Chemical compounds and reagents
Luteolin (≥ 98%), dihydroartemisinin (≥ 98%), FeCl3.6H2O (AR, 99%), 95% ethyl alcohol (chromatographically pure), lowered glutathione (98%) and hydrogen peroxide answer (GR, 30 wt% in H2O) have been bought from Shanghai Aladdin Biochemical Know-how Co. Methylene blue was bought from Shanghai McLean Biochemical Know-how Co. Hydroxyl radical detection equipment (O27, inexperienced fluorescence) was bought from Beijing Biolabs Know-how Co. Phosphate Buffer CellROX™ Deep Crimson was bought from Thermo Fisher Scientific. Cell Counting Equipment-8, Mitochondrial membrane potential assay equipment with JC-1, GSH and GSSG Assay Equipment and Lipid Peroxidation MDA Assay Equipment have been bought from Beyotime Biotechnology, Invitrogen™BODIPY™ 581/591 C11, CM-H2DCFDA, Hoechst 33,342, MitoTracker Inexperienced, LysoTracker Crimson, LIVE/DEAD™ Viability/Cytotoxicity Assay Equipment (Inexperienced/Deep Crimson), CellROX™ Deep Crimson have been bought from Thermo Fisher Scientific, U.S.A. Cy5, and RhoNox-1 have been bought from MedChemExpress.
Synthesis and characterization of Lut nps, FL NPs and FLD NPs
Lut NPs have been fabricated through the reprecipitation methodology. 100 µL 2 mg/mL luteolin (dissolved in 95% ethyl alcohol) and 100 µL methanol was dropwise added into the purified water below ultrasound (Optimised ultrasound occasions for 0.5, 1, 1.5 and a pair of h). Nitrogen was blown throughout sonication to take away natural reagents, and the answer was saved at room temperature. FL NPs have been ready through a coordination-induced self-assembly technique. Luteolin (10.0 mg/mL) and FeCl3·6H2O (20.0 mg/mL) have been blended, and optimum situations have been decided utilizing a single-variable methodology, optimizing synthesis mode (vortex, ultrasound, magnetic Stirring), molar ratio (3:1, 2:1, 1:1, 1:2), and PBS quantity (0, 100, 200, 400, 600, 1000 mL). The optimum synthesis situations have been decided by combining UV-Vis absorption spectroscopy to look at the absorbance values of the attribute peaks at 500–800 nm and the particle measurement. Subsequently, FLD NPs have been ready with a molar ratio of luteolin, Fe3+ and DHA at 2:1:1. Particularly, 20 µL of DHA answer (5.0 mg/mL) was added to ultrapure water, adopted 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 combination was subjected to vortex mixing to make sure uniform dispersion.
Particle measurement and zeta potential have been decided by dynamic gentle scattering (DLS, ZSU3100, MALVERN, UK). Morphology was noticed by transmission electron microscopy (TEM, FEI Talos F200S, USA). Ultraviolet-visible spectra (UV-Vis) have been decided utilizing a UV spectrophotometer (UV-2450, Japan). Fourier remodel infrared spectra (FT-IR) have been 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 Ok-Alpha, USA).
GSH depletion functionality and hydroxyl radical (·OH) technology assay
GSH depletion was evaluated utilizing MB answer. FL NPs (50 µg/mL) have been blended with MB, GSH (0.5, 1, 5 mM), and H2O2 (4 mM), and absorption spectra have been measured at 665 nm. The absorption spectra of the MB, MB + H2O2, the combination answer of MB, H2O2, and FL NPs (50 µg/mL) within the presence of GSH at 1 mM earlier than and after 808 nm laser irradiation (1.0 W/cm2, 5 min) have been measured.
Photothermal properties of nanoparticles in vitro
Temperature adjustments of FL NPs (50–400 µg/mL) below 1.8 W/cm² irradiation have been additionally measured. Photothermal stability was assessed by 5 cycles of 10 min laser irradiation (1.8 W/cm²) adopted by 10 min cooling, with temperatures recorded each 30 s.
Photoacoustic properties of nanoparticles in vitro
Ultrapure water, FeCl3·6H2O, Lut, Lut + FeCl3·6H2O, and FL NPs have been loaded into an ultrasonic coupler (TM100) to remove air bubbles. Samples have been scanned utilizing a photoacoustic (PA) computed tomography system throughout wavelengths of 680–970 nm. FL NPs at concentrations of 0, 50, 100, 150, and 200 µg/mL have been equally analyzed. PA stability of FL NPs was assessed in vitro below steady targeted gentle publicity on the optimum wavelength.
Tumor microenvironment response
FL NPs have been added to acidic aqueous options 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 individually and the absorbance adjustments at 500–800 nm have been noticed.
ROS generated by FLD NPs
FLD NPs have been added into acidic aqueous options of pH = 7.4, pH = 5.0 and pH = 3.0, respectively, after which incubated for 30 min at 37 °C with 20 µL of fifty µM CellROX™ Deep Crimson. The fluorescence was detected by an enzyme marker (Ex = 640 nm, Em = 665 nm) to find out the ROS stage. Subsequently, the ROS ranges have been examined at completely different time durations (0, 8, 16, 24, 32, 40, 48 h) and completely different concentrations of FLD NPs (0, 10, 20, 30, 40, 50 µg/mL).
Cytotoxicity assessments
Cytotoxicity was assessed by CCK8 assay. Darkish toxicity of Lut NPs was evaluated by incubating U251 cells with various concentrations for twenty-four h, adopted by CCK8 assay. To review the synergistic impact, U251 cells have been handled with completely different concentrations of FL NPs or FLD NPs for twenty-four h, irradiated with an 808 nm laser (1.0 W/cm², 5 min), respectively.
In vitro mobile uptake and localization assay
Mobile uptake: U251 and bEnd. 3 cells have been inoculated in 10 mm confocal dishes (1 × 104 cells per effectively). After 1, 2, and 4 h of incubation with FL@Cy5 NPs, tumor cells have been co-stained with Hoechst 33,342 for 15 min. Mobile localization: U251 cells have been inoculated in 10 mm confocal dishes. After 4 h of incubation with FL@Cy5 NPs, tumor cells have been co-stained with LysoTracker Crimson, MitoTracker inexperienced, 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 have been seeded on 0.4 μm transwell polycarbonate membrane inserts at 1 × 10⁵ cells/effectively and cultured for 4–6 days, with medium adjustments each different day till confluent. The higher chamber was stuffed with DMEM and DMEM-diluted FL@Cy5 NPs (FL@Cy5, Cy5: 2 µg/m L), respectively. The decrease chamber was added 1 mL PBS. PBS from the decrease chamber was collected at 0, 1, 2, 4, 6, and eight h, and fluorescence was measured utilizing the IVIS Lumina Sequence III (PerkinElmer, USA).
Luteolin organ-on-a-chip permeation check
After chip perfusion, the doorway and exit holes of the decrease channel have been sealed with polyurethane membrane, and the higher channel was perfused with low serum EBM-2 medium containing 50 µg/mL fluorescein thiocyanate-labeled glucan (10 kDa) at a price of 10 µL/min for 3 h. Then the higher channel was cleaned with PBS buffer, the PU membrane was eliminated, and the liquid within the decrease channel was collected (usually 1–2 mL). The fluorescence detection was carried out utilizing a microplate reader, and the outcomes have been in contrast with these of the empty chip management group. The permeability of the blood-brain barrier (PBBB) was calculated in line with the next method:
$$:{P}_{app}=frac{{V}_{al}.{C}_{al}}{A.{C}_{l}.t},:textual content{w}textual content{h}textual content{e}textual content{n}:tll:frac{{V}_{al}}{A.{P}_{app}};$$
$$:frac{1}{{P}_{app}}=frac{1}{{P}_{BBB}}+frac{1}{{P}_{0}}$$
The place, Val is the amount of tradition medium within the chip lumen, unit mL; Cal is the focus of analyte getting into the mind chip by way of the blood-brain barrier, in mol/mL; A is the world of the semipermeable membrane between the higher and decrease layers of the higher and decrease channels in cm2; Cl is the focus of analyte within the higher channel, mol/ml; t is the time of analyte perfusion, unit s; P0 is the drug permeability in cm/s of the empty chip coated solely by extracellular matrix and with out cell progress. Whereby the chip:
Val=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.
Cl=30 µg/mL.
In vivo biodistribution
Fluorescence imaging was performed 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 equal/kg, 0.1 mg Cy5 equal/kg) to evaluate BBB permeability and tumor concentrating on. Main organs (mind, coronary heart, liver, spleen, lungs, kidneys) have been imaged ex vivo. FL NPs (1 mg Lut equal/kg) have been injected intravenously, and PA imaging was carried out at 1, 2, 4, and eight h utilizing a Vevo LAZR-X system. After 4 h of PBS, FL NPs, or FLD NPs (1 mg Lut equal/kg) injection, tumors have been irradiated with an 808 nm laser (1.0 W/cm², 5 min), and thermal photographs have been captured.
Intracellular iron ranges, ROS technology and GSH assay
Intracellular Fe²⁺ was detected utilizing the RhoNox-1 fluorescent probe. U251 cells have been seeded in 10 mm confocal dishes and cultured for twenty-four h previous to nanoparticle remedy. After staining with RhoNox-1 for 20 min, cells have been washed twice with PBS, resuspended in 100 µL DMEM, and imaged utilizing CLSM.
U251 cells cultured in confocal dishes have been handled with nanoparticles adopted by laser irradiation (808 nm, 1.0 W/cm²). After 24 h incubation, cells have been stained with 6.25 µg/mL CM-H2DCFDA for 15 min at 37 °C to detect ROS. Following PBS washes, cells have been maintained in 100 µL DMEM for instant imaging utilizing CLSM. Intracellular ·OH ranges have been detected utilizing O27 below comparable situations.
The GSH content material in U251 cells was quantified utilizing business GSH/GSSG assay kits (Beyotime). Briefly, cells have been seeded in 6-well plates and cultured for twenty-four h previous to remedy. Following 24 h of experimental interventions, cells have been processed in line with the producer’s protocol for GSH willpower.
Intracellular lipid peroxidation stage
U251 cells have been inoculated in confocal dishes. After remedy with completely different nanoparticles, cells have been irradiated with a laser (1.0 W/cm2) after which washed twice with PBS. The cells have been then stained with Invitrogen™ BODIPY™ 581/591 C11 probe for 20 min, washed twice with PBS, and DMEM medium was added. Lastly, photographs have been acquired utilizing CLSM.
Mitochondrial membrane potential analysis
Mitochondrial membrane potential was evaluated utilizing the JC-1 fluorescent probe. U251 cells cultured in confocal dishes have been handled with nanoparticles, washed with PBS, and incubated with JC-1 staining answer (in DMEM) at 37 °C for 20 min. After PBS washes, cells have been maintained in DMEM and instantly analyzed by CLSM to observe JC-1 fluorescence shift.
Stay/lifeless cell staining assay
U251 cells have been seeded in 96-well plates for twenty-four h, adopted by completely different therapies. FL NPs and FLD NPs teams have been irradiated (808 nm, 1.0 W/cm², 5 min), washed with PBS, and stained with LIVE/DEAD Cell Exercise/Cytotoxicity Equipment (Inexperienced/Deep Crimson) (DMEM, 37 °C, 30 min). Apoptosis was analyzed through CLSM by quantifying stay/lifeless cells.
Western blot evaluation
After cell assortment, proteins have been extracted utilizing RIPA lysis buffer on ice. The lysates have been separated by SDS-PAGE and transferred to PVDF membranes. Following blocking at room temperature, membranes have been incubated with main antibodies in a single day at 4 °C and subsequently with HRP-conjugated secondary antibodies for 1 h at room temperature. Protein bands have been lastly visualized utilizing a ChemiDoc MP Imaging System.
Establishing glioblastoma animal fashions
All animals on this research are dealt with in line with the protocol authorised by the Institutional Animal Use and Care Committee of Shenzhen Institute for Drug Management (SZIDC-YL-20240331). BALB/c nude mice have been obtained from Very important River Laboratory Animal Know-how Co., Ltd. Orthotopic mind tumor fashions have been 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 carried out to observe the luc sign through bioluminescence mode to substantiate profitable tumor institution.
In vivo antitumor research
Ten days post-tumor induction, mice have been randomized into six teams: PBS (G1), Lut NPs (G2), FL NPs (G3), FL NPs + Laser (G4), FLD NPs (G5), and FLD NPs + Laser (G6). Every group (n = 3) acquired intravenous injections of PBS, Lut NPs, FL NPs, or FLD NPs (1 mg Lut equal/kg). Then G4 and G6 teams have been subjected to laser irradiation (808 nm, 1.0 W/cm2, 5 min). Following remedy, tumor-bearing mind tissues have been harvested, fastened in 4% paraformaldehyde and ready for histological evaluation.
Statistical evaluation
All quantitative information are expressed as imply ± normal deviation (SD). Statistical analyses have been carried out utilizing the two-tailed t-test (*p < 0.05, **p < 0.01, ***p < 0.001).
