Direct cytoplasmic delivery of gene editing nucleases such CRISPR/Cas9 systems and therapeutic proteins provides enormous opportunities in curing human genetic diseases, and assist research in basic cell biology. One approach to attain such a goal is through engineering nanotechnological tools to mimic naturally existing intra- and extracellular protein delivery/transport systems. Nature builds transport systems for proteins and other biomolecules through evolution-derived sophisticated molecular engineering. Inspired by such natural assemblies, I employed molecular engineering approaches to fabricate self-assembled nanostructures to use as intracellular protein delivery tools. Briefly, proteins and gold nanoparticles were co-engineered to carry complementary electrostatic recognition elements. When these materials were mixed together, they formed highly sophisticated, multi-layered, and hierarchical self-assembled nanostructures of few hundred-nanometer size. These structures carried a large number of engineered proteins, got fused to cell membrane upon incubation, and delivered the encapsulated protein content directly into cell cytoplasm. Using this technology, we delivered a wide range of proteins, and CRISPR/Cas9-ribonucleoprotein that resulted high efficient gene editing.

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.

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.

This book is devoted to the engineering of protein-based nanostructures and nanomaterials. One key challenge in nanobiotechnology is to be able to exploit the natural repertoire of protein structures and functions to build materials with defined properties at the nanoscale using “bottom-up” strategies. This book addresses in an integrated manner all the critical aspects that need to be understood and considered to design the next generation of nano-bio assemblies. The book covers first the fundamentals of the design and features of the protein building blocks and their self-assembly illustrating some of the most relevant examples of nanostructural design. Finally, the book contains a section dedicated to demonstrated applications of these novel bioinspired nanostructures in different fields from hybrid nanomaterials to regenerative medicine. This book provides a comprehensive updated review of this rapidly evolving field.

Nanomaterials are finding widespread use in biomedical applications. In particular, the ability of nanoparticles to penetrate into every corner of physiological systems suggests that they have applications in therapeutic strategies or as diagnostic tools. However, the potential benefits are not without risk. Interactions of nanoparticles with biological systems can have unintended or even lethal consequences. Therefore, understanding the interaction between these materials and biological media, from a physiochemical point of view, is important in the development of materials that are effective and safe. Furthermore, the ability to chemically engineer nanomaterials that may recognize, bind, and release particular biomacromolecules is of benefit for a variety of in vitro biotechnology applications. This dissertation aims to elucidate the important physiochemical attributes of N-isopropylacrylamide (NIPAm) based polymeric nanoparticles (NPs) that contribute to the interactions between NIPAm NPs and biomacromolecules such as proteins or peptides.A substantial collection of literature exists concerning the interactions between biological molecules and synthetic materials. These publications cover both fundamental evaluations of the interaction as well as practical biological applications. The first chapter of this dissertation is a brief review of the current literature concerning the interactions between synthetic materials and proteins and how this information applies to NIPAm NPs. A number of applications using these materials is also highlighted. In the second chapter, NIPAm NPs containing aromatic co-monomers are evaluated as synthetic affinity reagents for biological molecules. The ability of the NPs to bind and sequester a cytolytic peptide released by virulent strains of antibiotic resistant bacteria was evaluated in vitro with a quartz crystal microbalance, and in cell based assays. The aromatic components were shown to be significant contributors to the NP-peptide interaction and modification of the aromatic interaction was used to alter the affinity and capacity of the material to the target peptide. The third chapter provides a detailed study on the effects of monomer hydrophobicity, functional group proximity, and the NPs structure to the NIPAm NP-protein interaction. An important conclusion from this evaluation is that both the monomer distribution and NP structure are significant contributors to the NP-protein interaction, thus in the final chapter, the structure and co-monomer distribution within NIPAm NPs are evaluated using small angle neutron scattering (SANS)