Collaborative Research: Advanced biomanufacturing of functional bionanoparticles for biomedical engineering applications

1604826/1604925：Lei/Chen新型纳米材料的研究在过去十年中经历了爆炸式的增长。然而，从实验室规模到大规模生产的过渡的主要挑战，如高通量制造工艺，一致性，以及监测大批量产品质量的方法一直是实现其巨大潜力的瓶颈。该提案的目标是设计一种先进的制造工艺来制造基因工程多功能生物纳米颗粒（bio-NPs），并检查和验证其用于脑肿瘤癌症的非侵入性成像的实用性。如果成功，这将提供一个从NSF风格的基础科学到现实世界应用的极好演示。该项目将通过将先进的生物制造和生物成像模块整合到他们的教育和实验室培训中，对研究生，本科生和高中生的教育产生积极影响。将为学生提供一门新的生物制造研究型课程。本研究旨在通过基因工程的纳米囊泡形成菌发酵，合成新型的纳米级多功能外膜囊泡（OMV）。大肠杆菌中表达，并将修饰后的OMV用于脑肿瘤的无创生物成像。为了实现这一目标，重组DNA技术将首先用于设计用于生物成像的捕获和检测功能的新型基因工程蛋白质多功能生物纳米颗粒。生物纳米颗粒是具有均匀尺寸的基于脂质的OMV，双层的外小叶用新型工程蛋白融合物装饰，赋予多功能性。共展示多个拷贝的超活性NanoLuc荧光素酶（比常规萤火虫或海肾荧光素酶的活性高约150倍）的OMV将含有（i）用于锚定感兴趣的抗体的抗体结合结构域，和（ii）用于通过尺寸过滤简单纯化OMV的热响应性弹性蛋白样蛋白结构域。然后将设计一种与两级尺寸过滤集成的发酵工艺用于生产多功能OMV。最后，该项目将验证这些OMV用于脑肿瘤高性能生物成像的功能。这项研究将为生物制造提供一个新的视角，同时该产品可以极大地促进全球公共健康。这种新型的可扩展的基因工程制造平台可以推广到制备具有许多其他所需功能的OMV，适用于广泛的应用，包括生物修复，生物催化剂，生物传感，生物质转化，疫苗和药物递送。

1604826/1604925:Lei/ChenResearch on new nanomaterials has undergone explosive growth in the past decade. However, the main challenges of the transition from laboratory-scale to mass production, such as high throughput manufacturing processes, uniformity, and methodology of monitoring the quality of large-quantity products have been the bottlenecks to realize their tremendous potential. The goal of this proposal is to design an advanced manufacturing process to manufacture genetically engineered multi-functional bio-nanoparticles (bio-NPs) and to examine and validate their utility for non-invasive imaging of brain tumor cancer. If successful, this will provide an excellent demonstration from NSF-style basic science to real-world applications. This project will positively impact education of graduate, undergraduate and high school students by integrating advanced biomanufacturing and bioimaging modules into their educational and laboratory training. A new research-oriented course in Biomanufacturing will be offered to students. This multidisciplinary project aims to synthesize novel nano-sized multi-functional outer membrane vesicles (OMVs)decorated with engineered proteins through fermentation of genetically engineered nano-vesicle-forming E. coli and then apply the decorated OMVs for non-invasive bioimaging of brain tumor. To accomplish this, recombinant DNA technology will first be used to design novel genetically engineered protein multi-functional bio-NPs for capture and detection functions for bioimaging. The bio-NPs are lipid-based OMVs with a uniform size and the outer leaflet of the bilayer is decorated with novel engineered protein fusion, endowing multi-functionality. The OMVs, co-displaying multiple copies of super-active NanoLuc luciferase enzyme (~150-fold more active than that of conventional firefly or Renilla luciferase), will contain (i) an antibody-binding domain for anchoring antibodies of interest, and (ii) a thermo-responsive elastin-like protein domain for simple purification of the OMVs via size filtration. A fermentation process integrated with two-stage size filtration will then be designed for production of multi-functional OMVs. Finally, the project will validate the functionality of these OMVs for high performance bioimaging of brain tumor. The proposed research will offer a new perspective to biomanufacturing while the product can greatly promote global public health. This novel scalable genetically-engineered manufacturing platform can be generalized to prepare the OMVs with many other desired functions suitable for a wide range of applications including bioremediation, biocatalysts, biosensing, biomass conversion, vaccines, and drug delivery.