包裹光吸收子的、载药相变型光声/超声分子探针靶向多模态显像与治疗视网膜母细胞瘤研究

When microbubble contrast agents used for molecular imaging of tumor, there exist a fatal disadvantage, such as it cannot pass through the vascular endothelial gap and target to tumor cells for its micrometer diamete. Nano liquid fluorocarbon nanoparticles can pass through the endothelial gap and target to tumor cells, but its ultrasound imaging quality is not good, and can not make full use of UTMD for the release of drugs. Our previous study has proved that pulse laser can trigger the phase transition of nanoparticles made by perfluorohexan (PFH) and optical absorbers, rational utilization of the photoacoustic effect can achieve the aim of imaging and treatment to the tumor. Retinoblastoma (RB) is the most common intraocular malignant tumor in infants and young children. It is lack of early diagnosis and effective treatment. Laser can be widely used in the treatment of retinal diseases, because it can pass through clearing medium. So, this project proposed to develop a photoacoustic / ultrasonic molecular probe, which could target to the folate receptor of RB cells, and with perfluoro pentane (PFP) as the core, carrying adriamycin (ADM) and optical absorption of indocyanine green (ICG), lipid inclusions and has the ability of liquid gas phase transition. When used in vitro or in vivo, it could target to RB cells, and with pulse laser irradiation, the molecular probe will undergo a phase transition and produce microbubbles and photoacoustic effect, and release ADM from bubbles, make the chemotherapy combined with photo acoustic therapy efficiently, reduce its systemic toxicity, and make the photoacoustic / ultrasonic dual mode molecular imaging coming true.

微泡造影剂用于肿瘤分子显像存在致命缺点，其粒径为微米级、不能穿过血管内皮间隙实现靶向肿瘤细胞显影。纳米级液态氟碳纳米粒可穿过血管内皮间隙靶向肿瘤细胞，但其超声显影能力差，且无法通过超声空化效应实现控释药物。我们前期实验证实，脉冲激光能触发含印度墨水光吸收子的液态氟碳微球（PFH）相变，合理利用其光声效应，对肿瘤能达到显像和治疗的目的。视网膜母细胞瘤（RB）是婴幼儿最常见的眼内恶性肿瘤，缺乏早期诊断方法及有效治疗手段。结合激光被广泛用于治疗视网膜病变，本课题拟制备以RB细胞叶酸受体为靶点、以全氟戊烷（PFP）为核心、载阿霉素（ADM）及光吸收子吲哚菁绿（ICG）、脂质包裹的、具有液气相变能力的光声/超声分子探针，使其靶向聚集于RB细胞；通过脉冲激光辐照，促使其在肿瘤区域相变，产生微气泡及光声效应，靶向释放所载化疗药物ADM，使光声治疗与化疗相结合，减轻全身毒副作用，实现RB的光声/超声双模态显像。

本研究通过双乳法成功制备了包裹ICG、ADM和PFP的叶酸受体靶向的相变型纳米粒(ICG/FA/Pct-PFPNP:ADM)，透射电镜及光镜下可见纳米粒分布较均匀，呈圆形；粒径（374.4±23.48）nm，电位（-28±4.5）mV，紫外分光测得ICG和ADM的包封率分别为（91.85±2.98）%和（62.04±5.1）%；流式细胞检测显示纳米粒表面叶酸表达率为（98.83±0.17）%，无明显细胞毒性。ICG/FA/Pct-PFPNP:ADM具有良好光热效应，在浓度5 mg/ml、功率2 W/cm2 激光辐照180 s 时，其温度可升至60.8℃，纳米粒激光辐照后可发生液气相变。纳米粒具有超声及光声多模态成像能力，体外2 W /cm2激光作用3min后，B－mode 模式及Contrast 模式下均有较好的回声信号。体外光声信号随纳米粒浓度升高而增强，具有良好的线性关系，且较低浓度（0.1mg/ml）ICG/FA/Pct-PFPNP:ADM仍具有很好的光声显像能力。共聚焦显微镜显示ICG/FA/Pct-PFPNP:ADM纳米粒具有细胞寻靶能力，且体外激光治疗可对细胞起到杀伤作用。活体荧光实验可见ICG/FA/Pct-PFPNP:ADM组纳米粒注射后30min，肿瘤区域开始出现荧光信号，1h达到高峰，此后荧光信号逐渐减弱直至消失，荧光信号分布证实ICG/FA/Pct-PFPNP:ADM纳米粒具有体内寻靶能力。体内升温实验中，注射ICG/FA/Pct-PFPNP:ADM纳米粒的瘤鼠，在经过激光辐照后，肿瘤区域温度能在1min内上升至42℃。体内超声成像实验中可见ICG/FA/Pct-PFPNP:ADM组激光激发后可采集到超声增强图像。光声成像实验中，注射ICG/FA/Pct-PFPNP:ADM纳米粒后1h光声信号最强，持续至6h仍有光声信号。瘤鼠经治疗后，ICG/FA/Pct-PFPNP:ADM组瘤鼠肿瘤生长受到明显抑制，瘤体进行切片染色可见ICG/FA/Pct-PFPNP:ADM组治疗效果最佳。治疗后主要器官组织切片较对照组未见明显差异。本研究将眼科及超声治疗领域相结合，对视网膜母细胞瘤的靶向治疗具有指导作用和应用前景。