Intracellular Delivery Using Nanosecond-Laser Excitation of Large-Area Plasmonic Substrates

Abstract

Efficiently delivering functional cargo to millions of cells on the time scale of minutes will revolutionize gene therapy, drug discovery, and high-throughput screening. Recent studies of intracellular delivery with thermoplasmonic structured surfaces show promising results but in most cases require time- or cost-intensive fabrication or lead to unreproducible surfaces. We designed and fabricated large-area (14 × 14 mm), photolithography-based, template-stripped plasmonic substrates that are nanosecond laser-activated to form transient pores in cells for cargo entry. We optimized fabrication to produce plasmonic structures that are ultrasmooth and precisely patterned over large areas. We used flow cytometry to characterize the delivery efficiency of cargos ranging in size from 0.6 to 2000 kDa to cells (up to 95% for the smallest molecule) and viability of cells (up to 98%). This technique offers a throughput of 50000 cells/min, which can be scaled up as necessary. This technique is also cost-effective as each large-area photolithography substrate can be used to deliver cargo to millions of cells, and switching to a nanosecond laser makes the setup cheaper and easier to use. The approach we present offers additional desirable features: spatial selectivity, reproducibility, minimal residual fragments, and cost-effective fabrication. This research supports the development of safer genetic and viral disease therapies as well as research tools for fundamental biological research that rely on effectively delivering molecules to millions of living cells.

Analysis of poration-induced changes in cells from laser-activated plasmonic substrates

“…The pore radius sizes are in agreement with experiments delivering dextran molecules that have a Stokes radius in the range of 20 nm [21,34]. Membrane resealing occurs at this pore size [42,43].…”

“…The geometry consists of a polymer pyramid on a glass substrate with a 50-nm gold film on top. Please see [34] for detailed fabrication steps and simulation model.…”

“…Plasmonic substrate-based methods operate with the same physical poration mechanism as plasmonic nanoparticles but use lithography techniques to precisely pattern a surface of nanostructures that are not ingested by the cell. Recent studies have also shown that plasmonic substrates can deliver a broad range of cargo, including fluorescent probes, plasmids, bacteria, and proteins into cells [8,18,19,33,34]. Despite providing promising intracellular delivery results, the cellular changes induced by plasmonic substrates at the single cell level are not well understood [8,18,34].…”

“…Recent studies have also shown that plasmonic substrates can deliver a broad range of cargo, including fluorescent probes, plasmids, bacteria, and proteins into cells [8,18,19,33,34]. Despite providing promising intracellular delivery results, the cellular changes induced by plasmonic substrates at the single cell level are not well understood [8,18,34]. To better understand and optimize the delivery process, it is important to analyze the induced membrane pores and associated cellular changes.…”

A novel cell transfection platform based on laser optoporation mediated by Au nanostar layers

“…In contrast to the recent approaches, based on the addition of AuNPs to the transfection medium [25,31] in combination with laser irradiation, we use AuNS monolayers immobilized on the chemically activated bottoms of microplates [32]. Similar strategies using pulsed lasers and twodimensional plasmonic nanostructures were published recently [31,33,34]. However, to the best of our knowledge, none of them used AuNS monolayers in combination with CW laser for plasmonic cell optoporation.…”