Rj 0c107223

Hematotoxicity and hepatotoxicity evaluation were carried out to further monitor the in vivo biosafety of FeS₂-based NPs. Six-week-old female Balb/c mice were randomly divided into three groups and received one intravenous (i.v.) injection of 100 µL of PBS buffer, FeS₂ NPs and FeS₂@CP NPs, respectively. The dose of pyrite-based NPs was 60 mg/kg, and the mass referred to the nanoparticles based on FeS₂. The body weight curve of mice was recorded every 3 days during the 15-day monitoring with the observation of eating, drinking, and physical behavior. At the endpoint of monitoring, anticoagulant blood samples were collected in tubes with EDTA-Na₂, and clotted blood samples were obtained for serum collection. Finally, mice were sacrificed by overdose of isoflurane to collect main organs (heart, liver, spleen, kidney, lung) for organ index calculation and histomorphological observation via H&E staining. Organ index = organ weight (mg) / body weight (g). An automatic hematology analyzer (RJ-0C107223, Mindray, Shenzhen, China) was used to detect the blood routine values of anticoagulant blood samples. And a blood biochemical analyzer (BS-220, Mindray, Shenzhen, China) was used to detect the biochemical analysis of serum blood samples according to the manufacturer’s instructions.

The purity of catalase was examined by a size-exclusion column with a separation range of 10,000 Da–10,00,000 Da BioCore SEC-300) using a high-performance liquid chromatography (HPLC, 1,260 Infinity II system, Agilent) system. Transmission electron microscope (TEM, HT7700, Hitachi Ltd.) was employed to characterize the morphologies of CAT-PEG. Dynamic light scattering (DLS) measurements were performed on a Zetasizer Nano instrument (Malvern Instruments Ltd., United Kingdom) with a 10–mW helium–neon laser and thermoelectric temperature controller. Fluorescent intensity was measured with a Spectra Max M2 plate reader (Molecular Devices). The bioluminescent imaging of the mice was imaged with the IVIS Imaging System (IVIS Spectrum, PerkinElmer). H and E images were taken using inverted fluorescence microscope fluorescence microscope (BK-FL4, ChongQing Optec Instrument Co., Ltd.). TUNEL images were taken using a confocal laser scanning microscopy (Leica TCS SP8). The complete blood count and blood biochemical examination were performed using auto biochemistry analyzer (BS-420, Mindray) and auto hematology analyzer (RJ-0C107223, Mindray), respectively.