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Publication Date

2022-05-09

First Advisor

Sarah J. Moore

Second Advisor

Maren E. Buck

Document Type

Honors Project

Degree Name

Bachelor of Arts

Department

Biochemistry

Keywords

protein-polymer conjugates, targeted drug delivery, cancer therapy, side-chain functionalization, poly(2-vinyl-4, 4-dimethylazlactone), Fn3, Integrin

Abstract

Targeted delivery of therapeutics using protein-polymer conjugates (PPCs) is a promising strategy to address off-target effects of currently available anti-cancer drugs. Polymer provides increased circulation half-life and its chemical versatility allows loading of multiple drugs and conjugation to proteins, which guide the entire complex to the specific disease site. The goal of my thesis was to synthesize PPC via primary amine chemistry, optimize reaction conditions to achieve complete conjugation of protein, and purify conjugates to target the αvβ3 integrin receptors that are overexpressed on neovasculatures of growing tumors, and ultimately, contribute to the development of a more effective cancer treatment. In this study, the integrin receptor-targeting fibronectin type III (Fn3) protein was produced from transformed BL21(DE3) E. Coli. The azlactone-containing polymer poly(2-vinyl- 4,4-dimethylazlactone) (PVDMA) was synthesized via reversible addition-fragmentation chain transfer polymerization (RAFT), which allows tunable molecular weight and low dispersity. To solubilize hydrophobic PVDMA in aqueous solvent for conjugation to hydrophilic protein, the polymer was partially functionalized with a hydrophilic group triethylene glycol monomethyl ether (mTEG). Through a series of conjugation reactions with varying protein and polymer concentrations and protein-to-polymer molar ratios, the optimal conditions to achieve complete conjugation of Fn3 to PVDMA-mTEG were found using SDS-PAGE analysis. Conjugation reaction kinetics were also studied using protein gel and FT-IR spectroscopy. The conjugates were successfully isolated using size exclusion chromatography. The reaction kinetics of polymer hydrolysis was also studied for more accurate interpretation of SDS-PAGE analysis to assess conjugation efficiency. 2 Future work will involve assessing the binding affinity of the Fn3-PVDMA-mTEG conjugates to the avβ3 integrin receptor. Additionally, chemistries available for site-specific conjugation of polymer to protein will be developed.

Rights

©2022 Yeji Lee. Access limited to the Smith College community and other researchers while on campus. Smith College community members also may access from off-campus using a Smith College log-in. Other off-campus researchers may request a copy through Interlibrary Loan for personal use.

Language

English

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