Experimental

Materials

Aniline was obtained from Lingfeng Chemical Reagent Co., Ltd. (Shanghai, China). Benzil, 2,5-Thiophenedicarboxaldehyde, ammonium acetate, Benzophenone, 2-(Pyridin-4-yl)acetonitrilewere, 4-Methylbenzyl cyanide were purchased from Bide Pharmatech Co., Ltd. (Shanghai, China). Malononitrile, 4-Benzylpyridine were purchased from Yien Chemical Technology Co., Ltd. (Shanghai, China). Zinc powder was purchased from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). Acetic acid, THF, ethanol (EtOH) and TiCl4 were purchased from J&K Scientific Ltd. (Beijing, China). All other chemicals and solvents of high purity were purchased and used without further purification. All reactions dealing with air-sensitive compounds were performed under a nitrogen atmosphere.

Measurement

UV-Vis absorption spectra were measured using a Shimadzu UV-1900i spectrophotometer in the spectrofluorometer, with the excitation wavelength fixed at the absorption maximum of each sample and emission collected between 400 and 800 nm. 1H NMR spectra were obtained on a Bruker Avance 400 MHz spectrometer. High-resolution mass spectrometry (HRMS) data were acquired using an Agilent LC-QTOF-MS system. Cell imaging was conducted on a Zeiss LSM710 confocal microscope, and murine tissue imaging was performed on an IVIS LumiNova II system.

Synthesis of compound TPIT-CHO

A solution of aniline (558 mg, 6.0 mmol) and 2,5-thiophenedicarboxaldehyde (840 mg, 6.0 mmol) in acetic acid (50 mL) was stirred at room temperature for 1 h. Subsequently, benzil (1.26 g, 6.0 mmol) and ammonium acetate (3.23 g, 6.0 mmol) were added, and the reaction mixture was heated at 120 °C for 8 h. Upon completion, the solvent was removed under reduced pressure. The residue was extracted with dichloromethane (3 × 70 mL), and the combined organic layers were concentrated. The crude product was purified by column chromatography on silica gel using DCM as the eluent to afford 1.33 g of a yellow solid in 54.8% yield. 1H NMR (400 MHz, CDCl3) δ 9.80 (s, 1H), δ 7.58 (s, 1H), δ 7.51 (s, 1H), δ 7.47–7.38 (m, 3H), δ 7.28–7.19 (m, 9H), δ 7.17–7.14 (m, 2H), δ 6.98 (s, 1H). 13C NMR (101 MHz, CDC13) δ 182.86, 142.95, 142.10, 140.95, 139.32, 136.24, 135.90, 133.62, 132.64, 130.92, 129.93, 129.76, 129.68, 128.89, 128.51, 128.47, 128.31, 127.33, 127.14, 126.77, 77.38, 77.27, 77.07, 76.75. HRMS, m/z calcd. for C26H18N2OS: 406.1140; found 406.5030 (M + H)+.

Synthesis of compound TPIT-1

A mixture of TPIT-CHO (0.5 mmol, 203 mg), benzophenone (1.5 mmol, 273.3 mg), and zinc dust (4.8 mmol, 313.7 mg) in dry THF (15 mL) was stirred at −40 °C for 10 min. Subsequently, TiCl4 (2.4 mmol, 0.263 mL) was added dropwise slowly. The reaction mixture was then heated under reflux for 10–12 h. Upon completion, the mixture was cooled to room temperature and filtered under vacuum. The filtrate was extracted with dichloromethane (3 × 30 mL), and the combined organic layers were concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel using dichloromethane as the eluent to afford 111 mg of a yellow solid in 40% yield. 1H NMR (400 MHz, CDCl3) δ 7.55 (s, 1H), δ 7.53 (s, 1H), δ 7.41–7.39 (m, 3H), δ 7.36–7.33 (m, 2H), δ 7.30–7.06 (m, 20H), δ 6.62 (d, J = 3.8 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 139.09, 130.98, 130.05, 129 34, 128.89, 128.29, 128.20, 128.15, 127.47,127.37, 126.87, 120.55, 77.36, 77.24, 77.04, 76.72, 39.30. HRMS, m/z calcd. for C39H28N2S: 556.1973; found 557.2051 (M + H)+.

Synthesis of compound TPIT-2

To a solution of TPIT-CHO (0.4 mmol, 162.4 mg) and 4-methylacetonitrile (0.44 mmol, 57.6 mg) in dry EtOH, a solution of NaOH (50 mg) in EtOH was added dropwise at room temperature. The reaction mixture was further stirred for 4 h. Upon completion of the reaction. The crude product was purified by column chromatography on silica gel using dichloromethane as the eluent to afford 147.5 mg of a yellow solid in ​​73% yield. 1H NMR (400 MHz, CDCl3) δ 7.62–7.60 (m, 4H), δ 7.33–7.06 (m, 17H), 6.78 (d, J = 4.2 Hz, 1H), 2.37 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 148.5, 142.3, 138.7, 138.2, 137.5, 135.7, 134.3, 133.0, 131.7, 130.1, 129.6, 129.2, 128.7, 128.6, 127.9, 127.5, 126.4, 122.1, 118.8, 105.5. HRMS, m/z calcd. for C35H25N3S: 519.1769; found 520.1846 (M + H)+.

Synthesis of compound TPIT-3

A solution of TPIT-CHO (0.4 mmol, 162.4 mg) and malononitrile (0.44 mmol, 29 mg) in dry EtOH was refluxed for 10 h in the presence of one or two drops of piperidine as catalyst. Upon completion of the reaction. The crude product was purified by column chromatography on silica gel using dichloromethane as the eluent to afford 130 mg of a yellow solid in ​​72% yield​​. 1H NMR (400 MHz, CDCl3) δ 8.63 (s, 2H), δ 7.67 (s, 1H), δ 7.61 (d, J = 7.5 Hz, 2H), δ 7.55 (m, 1H), δ 7.48–7.39 (m, 5H), δ 7.33–7.11 (m, 10H),6.81 (d, J = 4.0 Hz, 1H). 13C NMR (101 MHz, CDCI3) δ 267.05, 151.00, 150.44, 141.49, 141.02, 139.37, 139.28, 136.94, 136.18, 136.03, 135.32, 134.03, 133.71, 132.49, 130.93, 130.84, 129.90, 129.75, 129.56, 128.96, 128.82, 128.50, 128.44, 128.29, 127.34, 127.26, 127.09, 126.74, 119.53, 117.06, 105.11, 77.40, 77.28, 77.09, 76.77. HRMS, m/z calcd. for C33H22N4S: 506.1565; found 507.1641 (M + H)+.

Synthesis of compound TPIT-4

A solution of TPIT-CHO (0.4 mmol, 162.4 mg) and 4-pyridineacetonitrile (0.44 mmol, 51.9 mg) in dry EtOH was refluxed for 10 h in the presence of one or two drops of piperidine as catalyst. Upon completion of the reaction. The crude product was purified by column chromatography on silica gel using dichloromethane as the eluent to afford 178.1 mg of a yellow solid in ​​88% yield​​. 1H NMR (400 MHz, CDCl3) δ 7.54 (s, 1H), δ 7.52 (m, 3H), 7.50 (m, 4H), δ 7.48 (m, 8H), 7.46 (d, J = 7.5 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 182.84, 149.95, 137.98, 135.51, 134.83, 130.82, 130.38, 129.98, 128.85, 128.70, 128.58, 128.35, 127.42, 127.39, 115.43, 114.19, 113.14, 77.34, 77.23, 77.02, 76.71, 29.71, 0.01. HRMS, m/z calcd. for C29H18N4S: 454.1252; found 455.1330 (M + H)+.

Synthesis of compound TPIT-5

4-Benzyl-1-methylpyridium iodide was first prepared through methylation of 4-benzylpyridine. A solution of TPIT-CHO(0.4 mmol, 162.4 mg) and 4-benzyl-1-methylpyridium iodide (0.4 mmol, 124.4 mg) in dry EtOH was refluxed for 12 h in the presence of one or two drops of piperidine as catalyst. Upon completion of the reaction. The crude product was purified by column chromatography on silica gel using dichloromethane as the eluent to afford 130 mg of a yellow solid in ​​72% yield​​. 1H NMR (400 MHz, CDCl3) δ 8.82 (d, J = 6.3 Hz, 2H), δ 8.37 (d, J = 6.3 Hz, 2H), δ 8.02 (s, 1H), δ 7.58 (m, 3H), δ 7.44 (m, 4H), 7.25 (m, 8H), δ 7.88 (m, 7H), 7.13 (d, J = 7.2 Hz, 2H), δ 6.52 (s, 1H), 4.47 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 157.0, 148.5, 146.3, 142.3, 140.0, 138.7, 138.2, 137.5, 133.0, 131.7, 130.1, 129.6, 129.2, 128.7, 128.6, 128.3, 128.2, 127.9, 127.5, 124.8, 122.1, 49.0 HRMS, m/z calcd. for C39H30N3S+: 572.2155; found 522.2157 M+.

Synthesis of compound TPIT-6

4-(cyanomethyl)-1-methylpyridin-1-ium iodide was first prepared through methylation of 2-(pyridin-4-yl)acetonitrile. A solution of TPIT-CHO (0.4 mmol, 162.4 mg) and 4-(cyanomethyl)-1-methylpyridin-1-ium iodide (0.4 mmol, 124 mg) in dry EtOH was refluxed for 12 h in the presence of one or two drops of piperidine as catalyst. Upon completion of the reaction. The crude product was purified by column chromatography on silica gel using dichloromethane as the eluent to afford 165.9 mg of a yellow solid in ​​64% yield. 1H NMR (400 MHz, CDCl3) δ 8.78 (d, J = 6.5 Hz, 2H), δ 8.29 (s, 1H), δ 7.83 (d, J = 6.5 Hz, 2H), δ 7.63 (m, 3H), δ 7.48–7.36 (m, 6H), δ 7.27 (m, 7H), δ 6.53 (s, 1H), 4.24 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 157.0, 148.5, 146.3, 142.3, 138.7, 138.2, 137.5, 133.0, 131.7, 130.1, 129.6, 129.2, 128.7, 128.2, 127.5, 124.8, 122.1, 49.0. HRMS, m/z calcd. for C34H25N4S+: 521.1794; found 521.1799 M+.

Preparation of nanoparticles

Lipophilic TPIT-6 (1 mg) and the amphiphilic polymer DSPE-PEG2000 (2 mg; 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000]) were dissolved together in 1.0 mL of tetrahydrofuran (THF). The resulting solution was then poured into 10 mL of ultrapure water under continuous ultrasonication for 10 min. Afterward, the residual THF was eliminated by flushing with nitrogen gas, and the obtained nanoparticle suspension was kept at 4 °C until further use. The concentration of the resulting nanoparticles (NPs) was determined to be 120 μmol·mL-1.

Cellular imaging

SCC7 mouse squamous cell carcinoma cells were obtained from the American Type Culture Collection (ATCC). Cell cultures were maintained at 37 °C in a humidified, 5% CO2 atmosphere using DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. For confocal imaging, SCC7 cells were plated into confocal dishes at a density of 2 × 105 cells/mL in RPMI-1640 medium and left to adhere overnight. After the medium was removed and the cells were washed three times with PBS, they were incubated with RPMI-1640 containing 10 μM TPIT-6 NPs or TPIT-6 NPs-Ab for 4 h. Following a further three PBS washes, the cells were resuspended in 1 mL of PBS and imaged using a confocal microscope (Zeiss LSM710) with 488 nm excitation and appropriate emission filters.

Cytotoxicity study

Smooth muscle cells (SMCs) in the logarithmic growth phase were maintained at 37 °C in a 5% CO2 atmosphere until they reached confluence in 96-well plates. Ten wells were randomly chosen and assigned to two groups, which were then treated with varying concentrations of TPIT-6 NPs (10–50 mmol·L-1, based on the chromophore standard) for 24 h under either light or dark conditions. Thereafter, 20 μL of MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5 mg·mL-1) was added to each well, and the plates were incubated for an additional 4 h. The culture medium was then removed, and 100 μL of DMSO was added to dissolve the formazan crystals. The plates were gently agitated on an orbital shaker for 10 min, and the absorbance at 550 nm was recorded using a microplate reader. The obtained data were plotted as bar graphs.

Ex vivo imaging

All animal experiments were approved by the ethics committee and conducted in accordance with relevant guidelines. Female C3H mice weighing 18–22 g were obtained from Beijing VITON-LH Experimental Technology Co., Ltd. The animals were maintained under a 12-h light/12-h dark cycle at 25 °C and 50%–60% relative humidity, with ad libitum access to drinking water and standard diet. The health status of the mice was checked daily, and all experiments commenced following a one-week acclimatization period. Subsequently, female C3H mice were used to establish SCC7 tumors by intraperitoneal administration of 100 μL of a cell suspension containing 3 × 106 SCC7 cells. Tumor resection was carried out when the tumors grew to approximately 10 mm. After tumor removal, tissue specimens suspected of containing tumor cells were harvested from the surgical margin and promptly immersed in PBS supplemented with 5% fetal bovine serum. Subsequently, they were transferred to PBS containing 50 μM TPIT-6 NPs-Ab and kept at ambient temperature for 15 min. After the incubation, the specimens were each washed twice with PBS to eliminate unbound probes, followed by imaging with an IVIS LumiNova II imaging system.