Fast Delivery of Multifunctional NIR‐II Theranostic Nanoaggregates Enabled by the Photoinduced Thermoacoustic Process

Abstract Multifunctional nanoaggregates are widely used in cancer phototheranostics. However, it is challenging to construct their multifunctionality with a single component, and deliver them rapidly and efficiently without complex modifications. Herein, a NIR‐absorbing small molecule named TBT‐2(TP‐DPA) is designed and certify its theranostic potentials. Then, their nanoaggregates, which are simply encapsulated by DSPE‐PEG, demonstrate a photothermal efficiency of 51% while keeping a high photoluminescence quantum yield in the NIR region. Moreover, the nanoaggregates can be excited and delivered by an 808 nm pulse laser to solid tumors within only 40 min. The delivery efficiency and theranostic efficacy are better than that of the traditional enhanced permeability and retention (EPR) effect (generally longer than 24 hours). This platform is first termed as the photoinduced thermoacoustic (PTA) process, and confirm its application requires both NIR‐responsive materials and pulse laser irradiation. This study not only inspires the design of multifunctional nanoaggregates, but also offers a feasible approach to their fast delivery. The platform reported here provides a promising prospect to boost the development of multifunctional theranostic drugs and maximize the efficacy of used medicines for their clinical applications.


Materials
Characterization UV-vis-NIR spectra were measured using Shimadzu (Japan) UV-2600 spectrophotometer. Fluorescence spectra were recorded by Hitachi (Japan) F-4600 fluorescence spectrophotometer. Absolute quantum yield was measured by Hamamatsu UV-NIR Absolute PL quantum yield spectrometer C13534. The size distribution of nanoparticles was determined by dynamic light scattering (DLS) using Malvern (UK) Nano ZS Zetasizer. Morphological structures of the nanoparticles were studied by transmission electron microscopy (TEM) using Hitachi (Japan) TEM-HT7700 at 100 kV accelerating voltage. In vivo NIR-II fluorescence imaging results were recorded on a Series III 900/1700-D NIR-II imaging system (Yingrui, Suzhou). In vivo photoacoustic imaging was performed by a commercial ORPAM system (NIR-VIS-50, PAOMTek Inc.). Flow cytometry analysis was acquired on a FACSCanto Analyzer.
The 808 nm laser was purchased from Changchun Laser Technology Co., Ltd (Changchun, China) and the infrared thermal images were acquired by FLIR E6 thermal imagers. Fluorescence confocal imaging was conducted on a Zeiss LSM980 confocal microscopy.

Preparation of TBT-2(TP-DPA) nanoparticles
The compound of TBT-2(TP-DPA) was first synthesized and purified according to modified reactions. In brief, TBT-2(TP-DPA) (1 mg) and DSPE-PEG2000 (1 mg) were dissolved in THF (1 mL) by sonication, then mixed with 9 mL of deionized water. The mixture was sonicated via an ultrasound probe (VCX150, Sonics) for 2 min at 75 W output. THF was removed through dialysis against DI water overnight in a dialysis bag (molecular weight cut off = 8000-14000 Da). The solution was filtered through a 0.2 μm syringe filter to yield nanoparticles suspended in water for further characterization.
Then the solutions of the nanoaggregates were imaged using an AR-PAM (acousticresolution photoacoustic microscopy) system with an 808 nm laser.

In vivo photoacoustic and NIR-II fluorescence imaging
4T1 tumor-bearing mice were intravenously injected with TBT-2(TP-DPA) PTA nanoaggregates (200 µL, 1 mg mL −1 ). The mice were placed on the imaging end of a nanoaggregates, five cycles of heating and cooling were recorded.

In Vitro Cytotoxicity
4T1 cells were seeded into a 96-well plate at a density of 1 × 10 4 cells and cultured at 5% CO2 and 37 °C for 24 h. Following, the initial medium was replaced with a fresh medium containing different concentrations (0, 10, 25, 50, 100, and 250 µg mL -1 ) of TBT-2(TP-DPA) PTA nanoaggregates to incubate the cells for another 24 h. Next, the culture media was replaced by fresh media containing 10% CCK-8 medium solution and cultured for 2 h. The cell viability was calculated by measuring the absorbance value on a microplate reader at 450 nm.

Live and Dead Cell Assay
4T1 cells were seeded into the confocal dish at a density of 1 × 10 5 cells and cultured at 5% CO2 and 37 °C for 12 h. TBT-2(TP-DPA) PTA nanoaggregates (0.05 mg mL −1 , 200 µL) were then added into the cell culture medium. After incubation of 12 h, the cells were washed and the culture media was replaced by fresh media. For PTT in vitro, the treatment groups were irradiated with 808 nm CW laser (1 W cm −2 ) for 10 min.
Then, the medium was removed and washed with 1 × PBS twice. The cells were successively incubated with Calcein-AM (100 µL, 5 µM) at 37 °C for 15 min and PI solution (100 µL, 50 µM) at room temperature for 15 min. The cells were gently washed and imaged by confocal microscopy.

Flow Cytometry Analysis
4T1 cells were seeded into 12-well microplates at a density of 1 × 10 5 cells and cultured at 5% CO2 and 37 °C for 12 h. TBT-2(TP-DPA) PTA nanoaggregates (1 mg mL −1 , 200 µL) were added into the cell culture plate and incubated with 4T1 cells for 12 h. For PTT, the treatment groups were irradiated with 808 nm CW laser (1 W cm −2 ) for 10 min. The supernatant was collected, and the cells were digested by 0.25% trypsin-EDTA solution and stained with annexin V-FITC/PI (Sangon Biotech), followed by analysis on FACSCanto Analyzer.

In Vivo Biosafety Analysis
The female BALB/c mice (4-6 weeks, ~20 g) were purchased from the Guangdong Medical Laboratory Animal Center. The animal procedures were approved by the Institutional Animal Care and Use Committee of Southern University of Science and Technology. The mice were treated with the TBT-2(TP-DPA) PTA nanoaggregates (1 mg mL −1 , 200 µL) through tail vein injection. The control group was injected with 1 × PBS buffer at the same volume. Before the mice were sacrificed, the blood was collected for into blood collection tubes immediately by enucleation of mouse eyes for haematology analysis. For biochemistry analysis, the rest of the blood sample was kept at room temperature for 2 h and then centrifuged at 4000 rpm for 10 min to collect the supernatant serum for use. The main organs (heart, liver, spleen, lung, and kidney) were collected at 0-, 7-, and 14-days post-injection, and were stained with H&E for histological analysis.

In Vivo Photothermal Therapy
4T1 cells (2 × 10 6 cells in 1 × PBS buffer) were injected subcutaneously into the flank of the female BALB/c nude mice (4 weeks). When the tumor volume reached 80 mm 3 , the mice were randomly divided into 5 groups (n = 3 each group) and given the following treatments: 1) TBT-2(TP-DPA) nanoaggregates + 808 nm pulse laser + 808 nm CW laser (PTA 40 min + PTT); 2) TBT-2(TP-DPA) nanoaggregates + 808 nm CW laser (EPR 24 h + PTT); 3) TBT-2(TP-DPA) nanoaggregates + 808 nm pulse laser (PTA); 4) 808 nm pulse laser + 808 nm CW laser; and 5) PBS, respectively. All the nanoaggregates were injected at the concentration of 1 mg mL-1 for a volume of 200 μL. For the PTA group, the tumors were irradiated by an 808 nm pulse laser for 40 min after injection of TBT-2(TP-DPA) PTA nanoaggregates. For PTA + PTT group, the tumors were irradiated by an 808 nm pulse laser for 40 min immediately after injection of TBT-2(TP-DPA) nanoaggregates, followed by irradiation of 808 nm CW laser (1 W cm −2 ) for 10 min. For EPR 24 h + PTT group, the tumors were directly irradiated by an 808 nm CW laser (1 W cm −2 ) for 10 min at 24 h post injection. For the PTA group, the tumors were only irradiated by an 808 nm pulse laser for 40 min without 808 nm CW laser irradiation, immediately post injection. The tumor volume was measured by a digital caliper every 2 days. The tumor volume = length × width 2 /2.

Statistical information
All results were expressed as mean ± standard deviation through at least three experiments. One-way analysis of variance (ANOVA) was used for the statistical analysis of data between 2 groups. p< 0.05 was considered statistically significant. 0.01 < *P < 0.05, 0.001 <**P < 0.01, ***P < 0.001. All the statistical calculations were conducted by Origin 2021b software.

Synthesis of 4-methoxy-N-(4-methoxyphenyl)-N-(4-(trimethylstannyl)phenyl)aniline (2).
To a solution of 4-bromo-N,N-bis(4-methoxyphenyl)aniline (1, 1.00 g, 2.60 mmol) in anhydrous THF (30 mL) was added n-BuLi (1.3 mL, 2.5 M in hexane) at -78°C under N2 atmosphere. The mixture was stirred at the same temperature for one hour, followed by the addition of trimethyltin chloride (3.4 mL, 3.38 mmol, 1.0 M in hexane). The reaction was allowed to slowly warm up to room temperaure and stirred overnight. The reaction mixture was quenched by aqueous KF solution, wahsed with water and brine, and dried over Na2SO4. The crude product was obtained by concentration under reduced pressure and used without futher purification.
After being cooled to room temperature, the reaction mixture was extracted with chloroform, washed with water and brine. After concontration under reduced pressure, the crude product was purified by column chromatography (stationary phase: silica gel; eluent: dichloromethane) to get the product as a white solid (741 mg, 62%
The crude product was obtained by concentration under reduced pressure and used without futher purification.
To a solution of compound 7 (600 mg, 1.55 mmol) in anhydrous THF (20 mL) was added n-BuLi (0.74 mL, 2.5 M in hexane) at -78°C under N2 atmosphere. The mixture was stirred at the same temperature for one hour, followed by the addition of trimethyltin chloride (2.01 mL, 2.01 mmol, 1.0 M in hexane). The reaction was allowed to slowly warm up to room temperaure and stirred overnight. The reaction mixture was quenched by aqueous KF solution, wahsed with water and brine, and dried over Na2SO4.
The crude product was obtained by concentration under reduced pressure and used without futher purification.