Materials
Cetyltrimethylammonium chloride (CTAC), triethanolamine (TEA), bis[3-(triethoxysilyl) propyl]tetrasulfide (BTES), tetraethyl orthosilicate (TEOS), (3-mercaptopropyl)trimethoxysilane (MPTES), ferrous acetylacetonate and transferrin were purchased from Sigma-Aldrich (MO, USA). Doxorubicin (DOX), deferiprone, urea, 3-(4,5Dimethylthiazol- yl)-2,5Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), N-(3-dimethylaminopropyl)-N′-ethylcarbodi imide hydrochloride (EDC), n-hydroxysuccinimide (NHS), L-Glutathione (GSH), silane–PEG-COOH (Mw = 2000) were purchased from Aladdin Reagent Database Inc (Shanghai, China). 4ʹ,6–15 diamidino-2-phenylindole (DAPI), indocyanine green (ICG) was obtained from Tokyo Chemical Industry (Tokyo, Japan). Fetal bovine serum (FBS) and Dulbecco’s Modified Eagle’s Medium (DMEM) were obtained from Gibco (USA). All other chemicals and solvents were of analytical or chromatographic grade. Deionized water (18.4 MΩcm) used in all experiments was prepared using a Milli-Q system (Millipore, Boston, USA) and was used in all experiments.
Synthesis of mesoporous organosilica nanoparticles (HMON NPs)
To start with, CTAC aqueous solution (20g, 10 wt. %) and TEA aqueous solution (3.5g, 10 wt.%) were first mixed and stirred at 80°C, and then TEOS (1ml) was added dropwise for 1h reaction. Subsequently, a mixture of TEOS (0.5ml) and BTES (1ml) was added for another 4h of reaction. The resultant products were collected after centrifugation and washed with ethanol several times. Afterward, the products were dispersed in methanol (30 mL) with NaCl (1 wt.%) to extract the template and the extraction procedure was repeated at least three times to guarantee the template was removed completely. For synthesis of HMON NPs, an ammonia-assisted selective etching strategy was used. The above products were dispersed in 30mL of water with a certain amount of ammonia solution and react for 3h at 95°C. The final HMON NPs were obtained by centrifugation and washed with ethanol several times.
Synthesis of Fe-doped hollow mesoporous organosilica nanoparticles (Fe-HMON NPs)
Then, HMON NPs (25mg) and ferrous acetylacetonate (200mg) were dissolved completely in urea ethanol solution (25ml) and homogenized for 5 min under ultrasound treatment. The mixture was transferred to a water bath and reacted for 12 h at 80°C. The resultant Fe-HMON NPs were collected by centrifugation and washed with an ethanol–deionized water solution (v: v 1:1) several times.
Synthesis of Fe-HMON-PEG NPs and Fe-HMON-PEG-Tf NPs
For the PEGylation of Fe-HMON NPs, Fe-HMON NPs (20mg) were dissolved into ethanol (30ml), followed by the addition of silane–PEG-COOH (30mg) with magnetic stirring under 78°C for 12 h. After the reaction, Fe-HMON-PEG NPs were obtained after centrifugation and washed with ethanol for several times. Then activating the -COOH groups to conjugate with transferrin. EDC (12mg) and NHS (15mg) were added to Fe-HMON-PEG NPs (20mg) suspended in 20ml PBS. The mixture was carried out in an Erlenmeyer flask under stirring at 37°C for 4h. The products were centrifuged with PBS three times to remove excess EDC and NHS polymer. Then added transferrin solution (200ul, 1mg/ml) to the products and reacted for 12h at 37°C with shaking. Fe-HMON-PEG-Tf NPs were collected by centrifugation and washed with PBS three times.
Characterization
The particle size and size distribution were measured by Dynamic light scattering (DLS, litesizer500, Anton-Paar, Austria). The morphology of the MSN NPs, HMON MSN NPs and Fe-HMON MSN NPs were observed by transmission electron microscopy (TME, JEM-1200EX, JEOL, Japan). X-ray diffraction (XRD) patterns were recorded by a D/MAX-2550 PC diffractometer using Cu Kα radiation with a 2θ range of 10°-80° (Rigaku Inc., Japan). Fourier transmission infrared (FTIR) spectra of nanoparticles were performed using a FTIR spectroscopy (VECTOR22, Bruker, Germany) in the range from 400 to 4000cm− 1. The distributions and proportions of Fe, O, Si were performed using energy-dispersive spectroscopy (EDS) elemental mapping (X-MAXn65 T, Oxford, UK). Nitrogen adsorption/desorption experiment was tested by using a Micromeritics Tristar II analyzer (Micromeritics, USA). The surface areas and average pore size distributions were calculated by Brunauer–Emmett–Teller (BET) and Barrett–Jyner–Halenda (BJH) methods. The valence state of iron analysis was performed on the x-ray photoelectron spectrometer (XPS, ESCALAB 250Xi, Thermo Fisher Scientific, UK).
Drug loading and release profiles
The encapsulation of DOX by HMON-Tf NPs, Fe-HMON-PEG NPs and Fe-HMON-Tf NPs were prepared by simply mixing the DOX (3mg) with nanoparticles s (10mg) in PBS solution for 24h under the dark conditions. After that the unloaded DOX was removed by centrifugation and the supernatants were reserved for the calculation of loading efficiency of drugs.
① Loading content =(TD − FD)/TN× 100%
② Encapsulation efficiency = (TD − FD)/TD× 100%
where TD is the total weight of DOX fed, FD is the weight of nonencapsulated free DOX, and TN is the weight of nanoparticles.
To study the dissociation of DOX@ Fe-HMON-Tf NPs in response to pH and GSH trigger. DOX@Fe-HMON-Tf NPs were dispersed into buffer solutions of different pH values (pH 7.4 and pH 5.5) and different GSH concentration (5mM and 10mM) at a concentration of 0.5 mg/ml. After predetermined periods of time, 0.2ml of the incubation solution was extracted and centrifuged at 12000 rpm for 15 min to remove undissolved nanoparticles. The concentration of DOX in the supernatant was detected by a fluorescence spectrophotometer. And the content of iron in the supernatant was determined by inductively coupled plasma mass spectrometry (ICP-MS) instrument (ICAPRQICPMS, Thermo Fisher, USA).
In vitro and in vivo MRI
The Fe concentration of Fe-HMON-Tf NPs were detetmined by ICP-MS. With deionized water as a control group, samples with various Fe concentrations (0, 0.036, 0.072, 0.288, 0.576, 1.152mM) were dispersed in deionized water. All these were placed in 2mL Eppendorf tubes and measured with a 3T MRI scanner (Discovery MR 750, GE, USA) to obtain T2-weighted MRI. Through fitting plots of the inverse relaxation times 1/T2 s− 1 vs Fe concentration (mM), the relaxation coefficients r2 was obtained.
T2-weighted MRI was performed on a 3 T MRI scanner with a small-animal coil. Anesthetized mice bearing tumors were scanned using a fast spin–echo sequence pre- and post-injection of nanoparticles via the tail vein, with the following scan parameters: repetition time 3000ms, time to echo 80ms, field of view 40×40mm, matrix size 250×250, and slice thickness 2mm.
MTT assay on the cytotoxicity of various nanosamples
HepG2 cell line and LO2 cell line were obtained from the Chinese Academy of Sciences cell bank (Shanghai, China). Cells were cultured in Dulbecco’s Modified Eagle Medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA) in a 37°C incubator with 5% CO2. Cells were subcultured regularly using trypsin/EDTA (Meilune, China). The mediums were refreshed every 2 days. The HepG2 cells were seeded onto a 96-well plate at density of 104 U per well and subsequently incubated overnight at 37°C under an atmospheric CO2 level of 5%. The incubation media were then replaced with fresh ones containing PBS, DOX, HMON-Tf NPs, Fe-HMON-Tf, NPs DOX@HMON-Tf NPs, DOX@Fe-HMON-PEG NPs and DOX@Fe-HMON-Tf NPs. The equivalent concentrations of DOX were maintained at 0.25,0.5,1,1.5,2,2.5,3,3.5µg/ml. Each nanosample group contained six wells, and the incubation periods were set to 24 h. Fresh media containing MTT agents (0.5mg/ml) were added into each well when the incubation was complete and incubated for 4h. Afterwards, unreacted dyes were carefully removed by aspiration, and 150µL DMSO was added to each well to dissolve the formazan crystals. After 10min of low-speed oscillation, the OD value was measured using a Microplate reader (Bio-Rad, Model 680, USA) at a wavelength of 570nm.
Evaluation on the cellular uptake of the nanosamples
For the cellular uptake evaluations, the HepG2 cells were seeded into a six-well plate at a density of 105 U per well and incubated in 2ml of medium overnight at 37°C under an atmospheric CO2 level of 5%. When the cell confluence reached around 70%, fresh media containing PBS, DOX, HMON-Tf NPs, Fe-HMON-Tf NPs, DOX@HMON-Tf NPs, DOX@Fe-HMON-PEG NPs and DOX@Fe-HMON-Tf NPs were used to replace the exhausted medium. The nanopsamples concentration were maintained at 20µg/ml and the equivalent concentrations of DOX were maintained at 2ug/ml, the incubation periods were set to 24h. When the incubation completed, the cells were washed three times with PBS, fixed with 4% paraformaldehyde solution, stained with DAPI and finally analyzed by confocal laser scanning microscopy (CLSM; SP8 TCS, Leica, Germany) observation.
Quantification of the intracellular iron level
HepG2 cells were first seeded onto six-well plates at a density of 105 U per well, and the incubation conditions were kept the same with the in vitro experiments above. When the cell confluence reached around 70%, the previously added mediums were replaced by new ones containing PBS, DOX, HMON-Tf NPs, Fe-HMON-Tf NPs, DOX@HMON-Tf NPs, DOX@Fe-HMON-PEG NPs, DOX@Fe-HMON-Tf NPs. The concentration of nanosamples was 20µg/ml, and the equivalent DOX concentration was maintained at 2µg/ml. The incubation would continue for 24h. Subsequently, the culture medium in all wells was removed, and the cells were washed three times with PBS. The cells were then detached by trypsin without EDTA-Na and purified twice by repetitive centrifugation and PBS washing. Cell lysate (containing 1% SDS, 1% Triton X-100, and 40mM tris acetate) was eventually added to lyse the cells, and the resultant solution was sonicated to ensure compete cell disintegration. The iron level was detected by ICP-MS as above.
Monitoring the level of H2O2 in tumor cells
The HepG2 cells were seeded into a six-well plate using exactly the same protocol as above. The medium in each well were replaced with fresh ones containing different concentrations of DOX (0, 0.125, 0.25, 0.5, 1, 2 and 5µg/ml) when the cell confluence reached around 70%. The incubation would last for 24 hours, then intracellular H2O2 level was examined using the standard Fluorimetric Hydrogen Peroxide Assay Kit (Sigma-Aldrich), for which the fluorescent probe was first added to each well and the cells were further incubated for 20min at 37°C. The red fluorescent product has an excitation wavelength of 540nm and an emission wavelength of 590nm, which were used for the observation of the intracellular H2O2 levels analyzed by CLSM observation.
Evaluation of the intracellular lipoperoxide
HepG2 cells were seeded into six-well plates as described above and treated with various samples when the cell confluence reached 70%. PBS, DOX, HMON-Tf NPs, Fe-HMON-Tf NPs, DOX@HMON-Tf NPs, DOX@Fe-HMON-PEG NPs, DOX@Fe-HMON-Tf NPs. The incubation lasted for 24 hours, the concentration of nanosamples was 20µg/ml and the equivalent DOX concentration was maintained at 2µg/ml. When the incubation was complete, the cells were washed twice with PBS and incubated with DOPIBY C11 (Lipoperoxide indicator; concentration, 5µM) for 30min. The intracellular level of lipoperoxides was monitored using a CytoFLEX flow cytometry system (Beckman Coulter). A same experimental setup was also used for the CLSM observations.
Monitoring the changes in mitochondrial membrane potential
HepG2 cells were seeded into six-well plates as described above and treated with PBS, DOX, HMON-Tf NPs, Fe-HMON-T NPs f, DOX@HMON-Tf NPs, DOX@Fe-HMON-PEG NPs, DOX@Fe-HMON-Tf NPs when the cell confluence reached 70%. The incubation lasted for 24h, and the concentration of nanosamples was 20µg/ml, and the DOX concentration was at 2µg/ml. When the incubation was complete, the tumor mitochondria were stained with JC-1 dye following the procedures provided in the user manual and then observed by CLSM.
Flow cytometric analysis of the cell apoptosis
HepG2 cells were seeded into a six-well plate at the density of 105U per well. When the cell confluence reached 70%, the mediums were replaced with fresh ones containing PBS, DOX, HMON-Tf NPs, Fe-HMON-Tf NPs, DOX@HMON-Tf NPs, DOX@Fe-HMON-PEG NPs, DOX@Fe-HMON-Tf NPs and the incubation would continue for 24h. The concentration for all nanoparticles were 20µg/ml, and the DOX concentration in each group was maintained at an equivalent level of 2µg/ml. The media were drained when the incubation was complete, and the cells were washed three times with PBS and subsequently detached using non–EDTA-Na–containing trypsin. The detached cells were purified twice by repetitive washing and centrifugation. The cell apoptosis was investigated by flow cytometry using the Annexin V-FITC/PI apoptosis detection kit (Sigma) via the protocol.
Intracellular GSH assay
HepG2 cells were seeded in six-well plates at the density of 105U per well and incubated until the cell confluence reached 70%. Then the mediums were replaced with fresh ones containing PBS, DOX, HMON-Tf NPs, Fe-HMON-Tf NPs, DOX@HMON-Tf NPs, DOX@Fe-HMON-PEG NPs, DOX@Fe-HMON-Tf NPs and the incubation would continue for 24h. Afterwards, the cells were harvested and washed with PBS thrice. Then cell lysates were collected and measured according to the instructions of GSH and GSSG Assay Kit. A UV–vis spectrophotometer (TU-1800PC, Beijing Purkinje General Instrument Co., Ltd., China) was used to measure the absorbance at 412nm to determine the GSH levels.
Intracellular GPX-4 activity assay
Intracellular GPX-4 activity was measured using a cellular glutathione peroxidase assay kit (Beyotime, Jiangsu, China). HepG2 cells were seeded in a six-well plate and cultured at 5% CO2, 37°C overnight. Then the mediums were replaced with fresh ones containing PBS, DOX, HMON-Tf NPs, Fe-HMON-Tf NPs, DOX@HMON-Tf NPs, DOX@Fe-HMON-PEG NPs, DOX@Fe-HMON-Tf NPs and the incubation would continue for 24h. The cell lysates were collected and measured according to the manufacturer’s instructions. The M5 full-band multi-function microplate reader (SynergyMx M5, Molecular Devices, USA) was used to measure the absorbance at 340nm.
Western blot assays
To determine the expression levels of the apoptotic protein of Caspase-3 and the ferroptosis-related proteins of GPX4. HepG2 cells were seeded into six-well plates at a density of 105U per well and incubated overnight at 37°C under an atmospheric CO2 level of 5% until the cell confluence reached around 70%. Subsequently, the mediums were replaced by fresh ones containing PBS, DOX, HMON-Tf NPs, Fe-HMON-Tf NPs, DOX@HMON-Tf NPs, DOX@Fe-HMON-PEG NPs, DOX@Fe-HMON-Tf NPs and further incubated for 24h. The concentration of nanosamples was 20µg/ml, and the DOX concentration was 2µg/ml. The cells were then lysed with Laemmli Sample Buffer (Bio-Rad), and the total protein was quantified by electrophoresis using a BCA protein kit (Beyotime) and 12% SDS–polyacrylamide gel electrophoresis. The proteins were then transferred from the gel onto polyvinylidene difluoride membrane (Immobilon P, Millipore) and blocked by primary and secondary antibodies. The images were captured on a molecular imager (ChemiDoc Touch Imaging System, Bio-Rad, USA).
Tumor model
Balb/c nude mice were (4–5 weeks, ~ 15g) were purchased from Shanghai Silaike Laboratory Animal Co., Ltd. All animal experiments were carried out in accordance with the National Institutes of Health (NIH, USA) guidelines for the care and use of laboratory animals in research. The surgical procedures and experiment protocols were approved by the Committee for Animal Experiments of Zhejiang University. The HepG2 tumor models were established by injecting 100µl of PBS containing 107U of HepG2 cells into the subcutaneous tissue of the mice. The tumor volume was calculated as Vtumor = LW2/2 (L, maximum diameter of the tumor; W, minimum diameter of the tumor, both were measured using a digital vernier caliper).
Fluorescence imaging for tracking nansamples in vivo
The evaluation of biodistribution was performed by using subcutaneous tumor bearing BALB/C nude mice (6 mice/group). The near infrared dye ICG was used to label the Fe-HMON-PEG NPs and Fe-HMON-Tf NPs before intravenous injection. The equivalent ICG concentration was maintained at 1mg/kg. The mice were anaesthetized at different time points, and then their fluorescent photographs were captured by the IVIS Spectrum Imaging System (Caliper, PerkinElmer, USA). In addition, the mice were euthanized by CO2 asphyxiation at 24h post-injection, and then their major organs (tumor, heat, liver, spleen, lung and kidney) were collected, which were then imaged to observe the fluorescence signals.
Tumor treatment effect and histology analysis
The therapeutic efficacy of different treatment was evaluated using subcutaneous HepG2 tumor-bearing mice. Various samples were then administrated when the tumor size reached 60mm3, and the initial weight of all mice was maintained at 18.2 ± 0.2g. Briefly, 42 HepG2 tumor-bearing mice were randomly divided into seven groups (each with six mice). The sample groups are PBS, DOX, HMON-Tf NPs, Fe-HMON-Tf NPs, DOX@HMON-Tf NPs, DOX@Fe-HMON-PEG NPs, DOX@Fe-HMON-Tf NPs. All samples were injected through the tail vein at an equivalent DOX concentration of 5mg/kg. The injection was repeated every other day, and the body weight and tumor volume of nude mice were both recorded. After 21 days of treatment, all mice were euthanized, and the tumors and major organs were harvested for the subsequent analysis. Typically, the organs and tissues were sectioned and embedded into paraffin after being fixed with 10% formalin at 4°C for 24h, and then, paraffin-embedded sections were stained with H&E to monitor the cytotoxicity induced by various nanoplatforms. In addition, the tumor sections were also stained by the colorimetric TUNEL Apoptosis Assay Kit to determine the therapeutic effect. Both the H&E- and TUNEL-stained tissue sections were then observed with a microscope.
For the survival analysis, 42 HepG2 tumor–bearing mice were treated using the above procedures. No more injection was given after 21 days, and the number of live mice in each group was recorded until day 60.
Statistical analysis
SPSS ver. 26.0 (IBM Inc., USA) and GraphPad Prism ver. 9.0 (GraphPad Software, USA) were used to process all data. Quantitative experimental data are recorded as means ± SD. We used the two-tailed t test or the Mann–Whitney U test to compare two groups and the Kruskal–Wallis test to perform multiple comparisons. Statistical significance was set at P < 0.05.