Self-Assembled, Peptide Based Biomaterials for Regenerative Medicine and Drug Delivery
- Author(s): Black, Katie Anna
- Advisor(s): Tirrell, Matthew V
- et al.
A focus of the field of biomaterials is to use directed design to create new materials which replicate and enhance the intricate functions of the human body. Nature's own building blocks, peptides, are an ideal material to create self-assembling biomaterials as they are biodegradable, relatively easy to synthesize, and can be designed with a wide array of functions. In this dissertation, self-assembling peptide materials were optimized for two important medical applications: regenerative medicine and drug delivery.
Peptide amphiphiles (PAs), peptides conjugated to fatty acid tails, can self-assemble into both spherical micelles and worm-like micelles. PA worm-like micelles are of particular interest for regenerative medicine applications for their ability to form viscoelastic hydrogels at high concentration. Here we created PA hydrogel systems with active formation and stabilization triggers that are amenable to in situ gelation. Two different methods of in situ gel formation in PA systems were investigated, shear force and pH.
Shear-induced formation of worm-like micelles is demonstrated in the PA termed C16-W3K. Before shearing, C16-W3K PAs form spherical micelles in solution and exhibit little to no viscoelasticity. As the solution is subjected to simple shear flow with increasing shear rate, spherical micelles form elongated worm-like micelles up to microns in length. In the C16-W3K PA system, shear force induced the change not only of the micelle structure but also of the peptide secondary structure simultaneously.
Worm-like micelle formation was also demonstrated using pH modulation, in the PA termed C16GSH, which was designed with a branched peptide headgroup of histidine and serine amino acids. At low pH, the histidine side chains are protonated and hydrogen bonding does not occur, creating weakly elastic hydrogels. At pH 7.4, above the pKa of the histidine imidazole group, cooperative hydrogen bonding occurs, stabilizing the self-assembled worm-like micelles and creating a strong viscoelastic hydrogel. This unique architecture of C16GSH makes it possible to create hydrogels spanning a wide range of stiffness (0.1-10 kPa). C16GSH were optimized in vitro and in vivo for the application of peripheral nerve regeneration. Peripheral nerve injury is a debilitating condition for which new bioengineering solutions are needed. One strategy to enhance regeneration inside nerve guide conduits is to fill the conduits with a hydrogel to mimic the native extracellular matrix found in peripheral nerves. C16GSH hydrogels were compared to a commercially available collagen gel, which has been previously investigated as a nerve guide filler gel. Schwann cells, a cell type important in the peripheral nerve regenerative cascade, were able to spread, proliferate and migrate better on C16GSH gels in vitro when compared to cells seeded on collagen gels. Moreover, C16GSH gels were implanted subcutaneously in a murine model and were found to be biocompatible, degrade over time, and support angiogenesis without causing inflammation or a foreign body immune response. Taken together, these results help optimize and instruct the development of a new synthetic, hydrogel as a luminal filler for conduit-mediated peripheral nerve repair.
In the second half of this dissertation, peptide based complex coacervates were optimized for delivery of protein therapeutics. Complex coacervation is a liquid-liquid phase separation based on the electrostatic association of two oppositely charged polymers in aqueous solution. Coacervation results in micron sized droplets of a dense polymer-rich phase (coacervate) which is separate from the dilute polymer-poor solution phase (aqueous phase). Complex coacervates based on synthetic polypeptides have many desirable features for therapeutic protein delivery. They can be synthetically produced, can be made to be biocompatible and biodegradable, and their formation can be tuned by a wide array of parameters. In this dissertation, a method to encapsulate proteins by complex coacervation using polypeptides is explored.
Protein encapsulation with a model protein system: bovine serum albumin (BSA) was demonstrated. Rheological properties were studied to determine the viscoelasticity which may have implications for cell internalization. It was demonstrated that there is tradeoff between loading efficiency and total loading. Therefore, depending on the application, high loading capacity, up to 1:3 molar ratio of protein to polypeptide, or 100% loading of the protein can be achieved, depending on the process and cost of the protein which is often high. Encapsulated BSA retained its secondary structure when encapsulated and was released under conditions of low pH due to disassembly of the coacervate. Lastly, protein loaded coacervates were shown to be non-toxic in a cell viability assay.
Polypeptide complex coacervates show promise at encapsulating proteins for therapeutic delivery, but it is difficult to control their size and stability to due dynamic rearrangement and coalescence. To control the size and stability of polypeptide coacervates, the crosslinker EDC was used to create a peptide bond between the amino acid side groups of poly(L-lysine) (PLys) and poly(D/L-glutamic acid) (PGlu). By changing the ratio of PGlu to PLys colloidal stability was achieved without the need for an additional excipient. Surface charge of the particles was also controlled by this method. Final particle size was controlled by both molecular weight and concentration of the polypeptides. A span of particle diameter from to 272nm to 1.3 µm was achieved. Lastly, stability at low pH, where non-crosslinked coacervates disassemble, was demonstrated. A simple and tunable method to control particle size, such as the one presented here provides a possible solution to a major limitation in the field of drug delivery, control of particle size.