Materials
PLGA-COOH(Mw12000kDa)was purchased from Jinan Daigang Biomaterial Co., Ltd (Jinan, China). Perfluoropentane (PFP) and agarose were obtained from Sigma-Aldrich Co., Ltd(St. Louis, MO). Chlorin e6 (Ce6) was purchased from Melone Pharmaceutical Co., Ltd (Dalian, China). Cell Counting Kit-8 (CCK-8) cytotoxicity assay kit was obtained from Dojindo Molecular Technologies (Tokyo, Japan). 2',7'-Dichlorodihydrofluorescein diacetate (H2DCFDA) and Calcein acetoxymethyl ester (Calcein-AM) were purchased from MedChemExpress Co., Ltd (NJ, USA). Propidium Iodide (PI) was obtained from Solarbio Science & Technology Co. Ltd (Beijing, China). Annexin V-FITC/PI were obtained from BD Biosciences (USA). Docetaxel (DTX) was purchased from MedChemExpress Co., Ltd (NJ, USA). All other reagents were analytical pure products without further purifications. Roswell Park Memorial Institute 1640 medium (DMEM), fetal bovine serum and tyrisin were purchased from Gibco (ThermoFisher Scientific, USA).
Synthesis of CPDP NPs
Ce6-PFP-DTX/PLGA nanoparticles (CPDP NPs) were prepared by a W/O/W double emulsion method according to the previous report [24]. Briefly, 2 mg Ce6 was firstly dissolved in 500 μl methanol. Next, 50 mg PLGA-COOH was dissolved in 4 ml dichloromethane and then the previous solution was simultaneously added into it. Then, 200 μl PFP and Docetaxel (2 mg) were added to the above solution. In consequence, the mixture was triggered by an ultrasonic probe (Sonics & Materials Inc., USA) to gain a first emulsion (5 s on and 5 s off, 3 min). To acquire the second emulsion, 8 ml of a poly (vinyl alcohol) (PVA) solution (w/v=4%) was added into the above emulsion, using the same ultrasonic probe for 2 min. After adding 10 ml 2% isopropyl alcohol into the final emulsion, the solution was mixed under room temperature mechanically for at least 4 h to make dichloromethane volatilized totally. Finally, CPDP NPs were centrifugated for three times (12000 rpm, 5 min) and then collected and stored at 4 ℃ for further use. The PDP NPs were prepared in the same way except for Ce6. All experimental processes were operated above ice and conducted strictly in the dark.
Characterization of CDPD NPs
The particle size and zeta potential of CPDP NPs and PDP NPs were realized by Malvern Zetasizer Nano instrument (Malvern, UK). The morphology was characterized by transmission electron microscopy (TEM) and optical microscopy. To evaluate the stability, CPDP NPs were dissolved in phosphate-buffered solution (PBS) and measured the sizes of 7 days respectively. Each sample was triplicate measured. The encapsulation efficiency of CPDP NPs were calculated through the following formula:
Encapsulation efficiency (%) = (Weight of loading DTX or Ce6/Weight of total DTX or Ce6) x 100%
Drug releasing rate of CPDP NPs
To evaluate the drug-releasing ability of Ce6 and DTX in CPDP NPs, two solutions with different pH (phosphate buffer solution, PBS: 7.4, acetate buffer solution, ABS: 5.6) were utilized to test the cumulative releasing efficiency. Briefly, CPDP NPs was firstly dispersed with 1ml PBS or ABS after the mixture was sealed into a dialysis bag (Mw:10000), the whole solution was then transferred into a glass bottle (total volume:150 ml) in which 149ml PBS or ABS was added to keep the total solution volume at 150ml. The glass bottle was then placed into a 37 ℃ constant temperature shaker, and at different periods (0.5, 1, 2, 4, 8, 12, 24, 48, 72 h), the solution was collected and immediately supplemented with the same volume of medium. Each group was repeated three times. Finally, the concentrations of Ce6 and DTX were measured by Synergy Hybrid Multi-Mode Read (BioTek, USA) at 403 and 229 nm respectively, and the drug-releasing rate at each time point was calculated.
In vitro ultrasound imaging
To investigate the ultrasonic capability of CPDP NPs, the emulsion (1 mg/ml) was firstly triggered by a low intensity focused ultrasound (LIFU) transducer equipment (Ronghai Ultrasonic Medical Engineering Research Center, Chongqing, China), and the conducting pattern was set as 50% duty cycle, 1 s pulse duration under different intensities (1-2 W/cm2) for different duration time. For ultrasound imaging, irradiated CPDP NPs were added into the previous prepared agarose model respectively, using Philips EPIQ5 ultrasound diagnostic instrument (probe frequency:12Mhz, MI:0.06) to observe both 2D and CEUS imaging of CPDP NPs. Meanwhile, ImageJ software was applied to analyze the gray scale value of each group.
Cell culture and in vitro ROS generation of CPDP NPs by LIFU irradiation
The murine breast cancer cell line 4T1 was obtained from Shanghai cell bank of the Chinese Academy of Sciences (Shanghai, China) and incubated in RPMI 1640 medium mixed with 10% FBS and 1% streptomycin/penicillin at 37 ℃ in a 5% CO2 humidified incubator.
4T1 cells were incubated as previous condition at a density of 1x104 cells per dish and separated into the following 5 groups: Control, CPDP NPs, LIFU, Ce6 + LIFU, CPDP NPs + LIFU. After 24 h conventional culture, the medium was replaced by CPDP NPs (200 μl, 0.8 mg/ml) or Ce6 solution (200 μl) respectively and the cells were co-incubated for another 3 h. Then LIFU irradiation (2 W/cm2, 120 s) was conducted respectively according to different groups. After co-incubation and LIFU treatment, 100 μl diluted DCFH-DA solution was added and each group was cultured in the previous incubator for 15 min. A confocal laser scanning microscope (CLSM) was used to confirm the result of reactive oxygen species production and the corresponding fluorescence intensities were measured by ImageJ software.
In vitro cytotoxicity and concerted treatment capability of CPDP NPs
A CCK-8 assay was applied to assess the cytotoxicity of CPDP NPs. Briefly, 4T1 murine breast cancer cells were incubated in a 96-well plate, with a density of 1x104 per well for 24 h. Then CPDP NPs were diluted with serum-free RPMI 1640 medium at various concentrations (0,0.2,0.4,0.6,0.8 mg/ml, n=3), with or without LIFU irradiation (2 W/cm2, 120 s). After another 6 h of cocultured process, the cell viability of 4T1 cells was performed.
To evaluate cell apoptosis efficiency of concerted treatment, 4T1 cells were cocultured as previous for 24h and then separated into five following groups: (1) Control (without any treatments), (2) LIFU (only with LIFU exposure at 2 W/cm2), (3) CPDP NPs (only with CPDP NPs solution at 0.8 mg/ml), (4) PDP NPs + LIFU and (5) CPDP NPs + LIFU. After various nanoparticles coincubation (200 μl) and LIFU exposure, each group was treated with Annexin V (5 μl) and Propidium Iodide (5 μl) double staining for 20 min and analyzed through a flow cytometry protocol.
In vitro inhibition of cell metastasis
To investigate the inhibition of cell metastatic ability, wound healing assay and transwell assay were designed. For wound healing assay, 4T1 cells were conventional cultured as previous in the 6-well plate. After cell growth to an 80% confluency, a pipette tip (10 μl) was applied to conduct a manmade scratch along the center of the 6-well plate. Then cells were treated in the same groups mentioned above. After a continuously coincubation for 24 hours, the cells were washed with PBS 3 times and observed under optical microscopy (Olympus, Japan).
For transwell assay, the top compartment of the transwell chamber (Corning, San Diego, USA) was primarily applied to imitate the extracellular matrix in vivo. 4T1 cells at a density of 1x105 cells per well were seeded into the upper chamber in a serum-free RPMI 1640 medium, while the bottom compartment was filled with a complete culture medium mixed with 10% FBS. Then cells were separated as the same as above groups and treated for 24 h respectively. After that, cells in the bottom surface were fixed with paraformaldehyde and stained with crystal violet. The results were observed with light microscopy (Olympus, Japan).
In vivo synergistic therapeutic efficiency of CPDP NPs
Healthy female BALB/c mice (4 weeks) and Kunming mice (4 weeks) were obtained from Ningxia Medical University Laboratory Animal Center. All animal experiments were conducted under the guideline approved by the Animal Welfare Ethics Review Committee of Ningxia Medical University. To establish the mice tumor-bearing model, BALB/c mice were inoculated with 4T1 breast cancer cells (1x107/ ml) at the right flank. The size of the tumor was recorded every two days, and the volume of the tumor was calculated by the formula as: Volume=1/2 x Length x Width2. Tumor size and mice body weight were recorded every 2 days while the pictures of tumor growth were recorded every 3 days. When tumor volume reached 60-80 mm3, mice with similar tumor size were randomly divided into the same 5 groups: Control, LIFU, CPDP NPs, PDP + LIFU, and CPDP NPs + LIFU (n=3). Each group was intravenously injected with various NPs (200 μl) via tail vein except for Control group (200 μl PBS instead). Twenty-four hours later, the tumor site was exposed with LIFU irradiation (2 W/cm2) for 120s. The whole SDT administration was repeated every 3 days and lasted for 18 days. The body weight and tumor volume of the mice were measured and calculated. After the treatment, mice were sacrificed and tumor tissues were sent to H&E, TUNEL, and PCNA for further histological analysis.
In vivo ultrasound imaging
Tumor-bearing BALB/c mice were injected with CPDP NPs (200 μl, 1 mg/ml) through the tail vein intravenously. After 24 hours, tumor sites of the mice were conducted with LIFU (2 W/cm2, 120 s), then the 2D and CEUS imaging were acquired through the Philips EPIQ5 ultrasound diagnostic instrument mentioned previously. The gray scale analysis was measured by ImageJ software.
Biosafety of CPDP NPs in vivo
To investigate the biosafety of CPDP nanoparticles in vivo, healthy female Kunming mice (n=3) were separated into the following 4 groups: Control, 5 mg/ml, 10 mg/ml, and 20 mg/ml. The CPDP NPs (200 μl) were injected through the mice tail vein, then the mice were free access to food and water without any further administration. The bodyweight of mice was measured every 2 days. After 30 days, mice were sacrificed and the blood samples were collected for blood cell and biochemistry analysis. The major organs (heart, liver, spleen, lung, and kidney) were collected and investigated for H&E staining respectively.
In vivo inhibition of lung metastasis
To evaluate the lung metastasis inhibition of each group, the whole process was conducted by observing the number of metastatic nodules in the lung as well as an H&E staining histology assessment. After all the mice were euthanized, the lung tissues were removed and fixed, then photographs of cancer nodules were taken and lung tissues were further analyzed with H&E staining.
Statistical analysis
Measurement data were all performed 3 times and expressed as mean ± standard deviation (SD) and analyzed by one-way ANOVA analysis or a standard Student’s t-test through the SPSS software (version:19.0), while p value < 0.05 was considered to be statistically significant.