Zwitterionic Dendrimer-modified PEG for Protein Conjugation

项目总结 聚合物-蛋白质结合物因其在生物医学领域的关键应用而吸引了人们的极大兴趣。 治疗许多疾病。作为一种具有高加工性的水溶性聚合物，聚乙二醇(PEGO)具有 被广泛用于蛋白质偶联，十几种聚乙二醇化的蛋白质已经商业化 各种疾病的临床治疗(包括关节炎、癌症、肝炎和痛风等)，每年一次 数十亿美元的销售额。然而，聚乙二醇会引起不良的免疫原性，降低蛋白质的生物活性。 两性离子聚合物(ZPs)在这些方面已经成为聚乙二醇的改进替代品，并可能导致 循环时间甚至更长，但通常线性ZP的加工性较低，这阻碍了它们的应用。 两性离子树状大分子(ZDS)比线性ZP具有更好的加工性，但蛋白质控制良好 直接用ZDS进行共轭是不可行的。在此R21提案中，我们的目标是将ZDS与PEGS集成在一起 ZD改性聚乙二醇酯(ZD-PEGS)作为新型蛋白质偶联共聚物的研究进展。中环 假设ZD-PEGS是PEGS和ZD-PEGylated蛋白质(ZD-PEG-PTEN)的改进替代品 可以保留蛋白质的生物活性，减轻聚乙二醇抗原性，并具有一系列良好的生物医学相关 属性。根据MPIS的互补专业知识，提出了以下两个具体目标：1) 开发ZD-PEGS和ZD-PEG-PTEN，以及2)了解ZD-PEG-PTEN的生物医学相关特性- PTEN。ZD-PEGS各自具有两性离子(ZW)功能化的基于聚酰胺胺的ZD单元和聚乙二醇酯 设计了带有ω-N-羟基丁二酰亚胺端子的积木。具有不同ZD的ZD-PEG文库 世代(G1至G4)、ZW型(羧基甜菜碱、磺基甜菜碱或磷酰胆碱)和聚乙二醇长(5K和 10K)将通过制备具有炔焦点官能团的ZDS来合成，然后偶联 ZDS与α-叠氮化物、ω-NHS通过炔-叠氮化物点击化学连接。以类猪尿酸酶为模型蛋白， 通过ω-NHS的酰胺化反应制备聚乙二醇化密度可控的ZD-PEG化尿酸酶 ZD-pegs的末端具有尿酸酶的胺功能。综合分析方法将是 用来表征ZD-PEGS和ZD-PEG-尿酸酶，以验证其良好控制的结构。系统化 将进行ZD-PEG-尿酸酶的性质研究，以实现对其结构的深入了解- 财产关系。ZD-聚乙二醇改性对其溶解性、抗生物污垢性能、酶活性的影响 尿酸酶的活性、免疫反应、循环时间、生物分布和毒性将通过 包括体外和体内研究。具体而言，ZW类型、ZD生成、聚乙二醇化长度和聚乙二醇化密度 将评估影响这些生物医学相关特性的因素。拟议的R21研究承诺不仅 建立了ZD-PEGS和ZD-PEG-PTEN的合成方法学，但也提供了对它们的关键见解 依赖于结构的生物医学相关特性。这些研究将为进一步的研究奠定坚实的基础。 ZD-PEG-PTEN作为多种临床应用的新型治疗药物的研究进展。

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.