Amyloid-β Deposits Target Efficient Near-Infrared Fluorescent Probes

Dec 30, 2015 - Smart D-π-A Type Near-Infrared Aβ Probes: Effects of a Marked π Bridge on Optical and Biological Properties. Kaixiang Zhou , Hongcun...
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Amyloid-# Deposits Target Efficient Near-Infrared Fluorescent Probes: Synthesis, in Vitro Evaluation and in Vivo Imaging Hualong Fu, Peiyu Tu, Liu Zhao, Jiapei Dai, Boli Liu, and Mengchao Cui Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04441 • Publication Date (Web): 30 Dec 2015 Downloaded from http://pubs.acs.org on December 31, 2015

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Amyloid-β Deposits Target Efficient Near-Infrared Fluorescent Probes: Synthesis, in Vitro Evaluation and in Vivo Imaging Hualong Fu,† Peiyu Tu,† Liu Zhao,† Jiapei Dai,‡ Boli Liu,† and Mengchao Cui*,† †

Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, People’s Republic of China ‡

Wuhan Institute for Neuroscience and Neuroengineering, South-Central University for Nationalities, Wuhan 430074, People’s Republic of China * Phone/Fax: +86-10-58808891. E-mail: [email protected] ABSTRACT: The formation of extracellular amyloid-β (Aβ) plaques is a common molecular change that underlies several debilitating human conditions, including Alzheimer’s disease (AD); however, the existing near-infrared (NIR) fluorescent probes for the in vivo detection of Aβ plaques are limited by undesirable fluorescent properties and poor brain kinetics. In this work, we designed, synthesized and evaluated a new family of efficient NIR probes that target Aβ plaques by incorporating hydroxyethyl groups to the ligand structure. Among these probes, DANIR 8c showed excellent fluorescent properties with an emission maximum above 670 nm upon binding to Aβ aggregates and also displayed a high sensitivity (a 629-fold increase in fluorescence intensity) and affinity (Kd = 14.5 nM). Due to the improved hydrophilicity that was induced by hydroxyls, 8c displayed increased initial brain uptake and a fast washout from the brain, as well as an acceptable biostability in the brain. In vivo NIR fluorescent imaging revealed that 8c could efficiently distinguish between AD transgenic model mice and normal controls. Overall, 8c is an efficient and veritable NIR fluorescent probe for the in vivo detection of Aβ plaques in the brain.

INTRODUCTION Cerebral dysfunction or disease, such as Alzheimer’s disease (AD), can result in damage to essential functions of the brain including memory loss, cognitive decline, as well as deficits in language and complex motor skills. According to the amyloid hypothesis, extracellular amyloid-β (Aβ) senile plaques (SPs) represent a characteristic pathology that is found in the brains of AD patients, and the accurate detection of these plaques has gained momentum in AD research, which provides clues to the early diagnosis and potential treatments of the disease.1,2 Currently, brain imaging for the early diagnosis of AD has strongly relied on positron emission tomography (PET) and magnetic resonance imaging (MRI) to achieve a sufficient penetration depth. However, these modalities are intrinsically faced with the shortcomings of long scanning times and high-cost facilities, thereby impairing their widespread application. Fluorescence is arguably an attractive and versatile tool in analytical sensing and optical imaging in terms of its exquisitely high sensitivity, rapid data acquisition, and technical simplicity.3,4 Recently, the marked advances in optical imaging have allowed for the evolution from “planar imaging” (i.e., fluorescence reflectance imaging, FRI) to three-dimensional imaging (i.e., fluorescence molecular tomography, FMT). Multiphoton microscopy imaging has a high three-dimensional spatial resolution that is afforded by the technique and allows for the chronic observation of individual Aβ plaques in the brain of living transgenic (Tg) mice. However, this modality requires probing through the cranial window, which results in it being an invasive and narrow-viewed imaging approach.5,6

Fortunately, near-infrared (NIR) imaging is a sensitive and non-invasive method that enables the real-time visualization of biomolecules in living systems. Particularly, the performance of this modality as a type of biological imaging necessarily depends on probes fluorescing in the NIR spectral range (“NIR window”, 650-900 nm), which is advantageous for its capacity to penetrate deep tissue and receive minimal interference from the autofluorescence of biomolecules.7 In addition, a viable fluorochrome for biological imaging should have additional characteristics including high fluorescent quantum yield (QY), biostability and proper pharmacokinetics.8 Furthermore, many molecular imaging probes possess dual functions of being a ligand and a fluorochrome, which allows for the design of small and compact NIR probes (MW < 600 Da), thereby, allowing for sufficient permeability across the bloodbrain barrier (BBB). Currently, according to these general requirements, AOI987 (Figure 1) was the first exploited probe for in vivo NIR imaging of Aβ plaques in the brain; however, its ionic nature resulted in the unfavorable BBB penetration.9 Moreover, Ran et al. have evaluated difluoroboron-derived curcumin analogues CRANAD-2 and -58 (Figure 1) as in vivo NIR imaging probes that target soluble or insoluble Aβ species.10,11 In vivo NIR imaging demonstrated that CRANAD-2 could distinguish between 19-month-old Tg2576 mice and wild-type (WT) mice. Furthermore, CRANAD-58 was capable of detecting soluble Aβ species in vivo. Nevertheless, the slow washout rate from the brain hindered their widespread use. To search for more practical Aβ NIR imaging probes, our group has developed a

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Figure 1. Chemical structures of reported probes for the detection of soluble/insoluble Aβ species and the newly designed NIR probes.

series of small molecules for Aβ imaging that center on electron donor-acceptor architecture.12-15 Among these probes, DANIR 2c (Figure 1, MW = 249.31) appears as the smallest Aβ probe with a high affinity (Kd = 26.9 nM) and significant fluorescence response to Aβ aggregates, and also displayed excellent BBB penetration.12 Nevertheless, DANIR 2c was not a “veritable” NIR probe because the emission wavelength (625 nm) shifted out of the “NIR window” after binding to Aβ aggregates. Subsequently, MCAAD-3 and BBTOM-3 (Figure 1) were reported and showed an ideal amount of fluorescence above 650 nm after incubated with Aβ aggregates.13,14 However, MCAAD-3 only had a moderate affinity (Ki = 106 nM), and BBTOM-3 showed poor BBB penetration. Our reported probes were also made unacceptable by a low QY, and accordingly, we generated a new family of NIR Aβ probes, DANIR 3a-e (Figure 1), by replacing the benzene ring of DANIR 2c with a naphthalene ring.15 DANIR 3b and 3c had excellent fluorescent properties, including emission wavelengths above 680 nm and high QYs of 30% and 9%, respectively. More importantly, DANIR 3b and 3c displayed high sensitivity and affinities (3b, Kd = 8.8 nM; 3c, Kd = 1.9 nM) to Aβ aggregates and have been well applied in in vivo NIR imaging. However, DANIR 3b had a short emission maximum (615 nm) when binding with Aβ aggregates. Due to high lipophilicity, DANIR 3c displayed poor brain kinetics; besides, DANIR 3c was also confirmed unstable in living mice. Inspired by previous experiences, our following efforts have targeted the generation of more efficient probes with better fluorescent and biological properties, including obtaining a emission maximum above 650 nm upon binding to Aβ aggregates and having good biostability. Thus, based upon the scaffold of DANIR 3a-e, we designed and synthesized a new family of NIR Aβ probes named DANIR 8a-c and 9a-c by incorporating hydroxyethyl groups to electron donor moiety (Figure 1) to improve the hydrophilicity as well as the brain kinetics of our probes. EXPERIMENTAL SECTION General Information. The synthetic trifluoroacetic acid salt forms of peptides Aβ1-42 was obtained from Osaka

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PEPTIDE INSTITUTE, Inc. (Osaka, Japan) and aggregated for in vitro studies using the previously reported procedures.16 All spectroscopic measurements were conducted using a spectrofluorophotometer (RF-5301PC, Shimadzu, Japan) and a UV spectrophotometer (UV-2450, Shimadzu, Japan). Fluorescent microscope observation was performed on the Axio Observer Z1 inverted fluorescence microscope (Zeiss, Germany) that was equipped with DAPI, AF488 and AF546 filter sets. Highperformance liquid chromatography (HPLC) analysis was performed on an Agilent 1260 Infinity Quaternary LC (Agilent Technologies, America) system using a Cosmosil packed column (5C18-AR-II, 4.6 × 150 mm, Nacalai Tesque, Japan) with a binary gradient elution system, while the mobile phases A and B were separately water and acetonitrile at a flow rate of 1.0 mL/min. Human brain sections were obtained from the Chinese Brain Bank Center. Normal ICR mice (5 weeks, male) that were used for BBB penetration test were purchased from the Vital River Laboratory Animal Technology Co. Ltd., Beijing, China. Tg mice (C57BL6, APPswe/PSEN1) and WT mice (C57BL6) that were used for in vitro histology, in vivo NIR imaging and ex vivo fluorescent staining were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences. All experiments requiring the use of mice were approved by the animal care committee of Beijing Normal University. Synthesis. The synthesis of the naphthyl aldehydes and newly designed DANIR probes were outlined in scheme 1 and S1 in Supporting Information, and the details of synthesis, 1H NMR, 13C NMR, MS, and HRMS spectra were described in Supporting Information. Spectroscopic Measurements. Fluorescence QYs were measured using an aqueous solution of Rhodamine 6G as a standard (Φ = 0.76).17 The fluorescence response of our probes with Aβ1−42 aggregates and bovine serum albumin (BSA) were measured in the same conditions by following our previously reported procedures.15 In Vitro Fluorescent Staining. Paraffin-embedded 8 µm brain sections from Tg mice (C57BL6, APPswe/PSEN1, 22 months old, male), two AD patients (female, 64 years old, dense-core plaques; female, 71 years old, diffuse plaques) and a cerebral amyloid angiopathy (CAA) patient (female, 68 years old) were used for the neuropathological staining. The brain sections from age-matched WT mice (C57BL6, 22 months old, male) were also used as a normal control. The staining was conducted by following the previously reported procedures.15 Molecule Modeling. The molecules were constructed with GaussView 5.0 and the ground-state geometries in water phase were optimized using a previously reported method.15 In Vitro Saturation Binding Studies. The saturation assays of NIR fluorochromes (NIRFs, 8b 8c, 9b and 9c) were performed by following the previously reported procedures.15 Blood-brain Barrier Penetration Determination. The BBB penetration test were conducted following previously established procedures.13 The details of the HPLC conditions were listed in Table S2 in Supporting Information. Partition Coefficient. The log P values of NIRFs were measured by following the previously reported procedures.15 In Vivo Near-Infrared Imaging. Tg mice (n = 4, C57BL6, APPsw/PSEN1, 16 months old, male) and age-matched WT mice (n = 3, C57BL6, 16 months old, male) were used for in

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vivo NIR imaging. The mouse with scalp hair removed was injected with a solution of 8c (0.2 mg/Kg, 15% DMSO and 85% propylene glycol, 50 µL). The imaging paradigm was performed on an IVIS Lumina III system (PerkinElmer, America) with proper filter sets (ex. at 580 nm and em. at 670 nm), and the fluorescence signals from the brain at various time points before and after dosing were collected. In the duration of imaging, the mice were under anesthesia with 2.5% isoflurane gas in an oxygen flow of 0.8 L/min. The acquired images were analyzed by drawing ROI with the area around the brain region using the Living Image software 4.5.2. Semiquantitative results were calculated from radiance efficiency. Ex Vivo Histology. A Tg mouse (C57BL6, APPsw/PSEN1, 12 months old, male) and an age-matched WT mouse (C57BL6, 12 months old, male) were i.v. injected with 8c (0.2 mg/kg, 15% DMSO, 85% propylene glycol, 50 µL) and sacrificed at 20 min after injection. We prepared frozen sections and conducted fluorescent microscopy following previously reported protocols.15 RESULTS AND DISCUSSION Synthesis. The key intermediates 1a and 1b with hydroxyethyl amino groups were prepared by the Bucherer reaction between 6-bromonaphthalen-2-ol and 2-(methylamino)ethanol or diethanolamine. The lithiation by n-butyllithium (n-BuLi) and the following formylation by N,N-dimethylformamide (DMF) were performed to give the primary aldehydes (4a and 4b). Naphthyl aldehydes with longer polyenic chains (5a, 5b, 7a and 7b) were prepared by the Wittig reaction. During these procedures, hydroxyl groups were protected by tetrahydro-2Hpyran (-OTHP, 2a, 2b, 3a, 3b, 6a and 6b), and the protective group (-OTHP) was then removed by acidification. The final DANIR probes were obtained by Knoevenagel condensation of naphthyl aldehydes and malononitrile within 30 min and separated as crystals without further purification. Scheme 1. Synthesis of newly designed DANIR probesa

a

Regents and conditions: (a) malononitrile, K2CO3, methanol, 5 - 30 min, rt. Spectroscopic Properties. In our previous studies, all imaging probes underwent intramolecular charge transfer (ICT) transitions, and the emission maxima displayed a bathochromic shift to the NIR spectral range with the extension of the π conjugation system.18,19 As anticipated, for the DANIR probes, noticeable red shifts of the absorption/emission maxima were observed with the prolonging of the π conjugated system (Table 1). The emission maxima of four probes (8b, 8c, 9b and 9c) in PBS red-shifted to the “NIR window” with the values ranging from 683 to 789 nm, which is consistent with their calculated HOMO-LUMO energy gaps (Table 1, Figure 3 and S7 in Supporting Information). Moreover, the emission maxima of these solvatochromic probes displayed significant solvent dependency and occurred at higher wavelengths with increasing solvent polarities (Figure S3, S4 and Table S1 in Supporting Information), indicative of the ongoing ICT transition with an increased dipole moment in the excited state when compared with that of the ground state.20-22 Notably, when compared with the DANIR 3b and 3c, these four

NIRFs maintained excellent fluorescent properties, including long

Figure 2. The emission spectra of 8a-c (left panel) and 9a-c (right panel) upon interaction with Aβ1−42 aggregates (10 µg/mL, red line) or BSA (10 µg/mL, black line). The spectra of the compound solutions in PBS (50 nM, blue line) and PBS alone (green line) were also measured under the same condition. emission maxima, high QYs (ranged from 7.1% to 22.3% in dichloromethane, Table 1), which are prerequisites of an optimal fluorochrome for in vivo NIR imaging. However, the excitation maxima of the NIRFs were less than 600 nm (Table 1), which limited the penetration depth of excitation light, and therefore, further modifications are necessary. In Vitro Evaluations. Another important feature of an NIR probe is the ability to trigger conspicuous fluorescence response upon binding to Aβ aggregates/plaques. As shown in Figure 2 and Table 1, marked fluorescence responses including the increase in fluorescence intensity (17- to 629-fold increase) and emission blue shifts (> 60 nm), were observed after the NIRFs binding to Aβ1−42 aggregates in PBS, thereby indicating that the probes were inserted into the hydrophobic cleavage resulting in a restrained double-bond isomerization or rotational relaxation. Notably, 8c was confirmed as the most sensitive probe with a 629-fold enhancement of the fluorescence intensity, comparable to that of DANIR 3b (716fold). Conversely, only weak fluorescence intensity was observed when the NIRFs alone or upon interacting with BSA in PBS. Furthermore, although the large blue shifts were observed upon binding to Aβ1−42 aggregates, the emission maxima of 8c and 9c were still retained in the NIR spectral range (678 and 673 nm, respectively), which signifies their potential application as veritable NIRFs for in vivo Aβ imaging. Moreover, the binding specificity of our probes was evaluated by histological fluorescent staining of the Aβ plaques in brain slices from double Tg mice and definitive AD/CAA patients. As seen in Figure 4a, 4b and S8 in Supporting Infor-

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mation, our probes (8a-c and 9a-c) could clearly distinguish the Aβ plaques in the brain sections from Tg mice, Table 1. Selected data of fluorescent properties and binding affinities. probe

λabs (nm)a

λex (nm)b

λem1 (nm)c

λem2 (nm)c

Φ (%)d

∆E (eV)e

foldf

Kd (nM)g

8a 8b

468 496

495 554

616 683

550 619

10.0 22.3

3.70 3.53

57 321

h 38.5 ± 6.9

8c

512

591

798

678

7.1

3.35

629

14.5 ± 3.9

9a

463

492

613

573

7.5

3.70

3

h

9b

492

517

685

642

15.7

3.53

17

197.0 ± 43.6

509 578 787 673 9.3 3.36 194 19.9 ± 4.5 9c a b c Absorbance (abs) measured in dichloromethane. Excitation maxima in PBS. λem1 and λem2 represent the emission maxima of the probes in the solution of PBS or upon binding with Aβ1−42 aggregates. dQYs were determined in dichloromethane. eThe HOMOLUMO energy gap calculated in the water phase with DFT at a B3LYP/6-31G level. fThe fold increase in the fluorescence intensity upon binding to Aβ1−42 aggregates. gAll values were measured in triplicate and reported as the mean ± SD. hNot observed. which agrees with the staining patterns of the adjacent sections by DANIR 3b,15 whereas no fluorescence signals were observed in the brain slices from WT mice following staining (Figure S10 in Supporting Information). Furthermore, evidences implies that Aβ deposits exist in three different forms, including highly compacted (dese-core) and diffuse plaques, as well as deposits in the walls of small arteries and arterioles within the leptomeninges and cortex, which is responsible for CAA.23,24 When tested on brain slices from AD patients, 8a-c could clearly mark the Aβ plaques in three different forms with high signal to background ratio (Figure 4c-h and S9 in Supporting Information); however, 9a-c only showed moderate staining of the dese-core plaques, and failed to label the diffuse plaques and the plaques in vessel walls (Figure S9 in Supporting Information). Although the quantitative binding studies revealed that 8c (Kd = 14.5 nM) and 9c (Kd = 19.9 nM) had similar affinities using synthetic Aβ1-42 aggregates, the Aβ deposits in the brain of AD patients exist in three different forms and the assemblies/folding of these deposits could have different tertiary and quaternary structures, which may explain the different fluorescent staining patterns of compound 8c and 9a-c. When considered together, the histological staining patterns suggested that the DANIR probes displayed a “turn-on”

phenomenon when associated with Aβ plaques. Furthermore, 8a-c displayed superior specificity by staining the plaques in different forms from Tg mice and AD/CAA patients. Molecule Modeling and Structure-Affinity Relationship Analysis. The optimized structures exhibited that the dicyanovinyl preferred to be planar and conjugated with the π

Figure 3. Optimized ground-state geometries of 8c (a) and 9c (b) in water phase and the frontier molecular orbitals of the HOMO and LUMO (c and d were for 8c and 9c, respectively) calculated with DFT at a B3LYP/6-31G(d) level.

Figure 4. Histological fluorescent staining on brain sections of Tg mouse (a and b) and AD patients (c-h) with 8b (red images, left panel) and 8c (purple images, right panel). The labeled plaques of AD patients were dense-core (c and d), diffuse

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plaques (e and f), and the deposits in vessel walls (g and h), magnification 5×. Table 2. Log P values and the brain uptakes (% ID/g) of the NIRFs at different time points. probe

log P

8b 8c 9b 9c

3.69 ± 0.019 4.30 ± 0.050 3.04 ± 0.0061 3.73 ± 0.023

time after injection (% ID/g) 2 min

10 min

30 min

60 min

16.27 ± 3.53 8.12 ± 0.91 1.65 ± 0.060 2.06 ± 0.26

12.05 ± 0.91 11.42 ± 2.27 0.95 ± 0.19 1.31 ± 0.55

7.27 ± 0.98 7.21 ± 0.60 0.99 ± 0.0052 1.42 ± 0.00084

1.50 ± 0.20 2.05 ± 0.37 0.36 ± 0.13 0.99 ± 0.16

brain2min/brain60min 10.8 5.6a 4.6 2.1

a

Brain10min/brain60min. system consisting of a naphthyl ring and polyenic chains (Figure 3 and S7 in Supporting Information), which is pivotal for Aβ binding.15,22 Furthermore, several studies have also indicated that the hydrophobic interactions may play a predominant role in the binding mode of small molecules and Aβ fibrils.13,15 As shown in Table 1, the quantitative binding studies have shown that the affinities of the probes were significantly improved with the continual enhanced hydrophobic interactions aroused by the extension of the π conjugation system, and that the NIRFs with longer π system displayed moderate to high affinities. Among these probes, 8c and 9c had the highest affinities of 14.5 and 19.9 nM, respectively, which were higher than those of MCAAD-3 (Ki = 106.0 nM) and DANIR 2c (Kd = 26.9 nM). This was mainly due to the larger hydrophobic contact surface area between the small molecules and Aβ fibrils. Furthermore, when compared with DANIR 3b and 3c, the probes had a loss in Aβ binding affinity, probably because the larger substitutions at the N position forced the molecules to prefer geometries with greater degrees of nonplanarity and, therefore, are inappropriate to fit the binding pocket.13,15,22

Figure 5. (a) Brains of ICR mice were dissected 2 min after dosing with NIRFs (2 mg/Kg, 8b, 8c, 9b and 9c) under visible light (top row) and ultraviolet light (365 nm, bottom row). (b) Brain uptakes of NIRFs (8b, 8c, 9b and 9c) at different time

points (2, 10, 30 and 60 min). The data of DANIR 3b and 3c are from ref 15. This was also confirmed by molecules with smaller substitutions at the N position (8b and 8c) displaying higher affinities than those containing larger substitutions (9b and 9c), which is also in accord with the histological staining patterns. Blood-brain Barrier Penetration Determination. The log P of the NIRFs was determined to have the values between 3.04 and 4.30 (Table 2). When compared with the log P values of DANIR 3b (log P = 4.10) and 3c (log P = 4.48), by introducing hydroxyl groups, the hydrophilicity of our NIRFs was considerably improved. Molecules 9b (log P = 3.04) and 9c (log P = 3.04), each with two hydroxyl groups displayed a higher hydrophilicity than 8b (log P = 3.69) and 8c (log P = 4.30), thereby suggesting that these NIRFs possessed appropriate lipophilicity to penetrate the BBB. The straightforward evaluation of BBB penetration capability of the NIRFs was conducted by ex vivo observation of the brains after injection of the probes. Figure 5a shows that 8b and 8c can efficiently penetrate the BBB, and that brains were stained intensely red in visible light and emitted red fluorescence under ultraviolet (UV) light of 365 nm. Conversely, no significant change in brain color was observed after dosing 9b and 9c, which indicated a limited BBB penetration. The quantitative determination of the brain pharmacokinetics was assessed by biodistribution assays on ICR mice, and the appearance of the probes was confirmed by an analysis of the brain extracts using HPLC. As shown in Table 2 and Figure 5b, 8b and 8c displayed a high initial brain uptake of 16.27% (brain2min) and 11.42% ID/g (brain10min), and washed out from the brain rapidly with the clearance ratios of 10.8 (brain2min/brain60min) and 5.6 (brain10min/brain60min), which is superior to that of DANIR 3c (brain10min = 4.44% ID/g, brain10min/brain60min = 3.3).15 However, 9b and 9c displayed poor brain uptakes (brain2min < 2.1% ID/g) and moderate brain egress (brain2min/brain60min < 5, Table 2), which impeded their further application for in vivo. Furthermore, HPLC profiles showed that our NIRFs had good biostability in the living brain (Figure S12 in Supporting Information), which is a prerequisite for showing good in vivo efficacy. Furthermore, in accordance with our previously reported results,15 a longer polyenic chain resulted in higher lipophilicity and undesirable brain kinetics; thus, 8c displayed a decreased brain uptake and slower brain washout rate than 8b. Moreover, the BBB permeability decreases conspicuously with the addition of hydrogen-bond-forming functional groups, especially hydroxyl groups, which act as both hydrogen bond donor and an acceptor.25,26 Thus, although they have a reduced lipophilicity, 9b and 9c had a loss in brain uptake due to the addition of hydrogen bonds that were induced by

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additional hydroxyl groups. Overall, 8c exhibited excellent brain kinetics and biostability, which demonstrated its great potential for in vivo imaging. Furthermore, our work provides new guidance to design the probes for brain imaging.

Figure 6. (a) Fluorescence images of the same Tg (top row) and WT (bottom row) mouse head at selected time points before or after i.v. injection of 8c. (b) Semi-quantitative analysis of the fluorescence signals from Tg and WT mice at various time points.

Figure 7. Ex vivo histology results of brain slices of cortex (b and d) and cerebellar regions (e and g) of Tg mice after dosing with 8c. The Aβ plaques were confirmed by staining the same sections with Th-S (c, d, g and h). Panels a, c, f and h are partial enlarged views of homologous sections. Scale bars: a, c, f and h, 10 µm; b, d, e and g, 100 µm. ences (p < 0.01, one-way ANOVA) in signals between Tg and In Vivo NIR Imaging and Ex Vivo Histology. The unique WT groups were most likely the result of the remarkable inadvantage of optical imaging is the exquisite detectable fluocrease in fluorescence intensity upon binding with Aβ plaques, rescence response under appropriate circumstances. According thereby suggesting that 8c indeed possessed the capability of to the above results, 8c displayed a striking increase in fluodetecting Aβ plaques in vivo. rescence intensity (629-fold) and a significant blue shift of the emission maximum (from 798 to 678 nm, total shift of 120 In vivo NIR imaging results were further confirmed by ex nm) after binding to Aβ aggregates, and the emission maxivivo histology on a Tg mouse and an age-matched WT mouse. mum (678 nm) was still in the NIR spectral range, which Fluorescent microscopy observations demonstrated that 8c could help to distinguish between the fluorescent signals of could selectively stain Aβ plaques in the cortex (Figure 7a and bound and unbound probes, thus providing a high signal-tob) and cerebellar region (Figure 7e and f), and the same fluonoise ratio. Moreover, the high affinity to Aβ and the excelrescence signals were observed when stained with thioflavin-S lent brain pharmacokinetics also make 8c the most ideal in (Th-S, Figure 7c, d, g and h), whereas the brain sections of a vivo NIR probe. Finally, the in vivo NIR imaging was perWT mouse showed no such plaques (Figure S13 in Supporting formed on Tg mice and age-matched WT mice. As shown in Information). The in vivo imaging result agrees with ex vivo Figure 6, after dosing, intense fluorescence signals in the histology, which showed that 8c could efficiently permeate the brains of both Tg and WT mice were observed 2 min after BBB and selectively bind to Aβ plaques in vivo. injection. Furthermore, the fluorescence signals of Tg mice CONCLUSION (brain compartment) were considerably higher than those of By employing the electron donor-acceptor scaffold, new WT mice (brain compartment) at all-time points that were NIR probes were discovered. Four of these probes (8b, 8c, 9b detected, and the signals of Tg mice displayed 1.33-, 1.24-, and 9c) functioned as NIRFs, with their emission maxima 1.35-, 1.40- and 1.44-fold of the signals of WT mice at 2, 12, falling into the “NIR window” in PBS (> 680 nm). Successful30, 60 and 90 min, respectively. Statistically significant differ-

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ly, 8c and 9c showed great potential as “veritable” NIRFs for Aβ detection by maintaining the emission maxima higher than 650 nm upon binding with Aβ1−42 aggregates. More importantly, 8c displayed a high sensitivity and affinity to Aβ1−42 aggregates. Furthermore, the BBB penetration test revealed excellent brain kinetics and biostability of 8c in the living brain. The ability of 8c to image Aβ plaques in vivo provided an ideal tool for real-time measurement of drug efficacy, and as a result, can accelerate the therapeutic discovery. The most similar technique to NIR that is used in current animal studies for detecting Aβ plaques is the FRI technique, which is an inherently surface-weighted qualitative planar imaging modality that has limited spatial resolution when imaging through the scalp and skull, and is insensitive to the alterations of dye distribution in deeper tissue. Thus, the newly developed three-dimensional FMT imaging that allows for improved localization and quantification in deep tissue has the greatest potential of authentically quantifying Aβ plaques in the brain.27 Additionally, the application of FMT necessarily relies on valid probes with both excitation and emission maxima in the NIR spectral range. Probe 8c was demonstrated to have great potential for FMT imaging of Aβ plaques, and our work would give valuable hints in developing such Aβ targeting ligands.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Additional scheme, tables and figures, 1H NMR, 13C NMR, MS, and HRMS spectra (PDF)

AUTHOR INFORMATION Corresponding Author *Phone/Fax: +86-10-58808891. E-mail: [email protected].

Author Contributions M. Cui, and H. Fu conceived and designed the experiments. H. Fu, M. Cui, P. Tu and L. Zhao performed the experiments. H. Fu, M. Cui, P. Tu and L. Zhao analyzed the data. M. Cui, J. Dai, and B. Liu contributed reagents, materials, and analysis tools. H. Fu and M. Cui wrote the paper.

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Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was funded by the National Natural Science Foundation of China (Grant No. 21571022 and 21201019), and the National Science and Technology Major Projects for Major New Drugs Innovation and Development (Grant No. 2014ZX09507007-002).

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