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Massively Parallel Delivery of Large-sized Cargo into Mammalian Cells with Light Pulses


Enabling technologies that transfer micron-sized objects into mammalian cells are needed to advance key applications in cell engineering. High-throughput delivery of organelles, modified intracellular pathogens, enzymes, proteins, and other types of cargo is required to obtain reliable data in settings of cellular heterogeneity. Delivered objects must also avoid entrapment in cell vacuoles or endosomes to retain functionality. Here, we report a massively parallel photothermal microfluidic platform for high-throughput delivery of large cargo directly into the cytosol of mammalian cells. Micron-sized cargo is reproducibly delivered in up to 100,000 cells on the platform in a minute, with parameters for cargo delivery tunable for each cell and cargo type. The delivery platform is a compact silicon chip on which hundreds of thousands of micron-sized cavitation bubbles expand and collapse within nanoseconds in response to pulsed laser excitation. Cavitation bubbles are formed by rapid heat transfer from nearby metallic nanostructures via a photothermal process enhanced by the lightning-rod effect. High speed, localized fluid flows near cavitation bubbles puncture contacting cell membranes with precision, resulting in micron-sized transient membrane pores. Applied pressure provides an active driving force to speed slow diffusing large cargo through these transient pores before membrane repair and resealing. We have reproducibly delivered large cargo including micron-sized bacteria, enzymes, antibodies, and functional nanoparticles into a variety of cell lines, including three different types of primary cells, with high efficiency and high cell viability. Our experiments involving delivery of functional enzymes, antibodies and vacuole escape-incompetent bacteria prove that cargo is delivered directly into the cell cytosol on this platform, bypassing endocytosis. Massively parallel and nearly simultaneous delivery of cargo into cells under the same physiological conditions enables reliable statistical measurements of cargo interactions with cells over time. To demonstrate its utility, we used this platform to explore the intracellular lifestyle of Francisella novicida and discovered that the iglC gene is unexpectedly required for intracellular replication even after vacuolar escape into the cell cytosol. Our photothermal platform approach, termed BLAST (Biophotonic Laser Assisted Surgery Tool), provides a reliable mechanism for high-throughput engineering of cells with micron-sized natural and man-made materials.

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