2.1 Preparation of USIONPs
Chemicals including acetone, ethanol, ferrous chloride, tris (acetylacetonato) iron(III), oleic, oleic acid, oil amine, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino (polyethylene glycol)-2000] (DSPE-PEG 2000) were purchased from Guoyao Chemical Incorporation (Shanghai, China).
As shown in Fig. 1, USIONPs(PION@E6) was synthesized according to our previously reported procedures [22]. Iron oxide nanoparticles (IONs) coated with oleic acid were prepared by means of high temperature pyrolysis. Briefly, 1.0 mmol FeCl2 solution, 6 mmol oleic acid and 6 mmol oil amine were added into 20 mL oleic by flushing the reaction medium with a nitrogen gas, then heated to 100–120℃ for 1 h. After that, 2.0 mmol Fe(acac)3 was added into the above mixture and heated to 180–220℃ for 30 min, then heated continuously for another 30 min to generate oleic acid coated IONs. After the reaction mixture cooled to room temperature, 75 mL anhydrous ethanol was added into the mixture to collect oleic acid coated IONs by magnetic separation. Following a washing with 35 mL acetone, the oleic acid coated IONs were collected by density-gradient centrifugation and then dissolved in 35 mL chloroform for the following preparation protocol.
To prepare USIONPs by the means of phase transfer, 50 mg DSPE-PEG 2000 and 10 mg chlorin e6(fluorescence tracer) were dissolved in 5 mL trichloromethane, and 10 mL the above collected oleic acid coated IONs was added into the mixture. After ultrasonic dispersion and addition of 5 mL deionized water, the mixture was rotarily evaporated to clear away the trichloromethane. After ultrasonic dispersion and cooling to room temperature, the supernatant aqueous phase solution containing USIONPs was collected via removing the aggregates by microfiltration and ultrafiltration.
2.2 Characterization of USIONPs
The hydrate particle sizes and core particle size of PION@E6 were measured using a Particle Analysis Device (Brookhaven, USA) and Philips Transmission Electron Microscope (TEM; EM300, Philips, Netherlands), respectively. The zeta potential of PION@E6 was measured using a Zeta Potential Device (Nanjing Fuxin Analysis, China), and the stability of PION@E6 dissolved in deionized water was determined by Colloid stability analysis. Ultraviolet and visible spectrometry analysis was carried out using UV2700 UV–VIS Spectrophotometer(Shimazdu, Kyoto, Japan).
2.3 Cell culture
Human GBM cell line U251 was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cells were cultured in Dulbecco’s Modified Eagle Medium(DMEM) supplemented with 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin in a humidified atmosphere of 5% CO2 incubator (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C. The cultivating media were refreshed every 3 d, and U251 cells in the logarithmic growth phase were used to conduct the experiments described as follows.
Rat glioma C6 cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA) and cultured in DMEM supplemented with 10% FBS at 37 °C in a humidified incubator (Thermo Fisher Scientific, Waltham, MA, USA) under 5% CO2/95% air. The cells were changed with complete media every 3 d and routinely sub-cultured when the density of cells reached 80% confluence.
2.4 Analysis of inhibition of cell proliferation
A cell-counting kit (CCK-8; Dojindo, Kumamoto, Japan) was used to measure cell proliferation according to the kit guideline. Optical densities (ODs) were measured at 450 nm using a microplate reader (Biorad, USA), and the cell inhibitory ratio was calculated according to the following formula:
$$\left[ {\left( {{1}{-}{\text{OD treated group}}} \right)/{\text{OD control group}}} \right]\, \times \,{1}00\%$$
2.5 Measurement of cell proliferation using 5-ethynyl-2′-deoxyuridine (EdU)
Fluor488 Click-iT EdU imaging detection kit (KGA331-100, Kegen, Nanjing, China) was used to measure cell proliferation. Briefly, the cells were cultured in DMEM supplemented with 10% FBS in 96-well plates for 24 h. After washing cells with phosphate buffer saline (PBS) for two to three times,100 μL EdU (50 μmol/L) was added to the culture media for 2 h and then the cells were fixed with 4% paraformaldehyde for 30 min. After addition of 100 μL 1 × Apollo® staining buffer (excitation wavelength of 495 nm and emission wavelength of 520 nm) for 30 min, the cells were washed and added to 100 μL TritonX-100(0.5%). Thereafter, the cells were counterstained with 1 × Hoechst33342 buffer (excitation wavelength of 350 nm and emission wavelength of 461 nm) and imaged using a high content cell imaging system (200 ×) (MD, USA).
2.6 Detection of lipid ROS
C11-BODIPY(Thermo, USA),as a fluorescent lipid peroxidation reporter molecule that shifts its fluorescence from red to green, was used to measure intracellular lipid ROS according to the instruction of manufacture. Briefly, after treatment with different concentrations of USIONPs for 48 h, the cells were then incubated with 100 μmol/L C11-BODIPY for 30 min at 37 °C. The samples were washed twice with PBS and then the fluorescence intensities were detected at an emission wavelength of 510 nm and an excitation wavelength of 488 nm using a Spectra Max M3 Fluorescence Microplate Reader (Molecular Devices, USA). Data were expressed as a percentage of the fluorescence intensity relative to vehicle controls.
2.7 Measurement of intracellular ROS level
2′,7′-dichlorodihydrofluorescein diacetate(DCFH-DA) staining (Sigma, USA) was used to measure intracellular ROS. Briefly, after treatment with different concentrations of USIONPs for 48 h, the cells were incubated with 20 μmol/L DCFH-DA for 30 min at 37 °C. Thereafter, the cells were washed twice with PBS and the fluorescence intensities at an emission wavelength of 535 nm and an excitation wavelength of 485 nm were detected using a Spectra Max M3 Fluorescence Microplate Reader (Molecular Devices, USA). Data were expressed as a percentage of the fluorescence intensity relative to vehicle controls.
2.8 Iron assay
Iron assay kit (MAK025, Sigma, USA) was used directly to detect both total and/or reduced iron concentrations in the samples after addition of acidic buffer. The released iron is reacted with a chromagen resulting in a colorimetric (593 nm) product, the intensity of which is proportional to the iron presented in the cells. After centrifugation at 16,000 × g for 10 min at 4 ℃, the supernatant was discharged. To measure total iron, 50 μL samples were supplemented with 5 μL iron reducer to reduce Fe3+ to Fe2+ and then adjusted to a final volume of 100 μL per well in a 96-well plate with assay buffer. Following a mixture on a horizontal shaker and incubation for 30 min at 25 ℃, 100 μL iron probe was respectively added to each well containing standard and test samples. Thereafter, the samples in the 96-well plate were mixed again and incubated for another 60 min at 25 ℃ in the dark. Finally, the absorbance was measured at 593 nm using a Spectra Max M3 Fluorescence Microplate Reader (Molecular Devices, USA).
2.9 Western blotting assay
Cells were homogenized in lysis buffer, separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to PVDF membranes (IPVH00010, EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% skimmed milk and then incubated with diluted primary antibodies including rabbit anti-FTH1(1:2000,abcam ab75973,UK), glutathione peroxidase 4(GPX4)(1:2000, ab125066, Aabcam, UK), Nrf2(1:1000, 16396–1-AP, Sanyin,Wuhan, China),Caspase-3(1:2000, ab184787, Abcam, UK),Bcl-2(1:2000, ab182858, Abcam, UK), Caspase-1(1:1000, ab207802,Aabcam, UK),NLRP3 (1:5000, ab210491, Aabcam, UK), Receptor interacting protein kinase (RIPK)3 (1:10,000, ab56164, Abcam UK), P62 (1:10,000; ab109012, Abcam, MA, USA), Beclin1(1:2000, ab207612, Abcam), LC3II (1:2000; ab192890, Abcam), LC3I (1:2000; ab192890, Abcam), and GAPDH (1:1000; ab181602, Abcam). The blots were washed three times with tris-buffered saline containing 0.1% (v/v) Tween-200 (TBST) and incubated with an appropriate goat peroxidase-conjugated secondary antibody (1:5000; KGAA35, Keygen Co., Nanjing, China) for 2 h. After washing three times with TBST, the blots were developed with the chemiluminescence method (ECL Luminata Crescendo, WBLUR0500, EMD Millipore).
2.10 Silencing of Beclin 1 and ATG5 by the interference of shRNA
The lentivirus(LV)expressing Beclin 1 shRNA (Sense: 5ˊ-CCCGTGGAATGGAATGAGATT- 3ˊ; Antisense: 5ˊ-AATCTCATTCCATTCCACGGG-3ˊ) and ATG5 shRNA (Sense:5ˊ-CCTGAACAGAATCATCCTTAA-3ˊ; Antisense: 5ˊ-TTAAGGATGATTCTGTTCAGG-3ˊ) were generated and produced by Keygen Inc.Co. Ltd (Nanjing, China). Meanwhile, the empty vector was used as a control. After confirmation of the corrected insertions of shRNA cassettes by direct DNA sequencing, the shRNA-expressing LV was transfected into U251 cells together with the LV helper plasmids to generate respective LVs. Infectious LVs were harvested 48 h post-transfection, centrifuged to remove cell debris, and then filtered through 0.45 µm cellulose acetate filters. The transfection efficiency was determined by monitoring green fluorescent protein (GFP) expression. Moreover, the ability of the LV-shRNA-Beclin 1 and LV-shRNA-ATG5 vectors to knock down Beclin1 and ATG5 was investigated using quantitative polymerase chain reaction(qPCR).
2.11 Overexpression of Beclin 1 and ATG5 by construction of lentivirus vectors
Human Beclin 1 and ATG5 genes were amplified by PCR and then cloned into the third- generation self-inactivating LV vector with cytomegalovirus promoter for driving constitutive expression of LV-Beclin 1 and LV-ATG5 vectors. The primers for Gene cloning were as follows, Beclin 1 (Sense: 5ˊ-TCCTCGAGACTAGTTaccatggaagggtctaagacgtc-3ˊ; Antisense:5ˊ-TAGTCCA TGGCGGCCgctttgttataaaattgtgagga-3ˊ), ATG5 (Sense:5ˊ-GATCTATTTCCGGTGaattcatgacagatg acaaagatgtg-3ˊ; Antisense: 5ˊ-TGGCGGCCGCTCTAGaatctgttggctgtgggatgatac-3ˊ). LVs expressing Beclin 1 and ATG5, and empty vector (as controls) were prepared by transient transfection in U251 cells. For all experiments, the cells were infected with LVs expressing wtα-syn at a multiplicity of infection of 40. After infection, the cells were cultured in a humidified, 5% CO2 atmosphere at 37 °C. All experiments were conducted in triplicate to ensure reproducibility.
2.12 Verification by Real Time-PCR (RT-PCR)
Total RNA, including miRNAs, was extracted from U251 cells using the TRIzol reagent (Invitrogen, San Diego, CA, USA) according to the manufacturer's protocol. RNAs were reverse-transcribed using a cDNA First Strand cDNA Synthesis Kit(TaKaRa RR036B, Japan). For quantification, the One Step TB Green™ PrimeScript™ RT-PCR Kit II (SYBR Green) (TaKaRa RR086B, Japan) were utilized to perform qPCR following the manufacturer's instructions with a fluorescence qPCR thermal cycler (Step one plus RT-PCR system, ABI, USA). The expression of mRNA was defined from the threshold cycle (Ct), and relative expression levels were calculated using the 2−ΔΔCt method after normalization to the expression of GAPDH. RT-PCR primers are Beclin1 (Sense: 5ˊ-AATGGTGGCTTTCCTGGACT-3ˊ; Antisense: 5ˊ-TGATGGAATAGGAG CCGCCA-3ˊ), ATG5 ( Sense: 5ˊ-TGACGTTGGTAACTGACAAAG TG-3ˊ; Antisense:5ˊ-ATGCCATTTC AGTGGTGTGC-3ˊ) and GAPDH(Sense: 5ˊ-CAAATTCCA TGGCACCGTCA-3ˊ; Antisense:5ˊ-AGCATCGCCCCACTTGATTT-3ˊ).
2.13 Statistical analysis
SPSS 13.0 (SPSS Inc., Chicago, USA) was used as the statistical analysis software. Statistical analyses were performed using two-way analysis of variance, and statistical values are presented as means ± standard error. P < 0.05 was considered significant.