INTELLECTUAL MERIT: The broad goal of this proposal is to develop polymeric micelles for biomedical applications from amphiphilic poly(ethylene glycol)- block-peptide copolymers with peptide segments (conjugates) that display lower critical solution temperatures (LCSTs). Amphiphilic block copolymers self-assemble to form stable micelles with hydrophobic cores to encapsulate hydrophobic drugs. With an LCST range of 22-35°C, these biomimetic polymers will undergo an additional assembly of the hydrophobic peptides, further stabilizing the micellar delivery vehicle. The relationships between copolymer structure, LCST, micellization behavior, and peptide secondary structure for this class of amphiphiles are not well understood. A fundamental study of these relationships is proposed by varying the length of each polymer block, the peptide identity, and the copolymer architecture. Peptides with known LCST behavior will be investigated, in particular, the elastin-like peptide (ELP) VPGVG, as well as hydrophobic peptides that demonstrate secondary structures, such as poly(valine), poly(proline), and poly(alanine). Both linear and branched copolymer architectures will be synthesized using a combination of standard peptide coupling techniques and reversible addition fragmentation chain transfer (RAFT) controlled polymerization methods. The proposed research has the following objectives: (1) Identify key structural requirements for micelle formation in PEG-ELP copolymers. (2) Discover new peptide sequences that form LCST-based biomaterials. (3) Evaluate the efficacy of PEG-peptide copolymers as drug delivery vehicles. By carrying out these experiments, the project will elucidate the effects of structure and polymer properties as they relate to performance of micellar drug delivery vehicles.
BROADER IMPACTS: Tunable vehicles for delivery of drugs, genetic material, and contrast agents have not yet been perfected. This proposal offers a new approach that is well worth pursuing. The proposal provides a model for integration of research and education at the undergraduate level. The project lends itself to being subdivided into modules differing in scope and complexity so that undergraduate students at all levels can participate effectively. Each undergraduate will be given an individual project, but they will work together to advance the overall aims of the proposal. Each student will be responsible for a primary literature search for relevant information concerning their own project. The PI provides clear and explicit detail about her laboratory management procedures and describes the group meeting experiences that will be provided to integrate the several components and to ensure that each student knows not only about his/her own component but understands how it fits with the overall goals. Reports, theses, manuscript drafting, and group meetings, along with opportunities to present at regional and national meetings, will allow the students to develop their skills at communicating the intellectual context and research results of the overall project.
Many current and potential medicinal substances have poor water solubility and low molecular weight, making delivery to the body a significant challenge. Among the various approaches offered to address these limitations, the use of self-assembled polymeric vehicles for both the protection and transport of these medicinal substances is particularly promising. Self-assembly occurs much like the phase separation of oil and water, where like segments prefer to associate with each other. Because these two segments, or blocks, are covalently bonded, the result is an aggregation of the polymer chains. By tuning the length and nature of each domain, a defined aggregation is obtained that can be useful in such materials as drug delivery vehicles. In aqueous solutions, these amphiphilic polymers self-assemble with the hydrophobic regions forming the core where hydrophobic drugs can be sequestered. The hydrophilic regions form the corona, solvating and shielding the drug from premature release, enzymatic degradation and unwanted protein binding interactions (Figure 1). Intellectual Merit The initial design for our amphiphilic copolymers utilized poly(ethylene glycol) (PEG) as the central segment. PEG is a hydrophilic non-toxic, non-immunogenic polymer that is FDA-approved for internal usage. Hydrophobic segments were added, each comprised of a short pentavaline sequence end capped with groups of varying hydrophobicity (Figure 2). These copolymers successfully self-assembled, with peptide secondary structure playing a role in aggregate formation. The peptide sequences themselves represent only about 10% of the copolymer mass, yet had a significant impact on the self-assembly behavior. These copolymers displayed low toxicity during in vitro cell studies with SW620 colorectal cancer cells. Two publications resulted from these studies. They showed importantly that self-assembly was possible with such short hydrophobic segments and that increasing end-group hydrophobicity decreases the critical aggregation concentration (CAC) to a viable value for drug delivery applications. Changes in the hydrophobic/hydrophilic ratios had little impact on the CAC value or aggregate size. These materials were successful at encapsulating hydrophilic small molecules. Carboxyfluorescein was used as a model drug compound, with 2-10 mol% loaded into the polymer vesicles. More significantly, these copolymers demonstrated incredible thermal stability. They remained self-assembled even at temperatures of 70 ?C. There was little to no disassembly of the beta sheet peptide structure. This robustness makes them highly attractive as drug delivery vehicles, as they would not require special storage conditions. Looking to also develop a polymeric delivery vehicle that could deliver proteins, we leveraged our polymer synthesis work to generate an initial bioconjugate of PEG covalently attached to trypsin as a model enzyme. The synthesis and stability of this bioconjugate was detailed in a third publication. PEG polymers of molecular weights (MWs) were activated using p-nitrophenyl chloroformate and reacted with the amine side chain of the amino acid lysine to yield a stable urethane linkage (Figure 3). For all MWs tested, 4-5 polymer chains decorated trypsin yet maintained the enzyme’s hydrolytic activity. Circular dichroism measurements demonstrated that the activity enhancement is due to preservation of trypsin’s secondary structure. The trypsin’s folded shape is retained at higher temperatures, keeping the active site intact. As well, dynamic light scattering measurements indicate that trypsin conjugated to 10 and 20 kDa PEG chains achieve the necessary hydrodynamic size to both evade renal filtration and exploit the EPR effect for improved treatments. Broader Impacts The scientific training in this award was directed at undergraduate students at Williams College. Students from all class years participated through summer research, thesis projects, independent study and winter study classes, and work-study opportunities. Roughly 35 students were involved in this research through 1 or more of these venues. The research was presented 16 times at national and local ACS conferences and a Gordon conference. Thirteen of these presentations were done by undergraduate students. Three of them won travel awards for their work. Demographics of the presenting students include three first generation college students, two minorities and six women (10 students total). Travel costs for these presentations were supported by this award. These students have gone on to higher studies in chemistry and molecular biology graduate schools, medical schools, as well as high school science teaching and industrial positions. Their research experiences have fueled their interest in chemistry, and they are now making their own contributions to the advancement of science. In addition, this award was used to engage younger students by implementing polymer workshops in conjugation with the Flying Cloud Institute. This group coordinates GIRLS!, an after school science club for girls in grades 3-6 in Berkshire County, MA. The students in both of these venues come from a wide range of educational and economic backgrounds. With the help of women high school mentors, the students were introduced to various aspects of polymers (synthesis, composition, characterization), allowing the girls to practice their quantitative and qualitative measuring skills. The students had a great time of it and loved the hands-on experiments.
