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.
“This technique could allow us to deliver a wide variety of
therapeutics that now cannot easily get into cells,” said Mark
Prausnitz, a professor in the School of Chemical and Biomolecular
Engineering at the Georgia Institute of Technology. “One of the
most significant uses for this technology could be for gene-based
therapies, which offer great promise in medicine, but whose
progress has been limited by the difficulty of getting DNA and RNA
into cells.”
The work is believed to be the first to use activation of
reactive carbon nanoparticles by lasers for medical applications.
Additional research and clinical trials will be needed before the
technique could be used in humans.
Researchers have been trying for decades to drive DNA and RNA
more efficiently into cells with a variety of methods, including
using viruses to ferry genetic materials into cells, coating DNA
and RNA with chemical agents or employing electric fields and
ultrasound to open cell membranes. However, these previous methods
have generally suffered from low efficiency or safety concerns.
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With their new technique, which was inspired by earlier work on
the so-called “photoacoustic effect,” Prausnitz and collaborators
Prerona Chakravarty, Wei Qian and Mostafa El-Sayed hope to better
localize the application of energy to cell membranes, creating a
safer and more efficient approach for intracellular drug
delivery.
Their technique begins with introducing particles of carbon
black measuring 25 nanometers – one millionth of an inch
– in diameter into the fluid surrounding the cells into which
the therapeutic agents are to be introduced. Bursts of
near-infrared light from a femotosecond laser are then applied to
the fluid at a rate of 90 million pulses per second. The carbon
nanoparticles absorb the light, which makes them hot. The hot
particles then heat the surrounding fluid to make steam. The steam
reacts with the carbon nanoparticles to form hydrogen and carbon
monoxide.
The two gases form a bubble which grows as the laser provides
energy. The bubble collapses suddenly when the laser is turned off,
creating a shock wave that punches holes in the membranes of nearby
cells. The openings allow therapeutic agents from the surrounding
fluid to enter the cells. The holes quickly close so the cell can
survive.
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.”