Platinum-coordinated graphitic carbon nitride ... - Springer Link

Nano Research 2016, 9(8): 2411–2423 DOI 10.1007/s12274-016-1127-5

Platinum-coordinated graphitic carbon nitride nanosheet used for targeted inhibition of amyloid β-peptide aggregation Meng Li1,2, Yijia Guan1,2, Zhaowei Chen1,2, Nan Gao1, Jinsong Ren1, Kai Dong1,2, and Xiaogang Qu1 () 1

Laboratory of Chemical Biology and State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China 2 University of Chinese Academy of Sciences, Beijing 100039, China

Received: 5 February 2016

ABSTRACT

Revised: 25 April 2016

Amyloid β-peptide (Aβ) aggregation is a critical step in the pathogenesis of Alzheimer’s disease (AD). Inhibition of Aβ production, dissolution of existing aggregates and clearance of Aβ represent valid therapeutic strategies against AD. Herein, a novel platinum(II)-coordinated graphitic carbon nitride (g-C3N4) nanosheet (g-C3N4@Pt) has been designed to covalently bind to Aβ and modulate the peptide’s aggregation and toxicity. Furthermore, g-C3N4@Pt nanosheets possess high photocatalytic activity and can oxygenate Aβ upon visible light irradiation, remarkably attenuating both the aggregation potency and neurotoxicity of Aβ. Due to its ability to cross the blood-brain barrier (BBB) and its good biocompatibility, g-C3N4@Pt nanosheet is a promising inhibitor of Aβ aggregation. This study may serve as a model for the engineering of novel multifunctional nanomaterials used for the treatment of AD.

Accepted: 29 April 2016 © Tsinghua University Press and Springer-Verlag Berlin Heidelberg 2016

KEYWORDS amyloid disease, platinum, graphitic carbon nitride nanosheet, photooxygenation, amyloid β-peptide (Aβ) inhibitors

1

Introduction

Alzheimer’s disease (AD) is a chronic neurodegenerative disorder characterized by dementia, cognitive impairment, and memory loss [1, 2]. Considerable evidence suggests that the formation of various aggregated forms of the amyloid β-peptide (Aβ), including oligomers, protofibrils and fibrils, is responsible for Address correspondence to [email protected]

the neuronal dysfunctions observed in AD patients [3–5]. Among them, the low molecular weight soluble oligomers of Aβ are the primary neurotoxic species [6–8]. Therefore, diverse therapeutic strategies that target the generation, disaggregation, and clearance of Aβ are being pursued. To date, a large number of peptide mimetics [9–11] and small organic molecules [12, 13] have been designed as inhibitors of Aβ

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aggregation. However, their poor targeting and the weakness of their noncovalent interactions with the aromatic side chains of the Aβ peptide result in these agents possessing only a moderate efficiency in inhibiting aggregation and limited ability to induce disaggregation. A novel strategy has recently emerged for inhibiting Aβ aggregation using inhibitors that can covalently bind to the peptide [14, 15]. Transition metal ions, which are highly concentrated in amyloid plaques, are known to exacerbate the formation of Aβ aggregates [16, 17]. Selectively occupying the metal binding site on Aβ (the imidazole N-donor of histidine residues) and preventing metal coordination can alter metal binding activity, effectively changing Aβ-metal interactions and rendering the peptide nontoxic. With this strategy in mind, complexes of various transition metals, copper(II) [18], platinum(II) [14, 15, 19, 20], and ruthenium(II) [21], have been designed to bind Aβ peptides and inhibit their aggregation. Although promising, a great number of these metal complexes suffer from many drawbacks, such as being more complex, therefore more difficult to manufacture. Furthermore, some of them exhibit a poor blood- brain barrier (BBB) permeability, while others exert toxic effects barring them from clinical application in AD treatment. To overcome these limitations, a range of nanomaterials for inhibiting Aβ aggregation have been employed, providing novel opportunities to intervene in the pathology of the disease [22–24]. However, most of these nanomaterials exert their inhibition effects via noncovalent interactions with Aβ, which makes them unsuitable for application in complex biological systems. Thus, there is still an urgent need for designing nanoinhibitors able to covalently bind Aβ. Graphitic carbon nitride (g-C3N4) is the most stable allotrope of carbon nitride under ambient conditions, and has attracted great interest because of its semiconductor properties [25–29]. Since first reported, g-C3N4 has been applied in many areas; however, it is mostly used in fields related to photocurrent, photoreactivity, and electrocatalysis [30–32]. Recently, 2D ultrathin g-C3N4 nanosheets, which possess a higher surfacearea-to-volume ratio compared to bulk g-C3N4, have been employed as fluorosensors in bioimaging

applications, due to their properties of high fluorescence, excellent biocompatibility, and nontoxicity [33, 34]. Although significant effort has been devoted to the goal, biomedical applications of g-C3N4 nanosheets are still limited. It has been recently demonstrated that the surface functionalities of g-C3N4, i.e., NH2/–NH–/=N–, are well-characterized ligands exhibiting high adsorption capacity for metal ions through chelation or redox reactions [35, 36]. We combined the advantages of the g-C3N4 nanosheets with those of metal complexes to fabricate platinum(II)-coordinated g-C3N4 nanosheets (g-C3N4@Pt) able to inhibit Aβ aggregation. In our study, Aβ40 was chosen as the protein model. Aβ40 is the most common isoform of Aβ, and has been widely used for inhibitor screening in vitro [22, 23]. As reported previously [33], g-C3N4 was found to adsorb molecules containing aromatic groups through π–π stacking; this was also true for Aβ40. Thus, the aromatic rings in g-C3N4@Pt nanosheets can bind the peptide through noncovalent π–π interactions, and the presence of the positively charged Pt2+ in the nanosheets should strengthen the binding by electrostatically interacting with the overall negatively charged Aβ40 peptide [37,38]. Thereafter, these noncovalent interactions promote the coordination of Aβ40 with the g-C3N4@Pt nanosheets, resulting in the fast platination of the Aβ40 peptide. More importantly, with a narrow band gap of 2.7 eV, g-C3N4 achieved functionality as a stable photocatalyst for the evolution of H2 and O2 via water splitting, and for the degradation of organic pollutants under visible light irradiation [39–42]. Futhermore, g-C3N4@Pt inherited the photocatalytic activity of g-C3N4, and can photooxidate Aβ40. To our knowledge, this is the first study where g-C3N4@Pt nanosheets are shown not only to act as potent inhibitors of the aggregation and the synaptotoxic effects of Aβ via coordination with the peptide but also to serve as photocatalysts for oxygenation of Aβ40 under visible light, transforming the highly aberrant Aβ40 to less toxic forms at the disease site (Fig. 1).

2 2.1

Experimental Materials

Dicyandiamide

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(C2H4N4),

potassium

platinum

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Figure 1 Schematic representation of g-C3N4@Pt that was designed to be used for AD treatment. The g-C3N4@Pt nanosheets not only act as potent inhibitors for Aβ40 aggregation and synaptotoxic activity via binding to the peptide but also as photocatalysts for oxygenation of Aβ40 under visible light, transforming the toxic, aberrant Aβ40 to less toxic forms at the disease site.

chloride(II) (K2PtCl4), and cis-diammineplatinum(II) dichloride (Pt(NH3)2Cl2) were purchased from Merck (Darmstadt, Germany). Aβ40 was obtained from American Peptide (Thermo Fisher Scientific, Pittsburgh, PA). All these reagents were used as received without further purification. The deionized water (18.2 MΩ·cm) that was used for all experiments was obtained from a Milli-Q system (Merck, Darmstadt, Germany). 2.2

Measurements

The samples were characterized by X-ray powder diffraction (XRD) using a Japan Rigaku D/MAX X-ray diffractometer (Rigaku Corporation, Tokyo, Japan) equipped with graphite monochromatized highintensity Cu-Kα radiation (λ = 1.54178 Å). Fluorescence spectra were measured on a JASCO FP-6500 spectrofluorometer (JASCO Applied Sciences, Halifax, Canada). Atomic force microscopy (AFM) measurements were performed using a Nanoscope V multimode atomic force microscope (Veeco Instruments Inc., Plainview, NY). Transmission electron microscopy

(TEM) images were recorded using a TECNAI G2 20 high-resolution transmission electron microscope (FEI, Hillsboro, OR) operating at 200 kV. X-ray photoelectron spectra (XPS) were acquired on an ESCALab220i-XL electron spectrometer (Thermo Fisher Scientific, Pittsburgh, PA) using 300 W Al Kα as the excitation source. The binding energies obtained in the XPS spectral analysis were corrected for specimen charging by referencing C1s to 284.8 eV. 2.3

Fluorescence titrations

The intrinsic tyrosine fluorescence was examined as previously described [43, 44]. The concentration of Aβ40 was 3 μM. The excitation wavelength was 278 nm, and the emission intensity at 306 nm was used for analysis. To obtain the proper fluorescence intensity values, fluorescence data must be corrected for changes due to the inner filter effect; this term refers to artificial decreases in the fluorescence intensities that are caused by the attenuation of the excitation beam and the emission signal, which in turn are the result of the

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absorption by quencher and fluorophore. This effect is corrected using known absorbance values from the corresponding spectra [45, 46]. The fluorescence of the system can be corrected using the following equation

(Veeco Instruments Inc., Plainview, NY). 2.6 Circular dichroism (CD) measurements

where Fcorr and Fobs are the corrected and observed fluorescence intensity, respectively. Aex is the absorbance value at the excitation wavelength and Aem is the absorbance value at the emission wavelength.

CD spectra were collected at 37 °C with a JASCO J-810 spectropolarimeter (JASCO Applied Sciences, Halifax, Canada) using a 1-mm path length quartz cell. The parameters were controlled at 0.1 nm intervals, using a 4-second response, and each sample was an average of three scans at a speed of 5 nm·min–1 over the wavelength range from 200 to 250 nm.

2.4

2.7

Fcorr = Fobs log–1[(Aex + Aem)/2]

(1)

Thioflavin T (ThT) fluorescence measurements

The kinetics of Aβ40 aggregation were monitored by using the ThT fluorescence assay. ThT is a benzothiazole dye that can selectively bind to Aβ40 fibrils, ignoring Aβ40 monomers. Upon binding with β-sheet regions present in Aβ40 fibrils, ThT undergoes a characteristic spectral alteration, which results in a great increase in its fluorescence. When ThT is added to samples containing β-sheet-rich protein deposits, it fluoresces strongly with excitation and emission maxima at approximately 444 and 482 nm, respectively. The reaction is initiated immediately upon mixing β-amyloid with ThT into an aqueous environment and is completed within 1 min. Therefore, the amount of ThT fluorescence is dependent on the formation of amyloid fibrils. Fluorescence measurements were carried out with a JASCO FP-6500 spectrofluorometer (JASCO Applied Sciences, Halifax, Canada). The fluorescence signal (excitation at 444 nm) was recorded between 460 and 650 nm; 10 nm slits were used for both emission and excitation measurements. The peptide and the ThT concentrations were 1 and 10 μM, respectively. At different times, aliquots from the solution were taken for fluorescence measurements. The fluorescence data were corrected for fluorescence changes due to the inner filter effect. 2.5

Atomic force microscopy

In order to perform AFM measurements, samples were diluted with deionized H2O to a final concentration of 1 μM. Then 20-μL aliquots were transferred onto freshly cleaved muscovite mica and allowed to dry. Data were acquired in tapping mode, on a Nanoscope V multimode atomic force microscope

Mass spectrometry

The nanosheets (100 μg·mL–1, final concentration) and Aβ40 peptide monomers (100 μM, final concentration) were mixed and diluted to a volume of 100 μL with a 10 mM ammonium acetate solution. The samples were analyzed by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDITOF MS). 2.8

NMR spectroscopy

Samples for NMR were run in aqueous Tris buffer (10 mM Tris in 150 mM NaCl, pH 7.3) containing 10% (v/v) 2H2O. Samples containing Aβ40 were run at 0.1 mM. The nanosheets were incubated with Aβ40 at 37 °C for 2 h. NMR measurements were carried out at 5 °C in a 500-MHz AVANCE NMR spectrometer (Bruker, Billerica, MA) equipped with a triple channel cryoprobe. The concentration of the g-C3N4@Pt nanosheets was 100 μg·mL–1.

3

Results and discussion

3.1 Synthesis and characterization of the g-C3N4@Pt nanosheets The g-C3N4 nanosheets were directly prepared by ultrasonication-assisted liquid exfoliation of bulk g-C3N4 [31]. As shown in the XRD patterns, the g-C3N4 nanosheets only showed a relatively weak (002) peak, which suggests that the layered bulk g-C3N4 was successfully exfoliated after the ultrasonication treatment (Fig. S1 in the Electronic Supplementary Material (ESM)) [47–49]. Transmission electron microscopy imaging (Fig. S2(a) in the ESM) of the exfoliated product


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revealed the presence of free-standing nanosheets with a diameter of ‫׽‬60 nm. Their relative small size enables them to penetrate the BBB. The near-transparency of the nanosheets indicate their ultrathin thickness. In addition, AFM (Fig. S3(a)) demonstrated that the g-C3N4 nanosheets were separated well with an average topographic height of ~2.5 nm, which means that the exfoliated nanosheet was comprised of a small number (about seven) of C–N layers. These results are consistent with those reported by Xie and co-workers [34]. After incorporating Pt2+, the fluorescence of the g-C3N4 nanosheet diminishes (Fig. S4(a) in the ESM). Figure S4(b) in the ESM shows the UV/visible absorption spectra of g-C3N4 before and after Pt2+ binding. The g-C3N4 nanosheets had a strong peak in optical absorption at 315 nm, which is typical for a semiconductor. After binding with the [PtCl2-(DMSO)] complex, the absorption peak of g-C3N4 at 315 nm changed, which is a result of the π–π* type transition that takes place in the Pt-coordinated nanosheets (π–π* type transition in Pt complexes occurred at this region) [48]. The metal-to-ligand charge-transfer (MLCT) band in the spectrum of g-C3N4@Pt (567 nm) was also observed [48, 49]. Further investigation into the surface interactions of Pt2+ with g-C3N4 was conducted by XPS measurements. As shown in Fig. S5(a) (in the ESM), the main peak of the N1s region (at 398.3 eV) corresponded to sp2-hybridized aromatic N bonded to carbon atoms (C–N=C). The presence of tertiary nitrogen (N–C3) and amino groups carrying hydrogen (C–N–H or C–N–H2) is confirmed by the peaks observed at 399.6 and 400.7 eV, respectively [35, 36]. After coordination with the [PtCl2-(DMSO)] complex two new peaks appeared at binding energies (399.3 and 401.9 eV) higher than those of C–N=C and C–N–H, respectively (Fig. S5(b) in the ESM) [35, 36]. This result could be explained by formation of complexes between PtCl2 and amino/imino groups. The coordination of N atoms with Pt2+ resulted in a decrease in electron density of the nitrogen atoms and an increase in N1s binding energy. Furthermore, the binding energy positions of the Pt4f peaks in g-C3N4@Pt nanosheets were assigned to the Pt2+ oxidation states, in g-C3N4@Pt nanosheets (Fig. S5(c) in the ESM). The coordination with Pt2+ did not change the morphology of the nanosheet (Figs. S2(b) and S3(b)). Quantification of the

amount of Pt2+ incorporated into the g-C3N4 nanosheets was performed using inductively coupled plasma mass spectrometry (ICP-MS); the Pt2+ content in g-C3N4@Pt was found to be 0.12 g·g–1. To the best of our knowledge, the physiological stability of nanomaterials is a determining factor for its successful application in biological systems. We measured the fluorescence and UV/visible absorption spectra of g-C3N4@Pt in culture medium (DMEM supplemented with 5% (v/v) fetal bovine serum and 10% (v/v) horse serum). As shown in Fig. S6(a) (in the ESM), the fluorescence of the g-C3N4 nanosheets did not change in the presence of two sera. Moreover, the MLCT band in the optical spectrum of g-C3N4@Pt was again observed (Fig. S6(b) in the ESM). ICP-MS was used to assess whether these conditions caused Pt to be released. Furthermore, the release of Pt from the g-C3N4@Pt was also monitored by ICP-MS. Leaching of Pt from the nanosheet was negligible after 12 h in the culture medium, and amounted to less than 4% of loaded Pt (Fig. S6(c) in the ESM), demonstrating that the g-C3N4@Pt nanosheets are stable enough to be used in biological systems. 3.2 Effect of the g-C3N4@Pt nanosheets on the kinetics of Aβ40 fibrillation process Having confirmed the successful fabrication of the g-C3N4@Pt nanosheets, we next investigated their inhibitory effect on Aβ40 aggregates formation. In order to evaluate the potential of g-C3N4@Pt nanosheet as inhibitors of Aβ40 aggregation, we first assessed the ability of the nanosheets to bind the Aβ40 using NMR spectroscopy (Fig. S7 in the ESM). The 1H signals of the aromatic moieties (F19F20) of Aβ40 underwent remarkable changes upon the addition of g-C3N4@Pt, which clearly suggests that these groups are associated with the Aβ40-nanosheet binding (Fig. 2(b)). The g-C3N4@Pt nanosheets can specifically bind to the hydrophobic core fragment of Aβ40 through π–π stacking (Fig. 2(a)). In addition, spectra recorded before and after the addition of g-C3N4@Pt to solutions of Aβ40, revealed a strong perturbation of the peaks corresponding to the C4H and C2H of the imidazole side chains of His-6, -13, and -14 [14, 15], a fact consistent with the nanosheets (Pt2+) coordinating with these residues (Fig. 2(b)).


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Figure 2 Binding sites between g-C3N4@Pt nanosheet and Aβ40. (a) The g-C3N4@Pt nanosheets coordinate Aβ40. (b) 1H NMR spectra of Aβ40 before and after the addition of the nanosheets. The spectra showed that the nanosheets were able to perturb the resonances because of the C4H protons of the imidazole side chains of the histidine residues (see box centered at 7.15 ppm), and decreased the protons signals of F19 and F20 (signals centered at 7.2 ppm).

It has been proposed that the inability of cisplatin to inhibit Aβ40 aggregation may be due to the lack of an aromatic moiety, which would allow for noncovalent interactions with the peptide through π–π stacking [14, 15, 19]. In order to evaluate the role of g-C3N4, cisplatin conjugated g-C3N4 nanosheets (g-C3N4@cis-Pt) were employed. This nanosheet does not possess a leaving group and cannot coordinate with the peptide, making it suitable for the investigation of noncovalent interactions between platinum-based nanosheets and Aβ40. As shown in Fig. S8(a) (in the ESM), the fluorescence intensity of Aβ40 was strongly quenched with the addition of either g-C3N4@cis-Pt or g-C3N4@Pt, a result that demonstrates that both these nanosheets can interact with Aβ40. In contrast, the g-C3N4 nanosheet itself had a weaker quenching effect on the fluorescence of Aβ40. These results indicate that the platinum-based nanosheets have much higher binding affinities to Aβ40 than the g-C3N4 nanosheet. This difference may be a result of the ability of nanosheetincorporated Pt2+ to electrostatically interact with the overall negatively charged Aβ40 peptide (pI = 5.3) [19]. The interaction between g-C3N4@cis-Pt and Aβ40 was also confirmed by NMR (Figs. S6 and S8(b) in the ESM); Aβ40 peaks decrease and become broader after treatment with g-C3N4@cis-Pt, especially the ones that correspond to aromatic amino acids, such as His, Phe, and Tyr. This result suggests that aromatic residues in Aβ40 are involved in the interaction with g-C3N4@cis-Pt. The decreased peaks of Tyr in NMR spectra (Fig. S7 in the ESM), supported by the

fluorescence quenching results, clearly indicate that the g-C3N4@cis-Pt nanosheets can interact with Aβ40. However, with no free coordination sites, g-C3N4@cis-Pt is unable to perturb the peaks corresponding to the imidazole side chains of histidine in Aβ40, so only the effects of the noncovalent interactions can be seen (Fig. S8(b) in the ESM). To examine the influence of g-C3N4@Pt nanosheets on the fibril formation of Aβ40 (Fig. 3(a)), incubation solutions of Aβ40 with and without g-C3N4@Pt were prepared. The kinetics of fibrillation were monitored by using a dye-binding assay with ThT, the fluorescent spectrum of which can be increased with the growth of fibrils [44, 50, 51]. When fresh Aβ40 alone incubated at 37 °C, ThT fluorescence as a function of incubation time displays a sigmoidal increase (Fig. 3(b)), which is consistent with the nucleation-dependent polymerization model [44]. However, in the presence of g-C3N4@Pt, ThT fluorescence did not increase, indicating that Aβ40 amyloid formation was suppressed. After coincubation with Aβ40 for 7 days, the g-C3N4@Pt nanosheets were able to decrease ThT fluorescence in a dose-dependent manner (Fig. S9 in the ESM). A control experiment demonstrated that g-C3N4@Pt nanosheets do not directly influence the ability of ThT to fluoresce (Fig. S10 in the ESM). In contrast, the precursor complexes, cisplatin and [PtCl2-(DMSO)] did not exert any inhibition on amyloid formation (Fig. S11 in the ESM). Aβ40 oligomers, protofibrils and fibrils, all share the common β-sheet structure, which drives Aβ40 aggregation and toxicity.


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Figure 3 The g-C3N4@Pt nanosheets can inhibit Aβ40 aggregation and photooxidate the targeted Aβ40 peptide. (a) Schematic representation of the inhibition effect of g-C3N4@Pt nanosheets on Aβ40 aggregation. (b) Aggregation properties of the native and oxygenated Aβ40. A Tris buffer solution (pH 7.3) containing Aβ40 (50 μM) and g-C3N4@Pt (25 μg·mL–1), with or without former light irradiation, was further incubated at 37 °C, and the incubated samples were analyzed at arbitrary time points. (c) CD spectra of Aβ40 in the presence of g-C3N4@Pt nanosheet with or without light irradiation. The mixture of 50 μM Aβ40 and 50 μg·mL–1 nanosheets was irradiated with visible light for 15 min and then further incubated for 7 days. (d) The morphology of Aβ aggregates was analyzed in AFM images. (1) 50 μM Aβ40. (2) 50 μM Aβ40 in the presence of 50 μg·mL–1 g-C3N4@Pt nanosheet. (3) 50 μM Aβ40 in the presence of 50 μg·mL–1 g-C3N4@Pt nanosheet with irradiation. Details are given in the experimental section.

CD studies demonstrated that addition of g-C3N4@Pt nanosheets to an Aβ40 solution can inhibit structural transition from the native Aβ40 random coil to the β-sheet conformation (Fig. 3(c)). The inhibitory effect of g-C3N4@Pt nanosheets on Aβ40 aggregation was further studied through AFM (Fig. 3(d)). AFM has been widely used to study the morphology of Aβ40 amyloid fibrils [52–54]. Samples of Aβ40 alone form typical unbranched Aβ40 amyloid fibrils, longer than 1 μm. In the presence of the g-C3N4@Pt nanosheets, almost no obvious aggregates or fibrils were observed. To examine whether or not other proteins can affect the inhibitory activity of g-C3N4@Pt, we performed the inhibition experiment in mice cerebrospinal fluid (CSF). The accumulation of Aβ in CFS is used both as a diagnostic criterion and a therapeutic target with

respect to AD [52]. Fluorescence titration experiments and ThT assays demonstrated that in CNF, which contains various proteins, the g-C3N4@Pt nanosheets still possess the ability to inhibit Aβ aggregation via binding and adsorbing Aβ monomers (Fig. S12 in the ESM). These results suggest that Aβ can bind on the surface of the g-C3N4@Pt nanosheets even in the presence of other proteins. 3.3 Photooxygenation nanosheets

of

Aβ40

by

g-C3N4@Pt

Interestingly, due to the photocatalytic activity of g-C3N4, g-C3N4@Pt nanosheets displayed a higher inhibitory efficiency upon irradiation with visible light at room temperature for 15 min, especially under low concentration levels. The experimental data were


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evaluated using ThT assay, AFM, and CD study (Fig. 3 and Fig. S9 in the ESM). The light-triggered oxygenation prevented the conformational transition of Aβ40 from a random coil to a β-sheet. Furthermore, when oxygenated Aβ40 was added to a freshly prepared solution containing native Aβ40 and incubated for 7 days, fewer long amyloid fibrils were observed (Fig. S13 in the ESM), suggesting that the oxygenated Aβ40 species could not form cross β-sheets with native Aβ40 monomers. It is worth noting that the g-C3N4@Pt photooxygenation of Aβ40 oligomers, which have been reported as being more toxic than larger and highly structured fibrils, also proceeded efficiently. The potency of oligomer Aβ40 to aggregate and form fibrils was almost completely abolished by the oxygenation (Fig. S14 in the ESM). The photocatalytic activity of the g-C3N4@Pt nanosheets gives them an excellent advantage over traditional platinum(II) complexes [14, 19, 20] with respect to inhibiting Aβ40 aggregation. More importantly, the g-C3N4@Pt nanosheets are kept stable during the photocatalytic reactions (Fig. S15 in the ESM), indicating that performing photooxygenation of Aβ40 using these nanosheets is a feasible strategy. The reactive oxidation species (ROS) formed upon visible light irradiation have been reported to account for the high photocatalytic performance of the g-C3N4 nanosheets [39–42]. To confirm the presence of ROS in the photooxidase reaction, we made use of 2',7'dichlorofluorescein diacetate (DCFH-DA). This dye is nonfluorescent, but readily reacts with free radicals producing dichlorofluorescein (DCF), a fluorophore [55–57]. DCF fluorescence intensity correlates with the abundance of reactive oxygen radicals. Exposure of the dye to 20 μg·mL–1 g-C3N4@Pt nanosheets, which had been previously photoirradiated for 15 min, caused

a 214% increase in fluorescence intensity relative to samples of nonirradiated g-C3N4@Pt (Fig. S16(a) in the ESM). Keeping the irradiation time stable at 15 min, we performed additional experiments, which demonstrated that the g-C3N4@Pt has a dose-dependent effect on fluorescence; the intensity increases monotonically with the concentration of g-C3N4@Pt (Fig. S16(a) in the ESM). No changes in fluorescence were detected when DCFH-DA was treated with g-C3N4@Pt under dark conditions, or under photo-irradiation but in the absence of g-C3N4@Pt (Fig. S16(b) in the ESM). It has been demonstrated that the four amino acids, histidine, methionine, tyrosine, and tryptophan, are susceptible to photochemical oxidation due to their sulfur-residues or aromatic groups [58]. Tyrosine (the 10th residue of Aβ40 peptide) is known to be able to donate an e– during O2 activation in several enzymatic reactions [59]. Therefore, because photoirradiation of Αβ40 in the presence of g-C3N4@Pt leads to the photooxygenation of the peptide, tyrosine may be damaged during the photoirradiation. As tyrosine possesses an intrinsic fluorescence, its destruction can be detected by measuring the fluorescence produced by the peptide. In order to verify this statement, we determined the fluorescence intensity of Aβ40 in the present of g-C3N4@Pt with photoirradiation. As shown in Fig. S17 (in the ESM), the intensity of the fluorescence in photoirradiated samples containing Αβ40 and g-C3N4@Pt decreases faster compared to the one in nonirradiated samples, indicating that the fluorescent amino acid residues of the Aβ40 molecule were destroyed to some extent during photoirradiation. To gain more detailed information about the affected structures, oxygenated Aβ40 was analyzed by MALDI-TOF MS. As seen in Fig. 4, a +17 Da

Figure 4 Evaluation of photooxygenated Aβ40. MS spectra of (a) native Aβ40 and (b) oxygenated Aβ40. | www.editorialmanager.com/nare/default.asp

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modification appeared in the oxygenated samples, indicating that the oxygenation may have occurred at these four sites, probably at the Tyr10 residue, yielding 3,4-dihydroxyphenylalanine (DOPA) [60]. 3.4 g-C3N4@Pt cytotoxicity

nanosheets

rescue

Aβ-induced

We then studied the effects of metal complexes on Aβ40-induced cytotoxicity using the MTT assay [50, 51]. Our experimental model was Rat pheochromocytoma PC12 cells that were forced to undergo neuronal differentiation. The resulting cells have been demonstrated to be able to mimic the neurons in the brain and are more sensitive to neurotoxicity caused by Aβ40 aggregation than undifferentiated PC12 cells [61, 62]. As seen in Fig. 5(a), the presence of aged Aβ40 led to a decrease of 42% in cellular reduction of MTT. In the presence of g-C3N4@Pt, the cell survival rate increased to about 84%. Aβ40-g-C3N4@Pt samples pretreated with photoirradiation had an even lower cytotoxicity, as the survival rate rose to about 92%, taking the experimental errors into account. Furthermore, g-C3N4@Pt, with or without irradiation, prevented cell death in a dose dependent manner, indicating that the decrease of cytotoxicity was due to the g-C3N4@Pt binding to the Aβ40 (Fig. S18(a) in the ESM). As seen in Fig. S18(b) (in the ESM), g-C3N4@Pt nanosheets alone, in our experimental conditions and within the used concentration range, had little effect on PC12 cell viability. The possibility

of the g-C3N4@Pt nanosheets having an effect on the efficiency of the MTT assay was ruled out by a control experiment, which demonstrated that the signal remained unchanged in the presence of g-C3N4@Pt (data not shown). The generation of ROS by Aβ aggregates is one of the proposed mechanisms of AD pathogenesis [63], as it can cause an increase in oxidative damage to cellular components such as DNA, lipids and proteins [64]. Since the g-C3N4@Pt nanosheets can inhibit Aβ aggregation, we investigated the effect of the nanosheets on ROS production caused by Aβ40 aggregation in PC12 cells. As shown in Fig. 5(b), the nanosheets effectively suppressed ROS production, and this provides further explanation as to why the nanosheets were able to enhance cell viability. Although the g-C3N4@Pt nanosheets can produce a small amount of ROS upon visible light irradiation, the formed ROS prefer to oxygenate the Aβ40 bound to the surface of the nanosheets, and do not contribute to oxidative stress and cytotoxicity (Fig. 18(b)). As suitable candidates for AD treatment, the multifunctional nanosheets should cross the BBB. To determine whether g-C 3 N 4 @Pt can passively accumulate in the brain of living animals, we used ICP-MS to measure the amount of g-C3N4@Pt in the brain 6 h after an intraperitoneal injection. A significant level of Pt was found in the brain of the mouse that was treated with g-C3N4@Pt, compared to in the control group. The efficiency of g-C3N4@Pt accumulation in

Figure 5 Protection effects of g-C3N4@Pt nanosheets on Aβ40-induced cytotoxicity of PC12 cells. (a) Cell viability was determined using the MTT method. (b) Effect of the nanosheets on ROS production in PC12 cells. Cells were treated with aged Aβ40 at a concentration of 5 µM in the absence or presence of g-C3N4@Pt nanosheets. Data represents mean ± SEM of at least three different experiments. (1) Control, (2) Aβ40 fibrils, (3) Aβ40 samples pre-incubated with g-C3N4@Pt nanosheets, (4) Aβ40 samples treated with g-C3N4@Pt nanosheets upon irradiation and then incubated for 7 days, (5) oxygenated Aβ40 samples pre-incubated with native Aβ40 peptide. Control: Aβ40-untreated cells, [Aβ40] = 5 μM, [g-C3N4@Pt nanosheet] = 10 μg·mL–1. www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano

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the brain was about 1.62% ± 0.21%, which was obvious higher than that of [PtCl2(DMSO)] (Table S1 in the ESM), indicating that g-C3N4@Pt has the potential to cross the BBB. We also examined the biodistribution of g-C3N4@Pt nanosheets. As illustrated in Fig. S19 (in the ESM), the nanosheets mainly accumulated in organs of the reticuloendothelial system (RES), such as the liver and the spleen, which were quite similar to the in vivo distribution of many other nanomaterials applied in biomedicine [65, 66]. Although we did not examine the in vivo inhibitory effect of g-C3N4@Pt, overall our results clearly indicate that g-C3N4@Pt nanosheets could act as promising therapeutic agents for AD treatment.

4 Conclusion In the present study, we have demonstrated that g-C3N4@Pt nanosheets can effectively inhibit Aβ aggregation through multiple mechanisms: noncovalent interactions, platinum coordination, and photooxygenation. These noncovalent interactions promote the binding of Aβ to this nanosheet, resulting in a fast platination of the peptide. This can effectively impede the intermolecular interactions between Aβ molecules, thereby inhibiting Aβ aggregation. Furthermore, due to its high photocatalytic activity, g-C3N4@Pt can also oxygenate Aβ upon visible light irradiation under physiological conditions. Through oxygenation, the aggregation potency and neurotoxicity of Aβ were markedly attenuated. More importantly, the accumulation of oxygenated Aβ can further inhibit the aggregation of native Aβ peptide, providing an enhanced inhibition effect. With the ability to cross the BBB and its good biocompatibility, g-C3N4@Pt nanosheet is a promising candidate for inhibiting Aβ aggregation. Our work may serve as an example of efficiently developing novel multifunctional nanomaterials used for the treatment of AD.

Acknowledgements This work was supported by the National Basic Research Program of China (Nos. 2012CB720602 and 2011CB936004), and the National Natural Science

Foundation of China (Nos. 21210002, 21431007, and 21533008). Electronic Supplementary Material: Supplementary material (experimental section and supporting figures) is available in the online version of this article at http://dx.doi.org/10.1007/s12274-016-1127-5.

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