中国肿瘤生物治疗杂志2025，Vol.32Issue(3)：247-256,10.DOI:10.3872/j.issn.1007-385x.2025.03.003
负载BMSC来源纳米囊泡的硒化透明质酸水凝胶的制备及其对胶质瘤GL261细胞的杀伤作用
Preparation of selenized hyaluronic acid hydrogel loaded with BMSC-derived nanovesicles and investigation of its cytotoxic effect on glioma GL261 cells
摘要
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.关键词
胶质瘤/纳米囊泡/GL261细胞/声动力疗法/吲哚菁绿/复合水凝胶Key words
glioma/nanovesicle/GL261 cell/sonodynamic therapy(SDT)/indocyanine green(ICG)/composite hydrogel分类
临床医学引用本文复制引用
张转转,周旋,陈翠敏,张停琳,高洁..负载BMSC来源纳米囊泡的硒化透明质酸水凝胶的制备及其对胶质瘤GL261细胞的杀伤作用[J].中国肿瘤生物治疗杂志,2025,32(3):247-256,10.基金项目
国家自然科学基金(No.82072051) （No.82072051）
