AMHERST, Mass. – Delivering proteins inside cells is a promising, fast-emerging field with potential uses in basic cell biology and therapeutics, say chemist Sankaran “Thai” Thayumanavan and colleagues at the University of Massachusetts Amherst. Now they have developed a new method of “shrink wrapping” bioactive proteins in a polymer coating that retains their shape and function, then dissolves away after the protein is delivered inside.
As Thayumanavan explains, many human diseases are due to a protein deficiency and patients would benefit from receiving the molecule they lack. But many proteins cannot be kept intact and will not be effective if delivered outside the target cell. “We could treat many disorders much more effectively if we had a way to get the specific protein delivered intact, inside the cell,” he says. “That’s what we set out to do.” Details appear now in the online edition of the Journal of the American Chemical Society, JACS.
“The way we thought about it is like shrink wrap,” Thayumanvan adds. “The idea is that you want to deliver the biologic in its original form, not degraded, and still able to perform its original function. We thought that a polymer shrink wrap might transport proteins across the cell membrane into its cytosol, where something in the microenvironment will cause the shrink wrap to fall apart, leaving the protein retaining all its original structure and function. We have actually shown it to work.”
To accomplish this, the researchers chose a specific functional group, an area on the cell wall where chemical reactions and bonds can take place, what they say is “a functional handle that is abundantly available on the surface of more than 85 percent of globular proteins.”
But where most others use an electrical charge to create a rather fragile interaction, Thayumanvan and colleagues, including Ph.D. student Kingshuk Dutta and research assistant professor Jiaming Zhang, decided to use a chemical bond. This gave them a stronger, more stable arrangement, “but the disadvantage is that it’s harder to break when you want it to let go,” Thayumanvan says.
“So between the protein and the polymer we put in a chemical linker that we can control. We can break it at will and release the protein back in its original form. This is really important because proteins are so finicky. If there is any small bit of shrink wrap left, they might misfire, misfold, and not work.”
They designed their chemical link to react to the presence of glutathione peptide, which is present in higher concentrations inside the cell and lower outside. When it encounters the peptide inside the cell, their shrink wrap falls apart. To demonstrate the technique’s effectiveness, they report on delivering five different, moderately complex, medium-sized proteins inside cells. They plan to expand this work soon to include both larger and smaller proteins.
The researchers write, “The versatility of the approach, demonstrated here, suggests that the strategy is compatible with a wide array of biologics.” Thayumanavan adds, “In my mind our accomplishment is quite astounding and here’s the reason: the best case scenario actually worked. It was quite gratifying. There are a variety of things we could do next. One is to work on the medical therapeutics and delivery of biologics, proteins and antibodies. But this work gets us over the biggest hurdle, especially for when the target is inside a cell.”
He says that in addition to therapeutics, “the other thing we are really excited about” is using this strategy for encapsulating proteins and its property of “traceless release in response to a specific trigger” to boost the magnitude of very small biological signals.
Thayumanavan notes, “Many proteins are enzymes, that is, they are catalysts of reactions but on a very small scale. Ideally you want to make the signal larger so you can notice it. With this new technique, now we have the opportunity to put in a catalyst that would enormously amplify a signal.”
This work was funded by the Army Research Office’s Multi-University Research Initiative (MURI).