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

Abstract

BackgroundAluminum 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.MethodsWe 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.ResultsIn 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.ConclusionsThe 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.

Non-linear dose-response of aluminium hydroxide adjuvant particles: Selective low dose neurotoxicity

“…Alhydrogel® biopersistence was confirmed in a variety of laboratory animal models up to 6-12 months post-injection, in both the injected muscle (Verdier et al, 2005;Authier et al, 2006;Khan et al, 2013;Eidi et al, 2015) and distant lymphoid organs . Particles traffic from an injected tissue to the dLNs is size-dependent, smaller particles (20-200 nm) being able to drain in a free form whereas medium-sized particles (0.5-2 µm) are exclusively subjected to cell transportation (Manolova et al, 2008).…”

“…Non-monotonic dose-response curves have been previously reported in the field of aluminium toxicology. Non-monotonic biphasic neurotoxic effects have been observed both in vitro (see for review Exley and Birchall, 1992;Platt et al, 1995;Eidi et al, 2015) and in vivo (Kumar, 1998;Tsunoda and Sharma, 1999;Kim, 2003;Roig et al, 2006) after oral Al administration. However, the dose-response curve of the present study was not biphasic.…”

“…We previously studied the fate of aluminium particles following im injections. Aluminium hydroxide is a highly hydrated crystalline compound composed of elementary nano-needles of approximately 2.2 nm x 4.5 nm x 10 nm (Mao et al, 2013) and displays a fibrous morphology at transmission electron microscopy (Shirodkar et al, 1990;Eidi et al, 2015). This compound spontaneously forms micron-sized agglomerates (Johnston et al, 2002), subjected to slight size variations after antigen adsorption and in vivo interactions with phosphate, organic acid and proteinaceous environments.…”

“…This compound spontaneously forms micron-sized agglomerates (Johnston et al, 2002), subjected to slight size variations after antigen adsorption and in vivo interactions with phosphate, organic acid and proteinaceous environments. A series of recent reports from our laboratory have shown that translocation of aluminium hydroxide may be specifically related to monocyte lineage cell uptake of this poorly biodegradable compound (Khan et al, 2013;Crépeaux et al, 2015;Eidi et al, 2015), likely resulting from phagocytosis or macropinocytosis (Mao et al, 2013).…”

“…Previous studies from our laboratory have shown that alum particles, as other poorly degradable particles, may not stay entirely localized in the injected tissue in mice, but can disseminate within phagocytic cells to regional lymph nodes and then to more distant sites and to the brain (Khan et al, 2013;Crépeaux et al, 2015;Eidi et al, 2015). In contrast to a previous belief, alum is characterized by striking biopersistence within immune cells in both the injected muscle, and the draining lymph nodes (dLNs) and spleen, where it may be found in conspicuous quantities 9 months after injection .…”