A Single-Molecule View at Nanoparticle Targeting Selectivity: Correlating Ligand Functionality and Cell Receptor Density

Abstract

Antibody-functionalized nanoparticles (NPs) are commonly used to increase the targeting selectivity toward cells of interest. At a molecular level, the number of functional antibodies on the NP surface and the density of receptors on the target cell determine the targeting interaction. To rationally develop selective NPs, the single-molecule quantitation of both parameters is highly desirable. However, techniques able to count molecules with a nanometric resolution are scarce. Here, we developed a labeling approach to quantify the number of functional cetuximabs conjugated to NPs and the expression of epidermal growth factor receptors (EGFRs) in breast cancer cells using direct stochastic optical reconstruction microscopy (dSTORM). The single-molecule resolution of dSTORM allows quantifying molecules at the nanoscale, giving a detailed insight into the distributions of individual NP ligands and cell receptors. Additionally, we predicted the fraction of accessible antibody-conjugated NPs using a geometrical model, showing that the total number exceeds the accessible number of antibodies. Finally, we correlated the NP functionality, cell receptor density, and NP uptake to identify the highest cell uptake selectivity regimes. We conclude that single-molecule functionality mapping using dSTORM provides a molecular understanding of NP targeting, aiding the rational design of selective nanomedicines.

Mapping the relationship between total and functional antibodies conjugated to nanoparticles with spectrally-resolved direct stochastic optical reconstruction microscopy (SR-dSTORM)

“…In accordance with a previous estimation reporting only 30% of functional Fabs, it was conrmed experimentally that indeed not all conjugated antibodies are functional to recognize their target. 11 Here, the single-molecule and single-particle quantication depicts that the experimental functionality percentage lies in a similar range (between 20-50% functionality). Furthermore, the results indicate that the functionality ratio also depends on the antibody conjugation concentration.…”

“…S3†). 11,21 SR-dSTORM localizations are proportional to the number of molecules on the NP, thus knowing how many localizations correspond to a single cetuximab or EGFR molecule during the imaging acquisition allows for the identification of the amount of functional and total cetuximab antibodies per NP. Finally, the total amount of Fab fragments per NP was calculated by multiplying the number of total cetuximab obtained by factor 2.…”

“…Finally, all NPs with their localizations were grouped (bandwidth 150 nm, minimum number of localizations 20) and ltered based on their size and elongation using a mean-shi clustering script (MATLAB) which was previously described. 11 To correlate the number of localizations with the number of cetuximab-CF680 or EGFR-AF647 molecules, a calibration was performed as previously described 11,21 to estimate the number of localizations obtained per singlemolecule under the same imaging conditions at a very low protein concentration (0.1 nM and 3.5 nM, respectively) attached to a cover glass (ESI Fig. S3 †).…”

“…Furthermore, recent reports indicate substantial heterogeneity in NP conjugation at a single-particle level. [9][10][11] Understanding NP ligand orientation and their functional heterogeneity are fundamental to achieving efficient active targeting.…”

“…Consequently, previous work focused only on the quantication of functional antibodyconjugated nanoparticles, while the relation between total and functional molecules remained to be elucidated at a singleparticle level. 11 Recently, multicolor approaches based solely on infrared dyes have been proposed, such as spectral demixing and imaging. 15 Spectral SMLM (sSMLM) congurations enable simultaneous optical super-resolution microscopy and spectroscopy at the single-molecule level.…”