Guided Intracellular Delivery using Precise Area Introduction and Transfection (PAINT) - PROJECT SUMMARY The objective of the proposed project is to develop a method called PAINT (Precise Area INtroduction and Transfection) for highly localized, versatile, and efficient biomolecule delivery for “direct-write” guided administration at spatial resolutions from single cell to hundreds of cells. One current approach that is commonly used to deliver biomolecules to cells involves viral or non-viral transfection. These methods do not offer the ability to target delivery of cargo to a subset of cells. Furthermore, they are limited in which cargoes they can deliver efficiently. In contrast, physical delivery methods offer control over the spatial distribution of cellular modification and are compatible with a wider range of cargo, but currently operation is too difficult, and versatility is too limited for them to gain widespread use. The proposed effort addresses these limitations with a new technology called PAINT, which will enable very precise control over the spatial distribution of cellular modification, i.e., introduction of nucleic acids (RNA, DNA), proteins, nanoparticles or viruses, using a novel combination of physical delivery means to transiently disrupt the cell membrane thus enabling cargo entry at a rate orders of magnitude greater than the current state of the art. The proposed new technology will be embodied in a PAINT “brush,” a stylus that delivers from one set of nozzles a cold plasma, and from a very closely located adjacent set (<< 1 mm away) an electro-kinetically generated jet of the cargo-containing solution along with a tightly focused coaxial gas jet for guided delivery. The plasma and coaxial gas jet remove any thin liquid layers that could impede transport of the cargo carrying liquid from the target area. The introduction of material is easily directed and confined to desired target areas so that PAINT permits guided production of heterogeneously modified bio-samples. The development of this new technology will be accomplished through completion of three key aims. The first specific aim is engineering design optimization and improvement and characterization of PAINT devices. The second aim is a careful characterization and optimization of the PAINT systems ability to deliver different cargos to human cells. The final aim is feasibility demonstrations in which PAINT is used to create biomimetic organoid systems in vitro. Because PAINT is a non-contact method, it is more versatile and simpler in implementation than micro-injection, and its electrokinetic jet can be caused to deliver cargo to targets as small as a cell or to modify areas hundreds of times bigger. As a result, PAINT permits simple, quick production of heterogeneously modified samples, including in vitro cell and tissue cultures.
