Quantitative mechanics of endothelial phagocytosis of silicon microparticles

Abstract

Endothelia, once thought of as a barrier to the delivery of therapeutics, is now a major target for tissue-specific drug delivery. Tissue-and disease-specific molecular presentations on endothelial cells provide targets for anchoring or internalizing delivery vectors. Porous silicon delivery vectors are phagocytosed by vascular endothelial cells. The rapidity and efficiency of silicon microparticle uptake lead us to delineate the kinetics of internalization. To discriminate between surface-attached and -internalized microparticles, we developed a double fluorescent/FRET flow cytometric approach. The approach relies on quenching of antibody-conjugated fluorescein isothiocyanate covalently attached to the microparticle surface by attachment of a secondary antibody labeled with an acceptor fluorophore, phycoerythrin. The resulting half-time for microparticle internalization was 15.7 min, with confirmation provided by live confocal imaging as well as transmission electron microscopy. (1), and first-stage targeting for multifunctional drug delivery vehicles (2). Distinct features of tumor blood vessels, i.e., vascular zip codes, can function as homing devices for intravascularly administered drug delivery vehicles labeled with appropriate peptides or antibodies (3). As a first step in designing delivery vectors for cancer therapeutics, we have fabricated porous silicon microparticles that are internalized by vascular endothelial cells via phagocytosis and macropinocytosis (4).The distinction between professional and nonprofessional phagocytes is attributed to an array of dedicated phagocytic receptors on the former population that broadens their target range. Nonprofessional phagocytes, such as vascular endothelial cells, are able to internalize large micron-sized particulates (4); however, in contrast to professional phagocytes, serum opsonization of particulates hinders rather than augments internalization. One approach to bypass this obstruction is to bioengineer the surface of drug delivery vehicles to reduce binding to these serum dysopsonins, favoring endothelial uptake of particulates (4).To create delivery vectors with physical characteristics that favor transient interactions with endothelia, we first predicted the optimal size and shape of our silicon particles by mathematical modeling (5). Before applying targeting ligands to our vectors, it is important to first establish techniques to measure binding and uptake of microparticles by endothelial cells. In this report, a comparison of existing methods, as well as a novel method, is presented that characterize the mechanics and kinetics of internalization. Because of the dependence of some of these techniques on fluores-

Brain microvascular endothelial cell association and distribution of a 5 nm ceria engineered nanomaterial

“…42 Most of the literature we are aware of showed cell line-and material-dependent ENM internalization half lives of more than a few minutes in vitro and in vivo. [42][43][44][45][46] In three different cancer cells, 14, 50, and 74 nm gold nanoparticles had uptake half-lives of more than 1 hour. 43 Flux of cationic arginine-vasopressin nanoparticles across the BBB started at 15 minutes through absorptive-mediated endocytosis in mice.…”

Physiologically based pharmacokinetic modeling of PLGA nanoparticles with varied mPEG content

“…21 However, in vitro cellular models have shown endothelial cell uptake of PLGA nanoparticles, with various capacities in different cell types, [22][23][24] with uptake half-lives much more than a few minutes or even hours. 25,26 These contradicting findings indicate that there are tremendous differences between in vivo and in vitro nanoparticle kinetics. The complexity of in vivo behavior may not be easily represented by cellular models.…”

Mesoporous silicon particles as intravascular drug delivery vectors: fabrication, in‐vitro, and in‐vivo assessments

“…In the assay, antibody-associated fluorescence from primary fluorophores on S1MPs bound to the cell surface is quenched by fluorophores attached to secondary binding antibodies. Based on the decrease in surface quenching that accompanies S1MP uptake, the half-time for their internalization is calculated to be 15.7 min [33]. This time course is similar for two Figure 7 Scanning (a) and transmission (b) electron micrographs of S1MPs loaded with iron oxide nanoparticles.…”

“…Images courtesy of Wiley Interscience, Serda et al [35]. subtypes of endothelial cells (umbilical vein and microvascular) and encompasses all stages of microparticle internalization, which includes adhesion, the latency period, emergence of pseudopodia and protrusion of pseudopodia to surround the S1MP, as well as the final phase in which the particle is pulled into the cell [33].…”