Two‐Photon Excited Near‐Infrared Phosphorescence Based on Secondary Supramolecular Confinement

Abstract Organic phosphorescence materials have received wide attention in bioimaging for bio‐low toxicity and large Stokes. Herein, a design strategy to achieve near‐infrared (NIR) excitation and emission of organic room‐temperature phosphorescence through two‐stage confinement supramolecular assembly is presented. Via supramolecular macrocyclic confinement, the host–guest complexes exhibit phosphorescence with two‐photon absorption (excitation wavelength up to 890 nm) and NIR emission (emission wavelength up to 800 nm) in aqueous solution, and further nano‐confinement assembly significantly strengthens phosphorescence. Moreover, the nano‐assemblies possess color‐tunable luminescence spanning from the visible to NIR regions under different excitation wavelengths. Intriguingly, the prepared water‐soluble assemblies maintain two‐photon absorption and multicolor luminescence in cells or vivo.


Section A. Materials and Methods
All reagents and solvents were purchased from commercialized suppliers and used as supplied except for specifying otherwise noted. 1 H NMR and 13 C NMR were performed with an Ascend 400 MHz instrument. NOESY and COSY spectra were measured on a ZhongKe-Oxford I-400 instrument. Highresolution mass spectrometry (HRMS) was recorded on a Q-TOF LC-MS in electrospray ionization mode. UV-vis absorption spectra were recorded on a Shimadzu UV-3600 spectrophotometer with a PTC-348WI temperature controller in a quartz cell (light path 10 mm) at 298 K. Photoluminescence (PL) spectra, time-correlated decay profiles, quantum efficiency were processed on an Edinburgh Instruments

Cytotoxicity experiments and Cell imaging
The human lung adenocarcinoma cells (A549) were obtained from the Cell Resource Center of China Academy of Medical Science in Beijing. The culture conditions for A549 cells: Ham's F12 nutrient medium supplemented with 10 % FBS and 1 % penicillin/streptomycin and humidified incubator with 5% CO2 atmosphere at 37 °C. A549 cells were seeded in 96-well plates for 12 h at 37 °C in 5% CO2.
The A549 cells were incubated with G/CB[8]/SC4AD at different concentrations in 96-well plates for 24 h. The relative cellular viability was determined by the CCK8 assay.
The A549 cells were seeded in a confocal petri dish and cultured in a humidified incubator at 37 °C with 5% CO2 for 24 hours. The well-cultured cells were incubated with G/CB[8]/SC4AD (2×10 -5 M) for 12 h. The cells were then repeatedly washed at least three times with PBS and observed with a confocal microscope. The two-photon imaging of G/CB[8]/SC4AD in cells was recorded with Leika S8 confocal microscope.

In vivo imaging
All experimental procedures with animals were carried out by licensed investigators. Animals: Onemonth-old male ICR mouse (20g), were obtained from the Department of Laboratory Animal Science, Peking University Health Science Center. Feeding conditions: All the animals were submitted to controlled temperature conditions (22~26 °C) humidity (50~60%), light (12 h light/12 h dark, 15~20 LX), adequate water, and adequate food. And all the animals were maintained on the standard laboratoryspecific pathogen-free (SPF). In vivo imaging: The living mice were anesthetized with Isoflurane.

Synthesis of compound 4:
Compound 4 was synthesized according to the literature. 1 4-(4-bromophenyl)pyridine (100 mg, 0.43 mmol) and 1,3-dibromopropane (0.35 g, 1.74 mmol) were dissolved in 25 mL CH3CN and heated at 60 °C for 12 h. After being cooled to room temperature, the reaction mixture was dispersed in 250 mL diethyl ether. The mixture was filtered and the solid was washed with acetone. The product was obtained as white solid (120 mg, 65%).

Synthesis of compound 6:
Compound 6 was synthesized according to the literature 3 and is similar to the description of compound 5. Product was obtained as white solid (65%).

Synthesis of compound 1:
Compound 5 (50 mg, 0.25 mmol) and compound 4 (100 mg, 0.23 mmol) were dissolved in 10 mL DMF and heated at 90 °C for 48 h. The residue was cooled to room temperature and evaporated to remove the solvent. The crude product was recrystallized with CH3CN to obtain the yellow powder with a yield of 42% (60 mg). 1