自组装载药纳米探针用于乳腺癌焦亡增敏及化学交换饱和转移成像研究

doi:10.3969/j.issn.1674-8115.2025.03.003

上海交通大学学报（医学版） ›› 2025, Vol. 45 ›› Issue (3): 271-281.doi: 10.3969/j.issn.1674-8115.2025.03.003

自组装载药纳米探针用于乳腺癌焦亡增敏及化学交换饱和转移成像研究

邓佳丽¹^,²(), 郭嘉婧², 王静怡¹^,², 丁心怡², 朱仪², 王中领²()

^1.上海理工大学健康科学与工程学院，上海 200093
^2.上海交通大学医学院附属第一人民医院放射科，上海 200080

收稿日期:2024-11-07 接受日期:2024-12-20 出版日期:2025-03-28 发布日期:2025-03-28
通讯作者: 王中领，主任医师，博士；电子信箱： zlwang138136@126.com。
作者简介:邓佳丽（1997—），女，硕士生；电子信箱： jali04512@163.com。
基金资助:
国家自然科学基金(82272057);上海交通大学医学院“双百人”项目(20191904)

Self -assembled drug -loaded nanoprobes for pyroptosis sensitization and chemical exchange saturation transfer imaging in breast cancer

DENG Jiali¹^,²(), GUO Jiajing², WANG Jingyi¹^,², DING Xinyi², ZHU Yi², WANG Zhongling²()

^1.School of Health Science and Engineering, University of Shanghai for Science and Technology, Shanghai 200093, China
^2.Department of Radiology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080, China

Received:2024-11-07 Accepted:2024-12-20 Online:2025-03-28 Published:2025-03-28
Contact: WANG Zhongling, E-mail: zlwang138136@126.com.
Supported by:
National Natural Science Foundation of China(82272057);“Two-hundred Talents” Program of Shanghai Jiao Tong University School of Medicine(20191904)

摘要/Abstract

摘要：

目的·制备具有化学交换饱和转移（chemical exchange saturation transfer，CEST）激活成像性能的自组装载药纳米探针，评价其体内外CEST激活成像效能及光动力增敏乳腺癌细胞焦亡治疗的价值。方法·采用自组装的策略构建吉西他滨（gemcitabine，Gem）与光敏剂二氢卟吩e6（chlorin e6，Ce6）共载的纳米探针（GC），通过扫描电镜（scanning electron microscope，SEM）和动态光散射（dynamic light scattering，DLS）等对其进行表征，并观察该纳米探针的体外CEST激活成像效能以及pH浓度和时间依赖的药物释放。GC联合激光照射处理小鼠乳腺癌4T1细胞后，采用2 ′，7 ′-二氯二氢荧光素二乙酸酯（2 ′，7 ′-dichlorodihydrofluorescein diacetate，DCFH-DA）探针检测活性氧（reactive oxygen species，ROS）的生成，采用酶联免疫吸附试验（enzyme-linked immunosorbent assay，ELISA）检测白细胞介素-1β（interleukin-1β，IL-1β）、IL-18等炎症因子的水平，采用免疫荧光检测钙网蛋白（calreticulin，CRT）和高迁移率族蛋白B1（high mobility group box 1 protein，HMGB1）评估焦亡介导的免疫源性细胞死亡（immunogenic cell death，ICD）效应。构建小鼠乳腺癌4T1肿瘤模型，观察GC纳米探针在体内的CEST特异性激活效应，并通过测量肿瘤体积及检测炎症因子和ICD标志物等评估其基于焦亡的抗肿瘤效果。结果·SEM、DLS分析结果显示GC纳米探针为均一的类球形结构。该纳米探针在体外模拟的酸性微环境中及在细胞水平上均具有良好的pH浓度及时间依赖性激活成像效应。该载药纳米探针在pH 5.0酸性条件下药物Gem的释放量高达80%，显著高于pH 7.4条件下的药物释放量（ P=0.003）。DCFH-DA荧光染色表明纳米探针介导的光增敏焦亡可生成大量的ROS。焦亡相关因子检测结果显示IL-1β、IL-18水平显著增加（均 P<0.05），ICD标志物CRT荧光表达增强、HMGB1荧光表达减弱。体内实验结果表明，尾静脉注射GC纳米探针4 h后肿瘤部位CEST信号显著增强并达到峰值；此外，体内抗肿瘤治疗结果显示GC联合激光照射的实验组与PBS处理的对照组相比，炎症因子IL-1β、IL-18水平增加，ICD标志物HMGB1、CRT均明显改变，肿瘤得到显著抑制（均 P<0.05）。结论·成功制备的GC纳米探针可特异性激活CEST成像，并引导光动力增敏乳腺癌肿瘤细胞焦亡，促进乳腺癌精准消融。

关键词: 化学交换饱和转移成像, 细胞焦亡, 纳米探针, 乳腺癌

Abstract:

Objective ·To prepare self-assembled drug-loaded nanoprobes with activatable chemical exchange saturation transfer (CEST) imaging capability, and evaluate their imaging performance and therapeutic potential for photodynamic-sensitized pyroptosis in breast cancer in vivo and in vitro. Methods ·GC nanoprobes co-loaded with gemcitabine (Gem) and chlorin e6 (Ce6) were constructed by using a self-assembly strategy. The physicochemical properties of the GC nanoprobes were characterized by scanning electron microscopy (SEM) and dynamic light scattering (DLS). The pH-/time-dependent CEST activation and drug release profiles were investigated. The 2 ',7 '-dichlorodihydrofluorescein diacetate (DCFH-DA) probe was used to detect the generation of reactive oxygen species (ROS), and enzyme-linked immunosorbent assay (ELISA) was used to detect the release of inflammatory factors such as interleukin-1β (IL-1β) and IL-18 in mouse breast cancer 4T1 cells after treatment with GC nanoprobes with synergistic laser irradiation. Immunofluorescence was performed to detect immunogenic cell death (ICD) markers, including calreticulin (CRT) and high mobility group box 1 protein (HMGB1). The 4T1 breast cancer mouse models were established to validate tumor-specific CEST activation and evaluate anti-tumor efficacy by measuring tumor volume and detecting inflammatory factors and ICD markers. Results ·SEM and DLS confirmed the uniform spherical morphology of the GC nanoprobes. The CEST imaging results showed that the nanoprobes had excellent pH-concentration and time-dependent activation imaging effects both in the simulated acidic microenvironment and at the cellular level in vitro. The drug release from this drug-loaded nanoprobe was 80% at pH 5.0, which was significantly higher than at pH 7.4 ( P=0.003). DCFH-DA fluorescence staining demonstrated that GC-mediated photodynamic therapy induced a significant generation of ROS. Analysis of pyroptosis-related factors revealed a marked increase in the release levels of IL-1β and IL-18 (both P<0.05), along with elevated fluorescence expression of CRT and HMGB1. The in vivo CEST imaging results showed that the CEST signal at the tumor site was significantly enhanced, peaking at 4 h with tail vein injection of GC. The GC nanoprobes with synergistic laser irradiation group showed markedly elevated inflammatory factors (IL-1β, IL-18), changed ICD biomarkers (HMGB1 and CRT), and significant tumor suppression, compared to the PBS control group (all P<0.05) . Conclusion ·The GC nanoprobes enables specific CEST imaging-guided photodynamic therapy, effectively inducing pyroptosis and precise ablation of breast cancer.

Key words: chemical exchange saturation transfer (CEST) imaging, pyroptosis, nanoprobe, breast cancer

中图分类号:

R445

邓佳丽, 郭嘉婧, 王静怡, 丁心怡, 朱仪, 王中领. 自组装载药纳米探针用于乳腺癌焦亡增敏及化学交换饱和转移成像研究[J]. 上海交通大学学报（医学版）, 2025, 45(3): 271-281.

DENG Jiali, GUO Jiajing, WANG Jingyi, DING Xinyi, ZHU Yi, WANG Zhongling. Self -assembled drug -loaded nanoprobes for pyroptosis sensitization and chemical exchange saturation transfer imaging in breast cancer[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(3): 271-281.

图/表 9

图1 GC纳米探针的表征、试剂水平 1O2 生成以及酸响应药物释放能力Note： A. SEM image of GC nanoprobes. B. Hydrodynamic size distribution of GC nanoprobes. C. Zeta-potential of GC nanoprobes. D. UV-visible absorption spectrum of Gem, Ce6, and GC nanoprobes. E. Fluorescence spectrum of GC nanoprobes and Ce6. F. UV-visible absorption spectrum of DPBF at different time points after laser irradiation in the water solution of GC nanoprobes. G/H. Release of Gem (G) and Ce6 (H) at pH 5.0 and pH 7.4. ①P=0.003, ②P=0.006.

Fig 1 Characterization of GC nanoprobes as well as generation capacity of 1O2 and drug release capacity at different pH values

图2 GC纳米探针联合激光照射对小鼠乳腺癌 4T1细胞增殖及细胞内 ROS生成的影响Note： A. Effects of different concentrations of GC nanoprobes combined with laser irradiation (0.4 W/cm 2) on the proliferation of 4T1 cells. B. ROS detection in 4T1 cells after different treatments. ①P=0.003, ②P<0.001.

Fig 2 Effects of GC nanoprobes combined with laser irradiation on the proliferation and intracellular ROS generation in mouse breast cancer 4T1 cells

图3 不同浓度 Gem及不同 pH值下 GC纳米探针的体外 CEST激活成像Note： A‒C. Z-spectra (A), MTRasym (B), and images of CEST signals (C) of free Gem at different concentrations. D‒F. Z-spectra (D), MTRasym (E), and images of CEST signals (F) of 20 mmol/L GC nanoprobes at pH 7.4 and pH 5.0.

Fig 3 In vitro CEST imaging of Gem at different concentrations and GC nanoprobes at different pH values

图4 GC纳米探针在小鼠乳腺癌 4T1细胞中的摄取Note： A/B. Flow cytometry (A) and fluorescence microscopy images (B) showing cellular uptake of GC nanoprobes in 4T1 cells.

Fig 4 Cellar uptake of GC nanoprobes in mouse breast cancer 4T1 cells

图5 GC纳米探针在肿瘤细胞和正常细胞的响应性 CEST激活成像Note： A. The representative images of CEST signals (A) and the corresponding MTRasym values (B) of GC nanoprobes incubated with 4T1 cells and MCF-10A cells. ①P=0.003, ②P=0.001.

Fig 5 Responsive CEST imaging of GC nanoprobes in tumor cells and normal cells

图6 PDT增敏下 GC纳米探针对 4T1细胞焦亡能力的诱导及免疫反应的触发Note： A. Morphological changes in 4T1 cells after treatment with PBS, Gem, Ce6, GC, Ce6+L and GC+L. B. Release levels of IL-1β, IL-18 and LDH in 4T1 cells after different treatments. C. CRT and HMGB1 immunofluorescence staining of 4T1 cells in different groups. ①P=0.001, ②P=0.003, ③P=0.002, ④P=0.013.

Fig 6 Pyroptosis-inducing ability and triggered immune responses of photosensitized GC nanoprobes with PDT sensitization in 4T1 cells

图7 GC纳米探针的体内 CEST成像研究Note： A/B. CEST imaging signals (A) and MTRasym values (B) of tumor-bearing mice after intravenous injection of GC nanoprobesor free Gem (50 mmol/L). ①P=0.002, ②P=0.000, ③P=0.003.

Fig 7 In vivo CEST imaging study of GC nanoprobes

图8 GC纳米探针的焦亡触发的体内抗肿瘤作用Note： A. Changes in tumor volume in Balb/C mice after different treatments. B/C. ELISA assay of IL-1β (B) and IL-18 (C) in mouse serum. D. CRT and HMGB1 immunofluorescence staining of tumor sections after 14 d of varying treatments (injection dose: 25 mg/kg of Gem). E. H-E staining, Ki67 and cleaved caspased-3 immunohistochemistry staining of tumor slices after treatments. ①P=0.003, ②P<0.001, ③P=0.002, ④P=0.001, ⑤P=0.005

Fig 8 Pyroptosis-mediated in vivo antitumor effects of GC nanoprobes

图9 GC纳米探针联合激光照射治疗后的小鼠主要器官的 H-E染色

Fig 9 H-E staining of major organs in mice after GC nanoprobes combined with laser irradiation therapy

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自组装载药纳米探针用于乳腺癌焦亡增敏及化学交换饱和转移成像研究

Self -assembled drug -loaded nanoprobes for pyroptosis sensitization and chemical exchange saturation transfer imaging in breast cancer

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