Preparation of PLGA nanoparticles
PLGA@5Fu/PFC NPs were prepared by a solid-in-oil-in-water (s/o/w) dual emulsion solvent evaporation as described [18]. Briefly, 50 mg of PLGA was dissolved in 2 mL of chloroform containing PFCs (2 mg). 5 mg of 5Fu was dissolved in 0.5 mL of aqueous solvent and then mixed with PLGA solution to generate the s/o primary solution. The emulsion was dispersed in 10 mL of aqueous solvent containing 2% w/v PVA to generate the final s/o/w emulsion. Free PLGA/PVA polymers were separated by centrifugation at 3,000 rpm for 15 min. EGF was modified onto PLGA NPs by N′-ethylcarbodiimide hydrochloride (EDC) method [19].
Characterization of nanoparticles
The shape of EGF-PLGA@5Fu/PFC NPs was examined by transmission electron microscopy (TEM) (Hitachi, Tokyo, Japan). A drop of NPs solution (0.5 mg/mL) was mounted on a carbon-coated copper grid. The samples were observed at an acceleration voltage of 75 kV. Size distribution and zeta potential were determined by dynamic light scattering (DLS, Zetasizer Nano ZS, Malvern Instruments Ltd, UK).
Drug encapsulation and in vitro release
The encapsulation efficiency of 5Fu in NPs was examined by UV-Vis spectrophotometer (1800, Shimadzu, Kyoto, Japan). Briefly, 1 mg of nanoparticles was dispersed in 1 mL of distilled water for the extraction of 5Fu. The solution was shaken gently for 12 h at 37°C, the obtained filtrates were diluted (1:10) with methanol and measured at λmax = 266 nm. The encapsulation efficiency and loading efficiency were calculated by below equations:
[Please see the supplementary files section to access the equations.]
The release profile of 5Fu from NPs was accessed at different pH values (5.0 or 7.4). 10 mg of NPs were dispersed in 10 mL PBS, then transferred into dialysis bag and placed into 50 mL media with stirring at 37°C. At predetermined time points, 2 mL of release medium was taken out and the equal volume fresh medium was added. The amount of 5Fu released was detected by UV-Vis spectrophotometer.
Cell lines and culture
Human colorectal cancer cell line SW620 was obtained from Chinese Academy of Science (Shanghai, China). SW620 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells were routinely cultured in a humidified cell incubator with 5% CO2 at 37°C.
In vitro cell uptake
SW620 cells were cultured in 12-well plate at a density of 2 × 104 cells/well. After 24 h, fresh DMEM medium with Cy5-labeled NPs (EGF-PLGA@5Fu/PFC and PLGA@5Fu/PFC) were added and cultured at 37°C for 2 h, 4 h and 6 h. Cells were washed with PBS, fixed with 4% paraformaldehyde and deposited with DAPI. At last, cells were observed by confocal laser scanning microscope (CLSM).
Cell cytotoxicity
SW620 cells were cultured in 96-well plate at a density of 2 × 103 cells/well. After 24 h, different formulations of NPs were added into cells and cultured for 48 h at 37°C. Then, 10 μL of CCK-8 was added to each well and incubated for another 4 h. The optical density was measured at 450 nm by a microplate reader (BioRad, Hercules, CA, USA).
Hoechst 33342 staining
SW620 cells (1×104 cells/well) were seeded in 24-well plate, and treated with different formulations of NPs for 48 h. Then, cells were washed with PBS for three times and stained with Hoechst (2 μg/mL) for 20 min at room temperature. Stained cells were observed under a fluorescent microscope (Nikon TE2000; Nikon Corporation, Tokyo, Japan) (magnification, ×100).
Cell apoptosis
SW620 cells (1×105 cells/well) were cultured in 6-well plate with different formulations of NPs for 48 h. Then, cells were washed with PBS twice, suspended in staining buffer containing propidium iodide (PI) (1 μg/mL) and annexin V-FITC (0.025 μg/mL) for 15 min at room temperature. Apoptotic cells were evaluated by using FACScalibur flow cytometer (BD Bioscience, Franklin lakes, NJ).
Animal model establishment
Female BALB/c mice (6-8 weeks, 20-22 g) were obtained from the Animal Laboratory of Nanjing University and were kept in the standard conditions with humidity 50%-60%, temperature 25 ± 2°C, 12-h dark/light cycle and access to free water and food. All the animal experiments were carried out in line with the Guidelines for Care and Use of Laboratory Animals of the University of Science and Technology of China and approved by the Animal Ethics Committee of Nanjing Medical University.
SW620 cells (3×106) were resuspended in 100 μL PBS and injected into the right flank of mice. When the average tumor volume reached about 100 mm3, mice were assigned into 6 groups (n=8) and administrated with saline, blank NPs (100 mg/kg), 5Fu (8 mg/kg), PLGA@5Fu (8 mg/kg of 5Fu), PLGA@5Fu&PFC (8 mg/kg of 5Fu) and EGF-PLGA@5FU&PFC (8 mg/kg of 5Fu) every two days. The tumor size was measured and volume was calculated as follows: volume (cm3) = Length (L) × Width2 (W2)/2. At the end, mice were sacrificed by intraperitoneal injection of sodium pentobarbital (100 mg/kg), tumor and major organs were isolated for further experiments.
In vivo fluorescence imaging
After 24 h of injection, mice were sacrificed and the biodistribution of different formulations of drugs in the tumor and major organs was determined using a fluorescence imaging system.
Histological analysis
Tissues from the mice were fixed in 10% formalin, embedded in paraffin and cut into 5-μm sections. The sections were stained with haematoxylin and eosin (H&E) to evaluate the histological changes of tumor and major organs. For TUNEL assay, tumor tissues were stained with an in situ apoptosis detection kit (Thermo Fisher Scientific) according to the manufacturer’s directions. For immunohistochemical staining (IHC), tumor tissues were incubated with primary antibody against Ki-67 (ab15580, Abcam). Images for H&E and IHC were obtained by a light microscope, images for TdT-mediated dUTP nick end labeling (TUNEL) were acquired by a fluorescent microscope.
Immunofluorescence staining
At the end, mice were intravenous injected with pimonidazole at the dose of 60 mg/kg for hypoxia staining. After 90 min, tumors were collected, imbedded and cut into 8-μm section. Subsequently, the tumor tissue was incubated with the primary antibody against pimonidazole (1:200, Hpoxyprobe-1 Plus Kit, Hypoxyprobe, Burlington) at 4°C overnight, followed by incubation with Alexa Fluo® 488 conjugated goat-anti-mouse antibody (1:500, ab150113, Abcam) for 1 h at 37°C. The nuclei were counterstained with DAPI for 3 min. Finally, images were captured under fluorescence microscope (magnification, ×200).
Statistical analysis
All data was analyzed by GraphPad Prism 5.0 and presented as mean ± standard deviation. One way ANOVA analysis followed by Tukey’s post hoc test was used to compare the difference between multiple groups. A p<0.05 was considered statistically significant.