背景:化疗,作为HER2阳性乳腺癌的辅助治疗策略,能有效改善临床症状,克服治疗性单克隆抗体的耐药性。核苷类似物是一类广泛应用于辅助治疗的传统化疗药物。然而,有许多关键问题限制了他们的临床效率,包括差的选择性和稳定性,严重的副作用和次优的治疗效果。因此,本文旨在开发一种新型的DNA纳米载体用于靶向药物递送以解决上述问题。
方法:合成4条41-merDNA链,并通过DNA固相合成将10个FUdR分子连接至每条DNA链的5'端。亲和体分子通过四股链之一中的接头连接到聚合物FUdR的末端。亲和体-FUdR-四面体DNA纳米结构(affi-F/TDNs)通过四条DNA链自组装,其中一个顶点连接到聚合物FUdR尾部末端的结合体,三个顶点仅是聚合物FUdR尾部。用共聚焦荧光显微镜和流式细胞术目视检查affi-F/TDN的体外细胞摄取,用MTT法研究affi-F/TDN对癌细胞的细胞毒性。AnnexinV-FITC/PI双染色法检测细胞凋亡。使用NOD/SCID(MusMusculus)小鼠模型,还评估了afi-F/TDN的靶向杀伤功效.
结果:FUdR在affi-TDN中的载药量按摩尔比为19.6%。体外结果表明,affi-F/TDNs对过表达HER2的乳腺癌BT474细胞具有高选择性和抑制作用(81.2%),对低HER2表达的MCF-7细胞具有低毒性。在体内应用期间,Affi-F/TDN在血液循环中表现出良好的稳定性,在肿瘤区域达到特异性积累和最佳抗肿瘤疗效(抑制率为58.1%),并表现出优异的生物相容性。
结论:亲和体-DNA四面体,作为一种简单有效的主动靶向递送纳米载体,为核苷类抗肿瘤药物的运输提供了新的途径。
BACKGROUND: Chemotherapy, as an adjuvant treatment strategy for HER2-positive breast cancer, can effectively improve clinical symptoms and overcome the drug resistance of therapeutic monoclonal antibodies. Nucleoside analogues are a class of traditional chemotherapeutic drugs that are widely applied in adjuvant therapy. However, there are many critical issues that limit their clinical efficiency, including poor selectivity and stability, severe side effects and suboptimal therapeutic efficacy. Hence, this work aims to develop a new DNA nanocarrier for targeted drug delivery to solve the above problems.
METHODS: Four 41-mer DNA strands were synthesized and 10 FUdR molecules were attached to 5\' end of each DNA strand by DNA solid-phase synthesis. An affibody molecule was connected to the end of polymeric FUdR through a linker in one of the four strands. The affibody-FUdR-tetrahedral DNA nanostructures (affi-F/TDNs) were self-assembled through four DNA strands, in which one vertex was connected to an affibody at the end of a polymeric FUdR tail and three vertices were only polymeric FUdR tails. In vitro cellular uptake of affi-F/TDNs was examined visually with confocal fluorescence microscopy and flow cytometry, and the cytotoxicity of affi-F/TDNs against cancer cells was investigated with MTT assay. Cell apoptosis was detected by Annexin V-FITC/PI double staining method. Using NOD/SCID (Mus Musculus) mice model, the targeted killing efficacy of affi-F/TDNs was also evaluated.
RESULTS: The drug-loading of FUdR in affi-TDNs was 19.6% in mole ratio. The in vitro results showed that affi-F/TDNs had high selectivity and inhibition (81.2%) for breast cancer BT474 cells overexpressing HER2 and low toxicity in MCF-7 cells with low HER2 expression. During the in vivo application, affi-F/TDNs displayed good stability in the blood circulation, achieved specific accumulation in tumor region and the best antitumor efficacy (inhibition ratio of 58.1%), and showed excellent biocompatibility.
CONCLUSIONS: The affibody-DNA tetrahedrons, as a simple and effective active targeting delivery nanocarrier, provided a new avenue for the transport of nucleoside antitumor drugs.