Restriction of microwave-induced amyloid fibrillar growth by gold nanoparticles

Journal Pre-proof Restriction of microwave-induced amyloid fibrillar growth by gold nanoparticles

Anang Kumar Singh, Susmita Bhattacharya, Krishna Halder, Swagata Dasgupta, Anushree Roy PII:

S0141-8130(19)37058-8

DOI:

https://doi.org/10.1016/j.ijbiomac.2020.02.128

Reference:

BIOMAC 14745

To appear in:

International Journal of Biological Macromolecules

Received date:

2 September 2019

Revised date:

12 February 2020

Accepted date:

12 February 2020

Please cite this article as: A.K. Singh, S. Bhattacharya, K. Halder, et al., Restriction of microwave-induced amyloid fibrillar growth by gold nanoparticles, International Journal of Biological Macromolecules(2018), https://doi.org/10.1016/j.ijbiomac.2020.02.128

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© 2018 Published by Elsevier.

Journal Pre-proof

Restriction of microwave-induced amyloid fibrillar growth by gold nanoparticles Anang Kumar Singh,1† Susmita Bhattacharya,1† Krishna Halder,2 Swagata Dasgupta,2 Anushree Roy1a 1

Department of Physics, Indian Institute of Technology Kharagpur.Kharagpur. Pin 721302. India.

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Department of Chemistry, Indian Institute of Technology Kharagpur.Kharagpur. Pin 721302. India.

Email: [email protected]

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Ph: +91 3222 283856 ORCID: 0000-0001-8298-0302

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Abstract: Microwave radiations from various electronic devices are of serious health concern. In

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this article, using spectroscopic measurements, we show that the microwave radiation of strength

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22 dBm and frequency 10 GHz facilitates protein fibrillation. However, this adverse effect of low-field radiation can be restricted by the presence of biocompatible citrate-capped gold

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nanoparticles. The dissipative field by metallic particles is able to disrupt the fibrillar network.

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We believe that the obtained results paved a way to find a therapeutic measure to combat

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fibrillation related diseases.

Keywords: gold nanoparticles, microwave radiation, amyloid fibrils

1 †Both authors contributed equally to this work

Journal Pre-proof 1. Introduction It is now well-established that the growth of amyloid fibrils due to the self-assembly of misfolded proteins is one of the root causes of incurable neurodegenerative diseases. There is a challenge in medical research to find a way to restrict the growth of these fibrils to the least if not to find a therapeutic measure to reverse the process of fibrillation. In the literature, metallic nanoparticles (NPs) are shown either as potential enhancers [1]

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or as inhibitors [2] of fibrillation. In a review article by Fei and Perrette [3] the diverse roles of

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metal NPs in amyloidogenesis are discussed. In general, two mechanisms govern the role of NPs

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in protein fibrillation. Either the NPs act as seeds in protein aggregation and enhance the

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fibrillation process [4,5], or they bind randomly to fibrils and modify the fibrillar structure [2,6,7]. Few other reports claim that these particles render better thermal resistance to the

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adsorbed protein corona and block the temperature mediated denaturation of the molecules [8].

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Though the high curvature of NPs, and hence, high surface energy, is expected to help the proteins to retain their native structure [3], a large number of reports in the literature demonstrate

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their diverse consequences to a variety of extents [9,10].

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The microscopic interaction mechanisms at the nano-bio interface are quite complex. The role of NPs in favor or against fibrillation is mainly governed by their physical and chemical characteristics, such as their morphology, surface charge, and catalytic activity [1,2,11]. The relative strength of various forces via electrostatic, hydrophobic, van der Waals interactions, hydrogen bonding, solvation forces render the observed effect of NPs on fibrillation [12]. In this regard, the stabilizing agent of metal particles plays a vital role [13-15]. While the citratereduced [13] and chitosan-stabilized [15] AuNPs are reported to interfere with the fibrillation, Lglutathione stabilized large AuNPs accelerates amyloidosis.

Interestingly, the same L2

Journal Pre-proof glutathione capped small particles inhibit the process [9]. The role of chirality of the capping molecule (glutamic acid) of AuNPs in suppressing the fibrillation of human serum albumin is discussed in Ref. [16]. Citrate-reduced AuNPs are easily available, biocompatible, and also an efficient carrier of drugs. They can be transported to different organs by the lymph and blood [17]. Thus, the use of citrate-capped AuNPs for therapeutic purposes is quite viable. Like metallic NPs, the whole spectrum of electromagnetic radiation also influences

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biological processes in different ways. Microwave radiation from electronic devices (for

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example, cellphones, TV antenna, etc.) is believed to have adverse effects on our health. In an

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earlier report [18], we have demonstrated the oscillatory behavior of structural rearrangement of

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lysozyme molecules with time, even under a super-low field of radiation. The high field microwave radiation affects the electrostatic interaction of the self-assembly process and results

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in diverse conformations of amyloid fibrils [19]. Furthermore, the thermal effect of radiation is

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shown to enhance the fibrillation process [20, 21]. The combined role of radiation and metal NPs has also been exploited to investigate amyloidogenesis. For example, an amplifying effect of

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high strength microwave radiation on amyloid aggregation in the presence of silver NPs has been

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demonstrated [22]. The above studies motivated us to explore whether the non-thermal effect of radiation can be exploited in restructuring or disintegrating the fibrillar structure. Lysozyme belongs to a class of enzymes called glycoside hydrolases. Its structural rearrangement, aggregation, and fibrillation draw attention because of their immunological importance. Hen egg white lysozyme (HEWL) is a model globular protein, which undergoes fibrillation under different environmental conditions (say at different pH and temperature). In this article, we have demonstrated that the non-thermal effect of super-low field microwave radiation enhances fibrillation. However, the presence of citrate-capped AuNPs can restrict the 3

Journal Pre-proof process. The chosen radiation parameters are very similar to the same we encounter in our daily life. The power dissipated by NPs in the presence of radiation is estimated and found to be enough to disintegrate the amyloid fibrillar network. Various optical spectroscopic techniques are employed to investigate the growth and disintegration of the structure of the fibrils in the presence or absence of AuNPs and under radiation. The study possibly prescribes a biocompatible therapeutic pathway to restrict the growth of amyloid fibrils.

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2. Materials and methods

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2.1 Materials

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HEWL, Thioflavin T (ThT) and Chloroauric acid (HAuCl4) were purchased from Sigma

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Chemical Co. (St. Louis, USA). Sodium citrate, sodium hydroxide, and other chemicals were obtained from SRL India.

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2.2 Preparation of AuNPs in colloidal solution

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Au colloid was prepared following the method of citrate reduction [23]. 240 mg of HAuCl4 was dissolved in 500 ml of water. The solution was heated at ~100oC and simultaneously stirred

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vigorously for 30 mins. Next, 1% sodium citrate (50 mL) was added to it, and the boiling was

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continued for an additional 30 mins. The pH of the as-prepared colloidal solution was 6. For our experimental purpose (to keep the pH same as of fibril solution), the pH of the colloidal solution was further adjusted to pH 12.75 by adding NaOH in the solution. As the addition of excess NaOH leads to an aggregation of particles in the sol, we have centrifuged (5000 rpm for 10 mins) the sol to remove the bigger particles. After centrifugation, the supernatant of pH 12.75 was collected. The excess citrate in the sol was removed by dialysis. Here we would like to mention that we refrained from using a buffer solution to adjust the pH of the colloidal solution, as it is known to destabilize the citrate capped AuNPs [24]. The use of NaOH for controlling the pH of 4

Journal Pre-proof the sol also destabilized the sol. However, it yielded better results than standard buffer solutions (say PBS). 2.3 Growth of HEWL fibrils in the presence and absence of AuNPs The charge state and hence the molecular structure of HEWL depend on the pH of the solution. The isoelectric point (pI) of HEWL is 11.35. At pH 12.75, the net charge of the protein is negative, and it attains a molten globular state. At this intermediate state, the hydrophobic

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surface of the molecule gets more exposed to the solvent, which promotes self-association. Thus,

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reasonably shorter incubation time scale [25, 26].

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under this condition fibrils can be grown even at 37oC, i.e., at physiological temperature, in

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The stock solution of protein was prepared in double-distilled water, and the pH of the solution was adjusted to 12.75 using the NaOH solution. For the fibrillation of HEWL, the final

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concentration of the protein in the solution was 150 μM. Further, to investigate the effect of

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AuNPs on fibrillation, the supernatant of the colloidal solution was added to the stock solution of HEWL in different volumetric ratios, 0.01, 0.15, and 0.45 v/v. HEWL and [HEWL–AuNP]

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solutions were then allowed to incubate for 72 hrs at ∼37◦C for the growth of the fibrils.

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2.4 Microwave radiation setup and experiments The details of the microwave setup are described elsewhere [18]. Briefly, a pair of rectangular waveguides (HEWLETT Packard ×281C (USA)) of dimension 2.3×1.0 cm2 (a×b) was used to obtain the well-defined radiation field distribution. An Agilent MXG analog signal generator (NS183A) with an operational range between 100 kHz–22 GHz was used as a microwave source. Microwave radiation of frequency 10 GHz and 22 dBm (0.15 Watt) power at the source waveguide end was fed into the system as an input.140 μl of all fibrillar samples under study, were irradiated. 5

Journal Pre-proof 2.5 Spectroscopic characterization Tryptophan (Trp) intrinsic fluorescence spectra and Thioflavin T (ThT) fluorescence spectra were measured using a spectrofluorimeter (Fluoromax-4, Horiba Jobin). Fluorescence was monitored using the wavelength of 295 nm for Trp fluorescence excitation. ThT fluorescence spectra were recorded using 450 nm as excitation wavelength. The final concentrations of protein and dye in the solution were 2M and 5 M respectively. Integration time and slit width for each

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incubation with ThT before fluorescence measurements.

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measurement were kept as 0.3 s and 5 nm, respectively. The solution was kept for 2 minutes

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The surface plasmon resonance band of AuNP and absorption band of Trp were recorded

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using an UV-vis spectrometer (Evolution-210, Thermofisher). Micro-Raman spectra were recorded in the back-scattering geometry. A 785 nm diode laser with 3 mW on the sample was

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used as the excitation source. An optical microscope with a 50L× objective lens was used to

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focus the incident light on the sample and also to collect the scattered radiation. The data acquisition time for each Raman spectrum was 300 sec. The spectrometer (iHR 550, Horiba

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USA).

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instruments. Inc.) was equipped with a Peltier cooled CCD detector (model-Synapse, Horiba JY,

2.6 Dynamic light scattering measurements Dynamic light scattering (DLS) measurements were carried out using Zetasizer Nano Zs (Malvern Panalytical‎ ,UK), equipped with a He-Ne laser of wavelength 632.8 nm, at room temperature (25C). The intensity-time autocorrelation was measured at a fixed scattering angle 173. We used 2ml of 150 M of each solution for the DLS measurements. The path length of the cuvette, used in this experiment, was 1cm. 2.7 Microscopic measurements 6

Journal Pre-proof Fluorescence microscopy images were recorded using a Leica DM 2500M (make Germany) microscope equipped with a fluorescence attachment. All images were captured using a 10× objective lens. For Transmission Electron Microscopy (TEM) measurements, the fibrillar solution (with 1:100 dilution in water) was drop-casted on carbon-filmed 300-mesh copper grids and then was allowed to dry in air. The images were recorded using FEI-TECNAI G2 20STWIN (make USA) microscope, operating at 80 kV.

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All experiments were performed thrice on new sets of freshly grown fibrils to check the

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reproducibility of the results.

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3. Results

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3.1 Characteristics of gold nanoparticles

The characteristic TEM image of the as-prepared AuNPs is shown in Fig. 1(a). Particles are

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nearly spherical in shape. The average size of the particles, estimated by measuring the diameter

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of 40 particles from many image frames, is 205 nm. The TEM image of the particles in the supernatant (inset of Fig. 1(a)) reveals the presence of particles, asymmetrical in shape, along

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with the spherical one. Though we removed the large agglomerates by centrifuge, few relatively

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small agglomerates remained in the sol. The surface plasmon resonance (SPR) bands of the asprepared AuNPs and particles in the supernatant are shown by red and black lines in Fig. 1 (b). The peaks of the SPR bands appear at 521 nm and 523 nm, respectively. Nearly unchanged SPR peak position reveals that the average size of the particles in the supernatant is nearly the same as in the as-prepared sol. The broadening of the SPR band of NPs in the supernatant (black line) indicates that the size distribution of the particles is slightly higher at pH 12.75 than in the asprepared colloidal sol, as also revealed from TEM measurements. From the DLS measurement

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(a)

as-prep (b)

pH 12.75

Absorbance (Arb. unit)

50 nm

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20 nm

500

600

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Wavelength (nm)

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Fig. 1. (a) TEM image of as-prepared AuNPs. The inset of the figure shows the same of the particles in the supernatant. (b) SPR bands of as-prepared AuNPs (red) and the same of NPs in the supernatant (black).

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the hydrodynamic radius of the particles is estimated to be 40 nm and zeta-potential of the sol to

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be -30 meV.

3.2 Microscopic evidence of fibrillation in the presence and absence of AuNPs

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The characteristic TEM images of as-prepared HEWL fibrils in the absence and in the presence

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of AuNPs, are shown in Fig. 2(a) and (b), respectively. The same of an as-prepared individual fibril is shown in Fig. 2(c). Fig. 2(d) shows the zoomed view of a fibril grown in the presence of AuNPs. We do not find any signature of binding of AuNPs to fibrils either in the image (b) or (d). In the fibrillar solution, agglomerated AuNPs are observed as dark patches, shown by arrows in Fig. 2(b) and also refer to Fig. 2(e). From the DLS measurements the hydrodynamic radius of the agglomerated particles is estimated to be 100 nm and the zeta potential of the sol (along with fibrils) as -20 meV.

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(a)

(c)

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(d) 100 nm

0.2 m

(e)

0.5 m

25 nm

0.5 m

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0.5 m

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0.2 m

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Fig. 2. TEM images of (a) as-prepared and (b) AuNP incubated HEWL fibrils. (c) The same of an individual as-prepared fibril. (d) Zoomed view of an individual fibril in presence of AuNPs. (e) Clustering of AuNPs in fibrillar solution. The arrows in (b) and (e) mark aggregated particles in the fibrillar solution. (f) and (g) ThT fluorescence images of the same samples as in (a) and (b), respectively.

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ThT is a dye molecule, fluorescence at 527 nm of which is commonly used to visualize fibrillar species resulting from misfolded proteins. Due to the quenching effect of the nearby excitation peak at 450 nm, the fluorescence intensity of ThT at 527 nm is, in general, weak. However, the intensity increases significantly upon binding to the protein aggregates [27]. The ThT images of the fibrils in the absence and in the presence of AuNPs are shown in Fig. 2(f) and (g). From these images we find that in the presence of AuNPs the fibrils form denser clusters than what is observed in the absence of the NPs. In the above results, the ThT fluorescence

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Journal Pre-proof images confirm the formation of the -sheet rich aggregates, and the fibrillar form is confirmed from the TEM images.

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2.0 1.5 1.0

480 520 560 Wavelength (nm)

Fib+AuNPs at 12.75

2.0

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(c)

1.6 0.00

0.5

600 350 400 450 500 550 Wavelength (nm) 0.18 AuNPs at 12.75

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(b)

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2.4 ×10

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0.15 0.30 0.45 Concentration (v/v)

0.16 0.14

(d)

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0.10 500 550 600 650 700 Wavelength (nm)

Absorbance (Arb. unit)

Native HEWL HEWL Fib Fib+AuNPs (0.01) Fib+AuNPs (0.15) Fib+AuNPs (0.45)

Native HEWL HEWL Fib Fib+AuNPs

Intensity (Arb.unit)

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10 × 2.5

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1.2 1.0 0.8 0.6 0.4 0.2

×10

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Intensity (Arb.unit)

ThT Intensity (Arb.unit)

3.3 Spectroscopic studies on the effect of AuNPs on the growth of fibrils

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Fig. 3. (a) ThT fluorescence of HEWL and HEWL fibrils in the presence and absence of AuNPs. (b) Trp fluorescence of HEWL fibrils incubated with different concentrations of AuNPs. The numbers in the bracket for the legends are the volume ratio of the colloidal sol and the fibrillar solution. For comparison, the same of the native HEWL molecules is shown by the solid black line in the figure. (c) The monotonic increase in fluorescence peak intensity of Trp with an increase in AuNP concentration. The solid line is a guide to the eyes. (d) SPR band of AuNPs at pH 12.75 (black line) and the same for its highest concentration (0.45 v/v) in fibrillar solution (red line) of the same pH. The growth of HEWL fibrils in the presence of AuNPs was further studied by spectroscopic techniques. As a controlled test (negative test), we recorded ThT fluorescence spectra of native HEWL at pH 6.0 (black line in Fig. 3(a)). The enhancement of the intensity of the ThT fluorescence band due to fibrillation in the presence and absence of AuNPs can be seen by 10

Journal Pre-proof following blue and purple lines in Fig. 3(a). The fibrillation can also be identified by measuring the intrinsic fluorescence intensity of the amino acid residues in the sequence [28,29]. For example, it has been shown that the fibrillation of insulin molecule, a globular protein like HEWL, can be monitored from the fluorescence intensity of the amino acid residues (say Tyr) in its structural sequence [25]. Fig. 3(b) plots the change in fluorescence intensity of the Trp molecules of the fibrils, incubated in different concentrations of AuNPs. Trp residues in native

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HEWL molecule exhibit fluorescence at 350 nm (shown by the solid black line in Fig. 3(b)). In

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the globular structure of the native protein molecule, out of the six Trp residues, three are buried

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in a hydrophobic domain. The unfolding of the molecular structure in fibrils exposes buried Trp

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residues [30] and enhances the Trp fluorescence intensity (see the solid blue line in Fig. 3(b)). We find that with the increase in concentration of AuNPs, the fluorescence intensity increases

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(follow the solid colored lines in Fig. 3(b) for different volumetric concentrations of AuNPs).

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The monotonic rise in fluorescence intensity of Trp with the increase in the concentration of AuNPs is shown in Fig. 3(c). The data points and the error bars correspond to the mean and

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standard deviation of the results obtained from three independent sets of measurements. Here we

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would like to mention that all spectra were recorded after incubation of fibrils in different concentrations of AuNPs for three days. With the increase in incubation time, we have observed a further increase in the intensity of the Trp band in all cases following the same sequence, as seen in Fig. 3(c). This observation prompted us to believe that, along with fibrils, protein monomers also exist in the solution. The solid black line in Fig. 3(d) describes the characteristic SPR band of AuNPs at pH 12.75 (same as shown in

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Fig. 4. Raman spectra of native HEWL (black symbols and lines) and HEWL fibrils (red symbols and lines) over the spectral range between (a) 450650 cm-1 and (b) 11401755 cm-1. Raman spectra of HEWL fibrils in the absence (red symbols and lines) and the presence (green symbols and lines) of AuNPs over the same spectral range are shown in (c) and (d), respectively. Difference spectra in all panels are shown by blue symbols. The solid blue lines are the difference in the fitted spectra. Fig. 1(b)). The resonance peak appears at 523 nm. The spectrum (shown in red line) of the

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AuNPs in the fibrillar solution exhibits the absorption peak at 544 nm.

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Furthermore, the molecular structure of HEWL fibrils incubated with AuNPs (0.45 v/v) is investigated using Raman spectroscopy. Fig. 4(a) plots Raman spectra of native HEWL (black + symbols) and HEWL fibrils (red + symbols) in the absence of AuNPs over the spectral range between 450 and 645 cm-1. The same over the spectral window between 1140 and 1755 cm-1 are shown in Fig. 4(b). All spectra are normalized with the total area under the curve. The expected Raman modes over the given spectral range are tabulated in Table 1 [31]. The solid lines are net fitted spectra obtained by deconvoluting each spectrum with the expected Raman modes by Lorentzian functions. The blue symbols follow the difference spectra. Below we discuss the 12

Journal Pre-proof main spectral features. In Fig. 4(a) the Raman spectrum of HEWL molecules exhibit vibrational modes of the S-S bonds at 520 and 570 cm-1. Fig. 4(b) is dominated by amide-III band between 1220 and 1380 cm−1 and amide-I band between 1635 and 1760 cm−1. Vibrational Raman peak of CH2 appears at 1447 cm−1. The sharp Raman peak at 1550 cm-1 is contributed by the Trp W3 mode. The difference spectrum (blue o symbols) in panel (a) reveals a shift in the S-S band in case of fibrils with respect to that of the native protein.

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Table 1. Assignment of vibrational bands of HEWL solutions in the absence and presence of AuNPs

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using Ref. [31]

Raman shift in cm-1 HEWL

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(S–S) Tyr , Phe Amide III CH2 deformation Trp W3 Amide I

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

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520 570 1210 1220-1380 1447 1550 1635–1760

Corresponding vibration

Such observation is a signature of a change in the molecular structure on fibrillation. The HEWL

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protein comprises of -helices, -sheets, and turns. Raman spectra of-helix, and -sheet of the amide-I band contribute over the spectral range of 1650–1660 cm-1 and 1665–1672 cm-1. In panel (b) we observe a clear signature of an increase in the -sheet content in case fibrils compared to the native protein. The increase in Raman intensity of Trp at 1550 cm -1 further confirms the exposure of Trp residues in fibrils, as concluded above from the measurements of fluorescence intensity of Trp in Fig. 3(b). Fig. 4(c) and (d) compare Raman spectra of HEWL fibrils in the absence (red symbols and solid lines) and in the presence (green symbols and solid 13

Journal Pre-proof lines) of AuNPs, for the highest concentration of the particles (0.45 v/v) in the fibrillar solution, over the spectral range of 450–650 cm-1 and 1140–1755 cm-1. We do not observe any significant change in the spectra in these two cases, indicating that the molecular structure of fibrils does not get affected significantly in the presence of AuNPs. For other concentrations of AuNPs, we observed very similar spectra, as described above. 3.4 Effect of microwave radiation on HEWL fibrils in the presence and absence of AuNPs

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We investigate the effect of low field microwave radiation on the fibrillation of HEWL in the

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presence (0.45 v/v) and the absence of AuNPs. First, we plot the intensity of π − π⋆ singlet-

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singlet absorption band of Trp at 275 nm of HEWL fibril in the presence (solid red line) and

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absence (solid black line) of AuNPs in Fig. 5(a). The increased intensity of the absorption band in the presence of AuNPs corroborates the results obtained in Fig. 3(b) (based on fluorescence

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measurements), indicating a higher concentration of fibrils in the solution in the presence of NPs.

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Next, we carry out the same spectroscopic measurements to study the effect of microwave radiation. The increase in absorption intensity of Trp in fibrillar solution (violet line in Fig. 5(a))

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indicates that the microwave radiation triggers the formation of fibrils (in the absence of AuNPs)

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in the solution (compare the black and violet solid lines in Fig. 5). However, in the presence of AuNPs, the growth appears to be restricted (compare the red and green solid lines in Fig. 5(a)). Fig. 5(b) summarizes the results obtained from absorption measurements. In the absence of AuNPs, microwave radiation enhances the intensity of the Trp absorption band by 80%. The data points and the error bars correspond to the mean and standard deviation of the results obtained from three independent sets of measurements. However, the presence of AuNPs halts this increment.

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Fib Irr

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Fib+AuNP

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(b)

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Fib+AuNP Irr

Fib

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% change in Trp absorption intensity

(a)

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Fig. 5. (a) UV absorption spectra of Trp for HEWL fibril before (black) and after (violet) microwave radiation of 30 mins. The spectra in the presence of AuNPs before and after radiation are shown by red and green lines. The inset shows the magnified view of the spectra, following the same color code, over the range between 300 nm and 580 nm. (b) Percentage change in Trp absorption intensity under different conditions.

The inset of Fig. 5(a) plots a magnified view of the absorption spectra between 300 nm and 600 nm. This range lies in the tail of the Trp band. Photoinduced charge transfer from/to specific amino acid residues and/or formation of intrinsically disordered structure gives rise to multiple absorption bands over this spectral range, and it increases the intensity of the tail of the 15

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Fig. 6. Raman spectra of HEWL fibrils before (black symbols) and after (red symbols) microwave radiation. (a) and (b) are spectra for fibrils in the absence of AuNPs. (c) and (d) are the same in the presence of AuNPs. Trp absorption band [32]. These high wavelength spectral signatures are a spectral characteristic

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of the formation of the protein oligomers [15]. In Fig. 5(a) and its inset, by comparing the

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absorption spectra of HEWL fibril in the presence (solid red lines) and in the absence (solid black lines) of AuNPs, we find that metallic particles facilitate the formation of both fibrils and protein oligomers. The microwave radiation alone also promotes fibrillation (compare black and violet lines). However, under the combined effect of AuNPs and radiation, the intensity of the absorption band of Trp band is the same as as-prepared fibrils, the spectral intensity over the range between 300 nm and 600 is appreciably high compared to that of as-prepared fibrils, indicating the formation of oligomers under radiation.

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Journal Pre-proof The characteristic normalized Raman spectra of irradiated (solid red lines) and unirradiated (solid black lines) fibrils in the presence and absence of AuNPs are shown in Fig. 6. Observed Raman modes are discussed earlier for Fig. 4. The panel 6(a) compares the spectral profile over the range between 450 and 650 cm-1 of the unirradiated and irradiated as-grown HEWL fibrils. The same for the AuNPs incubated fibrils are shown in Fig. 6(c).

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corresponding spectral profiles over the range between 1440 and 1750 cm-1 are shown in panel

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(b) and (d). From the difference spectra (blue symbols and lines), we do not find any significant

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change in the spectra on irradiation in both cases.

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4. Discussion

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Role of AuNPs on fibrillation

The gradual increase in the measured fluorescence intensity of Trp (of fibrils) with the increase

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in the concentration of AuNPs in the solution, as shown in Fig. 3(b), indicates that the presence

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of NPs aids fibrillation. In Fig. 5(a), we observe an increase in the intensity of the absorption band of Trp of fibrils in the presence of AuNPs. As mentioned earlier, the exposure of all six

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Trp residues of HEWL upon fibrillation, indeed, can increase the fluorescence absorption

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intensity. However, it is to be noted that in Fig. 3(b) and in Fig. 5(a) we do not observe any shift in the fluorescence or absorption band of the Trp residue of fibrils in the presence and absence of AuNPs. The binding of AuNPs at the Trp sites is expected to give rise to a change in the environment of the residue and hence, a shift in the fluorescence and absorption peaks due to charge transfer [33-35]. This suggests that particles do not bind to fibrils at the Trp sites. Though, this does not rule out the binding of particles with any other site of the fibrils. Further information can be obtained from a study of the SPR band of AuNPs in Fig. 3(d). The SPR band of metal NPs carries crucial signatures of the environment of the particles. For 17

Journal Pre-proof Mie scattering of light by particles, the absorption maximum of the SPR band depends on the particle-size and complex dielectric constant of the particles (0()) and the surrounding medium (m) [36]. The binding of the particles with the biological matrix or the change in the stabilizing surface charge of the particles in the solution can vary m; hence, it can result in a shift of the SPR maximum. The measured dielectric constant of the colloidal solution does not change appreciably in the presence of fibrils. Thus, we believe the change in the value of m is not the

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origin of the shift in the spectral peak. The observed aggregation of the particles in the fibrillar

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solution, as seen from the TEM images and also obtained from the DLS measurements, can give

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rise to a redshift of the absorption spectrum, as observed in Fig. 3(d). However, this does not

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irrefutably rule out the possibility of binding of the particles with fibrils, as both size effect and

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binding with fibrils can contribute to the observed spectral shift. Thus, it is still inconclusive to comment on whether the particles bind with HEWL fibrils in the solution.

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The role of AuNPs on fibrillation is further probed by studying the molecular structure of the fibrils in their presence and absence using Raman spectroscopic measurements. The

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vibrational frequencies of molecules probed by Raman spectroscopy are sensitive to their

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bonding environment. The binding of amino acid residues with AuNPs is expected to modify the bond strength and hence a shift in the Raman band. If we compare the Raman spectra of the fibrils in the presence and absence of AuNPs in Fig. 4(c) and (d), we do not observe any clear change in spectral features.

Thus, we believe that the particles act only as a catalyst for

fibrillation, which could be revealed from the fluorescence and absorption spectra of Trp (in Fig. 3(b) and Fig. 5(a)). AuNPs render an alternative pathway for fibrillation, possibly with lower activation energy. As the overall molecular structure of the fibrils remains the same, it is reasonable to believe that the thermodynamics of the process remains essentially the same. 18

Journal Pre-proof According to a recent report [37], pH 6 favors citrate interaction of AuNPs with HEWL (mainly with positively charged Lys and His residues). In contrast, at pH 9, the molecules are inert for electrostatic interaction with the particles. The catalytic effect of AuNPs in enhancing the fibrillation dynamics is reported in Ref. [38]. Effect of microwave radiation on fibrillation Next, we look into the effect of microwave radiation on protein fibrils. The increase in

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the intensity of the UV absorption band of Trp compared to that of native HEWL in Fig. 5(a)

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(compare black and violet lines) indicates that in the absence of AuNPs, the radiation facilitates

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the fibril formation. The unchanged Raman spectral profiles of the fibrils before and after

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radiation in Fig. 6(a) and (b) reveal that the molecular structure of fibrils, formed by radiation, essentially remains the same. Interestingly, we find in Fig. 5(a) (compare black and green lines)

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that the presence of AuNPs restricts the adverse increase in the fibrillar concentration under the

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microwave radiation.

We investigate the possible effects of microwave radiation on enhancing fibrillation, as

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observed experimentally. The radiation power P in a rectangular waveguide of dimension a×b

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depends on the field amplitude E0 by

P

ab E02 , where the wave impedance * 2 Z mn

* Z mn  Z 0 /(1  f r2 ) 0.5 , f r  f c / f with Z0=120π ohm as the free space wave impedance. fc is the

cut off frequency and is given by c/2a for the TE10 mode (c=3×108 m.s-1). f is the frequency of the radiation used. Using the above relation and other parameters (a =2.6 cm and b=1 cm of the waveguide, f=10 GHz, and P=22 dBm (= 0.15 Watt)), we estimate the field E0 as = 6 ×10-8 V/Å. Absorbed radiation increases the temperature of a system. For the power absorbed (Pabs) over a

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Journal Pre-proof period of time (dt), the rise in temperature (dT) is given by dT 

Pabs 2f E 2 tan  dt  dt, with c p c p

tan ′′′ as the dielectric loss factor.   and ′′ are the real and imaginary components of the dielectric constant of the material at a frequency f.  and cp are the density and specific heat capacity of the same material. At 10 GHz radiation, the values of   and   for HEWL in water are 5.7 and 1.96 [39]. Taking the value of cp as 4.5×103 J.Kg-1.oC-1 [40] and as 1000 Kg.m-3

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[41], the rise in temperature of the molecule can be estimated to be only 2oC, which is much

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lower than the temperature (70oC) required to denature a HEWL molecule [42]. Thus, it is

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reasonable to exclude any thermal effect to enhance fibrillation under microwave radiation,

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observed spectroscopically in Fig. 5(a). In a recent report [18] it has been shown by molecular dynamics simulation and spectroscopic measurements that super-low-field microwave radiation

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can result in a significant structural rearrangement of HEWL molecule. The native molecules

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attain a slacked structure under the low radiation field for a longer duration (~30 mins) in aqueous solution at pH 7. At pH 12.75 the molecules are partially unfolded. We believe that the

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further perturbative effect of radiation facilitates fibrillation in the absence of AuNPs.

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To find the possible reason for restricted fibrillation in the presence of AuNPs, we estimate the power dissipated, Pdis, by the particles for the given frequency and the electric field of radiation using the relation Pdis  K fE 20 . K is a constant (55.6310-12 Farad/meter) [43]. The calculated value of Pdis is 10-14 Joule/sec by each particle. The thermodynamics involved in the mechanism of unfolding and fibrillation of tetramer of Transthyretin (molecular weight ~14 kDa) has been studied by Hammarström et al. [44]. It has been shown that the activation energy required to disintegrate an amyloid structure is ~ 10-20 Joule [25]. The possibility of

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Fig. 7. A cartoon to show the effect of AuNPs and microwave radiation on fibrillation. The presence of AuNPs accelerates the formation of oligomers and fibrils from unfolded protein monomers. Microwave radiation also facilitates the fibrillation process. However, the same radiation disintegrates the fibrils to oligomers in the AuNP environment.

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disintegration of smaller amyloid fibrils by similar energy is also reported in the literature [45].

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We believe that in our case too, the dissipated power by AuNPs is sufficient enough to dissociate the HEWL (molecular weight ~14 kDa) fibrils and restrict their radiation-induced growth

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process. In the inset of Fig. 5(a), the higher intensity of the spectral feature over the range

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between 300 and 600 nm indicates that the radiation, most likely, breaks down the fibrils to oligomers in close proximity. The fibrils, which remain in the solution, retain their molecular structure as revealed from the Raman spectra in Fig. 6 (c) and (d). The role of local heat dissipated by AuNPs, attached to the amyloid beta protein (A), when irradiated by the gigahertz radiation field has been discussed by Marcelo et al [44]. In contrast, the silver nanoparticles are reported to facilitate the formation of aggregates under microwave radiation [22]. Our results strongly suggest the inhibitory role of the combined effect of metal nanoparticles and microwave radiation. We have discussed the possible reason behind 21

Journal Pre-proof it. The spectroscopic measurements indicate that the AuNPs do not bind to the fibrils; they act as a catalyst. HEWL is a medically significant molecule, as it causes hereditary systemic amyloidosis [8,9,46]. Its human counterpart fibrillates in a similar way under similar external conditions [47]. To establish the efficacy of AuNPs in restricting amyloidosis, it is mandatory to quantify the degree of fibrillation in their presence. Here we would like to mention that it is nontrivial to do so by spectroscopic measurements. The interaction (absorption or scattering) cross-

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sections of fibrils and electromagnetic radiation (light) need not be the same in the presence and

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absence of metal particles. The change in spectral intensity only renders a possible trend, which

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we have discussed in this article. We believe that our findings, exploiting microwave technology

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in conjunction with nanomaterials, open a possible pathway to be explored with more rigorous studies, to fight with these incurable medical problems, which causes prolonged human

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suffering.

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5. Summary

In this article, we have demonstrated that the microwave radiation, to which we are

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exposed to in our daily life, has a significant adverse effect—it expedites the process of

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fibrillation. The presence of citrate-stabilized AuNPs plays a multifaceted role in amyloid fibrillation, shown by a cartoon in Fig. 7. The NPs act as a catalyst and enhance fibrillation (along with oligomers). The microwave radiation also promotes fibrillation. However, the combined effect of radiation and AuNPs can curb amyloidogenesis.

Acknowledgements AR and SDG thank DST, Nanomission, Government of India, for financial assistance.

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Authors Statement

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Anang Kumar Singh: Experimental data collection, manuscript preparation Susmita Bhattacharya: Experimental data collection, manuscript preparation Krishna Halder: Fibril growth and DLS measurements Swagata Dasgupta: Conceptualization, writing, reviewing and editing Anushree Roy: Conceptualization, writing, reviewing and editing

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