Nanofontane probe
The cell was pierced with an electric nanofontane
Alexey Kozlov, N+1
American scientists have proposed delivering biological molecules inside individual cells using a nanofontane probe that pierces the cell membrane with a generated pulse and injects a substance into its cytosol. With the help of the probe, scientists were able to inject bovine serum albumin labeled with fluorescent protein deep into the cell, and in the future the technology will allow introducing almost any molecular cargo into the cell – for therapeutic or research purposes.
This is described in the journal Small (Nathamgari et al., Nanofountain Probe Electroporation Enables Versatile Single‐Cell Intracellular Delivery and Investigation of Postpulse Electropore Dynamics).
Drawings from the article in Small.
Molecules can be delivered into the cell in a variety of ways: for example, viral vectors or using chemical carriers – simple peptides or polymer nanocapsules. In addition, the cationic-lipid reagent lipofectamine or the method of volumetric electroporation is also used for this. Each method has its advantages, but also limitations: for example, viral vectors, for all their effectiveness, can cause adverse immune reactions or genotoxicity, and chemical carriers often turn out to be detrimental to the cell itself.
In addition, experiments with the delivery of substances into cells should be carried out on genetically homogeneous cell populations. Usually, in order to avoid genetically mixed populations of cells, it is required to grow and select individual cells, which will then produce lines of identical clones. However, this method of cell selection itself is extremely complex and time-consuming.
Scientists led by Horacio Espinosa from Northwestern University have figured out how to get around these problems. They proposed a method of electroporation of individual cells using an electric pulse generated by a nanofontane probe – a glass micropipette with a tip size of less than one micrometer. Similar microelectrodes have previously been used, for example, to fix the local potential on the cell membrane. Now it has been used for electroporation of the eukaryotic cell shell.
A model of molecular cargo delivery by a nanofontane probe.
First, scientists created a mathematical model of the interaction of the probe with a living surface in order to predict the behavior of the formed electrical circuit. So they were able to determine the optimal parameters of the electrical effect on the membrane, and only then applied them to living cells.
The experiment with puncturing the membrane and subsequent transport of molecules inside a particular cell took place in three stages: first, the membrane was charged, then an electric pulse from the electrode made a hole in it and, finally, the contents of the pipette were injected into the cytosol of the cell. According to the thickness of the membrane itself, the difference in electrical potentials on it and the resistance of the contact point of the electrode with the surface, scientists calculated the strength of the electric pulse that was required to create a pore and inject the substance.
With the help of a probe, it was possible to transfer a bovine serum albumin molecule labeled with a fluorescent protein inside a separate primary grown cell, as well as into a HeLa cell line. At the same time, the cells remained intact.
The researchers then compared the efficiency of transferring substances into cells using a nanofontane probe with a similar method of transfection using plasmids. They demonstrated that the new system in 95 percent of cases ensures the successful delivery of protein, DNA and RNA molecules to various "immortal" cell lines (such as HeLa cells) or to the culture of primary eukaryotic cells. At the same time, only 10 percent of cells died as a result of such manipulations – this, according to the authors, is not so much. Such efficiency and low invasiveness were ensured by fine-tuning the parameters of electrical action on the living system of the cell, which significantly reduces the likelihood of its death.
The new method is suitable for cell therapy and for the study of certain diseases. In the first case, we are talking about genome editing and immunotherapy, for example, for the treatment of oncological diseases by reprogramming T-lymphocytes. In the second case, it is about the control of the course of congenital genetic ailments. In addition, the probe allows you to study the dynamics of pore healing in the cell after it has been subjected to pulsed action.
By combining electroporation of a single cell and frame-by-frame fluorescence imaging, researchers are able to record the time it takes to close the temporarily formed pores. This is important for understanding the need for repeated electrical action on the membrane, for example, in order to take control samples of the substance from the cell and, if necessary, increase the volume of delivered molecular cargo.
The most promising application of the electric nanofontane probe is the delivery of pluripotency factors and monitoring of their concentrations during reprogramming of human stem cells. In addition, it can be used to strictly control the dosage of cas proteins in the CRISPR/Cas9 genome editing technology.
Recently, German researchers managed to deliver targeted medicine inside cancer cells using nanoparticles based on the glycoprotein mucin with a DNA molecule attached to it. You can read about it here.
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