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Archive > Volume 32 Issue 3 > 2025,32(3):247-256. DOI:10.3872/j.issn.1007-385X.2025.03.003 Prev Next
Basic Research
Preparation of selenized hyaluronic acid hydrogel loaded with BMSC-derived nanovesicles and investigation of its cytotoxic effect on glioma GL261 cells
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Abstract:
[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.
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