Aptamer-Modified Au Nanoparticles: Functional Nanozyme Bioreactors for Cascaded Catalysis and Catalysts for Chemodynamic Treatment of Cancer Cells

Polyadenine-stabilized Au nanoparticles (pA-AuNPs) reveal dual nanozyme catalytic activities toward the H2O2-mediated oxidation of dopamine to aminochrome and toward the aerobic oxidation of glucose to gluconic acid and H2O2. The conjugation of a dopamine-binding aptamer (DBA) to the pA-AuNPs yields aptananozyme structures catalyzing simultaneously the H2O2-mediated oxidation of dopamine to aminochrome through the aerobic oxidation of glucose. A set of aptananozymes consisting of DBA conjugated through the 5′- or 3′-end directly or spacer bridges to pA-AuNPs were synthesized. The set of aptananozymes revealed enhanced catalytic activities toward the H2O2-catalyzed oxidation of dopamine to dopachrome, as compared to the separated pA-AuNPs and DBA constituents, and structure–function relationships within the series of aptananozymes were demonstrated. The enhanced catalytic function of the aptananozymes was attributed to the concentration of the dopamine at the catalytic interfaces by means of aptamer–dopamine complexes. The dual catalytic activities of aptananozymes were further applied to design bioreactors catalyzing the effective aerobic oxidation of dopamine in the presence of glucose. Mechanistic studies demonstrated that the aptananozymes generate reactive oxygen species. Accordingly, the AS1411 aptamer, recognizing the nucleolin receptor associated with cancer cells, was conjugated to the pA-AuNPs, yielding a nanozyme for the chemodynamic treatment of cancer cells. The AS1411 aptamer targets the aptananozyme to the cancer cells and facilitates the selective permeation of the nanozyme into the cells. Selective cytotoxicity toward MDA-MB-231 breast cancer cells (ca. 70% cell death) as compared to MCF-10A epithelial cells (ca. 2% cell death) is demonstrated.


Instruments
Absorption spectra were recorded on a UV-2450 spectrophotometer (UV, Shimadzu). Kinetic measurements were performed at 25 °C using a Biotek Synergy H1 microplate reader, equipped with a Biotek dual dispensing unit, and using Corning 3696 96-hald-well plates. Dissociation constants were evaluated using Isothermal titration calorimetry (ITC) instrument (Malvern instruments MicroCal PEAQ-ITC). Fouriertransform infrared spectroscopy (FTIR) measurements were performed using a Nicolet iS50 FTIR Spectrometer. Transmission Electron Microscopy measurement were performed using a Tecnai G2 Spirit TWIN T12 apparatus. Electron paramagnetic resonance (EPR) measurements were carried out at room temperature using a Bruker ELEXYS E500 spectrometer operating at X-band frequencies (9.5 GHz) and a Bruker ER4102ST resonator.

Materials
Gold (III) chloride trihydrate (HAuCl4·3H2O), trisodium citrate dihydrate (Na3C6H5O7·2H2O), hydrogen peroxide (H2O2; 30 %), dopamine hydrochloride, ascorbic acid, Amplex red, and glucose were purchased from Sigma Aldrich. The series of modified DBA-pA sequences used to prepare the series of DBA-pA Au NPs are included in Table S1. The strands, HPLC pure, were provided by Integrated DNA Technologies.

Preparation of DBA-conjugated poly-adenosine DNA strands-modified Au NPs
The respective DBA-conjugated poly-adenosine DNA strands (1)-(5) and (2a), in 0.1 M sodium phosphate buffer (PBS, 0.1 M of NaCl, 10 mM of Na2HPO4 and NaH2PO4, pH 7.4) were incubated in 95°C for 5 min and then cooled to room temperature at a rate of 0.2 °C/min. The DNA hairpin scaffolds were prepared following this step. 10 nM of S3 DNA hairpin scaffolds were then incubated with 0.8 mM HAuCl4 for 5 h, which allowed the polyA-loop domains to associate with the enough AuCl4 − units. Then, same amount of trisodium citrate in PB buffer containing 500 mM Na + (pH = 6.0) was added to mixture DNA hairpin scaffolds solution under shaking continuously with 35°C. After incubating for 3 h, the pA-Au NPs was collected by centrifuge at 12000 g for 20 min and washed with deionized water for three times. Finally, the nanoparticle product was purified by electrophoretic separation on 3% agarose gel, followed by cutting out to appropriate band corresponding to the single modified DBA-pA-Au NPs. The resulting pA-Au NPs (aptananozyme) were achieved after elution by buffer extraction of the DBA-pA-Au NPs from the cut band and their concentration was determined by UV/vis spectroscopy at 521 nm (ε = 2.7 × 10 8 M −1 cm −1 ).

Kinetic measurements with aptananozymes.
Kinetic measurements were performed at 25 °C using a Biotek Synergy H1 microplate reader equipped with a Biotek dual dispensing unit and using Corning 3696 96-well plates. For dopamine oxidation, the aptananozymes (5 nM) were introduced into 5 mM MES buffer, pH 5.5, 5 mM MgCl2, 100 mM NaCl, and 10 μL of a dopamine, solution at variable concentrations which were added to the respective wells. Subsequently, 10 μL of H2O2 (final concentration 5 mM) or glucose (final concentration 50 mM) was dispensed into each well, and the absorbance values of the oxidized products (amidochrome, absorbance at 480 nm, ε = 3058 M −1 cm −1 ) were measured in the different wells for a time interval of 30 min. As for the L-DOPA and D-DOPA oxidation process, the aptananozyme IV (5 nM) was dissolved in 5 mM MES buffer, pH 5.5, 5 mM MgCl2, 100 mM NaCl, and 10 μL of DOPA, consisting of variable concentrations which were added to the respective wells. Subsequently, 10 μL of H2O2 (final concentration 10 mM) or glucose (final concentration 50 mM) was dispensed into S4 each well, and the absorbance values of oxidized products (absorbance at 475 nm, ε = 3600 M −1 cm −1 ) were measured in the different wells for a time interval of 30 min.

EPR measurements.
Radical specie such as ·OH, were detected using the EPR spin trapping technique coupled with a spin trap 3,4-dihydro-2-methyl-1,1-dimethylethyl ester-2H-pyrrole-2carboxylic acid-1-oxide (BMPO). Typically, the mixtures of H2O2 (5 mM) and aptananozyme II (5 nM), and glucose (50 mM) and aptananozyme II (5 nM), were prepared in 5 mM MES buffer, pH 5.5, 5 mM MgCl2, 100 mM NaCl, including BMPO cells/mouse were injected subcutaneously to the flack of each mouse. Tumor mass was generated after 7 days in a volume that is around 80-100 mm 3 , then the injections of the treatment were done intra-tumoral (IT) (2-3 times/week) in total 7 injections, by using Control-Au NPs with no aptamer and Control-Au NPs with random Aptamer for the control group, compared to Au-NPs with AS1411 aptamer. All particles were injected in a volume of 100 µl of the amount of 50µg/mouse. NPs were prepared in a final concentration of 1 mg/ml. For each group we used 4 mice. Tumor was majored every 2-3 days before the following injection to evaluate the width and the height, then tumor volume (mm 3 ) was measured using the equation of (Width 2 × Height)/2, the tumor S7 growth rate was a ratio of a tumor volume for each reading to the starting volume.
Toxicity of the treatment was evaluated by the mice weight change (g) that was measured twice a week. All results were presented as mean ± SEM.    The different DBA modified pA-Au nanoparticles, aptananozyme I-V, reveal glucose oxidase-like activities and catalyze the aerobic oxidation of glucose form gluconic acid and H2O2, eq.1. eq.1: This is exemplified in Figure S8 with the aerobic oxidation of glucose to gluconic acid and H2O2. The reaction is probed by monitoring at time intervals the generated H2O2 through the fluorescence changes of Resorufin generated by the H2O2-stimulated oxidation of Amplex Red (λex = 572 nm, λem = 583 nm), Figure S8A. eq.2: Using a calibration curve corresponding to the fluorescence intensities of Resorufin generated in the presence of known concentrations of H2O2, Figure S8B, the timedependent contents of H2O2 generated by the aptananozyme IV catalyzed aerobic oxidation of glucose, eq 1, were evaluated, Figure S8C.   To establish the advantages of the cascaded oxidation of dopamine to aminochrome through the H2O2, primary aerobic oxidation of glucose to gluconic acid and H2O2, followed by the catalyzed H2O2 oxidation of dopamine to aminochrome, using the aptananozyme IV bioreactor, the following control experiment (a random assembly of native glucose oxidase (GOx) and peroxidase (POx) enzymes) was performed. The aptananozyme IV conjugate, 5 nM, was probed toward the aerobic oxidation of glucose,