Photothermal nanoblade delivers super-sized cargo into living cells

Tools and approaches that enable biologists to deliver extracellular materials into living cells are desired for numerous biological and biomedical research applications. Current physical methods use electric currents, light, or sound to create temporary pores in the cell membrane. However, these methods limit the size of the cargo that can be delivered. This limitation arises from the slow diffusion speed of large objects and their inability to cross transiently opened membrane pores in these approaches.

Direct microinjection eliminates the slow diffusion issue by providing active pressure-driven delivery of cargo into a cell through a sharp glass pipette tip. However, to avoid severe cellular trauma and maintain cell viability, the size of the pipette tip usually must be kept below 200nm. During delivery, this small pipette nanochannel may clog near the tip. The method cannot be used to deliver cargo larger than the pipette tip's inner diameter, such as organelles (e.g., peroxisomes, mitochondria, nuclei), beads, or intracellular pathogens (e.g., bacteria). In addition, some encapsulation approaches, such as use of lipofectamine to enclose cargo in liposomes for delivery, can result in large cargo becoming trapped or degraded in endosomes or endolysosomes.

Recently, we demonstrated a new approach called a photothermal nanoblade, which can deliver cargo up to 2–3 microns in size.^1,2 The nanoblade utilizes an ultrahigh-speed, light-triggered cavitation bubble patterned by a metallic nanostructure to disrupt mammalian cell membranes and enable large cargo delivery (see Figure 1). To create the nanoblade, we coat the outer tip of a glass micropipette with a metallic thin film (approximately 100nm thick). Laser pulsing rapidly heats the nanoblade and triggers formation of a cavitation bubble that can be tuned to last for about 200ns. Rapid bubble expansion and collapse causes membrane cutting that locally disrupts the cell membrane when the pipette tip is close by or in soft contact with the membrane. During this process, the rest of the cell remains unperturbed.

Figure 1. High-speed membrane puncture mechanism using metallic nanostructure-guided cavitation to deliver small molecules and micron-sized cargo into somatic cells. Upon excitation by a nanosecond laser pulse, the titanium (Ti) blade heats up rapidly, producing an explosive cavitation bubble that expands and collapses in 200nsec, which locally disrupts the contacting cell membrane (three panels at right). Pressure-driven fluid flow injects the microcapillary contents into the cell in synchrony with the membrane disruption. SiO₂: Silicon dioxide.

After a transient membrane pore is opened, active pressure-driven fluid flow carries large cargo directly into the cytosol. By aiming the pipette tip toward the cell nucleus, we can simultaneously open both the plasma and nuclear membranes with a single cavitation explosion, allowing the nanoblade to inject genetic materials directly into the nucleus. Typically, more than 90% of cells remain viable following nanoblade delivery. In contrast to conventional microinjection techniques, the photothermal nanoblade never enters the cell during the delivery process, which may be key for retaining high cell viability.

The photothermal nanoblade has enabled the delivery of a broad range of cargo with sizes varying over three orders of magnitude. We successfully have delivered into living mammalian cells fluorescent dyes, plasmids, RNAs, polymer-conjugated quantum dots for imaging,³ various polystyrene and magnetic beads with sizes ranging from 200nm to 2μm, and live bacteria with efficiencies typically around 50%.⁴ Successful nanoblade delivery has been performed on harvested rat neurons in culture, human embryonic stem cells, and various mammalian cell lines, such as HeLa, IMR90, and HEK293T cells.

Overall, the photothermal nanoblade makes feasible the delivery of super-sized cargo into living cells, a technique that opens new possibilities in biological and biomedical research. Future development of the photothermal nanoblade includes improving its throughput. In its current pipette-based configuration, the nanoblade serially delivers cargo to about 200cells/hr. We are developing an ultrahigh-throughput nanoblade that aims to deliver cargo to about 100,000 cells in parallel within seconds.