[摘 要] 目的：构建纳米囊泡杂化透明质酸水凝胶（ICG-NV@SeHA）并探讨其联合声动力疗法（SDT）杀伤小鼠胶质瘤GL261 细胞的机制。方法：通过挤出法制备骨髓来源的间充质干细胞纳米囊泡（BMSC-NV），再将吲哚菁绿（ICG）掺入其中制备 ICG-NV。在EDC存在下用ED对HA进行氨基化，合成AHA，进一步通过亲核加成反应与γ-硒代丁内酯（SBL）连接，合成SeHA。 将AHA、ICG-NV和SBL 溶液混合，发生氧化交联获得ICG-NV@SeHA，对其进行物理表征。用DiD标记ICG-NV和 ICG-NV@SeHA后与GL261细胞共培养12 h，观察细胞内吞情况。用CCK-8法检测ICG-NV和ICG-NV@SeHA与GL261细胞和 小鼠海马神经元HT22细胞的生物相容性。将GL261细胞分为PBS + 超声处理（US）、ICG + US、IVG-NV + US和ICG-NV@SeHA + US组，Calcein-AM/PI染色法和DCFH-DA荧光探针标记法分别检测联合SDT对GL261细胞杀伤作用，以及对细胞内活性氧 （ROS）生成的影响；采用细胞免疫荧光实验检测对细胞表面钙网蛋白（CRT）表达的影响，酶联免疫吸附试验（ELISA）检测对细胞 高迁移率族蛋白1（HMGB1）和三磷酸腺苷（ATP）释放的影响。结果：成功制备BMSC-NV，粒径约154.3 nm；ICG成功被包入囊 泡中，包封率为40.6%；氨基成功连接在HA上，接枝率为32.5%。最后成功制备了ICG-NV@SeHA，透射电镜观察显示其具有疏 松的多孔结构，流变结果储能模量（G’）＞损耗模量（G”），均符合水凝胶特性，且具有剪切变稀特性。细胞实验结果显示，ICG NV可以被GL261细胞有效摄取。CCK-8实验和Calcein-AM/PI荧光染色实验结果显示，ICG-NV和ICG-NV@SeHA均具有良好 的生物相容性，对GL261和HT22细胞没有明显的细胞毒作用；而ICG-NV + US和ICG-NV@SeHA + US组细胞存活率较 ICG+US组显著降低（P < 0.01或P < 0.001）。ICG-NV + US和ICG-NV@SeHA + US组细胞DCFH-DA探针绿色荧光强度显著高于 PBS、PBS + US和ICG + US组（P < 0.000 1或P < 0.001），反映细胞内产生大量的ROS，且细胞表面CRT表达显著增加（P < 0.000 1）， 上清液中HMGB1和ATP 的释放也增多（P < 0.05或P < 0.01）。结论：成功制备具备优异的机械性能和可注射性的 ICG-NV@SeHA，生物相容性好，联合SDT能有效杀伤GL261细胞并诱导免疫原性细胞死亡（ICD），可能成为一种有效治疗胶质 瘤术后复发的新手段。

[Abstract] Objective: The nanovesicles-hybridized selenized hyaluronic acid hydrogel (ICG-NV@SeHA) was constructed, and its mechanism of action in synergistically killing glioma GL261 cells in mice when combined with sonodynamic therapy (SDT) was systematically investigated. Methods: BMSC-derived nanovesicles (BMSC-NVs) were prepared via the extrusion method, followed by the incorporation of indocyanine green (ICG) to fabricate ICG-NV. Under the presence of EDC, hyaluronic acid (HA) was aminated using ethylenediamine (ED) to synthesize aminated HA (AHA), which was further conjugated with γ-selenobutyrolactone (SBL) via nucleophilic addition to form selenized HA (SeHA). AHA, ICG-NVs, and SBL solutions were mixed and oxidatively cross-linked to obtain ICG-NV@SeHA, followed by physical characterization. DiD-labeled ICG-NVs and ICG-NV@SeHA were co-cultured with GL261 cells for 12 h to observe cellular internalization. The biocompatibility of ICG-NVs and ICG-NV@SeHA with GL261 cells and mouse hippocampal neuronal HT22 cells was evaluated using the CCK-8 assay.GL261 cells were divided into four groups: PBS + ultrasound (US), ICG + US, ICG-NV + US, and ICG-NV@SeHA + US. Calcein-AM/PI staining and DCFH-DA fluorescent probes were employed to assess the synergistic SDT-induced cytotoxic effects on GL261 cells and intracellular reactive oxygen (ROS) generation, respectively. Cellular surface calreticulin (CRT) expression was analyzed via immunofluorescence, while enzyme-linked immunosorbent assay (ELISA) was used to measure the release of high mobility group box 1 (HMGB1) and adenosine triphosphate (ATP). Results: BMSC-NVs were successfully prepared with an average particle size of approximately 154.3 nm. ICG was efficiently encapsulated into the nanovesicles with an encapsulation efficiency of 40.6%. HA was successfully aminated, achieving a grafting rate of 32.5%. Ultimately, the ICG-NV@SeHA hydrogel was successfully synthesized. Transmission electron microscopy (TEM) revealed a loose porous structure, and rheological analysis demonstrated that the storage modulus (G') exceeded the loss modulus (G''), consistent with hydrogel characteristics, along with shear-thinning behavior. Cellular experiments showed that ICG-NVs were effectively internalized by GL261 glioma cells. CCK-8 assays and Calcein-AM/PI fluorescence staining confirmed that both ICG-NVs and ICG NV@SeHA exhibited excellent biocompatibility with no significant cytotoxicity toward GL261 and HT22 cells. However, the cell viability in the ICG-NV + US and ICG-NV@SeHA + US groups was significantly reduced compared to the ICG + US group (P < 0.01 or P < 0.001) .DCFH-DA fluorescent probe assays revealed that the green fluorescence intensity in the ICG-NV + US and ICG NV@SeHA + US groups was markedly higher than in the PBS, PBS + US, and ICG + US groups (P < 0.000 1 or P < 0.001), reflecting substantial intracellular ROS production. Additionally, cell surface CRT expression was significantly upregulated (P < 0.000 1), and the release of HMGB1 and ATP in the supernatant increased (P < 0.05 or P < 0.01). Conclusion: The ICG-NV@SeHA hydrogel, which exhibits excellent mechanical properties and injectability, was successfully fabricated. Demonstrating favorable biocompatibility, this hydrogel, when combined with SDT, effectively kills glioma GL261 cells and induces immunogenic cell death (ICD). This strategy holds potential as a novel approach to prevent postoperative recurrence in glioma treatment.

国家自然科学基金（No. 82072051）