Tumor-targeted AIE polymeric micelles mediated immunogenic sonodynamic therapy inhibits cancer growth and metastasis

Tumor-targeted AIE polymeric micelles mediated immunogenic sonodynamic therapy inhibits cancer growth and metastasis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Tumor-targeted AIE polymeric micelles mediated immunogenic sonodynamic therapy inhibits cancer growth and metastasis Kai Deng, Yifeng Yu, Yong Zhao, Jia-Mi Li, Kun-Heng Li, Hong-Yang Zhao, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2330201/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Aggregation-induced emission luminogens (AIEgens) exhibit potent sonosensitivity in nanocarriers compared with conventional organic sonosensitizers owing to the strong fluorescence emission in the aggregated state. However, premature drug leakage and ineffective tumor targeting of current AIE nanosonosensitizers critically restrict their clinical application. Results: Here, an AIEgens-based sonosensitizer (AIE/Biotin-M) with excellent sonosensitivity was developed by assembling salicylaldazine-based amphiphilic polymers (AIE-1) and 4T1 tumor-targeting amphiphilic polymers (DSPE-PEG-Biotin) for the effective delivery of salicylaldazine to 4T1 tumor tissues, aiming to mediate immunogenic SDT. In vitro, AIE/Biotin-M were highly stable and generated plentiful singlet oxygen ( 1 O 2 ) under ultrasound (US) irradiation. After AIE/Biotin-M targeted accumulation in tumor, upon US irradiation, the generation of 1 O 2 not only leaded cancer cells death, but also elicited systemically immune response through causing immunogenic cell death (ICD) of cancer cells. In addition to mediate SDT, AIE/Biotin-M could chelate and reduce Fe 3+ , Cu 2+ and Zn 2+ by salicylaldazine for inhibiting neovascularization in tumor tissues. Ultimately, AIE/Biotin-M systemically inhibited tumor growth and metastasis upon US irradiation. Conclusions: This study presents a facile approach to the development of AIE nanosonosensitizers for cancer SDT. sonodynamic therapy polymeric micelles immunogenic cell death aggregation-induced emission immune response Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Sonodynamic therapy (SDT), features with non-ionizing radiation, high tissue-penetration (5–10 cm) and high spatiotemporal controllability, has emerged as a promising treatment modality for solid tumors [ 1 – 3 ]. During the typical process of SDT, ultrasound (US) locally activate sonosensitizers to generate a large number of reactive oxygen species (ROS), such as hydroxyl radical (·OH) and singlet oxygen ( 1 O 2 ), resulting in apoptosis or necrosis of cancer cells [ 4 , 5 ]. In addition to directly disrupting primary tumors, SDT can also noninvasively elicit systemically antitumor immune response to prevent metastasis [ 6 , 7 ]. During SDT, the generation of ROS induces immunogenic cell death (ICD) of cancer cells through the release of tumor-associated antigens (TAAs) and damage-related molecular patterns (DMAPs). The released TAAs and DMAPs serve as immune adjuvants to prompt maturation of dendritic cells (DCs), subsequently activating cytotoxic T cells to cause cancer cell death. It is worth noting that the therapeutic and immunogenic effects of SDT are primarily determined by sonosensitizers, which are irreplaceable elements for inducing cancer cell death under US irradiation [ 8 – 11 ]. Traditional organic sonosensitizers, including porphyrin, chlorophyll and curcumin, possess high biodegradability and excellent sonosensitivity, have been performed to destroy cancer cells by the US irradiation-mediated ROS production in vitro and in vivo [ 12 – 14 ]. However, these organic sonosensitizers with poor hydrophilicity and low tumor-targeting ability show insufficient accumulation, internalization and retention in cancer cells and tumor tissue, thus dramatically impairing the therapeutic effects of SDT [ 15 – 20 ]. To solve these problems, various nanocarriers, including polymeric micelles, nanoliposomes and inorganic nanoparticles, are developed to improve the concentration of organic sonosensitizers in tumor [ 21 – 26 ]. Although sonosensitizers loaded in nanocarriers have improved the antitumor effect of SDT to a certain degree in comparison to direct application of free sonosensitizers, accumulating evidence suggested that encapsulated sonosensitizers are not conducive to efficient SDT [ 27 – 32 ]. The hydrophobic sonosensitizers in nanocarriers lead to aggregation-caused fluorescence quenching (ACQ) effect, impairing the generation of ROS when irradiated with US [ 33 – 35 ]. On contrary to the traditional organic sonosensitizers, it has been demonstrated that aggregation-induced emission luminogens (AIEgens) exhibited potent sonosensitivity in aggregated state [ 36 – 38 ]. AIEgens encapsulated in polymeric micelles and biomimetic nanocarriers could generate a considerable concentration of ROS under US irradiation and exhibited commendable performance in mediating cancer SDT. However, these AIEgens-loaded nanoparticles suffer from premature drug release and lack of tumor-targeting, significantly restricting their clinical application. Recently, many studies have shown that nanomedicines consisting of amphiphilic polymer-drug conjuncts could be excellent drug carriers for inhibiting cancer growth owing to higher systemic stability, fewer premature drug leakage and more easily tumor-targeting modification in comparison with traditional nanomedicines [ 39 , 40 ]. Therefore, tumor-targeted AIE polymeric micelles consisted of amphiphilic polymer-AIEgens conjuncts have been widely applied for tumor therapy, e.g., photodynamic therapy, and achieved substantial therapeutic benefits against cancer [ 41 ]. However, there is no AIE polymeric micelles have been reported to mediate SDT. In our laboratory, a salicylaldazine-based amphiphilic polymer (AIE-1) was prepared and assembled into sub-20 nm AIE micelles (AIE-M) [ 42 ]. Based on this, we wondered if tumor-targeted AIE polymeric micelles composed of AIE-1 and tumor-targeted polymers might be able to become an effective AIEgens carrier for mediating cancer SDT. In this work, we developed highly stable AIE polymeric micelles (AIE/Biotin-M) to mediate immunogenic SDT for inhibiting 4T1 tumor growth and metastasis. As depicted in Fig. 1, AIE/Biotin-M was prepared from the assembly of AIE-1 and 4T1 tumor-targeting amphiphilic polymers (DSPE-PEG-Biotin). Our results demonstrated that AIE/Biotin-M is an excellent sonosensitizer with high stability and sonosensitivity. Under US irradiation, AIE/Biotin-M showed negligible fluorescence decay and generated large amounts of 1 O 2 . In vivo, AIE/Biotin-M actively accumulated in 4T1 tumor cells and triggered cells death under US irradiation. The dying cancer cells elicited ICD with exposure of calreticulin (CART) and release of high mobility group box 1 protein (HMGB1), which subsequently stimulated maturation of DCs and induced systemically antitumor immune response to inhibit 4T1 tumor growth and metastasis. Meanwhile, AIE/Biotin-M could inhibit neovascularization through chelating and reducing Fe 3+ , Cu 2+ and Zn 2+ by salicylaldazine in tumor microenvironment, restricted tumor growth in combination with SDT. Materials And Methods Materials 1-Bromohexadecane (98%), 2,4-dihydroxybenzaldehyde (99%), Cs 2 CO 3 (99%), methanesulfonyl chloride (98%), triethylamine (99.5%) and N 2 H 4 ·H 2 O (98%) were purchased from TCI. Methoxypolyethylene glycol (mPEG 2000 ) was obtained from Sigma-Aldrich and dried in toluene by azeotropic distillation for further use. 5,5-Dimethyl-1-pyrroline N-oxide (DMPO, 98%) was purchased from Sigma-Aldrich. 2,2,6,6-tetramethylpiperidine (TEMP, 99%) were acquired from Innochem. AIE-1 was prepared and characterized as our previously described. Breast-cancer cell line 4T1 were acquired and cultured with nutrient solution, and placed at 37 ℃ in a 5% CO 2− containing humid atmosphere. Beijing HFK provided female BALB/c mice (6–9 weeks old) with a body weight of 18–20 g. The mice were housed in a pathogen-free Animal Lab and subjected to experiments in accordance with the Animal Welfare Committee of the Animal Experiment Center of Wuhan University. The in vivo research was carried out on the subcutaneous 4T1 tumour model, which was generated by implanting 4T1 cells (150µL PBS, 3 × 106 cells per mice) into right flank of mice. Micelle Preparation And Characterization AIE-1 (50 mg) and DSPE-PEG 2000 -Biotin (0.5 mg) were dissolved in ultrapure water (5 mL) and treated with ultrasound for 15 min. The AIE-M was similarly prepared from AIE-1. The micelles solution was obtained and subsequently kept at 4°C for future use. Micelles were analyzed using Zeta-sizer Nano ZS (DLS, Malvern Instruments Ltd., UK) to determine their hydrodynamic size and polydispersity index (PDI). While, the morphological analysis was conducted via Field emission transmission electron microscopy (TEM, JEM-100CXII 100 kV). The aggregation-induced emission (AIE) characteristics of AIE/Biotin-M was measured as follows: AIE-1 (10 mg, 4 µmol) and DPPC-PEG 2000 -Biotin (0.1 mg) were separately dissolved in DMSO (1 mL) to prepare stock DMSO solution. A 0.1 mL aliquot of the DMSO stock solution was transferred to a 15 mL volumetric flask. After the addition of an appropriate amount of DMSO to maintain the final concentration of AIE-1 at 20 µM, water was added dropwise to furnish DMSO/water mixtures with water fractions of 0–99 vol%. Each sample was measured for fluorescence (excitation at 365 nm) after treated with ultrasound for 10 min. The ability of AIE/Biotin-M to chelate metal ions was determined as follows: A range of AIE/Biotin-M and AIE-M solution (20 µM) were separately prepared with additional CuCl 2 , Zn(ClO 4 ) 2 ·6H 2 O and FeCl 3 of different concentration (0–20 µM) together, and measured by fluorescence measurement (excitation at 365 nm) after stirring 15 min at room temperature. The Ultrasonic Stability Of Micelles The solution of AIE-M and AIE/Biotin-M were separately irradiated with US (1 MHz, 2 Wcm -2 ) for 5 and 10 min. After US irradiation, the fluorescence intensity and size distribution of AIE-M and AIE/Biotin-M were separately measured by fluorescence measurement (excitation at 365 nm) and DLS. The Sonosensitivity Of Micelles (Dup: Abstract ?) The detection of ROS generation from AIE-M and AIE/Biotin-M upon US irradiation was carried out by an electron paramagnetic resonance (ESR) spectrometer. To determine the generation of 1 O 2 , the solution of AIE-M and AIE/Biotin-M (100 µg/mL) were separately treated with US irradiation (2 Wcm − 2 , 1.0 MHz, 50% duty cycle) for 1 min in the presence of TEMP (50 µM), followed by detection through ESR spectrometer. The process of determination the generation of ·OH was same as 1 O 2 , but TEMP replaced with DMPO. A fluorescence spectrophotometer was used to conduct a quantitative analysis of ROS production. To quantitative measure the generation of 1 O 2 , the solution of AIE-M and AIE/Biotin-M (3950 µL, 100 µg/mL) were separately irradiated with US (2 Wcm − 2 , 1.0 MHz, 50% duty cycle) for 5 min and 10 min in the presence of singlet oxygen sensor green (SOSG, 50µL, 0.3 × 10 3 mM), and the fluorescence intensity was then recorded at 500–700 nm with 488 nm of excitation wavelength. The process of determination the generation of ·OH was same as 1 O 2 , but the SOSG was replaced with terephthalic acid (TA, 10 µg/mL), and then the fluorescence intensity at 350–500 nm was recorded at 325 nm of excitation wavelength. Intracellular Ros Production And Cellular Uptake 4T1 cells were seeded in 2 mL DMEM on confocal dishes at a density of 1.0 × 10 5 cells per well for 24 h. Then, for 8 and 24 h, AIE-M and AIE/Biotin-M were incubated separately with 4T1 cells at 200 g/mL concentrations. Next, the 4T1 cells were washed thrice with PBS to remove unabsorbed micelles. After that, they were stained with Hoechst 33258 for 15 minutes in a cell incubator after being fixed with 4% formaldehyde. Subsequently, cells were washed with PBS and examined using confocal laser scanning microscopy (CLSM). The estimation of micelles fluorescence intensity in different 4T1 cell structures and average micelles fluorescence intensity in 4T1cells was carried out using Imaging J software (National Institute of Health, USA). For 12 h, 4T1 cells were seeded in 2 mL of DMEM at a density of 2.0 × 10 4 cells per well in confocal dishes to detect the generation of intracellular ROS. Then AIE-M and AIE/Biotin-M were separately incubated with 4T1 cells at 200 µg/mL concentration for 8 h. After the free micelles were removed from dishes, 4T1 cells were stained with DCFH-DA concentration of 2 µM for 15 min, followed by washing with PBS to removed free DCFH-DA. Finally, 4T1 cells were irradiated with US (1.0 MHz, 2 Wcm − 2 , 50% duty cycle) for 1 min and 3 min and observed through CLSM. Imaging J software was used to calculate the fluorescence intensity of micelles in various 4T1 cell structures and the average fluorescence intensity of DCF in 4T1 cells. Determination of in vitro cytotoxicity MTT assay was used for the evaluation of the cytotoxicity of AIE-M and AIE/Biotin-M in the presence and absence of US. In 96-well plates, 4T1 cells were seeded with a density of 5 × 10 4 cells per well in cell incubator. After incubated for 24 h, AIE-M and AIE/Biotin-M were added separately and further incubated for 24 h. After removed free AIE micelles, 4T1 cells were irradiated with or without US (1.0 MHz, 2 W cm-2, 50% duty cycle) for 3 min. Then further subjected for 24 h incubated, DMEM was discarded and MTT solution was added into wells for 4 h. After that, each well was added 150 µL DMSO to replace the MTT solution. The microplate reader was then used to determine the cytotoxicity using the MTT assay, which relies on the absorbance value at 570 nm. The in vitro ICD effects 4T1 cells were seeded at a density of 5 × 10 5 cells in confocal dishes for 24 h. Then AIE-M and AIE/Biotin-M at 100 µg/mL concentration were separately incubated for 8 h with 4T1 cells, and then irradiated with US (1.0 MHz, 2 Wcm -2 , 50% duty cycle) for 3 min. For the detection of CART, US treated 4T1 cells were further incubated for 8 h and subjected to staining with individual primary antibodies against CART for 30 min. Finally, the exposure of CART was detected by CLSM. For the detection of HMGB1, US treated 4T1 cells were subjected to 12 h of further incubation followed by staining with individual primary antibodies against HMGB1 for 30 min. Finally, the exposure of HMGB1 was detected by CLSM. Intratumor Accumulation Of Micelles (Dup: Abstract ?) To evaluate the intratumoral accumulation of micelles, AIE/Ce6-M and AIE/Ce6/Biotin-M (Ce6 = 2.5 mg kg-1) with similar average size as AIE-M and AIE/Biotin-M were separately injected intravenously into 4T1 tumor-bearing BALB/c mice. Using an in vivo imaging system (IVIS® spectrum, PerkinElmer), real-time fluorescence images (excitation 640 nm, emission 680 nm) were obtained at different intervals following injection. In the meantime, tumours were collected by sacrificing the mice at various time intervals after injection which were sliced, and imaged the green fluorescence intensity of AIE micelles by CLSM. Detection of in vivo immunogenic effect The tumour-bearing 4T1 mice with an 80 mm 3 tumour size were weighed and randomly assigned into six groups (n = 9), including: (1) PBS, (2) US, (3) AIE-M, (4) AIE/Biotin-M, (5) AIE-M + US, (6) AIE/Biotin-M + US. After intravenously injected with AIE-M or AIE/Biotin-M (AIE-1 = 100 mg/mL) for 2 h, the tumors were irradiated with US (1.0 MHz, 2 Wcm − 2 , 50% duty cycle) for 5 min. The treatment was conducted three times every three days. After 24 h, 48 h, 72 h of last treatment, serum was collected and analyzed by an enzyme-linked immunosorbent assay (ELISA) kit for IFNγ, IL6, TNFα. And after 48 h of last treatment, one part of mice were sacrificed and collected tumours and tumour draining lymph node (TDLN) for further analysis. The one part of tumours were sliced and applied for CART immunofluorescence detection. The other parts of the tumour were dissociated to produce a suspension of single cells. Several fluorochrome-conjugated antibodies were used to stain the harvested cells; these include PE-anti-CD86, FITC-anti-CD11c, APC-anti-CD80, PE-anti-CD4, FITC-anti-CD3, and APC-anti-CD8 for analysing DCs and T cells by flow cytometer. In vivo SDT effect The 80 mm 3 tumour-bearing 4T1 mice were weighed and randomly divided into six groups (n = 9), including: (1) PBS, (2) US, (3) AIE-M, (4) AIE/Biotin-M, (5) AIE-M + US, (6) AIE/Biotin-M + US. After intravenously injected with AIE-M or AIE/Biotin-M (AIE-1 = 100 mg/mL) for 2 h, the tumours were irradiated with US (1.0 MHz, 2 Wcm-2, 50% duty cycle) for 5 min. The treatment was conducted three times every three days. The volumes of tumours and body weights of mice were measured every two days. Standard equations were used to determine the relative tumour volumes (RTV), tumour inhibitory rates (TIR, %), and relative mouse weights. Tumour tissues and major organs, including the liver, lung, heart, spleen, and kidney, were collected after all of the mice were sacrificed 16 days after treatment. For histological analysis, the collected tumours and major organs were weighed and stored at 4°C. Histological analysis of the proliferating cell nuclear antigen (PCNA), haematoxylin and eosin (H&E), platelet endothelial cell adhesion molecule-1 (CD31), terminal transferase dUTP nick-end labeling (TUNEL) and vascular endothelial growth factor (VEGF) was performed on paraffin-embedded tumour tissue sections that had been fixed in 4% formaldehyde. Lung and liver were applied for analysis of micrometastases. One part of tumor tissue and major organs were cut into pieces and heated in a solution of concentrated nitric acid to form transparent solution for analysing the concentration of metal ions by inductively coupled atomic emission spectrometer (ICP-AES). At last, the other part of the major organs was sliced and stained for H&E histological analysis. Statistical analysis The Student t-test was used for all statistical analyses. For all analyses, *(p < 0.05), **(p < 0.01) and ***(p < 0.001) were regarded as statistically significant. Data were expressed as means ± standard deviation (SD). Results And Discussion Preparation And Characterization Of Micelles In accordance with our previous reports, AIE-1 was synthesized and characterized [ 42 ]. The micelles AIE-M and AIE/Biotin-M were prepared through a simple ultrasonic treatment in the absence or presence of a tumor-targeting polymer (DSPE-PEG 2000 -Biotin). As depicted in Fig. 1A, the size distribution and average hydrodynamic diameter of AIE-M and AIE/Biotin-M, measured by DLS, were 19.2 nm and 20.5 nm with a narrow distribution (PDI = 0.098 and 0.103), respectively. Moreover, Fig. 1B showed that after being stored at 4°C for a week, both AIE-M and AIE/Biotin-M remained stable, with only minor changes in size distribution. The morphologies and diameter sizes of AIE-M and AIE/Biotin-M were observed in a dried state and calculated with TEM and image J. As shown in Fig. 1C and D, AIE-M and AIE/Biotin-M in a dried state were spherical in shape with 18.3 and 20.2 nm of average diameters, respectively. It was demonstrated that compared with AIE-M, AIE/Biotin-M endowed with tumor-targeted element exhibited negligible changes in diameter size and shape. These results showed that AIE/Biotin-M was suitable for cancer therapy. Our previous work has demonstrated that AIE-1 has no fluorescence emission when dissolved in a good solvent, while exhibiting strong fluorescence emission in an aqueous solution. As depicted in Fig. 1E, the photophysical properties of AIE-1/DPPC-PEG 2000 -Biotin in DMSO indicated that without fluorescence emission was detected at the excitation of 365 nm. However, the fluorescence intensity of AIE-1/DPPC-PEG 2000 -Biotin in DMSO was gradually enhanced with the increase of water fraction, which is a typical characteristic of AIEgens. Therefore, DPPC-PEG 2000 -Biotin in AIE/Biotin-M has negligible influence on the photophysical properties of AIE-M. Our previous work has demonstrated that chelating groups in AIE-M could form a stable complex with Cu 2+ , which impaired the fluorescence emission of this probe [ 42 ]. In addition to chelation with Cu 2+ , we further found that AIE-M and AIE/Biotin-M could chelate with Fe 3+ and Zn 2+ . The fluorescence emission of AIE-M and AIE/Biotin-M gradually changed with the addition of Fe 3+ and Zn 2+ , which indicated the formation of metal ion complexes with salen groups in AIE-M and AIE/Biotin-M. Notably, the fluorescence intensity of AIE-M-Zn and AIE/Biotin-M-Zn aqueous solution were gradually enhanced and reached a plateau with the addition of equivalent Zn 2+ . And after addition of Fe 3+ , the fluorescence intensity of AIE-M-Fe and AIE/Biotin-M-Fe aqueous solution gradually decreased and without completely quenched after the addition of equivalent Fe 3+ (Additional file 1: Figure S1). Meanwhile, metal ions (Fe 3+ , Cu 2+ , Zn 2+ ) have negligible influence on the average size of AIE-M and AIE/Biotin-M, Fig. 1F. These results revealed that AIE micelles could efficiently chelate with multiple metal ions (Fe 3+ , Cu 2+ , Zn 2+ ) and form stable complexes, exhibiting potentials for multiple metal ions interference cancer therapy. The Sonosensitivity Of Micelles To explore the sonodynamic performance of AIE/Biotin-M, the ultrasonic stability of micelles was firstly detected. AIE-M and AIE/Biotin-M have negligible change in fluorescence emission intensity and average hydrodynamic size after separately irradiated with US (1 MHz, 2 Wcm − 2 , 50% duty cycle) for 5 and 10 min (Fig. 2A-D), suggesting significant sono-stability of both AIE micelles. In the process of SDT, sonosensitizers irradiated with US can generate a large amount of ROS, such as 1 O 2 and ·OH. Here, we utilized 2, 2, dimethylpyrroline-1-oxide (DMPO) to separately differentiate 1 O 2 and ·OH in AIE micelles mediated SDT through electron spin resonance (ESR) technique. TEMP can efficiently capture 1 O 2 to yield 2, 2, 6, 6-tetramethylpiperidine-1-oxyl (TEMPO) and exhibit characteristic ESR signal of 1:1:1 triplet peak under static magnetic field. And DMPO can efficiently capture ·OH to yield DMPO-·OH and display a typical ESR signal of 1:2:2:1 under a static magnetic field. As depicted in Fig. 2E, both AIE micelles with US (1 MHz, 2 Wcm − 2 , 50% duty cycle) irradiation for 5 min exhibited a strong triplet peak of TEMPO indicated the generation of 1 O 2 . AIE-M and AIE/Biotin-M without US irradiation showed weak triplet peaks of TEMPO. And AIE-M and AIE/Biotin-M are equal in the triplet peak intensity of TEMPO after irradiated with US, which demonstrated tumor targeting Biotin-polymers have no effect on the sonosensitivity of AIE/Biotin-M. Furthermore, there was no characteristic ESR signal of DMPO-·OH was detected when AIE-M and AIE/Biotin-M treated with or without US irradiation (Fig. 2F), indicating no production of ·OH. To further qualitatively and quantitatively analyze ROS generation in AIE micelles mediated SDT, SOSG and TA were exploited to detect 1 O 2 and ·OH, respectively. SOSG with no fluorescence emission can be oxidized by 1 O 2 to form SOSG-endoperoxide (SOSG-EN) and show strong fluorescence emission at 525 nm; TA with no fluorescence emission can be oxidized by ·OH to form 2-hydroxyl terephthalic acid (TAOH) and show strong fluorescence emission at 435 nm. According to fluorescence emission (Fig. 2G-I) and Additional file 1: Figure S2, US (1 MHz, 2 Wcm − 2 , 50% duty cycle) treated AIE-M and AIE/Biotin-M exhibited highly characteristic SOSG-EN fluorescence signal, while no TAOH fluorescence signal could be observed. Meanwhile, the efficient generation of 1 O 2 positively corresponded to irradiation time, which was indicated by the SOSG-EN fluorescence signal increased over irradiation time. The ESR and fluorescence signal results demonstrated that AIE/Biotin-M could act as a highly efficient sonosensitizer to mediate sonodynamic cancer therapy by generating 1 O 2 under US irradiation in vitro and in vivo . Cellular uptake and cytotoxicity The tumor targeting ability of AIE/Biotin-M, which plays an important role in anti-tumor efficiency, was estimated towards 4T1 cells upon determination of intracellular AIEgens green fluorescence by confocal laser scanning microscope (CLSM). AIE-M and AIE/Biotin-M were separately treated with 4T1 cells for 8 h and 24 h. The green fluorescence of AIEgens in AIE-M and AIE/Biotin-M treated 4T1 cells were increased with coincubation time, Fig. 3A-C. Notably, the green fluorescence of AIEgens in AIE/Biotin-M treated 4T1 cells was higher than that in AIE-M treated cells. Quantitative analysis also showed that the green fluorescence of AIE micelles in AIE/Biotin-M treated 4T1 cells was 2.4-fold higher than that in AIE-M treated 4T1 cells after coincubation for 24 h. These results demonstrated that AIE/Biotin-M could effectively internalized by 4T1 cells with the high biotin receptor expression on cell membrane. Theoretically, high intracellular concentration of AIEgens sonosensitizers has positive effect on the US-mediated ROS generation. To qualitatively measure the generation of ROS in cells, DCFH-DA was selected as an intracellular ROS detection probe. DCFH, which enzymatically hydrolyzed from DCFH-DA in 4T1 cells, was oxidized by ROS to form DCF with green fluorescence emission. 4T1 cells pre-treated with AIE-M and AIE/Biotin-M exhibited negligible DCF green fluorescence in the absence of US irradiation (Fig. 3D-F). After US (1.0 MHz, 2 Wcm − 2 , 50% duty cycle) irradiation, both 4T1 cells pre-treated with AIE-M and AIE/Biotin-M exhibited DCF green fluorescence. And the DCF green fluorescence was increased with irradiation time, indicating intracellular generation of ROS was time-dependent in AIE micelles mediated SDT. Due to the high concentration of AIEgens in 4T1 cells pre-treated with AIE/Biotin-M, the DCF green fluorescence was stronger in AIE/Biotin-M treated cells than that in cells treated with AIE-M. Quantitative analysis demonstrated that after US irradiation, the average DCF green fluorescence intensity in 4T1 cells pre-treated with AIE/Biotin-M sharply increased 1.5-fold than that in 4T1 cells pre-treated with AIE-M. The results indicated that AIE/Biotin-M might show powerful performance on sonodynamic cancer therapy in vitro and in vivo. Due to AIE/Biotin-M showing excellent performance on cellular internalization and 1 O 2 generation, it can be speculated that AIE/Biotin-M have a significant effect on anti-tumor in vitro. The cytotoxicity of AIE/Biotin-M against 4T1 cells with or without US irradiation was evaluated by standard MTT assay. As shown in Fig. 3G, even at a high concentration of 200 µg·mL-1, neither AIE-M nor AIE/Biotin-M showed toxicity against 4T1 cells in the absence of US stimulation. Similarly, merely US irradiation has no toxicity to 4T1 cells. However, under US (1.0 MHz, 2 Wcm-2, 50% duty cycle) irradiation for 3 min, both AIE-M and AIE/Biotin-M significantly inhibited the growth of 4T1 cells in a concentration-dependent manner. Differently, the sonotoxicity of AIE/Biotin-M (IC 50 = 63.1 µg·mL-1) against 4T1 cells was significantly higher than that of AIE-M (IC 50 = 110.5 µg·mL-1), due to the excellent cellular endocytosis of AIE/Biotin-M. The results were consistent with the intracellular generation of ROS under US irradiation. The in vitro ICD effects Next, we investigated whether AIE/Biotin-M treated 4T1 cells could release DMAPs in the presence of US, such as CART and HMGB1, which are a crucial characteristic of ICD. CART, calcium-binding proteins in the endoplasmic reticulum (ER), exposed on the cell outer membrane act as an “eat me” signal, stimulating DCs and macrophages to recognize and engulf the dying tumor cells and tumor debris. As shown in CLSM measurement (Additional file 1: Figure S3A), after irradiation with US (1.0 MHz, 2 Wcm − 2 , 50% duty cycle) for 3 min, AIE-M and AIE/Biotin-M showed high CATR red fluorescence. Due to the effective intracellular endocytosis of AIE/Biotin-M, CATR red fluorescence in AIE/Biotin-M pre-treated 4T1 cells was higher than the 4T1 cells pre-treated with AIE-M. Other groups, such as only US, AIE-M, and AIE/Biotin-M without US irradiation exhibited negligible CATR red fluorescence intensity. HMGB1, a nuclear protein, could release from nuclear to cytoplasm and extracellular environment, stimulating inflammatory response and inducing DCs maturation. As depicted in Additional file 1: Figure S3B, upon US irradiation (1.0 MHz, 2 Wcm − 2 , 50% duty cycle) for 3 min, 4T1 cells pre-treated with AIE/Biotin-M also induced a large amount of HMGB1 transferring from nuclei to cytoplasm, exhibiting higher red fluorescence intensity in cytoplasm than other groups. Upon US irradiation, the strong expression of CART and HMGB1 in AIE/Biotin-M treated cells may due to cancer cells damage and death induced by a pronounced amount of ROS generation. These results confirmed that AIE/Biotin-M mediated SDT could efficiently cause immunogenic cancer cell death in vitro and exhibited potential in stimulating a systemically immune response in vivo . Intratumor Accumulation Of Micelles To optimize the experimental parameters for cancer SDT in vivo , this study applied a fluorescence imaging assay to track the in vivo behavior of AIE/Biotin-M. Due to the absorbance wavelength of AIE micelles below 600 nm, suffering from poor tissue penetration, we prepared Ce6-loaded AIE micelles to monitor the tumor accumulation of AIE-M and AIE/Biotin-M through in vivo real-time fluorescence imaging assay. The Ce6-loaded AIE/Ce6-M and AIE/Ce6/Biotin-M were prepared using the emulsification method. The average size of AIE/Ce6-M and AIE/Ce6/Biotin-M were quite consistent with that of AIE-M and AIE/Biotin-M (Fig. 4A). Therefore, Ce6-loaded micelles could be applied to explore the tumor accumulation of AIE-M and AIE/Biotin-M based on the fluorescence of Ce6. 4T1 tumor-bearing BALB/c mice were separately i.v. injected with free Ce6, AIE/Ce6/Biotin-M and AIE/Ce6-M (Ce6 2.5 mg/kg). At each time point (0–8 h), the in vivo fluorescence images were acquired after i.v. injection (Fig. 4B). Not surprisingly, free Ce6 exhibited low accumulation in the tumor, showing weak fluorescence intensity at each time point (0–8 h) after i.v. administration. While AIE/Ce6/Biotin-M and AIE/Ce6-M showed relatively strong fluorescence intensity in tumor tissues at 2 h post-injection due to enhanced permeability and retention effect (EPR). Notably, Fig. 4C quantitative analysis illuminated that AIE/Ce6/Biotin-M exhibited the highest fluorescence intensity in the tumor site and long-circulation time due to the high expression of the biotin receptor on the surface of 4T1 cells. As we all known, higher concentration of AIEgens would result in strong efficiency of SDT. Therefore, it was possible for AIE/Biotin-M to show an excellent SDT effect in vivo . We have demonstrated that the AIE micelles could chelate with the metal ions (Cu 2+ , Fe 3+ , Zn 2+ ) to enhance (Zn 2+ ) or impair (Cu 2+ , Fe 3+ ) the fluorescence emission of AIEgens. Therefore, we next explored the fluorescence change of AIE micelles in tumor tissue through excising tumor tissues at each time point (1–4 h) after i.v. injection of AIE-M and AIE/Biotin-M at AIE micelles dose of 100 mg·kg − 1 . The excised tumor tissues were sliced and observed by CLSM. As depicted in Fig. 4D-E, although the metal-ions (Cu 2+ , Fe 3+ , Zn 2+ ) were abundant in tumor tissues, there were detectable AIE micelles green fluorescence in tumor biopsies after i.v. injection for 1, 2, 3, 4 h. And at 2 h post-injection, AIE-M and AIE/Biotin-M exhibited high green fluorescence in tumor biopsies. Significantly, AIE/Biotin-M showed stronger green fluorescence than AIE-M in tumor biopsies at each time point (1–4 h) after i.v. injection, which was consistent with in vivo fluorescence imaging. As we illustrated that the high AIEgens green fluorescence in cells could contribute to significant SDT efficiency. Therefore, metal ions (Cu 2+ , Fe 3+ , Zn 2+ ) in tumor tissues had negligible influence on AIE/Biotin-M mediated SDT efficiency when AIE micelles at i.v. injection dose of 100 mg·kg − 1 . These results demonstrated that AIE/Biotin-M could not only chelate with multiple metal ions (Cu 2+ , Fe 3+ , Zn 2+ ) but also show excellent potential to mediate cancer SDT in vivo. The in vivo ICD effect and immune response Encouraged by in vitro results, we further investigated whether AIE/Biotin-M could induce ICD and immune response upon US irradiation in vivo . 4T1 tumour-bearing mice with tumour volume 80 mm 3 were randomly divided into six groups including PBS, PBS + US, AIE-M, AIE/Biotin-M, AIE-M + US and AIE/Biotin-M + US. Mice were intravenously injected with AIE-M and AIE/Biotin-M at AIE micelles dose of 100 mg/kg, once every 3 days for three times, respectively. Due to the high tumor accumulation of AIE-M and AIE/Biotin-M at 2 h post-injection, at this time point, tumors were treated with US (1 MHz, 2 Wcm − 2 , 50% duty cycle) for 5 min or without US. After the last irradiation for 6 h, tumors were excised, sliced and stained for immunofluorescence imaging of CART and HMGB1. As depicted in immunofluorescence imaging (Additional file 1: Figure S4), 4T1 tumors treated with AIE-M and AIE/Biotin-M plus US irradiation showed strong CART exposure on the cell outer membrane. In line with in vitro , 4T1 tumors treated with AIE/Biotin-M plus US irradiation induced the largest amount of CART exposure. Notably, without US irradiation, 4T1 tumors treated with AIE-M and AIE/Biotin-M slightly activated CART exposure may due to AIE micelles mediated metal ions (Cu 2+ , Fe 3+ , Zn 2+ ) depletion. Metal ions depletion in tumor tissues can reduce tumor angiogenesis, resulting in reducing/blocking the replenishment of nutrients for inhibition of tumor cells growth and metastasis. Consistent with CART, without US irradiation, 4T1 tumors treated with AIE-M and AIE/Biotin-M also slightly induced the release of HMGB1, which was demonstrated by the high signal of HMGB1. And 4T1 tumours treated with AIE/Biotin-M plus US irradiation induced the largest amount of HMGB1 release, which was demonstrated by the reduction of HMGB1 signal. These phenomena demonstrated that the combination of AIE/Biotin-M and US irradiation could potentiate the induction of tumor ICD, which would prominently activate immune response in vivo . The DMAPs released from dying cancer cells could stimulate immature DCs to engulf tumor antigens and debris. Subsequently, the immature DCs migrate to the nearby lymph nodes, where they underwent maturation and exposed the processed pro-inflammatory cytokines to naive T cells, thus inducing a systemically innate immune response. Therefore, the antitumor innate immunity induced by the combination of AIE/Biotin-M and SDT was evaluated by analysing the maturation of DCs and secretion of pro-inflammatory cytokines. 4T1 tumor-bearing mice with tumor size of 80 mm 3 were i.v. injection with AIE-M and AIE/Biotin-M at a concentration of 100 mg·kg − 1 , respectively, once every 3 days three times. Due to the high tumor accumulation of AIE-M and AIE/Biotin-M at 2 h post-injection, at this time point, tumours were treated with US for 5 min or without US. After the last US irradiation for 48 h, single-cell suspension of tumor-draining lymph nodes was stained with anti-CD86, anti-CD80 and anti-CD11c for FCM analysis. As depicted in Fig. 5A, AIE-M and AIE/Biotin-M without US irradiation just slightly promoted DCs maturation (CD80 + CD86 + ). However, in the presence of US, AIE-M and AIE/Biotin-M exhibited a high percentage of DCs maturation. Additionally, AIE/Biotin-M plus US significantly promoted DCs maturation (15.0%), which was consistent with the release of HMGB1 and CART. The matured DCs could secrete pro-inflammatory cytokines, such as IFN-γ, TNF-α, and IL-6. Although AIE-M and AIE/Biotin-M in the absence and presence of US irradiation cloud increase the TNF-α, IFN-γ, and IL-6 levels (Fig. 5B) in peripheral blood of mice, AIE/Biotin-M plus US induced highest production of pro-inflammatory cytokines. In addition, the highest maturation of DCs and production of pro-inflammatory cytokines suggested that AIE/Biotin-M plus US could induce an immune response and showed great potential in antitumor immunity. Thus, single-cell suspension of tumours was stained with anti-CD8, anti-CD4 and anti-CD3 for FCM analysis of infiltrated T cells (Fig. 5C). In the absence of US irradiation, the percentage of tumor-infiltrating activated T cells (CD4 + and CD8 + T cells) slightly elevated in AIE-M and AIE/Biotin-M, due to the metal ions chelation of AIE micelles. And the level of T cells was significantly increased in US treated AIE-M and AIE/Biotin-M. The highest tumor- infiltrating T cells in AIE/Biotin-M plus US was consistent with the DC maturation and pro-inflammatory cytokines release. These results revealed that AIE/Biotin-M plus US could activate the adaptive immune response and may potentially enhance anti-tumor therapy. The in vivo therapeutic effect Encouraged by the activation of systemically anti-tumor immune response by AIE/Biotin-M plus US, we further explored whether this combination could prevent breast cancer growth and metastasis in vivo. The mouse 4T1 tumor with poorly immunogenic, early metastasis and highly tumorigenic is analogous to human TNBC. Therefore, 4T1 tumor-bearing mice are regarded as an appropriate experiment model for simultaneously evaluating the therapeutic effect on inhibiting primary breast cancer growth and metastasis. 4T1 tumour-bearing mice with tumour volume 80 mm3 were randomly divided into six groups including PBS, PBS + US, AIE-M, AIE/Biotin-M, AIE-M + US and AIE/Biotin- M + US. Mice were intravenously injected with AIE-M and AIE/Biotin-M at AIE micelles dose of 100 mg/kg, respectively. After i.v. injection for 2 h, tumors were treated with US (1 MHz, 2 Wcm − 2 , 50% duty cycle) for 5 min or without US. As shown in Fig. 6A, merely US irradiation had no tumor inhibition compared to PBS treated mice, which validated that US irradiation has no damage to tissues. In the absence of US irradiation, AIE-M and AIE/Biotin-M showed slight tumor inhibition with average relative tumor volume (RTV) of = 8.5, 8.2 (Fig. 6A) and tumor inhibition rate (TIR) of = 33.1%, 36.3% (Fig. 6B) compared to PBS group due to the metal ions depletion in the tumor. Notably, in the presence of US irradiation, the generation of ROS and activation of immune response enhanced tumor inhibition of AIE-M with RTV = 4.3 and TIR = 64.4%. In the presence of US irradiation, Furthermore, the therapeutic ability of AIE/Biotin-M plus US irradiation on tumor tissues was conducted by histological, immunohistochemical, and immunofluorescent analysis of the tumor section at the end of therapy. To evaluate the apoptosis and/or necrosis, the excised tumors fixed, sectioned and stained to analyze H&E staining, TUNEL, and PCNA, Fig. 6F. The H&E staining results revealed the intact structure tumor cells in PBS and US treated tumors. Comparatively, tiny part of separated and sparse tumor cells was observed in AIE-M and AIE/Biotin-M group, which was lower than the AIE-M + US group. Notably, the most obvious damage of cells was observed in tumor tissue after being treated with AIE/Biotin-M plus US irradiation. Additionally, PCNA and TUNEL staining results further validated that AIE/Biotin-M plus US irradiation resulted in the weakest cell the TIR of AIE/Biotin-M was 1.3-fold higher than AIE-M due to tumor-targeting accumulation of AIE/Biotin-M triggered a strong generation of ROS and anti-tumor immunity. Meanwhile, weight measurements (Fig. 6C) and the photos (Fig. 6D) of tumors further validated that AIE/Biotin-M plus US effectively inhibited tumor growth. The six groups of mice were also measured for their body weights every two days, and no weight fluctuations were observed, demonstrating the biocompatibility of all treatments Fig. 6E. proliferation (weak red fluorescence in PCNA assay) and strongest cell apoptosis (strong green fluorescence in TUNEL assay). In addition to inhibition of tumor growth, reduction of tumor metastasis was also extraordinary significant in cancer therapy. As depicted in Fig. 6G, more significant amounts of tumor nodules were found in lungs and livers in PBS, US, AIE-M and AIE/Biotin-M than in AIE-M plus US irradiation, which was consistent with the histological examination. Compared to AIE-M plus US irradiation, AIE/Biotin-M plus US irradiation pronouncedly prevents liver and lung metastasis. Meanwhile, AIE/Biotin-M plus US irradiation could also result in the lowest vascular density and vascular endothelial growth factor (VEGF) in the tumor tissue (Additional file 1: Figure S5A). On the one hand, the generation of ROS during SDT could damage tumor vessels and VEGF. And the damage degree of vessel and VEGF was positively correlated with ROS production. On the other hand, AIE micelles with the ability of metal ions chelation could also inhibit angiogenesis and VEGF expression, which was demonstrated by the lowest concentration of Fe 3+ , Cu 2+ and Zn 2+ in AIE/Biotin-M treated tumour tissues, Fig. 7B. Although AIE/Biotin-M could induce metal ion depletion in tumors, negligible metal ions depletion was detected in other organs including liver, heart, lung, spleen, and kidney, Fig. 7B. Therefore, no apparent damage of cells was observed in liver, heart, lung, kidney, and spleen after tumor-free mice were i.v. injection of AIE-M and AIE/Biotin-M for 16 days demonstrated high biocompatibility of AIE/Biotin-M (Additional file 1: Figure S5B). Conclusion In conclusion, we firstly developed tumor-targeted AIE polymeric micelles (AIE/Biotin-M) for mediating immunogenic SDT. It was demonstrated that AIE/Biotin-M with high stability could actively accumulate into 4T1 tumor cells and cause no damage to mice. Upon US irradiation, AIE/Biotin-M efficiently damaged cancer cells and induced ICD of cancer cells through the local generation of ROS. The DMAPs and TAAs released from dying cancer cells induced maturation of DCs and activated antitumor immune response, systemically inhibiting tumor growth and metastasis. Meanwhile, the depletion of tumor metal ions (Fe 3+ , Cu 2+ and Zn 2+ ) by AIE/Biotin-M further enhanced the antitumor effect. In a word, this study offers a facile strategy to design AIE nanosonosensitizers for clinical application in the future. Abbreviations SDT: Sonodynamic therapy; AIE: Aggregation-induced emission; AIEgens: Aggregation-induced emission luminogens; US: Ultrasound; ROS: Reactive oxygen species; ·OH: hydroxyl radical; 1 O 2 : singlet oxygen; ICD: Immunogenic cell death; H&E: Hematoxylin and eosin stain; TUNNEL: TdT-mediated dUTP nick end labeling; DLS: Dynamic light scattering; DAMPs: Damage-associated molecular patterns; TEM: Transmission electron microscope; TAAs: Tumor-associated antigens; DCs: Dendritic cells; CART: Calreticulin; HMGB1: High mobility group box 1 protein; VEGF: Vascular endothelial growth factor. Declarations Acknowledgements We are grateful for the National Natural Science Foundation of China (52173137, 51873163, 82202132), Natural Science Foundation of Hubei Province (2021CFB055), Fundamental Research Funds for the Central Universities (2042021kf0153) and Youth Interdisciplinary Special Fund of Zhongnan Hospital of Wuhan University. Author contributions SH: Designed the project; KD, YY, JL, KL, and HZ: Performed research; KD and YY: Analyzed data; KD and MW: Wrote the paper. Funding This work was supported by the National Natural Science Foundation of China (52173137, 51873163, 82202132), Natural Science Foundation of Hubei Province (2021CFB055), Fundamental Research Funds for the Central Universities (2042021kf0153) and Youth Interdisciplinary Special Fund of Zhongnan Hospital of Wuhan University. Availability of data and materials All data generated or analyzed during the current study are included in this published article. Ethics approval and consent to participate All animal experiments were approved by the Experimental Animal Welfare Ethics Committee Zhongnan Hospital of Wuhan University (approval number: ZN2021232). Consent for publication Not applicable. Competing interests The authors declare that there are no competing interests in this published article. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2330201","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":156759272,"identity":"125293fb-e92e-4bc2-854d-65dbc485a3e1","order_by":0,"name":"Kai Deng","email":"","orcid":"","institution":"Zhongnan Hospital of Wuhan University, Wuhan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Deng","suffix":""},{"id":156759273,"identity":"5b2b42ff-c77e-4c92-b7eb-df03b1cf1a7a","order_by":1,"name":"Yifeng Yu","email":"","orcid":"","institution":"Zhongnan Hospital of Wuhan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yifeng","middleName":"","lastName":"Yu","suffix":""},{"id":156759274,"identity":"92bc2638-5192-46eb-b4fd-60cb33d0bfbb","order_by":2,"name":"Yong Zhao","email":"","orcid":"","institution":"Zhongnan Hospital of Wuhan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yong","middleName":"","lastName":"Zhao","suffix":""},{"id":156759275,"identity":"61cd4bbf-8934-4c59-9bd5-cf14975f36af","order_by":3,"name":"Jia-Mi Li","email":"","orcid":"","institution":"Wuhan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jia-Mi","middleName":"","lastName":"Li","suffix":""},{"id":156759276,"identity":"5659ccb7-d4a4-42af-a2ca-26d7848af31f","order_by":4,"name":"Kun-Heng Li","email":"","orcid":"","institution":"Wuhan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kun-Heng","middleName":"","lastName":"Li","suffix":""},{"id":156759277,"identity":"a678c837-9a19-4d36-af65-08838621aec4","order_by":5,"name":"Hong-Yang Zhao","email":"","orcid":"","institution":"Wuhan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hong-Yang","middleName":"","lastName":"Zhao","suffix":""},{"id":156759278,"identity":"0be98585-7397-4058-b56f-84e798b5c723","order_by":6,"name":"Meng Wu","email":"","orcid":"","institution":"Zhongnan Hospital of Wuhan University, Wuhan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Meng","middleName":"","lastName":"Wu","suffix":""},{"id":156759279,"identity":"2a6dc1a2-fb40-47af-8755-d0f07cd7a5f4","order_by":7,"name":"Shiwen Huang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYFACHjApxyABothI0GJMupbEBqK1yM/IPSbxc0dt+obbPQYMH8oOM/DPbsCvxeBGXppk75njuRvunDFgnHHuMIPEnQMEtEjnmEnwth3L3XYjx4CZt+0wg4FEAgGHzc4xk/zbdizdDKTlLzFaGG7nmEnzttUkgLUwEqPF4P4bY2vZtgOG+2+kFRzsOZfOI3GDkMN6zhjefNtWJy85I3njgx9l1nL8Mwg5DAIOg8kDDLBoIgLUEatwFIyCUTAKRiIAABTZQ39kYbtZAAAAAElFTkSuQmCC","orcid":"","institution":"Wuhan University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Shiwen","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2022-11-30 15:14:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2330201/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2330201/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":29925069,"identity":"f731e36e-f986-44c0-a1bc-891b3e51fdd1","added_by":"auto","created_at":"2022-12-05 20:25:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":86070,"visible":true,"origin":"","legend":"\u003cp\u003e(A) DLS measurements of the hydrodynamic size of AIE-M and AIE/Biotin-M. (B) Size changes of AIE-M (bule) and AIE/Biotin-M (red) after stored in PBS at 4 ℃. (C) TEM image of AIE-M and (D)AIE/Biotin-M. (E) Fluorescence spectra of 20 μM AIE-1 and DPPC-Biotin-PEG\u003csub\u003e2000\u003c/sub\u003e in a mixture solution containing DMSO and water. (F) Size changes of AIE-M and AIE/Biotin-M in a solution containing different concentrations of metal ions.\u003c/p\u003e","description":"","filename":"F1.png","url":"https://assets-eu.researchsquare.com/files/rs-2330201/v1/b256d7586a3b81f6c5c6daea.png"},{"id":29926233,"identity":"4e2e65b7-1cf7-4ffb-82c5-e7633e91237d","added_by":"auto","created_at":"2022-12-05 20:33:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":131392,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of AIE-M (A) and AIE/Biotin-M (B) fluorescence intensity after 5 and 10 min\u0026nbsp;of exposure to US. Size distribution of AIE-M (C) and AIE/Biotin-M (D) after irradiation with US for 5 and 10 min. (E) The generation of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e by PBS, AIE-M and AIE/Biotin-M with or without US irradiation determined by ESR. (F) The generation of ·OH by PBS, AIE-M and AIE/Biotin-M with or without US irradiation determined by ESR. (a) AIE/Biotin-M + US, (b) AIE-M + US, (c) AIE-M, (d) AIE/Biotin-M, (e) US (f) PBS. (G, H) The generation of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e by AIE-M and AIE/Biotin-M with US irradiation for different time was quantitatively determined by the fluorescence intensity of SOSG-EN. (I) The generation of ·OH by AIE/Biotin-M with US irradiation for different time was quantitatively determined by the fluorescence intensity of TAOH.\u003c/p\u003e","description":"","filename":"F2.png","url":"https://assets-eu.researchsquare.com/files/rs-2330201/v1/60e938afb8aefbf8b14b7ebf.png"},{"id":29925074,"identity":"2c055b52-3467-44ce-a83e-390f422e8655","added_by":"auto","created_at":"2022-12-05 20:25:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":119622,"visible":true,"origin":"","legend":"\u003cp\u003e(A) CLSM images of intracellular AIEgens fluorescence after incubation for different time with AIE-M and AIE/Biotin-M (scale bar = 25μm). (B, C) Average fluorescence intensity analysis and fluorescence intensity profiles for AIEgens in 4T1 cells from\u0026nbsp;Fig. 3A. (a) AIE-M 8 h, (b) AIE-M 24 h, (c) AIE/Biotin-M 8 h, (d) AIE/Biotin-M 24 h. (D) ROS-induced DCF fluorescence in 4T1 cells following the AIE-M and AIE/Biotin-M uptake and subsequent treatment with or without US irradiation (scale bar = 25μm). (E, F) ROS-induced DCF fluorescence intensity profiles and average fluorescence intensity analysis in 4T1 cells from Fig. 3D. (G) Cytotoxicity of AIE-M and AIE/Biotin-M against 4T1 cells without US or with US irradiation (1.0 MHz, 2 Wcm\u003csup\u003e-2\u003c/sup\u003e, 50% duty cycle). (a) PBS, (b) US, (c) AIE-M, (d) AIE/Biotin-M, (e) AIE-M + US (f) AIE/Biotin-M + US.\u0026nbsp;\u003c/p\u003e","description":"","filename":"F3.png","url":"https://assets-eu.researchsquare.com/files/rs-2330201/v1/737cadb9464329bdd196559f.png"},{"id":29925072,"identity":"0b9c58b6-6af1-491c-9614-b6dec1f13ec2","added_by":"auto","created_at":"2022-12-05 20:25:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":187346,"visible":true,"origin":"","legend":"\u003cp\u003eThe intratumor accumulation of AIE/Biotin-M. (A) Hydrodynamic size of AIE/Ce6-M (blue) and AIE/Biotin/Ce6-M (red) measured by DLS. (a) AIE/Ce6-M, (b) AIE/Biotin/Ce6-M. (B)\u003cem\u003e In vivo\u003c/em\u003e fluorescence images and (C) fluorescence intensity signals in 4T1 tumor tissues of mice after\u003cem\u003e i.v. \u003c/em\u003eseparate injection of free Ce6, AIE/Ce6-M and AIE/Biotin/Ce6-M. (a) free Ce6, (b) AIE/Ce6-M, (c) AIE/Biotin/Ce6-M. (D) CLSM images and (E) average fluorescence intensity analysis of excised tumor tissues\u003cem\u003e \u003c/em\u003eafter \u003cem\u003ei.v.\u003c/em\u003e separate injection of AIE-M and AIE/Biotin-M (scale bar = 100 μm). (a) AIE-M, (b) AIE/Biotin -M.Data are presented as the mean ± SD (n = 3).\u003c/p\u003e","description":"","filename":"F4.png","url":"https://assets-eu.researchsquare.com/files/rs-2330201/v1/8c11d7efaa7bf913a5e5ca29.png"},{"id":29925071,"identity":"a22291a4-c557-4785-9a99-674a12567037","added_by":"auto","created_at":"2022-12-05 20:25:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":133263,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e AIE/Biotin-M mediated SDT to induce DMAPs release, stimulate DC maturation, elicit proinflammatory cytokines expression and promote an immune response. 4T1 tumor-bearing mice were subjected to \u003cem\u003ei.v.\u003c/em\u003e separate injection of AIE-M and AIE/Biotin-M once every three days for three times, and then treated without or with US (1.0 MHz, 2 Wcm\u003csup\u003e-2\u003c/sup\u003e, 50% duty cycle) irradiation for 5 min at two h of post-injection. (A) The DC maturation in the 4T1 tumor-draining lymph nodes of BALB/c mice at 48 h after the last\u003cem\u003e \u003c/em\u003eUS irradiation. (B) The levels of TNF-α, IL-6 and IFN-γ in mice serum isolated at 24 to 72 h after last\u003cem\u003e \u003c/em\u003eUS irradiation. (D) The tumor infiltration of CD4 and CD8 T cells in the 4T1 tumors of BALB/c mice at 48 h after the last\u003cem\u003e \u003c/em\u003eUS irradiation. (a) PBS, (b) US, (c) AIE-M, (d) AIE/Biotin-M, (e) AIE-M + US (f) AIE/Biotin-M + US. Data are presented as the mean ± SD (n = 3).\u003c/p\u003e","description":"","filename":"F5.png","url":"https://assets-eu.researchsquare.com/files/rs-2330201/v1/3f4a7efe8cc19781142ac0a5.png"},{"id":29926234,"identity":"426e14b8-4a71-4bb4-b9c4-cb7f9988e509","added_by":"auto","created_at":"2022-12-05 20:33:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":406567,"visible":true,"origin":"","legend":"\u003cp\u003eThe inhibitory effect of AIE/Biotin-M mediated SDT on growth and metastasis of primary 4T1 tumor. 4T1 tumor-bearing mice were i.v. separate injection of AIE-M and AIE/Biotin-M once every 3 day for three times, and then treated without or with US (1.0 MHz, 2 Wcm-2, 50% duty cycle) irradiation at 2 h post-injection for 5 min. (A) Tumor growth curves of different groups after various treatments. (B) Tumor inhibitory rate and (C) tumor weight at 16 day post various treatments. (D) Photographs of excised primary 4T1 tumors at the end of treatments. (E) The relative body weight variation of mice after various treatments in 16 days. (F) The H\u0026amp;E staining, immunofluorescence staining for PCNA, and TUNEL staining of tumor tissue at the end of treatment. Photographs and H\u0026amp;E staining of hepatic (G) and pulmonary (H) metastatic tumor nodules at the end of treatments. (a) PBS, (b) US, (c) AIE-M, (d) AIE/Biotin-M, (e) AIE-M + US (f) AIE/Biotin-M + US. Data are presented as the mean ± SD (n = 5). The significance was determined using the Student's t-test. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"F6.png","url":"https://assets-eu.researchsquare.com/files/rs-2330201/v1/65d1c013cc752bd360376742.png"},{"id":29926235,"identity":"561dcd0f-4bc1-456c-b514-59a7736495bb","added_by":"auto","created_at":"2022-12-05 20:33:50","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":51034,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Metal ions (Fe\u003csup\u003e3+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e and Zn\u003csup\u003e2+\u003c/sup\u003e) concentration in tumor tissues at the end of treatment. (B) Metal ions (Fe\u003csup\u003e3+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e and Zn\u003csup\u003e2+\u003c/sup\u003e) concentration in major organs (heart, spleen, liver, kidney, and lung) at the end of treatment.\u003c/p\u003e","description":"","filename":"F7.png","url":"https://assets-eu.researchsquare.com/files/rs-2330201/v1/91bac265283b64de772d7743.png"},{"id":32225758,"identity":"9f6a45a1-6366-47bf-a25c-074cb02e0be0","added_by":"auto","created_at":"2023-01-30 18:14:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1507473,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2330201/v1/0bbf6e60-266b-428d-85c0-08e3c1cc7957.pdf"},{"id":29925076,"identity":"c4a62508-f588-4d8c-a9fd-f3f85b6b92bb","added_by":"auto","created_at":"2022-12-05 20:25:50","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1494899,"visible":true,"origin":"","legend":"","description":"","filename":"DengKaiSupplementarymaterialAdditionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-2330201/v1/45556573d18d6ad9d6360430.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Tumor-targeted AIE polymeric micelles mediated immunogenic sonodynamic therapy inhibits cancer growth and metastasis","fulltext":[{"header":"Background","content":"\u003cp\u003eSonodynamic therapy (SDT), features with non-ionizing radiation, high tissue-penetration (5\u0026ndash;10 cm) and high spatiotemporal controllability, has emerged as a promising treatment modality for solid tumors [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. During the typical process of SDT, ultrasound (US) locally activate sonosensitizers to generate a large number of reactive oxygen species (ROS), such as hydroxyl radical (\u0026middot;OH) and singlet oxygen (\u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e), resulting in apoptosis or necrosis of cancer cells [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In addition to directly disrupting primary tumors, SDT can also noninvasively elicit systemically antitumor immune response to prevent metastasis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. During SDT, the generation of ROS induces immunogenic cell death (ICD) of cancer cells through the release of tumor-associated antigens (TAAs) and damage-related molecular patterns (DMAPs). The released TAAs and DMAPs serve as immune adjuvants to prompt maturation of dendritic cells (DCs), subsequently activating cytotoxic T cells to cause cancer cell death.\u003c/p\u003e \u003cp\u003eIt is worth noting that the therapeutic and immunogenic effects of SDT are primarily determined by sonosensitizers, which are irreplaceable elements for inducing cancer cell death under US irradiation [\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Traditional organic sonosensitizers, including porphyrin, chlorophyll and curcumin, possess high biodegradability and excellent sonosensitivity, have been performed to destroy cancer cells by the US irradiation-mediated ROS production in vitro and in vivo [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, these organic sonosensitizers with poor hydrophilicity and low tumor-targeting ability show insufficient accumulation, internalization and retention in cancer cells and tumor tissue, thus dramatically impairing the therapeutic effects of SDT [\u003cspan additionalcitationids=\"CR16 CR17 CR18 CR19\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. To solve these problems, various nanocarriers, including polymeric micelles, nanoliposomes and inorganic nanoparticles, are developed to improve the concentration of organic sonosensitizers in tumor [\u003cspan additionalcitationids=\"CR22 CR23 CR24 CR25\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Although sonosensitizers loaded in nanocarriers have improved the antitumor effect of SDT to a certain degree in comparison to direct application of free sonosensitizers, accumulating evidence suggested that encapsulated sonosensitizers are not conducive to efficient SDT [\u003cspan additionalcitationids=\"CR28 CR29 CR30 CR31\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The hydrophobic sonosensitizers in nanocarriers lead to aggregation-caused fluorescence quenching (ACQ) effect, impairing the generation of ROS when irradiated with US [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. On contrary to the traditional organic sonosensitizers, it has been demonstrated that aggregation-induced emission luminogens (AIEgens) exhibited potent sonosensitivity in aggregated state [\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. AIEgens encapsulated in polymeric micelles and biomimetic nanocarriers could generate a considerable concentration of ROS under US irradiation and exhibited commendable performance in mediating cancer SDT. However, these AIEgens-loaded nanoparticles suffer from premature drug release and lack of tumor-targeting, significantly restricting their clinical application. Recently, many studies have shown that nanomedicines consisting of amphiphilic polymer-drug conjuncts could be excellent drug carriers for inhibiting cancer growth owing to higher systemic stability, fewer premature drug leakage and more easily tumor-targeting modification in comparison with traditional nanomedicines [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Therefore, tumor-targeted AIE polymeric micelles consisted of amphiphilic polymer-AIEgens conjuncts have been widely applied for tumor therapy, e.g., photodynamic therapy, and achieved substantial therapeutic benefits against cancer [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. However, there is no AIE polymeric micelles have been reported to mediate SDT.\u003c/p\u003e \u003cp\u003eIn our laboratory, a salicylaldazine-based amphiphilic polymer (AIE-1) was prepared and assembled into sub-20 nm AIE micelles (AIE-M) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Based on this, we wondered if tumor-targeted AIE polymeric micelles composed of AIE-1 and tumor-targeted polymers might be able to become an effective AIEgens carrier for mediating cancer SDT. In this work, we developed highly stable AIE polymeric micelles (AIE/Biotin-M) to mediate immunogenic SDT for inhibiting 4T1 tumor growth and metastasis. As depicted in Fig.\u0026nbsp;1, AIE/Biotin-M was prepared from the assembly of AIE-1 and 4T1 tumor-targeting amphiphilic polymers (DSPE-PEG-Biotin). Our results demonstrated that AIE/Biotin-M is an excellent sonosensitizer with high stability and sonosensitivity. Under US irradiation, AIE/Biotin-M showed negligible fluorescence decay and generated large amounts of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e. In vivo, AIE/Biotin-M actively accumulated in 4T1 tumor cells and triggered cells death under US irradiation. The dying cancer cells elicited ICD with exposure of calreticulin (CART) and release of high mobility group box 1 protein (HMGB1), which subsequently stimulated maturation of DCs and induced systemically antitumor immune response to inhibit 4T1 tumor growth and metastasis. Meanwhile, AIE/Biotin-M could inhibit neovascularization through chelating and reducing Fe\u003csup\u003e3+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e and Zn\u003csup\u003e2+\u003c/sup\u003e by salicylaldazine in tumor microenvironment, restricted tumor growth in combination with SDT.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003e1-Bromohexadecane (98%), 2,4-dihydroxybenzaldehyde (99%), Cs\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e (99%), methanesulfonyl chloride (98%), triethylamine (99.5%) and N\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO (98%) were purchased from TCI. Methoxypolyethylene glycol (mPEG\u003csub\u003e2000\u003c/sub\u003e) was obtained from Sigma-Aldrich and dried in toluene by azeotropic distillation for further use. 5,5-Dimethyl-1-pyrroline N-oxide (DMPO, 98%) was purchased from Sigma-Aldrich. 2,2,6,6-tetramethylpiperidine (TEMP, 99%) were acquired from Innochem. AIE-1 was prepared and characterized as our previously described.\u003c/p\u003e \u003cp\u003eBreast-cancer cell line 4T1 were acquired and cultured with nutrient solution, and placed at 37 ℃ in a 5% CO\u003csub\u003e2\u0026minus;\u003c/sub\u003econtaining humid atmosphere. Beijing HFK provided female BALB/c mice (6\u0026ndash;9 weeks old) with a body weight of 18\u0026ndash;20 g. The mice were housed in a pathogen-free Animal Lab and subjected to experiments in accordance with the Animal Welfare Committee of the Animal Experiment Center of Wuhan University. The in vivo research was carried out on the subcutaneous 4T1 tumour model, which was generated by implanting 4T1 cells (150\u0026micro;L PBS, 3 \u0026times; 106 cells per mice) into right flank of mice.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMicelle Preparation And Characterization\u003c/h3\u003e\n\u003cp\u003eAIE-1 (50 mg) and DSPE-PEG\u003csub\u003e2000\u003c/sub\u003e-Biotin (0.5 mg) were dissolved in ultrapure water (5 mL) and treated with ultrasound for 15 min. The AIE-M was similarly prepared from AIE-1. The micelles solution was obtained and subsequently kept at 4\u0026deg;C for future use.\u003c/p\u003e \u003cp\u003eMicelles were analyzed using Zeta-sizer Nano ZS (DLS, Malvern Instruments Ltd., UK) to determine their hydrodynamic size and polydispersity index (PDI). While, the morphological analysis was conducted via Field emission transmission electron microscopy (TEM, JEM-100CXII 100 kV).\u003c/p\u003e \u003cp\u003eThe aggregation-induced emission (AIE) characteristics of AIE/Biotin-M was measured as follows: AIE-1 (10 mg, 4 \u0026micro;mol) and DPPC-PEG\u003csub\u003e2000\u003c/sub\u003e-Biotin (0.1 mg) were separately dissolved in DMSO (1 mL) to prepare stock DMSO solution. A 0.1 mL aliquot of the DMSO stock solution was transferred to a 15 mL volumetric flask. After the addition of an appropriate amount of DMSO to maintain the final concentration of AIE-1 at 20 \u0026micro;M, water was added dropwise to furnish DMSO/water mixtures with water fractions of 0\u0026ndash;99 vol%. Each sample was measured for fluorescence (excitation at 365 nm) after treated with ultrasound for 10 min.\u003c/p\u003e \u003cp\u003eThe ability of AIE/Biotin-M to chelate metal ions was determined as follows: A range of AIE/Biotin-M and AIE-M solution (20 \u0026micro;M) were separately prepared with additional CuCl\u003csub\u003e2\u003c/sub\u003e, Zn(ClO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO and FeCl\u003csub\u003e3\u003c/sub\u003e of different concentration (0\u0026ndash;20 \u0026micro;M) together, and measured by fluorescence measurement (excitation at 365 nm) after stirring 15 min at room temperature.\u003c/p\u003e\n\u003ch3\u003eThe Ultrasonic Stability Of Micelles\u003c/h3\u003e\n\u003cp\u003eThe solution of AIE-M and AIE/Biotin-M were separately irradiated with US (1 MHz, 2 Wcm\u003csup\u003e-2\u003c/sup\u003e) for 5 and 10 min. After US irradiation, the fluorescence intensity and size distribution of AIE-M and AIE/Biotin-M were separately measured by fluorescence measurement (excitation at 365 nm) and DLS.\u003c/p\u003e\n\u003ch3\u003eThe Sonosensitivity Of Micelles (Dup: Abstract ?)\u003c/h3\u003e\n\u003cp\u003eThe detection of ROS generation from AIE-M and AIE/Biotin-M upon US irradiation was carried out by an electron paramagnetic resonance (ESR) spectrometer. To determine the generation of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e, the solution of AIE-M and AIE/Biotin-M (100 \u0026micro;g/mL) were separately treated with US irradiation (2 Wcm \u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, 1.0 MHz, 50% duty cycle) for 1 min in the presence of TEMP (50 \u0026micro;M), followed by detection through ESR spectrometer. The process of determination the generation of \u0026middot;OH was same as \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e, but TEMP replaced with DMPO.\u003c/p\u003e \u003cp\u003eA fluorescence spectrophotometer was used to conduct a quantitative analysis of ROS production. To quantitative measure the generation of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e, the solution of AIE-M and AIE/Biotin-M (3950 \u0026micro;L, 100 \u0026micro;g/mL) were separately irradiated with US (2 Wcm \u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, 1.0 MHz, 50% duty cycle) for 5 min and 10 min in the presence of singlet oxygen sensor green (SOSG, 50\u0026micro;L, 0.3 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e mM), and the fluorescence intensity was then recorded at 500\u0026ndash;700 nm with 488 nm of excitation wavelength. The process of determination the generation of \u0026middot;OH was same as \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e, but the SOSG was replaced with terephthalic acid (TA, 10 \u0026micro;g/mL), and then the fluorescence intensity at 350\u0026ndash;500 nm was recorded at 325 nm of excitation wavelength.\u003c/p\u003e\n\u003ch3\u003eIntracellular Ros Production And Cellular Uptake\u003c/h3\u003e\n\u003cp\u003e4T1 cells were seeded in 2 mL DMEM on confocal dishes at a density of 1.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well for 24 h. Then, for 8 and 24 h, AIE-M and AIE/Biotin-M were incubated separately with 4T1 cells at 200 g/mL concentrations. Next, the 4T1 cells were washed thrice with PBS to remove unabsorbed micelles. After that, they were stained with Hoechst 33258 for 15 minutes in a cell incubator after being fixed with 4% formaldehyde. Subsequently, cells were washed with PBS and examined using confocal laser scanning microscopy (CLSM). The estimation of micelles fluorescence intensity in different 4T1 cell structures and average micelles fluorescence intensity in 4T1cells was carried out using Imaging J software (National Institute of Health, USA).\u003c/p\u003e \u003cp\u003eFor 12 h, 4T1 cells were seeded in 2 mL of DMEM at a density of 2.0 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well in confocal dishes to detect the generation of intracellular ROS. Then AIE-M and AIE/Biotin-M were separately incubated with 4T1 cells at 200 \u0026micro;g/mL concentration for 8 h. After the free micelles were removed from dishes, 4T1 cells were stained with DCFH-DA concentration of 2 \u0026micro;M for 15 min, followed by washing with PBS to removed free DCFH-DA. Finally, 4T1 cells were irradiated with US (1.0 MHz, 2 Wcm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, 50% duty cycle) for 1 min and 3 min and observed through CLSM. Imaging J software was used to calculate the fluorescence intensity of micelles in various 4T1 cell structures and the average fluorescence intensity of DCF in 4T1 cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetermination of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ein vitro\u003c/span\u003e \u003cb\u003ecytotoxicity\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMTT assay was used for the evaluation of the cytotoxicity of AIE-M and AIE/Biotin-M in the presence and absence of US. In 96-well plates, 4T1 cells were seeded with a density of 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well in cell incubator. After incubated for 24 h, AIE-M and AIE/Biotin-M were added separately and further incubated for 24 h. After removed free AIE micelles, 4T1 cells were irradiated with or without US (1.0 MHz, 2 W cm-2, 50% duty cycle) for 3 min. Then further subjected for 24 h incubated, DMEM was discarded and MTT solution was added into wells for 4 h. After that, each well was added 150 \u0026micro;L DMSO to replace the MTT solution. The microplate reader was then used to determine the cytotoxicity using the MTT assay, which relies on the absorbance value at 570 nm.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ein vitro\u003c/span\u003e \u003cb\u003eICD effects\u003c/b\u003e\u003c/p\u003e \u003cp\u003e4T1 cells were seeded at a density of 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells in confocal dishes for 24 h. Then AIE-M and AIE/Biotin-M at 100 \u0026micro;g/mL concentration were separately incubated for 8 h with 4T1 cells, and then irradiated with US (1.0 MHz, 2 Wcm\u003csup\u003e-2\u003c/sup\u003e, 50% duty cycle) for 3 min. For the detection of CART, US treated 4T1 cells were further incubated for 8 h and subjected to staining with individual primary antibodies against CART for 30 min. Finally, the exposure of CART was detected by CLSM. For the detection of HMGB1, US treated 4T1 cells were subjected to 12 h of further incubation followed by staining with individual primary antibodies against HMGB1 for 30 min. Finally, the exposure of HMGB1 was detected by CLSM.\u003c/p\u003e\n\u003ch3\u003eIntratumor Accumulation Of Micelles (Dup: Abstract ?)\u003c/h3\u003e\n\u003cp\u003eTo evaluate the intratumoral accumulation of micelles, AIE/Ce6-M and AIE/Ce6/Biotin-M (Ce6\u0026thinsp;=\u0026thinsp;2.5 mg kg-1) with similar average size as AIE-M and AIE/Biotin-M were separately injected intravenously into 4T1 tumor-bearing BALB/c mice. Using an in vivo imaging system (IVIS\u0026reg; spectrum, PerkinElmer), real-time fluorescence images (excitation 640 nm, emission 680 nm) were obtained at different intervals following injection. In the meantime, tumours were collected by sacrificing the mice at various time intervals after injection which were sliced, and imaged the green fluorescence intensity of AIE micelles by CLSM.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetection of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ein vivo\u003c/span\u003e \u003cb\u003eimmunogenic effect\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe tumour-bearing 4T1 mice with an 80 mm\u003csup\u003e3\u003c/sup\u003e tumour size were weighed and randomly assigned into six groups (n\u0026thinsp;=\u0026thinsp;9), including: (1) PBS, (2) US, (3) AIE-M, (4) AIE/Biotin-M, (5) AIE-M\u0026thinsp;+\u0026thinsp;US, (6) AIE/Biotin-M\u0026thinsp;+\u0026thinsp;US. After intravenously injected with AIE-M or AIE/Biotin-M (AIE-1\u0026thinsp;=\u0026thinsp;100 mg/mL) for 2 h, the tumors were irradiated with US (1.0 MHz, 2 Wcm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, 50% duty cycle) for 5 min. The treatment was conducted three times every three days. After 24 h, 48 h, 72 h of last treatment, serum was collected and analyzed by an enzyme-linked immunosorbent assay (ELISA) kit for IFNγ, IL6, TNFα. And after 48 h of last treatment, one part of mice were sacrificed and collected tumours and tumour draining lymph node (TDLN) for further analysis. The one part of tumours were sliced and applied for CART immunofluorescence detection. The other parts of the tumour were dissociated to produce a suspension of single cells. Several fluorochrome-conjugated antibodies were used to stain the harvested cells; these include PE-anti-CD86, FITC-anti-CD11c, APC-anti-CD80, PE-anti-CD4, FITC-anti-CD3, and APC-anti-CD8 for analysing DCs and T cells by flow cytometer.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIn vivo\u003c/span\u003e \u003cb\u003eSDT effect\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe 80 mm\u003csup\u003e3\u003c/sup\u003e tumour-bearing 4T1 mice were weighed and randomly divided into six groups (n\u0026thinsp;=\u0026thinsp;9), including: (1) PBS, (2) US, (3) AIE-M, (4) AIE/Biotin-M, (5) AIE-M\u0026thinsp;+\u0026thinsp;US, (6) AIE/Biotin-M\u0026thinsp;+\u0026thinsp;US. After intravenously injected with AIE-M or AIE/Biotin-M (AIE-1\u0026thinsp;=\u0026thinsp;100 mg/mL) for 2 h, the tumours were irradiated with US (1.0 MHz, 2 Wcm-2, 50% duty cycle) for 5 min. The treatment was conducted three times every three days. The volumes of tumours and body weights of mice were measured every two days. Standard equations were used to determine the relative tumour volumes (RTV), tumour inhibitory rates (TIR, %), and relative mouse weights. Tumour tissues and major organs, including the liver, lung, heart, spleen, and kidney, were collected after all of the mice were sacrificed 16 days after treatment. For histological analysis, the collected tumours and major organs were weighed and stored at 4\u0026deg;C. Histological analysis of the proliferating cell nuclear antigen (PCNA), haematoxylin and eosin (H\u0026amp;E), platelet endothelial cell adhesion molecule-1 (CD31), terminal transferase dUTP nick-end labeling (TUNEL) and vascular endothelial growth factor (VEGF) was performed on paraffin-embedded tumour tissue sections that had been fixed in 4% formaldehyde. Lung and liver were applied for analysis of micrometastases. One part of tumor tissue and major organs were cut into pieces and heated in a solution of concentrated nitric acid to form transparent solution for analysing the concentration of metal ions by inductively coupled atomic emission spectrometer (ICP-AES). At last, the other part of the major organs was sliced and stained for H\u0026amp;E histological analysis.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe Student t-test was used for all statistical analyses. For all analyses, *(p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), **(p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and ***(p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) were regarded as statistically significant. Data were expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results And Discussion","content":"\u003ch3\u003ePreparation And Characterization Of Micelles\u003c/h3\u003e\n\u003cp\u003eIn accordance with our previous reports, AIE-1 was synthesized and characterized [\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e]. The micelles AIE-M and AIE/Biotin-M were prepared through a simple ultrasonic treatment in the absence or presence of a tumor-targeting polymer (DSPE-PEG\u003csub\u003e2000\u003c/sub\u003e-Biotin). As depicted in Fig. 1A, the size distribution and average hydrodynamic diameter of AIE-M and AIE/Biotin-M, measured by DLS, were 19.2 nm and 20.5 nm with a narrow distribution (PDI\u0026thinsp;=\u0026thinsp;0.098 and 0.103), respectively. Moreover, Fig. 1B showed that after being stored at 4\u0026deg;C for a week, both AIE-M and AIE/Biotin-M remained stable, with only minor changes in size distribution. The morphologies and diameter sizes of AIE-M and AIE/Biotin-M were observed in a dried state and calculated with TEM and image J. As shown in Fig. 1C and D, AIE-M and AIE/Biotin-M in a dried state were spherical in shape with 18.3 and 20.2 nm of average diameters, respectively. It was demonstrated that compared with AIE-M, AIE/Biotin-M endowed with tumor-targeted element exhibited negligible changes in diameter size and shape. These results showed that AIE/Biotin-M was suitable for cancer therapy. Our previous work has demonstrated that AIE-1 has no fluorescence emission when dissolved in a good solvent, while exhibiting strong fluorescence emission in an aqueous solution. As depicted in Fig. 1E, the photophysical properties of AIE-1/DPPC-PEG\u003csub\u003e2000\u003c/sub\u003e-Biotin in DMSO indicated that without fluorescence emission was detected at the excitation of 365 nm. However, the fluorescence intensity of AIE-1/DPPC-PEG\u003csub\u003e2000\u003c/sub\u003e-Biotin in DMSO was gradually enhanced with the increase of water fraction, which is a typical characteristic of AIEgens.\u003c/p\u003e\n\u003cp\u003eTherefore, DPPC-PEG\u003csub\u003e2000\u003c/sub\u003e-Biotin in AIE/Biotin-M has negligible influence on the photophysical properties of AIE-M.\u003c/p\u003e\n\u003cp\u003eOur previous work has demonstrated that chelating groups in AIE-M could form a stable complex with Cu\u003csup\u003e2+\u003c/sup\u003e, which impaired the fluorescence emission of this probe [\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e]. In addition to chelation with Cu\u003csup\u003e2+\u003c/sup\u003e, we further found that AIE-M and AIE/Biotin-M could chelate with Fe\u003csup\u003e3+\u003c/sup\u003e and Zn\u003csup\u003e2+\u003c/sup\u003e. The fluorescence emission of AIE-M and AIE/Biotin-M gradually changed with the addition of Fe\u003csup\u003e3+\u003c/sup\u003e and Zn\u003csup\u003e2+\u003c/sup\u003e, which indicated the formation of metal ion complexes with salen groups in AIE-M and AIE/Biotin-M. Notably, the fluorescence intensity of AIE-M-Zn and AIE/Biotin-M-Zn aqueous solution were gradually enhanced and reached a plateau with the addition of equivalent Zn\u003csup\u003e2+\u003c/sup\u003e. And after addition of Fe\u003csup\u003e3+\u003c/sup\u003e, the fluorescence intensity of AIE-M-Fe and AIE/Biotin-M-Fe aqueous solution gradually decreased and without completely quenched after the addition of equivalent Fe\u003csup\u003e3+\u003c/sup\u003e (Additional file 1: Figure S1). Meanwhile, metal ions (Fe\u003csup\u003e3+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e) have negligible influence on the average size of AIE-M and AIE/Biotin-M, Fig.\u0026nbsp;1F. These results revealed that AIE micelles could efficiently chelate with multiple metal ions (Fe\u003csup\u003e3+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e) and form stable complexes, exhibiting potentials for multiple metal ions interference cancer therapy.\u003c/p\u003e\n\u003ch3\u003eThe Sonosensitivity Of Micelles\u003c/h3\u003e\n\u003cp\u003eTo explore the sonodynamic performance of AIE/Biotin-M, the ultrasonic stability of micelles was firstly detected. AIE-M and AIE/Biotin-M have negligible change in fluorescence emission intensity and average hydrodynamic size after separately irradiated with US (1 MHz, 2 Wcm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, 50% duty cycle) for 5 and 10 min (Fig. 2A-D), suggesting significant sono-stability of both AIE micelles. In the process of SDT, sonosensitizers irradiated with US can generate a large amount of ROS, such as \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e and \u0026middot;OH. Here, we utilized 2, 2, dimethylpyrroline-1-oxide (DMPO) to separately differentiate \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e and \u0026middot;OH in AIE micelles mediated SDT through electron spin resonance (ESR) technique. TEMP can efficiently capture \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e to yield 2, 2, 6, 6-tetramethylpiperidine-1-oxyl (TEMPO) and exhibit characteristic ESR signal of 1:1:1 triplet peak under static magnetic field. And DMPO can efficiently capture \u0026middot;OH to yield DMPO-\u0026middot;OH and display a typical ESR signal of 1:2:2:1 under a static magnetic field. As depicted in Fig. 2E, both AIE micelles with US (1\u003c/p\u003e\n\u003cp\u003eMHz, 2 Wcm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, 50% duty cycle) irradiation for 5 min exhibited a strong triplet peak of TEMPO indicated the generation of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e. AIE-M and AIE/Biotin-M without US irradiation showed weak triplet peaks of TEMPO. And AIE-M and AIE/Biotin-M are equal in the triplet peak intensity of TEMPO after irradiated with US, which demonstrated tumor targeting Biotin-polymers have no effect on the sonosensitivity of AIE/Biotin-M. Furthermore, there was no characteristic ESR signal of DMPO-\u0026middot;OH was detected when AIE-M and AIE/Biotin-M treated with or without US irradiation (Fig.\u0026nbsp;2F), indicating no production of \u0026middot;OH.\u003c/p\u003e\n\u003cp\u003eTo further qualitatively and quantitatively analyze ROS generation in AIE micelles mediated SDT, SOSG and TA were exploited to detect \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e and \u0026middot;OH, respectively. SOSG with no fluorescence emission can be oxidized by \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e to form SOSG-endoperoxide (SOSG-EN) and show strong fluorescence emission at 525 nm; TA with no fluorescence emission can be oxidized by \u0026middot;OH to form 2-hydroxyl terephthalic acid (TAOH) and show strong fluorescence emission at 435 nm. According to fluorescence emission (Fig. 2G-I) and Additional file 1: Figure S2, US (1 MHz, 2 Wcm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, 50% duty cycle) treated AIE-M and AIE/Biotin-M exhibited highly characteristic SOSG-EN fluorescence signal, while no TAOH fluorescence signal could be observed. Meanwhile, the efficient generation of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e positively corresponded to irradiation time, which was indicated by the SOSG-EN fluorescence signal increased over irradiation time. The ESR and fluorescence signal results demonstrated that AIE/Biotin-M could act as a highly efficient sonosensitizer to mediate sonodynamic cancer therapy by generating \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e under US irradiation \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\n\u003cdiv class=\"Section2\" id=\"Sec13\"\u003e\n \u003ch2\u003eCellular uptake and cytotoxicity\u003c/h2\u003e\n \u003cp\u003eThe tumor targeting ability of AIE/Biotin-M, which plays an important role in anti-tumor efficiency, was estimated towards 4T1 cells upon determination of intracellular AIEgens green fluorescence by confocal laser scanning microscope (CLSM). AIE-M and AIE/Biotin-M were separately treated with 4T1 cells for 8 h and 24 h. The green fluorescence of AIEgens in AIE-M and AIE/Biotin-M treated 4T1 cells were increased with coincubation time, Fig.\u0026nbsp;3A-C. Notably, the green fluorescence of AIEgens in AIE/Biotin-M treated 4T1 cells was higher than that in AIE-M treated cells. Quantitative analysis also showed that the green fluorescence of AIE micelles in AIE/Biotin-M treated 4T1 cells was 2.4-fold higher than that in AIE-M treated 4T1 cells after coincubation for 24 h. These results demonstrated that AIE/Biotin-M could effectively internalized by 4T1 cells with the high biotin receptor expression on cell membrane.\u003c/p\u003e\n \u003cp\u003eTheoretically, high intracellular concentration of AIEgens sonosensitizers has positive effect on the US-mediated ROS generation. To qualitatively measure the generation of ROS in cells, DCFH-DA was selected as an intracellular ROS detection probe. DCFH, which enzymatically hydrolyzed from DCFH-DA in 4T1 cells, was oxidized by ROS to form DCF with green fluorescence emission. 4T1 cells pre-treated with AIE-M and AIE/Biotin-M exhibited negligible DCF green fluorescence in the absence of US irradiation (Fig.\u0026nbsp;3D-F). After US (1.0 MHz, 2 Wcm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, 50% duty cycle) irradiation, both 4T1 cells pre-treated with AIE-M and AIE/Biotin-M exhibited DCF green fluorescence. And the DCF green fluorescence was increased with irradiation time, indicating intracellular generation of ROS was time-dependent in AIE micelles mediated SDT. Due to the high concentration of AIEgens in 4T1 cells pre-treated with AIE/Biotin-M, the DCF green fluorescence was stronger in AIE/Biotin-M treated cells than that in cells treated with AIE-M. Quantitative analysis demonstrated that after US irradiation, the average DCF green fluorescence intensity in 4T1 cells pre-treated with AIE/Biotin-M sharply increased 1.5-fold than that in 4T1 cells pre-treated with AIE-M. The results indicated that AIE/Biotin-M might show powerful performance on sonodynamic cancer therapy in vitro and in vivo.\u003c/p\u003e\n \u003cp\u003eDue to AIE/Biotin-M showing excellent performance on cellular internalization and \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generation, it can be speculated that AIE/Biotin-M have a significant effect on anti-tumor in vitro. The cytotoxicity of AIE/Biotin-M against 4T1 cells with or without US irradiation was evaluated by standard MTT assay. As shown in Fig. 3G, even at a high concentration of 200 \u0026micro;g\u0026middot;mL-1, neither AIE-M nor AIE/Biotin-M showed toxicity against 4T1 cells in the absence of US stimulation. Similarly, merely US irradiation has no toxicity to 4T1 cells. However, under US (1.0 MHz, 2 Wcm-2, 50% duty cycle) irradiation for 3 min, both AIE-M and AIE/Biotin-M significantly inhibited the growth of 4T1 cells in a concentration-dependent manner. Differently, the sonotoxicity of AIE/Biotin-M (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;63.1 \u0026micro;g\u0026middot;mL-1) against 4T1 cells was significantly higher than that of AIE-M (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;110.5 \u0026micro;g\u0026middot;mL-1), due to the excellent cellular endocytosis of AIE/Biotin-M. The results were consistent with the intracellular generation of ROS under US irradiation.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eThe\u003c/strong\u003e \u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003ein vitro\u003c/span\u003e \u003cstrong\u003eICD effects\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eNext, we investigated whether AIE/Biotin-M treated 4T1 cells could release DMAPs in the presence of US, such as CART and HMGB1, which are a crucial characteristic of ICD. CART, calcium-binding proteins in the endoplasmic reticulum (ER), exposed on the cell outer membrane act as an \u0026ldquo;eat me\u0026rdquo; signal, stimulating\u003c/p\u003e\n \u003cp\u003eDCs and macrophages to recognize and engulf the dying tumor cells and tumor debris. As shown in CLSM measurement (Additional file 1: Figure S3A), after irradiation with US (1.0 MHz, 2 Wcm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, 50% duty cycle) for 3 min, AIE-M and AIE/Biotin-M showed high CATR red fluorescence. Due to the effective intracellular endocytosis of AIE/Biotin-M, CATR red fluorescence in AIE/Biotin-M pre-treated 4T1 cells was higher than the 4T1 cells pre-treated with AIE-M. Other groups, such as only US, AIE-M, and AIE/Biotin-M without US irradiation exhibited negligible CATR red fluorescence intensity. HMGB1, a nuclear protein, could release from nuclear to cytoplasm and extracellular environment, stimulating inflammatory response and inducing DCs maturation. As depicted in Additional file 1: Figure S3B, upon US irradiation (1.0 MHz, 2 Wcm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, 50% duty cycle) for 3 min, 4T1 cells pre-treated with AIE/Biotin-M also induced a large amount of HMGB1 transferring from nuclei to cytoplasm, exhibiting higher red fluorescence intensity in cytoplasm than other groups. Upon US irradiation, the strong expression of CART and HMGB1 in AIE/Biotin-M treated cells may due to cancer cells damage and death induced by a pronounced amount of ROS generation. These results confirmed that AIE/Biotin-M mediated SDT could efficiently cause immunogenic cancer cell death \u003cem\u003ein vitro\u003c/em\u003e and exhibited potential in stimulating a systemically immune response \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eIntratumor Accumulation Of Micelles\u003c/h3\u003e\n\u003cp\u003eTo optimize the experimental parameters for cancer SDT \u003cem\u003ein vivo\u003c/em\u003e, this study applied a fluorescence imaging assay to track the \u003cem\u003ein vivo\u003c/em\u003e behavior of AIE/Biotin-M. Due to the absorbance wavelength of AIE micelles below 600 nm, suffering from poor tissue penetration, we prepared Ce6-loaded AIE micelles to monitor the tumor accumulation of AIE-M and AIE/Biotin-M through \u003cem\u003ein vivo\u003c/em\u003e real-time fluorescence imaging assay. The Ce6-loaded AIE/Ce6-M and AIE/Ce6/Biotin-M were prepared using the emulsification method. The average size of AIE/Ce6-M and AIE/Ce6/Biotin-M were quite consistent with that of AIE-M and AIE/Biotin-M (Fig. 4A). Therefore, Ce6-loaded micelles could be applied to explore the tumor accumulation of AIE-M and AIE/Biotin-M based on the fluorescence of Ce6. 4T1 tumor-bearing BALB/c mice were separately \u003cem\u003ei.v.\u003c/em\u003e injected with free Ce6, AIE/Ce6/Biotin-M and AIE/Ce6-M (Ce6 2.5\u003c/p\u003e\n\u003cp\u003emg/kg). At each time point (0\u0026ndash;8 h), the \u003cem\u003ein vivo\u003c/em\u003e fluorescence images were acquired after \u003cem\u003ei.v.\u003c/em\u003e injection (Fig. 4B). Not surprisingly, free Ce6 exhibited low accumulation in the tumor, showing weak fluorescence intensity at each time point (0\u0026ndash;8 h) after \u003cem\u003ei.v.\u003c/em\u003e administration. While AIE/Ce6/Biotin-M and AIE/Ce6-M showed relatively strong fluorescence intensity in tumor tissues at 2 h post-injection due to enhanced permeability and retention effect (EPR). Notably, Fig. 4C quantitative analysis illuminated that AIE/Ce6/Biotin-M exhibited the highest fluorescence intensity in the tumor site and long-circulation time due to the high expression of the biotin receptor on the surface of 4T1 cells. As we all known, higher concentration of AIEgens would result in strong efficiency of SDT. Therefore, it was possible for AIE/Biotin-M to show an excellent SDT effect \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eWe have demonstrated that the AIE micelles could chelate with the metal ions (Cu\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e3+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e) to enhance (Zn\u003csup\u003e2+\u003c/sup\u003e) or impair (Cu\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e3+\u003c/sup\u003e) the fluorescence emission of AIEgens. Therefore, we next explored the fluorescence change of AIE micelles in tumor tissue through excising tumor tissues at each time point (1\u0026ndash;4 h) after \u003cem\u003ei.v.\u003c/em\u003e injection of AIE-M and AIE/Biotin-M at AIE micelles dose of 100 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The excised tumor tissues were sliced and observed by CLSM. As depicted in Fig.\u0026nbsp;4D-E, although the metal-ions (Cu\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e3+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e) were abundant in tumor tissues, there were detectable AIE micelles green fluorescence in tumor biopsies after \u003cem\u003ei.v.\u003c/em\u003e injection for 1, 2, 3, 4 h. And at 2 h post-injection, AIE-M and AIE/Biotin-M exhibited high green fluorescence in tumor biopsies. Significantly, AIE/Biotin-M showed stronger green fluorescence than AIE-M in tumor biopsies at each time point (1\u0026ndash;4 h) after \u003cem\u003ei.v.\u003c/em\u003e injection, which was consistent with \u003cem\u003ein vivo\u003c/em\u003e fluorescence imaging. As we illustrated that the high AIEgens green fluorescence in cells could contribute to significant SDT efficiency. Therefore, metal ions (Cu\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e3+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e) in tumor tissues had negligible influence on AIE/Biotin-M mediated SDT efficiency when AIE micelles at \u003cem\u003ei.v.\u003c/em\u003e injection dose of 100 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. These results demonstrated that AIE/Biotin-M could not only chelate with multiple metal ions (Cu\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e3+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e) but also show excellent potential to mediate cancer SDT \u003cem\u003ein vivo.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe\u003c/strong\u003e \u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003ein vivo\u003c/span\u003e \u003cstrong\u003eICD effect and immune response\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEncouraged by \u003cem\u003ein vitro\u003c/em\u003e results, we further investigated whether AIE/Biotin-M could induce ICD and immune response upon US irradiation \u003cem\u003ein vivo\u003c/em\u003e. 4T1 tumour-bearing mice with tumour volume 80 mm\u003csup\u003e3\u003c/sup\u003e were randomly divided into six groups including PBS, PBS\u0026thinsp;+\u0026thinsp;US, AIE-M, AIE/Biotin-M, AIE-M\u0026thinsp;+\u0026thinsp;US and AIE/Biotin-M\u0026thinsp;+\u0026thinsp;US. Mice were intravenously injected with AIE-M and AIE/Biotin-M at AIE micelles dose of 100 mg/kg, once every 3 days for three times, respectively. Due to the high tumor accumulation of AIE-M and AIE/Biotin-M at 2 h post-injection, at this time point, tumors were treated with US (1 MHz, 2 Wcm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, 50% duty cycle) for 5 min or without US. After the last irradiation for 6 h, tumors were excised, sliced and stained for immunofluorescence imaging of CART and HMGB1. As depicted in immunofluorescence imaging (Additional file 1: Figure S4), 4T1 tumors treated with AIE-M and AIE/Biotin-M plus US irradiation showed strong CART exposure on the cell outer membrane. In line with \u003cem\u003ein vitro\u003c/em\u003e, 4T1 tumors treated with AIE/Biotin-M plus US irradiation induced the largest amount of CART exposure. Notably, without US irradiation, 4T1 tumors treated with AIE-M and AIE/Biotin-M slightly activated CART exposure may due to AIE micelles mediated metal ions (Cu\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e3+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e) depletion. Metal ions depletion in tumor tissues can reduce tumor angiogenesis, resulting in reducing/blocking the replenishment of nutrients for inhibition of tumor cells growth and metastasis. Consistent with CART, without US irradiation, 4T1 tumors treated with AIE-M and AIE/Biotin-M also slightly induced the release of HMGB1, which was demonstrated by the high signal of HMGB1. And 4T1 tumours treated with AIE/Biotin-M plus US irradiation induced the largest amount of HMGB1 release, which was demonstrated by the reduction of HMGB1 signal. These phenomena demonstrated that the combination of AIE/Biotin-M and US irradiation could potentiate the induction of tumor ICD, which would prominently activate immune response \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eThe DMAPs released from dying cancer cells could stimulate immature DCs to engulf tumor antigens and debris. Subsequently, the immature DCs migrate to the nearby lymph nodes, where they underwent maturation and exposed the processed pro-inflammatory cytokines to naive T cells, thus inducing a systemically innate immune response. Therefore, the antitumor innate immunity induced by the combination of AIE/Biotin-M and SDT was evaluated by analysing the maturation of DCs and secretion of pro-inflammatory cytokines. 4T1 tumor-bearing mice with tumor size of 80 mm\u003csup\u003e3\u003c/sup\u003e were \u003cem\u003ei.v.\u003c/em\u003e injection with AIE-M and AIE/Biotin-M at a concentration of 100 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, once every 3 days three times. Due to the high tumor accumulation of AIE-M and AIE/Biotin-M at 2 h post-injection, at this time point, tumours were treated with US for 5 min or without US. After the last US irradiation for 48 h, single-cell suspension of tumor-draining lymph nodes was stained with anti-CD86, anti-CD80 and anti-CD11c for FCM analysis. As depicted in Fig.\u0026nbsp;5A, AIE-M and AIE/Biotin-M without US irradiation just slightly promoted DCs maturation (CD80\u003csup\u003e+\u003c/sup\u003eCD86\u003csup\u003e+\u003c/sup\u003e). However, in the presence of US, AIE-M and AIE/Biotin-M exhibited a high percentage of DCs maturation. Additionally, AIE/Biotin-M plus US significantly promoted DCs maturation (15.0%), which was consistent with the release of HMGB1 and CART.\u003c/p\u003e\n\u003cp\u003eThe matured DCs could secrete pro-inflammatory cytokines, such as IFN-\u0026gamma;, TNF-\u0026alpha;, and IL-6. Although AIE-M and AIE/Biotin-M in the absence and presence of US irradiation cloud increase the TNF-\u0026alpha;, IFN-\u0026gamma;, and IL-6 levels (Fig. 5B) in peripheral blood of mice, AIE/Biotin-M plus US induced highest production of pro-inflammatory cytokines. In addition, the highest maturation of DCs and production of pro-inflammatory cytokines suggested that AIE/Biotin-M plus US could induce an immune response and showed great potential in antitumor immunity. Thus, single-cell suspension of tumours was stained with anti-CD8, anti-CD4 and anti-CD3 for FCM analysis of infiltrated T cells (Fig. 5C). In the absence of US irradiation, the percentage of tumor-infiltrating activated T cells (CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells) slightly elevated in AIE-M and AIE/Biotin-M, due to the metal ions chelation of AIE micelles. And the level of T cells was significantly increased in US treated AIE-M and AIE/Biotin-M. The highest tumor-\u003c/p\u003e\n\u003cp\u003einfiltrating T cells in AIE/Biotin-M plus US was consistent with the DC maturation and pro-inflammatory cytokines release. These results revealed that AIE/Biotin-M plus US could activate the adaptive immune response and may potentially enhance anti-tumor therapy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe\u003c/strong\u003e \u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003ein vivo\u003c/span\u003e \u003cstrong\u003etherapeutic effect\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEncouraged by the activation of systemically anti-tumor immune response by AIE/Biotin-M plus US, we further explored whether this combination could prevent breast cancer growth and metastasis in vivo. The mouse 4T1 tumor with poorly immunogenic, early metastasis and highly tumorigenic is analogous to human TNBC. Therefore, 4T1 tumor-bearing mice are regarded as an appropriate experiment model for simultaneously evaluating the therapeutic effect on inhibiting primary breast cancer growth and metastasis. 4T1 tumour-bearing mice with tumour volume 80 mm3 were randomly divided into six groups including PBS, PBS\u0026thinsp;+\u0026thinsp;US, AIE-M, AIE/Biotin-M, AIE-M\u0026thinsp;+\u0026thinsp;US and AIE/Biotin-\u003c/p\u003e\n\u003cp\u003eM\u0026thinsp;+\u0026thinsp;US. Mice were intravenously injected with AIE-M and AIE/Biotin-M at AIE micelles dose of 100 mg/kg, respectively. After i.v. injection for 2 h, tumors were treated with US (1 MHz, 2 Wcm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, 50% duty cycle) for 5 min or without US. As shown in Fig. 6A, merely US irradiation had no tumor inhibition compared to PBS treated mice, which validated that US irradiation has no damage to tissues. In the absence of US irradiation, AIE-M and AIE/Biotin-M showed slight tumor inhibition with average relative tumor volume (RTV) of =\u0026thinsp;8.5, 8.2 (Fig. 6A) and tumor inhibition rate (TIR) of =\u0026thinsp;33.1%, 36.3% (Fig. 6B) compared to PBS group due to the metal ions depletion in the tumor. Notably, in the presence of US irradiation, the generation of ROS and activation of immune response enhanced tumor inhibition of AIE-M with RTV\u0026thinsp;=\u0026thinsp;4.3 and TIR\u0026thinsp;=\u0026thinsp;64.4%. In the presence of US irradiation, Furthermore, the therapeutic ability of AIE/Biotin-M plus US irradiation on tumor tissues was conducted by histological, immunohistochemical, and immunofluorescent analysis of the tumor section at the end of therapy. To evaluate the apoptosis and/or necrosis, the excised tumors fixed, sectioned and stained to analyze H\u0026amp;E staining, TUNEL, and PCNA, Fig. 6F. The H\u0026amp;E staining results revealed the intact structure tumor cells in PBS and US treated tumors. Comparatively, tiny part of separated and sparse tumor cells was observed in AIE-M and AIE/Biotin-M group, which was lower than the AIE-M\u0026thinsp;+\u0026thinsp;US group. Notably, the most obvious damage of cells was observed in tumor tissue after being treated with AIE/Biotin-M plus US irradiation. Additionally, PCNA and TUNEL staining results further validated that AIE/Biotin-M plus US irradiation resulted in the weakest cell the TIR of AIE/Biotin-M was 1.3-fold higher than AIE-M due to tumor-targeting accumulation of AIE/Biotin-M triggered a strong generation of ROS and anti-tumor immunity. Meanwhile, weight measurements (Fig. 6C) and the photos (Fig. 6D) of tumors further validated that AIE/Biotin-M plus US effectively inhibited tumor growth. The six groups of mice were also measured for their body weights every two days, and no weight fluctuations were observed, demonstrating the biocompatibility of all treatments Fig. 6E. proliferation (weak red fluorescence in PCNA assay) and strongest cell apoptosis (strong green fluorescence in TUNEL assay). In addition to inhibition of tumor growth, reduction of tumor metastasis was also extraordinary significant in cancer therapy. As depicted in Fig. 6G, more significant amounts of tumor nodules were found in lungs and livers in PBS, US, AIE-M and AIE/Biotin-M than in AIE-M plus US irradiation, which was consistent with the histological examination. Compared to AIE-M plus US irradiation, AIE/Biotin-M plus US irradiation pronouncedly prevents liver and lung metastasis. Meanwhile, AIE/Biotin-M plus US irradiation could also result in the lowest vascular density and vascular endothelial growth factor (VEGF) in the tumor tissue (Additional file 1: Figure S5A). On the one hand, the generation of ROS during SDT could damage tumor vessels and VEGF. And the damage degree of vessel and VEGF was positively correlated with ROS production. On the other hand, AIE micelles with the ability of metal ions chelation could also inhibit angiogenesis and VEGF expression, which was demonstrated by the lowest concentration of Fe\u003csup\u003e3+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e and Zn\u003csup\u003e2+\u003c/sup\u003e in AIE/Biotin-M treated tumour tissues, Fig. 7B. Although AIE/Biotin-M could induce metal ion depletion in tumors, negligible metal ions depletion was detected in other organs including liver, heart, lung, spleen, and kidney, Fig. 7B. Therefore, no apparent damage of cells was observed in liver, heart, lung, kidney, and spleen after tumor-free mice were i.v. injection of AIE-M and AIE/Biotin-M for 16 days demonstrated high biocompatibility of AIE/Biotin-M (Additional file 1: Figure S5B).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, we firstly developed tumor-targeted AIE polymeric micelles (AIE/Biotin-M) for mediating immunogenic SDT. It was demonstrated that AIE/Biotin-M with high stability could actively accumulate into 4T1 tumor cells and cause no damage to mice. Upon US irradiation, AIE/Biotin-M efficiently damaged cancer cells and induced ICD of cancer cells through the local generation of ROS. The DMAPs and TAAs released from dying cancer cells induced maturation of DCs and activated antitumor immune response, systemically inhibiting tumor growth and metastasis. Meanwhile, the depletion of tumor metal ions (Fe\u003csup\u003e3+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e and Zn\u003csup\u003e2+\u003c/sup\u003e) by AIE/Biotin-M further enhanced the antitumor effect. In a word, this study offers a facile strategy to design AIE nanosonosensitizers for clinical application in the future.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eSDT: Sonodynamic therapy; AIE: Aggregation-induced emission;\u0026nbsp;AIEgens:\u0026nbsp;Aggregation-induced emission\u0026nbsp;luminogens; US: Ultrasound; ROS: Reactive oxygen species; \u0026middot;OH: hydroxyl radical; \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e: singlet oxygen; ICD: Immunogenic cell death; H\u0026amp;E: Hematoxylin and eosin stain; TUNNEL: TdT-mediated dUTP nick end labeling; DLS: Dynamic light scattering; DAMPs: Damage-associated molecular patterns; TEM: Transmission electron microscope; TAAs: Tumor-associated antigens; DCs: Dendritic cells; CART: Calreticulin; HMGB1: High mobility group box 1 protein; VEGF: Vascular endothelial growth factor.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful for the\u0026nbsp;National Natural Science Foundation of China\u0026nbsp;(52173137, 51873163,\u0026nbsp;82202132), Natural Science Foundation of Hubei Province (2021CFB055), Fundamental Research Funds for the Central Universities (2042021kf0153) and Youth Interdisciplinary Special Fund of Zhongnan Hospital of Wuhan University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSH: Designed the project; KD, YY, JL, KL, and HZ: Performed research; KD and YY: Analyzed data; KD and MW: Wrote the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (52173137, 51873163, 82202132), Natural Science Foundation of Hubei Province (2021CFB055), Fundamental Research Funds for the Central Universities (2042021kf0153) and Youth Interdisciplinary Special Fund of Zhongnan Hospital of Wuhan University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during the current study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Experimental Animal Welfare Ethics Committee Zhongnan Hospital of Wuhan University (approval number: ZN2021232).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no competing interests in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eDepartment of Radiology, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, China. \u003csup\u003e2\u003c/sup\u003eDepartment of Orthopedic Trauma and Microsurgery, Zhongnan Hospital of Wuhan University, Wuhan 430071, China. \u003csup\u003e3\u003c/sup\u003eDepartment of Ultrasound, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, China. \u003csup\u003e4\u003c/sup\u003eKey Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University, Wuhan 430072, China. \u003csup\u003e5\u003c/sup\u003eWuhan Research Center for Infectious Diseases and Cancer, Chinese Academy of Medical Sciences, Wuhan 430071, China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNotes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDenkert C, von Minckwitz G, Darb-Esfahani S, Lederer B, Heppner BI, Weber KE, Budczies J, Huober J, Klauschen F, Furlanetto J, et al. 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Polym Chem. 2015;6:5185\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"sonodynamic therapy, polymeric micelles, immunogenic cell death, aggregation-induced emission, immune response ","lastPublishedDoi":"10.21203/rs.3.rs-2330201/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2330201/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Aggregation-induced emission luminogens (AIEgens) exhibit potent sonosensitivity in nanocarriers compared with conventional organic sonosensitizers owing to the strong fluorescence emission in the aggregated state. However, premature drug leakage and ineffective tumor targeting of current AIE nanosonosensitizers critically restrict their clinical application.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eHere, an AIEgens-based sonosensitizer (AIE/Biotin-M) with excellent sonosensitivity was developed by assembling salicylaldazine-based amphiphilic polymers (AIE-1) and 4T1 tumor-targeting amphiphilic polymers (DSPE-PEG-Biotin) for the effective delivery of salicylaldazine to 4T1 tumor tissues, aiming to mediate immunogenic SDT. In vitro, AIE/Biotin-M were highly stable and generated plentiful singlet oxygen (\u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e) under ultrasound (US) irradiation. After AIE/Biotin-M targeted accumulation in tumor, upon US irradiation, the generation of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e not only leaded cancer cells death, but also elicited systemically immune response through causing immunogenic cell death (ICD) of cancer cells. In addition to mediate SDT, AIE/Biotin-M could chelate and reduce Fe\u003csup\u003e3+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e and Zn\u003csup\u003e2+\u003c/sup\u003e by salicylaldazine for inhibiting neovascularization in tumor tissues. Ultimately, AIE/Biotin-M systemically inhibited tumor growth and metastasis upon US irradiation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eThis study presents a facile approach to the development of AIE nanosonosensitizers for cancer SDT.\u003c/p\u003e","manuscriptTitle":"Tumor-targeted AIE polymeric micelles mediated immunogenic sonodynamic therapy inhibits cancer growth and metastasis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-12-05 20:25:45","doi":"10.21203/rs.3.rs-2330201/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4331bd88-3484-4033-8409-e6e89612f8e4","owner":[],"postedDate":"December 5th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-01-30T18:14:12+00:00","versionOfRecord":[],"versionCreatedAt":"2022-12-05 20:25:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2330201","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2330201","identity":"rs-2330201","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}