Tuning Nanobodies’ Bioactivity: Coupling to Ultrasmall Gold Nanoparticles Allows the Intracellular Interference with Survivin

Abstract

Nanobodies are highly affine binders, often used to track disease‐relevant proteins inside cells. However, they often fail to interfere with pathobiological functions, required for their clinical exploitation. Here, a nanobody targeting the disease‐relevant apoptosis inhibitor and mitosis regulator Survivin (SuN) is utilized. Survivin's multifaceted functions are regulated by an interplay of dynamic cellular localization, dimerization, and protein–protein interactions. However, as Survivin harbors no classical “druggable” binding pocket, one must aim at blocking extended protein surface areas. Comprehensive experimental evidence demonstrates that intracellular expression of SuN allows to track Survivin at low nanomolar concentrations but failed to inhibit its biological functions. Small angle X‐ray scattering of the Survivin‐SuN complex locates the proposed interaction interface between the C‐terminus and the globular domain, as such not blocking any pivotal interaction. By clicking multiple SuN to ultrasmall (2 nm) gold nanoparticles (SuN‐N), not only intracellular uptake is enabled, but additionally, Survivin crosslinking and interference with mitotic progression in living cells are also enabled. In sum, it is demonstrated that coupling of nanobodies to nanosized scaffolds can be universally applicable to improve their function and therapeutic applicability.

The Molecular Footprint of Peptides on the Surface of Ultrasmall Gold Nanoparticles (2 nm) Is Governed by Steric Demand

“…Notably, the conjugation of larger ligands to a glutathione-azide-terminated gold surface via click chemistry (CuAAC) leads to fewer ligands with a higher molecular footprint. This was found for 2 nm gold nanoparticles carrying Cy3 molecules (5 per nanoparticle, 2.5 nm 2 each), Cy5 molecules (36 per nanoparticle; 0.35 nm 2 each), FAM molecules (6 per nanoparticle, 2.1 nm 2 each), , Alexa Fluor 647 molecules (11 to 18 per nanoparticle, 0.70 to 1.15 nm 2 each), , nanobodies (5 per nanoparticle, 2.5 nm 2 each), siRNA molecules (6 to 10 per nanoparticle, 1.4 to 2.0 nm 2 each), or molecular tweezers (11 to 30 per nanoparticle, 0.42 to 1.14 nm 2 each) . A quantitative assessment of the clicking efficiency on glutathione-azide-terminated 2 nm gold nanoparticles showed that only 6 to 11 out of 118 available azide groups were used for conjugation .…”

The Apoptosis Inhibitor Protein Survivin Is a Critical Cytoprotective Resistor against Silica-Based Nanotoxicity

“…We therefore compared specific differentiation markers of human lung tissue to those of mouse lung and cell culture models. For the first time, we showed that tissue cells of the human lung express the inhibitor of apoptosis protein, Survivin, which is critical for mitosis and proliferation [ 31 , 56 , 57 , 58 , 59 ]. Furthermore, we implemented cell culture models that reflect the necessary characteristics of lung tissue cells.…”

The Why and How of Ultrasmall Nanoparticles

“…However, the much smaller nanobodies (about 4 nm) 61 were successfully attached to ultrasmall gold nanoparticles (2 nm; carrying about 4 nanobodies each) and showed highly efficient targeting against the protein Survivin. 62 The ability to "dial in" nanoparticle-biosystem interactions fully extends to cells. HeLa cells were incubated with gold nanoparticles with 2, 4, and 6 nm cores and cationic, anionic, and zwitterionic ligands (Figure 7a), and the uptake was quantified by inductively coupled mass spectrometry (ICP-MS) (Figure 7b).…”

“…As antibodies are much larger (about 12 nm) than ultrasmall nanoparticles, it does not make sense to attach them as targeting moiety to their surface. However, the much smaller nanobodies (about 4 nm) were successfully attached to ultrasmall gold nanoparticles (2 nm; carrying about 4 nanobodies each) and showed highly efficient targeting against the protein Survivin …”