目的乳腺癌是世界范围内女性最高发的恶性肿瘤,其中20-30%乳腺癌表达HER2蛋白。HER2阳性乳腺癌患者预后较差,其存活期仅为HER2阴性乳腺癌患者的一半。目前针对HER2的靶向治疗主要是应用西妥昔单抗（赫赛汀）,但大多数患者在治疗一段时间后会对该抗体产生耐药性,因此需要开发新型的针对HER2的靶向治疗策略。本研究探索了对HER2阳性乳腺癌进行靶向热疗的可能性。既往肿瘤热疗所用的热敏剂不具备主动靶向功能,因而在肿瘤治疗中的应用有限。本课题构建了能与HER2阳性乳腺癌细胞靶向结合的铁热敏剂,并在体外初步评估其热疗效果。方法本研究通过生物素-链霉亲和素间的特异性结合反应,构建了HER2适配体与铁纳米粒的复合物（AptNPs）;通过流式细胞仪检测了HER2核酸适配体的结合特异性,以及该核酸适配体是否修饰到了纳米磁珠上；通过动态光散射粒径仪对构建的两亲性纳米粒进行了尺度表征；利用相差显微镜验证了该纳米复合物是否与HER2阳性乳腺癌细胞结合；利用MTS法检测了其在磁场下对抗肿瘤热疗效果的影响。结果我们通过流式细胞仪检测了HER2核酸适配体的结合特性,发现该适配体具有特异结合HER2阳性SK-BR-3乳腺癌细胞的能力。生物素标记的核酸适配体与链霉亲包被的铁纳米粒进行了反应；DNA探针杂交的结果显示,HER2适配体确实结合到了铁纳米粒的表面。纳米复合物的尺度呈单峰分布,平均值为333.7nm。与未经修饰的纳米粒相比,核酸适配体修饰的纳米粒（AptNPs）对靶细胞的亲和力显著提升。此外,AptNPs显著增强了对HER2阳性SK-BR-3乳腺癌细胞的热疗效应,但对HER2阴性MDA-MB-231细胞的疗效没有影响。上述结果提示,AptNPs所增强的抗肿瘤热疗效应具有HER2靶向性。结论AptNPs有可能靶向性地提升针对HER2阳性肿瘤细胞的热疗效率,在研发新型肿瘤靶向热疗方面具有潜在应用价值。目的乳腺癌是危害女性生命健康的最常见肿瘤,其中20%-30%表达HER2。HER2阳性乳腺癌的预后较差,其生存期仅为HER2阴性患者的一半。当前针对HER2的靶向治疗主要是通过西妥昔单抗（赫赛汀）来实现,但大多数患者在应用该单抗后会产生耐药,因此需要开发新型的针对HER2的靶向治疗策略。纳米科技的发展为肿瘤治疗提供了新的思路,并在抗肿瘤药物的靶向递送方面取得了进展。但迄今为止,纳米科技尚未被用来直接拉近淋巴细胞和肿瘤细胞,以靶向性地提升针对肿瘤的免疫反应。本课题构建了一种两亲性的纳米粒,可与NK细胞及HER2阳性乳腺癌细胞结合,拉近两种细胞,以靶向性地提升NK介导的抗乳腺癌免疫反应。方法本研究通过生物素-链霉亲和素间的特异性结合反应,将CD16和HER2核酸适配体按照5：1的比例修饰到纳米磁珠表面,构建了两亲性的纳米粒；通过流式细胞仪检测了CD16和HER2核酸适配体的细胞结合特性；以DNA探针评估了两种核酸适配体和纳米磁珠结合状况；通过激光共聚焦显微镜和动态光散射粒径仪表征了所构建两亲性纳米粒的尺度；利用相差显微镜研究了该两亲性纳米粒对PBMC和HER2阳性细胞的亲和特性,并评估了该两亲性纳米粒是否能将两种细胞在空间上拉近；利用MTS方法研究了该两亲性纳米粒是否影响NK对乳腺癌细胞的免疫杀伤。结果本研究构建了一种两亲性的纳米粒（HER2-NP-CD16）,其表面修饰了CD16和HER2核酸适配体,用于拉近CD16阳性NK细胞和HER2阳性肿瘤细胞。流式细胞仪检测发现,该HER2核酸适配体能特异结合HER2阳性SK-BR-3乳腺癌细胞,而CD16核酸适配体能结合富含NK的PBMC。DNA探针杂交实验显示,HER2及CD16核酸适配体确已装配到纳米粒上。两亲性纳米粒的平均尺度是350nm,呈单峰分布。对比空白的纳米粒,两亲性纳米粒对HER2阳性肿瘤细胞及PBMC的亲和能力有显著提升。相差显微镜显示,该两亲性纳米粒可使更多的PBMC附着于HER2阳性肿瘤细胞,拉近两种细胞间的距离。此外,该两亲性纳米粒能显著增强NK对HER2阳性肿瘤细胞（SK-BR-3）的免疫杀伤,但不影响对HER2阴性细胞（MDA-MB-231）的杀伤。此结果提示,该纳米结构能靶向性地增强针对HER2阳性乳腺癌细胞的免疫反应。结论CD16和HER2核酸适配体所修饰的两亲性纳米粒,能够拉近NK细胞和HER2阳性乳腺癌细胞,在体外增强NK对肿瘤细胞的免疫杀伤。该结果提示,核酸适配体所修饰的两亲性纳米结构,在研发针对HER2的新型靶向免疫治疗方面具有应用潜能。 

Objective   To   develop   aptamer-modified   nanoparticles   (AptNPs)   for   targeted enhancement of thermal damage to HER2-positive breast cancer cells.
Methods HER2 aptamer was connected to NPs via biotin-streptavidin reaction. AptNPs were characterized by Dynamic Light Scattering (DLS). The binding feature of the aptamer was evaluated by flow cytometry, and the affinity of AptNPs to target cells by phase-contrast microscopy. Thermal damage under alternative magnetic field was measured by MTS assay.
Results The average size of AptNPs was 333.7 nm. AptNPs exhibited strong binding to the HER2-positive but not the HER2-negative cells. Importantly, AptNPs enhanced the  thermal  damage  to  the  HER2-positive  tumor  cells,   but  not  that  to  the HER2-negative cells.
Conclusions Aptamer-guided iron particles may have potential utility in development of novel HER2-targeted thermal therapies.
Objective Breast cancer is the most common malignancy among women worldwide. HER2 is overexpressed in 20-30% of breast cancers. HER2-positive breast cancer is associated with high metastatic potential and poor prognosis. At present, HER2-targeted therapy was mainly achieved by the monoclonal antibody Trastuzumab (Herceptin). However, most Trastuzumab-treated patients will develop resistance to the antibody and become refractory to further treatment. Therefore, it is still necessary to explore new HER2-targeted therapeutic strategies. In this study, we explored the feasibility of applying nanotechnology to develop a new form of HER2-targeted therapy. Prior research on using nanotechnology for cancer treatment has mainly focused on improving the drug delivery to tumor cells. To date, nanotechnology has not been applied to directly pull together lymphocytes and tumor cells for targeted promotion of anticancer immune reaction. Here we designed an aptamer-based bispecific nanoparticle (NP) for pulling together the CD16-expressing natural killer (NK) cells and the HER2-positive breast cancer cells, to enhance the NK-mediated anticancer cytotoxicity.
Methods In this study, through biotin-streptavidin reaction, we constructed a bispecific nanoparticle (NP) by implanting CD16 and HER2 aptamers onto the surface of a nanobead at the ratio of 5:1. The binding feature of the CD16 and HER2 aptamers and whether this aptamers were connected onto nanobeads were evaluated by flow cytometry. The dynamic light scattering (DLS) instrument and the confocal microscopy were used to characterize the bispecific nanoparticle. Besides, the phase-contrast microscopy was used to study whether the bispecific NP could bind with the HER2-positive SK-BR-3 and CD16-expressing NK cells, And also the phase-contrast microscopy was used to evaluate whether SK-BR-3 and NK could pulled together by the bispecific nanoparticles. The NK cytotoxicity to the HER2-positive SK-BR-3 and HER2-negative MDA-MB-231 breast cancer cells was meassured by MTS assay. 
Results In this work, we designed a bispecific NP that was functionalized with both the CD16 and the HER2 aptamers, to pull together the CD16-expressing immunocytes and the HER2-positive tumor cells. The binding properties of the aptamers were verified with flow cytometry. The HER2 and CD16 aptamers was found capable of recognizing the HER2-positive SK-BR-3 cells and the CD16-positive immunocytes, respectively. These aptamers were planted onto the NP via biotin-streptavidin interaction, as confirmed by hybridizing DNA probes that could recognize the aptamers . The average diameter of the bispecific NPs was 351.7 nm, with a mono-peak size distribution. Compared with the blank NPs, the bispecific NPs had markedly increased affinity to both the NK and the HER2-positive cancer cells. The bespecific nanoparticles could pull together the CD16-expressing immunocytes and the HER2-positive tumor cells. Importantly, the bispecific NPs significantly enhanced the NK cytotoxicity to the HER2-positive SK-BR-3 breast cancer cells, but not that to the HER2-negative MDA-MB-231 cells, indicating that the enhanced anticancer immune reaction was HER2-targeted.
Conclusion A novel bispecific nanostructure was constructed by implanting CD16 and HER2 aptamers onto the surface of a nanoparticle. The bispecific NP could bind with both the CD16-expressing NK cells and the HER2-expresssing SK-BR-3 breast cancer cells. Moreover, the bispecific NP elicited a targeted enhancement of the NK-mediated cytotoxicity to HER2-expressing breast cancer cells in vitro. The results suggest that aptamer-based bispecific nanostructure may have application potential for induction of NK-mediated reaction against HER2-positive tumors