Potentiation of the activation of cholinergic receptors by multivalent presentation of ligands supported on gold nanoparticles.

Gold nanoparticles (NP) with a functionalized ligand shell offer the possibility to potentiate the action of agonists at the receptor site by multivalency. In order to find out whether this can be realized for the pharmacologically important class of cholinergic receptors known to be involved in the regulation of most organ functions, carbachol-functionalized gold NPs (Au-MUDA-CCh) with an average diameter of 14 nm were synthesized. As functional read-out, cholinergic agonist-induced anion secretion was measured as increase in short-circuit current (Isc) across rat proximal colon in Ussing chambers. Similarly to the corresponding native agonist acetylcholine, Au-MUDA-CCh induced a concentration-dependent increase in Isc, which represents chloride secretion across the epithelium. This response was inhibited by atropine and hexamethonium indicating the activation of muscarinic and nicotinic receptors by the functionalized NPs. A strong potentiation of ligand-receptor interaction was a key benefit of functionalized NPs over native agonists. This was observed with physiological approaches as measurements of changes in Isc revealed a nearly equivalent response evoked by 1 pM Au-MUDA-CCh and 500 nM native CCh. To better determine this potentiation at the receptor level, pharmacological approaches based on the signaling cascade of ACh-induced activation of muscarinic receptors were used. FRET (Förster Resonance Energy Transfer) measurements performed on HEK293T cells transiently transfected with M3-R, Gαq-YFP, Gβ1-wt and CFP-Gγ2, revealed that both Au-MUDA-CCh and native CCh activated G-proteins with EC50 amounting to 127 ± 0.44 fM and 224 ± 7.12 nM, respectively. Thus, the functionalization of the NPs with CCh yields a potentiation by over 106, a property that could find usage in specific targeting, activation and compensation of pathologically reduced expression of receptors of interest.