1. Introduction
Cancer has emerged as one of the paramount challenges confronting the contemporary medical landscape. Globally, millions are diagnosed with cancer annually, with pronounced disparities in incidence and mortality rates influenced by variables such as regional development, gender, and age demographics
[1],
[2]. Recent trends reveal a declining trajectory in the mortality rates associated with several cancer types
[1]. This favorable shift can be attributed to the monumental advancements realized in the domain of oncological research. Spanning several decades, meticulous research endeavors have significantly augmented the medical community's comprehension of the intricate processes governing tumor genesis, proliferation, metastasis, and the mechanisms engendering resistance to therapeutic interventions
[3],
[4],
[5],
[6],
[7]. This enriched understanding not only demystifies the inherent nature of cancer but also elucidates a spectrum of mechanisms potentially culminating in the eradication of malignant cells
[8],
[9],
[10]. Concurrent with the elucidation and refinement of these therapeutic mechanisms, there has been a proliferation of innovative treatment methodologies
[11],
[12],
[13]. These burgeoning approaches augur new therapeutic horizons for oncology patients and incessantly propel the medical fraternity towards the exploration and realization of treatment modalities that are more efficacious, targeted, and devoid of detrimental effects.
In the initial stages of cancer, the prospects for successful treatment markedly increase; however, achieving precise and effective early detection remains a profound challenge. To mitigate cancer-related mortalities, a punctual and exact diagnosis is paramount. The therapeutic arsenal for cancer is extensive, often encompassing surgical procedures, radiation therapy, and chemotherapy. Considering the myriad of cancer types and the distinct conditions of individual patients, it is imperative to devise tailored and targeted treatment strategies. Nanomedicine is devoted to pioneering medical systems at the nanoscale, designed to address pivotal challenges in diagnosing and treating clinical diseases
[14]. Unique nanosystems can cater to the diagnostic and therapeutic needs of a number of diseases. The creation of nanosystems with specialized responsive properties, controlled via physical methods, is imperative for propelling the evolution of precision nanomedicine
[15]. Traditional physical modulation techniques encompass light, radiation, magnetic fields, and ultrasound. Ultrasound, a mechanical wave with a frequency exceeding 20 kHz, presents advantages over light, radiation, and magnetic fields in medical applications due to its safety, visualization, non-invasiveness, and relatively affordable equipment cost
[16].
At present, ultrasound-based diagnostic and therapeutic protocols are extensively utilized in clinical settings. Ultrasound's parameters, such as frequency and power, alongside its emission techniques, are readily adjustable. By synergizing ultrasound with ultrasound-responsive nanosystems, a vast array of diagnostic and therapeutic applications can be achieved. This encompasses tumor molecular imaging, separation of tumor markers, facilitation of drug delivery across physiological barriers and into cells, precise drug release and activation, and a series of ultrasound-mediated therapeutic methods
[16],
[17],
[18],
[19],
[20],
[21],
[22]. These advanced capabilities stem from various ultrasound-induced phenomena, including direct thermal transmission, cavitation, acoustic streaming, and acoustic radiation forces. The intricate interactions among these phenomena can trigger structural vibrations, acoustic scattering, transmission of mechanical forces, elevated temperatures, and the generation of reactive oxygen species (ROS)
[23]. These phenomena form the fundamental mechanism of the interaction between ultrasound and ultrasound-responsive nanosystems.
To provide a comprehensive and systematic overview of this subject, we have structured our review around three core themes: comprehensive tumor diagnosis, precise drug delivery, and effective tumor therapy. Within this framework, we have extensively detailed the crucial role that ultrasound-enabled nanomedicine plays in these three major themes, further subdividing them based on their functionalities and objectives into tumor molecule imaging, tumor markers separation, physiological barrier penetration, cell membrane perforation, controlled drug release, responsive drug activation, and a range of ultrasound-induced anti-tumor therapeutic strategies (
Fig. 1). In every segment, we elucidated the mechanisms by which nanosystems interact with ultrasound and highlighted their innovative construction approaches. In conclusion, we broach discussions and offer insights into the challenges and promising horizons for ultrasound-enabled nanomedicine in refining precision oncology and its translational potential in clinical medicine.
2. Ultrasound-enabled nanomedicine for comprehensive tumor diagnosis
2.1. Tumor molecule imaging
Ultrasound imaging is a prevalent diagnostic modality within the realm of oncology, providing extensive data on cancer pathology. The advent of three-dimensional (3D) ultrasound facilitates the precise detection and localization of tumors, thereby providing information about their dimensions, morphology, and spatial orientation. Moreover, Doppler ultrasound and elastography imaging augment the diagnostic value of ultrasound, affording insights into the vascular dynamics and mechanical properties of tumor tissues, respectively
[24],
[25]. Ultrasound imaging presents a myriad of advantages, most notably its non-invasive modality, lack of ionizing radiation exposure, ability to penetrate deep tissue structures, and the capability to provide real-time imaging with enhanced contrast resolution
[15]. The foundational principle of ultrasound imaging hinges on the transmission of ultrasound waves from a transducer to a specified target region. Upon encountering tissues of varying densities and compositions, these waves produce distinct reflections. These reflected signals are then captured and subjected to digital processing, culminating in the generation of diagnostic visual images.
Conventional imaging modalities have proven efficacy in tumor identification. However, the quest for enhanced precision and a more holistic data acquisition has catalyzed the advent of molecular imaging techniques
[26],
[27]. Ultrasound-induced tumor molecular imaging combines ultrasound with molecular probes, specifically functionalized ultrasound contrast agents, to identify and visualize biomolecular activity. By incorporating diverse ligands such as antibodies, peptides, and carbohydrates into the structural framework of ultrasound contrast agents, these molecular probes are designed to selectively bind to tumor-associated biomarkers like specific proteins or cell surface antigens. Upon introduction into the physiological environment, these probes precisely target and adhere to their predetermined markers.
Fig. 2 [17],
[28],
[29],
[30],
[31] is the overview of the representative strategies of microbubbles, nanobubbles, nanodroplets, and gas vesicles for tumor molecular imaging. The ultrasound contrast agent exhibits a significant acoustic impedance difference compared to the surrounding tissues and can generate strong nonlinear echo signals (
Fig. 2(a))
[17]. This augmentation facilitates the sharp delineation of the highlighted zone, furnishing intricate, molecular-scale insights into the tumor's constitution.
In this section, we discuss the applications of four primary ultrasound contrast agents (microbubbles, nanobubbles, nanodroplets, and gas vesicles) in tumor molecular imaging. Additionally, it should be noted that gas-stabilizing nanoparticles also have the potential to serve as ultrasound contrast agents. These solid nanoparticles, equipped with hydrophobic surfaces or cavities, are adept at stabilizing gas pockets on their surfaces, furnishing heterogeneous nucleation sites. Consequently, when subjected to ultrasound, they induce the formation of micron-sized cavitation bubbles
[32]. More details will be elaborated in the sections that follow.
2.1.1. Tumor molecule imaging via microbubbles
Microbubbles, tiny entities resembling miniature balloons, are extensively utilized as contrast agents in ultrasound imaging. They are composed of a relatively stable gas core that resists dissolving in blood, encased by a shell made up of proteins, lipids, or polymers
[33]. The mechanical elasticity of these microbubbles is largely dictated by the material constituting their shell; the higher the elasticity of this material, the greater the acoustic energy the microbubble can endure
[34]. Microbubbles typically exhibit diameters between 1 and 10 µm. This size range enables them to traverse blood vessels unimpeded, without the risk of inducing thrombosis. Simultaneously, they offer superb acoustic contrast, enhancing the clarity of vascular imaging
[35],
[36].
Zhong and colleagues
[37] have developed a vascular endothelial growth factor receptor 2 (VEGFR2) targeted microbubble, utilizing C
3F
8 as the gas core and encapsulated within a shell created by the hybridization of VEGFR2-targeted peptides and liposomes. They discovered that this specialized microbubble, serving as an ultrasound molecular imaging probe, can distinguish cervical carcinoma (less than 3 mm in diameter) from the surrounding normal tissue. Interestingly, this VEGFR2-targeted microbubble demonstrated a heightened sensitivity in detecting extremely early tumors compared to larger ones. This research underscores the potential of using such targeted microbubble ultrasound molecular imaging for the detection of extremely early-stage cervical cancer. Furthermore, microbubbles targeting VEGFR2 have been reported to enhance the early detection rates of breast cancer, pancreatic cancer, and hepatic micrometastases. Further, multimodal imaging methods, which combine ultrasound imaging with other technologies, can provide more accurate and comprehensive diagnostic information. Pathak and colleagues
[38] encapsulated superparamagnetic iron oxide nanoparticles (SPIONs) within the multilayer polymer shell of microbubbles made from poly(
n-butylcyanoacrylate). Subsequently, they functionalized these SPION-embedded microbubbles (SPION-MBs) with cyclic Arg-Gly-Asp-
D-Phe-Lys (cRGDfK) or cyclic Arg-Ala-Asp-
D-Phe-Lys (cRADfK) peptides, leveraging a biotin–avidin coupling method. Through combined magnetic resonance imaging (MRI) and ultrasound imaging, it was demonstrated that cRGDfK-SPION-MBs could bind more rapidly and with higher retention in tumor neovasculature compared to cRADfK-SPION-MBs. Competitive blocking experiments further confirmed the specificity of cRGDfK-SPION-MBs' binding to alpha V beta 3 integrin (α
vβ
3) integrin receptors. This study suggests that cRGDfK-SPION-MBs can be used as a molecular contrast agent for MRI and ultrasound imaging, allowing for multiscale and multimodal molecular imaging of α
vβ
3 integrin expression in the neovascular system of malignant tumors.
The application of microbubbles in ultrasound molecular imaging is expanding its scope, including the detection of cellular activities and the evaluation of biological functions. These techniques are now being utilized for both
ex vivo quality evaluation and
in vivo surveillance of tissue-engineered vascular grafts
[39]. In an effort to delve deeper into the complex molecular crosstalk and synergistic interactions among various abnormally expressed proteins in tumor cells, as well as their dynamic spatial and temporal changes, Li and colleagues
[28] developed α
vβ
3 targeted α
vβ
3 integrin-targeted lipid microbubbles (L-MB
α) VEGFR2-targeted and lipid-poly(lactic-
co-glycolic acid) microbubbles (LP-MB
v). By leveraging the distinct acoustic collapse intensities of L-MB
α and LP-MB
v, they were able to differentiate between two specific biomarkers. Furthermore, they examined the impact of the α
vβ
3 integrin to VEGFR2 ratio on the invasive conduct of human MDA-MB-231 cell breast tumors (
Fig. 2(b) [28]).
Given the size constraints of microbubbles, they are unable to permeate blood vessel walls, thereby making them primarily useful for imaging tumor vessels. Moreover, due to the propensity of gas to diffuse easily and the clearance mechanisms of biological systems, the stability and longevity of microbubbles circulating within the body are relatively limited
[40],
[41]. In order to overcome the challenge of microbubbles' low stability, Deng and colleagues
[42] have ingeniously crafted ultra-stable protein air microbubbles (air@MBs) with a unique triple-layer structure through the ethanol–water exchange method. These protein air@MBs are comprised of an inner gas core, an intermediary protein layer, and an outer semipermeable membrane. The protein air@MBs can maintain stability in aqueous solutions over a prolonged duration, while demonstrating outstanding ultrasound imaging capabilities and drug release characteristics. This makes them ideal for applications in ultrasound-guided drug delivery. Liu and colleagues
[43] built cationic magnetic microbubbles (MB
M) by utilizing a biotin–avidin bridge to tether streptavidin-modified magnetic beads to the surface of cationic microbubbles. The MB
M demonstrated substantial stability during
in vivo blood circulation, and they showed the unique ability to selectively accumulate in the tumor vasculature via magnetic targeting. This led to amplified ultrasound imaging efficacy. Remarkably, this approach can also mitigate the existing challenge of suboptimal
in vivo circulation lifespan of microbubbles.
Despite microbubbles' incapacity to penetrate the vascular wall, Wang and colleagues
[44] showcased the promising endowment of ultrasound-induced microbubble destruction in advancing transvascular imaging. This technique can deliver cyclic Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys (iRGD)-functionalized liposomes loaded with quantum dots (QD), which are attached to microbubbles, into tumors. Subsequently, these QD-loaded iRGD-liposomes can bind with α
vβ
3 integrins, proteins present on the surface of breast cancer cells, thereby facilitating transvascular fluorescence imaging. The process of ultrasound-induced microbubble destruction will be elaborated upon in the next section.
2.1.2. Tumor molecule imaging via nanobubbles
Nanobubbles share a similar structure with microbubbles, consisting of a shell made up of surfactants, phospholipids, polymers, or proteins, and a core filled with gas. However, a crucial difference between nanobubbles and microbubbles is the type of gas they encapsulate. Nanobubbles typically contain perfluorocarbon, unlike microbubbles, which usually house air, nitrogen, or sulfur hexafluoride. This choice enhances nanobubbles' stability and prolongs their in-body circulation
[45],
[46].
Owing to their nanoscale size, nanobubbles can traverse tiny gaps between tumor blood vessel cells. This is aided by the enhanced permeability and retention effect, letting them accumulate in tumor masses. Further, their increased stability and circulation time also allow for greater tumor infiltration
[41]. In comparison to microbubbles, these unique attributes equip nanobubbles with the capacity to deliver more enduring and potent tumor imaging results at the tumor location
[45]. Recent research from Pellow and colleagues
[47] reveals that nanobubbles demonstrate superior acoustic pharmacokinetics and have the ability to passively infiltrate tumors in their entirety. The use of ultrasound stimulation can boost the extravasation of these nanobubbles, thereby enhancing their spatial bioavailability for extended imaging and therapeutic applications beyond the vasculature. Simultaneously, due to the superior permeability of nanobubbles, they can also be deployed for the early detection of other diseases that demonstrate alterations in vascular permeability
[48].
The acoustic response of bubbles is profoundly influenced by their size distribution. Monodispersed nanobubbles display enhanced uniformity in nonlinearity and superior signal-to-noise ratios, which in turn improve detection sensitivity and refine the quality of molecular imaging. Counil and colleagues
[49] convincingly showed that a standard mini-extruder setup can be employed to create these monodispersed nanobubbles both swiftly and efficiently. Significantly, these extruded nanobubbles yielded robust acoustic responses
in vitro and demonstrated remarkably strong and persistent enhancement of ultrasound signals in mice under non-linear contrast-enhanced ultrasound imaging. Reducing the size of nanobubbles not only compromises their stability but also diminishes the intensity of ultrasound echogenic signals. Consequently, the task of designing nanobubbles that possess a size smaller than 200 nm, while maintaining excellent imaging capability and high stability for long-term
in vivo imaging, presents a significant challenge. To address this, Gao and colleagues
[50] employed a core-template-free strategy to fabricate nanobubbles measuring approximately 100 nm. These nanobubbles were filled with perfluoropentane (PFP) and encapsulated within a shell composed of chitosan poly-acrylic acid. Remarkably, despite their diminutive size, these nanobubbles demonstrate exceptional ultrasound imaging capabilities and exhibit prolonged stability within the
in vivo environment.
Kumar and colleagues
[51] developed ultra-stable nanobubbles using a microfluidic-based reconstruction process, and they conjugated these nanobubbles with the tenth type III domain of human fibronectin, which targets human programmed death-ligand 1 (hPD-L1). This allowed for the assessment of hPD-L1 expression within the tumor microenvironment (TME). Using ultrasound imaging, they found a contrast signal triple that of non-targeted nanobubbles, overcoming previous limitations of microbubbles to intravascular imaging. Concurrently, antibodies can also be employed to tailor nanobubbles, thereby facilitating targeted ultrasound molecular imaging of tumor blood vessels
[52]. The dual-mode imaging strategy mentioned earlier for microbubbles can also be extended to nanobubbles. Zhu and colleagues
[53] incorporated SPIONs into an organic poly(lactic-
co-glycolic acid) shell and adorned them with polypeptides that specifically target prostate-specific membrane antigen. Through this approach, they developed nanobubble molecular imaging probes that possess the capability of dual-mode imaging, utilizing both MRI and ultrasound imaging, to detect prostate cancer. Notably, the researchers observed that nanobubble probes with smaller sizes demonstrated enhanced imaging performance, further highlighting the potential advantages of reducing the probe dimensions.
Certain cancer cells are devoid of unique markers, making traditional molecular imaging ineffective. To overcome this challenge, Jugniot and colleagues
[29] capitalized on the homotypic recognition pattern of cancer cells, using the inherent affinity of triple-negative breast cancer (TNBC) cell membranes as a foundation for targeted imaging. Through the application of microfluidic technology, they crafted the nanobubbles based on cancer cell membrane (NB
CCM) that are specifically targeted and based on the cancer cell membrane (CCM). Through ultrasound molecular imaging, it was demonstrated that the NB
CCM, when compared to non-targeted nanobubbles, exhibited a heightened level of extravasation and retention in the TNBC mouse model. The time–intensity graph further showcased a significant amplification of the NB
CCM signal. The innovative approach of biomimetic nanobubble imaging, crafted from the CCM, effectively addressed the challenges posed by TNBC's lack of unique cancer markers and its non-responsiveness to traditional targeting methods (
Fig. 2(c) [29]).
2.1.3. Tumor molecule imaging via nanodroplets
Nanodroplets represent another form of ultrasound contrast agents that achieve nanoscale dimensions. The primary distinction between nanodroplets and nanobubbles resides in the composition of their cores. Typically, nanobubbles incorporate a core made up of low boiling point perfluorocarbon gases, such as C
3F
8 and C
4F
10. In contrast, nanodroplets feature a core composed of high boiling point perfluorocarbon liquids, like C
5F
12 and C
6F
14 [17]. When subjected to high-intensity acoustic pulses, the perfluorocarbon liquid at the core of the nanodroplets rapidly evaporates, triggering a phase transition into microbubbles. This transformation process is accompanied by significant acoustic reflection and scattering, which in turn generates a potent ultrasound signal, beneficial for ultrasound imaging
[54].
Jandhyala and colleagues
[55] have published a study on the application of epidermal growth factor receptor antibody-functionalized phase-change perfluorohexane (PFH) core nanodroplets for ultrasound molecular imaging. Due to the 56 °C boiling point of the PFH core, these nanodroplets possess the ability to evaporate and condense multiple times, thus enabling repeated imaging and consequent enhancement of image contrast. Additionally, the relatively high boiling point of the PFH core contributes to enhanced stability and reduces the likelihood of spontaneous evaporation. Maghsoudinia and colleagues
[56] have developed smaller PFH core nanodroplets, which are functionalized with folic acid and loaded with gadolinium. These nanodroplets are designed for dual-modal imaging of mouse hepatocellular carcinoma cells, utilizing both ultrasound imaging and MRI. Remarkably, these nanodroplets can be triggered by clinical ultrasound, causing them to evaporate and metamorphose into microbubbles. Phase-change nanodroplets, as ultrasound contrast agents, have also demonstrated potential in the diagnosis of other diseases
[57].
Given that the transformation of high-boiling-point liquid perfluorocarbons into a gaseous state demands a significant amount of energy, there is potential promise in utilizing metastable liquid perfluorocarbons with lower boiling points, such as octafluoropropane and decafluorobutane, to fabricate phase-change nanodroplets. These nanodroplets, with their low boiling points, can undergo phase transitions under the pressure and frequency conditions provided by diagnostic ultrasound imaging. This process optimally minimizes the adverse biological impacts associated with high acoustic energy
[58]. Beyond the advantage of penetration due to their diminutive size, nanodroplets in their non-activated state wouldn't interfere with the acoustic rays that combine in the intended focal region. Upon activation and destruction via a sequence of acoustic pulses, they are able to deliver images of super-resolution
[59]. Dong and colleagues
[60] have introduced a novel type of blinking acoustic nanodroplets (BANDs). These nanodroplets are structured with a core filled with perfluorocarbon and a shell formulated from phospholipid materials and surfactant. Remarkably, these BANDs can be activated by acoustic pulses deemed clinically safe and provided by diagnostic ultrasound transducers. They exhibit a reversible vaporization and re-condensation process at an impressive rate of up to 5 kHz, a phenomenon described as “blinking.” This unique characteristic enables rapid, super-resolution ultrasound imaging of the vascular system within live animal muscle tissue.
Ultrasound isn't the only method to stimulate nanodroplets, and there are additional activation strategies available
[30],
[61],
[62],
[63]. Zhang and colleagues
[30] have developed a type of nanodroplet encapsulating PFP, pentafluorobutane (PFB), and doxorubicin (DOX) within a pH-sensitive polyamino acid polymersome. Compact in size (< 180 nm) during circulation, these nanoplatforms effectively localize within tumors. When exposed to the acidic TME (pH ≈ 6.8), they expand to 437 nm, decreasing intradroplet Laplace pressure and thus lowering the evaporation threshold of the enclosed perfluorocarbons. With local heating via infrared light or low-frequency ultrasound (LFUS), these perfluorocarbons transform into gas bubbles (> 500 nm), amplifying the ultrasound detectable echo, making these changes clearly visible (
Fig. 2(d) [30]).
Traditional nanodroplets are characterized by their chemical uniformity, whereas multiphase nanodroplets demonstrate a heightened level of complexity when compared to their traditional counterparts. The internal phases within multiphase nanodroplets facilitate chemical compartmentalization and precise control over the release of active ingredients. Consequently, the manipulation of various phases and internal morphologies within multiphase nanodroplets can also facilitate
in vivo bioimaging
[64]. Furthermore, harnessing the distinct properties of these internal phases allows for the attainment of specific objectives. Leveraging the ability of perfluorocarbon liquids to stabilize saline droplets into durable nanodroplets, Chen and colleagues
[65] have successfully synthesized nanodroplets containing hypertonic saline. Remarkably, these nanodroplets maintain their stability for at least two weeks, and they show significant potential in the area of cancer diagnosis, specifically for ultra-high-frequency radio-frequency acoustic molecular imaging.
2.1.4. Tumor molecule imaging via gas vesicles
Gas vesicles are unique nanostructures characterized by an inflatable core filled with gas and a protein shell. They are present in specific microorganisms, such as bacteria and archaea, serving as gas reservoirs that help regulate the buoyancy of these microorganisms in water
[66]. Unlike unstable microbubbles, gas vesicles effectively exclude surrounding water and allow gas to permeate freely through small pores on their protein shell, maintaining a state of equilibrium and ensuring their physical stability
[67]. The substantial mismatch in acoustic impedance between the gas inside the gas vesicles and the surrounding aqueous medium enables them to generate robust ultrasound contrast. Moreover, certain types of gas vesicles exhibit nonlinear contrast due to their buckling mechanical deformation under ultrasound, which aids in their detection within background tissues
[68]. The primary advantages of gas vesicles stem from their biological origin, as they typically demonstrate excellent biocompatibility and biodegradability within organisms. Additionally, the protein shell of gas vesicles can be chemically modified to serve as drug carriers or for targeted applications
[67],
[68].
Wang and colleagues
[69] have constructed polyethylene glycol (PEG) and hyaluronic acid-modified gas vesicles (PH-GVs) for tumor ultrasound molecular imaging. Uniquely, the PEG modification precludes macrophage internalization of the PH-GVs, demonstrating their capacity to avoid the reticuloendothelial system's clearance mechanism and consequently permitting an extended circulatory lifespan. Their minute size provides the advantage of penetration into the tumor's vascular network. In addition, the hyaluronic acid's targeting capabilities bestow the PH-GVs with superior tumor specificity and retention. PH-GVs generate strong ultrasound contrast at tumor sites in mice, with no noticeable side effects detected after venous injection. During the process of epithelial mesenchymal transition in tumor cells, there is a gradual decrease in the expression of E-cadherin and an increase in N-cadherin. Hao and colleagues
[31] have developed gas vesicles specifically targeting E-cadherin and N-cadherin by coupling antibodies against both proteins to bio-synthesized gas vesicles. These targeted gas vesicles have a particle size of approximately 200 nm and demonstrate remarkable capabilities in targeting tumor cells. They effectively generate strong ultrasound imaging signals, enabling the monitoring of E-cadherin and N-cadherin expression status in tumor cells throughout the progression of the disease (
Fig. 2(e) [31]). The complement component 1 Q subcomponent-binding protein, also known as p32, plays a significant role in tumor metastasis. Hao and colleagues
[70] utilized p32-targeting peptide-modified gas vesicles to conduct their study. Through the use of ultrasound molecular imaging, they observed a gradual and notable increase in the translocation of the p32 protein from the mitochondria to the plasma membrane during the progression of tumors.
Acoustic reporter genes (ARGs) are sophisticated gene constructs designed to encode gas vesicles. When these ARGs are present within cells, they enable the expression of gas vesicles, thereby facilitating their visualization through ultrasound imaging. Zhang and colleagues
[71] have engineered a strain of
Escherichia coli (
E. coli) to carry ARGs and genes that encode near-infrared fluorescent proteins. These
E. coli have the capability to autonomously infiltrate tumor locations, multiply within the tumor environment, and can be visualized using dual-mode optical and ultrasound imaging. This approach offers an alternative method for tumor targeting besides the conventional strategy of direct modification of gas vesicles. Moreover, gas vesicles obtained from various sources exhibit distinct properties and show variations in their ultrasound imaging capabilities
[72],
[73],
[74]. Hurt and colleagues
[72] conducted a phylogenetic screening of candidate gas vesicle gene clusters from a diverse spectrum of bacteria and archaea, leading to the identification of two unique gas vesicle gene clusters. Remarkably, these clusters, when expressed in either bacteria or mammalian cells, can not only give rise to strong non-linear signals, enhancing ultrasound contrast, but also maintain stable expression over extended periods. From these foundations, two ARGs were constructed: one tailored for bacteria and another for mammalian cells. The bacterial ARG empowers the non-invasive ultrasound imaging of bacteria that home to tumors, enabling the visualization of their microscale distribution within living organisms. On the other hand, the mammalian ARG facilitates non-invasive,
in situ microscale ultrasound imaging of human breast cancer cells. This allows for the tracking of tumor gene expression and the progression of tumor growth. Furthermore, it provides the capability for an ultrasound-guided biopsy of a genetically defined subpopulation within these tumor cells.
Gas vesicles also hold the potential to facilitate ultrasound imaging of a diverse range of biomolecules and biological processes
[75],
[76]. Moreover, the exploration of new ultrasound imaging techniques and the optimization of ultrasound imaging conditions can significantly improve the ultrasound imaging performance of gas vesicles, thereby broadening their potential applications
[77],
[78],
[79],
[80].
2.2. Tumor markers separation
Specific tumor markers are pivotal for the early detection of cancer, guiding treatment strategies, and evaluating therapeutic outcomes and patient prognosis. Liquid biopsy offers a streamlined approach to sampling tumor markers by isolating entities derived from tumors present in a cancer patient's body fluids. This includes circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and tumor-related extracellular vesicles (EVs). Once separated, the information these entities carry about the tumor is thoroughly analyzed, allowing for the continuous and dynamic tracking of tumor evolution
[81],
[82].
CTCs are tumor cells that originate from the primary tumor and subsequently enter the bloodstream. Their analysis, drawing from methodologies like single-cell genomics, transcriptomics, and proteomics, along with quantitative assessments based on cell enumeration, can significantly enhance clinical diagnostic accuracy
[83]. ctDNA emerges in the bloodstream as a result of tumor cell apoptosis or necrosis. By analyzing ctDNA, we can uncover cancer-specific genetic and epigenetic aberrations. This not only offers insights into the patient's specific cancer phenotype but also underscores ctDNA's role as a crucial marker for the early detection and prognostication of tumor growth and spread
[84],
[85]. EVs are minuscule nanovesicles expelled by cells into the surrounding extracellular matrix and bodily fluids. Notably, those EVs originating from cancer cells contain a myriad of tumor-specific molecules, including proteins, ribonucleic acid (RNA), and deoxyribonucleic acid (DNA). Such encapsulated information can shed light on the tumor's nature and any mutations it harbors
[86],
[87],
[88],
[89],
[90]. The combination of both quantitative and qualitative examinations of tumor markers sourced from liquid biopsies, along with the simultaneous detection of multiple markers, lays a solid foundation for diagnosing a range of cancers and formulating effective clinical decisions
[85],
[91],
[92],
[93],
[94].
Leveraging the non-contact characteristics, exceptional spatiotemporal resolution, and the notable biocompatibility of acoustic tweezer technology, it has been employed to isolate biological particles, especially extracting various tumor markers from the blood samples of oncology patients
[18],
[95]. The primary categories of acoustic tweezers encompass standing-wave tweezers, traveling-wave tweezers, and acoustic-streaming tweezers. While both the standing-wave and traveling-wave tweezers directly manipulate particles or fluids using the applied acoustic radiation force, the acoustic-streaming tweezers exert indirect control over particles through acoustically induced fluid flows. Each type of acoustic tweezer has its distinct strengths, finding applications in diverse contexts such as cell separation, cell patterning, manipulation and rotation of cells and droplets, as well as fluid mixing and pumping
[18].
Li and colleagues
[96] reported a microfluidic device leveraging standing surface acoustic waves to isolate CTCs from the peripheral blood samples of cancer patients. Utilizing interdigitated transducers (IDTs) angled relative to the fluidic microchannel, they established a standing wave field within the channel, leading to the formation of distinct pressure nodes and antinodes. As cells possessing varied physical attributes, such as size, density, and compressibility, navigate this field, they undergo different extents of acoustic radiation force, causing them to diverge along distinct trajectories for separation. Remarkably, this approach can distinctly segregate rare tumor cells, averaging diameters of 16 or 20 μm, from white blood cells, which are roughly 12 μm in diameter. The method boasts a cancer cell retrieval efficiency exceeding 83% (spanning 83%–96%), while achieving an impressive 90% removal rate for white blood cells. Harnessing the acoustic fields shaped by standing waves, the movement paths of target cells can be tailored, facilitating enrichment after separation
[97]. Additionally, by modulating the frequency of the standing wave, a spectrum of acoustic field patterns can be generated, simplifying the device's design
[98]. Similarly, devices based on traveling surface acoustic waves show promise in CTC separation. Geng and colleagues
[99] introduced a method using slanted IDT to generate a narrow and highly-focused surface acoustic wave beam. This beam can create an acoustic gradient that spans several tens of micrometres, leading to rapid and significant deflections that optimize sorting. The device they developed achieved an impressive efficiency of over 98% in separating CTCs from blood cells and reached a purity level of up to 93%. Importantly, the separated CTCs maintained their cellular vitality and growth potential. Acoustic streaming can also be harnessed for the selective capture of CTCs. The fluid flow triggered by acoustic waves near specially designed microscopic structures results in different flow directions, which guides the trajectory of CTCs, ensuring their separation
[100],
[101].
Several studies have focused on the separation of EVs
[102],
[103],
[104],
[105]. For instance, Wu and colleagues
[102] utilized two separation modules based on standing surface acoustic wave. Each of these modules utilizes a pair of IDTs to establish a standing wave field at a slanted angle, exploiting the different acoustic radiation forces on particles to achieve path deflection and separation. The primary module is designed to filter out miscellaneous blood components, concentrating the EVs, while the secondary module further refines this process by removing other EV subgroups, thereby enhancing the purity of the exosomes. Impressively, this apparatus showcases an outstanding efficiency in exosome isolation from whole blood, boasting a blood cell elimination rate that surpasses 99.999%. In a recent study, Gu and colleagues
[103] described a setup where a droplet is secured within a circular polydimethylsiloxane containment ring, situated between two slanted IDTs. This configuration capitalizes on the synergy between acoustic propulsion and droplet rotation dynamics, enabling the enrichment and separation of nanoparticles, resulting in the development of a droplet-based acoustofluidic centrifuge system. Surface acoustic waves can propel the droplet into rotation around its central axis, amplifying the internal streaming velocity and shear rates within the droplet by 10 to 100 times. By harnessing both acoustic radiation forces and the drag force exerted on the particles, they are driven in a spiral motion towards the center of the droplet, facilitating nanoparticle concentration. Remarkably, this mechanism offers swift concentration of entities ranging from DNA molecules to micrometer-scale particles. Furthermore, a sophisticated dual-droplet acoustofluidic centrifuge variant can segregate nanoparticles based on varied size distributions, such as specific exosome subpopulations. Certain tumors, like brain tumors, which are robustly shielded by barriers, tend to release fewer tumor markers into the bloodstream. By employing ultrasound, we can significantly boost the permeability of these barriers, leading to an increased release of associated tumor markers
[106],
[107],
[108]. We will delve into the specific mechanisms in subsequent chapters.
Acoustic tweezer technology employs the acoustic radiation force and acoustic streaming to separate and manipulate particles. At its core, this is a force resulting from the gradient present in the acoustic field. To delve into the underlying concepts of acoustics and fluid mechanics in sound wave-driven particle and fluid operations, as well as the relevant calculations, design principles, and a detailed overview of the construction of acoustic tweezer apparatuses, please consult the most recent review
[109],
[110],
[111]. Acoustic tweezers and acoustofluidic technologies showcase remarkable potential in areas such as cell analysis, drug screening and encapsulation, as well as direct cell manipulation
[112],
[113],
[114],
[115],
[116],
[117]. Beyond providing a novel breakthrough in ultrasound-enabled nanomedicine for tumor diagnosis, these advances also illuminate a vast realm of possibilities for the broader medical landscape in the future.
3. Ultrasound-enabled nanomedicine for precise drug delivery
3.1. Physiological barrier penetration
For effective tumor treatment, it is pivotal that the injected anti-tumor drugs reach the tumor site successfully. This delivery process entails navigating through the complex biological barriers constituted by vascular structures and heterogeneous tissue, while overcoming the robust defense mechanisms inherent in the tumor's immunosuppressive microenvironment. Achieving a therapeutically effective concentration of the drug in the vicinity of tumor cells is paramount for favorable treatment efficacy.
Options are limited for patients suffering from primary brain tumors or cerebral metastases. The conventional choices revolve around surgical removal, systemic chemotherapy, and radiotherapy. This paucity of viable alternatives largely owes itself to the strict biological protection encapsulating the central nervous system, which presents a daunting challenge to effective drug delivery efforts. This task grows even more daunting when confronted with the blood–brain barrier (BBB) and the blood–spinal cord barrier (BSCB)
[19],
[118]. The BBB is an intricate physiological construct composed of tightly adhered endothelial cells, which are further swathed and overlaid by pericytes and astrocyte end-feet. These units work in unison to form a highly ordered and distinctly selective structure known as the neurovascular unit. The BBB exerts strict control over the bi-directional traffic of substances between blood circulation and the central nervous system. It effectively precludes most molecular inhabitants of the blood from infiltrating the brain parenchyma, thus safeguarding the stability of the neural microenvironment
[19],
[119]. The BSCB, on the other hand, is built by closely sealed connections between epithelial cells overlaying the cerebrospinal fluid surface of the choroid plexus. It prevents the entrance of most substances in the blood to the cerebrospinal fluid, yet its permeability is elevated compared to the BBB's
[118].
Tumor progression can lead to the deterioration of the BBB, resulting in a highly variable and dynamically complex blood–tumor barrier (BTB). In comparison to the BBB, the BTB is of greater penetrability, yet this alone does not enable substantial drug accumulation at the tumor site
[6]. Following vascular wall traversal, the drug's obligation remains to traverse the TME, before finally landing at the tumor cells. However, the peculiarities of the TME—including its dense biomatrix constitution, profuse population of pro-tumor proliferative cells, amplified interstitial fluid pressure, and stymied immune cell infiltration—can further impede drug accumulation within the tumor. These cumulative characteristics introduce a convoluted barrier that complicates drug transport and distribution, potentially impairing the intended therapeutic impact of the drug
[120].
Ultrasound possesses the ability to reversibly breach biological barriers, enhancing the penetrability of drugs, and thus elevating their accumulation within tumor sites. The disruption of these biological barriers is primarily enabled by the cavitation of ultrasound contrast agents, facilitated by ultrasound waves. In environments with low acoustic pressure, the cavitation nuclei of these contrast agents oscillate in sync with the incoming ultrasound waves. They go through cyclical phases of expansion and contraction, generating what is known as stable cavitation. In the vicinity of the BBB, these cavitation nuclei exert mechanical force on adjacent endothelial cells—expansion mechanisms push against endothelial cells, while contraction induces invagination in the endothelial cell lining. Both these mechanisms serve to pry open the tight junctions between these endothelial cells (
Fig. 3(a))
[121],
[122]. However, under high acoustic pressure conditions, cavitation nuclei experience a magnified level of inertial cavitation, leading to violent collapses. This results in the release of potent shock waves, microjets, and shear stresses, which can lead to perforation of the cell membrane and significant opening of the BBB. Nonetheless, it is of paramount importance to note the potential adverse outcomes. Excessively intense inertial cavitation can result in harmful effects, such as cell death, neuroinflammation, and the formation of thromboses
[119],
[120].
Fig. 3 [121],
[123],
[124],
[125],
[126],
[122] is the overview of the representative strategies of ultrasound-induced physiological barrier penetration and cell membrane perforation.
Wu and colleagues
[123] have successfully enhanced the delivery of their specialized “all-in-one” nanoparticles, known as AMPTL (nanoparticles formed by self-assembly of paclitaxel (PTX)-thioketal linker (TL)-PEG-1000 (PEG
1K)-3-methyladenine (3-MA), 1,2-distearoyl-
sn-glycero-3-phosphoethanolamine-
N-[methoxy-(PEG)-2000] (DSPE-PEG
2K), and angiopep-2 peptide-modified DSPE-PEG
2K (DSPE-PEG
2K-angiopegp-2)), to intracranial glioma tissue. They achieved this by using focused ultrasound (FUS) and microbubbles to reversibly disrupt the BBB. AMPTL is loaded with autophagy inhibitor 3-MA and anti-tumor drug PTX. Moreover, by modifying these nanoparticles with the angiopep-2 peptide, they are uniquely equipped to target glioma cells that express the low density lipoprotein receptor-related protein 1 (LRP1). In response to ROS, AMPTL releases its drug payload, with 3-MA effectively obstructing autophagy. The result is an amplified effect on PTX-induced cell cycle arrest and apoptosis (
Fig. 3(b) [123]). Yang and colleagues
[127] harnessed FUS combined with microbubbles to breach the BBB locally. This allowed them to deliver a targeted clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated protein 9 (Cas9) gene-editing system, pinpointing temozolomide-resistance genes, directly into glioblastoma (GBM) cells. This approach consequently suppressed the expression of these resistant genes, thereby amplifying the therapeutic potency of temozolomide against GBM.
Liang and colleagues
[124] have developed indocyanine green-dopped microbubbles (MBs-ICG). Indocyanine green (ICG), with its distinctive bright fluorescence in the second near-infrared window (NIR-II), exemplifies superior photothermal capabilities. MBs-ICG showcase bright fluorescence within the NIR-II range, elevated ultrasound contrast, and properties of ultrasound-induced size transformation. When exposed to FUS, the MBs-ICG can penetrate the BBB, the course of which can be concurrently monitored due to the fluorescence in the NIR-II. Following this process, MBs-ICG transform into lipid-ICG nanoparticles, enhancing the efficiency of photothermal therapy (PTT) targeted at GBM cells (
Fig. 3(c) [124]). In response to the potential cellular damage that can be inflicted by intense inertial cavitation, Wang and colleagues
[125] have devised a monitoring system named microbubbles loaded with annexin V-conjugated albumin–ICG particles. They achieved this by encapsulating annexin V bound to the surface of albumin-ICG particles, configured as near-infrared nanoprobes, within the cavity of lipid-poly(lactic-
co-glycolic acid) hybrid microbubbles. This system is specifically designed to evaluate early cell apoptosis events that are triggered by the opening of the BBB, a process guided by FUS and microbubbles. Upon the collapse of the microbubbles, the annexin V-conjugated albumin–ICG nanoparticles (AV-ICG-NPs) are released. These particles have the ability to bind to the phosphatidylserine (PS) present on the membrane of apoptotic cells, an early biomarker of cell apoptosis. This binding allows for an indication of the extent of BBB damage. Additionally, antioxidants or neuroprotectants can be encapsulated within the microbubbles to suppress or reverse damage caused by cavitation (
Fig. 3(d) [125]).
The synergy of FUS and microbubbles has been clinically substantiated to aid in the permeation of the BBB and amplify drug delivery to the brain, thereby effectively augmenting the penetration of antibody-based and chemotherapy drugs into brain tumor territories
[128],
[129]. Presently, innovative devices tailored for FUS disruption of the BBB are under development, aiming to provide more precise and controllable disruption, thereby enhancing therapeutic outcomes for brain tumor patients
[130]. In addition to microbubbles, nanobubbles also possess the capability to facilitate the opening of the BBB
[131],
[132]. Compared to microbubbles, nanodroplets exhibit less intensified but more sustained inertial cavitation capacities. This unique characteristic perhaps enables them to orchestrate the opening of the BBB in a way that is not only safer but also more efficient
[133]. Curry and colleagues
[134] have successfully converted poly(
L-lactic acid) (PLLA), a biodegradable polymer, into biodegradable and biocompatible PLLA nanofibers with stable piezoelectric properties. These PLLA nanofibers can be implanted in the brain as ultrasound transducers, emitting ultrasound waves to stimulate the cavitation of microbubbles, thereby opening the BBB and enhancing drug delivery. Moreover, these PLLA nanofibers perform efficaciously within a defined lifespan, and in the end, disintegrate safely and naturally without any intervention.
As a tumor progresses, it manifests biological traits inherently divergent from those of normal tissues, such as escalated solid stress, increased interstitial fluid pressure, magnified hardness, alterations in material and tissue microstructural properties. These transitions markedly impinge on the efficacy of drug delivery
[135]. In terms of tumors that are robustly safeguarded by biological barriers, realizing successful drug delivery entails the surmounting of three primary hurdles: perfusion, extravasation, and extracellular matrix infiltration, which in turn requires devising relevant strategies
[136]. Such issues can be tackled by transforming the tumor's physiological parameters through either physical means such as heat, magnetic fields, ultrasound, and radiation, or chemical modalities including enzymes, cytokines, and small interfering RNA (siRNA), effectively dampening the aberrations of the TME. Furthermore, through the chemical modulation and regulation of tunable properties inherent to nanomedicines, we can purposefully boost the distribution of nanodrugs and elevate their delivery efficiency
[137].
The presence of the BBB and BTB, coupled with the high interstitial fluid pressure within tumors, poses significant challenges to the convective transport of drugs from the bloodstream into the tissue. Furthermore, the steric and adhesive interactions of the tumor cells' extracellular matrix also impede the dispersion of drugs. Curley and colleagues
[138] have found that by using MRI as a guide, the application of FUS and microbubbles to open the BTB and BBB significantly improves interstitial tumor flow. This enhancement, in turn, bolsters the transport and efficacy of drugs within the tumor tissues. Fusing the capabilities of FUS and microbubbles to mitigate vascular and interstitial transport impediments with the propensity of cationic nanoparticles to extend RNA circulation and heighten cellular uptake, Guo and colleagues
[139] have discovered a potent approach. By utilizing cationic lipid–polymer hybrid nanoparticles as siRNA carriers and leveraging FUS accompanied by microbubbles to augment drug penetration, they have created a composite strategy that markedly elevates the delivery of siRNA in the TME of brain tumors. Chen and colleagues
[140] have found that the disruption of the BBB, facilitated by FUS and microbubbles, could potentially trigger a shift from an immunosuppressive TME to an immunostimulatory one. This transformation could provide further therapeutic advantages.
As highlighted in the preceding text, enhanced tumor penetration has been observed with the synergistic use of ultrasound and microbubbles. However, the precise mechanisms underlying this improvement are still not fully understood. It's unclear whether the enhanced tumor infiltration is primarily due to an increase in vascular permeability or is a result of improvements in the TME. Further detailed research and literature support are required for clarification. An innovative strategy involves the infusion of high-purity oxygen into microbubbles, targeting tumor hypoxia reduction and consequently diminishing the impact of immune suppression
[141].
3.2. Cell membrane perforation
Cavitation nuclei, triggered by ultrasound, is capable of penetrating biological barriers, offering an effective means for drug accumulation in the vicinity of tumor cells. For some drugs that function within tumor cells, there is a need to cross the cell membrane barrier for absorption; an activity usually not initiated by tumor cells themselves. To address this, researchers have studied a series of physical penetration techniques developed specifically for efficient material delivery into cells
[142]. Sonoporation, one such technique, has exhibited immense potential and has been demonstrated to successfully ferry drugs, genes, and so forth into tumors
[20],
[143].
Sonoporation capitalizes on ultrasonically guided cavitation nuclei to breach the cell membrane. Under high acoustic pressure, cavitation nuclei undergo a collapse through inertial cavitation, generating potent shock waves and microjets. These shockwaves and microjets act upon adjacent cell membranes, causing perforations
[120],
[142],
[144]. However, as the cavitation nuclei collapse, high temperatures and ROS are produced, creating conditions that demand careful management to ensure cell viability and efficacy of drug delivery
[142]. In contrast, under conditions of low acoustic pressure, cavitation nuclei that maintain stable cavitation can induce fluid flow, generating shear stress. This shear stress is capable of triggering the formation of transient pores in the cell membrane. However, this mechanism necessitates precise control over bubble size and the spatial relationship between the bubble and the cell
[120],
[144],
[145].
Ultrasound and microbubble-mediated sonoporation has been shown to effectively catalyze the creation of pores within the cell membrane, thereby significantly boosting the efficiency of drug delivery. Achieving a deeper comprehension of the acoustic demeanor of microbubbles equips us with the capability to explore more favorable conditions for sonoporation, leading to the development of safer and more effective methods of drug delivery
[146],
[147],
[148]. As it stands, the amalgamation of ultrasound and microbubbles has proven to be an effective tool in amplifying the absorption of chemotherapy drugs within tumor cells
[149],
[150],
[151].
Recent research has revealed that sonoporation, which utilizes ultrasound and microbubbles, significantly enhances the delivery of various novel therapeutic systems. These include inorganic nanoparticles, organic nanoparticles, genes, and small molecule drugs
[152],
[153]. The intracellular cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) pathway plays an instrumental role in launching adaptive anti-tumor immunity via antigen-presenting cells (APCs). 2′,3′ cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) naturally triggers the cGAS-STING pathway, but its effectiveness is hindered by several challenges. It struggles to permeate cells, offers poor stability in serum, lacks specificity, and is quickly cleared from tissues. Li and colleagues
[154] bound cGAMP to a biocompatible branched cationic polymer to form a nanoparticle composite, which was then attached to microbubbles targeting APCs (modified with antibodies against cluster of differentiation (CD) 11b). When these microbubbles encountered ultrasound, they oscillated and instigated the formation of transient pores on the cell membrane. By harnessing sonoporation, they were able to enhance the infiltration of cGAMP into the cytosol of APCs. This action ignited the cGAS-STING pathway and subsequent downstream inflammatory responses, thereby driving the activation of tumor-specific T cells. Ilovitsh and colleagues
[155] have demonstrated that the oscillation and cavitation effects of microbubbles can be significantly amplified by applying LFUS at 250 kHz. During
in vivo investigations, microbubbles targeted at tumor cells were mixed with plasmids encoding interferon-beta (pIFN-β) and subsequently injected. As a result of the heightened cavitation exhibited by microbubbles under the influence of LFUS, the viability of the tumor cells was notably reduced. Concurrently, sonoporation allowed pIFN-β to effectively penetrate into the tumor cells. The subsequent secretion of IFN-β then greatly promoted the mobilization of immune cells, predominantly leading to an increase in macrophages and CD 8 positive T cells.
Other ultrasound contrast agents have also exhibited effective sonoporation capabilities, along with unique advantages distinct from microbubbles, such as the enhanced permeability of nanodroplets and the ability of gas vesicles to induce intracellular cavitation. Dong and colleagues
[156] designed a platform identified as plasmid-loadable magnetic/ultrasound-responsive nanodroplets (PMUNDs). These PMUNDs encapsulate SPIONs and PFP within a lipid layer providing a surface onto which plasmids can attach. Guided by an external magnetic field, PMUNDs find their way into tumors via a magnetically-induced permeation of the tumor's blood vessels, effectively focusing the treatment at the targeted locations. Upon being exposed to FUS, the PMUNDs undergo a remarkable phase transition, morphing into echogenic microbubbles. As a next step, these microbubbles, through inertial cavitation, facilitate the formation of transient pores on the cell membrane, a change that dramatically boosts the permeability of tumor cells to the plasmids. Xie and colleagues
[126] have developed a novel gene delivery technique, which employs ultrasound to facilitate the direct conveyance of plasmid DNA (pDNA) into the cell nucleus. This method utilizes cell internal cavitation, underpinned by gas vesicles. The cells can absorb the pDNA-binding gas vesicles (pDNA-GVs), and upon exposure to ultrasound, swiftly transport the pDNA into the nucleus. Intriguingly, in the absence of ultrasound, the pDNA-GVs maintain stability within the cytoplasm, thereby enabling a time-controlled delivery of nuclear genes. Through this innovative strategy, they have actualized the spatiotemporal control of the nuclear delivery of E-cadherin genes (
Fig. 3(e) [126]).
Sonoporation holds potential in various medical applications, not limited to tumor treatment. Belling and colleagues
[157] demonstrated a sonoporation-based acoustofluidic platform that enables high-throughput plasmid delivery to human T lymphocytes, peripheral blood mononuclear cells, hematopoietic stem, and progenitor cells. Cells treated by the acoustofluidic platform showed effective cytoplasmic DNA delivery. Therefore, the sonoporation-based acoustofluidic technology has the potential to serve as a platform for the production of genetically modified immune cells, promoting the development of cancer immunotherapies. Furthermore, the application of sonoporation can markedly enhance the efficiency of drug delivery in the treatment of various other diseases
[158].
3.3. Controlled drug release
At present, the development of stimulus-responsive drug delivery systems stands at the forefront of research, aiming to refine and optimize drug delivery for tumors. These innovative systems not only prolong the drug's circulation time in the bloodstream but also ensure a targeted, controllable release at tumor sites, minimizing potential adverse reactions
[159],
[160],
[161]. Such systems are adept at responding to both endogenous stimuli, like pH levels, ROS, glutathione, and enzymes, as well as exogenous triggers, including light, heat, ultrasound, and magnetic fields
[159]. Ultrasound, in particular, offers a unique advantage: it can pinpoint and target specific tissue regions from outside the body. Owing to its superior biocompatibility and minimal attenuation in tissues, ultrasound can channel ample energy directly to the desired area without compromising surrounding tissues. When combined with ultrasound-responsive drug delivery systems, it sets the stage for precise drug delivery, allowing for refined adjustments in dosage, spatial targeting, and release timing
[21],
[162].
Based on the different ultrasound-responsive characteristics of specific carriers, current work can mainly be divided into three major categories: bubble-based carriers, organic-based carriers, and inorganic-based carriers (
Table 1 [28],
[29],
[30],
[31],
[37],
[38],
[39],
[42],
[43],
[44],
[47],
[48],
[49],
[50],
[51],
[52],
[53],
[55],
[56],
[57],
[58],
[59],
[60],
[67],
[68],
[69],
[70],
[71],
[72],
[73],
[74],
[75],
[76],
[123],
[124],
[125],
[126],
[127],
[131],
[132],
[133],
[153],
[154],
[155],
[156],
[163],
[164],
[165],
[166],
[167],
[168],
[169],
[170],
[171],
[172],
[173],
[174],
[175],
[176],
[177],
[178],
[179],
[180],
[181],
[182],
[183],
[184],
[185],
[186],
[187],
[188],
[189],
[190],
[191],
[192],
[193],
[194],
[195],
[196],
[197],
[198],
[199],
[200],
[201],
[202],
[203],
[204],
[205],
[206],
[207],
[208],
[209]).
3.3.1. Controlled drug release via bubble-based carriers
Ultrasound-responsive bubble-based carriers primarily encompass microbubbles, nanobubbles, nanodroplets, and gas vesicles. These were originally deployed as ultrasound contrast agents, with their fundamental characteristics discussed in earlier chapters. Current cutting-edge research is exploring the use of cavitation to transform these entities into ultrasound-responsive drug delivery systems. Concurrently, these carriers hold the promise of unifying ultrasound imaging, barrier penetration, and drug delivery into one comprehensive system.
(1)
Microbubbles. Drugs can be directly dissolved in the components of the microbubble shell or the internal oil layer within the shell. Alternatively, they may be attached to the microbubble shell's surface through electrostatic forces, or they may be directly linked to the molecules of the microbubble shell
[23]. Utilizing microbubbles as carriers, the occurrence of acoustic pressure surpassing the critical amplitude may result in the rupture of the microbubble shell, subsequently triggering drug release
[120],
[143],
[145].
Beguin and colleagues
[163] harnessed biotin and avidin as bridging molecules to deposit the antimetabolite drug gemcitabine and the sonosensitizer rose bengal onto the surface of magnetically functionalized microbubbles. They synthesized a unified unit by integrating a magnetic array and an ultrasound transducer, aiming to concentrate and activate these microbubbles at the targeted site simultaneously. The
in vivo trials demonstrated that this combined approach significantly amplifies the efficacy of tumor drug delivery in comparison to using isolated devices or standalone microbubble treatments. Ingram and colleagues
[164] engineered a targeted, drug-loaded liposome–microbubble complex system. Their research demonstrated that the presence of microbubbles can substantially boost the concentration of chemotherapeutic drugs at tumor locations, while concurrently mitigating their bioavailability and toxicity in healthy tissues.
(2)
Nanobubbles. Due to the enhanced permeability brought by their smaller size, nanobubbles can more effectively deliver drugs, and in addition, they can carry multiple drugs at the same time to form a complex but stable system
[210],
[211].
Chan and colleagues
[132] encapsulated Fe–Pt nanoparticles and the DOX within the hydrophobic core of nanobubbles, further modifying them with transferrin to specifically target GBM cells overexpressing transferrin receptors. Fe–Pt not only boosts the resolution of MRI, but also employs its magnetic properties to steer the nanobubbles toward the tumor site. Exposure to high-intensity FUS (HIFU) induces nanobubble rupture, triggering cavitation to open the BBB and facilitating the infiltration of DOX into the tumor site.
(3)
Nanodroplets. The advantage of nanodroplets is twofold: they function as compact drug carriers and, following phase change, generate robust ultrasound contrast. Owing to their remarkable
in vitro and
in vivo multi-responsive and phase-changing capabilities, nanodroplets facilitate the integration of multiple functions, including imaging and drug delivery
[63],
[212].
Wang and colleagues
[165] developed size- and charge-transformable liposomal nanodroplets (SCGLNs) loaded with a gemcitabine prodrug and PFP. Upon entering tumor blood vessels, these SCGLNs, stimulated by ultrasound, undergo a liquid-to-gas phase transition due to PFP, transforming from nanodroplets to microbubbles. This process creates cavitation, leading to the permeabilization of the vascular wall. Subsequently, they morph back into liposomes and seep into the tumor periphery. As the acidic TME triggers the hydrolysis of dimethylmaleic amides within the liposome fraction, the SCGLNs' charge reverses from negative to positive. This rapid transformation allows the SCGLNs to penetrate deeper into the tumor tissue, thereby promoting a comprehensive delivery of gemcitabine throughout the tumor.
(4)
Gas vesicles. Gas vesicles, possessing distinct acoustic responsiveness and remarkable inertial cavitation capabilities, can be designed to target and disrupt specific cells by binding with their surface receptors. In parallel, bioengineered therapeutic bacteria, loaded with both gas vesicles and drug molecules, can undergo lysis triggered by the gas vesicle-mediated cavitation, which then leads to the release of the co-delivered drug molecules
[166]. Gas vesicles have demonstrated their potential as direct drug delivery carriers, with the capability to transfer genes into cell nuclei
[126]. Most notably, their inherent stability enables gas vesicles to function as oxygen carriers, providing a solution for tumor hypoxia. Meanwhile, micro/nanobubbles encounter a decrease in stability caused by the substantial pressure gradient and surface tension induced during oxygen encapsulation. However, gas vesicles are exempt from this complication
[213].
3.3.2. Controlled drug release via organic-based carriers
Ultrasound-responsive organic-based carriers primarily include liposomes, polymer micelles, polymersomes, and hydrogels. These carrier systems possess self-assembly characteristics and can encapsulate drugs within their structure. Different from bubble-based carriers, ultrasound-responsive organic-based carriers usually do not have a gas core. Their response to ultrasound depends on specific characteristics of their self-assembled structure
[23].
(1)
Liposomes. Liposomes serve as versatile nanoscale platforms for drug delivery, adept at encapsulating a broad spectrum of both hydrophobic and hydrophilic medications. They also readily lend themselves to functionalization with targeted ligands. Currently, a substantial number of liposome-based medications have not only been approved, but are also actively being employed in medical practices
[214].
Thébault and colleagues
[167] encapsulated combretastatin A4 phosphate (CA4P), a vascular disrupting agent, and maghemite (γ-Fe
2O
3) nanoparticles together in the aqueous core of a liposome, creating a ultra magnetic liposome charged with CA4P (CA4P-UML). By leveraging magnetic properties, they specifically directed the CA4P-UML towards tumor cells. They then employed HIFU to locally elevate the temperature to around 43 °C, the transition point of the lipids, thereby triggering the release of CA4P. The findings revealed that this highly efficient targeting and delivery system dramatically reduced the overall required dosage of the medication. Adopting a similar strategy, Zhang and colleagues
[168] achieved a release rate of 80% for resiquimod from thermosensitive liposomes within 5 min at 42 °C, demonstrating the impressive efficiency of the localized ultrasound heating. Furthermore, it has been shown that the refined MRI-guided FUS can boost the ultrasound reactivity of liposomes. Santos and colleagues
[169] have demonstrated that employing fractionated, ultrashort (∼30 s) thermal exposures (∼41 to 45 °C), as opposed to prolonged continuous high-temperature exposure, enhances the release of DOX from thermosensitive liposomes and facilitates its absorption by tumor tissues, thus significantly improving the efficacy of targeted hyperthermia treatments.
Ultrasound can also trigger the release of drugs from liposomes through various mechanisms based on non-thermal responses
[170],
[171],
[172],
[173]. As an example, Li and colleagues
[170] utilized the unique property of the hollow sono-sensitive TiO
2 shell, which generates ROS in response to ultrasound. This triggered the breakdown of the ROS-responsive liposome layer, which was wrapped around the outside of the hollow sono-sensitive TiO
2 shell, consequently leading to the release of the encapsulated drugs (
Fig. 4(a) [170]). In the research conducted by Batchelor and colleagues
[172], nanobubbles and drugs were jointly encapsulated within liposomes. They exploited the acoustic responsive capabilities of the nanobubbles to prompt the breakdown of the liposomes. The specific underlying mechanism for this could be attributed to the phase transformation induced by the mixed droplets present inside. Moreover, liposomes have demonstrated considerable promise in facilitating the delivery of anti-tumor drugs to the brain
[173],
[215].
(2)
Polymer micelles. Polymer micelles are formed through the self-assembly of amphiphilic polymers. A variety of hydrophobic and hydrophilic polymer blocks can be combined to achieve optimal loading, stability, and delivery to target cancer tissues. Drugs can be conjugated with the polymer at the distal ends to prepare a polymer system, which gives the conjugate enhanced solubility and stability
[216]. The primary factors that impact the ultrasound responsiveness of self-assembled block copolymer nanoparticles are the nature of the solvent and the ambient temperature. Spherical polymer micelles, being thermodynamically stable solid nanoparticles, are inherently insensitive to ultrasound
[217]. This challenge can be effectively tackled by formulating micelle molecules integrated with either ultrasound-responsive bonds or various other stimulus-responsive bonds
[174],
[175],
[176].
Shi and colleagues
[175] have developed polymer micelles utilizing amphipathic bottlebrush-like polymer dextran-(poly[(oligo(ethylene glycol) methyl ether methacrylate-
co-oligo(ethylene glycol) methacrylate)] (POEGMA))
9-
b-(poly[2-(methylthio)ethyl methacrylate] (PMTEMA))
22 (DOS), and have successfully co-loaded this micellar structure with DOX and sonosensitizer purpurin 18 (P18). The hydrophobic thioether chain within PMTEMA, a component of DOS, can respond to singlet oxygen produced by external ultrasound and endogenous H
2O
2, serving as a trigger switch for structural disassembly (
Fig. 4(b) [175]). Liang and colleagues
[176] developed a novel type of block copolymer micelles, responsive to ultrasound, based on a Ru(II) complex that links polypropylene glycol and PEG segments. The Ru(II)-terpyridine bond in the micelle acts as the ultrasound-responsive component. When exposed to HIFU, the structure breaks down, causing the micelles to dissolve and release encapsulated drugs. Acid-responsive polymer micelles, capable of achieving increased drug release in the acidic TME, represent a promising approach in the development of multi-responsive polymer micelle systems
[174]. Polymer micelles that respond to temperature changes likewise hold potential for their use in ultrasound-responsive drug delivery
[216].
(3)
Polymersomes. Contrary to polymer micelles which consist of a hydrophilic exterior encapsulating a hydrophobic core, polymersomes represent hollow structures that feature one or multiple lamellar bilayer shells encasing a hydrophilic core. Such polymersomes are capable of containing hydrophilic drugs within their inner void, whereas hydrophobic drugs integrate within the polymersome's membrane, a process akin to the drug encapsulation approach used by liposomes
[218],
[219]. Several modifiable characteristics of polymersomes, such as the comprehensive structure of the polymer segments, the topological configuration of the polymers, and the thickness of the membranes, contribute to their multi-functionality and adjustability. Evidence suggests that the drug release rate of polymersomes correlates with the molecular weight of the constituent block copolymers. Furthermore, these polymersomes are likely to demonstrate enhanced responsiveness in acidic environments
[177].
Wei and colleagues
[178] have engineered a multi-responsive polymersome by self-assembling poly(ethylene oxide)-
block-poly(2-(diethylamino)ethyl methacrylate)-
stat-poly(methoxyethyl methacrylate) (PEO-
b-P(DEA-
stat-MEMA)) block copolymer. In this construct, PEO serves as the hydrophilic component due to its superior biocompatibility and extended in-body circulation time, whereas MEMA is chosen as the hydrophobic component for its notable ultrasound responsiveness. The pH-sensitive DEA is utilized for the protonation of tertiary amine groups, enabling the capture of H
+ for endosomal escape. Through the application of external ultrasound, the delivery of the loaded anti-tumor drug, DOX, can be significantly expedited (
Fig. 4(c) [178]). A wide array of amphiphilic block copolymer designs is available, each specifically engineered to suit various stimulus-responsive strategies
[179]. Additionally, polymersomes have made significant strides in advancing the delivery of proteins and nucleic acids for a myriad of therapeutic applications
[220].
(4)
Hydrogels. Hydrogel is a network of polymers interlinked with each other. This network can either be established through covalently cross-linked polymer chains, be stabilized via metal ion-mediated electrostatic interactions, or be driven towards self-assembly in water due to non-covalent intermolecular forces. The hydrogel's 3D lattice structure can not only absorb significant amounts of water, but it also possesses the ability to encapsulate drugs
[221]. A variety of gel-based pharmaceutical delivery systems can significantly amplify the effectiveness of drug release and transportation with the support of ultrasound
[182],
[183].
Hydrogels crosslinked with calcium provide amplified drug release when subjected to ultrasound stimulation. In the absence of stimulation, the hydrogel network re-crosslinks, resulting in only a minimal baseline level of drug release. Emi and colleagues
[180] evaluated the impact of single and multiple pulse ultrasounds of various amplitudes and durations on the drug release performance of calcium-crosslinked alginate hydrogels loaded with various drug models. Their research indicated that multi-pulse ultrasound exposure can facilitate substantial drug release, while concurrently maintaining gel erosion and temperature rise at manageable levels to prevent compromise to the hydrogel's integrity. Kubota and colleagues
[181] prepared calcium alginate hydrogel microbeads encapsulating tungsten particles and drug model, enabling effective on-demand drug delivery in terms of time and dosage. The high acoustic impedance of tungsten particles allows them to act as an ultrasound-responsive release catalyst. These encapsulated tungsten particles induce local changes in the acoustic impedance of the hydrogel microbeads, conferring a high sensitivity to 20 kHz ultrasound. This sensitivity enables the microbeads to effectively respond to ultrasound, thereby enhancing the release rate of the drug model. Notably, these hydrogel microbeads maintain their effectiveness in releasing the drug model, even in cavitation-suppressed conditions, which typically hinder drug release. This indicates the dominant role of the ultrasound-responsive capabilities mediated by tungsten particles. Furthermore, an additional layer of poly-
L-lysine on the hydrogel microbeads can be employed to control the baseline leakage of the drug model (
Fig. 4(d) [181]).
3.3.3. Controlled drug release via inorganic-based carriers
The ultrasound-responsive inorganic-based carriers mainly include mesoporous silica nanoparticles (MSNs), gold nanoparticles (AuNPs), and iron oxide nanoparticles
[222]. These inorganic nanoparticles can be shaped into a diverse range of geometric shapes, structures, and dimensions
[223]. Drugs can adhere to or bind with the surfaces of these inorganic nanoparticle carriers. The presence of free dissolved gases or interfacial gaseous voids on their rough surfaces can serve as cavitation nuclei, facilitating the irreversible release of drugs through an ultrasound-induced cavitation
[23]. Furthermore, the development of stimulus-responsive drug delivery systems rooted in semiconductor materials is underway
[224].
(1)
MSNs. The salient advantage of using mesoporous nanoparticles for drug delivery lies in their adjustable, orderly, and consistent pore structure that spans from 2 to 50 nm, coupled with their large surface area, high porosity, and adequate pore volume, providing an abundance of adsorption sites for drug molecules. Among these, MSNs, the most extensively studied form of mesoporous nanoparticle drug delivery systems, are frequently utilized due to their superior attributes
[225],
[226].
MSNs predominantly exhibit a spherical configuration. This allows them to house a variety of drugs within their internal pores or to be tailored into hollow forms, encapsulating drugs at their centers
[184],
[185],
[227],
[228]. Additionally, they can serve as a coating, enhancing the attributes of other materials
[229],
[230]. Surface modifications or enveloping them with different substances can further amplify their capabilities
[231],
[232],
[233].
Conventionally, highly ordered MSNs are synthesized using surfactant-templating methods
[225],
[234]. Adjusting the conditions of the synthesis process allows for the modification of MSNs into various shapes. Factors such as particle size, porosity, and surface modifications of MSNs significantly impact their drug delivery behavior. Additionally, surface chemistry commands several factors such as the duration of circulation in the bloodstream, ensuing immune responses, and the efficiency of delivery via specific cellular uptake pathways
[234]. These are essential areas of focus for improvements in MSN-based drug delivery systems. Methods of improvement include incorporating various components like polymers for surface modifications, integrating different organic and metallic species to adjust the properties of the silica framework, and using advanced synthesis methods alongside new swelling agents for size adjustment of the mesopores
[234],
[235].
Stimulus-responsive MSN drug delivery systems have been widely applied in the diagnosis and treatment of numerous diseases
[226],
[235],
[236]. MSNs for drug delivery can be activated by ultrasound, releasing drugs on demand. The primary mechanism for this ultrasound-responsive release is a phase change
[184],
[185],
[186],
[187],
[188]. Xu and colleagues
[189] used SiO
2 as the template and 1,4-bis(triethoxysilyl)benzene as the organo-silica precursor to synthesize hollow mesoporous organosilica nanoparticles (HMONs). They then loaded these HMONs with DOX and liquid PFP to construct DOX/PFP-loaded HMONs (DPHs). Subsequently, the DPHs were internalized into macrophages (RAW 264.7 cells) and injected into tumor-bearing mice. By modulating the temperature, a controlled phase transition of PFP was achieved. At a temperature of 37 °C, PFP within the DPHs transforms into minuscule bubbles, facilitating real-time ultrasound visualization of tumor targeting steered by the RAW cells. Once concentrated within the tumor, the application of a 1 MHz HIFU prompted the liquid PFP to metamorphose into abundant large microbubbles. This process induced a mild hyperthermia, less than 45 °C, leading to the rupture of the macrophages and subsequent drug dispersion. Approximately 82% of DOX was immediately released after ultrasound treatment, in stark contrast to the minimal release observed in the absence of ultrasound intervention (
Fig. 4(e) [189]).
After filling the pores of mesoporous nanoparticles with molecules such as drugs, they can be sealed within the pores using nanocaps to prevent leakage. The thermal and mechanical effects of ultrasound can be used to stimulate the opening of various nanocaps, achieving ultrasound-responsive drug release
[236].
The bubbles produced by the phase change of fluorocarbon compounds wrapped in MSNs can provide good contrast for ultrasound imaging
[185],
[189],
[190]. Leveraging the intrinsic ultrasound reflection/scattering properties of silica nanoparticles, it's possible to develop ultrasound contrast agents that don't rely on bubbles
[193],
[194]. Inertial cavitation driven by bubbles can directly bolster anti-tumor treatments
[192],
[195],
[231]. Lee and colleagues
[195] integrated TiO
2 nanoparticles into colloidal MSNs, crafting mesoporous silica–titania nanoparticles (MSTNs) enriched with anatase-phased TiO
2. They then employed the silane-coupled method to affix PEG onto the surface, forming PEGylated MSTNs (P-MSTNs) designed for prolonged
in vivo circulation. In subsequent step, they encapsulated PFH into P-MSTNs through the oil-in-water emulsion process, yielding the PFH@P-MSTNs. Compared to P-MSTNs, the PFH bubbles in PFH@P-MSTNs can collapse upon ultrasound irradiation, generating powerful sonoluminescence. This energy is efficiently transferred to the TiO
2, enhancing ROS production and leading to a significant anti-tumor effect.
MSNs also possess the properties of gas-stabilizing nanoparticles. Gas-stabilizing nanoparticles can stabilize surface gas pockets and produce interfacial bubbles. These bubbles can enhance the contrast of ultrasound images and mediate strong inertial cavitation
[191],
[192],
[232]. Sabuncu and colleagues
[191] used hexamethyldisilazane (HMDS) to modify the surface of MSNs for hydrophobicity. They then mixed a solution of proteins approximately 50 nm in size (recombinant human serum albumin, mouse serum albumin, or 5% human or mouse plasma) with the hydrophobic MSNs (hMSNs) to create protein corona-stabilized hMSNs. The protein-coated hMSNs still exhibited strong cavitation activity at lower particle concentrations and could effectively ablate A375 human melanoma xenograft tumors at relatively low FUS intensities of 50 and 100 W, while other stabilizing layers rendered the hMSNs inactive in terms of cavitation. Furthermore, the protein-coated hMSNs exhibit good biodegradable properties (
Fig. 4(f) [191]).
(2)
AuNPs. Low-intensity pulsed ultrasound can effectively facilitate the release of DOX, which is electrostatically attached to the surface of AuNPs. The key parameters influencing this release behavior of DOX include the temperature, diameter, and surface potential of the AuNPs
[237]. AuNPs not only function as highly efficient drug carriers but also amplify the thermal and mechanical interplay associated with ultrasound stimulation. This dual function consequently enhances the sensitivity of tumor cells and promotes their drug absorption
[238]. AuNPs can be engineered into a variety of shapes to perform their functions, including but not limited to nanospheres, nanorods, nanocones, and nanoshells
[196],
[197],
[198],
[199],
[238]. Under the influence of lasers, AuNPs have the ability to facilitate bubble formation, thereby offering promising potential in enhancing ultrasound imaging and drug delivery
[239].
The surface cavities of gold nanocones (AuNCs) have the capacity to trap gas, thus providing cavitation nuclei essential for the ultrasound cavitation effect
[199]. Kip and colleagues
[196] explored the impact of low-dose cisplatin (Cis) chemotherapy, supported by ultrasound and AuNCs, on drug-resistant ovarian cancer cell line two/three dimensional (2/3D) models. AuNCs have the ability to enhance the mechanical effects of ultrasound, thereby increasing cell membrane permeability, effectively reversing Cis resistance, and boosting intracellular accumulation of Cis. This combined therapy demonstrated a 60% suppression of tumor spheroid formation within 3D ovarian cancer cell models. Leveraging the inherent ultrasound responsiveness of AuNPs, Bhargawa and colleagues
[198] introduced anisotropic gold nanorods during the fabrication of lysozyme microspheres (LyMs). The incorporation of these gold nanorods into LyMs instigates local density variations in the outer shell, leading to its rupture under ultrasound irradiation. The inclusion of gold nanorods elevates the release rate of LyMs from 70% to 95% when subjected to 200 kHz ultrasound exposure.
An and colleagues
[197] constructed polymer nanoparticles as cargo model, and coated them with gold. This protective layer of gold effectively blocks interactions between the cargo and various enzymes or ROS, thus curbing the biodegradation and potential leakage of the cargo post-injection. The resulting gold-clustered nanoparticles are utilized as both crosslinkers and sonothermal agents within hyaluronic acid hydrogel. Hyaluronic acid, when functionalized with dopamine, acts as the polymer chain and forms catechol coordination with the gold, promoting both interchain and intrachain crosslinking. The spiky surface structure of gold clusters facilitates the efficient absorption of ultrasound energy, which in turn induces apoptosis in cancer cells via the sonothermal effect. In subsequent stages, the gold clusters can disassociate, thereby triggering a controlled, on-demand release of the polymer cargo. Experiments have demonstrated that under the influence of 30 W ultrasound, the temperature of the gold-installed hyaluronic acid hydrogel significantly rises to 53.7 °C within a span of three minutes, precipitating the disassociation of the gold clusters and, consequently, the release of the cargo (
Fig. 4(g) [197]).
(3)
Iron oxide nanoparticles. Iron oxide nanoparticles have been employed in clinical practice for nearly 90 years, a period during which they have demonstrated their safety and multifaceted utility
[240]. Various sizes of iron oxide nanoparticles can be customized to cater to diverse needs. Those of ultra-small dimensions exhibit enhanced penetrative qualities, while maintaining their exceptional MRI capabilities
[153],
[200],
[201]. These nanoparticles can serve dual functions—they can either be enveloped by different materials, or be used to enhance the properties of other substances through modification
[202],
[203]. In addition, by directly modifying the iron oxide nanoparticles, they can be tailored to possess targeted functionalities and responsive behaviors towards specific stimuli
[153],
[201],
[204]. By incorporating iron oxide nanoparticles into various polymer systems, we can create drug delivery structures including core-shell nanoparticles, superparamagnetic polymer micelles, and superparamagnetic polymersomes
[241].
While research indicates that iron oxide nanoparticles possess ultrasound response abilities, such as augmenting the ultrasound-induced ROS production, the primary objective of integrating these nanoparticles into ultrasound-responsive drug delivery systems is to harness their potential in facilitating MRI, magnetic targeting, and inducing drug release through magnetic hyperthermia
[222]. An effective contrast agent for dual-mode MRI and ultrasound imaging can be crafted through the fusion of microbubbles, nanobubbles, or nanodroplets with SPIONs
[38],
[205],
[206]. Additionally, an ultrasound-responsive drug delivery system, integrated with SPIONs, can accumulate drugs at tumor locations due to its exceptional magnetic targeting and ultrasound-responsive release capabilities
[156],
[171]. Moreover, iron oxide nanoparticles have played a significant role in tumor treatment research based on ultrasound and ROS
[208],
[209].
Iron oxide nanoparticles possess a good capacity for drug loading and can be easily modified for targeted applications
[242],
[243]. Their drug-loading methods encompass either a covalent bonding of drug molecules with the nanoparticles' surface molecules or the adherence of drug molecules to the nanoparticles' surface, facilitated by physical interactions such as electrostatic, hydrophobic, and hydrophilic engagement. The nanoparticles' surface can be altered using polymers or coatings to enhance drug load efficiency
[244].
Using a solvothermal method, Jia and colleagues
[153] engineered ultra-small iron oxide nanoparticles, just 3.2 nm in size, stabilized by citric acid. Following this, they surface-modified the nanoparticles with ethylenediamine, adding amine groups. Then, they cross-linked these nanoparticles with
p-phthalaldehyde, resulting in nanoclusters endowed with acid pH-sensitive benzoic imine bonds. These nanoclusters can be used for DOX adsorption, along with its pH-responsive release. Ferumoxytol (FH) is a clinically approved nanoparticle, approximately 30 nm in diameter, composed of a magnetite–maghemite iron oxide core and enveloped by a shell of carboxymethyldextran polysaccharide chains. It is primarily used in treating iron deficiency anemia. Stater and colleagues
[207] have utilized FH as carrier, successfully encapsulating the vaccine adjuvant, monophosphoryl lipid A, within FH's carbohydrate shell without necessitating any further chemical adjustments. This method capitalizes on FH's passive targeting ability, ensuring targeted drug delivery to tumor-associated macrophages and other toll-like receptor 4 positive professional phagocytes.
3.4. Responsive drug activation
Mechanochemistry can be understood as a distinctive style of reaction that harnesses mechanical energy to propel the rearrangement of chemical bonds, bypassing traditional thermal or electrical activation methods
[245]. Capable of transmitting mechanical forces, ultrasound holds the potential for generating mechanical stimuli within the body, promoting the breakdown of chemical bonds and inducing changes to molecular structures
[246],
[247].
Taking advantage of polymer mechanochemistry, we can unite drug molecules to mechanically-responsive carriers that are sensitive to ultrasound, thereby enabling responsive release upon ultrasound stimulation
[22],
[248],
[249]. A prevalent design strategy for these carriers is the incorporation of labile bonds or structures into a relatively stable polymer framework. Responding to the potent shear stress exerted by ultrasound inertial cavitation, these labile bonds or structures are prone to fragmentation or transformation. By deliberately positioning these labile bonds or structures to serve as potential breaking points, the precise release of drug molecules embedded within the carrier can be realized through ultrasound activation
[22],
[250]. By adjusting variables—including the strength and conformation of the labile bonds, the molecular weight, degree of polymerization, and structure of the polymer—we can alter the specific responsive properties of the carrier to achieve a “customized” therapeutic result
[23],
[249].
Reported mechanochemical transformations span from simple conformational transitions across small activation barriers to the disruption of frail interactions such as van der Waals forces, π–π interactions, ionic and hydrogen bonds, and finally to coordination bonds and the severing of covalent bonds
[245]. This chapter provides an overview of the latest developments in responsive drug activation mediated by ultrasound, with a specific focus on ultrasound-responsive carriers premised on the scission of covalent or non-covalent bonds.
3.4.1. Responsive drug activation via covalent bond scission
The polymers utilized for ultrasound-responsive drug activation generally degrade into smaller oligomeric structures. By means of intelligent design, our objective is to harness this degradation process to liberate small, bioactive molecules
[22].
In a strategy centered on manipulating labile covalent bonds, Huo and colleagues
[251] integrated camptothecin, a monoterpene alkaloid known for its anti-tumor properties, into polymers designed around disulfide. They engineered the drug to affix itself to a carbonate linker in β-position to the disulfide. Following this precise assembly, mechanical forces generated by ultrasound triggered an intramolecular 5-
exo-
trig cyclization process, liberating camptothecin from its β-carbonate linker. By employing an analogous strategy, the release of gemcitabine can be triggered. This illustrates the broad applicability of a drug activation system that utilizes mechanochemically induced 5-
exo-
trig cyclization, which is responsive to ultrasound stimuli
[252].
Leveraging a variety of labile covalent bonds and bespoke structural designs, it is possible to attain the desired activation of small molecule drugs. Shi and colleagues
[253] discovered that when a disulfide-centered polymer fractures, the resulting sulfur element at the fracture point gives rise to a thiol. These thiol-terminated polymers, created through ultrasound-induced mechanochemistry, can engage in a Michael-type addition reaction within the Diels–Alder adducts of furylated drugs and acetylenedicarboxylate derivatives. A subsequent retro-Diels–Alder reaction can then catalyze the release of small-molecule drugs, such as furosemide and furylated DOX (
Fig. 5(a) [253]). The similar design can also be adapted for the direct release of small-molecule cargo, facilitated by ultrasound-induced mechanical force
[254]. Sun and colleagues
[255] reported a unique mechanochemical reaction in which ultrasound facilitates the release of carbon monoxide from norborn-2-en-7-one, suggesting the potential of employing analogous techniques for ultrasound-induced gas imaging and therapy (
Fig. 5(b) [255]).
3.4.2. Responsive drug activation via non-covalent bond scission
Currently, ultrasound-responsive drug activation systems, which are based on the scission of non-covalent bonds, predominantly depend on the superstructures of nucleic acid aptamers and supramolecular assemblies
[22].
In Huo's strategies
[251] focusing on the controlled scission of weak non-covalent bonds, the antibiotics neomycin B or paromomycin were cleverly enmeshed within high-molar-mass polyaptamers constructed from repeating units of nucleic acid aptamers. Subsequent application of mechanical forces via ultrasound disturbed the non-covalent interactions (such as hydrogen bonds or electrostatic interactions) within the polymers. This disruption instigated the scission of the covalent bonds and disaggregation of the phosphodiester RNA backbone, which was contemporaneous with the release of the drug. In another of their strategies, they used vancomycin (Van) and its hydrogen-bond complementary peptide target sequence Cys-Lys-Lys(Ac)-
D-Ala-
D-Ala (DADA) as the supramolecular binding motif. The poly(oligo(ethylene glycol) methyl ether methacrylate) polymer chains and AuNPs, or two AuNPs, were then connected using this supramolecular binding motif. Following this, mechanical forces induced by ultrasound disrupted the bonds between Van and DADA, prompting the subsequent release of the drug
[251]. Leveraging the mechanoresponsive pair of Van and DADA, the structure featuring grafted polymer brushes presents a promising strategy to augment drug loading capacity and enhance the efficiency of mechanochemical processes (
Fig. 5(c))
[256]. Moreover, AuNPs served a crucial function as transmitters of ultrasound shear forces
[258].
Based on supramolecular assemblies, Küng and colleagues
[257] described a supramolecular coordination cage. They employed six PEG-decorated bipyridine ligands to form an octahedral cage. The PEG chains present at each vertex of the octahedron serve as transmitters for the shear forces applied by ultrasound. Each of these octahedral cages is capable of enclosing a molecule of progesterone or two molecules of ibuprofen within its hydrophobic nanocavity. Activation occurs via the shear forces produced by ultrasound in aqueous solution, which triggers a full release of the encapsulated drug upon cage rupture (
Fig. 5(d) [257]).
Beyond its application in drug activation, the ultrasound-induced mechanochemical approach can be deployed to manipulate the structure and activity of large biomolecules, including enzymes and DNA
[259],
[260],
[261]. Zhou and colleagues
[261] effectively transmitted the mechanical force delivered by ultrasound to proteins by introducing an elastin-like polypeptide chain into the loop between the 10th and 11th β-strand of a mutated, more fold-stable protein superfolder green fluorescent protein (sfGFP) through genetic engineering. This chain serves as a long and extendable region responsive to shear forces. When exposed to ultrasound, the 11th β-strand in the β-barrel structure of the sfGFP becomes unstable, leading to the disappearance of green fluorescence, while the secondary structure of the protein remains intact. Employing this approach, they gained control over the enzymatic activity of trypsin. The strategy of using ultrasound-induced shear forces as a trigger to switch the activity of genetically engineered proteins on and off provides a valuable reference for the subsequent discussion on sonogenetic therapy.
4. Ultrasound-enabled nanomedicine for effective tumor therapy
4.1. Sonodynamic therapy (SDT)
SDT is a novel anti-tumor strategy that harnesses the interaction between sonosensitizers and low-intensity ultrasound to generate ROS, thereby inducing the apoptosis of tumor cells. In contrast to photodynamic therapy, SDT presents as a non-invasive therapeutic modality. It boasts superior spatiotemporal resolution, demonstrates deeper tissue penetration capabilities, and presents with milder post-treatment side effects. The salient benefits of SDT can be attributed to the precision focusing inherent to low-intensity ultrasound and the high-efficiency response dynamics of sonosensitizers towards ultrasound
[262]. To date, numerous sonosensitizers have been documented, ranging from inorganic types (noble metal-based, transition metal-based, carbon-based, and silicon-based) to organic ones (porphyrins and phthalocyanines, xanthene compounds, phenothiazine compounds, fluoroquinolone antibiotics, etc.)
[263],
[264].
The predominant mechanisms through which sonosensitizers produce ROS when exposed to ultrasound are sonoluminescence and pyrolysis. This production is linked to the swift collapse of microbubbles triggered by ultrasound cavitation, which results in both sonoluminescence and an exothermic process. As sonosensitizers capture energy from sonoluminescence, they transition from the ground state to a higher energy state, resulting in the formation of electron-hole (e
−-h
+) pairs, which in turn facilitates the production of ROS
[264],
[265]. The ensuing electrons and holes can act as catalysts, triggering the formation of ROS from O
2 and H
2O, including singlet oxygen (
1O
2), hydroxyl radicals (·OH), and superoxide anions (·O
2−)
[263]. Under the pyrolysis mechanism, the intense temperature and pressure stemming from the microbubble implosion can spawn free radicals through the thermal decomposition of water. Subsequently, these free radicals engage with other endogenous substrates, yielding a greater quantity of ROS
[263],
[266].
To prevent hydrophobicity-induced aggregation and to improve both delivery efficiency and targeting, sonosensitizers are typically encapsulated within nanocarriers or crafted directly into nanoparticles possessing sonosensitizer-like attributes for optimal delivery.
Dong and colleagues
[267] utilized CaCO
3 nanoparticles as a template, encapsulating them within a hollow metal-organic framework derived from the sonosensitizer meso-tetra(4-carboxyphenyl)porphyrin (TCPP) coordinated with ferric ions. During the preparation,
L-buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) biosynthesis, was simultaneously loaded, resulting in the formation of BSO-TCPP-Fe@CaCO
3. This compound responds to the acidic pH typical of the TME, leading it to dissociate and subsequently release Ca
2+ and BSO. This triggers a three-fold reaction: mitochondrial damage from elevated intracellular Ca
2+ levels, GSH depletion mediated by BSO, and SDT driven by TCPP and ultrasound. Together, these effects produce a potent synergistic increase in oxidative stress, causing significant cell death (
Fig. 6(a) [267]). Yin and colleagues
[268] incorporated the sonosensitizer protoporphyrin (PpIX) and
L-arginine (LA) into mesoporous organosilica nanoparticles (MONs). By harnessing the guanidino groups in LA, known for their ability to chemically bind with CO
2, they formulated the compound MON-PpIX-LA-CO
2. In the presence of the acidic TME or under the influence of low-intensity ultrasound, CO
2 dissociates from LA, leading to the formation of bubbles. These bubbles enhance the effects of ultrasound-induced inertial cavitation, thereby optimizing PpIX's potential to generate ROS upon ultrasound activation. Notably, the sustained inertial cavitation results in heightened drug retention within the tumor, subsequently enhancing ROS production and reinforcing the effectiveness of SDT against tumors (
Fig. 6(b) [268]). To address the low quantum yield of ROS in conventional sonosensitizer TiO
2 nanoparticles, Liang and colleagues
[269] applied a layer of Pt nanoparticles onto the surface of hollow mesoporous TiO
2 nanoparticles using the vacuum metal sputter deposition method. This was then further treated to form an oxygen-deficient layer on the TiO
2 nanoparticle surface. Following this, DOX, another sonosensitizer, was loaded into the cavity of TiO
2, resulting in the formation of hydrogenated hollow Pt–TiO
2 nanoparticles loaded with DOX (HPT–DOX). The oxygen-deficient layer combined with the Pt nanoparticles enhanced the separation of e
− and h
+ from the energyband structure of TiO
2 upon ultrasound irradiation, amplifying the quantum yield. In hypoxic conditions, the Pt nanoparticles functioned as enzyme-like catalysts, generating O
2. This not only addressed the tumor's oxygen shortage but also supplied an ample source of oxygen for ROS production. DOX, when inside cells, can penetrate the nucleus and induce cell death. Furthermore, under ultrasound irradiation, DOX serves as a sonosensitizer, generating
1O
2. When subjected to ultrasound, HPT–DOX demonstrated a significant ability to inhibit tumor growth.
The aforementioned work showcases the advanced design strategies of the SDT drug delivery system. It also illustrates the current directions for enhancing SDT's efficacy, which include alleviating hypoxia, enhancing cavitation effects, depleting GSH, and combining with chemotherapy drugs that are activated by hypoxia
[272]. Enhancing the efficacy of sonosensitizers and pairing them with cutting-edge ultrasound technology will propel SDT to even more effective therapeutic horizons
[273],
[274]. By synergizing with other tumor treatment modalities, SDT can amplify its strengths and offset its limitations
[275],
[276].
The multifunctional SDT drug delivery systems are a prevailing trend, with the goal of attaining multi-modal imaging guidance and enhanced penetration capabilities. Utilizing zeolitic imidazolate framework as a template, Pan and colleagues
[277] synthesized double-layer hollow manganese silicate nanoparticle (DHMS) via the
in-situ growth of Mn
2+. By facilitating e
−-h
+ separation, Mn boosts the generation efficiency of ROS. Additionally, it can serve as a catalyst, reacting with H
2O
2 in the TME to produce O
2. As a result, DHMS possesses robust ultrasound and MRI capabilities. The doping of Fe can also provide MRI capabilities
[278]. By utilizing the combined effects of ultrasound and microbubbles, the BBB becomes permeable, enabling the SDT drug delivery system to penetrate it effectively
[279]. Additionally, the SDT drug delivery system can utilize the BBB's inherent molecular recognition and transport pathways for efficient delivery
[280],
[281].
4.2. Sonogenetic therapy
Using biological components endowed with mechanical or thermal response properties as actuators, ultrasound can be applied to directly manipulate cellular activity. Stemming from this principle, a pioneering technique named sonogenetics has been developed, demonstrating its ability to directly modulate neuronal functions
[282],
[283]. Sonogenetics has the capability to modulate neural activity patterns, influencing whole-animal behaviors. Ibsen and colleagues
[284] harnessed a combination of low-pressure ultrasound and microbubbles to intensify ultrasound-induced mechanical stimuli. By doing so, they managed to specifically activate neurons that express the transient receptor potential channel 4 (TRP-4) ion channel, leading to the control of neural circuits and distinct behaviors in the
Caenorhabditis elegans. Notably, this research marked the inaugural introduction of the sonogenetics concept.
In a manner similar to optogenetics, which employs light to stimulate light-sensitive proteins, and chemogenetics, which harnesses small molecular compounds to activate specific receptors, sonogenetics taps into ultrasound-induced mechanical forces to activate mechanosensitive ion channels. This technique can regulate the activity of specific cells and selectively trigger cellular events. Unlike optogenetics, which requires the insertion of optical fibers, or chemogenetics, which demands the in-body delivery of small molecular compounds, sonogenetics employs non-invasive ultrasound. This ultrasound can be precisely directed to a target area spanning several cubic millimeters, with minimal energy loss even when passing through bones and deep tissues
[285],
[286],
[287].
The exact mechanism of sonogenetics is not yet fully understood. Current research suggests that the mechanism is associated with the response of mechanosensitive ion channels to the mechanical forces (such as shear forces) induced by ultrasound. This response leads to a structural transformation of these ion channels, allowing ions to flow in. Yoo and colleagues
[288] demonstrated that ultrasound activates neurons primarily through mechanical stimuli applied to them. By adjusting the ultrasound parameters, they excluded the influences of temperature elevation, cavitation, or large-scale cellular deformation. This mechanical exertion from the ultrasound prompts specific calcium-permeable mechanosensitive ion channels (transient receptor potential polycystin 1/2, transient receptor potential canonical 1, and Piezo 1) to open. Activation of these channels facilitates accumulation of calcium ions within the cells. These calcium ions, in turn, instigate the opening of additional ion channels (transient receptor potential melastatin 4, T-type channel), leading to amplified cellular responses. Duque and colleagues
[289] have discovered that a mammalian protein, human transient receptor potential ankyrin 1 (hsTRPA1), can confer ultrasound sensitivity to human embryonic kidney-293T cells and rodent neurons. This marks it as a promising mechanosensitive ion channel for implementing sonogenetic control in mammalian cells. Additionally, they highlighted the crucial role of the interactions between the N-terminal tip region of hsTRPA1 and membrane cholesterol, as well as the importance of an intact actin cytoskeleton in the target cells.
Sonogenetics can be further divided into mechano-sonogenetics and thermo-sonogenetics, based on whether ion channels respond primarily to ultrasound-induced mechanical stimuli or its thermal effects. In the practice of sonogenetics, several mechanosensitive ion channels have been identified that can undergo conformational changes in response to such stimuli. These channels include TRP-4, mechanosensitive channel with very large conductance (MscL), mechanosensitive channel with very small conductance, two-pore domain potassium channel family, Piezo, and certain voltage-gated channels
[290]. Conversely, ion channels like transient receptor potential vanilloid 1 are sensitive to the thermal effects of ultrasound
[291]. It's crucial to acknowledge that the activation of mechanosensitive ion channels is contingent on specific conditions. Various ultrasound parameters have been documented to activate the same type of these ion channels
[290]. Such discrepancies likely stem from the use of different ultrasound devices, highlighting the absence of a unified theoretical framework for predicting activation conditions. Implementing sonogenetics in mammalian cells demands careful consideration of several factors. These include the diversity in membrane compositions, the structural integrity of the cell membrane skeleton, the effectiveness of the ion channels' response, and the efficiency of ultrasound energy transfer
[289],
[292],
[293],
[294]. To enhance this methodology, potential approaches involve leveraging microbubbles or gas vesicles to amplify the ultrasound's mechanical force and engineering more responsive variants of the mechanosensitive ion channels
[292],
[294],
[295].
Owing to its promising potential in cell control, sonogenetics has been explored for tumor therapy. He and colleagues
[296] employed iron alginate nanogel as carrier to deliver MscL plasmids and the transfection reagent polyethylenimine into tumor cells. Their goal was to activate MscL using ultrasound, thereby inducing intracellular calcium ion overload and initiating apoptosis in the tumor cells. In melanoma mouse model, the apoptosis of tumor cells, induced by this sonogenetic therapy, effectively enhanced the activation of immune cells, subsequently leading to increased survival rate in mice. Wang and colleagues
[297] introduced the mutant of MscL (MscL I92L), which exhibit greater ultrasound sensitivity than the native MscL, into various tumor cell lines. Their findings suggest that sonogenetic therapy consistently enhances intracellular calcium ion levels, effectively inducing apoptosis in tumor cells.
In addition to inducing tumor cell death, sonogenetics has been harnessed to initiate specific responses in immune cells. Pan and colleagues
[292] developed a novel approach to engineer T cells. They equipped these cells with mechanosensitive Piezo 1 ion channels and physically paired them with microbubbles. Additionally, they modified the T cells by incorporating nuclear factor of activated T-cells (NFAT) response element upstream of the chimeric antigen receptor (CAR) gene sequence. When ultrasound is applied, the microbubbles amplify the ultrasound's shear stress. This amplified stress reaches the activation threshold of the Piezo 1 channels. Activated Piezo 1 channels then trigger an influx of calcium ions, which activates calcineurin. Calcineurin, in turn, dephosphorylates NFAT. Once dephosphorylated, NFAT binds to the region upstream of the CAR gene, initiating its expression. Through this sonogenetics-based method, these specialized T cells are primed with CAR, enabling them to target and destroy tumors effectively.
While mechano-sonogenetics has been explored for some time, the emerging thermo-sonogenetics has recently unveiled promising potential in tumor therapy. Leveraging temperature-dependent transcriptional repressors as biological switches, both Chen's team
[298] and Abedi's team
[299] crafted temperature-activated genetic circuits for interferon gamma (IFN-γ) or alpha cytotoxic T lymphocyte-associated antigen 4 (αCTLA-4) and alpha PD-L1 (αPD-L1) in bacteria, respectively. Following this, FUS was employed to attain the threshold temperature of the repressor, thereby enhancing gene expression. This line of research has forged a precise spatiotemporal gene expression strategy by combining the capabilities of bacteria and ultrasound.
Direct tumor cell eradication and the augmentation of immune cell function stand as the two primary objectives in tumor therapy. Thus far, sonogenetic therapy has exhibited significant advancements in both domains.
4.3. Other sonotherapies
Beyond SDT and sonogenetic therapy, tumor treatments directly driven by ultrasound also encompass sonomechanical therapy, sonopiezoelectric therapy, and sonothermal therapy
[16]. The principles and diverse applications of sonomechanical, sonopiezoelectric, and sonothermal effects have been discussed in previous sections. Specifically, mechanical forces induced by ultrasound can breach physiological barriers and cell membranes, and isolate tumor markers. The piezoelectric effect can generate a range of ultrasound specifically designed for tumor diagnosis and therapy. The heat delivered by ultrasound can promote drug release from their carriers and amplify the generation of ROS during SDT. Sonomechanical, sonopiezoelectric, and sonothermal effects can be directly applied to tumor treatment.
(1)
Sonomechanical therapy. Ultrasound-induced mechanical stimuli can produce a variety of forces, including sono-compression and tension from bubble oscillations, microstreaming-induced sono-compression, shear force and sono-compression arising from micro-jetting, and shear force generated by lipid membrane peroxidation. These forces have the potential to directly harm cancer cells, inducing either apoptotic or non-apoptotic cell death
[247]. The excellent ultrasound responsiveness of the ultrasound contrast agent makes it an ideal medium for mechanical force transmission in sonomechanical therapy
[300],
[301].
Bar-Zion and colleagues
[300] discovered that gas vesicles can act as nuclei for the formation and cavitation of free bubbles. By leveraging FUS, these gas vesicles, whether targeted at tumor cells or expressed in engineered bacteria, can be precisely activated. The cavitation caused by gas vesicles has shown a significant capability to kill tumor cells and lyse engineered bacteria. To showcase the potential of gas vesicles for sonomechanical therapy, the researchers engineered
E. coli Nissle 1917 (EcN) cells to express these gas vesicles. These modified EcN cells can penetrate and localize themselves within solid tumors. Upon activating the gas vesicles inside the EcN cells using ultrasound, localized cavitation occurs, leading to the mechanical destruction of the tumor tissue. This approach not only decelerates tumor growth but also extends the average survival time of mice.
(2)
Sonopiezoelectric therapy. Ultrasound-activated piezoelectric nanomaterials comprise a diverse category of both inorganic and organic dielectric compounds. These materials can become electrically polarized when subjected to mechanical stimulation, or experience strain in response to applied electric fields. In inorganic compounds, the piezoelectric effect originates from the relative displacement of ionic species within the material. In contrast, in organic materials, the effect is associated with the repositioning of molecular dipoles. Notable examples of these materials include ZnO, a non-ferroelectric piezoelectric material; BaTiO
3, a ferroelectric material; and poly(vinylidene) fluoride, a synthetic organic polymer
[302]. The ultrasound responsiveness of piezoelectric materials can also be harnessed to provide power for implantable medical devices
[303].
Zhu and colleagues
[304] harnessed ultrasound as a precise microscopic pressure source to stimulate piezoelectric tetragonal BaTiO
3 (T-BTO), thereby inducing the generation of ROS, and deployed this strategy in sonopiezoelectric therapy. Under the influence of periodic ultrasound vibration, which applies frequency/time-dependent microscopic pressure, the potential equilibrium between the polarization charges and accumulated screen charges in T-BTO is disrupted. e
−-h
+ pairs in T-BTO are continuously separated through piezoelectric effects, thereby establishing a potent built-in electric field and the surface charging. As a consequence of this dynamic process, surface electrons and holes are induced to serve as effective catalysts for the generation of ROS.
In vitro experiments have demonstrated that, under ultrasound triggering, T-BTO is capable of producing toxic hydroxyl (·OH) and superoxide radicals (·O
2−).
In vivo assessments using tumor xenograft models reveal that thermosensitive hydrogel laden with T-BTO nanoparticles can induce ultrasound-triggered cytotoxic effects and piezocatalytic tumor elimination, associated with a high level of therapeutic biosafety. In another example, Hoang and colleagues
[270] used ZnO nanorods coated with PEG and modified with AuNPs to serve as piezoelectric catalysts for sonopiezoelectric therapy. Under ultrasound irradiation, thermally excited electrons and holes within the ZnO nanorods are separated and subsequently accumulate on the surface by piezoelectric polarization, catalyzing the production of ROS. Additionally, the AuNPs deposited on the surface function as Fenton-like catalysts, enhancing the piezocatalytic generation of ROS. The integration of the pro-oxidant drug piperlongumine (PL) further validated the effectiveness of this combined chemo-piezocatalytic approach to therapy (
Fig. 6(c) [270]).
(3)
Sonothermal therapy. In cancer treatment, the application of heat has emerged as an effective therapeutic approach. Hyperthermia involves raising the temperature of the tumor site to between 39 and 45 °C, which serves to enhance the sensitivity of the tumor cells to radiotherapy and chemotherapy. In contrast, thermal ablation employs temperatures exceeding 50 °C to directly destroy tumor cells. Both of these strategies have gained widespread acceptance and are employed in clinical settings
[305]. HIFU represents a non-invasive thermal ablation technique that is presently being utilized in the clinical management of various solid malignant tumors. The integration of MRI and ultrasound imaging with HIFU provides clinicians with the ability to monitor the ablation process through real-time imaging, thereby enhancing the precision and safety of the treatment
[306]. Sonothermal therapy is designed to harness the sonothermal conversion capabilities of nanosystems, in conjunction with low-intensity ultrasound, to achieve highly targeted and precise thermotherapy within the body. This approach is crafted to minimize non-specific thermal damage to surrounding healthy tissues as much as possible.
Qi and colleagues
[271] employed a “B–H” interfacial-confined coordination strategy to synthesize HA-NC_Cu that are derived from a Zn (II) boron imidazolate framework, functionalized with hyaluronic acid, and feature uniformly dispersed single copper atoms. When exposed to low-intensity ultrasound irradiation, HA-NC_Cu demonstrates sonothermal conversion capabilities, facilitated by intermolecular lattice vibrations. In addition to this, HA-NC_Cu is capable of generating ·OH radicals by catalytically decomposing endogenous H
2O
2 within cells. Comprehensive
in vivo assessments have substantiated that the sonothermal-catalytic synergistic strategy orchestrated by HA-NC_Cu can effectively stifle the tumor proliferation in MDA-MB-231 tumor-bearing nude mice, while simultaneously enhancing their survival rate (
Fig. 6(d) [271]). Yang and colleagues
[307] synthesized oxygen-vacancy-rich BiO
2−x nanosheets. When exposed to LFUS, these nanosheets exhibit the piezoelectric effect, which effectively triggers the generation of ROS. Additionally, the LFUS stimulates electron motion within the BiO
2−x nanosheets. This, in turn, leads to phonon/lattice thermal vibrations, culminating in a rapid and significant sonothermal effect. Remarkably, this process orchestrates an integration of sonopiezoelectric therapy and sonothermal therapy.
5. Conclusions and perspectives
This review systematically summarizes the recent advancements in the integration of ultrasound technology with nanomedicine in the field of tumor diagnosis and treatment. Specifically, it focuses on prospective research that synergistically merges the strengths of ultrasound technology and nanomedicine. The review aims to provide readers with a clear and organized presentation of the innovative potential and application prospects of ultrasound-enabled nanomedicine, while additionally outlining potential research frameworks and directions for its future use in tumor diagnosis and treatment, in hopes of fostering further development and clinical translation in this interdisciplinary field. The integration of ultrasound technology with nanomedicine synergistically enhances the advantages of both approaches. By leveraging the exceptional capability of ultrasound to encode information and deliver targeted energy, ultrasound can effectively guide and activate specially designed nanosystems. This ultrasound-enabled nanomedicine facilitates the accomplishment of various critical objectives in the process of tumor diagnosis and treatment. It encompasses tumor molecular imaging, the separation of tumor markers, the penetration of physiological barriers, targeted cell membrane perforation, the controlled release and activation of therapeutic drugs, and the application of various sonotherapies powered by ultrasound energy. Together, these capabilities present a more comprehensive and potent set of solutions for the future of tumor diagnosis and treatment. From a long-term perspective, advancing the further development and clinical translation of ultrasound-enabled nanomedicine in tumor diagnosis and treatment requires substantial and sustained efforts. These efforts encompass several key areas, including but not limited to:
(1)Enhance the understanding of the physical properties of ultrasound to promote improvement of ultrasound equipment and application strategies, enabling more precise and efficient realization of diagnostic and therapeutic objectives. This entails:
• Advancing imaging and signal analysis techniques, such as harmonic imaging and contrast-enhanced imaging technologies. These advancements aim to markedly elevate image resolution and contrast, thereby boosting the overall quality of imaging. The ultimate goal is to incrementally establish dynamic, real-time imaging capabilities and sophisticated imaging at the molecular level of tumors.
• Advocating for the development and clinical integration of comprehensive multi-modal imaging systems. These systems aim to synergistically merge ultrasound imaging with cutting-edge technologies from various domains, including MRI and optical imaging. This design is intended to deliver more comprehensive and precise diagnostic information and to enable the manipulation of nanosystems in various physical forms.
• Improving the precision of ultrasound emission devices and optimizing the technology associated with the transmission and reception of ultrasound signals. These enhancements are designed to enable ultrasound to target and evaluate tissues with increased accuracy. This, in turn, minimizes the risk of undesired tissue heating and potential damage, thereby further augmenting the safety of ultrasound-based diagnostic and therapeutic procedures.
(2)Delve further into the mechanisms of the interaction between ultrasound and nanosystems, encompassing:
• Identifying the precise conditions under which ultrasound induces either stable cavitation or inertial cavitation in bubbles, and establishing the explicit correlations among critical parameters, such as bubble size and ultrasound intensity. Investigate strategies to regulate these parameters to engineer more sensitive and universally applicable bubble-based nanosystems, enabling control over cavitation intensity while minimizing collateral damage.
• Refining theoretical models for acoustic streaming and acoustic radiation forces associated with various ultrasound types, and optimizing the models that describe the influence of acoustic radiation forces on microscopic systems of differing sizes (e.g., tumor markers, nanoparticles, cells). Utilize these models to further inform the design and construction of ultrasound emission devices, facilitating precise manipulation of both extracorporeal and intracorporeal nanosystems and cells.
• Designing novel ultrasound-responsive drug carriers and investigating innovative ultrasound-responsive chemical bonds. By leveraging the multifaceted effects of ultrasound (e.g., phase transitions, pyrolysis, ROS generation), design more effective drug carriers and chemical bonds to enable diverse modes of ultrasound responsiveness. These new designs aim to yield enhanced drug release responsiveness or more controlled and stable drug delivery platforms.
• Engaging in comprehensive studies of the intricate mechanisms underlying sonodynamic, sonogenetic, sonomechanical, sonopiezoelectric, and sonothermal therapies. For instance, perform detailed analyses of the ROS generation mechanisms specific to different sonosensitizers, the ultrasound-responsive opening mechanisms of mechanosensitive ion channels, the conversion pathways from acoustic to mechanical energy, the mechanics of the sonopiezoelectric effect, and the sonothermal conversion processes. With these deep mechanistic insights, drive forward the development and application of highly efficient and precise sonotherapies.
(3)Deepen the investigation into the effects and kinetic behavior of nanosystems within biological organisms, including:
• Probing the distribution, transportation, and clearance mechanisms of nanosystems within biological organisms, with the objective of constructing a comprehensive lifecycle map of these nanosystems within the body. This endeavor necessitates a systematic assessment of the biocompatibility and potential toxicity of nanosystems over an extended timeframe, a critical component in propelling the clinical transition of nanomedicine.
• Investigating the interaction mechanisms between nanosystems and cellular components, as well as large biomolecules such as proteins and nucleic acids. This encompasses elucidating the cellular absorption processes of nanosystems and potential ensuing biological effects, analyzing how different surface modifications influence the selectivity and targeting capacity of nanosystems within various cells and tissues, and leveraging this information to inform the design of nanosystems with enhanced targeting and internalization capabilities.
• Undertaking a systematic study of the pharmacokinetics and pharmacodynamics associated with nanosystems, aiming to refine the prediction and optimization of dosing strategies and therapeutic outcomes of nanomedicine. Concurrently, probe the stability and biodegradability of nanosystems in complex biological environments, enabling precise evaluation of their long-term efficacy and sustainability as either drug delivery carriers or therapeutic instruments.
(4)Address the challenges in clinical translation of ultrasound-enabled nanomedicine, including:
• Biosafety remains a crucial concern in the clinical transition of ultrasound-enabled nanomedicine. It's imperative to closely monitor the distribution and potential toxic effects of nanosystems across different organs and tissues. Solutions may involve the adoption of clinically compliant formulations and manufacturing processes, enhancing the circulatory efficiency and targeting precision of nanosystems, and replacing high-dosage injections with more targeted controlled-release methods.
• The feasibility of employing ultrasound in clinical scenarios depends on its safety and effectiveness. A fundamental aspect is ensuring the precise control over the frequency and power of ultrasound equipment, complemented by real-time monitoring of the temperature and potential damage in the therapeutic area. Addressing these challenges could lead to the development of more adaptable, accurate, and cost-effective ultrasound equipment, as well as improving the nanosystems' responsiveness to ultrasound.
• Expanding and diversifying animal models is vital. Given that existing mouse models may not be entirely adequate, more extensive research using larger animal models is necessary. These models could provide a more accurate simulation of human responses to these treatments, yielding essential data on safety and efficacy that are crucial for clinical trials and the eventual clinical deployment.
We have great confidence in the promising future of the interdisciplinary fusion between ultrasound technology and nanomedicine. Ultrasound technology plays a vital role in various medical applications, including ultrasound imaging, ultrasound-guided punctures and biopsies, as well as HIFU-mediated ablation therapy. Years of extensive technological advancements have solidified the essential role of ultrasound in both diagnostic and therapeutic applications. Compared to optical, magnetic, and radioactive technologies utilized in clinical settings, ultrasound boasts distinctive advantages, such as exceptional safety, portability and flexibility of equipment, remarkable cost-effectiveness, painless and non-invasive features, and the capacity to deliver highly sensitive, real-time, and dynamic medical images. Nanomedicine is committed to crafting diagnostic and therapeutic systems on a nanoscale level, which allows for more precise and effective drug delivery than traditional approaches, optimizes drug bioavailability, and minimizes potential side effects. Various nanosystems, each possessing unique structures and properties, can be meticulously designed and modulated. This capability enables nanomedicine to construct treatments that are highly customized to specific diseases, distinct diagnostic and therapeutic objectives, and individual patient needs. By integrating the advantages of both, ultrasound-enabled nanomedicine is poised to inaugurate a new era in the realm of tumor diagnosis and treatment, offering powerful tools that support personalized and precision medical strategies.
Acknowledgments
This project was financially supported by the National Natural Science Foundation of China (32271440) and the Tianjin Health Research Project (TJWJ2023ZD001).
Compliance with ethics guidelines
Kairui Liu, Boyuan Jing, Jun Kang, Lei Han, and Jin Chang declare that they have no conflict of interest or financial conflicts to disclose.
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