Catalytic Bioswitch of Platinum Nanozymes: Mechanistic Insights of Reactive Oxygen Species Scavenging in the Neurovascular Unit

Oxidative stress is known to be the cause of several neurovascular diseases, including neurodegenerative disorders, since the increase of reactive oxygen species (ROS) levels can lead to cellular damage, blood–brain barrier leaking, and inflammatory pathways. Herein, we demonstrate the therapeutic potential of 5 nm platinum nanoparticles (PtNPs) to effectively scavenge ROS in different cellular models of the neurovascular unit. We investigated the mechanism underlying the PtNP biological activities, analyzing the influence of the evolving biological environment during particle trafficking and disclosing a key role of the protein corona, which elicited an effective switch-off of the PtNP catalytic properties, promoting their selective in situ activity. Upon cellular internalization, the lysosomal environment switches on and boosts the enzyme-like activity of the PtNPs, acting as an intracellular “catalytic microreactor” exerting strong antioxidant functionalities. Significant ROS scavenging was observed in the neurovascular cellular models, with an interesting protective mechanism of the Pt-nanozymes along lysosomal–mitochondrial axes.

. PtNPs sample reproducibility. Representative TEM images (top) and statistical size distribution analysis (bottom) for A) batch 1 and B) batch 2 of PtNPs. C) DLS measurements and D) agarose-gel assay (2.5%) for batches 1 and 2 of PtNPs.  Figure S4. Effect of protein concentration on protein corona formation and stability. A) Effect of increasing amounts of BSA on PtNPs-corona formation investigated with 3.5% agarose gel assay, (20 min run at 90V). The number of lanes refer to 1) PtNPs and 2-8) PtNPs incubated with increasing BSA concentrations: 2) 1.0 x 10 -3 mg/mL, 3) 1.0 x 10 -2 mg/mL, 4) 1.0 x 10 -1 mg/ml, 5) 2.5 x 10 -1 mg/mL, 6) 5.0 x 10 -1 mg/mL, 7) 1 mg/mL and 8) 10 mg/mL. The image was acquired using Gel-Doc with UV transillumination mode.    The CAT-like activity of PtNPs under acidic condition was increased more than 50% compared to neutral condition. B) Xanthine Oxidase activity in the absence of PtNPs at pH = 5 (purple) and pH = 7.4 (green) using SOD-assay kit. The strong decrease in the WST ox absorbance shows the inability of Xanthine Oxidase to work properly at acidic pH. Superoxide ions cannot be produced in this condition and thus SOD activity cannot be monitored. C) and D) pH and temperature CAT-like activity dependence of PtNPs and PtNPs-BSA exposed to 50% FBS measured by oxygen sensor. E) CATlike activity of PtNPs in different conditions as measured by colorimetric assay and O 2 sensor. Data of O 2 production are considered after 12 min of reaction. In general, the PtNPs activity is ca. doubled at pH=5 and T=37 °C; for this reason, data on pH are normalized, using the condition at pH = 5 as 100% of activity. Data on temperature are normalized, using the condition at T = 37 °C as 100% of activity. The graph is intended to show that H 2 O 2 consumption measured by the colorimetric assay actually corresponds to oxygen production, as for the catalase reaction.

Synthesis of 5 nm spherical PtNPs
The PtNPs were prepared as previously reported. 1 All reagents were dissolved in ultrapure water for the reaction. Briefly, 160 μL of hexachloroplatinic acid H 2 PtCl 6 0.5 M (BioXtra grade, Sigma-Aldrich) and 192 μL of trisodium citrate (BioUltra grade, Sigma-Aldrich) were consecutively added to 80 mL of ultrapure water (0.5 M) under stirring at room temperature (RT). After 2 min, 5.4 mL of sodium borohydride (NaBH 4 ) 0.06 M was added drop by drop. The vessel was then heated at 75° C under stirring. After 30 min, the solution was removed from the heat and let cool down at RT. The brownblack colloidal suspension was finally washed multiple time using a 2 mM sodium citrate to remove possible traces of the platinum precursor using Amicon centrifugal filters (MW 10KDa).

PtNPs-BSA preparation
PtNPs solution was adjusted to neutral using NaOH and were incubated at 37° C for 1 hour with 10 mg/mL of Bovine Serum Albumin (Sigma-Aldrich) in ultra-pure water. After incubation, the proteincoated NPs were washed three times by ultracentrifugation (25000 rpm, 30 min, 4° C) to remove the unbound free proteins from the solution. The pellet containing PtNPs corona was re-suspended in 200 µL of mQ H 2 O and then used for experiments.

Dynamic light scattering
The hydrodynamic diameter of PtNPs was determined by dynamic light scattering (Nano ZS Malvern Instruments, UK). The measurements are reported as the average of three independent measurements, consisting of an accumulation of 11 runs.

Agarose-gel assay
Agarose-gel assay was prepared and performed as previously reported. 4 The colloidal stability of PtNPs-BSA in water, PBS, complete cell culture media and 50% FBS was assessed via 2.5% or 3.5% agarose gel assay. 0.1 μM PtNPs were incubated for 48h before run. Protein corona degradation over time was evaluated after PtNPs-BSA incubation in Artificial Lysosomal Fluid. Gels were imaged with Gel Doc TM XR+ (BioRad) equipped with a UV filter and a white light source.

SDS-PAGE assay
Characterization of PtNPs-BSA was also performed via 2D SDS-PAGE gel shift assay. 10 µL of each sample were mixed with 10 μL of 2X loading buffer composed by denaturing solution (0.25 M dithiothreitol (DTT)) and 70 mM sodium dodecyl sulfate (SDS). The mixture was incubated for 15 min at 80 °C to complete the denaturation process. Conventional 10% SDS-PAGE was performed and run for about 1 hour at 130 V. 20 µL of each sample were loaded in a 10 wells SDS-PAGE gel. Pierce™ silver staining kit was used to visualize the protein bands. Gels were imaged with Gel Doc TM XR+ (BioRad) equipped with a UV and white light sources.

Proteomics analysis
From the SDS-Page, after the de-staining process, the band of interest was cut and proteins were digested and extracted from the gel following the procedure invented by Shevechenko et al. 5 The resulting peptides were then analyzed by high-resolution LC-MS on an Orbitrap Exploris 480 mass spectrometer (Thermo Scientific) equipped with a nanoelectrospray source and coupled with a nano-LC system. The peptides were eluted with a linear gradient of acetonitrile in water (3 to 50%). Both eluents were added with 0.1% formic acid. Data were acquired in data-dependent mode, selecting multiply charged stated (2+ to 6+) as precursors for MS/MS fragmentation. The resulting raw spectra were searched against the Bos Taurus reference proteomes (downloaded from UNIPROT, only reviewed entries) using the Proteome Discoverer software. The following parameters were used for a positive protein hit: 1% maximum false discovery rate (FDR) and a minimum of two peptides for a given protein sequence.

Evaluation of the enzyme-like activities of PtNPs / PtNPs-BSA
Evaluation of peroxidase-like and oxidase-like activity. POD-like and OX-like activities of PtNPs were evaluated by using 3,3',5,5' tetramethylbenzidine (TMB, BD Biosciences) chromogenic substrate. The TMB oxidation reaction is accompanied by a measurable color change of the solution from transparent to blue. POD-like activity protocol: PtNPs / PtNPs-BSA (100 µL, 0.1 nM) were added to a reaction mixture (480 µL) containing acetate buffer (10 mM, pH = 5), TMB (100 mM), and H 2 O 2 (40 mM). OX-like activity protocol: same procedure reported for POD-like activity, but using 5 nM PtNPs (initial concentration), and in absence of H 2 O 2 . In all tests, TMB oxidation was monitored recording the absorbance at λ = 652 nm for 20 minutes by using a UV/vis spectrophotometer. Samples without NPs and H 2 O 2 were used as control.
To evaluate the effect of protein corona using different protein concentrations, PtNPs-BSA were incubated in 10% or 50% FBS at a concentration of 50 nM. These samples were finally diluted 1:500 in water and analyzed as described above. After catalytic reactions in the desired conditions, a sample dilution was employed to avoid potential matrix effect (protein interference) during the detection step.
Evaluation of CAT-like activity. The CAT-like activity of PtNPs and PtNPs-corona was determined by using the PeroxiDetect Kit (Sigma-Aldrich). The assay is based on Fe 2+ conversion into to Fe 3+ ; Fe 3+ ions form a colored adduct with xylenol orange, which can be measured at 560 nm. The absorbance value was proportional to the amount of residual H 2 O 2 in the solution. The PtNPs, consuming H 2 O 2 , decrease the absorbance intensity by using a Tecan microplate reader (Spark). PtNPs or PtNPs-BSA (10 μL, 5 nM) were incubated with H 2 O 2 (40 μL, 10 mM) and the residual H 2 O 2 was determined after 0, 1, 4, 24 and 48 hours. The samples were diluted 1:100 and analyzed following the standard kit procedure. The sample were diluted 1:100 before analysis. The pH effect on nanozymes CAT-activity, was evaluated as described above but using 20 nM PtNPs (initial concentration) and determining the residual H 2 O 2 after 1 hour. The sample were diluted 1:100 before analysis. The comparative studies (Figures S8 and S9) were carried out following these conditions and using PtNPs-BSA exposed to in vivo-like conditions (hard corona). Evaluating the effect of protein corona using different protein concentration: PtNPs-BSA were incubated in 10% or 50% FBS at a concentration of 5 nM at 37 °C for at least 1h (to simulate physiological conditions). PtNPs-BSA in PBS at the same concentration was also incubated at 37 °C as a control. The samples were diluted 1:100 and analyzed following the kit standard procedure to avoid potential matrix effect (protein interference) during the detection step. The isolated hard corona complex formed by exposing PtNPs-BSA to 50% FBS for 48h, after removal of the free protein excess.
Oxygen sensor measurements: the experiments were performed in 4 mL glass vial closed with a lead with a septum. The pressure inside the system was kept in equilibrium with the atmospheric pressure through a needle inserted in the septum. The variation in O 2 partial pressure of the gas phase inside the vial was recorded with a FireSting®-O 2 sensor from Pyroscience, equipped with a fiber-optic needle sensor inserted in the vial through the septum. The vial was filled with 1.2 mL of a dispersion of PtNPs, PtNPs-BSA, or PtNPs-BSA-50% FBS hard corona (0.74 nM. The vial was left to equilibrate at RT or 37 °C for 5 minutes prior to the injection of 300 µL of H 2 O 2 (2.5M), starting the CAT-like reaction (final PtNPs concentration 0.59 nM). The H 2 O 2 solution was equilibrated at the same working temperature separately. The reaction was kept at the target temperature for the whole time of the experiment. The ΔO 2 partial pressure was obtained by subtracting the initial O 2 partial pressure from the readings. The relative activity was calculated as a percentage of the maximum ΔO 2 partial pressure recorded at a selected time point.
Evaluation of SOD-like activity. The SOD-like activity of PtNPs was evaluated by using the SOD Assay Kit-WST (Sigma-Aldrich). Superoxide anions (generated in situ by the enzyme xanthine oxidase) reacting with the water-soluble tetrazolium salt (WST) dye, lead to formazan formation, a dye strongly absorbing at 450 nm, inducing solution color change. The PtNPs SOD activity removes superoxide anions from the solution, decreasing formazan formation and, consequently, the absorbance intensity. When comparing samples in the main text, the data are plotted as relative activity (%) normalizing to 100% the more active compound. The assay was performed according to the manufacturer's instructions, using PtNPs or PtNPs-BSA 50 nM. The SOD activity was measured after 30 min of reaction by using an Infinite 200 Pro Tecan microplate reader. The samples for experiments related to effect of protein concentration, protein corona degradation and pH, were prepared as described the previous section (Evaluation of CAT-like activity). After catalytic reactions in the desired conditions, a sample dilution 1:100 was employed to avoid pH or protein interference during the detection. The obtained absorbance values were expressed as percentage of SOD activity with respect to the absorbance value of the initial amount of superoxide anions in solution.

Cell cultures
Mouse brain endothelial cell. Mouse brain endhotelial immortalized cell line (bEnd. Primary neurons. Primary cortical neurons were obtained from wild-type C57BL/6 mice (Charles River, Calco, Italy) Briefly, mice were sacrificed by CO 2 inhalation, and 18-day embryos (E18) were removed by cesarean section. Enzymatically dissociated cortical neurons were plated on polyd-lysine-coated (0.1 mg/mL, Sigma) glass coverslips at a total density of 80.000 cells/well. Cultures were incubated at 37 °C, 5% CO 2 , 90% humidity in medium consisting of Neurobasal (Gibco/Thermo-Fischer Scientific) supplemented to reach final concentration of 5% glutamine, 5% penicillin/streptomycin, and 10% B27 supplement (Gibco/Thermo-Fischer Scientific). All experiments were carried out in accordance with the guidelines established by the European Community Council (Directive 2010/63/EU of 22 September 2010) and were approved by the Italian Ministry of Health. All efforts were made to minimize suffering and reduce the number of animals used.
Primary astrocytes. Primary astrocytes cultures were prepared from wild-type C57BL/6 mice (Charles River, Calco, Italy), following the procedure described above. Cortices were incubated in 0.25% trypsin-EDTA for 30 min at 37 °C, and subsequently, the supernatant solution was removed.
The tissue was dissociated mechanically by adding Glial Medium (DMEM, Gibco/Thermo-Fischer Scientific) containing 5% glutamine, 5% penicillin/streptomycin and 10% Fetal Bovine Serum and plated onto poly-D-lysine-coated (0.01 mg/ml, Sigma) T75 culture flask. Cultures were maintained in Glial Medium at 37 °C in a 5% CO 2 humidified atmosphere. Astrocytes were grown until passage 3 before performing the experiments.

DHE assay
bEND.3, primary cortical neurons and primary cortical astrocytes were seeded at the density of 1 x 10 4 in 96-well plate (Falcon) in a final volume of 100 μL and grown under standard cell culture conditions. Cells were treated with PtNPs-BSA at the concentration of 50 μg/mL. After 48 hours of incubation, cells were incubated with 5 μM of Antimycin A (ThermoFisher) for 24 hours and the quantification of intracellular ROS levels was performed by Dihydroethidium assay kit (Abcam). The assay is based on the DHE oxidation to fluorescent ethidium in presence of superoxide anions. DHE was used at the concentration of 5 μM and incubated for 1 hour and 30 minutes with cells. Fresh FluoroBrite was added before measuring the DHE intensity via an Infinite 200 Pro Tecan microplate reader. The excitation filter was set at 485 nm and the emission filter at 590 nm. Results were normalized with respect to the untreated cells (negative controls). For all cell types, Antimycin A 5 μM was used as positive control.

Evaluation of apoptosis, lysosome acidification and mitochondria morphology
bEND.3, primary cortical neurons and primary cortical astrocytes were seeded at the density of 1 x 10 4 in 96-well plate in a final volume of 100 μL and grown under standard cell culture conditions. Cells were treated with PtNPs-BSA at the concentration of 50 μg/mL. After 48 hours of incubation, cells were incubated with 1 mM H 2 O 2 (according to the incubation time set for each cell type) or with 5 μM Antimycin A for 24 hours, followed by 30 minutes incubation with CellEvent TM Caspase 3/7 detection reagent (ThermoFisher), Lysotracker green (ThermoFisher) or Mytotracker Deep Red (ThermoFisher). H 2 O 2 and Antimycin A were removed and fresh FluoroBrite was added before measuring the Caspase fluorescence intensity via an Infinite 200 Pro Tecan microplate reader. The excitation filter was set at 502 nm and the emission filter at 530 nm. Results were normalized with respect to the untreated cells (negative controls). For all the three cell types, 1 mM H 2 O 2 and 5 μM Antimycin A were used as positive controls. For confocal imaging the same experiment was repeated using 96 glass bottom plates (CellVis).

Cytotoxicity assay
bEND.3 (1 x 10 5 cells/well) and primary cortical neurons (8 x 10 4 cells/well) were seeded in Petri dishes (Falcon) and grown under standard cell culture conditions. Cells were treated with PtNPs-BSA at the concentration of 50 μg/mL. After 48 hours of incubation, cells were stained for 5 min at RT with propidium iodide (PI, 1 µM, Sigma-Aldrich) for cell death quantification, calceinAM (1 µM, Sigma-Aldrich) for cell viability and Hoechst 33342 (1 µM, Sigma-Aldrich) for nuclei visualization. Cell viability was quantified using a Nikon Eclipse-80i upright epifluorescence microscope (Nikon, Tokio, Japan). At least 5 fields for conditions were imaged at 10X magnification. Untreated cells were used as negative control, while 1 mM H 2 O 2 was used as positive control. Analysis was performed using the Cell Counter plugin of ImageJ software considering the number of PI-positive cells over the total number of cells.

Statistical analysis
Values were presented as mean ± SEM of independent experiments performed in triplicate. For statistical analysis GraphPad Prism statistical analysis software was used (GraphPad Prism version 8.3.0 for Windows). P-Values were calculated using unpaired Student's t-test or One-way ANOVA/Tukey's test. Differences between the treated samples and controls were considered statistically significant for p-values < 0.05, ** = p < 0.01, *** = p < 0.001 and **** = p < 0.0001.