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J Am Coll Cardiol, 2005; 45:329-335, doi:10.1016/j.jacc.2004.08.067
© 2005 by the American College of Cardiology Foundation
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STATE-OF-THE-ART PAPER

Use of ultrasound contrast agents for gene or drug delivery in cardiovascular medicine

Raffi Bekeredjian, MD*, Paul A. Grayburn, MD, FACC{dagger},* and Ralph V. Shohet, MD*

* Department of Internal Medicine, Division of Cardiology, University of Texas Southwestern Medical Center, Dallas, Texas
{dagger} Baylor University Medical Center, Dallas, Texas

Manuscript received July 2, 2004; revised manuscript received August 16, 2004, accepted August 17, 2004.

* Reprint requests and correspondence: Dr. Paul A. Grayburn, Baylor Heart and Vascular Institute, 621 N. Hall Street, Suite H030, Dallas, Texas 75226 (Email: paulgr{at}baylorhealth.edu).


   Abstract
Top
 Abstract
Transport of substances by...
 Therapeutic concepts
 Adverse effects
 Summary and outlook
 References
 
The clinical utility of ultrasound contrast agents has been established in diagnostic echocardiography. Recently, the use of such agents has been promoted for transport and delivery of various bioactive substances, thus providing a technique for non-invasive gene therapy and organ-specific drug delivery. In this review, we give a critical update of published studies using ultrasound contrast agents for therapeutic use. We discuss the potential applications and limitations of this technique and suggest future applications in cardiovascular medicine.

Abbreviations and Acronyms
  DNA = deoxyribonucleic acid
  GFP = green fluorescent protein
  UTMD = ultrasound-targeted microbubble destruction
  VEGF = vascular endothelial growth factor

Ultrasound contrast agents have been used in diagnostic echocardiography for several decades. The development of newer, second-generation contrast agents that cross the pulmonary circulation has enabled the opacification of the left ventricular cavity and myocardium after intravenous injection (1–3). An important physical characteristic of such agents is that they oscillate during sonication. These oscillations can result in linear backscatter at low acoustic pressure, nonlinear signals with harmonic frequencies at medium acoustic pressure, and microbubble disintegration at high acoustic pressure (4). The destruction of microbubbles has been used in myocardial contrast echocardiography (MCE) to quantitate myocardial microperfusion using refill kinetics (5,6). Briefly, a high-power ultrasound pulse is used to destroy the microbubbles within the myocardial capillary bed, and then low-power ultrasound is used to measure the time course of replenishment of the capillary bed with microbubbles. Microbubble destruction by ultrasound has also raised questions about bioeffects in the vicinity of contrast agents. Early studies showed capillary rupture and local extravasation of red blood cells in skeletal muscle (7–9) after ultrasound-targeted microbubble destruction (UTMD). Although this phenomenon indicated that microbubble destruction can induce adverse effects, these bioeffects were seen only at much higher doses of microbubbles than are used clinically, as well as at local ultrasound pressures higher than would be expected in clinical imaging. Importantly, these studies demonstrated the potential for delivery of bioactive substances to ultrasound-accessible organs.

In recent years, several investigators have used ultrasound contrast agents as a tool for organ-specific drug and gene delivery. In this review, we give an overview of these studies and analyze the different strategies that have been used. After introducing the mechanical and chemical concepts of substance delivery with ultrasound contrast agents, we describe studies that have examined the use of UTMD for gene therapy, drug delivery, and direct mechanical bioeffects. Finally, we discuss potential risks of microbubble destruction and suggest future applications.


   Transport of substances by ultrasound contrast agents
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 Abstract
 Transport of substances by...
Therapeutic concepts
 Adverse effects
 Summary and outlook
 References
 
Most investigators who have used ultrasound contrast agents for therapeutic applications work with perfluorocarbon bubbles stabilized by an albumin or lipid shell. The advantages of this class of contrast agents include their fragility when exposed to moderate energy ultrasound and their ease of manufacture. Three concepts have been described: 1) microbubbles can be produced together with a bioactive substance, thus potentially incorporating it into the microbubble shell or lumen (10–13); 2) microbubbles can be incubated with a bioactive substance, thus attaching the substance to the microbubble shell, presumably by electrostatic or weak non-covalent interactions (14–16); or 3) microbubbles and the bioactive substance can be co-administered (8,9,17). Several considerations affect the choice of how to combine the microbubble and its bioactive component.

It is well known that deoxyribonucleic acid (DNA) is rapidly degraded after injection into the bloodstream by serum DNases (18) and removed by hepatic scavenger receptors (19). Thus, intravenous injection of plasmid DNA, even in the presence of ultrasound, does not lead to detectable transfection. Unpublished data from our own laboratory show that plasmid DNA that is incorporated within the microbubble shell (confirmed by fluorescent microscopy) is not significantly degraded by exposure to human whole blood. Therefore, it appears that DNA is protected from serum DNases if it is either attached to microbubbles or, preferably, integrated into the microbubble shell. However, the exact mechanisms of protection have not been elucidated.

Transport of a substance by microbubbles requires chemical compatibility with the microbubble shell. The most common component of microbubbles—albumin—can bind a wide range of substances, such as drugs, DNA, and virus particles (20). Cationic lipids have the advantage of binding negatively charged DNA. Thus, the use of such lipids in microbubbles has been promoted for DNA delivery (21,22).

Depending on the thermal stability of the therapeutic substance, sonication as a method to produce microbubbles may not be feasible, as temperatures reach more than 70°C. Therefore, the transport of a heat-labile virus or proteins requires either passive attachment to preformed bubbles (10,11) or lipid microbubble production by shaking (23). It is also important that sonication should not mechanically disrupt a substance. Integrity of plasmid DNA after sonication in an albumin solution has been shown to be unaltered (11).

A few studies have used ultrasound contrast agents that are not based on microbubbles, such as gas-filled poly(D,L-lactide-co-glycolide) microparticles produced with DNA/polymer complexes (24) or echogenic liposomes that are dehydrated, lyophilized, and then re-hydrated with a drug solution (25). However, ultrasound contrast agents that are not filled with gas cannot take advantage of important physical characteristics, such as oscillations, microstreaming, and microbubble disruption.

Advances in the chemistry of microbubble formulation could improve both the stability of the bubbles and their binding capacity for gene or drug delivery.

Delivery of substances by microbubble destruction.   The concept of delivering a bioactive substance by UTMD has two components. First, the transported substance can be released from the microbubbles by their destruction in the target region, thus increasing its local concentration. Second, the destruction of the bubbles may cause focal cavitation effects in the surrounding tissue that increase the permeability of biologic barriers (Fig. 1). This may be particularly important when using UTMD for gene delivery with naked DNA, because transfection efficiency across cell membranes would otherwise be very low.



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  		Figure 1 Schematic representation of substance delivery using ultrasound-targeted microbubble destruction. An ultrasound contrast agent with an attached or incorporated bioactive substance is administered into the vasculature and will distribute throughout the capillaries. Ultrasound can then destroy microbubbles in the target region, thus releasing the transported substance into the surrounding tissue.

 
Ultrasound alone has been shown to improve DNA uptake (26,27), drug (28), and protein (16) delivery into tissues without microbubbles. There are several potential mechanisms that could play a role in mediating this effect. It is known that ultrasound induces an oscillating convective motion to partially absorbing liquids (29). This might enhance diffusion of drugs or other substances in vivo. In addition, ultrasound heats tissue, which could theoretically enhance drug delivery. Finally, ultrasound can directly induce gas bubble formation and destruction (30). This process, known as cavitation, is associated with a number of secondary effects, which will be discussed.

Several studies have investigated the mechanisms for how UTMD enhances DNA delivery into cells. Electron microscopy has demonstrated pore formation on cell membranes immediately after destruction of microbubbles; this phenomenon is transient, with disappearance of the pores after 24 h (31–33). Such "sonoporation" effects may help facilitate gene or drug entry into the cell. In vitro studies on single bubbles have shown that even linear bubble oscillations are sufficient to achieve rupture of lipid membranes (34). Moreover, sudden violent collapse of microbubbles (inertial cavitation) can produce high-velocity fluid microjets that may penetrate adjacent membranes (35). In addition, inertial cavitation, which is dependent on microbubble shell composition, ultrasound frequency, pulse duration, and acoustic power (36,37), can lead to secondary shock waves (38,39), transient local high temperatures (40), and shear stress (41), all of which could potentially contribute to gene or drug delivery by UTMD.

The disruption force of microbubbles is greater when the ultrasound frequency used matches the resonant frequency of microbubbles (36,42–44). Even low acoustic pressures can result in microbubble destruction (44), but higher pressures (around 2 Mpa) will lead to more forceful reactions. A higher disruption force enhances delivery of a transported substance (42). Several studies have investigated various ultrasound modalities to identify optimal settings for highest delivery efficiencies. In the myocardium, triggered ultrasound has been shown to be superior to continuous ultrasound (44,45), presumably because the latter destroys most of the microbubbles within the cardiac chambers and the coronary arteries before they can reach the myocardial capillaries. No difference was seen between the ultraharmonic and power Doppler mode (44). However, this was investigated in small animal models that would show a very low level of ultrasound attenuation. In larger animals or humans, differences between ultraharmonic and power Doppler could be present.


   Therapeutic concepts
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 Abstract
 Transport of substances by...
 Therapeutic concepts
Adverse effects
 Summary and outlook
 References
 
Three delivery goals have been considered for UTMD. The most widely investigated application is for gene transfer/gene therapy. A second application is for drug and protein delivery. Finally, UTMD alone has been studied for therapeutic effects by itself (without any transported substance). Table 1 gives an overview of studies that have used ultrasound contrast agents to transport bioactive substances in vivo. It is evident from Table 1 that more data are currently available for delivery of genes rather than drugs or proteins.


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  		Table 1. Chronologic Overview of Studies That Have Used Ultrasound-Targeted Microbubble Destruction for Delivery of Substances to Muscle or Vessels
 
Gene transfer/gene therapy.   Increased knowledge about molecular mechanisms of diseases and physiologic regulation has raised expectations for gene therapy as a new treatment option. Despite substantial effort in cardiovascular medicine to develop and test gene therapy techniques, none has yet progressed beyond clinical studies (46). Use of UTMD provides many desirable characteristics for gene therapy, including low toxicity, low immunogenicity, low invasiveness, the potential for repeated application, organ specificity, and broad applicability to sonographically accessible targets. The main strategies for delivering a gene therapy vector by UTMD have been production of albumin microbubbles with DNA, production of neutrally charged or cationic lipid microbubbles with DNA or adenovirus, incubation of albumin microbubbles with adenovirus, and, most widely applied, incubation of DNA with Optison (GE Healthcare, Milwaukee, Wisconsin). The amount of DNA administered by UTMD in these studies varied from as little as 10 µg to as much as 2 mg. This demonstrates that further dose-finding studies with optimized microbubble compositions are still needed. Three main targets have been investigated: the myocardium, vessels, and skeletal muscle. The following section describes the most important in vivo, ex vivo, and in vitro studies (Table 1) that have been performed using UTMD for gene transfer.

The first study using UTMD for cardiac gene delivery used a cytomegalovirus beta-galactosidase–expressing adenovirus attached to albumin microbubbles (10). Although a 10-fold augmentation of cardiac beta-galactosidase expression was achieved in rats, adenoviral vectors have the disadvantage of low organ specificity (with a strong hepatic tropism) and immunogenicity that inhibits repeated applications. Subsequent studies extended this approach to the delivery of plasmid DNA to rats using sensitive reporter constructs to overcome the inefficiency that is the principal limitation of this method (42,47). In contrast to an adenoviral approach, the use of naked DNA resulted in a nearly 100% organ specificity, as DNA released into the circulation was rapidly digested by serum DNases, thus not transfecting other tissues. The temporal expression profile was investigated, showing highest transgene expression at day 4 after UTMD, with a rapid decline thereafter. Repeated treatments were able to prolong high-level luciferase expression (47). The ability to repeat the application provides the opportunity to control the duration of transgene expression, an important requirement for gene therapy of acquired diseases. Other myocardial gene transfer studies have been commenced to study biologically important treatment with antisense oligonucleotide delivery for tumor necrosis factor-alpha, resulting in reduced tumor necrosis factor-alpha production after ischemia in rats (12). There have also been initial efforts to scale this approach up to larger animals, using reporter gene delivery to canine hearts (21).

Blood vessels (either vascular walls or endothelium) seem to be obvious targets for microbubbles and ultrasound, because they are the first tissue exposed to the microbubbles. Several in vitro and in vivo studies have been performed to evaluate transfection and the physiologic response to UTMD in vessels. Cultured vascular smooth muscle cells and endothelial cells were transfected with plasmids and Optison, showing 3,000-fold higher expression than obtained with naked DNA alone (14). Porcine coronary arteries (ex vivo) could be transfected with antisense phosphorodiamidate morpholino oligomers, resulting in modest inhibition of c-myc, with the goal of reducing intimal proliferation (48). Rat carotid arteries were transfected with anti-oncogene (p53) plasmids and Optison after balloon injury, resulting in a significant reduction of intimal proliferation (49). Similarly, decoy oligodeoxynucleotides were used with Optison to reduce intimal proliferation in balloon-injured rat carotids (50). The transfection of endothelial nitric oxide synthase expression vectors to porcine carotids (ex vivo) resulted in detectable endothelial nitric oxide synthase expression and a blunted contractile response to prostaglandin (51).

Studies of UTMD in skeletal muscle are pertinent to cardiovascular treatment because of the similarity of cardiac and skeletal muscle as target tissues. Two different strategies have been described to transfect skeletal muscle. Direct injection of Optison and green fluorescent protein (GFP) encoding plasmids into the skeletal muscle with ultrasound application increased GFP expression compared with intramuscular naked plasmid injection alone and, at the same time, reduced muscle damage (in vivo) (52). This study also demonstrated an enhanced transfection of DNA by microbubbles alone (without ultrasound), although the mechanism for this finding was not elucidated. In a second approach, intravascular infusion of cytomegalovirus-luciferase encoding plasmids bound to cationic microbubbles with ultrasound was able to achieve luciferase expression in rat skeletal muscle, with intra-arterial application more efficient than intravenous infusion (22). Taniyama et al. (53) demonstrated increased capillary density in rabbit skeletal muscle using hepatocyte growth factor plasmid combined with Optison.

A few studies have even attempted to use UTMD to perform embryonic or fetal transfections. Chick embryos were transfected with direct injection of Optison and plasmids (beta-galactosidase, GFP, and the important developmental gene Sonic Hedgehog), together with ultrasound application (54). Reporter gene expression was identified and digit malformation was seen with Sonic Hedgehog (54). Another study used UTMD for fetal gene transfer in mice. Optison and plasmids (beta-galactosidase and GFP) were injected either intra-amniotically or into the peritoneum of the fetus. Gene expression in fetal tissues was 1,000-fold higher compared with injection naked DNA alone. Various organs could be transfected, including the brain, lung, heart, gut, and skin (55). These studies demonstrate the potential utility of this technique as a tool in studies of developmental biology.

Despite the promising results in gene transfer mediated by UTMD, its major limitation has been low transfection efficiency. Most studies have compared the gene expression after UTMD with application of naked plasmid DNA alone. In many studies, transgene expression was substantially higher (up to several 1,000-fold) compared with naked plasmid administration. However, considering the generally low transfection efficiency with naked DNA alone, this result still does establish that gene transfer efficiency will suffice for clinical applications. Several questions still have to be addressed. How many cells can be transfected in an organ? Is a beneficial physiologic response possible (such as angiogenesis, amelioration of heart failure, or modification of genetic defects)? And, can similar results be achieved in larger animals?

Protein and drug delivery.   The rationale for using UTMD for drug and protein delivery is based on the need for organ-specific pharmacotherapy. It is likely that many potent drugs with severe adverse effects may be used more beneficially if local concentrations could be increased while keeping systemic concentrations low. For example, this could be of great advantage in angiogenesis therapy for ischemic myocardium. A handful of studies have examined the potential of UTMD for such applications. Vascular endothelial growth factor (VEGF) bound to albumin microbubbles was delivered to the heart using UTMD. A 13-fold augmentation of cardiac VEGF uptake was seen compared with systemic VEGF administration (16). A study using lipid microbubbles with luciferase protein demonstrated a six- to seven-fold augmented cardiac uptake of luciferase compared with systemic administration (23). In vitro studies have shown that ultrasound contrast agents can also be used to deliver an antibiotic (25) or a radionuclide (56).

Direct therapeutic effects of microbubble destruction.   The mechanical effect of microbubble destruction has been promoted as potentially therapeutic, even without delivering a bioactive substance. Studies have shown that UTMD can mechanically declot thrombosed dialysis grafts in dogs (57,58). Microbubbles were able to further enhance thrombolysis in combination with a thrombolytic drug such as urokinase (59,60) or tissue plasminogen activator (61).

It has been suggested that UTMD in skeletal muscle can induce arteriogenesis and could therefore potentially be used for treatment of peripheral ischemia (62). In general, these applications of UTMD use higher energies of ultrasound than those used for diagnostic procedures or for typical bubble destruction for delivery of substances. Careful evaluation of potential tissue damage with these approaches will be required.


   Adverse effects
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 Abstract
 Transport of substances by...
 Therapeutic concepts
 Adverse effects
Summary and outlook
 References
 
If the destruction of microbubbles is able to create pores in cell membranes and deliver substances into tissues and cells, it is also possible to create cell and tissue damage that might outweigh the benefits of this technique. Many studies have been performed to investigate the risks of UTMD. The most common finding was capillary rupture and hemorrhage or dye extravasation (7,45,63,64). However, most of these studies were performed ex vivo, with bolus injections of microbubbles or with microbubble concentrations far higher than those used clinically. Other studies have not seen relevant adverse effects during in vivo applications of UTMD when using continuous microbubble infusions (65–67). It appears that the risks with this technique will be closely related to the concentration of the ultrasound contrast agent, the duration of the ultrasound application, and the ultrasound pressure amplitude in the tissue. It is not clear whether underlying disease processes might render specific tissues or organs more susceptible to the bioeffects of UTMD. It is likely that careful titration of all these factors will be necessary to keep the potential adverse effects low and have a maximum therapeutic benefit. However, further investigation, especially in larger animal models, is needed.


   Summary and outlook
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 Abstract
 Transport of substances by...
 Therapeutic concepts
 Adverse effects
 Summary and outlook
References
 
Therapeutic use of ultrasound contrast agents is an emerging technique with potential for a variety of targets. The most widely used application is directing gene expression to ultrasound-accessible organs. The method of UTMD has proven to be feasible for transport and delivery of plasmid DNA. It combines many favorable characteristics for gene therapy, including minimal invasiveness, organ specificity, low toxicity, no immunogenicity, repeatable applicability, and low costs. However, the limiting aspect remains poor efficiency. There has been inadequate quantitation of efficiency in many proof-of-principle studies, and it remains unknown how the results in rodent experiments will apply to larger animals. It does appear that technical improvements are necessary to achieve clinically relevant therapeutic efficiency. Despite this limitation, the potential of this technique is high, because there is still room for substantial optimization of microbubble composition (for maximal transport capacity of a given substance) and ultrasound application (such as a three-dimensional ultrasound field, which can transfect a whole organ instead of a slice). Greater collaboration among membrane chemists, ultrasound engineers, and biologists is required for further progress. Besides the options in gene therapy, drug and protein delivery as well as direct mechanical effects of microbubble destruction have exciting potential for future applications. Here again, efficiency will have to be improved and optimized.

Conclusions.   Ultrasound contrast agents may become an important therapeutic tool for targeted treatments. This application has grown out of contrast echocardiography and, at least in the first studies discussed here, shows great promise for cardiovascular applications.


   Footnotes
 
Dr. Grayburn was supported by a research grant from the National Institutes of Health (NIH), Bristol-Myers Squibb, and Point Biomedical. Dr. Shohet received a research grant from the NIH, the LeDucq Foundation, and the American Heart Association (AHA). Dr. Bekeredjian held a postdoctoral fellowship grant from the AHA, Texas Affiliate.


   References
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 Adverse effects
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