Multifunctional imaging of amyloid-beta peptides with a new gadolinium-based contrast agent in Alzheimer’s disease

Journal Pre-proof Multifunctional Imaging of Amyloid-Beta Peptides with a New Gadolinium-based Contrast Agent in Alzheimer’s Disease Garam Choi, Hee-Kyung Kim, Ah Rum Baek, Soyeon Kim, Min Jung Kim, Minsup Kim, Art E. Cho, Gang-Ho Lee, Hoesu Jung, Ji-ung Yang, Taekwan Lee, Yongmin Chang

PII:

S1226-086X(19)30626-4

DOI:

https://doi.org/10.1016/j.jiec.2019.11.031

Reference:

JIEC 4874

To appear in:

Journal of Industrial and Engineering Chemistry

Received Date:

10 September 2019

Revised Date:

8 November 2019

Accepted Date:

24 November 2019

Please cite this article as: Choi G, Kim H-Kyung, Baek AR, Kim S, Kim MJ, Kim M, Cho AE, Lee G-Ho, Jung H, Yang J-ung, Lee T, Chang Y, Multifunctional Imaging of Amyloid-Beta Peptides with a New Gadolinium-based Contrast Agent in Alzheimer’s Disease, Journal of Industrial and Engineering Chemistry (2019), doi: https://doi.org/10.1016/j.jiec.2019.11.031

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Multifunctional Imaging of Amyloid-Beta Peptides with a New

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Gadolinium-based Contrast Agent in Alzheimer’s Disease

Garam Choi, 1,2,† Hee-Kyung Kim,3 Ah Rum Baek,1 Soyeon Kim,1 Min Jung Kim,1 Minsup

Kim,4 Art E. Cho,4 Gang-Ho Lee,5 Hoesu Jung,6 Ji-ung Yang,1,6 Taekwan Lee,6,* and Yongmin

Department of Medical & Biological Engineering, Kyungpook National University, Daegu

41944, Republic of Korea

Department of R&D center, Myungmoon Bio Co., Hwaseong, Gyeonggi-do 18622,

Republic of Korea

Institute of Biomedical Engineering Research, Kyungpook National University, Daegu

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1

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Chang1,7,8,*

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41944, Republic of Korea 4

Department of Bioinformatics, Korea University Sejong Campus, Sejong 30019, Republic of

Korea 5

Department of Chemistry, College of Natural Science, Kyungpook National University,

Daegu 41944, Republic of Korea 1

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Laboratory Animal Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu

41061, Republic of Korea 7

Department of Molecular Medicine, School of Medicine, Kyungpook National University,

Daegu 41944, Republic of Korea. 8

Department of Radiology, Kyungpook National University Hospital, Daegu 41944, Republic

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of Korea

*Corresponding Authors: E-mail address: [email protected] (Y. Chang),

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[email protected] (T. Lee)

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Graphical abstract

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Abstract

Multifunctional imaging of the deposition of amyloid-beta (Aβ) aggregates in the brain is of great importance in diagnosing Alzheimer’s disease. Herein, we report a multifunctional Aβ-targeting small-molecular-weight gadolinium (Gd)-based contrast agent (CA), Gd-DO3A-Chal (a new Gdchelate conjugated with chalcone), that showed 8 times higher binding affinity to Aβ aggregates than a previously reported Gd-chelate conjugated with Pittsburgh compound B. Gd-DO3A-Chal showed

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multimodal imaging capability. As a new fluorescence imaging probe, Gd-DO3A-Chal showed a good match with immunostained images using 6E10 monoclonal antibodies for the detection of Aβ

aggregates in 5XFAD transgenic mouse brain sections. For in vivo magnetic resonance (MR) imaging without blood-brain barrier disruption, longitudinal relaxation time (T1)-weighted MR images after

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intravenous administration of Gd-DO3A-Chal showed signal enhancement of the Aβ distribution in living 5XFAD transgenic mouse brain. Therefore, in vivo MR images for Aβ detection in addition to

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fluorescent optical images of Aβ aggregates with high specificity and sensitivity using this new

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multifunctional Aβ-targeting CA were successfully demonstrated.

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Abbreviation: amyloid-beta, Aβ; contrast agent, CA; gadolinium ion, Gd(III); magnetic resonance, MR; alzheimer’s disease, AD; intravenous, IV; reticuloendothelial system, RES;

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blood-brain barrier, BBB; high resolution fast atom bombardment mass spectroscopy, HR-

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FABMS; tri-tert-butyl 1,4,7,10-Tetraazacyclododecane-1,4,7-triacetate hydrobromide, DO3A-(tBuO)3 ∙HBr; deionized water, D∙I water; water proton longitudinal relaxation rate, R1p; Dimethyl sulfoxide, DMSO; rapid acquisition with relaxation enhancement, RARE; multi-slice multi-echo, MSME ; inversion time, TI; echo time; TE; signal intensity, SI; normal goat serum, NGS; bovine serum albumin, BSA; Tris-buffered saline, TBS 3

Keywords: gadolinium complex, magnetic resonance contrast agent, chalcone derivative, multifunctional imaging, amyloid imaging

1. INTRODUCTION

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Alzheimer’s disease (AD) is a neurodegenerative disease that causes progressive and irreversible loss of cognitive functions and is the most common cause of dementia [1-3].

Although many evidences indicated that the amyloid hypothesis alone is not sufficient to explain the whole multifaceted features of AD, it is still accepted that abnormal levels of

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amyloid-beta (Aβ) aggregates play an important role [4, 5]. Excessive Aβ is known to

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progress to a variety of Aβ aggregates, which cause neuronal dysfunction, cell death, and the loss of synaptic connections [6-8], and they are the main component of Aβ plaques, which are

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the hallmark of AD. During AD, Aβ plaques show a characteristic distribution, appearing in some neocortical regions from which they propagate toward the whole neocortex and inner

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regions of the brain [9, 10]. Therefore, Aβ aggregates are undoubtedly a biomarker candidate for AD and it is of great importance to precisely detect them with the use of imaging

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techniques in the diagnosis of AD.

Multimodal imaging is an important approach to overcome the limitations of single

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imaging techniques in the diagnosis of disease [11-14]. For example, fluorescence imaging is widely used in the histologic examination of cells to detect the deposition of Aβ aggregates in brain sections with high sensitivity and accuracy. However, due to light scattering and attenuation, it is difficult for fluorescence imaging to provide depth penetration and in vivo tomographic imaging information. Therefore, by combining it with magnetic resonance (MR) 4

imaging, multimodal imaging can overcome the depth limitation of fluorescence imaging and achieve noninvasive in vivo imaging at greater depths of penetration with the sensitivity required for a more accurate diagnosis. For multimodal imaging, a single agent which can act as a dual or multiple imaging contrast- agent (CA) is preferred because a single multifunctional one provides imaging signals that may originate from the same location at the same time, thereby ensuring good quality imaging data using the same CA [15]. For MR Aβ imaging, several small-molecular-weight and nano-based targeted MR CAs

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have been proposed [16-23]. Although nanoscale Aβ-targeting MR CAs have already been reported, they have several limitations. For instance, when present in the bloodstream via

intravenous (IV) injection, 95% of the nanoparticles accumulate in the major organs of the

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reticuloendothelial system (RES), leaving only 5% available for targeting a specific organ

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[24]. Besides, this accumulation of nanoparticles in several RES organs raises the major issue of their toxicity, and there have been several attempts to modify the nanoparticles to

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minimize these limitations with only limited success [24]. In the case of small-molecularweight MR CAs, several Aβ-targeting small-molecular-weight gadolinium (Gd) CAs have

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also been reported, although they could only detect Aβ fibrils but not Aβ oligomers. According to the amyloid cascade hypothesis in AD, the Aβ targeting CAs should have the

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ability to detect Aβ oligomers because these could be the toxic factors during the very early stages of AD that perhaps even initiate the pathological cascade [25]. Furthermore, many of

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these previous studies using Aβ-targeting Gd CAs have only shown ex vivo imaging in postmortem brains or in vivo imaging after blood-brain barrier (BBB) breakdown by using mannitol or ultrasound. In this paper, we report a multifunctional Aβ-targeting small-molecular-weight Gd CA, which is capable of easy chemical modification and can be used to detect Aβ oligomers. 5

Furthermore, one of the benefits of the new CA is its multimodal imaging capability. That is to say, in addition to MR imaging, optical imaging is possible using the chalcone derivative as it shows good fluorescence properties comparable to commercial fluorophores. The chalcone moiety is known to have a significant binding affinity for Aβ aggregates and to can cross the BBB due to its high lipophilicity. It is known that lipophilicity is one of most important parameters to penetrate BBB which consists of lipid membrane environments [26]. In addition, electron-donating groups, especially dimethylamino groups is not only revealed

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crucial role for Aβ binding, but also make it be able to have fluorescence property [27].

Previously, a radiolabel imaging probe with chalcone derivative showed high brain uptake

rate [28]. In this study, in vivo MR imaging for Aβ detection using this new Aβ-targeting CA

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was successfully demonstrated in addition to fluorescent optical images of Aβ with high

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specificity. To deliver the probe into the brain without BBB breakdown, IV injection of the Aβ-targeting GdCA was used to detect Aβ aggregates in the brain of mice in a geratic AD

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animal model.

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2. EXPERIMENTAL SECTION 2.1 General Remarks All reagents were purchased from commercial sources and used without further purification. The 1H NMR data were acquired with a Bruker Advance 500 spectrometer at the Center for Instrumental Analysis, Kyungpook National University (KNU). Chemical shifts are given as δ values with reference to tetramethylsilane as an internal standard. The coupling constants are in Hz. Elemental analysis was carried out with a Flash 2000 Elemental

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Analyzer (ThermoFisher, USA), KNU. High resolution fast atom bombardment mass

spectroscopy (HR-FABMS) were obtained by using a JMS-700 model (Jeol, Japan) mass spectrophotometer at the Korea Basic Science Institute. Aβ1–42 was purchased from

2.2 Synthesis and Characterization.

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Bachem, Torrance, CA.

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2.2.1 (E)-1-(4-aminophenyl)-3-(4-(dimethylamino)phenyl)prop-2-en-1-one (Compound-1) 4′-aminoacetophenone (5.00 g, 36.99 mmol) was dissolved in ethanol (80 mL) and potassium

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hydroxide (6.00 g, 106.9 mmol) was added. 4-dimethylaminobenzaldehyde (5.85 g, 39.21 mmol) was added dropwise followed by changed orange solution was refluxed at 70℃ for 8

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h. The suspended mixture was poured into chilled water for precipitation and stirred for overnight at room temperature. Orange solid which gathered with filtration was washed with

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chilled ethyl ether to afford compound-1 (7.1 g, 72.1 %) as an orange powder and used in next step without further purification. 1H NMR (500 MHz, CDCl3, δ) 7.95 – 7.88 (m, 2H; ArH), 7.76 (d, J = 15.4 Hz, 1H; CHCO), 7.57 – 7.50 (m, 2H; ArH), 7.35 (d, J = 15.4 Hz, 1H; CHAr), 6.72 – 6.63 (m, 4H; ArH), 4.12 (s, 2H; NH2), 3.02 (s, 6H; -(CH3)2).

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2.2.2 (E)-2-chloro-N-(4-(3-(4-(dimethylamino)phenyl)acryloyl)phenyl)acetamide (Compound-2) To a solution of compound-1 (2.00 g, 7.51 mmol) and triethylamine (3.15 mL, 22.54 mmol) in chloroform (80 mL) was added chloroacetyl chloride (0.89 mL, 11.27 mmol) at 0 ℃. The mixture was refluxed for 1 h and washed with water three times after cooling down. The organic layer was dehydrated with magnesium sulfate and precipitated with chilled hexane for solidification. The compound-2 (2.03 g, 78.9 %) was obtained as brick red solid and used

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in next step without further purification. 1H NMR (500 MHz, acetone-d6, δ) 9.57 (s, 1H; NHCO-), 8.04 – 7.94 (m, 2H; ArH), 7.74 – 7.66 (m, 2H; ArH), 7.61 (d, J = 15.4 Hz, 1H;

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ArH), 4.16 (s, 2H; -CH2-), 2.92 (s, 6H; -(CH3)2).

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CHCO), 7.57 – 7.52 (m, 2H; ArH), 7.49 (d, J = 15.4 Hz, 1H; CHAr), 6.69 – 6.58 (m, 2H;

2.2.3 tri-tert-butyl 2,2',2''-(10-(2-((4-(3-(4-(dimethylamino)phenyl)acryloyl)phenyl)amino)-

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2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)(E)-triacetate (Compound-3) DO3A-(tBuO)3∙HBr (3.16 g, 5.30 mmol) was dissolved in acetonitrile (100 mL) and

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potassium carbonate (2.20 g, 15.92 mmol) was added. compound-2 (2.0 g, 5.83 mmol) solution in dimethylformamide (12 mL) was added dropwise and refluxed for 18h. After

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removal of inorganic salt, the crude compound was concentrated to precipitate with chilled

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ether and purified by column chromatography (elution, DCM/MeOH) to afford compound-3 (1.3 g, 30.0 %) as a yellow solid. 1H NMR (500 MHz, CDCl3, δ) 10.86 (s, 1H; -NHCO-), 8.11 – 8.03 (m, 2H; ArH), 7.92 – 7.85 (m, 2H; ArH), 7.75 (d, J = 15.4 Hz, 1H; CHCO), 7.56 – 7.49 (m, 2H; ArH), 7.33 (d, J = 15.3 Hz, 1H; CHAr), 6.73 – 6.66 (m, 2H; ArH), 3.72 (s, J = 34.1 Hz, 2H; -CH2-), 3.03 (s, 8

6H; ; -(CH3)2), 2.99 – 1.96 (m, 22H; -CH2- in DO3A backbone), 1.51 – 1.33 (m, 27H; C(CH3)3). ; HR-FABMS (m/z): [M+Na]+ calcd for C45H68N6O8Na, 843.4996; found, 843.4998; Element analysis (EA): anal. calcd for C45H68N6O8∙2HBr: C 54.99, H 7.18, N 8.55; found: C 55.04, H 7.03, N 8.21.

2.2.4 (E)-2,2',2''-(10-(2-((4-(3-(4-(dimethylamino)phenyl)acryloyl)phenyl)amino)-2oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (Compound-4)

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The reaction mixture with compound- 3 (0.9 g, 1.10 mmol) and trifluoroacetic acid (8 ml, excess) in 10 ml of dichloromethane was stirred for 24h at room temperature. The solvent was removed and the crude compound was purified by flash column on RP-18 silica gel

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(elution, H2O/MeOH) to yield compound-4 (0.4 g, 55%) as orange solid. 1H NMR (500 MHz,

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MeOD) δ 8.08 – 8.02 (m, 2H; ArH), 7.83 – 7.78 (m, 2H; ArH), 7.77 – 7.73 (m, 1H; CHAr), 7.64 – 7.60 (m, 2H; ArH), 7.51 (d, J = 15.4 Hz, 1H; -CHCO-), 6.81 – 6.76 (m, 2H; ArH),

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3.91 – 3.35 (m, 18H; -CH2- in cyclen and amide arm), 3.17 (brs, 6H; -CH2- in acetate arm), 3.06 (s, 6H; -(CH3)2). HR-FABMS (m/z): [M+H]+ calcd for C33H45N6O8, 653.3299; found,

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653.3302.

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2.2.5 [Gd(DO3A-Chal)(H2O)] (Gd-DO3A-Chal) Compound-4 (0.3 g, 0.46 mmol) was dissolved in D∙I water and gadolinium chloride

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hexahydrate (0.17 g, 0.46 mmol) was added. pH condition was adjusted with NaOH (1.0 N) in pH 7 followed by stirring for 24h at room temperature. Gd-DO3A-Chal with light orange color thus obtained was further purified by flash column on RP-18 silica gel and solidified by precipitation in water/acetone condition. The orange solid thus obtained was further purified by flash column on RP-18 silica gel (elution, H2O/MeOH) to yield Gd-DO3A-Chal (0.16 g, 9

40%) and solidified by precipitation in water/acetone condition. HR-FABMS (m/z): [M+H]+ calcd for C33H42GdN6O8, 808.2305; found, 808.2309.

2.3 MR relaxivity measurements Relaxation time measurement was performed at 3.0 T (GE Healthcare, SIGNA Architect. WI, U.S.) and 9.4 T (Bruker BioSpin, BioSpec 94/20 USR, Ettlingen, Germany). A fast spinecho sequence with variable inversion time and a T2-map sequence were used for the

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assessment of T1- and T2-relaxation times at 3.0 T. The parameters were as follows: repetition time = 2,000 ms, echo time = 10.2 ms, inversion time = 50–1,750 ms (35 different TI values), echo training length = 4 for T1 measurements, repetition time = 1,412 ms, echo time = 18.3–

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146.2 ms (8 different TE values), and echo training length = 16 for T2 measurements. An

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inversion recovery RARE (rapid acquisition with relaxation enhancement) sequence and an MSME (multi-slice multi-echo) sequences were used for measurement of the T1- and T2-

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relaxation times at 9.4 T. The parameters were as follows: repetition time = 10,000 ms, echo time = 10 ms, inversion time = 85–7,000 ms (11 different TI values), rare factor = 1 for T1

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measurements, repetition time = 10,000 ms, and echo time = 10–700 ms (70 different TE values) for T2 measurements. The relaxation time constant T1 and T2 were calculated based on

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the nonlinear least-squares fit of the mean pixel values in the region of interest and r1, r2 relaxivity values were computed using the linear fit as a reciprocal of relaxation time

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dependent on variable concentration (1, 0.5, 0.25, 0.125, and 0.0625 mM) All measurements were carried out in triplicate.

2.4 Kinetic stability and pH stability measurements

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The kinetic study refers to the literature method [29]. In brief, it is performed to measure the evolution of the water proton longitudinal relaxation rate (R1p) over time where a phosphate buffered solution (pH 7.4) contains equimolar Gd complex and zinc chloride. A diamagnetic Zn(II) leads to release Gd(III) by the transmetallation of a Gd complex, resulting to form insoluble GdPO4 salt, thus a decrease in the water proton longitudinal relaxation rate is observed [30, 31]. Gd-DO3A-Chal was prepared as a 1 mM solution with commercially available CAs: Gd-DO3A-BT, Gd-DOTA, Gd-BOPTA, and Gd-DTPA-BMA (Chart 1) for

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comparison purposes and immediately used after adding zinc chloride solution for the

measurement of T1 relaxation times for 72 hours. The results are displayed as a plot of the relative value of R1p at any time t, R1p(t)/R1p(0).

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The pH stability was given as the evolution of the water proton relaxation rate (R1p) like the

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preceding study for Gd-DO3A-Chal at various pH buffer solution values (pH 1, 3, 5, 7, 9, and 11) [32]. Gd-DOTA and Gd-DO3A-BT were used for comparison purposes. Experiments

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were performed by using a 3.0 T whole-body system (Magnetom TIM Trio, Siemens,

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Erlangen, Germany) at room temperature.

2.5 In sillico computational method

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Crystal structures of Amyloid beta monomer and fibrils were obtained from RCSB Protein Data Bank web sites (https://www.rcsb.org, PDB ID: 1IYT, 2BEG). For protein-ligand

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docking simulation, Glide application in Schrödinger suites was employed. Glide searches all possible ligand poses that have favorable interactions with the receptor using a series of hierarchical filters and evaluated the ligand poses using empirical scoring function called GlideScore. Compounds Gd-DO3A-Chal and Pittsburgh compound B (PiB) were prepared for docking simulation using LigPrep application in Schrödinger suites. Binding free energies 11

of docked ligands were calculated using Prime molecular mechanics/generalized born surface area (MM/GBSA) application in Schrödinger suites. OPLS3e force field and VSGB 2.0 solvation model were used in binding free energy calculation [33-35] .

2.6 Fluorescence measurement with Aβ1–42 oligomers The Aβ oligomers were prepared as described in a previous study [36]. The absorbance and emission wavelength were measured with a SpectraMax i3 (Molecular Devices, CA, U.S.).

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The emission fluorescence intensity was detected for Gd-DO3A-Chal (10 μM) with and without the Aβ oligomers (20 μM) in distilled water containing 10% DMSO [37].

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2.7 Saturation Binding study for determination binding constant, Kd

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A saturation binding study was used to determine the affinity of Gd-DO3A-Chal for Aβ oligomers. Gd-DO3A-Chal was prepared in dilutions to cover the desired concentration

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range (0.2, 0.5, 1, 1.5, 2, 2.5, 5, 10, 15, 20, 25, and 50 μM). The Aβ oligomers (final concentration 5 μM) in D2O containing 10% DMSO was split over a 96-microwell black

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plate and Gd-DO3A-Chal solution was mixed to attain variable concentrations. The emission fluorescence intensity was measured for the mixture at the excitation wavelength and the

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binding constant Kd was determined from the fitted curve using eq. (1): 𝐾𝑑 : = 𝐵𝑚𝑎𝑥 × 𝑥/(𝐾𝑑 + 𝑥)

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(1)

,where 𝑥 is the concentration of Gd-DO3A-Chal, y is the specific binding fluorescence

intensity, and Bmax corresponds to the apparent maximal observable fluorescence upon the binding of Gd-DO3A-Chal to Aβ1–42 oligomers [38-40].

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2.8 Aβ1–42 oligomer-binding MR study This study refers to a previous method with slight modifications [41]. Low and high molecular-weight Aβ1–42 oligomers were incubated in each media at room temperature for 24 h with Gd-DO3A-Chal and Gd-DO3A-BT for comparative purposes. The final concentration of the Gd complexes and Aβ42 oligomers was 100 μM as an equimolar ratio. After incubation, each tube was spun at 14,000 rpm for 30 min at 4 ℃ and the supernatant was removed followed by washing with distilled water. The gathered pellets were

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resuspended in distilled water containing 10% DMSO and MR acquisition was performed at 9.4 T (Bruker BioSpin, BioSpec 94/30 USR, Ettlingen, Germany). A RARE sequence with

the following parameters was performed for T1-weighted image acquisition: repetition time =

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670 ms, echo time = 7 ms, rare factor = 1, image size = 128ⅹ128, field of view = 21 mm, and

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slice thickness = 0.5 mm. The parameters for the T1- relaxation time measurements were as

2.9 AD Animal model

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mentioned previously.

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The 5XFAD transgenic mouse AD model was approved by the Institutional Animal Care and Use Committee of the Daegu-Gyeongbuk Medical Innovation Foundation (DGMIF) and

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in vivo experiments involving animals were performed according to their approved protocols. 9-month-old male 5XFAD transgenic mice was used and housed in a specific pathogen-free

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facility at the Laboratory Animal Center of the DGMIF before use.

2.10 In vivo MR imaging For in vivo MR imaging, a 9.4 T MR instrument was used with the same RARE sequence parameters for T1-weighted image as mentioned previously. An RF RES 400 1H 089/023 13

transmit/receive 23-mm volume coil (Bruker BioSpin, Ettlingen, Germany) was used for image acquisition. 5XFAD mice was anesthetized with isoflurane and after pre image acquisition, 100 mM of Gd-DO3A-Chal aqueous solution was administered via IV injection at a dose of 0.3 mmol Kg-1 followed by MR acquisition. The color-mapped image was given as a positive signal intensity (SI) value via eq. (2): ∆SI = 𝑆𝐼𝑝𝑜𝑠𝑡 − 𝑆𝐼𝑝𝑟𝑒

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(2) It is obtained by using python 3.6 and all scripts were composed by using the python library matplotblib.

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2.11 Immunofluorescence staining of the post mortem brain section for microscopy

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5XFAD transgenic mouse brain was harvested immediately after MR acquisition and placed in 4% paraformaldehyde. Embedding the tissue in paraffin blocks allowed the cutting

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of 5 μm thin sections and the sectioned brain tissue slides were deparaffinized for 1 h at 65 ℃ in an incubator with additional processing in xylene followed by rehydration through an

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ethanol series. The samples were permeabilized with 0.3% Triton™ X-100 for 15 min and then nonspecific protein binding was blocked with 5% normal goat serum (NGS) and bovine

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serum albumin (BSA) in Tris-buffered saline (TBS) for 1 h at room temperature. The slides were incubated overnight with rabbit polyclonal beta Aβ 1–42 antibodies at their respective

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dilution factors in 5% BSA/NGS solution and washed three times in TBS. After primary incubation, the slides were further incubated with secondary antibodies (anti-rabbit red fluorescence Alexa Fluor 555 IgG, Invitrogen, Carlsbad, CA) at room temperature for 2 h. Following secondary incubation, brain sections were co-stained with 1% Gd-DO3A-Chal aqueous solution for 2 h and mounted with VECTASHIELD® Mounting Medium with DAPI 14

(4′,6-diamidino-2-phenylindole) followed by cover-slipping. High-resolution microscopy of the brain sections was performed on an inverted microscope system (Eclipse Ti2; Nikon, Tokyo, Japan) using NIS-Elements BR 4.50 software. The sample was excited by using a Nikon FITC (fluorescein isothiocyanate) filter cube to visualize Gd-DO3A-Chal stained Aβ aggregates and a Nikon TRITC (tetramethylrhodamine) filter cube was used to visualize the Alexa Fluor 555 secondary antibody [42].

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2.12 Statistics

Curve fitting was performed with the MATLAB curve fitting tool and Python 3.6. Data are presented as the mean ± standard deviation or standard error, as mentioned in the main text.

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Statistical analysis was conducted via Mann−Whitney U tests with SPSS software.

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3. RESULTS AND DISCUSSION 3.1 Preparation of Gd-DO3A-Chal The structure of Gd-DO3A-Chal, a new Gd complex with a chalcone moiety including a dimethylamino group for Aβ MR imaging, and its synthetic procedure are displayed in Figure 1. 4′-aminoacetophenone and dimethylaminobenzaldehyde underwent base-catalyzed aldol condensation to form the chalcone structure (compound-1) and chloroacetyl chloride

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was used to make compound-2 which was able to couple with the DO3A-(tBuO)3∙HBr (tritert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7-triacetate) backbone, thereby playing the role of a chelate in the Gd complex, via amide bonding for increased solubility. The conjugation of DO3A-(tBuO)3∙HBr with compound-2 was achieved under previously published

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conditions; the deprotection of the DO3A tert-butyl group was conducted in trifluoroacetic

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acid and dichloromethane 1:1 (v/v). The pure ligand was obtained by using reverse-phase column chromatography. The Gd complex with the chalcone structure was prepared with

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GdCl3∙6H2O by adjusting the pH to 7, resulting in a 40% yield. The final purification was carried out via reverse-phase column chromatography, and all compounds including Gd-

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DO3A-Chal were evaluated using microanalytical and spectroscopic methods; e.g. 1H NMR, elemental analysis (EA), high-resolution fast-atom bombardment mass spectrometry (HR-

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FABMS; Supplementary data, Figure S1-3).

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3.2 MR and optical characteristics of Gd-DO3A-Chal The performance of Gd-DO3A-Chal as an MR imaging CA was evaluated through

relaxivity measurements at 3.0 T and 9.4 T on an MR instrument in comparison with GdDO3A-BT. Linear fittings of relaxivities for Gd complexes were well established as shown in Figure 2A,B. Gd-DO3A-Chal had relaxivity values of r1 = 4.95 ± 0.22 and r2 = 6.80 ± 0.07 16

mM-1s-1 at 3 T and r1 = 4.99±0.19 and r2 = 6.07±0.02 mM-1s-1 at 9.4 T (Figure 2C). The relaxivity data revealed that Gd-DO3A-Chal could work well with enough performance not only at 3.0 T but also at the ultrahigh field 9.4 T. These relaxivity results are quite promising for the application of Gd-DO3A-Chal with an ultrahigh field because the relaxivity of the Gd complex generally decreased along with increasing magnetic field strength. Because of the chalcone moiety conjugated with Gd, Gd-DO3A-Chal also showed good fluorescence capability which resulted in detecting Aβ aggregates such as Thioflavin S which

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are commonly used as a fluorescence probe for Aβ binding assays. As has been reported, the 4-dimethylaminochalcone structure exhibits absorption in the wavelength range 390 to 460

nm and emission in the range 450 to 620 nm [43]. The excitation and emission wavelengths

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of Gd-DO3A-Chal are confirmed and exhibited in Figure 3 as a significant red shift of 160

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nm (from 435 to 595 nm), which is advantageous for a fluorescence probe to allow easy

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separation of the excitation light from the emission light [37].

3.3 Transmetalation and pH stability

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Transmetalation of Gd complexes is known to be strongly associated with safety issues such as causing nephrogenic systemic fibrosis or deposition of Gd(III) in the brain [44, 45].

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In general, Gd complexes with high transmetalation stability are safer than those with low ones. Gd complexes can be kinetically labile and can thus suffer from transmetalation by

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endogenous ions in the human body, such as Cu(II), Ca(II), and Zn(II). Displacement of the paramagnetic Gd(III) can occur with these competitive ions, especially Zn(II), because of their high affinity toward the Gd-chelate at significant concentrations in human blood. Therefore, the transmetalation stability of Gd-DO3A-Chal was estimated under excess Zn(II). The changed longitudinal relaxation rate at evolved time t, R1p(t)/R1p(0), is a good 17

estimate for the extent of transmetalation, the results of which are displayed in Figure 4. Gd(III) acts as typical “hard” acids, then hard base is preferred for binding interaction which results in stable Gd-complexes. Gd-DOTA which has four carboxylate pendant arms, while as Gd-DO3A-BT and Gd-DO3A-Chal have three carboxylates with hydroxyl and carbonyl oxygen for each [31]. The stability of these three Gd complexes follows the order of, GdDOTA> Gd-DO3A-BT> Gd-DO3A-Chal, and unfortunately in 72h later, partial leaching (about 10%) of Gd(III) was observed for Gd-DO3A-Chal. This partial leaching cannot be

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considered negligible due to the high intrinsic toxicity of the free Gd(III) ion, compared to other metal ions. For possible in vivo application, this result therefore suggests an

improvement in design of new probe with greater thermodynamic stability and kinetic

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inertness.

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In addition, the longitudinal relaxation rate was measured at various pH values to observe pH stability. For comparison purposes, Gd-DO3A-BT and Gd-DOTA were used and the

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results was displayed as relaxation rate changes versus time for pH range 1 to 11 (Figure 5AC). Gd-DO3A-BT showed high relaxation rate changes, indicating the dissociation of Gd(III)

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from the chelates, while Gd-DO3A-Chal had no remarkable changes over the entire pH range. The stability of Gd-DO3A-Chal is compared favorably with Gd-DOTA and it also

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confirmed in Figure S4, which represented as relaxation rate changes versus different pH.

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3.4 In silico docking study of Gd-DO3A-Chal with Aβ 1–42 oligomers To rationalize the binding properties of Gd-DO3A-Chal with Aβ 1–42 oligomers, protein-

ligand docking simulation was performed with their solution structure (PDB ID: 1IYT) and 3D (oligomeric) structure (PDB ID: 2BEG). For the in silico study, the blind docking approach which covered the whole protein to identify the possible binding poses on each Aβ 18

conformation was employed and Pittsburgh compound B (PiB), an Aβ-targeting positron emission tomography imaging CA, was used as a positive control for comparison purposes. The output, a total of twenty docking poses for each Aβ conformation, was found by docking simulation (Figure S5, supplementary data) and the final binding combination was referred based on the binding free energy calculation for PiB in Figure 6A. The docking simulation demonstrated that PiB showed favorable binding free energy (ΔGbinding = -28.43 kcal mol-1) for Aβ solution and binding free energy (ΔGbinding = -47.51 kcal mol-1) for oligomeric Aβ by

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binding in the cleft of the U-shaped conformation the Aβ fibril. In Figure 6B, it can be seen that Gd-DO3A-Chal attained the predicted binding site and the results indicate that GdDO3A-Chal showed higher binding affinity than PiB with the values in Aβ solution

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(ΔGbinding = -32.95 kcal mol-1) and oligomeric Aβ (ΔGbinding = -61.01 kcal mol-1). The ligand

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interaction diagram in Figure 6C gives details on the interaction between Aβ protein and Gd-DO3A-Chal. The Gd complex was bound with Phe19 (D) and Phe19 (E) by π-π stacking

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on the benzene rings of chalcone moiety. Therefore, these data reveal that Gd-DO3A-Chal could bind to Aβ aggregates with high affinity, thereby helping the mechanism of action [46,

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47].

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3.5 Fluorescence spectra and binding affinity constant measurement with Aβ 1–42 oligomers

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Fluorescence spectra were measured with and without the Aβ 1–42 oligomers to test the

binding affinity of Gd-DO3A-Chal to them. Upon binding with the Aβ 1–42 oligomers, GdDO3A-Chal showed intensive emission through its fluorescence characteristics (specifically, a 5.76-fold fluorescence intensity increase) compared to the native fluorescence intensity, accompanied by a blue shift from 595 to 530 nm (Figure 7A). These fluorescence data are in 19

line with previous studies reporting a significant fluorescence intensity increase when the fluorescence probes were bound to the Aβ oligomers [38, 40]. For quantitative measurements, the apparent binding constants (Kd) were evaluated to 5 µM Aβ 1–42 oligomers in concentrations of 0.2–50 µM (Figure 7B). The equilibrium dissociation constant (Kd) is used to evaluate the order strengths of molecular interactions, indicating the smaller the value, the greater the binding affinity between the molecules. The measured Kd value of Gd-DO3A-Chal was 23.1 ± 4.31 µM and was much lower than the Kd value of

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Gd(DO3A-PiB), a previously reported MR probe for the visualization of Aβ aggregates [20]. Specifically, Gd-DO3A-Chal showed 8 times higher Aβ binding affinity than that of

Gd(DO3A-PiB). Therefore, when considering the µM concentrations of Aβ aggregates in AD

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[48-50]. the binding constant estimation revealed that the Aβ binding affinity of Gd-DO3A-

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Chal may have enough sensitivity to detect Aβ aggregates in AD in vivo.

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3.6 In vitro binding study with MR measurements

To confirm the binding capacity of Gd-DO3A-Chal to Aβ aggregates using MR

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measurements, an in vitro binding study was performed with low- (di-, tri-, tetramer) and high-molecular-weight (75–250 kDa) Aβ oligomers. The underlying principle of MR

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measurements is that the relaxivity is expected to increase upon binding of Gd complexes to Aβ oligomers due to the slow tumbling motion of the former [20, 51]. That is to say, once the

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Gd complex is bound to higher molecular-weight Aβ oligomers, its tumbling motion becomes slower and thus, higher relaxivity is expected. After incubation of the Gd complex with the Aβ oligomers for 24 h at 37 ℃, the mixture was separated out once via centrifugation and redissolved in D2O containing 10% dimethyl sulfoxide (DMSO). The Gd complex bound to the high molecular-weight Aβ oligomers was then evaluated via a longitudinal relaxation time 20

(T1)-weighted image in comparison with one of Gd-DO3A-BT (Figure 7C). A significant increase in MR signal intensity was observed in Gd-DO3A-Chal incubated with high molecular-weight Aβ oligomers, indicating a relevant Aβ binding affinity, whereas GdDO3A-BT, a nonspecific CA, did not show a significant increase in MR signal intensity with the Aβ oligomers. For quantification, the binding affinity with Gd-DO3A-Chal was performed with T1 measurements (Figure 7D) as relaxation rates (R1). For low molecularweight Aβ oligomers, the R1 of the Aβ oligomers was increased due to binding with Gd-

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DO3A-Chal. For the high-molecular-weight Aβ oligomers, the increase in R1 was more

significant when binding to Gd-DO3A-Chal due to the slow tumbling motion mentioned

previously. However, the R1 of the low- and high-molecular-weight Aβ oligomers with Gd-

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DO3A-BT did not show enough change, thus confirming that the Gd-DO3A-BT had no

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binding capacity for the Aβ oligomers.

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3.7 In vivo MR imaging and immunohistochemistry of Aβ aggregates In vivo T1-weighted images for the 5XFAD transgenic mouse model were acquired after IV

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administration of Gd-DO3A-Chal at a dose of 0.3 mmol Kg-1. The signal intensity difference in the T1-weighted image was shown as color mapped image at 4 hours after IV

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administration of Gd-DO3A-Chal to display the maximized signal enhancement. It is seemed that the increased signal was revealed in several brain areas including the cortex,

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hippocampus, and thalamus (Figure 8A). As shown in Table S1, the partition coefficient constant, LogP value, which indicates the lipophicity of compounds was higher rather than other non-specific commercial agents in spite of conjugation of hydrophilic DO3A backbone and it is supposed that this help to be able to acquire these signal enhancement in the brain. 21

Post mortem sections after MR imaging were double-stained with 6E10 monoclonal antibodies for the Aβ 1–42 peptide and Gd-DO3A-Chal by using its fluorescence property. The patterns of hyperintense spots in the T1-weighted MR images were similar to the Aβ distribution seen in the matched histological sections (see arrowheads in Figure 8B). However, due to a difference in slice thickness and orientation between the MR images (100 μm) and the tissue sections (40 μm), it was not possible to exactly match all of the dark spots seen in the MR image and the immunostained section.

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After binding with Aβ aggregates, immunostaining using 6E10 monoclonal antibodies showed the Aβ aggregates with red emission in the fluorescence imaging whereas GdDO3A-Chal showed them with green emission (Figure 8B). Therefore, although the

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structural effects influenced their intrinsic fluorescence properties, the chalcone exhibited

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similar absorption and emission properties with good fluorescence brightness even after conjugation with Gd-DO3A. The merged images of these two staining results in 5XFAD

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transgenic mouse brain sections showed co-localized orange colored spots indicating that the fluorescent images of Gd-DO3A-Chal showed a good match with the immunostaining

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images using 6E10 monoclonal antibodies on Aβ aggregates. Furthermore, the region of signal enhancement in the MR image of 5XFAD transgenic mouse brain was well matched

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with the fluorescence imaging data, suggesting that Gd-DO3A-Chal has good potential as an

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Aβ-targeting MR CA.

4. CONCLUSION Multifunctional Aβ-targeting small-molecular-weight Gd CA (Gd-DO3A-Chal) was

synthesized and tested for detection capability of Aβ oligomers and multimodal imaging efficacy. The in silico docking data predicted that Gd-DO3A-Chal possessed higher binding 22

free energy than that of clinically approved PiB. The docking simulation results were further evaluated by using fluorescence measurements. Binding affinity constant measurements with Aβ oligomers using fluorescence spectroscopy showed that Gd-DO3A-Chal had a smaller Kd value than that of previously reported Gd(DO3A-PiB), indicating that the former has almost 8 times higher affinity for Aβ oligomers than the latter. For multimodal imaging, GdDO3A-Chal exhibited strong fluorescence brightness, thereby demonstrating its efficacy as a good Aβ-targeting fluorescence imaging probe. Its Aβ-targeting efficacy was well

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demonstrated by a good match with immunostained images of Aβ aggregates. In the case of in vivo MR imaging, even though the relaxivity of the Gd complex was known to decrease

under a high magnetic field, Gd-DO3A-Chal exhibited sufficiently high signal enhancement

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at 9.4 T. Although an exact match with immunostaining data was not possible due to the

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differences in orientation and slice thickness in the matched histological sections, the MR images using Gd-DO3A-Chal showed signal enhancement in the Aβ distribution seen in the

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matched histological sections. Therefore, we successfully demonstrated in vivo MR images on Aβ detection in addition to fluorescent optical images of Aβ with high specificity using

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this new multifunctional Aβ-targeting CA. Funding

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This research was supported by the Brain Research Program (NRF-2018M3C7A1053217)

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funded by the Ministry of Science, ICT & Future Planning, Korea.

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 23

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Figure 1 Schematic procedure for synthesis of a chalcone conjugated gadolinium complex,

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Gd-DO3A-Chal and its 3D structure.

Figure 2 A) Linear fitting of r1 and r2 relaxivities for Gd-DO3A-BT (Black) and Gd-DO3AChal (Orange) at 3 T. B) Linear fitting of r1, r2 relaxivities for Gd-DO3A-BT (Black) and 26

Gd-DO3A-Chal (Orange) at 9.4 T. C) relaxivity values at 3 T and 9.4 T are represented in

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table (Data are presented as the mean ± standard error)

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Figure 3 Absorption (green) and emission (orange) spectra of Gd-DO3A-Chal.

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Figure 4 Transmetallation stability of Gd-DO3A-Chal with commercially available MR

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.

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agent in comparison

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Figure 5 pH stability of Gd-DO3A-Chal with commercially available MR agent, Gd-DO3A-

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versus time. (Data are presented as the mean ± standard error)

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BT (B) and Gd-DOTA (C), represented as the evolution of the water proton relaxation rate

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Figure 6 A) Binding sites of Pittsburgh compound B based on the best docking simulation for amyloid beta monomer and amyloid fibril model. B) Binding sites of Gd-DO3A-Chal based on the best docking simulation for amyloid beta monomer and amyloid fibril model. C) Ligand interaction diagram between amyloid beta protein and Gd-DO3A-Chal. 29

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Figure 7 A) The emission wavelength of Gd-DO3A-Chal (10 μM) with (red) and without

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(gray) Aβ aggregates (20 μM). B) curve fitting for determination binding constant Kd for Gd-

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DO3A-Chal to amyloid aggregates (5 μM). C) The color mapped T1-weighted image after incubation 24h at 37 ℃ with Gd complexes and high molecular amyloid oligomer, which

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shows signal enhancement due to amyloid binding. D) R1 relaxation rate changes with lowand high- molecular amyloid oligomer after incubation with Gd complexes 24h at 37 ℃. P <

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0.05 (*)

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Figure 8 A) The color mapped T1-weighted MR in vivo image after administration of GdDO3A-Chal with intravenous injection (post 4h, 0.3 mmol/Kg). B) immunofluorescence imaging on post-mortem brain after in vivo MR acquisition which co-stained with Gd-

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DO3A-Chal (green) and amyloid beta antibody (red).

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Chart 1 structures of the commercial MR agents in this study

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