Photoelectroporation: Biomacromolecule delivery via nanoscale light-amplified voltage generators

光电穿孔：通过纳米级光放大电压发生器传递生物大分子

• 批准号: 10538761
• 负责人: Julie Champion
• 金额: $ 21.56万
• 依托单位: GEORGIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10538761
• 关键词: Biological; Bioreactors; Biotechnology; Caliber; Cell Culture Techniques; Cell Line; Cell Survival; Cell Therapy; Cell membrane; Cells; Chemicals; Colon Carcinoma; Complex; Culture Media; Cytosol; DNA; Devices; Dextrans; Diffusion; Disease; Electronics; Electroporation; Elements; Filler; Future; Generations; Genes; Goals; HCT116 Cells; In Vitro; Length; Light; Measurement; Messenger RNA; Methods; Modeling; Molecular Weight; Nanomanufacturing; Nucleic Acids; Outcome; Oxides; Patients; Process; Production; Property; Proteins; Reagent; Research; Route; Series; Structure; Technology; Testing; Therapeutic; Time; Tissues; Work; base; biological research; biomacromolecule; chemical property; chimeric antigen receptor T cells; clinical application; crystallinity; density; electric field; fluorexon; human disease; in vivo; innovation; macromolecule; nanoparticle; nanoscale; nanowire; physical property; pre-clinical; success; targeted delivery; therapeutic gene; therapeutic protein; tool; voltage

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.

抽象的 生物大分子（例如蛋白质和核酸）受控有效的细胞内递送是一种 在实现其作为治疗和关键试剂中生产细胞和关键试剂的巨大挑战和 细胞产物疗法，例如CAR-T细胞。现有的生物学，化学和物理递送方法都有 排除它们在体内或大规模应用中使用的局限性。提出了光电子的（PEP） 克服这一挑战。包含单晶Si纳米线（直径约50 nm，长10 µm） 光电二极管是可以分散在细胞中并通过近红外激发的“光电子剂” （NIR）光在跨纳米线上产生电源和电流密度类似的电压 在传统和微观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。 NIR光可以穿透组织或生物反应器 在静态或流程构型中，无毒且无加热，具有出色的空间和临时控制。 pep 技术可以提供分布式或本地针对的交付，甚至在流量中，甚至在流量中， 将为需要生物，细胞或细胞衍生的疗法的患者带来重大好处。 Geode 已经开发了生产材料比常规方法多约105倍，最终使它多105倍 可行的不仅可以评估PEP的交付能力，还可以将其应用于体内或细胞处理用途 未来。该提案的目的是生产具有不同数量演讲和涂料的光电子 并在体外评估其PEP容量，以将模型和功能性生物ac纤维分子传递给没有 降低生存能力。已经设定了两个目标来实现这一目标。 （1）合成具有不同数字的SI纳米线 PN语音沿其长度和不同的涂层进行编程，并表征其物理化学 和照片属性。 （2）通过PEP证明将生物ac骨分子传递到细胞，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 了解分布式场内和分布场中的细胞是电穿孔的。功能蛋白，mRNA和 DNA货物将被输送到粘附和非粘附细胞中。这些结果将使PEP成为可行的 使用光线和分布式纳米线的大型功能性货物翻译可行的细胞的方法 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LINT- 用疾病相关的货物和靶细胞来缩放PEP。