{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,28]],"date-time":"2026-08-28T17:37:36Z","timestamp":1787938656291,"version":"build-2784847793"},"reference-count":62,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2023,4,18]],"date-time":"2023-04-18T00:00:00Z","timestamp":1681776000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:sec><jats:title>Background<\/jats:title><jats:p>Traditional emulsion adjuvants are limited in clinical application because of their surfactant dependence. Graphene oxide (GO) has unique amphiphilic properties and therefore has potential to be used as a surfactant substitute to stabilize Pickering emulsions.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>In this study, GO\u2013stabilized Pickering emulsion (GPE) was prepared and used as an adjuvant to facilitate an enhanced immune response to the <jats:italic>Chlamydia trachomatis<\/jats:italic> (<jats:italic>Ct<\/jats:italic>) Pgp3 recombinant vaccine. Firstly, GPE was prepared by optimizing the sonication conditions, pH, salinity, GO concentration, and water\/oil ratio. GPE with small-size droplets was characterized and chosen as the candidate. Subsequently, controlled-release antigen delivery by GPE was explored. Cellular uptake behaviors, M1 polarization, and cytokine stimulation by GPE + Pgp3 was considered in terms of the production of macrophages. Finally, GPE\u2019s adjuvant effect was evaluated by vaccination with  Pgp3 recombinant in BALB\/c mouse models.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>GPE with the smallest droplet sizes was prepared by sonication under 163 W for 2 min at 1 mg\/mL GO in natural salinity with a pH of 2 when the water\/oil ratio was 10:1 (w\/w). The optimized average GPE droplet size was 1.8 \u03bcm and the zeta potential was \u201325.0 \u00b1 1.3 mv. GPE delivered antigens by adsorption onto the droplet surface, demonstrating the controlled release of antigens both <jats:italic>in vitro<\/jats:italic> and <jats:italic>in vivo<\/jats:italic>. In addition, GPE promoted antigen uptake, which stimulated proinflammatory tumor necrosis factor alpha (TNF-\u03b1), enhancing the M1 polarization of macrophages <jats:italic>in vitro<\/jats:italic>. Macrophage recruitment was also significantly promoted by GPE at the injection site. In the GPE + Pgp3 treatment group, higher levels of immunoglobin (IgG), immunoglobin G1 (IgG1), immunoglobin G2a (IgG2a) sera, and immunoglobin A (IgA) were detected in vaginal fluid, and higher levels of IFN-\u03b3 and IL-2 secretion were stimulated, than in the Pgp3 group, showing a significant type 1 T helper (Th1)-type cellular immune response. <jats:italic>Chlamydia muridarum<\/jats:italic> challenging showed that GPE enhanced Pgp3\u2019s immunoprotection through its advanced clearance of bacterial burden and alleviation of chronic pathological damage in the genital tract.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>This study enabled the rational design of small-size GPE, shedding light on antigen adsorption and control release, macrophage uptake, polarization and recruitment, which enhanced augmented humoral and cellular immunity and ameliorated chlamydial-induced tissue damage in the genital tract.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fimmu.2023.1148253","type":"journal-article","created":{"date-parts":[[2023,4,18]],"date-time":"2023-04-18T06:14:27Z","timestamp":1681798467000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":12,"title":["Preparation of graphene oxide\u2013stabilized Pickering emulsion adjuvant for Pgp3 recombinant vaccine and enhanced immunoprotection against Chlamydia Trachomatis infection"],"prefix":"10.3389","volume":"14","author":[{"given":"Lanhua","family":"Zhao","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mingyi","family":"Shu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hongliang","family":"Chen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Keliang","family":"Shi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhongyu","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2023,4,18]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.1586\/14760584.2013.811188","article-title":"An update on safety and immunogenicity of vaccines containing emulsion-based adjuvant","volume":"12","author":"Fox","year":"2013","journal-title":"Exp Rev Vac"},{"key":"B2","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2017.01760","article-title":"AS03-and MF59-adjuvanted influenza vaccines in children","volume":"8","author":"Wilkins","year":"2017","journal-title":"Front Immunol"},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.2147\/IJN.S232677","article-title":"O\/W nanoemulsion as an adjuvant for an inactivated H3N2 influenza vaccine: based on particle properties and mode of carrying","volume":"15","author":"Zhao","year":"2020","journal-title":"Int J Nanomed"},{"key":"B4","doi-asserted-by":"publisher","first-page":"14","DOI":"10.1016\/j.smim.2018.05.001","article-title":"Correlates of adjuvanticity: a review on adjuvants in licensed vaccines","volume":"39","author":"Giuseppe","year":"2018","journal-title":"Semin Immunol"},{"key":"B5","doi-asserted-by":"publisher","first-page":"3","DOI":"10.1016\/j.cis.2016.03.002","article-title":"Natural emulsifiers-biosurfactants, phospholipids, biopolymers, and colloidal particles: Molecular and physicochemical basis of functional performance","volume":"234","author":"McClements","year":"2016","journal-title":"Adv Colloid Interface Sci"},{"key":"B6","doi-asserted-by":"publisher","DOI":"10.1016\/j.cis.2020.102117","article-title":"Recent advances of characterization techniques for the formation, physical properties and stability of Pickering emulsion","volume":"277","author":"Low","year":"2020","journal-title":"Adv Colloid Interface Sci"},{"key":"B7","doi-asserted-by":"publisher","DOI":"10.1038\/nmat5057","article-title":"Exploiting the pliability and lateral mobility of Pickering emulsion for enhanced vaccination","volume":"17","author":"Xia","year":"2018","journal-title":"Nat Mater"},{"key":"B8","doi-asserted-by":"publisher","DOI":"10.1039\/d0tb01318h","article-title":"A novel multiple emulsion enhanced immunity via its biomimetic delivery approach","volume":"8","author":"Zou","year":"2020","journal-title":"J Mater Chem B"},{"key":"B9","doi-asserted-by":"publisher","first-page":"3","DOI":"10.1016\/j.cis.2016.03.002","article-title":"Natural emulsifiers \u2013 biosurfactants, phospholipids, biopolymers, and colloidal particles: Molecular and physicochemical basis of functional performance","volume":"234","author":"McClements","year":"2016","journal-title":"Adv Colloid Interface Sci"},{"key":"B10","doi-asserted-by":"publisher","DOI":"10.2174\/13816128256661902011296290","article-title":"Comprehensive review on graphene oxide for use in drug delivery system","volume":"27","author":"Daniyal","year":"2020","journal-title":"Curr Med Chem"},{"key":"B11","doi-asserted-by":"publisher","DOI":"10.1016\/j.msec.2016.11.114","article-title":"Graphene oxide as a nanocarrier for controlled release and targeted delivery of an anticancer active agent, chlorogenic acid","volume":"74","author":"Barahuie","year":"2017","journal-title":"Mater Sci Eng C. Mater Biol Appl"},{"key":"B12","doi-asserted-by":"publisher","DOI":"10.1021\/acs.accounts.0c00336","article-title":"Synthetic particles for cancer vaccines: Connecting the inherent supply chain","volume":"53","author":"Xia","year":"2020","journal-title":"Acc Chem Res"},{"key":"B13","doi-asserted-by":"publisher","DOI":"10.1002\/adma.201400267","article-title":"Self-assembly of graphene oxide at interfaces","volume":"26","author":"Shao","year":"2014","journal-title":"Adv Mater"},{"key":"B14","doi-asserted-by":"publisher","DOI":"10.1021\/ja102777p","article-title":"Graphene oxide sheets at interfaces","volume":"132","author":"Kim","year":"2010","journal-title":"J Am Chem Soc"},{"key":"B15","doi-asserted-by":"publisher","DOI":"10.1021\/acsnano.5b06750","article-title":"Graphene oxides decorated with carnosine as an adjuvant to modulate innate immune and improve adaptive immunity in vivo","volume":"10","author":"Meng","year":"2016","journal-title":"ACS Nano"},{"key":"B16","doi-asserted-by":"publisher","DOI":"10.1039\/C5NR09208F","article-title":"Functionalized grapheme oxide serves as a novel vaccine nanoadjuvant for robust stimulation of cellular immunity","volume":"8","author":"Xu","year":"2016","journal-title":"Nanoscale"},{"key":"B17","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms14537","article-title":"PEGylated graphene oxide elicits strong immunological responses despite surface passivation","volume":"8","author":"Luo","year":"2017","journal-title":"Nat Commun"},{"key":"B18","doi-asserted-by":"publisher","DOI":"10.1016\/S1473-3099(18)30159-2","article-title":"Advancing the public health applications of Chlamydia trachomatis serology","volume":"18","author":"Woodhall","year":"2018","journal-title":"Lancet Infect Dis"},{"key":"B19","doi-asserted-by":"publisher","DOI":"10.1080\/14760584.2021.1899817","article-title":"Chlamydia trachomatis vaccines for genital infections: where are we and how far is there to go","volume":"20","author":"de la Maza","year":"2021","journal-title":"Expert Rev Vaccines"},{"key":"B20","doi-asserted-by":"publisher","DOI":"10.1016\/S1473-3099(19)30290-7","article-title":"First genital chlamydia vaccine enters in-human clinical trial","volume":"19","author":"Poston","year":"2019","journal-title":"Lancet Infect Dis"},{"key":"B21","doi-asserted-by":"publisher","DOI":"10.1016\/j.lfs.2021.119502","article-title":"Chlamydia trachomatis Pgp3 protein regulates oxidative stress via activation of the Nrf2\/NQO1 signal pathway","volume":"277","author":"Shu","year":"2021","journal-title":"Life Sci"},{"key":"B22","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2020.593295","article-title":"Chlamydia trachomatis plasmid protein pORF5 up-regulates ZFAS1 to promote host cell survival via MAPK\/p38 pathway, front","volume":"11","author":"Wen","year":"2020","journal-title":"Microbio."},{"key":"B23","article-title":"Identification of proteins interacting with pORF5 in the pathogenesis of c. trachomatis","volume":"10","author":"You","year":"2018","journal-title":"Am J Transl Res"},{"key":"B24","doi-asserted-by":"publisher","DOI":"10.1093\/femspd\/ftx111","article-title":"Chlamydia trachomatis pORF5 plasmid-encoded protein regulates autophagy and apoptosis of HeLa cells","volume":"33","author":"He","year":"2019","journal-title":"Biotechnol Biotec Equi"},{"key":"B25","doi-asserted-by":"publisher","DOI":"10.1007\/s11427-008-0130-9","article-title":"Immunization with chlamydial plasmid protein pORF5 DNA vaccine induces protective immunity against genital chlamydial infection in mice","volume":"51","author":"Li","year":"2008","journal-title":"Sci China C Life"},{"key":"B26","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2334-11-347","article-title":"Enhanced upper genital tract pathologies by blocking Tim-3 and PD-L1 signaling pathways in mice intravaginally infected with Chlamydia muridarum","volume":"11","author":"Peng","year":"2011","journal-title":"BMC Infect Dis"},{"key":"B27","doi-asserted-by":"publisher","DOI":"10.1021\/am400582","article-title":"Factors that affect Pickering emulsions stabilized by graphene oxide","volume":"5","author":"He","year":"2013","journal-title":"ACS Appl Mater Interfaces"},{"key":"B28","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jafc.6b04146","article-title":"Gelatin-based nanocomplex-stabilized Pickering emulsions: regulating droplet size and wettability through assembly with glucomannan","volume":"65","author":"Jin","year":"2017","journal-title":"Agric Food Chem"},{"key":"B29","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jafc.9b03356","article-title":"Current trends in Pickering emulsions: Particle morphology and applications","volume":"67","author":"Ortiz","year":"2019","journal-title":"J Agric Food Chem"},{"key":"B30","doi-asserted-by":"publisher","DOI":"10.1016\/j.jcis.2014.05.033","article-title":"Dispersion behaviour of graphene oxide and reduced graphene oxide","volume":"430","author":"Konios","year":"2014","journal-title":"J Colloid Interf Sci"},{"key":"B31","doi-asserted-by":"publisher","DOI":"10.1021\/jp500072a","article-title":"Graphene oxide-stabilized oil-in-water emulsions: pH-controlled dispersion and flocculation","volume":"118","author":"McCoy","year":"2014","journal-title":"J Phys Chem C"},{"key":"B32","doi-asserted-by":"publisher","DOI":"10.1021\/acs.analchem.1c00311","article-title":"Charge-based separation of acid-functional polymers by non-aqueous capillary electrophoresis employing deprotonation and heteroconjugation approaches","volume":"93","author":"Brooijmans","year":"2021","journal-title":"Anal Chem"},{"key":"B33","doi-asserted-by":"publisher","DOI":"10.1002\/slct.201601248","article-title":"And organic solvent dispersible gold nanorods that are pH responsive","volume":"17","author":"Imura","year":"2016","journal-title":"Talanta"},{"key":"B34","doi-asserted-by":"publisher","first-page":"24","DOI":"10.15761\/AMS.1000107","article-title":"Stabilization of pickering emulsions by iron oxide nano-particles","volume":"1","author":"Udoetok","year":"2016","journal-title":"Adv Mat Sci"},{"key":"B35","doi-asserted-by":"publisher","DOI":"10.1016\/j.jconrel","article-title":"Liposomes used as a vaccine adjuvant-delivery system: From basics to clinical immunization","volume":"303","author":"Wang","year":"2019","journal-title":"J Control Release"},{"key":"B36","doi-asserted-by":"publisher","DOI":"10.1016\/j.vaccine.2020.08.054","article-title":"Adjuvants for swine vaccines: Mechanisms of actions and adjuvant effects","volume":"38","author":"Charerntantanakul","year":"2020","journal-title":"Vaccine"},{"key":"B37","doi-asserted-by":"publisher","DOI":"10.1021\/acsbiomaterials.6b00232","article-title":"Adsorption behavior of silk fibroin on amphiphilic graphene oxide","volume":"2","author":"Tadepalli","year":"2016","journal-title":"ACS Biomater Sci Eng"},{"key":"B38","doi-asserted-by":"publisher","DOI":"10.1021\/acsami.5b08963","article-title":"Spontaneous protein adsorption on graphene oxide nanosheets allowing efficient intracellular vaccine protein delivery","volume":"8","author":"Li","year":"2016","journal-title":"ACS Appl Mater Interfaces"},{"key":"B39","doi-asserted-by":"publisher","DOI":"10.1021\/ac301569b","article-title":"Method for estimation of protein isoelectric point","volume":"84","author":"Pihlasalo","year":"2012","journal-title":"Anal Chem"},{"key":"B40","doi-asserted-by":"publisher","DOI":"10.3390\/polym14051032","article-title":"Graphene oxide-protein-based scaffolds for tissue engineering: recent advances and applications","volume":"14","author":"Biru","year":"2022","journal-title":"Polymers (Basel)"},{"key":"B41","doi-asserted-by":"publisher","DOI":"10.1016\/j.apsusc.2017.08.130","article-title":"Changing the activities and structures of bovine serum albumin bound to graphene oxide","volume":"427","author":"Zhang","year":"2018","journal-title":"Appl Surf Sci"},{"key":"B42","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.69951","article-title":"Vaccination induces rapid protection against bacterial pneumonia via training alveolar macrophage in mice","volume":"10","author":"Gu","year":"2021","journal-title":"Elife"},{"key":"B43","doi-asserted-by":"publisher","DOI":"10.1016\/j.cimid.2009.12.003","article-title":"Comparison between three adjuvants for a vaccine against canine leishmaniasis: In vitro evaluation of macrophage killing ability","volume":"33","author":"Trotta","year":"2010","journal-title":"Comp Immunol Microbiol Infect Dis"},{"key":"B44","doi-asserted-by":"publisher","DOI":"10.1021\/acsnano.5b04751","article-title":"Crucial role of lateral size for graphene oxide in activating macrophages and stimulating pro-inflammatory responses in cells and animals","volume":"9","author":"Ma","year":"2015","journal-title":"ACS Nano"},{"key":"B45","doi-asserted-by":"publisher","DOI":"10.1016\/j.jcis.2014.07.004","article-title":"In vitro evaluation of graphene oxide nanosheets on immune function","volume":"432","author":"Feito","year":"2014","journal-title":"J Colloid Interf Sci"},{"key":"B46","doi-asserted-by":"publisher","DOI":"10.1016\/j.jff.2015.03.009","article-title":"Squalene targets pro- and anti-inflammatory mediators and pathways to modulate over-activation of neutrophils, monocytes and macrophages","volume":"14","author":"C\u00e1rdeno","year":"2015","journal-title":"J Func Foods"},{"key":"B47","doi-asserted-by":"publisher","DOI":"10.1016\/j.yexcr.2019.02.007","article-title":"Lipopolysaccharide mediates time-dependent macrophage M1\/M2 polarization through the Tim-3\/Galectin-9 signalling pathway","volume":"376","author":"Zhang","year":"2019","journal-title":"Exp Cell Res"},{"key":"B48","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2020.01298","article-title":"Activation of M1 macrophages in response to recombinant TB vaccines with enhanced antimycobacterial activity","volume":"11","author":"Yang","year":"2020","journal-title":"Front Immunol"},{"key":"B49","doi-asserted-by":"publisher","DOI":"10.1016\/j.phrs.2020.105288","article-title":"Boosting immunity to treat parasitic infections: Asaia bacteria expressing a protein from wolbachia determine M1 macrophage activation and killing of leishmania protozoans","volume":"161","author":"Varotto-Boccazzi","year":"2020","journal-title":"Pharmacol Res"},{"key":"B50","doi-asserted-by":"publisher","DOI":"10.1021\/acsnano.8b03363","article-title":"Non-functionalized ultrasmall silica nanoparticles directly and size-selectively activate T cells","volume":"12","author":"Vis","year":"2018","journal-title":"ACS Nano"},{"key":"B51","doi-asserted-by":"publisher","DOI":"10.1002\/jsfa.10819","article-title":"The immunomodulatory effects of carapax trionycis ultrafine powder on cyclophosphamide-induced immunosuppression in BALB\/c mice","volume":"101","author":"Chen","year":"2021","journal-title":"J Sci Food Agric"},{"key":"B52","doi-asserted-by":"publisher","DOI":"10.1016\/j.biopha.2021.111799","article-title":"Squalene nanoemulsion reinforces mucosal and immunological fingerprints following intravaginal delivery","volume":"141","author":"Ho","year":"2021","journal-title":"BioMed Pharmacother"},{"key":"B53","doi-asserted-by":"publisher","first-page":"11520","DOI":"10.1038\/s41598-019-47885-z","article-title":"The droplet size of emulsion adjuvants has significant impact on their potency, due to differences in immune cell-recruitment and \u2013activation","volume":"9","author":"Shah","year":"2019","journal-title":"Sci Rep"},{"key":"B54","doi-asserted-by":"publisher","first-page":"897","DOI":"10.1080\/21691401.2016.1188396","article-title":"Construction of a stable w\/o nano-emulsion as a potential adjuvant for foot and mouth disease virus vaccine","volume":"45","author":"Chen","year":"2017","journal-title":"Artif Cells Nanomed Biotechnol"},{"key":"B55","doi-asserted-by":"publisher","DOI":"10.1016\/j.carbpol.2019.115128","article-title":"Polyethylenimine-coated PLGA nanoparticles-encapsulated angelica sinensis polysaccharide as an adjuvant to enhance immune responses","volume":"223","author":"Gu","year":"2019","journal-title":"Carbohydr Polym"},{"key":"B56","doi-asserted-by":"publisher","first-page":"55","DOI":"10.1016\/s0169-409x(00)00121-6","article-title":"Lymphatic targeting with nanoparticulate system","volume":"47","author":"Nishioka","year":"2001","journal-title":"Adv Drug Delivery Rev"},{"key":"B57","doi-asserted-by":"publisher","DOI":"10.3762\/bjnano.5.174","article-title":"Different endocytotic uptake mechanisms for nanoparticles in epithelial cells and macrophages","volume":"5","author":"Kuhn","year":"2014","journal-title":"Beilstein J Nanotechnol"},{"key":"B58","doi-asserted-by":"publisher","DOI":"10.1002\/adma.20200421","article-title":"Particulate alum via pickering emulsion for an enhanced COVID-19 vaccine adjuvant","volume":"32","author":"Peng","year":"2020","journal-title":"Adv Mater"},{"key":"B59","doi-asserted-by":"publisher","first-page":"14","DOI":"10.1016\/j.actbio.2020.06.009","article-title":"Recent progress of graphene oxide as a potential vaccine carrier and adjuvant","volume":"112","author":"Cao","year":"2020","journal-title":"Acta Biomater"},{"key":"B60","doi-asserted-by":"publisher","DOI":"10.1128\/CVI.00010-17","article-title":"The predominant CD4+ Th1 cytokine elicited to Chlamydia trachomatis infection in women is tumor necrosis factor alpha and not interferon gamma","volume":"24","author":"Jordan","year":"2017","journal-title":"Clin Vaccine Immunol"},{"key":"B61","doi-asserted-by":"publisher","DOI":"10.1128\/CMR.00105-13","article-title":"Chlamydia trachomatis: understanding the roles of innate and adaptive immunity in vaccine research","volume":"27","author":"Vasilevsky","year":"2014","journal-title":"Clin Microbiol Rev"},{"key":"B62","doi-asserted-by":"publisher","DOI":"10.1021\/acs.langmuir.8b02011","article-title":"Ionic liquid-containing pickering emulsions stabilized by graphene oxide-sased surfactants","volume":"34","author":"Lou","year":"2018","journal-title":"Langmuir"}],"container-title":["Frontiers in Immunology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2023.1148253\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,4,18]],"date-time":"2023-04-18T06:14:30Z","timestamp":1681798470000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2023.1148253\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,4,18]]},"references-count":62,"alternative-id":["10.3389\/fimmu.2023.1148253"],"URL":"https:\/\/doi.org\/10.3389\/fimmu.2023.1148253","relation":{},"ISSN":["1664-3224"],"issn-type":[{"value":"1664-3224","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,4,18]]},"article-number":"1148253"}}