What
is sonoporation and ultrasound-mediated transfection?
Sonoporation is
another name for ultrasound-mediated cell membrane permeabilisation or the
induction of membrane pores using ultrasound.
When the phenomenon is exploited to facilitate or promote entry of
nucleic acid into cells, this is called sonotransfection. In actual fact, sonoporation can also be
exploited in facilitating entry of other agents such as proteins and drugs into
living cells and as such it provides the researcher with a valuable tool for
applications such as drug screening or metabolic studies.
What
are microbubbles?
In the field of
sonoporation, it is believed that pores are formed in the membrane when
ultrasound brings about disturbance in the vicinity of that membrane. Formation of pores, (sonoporation) can occur
at low levels when cells are exposed to ultrasound. It can however be greatly enhanced by the addition
of microbubbles. These consist of a
stabilised lipid shell enclosing a gas (usually a perfluorocarbon gas). These agents are usually found to be
essential when using ultrasound for gene transfection because in their absence
transfection efficiency is relatively poor. Microbubbles, as the name suggests, are small
bubbles and they resonate (or vibrate) in the presence of an ultrasonic
field. The optimal resonant frequency of
the bubble is a function of the bubble size and so for optimal sonoporation, a
match must be achieved between the microbubble size and the ultrasound frequency
of the sonoporator device being used.
This is why SONIDEL Limited has designed its own MB101 microbubble to deliver
optimal gene transfection with its SP100
sonoporator platform emitting at ultrasound at a frequency of 1 MHz.
How
do microbubbles work?
Although many
studies have been carried out in order to answer this question it appears that
the microbubbles work in a combination of ways. It has been suggested that the bubble will
resonate at a specific frequency in an ultrasonic field. As the intensity or power density of the
ultrasound increases, the bubble resonates more violently so that eventually it
will collapse in a catastrophic manner.
This effect, if proximal to the cell membrane, will induce rupture of
the membrane and this will, in turn, allow reagents into the cell. Others have shown, using extremely high
speed image capture, that the bubble resonates in the ultrasonic field. In doing so, it forms very strange
projections from it surface and it has been suggested that these projections (like
needles) transiently rupture the cell membrane thereby permitting transmembrane
travel of extracellular substances.
How
does sonoporation compare with other transfection methods in terms of cell
viability?
When all parameters
are optimised, sonoporation causes little irreversible cell damage in most cell
lines. Cell membrane recover time has
been shown to be less than 10 seconds. The
factors that must be controlled to prevent irreversible cell damage and death
are:
- Concentrations
of transfection reagent or microbubbles
- Ultrasound
power output
- Ultrasound
application time
- Ultrasound
duty cycle
In
the published literature, everyone seems to be using different frequencies, which
is best?
Yes, this is very
true and indeed the field seems quite confused. This stems, primarily from the lack of
commercially-available instrumentation such as has been available to
researchers using electroporation over the past number of years. If one
reviews sonoporation in the scientific literature, one finds that
researchers have used different instruments, different ultrasound frequencies,
different ultrasound intensities or power densities, different treatment times,
different pulse regimes, different
exposure configurations and indeed different microbubble preparations. It really is extremely confusing. However, in delving through that body of
literature a number of aspects become clear.
One is that 1 MHz seems to yield the most consistent results and has
proven most successful for achieving superb membrane permeabilisation with
maximal cell viability. SONIDEL Limited
has harvested the best attributes indicated by this extensive body of
literature and used those to design its sonoporation platform and associated
range of products.
For consistent and
reproducible results SONIDEL Limited recommends:
