Inhibition of Beta-Amyloid Fibrillation by Luminescent Iridium(III) Complex Probes.

We report herein the application of kinetically inert luminescent iridium(III) complexes as dual inhibitors and probes of beta-amyloid fibrillogenesis. These iridium(III) complexes inhibited Aβ1-40 peptide aggregation in vitro, and protected against Aβ-induced cytotoxicity in neuronal cells. Furthermore, the complexes differentiated between the aggregated and unaggregated forms of Aβ1-40 peptide on the basis of their emission response.

Over the past few years, vast efforts have been dedicated to the development of imaging probes and inhibitors for the diagnosis and treatment of Alzheimer’s disease, which is a highly common neurodegenerative disorder characterized by the progressive loss of cognitive ability12. One of the strategies for the treatment of Alzheimer’s disease is the development of inhibitors that prevent the misfolding and self-assembling aggregation of monomeric Aβ peptides into neurotoxic fibrils1. Many inhibitors have been reported to inhibit Aβ peptide aggregation3456, such as short peptides78, organic molecules910111213141516, supramolecular cucurbit[7]uril17, polyoxometalates1819, nanoparticles20 and ligand-functionalized quantum dots21. Meanwhile, the development of new diagnostic probes for Aβ fibrillation has also been an important goal. The early diagnosis of Alzheimer’s disease may allow for palliative treatment to alleviate symptoms or to slow down the progression of the disease. A number of luminescent probes have been developed for the specific labeling and imaging of Aβ plaques, such as luminescent conjugated polythiophenes22 and common fluorescent dyes232425262728293031. Dual-role imaging agents and aggregation inhibitors of Aβ may serve a bifunctional purpose for Alzheimer’s disease, as the inhibition of Aβ fibrillogenesis may be monitored without the need for an extraneous labeling agent. However, to our knowledge, only a few examples of dual-function inhibitors and probes of Aβ have been reported in recent years. Organic dyes commonly used for labeling Aβ fibrils, including Congo Red and Thioflavine T (ThT), have been shown to inhibit Aβ aggregation at high concentrations of dyes32.

Luminescent transition metal complexes have found emerging use for the design of chemical and biological sensors3334 in view of their useful photophysical properties, such as tunable excitation and emission wavelengths (from blue to red), high luminescent quantum yields, and relatively long phosphorescent lifetimes33. At the same time, transition metal complexes have been increasingly regarded as a promising alternative to organic compounds as therapeutic agents for the treatment of human diseases35363738394041. Due to the well-defined three-dimensional structure of transition metal complexes, highly specific interactions between metal complexes and biomolecules can be obtained through modification of the steric and electronic nature of the organic ligands surrounding the metal centre. In the context of Alzheimer’s disease, metal complexes such as Pt(II)42434445464748, binuclear Ru(II)–Pt(II)49, Ru(II)505152, Co(III)53 and Ir(III) or Rh(III) solvato54 complexes have been reported to inhibit the aggregation of Aβ peptide555657. Furthermore, luminescent metal complexes such as Ru(II)5859, Re(I)60 complexes have also been used to monitor the Aβ fibrillation process3055. However, few transition metal complexes have been reported to be dual inhibitors and luminescent probes of Aβ. Moreover, while iridium(III) complexes have been widely used as probes for various biomolecules, their application as dual inhibitors and probes of beta-amyloid fibrillogenesis has not been previously described.

Our group has previously reported the application of Ir(III) and Rh(III) solvato complexes as inhibitors of amyloid fibrillogenesis and as luminescent probes for Aβ peptide54. Mass spectrometry experiments indicated that the Rh(III) solvato complex formed 1:1 covalent adducts with Aβ1–40, presumably through coordination of the N-donor histidine residue of Aβ1–40 to the Rh(III) center via displacement of the aqua ligands. Encouraged by our previous results with Group 9 solvato compounds, we sought to investigate the ability of kinetically inert Ir(III) complexes to function as dual imaging agents and inhibitors of Aβ peptide aggregation. We report herein the synthesis of a series of luminescent Ir(III) complexes containing various C^N and N^N ligands, and their ability to detect and inhibit Aβ fibrillation. We envisage that these kinetically inert Ir(III) complexes may be developed as a novel class of dual-purpose probes and inhibitors of Aβ aggregation for the effective diagnosis and/or treatment of Alzheimer’s disease.

Results

A library of twelve luminescent Ir(III) complexes (1–12, Fig. 1) were initially examined for their ability to interact with different forms of Aβ1–40 by emission titration. Of these twelve complexes, 12 bearing the 2-phenyl-1H-imidazo[4, 5-f][1,10]phenanthroline (phenyl-imidazo-phen) N^N ligand displayed the Ifibril/Imonomer ratio (>1.5), indicating that it possessed the highest distinguishing ability for detecting Aβ1–40 fibrils over Aβ1–40 monomers (Figure S1). However, 1–11 were unable to effectively discriminate between Aβ1–40 monomers and fibrils. Based on the structure of 12, we designed and synthesized 13 and 14. The characterization and photophysical properties of 1–14 are given in the ESI (Table S1).

Figure 1

Chemical structures of the luminescent Ir(III) complexes 1–14 which were synthesized and evaluated in this study.

Complexes 12–14 were investigated for their ability to inhibit the fibrillogenesis of Aβ1–40. Given that these complexes could detect Aβ1–40 fibrils over Aβ1–40 monomers, the kinetics of Aβ1–40 aggregation in the presence of the complexes could be monitored without the need for an external labeling agent. The results revealed typical sigmoidal growth curves in seed-mediated Aβ fibrillation when Aβ1–40 monomers was incubated with ThT or 12–14 (Figure S2a and S2b). However, fibrillogenesis was partial retarded by 13 and completely inhibited by 14, indicating that these two complexes were able to inhibit Aβ1–40 peptide aggregation. As both 13 and 14 contain the phenol-imidazo-phen N^N ligand, the effect of that ligand alone on Aβ1–40 peptide aggregation was investigated. The results showed that the presence of the N^N ligand alone had no inhibitory effect on Aβ1–40 peptide aggregation, highlighting the importance of the iridium(III) metal center in maintaining the octahedral structure of the active complexes (Figure S2c). Moreover, total internal reflection fluorescence microscopy (TIRFM) with laser excitation was used to observe the ThT-labelled fibrils with high sensitivity. In the absence of Ir(III) complexes, the fluorescence images of the Aβ1–40 peptides incubated by the seed-mediated method showed dense spots, corresponding to newly genereted Aβ1–40 fibrils (Fig. 2a). In contrast, these spots partially or completely disappeared when the samples were treated with 13 (Fig. 2b) or 14 (Fig. 2c), indicating that 13 and 14 could partially or completely inhibit the fibrillogenesis of amyloid peptides, respectively. Transmission electron microscopy (TEM) images further confirmed the ability of the Ir(III) complexes to inhibit seed-mediated Aβ aggregation. In the control experiment (Fig. 3a), Aβ1–40 grewinto thick, dense and long hair-like fibrils. However, when the sample was incubated with 13, the TEM image showed short flake-like fibris (Fig. 3b), indicating that 13 was able to partially inhibit the Aβ1–40 aggregation. Furthermore, incubation of the sample with 14 resulted in the complete absence of elongated fibrils, and only the preformed fibril seeds were observed (Fig. 3c), indicating the complete inhibition amyloid fibrillogenesis by 14. Moreover, the fibrillogenesis kinetics of Aβ1–40 in the presence of various concentrations of 14 showed that the inhibition of Aβ1–40 aggregation by 14 was concentration-dependent (Figure S3). At 50 μM of 14, the inhibition of 25 μM of Aβ1–40 peptides was nearly completely inhibited.

Figure 2

Inhibition of seed-mediated Aβ1–40 fibril growth by Ir(III) complexes.

TIRFM images of Aβ1–40 fibrils grown in the (a) absence and presence of (b) 13, (c) 14 after incubation at 37 °C for 1 h. The scale bar for TIRFM is 20 μm.

Figure 3

Inhibition of seed-mediated Aβ1–40 fibril growth by Ir(III) complexes.

TEM images of Aβ1–40 fibrils grown in the (a) absence and presence of (b) 13, (c) 14 after incubation at 37 °C for 1 h. The scale bar for TEM is 200 nm.

Encouraged by the promising activity of 13 and 14 against Aβ aggregation, we investigated that the luminescence behaviour of 13 and 14 towards different forms of Aβ1–40. The results showed that both complexes displayed a significantly enhanced luminescence response in the presence of the Aβ1–40 monomers or fibrils (Fig. 4). 13 exhibited ca. 6- and 12-fold emission enhancements at λmax = 540 nm in the presence of comparable mass concentrations of Aβ1–40 monomers and fibrils, respectively. On the other hand, 14 showed ca. 11- and 18-fold emission enhancements at λmax = 540 nm in the presence of Aβ1–40 monomers and fibrils. Taken together, these two Ir(III) complexes showed luminescence enhancement to the presence of Aβ1–40 monomers and fibrils to different extents. We presume that this behaviour of the Ir(III) complexes towards the Aβ1–40 peptide may be due to the ability of the complexes to bind to a hydrophobic region within the peptide, thus protecting the complexes from non-radiative decay by solvent quenching and thereby giving rise to an enhanced luminescence response. The differential luminescence response of the complexes towards Aβ1–40 monomers and fibrils may be due to the different microenvironments experienced by the Ir(III) complexes upon binding to the Aβ1–40 monomers or the fibrils.

Figure 4

Luminescence response of 2 μM of (a) 13 and (b) 14 in the absence or presence of 25 μM Aβ1–40 monomers or fibrils.

λEx = 360 nm.

ESI-TOF mass spectrometry experiments were performed to examine the binding of the Ir(III) complexes to Aβ1–40 peptide. The mass spectrum of the Aβ1–40 monomer in the absence of the Ir(III) complexes shows two characteristic peaks at 1083 and 1444, corresponding to the 4+ and 3+ ionization states of the Aβ1–40 monomer, respectively (Figure S4a). However, incubation of the Aβ1–40 peptide with 13 (Figure S4b) or 14 (Figure S4c) produced no new peaks in the mass spectra besides those corresponding to the free complex (813 for 13 and 861 for 14), suggesting that the Ir(III) complexes were not covalently bound to the Aβ1–40 peptide.

The cytotoxicity of the most potent Ir(III) complex 14 was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Figure S5). Neuroblastoma cells (SH-SY5Y) were incubated in the presence of different concentrations for 24 h and cell viability was examined using the MTT assay. The IC50 value of 14 was estimated to be >100 μM at 24 h of exposure. Notably, these IC50 values are significantly higher than the concentration of 14 required for complete inhibition of Aβ1–40 peptide aggregation, suggesting the presence of a therapeutic window whereby Aβ1–40 peptide aggregation can be controlled without significant damage to brain cells.

The effect of 14 on Aβ1–40-induced cytotoxicity in SH-SY5Y cells and mouse primary cortical cells was also investigated. The cytotoxicity of three different forms of Aβ1–40 peptide in the presence and the absence of 14 were examined: Aβ1–40 peptide monomer (M), Aβ1–40 peptide monomer with seeded fibrils (MS) and Aβ1–40 fibril (F) (Fig. 5). The results showed that treatment of cells with different forms of Aβ1–40 peptides caused toxicity to SH-SY5Y cells and mouse primary cortical cells (Fig. 4a,c,e,g). Encouragingly, 14 exhibited a neuroprotective effect against the cytotoxicity induced by all three forms of Aβ1–40 peptide at [Aβ1–40]/[14] ratios of 0.2, 1.0, or 5.0 for SH-SY5Y cells (Fig. 5a,b) or mouse primary cortical cells (Fig. 5e,f) after 2 h of incubation. The neuroprotective effects of 14 were still observable after 24 h of incubation of 14 (Fig. 5c,d,g,h). As a negative control, we also investigated the effect of 12, which showed no effect against amyloid aggregation, on Aβ1–40-induced toxicity. The results showed that 12 had no neuroprotective effect against cytotoxicity induced by all three forms of Aβ1–40 peptide at [Aβ1–40]/[12] ratios of 0.2, 1.0, or 5.0 in SH-SY5Y cells (Figure S6). Taken together, these data indicate that 14 displays neuroprotective effects against Aβ-mediated cytotoxicity when administered at a low enough dosage in SH-SY5Y cells and mouse primary cortical cells.

Figure 5

Neuroprotective effect of 14 against Aβ1–40 peptide-induced cytotoxicity towards (a–d) human neuroblastoma SH-SY5Y cells and (e–h) mouse primary cortical cells.

Cell viability is expressed as a percentage of control cells exposed to 0.5% DMSO. The histograms show the cell viability of various concentrations of Aβ1–40 peptide monomer (M), Aβ1–40 peptide with seeded fibril (MS), and fibrillar Aβ1–40 peptide (F), in the presence of 14. Various forms of Aβ1–40 peptide were incubated for (a,b,e,f) 2 h, and for (c,d,g,h) 24 h at [Aβ1–40]:[14] ratios of 0.2:1, 1:1, and 5:1.

Discussion

In conclusion, a library of 12 luminescent Ir(III) complexes containing various C^N and N^N ligands were initially screened as luminescent probes for Aβ1–40 peptide. Based on the ability of 12 for distinguishing Aβ1–40 fibrils over monomers, 13 and 14 were further synthesized and tested. The novel Ir(III) complex 14 emerged as the most potent candidate and was shown to inhibit Aβ1–40 peptide aggregation as revealed by a luminescence assay, as well as TIRFM and TEM imaging. Notably, Aβ1–40 peptide aggregation was nearly completely inhibited at 50 μM of 14. A neuroprotective effect of 14 against Aβ1–40-induced cytotoxicity in SH-SY5Y cells and mouse primary cortical cells was also demonstrated. Using ESI-TOF mass spectrometry, we also showed 14 was not covalently bound to the Aβ1–40 peptide. Non-covalent probes may have a better safety index, lower cross-reactivity, and lower immunogenicity compared to covalently-binding molecules616263. We envision that this work would open up new avenues for the development of dual-role imaging agents and aggregation inhibitors of Aβ for the treatment of Alzheimer’s disease.

Methods

Chemicals and materials

Reagents, unless specified, were purchased from Sigma Aldrich (St. Louis, MO) and used as received. Iridium chloride hydrate (IrCl3·xH2O) was purchased from Precious Metals Online (Australia).

General experiment

Mass spectrometry was performed at the Mass Spectroscopy Unit at the Department of Chemistry, Hong Kong Baptist University, Hong Kong (China). Deuterated solvents for NMR purposes were obtained from Armar and used as received.

1H and 13C NMR were recorded on a Bruker Avance 400 spectrometer operating at 400 MHz (1H) and 100 MHz (13C). 1H and 13C chemical shifts were referenced internally to solvent shift (acetone-d6: 1H δ 2.05, 13C δ 29.8). Chemical shifts (δ are quoted in ppm, the downfield direction being defined as positive. Uncertainties in chemical shifts are typically ±0.01 ppm for 1H and ±0.05 for 13C. Coupling constants are typically ±0.1 Hz for 1H-1H and ±0.5 Hz for 1H-13C couplings. The following abbreviations are used for convenience in reporting the multiplicity of NMR resonances: s, singlet; d, doublet; t, triplet; m, multiplet. All NMR data was acquired and processed using standard Bruker software (Topspin).

Photophysical measurement

Emission spectra and lifetime measurements for complexes were performed as previously reported method64.

Transmission electron microscopy (TEM) imaging

8 μL of diluted sample solution was applied to a carbon-coated copper grid (T200H-Cu Electron Microscopy Sciences, Washington, USA), dried, and was then negatively stained with 2% uranyl acetate. The stained sample was then allowed to be dried. Transmission electron micrographs were recorded using a Technai G2 Transmission Electron Microscope (FEI, USA) with an acceleration voltage of 200 kV.

Total internal reflection fluorescence microscopy (TIRFM) imaging

This experimental system and operation was as previously described52. After the incubation period, the organic dye ThT (the mole ratio of Aβ1–40 and ThT is 1:2) was added to label the Aβ1–40 fibrils. Then, a 445 nm diode laser (50 mW, LQC445-40E, Newport, USA) was used for the excitation of the ThT-labeled Aβ1-40. Images were obtained by using the WinSpec/32 software (Princeton Instruments, Version 2.5.22.0, Downingtown, PA).

Preparation of stock solution of tested Ir(III) complexes

The stock solution of all the tested Ir(III) complexes are 10 mM in DMSO.

Emission measurement in buffered solution

Ir(III) complexes (2 μM) and different concentrations of Aβ1–40 monomer/fibril were added into phosphate buffer (50 mM Na2HPO4, pH 7.4). The mixtures were allowed to equilibrate at room temperature for 3 min without degassing. Emission spectra were recorded on PTI TimeMaster C720 Spectrometer.

Preparation of Aβ1–40 fibrils for seeding

The incubation of Aβ1–40 fibrils and seeds used the previously reported method52.

ThT and Ir(III) complexes luminescence binding assays

50 μM of ThT/complex solution (1%) was incubated with 25 μM Aβ1–40 monomer solution in phosphate buffer at 37 °C. The luminescence intensity at different time points was measured on a PTI TimeMaster C720 Spectrometer.

Mass spectrometry experiments

A solution of containing 13 or 14 (25 μM, final concentration) (final DMSO concentration ≤1%) and Aβ1–40 peptide monomers (50 μM, final concentration) were mixed in 1 mM ammonium acetate (pH 7.6), and injected into the ESI-TOF-MS at a rate of 3 L/min. ESI-TOF-MS experiments were conducted in the negative-ion mode with a Bruker MicrOTOFQ mass spectrometer. The capillary voltage was set at +3500 V, and the dry N2 gas flow was 4.0 L/min at 100 °C. Data were analyzed by the software Bruker Daltonics Data Analysis.

Cell viability analysis

The cell viability analysis is referenced from a reported method52. Specifically, SH-SY5Y cells or primary cortical cells were plated into 96-well plates at a density of 5 × 104 cells/well in cell culture medium. At the next day, the culture medium was replaced with the same medium with 0.2% serum and containing 10 μM 12 and 14 together with monomer, mixture of monomer and seed, and seeding fibril respectively in 1-to-0.2, 1-to-1 or 1-to-5 ratio (final concentration of DMSO ≤ 1.6%). Cells were incubated at 37 °C for 2 and 24 h, respectively. The medium of each well was replaced with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/mL) and further incubated for 3 h at 37 °C. The MTT solution was aspirated off and then 100 μL of DMSO was added to each well to dissolve the formazan crystals. The plates were agitated on a plate shaker for 15 min and the absorbance was measured at 570 nm using a SpectraMax M5 microplate reader (Molecular Devices). Wells without cells were used as blanks and were subtracted as background from each sample. Results were expressed as a percentage of control.

Additional Information

How to cite this article: Lu, L. et al. Inhibition of Beta-Amyloid Fibrillation by Luminescent Iridium(III) Complex Probes. Sci. Rep. 5, 14619; doi: 10.1038/srep14619 (2015).