Design of AIEgens for near-infrared IIb imaging through structural modulation at molecular and morphological levels

Fluorescence imaging in near-infrared IIb (NIR-IIb, 1500–1700 nm) spectrum holds a great promise for tissue imaging. While few inorganic NIR-IIb fluorescent probes have been reported, their organic counterparts are still rarely developed, possibly due to the shortage of efficient materials with long emission wavelength. Herein, we propose a molecular design philosophy to explore pure organic NIR-IIb fluorophores by manipulation of the effects of twisted intramolecular charge transfer and aggregation-induced emission at the molecular and morphological levels. An organic fluorescent dye emitting up to 1600 nm with a quantum yield of 11.5% in the NIR-II region is developed. NIR-IIb fluorescence imaging of blood vessels and deeply-located intestinal tract of live mice based on organic dyes is achieved with high clarity and enhanced signal-to-background ratio. We hope this study will inspire further development on the evolution of pure organic NIR-IIb dyes for bio-imaging.

2) This contribution lacks the traceable measurements that are needed to draw the conclusions and speculations on the clinical applications that are made throughout the contribution. Comparison is made with preclinical tests of 1100 nm LP (8 ms, 37 mW/cm2), 1200 nm LP (10 ms, 37 mW/cm2), 1500 nm LP filter (75 ms, 110 mW/cm2), but excited at 808 with emission collected (non-optimally) at >1500 nm at different powers and time points. The exposure times of 70 ms are quite long compared to video rate NIR-II (8~10 ms ) measurements routine. The authors make conclusions that are not substantiated by the results. The contribution is interesting, but not convincing as more rigorous testing is needed.
3) The authors demonstrate visualization of the intestinal tract located deep within at ~5 mm depth and the individual small bowel diverticula (~1 mm) -this is within the range of visible and NIR-I, NIR-II fluorophores and does not demonstrate the advantages of using NIR-II -the authors should use a larger animal model to show great depth of penetration. 4) The highest spatial resolution of the vessels for 1100, 1200, and 1500 nm LP was 0.58, 0.56 and 0.41 mm. Those were much lower than that of NIR-II agents in the literature (Biomaterials, 2018, 171, 153;Theranostics, 2019, 9, 3866, Nat. Commun. 2018, 9, 11712014, 5, 4206, Nat. Med. 2012, 18, 1841, Did the presented data have better results compared with other studies? 5) The compounds were poorly characterized. Based on the NMR data of these compounds, they were not pure. The NMR spectra data of the samples were not completely interpreted and assigned in the Supplementary information. The purity of these compounds must be insured, as this significantly affects the optical properties of the compounds. Thus, it is required to purify the compounds again and re-check all the related measurements including optical spectra, quantum yields et al.

Reviewer #3:
Remarks to the Author: This manuscript reports the preparation of organic nanofluorophores for biological imaging in the NIR-IIb window. The nanofluorophores were formed by encapsulation of D-A typed AIEgens with alkyl thiophene bridge into amphiphilic copolymers, and displayed fluorescence emission tail extending to 1600 nm. The alkyl chain was tuned to enhance the quantum yield (QY), and the optimized nanofluorophore 2TT-oC26B was utilized for blood vasculature and intestinal tract imaging at NIR-IIb window, demonstrating high imaging quality. The importance of this work lies in the discovery of NIR-IIb emission based on D-A typed organic molecules for bioimaging. This is a remarkable result and has great general interest. It is strongly recommended for publication in the journal after addressing the following points: 1) The current discussion on the molecule design seems problematic and needs improvement. The authors mentioned "…twisted molecular rotors for enhanced molecular motion…" and "…molecular aggregation restrict the intramolecular motion…". These two points sound contradictory.
2) The authors claimed that the buckier alkyl chain could promote intramolecular motion, "which is conductive to the formation of dark TICT state". Then, how to explain that 2TT-oC6B with the least distortion shows the longest peak emission in Fig 2c? 3) Detailed optical data should be provided. For example, the PL spectra in Fig. 2a, S9 and Fig. S11 just cover up to 1300(1200) nm. Longer wavelength data in these conditions are helpful to understand the NIR-IIb emission in Fig 2c. The absorption coefficient and fluorescence quantum yields in organic solvents should also be given. 4) The QY calculation in confusing. Base on "Methods", the fluorescence signals from 900-1500 were used for QY calculation. However, the authors provided the QYs of 1000-1600 nm and 1500-1600 nm. The authors are recommended to provide the original data for these calculations. Fig 2d is strange as the integrated fluorescence for IR26 is negative, why? The authors may provide the original PL spectra with the appropriate unit and scale bar. 5) The way to calculate NIR-IIb QY based on the emission integration ratio may not be accurate. As the emission in NIR-IIb region is very weak and could be significantly affected by the background. Based on Fig 2d, the background signal is substantial. It is better to use the slope of multiple points to get a more accurate NIR-IIb QY. 6) Line 24 "inorganic counterparts" should be "organic counterparts". 7) There is a recent paper on J-aggregate of cyanine dye for NIR-IIb imaging (DOI: 10.1021/jacs.9b10043). The authors may modify the claim on the "first" organic NIR-IIb fluorophore.

To Reviewer I:
This is a very nice contribution that is certainly worthy of publication in Nature Communications. it refers to a non-emissive, or poorly emissive S1 excited state in which the molecular configuration is twisted relative to S0.
Reply: Thanks for your kind suggestion. The "dark TICT state" refers to the weakly emissive S1 excited state, as it efficiently quenches by various nonradiative processes. We have added the explanation in the revised manuscript on Page 3 Paragraph 1 Line 1.

The logic for molecular design is very well articulated on Line 88.
Reply: Thank you exceedingly for your positive comment.

On
Line 153: the authors claim that the use of copolymer to stabilize chromophore suspensions lead to "desirable circulation time". That is not obvious to me why it should be so.
There should be some rationale, or a reference to previous work that demonstrates this to be the case.
Reply: Thank you immensely for your valuable suggestion. We used the amphiphilic copolymer 6. Line 170: should be "spherical" structure.
Reply: Thank you very much for your kind reminder. The mistake has been corrected in the revised manuscript.

To Reviewer II:
This is one of a number of recent contributions to explore the use of the NIR-II wavelength range (1000 nm-1700 nm) in qualitative preclinical imaging. The authors have reported a NIR-IIb agent 2TT-oC26B with excitation at ~1000 nm and collection of emission light >1500 nm and test it in preclinical studies of the blood vessels and the intestinal tract. I believe that the authentic NIR-IIb dye (maximum emission wavelength >1500 nm) will be of great importance for in-vivo molecular imaging and image-guided diagnostics and surgery due to almost zero autofluorescence and much lower photon scattering. Overall, the manuscript is well organized and written, so I recommend publication after major revision. Reply: Thank you very much for your valuable comment. Since the QY of 2TT-oC26B nanoparticles in the NIR-IIb region (1500-1700 nm) is much lower than that of 1100-1700 nm and 1200-1700 nm, it is inevitable to choose higher power density and longer exposure time. It is worth mentioning that the comparisons are conducted in their optimal conditions. Even if the power density or exposure time is increased, the resolution will not improve as much.
According to the review's suggestion, to make the manuscript more rigorous, we have reduced   Reply: Thank you very much for your valuable suggestion. It is no doubt that imaging in visible, NIR-I, and NIR-II can visualize deep tissue to some extent. However, the limited resolution and SBR are their serious drawbacks in such depth. To further compare the bio-imaging ability of 2TT-oC26B NPs in different NIR windows, the capillary tube filled with 2TT-oC26B NPs is immersed in 1% Intralipid solution at pointed phantom depths. As shown in Fig. R2, even at 6 mm immersion depth, the clear tube boundary can be distinguished in the NIR-IIb region, but the tube is blurred and invisible in the NIR-I region. Although the brightest image is recorded in the NIR-II region due to the highest QY, its SBR (1.8) and resolution according to the Gaussianfitted full width at half maximum (FWHM=0.86 cm) is significantly lower than those of in NIR-IIb (SBR=3.1 and FWHM=0.32 cm) based on the advantages of almost zero autofluorescence and much lower photon scattering. The results suggested that imaging in the NIR-IIb region exhibited the highest advantage than in the NIR-I and NIR-II region. We have updated this in the revised manuscript (Page 8) and Supplementary Information (Supplementary Fig. 20). According to the review's suggestion, a large animal (rat) was imaged after being gavage with the 2TT-oC26B NPs. Its intestine structure can be distinguished clearly in the NIR-IIb region at a depth ~8mm while it is difficult to discriminate in the NIR-I and NIR-II region (Fig. R3). We have updated this in the revised manuscript (Page 12) and Supplementary Information   (Supplementary Fig. 25). 4. The highest spatial resolution of the vessels for 1100, 1200, and 1500 nm LP was 0.58, 0.56 and 0.41 mm. Those were much lower than that of NIR-II agents in the literature (Biomaterials, 2018, 171, 153;Theranostics, 2019, 9, 3866, Nat. Commun. 2018, 9, 11712014, 5, 4206, Nat. Med. 2012, 18, 1841, Did the presented data have better results compared with other studies?
Reply: Thank you very much for your valuable suggestion, which is vital to improve the quality of our manuscript. In our previous work, we focused to demonstrate the imaging advantage of the NIR-IIb region, so we only conducted the whole-body imaging macroscopically but ignored monitoring the small vessel structure.
In the revised manuscript, cerebral vasculature of Balb/c nude mouse through the intact scalp and skull was observed in vivo after intravenous injection of 2TT-oC26B NPs (Fig. R4 a-c). The cerebral vessel with an apparent width (i.e., FWHM) of approximately 71.6 μm was distinctly recognized. Although the resolution is slightly lower than that (43.65 μm) in the reported work (Biomaterials, 2018, 171, 153), it is comparable and is the highest reported in the NIR-IIb region by organic materials. Meanwhile, the limited resolution in our work is attributed to the InGaAs camera (TEKWIN), whose pixel size is 25 μm. However, in the aforementioned work (Biomaterials, 2018, 171, 153), the pixel size was as small as 15 μm.
To improve the spatial resolution, high-magnification through-skull microscopic vessel imaging of the brain was also conducted. As shown in Fig. R4 d-

The compounds were poorly characterized. Based on the NMR data of these compounds, they
were not pure. The NMR spectra data of the samples were not completely interpreted and assigned in the Supplementary information. The purity of these compounds must be insured, as this significantly affects the optical properties of the compounds. Thus, it is required to purify the compounds again and re-check all the related measurements including optical spectra, quantum yields et al.

Reply:
Thanks for your critical comment. As you suggested, the compounds were purified, and their NMR spectra were provided, interpreted, and assigned in detail in Supplementary Fig. 1-6. After ensuring the purity of the compounds, all the optical spectra, quantum yields were remeasured and recalculated in the manuscript (Fig. 2, Supplementary Fig. 8-17 and Supplementary Table 1).

To Reviewer III:
This manuscript reports the preparation of organic nanofluorophores for biological imaging in This is a remarkable result and has great general interest. It is strongly recommended for publication in the journal after addressing the following points: 1. The current discussion on the molecule design seems problematic and needs improvement.
The authors mentioned "…twisted molecular rotors for enhanced molecular motion…" and "…molecular aggregation restrict the intramolecular motion…". These two points sound contradictory.
Reply: Thanks for your kind reminder. However, these two points are not in conflict. Compared with traditional planar dyes, AIE luminogens (AIEgens) are often propeller-like in shape and, therefore, have molecular mobility even in the solid-state (ACS Materials Lett. 2019, 1, 425-431).
In the aggregate state, the twisted NIR-IIb emitters are favorable for molecular motion compared to planar molecules, which in turn promotes formation of the TICT state. On the other hand, molecular aggregation partially restricts the intramolecular motions, giving strong fluorescence.
It should be noted that AIEgens has molecular mobility in the aggregate state. Thereof, the TICT state and high fluorescence efficiency can be achieved simultaneously in the aggregate state.
Thus, these two points are not contradictory. As suggested, we have added the above explanations in the conclusion section of the revised manuscript. Reply: Thank you for your kind suggestion. According to your comments, all the PL in Fig. 2a,   S9, S11 spectra were remeasured extended to 1600 nm in the revised manuscript. The molar absorption coefficient of 2TT-oC6B, 2TT-oC26B and 2TT-oC610B molecule in THF is 2.49⨯10 4 , 2.25⨯10 4 , 2.13⨯10 4 L mol -1 cm -1 , respectively. The fluorescence quantum yields in organic solvents were measured and calculated ( Supplementary Fig.17  Reply: Thanks for your suggestion. We are sorry for miswriting 900-1500 nm as 1000-1600 in the manuscript. In our previous version, we calculated the whole NIR-II QY (900-1500) as the NIR-II QY according to the reference (Nat. Commun., 2014, 5, 4206). The QYs of 1500-1600 nm were calculated according to the integration area ratio (QY 1500-1600 nm =QY NIR-II *A 1500-1600nm /A 900-1500 ). As you suggested, the original data used for calculation the QY was provided in Fig.   R5, and the source data was provided as a Source Data file. The negative integrated fluorescence of IR26 is because we used the same conditions to measure the PL spectra of the sample. Hence the negative noise of IR26 is evidently due to the detector of the PL machine. Thus, the integrated fluorescence of IR26 is negative. However, the negative, integral area does not affect the slope calculation, so we did not calibrate the curve in our To reduce errors, we used a new PL instrument (Horiba iHR 320 with InGaAs detector) whose baseline was calibrated well. The QYs from 1000-1600 nm as NIR-II QY and 1500-1600 nm as NIR-IIb QY were all remeasured using the slope of multiple points (Fig. 2d, Supplementary Fig.   15-16). The value of QYs was concluded in Supplementary Table 1. The original data was provided as a Source Data file.

The way to calculate NIR-IIb QY based on the emission integration ratio may not be accurate.
As the emission in NIR-IIb region is very weak and could be significantly affected by the background. Based on Fig 2d, the background signal is substantial. It is better to use the slope of multiple points to get a more accurate NIR-IIb QY.
Reply: Thanks for your kind suggestion. We have prepared the samples and measured its PL spectra. According to your advice, to get a more accurate result, the QYs of 1000-1600 nm and 1500-1600 nm were calculated using the slope of multiple points (Fig. 2d, Supplementary Fig.   14-16). The value of QYs was concluded in Supplementary Table 1. 6. Line 24 "inorganic counterparts" should be "organic counterparts".
Reply: Thanks for your kind reminder. The mistake has been corrected in the revised manuscript.
7. There is a recent paper on J-aggregate of cyanine dye for NIR-IIb imaging (DOI: 10.1021/jacs.9b10043). The authors may modify the claim on the "first" organic NIR-IIb fluorophore.
Reply: Thanks for your suggestion. This excellent paper attracted our attention when the manuscript was under consideration. According to your suggestion, we have revised the manuscript and cited the paper.