一种新型靶向肿瘤αvβ3双功能近红外量子点纳米脂质体探针的制备及其应用

Molecular imaging and preferential drug delivery system targeting to integrins such as ανβ3 in tumor plays a very important role in diagnosis and treatment of tumor. In this study, membrance materials such as cholesterol (CHOL), dipalmitoylphosphatidyl choline (DPPC), and maleimido-polyethylene glycol2000-distearyl phosphatidyl ethanolamine (Mal-PEG2000-DSPE) are used to prepare a nano-liposome which contain hydrophilic near-infrared quantum dot (Qdot 800) and chemical treatment drug (Epirubicin, EPB) against breast cancer. The liposome is modified with targeting peptide (P1c) with my own intellectual property. Finally, a targeting nano-liposome probe which can be used in both diagnosis and treatment is obtained by optimization of P1c density on liposome surface and nanoparticle size. The characteristics of the probe on human breast cancer lines (MDA-MB-231, MCF-7) and human umbilical vein endothelial cells (HUVECs) such as in vitro specific binding, sub-cellular localization, competitive inhibition, and damage or destructive effect of the probe are investigated by the methods of flow cytometry, Laser confocal microscopy and near-infrared fluorescence(NIRF) imaging. Human breast cancer models with ανβ3 positive and negative expression are established. Treatment effect and NIRF molecular imaging of targeting probe in tumor-bearing nude mice is observed. The differences of imaging and treatment effect between two models and the relationship between targeting of probe and P1c density on liposome surface as well as nanoparticle size are compared. The feasibility that the same probe can have both function of diagnosis and treatment is explored by in vitro and in vivo study.

基于肿瘤整合素ανβ3的靶向分子成像和药物运载系统的深入研究为临床应用奠定了基础。本研究拟以胆固醇、二棕榈酰磷脂酰胆碱及马来酰亚胺-聚乙二醇-二硬脂酰磷脂酰乙醇胺作为膜材，将近红外量子点（Qdot 800）、乳腺癌化疗一线药物表阿霉素（EPB）包裹其中，用拥有自主知识产权的靶向肽P1c进行修饰；优化靶向肽表面密度及纳米脂质体粒径，制备具有诊断和治疗双重功能的靶向量子点纳米脂质体探针。通过激光共聚焦显微镜、流式细胞术、近红外成像等对MDA-MB-231、MCF-7人乳腺癌细胞及脐静脉内皮细胞进行体外结合、亚细胞定位、竞争抑制及细胞杀伤研究；建立ανβ3阳性及阴性人乳腺癌裸鼠模型，用靶向探针实现肿瘤的在体近红外分子成像，观察对荷瘤裸鼠的治疗效果，比较探针在两种模型中的治疗和成像差异；评价靶向肽表面密度及纳米粒径与靶向性的关系，探索同一探针实现诊断与治疗双重作用的可能性。

根据项目研究计划及国家自然基金委的要求，本课题组按照年度计划开展了相应的系列研究，其中包括：ανβ3穿膜靶向肽的设计和优化、靶向载药脂质体及自组装纳米探针的制备及表针、探针的免疫学活性及特异性鉴定、探针体外细胞学结合研究、荷瘤裸鼠模型的建立、活体肿瘤近红外成像及疗效评估、细胞凋亡相关机理研究等。总结如下： .1）基于能量共振转移（FERT）分子信标的设计及应用。根据能量共振转移原理，我们设计了4色分子信标，构建了在单各体系中可同时检测4种肿瘤基因的实时PCR系统，并已成功用于临床标本的检测，对临床非小细胞肺癌个体化用药的选择及疗效评估具有重要意义。2）基于P1c肿瘤靶向及穿膜双功能小肽的构建及在肿瘤成像中作用的研究。我们设计构建了一个ανβ3靶向穿膜肽PTS，该肽在30 min内可将标记荧光分子穿膜进入细胞浆，并成功实现乳腺癌裸鼠模型的肿瘤成像研究。3）抗CTGF scFv二聚体及VHH抗体库的构建及作用。通过基因工程和噬菌体展示技术获得scFv二聚体及VHH抗体，两种抗体可明显抑制CTGF介导的ASM细胞的增殖，该作用是通过抑制Akt和mTOR的磷酸化而实现。4）靶向脂质体纳米运载系统在治疗中作用的研究。通过脂质体制备技术获得靶向载药脂质体，该脂质体具有明显靶向协同抗耐药菌能力。5）靶向肽载药自组装纳米用于肿瘤凋亡研究。我们设计构建了一个自组装杂合肽，用DOX可迅速激发形成平均粒径50 nm的载药球形纳米，该纳米能显著增强乳腺癌细胞株MDA-MB-231的凋亡。6）基于肽电荷反转的诊疗一体化探针的制备及应用。我们设计构建了一个酸敏释放的集肿瘤荧光成像及细胞核靶向药物递送的诊疗一体化探针，通过大量实验验证了该探针的可行性和实用性。7）靶向噬菌体近红外探针用于体内血栓成像研究。利用噬菌体展示技术制备了靶向噬菌体，通过修饰近红外染料制成靶向荧光探针可实现血栓的在体成像。8）靶向抗体在治疗结肠癌中作用及相应机制研究。VEGF和EGFR单抗单独或联合干预，可显著抑制结肠癌细胞的增值、迁移及肿瘤的血管生成，该效应是通过激活c-Myc, NF-κB/p65 和 IκBα信号通路、抑制STAT3的磷酸化而实现。.目前已完成合同要求的全部研究内容，发表论文12篇，其中SCI论文10篇（平均IF 3.9）；申请专利4项，授权1项；培养研究生6名。