2.1 Cell Culture
4T1 murine breast cancer cells were preserved in our laboratory (Hubei Province Key Laboratory of Molecular Imaging) and propagated in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco, Gaithersburg MD, USA) supplemented with 10% v/v fetal bovine serum (Gibco, USA) and 1% v/v penicillin–streptomycin solution (Solarbio, Beijing, China). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2.
2.2 Isolation of GEV
GEV were isolated using sequential ultracentrifugation of fresh commercial goat milk. First, goat milk was ultracentrifuged at 8,000 × g for 30 min at 4°C to remove lipid and cell debris. Residual lipid and dead cells in the skim milk supernatant were removed using ultracentrifugation at 13,000 × g for 60 min. Next, the casein was removed from the supernatant by adding 0.025 g/L chymosin and incubating at 37°C for 4–6 h. The resulting whey was filtered through a 0.45 µm membrane to remove excess cell debris. Finally, the preliminary GEV products were centrifuged at 120,000 × g for 90 min, and the resulting GEV in the pellet were resuspended in phosphate buffered saline (PBS; Gibco, USA), passed through a 0.22 µm membrane, and quantified according to their surface protein content using a BCA Protein Assay Kit (Beyotime, Shanghai, China).
2.3 Synthesis of Ce6@GEV
When used as a carrier of the hydrophobic drug curcumin, EVs improved its solubility, stability, and bioavailability[26]. In a similar way, the Ce6 photosensitizer was loaded into GEV by co-incubation to obtain Ce6@GEV. In brief, Ce6 (5 mg/mL in DMSO) was mixed with GEV (2.12 mg/mL final concentration) in a 1:1 volume ratio. Then, the mixture was stirred at 2500 rpm overnight in the dark at 37°C. Next, free Ce6 molecules were removed using ultrafiltration (50 mL, 100 kDa, Millipore, USA). The Ce6@GEV, obtained as a green pellet, were dispersed in PBS.
2.4 Characterization of GEV and Ce6@GEV
The morphologies of GEV and Ce6@GEV were characterized using transmission electron microscopy (JEM-1400Plus, Japan). Hydrodynamic diameters and zeta potentials of GEV and Ce6@GEV were measured using dynamic light scattering (Brookhaven Instruments, USA) at room temperature and continuously monitored for 7 d to assess their stability in vitro. Representative cell surface markers (Tsg101, CD63, and CD9) of GEV and Ce6@GEV were analyzed using western blotting. To measure Ce6 encapsulation efficiency, a standard curve was constructed from the UV-vis-NIR spectra collected using a microplate reader (Bio-Rad, USA). Cell Counting Kit-8 (CCK-8) (HY-K0301, MCE, USA) was used to assess the cytotoxicity of different GEV and Ce6@GEV concentrations to 4T1 cells.
2.5 In vitro cell uptake and penetration of Ce6@GEV
Confocal dishes were each seeded with 2 × 105 4T1 cells and incubated at 37°C for 36 h. Then, 500 µL serum-free medium was added to each dish, followed by 0.22 µm-filtered Ce6@GEV (10 µL) or Ce6 (3 µL) to yield a final concentration of 1 µg/mL Ce6. After 4 h incubation, the treated culture medium was aspirated, then the cells were washed twice with PBS (pH 7.4) and fixed with paraformaldehyde. In addition, the cytoskeleton was stained with FITC-phalloidin (Solarbio, Beijing, China), and the nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI; Boster, Wuhan, China). Finally, the tumor cells were observed using a fluorescence confocal microscope (LSM 880, ZEISS, Germany) or analyzed using flow cytometry (FACSCalibur, BD, USA).
2.6 CL-induced PDT effect in cells
2.6.1 Cell viability experiment
4T1 cells were seeded in 96-well plates (5 × 103 cells/well) and then incubated under normoxic conditions (37°C, 5% CO2) for 24 h to allow them to adhere. 18F-FDG, GEV, Ce6, or Ce6@GEV were added to the medium and incubated for another 12 h. In addition, to evaluate 18F-FDG-induced PDT effect, a combination of Ce6 or Ce6@GEV (0, 0.1, 0.2, 0.5, 1, 2, 4, 10, 20, or 50 µg/mL) and 18F-FDG (0, 0.185, 0.37, 0.74, 1.48, 2.96, 3.7, or 7.4 MBq/well) was added to the culture medium. After 12 h incubation, the treated medium was removed and the cells were washed twice with PBS, then 10 µL CCK-8 working solution (HY-K0301, MCE, USA) and 100 µL serum-free medium were added to each well. After 1 h incubation, the 450 nm absorbance of each well was measured using a microplate reader (Bio-Rad, USA).
2.6.2 Intracellular ROS detection
Briefly, confocal dishes were each seeded with 2 × 105 4T1 cells and incubated for 36 h. Next, 1000 µL serum-free medium was added to each dish, followed by Ce6@GEV (20 µL) or Ce6 (6 µL) to yield a final concentration of 1 µg/mL Ce6. After 6 h incubation, the treated medium was aspirated and the cells were washed twice with PBS (pH 7.4), then 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA; Yeasen, Shanghai, China) was added to each dish and the cells were incubated for 30 min at 37°C. To stimulate intracellular accumulation of Ce6, 18F-FDG (3.7 MBq) was added to each dish. After two half-lives, the cell nuclei were stained with DAPI. Finally, antifade mounting medium (Beyotime, Shanghai, China) was added to prevent fluorescence quenching, and a fluorescence confocal microscope was used to detect the fluorescence of dichlorofluorescein (DCF, Ex/Em = 488/525 nm), Ce6 (Ex/Em = 405/680 nm), and DAPI (Ex/Em = 364/454 nm). The above experiments were carried out under dark conditions.
2.6.3 Cell apoptosis assay
The effects of each treatment were examined using an apoptosis kit (Calcein-AM/PI; Meilunbio, Dalian, China). 4T1 cells were inoculated into 6-well plates (2 × l04 cells/well), incubated overnight, then washed twice with PBS. The wells were then supplemented with untreated culture medium or medium containing 2 µg/mL Ce6@GEV or Ce6. After 6 h incubation, 2.96 MBq 18F-FDG was added to each well, and after two half-lives the cells were digested and stained with 200 µL PBS containing 2 µM calcein-AM and 8 µM propidium iodide for flow cytometry analysis (FACSCalibur, BD, USA). The apoptosis rate was calculated as the percentage of cells undergoing early or late apoptosis.
2.7 Imaging Cerenkov radiation and CRET In Vitro
To detect CRET, we used the small-animal In Vivo Imaging System (IVIS; PerkinElmer Inc. USA) to capture 18F emission in response to different treatments (PBS, GEV, Ce6, 18F-FDG, 18F-FDG + Ce6, or 18F-FDG + Ce6@GEV). To image the radioactivity-dependence of 18F-FDG emissions, CL was measured in response to 0, 0.037, 0.37, 1.85, 3.7, and 7.4 MBq 18F-FDG, using open, 520, 620, 670, and 710 nm filters. To image the concentration-dependence of Ce6@GEV emissions, 1 mg/mL Ce6@GEV stock solution was diluted in 100 µL PBS in a black 96-well plate to achieve final Ce6 concentrations of 0, 10, 25, 50, 100, and 200 µg/mL. 18F-FDG was added (1.48 MBq/well) at the time of CL imaging, yielding a final volume of 150 µL. Images were captured using open, 520, 570 620, 670, and 710 nm emission filters, a binning parameter of 8, and a 60 s exposure. CRET ratios were calculated by manually delineating the average radiance (photons/s/cm2/sr) of the regions of interest as follows[27]:
$$CRETx=\frac{Cerenkov+{Fluorophore}_{x}}{Cerenkov+{Fluorophore}_{y}}-\frac{{Cerenkov}_{x}}{{Cerenkov}_{y}}\text{ }\text{ }\text{(1)}$$
where Cerenkov + Fluorophorex and Cerenkov + Fluorophorey are the radiances detected within spectral windows x and y centered on the fluorophore and Cerenkov radiation emission wavelengths, respectively, and Cerenkovx and Cerenkovy are the corresponding radiances in the absence of a fluorophore. In the case of 18F-FDG and Ce6@GEV, Eq. 1 becomes:
$${CRET}_{>620}={\left[\frac{Cerenkov+{Ce6@GEV}_{>620}}{Cerenkov+{Ce6@GEV}_{<520}}\right]}_{ave}- {\left[\frac{{Cerenkov}_{>620}}{{Cerenkov}_{<520}}\right]}_{ave} \left(2\right)$$
where ave denotes the average value.
2.8 Tumor-bearing mouse models
All animal experiment protocols were reviewed and approved by the Animal Care and Use Committee of Tongji Medical College, Huazhong University of Science and Technology. 4T1 cells (5 × 106) suspended in 100 µL PBS were subcutaneously injected into the right front limb of BALB/c mice (female, 4–5 weeks old, Beijing Vital River Laboratory Animal Technology Co., Ltd, China) fed in a pathogen-free environment. Imaging studies were performed when the tumors reached approximately 5 mm in diameter, and treatment experiments started when tumor volumes reached 60–80 mm3.
2.9 Fluorescent imaging in vivo
When the tumor diameters reached 5 mm, the tumor-bearing mice were injected with 25 mg/kg Ce6@GEV or Ce6 in a volume of 100 µL via the tail vein. In vivo fluorescence imaging was conducted 1, 2, 4, 6, 8, 16, 24, and 48 h after injection, using the IVIS equipped with a fluorescence filter set (Ex/Em = 680/790 nm). Tumors and organs of interest (muscle, large intestine, small intestine, kidneys, spleen, liver, and stomach) were resected and imaged 24 h later.
2.10 Positron emission tomography (PET)/computed tomography (CT) and CL imaging in vivo
Mice were anesthetized using 2% isoflurane 0.5, 1, and 2 h after injection of 18F-FDG, and images were acquired using a micro-PET/CT scanner (Novel Medical, InliView-3000B, China). For CL imaging, 6 h after Ce6@GEV administration, 18F-FDG (5.55 MBq) was injected and CL imaging (binning parameter, 16; exposure, 300 s) was performed 0.5, 1, and 2 h later using the IVIS without a fluorescence filter set. The control group was not injected with Ce6@GEV before 18F-FDG injection (n = 3 per group).
2.11 CL-induced PDT in animals
Six groups of tumor-bearing mice were randomly divided (n = 5 per group) for various treatments (Ce6@GEV + 18F-FDG, Ce6 + 18F-FDG, 18F-FDG, Ce6@GEV, Ce6, or PBS). Mice were intravenously injected with 25 mg/kg Ce6@GEV or Ce6. Twelve hours later, 37–40.7 MBq of 18F-FDG (100 µL) was intravenously injected to trigger CL-induced PDT. The night before the experiment, the mice were fasted to reduce the physiological uptake of 18F-FDG in the gastrointestinal tract. Tumor sizes were measured every 2 d using a caliper, and tumor volumes were calculated using the formula (tumor length) × (tumor width)2/2. The survival rate was assessed using the Kaplan–Meier method. The following endpoints were used: tumor volume > 1500 mm3; ulcerated tumor tissue; mortality; weight loss > 15%. After 7 d or 20 d of treatment, the tumors from each group were paraffin sectioned for hematoxylin and eosin (H&E) staining and immunohistochemistry staining.
2.12 In vivo toxicity evaluation
Experiment groups and treatment methods were as described above (n = 5 per group). The general state of the mice was monitored daily, and they were weighed every other day. On days 7 and 20 of treatment, blood samples were collected for hematological analysis. A blood biochemical analyzer (AU-480, Beckman, USA) was used to evaluate markers of liver and kidney function, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), blood urea nitrogen (BUN), and creatinine (CRE). On day 20 of treatment, organs of interest (heart, liver, spleen, lungs, and kidneys) were collected for H&E staining and then examined using an optical microscope (IX73, Olympus, Japan).