包裹ICG的相变型阳离子分子探针靶向显像及治疗视网膜母细胞瘤的研究

Ultrasonic molecular imaging and targeted therapy in the experimental study shows attractive application prospect. However, ordinary microbubbles carry either a net neutral or slightly negative surface charge, while the cell surface also are negatively charged in plasma, so ordinary targeted microbubbles minimizes the interactions with the cell, leading to the limitation of target ability. Therefore, cationic microbubbles (CMB) were developed. 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. CD105 is highly expressed in endothelial cells in retinoblastoma (Rb). This project proposed to develop a indocyanine green (ICG) incorporated molecular probes with a positive surface Charge lipid membrane which carry CD105 antibody , liquid fluorocarbon core(PFP) and the capacity of the liquid-gas phase transition. which leading to gathering of probes on endothelial cells in Rb. With pulse laser irradiation, the molecular probes will undergo a phase transition and produce microbubbles and photoacoustic effect in situ in tumor, so as to realize the ultrasound and photoacoustic molecular imaging and targeted therapy of Rb. By exploring imaging and treatment conditions in vitro and in vivo, the mechanism of molecular probe liquid-gas phase transition as well as tumor therapy, to establish a highly efficient, safe mode of tumor molecular imaging and treatment.

靶向超声分子显像及治疗在实验研究中展示了诱人的应用前景。但是，常用的靶向超声分子探针表面常为中性或负电荷，而血浆中的细胞表面也为负电荷，这不利于靶向微泡与细胞的连接，致使其靶向能力受限，因此，带阳性电荷的靶向微泡的研发成为热点。我们的前期研究证实：脉冲激光能触发含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。因视网膜母细胞瘤（Rb）血管内皮细胞高表达CD105，本课题拟以带阳性电荷的连接CD105抗体的脂质为膜材，以PFP为核心，研发一种包裹吲哚菁绿（ICG）的、具有液气相变能力的分子探针，使其靶向聚集于Rb血管内皮；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，从而实现Rb的超声及光声分子显像与靶向治疗。通过探索体内外显像与治疗的条件，液气相变以及肿瘤治疗机制，为建立一种高效、安全的肿瘤分子显像与治疗方式开辟新的思路。

本研究通过双乳法、链酶亲和素连接法成功制备阳离子靶向相变型ICG脂质纳米粒（INPT），透射电镜及光镜下可见纳米粒分布较均匀，呈圆形；粒径（354.20±93.85）nm，电位（25.20±3.29）mV，紫外分光测得ICG 包封率为（89．56±0．79）％；流式细胞检测显示抗体连接率为99.89％，激光共聚焦下可见荧光基本重合，表明抗体与纳米粒连接率高，武鸣县细胞毒性。INPT的具有良好光热效应，一定浓度下不同功率激光辐照，温度随功率增大升高，在浓度5 mg/ml、功率2 W/cm2 激光辐照180 s 时，其温度可升至63℃。在同一激光功率条件下，辐照不同浓度INP，温度随浓度增加而升高,纳米粒激光辐照后可发生液气相变。纳米粒具有光声及超声多模态成像功能，体外低强度聚焦超声3 W 功率作用0.5、2.5、5、7.5 min，B－mode 模式及Contrast 模式下均显示作用5.0 min 时回声强度升至最高。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）INPT 仍具有很好的光声显像能力。共聚焦显微镜显示INPT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，纳米粒浓度越高，细胞杀伤力越强。活体荧光实验可见INPT组纳米粒注射后15min出现血管一过性现象，30min时荧光信号较15min低，又逐渐增强，2h达到高峰，此后荧光信号逐渐减弱直至消失，表明注射2h后纳米粒达到高峰，在体内具有较长滞留期，荧光信号分布证实INPT纳米粒具有体内寻靶能力。体内光声成像实验中注射INPT纳米粒后2h光声信号最强，持续至6h仍有较高信号，12h时光声信号可见明显衰减。超声成像实验中可见INPT组激光激发后可采集到超声增强图像。瘤鼠经治疗后，INPT组瘤鼠肿瘤生长受到明显抑制，瘤体进行病理切片染色可见INPT组治疗效果最佳。治疗后瘤鼠生化指标较对照组无统计学差异，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。