Engineered nanoparticles provide a powerful scaffold for interfacing with proteins. The nanoparticle surface can be tailored to present recognition elements, providing surface complementarity to interact with protein surfaces. In this thesis, I have explored both the fundamental and the applied aspects of this interaction. On the fundamental side, I have co-engineered the nanoparticles and the proteins to generate robust dyads with strong binding affinity even at high salt concentration. Fluorescence titrations and docking studies were carried out to quantify the binding properties of the nanoparticles and proteins. Those studies revealed the prospect of tuning the affinity between the nanoparticles and proteins by co-engineering. On the application side, I have employed nanoparticle-protein interaction to fabricate self-assembled nanostructures to be used as intracellular protein delivery tools. In the first segment, nanoparticles and proteins were assembled to form nanoparticle stabilized capsules (NPSCs) for nuclear trafficking of proteins. The first non-peptide synthetic nuclear localization signal based on boronate was discovered, as well, using NPSC delivery platform. In the second segment, proteins and nanoparticles were co-engineered to self-assemble into hierarchical multi-layered nanostructures. These nanostructures were employed to deliver encapsulated proteins into cell cytosol, establishing a general strategy for protein delivery. Using this technology, I have delivered CRISPR/Cas9-ribonucleoprotein that resulted in highly efficient gene editing. Further, I have created an integrated nanotechnology/biology approach to engineer macrophages in vitro, thus, greatly enhancing their ability to phagocytose tumor cells, providing a new immunotherapeutic strategy for cancer therapy.

In recent years, the fabrication of nanomaterials and exploration of their properties have attracted the attention of various scientific disciplines such as biology, physics, chemistry, and engineering. Although nanoparticulate systems are of significant interest in various scientific and technological areas, there is little known about the safety of these nanoscale objects. It has now been established that the surfaces of nanoparticles are immediately covered by biomolecules (e.g. proteins, ions, and enzymes) upon their entrance into a biological medium. This interaction with the biological medium modulates the surface of the nanoparticles, conferring a “biological identity” to their surfaces (referred to as a “corona”), which determines the subsequent cellular/tissue responses. The new interface between the nanoparticles and the biological medium/proteins, called “bio-nano interface,” has been very rarely studied in detail to date, though the interest in this topic is rapidly growing. In this book, the importance of the physiochemical characteristics of nanoparticles for the properties of the protein corona is discussed in detail, followed by comprehensive descriptions of the methods for assessing the protein-nanoparticle interactions. The advantages and limitations of available corona evaluation methods (e.g. spectroscopy methods, mass spectrometry, nuclear magnetic resonance, electron microscopy, X-ray crystallography, and differential centrifugal sedimentation) are examined in detail, followed by a discussion of the possibilities for enhancing the current methods and a call for new techniques. Moreover, the advantages and disadvantages of protein-nanoparticle interaction phenomena are explored and discussed, with a focus on the biological impacts.

Engineering the surface functionality of nanomaterials is the key to investigate the interactions between nanomaterials and biomolecules for potent biological applications such as therapy, imaging and diagnostics. My research has been orientted to engineer both of the surface monolayers and core materials to fabricate surface-functionalized nanomaterials through the synergistic multidisciplinary approach that combine organic chemistry, materials science and biology. This thesis illustrates the design and synthesis of the surface-funcitonalized quantum dots (QDs) and gold nanoparticles (AuNPs) for the fundamental studies and practical applications. For QDs, A new class of cationic QDs with quaternary ammonium derivatives was synthesized to provide permanent positive charge. The toxicity and stability of these cationic QDs were systematically investigated in cells. Furthermore, these cationic QDs were employed in the design of biosensor and fabrication of functional nanofibers. QDs with different surface functionalities were also used in the studies of controllable cellular uptake of nanoparticles and the construction of multifunctional nanocapsules. For AuNPs, cationic AuNPs were used to interact with proteins. The monolayer on AuNP surface can be tailored through host-guest chemistry to modulate the protein behaviors. Also, mass spectrometry was applied to detect the templation of AuNP monolayers to protein surface through molecular recognition. Taken together, nanomaterials with desired properties can be fabricated through surface functionalization approach to facilitate their use in biological system.

Nanoparticle-protein conjugates have a variety of applications in imaging, sensing, assembly and control. The nanoparticle-protein interface is made of numerous complex interactions between protein side-chains and the nanoparticle surface, which are likely to affect protein structure and compromise activity. Ribonuclease S and cytochrome c are covalently linked to nanoparticles via attachment to a specific surface cysteine, with the goal of optimizing protein structure and activity, and understanding conditions that minimize non-specific adsorption. Protein behavior is explored as a function of the nanoparticle surface chemistry and material, the density of proteins on the nanoparticle surface, and the position of the labeled site. Ribonuclease S is attached to Au nanoparticles by utilizing its two-piece structure. Enzymatic activity is determined using RNA substrate with a FRET pair. Conjugation lowers the ribonucleatic activity, which is rationalized by the presence of negative charges and steric hindrance which impede RNA in reaching the active site. Cytochrome c is linked to Au and CoFe204 nanoparticles. The protein is denatured when the nanoparticle ligands are charged, but remains folded when neutral. The presence of salt in the buffer improves folding. This indicates that electrostatic interactions of charged amino acids with the charged ligands are prone to lead to protein denaturation. The attachment site can be controlled by mutations of surface residues to cysteines. Protein unfolding is more severe for nanoparticle attached in the vicinity of charged amino acids. Molecular dynamics simulations of the conjugate reveal that electrostatic interactions with· the nanoparticle ligand lead to local unfolding of [alpha]-helices of cyt c. Furthermore, the nanoparticle induces more structural disturbance when it is attached on the N- and C-terminal [alpha]-helices foldon, which is the most stable motif of cyt c and the most essential for folding.

Nanoparticles have numerous biomedical applications including drug delivery, bone implants and imaging. A protein corona is formed when proteins existing in a biological system cover the nanoparticle surface. The formation of a nanoparticle–protein corona, changes the behaviour of the nanoparticle, resulting in new biological characteristics and influencing the circulation lifetime, accumulation, toxicity, cellular uptake and agglomeration. This book provides a detailed understanding of nanoparticle–protein corona formation, its biological significance and the factors that govern the formation of coronas. It also explains the impact of nanoparticle–protein interactions on biological assays, ecotoxicity studies and proteomics research. It will be of interest to researchers studying the application of nanoparticles as well as toxicologists and pharmaceutical chemists.

Nanoparticles for Biomedical Applications: Fundamental Concepts, Biological Interactions and Clinical Applications brings into one place information on the design and biomedical applications of different classes of nanoparticles. While aspects are dealt with in individual journal articles, there is not one source that covers this area comprehensively. This book fills this gap in the literature. Outlines an in-depth review of biomedical applications of a variety of nanoparticle classes Discusses the major techniques for designing nanoparticles for use in biomedicine Explores safety and regulatory aspects for the use of nanoparticles in biomedicine