Particle characterization

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 Diameter distribution and zeta potential values of mfND, Alhydrogel®, AluDia and ENGERIX® alum particles Full size table

Fig. 1 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 Full size image

The Alhydrogel® and mfND ratio in the AluDia complex was determined on the basis of different experimental assays. The respective proportions of the suspensions Alhydrogel® and mfNDs were varied from 1/0.25 to 1/100 and the resulting complex observed with fluorescent microscopy and characterized by TEM. The final value of 1/17 was chosen as the optimal compromise between these two distant values. With the 1/0.25 ratio, mfNDs were difficult to detect in the large amount of Alhydrogel® during TEM observations. At the inverse, with the 1/100 ratio, Alhydrogel® became almost the minor component. The preparations of the AluDia complex were based on the simple mixing of both solutions and their agitation to favor the complex formation. The formed particles had a micrometric size and they fell down rapidly to the bottom of the eppendorf. However, free mfNDs gave stable suspensions and should then remain in the supernatant. The analysis of the latter in the mixture case used in this study showed no free mfNDs.

HR-TEM imaging showed that mfNDs have a rounded to polygonal shape (Fig. 1a). In addition to the mfND size indicated by dynamic light scattering analysis, very small mfNDs (a few nm) were detected within the mfND aggregates by TEM. When excited by a laser at 532 nm, mfNDs displayed a red fluorescence (Fig. 1b) with a specific luminescence spectrum peaking at 700 nm (Fig. 1c).

We attribute the favorable interactions created between alum and mfNDs to the presence of numerous hydroxyl groups on the polyglycerol chains synthesized at the surface of mfNDs. Polyols were reported to be adsorbed strongly at the surface of boehmite (AlOOH) particles and hydrogen bonds were created between their hydroxyl groups and AlOOH [21]. Thanks to the hyperbranched structure of polyglycerol, numerous hydroxyl groups are present, along and at the end of the HPG chains, and can thus create such hydrogen interactions with alum (Fig. 1d). Although electrostatic interactions due to opposite zeta potential values between mfNDs and Alhydrogel® cannot be completely excluded, the contribution of this second type of interaction to the stability of the complex should almost disappear in the salty solutions (PBS 1x) that we use due to the charge screening effect.

Both alum and vaccine particles displayed a nano-fibrous morphology (Fig. 1e, f), as previously reported in the literature [22]. AluDia were distinctly composed of nano-fibrous agglomerates of alum randomly decorated by polygonal, non-fibrous, electron dense mfNDs (Fig. 1g, h). This was confirmed by X-ray microanalysis and high resolution transmission electron microscopy (data not shown, see below). In bright field TEM images, the mfNDs have various crystal orientations with respect to the incident electron beam. The darker particles correspond to nanocrystals in Bragg’s position on which the electron beam has been diffracted and then stopped by the objective aperture (Fig. 1a, g and h). AluDia particles stained by Morin to detect aluminum showed colocalization of the red fluorescence of mfNDs with the green fluorescence of the Morin-alum complex, without alteration of the typical mfND emission spectrum (Fig. 1i-l). Thus, the association of mfNDs with alum does not disturb the fluorescent signature of mfNDs. Taken together, these results and the persistent colocalization observed at distant places from the injection point in mice (as will be shown later) strongly suggest that stable attractive interactions are created between aluminum oxyhydroxide and mfNDs. Moreover, fluorescence images (Fig. 1i-k) show clearly that mfNDs are much more fluorescent than alum stained by Morin which makes these particles useful to detect isolated alum particles.

Morin stain was previously used in the paper from our lab showing systemic translocation of Al particles [3]. However, Morin stain was reported to be not entirely specific for Al. Browne et al. showed that Morin could stain other metals but Al has the most of affinity to Morin according to the following order: Al > Fe(III) > Cu > Fe(II) > Ca > Mg > Mn = Zn [23]. Lumogallion was reported as an interesting dye for aluminum studies [24, 25]; however, it has limitations, too. Lumogallion specificity for Al is higher than that of Morin but Lumogallion also stains gallium, in addition to Al [26] and other metals, such as Fe [27, 28]. Furthermore, both the Lumogallion affinity for Al and fluorescence signal intensity are much lower than those of Morin [29] limiting its use when the detection of trace amounts of Al in tissues is concerned, e.g. in brain. Additionally, the Lumogallion red/orange fluorescence emission spectrum is very close to that of the mfNDs we used, precluding its use in place of Morin in the present study.

In vivo observations

Granuloma formation at the injection site

At 21 days after i.m. injection, AluDia particles accumulated into the injected muscle similarly to vaccine particles [3]. Indeed, granuloma mainly composed of CD11b+ monocyte- macrophage lineage cells filled with AluDia was formed in the endomysium, i.e. in between myofibers, at the injection site (Fig. 2a). Non Morin-stained AluDia particles in muscles have the same fluorescent signature as compared to those of mfNDs (Fig. 2b,c). These particles do not display any fluorescence when they are excited at 420nm as compared to Morin-stained AluDia (Fig. 2c). The phase contrast image shows AluDia particles within the granuloma region in muscle section (Fig. 2d). Morin stain for aluminum confirmed that macrophages contained stably associated AluDia particles as assessed by both red and green fluorescence (Fig. 2e-g). Importantly, photostability of mfNDs upon long laser exposure made AluDia detection very easy without background fluorescence whereas the detection of Morin stain was commonly disturbed by its bleaching and a strong tissue fluorescent background (Fig. 2h-j). Serial sectioning of the injected muscle at day 45, day 135, day 180 and day 270 after AluDia injection showed progressive shrinkage of muscle granulomas (Table 2), as previously reported in rats [30]. At 270 days post-injection, one out of three tested mice was completely free of muscle granuloma, and the other two mice only had small residual muscle granulomas.

Fig. 2 AluDia translocation and biopersistence in injected muscle after 21 days. a: immunohistochemistry analysis: AluDia particle accumulation in the injected muscle inducing a granuloma of CD11b+ monocyte-macrophage lineage cells filled with AluDia in the endomysium area. The green fluorescence corresponds to CD11b protein immunolabeling. b-d: AluDia particle detection (b) in injected muscle non-stained with Morin. Tissue section was observed using the 420 nm excitation for Morin (c) and phase contrast (d). e-g: the stable association between the mfND component as assessed by the red fluorescence obtained with the 532 nm laser excitation (e) and alum after Morin staining observed with 420 nm excitation (f) and phase contrast image (g). h: mfND component of AluDia complex (red fluorescence) with alum component stained with Morin and disturbed by the high fluorescent background of muscle cells (i) at the injection site in muscle section observed with phase contrast microscopy analysis (j). AluDia Alhydrogel® and mfND complex, mfNDs modified fluorescent nanodiamonds Full size image

Table 2 Semi-quantitative study of the progressive decrease of granuloma size in injected muscle with AluDia complex. AluDia Alhydrogel® and mfND complex, TA tibialis anterior Full size table

AluDia translocation from the injected muscle to distant organs

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 AluDia particle systemic distribution at 7 and 21 days after injection of an equivalent of 400 μg/Kg aluminum in the tibialis anterior muscle. AluDia Alhydrogel® and mfND complex, dLNs draining lymph nodes, mfNDs modified fluorescent nanodiamonds Full size table

Fig. 3 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 Full size image

Four brains were examined at day 21 after i.m. injection of AluDia. Consistent with the low level of cerebral incorporation of particle previously noted at this early time point [3], each of the four brains contained 15 ± 4 AluDia particles, usually forming small clusters in the cerebellum or cerebral cortex. As shown in Fig. 3j-l, Morin stain for aluminum revealed that mfNDs and alum were colocalized in most particles, whereas occasional particles were solely positive for either Morin+ or fND+. Since the labeling of Alhydrogel® with mfNDs confers physicochemical properties to the neo-particle that are very similar to whole vaccine particles, these data definitely establish that bona fide alum adjuvants of vaccines can penetrate in the brain [3]. This occurs in the particulate form and mimics brain translocation of infectious particles, such as intracellular bacteria, HIV and other pathogens [32–34].

Finally, AluDia particles confirmed the biodistribution modalities of poorly degradable particles. In addition, AluDia particles were well characterized whereas previously used AlRho particles had undetermined size, zeta potential, ultrastructure and proportion relative to alum. In addition to good relevance to vaccine, AluDia particles allowed administration of precise amounts of aluminum.

In vitro observations

Cytotoxicity

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 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 Full size image

In contrast to mfNDs, alum particles were toxic or severely toxic at all doses used (Fig. 4b), and the same was observed with AluDia (Fig. 4c). AluDia had no supplemental toxicity compared to Alhydrogel® particles alone. Interestingly, with both Alhydrogel® and AluDia, cytotoxity did not show a linear dose–response, since higher doses tended to be less toxic than intermediate doses, as previously noted for particle toxicity [46–49]. Alhydrogel®in vitro toxicity for neuronal cells is in keeping with mouse studies showing in vivo neurotoxic effects of subcutaneously administered Alhydrogel®, including neural apoptosis and both motor and behavioral deficits [50].

Ultrastructural studies

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 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 Full size image

Another ultrastructural finding after 24-hour AluDia exposure consisted in intracellular particles encircled by double membranes highly suggestive of autophagophores, thus assessing active autophagy (Fig. 5d). Cells coping with microbes use a dedicated form of autophagy termed “xenophagy” as a host defense mechanism to engulf and degrade intracellular pathogens. The same holds true for inert particles subjected to phagocytosis/endocytosis [59]. Eidi et al. reported that the free internalized particles in cell cytoplasm could induce stress of mitochondria or other intracellular organelles resulting in autophagy activation [56]. As mentioned above, alum particles are toxic to membranes which destabilizes phagosomes and lysosomes, triggers inflammasome assembly, and impedes the autophagy pathways [52–55]. It seems possible that macrophages that perceive the foreign particles in their cytosol, just like senescent organelles or bacteria, will attempt to reiterate the autophagic process until they dispose of the alien materials. The compartmentalization of particles within double membrane and subsequent fusion of autophagosomes with repaired and re-acidified lysosomes could expose alum particles to lysosomal acidic pH, a crucial factor in the alum solubilization process. Notably, Li et al. reported that alpha-alumina nanoparticles activate the autophagy in dendritic cells much more efficiently than alum particles [60]. To our knowledge, no study in the literature has reported autophagy activation by mfNDs.

It is difficult to visualize moderate amounts of alum within cells by TEM, and one has to use X-ray microanalysis (EDX) to assess the presence of aluminum by detection of a specific emission peak (Fig. 5e). This approach has limitations since the sample is always at risk of being contaminated by extrinsic aluminum present in the air or incorporated into the sample during its processing. The use of AluDia may help in tracking alum in resin embedded material. MfNDs cannot represent contaminants and their specific and highly photostable fluorescence can be detected in semi-thin sections. Moreover, high resolution TEM can reliably identify the specific crystalline structures. As shown in Fig. 5f-i, high resolution TEM of the endosome content of THP1 cells exposed to AluDia identified the specific crystal periodicity of both mfNDs (red pseudo-color) and Alhydrogel® (green pseudo-color), as well as superimposition of the two crystalline structures (yellow pseudo-color). This confirmed the stability of AluDia after internalization by immune cells. Using mfNDs as a tag mimicing vaccine antigen, the same approach could be used to assess if and how the alum adjuvant dissociates from compounds adsorbed at its surface over a long time in vivo.