Photoelectroporation: Biomacromolecule delivery via nanoscale light-amplified voltage generators

摘要 生物大分子（例如蛋白质和核酸）的受控和有效的细胞内递送是一种有效的方法。 在实现其作为治疗剂和用于制造细胞关键试剂的潜力方面存在重大挑战， 细胞产品疗法，如CAR-T细胞。现有的生物、化学和物理输送方法都具有 这些限制妨碍了它们在体内或大规模应用中的使用。提出了光电穿孔技术（PEP） 来克服这个挑战。单晶Si纳米线（直径约50 nm，长10 µm），包含 光电二极管是“光电穿孔器”，其可以分散在细胞中并由近红外激发。 (NIR)光以在纳米线上产生电压，计算出的电场和电流密度类似于 在传统和微尺度电穿孔中实现的那些，其足以驱动细胞膜孔 形成并使大分子扩散到胞质溶胶中。近红外光可以穿透组织或生物反应器 在静态或流动配置中，无毒且不加热，并且具有出色的空间和时间控制。PEP 技术可以提供大批量或小批量的分布式或局部目标交付，甚至是流动式交付， 将为需要生物、细胞或细胞衍生疗法的患者提供显著益处。这个晶洞 一种新的生产工艺已经发展到比传统方法多生产105倍的材料， 不仅可以评估PEP的递送能力，而且可以将其应用于体内或细胞加工用途， 未来本提案的目标是生产具有不同数量的二极管和涂层的光电转化器 并评估其PEP在体外将模型和功能性生物大分子递送至细胞而不 降低生存能力。为实现这一目标，确定了两个目标。(1)合成不同数目的硅纳米线 的pn二极管编程沿着其长度和不同的涂层，并表征其物理，化学 和照片属性。(2)演示通过PEP向细胞递送生物大分子，包括识别 具有最大效率和细胞活力的纳米线性质和PEP参数，以及 了解分布电场附近和分布电场内的细胞如何被电穿孔。功能蛋白、mRNA和 DNA货物将被递送至粘附细胞和非粘附细胞。这些结果将建立PEP作为一个可行的 用大型功能性货物转染活细胞的方法，该方法使用光和分布式纳米线， 克服了其他方法的限制，使未来的临床前工作，包括在体内PEP和升- 用疾病相关的货物和靶细胞来缩放PEP。

ABSTRACT Controlled and efficient intracellular delivery of biomacromolecules, such as proteins and nucleic acids, is a significant challenge in realizing their potential as therapeutics and critical reagents for manufacture of cell and cell product therapies, such as CAR-T cells. Existing biological, chemical, and physical delivery methods all have limitations that preclude their use in in vivo or large scale applications. Photoelectroporation (PEP) is proposed to overcome this challenge. Single-crystalline Si nanowires (~50 nm diameter, 10 µm long) containing photodiodes are the “photoelectroporators” that can be dispersed amongst cells and excited by near-infrared (NIR) light to generate a voltage across nanowires with calculated electric fields and current densities similar to those achieved in traditional and microscale electroporation, which are sufficient to drive cell membrane pore formation and enable diffusion of macromolecules into the cytosol. NIR light can penetrate tissue or bioreactors in static or flow configurations, is non-toxic and non-heating, and has excellent spatial and temporal control. PEP technology could provide distributed or locally targeted delivery, in large or small volumes, even in flow, and would offer significant benefits to patients in need of biologic, cellular, or cell derived therapies. The Geode process has been developed to produce ~105 times more material than conventional methods, finally making it feasible not only to evaluate the delivery ability of PEP but to apply it to in vivo or cell processing uses in the future. The goal of this proposal is to produce photoelectroporators with different numbers of diodes and coatings and evaluate their PEP capacity in vitro to deliver model and functional biomacromolecules to cells without reducing viability. Two aims have been set to meet this goal. (1) Synthesize Si nanowires with different numbers of pn diodes programmed along their length and with different coatings and characterize their physical, chemical and photo properties. (2) Demonstrate delivery of biomacromolecules to cells via PEP, which includes identifying the nanowire properties and PEP parameters with the greatest efficiency and cell viability as well as understanding how cells near and within a distributed field are electroporated. Functional protein, mRNA and DNA cargo will be delivered to both adherent and non-adherent cells. These results will establish PEP as a viable method to transfect viable cells with large, functional cargoes that uses light and distributed nanowires to overcome the constraints of other methods and enable future preclinical work, including in vivo PEP and liter- scale PEP with disease relevant cargoes and target cells.