Photoelectroporation: Biomacromolecule delivery via nanoscale light-amplified voltage generators

摘要 生物大分子，如蛋白质和核酸的受控和有效的细胞内输送是一种 在实现其作为治疗药物和关键试剂的潜力方面面临重大挑战 细胞产物疗法，如CAR-T细胞。现有的生物、化学和物理递送方法都有 限制了它们在体内或大规模应用中的使用。提出了光电穿孔(PEP)技术 来克服这一挑战。单晶硅纳米线(直径约50 nm，长10微米)，包含 光电二极管是一种“光电穿孔器”，它可以分散在细胞中，并被近红外线激发。 (近红外)光在纳米线上产生电压，计算出的电场和电流密度类似于 那些在传统和微型电穿孔中实现的，足以驱动细胞膜穿孔 形成并使大分子能够扩散到细胞质中。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、不加热，并具有良好的空间和时间控制。佩普 技术可以提供分布式或本地定向交付，无论是大容量还是小容量，甚至是流动的，以及 将为需要生物、细胞或细胞衍生疗法的患者提供显著的好处。吉奥德 已经开发出一种工艺，可以生产出比传统方法多105倍的材料，最终使它 不仅可以评估PEP的传递能力，而且可以将其应用于体内或细胞处理用途 未来。这项计划的目标是生产具有不同数量的二极管和涂层的光电穿孔器 并在体外评估其PEP将模型和功能生物大分子输送到细胞的能力 降低了生存能力。为了实现这一目标，已经设定了两个目标。(1)合成不同数量的硅纳米线 沿其长度方向编程并具有不同涂层的pn二极管，并表征其物理、化学 和照片属性。(2)演示通过PEP将生物大分子传递到细胞，包括识别 具有最高效率和细胞活力的纳米线特性和PEP参数以及 了解分布场附近和内部的细胞是如何被电穿孔的。功能蛋白、mRNA和 DNA货物将被运送到贴壁细胞和非贴壁细胞。这些结果将使PEP成为一种可行的 一种利用光和分布的纳米线将功能强大的货物导入活细胞的方法 克服其他方法的限制，使未来的临床前工作成为可能，包括体内PEP和LITER- 测量PEP与疾病相关的货物和靶细胞。

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.