Emerging diagnostic and therapeutic technologies based on ultrasound-triggered biomaterials

Ultrasound (US) is a kind of acoustic wave with frequency higher than 20 kHz. Learning from the echo detection ability of bats and dolphins, scientists applied US for clinical imaging by sending out US waves and detecting echoes with shifted intensities and frequencies from human tissue. US has long played a critical role in noninvasive, real-time, low-cost and portable diagnostic imaging. With the in-depth study of US in multidisciplinary fields, US and US-responsive materials have shown practical value in not only disease diagnosis, but also disease treatment. In this review, we introduce the recently proposed and representative US-responsive materials for biomedical applications, including diagnostic and therapeutic applications. We focused on US-mediated physicochemical therapies, such as sonodynamic therapy, high-intensity focused US ablation, sonothermal therapy, thrombolysis, etc, and US-controlled delivery of chemotherapeutics, gases, genes, proteins and bacteria. We conclude with the current challenges facing the clinical translation of smart US-responsive materials and prospects for the future development of US medicine.


Introduction
Ultrasound (US) refers to sound wave whose frequency exceeds the highest threshold of human hearing (20 kHz) [1]. Although humans cannot hear US, various animals have * Author to whom any correspondence should be addressed.
Original content from this work may be used under the terms of the Creative Commons Attribution 4.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. the ability to sense and produce US. Bats and dolphins, for instance, can send out ultrasonic waves and use the intensity, delay and frequency shift of the echo to determine the approximate type, distance and speed of the unknown objects ahead. Inspired by these animals, by processing information generated by the interaction between the physical properties of US waves and the acoustic properties of human tissues to form graphs, curves or other data, the first generation of B-mode US came out in the 1950s, marking the beginning of modern ultrasonic diagnostic technology [2]. Due to its high-spatial-resolution, low cost, excellent biosafety and portability, ultrasonography has long been a popular and widely applied imaging method [3][4][5][6][7]. Based on the grayscale ultrasonography, scientists further developed numerous applicable US imaging modalities to facilitate diagnosis, including Doppler imaging, elastography, contrast-enhanced US (CEUS), etc [8][9][10][11][12][13][14].
Then, the exploration of various physicochemical effects of US broadens the biomedical potential of US [15][16][17][18]. For example, when a liquid medium is exposed to US, a region in the liquid will form a local temporary negative pressure area, causing the gas that was dissolved in the liquid to supersaturate and escape from the liquid to form small bubbles. This process is referred to as the cavitation effect. Generally, the cavitation effect includes inertial and non-inertial cavitation. Non-inertial cavitation is also referred to as stable cavitation, which describes the oscillation of microbubbles. However, during inertial cavitation, microbubbles tend to expand rapidly in areas of negative pressure created by longitudinal ultrasonic propagation. Finally, the bubbles burst instantaneously, accompanied by the release of large amounts of heat and pressure, causing further effects, such as sonoporation, sonoluminescence, pyrolysis, etc [19][20][21][22][23][24]. Since then, the exploration of US has covered more and more fields. The rapid development of computer technology and electronics has greatly broadened the application potential of US in multidisciplinary areas, such as chemistry, engineering, biology, metrology, materials science, etc. Moreover, in terms of improving human health, US is not only a medical imaging technique, but also a potential therapeutic tool.
In recent decades, to enlarge and broaden the US functionalities, attention has been focussed on smart materials that can be responsive to US stimulus. With the assistance of these US-responsive materials, US shows more potential in biomedical applications. In this review, we introduce various US-responsive materials for diagnostic applications, such as endovascular imaging and endogenous biosensing. Moreover, the therapeutic applications of the US-responsive matters were demonstrated from two aspects, one was the US-mediated physicochemical therapies, and the other was US-controlled therapeutics delivery. The first aspect included sonodynamic therapy (SDT), high-intensity focused US (HIFU) ablation, sonothermal therapy, thrombolysis, and the second aspect illustrated the delivery of chemotherapeutics, gases, genes, proteins and bacteria with detailed examples. Finally, we conclude with the current progress and challenges facing the clinical translation of US-responsive materials and prospects for the future development of US medicine.

Diagnostic applications
As a result of the biosafety, great penetrability and low cost of US, more and more biomedical applications based on US and US-responsive matters have been explored during recent decades. Initially, US was mainly employed in clinical imaging. Compared with computerized tomography (CT) and magnetic resonance imaging (MRI), US can display in vivo conditions in a real-time and non-irradiated manner. With the advanced progress of US contrast-enhanced agents, CEUS has played a more and more indispensable role in clinical diagnosis. Furthermore, US-propelled micro-and nanorobots have not only shown possibilities in microsurgery and drug delivery, but have also attracted attention in fast and ultrasensitive biosensing.

Imaging
Based on harmonic signals reflected from microbubbles or nanoparticles upon US excitation in vivo, CEUS has been broadly applied clinically. By intravenously injecting contrastenhanced agents, the vascular distribution inside tissues can be depicted clearly, contributing to clinical diagnosis. As the second generation of contrast agents, SonoVue has shown epoch-making significance. The recently developed commercial ultrasonic contrast agent Sonazoid, with its ability to target Kupffer cells, has exhibited practical significance for molecular imaging in clinical translation. Despite the wide applications of SonoVue and Sonazoid, the fast clearance and low stability of these lipid microbubbles hinders the sensitivity and duration of detecting lesions. Thus, researchers have focussed on the development of multifunctional and multimodal ultrasonic contrast agents.
With their excellent biocompatibility and blood/tissue permeation, perfluorocarbon (PFC) nanodroplets have been proven to be promising contrast agents [25]. Progress has been made in exploring materials to stabilize PFC droplets and enhance US sensitivity [26,27]. Learning from the application of red blood cells (RBCs) in pharmaceutical engineering, Xu et al coated liposomal dodecafluoropentane (lipoDFP) with multilayer RBC membrane (RBCm), namely Sonocyte (figure 1(A)) [28]. The multilayer RBCm endowed the lipoDFP with significantly improved stability, which is reflected in the sustained blood circulation duration and reduced cellular uptake, contributing to enhanced targeted lesion accumulation. By detecting the hepatic portal vein of the rat via power Doppler imaging, Sonocyte showed higher US signal than lipoDFP and imaging profile similar to commercial SonoVue. In terms of detecting bacterial liver necrosis, Sonocyte and SonoVue showed similar diagnostic capacity. SonoVue ′ s large diameter of microbubbles, which is micron-sized, limited its penetrability into tumor interstitium. However, nanoscale lipoDFP and Sonocyte can address this problem. As demonstrated by animal fluorescence imaging, compared with lipoDFP, Sonocyte can accumulate in tumor tissues more effectively. Notably, the diagnostic efficacy of Sonocyte was elucidated to be significantly higher than lipoDFP, indicating that the RBCm coating contributed tremendously to the stability and targeted diffusion of PFC nanodroplets.
The US responsiveness of the micro/nano matter plays an indispensable role in clinical imaging, not only in ultrasonography, but also in fluorescent imaging, CT and MRI. Imaging agents for ultrasonography and multimodal imaging. RBC membrane-coated phase-changeable Sonocyte for assessing liver lesions. Reprinted from [28], Copyright (2020), with permission from Wiley-VCH.
Due to the diverse demand in clinical practice, multimodal imaging is urgently needed nowadays. Traditional CEUS agents are blood-pool contrast agents, such as SonoVue, which are limited to imaging vascularity only. The emergence of SonaZoid, which is able to consume and display the Kupffer cells, has achieved new advances in molecular US imaging. Interestingly, Koshkina et al presented a novel type of multimodal imaging nanoparticle containing PFC (PFCE-PLGA) for in vivo cell tracking [29]. Unlike the PFC assemblies mentioned above, the PFCE-PLGA will not cause liquidto-gas phase transition under US irradiation, contributing to its repeated and long-term in vivo imaging. The strong stability of PFCE-PLGA is ascribed to its fractal core-shell structure. Researchers applied these nanoparticles for tracking cells in dendritic cell therapy that was employed in clinical trials [30]. The enhanced contrast signals were confirmed in the inguinal lymph nodes and popliteal lymph nodes that were injected with nanoparticle-labeled therapeutic dendritic cells via clinical US scanner (frequency of 7 MHz), highresolution small animal scanner (frequency of 48 MHz), fluorescence imaging and 1 H/ 19 F MRI. Furthermore, the acoustic signals can last for days, realizing long-term US imaging. This study paves the way for sustained multimodal imaging, and future clinical translation is expected. In addition to simple imaging, researchers have been working on multifunctional microbubbles that feature not only in imaging, but also therapeutic functions.

Biosensing
Genetic materials in cells control cellular growth and apoptosis. Studies have confirmed that many diseases are caused by abnormal microRNA (miRNA) expression, from diabetes to cancers [31]. The detection of intracellular miRNA contributes to the disease diagnosis and guides the development of therapeutic plans. Nowadays, the existing miRNA detection technologies require a large number of cells and considerable analyzing duration. With the rapid development of microand nano-robots, scientists have devoted effort to the employment of fuel-free robots for biosensing. As a mechanical wave with temporal and spatio-controllability, US has shown great potential in propelling micro-and nano-robots [32,33]. As a classic case, Esteban-fernández deÁvila et al proposed a US-propelled nano-motor for real-time intracellular miRNA detection (figure 2(a)) [34]. The presented novel nanomotor is composed of a graphene oxide (GO)-decorated gold nanowire (GO-AuNWs) and a dye-labeled single-stranded DNA (ssDNA), which has a preferential binding with miRNA-21. The miRNA-21 has been found to be overexpressed in most tumors [35]. Using the metastatic breast-cancer cell line (MCF-7), researchers found that, under the excitation of US, the ssDNA functionalized GO-AuNWs can rapidly internalize the cell, accelerating the hybridization process between miRNA-21 and DNA probe. Most importantly, this method can realize miRNA detection at single-cell level in a few minutes, greatly improving the detection efficiency and sensitivity.
In contrast, endogenous metabolites often reflect the physiological state of cells. Compared with the commercially used microbubble contrast agents, diverse inorganic nano-materials and enzymes have been developed for the endogenous detection of hydrogen peroxide [36][37][38]. For example, Walker et al designed a nano-sensor via a layerby-layer fabrication method for endogenous pH monitoring though US contrast imaging [39]. Recently, Zhu's group presented a pH-sensitive nanoparticle that can catalyze endogenous hydrogen peroxide into oxygen, which further contributes to US imaging, focused US ablation therapy and hypoxia alleviation during chemotherapeutic treatment against 4T1 tumors (figure 2(b)) [36]. Experiments conducted on gel phantoms and animal tissue both demonstrated a significantly increased contrast-enhanced ultrasonography when pH was reduced below a healthy level. Moreover, compared to normal microbubble contrast agents, these multi-layer nanoparticles show sustained imaging capacity, allowing for long-time monitoring.

Therapeutic applications
Due to the numerous chemical and mechanical effects of US, this biofavorable mechanical wave has been widely explored in therapeutic applications. Based on numerous US-induced biomechanical effects, US can not only exert therapeutic functions directly, but also be used as a stimulant for responsive drug release. The sonocatalytic effect between US and sonosensitizers leads to the production of a large amount of reactive oxygen species (ROS), which could cause oxidative damage to cancer cells, bacteria, etc, and this is defined as SDT. The energy conversion after the interaction between US and specific matter, which could convert the mechanical vibration into hyperthermia, contributed significantly to HIFU tumor ablation therapy and sonothermal pathogen elimination. In addition, the great biofavorability of US ensures biosafety and patient-tolerance during treatment. More importantly, the penetrability and temporal accuracy of US enables enhanced therapeutic efficacy. Compared with photodynamic therapy (PDT), SDT can target lesions that are located deeper; compared with microwave and radiofrequency ablation therapy, HIFU ablation therapy exhibits higher targetability and biosafety; compared with external stimuli, such as magnetic field and near inferred light, US can penetrate multilayer tissues and trigger drug release accurately. In this section, we will present numerous US-responsive matter-based therapeutic applications and clinical translations. Since Umemura et al raised the use of SDT in 1990, tremendous progress has been witnessed during recent decades [40]. Despite the positive efficacy and significant advantages of SDT, few clinical trials have been promoted. However, recent decades have witnessed numerous advanced studies that have paved the way for the clinical translation of SDT. Sun et al elucidated the ideal anticancer efficacy of SDT combined with antibody treatment, using US-responsive microbubbles (TP MBs) loaded with sonosensitizer pyropheophorbide-lipid and therapeutic antibody trastuzumab simultaneously (figure 3(a)) [41]. The conjugated trastuzumab, which is a monoclonal antibody against the HER2 gene, endowed the microbubbles with the targeting ability of the HER2 receptor, enhancing the tumor accumulation of TP MBs. Thus, the TP MBs also realized the targeted in vivo CEUS imaging. It was first demonstrated in vitro that TP MBs are sensitive to US and can be effectively destroyed by US stimulation, contributing to US-controllable drug release. In the HER2-positive gastric cancer model established on mice, the excellent CEUS imaging capacity and tumor-killing effects were further elucidated. Recently, Huang et al combined SDT with immunotherapy in a single nanoregime and achieved ideal anticancer efficacy against melanoma (figure 3(b)) [42]. The synthesized micellar nanocarrier has a core of sonosensitizer Ce6 and an interlayer conjugated anti-PD-L1 antibody, along with a pH-sheddable shell. Upon reaching the tumor site, the nanocarrier showed dual sensitivity according to the low pH at the tumor site and the external US excitation, leading to the immuno-SDT.
Similar to the anticancer mechanism of SDT, PDT fights tumors by generating ROS from the reaction between photosensitizers and low-power lasers. The rapid and large consumption of oxygen during SDT and PDT commonly results in tumor hypoxia, which can stimulate the tumor resistance and accelerate tumor metastasis. Studies showed the necessity of normalizing tumor vasculature during anti-tumor treatment. For instance, irinotecan can enhance vascular function when fighting cancer [44,45]. Thus, the combination of SDT/PDT and chemotherapy could be a promising strategy for enhanced anticancer efficacy. Chen et al realized PDT and chemotherapy in a single microbubble platform (PCF-MBs), which was synthesized in situ via local  [41]. Reprinted from [41], Copyright (2021), with permission from the American Chemical Society. (b) Immuno-sonodynamic therapy based on US-responsive micellar nanocarrier [42]. Reprinted from [42], Copyright (2021), with permission from Elsevier. (c) PCF-NPs fabricated via US-mediated microbubble destruction (i) realized the combination of PDT and chemotherapy (ii) [43]. Reprinted from [43], Copyright (2018), with permission from the American Chemical Society.
US-mediated microbubble destruction (figure 3(c)) [43]. The microbubbles were fabricated based on the cavitation of the perfluoropropane core. The photosensitizer porphyrin grafted lipid and chemotherapeutic camptothecin-floxuridine conjugate self-assembled into microbubbles, not only realizing the combinational treatment, but also showing fluorescence imaging and CEUS capacity.
The piezocatalytic redox reaction between piezoelectric materials and US effectively produces ROS, which can exert tumor killing efficacy in anti-cancer treatments. Notably, piezoelectric nanomaterials feature excellent biocompatibility, contributing to their potential in therapeutic applications [46]. The practical application of organic and inorganic sonosensitizers in anti-tumor therapy has been widely explored. However, due to the limited bioavailability and fast in vivo clearance of organic sonosensitizers as well as the relatively low production of ROS and limited stability of inorganic sonosensitizers, studies have been carried out on developing effective and multifunctional drug carriers to deliver sonosensitizers and enhance SDT efficacy simultaneously. Due to their abundant surface area and desired biocompatibility, hollow mesoporous organosilica nanoparticles (HMONs) have shown promising potential in drug loading and delivery. By loading organic sonosensitizer IR780, which is a kind of NIR fluorescence dye and also a photosensitizer, Chen et al developed a composite nano-sonosensitizer for treating pancreatic cancer through SDT (figure 5(a)) [49]. In addition to traditional HMONs, fluorocarbon-chain-functionalized HMONs (FHMONs) not only show excellent drug loading capacity but also play the role of biocompatible oxygen carrier (IR780@O 2 -FHMON). This US-responsive oxygen-selfproduced nanocomposite showed efficient hypoxia alleviation and anti-tumor efficacy against pancreatic tumors both in cytotoxic experiments conducted on PANC-a cells and in animal experiments conducted on tumor-bearing mice. The advanced results achieved by Chen's group paved the way  for the co-delivery of oxygen and organic sonosensitizers in a single regime.
Titanium dioxide (TiO 2 ) is a typical and representative inorganic nano-sonosensitizer. However, its limited quantum yield of ROS hinders its application in SDT. Numerous efforts have been focussed on the improvement of the band structure of TiO 2 . For example, Dai et al recombined TiO 2 with GO nanosheets with in situ attached MnO x nanoparticles (MnO x /TiO 2 -GR) ( figure 5(b)) [50]. The novel MnO x /TiO 2 -GR not only showed excellent imaging monitoring, but also featured enhanced ROS production efficiency due to the high electroconductivity and large surface chemistry of GO nanosheets. Moreover, the photothermal-conversion property of GO nanosheets contribute to the realization of photo-SDT. Han et al established an oxygen-deficient layer on black TiO 2 (B-TiO 2−x ) through an aluminum reduction procedure [52]. The crystallographic texture of B-TiO 2−x features oxygen defects, which facilitate the split of electrons and holes, contributing to the increased ROS production. Moreover, progress hasve been made in combining TiO 2 with noble metals, such as Au, Ag, Pt, etc [53]. For example, Liang's group developed Pt-decorated TiO 2 Janus nanoparticles with a hydrogenated hollow core (H-Pt-TiO 2 ) to simultaneously alleviate tumor hypoxia, improve SDT therapeutic efficacy, and realize chemo-SDT (figure 5(c)) [51]. The hollow architecture of H-Pt-TiO 2 Janus particles endowed the nanoplatform with drug loading capacity. Thus, after loading doxorubicin (DOX) into H-Pt-TiO 2 Janus particles, which is a chemotherapeutic but also a sonosensitizer, significant apoptosis of 4T1 cells can be observed with the assistance of US irradiation. The anti-tumor efficacy was further elucidated in in vivo experiments. The abovementioned advances in modifying organic and inorganic sonosensitizers highlight the improvement of traditional sonosensitizers.  [54]. Compared with simple T-BTO nano-cubes, Au@BTO displays enhanced ROS generating properties due to the piezoelectric/metal interface between Au and T-BTO. t Due to the biocompatibility of Au and T-BTO, the desired biosafety of Au@BTO allows biomedical applications, such as infected wound dressing. In vitro antibacterial experiments showed the outstanding bacterial elimination capacity of Au@BTO on E. coli and Staphylococcus aureus, with a more effective antibacterial effect on S. aureus. Therefore, researchers applied the Au@BTO nanocomposite to the surface of the S. aureus-infected wounds, followed by the irradiation of US. Results showed that ROS generated from the piezocatalytic reaction between US and Au@BTO can effectively fight the bacteria, and the US wave exerted a positive effect on cell migration. Thus, these two processes contributed to the healing of infected wounds. Recently, our group employed 3D printing technology to fabricate a novel Janus patch encapsulating Au@BTO nanocubes in the top layer while loading vascular growth factors in the bottom layer, realizing US-triggered bacteria elimination and wound dressing [46].
Although the application of antibiotics effectively combats bacteria, the emergence of multidrug-resistant bacteria is another obstacle to antimicrobial treatment. Concerning methicillin-resistant S. aureus (MRSA), for instance, challenges remain in clinical practice. SDT-induced antibacterial therapy has shown promising potential in eliminating MRSA. Sun et al conjugated the sonosensitizer T790 with the oxygenproducing nanocomposite Pd@Pt (Pd@Pt-T790) to successfully treat MRSA-infected myositis ( figure 6(b)) [55]. Pd@Pt can be activated under US irradiation, thus generating oxygen through the enzyme catalase. Moreover, Pd@Pt played the role of drug carrier of T790 due to its excellent liquid dispersity and large surface area. The antibacterial efficacy of US-activated Pd@Pt-T790 nanocomposite was demonstrated in vitro. In the MRSA-infected mice model, the Pd@Pt-T790 nanocomposites accumulate effectively at the myositis site after intravenous injection, which can be monitored through photoacoustic imaging, CT and MRI, indicating the triplemodal imaging capacity of Pd@Pt-T790. After 14 d treatment, the mice treated with Pd@Pt-T790 along with US irradiation showed excellent eradication of MRSA and significantly decreased level of TNF-α and IL-6.

Others.
Due to the excellent tissue penetration of US, researchers explored the application of US in treating deep-sited inflammatory in joints. ROS generated from the reaction between sonosensitizers and US contributes to the reduction of synovial hyperplasia, benefitting anti-fibroblasts and anti-angiogenesis. As an FDA-proved fluorescent dye, indocyanine green (ICG) shows significant practicability in biomedical applications. In addition, to its US-activated ROS generation in fighting tumors, Tang et al for the first time found that ICG could be applied in not only US-excited ROS production, but also to early inflammatory arthritis synovitis fluorescent imaging [56]. In vitro experiments demonstrated that mitochondrial impairment and ROS generation contributed to the cytotoxicity of fibroblast-like synoviocytes. This study casts light on the application of SDT in treating rheumatoid arthritis (RA).
Considering that the hypoxic microenvironment of the joint contributes to the angiogenesis, which accelerates the RA progression, and hinders ROS production during SDT, it is anticipated that sonosensitizers be combined with hypoxia-relieved regimes. Recently, Li et al proposed a rhodium nanozyme (Rh/SPX-HSA), which features a concave-cubic structure and load sonosensitizers, for the treatment of RA ( figure 7(a)) [57]. The employed sonosensitizer was spafloxacin (SPX), which is one of the US-sensitive second-generation fluoroquinolone antibiotics. Compared with other nanomaterials that feature enzyme catalytic activity, the Rh nanozyme showed peroxidase and catalase activities, contributing to the enhanced antifibroblasts and anti-angiogenesis. In addition, since the synovial cells in RA joints showed up-grated metabolism, the requirement of albumin is higher in RA joints than healthy tissue. By modifying Rh/SPX with human serum albumin (HSA), the nanocomposite could target the RA joint, improving the therapeutic efficacy and avoiding side effects.
In vitro experiments validated the ROS generation and mitochondrial damage ability of Rh/SPX-HAS with US irradiation ( figure 7(b)). The therapeutic efficacy of Rh/SPX-HAS against RA joints was further evaluated in a mouse model. By analyzing the micro-CT images, immunohistochemical staining and western blotting results of the joints from different groups, it can be observed that the RA joints treated with Rh/SPX-HAS with US showed a smoother surface, more degradation of cartilage, alleviation of hypoxic microenvironment and decreased anagenesis.
In recent years, SDT has been elucidated to show the therapeutic effect on atherosclerosis [58][59][60][61][62][63][64]. Studies have shown the SDT-induced foam-cell apoptosis, iron retention alleviation and cytokine secretion inhibition contributed to the stabilization in advanced plaques by using 5aminolevulinic acid, sinoporphyrin sodium, etc [59,61]. Advanced progress has been achieved in clinical trials [65]. The latest work on developing theragnostic materials (PFP-HMME@PLGA/MnFe 2 O 4 -ramucirumab) for atherosclerotic plaque neovascularization was reported by Yao et al [64]. Thus, MnFe 2 O 4 is a kind of metal spinel ferrite nanoparticle, which played the role of an MRI nanoprobe. As a biocompatible organic sonosensitizer, hematoporphyrin monomethyl ether (HMME) showed the desired ROS producing property. Ramucirumab is an antibody against VEGFR-2 to inhibit tumor neovascularization. Notably, the combination of MnFe 2 O 4 , HMME and perfluoropentane (PFP) endowed the nanocomposite with multimodal imaging capacity. It was demonstrated that ROS produced under US excitation (1.5 W cm −2 , 1.0 MHz) mediated the mitochondrial-caspase apoptosis in rabbit aortic endothelial cells. Together with the ideal targeted properties and cytotoxicity of this nanomaterial, experiments conducted on rabbits with advanced plaques demonstrated that the PFP-HMME@PLGA/MnFe 2 O 4 -ramucirumab can significantly reduce the neovessel density, inhibit intraplaque hemorrhage, and subsequently lead to the stabilization of atherosclerotic plaques.

HIFU ablation.
As an innovative strategy for targeted ablation, HIFU is able to increase the local temperature up to 65 • C-100 • C within 1 s by converting the mechanical energy of US into heat. To date, various clinical trials have elucidated its practical significance [66][67][68]. Despite the accurate deposition of HIFU, the ablation area is relatively small, leading to the incomplete eradication of lesions. Meanwhile, the hyperthermia-mediated necrosis leads to insufficient blood supply, which would accelerate tumor progress through hypoxia and drug resistance. Thus, HIFU synergists have been developed to address these problems [69][70][71][72][73]. In addition, although the hyperthermia caused by HIFU contributed to the tumor eradication, the sonothermal damage is not desired for drug delivery. Thus, numerous researches have focussed on triggering drug release by a relatively low temperature caused by HIFU [72,73]. Ma et al proposed an oxygen and chemotherapeutics DOX co-delivery cerasome as a neoadjuvant strategy for HIFU treatment against triple-negative breast cancer ( figure 8(a)) [72]. The designed cerasome had an oxygen-sufficient PFC core, and a pH-sensitive peptide-decorated polyorganosiloxane shell. The mild-temperature HIFU (M-HIFU) was elucidated to excite the release of oxygen and DOX, contributing to the synergistic therapeutic efficacy of hyperthermia ablation and chemotherapy. Studies in vitro and in vivo conducted on 4T1 cells further verified the hypoxia alleviation. Moreover, the gas-phase PFC core benefited the CEUS during HIFU treatment. Similarly, as reported by Cheng's group, a mesoporous silica nanoparticle loaded with MRI contrast-enhanced agent gadopentetate dimeglumine (Gd-DTPA) in the nanopores and with PEG covering the pores was presented for the real-time tracking of nanocarriers and HIFU-mediated drug release ( figure 8(b)) [73]. Using an agarose phantom that can mimic the acoustic properties of in vivo tissue, it was demonstrated that the HIFU energy can trigger the release of Gd-DTPA and realize MRI contrast imaging. Furthermore, the HIFU-responsive imaging capacity was elucidated with a piece of chicken breast tissue. This study combines the advantages of MRI and HIFU, both of which have been applied clinically and are noninvasive, casting light on non-ionizing radiation real-time multi-modal imaging strategies.

Sonothermal therapy.
In addition to the antibacterial treatment based on US-excited ROS generation, Guan's group proposed a sonothermal therapy, which was based on titanium (Ti)-deposited red phosphorus (RP) (Ti-RP) (figure 9(a)) [74]. Compared with simple Ti, the synthesized Ti-RP showed obvious heating capacity under US excitation (1.0 W cm -2 , 1 MHz), from 22.30 • C to 44.90 • C ( figure 9(b)). It is elucidated that the electron motion induced by the US vibration can further result in phonon production and heat generation. Moreover, the metal/semiconductor interface provided by the heterojunction between Ti and RP significantly contributed to the activation of electron motion [75,76]. To make the sonothermal antibacterial treatment more meaningful and practical, researchers further modified the Ti-RP with nitric oxide (NO)coated mesoporous silicon nanoparticles (Ti-RP-SNO). The synergistic therapeutic ability of Ti-RP-SNO was confirmed in vitro by fighting MRSA. It was demonstrated that the USinduced hyperthermia could effectively trigger the release of NO and realize the combination of sonothermal and NO treatments. This advanced achievement addresses the limitations of traditional nonspecific sonothermal effects and proposes the promising composite substrates that can be applied for antibacterial treatment.  [77]. Due to the nanoscale, which was stabilized by the PLGA shell, the PFH droplets were endowed with the ability to penetrate the internal regions of tissues without being captured by the immune system. In vitro experiments conducted via CEUS and B-mode US showed the imaging properties of these novel nanoparticles due to the US-sensitivity of PFH. Furthermore, it can be observed from the microscope images that the volume of these PFH nanodroplets increased up to 300-fold after US irradiation, indicating the feasibility of these nanoparticles for mechanical thrombolysis ( figure 10(b)). In vitro experiments verified their thrombolytic efficacy. As illustrated in the H&E staining images, cavities with diameters of approximately 300 µm can be detected around the blood clots, indicating that microstreaming caused by the expansion and explosion of PFH nanodroplets can lead to the mechanical destruction of blood clots ( figure 10(c)). Along with the low-intensity US-triggered sonothrombolysis [78][79][80][81][82], which was based on the cavitation and sonoporation effects, the thrombolytic therapy was successfully realized in vivo with the assistance of Fe 3 O 4 -PLGA-PFH-CREKA NPs.

US-controlled therapeutic agents' activation and release
To avoid the untargeted drug release or the unwanted systematic effects during the drug administration process, controllable drug delivery systems have been explored for decades [83][84][85]. To trigger these intelligent drug carriers, diverse internal stimuli, such as pH, temperature, alkalinity or acidity, and external stimuli, such as NIR light, magnetic field, radiation, electric field, acoustic wave, et,c can be employed. Considering the desired biosafety, efficiency and penetration ability, US showed outstanding advantages in the remote control of drug release. However, there are some limitations in the application site of US since it is difficult to pass through the cavities and bones in the body.

Chemotherapeutics.
Having learnt from the in situ bursting of CEUS microbubbles, efforts have been focussed on delivering drugs via microbubbles and targeted controlled release of drugs with the excitation of US. Microbubbles can deliver anticancer drugs in diverse ways, such as encapsulating drugs in shells, cross-linking the drugs with polymer side chains or lipids in shells, encasing therapeutic gases in the core, and so on. By functionalizing chemotherapeutics with biotin, the conjugation between chemotherapeutics and lipid shells could be realized. For example, McEwan et al fabricated a type of oxygen-loaded microbubble with a shell decorated with biotin functionalized 5-fluorouracil, which is an anti-metabolite against pancreatic cancer [86]. It was demonstrated that the external US excitation could trigger the release of oxygen and 5-fluorouracil, contributing to tumor elimination. In addition, Nesbitt et al proposed a biotinylated gemcitabine ligand-attached lipid-shell microbubble, which carried oxygen to relieve the hypoxic microenvironment of pancreatic cancer [87]. These microbubble platforms show outstanding multi-agent delivery and US-triggerable release capacity, and a combination of more therapeutic strategies is still anticipated.
In addition to the microbubbles, liposomes that contain phospholipid shells feature not only biocompatibility, but also enhancement of chemotherapeutic pharmacodynamics. Among the developed nanotechnology vehicles, lysothermosensitive liposomal DOX (LTLD) is a kind of thermosresponsive triggerable drug delivery platform that has been widely explored. It has been elucidated that the LTLDs can release the loaded DOX when the temperature increases to 39.5 • C, and that the liposome-carried DOX has a much longer in vivo plasma half-life compared with the free DOX [88]. Due to the thermosensitivity of LTLDs, they have been employed in clinical trials and applications for a long time. For example, a phase 3 trial study where LTLDs were administered systematically while radio-frequency ablation was operated in a hepatocellular carcinoma showed that thermosensitive LTLDs could effectively improve the therapeutic efficacy of tumor margins during ablation treatment [89]. Despite the positive results in clinical trials, most radio-frequency ablation treatments are invasive. In contrast, focussed and high-intensity US realizes the generation of regulable hyperthermia and noninvasiveness. Thus, Paul et al started a phase 1 clinical trial where focussed US was applied to trigger the located drug release of LTLDs to treat hepatocellular carcinoma [90]. The enrolled patients had primary or secondary hepatic tumors that were refractory to traditional chemotherapy. The post-treatment biopsy of the tumors verified that US could effectively trigger the release of DOX and enhance the intracellular drug content. In addition, this therapeutic plan was elucidated to be safe and feasible. Therefore, further clinical trials are anticipated.

Gas.
The maneuverable core-shell structure of microbubbles enables the multi-drug loading capacity in a single regime, contributing to the synergistic anticancer treatment. For example, by loading chemotherapeutics in the polymer shell of microbubbles and simultaneously encasing anti-tumor gas-signaling hydrogen sulfide (H 2 S) molecules in the core, it can realize the combinational therapy against pancreatic cancer through in situ administration and external US triggering [91].
Since the ROS that plays a major role in SDT is basically produced from the interaction between US, sonosensitizers and oxygen, the relatively hypoxic environment at the tumor site usually hampers the SDT efficacy. Along with the rapid consumption of oxygen during SDT, the oxygen levels at the tumor site would be further reduced. Thus, efforts have been made to enhance the amount of oxygen during SDT against tumors. Based on the precise controllability and maneuverability of microfluidic technology [92][93][94][95], our group proposed a multi-drug co-loaded microcarrier, whose shell carries chemotherapeutics and sonosensitizer, while core loading oxygen-enriched PFC (PO/GI-MCs) ( figure 11(a)) [96]. The presented PO/GI-MCs showed not only ideal tumor inhibitory effect on pancreatic cancer, but also effectively alleviated the hypoxic microenvironment at the tumor site. Moreover, due to the polymer shell of the microcapsules, sonosensitizers ICG and chemotherapeutic gemcitabine were co-loaded into the shell. Upon in situ injection into the tumor site, the drugs would be rapidly released.Along with the external US irradiation, the ICG is excited to higher energy levels, followed by the generation of ROS. In addition, oxygen in the core can be released in response to the US, which could enhance the production of ROS as well as improve the hypoxia of pancreatic tumors. Furthermore, numerous oxygen delivery platforms have been developed for the enhancement of ROS production during SDT, such as oxygen-loaded microbubbles, oxygen-produced nanoplatforms, PFC-assisted oxygen-carrying nanoparticles, etc.
Although these oxygen delivery platforms did address the oxygen-insufficient problems during SDT, the uncontrollable oxygen release might cause over production of ROS, which might damage the normal tissue. Thus, materials that can respond to the oxygen concentrations might become a feasible solution [97]. Recently, Yang et al proposed a hypoxia-responsive nanoplatform for enhancing oxygen levels and improving SDT therapeutic efficacy ( figure 11(b)) [98]. These nano-assemblies contain hydrophobic 2-nitroimidazoles, which can be converted into hydrophilic 2-aminoimidazoles in hypoxic environments. Therefore, by creating a hypoxic environment imitating tumor microenvironment using NADH and nitro-reductase in nitrogen, these nano-assemblies dissociated completely, indicating its hypoxia-responsive capacity. Researchers loaded ALA, a precursor of PpIX, into the hollow cavity of hMVs, and encapsulated manganese ferrite nanoparticles into the polymer layer of the nano-assemblies (ALA-hMVs). The synthesized ALA-hMVs showed excellent hypoxia-triggered ALA release and oxygen generation. By employing the ALA-hMVs along with US in the B16 tumor-bearing mice model, the ideal tumor-inhibiting efficacy is elucidated. Oxygensensitive materials are seldom explored. Thus, the findings reported by Yang et al have significant meaning for the augmentation of SDT.

Gene.
During recent decades, the modification of gene expression through externally added genes and proteins has become a promising strategy in treating cancers, metabolic diseases, neurodegenerative diseases, etc [99]. Despite the efficient gene transfection led by viral vectors, the unsafety of the virus might result in adverse effects, such as immunological inflammation [100][101][102][103][104]. In addition, the blood-brain barrier (BBB) significantly hampered the delivery efficiency of the therapeutic genes [105]. In contrast, the micro-streaming and pressure explosion caused by the US-mediated microbubble destruction leads to the reversible micropores on cell membranes, contributing to the penetration of BBB for therapeutic agents administered systematically, especially the intracellular delivery of genes and proteins. Thus, numerous studies have been focussed on designing gene and protein-loaded micro/nano-bubble/particles for USmediated targeted delivery. For example, Zhang et al fabricated a plasmid deoxyribonucleic acid encoding glucagon-like peptide 1 (GLP-1)-loaded microbubble for the US-induced targeted delivery of genes, contributing to the treatment of type 2 diabetes [106]. The enhanced intracellular delivery of GLP-1 significantly improved the regeneration of pancreatic beta cells. Moreover, Lin's group proposed a kind of neurotrophic factor (NF)-loaded microbubble for the treatment of Parkinson's disease ( figure 12(a)) [107]. The results exhibited the efficient delivery of brain-derived NFs and glia cell line-derived NFs and the significant protection of dopaminergic neurons under US excitation. Liu et al also elucidated the US-triggered BBB penetration of quercetin-modified sulfur nanoparticle-loaded microbubbles and its therapeutic efficacy for Parkinson's disease [108]. Interestingly, Ryu et al developed a Cas9/sgRNA-loaded microbubble-nanoliposomal particle that could be responsive to US excitation, leading to the targeted release of functional proteins and treatment of androgenic alopecia ( figure 12(b)) [109]. Compared to the delivery of genes, the external supply of proteins showed more advantages, such as higher biosafety and less off-targeting effects. The confocal laser scanning microscopy (CLSM) fluorescent images and H&E staining images of the mouse skin showed that the mice treated with US-triggered Cas9/sgRNA release contributed significantly to the growth of hair.

Protein.
Therapeutic protein delivery has become a potent precision treatment nowadays, such as monoclonal antibodies [110]. Restricted by the hydrophilic nature and large size of the proteins, the efficient and precise transduction of therapeutic proteins seems to be a significant challenge. Carriers fabricated from lipids and polymers have become promising approaches to enhance targeted cellular uptake [111]. Recently, Sloand et al developed a type of US-responsive protein-loaded PFC nanodroplet that could realize the monitored delivery and spatiotemporal control of the targeted protein release at the same time (figure 13(a)) [112]. By assembling cell-targeted peptides onto a shell of nanodroplets, the nano-vehicles can localize targeted cells by binding to the extracellular integrins. The US stimulus contributes to the gas phase transition and microbubble generation, and thus leads to the collapse of bubbles. The destruction of the bubbles not only triggers the therapeutic protein release, but also results in the generation of reversible pores in the cell membrane, referred to as the sonoporation effect, which facilitates the intracellular protein transduction ( figure 13(b)). Attributed to the US-induced liquid-to-gas phase transition of PFC nanodroplets, they are also endowed with the function of imaging-guided carriers ( figure 13(c)). This work develops a novel antibody-loaded emulsion nanocarrier to realize the spatiotemporal control and responsive release of proteins in a single platform, showing the great potential of the combination of therapeutic and diagnostic applications of US.

Engineered bacteria.
Nowadays, engineered microbes have served as significant therapeutic agents for various therapeutic applications [113][114][115][116][117]. Several internal and external stimuli can activate the colonization and payload release of these administered bacteria [118,119]. As a biofavorable mechanical wave, US has also been explored to trigger the therapeutic functions of targeted bacteria. In the last few years, Shapiro's group has achieved tremendous advances in thermal-triggering in vivo activation of engineered microbes for therapeutic purposes via using focussed US [118,120]. They first explored two types of thermal logic circuits that could respond to different temperatures by engineering two novel thermal bioswitches, namely, TlpA and TcI. In Piraner's study, TlpA was encoded to regulate the expression of green fluorescent protein (GFP), while TcI was encoded to modulate the expression of red fluorescent protein (RFP) [120]. In one of the two circuits, the TlpA and TcI genes were activated independently, leading to the GFP expression at 37 • C and RFP expression at 42 • C. In another circuit, the TcI gene was modulated through the lambda promoter, resulting in the activation of RFP expression at 40 • C and the inhibition of RFP expression at 45 • C, accompanied by the activation of GFP expression. By applying a tofu phantom to mimic biological tissues, the MRI-guided focussed US could effectively trigger the patterned GFP and RFP expressions. The in vivo fluorescence imaging consistently showed significant GFP expression at the US-irradiated site on the nude mouse. In another study reported by Abedi et al, a temperaturesensitive genetic bioswitch was developed to realize sustained activation of engineered bacteria for lasting release of therapeutic payloads [118].

Future perspectives
With the wide exploration of US and US-responsive materials, the application potential of US in the field of biomedicine has been widely exploited. The practical application of US is no longer limited to imaging diagnosis, but also shows great potential in molecular biosensing and disease treatment. Interestingly, numerous smart materials have been developed to realize the integration of multi-mode imaging and therapeutic value. Despite the advanced achievements, clinical translation of these US-responsive materials is seldom reported. Various clinical trials based on US-triggered drug delivery microbubbles and multimodal US imaging contrast agents are under way and have shown positive results. Although many of the above-mentioned US-responsive materials showed more functions and more significant therapeutic effects, their complicated preparation process, expensive materials, inconvenient preservation and transportation make them difficult to be clinically transformed. Moreover, these US-responsive materials require stimulation by US waves with specific frequency and intensity, which should also be biofavorable (table 1). However, US waves with precise parameters used in the above-mentioned studies were mainly produced by large, complex, specialized acoustic instruments that are not easy to use at the bedside, hindering their clinical translation. Therefore, multidisciplinary cooperation and communication are of practical significance for the development and synthesis of more biocompatible and versatile US-responsive materials for future clinical translation. The development of more intelligent US transmitters and new sonosensitizers that can efficiently generate ROS are also the main research directions in the future. In the field of mechanical engineering and acoustic physics, the development of portable ultrasonic emission instruments that can flexibly adjust various ultrasonic parameters can also promote the further development of US medicine. The ultimate goal of the research and exploration of US-responsive materials is to improve human health. Therefore, the active cooperation and promotion of clinicians is also crucial for the development of US medicine. With the addressing of current challenges and the proposed innovative US-responsive materials, the practical application of US medicine is expected to be further expanded.