Recent Progress Toward Imaging Application of Multifunction Sonosensitizers in Sonodynamic Therapy

Introduction

Worldwide, cancer leads the list of causes of death, and the mortality and morbidity associated with cancer are on the rise.¹ Although cancer research is expensive, cancer’s variety, heterogeneity and complexity limit therapeutic choices.² Surgery, radiation and chemotherapy have systemic toxicity, selectivity, pharmacoresistance and possible long-term effects. Various remedies have been explored to compensate for these shortcomings.³ SDT is a rapidly evolving, updated therapy with excellent development potential.

SDT employs low-frequency ultrasound to irradiate sonosensitive substances concentrated in the treatment region to achieve the therapeutic goal of destroying aberrant cells via the toxic and physical effects.⁴ Ultrasound (US) has a high tissue penetration rate and can operate on tumor cells deep into biological tissues. The therapeutic impact can be improved by using concentrated ultrasonic energy and a dynamic effect.³ Ultrasonic waves can cause the formation of bubbles in the liquid. The bubbles then sharply shrink and break to produce mechanical damage, local high temperature and sonoluminescence, which is called inertial cavitation.^5,6 Inertial cavitation can produce reactive oxygen species (ROS) by directly decomposing the sonosensitizers or through pyrolysis with water.⁷ The resulting ROS will further react with other endogenous substrates to form alkoxy and peroxyl radicals.⁸ All of these adverse factors can lead to cell death, including autophagy, apoptosis, necroptosis, and pyroptosis, and limit tumor spread in sonodynamic treatment for various malignancies (Figure 1).^9–15 This method, in particular, allows for precision tumor targeting while causing minimal harm to the surrounding normal tissue.^16–19

Figure 1 Schematic illustration of the mechanism of sonodynamic therapy. The stable cavitation induced by ultrasonic irradiation promoted the sonosensitizer to enter the target area from the blood vessel, while the irradiation caused the sonosensitizers to produce ROS to kill tumour cells.

On the other hand, due to the rapid advancement of diagnostic imaging technology, molecular imaging has emerged as a novel diagnostic tool with excellent resolution and sensitivity.²⁰ Some diagnostic imaging methods, such as photoacoustic (PA) imaging, computed tomography (CT), and magnetic resonance (MR) imaging, have high spatial resolution and penetrate deep into the tissue.^21–23 Therefore, many novel sensitizers developed by researchers combine diagnostic imaging capabilities with treatment capabilities. Nanoparticles can be treated utilizing ultrasound and may also be used to enhance the efficacy of therapy through imaging techniques to observe drug distribution, which can help calculate the best time to start treatment to develop the therapeutic effect.^24,25 As a result, the development of multifunctional drugs that combine tumour targeting, imaging, and therapeutic capabilities is critical for precision oncology.^26–28

This paper outlines the recent studies using imaging in sonodynamic treatment.

Sonosensitizers are presented in Table 1, summarising the imaging properties of the given article for the reader’s ease of “browsing.”

Table 1 Imaging Characteristics of the Multifunctional Sonosensitizers

Sonosensitizers with Various Imaging Functions

Significance of Imaging Applications

The sensitivity, spatial resolution and imaging depth of non-invasive diagnostic imaging methods such as FL imaging, ultrasonography, CT and MR imaging vary.²⁹ They are all able to detect the location of the disease early. When the sonosensitizer has an imaging function, it may be led by the imaging function to carry out therapy, resulting in the integration of diagnostic and treatment. On the other hand, by utilizing bioimaging in the exact focus of the US and time-dependent tracking of drug retention in the tumour, we can determine the ideal irradiation period during therapy to achieve higher SDT accuracy and limit potential harm surrounding normal tissues.³⁰ Sonosensitizers with imaging capabilities will be discussed in the following sections.

MR Imaging

MRI is an imaging paradigm that is commonly used in disease diagnosis and localization with excellent spatial resolution.³¹ MRI measures such as longitudinal relaxation (T1), transverse relaxation (T2), and proton density (PD) may identify human tissues. In clinical practice, T1/T2 contrast pictures can be obtained by changing the MRI scanner settings to emphasize or saturate tissue image intensity.³² The acquired data can identify histology, quantify changes in disease severity, and objectively monitor therapy.³³

Metal ions have been shown to have MRI imaging capabilities, and sonosensitizers can create regulated therapeutic effects. Based on this function, metal-containing nano-sonosensitizers with sonodynamic therapeutic benefits have been developed extensively.³⁰ Zhang et al created OCN-PEG-(Ce6-Gd³⁺)/BNN6 nanocomposite by organically combining Ga³⁺ with sonosensitive agent e6(Ce6) and loading the N, N’ -di-sec-butyl-N, N’ -dinitro-1, 4-phenylenediamine (BNN6).³⁴ The brightness of T1-weighted images was correlated with the Gd³⁺ concentration in a 0.5T MRI system. The longitudinal relaxation rate (r1) and transverse relaxation rate (r2) are 89.0 and 103.5 mm⁻¹s⁻¹, respectively, much higher than the routinely employed gadolinium chelating contrast agent. After injection (4 mg/mL) into mice, MR images of tumour sites increased in brightness at 12 h and 24 h. Sub-5 nm Gd³⁺-hemoporfin framework nanodots (GDHF-NDs) were created by Geng et al as multifunctional therapeutic nanoagents for malignancies.³⁵ In a 3T MR scanner, the MRI images of the solution got brighter and brighter as its concentration (0–0.1 mg/mL) increased. After intravenous injection (150 L, 2.0 mg/mL), the signal intensity of tumour tissue increased from 751.7 ± 33 to 1193.5 ± 56 after 3 h and then decreased to 1118.3 ± 21 after 6 h. It was worth mentioning that the size of GDHF-ND was less than 5 nm and belonged to ultrafine nanomaterials (10 nm), which could be successfully filtered through the kidney and contribute to biosafety due to its fast clearance rate.^36–38 This was also supported by the time-dependent biodistribution study in this research. Geng et al created nanomaterials Mn (III) -HMME frameworks (Mn (III) -HFs) with MRI imaging and SDT effect employing Mn (III) ions as metal nodes and hemorphyrin monomethyl ether (HMME) as sonosensitized ligands.³⁰ The T1 signal intensity of Mn (III) -HFS/PEG increased with concentration (0–0.2mm), while the T2 signal intensity decreased. In particular, Mn (III) is converted to Mn (II) after interacting with GSH, and Mn (II) ion contains more unpaired electrons, resulting in a slight improvement in T1/T2-weighted MRI image. In CT26 tumour-bearing mice (150 L, 2 mg/mL), the intensity of the T1 signal intensity reached a high of 2603.1 ± 250.9 at 4 h, but the T2 signal intensity dropped from 2647.2 ± 269.9 before injection to 1696.1 ± 111.3 4 h later and 1905.1 ± 140.9 8 h later. Du et al developed a unique nanoparticle DOX/Mn-TPPS@RBCs, an oxygen-producing RBC carrier system for combinatorial sonodynamic and chemotherapy in breast cancer.³⁹ In vitro investigations using a 3T MRI system revealed a concentration-dependent whitening response for nanomaterials with varied concentrations. The r1 value was 18.32 mm⁻¹s⁻¹. MR images of tumour-bearing mice (Mn-TPPS: 3.5 mg/kg) demonstrated the strongest signal of 118 a.u. At 12h after injection, the signal lasted for 24h. Zhang et al used glucose oxidase (GOx) and mesoporous MnO₂ NPs to construct a cascade catalytic nanoplatform GOx-MnO₂/HMME, which could play a synergistic role in SDT and starvation therapy on breast cancer tumours.⁴⁰ After incubating GOx-MnO₂/HMME solutions with glucose (1 mg/mL) for 1 hour, the was greater in the MnO₂ NPs group (r1 = 2.88 mm⁻¹s⁻¹) than in the untreated group (r1 = 0.86 mm⁻¹s⁻¹). Subsequently, tumour-bearing mice were injected with nanomaterials ([MnO₂] = 10 mg/kg) for 3 h, followed by T1-weighted imaging using a 3.0T clinical scanner, which demonstrated an increased signal at the tumour site. Bai et al’s ultra-fine iron-doped titanium dioxide nanodots (Fe-TiO₂ NDs) conducted combination therapy of CDT and SDT under the dual direction of MR imaging for breast cancer treatment.⁴¹ The r1 value was estimated to be 4.71 mM⁻¹s⁻¹ (Figure 2A). Images and quantitative analysis of the MR signal indicated that the intensity of the T1-weighted MR signal in the tumour region was 1.87 times that of the tumour preinjection (Figure 2B and C).

Figure 2 Imaging properties of Fe-TiO2 NDs. (A) MR images of Fe-TiO2 solutions with different concentrations and the relative T1 relaxation rates. (B) MR imaging of 4T1 tumour-bearing mice before and after injection of Fe-TiO2 NDs for 24 h. (C) Quantification of MR signals from the tumours in (B). Reprinted with permission from Bai S, Yang N, Wang X, et al. Ultrasmall iron-doped titanium oxide nanodots for enhanced sonodynamic and chemodynamic cancer therapy. ACS Nano. 2020;14(11):15119–15130. Copyright [2020] American Chemical Society .41

CT

CT scans employ computer-processed combinations of multiple X-ray images obtained from various angles to construct an image of the anatomy of the scanned item.⁴² CT has been widely used in clinical practice and medical procedures for its ability to observe what is happening in a patient’s body in a non-invasive manner. CT has been used to aid the precise targeting of probes for diagnosis and treatment. Some researchers have conducted studies on the collaboration of probe and CT technology in response to a diverse microenvironment to enhance the tumour microenvironment and achieve tumour therapies.⁴³

Zhang et al created enteric-soluble nanoparticle Au@mSiO2/Ce6/DOX/SLB-FA@CMC (GMCDS-FA@CMC) capable of pH/ultrasound dual response, CT imaging, and sonodynamic-chemotherapy for colorectal cancer treatment.²⁵ The gold in mesoporous silicon inside the GMCDS-FA@CMC could be utilized for CT imaging. CT imaging was performed on orthotopic colorectal cancer model mice after oral administration of GMCDS-FA@CMC. The clear CT signal of the tumour was noticed, and the clear signal persisted for 7–9 hours. Findings indicate that GMCDS-FA@CMC has a strong CT imaging impact on guiding sonodynamic chemotherapy.

CEUS

For many years, CEUS has been routinely utilized to improve imaging of the heart’s circulatory system and other organs.⁴⁴ CEUS achieves its contrast enhancement effect by the high scattering of ultrasonic pulses by stable microbubbles (MBs) or nanobubbles (NBs) for persistence and echogenicity during intravenous administration.^45,46 MBs and NBs can be used as ultrasound contrast agents and synergistic non-invasive imaging and therapy by using ultrasound to destroy therapeutic genes and drugs encapsulated in the lesion region.^47–50 Consequently, MBs and NBs have a promising future as integrated diagnostic and treatment providers.

Zhang et al recommended using all-in-one nanoparticles (CPDP NPs) to construct a diagnosis and treatment system for breast cancer based on SDT-oriented collaborative therapy combined with chemotherapy and US imaging.⁵¹ Acoustic nanodroplets with a liquid core can be transformed to MBs via acoustic droplet vaporization (ADV) when exposed to US irradiation.^52,53 Additionally, ADV-generated bubbles (ADV-Bs) may have the same properties as microbubbles (MBs).^54,55 The acoustic impedance values of perfluorobutane (PFP) are similar to those of adjacent tissues, so liquid PFP droplets must evaporate into bubbles to become effective US contrast agents.^56,57 The nanodroplet of PFP has a strong phase transition capacity and can go from liquid to gas phase by mediating ADV.^53,58,59 Using a LIFU transducer (50% duty cycle, 1–2 W/cm², 1 s pulse) to irradiate 1 mg/ mL emulsion, it was found that 2D and CEUS images were most significant when LIFU intensity reached 2 W/cm² and duration was 120 seconds (Figure 3A). In vivo, LIFU irradiation strengthened the images of tumours in mice (Figure 3B). The images’ findings were consistent with the trend of data obtained from quantitative analysis (Figure 3C and D). Ho et al developed superhydrophobic mesoporous silica nanoparticles (FMSNs-DOX) loaded with the antitumor medication doxorubicin to treat prostate cancer in their investigation.⁶⁰ The hydrophobic surface of mesoporous silica nanoparticles (MSNs) was modified to enhance the concentration of air nanoparticles (NBs) at the interface between the water and the hydrophobic MSNs surface in the previous research.^61,62 These interfacial nanobubbles (INBs) are confined in the cavities supplied by the mesopores of hydrophobic MSNs, allowing them to be employed as nanoscale ultrasonic contrast agents.^61,63–66 Ho et al used perfluorodecyltriethoxysilane (PFDTS) with moderate surface energy and superhydrophobicity as the surface coating of MSNs to make MSNs superhydrophobic in order to improve image contrast by enhancing INB accumulation.^67–69 Analysis of mouse tumours revealed that INBs could be repeated and cavitated from day 1 to day 9 to generate significant contrast enhancement within the tumour. The quantitative findings revealed that the US imaging was steady (3.84 ± 0.47 dB) from day 1 to day 9 and declined with time after day 9. Zhang et al investigated a nanosystem (BBC-HPBS/HMME/PFH) for breast cancer that achieved precision drug administration guided by US imaging and local sonodynamic treatment.⁷⁰ Like PFH, PFH was a phase change material that could be converted from liquid to gas to become a US contrast agent when the pressure at the tumour tissue was decreased to the vaporization pressure threshold by an external input of energy.^71–73 In vitro, the aqueous dispersion of RBC-HPBs/HMME/PFH was irradiated with ultrasound (3 MHz, 5.0 W/cm2, 30s). Furthermore, the images exhibited concentration-dependent ultrasonic signal amplification. Then, RBC-HPBs/HMME/PFH (5 mg/kg) were administered intravenously into tumour-bearing mice, followed by ultrasonic irradiation (3 MHz, 5.0 W/cm2, 30s). A distinct US signal was discovered, and the signal was most outstanding at 8h. Feng et al reported the TPZ/ HMTNPs-SNO therapeutic agent delivery system, which is made by combining tirapazamine (TPZ) with S-nitrosothiol (R-SNO) modified hollow mesoporous titanium dioxide nanoparticles (HMTNPs).⁷⁴ Here, Feng et al applied R-SNO, a ROS-sensitized NO donor, to the surface of HMTNPs. Following US activation of HMTNPs to form ROS, the -SNO group would interact with ROS and trigger S-N bond homolysis to release NO.⁷⁵ The released NO would exhibit blistering behaviour, thus acting as a US contrast enhancer. In vitro, US images of HMTNPS-SNO after US irradiation (1 W/cm2) indicated that the improvement of acoustic contrast was consistent with the increase in NO yield data. In vivo, the HMTNPs-SNO group (20 mg/kg) demonstrated elevated spots at the tumour location following US stimulation (1 W/cm2) and increased ultrasonic signal over time.

Figure 3 Imaging properties of CPDP NPs. (A) Ultrasound images of 2D and CEUS under different LIFU intensities and duration times. (B) 2D and CEUS images with and without LIFU irradiation. (C) The corresponding grayscale intensity (**p < 0.01, *p < 0.05, n = 3). After the H&E, PCNA, and TUNEL staining, the proliferate rate of PCNA in CPDP NPs + LIFU group was only 20.50%. The TUNEL results indicated CPDP NPs + LIFU group exhibited an obvious apoptosis index of 72.86%. Reproduced from Zhang Q, Wang W, Shen H, Tao H, Wu Y, Ma L, Yang G, Chang R, Wang J, Zhang H, Wang C, Zhang F, Qi J, Mi C. Low-Intensity Focused Ultrasound-Augmented Multifunctional Nanoparticles for Integrating Ultrasound Imaging and Synergistic Therapy of Metastatic Breast Cancer. Nanoscale Res Lett. 2021;16(1):73. To view a copy of this licence, visit https://creativecommons.org/licenses/by/4.0/.51

Optical Imaging

PA and FL imaging are non-invasive optical imaging techniques.⁷⁶ PA imaging is a new hybrid imaging method based on the PA effect.⁷⁷ By irradiating endogenous chromophores and exogenous contrast agents with near-infrared (NIR) light at 650–1200 nm, the photon energy is converted into sonic pressure waves to achieve deep tissue penetration of non-ionizing imaging technology that provides high contrast depth imaging and molecular imaging.^59,78,79 Therefore, PA imaging promises to provide practical information about biological tissues, such as vascular networks and oxygenation status in the tumour region.^80,81 Fluorescence imaging is also an exciting diagnostic technique. NIR irradiation or the physiological milieu of a biological condition can activate fluorescent probes, resulting in light emission imaging.^82,83 FL imaging allows for real-time monitoring of imaging probe dispersion throughout the body, including malignancies.^84–88

Melanin nanoparticles (MNPs) produced from natural biopolymers are excellent contrast agents for PA imaging because of their high light absorption.^89–92 Huang et al designed and synthesized FA-HMME-MNPs-PLGA nanoparticles with core/shell structure utilizing MNPs-linked aromatic structure, and the imaging capacity of MNPs was used to improve the PA image-guided SDT.⁹³ PA images of FHMP NPs in vitro indicated that they performed best under stimulation at 700 nm. In MDA-MB-231 tumour-bearing mice, the PA signal in the tumour region reached a peak at 2 h after injection of FHMP NPs (200 µL, 10 mg/mL). Dong et al assembled nanoscale sonosensitizer Ce6-PTX@IR783 by merging two organic dyes (Ce6 and IR783) with the anticancer agent paclitaxel (PTX).⁹⁴ IR783 is a gorgeous hydrophilic heptamethine cyanine dye with excellent optical imaging and tumour targeting properties.^95–97 Ce6-PTX@IR783 reaches its peak PA value at 795 nm. There was a linear relationship between the PA value and the concentration of Ce6-PTX@IR783. After intravenous injection into 4T1 tumour-bearing mice, the PA value peaked at 7 h.

Since Ag₂S quantum dots (QD) have a tiny bandgap (0.9 eV), they emit light in the near-infrared (NIR).⁹⁸ Additionally, Ag₂S QDs, such as their broad absorbance-narrow emission profile, adjustable emission wavelength, and extended luminescence lifetime, make them excellent for FL imaging in biological tissues.⁹⁹ Li et al modified Ag₂S QDs with Pluronic F-127 and coated them with RBC vesicles to form biomimetic agent (QD@P) Rs for enzyme-enhanced SDT.¹⁰⁰ The FL signal at the tumour sites peaked at 6~9 h after (QD@P) Rs were injected into C26 xenograft mice, then a faint FL signal was obtained 24 h later. Zhang et al constructed a hypocrellin derivative sonosensitizer (APHB NPs) to improve the depth of tumour therapy.¹⁰¹ Hypocrellins is a water-soluble metabolite produced by the traditional Chinese medicine fungus Hypocrella Bambusae, with high singlet oxygen production and broad spectrum absorption in the NIR.^102,103 In this experiment, the modification of hypocrellin B by 1, 2-diaminopropane made the obtained APHB possess excellent FL imaging ability and water solubility (Figure 4A). After 638 nm laser irradiation of 4T1 tumour mice, intratumoral FL reached a plateau 7 h after injection of APHB NPs, and diminished substantially 48 h later (Figure 4B and C). Ex Vitro FL images indicated that FL signals in tumors were remarkably higher than in other tissues (Figure 4D and E).

Figure 4 Synthesis and imaging properties of APHB NPs. (A) The synthetic route of APHB. (B) FL images of the mice and (C) FL intensities of the tumour at different time points post-injection of APHB NPs. Ex vivo (D) FL images and (E) FL intensities of major organs and tumours at 48 h post-injection of APHB NPs. Approximately 90% cell death is found at the concentration of 100 μg/mL under ultrasound stimulation (0.6 W/cm, 60s). Notes: Reprinted with permission from Zheng X, Liu W, Ge J, et al. Biodegradable natural product-based nanoparticles for near-infrared fluorescence imaging-guided sonodynamic therapy. ACS Appl Mater Interfaces. 2019;11(20):18178–18185. Copyright 2019 American Chemical Society.101

Multimodal Imaging

Advantages of Multimodal Imaging

It is well-known that multimode imaging supplies more reliable physiological information for the early detection and therapy of cancer by incorporating the advantages of individual diagnostic imaging.¹⁰⁴ Due to equipment limitations, the data provided by single-mode imaging is often short of the high precision and reliability required for diagnosing, surgical guidance and prognostic assessment.¹⁰⁵ Ultrasound imaging, for example, is hindered by weak penetration in gas. MRI is much less sensitive than other types of imaging. Soft tissue contrast is frequently a limitation of CT imaging. Due to the scattering of solid light in cells or tissues, the resolution of fluorescence imaging diminishes significantly as the imaging depth increases, limiting the technique’s practical imaging depth. Many multimodal imaging materials based on nanoparticles have been produced to date in order to achieve better imaging and early treatment.^29,106–109

Dual-Mode Imaging

Porphyrins could generate fluorescence in the red wavelength part of the spectrum after UV or violet stimulation, used for PA and FL imaging.^110,111 Moreover, nanoparticles constituted of varying metal ions (Mn and Cu ions) could redox with glutathione (GSH), thus releasing metal ions to induce MRI.^112–115 When porphyrin was synthesized with metal to form the metal-porphyrin complex, it would become a dual mode contrast agent. Such potent nanoparticles have been used to design nanoparticles with specific therapeutic properties.^116–118 Wang et al created UPFB nanocomposites with Janus nanostructures made of UCNPs and porphyrin-based MOFs [PCN-224 (Fe)] to achieve direct photodynamic and sonodynamic co-therapy.¹¹⁹ The FL signal in the tumour started to arise at 1 h after intravenous infusion, with the brightest signal released at 4 h (Figure 5A and B). Then the intensity progressively diminished 12 h later, but the intensity in the liver and kidney climbed (Figure 5C). The r2 of UPFB in the GSH solution was 55.17 mM⁻¹s⁻¹. This implied that UPFB can react with GSH to convert Fe3+ into T2-weighted MRI contrast agent Fe2+. In addition, after the addition of H₂O₂ and GSH, r2 was 81.06 mM⁻¹s⁻¹, which was because neutral conditions facilitated the transformation of catalase-like Fe³⁺ into Fe²⁺. The 1.2T MR scanner showed that the tumour site in the UPFB-treated group darkened over time compared to the mice injected with UPF (Figure 5D). Wang et al created MG@P NPs for synergistic starvation treatment and SDT by encapsulating GOx and 5, 10, 15, 20-tetrakis (4-chlorophenyl) porphyrin) Cl (denoted PMnC) in poly (lactic-co-glycolic) acid (PLGA) NPs.¹²⁰ The increase of T1-weighted and PA signal intensity was positively correlated with MG@P NPs concentration. The semi-quantitative T1 signal intensity reached the peak at 24 h after injection in 4T1 tumour-bearing mice. The peak of the PA signal occurred around 24h, which corresponded to the strongest signal in MR imaging. Similarly, Ma et al designed HSA-wrapped metal-porphyrin complex (MnTTP-HSAs) by utilizing Mn ion and 4-methylphenylporphyrin (TTP) ligand, which can conduct MR/PA image-guided SDT in deep tumour tissues.¹²¹ The image findings are shown in Table 1. Dong et al created AgBiS2@DSPE-PEG2000-FA(ABS-FA), targeted multifunctional hydrophilic nanomicelles and used them to treat breast cancer.⁴³ Ternary alloy silver bismuth sulfide (AgBiS₂, ABS) nanodots exhibited excellent X-ray attenuation, high near-infrared absorption, and photothermal conversion efficiency, making them an ideal CT/IR bimodal imaging agent (Figure 6A and B).^122,123 After the injection of ABS-FA (300 μL, 5 mg/mL), CT signals appeared at the tumour site and increased steadily, reaching a peak at 6–12 h (Figure 6C). Furthermore, in vivo low power (0.35W/cm²) infrared thermal imaging results were consistent with CT, with the thermal signal at 2 h and the maximum thermal signal at 10 h (Figure 6D). Zhang et al used PLGA as a carrier to add IR780 and HMME to the shell and wrapped GOx to synthesize IHG@P NPs with mitochondrial targeted synergistic SDT and starvation therapy effects.¹²⁴ Previous research has demonstrated that HMME can be utilized in PA imaging of malignancies.^125,126 At the same time, IR780 has a significant near-infrared absorption peak and generates high-intensity fluorescence in the 807–823 nm wavelength region, making it suitable for PA and FL imaging applications.^127–129 In vitro, PA imaging of the IHG@P solution determined an optimal excitation wavelength of 800 nm. At 24 h after intravenous administration of IHG@P NPs (5 mg/mL, 200 µL) to 4T1 xenograft mice, PA signal intensity in the tumour region increased 3.02 times before injection. For FL imaging, the signal could be observed at the tumour sites 1h after injection and reached a peak at 24h. Zhao et al created the LIP3 liposome nanosystem, which consists of a lipid bilayer of HMME as the shell, PFH and water-soluble prodrug banoxantrone (AQ4N) as the core.¹³⁰ CEUS imaging revealed that when AI was 6 W/cm2, the echo signal attained its maximum value after 3 min. In PA imaging, the PA signal increased with increasing concentration, and the linear correlation coefficient between them was R = 0.992. Hou et al constructed an amphiphilic Rose Bengal (ARB) and encapsulated it in fluorine gas to create a new sonosensitizer (RB-MBs).¹³¹ The fluorescent and photosensitive Bengal Rose was coupled with dihexalkylamine via a stable amine link to generate the ARB, employed as a sonosensitizer and FL imaging agent.^132,133 Simultaneously, CEUS would be performed using MBs generated by fluorinated gas encapsulated by ARB. There was a distinct ultrasonic signal when the concentration of RB-MBs reached 6.2×10⁻⁶ MBs/mL in vitro. In mice with HT-29 tumours, an instant increase in US signal was seen at the tumour site and lasted more than 4 minutes. Furthermore, clear fluorescence signals were observed at the tumour site using 523nm and 610nm excitation wavelength filters. Lei et al reported iron-doped vanadium disulfide nanosheets (Fe-VS₂ NSs) for sonodynamic/chemodynamic combination therapy.¹³⁴ Vanadium disulfide (VS₂) has demonstrated promising MR/PA multimodal imaging.¹³⁵ Simultaneously, metal doping in TMDC can optimize its physical properties, allowing them to adapt to a wider range of applications.¹³⁶ PA images of 4T1 xenograft mice after injection of FE-VS₂-PEG NSs (7.5 mg/kg) showed an obvious PA signal in the tumour area. The r1 was 19.968 mm⁻¹s⁻¹, which was greater than the r1 of the VSx nanoplatform studied in the past, indicating that the addition of Fe³⁺ might enhance the MRI ability of the V element. According to the PA imaging results, the intensity of the MR signal in mice rose to 2.04 times that before injection. Indocyanine green (ICG) has been verified to have moderate fluorescence quantum yield and absorption in the NIR, which could be used in optical (PA/FL) imaging.^137–139 Because it also functions as a sonosensitizer, it is frequently employed to build novel multifunctional sonosensitive agents. Yang et al assembled the sonosensitizer ICG and TPGS-PEM prodrug (D-α-tocopheryl polyethylene 1000 succinate (TPGS) modified by pemetrexed (PEM)) into dual-mode imaging and active targeting TPGS-PEM-ICG nanoplatform (TPI).¹⁴⁰ The FL signal of HeLa tumour-bearing mice was the highest at 6 h and lasted until 24 h after TPI injection through the tail vein. The peak PA signal in the tumour area reached the peak 6 h after injection. Chen et al prepared oxygen-carrying PLGA nanoparticles (PIO_NPs) using PLGA as the shell loaded of ICG and PTX to encapsulate PFP and oxygen.¹⁴¹ Following laser irradiation, ICG wrapped in a PFP droplet will generate an immediate temperature rise, leading to evaporation of the PFP droplet into the gas phase and subsequently producing US and PA imaging functions. Meanwhile, The thermal tissue expansion caused by the temperature rise of ICG will generate an ultrasonic pressure wave, which magnifies the US/PA signal.^77,142 In vivo, the intensity of the PA signal in the tumour was heightened at 2 h, peaked at 6h and lasted until 12h. So, the optimal imaging time was 6 h. At this time, the average PA value (a.u.) increased from 0.26 ± 0.02 to 0.37 ± 0.03 after near-infrared irradiation (1.5 W/cm², 5 min). The EI of B-mode was also increased substantially. Analogously, Xie et al and Zheng et al created OI_NPs and OIX_NPs that were similar to PIO_NPs in that they encapsulated PFP with PLGA and loaded ICG, and they used the imaging features of the ICG and PFP bubbles to develop the PA/US imaging performance of sonosensitive nanoparticles.^143,144 Liu et al loaded ICG and folic acid target molecules into the shell using lipids as carriers and encapsulated oxygen-carrying PFH to synthesize nanoparticles (FA-OINPs) to achieve dual-mode imaging monitoring for ovarian cancer treatment.¹⁴⁵ All groups were irradiated with a 1.5 W/cm² laser for 5 min. In vitro, the EI of CEUS increased to 22.49 times that before the irradiation. The EI of B-mode increased from (33.05 ± 2.78) a.u. to (83.09 ±12.36) a.u. Consistently, the PA values increased from (0.31±0.02) a.u. to (0.72±0.08) a.u. In vivo, the optimal imaging time for US and PA imaging was 6 h after injection (64 μg/mL of ICG, 200 μL). He et al synthesized multifunctional nanocomposites (mTiO₂@PPYs) by combining mesoporous TiO₂ nanoparticles (mTiO₂s) with photothermal polypyrrole (PPY), which has a synergistic therapeutic effect on tumours.¹⁴⁶ In this research, the author discovered that pure mTiO2 could play the role of US imaging in vivo and in vitro, which was not found in other articles. The outer PPY layer is a typical conjugated polymer with a larger length than polycyclic aromatic compounds (such as porphyrins), which leads to an increase in absorption coefficient and transfer of absorption to higher absorption wavelengths, resulting in better PA imaging performance.^147–149 In vivo, the intensity of the US signal increases with increasing concentration of mTiO2@PPY. The US and PA signals were also detected in vivo in the tumour region of tumour-bearing mice in the mTiO2@PPY group. Li et al designed a nanoplatform (F3-PLGA@MB/Gd NPs) that effectively coloureds sonosensitizer (methylene blue, MB) and Gd-DTPA-BMA, integrating dual-mode imaging (PA and MRI) with dual-mode treatment (SDT and HIFU ablation).¹⁵⁰ Methylene blue (MB) is a hydrophilic phenothiazine derivative that may be employed as a sonosensitizer and a PA contrast agent.^151–154 At the same time, Gd-DTPA-BMA as an MRI contrast agent and MB work together to improve imaging ability. With the increase in concentration, the intensity of the T1 signal and PA signal became clearer at a wavelength of 700 nm. High-resolution PA and MR tumour images appeared at 6 h after injection of F3-PLGA@MB/Gd NP solution (0.2 mL, 10 mg/mL) in MDA-MB-231 tumour mice.

Figure 5 Imaging properties of UPFB. (A) FL images of U14-tumour-bearing mice taken after i.v. injection of UPF and UPFB. (B) Ex vivo fluorescence images of major organs and tumours at 4 h post-injection with UPF and UPFB. (C) FL intensity of the major organs and tumours at different times. (D) T2-MRI of a tumour-bearing mouse with i.v. Injection of UPF or UPFB at different time intervals. The tumour site was labelled with a red ellipse. The high contents of UPFB without DMTU upon irradiation with the 808 nm laser and US exhibited the lowest cell viability rates (16.7%). Notes: Reprinted with permission from Wang Z, Liu B, Sun Q, et al. Upconverted metal-organic framework janus architecture for near-infrared and ultrasound co-enhanced high performance tumor therapy. ACS Nano. 2021. Copyright [2021] American Chemical Society.119

Figure 6 Synthesis and imaging properties of ABS-FA. (A) Schematic illustration for the synthesis of ABS-FA. (B) surgical navigation during tumour treatment. (C) CT imaging of HeLa tumour-bearing mice after injection of ABS-FA and ABS; red dotted circle: the tumour site. (D) Low-power (0.35 W/cm2) infrared thermal imaging of tumour-bearing mice at different time points. When HeLa cells underwent NIR irradiation and ultrasound treatment simultaneously, the survival rate when cultivated with ABS-FA was only 8.99%. Notes: Reprinted with permission from Cheng K, Zhang RY, Yang XQ, et al. One-for-all nanoplatform for synergistic mild cascade-potentiated ultrasound therapy induced with targeting imaging-guided photothermal therapy. ACS Appl Mater Interfaces. 2020;12(36):40052–40066. Copyright [2020] American Chemical Society.43

Triple Mode Imaging

Liu et al constructed a nanoparticle (ANG-IR780-MnO₂-PLGA, AIMP) with multimode (PA/FL/MR) imaging capability utilizing IR780 and MnO_2.¹⁵⁵ In vitro, the FL, PA signal and 1/T1 value were concentration-dependent. The r1 was 6.119 under simulated TEM settings (pH 6.5, 2 mM GSH, 100 μM H2O2), which was considerably higher than the pH 7.4 (physiological environment) group, indicating that AIMP NPs may be employed as TME stimuli-responsive T1 MRI contrast agents. After injection of AIMP NPs (2 mg/mL, 300 µL) into U87 MG xenograft mice through the vein, FL signalling at the tumour site peaked at 4 h and kept high until 24 h. After administration, the changing trend of the PA signal was consistent with FL imaging data. However, the maximum strength of the MR signal arrived later, at 6h. However, due to the hyperoxic level of the tumor site, 6h may be the best time for LIFU irradiation. Zhang et al described the construction of nanodroplets (IR780-NDs) utilizing IR780 and PFP for multimode (FL/PA/US) imaging-guided SDT.¹⁵⁶ It was observed under an optical microscope that a large amount of IR780-NDs was transformed into MB with an average size of about 2 μm after 3 min of irradiation at 2.4 W/cm². A significant increase in US intensity was also observed (Figure 7A and B). Under irradiation with a 780 nm laser, the PA signal expanded linearly with the IR780-NDs concentration (Figure 7C). In 4T1 tumour-bearing mice, the US signal in CEUS mode was bright 24 h after injection (Figure 7D). Furthermore, the quantitative biodistribution analysis showed that the EI was considerably higher after injection than before (Figure 7E). Meanwhile, the PA signal peaked at about 24 h after injection (Figure 7F and G). FL signal was prominent at 0.5–24 h (Figure 7H). Ex Vitro FL images showed that FL signals of tumours were more significant than in other organs (Figure 7I).

Figure 7 Imaging properties of IR780-NDs. (A) The US images (CEUS and B-mode) and corresponding quantitative analysis (B) of the echo intensities. (C) PA images and PA values at different concentrations. (D) CEUS and B-mode imaging before and after US irradiation. (E) Corresponding echo intensities of tumours. (F) Changes in PA signal intensities and images (G) at the tumour regions at the corresponding time points. (H) FL images of tumours in 4T1 tumour-bearing mice at different time points. (I) Ex vivo FL images of major organs and tumours dissected from mice 24 h post-injection. 91.52% of cells died in the IHG@P group. The lowest tumor volume was recorded in the IHG@P + US group (1.94-fold increase). Notes: Reprinted with permission from Zhang L, Yi H, Song J, et al. Mitochondria-targeted and ultrasound-activated nanodroplets for enhanced deep-penetration sonodynamic cancer therapy. ACS Appl Mater Interfaces. 2019;11(9):9355–9366. Copyright [2019] American Chemical Society.156

Reflection of Multimodal Imaging Nanoparticles

Hybrid imaging approaches provide the benefits of high temporal and spatial sensitivity and anatomical and functional information by integrating single imaging modes. Scan time and dosage can be lowered by injecting multimode probes, and the enhanced imaging signal can be used for diagnosis, localization, and therapy guiding. It should be noted that significant differences in sensitivity between different techniques mean that multimode optimized seekers require a large number of low-sensitivity seekers to meet the ideal probe concentration for each imaging mode or multimode probes that can overcome sensitivity differences need to be designed to achieve synchronous data acquisition. The crucial question is whether the properties of each mode in the multimode nanoparticle are comparable sufficiently to those of the single-mode nanoparticle that each portion of the combined injection may offer the same information as the corresponding single probe. This challenge cannot be overcome by solely raising probe concentrations for different forms of imaging or by integrating various types of imaging probes. In a nutshell, the fusing of nanoparticles with varied functionalities without compromising the individual efficiency of each function is a problem to resolve when establishing novel integrated diagnostic and therapeutic vectors. On this basis, we briefly summarized the main synthesis strategies of sonosensitizers in Table 2, which can provide new ideas and guidance for the development of sonosensitizers in the future.

Table 2 Synthesis Strategies of Sonosensitizers

Sonosensitizers Beyond Imaging

With the expansion of basic research, people are paying more and more attention to the genetic level of treatment and monitoring methods to find a cure for cancer.¹⁵⁷ The delivery function of sonosensitizers could transport therapeutic genes to the target to achieve gene therapy for tumours. Multiple studies have supported that multifunctional sonosensitizers could alter gene expression in cancer and immune-related cells and revealed therapeutic target genes in various signaling pathways by the data analysis.^158–161 For example, Wu et al designed IR820@NCP using RAS inhibitor Farnesyl-Thiosalicylic acid (FTS) to demonstrate that SDT combined with FTS predominantly achieved anti-tumour effects by inhibiting endothelial cells and simulating host immunity.¹⁶⁰ In Chen et al’s research on the treatment of liver cancer by RSL3@O2-ICG NBs, essential genes were identified to affect the sensitivity of hepatocellular carcinoma to iron death.¹⁵⁸ In addition to molecular imaging techniques, such gene and protein level monitoring could be achieved by reporter imaging techniques that combined reporter proteins with corresponding imaging probes.¹⁶² Because the reporter gene is inserted directly into the cell’s DNA, the imaging is generated only in the living cell and can retain accurate imaging for a long time as the cell divides and is passed on to daughter cells.^163,164 Various molecular imaging modalities, including FLI, PAI and MRI, could be used for reporter gene imaging for gene activity monitoring, cell tracing and gene therapy monitoring. The combination of sonosensitizer and reporter gene imaging technology could simultaneously play a role in the precise localization of cancer cells and gene therapy. This new therapy model may hold great promise.

Conclusions and Outlook

There is no doubt that SDT has become a perfect cancer treatment due to its excellent therapeutic effectiveness, deep tissue penetration and low lateral injury. Since SDT was initially described in 1989, significant efforts have been made to use SDT for cancer treatment and to produce relevant sonosensitizers. Now, SDT combined imaging has opened the door to a new therapeutic approach and has become an attractive trend. Such multifunctional, integrated nanosystems are considered a potential candidate for innovative non-invasive therapeutics, presenting a prominent bright spot for enhancing nanosystems’ applicability in the biomedical area. Imaging technology helps diagnose cancer earlier and more correctly, provides precise and potent therapy interventions for tumours, and improves prognosis and survival. Considering the limitations of existing therapies, SDT combination therapy may have higher clinical potential. Hence, creating new sonosensitizers with better diagnostic capabilities, especially those active solely in TME, maybe a prospective approach in sonodynamic research.

However, from a clinical point of view, there are still many elements to optimize the imaging quality. In order to further the research and development of tumour imaging and acoustic power integrated therapy, we must focus on novel imaging target identification and validation and investigate other features. Imaging probe production and characterization require the development of high sensitivity, high-resolution imaging equipment, hybrid equipment and improved image reconstruction techniques. Academic researchers, doctors, and the pharmaceutical sector must collaborate closely to bring integrated tumour imaging and dynamic treatment probes into cancer management.

We assume that by emphasizing the most recent achievements in this field, we can stimulate the scientific community to make significant contributions to cancer therapy. Based on the promising advances described in this research, we anticipate that these integrated nanoparticles for detection and therapy will shortly provide a new perspective for cancer therapeutic development.