Supramolecular regulation of bioorthogonal catalysis in cells using nanoparticle-embedded transition metal catalysts

Abstract

Bioorthogonal catalysis broadens the functional possibilities of intracellular chemistry. Effective delivery and regulation of synthetic catalytic systems in cells are challenging due to the complex intracellular environment and catalyst instability. Here, we report the fabrication of protein-sized bioorthogonal nanozymes through the encapsulation of hydrophobic transition metal catalysts into the monolayer of water-soluble gold nanoparticles. The activity of these catalysts can be reversibly controlled by binding a supramolecular cucurbit[7]uril 'gate-keeper' onto the monolayer surface, providing a biomimetic control mechanism that mimics the allosteric regulation of enzymes. The potential of this gated nanozyme for use in imaging and therapeutic applications was demonstrated through triggered cleavage of allylcarbamates for pro-fluorophore activation and propargyl groups for prodrug activation inside living cells.

Figure 1. Bioorthogonal nanozyme design and supramolecular regulation of intracellular catalysis

a, AuNP, catalyst-embedded AuNP, and CB[7]-capped catalyst-embedded AuNPs used in study. b, Endosomal uptake of nanozymes. c, Intracellular catalysis with NP_Ru converting substrate into product. d, CB[7] complexation with the ligand headgroup to provide NP_Ru_CB[7] inhibits catalyst activity. e, Nanozyme activity is restored through addition of the competitive guest 1-adamantylamine (ADA). f, Structures of the NP platform with the surface ligand bearing a dimethylbenzylammonium headgroup, CB[7] gatekeeper, and ADA, a competitive guest molecule for CB[7] binding. f, Structures of the pro-fluorescent substrate (rhodamine 110 derivative), fluorescent product (rhodamine 110) obtained after catalysis, and embedded catalyst for allylcarbamate cleavage.

Figure 2. Catalytic activity of nanozymes in solution

a, Fluorescence was generated by NP_Ru after the cleavage of profluorophore bis-Alloc-rhodamine 110, while NP_Ru_CB[7] showed no significant change. b, After adding ADA, catalytic activity of NP_Ru_CB[7] was restored and no significant effect was observed for the activity of NP_Ru. c, The reaction rates of NP_Ru and NP_Ru_CB[7] before and after adding ADA showing catalytic activity for NP_Ru_CB[7] was fully recovered after the addition of ADA. The reaction rate experiments were performed in triplicate. Error bars represent standard deviations of these measurements. d, Photo of the reaction mixtures in water with NP_Ru and NP_Ru_CB[7] under UV light.

Figure 3. Lineweaver-Burk plot showing competitive binding of CB[7] to nanozyme

Kinetic studies of NP_Ru and NP_Ru_CB[7] in sodium phosphate buffer (5 mM, pH 7.4) indicate that CB[7] inhibits catalyst activity through a competitive inhibition mechanism, with CB[7] affinity and stoichiometry consistent with ITC binding studies. Numbers calculated on a per particle basis. Kinetic experiments with each nanozyme were repeated in triplicate. Error bars represent standard deviations of these measurements.

Figure 4. Triggered allylcarbamate cleavage in living cells using gated nanozymes

a, Flow cytometry of NP_Ru, NP_Ru_CB[7], and controls (only cell and NP) revealing NP_Ru showed significant increase in fluorescence while NP_Ru_CB[7] was completely inhibited. b, Addition of ADA to NP_Ru_CB[7] treated cells recovered the catalysis and resulted in increase in fluorescence. c–f, Confocal microscopy images of HeLa cells showing the supramolecularly regulated intracellular chemical reactions. A punctate fluorescence was observed for NP_Ru and NP_Ru_CB[7] + ADA treated cells as the indication of catalysis while no fluorescence was obtained for only substrate and NP_Ru_CB[7] (scale bars = 10 μm).

Figure 5. Prodrug activation in living cells using supramolecularly controlled nanozymes

a, Structures of pro-5FU, 5FU and the palladium catalyst used for prodrug activation. b, Viability of cells treated with 5FU and pro-5FU at various concentrations, showing a nice therapeutic window was obtained between 5FU and pro-5FU. c, NP_Pd and ADA treated NP_Pd_CB[7] showed increasing intracellular toxicity as a result of more conversion of prodrug into 5FU drug at higher pro-5FU concentrations, while NP_Pd_CB[7] did not show any toxicity at any prodrug concentration used due to the blocking catalysis. Also, only prodrug, NP_Pd, NP_Pd_CB[7], and NP_Pd_CB[7] + ADA did not cause any toxicity into the system at zero prodrug concentration. Cell viability experiments were performed in triplicate. Error bars represent standard deviations of these measurements.