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With New Microfludic Technique, MIT Team Aims to 'Squeeze' siRNAs into Cells

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Researchers from the Massachusetts Institute of Technology last week reported on the development of a new microfluidic-based approach to delivering macromolecules, including functional siRNAs, into cells without the need for a vector.

According to the investigators, who published their findings in the Proceedings of the National Academy of Sciences, the technique involves compressing cells by passing them through a constriction, which opens up temporary holes in their membranes that permit the diffusion of materials in surrounding buffer to enter the cytosol.

“By providing flexibility in application and obviating the need for exogenous materials or electrical fields, this method could potentially enable new avenues of disease research and treatment,” they wrote.

Although intracellular delivery of macromolecules is a key step in therapeutic and research applications, the cellular membrane is largely impermeable to such compounds, according to the PNAS paper. Existing methods to overcome this hurdle, which has proven to be a major stumbling block for RNAi drugs, typically involve the use of polymeric nanoparticles, liposomes, or chemical modifications of the target molecules to facilitate membrane poration or endocytotic delivery.

When it comes to nucleic acids, which are relatively structurally uniform, these approaches can be efficient. Still, the “endosome escape mechanism that most of these methods rely on is often inefficient; hence, much material remains trapped in endosomal and lysosomal vesicles,” the MIT team pointed out. “More effective gene delivery methods, such as viral vectors, however, often risk chromosomal integration.”

Meantime, electroporation has proven effective, even in difficult to transfect primary cells, but has limited applicability and can cause cell death. Microinjection, too, has certain advantages in settings such as the creation of transgenic organisms, but its low throughput hamstrings many therapeutic and research applications, the researchers noted.

To overcome the limitations of existing delivery techniques, the MIT group had initially been attempting to “shoot” molecules of interest into cells, Armon Sharei, an MIT graduate student in chemical engineering and lead author of the PNAS paper, told Gene Silencing News.

“That system had its own challenges, and through the course of that work, we stumbled upon this effect where if you squeeze the cells rapidly enough, it will temporarily disrupt their membrane,” he said.

More specifically, the researchers found that the “rapid mechanical deformation of a cell, as it passes through a constriction with a minimum dimension smaller than the cell diameter, results in the formation of transient membrane disruptions or holes,” they wrote in PNAS. “The size and frequency of these holes would be a function of the shear and compressive forces experienced by the cell during its passage through the constriction. Material from the surrounding medium may then diffuse directly into the cell cytosol throughout the life span of these holes.”

To test this idea, the researchers constructed devices, each consisting of 45 identical, parallel microfluidic channels containing one or more constrictions, etched onto a silicon chip and sealed in glass. The width of each constriction ranged from 4 to 8 micrometers, and the lengths ranged from 10 to 40 micrometers.

“Before use, the device is first connected to a steel interface that connects the inlet and outlet reservoirs to the silicon device,” the researchers wrote. “A mixture of cells and the desired delivery material is then placed into the inlet reservoir and Teflon tubing is attached at the inlet. A pressure regulator is then used to adjust the pressure at the inlet reservoir and drive the cells through the device. Treated cells are collected from the outlet reservoir.”

The system was tested with a variety of molecules, including carbon nanotubes and proteins, as well as siRNAs targeting GFP. According to Sharei, when the siRNAs were delivered into GFP-expressing HeLa cells using the microfluidic platform, the investigators were able to achieve 80 to 90 percent target knockdown.

He noted that the knockdown effects weren't as robust as with Lipofectamine 2000, but “we were still encouraged because something like Lipofectamine is known to be toxic and therefore inapplicable for humans.” Notably, the microfluidic device and operating parameters were not optimized for siRNAs, further limiting its ability to compete with the transfection reagent in these studies.

“The other good thing was that we seem to work just as well for primary cells, whereas existing methods like Lipofectamine don't translate well once you start moving out of the standard cell models you have in the lab,” he added.

The MIT team also successfully delivered 3 kilodalton dextran molecules — which are approximately the same size as a standard siRNA molecule and a “pretty accurate” surrogate for the gene-silencing molecules — into newborn human foreskin fibroblasts, primary murine dendritic cells, and embryonic stem cells, suggesting that the method could be used with siRNAs into a variety of cell types, Sharei said.

Buoyed by the positive data, he and his colleagues are now further testing the platform with siRNAs against “easy readout genes” in primary cells including immune cells and stem cells, he said. “Once we establish that, we'd try to go for an application where there's an siRNA that's going to knock down something functional.

“I can't say exactly what we've been up to because it's not published, but it has been going pretty well,” he added.

Ultimately, the MIT group aims to develop the microfluidic platform not only for research applications, where it could be “incorporated into a larger integrated system consisting of multiple pre-treatment and post-treatment modules” that could take advantage of its average throughput rate of 20,000 cells a second, but also therapeutic ones, too.

A number of investigational stem cell-based therapies, for instance, involve the ex vivo manipulation of the cells, Sharei said. The delivery platform could theoretically be used to “enhance or facilitate that manipulation.”

“Such an approach would take advantage of the potentially increased delivery efficiency of therapeutic macro- molecules and could be safer than existing techniques because it would obviate the need for potentially toxic vector particles and would mitigate any potential side effects associated with reticuloendothelial clearance and off-target delivery,” the study authors wrote in PNAS.

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