Comparison of Gold Nanoparticle Mediated Photoporation: Vapor Nanobubbles Outperform Direct Heating for Delivering Macromolecules in Live Cells

Abstract

There is a great interest in delivering macromolecular agents into living cells for therapeutic purposes, such as siRNA for gene silencing. Although substantial effort has gone into designing nonviral nanocarriers for delivering macromolecules into cells, translocation of the therapeutic molecules from the endosomes after endocytosis into the cytoplasm remains a major bottleneck. Laser-induced photoporation, especially in combination with gold nanoparticles, is an alternative physical method that is receiving increasing attention for delivering macromolecules in cells. By allowing gold nanoparticles to bind to the cell membrane, nanosized membrane pores can be created upon pulsed laser illumination. Depending on the laser energy, pores are created through either direct heating of the AuNPs or by vapor nanobubbles (VNBs) that can emerge around the AuNPs. Macromolecules in the surrounding cell medium can then diffuse through the pores directly into the cytoplasm. Here we present a systematic evaluation of both photoporation mechanisms in terms of cytotoxicity, cell loading, and siRNA transfection efficiency. We find that the delivery of macromolecules under conditions of VNBs is much more efficient than direct photothermal disturbance of the plasma membrane without any noticeable cytotoxic effect. Interestingly, by tuning the laser energy, the pore size could be changed, allowing control of the amount and size of molecules that are delivered in the cytoplasm. As only a single nanosecond laser pulse is required, we conclude that VNBs are an interesting photoporation mechanism that may prove very useful for efficient high-throughput macromolecular delivery in live cells.

Cytosolic Delivery of Nanolabels Prevents Their Asymmetric Inheritance and Enables Extended Quantitative in Vivo Cell Imaging

“…Note that a 1 h incubation time was selected since QD induced toxicity became too high for longer incubation times ( Figure S3). Photoporation was performed with a fixed concentration of contrast agents (2 mg/ml for FD and 1 µM for CdSe QD), but with increasing laser fluence settings (all above the vapour nanobubble generation threshold of ~1 J/cm 2 ) 43 . From the confocal images in Fig.…”

“…For a more detailed theoretical framework the reader is referred to the work by Pustovalov et al 42 . Finally, the setup has a time-response and light scattering mode to allow detection of VNB formation 43 . Finally, the relative MFI of a cell population was calculated by dividing the MFI at a certain time after labelling by the MFI of untreated cells at the same time point.…”

Optical delivery of multiple opsin-encoding genes leads to targeted expression and white-light activation

“…Traditional methods of targeted delivery of impermeable molecules into cells require microinjection using a mechanical needle or microelectrodes, which often causes cell death and compromises sterility. Carbon 28,29 and gold 30,31 nanoparticles in conjunction with pulsed 28 or continuous-wave 29 laser beams have been employed for the delivery of exogenous small molecules to cells; this approach requires the presence of the nanoparticles along with the molecules to be delivered in the vicinity of the membrane of the target cell. Furthermore, in viral delivery methods, the size of the plasmids that can be packaged for delivery is limited, which may restrict the use of these methods to the delivery of only single-opsin plasmids rather than multiple opsins (such as White-opsin).…”

In vitro and ex vivo strategies for intracellular delivery

“…Upon laser irradiation local perturbation effects may be due to thermal, chemical, or cavitation-mediated fluid shear phenomena. Recent efforts indicate that tuning the laser pulse parameters and properties of the absorbing particles can bias the mechanism towards a particular mode 95,96 . Although parameters are still being explored, a proof-of-concept study demonstrated conditions under which irradiation of gold nanoparticles was proposed to trigger localized thermal damage that permitted more than half of the treated cells to take up labelled antibodies 97 .…”