Polarity-active NIR probes with strong two-photon absorption and ultrahigh binding affinity of insulin amyloid fibrils

Amyloid fibrils are associated with many neurodegenerative diseases. In situ and in vivo visualization of amyloid fibrils is important for medical diagnostics and requires fluorescent probes with both excitation and emission wavelengths in the far-red and NIR region, and simultaneously with high binding-affinity to amyloid fibrils and the ability to cross the blood–brain barrier, which, however, remain a challenge. Here, we rationally design and synthesize an excellent polarity-sensitive two-photon excited NIR fluorophore (TZPI) based on a donor (D)–acceptor (A)-ion compound. The electron-rich carbazole group and the ionic pyridinium bromide group, linked by an electron-poor π-conjugated benzothiadiazole group, ensure strong near infrared (NIR) emission. Furthermore, the lipophilic carbazole together with the benzothiadiazole group facilitates docking of the probe in the hydrophobic domains of amyloid aggregates with the dissociation constant Kd = 20 nM and 13.5-fold higher binding affinity to insulin fibrils than the commercial probe ThT. On association with the amyloid fibrils, the tiny decrease in polarity leads to a large increase in its NIR emission intensity with an on–off ratio > 10; meanwhile, the TZPI probe exhibits a quantum yield of up to 30% and two-photon absorption cross-section values of up to 467.6 GM at 890 nm. Moreover, the application of TZPI in two-photon imaging is investigated. The ultrahigh binding affinity, the strong NIR emission, the good two-photon absorption properties, the high photo-stability, the appropriate molecular mass of 569 Da and the lipophilicity with log P = 1.66 ± 0.1 to cross the BBB make TZPI promising as an ideal candidate for detecting amyloid plaques in vivo.


The partition coefficient in n-octanol experimental determination of log P
According to published articles, 2 a certain amount of TZPI in octanol 2.0 mL was subjected to partition with octanol-saturated water 2.0 mL. The resulting mixture was stirred vigorously for 5 min., and centrifuged at 2,000 rpm for 5 min. The octanol layer was separated from water layer, and its fluorescence spectrum was recorded as FL-1. The above water layer was partitioned with water-saturated octanol 2.0 mL, and the octanol layer was separated after 5 min. vigorous stirring and 5 min. centrifuge at 2,000 rpm, and its spectrum was recorded as FL-2. As shown in Fig. S2, the emission peaks of the two spectra at 650 nm are 14820 and 410, respectively. The log P value was calculated by the fluorescence intensity ratio at 650 nm for the above two octanol

Synthesis and characterization of TZPI
Scheme S1. Synthetic route of TZCHO.
Then the reaction mixture was poured into water and extracted with CH 2 Cl 2 and dried over anhydrous Mg 2 SO 4 . The crude product was purified by column chromatography on silica gel(petroleum ether/ DCM = 1/2)to give a orange red powder of TZCHO (257 mg, 72% of the yield). 1

Fibrils formation
Insulin amyloid fibrils were prepared according to the protocols described elsewhere. 3 A stock solution containing Insulin (5 mg·mL -1 ) and HCl (0.01 M, pH = 2) were prepared as shown in Scheme S3a.
The solutions were filtered through a 0.2 μm filter and then incubated in an orbital thermomixer with constant agitation at 600 rpm at 60℃ for 24 h. After heating, the solutions were cooled and then centrifuged (Jouan SA A14 centrifuge) at 3000 rpm for 3 min to remove any globular particulates. The supernatant liquid containing the fibrils was removed and stored at 4℃ until needed; this was used as the stock fibril solution. Fibril solutions were not used past 1 week.

Optical Measurement
The steady-state absorption spectra were measured on a Shimidazu UV-3600 UV-VIS-NIR spectrophotometer. The stationary fluorescence spectra and absolute fluorescence quantum yields were performed on a Horiba FluoroMax-4-NIR spectrophotometer equipped with an integrating sphere. The fluorescence lifetime was measured using an Edinburgh Instrument FLS980 Integrating sphere (FLS980 Series of Fluorescence Spectrometers).
In the study of using TZPI and ThT as an ex situ probe in scheme S3a, an aliquot of the insulin solution taken out from the incubation mixture at a defined time was diluted with Tris-HCl buffer, followed by the addition of the probe. The final concentrations of insulin, TPZI and ThT were 10 μg/mL, 20 nM and 20 nM, respectively.
In the study of using TZPI and ThT as an in situ probe in scheme S3b，TZPI or ThT was added to insulin solution prior to incubate at 60 °C with constant agitation at 600 rpm. Then an aliquot of the insulin solution with the probe taken out from the incubation mixture at a defined time was diluted with Tris-HCl buffer. The final concentrations of insulin, TZPI and ThT were 10 μg/mL, 20 nM and 20 nM, respectively.
Under the condition of avoiding light, we shake the solution by hand for 10 seconds to mix well and make the fluorescence probe fully combine with the substance, and then the and then the spectra were measured on spectrophotometer.

Transmission electron microscopy (TEM) experiment
In order to confirm the existence of fibrils, the solution of insulin was incubating for

Two-photon fluorescence imaging experiment
The two-photon imaging was investigated by a commercial microscope (TCS                Furthermore，the K d for TZPI/ Aβ 42 fibrils was derived by a fluorescence titration method and the value was found to be 58 nM, indicating a high binding affinity.