Synthetic mimetics of the endogenous gastrointestinal nanomineral: Silent constructs that trap macromolecules for intracellular delivery

Abstract

Amorphous magnesium-substituted calcium phosphate (AMCP) nanoparticles (75-150 nm) form constitutively in large numbers in the mammalian gut. Collective evidence indicates that they trap and deliver luminal macromolecules to mucosal antigen presenting cells (APCs) and facilitate gut immune homeostasis. Here, we report on a synthetic mimetic of the endogenous AMCP and show that it has marked capacity to trap macromolecules during formation. Macromolecular capture into AMCP involved incorporation as shown by STEM tomography of the synthetic AMCP particle with 5 nm ultra-fine iron (III) oxohydroxide. In vitro, organic cargo-loaded synthetic AMCP was taken up by APCs and tracked to lysosomal compartments. The AMCP itself did not regulate any gene, or modify any gene regulation by its cargo, based upon whole genome transcriptomic analyses. We conclude that synthetic AMCP can efficiently trap macromolecules and deliver them to APCs in a silent fashion, and may thus represent a new platform for antigen delivery.

Imaging flow cytometry assays for quantifying pigment grade titanium dioxide particle internalization and interactions with immune cells in whole blood

“…The darkfield scattered light signals of TiO 2 particles appeared as small punctate clusters within the CD14 1 monocytes (Fig. 3D), as would be expected of particles grouped together inside endo-lysosomal compartments within the cells, after active particle uptake by phagocytic cell types (29)(30)(31).…”

Formation and properties of amorphous magnesium-calcium phosphate particles in a simulated intestinal fluid

“…We also observed that AMCPs are able to incorporate the organic molecules present in the fluid in a time-dependent manner, as shown by means of both FT-IR spectroscopy and thermal analysis. This feature, which is a characteristic of endogenous AMCPs [5], is retained in vitro in agreement with previous literature reports [6]. The nanoscale structure of the amorphous particles was further inspected, and TEM analysis revealed a very peculiar structure in AMCPs_SIF: together with clustered particles, it was possible to observe some core-shell structures, with a 10 nm-core and an electron-dense 3 nmshell.…”

“…orally fed protein antigens and bacterial peptidoglycans, and transport them to the immune cells of the intestinal tissue, promoting the immuno-surveillance mechanisms. Endogenous AMCPs are porous and composed by agglomerates of small nanoparticulate structures; the amorphous nature allows for the effective incorporation of the organic cargo, as it was shown during in vitro experiments [6].…”

“…A peculiar feature of the endogenous AMCPs is their ability to trap antigens and peptidoglycans in the lumen, acting like a shuttle for delivering the organic cargo to the immune cells in the intestinal wall lining [5,45]. The ability of AMCP-like nanoparticles to trap macromolecules during formation was investigated in vitro by Pele et al, who found macromolecule-incorporation properties similar to their in vivo counterpart [6]. As we observed that the organic molecules present in the SIF affect the morphology of the synthesized particles, we inspected their incorporation/adsorption on AMCPs as a function of time.…”

Calcium phosphate nanoparticles as intrinsic inorganic antimicrobials: In search of the key particle property

“…One such immune role was ascribed to Mg-enriched ACP nanoparticles forming in the ileum due to endogenous secretion of calcium and phosphate ions from the distal small intestine to the lumen, where they trap orally entered antigens and bacterial peptidoglycans and chaperone them to the immune cells of the intestinal tissue for immunosurveillance. 3,4 Being less stable and more chemically active than HAp, ACP has been used in some dental formulations to remineralize enamel, 5 while binding with high affinity to S. mutans 6 and exhibiting mild anticariogenic properties whose origins are yet to be elucidated. Most of these formulations apply ACP in combination with casein phosphopeptides, providing for a rudimentary mimicry of sophisticatedly structured casein micelles stabilized in milk by ∼2 nm sized ACP nanoparticles lying along the internal micelle membrane.…”