Poster W14 - Side Chain Reactive Protein-Polymer Conjugates for Targeted Drug Delivery

Introduction: There is a critical need for efficient, site-specific protein to polymer coupling methods to synthesize bioconjugate structures that can be tailored for numerous biomedical applications. This work ¬¬demonstrates a high-throughput combinatorial approach to develop side chain reactive polymers which can be easily modified post-polymerization by varying hydrophilic side chains and number of reactive side chains, which greatly increases both the loading capacity of the polymers and hydrophilic tunability. Reactive side chains can be used to attach small molecules, such as drugs or fluorophores, as well as graft proteins to the polymer to make protein-polymer conjugates (PPCs). This has implications in developing targeted drug delivery¬¬ molecules.

Materials and

Methods: We use RAFT polymerization to synthesize poly(tetrafluorophenyl) acrylate (PTFPA) and a library of hydrophilic, side chain reactive polymers for protein conjugation in aqueous conditions. We quantify degree of polymerization and molecular weight through gel permeation chromatography (GPC). Functionalizing our PTFPA with varying degrees of triethylene glycol monomethyl ether (mTEG), glucamine, and hydroxypropyl amine (HPA) allows us to determine the effect of side chain chemistry and quantity on conjugation efficiency. We monitor the functionalization process through nuclear magnetic resonance (NMR). Post functionalization, the polymer is conjugated to an engineered fibronectin type III (Fn3) protein through amine chemistry that selectively recognizes biological targets including tumor biomarker integrin αvβ3. To verify conjugation, we perform gel electrophoresis and western blot and measure conjugation efficiency by quantifying how much protein levels decrease from control, indicating that it has successfully conjugated to polymer. We characterize the protein-polymer conjugates using dynamic light scattering, size exclusion chromatography, and mass spectrometry. Binding and internalization assays are performed using flow cytometry.

Results, Conclusions, and Discussions: mTEG, glucamine, and HPA functionalized polymer show varying degrees of conjugation efficiencies. Glucamine, a sugar derivative, shows high conjugation efficiency at a lower functionality, given its large size and steric hindrance. mTEG, a PEG-like molecule, and HPA, a small molecule, conjugate efficiently at higher percent functionalization. Increased functionalization leads to better polymer hydrophilicity with little to no observed cell death in human glioblastoma (U87MG) cells. Overall, higher side chain functionalization improves hydrophilicity while retaining the ability to conjugate disease targeting proteins such as Fn3 that will bind to and be internalized by αvβ3 containing cells in vitro. Developing a side chain functionalized polymer library is an essential step in developing new classes of small molecule and protein-based therapeutics to address the needs of targeted delivery for multiple disease types. We show that side chain reactive polymers provide a viable approach to the synthesis of diversely functionalized bioconjugates.

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