Rapid Dissolution of Amyloid Î² Fibrils by Silver Nanoplates

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Rapid dissolution of amyloid # fibrils by silver nanoplates Swathi Sudhakar, and Ethayaraja Mani Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b00080 • Publication Date (Web): 27 Apr 2019 Downloaded from http://pubs.acs.org on April 27, 2019

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Rapid dissolution of amyloid β fibrils by silver nanoplates†

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Swathi Sudhakar and Ethayaraja Mani*

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Polymer Engineering and Colloid Science Laboratory, Department of Chemical Engineering, Indian

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Institute of Technology, Madras-600 036, India

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ACS Paragon Plus Environment

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ABSTRACT

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Plaques of amyloid beta (Aβ) protein is associated with neurodegenerative diseases, and

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preventing their formation and dissolution of plaques are essential to the development of

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therapeutics. In this study, silver triangular nanoplates (AgTNP) are shown to dissolve mature

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Aβ fibrils due to their plasmonic photothermal property. Mature Aβ fibrils treated with

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AgTNP under near infra red (NIR) illuminated conditions are dissolved in less than 1 hr

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while equal concentration of silver spherical nanoparticles (AgSP) took about 70 hr.

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Concentration of the fibrils decreased from 10 µM to 0.3 µM upon treating the amyloid

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fibrils with AgTNP under NIR. AgTNP are also shown to prevent the formation of Aβ fibrils

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by selective binding to the positively charged amyloidogenic sequence of Aβ monomer. The

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kinetics of inhibition by AgTNP follows the predictions of the detailed kinetic model

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(Ramesh et al., Langmuir 2018, 34, 4004−4012). The kinetics of dissolution and inhibition

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are characterized by Congo red/ThT assay, Transmission Electronic Microscopy (TEM),

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Atomic Force Microscopy (AFM) and Attenuated Total Reflectance Fourier Transform -

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Infrared Spectroscopy (ATR-FTIR). Cell viability studies on SH-SY5Y and BE-(2)-C cells

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using MTT and LDH assy show that the viability of the cells increased from 33% to 70% on

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treating the cells with AgTNP-incubated Aβ fibrils compared to the mature Aβ fibrils. The

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study provides new insights to design plasmonic nanoparticle-based therapeutics to cure

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neurodegenerative diseases.

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INTRODUCTION

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Escalation of Alzheimer disease (AD) among aging people poses a great challenge to health

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care system and policies due the lack of therapeutics for rapid recovery and cure to

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AD.1,2,3AD is one of the most complicated neurodegenerative disorders and characterized by

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loss of cortical neurons, synaptic dysfunction associated with disruption of the neural circuit

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communications leading to the loss of cognitive functions and memory.4,5 Even though the

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molecular mechanisms leading to AD are not fully understood and alternate hypotheses have

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been proposed, massive evidence suggest that deposition of amyloid-beta (Aβ) aggregates

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(fibrils and plaques) on external surface of neurons6,7 and the presence of neurofibrillary

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tangles within the neurons are considered as two critical steps in the pathogenesis of the

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disease.8,9

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Several anti-AD agents, such as small molecules,10 surfactants,11 Cu/Zn chelators,12

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curcumin,13 transhinones,14 Rifampicin,15 hydroxyl indole derivatives,16

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peptides18 and metal oxides19 show inhibitory effect on the formation of Aβ fibril, but they

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are sluggish in dissociating mature fibrils. Numerous kinds of nanoparticles such as gold,

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silver, graphene, carbon nanotubes with extensive applications in biomedical areas such as

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cellular imaging,16 and tumour therapy,20,

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therapies. These studies reported that a particular nanostructure, especially spherical particles

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(such as inorganic nanoparticles and polymeric nanomaterials), decreased the conversion of

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Aβ monomers into fibrils.22,23 Contrarily, some studies have reported that nanoparticles act as

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an catalysts and accelerate the process of fibril formation.24 Whether a given nanoparticle

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inhibit or accelerate the formation of fibrils depend on size, charge, shape, concentration and

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surface functional groups.

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polymers,17

have been considered in developing anti-AD

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A few studies have reported that spherical nanoparticles are capable of dissolving mature

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fibrils very slowly.25 One of the strategies to expedite the dissolution of mature Aβ fibrils can

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be the use photo-thermal property of anisotropic nanostructures, taking cue from cancer

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photo-thermal therapy. For instance, Lin et al26 reported that femtosecond laser-irradiated

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gold nanorods destructed Aβ fibrils by breaking the fibrils into shorter fragments. Sudhakar

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et al27 reported on the role of charge and aspect ratio of gold nanorods in dissolving Aβ fibrils

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and showed the time scale of dissolution was about 25 hr under NIR illumination and 70 hr

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without illumination. Yagi et al28 reported that sole laser irradiation destructed the mature

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fibrils (without nanoparticles), but irradiating the sample in the growth stage of fibrils

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enhanced the rate of formation, thus demonstrating the time of irradiation is crucial. Yang et

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al29 reported that graphene sheets destroyed the amyloid fibrils by penetrating and extracting

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a large number of peptides from the preformed fibrils. Thioflavin-modified graphene oxide

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(GO), under near infrared (NIR) laser irradiation, is shown to dissolve Aβ deposits in mice

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cerebrospinal fluid.30

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While gold nanorods and GO under NIR illumination have been used to dissolve mature Aβ

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fibrils, the photo-thermal properties of silver triangular nanoplates (AgTNP) have largely

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been untapped in this context. AgTNP show near infrared absorption that is tunable by the

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edge length. The ease of synthesis of AgTNP and superior absorption properties in NIR

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wavelengths make AgTNP an ideal therapeutic agent for AD. We note that irradiated AgNTP

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has been used to kill E. coli cells31 and cancer cells32, thanks to their photo-thermal

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properties. The aim of this study is to design biocompatible AgTNP in nanomolar

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concentrations to inhibit the Aβ fibril formation and to destroy the mature Aβ fibrils rapidly.

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In this report, we show that NIR-irradiated AgTNP dissolves mature Aβ fibrils in about 1 hr

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while equal concentration of spherical silver nanoparticles (AgSP) and NIR-irradiated gold

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nanorods take 70 hr and 25 hr, respectively. This tremendous reduction of time scale in

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dissolving Aβ fibrils may pave ways to use AgTNP as a potential therapeutic agent for

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patients with advanced stage of AD. Further, we also show that AgTNP can inhibit the

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formation of Aβ fibrils if treated in the incipient stage of fibril formation. We use a

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combination of experimental tools such as Congo red absorbance assay, transmission electron

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microscopy, optical microscopy, atomic force microscopy, attenuated total reflectance

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Fourier transform infrared spectroscopy (ATR-FTIR) to demonstrate the inhibitory effect and

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dissolution ability of irradiated AgTNP. To the best of our knowledge, this is the first study

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that investigates AgTNP as a potential anti-AD agent.

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EXPERIMENTAL SECTION

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Silver nitrate was purchased from Alfa Aesar, ascorbic acid, PVP, Congo red were purchased

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from Sigma-Aldrich. Amyloid β (Aβ[1-40]) protein with >98% purity was purchased from

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Sigma. Zeta potential of protein monomer was measured using dynamic light scattering

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(DLS, Horiba and zetasizer).

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Preparation of Aβ fibrils

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The amyloid fibrils were grown by following the method mentioned elsewhere.4 The fibrils

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were synthesized by dissolving 1 mg of Aβ monomers in 600 μL of 0.02% ice cold ammonia

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solution and the stock solution is stored at −80 °C. Then 50 μL of the stock solution was

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diluted to 50 μM with phosphate buffer. The solution was incubated at 37 °C for 24 h at 250

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rpm and stored at 4 °C. To prepare second generation fibrils 5 μL of monomeric Aβ protein

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was added to the 2 μL of the well grown fibrils and diluted to 50 μL with phosphate buffer

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solution. Then the mixture was incubated at 37 °C for 10 h and stored in 4 °C for further use.

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Congo red assay

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Synthesis of negatively charged AgSP and AgTNP and their characterization are given in

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detail in the Supporting Information (Figure S1 and S2). The AgSP and AgTNP solutions of

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concentration in the range of 3 - 30 nM were taken and mixed with Aβ solution in a 1:1

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volume ratio. 100 µL of 10 µM protein samples was mixed with 100 µL of 30 µM Congo red

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prior to absorbance measurement.43 Absorbance measurement was conducted using a

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multiplate reader (JASCO) at room temperature. The concentration of the Aβ fibrillar species

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in peptide solutions was determined every 1h up to 40 h using the following equation33

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[fibrillar Aβ] = A540/4780 -A405/6830

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Where [fibrillar Aβ] is the concentration of Aβ fibril. A540 and A405 are the absorbance of the

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Congo red with peptide sample and the Congo red alone in the sample, respectively. Further

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Figure S3 in the Supporting Information shows the absorbance of aqueous solution of

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AgTNP and Congo red dye. It can be seen that the absorbance value of AgTNP is

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insignificant compared to that of Congo red at 540 nm, which corresponds to the calculation

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of fibril concentration. AgTNP did not influence the detection of amyloid fibrils by Congo

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red. Additionally, NIR irradiation did not change the absorption of Congo red.

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Atomic force microscopy (AFM) and optical microscopy.

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For AFM imaging, 3 μL of the sample was deposited on clean glass surface (2 × 2 cm2). The

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droplet was allowed to dry in room temperature and all the measurements were done using

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AFM (Asylum MFP-3D classic AFM) in AC and tapping mode within 1 h after sample

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preparation. The same sample preparation was followed for optical imaging of the samples

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and visualised using optical microscopy (Leica DMI3000B).

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Transmission electron microscopy (TEM).

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About 5 μL of AgSP and AgTNP treated fibrils and 5 μL of AgSP and AgTNP solutions were

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dropped on a carbon-coated copper grid (icon) and dried under room temperature for 20 min.

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Then the fibrils were further negatively stained with 0.05% of uranyl acetate for another 1

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min. The sample was washed with water, dried, and then visualized by using a Technai G2

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transmission electron microscope (FEI) with an acceleration voltage of 200 kV.

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ATR-FTIR measurements.

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The secondary structure content and conformational changes that occurred in Aβ due to the

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AgSP and AgTNP was characterized by ATR FTIR (Bruker) spectroscopic technique. At

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different time intervals, 20 μL of the sample was collected and placed in the sample holder

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with the help of a capillary tube. The spectra were read in the wavenumbers ranging from

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1600 to 1700 cm−1.

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Cell viability assay.

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The MTT (3-[4,5-Dimethy-lthi-azol-2-yl]-2,5-diphenyl-tetrazdium bromide) assay was

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employed to examine the cytotoxicity. The human neuroblastoma

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(ATCC#CRL-2266) and BE-(2)-C (ATCC#CRL-2268) were incubated at 37 ° C under 5%

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CO2 and were cultured in RPMI media with 10% fetal bovine serum (FBS) in a humidified

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camber. A total of 8 × 104 cells were seeded overnight in the growth medium in a

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polystyrene 96-well plate. After discarding the growth medium the cells were washed once

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by RPMI media without FBS. Then the FBS-free RPMI of about 50 μL was added into

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each well. Followed by that the cells were treated with 25 μL of the end-point products.

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Cells were then incubated for about 6, 24, and 48 h. Subsequently 5 μL of MTT solution, 4.5

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mg/mL in Dulbecco’s phosphate-buffered saline was added into each well and incubated for

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another 4 h. After the incubation the media was discarded and Dimethyl sulfoxide (DMSO)

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cells like SH-SY5Y

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was used to lyze the cells until the purple crystals were fully dissolved and the absorbance

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was measured at 570 nm by a ELISA multimode microplate reader (Biotek, Epoch). Cell

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viability calculation was done using a standard protocol. Lactate dehydrogenase (LDH) assay

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test also performed in addition to MTT assay to confirm the results. In LDH assay about 50

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µL of the cell cultures treated were mixed with 50 µL of the LDH assay solution and

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incubated in the dark for 30 min, and the absorbance was read on a plate reader at 510 nm.

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Cell viability assays were represented as means ± Standard Error Mean (SEM) of four

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independent experiments in triplicate. Comparisons between the control cell group and cell

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group treated with the AgTNP and AgSP were performed using analysis of variance

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(ANOVA). The statistical data was analysed with the help of KaleidaGraph version 4.1.3

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Synergy software. The difference in means between the test control cell group and the

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corresponding cells treated with AgTNP, AgSP, and AgTNP with NIR was statistically

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significant for all the time periods with the p-value is around 0.0012 which is less than 0.01.

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NIR laser irradiation.

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In the NIR irradiation experiments, 200 μL of Aβ fibrils were added with 30 nM of AgTNP

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and irradiated with near infrared laser (Insight DeepSee Laser System, 800 nm) with power

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density 200 mW/mm2. The solution was then transferred to a 10 mm × 10 mm quartz cuvette.

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And the samples were irradiated for 1 min with the laser at 800 nm with an unfocused spot

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diameter of about 2.0 mm. The quartz cuvette was scanned in a way that the whole sample

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would be exposed to irradiation.

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RESULTS AND DISCUSSION

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Photothermal dissolution of Aβ fibrils by AgTNP

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Silver triangular nanoplates (AgTNP) and silver spherical nanoparticles (AgSP) are

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synthesized and characterized (Figure S1 and S2 in Supporting Information) to study the

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dissolution of mature fibrils. AgTNP of edge length 70 ± 8 nm and AgSP of diameter 22 ± 5

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nm are used in the experiments. Both nanoparticles are capped with poly(vinyl) pyrrolidone

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[PVP] polymer to ensure colloidal stability.34,35 The zeta potential of AgSP and AgTNP are

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found to be -40 ± 0.8 mV and -41 ± 1.4 mV, respectively. Mature fibrils grown in control

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experiments are incubated with different concentration of AgTNP or AgSP and the fibril

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content is quantified using Congo red assay. Computational studies show that Congo red

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binds parallel to the fibril axis.36,37 The UV-VIS absorbance of Congo red at 540 nm is

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proportional to the amount of fibrils (see Experimental section). Up to 2.64 moles of Congo

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red is required to bind to 1 mole of amyloid fibrils (See Figure S4-S6 for details of

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calibration). All the experiments are carried out with 30 µM of Congo red dye for 10 µM of

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amyloid protein, so that Congo red dye is in excess compared to the stoichiometric

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requirement.

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Figure 1a shows the main result of our study. Mature fibrils are themselves quite stable and

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the corresponding Congo red absorbance of pure mature fibrils remains nearly constant with

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time. Irradiating the sample with NIR did not show any significant change in the Congo red

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absorbance suggesting the NIR treatment alone did not cause any reduction in the fibril

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content. However, when the sample is treated with AgTNP of different concentration and

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irradiated with NIR, remarkable reduction in the fibril content at very short time is observed.

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For instance, the sample treated with 30 nM of AgTNP showed nearly complete dissolution

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of mature fibrils under illuminated conditions within 1 hr. Nevertheless, samples treated with

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lesser concentration of AgTNP dissolved mature fibrils at slightly longer times under

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illuminated conditions. These results imply that the NIR absorption by AgTNP produces 9


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local hot spots and increases temperature around the mature fibrils and facilitate faster

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dissolution. This mechanism is similar to the destruction of cancerous cells by photothermal

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effect of gold nanorods.38,39,40,41 For this effect to be efficient, protein fibrils and AgTNP

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should interact and this is ensured by using negatively charged AgTNP in our study. It is

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well-known that negatively charged nanomaterials (spheres, rods) electrostatically attract Aβ

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fibrils.26,29 We note that AgTNP did not undergo any morphological changes upon irradiation

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of continuous NIR for 1 min at 200 mW/mm2, although Yang et al38 reported that gold

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nanorods have been interconverted into spheres on passing femtosecond IR laser for 10 min

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at 250 mW/mm2. This is probably due to longer exposure time than that of the current study.

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The longitudinal surface plasmon resonance (LSPR) of AgTNP is attributed to the expedition

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of dissolution of fibrils. As shown in Figure S2, the LSPR of AgTNP correspond to one at

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about 400 nm (out-of-plane dipole resonance) and the other centered around 810 nm (in-

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plane dipole resonance). Therefore, a laser operating at 800 nm should be absorbed by

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AgTNP, thus producing a local hot spot or hyperthermic condition. The electrons present in

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the AgTNP absorbs the incident photons coupling to the lattice phonons resulting in heat

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generation where the light energy is transferred into heat. When mature fibrils and AgTNP

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are incubated together and when a 800 nm laser is irradiated, the dissolution process can be

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expedited due to increased thermal energy around the AgTNP. Thus, after irradiating the

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sample with NIR-laser for 1 min at power densities of 200 mW/mm2 at 800 nm wavelength,

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nearly all the fibrils are dissolved within 1 h (Figure 1a). As a negative control, the sample

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irradiated with NIR-laser in the absence of AgTNP did not show any sign of dissociation of

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fibrils, thus demonstrating the importance of photo-thermal properties of AgTNP in the

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dissolution kinetics of Aβ fibrils.

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While the combination of AgTNP and NIR illumination rapidly dissolved the mature fibrils

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in about 1 hr, AgTNP alone (without NIR illumination) are also shown to be effective in 10


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dissolution, although the dissolution time is much longer. Figure 1b shows the dissolution

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kinetics of fibril samples treated with different concentration of AgTNP without NIR

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illumination. Nearly complete dissolution takes about 70 hr at highest concentration, i.e 30

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nM, and longer for lower concentrations. This data suggests that AgTNP with their sharp

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edges and tips are able to cleave the mature fibrils and fragment them similar to graphene

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oxide sheets. But the best effect results when photothermal property of AgTNP is exploited.

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For comparison, the effect of AgSP on the dissolution of mature fibrils is shown in Figure 1c.

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Dissolution kinetics of AgSP depend on the concentration of AgSP, similar to AgTNP, and

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up to 70 hr, 90% of fibrils is dissolved at highest concentration and about 50% dissolved at

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the lowest concentration. In the case of the AgTNP and AgSP, without NIR irradiation, the

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negatively charged nanoparticles interact with aspartate and lysine residues thus breaking the

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salt bridge formation between the beta sheets thus cleaving them to small aggregates. A

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similar reduction was seen when matured fibrils were incubated with negatively charged

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spherical gold nanoparticles and gold nanorods for 70 h.25,27 Comparison of Figures 1a, b and

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c clearly suggest that the AgTNP under NIR illuminated conditions brings the dissolution

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time within 1 hr, demonstrating the rapidity of the process. It may be noted that 30 nM gold

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nanorods under NIR illuminated conditions took 25 hr to dissolve 90% of mature fibrils.27

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Control experiments in which mature fibrils were treated with either PVP (0.4 nM) or Congo

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red (30 µM) showed that neither PVP nor congo red dissolved fibrils themselves.

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To corroborate the Congo red assay and optical microscopic analysis (Figure S7), TEM

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images of the sample are recorded and shown in Figure 2. The top and bottom panels show

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samples treated with nanoparticles for 1 hr and 24 hr, respectively, with different kinds of

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nanoparticles. From these images, the binding of the AgTNP to amyloid fibrils can be

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visualized.

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significant reduction in the fibril content at 1 hr and a slight reduction is observed at 24 hr.

Figure 2a, 2d show samples treated with AgSP and it is observed that no

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Figure 2b, 2e show samples treated with AgTNP, and it is observed that at 24 hr significant

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reduction in the fibril content is observed yet incomplete. Finally Figure 2c, 2f show the

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images of samples treated with AgTNP under NIR irradiation, and within 1 hr itself fibrils are

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completely removed.

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Dynamic light scattering (DLS) analysis of the sample is carried out to get quantitative data

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on the dissolution kinetics and fibril length distribution, apart from TEM studies. Figure S9 in

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the Supporting Information shows the size distribution of the mature fibrils and the fibrils

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treated with the silver nanoplates at different time intervals. The mature amyloid fibrils show

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a bimodal distribution with a peak centered around 400 nm and another centered around 2.5

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µm. After treating the fibrils with silver nanoplates and NIR irradiation, the peak in the fibril

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length distribution at 30 min is observed around 100 nm with the absence of the peak in 2.5

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µm range, corresponding to the dissolution of fibrils into smaller aggregates. After 1 h, the

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peak shifted to 50 nm suggesting the appearance of much smaller aggregates compared to the

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initial mature fibrils. From DLS and TEM/spectroscopic studies, it may be concluded that

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dissolution of fibrils into shorter aggregates occurred within 1 h in the presence of NIR

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irradiated silver nanoplates. Thus, Congo red absorbance measurement, real space imaging by

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optical microscopy and transmission electron microscopy analysis collectively suggest the

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rapid dissolution of mature Aβ fibrils using AgTNP and NIR illumination.

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Inhibition kinetics of Aβ fibrillation by AgTNP

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In the previous section, the rapid dissolution of mature Aβ fibrils by AgTNP under NIR

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illumination is demonstrated. In this section, the inhibitory role of AgTNP and AgSP in the

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formation of Aβ fibrils is described. This is to test whether the formation of fibrils can be

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prevented if nanoparticles are administered in the incipient stage of Aβ fibrillation. We note

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that the size and zeta-potential of the Aβ (1-40) monomer, measured using Dynamic Light

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Scattering (DLS, Horiba and zetasizer), are found to be 3 ± 1 nm and -20 mV, respectively, at

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pH 7. These values are consistent with the previous reports.42 Although Aβ (1-40) monomer

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contains both negatively-charged and positively-charged residues, the net charge on the

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protein was found to be negative, whereas the charges on the residues (KLVFF)

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corresponding to beta-sheet formation are positively charged.

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The control studies were carried out without AgSP and AgTNP and a sigmoidal-shaped curve

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was observed with initial lag phase followed by a rapid elongation to form fibrils and finally

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reaching the stationary phase, where almost all the monomer is converted to fibrils (Figure 2).

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For the sample incubated with AgSP and AgTNP, the fibrillation kinetics shows a decreased

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fibril concentration at longer times, which confirms the inhibition of fibril formation. When

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compared with the AgSP, AgTNP showed better inhibitory effect for the same concentration.

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This may be due to larger surface area of AgTNP leading to the adsorption of more number

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of Aβ monomers on to the AgTNP thus a larger reduction in fibril content is observed. The

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presence of AgTNP of 30 nM concentration has decreased the fibrils content to less than 1

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µM. It is observed that fibril concentration decreased with increase in concentration of

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nanoparticles. Both AgSP and AgTNP actively interfere in the nucleation phase, thus

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prolonging the lag phase and thus inhibiting the growth of the fibrils. The fact that we see a

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difference in the fibrillation kinetics for AgSP and AgTNP suggest that the shape and surface

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area play a role in fibrillation. In addition to the Congo red assay, optical microscope, atomic

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force microscopy (AFM), and transmission electron microscopy (TEM) were used to confirm

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the inhibitory role of AgSP and AgTNP.

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AFM (top panel) and optical microscopic (bottom panel) images of the samples are shown in

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Figure 4. Figure 4a and 4d show the images of control sample that is pure Aβ protein,

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incubated for 24 h. The presence of mature fibrils is evident in these images and corroborates

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with the Congo red assay measurements (Figure 3). Figure 4b and 4e show images of the 13


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protein samples treated with 30 nM of AgSP for 24 h, and Figure 4c and 4f show images of

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the protein samples treated with 30 nM of AgTNP for 24 h. From these images, it is observed

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that the inhibition of fibrillation is better with AgTNP than AgSP as lesser fibrils are found in

4

the images of the samples treated with AgTNP (Figure 4c, 4f).

5

TEM images of the samples incubated with AgTNP of different concentration are shown in

6

Figure 5. Figure 5a shows the presence of mature fibrils entangled throughout the image in

7

the control sample. Figure 5b-f shows the TEM images of the sample incubated with AgTNP

8

of various concentration of 3 nM, 6 nM, 8 nM, 10 nM and 30 nM respectively, for 24 h. TEM

9

images of the samples incubated with AgSP of different concentration are shown in Figure

10

S8. Comparison of Figure S8b-f with 5b-f show that increasing the concentration of AgTNP

11

reduced fibrils content better than AgSP. Thus, the role of shape and concentration in

12

reducing is evidently seen in these analyses.

13

We have previously addressed the mechanism of inhibition of Aβ fibrillation by negatively

14

charged gold nanoparticles using Molecular Dynamics simulation and FTIR-ATR

15

spectroscopy.25 The same mechanism may be expected in the current study involving AgSP

16

and AgTNP. It has been reported that the residue cluster KLVFF and the salt-bridge between

17

residues Asp23 and Lys28 are involved in the β-sheet structure.16 The ability to interfere with

18

these residues is the key requirement for an inhibitor. As the AgSP and AgTNP used in our

19

present study are of negatively charged, they preferentially bind with Lys16 and Lys28

20

(positively charged residues) due to electrostatic attraction thus inhibiting the formation of the

21

fibrils. The proposed mechanism for inhibition of the fibril formation have been supported by

22

MD simulation study.25

23

We have recently published a detailed kinetic model for the inhibitory effect of charged

24

nanoparticles on the amyloid fibrillation.43 The model considers nucleation, growth,

14


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fragmentation and the effect of nanoparticles on the conformational change of the protein

2

monomer. Although the model results were compared with experimental data available for

3

spherical nanoparticles, the model is generally applicable for other shapes as well. We have

4

now used the model to compare with the experimental data of the present work, and the

5

results are shown in Figure S10 in the Supporting Information. A good match between the

6

model and experiments can be noted. Parameters used in the model are listed in Table S1 in

7

the Supporting Information.

8

Conformational changes induced by AgSP and AgTNP in Aβ

9

The inhibitory and dissolution aspects of AgTNP and AgSP are characterized by Congo red

10

assay and various imaging techniques, but these techniques do not offer any information on

11

the internal structural detail of protein aggregates. To this end, we use attenuated total

12

reflectance Fourier transform infrared spectroscopy (ATR-FTIR). As the vibration bands for

13

beta sheet and alpha helix are significantly deconvoluted, the relative absorbance at these

14

bands provide information on the relative amount of beta sheets and alpha helix secondary

15

structure. Figure 6 shows the ATR-FTIR spectra of Aβ sample obtained in the absence and

16

presence of AgNP and AgTNP in the wavenumber range from 1600-1700cm−1, where most

17

of the protein’s secondary structure information can be obtained. The vibrations

18

corresponding to beta sheet structure is due to amide I band around 1632 cm−1 and for alpha

19

helix, it is due to amide II band around 1652 cm−1.44,45 After treating the sample with the

20

AgSP, there is decrease in the beta sheet structure from 68% to 32% with a corresponding

21

increase in the alpha helical structure content from 22% to 33% after 24 h of incubation time.

22

Similarly, for the Aβ treated with AgTNP, the beta sheet content decreased from 68% to

23

15%, whereas the alpha helical content increased 22% to 48%. Significant increase in the

24

random coil was also observed in the samples treated with AgTNP than that of AgSP. The

25

random coil content is obtained by subtracting the percentages of beta sheet and alpha helical 15


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1

content from 100%.

From the results, it may be inferred that AgTNP is a more effective

2

inhibitor than AgSP. A similar increase in alpha helix structure content at the expense of beta

3

sheet content was observed when spherical Au nanoparticles (AuNP) and gold nanorods

4

(AuNR) were used as an inhibitor.25 Although the molecular mechanism of this conversion

5

process facilitated by AgSP and AgTNP is not understood, a probable reason could be that

6

the negatively charged silver nanoparticles may act as nanochaperones and assist the

7

misfolded protein to retain to its native disordered state.25

8

To substantiate the structural analysis further, Circular Dichroism (CD) spectra for the Aβ

9

(1– 40) peptides were recorded on a JASCO J-1700 series spectrometer. A 0.1 cm quartz cell

10

was used for far UV measurements (190 – 260 nm). Three scans for each sample were

11

measured and averaged. The secondary structure contents (alpha helix, beta sheet, turn, and

12

random coil) of the Aβ peptide samples were estimated using the CDPro software from the

13

spectrum, and shown in Figure S11 in the Supporting Information. The CD spectrum of the

14

mature Aβ(1–40) fibrils alone exhibited a characteristic peak with an absorption minimum

15

around 218 nm confirming β-sheet conformation. After the treatment with AgSP and AgTNP,

16

CD ellipticity signal at 218 nm decreased, indicating beta-sheet formation was inhibited by

17

the AgSP and AgTNP. The beta sheet percentage decreased from 60% to 22% on treating

18

mature fibrils with AgSP and to 15% while treating with AgTNP.

19

Cell viability assays

20

Though the Congo red assay and TEM studies showed that the AgSP and AgTNP are

21

potential candidates for the inhibition of fibrillation and for dissolution of mature fibrils, the

22

viability of the cells need to be studied. The MTT assay was employed to determine the cell

23

viability on the neuroblastoma cells (SH-SY5Y). Figure 7 shows cell viability in percentage

24

when the cells are treated with mature fibrils, fibrils treated with AgSP and AgTNP for two

16


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different times (24hr and 48 hr). We find that the mature fibrils are toxic to the cells (control)

2

as only 30% of the cells are viable after 24 h. But when the cells incubated with the mature

3

fibril sample, which has been treated with AgSP and AgTNP of 30 nM concentration for 24 h

4

and 48 h, the cell viability increased to 52% and 65%, respectively. This significant increase

5

in cell viability is due to reduced number of fibrils in these samples compared to the control.

6

Further, cells incubated with the fibril sample, which has been incubated with AgTNP after

7

NIR laser illumination show cell viability of 80 % and 79 % respectively. As it has been

8

shown previously that NIR irradiation decreased the fibrils content to a larger extent

9

compared to no-irradiation case (Figure 1a), the cell viability has greatly improved upon

10

irradiation. This result confirms that the cell viability is enhanced when the sample is treated

11

with AgTNP under illuminated condition compared to mature fibrils alone. LDH assay on

12

SH-SY5Y cells also show similar results. MTT and LDH assay on the viability of BE-(2)-C

13

cells in the solution of amyloid ﬁbrils, and in the protein solution treated AgSP, AgTNP, and

14

the NIR treated AgTNP yielded similar results (Figure S12 in the Supporting Information).

15

Further MTT and LDH assay are performed to determine the cell viability of SH-SY5Y cells

16

in the inhibition stage. Figure S13 in the Supporting Information shows the viability of the

17

cells incubated with the protein solution treated with AgSP and AgTNP at different times. It

18

is observed that AgTNP has shown a fibrillation inhibitory effect, and therefore the cell

19

viability increased from 30% to 70% at 6 h, 25% to 67% at 24 h, 15% to 63% at 48 h, and

20

15% to 60% at 72 h. From the result, it can be inferred that silver nanoparticles have both

21

inhibitory and destructive effects on fibrils.

22

The size distribution of the aggregates resulting from dissolution of fibrils (Figure 2 and S7)

23

shows that the fibrils are not necessarily converted to oligomers or monomers. The mature

24

fibrils are converted to small amorphous aggregates (~50 nm) which are nontoxic, suggesting

25

that protein might have undergone conformational changes resulting in aggregates without 17


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1

beta sheets. CD and ATR/FTIR structural analysis shows reduction in beta sheet in the

2

aggregates resulting from fibril dissolution. These are consistent with our previous studies on

3

the effect of gold nanorods on the dissolution of amyloid fibrils.27,29

4

ThT assay can also be used to detect inhibition of amyloid fibrillation and dissolution of

5

amyloid fibrils.27,29 Figure S14 in the Supporting Information shows the inhibition and

6

dissolution kinetics measured using ThT fluorescence assay. While ThT fluorescence assay is

7

often used in the literature to study the kinetics of fibrillation of pure amyloid, the use of

8

plasmonic nanoparticles in the system makes the use of ThT assay bit undesirable. This is

9

because of the inner filter effect: the fluorescence intensity of ThT may be quenched by the

10

presence of plasmonic particles. For instance, the emission wavelength of ThT is around 480

11

nm, while the absorbance of the AgSP is around 420 nm and of AgTNP are around 400 nm

12

and 810 nm. These overlaps may cause a decrease in the fluorescence intensity of ThT, which

13

could be wrongly inferred as a decrease in the amount of fibrils. We have systematically

14

studied this effect in our previous works and reported that when nanoparticles are used in

15

nanomolar concentrations, these effects are negligible.27,29 However, at higher concentration

16

of nanoparticles (in µM range) the inner filter effect may not be neglected. For this reason,

17

we have used congo red assay for the present study.

18

One of the main challenges in developing nanotherapy for AD is that the particles used in the

19

therapy should able to cross blood brain barrier. The sizes of the AgTNP and AgSP used in

20

our study are within the range of 15−100 nm, which are well-suited for crossing the blood-

21

brain barrier46,47. Further AgTNP and AgSP should be biocompatible to make them practice

22

in clinical trials. The safety limit for uptake of silver nanoparticle into the cell is reported to

23

as 2.5 mM.48,49 The concentrations used in our study are in nanomolar range and is much

24

lower compared to this safety limit. Direct injection of nanoparticles into the brain using a

25

catheter, (used for treating brain tumours) is one possible ways of direct delivery of the 18


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1

nanoparticle to the brain. After delivery of nanoparticles in vivo, the next challenge is the

2

formation of protein corona on the nanoparticles, thus making them ineffective for AD

3

therapy. We have reported earlier that, for AuNR and AuNPs smaller than 100 nm the protein

4

corona formation is insignificant.25 The next bottleneck is whether NIR rays can penetrate the

5

skull. Siegel et al50 have recently studied the penetration of NIR in human soft tissue, skull

6

bone and in brain and their findings proved that NIR can penetrate to neural tissues in the

7

brain. However, the penetration efficiency is less, which will be a major hurdle in developing

8

the technology. In this case, the alternative way is that NIR can be passed to the amyloid

9

specific region through optical fibers, thus the efficacy of NIR in dissociating the preformed

10

fibrils can be increased substantially.

11

CONCLUSION

12

In this study, we have demonstrated the use of PVP-stabilized, negatively charged silver

13

nanoplates inhibited the formation of Aβ (1-40) fibrils at nanomolar concentrations. Further it

14

has been shown that AgTNP dissolved mature Aβ fibrils under NIR illuminated conditions

15

due to hyperthermic effect caused by in-plane dipole resonance. Laser irradiation is carried

16

out for 1 min. The findings are supported by complementary experimental techniques such as

17

absorbance assay, optical microcopy, atomic force microscopy, transmission electron

18

microscopy and attenuated total reflectance Fourier transform spectroscopy. Most

19

importantly, the dissolution time scale has been reduced to within 1 hr and we claim that this

20

is the fastest dissolution compared to other methods reported earlier. Although the effect of

21

concentration of AgTNP on the dissolution kinetics have been studied here, additional studies

22

are required to investigate the role of edge length of AgTNP, PVP molecular weight and

23

capping thickness, NIR laser wavelength, power density and duration of irradiation in the

24

efficiency of dissolution process. Numerical modeling is needed to calculate the temperature

25

distribution around Aβ fibrils in the presence of AgTNP under NIR illumination to optimize 19


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1

the laser power and illumination duration. AgTNP qualifies as a potential AD therapeutic as

2

it satisfies the safe limit of concentration, size constraints to cross blood-brain barrier and

3

non-cytotoxicity. Augmented with innovation in biomedical devices, AgTNP can be

4

developed as a potential anti-AD agent aided by NIR illumination.

5

ASSOCIATED CONTENT

6

The supporting Information is available free of charge on the ACS Publication website at

7

www.pubs.acs.org.

8

Synthesis and characterization of silver nanospheres and silver triangular nanoplates,

9

calibration of Congo red absorbance to measure fibril content, DLS analysis of dissociated

10

fibrillar aggregates, comparison between model and experiments on the kinetics of

11

fibrillation, structural analysis of amyloid aggregates by cyclic dichroism, cell viability

12

results and ThT assay for inhibition of fibrillation and dissolution of mature fibrils by

13

AgTNP.

14

AUTHOR INFORMATION

15

Corresponding Author

16

*Email: [email protected]

17

ORCID

18

Ethayaraja Mani: 0000-0002-4091-1576

19

Author Contributions

20


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The manuscript was written through contribution of both the authors.

2

Notes

3

The authors declare no competing financial interest.

4

ACKNOWLEDGMENTS

5

The authors thank Nirmal Kumar Ramesh for his help in the simulation of kinetic

6

model and Prof. Erik Schaefer for the equipment facility availed. This work was

7

supported by Department of Science and Technology, India via the research grant

8

EMR/2016/000532. Some part of the work was also supported by the PhD Network:

9

"Novel nanoparticles: from synthesis to biological applications" of the University of

10

Tübingen, Germany.

11

REFERENCES

12

(1)

(1)

Harper, J. D.; Lansbury, P. T. Models of amyloid seeding in alzheimer’s

13

disease and scrapie:Mechanistic Truths and Physiological Consequences of the Time-

14

Dependent Solubility of Amyloid Proteins. Annu. Rev. Biochem. 1997, 66 (1), 385–

15

407. https://doi.org/10.1146/annurev.biochem.66.1.385.

16

(2)

Gregersen, N.; Bross, P.; Vang, S.; Christensen, J. H. Protein Misfolding and Human

17

Disease.

18

https://doi.org/10.1146/annurev.genom.7.080505.115737.

19

(3)

Annu.

Rev.

Genomics

Hum.

Genet.

2006,

7

(1),

103–124.

Holtzman, D. M.; Morris, J. C.; Goate, A. M. Alzheimer’s Disease: The Challenge of

21


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

the

2

https://doi.org/10.1126/scitranslmed.3002369.

3

(4)

Second

Century.

Science

Approaches.

5

https://doi.org/10.1002/anie.200802808.

(5)

(6)

Angew.

Chem.

Int.

Ed.

Engl.

2009,

48

(17),

3030–3059.

Mann, D. M. Pyramidal Nerve Cell Loss in Alzheimer’s Disease. Neurodegeneration

Norfray, J. F.; Provenzale, J. M. Alzheimer’s Disease: Neuropathologic Findings and

https://doi.org/10.2214/ajr.182.1.1820003.

10

(7)

Selkoe, D. J. Alzheimer’s Disease is a Synaptic Failure. Science.. 2002, 298 (5594), 789–791. https://doi.org/10.1126/science.1074069.

12

13

2011.

Recent Advances in Imaging. Am. J. Roentgenol. 2004, 182 (1), 3–13.

9

11

Medicine.

1996, 5 (4), 423–427. https://doi.org/10.1006/neur.1996.0057.

7

8

Translational

Jakob-Roetne, R.; Jacobsen, H. Alzheimer’s Disease: From Pathology to Therapeutic

4

6

Page 22 of 35

(8)

Lee, J.; Culyba, E. K.; Powers, E. T.; Kelly, J. W. Amyloid-β Forms Fibrils by

14

Nucleated Conformational Conversion of Oligomers. Nat. Chem. Biol. 2011, 7 (9),

15

602–609. https://doi.org/10.1038/nchembio.624.

16

(9)

1997, 275 (5300), 630–631. https://doi.org/10.1126/science.275.5300.630.

17

18

Selkoe, D. J. Alzheimer’s Disease--Genotypes, Phenotype, and Treatments. Science.

(10)

Soto, C.; Sigurdsson, E. M.; Morelli, L.; Asok Kumar, R.; Castaño, E. M.; Frangione,

19

B. β-Sheet Breaker Peptides Inhibit Fibrillogenesis in a Rat Brain Model of

20

Amyloidosis: Implications for Alzheimer’s Therapy. Nat. Med. 1998, 4 (7), 822–826.

22


Page 23 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

https://doi.org/10.1038/nm0798-822.

1

2

(11)

Sabaté, R.; Estelrich, J. Stimulatory and Inhibitory Effects of Alkyl Bromide

3

Surfactants on β-Amyloid Fibrillogenesis. Langmuir 2005, 21 (15), 6944–6949.

4

https://doi.org/10.1021/la050472x.

5

(12)

Bush, A. I. Metal Complexing Agents as Therapies for Alzheimer’s Disease.

6

Neurobiol.

7

4580(02)00120-3.

8

(13)

2002,

23

(6),

1031–1038.

https://doi.org/10.1016/S0197-

Ono, K.; Hasegawa, K.; Naiki, H.; Yamada, M. Curcumin Has Potent AntiAmyloidogenic Effects for Alzheimer’s β-Amyloid Fibrils In Vitro. J. Neurosci. Res.

9

2004, 75 (6), 742–750. https://doi.org/10.1002/jnr.20025.

10

11

Aging

(14)

Dong, M.; Zhao, W.; Hu, D.; Ai, H.; Kang, B. N ‑ Terminus Binding Preference for

12

Either Tanshinone or Analogue in Both Inhibition of Amyloid Aggregation and

13

Disaggregation of Preformed Amyloid Fibrils - Toward Introducing a Kind of Novel

14

Anti-Alzheimer Compounds. 2017. https://doi.org/10.1021/acschemneuro.7b00080.

15

(15)

Tomiyama, T.; Shoji, A.; Kataoka, J.; Suwa, Y.; Asano, S.; Kaneko, H.; Endo, N.

16

Inhibition of Amyloid β Protein Aggregation and Neurotoxicity by Rifampicin. J. Biol.

17

Chem. 1996, 271 (12), 6839–6844. https://doi.org/10.1074/jbc.271.12.6839.

18

(16)

and Cytotoxicity by Hydroxyindole. 2006, 4727–4735.

19

20

August, R. V; Re, V.; Recei, M.; February, V. Inhibition of Amyloid Fibril Formation

(17)

Cabaleiro-Lago, C.; Quinlan-Pluck, F.; Lynch, I.; Lindman, S.; Minogue, A. M.;

23


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 35

1

Thulin, E.; Walsh, D. M.; Dawson, K. A.; Linse, S. Inhibition of Amyloid β Protein

2

Fibrillation by Polymeric Nanoparticles. J. Am. Chem. Soc. 2008, 130 (46), 15437–

3

15443. https://doi.org/10.1021/ja8041806.

4

(18)

Frydman-Marom, A.; Rechter, M.; Shefler, I.; Bram, Y.; Shalev, D. E.; Gazit, E.

5

Cognitive-Performance Recovery of Alzheimer’s Disease Model Mice by Modulation

6

of Early Soluble Amyloidal Assemblies. Angew. Chemie - Int. Ed. 2009, 48 (11),

7

1981–1986. https://doi.org/10.1002/anie.200802123.

8

(19)

Barnham, K. J.; Kenche, V. B.; Ciccotosto, G. D.; Smith, D. P.; Tew, D. J.; Liu, X.;

9

Perez, K.; Cranston, G. a; Johanssen, T. J.; Volitakis, I.; et al. Platinum-Based

10

Inhibitors of Amyloid-Beta as Therapeutic Agents for Alzheimer’s Disease. Proc.

11

Natl.

12

https://doi.org/10.1073/pnas.0800712105.

13

(20)

Sci.

U.S.A.

2008,

105

(19),

6813–6818.

Cai, W. Applications of Gold Nanoparticles in Cancer Nanotechnology. Nanotechnol. Sci. Appl. 2008, 1, 17–32. https://doi.org/10.2147/NSA.S3788.

14

15

Acad.

(21)

Lei, Y.; Hamada, Y.; Li, J.; Cong, L.; Wang, N.; Li, Y.; Zheng, W.; Jiang, X. Targeted

16

Tumor Delivery and Controlled Release of Neuronal Drugs with Ferritin Nanoparticles

17

to Regulate Pancreatic Cancer Progression. J. Control. Release 2016, 232, 131–142.

18

https://doi.org/10.1016/j.jconrel.2016.03.023.

19

(22)

Liao, Y.; Chang, Y.; Yoshiike, Y.; Chang, Y.; Chen, Y. Negatively Charged Gold

20

Nanoparticles Inhibit Alzheimer ’ s Amyloid- β Fibrillization , Induce Fibril

21

Dissociation

22

https://doi.org/10.1002/smll.201201068.

,

and

Mitigate

24

Neurotoxicity.


2012,

1–9.

Page 25 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Langmuir

(23)

Cabaleiro-Lago, C.; Szczepankiewicz, O.; Linse, S. The Effect of Nanoparticles on

2

Amyloid Aggregation Depends on the Protein Stability and Intrinsic Aggregation Rate.

3

Langmuir 2012, 28 (3), 1852–1857. https://doi.org/10.1021/la203078w.

4

(24)

Das, P.; Kang, S. G.; Temple, S.; Belfort, G.; Kim, Y.; Park, J.-H.; Lee, H.; Nam, J.-

5

M. How Do the Size, Charge and Shape of Nanoparticles Affect Amyloid β

6

Aggregation on Brain Lipid Bilayer? Sci. Rep. 2016, 6 (7), 19548-19555.

7

https://doi.org/10.1038/srep19548.

8

(25)

Inhibitors on Amyloid Beta Fibrillation. J. Phys. Chem. C 2017, 121 (11), 6339–6348.

9

https://doi.org/10.1021/acs.jpcc.6b12307.

10

11

Sudhakar, S.; Kalipillai, P.; Santhosh, P. B.; Mani, E. Role of Surface Charge of

(26)

Lin, D.; He, R.; Li, S.; Xu, Y.; Wang, J.; Wei, G.; Ji, M.; Yang, X. High-Efficient

12

Destruction of Amyloid-β Fibrils by Femtosecond Laser-Induced Nano-Explosion of

13

Gold

14

https://doi.org/10.1021/acschemneuro.6b00244.

15

(27)

Nanorods.

ACS

Chem.

Neurosci.

2016,7(12),

1728-1736.

Sudhakar, S.; Santhosh, P. B.; Mani, E. Dual Role of Gold Nanorods : Inhibition &

16

Dissolution of Aβ Fibrils Induced by Near IR Laser. 2017, 8(10),2325–2334.

17

https://doi.org/10.1021/acschemneuro.7b00238.

18

(28)

Yagi, H.; Ozawa, D.; Sakurai, K.; Kawakami, T.; Kuyama, H.; Nishimura, O.;

19

Shimanouchi, T.; Kuboi, R.; Naiki, H.; Goto, Y. Laser-Induced Propagation and

20

Destruction of Amyloid β Fibrils. J. Biol. Chem. 2010, 285 (25), 19660–19667.

21

https://doi.org/10.1074/jbc.M109.076505.

25


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

(29)

Page 26 of 35

Yang, Z.; Ge, C.; Liu, J.; Chong, Y.; Gu, Z.; Jimenez-Cruz, C. A.; Chai, Z.; Zhou, R.

2

Destruction of Amyloid Fibrils by Graphene through Penetration and Extraction of

3

Peptides.

4

https://doi.org/10.1039/C5NR01172H.

5

(30)

Nanoscale

2015,

7

(44),

18725–18737.

Li, M.; Yang, X.; Ren, J.; Qu, K.; Qu, X. Using Graphene Oxide High Near-Infrared

6

Absorbance for Photothermal Treatment of Alzheimer’s Disease. Adv. Mater. 2012, 24

7

(13), 1722–1728. https://doi.org/10.1002/adma.201104864.

8

(31)

D’Agostino, A.; Taglietti, A.; Desando, R.; Bini, M.; Patrini, M.; Dacarro, G.; Cucca,

9

L.; Pallavicini, P.; Grisoli, P. Bulk Surfaces Coated with Triangular Silver Nanoplates:

10

Antibacterial Action Based on Silver Release and Photo-Thermal Effect.

11

Nanomaterials 2017, 7 (1), 7. https://doi.org/10.3390/nano7010007.

12

(32)

Potara, M.; Boca, S.; Licarete, E.; Damert, A.; Alupei, M.-C.; Chiriac, M. T.; Popescu,

13

O.; Schmidt, U.; Astilean, S. Chitosan-Coated Triangular Silver Nanoparticles as a

14

Novel Class of Biocompatible, Highly Sensitive Plasmonic Platforms for Intracellular

15

SERS

16

https://doi.org/10.1039/c3nr00005b.

17

(33)

Sensing

and

Imaging.

Nanoscale

2013,

5

(13),

6013.

Wang, S. S. S.; Chen, Y. T.; Chou, S. W. Inhibition of Amyloid Fibril Formation of β-

18

Amyloid Peptides via the Amphiphilic Surfactants. Biochim. Biophys. Acta - Mol.

19

Basis Dis. 2005, 1741 (3), 307–313. https://doi.org/10.1016/j.bbadis.2005.05.004.

20

(34)

Kittler, S.; Greulich, C.; Köller, M.; Epple, M. Synthesis of PVP-Coated Silver

21

Nanoparticles and Their Biological Activity towards Human Mesenchymal Stem Cells.

22

Material and Technology2009. https://doi.org/10.1002/mawe.200800437. 26


Page 27 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Langmuir

(35)

Mirzaei, A.; Janghorban, K.; Hashemi, B.; Bonyani, M.; Leonardi, S. G.; Neri, G.

2

Characterization and Optical Studies of PVP-Capped Silver Nanoparticles. J.

3

Nanostructure Chem. 2017. https://doi.org/10.1007/s40097-016-0212-3.

4

(36)

Khurana, R.; Uversky, V. N.; Nielsen, L.; Fink, A. L. Is Congo Red an Amyloid-

5

Specific

6

https://doi.org/10.1074/jbc.M011499200.

7

(37)

J.

Biol.

Chem.

2001,

276

(25),

22715–22721.

Klunk, W. E.; Pettergrew, J. W.; Abraham, D. J. Quatitative Evaluation of Congo Red Binding with a Beta-Pleated To. J. Histochem. Cytochem. 1989, 37 (8), 1273–1281.

8

9

Dye?

(38)

Huang, X.; El-Sayed, I. H.; Qian, W.; El-Sayed, M. A. Cancer Cell Imaging and

10

Photothermal Therapy in the Near-Infrared Region by Using Gold Nanorods. J. Am.

11

Chem. Soc. 2006, 128 (6), 2115–2120. https://doi.org/10.1021/ja057254a.

12

(39) Gui, C.; Cui, D.-X. Functionalized Gold Nanorods for Tumor Imaging and Targeted

13

Therapy.

14

3941.2012.04.002.

15

(40)

Cancer

Biol.

Med.

2012.

https://doi.org/10.7497/j.issn.2095-

Dickerson, E. B.; Dreaden, E. C.; Huang, X.; El-Sayed, I. H.; Chu, H.; Pushpanketh,

16

S.; McDonald, J. F.; El-Sayed, M. A. Gold Nanorod Assisted Near-Infrared Plasmonic

17

Photothermal Therapy (PPTT) of Squamous Cell Carcinoma in Mice. Cancer Lett.

18

2008, 269 (1), 57–66. https://doi.org/10.1016/j.canlet.2008.04.026.

19

(41)

Huang, X.; El-Sayed, I. H.; El-Sayed, M. A. Applications of Gold Nanorods for

20

Cancer

Imaging

and

Photothermal

21

https://doi.org/10.1007/978-1-60761-609-2_23.

27

Therapy.


Methods

Mol.

Biol.

2010.

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

(42)

Page 28 of 35

Ahmed, M.; Davis, J.; Aucoin, D.; Sato, T.; Ahuja, S.; Aimoto, S.; Elliott, J. I.; Van

2

Nostrand, W. E.; Smith, S. O. Structural Conversion of Neurotoxic Amyloid-Beta(1-

3

42) Oligomers to Fibrils. Nat. Struct. Mol. Biol. 2010, 17 (5), 561–567.

4

https://doi.org/10.1038/nsmb.1799.

5

(43)

Ramesh, N. K.; Sudhakar, S.; Mani, E. Modeling of the Inhibitory Effect of

6

Nanoparticles on Amyloid β Fibrillation. Langmuir 2018 34(13), 4004-4012.

7

https://doi.org/10.1021/acs.langmuir.8b00388.

8

(44)

Transition in Stretched and Compressed Hydrated Fibrin Clots. Biophys. J. 2012, 103

9

(5), 1020–1027. https://doi.org/10.1016/j.bpj.2012.07.046.

10

11

Litvinov, R. I.; Faizullin, D. A.; Zuev, Y. F.; Weisel, J. W. The α-Helix to β-Sheet

(45)

Sarroukh, R.; Goormaghtigh, E.; Ruysschaert, J. M.; Raussens, V. ATR-FTIR: A

12

“Rejuvenated” Tool to Investigate Amyloid Proteins. Biochim. Biophys. Acta -

13

Biomembr.

14

https://doi.org/10.1016/j.bbamem.2013.04.012.

15

(46)

2013,

1828

(10),

2328–2338.

Teleanu, D. M.; Chircov, C.; Grumezescu, A. M.; Volceanov, A.; Teleanu, R. I.

16

Blood-Brain Delivery Methods Using Nanotechnology. Pharmaceutics 2018, 10 (4),

17

1–16. https://doi.org/10.3390/pharmaceutics10040269.

18

(47)

Shilo, M.; Sharon, A.; Baranes, K.; Motiei, M.; Lellouche, J. P. M.; Popovtzer, R. The

19

Effect of Nanoparticle Size on the Probability to Cross the Blood-Brain Barrier: An in-

20

Vitro

21

https://doi.org/10.1186/s12951-015-0075-7.

Endothelial

Cell

Model.

28

J.


Nanobiotechnology

2015.

Page 29 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Langmuir

(48)

Składanowski, M.; Golinska, P.; Rudnicka, K.; Dahm, H.; Rai, M. Evaluation of

2

Cytotoxicity, Immune Compatibility and Antibacterial Activity of Biogenic Silver

3

Nanoparticles. Med. Microbiol. Immunol. 2016. https://doi.org/10.1007/s00430-016-

4

0477-7.

5

(49)

Rizzo, A.; Inganäs, O.; Solin, N. Preparation of Phosphorescent Amyloid-like Protein

6

Fibrils.

7

https://doi.org/10.1002/chem.201000146.

8 9 10

(50)

Chem.

-

A

Eur.

J.

2010,

(14),

4190–4195.

Jagdeo, J. R.; Adams, L. E.; Brody, N. I.; Siegel, D. M. Transcranial Red and Near Infrared Light Transmission in a Cadaveric Model. 2012, 7 (10), 1–10. https://doi.org/10.1371/journal.pone.0047460.

11

12

13

16

FIGURES

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Aβ fibrils Aβ fibrils + NIR Aβ fibrils + NIR + 3 nM AgTNP Aβ fibrils + NIR + 6 nM AgTNP Aβ fibrils + NIR + 10 nM AgTNP Aβ fibrils + NIR + 30 nM AgTNP

(a)

(b)

(c)

1 2

Figure 1: Dissociation kinetics of mature Aβ fibrils treated with different concentrations of

3

nanoparticles: a) AgTNP, NIR irradiated b) AgTNP in the absence of NIR irradiation and c)

4

AgSP.

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a

b

c

2

b

3 4 5

d

e

b

b

f b

6 7 8 9

Figure 2. TEM images of fibrils treated with nanoparticles for 1 h (top panel) and 24 hr

10

(bottom panel): (a,d) AgSP; (b,e) AgTNP without NIR and (c, f) AgTNP with NIR Scale bar:

11

500 nm. Concentrations of AgTNP and AgSP are 30 nM.

12 13 14 15

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(a)

1

(b)

2 3

Figure 3. Fibrillation kinetics of Aβ protein incubated with a) AgSP and b) AgTNP of

4

different concentration measured in terms of Congo red absorbance. The curve corresponding

5

to control refers to the growth of fibrils in the absence of AgSP and AgTNP.

6 7

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Figure 4. AFM images (top panel) and optical microscopic images (bottom panel) of 10 μM

3

Aβ sample incubated for 24 hr. (a, d) pure Aβ protein sample; (b, e) protein sample incubated

4

30 nM AgSP; and (c, f) protein sample incubated 30 nM AgTNP. Scale bar: 1 μm.

5

a

b

c

e

f

(a)

d

6 7

Figure 5. Aβ fibrils were incubated with different concentration of AgTNP for 24 h (a) 0 nM

8

(Control) (b) 3 nM (c) 6 nM and (d) 8 nM (e) 10 nM (f) 30 nM. Scale bar: 200 nm.

9

(a) (a)

10 11

(b)

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1 2 3

(b)

4 5 6 7 8

Figure 6. (a) ATR-FTIR spectra in the region 1600-1700 cm−1 and (b) Secondary structure

(b)

(a)

9

content of amyloid treated with AgSP and AgTNP of 30 nM concentration for 24 h.

10

Figure 7. SH-SY5Y cell viability (percentage) in the solution of amyloid ﬁbrils, in the

11

protein solution treated AgSP, AgTNP and the NIR treated AgTNP. (a) MTT assay (b) LDH

12

assay.

13

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Rapid Dissolution of Amyloid Î² Fibrils by Silver Nanoplates

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