In this study, HER2 RNA aptamers were conjugated to mertansine (DM1) and the anti-cancer effectiveness of the conjugate was evaluated in HER2-overexpressing breast cancer models. The conjugate of HER2 aptamer and anticancer drug DM1 (aptamer-drug conjugate, ApDC) was prepared and analyzed using HPLC and mass spectrometry. The cell-binding affinity and cytotoxicity of the conjugate were determined using confocal microscopy and WST-1 assay. The in vivo anti-tumoral efficacy of ApDC was also evaluated in mice carrying BT-474 breast tumors overexpressing HER2. The synthesized HER2-specific RNA aptamers were able to specifically and efficiently bind to HER-positive BT-474 breast cancer cells, but not to HER2-negative MDA-MB-231 breast cancer cells. Also, the HER2-specific ApDC showed strong toxicity to the target cells, BT-474, but not to MDA-MB-231 cells. According to the in vivo analyses drawn from the mouse xenografts of BT-747 tumor, the ApDC was able to more effectively inhibit the tumor growth. Compared to the control group, the mice treated with the ApDC showed a significant reduction of tumor growth. Besides, any significant body weight losses or hepatic toxicities were monitored in the ApDC-treated mice. This research suggests the HER2 aptamer-DM1 conjugate as a target-specific anti-cancer modality and provides experimental evidence supporting its enhanced effectiveness for HER2-overexpressing target tumors. This type of aptamer-conjugated anticancer drug would be utilized as a platform structure for the development of versatile targeted high-performance anticancer drugs by adopting the easy deformability and high affinity of aptamers.

Efficient evaluation of aptamer candidates after systematic evolution of ligands by exponential enrichment (SELEX) remains limited by the time and cost required for direct kinetic characterization. We developed a centrifugal microspin filtration (MSF)-based MSF–k_off assay for retention-based, dissociation-sensitive pre-screening of aptamer candidates. Repeated washing preferentially removes rapidly dissociating aptamers from target-immobilized magnetic beads, and the retained aptamers are quantified by quantitative polymerase chain reaction and expressed as Wash 0-normalized retention (W0-NRT). In a thrombin aptamer panel, W0-NRT showed a strong inverse relationship with SPR-derived k_off. A preliminary analysis of streptavidin aptamers likewise showed a W0-NRT-based ranking consistent with the literature-reported k_off values, although the two target systems exhibited distinct empirical relationships. In a wash-based thrombin sandwich assay, two affinity-matched sensor aptamers differing 1.76-fold in k_off were ranked in the same order by post-wash retention as by W0-NRT. The agreement was in rank order rather than in the magnitude of retention. The parallel microspin filter format was also estimated to offer advantages in analysis time and assay-related cost. The MSF–k_off assay therefore provides a simple post-SELEX approach for prioritizing candidates with higher retention under dissociation-sensitive washing before detailed kinetic characterization.