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.

NARRATIVE Many proteins and genes have potential to treat or cure critical human diseases but cannot be used due to their inability to be delivered inside cells. This proposal describes a new method that uses light and nanoscale wires to briefly and safely make pores in cell membranes to enable proteins or genes to enter. Unlike other pore-forming approaches, this method could be used directly in patients or in large scale manufacturing that make cell therapies.