EAPSI: Development of Amphiphilic, pH Responsive Cationic Copolypeptoids for Nonviral Cell Transfection in Serum

通过细胞转染法(CT)进行的基因治疗实践是解决罕见疾病和遗传性疾病(如血友病、SCID和Kippel Trenauny)的一种有前途的方法。在非病毒基因载体中，CT聚合物受到了极大的关注，主要是因为它们的化学通用性、大规模生产和细胞相容性使其具有潜在的生物医学应用前景。然而，低转染率和大小相关的细胞毒性等缺点限制了这些系统的充分利用。在这项研究中，将开发响应性阳离子多肽共聚物。在苏州大学细胞转染学专家尹立臣教授的指导下，合成的共聚物将用于研究核酸凝聚多聚体在体外细胞转染过程中的颗粒稳定性和行为。这项研究将提供一种在细胞转染过程中稳定遗传物质的策略。这种方法还允许通过内体逃逸在细胞内有效地释放遗传物质。聚(N-取代甘氨酸)是一类特殊的模拟肽聚合物，其主链上的氮原子上含有取代基。这类聚合物通常被称为多肽，以其最小的细胞毒性、增强的蛋白质降解稳定性和结构可调性而闻名，使其成为各种生物医学和生物技术应用的潜在候选者，包括智能涂层、抗菌剂和药物输送。目前，由于多肽/DNA复合体在血清中的不稳定性，使得多肽/DNA复合体在含血清介质中的细胞转导存在问题。电荷屏蔽的表现形式，其中与阴离子血清成分(脂质、白蛋白、盐等)相互作用。减少聚合物表面电荷，是造成这一结果的主要原因。为了应对这一挑战，本项目将开发含有亲水和疏水链段的嵌段多肽共聚物。在这项研究中，将测试合成的共聚物在体外核酸酶细胞转染过程中有效保护和运输治疗基因P53的能力。在允许电荷屏蔽的细胞外条件下(pH=5.0-5.5)，预计共聚物将通过采用胶束结构来响应这种生物刺激，胶束结构具有带电的疏水内核和电荷中性的外壳。殷博士在细胞转基因领域的团队熟练程度将对理解和阐明这些聚合系统在生物环境中的行为做出重大贡献。该奖项由东亚和太平洋暑期学院项目设立，资助一名美国研究生的暑期研究，由美国国家科学基金会和中国科技部联合资助。

Carried out by the process of cell transfection (CT), the practice of gene therapy is a promising approach in addressing rare diseases and genetic disorders (e.g. hemophilia, SCID, and Kippel Trenauny). Among non-viral gene carriers used for CT polymers have received significant attention primarily because their chemical versatility, large-scale production, and cytocompatibility make them potential candidates for biomedical applications. However, drawbacks such as low transfection efficiency and size related cytotoxicity limiting full usage of these systems. In this research, responsive, cationic polypeptoid copolymers will be developed. Under the guidance of Professor Lichen Yin, an expert in cell transfection at Soochow University, synthesized copolymers will be used to investigate the particle stability and behavior of nucleic acid condensed polyplexes during in vitro cell transfection. This research will provide a strategy to stabilize genetic material during cell transfection. This approach will also permit the effective release genetic material intracellularly via endosomal escape.Poly(N-substituted glycines) are a special class of peptidomimetic polymers bearing substituents on the nitrogen present in the backbone. Commonly referred to as polypeptoids, this group of polymers are known for their minimal cytotoxicity, enhanced proteolytic stability and structural tunability, making them potential candidates for a variety of biomedical and biotechnological applications including smart-coatings, antimicrobial agents, and drug delivery. Currently, the cell transfection of polypeptoid/DNA complexes in serum-containing media is problematic due to the instability of the complexed particles in serum. The manifestation of charge shielding, in which interaction with anionic serum components (lipids, albumin, salts, etc.) reduces polyplex surface charge, is mainly responsible for this outcome. To address this challenge, block polypeptoid copolymers containing hydrophilic and hydrophobic segments will be developed in this project. In this research synthesized copolymers will be tested for their ability to effectively protect and transport therapeutic gene p53 during in vitro cell transfection in nuclease. Under extracellular conditions where charge shielding is permissible (pH= 5.0-5.5), it is anticipated that copolymers will respond to this biological stimuli by adopting a micellar structure with a charged, hydrophobic inner core and charge neutral outer shell. Dr. Yin's group proficiency in the field of cell transfection will contribute significantly towards efforts to understand and elucidate the behavior of these polymeric systems in biological environments. This award, under the East Asia and Pacific Summer Institutes program, supports summer research by a U.S. graduate student and is jointly funded by NSF and the Chinese Ministry of Science and Technology.