Engineering the Inorganic-Living Interface

Surface functionalization of nanocrystals is essential for their practical application in living systems, but introducing biocompatibility can come at the cost of diminished nanoparticle optics, diminished colloidal stability, or massive increases in probe size. In addition, the cell presents a remarkably complex chemical environment, rich in metal ions, amines, thiols, and other compounds that can disrupt nanoparticle complexes that are perfectly stable <i>in vitro</i>. Inorganic nanocrystals such as quantum dots (QDs),^1,2 upconverting nanoparticles (UCNPs),³ and avalanching nanoparticles (ANPs)^4,5 are uniquely suited for quantitative live-cell imaging and are typically functionalized with ligands to study specific receptors or cellular targets. Antibodies are among the most useful targeting reagents owing to their high affinities and specificities, but common nanocrystal labeling methods may orient antibodies incorrectly, be reversible or denaturing, or lead to antibody-nanoparticle complexes too large for some applications. We have shown that SpyCatcher proteins, which bind and spontaneously form covalent isopeptide bonds with cognate SpyTag peptides, can conjugate engineered antibodies to nanoparticle surfaces with control over stability, orientation, and stoichiometry.⁶ Compact SpyCatcher-functionalized QDs and UCNPs may be labeled with short-chain variable fragment antibody (scFv) engineered to bind urokinase-type plasminogen activator receptors (uPAR) that are overexpressed in many human cancers. Confocal imaging of anti-uPAR scFv-QD conjugates shows the antibody mediates specific binding and internalization by breast cancer cells expressing uPAR. Time-lapse imaging of photostable scFv-UCNP conjugates shows that antibody binding causes uPAR internalization with a ∼20 min half-life on the cell surface, and uPAR is internalized to endolysosomal compartments distinct from general membrane stains and without significant recycling to the cell surface. With Tm³⁺-doped upconverting ANPs functionalized with these scFv antibodies, the extreme nonlinearly of ANP emission leads to sub-100 nm spatial resolution using only simple scanning confocal microscopy and before any computational analysis. Quantification of ANP spot intensity across multiple <i>z</i> planes then yields a high-resolution 3D map of uPAR distribution on the cell surface. The controlled and stable conjugation of engineered antibodies to nanoparticles enables targeting of diverse receptors for quantitative live-cell study of their distribution, trafficking, and physiology<br/><br/>1. Mann, V. R., Powers, A. S., Tilley, D. C., Sack, J. T. & Cohen, B. E. Azide–Alkyne Click Conjugation on Quantum Dots by Selective Copper Coordination. <i>ACS Nano</i> <b>12</b>, 4469–4477 (2018).<br/>2. Wichner, S. M. <i>et al.</i> Covalent Protein Labeling and Improved Single-Molecule Optical Properties of Aqueous CdSe/CdS Quantum Dots. <i>ACS Nano</i> <b>11</b>, 6773–6781 (2017).<br/>3. Tian, B. <i>et al.</i> Low irradiance multiphoton imaging with alloyed lanthanide nanocrystals. <i>Nat. Commun.</i> <b>9</b>, 3082 (2018).<br/>4. Lee, C. <i>et al.</i> Giant nonlinear optical responses from photon-avalanching nanoparticles. <i>Nature</i> <b>589</b>, 230–235 (2021).<br/>5. Lee, C. <i>et al.</i> Indefinite and bidirectional near-infrared nanocrystal photoswitching. <i>Nature</i> (2023) doi:10.1038/s41586-023-06076-7.<br/>6. Pedroso, C. C. S. <i>et al.</i> Immunotargeting of nanocrystals by SpyCatcher conjugation of engineered antibodies. <i>ACS Nano</i> <b>15</b>, 18374–18384 (2021).