Effect of crude and carboxyl-functionalized carbon nanotubes on protein–protein interaction

Abstract

Carbon nanotubes (CNTs) could serve as nanovehicle for drug delivery. It is proposed that this class of emerging materials is capable to penetrate into cells without causing cell death. However, relatively little is known about the potential biological risks from CNTs. Nanoparticles present enormous surface areas and are found to enhance the rate of protein fibrillation by decreasing the lag time for nucleation. Protein fibrillation is associated with protein misfolding diseases including Type II Diabetes, Alzheimer’s, Parkinson's, Creutzfeld-Jacob disease and Dialysis-related amyloidosis. In the present study, via Congo red binding assay and circular dichroism (CD) spectroscopy we investigated the amyloidogenic effects of crude and carboxylated-CNT on the in-vitro fibrillogenesis of hen-egg wight lysozyme at acidic condition. Our results showed that the formation of lysozyme amyloid fibrils at pH 2.0 and 55°C was remarkably increased by the addition of crude unmodified CNTs, whereas carboxylated CNTs did not enhance the probability of appearance of a critical nucleus for nucleation of protein fibrils. Introduction: Recently, considerable effort has been generated to characterize intermediate structures accumulated during protein folding and unfolding (1). Interestingly growth of toxic amyloid structures, is supposed to be associated with various late-onset neurodegenerative disorders, initiated from intermediate structures interaction (2, 3). The potential of nanoparticles to promote protein fibrillation is demonstrated (4). Previous investigations by different research groups showed that exposure of mammalian cells to CNTs caused inhibition of cell proliferation (5), activation of nuclear transcription factor-κB (6) and also increase in oxidative stress and accumulation of peroxidative products (7). Our previous results showed no significant decrease in viability of cells exposed to different concentrations of carboxylated-CNTs (8). Additionally; no significant increase in ROS was seen at CNT concentrations ranging from 1 to 15μgml−1 (8). Also, our finding confirmed that carboxylated-CNTs probably have no interaction with the expression of the iNOS protein (8). In the present study, acidic fibrillation of hen-egg wight lysozyme (HEWL) was analyzed in the presence of prepared crude and carboxylfunctionalized nanotubes. Materials & Methods: Preparation of carbon nanotubes: As-received multi-walled CNTs (10 mg) were suspended in concentrated H2SO4:HNO3 solution (3:1 v/v) and sonicated in a sonication bath for 4 h at 40°C. The CNT suspension was diluted with distilled water and a CNT mat was obtained after filtration through a 0.45 μm polycarbonate membrane. The CNT mat was washed until no acid was detected and then was suspended in 25 ml deionized water (di-H2O) by sonication. IR-spectroscopy was used to verify the CNT functionalization. Amyloid fibril formation condition: A sample solution of 2 mg/ml HEWL in di-H2O (pH 2.0) with salts (136 mM NaCl, 2.7 mM KCl) was prepared. Nanotubes at two final concentrations (2.5 and 10 μg/ml) were added to HEWL solution. HEWL sample solutions were incubated at 55°C for different times. Congo red binding assay: Increase in the absorbance intensity combined with a red shift in the absorbance spectrum of Congo red, is a method of choice to characterize amyloid fibrillation (9). The absorbance spectrum was acquired on Shimadzu UV-visible Spectrophotometer (Kyoto, Japan) in the 400to 600-nm region. Far UV-Circular Dichroism (CD): Far-UV CD spectra were acquired on Aviv 215 spectropolarimeter (Aviv Associates, Lakewood, NJ). A quartz cell with 0.1 mm light path and a protein concentration of 0.6 mg/ml was used to record CD spectrum from 190 to 260 nm. Results and Discussion: The IR spectrum of the acid-treated multi-walled CNTs showed peaks at 3424, 1719 and 1626cm attributed to the hydroxyl, carboxyl and carbonyl functional groups, respectively. Nanoparticles present enormous surface areas and are found to enhance the rate of protein fibrillation by decreasing the lag time for nucleation (4). In this study, effect of nano tubes with polar and non-polar surfaces on the process of amyloid fibrillation was investigated. Results from Congo red absorption spectroscopy demonstrated the formation of fibrillar amyloid structures at 72 hours after the initiation of HEWL incubation in amyloidogenic condition. In addition, CD spectra of HEWL exhibited a characteristic pattern of β-sheet conformation (Fig. 1). In exploring if CNT exerted a stimulatory action on the formation of HEWL fibrils, we monitored the changes of Congo red binding and CD spectra in amyloidogenic conditions. Our observations indicated that crude CNT could outstandingly enhance amyloid fibril formation while CNT in carboxylated form had no significant effect on fibrillation process. It is concluded, introduction of polar groups on the hydrophobic surfaces may play an important inhibitory role on surface-assisted nucleation.