title: 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.
authors.forename: Nabiha
authors.identifier: 0000-0002-6336-080X
authors.initials: N
authors.lastname: Saklayen
authors.affiliation: Department of Physics, Ludwig Maximilian University of Munich , 80539 Munich, Germany.
authors.forename: Marinus
authors.initials: M
authors.lastname: Huber
authors.forename: Marinna
authors.initials: M
authors.lastname: Madrid
authors.affiliation: ECE Paris Ecole d'Ingénieurs , 75015 Paris, France.
authors.forename: Valeria
authors.initials: V
authors.lastname: Nuzzo
authors.forename: Daryl I
authors.initials: DI
authors.lastname: Vulis
authors.forename: Weilu
authors.initials: W
authors.lastname: Shen
authors.forename: Jeffery
authors.initials: J
authors.lastname: Nelson
authors.forename: Arthur A
authors.initials: AA
authors.lastname: McClelland
authors.affiliation: Institute for Quantum Optics, Leibniz University Hannover , 30167 Hanover, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Heisterkamp
authors.forename: Eric
authors.identifier: 0000-0003-3194-9836
authors.initials: E
authors.lastname: Mazur
mesh_terms: D002470:Cell Survival; D016503:Drug Delivery Systems; D005434:Flow Cytometry; D006046:Gold / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D007834:Lasers; D053768:Metal Nanoparticles / Q000737:chemistry*; D010316:Particle Size; D055668:Photochemical Processes; D013499:Surface Properties; D013696:Temperature; D013997:Time Factors
chemical_list: D006046:Gold
keywords: flow cytometry; plasmonic intracellular delivery; pulsed lasers; template-stripping; thermoplasmonic substrates