Zwitterionic Dendrimer-modified PEG for Protein Conjugation - PROJECT SUMMARY Polymer-protein conjugates have attracted significant interest because of their critical biomedical applications in treating many diseases. As a water-soluble polymer with high processability, poly(ethylene glycol) (PEG) has been widely used for protein conjugation, and over a dozen PEGylated proteins have been commercialized for the clinical treatment of various diseases (including arthritis, cancer, hepatitis, and gout, etc.), with an annual sale of multi-billion dollars. However, PEG can induce unfavorable immunogenicity and reduce protein bioactivity. Zwitterionic polymers (ZPs) have emerged as improved alternatives for PEG on these aspects and may lead to even longer circulation time, but typically linear ZPs have low processability, which hinders their applications. Zwitterionic dendrimers (ZDs) have improved processability than linear ZPs, but well-controlled protein conjugation directly by ZDs is not feasible. In this R21 proposal, we aim to integrate ZDs with PEGs for the development of ZD-modified PEGs (ZD-PEGs) as novel copolymers for protein conjugation. The central hypothesis is that ZD-PEGs are improved alternatives of PEGs, and ZD-PEGylated proteins (ZD-PEG-PTENs) can retain protein bioactivity, mitigate PEG antigenicity, and possess a range of favorable biomedical-relevant properties. Based on the complementary expertise of MPIs, the following two specific aims are proposed: 1) to develop ZD-PEGs and ZD-PEG-PTENs, and 2) to understand the biomedical-related properties of ZD-PEG- PTENs. ZD-PEGs each having a zwitterion (ZW)-functionalized poly(amidoamine)-based ZD unit and a PEG block with ω-N-hydroxysuccinimide (NHS) terminal are designed. A library of ZD-PEGs with varied ZD generation (G1 to G4), ZW type (carboxybetaine, sulfobetaine, or phosphorylcholine), and PEG length (5K and 10K) will be synthesized by the preparation of ZDs with an alkyne focal functionality, followed by coupling the ZDs with α-azide,ω-NHS PEGs via alkyne-azide click chemistry. Using porcine-like uricase as a model protein, ZD-PEG-uricases with controlled PEGylation density will be prepared through amidation reactions of ω-NHS terminals of ZD-PEGs with amine functionalities of uricase. Comprehensive analytical approaches will be employed to characterize ZD-PEGs and ZD-PEG-uricase to verify their well-controlled structures. Systematic property studies of ZD-PEG-uricases will be performed to achieve an insightful understanding of their structure- property relationships. The effects of ZD-PEG modification on the solubility, anti-biofouling property, enzyme activity, immune response, circulation time, biodistribution, and toxicity of uricase will be investigated through both in vitro and in vivo studies. Specifically, how ZW type, ZD generation, PEG length, and PEGylation density affect these biomedical-related properties will be assessed. The proposed R21 studies promise to not only establish the synthetic methodology for ZD-PEGs and ZD-PEG-PTENs, but also provide critical insights into their structure-dependent biomedical-relevant properties. These studies will lay a solid foundation for the further development of ZD-PEG-PTENs as novel therapeutics for many clinical applications.
