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Synthesis and evaluation of novel two-photon fluorescence probes for in vivo imaging of amylin aggregates in the pancreas
Dyes and Pigments 170 (2019) 107615
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Synthesis and evaluation of novel two-photon fluorescence probes for in vivo imaging of amylin aggregates in the pancreas
T
Hiroyuki Watanabe∗, Yusuke Miki, Yoichi Shimizu, Hideo Saji, Masahiro Ono∗∗ Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto, 6068501, Japan
A R T I C LE I N FO
A B S T R A C T
Keywords: Amylin Two-photon fluorescence imaging Type 2 diabetes mellitus Fluorescence probe
The deposition of islet amyloid consisting of amylin aggregates is one of the hallmarks of type 2 diabetes mellitus (T2DM). Therefore, the amylin aggregate is regarded as an attractive target for diagnosis, treatment and elucidation of the pathogenic mechanism of T2DM. However, no fluorescence probe for in vivo imaging of amylin aggregates has been reported. In this study, we evaluated four fluorescent probes (DANIRs) including two novel compounds (DANIR-OH 2b and DANIR-OH 2c) for two-photon fluorescence imaging of amylin aggregates in the pancreas. All DANIRs showed binding affinity for amylin aggregates and clearly stained amylin aggregates in pancreatic sections of the islet amylin model mice and T2DM patients in in vitro. In an ex vivo fluorescence staining study, these probes also bound to amylin aggregates in the pancreas of the islet amylin model mice. Furthermore, in an in vivo two-photon fluorescence imaging study, we clearly imaged the amylin aggregates after injecting DANIRs via the tail vein of the islet amyloid model mice. The results suggest that DANIRs may be useful for two-photon fluorescence imaging of amylin aggregates in the living mouse pancreas.
1. Introduction The number of patients with diabetes mellitus worldwide will increase from 425 million in 2015 to 629 million in 2045 [1]. More than 90% of diabetes patients have type 2 diabetes mellitus (T2DM), characterized by insulin resistance and β-cell dysfunction [2,3]. The deposition of islet amyloid is one of the hallmarks of T2DM. Islet amyloid mainly consists of amylin, also known as islet amyloid polypeptide, which is a 37-amino-acid residue peptide hormone co-secreted and colocalized with insulin from pancreatic β-cells [4]. The secretion of amylin monomers is a part of the normal physiological response to increases in blood glucose concentrations. Some reports suggest that amylin aggregates are related to β-cell failure in T2DM [5,6]. Several mechanisms including membrane permeabilization, mitochondrial damage, endoplasmic reticulum stress, and inflammation have been proposed [7,8]. In addition, the amount of islet amyloid formation has been reported to be correlated with a β-cell mass reduction, but the relationship between T2DM and amylin is not completely clear [4,9]. Therefore, in vivo imaging of amylin aggregates is highly desirable for diagnosis, treatment, and the elucidation of the pathogenic mechanism of T2DM. The deposition of β-amyloid (Aβ) peptides in the brain is widely ∗
accepted as a biomarker of an initial and specific event in Alzheimer's disease (AD). Therefore, Aβ is the major target of diagnosis and treatment for AD [10]. Both Aβ and amylin aggregates have a β-sheet structure [11,12]. Since Aβ imaging probes are designed to bind to the β-sheet structure of Aβ aggregates, they are expected to bind not only to Aβ but also to amylin aggregates. Within a decade, many radiolabeled benzofuran derivatives have been developed that show high binding affinity for Aβ aggregates in vitro and in vivo [13–16]. Aβ aggregates are not deposited in the pancreas. Therefore, amylin imaging probes do not need a selective binding affinity for amylin aggregates versus Aβ aggregates. More recently, we tried to apply pyridyl benzofuran (PBF) derivatives as amylin imaging probes [17,18]. These probes showed binding affinity for amylin aggregates in vitro. Among them, a 99mTclabeled PBF derivative ([99mTc]1) bound islet amyloid in the model mouse pancreas. These results suggested that such Aβ imaging probes could be applied to amylin imaging. Optical imaging combined with an appropriate molecular probe is a versatile tool that offers real-time, operationally simple, inexpensive, nonradioactive, and high-resolution imaging [19,20]. However, no fluorescence imaging probes for in vivo imaging of amylin aggregates have been reported. We previously reported some fluorescence probes for imaging of Aβ aggregates [21–24]. Among them, DANIR 2b and
Corresponding author. Corresponding author. E-mail addresses: [email protected] (H. Watanabe), [email protected] (M. Ono).
∗∗
https://doi.org/10.1016/j.dyepig.2019.107615 Received 10 January 2019; Received in revised form 2 May 2019; Accepted 2 June 2019 Available online 04 June 2019 0143-7208/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. Chemical structures of DANIRs evaluated in this study.
solvent was removed in a vacuum, and a yellow oily liquid (1.79 g, 93.2%) was obtained after silica gel chromatography (CHCl3: MeOH = 19 : 1). 1H NMR (400 MHz, CDCl3) δ 1.57 (m, 6H), 3.12 (s, 3H), 3.62 (m, 1H), 3.66 (m, 4H), 3.94 (m, 1H), 6.76 (d, J = 9.2Hz, 2H), 7.73 (d, J = 8.8 Hz, 2H), 9.74 (s, 1H). MS (ESI) m/z 264.2 [MH+].
DANIR 2c could visualize Aβ aggregates in vitro and in vivo (Fig. 1) [23]. In addition, two-photon absorption cross-section values of these probes were previously calculated. Since these values were sufficient for twophoton fluorescence imaging in the near-infrared region (DANIR 2b; 201 GM at 876 nm, DANIR 2c; 503 GM at 984 nm), DANIR 2b and DANIR 2c showed potential for application to in vivo two-photon fluorescence imaging [25]. Compared to one-photon fluorescence imaging, fluorescence imaging with two-photon microscopy (TPM) has a number of advantages, including greater penetration depth, minimal background autofluorescence interference, lower photodamage to tissue, and lower photobleaching [20,26,27]. In vivo fluorescence imaging by TPM has great potential not only for biological research such as understanding of the dynamic aspect of the metastatic process but also for clinical applications including early diagnosis, monitoring therapy, and precise treatments [27–30]. In vivo imaging of amylin aggregates with TPM is a more attractive tool for the diagnosis and elucidation of the pathogenic mechanism of T2DM than imaging with one-photon confocal microscopy. Taken together, it is reasonable to apply DANIR 2b and DANIR 2c to two-photon fluorescence imaging of amylin aggregates. However, DANIR 2b and DANIR 2c must be dissolved in an organic solvent (DMSO: propylene glycol = 2 : 8) [23]. Because organic solvents are frequently toxic to living subjects, we newly designed two water-soluble DANIRs, DANIR-OH 2b and DANIROH 2c, based on DANIR 2b and DANIR 2c, as fluorescence imaging probes targeting amylin aggregates (Fig. 1). In the present study, we synthesized these two novel DANIRs and evaluated the binding affinity for amylin aggregates of four DANIRs (DANIR 2b, DANIR 2c, DANIROH 2b, and DANIR-OH 2c). Furthermore, we tried to apply all four probes to the in vivo two-photon fluorescence imaging of amylin aggregates.
2.1.4. (E)-3-(4-((2-Hydroxyethyl)(methyl)amino)phenyl)acrylaldehyde (2) To a stirred solution of 1 (1.8 g, 7.4 mmol) in anhydrous THF (50 mL) was added (1,3-dioxolan-2-ylmethyl)triphenylphosphonium bromide (6.05 g, 14 mmol) and 18-crown-6 (26 mg, 0.1 mmol). After stirring for 30 min at room temperature, NaH (60% dispersion in mineral oil, 2.5 g) was added, and stirring was continued for 3 h. The reaction was quenched with water and the mixture was extracted with AcOEt (3 × 100 mL). After the combined organic phase was dried over anhydrous sodium sulfate and the solvent was removed in a vacuum, the residue was purified by silica gel chromatography (AcOEt: Hexane = 3 : 1) to give a yellow oily liquid. THF (20 mL) and HCl (0.7 M) were added and stirred for 3 h at room temperature. After washing the water phase with AcOEt (2 × 50 mL), the mixture was neutralized with Na2CO3 and extracted with AcOEt (2 × 100 mL). The combined organic phase was dried over anhydrous sodium sulfate and removed in a vacuum, and the residue was purified by silica gel chromatography (CHCl3: MeOH = 9 : 1) to give 2 (820 mg, 37.1%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 3.06 (s, 3H), 3.67 (t, J = 5.8 Hz, 2H), 4.26 (t, J = 5.4 Hz, 2H), 6.54 (q, J = 8.0 Hz, 1H), 6.73 (d, J = 7.2 Hz, 2H), 7.37 (d, J = 15.6 Hz, 1H), 7.45 (d, J = 9.2 Hz, 2H), 9.59 (d, J = 8.0 Hz, 1H). MS (ESI) m/z 206.1 [MH+]. 2.1.5. (E)-2-(3-(4-((2-Hydroxyethyl)(methyl)amino)phenyl)allylidene) malononitrile (3, DANIR-OH 2b) To a solution of 2 (170 mg, 0.8 mmol) in chloroform (20 mL) was added malononitrile (720 mg, 11 mmol), and then triethylamine (0.1 mL) was added as a catalyst. After stirring for 30 min at room temperature, the solvent was removed in a vacuum. The residue was purified by silica gel chromatography (AcOEt: Hexane = 2 : 1) and RPHPLC (MeCN: H2O = 6 : 4) to give 3 (15.6 mg, 7.6%) as a dark red solid. 1H NMR (400 MHz, CDCl3) δ 3.13 (s, 3H), 3.63 (t, J = 5.6 Hz, 2H), 3.87 (t, J = 5.0 Hz, 2H), 6.75 (d, J = 8.8 Hz, 2H), 7.03 (dd, J = 16.8 Hz, 1H), 7.17 (d, J = 14.8 Hz, 1H), 7.49 (m, 3H). 13C NMR (100 MHz, CDCl3) δ 39.20, 54.31, 60.19, 112.11, 113.00, 114.91, 117.46, 131.71, 151.32, 152.52, 160.51. HRMS (FAB+) m/z calcd for C15H16N3O 254.1525 found 254.1293 [MH+].
2. Material and methods 2.1. Chemistry 2.1.1. General All reagents were obtained commercially and used without further purification unless otherwise indicated. W-Prep 2XY (Yamazen, Osaka, Japan) was used for silica gel column chromatography on a Hi Flash silica gel column (40 μm, 60 Å, Yamazen). 1H and 13C NMR spectra were obtained on a JEOL JNM-ECS400 (JEOL Ltd., Tokyo, Japan) spectrometer with TMS as an internal standard. Coupling constants are reported in hertz. Multiplicity was defined by s (singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). Low-resolution mass spectra (LRMS) were obtained using a SHIMADZU LCMS-2020 (SHIMADZU, Kyoto, Japan). High-resolution mass spectrometry (HRMS) was conducted with a JEOL JMS-700.
2.1.6. (E)-3-(4-((2-((Tetrahydro-2H-pyran-2-yl)oxy)ethyl)(methyl) amino)phenyl)acrylaldehyde (4) The same reaction described above to prepare 1 was used, and 4 was obtained as a yellow oily liquid (600 mg, 70.9%). 1H NMR (400 MHz, CDCl3) δ 1.54 (m, 4H), 3.09 (s, 3H), 3.49 (m, 1H), 3.63 (m, 3H), 3.78 (m, 1H), 3.91 (m, 1H), 4.58 (t, J = 3.4 Hz, 1H), 6.54 (q, J = 7.9 Hz, 1H), 6.72 (d, J = 9.2 Hz, 2H), 7.37 (d, J = 15.6 Hz, 1H), 7.44 (d, J = 9.2 Hz, 2H), 9.59 (d, J = 8.0 Hz, 1H). MS (ESI) m/z 290.2 [MH+].
2.1.2. Synthesis of DANIR 2b and DANIR 2c DANIR 2b and DANIR 2c were synthesized according to a previously reported method [23]. 2.1.3. N-Methyl-N-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)-4aminobenzaldehyde (1) To a solution of N-methyl-N-(2-hydroxyethyl)-4-aminobenzaldehyde (1.14 g, 6.4 mmol) in anhydrous CH2Cl2 (20 mL) was added 3,4dihydro-2H-pyran (2.0 g, 24 mmol) and pyridinium toluene-4-sulphonate (400 mg, 1.6 mmol). The mixture was stirred at 40 °C for 3 h. The
2.1.7. (2E,4E)-5-(4-((2-Hydroxyethyl)(methyl)amino)phenyl)penta-2,4dienal (5) The same reaction described above to prepare 2 was used, and 5 was obtained as a yellow solid (102 mg, 21.3%). 1H NMR (400 MHz, CDCl3) δ 3.06 (s, 3H), 3.57 (t, J = 5.6 Hz, 2H), 3.85 (t, J = 5.6 Hz, 2H), 6.18 (q, 2
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2.6. In vitro fluorescent staining of islet amyloid deposits in human pancreas
J = 7.7 Hz, 1H), 6.82 (m, 4H), 7.25 (m, 1H), 7.40 (d, J = 8.8 Hz, 2H), 9.56 (d, J = 8.0 Hz, 1H). MS (ESI) m/z 232.1 [MH+].
Paraffin-embedded pancreas tissue sections from a T2DM patient (69-year-old woman) were purchased from BioChain Institute Inc. (Hayward, CA, USA). The sections were subjected to two 15-min incubations in xylene, two 1-min incubations in 100% EtOH, two 1-min incubations in 90% EtOH, and one 1-min incubation in 70% EtOH to completely deparaffinize them, followed by two 2.5-min washes in water. The sections were incubated with 20% or 50% solutions of DANIR derivatives for 30 min at room temperature. After washing in 20% EtOH for 10 min, fluorescent observation was performed using an FSX100 inverted microscope equipped with a U-MWIG3 cube. The location of islet amyloid was comfirmed by staining the same sections with thioflavin S and observing with the microscope equipped with a UMWIBA3 cube.
2.1.8. 2-((2E,4E)-5-(4-((2-Hydroxyethyl)(methyl)amino)phenyl)penta2,4-dien-1-ylidene)malononitrile (6, DANIR-OH 2c) The same reaction described above to prepare 3 was used, and 6 was obtained as a dark-purple solid (15.5 mg, 12.6%). 1H NMR (400 MHz, CDCl3) δ 3.10 (s, 3H), 3.60 (t, J = 5.2 Hz, 2H), 3.86 (t, J = 5.2 Hz, 2H), 6.78 (m, 4H), 6.97 (d, J = 15.2 Hz, 1H), 7.07 (dd, J = 10.8 Hz, 14.4 Hz, 1H), 7.40 (d, J = 8.8 Hz, 2H), 7.44 (d, J = 12.0 Hz, 1H) 13C NMR (100 MHz, CDCl3) δ 39.05, 54.47, 60.29, 112.20, 112.67, 122.36, 123.47, 123.89, 130.06, 146.03, 151.30, 151.94, 159.59. HRMS (FAB +) m/z calcd for C17H18N3O 280.1372 found 280.1450 [MH+]. 2.2. Fluorescence measurement in CHCl3 Absorption, fluorescence excitation and emission wavelengths, and quantum yields were determined with 10 μM of the compounds in CHCl3 (UV-1800, SHIMADZU, RF-6000, SHIMADZU, or Fluorolog-3, HORIBA Jobin Yvon Inc., Kyoto, Japan).
2.7. Solubility assay of DANIRs Solubility assay was performed by measuring the absorbance of DANIRs (0–500 μM) in each solution (DMSO: propylene glycol = 2 : 8 or saline containing 5 v/v% DMSO). After voltex and centrifugation (1 min), the absorbance of 200 μL of each solution was measured at 254 nm with a UV-spectrophotometer (UV-1800).
2.3. Fluorescence measurement using amylin aggregates A solid form of amylin was purchased from Peptide Institute (Osaka, Japan). Aggregation was carried out according to the method reported previously [17]. A mixture (10% DMSO) containing DANIRs (2.0 μM) and amylin aggregates was incubated at room temperature for 30 min. After incubation, fluorescence emission spectra of DANIRs were collected with excitation at 525 nm (DANIR 2b), 560 nm (DANIR 2c), 510 nm (DANIR-OH 2b), and 550 nm (DANIR-OH 2c), which are the maximum respective excitation wavelengths in the presence of amylin aggregates (Infinite M200PRO, Tecan, Männedorf, Switzerland).
2.8. Ex vivo fluorescent staining of amylin aggregates in islet amyloid model mouse Islet amyloid model and normal mice were intravenously injected with DANIRs (500 μM (DMSO: propylene glycol = 2 : 8 for DANIR 2b and DANIR 2c, saline containing 5 v/v % DMSO for DANIR-OH 2b and DANIR-OH 2c), 50 μL). The mice were sacrificed by decapitation at 30 min postinjection. The pancreas was removed and fixed with 4% paraformaldehyde in phosphate buffer. Sections were prepared and observed using the same method as described for the in vitro fluorescent staining study. The locations of aggregates were confirmed by staining the same sections with thioflavin S.
2.4. Saturation binding assay using amylin aggregates A solution of 40 μL of amylin aggregates (final conc., 2.0 μM) was added to the mixture containing 40 μL of DANIRs, and thioflavin T (final conc., 3.9 nM - 10 μM in DMSO) and 320 μL of the buffer (20 mM Tris/HCl, 100 mM NaCl, pH 7.5) in a final volume of 400 μL. Nonspecific binding was defined without amylin aggregates. After the mixture was incubated for 30 min at room temperature, the solution was transferred to a black 96-well plate, and the fluorescence intensity was measured using a microplate reader (Infinite M200PRO). The dissociation constants (Kd) were determined using GraphPad Prism 6.0.
2.9. In vivo pancreas imaging in islet amyloid model mice using two-photon microscopy Islet amyloid model mice were anesthetized and left-flank incised. A solution of DANIRs (500 μM (DMSO: propylene glycol = 2 : 8 for DANIR 2b and DANIR 2c, saline containing 5 v/v % DMSO for DANIROH 2b and DANIR-OH 2c), 50 μL) was injected intravenously into the tail vein. Each mouse was placed and fixed in the lateral position on an electric heating pad maintained at 37 °C while maintaining anesthesia. To stabilize the exposed pancreas, a vacuum-stabilized imaging window was placed over the pancreas. At 30 min postinjection, Z-stack images of fluorescence accumulation were acquired from the surface inward at 2 μm intervals and at a scan speed of 4 μs per pixel with a FV1200MPE Laser Scanning Microscope (Olympus) and BX61WI Upright Microscope (Olympus) equipped with mCherry (Ch1, 570–615 nm) and iRFP (Ch2, 675–740 nm) as emission filters and with excitation at 980 nm (DANIR 2b), 840 nm (DANIR 2c), 1000 nm (DANIR-OH 2b), and 1120 nm (DANIR-OH 2c). The excitation wavelengths were determined in a phantom study of DANIRs solution. Recorded images were processed with MetaMorph software (Moleculer Devices, Sunnyvale, CA).
2.5. In vitro fluorescent staining of amylin aggregates in islet amyloid model mice The experiments with animals were conducted in accordance with our institutional guidelines and approved by the Kyoto University Animal Care Committee. Islet amyloid model mice were prepared as reported previously [17]. Each islet amyloid model mouse was sacrificed by decapitation and the pancreas was immediately removed and fixed with 4% paraformaldehyde in phosphate buffer. After fixing for 1 day, the pancreas was embedded in Super Cryoembedding Medium (SCEM) Compound (Section-Lab Co Ltd., Hiroshima, Japan) and then frozen in a dry ice/hexane bath. Frozen sections were prepared at a thickness of 20 μm. Next, they were incubated with 20% or 50% DMSO solution of DANIRs (100 μM) for 30 min. The location of aggregates was confirmed by staining adjacent sections with thioflavin S (100 μM). Finally, the sections were washed with 20% DMSO. Fluorescence observation was performed using an FSX100 inverted microscope (Olympus, Japan) equipped with an Olympus U-MWIBA3 cube for thioflavin S or an Olympus U-MWIG3 cube for DANIRs. The obtained images were recorded and processed with CellSens Dimension Desktop (Olympus).
3. Results and discussion 3.1. Synthesis of DANIRs The synthesis of DANIR-OH 2b and DANIR-OH 2c is outlined in Scheme 1. The hydroxy group of N-methyl-N-(2-hydroxyethyl)-4-aminobenzaldehyde was protected with a 2-tetrahydropyranyl group (THP). Then, 2 and 5 were obtained by the Wittig reaction and 3
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Scheme 1. Synthetic route of DANIRs. Reagents: (a) 3,4-dihydro-2H-pyran, pyridinium toluene-4-sulphonate, CH2Cl2; (b) (1) (1,3-dioxan-2-yl-methyl)triphenylphosphoniumbromide, 18-crown-6, NaH, THF, (2) HCl, THF; (c) malononitrile, triethylamine, CHCl3.
deprotection of THP (37 and 21% yields). 3 (DANIR-OH 2b) and 6 (DANIR-OH 2c) were prepared by condensation of the corresponding aldehydes with malononitrile in chloroform (7.6 and 13% yields).
3.3. Binding affinity for amylin aggregates in vitro Next, we performed saturation binding experiments and calculated the apparent binding dissociation constant (Kd) to quantitatively evaluate the binding affinity for amylin aggregates. The Kd values estimated for DANIR 2b, DANIR 2c, DANIR-OH 2b, and DANIR-OH 2c were 500, 80.5, 1220, and 695 nM, respectively (Table 2). These values were much higher than for IPBF (Kd = 8.3 nM), but the experimental method was different, so it is not appropriate to directly compare the values. Because the Kd value of thioflavin T was 791 nM for amylin aggregates, the binding affinity of DANIRs except for DANIR-OH 2b may be sufficient to visualize amylin aggregates. Compared to the binding affinity for Aβ aggregates (DANIR 2b: 36 nM, DANIR 2c: 27 nM) [23], the binding affinity of DANIR 2c for amylin aggregates was similar to that for Aβ aggregates, but the affinity of DANIR 2b for amylin aggregates was lower than that for Aβ aggregates. This phenomenon may be due to the differences in amino acids constituting the β-sheet structure between Aβ and amylin aggregates.
3.2. Fluorescence characterization First of all, we measured the spectra of absorbance, excitation, emission, quantum yield, and excitation coefficient in CHCl3 (Table 1 and Figure S1). These properties of DANIR-OH 2b and DANIR-OH 2c were similar to those of DANIR 2b and DANIR 2c, respectively. In general, extension of the π-electron system increases absorption coefficient, but absorption coefficient of DANIRs does not correlate with increasing the number of conjugated double bonds. Although the reason is not clear, the absorption coefficients of previously reported probes with similar structure also showed same trend [31–34]. A change in fluorescence intensity upon binding to amylin aggregates is one of the essential properties for fluorescence probes targeting amyloid aggregates [35, 36]. Therefore, we initially evaluated the fluorescence intensity of DANIRs with or without amylin aggregates (Fig. 2). In a solution containing amylin aggregates, their fluorescence intensities significantly increased with the concentration of aggregates. These results suggested that all four DANIRs have the potential to image amylin aggregates in vitro and in vivo. Compared to thioflavin-T and thioflavin-S, a dye commonly used to stain amyloid aggregates, DANIRs showed longer emission wavelengths. Notably, the emission wavelengths of DANIR 2c and DANIR-OH 2c were in the near-infrared region (650–900 nm). The background autofluorescence is low in this region, suggesting that these probes may be more suitable for imaging of amylin aggregates in the pancreas.
3.4. In vitro fluorescent staining of amylin aggregates in islet amyloid model mouse In order to confirm the affinity of DANIRs for amylin aggregates in the mouse pancreas, we performed in vitro fluorescence staining with DANIR 2b, DANIR 2c, DANIR-OH 2b, and DANIR-OH 2c using pancreas sections from islet amyloid model mice having amylin aggregates transplanted in the pancreas [18]. Several fluorescence positive regions were observed in mouse pancreas sections (Fig. 3A, 3B, 3C, and 3D). The staining patterns of these probes were consistent with that observed with thioflavin S, a dye commonly used to stain amyloid aggregates including amylin aggregates (Fig. 3E). This result suggested that all four DANIRs specifically bind to amylin aggregates in the mouse pancreas.
Table 1 Fluorescence profiles of DANIRs. Compound
DANIR 2b DANIR 2c DANIR-OH 2b DANIR-OH 2c
Abs (nm)a
490 521 486 515
Ex (nm)a
493 520 488 517
3.5. In vitro fluorescent staining of islet amyloid deposits in human pancreas Em (nm)a
559 644 558 644
Quantum yield (%)
Absorption coefficient (M−1cm−1)
0.3 1.1 0.4b 1.1c
54200 49067 51267 37333
The depositions of islet amyloid in human pancreas were composed of various molecules, including amylin. To confirm the affinity for islet amyloid deposited in human pancreas, we also carried out in vitro fluorescence staining with DANIR 2b, DANIR 2c, DANIR-OH 2b, and DANIR-OH 2c using post-mortem human pancreatic sections from a T2DM patient. For all DANIRs, many fluorescence spots were observed (Fig. 4A, 4C, 4E, and 4G), and this staining pattern correlated well with that observed with thioflavin S in the same sections (Fig. 4B, 4D, 4F, and 4H). These results suggested that all four DANIRs could bind to and visualize amylin aggregates in islet amyloids deposited in the pancreas in T2DM patients.
a
Wavelengths of absorption maxima, wavelengths of excitation maxima, and wavelengths of emission maxima of DANIRs were determined with 10 μM of the compounds in CHCl3. b DANIR 2b was taken as a reference for determining quantum yields. c DANIR 2c was taken as a reference for determining quantum yields. 4
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Fig. 2. Fluorescence intensities of DANIR 2b (A), DANIR 2c (B), DANIR-OH 2b (C), and DANIR-OH 2c (D) in interaction with amylin aggregates.
3.6. Solubility assay of DANIRs
Table 2 Apparent binding constants (Kd) of DANIRs and Thioflavin T. Compound
Kd (nM)a
DANIR 2b DANIR 2c DANIR-OH 2b DANIR-OH 2c Thioflavin T
500 ± 93 80.5 ± 6.2 1220 ± 284 695 ± 118 791 ± 91
Before in vivo experiments, we confirmed the solubility of DANIRs. First, we evaluated the solubility of the organic solvent (DMSO: propylene glycol = 2 : 8), which was previously used in an in vivo study with DANIR 2b and DANIR 2c [23]. For all DANIRs, the absorbance correlated well with the concentration (R2 ≧ 0.98) (Fig. 5A), indicating that all four DANIRs were soluble in the organic solvent at the concentration of 500 μM. Although DANIR 2b and DANIR 2c were not soluble (R2 = 0.031 and 0.637, respectively) in saline containing 5 v/v % DMSO, the solubilities of DANIR-OH 2b (R2 = 0.984) and DANIR-OH 2c (R2 = 0.996) were markedly higher in this aqueous media (Fig. 5B).
a
Values are the mean ± standard error of the mean for three independent experiments performed in triplicate.
Fig. 3. In vitro fluorescence staining of DANIR 2b (A), DANIR 2c (B), DANIR-OH 2b (C), and DANIR-OH 2c (D) in 20 μm sections from islet amyloid model mice. Labeled plaques were confirmed by staining of the adjacent sections with thioflavin S (E). Scale bars indicate 1 mm. 5
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Fig. 4. In vitro fluorescence staining of DANIR 2b (A), DANIR 2c (C), DANIR-OH 2b (E), and DANIR-OH 2c (G). The presence of amylin aggregates was confirmed by staining the same sections with thioflavin S (B, D, F, and H). Scale bars indicate 500 μm.
with little autofluorescence at 30 min after injection of all four DANIRs through the tail vein. Based on the results of the ex vivo fluorescence staining study after in vivo two-photon fluorescence imaging (Fig. S2), the fluorescence signals obtained with TPM were considered to indicate amylin aggregates. These results suggested that all four DANIRs may be useful for in vivo two-photon fluorescence imaging of amylin aggregates in the mouse pancreas. In the in vitro binding study, DANIR-OH 2b showed lower affinity for amylin than the other DANIRs, but all DANIRs clearly visualized amylin aggregates in the in vivo two-photon fluorescence imaging study. This result suggested that the Kd value (1220 nM) of DANIR-OH 2b for amylin aggregates was sufficient for in vivo twophoton fluorescence imaging. DANIRs are the first probes for in vivo imaging of amylin aggregates with TPM. This technique is needed when preparing to surgically expose the pancreas. Because the surgical preparation may cause a great deal of strain to the mice, it is one of the disadvantages for in vivo imaging of pancreas with TPM. Although this disadvantage was not eliminated, two-photon fluorescence imaging of amylin aggregates may contribute considerably in elucidating the relationship between T2DM and amylin.
Because organic solvents are frequently toxic in living subjects, these results suggested that DANIR-OH 2b and DANIR-OH 2c may be more appropriate probes for in vivo imaging than DANIR 2b and DANIR 2c. Based on the results of the solubility study, we used the organic solvent (DMSO: propylene glycol = 2 : 8) for DANIR 2b and DANIR 2c, and the aqueous media (saline containing 5 v/v% DMSO) for DANIR-OH 2b and DANIR-OH 2c in the ex vivo and in vivo imaging studies. 3.7. Ex vivo fluorescent staining of amylin aggregates in islet amyloid model mice Ex vivo fluorescence staining was performed to test the labeling of amylin aggregates by DANIRs in vivo. We removed each pancreas at 30 min after the injection of DANIRs, prepared frozen pancreatic sections, and observed them with a one-photon fluorescence microscope. High fluorescence regions were observed in the pancreas sections of the islet amyloid model mice (Fig. 6A, 6C, 6E, and 6G). Because these regions were clearly stained with thioflavin S (Fig. 6B, 6D, 6F, and 6H), all four DANIRs could bind to and image the transplanted amylin aggregates in the living mouse pancreas.
4. Conclusions 3.8. In vivo imaging of pancreas in islet amyloid model mouse using twophoton microscopy
We newly designed and synthesized DANIR-OH 2b and DANIR-OH 2c and evaluated four DANIRs (DANIR 2b, DANIR 2c, DANIR-OH 2b, and DANIR-OH 2c) for two-photon fluorescence imaging of amylin aggregates in the pancreas. All DANIRs showed binding affinities with amylin aggregates in the in vitro fluorescent saturation binding assay and the fluorescent staining study. In the in vivo two-photon
Finally, to evaluate the potential of DANIRs for in vivo two-photon fluorescence imaging targeting amylin aggregates, we performed twophoton fluorescence imaging experiments using living islet amyloid model mice. As shown in Fig. 7, clear and bright images were obtained
Fig. 5. Correlation between absorbance and concentration of DANIR 2b, DANIR 2c, DANIR-OH 2b, and DANIR-OH 2c in organic solvent (DMSO: propylene glycol = 2 : 8) (A) and aqueous media (saline containing 5 v/v% DMSO) (B). 6
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Fig. 6. Ex vivo fluorescence observation of pancreas sections from islet amyloid model mice after injection of DANIR 2b (A), DANIR 2c (C), DANIR-OH 2b (E), and DANIR-OH 2c (G). The presence of amylin aggregates was confirmed by staining the same sections with thioflavin S (B, D, F, and H). Scale bars of A, B, C, D, E, and F indicate 500 μm, and those of G and H are 1 mm.
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Fig. 7. In vivo images of amylin aggregates with two-photon excitation fluorescence microscopy after injection of DANIR 2b (A), DANIR 2c (B), DANIR-OH 2b (C), and DANIR-OH 2c (D) into islet amyloid model mice. The XY plane is a square, 507.9 μm × 507.9 μm. Z is 108 μm (A), 84 μm (B), 46 μm (C), and 164 μm (D), respectively.
fluorescence imaging studies using islet amyloid model mice, we successfully obtained clear images of amylin aggregates after injection via the tail vein. In addition, in the ex vivo fluorescence staining study, we confirmed that these probes bound to amylin aggregates in the model mouse pancreas. These results suggested that the DANIRs tested in this study may be useful for two-photon fluorescent imaging of amylin aggregates. Among the DANIRs, because DANIR-OH 2b and DANIR-OH 2c can be used in aqueous media, these probes are more appropriate for in vivo imaging studies.
Acknowledgements This work was supported by the Nakatani Foundation for Advancement of Measuring Technologies in Biomedical Engineering, Takeda Science Foundation, and Kyoto University Live Imaging Center.
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.dyepig.2019.107615.
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Dyes and Pigments journal homepage: www.elsevier.com/locate/dyepig
Synthesis and evaluation of novel two-photon fluorescence probes for in vivo imaging of amylin aggregates in the pancreas
T
Hiroyuki Watanabe∗, Yusuke Miki, Yoichi Shimizu, Hideo Saji, Masahiro Ono∗∗ Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto, 6068501, Japan
A R T I C LE I N FO
A B S T R A C T
Keywords: Amylin Two-photon fluorescence imaging Type 2 diabetes mellitus Fluorescence probe
The deposition of islet amyloid consisting of amylin aggregates is one of the hallmarks of type 2 diabetes mellitus (T2DM). Therefore, the amylin aggregate is regarded as an attractive target for diagnosis, treatment and elucidation of the pathogenic mechanism of T2DM. However, no fluorescence probe for in vivo imaging of amylin aggregates has been reported. In this study, we evaluated four fluorescent probes (DANIRs) including two novel compounds (DANIR-OH 2b and DANIR-OH 2c) for two-photon fluorescence imaging of amylin aggregates in the pancreas. All DANIRs showed binding affinity for amylin aggregates and clearly stained amylin aggregates in pancreatic sections of the islet amylin model mice and T2DM patients in in vitro. In an ex vivo fluorescence staining study, these probes also bound to amylin aggregates in the pancreas of the islet amylin model mice. Furthermore, in an in vivo two-photon fluorescence imaging study, we clearly imaged the amylin aggregates after injecting DANIRs via the tail vein of the islet amyloid model mice. The results suggest that DANIRs may be useful for two-photon fluorescence imaging of amylin aggregates in the living mouse pancreas.
1. Introduction The number of patients with diabetes mellitus worldwide will increase from 425 million in 2015 to 629 million in 2045 [1]. More than 90% of diabetes patients have type 2 diabetes mellitus (T2DM), characterized by insulin resistance and β-cell dysfunction [2,3]. The deposition of islet amyloid is one of the hallmarks of T2DM. Islet amyloid mainly consists of amylin, also known as islet amyloid polypeptide, which is a 37-amino-acid residue peptide hormone co-secreted and colocalized with insulin from pancreatic β-cells [4]. The secretion of amylin monomers is a part of the normal physiological response to increases in blood glucose concentrations. Some reports suggest that amylin aggregates are related to β-cell failure in T2DM [5,6]. Several mechanisms including membrane permeabilization, mitochondrial damage, endoplasmic reticulum stress, and inflammation have been proposed [7,8]. In addition, the amount of islet amyloid formation has been reported to be correlated with a β-cell mass reduction, but the relationship between T2DM and amylin is not completely clear [4,9]. Therefore, in vivo imaging of amylin aggregates is highly desirable for diagnosis, treatment, and the elucidation of the pathogenic mechanism of T2DM. The deposition of β-amyloid (Aβ) peptides in the brain is widely ∗
accepted as a biomarker of an initial and specific event in Alzheimer's disease (AD). Therefore, Aβ is the major target of diagnosis and treatment for AD [10]. Both Aβ and amylin aggregates have a β-sheet structure [11,12]. Since Aβ imaging probes are designed to bind to the β-sheet structure of Aβ aggregates, they are expected to bind not only to Aβ but also to amylin aggregates. Within a decade, many radiolabeled benzofuran derivatives have been developed that show high binding affinity for Aβ aggregates in vitro and in vivo [13–16]. Aβ aggregates are not deposited in the pancreas. Therefore, amylin imaging probes do not need a selective binding affinity for amylin aggregates versus Aβ aggregates. More recently, we tried to apply pyridyl benzofuran (PBF) derivatives as amylin imaging probes [17,18]. These probes showed binding affinity for amylin aggregates in vitro. Among them, a 99mTclabeled PBF derivative ([99mTc]1) bound islet amyloid in the model mouse pancreas. These results suggested that such Aβ imaging probes could be applied to amylin imaging. Optical imaging combined with an appropriate molecular probe is a versatile tool that offers real-time, operationally simple, inexpensive, nonradioactive, and high-resolution imaging [19,20]. However, no fluorescence imaging probes for in vivo imaging of amylin aggregates have been reported. We previously reported some fluorescence probes for imaging of Aβ aggregates [21–24]. Among them, DANIR 2b and
Corresponding author. Corresponding author. E-mail addresses: [email protected] (H. Watanabe), [email protected] (M. Ono).
∗∗
https://doi.org/10.1016/j.dyepig.2019.107615 Received 10 January 2019; Received in revised form 2 May 2019; Accepted 2 June 2019 Available online 04 June 2019 0143-7208/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. Chemical structures of DANIRs evaluated in this study.
solvent was removed in a vacuum, and a yellow oily liquid (1.79 g, 93.2%) was obtained after silica gel chromatography (CHCl3: MeOH = 19 : 1). 1H NMR (400 MHz, CDCl3) δ 1.57 (m, 6H), 3.12 (s, 3H), 3.62 (m, 1H), 3.66 (m, 4H), 3.94 (m, 1H), 6.76 (d, J = 9.2Hz, 2H), 7.73 (d, J = 8.8 Hz, 2H), 9.74 (s, 1H). MS (ESI) m/z 264.2 [MH+].
DANIR 2c could visualize Aβ aggregates in vitro and in vivo (Fig. 1) [23]. In addition, two-photon absorption cross-section values of these probes were previously calculated. Since these values were sufficient for twophoton fluorescence imaging in the near-infrared region (DANIR 2b; 201 GM at 876 nm, DANIR 2c; 503 GM at 984 nm), DANIR 2b and DANIR 2c showed potential for application to in vivo two-photon fluorescence imaging [25]. Compared to one-photon fluorescence imaging, fluorescence imaging with two-photon microscopy (TPM) has a number of advantages, including greater penetration depth, minimal background autofluorescence interference, lower photodamage to tissue, and lower photobleaching [20,26,27]. In vivo fluorescence imaging by TPM has great potential not only for biological research such as understanding of the dynamic aspect of the metastatic process but also for clinical applications including early diagnosis, monitoring therapy, and precise treatments [27–30]. In vivo imaging of amylin aggregates with TPM is a more attractive tool for the diagnosis and elucidation of the pathogenic mechanism of T2DM than imaging with one-photon confocal microscopy. Taken together, it is reasonable to apply DANIR 2b and DANIR 2c to two-photon fluorescence imaging of amylin aggregates. However, DANIR 2b and DANIR 2c must be dissolved in an organic solvent (DMSO: propylene glycol = 2 : 8) [23]. Because organic solvents are frequently toxic to living subjects, we newly designed two water-soluble DANIRs, DANIR-OH 2b and DANIROH 2c, based on DANIR 2b and DANIR 2c, as fluorescence imaging probes targeting amylin aggregates (Fig. 1). In the present study, we synthesized these two novel DANIRs and evaluated the binding affinity for amylin aggregates of four DANIRs (DANIR 2b, DANIR 2c, DANIROH 2b, and DANIR-OH 2c). Furthermore, we tried to apply all four probes to the in vivo two-photon fluorescence imaging of amylin aggregates.
2.1.4. (E)-3-(4-((2-Hydroxyethyl)(methyl)amino)phenyl)acrylaldehyde (2) To a stirred solution of 1 (1.8 g, 7.4 mmol) in anhydrous THF (50 mL) was added (1,3-dioxolan-2-ylmethyl)triphenylphosphonium bromide (6.05 g, 14 mmol) and 18-crown-6 (26 mg, 0.1 mmol). After stirring for 30 min at room temperature, NaH (60% dispersion in mineral oil, 2.5 g) was added, and stirring was continued for 3 h. The reaction was quenched with water and the mixture was extracted with AcOEt (3 × 100 mL). After the combined organic phase was dried over anhydrous sodium sulfate and the solvent was removed in a vacuum, the residue was purified by silica gel chromatography (AcOEt: Hexane = 3 : 1) to give a yellow oily liquid. THF (20 mL) and HCl (0.7 M) were added and stirred for 3 h at room temperature. After washing the water phase with AcOEt (2 × 50 mL), the mixture was neutralized with Na2CO3 and extracted with AcOEt (2 × 100 mL). The combined organic phase was dried over anhydrous sodium sulfate and removed in a vacuum, and the residue was purified by silica gel chromatography (CHCl3: MeOH = 9 : 1) to give 2 (820 mg, 37.1%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 3.06 (s, 3H), 3.67 (t, J = 5.8 Hz, 2H), 4.26 (t, J = 5.4 Hz, 2H), 6.54 (q, J = 8.0 Hz, 1H), 6.73 (d, J = 7.2 Hz, 2H), 7.37 (d, J = 15.6 Hz, 1H), 7.45 (d, J = 9.2 Hz, 2H), 9.59 (d, J = 8.0 Hz, 1H). MS (ESI) m/z 206.1 [MH+]. 2.1.5. (E)-2-(3-(4-((2-Hydroxyethyl)(methyl)amino)phenyl)allylidene) malononitrile (3, DANIR-OH 2b) To a solution of 2 (170 mg, 0.8 mmol) in chloroform (20 mL) was added malononitrile (720 mg, 11 mmol), and then triethylamine (0.1 mL) was added as a catalyst. After stirring for 30 min at room temperature, the solvent was removed in a vacuum. The residue was purified by silica gel chromatography (AcOEt: Hexane = 2 : 1) and RPHPLC (MeCN: H2O = 6 : 4) to give 3 (15.6 mg, 7.6%) as a dark red solid. 1H NMR (400 MHz, CDCl3) δ 3.13 (s, 3H), 3.63 (t, J = 5.6 Hz, 2H), 3.87 (t, J = 5.0 Hz, 2H), 6.75 (d, J = 8.8 Hz, 2H), 7.03 (dd, J = 16.8 Hz, 1H), 7.17 (d, J = 14.8 Hz, 1H), 7.49 (m, 3H). 13C NMR (100 MHz, CDCl3) δ 39.20, 54.31, 60.19, 112.11, 113.00, 114.91, 117.46, 131.71, 151.32, 152.52, 160.51. HRMS (FAB+) m/z calcd for C15H16N3O 254.1525 found 254.1293 [MH+].
2. Material and methods 2.1. Chemistry 2.1.1. General All reagents were obtained commercially and used without further purification unless otherwise indicated. W-Prep 2XY (Yamazen, Osaka, Japan) was used for silica gel column chromatography on a Hi Flash silica gel column (40 μm, 60 Å, Yamazen). 1H and 13C NMR spectra were obtained on a JEOL JNM-ECS400 (JEOL Ltd., Tokyo, Japan) spectrometer with TMS as an internal standard. Coupling constants are reported in hertz. Multiplicity was defined by s (singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). Low-resolution mass spectra (LRMS) were obtained using a SHIMADZU LCMS-2020 (SHIMADZU, Kyoto, Japan). High-resolution mass spectrometry (HRMS) was conducted with a JEOL JMS-700.
2.1.6. (E)-3-(4-((2-((Tetrahydro-2H-pyran-2-yl)oxy)ethyl)(methyl) amino)phenyl)acrylaldehyde (4) The same reaction described above to prepare 1 was used, and 4 was obtained as a yellow oily liquid (600 mg, 70.9%). 1H NMR (400 MHz, CDCl3) δ 1.54 (m, 4H), 3.09 (s, 3H), 3.49 (m, 1H), 3.63 (m, 3H), 3.78 (m, 1H), 3.91 (m, 1H), 4.58 (t, J = 3.4 Hz, 1H), 6.54 (q, J = 7.9 Hz, 1H), 6.72 (d, J = 9.2 Hz, 2H), 7.37 (d, J = 15.6 Hz, 1H), 7.44 (d, J = 9.2 Hz, 2H), 9.59 (d, J = 8.0 Hz, 1H). MS (ESI) m/z 290.2 [MH+].
2.1.2. Synthesis of DANIR 2b and DANIR 2c DANIR 2b and DANIR 2c were synthesized according to a previously reported method [23]. 2.1.3. N-Methyl-N-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)-4aminobenzaldehyde (1) To a solution of N-methyl-N-(2-hydroxyethyl)-4-aminobenzaldehyde (1.14 g, 6.4 mmol) in anhydrous CH2Cl2 (20 mL) was added 3,4dihydro-2H-pyran (2.0 g, 24 mmol) and pyridinium toluene-4-sulphonate (400 mg, 1.6 mmol). The mixture was stirred at 40 °C for 3 h. The
2.1.7. (2E,4E)-5-(4-((2-Hydroxyethyl)(methyl)amino)phenyl)penta-2,4dienal (5) The same reaction described above to prepare 2 was used, and 5 was obtained as a yellow solid (102 mg, 21.3%). 1H NMR (400 MHz, CDCl3) δ 3.06 (s, 3H), 3.57 (t, J = 5.6 Hz, 2H), 3.85 (t, J = 5.6 Hz, 2H), 6.18 (q, 2
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2.6. In vitro fluorescent staining of islet amyloid deposits in human pancreas
J = 7.7 Hz, 1H), 6.82 (m, 4H), 7.25 (m, 1H), 7.40 (d, J = 8.8 Hz, 2H), 9.56 (d, J = 8.0 Hz, 1H). MS (ESI) m/z 232.1 [MH+].
Paraffin-embedded pancreas tissue sections from a T2DM patient (69-year-old woman) were purchased from BioChain Institute Inc. (Hayward, CA, USA). The sections were subjected to two 15-min incubations in xylene, two 1-min incubations in 100% EtOH, two 1-min incubations in 90% EtOH, and one 1-min incubation in 70% EtOH to completely deparaffinize them, followed by two 2.5-min washes in water. The sections were incubated with 20% or 50% solutions of DANIR derivatives for 30 min at room temperature. After washing in 20% EtOH for 10 min, fluorescent observation was performed using an FSX100 inverted microscope equipped with a U-MWIG3 cube. The location of islet amyloid was comfirmed by staining the same sections with thioflavin S and observing with the microscope equipped with a UMWIBA3 cube.
2.1.8. 2-((2E,4E)-5-(4-((2-Hydroxyethyl)(methyl)amino)phenyl)penta2,4-dien-1-ylidene)malononitrile (6, DANIR-OH 2c) The same reaction described above to prepare 3 was used, and 6 was obtained as a dark-purple solid (15.5 mg, 12.6%). 1H NMR (400 MHz, CDCl3) δ 3.10 (s, 3H), 3.60 (t, J = 5.2 Hz, 2H), 3.86 (t, J = 5.2 Hz, 2H), 6.78 (m, 4H), 6.97 (d, J = 15.2 Hz, 1H), 7.07 (dd, J = 10.8 Hz, 14.4 Hz, 1H), 7.40 (d, J = 8.8 Hz, 2H), 7.44 (d, J = 12.0 Hz, 1H) 13C NMR (100 MHz, CDCl3) δ 39.05, 54.47, 60.29, 112.20, 112.67, 122.36, 123.47, 123.89, 130.06, 146.03, 151.30, 151.94, 159.59. HRMS (FAB +) m/z calcd for C17H18N3O 280.1372 found 280.1450 [MH+]. 2.2. Fluorescence measurement in CHCl3 Absorption, fluorescence excitation and emission wavelengths, and quantum yields were determined with 10 μM of the compounds in CHCl3 (UV-1800, SHIMADZU, RF-6000, SHIMADZU, or Fluorolog-3, HORIBA Jobin Yvon Inc., Kyoto, Japan).
2.7. Solubility assay of DANIRs Solubility assay was performed by measuring the absorbance of DANIRs (0–500 μM) in each solution (DMSO: propylene glycol = 2 : 8 or saline containing 5 v/v% DMSO). After voltex and centrifugation (1 min), the absorbance of 200 μL of each solution was measured at 254 nm with a UV-spectrophotometer (UV-1800).
2.3. Fluorescence measurement using amylin aggregates A solid form of amylin was purchased from Peptide Institute (Osaka, Japan). Aggregation was carried out according to the method reported previously [17]. A mixture (10% DMSO) containing DANIRs (2.0 μM) and amylin aggregates was incubated at room temperature for 30 min. After incubation, fluorescence emission spectra of DANIRs were collected with excitation at 525 nm (DANIR 2b), 560 nm (DANIR 2c), 510 nm (DANIR-OH 2b), and 550 nm (DANIR-OH 2c), which are the maximum respective excitation wavelengths in the presence of amylin aggregates (Infinite M200PRO, Tecan, Männedorf, Switzerland).
2.8. Ex vivo fluorescent staining of amylin aggregates in islet amyloid model mouse Islet amyloid model and normal mice were intravenously injected with DANIRs (500 μM (DMSO: propylene glycol = 2 : 8 for DANIR 2b and DANIR 2c, saline containing 5 v/v % DMSO for DANIR-OH 2b and DANIR-OH 2c), 50 μL). The mice were sacrificed by decapitation at 30 min postinjection. The pancreas was removed and fixed with 4% paraformaldehyde in phosphate buffer. Sections were prepared and observed using the same method as described for the in vitro fluorescent staining study. The locations of aggregates were confirmed by staining the same sections with thioflavin S.
2.4. Saturation binding assay using amylin aggregates A solution of 40 μL of amylin aggregates (final conc., 2.0 μM) was added to the mixture containing 40 μL of DANIRs, and thioflavin T (final conc., 3.9 nM - 10 μM in DMSO) and 320 μL of the buffer (20 mM Tris/HCl, 100 mM NaCl, pH 7.5) in a final volume of 400 μL. Nonspecific binding was defined without amylin aggregates. After the mixture was incubated for 30 min at room temperature, the solution was transferred to a black 96-well plate, and the fluorescence intensity was measured using a microplate reader (Infinite M200PRO). The dissociation constants (Kd) were determined using GraphPad Prism 6.0.
2.9. In vivo pancreas imaging in islet amyloid model mice using two-photon microscopy Islet amyloid model mice were anesthetized and left-flank incised. A solution of DANIRs (500 μM (DMSO: propylene glycol = 2 : 8 for DANIR 2b and DANIR 2c, saline containing 5 v/v % DMSO for DANIROH 2b and DANIR-OH 2c), 50 μL) was injected intravenously into the tail vein. Each mouse was placed and fixed in the lateral position on an electric heating pad maintained at 37 °C while maintaining anesthesia. To stabilize the exposed pancreas, a vacuum-stabilized imaging window was placed over the pancreas. At 30 min postinjection, Z-stack images of fluorescence accumulation were acquired from the surface inward at 2 μm intervals and at a scan speed of 4 μs per pixel with a FV1200MPE Laser Scanning Microscope (Olympus) and BX61WI Upright Microscope (Olympus) equipped with mCherry (Ch1, 570–615 nm) and iRFP (Ch2, 675–740 nm) as emission filters and with excitation at 980 nm (DANIR 2b), 840 nm (DANIR 2c), 1000 nm (DANIR-OH 2b), and 1120 nm (DANIR-OH 2c). The excitation wavelengths were determined in a phantom study of DANIRs solution. Recorded images were processed with MetaMorph software (Moleculer Devices, Sunnyvale, CA).
2.5. In vitro fluorescent staining of amylin aggregates in islet amyloid model mice The experiments with animals were conducted in accordance with our institutional guidelines and approved by the Kyoto University Animal Care Committee. Islet amyloid model mice were prepared as reported previously [17]. Each islet amyloid model mouse was sacrificed by decapitation and the pancreas was immediately removed and fixed with 4% paraformaldehyde in phosphate buffer. After fixing for 1 day, the pancreas was embedded in Super Cryoembedding Medium (SCEM) Compound (Section-Lab Co Ltd., Hiroshima, Japan) and then frozen in a dry ice/hexane bath. Frozen sections were prepared at a thickness of 20 μm. Next, they were incubated with 20% or 50% DMSO solution of DANIRs (100 μM) for 30 min. The location of aggregates was confirmed by staining adjacent sections with thioflavin S (100 μM). Finally, the sections were washed with 20% DMSO. Fluorescence observation was performed using an FSX100 inverted microscope (Olympus, Japan) equipped with an Olympus U-MWIBA3 cube for thioflavin S or an Olympus U-MWIG3 cube for DANIRs. The obtained images were recorded and processed with CellSens Dimension Desktop (Olympus).
3. Results and discussion 3.1. Synthesis of DANIRs The synthesis of DANIR-OH 2b and DANIR-OH 2c is outlined in Scheme 1. The hydroxy group of N-methyl-N-(2-hydroxyethyl)-4-aminobenzaldehyde was protected with a 2-tetrahydropyranyl group (THP). Then, 2 and 5 were obtained by the Wittig reaction and 3
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Scheme 1. Synthetic route of DANIRs. Reagents: (a) 3,4-dihydro-2H-pyran, pyridinium toluene-4-sulphonate, CH2Cl2; (b) (1) (1,3-dioxan-2-yl-methyl)triphenylphosphoniumbromide, 18-crown-6, NaH, THF, (2) HCl, THF; (c) malononitrile, triethylamine, CHCl3.
deprotection of THP (37 and 21% yields). 3 (DANIR-OH 2b) and 6 (DANIR-OH 2c) were prepared by condensation of the corresponding aldehydes with malononitrile in chloroform (7.6 and 13% yields).
3.3. Binding affinity for amylin aggregates in vitro Next, we performed saturation binding experiments and calculated the apparent binding dissociation constant (Kd) to quantitatively evaluate the binding affinity for amylin aggregates. The Kd values estimated for DANIR 2b, DANIR 2c, DANIR-OH 2b, and DANIR-OH 2c were 500, 80.5, 1220, and 695 nM, respectively (Table 2). These values were much higher than for IPBF (Kd = 8.3 nM), but the experimental method was different, so it is not appropriate to directly compare the values. Because the Kd value of thioflavin T was 791 nM for amylin aggregates, the binding affinity of DANIRs except for DANIR-OH 2b may be sufficient to visualize amylin aggregates. Compared to the binding affinity for Aβ aggregates (DANIR 2b: 36 nM, DANIR 2c: 27 nM) [23], the binding affinity of DANIR 2c for amylin aggregates was similar to that for Aβ aggregates, but the affinity of DANIR 2b for amylin aggregates was lower than that for Aβ aggregates. This phenomenon may be due to the differences in amino acids constituting the β-sheet structure between Aβ and amylin aggregates.
3.2. Fluorescence characterization First of all, we measured the spectra of absorbance, excitation, emission, quantum yield, and excitation coefficient in CHCl3 (Table 1 and Figure S1). These properties of DANIR-OH 2b and DANIR-OH 2c were similar to those of DANIR 2b and DANIR 2c, respectively. In general, extension of the π-electron system increases absorption coefficient, but absorption coefficient of DANIRs does not correlate with increasing the number of conjugated double bonds. Although the reason is not clear, the absorption coefficients of previously reported probes with similar structure also showed same trend [31–34]. A change in fluorescence intensity upon binding to amylin aggregates is one of the essential properties for fluorescence probes targeting amyloid aggregates [35, 36]. Therefore, we initially evaluated the fluorescence intensity of DANIRs with or without amylin aggregates (Fig. 2). In a solution containing amylin aggregates, their fluorescence intensities significantly increased with the concentration of aggregates. These results suggested that all four DANIRs have the potential to image amylin aggregates in vitro and in vivo. Compared to thioflavin-T and thioflavin-S, a dye commonly used to stain amyloid aggregates, DANIRs showed longer emission wavelengths. Notably, the emission wavelengths of DANIR 2c and DANIR-OH 2c were in the near-infrared region (650–900 nm). The background autofluorescence is low in this region, suggesting that these probes may be more suitable for imaging of amylin aggregates in the pancreas.
3.4. In vitro fluorescent staining of amylin aggregates in islet amyloid model mouse In order to confirm the affinity of DANIRs for amylin aggregates in the mouse pancreas, we performed in vitro fluorescence staining with DANIR 2b, DANIR 2c, DANIR-OH 2b, and DANIR-OH 2c using pancreas sections from islet amyloid model mice having amylin aggregates transplanted in the pancreas [18]. Several fluorescence positive regions were observed in mouse pancreas sections (Fig. 3A, 3B, 3C, and 3D). The staining patterns of these probes were consistent with that observed with thioflavin S, a dye commonly used to stain amyloid aggregates including amylin aggregates (Fig. 3E). This result suggested that all four DANIRs specifically bind to amylin aggregates in the mouse pancreas.
Table 1 Fluorescence profiles of DANIRs. Compound
DANIR 2b DANIR 2c DANIR-OH 2b DANIR-OH 2c
Abs (nm)a
490 521 486 515
Ex (nm)a
493 520 488 517
3.5. In vitro fluorescent staining of islet amyloid deposits in human pancreas Em (nm)a
559 644 558 644
Quantum yield (%)
Absorption coefficient (M−1cm−1)
0.3 1.1 0.4b 1.1c
54200 49067 51267 37333
The depositions of islet amyloid in human pancreas were composed of various molecules, including amylin. To confirm the affinity for islet amyloid deposited in human pancreas, we also carried out in vitro fluorescence staining with DANIR 2b, DANIR 2c, DANIR-OH 2b, and DANIR-OH 2c using post-mortem human pancreatic sections from a T2DM patient. For all DANIRs, many fluorescence spots were observed (Fig. 4A, 4C, 4E, and 4G), and this staining pattern correlated well with that observed with thioflavin S in the same sections (Fig. 4B, 4D, 4F, and 4H). These results suggested that all four DANIRs could bind to and visualize amylin aggregates in islet amyloids deposited in the pancreas in T2DM patients.
a
Wavelengths of absorption maxima, wavelengths of excitation maxima, and wavelengths of emission maxima of DANIRs were determined with 10 μM of the compounds in CHCl3. b DANIR 2b was taken as a reference for determining quantum yields. c DANIR 2c was taken as a reference for determining quantum yields. 4
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Fig. 2. Fluorescence intensities of DANIR 2b (A), DANIR 2c (B), DANIR-OH 2b (C), and DANIR-OH 2c (D) in interaction with amylin aggregates.
3.6. Solubility assay of DANIRs
Table 2 Apparent binding constants (Kd) of DANIRs and Thioflavin T. Compound
Kd (nM)a
DANIR 2b DANIR 2c DANIR-OH 2b DANIR-OH 2c Thioflavin T
500 ± 93 80.5 ± 6.2 1220 ± 284 695 ± 118 791 ± 91
Before in vivo experiments, we confirmed the solubility of DANIRs. First, we evaluated the solubility of the organic solvent (DMSO: propylene glycol = 2 : 8), which was previously used in an in vivo study with DANIR 2b and DANIR 2c [23]. For all DANIRs, the absorbance correlated well with the concentration (R2 ≧ 0.98) (Fig. 5A), indicating that all four DANIRs were soluble in the organic solvent at the concentration of 500 μM. Although DANIR 2b and DANIR 2c were not soluble (R2 = 0.031 and 0.637, respectively) in saline containing 5 v/v % DMSO, the solubilities of DANIR-OH 2b (R2 = 0.984) and DANIR-OH 2c (R2 = 0.996) were markedly higher in this aqueous media (Fig. 5B).
a
Values are the mean ± standard error of the mean for three independent experiments performed in triplicate.
Fig. 3. In vitro fluorescence staining of DANIR 2b (A), DANIR 2c (B), DANIR-OH 2b (C), and DANIR-OH 2c (D) in 20 μm sections from islet amyloid model mice. Labeled plaques were confirmed by staining of the adjacent sections with thioflavin S (E). Scale bars indicate 1 mm. 5
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Fig. 4. In vitro fluorescence staining of DANIR 2b (A), DANIR 2c (C), DANIR-OH 2b (E), and DANIR-OH 2c (G). The presence of amylin aggregates was confirmed by staining the same sections with thioflavin S (B, D, F, and H). Scale bars indicate 500 μm.
with little autofluorescence at 30 min after injection of all four DANIRs through the tail vein. Based on the results of the ex vivo fluorescence staining study after in vivo two-photon fluorescence imaging (Fig. S2), the fluorescence signals obtained with TPM were considered to indicate amylin aggregates. These results suggested that all four DANIRs may be useful for in vivo two-photon fluorescence imaging of amylin aggregates in the mouse pancreas. In the in vitro binding study, DANIR-OH 2b showed lower affinity for amylin than the other DANIRs, but all DANIRs clearly visualized amylin aggregates in the in vivo two-photon fluorescence imaging study. This result suggested that the Kd value (1220 nM) of DANIR-OH 2b for amylin aggregates was sufficient for in vivo twophoton fluorescence imaging. DANIRs are the first probes for in vivo imaging of amylin aggregates with TPM. This technique is needed when preparing to surgically expose the pancreas. Because the surgical preparation may cause a great deal of strain to the mice, it is one of the disadvantages for in vivo imaging of pancreas with TPM. Although this disadvantage was not eliminated, two-photon fluorescence imaging of amylin aggregates may contribute considerably in elucidating the relationship between T2DM and amylin.
Because organic solvents are frequently toxic in living subjects, these results suggested that DANIR-OH 2b and DANIR-OH 2c may be more appropriate probes for in vivo imaging than DANIR 2b and DANIR 2c. Based on the results of the solubility study, we used the organic solvent (DMSO: propylene glycol = 2 : 8) for DANIR 2b and DANIR 2c, and the aqueous media (saline containing 5 v/v% DMSO) for DANIR-OH 2b and DANIR-OH 2c in the ex vivo and in vivo imaging studies. 3.7. Ex vivo fluorescent staining of amylin aggregates in islet amyloid model mice Ex vivo fluorescence staining was performed to test the labeling of amylin aggregates by DANIRs in vivo. We removed each pancreas at 30 min after the injection of DANIRs, prepared frozen pancreatic sections, and observed them with a one-photon fluorescence microscope. High fluorescence regions were observed in the pancreas sections of the islet amyloid model mice (Fig. 6A, 6C, 6E, and 6G). Because these regions were clearly stained with thioflavin S (Fig. 6B, 6D, 6F, and 6H), all four DANIRs could bind to and image the transplanted amylin aggregates in the living mouse pancreas.
4. Conclusions 3.8. In vivo imaging of pancreas in islet amyloid model mouse using twophoton microscopy
We newly designed and synthesized DANIR-OH 2b and DANIR-OH 2c and evaluated four DANIRs (DANIR 2b, DANIR 2c, DANIR-OH 2b, and DANIR-OH 2c) for two-photon fluorescence imaging of amylin aggregates in the pancreas. All DANIRs showed binding affinities with amylin aggregates in the in vitro fluorescent saturation binding assay and the fluorescent staining study. In the in vivo two-photon
Finally, to evaluate the potential of DANIRs for in vivo two-photon fluorescence imaging targeting amylin aggregates, we performed twophoton fluorescence imaging experiments using living islet amyloid model mice. As shown in Fig. 7, clear and bright images were obtained
Fig. 5. Correlation between absorbance and concentration of DANIR 2b, DANIR 2c, DANIR-OH 2b, and DANIR-OH 2c in organic solvent (DMSO: propylene glycol = 2 : 8) (A) and aqueous media (saline containing 5 v/v% DMSO) (B). 6
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Fig. 6. Ex vivo fluorescence observation of pancreas sections from islet amyloid model mice after injection of DANIR 2b (A), DANIR 2c (C), DANIR-OH 2b (E), and DANIR-OH 2c (G). The presence of amylin aggregates was confirmed by staining the same sections with thioflavin S (B, D, F, and H). Scale bars of A, B, C, D, E, and F indicate 500 μm, and those of G and H are 1 mm.
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Fig. 7. In vivo images of amylin aggregates with two-photon excitation fluorescence microscopy after injection of DANIR 2b (A), DANIR 2c (B), DANIR-OH 2b (C), and DANIR-OH 2c (D) into islet amyloid model mice. The XY plane is a square, 507.9 μm × 507.9 μm. Z is 108 μm (A), 84 μm (B), 46 μm (C), and 164 μm (D), respectively.
fluorescence imaging studies using islet amyloid model mice, we successfully obtained clear images of amylin aggregates after injection via the tail vein. In addition, in the ex vivo fluorescence staining study, we confirmed that these probes bound to amylin aggregates in the model mouse pancreas. These results suggested that the DANIRs tested in this study may be useful for two-photon fluorescent imaging of amylin aggregates. Among the DANIRs, because DANIR-OH 2b and DANIR-OH 2c can be used in aqueous media, these probes are more appropriate for in vivo imaging studies.
Acknowledgements This work was supported by the Nakatani Foundation for Advancement of Measuring Technologies in Biomedical Engineering, Takeda Science Foundation, and Kyoto University Live Imaging Center.
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.dyepig.2019.107615.
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