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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

Particle characterization by microscopy. a: TEM analysis of fND aggregates that contained very small mfNDs (few nm). b: The red specific fluorescence of mfNDs excited by a 532 nm laser source. c: mfNDs luminescence spectrum with a specific peak at 700 nm. d: Schematic representation of the possible hydrogen bonds between the hydroxyl groups of the HPG chains and that of alum. e and f: The nanofibrous morphology of alum and vaccine aggregates (ENGERIX B®) by TEM analysis. g and h: The AluDia complex analyzed by TEM in which alum keeps its nanofibrous morphology and is loaded by non-fibrous, electron dense mfNDs. i-k: Fluorescence microscopy observation of an AluDia agglomerate. Red fluorescence of AluDia particles excited by a 532 nm laser source (i). Alum of AluDia complex stained with Morin and detected by a green fluorescence with a characteristic 520 nm emission when excited at 420nm (j), colocalization of the red fluorescence of mfNDs and the green Morin fluorescence of alum (k), without alteration of the typical mfND emission spectrum (l). AluDia Alhydrogel® and mfND complex, HPG hyperbranched polyglycerol, mfNDs modified fluorescent nanodiamonds, TEM transmission electron microscope
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Fig1: Particle characterization by microscopy. a: TEM analysis of fND aggregates that contained very small mfNDs (few nm). b: The red specific fluorescence of mfNDs excited by a 532 nm laser source. c: mfNDs luminescence spectrum with a specific peak at 700 nm. d: Schematic representation of the possible hydrogen bonds between the hydroxyl groups of the HPG chains and that of alum. e and f: The nanofibrous morphology of alum and vaccine aggregates (ENGERIX B®) by TEM analysis. g and h: The AluDia complex analyzed by TEM in which alum keeps its nanofibrous morphology and is loaded by non-fibrous, electron dense mfNDs. i-k: Fluorescence microscopy observation of an AluDia agglomerate. Red fluorescence of AluDia particles excited by a 532 nm laser source (i). Alum of AluDia complex stained with Morin and detected by a green fluorescence with a characteristic 520 nm emission when excited at 420nm (j), colocalization of the red fluorescence of mfNDs and the green Morin fluorescence of alum (k), without alteration of the typical mfND emission spectrum (l). AluDia Alhydrogel® and mfND complex, HPG hyperbranched polyglycerol, mfNDs modified fluorescent nanodiamonds, TEM transmission electron microscope

Mentions: As assessed by dynamic light scattering of particles in PBS at pH7.2, mfNDs appeared to be of nanometric size (about 80 nm) whereas agglomerates of alum alone, 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). Thus, in the physiological conditions we used, AluDia particle size and zeta potential were very similar to those of alum alone or alum adsorbed with HBV antigen. In addition, the size and charge of the AluDia particles remained stable during 15 days in PBS as well as in DMEM culture medium supplemented with 5 % (v/v) fetal bovine serum at 4 °C (data non shown). Thus, the physico-chemical properties of the AluDia complex were as close as possible to those of the HBV vaccine, making mfNDs relevant for further investigation as a tag for alum particle tracking.Table 1


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)

Particle characterization by microscopy. a: TEM analysis of fND aggregates that contained very small mfNDs (few nm). b: The red specific fluorescence of mfNDs excited by a 532 nm laser source. c: mfNDs luminescence spectrum with a specific peak at 700 nm. d: Schematic representation of the possible hydrogen bonds between the hydroxyl groups of the HPG chains and that of alum. e and f: The nanofibrous morphology of alum and vaccine aggregates (ENGERIX B®) by TEM analysis. g and h: The AluDia complex analyzed by TEM in which alum keeps its nanofibrous morphology and is loaded by non-fibrous, electron dense mfNDs. i-k: Fluorescence microscopy observation of an AluDia agglomerate. Red fluorescence of AluDia particles excited by a 532 nm laser source (i). Alum of AluDia complex stained with Morin and detected by a green fluorescence with a characteristic 520 nm emission when excited at 420nm (j), colocalization of the red fluorescence of mfNDs and the green Morin fluorescence of alum (k), without alteration of the typical mfND emission spectrum (l). AluDia Alhydrogel® and mfND complex, HPG hyperbranched polyglycerol, mfNDs modified fluorescent nanodiamonds, TEM transmission electron microscope
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Particle characterization by microscopy. a: TEM analysis of fND aggregates that contained very small mfNDs (few nm). b: The red specific fluorescence of mfNDs excited by a 532 nm laser source. c: mfNDs luminescence spectrum with a specific peak at 700 nm. d: Schematic representation of the possible hydrogen bonds between the hydroxyl groups of the HPG chains and that of alum. e and f: The nanofibrous morphology of alum and vaccine aggregates (ENGERIX B®) by TEM analysis. g and h: The AluDia complex analyzed by TEM in which alum keeps its nanofibrous morphology and is loaded by non-fibrous, electron dense mfNDs. i-k: Fluorescence microscopy observation of an AluDia agglomerate. Red fluorescence of AluDia particles excited by a 532 nm laser source (i). Alum of AluDia complex stained with Morin and detected by a green fluorescence with a characteristic 520 nm emission when excited at 420nm (j), colocalization of the red fluorescence of mfNDs and the green Morin fluorescence of alum (k), without alteration of the typical mfND emission spectrum (l). AluDia Alhydrogel® and mfND complex, HPG hyperbranched polyglycerol, mfNDs modified fluorescent nanodiamonds, TEM transmission electron microscope
Mentions: As assessed by dynamic light scattering of particles in PBS at pH7.2, mfNDs appeared to be of nanometric size (about 80 nm) whereas agglomerates of alum alone, 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). Thus, in the physiological conditions we used, AluDia particle size and zeta potential were very similar to those of alum alone or alum adsorbed with HBV antigen. In addition, the size and charge of the AluDia particles remained stable during 15 days in PBS as well as in DMEM culture medium supplemented with 5 % (v/v) fetal bovine serum at 4 °C (data non shown). Thus, the physico-chemical properties of the AluDia complex were as close as possible to those of the HBV vaccine, making mfNDs relevant for further investigation as a tag for alum particle tracking.Table 1

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