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超顺磁性氧化铁纳米颗粒标记靶向癌胚抗原的CAR-T细胞及体外磁共振成像研究
Labeling of carcinoembryonic antigen-specific CAR-T cells with superparamagnetic iron oxide nanoparticles and
in vitro magnetic resonance imaging- 陆军军医大学学报 2024年46卷第17期 页码：1951-1958
- 作者机构：
1.400038 重庆，陆军军医大学(第三军医大学)第一附属医院放射科
2.401121 重庆，重庆市食品药品检验检测研究院
- 作者简介：
[徐豆豆，E-mail：xudoudou@cqifdc.org.cnXU Doudou, E-mail: xudoudou@cqifdc.org.cn
- 基金信息：重庆市自然科学基金面上项目(cstc2021jcyj-msxmX1078);重庆市博士后科研项目特别资助项目(2020年);重庆市科研机构绩效激励引导专项(cstc2022jxjl120006)
DOI：10.16016/j.2097-0927.202404042
中图分类号： R392.12;R445.2;R981.9收稿：2024-04-12，
修回：2024-06-28，
纸质出版：2024-09-15
- 稿件说明：
移动端阅览
何坤高, 蒋波, 郭牡丹, 等. 超顺磁性氧化铁纳米颗粒标记靶向癌胚抗原的CAR-T细胞及体外磁共振成像研究[J]. 陆军军医大学学报, 2024,46(17):1951-1958.
Kungao HE, Bo JIANG, Mudan GUO, et al. Labeling of carcinoembryonic antigen-specific CAR-T cells with superparamagnetic iron oxide nanoparticles and
in vitro magnetic resonance imaging[J]. Journal of Army Medical University, 2024, 46(17): 1951-1958.何坤高, 蒋波, 郭牡丹, 等. 超顺磁性氧化铁纳米颗粒标记靶向癌胚抗原的CAR-T细胞及体外磁共振成像研究[J]. 陆军军医大学学报, 2024,46(17):1951-1958. DOI： 10.16016/j.2097-0927.202404042.
Kungao HE, Bo JIANG, Mudan GUO, et al. Labeling of carcinoembryonic antigen-specific CAR-T cells with superparamagnetic iron oxide nanoparticles and
in vitro magnetic resonance imaging[J]. Journal of Army Medical University, 2024, 46(17): 1951-1958. DOI： 10.16016/j.2097-0927.202404042.
摘要
目的
2
采用超顺磁性氧化铁纳米颗粒(superparamagnetic iron oxide nanoparticles
SPIONs)标记靶向癌胚抗原(carcinoembryonic antigen
CEA)的嵌合抗原受体T细胞(chimeric antigen receptor T cells
CAR-T细胞)，并进行体外磁共振成像研究，为监测CEA CAR-T细胞的体内回输治疗提供依据。
方法
2
将ferumoxytol、肝素钠和硫酸鱼精蛋白分别以高(ferumoxytol 100 μg/mL，肝素钠4 IU/mL，硫酸鱼精蛋白120 μg/mL)、中(ferumoxytol 50 μg/mL，肝素钠2 IU/mL，硫酸鱼精蛋白60 μg/mL)、低(ferumoxytol 25 μg/mL，肝素钠1 IU/mL，硫酸鱼精蛋白30 μg/mL)剂量混合形成SPIONs复合物FHP(ferumoxytol/heparin/protamine)，然后与CEA CAR-T细胞共孵育进行细胞标记。CCK-8、EdU和流式细胞术检测FHP对细胞的生物相容性，普鲁士蓝染色检测细胞对FHP的摄入情况，电感耦合等离子体-质谱法(inductively coupled plasma-mass spectrometry
ICP-MS)定量检测细胞中Fe含量，流式细胞术检测FHP标记的CEA CAR-T细胞对肿瘤细胞的杀伤作用，磁共振成像(magnetic resonance imaging
MRI)扫描FHP标记的CEA CAR-T细胞。
结果
2
高、中、低剂量的FHP对CEA CAR-T细胞的活力没有明显影响，CCK-8实验显示细胞活性均在100%以上；EdU实验显示各组细胞DNA复制活性均在94.3%以上。普鲁士蓝染色显示CEA CAR-T细胞可摄取FHP，且随FHP浓度的增加，摄取量增大。ICP-MS进一步检测显示胞内Fe含量为(440.23±189.36)ng/mL。肿瘤细胞杀伤实验表明FHP标记的CEA CAR-T细胞的杀伤肿瘤细胞能力良好。MRI扫描显示随着FHP浓度的增高，FHP标记的CEA CAR-T细胞T2WI信号呈明显降低趋势(
P
<
0.01)。
结论
2
SPIONs复合物FHP具有良好的生物相容性并能够有效标记CEA CAR-T细胞，可作为CEA CAR-T细胞的磁标记物，为临床提供一种可行的MRI示踪方法。
Abstract
Objective
2
To use superparamagnetic iron oxide nanoparticles (SPIONs) to label chimeric antigen receptor (CAR) T cells targeting carcinoembryonic antigen (CEA)
and perform magnetic resonance imaging (MRI) to real time trace CEA CAR-T cells
in vivo
.
Methods
2
Appropriate amount of ferumoxytol
heparin sodium and protamine sulfate were mixed at high (ferumoxytol 100 μg/mL
heparin sodium 4 IU/mL
protamine sulfate 120 μg/mL)
medium (ferumoxytol 50 μg/mL
heparin sodium 2 IU/mL
protamine sulfate 60 μg/mL)
and low (ferumoxytol 25 μg/mL
heparin sodium 1 IU/mL
protamine sulfate 30 μg/mL) concentrations to form a SPIONs complex ferumoxytol/heparin/protamine (FHP)
and then co-incubated with CEA CAR-T cells for cell labeling. The biocompatibility of FHP was detected by CCK-8 assay
EdU assay and flow cytometry. The uptake of FHP was detected by Prussian blue staining
and SPIONs content in the cells was quantitatively detected by inductively coupled plasma-mass spectrometry (ICP-MS). Flow cytometry was used to detect the lytic effect of FHP-labeled CEA CAR-T cells on tumor cells
and MRI was employed to scan FHP-labeled CEA CAR-T cells.
Results
2
FHP at high
medium
and low concentrations had no significant effect on the activity of CEA CAR-T cells
with cell activity above 100% determined by CCK-8 assay. DNA proliferation was above 94.3% in EdU assays. Prussian blue staining showed that CEA CAR-T cells could take FHP up
with the uptake increased with the increment of FHP concentration. ICP-MS showed that the intracellular Fe content was 440.23±189.36 ng/mL. Tumor cell killing experiment showed that FHP-labeled CEA CAR-T cells had excellent killing capability against tumor cells. MRI scans indicated that T2WI signals of FHP-labeled CEA CAR-T cells were significantly reduced with increasing FHP concentration (
P
<
0.01).
Conclusion
2
SPIONs complex FHP shows good biocompatibility and can effectively label CEA CAR-T cells. SPIONs complex FHP can be used as a magnetic marker for CEA CAR-T cells and a feasible MRI tracer for clinical application.
关键词
Keywords
references
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