波谱学杂志 ›› 2025, Vol. 42 ›› Issue (3): 231-248.doi: 10.11938/cjmr20253149cstr: 32225.14.cjmr20253149
隋美菊1,2, 张磊1,2, 王瑞芳1,2, 骆盈盈1, 李莎1, 丘茂松1, 徐秋怡1,2, 陈代钦1,2, 陈世桢1,2,3,*(
), 周欣1,2,3
收稿日期:2025-03-14
出版日期:2025-09-05
在线发表日期:2025-05-06
通讯作者:
* Tel: 027-87198631, E-mail: chenshizhen@apm.ac.cn.基金资助:
SUI Meiju1,2, ZHANG Lei1,2, WANG Ruifang1,2, LUO Yingying1, LI Sha1, QIU Maosong1, XU Qiuyi1,2, CHEN Daiqin1,2, CHEN Shizhen1,2,3,*(), ZHOU Xin1,2,3
Received:2025-03-14
Published:2025-09-05
Online:2025-05-06
Contact:
* Tel: 027-87198631, E-mail: chenshizhen@apm.ac.cn.摘要：
免疫抑制性肿瘤微环境（ITME）对抗肿瘤免疫治疗构成了重大障碍．为应对这一挑战，本研究基于Mn2+和内源性血红素制备了一种新型纳米酶，其具有多重类酶催化活性，可有效逆转ITME并激活抗肿瘤免疫应答，从而协同增强免疫检查点抑制剂（Immune checkpoint inhibitor，ICI）αPD-L1对肿瘤生长和转移的抑制作用．同时，该纳米酶兼具磁共振成像功能，可实现对肿瘤及纳米酶分布的可视化追踪．
中图分类号:
隋美菊, 张磊, 王瑞芳, 骆盈盈, 李莎, 丘茂松, 徐秋怡, 陈代钦, 陈世桢, 周欣. MRI示踪的纳米酶用于级联反应增强的免疫治疗[J]. 波谱学杂志, 2025, 42(3): 231-248.
SUI Meiju, ZHANG Lei, WANG Ruifang, LUO Yingying, LI Sha, QIU Maosong, XU Qiuyi, CHEN Daiqin, CHEN Shizhen, ZHOU Xin. MRI-traceable Nanoenzyme for Cascade Catalysis-enhanced Immunotherapy[J]. Chinese Journal of Magnetic Resonance, 2025, 42(3): 231-248.
表1
实验试剂与材料
| 试剂/材料名称 | 生产厂商 | 规格 | ||
|---|---|---|---|---|
| 氢氧化钠 | 中国医药集团有限公司 | 500 g | ||
| 浓盐酸 | 中国医药集团有限公司 | 500 mL | ||
| 四水合氯化锰 | 上海阿拉丁生化科技股份有限公司 | 500 g | ||
| 血红素 | 上海迈瑞尔生化科技有限公司 | 25 g | ||
| 谷胱甘肽 | 上海麦克林生化科技有限公司 | 25 g | ||
| 十二水合磷酸氢二钠 | 中国医药集团有限公司 | 500 g | ||
| 二水合磷酸二氢钠 | 中国医药集团有限公司 | 500 g | ||
| 5,5'-二硫代双(2-硝基苯甲酸)(DTNB) | 上海毕得医药科技有限公司 | 10 g | ||
| 3,3',5,5'-四甲基联苯胺（TMB） | 西格玛奥德里奇贸易有限公司 | 250 mg | ||
| 5,5-二甲基-1-吡咯啉-N-氧化物(DMPO) | 西格玛奥德里奇贸易有限公司 | 100 mg | ||
| 吲哚菁绿（ICG） | 上海麦克林生化科技股份有限公司 | 100 mg | ||
| 4',6-二脒基-2-苯基吲哚（DAPI）染色液 | 上海碧云天生物技术股份有限公司 | 50 mL | ||
| 2,7-二氯荧光素二乙酸酯（DCFH-DA） | 北京索莱宝科技有限公司 | 25 mg | ||
| 无菌PBS | HyClone | 500 mL | ||
| 胎牛血清 | 长沙赛尔博克斯生物科技有限公司 | 50 mL | ||
| RPMI 1640基础培养基 | Gibco Life Sciences公司 | 500 mL | ||
| DMEM基础培养基 | Gibco Life Sciences公司 | 500 mL | ||
| 青霉素-链霉素溶液 | Gibco Life Sciences公司 | 100 mL | ||
| 胰蛋白酶 | Gibco Life Sciences公司 | 100 mL | ||
| 抗体：CD80、CD86 | 赛默飞世尔科技有限公司 | 100 μg | ||
| 小鼠αPD-L1抗体 | BioXcell | 5 mg | ||
| IFN-β ELISA检测试剂盒 | 武汉贝茵莱生物科技有限公司 | 96 T | ||
| TNF-α ELISA检测试剂盒 | 武汉贝茵莱生物科技有限公司 | 96 T | ||
| 4T1、BEAS-2B细胞 | 中国科学院细胞库 | 1瓶 | ||
| Balb/c小鼠 | 湖北贝恩特生物科技有限公司 | 6周龄雌鼠 | ||
表2
实验仪器
| 仪器名称 | 生产厂商 | 型号 |
|---|---|---|
| pH计 | METTLER TOLEDO | FE20 |
| 磁力加热搅拌器 | IKA | HS 7 |
| 高速离心机 | Beckman Coulter | Advanti J-25 |
| 动态光散射仪 | Malvern | ZETASIZER Nano-ZS90 |
| 透射电子显微镜 | JEOL | JEM-2100 |
| 扫描电子显微镜 | Carl Zeiss AG | Zeiss SIGMA |
| 紫外-可见吸收光谱仪 | Thermo Fisher Scientific | Evolution 220 |
| 傅里叶变换红外吸光谱仪 | Thermo Fisher Scientific | Nicolet iS10 |
| X射线光电子能谱仪 | Thermo Fisher Scientific | ESCA Lab 250Xi |
| 电子顺磁共振波谱仪 | Bruker | Elexsys E580-10/12 |
| 808 nm激光器 | 北京镭志威 | LWIRL808 |
| 红外热成像仪 | 武汉红视热像科技 | HS160 |
| 溶氧仪 | 上海雷磁 | JPB-607A |
| 电感耦合等离子体质谱仪 | 德国耶拿分析仪器有限公司 | PQ-MS |
| 细胞培养箱 | Thermo Fisher Scientific | HERACELL 150i |
| 细胞计数仪 | 瑞沃德 | C100-SE/C100 |
| 无菌操作台 | Thermo Fisher Scientific | MCS ADVANTAGE |
| 共聚焦激光扫描显微镜 | Nikon | A1R/A1 |
| 流式细胞仪 | Beckman Coulter | CytoFLEX |
| 小动物麻醉机 | 瑞沃德 | R450 |
| 小动物光声成像仪 | iThera Medical GmbH | MOST inVision 256-TF |
| 小动物活体荧光成像仪 | Perkin Elmer | IVIS spectrum imaging system |
| 7T磁共振成像仪 | Bruker | BioSpec 70/20 USR |
图1
MH NPs的表征. (a) MH NPs的TEM和TEM mapping图；(b) MH NPs的粒径分布；(c) MH NPs的ζ电势；(d) MH NPs中Fe元素的X射线光电子能谱；(e) MH NPs中Mn元素的X射线光电子能谱；(f)血红素（Hemin）和MH NPs的紫外-可见吸收光谱；(g)血红素（Hemin）和MH NPs的傅里叶变换红外吸收光谱
图2
MH NPs的类酶催化性能. (a) MH NPs催化过程示意图；(b) GSH与不同浓度MH NPs（12.5、25、50、100和200 μg·mL-1）反应后DTNB溶液的紫外-可见吸收光谱和照片；(c) H2O2与不同浓度MH NPs（0、10 、20 、30 、40和50 μg·mL-1）反应O2随时间的生成情况；(d) H2O2分别与MH NPs（90 μL, 25 μg·mL-1）、Fe2+和H2O反应的电子顺磁共振谱图；(e)不同反应体系[TMB、TMB + H2O2和TMB + H2O2 + MH NPs（10 μL, 0.1 mg·mL-1）]的紫外-可见吸收光谱；(f)反应体系[TMB + H2O2 + MH NPs（10 μL, 0.1 mg·mL-1）]在不同时间的紫外-可见吸收光谱
图3
MH NPs的光热和光声性能. (a)近红外激光（808 nm激光，500 mW·cm-2）照射下MH NPs溶液（500 μg·mL-1）和H2O的升温曲线和红外热像图；(b) 808 nm激光（500 mW·cm-2）照射下不同浓度MH NPs溶液的温度变化曲线；(c)不同功率密度808 nm激光照射下MH NPs溶液（500 μg·mL-1）的温度变化曲线；(d) MH NPs光热转换效率的计算；(e) 808 nm激光（500 mW·cm-2）照射循环下MH NPs溶液的温度变化曲线；(f)光声（PA）信号强度与MH NPs溶液浓度的相关性以及不同浓度MH NPs溶液的光声图像
图4
MH NPs的T2加权磁共振成像能力. (a)不同pH条件下MH NPs的T2 MRI图像；(b)不同pH条件下MH NPs的横向弛豫率与锰浓度之间的线性关系；(c)不同pH（pH = 7.4、6.5和5.5）条件下，MH NPs中Mn2+的体外释放曲线；(d) MH NPs与不同浓度GSH反应后的T2 MRI图像；(e) 不同GSH条件下，MH NPs的横向弛豫率与锰浓度之间的线性关系；(f)与不同浓度GSH反应后，MH NPs中Mn2+的体外释放曲线
图5
MH NPs介导光热作用增强的抗肿瘤催化治疗的体外评价. (a)不同处理后4T1细胞内ROS水平的共聚焦荧光图像（比例尺：100 μm）；(b)不同处理后4T1细胞内ROS水平的半定量分析；(c)不同处理后4T1细胞内LPO水平的共聚焦荧光图像（比例尺：100 μm）；(d)不同处理后4T1细胞内LPO水平的半定量分析；(e)不同处理后4T1细胞中STING通路相关蛋白表达的Western blot结果；(f)不同处理后4T1细胞中STING信号通路相关蛋白表达的半定量分析；(g)利用transwell系统探究不同处理对4T1细胞诱导的树突状细胞熟化情况示意图；(h)细胞上清液中IFN-β的表达水平；(i)细胞上清液中TNF-α的表达水平；(j)树突状细胞熟化水平的流式分析结果
图6
MH NPs的活体生物分布和磁共振、光声成像. (a)静脉注射MH@ICG NPs不同时间点4T1荷瘤小鼠的活体荧光图像；(b)静脉注射MH@ICG NPs后不同时间点4T1荷瘤小鼠肿瘤部位的荧光变化曲线；(c)MH@ICG NPs静脉注射72 h后4T1荷瘤小鼠主要脏器的荧光图像；(d)静脉注射72 h后4T1荷瘤小鼠主要脏器中MH@ICG NPs的半定量分析；(e)静脉注射MH NPs后不同时间点4T1荷瘤小鼠的T2 MRI图像；(f)静脉注射MH NPs后不同时间点4T1荷瘤小鼠的光声图像；(g)静脉注射MH NPs后不同时间点4T1荷瘤小鼠肿瘤部位T2 MRI信号变化曲线；(h)静脉注射MH NPs后不同时间点4T1荷瘤小鼠肿瘤部位光声信号变化曲线
图7
光热增强MH NPs介导的抗肿瘤催化免疫治疗联合αPD-L1对4T1移植瘤的治疗效果. (a)光热增强MH NPs介导的抗肿瘤催化免疫治疗联合αPD-L1的治疗示意图；(b, e)接受不同治疗的4T1荷瘤小鼠原发（Primary）和远端（Distant）肿瘤的生长曲线；(c, f)接受不同治疗的4T1荷瘤小鼠原发和远端肿瘤的重量；(d, g)接受不同治疗的4T1荷瘤小鼠原发和远端肿瘤的照片；(h)接受不同治疗的4T1荷瘤小鼠原发肿瘤的H&E和TUNEL组织染色分析
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