Development of an MRI contrast agent for both detection and inhibition of the amyloid-β fibrillation process

A curcumin derivative conjugated with Gd-DO3A (Gd-DO3A-Comp.B) was synthesised as an MRI contrast agent for detecting the amyloid-β (Aβ) fibrillation process. Gd-DO3A-Comp.B inhibited Aβ aggregation significantly and detected the fibril growth at 20 μM of Aβ with 10 μM of probe concentration by T1-weighted MR imaging.


Experimental Procedure
All the solvents used were in analytical standard grade. The NMR spectra were measured on a Bruker biospin AVANCE II (400 MHz for 1 H and 100 MHz for 13 C) or a Bruker biospin AVANCE III (500 MHz for 1 H, 125 MHz for 13 C, and 470 MHz for 19 F). Chemical shift (δ) was reported in ppm relative to internal tetramethylsilane. The HRMS data were recorded on Bruker ESI-TOF-MS micrOTOF II instrument with sodium formate as the calibration standard. Vanilin, 3-bromopropylamine hydrobromide, potassium carbonate, potassium bicarbonate, morpholine, boric acid, and 10% Pd/C were purchased from Wako Chemical (Japan). The 1,4,7,10-tetraazacyclododecane were purchased from Accela (USA). Benzyl chloroformate, tert-butyl bromoacetate, PyBOP, and HOBt were purchased from Tokyo Chemical Industry (Japan). Microwave for synthesis was conducted on Biotage® Initiator+ instrument. Column chromatography was performed on silica gel Chromatorex (Japan). Purity analysis was determined by HPLC analysis using Inertsil ODS-3 5 µm (4.6 × 75 mm; GL Science) with a linear gradient of 0.1% formic acid in water/0.1% formic acid in MeCN detected by UV lamp for 20 min. Amyloid β (Aβ42) peptide was purchased from Peptide Institute (Japan). Thioflavin T (ThT) for fluorescence detection of Aβ42 was purchased from Sigma (USA).
Compound 7 (2 mmol) was added then followed by morpholine (2 mmol). The mixture was irradiated in microwave at 100 o C for 10 minutes. The reaction mixture was quenched by HCl 0.1 N and extracted using ethyl acetate. The organic phase was dried using MgSO4. The crude product was purified by column chromatography (silica gel, hexane:ethyl acetate 4:1) to obtain the desired compound.

Aβ Preparation for Aggregation Study
To prepare the Aβ monomer stock, in amount 0.5 mg of the lyophilized Aβ42 (Peptide Institute) was dissolved in DMSO by gently mixing without vortexing to obtain 500 µM as final concentration. The solution was centrifuged at 13,200 rpm at 4 o C for 10 minutes. The supernatant was collected and stored in −80 o C until used. The Aβ stock was diluted into 100 µM in 2 mM NaOH freshly before used.

Congo Red and Thioflavin T Assay
Congo Red or Thioflavin T stock at concentration 2 mM was freshly prepared in tris glycine 10 mM pH 8.5, then diluted in PBS at pH 7.4 to reach final concentration 40 µM. Gd probes was added at final concentration 10 µM followed by Aβ to reach final concentration 20 µM then was incubated at 37 o C. After 24 h, the mixture was transferred in 384-well plate black bottom non-binding to be scanned the fluorescence S9 intensity with an excitation of 430 nm and emission range from 450 to 600 nm using microplate reader (SpectraMax iD5, Molecular Device, USA).

Negative-Staining TEM Imaging
Elastic carbon grids (ELS-C10, STEM, Japan) was hydrolysed by ion coater (IB-2, Eiko, Japan) with 3 mA of plasma current for 40 seconds before applying sample solution. Briefly, 5 µL of the Aβ only or containing Gd probes after 24 h incubation was applied to a hydrophilic grid and incubated for one minute at RT. After gently dried with filter paper, the grid sample was washed with Milli-Q water and dried again with filter paper two times. Finally, the grid was incubated with 5 µL of 1% Nano-W negative staining solution (NY, USA) for one minute followed by complete drying using filter paper. The negative stained sample was observed using TEM H-8100 (Hitachi) operated at 200 kV.

Nuclear Magnetic Resonance (NMR) Relaxometry
Longitudinal relaxation time (T1) was measured using a Spinsolve ULTRA 43 MHz 1 H-NMR (Magritek Ltd., Wellington, New Zealand) in PBS at pH 7.4. The inversion-recovery (IR) was used to measure T1. The parameters in the IR pulse sequence were as follows: number of scans = 2, acquisition time = 1.6 s, repetition time = 7 s, maximum inversion time = 5 s, number of steps = 21. To confirm inhibitory effect on fibril growth, 20 µM of monomeric Aβ was mixed with 10 µM Gd probes at 37 o C and the T1 was measured at 0.5, 1, 2, and 24 h after preparing the samples. The fibrillation growth was detected as follows: 20 µM of monomeric Aβ was pre-incubated at 37 o C for 1, 3, 6, 12, and 24 h to cause the fibril formation in different growth stages. Then the 10 µM Gd probes were added into the fibril at each incubation time and T1 was measured without further incubation. The T1 change (ΔT1) was calculated by the equation as follow; where T1,(t) is T1 of the Gd probe solution with Aβ at t hour while T1,(0) was T1 of the Gd probe solution without Aβ at 0 hour.