Materials
PLGA (50:50, inherent viscosity 0.20 dL/g, MW = 15,000) was obtained from the Shandong Institute of Medical Instruments (Jinan, Shandong Province, CHN). Poly(vinyl alcohol) (PVA, MW = 30,000 - 70,000, 78-90% hydrolysed) and ICG were purchased from Sigma-Aldrich (St Louis, MO, US). RPMI 1640 cell culture medium and foetal bovine serum (FBS) were obtained from Gibco (Thermo Fisher Scientific, Shanghai, CHN). The SDS-PAGE, DCFH-DA, and CCK-8 kits were obtained from Beyotime (Shanghai, CHN). The formaldehyde solution (4%), HE staining solution, and AO were purchased from Solarbio (Beijing, CHN). Deionized water was obtained from a Milli-Q water purification system.
Preparation of PINPs and PINPs@PM
The ICG-loaded PLGA nanoparticles (PINPs) were prepared using a reported water-in-oil-in-water double emulsion method with modifications [16, 40]. Briefly, PLGA (120 mg) was dissolved in 2.5 ml of methylene chloride. ICG solution (7.5 mg/ml, 200 μl) was added, and the mixture was emulsified by sonication (25% amplitude, 2 min) using a Digital Sonifier S-250D (Branson Ultrasonic, CT, US) in an ice bath. The primary emulsion (first emulsion) was poured into 10 ml of PVA solution (3%, w/v) and sonicated for another 3 min, obtaining a double emulsion (second emulsion). The obtained product was stirred to evaporate the organic solvent and was then centrifuged at 1,200 rpm for 12 min to remove the non-encapsulated ICG.
PM camouflage was performed as we recently described [4]. Mouse platelets were collected by gradient centrifugation, frozen at -80 °C and thawed at room temperature. After three freeze-thaw cycles, the membrane was obtained by centrifugation at 8,000 rpm for 10 min, washed with PBS containing protease inhibitor and sonicated for 5 min. PINPs (300 μg, based on the content of ICG) dissolved in PBS (1 ml) were added to equal volumes of PM. The mixture was then sonicated on ice for 2 min and maintained at 4 °C overnight. Large fragments of PM were removed using a filter membrane with a pore size of 400 nm. The PINPs@PM was collected by high-speed centrifugation at 10,000 rpm for 15 min. A BCA assay was employed to determine the efficiency of membrane coating. Animals were cared for and treated in accordance with the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals (NIH Publication No. 85e23 Rev. 1985) as approved by the Animal Experimental Ethics Committee of TMMU.
Characterization analysis
The morphologies of the PINPs and PINPs@PM were obtained by TEM (Tecnai G2 F20 U-TWIN, FEI, Hillsboro, OR, US). To confirm the PM camouflage, PINPs@PM was denatured and resolved via 10% SDS-PAGE. The protein bands were visualized by Coomassie blue staining. The DLS and zeta potential experiments were determined by a Nano-ZS (Malvern, Worcestershire, UK) at room temperature. The temperature alteration of PINPs@PM exposed to NIR at 808 nm for different times (1.0 W/cm2, Laserwave, Beijing, CHN) was recorded with a thermoelectric thermometer (HH806W, Omega, US).
Toxicological evaluation
HUVECs and 4T1 cells obtained from the Chinese Academy of Sciences (Shanghai, CHN) were cultured in RPMI 1640 medium containing 10% FBS. Ten thousand cells cultured to the logarithmic phase were incubated with different concentrations of PINPs@PM (0, 10, 20, 30, 40, and 50 mg/ml, based on the dose of ICG) for 24 h. The cell viability was determined by CCK-8 assay. Mouse erythrocytes diluted in saline solution (2%, v/v, 300 ml) were incubated with 1.2 ml of PINPs@PM at 37 °C for 2 h. The absorbance of the supernatant was determined at 405 nm. These experiments were conducted in triplicate and repeated twice. The toxicity of PINPs@PM in vivo was evaluated by a mouse experiment. Animals were cared for and treated as demonstrated in the preparation of PINPs@PM. PINPs@PM (60 μg, based on the content of ICG) was intravenously injected. The mice were sacrificed after 7 days, and their hearts, livers, spleens, lungs, and kidneys were obtained by surgery. The pathological changes were observed with an Olympus DX51 optical microscope (Tokyo, JPN) after HE staining.
Flow cytometry-based endocytic assay
4T1 cells were seeded in a 6-well plate at a density of 2×105 CFU per well and cultured overnight in RPMI 1640 medium containing 10% FBS. PINPs@PM (30 μg/ml, based on the content of ICG) was co-incubated with 4T1 cells for 6 and 12 h in the presence and absence of a 4-Gy X-ray irradiation pretreatment with an RS2000 X-ray irradiator (1.0 Gy/min, Rad Source, Suwanee, GA). The counts of fluorescent cells uptaking ICG-loaded PINPs@PM excited at 780 nm and the fluorescence intensity were determined with a BD flow cytometry system (Franklin Lakes, NJ, US), in which 15,000 events per sample were obtained. The experiment was repeated three times, and the data were processed using FlowJo software (version 7.6.1).
Cell cycle analysis
4T1 cells (2 × 105 CFU) were cultured overnight in a 6-well plate and irradiated with a 4-Gy X-ray. After further incubation in RPMI 1640 medium containing 10% FBS for 6 and 12 h, cells were harvested by trypsinization and fixed with 70% ethanol at 4 °C for 24 h. The cells were then stained with PI for 30 min after resuspension in RNase A buffer. The cell cycle was determined with a BD C6 flow cytometer. The data were processed using FlowJo software (version 7.6.1). This experiment was repeated three times on different days.
Western blot
4T1 cells irradiated with a 4-Gy X-ray and incubated for 6 and 12 h were collected and processed with RIPA lysis and extraction buffer (89900, Thermo Fisher Scientific). The cell extracts were denatured and resolved via 10% SDS-PAGE. A primary anti-Caveolin-1 rabbit monoclonal antibody (Abcam, ab32577, 1:200, Shanghai, CHN) and a goat anti-rabbit secondary antibody (Abcam, ab205718, 1:1000) were employed to detect Caveolin-1. b-actin determined by a mouse monoclonal antibody (AA128, Beyotime, 1:1000) was used as a reference. This assay was repeated three times on different days.
Antitumor cell experiment
4T1 cells seeded in a 96-well plate at a density of 1×104 CFU per well were cultured overnight and incubated with 30 μg/ml of PINPs@PM (based on the dose of ICG) in the presence and absence of a 4-Gy X-ray irradiation pretreatment. NIR (808 nm, 1.0 W/cm2, 10 min) was performed after 12 h of co-incubation. Cells were then washed with sterile PBS, stained with 100 μl of DCFH-DA (10 μM) and AO dyes (5 μg/ml) according to the manufacturer’s instructions and observed with an Olympus IX70 inverted fluorescence microscope. Otherwise, the cells were further incubated for 16 h followed by NIR treatment and the survival was detected by a CCK-8 assay. The experiment was conducted in duplicate and repeated three times.
Tumor targeting evaluation
Female 8-week-old BALB/c mice were subcutaneously injected with 2 × 109 CFU 4T1 cells in the right hind leg. Animals were cared for and treated as demonstrated in the preparation of PINPs@PM. A 4-Gy X-ray local irradiation was performed when the tumor reached approximately 75 mm3. PINPs@PM (60 μg, based on the content of ICG) was intravenously administered 6 h later. The accumulation of PINPs@PM in the tumor-bearing mice was observed with an IVIS spectrum in vivo imaging system (Perkin Elmer, Shanghai, CHN) at 12 and 24 h post-treatment. A Fortric 226s thermal imager (Shanghai, CHN) was used to obtain the infrared thermal images and record the temperature variations of the tumors along with the increasing NIR exposure times.
Antitumor assay in vivo
The tumor-bearing mice (n=5) were prepared and cared for as described in the tumor targeting evaluation. PINPs@PM (60 μg, based on the content of ICG) was given by tail vein injection in the presence and absence of a 4-Gy X-ray local irradiation. NIR treatment was continued for 10 min after 24 h. The tumor volumes were monitored for three weeks. The tumor tissues were obtained by surgery for weigh and TUNEL and HE staining.
Statistical analysis
The significant differences (P) were calculated by SPSS 17.0 using the Student’s two-tailed t test and LSD multiple-comparison test. A P value lower than 0.05 was defined as statistically significant.