(s) Yan Yu ( [email protected] ) Close Corresponding author: Yan Yu Competing interests: No competing interests were disclosed. Grant information: This work was financially supported by the U.S. National Science Foundation under Award No. 2153891 (to Y.Y.). H.R. was supported by a predoctoral fellowship from the Graduate Training Program in Quantitative and Chemical Biology supported by National Institutes of Health (T32GM131994) and Indiana University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2024 Johnson B et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Johnson B, Peck M, Richman H et al. Quantitative cytotoxicity analysis of antibacterial Janus nanoparticles in immune and cancer cells [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2024, 13 :1340 ( https://doi.org/10.12688/f1000research.156592.1 ) First published: 08 Nov 2024, 13 :1340 ( https://doi.org/10.12688/f1000research.156592.1 ) Latest published: 08 Nov 2024, 13 :1340 ( https://doi.org/10.12688/f1000research.156592.1 ) Introduction The rise of multi-drug resistant bacteria poses a significant threat to human health. In 2019 alone, antimicrobial resistance (AMR) caused an estimated 1.27 million deaths globally. 1 The AMR crisis is expected to worsen with the slowdown in new antibiotic development. To address the urgent need for effective antibacterial agents, nanoparticles (NPs) have emerged as a promising alternative to traditional antibiotics. 2 – 4 Owing to their unique physicochemical properties, antibacterial NPs are known to interact with bacterial cells through a combination of multiple mechanisms, including physical disruption of cell membranes and oxidative stress induction, which collectively minimize the likelihood of bacterial resistance development. 5 – 8 However, alongside their potential benefits, the cytotoxicity of antibacterial NPs, particularly their impact on mammalian cells, must be thoroughly evaluated, to ensure their therapeutic viability and safety. Previous research has shown that nanoparticle cytotoxicity can vary widely depending on their physiochemical properties, such as size, shape, surface charge, and composition. 9 , 10 For instance, NPs with certain surface chemistries, including positive charges and hydrophobicity, may induce oxidative stress or inflammation in mammalian cells, potentially leading to adverse effects. 11 – 14 Our previous work demonstrated that amphiphilic Janus NPs, designed with hydrophobic and polycationic ligands on separate hemispheres, are highly effective against both gram-positive and gram-negative bacteria. 15 We showed that these Janus NPs effectively inhibit bacterial growth by physically disrupting the bacterial outer membranes and inducing oxidative stress in bacterial cells. Building upon these findings, the current study extends our investigation to assess the cytotoxicity of these NPs in mammalian cell cultures, focusing on immune cells and cancer cells. Using quantitative cytotoxicity assays with single-cell precision, we establish the relationship between nanoparticle concentration and cytotoxicity in vitro. Methods Reagents and cells RAW264.7 macrophage cells and HeLa cells were purchased from ATCC. Gibco advanced Dulbecco’s Modified Eagle Medium (DMEM) 12491015, Gibco FluoroBrite™ Dulbecco’s Modified Eagle Medium (FluoroBrite DMEM) A1896701, Gibco L-Glutamine 200 mM (100x) 25030081, propidium iodide (PI) P21493 and Hoechst ™ H21486 fluorescent dyes were purchased from ThermoFisher™ (Waltham, MA, USA). 12-well glass-bottomed cell culture plates (P12G-1.5-11-F) were purchased from Mattek (Ashland, MA, USA). 100x Penicillin-streptomycin (10,000 mg/mL) MT30001CI and fetal bovine serum (MT35010CV) were purchased from Corning (Corning, NY, USA). Amine-functionalized silica nanoparticles (100 nm) SISN100-25M were procured from Nanocomposix (San Diego, CA, USA). Gold (99.99% purity) EVMAU40QXQ and chromium (99.99% purity) EVMCR35 were purchased from Kurt J. Lesker, Co. (Jefferson Hills, PA, USA). Octadecanethiol (L04123-22), colistin sulfate, were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ultrapure water (18 MΩ·cm) was used for all experiments. Janus nanoparticle (NP) fabrication Amphiphilic cationic/hydrophobic Janus NPs (+/pho JPs) were fabricated as described below. Briefly, a sub-monolayer of 100 nm cationic silica NPs was coated on piranha-etched microscope slides. Chromium (5 nm thickness) and gold (25 nm thickness) were coated onto one hemisphere of the particles using an Edwards thermal evaporation system. The metal-coated NPs were immediately functionalized with 2 mM 1-octadecanethiol in ethanol to render the gold side hydrophobic. The resulted NPs were sonicated off the slides within 24 h before use. Aggregates from metal bridging were removed by two steps of differential centrifugation (100 rcf four times, 500 rcf four times). For colistin conjugation, Janus NPs were washed 3 times with 10 mM HEPES buffer (pH 7.40) and resuspended with 10 % glutaraldehyde in 10 mM HEPES buffer for 90 min at room temperature under gentle rotation. The particles were then washed three times with 10 mM HEPES buffer to remove excess glutaraldehyde. Particles were then resuspended and incubated in 1 mg/mL colistin in 10 mM HEPES buffer for 60 min at room temperature under gentle rotation, followed by washing three times in 10 mM HEPES and then three times in 70% ethanol to remove excess colistin. Functionalized Janus NPs were then stored in 70% ethanol at 4°C until use, when ethanol was replaced with cell imaging buffer. Hydrodynamic size and zeta potential of colistin conjugated amphiphilic Janus NPs (col/pho JPs) were characterized using Dynamic Light Scattering (DLS) and Malvern Zetasizer). Gold coating on the NPs were assessed using scanning electron microscopy. For more detail on the fabrication and properties of col/pho Janus NPs, see methods from Wiemann et al. 15 Dual-staining cytotoxicity assay Mammalian cells (RAW264.7 macrophage and HeLa) were seeded onto glass-bottomed 120-well cell culture plates at a concentration of 0.125 million cells/mL (RAW264.7 cells) or 0.0156 million cells/mL (HeLa cells) and grown for 24h at 37 °C, 5% CO 2 . Both mammalian cells lines were grown in 400 μL of DMEM α containing advanced DMEM with penicillin-streptomycin (1x), L-glutamine (2 mM), and fetal bovine serum (10% v/v). Following 24h incubation, cell media was replaced with serum free media (advanced DMEM with no fetal bovine serum) containing various concentrations of Janus NPs. Janus NPs were stored in 70% ethanol and washed multiple times with DMEM β containing advanced DMEM with penicillin-streptomycin (1x) and L-glutamine (2 mM) before addition to cell samples. Janus NPs were incubated with cells for 24h before the dual-staining cytotoxicity assay. Following exposure to Janus NPs, cells were washed once with DMEM. Then a total volume of 400 μL staining mixture containing propidium iodide (2.5 μg/mL) and Hoechst (25 μg/mL) was added to the cell media in each well. Approximately 10 min after addition of the staining mixture, fluorescence and differential interference contrast (DIC) images were acquired using a Nikon Ti-E inverted microscope equipped with a 40x/0.95 NA Nikon air objective. Hoechst was imaged at excitation/emission 350/450 nm and propidium iodide was imaged at excitation/emission 535/615 nm. Cell samples were incubated at 37 °C, 5% CO 2 throughout imaging process using a UNO-T-H-CO2 stage top incubator. Results and Discussion The amphiphilic Janus NPs (100 nm in diameter) have distinct surface coatings on two hemispheres ( Figure 1A ), same as we reported previously, and were fabricated using our established protocol. 15 , 16 Briefly, we first coated one hemisphere of aminated silica NPs with thin chromium (5 nm thickness) and gold layers (25 nm thickness) via directional vapor deposition, and then conjugated octadecanethiol on the gold cap to render it hydrophobic. The other hemisphere of the aminated silica NPs was conjugated with colistin using glutaraldehyde crosslinking chemistry. Colistin, typically reserved as a last-resort antibiotic, functions here as a polycationic ligand that enhances the strong electrostatic interaction of Janus NPs with bacterial membranes, as we reported previously. 15 Figure 1. Schematic illustration of Janus nanoparticles (NPs) and the cell viability assay. (A) Amphiphilic Janus NPs exhibit distinct surface makeups on two hemispheres. One side is conjugated with hydrophobic octadecane thiol ligands on a gold cap, while the other side is conjugated with the polycationic ligand colistin. (B) In the cytotoxicity assay, nuclei of all cells are stained with membrane permeable Hoechst dye (green). (C) Nuclei of non-viable cells with damaged plasma membrane are stained with the membrane impermeable dye propidium Iodide (red). Based on our previous findings demonstrating the effective disruption of model lipid membranes and bacterial outer membranes by Janus NPs, 15 , 17 – 20 we proceeded to investigate their impact on plasma membrane integrity and subsequent cell viability. This investigation utilized a dual staining protocol involving propidium iodide and Hoechst, a widely accepted method for assessing cell cytotoxicity. 21 , 22 The assay was conducted in HeLa cells and RAW264.7 macrophage cells, two commonly used cell lines to represent cancer and immune cells, respectively. In this protocol, Hoechst dye, which penetrates intact membranes, stains the DNA of all cells, whether alive or dead ( Figure 1B ). Conversely, propidium iodide, a membrane-impermeable dye that binds to nucleic acids, selectively stains non-viable cells with compromised membranes. This approach allows us to not only identify cells with nanoparticle-induced plasma membrane permeabilization, but also quantify cytotoxic effects by comparing the number of propidium iodide-stained non-viable cells to the total Hoechst-stained cell population. Initially, HeLa cells were incubated for 24h with Janus NPs at concentrations ranging up to 64 pM. The 0-64 pM concentration range was selected because it covers and exceeds the range of half maximal effective concentrations (EC50) of these NPs against four different bacterial strains (10-47 pM). 15 We previously demonstrated that 64 pM Janus NPs induce substantial membrane damage and production of reactive oxygen species in almost all E. coli cells. 15 Using both differential interference contrast (DIC) and widefield fluorescence microscopy, we observed that the morphology of HeLa cells remained unchanged in terms of both shape and size following exposure to Janus NPs ( Figure 2A, C ). There was a slight increase in the number of cells stained with propidium iodide, as nanoparticle concentration increased, indicating minor damage to the cell plasma membrane ( Figure 2B ). To quantify the impact on cell viability, we calculated the percent viability at each particle concentration using the equation: Percent Viability = Number of Viable Cells Number of Total Cells × 100 % . As shown in Figure 2D , even with 64 pM NPs, approximately 94.8% of cells remained viable. This reduction in cell viability is much less than the cytotoxic threshold defined by EN ISO 10993-5 as a reduction in cell viability by more than 30% compared to an untreated control. 23 Therefore, Janus NPs exhibited minimal effect on overall viability of HeLa cells. Figure 2. Viability assay results of HeLa cells after interaction with varying concentrations of Janus NPs. Differential interference contrast (DIC) (A) and widefield epi-fluorescence (B) images showing cells stained with Hoechst (green) and propidium iodide (red) after 24h incubation with Janus NPs at varying concentrations. (C) Zoomed-in DIC images of areas outlined in red rectangles in (A). Quantification of viability of HeLa cells as a function of Janus nanoparticle concentration (D). Each data point represents the average of two independent samples and each error bar represents the standard error between samples. Scale bars: 40 μm. After assessing the impact of JNP treatment on HeLa cells, we investigated the cytotoxicity of the Janus NPs to professional phagocytes, a type of immune cells specialized in internalizing and digesting foreign particles including pathogens and synthetic NPs. 24 – 28 To this end, we evaluated the cytotoxicity of the NPs against RAW264.7 cells using the same dual staining protocol. Using both DIC and widefield fluorescence imaging, we observed that some NPs were internalized by the macrophage cells, resulting in regions immediately adjacent to cells showing almost no NPs compared to background levels ( Figure 3A, C ). This phenomenon is more pronounced at higher nanoparticle concentrations. Meanwhile, the cell morphology changed slightly upon nanoparticle exposure. Post-interaction with NPs, cells exhibited increased heterogeneity in shape, characterized by enhanced membrane protrusions and asymmetry ( Figure 3C ). Figure 3. Viability assay results of RAW264.7 macrophage cells after interaction with varying concentrations of Janus NPs. Differential interference contrast (DIC) images (A) and Widefield epi-fluorescence images (B) showing cells stained with Hoechst (green) and propidium iodide (red) after 12h interaction with Janus NPs of varying concentrations. (C) Zoomed-in DIC images of areas outlined in red rectangles in (A). Quantification of viability of RAW264.7 macrophage cells as a function of Janus nanoparticle concentration (D). Each data point represents the average from three independent samples and each error bar represents the standard error between samples. Scale bars: 40 μm. Consistent with these observations, there was a slight increase in the number of cells stained with propidium iodide at higher nanoparticle concentrations ( Figure 3B ), indicating concentration-dependent damage to the cell plasma membrane and subsequent cell death. As shown in Figure 3D , Janus NPs up to 16 pM had negligible effects on macrophage cell viability. However, cell viability decreased by 22.9% and 26.6% after exposure to 32 pM and 64 pM NPs, respectively. It is important to note that, even though higher concentrations of Janus NPs reduced cell viability, it did not fall below the threshold of a 30% reduction, which is considered cytotoxic as defined by the EN ISO 10993-5 standard. 23 By comparing the results from HeLa cells and RAW264.7 macrophage cells, it is evident that the cytotoxic effect of the Janus NPs is more pronounced in phagocytic immune cells, which inherently internalize a larger number of NPs. This is supported by viability quantification and our observation of changes in macrophage cell morphologies. This finding aligns with previous reports that internalization is a contributing factor in the cytotoxicity of NPs. 29 – 32 Importantly, neither cell type shows a significant cytotoxic effect from the Janus NPs, based on the established threshold of a 30% reduction in cell viability as indicative of toxicity. The nanoparticle concentrations we tested ranged from 0 to 64 pM, exceeding the EC50 values of the NPs against various bacteria (e.g., an EC50 of ~10 pM for E. coli). 15 These results validate the potential of Janus NPs as antibacterial agents. This research opens doors to further studies, including more comprehensive assessments of both in vitro and in vivo cytotoxicity of the NPs using additional markers, such as the generation of reactive oxygen species and the inflammatory response of immune cells. Author contribution statement B.J., M.P., H.R., and S.B. carried out the experiments. All authors wrote the manuscript. Y.Y. conceived the project idea and supervised the project. Ethics & consent Ethical approval and consent were not required. Data availability statement All study data are included in the manuscript and/or supporting information. Raw data used to generate main figures and source code for simulation have been deposited at https://doi.org/10.6084/m9.figshare.27092086.v3 . FigShare: Janus-particle Cytotoxicity Raw Data, https://doi.org/10.6084/m9.figshare.27092086.v3 . 33 This project contains the following underlying data: • Raw Data. (All Raw264.7 and HeLa cell microscopy images used for figures and analyzed for graphs) Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication). Acknowledgements We thank Prof. J. P. Gerdt at Indiana University for providing E. coli culture used in this study, Dr. Yi Yi and Dr. Jun Chen at IUB Nanoscale Characterization Facility for assistance with instrument use. References 1. 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Publisher Full Text Comments on this article Comments (0) Version 1 VERSION 1 PUBLISHED 08 Nov 2024 ADD YOUR COMMENT Comment Author details Author details 1 Department of Chemistry, Indiana University, Bloomington, IN, 47405, USA Benjamin Johnson Roles: Data Curation, Formal Analysis, Writing – Original Draft Preparation Matthew Peck Roles: Data Curation, Formal Analysis, Writing – Original Draft Preparation Hunter Richman Roles: Data Curation, Formal Analysis, Writing – Original Draft Preparation Swagata Bhattacharyya Roles: Data Curation, Formal Analysis, Writing – Original Draft Preparation Yan Yu Roles: Conceptualization, Project Administration, Supervision, Writing – Review & Editing Competing interests No competing interests were disclosed. Grant information This work was financially supported by the U.S. National Science Foundation under Award No. 2153891 (to Y.Y.). H.R. was supported by a predoctoral fellowship from the Graduate Training Program in Quantitative and Chemical Biology supported by National Institutes of Health (T32GM131994) and Indiana University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Article Versions (1) version 1 Published: 08 Nov 2024, 13:1340 https://doi.org/10.12688/f1000research.156592.1 Copyright © 2024 Johnson B et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Johnson B, Peck M, Richman H et al. Quantitative cytotoxicity analysis of antibacterial Janus nanoparticles in immune and cancer cells [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2024, 13 :1340 ( https://doi.org/10.12688/f1000research.156592.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 1 VERSION 1 PUBLISHED 08 Nov 2024 Views 0 Cite How to cite this report: Lee WH. Reviewer Report For: Quantitative cytotoxicity analysis of antibacterial Janus nanoparticles in immune and cancer cells [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2024, 13 :1340 ( https://doi.org/10.5256/f1000research.171921.r364567 ) The direct URL for this report is: https://f1000research.com/articles/13-1340/v1#referee-response-364567 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 22 Feb 2025 Wing-Hin Lee , Universiti Kuala Lumpur, Kuala Lumpur, Federal Territory of Kuala Lumpur, Malaysia Not Approved VIEWS 0 https://doi.org/10.5256/f1000research.171921.r364567 This manuscript is not suitable for indexing. This is due to multiple reasons: 1) No bacteria experiment works are included in this manuscript. 2) This manuscript is unclear as to why HeLa and Raw cells were used. ... Continue reading READ ALL This manuscript is not suitable for indexing. This is due to multiple reasons: 1) No bacteria experiment works are included in this manuscript. 2) This manuscript is unclear as to why HeLa and Raw cells were used. 3) The amount of drug loaded was not reported. 4) In general, the main objective of this manuscript was not clear. Is the work clearly and accurately presented and does it cite the current literature? No Is the study design appropriate and is the work technically sound? No Are sufficient details of methods and analysis provided to allow replication by others? No If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? No Are the conclusions drawn adequately supported by the results? Partly Competing Interests: No competing interests were disclosed. Reviewer Expertise: Drug delivery I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Lee WH. Reviewer Report For: Quantitative cytotoxicity analysis of antibacterial Janus nanoparticles in immune and cancer cells [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2024, 13 :1340 ( https://doi.org/10.5256/f1000research.171921.r364567 ) The direct URL for this report is: https://f1000research.com/articles/13-1340/v1#referee-response-364567 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Roy AS. Reviewer Report For: Quantitative cytotoxicity analysis of antibacterial Janus nanoparticles in immune and cancer cells [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2024, 13 :1340 ( https://doi.org/10.5256/f1000research.171921.r362489 ) The direct URL for this report is: https://f1000research.com/articles/13-1340/v1#referee-response-362489 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 13 Feb 2025 Atanu Singha Roy , Department of Chemical and Biological Sciences, National Institute of Technology Meghalaya, Shillong, India Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.171921.r362489 The authors have fabricated the nanoparticles. In this regard, no data/plots are provided at least as the supplementary information. JNPs exhibits interesting features and I want to get the following details related to the characterization of the JNPs: ... Continue reading READ ALL The authors have fabricated the nanoparticles. In this regard, no data/plots are provided at least as the supplementary information. JNPs exhibits interesting features and I want to get the following details related to the characterization of the JNPs: 1. PXRD data before and after fabrication of all the NPs systems. 2. FT-IR related to surface fabrication. 3. XPS to get the details after fabrication related to existence of various metals in JNPs. 4. TEM/FESEM images of the NPs (if possible with EDX). These results/plots etc as applicable must be supplied in the supplementary information. Also the results from DLS and Zeta potential are not mentioned. It is also needed to be added in the SI. Is there any changes of zeta potential after interaction with the cell lines? Is it determined? Any related discussion is possible? Is the work clearly and accurately presented and does it cite the current literature? No Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? No If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Biophysical Chemistry I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Roy AS. Reviewer Report For: Quantitative cytotoxicity analysis of antibacterial Janus nanoparticles in immune and cancer cells [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2024, 13 :1340 ( https://doi.org/10.5256/f1000research.171921.r362489 ) The direct URL for this report is: https://f1000research.com/articles/13-1340/v1#referee-response-362489 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Comments on this article Comments (0) Version 1 VERSION 1 PUBLISHED 08 Nov 2024 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 Version 1 08 Nov 24 read read Atanu Singha Roy , National Institute of Technology Meghalaya, Shillong, India Wing-Hin Lee , Universiti Kuala Lumpur, Kuala Lumpur, Malaysia Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Lee W. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 22 Feb 2025 | for Version 1 Wing-Hin Lee , Universiti Kuala Lumpur, Kuala Lumpur, Federal Territory of Kuala Lumpur, Malaysia 0 Views copyright © 2025 Lee W. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Not Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions This manuscript is not suitable for indexing. This is due to multiple reasons: 1) No bacteria experiment works are included in this manuscript. 2) This manuscript is unclear as to why HeLa and Raw cells were used. 3) The amount of drug loaded was not reported. 4) In general, the main objective of this manuscript was not clear. Is the work clearly and accurately presented and does it cite the current literature? No Is the study design appropriate and is the work technically sound? No Are sufficient details of methods and analysis provided to allow replication by others? No If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? No Are the conclusions drawn adequately supported by the results? Partly Competing Interests No competing interests were disclosed. Reviewer Expertise Drug delivery I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. reply Respond to this report Responses (0) Lee WH. Peer Review Report For: Quantitative cytotoxicity analysis of antibacterial Janus nanoparticles in immune and cancer cells [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2024, 13 :1340 ( https://doi.org/10.5256/f1000research.171921.r364567) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-1340/v1#referee-response-364567 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Roy A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 13 Feb 2025 | for Version 1 Atanu Singha Roy , Department of Chemical and Biological Sciences, National Institute of Technology Meghalaya, Shillong, India 0 Views copyright © 2025 Roy A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The authors have fabricated the nanoparticles. In this regard, no data/plots are provided at least as the supplementary information. JNPs exhibits interesting features and I want to get the following details related to the characterization of the JNPs: 1. PXRD data before and after fabrication of all the NPs systems. 2. FT-IR related to surface fabrication. 3. XPS to get the details after fabrication related to existence of various metals in JNPs. 4. TEM/FESEM images of the NPs (if possible with EDX). These results/plots etc as applicable must be supplied in the supplementary information. Also the results from DLS and Zeta potential are not mentioned. It is also needed to be added in the SI. Is there any changes of zeta potential after interaction with the cell lines? Is it determined? Any related discussion is possible? Is the work clearly and accurately presented and does it cite the current literature? No Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? No If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise Biophysical Chemistry I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (0) Roy AS. Peer Review Report For: Quantitative cytotoxicity analysis of antibacterial Janus nanoparticles in immune and cancer cells [version 1; peer review: 1 approved with reservations, 1 not approved] . F1000Research 2024, 13 :1340 ( https://doi.org/10.5256/f1000research.171921.r362489) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/13-1340/v1#referee-response-362489 Alongside their report, reviewers assign a status to the article: Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. 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