Human Microphysiological System-based SELEX Technology for Robust Identification of BBB-Shuttling Aptamers for CNS Drug Delivery

Introduction:: The Blood-Brain Barrier (BBB) is a selective barrier that regulates the movement of molecules and ions between the blood and the brain in the microvasculature of the central nervous system (CNS). However, the BBB is a significant obstacle to drug development for the CNS due to its function of filtering out foreign substances from entering the brain. To overcome this challenge, there has been a lot of effort to discover BBB-specific ligands that enable BBB penetration for brain drug delivery. Aptamers, which are short DNA or RNA nucleotides that can bind to target molecules in a 3D structure, have been proposed as potential BBB shuttle molecules because of their practical advantages over conventional molecules such as antibodies or peptides.¹ Aptamer screening has traditionally been conducted through the Systemic Evolution of Ligands by EXponential Enrichment (SELEX) process, which uses two-dimensional cell-based culture models or animal models. However, traditional screening models have limitations in accurately reflecting the human in vivo environment.² This limited the robust screening of small-sized oligonucleotides with a strong BBB shuttling capacity. To address this issue, a novel human microphysiological system-based SELEX (MPS-SELEX) technology has been developed for screening aptamers with robust functionality.³





Materials and Methods:: A physiologically relevant human BBB MPS was reconstituted in a poly(dimethylsiloxane) (PDMS) device with upper brain and lower blood channels separated by a highly permeable PET membrane. The blood channel is lined with human iPS-derived BMECs interfaced with a brain channel where human primary astrocytes and pericytes are cultured. Using BBB MPS, we successfully screened novel aptamers that can recognize human brain microvascular endothelial cells (BMEC) and penetrate the BBB under physiological conditions (Figure 1). The MPS-SELEX process involved three major steps: 1) dosing of the single-stranded DNAs (ssDNA) library through the vascular channel under fluid flow, 2) collecting the BBB penetrating ssDNA from the brain channel effluent, and 3) amplification of the ssDNA pool for the next round by polymerase chain reaction (PCR). After five rounds of SELEX, aptamer candidates were listed by conducting next-generation sequencing (NGS) and further investigated for BBB penetration efficacy using in vitro BBB transwell and MPS. The aptamer with the highest functionality was selected as the human BBB shuttling aptamer, named hBS01, and was further analyzed for BBB penetration mechanism and validated for its performance in an animal model.


Results, Conclusions, and Discussions:: The MPS-SELEX technique effectively identified a functional BBB shuttle aptamer, hBS01, which binds to the target present on BMECs and efficiently crosses the brain through the clathrin-mediated endocytosis (CME) pathway. hBS01 shows ~2.4 folds higher BBB penetrating efficacy compared to the control random aptamer group (Figure 2). We evaluated the penetration efficacy of hBS01 in various endothelial cell models to demonstrate its organ-specific shuttling capability. Interestingly, it exhibited enhanced brain targetability, with increased cellular uptake and penetration across the human BBB, but not to endothelial cells from other organs (Figure 3). The robust BBB shuttling capacity of hBS01 under physiological conditions was further confirmed in a mouse in vivo model, demonstrating the aptamer's brain targetability while circulating in the bloodstream. Upon tail vein injection of the aptamer conjugated to a fluorescent-labeled nanoconstruct (hBS01-SA-Cy5.5), we observed a significantly high brain signal when hBS01 was employed (Figure 4). Moreover, the images of mouse brain sections revealed higher aptamer particle localization and accumulation ratio in the cortex and hippocampal regions (Figure 5). Through the comprehensive investigation of the MPS-SELEX approach using human MPS, we have established a reliable platform for the selection of BBB shuttle aptamers under physiologically relevant conditions. The human BBB MPS effectively mimics the in vivo BBB environment, displaying physiologically relevant barrier functions and selective transcytosis of molecules, allowing for the identification of aptamers with high BBB penetration potential. Although this study primarily utilized BBB MPS for screening BBB-penetrating aptamers, the approach can be further improved by integrating additional organ models, such as liver and kidney MPS, into the MPS-SELEX process to exclude aptamers targeting other organs. Consequently, our proposed method holds promise for the development of aptamer-based drug delivery systems for a range of brain disorders and can potentially be extended to various organ MPSs.


Acknowledgements (Optional): : This work was supported by a National Research Foundation of Korea (NRF) grant (NRF- 2020R1C1C1014753), the Korea Health Technology R&D Project (HU20C0094020201001), and UNIST research grant (1.220023.01).


References (Optional): : (1) Chen, K.; Liu, B.; Yu, B.; Zhong, W.; Lu, Y.; Zhang, J.; Liao, J.; Liu, J.; Pu, Y.; Qiu, L. Advances in the development of aptamer drug conjugates for targeted drug delivery. Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology 2017, 9 (3), e1438.

(2) Sola, M.; Menon, A. P.; Moreno, B.; Meraviglia-Crivelli, D.; Soldevilla, M. M.; Cartón-García, F.; Pastor, F. Aptamers against live targets: is in vivo SELEX finally coming to the edge? Molecular Therapy-Nucleic Acids 2020, 21, 192-204.

(3) Jiang, H.; Xue-Fei, L.; Ke-Xin, Z. Progress of aptamer screening techniques based on microfluidic chips. Chinese Journal of Analytical Chemistry 2020, 48 (5), 590-600.