title: Fluorescent nanodiamonds as a relevant tag for the assessment of alum adjuvant particle biodisposition.
abstract: BACKGROUND
  Aluminum oxyhydroxide (alum) is a crystalline compound widely used as an immunologic adjuvant of vaccines. Concerns linked to alum particles have emerged following recognition of their causative role in the so-called macrophagic myofasciitis (MMF) lesion in patients with myalgic encephalomyelitis, revealing an unexpectedly long-lasting biopersistence of alum within immune cells and a fundamental misconception of its biodisposition. Evidence that aluminum-coated particles phagocytozed in the injected muscle and its draining lymph nodes can disseminate within phagocytes throughout the body and slowly accumulate in the brain further suggested that alum safety should be evaluated in the long term. However, lack of specific staining makes difficult the assessment of low quantities of bona fide alum adjuvant particles in tissues.
  
  
  METHODS
  We explored the feasibility of using fluorescent functionalized nanodiamonds (mfNDs) as a permanent label of alum (Alhydrogel(®)). mfNDs have a specific and perfectly photostable fluorescence based on the presence within the diamond lattice of nitrogen-vacancy centers (NV centers). As the NV center does not bleach, it allows the microspectrometric detection of mfNDs at very low levels and in the long-term. We thus developed fluorescent nanodiamonds functionalized by hyperbranched polyglycerol (mfNDs) allowing good coupling and stability of alum:mfNDs (AluDia) complexes. Specificities of AluDia complexes were comparable to the whole reference vaccine (anti-hepatitis B vaccine) in terms of particle size and zeta potential.
  
  
  RESULTS
  In vivo, AluDia injection was followed by prompt phagocytosis and AluDia particles remained easily detectable by the specific signal of the fND particles in the injected muscle, draining lymph nodes, spleen, liver and brain. In vitro, mfNDs had low toxicity on THP-1 cells and AluDia showed cell toxicity similar to alum alone. Expectedly, AluDia elicited autophagy, and allowed highly specific detection of small amounts of alum in autophagosomes.
  
  
  CONCLUSIONS
  The fluorescent nanodiamond technology is able to overcome the limitations of previously used organic fluorophores, thus appearing as a choice methodology for studying distribution, persistence and long-term neurotoxicity of alum adjuvants and beyond of other types of nanoparticles.
authors.affiliation: Institut National de la Santé et de la Recherche Médicale (INSERM) - UMR 1204, Université Evry-Val d'Essonne, Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, Evry, France. housam.eidi@gmail.com.|Inserm - U955, Université Paris Est, Faculté de Médecine, Créteil, France. housam.eidi@gmail.com.
authors.forename: Housam
authors.initials: H
authors.lastname: Eidi
authors.affiliation: Institut National de la Santé et de la Recherche Médicale (INSERM) - UMR 1204, Université Evry-Val d'Essonne, Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, Evry, France. MO.David@iut.univ-evry.fr.
authors.forename: Marie-Odile
authors.initials: MO
authors.lastname: David
authors.affiliation: Inserm - U955, Université Paris Est, Faculté de Médecine, Créteil, France. guillemette.crepeaux@gmail.com.
authors.forename: Guillemette
authors.initials: G
authors.lastname: Crépeaux
authors.affiliation: Institut National de la Santé et de la Recherche Médicale (INSERM) - UMR 1204, Université Evry-Val d'Essonne, Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, Evry, France. laetitia.henry@wanadoo.fr.
authors.forename: Laetitia
authors.initials: L
authors.lastname: Henry
authors.affiliation: Institut National de la Santé et de la Recherche Médicale (INSERM) - UMR 1204, Université Evry-Val d'Essonne, Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, Evry, France. vandana.joshi@univ-evry.fr.
authors.forename: Vandana
authors.initials: V
authors.lastname: Joshi
authors.affiliation: Laboratoire Pierre-Marie Fourt, Centre des Matériaux de l'Ecole des Mines de Paris and CNRS UMR 7633, Evry, France. marie-helene.berger@mines-paristech.fr.
authors.forename: Marie-Hélène
authors.initials: MH
authors.lastname: Berger
authors.affiliation: Laboratoire Pierre-Marie Fourt, Centre des Matériaux de l'Ecole des Mines de Paris and CNRS UMR 7633, Evry, France. mohamed.sennour@ensmp.fr.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Sennour
authors.affiliation: Inserm - U955, Université Paris Est, Faculté de Médecine, Créteil, France. josette.cadusseau@inserm.fr.|Faculté des Sciences et Technologie UPEC, Créteil, France. josette.cadusseau@inserm.fr.
authors.forename: Josette
authors.initials: J
authors.lastname: Cadusseau
authors.affiliation: Inserm - U955, Université Paris Est, Faculté de Médecine, Créteil, France. romain.gherardi@hmn.aphp.fr.
authors.forename: Romain K
authors.initials: RK
authors.lastname: Gherardi
authors.affiliation: Institut National de la Santé et de la Recherche Médicale (INSERM) - UMR 1204, Université Evry-Val d'Essonne, Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, Evry, France. pcurmi@univ-evry.fr.
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Curmi
mesh_terms: D000276:Adjuvants, Immunologic / Q000009:adverse effects; D000328:Adult; D000534:Alum Compounds / Q000009:adverse effects*; D005208:Fasciitis / Q000139:chemically induced; D005260:Female; D005456:Fluorescent Dyes / Q000494:pharmacology*; D006801:Humans; D009220:Myositis / Q000139:chemically induced; D058612:Nanodiamonds; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D000276:Adjuvants, Immunologic; D000534:Alum Compounds; D005456:Fluorescent Dyes; D058612:Nanodiamonds; C041524:aluminum sulfate