Advisor:

Julie Champion, PhD

Committee Members:

Julie Champion, Ph.D.

Shuichi Takayama, Ph.D.

Ravi Kane, Ph.D.

Valeria Milam, Ph.D.

James Dahlman, Ph.D.

Engineering Recombinant Protein Vesicles for Delivery Applications

Recombinant proteins have emerged as promising building blocks for self-assembly of nanoparticles. Their versatility, accessibility through genetic manipulation, and biocompatibility are key advantages compared with synthetic block copolymers. One example of recombinant protein materials is hollow protein vesicles self-assembled from recombinant fusion proteins containing thermoresponsive elastin-like polypeptide (ELP). While synthetic nanoparticles typically require chemical conjugation or physical adsorption to incorporate biofunctional proteins, protein vesicles are made directly from biofunctional proteins. This prevents loss of protein structure and activity, and enables control over protein orientation. Vesicles use high-affinity leucine zippers, ZE and ZR, to enable a range of different biofunctional proteins to be displayed on the surface at a controlled density. The overall goal of this work is to translate protein vesicles into biofunctional materials made from bioactive proteins with the required physical and biological properties for use as delivery vehicles. For increased stability at physiological conditions, a photo-crosslinkable unnatural amino acid is incorporated into the ELP domain. Additionally, ELP hydrophobicity and length are engineered for desired size and stability. Vesicle size was reduced from micron-scale to nano-scale by tuning ionic strength, ELP hydrophobicity and length. To demonstrate the therapeutic potential of protein vesicles, a small molecule cancer treatment drug, doxorubicin, is encapsulated in the vesicle lumen and delivered into cancer cells. In addition to small molecule cargo in the lumen, a model antigen protein, ovalbumin, is fused ZE and incorporated into the surface of self-assembled vesicles with a controlled size and antigen density. The resulting antigen-displaying protein vesicles induce antigen-specific humoral and cellular immune responses in a mice model. This work is the first to make therapeutic protein vesicles and demonstrates the value of this platform in delivering a wide range of cargos with vastly different properties, ranging from small hydrophobic molecules to large, folded proteins.