2.10 Cell culture
Human OS cells (MNNG/HOS) were obtained from Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The cells were routinely cultured and maintained in α-MEM (Hyclone) containing 10% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific), 1% antibiotics (penicillin and streptomycin). All cells were incubated at 37°C in a humidified incubator with 5% CO2.
2.11 Cellular uptake
The intracellular uptake and targeted cellular uptake ability of GNS@MSNs-FA were investigated by a fluorescence microscopy. In brief, MNNG/HOS cells were seeded in six-well plates at a density of 1×105 cells per well and grown overnight at 37°C. Afterward, the cells were incubated with ICG-loaded nanoparticles at 37°C in a humidified atmosphere of 5% CO2 for 4 h. Meanwhile, for competitive inhibition experiments, other two groups of cells were pre-incubated with 2 mM of FA at 37°C for 2 h before the GNS@MSNs-FA was added. After that, the cells were washed carefully with PBS three times and the nucleus were stained with Hoechst 33342 (10 µg mL− 1) for 10 min. Finally, the cells were carefully washed and observed under fluorescence microscopy (Olympus Corporation, Tokyo, Japan).
2.12 Cell viability assay
Cell counting kit-8 (CCK-8, Dojindo, Kyushu Island, Japan) assay was performed to assess cell viability according to the manufacturer’s protocol. Briefly, cells were seeded in a 96-well plate at a density of 5,000 cells per well and allowed to grow overnight. After the corresponding treatment, the cells were cultured at 37°C in a humidified atmosphere of 5% CO2 for 24 h. Afterwards, the culture medium was replaced with 100 µL medium containing 10% CCK-8 solution and incubated in dark at 37°C for 2 h. The absorbance of individual wells at 450 nm was measured by a microplate reader (Biotek, Winooski, VT, USA).
2.13 Detection of intracellular reactive oxygen species (ROS)
Intracellular ROS production was detected by using the ROS assay kit (Beyotime Company, Shanghai, China) according to the manufacturer’s instructions. Briefly, cells were seeded in a six-well plate at a density of 1×105 cells per well. After 24 h incubation under a humidified atmosphere of 5% CO2 at 37°C, the cells were divided into four groups according to the samples added: control group (PBS), free Ly (0.5 µg/mL), GNS@MSNs-FA/ group (100 µg/mL), and GNS@MSNs-FA/Ly group (100 µg/mL, equivalent Ly dosage of 0.5 ug/mL) was added to each well. After 4 h incubation, GNS@MSNs-FA group and GNS@MSNs-FA/Ly group were treated with 808 nm NIR laser for 5 min at 1.5 W cm− 2. After cultured for another 20 h, the cells were washed twice with PBS, incubated with DCFH-DA reagent (10 µM) in medium without FBS at 37°C for 30 min, and then washed with PBS three times. The fluorescence intensity of the cells was observed using a fluorescence microscope.
2.14 Cell apoptosis
In brief, the cells were seeded in a six-well plate (1×105 cells/well), and cultured at 37 ºC in a humidified 5% CO2 atmosphere. After culturing for 24h, the cells were treated according to the description aforementioned. At the end of incubation, all cells were trypsinized, harvested and washed twice with PBS. Then the cells were stained with Annexin V- fluorescein isothiocyanate (FITC) / propidium iodide (PI) dual staining (Nanjing Keygen Biotech, Nanjing, China) according to the manufacturer’s protocol. After incubation in the dark for 20 min at room temperature, the cells were examined by flow cytometry (Becton Dickinson, Franklin Lakes, New Jersey, USA).
Measurement of ATP levels
The intracellular ATP levels were assessed using an ATP determination kit (Beyotime, China). Briefly, after 24 h of treatment, the cells were harvested and lysed with lysis buffer on ice. After centrifugation for 5 min at 12000 g at 4°C, the supernatant was collected. After a standard curve was generated according to the manufacturer’s protocol, the ATP concentration of the sample was calculated.
Assessment of intracellular GSH
The intracellular GSH was measured with Ellman’s reagents . In Brief, the treated cells were lysed by repeated cycle of freezing and thawing. After centrifugation, the supernatants were collected for the measurement of GSH concentration based on a standard curve.
Determination of intracellular Ca 2+ ions
The calcium probe Fluo-3/AM (Dojindo Laboratories Co., Ltd., Kumamoto, Japan) was used to detect the changes of intracellular Ca2+ ions according to the manufacturer’s protocol. Briefly, cells were harvested after various treatment and then incubated with 5 µM Fluo-3/AM for 30 min at 37°C. Then, the cells were washed with PBS to remove excess Fluo-3/AM and the fluorescence intensity was detected by using a flow cytometer.
2.15 Western blotting analysis
Briefly, the treated cells lysed in RIPA buffer (Thermo Fisher Scientific) containing protease inhibitors and phosphatase inhibitors. Subsequently, the lysates were then centrifuged at 12,000 rpm for 15 min at 4°C and the supernatant was carefully collected. The protein concentrations were measured by using the BCA Protein Assay Kit (Beyotime Biotechnology Co. Ltd). Equal amounts of lysates (20 µg) were electrophoresed by sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA). The membranes were then blocked with 5% non-fat milk in Tris-buffered saline plus Tween-20 (TBST) buffer for 1 h at room temperature and incubated with the respective primary antibodies at 4°C overnight. After being washed three times with a TBST buffer, the membranes were incubated with horseradish peroxidase conjugated secondary antibody for 1 h at room temperature. Subsequently, the membranes were washed three times with TBST buffer and detected by using electrochemiluminescence detection reagent (EMD Millipore) according to the manufacturer’s instructions.
2.16 In vivo imaging and biodistribution analysis.
All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Tongji Medical College, Huazhong University of Science and Technology (IACUC Number: S2374). In brief, a volume of 200 µL MNNG/HOS cell suspension (density of 1×107 cells/mL) in cold PBS was subcutaneously injected into the right flank of nude mice.
When the volume of tumors reached 60–100 mm3, 150 µL GNS@MSNs-FA (2mg mL− 1) labelled with ICG (1mg mL− 1) was intravenously injected through the tail vein into the mice bearing the tumor. At the given time intervals (0 h, 1 h, 3 h, 6 h, 12 h and 24 h), in vivo tumor imaging was obtained by IVIS small animal imaging system (PerkinElmer Inc., Waltham, USA). After 24 h of observation, the mice were sacrificed and main organs (heart, lung, liver, spleen, and kidney) as well as tumors were harvested for ex vivo imaging to study nanoparticles tissue distribution.
2.17 In vivo antitumor efficacy and biosafety
When the tumor could be palpated subcutaneously (5 days after inoculation), the mice were randomly allocated into four groups: (1) PBS; (2) free Ly; (3) GNS@MSNs-FA + NIR; and (4) GNS@MSNs-FA/Ly + NIR (n = 3 per group). 100 µL free Ly (10 mg kg− 1) was injected intraperitoneally every three days. Other therapeutic agents were intravenously injected into the mice via the tail vein every three days. After 6 h of injection, the NIR laser treatment groups were irradiated with an 808 nm laser for 5 min (1.0 W cm− 2).
The body weights and tumor sizes were measured every 2 days to observe the growth of the tumors. At the end of the experiment (12 days after treatment), all mice were sacrificed, and tumor tissues as well as major organs (heart, liver, spleen, lung, and kidney) were collected and stained with hematoxylin and eosin (H&E) for histological observation and morphometric analysis. In addition, the tumors were sliced for immunofluorescence-stain with Ki67 antibody and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining according to the manufacturer's instruction. Images of sections were obtained using a light microscope. To further affirm the biocompatibility and biosafety of all treatment, the liver or kidney function was evaluated by determining the serum level of alanine aminotransferase (ALT) or urea nitrogen (BUN).
2.18 Statistical analysis
All data were expressed as mean ± standard deviation (SD) from at least three independent experiments under the same experimental conditions. Statistical analysis was conducted with Student’s t-test and one-way analysis of variance (ANOVA) by using GraphPad Prism version 6.01 for Windows. A P-value < 0.05 was considered to show statistically significant differences.