Dithieno[3,2-b:2′,3′-d]silole-based conjugated polymers for bioimaging in the short-wave infrared region

The short-wave infrared window (SWIR, 900–1700 nm) fluorescence imaging has been demonstrated to have excellent imaging performance in signal/noise ratio and tissue penetration compared to the conventional NIR biological window (NIR-I, 700–900 nm). Conventional organic SWIR fluorescent materials still suffer from low fluorescence quantum efficiency. In this work, a donor unit with sp3 hybrid configuration and an acceptor unit with small hindered alkyl side chains are employed to construct donor–acceptor (D–A) type conjugated polymers P1 and P2, which were substituted with one or two fluorine atoms. These structural features can alleviate the aggregation-caused quenching (ACQ) and contribute to charge transfer, resulting in a significantly improved fluorescence quantum efficiency. The SWIR fluorescent quantum efficiencies of P1 and P2 nanoparticles are 3.4% and 4.4%, respectively, which are some of the highest for organic SWIR fluorophores reported so far. Excellent imaging quality has been demonstrated with P2 nanoparticles for SWIR imaging of the vascular system of nude mice. The results indicate that our design strategy of introducing sp3 hybrid configuration and small hindered alkyl side chains to fabricate conjugated polymers is efficient in improving the fluorescent quantum efficiency as SWIR fluorescent imaging agents for potential clinical practice.


General Synthetic Procedure of Polymers
Compound DTS [1] , 1 [2] and 2 [3] were synthesized according to previously reported methods. The polymers were prepared by the same procedure through

Preparation of nanoparticles
The schematic illustration of nanoprecipitation was shown in Fig. S1. Nanoparticles were prepared by nanoprecipitation. 2 mg of P1 (or P2) and 10 mg of DSPE-mPEG2000 were dissolved in 8 mL of tetrahydrofuran, and the solution was quickly added to 72 mL of distilled water under strong ultrasonication, followed by distillation under reduced pressure to remove tetrahydrofuran and most of the water to obtain 4 mL of a concentrated nanoparticles solution at a concentration of 500 μg·mL -1 .

Calculation of quantum yield [4]
IR-26 was used as a standard sample, and its absorption spectrum was measured by the UV-Vis-NIR to obtain five concentrations of solutions having absorbances at 808 nm of 0.023, 0.040, 0.053, 0.062, and 0.083. (Fig. S3a) The above five concentrations of IR-26 were excited by laser at 808 nm to obtain their fluorescence spectra (Fig.   S3d). The range of 900-1500 nm in the fluorescence spectrum was integrated using Origin Lab, and the results were plotted according to the absorbance-integral area to obtain the intercept and slope of the curve (Fig. S3g).
Similarly, different concentrations of P1 and P2 nanoparticles solutions (Fig. S3b, c) were excited at 808 nm, and the above solkutions were excited by laser at 808 nm to obtain their fluorescence spectra (Fig. S3e, f). The range of 900-1500 nm in the fluorescence spectrum was integrated using Origin Lab, and the results were plotted according to the absorbance-integral area to obtain the intercept and slope of the curve (Fig. S3h, i).
Quantum yield is calculated by the following formula:

Cytotoxicity test
Bcap 37 cells were used to test for cytotoxicity. The nanoparticles of P1 and P2 were dissolved in the culture medium at a concentration of 20 μg·mL -1 , 40 μg·mL -1 , 60 μg·mL -1 , 80 μg·mL -1 and 100 μg·mL -1 . Five concentrations of the solution were used to test cytotoxicity. Ten groups were tested in parallel for each concentration. After the highest and lowest values were removed, the average was obtained and the final result was obtained.

SWIR fluorescence imaging of nude mice
The nude mice used were 4 weeks old, and a laser of 635 nm was used as an excitation light source. 50 μL of P2 nanoparticles solution (200 μg mL -1 ) was injected into the nude mice. Nude mice were anesthetized with ether after injection of nanoparticles and then placed under a camera lens for imaging.

Photoacoustic imaging of nude mice
The nude mice used were 4 weeks old, the pulsed laser had a wavelength of 530 nm, the power of the laser is 1 W. The laser is irradiated within a circle with a radius of about 5 cm, and the laser intensity is about 1/(3.14*5*5)=0.013 W cm -2 . 50 μL of P2 nanoparticles solution (200 μg mL -1 ) was injected into the nude mice. Nude mice were anesthetized with ether after injection of P2 nanoparticles solution, and then their limbs were fixed on a wooden board. The abdomen of the nude mouse is coated with ultrasonic glue, the ultrasonic glue is in contact with the lower surface of the plastic wrap, the water is directly contacted above the wrap film, and the signal receiver receives the acoustic signal in the water.

Measurements
A Bruker Advance III HD 400 (400 MHz) system was used to record the 1H NMR spectra of the organics. A Hitachi UH4100 system was used to record the UV-Vis-NIR absorption spectrum of the polymer at room temperature. An FLS980 system was used to record the fluorescence spectrum of the polymer. The photothermal data were measured by a FOTRIC 226 system. A Uninano-NIR II system was used for SWIR fluorescence imaging in nude mice.