Microfluidic Synthesis of Nanoparticles for Cell Reprogramming

在正常生活中，身体不同部位的细胞利用它们的遗传遗产来执行创造人类健康的任务。在疾病期间，某些细胞可能无法正确执行这些任务。由于患者本身的基因组差异，适当和最佳的治疗方法可能会有所不同，因此使用患者自己的细胞来正确执行他们指定的任务将非常有帮助。在通过适当的遗传编程成功地将细胞的身份从一种类型改变为另一种类型后，这可能变得可行。最近，通过将成人皮肤细胞转化为其他理想的细胞类型，这一领域取得了突破性的进展。这样的细胞重编程方法更容易被接受，因为修改后的细胞来自患者，变化不会传递给其他人或后代。不幸的是，目前用于细胞重编程的传递工具非常慢，而且效率太低，无法用于治疗。该奖项研究了一种新的微流控方法来合成和制造用于高效基因传递的新型聚合物纳米颗粒。由于其连续的流程操作，这种新的方法以低成本大量生产这样的基因载体。此外，产生的高质量纳米颗粒可以显著提高细胞重新编程的成功率，以高产率生产所需类型的患者细胞。它的纳米制造潜力可能会加速这种患者匹配细胞未来的临床和制药用途。这项研究还将培训研究生在科学和工程方面的多学科技能。这些成果将被整合到工程课程中，通过在生物和纳米制造、细胞治疗、化学、表征和工艺开发方面的实践机会，激励、教育和留住本科生和少数族裔学生。虽然持续的细胞重编程是可行的，但低成功率与其瞬时转染行为有关，即关键转录因子的传递效率低，细胞存活率低，允许重复传递，以及在生产所需的基因载体和重新编程的细胞方面缺乏适当的制造工艺。本研究项目研究一种混合场微流控过程，通过限制流体界面的络合作用来生产均匀的复合纳米颗粒，以更好地保护、凝聚和释放遗传探针。金纳米颗粒载体有助于在聚合物解离后固定内化的聚合物分子，以降低细胞毒性。此外，电穿孔治疗还促进了遗传物质向细胞核的运输。这一组合确保了更高的传递效率，并允许重复转染以维持所需重新编程因子的表达。混合式微流控系统--S流动操作--允许以高通量和良好的质量控制连续生产基因载体和后来的患者匹配的细胞系。该项目的成功不仅为纳米探针的合成提供了一条强大的制造路线，也为潜在的诊断和治疗协同作用提供了有效的工具，以推进当前的基因/药物输送和基于细胞的生物制造过程。

During normal life, the cells in different parts of the body use their genetic legacy to perform tasks that create human health. During illnesses certain cells may not properly perform these tasks. As appropriate and optimal therapies may vary among patients due to their own genome difference, it would be very helpful to use the patient's own cells to correctly perform their designated tasks. This could become feasible after the identity of a cell is successfully changed from one type to another by proper genetic programming. Ground-breaking progress was made in this field recently by converting adult skin cells into other desirable cell types. Such cell reprogramming methods are more acceptable because the cells that are modified come from the patient and the changes cannot be transmitted to others or to offspring. Unfortunately, current delivery tools for cell reprogramming are very slow and too inefficient to use in therapy. This award investigates a new microfluidic approach to synthesize and manufacture novel polymeric nanoparticles for efficient gene delivery. With its continuous flow operation, this novel approach produces such gene carriers in large quantities and at low cost. Moreover, the high-quality nanoparticles that are generated could significantly increase the success rate of cell reprogramming to produce desired types of a patient's cells with high yield. Its nanomanufacturing potential could accelerate future clinical and pharmaceutical uses of such patient-matched cells. This research will also train graduate students in multidisciplinary skills in science and engineering. The results will be integrated into engineering courses to inspire, educate, and retain undergraduate and minority students through hands-on opportunities in bio- and nanomanufacturing, cell therapy, chemistry, characterization, and process development. Although sustained cell reprogramming is feasible, the low success rate is tied to its transient transfection behavior, namely, poor delivery efficiency of the key transcription factors and low cell survival to allow for repeated delivery, and the lack of appropriate manufacturing process in the production of desired gene carriers and reprogrammed cells. This research project studies a hybrid field microfluidic process to produce homogeneous polyplex nanoparticles for better protection, condensation, and release of genetic probes by limiting the complexation at the fluid interface. Gold nanoparticle carriers help fix the internalized polymer molecules after the dissociation of the polyplex to reduce cytotoxicity. In addition, electroporation treatment promotes transport of genetic material to the nucleus. The combination ensures increased delivery efficiency and the allowance for repeated transfection to sustain the expression of the needed reprogramming factors. The hybrid microfluidic system?s flow operation allows continuous production of gene carriers and later patient-matched cell lines with high throughput and good quality control. The project's success provides not only a powerful manufacturing route in nanoprobe synthesis, but also an effective tool for potential synergy of diagnosis and therapeutics to advance current gene/drug delivery and cell-based bio-manufacturing processes.