基于外泌体的双模态成像探针联合光热治疗的肿瘤诊治研究

We have found that the stability and biocompatibility of tumor imaging probe in vivo are usually poor in previous studies, in the mean while, there are limitations in the single mechanism of imaging. In order to obtain a new type of drug delivery carriers with long circulation, low toxicity and super stability in vivo, extracellular vesicles are used recently. In this proposal, we design an imaging probe targeted to the tumor tissue via exosome (a subgroup of extracellular vesicles). The exosomes we plan to use will be secreted from imDC of human source. Because there are limitations in the single mechanism of imaging, in this project, we are going to combine two models of imaging with different mechanisms to overcome the limitations and improve the accuracy of imaging in tumor. Gd-DTPA is a kind of MRI contrast agent. IR-780 is a kind of near-infrared fluorescence (NIR) dye. We are going to load Gd-DTPA and IR-780 in exosome at the same time to construct a dual-modals imaging probe. As there is photothermal property of IR-780, the probe could guide targeting photothermal therapy (PTT) for cancer. To gain targeting efficiency, the exosomes are self-assemblied with the coupling conjugate of phospholipid-aptamer against PSMA. To improve the stability of aptamer in vivo, we take chemical modification on bases. The constructed exosome-based probe of diagnosis and treatment can deliver Gd-DTPA and IR-780 to the tumor area due to the targeting ability of aptamer to PSMA. We will investigate the optimized conditions for the production and isolation of exosomes, the coupling of phospholipid and aptamer, the self-assembly of phospholipid-aptamer conjugate into the membrane of exosomes. The targeting efficiency and the in vivo dynamic distribution of exosome-based probe will also be investigated, especially the clearance mechanism. In vitro and in vivo MRI and NIR imaging will be systematically investigated. It is expected to reveal the general mechanism of construction and in vivo imaging of dual-modal probe based on exosome with stability. This study will also open a new field for developing new type of dual-modal probe guided PTT cancer therapy based on exosome as carrier and different targeting aptamers for diagnosis and treatment of various cancers.

在先前对MRI和NIR成像探针的研究中，申请人发现肿瘤成像分子探针在体内成像中通常存在稳定性和生物相容性的问题，并且单一成像技术具有局限性。研究发现细胞外囊泡作为药物递送载体，具有活体稳定性高、毒性低、循环时间长等优势。故本项目利用细胞外囊泡中的外泌体亚群作为载体，拟设计靶向于肿瘤组织的成像探针；将靶向于前列腺特异膜抗原（PSMA）的核酸适配体修饰于外泌体表面，实现载体在肿瘤组织高富集，并通过对适配体进行碱基的化学修饰提高其在体内的稳定性；将MRI造影剂Gd-DTPA和NIR荧光染料IR-780装载进外泌体内部，通过联合两种成像机制，提高肿瘤检测的准确性；并利用近红外荧光染料的光热性能，达到成像探针可视化指导治疗的目的；同时，本项目通过体内外实验研究，分析该诊疗体系对于肿瘤部位成像的灵敏度、准确性、靶向效率和光热治疗效果等，并揭示活体内外泌体作为载体的诊疗体系的构建的一般规律和内在机理。

本项目构建了基于外泌体的诊疗一体的磁共振成像探针，同时指导光热治疗。主要研究内容如下：制备出分散度好的聚多巴胺（PDAFe）纳米颗粒，利用MCF-7细胞分泌的外泌体靶向于肿瘤组织的功能，将PDAFe装载于MCF-7细胞分泌的外泌体中，构建了基于外泌体的靶向于肿瘤部位的磁共振成像探针PDAFe@Exo。由于PDAFe在808nm激光照射下具有光热作用，该体系也具有光热治疗的功能。为了增加PDAFe在外泌体中的装载量，将自噬腺病毒LC3B转染进MCF-7细胞，加入自噬激活剂，提高了外泌体对于PDAFe的装载浓度。对于PDAFe@Exo进行物理化学表征和生物安全性考察，扫描电镜图可知，制备出的PDAFe为规则的球形，大小在90nm左右，分散性较好，测量PDAFe的弛豫率r2为281.18mM-1S-1。WB结果显示外泌体探针样品中含有2个阳性蛋白TSG101和CD63，不含阴性对照蛋白Calnexin，证明收集到的细胞外囊泡为外泌体，NTA表征外泌体探针尺寸为152.9nm。应用外泌体探针对于人肝癌细胞huh7和人肝上皮细胞thle两种细胞进行体外光热治疗，CCK-8处理后实验结果显示，其降低肿瘤细胞的增殖及活性的能力与其浓度正相关。共聚焦显微镜下观察huh7和thle两种细胞对于PDAFe@Exo的摄取，结果表明肝癌细胞huh7对于MCF-7细胞分泌的外泌体在不同时间点均有明显摄取，肝上皮细胞thle在每个时间点均摄取极少。原位肝脏肿瘤模型裸鼠的体内实验中，在0-6h期间考察其肝脏部位MR成像信号变化，发现静脉注射外泌体探针1h后成像信号最强。激光照射治疗后，磁共振成像及肝脏解剖结果表明治疗后肿瘤体积明显减小，对试验后的裸鼠肿瘤及主要脏器做HE切片染色观察，不同组别均未见明显毒性影响。在对裸鼠进行光热治疗期间对其进行称重，体重未见明显减轻。本项目基于外泌体构建的靶向性磁共振成像探针指导光热治疗的体系，提高了肿瘤检测的靶向性和准确性，由磁共振成像信号指导的肝脏肿瘤原位模型的光热治疗，为细胞外囊泡作为递送系统在肿瘤诊疗方面的应用提供参考。