UCLA

Peptide and protein therapeutics are highly effective for the treatment and management of numerous diseases. Despite this, their clinical potential is underutilized mainly due to drawbacks inherent to many native proteins such as poor stability, immunogenicity, and short pharmacokinetics. These issues cause a multitude of challenges in manufacturing, formulation, transportation, and administration. Ideally, peptide and protein therapeutics are shelf stable and are able to be administered in its native state through a minimally invasive method. My research focuses on 1) the exploration of different polymeric models in response to various stimuli to elucidate mechanisms behind certain drug delivery vehicles, 2) the sustained release of therapeutic peptide glucagon through a glucose-responsive hydrogel for the prevention of hypoglycemia, 3) the site-selective conjugation of a degradable, zwitterionic polymer to a model protein, and 4) the investigation of how polymer tacticity affects biological function through the conjugation of regio-and sequence-defined macromolecules to a model protein. Chapter 1 discusses three different strategies

Stimuli-responsive nanoparticles, particularly those that respond to two different environmental cues, are useful materials in drug delivery. In chapter 2, the size-response of nanogels based on two different polymers, poly(N-isopropylacrylamide) p(NIPAM) and poly(oligo(ethylene glycol) methyl ether methacrylate) (PEGMA), to temperature and glucose concentration changes was investigated. The nanogels were prepared by precipitation polymerization, 114 nm and 169 nm for pNIPAM and PEGMA at 37 �C, respectively, and characterized by proton nuclear magnetic resonance and infrared spectroscopies. Both nanogels underwent a volume phase transition in biologically relevant ranges upon heating. Incorporation of 2-aminophenylboronic acid enabled glucose-binding, resulting in a shift of the volume phase transition temperature (VPTT) of both nanogels as assessed by differential scanning calorimetry (DSC) and turbidity measurements. P(NIPAM) nanogel demonstrated a predictable decrease in size in response to both increase of temperature and glucose. PEGMA nanogel showed a predictable decrease in size to increasing temperature and but exhibited a surprising increase then decease in size to increasing concentrations of glucose.Glucagon is a peptide hormone used in the treatment of hypoglycemia. Unlike its counterpart insulin, glucagon has only started garnering interest in the past few decades. While recent advances have introduced user friendly formulations for glucagon administration, there remain no glucagon formulations on the market intended for combating nocturnal hypoglycemia. Chapter 3 details the sustained release of native glucagon using a glucose- and thermo-responsive hydrogel. RAFT polymerization was used to create PEG-b-p(NIPAM-co-2-APBA) polymers which were subsequently crosslinked using glucose. Polymer size, PEG length, boronic acid incorporation, and polymer wt % were varied to optimize hydrogel sensitivity to 1 mg/mL glucose at 37 ⁰C, which correspond to normoglycemia. Glucagon release was tested over 48 hours in which the hydrogel released up to 80% of the payload, 40% more than its control. Rheological measurements demonstrate the shear-thinning property of the hydrogel. Finally, viscosity measurements in conjunction with injectability calculations show that the hydrogel can be formulated as an injectable. Chapter 4 describes the exploration of a degradable zwitterionic polymer as a PEG-alternative. Zwitterionic polymers have gained rising interest for their ability to stabilize proteins, increase circulation time, and retain bioactivity. While polyethylene glycol (PEG) still exists as the gold standard polymer used to mitigate challenges associated with native proteins, there are merits to investigating alternative polymers for this purpose. In this work, we report the site-selective conjugation of degradable zwitterionic poly(caprolactone-carboxybetaine) (pCLZ) to growth hormone receptor antagonist (GHA) B2036-alkyne and study bioactivity, pharmacokinetics, and immunogenicity. Azide-containing pCLZs of 5, 20, and 60 kDa are conjugated to GHA B2036-Alkyne via copper-catalyzed click reaction and their in vitro bioactivity is compared to PEGylated GHA B2036 5, 10, and 20 kDa, of matching hydrodynamic radii. The ability of pCLZs to elicit IgG and IgM antibody production was tested in vivo and no measurable antibody production was detected. Herein, we report that pCLZs demonstrate a high retention of bioactivity, as measured by half-maximal inhibitory concentrations in vitro, as well as low immunogenicity in vivo. Using 18F labeled PET/CT imaging, pharmacokinetics of our pCLZ conjugates show a significant increase in circulation time by 23 min compared to that of GHA B2036. Finally, in chapter five, a series of uniform, stereospecific protein-polymer conjugates were synthesized through copper-mediated click chemistry. Discrete, uniform polymers were provided by Wencong Wang from Prof. Jeremiah Johnson’s lab at the Massachusetts Institute of Technology. The impact of molecular features of the conjugated polymers were evaluated through activity studies of growth hormone antagonist. Preliminary attempts at crystallizing the protein, polymer, and protein-polymer conjugates are demonstrated. Crystallization screening and crystallography model fitting was done largely with help from Genesis Falcon, Niko Vlahakis, and Dr. Michael Sawaya.