A controllable self-amplifying oxidative stress strategy for boosting noninvasive sonodynamic therapy and synergistic immunotherapy.

Recent advances in nanoparticles-based sonodynamic and immune checkpoint blockade synergistic therapy for pancreatic ductal adenocarcinoma

Open AccessCCS ChemistryRESEARCH ARTICLE1 Feb 2022Sonosensitized Aggregation-Induced Emission Dots with Capacities of Immunogenic Cell Death Induction and Multivalent Blocking of Programmed Cell Death-Ligand 1 for Amplified Antitumor Immunotherapy Shaorui Jia†, Zhiyuan Gao†, Zelin Wu, Heqi Gao, He Wang, Hanlin Ou and Dan Ding Shaorui Jia† Key Laboratory of Bioactive Materials Ministry of Education, College of Life Sciences, State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin 300071 , Zhiyuan Gao† Key Laboratory of Bioactive Materials Ministry of Education, College of Life Sciences, State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin 300071 , Zelin Wu Key Laboratory of Bioactive Materials Ministry of Education, College of Life Sciences, State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin 300071 , Heqi Gao Key Laboratory of Bioactive Materials Ministry of Education, College of Life Sciences, State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin 300071 , He Wang Department of Urology, First Affiliated Hospital of Soochow University, Suzhou 215006 , Hanlin Ou Key Laboratory of Bioactive Materials Ministry of Education, College of Life Sciences, State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin 300071 and Dan Ding *Corresponding author: E-mail Address: [email protected] Key Laboratory of Bioactive Materials Ministry of Education, College of Life Sciences, State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin 300071 https://doi.org/10.31635/ccschem.021.202101458 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail The combination of immunogenic cell death (ICD) induction and immune checkpoint blockade has emerged as a major direction of cancer immunotherapy. Among currently available ICD inducers, sonosensitizers that produce reactive oxygen species (ROS) under an external trigger to evoke ICD of tumor cells have shown great promise. However, a highly efficient sonosensitizer-based ICD inducer with an aggregation-induced emission (AIE) characteristic has yet to be developed. Herein, a novel AIE sonosensitizer with a twisted molecular structure, very small energy gap between the singlet and triplet excited states (ΔEST), and efficient ROS generation ability, which can serve as an effective ICD inducer, is reported for sonodynamic processes in cancer immunotherapy. Furthermore, an AIE sonosensitizer-based nanosystem with surface modification of anti-PD-L1 peptide is constructed for boosting antitumor immunotherapy. In this system, AIE sonosensitizer-mediated sonodynamic therapy can successfully convert a hypoimmunogenic cold tumor to a hot one and further facilitate the multivalent blocking of programed death ligand (PD-L1) by anti-PD-L1 peptides. Such an advanced nanosystem could effectively initiate the activation of antitumoral immune reactions and modulation of an immunosuppressive microenvironment, contributing to systemic antitumor effects to further inhibit the growth of distant tumors. Download figure Download PowerPoint Introduction Antitumor immunotherapy, which boosts specific cytotoxic T cells to eliminate tumor cells, is recognized as an effective cancer treatment strategy.1 Currently, inducing immunogenic cell death (ICD) of tumor cells is an effective strategy for enhancing recruitment and infiltration of specific cytotoxic T cells into solid tumors (e.g., triple negative breast cancer), converting a cold tumor, with a paucity of T cell infiltration, to hot.2–4 During ICD of tumors, tumor-associated antigens (TAAs) and damage-associated molecular patterns (DAMPs) are generated, featuring surface-exposed calreticulin (ecto-CRT), adenosine triphosphate (ATP) secretion, and release of high-mobility group protein B1 (HMGB1) and heat shock protein 70 (HSP70).5,6 DAMPs act as a vital signal and a natural adjuvant to stimulate the presentation of TAA by antigen-presenting cells (APCs) (such as dendritic cells (DCs)) to T cells, which lead to the further activation of tumor-specific T cell-mediated immune response.7,8 Because of the pivotal role of ICD in antitumor immunotherapy, the development of highly effective ICD inducers with few side effects has attracted great interest during the past decade.5,9 Among currently available ICD inducers, photosensitizers evoke ICD of tumor cells by producing reactive oxygen species (ROS) upon light irradiation, which has showed conspicuous spatiotemporal precision and excellent biocompatibility.10 Our previous work has demonstrated that an aggregation-induced emission (AIE) photosensitizer with high ROS production capacity can massively induce sufficient ICD to evoke antitumor immunity.11,12 However, the limited penetration depth of light is a major challenge for the application of photosensitizer-based photodynamic therapy (PDT) in vivo.13 Sonodynamic therapy (SDT), derived from PDT, which utilizes ultrasound (US) to activate the sonosensitizer to produce ROS, enables deep tissue penetration, provides a more effective and safer strategy for the treatment of deep tumors, and shows unique advantages and great potential in antitumor immunotherapy.14–16 The mechanism of SDT is a complex, combinational output of different mechanisms.17–19 Among them, sonoluminescence was proven important to generate ROS.15,20 Sonoluminescence refers to the light emitted by the collapse of a bubble that is generated by US irradiation, which can activate the energy-matching photoactive sonosensitizers and generate the same photochemical reaction like PDT.21,22 For traditional organic photosensitizers with planar molecular structure, strong intermolecular interactions (e.g., π–π stacking) facilitate a nonradiative pathway of excited states, and the aggregation of photosensitizers within nanoparticles (NPs) causes quenching of the emission and ROS generation.23,24 At present, many organic photosensitizers that can be used as sonosensitizers to produce ROS, kill tumor cells, and induce ICD in tumor cells have been reported in SDT.25–27 Similar to photosensitizers, the aggregation-caused quenching (ACQ) effect also occurs when they are used as sonosensitizers in the aggregate state.28,29 Fortunately, sonosensitizers with AIE properties provide a good solution for the ACQ problem.30,31 AIE sonosensitizers have peripheral intramolecular motion units (e.g., rotating benzene rings) and a three-dimensional (3D) molecular structure.32,33 The 3D molecular structure significantly reduces the intermolecular interactions in NPs and aggregates. Meanwhile, the steric hindrance restricts the molecular motion in the excited state, which allows as much absorbed excitation energy as possible to be used in fluorescence emission or ROS generation.34,35 To the best of our knowledge, however, the ICD effect of AIE in SDT has never been studied. The reason may be the lack of appropriate strategies and AIE sonosensitizers with high efficiency. The application of SDT in immune system activation also faces challenges. Therefore, the design of an efficient AIE sonosensitizer is urgently needed to explore whether AIE can build an effective platform for sonosensitivity-based ICD inducers. Additionally, after successfully eliciting antitumor immune response by the induction of ICD, tumors may express autoprotective checkpoint molecules such as programmed cell death-ligand 1 (PD-L1) to bind with programmed death-1 (PD-1) on activated T cells, escaping attack from the immune system.36–38 Thus, an immune checkpoint blockade (ICB), such as the PD-1/PD-L1 blockade, has been employed to block PD-1/PD-L1 interaction to improve the tumor cell killing effect of T cells, thereby enhancing the inhibition efficacy against ICD-induced hot tumors.39–43 Although ICB holds great promise in cancer treatment, its therapeutic effect is largely limited by the insensitive response and insufficient T cells in cold tumors.44,45 Moreover, the synergistic anticancer efficacy of the integrated ICD induction and ICB is superior to monotherapy with only one of them.37,46,47DPPA (CNYSKPTDRQYHF, D-type PD-L1 peptide antagonists) can effectively block the PD-1/PD-L1 pathway, offering salient advantages such as higher stability, lower cost, and easier modification as compared with clinical anti-PD-L1 monoclonal antibodies.48,49 Consequently, for tremendously amplifying the effect of antitumor immunotherapy, the strategy of combing anti-PD-L1 peptide and ICD inducer in a single nanosystem is valuable and highly desirable but currently rare.50,51 Herein, we report sonosensitized AIE ICD inducer-based dots modified with anti-PD-L1 peptides on the surface for considerable improvement of antitumor immunotherapy outcomes. We designed and synthesized three new AIE luminogens (AIEgens): diphenylamino (DPA)-tetraphenylethylene (TPE)-(4-styryl-cyano)pyridinium salt (SCP), triphenylamine (TPA)-Ph-SCP, and TPA-2Ph-SCP). They have D–π–A structure and abundant intramolecular movement unit. Compared with TPA-Ph-SCP and TPA-2Ph-SCP, DPA-TPE-SCP has a more twisted molecular structure and very small ΔEST, which lead to stronger AIE activity, weaker intermolecular interactions of aggregates, and more efficient ROS generation. By comparison, DPA-TPE-SCP is not only the best photosensitizer, but also the best sonosensitizer. AIE sonosensitizers under US excitation showed superior ICD induction capability when compared with the reported sonosensitizing ICD inducer hematoporphyrin monomethyl ether (HMME), and successfully transformed a cold tumor into a hot one that was sensitized to PD-L1 blocking in vivo. Subsequently, surface modification of sonosensitized AIE dots with anti-PD-L1 peptides endowed them complementary advantages. In vivo experiments demonstrate that such function-cooperative dots significantly enhanced immune responses and relieved immune suppression, triggering a systemic antitumor immune therapeutic effect by virtue of the excellent ICD induction and multivalent PD-L1 blockade. Thus, this work not only provides a molecular guideline to design advanced sonosensitizer-based ICD inducers but also introduces new insights into the combination of ICD induction and ICB for synergistic antitumor immunotherapy (Scheme 1). Scheme 1 | Synthesis of AIE-dots-DPPA and the mechanism of antitumor immune responses induced by AIE-dots-DPPA-mediated sonodynamic therapy. Download figure Download PowerPoint Experimental Methods Computational method Structures of TPA-Ph-SCP, TPA-2Ph-SCP, and DPA-TPE-SCP were optimized in the water phase with the B3LYP method and 6-311G (d, p) basis set, Gaussian 09 program. Energy levels of S1–S6 and T1–T6 were calculated by the vertical excitation of the above optimized structures, with the same method of B3LYP/6-311G (d, p). Preparation of nano-sonosensitizer Nano-sonosensitizer was formulated by a nanoprecipitation approach using an amphiphilic co-polymer, DSPE-PEG2000. In brief, sonosensitizer (1 mg) and DSPE-PEG2000 (3 mg) were dissolved in 1 mL of tetrahydrofuran (THF); the solution was added dropwise into 9 mL of water undergoing sonication (3 min) by a microtip probe sonicator (XL2000, Misonix). An air pump was subsequently used to volatilize THF in the mixture to obtain a sonosensitizer-encapsulated dot aqueous solution. Then, the sonosensitizer-encapsulated dot solution was purified by 5000 rpm ultrafiltration for 10 min and filtration using a 0.45 μm syringe driven filter. Preparation of DPPA-conjugated dots DPPA peptides were modified on the AIE dots. First, DPPA peptides were added to the dots suspension, and then the suspension was stirred for 12 h to couple the thiol group of the peptide with the maleimide group of PEG2000 via the click reaction. Peptides that were not conjugated to the dots were then removed by centrifugation. ROS detection in 4T1 cells The 4T1 cancer cells were incubated in special confocal chambers with each nano-sonosensitizer (10 μg/mL of DPA-TPE-SCP dots, 10 μg/mL of HMME NPs) for 4 h at 37 °C. Subsequently, the cells were washed with 1× phosphate-buffered saline (PBS) three times, and then incubated with 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA; 20 μM) in FBS free culture medium. The above-mentioned process was performed in the dark. Then, under US exposure (1 MHz, 50% duty cycle, 0.5 W/cm2, 3 min), the cells were imaged with confocal laser scanning microscopy (CLSM) for DCF detection (Ex: 488 nm, Em: 530 ± 20 nm). Ecto-CRT staining in 4T1 cancer cells After the 4T1 cancer cells were incubated with each ICD inducer (10 μg/mL of DPA-TPE-SCP dots, 10 μg/mL of HMME NPs) for 4 h at 37 °C, the cells were washed and irradiated by US for 3 min (1 MHz, 50% duty cycle, 0.5 W/cm2). After 12 h, the cells were washed by precooling 1× saline, fixed with 4% paraformaldehyde on ice for 20 min, and then successively incubated with anticalreticulin antibody (ab2907, 1:200 dilution with 1× PBS) for 2 h and stained with Alexa Fluor 633-conjugated secondary antibody (1:200 dilution with 1× PBS) for another 2 h. The ecto-CRT expression of each ICD inducer-treated cancer cell was visualized by the ecto-CRT immunofluorescence using CLSM with the excitation at 633 nm and signal acquisition in the range from 640 to 670 nm. Detection of extracellular ATP, HMGB1, and HSP70 The 4T1 cancer cells (1 × 105 cells mL−1) were cultured in a black 12-well plate. After adherence, the 4T1 cancers cells were incubated with 10 μg/mL of DPA-TPE-SCP dots and 10 μg/mL of HMME NPs for 4 h at 37 °C, respectively. Next, the treated cells were washed and irradiated by US for 3 min (1 MHz, 50% duty cycle, 0.5 W/cm2). After 12 h, the supernatants were collected and then subjected to centrifugation using 12,000 rpm for 10 min at 4 °C, which was followed by addition of protease and phosphatase inhibitors into the supernatants. Furthermore, the cancer cells in each group were harvested and lysed, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. Finally, the extracellular levels of HMGB1 and HSP70 were analyzed by western blot with the anti-HMGB1 antibody (1:500, Abcam, ab79823) and anti-HSP70 antibody (1∶1000, Abcam, ab181606), respectively. The level of secreted ATP was quantitatively determined by ATP Bioluminescent Assay Kit per manufacturer's instruction. In vivo SDT treatment 4T1 cells (1 × 106) dispersed in 50 μL of 1× PBS were injected into the second breast fat pad on the right side of each BALB/c mouse. The mice bearing 4T1 tumors were randomly divided into four groups when their tumors reached about 50 mm3. The AIE dots were intravenously injected into the mice three times on day 2, 4 and 6, respectively (once a day for three days (2, 4, and 6)). The tumors were exposed to US irradiation (1.0 MHz, 50% duty cycle, 1.5 W/cm2, 5 min) at 6-h post each injection. The tumor volume was measured by a caliper and determined with the following formula: Volume = Width2 × Length/2. The mice with tumors reaching 1500 mm3 were euthanized due to the standard animal protocol in this work. To establish a bilateral 4T1 orthotopic tumor model, 4T1 cells (1 × 106) were injected into the second breast fat pad on the right side of each female BALB/c mouse as primary tumor. Four days later, 4T1 cells (2 × 105) were injected into the second breast fat pad on the left side of mice as a distant tumor. The mice bearing ∼50 mm3 of primary tumors were randomly divided into four groups. The AIE dots were intravenously injected into the mice three times on day 2, 4 and 6, respectively (once a day for three days (2, 4, and 6)). At 6 h after each injection, the tumors were exposed to US irradiation for 5 min (1.0 MHz, 50% duty cycle, 1.5 W/cm2). The tumor volume was measured by a caliper and determined with the following formula: Volume = Width2 × Length/2. Flow cytometry analysis At designated time points, lymph nodes and tumors were harvested and homogenized to single-cell suspensions. After that, cell suspensions of lymph nodes were co-stained with anti-CD11c-FITC, anti-CD86-APC, and anti-CD80-PE for flow cytometry analysis of DCs. Single-cell suspensions in tumors were co-stained with different antibodies for flow cytometry analysis of CD8+ T cells (anti-CD3-FITC, anti-CD8-PE, or anti-CD8-APC), PD-L1+ tumor cells (anti-CD45-PE and anti-PD-L1-APC), Treg cells (anti-CD3-FITC, anti-CD4-APC, anti-CD25-PerCP/Cyanine5.5, and anti-Foxp3-PE), and IFN-γ+ of CD8+ T cells (anti-CD3-FITC, anti-CD8-APC, and anti-IFN-γ-PE). Statistical analysis Quantitative data are shown as mean ± standard deviation (SD). All the experiments were repeated at least three times. Statistical comparisons were made by ANOVA analysis and two-sample Student's t-test. P value < 0.05 was considered statistically significant. Results and Discussion Design, synthesis, and characterization of AIEgens Based on our design strategy, the phenylethylene core was used as a π-linker, and compared to triphenylethylene and diphenylethylene, TPE is an excellent AIE skeleton. The D–π–A structure was constructed using a DPA block as an electronic donor (D) and the pyridinium as an electronic acceptor (A). The compounds DPA-TPE-SCP, TPA-2Ph-SCP, and TPA-Ph-SCP were designed and synthesized according to the synthetic route shown in Figure 1a. Compound 1 was prepared through a Horner–Wadsworth–Emmons reaction. After quenching with dimethylformamide (DMF), compound 1 containing bromine was functionalized to afford the corresponding aldehyde derivatives, and DPA-TPE-SCP, TPA-2Ph-SCP, and TPA-Ph-SCP were obtained from the Knoevenagel condensation of compound 2 and SCP under basic conditions (for detailed synthetic routes of all compounds see Supporting Information Scheme S1). The intermediates were characterized by 1H NMR, and DPA-TPE-SCP, TPA-2Ph-SCP, and TPA-Ph-SCP were characterized by 1H NMR, 13C NMR, and high-resolution mass spectrometry (HRMS) ( Supporting Information Figures S1–S17). Figure 1 | (a) Synthetic route to DPA-TPE-SCP, TPA-2Ph-SCP, and TPA-Ph-SCP. (b) Plot of ln(A0/A) against light exposure time, where A0 and A are the ABDA absorbance (378 nm) before and after irradiation, respectively. (c) Jablonski diagrams displaying the photophysical properties of the DPA-TPE-SCP. (d) HOMO–LUMO distributions by DFT calculations of DPA-TPE-SCP. (e) Energy levels of S1–S6 and T1–T6 calculated by the vertical excitation of the optimized structures in (f). (f) The dihedral angles of DPA-TPE-SCP, TPA-2Ph-SCP, and TPA-Ph-SCP by DFT calculations. Download figure Download PowerPoint Photophysical properties The absorption and photoluminescence (PL) spectra of DPA-TPE-SCP, TPA-2Ph-SCP, and TPA-Ph-SCP are shown in Supporting Information Figure S18. The conjugated structure of TPA-Ph-SCP, TPA-2Ph-SCP, and DPA-TPE-SCP becomes more and more distorted, so that their absorption peaks show hypochromatic shifts at 447, 382, and 350 nm; gradual red-shifting maximum emission wavelengths are located at 628, 630, and 638 nm. The PL quantum yields (QYs) of DPA-TPE-SCP, TPA-2Ph-SCP, and TPA-Ph-SCP in the solid state were 12.4%, 7.9%, and 2.6%, respectively. When a poor solvent (toluene) was gradually added to the good solution [dimethyl sulfoxide (DMSO)], aggregate formation occurred, and the PL intensities of DPA-TPE-SCP, TPA-2Ph-SCP, and TPA-Ph-SCP were significantly intensified, which prove that DPA-TPE-SCP, TPA-2Ph-SCP, and TPA-Ph-SCP have an obvious AIE feature. Notably the DPA-TPE-SCP showed the best AIE property when comparing the PL intensity of DPA-TPE-SCP, TPA-2Ph-SCP, and TPA-Ph-SCP in a DMSO/toluene mixture with 99% toluene fraction to that in pure DMSO ( Supporting Information Figure S18). Because high ROS generation capacity is a prerequisite for an ICD inducer, we next studied and compared the ROS production of DPA-TPE-SCP, TPA-Ph-SCP, and in a 10 μM) under light irradiation (10 with as the ROS shown in Figure the ABDA of DPA-TPE-SCP, TPA-2Ph-SCP, and TPA-Ph-SCP were and that DPA-TPE-SCP is an mechanism Based on the Jablonski the fixed absorption energy excited by light is through three fluorescence through followed by production of ROS or and nonradiative one or energy as much absorbed energy as possible on the is an effective to the efficacy of an To the mechanism of the superior ROS production of DPA-TPE-SCP compared to and TPA-Ph-SCP, calculations were The on electronic structures in the state that DPA-TPE-SCP, TPA-2Ph-SCP, and TPA-Ph-SCP a with the molecular on the and the molecular located on the and Supporting Information Figure in Figure TPA-Ph-SCP has a gap between the singlet and triplet excited of However, using the TPE as the π-linker, DPA-TPE-SCP has a very small value of to the highly ΔEST, was which for the superior ROS generation capability of DPA-TPE-SCP. shown in Figure the optimized structures calculated by DFT that compared with and TPA-Ph-SCP, DPA-TPE-SCP has a much molecular a TPE group used as a π-linker, the structure becomes more For the same dihedral between and to and in DPA-TPE-SCP from the of groups ( Supporting Information Figure The more twisted molecular structure the intermolecular interactions such as π–π and nonradiative in the or solid state, the absorbed excitation energy flow to the Therefore, successfully DPA-TPE-SCP showed higher fluorescence and ROS generation and TPA-Ph-SCP is for DPA-TPE-SCP, TPA-2Ph-SCP, and Such excellent of DPA-TPE-SCP is to our molecular design structure, of intramolecular motion units on π-linker, 3D twisted molecular which the process and the ROS generation efficiency. Preparation of AIE dots and detection of ROS in dots The AIE dots were prepared by the method The amphiphilic was used to the microscopy that DPA-TPE-SCP dots with of about nm, respectively dot has on the ROS generation of DPA-TPE-SCP, as by the same ABDA of DPA-TPE-SCP (10 μM) solution and DPA-TPE-SCP dots (10 on upon light ( Supporting Information Figure shown in Figure the reported dots and dots with excellent ROS production and the available sonosensitized compound HMME NPs were used to the ROS production of DPA-TPE-SCP dots dots and TPA-Ph-SCP dots under US irradiation (1 MHz, 50% duty cycle, 1.5 W/cm2). DPA-TPE-SCP dots showed higher ROS generation the sonosensitizers The of ROS production the ROS that the mechanism of DPA-TPE-SCP dots producing ROS may be activated by Next, was used to the species of The was with water and DPA-TPE-SCP dots, followed by US DPA-TPE-SCP dots showed an obvious For was used as the under detection and the that the dots could also generate post US irradiation Consequently, DPA-TPE-SCP dots were then as AIE dots for the following sonosensitized ICD induction Figure 2 | (a) The of sonosensitized AIE dots. (b) and of DPA-TPE-SCP dots. (c) The structure of and (d) Plot of ln(A0/A) against ultrasound (US) exposure time, where A0 and A are the ABDA absorbance (378 nm) before and after irradiation, respectively. (e) generation by DPA-TPE-SCP dots and water with US using (f) generation by DPA-TPE-SCP dots and water with US using Download figure Download PowerPoint In SDT effect and ICD induction by AIE dots has been that SDT can evoke ICD of cancer cells by producing First, of DPA-TPE-SCP dots by 4T1 breast cancer cells within 4 h was analyzed by flow cytometry ( Supporting Information Figure Next, the ROS generation level in 4T1 cells was by of by ROS to the upon US irradiation for 3 min (1 MHz, 0.5 the CLSM of the 4T1 cells treated with DPA-TPE-SCP dots showed the fluorescence and Supporting Information Figure the fluorescence intensity of dots is higher that of NPs ( Supporting Information Figure the excellent ROS production capacity of DPA-TPE-SCP dots. Then, was used to the therapeutic effect of SDT on 4T1 cells in and The of DPA-TPE-SCP dots in 4T1 cells was at different US the great of DPA-TPE-SCP dots. After exposure to US for 3 min (1 MHz, 0.5 the of 4T1 cells as the of DPA-TPE-SCP dots Furthermore, under different US irradiation time, the AIE 4T1 cancer cells more cell that of HMME All demonstrate that DPA-TPE-SCP dots can produce highly efficient ROS in 4T1 cells, a superior SDT effect by DPA-TPE-SCP dots in Figure 3 | (a) CLSM the ROS levels in 4T1 cancer cells after was used as the ROS 50 (b) Cell of 4T1 cells incubated with different of DPA-TPE-SCP dots with or US (c) Cell of 4T1 cells after with DPA-TPE-SCP dots and HMME followed by US irradiation with different (d) CLSM of ecto-CRT on 4T1 cells surface The cell were stained by 50 (e) Quantitative of ATP in the supernatants of 4T1 cells after different (f) the protein levels of HMGB1 and HSP70 in the 4T1 cell

Amorphous 2D Mn-doped CoMo-layered double hydroxide nanosheets for magnetic resonance imaging-guided sonodynamic cancer therapy

Breast cancer remains the leading cause of cancer-related deaths among women worldwide, necessitating more effective treatment strategies. Chemotherapy combined with immunotherapy is the first-line treatment for breast cancer, but it still suffers from limited therapeutic efficiency and serious side effects, which are usually due to the poor delivery efficiency, drug resistance of tumor cells, and immunosuppressive tumor microenvironment. This study explores the development of ultrasound-responsive nanobubbles (Ce6/PTX Nbs) for targeted imaging and sonoimmunotherapy in breast cancer treatment. By integrating sonodynamic therapy (SDT), chemotherapy, and immunotherapy, the nanobubbles aim to address challenges such as poor drug delivery, systemic toxicity, and immune suppression in conventional therapies. The nanobubbles, composed of sonosensitizer chlorin e6 (Ce6)-modified phospholipid and loaded with the chemotherapeutic agent paclitaxel (PTX) enhancing drug-loading capacity, are designed to precisely target tumor sites via cyclic-RGD peptides. Upon ultrasound activation, Ce6 induces reactive oxygen species (ROS), promoting immunogenic cell death (ICD), while PTX disrupts tumor cell mitosis, enhancing the immune response. The nanobubbles' ultrasound responsiveness facilitates real-time imaging and controlled drug release, maximizing therapeutic efficacy while minimizing side effects. Key findings demonstrate that Ce6/PTX Nbs significantly reduced tumor growth in a 4T1 breast cancer model, enhanced immune activation via the cGAS-STING pathway, and increased the infiltration of CD8+ T cells in both primary and distant tumors. In combination with anti-PD-L1 checkpoint inhibitors, the treatment achieved a substantial suppression of tumor metastasis. This innovative approach offers a highly targeted, effective, and minimally toxic breast cancer treatment with potential for clinical translation due to its dual imaging and therapeutic capabilities.

Sonodynamic cancer therapy by a nickel ferrite/carbon nanocomposite on melanoma tumor: In vitro and in vivo studies.

Hypoxia in the tumor microenvironment (TME) severely compromises the effectiveness of sonodynamic therapy (SDT) and disrupts the process of cuproptosis. SDT generates insufficient reactive oxygen species (ROS) under low oxygen levels, while cuproptosis is impeded by hypoxia-induced mitochondrial respiration suppression. To address these limitations, we develop a CaO2-based self-oxygenating nanosonosensitizer incorporating a copper-based metal-organic framework (MOF) shell with loaded disulfiram (DSF), named CaO2-MD, which undergoes TME-responsive disassembly to generate O2 and release the drug. Upon ultrasound (US) irradiation, CaO2-MD generates ROS via SDT and simultaneously triggers cuproptosis through the release of copper ions and DSF. In vivo and in vitro experiments indicate that CaO2-MD can effectively alleviate tumor hypoxia, thereby synergistically activating cuproptosis and boosting SDT performance. In the 4T1 tumor model, CaO2-MD with US irradiation achieves potent tumor suppression and a 40% cure rate which further increases to 80% when combination with PD-L1 blockade therapy. The durable immune memory is established to effectively prevent recurrence. This work breaks through the hypoxic TME limitations constraining both SDT and cuproptosis, offering a promising platform for developing effective cancer therapies based on TME reconstruction.

Nanodroplet-enhanced sonodynamic therapy potentiates immune checkpoint blockade for systemic suppression of triple-negative breast cancer.

Sonodynamic therapy (SDT) benefiting from its intrinsic merits, such as noninvasiveness and deep tissue penetrability, is receiving increasing considerable attention in reactive oxygen species (ROS)-based tumor treatment. However, current sonosensitizers usually suffer from low tumor lesion accumulation, insufficient ROS generation efficiency under ultrasound, and non-biodegradability, which seriously impede the therapeutic outcomes. Additionally, it is difficult that SDT alone can completely eradicate tumors because of the complex and immunosuppressive tumor microenvironment (TME). Herein, we simultaneously employ sonosensitive porphyrin building blocks and glutathione (GSH)-responsive disulfide bonds to construct a novel degradable multifunctional porphyrin-based hollow porous organic polymer (POP) nanosonosensitizer (H-Pys-HA@M/R), which combine SDT, "on-demand" chemotherapy, and immunotherapy. Taking the unique advantages of POPs with designable structures and high specific surface area, this H-Pys-HA@M/R nanosonosensitizer can achieve tumor target accumulation, GSH-triggered drug release, and low-frequency ultrasound-activating ROS generation with encouraging results. Furthermore, this multifunctional nanosonosensitizer can effectively evoke immunogenic cell death (ICD) response through the combination of SDT and chemotherapy for both primary and distal tumor growth suppression. Meanwhile, H-Pys-HA@M/R exhibits favorable biodegradation and biosafety. Therefore, this study provides a new strategy for reasonably designing and constructing POP-related sonosensitizers combining SDT/chemotherapy/immunotherapy triple treatment modalities to eradicate malignant tumors.

Sonodynamic therapy, with advantages in large tissue penetration depth and great controllability, is a promising type of non-invasive cancer treatment method. Developing sonosensitizers with high reactive oxygen species (ROS) quantum yield and the ability to regulate tumor microenvironment to achieve enhanced performances in sonodynamic therapy would thus be rather attractive. Herein, vanadium (V) doped TiO2 (V-TiO2) nanospindles with glutathione-depleting properties are fabricated for enhanced sonodynamic cancer therapy. Due to doping of the V element, the bandgap of V-TiO2 nanospindles is reduced, increasing the efficiency of ultrasound-triggered ROS production compared to that of pure TiO2 nanoparticles. More interestingly, the doping of V also makes V-TiO2 nanospindles an effective Fenton-like agent, which can catalyze the generation of highly toxic hydroxyl radicals (•OH) from endogenous H2O2 in the tumor, thus enabling cancer-killing through chemodynamic therapy. In addition, the V doping also endows V-TiO2 nanospindles with the function of glutathione depletion, further amplifying the oxidative stress generated by chemodynamic-sonodynamic therapy. In vitro cell experiments and in vivo animal experiments demonstrate that V-TiO2 nanospindles can effectively kill cancer by the combined chemodynamic-sonodynamic therapy, significantly improving the tumor treatment outcomes. Importantly, V-TiO2 with the ultrasmall spindle morphology can be quickly excreted from the body, without causing any long-term toxicity. This work illustrates that doping TiO2 with other special elements is a meaningful strategy to fabricate nanostructures with interesting functions useful in biomedicine.

Mitochondria play an important role in regulating tumor cell death and metabolism so that they can be potential therapeutic targets. Sonodynamic therapy (SDT) represents an attractive antitumor method that induces apoptosis by producing highly toxic reactive oxygen species (ROS). Mitochondria-targeting SDT can cause oxidative damage and improve the efficiency of tumor therapy. However, due to the nonselective distribution of nanosystems and the anti-apoptotic mechanism of cancer cells, the therapeutic effect of SDT is not ideal. Therefore, we proposed a novel mitochondria-targeting nanosystem (‘Mito-Bomb’) for ferroptosis-boosted SDT. Sonosensitizer IR780 and ferroptosis activator RSL-3 were both encapsulated in biocompatible poly(lactic‐co‐glycolic acid) (PLGA) nanoparticles to form ‘Mito-Bomb’ (named IRP NPs). IR780 in this nanosystem was used to mediate mitochondria-targeting SDT. RSL-3 inhibited the activity of GPX4 in the antioxidant system to induce ferroptosis of tumor cells, which could rewire tumor metabolism and make tumor cells extremely sensitive to SDT-induced apoptosis. Notably, we also found that RSL-3 can inhibit hypoxia inducible factor-1α (HIF-1α) and induce ROS production to improve the efficacy of SDT to synergistically antitumor. RSL-3 was applied as a ‘One-Stone-Three-Birds’ agent for cooperatively enhanced SDT against triple-negative breast cancer. This study presented the first example of RSL-3 boosting mitochondria-targeting SDT as a ferroptosis activator. The ‘Mito-Bomb’ biocompatible nanosystem was expected to become an innovative tumor treatment method and clinical transformation.

Titanium-based sonosensitizers for sonodynamic cancer therapy

MXene-based nanozymes remodel tumor microenvironment for heterojunction-enhanced sonodynamic and chemodynamic therapy to boost robust cancer immunotherapy

Reactive oxygen species (ROS), generated by sonosensitizers, play a pivotal role in tumor cell apoptosis during sonodynamic therapy (SDT), particularly for tumors located deep within tissues. Nevertheless, conventional sonosensitizers present limitations including inadequate ROS generation, insufficient tumor-specific accumulation, and associated adverse effects, significantly restricting their clinical applicability. To address these limitations, novel drug-free multifunctional nanoparticles (designated HGMP NPs) were synthesized. These NPs consist of mesoporous polydopamine (MPDA)-loaded protoporphyrin IX (PpIX), further surface-modified with glucose oxidase (GOx) and hyaluronic acid (HA), to achieve integrated photothermal, sonodynamic, and starvation-based tumor therapy. Upon exposure to near-infrared (NIR) irradiation (808nm) combined with ultrasound (US), HGMP NPs exhibited pronounced synergistic anticancer effects. Specifically, the photothermal effect triggered by NIR irradiation effectively enhanced local oxygen supply within tumor sites, thus significantly augmenting ROS production and improving the therapeutic outcomes of SDT. Concurrently, GOx-mediated glucose depletion induced tumor starvation and produced hydrogen peroxide (H2O2), further exacerbating oxidative stress within the tumor microenvironment. Transcriptomic analysis revealed that ROS and TNF signaling pathways represented key mechanisms underlying tumor elimination by this multimodal synergistic strategy. Real-time PCR analysis and ELISA assays further validated activation of the TNF signaling pathway. Importantly, this study first confirmed the high biocompatibility and biosafety of HGMP NPs via serum metabolomics, demonstrating no detectable systemic metabolic perturbations. Collectively, the prepared HGMP NPs provide a rational paradigm for synergistic anticancer therapy. These findings highlight the potential of HGMP NPs as an exceptionally safe and effective nanoplatform for cancer treatment, offering valuable insights into future developments in cancer nanomedicine.

Lipid bilayer-based biological nanoplatforms for sonodynamic cancer therapy

Polymer-based nanodrugs enhance sonodynamic therapy through epigenetic reprogramming of the immunosuppressive tumor microenvironment.