99mTc标记树状大分子包裹金纳米颗粒偶联Duramycin对肿瘤化疗诱导细胞凋亡的分子影像学研究

Apoptosis is a fundamental biologic process of regulated cell death, having a close relationship with treatment of tumor. Apoptosis imaging can noninvasively and dynamically evaluate the effect and prognosis of chemotherapy of malignant tumor in vivo. Phosphatidylethanolamine (PE) is the second most abundant phospholipid species in mammalian cellular membranes and can be exposed to the outlet of cell membrane at the early stage of apoptosis. Duramycin, a 19 amino acid lantibiotic peptide, can bind PE with high affinity and specificity. In the former work of our group, we synthesized Cy5.5-Duramycin and studied the value of Cy5.5-Duramycin in evaluating the effect of chemotherapy in U87MG glioma model. In vivo and in vitro study showed the uptake of treated U87MG glioma was significant higher than control group, but optical imaging has the limitation such as weak transmission of signal and hard clinical translation. Based on the previous study, we aim to combine 99mTc-Duramycin with dendrimer-entrapped gold nanoparticles, demonstrate its binding efficiency with tumor cell apoptosis in vitro and in vivo and display its value in monitoring treatment efficiency. This dual-modality molecular probe will provide a novel molecular imaging biomarker for in vivo real-time monitoring cancer therapy.

肿瘤化疗疗效与细胞凋亡有密切关系，利用凋亡显像可在活体内实现肿瘤化疗疗效及预后的动态监测,指导肿瘤患者个体化治疗。细胞凋亡早期可出现特征性细胞膜磷脂酰乙醇胺（PE）外翻，小分子多肽耐久霉素（Duramycin）可与PE高亲和力、特异性结合。课题组前期利用荧光染料Cy5.5标记Duramycin对胶质瘤化疗前后进行了体内及体外实验，光学显像结果显示治疗后肿瘤对Cy5.5-Duramycin的摄取明显高于治疗前，体外实验证实凋亡细胞对Cy5.5-Duramycin的摄取明显高于正常细胞，但光学显像存在信号穿透力弱、难以临床转化的缺点。本课题在前期研究基础上，拟用99mTc标记树状大分子包裹金纳米颗粒偶联Duramycin，制备成SPECT/CT双模态纳米凋亡显像剂，在体外及活体肿瘤模型上研究其靶向凋亡细胞成像及监控化疗疗效的价值，为活体实时无创监测肿瘤疗效提供新的分子影像学方法。

肿瘤细胞凋亡与化疗疗效密切相关，利用凋亡显像可在活体内监测肿瘤化疗疗效。细胞凋亡早期出现特征性细胞膜磷脂酰乙醇胺（PE）外翻，可作为细胞凋亡显像靶点。耐久霉素（duramycin）可与PE高亲和力、特异性结合。本课题选择第五代PAMAM树状大分子（G5.NH2)作为载体材料，内部包裹金，表面修饰靶向多肽duramycin，并用放射性核素99mTc进行标记，制成新型细胞凋亡显像剂。1H核磁共振谱确认了靶向材料（Au DENPs-duramycin）和非靶向材料（Au DENPs），动态光散射测得靶向材料水动力半径为327nm，电势为11.933mV；非靶向材料水动力半径为287.9 nm，电势为-0.107mV。透射电子显微镜结果显示靶向及非靶向材料均具有良好的尺寸分布。细胞毒性实验结果显示靶向材料和非靶向材料对胶质瘤细胞的生长均无明显抑制，证明纳米材料具有良好的细胞相容性。流式细胞术结果显示经阿霉素诱导凋亡的胶质瘤细胞对靶向材料Au DENPs-duramycin 的摄取明显高于非靶向材料Au DENPs 和对照组PBS，证实Au DENPs-duramycin 对凋亡细胞具有靶向性。靶向材料99mTc-Au DENPs-duramycin的标记率为60.4±5.4%（n=3），非靶向材料99mTc-Au DENPs的标记率为64.5±6.8%（n=3），放化纯均大于99%，靶向材料和非靶向材料在PBS中均具有良好的稳定性。正常大鼠的体内分布结果显示，两种纳米显像剂主要聚集在肝、脾中，可经肾脏、膀胱排泄，在甲状腺、胃肠道及软组织中均无明显摄取。SPECT显像结果显示，荷胶质瘤裸鼠在治疗前经尾静脉注射靶向材料99mTc-Au DENPs-duramycin后，肿瘤部位无放射性摄取；经阿霉素治疗后3天，肿瘤部位可见放射性摄取；非靶向材料在治疗前及治疗3天，肿瘤部位均无明显放射性摄取，表明靶向材料99mTc-Au DENPs-duramycin具有良好的肿瘤凋亡细胞靶向性。H&E染色及TUNEL染色进一步证明了阿霉素能够诱导胶质瘤细胞的凋亡。99mTc-Au DENPs-duramycin有望成为一种新型细胞凋亡显像剂，为活体内实时、无创监测肿瘤疗效提供新的分子影像学方法。