EAGER: Three-Dimensional Printing of Functional Nanobots for Precision Gene Delivery

超过10万种人类疾病是由基因组的基因改变引起的，这些疾病中只有很小一部分是可以治愈的。基因编辑作为一种纠正基因组内缺陷和突变的强大工具，代表了疾病治疗学的关键发展。特别是，聚集规则间隔短回文重复序列（CRISPR） Cas9代表了精确、有针对性的基因组变化能力的范式转变。最近，已经开发了几种用于细胞内递送CRISPR/Cas9复合物的方法。虽然这些方法取得了一定程度的成功，但要实现高效的细胞内CRISPR/Cas9传递仍然极具挑战性。这项探索性研究的早期概念资助（EAGER）资助支持设计、制造和测试纳米机器人或纳米级机器人的研究，这些机器人可以精确地靶向并将CRISPR/Cas9传递到患病细胞，并以受控的方式释放基因编辑机构。纳米机器人的三维纳米打印技术涉及多种材料的打印，可以为各种应用的功能纳米机器的可扩展纳米制造提供有力工具。纳米机器人可以彻底改变基因或药物传递，以修复许多人类疾病的遗传紊乱，这将对人类健康产生重大影响。该项目为研究生和本科生提供了令人兴奋的跨学科培训，整合了从制造到生物材料到纳米机器到治疗学的内容。纳米打印和纳米机器人是实验室演示的极好工具，可以吸引高中学生和教师、女性和未被充分代表的少数民族研究人员进入科学和工程领域。本项目旨在研究一种新型纳米机器人系统的纳米制造工艺，用于单细胞水平的靶向基因或药物递送。合作研究团队使用生物相容性材料设计纳米机器人，并使用纳米级3D打印系统制造它。该纳米机器人由磁性纳米马达和可生物降解的纳米载体组成。这种纳米马达通常是200纳米圆，400纳米长，是通过在水凝胶中嵌入氧化铁磁性纳米颗粒来3D打印的。这种尺寸相似的纳米货物也是通过将CRISPR/Cas9包裹在可生物降解的水凝胶中进行3D打印的，这样CRISPR/Cas9进入细胞后就可以通过生物降解释放出来。基础研究的重点是研究材料组成和性能、纳米制造工艺参数对纳米机器人性能的影响。该团队还测试了纳米机器人将CRISPR/Cas9传递到癌细胞中抑制肿瘤的功效。纳米制造工艺的可扩展性通过一系列纳米机器人的可重复性制造得到证明。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。

Over 100,000 human diseases are caused by genetic alterations in the genome, and only a very small portion of these diseases can be cured. Gene editing represents a pivotal development in disease therapeutics as a powerful tool to correct defects and mutations within the genome. In particular, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cas9 represents a paradigm shift in the ability to make precise, targeted genomic change. Recently, a few approaches have been developed for intracellular delivery of CRISPR/Cas9 complexes. While these approaches have some degree of success, it remains extremely challenging to achieve highly effective and efficient intracellular CRISPR/Cas9 delivery. This EArly-concept Grants for Exploratory Research (EAGER) grant supports research to design, manufacture, and test nanobots or nanoscale robots that can precisely target and deliver CRISPR/Cas9 to diseased cells and release the gene-editing agencies in a controlled fashion. The three-dimensional nanoscale printing method for fabricating the nanobots involves multi-materials printing and could be a powerful tool for scalable nanomanufacturing of functional nanoscale machines for a variety of applications. The nanobots could revolutionize gene or drug delivery to repair genetic disorder of many human diseases, which would have a strong impact on human health. The project offers exciting interdisciplinary training that integrates content from manufacturing to biomaterials to nanomachines to therapeutics for a diverse group of graduate and undergraduate students. Nanoscale printing and nanobots are excellent tools for laboratory demonstrations to attract high school students and teachers, and women and underrepresented minority researchers to science and engineering fields.This project aims to investigate the nanomanufacturing processing of a novel nanobot system for targeted gene or drug delivery at the single cell level. The collaborative research team designs the nanobot using biocompatible materials and uses a nanoscale 3D printing system to fabricate it. The nanobot consists of a magnetic nanomotor and a biodegradable nano-cargo. The nanomotor, which is typically 200 nm round and 400 nm long, is 3D printed by embedding iron oxide magnetic nanoparticles in hydrogel. The nano-cargo, which is of similar dimensions, is also 3D printed by encapsulating CRISPR/Cas9 in a biodegradable hydrogel, so that CRISPR/Cas9 can be released through biodegradation once inside the cell. Fundamental research focuses on investigating the effects of material composition and properties, and nanomanufacturing processing parameters on the nanobot performance. The team also tests the efficacy of the nanobot to deliver CRISPR/Cas9 into cancer cells for tumor suppression. The scalability of the nanomanufacturing process is demonstrated through the reproducible fabrication of an array of nanobots.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.