L-phosphatidylcholine (PC), cholesterol (Chol), 1,2-distearoyl-sn-glycero-3-phospho- ethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), Bionic TM buffer, phosphate buffer saline (PBS), melanin, perfluorohexane (PFH), 5-flurouracil (5-FU), Triton-X, and phosphotungstic acid were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle’s culture Medium (DMEM), fetal bovine serum (FBS), Roswell Park Memorial Institute Medium (RPMI 1640), and antibiotic antimycotic solution were purchased from WELGENE (Daegu, Korea). All other reagents were used without purification.
2.2. Preparation of perfluorohexane (PFH) emulsion
PFH emulsion was prepared with a previously reported method  with some modifications. Briefly, 10 mg of PC was dissolved in 2 ml of ethanol, transferred to a round flask, and vacuum dried. Thereafter, the obtained thin film was hydrated with 110 mM ammonium sulfate buffer. PFH liquid was then added to the flask at a 1:1 ratio of the above solution. To prevent evaporation of PFH, it was stored in an ice-water bath while waiting for the next manufacturing step. To homogenize the emulsion, sonication was performed three times for 30 seconds and cooled in each cycle. Finally, the emulsion solution was extruded 11 times through a 200 nm polycarbonate filter using an Avanti Mini Extruder (Alabaster, AL, USA).
2.3. Preparation of [email protected]@5-FU-liposomes
[email protected]@5-FU-liposomes were prepared by the thin film method according to a previously reported method . First, PC (10 mg), Chol (1.5 mg), and DSPE-PEG (3 mg) were dissolved in ethanol. Melanin (1 mg) was dissolved in 1 ml of bioionic buffer. These solutions were mixed in a round flask and sufficiently evaporated under vacuum. The formed thin lipid film was hydrated with a solution of 5-FU (2 mg) dissolved in 2 ml of PBS (pH 7.4). [email protected] solution was then sonicated for 9 min (on 3 s, off 1 s) in ice-water conditions using a sonicator (SCIENTZ-IID, SCIENIZ, Zhejiang, China). After sonication, the [email protected] solution was extruded 11 times through a 200 nm polycarbonate filter using an Avanti Mini Extruder (Alabaster, AL, USA). Free 5-FU was then separated from the [email protected] solution using a PD-10 column (PD-10 desalting column, GE Healthcare Life Sciences). Finally, the prepared liposome suspension was mixed with PFH emulsion at a 1:1 ratio and sonicated for 90 seconds. The obtained [email protected]@5-FU-liposome was extruded 11 times through a 200 nm filter.
Dynamic light scattering (DLS, Zetasizer NanoZS90, Malvern Instruments Ltd., Worcestershire, United Kingdom) was used to confirm size distributions of various amounts of PFH emulsion and [email protected]@5-FU-liposome. The morphology of [email protected]@5-FU-liposome was observed using a transmission electron microscope (TEM, H-7500, Hitachi Ltd., Tokyo, Japan). Phosphotungstic acid was used as a negative stain agent for TEM observation.
2.5. Photothermal conversion and bubble generation ability
To evaluate the photothermal conversion ability of [email protected]@5-FU-liposome in vitro, the solution was transferred to a disposable cuvette and then irradiated with an 808 nm NIR laser at a power density of 1.5 W/cm2 for 10 min. An infrared (IR) thermal imaging camera (C2, FLIR System Inc., Sweden) was used to record temperature changes every minute. Distilled water was used as a control group to compare temperature changes. To evaluate bubble generation ability, when [email protected]@5-FU-liposome solution was irradiated with an 808 nm NIR laser at an intensity of 1.5 W/cm2 for 10 min, bubble generation was visually observed. Specifically, to observe bubble formation, the phantom model used a latex tube with a diameter of 5 mm. The [email protected]@5-FU-liposome solution (3 ml) was then transferred to a latex tube. In the same manner as described above, while irradiating [email protected]@5-FU-liposome with a laser, bubble generation was observed using an ultrasound imaging system (SONON, B-mode, Healcerion, Seoul, Korea).
2.6. In vitro drug release behavior
Drug release behavior of [email protected]@5-FU-liposome induced by bubble generation of PFH due to the photothermal effect of melanin was evaluated with or without near-infrared laser irradiation. In detail, [email protected] and [email protected]@5-FU-liposome were transferred to disposable cuvettes and irradiated with an 808 nm NIR laser at a power density of 1.5 W/cm2 for 10 min. Thereafter, each solution was placed in a dialysis bag (MWCO 12,000 Da), immersed in 5 ml of PBS and then placed in a water bath adjusted to 37°C. Released medium was collected at predetermined time points. The absorbance of the drug was measured using a UV-spectrophotometer (T60U, PG Instruments Limited, UK) at a wavelength of 265 nm. The concentration of drug released was calculated using a standard calibration curve. To measure drug loading efficiencies of [email protected] and [email protected]@5-FU-liposome, these liposome membranes were disrupted using Triton-X to induce the release of the encapsulated drug. The absorbance of 5-FU was then measured using the UV-spectrophotometer.
2.7. Cytotoxicity and PTT-mediated cell death
HaCaT and CT26 cell lines were used to evaluate the cytotoxicity and chemo-photothermal-mediated cell death effect of [email protected]@5-FU-liposome. HaCaT and CT26 cells were cultured in DMEM and RPMI1640 medium containing 10% fetal bovine serum in a CO2 cell incubator (37°C, 5% CO2), respectively. To evaluate the cytotoxicity of [email protected]@5-FU-liposome, cell viability was measured using an MTT method. Briefly, in a 48-well plate, 4×104 cells per well were treated with various amounts of [email protected]@5-FU-liposome and cultured in a CO2 cell incubator for 24 h. Thereafter, cells were treated with a MTT reagent (40 µl). After incubating 37°C for 3 h, the medium containing the MTT reagent was removed. Finally, the purple crystal product formed inside living cells was dissolved in DMSO and the absorbance of the mixture was measured at 570 nm to calculate cell viability. To confirm chemo-photothermal-mediated cell death, 2×105 cells per well were treated with various amounts of [email protected]@5-FU-liposome and then cultured in a CO2 cell incubator for 6 h. In the same manner as described above, the medium containing [email protected]@5-FU-liposome was removed and replaced with a new medium. Thereafter, the plate was irradiated with an 808 nm laser at an intensity of 1.5 W/cm2 for 10 min. After laser irradiation, cells were cultured for 24 h. MTT assay was then performed.
2.8. Tumor therapy in vivo
To investigate the tumor therapeutic effect of [email protected]@5-FU-liposome, we established a CT26 xenograft tumor model. Mice (male, 6 weeks old, Orient Bio Inc., Seoul, Korea) were anesthetized with isoflurane. Then 0.1 ml of 5×105 CT26 cells suspended in Matrigel was inoculated subcutaneously into each mouse to prepare the animal model. After preparing the mouse tumor model, mice were randomly divided into three groups: (1) control group, (2) [email protected]@5-FU-liposome injection group, and (3) [email protected]@5-FU-liposome injection with laser irradiation group. First, 200 µl of [email protected]@5-FU-liposome was intravenously injected into the tail vein of each mouse in the group corresponding to sample injection. Laser irradiation (808 nm, 1.5 W/cm2, 10 min) was performed at 1 h, 4 h, and 12 h after intravenous injection. After laser irradiation, temperature change in the tumor was checked using an IR thermal imaging camera. The presence or absence of bubble formation was observed using a portable ultrasound imaging system. The length and width of the tumor were measured using digital calipers. Tumor volume was calculated according to the following formula: (tumor length × tumor width2)/2.
2.9. Histological analysis
Mice were sacrificed at 14 days after treatment procedures in various groups. Major organs (heart, kidney, liver, lung, and spleen) including tumor tissues were removed from mice and stained with hematoxylin and eosin (H&E). Observation of stained tissues was performed using an optical microscope (FX-II, Olympus Inc., Tokyo, Japan).
2.10. Statistical analysis
Statistical comparisons among multiple treatment groups were performed using analysis of variance (ANOVA). Probability (p) value less than < 0.05 was regarded as statistically significant. Experimental results are presented as means ± standard deviation (S.D.).