新型放射性靶向长循环纳米药物对耐药乳腺癌的治疗作用及其机制研究

Breast cancer is the leading contributor to cancer incidence and one of the most common causes of cancer death in women. But many breast cancer patients died from multidrug resistance (MDR) in the clinic. Overexpression of P-glycoprotein, the product of the multiple drug resistance type 1 (MDR1) gene, which functions as an energy-dependent pump driving potentially toxic species out of cells, might play a very important role in multidrug resistance. Nanocarriers offer numerous advantages: small particle size, narrow size distribution, surface features for target specific localization, protective insulation of drug molecules to enhance stability, opportunity to develop nanocarriers that respond to physiological stimuli, feasibility for delivery of multiple therapeutic agents in a single formulation, combination of imaging and drug therapy to monitor effects in real time, and the opportunity to combine drugs with energy (heat, light, and sound) delivery for synergistic therapeutic effects. The unique properties of nanocarriers offer promising opportunities for the treatment of MDR tumors. Integrin αvβ3 is upregulated on the activated tumor endothelial cells and also highly expressed on breast cancer. We design a novel multifunctional nanocarrier with a superparamagnetic Fe3O4 core and a small amount of surface-conjugated cRGD. We try to investigate the effect of targeted therapy in multidrug resistant breast cancer and its mechanism on P-glycoprotein downregulation by 131I labelled cRGD-Fe3O4 nanoparticles using Bcap37/MDR1 cells. We hope this nano-drug system will serve as a new strategy for treatment of multidrug resistant breast cancer.

乳腺癌常因P-pg的过表达产生耐药而导致治疗失败，现存的各种逆转耐药的方法存在着缺陷，而纳米颗粒可绕过P-pg通路，修饰后可靶向输送药物。能与乳腺癌细胞高表达的整合素αvβ3受体结合的cRGD常用做靶向多肽。前期试验中我们构建了125I-cRGD-USPIO，证实其在乳腺癌肿瘤中有高效靶向和长时间滞留的特性。为此，我们提出假说：131I-cRGD-USPIO可靶向内照射治疗耐药乳腺癌，并且通过131I和cRGD作用抑制P-pg的表达。为验证该假说，首先，我们用131I-cRGD-USPIO在通过MDR1基因导入建立的耐药乳腺癌的细胞及动物实验，验证其对耐药乳腺癌的治疗作用；其次，通过检测信号通路相关因子研究131I产生的ROS通过JNK/cJun/NF-κB通路和cRGD拮抗ανβ3受体阻断PI3K/Akt/NF-κB通路而抑制P-pg表达的可能性及其机制，为耐药乳腺癌的治疗提供新的思路。

乳腺癌常出现耐药等而导致治疗失败，及早准确地诊断和针对耐药肿瘤的有效治疗是良好预后的关键，因此本研究尝试研制放射性碘标记的以Fe3O4为核心的多模态磁性纳米药物，实现乳腺癌等多种肿瘤的多模态显像及放射性治疗的诊疗一体化药物，同时尝试治疗耐药乳腺癌并探讨其作用机制。方法：1. 自行设计并合成新型cRGD环肽，验证其靶向显像能力；2. 将Fe3O4纳米颗粒表面修饰后连接cRGD环肽；3. 利用放射性碘标记cRGD环肽耦联的Fe3O4纳米颗粒，验证其靶向能力和SPECT/MR双模态显像能力；4. 优化磁性纳米材料，建立T1、T2同时增强的纳米药物，合成Fe3O4-Ag双金属纳米药物，并验证其双模态显像能力和体内毒性；5. 建立耐药乳腺癌细胞株，并验证其对多模态药物的摄取能力；6. 验证放射性碘标记cRGD环肽耦联的Fe3O4纳米颗粒对乳腺癌的治疗能力。结果：1. 成功实现了放射性碘标记cRGD环肽耦联的Fe3O4纳米颗粒，检测了其表征；2. 证实了放射性碘标记cRGD环肽耦联的Fe3O4纳米颗粒可以用于乳腺癌的SPECT/MR双模态显像，SPECT/CT显像结果示实验组小鼠肿瘤部位放射性明显浓聚，对照组肿瘤部位未见明显放射性浓聚。实验组在4h达到峰值(4.27±0.41)%ID/g，是对照组的3倍；在48h仍有(1.61±0.70)%ID/g，是对照组的2.9倍。MRI结果显示，肿瘤部位T1加权像呈明显不均匀强化，T2加权像呈显著的信号降低且在4h效果最佳。注射后4h，实验组T1信号强度的变化量比对照组高约6.3倍，T2信号强度的变化约为对照组的2.87倍。 3. 合成了Fe3O4-Ag双金属纳米药物，证实了其双模态显像能力，并研究了其体内急性毒性；4. 建立了耐药乳腺癌细胞株，验证了其对18F-FDG的摄取能力和机制；5 放射性碘标记cRGD环肽耦联的Fe3O4纳米颗粒可用于乳腺癌的治疗，与对照组相比，131I-cRGD-USPIO组抑制率明显增高；治疗组HE染色显示肿瘤治疗后坏死区域增大，肿瘤细胞坏死胞质浓缩，体积缩小；与未治疗组比较，131I-cRGD-USPIO治疗后，Ki67及CD31表达均降低。结论：合成了新型的放射性碘标记的以Fe3O4为核心的多模态磁性纳米药物，可实现乳腺癌的多模态显像及放射性治疗的诊疗一体化。