Anti-amyloidogenic property of gold nanoparticle decorated quercetin polymer nanorods in pH and temperature induced aggregation of lysozyme

Quercetin is an abundant plant polyphenol effective against several diseases due to its antioxidant and anti-inflammatory activity. Herein, we report novel polymeric quercetin nanorods and the former decorated with gold nanoparticles for the first time. The prepared conjugates quercetin-polyvinylpyrrolidone (Q-PVP) and quercetin-polyvinylpyrrolidone-gold nanoparticles (Q-PVP-Au) were characterized by UV-visible spectroscopy, Fourier transform infrared, dynamic light scattering, and zeta potential measurements. The surface morphology of conjugates was analyzed by field emission scanning electron microscopy. These conjugates exhibit harmonized rod-like morphology with a narrow size distribution. Furthermore, the quercetin conjugates with nanorod morphology exhibited enhanced and prolonged drug release over a long period. The synthesized conjugates were investigated for lysozyme aggregation kinetics. ThT binding assay, fibril size measurement, and electron microscopy results revealed that conjugates could suppress fibrillogenesis in lysozyme. The highest amyloid aggregation inhibition activity (IC50) was obtained against Q-PVP and Q-PVP-Au at 32 μg mL−1 and 30 μg mL−1 respectively. The amyloid aggregate disintegration activity (DC50) obtained against Q-PVP and Q-PVP-Au was 27 μg mL−1 and 29 μg mL−1 respectively. The present quercetin conjugates exhibit enhanced bioavailability and stability. They were potent inhibitors of lysozyme aggregation that may find applications as a therapeutic agent in neurological diseases like Alzheimer's and Parkinson's.


Anti-amyloidogenic property of gold nanoparticle decorated quercetin polymer nanorods in pH and temperature induced aggregation of lysozyme
Pranita Rananaware a , Parimal Pandit a , Seekha Naik b , Monalisa Mishra b , Rangappa S. Keri a , Varsha P. Brahmkhatri a * a Nanomaterials for Drug delivery and Therapeutics (NDT-Lab), Centre for Nano and Material Science, Jain University, Jain Global Campus, Bengaluru,562112, Karnataka, India b Neural Developmental Biology Lab Department of Life Science NIT Rourkela,Rourkela Odisha,brahmkhatri.varsha@gamil.com

Synthesis of citrate capped gold nanoparticle (Au nanoparticle)
Citrate capped AuNPs synthesized by J. Turkevich method 1 . Chloroauric acid (HAuCl4) is a metallic nanoparticle precursor and tri-sodium citrate used as reducing agent. 100ml of 1mM HAuCl4 was boiled under reflux condenser with continue stirring for 20 min. after boiling 10ml of 38.8mM tri sodium citrate was added dropwise into the boiling solution. Colour of the solution has been changed from pale yellow to colorless and finally dark reddish within few minutes.

Preparation of standard stock solution:
The standard stock solution of quercetin was prepared by dissolving 2 mg drug in 50 ml of methanol, in volumetric flask to get a solution containing 40µg/ml of drug. Series of solution (1µg/ml, 2µg/ml, 4µg/ml, 8µg/ml, 12µg/ml, 16µg/ml, 20µg/ml, 24µg/ml, 28µg/ml, 32µg/ml, 36µg/ml, 40µg/ml) for the calibration curve prepared in volumetric flask by taking appropriate aliquots of working standard solution of quercetin withdrawn and diluted up to 10 ml with methanol.
Electronic Supplementary Material (ESI) for RSC Advances. This journal is © The Royal Society of Chemistry 2022

Construction of calibration curve:
Series of solution (1µg/ml, 2µg/ml, 4µg/ml, 8µg/ml, 12µg/ml, 16µg/ml, 20µg/ml, 24µg/ml, 28µg/ml, 32µg/ml, 36µg/ml, 40µg/ml) for the calibration curve prepared in volumetric flask by taking appropriate aliquots of working standard solution of quercetin withdrawn and diluted up to 10 ml with methanol. Quercetin shows absorption maxima at 372 nm. The concentration of drug in unknown samples from drug release studies were determined from the absorbance to concentration of the drug from Figure S1.

FTIR stretching frequencies for Q, AuNP, Q-PVP and Q-PVP-Au
The new FTIR spectra for Q, AuNP, Q-PVP and Q-PVP-Au for the confirmation of additional group. We noted some additional groups are present. We observed the peak and intensity changes due to the conjugation of PVP and AuNP with quercetin.

Stability profile of Quercetin.
Stability profile of Q was studied by the UV-visible spectroscopy for the several days. Stability of Q examined with a specific time interval by dissolving the free Q in Milli Q water at room temperature.

Drug loading at different morphology of Q-PVP and Q-PVP-Au
We have calculated the loading amount of Q under different morphology of Q-PVP, Q-PVP-Au. The Q drug loading for the different morphology in Q-PVP have been included for the table S3 and the Q drug loading for the different morphology for Q-PVP-Au have been included in the table S4 along with specific time.

Fourier transform infrared (FT-IR) measurement of lysozyme aggregates
FTIR spectra for validation of functional groups of lysozyme aggregates treated by PVP, Quercetin, Au NP, Q-PVP and Q-PVP-Au. β-sheet structure is likely to form mature fibrils in HEWL, hence forth the secondary structure of HEWL was verified by FT-IR spectroscopy. FTIR analysis was used to characterize the secondary structure of HEWL aggregates. HEWL, 0.2 mg/mL was dissolved in 2mL of 0.1% HCl (pH 1.6) and heated up to 65°C for 24h and stirred at 550 rpm. Then, samples were mixed with the KBr to make a pallet, followed by air drying before FTIR measurements. HEWL samples with and without nanoconjugates were taken and analyzed for FT-IR measurement. Figure S3: A) FTIR spectra for validation of functional groups of lysozyme aggregates treated by PVP (red line), Quercetin (green line), Au NP (pink line), Q-PVP (purple line), Q-PVP-Au (blue line); B) Shows the same spectra but zoomed into area of interest from 2000 cm-1 to 500 cm-1 to assign specific transmission bands to some functional groups.