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′-ethylcarbodiimide hydrochloride (EDC), n-hydroxysuccinimide (NHS), L-Glutathione (GSH), silane–PEG-COOH (Mw = 2000) were purchased from Aladdin Reagent Database Inc (Shanghai, China). 4ʹ,6–diamidino-2-phenylindole (DAPI), indocyanine green (ICG) was obtained from Tokyo Chemical Industry (Tokyo, Japan). Fetal bovine serum (FBS), phosphate buffered saline (PBS), trypsin-EDTA and Dulbecco’s modified eagle’s medium (DMEM) were obtained from Gibco (USA). All other chemicals and solvents were of analytical or chromatographic grade.
Synthesis of mesoporous organosilica nanoparticles (HMON NPs)
To start with, CTAC aqueous solution (20g) and TEA aqueous solution (3.5g) were first mixed and stirred at 80°C for 15min and TEOS (1ml) was added dropwise for 1h reaction. Subsequently, add a mixture of TEOS (0.5ml) and BTES (1ml) for another 4h. Afterwards, the products were washed with ethanol several times and dispersed in methanol (30 mL) with NaCl (1 wt.%) to extract the template. This step was repeated at least three times, each time at least 12h to ensure the template was removed completely. The final HMON NPs were obtained by ammonia-assisted selective etching strategy reacting for 3h at 95°C with a certain amount of ammonia solution and washed with ethanol several times.
Synthesis of Fe-doped hollow mesoporous organosilica nanoparticles (Fe-HMON NPs)
HMON NPs (25mg) and ferrous acetylacetonate (200mg) were dissolved completely in urea ethanol solution (25ml) and homogenized for 5 min under ultrasound treatment. Then, the mixture was reacted for 12h with stirring at 80°C. The resultant Fe-HMON NPs were collected by centrifugation and washed with ethanol–deionized water solution several times.
Synthesis of Fe-HMON-PEG-Tf NPs
Fe-HMON NPs (20mg) were dissolved into ethanol (30ml), followed by the addition of silane–PEG-COOH (30mg) with stirring under 78°C for 12 h. After the reaction, Fe-HMON-PEG NPs were obtained after centrifugation and washed with ethanol several times. Then EDC (12mg) and NHS (15mg) were added to Fe-HMON-PEG NPs (20mg) suspended in 20ml PBS to activate the -COOH groups to bind to transferrin. The mixture was carried out in an Erlenmeyer flask with stirring at 37°C for 4h. The products were centrifuged with PBS three times to remove excess EDC and NHS. Then added transferrin solution (200ul, 1mg/ml) to the products and reacted for 12h at 37°C with stirring. 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, D/MAX-2550 PC, Rigaku Inc., Japan) patterns were using Cu Kα radiation with a 2θ range of 10°-80°. The valence state of iron analysis was performed on the x-ray photoelectron spectrometer (XPS, ESCALAB 250Xi, Thermo Fisher Scientific, UK). Fourier transform infrared spectroscopy (FTIR, VECTOR22, Bruker, Germany) of nanoparticles were performed 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). The 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–Joyner–Halenda (BJH) methods.
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 mixing the DOX (3mg) with nanoparticles (10mg) in PBS solution under the dark conditions for 24h. After that, the unloaded DOX was removed by centrifugation and the supernatants were reserved for the calculation of loading efficiency of drugs.
Loading content =(TD − FD)/TN× 100%
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 investigate the dissociation of DOX@ Fe-HMON-Tf NPs in response to pH and GSH trigger. A certain concentration of DOX@Fe-HMON-Tf NPs was dispersed into buffer solutions with different pH (pH 7.4 and pH 5.5) and different GSH concentrations (5mM and 10mM). At predetermined time points, undissolved nanoparticles were removed by centrifuging at 12000 rpm for 15 min and the concentration of DOX in the supernatant was detected using a fluorescence spectrophotometer. And the content of iron in the supernatant was measured by inductively coupled plasma mass spectrometry instrument (ICP-MS, ICAPRQICPMS, Thermo Fisher, USA).
In vitro and in vivo MRI
The Fe concentration of Fe-HMON-Tf NPs determined by ICP-MS. Various Fe concentrations (0, 0.036, 0.072, 0.288, 0.576, 1.152mM) were dispersed in deionized water in 1mL Eppendorf tubes and measured with a 3T MRI scanner (Discovery MR 750, GE, USA) same time to obtain T2-weighted imagines. The relaxation coefficients r2 was obtained by fitting plots of the inverse relaxation times 1/T2 (s − 1) and Fe concentration (mM).
T2-weighted MRI was performed on a 3 T MRI scanner with a small-animal coil. The tumours-bearing mice need to be anaesthetized before and after the tail vein injection of nanoparticles. Then using a fast spin-echo sequence to scan with repetition time 3000ms, time to echo 80ms, the field of view 40×40mm, matrix size 250×250 and slice thickness 2mm.
MTT assay on the cytotoxicity of various nanosamples
HepG2 cell line was obtained from the Chinese Academy of Sciences cell bank (Shanghai, China). The HepG2 cells were plated in 96-well plates at a density of 104 U per well and cultured at 37°C with 5% CO2 overnight. The culture media 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 cells were cultured for 24h. The equivalent concentrations of DOX were maintained at 0.25,0.5,1,1.5,2,2.5,3,3.5µg/ml. Then 20ul MTT solution (5mg/ml) was added into each well and incubated for 4h. Afterwards, carefully removed the media and added 50µL DMSO to each well with low-speed oscillation for 15min to dissolve the formazan crystals. The OD value was measured using a Microplate reader (Bio-Rad, Model 680, USA) at a wavelength of 570nm.
Assess cellular uptake and intracellular iron levels of different nanosamples
The HepG2 cells were plated into 24-well plates at a density of 105 U per well and cultured at 37°C with 5% CO2 overnight. 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 concentrations were maintained at 20µg/ml and the equivalent concentrations of DOX were maintained at 2ug/ml, the culture periods were set to 24h. The cells were washed with PBS three times after the culture was completed. Then fixed with 4% paraformaldehyde solution for 0.5h, stained with DAPI for 15min. Finally, confocal laser scanning microscopy (CLSM; SP8 TCS, Leica, Germany) was used for observation and analysis.
For the determination of intracellular iron levels, repeated the above steps and separated the cells by trypsin without EDTA-Na, then added cell lysate to lyse the cells and sonicated the resultant solution to ensure the cells are completely broken down. The iron level was detected by ICP-MS as above.
Evaluation the level of H2O2 in tumour cells
Using the same protocol as above to plated HepG2 cells into 24-well plates. When the cell confluence reached around 70%, the media was replaced with fresh ones containing different concentrations of DOX (0, 0.125, 0.25, 0.5, 1, 2 and 5µg/ml) in each well and cultured for 24 h. Then the fluorescent probe was added to each well except the blank control and further cultured for 20min at 37°C. The intracellular H2O2 level was examined using the standard Fluorimetric Hydrogen Peroxide Assay Kit (Sigma-Aldrich) that the red fluorescent product had an excitation wavelength of 540nm and an emission wavelength of 590nm, which could be used for the observation and analysis by CLSM.
Evaluation of the intracellular lipid peroxides
HepG2 cells were plated into 12-well plates as above. When the cell confluence reached 70%, the media was 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 cultured for 24h. The concentration of nanosamples was 20µg/ml and the equivalent DOX concentration was kept at 2µg/ml. The intracellular level of lipid peroxides was monitored by culturing with DOPIBY C11 (5µM) for 30min and measured by the CytoFLEX flow cytometry system (Beckman Coulter). The CLSM observation also used the same experimental protocol.
Evaluation of the mitochondrial membrane potential
HepG2 cells were plated into 24-well plates and dealt with PBS, DOX, HMON-Tf NPs, Fe-HMON-T NPs, DOX@HMON-Tf NPs, DOX@Fe-HMON-PEG NPs, DOX@Fe-HMON-Tf NPs when the cell confluence reached 70%. The concentration of nanosamples and DOX were the same as above and cultured for 24h. After the incubation, the mitochondria were stained with JC-1 dye and observed by CLSM. When the mitochondrial membrane potential was high, JC-1 dye aggregated in the matrix to form polymers, which can produce red fluorescence (Ex/Em = 585/590nm); when the mitochondrial membrane potential was low, JC-1 dye existed as monomers and produced green fluorescence (Ex/Em = 510/527 nm).
Analysis of the cell apoptosis
HepG2 cells were plated into 12-well plates at the density of 105U per well. When the cell confluence reached 70%, the cells were dealt with fresh media 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 lasted for 24h. The concentration of nanosamples and DOX were the same as above. The Annexin V-FITC/PI apoptosis detection kit (Sigma) was used as the protocol to study cell apoptosis by flow cytometry.
Determination of intracellular GSH and GPX-4 activity
HepG2 cells were plated in 12-well plates at the density of 105U per well and cultured at 37°C with 5% CO2 overnight. The cells dealt with fresh media 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 continued for 24h. The concentration of nanosamples and DOX were the same as above. Then cell lysates were collected and measured according to the instructions of GSH and GSSG Assay Kit. The UV–vis spectrophotometer (TU-1800PC, Beijing Purkinje General Instrument Co., Ltd., China) was used to measure the absorbance at 412nm to determine the GSH level.
For the determination of intracellular GPX-4 activity, repeated the above steps and collected the cell lysates. 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 blotting analysis
HepG2 cells were plated into six-well plates at a density of 105U per well and cultured until the cell confluence reached around 70%. Afterwards, the cells dealt with fresh media 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 culture continued for 24h. The concentration of nanosamples and DOX were the same as above. To determine the expression levels of Caspase-3 apoptotic proteins, DOX-activated related proteins NOXs and GPX4 ferroptosis-related proteins. Then the cells were lysed, and the total protein was quantified by electrophoresis using the BCA protein kit (Beyotime) and 12% SDS-polyacrylamide gel electrophoresis. The protein was then transferred from the gel to a polyvinylidene fluoride membrane (Immobilon P, Millipore) and blocked with primary and secondary antibodies. The image was captured on a molecular imager (ChemiDoc Touch Imaging System, Bio-Rad, USA).
Fluorescence imaging for tracking nansamples in vivo
The tumour model establishment plan was as follows. The model mice were divided into two groups (6 mice/group). Fe-HMON-PEG NPs and Fe-HMON-Tf NPs were labelled with ICG, and the equivalent ICG concentration of the nano-samples injected into the tail vein was maintained at 1 mg/kg. The mice were anaesthetized at different time points, and the in vivo distribution was observed through the IVIS spectral imaging system (Caliper, PerkinElmer, USA). Furthermore, the mice were euthanized after 24 hours, and the major organs were collected and observed the fluorescence signals, including tumour, heart, liver, spleen, lung and kidney.
Tumour treatment and histology analysis
All animal experiments were performed following the care and use guidelines of the National Institutes of Health (NIH, USA) and approved by the Animal Experiment Committee of Zhejiang University. Balb/c nude mice (4–5 weeks) were purchased from Shanghai Silaike Laboratory Animal Co., Ltd. The HepG2 tumour models were established by injecting 100µl of PBS containing 107U of HepG2 cells into the subcutaneous tissue of the mice. When the tumour size reached 60mm3, 42 HepG2 tumour-bearing mice were randomly divided into seven groups (6 mice/group). The tumour volume was calculated as V = LW2/2 (L, the maximum diameter of the tumour; W, the minimum diameter of the tumour). Then, all samples were injected through the tail vein at an equivalent DOX concentration of 5mg/kg, including 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 injection was repeated every other day for several cycles, and the body weight and tumour volume of nude mice were recorded daily. All mice were euthanized After 21 days, and the tumours and major organs were collected and fixed with 10% formalin for 24 hours. The paraffin-embedded sections were stained with hematoxylin and eosin (H&E) to monitor the cytotoxicity induced by various nanosamples and tumour sections performed terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining to determine the treatment effect. For the survival analysis, 42 HepG2 tumour–bearing mice were processed using the above procedures. No injections were 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.