Better delivery through compression

CAMBRIDGE, Mass.—Nanoparticles and other large moleculesenjoy a lot of popularity for their potential, but researchers have alwaysfaced an issue when it comes to delivering the molecules past the cellularmembrane because of their size. But now, a research team from the MassachusettsInstitute of Technology (MIT) has found that if cells are squeezed through anarrow passage, small holes open temporarily in the membrane that largemolecules can pass through easily.

Existing methods for delivering large molecules are variedand successful, but all of them face drawbacks. The method of packaging DNA orRNA into viruses allows for easy bypassing of the membrane, but runs the riskthat the viral DNA will be absorbed into the host cell. Tagging large moleculeswith short proteins that can bypass the membrane and pull the large-moleculepayload along with it, or packing DNA or proteins into nanoparticles, are twoother options, but both often require re-engineering based on cell and large-molecule type, and nanoparticle fragments can end up trapped in the cell'sendosomes with toxic side effects. Electroporation also works, by applyingelectricity to cells to open the membrane, but obviously runs the risk ofdamaging the cells and the material earmarked for delivery.

The MIT team's new method, however, faces no such sideeffects; the distortion of the membrane is temporary and causes no irreparabledamage to the cell, and cellular functions are maintained after the treatment.

"It's very useful to be able to get large molecules intocells. We thought it might be interesting if you could have a relatively simplesystem that could deliver many different compounds," Klavs Jensen, a seniorauthor of the paper that describes this discovery, said in a press release.Jensen is the Warren K. Lewis Professor of Chemical Engineering and a professorof materials science and engineering at MIT.

The team tested this squeezing technique in a variety ofcell types, says chemical engineering graduate student Armon Sharei, a leadauthor 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 adultcells back to stem cells with the same differentiation potential as embryonicstem cells. The researchers were able to deliver reprogramming proteins andgenerate induced pluripotent stem cells, with a success rate roughly 10 to 100times better than that of existing methods.

The idea for this approach came from previous work in thelabs of Jensen and Robert Langer, the David H. Koch Institute Professor at MITand a senior author of the paper, that used microinjection to force largemolecules into cells as they passed through a microfluidic device. Theresearchers discovered during their work that when a cell is squeezed through anarrow tube, small holes open in the membrane to allow diffusion by nearbymolecules.

By building microfluidic chips roughly the size of a quarterwith 40 to 70 channels, the team set out to test the new method. Cells areplaced in a solution that also contains the material earmarked for delivery,and the solution is then flowed through the channel at approximately one meterper second. Halfway through, the channel constricts to a size 30 to 80 percentsmaller than the cells' diameter, opening the holes in the membrane.

Sharei notes that this could have significant therapeuticpotential 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 anex-vivo treatment, so you can imaginesomething like a dialysis machine, but instead of dialysis it deliversmaterials to your cells," Sharei explains. "So one idea would be, that we'rekind of working on, is you can take the immune cells from a patient out oftheir blood, process it through the machine and deliver drugs directly to theimmune cells to try to affect their function or signal to them to attack acancer, and then put those back into the patient."

Moving forward, Sharei says they will be pursuing thisapproach's potential in both stem cell manipulation and as a therapeutic,calling it a matter of realizing its advantages and applications. Having provedthe effectiveness in transforming human fibroblasts into pluripotent stemcells, the next step will be to look further down the line and use this methodto deliver the necessary proteins for differentiating stem cells into specificcell or tissue types. Work will continue on the 'dialysis' approach as well, tosee if cells can be trained or signaled to go after certain targets. Shareiexpects this approach to have "a significant advantage" in delivering proteinsand siRNAs.

"We're using it with our collaborators as a platform to tryto understand disease mechanism," Sharei adds, "so by delivering nanomaterialslike quantum dots or carbon nanotubes, you can try to sense intracellularenvironment or track intracellular components and really start to understandwhat's going on in a disease case."

Quantum dots—nanoparticles composed of semi-conductingfluorescing metals—allow users to label individual proteins or molecules withincells, but they often get trapped in the endosomes. In a paper published inNovember 2012, the team worked with MIT graduate student Jungmin Lee andchemistry professor Moungi Bawendi to demonstrate that they could deliverquantum dots into lab-grown human cells without the particles getting trapped.

Thepaper, "A vector-free microfluidic platform for intracellular delivery," waspublished online ahead of print Jan. 22 by the Proceedings of the National Academy of Sciences. The U.S. NationalInstitutes of Health and its National Cancer Institute both provided fundingfor the research.