Developing Multi-functional Nanoplatforms Using Protein Architectures

Abstract

A variety of nano-sized materials are developed in the biotechnology fields. Developing nano-sized particles have become a critical issue in biomedical applications because they are closely related to quantum yield, large surface area and EPR effects. Despite these advantages of nanoparticles, it has become indispensable to use more advanced nanoparticles due to their chemical complexity, heterogeneity, difficulty in precisely controlling the size, and toxicity in vivo applications. In this regard, protein-based nanoparticles have biocompatibility, uniform size, shape, composition and stability, and they are quite suitable as multifunctional nanoplatforms. In addition, the structures of protein nanoparticles are based on the atomic resolution crystal structure allowing genetic and chemical modifications at the molecular level. The aim of this thesis was to describe of developing the multi-functional protein nanoparticles using protein cages and monomeric fusion proteins. Thus, Thermotoga maritima encapsulin protein cage whose outer diameter 24 nm was developed as in vitro theranostic nanoplatform. A novel protein cage, encapsulin have not been used for targeted delivery system before, and was prepared as a versatile template for targeted delivery through SP94 peptide insertion which known to bind with hepatocellular carcinoma cells. Functional plasticity and versatility of the engineered encapsulin allow us to apply for specifically detecting and effective treatment of diseases. Relatively small lumazine synthase which isolated from Aquifex aeolicus (AaLS) protein cage nanoparticles with outer diameter of 15.4 nm have been utilized to develop as uniform layer by layer assemblies. High ordered structures of two complementary AaLS protein cages were successfully constructed using simple recognition of histag and Ni-NTA. Furthermore, fluorescent imaging modular toolkits were established using monomeric fusion proteins and ligation proteins by giving cancer cell targeted capability of affibody and visualizing cancer cells of fluorescent proteins. These affibody-fluorescent protein conjugations are post-translationally generate, allowing simple and rapid binding between affibodies and fluorescent proteins. A variety of protein nanoparticles demonstrated that they have potential to be utilized as a multifunctional nanoplatforms in biomedical and biotechnology fields.