Intracellular trafficking of silicon particles and logic-embedded vectors

Abstract

Mesoporous silicon particles show great promise for use in drug delivery and imaging applications as carriers for second-stage nanoparticles and higher order particles or therapeutics. Modulation of particle geometry, surface chemistry, and porosity allows silicon particles to be optimized for specific applications such as vascular targeting and avoidance of biological barriers commonly found between the site of drug injection and the final destination. In this study, the intracellular trafficking of unloaded carrier silicon particles and carrier particles loaded with secondary iron oxide nanoparticles was investigated. Following cellular uptake, membrane-encapsulated silicon particles migrated to the perinuclear region of the cell by a microtubule-driven mechanism. Surface charge, shape (spherical and hemispherical) and size (1.6 and 3.2 microm) of the particle did not alter the rate of migration. Maturation of the phagosome was associated with an increase in acidity and acquisition of markers of late endosomes and lysosomes. Cellular uptake of iron oxide nanoparticle-loaded silicon particles resulted in sorting of the particles and trafficking to unique destinations. The silicon carriers remained localized in phagosomes, while the second stage iron oxide nanoparticles were sorted into multi-vesicular bodies that dissociated from the phagosome into novel membrane-bound compartments. Release of iron from the cells may represent exocytosis of iron oxide nanoparticle-loaded vesicles. These results reinforce the concept of multi-functional nanocarriers, in which different particles are able to perform specific tasks, in order to deliver single- or multi-component payloads to specific sub-cellular compartments.

Fig 1

Perinuclear trafficking of hemispherical silicon particles A) TEM micrographs of APTES-modified silicon particles 6hrs after introduction to HMVEC cells (top: 3.2μm; bottom: 1.6μm particles). B) Confocal images of particles-treated control or nocodazole (150 nM)-treated HMVECs visualized with DRAQ5 (nuclei) and FITC-conjugated α-tubulin antibody, 2 hrs after particles addition. The localization of particles with respect to the nucleous was quantified by dividing portions of cells in quadrants, having defined distances from the nucleus.

Fig 2

Colocalization of silicon particles with NPC1 protein and acidic vesicles. A) HMVECs were incubated with 3.2 μm discoidal silicon particles conjugated with a pH sensitive dye (pH-Rodo). Mean fluorescence was monitored over 16 hrs by flow cytometry. B–C) Confocal images of GFP-NPC1 transfected HMVECs incubated 4hours with Dylight 594-labelled B) or un-labelled C) silicon particles. The images are reported in separated and merged channels.

Fig 3

Effect of size and shape on the rate of particle migration. A) Manual tracking from the point of uptake to perinuclear region was done by confocal imaging of cells every 5 min (bar 10μm). B) 1) An example of MDS curve fittings (top) and 2), and box charts showing the calculated rates of migration toward the perinuclear region for different types of particles[oxidized and APTES are modified hemispherical silicon particles (two sizes), and beads are spherical silica particles (three sizes)]. Population were not significantly different based on ANOVA test with P=0.05.

Fig 4

particles characterization and loading FTIR spectra A) and XPS elemental analysis B) of carboxylated (IONP-COOH) and amine-PEG (IONP-NH) SPIONs (IO=Iron Oxide). C) Zeta potential and dynamic light scattering (DLS) measuraments for SPIONs and silicon particles in phosphate and borate buffers. D) TEM (left) and SEM (right) micrographs showing 3.2 μm discoidal particles loaded with amino-PEG SPIONs.

Fig 5

TEM micrographs of silicon particles loaded with amine-PEG SPIONs 24 hrs after internalization by HMVECs showing endosomal sorting of SPIONs into multi-vesicular bodies (MVB) and the process of MVB budding into separate membrane bound compartments.

Fig 6

Endosomal sorting and secretion of SPIONs A) TEM image showing a HMVEC cell containing a membrane-bound silicon particles with the majority of SPIONs released 7 days after LEV introduction. B) SPIONs secretion from cells over 7 days following treatment with free or silicon particles-delivered SPIONs (GP are silicon particles, %NH is amine-PEG coated SPIONs)