FeS@BSA Nanoclusters to Enable H2S‐Amplified ROS‐Based Therapy with MRI Guidance

Abstract Therapeutic systems to induce reactive oxygen species (ROS) have received tremendous success in the research of tumor theranostics, but suffered daunting challenges in limited efficacy originating from low presence of reactants and reaction kinetics within cancer cells. Here, ferrous sulfide‐embedded bovine serum albumin (FeS@BSA) nanoclusters, in an amorphous nature, are designed and synthesized via a self‐assembly approach. In acidic conditions, the nanoclusters degrade and simultaneously release H2S gas and Fe2+ ions. The in vitro study using Huh7 cancer cells reveals that Fe2+ released from FeS@BSA nanoclusters induces the toxic hydroxyl radical (·OH) effectively via the Fenton reaction. More interestingly, H2S gas released intracellularly presents the specific suppression effect to catalase activity of cancer cells, resulting in the promoted presence of H2O2 that facilitates the Fenton reaction of Fe2+ and consequently promotes ROS induction within the cells remarkably. After intravenous administration, the nanoclusters accumulate in the tumors of mice via the enhanced permeability and retention effect and present strong magnetic resonance imaging (MRI) signals. The findings confirm this therapeutic system can enable superior anti‐tumor performance with MRI guidance and negligible side effects. This study, therefore, offers an alternative gas‐amplified ROS‐based therapeutic platform for synergetic tumor treatment.

As a comparison, Fe 2+ ions contained BSA nanoparticles (Fe 2+ @BSA) were prepared following the similar procedure of FeS@BSA nanoclusters, except replacing Na 2 S solution with equal volume of deionized water. Furthermore, crystalline FeS nanoparticles were also synthesized according to a typical approach reported previously. [1] In brief, 0.225 g thioacetamide, 1.176 g ammonium ferrous sulfate and 0.729 g ammonium citrate were added into 30 mL water under stirring. After removing oxygen from the solution by N 2 bubbling, the solution was placed in oil bath at 85 °C for 6 h. The final crystalline FeS was obtained by centrifugation, and washed with water and ethanol for several times.
Characterization: The microstructure of samples was examined using a field-emission scanning electron microscopy (FESEM, Hitachi SU-70) and transmission electron microscopy (TEM, Tecnai F20, FEI). The hydrodynamic size was determined by dynamic light scattering (DLS) using Zetasizer (Zetasizer Nano-ZS, Malvern). The freeze-dried powder sample was characterised via an X-ray diffraction (XRD) on a X'pert PRO MPD, scanning 2θ from 10° to 80°. The CD spectra were measured by CD Spectropolarimeter (Jasco-1500), and the UV-vis spectra were recorded by a TU-1810 UV-vis spectrophotometer. X-ray photoelectronspectroscopy (XPS) was performed on a AXIS Supra, Kratos.
Exploration of pH-triggered H 2 S release phenomenon: FeS@BSA nanoclusters were dispersed in PBS solutions with varied pH (7.4, 6.5 and 5.5) under gentle shaking at 37 ℃.
The concentration was set at 1 mg mL -1 . Subsequently, 2 mL FeS@BSA solution was collected at the time intervals of 0.2, 0.5, 1, 2, 3, 4, 6, 8, 24, 36, 48 and 72 h. The unreacted FeS@BSA nanoclusters were removed by ultrafiltration to avoid the possible interference for H 2 S measurement. The concentration of H 2 S was measured with a standard method as described previously. [2] Briefly, 1.5 mL of solution was mixed with zinc acetate/sodium acetate mixture (4:1 mass ratio, 1 mL). Methylene blue was then formed by the addition of N, N-dimethyl-p-phenylenediamine dihydrochloride and FeCl 3 . After incubation for 15 minutes, the absorbance at 665 nm was examined, and the concentration of H 2 S was determined using a standard curve of Na 2 S ( Figure S17, Supporting Information).
Examination of hydroxyl radicals (·OH) induction: 1,3-diphenylisobenzofuran (DPBF) was used to examine the ·OH production. [3] Briefly, DPBF in DMSO was mixed with FeS@BSA In vitro cytotoxicity assays: WRL-68 normal cells and Huh7 cancer cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1% penicillin and streptomycin in a 37 °C incubator with 5% CO 2 . Cell Counting Kit-8 (CCK-8) assay was used to determine the cell viability. Typically, cells (5000 cells/well) were seeded in 96-well plates and incubated at 37 °C for 24 h. Then the medium was replaced with fresh culture medium containing FeS@BSA nanoclusters at different concentrations. After incubation for 24 or 48 h, the medium was discarded and CCK-8 solution was added to each well.
Incubating for 1-4 hours in the incubator and mixing gently on an orbital shaker for 1 minute, the absorbance at 450 nm was measured using a microplate reader (Tecan 50, The Switzerland).
Furthermore, live/dead cell staining was also conducted to evaluate cytotoxicity. Briefly, cells were cultured in 6-well plate for 24 h, and then treated with PBS, Na 2 S, Fe 2+ @BSA and Injections were carried out once a day for 10 days, and the dosage for S 2and Fe 2+ of all the formulations was equivalent for each injection (7 mg kg -1 FeS). The tumor volume and body weight were measured every day. Tumor volume was calculated according to the following formula: width 2 × length/2. After the treatment course, all mice were sacrificed and tumors were peeled for histopathological analysis including H&E staining and Ki-67 staining.
Bio-safety assessement: For histopathological analyses of major organs, the mice were sacrificed after the treatment to collect the major organs (heart, liver, spleen, lung and kidney).               S16. H&E stained images of the major organs (heart, liver, spleen, lung and kidney) of mice collected 2 days after receiving Na 2 S and Fe 2+ @BSA treatment. No clear tissue damage or inflammatory lesion was observed in these major organs, demonstrating low side effect after Na 2 S and Fe 2+ @BSA treatments. Scale bar is 200 μm.