- Main
Polymeric Materials for Enhancing Therapeutic Biologics
- Puente, Ellie Grace
- Advisor(s): Maynard, Heather D
Abstract
Peptide and protein therapeutics are a growing and diverse field of medicine for thetreatment of numerous diseases. Compared to small molecule therapeutics, protein and peptide therapeutics offer high specificity towards their target, minimizing off-target effects. However, translation of these therapeutics to the clinic is limited by their poor stability, pharmacokinetics, and immunogenetic concerns. Polymeric materials have been used to stabilize and deliver these biologics as excipients, conjugates, and nanoparticles. Herein, challenges with the stability of protein and peptide therapeutics will be discussed as well as polymeric delivery strategies with an iii emphasis on polymeric nanoparticles, covalent conjugation of PEG, known as PEGylation, and biodegradable polymers. My research focuses on 1) the exploration of polymeric nanoparticles to stabilize and deliver the therapeutic peptide glucagon, 2) the comparison of polymer protein homo dimerization via cysteine bioconjugation, 3) the comparison of polymer protein multimerization with Au (III) reagents via cysteine bioconjugation, and 4) the synthesis and characterization of degradable sulfonated polymers. Glucagon is a peptide hormone that acts via receptor-mediated signaling predominantly in the liver to raise glucose levels by hepatic glycogen breakdown or conversion of noncarbohydrate, 3 carbon precursors to glucose by gluconeogenesis. Glucagon is administered to reverse severe hypoglycemia, a clinical complication associated with type 1 diabetes. However, due to low stability and solubility at neutral pH, there are limitations in the current formulations of glucagon. In Chapter 2, trehalose methacrylate-based nanoparticles were utilized as the stabilizing and solubilizing moiety; glucagon was site-selectively modified to contain a cysteine at amino acid number 24 to covalently attach to the methacrylate-based polymer containing pyridyl disulfide side chains. PEG2000 dithiol was employed as the crosslinker to form uniform nanoparticles. Glucagon nanogels were monitored in Dulbecco's phosphate-buffered saline (DPBS) pH 7.4 at various temperatures to determine its long-term stability in solution. Glucagon nanogels were stable up to at least 5 months by size uniformity when stored at −20 °C and 4 °C, up to 5 days at 25 °C, and less than 12 hours at 37 °C. When glucagon stability was studied by either HPLC or thioflavin T assays, the glucagon was intact for at least 5 months at −20 °C and 4 °C within the nanoparticles at −20 °C and 4 °C and up to 2 days at 25 °C. Additionally, the glucagon nanogels were studied for toxicity and efficacy using various assays in vitro. The findings indicate that the nanogels were nontoxic to fibroblast cells and nonhemolytic to red blood cells. The glucagon in iv the nanogels was as active as glucagon alone. These results demonstrate the utility of trehalose nanogels towards a glucagon formulation with improved stability and solubility in aqueous solutions, particularly useful for storage at cold temperatures. Protein self-assembly into dimers and higher order structures in biological systems can be essential for protein function and activity, however, many of these complexes are unstable in vivo. Polymeric linkers with reactive handles for protein bioconjugation can stabilize protein higher order structures and improve their pharmacokinetics. In Chapter 3, cysteine bioconjugation strategies are explored with poly(ethylene) glycol (PEG) reagents to dimerize the model protein T4 lysozyme (V131C), containing a single surface exposed cysteine. Dimer conversion with dicyclohexylphosphine (PCy2) P,N ligated PEG2000 Au(III), di-1-adamantylphophine (PAd2) P,N ligated PEG2000 Au(III), (maleimide)2, and (vinyl sulfone)2 bifunctionalized PEG were compared at pH 6.0, pH 7.5, and 9.0 and their stability evaluated. This work adds to the growing body of literature on protein dimerization. The work towards investigating higher order oligomeric protein polymer structures is expanded on utilizing the therapeutic protein basic fibroblast growth factor 2 (FGF-2) in Chapter 4. The multimerization conversion from monomeric FGF-2 is investigated at various equivalents, temperatures, and reaction times. This work adds to the growing body of literature on protein multimerization. Polycaprolactone is a widely used biocompatible and degradable polymer. However, the polymer is hydrophobic, and not soluble in water. There are advantages to rendering the polymer soluble in aqueous solutions. In Chapter 5, allyl functionalized caprolactone underwent anionic ring-opening polymerization (ROP) and post-polymerization modification via thiol-ene click chemistry with 3-mercaptopropane sulfonate. ROP of ally caprolactone to yield poly (allylv caprolactone) with molecular weights from 6.3 - 81.2 kDa and poly (sulfonate-caprolactone) with molecular weights from 12.2 - 163.3 kDa after functionalization via thiol-ene. The synthetic approaches taken to access high molecular weights of poly (sulfonate-caprolactone), mechanical properties, and degradability of these materials are discussed. Chapter 2 is published as: Puente, E. G.; Sivasankaran, R.; Vinciguerra, D.; Yang, J.; Lower, H. C.; Hevener, A.; Maynard, H. D. “Uniform Trehalose Nanogels for Glucagon Stabilization.” RSC Appl. Polym. 2024, 2, 473. Chapter 3 is in preparation for publication as: Puente, E. G.; Polite, M. F.; Meckes, F. A.; Spokoyny, A. M.; Maynard, H. D. “Comparison of Polymer Protein Homo Dimerization via Cysteine Bioconjugation.” In Preparation. Chapter 5 is in preparation for publication as: Puente, E. G.; Snell, K. M.; Maynard, H. D. “Degradable Sulfonate Polymers by Thiol-ene Click Chemistry.” In Preparation.
Main Content
Enter the password to open this PDF file:
-
-
-
-
-
-
-
-
-
-
-
-
-
-