Gene Delivery Using Physical Methods - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 103

second-generation contrast agents (e.g., Optison) because of their enhanced transfec-
tion efficiency. These new formulations employ special coating materials to stabilize
the bubbles, as well as insoluble gases (such as perfluorocarbon), and they provide
much better performance. Taniyama et al. demonstrated that electron micrographs of
cells treated with Optison followed by using ultrasound energy in the cell culture
suggested transient pores formation in the cell membrane opened up immediately
following ultrasound treatment [223] .
�ike EP, sonoporation also induces transient formation of small nanosized
pores in the cell membrane that allow direct transfer of genetic material into cells
[223,224] , and several cell types have been successfully transfected in vitro [225] .
Recently, in vivo gene delivery mediated by ultrasound has also been reported,
first using lithotripter [226] and then using focused sinusoidal sonoporation [227] .
Sonoporation has been broadly used in gene transfection in different tissues, includ-
ing solid tumors [228] , muscle [223,229] , and vasculature [230,231] . Intratumoral
injection of DNA followed by targeted ultrasound has resulted in a 10- to 15-fold
increase in the reporter gene expression [227] . Taniyama et al. have used low-dose
(1 min at 1 MHz, 2.5 W/cm 2 ) ultrasound to improve gene delivery in skeletal muscle
[233] and the carotid artery [229] . Other scientists have used a similar approach for
gene transfer into skeletal muscle [229,232,233] and the heart [234,235] . In contrast,
Huber et al. used HIFU (1 min at 0.85 MHz, 155 W over a 2 mm width) to perform
gene transfer to the carotid artery on the hypothesis that this treatment will allow
specific localization of the enhanced gene transfer [231] . They demonstrated an
eightfold increase in total reporter gene expression with HIFU alone and a 17.5-fold
increase when ultrasound contrast bubbles were used. HIFU and lithotripter shock
waves have also been used to simultaneously destroy tumors by high-energy transfer
and perform gene transfer into the residual mass, but gene expression was very vari-
able, possibly due to variable degrees of tumor destruction [228] .
Optimization of ultrasound-mediated gene transfer depends upon several fac-
tors, including transducer frequency, acoustic pressure, pulse duration, and exposure
duration. Additional factors are also important, such as the ultrasound contrast agent
concentration and its formulation [236] . Both diagnostic equipment and specific
ultrasound devices have been used for sonoporation. The latter comprise a piezo-
electric transducer, which is air-backed, focused or not, together with a generator and
amplifier. This type of system permits enhancement of transfection with subtle modi-
fications in the range of physical settings possible. A commercial device is available
both for in vitro and in vivo gene therapy (Sonitron 1000, manufactured by Rich Mar
Corporation, Protech International, Inc., San Antonio, TX, USA).
Enhanced gene expression in various cell lines by application of ultrasound has
been shown in Figure 3.4 . The most marked enhancement of gene expression has been
observed in cell lines that are typically difficult to transfect. Ultrasound energy has
been used in concert with liposomal DNA delivery systems, naked DNA, and peptide-
based delivery systems. The major steps in conducting DNA delivery to cells with
ultrasound are similar to conventional expression systems. The cells are plated into a
6-well culture dish and grown to 80% confluence. The transfection mixture is added
to the wells. At this point, the system is treated with ultrasound. Ultrasound energy