Chemicals and materials

(2E,2’E)-2,2’-(2,8-bis(2-decyltetradecyl)-1,3,7,9-tetraoxo-1,2,3,7,8,9-hexahydro-dithiolo[4’,5’:5,6]benzo[1,2,3,4-lmn] [1, 3]dithiolo[4,5-f] [3, 8]phenanthroline-5,11-diylidene)bis(2-bromoacetonitrile) (NDTA-2Br) and 2,5-bis(2-butyloctyl)-3,6-bis(5-(trimethylstannyl)thiophen-2-yl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione (TDPP-2Sn) were purchased from SunaTech Inc. Poly(ethylene glycol) 2000-distearoylphosphatidylethanolamine (DSPE-PEG2000) was purchased from Xi’an Ruixi Biological Technology Co, Ltd. Pd2(dba)3, P(o-tol)3, and anhydrous toluene were purchased from J&K Scientific Ltd. Methanol, acetone, petroleum ether, and chloroform were purchased from Shanghai Macklin Biochemical Co., Ltd. Nitrogen gas (N2) was purchased from Shenzhen Shente Industry Gas Co., Ltd. All reagents and solvents were used without further purification.

Synthesis of NT polymer

The Pd-catalyzed Stille polymerization was carried out to synthetize a polymer with NIR-II absorption. Briefly, NDTA-2Br (132.3 mg, 1.0 eq.), TDPP-2Sn (96.2 mg, 1.0 eq.), Pd2(dba)3 (4.67 mg, 0.05 eq.), P(o-tol)3 (3.04 mg, 0.1 eq.) and anhydrous toluene (10 mL) were added to a dried Schlenk tube (25 mL). All the above operations were carried out in a glove box filled with N2. The reaction medium was stirred for 24 h at 120 °C under N2. Afterward, 1.0 mL of concentrated hydrochloric acid was added to quench the reaction and stirred for another 12 h. After cooling to room temperature, the polymer was precipitated into cold methanol (300 mL), filtered, and then dried in a vacuum drying oven. The polymer was successively washed in a Soxhlet extractor with acetone, petroleum ether, and chloroform overnight. The final product was collected from chloroform, precipitated into cold methanol again, and dried under reduced pressure at room temperature to obtain a black solid, namely, the NT polymer.

Fabrication of SPD

SPD was fabricated by a nanoprecipitation method. Briefly, NT polymer (0.5 mg) and DSPE-PEG2000 (2.5 mg) were first dissolved in dimethyl sulfoxide (DMSO, 0.5 mL) solution followed by sonication for 10 min to obtain a uniform mixture. Then, the mixture solution was injected quickly into 5 mL of ultrapure water under vigorous stirring for 2 min. The as-prepared products were then purified by dialysis (molecular weight cutoff: 100 kDa) against ultrapure water for a day to obtain the SPD solution. Finally, the SPD solution was concentrated by ultrafiltration, and the concentration was quantified according to the standard curve of SPD in DMSO. The collected SPD was dispersed into ultrapure water and stored at 4 °C in the dark.

Characterization

Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance III 500 MHz NMR spectrometer using tetramethylsilane (TMS; δ = 0 µg/ml) as an internal standard. Gel permeation chromatography (Alliance e2695) was used to evaluate the size range of the polymer. Zetasizer NanoZS90 equipment was employed to record the hydrodynamic size and zeta potential of SPD. Transmission electron microscopy (TEM) images were obtained on a Hitachi HT-7700 electron microscope. Absorption spectra were recorded on an Agilent 4100 spectrometer.

Measurement of the photothermal effect of SPD

The photothermal response of SPD was investigated on a simple setup in the air. Briefly, a centrifuge tube containing 500 µL of SPD at different concentrations was fixed on an optical holder. Ultrapure water was used as a blank control. 1064 nm CW laser with a 1 W/cm2 power density was used to irradiate the sample dispersion from the top. An infrared thermal camera (FLIR A300) was operated for temperature recording. The heat conversion efficiency (HCE) was calculated based on Eqs. 1–4 [9].

$$\eta =\frac} - }) - }}}^}}})}}$$

(1)

$$\theta =\frac}}}} - }}}$$

(4)

where \(\text\) is the heat transfer coefficient, \(\text\) is the surface area of the container, and the value of \(\text\) can be obtained according to the natural cooling curve. \(_\) is the steady-state temperature of PTA under laser irradiation while \(_\) is the surrounding temperature. \(_\) represents the energy absorbed by the container and solvent. \(\text\) is the power of incident laser power, while \(_\) is the absorbance of photothermal agents at 1064 nm. mD and CD are the mass and specific heat capacity of the sample dispersion, respectively.

Theory of PA thermometry

The PA signal of the PTA can be generated based on the thermoelastic effect, if the width of the irradiating pulsed laser meets the thermal and stress confinements [25]. After propagation in the medium, the transducer-detected ultrasonic pressure can be described by

where Γ is the Grueneisen parameter (dimensionless), µa is the specific absorption coefficient, and F is the optical fluence. Here Γ can be expressed as

$$\Gamma =\frac^}}}}=f(T)$$

(6)

where Cp denotes the specific heat capacity at constant pressure, β is the volume expansion coefficient, and cs is the acoustic velocity. Because the volume expansion coefficient and the acoustic speed are both dependent on the temperature for water-based and fatty tissues between 10 and 55 °C [15, 26], Γ can be derived as a function of temperature T,

Thus, the measured PA amplitude is directly dependent on the temperature if µa and F are invariable during heating. Note that here, the temperature refers to the base temperature induced by the photothermal laser, whereas the instantaneous temperature increase due to the thermoelastic effect of the absorber is negligible [27].

Experimental setup for PA thermometry

The platform had three functional modules, including the PA system, PTT system and sample platform. In the PA system, a Nd:YAG pulsed laser (I-20, Surelite) was used. The pulse repetition rate was 20 Hz with an 8 ns width, and the wavelength was tuned at 1064 nm to ensure both optical penetration and absorption of SPD. The energy of the laser pulse was 18 mJ/cm2, which was below the American National Standards Institute (ANSI) safety standard (100 mJ/cm2). To ensure the consistency of the laser intensity of each pulse, a photodiode (SM1PD1A, Thorlabs) was used to monitor the energy fluctuation between the pulses. An ultrasound transducer (V309-SU, Olympus) with a fixed 25.4 mm focal depth and 5 MHz center frequency was used to detect the PA signal. The PA signal varying with increasing or decreasing temperature was amplified (DPR300 Pulser/Receiver, Imaginant Inc.) and sampled by a 12-bit data acquisition card (ATS9371, Alazar Technologies Inc.) with a sampling rate of 50 MHz. The custom-complied LabVIEW program and MATLAB were separately used to record and analyze the data. The fixed delay output trigger from the pulsed laser synchronized the laser radiation and the data acquisition. In the PTT system, a 1064 nm CW mode laser (GX-1064, Leishi Inc.) was used, and the energy of the CW laser was 1 W/cm2. ITC was used to standardize the temperature rise in the PTT process. In the sample platform, water tank with two optical windows was used. A polyethylene tube with a 0.58 mm internal diameter was attached to the optical window for loading SPD at a 100 µg/ml concentration. The pulsed laser, CW laser and ITC were calibrated to the same area. Different insertions of distilled water, gelatin phantom (with 3% intralipid), and intact skull of a mouse were placed in the PA signal propagation path. The CW laser was switched on/off for multiple short-term or the long-term therapeutic periods for different heating requirements.

For 2D PA signal acquisition in vivo, a multichannel ultrasound data transceiver platform (Vantage128, Verasonics, WA, USA) with a linear array ultrasound transducer (L11-4, Verasonics, WA, USA) was used. The system can realize multiangle plane wave ultrasound imaging in transmit-receive mode with PA imaging functions [28].

Cytotoxicity evaluation

The in vitro cytotoxicity of SPD was evaluated by coincubation with mouse glioma 261 (GL261), breast cancer (MCF-7), breast cancer epithelial (HS578T), human embryonic kidney 293 (293T) cells, and mouse breast tumor (4T1) cells. All cells were seeded in 96-well plates at a density of 5000 cells per well and grown to nearly 80% confluence. Then, the culture medium was replaced by fresh culture medium containing SPD at various concentrations (0, 0.5, 1, 2, 5, 10, 20, 50, and 100 µg/ml). After 24 h of incubation, the cell viability was determined using a Cell Counting K-8 assay.

Establishment of the 4T1 tumor xenograft model

BALB/c male mice (NO.44,007,200,082,293) were purchased from the Guangdong Medical Laboratory Animal Center. All animals were kept in the Experimental Animal Center of Shenzhen University Medical School. The 4T1 tumor model was established by subcutaneous injection of 100 µL of 4T1 suspension solution (107 cells/mL) into the right underarm of mice. The tumors grew for approximately 10 days and reached a size of approximately 100 mm3 before use. The animal experiments conformed to the guidelines of the University Animal Care and Use Committee, according to protocol No. AEWC-202,300,019.

In vivo experiments

To study the biodistribution of the SPD, the tumor-bearing mice were intravenously injected with 0.2 mL of SPD at a 100 µg/ml concentration. Ultrasound imaging and 2D PA signal acquisition were performed in tumor locations before or after the injection of SPD. Twenty-four hours after injection, the mice were sacrificed and major organs including the tumor, spleen, liver, intestine, bladder, kidney, lung and heart were harvested. Then the ex vivo imaging of the organs was conducted by ultrasound imaging and 2D PA signal acquisition. To study the performance of PA thermometry in vivo, the tumor-bearing mice were intratumorally injected with 20 µL SPD at a 100 µg/ml concentration. Then, ultrasound imaging and 2D PA signal acquisition were performed in tumor locations with or without CW laser irradiation.

To investigate the biocompatibility, BALB/C mice of six-week-old were injected with SPD of 10 mg/kg or PBS of 200 µL. Each group contains five mice. Then, the experiments were performed, including body weight recording, hemolysis assays, blood routine, blood biochemistry, and histological analysis of major organs (heart, liver, spleen, lung, and kidney), respectively.

Statistical analysis

Data were presented as means ± SD. Statistical analysis was performed using ANOVA. The statistical significance was examined by Student’s t-test when two groups were compared. Statistical analysis was considered significant differences when P values less than 0.05.