Platinum acetylacetonate (Pt(acac)2, 97%) was purchased from Acros Organics and used as received. Iron pentacarbonyl (Fe(CO)5, 99%), hexadecane-1,2-diol (90%), oleyl amine (80–90%), oleic acid (70%), dioctyl ether (90%), 1-octadecene (90%), 3-mercaptopropionic (3-MPA, 97%), pyrrole (Py, reagent grade, 98%), polyvinyl alcohol (PVA, Mw: 9000–10,000), ammonium persulfate ((NH4)2S2O8, 98%), sodium dodecyl sulfate (SDS), potassium ferrocyanide, hydrochloric acid, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich and used as received during experiments. Cellular staining reagents including trypan blue, propidium iodide (PI), and Hoechst 33342 were also purchased from Sigma-Aldrich. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, 1× trypsin, and phosphate-buffered saline (PBS) were purchased from HyClone (South Logan, UT, USA). Distilled water (DI) was used for all experiments.
Synthesis of FePt@PPy NPs
The synthesis of FePt@PPy NPs was performed through three steps which were described in Scheme 1.
Step 1—Synthesis of Hydrophobic FePt NPs
The synthesis of hydrophobic FePt NPs was done according to the reported scheme . In short, 97-mg Pt(acac)2, 4-mL dioctyl ether, 66-μL Fe(CO)5, 195-mg 1,2-hexadecandiol, 100-μL oleyl amine, and 100-μL oleic acid were loaded into a 50-mL three-neck round-bottom flask. The reaction mixture was heated to 240 °C with a heating rate of 15 °C/min under Argon gas. After 30 min, the product was cooled to room temperature. The FePt NPs were collected by centrifugation (15,000 rpm, 30 min) and washed several times with hexane. The final nanoparticle solution was stored in hexane.
Step 2—Ligand Exchange
The ligands on the surface of hydrophobic FePt NPs were exchanged with 3-Mercaptopropionic acid (3-MPA) as reported in articles . Moreover, 1 mL of 3-MPA and 1 mL of cyclohexanone were loaded in a centrifuge tube, and then, 0.5 mL of hydrophobic FePt NPs dispersed in hexane (~ 10 mg) was added to the above solution and shaken by using a vortex. After 30 min, the FePt NPs started precipitating, and all nanoparticles precipitated after 1 h. The hydrophilic FePt NPs were collected by centrifugation (3500 rpm, 5 min). The product was washed with cyclohexanone, ethanol, and acetone, respectively. Finally, the hydrophilic FePt NPs diluted in DI with the addition of NaOH.
Step 3—Coating Hydrophilic FePt NPs with PPy
Five milligrams of hydrophilic FePt NPs was dissolved in 200-mL barker containing 60-mL DI and was continuously sonicated for 10 min. Then, 6 mL of 40-mM SDS was added to the above solution. Next, 1-g PVA that was completely dissolved in hot water was added to the above solution. The resulting mixture was then stirred at 500 rpm. Next, 10 mL of 6-mM (NH4)2S2O8 was added to the stirred solution. After 1 h of equilibration, 6 mL of 100-mM Py was added into the above solution. After several minutes, the solution gradually turned to black. After 2 h of polymerization, the resulting nanoparticles were separated by centrifugation (12,000 rpm, 30 min) and were washed several times with hot water to remove impurities. The obtained FePt@PPy NPs were resuspended with PBS by ultrasonication for 3 min.
The morphology of nanoparticles was observed using field-emission transmission electron microscopy (FETEM; JEM-2100F, JEOL, Japan). The atomic composition was analyze by energy-dispersive spectroscopy (EDS). The chemical functional groups of the nanoparticles were analyzed using a Fourier-transform infrared spectroscopy (FTIR) spectrometer (Perkin Elmer 1320 FTIR spectrophotometer). The nanoparticle diameter was determined by the dynamic light scattering method by using electrophoretic light scattering spectrophotometer (ELS-8000, OTSUKA Electronics Co. Ltd., Japan). UV-Vis-NIR spectra were measured by using UV-Vis-NIR spectroscopy (Thermo Biomate 5 Spectrophotometer). Laser irradiation was performed using a power-tunable 808-nm laser (continuous wave, maximal power = 5 W, Hi-TechOptoelectronics Co., Beijing, China).
For measuring the photothermal performance of as-prepared NPs, a suspension (1 mL) containing the FePt@PPy NPs with specific concentrations (20, 30, 50, 70, 100, and 120 μg/mL) was added into a 12-well plate. Then, each well was exposed by an 808-nm laser at a power density of 1 W/cm2 for 5 min. In addition, the increasing temperature of irradiated FePt@PPy NPs at different power densities of the 808-nm laser was also recorded. Briefly, 50-μg/mL FePt@PPy NP solution was irradiated by the NIR laser at the desired power density of 0.5, 1, and 1.5 W/cm2 for 6 min. The temperature was recorded by a thermometer (MASTECH, CA, USA) via a thermal fiber.
50-μg/mL FePt@PPy NPs was exposed to the 808-nm laser at a power density of 1 W/cm2 till the highest temperature was achieved, and then, it was allowed to return to room temperature by turning the laser off. The heating and cooling cycles were repeated six times. The UV-Vis spectrum of the irradiated sample was recorded to compare with the irradiated sample.
Long-Term Storage Test
The aqueous suspension FePt@PPy NPs at 120 μg/ml concentration was stored at 4 °C for 30 days to evaluate its stability in long-term storage. For the comparison, the UV-Vis absorption spectra and the particles size of FePt@PPy NPs were observed for the 1st day and the last day. In addition, FePt@PPy NPs on different media including DI, DMEM media plus FBS, and PBS were stored at 4 °C for 30 days to evaluate the stability of prepared FePt@PPy NPs.
Cytotoxicity Assay of FePt-PPy NPs
A standard MTT assay  was used to quantify the cell cytotoxicity. The MDA-MB-231 breast cancer cells were used as model cancer cells to test the biocompatibility of FePt@PPy NPs. FePt NP-treated cancer cells were used as a control. The MDA-MB-231 cell line was cultured in a DMEM medium supplemented 10% FBS and 1% antibiotics in a humidified atmosphere at 37 °C and 5% CO2. The MDA-MB-231 cells were seeded in 96-well microplate at a density of 1 × 104 cells/well. After 24 h, the DMEM media containing FePt@PPy NPs (or FePt NPs) with different concentrations (0, 20, 30, 50, 70, 100, and 120 μg/mL) were added to cell plates, and the treated cells were then incubated for 48 h. Note that the amount of FePt is the same for the two tested nanoparticles, including FePt NPs and FePt-PPy NPs. Next, 100-μL MTT dissolved in PBS at 0.5 mg/mL was added to each well, and the cell plates were further incubated for 4 h. The dehydrogenase enzyme, which is present in the mitochondria of the alive cells, converted the soluble MTT to insoluble purple formazan. Next, 100 μL DMSO was added to dissolve the insoluble purple formazan. Subsequently, the absorption of purple formazan was recorded at 570 nm using a plate reading spectrophotometer to quantify the percentage of cell viability.
Prussian blue staining was used to check the cellular uptake of FePt@PPy NPs in the MDA-MB-231 cell . The cells were seeded at a density of 1 × 105 cells/mL in 12-well plates and incubated for 24 h. Next, 200-μg/mL FePt@PPy NPs was added to the cell plates and incubated for another 24 h. After that, the cells were fixed with cold formaldehyde for 15 min. And then, 10% potassium ferrocyanide and 20% aqueous solution of hydrochloric acid (50:50 v/v) were added to the cell plates and incubated for 1 h. The result was observed using optical microscopy.
In Vitro Photothermal Therapy
The MTT assay was performed to quantify the efficacy of FePt@PPy NPs on the killing capability of MDA-MB-231 breast cancer cells. Briefly, the MDA-MB-231 cells were cultured in a 96-well microplate at a density of 1 × 104 cells/well. On the next day, the FePt@PPy NP solutions with specific concentration (0, 10, 20, 30, 50, 70, and 100 μg/mL) were added to the cell plates, and the treated cells were incubated for another 24 h. Then, PBS was used to wash the unbound nanoparticles. Subsequently, the microplates were exposed to the NIR laser at a power density of 1 W/cm2 for 4 and 6 min, respectively. To obtain the results, the following steps were conducted in accordance with the cell cytotoxicity assay in the “Cytotoxicity Assay of FePt-PPy NPs” section.
Double staining of Hoechst 33342 and PI was also used to detect the damaged and dead cells as a result of the photothermal treatment using FePt@PPy NPs. Concretely, the MDA-MB-231 cells were seeded in a 12-well plate at a density of 1 × 105 cells/well. After 24 h, the cells were treated with the FePt@PPy NPs (0, 50, 70, and 100 μg/mL) and continuously incubated for another 24 h at 37 °C. Next, the unbound nanoparticles were removed by washing gently with PBS. Subsequently, the cell plates were exposed to the NIR laser at a power density of 1 W/cm2 for 6 min. Next, the cell culture plates were kept for 24 h in the incubator, and then, the irradiated cells were stained with Hoechst 33342 and PI. Note that 1.5-mL Hoechst 33342 (10 μg/mL) was added in the cell culture plate and then kept in the incubator for 20 min. Then, the cells were washed with three-time PBS to remove the excess stain. Then, the cells were continuously stained with 1.5-mL PI (10 μg/mL) and incubated at room temperature for 5 min. Finally, the cells were again washed with PBS, and the fluorescent images were captured by a fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany).
To perform an in vivo test of the photothermal properties of FePt@PPy NPs, a 6-week-aged female BALB/c nude mouse was subcutaneously injected with 100 μL of 100 μg/mL FePt@PPy NPs in PBS. Another nude mouse without injection was used as a control. Afterwards, the injected area of the mice was irradiated with an 808-nm laser at 1 W/cm2 for 6 min. The experimental procedures with animals were approved by the animal care and use committee of Pukyong National University and performed according to the guiding principles for the care and use of laboratory animals.
In Vitro Photoacoustic Imaging
PAI on phantom was performed to evaluate the PA signal of FePt@PPy NPs. Our group has developed the noninvasive PAI system as reported in the previous study . The schematic diagram of PAI setup was shown in Fig. 11. An optical system embedded with a pulsed Nd-YAD Q-switched laser (Surelite III, CA, USA) was employed. The laser was set at 808-nm wavelength and 10-Hz frequency with 5-ns pulse operation. The input optical fiber having a focal length of 50 mm (Thorlabs, Newton, NJ, USA) was connected to a plano-convex lens. The output optical fiber was linked to a focused transducer (Olympus NDT, USA) and adjusted to the illuminated zone’s center. To record PA signals, the data was digitized and stored via a DAQ (data acquisition) system integrated with the laser system. Subsequently, the recorded data was used to reconstruct 2D images of the phantom by a LabVIEW program.
The PVA phantom was prepared with 8% PVA to mimic the tissue. The preseeded MBA-MD-231 cancer cells were treated with different concentrations of FePt@PPy NPs (50, 100, and 200 μg/mL) for 24 h, and then, the cells were harvested and mixed with 4% gelatin on the phantom (Fig. 12a). Then, the phantom was covered by a small layer of 4% gelatin and allowed to solidify. Finally, the phantom was fixed on the water tank for PAI processing.