**2.1 Sonoporation**

When ultrasound is irradiated locally with certain energy, the cavitation nuclei, such as ultrasound contrast agents and bubbles, could alternately occur expansion, contraction, splitting, fusion, and even rupture. This physical process is called cavitation effect. Accompanied by the cavitation effect, acoustic microstreaming, micro-jet, high temperature, and shockwave will occur in the medium, resulting in the formation of some temporary, reversible pores on the cell membrane, which is sonoporation [6, 7]. Generally, it is an accepted notion that the sonoporation from cavitation effect allows genes and drugs to enter cells [8].

There are a large number of studies, which have confirmed that sonoporation can increase the efficiency of gene delivery through enhancing the permeability of the cell membrane [9–12]. The number of pores, having a high impact on the gene delivery efficiency, can be affected by a lot of factors, such as acoustic pressure, irradiation duration time, and pulse repetition frequency [13–15]. Sonoporation pores trend to be larger along with the increase of acoustic pressure and irradiation time, which also enhance gene transfection efficiency [16]. However, excessive acoustic pressure or ultrasonic duration may reduce cell viability and even cause cell death, vascular rupture, and other side effects [17–19]. Therefore, to achieve a

**101**

*Recent Advances about Local Gene Delivery by Ultrasound*

high gene transfection efficiency and remain a cell viability as much as possible, it is important to optimize ultrasound irradiation parameters during gene transfection.

In addition to sonoporation, cavitation effect can change the cell membrane structure through microstreaming and shear force. The mechanical force may cause cytoskeleton rearrangement and regulate various downstream cellular signaling pathways, helping the endocytosis of genetic cargo [20, 21]. Generally, there are three forms of endocytosis, including macropinocytosis, clathrin-mediated endocytosis, and caveolae-mediated endocytosis [22]. After ultrasound irradiation, the reactive oxygen species are produced to stimulate the calcium influx and induce the occurrence of endocytosis [23]. In addition, cavitation effect and shear force induced by ultrasound can change cell structure and influence endocytosis through mechanosensors and signaling cascade [24]. Meijering et al. demonstrated that endocytosis was involved in the uptake of the macromolecular substances, while small molecules enter cells mainly through the pores of the membrane surface [25].

Recently, Cock et al. put forward a new viewpoint on the mechanism of ultrasound-mediated gene delivery [26]. By using the real-time scanning confocal microscopy, they found that nanoparticle-loaded microbubbles could deposit the nanoparticles in patches onto the cell membrane during ultrasound irradiation and promote the particles that enter cell through the fluidity of the membrane. In their opinion, this method, termed sonoprinting, is neither the traditional sonoporation nor the material swallowing. The underlying mechanisms still need to be explored.

Genes administrated by the intravenous route are easily be degraded. Conventionally, genes such as plasmids, mRNA, siRNA, and miRNA need to be protected from degradation by extracellular and intracellular barriers **Figure 2**. The ideal gene vectors should have the following characteristics: (1) safe and nontoxic, long cycle time in vivo, protecting the nucleic acid molecules from being destroyed by extracellular nucleic acid enzymes; (2) possessing the characteristics of a targeting ability and delivering the gene to target tissue or target cells; (3) high gene-carrying capacity; (4) promoting the gene to enter cytoplasmic or nucleus and stable expressing; (5) ensuring the controllability of gene function; and (6) noninvasive evaluation of gene delivery effectiveness. In the field of ultrasoundmediated gene delivery, many ultrasound contrast agents, including microbubbles, nanobubbles, nanodroplets, and some nanoparticles, are being developed into gene

The gene vector may help them to avoid degradation by extracellular and intracellular barriers, including serum endonucleases, immune detection, and endosome

Microbubbles are small, gas-filled microspheres with the particle size of 1–3 μm.

As gene vectors, they not only can protect the genes from nucleic acid enzyme degradation and from reticuloendothelial system clearance but may also enhance

**3. Type of ultrasound contrast agents as gene vector**

vectors in gene delivery mediated by ultrasound.

(Quoted from: Yin et al. [2]).

**3.1 Microbubbles**

*DOI: http://dx.doi.org/10.5772/intechopen.80036*

**2.2 Endocytosis**

**2.3 Sonoprinting**

high gene transfection efficiency and remain a cell viability as much as possible, it is important to optimize ultrasound irradiation parameters during gene transfection.
