Materials
Anti 3-nitrotyrosine (3-NT), anti-GPX4, anti-integrin α4, anti-γ-H2AX, and DAR-1 were provided by Abcam (Cambridge, UK). The organic solvents used in synthesis procedure were obtained from Adamas (Shanghai, China). 5,5’-Dithiobis-2-nitrobenzoic acid (DTNB), N-Boc-ethylenediamine, and N, N-diisopropylethylamine (DIPEA) were came from Aladdin Biochemical Technology Co., Ltd (Shanghai, China) and the other chemicals for synthesis were brought from Bidepharm (Shanghai, China). Ferrostain-1 (Fer-1), Necrostatin-1 (Nec-1), and Z-VAD-Fmk were provided by Bidepharm (Shanghai, China). Cell counting kit-8 (CCK-8) assay kit, Bicinchoninic acid (BCA) assay kit, D-luciferin potassium salt, 1’-dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine,4-chlorobenzenesulfonate salt (DID), 2-(4-amidinophenyl)-6-indolecarbamidine (DAPI), DAF-FM, and Evans Blue were obtained from Dalian Meilun Biotechnology Co., Ltd (Dalian, China). Cell lysis buffer, phenylmethanesulfonylfluoride (PMSF), GSH, membrane and cytosol protein extraction kit, chemokine C-C motif receptor 2 (CCR2) antibody, 2,7-dichlorofluorescin diacetate (DCFH-DA), anti-β-actin, and Triton X-100 (10%) were bought from Beyotime Biotechnology Co., Ltd (Shanghai, China). GelNest™ was from NEST Biotechnology (Wuxi, China). Egg phosphatidylcholine and cholesterol (for injection) were obtained from AVT (shanghai) Pharmaceutical Tech Co., Ltd. Glutaraldehyde stationary liquid (2.5%) and 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylimidacarbocyanine (JC-1) assay kit were bought from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). GSH assay kit (abs580006-96T) was purchased from Absin (Shanghai, China). Malonaldehyde (MDA) assay kit came from Jiancheng Bioengineering Institute (Nanjing, China). O58 probe was supplied by BestBio (Nanjing, China). C11-BODIPY581/591 was obtained from Thermo Fisher Scientific Inc (MA, USA). FITC Rabbit anti-goat IgG (H + L) antibody (K1213) was from APExBIO (Houston, USA).
Cells and animals
Macrophage cell line (RAW 264.7), Lewis lung carcinoma line (LLC), and lung epithelial cell line (MLE-12) were provided by Shanghai Cell Bank, Chinese Academy of Sciences (Shanghai, China). RAW 264.7 cells, LLC cells, and LLC cells stably expressing firefly luciferase (LLC-Luc) were incubated at 37℃ in high glucose Dulbecco’s medium (DMEM) medium (HyClone) containing 10% fetal bovine serum (FBS, Biochannel, BC-SE-FBS01), 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco). MLE-12 cells were maintained at 37℃ in DMEM/F12 supplemented with 5 µg/mL insulin, 10 µg/mL transferrin, 30 nM sodium selenite, 10 nM hydrocortisone, 10 nM β-estradiol, 2 mM L-glutamine, and 2% FBS.
Male C57B/6 (4 ~ 6 weeks) mice were purchased from the Laboratory Animal Center of Fudan University (Shanghai, China) and raised in specific pathogen-free conditions. All the mice were acclimated to environment for 7 days prior to experiments. All the animal experimental procedures were in accordance with the guidelines for the Care and Use of Laboratory Animals of Fudan University and approved by the Institutional Animal Care and Use Committee of the School of Pharmacy, Fudan University.
Isolation of RAW 264.7 cell membrane
RAW 264.7 cells in logarithmic growth phase were washed three times with pre-chilled PBS, harvested, and centrifuged to collect cell precipitation. According to the protocol of the membrane and cytosol protein extraction kit, cell precipitation was resuspended in reagent A containing 1 mM PMSF, placed in ice-water bath for 15 min, and homogenized 50 times by a pre-cold handheld glass homogenizer. The homogenate was then centrifuged (4 °C, 1000 g) for 10 min to remove cell nuclei and unbroken cells and the supernatant was then centrifuged (4 °C, 14000 g) for 0.5 h to pellet cell membrane fragments. After resuspending in PBS, the suspension was extruded through 800 nm and 200 nm porous polycarbonate filters for 11 times to prepare RAW 264.7 cell-membrane nanovesicles (RCM). The protein content of RCM was determined using a BCA assay kit.
Preparation of DHA-N@M
To investigate the optimal ratio, RCM and DHA-SNO were mixed in PBS at different weight ratios and incubated for 0.5 h in a 37 °C water bath to form DHA-SNO-inserted RCM nanoreactors (DHA-N@M), followed by wash with PBS to remove free DHA-SNO. After extracting DHA-SNO with methanol from DHA-N@M, the encapsulation efficiency and drug loading efficiency were determined by high performance liquid chromatography (HPLC, Agilent 1260 infinity, USA) method: column, Eclipse Plus C18, 4.6 × 250 mm, 5 Micro; mobile phase, acetonitrile, tetrahydrofuran, and 0.4% acetic acid solution (77:3:20); flow rate, 1.0 mL/min; column temperature, 30 °C; sample volume, 18 µL; detection wavelength, 210 nm.
Characterization of DHA-N@M
The structure of RBC and DHA-N@M was observed using a Cryo-transmission electron microscopy (Cryo-TEM, Tecnai G2 F20, Fei, USA). The particle size, polydispersity index (PDI), and Zeta potential were measured by the dynamic light scattering (DLS) performed on the Zetasizer Nano ZS device. Fourier transform infrared spectroscopy (FT-IR, Nicolet™ iS5, Thermo Fisher Scientific Inc, USA) was employed to determine the functional groups of nanoreactors.
To test stability in typical storage condition, DHA-N@M was dispersed in PBS and stored at 4 °C. Daily measurements were carried out to monitor changes in particle size, PDI, and drug loading content after centrifugation to remove leak DHA-SNO. Additionally, the particle size of DHA-N@M incubated in PBS (pH 7.4) and PBS (pH 7.4) with 10% FBS after 24 h were determined to assess the stability in physiological fluid conditions.
The protein profiles in RAW 264.7, RCM, DHA-SNO, and DHA-N@M were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The typical markers of integrin α4 and CCR2 were identified by western blot analysis.
Next, we evaluated the GSH-responsiveness of DHA-N@M. DHA-N@M was respectively suspended in PBS (pH 7.4) and PBS (pH 7.4) + 10 mM GSH containing a NO fluorescent probe of DAR-1 (5 µM) with or without X-ray (6 Gy) and kept in a 37 °C shaker. The fluorescence values of the solution (Ex = 560 nm, Em = 595 nm) were measured at predetermined time points using a microplate reader (Synergy 2, BioTek Instruments Inc, USA). Furthermore, the morphology and the particle size of DHA-N@M were monitored after incubation in PBS (pH 7.4) and PBS (pH 7.4) + 10 mM GSH with or without X-ray (6 Gy).
The GSH consumption ability of DHA-N@M was determined using DTNB method. DHA-N@M was redispersed in PBS (pH 7.4) containing 10 mM GSH mimicking tumor intracellular high-reduction environment. After vibrating with shaker at 37 °C for 12 h, the supernatant was collected via centrifugation, reacted with DTNB, and the optical density (OD) at 405 nm was measured using a microplate reader.
Cellular uptake
The preparation of rhodamine-B-fluorescent-group-modified-DHA@M (D-RhB@M) was similar as that of DHA-N@M. For the stability test, D-RhB@M and DHA-N@M were incubated in cell culture media with or without 10%FBS, and cargo leakage was evaluated using spectrofluorometer or HPLC.
RAW 264.7 cells, LLC cells and MLE-12 cells were counted by automatic cell counter (Countstar BioTech, Shanghai, China) and seeded in confocal dishes with incubation overnight. Then the cells were incubated with free D-RhB and D-RhB@M with equal concentration of D-RhB for 4 h, rinsed three times with cold PBS to remove free drug, and visualized under confocal laser scanning microscopy (CLSM, LSM 710, Zeiss, Germany). Meanwhile, the treated cells were collected, made into single-cell suspension, and then subjected to flow cytometry (FCM) analysis (CytoFlex S, Beckman, USA) to quantify cellular uptake.
Three different DID-labeled nanovesicles were prepared as follow. (1) DID-labeled RCM (DID@M) was prepared as previously depicted and the stability test of DID@M was similar as that of D-RhB@M. (2) To confirm the role of integrin α4 of macrophage membrane in mediating cellular uptake, DID@M was mixed with anti-integrin α4 antibody, which was designated as the blocked DID@M group. (3) To further explore the superiority of RCM-based nanovesicles, liposome, a classical dosage form, was served as a formulation control. Egg phosphatidylcholine and cholesterol were dissolved at molar ratio of 60%/40% in 1 mL chloroform, and DID at 1% molar ratio was added. The mixture was evaporated to form a uniform blue film at the bottom of the round-bottom flask, which was then hydrated in 1 mL PBS, sonicated and passed repeatedly through a 200 nm porous polycarbonate filter to obtain the DID-labeled liposome (DID@Lipo) with equivalent particle size to DID@M. Following an overnight culture, the cells were respectively incubated DID@M, DID@Lipo, and blocked DID@M carrying equal fluorescence dye for 4 h. Nuclei were stained with DAPI and observed by CLSM.
In vitro cytotoxicity study
The viability of LLC cells was investigated by CCK-8 assay. In brief, LLC cells were seeded in 96-well plates at 10,000 cells/well and cultured overnight. Then, DHA, DHA@M, SNO-grafted behenic acid (BA-SNO), BA-N@M, DHA-SNO, and DHA-N@M with different concentrations were respectively added to the culture media for 12 h, while the cells in radiation groups were exposed to 6 Gy X-ray (RS 2000, Rad Source Technologies, USA). After culture for another 12 h, 10% CCK-8 solution was added to each well for 1 h incubation and the OD value at 450 nm was recorded by a microplate reader.
MLE-12 cells were used to assess the possible cytotoxicity of DHA-N@M on normal lung cells. Following overnight incubation to adherent, cells were treated with DHA-N@M at concentrations ranging from 1 µM to 100 µM for 24 h, and the cell viability was determined by CCK-8 assay.
To investigate the cell death pathway, various cell death pathway inhibitors were used to rescue cell death mediated by DHA-N@M + X-ray. In brief, LLC cells were seeded in 96-well plates and grown to 70 ~ 80% confluency. Then cells were exposed to X-ray (6 Gy) at 12 h of incubation with 50 µM DHA-N@M in the absence or presence of 25 µM Nec-1, 50 µM Z-VAD-Fmk, or 1 µM Fer-1. Another 12 h after cell culture, cell viability was determined using CCK-8 assay.
NO, ROS, and ONOO– production in LLC cells
To detect the intracellular NO, ROS, and ONOO–, LLC cells were seeded in glass-bottom 24-well plates (NEST Biotechnology Co., Ltd, Wuxi, China) for CLSM imaging or 12-well plates for FCM analysis and cultured overnight prior to studies. DHA-SNO, DHA@M, BA-N@M, and DHA-N@M at 50 µM suspended in DMEM were added and incubation for 12 h. Afterwards, cells were loaded with probes at working concentration (NO probe: 3-amino-4-aminomethyl-2’,7’-difluorescein diacetate, DAF-FM DA, at 5 µM; ROS probe: DCFH-DA at 10 µM; ONOO– probe: O58 at 1:1000) for 30 min, with a three-time wash using PBS to remove excess or non-specific probes and a replacement with fresh DMEM. Subsequently, cells in radiation groups were irradiated (6 Gy) and their nucleus were identified by Hoechst 33342 staining (10 µg/mL) for CLSM imaging.
Meanwhile, the cells following the same treatment were harvested, processed into single-cell suspensions, and subjected to FCM analysis to quantify the level of intracellular NO, ROS, and ONOO–.
Detection of GSH and GPX4
LLC cells were at a density of 3 × 105 cells per well in 6-well plates. After incubation overnight and attachment, DHA-SNO, DHA@M, BA-N@M, and DHA-N@M at 50 µM were added and co-incubation with cells for 12 h, following with 6 Gy X-ray irradiation. Then cells were harvested 12 h post-culture, washed with PBS and lysed in lysis buffer containing 1 mM PMSF. GSH content in cell lysates were performed by the reduced GSH assay kit according to the instructions, which were normalized to total protein content using a BCA assay.
Additionally, after denaturation by boiling in loading buffer, the total protein in cell lysate was separated by 15% SDS-PAGE, transferred from gel to nitrocellulose membrane (Millipore, USA) at 250 mA for 2 h, and blocked with by 5% non-fat milk for 1 h. Subsequently, specific primary antibodies (rabbit anti-GPX4, dilution 1:1000; mouse anti-β-actin, dilution 1:1000) were added for overnight incubation at 4 °C and the antibody-bound proteins were developed with the enhanced chemiluminescence reagent coupled with horseradish peroxidase-conjugated secondary antibody.
Measurement of intracellular LPO and MDA formation
Next, the levels of LPO and MDA, as the characteristic indicators of ferroptosis, were estimated to reflect the effect of DHA-N@M + X-ray on ferroptosis. For LPO staining, LLC cells grown in confocal culture plates were incubate with different formulations including DHA-SNO, DHA@M, BA-N@M, and DHA-N@M for 12 h with or without X-ray exposure. After continuous culture for 12 h, 10 µM C11-BODIPY581/591 staining was carried out for 0.5 h at 37 °C, protected from light. After Hoechst 33342 staining and wash steps, the intracellular oxidation of probe by LPO was visualized under CLSM.
Lysates from LLC cells treated with different formulations were prepared as described above and used for measuring MDA content in accordance with the kit instructions.
Mitochondrial membrane potential (ΔΨm) test
The change of ΔΨm is indicated by the green/red fluorescence intensity ratio of JC-1. LLC cells in logarithmic growth phase were seeded at a density of 1.2 × 105 cells/well in 24-well glass bottom confocal plates. After overnight incubation, various formulations were added: control medium, DHA-SNO, DHA@M, BA-N@M, and DHA-N@M. Cells in the radiation therapy group were incubated with the formulations for 12 h before receiving a single dose of X-ray irradiation. Following standard culture for an additional 12 h, JC-1 probe was added at the working concentration according to the kit instructions. After incubation at 37 °C in the dark for 0.5 h, cells were washed twice with pre-chilled staining buffer and then with Hank’s buffer. Intracellular fluorescence was immediately observed using CLSM.
Observation of mitochondria morphology
Bio-TEM was applied to observe the morphology of mitochondria. After overnight cultivation in 10-cm dish, LLC cells were incubated with DMEM containing DHA-N@M at 50 µM for 12 h before being subjected to 6 Gy X-ray irradiation. In contrast, the untreated and unirradiated LLC cells were performed as control. Following an additional 12 h incubation, cells were collected using a cell scraper and centrifuged at 1500 rpm for 10 min to harvest cell pellet, which was then fixed with pre-cold 2.5% glutaraldehyde stationary liquid. After overnight fixation at 4 °C, the cell samples were processed as the standard sample preparation and bio-TEM observation.
DNA damage evaluation
Immunofluorescence assay for γ-H2AX was performed to assess the DNA damage. LLC cells were incubated with different formulations for 12 h without or with X-ray irradiation. One hour after X-ray treatment, cells were fixed with 4% paraformaldehyde for 15 min at room temperature. After being rinsed with PBS, buffer supplemented with 5% BSA and 0.3% Triton X-100 was used to block and permeabilize. Next, cells were incubated overnight at 4 °C with rabbit anti-γ-H2AX (1:200). After washing, the secondary antibody with FITC labeling was added for 1 h followed by nuclear DAPI counterstain and CLSM observation.
Target lipidomics study
Target lipidomics study was performed by Shanghai Bioprofile Technology Co., Ltd. In detailed, LLC cells were exposed to 6 Gy X-ray after a 12-hour incubation with DHA-N@M. Cells without treatment were taken as the control. All cells were washed, collected with scrapers, counted, and centrifuged. The obtained cell samples were subjected to extraction by adding 200 µL of pre-chilled 75% methanol solution and 825 µL tert-butyl methyl ether (MTBE), followed by shaking for 60 min. Subsequently, the samples were sonicated in an ice-bath for 30 min, then 200 µL of water was added and mixed, followed by incubation at room temperature for 10 min. After centrifugation at 4 °C and 16,000 g for 20 min, the precipitated proteins were resuspended in 300 µL of SDT buffer. The solution containing an equal number of cells was subjected to vacuum drying, reconstituted in 120 µL of DCM/methanol (1:1, v/v), centrifugated to collect supernatant for UPLC-MS/MS analysis. Of note, the sample processing was conducted at 4 °C throughout.
Samples were separated by ultra high-performance liquid chromatography (UPLC; Nexera X2 LC-30AD, Shimadzu) on an Acquity UPLC BEH HILIC column (130Å, 1.7 μm, 2.1 mm × 100 mm, Waters) column followed by mass spectroscopy performed on QTRAP 5500 (AB SCIEX). Mobile phase A (water/acetonitrile (50:50, v/v) with 10 mM ammonium acetate, pH 8.0) and mobile phase B (acetonitrile) were used for gradient elution: 0–0.1 min, 85% mobile phase B; 0.1–7.5 min, a linear gradient of B from 85 to 65%; 8.5–11 min, a linear gradient of B from 65 to 5%; 11–11.1 min, a linear gradient of B from 5 to 85%; 11.1–15 min, 85% mobile phase B. Flow rate, 300 µL/min; Column temperature, 40 °C; Sample volume, 2 µL; Sample temperature, 4 °C.
Electrospray ionization conditions in positive ion mode: Source Temperature 550℃, Ion Source Gas1 (GAS1): 40, Ion Source Gas2 (GAS2): 50, Curtain Gas (CUR): 35, Ion Spray Voltage Floating (ISVF) 5500 V.
Electrospray ionization conditions in negative ion mode: Source Temperature 550℃, Ion Source Gas1 (GAS1): 40, Ion Source Gas2 (GAS2): 50, Curtain Gas (CUR): 35, Ion Spray Voltage Floating (ISVF) -4500 V.
Assessment on the feasibility of RCM inhalation
The particle size, PDI, and the DHA-SNO leakage of DHA-N@M before and after aerosolization by liquid aerosol device (HY-LWH03, YSKD bio-technology co., LTD, China) consisted of a micro-sprayer and a high-pressure syringe.
A next-generation impactor (NGI; Copley Scientific, UK) was employed to analyze the aerosol particle size distribution (APSD). The flow rate was set to 15 L/min (± 5%) and the experiment was performed in an NGI cooler at 5 ± 1.5 °C with at least 90 min pre-cooling time. DHA-N@M solution was loaded into the nebulizer cup, and the apparatus was assembled according to the manufacturer’s instructions, followed by simultaneous activation of the flow pump and nebulizer (PARI Turboboy N compressor/LC Plus nebulizer, PARI GmbH, Starnberg, Germany). Upon completion of nebulization, nanoreactors deposited in the induction port and impaction cups were respectively collected for HPLC quantification. APSD parameters, including fine particle fraction (FPF), mass median aerodynamic diameter (MMAD), and geometric standard deviation (GSD), were calculated by Copley Inhaler Testing Data Analysis Software (version 3.10 EIBU).
Under cold light source, locating the glottis of mice using a small animal Laryngoscope (HY-SHJ01, YSKD bio-technology co., LTD, China). Then the liquid aerosol device was inserted into the trachea via the oral cavity and glottis, and immediately 25 µL of Evans Blue was spayed into lungs. Afterwards, the mice were transcardially perfused with 0.9% NaCl (15 mL), after which lungs were excised to observe distribution of the blue dye.
Orthotopic lung cancer model
LLC-Luc cells were resuspended in PBS to a concentration of 6.6 × 105 cells/mL and mixed with an equal volume of GelNest™, kept on ice-water bath to maintain cell viability and fluidity of GelNest™. C57BL/6 mice were anesthetized, shaved, and a small skin incision was made on the left chest to expose lungs. About 105 cells in 30 µL suspension were injected into the left lung to a depth of 4 mm, followed by immediate surgical glue (3M, USA) application to stitch the incision. After inoculation, the mice were placed at the right lateral decubitus position in a heating pad and monitored until fully awaken from narcosis. The tumor burden was recorded based on luciferase bioluminescence.
Biodistribution study
The biodistribution experiment was carried out in orthotopic LLC-Luc tumor bearing mice, who were randomly divided into three groups (three mice per group). The mice were i.t. administered with DID@M or DID@Lipo, or i.v. injected with DID@M at an equal dose of 0.2 mg/kg of DID. At 6, 12, 24, and 48 h post-administration, mice were received intraperitoneal injection of D-luciferin potassium salt at 150 mg/kg and euthanized 12 min later. The heart, liver, spleen, lung, kidney, and blood samples (20 µL) were collected for fluorescence imaging (IVIS Spectrum, USA).
To determine the localization of nanovesicles in orthotopic lung tumors, the lung tissues with tumors were excised at 12 h after administration, rapidly frozen, optimal cutting temperature (OCT)-embedded, and cryo-sectioned. After the slides were counterstained by DAPI to visualize nuclei, the microscopic distribution of DID-labeled nanovesicles in lung and tumor was observed using CLSM.
In vivo therapy
The therapeutic efficacy of DHA-N@M mediated ferroptosis-radiotherapy was examined in orthotopic LLC-Luc lung cancer model. Five days after tumor cell inoculation (set as Day 0), the tumor-bearing mice were randomly divided into seven groups (Day 0) and respectively i.t. injection with 25 µL of PBS (G1), DHA-N@M (G2), PBS (G3), DHA-SNO (G4), DHA@M (G5), BA-N@M (G6), and DHA-N@M (G7) at 7.5 µmol/kg (Day 1). Of these, mice in G3 ~ G7 were received a single dose of 6 Gy X-ray 12 h after nebulization. During irradiation, mice were with a small animal gas anesthesia machine (ABS, Yuyanbio) and placed in a dedicated container for local irradiation while lying on their right side. A lead plate covered other parts of the mice, exposing only the lung region. The treatment was repeated on Day 7.
The mice were weighted every 2 days and bioluminescence-imaged to monitor the tumor progression on Day 0, 3, 6, 9, 12, 15, and 20. The relative tumor progression was normalized to the initial total flux on Day 0. The tumor inhibition rate was calculated as follows:
$$\:\text{Tumor inhibition rate=1-}\frac{\text{Relative tumor total flux(treatment)}}{\text{Relative tumor total flux(control)}}$$
On Day 20, the mice were sacrificed to collect the heart, liver, spleen, lung with tumor, kidney, and trachea. The lungs with tumors were prepared for hematoxylin and eosin (H&E) staining to observe the tumor cellularity. H&E staining was also applied on the other organs to preliminarily evaluate systemic toxicity of the treatment regimen.
Immunofluorescence staining
LLC-Luc tumor-bearing mice were treated as described above. One day (24 h) after the last X-ray irradiation, the lungs with tumors were excised to prepare paraffin-embedded sections which were stained with antibodies or probe (anti-GPX4, dilution 1:600; anti-3-NT, dilution 1:200; anti-γ-H2AX, dilution 1:1000; C11-BODIPY581/591).
Statistical analysis
Data were presented as mean ± standard deviation (SD). Student’s t-test, one-way ANOVA, or two-way ANOVA was used to determine statistical significance. n.s. means no significance, and the difference was considered significant as *P < 0.05, **P < 0.01, and ***P < 0.001.
