Biocompatible Cobalt Oxide Nanoparticles for X-ray Fluorescence Microscopy

Biocompatible Cobalt Oxide Nanoparticles for X-ray Fluorescence Microscopy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Biocompatible Cobalt Oxide Nanoparticles for X-ray Fluorescence Microscopy Christian Scott, Sophia Miller, Pierre Moenne-Loccoz, Craig Barnes, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4312367/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Oct, 2024 Read the published version in Journal of Nanoparticle Research → Version 1 posted 7 You are reading this latest preprint version Abstract The synthesis of water-soluble nanoparticles is a well-developed field for ferrite-based nanoparticles with the majority consisting of iron oxide or mixed metal iron oxide nanoparticles. However, the synthesis of non-agglomerated non-ferrite metal/metal oxide NPs is not as well established. The synthesis and characterization of uniform 20 nm, biologically compatible cobalt oxide (CoO) nanoparticles (NPs) is described. These nanoparticles have two principle components: 1) a CoO core of suitable size to contain enough cobalt atoms to be visualized by X-ray fluorescence microscopy (XFM) and 2) a robust coating that inhibits NP aggregation as well as renders them water-soluble and biocompatible (i.e. stealth coatings). Stable cobalt oxide NPs are obtained with octadecyl amine coatings as reported by Bhattacharjee. Two strategies for solubilizing these NPs in water were investigated with varying degrees of success. Exchanging the octadecyl amine coating for a nitrodopamine anchored PEG coating yielded the desired water-soluble NPs but in very low yield. Alternately, leaving the octadecyl amine coating on the NP and interdigitating this with a maleic anhydride-vinyl copolymer with different hydrophobic sidechains followed by opening the maleic anhydride ring with amine substituted PEG polymers (the water solubilizing component), yielded the desired water soluble NPS were obtained in good yield. Characterization data for the nanoparticles and the components of the coatings required for bioorthogonal reactions to ligate them with biotargeting agents are also described. cobalt oxide nanoparticles biocompatible stealth coating X-ray fluorescence microscopy bioorthogonal chemistry click chemistry Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The applications of metal-based nanoparticles are expanding rapidly as their synthesis and properties become better understood.[ 1 , 2 ] One area of application involves utilization of nanoparticles conjugated with biotargeting agents to perform correlative imaging such as electron microscopy or synchrotron-based X-ray fluorescence microscopy (XFM). XFM has emerged as a powerful tool to quantitatively image trace elemental distributions in biological specimen.[ 3 ] Currently, beamlines at synchrotrons offer spatial resolutions ranging from the tens of microns to fifty nanometers and beyond. Sensitivity and resolution of XFM measurements are sufficient to distinguish cell types and organelles, but straightforward tools to identify cells and organelles simultaneously within one XFM scan are lacking. This technical in the in the interpretion of XFM data is expected to be further exacerbated by furture upgrades of synchrotron sources. Several correlative approaches have been developed to match metal distributions to their precise location in different materials. One of these uses gold (Au) nanoparticles that are conjugated to 2° antibodies which in turn bind to 1° antibodies[ 4 , 5 ]. XFM detection of the Au L-line fluorescence is then used to indirectly localize the proteins of interest and match it to the X-ray fluorescent signature of the desired element(s). However, Au X-ray fluorescence requires excitation energies above 10 keV (Au L-electron binding energies > 11,900 eV [ 6 ]), where beamline instrumentation optimized for biological specimen is much less efficient. Moreover, the significant overlap of the Zn K β and Au L α1 emission lines can make imaging of Zn distributions difficult.[ 5 ] The synthesis of NPs containing first row transition metal atoms is dominated by those containing iron in the form of iron oxide-based particles or containing endogenous metals such as copper, zinc or manganese.[ 7 , 8 ] Because of the ubiquitous occurrence of iron in many biological systems we could not avail ourselves of iron or Fe-Co oxide-based NPs. Therefore, cobalt nanoparticles are ideal for XFM imaging in biological specimens because only trace amounts of endogenous cobalt are present, and the cobalt K α emission line falls well within the ~ 10 keV excitation window and doesn’t show any significant overlap with other biologically relevant elements. We therefore sought to develop an efficient synthesis of biologically compatible, cobalt-based nanoparticles. Beyond simple biocompatibility, it must also be possible to ligate such NPs with biotargeting molecules and other spectroscopic tags to study the actions of the NPs both in vivo and in vitro experiments. This latter goal is frequently accomplished by inserting chemical groups into the nanoparticle coating for bioorthogonal coupling strategies such as click reactions.[ 9 ] Here, we incorporated azidoe groups into our NP coating as shown in Fig. 1 . A few syntheses of cobalt and mixed metal iron-cobalt oxide nanoparticles have been reported [ 10 – 12 ] but, to our knowledge, none for cobalt alone with coatings that make them water soluble, nonaggregated, and tailored to inhibit immune responses in living systems. Furthermore, most pure cobalt oxide NP syntheses yield small (5–10 nm) NPs which would not contain enough cobalt to produce a detectable fluorescence signal for typical XFM conditions.[ 13 ] Methods and Results Recently, Purkayastha and coworkers described a simple, high yield synthesis of unaggregated, CoO NPs using octadecylamine as both a solvent and surfactant coating (Fig. 2 ).[ 14 ] The size distribution for these hydrophobic nanoparticles was reported to be centered around 20 nm, which is ideal for XFM visualization applications. The identity and phase purity of the cobalt oxide-based NPs was verified by PXRD. Both PXRD and TEM data indicate that the NP size distribution is centered around 18–20 nm (electronic material). The hydrophobic octadecyl amine coating renders these initial nanoparticles completely water insoluble. Two strategies were investigated to make them water soluble: 1) exchanging the amine-anchored coating with a water solubilizing polyethylene (PEG)-based coating having a nitro dopamine (nDOPA) anchoring group, and 2) leaving the amine-anchored coating in place and adding a bipolar copolymer that interdigitates with the octadecyl chains while providing a hydrophilic outer layer to solubilize the particles (Fig. 3 ). For the ligand exchange strategy, we chose the nitro dopamine (nDOPA) group to anchor the new PEG-based coating to the cobalt oxide surface due to its reported wide range of pH tolerance and nearly irreversible binding to oxide surfaces.[ 15 , 16 ] Sonication of the hydrophobic nanoparticles with an excess of nDOPA-PEG 1000 -OMe (50–90%) and nDOPA-PEG 2000 -N 3 (50 − 10%) followed by extraction into water was successful in creating water soluble nanoparticles but in very low yields (1–5%). The final, water soluble, PEG coated CoO nanoparticles were analyzed by cobalt elemental analysis (ICP-OES/MS), single particle ICPMS (spICPMS), PXRD, and TEM (Fig. 4 ) (see experimental details in EM to this Letter). Although the synthesis is direct (one step) and leads to very stable (months in aqueous solution) hydrophilic NPs, the very low yields in the ligand exchange step led us to search for higher yielding synthetic strategies. Schieber and coworkers recently described an interdigitation strategy for producing hydrophilic azide-modified CdSe/ZnS Core–shell quantum dots. [ 17 ] This interdigitation strategy involves two steps (Fig. 5 ). First, the C 18 NH 2 -coated NPs are exposed to a commercially available vinyl-maleic anhydride copolymer. The hydrophobic sidechains (phenyl, isobutylene, or octadecyl) of the co-polymer interdigitate or intertwine with the existing octadecyl amine coating developing a double coated nanoparticle. In a second in situ step, the maleic anhydride is ring-opened with an appropriately functionalized short-chain PEG polymer thus creating the desired, outer solubilizing, hydrophilic coating. The final product is extracted into water and purified by filtering through a 100k molecular weight centrifugation filter (Spin-X). Three commercially available maleic anhydride copolymers were tested as well as several different amine PEG polymers (ethanol amine, H 2 N-PEG 150 -OMe, and H 2 N-PEG 550 -OMe) to determine the optimum combination that produced the desired water-soluble nanoparticles. No combination of PEG-amine and either the maleic anhydride-co-styrene or -co-isobutylene polymers produced water-soluble NPs. Only the combination of maleic anhydride-co-octadecyl vinyl ether (mal-C 18 ) successfully interdigitated with the octadecylamine NP coating, and ultimately produced water soluble nanoparticles. After opening of the maleimide ring with ethanol amine and H 2 N-PEG 150 -OMe only very low amounts of water-soluble NPs were obtained. However, the longer PEG amine (H 2 N-PEG 550 -OMe) succeeded in producing much more concentrated aqueous solutions of nanoparticles. This combination gave a desired water-soluble cobalt oxide nanoparticle product in good yield (50% wt). TEM images of the water-soluble interdigitated NPs showed that the sizes of the cobalt oxide cores of the NPs had not significantly changed ( = 20 nm; Fig. 6 ). Transmittance IR spectra of aqueous solutions of the NPs with different amounts of the PEG-azide in their coating were obtained. Clear evidence for the presence of the azide group was observed (N 3 stretch at ~ 2110–2120 cm − 1 ) down to 20% loading of the PEG-azide with the bulk of the coating made up of PEG-OMe chains. Discussion-Nanoparticle Stability The shelf stability of the ligand exchange NPs and interdigitation NPs is on the order of months with no visual evidence of aggregation leading to precipitation of the NPs. Additionally, nanoparticle batches may be dried and resuspended with sonication with no visual signs of aggregation. This long-term stability shows that both types of nanoparticles are quite stable and can be prepared and stored for long periods of time. Summary and Future Work The successful synthesis of functionalized cobalt oxide nanoparticles through two pathways provides a convenient framework with which to further elaborate these NPs for use as bio-applicable XFM probes. The azide groups terminating PEG chains in the outer coatings around the NPs are often used for this purpose via bioorthogonal click reactions. Current work is now focused on characterizing different nanoparticle-biotargeting agent constructs (e.g. antibodies, nanobodies) and developing protocols for in vitro and in vivo X-ray fluorescence microscopy experiments. Declarations Acknowledgments and Funding: Research in this publication was supported by the National Institutes of Health award (R21 GM129592) to Martina Ralle, and Christian Scott. Sophia Miller was supported by the Medical Research Foundation of Oregon. ICPMS measurements were performed in the OHSU Elemental Analysis Core with partial support from the National Institutes of Health (S10OD028492). Electron microscopy was performed at the Multiscale Microscopy Core, a member of the OHSU University Shared Resource Cores. Electronic Materials: Detailed synthetic procedures, NMR spectra, ICPMS, PXRD, TEM data; biochemical protocols Statement of Conflict of Interest: The authors declare no competing financial interest. Data Availability: All data is provided in the electronic supplementary material in addition to their corresponding references References Taiariol, L., et al., Click and Bioorthogonal Chemistry: The Future of Active Targeting of Nanoparticles for Nanomedicines? Chemical Reviews, 2022. 122(1): p. 340–384. Kapate, N., J.R. Clegg, and S. Mitragotri, Non-spherical micro- and nanoparticles for drug delivery: Progress over 15 years . Advanced Drug Delivery Reviews, 2021. 177: p. 113807. Leary, S.C. and M. Ralle, Advances in visualization of copper in mammalian systems using X-ray fluorescence microscopy . Curr Opin Chem Biol, 2020. 55: p. 19–25. Pushkar, Y., et al., Aging results in copper accumulations in GFAP-positive cells in the subventricular zone . Aging Cell, 2013. McRae, R., et al., Correlative microXRF and optical immunofluorescence microscopy of adherent cells labeled with ultrasmall gold particles . J Struct Biol, 2006. 155(1): p. 22–9. Center for X-ray Optics and Advanced Light Source. X-ray Data Booklet . 2009; Available from: http://xdb.lbl.gov/ . Chandrakala, V., V. Aruna, and G. Angajala, Review on metal nanoparticles as nanocarriers: current challenges and perspectives in drug delivery systems . Emergent Materials, 2022. 5(6): p. 1593–1615. Idiago-López, J., et al., Nanoparticles and bioorthogonal chemistry joining forces for improved biomedical applications . Nanoscale Advances, 2021. 3(5): p. 1261–1292. Marques, A.C., et al., Functionalizing nanoparticles with cancer-targeting antibodies: A comparison of strategies . Journal of controlled release., 2020. 320: p. 180–200. Mahin, J., et al., Green, scalable, low cost and reproducible flow synthesis of biocompatible PEG-functionalized iron oxide nanoparticles . Reaction Chemistry & Engineering, 2021. 6(10): p. 1961–1973. Salunkhe, A.B., et al., Water dispersible superparamagnetic Cobalt iron oxide nanoparticles for magnetic fluid hyperthermia . Journal of Magnetism and Magnetic Materials, 2016. 419: p. 533–542. Sun, S., et al., Monodisperse MFe2O4 (M = Fe, Co, Mn) Nanoparticles . Journal of the American Chemical Society, 2004. 126(1): p. 273–279. Wolf, M., N. Fischer, and M. Claeys, Surfactant-free synthesis of monodisperse cobalt oxide nanoparticles of tunable size and oxidation state developed by factorial design . Materials chemistry and physics, 2018. 213: p. 305–312. Purkayastha, D.D., B. Sarma, and C.R. Bhattacharjee, Surfactant-assisted low-temperature synthesis of monodispersed phase pure cubic CoO solid nanoparallelepipeds via thermal decomposition of cobalt(II) acetylacetonate . Materials Letters, 2013. 107: p. 71–74. Amstad, E., M. Textor, and E. Reimhult, Stabilization and functionalization of iron oxide nanoparticles for biomedical applications . Nanoscale, 2011. 3(7): p. 2819–2843. Cencer, M., et al., Effect of Nitro-Functionalization on the Cross-Linking and Bioadhesion of Biomimetic Adhesive Moiety . Biomacromolecules, 2015. 16(1): p. 404–410. Schieber, C., et al., Conjugation of transferrin to azide-modified CdSe/ZnS core-shell quantum dots using cyclooctyne click chemistry. Angewandte Chemie (International ed. in English), 2012. 51(42): p. 10523. Additional Declarations No competing interests reported. Supplementary Files BiocompatibleCobaltOxideNanoparticlesforXrayFluorescenceMicroscopyEM.docx Cite Share Download PDF Status: Published Journal Publication published 03 Oct, 2024 Read the published version in Journal of Nanoparticle Research → Version 1 posted Editorial decision: Revision requested 25 Jun, 2024 Reviews received at journal 16 Jun, 2024 Reviewers agreed at journal 04 Jun, 2024 Reviewers invited by journal 28 May, 2024 Editor assigned by journal 19 May, 2024 Submission checks completed at journal 17 May, 2024 First submitted to journal 23 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4312367","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":307880911,"identity":"47378e36-cd61-48e2-91de-e4882e7e660f","order_by":0,"name":"Christian Scott","email":"","orcid":"","institution":"University of Tennessee at Knoxville","correspondingAuthor":false,"prefix":"","firstName":"Christian","middleName":"","lastName":"Scott","suffix":""},{"id":307880912,"identity":"f036b8f1-2a56-4948-970c-fc68ec92593b","order_by":1,"name":"Sophia Miller","email":"","orcid":"","institution":"Oregon Health \u0026 Science University","correspondingAuthor":false,"prefix":"","firstName":"Sophia","middleName":"","lastName":"Miller","suffix":""},{"id":307880913,"identity":"559a5257-a463-4141-9b5c-cc84ba7f5cf1","order_by":2,"name":"Pierre Moenne-Loccoz","email":"","orcid":"","institution":"Oregon Health \u0026 Science University","correspondingAuthor":false,"prefix":"","firstName":"Pierre","middleName":"","lastName":"Moenne-Loccoz","suffix":""},{"id":307880914,"identity":"7ce577ac-44be-4bad-a0de-1507e1460516","order_by":3,"name":"Craig Barnes","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuklEQVRIiWNgGAWjYFACHgjFz8DAzADBxGqRbGAmVYvBAWK18Lf3HvxcucPG3vhG/mEDhgrrxAZCWiTOnEuWPHsmLXHbjWTmBIYz6YS1GEjkGEg2th1OMANqOcDYdpgILfJvjH82tv23N54B0vKPGC0SPGZAWw4wbpAAOoyxgQgtEmfy0iwb25ITZ5x5bGyQcCzdmKAW/vazh282ttnZ87cnPpb4UGMtS1ALKkggTfkoGAWjYBSMAlwAAFPbO/IjZsQxAAAAAElFTkSuQmCC","orcid":"","institution":"University of Tennessee at Knoxville","correspondingAuthor":true,"prefix":"","firstName":"Craig","middleName":"","lastName":"Barnes","suffix":""},{"id":307880915,"identity":"f5f15162-e2f8-4a18-b6a5-369f40e834e9","order_by":4,"name":"Martina Ralle","email":"","orcid":"","institution":"Oregon Health \u0026 Science University","correspondingAuthor":false,"prefix":"","firstName":"Martina","middleName":"","lastName":"Ralle","suffix":""}],"badges":[],"createdAt":"2024-04-23 13:40:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4312367/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4312367/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11051-024-06134-9","type":"published","date":"2024-10-03T15:57:40+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":57629211,"identity":"8b0a6aaf-f711-420e-88c1-befc28fd5d73","added_by":"auto","created_at":"2024-06-03 14:36:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":263036,"visible":true,"origin":"","legend":"\u003cp\u003eIllustration of a nanoparticle metal-metal oxide X-ray fluorescent spectroscopic probe biocompatible nanoparticle. The nanoparticle must be stabilized and made biocompatible via a robust coating of polymer molecules such as PEG chains. Conjugation with biotargeting agents such as antibodies, nanobodies or proteins is made possible by including functionalized PEG chains in the coating, in this case azide groups for bioorthoganol click reactions.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4312367/v1/2182b809b0563a5d22d5880f.png"},{"id":57629210,"identity":"9c507cce-796f-4143-bc77-97b8dfc3f565","added_by":"auto","created_at":"2024-06-03 14:36:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":275746,"visible":true,"origin":"","legend":"\u003cp\u003eSynthesis of the initial hydrophobic cobalt oxide nanoparticle. The polymer coating is composed of octadecyl amine molecules.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4312367/v1/575ebde102afd2d9dff81d9e.png"},{"id":57629213,"identity":"ace4cc0d-293a-436d-810d-796cb9cf0570","added_by":"auto","created_at":"2024-06-03 14:36:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":874187,"visible":true,"origin":"","legend":"\u003cp\u003eTwo strategies for transforming initially synthesized hydrophobic NPs into biocompatible hydrophilic nanoparticles. The ligand exchange strategy involves replacing the initial hydrophobic coating while the interdigitating approach involves developing a bilayer-like arrangement with an outer hydrophilic component (PEG) making the nanoparticles water soluble.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4312367/v1/90738ecc472da99d2e855860.png"},{"id":57629215,"identity":"19b6f410-e5e4-4ddc-9412-eb3aa59e8b16","added_by":"auto","created_at":"2024-06-03 14:36:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1265767,"visible":true,"origin":"","legend":"\u003cp\u003eTEM image (\u003cstrong\u003eA\u003c/strong\u003e) and particle size distribution plot (\u003cstrong\u003eB\u003c/strong\u003e) derived from single particle ICPMS measurements of water-soluble CoO-nDOPA-PEG nanoparticle samples. Particle size distributions from both measurements maximized at 18 – 20 nm.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4312367/v1/9710b7ad0599d171c0ab41d4.png"},{"id":57629212,"identity":"269f333f-4155-40c5-a71e-6adeb1b5505f","added_by":"auto","created_at":"2024-06-03 14:36:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":810826,"visible":true,"origin":"","legend":"\u003cp\u003eThe interdigitation strategy leaves the initial octadecyl amine coating on the NP in place and involves two steps: First, exposure to the maleic anhydride-vinyl copolymer with a long hydrophobic sidechain causes the two hydrophobic polymers to mix (interdigitate) leaving the maleic anhydride ring exposed. In a second step, reaction with an amine functionalized PEG molecule opens the ring producing a hydrophilic outer coating containing both carboxylic acid groups and PEG chains. This outer coating renders the nanoparticles water soluble and biocompatible.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4312367/v1/d485851bea3d192d4126beb7.png"},{"id":57630212,"identity":"3a1953ae-cbfb-4c22-8826-b672fbc49e69","added_by":"auto","created_at":"2024-06-03 14:44:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1756970,"visible":true,"origin":"","legend":"\u003cp\u003eRight: TEM image the C18 interdigitated cobalt oxide nanoparticles. Left: Transmittance IR data for aqueous solutions of different PEG-azide loadings in the outer coating of the interdigitated nanoparticles. The azide stretch can be observed down to ~20% loading in the PEG outer coating around each nanoparticle.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4312367/v1/4e57923f9b40a3fc23be558b.png"},{"id":66097558,"identity":"914602c7-5b83-47c2-b6e8-e0a64cff7fb7","added_by":"auto","created_at":"2024-10-07 16:14:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6283035,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4312367/v1/241adb6e-f29b-4dfa-974b-389b40476019.pdf"},{"id":57629216,"identity":"c26b8567-8ad9-41a5-94c7-d6c2fbbe0303","added_by":"auto","created_at":"2024-06-03 14:36:07","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":20621270,"visible":true,"origin":"","legend":"","description":"","filename":"BiocompatibleCobaltOxideNanoparticlesforXrayFluorescenceMicroscopyEM.docx","url":"https://assets-eu.researchsquare.com/files/rs-4312367/v1/23543a17871a453f916abc7a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biocompatible Cobalt Oxide Nanoparticles for X-ray Fluorescence Microscopy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe applications of metal-based nanoparticles are expanding rapidly as their synthesis and properties become better understood.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] One area of application involves utilization of nanoparticles conjugated with biotargeting agents to perform correlative imaging such as electron microscopy or synchrotron-based X-ray fluorescence microscopy (XFM). XFM has emerged as a powerful tool to quantitatively image trace elemental distributions in biological specimen.[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] Currently, beamlines at synchrotrons offer spatial resolutions ranging from the tens of microns to fifty nanometers and beyond. Sensitivity and resolution of XFM measurements are sufficient to distinguish cell types and organelles, but straightforward tools to identify cells and organelles simultaneously within one XFM scan are lacking. This technical in the in the interpretion of XFM data is expected to be further exacerbated by furture upgrades of synchrotron sources.\u003c/p\u003e \u003cp\u003eSeveral correlative approaches have been developed to match metal distributions to their precise location in different materials. One of these uses gold (Au) nanoparticles that are conjugated to 2\u0026deg; antibodies which in turn bind to 1\u0026deg; antibodies[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. XFM detection of the Au L-line fluorescence is then used to indirectly localize the proteins of interest and match it to the X-ray fluorescent signature of the desired element(s). However, Au X-ray fluorescence requires excitation energies above 10 keV (Au L-electron binding energies\u0026thinsp;\u0026gt;\u0026thinsp;11,900 eV [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]), where beamline instrumentation optimized for biological specimen is much less efficient. Moreover, the significant overlap of the Zn K\u003csub\u003eβ\u003c/sub\u003e and Au L\u003csub\u003eα1\u003c/sub\u003e emission lines can make imaging of Zn distributions difficult.[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eThe synthesis of NPs containing first row transition metal atoms is dominated by those containing iron in the form of iron oxide-based particles or containing endogenous metals such as copper, zinc or manganese.[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] Because of the ubiquitous occurrence of iron in many biological systems we could not avail ourselves of iron or Fe-Co oxide-based NPs. Therefore, cobalt nanoparticles are ideal for XFM imaging in biological specimens because only trace amounts of endogenous cobalt are present, and the cobalt K\u003csub\u003eα\u003c/sub\u003e emission line falls well within the ~\u0026thinsp;10 keV excitation window and doesn\u0026rsquo;t show any significant overlap with other biologically relevant elements. We therefore sought to develop an efficient synthesis of biologically compatible, cobalt-based nanoparticles.\u003c/p\u003e \u003cp\u003eBeyond simple biocompatibility, it must also be possible to ligate such NPs with biotargeting molecules and other spectroscopic tags to study the actions of the NPs both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e experiments. This latter goal is frequently accomplished by inserting chemical groups into the nanoparticle coating for bioorthogonal coupling strategies such as click reactions.[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] Here, we incorporated azidoe groups into our NP coating as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA few syntheses of cobalt and mixed metal iron-cobalt oxide nanoparticles have been reported [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] but, to our knowledge, none for cobalt alone with coatings that make them water soluble, nonaggregated, and tailored to inhibit immune responses in living systems. Furthermore, most pure cobalt oxide NP syntheses yield small (5\u0026ndash;10 nm) NPs which would not contain enough cobalt to produce a detectable fluorescence signal for typical XFM conditions.[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/p\u003e"},{"header":"Methods and Results","content":"\u003cp\u003eRecently, Purkayastha and coworkers described a simple, high yield synthesis of unaggregated, CoO NPs using octadecylamine as both a solvent and surfactant coating (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] The size distribution for these hydrophobic nanoparticles was reported to be centered around 20 nm, which is ideal for XFM visualization applications. The identity and phase purity of the cobalt oxide-based NPs was verified by PXRD. Both PXRD and TEM data indicate that the NP size distribution is centered around 18–20 nm (electronic material).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe hydrophobic octadecyl amine coating renders these initial nanoparticles completely water insoluble. Two strategies were investigated to make them water soluble: 1) exchanging the amine-anchored coating with a water solubilizing polyethylene (PEG)-based coating having a nitro dopamine (nDOPA) anchoring group, and 2) leaving the amine-anchored coating in place and adding a bipolar copolymer that interdigitates with the octadecyl chains while providing a hydrophilic outer layer to solubilize the particles (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor the ligand exchange strategy, we chose the nitro dopamine (nDOPA) group to anchor the new PEG-based coating to the cobalt oxide surface due to its reported wide range of pH tolerance and nearly irreversible binding to oxide surfaces.[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] Sonication of the hydrophobic nanoparticles with an excess of nDOPA-PEG\u003csub\u003e1000\u003c/sub\u003e-OMe (50–90%) and nDOPA-PEG\u003csub\u003e2000\u003c/sub\u003e-N\u003csub\u003e3\u003c/sub\u003e (50 − 10%) followed by extraction into water was successful in creating water soluble nanoparticles but in very low yields (1–5%). The final, water soluble, PEG coated CoO nanoparticles were analyzed by cobalt elemental analysis (ICP-OES/MS), single particle ICPMS (spICPMS), PXRD, and TEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) (see experimental details in EM to this Letter). Although the synthesis is direct (one step) and leads to very stable (months in aqueous solution) hydrophilic NPs, the very low yields in the ligand exchange step led us to search for higher yielding synthetic strategies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSchieber and coworkers recently described an interdigitation strategy for producing hydrophilic azide-modified CdSe/ZnS Core–shell quantum dots. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] This interdigitation strategy involves two steps (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). First, the C\u003csub\u003e18\u003c/sub\u003eNH\u003csub\u003e2\u003c/sub\u003e-coated NPs are exposed to a commercially available vinyl-maleic anhydride copolymer. The hydrophobic sidechains (phenyl, isobutylene, or octadecyl) of the co-polymer interdigitate or intertwine with the existing octadecyl amine coating developing a double coated nanoparticle. In a second \u003cem\u003ein situ\u003c/em\u003e step, the maleic anhydride is ring-opened with an appropriately functionalized short-chain PEG polymer thus creating the desired, outer solubilizing, hydrophilic coating. The final product is extracted into water and purified by filtering through a 100k molecular weight centrifugation filter (Spin-X).\u003c/p\u003e \u003cp\u003eThree commercially available maleic anhydride copolymers were tested as well as several different amine PEG polymers (ethanol amine, H\u003csub\u003e2\u003c/sub\u003eN-PEG\u003csub\u003e150\u003c/sub\u003e-OMe, and H\u003csub\u003e2\u003c/sub\u003eN-PEG\u003csub\u003e550\u003c/sub\u003e-OMe) to determine the optimum combination that produced the desired water-soluble nanoparticles. No combination of PEG-amine and either the maleic anhydride-co-styrene or -co-isobutylene polymers produced water-soluble NPs. Only the combination of maleic anhydride-co-octadecyl vinyl ether (mal-C\u003csub\u003e18\u003c/sub\u003e) successfully interdigitated with the octadecylamine NP coating, and ultimately produced water soluble nanoparticles.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter opening of the maleimide ring with ethanol amine and H\u003csub\u003e2\u003c/sub\u003eN-PEG\u003csub\u003e150\u003c/sub\u003e-OMe only very low amounts of water-soluble NPs were obtained. However, the longer PEG amine (H\u003csub\u003e2\u003c/sub\u003eN-PEG\u003csub\u003e550\u003c/sub\u003e-OMe) succeeded in producing much more concentrated aqueous solutions of nanoparticles. This combination gave a desired water-soluble cobalt oxide nanoparticle product in good yield (50% wt).\u003c/p\u003e \u003cp\u003eTEM images of the water-soluble interdigitated NPs showed that the sizes of the cobalt oxide cores of the NPs had not significantly changed (\u0026lt; \u003cem\u003ed\u003c/em\u003e \u0026gt; = 20 nm; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Transmittance IR spectra of aqueous solutions of the NPs with different amounts of the PEG-azide in their coating were obtained. Clear evidence for the presence of the azide group was observed (N\u003csub\u003e3\u003c/sub\u003e stretch at ~ 2110–2120 cm\u003csup\u003e− 1\u003c/sup\u003e) down to 20% loading of the PEG-azide with the bulk of the coating made up of PEG-OMe chains.\u003c/p\u003e "},{"header":"Discussion-Nanoparticle Stability","content":"\u003cp\u003e \u003c/p\u003e\u003cp\u003eThe shelf stability of the ligand exchange NPs and interdigitation NPs is on the order of months with no visual evidence of aggregation leading to precipitation of the NPs. Additionally, nanoparticle batches may be dried and resuspended with sonication with no visual signs of aggregation. This long-term stability shows that both types of nanoparticles are quite stable and can be prepared and stored for long periods of time.\u003c/p\u003e"},{"header":"Summary and Future Work","content":"\u003cp\u003eThe successful synthesis of functionalized cobalt oxide nanoparticles through two pathways provides a convenient framework with which to further elaborate these NPs for use as bio-applicable XFM probes. The azide groups terminating PEG chains in the outer coatings around the NPs are often used for this purpose via bioorthogonal click reactions. Current work is now focused on characterizing different nanoparticle-biotargeting agent constructs (e.g. antibodies, nanobodies) and developing protocols for \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e X-ray fluorescence microscopy experiments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments and Funding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResearch in this publication was supported by the National Institutes of Health award (R21 GM129592) to Martina Ralle, and Christian Scott. Sophia Miller was supported by the Medical Research Foundation of Oregon. ICPMS measurements were performed in the OHSU Elemental Analysis Core with partial support from the National Institutes of Health (S10OD028492). Electron microscopy was performed at the Multiscale Microscopy Core, a member of the OHSU University Shared Resource Cores.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eElectronic Materials:\u003c/strong\u003eDetailed synthetic procedures, NMR spectra, ICPMS, PXRD, TEM data; biochemical protocols\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatement of Conflict of Interest: \u0026nbsp;\u003c/strong\u003eThe authors declare no competing financial interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u003c/strong\u003e All data is provided in the electronic supplementary material in addition to their corresponding references\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTaiariol, L., et al., \u003cem\u003eClick and Bioorthogonal Chemistry: The Future of Active Targeting of Nanoparticles for Nanomedicines?\u003c/em\u003e Chemical Reviews, 2022. 122(1): p. 340\u0026ndash;384.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKapate, N., J.R. Clegg, and S. Mitragotri, \u003cem\u003eNon-spherical micro- and nanoparticles for drug delivery: Progress over 15 years\u003c/em\u003e. Advanced Drug Delivery Reviews, 2021. 177: p. 113807.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeary, S.C. and M. Ralle, \u003cem\u003eAdvances in visualization of copper in mammalian systems using X-ray fluorescence microscopy\u003c/em\u003e. Curr Opin Chem Biol, 2020. 55: p. 19\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePushkar, Y., et al., \u003cem\u003eAging results in copper accumulations in GFAP-positive cells in the subventricular zone\u003c/em\u003e. Aging Cell, 2013.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcRae, R., et al., \u003cem\u003eCorrelative microXRF and optical immunofluorescence microscopy of adherent cells labeled with ultrasmall gold particles\u003c/em\u003e. J Struct Biol, 2006. 155(1): p. 22\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCenter for X-ray Optics and Advanced Light Source. \u003cem\u003eX-ray Data Booklet\u003c/em\u003e. 2009; Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://xdb.lbl.gov/\u003c/span\u003e\u003cspan address=\"http://xdb.lbl.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChandrakala, V., V. Aruna, and G. Angajala, \u003cem\u003eReview on metal nanoparticles as nanocarriers: current challenges and perspectives in drug delivery systems\u003c/em\u003e. Emergent Materials, 2022. 5(6): p. 1593\u0026ndash;1615.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIdiago-L\u0026oacute;pez, J., et al., \u003cem\u003eNanoparticles and bioorthogonal chemistry joining forces for improved biomedical applications\u003c/em\u003e. Nanoscale Advances, 2021. 3(5): p. 1261\u0026ndash;1292.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarques, A.C., et al., \u003cem\u003eFunctionalizing nanoparticles with cancer-targeting antibodies: A comparison of strategies\u003c/em\u003e. Journal of controlled release., 2020. 320: p. 180\u0026ndash;200.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMahin, J., et al., \u003cem\u003eGreen, scalable, low cost and reproducible flow synthesis of biocompatible PEG-functionalized iron oxide nanoparticles\u003c/em\u003e. Reaction Chemistry \u0026amp; Engineering, 2021. 6(10): p. 1961\u0026ndash;1973.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalunkhe, A.B., et al., \u003cem\u003eWater dispersible superparamagnetic Cobalt iron oxide nanoparticles for magnetic fluid hyperthermia\u003c/em\u003e. Journal of Magnetism and Magnetic Materials, 2016. 419: p. 533\u0026ndash;542.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun, S., et al., \u003cem\u003eMonodisperse MFe2O4 (M\u0026thinsp;=\u0026thinsp;Fe, Co, Mn) Nanoparticles\u003c/em\u003e. Journal of the American Chemical Society, 2004. 126(1): p. 273\u0026ndash;279.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWolf, M., N. Fischer, and M. Claeys, \u003cem\u003eSurfactant-free synthesis of monodisperse cobalt oxide nanoparticles of tunable size and oxidation state developed by factorial design\u003c/em\u003e. Materials chemistry and physics, 2018. 213: p. 305\u0026ndash;312.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePurkayastha, D.D., B. Sarma, and C.R. Bhattacharjee, \u003cem\u003eSurfactant-assisted low-temperature synthesis of monodispersed phase pure cubic CoO solid nanoparallelepipeds via thermal decomposition of cobalt(II) acetylacetonate\u003c/em\u003e. Materials Letters, 2013. 107: p. 71\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmstad, E., M. Textor, and E. Reimhult, \u003cem\u003eStabilization and functionalization of iron oxide nanoparticles for biomedical applications\u003c/em\u003e. Nanoscale, 2011. 3(7): p. 2819\u0026ndash;2843.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCencer, M., et al., \u003cem\u003eEffect of Nitro-Functionalization on the Cross-Linking and Bioadhesion of Biomimetic Adhesive Moiety\u003c/em\u003e. Biomacromolecules, 2015. 16(1): p. 404\u0026ndash;410.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchieber, C., et al., \u003cem\u003eConjugation of transferrin to azide-modified CdSe/ZnS core-shell quantum dots using cyclooctyne click chemistry.\u003c/em\u003e Angewandte Chemie (International ed. in English), 2012. 51(42): p. 10523.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanoparticle-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nano","sideBox":"Learn more about [Journal of Nanoparticle Research](http://link.springer.com/journal/11051)","snPcode":"11051","submissionUrl":"https://submission.nature.com/new-submission/11051/3","title":"Journal of Nanoparticle Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"cobalt oxide nanoparticles, biocompatible, stealth coating, X-ray fluorescence microscopy; bioorthogonal chemistry, click chemistry","lastPublishedDoi":"10.21203/rs.3.rs-4312367/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4312367/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe synthesis of water-soluble nanoparticles is a well-developed field for ferrite-based nanoparticles with the majority consisting of iron oxide or mixed metal iron oxide nanoparticles. However, the synthesis of non-agglomerated non-ferrite metal/metal oxide NPs is not as well established. The synthesis and characterization of uniform 20 nm, biologically compatible cobalt oxide (CoO) nanoparticles (NPs) is described. These nanoparticles have two principle components: 1) a CoO core of suitable size to contain enough cobalt atoms to be visualized by X-ray fluorescence microscopy (XFM) and 2) a robust coating that inhibits NP aggregation as well as renders them water-soluble and biocompatible (i.e. stealth coatings). Stable cobalt oxide NPs are obtained with octadecyl amine coatings as reported by Bhattacharjee. Two strategies for solubilizing these NPs in water were investigated with varying degrees of success. Exchanging the octadecyl amine coating for a nitrodopamine anchored PEG coating yielded the desired water-soluble NPs but in very low yield. Alternately, leaving the octadecyl amine coating on the NP and interdigitating this with a maleic anhydride-vinyl copolymer with different hydrophobic sidechains followed by opening the maleic anhydride ring with amine substituted PEG polymers (the water solubilizing component), yielded the desired water soluble NPS were obtained in good yield. Characterization data for the nanoparticles and the components of the coatings required for bioorthogonal reactions to ligate them with biotargeting agents are also described.\u003c/p\u003e","manuscriptTitle":"Biocompatible Cobalt Oxide Nanoparticles for X-ray Fluorescence Microscopy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-03 14:36:02","doi":"10.21203/rs.3.rs-4312367/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-25T21:51:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-16T06:42:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"223694995132990985735505920200737356107","date":"2024-06-04T19:24:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-28T16:39:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-19T20:13:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-17T08:00:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanoparticle Research","date":"2024-04-23T12:37:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanoparticle-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nano","sideBox":"Learn more about [Journal of Nanoparticle Research](http://link.springer.com/journal/11051)","snPcode":"11051","submissionUrl":"https://submission.nature.com/new-submission/11051/3","title":"Journal of Nanoparticle Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d59d8fd9-26fd-4185-be25-001ff31947d6","owner":[],"postedDate":"June 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-07T16:10:31+00:00","versionOfRecord":{"articleIdentity":"rs-4312367","link":"https://doi.org/10.1007/s11051-024-06134-9","journal":{"identity":"journal-of-nanoparticle-research","isVorOnly":false,"title":"Journal of Nanoparticle Research"},"publishedOn":"2024-10-03 15:57:40","publishedOnDateReadable":"October 3rd, 2024"},"versionCreatedAt":"2024-06-03 14:36:02","video":"","vorDoi":"10.1007/s11051-024-06134-9","vorDoiUrl":"https://doi.org/10.1007/s11051-024-06134-9","workflowStages":[]},"version":"v1","identity":"rs-4312367","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4312367","identity":"rs-4312367","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}