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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

Cell viability assayed by mitochondrial metabolism assessment (MTT test). NSC-34 neuron-like cells were incubated with different concentrations of mfNDs, alum and AluDia particles for 72 hours. AluDia concentrations tested in (c) = mfNDs concentrations in (a) + alum concentrations in (b). a: mfNDs particles are non-toxic, except at the highest dose. b: alum particles display a toxic or severely toxic effect at all doses used. c: AluDia particles have no supplemental toxicity compared to alum alone. Viability was normalized to the value determined in untreated cells. Results are expressed as mean ± standard deviation. Cells were obtained from four different cultures to realize four biological replications (n = 4). Viability measurement of each concentration point was repeated 12 times. *Significant difference at p < 0.05. AluDia Alhydrogel® and mfND complex, mfNDs modified fluorescent nanodiamonds
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Fig4: Cell viability assayed by mitochondrial metabolism assessment (MTT test). NSC-34 neuron-like cells were incubated with different concentrations of mfNDs, alum and AluDia particles for 72 hours. AluDia concentrations tested in (c) = mfNDs concentrations in (a) + alum concentrations in (b). a: mfNDs particles are non-toxic, except at the highest dose. b: alum particles display a toxic or severely toxic effect at all doses used. c: AluDia particles have no supplemental toxicity compared to alum alone. Viability was normalized to the value determined in untreated cells. Results are expressed as mean ± standard deviation. Cells were obtained from four different cultures to realize four biological replications (n = 4). Viability measurement of each concentration point was repeated 12 times. *Significant difference at p < 0.05. AluDia Alhydrogel® and mfND complex, mfNDs modified fluorescent nanodiamonds

Mentions: Since little has been reported about toxic effects of alum in vitro, we examined next whether AluDia particles could be used in vitro to study what could be the impact of alum on cultured cells. In order to compare the cytotoxicity of alum and AluDia particles, we incubated NSC-34 neuronal lineage cells with different concentrations of mfNDs, Alhydrogel®, and AluDia particles for 72 hours. Particle toxicity was evaluated based on cell viability assessed by MTT assay relative to controls, as proposed by Kong et al. [35]: (1) non-toxic >90 % cell viability; (2) slightly toxic = 65–90 %; (3) toxic = 35–65 %; (4) severely toxic ≤35 %. mfNDs appeared non-toxic, except at the highest dose (Fig. 4a), confirming previous reports on the lack of toxicity of nanodiamonds [36, 37]. Paget et al. showed that mfNDs are neither cytotoxic nor genotoxic on six human cell lines representative of potential target organs: HepG2 and Hep3B (liver), Caki-1 and Hek-293 (kidney), HT29 (intestine) and A549 (lung) [38]. These authors did not check mfNDs cytotoxicity on neural cell lines, but Hsu et al. reported that mfNDs disturb neither neuronal differentiation nor neuron functions [14]. In addition, mfNDs were shown to be non-toxic in vivo, in both Caenorhabditis elegans and mouse [39, 40]. However, other studies showed slight toxic and genotoxic effects of nanodiamonds in vitro and in vivo [41–43]. Only a few studies reported serious toxic effects in vivo [44, 45].Fig. 4


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)

Cell viability assayed by mitochondrial metabolism assessment (MTT test). NSC-34 neuron-like cells were incubated with different concentrations of mfNDs, alum and AluDia particles for 72 hours. AluDia concentrations tested in (c) = mfNDs concentrations in (a) + alum concentrations in (b). a: mfNDs particles are non-toxic, except at the highest dose. b: alum particles display a toxic or severely toxic effect at all doses used. c: AluDia particles have no supplemental toxicity compared to alum alone. Viability was normalized to the value determined in untreated cells. Results are expressed as mean ± standard deviation. Cells were obtained from four different cultures to realize four biological replications (n = 4). Viability measurement of each concentration point was repeated 12 times. *Significant difference at p < 0.05. AluDia Alhydrogel® and mfND complex, mfNDs modified fluorescent nanodiamonds
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Cell viability assayed by mitochondrial metabolism assessment (MTT test). NSC-34 neuron-like cells were incubated with different concentrations of mfNDs, alum and AluDia particles for 72 hours. AluDia concentrations tested in (c) = mfNDs concentrations in (a) + alum concentrations in (b). a: mfNDs particles are non-toxic, except at the highest dose. b: alum particles display a toxic or severely toxic effect at all doses used. c: AluDia particles have no supplemental toxicity compared to alum alone. Viability was normalized to the value determined in untreated cells. Results are expressed as mean ± standard deviation. Cells were obtained from four different cultures to realize four biological replications (n = 4). Viability measurement of each concentration point was repeated 12 times. *Significant difference at p < 0.05. AluDia Alhydrogel® and mfND complex, mfNDs modified fluorescent nanodiamonds
Mentions: Since little has been reported about toxic effects of alum in vitro, we examined next whether AluDia particles could be used in vitro to study what could be the impact of alum on cultured cells. In order to compare the cytotoxicity of alum and AluDia particles, we incubated NSC-34 neuronal lineage cells with different concentrations of mfNDs, Alhydrogel®, and AluDia particles for 72 hours. Particle toxicity was evaluated based on cell viability assessed by MTT assay relative to controls, as proposed by Kong et al. [35]: (1) non-toxic >90 % cell viability; (2) slightly toxic = 65–90 %; (3) toxic = 35–65 %; (4) severely toxic ≤35 %. mfNDs appeared non-toxic, except at the highest dose (Fig. 4a), confirming previous reports on the lack of toxicity of nanodiamonds [36, 37]. Paget et al. showed that mfNDs are neither cytotoxic nor genotoxic on six human cell lines representative of potential target organs: HepG2 and Hep3B (liver), Caki-1 and Hek-293 (kidney), HT29 (intestine) and A549 (lung) [38]. These authors did not check mfNDs cytotoxicity on neural cell lines, but Hsu et al. reported that mfNDs disturb neither neuronal differentiation nor neuron functions [14]. In addition, mfNDs were shown to be non-toxic in vivo, in both Caenorhabditis elegans and mouse [39, 40]. However, other studies showed slight toxic and genotoxic effects of nanodiamonds in vitro and in vivo [41–43]. Only a few studies reported serious toxic effects in vivo [44, 45].Fig. 4

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