Supporting Information rganic Semiconducting anoparticles as Efficient Photoacoustic Agents for Lightening Early Thrombus and Monitoring Thrombolysis in Living Mice Cao Cui,, Zhen Yang,, Xiang Hu, Jinjun Wu, Kangquan Shou, Hengheng Ma, Chao Jian, Yong Zhao, Baiwen Qi, Xiaoming Hu, Aixi Yu*,, Quli Fan*
Synthesis Scheme and procedures for the crgd-pdi Ps. Br C 20 -H 2 Br Pyrrolidine ah Br MP Br Isopropanol 1 2 3 BCH H 2 48 48 H 2- PEG 2000 -HBC MP TFA CHCl 3 4 5 6 Self-assemble in water PDI Ps Sulfo-SMCC, crgd-sh crgd-pdi Ps Scheme S1. Synthetic route to crgd-pdi Ps. Synthesis of 2: A suspension of 1 (0.85 g, 1.55 mmol), 1 2-n-ctyl-1-dodecylamine (1.53 g, 5.1 mmol), and acetic acid (5 g, 8.33 mmol) in -methyl-2-pyrrolidinone (MP; 100 ml) was stirred at 85 C under 2
for 8 h. After cooling the mixture to room temperature, it was poured into aqueous 1 HCl and the precipitate was separated by suction filtration, washed with deionized water until ph 7, and dried under vacuum. The crude product was purified by silica gel column chromatography with CH 2 Cl 2 /petroleum ether (4:1, v/v, Rf = 0.5) as eluent. The orange band was collected and 2 was obtained after evaporation of the solvent as a brown powder (1.60 g, 90%). The regioisomeric 1,7- and 1,6-dibromoperylene diimide could not be separated by column chromatography. 1 H MR (400 MHz, CDCl 3 ): δ = 9.50 (m, 2 H), 8.94 (d, 2 H), 8.71 (s, 2 H), 4.26 4.19 (m, 4 H), 1.79 1.70 (m, 4 H), 1.50 1.43 (m, 66 H), 1.00 (t, 12 H) ppm. HRMS: calcd. for C 64 H 88 Br 2 2 4 [M + H]+ 1106.5101; found 1106.5132. Synthesis of 3: A mixture of 2 (331.8 mg, 0.3 mmol) and 8 ml pyrrolidine was heated to 55 C under 2. The reaction mixture was kept at 55 C for about 24 h and then the solvents were evaporated using rotary evaporators. The residue was purified by column chromatography on silica gel with CHCl 3 (Rf = 0.42) as eluent. The regioisomeric 1,7- and 1,6-dibromoperylene diimide could be separated by column chromatography at this step. The green fraction was collected and after evaporation of the solvent, 3 was collected as a green powder (248 mg, 75 %). 1 H MR (400 MHz, CDCl 3 ): δ = 8.28 (d, 4 H), 7.53 (s, 4 H), 4.25 4.19 (t, 4 H), 3.67 (m, 4 H), 2.65 2.51 (m, 4 H), 2.03 1.72 (m, 12 H), 1.48 (m, 66 H), 1.00 (t, 12 H) ppm. HRMS: calcd. for C 72 H 104 4 4 [M + H]+ 1088.8111; found 1088.8134. Synthesis of 4: A solution of 2 (1.09 g, 1.00 mmol) and ah (4.68 g, 83.40 mmol) in isopropanol (36 ml) was heated to reflux. After stirred for 0.5 h, the reaction mixture was poured into acetic acid (50 ml) and stirred over night at room temperature. After filtration, the precipitate was washed with a large amount of H 2 then MeH. The crude product was purified by silica gel column chromatography with CHCl 3 (Rf = 0.3) as eluent. The green band was collected and 4 was obtained after evaporation of the solvent as a green powder (1.60 g, 70%). 1 H MR (400 MHz, CDCl 3 ) 8.45-8.32 (m, 4H), 7.46-7.53 (d, 2H), 4.22 (d, 2H), 3.85-3.65 (m, 4H), 2.85-2.65 (m, 4H), 2.25-1.95 (m, 8H), 1.48 (m, 33 H), 0.80 (t, 6 H) ppm. HRMS: calcd. for C 52 H 63 3 5 [M + H]+
809.4876; found 809.4844. Synthesis of 5, 6: A mixture of 4 (404.74 mg, 0.50 mmol), tbc-h-poly(ethylene glycol)-h 2 (1.155 g, 0.550 mmol), zinc acetate dihydrate (21.9 mg, 0.1 mmol), and imidazole (20 g) was heated at 140 C for 3 h. The resulting mixture was poured into deionized water (150 ml). The precipitate was separated by suction filtration, and washed with deionized water until ph 7. The residue was subjected to the silica gel column chromatography (CH 2 Cl 2, then MeH/CH 2 Cl 2 =2/1, then MeH /ethyl acetate = 1/1). The green powder was collected and 5 was obtained after evaporation of the solvent as a green powder (1.017 g, 70%). 5 was dissolved in a mixture of CHCl 3 (10 ml) and trifluoroacetic acid (10 ml) with stirring over night at room temperature. After evaporation of the solvent, the resulting was then precipitate in diethyl ether and then green powder 6 was obtained by filtering and dried under vacuum. Synthesis of crgd-pdi Ps: The 4-(maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo--hydroxysuccinimide ester sodium salt (sulfo-smcc) (2.2 mg) was first dissolved in 36 μl of dimethyl sulfoxide (DMS). The PEG-PDI Ps [1 mg in 1 ml of PBS (ph = 7.2)] was incubated with the above cross-linker solution for 2 h at room temperature. The resultant ran through a PD-10 column prewashed with PBS (ph = 7.2, 10 mm) to remove the excessive sulfo-smcc and byproducts. The crgd stock solution (200 μl of 5 mm in the degassed water) was added to the above solution with stirring. The conjugation reaction proceeded for 24 h at 4 C. The uncoupled crgd peptide was removed through a PD-10 column. The number of coupled crgd on one PDI Ps was then calculated by MALDI-TF. The resultant product were stored at 4 C for one month without losing targeting activity. The final crgd-pdi Ps were reconstituted in PBS and filtered through a 0.22 μm filter for cell and animal experiments. The crad-pdi Ps were also obtained and characterized through the same route as crgd-pdi Ps. After freeze-drying of the Ps, we further tested the 1 H MR for the mix PDIs to confirm the crgd and crad linked to 6. The number of PDI molecule in one PDI P was calculated as follows: 2
Because of the low contrast of PEG, PEG shell on PDI Ps cannot be observed in TEM. Thus, the PDI Ps observed in TEM (Figure 2b) was attributed to the PDI core in crgd-pdi, the diameter of which was about 40 nm. Considering the density of PDI is 1.4 g/cm 3, the number of PDI molecule in one crgd-pdi can be finally calculated to be about 3.48 10 4. The equation was listed as follows: PDI = w PDIcore ρ PDI A = (4 3)πr 3 ρ PDI A M PDI M PDI In this equation, PDI is the number of PDI molecule in one crgd-pdi P, W PDIcore is the weight of the PDI core in one crgd-pdi, V PDI is the volume of the PDI core in one crgd-pdi, M PDI is the molar mass of PDI (810 g/mole), r is the radius of PDI core (20 nm), is the density of PDI (1.4 g/cm 3 ), A is Avogadro's constant (6.02 10 23 ). Figure S1. 1 H MR spectrum (400 MHz, chloroform-d, room temperature) of 2.
Figure S2. 13 C MR spectrum (100 MHz, chloroform-d, room temperature) of 2. Figure S3. 1 H MR spectrum (400 MHz, chloroform-d, room temperature) of 3.
Figure S4. 13 C MR spectrum (100 MHz, chloroform-d, room temperature) of 3. Figure S5. 1 H MR spectrum (400 MHz, chloroform-d, room temperature) of 4.
Figure S6. 13 C MR spectrum (100 MHz, chloroform-d, room temperature) of 4. Figure S7. 1 H MR spectrum (400 MHz, chloroform-d, room temperature) of 5.
Figure S8. 1 H MR spectrum (400 MHz, chloroform-d, room temperature) of 6. The significant decrease of Chemical shift peak around 1.4 showed the successful removal of BC protecting group. Figure S9. 1 H MR spectrum (400 MHz, chloroform-d, room temperature) of crgd-pdi
Ps. Figure S10. MALDI-TF mass spectrum of crgd-pdi Ps.
Figure S11. 1 H MR spectrum (400 MHz, chloroform-d, room temperature) of crad-pdi Ps. Figure S12. MALDI-TF mass spectrum of crad-pdi Ps. Figure S13. (a) Representative TEM images and (b) DLS of crad-pdi Ps in PBS (PH=7.4) solution.
Figure S14. Stability of the crgd-pdi Ps hydrodynamic size during different incubation periods with serum. Figure S15. Toxicity evaluation of crgd-pdi Ps. MTT assay IH/3T3 fibroblast cells with crgd-pdi Ps concentration 0.001, 0.01, 0.1, 1, 10, 100, 200 nm after 24 h incubation at 37 o C.
Figure S16. Real-time monitoring of the PA intensity changes of jugular veins in normal mice after injection of crgd-pdi Ps and crad-pdi Ps (300 μl, 3.33 mg ml -1 ). Figure S17. In vivo PA detection of early thrombus under laser irradiation at two different wavelengths. 700 nm and 850 nm wavelengths were used to detect the mouse jugular veins with early thrombus after injection of crgd-pdi Ps. (a) Under 850 nm laser irradiation, the sites of thrombus were marked with yellow circle, and (b) under 700 nm laser irradiation, the sites of thrombus were enveloped by white circle.
Figure S18. Ex vivo PA investigation of major organs embedded in agarose gel. (a) Photographic and PA image of major organs collected after 2 d crgd-pdi Ps injection. From left to right in the top row: liver, spleen, kidney, lung, and stomach. From left to right in the bottom row: intestine, heart, muscle, bone, and skin. (b) PA signal intensity of major organs after 2 d injection of crgd-pdi Ps, crad-pdi Ps and saline. REFERECES 1. Wurthner, F.; Stepanenko, V.; Chen, Z.; Saha-Moller, C. R.; Kocher,.; Stalke, D. Preparation and Characterization of Regioisomerically Pure 1,7-Disubstituted Perylene Bisimide Dyes. J. rg. Chem. 2004, 69, 7933-7939. 2. Cheng, K.; Kothapalli, S. R.; Liu, H.; Koh, A. L.; Jokerst, J. V.; Jiang, H.; Yang, M.; Li, J.; Levi, J.; Wu, J. C.; Gambhir, S. S.; Cheng, Z. Construction and Validation of ano Gold Tripods for Molecular Imaging of Living Subjects. J. Am. Chem. Soc. 2014, 136, 3560-3571.