Tunable Nanoparticles with Aggregation‐Induced Emission Heater for Precise Synergistic Photothermal and Thermodynamic Oral Cancer Therapy of Patient‐Derived Tumor Xenograft

Abstract The fluorophores in the second near‐infrared (NIR‐II) biological window (1000 – 1700 nm) show great application prospects in the fields of biology and optical communications. However, both excellent radiative transition and nonradiative transition cannot be achieved simultaneously for the majority of traditional fluorophores. Herein, tunable nanoparticles formulated with aggregation‐induced emission (AIE) heater are developed rationally. The system can be implemented via the development of an ideal synergistic system that can not only produce photothermal from nonspecific triggers but also trigger carbon radical release. Once accumulating in tumors and subsequently being irradiated with 808 nm laser, the nanoparticles (NMB@NPs) encapsulated with NMDPA‐MT‐BBTD (NMB) are splitted due to the photothermal effect of NMB, leading to the decomposition of azo bonds in the nanoparticle matrix to generate carbon radical. Accompanied by second near‐infrared (NIR‐II) window emission from the NMB, fluorescence image‐guided thermodynamic therapy (TDT) and photothermal therapy (PTT) which significantly inhibited the growth of oral cancer and negligible systemic toxicity is achieved synergistically. Taken together, this AIE luminogens‐based synergistic photothermal‐thermodynamic strategy brings a new insight into the design of superior versatile fluorescent NPs for precise biomedical applications and holds great promise to enhance the therapeutic efficacy of cancer therapy.


Synthesis of compound 4.
To the solution of compound 3 (421 mg, 1.0 mmol) in the 20 mL dry THF, then the mixture was stirred and cooled down to -80 ℃ under nitrogen. After that, the n-butyl lithium (0.5 mL, 2.4 mM) was added in the mixture and stirred for 2 h. Then the tributyltin chloride (325 mg, 1.0 mmol) was added in the solution and stirred at room temperature for 8 h. Then the mixture was concentrated under reduced pressure. The resultant product was directly used in the next step reaction without purification.

Calculation of photothermal conversion efficiency.
NMB@NPs, NPs and deionized water were upon 808 nm laser at a power density of 1.0 W/cm 2 for 10 min. The real-time temperature was recorded during this process and lasted until the dispersions returned to initial temperature. The photothermalconversion efficiency of NMB@NPs was determined according to the previous method. [1][2] Details are as follows: Where h is the heat transfer coefficient, S is the surface area of the container, and the value of hS can be obtained from Equation S2, S3, and S4. Where T is the temperature of NMB@NPs, T max is the maximum system temperature and T surr is the initial temperature. I is the laser power and A 808 means the absorbance of nanoparticle at the wavelength of 808 nm. Q g (0.0251 J/s) is the energy that absorbed by the solvent. Where s is the sample system time constant. m D and C D are the mass (1.09 g) and heat capacity (4.2 J/g) of deionized water, respectively.

Cellular uptake.
Cal-27 cells were cultured in DMEM supplemented with 15% (v/v) fetal bovine serum, 1% (v/v) penicillin, and 1% (v/v) streptomycin, respectively. Cells were incubated in a humidified incubator at 37 °C with 5% CO 2 . Cancer cells were seeded on a glass bottom petri dish for 12 h before diverse treatments and then examined with CLSM imaging. Hoechst 33342 and Nile red was used to label nucleus and NPs, respectively. Cells were incubated with Nile red-loaded NPs (Nile red@NPs) (300 g/mL) for 0.5, 1.5, 3.5, and 5.5 h, before incubation with Hoechst 33342 solution for 0.5 h. Afterward, the cells were rinsed with cold PBS to remove free Hoechst 33342 and Nile red@NPs samples. Finally, the stained cells were imaged by the CLSM system.
For flow cytometry analysis, the culture medium was replaced with Nile red@NPs and further incubated for 1, 2, 4, and 6 h, respectively. All the cells were washed with PBS and collected by trypsinization. After that, the cells were resuspended and analyzed by flow cytometry.
The exploration for endocytic inhibition was performed as follows. Cal-27 cells were pretreated with 0.1% NaN 3 /50 mM 2-deoxyglucose (DOG) in serum-free DMEM for 1 h to suppress the energy-dependent endocytosis. Cells were preincubated in serum-free DMEM containing 5 mM methyl -cyclodextrin (MCD) for 15 min at 37 ºC/5% CO 2 to inhibit MCD. Cells were pretreated with the 225 mM sucrose in serum-free DMEM for 30 min at 37 ºC/5% CO 2 to inhibit the clathrin-mediated endocytosis. After exposure to the respective inhibitors and Nile red@NPs 2 h for all endocytic inhibition tests, the cells were washed with cold PBS and followed by quantifying the fluorescence intensity.

Intracellular free-radicals detection.
The intracellular generation of free radicals was monitored using H 2 DCFH-DA