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

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 vectors in gene delivery mediated by ultrasound.

The gene vector may help them to avoid degradation by extracellular and intracellular barriers, including serum endonucleases, immune detection, and endosome (Quoted from: Yin et al. [2]).

### **3.1 Microbubbles**

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

#### **Figure 2.** *Schematic model of transfection process of genes in carriers.*

their local delivery through active and passive targeting. Traditional membrane materials consist of microbubbles, which include albumin, lipid, polymers, and surfactants. Different shell compositions have various characteristics. Albumin is commonly used in the preparation of commercial ultrasound contrast agents, but it is susceptible to degeneration due to temperature change. In addition, it is expensive and easy to cause immune response. The synthetic phospholipids are good and biocompatible, but their half life is short in vivo. Polymers are slightly inferior in biocompatibility, but it possesses better stability.

It has been proved that the application of ultrasound combined with commercial microbubbles and gene mixture could regulate gene expression and achieve therapeutic effect [27–30]. Wang et al. compared the effect of gene delivery by three kinds of typical commercial microbubbles—Optison, Sonovue, and Levovist. The mixture of microbubbles and plasmid DNA encoding green fluorescent protein was injected into tibialis anterior muscle of mice. After ultrasound irradiation,

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ultrasound irradiation [47].

*Recent Advances about Local Gene Delivery by Ultrasound*

the number of GFP-positive fibers was significantly increased in Optison- and Sonovue-treated groups, proving the efficiency of gene transfection by ultrasound combined with commercial microbubbles [31]. However, DNA is anionic molecules, and most microbubbles are negatively charged on the surface, which bring some difficulty for the formation of DNA/microbubble complexes. In order to address this issue, some cationic microbubbles are developed and applied as gene vector to enhance the gene-carrying capacity [32–37]. Wang et al. evaluated the difference of gene transfection rate between cationic microbubbles and neutral microbubbles in combination with ultrasound. Their results demonstrated that the expression of reporter gene in cationic microbubble group was 20-fold higher in vitro and 3-fold higher in tumor model than neutral bubbles [34]. Recently, Wei et al. applied Targesphere, a kind of commercial cationic microbubbles, as short hairpin (shRNA) vector for connective tissue growth factor (CTGF). It was showed that the expression of CTGF was decreased in renal fibrosis mouse model after ultrasound irradiation, which proved the great potential in gene delivery mediated by ultrasound

Nanoscale ultrasound contrast agents, with the particle size from 100 to 600 nm, are also developed in the recent years. Compared with traditional microbubbles, nanoscale contrast agents have smaller size and stronger penetrating ability. In addition, nanoscale contrast agents possess greater gene-carrying capacity due to their larger surface area. Common nanoscale ultrasound contrast agents include nanobubbles, solid nanoparticles, and liquid fluorocarbon nanoparticles. Most of the shell membrane of nanobubbles are lipid or polymer, and the core could be gas or liquid. Nanobubbles can cross through the blood vessels and aggregate in the tumors through the enhanced permeability and retention (EPR) effect [39]. It was proved that nanobubbles could achieve ideal gene transfection efficiency when combined with ultrasound [40, 41]. Horie et al. applied ultrasound combined with nanobubbles mediating tumor necrosis factor (TNF-α) DNA delivery to treat tumor-bearing mice and resulted in the decrease of the tumor vessel density and inhibition of tumor growth [42]. To enhance the gene-carrying capacity and local transfection efficiency, cationic nanobubble or targeted nanobubbles have been applied and showed excellent therapeutic effect in vitro and in vivo [43–45]. Yin et al. developed a new kind of siRNA-nanobubble, through a nanoparticle heteroassembly of siRNA-loaded polymeric micelles and liposomes, demonstrating their ideal therapeutic effect in cancer treatment [46]. Xie et al. used cell-permeable peptides (CPPs) to enhance the transferring rate of siRNA. They developed CPP-siRNA that targets oncogene c-myc and encapsulated it into nanobubbles. It was shown that the expression of c-myc mRNA was significantly decreased, and the growth of tumor was significantly inhibited after

Recently, liquid fluorocarbon nanodroplets have attracted wide attentions in the ultrasound-mediated gene delivery. These nanodroplets prepared from a lipid or a polymer shell can encapsulate liquid fluorocarbon emulsion (perfluoropentane, etc.). The liquid core would occur "acoustic droplet vaporization" (ADV) under ultrasound irradiation, which makes the nanodroplet transform into gas-containing microbubbles, greatly enhancing the cavitation effect of ultrasound **Figure 3**. Although nanodroplets have shown its therapeutic effect in high-intensity focused ultrasound (HIFU) and drug delivery, its application in gene delivery is still rare. Gao et al. synthesized a novel tumor-targeting cationic nanodroplet and applied it as gene vector to treat Her2-positive breast cancer. The results in their study

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

combined with cationic microbubbles [38].

**3.2 Nanoparticle, nanodroplet, and nanobubble**

*Recent Advances about Local Gene Delivery by Ultrasound DOI: http://dx.doi.org/10.5772/intechopen.80036*

*Gene Expression and Control*

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**Figure 2.**

their local delivery through active and passive targeting. Traditional membrane materials consist of microbubbles, which include albumin, lipid, polymers, and surfactants. Different shell compositions have various characteristics. Albumin is commonly used in the preparation of commercial ultrasound contrast agents, but it is susceptible to degeneration due to temperature change. In addition, it is expensive and easy to cause immune response. The synthetic phospholipids are good and biocompatible, but their half life is short in vivo. Polymers are slightly inferior in

It has been proved that the application of ultrasound combined with commercial microbubbles and gene mixture could regulate gene expression and achieve therapeutic effect [27–30]. Wang et al. compared the effect of gene delivery by three kinds of typical commercial microbubbles—Optison, Sonovue, and Levovist. The mixture of microbubbles and plasmid DNA encoding green fluorescent protein was injected into tibialis anterior muscle of mice. After ultrasound irradiation,

biocompatibility, but it possesses better stability.

*Schematic model of transfection process of genes in carriers.*

the number of GFP-positive fibers was significantly increased in Optison- and Sonovue-treated groups, proving the efficiency of gene transfection by ultrasound combined with commercial microbubbles [31]. However, DNA is anionic molecules, and most microbubbles are negatively charged on the surface, which bring some difficulty for the formation of DNA/microbubble complexes. In order to address this issue, some cationic microbubbles are developed and applied as gene vector to enhance the gene-carrying capacity [32–37]. Wang et al. evaluated the difference of gene transfection rate between cationic microbubbles and neutral microbubbles in combination with ultrasound. Their results demonstrated that the expression of reporter gene in cationic microbubble group was 20-fold higher in vitro and 3-fold higher in tumor model than neutral bubbles [34]. Recently, Wei et al. applied Targesphere, a kind of commercial cationic microbubbles, as short hairpin (shRNA) vector for connective tissue growth factor (CTGF). It was showed that the expression of CTGF was decreased in renal fibrosis mouse model after ultrasound irradiation, which proved the great potential in gene delivery mediated by ultrasound combined with cationic microbubbles [38].

#### **3.2 Nanoparticle, nanodroplet, and nanobubble**

Nanoscale ultrasound contrast agents, with the particle size from 100 to 600 nm, are also developed in the recent years. Compared with traditional microbubbles, nanoscale contrast agents have smaller size and stronger penetrating ability. In addition, nanoscale contrast agents possess greater gene-carrying capacity due to their larger surface area. Common nanoscale ultrasound contrast agents include nanobubbles, solid nanoparticles, and liquid fluorocarbon nanoparticles. Most of the shell membrane of nanobubbles are lipid or polymer, and the core could be gas or liquid. Nanobubbles can cross through the blood vessels and aggregate in the tumors through the enhanced permeability and retention (EPR) effect [39]. It was proved that nanobubbles could achieve ideal gene transfection efficiency when combined with ultrasound [40, 41]. Horie et al. applied ultrasound combined with nanobubbles mediating tumor necrosis factor (TNF-α) DNA delivery to treat tumor-bearing mice and resulted in the decrease of the tumor vessel density and inhibition of tumor growth [42]. To enhance the gene-carrying capacity and local transfection efficiency, cationic nanobubble or targeted nanobubbles have been applied and showed excellent therapeutic effect in vitro and in vivo [43–45]. Yin et al. developed a new kind of siRNA-nanobubble, through a nanoparticle heteroassembly of siRNA-loaded polymeric micelles and liposomes, demonstrating their ideal therapeutic effect in cancer treatment [46]. Xie et al. used cell-permeable peptides (CPPs) to enhance the transferring rate of siRNA. They developed CPP-siRNA that targets oncogene c-myc and encapsulated it into nanobubbles. It was shown that the expression of c-myc mRNA was significantly decreased, and the growth of tumor was significantly inhibited after ultrasound irradiation [47].

Recently, liquid fluorocarbon nanodroplets have attracted wide attentions in the ultrasound-mediated gene delivery. These nanodroplets prepared from a lipid or a polymer shell can encapsulate liquid fluorocarbon emulsion (perfluoropentane, etc.). The liquid core would occur "acoustic droplet vaporization" (ADV) under ultrasound irradiation, which makes the nanodroplet transform into gas-containing microbubbles, greatly enhancing the cavitation effect of ultrasound **Figure 3**. Although nanodroplets have shown its therapeutic effect in high-intensity focused ultrasound (HIFU) and drug delivery, its application in gene delivery is still rare. Gao et al. synthesized a novel tumor-targeting cationic nanodroplet and applied it as gene vector to treat Her2-positive breast cancer. The results in their study

demonstrated that this nanodroplet could achieve better gene transfection efficiency, showing its potential in gene delivery by ultrasound [48, 49].

**Figure 3.** *Schematic model of acoustic droplet vaporization (ADV).*

(A) Nanoparticles penetrate the tissue through the EPR effect; (B) droplets vaporize into microbubbles through ADV under certain acoustic pressure, which enhances the cavitation effect and changes the structure of tumor vessels. (Quoted from: Ho et al. [50]).

Nanoparticles commonly used in gene transfection include liposomes, polymer, and magnetic nanoparticles. Studies demonstrated that the cavitation effect produced by UTMD could increase the concentration of nanoparticles in targeted tissue and improved gene transfection efficiency. In the field of ultrasound-mediated gene delivery, liposome and polyethylenimine (PEI) are the most popular gene vectors.

Liposome is used as a nanocarrier for gene transfection, with high gene-carrying capacity and transfection efficiency. Taking advantages of UTMD, researchers have demonstrated that the accumulation of gene-carrying liposomes can be improved in targeting cells or tissue [51, 52]. Yoon et al. proved that ultrasound combined with microbubbles and gene-carrying liposomes could be a superior gene transfection system [53]. Recently, Chertok et al. modified heparin on the surface of liposome to increase the accumulation of gene in tumor site and reduce the off-target effect. Compared with nonheparinized DNA-carrying liposomes, modified liposomes combined with UTMD could significantly enhance the gene transfection rate in tumor in vivo [54].

PEI is another commonly used gene vector with high-density positive charge. It can form stable complex with genes through electrostatic adsorption. Also, utilization of PEI can avoid DNA degradation by nucleic acid enzyme and improve the stability and integrity of genes in vivo. Meanwhile, PEI can assist gene delivery into nuclei through proton sponge mechanism and endosomal escape, which will enhance the expression of targeting gene [55] **Figure 4**. However, the cell toxicity is inevitable because of its strongly positive charge. UTMD may function as an effective method to balance the cytotoxicity and transfection efficiency of PEI. UTMD could not only temporarily mediate the opening of cell membrane and promote the PEI-DNA complex entering the cell but also improve the level of intracellular calcium and PKC protein expression, which can enhance the effect of endocytosis. It was confirmed that UTMD combined with PEI or chemical modified PEI could be an effective and safety gene transfection strategy in vitro or in vivo [56, 57]. Dang et al.

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Cl−

**Figure 4.**

**4.1 Tumor**

*Recent Advances about Local Gene Delivery by Ultrasound*

demonstrated that UTMD combined with PEI could achieve the same transfection efficiency as Lipofectamine 2000 and lower cytotoxicity [58]. Deshpande et al. found that ultrasound combined with PEI could enhance the DNA transfection rate up to 200-fold than naked DNA plasmids [59]. Park et al. applied UTMD combined with PEI mediating the adenine nucleotide translocase-2 (ANT2) shRNA to successfully increase the survival rate of xenograft mice and induce the tumor regression [60].

PEI binds with cell membrane and is endocytosed. When they enter lysosome, the unsaturated amino groups are able to capture protons and cause the retention of

ion and water molecule, which will make lysosomal swelling and rupture, and

Tumor is a kind of genetically related disease. Its occurrence, development, and recurrence are closely related to the mutation and deletion of the gene. With the development of molecular biology, gene therapy has shown a great potential in cancer treatment. At present, the common strategy of gene therapy is to transfer tumor suppressor gene into tumor cells to restore normal phenotype of cells. Nande et al. applied UTMD to mediate tumor suppressor genes, including p53, Rb, p130, and significantly reduced tumor growth [61]. Chang et al. utilized the p53-loaded targeted microbubbles for ovarian cancer treatment and achieved higher transfection

then release the lysosomal content. (Quoted from: Nel et al. [55]).

**4. Application of local gene delivery by ultrasound**

*Schematic model of the proton sponge effect by cationic nanoparticles.*

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

*Gene Expression and Control*

from: Ho et al. [50]).

*Schematic model of acoustic droplet vaporization (ADV).*

**Figure 3.**

tumor in vivo [54].

demonstrated that this nanodroplet could achieve better gene transfection effi-

(A) Nanoparticles penetrate the tissue through the EPR effect; (B) droplets vaporize into microbubbles through ADV under certain acoustic pressure, which enhances the cavitation effect and changes the structure of tumor vessels. (Quoted

Nanoparticles commonly used in gene transfection include liposomes, polymer, and magnetic nanoparticles. Studies demonstrated that the cavitation effect produced by UTMD could increase the concentration of nanoparticles in targeted tissue and improved gene transfection efficiency. In the field of ultrasound-mediated gene delivery, liposome and polyethylenimine (PEI) are the most popular gene vectors. Liposome is used as a nanocarrier for gene transfection, with high gene-carrying capacity and transfection efficiency. Taking advantages of UTMD, researchers have demonstrated that the accumulation of gene-carrying liposomes can be improved in targeting cells or tissue [51, 52]. Yoon et al. proved that ultrasound combined with microbubbles and gene-carrying liposomes could be a superior gene transfection system [53]. Recently, Chertok et al. modified heparin on the surface of liposome to increase the accumulation of gene in tumor site and reduce the off-target effect. Compared with nonheparinized DNA-carrying liposomes, modified liposomes combined with UTMD could significantly enhance the gene transfection rate in

PEI is another commonly used gene vector with high-density positive charge. It can form stable complex with genes through electrostatic adsorption. Also, utilization of PEI can avoid DNA degradation by nucleic acid enzyme and improve the stability and integrity of genes in vivo. Meanwhile, PEI can assist gene delivery into nuclei through proton sponge mechanism and endosomal escape, which will enhance the expression of targeting gene [55] **Figure 4**. However, the cell toxicity is inevitable because of its strongly positive charge. UTMD may function as an effective method to balance the cytotoxicity and transfection efficiency of PEI. UTMD could not only temporarily mediate the opening of cell membrane and promote the PEI-DNA complex entering the cell but also improve the level of intracellular calcium and PKC protein expression, which can enhance the effect of endocytosis. It was confirmed that UTMD combined with PEI or chemical modified PEI could be an effective and safety gene transfection strategy in vitro or in vivo [56, 57]. Dang et al.

ciency, showing its potential in gene delivery by ultrasound [48, 49].

**104**

demonstrated that UTMD combined with PEI could achieve the same transfection efficiency as Lipofectamine 2000 and lower cytotoxicity [58]. Deshpande et al. found that ultrasound combined with PEI could enhance the DNA transfection rate up to 200-fold than naked DNA plasmids [59]. Park et al. applied UTMD combined with PEI mediating the adenine nucleotide translocase-2 (ANT2) shRNA to successfully increase the survival rate of xenograft mice and induce the tumor regression [60].

**Figure 4.** *Schematic model of the proton sponge effect by cationic nanoparticles.*

PEI binds with cell membrane and is endocytosed. When they enter lysosome, the unsaturated amino groups are able to capture protons and cause the retention of Cl− ion and water molecule, which will make lysosomal swelling and rupture, and then release the lysosomal content. (Quoted from: Nel et al. [55]).
