Rapid in vivo screening of tumor-targeting aptamers using zebrafish larvae

Although in vitro methods for aptamer screening are well established, quantitative systematic in vivo screening of multiple aptamers remains challenging due to time and cost constraints associated with rodent models. To address this bottleneck, we developed a strategy for rapid screening of tumor-targeting aptamers using light-sheet fluorescent imaging after systemic administration of aptamers in a zebrafish larvae neuroblastoma model. Correlated imaging and qPCR-based aptamer detection identified an optimal screening window of 30-90 min post-injection, enabling rapid in vivo evaluation of five aptamers and one negative control oligonucleotide. We compared this strategy with in vitro screening of the same aptamers against a panel of neuroblastoma cell lines and found that zebrafish larvae screening ranked lead aptamers differently from in vitro assays, suggesting that in vivo screening may identify more physiological tumor-preferential aptamers for therapeutic development than in vitro screening alone. To demonstrate the therapeutic potential, the lead aptamer was conjugated to fusogenic nanovesicles and showed reproducible neuroblastoma-preferential accumulation 1-2 h post-injection. This strategy enables rapid in vivo evaluation of aptamers selected in vitro to identify candidates with tumor-targeting properties. Importantly, this in vivo approach can also assess off-target binding to healthy tissues without the time and cost associated with rodent models.