Rational design and facile synthesis of AIE-CNPy-AD. We make full use of the “all-in-one” strategy to pursue high-performance probes for early in-situ tracing of Aβ fibrils in vivo. As introduced above, extended D-π-A electronic architecture is built by utilizing the dimethylamino as electron-donor, the acetonitrile and pyridyl group as electron-acceptor, single bonds as linkers, and benzyl groups and C=C double bond as π-bridges, respectively. Red or even NIR emission and relatively long-wavelength excitation is supposed to result from this D-π-A structured backbone. Such a rod-like geometric configuration is envisioned to bestow the probe with strong binding affinity to Aβ fibril/plaque. Moreover, according to the principle of restriction of intramolecular motions (RIM), the multiple rotors in the skeleton would efficiently consume the excited-state energy and lead to weak or even no emission in the unconstrained state, while the distorted 3D conformation would prevent π-π stacking and self-quenching in the constrained state. It means such an AIE-active probe would give light-up response to the target species with satisfactory SNR under suitable conditions. To further reinforce the SNR, hydrophilic propanesulfonate group is incorporated to the hydrophobic skeleton to for one thing enhance the solubility of the probe in aqueous media and thus reduce the background/noise signal, and for another to strengthen the binding between the probe and Aβ fibrils/plaques via multiple electrostatic interactions. Moreover, the considerable lipophilicity of the AIE-CNPy-AD is anticipated to be sufficient to ensure desirable BBB penetrability. The elaborately designed rod-like amphiphilic NIR-emissive zwitterionic AIE probe AIE-CNPy-AD was conveniently synthesized according to the synthetic route shown in Fig. S1 in the Electronic Supporting Information (ESI) with commercially available cheap raw materials. AIE-CNPy-AD was fully characterized with the help of 1H (Fig. S2), 13C NMR (Fig. S3) and high-resolution mass spectrometry (HRMS, Fig. S4).
Excellent photophysical properties facilitating the detection of Aβ fibrils/plaques. AIE-CNPy-AD showed an absorption maximum at 455 nm and an emission maximum at 720 nm (10–4 M) in DMSO solution (Fig. 2A), with the Stokes shift of as large as 265 nm. As AIE-CNPy-AD is soluble in highly polar solvents but aggregates in less polar solvents, DMSO was chosen as a good solvent, and THF was selected as a poor solvent to evaluate the AIE behaviors of AIE-CNPy-AD (Fig. 2 and Fig. S5). As anticipated, with the addition of THF, fluorescence enhancement was clearly observed, with the emission peak blue-shifted from 720 nm (DMSO solution) to 675 nm (DMSO/THF = 1/99, v/v), manifesting the synergistic effect of AIE and intramolecular charge transfer (ICT) (Fig. 2B). The emission intensity continuously and slowly increased when the THF fraction (fTHF) was no more than 60 vol%, while boosted sharply once fTHF reached 70 vol% (Fig. 2C). Remarkably, AIE-CNPy-AD was hardly affected by the pH value varying from 3 to 10 which covers the normal pH range of human body (Fig. S6 and Fig. 2D), suggesting the good pH stability of the fluorescence properties of our probe. Taken together, all these results undoubtedly demonstrated the remarkable AIE feature, strong ICT effect, and the stable NIR fluorescence in the aggregated state of AIE-CNPy-AD, which are conducive to the imaging of Aβ deposits.
Superb specificity and high affinity of AIE-CNPy-AD to Aβ fibrils. The binding affinity of AIE-CNPy-AD to hen egg white lysozyme (HEWL), a common model protein for amyloid studies, was evaluated first. As shown in Fig. 3A, dramatic fluorescence enhancement at 620 nm was clearly observed with the increasing concentration of fibrillar HEWL in the PBS solution. In sharp contrast, AIE-CNPy-AD displayed much less marked fluorescence response to native HEWL at the same concentration level (Fig. S7A). As compared to the emission in DMSO solution, the probe bound to HEWL showed an emission peak blue-shifted from 720 to 620 nm, which might be attributed to the ICT effect and less polar microenvironment of the protein pockets. To our satisfaction, AIE-CNPy-AD has much higher ability to distinguish fibrillar HEWL from native ones, as ThT had the same response to fibrillar and native HEWL (Fig.s 3B, 3C, and Fig. S7B). Moreover, due to the strong ACQ effect of ICG, just a small amount of fibrillar HEWL would prompt the quenching of fluorescence (Fig. S7C and Fig. 3C), making it unworkable in the detection of fibrillar HEWL. These results suggest that AIE-CNPy-AD possess higher possibility to function as a highly specific probe to Aβ fibrils in comparison to clinically used gold standard fluorescent probes such as ThT and ICG.
We then studied whether this AIE-active NIR probe could have a specific fluorescence response to Aβ fibrils. Aβ1‒42 peptide was fully incubated to afford Aβ1‒42 fibrils with expected fibrous or filamentous structure (Fig. 3D). When the Aβ1‒42 fibrils were added, emission intensity of AIE-CNPy-AD increased consistently with the emission peak blue-shifted to 620 nm like the situation of binding to HEWL (Fig. 3E). Obviously, the background from AIE-CNPy-AD is far lower than that of ThT, which is merely 1/35 times that of ThT. Whereas, the signal of AIE-CNPy-AD upon interaction with Aβ1‒42 fibrils is much higher than that of ThT under parallel conditions, which is about 1.4 times that of ThT (Fig. S8). In consequence, the SNR of AIE-CNPy-AD reached 60, which is 10 times to that of ThT (SNRThT = 6; Fig. 3F). The significantly enhanced SNR of AIE-CNPy-AD achieved by the minimization of background and amplification of signal could be interpreted as follows: (i) on one hand, the right hydrophilicity makes the AIE-CNPy-AD well dispersed in aqueous solution and the vigorous intramolecular motions efficiently exhaust the excited state, resulting in minimal emission; (ii) on the other hand, the strong binding of AIE-CNPy-AD to Aβ1‒42 fibrils greatly hampered the intramolecular motions and activated the radiative decay channels, maximizing the fluorescence signal as a consequence of AIE effect. Ultrasensitive detection of Aβ1‒42 fibrils could be expected.
In addition to the ultra-high SNR, AIE-CNPy-AD also has high specificity to Aβ1‒42 fibrils. A large variety of biological species including carbohydrates, amino acids, peptides, and other proteins were employed to assess specificity of AIE-CNPy-AD to Aβ1‒42 fibrils (Fig.3G, Fig. S9). It is evident that AIE-CNPy-AD not only hardly has response to interfering small molecular species, but also has low response to potentially competitive peptides and enzymes with large molecular weight, especially Aβ1‒42 monomer (Fig. 3G and Fig. S9).
Apart from specificity, binding affinity of the probe to analyte is also a vital parameter that ensures accurate tracing of the Aβ1‒42 fibrils. Displacement assay of AIE-CNPy-AD against ThT-bound Aβ1‒42 fibrils (Fig. S10 and Fig. 3H) was then carried out to investigate the binding affinity. Fluorescence intensity of the pre-prepared ThT/Aβ1‒42 fibrils complex was firstly recorded with excitation at 420 nm. AIE-CNPy-AD solution was subsequently added stepwise into the ThT/Aβ1‒42 complex, and the emission intensities of these two probes were measured under excitation at their corresponding maximum absorption wavelengths. It is observed that the emission intensity of ThT at 482 nm decreased continuously with increasing concentration of AIE-CNPy-AD; and in the meantime, fluorescence of AIE-CNPy-AD peaked at 620 nm emerged and was enhanced accordingly. Remarkably, it is indicated that AIE-CNPy-AD displaced ThT from ThT/Aβ fibrils complex to generate the more strongly bound AIE-CNPy-AD/Aβ fibrils complex in the solution. Besides, dissociation constant (Kd)41 of AIE-CNPy-AD was calculated to be 185 nM (Fig. S11), considerably smaller than that of ThT (890 nM)42. Sufficient evidences proved that AIE-CNPy-AD has much higher binding affinity to Aβ1‒42 fibrils than ThT.
High-contrast and high-resolution in-vitro fluorescent staining of paraffin slices of mice brains. It is confirmed that AIE-CNPy-AD, with high specificity, binding affinity and SNR, exhibits excellent performance on precise detection of Aβ1-42 fibrils in solution. To explore the ability of AIE-CNPy-AD to label Aβ plaques in brain tissues, in-vitro fluorescent staining of paraffin mice brain slices resected from 5*FAD transgenic mice, APP/PS1 transgenic mice and age-matched wild-type mice was conducted. False signals originated from the binding of AIE-CNPy-AD or antibody with proteins or interfering species in brain cells could be identified through the localization of nuclei with Hoechst 33342. Notably, as displayed in Fig. 4 and Fig. S12, no matter in 2.5-month-old 5*FAD transgenic mice or in 6-month-old APP/PS1 transgenic mice, Aβ plaques were unambiguously visualized with bright red fluorescence. Specific labeling of Aβ plaques with high contrast and high resolution in these mice indicates that AIE-CNPy-AD is universal to label Aβ plaques in different strains of mice. Moreover, it can be easily seen from the CLSM images, the Aβ plaques in 2.5-month-old 5*FAD transgenic mice (Fig. 4A and Fig. S12A-S12C) are larger and more densely distributed than that in 6-month-old APP/PS1 transgenic mice (Fig. 4F and Fig. S12D-S12F). The experimental results agreed well with situation of early plaque formation at the time point in these two strains of mice43,44, implying the reliability of AIE-CNPy-AD in fluorescent staining of Aβ plaques in slices of mice brain. To our delight, there was no observable intracellular fluorescence signal, which implied that our probe is preferentially bound to the Aβ plaques generally forming extracellularly, eliminating the false signal from intracellular species. Simultaneously, the signals in the red channel agree fairly well with those in the green channel, manifesting that AIE-CNPy-AD has very high specificity comparable to that of antibody, to Aβ plaques (Fig. 4B-4E, 4G-4J). In contrast with transgenic mice, no plaques were found in the age-matched wild-type mice no matter observing from red or green channel (Fig. S13), which not only verified the high specificity of AIE-CNPy-AD to Aβ plaques for a second time, but also suggested the fairly high fidelity of the designed probe in real brain tissues.
In-vivo imaging of Aβ plaques in live mice with outstanding BBB penetrability. Inspired by the superb red fluorescence light-up Aβ plaque-specific response in the brain slices of mice, we further evaluated the biocompatibility and BBB penetrability of AIE-CNPy-AD which is indispensable before in-vivo imaging. Cell viability experiments were first carried out to ascertain the biocompatibility of AIE-CNPy-AD by CCK-8 assays. Human neuroblastoma cells (SH-SY5Y), mouse brain neuroblastoma cells (Neuro-2a), mouse breast cancer cells (4T1) and human breast cancer cells (MCF-7) were incubated with different concentrations of AIE-CNPy-AD for 24 h, respectively. The cell viabilities of all these four cell lines both kept at a level close to 100% even at a probe concentration of 32 µM (Fig. S14A‒Fig. S14D). The results provided strong evidence to the fact that AIE-CNPy-AD has low cytotoxicity and favorable biocompatibility to various cells, demonstrating its high applicability to live animals. The oil-water partition coefficient (log P) is often used as an index indicating the possible ability of penetrating BBB45. The log P of AIE-CNPy-AD was determined to be 1.24 by shaking-flask method, which is far larger than that of ThT (0.16),8 suggestive of the higher lipophilicity and BBB penetrating potential of AIE-CNPy-AD as compared with ThT.
The feasibility of AIE-CNPy-AD tracking Aβ plaques in vivo was confirmed using 2.5-month-old 5*FAD mice, 6-month-old APP/PS1 mice and age-matched wild-type mice as model mice. Live imaging of these mice was performed after the tail vein injection of AIE-CNPy-AD (Fig. 5). Almost all the fluorescence signals were clearly witnessed in the center of brain compartments and able to be efficiently captured. Apparently, the fluorescence signals in the brain regions of 2.5-month-old 5*FAD mice (Fig. 5A) were already much stronger than those in the wild-type mice at 5 min post injection (Fig. 5B). Particularly, with the decay of signal, difference in signal intensity recorded from 5*FAD and wild-type mice was enlarged as indicated by the semi-quantitative analysis of the images (Fig. 5E). Similarly, intense fluorescence signal is readily visible from the brain area of 6-month-old APP/PS1 mice after the injection of AIE-CNPy-AD. Moreover, the contrast between the fluorescence signals from APP/PS1 and 6-month-old wild-type mice is quite dramatic. Compared with 5*FAD mice, difference in the signal intensity between APP/PS1 mice and age-matched wild-type mice was 4.7 times larger than that between 5*FAD mice and the corresponding wild-type mice at 5 min after being injected with AIE-CNPy-AD (Fig. 5G, 5H, and 5K). It might be because the formation of Aβ deposits in APP/PS1 mice is slower than that in 5*FAD mice. In addition, fluorescence signal intensity of APP/PS1 mice declined faster than that of 5*FAD mice after being injected with AIE-CNPy-AD for 30 min, as it can be seen from the semi-quantitative analysis of the images (Fig. 5K). It is possibly because that there are larger and more Aβ plaques in 2.5-month-old 5*FAD mice than in 6-month-old APP/PS1 mice, which slows down the clearance of AIE-CNPy-AD from the brain. Notably, whether in 5*FAD mice or APP/PS1 mice at 1 h post probe injection, F(t)/F(Pre) value of transgenic mice was at least 1.75 times that of wild-type mice, indicative of the potential of AIE-CNPy-AD to realize long-term tracking of Aβ plaques in vivo.
The case as for ThS is very different from that of AIE-CNPy-AD. As shown in Fig. 5C, 5D, 5F, 5I, 5J, and 5L, hardly any valid signal was found from the 5*FAD mice, APP/PS1 mice and wild-type mice administrated with ThS, clearly revealing the powerlessness of ThS in in-vivo imaging of Aβ plaques. This most probably results from the combination of its poor BBB permeability, short excitation and emission wavelengths which cannot penetrate the skull of mice, and the ACQ effect46. Undoubtedly, these visualization results directly validated that AIE-CNPy-AD is capable of penetrating the BBB and imaging Aβ fibrils/plaques in vivo with high contrast and fidelity.
In-vivo tracking of Aβ plaques in APP/PS1 mice at a very early stage. It has confirmed that AIE-CNPy-AD exhibits significant signal difference between transgenic mice and wild-type mice in a relatively long time-period post probe administration. It can be envisaged that AIE-CNPy-AD might be promising for early diagnosis of AD in transgenic mice by virtue of the Aβ plaques-sepecific in-vivo imaging ability of AIE-CNPy-AD. Young APP/PS1 transgenic mice of 2-month-, 3-month-, 4-month-, and 6-month-old and age-matched wild-type (WT) mice were thus employed to systematically assess the performance of AIE-CNPy-AD in early diagnosis of AD (Fig. 6 and Fig. S15). It was observed that the difference in the fluorescence signal intensity of APP/PS1 transgenic mice and wild-type mice already became very evident at the age of 4 month. In the meantime, the fluorescence signal intensity is positively correlated with the age of transgenic AD mice (Fig. 6A). More specifically, at 20 min post injection, the F(t)/F(Pre) value of the 4-month-old APP/PS1 transgenic mice was 1.86 times that of the age-matched WT mice, and the F(t)/F(Pre) value of 6-month-old APP/PS1 transgenic mice was 2.06 times that of the age-matched WT mice (Fig. 6B). Meanwhile, there was no apparent signal difference in WT mice of different months (Fig. 6C), and the F(t)/F(Pre) values of 2-month-old and 3-month-old APP/PS1 transgenic mice were almost the same as those of the age-matched WT mice (Fig. 6D). These imaging results especially those acquired with APP/PS1 transgenic mice at an age of 4-month-old clearly showed that AIE-CNPy-AD is competent for the precise diagnosis of AD at a super-early stage. It has been speculated that the reason for the failure of early drug intervention on AD is probably that intervention in the phase is not early enough, which in turn is greatly related to the efficient capture of biological manifestations rather than clinical manifestations in the early diagnosis47,48. The experimental results obtained with AIE-CNPy-AD is of great significance because APP/PS1 transgenic mice are found to exhibit memory deficits from 5 months old on which is early clinical presentations, confirming that AIE-CNPy-AD can diagnose AD of APP/PS1 transgenic mice during the period of early biological manifestations, prior to the appearance of clinical presentations43,47,49. Furthermore, as shown in Fig. S16, ThS could neither give discriminatory signals between the APP/PS1 transgenic mice and wild-type mice nor provide distinct signals among mice of different ages, manifesting that our probe outperforms the clinically used gold-standard probe and could be used as an upgraded alternative to the commercially available ones.
It is worth mentioning that AIE-CNPy-AD does not only hold satisfactory cytocompatibility with negligible toxicity to diverse cells including neuronal cell lines but also has outstanding in-vivo biocompatibility as suggested by Fig. S17. The H&E staining results of the heart, liver, spleen, lung, kidney, and brain slices resected from 5*FAD (2-month-old), APP/PS1 (6-month-old) transgenic mice, and wild-type mice (6-month-old) at 24 h post the administration with AIE-CNPy-AD via tail vein injection are almost identical to those injected with PBS buffer under parallel conditions. It manifests that AIE-CNPy-AD does not cause obvious necrosis to tissues, suggesting the superb in-vivo biocompatibility of the present probe. The Tunel results of the brain slices further verifies this as there is no evident apoptosis being observed in all the samples. Therefore, in view of the very low toxicity to cells and tissues, the AIE-CNPy-AD is proven to be highly suitable for in-vivo detection and has great potential in clinical use.
Ex-vivo observation of frozen brain slices of mice pre-administrated with AIE-CNPy-AD further validates its high fidelity of Aβ plaques-specific imaging. To further examine whether the AIE-CNPy-AD binds exclusively to Aβ plaques in the brain of live mice, the APP/PS1 transgenic mice of different ages and age-matched wild-type mice were injected with AIE-CNPy-AD and sacrificed at 15 min post-administration. The frozen slices of these mice brain were then stained with rabbit anti-mouse primary antibody (ab201060), Alexa Fluor® 488-labeled goat anti-rabbit secondary antibody (ab150077), and Hoechst 33342 in sequence. As shown in Fig. 7A, 7F, and Fig. S17, the Aβ plaques were imaged with high resolution and high contrast. Moreover, it can be clearly observed that the Aβ plaques in brain slices of 6-month-old APP/PS1 transgenic mice were larger and richer in number than those of 4-month-old APP/PS1 transgenic mic, while no significant Aβ plaques were found in the brain slices of 2-month-old, 3-month-old APP/PS1 transgenic mice (Fig. S18), and all the age-matched WT mice (Fig. S19). Manifestly, AIE-CNPy-AD indeed labelled Aβ plaques in vivo with ultrahigh fidelity and specificity, as suggested by the very good overlap between the green (antibody) and red channels (AIE-CNPy-AD; Fig. 7B-7E and 7G-7J).
Elucidation of the working mechanism with molecular docking simulations. We believe that the rod-like architecture, extended D-π-A electronic structure, flexible 3D conformation, the amphiphilic and zwitterionic molecular structure collectively contribute to the outstanding performance of AIE-CNPy-AD in Aβ fibrils/plaques-specific detection and imaging. As above mentioned, the rod-like structure is supposed to favors the recognition of β sheets. Moreover, the hydrophobic π-conjugated backbone and the pyridyl and sulfonate groups are envisaged to benefit the binding of AIE-CNPy-AD to Aβ species possibly via hydrophobic interaction, π-π interaction, and electrostatic interaction. When in solution or molecularly dispersed, the AIE-CNPy-AD molecules undergo active intramolecular motions, leading to an OFF state and a low background. When coexists with Aβ monomer, intramolecular motions of AIE-CNPy-AD are merely weakly restricted, rendering weak red emission released (near-OFF state), due to the weak binding affinity of AIE-CNPy-AD and Aβ monomer. On the contrary, strong interactions between AIE-CNPy-AD and fibrils impose severe restriction on the intramolecular motions, which activates the AIE process50,51, and switches AIE-CNPy-AD from OFF to ON state to emit strong red/NIR fluorescence. Thus, AIE-CNPy-AD can distinguish Aβ fibrils from Aβ monomers precisely (Fig. 8A).
Molecular docking simulations were carried out to unveil the interactions between AIE-CNPy-AD and Aβ fibrils or monomer and to elucidate the working mechanism of specific detection of Aβ fibrils. As revealed by the three top docking conformations with the lowest binding free energies shown in Fig. 8B, 8C and Fig. S20, the most preferable binding direction of AIE-CNPy-AD to Aβ fibrils is consistent with the orientation of the β-sheets of Aβ fibrils, which proves that rod-shaped geometric structure of AIE-CNPy-AD is very helpful to the binding with Aβ fibrils53. Simultaneously, the hydrophobic PHE-19 residue on the β-sheets of Aβ fibrils has strong C-H···π interaction (2.6 Å) with the phenyl ring of AIE-CNPy-AD, and the ALA-21 residue exhibits very strong interaction with the cyano group of AIE-CNPy-AD (2.2 Å). To our astonishment, the GLY-25 residue and two LYS-28 residues have strong hydrogen bonding interactions with the sulfonate group of AIE-CNPy-AD (1.7−2.1 Å). It is obvious that sulfonate not only improves water solubility of AIE-CNPy-AD, but also further enhances the binding ability of AIE-CNPy-AD to Aβ fibrils via H-bonding and electrostatic interactions. Collectively, the strong intermolecular interactions between AIE-CNPy-AD and Aβ fibrils greatly hinder the intramolecular motions of AIE-CNPy-AD to give out the “lighted-up” fluorescent response54.
In contrast, only the LYS-28 residue of Aβ monomer shows hydrogen bonding interaction with the sulfonate unit of AIE-CNPy-AD and no other interactions exist, which results in weaker restriction on the intramolecular motions and weak fluorescence (Fig. 8D and Fig. 8E). The inhibition constant (Ki)55 of AIE-CNPy-AD and Aβ fibrils (935 nM) is far smaller than that of AIE-CNPy-AD and Aβ monomer (45.0 μM). Simultaneously, the lowest docking energy of AIE-CNPy-AD and Aβ fibrils was calculated to be -8.23 kcal/mol, which is substantially lower than that of AIE-CNPy-AD and Aβ monomer (-5.93 kcal/mol). As exhibited in Table S1, all the ten best binding poses with the lowest energies of AIE-CNPy-AD and Aβ fibrils consistently display lower docking energies than those of AIE-CNPy-AD and Aβ fibrils. These data sufficiently suggested that binding affinity of AIE-CNPy-AD to Aβ fibrils is much stronger than that of AIE-CNPy-AD to Aβ monomer. Moreover, the binding energy of AIE-CNPy-AD and Aβ fibrils is lower than that of ThT and Aβ fibrils (-7.18 kcal/mol)56, indicating that the AIE-CNPy-AD possesses higher affinity to Aβ fibrils as compared to ThT. The molecular docking simulation results are powerful proofs to our experiment results and further verify the rationality and feasibility of our design strategy.