How Carrier Size and Valency Modulate Receptor-Mediated Signaling: Understanding the Link between Binding and Endocytosis of ICAM-1-Targeted Carriers

Abstract

Targeting of drug carriers to endocytic cell-receptors facilitates intracellular drug delivery. Carrier size and number of targeting moieties (valency) influence cell binding and uptake. However, how these parameters influence receptor-mediated cell-signaling (the link between binding and uptake) remains uncharacterized. We studied this using polymer carriers of different sizes and valencies, targeted to endothelial intercellular adhesion molecule-1 (ICAM-1), a marker overexpressed in many pathologies. Unexpectedly, induction of cell-signals (ceramide and PKC enrichment and activation) and uptake, were independent of: carrier avidity, total number of carriers bound per cell, cumulative cell-surface area occupied by carriers, number of targeting antibodies at the carrier-cell contact, and cumulative receptor engagement by all bound carriers. Instead, “valency density” (number of antibodies per carrier surface area) ruled signaling, and carrier size independently influenced uptake. These results are key to understanding the interplay between carrier design parameters and receptor-mediated signaling conducive to endocytosis, paramount for intracellular drug delivery.

ICAM-1 targeting, intracellular trafficking, and functional activity of polymer nanocarriers coated with a fibrinogen-derived peptide for lysosomal enzyme replacement

“…Where indicated, ASM was pre-labelled with 125 Iodine to quantify loading of this therapeutic cargo on the NC surface using 125 I-ASM specific activity and a gamma-radiation counter (Wizard 2 ; PerkinElmer) [9]. Non-coated proteins were removed by centrifugation and coated NCs were resuspended at ≈10 9 NCs/μL in 1% bovine serum albumin (BSA)-supplemented phosphate buffered saline (PBS) and sonicated [31]. The enzyme loading efficiency was calculated as the enzyme that coated nanoparticles (recovered in the pellet) compared to the total amount of enzyme added to the coating reaction, expressed as a percentage.…”

“…The enzyme loading efficiency was calculated as the enzyme that coated nanoparticles (recovered in the pellet) compared to the total amount of enzyme added to the coating reaction, expressed as a percentage. The absolute amount of enzyme coated per nanoparticles was calculated from the amount of 125 Iodine in the nanoparticle pellet, the measured 125 Iodine cpm per mass of enzyme, and the known number of nanoparticles in the coating mixture, as previously described [31]. The diameter of the resulting NCs, the polydispersity index (PDI), and the Z potential were determined by dynamic light scattering (Zetasizer NanoZS90, Malvern Instruments, Westborough, MA, USA).…”

“…Images were taken using an ORCA-1CCD camera (Hamamatsu Corporation, Bridgewater, NJ) and analyzed using Image-Pro 6.3 software (Media Cybernetics, Bethesda, MD), to quantify the number of NC bound per cell. This is automatically assessed using a previously described algorithm that detects and counts fluorescent objects whose pixel size is equivalent that of NCs and which amply surpass the fluorescence intensity of the surrounding background [31]. …”

“…Cells were washed, fixed, and incubated with Texas Red-labeled goat anti-mouse IgG, which can bind to tracer IgG on the NC coat only if NCs are located on the cell surface, not if they are internalized within cells. This enables differential visualization of cell surface-bound NCs as yellow objects (green + red) vs. internalized counterparts which appear green [31]. Samples were imaged by phase-contrast and fluorescence microscopy to quantify the percentage of internalized NCs from the total number of NCs associated to cells, as described [31].…”

“…This enables differential visualization of cell surface-bound NCs as yellow objects (green + red) vs. internalized counterparts which appear green [31]. Samples were imaged by phase-contrast and fluorescence microscopy to quantify the percentage of internalized NCs from the total number of NCs associated to cells, as described [31]. …”