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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

Ultra-structure observations by TEM of AluDia interaction with THP-1 monocyte cell line. Cells were treated with 20 μg/mL of AluDia particles for 3 (a) or 24h (b-i). a and b: AluDia particle internalization by THP-1 cells. Arrows indicate endosome membranes. c: AluDia particles inside macropinosome. Black (b,c) and white (c) arrows indicate macropinosome membrane and its absence, respectively. d: Intracellular AluDia particles encircled by double membrane autophagosome (arrow) indicating the autophagy activation. e: Detection of aluminum specific emission peak of (h) region, of internalized AluDia, by the X-ray microanalysis (EDX). f: macropinosome filled with AluDia particles. g- i: High resolution TEM analysis of the endosome content identified the specific crystal periodicity of mfNDs, (h): red (mfNDs) and green (alum) pseudo-colors, are superimposed to show the two crystalline structures of the AluDia complex (yellow pseudo-color, i). AluDia Alhydrogel® and mfND complex, mfNDs modified fluorescent nanodiamonds, TEM transmission electron microscopy
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Fig5: Ultra-structure observations by TEM of AluDia interaction with THP-1 monocyte cell line. Cells were treated with 20 μg/mL of AluDia particles for 3 (a) or 24h (b-i). a and b: AluDia particle internalization by THP-1 cells. Arrows indicate endosome membranes. c: AluDia particles inside macropinosome. Black (b,c) and white (c) arrows indicate macropinosome membrane and its absence, respectively. d: Intracellular AluDia particles encircled by double membrane autophagosome (arrow) indicating the autophagy activation. e: Detection of aluminum specific emission peak of (h) region, of internalized AluDia, by the X-ray microanalysis (EDX). f: macropinosome filled with AluDia particles. g- i: High resolution TEM analysis of the endosome content identified the specific crystal periodicity of mfNDs, (h): red (mfNDs) and green (alum) pseudo-colors, are superimposed to show the two crystalline structures of the AluDia complex (yellow pseudo-color, i). AluDia Alhydrogel® and mfND complex, mfNDs modified fluorescent nanodiamonds, TEM transmission electron microscopy

Mentions: Electron microscopy was performed on THP-1 monocyte/macrophage lineage cells incubated with AluDia particles. Particles were internalized by THP-1 cells within hours (Fig. 5a and b). After 24 hours AluDia particles were often found in large intracellular structures that may suggest macropinosomes, as was reported by Alhaddad et al. for siRNA delivery by nanodiamonds [51]. However, macropinosomes filled with AluDia particles often have damaged membranes (Fig. 5c and f). This observation was in line with the toxicity of alum for membrane lipid bilayers [52–54]. It seems possible that alum crystals directly aggress membranes [55], and this may play a crucial role in its adjuvant effect by inducing lysosomal function blockade [52–55]. Another mechanism of endosomal membrane damage may be related to nanomaterial-induced oxidative stress [56], and, indeed, aluminum [57], but not nanodiamonds [58], induces significant oxidative stress.Fig. 5


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)

Ultra-structure observations by TEM of AluDia interaction with THP-1 monocyte cell line. Cells were treated with 20 μg/mL of AluDia particles for 3 (a) or 24h (b-i). a and b: AluDia particle internalization by THP-1 cells. Arrows indicate endosome membranes. c: AluDia particles inside macropinosome. Black (b,c) and white (c) arrows indicate macropinosome membrane and its absence, respectively. d: Intracellular AluDia particles encircled by double membrane autophagosome (arrow) indicating the autophagy activation. e: Detection of aluminum specific emission peak of (h) region, of internalized AluDia, by the X-ray microanalysis (EDX). f: macropinosome filled with AluDia particles. g- i: High resolution TEM analysis of the endosome content identified the specific crystal periodicity of mfNDs, (h): red (mfNDs) and green (alum) pseudo-colors, are superimposed to show the two crystalline structures of the AluDia complex (yellow pseudo-color, i). AluDia Alhydrogel® and mfND complex, mfNDs modified fluorescent nanodiamonds, TEM transmission electron microscopy
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Ultra-structure observations by TEM of AluDia interaction with THP-1 monocyte cell line. Cells were treated with 20 μg/mL of AluDia particles for 3 (a) or 24h (b-i). a and b: AluDia particle internalization by THP-1 cells. Arrows indicate endosome membranes. c: AluDia particles inside macropinosome. Black (b,c) and white (c) arrows indicate macropinosome membrane and its absence, respectively. d: Intracellular AluDia particles encircled by double membrane autophagosome (arrow) indicating the autophagy activation. e: Detection of aluminum specific emission peak of (h) region, of internalized AluDia, by the X-ray microanalysis (EDX). f: macropinosome filled with AluDia particles. g- i: High resolution TEM analysis of the endosome content identified the specific crystal periodicity of mfNDs, (h): red (mfNDs) and green (alum) pseudo-colors, are superimposed to show the two crystalline structures of the AluDia complex (yellow pseudo-color, i). AluDia Alhydrogel® and mfND complex, mfNDs modified fluorescent nanodiamonds, TEM transmission electron microscopy
Mentions: Electron microscopy was performed on THP-1 monocyte/macrophage lineage cells incubated with AluDia particles. Particles were internalized by THP-1 cells within hours (Fig. 5a and b). After 24 hours AluDia particles were often found in large intracellular structures that may suggest macropinosomes, as was reported by Alhaddad et al. for siRNA delivery by nanodiamonds [51]. However, macropinosomes filled with AluDia particles often have damaged membranes (Fig. 5c and f). This observation was in line with the toxicity of alum for membrane lipid bilayers [52–54]. It seems possible that alum crystals directly aggress membranes [55], and this may play a crucial role in its adjuvant effect by inducing lysosomal function blockade [52–55]. Another mechanism of endosomal membrane damage may be related to nanomaterial-induced oxidative stress [56], and, indeed, aluminum [57], but not nanodiamonds [58], induces significant oxidative stress.Fig. 5

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