Auger Electrons Constructed Active Sites on Nanocatalysts for Catalytic Internal Radiotherapy

Abstract Excess electrons play important roles for the construction of superficial active sites on nanocatalysts. However, providing excess electrons to nanocatalysts in vivo is still a challenge, which limits the applications of nanocatalysts in biomedicine. Herein, auger electrons (AEs) emitted from radionuclide 125 (125I) are used in situ to construct active sites in a nanocatalyst (TiO2) and the application of this method is further extended to cancer catalytic internal radiotherapy (CIRT). The obtained 125I‐TiO2 nanoparticles first construct superficial Ti3+ active sites via the reaction between Ti4+ and AEs. Then Ti3+ stretches and weakens the O—H bond of the absorbed H2O, thus enhancing the radiolysis of H2O molecules and generating hydroxyl radicals (•OH). All in vitro and in vivo results demonstrate a good CIRT performance. These findings will broaden the application of radionuclides and introduce new perspectives to nanomedicine.


Synthesis of TiO 2 -tyr.
The procedure was based on a typical solvothermal method. Firstly, Oleic acid-coated TiO 2 NPs (TiO 2 -OA) were prepared. Tetrabutyl titanate (5 mmol, 1.7 g), oleic acid (25 mmol, 7.06 g), oleylamine (25 mmol, 6.69 g) and ethyl alcohol (3.20 g) were mixed homogeneously and stirred for 10 min. Ethyl alcohol (15.15 g) and DI water (0.8 g) were then added for another 5 min stirring. The mixture was then transferred to a hydrothermal synthesis reactor, and maintained at 180 ℃ for 18 h. After washed 3 times with ethanol and cyclohexane, the product was dispersed in cyclohexane (20 mL).
Secondly, to produce citric acid-coated TiO 2 NPs (TiO 2 -COOH), diethylene glycol (30 mL) and sodium citrate (4 mmol) were mixed with the above solution (5 mL), heated to 160 ℃ and stirred for 3 h under argon (Ar) gas protection. After the reaction system was cooled down, the superfluous diethylene glycol was removed by centrifugation at 19,000 revolutions per minute (rpm) for 30 min. The obtained sediment was redissolved in DI waster for a second centrifugation at 19,000 rpm for 30 min, and the collection was redispersed in ethyl alcohol for a third centrifugation at 19,000 rpm for 10 min. The mixture was clear and transparent after redispersed in 10 ml DI water.
Finally, for tyramine modification of TiO 2 NPs (TiO 2 -tyr), the above solution (10 mL) was added with EDC·HCl (0.1 mmol) and NHS (0.3 mmol) and fully stirred for 30 min, and then reacted with tyramine (1 mmol) in the dark overnight. The final product of TiO 2 -tyr was rinsed for several times and re-suspended in DI water for further use.
Characterization of TiO 2 NPs. Transmission electron microscopy (TEM) morphology of the TiO 2 NPs was confirmed by a FEI Tecnai G2 F30. X-ray diffraction (XRD) patterns were performed on a Rigaku D/MAX-2250V diffractometer at Cu Kα (λ = 0.154056 nm) with the scanning speed of 20° min -1 and range from 20° to 80°. Characteristic UV-Vis absorption spectra of TiO 2 NPs after the surface modification of oleic acid (TiO 2 -OA), citric acid (TiO 2 -COOH) and tyramine (TiO 2 -tyr) were determined by a UV-Vis spectrophotometer (UV-3600, Shimadzu, Japan). Fourier transform infrared (FT-IR) spectra was obtained from a Bruker TENSOR II FTIR Spectrometer (Bruker Corporation) using KBr pellets. Zeta potential and the hydrodynamic size of TiO 2 NPs with different modifications were measured by Nanotrac Wave II Q Nanoparticle Size Analyzer (Microtrac Inc., USA). X-ray photoelectron spectra (XPS) were recorded using a VG ESCALAB 250Xi instrument. 125 I labeling rate and stability tests. 125 I was labeled via a standard Iodogen-catalyzed method: [1] Free 125 I (Na 125 I solution in PBS) was quickly added into two Eppendorf (EP) tubes with each coated with 50 µg Iodogen, and slowly oscillated for 15 min. Next, TiO 2 -tyr (144 ug, 7.3 mg mL -1 ) was added into the tubes followed with another 30 min reaction at room temperature. The tubes were manually shook every 15 min for sufficient reaction.
The labeling rate and stability were monitored by radio thin layer chromatography (radio-TLC, Mini-scan, B-MS-1000F, Eckert & Ziegler radiopharma, Inc. MA, USA) with a γ-detector, and Whatman chromatography paper and 0.9% NaCl solution were applied as the stationary and mobile phase, respectively. The in vitro stability of the labeling production 125 I-TiO 2 was tested by incubating with PBS solution containing 0.1% (v %) FBS at 37 °C for 24 h.
Calculated simulations. The first-principles calculations were carried out based on density functional theory. The generalized gradient approximation (GGA) of Perdew-Burke-Ernzerhof (PBE) was used to describe the exchange and correlation terms, and the Projector-Augmented Wave (PAW) pseudopotential method was also adopted in our system. The TiO 2 surfaces with the Ti 3+ and Ti 4+ structures have been established. In addition, the cut-off energy is set as 400 eV, and the Brillouin zone is sampled by a 3 × 3 × 1 k-grid. Finally, the optimization of the surface structure with adsorbed H 2 O was continued until forces on each atom were smaller than 0.03 eV/Å. The cell toxicity was evaluated through CCK-8 assay. Firstly, SW1990 cells were seeded on four 96-well plates (2 × 10 4 cells per well), each formed a matrix with 9 columns (concentration gradient of NPs) multiplying 3 lines (three replication wells). The cells were adhesive 24 h later, and the culture medium was changed to TiO 2 solution in DMEM, with the concentration of TiO 2 ranging from 0 μg mL -1 and 7.8 μg mL -1 doubled to 1000 μg mL -1 .

In vitro cytotoxicity of TiO
After 24 h and 48 h co-incubation, respectively, CCK-8 solution (15 μL) was added to each well and stained for 3 h. Finally, the iMark™ microplate reader (Bio-Rad, Hercules, CA, USA) was applied to test the absorbance value at 450 nm, and the cell viability was obtained by calculating the absorbance percentage of each group relative to that of the control group. Cellular uptake of TiO 2 NPs. The intracellular uptake and distribution of TiO 2 NPs were observed by bio-TEM scanning. In detail, adherent cells that were pre-seeded in six-well plate (about 10 5 cells per well) were treated with TiO 2 (2,400 μg) in DMEM solution for 30 min, 1.5 h, 3 h or 5 h, respectively. Next, the cells were trypsinized, centrifugated and collected, followed with fixed by electron microscopy fixative (2.5% glutaraldehyde) at 4 ℃ for 3 h.
Then, the cells were dehydrated with ethyl alcohol and acetone, osmosed with acetone and 812 embedding medium, embedded at 60 ℃ for 48 h and sliced into 60 nm-sick slices. After stained by uranium salts and lead salts for 15 min and dried overnight at room temperature, the slices were finally imaged under a bio-TEM system (Hitachi, HT7700, 80.0 kv).
Cellular experiments. For in vitro therapeutic experiments except for clone formation assay, basic treatments of cells prior to the final assay tests were consistent: In detail, SW1990 cells (three groups, three duplications for each group) were seeded onto six-well culture plates, except that the cells for •OH test by fluorescence assay were grown on the preset cell culture slides placed in the wells. After adherence, the medium was substituted by DMEM, or DMEM containing Na 125 I or 125 I-TiO 2 (600 μCi mL -1 of 125 I corresponding to 144 μg mL -1 of TiO 2 ). The treatment lasted for 24 h, then cells were trypsinized, centrifugated, collected and resuspended in PBS for western blotting assay, or fixed by 4% paraformaldehyde for immunofluorescence tests. Specially, for cellular •OH detection by fluorescence assay, the cells adhered to slices were rinsed by PBS for direct test under the microscope. The subsequently detailed testing process of above collected samples was depicted in the following.

Cell apoptosis analysis by TUNEL. The fixed SW1990 cells (about 2 × 10 6 cells per sample)
were collected and smeared, permeabilized with Triton X-100 (0.5%, 75 μL) for 20 min, incubated with the TUNEL kit (TdT : dUTP = 1 : 9) for 2 h, counterstained with DAPI for 10 min, and finally mounted by antifade mounting medium. All of the slices were imaged by fluorescence microscope (Nikon Eclipse CI, Japan), with exciting light for DAPI and FITC at 355 nm and 480 nm, respectively. The positive rates were quantified by an Image-pro plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) software. Finally, membranes were exposed to electrochemiluminescence (ECL) reagent kit (Servicebio) before developing and fixing process. Bands images were acquired via an Epson scanner (Perfection V 300, China). γ-H2AX foci of cells (n = 10) and evaluating tumor growth of mice (n = 5). All data were tested for their normality and homogeneity of variance before determining the specific statistical method. Statistical differences between two groups were analyzed by unpaired Student's t test for data simultaneously conform to normal distribution and homogeneity of variance, or a Mann-Whitney U test would be performed. Statistical differences among three groups was tested by one-way ANOVA followed with LSD-t post-hoc test for normally distributed sets with equal variance, or Kruskal-Wallis 1-way ANOVA test for those with non-normal distribution. With a testing level of α = 0.05, the two-sided P value less than 0.05 was considered as statistically significant. In a detailed presenting form, the symbol P was labeled with different number of asterisks (*) according to its actual value (*P < 0.05; **P < 0.01; ***P < 0.001). All the statistical processes were performed via SPSS 21.0 software (IBM Corp., Armonk, NY, USA). Figure S1. UV-Vis spectra of TiO 2 -OA, TiO 2 -COOH and TiO 2 -tyr.        Figure S10. Time-dependent body weight change of mice within 20 days after different treatments (n = 5, mean ± s.d). The relative body weight (W W 0 -1 ) was given when the weight (W) measured at the given time was divided by the initial weight (W 0 ) at 0 d P.I..