Using chemical "nanoblasts" that punch tiny holes in the protective membranes of cells, researchers have demonstrated a new technique for getting therapeutic small molecules, proteins and DNA directly into living cells.
Carbon nanoparticles activated by bursts of laser light trigger the tiny blasts, which open holes in cell membranes just long enough to admit therapeutic agents contained in the surrounding fluid. By adjusting laser exposure, the researchers administered a small-molecule marker compound to 90 percent of targeted cells – while keeping more than 90 percent of the cells alive.
The research was sponsored by the National Institutes of Health and the Institute of Paper Science and Technology at Georgia Tech. It will be reported in the August issue of the journal Nature Nanotechnology.
The researchers have demonstrated that they could get the small molecule calcein, the bovine serum albumin protein and plasmid DNA through the cell membranes of human prostate cancer cells and rat gliosarcoma cells using this technique. Calcein uptake was seen in 90 percent of the cells at laser levels that left more than 90 percent of the cells alive.
"We could get almost all of the cells to take up these molecules that normally wouldn't enter the cells, and almost all of the cells remained alive," said Prerona Chakravarty, the study's lead author. "Our laser-activated carbon nanoparticle system enables controlled bubble implosions that can disrupt the cell membranes just enough to get the molecules in without causing lasting damage."
To assess how long the holes in the cell membrane remained open, the researchers left the simulated therapeutics out of the fluid when the cells were exposed to the laser light, then added the agents one second after turning off the laser. They saw almost no uptake of the molecules, suggesting that the cell membranes resealed themselves quickly.
To confirm that the carbon-steam reaction was a critical factor driving the nanoblasts, the researchers substituted gold nanoparticles for the carbon nanoparticles before exposure to laser light. Because they lacked the carbon needed for reaction, the gold nanoparticles produced little uptake of the molecules, Prausnitz noted.
Similarly, the researchers substituted carbon nanotubes for the carbon nanoparticles, and also measured little uptake, which they explained by noting that the nanotubes are less reactive than the carbon black particles.
Experimentation further showed that DNA introduced into cells through the laser-activated technique remained functional and capable of driving protein expression. When plasmid DNA that encoded for luciferase expression was introduced into the cancer cells, production of luciferase increased 17-fold.
For the future, the researchers plan to study use of a less expensive nanosecond laser to replace the ultrafast femtosecond instrument used in the research. They also plan to optimize the carbon nanoparticles so that nearly all of them are consumed during the exposure to laser light. Leftover carbon nanoparticles in the body should produce no harmful effects, though the body may be unable to eliminate them, Prausnitz noted.
"This is the first study showing proof of principle for laser-activation of reactive carbon nanoparticles for drug and gene delivery," he said. "There is a considerable path ahead before this can be brought into medicine, but we are optimistic that this approach can ultimately provide a new alternative for delivering therapeutic agents into cells safely and efficiently." ###
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