1 Introduction

Medical ultrasound imaging has been firmly established as a critical diagnostic tool due to its real-time capabilities, portability, safety, and functional imaging capabilities. These features are prominent advantages over other imaging modalities, including magnetic resonance imaging (MRI) and computer tomography (CT), making ultrasound imaging the second most popular imaging modality in the medical market. According to a survey in 2015, the world-wide market for ultrasound imaging devices will reach approximately 8 billion dollars by the year 2020 [1]. The major clinical applications of ultrasound imaging are cardiology (abdominal, thyroid, heart, and blood vessel-related) and obstetrics, mainly for the diagnosis of various diseases including cancers. Due to technical advancements, ultrasound has recently drawn attention in the fields of brain imaging [2, 3], fusion imaging [4,5,6,7,8], cellular biophysics [9,10,11], and theragnosis [12,13,14,15]. Contrast agents are a big part of this trend as they allow for maximizing the usefulness of ultrasound. Among the various contrast agents available, microbubbles have been developed for contrast enhancement of ultrasound imaging and theragnosis [16].

Microbubbles generally consist of a shell that surrounds a core gas. Materials that often comprise microbubble shells include lipids [17], proteins [18], and polymers [19]. Air [20], nitrogen [21], and perfluorocarbon [22] are typically used as the core gas (Fig. 1). To increase the contrast resolution of an ultrasound image, microbubbles exposed to a certain frequency experience a resonance phenomenon [23,24,25,26]. The resonant frequency of microbubbles with diameters of 1–7 μm lies within the 2–15 MHz range, and this is the ultrasound frequency range used in clinical diagnosis [27]. When used as a contrast agent, microbubbles can be detected by an ultrasound device when they possess acoustical characteristics that differ from the surrounding medium. The microbubbles act as echo-enhancers by essentially the same mechanism of echo-scattering found in all other cases of diagnostic ultrasound: by increasing ultrasound impedance such that there is a mismatch between the microbubbles and the surrounding tissues.

Fig. 1
figure 1

Schematic illustration of a microbubble. a Representative illustration of a lipid shell, b representative illustration of a protein shell, c representative illustration of a polymer shell. Various kinds of gas (ex. perfluorocarbon, nitrogen, air) can be used in various kind of shell

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Theragnostic applications are achieved through the mechanisms of cavitation [28] and sonoporation [29], which are the combined effects of ultrasound and microbubbles. This will be discussed in detail in later sections. Sonoporation refers to a technique capable of controlling drug delivery efficiency by maximizing the drug permeability of surrounding tissues or cells using a reaction mechanism between microbubbles and ultrasonic waves [30, 31]. In addition, it is possible to not only deliver drugs together with very high efficiency, but also to minimize the adverse effects via time- and site-specific therapeutic strategies. Furthermore, the microbubbles not only have a therapeutic effect when combined with ultrasound as mentioned above, but also can themselves act as the drug delivery vehicles [32,33,34]. For example, for microbubbles composed of lipids, hydrophobic anticancer drugs can be very efficiently loaded onto the hydrophobic tail of the lipid found between the gas core and the shell [35]. Thus, the loaded drug can be safely delivered to the target tissue without being attacked by various enzymes present in the blood and can then be released in response to ultrasonic waves as an external stimulus-triggered drug release strategy [36].

In this review, we introduce the basic principles of microbubbles as ultrasound contrast agents and provide a brief description of commercialized products, as well as research on and applications of microbubbles and ultrasound for the treatment of various diseases. In particular, we focus on the study of microbubbles used in the treatment of various diseases and discuss the limitations and development directions of the present microbubble technology.

2 Commerical ultrasound contrast agents

Microbubbles with a size of 1–10 µm are common ultrasound contrast agents, and many kinds of microbubbles have been developed for use in ultrasound imaging and as a medical treatment technique [37, 38]. Microbubbles consist of a shell and a gas core. As technology gradually develops, the creation of small microbubbles with uniform size became possible [39]. For example, small or highly elastic microbubbles can pass through capillary vessels, thereby becoming able to circulate to the entire body and possibly reach the target organs [40]. For this reason, many studies have tested several shell materials and core gases with high molecular weights and low solubilities. Shell materials like proteins, lipids, and polymers disperse in an aqueous solution with a surfactant, or a combination of two substances can be used for stability [41]. A representative sample of commercially used microbubbles including Levovist®, Definity®, Optison®, Sonazoid® and SonoVue® [42,43,44,45,46] are listed in the order of discussion in the following subsections. The physical properties of these microbubbles are also summarized in Table 1.

Table 1 Type and physical properties of ultrasound contrast agents
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2.1 Levovist®

Levovist microbubbles are the first generation of ultrasound contrast agents. They have a shell composed of 99.9% galactose, which is biocompatible, and a core of 0.1% palmitic acid mixed with air [47]. Levovist microbubbles have harmonic signals that enhance ultrasound imaging by decreasing noise signals. These microbubbles can only be destroyed during high acoustic power harmonic imaging. So, a transducer with low acoustic power is somewhat inadequate for use with Levovist microbubbles [48].

2.2 Optison®

Optison microbubbles consist of an octafluoropropane core covered by an outer protein shell composed of human serum albumin. Together with galactose, microbubbles with albumin shells have been consistently studied because of their stability in coronary blood flow or systemic hemodynamics [49,50,51]. Although albumin-based microbubbles are phagocytosed by Kupffer cells, which are a component of the liver, they still retain their acoustic properties. Kupffer cells phagocytose contrast agents, and the imaging difference is so clearly evident between normal tissue and cancer tissue that lesion location can be precisely identified [49, 52]. This phenomenon enables continuous imaging during and after the microbubbles circulate throughout the entire body [53].

2.3 SonoVue®

SonoVue microbubbles consist of an SF6 core covered by a phospholipidic monolayer outer shell, which has a low solubility [54, 55]. SonoVue microbubbles can be restored after lyophilization and has low cytophagy property to peripheral cells. Above all, SonoVue microbubbles are stable in the presence of several surfactants, such as polyethylene glycol, phospholipids, and palmitic acid, and can be maintained in the vial for at least 6 h [56]. Furthermore, because SF6 has a low solubility, SonoVue microbubbles have high and prolonged stability in the peripheral blood, which improves harmonic behavior at low acoustic power [57].

2.4 Definity®

Definity microbubbles consist of an octafluoropropane core covered by a shell composed of various lipids. This product is currently distributed mainly in Europe and North America. The use of octafluoropropane facilitates the use of low acoustic power modes. Additionally, due to the use of several lipids in the shell, these microbubbles are highly stable when exposed to ultrasound [58].

2.5 Sonazoid®

Sonazoid microbubbles consist of a perfluorobutane core covered by a lipid shell. Perfluorocarbon has low reactivity with other molecules because of its strong carbon–fluorine bonding. Sonazoid microbubbles are stable for a longer time and have fewer side effects compared to other microbubbles [52]. Additionally, Sonazoid has been used for the detection of focal liver lesions and can diagnosis malignant tumors in the liver [59]. Similar to the Optison microbubbles, Sonazoid microbubbles are also used with Kupffer cells.

3 Theory of ultrasound imaging with microbubbles

Microbubbles were invented for use as an ultrasound contrast agent to enhance image resolution to distinguish vessels clearly and minimize noise and background signals (Fig. 2) [60]. In this section, we will review the theory of microbubbles within the framework of ultrasound imaging.

Fig. 2
figure 2

Ultrasound images using microbubbles a in vitro images of microbubbles prepared via a microfluidic system and b in vivo images of a tumor before and after injection of microbubbles

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3.1 Ultrasound imaging with microbubbles

Since Gramiak and Shah discovered that an ultrasound signal is altered when passing through the saline that is injected into the micro-vessels [61], microbubbles have been studied as a way to enhance ultrasonic signals. A shelled microbubble resonates in response to a transmitted acoustic wave, and its linear resonant frequency f r is determined by its radius r, the density of the surrounding medium ρ, the equilibrium pressure inside the microbubble \( p_{e} \), the polytropic index of gas κ, the shear modulus of the shell \( G_{s} \), and instantaneous shell thickness \( d_{s} \) as follows as follows [62].

$$ f_{r} = \frac{1}{2\pi r}\sqrt {\frac{{3\kappa p_{e} + 12G_{s} \frac{{d_{s} }}{r}}}{\rho }} $$
(1)

Equation (1) indicates that the characteristics of the shell influence the resonant frequency of a microbubble although the resonant frequency is typically inversely proportional to the size of the microbubble. In general, the resonant frequency of encapsulated microbubbles is higher than that of gas bubbles at a given size. Note that an ultrasound transducer with an operating frequency similar to the resonant frequency of microbubbles is generally used in clinics to maximize the energy of echoes from the microbubbles. As the amplitude of the transmitted ultrasound wave increases, the microbubble nonlinearly responds; it expands well in response to the rarefactional pressure of the ultrasound wave but then contracts poorly in response to the wave’s compressional pressure. As such, the microbubble generates a harmonic frequency components of transmitted ultrasound and their amplitude is considerably higher than that of the harmonic frequencies produced by the tissues when imaged. When the acoustic pressure exceeds some threshold, the microbubble bursts. This is called the cavitation effect. This threshold is associated with the amplitude of peak rarefactional pressure generated by an ultrasound transducer.

Due to the high echogenicity of microbubbles even under low MI, contrast-enhanced ultrasound images can be acquired when microbubbles are used. After microbubbles are administered to a blood vessel by intravenous injection, they are diffused to blood vessels and organs. Thus, it is possible to acquire contrast-enhanced ultrasound images of blood vessels and organs (e.g., the heart). Therefore, with this imaging technique, the accuracy with which cancer [63], metastasis [64], and cardiovascular diseases [65] are diagnosed can be improved. Additionally, the nonlinear behavior of microbubbles (i.e., generation of harmonic frequency components) can be used to increase the spatial resolution of ultrasound images; the center frequency and spectral bandwidth of ultrasound are the major factors determining the spatial resolution. The harmonic components generated by microbubbles have higher frequency than incident ultrasound (or their resonant frequency). For high spatial resolution images, therefore, one of harmonic components is extracted from the echoes received by an ultrasound transducer and used to construct an image, called ultrasound harmonic imaging. In fact, this harmonic imaging technique was developed to simultaneously improve both spatial and contrast resolutions of tissue images by using the nonlinear behavior of ultrasound in tissue [66, 67]. However, the improvement of image quality by the harmonic imaging technique is limited in the case of general tissue imaging because only the second harmonic component is available to construct ultrasound images of the tissues. This is because the other harmonic components induced in the tissue have a very low amplitude. In contrast, because the amplitude of the third or even higher harmonic components generated by microbubbles is high enough for ultrasound imaging, super-harmonic imaging [68] and second harmonic imaging are viable [69].

Recently, it has been reported that the ultrafast ultrasound imaging method using plane waves is capable of detecting the slow movement of microbubbles with sub-wavelength size [3]. Since the frame rate of the plane wave imaging method is higher than 500 frames per second, each frame can contain the information about microbubble displacement smaller than the wavelength and displacement vectors are obtained by comparing the positions of microbubbles in adjacent frame images. Velocity vectors corresponding to the speed of blood in vessels can be calculated from the displacement vectors based on the prior knowledge of the time interval between the image frames. The velocity map provides a super-resolution image of blood vessel as well as information about blood velocity in the vessel.

4 Theory of therapeutic effects of microbubbles

Microbubbles have also recently been recognized for their therapeutic abilities. These attractive characteristics led many researchers to study the mechanism of microbubbles and use them in their research. In this section, underlying theories of ultrasound and microbubbles as therapeutic agents will be reviewed [70].

4.1 Cavitation

One of the most attractive capabilities of ultrasound-mediated therapy is the enhancement of the cell membrane’s permeability, which is induced by microbubble cavitation (Fig. 3) [32]. In general, the cavitation of microbubbles by ultrasonic waves is a phenomenon involving both oscillation and destruction, thereby forming jet streaming and physical effects on surrounding tissues. However, here we will focus only on aspects related to the therapeutic effect of ultrasound-induced microbubble cavitation. Especially, it is classified into stable and inertial cavitation due to the degree of deformation according to ultrasonic intensity applied to the microbubble. Stable cavitation induces a pushing and pulling of the cell membrane that generates microstreaming near the adjacent cells, resulting in increased permeability of the cell membrane. On the other hand, inertial cavitation can induce collapse of the microbubble and jet streaming towards the cell membrane, which can make temporary pores in the cell membrane [28, 29, 71]. Furthermore, when a fluid with gas bubbles is irradiated by an acoustic field and the gas bubbles are under the primary radiation force experienced by single particles, microbubbles can be transported across the cell membrane. The application of cavitation as a therapeutic strategy will be discussed in the following section.

Fig. 3
figure 3

The cavitation of a microbubble according to incident acoustic pressure

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4.2 Sonoporation effect

When a microbubble collapses via inertial cavitation, surrounding cell or tissue membranes are ruptured due to the generated jet streaming and shock wave (Fig. 4) [29, 72]. Many researchers have investigated the mechanism of sonoporation [73, 74], though the exact mechanism is still unclear. Recently, Fan et al. [75] reviewed potential mechanisms of sonoporation classified by several acoustic properties. Sonoporation allows the microbubble itself to behave as a therapeutic agent and to deliver therapeutic agents to the target cells or blood vessels at the site of ultrasound irradiation by generating temporary pores in cell membranes [76]. It is a temporary phenomenon and can extravasate large molecules such as DNA so that they pass through extravascular tissue [77]. Using the sonoporation effect of microbubbles for disease therapy has many advantages. Injecting microbubbles intravenously can minimize drug side effects that may cause nonspecific delivery of drugs into healthy organs because the microbubbles only collapse in specific diseased areas due to focused ultrasound irradiation. Furthermore, this technique can improve the bioavailability of drugs due to effective drug delivery, thereby requiring a lower dosage [78].

Fig. 4
figure 4

Therapeutic use of the sonoporation effect of microbubble

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4.3 Studies on factors affecting sonoporation effect

Kooiman et al. [79] have demonstrated that microbubbles that are conjugated with antibodies, referred to as targeted microbubbles, amplify the sonoporation effect under relatively low peak negative acoustic pressures. In the example of epithelial cancer, microbubbles become attached to the cell membrane due to the affinity between the antibody and CD31, which is expressed on the surface of all endothelial cancer cells. This results in an improved sonoporation effect on the target cells compared to those in a control group. As a result, 30, 20, and 83% of the cells had taken up fluorescent dye at 80, 120, and 200 kPa, respectively. This result also demonstrates that the peak negative acoustic pressure also affects the sonoporation effect when delivering therapeutic agents. Another group, Karshafian et al., showed similar results regarding how the sonoporation effect is influenced by the peak negative pressure [80]. Under ultrasound irradiation, cells accommodate various molecule sizes depending on the peak negative pressure. They showed that the sonoporation effect of Definity® and Optison® microbubbles causes changes in the ability of the cell to uptake FITC-dextran with different molecular weights according to different peak negative pressures. These results showed that larger-sized FITC-dextran (from 10 kDa to 2 MDa) can enter the cells when the acoustic pressure increases from 125 to 570 kPa. Additionally, another factor affecting sonoporation has also been proposed. Liao et al. showed that the evaluations of the penetration depth was studied with microbubbles of different sizes (1.4–3.5 μm). As a result, when exposing the ultrasound of the same intensity, larger microbubbles result in more uniform penetration depths, which demonstrates that large microbubbles can increase the effective drug delivery and improve the sonoporation effect [81].

5 Therapeutic applications of microbubbles

Microbubbles are also used in therapeutic applications based on the intrinsic sonoporation ability of the encapsulated drugs. In this section, various therapeutic applications of microbubbles are summarized, organized by target tissues; blood–brain barrier, cancers, cardiovascular system. First, cavitation and sonoporation are used for overcoming biological drug delivery barriers, such as the blood–brain barrier. Next, several studies on microbubble sonoporation effects as cancers and cardiovascular disease therapy will be reviewed.

5.1 Blood–brain barrier

The blood–brain barrier (BBB) is a physical barrier composed of endothelial cells linked by tight junctions. The BBB selectively transports several molecules into the central nervous system (CNS) and protects the brain from external substances, such as toxic compounds or pathogens [82]. To achieve effective drug delivery, it is important to loosen the tight junctions between the epithelial cells. Recently, microbubbles coupled with ultrasound exposure have been studied for opening the BBB and delivering drugs [36, 83, 84]. Hynynen et al. [85] showed that local and reversible BBB disruption by non-invasive FUS results in focal disruption of the BBB, necessitating future research on the brain and diagnostic and therapeutic effects. Also, Konofagou et al. showed that local and reversible BBB opening by FUS in non-human primates (NHP). Microbubble’s diffusion patterns revealed important information of the FUS-induced BBB opening following the patterns of the underlying brain structures [86]. Stable cavitation is thought to be the major mechanism of opening the BBB. Mechanical and shear stress, generated by stable cavitation of microbubbles, increases the permeability of blood vessel walls in the brain, resulting in the reduction of tight junctions between the vascular endothelial cells. Furthermore, microbubbles under ultrasound exposure affect the efflux transporters in the BBB, which act as a functional barrier [87]. In a study of the BBB in rat brains, Cho et al. revealed a correlation between P-gp expression and the opening of the BBB. Although there are some limitations, such as the time difference between P-gp fluorescence imaging and ultrasound irradiation, the results showed that ultrasound and microbubbles inhibited expression of P-gp. Although the exact mechanism was not demonstrated in this study, it suggests that ultrasound and microbubbles can potentially be used to control the drug efflux ability of the BBB.

5.2 Cancers

Since the applicability of ultrasound contrast agents has been spotlighted, ultrasound contrast agents has attracted great attention in cancer diagnostic and therapy. Ultrasound contrast agents including microbubbles with lipid-, protein-, and polymer-based shells have been developed and also used as drug loaded-therapeutic agent by many groups. These microbubbles were used as ultrasound-guided cancer therapy. For examples, numerous studies have reported that ultrasound-guided cancer therapy using microbubbles has high potentials to enhance the therapeutic effects on lethal cancer types such as breast cancer [88], liver cancer [89,90,91], and pancreatic cancer [92, 93].

Sorace et al. [94] developed contrast-enhanced ultrasound and targeted microbubble for assessing the early tumor response to antiangiogenic therapy in breast cancer. To visualize molecular US imaging of angiogenic markers in breast cancer, multitargeted microbubbles were fabricated. Antibodies against mouse αvβ3 integrin, P-selectin, and VEGFR2 were conjugated to the surface of microbubbles. In this research, multitargeted microbubbles showed that the evaluation and assessment of the early response to antiangiogenic treatment can be utilized in in vivo breast cancer animal model.

In addition, other research groups studied the use of nanomedicine technology combined with ultrasound-mediated microbubble destruction techniques for cancer therapy. Among them, Bai et al. [88] showed that the enhanced therapeutic effect of Adriamycin on multidrug resistant breast cancer was achieved by combining siRNA silencing ABCG2-loaded mPEG-PLGA-PLL nanoparticles (PEAL NPs) and ultrasound-targeted microbubble destruction. Multidrug resistance, the principal mechanism by which many cancers develop resistance to chemotherapy, is induced by a novel protein, ABCG2 [95], which is a member of the ATP-binding cassette (ABC) transporter family [96]. To overcome the drug resistance obstacle in breast cancer therapy, Bai M et al. fabricated the siRNA silencing ABCG2-loaded mPEG-PLGA-PLL nanoparticles (PEAL NPs) and then combined these developed nanoparticles with ultrasound-targeted microbubble destruction (UTMD). Microbubbles facilitate the enhanced penetration of PEAL NPs into the multidrug resistant breast cancer cells. Therefore, more anti-ABCG2 siRNA, dissociated from PEAL NPs, was accumulated in the cell’s cytosol and showed an enhanced gene silencing effect compared to PEAL NP without UTMD. This resulted in increased Adriamycin accumulation in multidrug resistant breast cancer cells. Ultimately, this therapeutic strategy exhibited effective, feasible inhibition of drug resistance in breast cancer.

The followings are examples of pancreatic cancer. Kotopoulis et al. [92] showed that a custom-made ultrasound transducer has been developed to induce the sonoporation effect in pre-clinical studies and to create a bioluminescent model of pancreatic adenocarcinoma (PA). The aim of this study was to increase the delivery of gemcitabine to pancreatic tumors by confirming the effect of sonoporation for local cancer therapy in MIA PaCa-2 human pancreatic cancer cells via the inoculated mice model. The research showed in detail that a custom-made single-element ultrasound transducer can validate the beam profile, with a focus that is 4.0 mm in diameter and 22.0 mm in length to induce sonoporation. The therapeutic transducer can control the sound field with high precision in the focused treatment area. In this research, the tumors of inoculated mice (n = 10) were measured using 3D ultrasound and bioluminescent imaging to evaluate the safety and efficacy of sonoporation of gemcitabine combined with microbubbles. As a result, the combined treatment group showed a significant therapeutic effect compared to the control (untreated) and gemcitabine groups. Based on these results, ultrasound-guided cancer therapy with microbubbles exhibited remarkable therapeutic efficacy.

Moreover, the Du research group used combined nanomedicine and ultrasound-mediated microbubble destruction techniques to enhance the therapeutic effect on pancreatic cancer [93]. Due to the limitations of conventional chemotherapeutic treatment, pancreatic cancer is considered one of the most lethal human malignancies among numerous cancers. This research showed that pancreatic cancer-targeting, three-block copolymer methoxy polyethylene glycol-polylacticco-glycolic acid-polylysine (mPEG-PLGA-PLL) NPs, which were modified with anti CA19-9 antibodies and encapsulated paclitaxel (PTX) and combined with ultrasound-mediated microbubble destruction (UMMD) significantly increased the cellular uptake in vitro and drug retention in vivo, suggesting a promising strategy for cancer therapy. Their study demonstrated that UMMD technology could significantly increase cellular uptake in vitro and enhance the EPR effect in vivo, thereby improving the therapeutic efficacy of pancreatic cancer therapy.

In addition, liver cancer is also one of the major target tumors for treatment with microbubbles and ultrasound. As in the previous case of breast and pancreatic cancers, a strategy referred to as UTMD or UMMD can be equally applied to liver cancer [91].

5.3 Cardiovascular system

Stroke and myocardial infarction are among the most common causes of death [97, 98]. One of the first therapeutic strategies developed using ultrasound and microbubbles was targeted at treatment of these cardiovascular diseases. One major therapeutic strategy targeted at the cardiovascular system is sonothrombolysis, whose underlying principle is based on cavitation effects [99]. Sonothrombolysis has been studied as a treatment for both acute ischemic stroke and acute myocardial infarction. Although ultrasound itself is well known to increase the dissolution rate of clots because elimination of the no-reflow region in the circulatory bed results in the recovery of blood flow, microbubbles can be incorporated into cardiovascular-therapeutic applications of ultrasound for increased efficiency via the mechanisms of cavitation and sonoporation.

In sonothrombolysis, the thrombus, which causes cardiovascular diseases, is one of the main targets. Fibrinolytic drugs, such as streptokinase, which is one of the plasminogen activators (PAs), are used in the treatment of thrombi. However, PAs can induce side effects (e.g., bleeding complications and neurotoxicity) during pharmacological therapy of thrombi, and a rapid, non-invasive imaging technology is not available. Wang et al. [100] investigated a novel technology in which thrombus-targeting microbubbles, which have fibrinolytic drugs on the surface, can overcome this problem. Specifically, anti-GPIIb/IIIa single-chain antibodies, which bind specifically to the main fibrinogen/fibrin receptor-mediating platelet aggregation, and single-chain urokinase plasminogen activators are conjugated onto the microbubble for enhanced and localized delivery of therapeutic agents. Using this technique, they reduced the size of the thrombus via ultrasound imaging without prolonging bleeding time.

Additionally, Hagisawa et al. [101] demonstrated significant improvements in ultrasonic thrombus disruption in vitro and in vivo via thrombus-targeted perfluorocarbon-bubble liposomes (BLs) and an external low-frequency ultrasound system. Thus, it is highly possible that a non-invasive therapy combining low-frequency ultrasound with thrombus-targeted BLs could be developed. Consequently, sonothrombolysis and drug delivery methods have been reported to recanalize canine iliofemoral and coronary arteries without tissue damage. These studies demonstrate that clinically available diagnostic low-frequency ultrasound systems can be used for thrombus dissolution with thrombus-targeted microbubbles.

6 Limitations of microbubbles

Microbubbles take center stage in ultrasound imaging and therapy because of their sensitive contrast and therapeutic efficiency. However, stability limitations, caused by diffusion of the core gas across the shell, still exist. Many researchers have tried to overcome this limitation using mixed materials, such as PEG, for shells or by blending core gases such as nitrogen and perfluorocarbon [102]. Improved shell stability can substantially enhance microbubble functionality and in vivo therapeutic application strategies. Furthermore, the large size distribution of microbubbles presents a limitation for utilizing advanced ultrasound imaging via resonant frequency properties. Due to the large size distribution of microbubbles, a weak subharmonic response is induced at high frequency, resulting in subharmonic imaging with a low sensitivity [103]. Another limitation of the microbubble is the scale of its size when used in drug delivery to tumor tissues. The micrometric proportions of microbubbles limit their ability to penetrate through the intercellular junctions. Consequently, most microbubble applications are limited to blood vessels.

7 Future perspective of microbubbles

Currently, microbubble size is delicately controlled via several methods, including centrifugation [104] and microfluidics [105]. Microbubble size is a significant property that merits future research (e.g., resonance frequency [106]). If research on size-dependent properties is broadened, subsequent mechanistic studies will follow. Furthermore, to overcome the limitation about microbubble size, many researchers have developed gas-generating or evaporation strategies, in which nanometer-scale particles or bubbles containing liquid can evaporate under biological conditions or as an external stimuli, such as a laser, increases temperature [107]. Min et al. [108] have constructed doxorubicin-loaded calcium carbonate (CaCO3) hybrid nanoparticles (DOX-CaCO3-MNPs) through a block copolymer templated in situ with the mineralization approach. This nanoparticle can generate CO2 bubbles at weak acidic tumoral pH (pH 6.8-7.2) levels and release loaded drugs into tumor tissues. Consequently, passive tumor targeting is expected due to the enhanced permeability and retention (EPR) effect of nanoparticles, resulting in efficient drug delivery to tumor sites and subsequent ultrasound imaging by the generated gas. In this review, we focused on the therapeutic application through the sonoporation effect of microbubbles, but studies on the functionalization of microbubbles, such as the above example, are also actively conducted. These studies such as coating microbubbles with nanoparticles [109], US/MR dual-modal microbubbles [19], microbubbles for targeted imaging [110] can enhance the potential to overcome the limitations of microbubbles and extend their applications.

8 Conclusion

Overall, microbubbles, as functional ultrasound contrast agents, have enormous potential for use in advanced ultrasound imaging and therapeutic strategies. Beyond the currently used basic ultrasound contrast agents, many researchers have developed functionalized microbubbles for specific molecule-targeted imaging and therapeutic use in the treatment of various diseases. Recent studies on the combined effects of ultrasound and microbubbles have shown positive results for clinical translation. Even considering the limitations mentioned in the previous section, it is highly expected that advanced next-generation microbubbles will be used clinically in the near future.