Ultrasound technology for molecular imaging: From contrast agents to

Review Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Ultrasound Technology for Molecular Imaging: From Contrast Agents to Multimodal Imaging Yue Li,‡ Yuhao Chen,‡ Meng Du,‡ and Zhi-Yi Chen*,‡ ‡

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Department of Ultrasound Medicine, Laboratory of Ultrasound Molecular Imaging, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou 510150, People’s Republic of China ABSTRACT: Ultrasound (US) takes advantage of ultrasound contrast agents (UCAs) to further increase the sensitivity and specificity of monitoring at the cellular level, which has had a considerable effect on the modern molecular imaging field. Gas-filled microbubbles (MBs) as UCAs in the bloodstream generate resonant volumetric oscillations in response to rapid variations in acoustic pressure, which are related to both the acoustic parameters of applied ultrasound and the physicochemical properties of the contrast agents. Nanoscale UCAs have been developed and have attracted much attention due to their utility in detecting extravascular lesions. Ultrasound molecular assessment is achieved by binding disease-specific ligands to the surface of UCAs, which have been designed to target tissue biomarkers in the area of interest, such as blood vessels, inflammation, or thrombosis. Additionally, the development of multimodal imaging technology is conducive for integration of the advantages of various imaging techniques to acquire additional diagnostic information. In this review paper, the present status and the critical issues for developing ultrasound contrast agents and multimodal imaging applications are described. Conventional MB UCAs are first introduced, including their research material, diagnostic applications, and intrinsic limitations. Then, recent progress in developing targeted UCAs and phase-inversion contrast agents for diagnostic purposes is discussed. Finally, we review the present status and the critical issues for developing ultrasound-based multimodal imaging applications and summarize the existing challenges and future prospects. KEYWORDS: ultrasound, molecular imaging, contrast agent, microbubbles, multimodal imaging

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INTRODUCTION Ultrasound molecular imaging is a discipline that combines ultrasound medicine, molecular imaging, material science, and nanotechnology with the unique advantages of noninvasiveness, precision and repeatability. Echogenic particles for ultrasound, also known as ultrasound contrast agents (UCAs), are a class of imaging agents that can significantly enhance the echoing signals in detection, improve the quality of ultrasound images, and increase the sensitivity and specificity of ultrasound diagnosis.1 Conventional ultrasound imaging is enhanced by the gas-filled microbubbles (MBs). These echogenic particles are distributed in the body with the blood flow and generate linear backscatter and nonlinear signals with harmonic frequencies under different acoustic pressure,2 and the contrast-enhanced image is conducive to the dynamic observation of blood vessels and organs. At a high acoustic power, the MBs collapse and dissolve, resulting in intermittent triggered imaging for per-fusion quantification and therapeutic ultrasound technologies utilizing various bioeffects. In addition, the surface of the UCAs needs to be decorated with targeting ligands to tumor angiogenesis, inflammation, and thrombosis. Using such targeted MBs or nanoparticles, ultrasound imaging can be transformed from traditional nonspecific tissue imaging into specific molecular imaging © XXXX American Chemical Society

and from general morphology into molecular imaging, which can vividly display changes in the tumor and sites of inflammation at cellular and subcellular levels. In recent years, research and development of multifunctional UCAs, updating of multimodal imaging systems, and in-depth studies regarding targeted delivery of therapeutic substances have all contributed to the development of ultrasound molecular imaging. UCAs represent the core and key for ultrasound-targeted delivery technology. Among them, multifunctional UCAs not only possess the function of sensitive molecular imaging but also achieve ideal therapeutic effects via substance delivery and cavitation, which is currently a strong research focus.

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CONVENTIONAL ULTRASOUND CONTRAST AGENT UCAs can be divided into MBs and nanoparticles according to size. MBs with semisynthetic phospholipids as the shell materials are the most common, demonstrating good biocompatibility but a short half-life in the body. Table 1 Received: April 5, 2018 Accepted: July 5, 2018 Published: July 5, 2018 A

DOI: 10.1021/acsbiomaterials.8b00421 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Review

ACS Biomaterials Science & Engineering Table 1. Commercial Ultrasound Contrast Agent name

shell

gas

mean size (μm)

application

half-life (min)

Optison Sonazoid SonoVue/Lumason Definity/Luminity a Imagent/Imavist a Levovist a Albunex

albumin lipid lipid lipid lipid galactose albumin

C3F8 C4F10 SF6 C3F8 C3F8, N2 air air

2.0−4.5 2.1 2.0−3.0 1.1−3.3 6.0 2.0−4.0 4.3

left ventricular liver, breast cancer heart, abdomen heart, abdomen echocardiography Doppler imaging trans-pulmonary imaging

2.5−4.5 60−120 3−6 2−10 60−180 2−5 1−2

a

No longer used clinically.

summarizes typical commercial UCAs for clinical use. To avoid causing embolic events by intravenous administration, the MB size must remain under 10 μm so that clinical UCAs can recirculate freely, similar to red blood cells. The bioinert heavy gases decrease the gas diffusion coefficient from the inner core of bubbles, and the biomembrane shell can escape from the immune system by avoiding activation of the monocytemacrophage system, showing a longer life in the body. Although the biocompatibility of synthetic polymer material is slightly inferior, it has a longer half-life in the body because of its slower degradation rate. MBs in the bloodstream generate resonant volumetric oscillations in response to the rapid variations of acoustic pressure, which are emitted by ultrasound transducers, leading to size changes in MB in a process termed cavitation. Cavitation may be accompanied by increasing pressure and temperature within MBs, which leads to mechanical stress and nonthermal bioeffects on surrounding tissues or generation of free radicals. The propensity for cavitation to occur leads to unamiable bioeffects, such as microvascular rupture or petechiae due to the transmitting frequency of the ultrasound probe, with cavitation less likely to occur at high frequencies (low mechanical index, MI). Specifically, at low MI (15 MHz) for contrast-enhanced ultrasound imaging. The gas core consisted of C4F10, and the main coating lipid was either DSPC or DPPC. They found that mechanical agitation resulted in UCAs with smaller MBs than sonication and the DPPC-based UCAs formulations had higher nonlinear responses at both the fundamental and subharmonic frequencies in vitro. These results suggested that DSPCbased MBs produced by mechanical agitation resulted in small MBs with high nonlinear responses suitable for high-frequency contrast-enhanced ultrasound imaging. High Molecular Weight Polymer. As a membraneforming material, polymers have the advantages of a strong shell, good compression resistance, and good stability. Polymeric UCAs often use synthetic or natural macromolecules as membrane-forming materials, such as poly(lactic-co-glycolic acid) (PLGA), polyethylene glycol (PEG), and chitosan. PLGA is a commonly used polymer with good biodegradability and biocompatibility and can be hydrolyzed to lactic acid and glycolic acid. Lactic acid is a naturally generated metabolite, whereas glycolic acid can be degraded into CO2 and water in vivo by an endogenous enzyme. The U.S. Food and Drug Administration (FDA) has confirmed the good biocompatibility and safety of PLGA. Luo et al.15 used an amphiphilic polymer to prepare nanoscale UCAs with lactoferrin connected to the surface, and ultrasound imaging was evaluated in in vitro and in vivo experiments. The contrast agent showed a superior imaging performance for tumors, with a longer imaging time. Chitin is an organic substance that is found widely in nature, and it is the main component of crustacean exoskeletons. Chitin can be deacetylated to obtain chitosan. Chitosan is a copolymer of N-acetyl-amido monosaccharides, which are derived from natural sources, and has the advantages of good biocompatibility, biodegradability, low toxicity, and high cationic charge. Min et al.16 linked a β-cholic acid onto the natural polymer polyethylene glycol chitosan and successfully synthesized an amphiphilic chitosan derivative. The material was used to prepare UCAs encapsulating liquid fluorocarbons and doxorubicin with a particle size of 432 nm. In vivo experiments in mice showed that the nanodrug-loaded UCAs could permeate into the interior of the tumor through the enhanced permeability and retention (EPR) effect, and the drug release can be controlled by ultrasound to enhance the uptake of the drug by tumor cells. In addition, nanoscale UCAs has a small particle size, and they are cleared from the body mainly by macrophage phagocytosis of the liver. Liver cells can be roughly divided into parenchymal cells and nonparenchymal cells. The parenchymal cells include hepatocytes and cholangiocytes, whereas the nonparenchymal cells include liver sinusoidal endothelial cells (LSECs), Kupffer cells, and hepatic stellate cells (HSCs).17 Park et al. investigated the distribution following spleen injection of PLGA nanoparticles in liver cells. In the experiment, various types of liver cells were isolated, and the uptake of the nanoparticles was tested. The results showed that the proportions of nanoparticles in Kupffer cells, LSECs, HSCs, hepatic parenchymal cells, and nano-

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PHASE-INVERSION ULTRASOUND IMAGING

MB ultrasound imaging and treatment are mostly confined to blood vessels due to the micron-scale sizes of MBs. In contrast, nanoscale contrast agents, such as nanobubbles and nanodroplets, have the advantages of a smaller particle size and stronger permeability, which are especially beneficial for the detection of extravascular lesions. However, some additional problems remain to be solved. For example, the particle size and acoustic intensity of a contrast agent are contradictory factors. A smaller particle size results in a stronger penetrating ability but a weaker acoustic reflection, and vice versa. To solve this problem, researchers have proposed a strategy of “small to large,” known as acoustic droplet vaporization (ADV), in which the engineered nanodroplets are small enough to permeate into the tumor interstitium from cancerous vasculature.18 Under ultrasonic irradiation or other energy delivery, liquid fluorocarbons can transform into micron-sized bubbles, thereby enhancing their performance in ultrasound imaging and treatment. Liu et al.19 prepared folic-acid-modified nanodroplets (FA-NDs) with phase-change ability via the thinfilm rehydration method. FA-NDs undergo a liquid−gas phase transition under low-intensity focused ultrasound (LIFU) irradiation, and the resultant micron-sized MB can enhance the ultrasound imaging signals. After the fluorescently labeled FA-NDs were injected into the mice model of ovarian cancer via the tail vein, the contrast agent was enriched in the tumor area under LIFU irradiation, and the signal was significantly enhanced. The imaging effect was significantly improved compared with the unmodified nanoparticles. The results of this study suggest that the combination of FA-NDs with LIFU may allow ultrasound imaging of tumors overexpressing folate receptors, thus improving the sensitivity of tumor diagnosis. Teng et al.20 prepared magnetic mesoporous nanoparticles with a liquid fluorocarbon-filled core, which can convert magnetic energy into heat energy in vivo and in vitro, causing liquid−gas phase transitioning of the liquid fluorocarbon in the core, such that the ultrasonic imaging signal is enhanced while the tumor is eradicated in a highly efficient manner. These findings could provide new insights into the application of a phase-inversion contrast agent in ultrasound-targeted diagnosis and treatment.

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ULTRASOUND CONTRAST AGENT FOR TARGETED MOLECULAR IMAGING The types of targeting are commonly divided into passive targeting and active targeting, according to their targeting mechanisms. The former uses the EPR effect of tumors or acoustic radiation to deliver the contrast agent, while the latter allows the UCAs to function in active targeting to the tissue or organ by binding the ligand or peptide on the surface of the UCAs, which is more commonly used in experimentation and research. In addition to contrast agent ligand modification to achieve active targeting, researchers have developed a wide range of targeted UCAs for different uses to adapt to the complex internal environment of the body, thus improving the function and application value of the contrast agent. Targeted delivery by the EPR effect of the tumor or inflammatory site is C


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ACS Biomaterials Science & Engineering

expected to be further used in antiangiogenic therapy and efficacy evaluation. The RGD peptide has a high affinity for integrin αvβ3 and can actively guide the drug/gene loaded UCAs through tumor blood vessels. The modified contrast agent can specifically bind to target tumor blood vessels and effectively inhibit the immune response induced by the biotin−avidin system in the body.25 Yan et al. modified the lipid UCAs using the integrin αvβ3 ligand and transmembrane peptide iRGD, followed by the combination of fluorescence imaging and molecular ultrasound imaging technologies to observe the targeted imaging effects of the lipid contrast agent in breast cancer neovascularization. It was demonstrated that the targeted lipid contrast agent could accurately locate the breast cancer tissue and effectively enhance the detection of breast cancer neovascularization. In another aspect, US super-resolution techniques have activated the potential application for vascular structure imaging and found a way out of the diffraction limit at depths. Ultrafast ultrasound localization microscopy captures the transient single echoes from individual microbubbles and forms a super-resolved image from the superposition of all localizations in tens of thousands of frames.26−28 Precise tracking of microbubble positions allows both noninvasive structural imaging and hemodynamic quantification of microvessels. These promising techniques open avenues for the fundamental understanding and diagnostics of various disease processes in the clinic. Inflammation. On the basis of the characteristics of the specifically expressed inflammatory markers during the inflammatory response in the body, the corresponding targeted UCAs can be designed. The common inflammatory markers include intercellular adhesion molecule-I,29 vascular endothelial cell adhesion molecule-I,30 E-selectin,31 and P-selectin.32 Selectin, as a specific cell surface adhesion molecule, can recognize and adhere to inflammatory cells and vascular endothelial cells, playing an important role in inflammation. PSGL-1, an important molecule that mediates leukocyte adhesion, is a common ligand of E/P-selectin. Its binding to selectin can reduce the flow of inflammatory cells in the bloodstream and facilitate its passing across the blood vessel wall to reach the site of inflammation. Machtaler et al.33 prepared rPSGL-Ig-MBs targeting E/P-selectin, and these rPSGL-Ig-MBs were injected into mice with chronic enteritis. After causing an acute onset of chronic enteritis, the area of colorectal inflammation in mice was observed by focused ultrasound, and good images of colorectal inflammation in mice were obtained using focused ultrasound-mediated rPSGL-Ig-MBs. In addition to validating the targeting performance of the targeted UCAs, the relationship between the imaging effect and the targeted ligand binding was further investigated. Wu et al.34 used the magnetic bridged streptavidin method and ordinary bridged streptavidin method to modify the UCAs with anti-P-selectin antibodies and performed in vivo imaging of the abdominal aortitis in the mouse model with ultrasound to identify the modified targeted contrast agents. The results showed that the magnetic-targeted UCAs, which were modified in different ways, could significantly improve the targeting performance of the contrast agent, enhance the imaging of the disease area, and effectively improve the detection rate of the abdominal aortitis, for which superior imaging was achieved using targeted UCAs that were modified using the magnetic bridged streptavidin method.

a type of passive targeting. In such lesions, there is often a gap between vascular endothelial cells, with a width up to 780 nm.21 Some nanoscale molecules can then exude from the blood vessel at the tumor site or the inflammation area, where these foreign molecules tend to accumulate due to the tumorassociated obstruction of lymphatic drainage, leading to backflow and slow blood flow.22 Therefore, nanoscale UCAs can be exuded from and can accumulate outside the blood vessel by the EPR effect to achieve imaging of the tumor tissue (Figure 2). Active targeted UCAs have been used in the

Figure 2. Active targeting by the coupling of ligands to MB that bind to intravascular receptors on tumor endothelium and the infiltration of nanoparticles to the tumor tissue through the EPR effect.

diagnosis and treatment of various diseases, such as blood vessels, inflammation, and thrombosis. This type of targeting can carry and release drugs or genes during imaging, thereby achieving the purpose of diagnosis and imaging integration. Tumor Angiogenesis. Vascular formation and growth are crucially linked to tumor growth and metastasis. In the process of angiogenesis, endothelial cells can specifically or highly express certain receptors, which are important targets for targeted therapy and targeted imaging. The expressions of vascular endothelial growth factor (VEGF), integrin αvβ3, and endoglin in tumor neovascularization are significantly upregulated. Fan et al.23 prepared lipid MB targeting the VEGF receptor based on the overexpressed VEGF-A in glioma tissue. The MBs were intravenously injected into mouse bregma, which is irradiated by focused ultrasound (FUS) for therapy and tumor growth monitoring. The results of their study demonstrated that the lipid microvesicles targeting the VEGF receptor could specifically accumulate in the area of tumor angiogenesis, and the amount of the contrast agent was significantly greater than that in normal brain tissue. Wang et al.24 cross-linked the MB with vascular growth factor VEGF121 (MBVEGF) to target neovascularization. After confirming its ability to specifically bind to neovascular endothelial cells in vitro, MBVEGF was injected into mice with squamous cell carcinoma to investigate its imaging performance. The results showed that compared with the control group, MBVEGF significantly enhanced the ultrasound signal of the tumor neovascularization (3.8 ± 4.4 dB vs 17.3 ± 9.7 dB, p < 0.05), achieving an ideal imaging result. These findings demonstrate that the MB combined with targeted angiogenesis receptors can achieve sensitive and specific tumor angiography, and it is D


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Figure 3. Preparation of exosome-like silica nanoparticles and ultrasound images and quantification of cells echogenicity in vivo. (a) Schematic of the ELS nanoparticle fabrication and morphology. TSPA (red) changed the overall stiffness of the silica shell and rendered them more elastic to form the ELS nanoparticles. (b−e) TEM images of silica products made with (b) 0%, (c) 20%, (d) 40%, and (e) 100% TSPA (red). (b) Hollow spheres were obtained when no TSPA was added; (e) a silica gel was formed with only TSPA. (f) Ultrasound intensity analysis of ELS with other silica nanoparticles. SSN, MSN, and MCF also increased the echogenicity of hMSCs but not as strong as the ELS nanoparticles. (g) TEM images of ELS-labeled hMSCs indicated aggregation of ELS inside the cells. ELS located both inside and on the cells. Arrows indicate the ELS nanoparticles, and Nuc indicates the nucleus. (h) This higher-magnification TEM image indicated that the ELS retained the unique curvature after entering the hMSCs. (i) Epifluorescence microscopy with hMSCs nucleus in blue and ELS nanoparticles fluorescently tagged in green. (j) The ELS-labeled hMSCs were subcutaneously injected with a Matrigel carrier into nude mice. The majority of ELS was specifically bound to the hMSCs. In vivo ultrasound images of (k) PBS, (l) 1 million ELS-labeled hMSCs, (m) 0.2 million unlabeled hMSCs, and (n) 0.2 million ELS-labeled hMSCs. Reproduced with permission from ref 45. Copyright 2017 Royal Society of Chemistry.

Thrombosis. Thromboembolism is a major cause of sudden-onset acute vascular embolism, myocardial infarction, pulmonary embolism, and stroke. Early monitoring, diagnosis, and treatment of thromboembolism are particularly important in clinical practice. Studies have shown that thrombosis is correlated with the high expression of platelet glycoprotein IIbIIIa. For the diagnosis of thrombosis, if the UCAs are modified with a ligand specific for the receptor of glycoprotein IIb-IIIa to target a thrombus, then the detection rate of thrombosis can be effectively improved.35−37 Levels of plasminogen activators such as platelet glycoprotein IIb-IIIa and urokinase plasminogen activator (uPA) are elevated during thromboclasis. Wang et al.38 used antiplatelet glycoprotein IIb-IIIa scFv and recombinant urokinase plasminogen activator scuPA for dual modification to form targeted theranostic MBs (TT-MBs), and diagnosis of the thrombosis and treatment effects were observed before and 45 min after the administration of MBs, with antiplatelet glycoprotein IIb-IIIa scFv-modified singletargeted MBs serving as a control. TT-MBs were shown to accurately image the thrombus and significantly reduce the thrombus volume, and their thromboclasis ability was similar

to that of urokinase. Therefore, TT-MBs can be used as an effective tool for the early diagnosis and treatment of thrombosis, in addition to evaluation of the treatment efficacy. Stem Cells. Stem cells are one of the promising cell-based therapy strategies that play an important role in developing new treatments for regenerative medicine.39 Although there have been increasingly many clinical trials regarding stem-cellbased therapies in recent years,40−43 the long-term risks and benefits of these therapies are still unknown. Moreover, detailed studies and a better understanding of stem cells are needed, including the distribution, differentiation, and interaction of stem cells with their microenvironment. As an approach for noninvasive cell tracking engrafted cells in real time, MBs are a powerful tool for determining the efficacy of stem-cell-based therapies. These MBs are clinically approved and have been successfully used in stem cell labeling and tracking with imaging modalities via ultrasound imaging. A single MB can induce acoustic impedance mismatch with tissues, and thus, a single cell can be detected with this imaging modality.44 Cui et al. presented the successful labeling of NPCs with lipid shell MBs and performed the assessment of E


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using MBKDR is safe and allows assessment of KDR expression and using immunohistochemistry (IHC) as the gold standard. Twenty-four women with focal ovarian lesions and 21 women with focal breast lesions were injected intravenously with MBKDR (0.03 to 0.08 mL/kg of body weight), and USMI of the lesions was performed starting 5 min after injection and lasting up to 29 min. The result showed that the KDR-targeted USMI signal matches well with KDR expression on IHC, and the targeted signal could be observed for up to 29 min after intravenous injection. None of the experienced adverse events led to discontinuation of the study, and there were no abnormalities or significant changes or trends in vital signs, ECG, or measured laboratory tests. These results suggest, for the first time, that ultrasound molecular imaging is safe and feasible and allows for the noninvasive assessment of KDR expression in patients, and they demonstrate the successful clinical translation of USMI with MBKDR, which is currently the only available clinical-grade molecularly targeted contrast agent.

internalized MB survival to determine the viability of this approach in vivo. Internalized MBs were less sensitive to destruction by ultrasound irradiation and remained visible in vivo for days compared to minutes when free. The study also demonstrated the ability to image these MB-loaded NPCs with US, defined as the minimum number of cells that can be detected, and documented these observations in vivo. The extended longevity provides ample time to allow cells to reach their intended target. Recently, novel UCAs are also exploited to improve contrast imaging. Chen et al.45 synthesized novel exosome-like silica nanoparticles (ELS) that have significant ultrasound impedance mismatches for labeling stem cell imaging. These novel ELS nanoparticles of approximately 140 nm were prepared via an emulsion template method (Figure 3a), and they were synthesized with different ratios. Finally, the study characterized and selected ELS nanoparticles made with 40% TSPA for stem cell imaging (Figure 3b−e). The ultrasound intensity analysis and TEM revealed that the positive charge of silica nanoparticles facilitates cell uptake and inherently increased echogenicity (Figure 3f−h). In vivo ultrasound images revealed significant increases in the echogenicity of the transplanted ELS-labeled stem cells compared to the unlabeled cells (Figure 3k−n), indicating that these novel ELS nanoparticles can increase the cell contrast and enable real-time cell tracking/imaging via affordable ultrasound. Clinical-Grade Targeted Ultrasound Contrast Agents. In recent years, with the in-depth study of tumor targets, many tumor-specific receptors have been identified. Using these tumor targets, targeted tumor drugs could be synthesized for antitumor therapy with high efficiency and low toxicity, and targeted contrast agents could be prepared for early and accurate tumor diagnosis. BR55 is a specific clinical-grade UCAs with targeted binding to vascular endothelial growth factor receptor 2 (VEGFR2), demonstrating good prospects for tumor imaging in a variety of preclinical models. Hackl et al.46 performed animal experiments to confirm that the application of high-resolution BR55 technology could achieve the noninvasive and sensitive detection of early tumor micrometastases, showing the potential for the early diagnosis of tumors. With further experiments in vitro and in vivo, BR55 began to be used in clinical trials. In 2017, Smeenge et al.47 investigated, for the first time, the feasibility and safety of the BR55 contrast agent for the detection of prostate cancer in humans. The researchers selected 24 patients who were diagnosed with prostate cancer by biopsy, and they performed BR55 contrast-mediated imaging using clinical low-acoustic intensive ultrasound. As the first in vivo clinical trial, the dosages of the contrast agent and the imaging procedures were carefully considered. This experiment combined the imaging data with histopathology results after radical prostatectomy, and the detection rate of ultrasound imaging with this contrast agent was analyzed. The results showed that 0.03 and 0.05 mL/kg contrast agents were sufficient to obtain a contrastenhanced image for 30 min. Safety was monitored during the process of contrast agent injection and after injection, and no serious side effects were observed. Kinase insert domain receptor (KDR) is one of the key regulators of neoangiogenesis in cancer. Willmann et al.48 performed a first-in-human clinical trial on ultrasound molecular imaging in patients with breast and ovarian lesions using a clinical-grade KDR-targeted contrast MBs (MBKDR). They aimed to assess whether ultrasound molecular imaging

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MULTIMODAL ULTRASOUND IMAGING TECHNOLOGY Multimodal UCAs are particularly used to reveal the occurrence, proliferation, and metastasis of tumors at the molecular level by hybrid imaging of the fate of MBs and other imaging agents, such as superparamagnetic iron oxide (SPIO) or fluorescent dyes. Multimodal UCAs make use of the entrapment, attachment, or adsorption of imaging agents in or on the shells of MBs for biodistribution and histological quantitation studies. Song et al.49 utilized the modified double emulsion solvent evaporation process using carbodiimide technology to prepare herceptin-decorated and ultrasmall SPIO (USPIO)/paclitaxel (PTX)-embedded nanobubbles (PTX-USPIO-HER-NBs) that target breast cancer. These nano-MBs can be used for trimodal ultrasound imaging, magnetic resonance imaging, and photoacoustic imaging, and they can rapidly release paclitaxel after low-frequency ultrasound irradiation, demonstrating their potential application value in imaging and drug delivery for breast cancer. In addition, multimodal imaging can also be applied in highintensity focused ultrasound (HIFU) ablation for real-time guidance, manipulation, and evaluation of cancer therapy. Zhou et al.50 prepared folate-targeted perfluorohexane lipid nanoparticles carrying bismuth sulfide (FLBS-PFH-NPs) that can simultaneously enhance ultrasound and CT imaging. Modification with PEG and folic acid on the surface of FLBSPFH-NPs gives them good stability, biocompatibility, and the ability to target tumor cells. The results show that the liquid fluorocarbon inside nanoparticles can enhance the cavitation effect in the HIFU area after liquid−gas-phase transformation and can significantly increase the coagulation necrosis volume of the tumor. FLBS-PFH-NPs, as a new dual-modal contrast agent, can improve ultrasound/CT imaging and HIFU ablation treatment by which diagnosis and treatment are integrated. The development of multimodal ultrasound imaging technology maintains the advantage of US (noninvasive, affordable, and broad diagnostic applicability) and is conducive to integration of the advantages of various imaging techniques to acquire additional diagnostic information. Ultrasound/Photoacoustic Imaging. Photoacoustic (PA) imaging is a technology that combines ultrasound imaging with optical imaging and captures significant attention in bioimaging. It is a hybrid modality, combining the F


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Figure 4. Photoacoustic nanodroplets for laser-activatable ultrasound imaging. (a) Structure and design of the PS−PDIPAnDs. PSs and PFC were coencapsulated by the self-assembled amphiphilic PDI molecules. The PSs were uniformly dispersed at the interface of the PDI shell and PFC core. (b) TEM image showing a uniformly distributed core−shell structure. (c) Dynamic light scattering analysis indicating a diameter of 113.5 ± 24.2 nm. (d) Optical images of the PS−PDI−PAnDs before and after laser irradiation. (e) B-mode US images of the PS−PDI−PAnDs at different concentrations before and after the laser irradiation. (f) Schematic illustration of PS−PDI−PAnD. The PDI shell will convert light energy into heat when irradiated with a 671 nm laser, vaporize the PFC core for contrast-enhanced US imaging, and kill cancerous cells by photothermal effect. Meanwhile, the encapsulated PSs transferred light energy to the oxygen and promoted O2 generation and enhanced PDT effect. (g) Representative fluorescence images taken at different time points after the PS−PDI−PAnDs injection (label with IRDye800). (h) PA images of U87MG tumorbearing mice pre- and post-i.v. injection of PS−PDI−PAnDs and PS−PDI−PAnPs. The PS−PDI−PAnDs and PS−PDI−PAnPs exhibit similar tumor accumulation, which may be because of their similar particle size and surface chemistry. (i) B-mode US images of U87MG tumor-bearing mice before and after photoirradiation. Reproduced with permission from ref 64. Copyright 2018 American Chemical Society.

ability and requires low laser energy for phase inversion. In addition, phase-inversion nanoparticles have been demonstrated to be capable of both destroying cancer cells and being used for photoacoustic-ultrasound dual imaging in vivo, in addition to exhibiting great potential application in cancer molecular imaging and therapy.61−63 Tang et al.64 stabilized the perfluorocarbon droplet with a photoabsorber and photoacoustic agent of perylene diimide (PDI) molecules and coencapsulated the droplet with photosensitizers (PS) of ZnF16Pc molecules (PS−PDI−PAnDs). An optical droplet vaporization process used via laser irradiation was a safer and controllable method to achieve dual-modal PA/US imaging contrast and photothermal therapy. In addition, PFC can serve as an O2 reservoir to continuously generate cytotoxic O2 in photodynamic therapy (PDT; Figure 4f). When intravenously injected into tumor-bearing mice, the PS−PDI−PAnDs exhibit high tumor accumulation via the EPR effect. With single laser irradiation, PS−PDI−PAnDs exhibit a dual-modal PA/US imaging-guided (Figure 4h,i) synergistic photothermal and oxygen self-enriched photodynamic treatment, resulting in complete tumor eradication and minimal side effects. Ultrasound/Magnetic Resonance Imaging. Magnetic resonance imaging (MRI) is an imaging technology with high spatial resolution that can sensitively reflect the contrast of soft tissues, but it has the disadvantages of a long data acquisition time and inconvenient operation. The MRI signal is created

characteristics of high penetration and high spatial resolution of ultrasound imaging with high-contrast optical imaging.51 PA imaging is capable of providing high-resolution structural, functional, and molecular imaging52,53 in vivo in optically scattering biological tissue. The main parameter of PA is the light absorption coefficient, which is closely related to the chemical composition of the body. The PA signal detected by the ultrasonic detector is directly related to the thermal energy generated by the accumulation of light energy in the tissue, and the accumulation of light energy is related to many physical characteristics of the tissue, such as scattering, absorption characteristics, thermal characteristics, and elasticity.54−56 In recent years, many studies have reported on the research and development of ultrasound/photoacoustic dual-modal imaging contrast agents and their use in diagnosis57,58 and treatment. Huynh59 presented the first trimodal phospholipid MBs by incorporating a dense concentration of porphyrin molecules within a MB shell, enabled by the use of a single porphyrinlipid component. These MBs possessed ultrasound, photoacoustic, and fluorescence properties that were demonstrated in vitro and in a mouse tumor xenograft model. Jian et al.60 used PLGA polymer materials as the shell and incorporated multifunctional phase-transition nanodroplets to prepare nanoemulsion as a contrast agent offering excellent contrasts for photoacoustic and ultrasound dual-modality imaging. This contrast agent has good stability and strong light absorption G


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Figure 5. Schematic illustration of the transarterial chemoembolization enhancing the nanoparticle-based synergistic cancer (HCC) surgery of HIFU and the microstructure of Fe3O4-PFH/PLGA composite nanocapsules. Reproduced with permission from ref 72. Copyright 2016 Royal Society of Chemistry.

while also achieving ultrasound/magnetic resonance dualmodal imaging. In the experiment, the BBB was transiently opened, the drugs were released by focused ultrasound, and a magnet was placed on the scalp of the mice to establish a magnetic field for magnetic targeting. Finally, T2-weighted imaging (T2WI) and susceptibility-weighted imaging (SWI) were used to detect the concentration of SPIO nanoparticles. The results showed that this system not only had stable ultrasound/MR imaging characteristics but could also quickly open the BBB for effective drug delivery. The magnetic targeting increased the SPIO concentration at the tumor site by 22.4%, which provided an effective strategy for the treatment of glioma. The combination of imaging with treatments such as HIFU, in particular, is not only capable of performing acoustic imaging but also has been extensively applied to treat some primary solid tumors and even metastatic cancer,68−71 thus improving the therapeutic effect and achieving the integration of diagnosis and treatment. HIFU synergistic agents are conducive to improving its deficiencies of long treatment duration and high operating energy. You et al.72 designed a multifunctional magnetic Fe3O4−PFH/PLGA nanocapsule and applied it in the treatment of liver cancer. In animal experiments, the Fe3O4−PFH/PLGA nanocapsule complex was injected together with lipiodol through the hepatic artery into the tumor tissue, and it was combined with a highintensity focused ultrasound for treatment. The transarterial chemoembolization assisted by lipiodol could substantially retain the synergistic agents within tumor tissues for subsequent HIFU cancer surgery, and a better therapeutic effect was obtained through two HIFU irradiations. During irradiation, a phase transition of the perfluorohexane occurred in the nanocapsule complex, and a synergistic effect of the enhanced HIFU ablation antitumor was successfully achieved in rabbits by VX2 liver xenograft (Figure 5). In addition, the nanocapsule could simultaneously perform imaging using three modalities, ultrasound, magnetic resonance, and photoacoustic

through the interaction of the total water signal and the magnetic properties.65 This provides an opportunity for the development of contrast agents that can be used for both positive and negative enhancement. In addition, the ability to combine high-resolution anatomical images with molecular information from targeted contrast agents using a single imaging system would greatly improve the detection of disease. Ultrasound technology combined with MRI effectively improves the rate of disease diagnosis and leads to a larger area of application in disease diagnosis and treatment. Superparamagnetic iron oxide (SPIO) nanoparticles are a type of magnetic nanobiomaterial that is mainly composed of Fe3O4 and Fe2O3. The SPIO nanoparticle surface has many binding sites to facilitate its modification into a multimodal imaging probe, and it can also enhance backscatter signals to improve ultrasound imaging by influencing the acoustic impedance of the tissue, resulting in its broad application in ultrasound imaging combined with MRI imaging. John et al.66 encapsulated SPIO with a protein shell to synthesize a specialized targeted protein microsphere, the RGD functionalized Nile-Red-superparamagnetic iron oxide encapsulated (RGD-NR-SPIO) microsphere. The surface of this microsphere is modified with an arginine-glycine-aspartate (RGD) tripeptide ligand, enabling it to specifically bind to the ανβ3 integrin receptor. It is confirmed that RGD-NR-SPIO can effectively enhance ultrasound and magnetic resonance imaging and thus achieve tumor angiography. Currently, the application for diagnosis and treatment in central nervous system diseases using ultrasound/MR imaging technology is also developed. Confocal ultrasound has attracted a large amount of attention because of its ability to noninvasively open the blood−brain barrier (BBB). During the treatment of brain diseases, the combination of confocal ultrasound and MR can guide and monitor treatment in real time. Fan et al.67 prepared DOX-loaded and SPIO-conjugated MBs (DOX-SPIO-MBs) by linking SPIO to doxorubicinloaded MBs (DOX-MBs) to achieve targeted drug delivery H


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Figure 6. Preparation and in vivo study of IHM. (a) Scheme of IHM for tumor recognition and O2 evolving for effective PDT under NIR laser illumination. (b) Design of IHM. MnO2 NPs were encapsulated into the reel of thread such as HANP with ICG covalently conjugated on the surface. MnO2 reacted with H2O2 and produced rich oxygen for increasing PDT efficiency. (c) Dynamic light scattering analysis of HANP (180 ± I


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ACS Biomaterials Science & Engineering Figure 6. continued

4.5 nm), MnO2 (35 ± 2.5 nm), and IHM (239 ± 5.8 nm). TEM image for IHM is inserted. Scale bar equal to 200 nm. (d) Ultrasound images (white color) observed the O2 generation of IHM in different amounts of H2O2 (0, 2, 10, 50, and 100 mM). No O2 (black color) was detected in the IHNAP with the H2O2 group. (e) Detection of SO generated by IHM in SCC7 cells. (f−k) Noninvasive in vivo imaging monitoring the tumor accumulations of IHM. (f) NIR fluorescent imaging of intravenously injected IHM accumulation in SCC7 tumor-bearing mice at different times. (g) Photoacoustic imaging of intravenously injected IHM accumulation in tumor-bearing mice at different times. (h) Ultrasound imaging monitoring the generation of O2 in tumors after intravenously injected IHM. (i) Tumor/muscle (T/M) ratio of SCC7 tumor-bearing mice administered IHM and ICG, suggesting IHM peaked in tumor at 6 h p.i. (j) Normalized PA signals of SCC7 tumor-bearing mice administered IHM and ICG, suggesting IHM peaked in tumor at 6 h p.i. (k) Relative echo intensity of SCC7 tumor-bearing mice administered IHM and ICG, suggesting the O2 amount peaked at 6 h p.i. Reproduced with permission from ref 78. Copyright 2017 Elsevier.

ultrasound/photoacoustic imaging technique was used to observe the generation of oxygen in the tumor area (Figure 6d). The oxygen content in the tumor was elevated 2.25 ± 0.07 times compared to the control group, as ultrasound imaging confirmed during the study. After laser irradiation, significant tumor growth inhibition was observed in the ICGHANP/MnO2-treated group compared to the ICG-HANPtreated group; this difference was attributed to the beneficial oxygen-generating property of IHM for PDT (Figure 6f−k).

imaging, thus demonstrating its potential clinical value for treatment and diagnosis. Ultrasound/Fluorescence Imaging. A hybrid modality of ultrasound and fluorescence imaging such as ultrasoundmodulated fluorescence (UMF) imaging has been applied to provide fluorescent contrast while maintaining ultrasound resolution in detecting distinct biochemical states and tumor microenvironment. UMF was experimentally demonstrated based on contrast agents such as fluorophore-labeled MBs or nanoparticles that can be tuned to provide information about local tissue properties.73−75 Fluorescence intensity modulation was demonstrated at the ultrasound driving frequency, and the spectrum of the detected fluorescence can inform about relative tissue oxygenation, pH, and other properties.76 Fluorescent MBs have been fabricated with the capacity to have their emission modulated by ultrasound. In recent years, extensive studies about ultrasound/fluorescence dual-modal imaging have been conducted, including image fusion technology, imaging systems, and contrast agents, in particular. These contrast agent particles could potentially be used to extract fluorescence modulation from a strong light background to increase imaging depth and resolution in scattering media. Liu et al.77 linked the fluorescent N-hydroxysuccinimide (NHS) ester to the surface of an ultrasonic contrast agent. By controlling the concentration of the fluorophore through ultrasound, the fluorescence quenching effect was observed when no ultrasound was applied. The fluorescence emission intensity is modulated by ultrasound-induced MB oscillations and is characterized by UMF. This study demonstrated that the UMF signal largely depends on the oscillation amplitude of the MBs and the initial quenching state of the surface fluorophore. Ultrasound can be used to control the intensity of the fluorescent contrast agent by causing the MB oscillation to achieve molecular imaging based on fluorescence imaging. Moreover, to optimize the therapeutic time window, extensive preclinical studies have taken advantage of multimodal imaging to monitor the tumor accumulation of molecular probes and realize the integration of diagnosis and treatment. PDT, which we mentioned above, is a promising therapeutic modality for tumor that can convert oxygen into cytotoxic single oxygen via photosensitizer to ablate tumor growth. Gao et al.78 designed an oxygen-generating PDT nanocomplex (the ICG-HANP/ MnO2 nanocomplex) by encapsulating a manganese dioxide nanoparticle (MnO2 NP) in an indocyanine green (ICG) modified hyaluronic acid nanoparticle (HANP). The efficiency of PDT is by improved by exploiting the high reactivity of MnO2 NP and H2O2 to produce oxygen. ICG as a photosensitizer was conjugated onto the surface of tumortargeted HANP (Figure 6a−c). The tumor accumulation of the ICG-HANP/MnO2 nanocomplex was monitored via fluorescent imaging and photoacoustic imaging (Figure 6e). The

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CONCLUSIONS AND FUTURE TRENDS Ultrasound technology for molecular imaging is a comprehensive discipline of biology, chemistry, physics, computer engineering, and clinical medicine, with a particular need for interdisciplinary cooperation and development. It is believed that in the near future, the increase in interdisciplinary and cross-disciplinary cooperation will further promote the development of ultrasound molecular imaging, with more achievements in basic research and clinical trials and consequent far-reaching effects on human health. Research and development of UCAs for ultrasound molecular imaging represent the core of the development of ultrasound-targeted technology and the driving force for the development of ultrasound molecular imaging. Advancing preclinical experimental research to clinical application is the main task of current ultrasound molecular imaging. In addition, the clinical application of new knowledge and methods in the field of ultrasound molecular imaging to benefit patients is a challenge for future studies of ultrasound molecular imaging. With the development of related technologies and further research regarding the pathogenesis of diseases, it is believed that more achievements will be applied in clinical practice.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86-020-81292115. Fax: +86-4006981163-30863. Email: [email protected]. ORCID

Zhi-Yi Chen: 0000-0002-5498-5349 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the Research Projects of National Natural Science Foundation of China (No. 81671707), the Natural Science Foundation of Guangdong Province (No. 2016A030311054), the Research Projects of Guangzhou Technology Bureau (No. 201607010201), and the Research Fund for Lin He’s Academician Workstation of New Medicine and Clinical Translation. J


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nanobubbles for tumor-targeting ultrasonic imaging. Int. J. Nanomed. 2015, 10, 5805−5817. (16) Min, H. S.; You, D. G.; Son, S.; Jeon, S.; Park, J. H.; Lee, S.; Kwon, I. C.; Kim, K. Echogenic glycol chitosan nanoparticles for ultrasound-triggered Cancer Theranostics. Theranostics 2015, 5 (12), 1402−1418. (17) Park, J. K.; Utsumi, T.; Seo, Y. E.; Deng, Y.; Satoh, A.; Saltzman, W. M.; Iwakiri, Y. Cellular distribution of injected PLGAnanoparticles in the liver. Nanomedicine 2016, 12, 1365−1374. (18) Torchilin, V. Tumor delivery of macromolecular drugs based on the EPR effect. Adv. Drug Delivery Rev. 2011, 63 (3), 131−135. (19) Liu, J.; Shang, T.; Wang, F.; Cao, Y.; Hao, L.; Ren, J.; Ran, H.; Wang, Z.; Li, P.; Du, Z. Low-intensity focused ultrasound (LIFU)induced acoustic droplet vaporization in phase-transition perfluoropentane nanodroplets modified by folate for ultrasound molecular imaging. Int. J. Nanomed. 2017, 12, 911−923. (20) Teng, Z.; Wang, R.; Zhou, Y.; Kolios, M.; Wang, Y.; Zhang, N.; Wang, Z.; Zheng, Y.; Lu, G. A magnetic droplet vaporization approach using perfluorohexane-encapsulated magnetic mesoporous particles for ultrasound imaging and tumor ablation. Biomaterials 2017, 134, 43−50. (21) Maeda, H.; Sawa, T.; Konno, T. Mechanism of tumor-targeted delivery of macromolecular drugs, including the EPR effect in solid tumor and clinical overview of the prototype polymeric drug SMANCS. J. Controlled Release 2001, 74 (1−3), 47−61. (22) Danhier, F.; Feron, O.; Préat, V. To exploit the tumor microenvironment: Passive and active tumor targeting of nanocarriers for anti-cancer drug delivery. J. Controlled Release 2010, 148 (2), 135− 146. (23) Fan, C. H.; Ting, C. Y.; Liu, H. L.; Huang, C. Y.; Hsieh, H. Y.; Yen, T. C.; Wei, K. C.; Yeh, C. K. Antiangiogenic-targeting drugloaded microbubbles combined with focused ultrasound for glioma treatment. Biomaterials 2013, 34 (8), 2142−2155. (24) Wang, J.; Qin, B.; Chen, X.; Wagner, W. R.; Villanueva, F. S. Ultrasound Molecular Imaging of Angiogenesis Using Vascular Endothelial Growth Factor-Conjugated Microbubbles. Mol. Pharmaceutics 2017, 14 (3), 781−790. (25) Yan, F.; Xu, X.; Chen, Y.; Deng, Z.; Liu, H.; Xu, J.; Zhou, J.; Tan, G.; Wu, J.; Zheng, H. A Lipopeptide-Based αvβ3 IntegrinTargeted Ultrasound Contrast Agent for Molecular Imaging of Tumor Angiogenesis. Ultrasound Med. Biol. 2015, 41 (10), 2765−2773. (26) Errico, C.; Pierre, J.; Pezet, S.; Desailly, Y.; Lenkei, Z.; Couture, O.; Tanter, M. Ultrafast ultrasound localization microscopy for deep super-resolution vascular imaging. Nature 2015, 527 (7579), 499− 502. (27) Lin, F.; Tsuruta, J. K.; Rojas, J. D.; Dayton, P. A. Optimizing Sensitivity of Ultrasound Contrast-Enhanced Super-Resolution Imaging by Tailoring Size Distribution of Microbubble Contrast Agent. Ultrasound Med. Biol. 2017, 43 (10), 2488−2493. (28) Opacic, T.; Dencks, S.; Theek, B.; Piepenbrock, M.; Ackermann, D.; Rix, A.; Lammers, T.; Stickeler, E.; Delorme, S.; Schmitz, G.; Kiessling, F. Motion model ultrasound localization microscopy for preclinical and clinical multiparametric tumor characterization. Nat. Commun. 2018, 9 (1), 1527. (29) Kaufmann, B. A.; Sanders, J. M.; Davis, C.; Xie, A.; Aldred, P.; Sarembock, I. J.; Lindner, J. R. Molecular imaging of inflammation in atherosclerosis with targeted ultrasound detection of vascular cell adhesion molecule-1. Circulation 2007, 116 (3), 276−284. (30) Suzuki, J.; Ogawa, M.; Takayama, K.; Taniyama, Y.; Morishita, R.; Hirata, Y.; Nagai, R.; Isobe, M. Ultrasound-microbubble-mediated intercellular adhesion molecule-1 small interfering ribonucleic acid transfection attenuates neointimal formation after arterial injury in mice. J. Am. Coll. Cardiol. 2010, 55 (9), 904−913. (31) Spivak, I.; Rix, A.; Schmitz, G.; Fokong, S.; Iranzo, O.; Lederle, W.; Kiessling, F. Low-Dose Molecular Ultrasound Imaging with ESelectin-Targeted PBCA Microbubbles. Mol. Imaging Biol. 2016, 18 (2), 180−190. (32) Bettinger, T.; Bussat, P.; Tardy, I.; Pochon, S.; Hyvelin, J. M.; Emmel, P.; Henrioud, S.; Biolluz, N.; Willmann, J. K.; Schneider, M.;

ABBREVIATIONS UCAs, ultrasound contrast agents; MB, microbubble; MI, mechanical index; EPR, enhanced permeability and retention; PA, photoacoustic; PDT, photodynamic therapy.

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Ultrasound technology for molecular imaging: From contrast agents to

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