新型多模态纳米微泡超声造影剂的构建及其介导胰腺癌多方位联合靶向治疗的实验研究

Ultrasound molecular imaging technology has broad application prospects in the early diagnosis and effective treatment of pancreatic cancer due to no radiation pollution and real-time dynamic display. The high expression of folate receptor (FR) plays an important role in the occurrence, development and metastasis of pancreatic cancer. Taking FR as a target for early ultrasound molecular imaging and treatment of pancreatic cancer has important clinical significance. However, used as conventional ultrasound contrast agents, microbubbles have many defects such as large particle size, being difficult to penetrate through the tumor vasculature, poor target, single therapeutic molecule and so on. In previous studies, we prepared pure nano-sized bubbles as ultrasound contrast agents which can specially target to HER2 molecule in tumor, and the in vivo experiments verified that the nanobubbles can directly penetrate through the leaks of tumor vessels and perform molecular targeted ultrasonic imaging. In this study, the FR molecule in pancreatic cancer was used as the breakthrough point, our aim is to prepare multi-mode nanobubbles with the function of dual-mode targeting, dual-mode imaging and various methods combined targeting-treatment, and to identify the effect of early detecting and effective killing for pancreatic cancer primary lesions and micrometastasis from the in vitro and the in vivo level. The study can provide experimental basis for early accurate diagnosis and effective targeted therapy of pancreatic cancer and has profound research significance.

超声分子影像技术由于无辐射污染、能实时动态显示等优势，在胰腺癌的早期诊断和有效治疗中具有广泛应用前景。叶酸受体（FR, folate receptor ）高表达对胰腺癌的发生、发展及转移起着重要作用。以FR为靶点进行胰腺癌的早期超声分子靶向成像和治疗具有重要的临床意义。然而，目前使用的微米级超声造影剂存在粒径大、难以透过肿瘤血管壁及靶向和治疗分子单一等诸多缺陷。我们在前期研究中成功制备了能特异靶向肿瘤HER2分子的纯粒径纳米微泡超声造影剂，在动物体内证实其能穿透肿瘤血管壁裂隙到达肿瘤组织实现超声分子靶向成像。本研究拟以胰腺癌FR作为切入点，构建一种兼具双重靶向、双重成像及多方位联合靶向治疗功能的多模态纳米微泡超声造影剂，从体外细胞和动物体内水平探讨其对胰腺癌原发病灶及微小转移灶的早期探查及有效杀伤作用。为胰腺癌的早期精准诊断和有效靶向治疗奠定实验基础，具有深远的研究意义。

胰腺癌由于恶性度高、易早期转移、手术预后差以及对传统放/化疗不敏感，使得当前临床治疗非常棘手。 分子靶向超声造影由于无辐射、能实时动态显示及操作简便等优势，有望突破这一瓶颈。实现该技术的关键是制备性能优良的靶向纳米级超声造影剂。本课题拟以胰腺癌叶酸受体作为靶点，构建一种兼具双重靶向、双模态成像及多方位联合靶向治疗功能的诊疗一体化纳米微泡造影剂，从体外细胞和动物体内水平探讨其对胰腺癌早期原发病灶及微小转移灶的精准探查及有效杀伤能力。研究主要取得如下进展：1、当纳米微泡壁脂质壳膜携载较多元件时，微泡粒径会明显增加（接近1000nm），稳定性相应减小。用高速均质剪切机将脂质壳膜剪切成较小独立分散壳膜，随后制作的微泡粒径会明显变小（349.8±159.1 nm），达到理想要求；2、制备出诊疗一体化复合纳米微泡IR780/FA-NBs-DTX，探索IR-780（0.15 mg）和DTX (1.0mg )的最佳初始剂量可获得两者在微泡壳膜的合适载量。微泡最佳超声显影浓度为6×106 微泡/mL，稳定性及生物安全性良好，在体外对胰腺癌细胞的靶向结合率显著（95.6±1.7%）。荷瘤动物体内治疗研究显示，IR780/FA-NBs-DTX介导的靶向光热消融联合化疗可致肿瘤在治疗后第18天几乎消失。3、进一步拓展研究，制备了靶向复合纳米液滴造影剂Nds-IR780、IR780/FA-Nds-DTX，粒径小而均一（200nm），体外及体内稳定性较纳米微泡更好。不足之处在于当液滴进入体内靶向到肿瘤局部时，需要先被实施“液—气”相变，再进行超声造影成像。4、研究中发现，靶向造影剂进入体内容易被巨噬细胞吞噬系统吞噬，只有少部分能到达预期的肿瘤靶点，我们尝试用自体红细胞膜作为微泡/液滴壳膜解决这一问题。制备了仿生靶向诊疗一体化纳米液滴IR780-RBCM@NDs，在体外实验中验证了巨噬细胞对其吞噬作用明显低于脂质壳膜纳米液滴。综上研究表明，本课题新制备的诊疗一体化多功能靶向纳米级造影剂，对早期胰腺癌微小病灶及转移灶有精准靶向诊疗能力。对胰腺癌这一难治性“癌中之王”的有效治疗奠定了实验基础，提出了新的思路。