Researchers have demonstrated an innovative method for transferring small biologically active particles, proteins and DNA directly into the interior of living cells using chemical "nano-jets", which pierce tiny holes in the cells' protective envelope
Carbon nanoparticles, activated by laser beam bursts, create the tiny jets and these, in turn, cause holes in the cell membrane to open for a period of time sufficient to introduce medical substances found in the extracellular fluid into the cell interior. By adjusting the exposure time to the laser beam, the researchers were able to inject a small marker compound into ninety percent of the target cells - while maintaining the healthy activity of more than ninety percent of the cells.
The research, carried out under the auspices of the US National Institutes of Health (NIH and a research institute at Georgia Tech., was published in the scientific journal Nature Nanotechnology.
This method will allow us to deliver a wide variety of medical substances that are currently quite difficult to introduce into the interior of cells," said Mark Prausnitz, a professor in the School of Chemical and Biomolecular Engineering at the Georgia Institute of Technology. "One of the most important applications of this technology could be in the development of gene-based healing methods, which provide great hope in medicine, and whose progress has been hindered by the difficulty of introducing DNA or RNA into the interior of cells."
It is claimed that this method is the first to use the activation of activated carbon nanoparticles with a laser for medical applications. Further research and medical trials will still be required before introducing the method to human treatment.
For decades scientists have been trying to introduce DNA and RNA into the interior of cells more and more effectively through a variety of methods, including the use of viruses as a means of transferring genetic materials, coating DNA and RNA with chemical substances or using electric fields and ultrasonic waves to Fission of cell membranes. However, these methods suffered from poor efficacy or human safety concerns.
The innovative method, based on previous work by a team of researchers who discovered what they called the photoacoustic effect, begins by introducing carbon nanoparticles (25 nanometers in size) into the extracellular fluid that surrounds the cells, and which where the medical materials will be found. In the next step, bursts of near-infrared radiation from a femtosecond laser are applied to the liquid at a rate of 90 million pulses per second.
The carbon nanoparticles absorb the energy and subsequently heat up. The hot particles then cause the liquid surrounding them to heat up and turn it into steam. The hot steam reacts with the carbon nanoparticles to form the gases hydrogen and carbon monoxide. The two gases form a bubble that grows larger as long as it receives energy from the activated laser. When the laser is turned off, the bubble immediately collapses and creates a shock wave that punctures holes in the membranes of the adjacent cells. This perforation allows the penetration of medical substances found in the extracellular fluid into the cell interior. The tiny nozzles close quickly and thus the cell is able to live and remain active.
The researchers demonstrated that they are able to inject the marker substance Calcein, the albumin protein in mammalian serum and plasmid DNA through the membranes of human prostate cancer cells and rat cells infected with malignant tumors using the innovative method. The absorption of the Calcein marker was detected in ninety percent of the cells when the laser was activated at levels that kept ninety percent of them alive.
"We showed that almost all the cells are able to absorb these substances, which normally do not penetrate the cell, and that almost all the cells remained alive," explains one of the researchers. "Our system of carbon nanoparticles, which are activated by means of a laser, allows for a controlled collapse of bubbles that causes the cell membranes to be punctured to such an extent that allows penetration of the materials without causing damage to the cells themselves."
In order to test how long the nozzles are formed by the cell membrane, the researchers punctured cells without the medical substances in their environment, waited one second after turning off the laser, and then added them to the system. They discovered that almost no penetration of the active substances was possible, which suggests that the cell membranes "sealed" themselves quickly.
In order to verify the hypothesis that the evaporation reaction of the carbon particles is the decisive factor in the formation of the nano-jets, the researchers replaced them with gold nanoparticles before activating the laser radiation. The gold nanoparticles caused very little absorption of the active substances, due to the fact that they did not contain the carbon necessary for the reaction itself. Similarly, the researchers tried their method for carbon nanotubes instead of nanoparticles and in this situation they also measured only a little absorption, due to the fact that nanotubes are less active than carbon nanoparticles.
Additional experiments showed that DNA fragments do succeed in penetrating the cell with this method and remain active in it, and are able to cause protein expression in the cell. When plasmid DNA encoding the expression of the substance luciferase was introduced into cancer cells, its production increased by seventeen orders of magnitude.
In the next stages of the research, the scientists intend to test the effectiveness of the method also using a nanosecond laser, which is less expensive than the femtosecond equipment they use today. Also, they intend to improve the carbon nanoparticles so that almost all of them will react during exposure to the laser radiation. Although these nanoparticles are not supposed to endanger human health, the body may not be able to excrete them, the researcher points out.
"This is the first study ever to prove the principle of activating carbon nanoparticles using a laser to transfer drugs and genes," said the researcher. "There is still a long way to go before this method can be used in everyday medicine, but we are extremely optimistic that this approach can, in the end, provide a new alternative for transferring medical substances into the interior of cells in an efficient and safe manner."
