Molecular Engineering-Boosted High-Performance Sonosensitizer for Cancer Sonodynamic-Augmented Immunotherapy

Molecular Engineering-Boosted High-Performance Sonosensitizer for Cancer Sonodynamic-Augmented Immunotherapy | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL Aggregate This is a preprint and has not been peer reviewed. Data may be preliminary. 3 January 2025 V1 Latest version Share on Molecular Engineering-Boosted High-Performance Sonosensitizer for Cancer Sonodynamic-Augmented Immunotherapy Authors : Chao Fu , Wei Zhao , Xionglei Wang , Xia He , Yuting Yin , Jiayi Li , Qiyun Deng , Caihong Yan , Yuli Yin , Zhiming Wang 0000-0002-3047-3285 , and Rong Hu 0000-0002-3246-6468 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.173586982.23529830/v1 Published Aggregate Version of record Peer review timeline 484 views 254 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Immunogenic cell death (ICD) has been demonstrated as a reliable approach to improve therapeutic effect in cancer treatment by triggering antitumor immunity. However, the trigger of ICD based on chemotherapy and phototherapy meets the obstacles of serious side effect and poor penetration ability, which seriously impedes the therapeutic effect. The development of sonodynamic immunotherapy with the evoking of ICD presents high promise for cancer treatment with high efficacy. Herein, high performance aggregation-induced emission (AIE) sonosensitizer is constructed based on the engineering structure modulation for sonodynamic-augmented immunotherapy. By regulating the intermolecular interaction and pull-push electronic effect, sonosensitizer bearing AIE feature and amplified sono-sensitizing effect is developed. In addition, in vitro observation demonstrated that thiolate-substituted segment incorporation endows the molecules with enhanced cellular uptake efficiency and improved tumor cell eradication ability. More importantly, the developed sonosensitizer could efficiently evoke ICD upon the trigger of ultrasound, which allows for the efficient tumor eradication both at cellular level and in solid tumor. The inhibition of primary tumor and further boost systemic immunity response with the complete elimination towards the distant tumor is achieved. The investigation highlights the promise of utilizing AIE sonosensitizers in sonodynamic immunotherapy to conquer the current limitation of immunotherapy in solid tumor treatment. Molecular Engineering-Boosted High-Performance Sonosensitizer for Cancer Sonodynamic-Augmented Immunotherapy Chao Fu 1,# , Wei Zhao 1,# , Xionglei Wang 1 , Xia He 1 , Yuting Yin 1 , Jiayi Li 1 , Qiyun Deng 2 , Caihong Yan 3 , Yuli Yin *1 , Zhiming Wang 2 , Rong Hu *1 1 School of Chemistry and Chemical Engineering, University of South China, Hengyang, China. 2 Center for Aggregation-Induced Emission, AIE institute, State Key Laboratory of Luminescent Materials and Devices, Guangdong Provincial Key Laboratory of Luminescence from Molecular Aggregates, Center for Aggregation-Induced Emission, South China University of Technology, Guangzhou, PR China. 3 The Second Affiliated Hospital, Hengyang Medical School, University of South China, Hengyang, Hunan, China. # These authors contributed equally. Correspondence : Yuli Yin ( [email protected] ); Rong Hu ( [email protected] ) Keywords: organic sonosensitizers, molecular engineering, sonodynamic therapy, immunotherapy, aggregation-induced emission ABSTRACT Immunogenic cell death (ICD) has been demonstrated as a reliable approach to improve therapeutic effect in cancer treatment by triggering antitumor immunity. However, the trigger of ICD based on chemotherapy and phototherapy meets the obstacles of serious side effect and poor penetration ability, which seriously impedes the therapeutic effect. The development of sonodynamic immunotherapy with the evoking of ICD presents high promise for cancer treatment with high efficacy. Herein, high performance aggregation-induced emission (AIE) sonosensitizer is constructed based on the engineering structure modulation for sonodynamic-augmented immunotherapy. By regulating the intermolecular interaction and pull-push electronic effect, sonosensitizer bearing AIE feature and amplified sono-sensitizing effect is developed. In addition, in vitro observation demonstrated that thiolate-substituted segment incorporation endows the molecules with enhanced cellular uptake efficiency and improved tumor cell eradication ability. More importantly, the developed sonosensitizer could efficiently evoke ICD upon the trigger of ultrasound, which allows for the efficient tumor eradication both at cellular level and in solid tumor. The inhibition of primary tumor and further boost systemic immunity response with the complete elimination towards the distant tumor is achieved. The investigation highlights the promise of utilizing AIE sonosensitizers in sonodynamic immunotherapy to conquer the current limitation of immunotherapy in solid tumor treatment. Chao Fu and Wei Zhao contributed equally to this study This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. ©2024 The Author(s). Aggregate published by SCUT, AIEI, and John Wiley & Sons Australia, Ltd. Introduction Metastasis, as the significant hallmark of cancer, is responsible for the majority of cancer-related deaths worldwide [1]. Compared to the traditional therapeutic strategy of surgery, chemotherapy and radiotherapy, cancer immunotherapy has aroused increasing attention as emerging treatment modalities by mobilizing and enhancing immune response to avoid the metastasis and residue of cancer cells. The trigger of immunogenic cell death (ICD) as a promising and critical step of cancer immunotherapy has raised a significant issue [2-3]. The activation of ICD will result in the release of damage-associated molecular patterns (DAMPs), followed with the trigger of immune recognition and the promotion of dendritic cell maturation to initiate adaptive immunity. It has been reported that chemotherapy [4], photodynamic therapy (PDT) [5-7], photothermal therapy (PTT) [11-12] and radiotherapy [6] could induce ICD of cancer cells with the realization of tumor inhibition based on synergetic immunotherapy. However, radiotherapy and chemotherapy meet the high risk of side effect, limited the treatment efficacy. PDT and PTT have been widely employed for multiple malignancies treatment as non-invasive tools, while, the poor tissue penetration depth and limited therapeutic efficiency restrict the related development as ICD inducer [13]. The development of efficient approach to induce ICD for synergetic cancer immunotherapy is highly desirable. Sonodynamic therapy (SDT), as an emerging non-invasive cancer treatment, attracted extensive attention owing to the great superiority of penetrating ability [14]. Upon low-intensity ultrasound (US), sonosensitizers could generate reactive oxygen species (ROS) and cavitation, resulting in the cancer cell eradication. Particularly, increasing evidences have proved that SDT can trigger ICD for sonodynamic immunotherapy, which can address the limitation of poor penetrating ability of phototherapeutic approaches [15-18]. Given to the pivotal role of SDT, various sonosensitizers, including organic sonodynamic materials and inorganic ones, have been constructed in recent years [19-23]. And desirable tumor therapeutic effect was acquired by inducing ICD to realize synergetic immunotherapy, demonstrating that SDT presents high promise for synergistic treatments combined with immunotherapy. Nevertheless, the good processability and promising biosafety are highly desirable to meet the diverse demands during practical applications. Organic sonosensitizers offer the superiorities of good biocompatibility, desirable processability, excellent biodegradability and metabolism for diagnosis and therapeutic applications. Tremendous studies have been made to prepare organic sonodynamic materials with diverse structures and functions, including porphyrin derivatives, cyanine derivatives, chemotherapeutic drugs and conjugated polymers for sonodynamic immunotherapy [20, 24-35]. For example, Pu et al. reported a semiconducting polymer for deep-tissue activatable SDT strategy for precision cancer sonodynamic immunotherapy [36]. Kim et al. constructed an imiquimod-based sonosensitizer with glutathione activatable property for synergistic treatments of SDT and immunotherapy towards cancer with high selectivity [37]. Ding et al. prepared an aggregation-induced emission (AIE) sonosensitizers with the successful trigger of ICD for boosting antitumor immunotherapy [38]. Even though the reported organic sonosensitizers afforded desirable synergetic therapy effect, the ROS generation ability should be further improved to achieve efficient therapeutic behavior. Moreover, owing to the relatively hydrophobic structure, the majority organic sonosensitizers show poor cellular uptake efficiency, inhibiting the activation of ICD and tumor ablation effect. In this study, AIE sonosensitizers with amplified ROS generation capacity and enhanced cellular uptake effect were designed and established for tumor sonodynamic immunotherapy (Scheme 1). Based on the molecular engineering, the fabrication of D-π-A-π-D structure enables the developed molecules with accelerated intersystem crossing process and reduced intermolecular interaction, resulting in the amplified sono-sensitizing effect and AIE feature. In vitro results demonstrated that thiolate-substituted segment incorporation endows the molecules with enhanced cellular uptake efficiency and improved tumor cell eradication ability. Moreover, the different cellular uptake and sono-sensitizing behaviors of the developed sonosensitizers resulted in controllable inhibition pathway of ferroptosis and necrocytosis to ablate tumor cells. Finally, the efficient trigger of ICD was achieved both in vitro and in vivo based on the SDT treatments, and the inhibition of primary tumor and further boost systemic immunity response with the complete elimination towards the distant tumor, demonstrating the great potential for metastatic tumor treatment in practical. SCHEME 1. Illustration of sonodynamic immunotherapy based on AIE sonosenitizer. Results 2.1 Sonosensitizer construction and characterization To develop sonosensitizers with high ROS generation capacity and good biosafety, series of novel organic materials, named ffBT-EDOT, ffBT-TVT, and BSM-TVT, were synthesized by rational molecular design and structural modification (Figure 1a). In this design, difluoro-substituted (ffBT) was recommended as electron acceptor core to fabricate D-π-A-π-D conjugated architecture considering that the strong electron-withdrawing ability is advantageous to amplify ROS generation. In addition, tetraphenylethylene (TPE) worked as an electron donor and molecular rotor to guarantee AIE characteristics. FIGURE 1. (a) Chemical structures and molecular design. (b) Normalized absorption and emission spectra of ffBT-EDOT, ffBT-TVT and BSM-TVT in THF and their corresponding NPs in water. (c-e) AIE curves for ffBT-EDOT, ffBT-TVT, and BSM-TVT. (f) The absorption intensity of DPBF in the presence of ffBT-EDOT, ffBT-TVT and BSM-TVT. (g-i) Dynamic laser scattering (DLS) of ffBT-EDOT, ffBT-TVT and BSM-TVT NPs in water. (c-e) AIE curves for ffBT-EDOT, ffBT-TVT, and BSM-TVT. (f) The absorption intensity of DPBF in the presence of ffBT-EDOT, ffBT-TVT, and BSM-TVT. (g-i) Dynamic laser scattering (DLS) of ffBT-EDOT, ffBT-TVT, and BSM-TVT. Meanwhile, introducing appropriate π-bridge that link electron donor with electron acceptor unit could not only further redshift the absorption wavelength but facilitate triplet sensitization to accelerate the intersystem crossing process in the excited state, leading to superior photophysical property and sono-sensitization ability. Firstly, by using 3,4-ethylenedioxythiophene (EDOT) as π-bridge, ffBT-EDOT was developed. Based on π-bridge engineering, (E)-1,2-Di(thiophen-2-yl) ethene (TVT) was adopted to provide ffBT-TVT with reduced intermolecular interaction. To further enhance the intramolecular charge transfer effect, thiolate-substituted BT (BSM) bearing stronger electron-withdrawing group (EWG) was utilized to obtain BSM-TVT. The molecular structures of the targeted compounds have been fully characterized, and the experiment details are provided in the supporting information (Figure S1-S8). 2.2 Photophysical Properties The photophysical properties of the above molecules were subsequently studied. As shown in Figure 1b, the maximum absorption wavelengths of ffBT-EDOT, ffBT-TVT and BSM-TVT in THF solution were measured at 469, 530, and 552 nm, respectively, with respective molar coefficient value of 1.1 ×10 4 , 3.1×10 4 and 1.6×10 4 M−1 cm−1 (Figure S9). PL spectra suggested that ffBT-EDOT, ffBT-TVT and BSM-TVT displayed emission peaks at 640, 670, and 741 nm, respectively. The gradually red-shifted absorption and emission spectra indicated that both improving the electron-accepting ability of the core and regulating the π-bridge can efficiently optimize the D-A interaction, which is well consistent with our design principle. To improve the aqueous dispersion and reduce non-specific interactions, ffBT-EDOT, ffBT-TVT, and BSM-TVT nanoparticles (NPs) were prepared for in vitro and in vivo experiments, respectively. Compared with those molecules in THF solution, both NPs exhibited red-shifted absorption, consistent with the stronger π-π stacking in the aggregated state. Then, the luminescent behaviors of the three sonosensitizer in aggregated state were investigated in the THF/hexane mixture with different hexane fractions ( f h ). As shown in Figure 1c and Figure S10, ffBT-EDOT exhibited a typical ACQ phenomenon with the character of obviously decreased fluorescence intensity of the aggregates in comparison with the isolated molecules, suggesting that only twisted TPE rotor is not sufficient to achieve highly emissive aggregates. Interestingly, this f h -dependent fluorescence of ffBT-TVT with TVT bridge highlighted its silent emission in THF solution (in the molecular state) and strong NIR emission in the aggregation state (Figure 1d). Although BSM-TVT displayed a relatively weak AIE feature compared to that of ffBT-TVT due to the stronger twisted intramolecular charge transfer (TICT) effect, the desired AIE property can be ultimately retained (Figure 1e). To sum up, by virtue of the molecular engineering strategy, the molecules with TVT bridge demonstrated the ACQ-to-AIE transformation, which is an effective manner to obtain highly bright emitters for image-guided sonodynamic therapy. To confirm the potential therapeutic efficacy of the sonosensitizers NPs under US, DPBF was employed as an indicator to evaluate the extracellular ROS generation capacity. Generally, the singlet oxygen ( 1 O 2 ) could oxidate DPBF to an endoperoxide, in which the decreased absorption could monitor the 1 O 2 production. [39] As depicted in Figure 1f, the absorption of DPBF decreased significantly in the presence of sonosensitizers upon US trigger. Moreover, the generation efficiency of 1 O 2 was correlated with the gradually reduced band gap of the sonosensitizers and was in the sequence of ffBT-EDOT < ffBT-TVT < BSM-TVT. Notably, ffBT-TVT and BSM-TVT exhibited superior ROS generation ability compared to the commercially available sensitizer, Ce6, showing surprising SDT performance. In addition, dynamic laser scattering (DLS) revealed that the average hydrodynamic diameters of ffBT-EDOT, ffBT-TVT, and BSM-TVT NPs were about 44, 56, and 64 nm, respectively, which is optimal for accumulation in tumors derived from the enhanced permeability and retention (EPR) effect (Figure 1g-i). In order to gain insight into the structural information of these sonosensitizers, density functional theory (DFT) calculations were performed at the B3LYP/6-31 G(d) level using the Gaussian 09 program. According to Figure 2a, the optimized geometry of the ground state suggested ffBT-EDOT has a distorted configuration with a larger dihedral angle of 41.5o between ffBT core and EDOT bridge. After π-bridge engineering, the dihedral angle between acceptor core and TVT bridge in ffBT-TVT reached an astonishing 0.1° owing to the intramolecular F⋯H noncovalent interaction, far smaller than that of ffBT-EDOT. Furthermore, the acceptor core engineering destroyed the intramolecular conformational lock, which eventually increases this dihedral angle to 37.0° for BSM-TVT. Therefore, ffBT-TVT had the best molecular flatness and the highest molar extinction coefficient, consistent well with the experimental results (Figure S9). This result raises an intriguing question, namely, why these sonosensitizers displayed opposite photophysical properties by considering the more coplanar backbone would strengthen intermolecular π-π stacking interactions and nonradiative decay to empirically produce the ACQ effect? [40-42] To clarify the reasons, the molecular electrostatic potential surface maps (ESPs) and dipole moment of these molecules were calculated to help understand the molecular packing modes. For ffBT-EDOT, the positive electrostatic potential (red color) was mainly located on the EDOT bridge, while the negative electrostatic potential (blue color) was observed on the acceptor core, showing a distinct ESP difference between the electron donor and electron acceptor groups (Figure 2b). The larger ESP difference drives ffBT-EDOT to form stronger D-A interactions between the adjacent molecules, [43-45] which further endows ffBT-EDOT with ACQ characteristics. However, compared with ffBT-EDOT, the positive areas of ESP distribution and corresponding values of ffBT-TVT were reduced by π-bridge engineering, which indicated that the strong intermolecular interaction and the ACQ effect were largely weakened, mitigating emission quenching. Moreover, a much stronger electron acceptor substent can increase the electron density, leading to a stronger intermolecular π-π stacking interaction, which is highly consistent with the relatively weak AIE property of BSM-TVT. Meanwhile, the dipole moment of ffBT-TVT (0.43 Debye) was much smaller than that of ffBT-EDOT (5.27 Debye) and BSM-TVT (7.98 Debye) due to its flattened molecular configuration (Figure. S11). The higher dipole moments of ffBT-EDOT and BSM-TVT were expected to induce an enhanced dipole-dipole interaction, which could induce stronger intermolecular π-π stacking interactions and agree well with the calculated ESP results for DFT optimized molecules. Moreover, the electron clouds of the lowest unoccupied molecular orbitals (LUMOs) were found to be primarily distributed at the electron-withdrawing acceptor core, while the highest occupied molecular orbitals (HOMOs) were delocalized along whole molecular skeletons, indicating an obvious D-A interaction and inherent intramolecular charge-transfer feature (Figure 2c and 2d). Since ffBT-TVT and BSM-TVT containing TVT bridge have longer π-conjugation lengths than those containing EDOT bridge, the reduced LUMO-HOMO energy gap could be observed, which is expected to provide superior ROS-generating capacity. 2.3 In vitro imaging and in vitro sonodynamic efficiency. Given to the outstanding photophysical properties of the developed sonosensitizers, we further explored their bioimaging behaviors, and 4T1 cells were selected as the representative tumor cell lines. Bright fluorescence could be detected in cells treated by ffBT-EDOT, ffBT-TVT and BSM-TVT NPs, revealing the efficient uptake of 4T1 cells (Figure S12). Further colocalization analysis showed that the emission of ffBT-EDOT, ffBT-TVT, and BSM-TVT NPs merged well with Lysotracker with the Pearson correlation coefficients of 0.92, 0.84, and 0.65, respectively (Figure 3a), indicating the enrichment of lysosome. Furthermore, the cellular uptake efficiency of these sonosensitizers was evaluated by examining the related absorption of intracellular components. As exhibited in Figure 3b, a significant enhancement in the cellular uptake of BSM-TVT was observed compared to that of both ffBT-TVT and ffBT-EDOT. Moreover, BSM-EDOT with similar acceptor moiety was constructed, which also exhibited enhanced cellular uptake efficiency (Figure S13). The combined results indicated that the incorporation of thiolate-substituted segment could enable the improved cellular affinity and uptake efficiency. FIGURE. 2 ( a) Optimized ground -state geometries. (b) ESP maps. (c-d) Calculated LUMOs and HOMOs of ffBT-EDOT, ffBT-TVT, and BSM-TVT. Benefiting from the ultra-strong sono-sensitizing ability, the intracellular ROS level of 4T1 cells treated with these nanoparticles in the absence and presence of US irradiation was investigated. As shown in Figure 3c and Figure S14, neither sonosensitizers treatments nor US irradiation would cause the ROS expression inside cells, since none obvious fluorescence of DCFH could be detected. Meanwhile, bright green fluorescence was observed inside cells treated with these three nanoparticles under US irradiation, proving the ROS generation under sonodynamic treatments and presenting the high promise for SDT applications. FIGURE. 3 (a) Colocalization images of 4T1 cells co-stained by ffBT-EDOT, ffBT-TVT and BSM-TVT NPs with Lyso-tracker. [Lyso-Tracker Blue] = 150 mM, λex = 405 nm, λem = 429~474 nm. (b) Absorption of intracellular components of ffBT-EDOT, ffBT-TVT and BSM-TVT. (c) Intracellular ROS generation (green fluorescence) of 4T1 cells with different treatments. (d-f) Cell viabilities of 4T1 cells treated different NPs SDT with Fer-1 and RIPI. (g) Levels of MDA after different nanoparticle treatments. (h) TEM of ffBT-TVT and BSM-TVT NPs with and without US. (i) Necroptosis detection kits for BSM-EDOT, BSM-TVT NPs and necroptosis inducer treated with and without US conditions. Hence, the cell viability of 4T1 cells treated by the sonosensitizer before and after US irradiation were determined via typical methyl thiazolyl tetrazolium (MTT) assay. As depicted in Figure 3d-3f, all these nanoparticles exhibited good biocompatibility with ignorable effect the cell growth. However, the cell viability markedly decreased upon the these sonosensitizers-based SDT treatment, demonstrating the perfect cell ablation effect of the developed strategies. Furthermore, the inhibitors of fer-1 and RIPI were utilized to discriminate the inhibition pathway of ferroptosis and necrocytosis during SDT treatment, respectively. It should be noted that these two inhibitors showed faint effect on growth of 4T1 cells (Figure S15). While, the cell viability of 4T1 cells treated by both ffBT-EDOT and ffBT-TVT was apparently recovered in the presence of fer-1, and none detectable recovery could be observed with the treatment of RIPI (Figure S16), which proved the eradication of tumor cells by ffBT-EDOT- and ffBT-TVT-dependent SDT mainly relied on the ferroptosis pathway. Meanwhile, the incubation of fer-1 exhibited faint effect on the growth of BSM-TVT-treated 4T1 cells. However, the remarkable recovery was observed in the cells treated by BSM-TVT with the treatment of RIPI, indicating the necrosis pathway played the critical role in the growth inhibition of tumor cells based on BSM-TVT-dependent SDT approach. The massive generation of ROS will cause the cellular peroxidation, followed with the activation of ferroptosis. Therefore, we first examined the expression level of malonaldehyde (MDA), one type of lipid peroxides, in cells with ffBT-EDOT and ffBT-TVT-based SDT treatments to verify the inhibition mechanism. Herein, cells treated with erastin, known as the ferroptosis inducer, were set as the positive control group. As show in Figure 3g, significant increase in the level of MDA was detected for cells treated with ffBT-EDOT NPs and ffBT-TVT NPs in the presence of US irradiation, and the obvious enhancement of MDA was also observed in cells incubated with erastin. The results demonstrated that ffBT-EDOT and ffBT-TVT-based SDT resulted in the accumulation of lipid peroxides. In addition, biological transmission electron microscopy (Bio-TEM) revealed that the mitochondria morphology of cells with SDT treatments collapsed significantly, which demonstrated the presence of the intracellular oxidative stress (Figure 3h). The above results verified that ffBT-EDOT and ffBT-TVT-based SDT treatments could inhibit the growth of tumor cells via causing oxidative stress and inactivating mitochondrial to trigger ferroptosis. In addition, necrotic staining of Hoechst 33342 and propidium iodide (PI) was used to explore the presence of cellular necroptosis in the BSM-TVT -treated cells, and the necroptosis inducer was employed as the positive control group. As shown in Figure 3i, in the absence of US irradiation, none red fluorescence of PI could be tested inside 4T1 cells incubated with BSM-TVT. Nevertheless, the fluorescence intensity of PI was significantly improved inside BSM-TVT-treated cells after exposing towards US to trigger SDT treatment, which further proved that necroptosis was the main pathway that inhibited the growth of 4T1 cells based on BSM-TVT-based SDT strategy. To facilitate the observation of the therapeutic effect, Calcein AM was employed to distinguish the live (green) cells after the SDT treatments. As shown in Figure S17, bright green fluorescence could be detected inside cells treated by sonosensitizers, which was significantly decreased after the US irradiation. The results were well consistent with the cell viability observation, verifying the effective therapeutic effect of the SDT treatment based on these sonosensitizers. Combined with the sono-sensitization ability and the cellular uptake efficiency observation of the sonosensitizers, we can figure out that the ffBT-EDOT and ffBT-TVT with moderate ROS generation ability under US will trigger ferroptosis to realize tumor cell ablation, mean-while, BSM-TVT possessing ultra-strong sono-sensitizing effect and improved cellular binding ability can eradicate tumor cells by activating necrocytosis pathway. FIGURE. 4 CLSM images of 4T1 cells with immunofluorescence staining of (a) HMGB1 (green fluorescence) and (b) CRT (green fluorescence) after different treatments. Increasing evidence have proved that SDT can effectively induce immunogenic cell death (ICD) of tumor cells to activate immunotherapy. Given to the promising SDT effect, we further investigated the potential of ffBT-EDOT, ffBT-TVT and BSM-TVT in immunotherapy. Upon the trigger of ICD, damage associated molecular patterns (DAMPs) will be released and further bind to anti-gen-presenting cells (e.g., dendritic cell DC cells), recognizing and phagocytosing dead cell antigens. Moreover, adaptive immune responses will be activated. The presence of high expression of HMGB 1 protein and the CRT on the cell membrane are typical feature of ICD, which will enhance the uptake of tumor antigens and anti-body expression by DCs to further activate the immune response. As shown in Figure 4a, based on immunofluorescence analysis, only weak green fluorescence was observed in cells treated with ffBT-EDOT, ffBT-TVT, and BSM-TVT NPs, suggesting these sonosensitizers will induce the expression of HMGB1 to some extent. Meanwhile, increased expression of HMGB1 was obtained in cells treated with the sonosensitizers after exposure to US, suggesting the upregulating expression of HMGB1 under SDT treatment. In addition, the expression of CRT was investigated as well, and the similar phenomenon was observed compared to that of HMGB1 (Figure 4b). The combined results verified that the developed sonosensitizers could provoke the ICD of tumor cells, which would further activate the immune response for sonodynamic immunotherapy applications. 2.4 In vivo tumor suppression Based on the satisfactory therapeutic effects and the successful trigger of ICD at the cellular level, we further explored the associated in vivo tumor suppression capabilities. As shown in Figure 5a, a mouse model was constructed by subcutaneously injecting 4T1 cells bilaterally into the buttocks of female BALB/c mice on days -8 and -1, respectively. Herein, the first and second inoculations were utilized to establish the primary and distant tumors, respectively. The body weight monitoring revealed that there was no significant variation in the body weight of mice in all experimental groups compared with the blank group, indicating that neither nanoparticles nor SDT treatments would cause any damage to mice (Figure 5b). In vivo fluorescence monitoring was performed to optimize the appropriate accumulation and distribution of the sonosensitizers. As shown in Figure 5c and Figure 5d, the fluorescence of ffBT-EDOT NPs in the tumor region gradually enhanced and reached the maximum at 24 h after systemic administration, which indicated that the enrichment of ffBT-EDOT in the solid tumors was ideal based on the enhanced permeability and retention (EPR) effect. The similar phenomenon and trend were observed in mice injected after the systemic administration of ffBT-TVT and BSM-TVT NPs (Figure 5e-h), in which the maximum fluorescence could be obtained in the tumors after 84 h and 30 h, respectively. Thereafter, the mice were sacrificed and the distribution of nanoparticles in major organs and tumor tissues was investigated by measuring the fluorescence intensity of ffBT-EDOT, ffBT-TVT, and BSM-TVT NPs after tail vein injection. By analyzing the fluorescence intensity (Figure 5i), it was found that the nanoparticles were mainly accumulated in tumors and liver. These results confirmed the high selectivity of the sonosensitizers for solid tumors in vivo. Then, to evaluate the in vivo tumor inhibition effect on both primary and distant tumors, tumor growth of mice was monitored for 15 days in mic with different treatments. As shown in Figure 5k, sonosensitizers treatment alone presented faint inhibition effect towards solid tumors, while, significant inhibitory behavior was achieved with the systemic administration of sonosensitizers upon US irradiation. It should be noted that complete ablation of tumors could be acquired for mice with BSM-TVT-based SDT treatment. Moreover, compared to blank group, the significant inhibition effect towards the distant tumor could be observed for SDT-treated mice (Figure 5l), which was owing to the successful trigger of ICD. Meanwhile, the most efficient inhibition efficacy was achieved in mice treated by BSM-TVT-based SDT due to the enhanced cellular uptake efficiency and sono-sensitizing effect. Besides, the results displayed that sonosensitizers-treated mice presented slight suppression behavior towards distant tumor growth, benefiting from the related trigger of ICD to some extent. Afterwards, both primary and distant tumors were collected and weighed (Figure 5j), which showed a similar trend to the that of tumor volume with different treatments. FIGURE.5 (a) Treatment schedule for in vivo antitumor study. (b) Body weight of 4T1-tumor-bearing mice with different treatments. (c-h) In vivo fluorescence imaging and intensity of primary tumor in 4T1-tumor-bearing mice after intravenous injection of ffBT-EDOT, ffBT-TVT and BSM-TVT NPs. (i) In vivo fluorescence imaging of dissected main organs and primary tumors of the mice sacrificed post intravenous injection of ffBT-EDOT, ffBT-TVT and BSM-TVT NPs. (j) Excised primary and distant tumor after different treatments. (I: Sterilized PBS without US. II: Sterilized PBS with US for 5 min. III: ffBT-EDOT NPs without US. IV: ffBT-EDOT NPs with US for 5 min. V: ffBT-TVT NPs without US. VI: ffBT-TVT NPs with US for 5 min. VII: BSM-TVT NPs without US. VIII: BSM-TVT NPs with US for 5 min, n=5). (k-l) Relative primary and distant tumor volume changes of 4T1 tumor-bearing mice with different treatments, n=5. (m) CD4+CD8+ T cells in spleen after different treatments, n=3. To essentially clarify the underlying mechanisms of anti-tumor and anti-metastatic efficacy, the immune mechanism of SDT-mediated immunotherapy was performed based on immunological analysis of the activated immune cells. Different types of T cells in spleen were investigated by determining the corresponding biomarkers of CD3, CD4 and CD8 via flow cytometry. CD3 + CD4 + and CD3 + CD8 + T lymphocytes, acting as T-helper lymphocytes and T-suppressor cells, play important roles in immune system. Take CD4 + CD8 + T lymphocytes as example, it will generate a message of confrontation to the immune system and play a pivotal role in the immune response. As depicted in Figure 5m, CD4 signal increased slightly in ffBT-EDOT, ffBT-TVT, and BSM-TVT NPs groups in the absence of US irradiation. While, the percentage of CD3 + CD4 + T lymphocytes increased from 57.4% to 65.0% based on the ffBT-EDOT-based SDT treatment compared to the blank group. And the similar trend could also be detected in the spleen of mice bearing ffBT-TVT and BSM-TVT-based SDT treatments with the percentage CD3 + CD4 + T lymphocytes of 75.6% and 66.6%, respectively. In contrast, CD8 signal in sonosensitizer-treated mice without US irradiation showed negligible change compared to blank ones. While, the percentage of CD3 + CD8 + T lymphocytes decreased from 39.6% to 24.0% for mice with ffBT-EDOT-based SDT treatment, meanwhile, the percentage of CD3 + CD8 + T lymphocytes in ffBT-EDOT and BSM-TVT-based SDT treated mice decreased to 15.4% and 25.0%, respectively. The remarkably enhanced in the expression of CD3+CD8+ T lymphocytes upon the trigger of SDT demonstrated the formation of helper T cells and activation of immune response, showing great promise for immunotherapy. Based on the excellent therapeutic efficacy, hematoxylin-eosin (H&E) and immunofluorescent staining were employed to reveal the detail mechanism of cell death in primary and distant tumors based on SDT treatments. Herein, TdT-mediated dUTP nick end labeling (TUNEL) staining was performed, and the results showed that only massive apoptotic cells could be observed in the primary tumors for mice treated with ffBT-EDOT, ffBT-TVT, and BSM-TVT NPs-based SDT treatments, whereas no significant cell death was detected in the other groups, including distant tumors with different treatments (Figure S18). Finally, the in vivo biosafety was further verified. H&E staining of major organs was examined and no damage was detected (Figure S19). We also assessed the functional biomarker levels of albumin (ALB), alkaline phosphatase (ALP) of liver, and blood urea (UREA) of kidney based on routine analysis and biochemical analysis. The results revealed that the tested parameters in all experimental groups were generally consistent with those of blank groups (Figure S20). These results suggest that this SDT strategy has an outstanding tumor suppression effect and a good biosafety. 3.Conclusion In summary, our study sheds light on the construction of AIE sonosensitizer for sonodynamic-immunotherapy. By adjusting the electron-donating capability of π bridges to inhibit π-π stacking, the developed sonosensitizers bearing TVT as π bridge shared the desired AIE feature. Moreover, the incorporation of thiolate-substituted acceptor core enables the development of sonosensitizers to further improve ROS generation ability. Our findings reveal that compared to ffBT-EDOT and ffBT-TVT, BSM-TVT facilitates the enhanced cellular uptake efficiency and improved ultrasonic-trigger ROS generation, underscoring powerful sonodynamic impact towards tumor cells with the eradication of tumor cells via ferroptosis pathway. 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Collection Aggregate Keywords aggregation-induced emission immunotherapy molecular engineering organic sonosensitizers sonodynamic therapy Authors Affiliations Chao Fu University of South China View all articles by this author Wei Zhao University of South China View all articles by this author Xionglei Wang University of South China View all articles by this author Xia He University of South China View all articles by this author Yuting Yin University of South China View all articles by this author Jiayi Li University of South China View all articles by this author Qiyun Deng South China University of Technology View all articles by this author Caihong Yan University of South China Hengyang Medical School View all articles by this author Yuli Yin University of South China View all articles by this author Zhiming Wang 0000-0002-3047-3285 South China University of Technology View all articles by this author Rong Hu 0000-0002-3246-6468 [email protected] University of South China View all articles by this author Metrics & Citations Metrics Article Usage 484 views 254 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Chao Fu, Wei Zhao, Xionglei Wang, et al. Molecular Engineering-Boosted High-Performance Sonosensitizer for Cancer Sonodynamic-Augmented Immunotherapy. Authorea . 03 January 2025. DOI: https://doi.org/10.22541/au.173586982.23529830/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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