Title

Self-assembling Polypeptide Nanoparticles: Their Biophysical Properties, Refolding Conditions, And Potential Biomedical Applications

Date of Completion

January 2010

Keywords

Nanotechnology|Biophysics, General|Engineering, Materials Science

Degree

Ph.D.

Abstract

Bionanoparticles such as virus-like particles are finding enormous biomedical applications in vaccination, bioimaging, drug delivery and targeting, and other technologies. In this work, a type of self-assembling polypeptide nanoparticles (SAPNs) based on coiled coil oligomerization domains are investigated, and their potential biomedical applications are also explored.^ The design principle of the SAPNs is inspired by the structure of viral capsids with icosahedral symmetry. An icosahedron is composed of 60 asymmetric units and has two-, three- and five-fold rotational symmetry axes. A nanoparticle with icosahedral symmetry can be generated by superposition of appropriate protein oligomerization domains onto the symmetry axes. Coiled coils are the most ubiquitous and versatile oligomerization motifs in protein, therefore used as building blocks for the design of SAPNs. In this work, polypeptides with pentameric and trimeric coiled-coil domains are designed and self-assemble into SAPNs. ^ The biophysical properties and refolding conditions of SAPNs are investigated using the P6c peptide as a model. Both scanning transmission electron microscopy and analytical ultracentrifugation analysis reveal that the majority of P6c SAPNs have T = 3 icosahedral structure that accommodate 180 peptide chains, although P6c SAPNs are composed of species with multiple oligomerization states. The refolding of SAPNs is influenced by factors such as ionic strength, pH, temperature, and glycerol concentration. The average sizes of SAPNs increase with increasing salt concentrations in their refolding buffers. And there exist optimal pH and glycerol conditions for the formation of regular P6c SAPNs. P6c SAPNs are also shown to be thermally robust. Furthermore, the formation of regular SAPNs is influenced by their primary sequence, especially the residues near the link region and the surface-presenting epitopes. ^ Several potential biomedical applications of SAPNs are explored in this work. SAPNs can be used as repetitive antigen display platform for vaccine development, for instance, malaria vaccines. Multiple functions can be obtained for drug delivery and targeting applications by co-assembling polypeptides bearing different functional groups, such as PEG and cancer cell targeting peptide Bombesin. In addition, the central cavity of SAPNs can be utilized for bioimaging applications, by encapsulating inorganic nanoparticles, such as gold nanoparticles and quantum dots. ^