Gene Delivery Using Physical Methods - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 101

techniques of DNA delivery have also been studied for their ability to enhance
the delivery of plasmid DNA inside cells by employing external physical force.
Sonoporation is a nonviral gene delivery system for enhancing plasmid DNA trans-
fer across the biological cell membranes. It works by transient permeabilization of
the cell membrane and by transferring therapeutic DNA effectively across tissue and
into cells by application of ultrasound energy. The ultrasound technique has been
routinely used clinically for both therapeutic and diagnostic purposes, and it covers
a broad range of frequencies and waveforms. However, therapeutic application has
mainly been achieved through sonoporation using sinusoidal probes at megahertz fre-
quencies. �ower frequencies (e.g., 20 kHz) have been used for cell lysis, cell disrup-
tion, and diagnostic imaging, whereas high-intensity shock waves are employed for
lithotripsy of kidney stones and gall bladder stones. The phenomenon of cell lysis by
high-intensity focused ultrasound (HIFU) has also been used for tumor regression and
destruction by the thermal energy produced by HIFU [209] .
The ultrasound technique has shown the advantages of simplicity, noninvasive-
ness, and high-safety profile, along with enhanced gene transfection in vitro and
in vivo by 10- to 15-folds when compared with naked DNA injection. The facility of
imaging observed during ultrasound therapy has also been found to be advantageous.
Because ultrasound penetrates soft tissue and can be applied to a specific area, it
could become an ideal method for noninvasive gene transfer into cells of the internal
organs. However, as compared to EP, it shows comparatively less DNA transfection.
The efficacy and toxicity of this method is still not widely evaluated, and hence fur-
ther investigations are required.
The ultrasound technique is based on the principle of cavitation and microbubble
formation. After applying ultrasound energy to a liquid during in vitro and in vivo
conditions, formation of vapor-filled bubbles or cavities in the solution takes place.
The diameter of these bubbles enlarges on applying energy, but on acquiring pres-
sure, the bubbles later collapse. The collapse of these microbubbles releases a large
amount of energy, which significantly permeabilizes the cell wall, alters the cell struc-
ture transiently, and enhances the entry of the macromolecules, including DNA, into
the cytoplasm from extracellular milieu. This formation and collapsing of the ultra-
sound-induced microbubbles is called cavitation. This sound-mediated, or acoustic,
cavitation is the most probable mechanism involved in the process of sonoporation.
The effect of ultrasound on microbubbles is explained in Figure 3.4 .
The application of low frequency ultrasound effectively delivers macromolecules
both in vitro and in vivo [210,211] and is well documented. Applying 20 kHz ultra-
sound to a suspension of yeast cells by a sonicator facilitated the delivery of plas-
mid DNA inside cells [212] . The sonication showed no deteriorating effect on the
structural integrity of the plasmid DNA. The transfection efficiency of ultrasound-
mediated gene delivery is dependent on several factors such as frequency and output
strength of the ultrasound energy applied, the duration of ultrasound treatment [213] ,
and the amount of plasmid DNA used. The initial experiments transfecting mam-
malian cells as human prostate cancer cell lines, �nCaP, and PC-3 by low-intensity
ultrasound signals showed differential gene transfer and transient expression of the
GFP [214] . The technique has shown no significant adverse effects when focused