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

Eidi H, David MO, Crépeaux G, Henry L, Joshi V, Berger MH, Sennour M, Cadusseau J, Gherardi RK, Curmi PA - BMC Med (2015)

Bottom Line: As the NV center does not bleach, it allows the microspectrometric detection of mfNDs at very low levels and in the long-term.Expectedly, AluDia elicited autophagy, and allowed highly specific detection of small amounts of alum in autophagosomes.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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus

Assessment of AluDia biodistribution following its injection in tibialis anterior muscle at day 21. a-c: AluDia translocation in inguinal lymphatic nodes which appeared mostly empty at day 21 (cf. Table 2). d-l: AluDia particles reach liver, spleen and brain forming small clusters. In all observations, Morin stain of aluminum revealed that mfNDs and alum were co-localized in most particles. AluDia Alhydrogel® and mfND complex, mfNDs modified fluorescent nanodiamonds
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Fig3: Assessment of AluDia biodistribution following its injection in tibialis anterior muscle at day 21. a-c: AluDia translocation in inguinal lymphatic nodes which appeared mostly empty at day 21 (cf. Table 2). d-l: AluDia particles reach liver, spleen and brain forming small clusters. In all observations, Morin stain of aluminum revealed that mfNDs and alum were co-localized in most particles. AluDia Alhydrogel® and mfND complex, mfNDs modified fluorescent nanodiamonds

Mentions: AluDia injected in the mouse tibialis anterior muscle was followed by lymphatic and systemic particle biodistribution (Table 3), as previously reported with other fluorescent particles [3]. Alum and mfNDs remained clearly colocalized in a large majority of particles detected remote from the injection site as assessed by Morin stain (Fig. 3a-i). Actually, our data of particle counting in sections from various tissues showed that 88 ± 4 % of observed nanodiamonds were close to those of alum. Similarly to alum-rhodamine nanohybrid particles (AlRho) used by Khan et al. [3], AluDia reached the inguinal dLN, as observed at day 7, and then left the dLN which partially emptied at day 21. One striking observation was the marked increase of AluDia particles in spleen at day 7 (54,500 particles) with a decrease at day 21 (7,000 particles). This massive alum access to spleen at day 7 was not previously noted in the Khan et al. study [3] which had no intermediate time points between day 4 and day 21. This observation is in keeping with the time frame of a primary immune response in the lymphoid organs. Particles were also detected in the liver, an organ not studied by Khan et al. [3], but previously shown to incorporate alum adjuvants from the circulation [31]. Besides additional insights provided by the evaluation of different time points and additional organs, the use of AluDia allowed us to substantiate our previous contention that alum particles translocate from the injected muscle to dLNs and then to distant organs unplugged to lymphatic vessels [3].Table 3


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

Eidi H, David MO, Crépeaux G, Henry L, Joshi V, Berger MH, Sennour M, Cadusseau J, Gherardi RK, Curmi PA - BMC Med (2015)

Assessment of AluDia biodistribution following its injection in tibialis anterior muscle at day 21. a-c: AluDia translocation in inguinal lymphatic nodes which appeared mostly empty at day 21 (cf. Table 2). d-l: AluDia particles reach liver, spleen and brain forming small clusters. In all observations, Morin stain of aluminum revealed that mfNDs and alum were co-localized in most particles. AluDia Alhydrogel® and mfND complex, mfNDs modified fluorescent nanodiamonds
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4482291&req=5

Fig3: Assessment of AluDia biodistribution following its injection in tibialis anterior muscle at day 21. a-c: AluDia translocation in inguinal lymphatic nodes which appeared mostly empty at day 21 (cf. Table 2). d-l: AluDia particles reach liver, spleen and brain forming small clusters. In all observations, Morin stain of aluminum revealed that mfNDs and alum were co-localized in most particles. AluDia Alhydrogel® and mfND complex, mfNDs modified fluorescent nanodiamonds
Mentions: AluDia injected in the mouse tibialis anterior muscle was followed by lymphatic and systemic particle biodistribution (Table 3), as previously reported with other fluorescent particles [3]. Alum and mfNDs remained clearly colocalized in a large majority of particles detected remote from the injection site as assessed by Morin stain (Fig. 3a-i). Actually, our data of particle counting in sections from various tissues showed that 88 ± 4 % of observed nanodiamonds were close to those of alum. Similarly to alum-rhodamine nanohybrid particles (AlRho) used by Khan et al. [3], AluDia reached the inguinal dLN, as observed at day 7, and then left the dLN which partially emptied at day 21. One striking observation was the marked increase of AluDia particles in spleen at day 7 (54,500 particles) with a decrease at day 21 (7,000 particles). This massive alum access to spleen at day 7 was not previously noted in the Khan et al. study [3] which had no intermediate time points between day 4 and day 21. This observation is in keeping with the time frame of a primary immune response in the lymphoid organs. Particles were also detected in the liver, an organ not studied by Khan et al. [3], but previously shown to incorporate alum adjuvants from the circulation [31]. Besides additional insights provided by the evaluation of different time points and additional organs, the use of AluDia allowed us to substantiate our previous contention that alum particles translocate from the injected muscle to dLNs and then to distant organs unplugged to lymphatic vessels [3].Table 3

Bottom Line: As the NV center does not bleach, it allows the microspectrometric detection of mfNDs at very low levels and in the long-term.Expectedly, AluDia elicited autophagy, and allowed highly specific detection of small amounts of alum in autophagosomes.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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus