Our interest in nanoemulsions is in the formation of nano- or micro-bubbles for drug and gene delivery, particularly bubbles that will oscillate in size within an acoustic field. Bubbles undergoing oscillatory movements in an acoustic field are called cavitation bubbles, and the phenomena they create are called cavitation events. Cavitating bubbles are known to create strong local shear forces that can perturb cell membranes and lead to increased drug and gene uptake by these cells. [1] Ultrasonic cavitation is also known to stimulate cell growth. [2] The total collapse of bubbles down to a very small volume, called collapse cavitation, is thought to be more effective in drug delivery. [3] We hypothesized that stable liquid nanoemulsions could be induced to form collapse cavitation events by application of low frequency ultrasound of sufficient amplitude. An emulsion, whose organic phase has a boiling point slightly above ambient temperature, would begin to transform into the gas phase when the ultrasonic pressure wave reduced the local pressure below the organic phase vapor pressure at the local temperature.
EXPERIMENTAL Nanoemulsions of perfluorohexane (with a boiling point of 56°C) were formed using perfluorooctanoic acid as a stabilizing surfactant. Size distribution was measured by dynamic light scattering. Ultrasound was applied at 20 kHz and 500 kHz, at various intensities, and at 20° and 37°C. Controls were done in degassed water, and degassed water containing the surfactant only. The formation of gas bubbles was detected by listening with a hydrophone to the acoustic emission from the insonated sample. The emission was Fourier transformed to provide the frequency spectrum of the sample. A single frequency at the applied frequency (20 or 500 kHz) indicated the absence of gas bubbles. Higher harmonics indicated the presence of an oscillating gas bubble. The appearance of broadband background noise indicated the presence of shock waves from bubble collapse.
RESULTS At both 20 and 500 kHz as the acoustic intensity was increased slowly on the nanoemulsions, the fundamental frequency (indicative of no bubbles) was joined by higher harmonics (cavitating bubbles) and eventually by a sudden jump in broadband noise (collapse cavitation). At 20 kHz ultraharmonics were also observed just below the intensity require to broadband noise. At 500 kHz ultraharmonics were observed at the same or higher intensities than the onset of broadband noise. The same types of signals were observed in the surfactant solution and degassed water, but the thresholds for the onset of these signals were at a higher intensity for surfactant solution, and even higher for water. The thresholds for the onset of harmonics and broadband noise were higher at 500 kHz than at 20 kHz. Furthermore, the thresholds were higher at 20° than at 37°C.
DISCUSSION These results indicate that ultrasound is capable of inducing a phase transition from an emulsion liquid to the gas state, even when the temperature of the system is below the boiling point of the liquid. Once these gas bubbles form, we believe that some gas remains stable as a gas bubble because the appearance of higher harmonic frequencies in the acoustic spectrum is indicative of a gas bubble that oscillates stably without collapsing back to the liquid phase. The surfactant probably stabilizes these gas bubbles.
At sufficiently high intensities, these gas bubbles are collapsing, as indicated by the signature of the shock waves in the background noise. These shock waves are the cavitation events that are thought to be responsible for cell membrane permeability, and increased drug and gene uptake.
It is consistent with physical mechanisms that a lower frequency (at the same acoustic pressure amplitude) is better able to transform the liquid into gas. Using the simplified model that the liquid begins to boil as soon as the oscillating local pressure drops below the local vapor pressure, one can argue that a lower frequency will allow a larger time window for part of the liquid to boil off. Once the liquid begins to boil, the emulsion droplet and its surrounding are immediately cooled, thus lowering the vapor pressure and hindering further boil-off. At lower frequencies there is more time for heat transfer from the surrounding liquid to warm the emulsion droplet sufficiently to continue to boil. At 20°, the vapor pressure of the perfluorohexane is lower than at 37°, and so more acoustic intensity is required.
We believe that such an ultrasonic-induced phase transition can have application in many areas, including drug and gene delivery. Nanoemulsions can carry hydrophobic drugs in their interior, whereas preformed gas bubbles cannot. Upon transformation the drug would be deposited in the same region in which the bubble is cavitating, thus providing very high local concentrations of drug adjacent to cells subjected to membrane shear stress. DNA with its negative charge could be decorated onto nanoemulsions employing a cationic surfactant, and would also be released at the point of the cavitation event. Thus these nanoemulsions carrying drugs or DNA could be injected into the body, and would retain their payload until they flowed through an acoustic field focused on a particular target region or tissue in the body. Ultrasound is easily and painlessly applied and can be focused onto a delivery site in the body, thus sparing the rest of the body from any side effect of the drug or DNA.
REFERENCES
[1] R. K. Schlicher, H. Radhakrishna, T. P. Tolentino, R. P. Apkarian, V. Zarnitsyn, and M. R. Prausnitz, "Mechanism of intracellular delivery by acoustic cavitation," Ultrasound in Medicine and Biology, vol. 32, pp. 915-924, 2006.
[2] W. G. Pitt and S. A. Ross, "Ultrasound increases the rate of bacterial cell growth," Biotechnol Prog, vol. 19, pp. 1038-44., 2003.
[3] G. A. Husseini, M. A. Diaz, E. S. Richardson, D. A. Christensen, and W. G. Pitt, "The Role of Cavitation in Acoustically Activated Drug Delivery," J. Controlled Release, vol. 107, pp. 253-261, 2005.