Regulation of CTLs/Tregs via Highly Stable and Ultrasound-Responsive Cerasomal Nano-Modulators for Enhanced Colorectal Cancer Immunotherapy.

Immunotherapy is showing good potential for colorectal cancer therapy, however, low responsive rates and severe immune-related drug side effects still hamper its therapeutic effectiveness. Herein, a highly stable cerasomal nano-modulator (DMC@P-Cs) with ultrasound (US)-controlled drug delivery capability for selective sonodynamic-immunotherapy is fabricated. DMC@P-Cs' lipid bilayer is self-assembled from cerasome-forming lipid (CFL), pyrophaeophorbid conjugated lipid (PL), and phospholipids containing unsaturated chemical bonds (DOPC), resulting in US-responsive lipid shell. Demethylcantharidin (DMC) as an immunotherapy adjuvant is loaded in the hydrophilic core of DMC@P-Cs. With US irradiation, reactive oxygen species (ROS) can be effectively generated from DMC@P-Cs, which can not only kill tumor cells for inducing immunogenic cell death (ICD), but also oxidize unsaturated phospholipids-DOPC to change the permeability of the lipid bilayers and facilitate controlled release of DMC, thus resulting in down-regulation of regulatory T cells (Tregs) and amplification of anti-tumor immune responses. After intravenous injection, DMC@P-Cs can efficiently accumulate at the tumor site, and local US treatment resulted in 94.73% tumor inhibition rate. In addition, there is no detectable systemic toxicity. Therefore, this study provides a highly stable and US-controllable smart delivery system to achieve synergistical sonodynamic-immunotherapy for enhanced colorectal cancer therapy.

Nanomaterials augmented bioeffects of ultrasound in cancer immunotherapy.

Immunotherapy as a milestone in cancer treatment has made great strides in the past decade, but it is still limited by low immune response rates and immune-related adverse events. Utilizing bioeffects of ultrasound to enhance tumor immunotherapy has attracted more and more attention, including sonothermal, sonomechanical, sonodynamic and sonopiezoelectric immunotherapy. Moreover, the emergence of nanomaterials has further improved the efficacy of ultrasound mediated immunotherapy. However, most of the summaries in this field are about a single aspect of the biological effects of ultrasound, which is not comprehensive and complete currently. This review proposes the recent progress of nanomaterials augmented bioeffects of ultrasound in cancer immunotherapy. The concept of immunotherapy and the application of bioeffects of ultrasound in cancer immunotherapy are initially introduced. Then, according to different bioeffects of ultrasound, the representative paradigms of nanomaterial augmented sono-immunotherapy are described, and their mechanisms are discussed. Finally, the challenges and application prospects of nanomaterial augmented ultrasound mediated cancer immunotherapy are discussed in depth, hoping to pave the way for cancer immunotherapy and promote the clinical translation of ultrasound mediated cancer immunotherapy through the reasonable combination of nanomaterials augmented ultrasonic bioeffects.

Ultrasound-Induced Piezocatalysis Triggered NO Generation for Enhanced Hypoxic Tumor Therapy.

Conventional NO gas generation based on l-arginine (l-Arg) is usually dependent on H2O2 and O2, both of which are very limited within the tumor microenvironment, thus greatly limiting l-Arg's therapeutic effect. Herein, a novel nanoplatform for efficiently triggering NO production based on ultrasound-induced piezocatalysis was developed, which was fabricated by coating amphiphilic poly-l-arginine (DSPE-PEG2000-Arg, DPA) on the piezoelectric material of barium titanate (BTO). The resulting BTO@DPA nanoparticles can efficiently generate H2O2, 1O2, and O2 via ultrasound-induced piezocatalysis based on BTO and oxidize the surface arginine to produce NO, which can even further interact with the reactive oxygen species (ROS) to produce more reactive peroxynitrite, thus inducing serious tumor cell apoptosis both in hypoxia and normoxia. After intravenous injection, BTO@DPA accumulated well at the tumor tissue at 4 h postinjection; later, ultrasound irradiation on the tumor not only achieved the best tumor inhibition rate of ∼70% but also completely inhibited tumor metastasis to the lungs via the alleviation of tumor hypoxia. Such a strategy was not dependent on the tumor microenvironment and can be well controlled by ultrasound irradiation, providing a simple and efficient therapy paradigm for hypoxic tumor.

Ultrasound-Triggered Piezocatalysis for Selectively Controlled NO Gas and Chemodrug Release to Enhance Drug Penetration in Pancreatic Cancer.

Nitric oxide (NO) is drawing widespread attention in treating pancreatic ductal adenocarcinoma (PDAC) as a safe and therapeutically efficient technique through modulating the dense fibrotic stroma in the tumor microenvironment to enhance drug penetration. Considerable NO nanogenerators and NO releasing molecules have been developed to shield the systemic toxicity caused by free diffusion of NO gas. However, on-demand controlled release of NO and chemotherapy drugs at tumor sites remains a problem limited by the complex and dynamic tumor microenvironment. Herein, we present an ultrasound-responsive nanoprodrug of CPT-t-R-PEG2000@BaTiO3 (CRB) which encapsulates piezoelectric nanomaterials barium titanate nanoparticle (BaTiO3) with amphiphilic prodrug molecules that consisted of thioketal bond (t) linked chemotherapy drug camptothecin (CPT) and NO-donor l-arginine (R). Based on ultrasound-triggered piezocatalysis, BaTiO3 can continuously generate ROS in the hypoxic tumor environment, which induces a cascade of reaction processes to break the thioketal bond to release CPT and oxidize R to release NO, simultaneously delivering CPT and NO to the tumor site. It is revealed that CRB shows a uniform size distribution, prolonged blood circulation time, and excellent tumor targeting ability. Moreover, controlled release of CPT and NO were observed both in vitro and in vivo under the stimulation of ultrasound, which is beneficial to the depletion of dense stroma and subsequently enhanced delivery and efficacy of CPT. Taken together, CRB significantly increased the antitumor efficacy against highly malignant Panc02 tumors in mice through inhibiting chemoresistance, representing a feasible approach for targeted therapies against Panc02 and other PDAC.

Genetically Engineering Cell Membrane-Coated BTO Nanoparticles for MMP2-Activated Piezocatalysis-Immunotherapy.

Tumor immunotherapy based on immune checkpoint blockade (ICB) still suffers from low host response rate and non-specific distribution of immune checkpoint inhibitors, greatly compromising the therapeutic efficiency. Herein, cellular membrane stably expressing matrix metallopeptidase 2 (MMP2)-activated PD-L1 blockades is engineered to coat ultrasmall barium titanate (BTO) nanoparticle for overcoming the immunosuppressive microenvironment of tumors. The resulting M@BTO NPs can significantly promote the BTO's tumor accumulation, while the masking domains on membrane PD-L1 antibodies are cleaved when exposure to MMP2 highly expressed in tumor. With ultrasound (US) irradiation, M@BTO NPs can simultaneously generate reactive oxygen species (ROS) and O2 based on BTO mediated piezocatalysis and water splitting, significantly promoting the intratumoral infiltration of cytotoxic T lymphocytes (CTLs) and improving the PD-L1 blockade therapy to the tumor, resulting in effective tumor growth inhibition and lung metastasis suppression in a melanoma mouse model. This nanoplatform combines MMP2-activated genetic editing cell membrane with US responsive BTO for both immune stimulation and specific PD-L1 inhibition, providing a safe and robust strategy in enhancing immune response against tumor.

In Situ Micro-Nano Conversion Augmented Tumor-Localized Immunochemotherapy.

The immune checkpoint blockade (ICB) therapy based on monoclonal antibodies still suffers from a lower immune response rate and severe immune-related side effects, which greatly compromise its therapeutic benefits. Herein, ultrasound (US) microbubbles (MBs) that locally delivered the camptothecin-floxuridine (CF) drug combination and anti-PD-L1 blocking antibody (αPD-L1) to tumors were developed to improve ICB therapy. The resulting αPCF MBs exhibited good stability, allowing their use as US imaging contrast agents to trace the drug delivery in vivo. Furthermore, the combination of αPCF MBs treatment and disrupted US irradiation triggered tumor in situ conversion of αPCF MBs to αPCF NPs while promoting higher tumor cell uptake and deeper tumor penetration as confirmed by the US/fluorescence bimodal imaging. Camptothecin (CPT) and floxuridine (FUDR) were further released at a fixed 1:1 molar ratio within the tumor microenvironment (TME) to synergistically elicit an immunogenic tumor phenotype and sensitize tumors to αPD-L1-mediated ICB therapy, while the local simultaneous delivery of immunotherapeutic αPD-L1 further reversed the immunosuppressive tumor microenvironment and promoted the infiltration of cytotoxic T lymphocytes (CTLs), thus achieving a synergistic therapeutic effect of chemotherapy and immunotherapy in the CT26 tumor-bearing mice. Thus, αPCF MBs + US mediated local co-delivering of the drug combination and αPD-L1 well augmented the ICB therapy while effectively minimizing the off-target side effects, providing a safe and universal therapeutic strategy for tumor immunotherapy.

Magnetic nanocluster-mediated photothermal effect and macrophage modulation for synergistic photothermal immunotherapy of cancer.

In the tumor microenvironment, macrophages predominately exhibit M2-type functionalities which promote malignant progression and cancer metastasis, thus posing a big hurdle for current anticancer strategies. Different approaches have been exploited to reverse the macrophages towards the M1 pro-inflammatory phenotype; however, it is hard to achieve tumor regression with this macrophage modulation alone. Herein we synthesized photothermal magnetic nanoclusters (MNCs) to test their capability for reprograming M2 macrophages towards the M1 phenotype. We demonstrated that these photothermal MNCs themselves can effectively trigger this desired macrophage repolarization, increase the phagocytosis of macrophages at the tumor site, and cause subsequent T cell activation with enhanced systemic cytokine release, leading to cancer cell killing both in vitro and in vivo. More interestingly, it was found that the photothermal effect can further facilitate this immune activation to a greater extent, inducing much higher level of macrophage repolarization and T cell infiltration into the tumor site as well as eliciting a much more efficient antitumor effect compared with MNCs alone. Our findings demonstrate that MNCs combined with their photothermal effect can reverse the local suppressive environment of the tumor by macrophage and T cell modulation, providing an effective approach to trigger systemic immune responses that contribute to an enhanced overall anticancer outcome and the suppression of cancer metastasis.

Charge Reversal Polypyrrole Nanocomplex-Mediated Gene Delivery and Photothermal Therapy for Effectively Treating Papillary Thyroid Cancer and Inhibiting Lymphatic Metastasis.

As a traditional treatment for papillary thyroid cancer (PTC), surgical resection of diseased tissues often brings lots of inconveniences to patients, and the tumor recurrence and metastasis are difficult to avoid. Herein, we developed a gene and photothermal combined therapy nanosystem based on a polypyrrole (Ppy)-poly(ethylene imine)-siILK nanocomplex (PPRILK) to achieve minimally invasive ablation and lymphatic metastasis inhibition in PTC simultaneously. In this system, gelatin-stabilized Ppy mainly acted as a photothermal- and photoacoustic (PA)-responsive nanomaterial and contributed to its well-behaved photosensitivity in the near-infrared region. Moreover, gelatin-stabilized Ppy possessed a charge reversal function, facilitating the tight conjunction of siILK gene at physiological pH (7.35-7.45) and its automatic release into acidic lysosomes (pH 4.0-5.5); the proton sponge effect generated during this process further facilitated the escape of siILK from lysosomes to the cytoplasm and played its role in inhibiting PTC proliferation and lymphatic metastasis. With the guidance of fluorescence and PA bimodal imaging, gene delivery and Ppy location in tumor regions could be clearly observed. As a result, tumors were completely eradicated by photothermal therapy, and the recurrences and metastases were obviously restrained by siILK.

Cascade Drug Delivery through Tumor Barriers of Pancreatic Cancer via Ultrasound in Combination with Functional Microbubbles.

The abundant desmoplastic stroma and the lack of sufficient targets on pancreatic cancer cells render poor drug penetration and cellular uptake, which significantly compromise the chemotherapy efficacy. Herein, we reported a three-step cascade delivery strategy for selective delivery of paclitaxel (PTX) to achieve a targeted therapy for pancreatic cancer. cRGD and cCLT1 peptides, which could target the integrin and fibronectin, respectively, overexpressed in pancreatic cancer cells and stroma, were decorated on PTX-loaded microbubbles, resulting in the formation of dual-targeting PTX-RCMBs. In this strategy, ultrasound in combination with PTX-RCMBs first enhanced the permeability of tumor vessels via cavitation effects and simultaneously helped the generated PTX-RCNPs penetrate into the stroma. Then, the cCLT1 peptide modified on PTX-RCNPs selectively bound the fibronectin highly expressed in the stroma and later targeted the integrin (α5ß1) on the cell surface. Finally, another targeting cRGD peptide modified on PTX-RCNPs would further promote PTX uptake via targeting the integrin (αvß3) on the cell surface. This strategy significantly increased the delivery of PTX into tumor tissues. Moreover, the in vivo effective accumulation of PTX was monitored by ultrasound and fluorescence bimodal imaging. The tumor growth inhibition was investigated on subcutaneous tumor mouse models with 89.8% growth inhibition rate during 21 days of treatment, showing great potential for improving pancreatic cancer therapy.

Ultrasound-induced biophysical effects in controlled drug delivery.

Ultrasound is widely used in biomedical engineering and has applications in conventional diagnosis and drug delivery. Recent advances in ultrasound-induced drug delivery have been summarized previously in several reviews that have primarily focused on the fabrication of drug delivery carriers. This review discusses the mechanisms underlying ultrasound-induced drug delivery and factors affecting delivery efficiency, including the characteristics of drug delivery carriers and ultrasound parameters. Firstly, biophysical effects induced by ultrasound, namely thermal effects, cavitation effects, and acoustic radiation forces, are illustrated. Secondly, the use of these biophysical effects to enhance drug delivery by affecting drug carriers and corresponding tissues is clarified in detail. Thirdly, recent advances in ultrasound-triggered drug delivery are detailed. Safety issues and optimization strategies to improve therapeutic outcomes and reduce side effects are summarized. Finally, current progress and future directions are discussed.

Ultrasound Microbubbles Mediated Sonosensitizer and Antibody Co-delivery for Highly Efficient Synergistic Therapy on HER2-Positive Gastric Cancer.

Trastuzumab combined with chemotherapy is the first-line treatment for advanced HER2-positive gastric cancer, but it still suffers from limited therapeutic efficiency and serious side effects, which are usually due to the poor delivery efficiency and the drug resistance of tumor cells to the chemotherapeutic drugs. Herein, a type of ultrasound microbubble for simultaneous delivery of sonosensitizers and therapeutic antibodies to achieve targeting combination of sonodynamic therapy and antibody therapy of HER2-positive gastric cancer was constructed from pyropheophorbide-lipid followed by trastuzumab conjugation (TP MBs). In vitro and in vivo studies showed that TP MBs had good biological safety, and their in vivo delivery can be monitored by ultrasound/fluorescence bimodal imaging. With ultrasound (US) located at the tumor area, TP MBs can be converted into nanoparticles (TP NPs) in situ by US-targeted microbubble destruction; plus the enhanced permeability and retention effects and the targeting effects of trastuzumab, the enrichment of sonosensitizers and antibodies in the tumor tissue can be greatly enhanced (∼2.1 times). When combined with ultrasound, TP MBs can not only increase the uptake of sonosensitizers in HER2-positive gastric cancer NCI-N87 cells but also efficiently generate singlet oxygen to greatly increase the killing effect on cells, obviously inhibiting the tumor growth in HER2-positive gastric cancer NCI-N87 cell models with a tumor inhibition rate up to 79.3%. Overall, TP MBs combined with US provided an efficient way for co-delivery of sonosensitizers and antibodies, greatly enhancing the synergistic therapeutic effect on HER2-positive gastric cancer while effectively reducing the side effects.

Ultrasmall Barium Titanate Nanoparticles for Highly Efficient Hypoxic Tumor Therapy via Ultrasound Triggered Piezocatalysis and Water Splitting.

Hypoxia in a solid tumor microenvironment (TME) can lead to the overexpression of hypoxia-inducible factor-1α (HIF-1α), which correlates to tumor metastasis. Reactive oxygen species (ROS) induced tumor cell apoptosis is becoming a promising method in tumor treatment. Currently, the ROS generating systems, e.g., photodynamic treatment and sonodynamic treatment, highly depend on oxygen (O2) in the tumor microenvironment (TME). However, the level of O2 in TME is too low to produce enough ROS. Herein, we developed an ultrasmall DSPE-PEG2000 coated barium titanate nanoparticle (P-BTO) for tumor treatment based on ultrasound triggered piezocatalysis and water splitting. Interestingly, irradiated by ultrasound, the surface of ultasmall P-BTO nanoparticles produced imbalance charges, which induced a cascade of redox reaction processes to simultaneously generate ROS and O2, the latter one was hardly generated in large-sized barium titanate nanoparticles. The as-synthesized P-BTO reached the highest accumulation in the tumor site at 4 h after intravenous injection. The results showed that the produced O2 significantly alleviated the hypoxia of TME to down-regulate the expression of HIF-1α, and the produced ROS can efficiently kill tumor cells. Moreover, the tumor metastasis was also inhibited, providing a different way to treat triple-negative breast cancer, which was easily metastatic and lacked effective treatments in the clinic.

Anti-Tumor Metastasis via Platelet Inhibitor Combined with Photothermal Therapy under Activatable Fluorescence/Magnetic Resonance Bimodal Imaging Guidance.

Photothermal therapy (PTT) is a promising tumor therapy strategy; however, heterogeneous heat distribution over the tumor often exists, resulting in insufficient photothermal ablation and potential risk of cancer metastasis, which has been demonstrated to be associate with platelets. Herein, a near-infrared (NIR) photothermal agent of IR780 was conjugated with MRI agent of Gd-DOTA via a disulfide linkage (ICD-Gd), which was coassembly with lipid connecting tumor-homing pentapeptide CREKA (Cys-Arg-Glu-Lys-Ala) (DSPE-PEG-CREKA) to encapsulate a platelet inhibitor of ticagrelor (Tic), affording a multistimuli-responsive nanosystem (DPC@ICD-Gd-Tic). The nanosystem with completely quenching fluorescence could specifically target the tumor-associated platelets and showed pH/reduction/NIR light-responsive drug release, which simultaneously resulting in dis-assembly of nanoparticle and fluorescence recovery, enabling the drug delivery visualization in tumor in situ via activatable NIR fluorescence/MR bimodal imaging. Finally, DPC@ICD-Gd-Tic further integrated the photoinduced hyperthermia and platelet function inhibitor to achieve synergistic anticancer therapy, leading to ablation of primary tumor cells and effectively suppressed their distant metastasis. The number of lung metastases in 4T1 tumor bearing mice was reduced by about 90%, and the size of tumor was reduced by about 70%, while half of the mouse was completely cured by this smart nanosystem.

Design and application of inorganic nanoparticles for sonodynamic cancer therapy.

As an alternative to photodynamic therapy (PDT), ultrasound-triggered tumor sonodynamic therapy (SDT) has garnered significant attention, owing to its high tissue penetration, few side effects, and reliable patient compliance. A sonosensitizer is the most important component in SDT, and high-quantum-yield safe sonosensitizers are crucial for SDT. Existing sonosensitizers mainly include organic sonosensitizers and inorganic sonosensitizers. Organic sonosensitizers, mainly some small dye molecules, have been widely studied. However, organic sonosensitizers have limited utility owing to their poor stability, rapid blood clearance, and potential phototoxicity. In contrast, inorganic sonosensitizers have stable chemical properties, long circulation time in the blood and can effectively reduce phototoxicity. In addition to their utilization as sonosensitizers, some inorganic nanoparticles can also operate as carriers for delivering organic sonosensitizers, effectively overcoming the inherent shortcomings of organic small-molecule sonosensitizers. This review mainly focuses on inorganic nanomaterial-based SDT, the possible mechanisms of SDT, and newly developed inorganic sonosensitizers, as well as the challenges and possible solutions associated with their clinical translation are introduced.

Applications of Micro/Nanotechnology in Ultrasound-based Drug Delivery and Therapy for Tumor.

Ultrasound has been broadly used in biomedicine for both tumor diagnosis as well as therapy. The applications of recent developments in micro/nanotechnology promote the development of ultrasound-based biomedicine, especially in the field of ultrasound-based drug delivery and tumor therapy. Ultrasound can activate nano-sized drug delivery systems by different mechanisms for ultrasound- triggered on-demand drug release targeted only at the tumor sites. Ultrasound Targeted Microbubble Destruction (UTMD) technology can not only increase the permeability of vasculature and cell membrane via sonoporation effect but also achieve in situ conversion of microbubbles into nanoparticles to promote cellular uptake and therapeutic efficacy. Furthermore, High Intensity Focused Ultrasound (HIFU), or Sonodynamic Therapy (SDT), is considered to be one of the most promising and representative non-invasive treatment for cancer. However, their application in the treatment process is still limited due to their critical treatment efficiency issues. Fortunately, recently developed micro/nanotechnology offer an opportunity to solve these problems, thus improving the therapeutic effect of cancer. This review summarizes and discusses the recent developments in the design of micro- and nano- materials for ultrasound-based biomedicine applications.

Smart strategies to overcome tumor hypoxia toward the enhancement of cancer therapy.

Hypoxia, as a typical factor in a tumor microenvironment, plays a vital role in tumor treatment resistance, tumor invasion and migration. Hypoxia inducible factor (HIF), as the vital response element of hypoxia, mediates these untoward effects through a series of downstream reactions. Cancer treatments such as photodynamic therapy (PDT), radiotherapy (RT) and chemotherapy are severely hindered by hypoxia and HIF, back, however, could be intelligently manipulated through nanocomposite materials for their great potentiality to combine different functions. Herein, we reviewed the smart strategies in emerging research studies to overcome hypoxia toward the enhancement of tumor therapy.

Treating tumors with minimally invasive therapy: A review.

With high level of morbidity and mortality, tumor is one of the deadliest diseases worldwide. Aiming to tackle tumor, researchers have developed a lot of strategies. Among these strategies, the minimally invasive therapy (MIT) is very promising, for its capability of targeting tumor cells and resulting in a small incision or no incisions. In this review, we will first illustrate some mechanisms and characteristics of tumor metastasis from the primary tumor to the secondary tumor foci. Then, we will briefly introduce the history, characteristics, and advantages of some of the MITs. Finally, emphasis will be, respectively, focused on an overview of the state-of-the-art of the HIFU-, PDT-, PTT-and SDT-based anti-tumor strategies on each stage of tumor metastasis.

Bubble-Manipulated Local Drug Release from a Smart Thermosensitive Cerasome for Dual-Mode Imaging Guided Tumor Chemo-Photothermal Therapy.

Thermosensitive liposomes have demonstrated great potential for tumor-specific chemotherapy. Near infrared (NIR) dyes loaded liposomes have also shown improved photothermal effect in cancer theranostics. However, the instability of liposomes often causes premature release of drugs or dyes, impeding their antitumor efficacy. Herein, we fabricated a highly stable thermo-responsive bubble-generating liposomal nanohybrid cerasome with a silicate framework, combined with a NIR dye to achieve NIR light stimulated, tumor-specific, chemo-photothermal synergistic therapy. Methods: In this system, NIR dye of 1,1'-Dioctadecyl-3,3,3',3'- Tetramethylindotricarbocyanine iodide (DiR) with long carbon chains was self-assembled with a cerasome-forming lipid (CFL) to encapsulate ammonium bicarbonate (ABC), which was further used for actively loading doxorubicin (DOX), affording a thermosensitive and photosensitive DOX-DiR@cerasome (ABC). Results: The resulting cerasome could disperse well in different media. Upon NIR light mediated thermal effect, ABC was decomposed to generate CO2 bubbles, resulting in a permeable channel in the cerasome bilayer that significantly enhanced DOX release. After intravenous injection into tumor-bearing mice, DOX-DiR@cerasome (ABC) could be efficiently accumulated at the tumor tissue, as monitored by DiR fluorescence, lasting for more than 5 days. NIR light irradiation was then performed at 36h to locally heat the tumors, resulting in immediate CO2 bubble generation, which could be clearly detected by ultrasound imaging, facilitating the monitoring process of controlled release of the drug. Significant antitumor efficacy could be obtained for the DOX-DiR@cerasome (ABC) + laser group, which was further confirmed by tumor tissue histological analysis.

Evaluating HIFU-mediated local drug release using thermal strain imaging: Phantom and preliminary in-vivo studies.

PURPOSE: High-intensity focused ultrasound (HIFU)-mediated drug release becomes a promising therapeutic technique for treatment of cancer, which has merits of deep penetration, noninvasive approach and nonionizing radiation. However, conventional thermocouple-based approach for treatment monitoring would encounter big challenges such as the viscous heating artifact and difficulty in monitoring in the deep region. In this study, we develop an effective method based on thermal strain imaging (TSI) for the evaluation of HIFU-mediated drug release. METHODS: Both phantom experiments and preliminary animal experiments were performed to investigate the feasibility of the proposed approach. Doxorubicin (DOX)-loaded cerasomes (HIFU and temperature-sensitive cerasomes, HTSCs) were prepared. In the phantom experiments, the HTSC solution is contained inside a cylindrical chamber within a tissue-mimicking phantom. In the animal experiments, the HTSCs are intravenously injected into tumor-bearing mice. An HIFU transducer is used to trigger DOX release from the HTSCs within the phantom or mice, and TSI is performed during HIFU heating. In the phantom experiments, the accuracy of temperature estimation using TSI is validated by measuring with a thermocouple. In animal experiments, the spatial consistency between the distribution of DOX released within the tumor and the location of the heating region estimated by TSI is validated using a spectrofluorophotometer. RESULTS: In the phantom experiments, the HTSCs show a burst release of DOX when the temperature of the HTSC solution estimated by TSI reaches about 42°C, which is in agreement with the condition for drug release from the HTSCs. The temperature estimation using TSI has high accuracy with error below 2.5%. In animal experiments, fluorescence imaging of the tumor validates that the heating region of HIFU could be localized by the low-strain region of TSI. CONCLUSION: The present framework demonstrates a reliable and effective solution to the evaluation of HIFU-mediated local drug delivery.