Recent Advances about Local Gene Delivery by Ultrasound

Gene therapy has been widely explored as a pharmacological approach, with a great potential to treat various diseases. Generally, many diseases have definite lesion’s site, especially for tumors. This feature results in a great demand on the delivery of therapeutic gene to the local lesion’s site. Ultrasound combined with microbubbles provides a promising platform to deliver gene in a spatiotemporally controlled way. Ultrasound beam can be positioned and targeted onto the deepseated lesion’s site of diseases by an external mobile transducer. Microbubbles can serve as vehicles for carrying genetic cargo and can be destructed by ultrasound, resulting in the local release of genetic payload. Meanwhile, sonoporation effect will occur upon which the bubbles are exposed to the appropriate ultrasonic energy, producing the transient small holes on the adjacent cell membrane and thus increasing the vascular and cellular permeability. In this chapter, we will review the recent advances about local gene delivery by ultrasound.


Introduction
Gene therapy, designed to deliver nucleic acid into cells to compensate for abnormal genes, is now considered a promising treatment option for some human diseases [1]. With the development of modern medicine and precise medicine, there is an increasing trend to change the traditional gene delivery mode into local gene delivery. At present, there are mainly two gene delivery approaches, virusmediated transfection and nonvirus-mediated transfection [2]. The former method has high-transfection efficiency, but the preparation procedure of recombinant viruses is sophisticated, and their clinical application is restricted due to biosafety concerns [3]. Nonviral vector approaches, such as liposome-mediated methods and electroporation techniques, are relatively safe. However, poor targeting and low-transfection efficiencies limit their widespread use [4]. It is a current research hotspot to look for an effective and safe method to mediate gene delivery for biomedical application.
Ultrasound is a widely used diagnostic technique in clinic, which possesses the advantages of safety, real-time monitoring, and low cost. Recently, with the development of ultrasound contrast agents, ultrasound has evolved from a diagnostic tool to a treatment application for delivering locally therapeutic substances into the lesion's sites. Ultrasound-targeted microbubble destruction (UTMD) provides a promising platform to deliver genes in a spatiotemporally controlled way. 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][28][29][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, 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][33][34][35][36][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].

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 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.  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 Cl − ion and water molecule, which will make lysosomal swelling and rupture, and then release the lysosomal content. (Quoted from: Nel et al. [55]).

Application of local gene delivery by ultrasound 4.1 Tumor
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 efficiency than conventional nontargeted microbubbles [62]. Mishel et al. used ultrasound to mediate hSef-b delivery, another kind of human tumor suppressor gene, and demonstrated the efficacy of gene therapy mediated by ultrasound [63]. Recently, gene delivery by ultrasound was also applied in gene-directed enzyme prodrug therapy (GDEPT). The key process of GDEPT is effectively transferring gene encoding enzyme, which can convert a nontoxic prodrug into an activated cytotoxic agent [64]. Devulapally et al. used PEGylated-PLGA-PEI nanoparticles and mediated TK-NTR fusion gene delivery in tumor xenograft mice. Their results showed that the tumor size was reduced by 2.3-fold when compared with untreated mice [65].
In the field of tumor therapy, RNAi could selectively inhibit the expression of key genes in the development of cancer. It has been proved that co-delivery of siRNA and chemotherapy drugs by ultrasound could improve the therapeutic effect of tumor and reduce the dosage chemotherapy drugs [66][67][68][69]. Zhao et al. synthesized cationic porphyrin microbubbles for the delivery of FOXA1-siRNA, achieving an excellent therapeutic effect for breast cancer [70]. Cancer stem cells (CSCs), a group of tumor cells with self-renewal, multidirectional differentiation potential, are thought to be the key of tumor recurrence, metastasis, and drug resistance. Specific markers, such as CD133, are important targets for gene therapy. Liu et al. used UTMD to deliver shRNA-CD133 to liver CSCs and reversed the process of epithelial-mesenchymal transition [71].

Cardiovascular disease
Atherosclerosis is the main cause of coronary heart disease, cerebral infarction, and peripheral vascular disease. Studies demonstrated that ultrasound combined with microbubbles can deliver angiogenic genes to the ischemic region of the myocardium and enhance expression of angiogenesis-related factors and thus improve myocardial blood supply [72,73]. Du et al. utilized UTMD and cationic microbubbles to mediate delivery of growth differentiation factor 11 (GDF11) plasmid to aged heart. Their results suggested that ultrasound could enhance GDF11 expression, increase the cardiac stem cell (CSC) proliferation, and rejuvenate the senescent heart from ischemic injury [74]. Castle et al. successfully enhanced the level of ApoA-I and high-density lipoprotein cholesterol (HDL-C) in vivo through delivering human apolipoprotein ApoA-I plasmids by ultrasound [75].
Heart failure, caused by various cardiac structures and functional disorders, will impair ventricular filling and ejection function and eventually cause cardiac output unable to meet body tissue metabolic needs. Lee et al. delivered survivin gene to cardiomyocyte by UTMD and observed its efficacy on cardiac function. The apoptosis rate of cadiomyocyte was significantly decreased, and the left ventricular systolic dysfunction was attenuated after 6 weeks, demonstrating that ultrasoundmediated gene delivery can be an effective treatment in heart failure [76].

Central nervous system diseases
Blood-brain barrier (BBB) is an important obstacle for central nervous system (CNS) diseases. BBB is mainly composed of cerebral capillary endothelial cells and their cells, matrix, astrocytes, and extracellular matrix [77]. To cross the BBB, researchers have tried various methods, including invasive surgery, hypertonic drugs, chemical modification of drugs to target delivery to brain, and micro-carriers [78][79][80]. Recently, ultrasound mediating BBB opening has attracted researchers' attention due to its characteristic of noninvasive, reversible, and targeted delivery. Hynymen et al. proved that microbubbles could be applied as cavitation nuclei to reduce the ultrasonic energy to open the BBB, reducing the risk of tissue damage and bleeding [81]. Based on this, numerous studies are exploring the therapeutic effect of gene delivery mediated by ultrasound in CNS diseases [82,83], such as glioma, Parkinson's disease, and Alzheimer's disease.
Glioma is the most common malignant tumor of the central nervous system. UTMD has a wide application prospect in the treatment of brain glioma. In 2016, Carpentier et al. developed an implantable ultrasonic irradiation system, named SonoCloud. They used this system to open the local area of BBB with microbubbles. In their study, 15 patients with recurrent brain glioma were selected to test the therapeutic effect of UTMD-mediated BBB opening. After intravenous administration of carboplatin and Sonovue combined with ultrasound treatment, it was proved that the BBB could be safely opened, and 9 of 15 patients showed no further tumor growth [84]. Fan [90,91].
In addition, ultrasound-mediated gene delivery was also applied in other CNS diseases. Song et al. developed PLGA nanobubbles for NGF delivery. NGF expression was significantly enhanced, and neuronal apoptosis in injured spinal cords was inhibited after ultrasound irradiation [92]. Wang et al. demonstrated that UTMD could successfully mediate VEGF gene delivery into brain and decreased infarct areas in a cerebral ischemic injury model [93].

Musculoskeletal disease
Arthritis is a common chronic inflammatory disease. Of these, the most common type is osteoarthritis and rheumatoid arthritis (RA). At present, the main treatment of arthritis is drug, including nonsteroidal anti-inflammatory drugs, cytotoxic drugs, and hormones. However, there are some drawbacks such as low local concentration and systemic side reaction. Ultrasound-mediated gene delivery has been proved to be effective in arthritis therapy. Xiang et al. applied UTMDmediated enhanced green fluorescent protein (EGFP) gene delivery in antigeninduced arthritis rabbit model, and the significantly enhanced expression remained detectable for 40 days in the synovial pannus [94]. Tumor necrosis factor α (TNFα) secreted by synovial fibroblasts plays an important role in the progression of RA, which can cause bone destruction and joint dysfunction. Inue et al. transferred siRNA-TNFα to the articular synovial membrane of the rat through UTMD technique. They found that the expression of TNFα was inhibited, resulting in a significant remission of paw swallowing in comparison to control group [95].
In the field of fracture healing, bone morphogenetic protein-2 (BMP-2) is an ideal osteoinduction factor, which possesses the function of inducing cartilage and bone formation [96]. Some studies have confirmed that the transfection rate of BMP-2 gene in skeletal muscle cells and fibroblast cells could be enhanced by UTMD [97]. Osawa et al. delivered BMP-2 gene to the skeletal muscle in vivo, confirming the therapeutic effect of UTMD mediating gene transfection [98].
Tendon injury is a common disease in orthopedics with a significant impact on the quality of patient's life. Regulation of the expression of local cytokines in Achilles tendon by gene therapy is a potential therapeutic method to improve the prognosis of patient. Studies demonstrated that UTMD could increase the expression of genes in the Achilles tendon [99][100][101]. For example, Tang et al. transfected injured Achilles tendons of mice with insulin-like growth factor-1 (IGF-1) cDNA, showing that the maximum load, stiffness, and ultimate stress of treated Achilles tendons were higher than control group [102]. Bez et al. transferred BMP-6 encoding DNA by UTMD in Yucatan mini-pigs, showing the significantly enhanced osteointegration of all pigs after 8 weeks [103].

Ocular disease
For the treatment of ocular diseases, the most common method of drug delivery is surface administration or systemic administration. However, due to the unique structure of the eye, traditional drugs are difficult to enter the posterior eye segment, causing low bioavailability of drugs. As for ultrasound mediated gene delivery in ocular diseases, recent researches mainly focus on the cornea, retinoblastoma, and retinal neovascularization.
Cornea is a transparent tissue without blood vessels, which is an ideal target tissue for gene therapy because of its superficial position, transparent organization, and easy observation. Sonoda et al. confirmed that UTMD could mediate eGFP gene transfection to cornea epithelial cells of rabbit. In their study, they injected plasmid and microbubbles into the cornea of the rabbit and irradiated the eyes with ultrasound. They found that the corneal cells with GFP-positive expression were distributed around the injected region. No obvious tissue damage was observed in their study [104]. To optimize the gene transfection efficiency, Yamashita et al. developed a novel lipid microbubble, composed of polyethylene glycol (PEG) modified liposomes and perfluoropropane gas, and achieved a 27% gene transfection rate [105].
In the retina, there is a biological barrier similar to the blood-brain barrier named blood-retinal barrier (BRB), which is composed of tight connection between retinal endothelial cells and retinal pigment epithelial (RPE) cells. The presence of BRB prevents most systemically administered genes entering the retina, reducing the effectiveness of treatment. Park et al. demonstrated that UTMD could mediate BRB reversible opening without retinal damage [106]. Some studies have confirmed the effect of UTMD-mediated gene delivery into retinal in vitro and in vivo [107][108][109][110].
Retinoblastoma (RB) is a common ocular malignancy. Local treatment not only can retain part of the vision but also reduce the toxic side effects. Luo et al. applied wild-type 53 (wtp53) as a therapeutic gene. The in vitro experiment showed that the apoptosis rate of RB cells was higher (25.58%) than control group after ultrasound treatment [111]. To prove the therapeutic effect of gene delivery by ultrasound in vivo, Gao et al. transferred both wtp53 and Rb94 by UTMD to treat tumorbearing mice. RB tumor growth was significantly inhibited, along with the decrease of the level of vascular endothelial factor and microvessel density [112].
Retinal neovascularization (RNV) is caused by hypoxic-ischemic ocular fundus diseases, characterized by retinal fibrous hyperplasia, retinal detachment, and even loss of vision. It has been reported that endostatin can be used for treating RNV because of its excellent antiangiogenic effect [113]. Xu et al. significantly enhanced the expression of endostatin by using cationic microbubbles to deliver endostatin gene under ultrasound irradiation. As a result, the growth of human retinal vascular endothelial cell was inhibited, suggesting that endostatin gene delivery mediated by UTMD may be a useful tool for RNV therapy [110].

Nephropathy
The blood flow of the normal kidney accounts for one-fourth to one-fifth of the total circulating blood volume. Therefore, a large number of microbubbles could enter the kidney blood vessels, which could be applied for ultrasound contrast imaging or targeted treatment. Based on this feature, some researchers applied UTMD to deliver genes to treat nephropathy, including diabetic nephropathy, hypertensive nephropathy, and renal fibrosis. Zhang et al. found that UTMD could increase the renal interstitial capillary permeability in diabetic nephropathy rat models [114]. Transforming growth factor β (TGF-β) is the key cytokine to promote the development of renal fibrosis. It can induce apoptosis of the podocytes on glomerular filtration membranes and promote the activation and proliferation of interstitial fibroblasts through TGF-β/SMAD signaling pathway. Lan et al. enhanced the expression of Smad7 in rat unilateral ureteral obstruction model by ultrasound as the gene delivery system, greatly attenuating tubulointerstitial fibrosis [115]. The therapeutic effect of UTMD-mediated Smad7 gene delivery in renal fibrosis was also proved in Smad7 gene knockout mice [116], diabetic nephropathy model mice [117], and angiotensin II-mediated hypertensive nephropathy [118].
In addition to the TGF-β/SMAD signal pathway, researchers have also explored the use of other signaling pathways in the treatment of renal fibrosis. RAP1 is a small molecule G protein that participates in the regulation of cell proliferation, differentiation, and intercellular adhesion [119]. Xiao et al. treated diabetic model rats with Rap1 gene delivery by ultrasound and microbubble (Optison). It was demonstrated that this treatment could protect the mitochondrial function of renal tubules and reduce the interstitial fibrosis [120]. In diabetic nephropathy, Yiu et al. confirmed the therapeutic effect of Kallistatin, which possesses the function of antioxidative and anti-inflammatory. The glomerulosclerosis and renal fibrosis were attenuated, and the renal function was improved after Kallistatin gene delivery by UTMD [121].
Recently, RNAi combined with UTMD therapy has been applied to the treatment of renal diseases. miR-29b is low expression in diabetes [122] and can function as a therapeutic targeting [123]. Chen et al. delivered miR-29b in diabetic mice by ultrasound combined with SonoVue. The results showed that this treatment could inhibit the inflammation induced by NF-κB/p63 and delay the progress of renal fibrosis [28]. Zhong et al. found that the level of miR-21 is highly associated with the development of renal fibrosis in diabetic mice and effectively improved renal fibrosis and inflammation by using UTMD-mediated miR-21 shRNA delivery [29]. Wei et al. applied UTMD combined with shRNA-CTGF to treat mouse models of renal fibrosis; the level of CTGF was significantly lower; and the renal fibrosis was attenuated, accompanied by the reduction of TGF-β and Type I collagen [38].

Conclusion and prospect
With the development of ultrasound contrast agents and the understanding of the biological effects of ultrasound, ultrasound-mediated gene delivery has been proven the great potential in the treatment of various diseases. Ultrasound contrast agents, including microbubbles, nanoparticles, and nanobubbles, can be used as gene vectors through intravenous or local injection into lesion site. With ultrasound irradiation at a certain level of acoustic intensity, the cavitation effect, sonoporation, and thermal effects occur, which can enhance the permeability of local tissue and promote the gene delivery into the pathological tissue.
Although ultrasound-mediated gene delivery has a broad application in animal study, there is still a long way for its application in human body. The main © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. problems, which need to be solved, may include the following aspects. First, many cationic materials are applied for the preparation of the ultrasound contrast agents. They have high gene-carrying capacity, but their biocompatibility is still doubtful. Second, ultrasound security is also an important concern. Unlike diagnostic ultrasound energy, the intensity applying in gene delivery is greater. Studies have shown that severe cavitation effects can lead to membrane rupture, DNA rupture, nuclear fragmentation, endothelial cell damage, microvascular leakage, hemolysis, myocardial injury, and even left ventricular function [19,124]. More investigations need to be made to optimize the ultrasonic parameters so as to maximize the gene transfection efficiency and reduce the adverse side effects on the normal tissues and organs. In addition, the different types of ultrasound equipment used in various laboratories also bring some difficulties for the repeatability, which hinder the progress of ultrasound-mediated gene transfection technology to some degree. At the same time, it is believed that ultrasound will make more progress in gene delivery and bring about greater medical revolution in the future.

Author details
[5] Sirsi SR, Borden MA. Advances in ultrasound mediated gene therapy using microbubble contrast agents.