Spectroscopic investigation of protein-carbon nanoparticle and protein-protein interactions

Abstract

With the recent advances in nanotechnology in the field of pharmaceutical and medical diagnostics, there is growing interest in studying the interaction of various nanoparticles (NPs) with proteins. The dynamic layer of proteins at the NP surface is known as “protein corona,” which determines the ultimate interaction of NPs with living systems. It has often been observed that the physicochemical properties of these protein-coated NPs differ significantly as compared to the bare NPs which may influence the cell uptake as well as its distribution. On the other hand, various proteins have been found to lose their native-like secondary structure and activity when in contact with the NP surface. Therefore, it is utmost important to understand the mechanistic aspects of protein adsorption on the NP surface before their direct introduction into the cellular environments. Semiconductor NPs or quantum dots (QDs) have been widely utilized as a biological optical marker because of their superior and unique photoluminescence (PL) properties as compared to organic dyes. However, these heavy metal-based core-shell QDs do have a significant drawback in biomedical applications due to their cytotoxicity (leaching of toxic metals).Recently, small (<10 nm) carbon nanoparticles (CNPs) have emerged as photoactive metal-free luminescent nanoparticles for biomedical imaging applications due to their low cytotoxicity, smaller size, and easy surface functionalization with comparable PL properties to core-shell QDs. They show unique optoelectronic properties like high photostability, excitation wavelength (λex)-dependent PL, high PL quantum yield, and no PL blinking. These luminescent CNPs can be easily synthesized from various sources and show diverse physicochemical properties. On the basis of their intrinsic structure and composition, they have been sub-classified as graphene quantum dots (GQDs), carbon quantum dots (CQDs), carbon dots (CDs), and polymer nanodots (PNDs).In this thesis, luminescence properties of CDs and PNDs, as well as their interaction mechanism with various proteins have been investigated. An important and unifying feature of the PL of CDs is the λex-dependence of the emission wavelength and intensity. The origin behind the λexdependent PL has been poorly understood in the literature. In the present thesis, the origin behind the λex-dependent PL of CDs has been demonstrated by using a protein nanocage, namely, ferritin. The pHdependent interaction of CDs with an iron transport protein, transferrin have been investigated by means of spectroscopic and single particle imaging techniques. Moreover, the intrinsic PL properties of PND and its interaction with human serum albumin (HSA) with its free and ligandbound state have been investigated. It is important to note that the interaction between PND and HSA was performed with dilute concentration of protein. However, proteins often experience highly crowded environments inside the cellular systems, and as a consequence proteins are always in contact with like proteins or different proteins which affect their structure and activity.At high concentration, proteins tend to self-associate or aggregate through soft protein-protein interactions. It is known that irreversible aggregation of proteins may lead to various kinds of human diseases. Therefore, it is important to know the influence of concentration on the structure and stability of proteins at physiological conditions. In this thesis, the concentration-dependent self-association of serum albumins has been investigated at physiological conditions by means of spectroscopic and microscopic techniques. Moreover, the molecular origin behind the concentration-dependent intrinsic blue fluorescence from HSA has also been explored.