Enhanced Photoresponsive Graphene oxide-modified g-C3N4 for

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Biological and Medical Applications of Materials and Interfaces

Enhanced Photoresponsive Graphene oxidemodified g-C3N4 for Disassembly of Amyloid # Fibrils Jie Wang, Zhongyang Zhang, Hongxing Zhang, Chenglong Li, Menglin Chen, Lei Liu, and Mingdong Dong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b10343 • Publication Date (Web): 11 Dec 2018 Downloaded from http://pubs.acs.org on December 12, 2018

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ACS Applied Materials & Interfaces

Enhanced Photoresponsive Graphene oxide-modified g-C3N4 for Disassembly of Amyloid β Fibrils Jie Wang,†,‡ Zhongyang Zhang,‡ Hongxing Zhang,† Chenglong Li,† Menglin Chen‡, Lei Liu,†,‡* Mingdong Dong‡,* †Institue

for Advanced Materials, School of Material Science and Engineering, Jiangsu University,

Zhenjiang 212013, China ‡Interdisciplinary

Nanoscience Center (iNANO), Aarhus University, DK-8000 Aarhus C,

Denmark Supporting information for this article is given via a link at the end of the document. KEYWORDS ： Amyloid β disassembly, Photo-degradation, Atomic force microscopy, Quartz crystal microbalance, Neurodegenerative disease

ABSTRACT Protein misfolding and abnormal self-assembly lead to the aggregates of oligomer, fibrils, or senior amyloid β plaques, which is associated with the pathogenesis of many neurodegenerative diseases. Progressive cerebral accumulation of amyloid β-protein (Aβ) was widely proposed to explain the cause of Alzheimer's disease, for which one promising direction of preclinical study is to convert the pre-formed β-sheet structure of amyloid β aggregates into

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innocent structures. However, the conversion is even harder than the modulation of amyloidosis process. Herein, graphene oxide/carbon nitride (GO/g-C3N4) composite was developed as a good photocatalyst for irreversibly disassembling the amyloid β aggregates of Aβ (33-42) under UV. Quartz crystal microbalance, circular dichroism spectrum, atomic force microscopy, fluorescent spectra, and mechanical property analysis were performed to analyze this photo-degradation process from different aspects for fully understanding the mechanism, which may provide an important enlightenment for the relevant research in this field and neurodegenerative disease study.

1. INTRODUCTION Amyloid β protein misfolding and abnormal self-assembly into amyloid β aggregates is associated with many types of diseases, such as Alzheimer’s diseases (AD), one of the most prevalent neurodegenerative diseases. The pathogenesis of AD can be characterized by the accumulation of extracellular amyloid β fibril plaques in the brain.1 The formation of senile plaques is a complex self-assembly process of amyloid β-protein (Aβ). In the process Aβ peptides abnormally aggregate from their soluble unstructured monomers into β-sheet rich oligomers, protofibrils and insoluble amyloid β fibrils.2-4 On the other hand, impaired clearance of Aβ (e.g. Aβ40 and Aβ42) in the AD patients could also result in the over accumulation.5 Despite of the unclear molecular mechanism of pathologic process, in principle, a modulator which could reduce, inhibit, or even reverse the amyloid β aggregation of β-sheet-rich structure is promising in pathogenesis investigation and potential therapeutic treatment for neurodegenerative diseases. Specifically, any intermediate during the process of fibrillogenesis can be considered as an attractive therapeutic target to treat AD.


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To the best of our knowledge, many studies on interfering the formation of amyloid β fibrillization have been reported,6-9 while several types of modulators for the assembly of Aβ peptides have been developed, including organic molecules, well-designed peptide motifs10-12 and inorganic nanoparticles (such as fullerene,13 quantum dots,14 carbon nanotube (CNT)15-17 and graphene oxide (GO)18-19). Especially, GO shows strong modulation effect on amyloid β assembly. Although those previously reported amyloid β modulators could mostly inhibit the aggregation process of peptide monomers, they were still unable to disassemble or degrade mature amyloid β fibrils deposits. Meanwhile, several enzymes including neprilysin20, insulin-degrading enzyme21 and endothelinconverting enzyme,22 etc. have been identified the ability to degrade Aβ(33-42) monomers, but the complicated structure and expensive cost hinder their practical use in large-scale. More importantly, most of these enzymes are relatively insensitive to amyloid β oligomers, which exactly contribute to the major cytotoxicity of amyloid β proteins in human body.23 Therefore, it is critical to develop new methods to enhance the clearance of amyloid β plaques for the pathogenesis and treatment research. Photo-degradation is mainly used in disinfection technologies and pollutants treatment as well. Recently, degradation of amyloid β proteins such as Aβ42 were achieved by using polyoxometalates,23 fullerene derivatives,24-25 light-activatable organic molecules,26 or porphyrin derivatives27 under photoirradiation. Laser irradiation could also seperately serve as a means to decrease the pre-formed amyloid β fibrils.28 Graphitic carbon nitride (g-C3N4), as an intriguing visible light photocatalyst, possesses a two-dimensional structure, tunable electronic structure, excellent chemical stability and good stablility in a wide pH range under light irradiation.29 Recently, g-C3N4 and its derivatives have been used in amyloid β researches.30-31 As demonstrated before, GO could adsorb plenty of amyloid β peptide molecules so as to inhibit the aggregation of


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amyloid β peptide due to its high surface area and abundance of functional groups.18-19, 32-33 We suppose that when combined with g-C3N4 in a composite, GO could act as an amyloid β collector and g-C3N4 could serve as the cleaner; the cooperation of them could be used to eliminate the preexisting amyloid β aggregates. It is noteworthy that the efficiency of photo-degradation in the composite could remain high, because of the heterojunction between GO/g-C3N4 helps to separate photoexcited electron-hole pairs.32 Here, as shown in Scheme 1, we utilized GO/g-C3N4 composite to photo-degrade the aggregates of Aβ(33-42) that is the key fragment of Aβ. High-resolution atomic force microscopy (AFM), quantitative quartz crystal microbalance (QCM), fluorescent spectra (FL), circular dichroism (CD) spectra and polyacrylamide gel electrophoresis (PAGE) and AFM quantitative nanomechanical property mapping (QNM) analysis were performed to investigate the disassembly and photodegradation of Aβ (33–42) aggregates. The results showed GO/g-C3N4 could be used to eradicate the amyloid β aggregates. Scheme 1. The illustration for the degradation of amyloid β aggregates by GO/g-C3N4 under light irradiation.


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2. EXPERIMENTAL SECTION 2.1 GO/g-C3N4 preparation. We prepared GO suspension by using a modified Hummer’s method.33-34 Bulk g-C3N4 powder was prepared by sinterring melamine at 600 ℃ for 2 h in air. Then we prepared the ultrathin g-C3N4 nanosheets by liquid exfoliating of the above-mentioned bulk g-C3N4. For example, 5 mg of bulk g-C3N4 powder was added into 2 mL water and sonicated for 20 hours. After that the suspension of g-C3N4 was centrifuged to remove unexfoliated particles.35 GO/g-C3N4 composite was synthesized via sonochemical approach. The mixture suspension containing 4 mg GO and 20 mg g-C3N4 nanosheets was ultra-sonicated for 10 hours and then dried


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at 60 ℃. The characterization was done by AFM/TEM/XRD/XPS/FTIR/UV-VIS shown in the figure S1-S4 of supporting information. 2.2 Preparation of Aβ(33-42) solution and photo-degradation process by GO/g-C3N4. To prepare Aβ(33-42) solution, 2 mg Aβ(33-42) was dissolved in 1 mL 1,1,1,3,3,3-hexafluoro-2propanol (amino acid sequence: NH2-GLMVGGVVIA-COOH). Later the solution was sonicated and vortexed for 3 times and finally incubated for 10 hours at 25 °C on a thermo-shaker to obtain the Aβ(33-42) HFIP solution. To obtain Aβ(33-42) aggregates, 100 μL of Aβ(33-42) HFIP solution was sealed with a patch of parafilm into a 1.5 mL centrifuge tube for 2 hour at 25 °C in a vacuum drying oven to eradicate the HFIP solvent. Then 200 μL Milli-Q water was added into the tube to dissolve the peptide film on the tube wall and after that the solution was sonicated and vortexed for several times until the solution was clear. The clear Aβ(33-42) water solution was transferred onto a thermo-shaker and incubated at 37 °C at 350 rpm/min for 10 hour to produce Aβ(33-42) fibrils. Finally the pre-formed Aβ(33-42) aggregate was mixed with GO/g-C3N4 composite and applied in the photo-degradation experiment under UV irradiation with a wavelength of 365 nm (FUV 6 BK, Suzhou BANGWO Co., Ltd., China). 2.3 Atomic Force Microscopy. After photo-degradation experiment under UV irradiation, 20 μL mixture suspension of Aβ(33-42) and GO/g-C3N4 composite was taken out and deposited on freshly cleaved mica for 10 min. All AFM measurements were performed on an apparatus (MFP-3D-SA, Asylum Research, Santa Barbara, USA) with a silicon cantilevers (the nominal spring constant at 26 N/m, OMCL-AC160TS-R3, Olympus). In the experiment, the scan rate was 1 Hz. The AFM QNM measurement was accomplished on an equipment (Multimode 8, Bruker Co., Ltd., USA) and we utilized AFM tips (Tap525A, P/N MPP-13120-10, Bruker, USA). In the


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process of QNM study, we used a standard sample of Sapphire-15M to calibrate the Deflection sensitivity and another standard sample of PSFILM (Polystyrene) to calibrate the parameter of tip. 2.4 Raman Spectroscopy. Raman measurements were performed on a Raman apparatus at a wavelength of 532 nm (DXR, Thermo Fisher, USA). In experiment, 50X objective len was used to focus the laser beam with a power of 10 mW. 2.5 Circular dichroism (CD) spectroscopy. CD spectra measurement was accomplished on a apparatus (JASCO, Hachioji City, Japan) which had a PTC-348W1 model. For all the measurements, samples were put into a 0.1 cm quartz cuvette and at a scan speed of 100 nm/min and with a slit-width of 2 nm. The obtained signals in the range of 190–250 nm were analyzed with the signal from Milli-Q subtracted as the baseline. In all the measurements, the volume of sample was 300 μL. 2.6 Quartz crystal microbalance (QCM). The QCM measurements were performed on a equipment (Q-Sense E4, Biolin Scientific, Sweden). We let the sample solution flow through a QSense window module under UV irradiation and the flow rate was set at a speed of 50 μL/min. The hybrids of Aβ(33-42) aggregates and GO/g-C3N4 adsorbed on the QCM chip and the shift of mass of disassembled amyloid aggregates was in realtime monitored by the QCM frequency shift and energy dissipation signals. 2.7 The X-ray diffraction (XRD). The XRD characterization was carried out on a apparatus (D/MAX-2500PC, Rigaku, Japan) which uses Cu Ka radiation (l = 1.5406 Å). In the experiment, the scan angle is in the range of 5°

Enhanced Photoresponsive Graphene oxide-modified g-C3N4 for

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