Engineering Responsive Chemical Heterogeneity in Protein Vesicles for Simultaneous Delivery of Diverse Cargoes

非技术概述这里提出的工作是创造蛋白质囊泡，这是一种由蛋白质制成的微小中空容器，可以结合到特定类型的细胞上，并在细胞内携带三种不同的治疗药物。这一概念的功能演示将针对乳腺癌细胞，并提供一种蛋白质、核酸和化疗分子的组合，这些分子可能对它们具有独特的有效作用。这一点意义重大，因为这些囊泡有可能通过同时传递三重药物来应对耐药和转移的挑战。除了设计蛋白质囊泡，这项拟议的工作将提出基本和实际问题，这些问题将建立囊泡组装过程和性质的关键知识，并将这项工作扩展到其他领域和由其他类型的分子制成的囊泡。蛋白质囊泡的优势之一是它们能够在表面呈现一系列蛋白质，这些蛋白质可以靶向不同类型的细胞，并携带各种货物，因此可以设想许多其他药物输送应用，如疫苗和免疫疗法。广泛的影响也可以通过其他用途实现，包括生物传感器、人造细胞和小型生物反应器。除了关键疾病研究的社会益处外，国际和平研究所还将开展活动，招募和留住妇女，特别是来自代表性不足群体的妇女，在从中学到毕业的教育水平上。作为夏令营和实地考察的一部分，中学女生将参观实验室。这项研究将为女性研究生和本科生的技术培训和关键的个人辅导提供依据。它还将为亚特兰大代表性不足的高中生提供有意义的研究经验、专业发展和指导。这项工作的结果将在佐治亚理工学院的化学工程入门课程和PI的蛋白质工程选修课上分享，并在K-12学校访问中分享，以告知和激励学生追求并坚持STEM学位。技术总结本研究的目标是理解、设计和使用由球形、功能蛋白质制成的自组装蛋白质囊来进行治疗。具体地说，这些囊泡将由全尺寸治疗性或靶向性蛋白质制成，这些蛋白质还在管腔或膜中携带三种不同类别的治疗性货物：核酸、小分子和额外的蛋白质。目标1将通过改变囊泡构建块蛋白质的氨基酸序列，将响应元件和不同的化学环境结合到蛋白质囊泡中。使用天然和非天然氨基酸修改蛋白质序列的能力将使调整能够确保货物在需要的时候和地点释放并发挥作用。目的2利用动态光散射、透射电子显微镜、吸光度和荧光光谱等技术，了解这些序列变化如何影响囊泡的自组装、性质和稳定性。这些知识将帮助研究人员合理地设计合成、重组或杂化分子，这些分子能够形成具有响应性和装载量的自组装结构。目的3将利用这一知识在体外创造能够同时将蛋白质(YopJ)、核酸(抗p-糖蛋白siRNA)和小分子(阿霉素)运送到乳腺癌细胞(表皮生长因子受体阳性)的功能性蛋白囊泡作为概念的证明。交付将通过流式细胞术、共聚焦显微镜、细胞活力和功能分析来证实。这项研究将为女研究生、本科生和亚特兰大高中生的技术培训和关键的个人指导提供基础。PI还将在STEM从中学到毕业的教育水平上实施招募和留住妇女的活动，特别是来自代表性不足群体的妇女。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Non-Technical SummaryThe work proposed here creates protein vesicles, tiny hollow containers made from proteins, that can bind to specific types of cells and carry three different kinds of therapeutic drugs inside cells. The functional demonstration of this concept will target breast cancer cells and deliver a combination of protein, nucleic acid and chemotherapeutic molecules that could be uniquely effective against them. This is significant because these vesicles have the potential to address challenges with drug resistance and metastasis by simultaneous triple drug delivery. Beyond engineering protein vesicles, the proposed work will ask both fundamental and practical questions that will build key knowledge of the vesicle assembly process and properties and extend this work into other areas and vesicles made from other types of molecules. One of the advantages of protein vesicles is their ability to present a range of proteins on the surface, which can target different types of cells, and carry a variety of cargoes, so that many other drug delivery applications could be envisioned, such as vaccines and immunotherapies. Broad impact could also be realized by other uses including biosensors, artificial cells, and small bioreactors. In addition to societal benefits of the research on critical diseases, the PI will implement activities to recruit and retain women, especially women from underrepresented groups, in STEM at education levels ranging from middle school to graduate. Middle school girls will visit the lab as part of summer camps and field trips. The research will provide the basis for technical training and critical personal mentoring of female graduate and undergraduate students. It will also provide meaningful research experience, professional development, and mentoring to underrepresented Atlanta high school students. The results from this work will be shared in the chemical engineering introductory course and the PI’s protein engineering elective at Georgia Tech, and at K-12 school visits, to inform and inspire students to pursue and persist in STEM degrees.Technical SummaryThe goal of this research is to understand, engineer, and employ self-assembled protein vesicles made from globular, functional proteins for therapeutic delivery. Specifically, the vesicles will be made from full-size therapeutic or targeting proteins that also carry three different classes of therapeutic cargos in the lumen or membrane: nucleic acids, small molecules, and additional proteins. Objective 1 will incorporate responsive elements and distinct chemical environments into protein vesicles by modifying the amino acid sequences of the vesicle building block proteins. The ability to modify the protein sequence, with both natural and non-natural amino acids, will enable tuning to ensure the cargos are released and function when and where needed. Objective 2 will understand how these sequence changes affect vesicle self-assembly, properties and stability using techniques including dynamic light scattering, transmission electron microscopy, and absorbance and fluorescence spectroscopy. This knowledge will help researchers rationally design synthetic, recombinant, or hybrid molecules capable of forming self-assembled structures with responsive properties and cargo loading. Objective 3 will use this knowledge to create functional protein vesicles capable of simultaneous, receptor targeted delivery of protein (YopJ), nucleic acid (anti-p-glycoprotein siRNA) and small molecule (doxorubicin) cargoes to breast cancer cells (epidermal growth factor receptor positive) in vitro as proof of concept. Delivery will be demonstrated via flow cytometry, confocal microscopy, and cell viability and function assays. The research will provide the basis for technical training and critical personal mentoring of female graduate, undergraduate, and Atlanta high school students. The PI will also implement activities to recruit and retain women, especially women from underrepresented groups, in STEM at education levels ranging from middle school to graduate.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.