Ethical statement
The current study was approved by the clinical research Ethics Committee (PR003/13) of Tongji University School of Medicine, and conformed with the guidelines of the Declaration of Helsinki. Signed informed consents were obtained from all participants prior to specimen collection. Animal experiments were performed in accordance with the standard of the Guide for the Care and Use of Laboratory animals published by the National Institutes of Health. Animal experiment protocols were reviewed and granted by the animal ethics committee of Tongji University School of Medicine. Extensive efforts were made to minimize the suffering of the included animals.
Synthesis of Fe
3
O
4
MNPs
Fe3O4 MNPs were synthesized with the help of an oxidative hydrolysis method. First, to synthesize Fe3O4, FeCl3 deionized aqueous solution (1.66 g; 20 mL) was mixed with FeCl2 4H2O (1.00 g; 20 mL) in deionized water. A black precipitate was observed after the addition of NH4OH solution (25%, 20 mL). The solution was stirred for 30-min to complete the reaction. Next, the solution was placed on a magnet, which allowed the magnetite to be absorbed to the surface. After the reaction solution was poured off, the magnetite dispersion was added with oleic acid solution in hexane (2.5–10 wt% solution in 20 mL hexane) under constant stirring. Concentration was subsequently performed by evaporation of hexane from the dispersion, and a black magnetite concentrate was obtained. Meanwhile, a stannous octoate catalyst was used for the polyethylene glycol lactide (PEG-LAC) copolymerization reactions. A total of 1.00 g lactide and 0.2 g PEG2000 were mixed in 9.00 mL toluene containing 0.005 g stannous octoate. The reaction balloon was then placed in a hot oil bath (140 °C) to react for 6 h. After the reaction mixture was cooled at room temperature, the copolymer was precipitated with icy diethyl ether liquid and dried in a vacuum oven. To cover the magnetite core surface with PEG-LAC layer, 20 mL of magnetite hexane dispersion was added to 20 mL of copolymer aqueous solution (1 wt% copolymer). Afterwards, the mixture was sonicated for 4 h to transfer the particles from the hexane top layer to the aqueous bottom layer. The residual dispersion in the aqueous phase was then dialyzed using a dialysis bag (1200) and lyophilized. To cover the chitosan layer on the surface of the previously synthesized particles, 0.05 g magnetite particles were dissolved in 1 mL dichloromethane, and then dripped onto an aqueous solution containing 0.2 mL acetic acid and 0.05 g chitosan. After overnight stirring, the solution was filtered through a 0.22 mm filter and lyophilized. Additionally, 0.02 g branched polyethyleneimine (PEI; 25 kDa) was dissolved in 10 mL deionized water, and added with the same amount (0.02 g) of product from the preparation of chitosan layer while maintaining stirring. Finally, the mixture was placed on a stirrer for 24 h.
Characterization of Fe
3
O
4
magnetic materials
Fourier transform infrared reflection (FTIR) spectroscopy analysis was performed for Characterization of Fe3O4 magnetic materials. The samples (2 mg) and 200 mg potassium bromide were mixed and ground for 3 min. The mixture was then pressed into pellets for measurement. FTIR spectra were recorded using a FTIR spectrophotometer (Bruker tensor 27 spectrometer, Billerica, MA, USA) at the range of 400–4000 cm−1.
Synthesis of NPs
The experimental materials for the synthesis of NPs were provided by Dharmacon (Lafayette, CO, USA). Scrambled control FAM-siBIRC5-NC (5′-CAGUCGCGUUUGCGACUGGUUdTdT-3′; 3′-dTdTGUCAGCGCAAACGCUGACCAA-5′) and FAM-siBIRC5 (5′-GGCUGGCUU CAUCCA-CUG-CdTdT-3′; 3′-dTdTCCGACCGAAGUAGGUGACG-5′) were synthesized by Integrated DNA Technologies (Coralville, IA, USA). In addition, FAM-AS-ODN (anti-sense: 5′-CCCAG-CCTTCCAGTCCCTTG-3′) and FAM-AS-ODN-NC (sense: 5′-CAAGGGACTGGAAGGCTGGG-3′) were purchased from Dharmacon (Lafayette, CO, USA).
To synthesize the Fe3O4 MNPs loaded with siBIRC5 solution and AS-ODN, the solution of siBIRC5 (100 ng) in Opti-minimum essential medium (MEM) and AS-ODN (200 ng) was added to the Fe3O4 MNPs. Subsequently, NPs were prepared after vortexing the resulting solution for 10 s and a 30-min incubation at room temperature.
Size morphology characterization of NPS
NPs size and morphology were observed and analyzed with the help of transmission electron microscopy (TEM, Philips, Eindhoven, the Netherlands) and scanning electron microscopy (SEM, Philips, XL30 microscope instrument, the Netherlands). Particle size was analyzed using the ImageJ software. In addition, Zeta potential and hydrodynamic size of NPs were analyzed by dynamic light scattering (DLS, Mastersizer 2000, Malvern, Worcestershire, UK). NPs prepared with different mass ratios (Fe3O4 MNPs: siBIRC5: AS-ODN) were subjected to 3% agarose gel electrophoresis at 90 mV for 45 min, after which the gels were stained with ethidium bromide solution and observed under a GelDoc imaging system (UV doc-008, UVP, Upland, CA, USA).
Clinical samples
Fresh squamous cell lung adenocarcinoma tissues and adjacent non-tumor lung tissues were collected from patients during resection surgery performed at the Tongji University School of Medicine. Histological typing was performed by the pathology department of the Tongji University School of Medicine. All human tissues were stored in RNAlater™ Medium, and stored in liquid nitrogen before processing. The selected samples were subsequently subjected to reverse transcription quantitative polymerase chain reaction (RT-qPCR). The expression patterns of BIRC5 were detected by Western blotting.
Cell culture and transfection
Lung adenocarcinoma cells A549 (CCL-185, ATCC, Manassas, VA, USA) and H460 (HTB-177, ATCC, Manassas, VA, USA) were cultured in Dulbecco’s modified eagle medium (DMEM; CAT#01–055, Biological industries, Beit HaEmek, Israel). Meanwhile, human embryonic lung fibroblasts HFL-1 (CC-Y1584, EK-Bioscience, Shanghai, China) were cultured in Ham’s F-12 (CAT#01–095, Biological industries). All media were then supplemented with 10% fetal bovine serum (FBS; GIBCO™, CAT# 10270106, Life Technologies, San Jose, CA, USA), 100 units/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine, which were all purchased from Biological industries (Kibbutz Beit Haemek, Israel). Non-essential amino acids (NEAA; CAT# X0557, 1: 100, Biowest, Logan, Utah, USA) were employed for HFL-1 culture. Afterwards, the cells were cultured in a humidified incubator (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 37 °C with 5% CO2.
A549 cells were transfected with the HA-BIRC5 expression vector (pcDNA3-HA-BIRC5) and Myc-DR5 expression vector (DR5 Myc-tag) or corresponding empty vector (pcDNA) following the instructions of the Lipofectamine 2000 transfection reagent (CAT# 11668019, Invitrogen, Carlsbad, CA, USA). Cells were transfected with 1 μg of pcDNA3-HA-BIRC5 and DR5 Myc-tag or the corresponding empty vector pcDNA. After a 24-h period of transfection, the cells were treated with NPs for subsequent analyses.
Western blotting
To detect the expression patterns of related proteins, highly efficient radio immunoprecipitation (RIPA) lysis buffer (R0010, Solarbio, Beijing, China) was adopted to extract the total protein content from the cells or tissues according to the manufacturer’s instructions. The supernatant was collected after 15-min lysis at 4 °C and 15-min centrifugation at 12,000 rpm. Protein concentration in the samples was subsequently determined using a bicinchoninic Acid (BCA) Kit (20201ES76, Yeasen Biology, Shanghai, China). Next, the proteins were mixed with sodium dodecyl sulfate (SDS) loading buffer after quantification, and samples were incubated at 10 °C for 5 min and cooled down naturally at room temperature. Samples and protein markers were then separated using SDS–polyacrylamide gel electrophoresis (PAGE; Solarbio). After electrophoresis, the gel was rinsed with deionized water and then transferred onto a polyvinylidene fluoride membrane. After the transfer, the membranes were blocked with 5% skimmed milk in tris-buffered saline tween (TBST) for 1 h at room temperature. To visualize the proteins on the membranes, the membranes were incubated with mouse anti-BIRC5 (dilution ratio of 1: 500, sc-17779, Santa Cruz biotechnology, CA, USA), mouse-anti-DR5 (dilution ratio of 1: 500, sc-166624, Santa Cruz biotechnology), and mouse anti-human caspase-3 antibodies (dilution ratio of 1: 500, sc-56053, Santa Cruz biotechnology) overnight at 4 °C. The following day, the membranes were then incubated with the horseradish peroxide (HRP) conjugated secondary antibody (dilution ratio of 1: 4000; Southern biotech, Birmingham, Alabama, US) for 1 h at room temperature. Afterwards, the membrane was detected using an enhanced chemiluminescence immunoblotting detection kit. In addition, mouse anti-β-actin antibody (dilution ratio of 1: 2000, sc-8432, Santa Cruz biotechnology) was treated as described previously. For absorbance analysis, scanned photographs were quantified using the AlphaEasy FC software (Alpha Innotech, San Leandro, CA, USA). Each experiment was repeated three times to obtain the mean value.
RT-qPCR
Total RNA content was extracted the cells or tissues using RNeasy Mini kits (CAT#74104, Qiagen, Hilden, Germany) following the instructions to determine the transcription expression levels of genes. Complementary DNA (cDNA) was then reverse-transcribed with the help of a reverse transcription kits (Promega, Madison, WI). Subsequently, mRNA expression levels of related factors were determined using an ABI PRISM 7500 sequence detection system (Applied Biosystems, Foster City, CA). All primer sequences are shown in Additional file 3: Table S1.
Cellular fluorescence imaging
To explore the uptake of siBIRC5 and AS-ODN, the cells were seeded in a 24-well culture plate and incubated for 12 h. A magnet (magnetic field: 0.5 T) was placed under the center position of the culture plate, and then incubated with AS-ODN (concentration of 200 nM) and siBIRC5 (concentration of 100 nM). Sense-ODN and scrambled siBIRC5 were transfected performed using the Lipofectamine 3000 transfection reagent (l300001, Invitrogen) as a positive control. FAM labeling was employed to observe the cellular uptake of siBIRC5 and AS-ODN with the help of fluorescence microscopy (observer A1, Carl Zeiss, Oberkochen, Germany). Fe3O4 MNPs loaded siBIRC5 as p-siBIRC5, while Fe3O4 MNPs loaded AS-ODN as p-AS-ODN. Simultaneously, sense-ODN and scrambled siBIRC5 were employed as the negative control (NC).
Immunofluorescence staining
Five sterile round coverslips (1 cm × 1 cm) were placed on 6-well plates. Cells (1 × 105 per well) were then seeded into each well. Following treatment with each preparation and magnetic field (0.5 T), the cells were fixed with 4% paraformaldehyde for 20 min, and then permeabilized with 0.2% Triton X-100 for 10 min. Next, the cells were blocked with 3% horse serum for 1 h at room temperature, and incubated with mouse anti-BIRC5 and mouse-anti-DR5 primary antibodies for 1 h. Afterwards, the cells were incubated with the anti-mouse secondary antibody labeled with tetramethylrhodamine for 1 h. Later, the cells were mounted with DAPI (Sigma Aldrich Co., St Louis, MO, USA) before imaging with a confocal microscope.
γ-H2AX immunofluorescence staining
Cells were seeded in a 96-well plate with transparent black bottom (Corning, NY, USA) at 104 cells per well, and incubated overnight at 37 °C in 5% CO2. Next, the cells were assigned into the control group, the NPs group (siBIRC5 with concentration of 500 nM, AS-ODN with concentration of 1000 nM), the RT group, and the NPs + RT group. After 24 h, the cells were fixed with 4% paraformaldehyde for 20 min. Fixed cells were then permeabilized with 0.1% Triton X-100 in PBS for 5 min, blocked with 1% bovine serum albumin (BSA) in PBS (blocking solution) for 30 min, and incubated overnight at 4 °C with mouse monoclonal anti-γ-H2AX antibody (dilution ratio of 1: 400 in blocking solution). Later, the cells were incubated in dark conditions with the FITC conjugated anti mouse antibody (dilution ratio of 1: 200 in blocking solution) for 1 h at room temperature and counterstained with 0.1 µg/mL DAPI for 1 min. Fluorescence pictures were taken on a Nikon A1R spectral confocal microscope and cytation 3 imaging multimode plate reader (BioTek, Winooski, VT, USA).
Flow cytometry
Cellular uptake efficiency was determined. Following treatment with each preparation and magnetic field (0.5 T), the cells were rinsed twice with cold PBS. Next, the cells were trypsinized and then analyzed using a flow cytometer (BD facsverse, BD Biosciences, San Jose, CA, USA).
$${\text{Cellular}}\,{\text{uptake}}\,{\text{efficiency}}\,(\% )\, = \,\frac{{{\text{Cellular}}\,{\text{uptake}}\,{\text{of}}\,{\text{siBIRC5}}\,{\text{and}}\,{\text{AS - ODN}}}}{{{\text{siBIRC5}}\,{\text{and}}\,{\text{AS - ODN}}\,{\text{added}}\,{\text{to}}\,{\text{the}}\,{\text{cell}}}}$$
The expression patterns of DR5 were determined after cells were treated with each preparation and magnetic field (0.5 T) and trypsinized. Next, the cells were incubated with the PE conjugated anti-human-DR5 (CD262) antibody (Biolegend®, San Diego, CA, USA) for 30 min. The level of DR5 expression on the cell membrane was subsequently reflected by measuring the PE levels using flow cytometry (lsrfortessa™, BD Biosciences, San Jose, CA, USA).
Cell apoptosis was measured. Briefly, lung adenocarcinoma cell lines at the logarithmic phase of growth were seeded in 6-well plates at 2.0 × 105 cells in each well. Four groups (control group, RT group, NPs group and the NPs + RT group) of cells were collected and counted. Annexin V-FITC/PI apoptosis detection kits were adopted for testing. Cell pellets were resuspended in 195 μL binding buffer and stained with 5 μL each of annexin V-FITC and PI staining solution for 10 min at room temperature in dark conditions. Flow cytometry was then performed on a FACScan system with CellQuest software. The apoptosis rate was calculated as follows: (number of apoptosis in each group / total number of cells in each group) × 100%. Three parallels were set for each group to obtain the mean value.
Enzyme-linked immunoassay (ELISA)
To measure BIRC5 protein, we seeded cells (1 × 105 per well) into 6-well plates. After cells were treated with each preparation and magnetic field (0.5 T), cells were treated with 200 µL of 0.5% Triton X-100 lysis buffer for 30 min in an ice bath. Cell lysates were collected, vortexed briefly, and then incubated on ice for an additional 15 min. Cell debris was removed by centrifugation at 2000 g for 5 min, diluted with 1% BSA in PBS and protein concentration was determined by Coomassie blue assay (Bradford method). A 10 mg sample of total protein was added to a 96-well plate precoated with capture antibody, and BIRC5 protein was determined by ELISA Kit (R&D systems, Minneapolis, MN, USA).
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
To evaluate cell proliferation, cells were seeded in 96-well plates at 5000 cells/well. The medium was removed the following day. Subsequently, NPs containing 0, 250, 500, 750, 1000 μM of siBIRC5 were incubated with cells under a magnetic field (0.5 T). After 24 h, the cells were irradiated at doses of 0 Gy (no radiation), 2 Gy, 4 Gy, 6 Gy, and 8 Gy, and incubation was continued for another 48 h. The wells were added with a total of 0.5 mg/mL MTT reagent and treated for 1 h, and then with dimethyl sulfoxide (DMSO, Serva electrophoresis GmbH, Heidelberg, Germany). The absorbance of the samples was measured at 570 nm using a synergy HTX microplate reader (BioTek).
Plate clonogenic assay
To evaluate cell proliferation, cells with or without NPs (siBIRC5 with concentration of 500 nM; AS-ODN with concentration of 1000 nM) under a magnetic field (0.5 T), which were irradiated at room temperature with γ-ray at doses of 0, 2, 4, 6, and 8 Gy. Subsequently, using a 137Cs light source (Mark 1–68 irradiator, JL Shepherd & Associated, San Fernando, CA, USA), the cells were irradiated at a dose rate of 3.66 Gy/min and further incubated for another 6 h. Next, the cells were trypsinized and counted, and then the cells were cultured in drug-free medium to analyze the colony forming ability. After 2 weeks of culture, the cells were fixed and stained with PBS containing 4% formaldehyde and 0.05% crystal violet, and colonies with greater than 50 cells were counted. Three parallels were set for each group.
Scratch test
To measure cell migration, cells were added in triplicate in 6-well plates and incubated until cell grew adherent to the wall. Cells were scratched using a 10 μL pipette. Subsequently, the control, the NPs (siBIRC5 with concentration 500 nM; AS-ODN with concentration 1000 nM), the radiotherapy (with radiation at 2 Gy dose), as well as the NPs + RT group were set up. Cells were first incubated in the presence or absence of NPs under a magnetic field (0.5 T), and then treated with or without radiation. After a 24-h period of incubation, the samples were observed using a phase contrast microscope.
Xenograft tumor in nude mice
BALB/C female nude mice (aged 5–6 weeks old) were housed in a specific-pathogen-free grade animal room with room temperature of 25 °C and humidity of 70%, 12-h light/dark cycle with ad libitum access to water and food. Animal experiments were performed by subcutaneous injection of A549 and H460 cell suspensions (2 × 106 cells/100 μL) into the right rear flank of mice, respectively. The experiment was conducted when the tumor volume grew to 100 mm3. Tumor volume = 0.5 × A2 × B (A = width, B = length).
In vivo radiotherapy sensitization
To explore the effects of radiotherapy in vivo, A549 and H460 subcutaneous tumor bearing mice were randomly divided into the following four groups: (a) the control group (P-NC), (b) the NPs injection group, (c) the radiotherapy alone group (RT) and (d) the NPs combined with RT group (NPS + RT). Following intravenous injection of NPs (25 mg/kg/day), a magnet (magnetic field: 0.5 T) was fixed at the tumor site of the mice. After 24 h, radiotherapy (2 Gy/day) was performed for 5 consecutive days. Tumor volume (mm3) was measured every other day, and mouse body weight was recorded. Simultaneously, mice in the control group and the RT group were injected with 0.9% NaCl solution alone at the same time point. On the 30th day, the mice were euthanized, and the tumor tissues were removed for photographing and weight measurement.
Another group of mice were euthanized, and the tumor tissues from tumor bearing mice were removed on day 15 from the (a) control (P-NC), (b) NPs injection group, (c) radiotherapy alone group (RT) and (d) NPs combined with RT group (NPS + RT) for RT-qPCR, Western blotting, and immunohistochemical staining.
Immunohistochemistry and Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) staining
For immunohistochemical staining, after deproteinization, hydration, and antigen retrieval, tumor tissue sections were treated with endogenous peroxidase in 0.3% H2O2 for 10 min. Next, the sections were blocked with PBS containing 1.5% blocking serum, and then incubated with the primary antibodies rabbit polyclonal anti-DR5, rabbit polyclonal anti-BIRC5, or rabbit monoclonal anti-Ki67 overnight at 4 °C. Later, the sections were added with appropriate amounts of goat anti-rabbit or anti-mouse secondary antibody working solution (ZSGB-Bio, Guangzhou, China) for 1 h incubation at 37 °C. Color was developed using diaminobenzidine (DAB) (ZSGB-Bio) for 3–5 min. Afterwards, the sections were counterstained with hematoxylin, while sections without incubation with primary antibody were regarded as the NC. The results were observed microscopically and evaluated by quantifying the staining intensity and the percentage of stained tumor cells. Expressions were analyzed by two independent investigators blinded to clinical data using a multi-headed microscope.
TUNEL staining was performed according to the manufacturer’s instructions (Roche Applied Sciences, Germany) and previously established methods [15].
Evaluation of NP biocompatibility
To evaluate the NP biocompatibility, healthy BALB/c mice were injected with NPs (25 mg/kg/day), and changes in body weight were observed over 15 days. Another batch of healthy mice was taken, and blood samples were collected for liver and kidney function indexes and blood routine analysis on days 1, 7, and 15 after injection. Mice were euthanized on day 15. Major organs, including heart, lung, kidney, liver, and spleen, were excised, while tissues were fixed using formalin, embedded with paraffin, and then sectioned. Later, the Sections (4 μm) were stained with hematoxylin–eosin for histological examination.
Statistical analysis
Statistical analyses were performed using the SPSS21.0 software (IBM SPSS statistics, Armonk, NY, USA). Measurement data were expressed as mean ± standard deviation, and two group data were compared following an unpaired design with normal distribution and homogeneous variance, using unpaired t-test. Data comparisons among multiple groups were performed by one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. Survival was calculated using the Kaplan–Meier method. A value of p < 0.05 was regarded statistically significant.