Technique instantly delivers controlled doses directly into cytosol with no reliance on endocytosis or effects on cell viability, the team reports.
Scientists report on the development of a nanochannel electroportation (NEP) device that can deliver precisely monitored amounts of either large or small transfection agents into individual living cells, without affecting cell viability. The approach effectively involves capturing a cell in a microchannel abutting a nanochannel, and then generates an intense electric field over a very small area on the cell membrane, which drives the transfection agent in the nanochannel directly through a pore created in the cell membrane and into the cytosol.
The Ohio State University team claims that while delivery methods based on bulk electroporation (BEP), or even microfluidics-based electroporation (MEP), rely on endocytic processes to complete the delivery of large biomolecules into cells, the NEP device can electroporetically drive even large transfection agents directly into the cell cytosol. L. James Lee, Ph.D., Pouyan E. Boukany, Ph.D., and colleagues describe their NEP device and how it works in Nature Nanotechnology. Their paper is titled “Nanochannel electroporation delivers precise amounts of biomolecules into living cells.”
BEP is the most widely used of the physical methods for transfecting cells because its technically simple, enables fast delivery, and has virtually no limits in terms of cell type and size, the Ohio State investigators explain. More recently developed MEP techniques for transfecting individual cells involves placing a cell next to a small aperture (generally a few microns across), which focuses the porating electric field to a corresponding area on the cell membrane.
However, while MEP demonstrates advantages of BEP such as lower poration voltages, and better transfection efficiency with higher cell mortality, both methods are diffusion-dominated, the team notes. This means that the delivery of large biomolecules, such as nucleic acids, is effected by attachment of the transfection agent onto the cell membrane, followed by an endocytosis-like process. In contrast, NEP delivers the agent directly into the cell cytosol during poration, with negligible diffusion after poration, precise dose control, and without causing cell death.
The NEP device basically consists of microchannels that are connected by a nanochannel (of about 90 nm in the reported study). The target cell is placed in one microchannel (using optical tweezers), lying against the nanochannel, and the second microchannel is filled with the agent to be delivered. One or more voltage pulses lasting milliseconds is delivered between the two microchannels, causing poration over a nanoscale area of the cell membrane to enable transfection. Dose control involves simply changing the duration and number of pulses, or changing the voltage and/or agent concentration.
The structure of the NEP device is generated using a simple, low-cost DNA combing and imprinting method developed by the team, which they used to construct a sealed array of laterally ordered nanochannels interconnected to microchannels. Gold-coated “combed” DNA strands are used as templates to stamp the nanochannels into a microridged polymer, after which the templates are peeled off, and the polymer hardened: the diameter of the resulting nanochannel imprints thus depends on the thickness of the gold coating. The associated micro- and macroscale inlets/outlets are formed in the fabrication process. Each of the resulting two rows of microchannels leads to a reservoir into which cells and the transfection material are respectively loaded, and an electrode is placed in each reservoir to carry out transfection using voltage pulses varying between 150 and 350 V.
To test the efficiency, repeatability, and dosage control capabilities of the system, the researchers first used it to successfully transfect Jurkat cells with a fluorescently 18-mer oligodeoxynucleotide (ODN G3139), and a GAPDH molecular beacon (GAPDH-MB): an mRNA probe with a fluorophore at one end and a quencher at the other end of a stem-hairpin structure. K562 cancer cells were then transfected with siRNA(Mcl-1), to verify dosage control using the NEP system, by determining the critical dosage of siRNA(Mcl-1) required to kill K562 cells.
The results showed that the amount of ODN transfected was directly linearly related to the duration of voltage pulse. A similar dose versus pulse length dependence was also seen for leukemia-patient cells as small as 8 μm in diameter. In repeated experiments using an array of five cells simulatenously transfected, the cell-to-cell variation in the amount of ODN delivered was just +/- 10–12%. In the siRNA(Mcl-1) test, pulses longer than 5 ms at 220 V/2 mm, delivered a fatal dose of siRNA(Mcl-1) to cells, whereas most cells remained viable for pulses shorter than 2 ms.
The researchers put forward their theoretical understanding of how the NEP process works. On applying a voltage pulse, the potential across the membrane abutting the nanochannel (membrane 1) rapidly increases, reaches a critical voltage within nanoseconds and porates. Assuming that the porated membrane 1 has neglible resistance, the opposing side of the cell membrane (the outer membrane, 2), starts charging, and within tens of microseconds, a steady-state current is reached. This means that for some 99% of the pulse, the cell has a steady-state, nanochannel-limited current flowing through it. “Assuming the transfection agent makes up a constant fraction of this current, this explains the near-linear relationship between pulse length and dose,” they state.
During the pulse, almost the entire applied voltage appears across the nanochannel, which means the generated electric fields are huge, roughly 70 MV m-1 for a 200 V pulse. This produces large electrophoretically driven draft velocities, reaching 700 mm ms-1 typically for quantum dots, for example. The upshot is that charged transaction agents pass into the nanochannel through a combination of drift and diffusion, and within microseconds are moved into the cell.
Critically, the mechanism that creates the pore in NEP appears to be different from that of BEP and MEP, the investigators state. Experimentally, using NEP results in large agents such as DNA and quantum dots appearing almost instantly inside the cell, which doesn’t happen using the other methods, and indicates that in NEP, large pores are generated in the cell membrane adjacent to the nanochannel during the electrical pulse. This notion was supported by tests carried out to visualize the uptake of fluorescent dyes into cells using NEP, BEP, and MEP. For both BEP and MEP, uptake was predominantly a diffusion-dominated process that occured largely subsequent to electrical pulsing. In contrast, NEP resulted in a sudden peak of fluorescence in the middle of the cell occurring simultaneously with the electrical pulse, and no further dye diffusion into the cell was seen after poration.
Delivery of large particles such as nanoprobes into cells using current techniques can generate unintended side effects, the team continues. “Most such methods rely on a nonspecific endocytosis process and have the disadvantage that internalized quantum dots are often trapped within endosomal compartments, which may block their ability to reach specific intracellular targets.”
The team therefore compared the abilities of BEP, MEP, and NEP to transfect Juarkat cells with quantum dots conjugated to a COOH group. They found with BEP and MEP, the nanoparticles basically stuck to the cell membrane (although some might subsequently have entered the cell by endocytosis). However, NEP resulted in quantum dots being rapidly internalized into the cells, and spatially distributed throughout the cell uniformly.
NEP, BEP, and MEP were then used to introduce a 3.5 kbp Cy3-labeled GFP plasmid into Jurkat cells to visualize a gene transfection process. Using BEP and MEP, DNA complexes formed on the outside of the porated cell, followed by endocytosis-like passage of the constructs into the cytoplasm, which for BEP took over an hour. Migration to the nucleus and subsequent transcription occurred only over a period of many hours, and GFP fluorescence wasn’t observed even 18 hours after transfection. In contrast, NEP effectively “injected” the plasmid directly into the cytoplasm, and significant Cy3 fluorescence was observed inside the cell membrane within 40 seconds. Migration of the DNA to the nucleus and transcription took just six hours. Gene transfection and strong GFP expression using NEP was even faster when nanoparticles such as gold or quantum dots were used to facilitate delivery, which has an effect roughly analogous to the role of the needle in microinjection, the authors remark.
“Whereas BEP and MEP create large numbers of small pores over a significant fraction of a cell surface, NEP appears to create either a single very large pore or several large pores in the cell membrane adjacent to the nanochannel,” they note. “The large NEP pore(s) allowed efficient transfection of relatively large agents directly into the cell cytosol, thus avoiding the endocytosis-to-endosome route on which other electroporation methods rely.” And importantly, because NEP affects a very limited area of the cell membrane, equivalent to less than 1% of that affected by even a small-area MEP device, all the test cells survived.
While the reported research was carried out using just a few cells at a time on the small scale NEP device, the Ohio State team is currently developing a mechanical loading system that would allow the transfection of 100,000 cells. “In the future, both experimental work and improved modeling will be needed to better characterize the NEP process,” they admit. However, “absolute quantitative calibration of dosage may be accomplished by comparing fluorescence measurements with single-cell polymerase chain reaction.”