Functionalisation of Polyvinylpyrrolidone on Gold Nanoparticles Enhances Its Anti-Amyloidogenic Propensity towards Hen Egg White Lysozyme

Protein amyloids are characterized by aggregates that usually consist of fibres containing misfolded proteins and having a cross β-sheet conformation. These aggregates can eventually lead to several degenerative diseases like Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), Huntington’s disease and Parkinson’s disease. The present study describes the effect of chemically synthesized polyvinylpyrrolidone (PVP)-conjugated gold nanoparticles (PVP-AuNps) on hen egg white lysozyme (HEWL) amyloids. The synthesized nanoparticles have been characterized using various biophysical techniques like Ultraviolet-Visible (UV-Vis) Spectroscopy, Transmission electron microscopy (TEM), X-ray diffraction (XRD) analysis, dynamic light scattering (DLS), zeta-potential measurement and Fourier transform infrared spectroscopy (FTIR). The aggregation studies showed that PVP acts as a partial inhibitor of HEWL amyloidogenesis. However, when conjugated to gold nanoparticle surface, it leads to complete inhibition of amyloid formation. Apart from inhibition, PVP-conjugated gold nanoparticles also exhibited a significant disaggregation effect on mature amyloids and hence can be exploited as an effective therapeutic agent against hereditary systemic amyloidosis.


Particle Size Analysis by Dynamic Light Scattering (DLS)
This measures the hydrodynamic size that can be defined as the size of a sphere that has the same translational diffusion co-efficient as that of the particle [1]. From the data ( Figure 2) it is evident that for the majority of the nanoparticles, size ranges from 50 to 150 nm with peak at 120.7 nm. In addition there are certain particles of size less than 20nm. Thus, it can be concluded that the solution has particles of different sizes (both large and small sized nanoparticles). The Z-average value (d.nm)(average gold nanoparticle size) and the polydispersity index (PDI) value from the report are 95.04 nm and 0.406 respectively ( Table 1). The PDI value is less than 0.7 thereby indicating that the sample does not have a very broad size distribution and thereby ideal for dynamic light scattering (DLS) technique.

Zeta Potential Distribution Study
Electrostatic stabilization can be estimated by measuring the zeta potential, which is the charge at the surface of the electrical double layer [2].The zeta potential value from the report is -7.32 (figure 3a) which indicates that PVP-capped nanoparticles have low magnitude of zeta potential. A negative zeta potential value indicates that the particles are negatively charged. Thus, the nanoparticles coated with PVP remain dispersed in part by zeta potential and in part due to the steric hindrance created by

X-Ray Diffraction (XRD) Analysis
The XRD pattern shown in Figure 4 indicates fingerprints of both PVP and gold. The broad peaks at 2Ɵ=9-15˚ and 2Ɵ=19-25˚ are associated with the amorphous nature of PVP [3]. Diffraction peaks corresponding to (111), (200), (220) and (311) planes (Table 2) correspond to the face-centeredcubic (FCC) structure of gold. This depicts that the product is composed of pure crystalline gold and PVP which may be existing together while interacting with each other via covalent bonds. The crystal size can also be calculated from the XRD data using the Scherrer equation (1) [4], where Dp is average crystallite size, β is line broadening in radians, θ is Bragg angle, and λ is X-ray wavelength. (1) Considering the wavelength (ʎ) = 1.54056 and the peak corresponding to 2Ɵ=38.56, full width at half maxima (β) can be calculated. The β value can be calculated from the graph (=0.2 degrees). Thus, the average crystal size can be calculated from the above equation which comes to a value of 43.97nm.

Fourier Transform Infrared (FTIR) Spectroscopy
The FTIR spectra of pure PVP and PVP-AuNps are shown in Figure 5. The absorption peaks at 1463cm -1 and 1424cm -1 were characteristic absorptions peaks of the pyrrolidinyl group of PVP. Also, peaks at 1643cm -1 and 1283cm -1 are due to the C O and C-N vibrations in PVP [5]. The typical peaks in pure PVP also existed in PVP-AuNps indicating that PVP molecules were adsorbed onto the surface of gold nanoparticles. Thus, it can be concluded that appropriate functionalisation has taken place. The FTIR spectra of bare AuNps was also taken but no significant peaks were found in this range (data not shown).

Transmission Electron Microscopy (TEM)
The morphology of PVP-coated Au NPs can be studied with TEM. Figure

X-Ray Energy Dispersive Spectrometry (EDS)
Chemical analysis can be done in a TEM microscope by X-rays energy dispersive spectrometry (EDS). It can be used for the identification and quantification of chemical elements [6]. An EDS spectrum has peaks characteristic of chemical elements. EDS Spectrum is shown in Figure 7.