Nanocomposite-based dual enzyme system for broad-spectrum scavenging of reactive oxygen species

A broad-spectrum reactive oxygen species (ROS)-scavenging hybrid material (CASCADE) was developed by sequential adsorption of heparin (HEP) and poly(L-lysine) (PLL) polyelectrolytes together with superoxide dismutase (SOD) and horseradish peroxidase (HRP) antioxidant enzymes on layered double hydroxide (LDH) nanoclay support. The synthetic conditions were optimized so that CASCADE possessed remarkable structural (no enzyme leakage) and colloidal (excellent resistance against salt-induced aggregation) stability. The obtained composite was active in decomposition of both superoxide radical anions and hydrogen peroxide in biochemical assays revealing that the strong electrostatic interaction with the functionalized support led to high enzyme loadings, nevertheless, it did not interfere with the native enzyme conformation. In vitro tests demonstrated that ROS generated in human cervical adenocarcinoma cells were successfully consumed by the hybrid material. The cellular uptake was not accompanied with any toxicity effects, which makes the developed CASCADE a promising candidate for treatment of oxidative stress-related diseases.

For the cellular experiments, all the materials were of cell biological purity. Dulbecco's PBS was acquired from Lonza, RIPA lysis buffer from Merck, cOmplete ULTRA tablets from Roche, reactive oxygen species (ROS) sensitive dye 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) from Thermo Fisher Scientific. Blue-green-red apoptosis/necrosis full assay kit (ab176749) was purchased from Abcam.
The HEPES buffer used for cell experiments contained physiological components, i.e., NaCl (140 mM), KCl (5 mM), CaCl2 (5 mM), MgCl2 (1 mM) and glucose (10 mM). The HEPES S3 content was 10 mM and the pH was adjusted to 7.5 with NaOH, if necessary. All the experiments were performed at room temperature and the samples were prepared using ultrapure water (Millipore).
Preparation of the carrier layered double hydroxide. The layered double hydroxide (LDH) material was synthesized by the flash co-precipitation method 1 , as detailed elsewhere 2 . Briefly, Mg(NO3)2×6H2O and Al(NO3)3×9H2O were dissolved in 100 mL ultrapure water with a stoichiometric ratio of 2:1. A second solution containing 20 mL of 1 M NaOH and Na2CO3 in a 1:1 stoichiometric ratio to Mg(NO3)2×6H2O was prepared. These two solutions were rapidly mixed together and the mixture was stirred for 24 h. The pH of the obtained slurry was monitored and kept at 9. The solid product was filtered on 0.2 μm pore size nylon filters (Millex) and washed thoroughly with ultrapure water. The solid material was dried overnight in an oven at 60 °C prior to dispersing in water to get 4 wt% dispersions. These samples were transferred to an autoclave with PTFE inlet (Col-Int Tech) for hydrothermal treatment for 24 h at 120 °C.
The final product was washed several times with ultrapure water and re-dispersed as 1 wt% dispersion that served as stock.
Preparation of CASCADE. 1000 mg/L stock solutions of HPR, SOD, PLL and HEP were prepared, while for LDH, a stock aqueous dispersion with a concentration of 10000 mg/L was used. In the sequential adsorption method, 900 µL of ultrapure water were added first to 100 µL of bare LDH stock and the sample was vortexed for 10 s and left standing for 10 min prior to the addition of 50 µL HEP. The same procedure was repeated with calculated volumes of HRP, PLL, SOD and again HEP stock solutions to achieve the desired composition in CASCADE (HEP1: 50 mg/g, HRP: 10 mg/g, PLL: 200 mg/g, SOD: 10 mg/g and HEP2: 100 mg/g). The sample preparation was finalized by adding ultrapure water to reach the final volume of 1350 µL.

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Determination of the stability ratio. Time-resolved dynamic light scattering (DLS) measurements were performed to determine stability ratio ( ) values. For this, the evolution of the hydrodynamic radius ( ) was followed in time ( ) under different experimental conditions (e.g., ionic strength and polyelectrolyte dose) and was calculated as follows 3 : where (0) is the hydrodynamic radius of the primer particle, is the apparent By plotting the inhibition values as a function of the enzyme concentration, the enzyme content that causes 50% of the maximum inhibition was determined. This is the so-called value, which is used to compare the activity of different enzymatic systems. Note that the activity of enzymes from different sources or batches may alter, therefore tuning the final concentrations may be necessary.

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HRP assay. The guaiacol assay was employed to determine the HRP activity of the biocatalytic materials and to calculate the characteristic kinetic data 6