Design, Synthesis And Application Of A Novel Protein Nanocapsule

Protein drugs are one of the most important biological drugs in the field of medical treatments and tissue engineering today. They have high and specific activities, low toxicity, clear biofunctions and wild usages, which can realize the diagnostication, prevention and treatment on protein molecular level. This dissertation focuses on two platforms for intracellular and extracellular delivery separately, which are based on the pathways of protein intercellular-function and protein cell- membrane-function.A noncovalent-bond in-situ polymerization in aqueous solution is used to encapsulate model proteins into a polymer nanocapsule with uniform core-shell structure. The effect of monomer kinds and ratios on sizes and charges is investigated, as well as the relation between the positive charge of nanocapsules and protein delivery in cells is built up. The nanocapsules with small size and high positive charge are synthesized with spermine-V monomers. With high positive charge, nanocapsules can be delivered into cells with high efficiency; however, when the charge is above +8 mV, the interaction between nanocapsules and cell membrane is so strong that the membrane can be disrupted. To targeting deliver the ricin nanocapsules into cells, a platform to conjugate nanocapsules is demonstrated by conjugation betweenαCD4/αCD40 and ricin nanocapsules, the effience of which can be increased about 4 times.Growth factor nanocapsules (Vascular endothelial growth factor, VEGF and Bone morphogenetic protein, BMP-2), which are responsive to environmental changes, are successfully prepared by the noncovalent-bond in-situ polymerization platform. VEGF nanocapsules with peptide as crosslinkers can controlled release VEGF with the specific enzyme stimulation; while BMP-2 nanocapsules with ester crosslinkers are able to response to pH increase during the bone-cell-differentiation process. The polymer shell with proper charge and thickness can protect inner growth factors from denaturing during operating procedures or in protease-containing condition. The covalently binding between the double bonds or amine groups on the surface of nanocapsules and the supports (hyaluronan hydrogel or collagen) prevents protein leaching during the support swelling and in the solution washing. The leaching amount of the nanocapsules maintain 1/4~1/10 of that of the naked protein. The polymer nanocapsules improve the activity and efficiency of the growth factor cell growth and differentiation in tissue engineering.A two-step in-situ polymerization is improved to obtain more stable single protein nanocapsules, in which the first step is to generate vinyl groups on the protein surface by acryloylation and the second step is polymerization based on the anchors on the protein surface. The polymer shell encapsulated Horseradish Peroxidase (HRP) in the core not only maintains the natural activity of this enzyme, but also improves the thermal stability by 20°C. After conjugated withαCD4 on the surface of the nanocapsules, obvious enhancement on tumor targeting delivery about six times is achieved with the Indole-3-acetic acid/HRP nanocapsules pro-drug system.