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

这里提出的工作创造了蛋白质囊泡，一种由蛋白质制成的微小中空容器，可以与特定类型的细胞结合，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以乳腺癌细胞为目标，并提供一种蛋白质、核酸和化疗分子的组合，这种组合可能对乳腺癌细胞具有独特的疗效。这是很重要的，因为这些囊泡有潜力通过同时三重给药来解决耐药和转移的挑战。除了工程蛋白囊泡之外，拟议的工作将提出基础和实际问题，这些问题将建立囊泡组装过程和特性的关键知识，并将这项工作扩展到其他领域和由其他类型分子制成的囊泡。蛋白质囊泡的优点之一是它们能够在表面呈现一系列蛋白质，这些蛋白质可以针对不同类型的细胞，并携带各种货物，因此可以设想许多其他药物递送应用，例如疫苗和免疫疗法。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。除了重大疾病研究的社会效益外，该倡议还将开展活动，在从中学到研究生的教育水平上招募和留住妇女，特别是来自代表性不足群体的妇女。中学女生将参观实验室作为夏令营和实地考察的一部分。该研究将为女性研究生和本科生的技术培训和批判性个人指导提供依据。它还将为代表性不足的亚特兰大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乔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中分享，并在K-12学校访问中分享，以告知和激励学生追求和坚持STEM学位。本研究的目的是了解、设计和使用由球形功能蛋白制成的自组装蛋白囊泡用于治疗递送。具体来说，囊泡将由全尺寸的治疗性或靶向性蛋白质制成，这些蛋白质在管腔或膜中也携带三种不同类型的治疗货物：核酸、小分子和附加蛋白质。目的1将通过修饰囊泡构建块蛋白的氨基酸序列，将响应元件和不同的化学环境纳入蛋白质囊泡中。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间和地点释放和发挥作用。目的2将利用动态光散射、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。这些知识将有助于研究人员合理地设计合成、重组或杂交分子，这些分子能够形成具有响应特性和载货能力的自组装结构。目标3将利用这一知识创建功能性蛋白囊泡，能够同时将蛋白（YopJ）、核酸（抗p糖蛋白siRNA）和小分子（阿霉素）的受体靶向递送到体外的乳腺癌细胞（表皮生长因子受体阳性），作为概念的证明。将通过流式细胞术、共聚焦显微镜、细胞活力和功能测定来证明递送。本研究将为女性研究生、本科生和亚特兰大高中学生提供技术训练和批判性个人指导的基础。PI还将开展活动，在从中学到研究生的教育水平上招募和留住女性，特别是来自代表性不足群体的女性。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值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.