MPO响应性新型近红外发光纳米探针的构建及其在乳腺癌肺部转移早期诊断中的应用研究

The early diagnosis of metastasis is still a crucial problem for the control of breast cancer. In recent studies, researchers have been proved that the neutrophils were playing an important role in lung metastasis of breast cancer. Which suggested that neutrophils could be considered as a biomarker for diagnosis of metastasis. Although neutrophils could be real-time monitored by the method of self-luminescent imaging, for instance chemiluminescence. The available luminescent nanoprobe owned obvious defects, such as non-specific distribution, poorer depth-penetration capability and shorter duration time. In our previous findings, the nanoparticle, which have been established with covalent conjugate of β-cyclodextrin and luminol (LCD), possessed longer luminescence time. And it was clarified that the variations of neutrophils at focus could be monitored by luminescent intensity of nanoparticles in different mice models. Therefore, the hypothesis is proposed that the near-infrared luminescent nanoprobe will be established with LCD and another molecular based on the effect of chemiluminescence resonance energy transfer (CRET). Then the target units will be introduced onto the surface of near-infrared nanoprobe. Attributing to the CRET effect and the active targeting function, the imaging ability of the nanoprobe will be improved and strengthened. To prove the assumption above mentioned, a novel near-infrared nanoprobe will be established with LCD and a special near-infrared conjugated polymer showing aggregation-induced emission (AIE). The aptamer AS1411 is utilized as a active target unit to modify the surface of the nanoprobe. The physicochemical properties of which will be explored. Additionally, the study including the imaging capability and target function of the nanoprobe in vitro and vivo will be also investigated. The successful establishment of the near-infrared nanoprobe will be expected to provide an attractive strategy for the early diagnosis of breast cancer metastasis.

早期乳腺癌转移诊断仍是其有效防治的瓶颈。中性粒细胞在乳腺癌肺部转移中发挥重要作用，故有望作为诊断的细胞靶标。化学发光等自发光成像能实时监测中性粒细胞数，但目前可用发光探针存在非特异性分布、发光时间短、组织穿透性差等缺陷。申请人前期研究发现，以鲁米诺键合环糊精材料（LCD）构建的纳米粒，其发光时间长，且发光强度与中性粒细胞数呈正相关。基于此，本课题提出假设：以LCD为化学发光载体负载可实现CRET效应的分子构建近红外发光纳米探针，并在其表面修饰靶向单元，通过CRET介导近红外发光的组织穿透增强和主动靶向实现协同效应，提高其对乳腺癌早期转移的成像效果。为证明该假设，拟选择具有AIE特性的聚合物DPA-CN-PPV为近红外荧光探针，以适配体AS1411为主动靶向单元，构建靶向性近红外发光纳米探针；表征其理化特性，体内外评价其靶向性和成像功能。项目的实施能为早期诊断乳腺癌肺部转移提供新的成像策略。

乳腺癌是全世界范围内女性易患癌症之一，原位乳腺癌并不致命，乳腺癌导致的大部分死亡主要归因于转移相关并发症，肺是乳腺癌转移的主要器官，由于转移早期癌细胞很少，乳腺癌肺转移的早期诊断和确证仍具有很大的挑战性。研究表明，中性粒细胞在乳腺癌肺转移进程中发挥了重要作用，与肿瘤细胞的侵袭、增殖等密切相关，基于此，我们提出通过设计一种响应性自发光纳米探针对肺中性粒细胞浸润进行发光成像来早期诊断乳腺癌肺转移的新策略。首先，以小分子化合物luminol与环糊精共价键合合成具有良好生物相容性和中性粒细胞响应性的自发光材料，并将其与聚集诱导发光化合物结合制备发光纳米探针(LAD NPs)。通过体外实验，对LAD NPs的化学发光共振能量转移 (CRET) 效应和发光特性进行表征；同时，使用小鼠腹腔中性粒细胞，评价了LAD NPs的体外发光特性，包括探针发光的响应性、组织穿透深度和持续性发光能力等，由于CRET效应，LAD NPs显示出更好的组织穿透能力和更持久的发光特性。通过建立4T1 乳腺癌肺转移小鼠模型，对LAD NPs进行了体内发光成像和相关性分析，此外，通过用中性粒细胞靶向肽PGP对LAD NPs进行表面修饰，开发了一种活性靶向纳米探针，并通过体外和体内研究对该探针也进行了系统评估。结果显示，在4T1乳腺癌肺转移的小鼠模型中，我们发现中性粒细胞和肺中的肿瘤细胞之间存在理想的相关性，证明了新开发的LAD NPs对转移前微环境进行早期成像的有效性；而主动靶向纳米探针在早期检测乳腺癌肺转移中，显示出进一步增强的发光成像能力。值得注意的是，在所检查的小鼠模型中，基于靶向纳米探针的发光成像策略显著优于PET/CT成像模式。此外，通过细胞和动物实验对LAD NPs的安全性进行了初步评价，数据表明，纳米探针具有良好的安全性。因此，基于新开发的发光纳米探针对中性粒细胞靶向成像策略可作为有前景的早期诊断乳腺癌肺转移的方式。