Materials
Iron, red phosphorus, sulfur and iodine powders (99.99%), and NMP (N-Methyl-2-pyrrolidone) were bought from Aladdin Reagents. PLL-PEG, PLL-PEG-FA were purchased from Ruixi Co., Ltd (Xi’an, China). Anti-miR-NC, the negative control of microRNAs inhibitor, miR-19a inhibitor (anti-miR-19a), and Cy5.5-labeled anti-miR-19a were from RiboBio Co., Ltd (Guangzhou, China). FITC and Cy5.5 fluorescence dyes were obtained from Lumiprobe (Maryland, USA). The cell culture reagents including DMEM mediem and fetal bovine serum (FBS) and so on were from Gibco (AG, Switzerland). Beyotime (Shanghai, China) provided Calcein-AM, PI and DAPI staining solutions, and Cell Counting Kit-8 (CCK-8). The antibodies for immunostaining were from Cell Signaling Technology (Maryland, USA). Other chemical reagents at analytical reagent grade were directly used.
Synthesis Of Feps Crystals And Nanosheets
The FePS3 crystals were prepared using chemical vapor transport (CVT) technique. High-purity iron, red phosphorus and sulfur powders with the molar ratio of 1:1:3 (around 1.37 g in total), and the transport agent (iodine, 20 mg) were filled together in a quartz ampule with dimeter of 18 mm, length of 100 mm, wall thickness of 2 mm followed by seal under high vacuum (below 5 × 10− 4 Torr). Subsequently, the sealed quartz ampule was placed in a two-zone furnace and heated to 700°C for 5 days. Finally, the two-zone furnace was cooled to room temperature in 8 h, and the bulk FePS3 crystals were obtained.
The FePS NSs were synthesized from bulk FePS3 crystals by a liquid exfoliation strategy. Briefly, 100 mg of FePS3 crystals were fully ground and then exfoliated by probe sonication in 100 mL of NMP for 12 h in a bath at 6°C. After sonication, the precipitate between 9000–14000 rpm was collected to obtain FePS NSs. Before using, washing with ethanol and water three times each was carried out.
Functionalization Of Feps Nss
1 mg of PLL-PEG or PLL-PEG-FA was mixed with 200 µg of FePS NSs dispersed in 5 mL water, sonicated for 30 min followed by stirring for 3 h. The obtained FePS@PP and FePS@PPF were washed to remove the excess PLL-PEG and PLL-PEG-FA. Afterwards, 0.6 nmol of anti-miR-NC or anti-miR-19a was added to 100 µg of FePS@PPF in 2 mL water, and magnetically stirred for 4 h at room temperature. Ultimately, functionalized anti-miRNA/FePS@PPF was collected after washing and centrifugating.
To synthesize FITC-labeled FePS@PPF, 10 mg of FePS@PPF and 1.0 mg of FITC dye were dispersed in 10 mL of water, magnetically stirred for 4 h. Then the mixture was washed with water to remove unreacted FITC.
Characterizations
JEM-3200FS (JEOL, Japan) was used to take the transmission electron microscopy (TEM) images at 200 kV. Size distribution and zeta potential were determined using Zetasizer 3000 HAS (Malvern Ltd., UK). XRD (X-ray diffraction) analysis was performed by the SmartLab X-ray diffractometer (Rigaku, Japan). The UV-Vis-NIR absorption were carried out by U-3900 spectrophotometer (Hitachi, Japan). The concentration of FePS NSs was measured with ICP-OES (7000DV, PerkinElmer, USA). Fourier Transform infrared spectroscopy (FTIR) spectra were detected by MDTC-EQ-M13-01 (Thermo, USA).
Photothermal Effects
The FePS@PPF dispersed in water (0, 15, 25, 50 µg/mL) were exposed to 1064 nm laser for 10 min with a power density of 1.0 W/cm2. The temperature was monitored using the Ti27 infrared thermal imager (Fluke, USA). Additionally, 50 µg/mL of FePS@PPF solution was irradiated at 0.5, 1.0, and 1.5 W/cm2. The temperature changes during the rise and natural cooling processes were recorded.
Photothermal Conversion Efficiency Of Feps@ppf
The photothermal conversion efficiency (η) can be calculated by equations 1–4.
η = (hS(Tmax-Tsurr)-Qdis)/I(1–10− A) (1)
hS = ∑mCp/τS (2)
τS = - t/lnθ (3)
θ = (T - Tsurr)/(Tmax - Tsurr) (4)
where h is heat transfer coefficient, S is the area of container, τS is the time constant of system heat transfer, m is mass of 1 g, Cp (4.2 Jg–1 °C–1) is specific heat capacity of water, and τS = 263.77 s is obtained from Fig. 3f. hS is obtained from Eqs. 2 (hS = 1*4.2/263.77 = 15.92 mW/°C), Qdis is measured independently to be 74.84 mW, Tmax is the equilibrium temperature of FePS@PPF and Tsurr is the ambient temperature. I is 1.0 W/cm2 and A refers to absorbance of FePS@PPF at 1064 nm (A1064 = 0.568). Accordingly, η = {[15.92*(49.0-27.4)-74.84]/[1000*(1–10− 0.568)]}*100%= 47.1%.
Intracellular Uptake
5 × 104 cells per dish of the HOS cells were seeded and cultured in confocal dishes overnight. After incubation with 25 µg/mL of FITC-labeled FePS@PPF or Cy5.5-labeled anti-miR-19a/FePS@PPF for 6 hours, washing with PBS was carried out and fixing the cells with 4% PFA. The nuclei were stained by DAPI. The images were taken using confocal microscope (Leica stellaris 5, GER).
In vitro antitumor efficiency
HOS and MG63 cells were seeded with 1 × 104 cells per well in 96-well plates and cultured overnight. Different concentrations of FePS@PPFs (0, 6, 12, 25 and 50 µg/mL) were added and treated cells for 48 h. Afterwards, a CCK-8 assay was used to determine cell viabilities. For antitumor assay, the medium containing 25 µg/mL (FePS@PPF concentration) of anti-miR-NC/FePS@PPF or anti-miR-19a/FePS@PPF was used to treat cells for 6 h. Then, the samples were removed by adding fresh medium, and exposed to 1064 nm laser for 10 min, 1.0 W/cm2. Incubation for another 48 h was performed, and CCK-8 assay was conducted to analyze cell viability. In addition, cells were treated as described above and co-stained with Calcein-AM/PI (5 µg/mL) at 37 oC for 10 min. Subsequently, fluorescent images indicating live/dead cells were taken by IX71 (Olympus, Japan). The cell apoptosis after different treatments was measured by flow cytometry (BD FACSCelesta) using the Annexin V-FITC Apoptosis Kit.
Western Blot
Cells were lysed and the protein was extracted with radio immunoprecipitation lysis buffer on ice. BCA assay was used to determine the protein concentrations and Western blot was conducted using 12% SDS-PAGE. Bovine serum albumin was used to block the polyvinylidene fluoride membranes (0.45 mm) and then the membranes were incubated with different primary antibodies overnight at 4°C. After washing, the primary antibodies-coated membranes were conjugated with secondary antibody at room temperature for 1 h. Finally, membranes were detected by chemiluminescence imaging (Bio-Rad, Singapore) and the bands were normalized to beta-actin level.
Animals And Construction Of Xenograft Tumor Models
Balb/c nude mice, which were female and about 4–6 weeks old were purchased from a company of Laboratory Animal Technology (Charles River Co., Ltd., China) and raised in SPF laboratory. The animal experiments were approved by Administrative Committee of SIAT (Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences) responsible for supervising of animal research.
HOS cells (1×108/mL) were dispersed in PBS and the cell suspension (100 µL) was seeded into the right back side of mice. The formula: volume (V) = length × width2/2 was used to calculate the volume of tumor.
In vivo biodistribution and photothermal effects
Cy5.5-labeled FePS@PP or FePS@PPF was intravenously injected with 10 mg/kg FePS NSs for tumor-bearing mouse. The in vivo fluorescence images were acquired by Caliper IVIS Spectrum (PerkinElmer, USA) at designed time point (3, 6, 12, 24, 48 h). The ex vivo fluorescence of heart, liver, spleen, lung, kidney and tumor was detected at 24 h post-administration. All images were analyzed and calculated by Living Image software.
Mice were anaesthetized after 24 h-injection of PBS, FePS@PP or FePS@PPF, and the tumor sites were irradiated by 1064 nm laser for 5 min, 1.0 W/cm2. The temperature of tumor sites were monitored using an infrared thermal imager.
In vivo synergistic anti-tumor effects
When the tumor models were established, the mice were divided randomly into six groups with 5 mice in each group: (1) PBS (control), (2) anti-miR-NC/FePS@PPF, (3) anti-miR-19a/FePS@PPF, (4) PBS + NIR, (5) anti-miR-NC/FePS@PPF + NIR, (6) anti-miR-19a/FePS@PPF + NIR. On day 0, 100 µL of PBS, anti-miR-NC/FePS@PPF or anti-miR-19a/FePS@PPF was injected via the tail vein at the concentration of 10 mg/kg FePS NSs once a week. On day 1 and day 7, mice in group (4), (5) and (6) were anaesthetized and exposed to 1064 nm laser (1.0 W/cm2, 5 min). The tumor length, width and body weight were measured every 2 days. On day 14, mice were sacrificed, tumor as well as main organs (heart, liver, spleen, lung, kidney) were collected and fixed in 4% PFA for histopathological and immunohistochemical analyses.
In vivo clearance and biosafety evaluation
At 0, 1-, 3-, 7- and 14-days after intravenous injection of FePS@PPF (10 mg/kg), the main organs were removed, digested in HNO3 and analyzed by ICP-OES. The concentrations of elements Fe, P and S in the main organs were determined to assess the biodegradability of FePS@PPF.
On day 14, 0.6 mL of blood sample was collected from venous plexus of eye socket into heparinized tubes. The serum samples were obtained by centrifugation at 3000 rpm, 15 min, 4 ℃. The blood biochemistry assay which is related with liver and renal function was detected at Wuhan Servicebio Technology Co., Ltd. The H&E staining of sections were performed to observe the tissue morphology.
Statistical analysis
All quantitative results were presented as mean ± SD from three independent experiments. Statistical comparisons were analyzed by SPSS software (Chicago, USA) by Tukey’s post-test and one-way ANOVA analysis. *p < 0.05, **p < 0.01.