Reagents
Foetal bovine serum (FBS), Dulbecco's modified Eagle's medium (DMEM), and trypsin were purchased from BI, the Netherlands. 1,2-Dipalmitoyl-glycero-3-phosphate (DPPA), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (DPPE) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were purchased from Corden Pharma, Switzerland. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-PEG-2000 (DSPE-PEG2000) was purchased from NANOCS, USA. CCK-8 reagents and the TUNEL kit were purchased from Shanghai Biyuntian Biotechnology Co., Ltd. Paclitaxel was procured from Beijing Suolaibao Co., Ltd. Indocyanine Green was purchased from Sigma, USA. Perfluoropropane was purchased from Tianjin Institute of Physics and Chemistry. The aspartate aminotransferase test kit, phenylalanine aminotransferase test kit, serum creatinine test kit and blood urea nitrogen test kit were procured from Jiancheng Bioengineering Institute, Nanjing, China. Male nude mice were purchased from Beijing Huakangfu Biotechnology Co., Ltd.
Cell culture and establishment of xenograft models
The human prostate cancer cell line PC-3 was kindly provided by the Stem Cell Bank, Chinese Academy of Sciences. Cells were cultured in DMEM containing 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin at 37 °C with 5% CO2. Cells in the exponential growth phase were digested with 0.25% trypsin and passaged or used for experiments. A 200 µL suspension of 5×106/mL PC-3 cells was injected into the dorsal side of 4 to 5-week-old male nude mice. Animal experiments were performed when the tumour volume reached 100 mm3 (tumour volume = a × b2 / 2 where a is the long diameter and b is the short diameter). All animal experimental protocols were approved by the Animal Ethics Committee of the Third Military Medical University.
Preparation and characterization of ICG-PTX NBs
According to the ratio 3:3:3:1:1, a total of 11 mg of DPPC, DPPE, DPPG, DPPA, and DSPE-PEG2000 as well as 2 mg of paclitaxel and 500 µg of ICG were dissolved in 1000 µL of PBS/glycerol solution and heated for 20 min at 45 °C. The contents were transferred to another vial, the air in the vial was replaced using perfluoropropane, and the vial was then shaken in an ST amalgam capsule blender (AT&M, China) for 90 s and allowed to stand overnight at 4 °C. All the liquid in the vial was then transferred to a 10 mL centrifuge tube to be centrifuged at 300 rpm for 3 min, and the intermediate emulsion was collected into an Eppendorf tube to finish the preparation of ICG-PTX NBs. Another 11 mg of lipids was used to prepare blank nanobubbles following the above steps (Blank NBs).
The concentrations of ICG-PTX NBs and Blank NBs were calculated using a haemocytometer. The particle size, particle size distribution, and surface zeta potential were measured with a Zetasizer nano ZS90 particle size detector (Malvern, UK). Each measurement was repeated three times. The shape, size and distribution of the nanobubbles were observed by optical microscopy (Olympus, Japan) and transmission electron microscopy (JEOL, Japan). The nanobubbles were stored at 4 °C, and changes in the particle size and fluorescence intensity of ICG-PTX NBs were measured on days 0, 1, 3, 5, and 7 to analyse the stability of the ICG-PTX NBs. An ultraviolet-visible spectrophotometer (Thermo Fisher, USA) was used to obtain the UV absorption spectra of ICG-PTX NBs, Blank NBs, ICG and double distilled water. The standard curves of ICG and PTX were plotted, and the encapsulation efficiency (EE) and drug loading efficiency (LE) of ICG and PTX in ICG-PTX NBs were calculated. Calculation of EE and LE:
EE = (amount of ICG encapsulated in nanobubbles/total amount of ICG added) × 100%
LE = (amount of ICG encapsulated in nanobubbles/the total amount of lipids used for the preparation of the nanobubbles) × 100%
Haemolytic action of ICG-PTX NBs
After the microhematocrit blood tube was inserted into the inner corner of the eye, fresh blood was collected in an anticoagulant tube, and transferred to a 10 mL centrifuge tube. A 3× volume of PBS was added, and the blood samples were centrifuged at 2000 rpm for 10 min to remove the supernatant. This washing was repeated 3 times until the supernatant was colourless and transparent. An appropriate amount of red blood cells (RBCs) and PBS were used to prepare a 2% RBC suspension followed by incubation with 1.0×108/mL ICG-PTX NBs and Blank NBs for 1 h. Double distilled water was used as a positive control, and PBS was used as a negative control. The absorbance at 545 nm was measured by an ultraviolet-visible spectrophotometer, and the haemolysis rate was calculated using the following formula:
[(sample absorbance value - absorbance value of the negative control group)/(absorbance value of the positive control group - absorbance value of the negative control group)] × 100%
In vitro ultrasound, photoacoustic and fluorescence imaging using ICG-PTX NBs
Different concentrations of ICG-PTX NBs were placed in a cavity model made of 1% agarose gel (1.0×108/mL, 5.0×107/mL, 1.0×107/mL, 5.0×106/mL, and 1.0×106/mL), and the corresponding ultrasound images in B-mode were acquired using a Vevo 2100 small animal ultrasound imaging system (VisualSonics, Canada) (centre frequency 18 MHz, gain 40 dB). Then, 1.0×108/mL ICG-PTX NBs were mechanically blasted using ultrasonic waves of high mechanical index, and the ultrasonic images before and after blasting were analysed. The DFY-type diagnostic instrument for the quantitative analysis of ultrasonic images (Chongqing Institute of Ultrasound Molecular Imaging, China) was used to quantitatively assess the images and calculate the grey value for statistical analysis. The imaging parameters of the Vevo LAZR photoacoustic imager (VisualSonics, Canada) were set (centre frequency 21 MHz, gain 40 dB), and the optimal photoacoustic excitation wavelength for ICG-PTX NBs was measured by full wavelength scanning. Different concentrations of ICG-PTX NBs (1.0×108/mL, 5.0×107/mL, 1.0×107/mL, 5.0×106/mL, and 1.0×106/mL ) were placed in a cavity model made from 1% agarose gel for photoacoustic imaging at a wavelength of 825 nm, and a Vevo LAZR photoacoustic imager was used to collect photoacoustic images and quantitatively analyse the photoacoustic signals. ICG-PTX NBs (1.0×108/mL) and ICG solution (0.35 mg/mL) with equal concentrations of ICG and double distilled water were added to Eppendorf tubes and then placed in the IVIS Spectrum living animal imaging system (PerkinElmer, USA). Under irradiation conditions at an excitation wavelength of 740 nm and emission wavelength of 820 nm, the Eppendorf tubes were scanned, and their fluorescence intensities were quantified.
In vivo ultrasound, photoacoustic and fluorescence imaging using ICG-PTX NBs
When the volume of the subcutaneous tumour reached approximately 1 cm3, tumour blood flow was observed using ultrasound examination, and tumours with rich blood flow were selected. After anaesthetizing with isoflurane, the nude mice were fixed in the prone position and scanned using a Vevo 2100 small animal ultrasound imaging system and an MS250 high frequency probe. The sections with the optimal imaging effect were selected, and the optimal values of imaging parameters were determined (centre frequency of the probe 18 MHz, gain 40 dB). After injecting 200 µL of ICG-PTX NBs (1.0×108/mL) through the orbital vein, the ultrasound imager was used to continuously acquire images at different time points after the injection of the contrast agent, and dedicated software (Vevo 2100 onboard software, VisualSonics, Canada) was used to analyse the time-intensity curve. After the contrast-enhanced echo had subsided, the “burst” button was used to blast the residual contrast agent. Blank NBs (200 µL, 1.0×108/mL) were injected in the same way after the echo had completely subsided.
Chloral hydrate (4%, 0.20 mL/20 g) was intraperitoneally injected into nude mice as anaesthesia. The nude mice were fixed in the prone position, and the values of imaging parameters were adjusted (laser wavelength 825 nm, central frequency of the probe 21 MHz, gain 40 dB). The Vevo LAZR photoacoustic imager was used to collect photoacoustic images of the tumour before injection of the contrast agent. A total of 200 µL of ICG-PTX NBs (1.0×108/mL) and ICG solution (0.35 mg/mL) of equal concentration were injected into the orbital vein. The photoacoustic imager was used to observe and store the dynamic images. Quantitative analysis of the photoacoustic signal in the region of interest was performed, and the time-photoacoustic signal intensity curve was plotted.
After anaesthetizing with isoflurane, the nude mice were fixed in the lateral position, and the parameters of the IVIS Spectrum living animal imaging system (with excitation and emission wavelengths set to 740 nm and 820 nm, respectively) were adjusted to collect fluorescence images of the nude mice before injection of the contrast agents. A total of 200 µL of ICG-PTX NBs (1.0×108/mL) and ICG solution (0.35 mg/mL) of equal concentration were injected into the nude mice through the orbital vein, and fluorescence images were collected at different time points (3 min, 5 min, 10 min, 15 min, 30 min, and 60 min) for quantitative analysis of the metabolism of ICG-PTX NBs and ICG in tumour-bearing mice. Two hours after injection of the contrast agents, the animals were sacrificed, and the heart, liver, spleen, lung, kidney and tumour tissues were isolated for fluorescence imaging. The fluorescence intensity of each organ was quantitatively analysed by IVIS fluorescence analysis software to determine the level of residual ICG-PTX NBs in different tissues and organs.
Effect of indocyanine green on the cavitation of nanobubbles
After cells in the logarithmic growth phase were seeded in a 96-well plate and cultured for 24 h, they were divided into 8 groups, namely, the blank nanobubbles group (Blank NBs), indocyanine green nanobubbles group (ICG NBs), paclitaxel nanobubbles group (PTX NBs), indocyanine green and paclitaxel nanobubbles group (ICG-PTX NBs), blank nanobubbles plus ultrasound irradiation group (Blank NBs+US), indocyanine green nanobubbles plus ultrasound irradiation group (ICG NBs+US), paclitaxel nanobubbles plus ultrasound irradiation group (PTX NBs+US), indocyanine green and paclitaxel nanobubbles plus ultrasound irradiation group (ICG-PTX NBs+US). The preparation method of the ICG NBs and PTX NBs was the same as that of the ICG-PTX NBs. Appropriate amounts of Blank NBs, ICG NBs, PTX NBs, and ICG-PTX NBs at concentrations of 1.0×108/mL, 5.0×107/mL, 1.0×107/mL, and 5.0×106/mL were added to the cells. After ultrasound irradiation intervention (1 W/cm2 irradiation for 20 s), cells were cultured for another 24 h, the optical density value (OD value) of each group was determined by CCK-8 assay, and the inhibition rate of cell proliferation of each group was calculated according to the following the formula:
inhibition rate of cell proliferation (%) = [(OD experimental group - OD blank group)/(OD control group - OD blank group)] × 100%
After the cells in the logarithmic growth phase were inoculated in a 6-well plate and cultured for 24 h, treatments were given following the above groupings, and the cells were cultured for another 24 h. The cells were then collected to prepare a cell suspension with staining buffer and stained with fluorescein isothiocyanate (FITC)-labelled Annexin V (Annexin V-FITC) and propidium iodide (PI) for 5 to 15 min at room temperature in the dark. Flow cytometry (ACEA, USA) was used to analyse the apoptotic effects in each group and to further analyse the effects of ICG on the cavitation of the nanobubbles.
Cytocompatibility assay of ICG-PTX NBs
PC-3 cells in the logarithmic growth phase were resuspended to prepare a single-cell suspension after digestion, inoculated into a 96-well plate at a density of 1×104 cells/well, and cultured at 37 °C in an incubator with 5% CO2 and saturated humidity for 24 h. The cells were then divided into six groups: blank control group (PBS), ultrasound irradiation group (US), paclitaxel group (PTX), paclitaxel plus ultrasound irradiation group (PTX+US), ICG-PTX NBs group and ICG-PTX NBs plus ultrasound irradiation group (ICG-PTX NBs+US). Reagents were administered to the PTX group, PTX+US group, ICG-PTX NBs group, and ICG-PTX NBs+US group at the IC50 concentration. After drug administration, ultrasound irradiation at 1 W/cm2 for 20 s was administered to the US group, PTX+US group and ICG-PTX NBs+US group. After the stipulated intervention for each group, cells were cultured for another 24 h; then, the OD value of each group was determined by CCK-8 assay, and the rate of inhibition of cell proliferation was calculated. PC-3 cells in the logarithmic growth phase were inoculated into 6-well plates at 5×104 cells/well and cultured for 24 h. After the intervention for each group, the cells were cultured for another 24 h. After digestion and centrifugation, 50,000-100,000 cells were collected and resuspended in Annexin V-FITC solution, followed by staining with Annexin V-FITC and PI for 5 to 15 min at room temperature in the dark. The apoptotic effects of each group were analysed by flow cytometry.
Inhibition effects of ICG-PTX NBs on prostate tumour growth and their in vivo safety
When tumour volumes reached 100 mm3, the tumour-bearing nude mice were randomly divided into six groups (n = 5): PBS, US, PTX, PTX+US, ICG-PTX NBs, and ICG-PTX NBs+US groups. All treatment groups were administered drugs through orbital vein injection every 3 days. Nude mice in the PTX, PTX+US, ICG-PTX NBs and ICG-PTX NBs+US groups received paclitaxel at a total dose of 20 mg/kg, and the US group, PTX+US group and ICG-PTX NBs+US group were subjected to ultrasound irradiation at 1 W/cm2 for 60 s. Drugs were administered for over a period of 18 days. The tumour volumes and body weights of the mice were measured before each administration, and the tumour growth curve and changes in mouse body weight were plotted until the last administration. One day before treatment and 1 day after the end of treatment, the maximum sections of the tumours in each group were scanned by contrast-enhanced ultrasound with a dosage of 200 µL 1×108/mL Blank NBs, and the tumour growth before and after treatment in each experimental group was recorded. After congestion of the retroorbital venous plexus, the microhematocrit blood tube were inserted into the corner of the eye, the blood was collected using an anticoagulation tube, and blood biochemical indicators such as aspartate aminotransferase (AST), phenylalanine aminotransferase (ALT), serum creatinine (CRE) and blood urea nitrogen (BUN) were analysed to assess the toxic effects and side effects of the drugs on liver and kidney function in the tumour-bearing mice. At the end of the experiment, the mouse's head was held with the left hand, the mouse's tail was grasped with the right hand, the nude mouse was sacrificed by spinal dislocation, and the tumour tissues were routinely fixed, embedded, sectioned and subjected to haematoxylin-eosin (H&E) and TUNEL immunohistochemical staining to observe the morphological changes and apoptosis in each group. In addition, H&E staining was performed in the heart, liver, spleen, lung, kidney, etc. tissues obtained from the mice in each experimental group, and the biosafety of the drugs was analysed.
Statistical analysis
One-way ANOVA and paired t-tests were performed using the Social Pack for Social Sciences 22.0, and the analysed data are expressed as the mean ± standard deviation. The LSD test was used for comparisons between groups. P < 0.05 indicates a significant difference in statistical analysis; * indicates P < 0.05 and ** indicates P < 0.01.