Ultrasound: Mechanical gene transfer into plant cells by sonoporation

Biotechnology Advances 24 (2006) 1 – 16 www.elsevier.com/locate/biotechadv

Research review paper

Ultrasound: Mechanical gene transfer into plant cells by sonoporation Y. Liu a,*, H. Yang a, A. Sakanishi b a b

School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, PR China Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma University, Kiryu, Gunma 376-8515, Japan Received 26 January 2005; accepted 1 April 2005 Available online 1 June 2005

Abstract Development of nonviral gene transfer methods would be a valuable alternative of gene therapy or transformation. Ultrasound can produce a variety of nonthermal bioeffects via acoustic cavitation. Cavitation bubbles can induce cell death or transient membrane permeabilization (sonoporation) on cells. Application of sonoporation for gene transfer into cells or tissues develops quickly in recent years. Many studies have been performed in vitro exposure systems to a variety of cell lines transfected successfully. In vivo, cavitation initiation and control are more difficult, but can be enhanced by ultrasound contrast agents (microbubbles). The use of ultrasound for nonviral gene delivery has been applied for mammalian systems, which provides a fundamental basis and strong promise for development of new gene therapy methods for clinical medicine. In this paper, ultrasound applied to plant cell transformation or gene transfer is reviewed. Recently, most researches are focused on sonication-assisted Agrobacterium-mediated transformation (SAAT) in plant cells or tissues. Microbubbles are also proposed to apply to gene transfer in plant cells and tissues. D 2005 Elsevier Inc. All rights reserved. Keywords: Ultrasound; Cavitation; Gene transfer; SAAT; Plant cell; Microbubbles

Contents 1. 2.

3. 4.

Introduction. . . . . . . . . . . . . . . . . . . . Physical mechanisms of ultrasound . . . . . . . 2.1. Thermal effect . . . . . . . . . . . . . . . 2.2. Acoustic cavitation . . . . . . . . . . . . 2.3. Mass transfer enhancement . . . . . . . . 2.3.1. Boundary layer . . . . . . . . . . 2.3.2. Cell membrane and cell wall . . . 2.3.3. Cytosol . . . . . . . . . . . . . . Significances of gene transfer into plants . . . . Application of ultrasound in plant transformation

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* Corresponding author. Tel./fax: +86 28 8320 6124. E-mail address: [email protected] (Y. Liu). 0734-9750/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2005.04.002

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Y. Liu et al. / Biotechnology Advances 24 (2006) 1–16

5. Ultrasound contrast agents (microbubbles) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Ultrasound has been used for diagnostic imaging in clinical fields without producing any significant adverse effects. Recently ultrasonic methods have considerable potential for the introduction of macromolecules or DNA into cells (Wyber et al., 1997). Several methods have been developed for the delivery of DNA into cells (Fig. 1). (1) Chemically facilitated methods, e.g., calcium phosphate and DEAE-dextran. (2) Vector-mediated methods, e.g., liposome and retroviruses. (3) Mechanical methods, e.g., particle bombardment (gene gun), microinjector and electroporation. All the transfection techniques can be divided into two broad categories—viral and nonviral. The two major forms of viral transfection are the use of retroviruses and adenoviruses. Retroviruses have proven to be a very effective means of stably delivering therapeutic nucleotides to cells. However, retroviruses have several shortcomings including the inability to infect nonproliferating cells, the random incorporation of the viral DNA into the genomic DNA of the cell causing the potential for insertional mutagenesis and malignancy, and the potential for the replication competent viral

Eukaryotic virus infection

particles to emerge, causing cell death (Sokol and Gewirtz, 1996). Adenoviruses also have proven to be quite effective in the transfection of cells with therapeutic nucleotides, but they also have major shortfalls. Adenoviruses induce a short expression time of the delivered nucleotides, which is probably caused by the adenoviral delivery system itself, and also are not useful for re-treatment without major efficiency decrease due to immunological response, two potentially insurmountable problems for in vivo application. Both adenoviral and retroviral methods of transfection also suffer from nonspecificity of delivery systems. If viral particles transfect non-target cells, deleterious side effects will be caused. Forms of nonviral transfection are represented by lipofection, electroporation, particle bombardment, and sonoporation (ultrasound). Lipofection is one of the most widely used nonviral methods of transferring genetic material to living cells. Essentially, cationic lipids encapsulate the negatively charged DNA and facilitate transfer of the gene through the cell membrane (Gershon et al., 1993). This method allows high transfection efficiency with minimal cellular toxicity, but as with the viral vectors, it does not allow control of spatial or temporal specificity of delivery. Electroporation refers to the transfer of DNA through membrane pores formed in high-voltage electric fields. This method allows some spatial targeting, but requires electrode

Direct injection (microinjection)

Gene-gun (particle bombardment)

nucleus

Electroporation

Sonoporation

13 15 15

DNA-calcium phosphate coprecipitate

Liposome fusion

Fig. 1. The usually used methods for gene transfer into cells.

Y. Liu et al. / Biotechnology Advances 24 (2006) 1–16

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Fig. 2. Marker gene expression 24 h after lipoplex-mediated transfection of enhanced green fluorescent protein marker gene in 9 L rat brain tumor cells grown on 12-well plates. (A) Fluorescence of living control cells without ultrasound treatment; and (B) fluorescence of living cells treated for 60 s with ultrasound. Original magnification 240.

placement, which can be invasive. Particle bombardment represents yet another way of binjectingQ foreign DNA into cells, this time by coupling the gene to projectiles that are made to penetrate the membrane at high speed. This method also allows accurate placement of DNA delivery, but appears to be limited to surface (e.g., skin) applications. As mentioned above, each method has inherent limitations, such as low efficiency, complex protocols and/ or high cost; in particular, many cells are only responsive to one or a few specialized methods. In this respect, mechanical methods are often more versatile, as they are based on disruption of the cell membrane and are less dependent on cell type. In recent years there are intense interests to have an efficient but simple way to deliver DNA into cells for genetic transformation (Choudgary and Chin, 1995). It

has been shown that sonication (ultrasound) can alter the transient permeability of plasma membrane to facilitate uptake (Tachibana et al., 1999) (Fig. 12). Compared to other direct DNA delivery methods, such as particle gun bombardment, electroporation and microinjection, the ultrasound treatment may be simpler to carry out. Sonication, however, could cause cell damage or ever rupture. If sonication is used to facilitate uptake, it is important to optimize the conditions for uptake without causing damage to the cells. Mild ultrasound irradiation has been proved an efficient method for transfection in animal cells and tissues in vitro and in vivo (Bao et al., 1997; Miller et al., 1999; Huber and Pfisterer, 2000). The ability of ultrasound to load cells with large molecules, which survive for subsequent culture, opens the possibility of DNA transfection and expression of foreign gene products in vitro and in vivo.

35

Transfected cells (%)

30 25 20 15 10 5 0

0 sec

30 sec

60 sec

90 sec

Ultrasound treatment time Fig. 3. Graph showing rate of transfection (percentage of transfected cells) after ultrasound treatment of lipoplex-induced 9 L cells growth on 12well plates.

Y. Liu et al. / Biotechnology Advances 24 (2006) 1–16

γ

Transfection efficiency w / %

0.9

10% 20% 30%

0.6

0.3

0

0

20

40

60

Apparent exposure time t' / s Fig. 4. Transfection efficiency of HeLa-S3 cells exposed by 10%, 20% and 30% duty cycle ultrasound vs. the total exposure time t.

100 90 Light units per µg protein

For in vitro transfection, ultrasound treatment of 9 L gliosarcoma cells grown in 12-well plates caused a significant increase of lipoplex (liposome/plasmidDNA complex)-mediated transfection rates (Figs. 2 and 3). The mean values of transfection rates were 13.2%, 20.9% and 32.7% for ultrasound exposure of 30, 60 and 90 s, while 7.4% for control (Koch et al., 2000). In our former research (Liu et al., 2005), the transfection of HeLa-S3 cells was investigated in vitro by 10%, 20% and 30% duty cycle ultrasound exposures. It was found that the optimal transfection efficiency (0.80%) was at about 3.8 s of effective exposure time (Fig. 4) and 3.4-fold higher than the control (0.24%). Lawrie and co-workers (1999) reported that cultured porcine vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) were transfected with naked or liposome-complexed luciferase reporter plasmid. Ultrasound exposure for 60 s at 1 MHz and 0.4 W/cm2 enhanced luciferase activity 48 h later by 7.5-fold and 2.4-fold, respectively (Fig. 5). Ultrasound exposure caused only minor acute damage to the cell monolayer and has no effect on naked or Tfx-50-complexed plasmid integrity. At present, in order to increase the transfection efficiency, ultrasound contrast agents (microbubbles) were used in the ultrasound-mediated microbubbles for gene transfer or therapy, which hold considerable promise in vitro and in vivo (Frenkel et al., 2002). This finding opens tremendous opportunities for targeted transfection. Fig. 6 shows the ultrasound-OptisonR-mediated transfection (Taniyama et al., 2002). Other studies are reported that ultrasound with microbubbles can obvi-

A

*

80 70 60 50 40 30 20 *

10 0 DNA

DNA + 1MHz USE

DNA + Heat

Tfx/DNA

Tfx/DNA Tfx/DNA + + 1MHz USE Heat

Transfection conditions

100 90 Light units per µg protein

4

B *

80 70 60 50 40 30 20 10 0

DNA

DNA Tfx/DNA Tfx/DNA + + 1MHz USE 1MHz USE

Transfection conditions

Fig. 5. Porcine VSMCs (A) and ECs (B) were transfected for 3 h with naked or liposome (Promega Tfx-50) complexed luciferase DNA (n = 12), and luciferase activity in cell lysates was assayed after 48 h at 37 8C (A and B). Ultrasound exposure (USE, 1 MHz, continuous wave, 0.4 W/cm2) was performed for 60 s. Asterisks indicate significant differences between control and ultrasound-exposed cells. *P b 0.05.

ously produce tumor growth reduction or inhibit tumor growth in vivo (Miller and Song, 2002, 2003). Up to now, maybe it is difficult to carry out, so there are few gene transfer studies in vivo by ultrasound. Kim et al. (1996) tested the possible extension of their in vitro experiments to in vivo conditions. The h-galactosidase reporter plasmid was injected into both knee joints of rats, and one joint was treated with 1 MHz plus 30 kHz ultrasound at 0.4 MPa and kPa, respectively, for 1 min. Three of four treated knees showed reporter gene expression after 4 days, while none was detected in the unexposed joints, as shown in Fig. 7. Unger et al. (2001a,b) injected the gene for interleukin2 (IL-2) using a lipid vector (in this case not a microbubble) into the tumors of mice in vivo. These mice

Y. Liu et al. / Biotechnology Advances 24 (2006) 1–16

% increase in luciferase activity

10000

5

P< 0.01

**

5000

**

100 Control

Plasmid+ Plasmid + Ultrasound Optison +Ultrasound

Fig. 6. Comparison of luciferase activity achieved by transfection of naked plasmid DNA alone, naked plasmid DNA using ultrasound and naked plasmid DNA using ultrasound with Optison in human cultured skeletal muscle cells. Control, cells transfected with naked luciferase plasmid DNA alone; plasmid + ultrasound, cells transfected with naked luciferase plasmid DNA using ultrasound; plasmid + Optison + ultrasound, cells transfected with naked luciferase plasmid DNA using ultrasound with Optison. Values are expressed as percent increase in luciferase activity as compared with control. n = 8 for each group calculated from eight independent experiments. **P b 0.01 vs. control.

were killed 72 h later; the tumors were excised, and the cells were obtained from the tumor and were grown in cell culture. As shown in Fig. 8, ultrasound increased the gene expression in the tumors. Ultrasound for transfection has been widely applied in animal cells or tissues. However, there are few researches about ultrasound in plant cells or tissues. It may be that the cell wall of plant cell is the big obstacle for gene transfer. The present study is to review application of ultrasound for gene transfer in animal and plant cells. To better present the application of ultrasound for transfection, the physical mechanisms of

Fig. 8. Effect of ultrasound on gene therapy to mice tumors in vivo. In these experiments, the gene for cytokine interleukin-2 (IL-2) was injected into the tumor using a lipid carrier (DMRIE:DOPE). Ultrasound caused a several-fold increase in gene expression in the tumors, resulting in increased production of IL-2 in the tumors.

ultrasound are firstly described. Finally, the perspectives are put forward to increase the transfection efficiency in plants. 2. Physical mechanisms of ultrasound Ultrasound, or sound of high frequency above 20 kHz, is inaudible to the human ear. For changes in living systems caused by exposure to ultrasound, the potential bioeffects of ultrasound result from two major mechanisms: thermal effect and mechanical effect (nonthermal). 2.1. Thermal effect When ultrasonic waves propagate in medium, its energy will be absorbed in part by the medium, and the temperature of the medium increases. Increasing

Fig. 7. Photomicrograph of rat knee joints after injection of a h-galactosidase plasmid and exposure to ultrasound in the presence of the contrast agent AlbunexR. Three out of four exposed knees showed h-galactosidase expression (A), while no expression was seen in control, unexposed (B) joints.

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Fig. 9. Schematic representation of variation in bubble size at transient cavitation (A) and stable cavitation (B) in a liquid irradiated with ultrasound.

temperature of the medium by ultrasound cannot be the main activating effect of ultrasound in biotechnology, but it could form an important part. On the other hand, the thermal deactivation is an important mechanism in the denaturation of enzymes. 2.2. Acoustic cavitation The cavitation phenomenon is the largest nonthermal effect generated by ultrasound, and it also is a dynamic process in the medium (Doktycz and Suslick, 1990). Cavitation and, in particular, inertial cavitation, is considered a major mechanism for causing alterations to biological tissues, especially increased membrane permeability (Bommannan et al., 1992). A distinction may be made between two types of cavitations (Fig 9). The first is non-inertial (or stable) cavitation, where bubbles exist for a considerable number of acoustic cycles, and where the radius of each bubble varies about an equilibrium value. The second is inertial (or transient) cavitation, where bubbles oscillate in an unstable manner about their equilibrium, expanding to 2 or 3 times their resonant size before collapsing violently during a single compression half cycle (Riesz and Kondo, 1992; Suslick, 1988). Although damage to biological tissues has also been proposed to result from stable cavitation

bubbles (Lewin and Bjorno, 1981, 1982), transient cavitation effects are considered to be the primary mechanism of damage to intact cells (Riesz and Kondo, 1992). When the cavitation bubbles grow the critical state and collapse, it will generate high temperature and pressure. Cavitation bubble collapse is a remarkable phenomenon and generates temperatures of about 4000 K and pressures in excess of 1000 atm (Figs. 10 and 11). 2.3. Mass transfer enhancement Ultrasound, at a low intensity levels, enhances the movement of the liquid medium, favouring mass transfer and reaction rates in both multiphase and homogeneous systems (Bar, 1988). There are three different zones where this process take place: the boundary layer, the membrane and/or cell wall and in the cytosol. 2.3.1. Boundary layer It is well known that the vibratory gas bubble in an acoustic field generates around it a circulatory liquid motion referred to as a microstreaming. This fact favours the flux of reagents to the active site of the enzyme or to the cell and of the reaction products to the medium, increasing the turnover number, and thus increasing the rate of the process.

Fig. 10. Generating process of an acoustic bubble. Bubble collapse is a remarkable phenomenon induced throughout the liquid by ultrasound, and will generate many chemical and mechanical effects.

Y. Liu et al. / Biotechnology Advances 24 (2006) 1–16

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brane or of temperature variation in the medium. Similar effects have been reported in the case of potassium oxalate and in other solutions.

Fig. 11. The dynamic formation of cavitation bubbles induced by ultrasound.

2.3.2. Cell membrane and cell wall It has been proved that ultrasound favours mass transfer through both artificial and biological membranes (Fig. 12), e.g., the rate of salt transfer from 5% saline solution through a cellophane membrane into distilled water increases by 100% when ultrasound was applied in the direction of diffusion but was very poor when the irradiation was opposite to that of diffusion. It has been proved that this variation in the rate diffusion is not a result of damage to the mem-

2.3.3. Cytosol Ultrasound has the potential for enhancing mass transfer within cells. At an appropriate intensity level of ultrasound, intracellular microstreaming has been observed inside animal and plant cells with rotation of organelles and eddying motions in vacuoles of plant cells (Nyborg, 1982). A membrane permeation enhancing effect of ultrasound has been noted already. Extremely high frequency ultrasound (N 1 MHz) has been used to repeatedly harvest vacuole-located secondary metabolites from in vitro growth plant cells (Kilby and Hunter, 1990, 1991). All these effects can produce an increase in the metabolic functions of the cell that are of use in biotechnology. 3. Significances of gene transfer into plants Plants are important resources that have been providing us foods and medicines from the earliest times. The rapid advances that have taken place in plant

Fig. 12. Scanning electron microscopic images of HL-60 cells. Irradiated with ultrasound in the presence of MC 540 (A to D), untreated intact cells (E), cells irradiated with ultrasound alone (F).

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genetic engineering have made it possible to modify plants to increase food production and produce pharmaceuticals or chemicals (Misawa, 1994). Recombinant DNA technology in higher plants has opened up a new field of basic research and application in plant science. Foreign genes can be basically introduced into any kind of plant and it is possible to perform metabolic engineering of plants whose metabolic pathway and activity have been changed by foreign genes (Yoshida and Shinmyo, 2000). The purposes or significances for gene transfer in plants are mainly in two aspects: Trait improvement of economical crops and therapeutic proteins or chemicals production (Table 1). Biotechnology is transforming world agriculture, adding new traits to crop plants at a greatly accelerated rate. The improvement of crops with the use of genetics has been occurring for years. Traditionally, crop improvement was accomplished by selecting the best looking plants/seeds and saving them to plant for the next year (Gleba et al., 1999). Once the science of genetics became better understood, plant breeders Table 1 The production in transgenic plants of biopharmaceuticals for human health (Daniell et al., 2001) Plant host Potential application or human protein Anticoagulant Tobacco Thrombin inhibitor Canola (Brassica napus) Neutropenia Tobacco Growth hormone Tobacco Anemia Tobacco Antihyperanalgesic Arabidopsis by opiate activity Wound repair and Tobacco control of cell proliferation Hepatitis C and B Rice, turnip Tobacco Liver cirrhosis, Tobacco burns, surgery Blood constitute Tobacco Collagen Tobacco Cystic fibrosis, liver Rice disease Antimicrobial Non-human proteins Hypertension

Potato

HIV therapies

Tobacco, tomato Tobacco

Gaucher’s disease

Tobacco

Protein Protein C Hirudin Granulocyte-macrophage Somatropin, chloroplast Erythropoietin Enkephalins Epidermal growth

Interferon-a Interferon-h Serum albumin Hemoglobin a, h Homotrimeric collagen a-1 Antitrypsin trypsin inhibitor for transplant surgery maize aprotinin Lactoferrin

Angiotensin-converting enzyme a-Tricosanthin from TMV-U1 sub-genomic coat protein Glucocerebrosidase

used what they knew about the genes of a plant to select for specific desirable traits. This type of genetic modification, called traditional plant breeding, modifies the genetic composition of plants by making crosses and selecting new superior genotype combinations. Traditional plant breeding has been going on for hundreds of years and is still commonly used today. Plant breeding is an important tool, but has limitations. First, breeding can only be done between two plants that can sexually mate with each other. This limits the new traits that can be added to those that already exist in that species. Second, when plants are mated (crossed), many traits are transferred along with the trait of interest including traits with undesirable effects on yield potential. Genetic engineering is a new type of genetic modification. It is the purposeful addition of a foreign gene or genes to the genome of an organism. A gene holds information that will give the organism a trait, such as insect resistance, herbicide resistance, and salt tolerance. Genetic engineering is not bound by the limitations of traditional plant breeding. Genetic engineering physically removes the DNA from one organism and transfers the gene(s) for one or a few traits into another. Since crossing is not necessary, the dsexualT barrier between species is overcome. Therefore, traits from any living organism can be transferred into a plant. This method is also more specific in that a single trait can be added to a plant (Giri and Laxmi, 2000). On the other hand, plants have considerable potential for the production of biopharmaceutical proteins and peptides because they are easily transformed and provide a cheap source of protein (Cramer et al., 1996). The use of plants for medicinal purposes dates back thousands of years but genetic engineering of plants to produce desired biopharmaceuticals is much more recent. As the demand for biopharmaceuticals is expected to increase, it would be wise to ensure that they will be available in significantly larger amounts, on a costeffective basis. Currently, the cost of biopharmaceuticals limits their availability. Plant-derived biopharmaceuticals are cheap to produce and store, easy to scale up for mass production, and safer than those derived from animals. At present, transgenic plant cell cultures are applied to produce high-value secondary metabolites including pharmaceuticals (e.g., recombinant proteins), chemicals, antibodies and food additives (Daniell et al., 2001; Lessard et al., 2002). For example, one transgenic plant-derived biopharmaceutical, hirudin, is now being commercially produced in Canada for the first time, (Giddings et al., 2000). Some of the most expensive biopharmaceuticals of restricted availability,

Y. Liu et al. / Biotechnology Advances 24 (2006) 1–16

such as glucocerebrosidase, could become much cheaper and more plentiful through production in transgenic plants. The production of recombinant proteins in plants has many potential advantages for generating biopharmaceuticals relevant medicine. First, plant systems are more economical than industrial facilities using fermentation or bioreactor systems (Fig. 13). Second, the technology is already available for harvesting and processing plants and plant products on a large scale. Third, the purification requirement can be eliminated when the plant tissue containing the recombinant protein is used as food (edible vaccines). Fourth, plants can be directed to target proteins into intracellular compartments in which they are more stable, or even to express them directly in certain compartments (chloroplasts). Fifth, the amount of recombinant product that can be produced approaches industrial-scale levels. Last, potential human pathogens or toxins are minimized. 4. Application of ultrasound in plant transformation As we know, there are hard and thick cell wall in plant cell. Sonication is a novel method for gene transfer into plant protoplasts and intact plant cells (Fechheimer et al., 1987; Fechheimer and Taylor, 1987). Although the procedures for gene transfer into plant cells have proved to be quite successful during recent years, some species are still recalcitrant to transformation and some methods for gene transfer are tedious and/or require sophisticated equipment. There-

100 000

Mammalian cell culture

10 000

US $ g

-1

Transgenic goats

1000 7.5 tonne ha -1

100

Transgenic plants 120.0 tonne ha -1

10 1

10

20

50

100

300

500

700 1000 5000

Expression (mg l-1 cell culture, mg l-1 milk, mg Kg-1 plant)

Fig. 13. Costs per gram for purified immunoglobulin A (IgA) produced by different expression systems.

9

fore, further progress is desirable in this field. Many research have found the use of ultrasound to facilitate uptake of nucleic acids into plant cells and protoplasts (Joersbo, 1990; Joersbo and Brunstedt, 1990a; Zhang et al., 1991). Apparently, this technique provides an attractive alternative to traditional methods, particularly because intact plant cells can readily be transformed by ultrasound. Ultrasound has been reported to mediate gene uptake in plant protoplast, suspension cells and intact pieces of tissues. Gene transfer by ultrasonication employs the same simple procedure irrespective of the nature of the plant material to be transformed. The protoplasts, suspension cells or small pieces of tissue are suspended in a few milliliters of sonication medium in a microcentifuge tube. Plasmid DNA (and possibly carrier DNA) is then added, and after rapid mixing the samples are ready for sonication. The pulses of ultrasound are delivered by ordinary machines (e.g., BRANSON Ultrasonic Corporation, 20 kHz Digital Sonifier S-250 D) used for homogenization of various tissues. The microtip (normally tapered) is immersed 2–3 mm into the suspension and pulses of selected intensity and duration are delivered. The cells are finally transferred to fresh growth medium. Joersbo and Brunstedt (1990a) found that plasmid DNA could be introduced efficiently into sugar beet and tobacco protoplasts by a brief exposure (500–900 ms) to 20 kHz ultrasound at 0.5–1.5 W/cm2 of acoustic power. Successful transformation was evidenced by transient expression of the introduced gene for chloramphenicol acetyltransferase (CAT). Optimal transient expression was obtained at rather high plasmid DNA concentrations (40–100 mg/l) and sucrose concentrations (21–28%) in the sonication medium. In sugar beet protoplasts transient expression was reported to be 7- to 15-fold higher than the expression obtained by electroporation (Joersbo and Brunstedt, 1990b). Using almost identical ultrasonic conditions as for DNA transformation, Joersbo and Brunstedt (1990b) showed that intact virus particles (beet necrotic yellow vein virus) could be encapsulated by sugar beet protoplasts leading to high levels of infection, as measured by quantification of the amount of expressed coat protein by ELISA. At present the mechanism for acoustic permeabilization is not understood. However, two possibilities should be considered. The first regards the violent collapse of cavitation bubbles, generating high pressure and high temperature shock waves which could possibly cause localized rupture of the plasmalemma and lead to uptake of exogenous solutes followed by rees-

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Y. Liu et al. / Biotechnology Advances 24 (2006) 1–16

Table 2 Transient expression in suspension cells of tobacco and sugar beet sonicated at various acoustic powers and durations in the presence of 100 mg/l of the plasmid CaMVCN which encodes a chloramphenicol acetyltransferase (CAT) gene Acoustic power (W/cm2)

Pulse duration (ms)

Transient expression (%) Tobacco

Sugar beet

0 1.28 1.60 1.92 2.24 1.60 1.60

0 800 800 800 800 570 1000

0.03 – 0.13 – – 0.06 0.07

0.02 0.06 0.21 0.17 0.07 – –

Percentages indicate the level of acetylated chloramphenicol, which is proportional to the enzyme activity expressed by the transferred gene (Joersbo, 1990).

tablishment of membrane integrity. The second hypothetical possibility originates from the electro-mechanical model presented by Zimmermann et al. (1974) (for a review, Joersbo and Brunstedt, 1992) predicting the existence of a critical hydrostatic pressure at which the intrinsic membrane potential is sufficiently high to induce mechanical breakdown of the membrane. Reversible mechanical breakdown has been observed in human erythrocytes subjected to high pressures (60– 100 MPa) in a hyperbaric chamber (Zimmermann et al., 1980). Consequently, it is possible that either the high

oscillating pressure generated by the ultrasonic field and/or the high pressure shock waves originating from collapsing cavitation could produce such high hydrostatic pressures that reversible membrane breakdown would occur. The two above-mentioned possibilities are closely related and may act in concert. Joersbo and Brunstedt (1990b) found that infection of sonicated sugar beet protoplasts with virus particles was gradually reduced 1 h after sonication, indicating that the state of increased permeability towards macromolecules may persist for a considerable time after sonication. This is in contrast to electroporation where uptake of nucleic acids occurs almost exclusively during delivery of the electric pulse and not afterwards. Sonication has also been shown to be able to facilitate uptake of plasmid DNA into intact suspension cells of sugar beet and tobacco (see Table 2; Joersbo, 1990). Ultrasound intensity and duration were 2- to 3fold higher than for protoplast transfection. As a result of sonication the majority of the cell aggregates were dissociated into single cell or small clusters of 1–5 cells providing a quasi-single cell system. The mechanism for DNA uptake in sonicated cells remains to be established but formation of small breaks in the cell wall could possibly be involved in concert with the plasma membrane alternation hypothesized above. Choudgary and Chin (1995) have studied the effects of ultrasound on the uptake of foreign sub-

24 21 62 kHz Fluorescent protoplast (%)

18 80 kHz 15 12 9 6 3 0

0

2

4

6

8

10

Exposure time (Sec) Fig. 14. Effect of ultrasound intensity on the delivery of calcein into petunia protoplasts. Two ultrasound sources, 62 kHz and 80 kHz at 0.3 W/cm2 and 0.9 W/cm2, respectively, were used.

Y. Liu et al. / Biotechnology Advances 24 (2006) 1–16 Table 3 Transient expression of GUS with and without SAAT in various plant tissues (Trick and Finer, 1997) Plant/tissue type

SAAT GUS expressionb a duration (s) SAAT +SAAT

Soybean/immature 2 cotyledons ( AS)c Soybean/immature 2 cotyledons (+AS)c Cowpea/leaflets 60 Wheat/seedlings 100 Maize/immature 30 embryos White spruce/seedlings 50 Soybean/embryogenic 60 suspension Buckeye/embryogenic 20 suspension

0.05 F 0.16% 18.6 F 14.4% 0.44 F 0.72% 44.0 F 21.3% 0.5% 0 foci 0.6 F 0.06%

42% 28 foci 80.3 F 8.3%

0 foci 0 foci

26 foci 1807 foci

0 foci

112 foci

a

Conditions are representative and are not fully optimized. Percentage values represent the proportion of surface area expressing GUS; foci is a count of GUS-positive foci; each value F standard deviation represents the mean of 10–30 replications. c Co-cultivated without ( ) and with (+) acetosyringone (AS). b

stance by Petunia protoplasts using calcein. Treatment with ultrasound facilitated the uptake of calcein. The ultrasound uptake was dependent on sound frequency

11

Table 4 Time course of SAAT on transient expression in immature cotyledons of soybean (Trick and Finer, 1997) SAAT duration (s)

Surface area expressing GUS (%)a

0 0.2 1.0 2.0 5.0 10.0

0.6 F 1.8 24.2 F 22.6 48 F 23 67 F 35 62 F 23 79.9 F 9

a Each value represents F standard deviation represents the mean of 10–30 replications.

and duration of treatment, uptake increased with treatment time but prolong treatment caused the rupture of protoplasts (Fig. 14). It was found that the optimal treatment time was about 1 s. On the other hand, the effect of ultrasound on protoplasts integrity was also investigated. Zhang et al. (1991) reported stable transformation of tobacco by sonicating small pieces (4  8 mm) of leaf tissue. Sonication was performed at 0.5 W/cm2 for 30 min, which was approximately the same intensity as used for protoplasts, but the exposure time was 1500- to 2000-fold longer. Addition of carrier DNA and dimethylsulphoxide increased the transformation rate

Fig. 15. Transient GUS expression in immature soybean cotyledons, cowpea leaves and immature maize embryos with and without SAAT. (A) Soybean with Agrobacterium alone (bar = 1 mm). (B) Soybean with SAAT (bar = 1 mm). (C) Cowpea with Agrobacterium alone (bar = 500 Am). (D) Cowpea with SAAT (bar = 500 Am). (E) Maize with Agrobacterium alone (bar = 1 mm). (F) Maize with SAAT (bar = 1 mm).

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Fig. 16. Scanning electron micrographs of non-sonicated immature soybean cotyledons (A and C) and 5 s SAAT-treated samples with thousands of micro-wounds caused by sonication (B and D). A and B, bar = 500 Am; C and D, bar = 5 Am.

individual foci could not be distinguished (Fig. 15) and it was necessary to estimate the percent of the surface that was GUS-positive. But tissues responded to a wide range of SAAT durations (Table 4). The treatments that yielded high levels of GUS expression with a minimum SAAT duration were most desirable. As tissues differed in their response to SAAT (Table 3), the best treatment for each tissue needs to be empirically determined. In order to determine the basis for increase in transient GUS expression from SAAT, scanning electron microscopy revealed the formation of large numbers of reparable micro-wounds on the surface of the SAAT-treated tissues, while the surface of the non-treated tissue was smooth and intact (Fig. 16). Recently, many experiments have been demonstrated that SAAT tremendously improved the efficiency of Agrobacterium infection by introducing large numbers

significantly. The transferred genes were inherited in a Mendelian fashion. However, at present most researches are focused on sonication-assisted Agrobacterium-mediated transformation (SAAT) in plant cells or tissues (Horsch et al., 1985; Trick and Finer, 1997, 1998; Santare´m et al., 1998; Weber et al., 2003). SAAT is a new technology and method involves subjecting the plant tissue to brief periods of ultrasound in the presence of Agrobacterium. Trick and Finer (1997) have reported that, in all tissues tested, the SAAT treatment greatly enhanced the levels of transient expression (Table 3; Fig. 15). In most tissues, transient expression without sonication was very low, with no h-glucuronidase (GUS) expression being observed in tissues of wheat and white spruce seedlings as well as soybean and buckeye suspension cultures. With SAAT, transient expression was so high in areas of some tissues that 25 a

Transient expression (%)

a

a

20 bc

ab bc

15 10

c

5 d

0

0

0.1

0.5

1

2

5

7

10

Treatment duration (s) Fig. 17. Effect of sonication duration on the frequency of transient GUS expression of SAAT-treated immature soybean cotyledons using 0.1 OD600.

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of micro-wounds into the target plant cells or tissues (Santare´m et al., 1998; Tang, 2003). It was reported that a tremendous enhancement of GUS expression was observed when sonication was applied along with Agrobacterium (Fig. 17), treatments ranging from 0.5 s to 2 s gave the highest transient expression (Santare´m et al., 1998). In summary, at present, a number of techniques exist for transferring genes into plant protoplasts and cells. Some of the methods (e.g., electroporation, microinjection and polyethylene glycol) are confined to transformation of protoplasts because the cell wall, which is largely impassible to plasmid DNA, is generally not sufficiently permeabilized to permit uptake of DNA. As protoplasts of many species are often difficult to regenerate efficiently into fertile plants, the major asset of those methods is as analytical tools, for example, in testing functionality of gene constructs. Other direct gene transfer methods (e.g., particle bombardment) are designed for transformation of intact tissues. Transgenic plants of several species have been obtained by this technique. However, most of the methods listed require sophisticated and costly equipment which furthermore do not allow the combination of both accurate quantitative estimates of gene expression and efficient generation of transgenic plants. Ultrasound transformation is unique among existing methods because of its versatility: both plant protoplasts, cells in suspension and intact pieces of plant tissue can be readily transformed. This future, along with the fact that the equipment for ultrasound transformation is simple, cheap and multifunctional, should allow for substantial future applications.

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5. Ultrasound contrast agents (microbubbles) Encapsulated gas microbubbles are well known as ultrasound contrast agents for diagnostic imaging. Recently, the encapsulated gas microbubbles were also used as gene/drug carriers (Blomley et al., 2001). Ultrasound microbubbles consist of micron-sized gas bubbles coated with a stabilizing layer of a surfactant fluid or biocompatible solid such as denatured serum albumin. Microbubble can increase absorption of sonic energy and lower the threshold of energy for cavitation. In cavitation, ultrasound energy is concentrated into micro-domain. Cavitation creates small shock waves, which will increase cell permeability. Cavitations destroy the microbubbles and release the genes/drugs trapped within the microbubbles or coated onto the surface of the microbubbles (Fig. 18) (Unger et al., 2001a,b). Fig. 19 shows two different designs that microbubbles may entrap different DNA or drugs (Unger et al., 2002). DNA or drugs may be incorporated into the microbubbles or the surface of the microbubbles. The advantages of this approach are that when cavitation occurs, the microbubbles themselves release the DNA or drugs and cavitation optimally delivers the drug locally into the tissue. Now microbubbles are commercially produced by many companies and widely applied to increase the transfection efficiency in animal cells, such as AlbunexR, LevovistR and OptisonR (Klibanov, 1999; Miller and Song, 2002). Fig. 20 is a kind of microbubble developed by ImaRx and can effectively deliver gene to skin, lung and other tissues. For the ultrasound-assisted delivery of nucleic acids to the cells or tissues, plasmid DNA can be either added

Fig. 18. Gene delivery using ultrasound and microbubbles. The presence of gas in the gene-filled microbubble allows ultrasound energy to bpopQ the bubble. An energetic wave is then created which allows the genetic material to enter surrounding cells.

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Fig. 19. Two different designed microbubbles can transport DNA or drugs used in animal cells and tissues. DNA/drugs can be incorporated into a layer of oily material that forms a film around the microbubble, which is then surrounded by a stabilizing membrane (A); in this example, a targeting ligand is incorporated on the membrane allowing targeted delivery of the drug. DNA/drugs may also be attached to the membrane surrounding the microbubble (B).

to the cell culture medium or attached to the positively charged liposomes via a charge attraction. Treatment with ultrasound improves the transfection efficacy. The presence of microbubbles improves transfection and intracellular delivery of macromolecules even further (Bao et al., 1997). Positively charged microbubbles can be prepared in a manner similar to positively charged liposome transfection reagents. Transfection of cells with the plasmids carrying reporter genes with the aid of reagents was demonstrated in vitro even in the absence of the ultrasound field. Positively charged

particles bind plasmid DNA; they also attach efficiently to the negatively charged cell surface, and enhance DNA delivery. Further improvement of transfection efficacy in this system via insonification was demonstrated; probably, rapid vibration of microbubbles on the surface of the cells, or microstreaming, allows enhanced delivery of DNA across the cell membrane (Greenleaf et al., 1998). In the latest research it was reported that microbubbles could significantly improve the transfection efficiency even in the absence of ultrasound (Lu et al., 2003).

Fig. 20. Liquid perfluorocarbon gene carrier microbubbles (A). The outer surface is stabilized by amphipathic lipid. Targeting ligands have been incorporated onto the head groups of the lipids. The genetic material is stabilized by cationic lipids. Electron microscopy studies have shown that the DNA is condensed as an electron-dense granule within the center of the nanoparticle. The diameter of these particles is about 100–200 nm. Figure (B) is the micrograph by scanning electron microscopy.

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6. Conclusions and perspectives Ultrasound has been demonstrated to be a successful method in gene transfer for animal in vitro and in vivo, and the transfection efficiency is also high. Ultrasound as a mechanical method is often more versatile and less dependent on cell types. On the other hand, transgenic plants have significant potential in the bioproduction of complex human therapeutic proteins due to ease of genetic manipulation, lack of potential contamination with human pathogens, conservation of eukaryotic cell machinery mediating protein modification, and low cost of biomass production. Many approaches have been tried to transfer genes into plant cells or tissues. Under control conditions, ultrasound is an effective means of delivering DNA or nucleic acids into cells. The subsequent expression of DNA molecules in cells depends upon a balance between transient cell damage and cell death. Recently, ultrasound contrast agents (microbubbles) have been produced commercially, which have been shown to provide stable gas bodies that nucleate cavitational events to increase the cell membrane permeability. Microbubbles have been successfully applied to increase the transfection efficiency in animal cells. A synergistic effect is attained with the use of microbubbles and ultrasound. The concentration of microbubble as well as the exposure duration and ultrasonic intensity play a key role in transfection efficiency. Their optimal conditions are also proposed to determine by trials. References Bao S, Thrall BD, Miller DL. Transfection of a reporter plasmid into cultured cells by sonoporation in vitro. Ultrasound Med Biol 1997;23:953 – 9. Bar R. Ultrasound-enhanced bioprocesses: cholesterol oxidation by Rhodococcus erythropolis. Biotechnol Bioeng 1988;32:655 – 63. Blomley MJK, Cooke JC, Unger EC, Monaghan kJ, Cosgrove DO. Microbubble contrast agents: a new era in ultrasound. Br Med J 2001;322:1222 – 5. Bommannan D, Menon GK, Okuyama H, Elias PM, Guy RH. Sonophoresis: II Examination of the mechanism(s) of ultrasound enhanced transdermal drug delivery. Pharm Res 1992;9:1043 – 7. Choudgary ML, Chin CK. Ultrasound mediated delivery of compounds into petunia protoplasts and cells. J Plant Biochem Biotech 1995;4:37 – 9. Cramer CL, Weissenborn DL, Oishi KK, Grabau EA, Bennett S, Ponce E, et al. Bioproduction of human enzymes in transgenic tobacco. Ann N Y Acad Sci 1996;792:62 – 71. Daniell H, Streatfield SJ, Wycoff K. Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci 2001;6:219 – 26. Doktycz SJ, Suslick KS. Interparticle collisions driven by ultrasound. Science 1990;247:1067 – 9.

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