Fluorescent Imaging of β-Amyloid Using BODIPY Based Near-Infrared

Sep 6, 2018 - Fluorescent imaging of β-amyloid (Aβ) is one of the most promising methods for Alzheimer's disease diagnosis. Several fluorescent prob...
0 downloads 0 Views 4MB Size
Download PDF
Article Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX

pubs.acs.org/bc

Fluorescent Imaging of β‑Amyloid Using BODIPY Based NearInfrared Off−On Fluorescent Probe Wenming Ren,#,† Jingjing Zhang,#,‡,⊥ Cheng Peng,#,‡,∥,⊥ Huaijiang Xiang,§ Jingjing Chen,⊥ Chengyuan Peng,† Weiliang Zhu,‡,∥,⊥ Ruimin Huang,*,†,⊥ Haiyan Zhang,*,†,‡,⊥ and Youhong Hu*,†,⊥ State Key Laboratory of Drug Research, ‡CAS Key Laboratory of Receptor Research, and ∥Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China § College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China ⊥ University of Chinese Academy of Sciences, Beijing, 100049, China Downloaded via UNIV OF SOUTH DAKOTA on September 23, 2018 at 07:38:46 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

S Supporting Information *

ABSTRACT: Fluorescent imaging of β-amyloid (Aβ) is one of the most promising methods for Alzheimer’s disease diagnosis. Several fluorescent probes have been reported to detect Aβ both in vitro and in vivo. However, highly sensitive and highly selective probes with low background signals are still greatly needed. Herein, we rationally designed and synthesized a PIET quenched near-infrared probe QAD-1 to detect Aβ. This probe contains BODIPY as fluorophore and tetrahydroquinoxaline as the quenching group. QAD-1 exhibited significant fluorescent switch-on after binding to soluble and insoluble Aβ species, and the probe had the benefit of low background signal to stain Aβ plaques without the need of wash-out procedures in vitro, which was specially found by the fluorescence off−on probe. QAD-1 could identify the overproduced Aβ in transgenic (APPSWE/ PSEN 1dE9) AD mice as early as 6 months old in vivo, which indicated that QAD-1 may be a potential probe for monitoring Aβ species at an early stage of AD.

■

and utilized for Aβ detection in vivo (Figure 1).16−20 However, there are still many obstacles in the development of Aβ fluorescent probes, such as high background signals due to the unspecific binding21 and lack of the ability to monitor AD at an early stage. It is important to develop novel fluorescent probes

INTRODUCTION Alzheimer’s disease (AD) is the most common type of dementia that causes problems with memory, thinking, and behavior. According to the Alzheimer’s association report, 1 in 10 people aged 65 and older has suffered from Alzheimer’s dementia. The hallmark pathologies of AD are the progressive accumulation of β-amyloid (Aβ) and twisted strands of tau.1 Even though many Aβ-targeted drug candidates failed in clinical trials, it is believed that Aβ accumulation is the earliest biomarker in the malignant neurodegenerative processes of AD.2−6 Diagnosis, especially early diagnosis, appears to be critically urged for AD therapies and drug development. Up to now, FDA and EMA have approved three amyloid-based radiolabeled tracers (Florbetapir,7 Florbetaben,8 and Flutemetamol9) to measure Aβ level in the brain for positron emission tomography (PET) imaging. The National Institute on Aging and the Alzheimer’s Association also developed PET imaging of Aβ plaques as part of diagnostic criteria to categorize the brain changes. As an alternative to isotope imaging, fluorescence imaging is an ideal method to detect biomolecules due to its low cost, real time, and highly sensitive detection. During the past decade, a variety of fluorescent probes have been developed for Aβ fluorescent imaging in vitro,10 and several near-infrared fluorescent probes such as NIAD-4,11 AOI-987,12 THK265,13 CRANAD-2,14 and DANIR-2c15 have been developed © XXXX American Chemical Society

Figure 1. Typical NIR fluorescent probes reported for the detection of Aβ. Received: September 4, 2018 Published: September 6, 2018 A

DOI: 10.1021/acs.bioconjchem.8b00623 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry

Figure 2. A. Design of the NIR fluorescence off−on probe QAD-1. B. Frontier molecular orbital energy (eV) and electron density distribution in HOMO and LUMO of probe QAD-1 and tetrahydroquinoxaline by M06-2X/6-31G (d, p) calculation.

In order to design a NIR fluorescence quenching probe via a PIET process, theoretical prediction of fluorescent probes was performed based on DFT and TDDFT at the M06-2X/6-31G (d, p) level. As the energy gap between highest occupied molecular orbital energy (EHOMO) of the fluorophore and the electron donor plays a vital role in a PIET process,30 we calculated molecular orbital energy that was shown in Figures S1 and 2B. It was found that the EHOMO of N-methyl aniline (−6.07 eV) was lower than that of probe D1 while its lowest unoccupied molecular orbital energy (ELUMO) was higher than that of D1. This led to a prohibited PIET effect from N-methyl aniline moiety to fluorophore of D1. The EHOMO of tetrahydroquinoxaline (−4.76 eV) was higher than N-methyl aniline and between the EHOMO (−5.90 eV) and ELUMO (−2.15 eV) of QAD-1, which meant that the tetrahydroquinoxaline group was able to serve as an electron donor making the electron transition to the HOMO of fluorophore of QAD-1, and quenching the fluorescence (Figure 2). As a result, QAD-1 was rationally designed with thiophene vinyl BODIPY as the fluorophore and tetrahydroquinoxaline as the quenching moiety. Synthesis of Probe QAD-1. QAD-1 was synthesized as following (Scheme 1): condensation of previously reported dye 2 with aldehyde 3 synthesized fluorescent dye 4; Oxidation of 4 using pyridine sulfur trioxide in DMSO/DCM gave intermediate 5; Borch reduction of 5 with tetrahydroquinoxaline achieved probe QAD-1. All products were confirmed by 1 H NMR, 13C NMR, and high resolution mass spectrometry (Supporting Information). Photophysical Properties of Probe QAD-1. The photophysical properties including absorption, excitation, and emission spectra of QAD-1 in organic solvents DMSO and chloroform were evaluated (Figures 3, S2 and S3). QAD-1 exhibited the maximum absorption and emission wavelengths of 645 and 755 nm in DMSO, respectively, which fall into the best range for NIR probes. The fluorescence quenching property was also measured by comparing the emission spectra of QAD-1 with dye 4 which was an unquenched probe (always-on control). As shown in Figure 3B, the fluorescence intensity of QAD-1 was 10-fold lower than dye 4, which confirmed the quenching effect of the tetrahydroquinoxaline group. Binding Affinity and Fluorescence Intensities of QAD-1 to Aβ. There are many conformational altered Aβ species formed during the AD processes, such as insoluble fibrils, soluble monomers, and oligomers.31 First, fluorescent

for high sensitivity, high selectivity, and early detection of AD. Recently, Ran’s group did a lot of excellent work for the selective Aβ imaging based on the curcumin scaffold, which were capable of distinguishing the soluble and insoluble Aβ species for application in animal models of AD disease at the different stages.19,22−24 Boron dipyrromethane (BODIPY) is one of the most widely used small molecule organic fluorophores in bioimaging. Saji’s group reported several probes based on BODIPY for Aβ imaging, such as BAP-1, BAP-2, and EUA-4.20,21,25,26 Chang also developed the probe BD-Oligo for Aβ oligomers detection.27 BODIPY-based probes, however, have not yet been reported to image Aβ in vivo due to high background signal.21 In our previous work, the fluorescence off-on probe 1 was developed for staining Aβ plaques, and this probe exhibited low background signal and high sensitivity as a result of the unique photoinduced electron transfer (PIET) quenching mechanism.28 Here in, we reported a BODIPYbased fluorescent off−on probe QAD-1 to identify the overproduced Aβ in transgenic (APPSWE/PSEN 1dE9) AD mice as early as 6-month-old in vivo. This is the first PIETquenching mechanism based near-IR probe to detect both soluble and insoluble Aβ species. The probe also exhibited the benefits of low background signal as staining Aβ plaques without the need for wash-out procedures in vitro.

■

RESULTS AND DISCUSSION Probe Design. It is known that marked red-shift of the emission maxima could be achieved through styryl substitution at the α-position of BODIPY.29 BAP-2 is the most potential fluorophore for Aβ detection reported by Saji’s group, which possesses many excellent features such as NIR fluorescence emission, high quantum yield, high affinity for Aβ aggregates in vitro, and the ability to rapidly cross the blood-brain barrier (BBB) and then enter the brain. However, BAP-2 could not image Aβ in vivo due to the rapid accumulation in the scalp and then emit strong noise fluorescence which shielded signal fluorescence from the brain. In our previous work, BODIPYbased probe 1 for staining Aβ plaques in the brain tissues with low unspecific noise fluorescence without the need of postwashing procedures was discovered. We believed that the high staining contrast was due to the unique off−on mechanism which was different from other reported Aβ probes.28 So we tried to combine the fluorophore of BAP-2 and off−on framework of probe 1 to design a novel NIR fluorescence probe (Figure 2A). B

DOI: 10.1021/acs.bioconjchem.8b00623 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry Scheme 1. Synthetic Route of QAD-1a

which elongates the distance between BODIPY and tetrahydroquinoxaline to turn on the fluorescence.28 The binding constants of QAD-1 with Aβ(1−42) were measured by saturation binding assay (Figure S5). Different concentrations of QAD-1 were applied to Aβ(1−42) mixture and the Kd values were 18 nM, 6 nM, and 27 nM for monomers, oligomers, and fibrils, respectively. Compared with binding to Aβ(1−42) fibrils, stronger affinity of QAD-1 was observed when binding to soluble Aβ(1−42) species. This is probably due to the stereohindrance of tetrahydroquinoxaline in probe33 or different binding modes of probe between monomers and aggregates. Overall, those results indicate that QAD-1 is an excellent Aβ fluorescent tracer for both soluble monomers, oligomers, and insoluble aggregates. Staining of Aβ Plaques in Brain Tissues. The capability of QAD-1 to detect Aβ plaques was tested by fluorescent staining of brain slices from a 15-month-old transgenic mouse (Figure 5). To evaluate its unique switch-on feature as a probe with off−on characteristics, we tested it without washing procedures. Results showed that high-contrast fluorescent spots (red) were observed in both cortex and hippocampus regions due to the low background signal of PIET probe. These spots were confirmed to be Aβ plaques by colocalization with thioflavin S (ThS) staining (green). Then we conducted negative control experiments by staining brain slices from agematched wild-type mice where Aβ plaques were absent to test the specificity of binding Aβ rather than other biomolecules. Compared to the obvious spots observed on APPSWE/PSEN 1dE9 transgenic mice, no spots were observed on the brain sections from wild-type mice (Figure S6). In fluorescence images of brain tissue, QAD-1 seems to stain broader regions other than Aβ plaques compared with ThS. In order to interpret the different staining patterns of ThS and QAD-1, we performed a triple staining assay (ThS, QAD-1, and Aβ antibody ab2454) in the same brain slice of APPSWE/ PSEN 1dE9 transgenic mice. As shown in Figure 6, we further verified that QAD-1 stained broader regions other than that of ThS staining (Figure 6D). In contrast, QAD-1 staining exhibited a similar staining pattern as that of ab2454 antibody staining (Figure 6E). ThS is widely known to mainly stain fibrilized Aβ, while ab2454 antibody could recognize both Aβ monomer and oligomers besides Aβ fibrils.34,35 It indicated that the broader staining region of QAD-1 found in our study may due to the interaction between QAD-1 and soluble species of Aβ, including Aβ monomers and oligomers.

a Reaction conditions: (a) CH3COOH/piperidine, toluene/CHCl3, reflux; (b) Py.SO3, TEA, DMSO/DCM, room temperature; (c) tetrahydroquinoxaline, NaBH3CN, CH3COOH (cat.), DCE/MeOH, room temperature. Py: pyridine, TEA: trimethylamine, DMSO: dimethyl sulfoxide, DCM: dichloromethane, DCE: 1,2-dichloroethane.

spectra of QAD-1 upon interaction with Aβ(1−42) fibrils were tested.28 As shown in Figure 4A, QAD-1 (5 μM) exhibited faint fluorescence in PBS (pH 7.4). When different concentrations of Aβ(1−42) fibrils (final concentration: 2 μM, 4 μM, 6 μM, and 8 μM) were added, fluorescence intensities of the probe were increased remarkably. On the other hand, no obvious change was observed when QAD-1 incubated with BSA (35 μg/mL), indicating QAD-1 could bind to Aβ selectively. Then different concentrations of Aβ(1−42) monomers and oligomers (2 μM, 4 μM, 6 μM, and 8 μM) were incubated with the probe.19,32 To our delight, QAD-1 could increase the fluorescence intensities in a dose-dependent manner upon binding to Aβ(1−42) monomers or oligomers (Figures 4B and 4C). Notably, the maximum emissions wavelengths of QAD-1 were blue-shifted 50 nm for fibrils and oligomers, 70 nm for monomers. Based on our previous work, we speculate that the fluorophore of QAD-1 would bind to Aβ,

Figure 3. Absorption spectra (A) and fluorescence (B) spectra of dye 4 (black) and QAD-1 (red). Dye concentration: 5 μM, excitation at 640 nm. C

DOI: 10.1021/acs.bioconjchem.8b00623 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry

Figure 4. Fluorescent spectra of QAD-1 (5 μM) upon interaction with Aβ(1−42) fibrils (A), oligomers (B), and monomers (C) in PBS (10 mM, pH 7.4) with 10% EtOH. Excitation at 645 nm.

Figure 5. Fluorescent staining of Aβ deposits with QAD-1 and ThS in the cortex and hippocampus of 15-month-old APPSWE/PSEN 1dE9 mice. Excitation channel = 635 nm, emission channel = 700 nm for QAD-1; excitation channel = 488 nm, emission channel = 550 nm for ThS; scale bar = 250 μm.

Figure 7. Fluorescent staining of Aβ deposits with QAD-1 and ThS in the cortex and hippocampus of 6-month-old APPSWE/PSEN 1dE9 mice. Excitation channel = 635 nm, emission channel = 700 nm for QAD-1; excitation channel = 488 nm, emission channel = 550 nm for ThS; scale bar = 250 μm.

Determination of Plasma and Brain QAD-1 Concentrations. The biodistribution experiments of QAD-1 were conducted to test the uptake into and washout from the brain. Healthy ICR mice were used for this study at a dose of 10 mg/ kg and LC-MS/MS was used as the quantitative analysis method. The result was shown in Figure 8, QAD-1 showed fast

Figure 6. Fluorescent staining of β-amyloid deposits with ThS, QAD1, Aβ antibody in the brain slice of 12-month-old APPSWE/PSEN 1dE9 mice. A: ThS staining. B: QAD-1 staining. C: Aβ antibody (ab2454) staining. D: the merge of ThS and QAD-1. E: the merge of QAD-1 and ab2454. Excitation channel = 488 nm, emission channel = 550 nm for ThS; excitation channel = 635 nm, emission channel = 700 nm for QAD-1; excitation channel = 546 nm, emission channel = 573 nm for ab2454; scale bar = 10 μm.

Figure 8. Brain and plasma distribution of QAD-1

uptake to the brain and the max concentration was 911 ng/g at 5 min post-injection and 90% of the probe was eliminated at 30 min. The results showed that QAD-1 is suitable for further imaging experiments in double transgenic AD mice. In Vivo Imaging. The in vivo Aβ imaging ability of QAD-1 was evaluated in APPSWE/PSEN 1dE9 mice. As previously described, 6-month-old male APPSWE/PSEN 1dE9 mice were used with age-matched wild type littermates (C57BL6) served as controls (Figure 9). Images were recorded after i.v. injection of QAD-1 at 2.0 mg/kg dosage. The results obtained from 4 independent groups showed that the fluorescent signals of APPSWE/PSEN 1dE9 transgenic mice were higher (1.5−1.6-

Moreover, as QAD-1 could detect both Aβ soluble species and insoluble aggregates in vitro, the capability of QAD-1 to detect Aβ in the brain slices from transgenic mice was also assessed to investigate the ability of early detection. Generally, transgenic mice of 6-month-old were just about to appear sporadic Aβ plaques without obvious cognitive behavioral abnormalities.36 As shown in Figure 7, sporadic spots were stained by QAD-1, which were confirmed to be Aβ plaque by costaining with ThS. D

DOI: 10.1021/acs.bioconjchem.8b00623 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry

Figure 9. In vivo imaging of Aβ deposits using QAD-1. (A) Images from 6-month-old APPSWE/PSEN 1dE9 transgenic (Tg) and wild type (WT) mice after intravenous administration of QAD-1 (2 mg/kg), images from left to right: right before administration and 5 min, 15 min, and 30 min after administration. (B) Radiant efficiency of the brains of 6-month-old mice at 5, 15, and 30 min after i.v. administration of QAD-1. (C) Radiant efficiency of the brains of 6 months mice at 5 min after i.v. administration of QAD-1. Data were shown as means ± SEM from four independent experiments; *p < 0.05 vs WT group.

fold) than that of wild type mice. As shown in Figure 9C, significant difference between APPSWE/PSEN 1dE9 transgenic mice and wild type littermates was observed 5 min post injection, which is matched the characteristic of rapid BBB penetration shown in the distribution data above. These results suggest that QAD-1 is able to identify Aβ in transgenic (APPSWE/PSEN 1dE9) AD mice as early as 6 months old in vivo. Ex Vivo Histology. Mice were sacrificed after in vivo observation, and brains were collected for histological examination. The binding ability of QAD-1 to central Aβ deposits was confirmed by ex vivo staining. As shown in Figure 10, sporadic spots in the brain slices from transgenic mice were

observed. However, no spots were found in the age-matched control mice (Figure S8). The spots were further confirmed to be Aβ plaque by costaining with ThS. These results further confirmed the in vivo imaging data that QAD-1 could label Aβ at an early stage.

■

CONCLUSIONS In conclusion, the first NIR fluorescence off-on probe QAD-1 was rationally designed and synthesized for in vivo detection of Aβ. The probe has following features: (1) QAD-1 is a BODIPY-based probe with the max emission wavelength at 755 nm and the fluorescence of probe was quenched by PIET mechanism; (2) QAD-1 exhibits significant fluorescent switchon after binding to Aβ and showed high affinity for both Aβ fibrils and soluble species ; (3) QAD-1 has the capability to detect Aβ plaques in vitro and diagnose transgenic (APPSWE/ PSEN 1dE9) mice in vivo; (4) QAD-1 shows rapid BBB penetration ability and exhibits significant fluorescent difference as fast as 5 min after administration, which are vital characteristics to an ideal fluorescent probe for Aβ of Alzheimer disease. Also, in vivo imaging showed that the NIRF signal of QAD-1 could diagnose AD mice as early as 6 months old, indicating that it may be a potential probe for monitoring Aβ species at an early stage of AD. Taken together, this work provided a highly sensitive and rapidly detectable probe for in vivo Aβ imaging.

■

MATERIALS AND METHODS Quantum Calculation of Fluorescent Probes. The optimum geometries of fluorescent probes at the ground state (S0) and the excited state (S1) were determined by density functional theory (DFT) and time-dependent DFT (TDDFT)

Figure 10. Ex vivo microscopic images of the cortex and hippocampus from 6-month-old APPSWE/PSEN 1dE9 mice after intravenous administration of QAD-1. Excitation channel = 635 nm, emission channel = 700 nm for QAD-1; excitation channel = 488 nm, emission channel = 550 nm for ThS; scale bar = 250 μm. E

DOI: 10.1021/acs.bioconjchem.8b00623 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry methods, receptively, with the M06-2X functional37 and 6-31G (d,p) basis set. All calculations were performed in vacuum using the Gaussian 09 program.38 Synthesis of Probe QAD-1. Information for the synthesis of probe QAD-1 and other compounds were detailed in the Supporting Information. Fluorescence Spectral Testing of QAD-1 with Aβ(1−42) Monomers, Oligomers, Aggregates, and BSA. To test interactions of QAD-1 with Aβ species, a 50 μM stock solution of Aβ(1−42) fibrils were prepared as previous reported procedures,28 and a 50 μM solution of Aβ(1−42) monomers were freshly made by redissolving the purified monomers (0.1 mg) with 11 μL DMSO, followed by 432 μL phosphate buffer solution (PBS, 10 mM, pH = 7.4).19 For Aβ oligomers preparation, the prepared solution of Aβ monomers were further incubated at room temperature for 20 min followed by centrifugation at 14,000 g for 15 min. The supernatant fraction was transferred in an Eppendorf tube and incubated for 24 h at room temperature.32 The structures of those samples were confirmed using transmission electron microscopy (TEM). The BSA stock solution was prepared by dissolving 225 μg BSA in 1 mL PBS. Parameters of the fluorospectrometer (JASCO FP 6500 for Aβ fibrils and monomers, Horiba FluoroMax 4 for Aβ oligomers) were as follows: Excitation at 645 nm, Emission at 650−800 nm. A solution of Aβ(1−42) (0, 24, 48, 72, 96 μL of Aβ stock solutions, 0, 2, 4, 6, 8 μM in the final assay mixture) or BSA (96 μL of BSA stock solution) was added to the mixture containing QAD-1 (60 μL, 50 μM in EtOH), and then PBS (10 mM, pH = 7.4) was added to make the final volume of 600 μL. The mixture solutions were incubated for 30 min at room temperature, and then transferred to a quartz cuvette. The fluorescence emission spectra were recorded by the fluorospectrometer. In Vitro Saturation Binding Studies. Fluorescence test was employed to measure the Kd of QAD-1. A solution of Aβ(1−42) (12 μL, 1 μM in the final assay mixture) was added to the mixture containing 60 μL of QAD-1 (0 nM to 200 nM in EtOH) and 428 μL of PBS (10 mM, pH = 7.4) in a final volume of 600 μL. The mixture solutions were incubated for 30 min at room temperature, and then transferred to a quartz cuvette. The fluorescence emission spectra were recorded with the same parameters above. Fluorescent intensities (FI) at 705 nm for Aβ fibrils and oligomers, 680 nm for Aβ monomers were read. The Kd value was calculated with PRISM software (nonlinear regression, one site-binding). In Vitro Fluorescence Staining of Brain Slices from APPswe/PSEN 1dE9 Mice. APPswe/PSEN 1dE9 transgenic mice and their age-matched littermates (wild type mice, male, 15 months) were sacrificed for testing the Aβ plaque staining ability of QAD-1. Frozen sections were made after perfusion and gradient dehydration of APPswe/PSEN 1dE9 mice brains. Brain slices were incubated with ThS (10 mg/mL) as positive control of this experiment. After 3× washing by 50% ethanol (1 min per wash), the same slice was incubated with QAD-1 (100 μM) for 20 min at room temperature. After removing the residual liquid with dust free paper, the slice was then mounted with coverslips and imaged using a Leica confocal microscope (TCS SPS CFSMP). Three independent experiments were conducted for fluorescence staining. For triple staining assay, the same brain slices were incubated with ThS (10 mg/mL), Aβ antibody (ab2454), and QAD-1 (100 μM) in sequence. As

for the Aβ antibody staining, brain slices were incubated with ab2454 (Cell Signaling Technology; 1:500) overnight at 4 °C, then washed 3 times (5 min each time) with PBS and incubated with fluorescent secondary antibody (Invitrogen; 1:500) for 1 h at room temperature. The prepared slices were imaged using confocal microscopy under different channels (excitation channel = 488 nm, emission channel = 550 nm for ThS; excitation channel = 635 nm, emission channel = 700 nm for QAD-1; excitation channel = 546 nm, emission channel = 573 nm for ab2454). In Vivo NIRF Imaging of APPswe/PSEN 1dE9 Transgenic Mice. IVIS Spectrum imaging system (PerkinElmer) was used to assess the in vivo NIR imaging ability of QAD-1. The heads of APPswe/PSEN 1dE9 transgenic mice and their age-matched littermates (wild type mice, male, 6 months) were shaved before imaging (6 months old). All animals were anaesthetized by isoflurane supplied by gas anesthesia system inside and outside the imaging device. Both transgenic and wild type mice were injected with 2 mg/kg of freshly prepared QAD-1 (i.v., 5% DMSO, 10% Cremophor EL, and 85% saline, 0.4 mg/mL). Fluorescent signals in the brains were acquired sequentially with filter sets Ex = 635 nm and Em = 720 nm, and the circular regions of interest were analyzed by Living Imaging Software. Ex Vivo Fluorescent Staining of QAD-1 to Aβ Plaques in the Brain of APPswe/PSEN 1dE9 Mice. After in vivo imaging, both APPswe/PSEN 1dE9 transgenic mice and their age-matched wild type mice were sacrificed and perfused in cold saline. Brains were immediately removed, dissected, and fixed in 4% paraformaldehyde overnight, and then dehydrated in 30% sucrose until they sank to the bottom. Then the tissues were OCT (optimum cutting temperature compound) − embedded on trays under −10 °C and were sliced into serial sections (20 μm thick) in frozen section machine. After mounting the coverslips, the slides were imaged using a Leica confocal microscope (TCS SPS CFSMP).

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.8b00623.

■

Experimental details and compound characterization; Frontier molecular orbital energy of probe; Optical properties; Aβ preparation; Binding curves; Fluorescent staining of brain slices; NMR spectra (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Ruimin Huang). *E-mail: [email protected] (Haiyan Zhang). *E-mail: [email protected] (Youhong Hu). ORCID

Youhong Hu: 0000-0003-1770-6272 Author Contributions #

W. Ren, J. Zhang, and C. Peng contributed equally to this work. Notes

The authors declare no competing financial interest. F

DOI: 10.1021/acs.bioconjchem.8b00623 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry

■

Testing of Difluoroboron-Derivatized Curcumins as Near-Infrared Probes for in Vivo Detection of Amyloid-β Deposits. J. Am. Chem. Soc. 131 (42), 15257−15261. (15) Cui, M., Ono, M., Watanabe, H., Kimura, H., Liu, B., and Saji, H. (2014) Smart Near-Infrared Fluorescence Probes with Donor− Acceptor Structure for in Vivo Detection of β-Amyloid Deposits. J. Am. Chem. Soc. 136 (9), 3388−3394. (16) Raymond, S., Skoch, J., Hills, I., Nesterov, E., Swager, T., and Bacskai, B. (2008) Smart optical probes for near-infrared fluorescence imaging of Alzheimer’s disease pathology. Eur. J. Nucl. Med. Mol. Imaging 35 (1), 93−98. (17) Lee, Y. O., Shin, J.-W., Yi, C., Lee, Y. H., Sohn, N.-W., Kang, C., and Kim, J. S. (2014) Detection of A[small beta] plaques in mouse brain by using a disaggregation-induced fluorescence-enhancing probe. Chem. Commun. 50 (43), 5741−5744. (18) Voropai, E. S., Samtsov, M. P., Kaplevskii, K. N., Maskevich, A. A., Stepuro, V. I., Povarova, O. I., Kuznetsova, I. M., Turoverov, K. K., Fink, A. L., and Uverskii, V. N. (2003) Spectral Properties of Thioflavin T and Its Complexes with Amyloid Fibrils. J. Appl. Spectrosc. 70 (6), 868−874. (19) Zhang, X., Tian, Y., Li, Z., Tian, X., Sun, H., Liu, H., Moore, A., and Ran, C. (2013) Design and Synthesis of Curcumin Analogues for in Vivo Fluorescence Imaging and Inhibiting Copper-Induced CrossLinking of Amyloid Beta Species in Alzheimer’s Disease. J. Am. Chem. Soc. 135 (44), 16397−16409. (20) Ono, M., Watanabe, H., Kimura, H., and Saji, H. (2012) BODIPY-Based Molecular Probe for Imaging of Cerebral β-Amyloid Plaques. ACS Chem. Neurosci. 3 (4), 319−324. (21) Watanabe, H., Ono, M., Matsumura, K., Yoshimura, M., Kimura, H., and Saji, H. (2013) Molecular imaging of β-amyloid plaques with near-infrared boron dipyrromethane (BODIPY)-based fluorescent probes. Mol. Imaging 12 (5), 338−347. (22) Mawuenyega, K. G., Sigurdson, W., Ovod, V., Munsell, L., Kasten, T., Morris, J. C., Yarasheski, K. E., and Bateman, R. J. (2010) Decreased clearance of CNS beta-amyloid in Alzheimer’s disease. Science 330 (6012), 1774. (23) Nordberg, A. (2011) Molecular imaging in Alzheimer’s disease: new perspectives on biomarkers for early diagnosis and drug development. Alzheimer's Res. Ther. 3 (6), 34. (24) Zhang, X., Tian, Y., Zhang, C., Tian, X., Ross, A. W., Moir, R. D., Sun, H., Tanzi, R. E., Moore, A., and Ran, C. (2015) Near-infrared fluorescence molecular imaging of amyloid beta species and monitoring therapy in animal models of Alzheimer’s disease. Proc. Natl. Acad. Sci. U. S. A. 112 (31), 9734−9739. (25) Ono, M., Ishikawa, M., Kimura, H., Hayashi, S., Matsumura, K., Watanabe, H., Shimizu, Y., Cheng, Y., Cui, M., Kawashima, H., et al. (2010) Development of dual functional SPECT/fluorescent probes for imaging cerebral beta-amyloid plaques. Bioorg. Med. Chem. Lett. 20 (13), 3885−8. (26) Sozmen, F., Kolemen, S., Kumada, H.-O., Ono, M., Saji, H., and Akkaya, E. U. (2014) Designing BODIPY-based probes for fluorescence imaging of [small beta]-amyloid plaques. RSC Adv. 4 (92), 51032−51037. (27) Teoh, C. L., Su, D., Sahu, S., Yun, S.-W., Drummond, E., Prelli, F., Lim, S., Cho, S., Ham, S., Wisniewski, T., et al. (2015) Chemical Fluorescent Probe for Detection of Aβ Oligomers. J. Am. Chem. Soc. 137 (42), 13503−13509. (28) Ren, W., Xu, M., Liang, S. H., Xiang, H., Tang, L., Zhang, M., Ding, D., Li, X., Zhang, H., and Hu, Y. (2016) Discovery of a novel fluorescent probe for the sensitive detection of β-amyloid deposits. Biosens. Bioelectron. 75, 136−141. (29) Lu, H., Mack, J., Yang, Y., and Shen, Z. (2014) Structural modification strategies for the rational design of red/NIR region BODIPYs. Chem. Soc. Rev. 43 (13), 4778−4823. (30) Boens, N., Leen, V., and Dehaen, W. (2012) Fluorescent indicators based on BODIPY. Chem. Soc. Rev. 41 (3), 1130−1172. (31) Morgan, C., Colombres, M., Nuñez, M. T., and Inestrosa, N. C. (2004) Structure and function of amyloid in Alzheimer’s disease. Prog. Neurobiol. 74 (6), 323−349.

ACKNOWLEDGMENTS This work was supported by the funds from National Natural Science Foundation of China (No. 81522045, 81771890), China Postdoctoral Science Foundation Grant (No. 2017M611638) and “Personalized Medicines-Molecular Signature-based Drug Discovery and Development”, Strategic Priority Research Program of the Chinese Academy of Sciences, (Grant No. XDA12040207), Institutes for Drug Discovery and Development, Chinese Academy of Sciences (CASIMM0120163010), and One Hundred Talent Program of Chinese Academy of Sciences. The calculations were performed at TianHe 2 supercomputer in Guangzhou, supported by Special Program for Applied Research on Super Computation of the NSFC-Guangdong Joint Fund (the second phase) with Grant No.U1501501.

■

REFERENCES

(1) Alzheimer’s Association (2017) 2017 Alzheimer’s disease facts and figures. Alzheimer's Dementia 13 (4), 325−373. (2) Jack, C. R., Knopman, D. S., Jagust, W. J., Shaw, L. M., Aisen, P. S., Weiner, M. W., Petersen, R. C., and Trojanowski, J. Q. (2010) Hypothetical model of dynamic biomarkers of the Alzheimer’s pathological cascade. Lancet Neurol. 9 (1), 119−128. (3) Rosa-Neto, P., Hsiung, G. Y., and Masellis, M. (2013) Fluid biomarkers for diagnosing dementia: rationale and the Canadian Consensus on Diagnosis and Treatment of Dementia recommendations for Canadian physicians. Alzheimer's Res. Ther. 5, S8. (4) Panza, F., Logroscino, G., Imbimbo, B. P., and Solfrizzi, V. (2014) Is there still any hope for amyloid-based immunotherapy for Alzheimer’s disease? Curr. Opin. Psychiatr. 27 (2), 128−137. (5) Hawkes, N. (2016) Amyloid plaques are still main target for Alzheimer’s drugs. BMJ. 352, i214. (6) Staderini, M., Martin, M. A., Bolognesi, M. L., and Menendez, J. C. (2015) Imaging of β-amyloid plaques by near infrared fluorescent tracers: a new frontier for chemical neuroscience. Chem. Soc. Rev. 44 (7), 1807−1819. (7) Camus, V., Payoux, P., Barre, L., Desgranges, B., Voisin, T., Tauber, C., La Joie, R., Tafani, M., Hommet, C., Chetelat, G., et al. (2012) Using PET with 18F-AV-45 (florbetapir) to quantify brain amyloid load in a clinical environment. Eur. J. Nucl. Med. Mol. Imaging 39 (4), 621−31. (8) Richards, D., and Sabbagh, M. N. (2014) Florbetaben for PET Imaging of Beta-Amyloid Plaques in the Brain. Neurol. Ther. 3 (2), 79−88. (9) Heurling, K., Leuzy, A., Zimmer, E. R., Lubberink, M., and Nordberg, A. (2016) Imaging beta-amyloid using [(18)F]flutemetamol positron emission tomography: from dosimetry to clinical diagnosis. Eur. J. Nucl. Med. Mol. Imaging 43 (2), 362−73. (10) Xu, M.-m., Ren, W.-m., Tang, X.-c., Hu, Y.-h., and Zhang, H.-y. (2016) Advances in development of fluorescent probes for detecting amyloid-β aggregates. Acta Pharmacol. Sin. 37 (6), 719−730. (11) Nesterov, E. E., Skoch, J., Hyman, B. T., Klunk, W. E., Bacskai, B. J., and Swager, T. M. (2005) In Vivo Optical Imaging of Amyloid Aggregates in Brain: Design of Fluorescent Markers. Angew. Chem., Int. Ed. 44 (34), 5452−5456. (12) Hintersteiner, M., Enz, A., Frey, P., Jaton, A. L., Kinzy, W., Kneuer, R., Neumann, U., Rudin, M., Staufenbiel, M., Stoeckli, M., et al. (2005) In vivo detection of amyloid-beta deposits by nearinfrared imaging using an oxazine-derivative probe. Nat. Biotechnol. 23 (5), 577−83. (13) Okamura, N., Mori, M., Furumoto, S., Yoshikawa, T., Harada, R., Ito, S., Fujikawa, Y., Arai, H., Yanai, K., and Kudo, Y. (2011) In vivo Detection of Amyloid Plaques in the Mouse Brain using the Near-Infrared Fluorescence Probe THK-265. J. Alzheimer's Dis. 23 (1), 37−48. (14) Ran, C., Xu, X., Raymond, S. B., Ferrara, B. J., Neal, K., Bacskai, B. J., Medarova, Z., and Moore, A. (2009) Design, Synthesis, and G

DOI: 10.1021/acs.bioconjchem.8b00623 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry (32) Kayed, R., Head, E., Thompson, J. L., McIntire, T. M., Milton, S. C., Cotman, C. W., and Glabe, C. G. (2003) Common Structure of Soluble Amyloid Oligomers Implies Common Mechanism of Pathogenesis. Science 300 (5618), 486−489. (33) Li, Y., Yang, J., Liu, H., Yang, J., Du, L., Feng, H., Tian, Y., Cao, J., and Ran, C. (2017) Tuning the stereo-hindrance of a curcumin scaffold for the selective imaging of the soluble forms of amyloid beta species. Chem. Sci. 8 (11), 7710−7717. (34) Biancalana, M., and Koide, S. (2010) Molecular mechanism of Thioflavin-T binding to amyloid fibrils. Biochim. Biophys. Acta, Proteins Proteomics 1804 (7), 1405−1412. (35) Um, J. W., Nygaard, H. B., Heiss, J. K., Kostylev, M. A., Stagi, M., Vortmeyer, A., Wisniewski, T., Gunther, E. C., and Strittmatter, S. M. (2012) Alzheimer amyloid-β oligomer bound to postsynaptic prion protein activates Fyn to impair neurons. Nat. Neurosci. 15, 1227. (36) Jankowsky, J. L., Fadale, D. J., Anderson, J., Xu, G. M., Gonzales, V., Jenkins, N. A., Copeland, N. G., Lee, M. K., Younkin, L. H., Wagner, S. L., et al. (2004) Mutant presenilins specifically elevate the levels of the 42 residue β-amyloid peptide in vivo: evidence for augmentation of a 42-specific γ secretase. Hum. Mol. Genet. 13 (2), 159−170. (37) Zhao, Y., and Truhlar, D. G. (2008) The M06 suite of density functionals for main group thermochemistry, thermochemical kinetics, noncovalent interactions, excited states, and transition elements: two new functionals and systematic testing of four M06class functionals and 12 other functionals. Theor. Chem. Acc. 120 (1), 215−241. (38) Frisch, M. J., Trucks, G. W., Schlegel, H. B., Scuseria, G. E., Robb, M. A., Cheeseman, J. R., Scalmani, G., Barone, V., Mennucci, B., and Petersson, G. A. (2009) Gaussian 09, Revision C.01, Wallingford, CT.

H

DOI: 10.1021/acs.bioconjchem.8b00623 Bioconjugate Chem. XXXX, XXX, XXX−XXX