靶向载金纳米棒PH敏感型脂质纳米超声造影剂光-声双模态显像乳腺癌M2型TAM的研究

The steps of macrophage trafficking to the tumor siteis guided by the presence of chemotactic and chemostatic molecules leading to the mobilization in different regions of the breast tumor.Tumor-associated macrophages (TAMs) are a set of macrophages residing in the tumor microenvironment, which are recruited to the tumor site from the bone marrow in response to signals released by both tumor and stromal cells. M2-polarzied TAMs promote tumor cell migration and invasion by secreting cytokines and chemokines. Increasing evidence from clinical and preclinical studies has indicated that TAMs promote tumor cell invasion, proliferation, survival, and metastasis to local and distant sites.Several clinical studies have demonstrated that increased macrophage infiltration into tumors confers metastatic potential and poor prognosis in breast cancer. M2-like macrophage populations are found at the invasive edge of breast tumors. In many cancers, including breast cancer, the increased presence of TAMs is associated with increased aggressiveness of tumors and decreased patient’s survival.Macrophage mannose receptor (MMR; CD206) is not expressed in M1 macrophages; therefore, CD206 serves as a useful marker to distinguish M2 from M1 macrophages.This study is based on our previous research on the preparation of COOH ended long circulating nano lipid microbubbles. Furthermore, we try to prepare nanobubble which contain COOH group.PFP is contained in the Nanobubble, whicn can change into gas . Gold nanorods were also added in order to enhance ability of PFP phase- transition.By observing the proportion of materials, ultrasonic oscillation of time and energy on the diameter, Zeta potential, stability of the nanomicelles, a best proposal to produce the gold nanorods loading nanobubbles would be found. By carbodiimide method(EDC/NHS), CD206 is connected to the COOH end of the nanobubbles. Then testing the targeting effect in vitro and in vivo. It has important significance to evaluate the progesss of the breast cancer

研究证实M2型肿瘤相关巨噬细胞（TAM）分泌一些细胞因子及趋化因子而促进肿瘤细胞的增殖、侵袭及转移；且TAM侵袭增加与乳腺癌的耐药、远处转移及预后差密切相关。在乳腺癌进展的不同时期及治疗过程中TAM聚集的位置及数量会发生改变，如肿瘤快速进展时更多聚集在邻近血管生成部位且数量增多；治疗有效则数量减少。因此，通过显示出TAM能否有助于临床评估乳腺癌的进展情况值得深入研究。甘露醇受体（CD206 ）为仅表达于TAM的特异性受体，CD206有望成为TAM显影靶点。本研究拟在我们前期制备含-COOH末端纳米靶向脂质微泡的研究基础上在成膜材料中加入PH敏感脂质材料及金纳米棒，以液态氟碳（PFP）为成像气体；通过碳化二亚胺的方法将CD206连接至造影剂表面；金纳米棒能进行光热转换辅助PFP液气相变。期望利用其PH响应性及主动靶向性显示乳腺癌中TAM，为临床评估乳腺癌进展情况提供科学依据。

乳腺癌的发病有逐年增多且年轻化的趋势，在乳腺癌微环境中存在大量肿瘤相关巨噬细胞( TAM)，是肿瘤基质中的主要炎症细胞群，且M2型TAM与肿瘤预后呈负相关。巨噬细胞在肿瘤的周边分布较多，而在肿瘤的间质中较少。在肿瘤进展的不同时期 TAM 聚集的位置会发生改变，如肿瘤快速进展时更多聚集在邻近血管生成部位且数量增多；因此通过显示 M2 型TAM 为临床评估保乳手术的安全边界，肿瘤的转移风险、肿瘤治疗效果以缩短治疗时间从而监督肿瘤的进展提供指引。本项目通过点击化学和糖代谢工程的联合应用，筛选出能标记巨噬细胞的叠氮乙酰半乳糖胺（AAG），制备出甘露糖修饰的包载AAG 脂质体，粒径约126nm，其糖包载量约89%，较低的细胞毒性。通过与巨噬细胞共孵育24h，通过共聚焦显微镜观察可发现巨噬细胞成功标记叠氮糖，通过通过小鼠活体成像技术我们在4T1荷瘤BALB/c小鼠体内测定了靶向脂质体的肿瘤靶向特性。甘露糖修饰的包载AAG 脂质体较非靶向脂质体更多在肿瘤部位聚集，在其他正常组织中聚集较少。同时通过组织切片可见脂质体和巨噬细胞可以很好的共定位。为了进一步探究巨噬细胞在体内的成像效率，我们通过点击化学研究了甘露糖修饰载AAG -脂质体在体内的选择性标记能力，通过体内荧光成像技术在小鼠体内注射甘露糖修饰载AAG -脂质体后12h，再注射DBCO-Cy5进行观察，肿瘤组织可很好显影。