Materials
Liposome includes 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000](DSPE-PEG2000), 1, 2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DSPG) and cholesterol were purchased from Avanti Polar Lipids (Alabaster, AL, USA) Perfluoropentane (PFP), indocyanine green (ICG), propidium iodide (PI) were obtained from Sigma Aldrich (St. Louis, MO, USA).
Reactive Oxygen Species Assay Kit (20,70-dichlorofluorescin diacetate, DCFH-DA), 1,10-dioctadecyl-3,3,30,30-tetramethylindocarbocyanine perchlorate (DiI) were purchased from Santa Cruz Biotechnology (TX, USA). All reagents were of analytical grade and used without further purification.
Preparation of LIP-ICG-PFP-cRGD, LIP-ICG-PFP
The liposomes loaded with ICG and PFP, referred to as Lip-ICG-PFP-cRGD, were synthesized by a two-step emulsion method as described previously (Jian et al. 2014). Briefly, 5 mg of DPPC, 2.0 mg of DSPG, 1.5 mg of DSPE-PEG-cRGD, and 1.5 mg of cholesterol were dissolved in 10 mL of methanol and 10 mL of chloroform. The solution was then transferred to a round bottom flask to form lipid films by rotary evaporation. After that, 2 mL of phosphate buffer saline (PBS) was added to the flask, sonicating the mixture to hydrate the lipid films for 5 min using ultrasound cleaner. Next, 0.05 mL of ICG aqueous solution (10 mg/mL) and 0.2 mL of PFP were added into lipid films and emulsified in an ice bath for 3 min using a sonicator (Heat System Inc, USA). Following that, a solution of LIP-ICG-PFP-cRGD was purified three times by centrifugation (8000 rpm, 5 min), and the supernatant was removed. Finally, 2 mL of PBS were added to the precipitate for further experimentation. The LIP-ICG-PFP liposomes were synthesized by sonicating the hybrid of lipid films without cRGD and PFP in an ice bath for 3 min and then purified three times by centrifugation (8000 rpm, 5 min).
Characterization of liposome
Nanoparticles (NPs) carrying PFP and ICG were successfully synthesized using DSPG, DSPE-PEG2000, DPPC, and cholesterol, and were called LIP-ICG-PFP-cRGD, generating a US-mediated cavitation effect. The diameter, zeta potential, and polydispersity index (PDI) of different kinds of NPs or NBs were measured by Zetasizer Nano ZS unit (Malvern Instruments, Malvern, UK) and their morphology and structure were observed by transmission electron microscopy (TEM). Ultraviolet-visible (UV-vis) spectrophotometer with scanning wavelength ranging from 200 to 900 nm was used to determine the absorption spectra of different nanoparticles. UV-vis spectrophotometer (260-Bio, Thermo Fisher Scientific) was used to evaluate the entrapment efficiency and loading of ICG, and entrapment efficiency and loading content were then calculated.
PA and US Imaging In Vitro
The agar-gel model (3% agar w/v in double-distilled water) was utilized to evaluate LIP-ICG-PFP-cRGD capacity, which acts as contrast agents for dual-modal imaging. PA imaging of different concentrations of nanoparticles was shown on VEVO LASER PA imaging system (VEVO 2100, Canada).
For US imaging, liposomes were exposed to low-intensity focused ultrasound (2-6 W) for 100-300 s. The contrast-enhanced ultrasound (CEUS) and B-mode images were obtained by an ultrasonic diagnostic instrument (MyLab 90; Esaote, Italy).
Detection of ROS
1, 3-Diphenylisobenzofuran (DPBF) was used to determine ROS generation in nanoparticles. A total of 60 µL of DPBF (4 mg/mL), 50 µL of LIP-ICG-PFP-cRGD, and 1 mL of double distilled water were added into Eppendorf tube and exposed to low-intensity ultrasound with 4 W/cm2 for 0-10 min in dark. Then, a multimode reader was used to evaluate ROS production by measuring the absorption at 410 nm.
To detect intracellular ROS generation, 2', 7'-dichlorofluorescin diacetate (DCFH-DA) was employed as an indicator. In brief, 1 x 105 ID8 cells were seeded in 12-well plates and co-incubated with LIP-ICG-PFP-cRGD (the concentration of each group was 0.4 mg/mL) for 3 h. All the wells were divided into six groups: control group, NPs group, only LIFU group (4 W/cm2 for 60 s), and NPs combined with LIFU group (4 W/cm2 for 60 s). Then, each of the wells was washed three times with PBS. Subsequently, the cells were cultured with diluted DCFH-DA for 30 min in dark. The ROS generation was evaluated by flow cytometry.
ID8 cells were seeded in confocal dishes at a density of 1 x 105 for 24 h, and the rest of routine was as described above. Finally, laser scanning confocal microscopy (LSCM, Nikon AJR, Japan) was used to detect ROS generation.
Determination of Cellular Uptake
ID8 cells were seeded onto confocal dishes at a density of 1 x 105 for 24 h. Next, the original media was replaced by media containing LIP-ICG-PFP or LIP-ICG-PFP-cRGD labeled with Dil dye for co-incubation with different periods (1.0, 2.0, and 3.0 h). Then, the cells were washed with PBS and cultured with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) for 10 min to stain cell nuclei. Subsequently, PBS was used to wash the cells three times, and 4% paraformaldehyde was utilized to fix the cells. Finally, the cellular uptake ability was evaluated by confocal laser scanning microscopy.
In vitro cell viability and Cell death assay using annexin V/PI staining
To assess cytotoxicity in vitro, ID8 cells were seeded in a 96-well plate for 24 h, and then, 100 µL of LIP-ICG-PFP-cRGD nanoparticles with various concentrations were added and cultured for another 24 h. Cell viabilities were tested by CCK-8 assay.
To evaluate the therapeutic effects, ID8 cells were seeded in a 96-well plate and co-cultured with LIP-ICG-PFP-cRGD nanoparticles (different concentrations were divided into five groups) for 3 h. PBS was utilized to wash the wells three times, and the media in each well were displaced with fresh media. Next, the groups were exposed to low-intensity ultrasound with various power (4.0 W/cm2) for 200 s in dark, and cell viabilities were determined using CCK-8 method.
ID8 cells were cultured in 6-well dishes for 24 h. Additionally, cells were incubated with liposomes and free ICG for 3 h and washed with PBS. Then, cells were trypsinized and re-suspended with 400 µL of 1% FBS containing annexin V binding buffer. After that, the cells were exposed to LIFU for 200 s (power 4.0 W/cm2). The cell viabilities were determined using CCK-8 method or further incubated for 1 h and stained with FITC annexin V and PI for 10 min. Finally, the cells were observed using a fluorescence microscope and analyzed with flow cytometry.
The CRT expression and HMGB1 Release Assay
The ID8 cells were co-cultured with nanoparticles (LIP-ICG-PFP-cRGD 0.8 mg/mL) in 6-well plates for 3 h. All the wells were divided into four groups: control group, only LIFU group, only NPs group, and LIP-ICG-PFP-cRGD plus LIFU group (4 W/cm2 for 200 s). The cells were cultured for another 18 h, and the supernatant was added into another 6-well plate which was cultured with dendritic cells (DC) for 6 h. Subsequently, the cells and supernatants from DC co-cultured were collected to examine the CRT and HMGB1 level by ELISA (LifeSpan BioSciences), according to manufacturer’s instructions.
The expression of CD86 in dendritic cells
To test the expression of CD86 (a marker for DC maturation), ID8 cells were seeded and co-incubated with different nanoparticles. Following exposure to different treatments, the supernatant was collected separately and added into another 12-well plate cultured with DC for 24 h. After washing with PBS and staining with FITC-conjugated anti-CD86 for 60 min in dark, the samples were analyzed using flow cytometry to quantify CD86 expression in DC.
Statistical analysis
The experimental data were analyzed using GraphPad Prism (version 8.0) with the unpaired Student’s t-test, or the paired Student’s t-test. P-values < 0.05 indicated statistically significant difference (*p < 0.05, **p < 0.01).