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Better delivery through compression
CAMBRIDGE, Mass.—Nanoparticles and other large molecules enjoy a lot of popularity for their potential, but researchers have always faced an issue when it comes to delivering the molecules past the cellular membrane because of their size. But now, a research team from the Massachusetts Institute of Technology (MIT) has found that if cells are squeezed through a narrow passage, small holes open temporarily in the membrane that large molecules can pass through easily.
Existing methods for delivering large molecules are varied and successful, but all of them face drawbacks. The method of packaging DNA or RNA into viruses allows for easy bypassing of the membrane, but runs the risk that the viral DNA will be absorbed into the host cell. Tagging large molecules with short proteins that can bypass the membrane and pull the large-molecule payload along with it, or packing DNA or proteins into nanoparticles, are two other options, but both often require re-engineering based on cell and large-molecule type, and nanoparticle fragments can end up trapped in the cell's endosomes with toxic side effects. Electroporation also works, by applying electricity to cells to open the membrane, but obviously runs the risk of damaging the cells and the material earmarked for delivery.
The MIT team's new method, however, faces no such side effects; the distortion of the membrane is temporary and causes no irreparable damage to the cell, and cellular functions are maintained after the treatment.
"It's very useful to be able to get large molecules into cells. We thought it might be interesting if you could have a relatively simple system that could deliver many different compounds," Klavs Jensen, a senior author of the paper that describes this discovery, said in a press release. Jensen is the Warren K. Lewis Professor of Chemical Engineering and a professor of materials science and engineering at MIT.
The team tested this squeezing technique in a variety of cell types, says chemical engineering graduate student Armon Sharei, a lead author of the paper. Among the types tested are a variety of cancer cell lines, patient-derived cells, immune cells such as T and B cells, macrophages, embryonic stem cells and fibroblasts.
This technique also has potential in reprogramming adult cells back to stem cells with the same differentiation potential as embryonic stem cells. The researchers were able to deliver reprogramming proteins and generate induced pluripotent stem cells, with a success rate roughly 10 to 100 times better than that of existing methods.
The idea for this approach came from previous work in the labs of Jensen and Robert Langer, the David H. Koch Institute Professor at MIT and a senior author of the paper, that used microinjection to force large molecules into cells as they passed through a microfluidic device. The researchers discovered during their work that when a cell is squeezed through a narrow tube, small holes open in the membrane to allow diffusion by nearby molecules.
By building microfluidic chips roughly the size of a quarter with 40 to 70 channels, the team set out to test the new method. Cells are placed in a solution that also contains the material earmarked for delivery, and the solution is then flowed through the channel at approximately one meter per second. Halfway through, the channel constricts to a size 30 to 80 percent smaller than the cells' diameter, opening the holes in the membrane.
Sharei notes that this could have significant therapeutic potential for patients, though it would most likely not be an in-vivo approach.
"I think what we really envision at this point is kind of an ex-vivo treatment, so you can imagine something like a dialysis machine, but instead of dialysis it delivers materials to your cells," Sharei explains. "So one idea would be, that we're kind of working on, is you can take the immune cells from a patient out of their blood, process it through the machine and deliver drugs directly to the immune cells to try to affect their function or signal to them to attack a cancer, and then put those back into the patient."
Moving forward, Sharei says they will be pursuing this approach's potential in both stem cell manipulation and as a therapeutic, calling it a matter of realizing its advantages and applications. Having proved the effectiveness in transforming human fibroblasts into pluripotent stem cells, the next step will be to look further down the line and use this method to deliver the necessary proteins for differentiating stem cells into specific cell or tissue types. Work will continue on the 'dialysis' approach as well, to see if cells can be trained or signaled to go after certain targets. Sharei expects this approach to have "a significant advantage" in delivering proteins and siRNAs.
"We're using it with our collaborators as a platform to try to understand disease mechanism," Sharei adds, "so by delivering nanomaterials like quantum dots or carbon nanotubes, you can try to sense intracellular environment or track intracellular components and really start to understand what's going on in a disease case."
Quantum dots—nanoparticles composed of semi-conducting fluorescing metals—allow users to label individual proteins or molecules within cells, but they often get trapped in the endosomes. In a paper published in November 2012, the team worked with MIT graduate student Jungmin Lee and chemistry professor Moungi Bawendi to demonstrate that they could deliver quantum dots into lab-grown human cells without the particles getting trapped.