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.
Highlights
Aluminum oxyhydroxide is a crystalline compound widely used as an immunologic adjuvant of vaccines
Particle characterization As assessed by dynamic light scattering of particles in Phosphate buffered saline (PBS) at pH7.2, mfNDs appeared to be of nanometric size whereas agglomerates of alum alone, Alhydrogel® and mfND complex (AluDia) and Engerix® alum formed particles of micrometric diameter with peaks from 2,900 nm to 3,800 nm (Table 1 and Fig. 1)
Our characterization data showed that the zeta potential of mfNDs is slightly negative (−29 mV) whereas those of alum, AluDia, and ENGERIX® particles are slightly positive, ranging from +25 to +30 mV (Table 1)
Summary
Preparation and characterization of the AluDia complex Reagent grade chemicals were purchased from Sigma– Aldrich (France) and used as received. Size and zeta potential measurements We characterized the size distribution and zeta potential of mfNDs, alum and AluDia as well as of the particles present in a commercially available anti-hepatitis B vaccine (HBV, ENGERIX B® GlaxoSmithKline Inc., Evreux, France) using dynamic light scattering. In vitro and in vivo experiments Cell culture We used THP1 and NSC-34 as in vitro models These cell lines were used to assess particle internalization, intracellular colocalization of alum and mfNDs particles and their cytotoxicity. Cells were treated with different doses of alum, mfNDs and AluDia particles two days after the beginning of the culture. The viabilities of cultured cells were assessed 72 hours after treatment with mfNDs, alum and AluDia particles following the manufacturer’s protocol. All statistical analyses were performed with the SigmaStat 3.11 software package (Sistat Inc., USA)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
