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.
