Quadruple synergistic amplification of ferroptosis for precision gliob…

Chemicals and reagents

Luteolin (≥ 98%), dihydroartemisinin (≥ 98%), FeCl₃.6H₂O (AR, 99%), 95% ethyl alcohol (chromatographically pure), reduced glutathione (98%) and hydrogen peroxide solution (GR, 30 wt% in H₂O) were purchased from Shanghai Aladdin Biochemical Technology Co. Methylene blue was purchased from Shanghai McLean Biochemical Technology Co. Hydroxyl radical detection kit (O27, green fluorescence) was purchased from Beijing Biolabs Technology Co. Phosphate Buffer CellROX™ Deep Red was purchased from Thermo Fisher Scientific. Cell Counting Kit-8, Mitochondrial membrane potential assay kit with JC-1, GSH and GSSG Assay Kit and Lipid Peroxidation MDA Assay Kit were purchased from Beyotime Biotechnology, Invitrogen™BODIPY™ 581/591 C11, CM-H₂DCFDA, Hoechst 33,342, MitoTracker Green, LysoTracker Red, LIVE/DEAD™ Viability/Cytotoxicity Assay Kit (Green/Deep Red), CellROX™ Deep Red were purchased from Thermo Fisher Scientific, U.S.A. Cy5, and RhoNox-1 were purchased from MedChemExpress.

Synthesis and characterization of Lut nps, FL NPs and FLD NPs

Lut NPs were fabricated via the reprecipitation method. 100 µL 2 mg/mL luteolin (dissolved in 95% ethyl alcohol) and 100 µL methanol was dropwise added into the purified water under ultrasound (Optimised ultrasound times for 0.5, 1, 1.5 and 2 h). Nitrogen was blown during sonication to remove organic reagents, and the solution was stored at room temperature. FL NPs were prepared via a coordination-induced self-assembly strategy. Luteolin (10.0 mg/mL) and FeCl₃·6H₂O (20.0 mg/mL) were mixed, and optimal conditions were determined using a single-variable method, optimizing synthesis mode (vortex, ultrasound, magnetic Stirring), molar ratio (3:1, 2:1, 1:1, 1:2), and PBS volume (0, 100, 200, 400, 600, 1000 mL). The optimal synthesis conditions were determined by combining UV-Vis absorption spectroscopy to observe the absorbance values of the characteristic peaks at 500–800 nm and the particle size. Subsequently, FLD NPs were prepared with a molar ratio of luteolin, Fe³⁺ and DHA at 2:1:1. Specifically, 20 µL of DHA solution (5.0 mg/mL) was added to ultrapure water, followed by the addition of 20 µL luteolin (10.0 mg/mL) and 5 µL FeCl₃·6 H₂O (20.0 mg/mL). Following the addition of 400 µL PBS, the mixture was subjected to vortex mixing to ensure uniform dispersion.

Particle size and zeta potential were determined by dynamic light scattering (DLS, ZSU3100, MALVERN, UK). Morphology was observed by transmission electron microscopy (TEM, FEI Talos F200S, USA). Ultraviolet-visible spectra (UV-Vis) were determined using a UV spectrophotometer (UV-2450, Japan). Fourier transform infrared spectra (FT-IR) were obtained by FT-IR spectrometer (Thermo Scientific iN10, USA). The valence state of iron (Fe) was analysed by X-ray photoelectron spectroscopy (XPS, Thermo Scientific K-Alpha, USA).

GSH depletion capability and hydroxyl radical (·OH) generation assay

GSH depletion was evaluated using MB solution. FL NPs (50 µg/mL) were mixed with MB, GSH (0.5, 1, 5 mM), and H₂O₂ (4 mM), and absorption spectra were measured at 665 nm. The absorption spectra of the MB, MB + H₂O₂, the mixture solution of MB, H₂O_2, and FL NPs (50 µg/mL) in the presence of GSH at 1 mM before and after 808 nm laser irradiation (1.0 W/cm², 5 min) were measured.

Photothermal properties of nanoparticles in vitro

Temperature changes of FL NPs (50–400 µg/mL) under 1.8 W/cm² irradiation were also measured. Photothermal stability was assessed by five cycles of 10 min laser irradiation (1.8 W/cm²) followed by 10 min cooling, with temperatures recorded every 30 s.

Photoacoustic properties of nanoparticles in vitro

Ultrapure water, FeCl₃·6H₂O, Lut, Lut + FeCl₃·6H₂O, and FL NPs were loaded into an ultrasonic coupler (TM100) to eliminate air bubbles. Samples were scanned using a photoacoustic (PA) computed tomography system across wavelengths of 680–970 nm. FL NPs at concentrations of 0, 50, 100, 150, and 200 µg/mL were similarly analyzed. PA stability of FL NPs was assessed in vitro under continuous focused light exposure at the optimal wavelength.

Tumor microenvironment response

FL NPs were added to acidic aqueous solutions of pH = 7.4, pH = 5.0 and pH = 3.0, and observe the absorbance change at 500–800 nm. Then GSH (5 mM) was added separately and the absorbance changes at 500–800 nm were observed.

ROS generated by FLD NPs

FLD NPs were added into acidic aqueous solutions of pH = 7.4, pH = 5.0 and pH = 3.0, respectively, and then incubated for 30 min at 37 °C with 20 µL of 50 µM CellROX™ Deep Red. The fluorescence was detected by an enzyme marker (Ex = 640 nm, Em = 665 nm) to determine the ROS level. Subsequently, the ROS levels were examined at different time durations (0, 8, 16, 24, 32, 40, 48 h) and different concentrations of FLD NPs (0, 10, 20, 30, 40, 50 µg/mL).

Cytotoxicity assessments

Cytotoxicity was assessed by CCK8 assay. Dark toxicity of Lut NPs was evaluated by incubating U251 cells with varying concentrations for 24 h, followed by CCK8 assay. To study the synergistic effect, U251 cells were treated with different concentrations of FL NPs or FLD NPs for 24 h, irradiated with an 808 nm laser (1.0 W/cm², 5 min), respectively.

In vitro cellular uptake and localization assay

Cellular uptake: U251 and bEnd. 3 cells were inoculated in 10 mm confocal dishes (1 × 10⁴ cells per well). After 1, 2, and 4 h of incubation with FL@Cy5 NPs, tumor cells were co-stained with Hoechst 33,342 for 15 min. Cellular localization: U251 cells were inoculated in 10 mm confocal dishes. After 4 h of incubation with FL@Cy5 NPs, tumor cells were co-stained with LysoTracker Red, MitoTracker green, and Hoechst 33,342 for 15 min. The fluorescence imaging of U251 cells was imaged by CLSM.

In vitro BBB permeability assay

bEnd.3 cells were seeded on 0.4 μm transwell polycarbonate membrane inserts at 1 × 10⁵ cells/well and cultured for 4–6 days, with medium changes every other day until confluent. The upper chamber was filled with DMEM and DMEM-diluted FL@Cy5 NPs (FL@Cy5, Cy5: 2 µg/m L), respectively. The lower chamber was added 1 mL PBS. PBS from the lower chamber was collected at 0, 1, 2, 4, 6, and 8 h, and fluorescence was measured using the IVIS Lumina Series III (PerkinElmer, USA).

Luteolin organ-on-a-chip permeation test

After chip perfusion, the entrance and exit holes of the lower channel were sealed with polyurethane membrane, and the upper channel was perfused with low serum EBM-2 medium containing 50 µg/mL fluorescein thiocyanate-labeled glucan (10 kDa) at a rate of 10 µL/min for 3 h. Then the upper channel was cleaned with PBS buffer, the PU membrane was removed, and the liquid in the lower channel was collected (generally 1–2 mL). The fluorescence detection was performed using a microplate reader, and the results were compared with those of the empty chip control group. The permeability of the blood-brain barrier (P_BBB) was calculated according to the following formula:

Where, V_al is the volume of culture medium in the chip lumen, unit mL; C_al is the concentration of analyte entering the brain chip through the blood-brain barrier, in mol/mL; A is the area of the semipermeable membrane between the upper and lower layers of the upper and lower channels in cm²; C_l is the concentration of analyte in the upper channel, mol/ml; t is the time of analyte perfusion, unit s; P₀ is the drug permeability in cm/s of the empty chip covered only by extracellular matrix and without cell growth. Wherein the chip:

V_al=0.8 cm×0.05 cm×0.01 cm = 0.0004 mL.

A=(1 × 0.04 cm²)×(2 × 10⁶ pores/cm²)×(π×(0.2 × 10⁻⁴)²)cm²) = 1 × 10⁻⁴ cm².

t = 3 × 60 × 60 = 10,800 s.

C_l=30 µg/mL.

In vivo biodistribution

Fluorescence imaging was conducted in U251 tumor-bearing mice at 1, 2, 4, 8, 12, and 24 h post-injection of free Cy5, Lut@Cy5 NPs, and FL@Cy5 NPs (1 mg Lut equivalent/kg, 0.1 mg Cy5 equivalent/kg) to assess BBB permeability and tumor targeting. Major organs (brain, heart, liver, spleen, lungs, kidneys) were imaged ex vivo. FL NPs (1 mg Lut equivalent/kg) were injected intravenously, and PA imaging was performed at 1, 2, 4, and 8 h using a Vevo LAZR-X system. After 4 h of PBS, FL NPs, or FLD NPs (1 mg Lut equivalent/kg) injection, tumors were irradiated with an 808 nm laser (1.0 W/cm², 5 min), and thermal images were captured.

Intracellular iron levels, ROS generation and GSH assay

Intracellular Fe²⁺ was detected using the RhoNox-1 fluorescent probe. U251 cells were seeded in 10 mm confocal dishes and cultured for 24 h prior to nanoparticle treatment. After staining with RhoNox-1 for 20 min, cells were washed twice with PBS, resuspended in 100 µL DMEM, and imaged using CLSM.

U251 cells cultured in confocal dishes were treated with nanoparticles followed by laser irradiation (808 nm, 1.0 W/cm²). After 24 h incubation, cells were stained with 6.25 µg/mL CM-H2DCFDA for 15 min at 37 °C to detect ROS. Following PBS washes, cells were maintained in 100 µL DMEM for immediate imaging using CLSM. Intracellular ·OH levels were detected using O27 under similar conditions.

The GSH content in U251 cells was quantified using commercial GSH/GSSG assay kits (Beyotime). Briefly, cells were seeded in 6-well plates and cultured for 24 h prior to treatment. Following 24 h of experimental interventions, cells were processed according to the manufacturer’s protocol for GSH determination.

Intracellular lipid peroxidation level

U251 cells were inoculated in confocal dishes. After treatment with different nanoparticles, cells were irradiated with a laser (1.0 W/cm²) and then washed twice with PBS. The cells were then stained with Invitrogen™ BODIPY™ 581/591 C11 probe for 20 min, washed twice with PBS, and DMEM medium was added. Finally, images were acquired using CLSM.

Mitochondrial membrane potential evaluation

Mitochondrial membrane potential was evaluated using the JC-1 fluorescent probe. U251 cells cultured in confocal dishes were treated with nanoparticles, washed with PBS, and incubated with JC-1 staining solution (in DMEM) at 37 °C for 20 min. After PBS washes, cells were maintained in DMEM and immediately analyzed by CLSM to monitor JC-1 fluorescence shift.

Live/dead cell staining assay

U251 cells were seeded in 96-well plates for 24 h, followed by different treatments. FL NPs and FLD NPs groups were irradiated (808 nm, 1.0 W/cm², 5 min), washed with PBS, and stained with LIVE/DEAD Cell Activity/Cytotoxicity Kit (Green/Deep Red) (DMEM, 37 °C, 30 min). Apoptosis was analyzed via CLSM by quantifying live/dead cells.

Western blot analysis

After cell collection, proteins were extracted using RIPA lysis buffer on ice. The lysates were separated by SDS-PAGE and transferred to PVDF membranes. Following blocking at room temperature, membranes were incubated with primary antibodies overnight at 4 °C and subsequently with HRP-conjugated secondary antibodies for 1 h at room temperature. Protein bands were finally visualized using a ChemiDoc MP Imaging System.

Establishing glioblastoma animal models

All animals in this study are handled according to the protocol approved by the Institutional Animal Use and Care Committee of Shenzhen Institute for Drug Control (SZIDC-YL-20240331). BALB/c nude mice were obtained from Vital River Laboratory Animal Technology Co., Ltd. Orthotopic brain tumor models were established by implanting U251 cells transfected with the luc-cogfp virus (U251-cogfp-luc) into the brains of 6-week-old BALB/c nude mice. After modeling, in vivo fluorescence imaging was performed to monitor the luc signal via bioluminescence mode to confirm successful tumor establishment.

In vivo antitumor study

Ten days post-tumor induction, mice were randomized into six groups: PBS (G1), Lut NPs (G2), FL NPs (G3), FL NPs + Laser (G4), FLD NPs (G5), and FLD NPs + Laser (G6). Each group (n = 3) received intravenous injections of PBS, Lut NPs, FL NPs, or FLD NPs (1 mg Lut equivalent/kg). Then G4 and G6 groups were subjected to laser irradiation (808 nm, 1.0 W/cm², 5 min). Following treatment, tumor-bearing brain tissues were harvested, fixed in 4% paraformaldehyde and prepared for histological analysis.

Statistical analysis

All quantitative data are expressed as mean ± standard deviation (SD). Statistical analyses were performed using the two-tailed t-test (*p p p