In Vivo Detection of Cerebral Amyloid Fibrils with Smart

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In Vivo Detection of Cerebral Amyloid Fibrils with Smart Dicynomethylene-4H-Pyran Based Fluorescence Probe Yan Cheng, Biyue Zhu, Yue Deng, and Zhirong Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b00017 • Publication Date (Web): 15 Apr 2015 Downloaded from http://pubs.acs.org on April 24, 2015

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Analytical Chemistry

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1

In Vivo Detection of Cerebral Amyloid Fibrils with Smart Dicynomethylene-4H-Pyran Based Fluorescence Probe Yan Cheng*, Biyue Zhu, Yue Deng, Zhirong Zhang* Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, Sichuan University, Chengdu 610041, China

*To whom correspondence should be addressed: Phone: +86-28-85501566, Fax: +86-28-85501615.

E-mail:

[email protected]

for

Y.

Cheng.

Phone:

+86-28-85501566, Fax: +86-28-85501615, e-mail: [email protected] for Z. Zhang.

Key words: Alzheimer’s disease, β-amyloid, fluorescence probe, in vivo optical imaging


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ABSTRACT In vivo detection of cerebral β-amyloid fibrils may facilitate the monitoring of β-amyloidosis in the brain and effectiveness of anti-amyloid therapies. Thioflavin T (ThT) is a widely used dye for the spectroscopic determination of β-amyloid fibrils, but its ability to detect cerebral β-amyloid fibrils in vivo is limited due to the charged molecule. To this end, a smart dicynomethylene-4H-pyran (DCM) fluorophore, namely

(E)-2-(2-(4-(dimethylamino)styryl)-6-methyl-4H-pyran-4-ylidene)

malononitrile (PAD-1), was evaluated for in vivo fluorescence imaging of cerebral β-amyloid fibrils. PAD-1 rapidly entered the brain with high initial brain uptake after intravenous injection, which is highly desirable for in vivo detection of β-amyloid fibrils. PAD-1 displayed a turn-on effect, showing significant enhancement in fluorescence when bound to the aggregated β-amyloid fibrils. It also showed specific labelling of β-amyloid deposits in APP/PS1 transgenic mouse brains. Thus, PAD-1 proved to be a valuable alternative to ThT for cerebral β-amyloid detection and may enable quantitative imaging in vivo.



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3 INTRODUCTION The formation of insoluble amyloid fibrils in the brain is related to a broad range of human pathologies, especially the neurodegenerative diseases including Alzheimer’s disease, prion-based disease, Parkinson’s disease, and other forms of amyloidosis.1-3 Empirical observations from X-ray fiber diffraction, electron microscopy, solid state NMR, Circular dichroism spectroscopy, Fourier-transform infrared spectroscopy and specific chemical staining indicate that amyloid fibrils share a characteristic β-sheet structure, despite the fact that their soluble precursor proteins showed no obvious common folding patterns.4-8 A rational understanding of β-amyloid fibril deposition in vivo may facilitate monitoring of disease progression and identification of therapeutic therapies to prevent or cure diseases associated with amyloidosis. Techniques such as magnetic resonance imaging (MRI), positron emission tomography (PET), single photon emission computed tomography (SPECT) and optical imaging would provide a powerful tool for noninvasive detection of β-amyloid fibrils in vivo, greatly accelerating preclinical therapeutic research in animal models and diagnosis in humans. To date, a number of groups have worked on in vivo β-amyloid imaging. Amyloid identification using MRI showed low sensitivity and the development of contrast agents that specially enhance amyloid signals are needed.9-12 Notably, remarkable progress in PET imaging of β-amyloid fibrils has brought promise to in vivo detection of early amyloidosis. [18F]AV-45 is the first β-amyloid imaging probe approved by FDA. 13-15 However, broad usage might be hampered by limited availability of radioisotopes and exposure to radioactivity. By contrast, optical imaging using specific fluorescent contrast agents is an attractive


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4 alternative, providing simple, convenient, and cost-effective methods for in vivo detection of cerebral β-amyloid fibrils. Congo red (CR) and Thioflavin T (ThT) provide the most standardized way of staining β-amyloid fibrils. ThT is also used in analysis of aggregated amyloid proteins. It exhibits a green fluorescence which becomes thousand times brighter upon binding to amyloid fibrils.16 However, the negatively charged CR’s sulfonate group or the positively charged ThT’s benzothiazolium group limits the molecule’s blood-brain barrier permeability, which is critical for cerebral imaging. To design an effective imaging probe for cerebral β-amyloid fibrils requires consideration of both the probe and binding pocket characteristics. Although the exact structure of β-amyloid fibrils is unknown, the following properties are listed as empirical requirements: 1) specific labeling of cerebral β-amyloid fibrils; 2) changes in fluorescence properties upon binding to β-amyloid fibrils (fluorescence intensity, emission wavelength, etc.); 3) a sufficient amount to enter the brain with rapid clearing of the unbound probe from the brain; 4) a suitable emission wavelength for imaging cerebral β-amyloid fibrils in vivo.16, 17 A major drawback of conventional fluorescence probes targeting β-amyloid fibrils is that the emission wavelength of the probes for optical imaging is usually less than 550 nm, which will interfere with autofluorescence from biological matter in vivo and limit their usage for imaging of deep living tissues. Fluorescent contrast agents with long emission wavelength (600-900 nm) allow the low auto-fluorescence of biological matter and longer-wavelength light penetration through the living tissues necessary for deep tissue imaging with minimized photodamage. A number of



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5 commercially available fluorophores with long emission wavelength, including Rhodamine dyes, Cyanine dyes (Cy), Indocyanine Green dyes (ICG), IR dyes and Alexa Fluor dyes, have been used for labeling biologically active proteins for targets other than the brain, due to the large molecular weight and charged molecules of these dyes.18 Thus, there is a growing interest towards small, compact fluorophore with the capability to produce noninvasive in vivo imaging. Initial study with oxazine derivative AOI987 suggested the feasibility of efficient imaging of cerebral β-amyloid deposits in vivo; however, AOI987 displayed moderate affinity and slight fluorescence intensity decrease instead of significant fluorescence intensity increase upon binding to β-amyloid aggregates.19 More recently, fluorescence probes such as NIAD-4,16 CRANAD-2,20 THK-265,21 ANCA-11,22 BMAOI-14,23 DANIR-2c,18 and BODIPY derivatives (BODIPY-7, BAP-1, BAP-2)24-26 have been characterized for detecting β-amyloid deposits in vivo (Figure 1). To date, none of the reported probes meet all the criteria above. ---------Figure 1 ---------Recently, the interest in organic push-pull fluorophores has been highly activated for the development of smart fluorescence probes for cerebral β-amyloid fibrils. Typical one-component organic push-pull architecture consists of a π-conjugated system end-caped with strong donor and acceptor moieties (donor-π-acceptor). Dicynomethylene-4H-pyran (DCM) chromophore has typical push-pull architecture


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6 with terminal donor and acceptor moieties that are interconnected by π-conjugated system. We hypothesized that, by modifying the structure of DCM, it would be possible to develop a probe with good binding affinity to β-amyloid fibrils and suitable emission wavelength for fluorescence imaging. We screened DCM derivatives

for

use

as

amyloid

imaging

agents

and

discovered

(E)-2-(2-(4-(dimethylamino)styryl)-6-methyl-4H-pyran-4-ylidene)malononitrile (PAD-1) as a candidate fluorescence probe for cerebral β-amyloid fibrils (Figure 2). To our knowledge, this is the first report on DCM-based probes for detection of β-amyloid fibrils in the brain. Herein, we report the biological evaluation of PAD-1. ---------Figure 2 ---------EXPERIMENTAL SECTION Preparation of Aggregated β-Amyloid Fibrils. Aβ1-42 peptide was purchased from Amresco (USA). Aggregation was carried out by gently dissolving the peptide (0.25 mg/mL) in PBS solution (pH 7.4) containing 1 mM EDTA. The solution was incubated at 37˚C for 42 h with gentle and constant shaking. Spectroscopic Measurements. UV/VIS and fluorescence spectra were recorded and analyzed

using

UV/VIS

(UV-3600,

Shimadzu,

Japan)

and

fluorescence

spectrophotometers (RF5301PC, Shimadzu, Japan), respectively. PAD-1 (CAS No: 51328-91-8) was purchased from ACROS Organics (New Jersey, USA) and used without further purification. Rhodamine B (Sigma, USA) was taken as a reference for



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7 determining quantum yields.27 PAD-1 (1.0 µM, final concentration) was dissolved in PBS solution alone or a prepared solution of Aβ1-42 aggregates (2.75 µM, final concentration) in PBS. The absorption spectra, excitation spectra, emission spectra, molar absorption coefficients and quantum yields were measured. Binding Assays Using the Aggregated β-amyloid fibrils in Solution. A solution of 100 µL of Aβ1-42 aggregates (5.5 µM) was added to the mixture containing 20 µL of PAD-1 (5 nM - 10000 nM in ethanol) and 210 µL of PBS solution in a final volume of 330 µL. The mixture was incubated at room temperature for 1 h. The mixture was then transferred to a black 96-well plate, and the fluorescent intensity was measured (Ex: 490 nm, Em: 570 nm) by a multifunction microplate reader (Thermo Scientific Varioskan Flash, USA). The fluorescence intensity of Aβ1-42 aggregates in PBS solution or PAD-1 without the presence of Aβ1-42 aggregates in PBS solution was also measured under the same condition to normalize specific binding signals within the range of the above tested concentrations. Value for dissociation constant (Kd) was determined from curves of three independent experiments using GraphPad Prism 5.0 with nonlinear one-site binding regression (GraphPad Software, Inc., USA). Fluorescence Staining. Brain sections (10 µm) of APP/PS1 transgenic mouse (male, 12-month-old) and wild-type mouse (male, 12-month-old) were incubated with PAD-1 (33 nM in 20% ethanol) for 10 min at room temperature. The sections were then dipped in 40% ethanol (two 2-min washes), washed with 40% ethanol (one 2-min wash), and rinsed with water for 30 s. The adjacent brain sections were stained by Congo red to confirm the presence of β–amyloid plaques. For the staining of


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8 Congo red, sections were immersed in a 0.1% Congo red solution containing 25% ethanol for 3 min, washed in 40% ethanol (two 2-min washes), and rinsed with water for 30 s. After drying, all brain sections were examined using a microscope (Aviovert 40 CFL, Zeiss, Germany) equipped with a FITC filter set. In Vivo Fluorescence Imaging on Normal Mice. Kunming mice (male, 6-week-old) were provided by the Laboratory Animal Center of Sichuan University (Chengdu, China). All animal study procedures were approved by the Sichuan University Animal Ethical Experimentation Committee according to the requirements of the National Act on the use of experimental animals (China). The mice were anaesthetized and shaved under isoflurane and injected directly into the tail vein with PAD-1 (5.0 mg/kg, 10% polysorbate 80, 20% DMSO, and 70% PBS solution). Fluorescence images were obtained with a QuickView 3000 imaging system (BioReal, Austria) with a customized filter set (excitation, 474 nm; emission, 586 nm).The imaging parameters were: exposure time, 1 sec; vertical pixel shift speed, 6.5; horizontal pixel shift RO speed, 1MHz; EM gain, 0; binning, 1×1. Ex Vivo Fluorescence Imaging on Normal Mice. Kunming mice (male, 6-week-old) were injected directly into the tail vein with PAD-1 (5.0 mg/kg, 10% polysorbate 80, 20% DMSO, and 70% PBS solution). The mice were sacrificed at 5, 30, 60, and 120 min postinjection and the brains were removed and weighed (n = 5 at each time point). Fluorescence images of the brains were obtained with a QuickView 3000 imaging system (BioReal, Austria) with a customized filter set (excitation, 474 nm; emission, 586 nm).The imaging parameters were: exposure time, 1 sec; vertical pixel shift speed,



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9 6.5; horizontal pixel shift RO speed, 1MHz; EM gain, 0; binning, 1×1. ROI was drawn around the brain region to calculate fluorescence signals. Brains without injection of PAD-1 were used to adjust the background signal (sum = 0). The fluorescence signals were analyzed using QuickView 3000 software. The fluorescence intensity was expressed as signal per organ weight (photon/g). In Vivo Fluorescence Imaging on Transgenic Mice and Wild-Type Mice. APP/PS1 transgenic mice (male, 13-month-old) were provided by Nanjing Biomedical Research Institute of Nanjing University (Nanjing, China). The mice were anaesthetized and shaved under isoflurane and injected directly into the tail vein with PAD-1 (5.0 mg/kg, 10% polysorbate 80, 20% DMSO, and 70% PBS solution). Fluorescence images were obtained with a QuickView 3000 imaging system (BioReal, Austria) with a customized filter set (excitation, 474 nm; emission, 586 nm).The imaging parameters were: exposure time, 1 sec; vertical pixel shift speed, 6.5; horizontal pixel shift RO speed, 1MHz; EM gain, 0; binning, 1×1.

RESULTS AND DISCUSSION Spectral Properties of PAD-1. PAD-1 is a dark-red compound which exhibits a bright red fluorescence in methanol solution. The emission wavelength of PAD-1 displayed a typical solvent-dependency (603 nm in methanol to 617 nm in PBS solution) The fluorescence quantum yield of PAD-1 in PBS solution alone is very low (0.5%) , which may be in part due to the greater stabilization in aqueous media of the non-emissive intramolecular charge transfer state as opposed to the emissive planar


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10 excited state.16 The quantum yield increased to 13% upon mixing with aggregated β-amyloid 1-42 fibrils (Aβ1-42 aggregates) (Table 1). In vitro spectral study of PAD-1 with Aβ1-42 aggregates showed about 7.0-fold increase in fluorescence intensity along with a hypochromatic shift in the emission spectra of 46 nm, compared to the aqueous solutions of PAD-1 without Aβ1-42 aggregates (Figure 3). It is likely that the phenomenon is a consequence of binding between PAD-1 and β-amyloid fibrils. The significant enhancement in fluorescence may come from the loss of rotational degrees of conformational freedom upon binding to groove-like β-amyloid fibrils.16, 28 ---------Table 1 ------------------Figure 3 ---------Binding Affinity for β-Amyloid Fibrils. Although new approaches for determination of binding for β-amyloid fibrils have been proposed,29,

30

binding

affinity of PAD-1 for β-amyloid fibrils was determined by using saturation assays reported previously, enabling a comparison with reported amyloid-specific fluorophores.18,

20, 25

The results revealed that PAD-1 displays high affinity for

β-amyloid fibrils with the dissociation constant (Kd) value of 58.9 nM. This affinity is much higher than ThT (Ki = 580 nM). Compared to amyloid-specific fluorophores reported previously, the affinity was higher than that of AOI987 (Kd = 220 nM),



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11 THK-265 (Kd = 97 nM) and BODIPY-7 (Kd = 108 nM), and was similar to that of CRANAD-2 (Kd = 38.9 nM), BAP-1 (Kd = 44.1 nM), and BAP-2 (Kd = 55 nM), indicating that PAD-1 binds to the same site as other high-affinity fluorophores. Fluorescence Staining of Brain Tissue. Specific labeling of β-amyloid deposits in the brain was further confirmed using sections of brain tissue from APP/PS1 transgenic mice and control subjects. APP/PS1 transgenic mice co-express the Swedish mutation of APP (APPswe) with two FAD-PS1 variants that differentially accelerate amyloid pathology in the brain.31 Fluorescent images showed distinctive staining of β-amyloid deposits in the APP/PS1 transgenic mouse brain (Figure 4A, C), while wild-type mouse brain showed no labeling (Figure 4B). β-Amyloid deposits were confirmed present by staining the adjacent brain sections with CR (Figure 4D). The results suggested that PAD-1 showed affinity for β-amyloid deposits in addition to aggregated β-amyloid fibrils. This is the first report that a DCM-based probe has successfully labeled β-amyloid deposits. ---------Figure 4 ---------Blood-Brain Barrier Permeability. To evaluate blood-brain barrier permeability, in vivo imaging was performed in normal mice. The calculated logP value for PAD-1 is 2.06 (ChemDraw Ultra 8.0), which is in an optimal log P range to penetrate the blood-brain barrier by passive diffusion.17 As expected, the mice showed high fluorescence intensity immediately after intravenous injection of PAD-1, suggesting


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12 good blood-brain barrier penetration (Figure 5). Ex vivo imaging of brain signals at each timepoint revealed that PAD-1 crossed the blood-brain barrier with a high initial uptake, peaked at 5 min postinjection, and washed out rapidly from the brain (77% cleared at 60 min postinjection) (Figure 6). Since normal brain tissue has no β-amyloid to trap PAD-1, the fluorescence intensity should decrease quite rapidly. Therefore, the rapid clearance of PAD-1 from normal brain is appropriate for in vivo detection of β-amyloid deposits in the brain. The results showed that interference with the imaging from unbound probe is expected to be relatively minor. ---------Figure 5 ------------------Figure 6 ---------In Vivo Imaging. Finally, APP/PS1 transgenic mice and wild-type controls were used to assess the feasibility of PAD-1 for specific β-amyloid imaging in vivo using fluorescence imaging. In this study, fluorescence reflectance modality was used as it is suitable for fast imaging, despite that it has limited penetrating depth and resolution. Mice images were recorded after intravenous injection of PAD-1 at 5.0 mg/kg dosage. The fluorescence signals in the brain regions of the transgenic group were higher than those of the control group at 60 min postinjection (Figure 7). These results further



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13 confirmed that PAD-1 could cross the blood-brain barrier and label β-amyloid fibrils specifically in vivo. ---------Figure 7 ---------Nilsson and hammarström have recently reported a series of luminescent oligothiophene derivatives which afford a spectroscopic signature for individual β-amyloid conformation. These molecular probes showed promise in obtaining information on the progression of intermediate states by monitoring fluorescence parameters, such as anisotropy, and quantum efficiency changes upon β-amyloid binding.32-36 Among these probes, pentameric thiophene derivative p-FTAA, with a two-photon absorption cross-section in the wavelength range 775-850 nm, also showed specific labeling of β-amyloid deposits in the living brain using multiphoton microscopy, enabling the in vivo visualization of the populated oligomeric intermediates and fibril structures of β-amyloid.35, 36 Despite the inspiring progress in visualization of β-amyloid in the brains of living animals, there are several limitatons of p-FTAA. Firstly, although p-FTAA crossed the blood-brain barrier after intravenous injection, sufficient brain uptake might be greatly hampered by the bulky and fairly negatively charged molecule. PAD-1 has a small, compact and neutral molecule which facilities the efficient permeability of blood-brain barrier by passive diffusion, enabling the high uptake in the brain. Secondly, multiphoton microscopy is a laser-scanning modality that provides optical sectioning deep within tissue; however,


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14 the modality involves an invasive installation of a cranial window overlying the parietal cortex of the mice.35-38 PAD-1 thus may provide a practical noninvasive alternative, as no craniotomy is needed for in vivo imaging of cerebral β-amyloid fibrils, facilitating fast monitoring of β-amyloidosis in the brain and screening of effective anti-amyloid therapies in animal models.

CONCLUSION In vivo optical imaging for β-amyloid fibrils holds promise for basic disease research and potential clinical application. Smart fluorescence probes coupled with advances in imaging provide means to facilitate accelerated research in animal models. In this study, PAD-1 was evaluated as a novel fluorescence probe for cerebral β-amyloid fibrils. PAD-1 showed high affinity for aggregated β-amyloid fibrils and specific labeling of β-amyloid deposits in sections of APP/PS1 transgenic mouse brain. The fluorescence of PAD-1 enhanced significantly upon binding to β-amyloid fibrils. It is likely that PAD-1 used a similar mechanism as ThT to bind to groove-like β-amyloid fibrils and the structural constraints for binding enhanced fluorescence. In vivo fluorescence imaging confirmed the specific interaction of PAD-1 with β-amyloid fibrils in vivo. PAD-1 is the first example of DCM-based fluorophore for in vivo imaging for small animals, which provides a new type of fluorescence probe for cerebral β-amyloid fibrils. In summary, PAD-1 may become an attractive probe for the detection of β-amyloid fibrils both in vitro and in vivo.



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15 ACKNOWLEDGMENTS This research was funded by the National Natural Science Foundation of China (No. 81402891), Ph.D. Programs Foundation of Ministry of Education of China (No. 20130181120114), Scientific Research Foundation for Youth Scholars from Sichuan University (No. 2012SCU11091), and China Postdoctoral Science Foundation Grant (No. 2014M560723). The authors give special thanks to Dr. Mengchao Cui (College of Chemistry, Beijing Normal University) for assistance in the preparation of APP/PS1 transgenic mouse brain sections.

Author Contributions Y.C. and Z.Z. designed all experiments. Y.D., B.Z. and Y.C. performed in vitro experiments. Y.C. and B.Z. performed in vivo experiments. Y.C. completed data analysis, writing, and editing.

Notes The authors declare no competing financial interest.

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20 Table 1. Fluorescence excitation and emission profile of PAD-1 (1 µM). λex λem ε ΦF λabs (nm) (nm) (nm) (cm-1/M) PAD-1 (in methanol) 467 480 603 44936 0.43a PAD-1 (in PBS) 463 464 617 8300 0.005 PAD-1 + Aβ1-42 (in PBS)b 468 490 570 35012 0.13 a b Data from Reference 39. Fluorescence excitation and emission of PAD-1 were determined with Aβ1-42 aggregates (2.75 µM).



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21

F

N

N+

O

O

O

F F B- F F

S

N N B F F

S

2

HN S

NH N H

O

S S

N

S

N H

DANIR-2c

O

OH

O

ONa O

NaO

O

O

O

S

OH

N ONa

HO

CH3 OH

N N

H N 6

H

H3C H N

H 2 O

O

p-FTAA

CN

THK-265

ANCA-11

S

O CN

O

O

S

N

BAP-2

O O

CN

S

N N B F F

N

BAP-1

O

NaO

N

CRANAD-2

N N B F F

I

O

N

N

NIAD-4

BODIPY-7

O

CN

O

AOI-987

O

CN

S HO

F B

H

O

BMAOI-14

Figure 1. Chemical structure of fluorescence probes targeting cerebral β-amyloid fibrils.


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22 NC

CN

O N

donor-π-acceptor Figure 2. Chemical structure of PAD-1.



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23

Figure 3. Fluorescence “turn-on” of PAD-1 (1 µM) induced by aggregated β-amyloid fibrils (red dashed line); PAD-1 alone in PBS (blue solid line).


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24

Figure 4. Fluorescent staining of mouse brain sections with PAD-1. Intensive labeling of β-amyloid deposits in brain tissue from APP/PS1 transgenic model mouse is shown (A, C). Wild-type mouse brain exhibits no labeling by this probe (B). β-Amyloid deposits were confirmed by staining the adjacent brain section with Congo red, a common pathological dye for β-amyloid fibrils (D).



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25

Figure 5. In vivo fluorescence imaging at different time points after intravenous injection of PAD-1 (5.0 mg/kg) in normal mice (Ex 474 nm, Em 586 nm).


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26

Figure 6. Fluorescence intensity in normal mouse brains (n = 5 at each time point) after PAD-1 administration (5.0 mg/kg).



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Figure 7. Comparison of the fluorescent image at 60 min after intravenous injection of 5.0 mg/kg of PAD-1 into APP/PS1 transgenic mouse (left) and wild-type mouse (right).


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28 Graphical Abstract (for TOC only)


In Vivo Detection of Cerebral Amyloid Fibrils with Smart

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