Nanocapsule-based Platforms for Protein Delivery and Protein Therapy

Author(s): Liu, Yang | Advisor(s): Lu, Yunfeng | Abstract: In living organisms, numerous biochemical reactions occur synergistically, allowing the organisms to grow and reproduce, convert food to energy, maintain the structure, and response to the environment – all activities that we called “life”. As one of the most important types of molecules for any living organism, protein controls and mediates all these biological processes; malfunction of protein often induces the body to an abnormal state namely disease. Viewed from the perspective of disease mechanisms, protein therapy, which treats diseases by delivering therapeutic proteins directly to redress disorders, represents a tremendous opportunity to alleviate many incurable diseases. However, intrinsic problems, including low stability, low permeability, and high immunogenicity, hamper the clinic application of proteins. Development of advanced protein delivery systems, which can properly address these issues, has been considered as a practical solution that could enable the therapeutic use of a wide scope of proteins in disease treatments.In this dissertation, novel delivery systems have been developed based on the protein nanocapsule technology, providing several practical methods to overcome essential issues that hinder the application of therapeutic proteins, but have not yet been properly resolved by current protein delivery systems. These common issues include the efficiency of protein intracellular delivery, co-delivery of multiple proteins, and the plasma half-life and immunogenicity of proteins when delivering systemically. Based on these issues, the dissertation research can be outlined briefly with the following three topics:1. Intracellular delivery of any protein(s) with optimal efficiency. This part of work demonstrated a polymer-based nano-carrier that was constructed by utilizing multiple weak interactions, which offers the feasibility of loading and delivering proteins without any chemical modification, as well as finely tuning the surface properties of the nano-carriers to achieve optimal delivery efficiency. The intracellular delivery capability of this nano-carrier was first demonstrated by delivering bioactive transcription factors into cells (Chapter 3). Combining with single-protein nanocapsule and microfluidic platform, any protein can be intracellularly delivered using this nano-carrier and the delivery efficiency can be optimized with a high-throughput systhesis and screening system (Chapter 5), providing a fundamental platform for researches on stem cell reprogram and regenerative medicine.2. Co-delivery of multiple proteins that function synergistically. In eukaryotic cells, most biological processes are accomplished by multiple enzymes or proteins, which are usually spatially confined with precise ratio control in order to function correctly and optimally. Demonstrated in this part of work (Chapter 1), defined types and amount of enzymes were assembled and co-encapsulated into enzyme nanocomplex, which ensures those enzyme functioning effectively no mater being delivered into local tissues or diluted system like blood stream. This strategy provides a feasible method to construct protein-based multi-functional nanostructure as needed without engineering protein sequence. From the perspective of developing protein therapeutics, this system offers a practical method to detoxify the by-product of many enzymes, enabling safe use of them for therapeutic purposes.3. Prolong circulation time and reduce immunogenicity of therapeutic proteins. One major problem of most therapeutic proteins is the fast clearance by immune system, which significantly reduces or completely diminish their therapeutic effects. In this part (Chapter 3), we designed a general method that can prolong the plasma half-life of most proteins significantly by minimizing their interactions with serum proteins, blood cells, tissues and organs with a protein-adsorption-resistant polymer shell. With this method, the overall therapeutic effect was enhanced after systemic administration, which was demonstrated with uricase as model therapeutic protein. This strategy also inhibits immune responses against exogeneous proteins, which can broaden the scope of proteins with therapeutic potentials.Overall, this dissertation research established various methods to endow new surface properties to proteins and enzymes as needed. Based on these work, one can expect that increasing number of protein therapeutics will be developed and widely applied for curing diseases in the near future.