In Cellulo Bioorthogonal Catalysis by Encapsulated AuPd Nanoalloys: Overcoming Intracellular Deactivation

Abstract

Bioorthogonal metallocatalysis has opened up a xenobiotic route to perform nonenzymatic catalytic transformations in living settings. Despite their promising features, most metals are deactivated inside cells by a myriad of reactive biomolecules, including biogenic thiols, thereby limiting the catalytic functioning of these abiotic reagents. Here we report the development of cytocompatible alloyed AuPd nanoparticles with the capacity to elicit bioorthogonal depropargylations with high efficiency in biological media. We also show that the intracellular catalytic performance of these nanoalloys is significantly enhanced by protecting them following two different encapsulation methods. Encapsulation in mesoporous silica nanorods resulted in augmented catalyst reactivity, whereas the use of a biodegradable PLGA matrix increased nanoalloy delivery across the cell membrane. The functional potential of encapsulated AuPd was demonstrated by releasing the potent chemotherapy drug paclitaxel inside cancer cells. Nanoalloy encapsulation provides a novel methodology to develop nanoreactors capable of mediating new-to-life reactions in cells.

Keywords: bioorthogonal; catalysis; gold; nanoalloys; nanoencapsulation; palladium.

Figure 1

(a) Analysis of the conversion of nonfluorescent PocRho (100 μM) to fluorescent Rho after incubation with single-metal or alloyed NPs (20 μg metal/mL) in PBS and 10% FBS in PBS for 14 h. The fluorescence was measured at λ_ex/em 480/535 nm. Error bars: ±SD, n = 3. (b) A549 cell viability study after treatment with Pd, PdPt, PdRu, and AuPd NPs over a range of concentrations. Cell viability measured at day 5 using PrestoBlue reagent. Error bars: ±SD, n = 3. (c) Representative TEM (left) and HAADF-STEM (right) images of AuPd PLGA at different magnifications. (d) Elemental analysis of an AuPd NP embedded in PLGA by energy-dispersive X-ray spectroscopy (EDS). Analysis was carried out in the marked area of the HAADF-STEM image. (e) Representative TEM (left) and HAADF-STEM (right) images of AuPd SiO₂ at different magnifications. (f) Schematic display of a SiO₂ scaffold decorated with AuPd NPs and elemental mapping analysis of an AuPd NP by STEM-EDS (scanning mode).

Figure 2

Analysis of the conversion of PocRho (100 μM) into Rho after incubation with naked AuPd NPs, AuPd PLGA, and AuPd SiO₂ (20 μg metal/mL) in PBS and 10% FBS in PBS for 14 h. Fluorescence was measured at λ_ex/em 480/535 nm. Error bars: ±SD, n = 3.

Figure 3

Nanoalloy internalization studies in lung cancer A549 cells. (a) Representative TEM image of the ultrathin cross-section of a cell treated with AuPd PLGA, showing the membrane and the cytoplasm. A NP is indicated with an orange arrow. Scale bar = 1 μm. (b) Representative TEM image of the ultrathin cross-section of a cell treated with AuPd SiO₂, showing the membrane and the cytoplasm. Two laterally imaged NPs are indicated with red arrows. A transversally imaged NP is indicated with a blue arrow. Scale bar = 0.5 μm. (c) Quantification of Au and Pd content inside the cell (pmol of metal/cell). Analysis performed by ICP-OES. Error bars: ±SD, n = 3.

Figure 4

(a) AuPd-mediated conversion of Pro-PTX to PTX. (b) Intracellular prodrug activation assay in A549 lung cancer cells. Cells were treated with uncoated and encapsulated AuPd for 30 min prior to prodrug addition. Cell viability was measured at day 5. Experiments: 0.1% DMSO (untreated control); AuPd (20 μg metal/mL, −ve control); Pro-PTX (1 μM, −ve control); PTX (1 μM, +ve control); and AuPd + 1 μM Pro-PTX (activation assays). Error bars: ±SD, n = 3. Significance was determined by one-way analysis of variance (ANOVA): ***P < 0.001. (c) Immunofluorescence study of the microtubule distribution in A549 cells labeled with AuPd PLGA or AuPd SiO₂ and treated with Pro-PTX (1 μM). Cells were incubated with encapsulated AuPd for 30 min prior to prodrug addition. Negative controls: Pro-PTX (1 μM), AuPd PLGA, and AuPd SiO₂. Positive control: PTX (1 μM). 48 h after treatment, cells were fixed and stained for microtubules (green), actin filaments (red), and cell nuclei (blue). The panel shows the merged image of the three channels as maximal projections. Scale bar = 10 μm.