Photoelectroporation: Biomacromolecule delivery via nanoscale light-amplified voltage generators

摘要 生物大分子（例如蛋白质和核酸）的受控且有效的细胞内递送是一种 实现其作为治疗剂和细胞制造关键试剂的潜力是一项重大挑战 细胞产品疗法，例如 CAR-T 细胞。现有的生物、化学和物理递送方法均具有 限制其在体内或大规模应用中的使用。提出光电穿孔（PEP） 来克服这一挑战。单晶硅纳米线（直径约 50 nm，长 10 µm）包含 光电二极管是可以分散在细胞中并被近红外激发的“光电穿孔器” (NIR) 光在纳米线上产生电压，计算出的电场和电流密度类似于 在传统和微型电穿孔中实现的那些足以驱动细胞膜孔 形成并使大分子扩散到细胞质中。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 技术可以提供分布式或局部有针对性的交付，无论是大批量还是小批量，甚至是流动的，并且 将为需要生物、细胞或细胞衍生疗法的患者带来显着的益处。晶洞 已开发出比传统方法多生产约 105 倍材料的工艺，最终使其成为 不仅可以评估 PEP 的递送能力，还可以将其应用于体内或细胞处理用途。 未来。该提案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 并评估其 PEP 在体外将模型和功能性生物大分子递送至细胞的能力，而无需 降低生存能力。为了实现这一目标，我们设定了两个目标。 (1) 合成不同根数的硅纳米线 沿其长度和具有不同涂层的 pn 二极管进行编程，并表征其物理、化学特性 和照片属性。 (2) 展示通过PEP将生物大分子递送至细胞，其中包括识别 具有最高效率和细胞活力的纳米线特性和 PEP 参数以及 了解分布场附近和分布场内的细胞如何被电穿孔。功能性蛋白质、mRNA 和 DNA 货物将被递送至贴壁细胞和非贴壁细胞。这些结果将使 PEP 成为一种可行的方法 用大型功能性货物转染活细胞的方法，使用光和分布式纳米线 克服其他方法的限制并实现未来的临床前工作，包括体内 PEP 和 Lite- 使用疾病相关货物和靶细胞扩展 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.