Fluorescent nanodiamonds as a relevant tag for the assessment of alum adjuvant particle biodisposition

Eidi et al. BMC Medicine (2015) 13:144 DOI 10.1186/s12916-015-0388-2 RESEARCH ARTICLE Open Access Fluorescent nanodiamonds as a relevant tag for the assessment of alum adjuvant particle biodisposition Housam Eidi1,3*, Marie-Odile David1, Guillemette Crépeaux3, Laetitia Henry1, Vandana Joshi1, Marie-Hélène Berger2, Mohamed Sennour2, Josette Cadusseau3,4, Romain K. Gherardi3† and Patrick A. Curmi1† 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. Keywords: Alum, fluorescent nanodiamonds, vaccine adjuvant, biodisposition * Correspondence: † Equal contributors 1 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 3 Inserm - U955, Université Paris Est, Faculté de Médecine, Créteil, France Full list of author information is available at the end of the article © 2015 Eidi et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Eidi et al. BMC Medicine (2015) 13:144 Background The understanding of how the body handles small particles in the long-term, especially those which interact with the immune system, is a major objective of recent research [1]. For example, concerns linked to the use of aluminum particles as vaccine adjuvants [aluminum oxyhydroxide (“alum”)] have emerged following recognition of their role at the origin of the focal lesion called macrophagic myofasciitis (MMF). This revealed a fundamental misconception of the fate of alum in the organism pointing out its unexpectedly long-lasting biopersistence within immune cells [2]. It also demonstrated their capacity to migrate to the lymphoid organs, to disseminate throughout the body within monocyte-lineage cells, and to slowly accumulate in the brain [3]. Millions of humans have received vaccines adjuvanted with alum. Overall safety of these vaccines has been regarded as excellent at the level of the population [4], but adverse effects have also been reported [5, 6]. It seems very likely that a small proportion of presumably susceptible individuals exposed to particulate materials with adjuvant effects, e.g. alum adjuvants or breast implant-derived silicone, may develop progressive systemic and neurologic autoimmune/inflammatory manifestations or “ASIA” [7]. These individuals typically show long-term persistence of particles within the monocyte-lineage cells at either the site of previous immunization with alum-containing vaccines, i.e. MMF, or in the vicinity of leaky breast implants [8]. Alum particles have neither fluorescent nor magnetic properties. Their detection in tissues therefore represents a difficult challenge. Khan et al. [3] analyzed biodisposition of alum particles in mice by tracking fluorescent alum surrogates, such as alum-like hybrids which were composed of a rhodamine core coated with precipitated aluminum hydroxide. This approach has limitations since the precipitated aluminum hydroxide used by Khan et al. [3] is similar but not strictly identical to the aluminum oxyhydroxide used in vaccines [9]. Indeed, particles may exhibit strikingly different properties according to their physicochemical properties, the main parameters being their size, shape, zeta potential and chemical composition [10]. The present study aimed at evaluating the possibility of constructing a fluorescent complex highly relevant to vaccine by tagging the alum adjuvant itself (Alhydrogel®) using modified fluorescent nanodiamonds (mfNDs). MfNDs have unique fluorescence properties, which allow their detection at very low levels and over a very longterm period [11–13]. Indeed, their fluorescence, based on the presence of nitrogen-vacancy centers (NV centers) within the nanodiamond crystal lattice, is perfectly photostable with neither bleaching nor blinking. mfNDs were reported as biocompatible fluorescent particles with very low toxicity [14]. These properties overcome Page 2 of 13 the limitations of organic fluorophores or quantum dots, i.e. photobleaching and toxicity [15–17]. In our last paper we showed that fNDs functionalized with hyperbranched polyglycerol (mfNDs) could be promising tools for biomedical research [18]. In the present study, Alhydrogel® used in vaccines was tagged with mfNDs forming the AluDi (...truncated)