(711d) Thermo-Reversible Nanoparticle Formation Using a Novel Elastin-Like Three-Armed Star Polypeptide

Elastin like polypeptides are responsive materials that have been studied for the past three decades because of their promising characteristics which can be exploited in many different areas including targeted drug delivery, tissue engineering, surface modification, protein purification and more. These materials are a member of a class of materials that show lower critical solution temperature (LCST) and if crosslinked, they produce thermally responsive hydrogels. They can be designed in such a way to respond to different environmental stimuli including temperature, pH, ionic strength and light. The primary sequence of these polypeptides is GVGVP in which the two valines can be substituted with most of the naturally occurring amino acids to change the polypeptide hydrophobicity and consequently its transition temperature. In recent years and thanks to the technical advances in molecular biology, it has become possible to design and construct these materials at the gene level with total certainty regarding their sequence and length. As a result, the transition temperature of these materials can be controlled.

In this study, we successfully designed and expressed a new three-armed star polypeptide, based on pentapeptide repeats of GVGVP followed by a trimer-forming oligomerization domain at the C-terminus of the ELP. These new polypeptides were expressed in E.coli and purified by thermal transition cycling. By characterizing the purified protein using SDS-PAGE and circular dichroism (CD) spectroscopy, we confirmed the formation of the three-armed star polypeptide.

By heating up this star polypeptide above its transition temperature we managed to get stable small micellar particles with the hydrophobic ELP tails being stabilized by the charged oligomerization domains as the head groups. These micelles showed thermal stability without any measurable aggregation up to the temperatures well above the ELP transition temperature and even higher than the oligomerization domain stability temperature. This is a very interesting and different response in comparison to all the typical ELP molecules. Normally for an ELP at the transition temperature, the chains would aggregate to very big random particles which is the start of a phase separation process which eventually leads to soluble and coacervate phases if the solution is kept above transition temperature for a longer time. But the newly designed molecules are stable in their micellar form independent of the time they are kept above their transition temperature without forming bigger aggregates or phase separation.

The transition temperature of the these novel three-armed star polypeptides can be tuned during the gene synthesis by changing the length of ELP molecule or by changing the hydrophobicity of the molecule using different amino acids.

Since these nano-particles can be designed to respond to temperature and pH changes and considering their very small controllable size and their perfectly controllable transition temperature they have a very good potential of being used especially in drug delivery of poorly soluble drugs and modified for targeted drug delivery.