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

Abstract

Laser-exposed plasmonic substrates permeabilize the plasma membrane of cells when in close contact to deliver cell-impermeable cargo. While studies have determined the cargo delivery efficiency and viability of laser-exposed plasmonic substrates, morphological changes in a cell have not been quantified. We porated myoblast C2C12 cells on a plasmonic pyramid array using a 532-nm laser with 850-ps pulse length and time-lapse fluorescence imaging to quantify cellular changes. We obtain a poration efficiency of 80%, viability of 90%, and a pore radius of 20 nm. We quantified area changes in the plasma membrane attached to the substrate (10% decrease), nucleus (5 -10% decrease), and cytoplasm (5 -10% decrease) over 1 h after laser treatment. Cytoskeleton fibers show a change of 50% in the alignment, or coherency, of fibers, which stabilizes after 10 mins. We investigate structural and morphological changes due to the poration process to enable the safe development of this technique for therapeutic applications. Brown, "Glass needle-mediated microinjection of macromolecules and transgenes into primary human blood stem/progenitor cells," Blood 95(2),

Intracellular Delivery by Membrane Disruption: Mechanisms, Strategies, and Concepts

“…In standard calcium conditions recovery of microtubule integrity has been reported to take minutes up to an hour 463,464,467 . In some cases, membrane disruption has also appears to cause depolymerization of F-actin and intermediate filaments 464,469 .…”

A comparison of inverted and upright laser-activated titanium nitride micropyramids for intracellular delivery

“…As fluence increases, the membranes become more permeable, leading to greater molecular uptake. After the fluence increases beyond the optimum fluence, efficiency drops, as calcein leaves the dead cells through their highly porous, leaky membranes, a phenomenon seen in other intracellular studies 34 , 38 . As all porated cells are viable, the viability and poration efficiency curves match each other with increasing laser fluence.…”

“…This methodology can be traced back to pioneering studies using immobilized gold nanoparticles and metallic films on top of substrates such as glass and silicon 33 , 34 . More recently, a very promising, novel intracellular delivery platform uses structured, thermoplasmonic substrates 35 – 38 . These substrates are patterned with an array of gold, pyramid-shaped microstructures.…”

Plasmonic Intracellular Delivery

“…However, there is still a need for a high-efficiency, high-throughput, low-toxicity, cost-effective intracellular delivery technique that is applicable to a range of cells types for a range of cargoes. Plasmonic nanostructured surfaces may be a promising alternative to the currently available intracellular delivery techniques and utilize the unique ability of plasmonic structures to absorb laser light energy and transfer the energy to a confined volume within the nearby surrounding medium [31,39,[49][50][51][52]. Upon illumination with a short laser pulse, the laser light energy is strongly absorbed by the plasmonic nanostructures, resulting in a rise in temperature [1,[57][58][59].…”

“…Laser-activated nanostructured substrates bypass this potential toxicity problem, as cells can be cultured on the substrates, porated, and removed from the substrates (which remain intact) after intracellular delivery without leaving metallic particles within the cells [31,39,[49][50][51][52]. In this thesis we explore the fabrication of various thermoplasmonic nanostructured substrates for intracellular delivery and use the fabricated substrates to deliver a wide range of membrane-impermeable cargoes (dyes, dextrans, proteins, etc.)…”