Poking cells for efficient vector-free intracellular delivery

Abstract

Techniques for introducing foreign molecules and materials into living cells are of great value in cell biology research. A major barrier for intracellular delivery is to cross the cell membrane. Here we demonstrate a novel platform utilizing diamond nanoneedle arrays to facilitate efficient vector-free cytosolic delivery. Using our technique, cellular membrane is deformed by an array of nanoneedles with a force on the order of a few nanonewtons. We show that this technique is applicable to deliver a broad range of molecules and materials into different types of cells, including primary neurons in adherent culture. Especially, for delivering plasmid DNAs into neurons, our technique produces at least eightfold improvement (B45% versus B1-5%) in transfection efficiency with a dramatically shorter experimental protocol, when compared with the commonly used lipofection approach. It is anticipated that our technique will greatly benefit basic research in cell biology and also a wide variety of clinical applications.

Nanoneedle-Based Sensing in Biological Systems

“…Suspensions of cells can be centrifuged over nanoneedle arrays 62 , or vice versa nanoneedles can be centrifuged over cells adherent to a substrate 2 . Hypergravity values ranging from 12.8g to 35.5g yield improve cell interfacing from 5% to 80%, as measured through delivery efficiency, while preserving viability 60 . A custom-design high speed linear actuator for nanoneedle arrays enables controlled interfacing 61 .…”

“…In general, the vast majority of the systems available have all reached a degree of optimization such that they present minimal toxicity and invasiveness for the biological systems they have been tested with. In vitro, AFM operated nanoneedles (Si) display a threshold for a sharp increase in toxicity at 400 nm in diameter 60 . Despite cytotoxicity is not reported in all instances, several strategies for forceful application of nanoneedles, including hypergravity 60 , mechanical actuation 43 , and piezoelectric oscillation 61 retain cell viability comparable to controls.…”

“…In vitro, AFM operated nanoneedles (Si) display a threshold for a sharp increase in toxicity at 400 nm in diameter 60 . Despite cytotoxicity is not reported in all instances, several strategies for forceful application of nanoneedles, including hypergravity 60 , mechanical actuation 43 , and piezoelectric oscillation 61 retain cell viability comparable to controls. Similarly, nanostraws (Al 2 O 3 ) are not toxic to primary cells, despite being able to extract up to 10% of intracellular reporter proteins 19 .…”

“…These strategies are are varied, and include hypergravity through centrifugation 60 , manual application of forces 18 , linear actuation 61 , and high-frequency oscillating piezoelectric movements for rapid, repeated interfacing 61 . All these strategies improve the ability of nanoneedles to sense from and delivery within the cell, but, similarly to what observed for cell seeding on nanoneedle arrays, there is no direct evidence that any of them can insert nanoneedles within cells.…”

“…In vitro, AFM operated nanoneedles (Si) display a threshold for a sharp increase in toxicity at 400 nm in diameter 60 . Despite cytotoxicity is not reported in all instances, several strategies for forceful application of nanoneedles, including hypergravity 60 …”