(I-344) Design and Construction of a Mechanically-Switchable Aptamer-Based Biotherapeutic Conjugate.

Stimulation responsive biomolecules undergo structural changes when activated by external signals. Among many stimuli, flow induced stress represents an important factor regulating many physiological functions.^1-4 Designing materials that can respond to flow has been a subject of diligent research. Despite extensive research on polymer rheology and shear responsive gels, knowledge of how single molecules behave in flow is still insufficient. To our knowledge, single-molecule based shear-switchable material has yet to be demonstrated, impeded by limited understanding of the dynamic behavior of biomolecules in flow as only a few selected proteins and DNA have been charactrized.^5-6 This study aims to design Single-MOlecule based materials with structures and functions REsponsive to Shear (SMORES), inspired by the function of von Willebrand factor (VWF), a shear-responsible coagulation protein. VWF changes its conformation drastically at the injury site of high flow rates resulting in exposing the platelet and collagen binding sites to form blood clots. To mimic the function of VWF, the SMORES material uses a microparticle to sense and amplify hydrodynamic forces, with aptamer molecules serving as the flow transducer/switch. The drag force experienced by the particle is transduced to the aptamer molecule and alters its conformation, which modulates the structure of the aptamer and the bio-availability of its target, VWF-A1 domain, achieving single-molecule flow responses similar to VWF (Fig.1). The mechanical properties of the aptamer and shear response of the engineered SMORES material have been characterized in this work.

To investigate the mechanical properties of the selected aptamer as a molecular force transducer, we first characterized the unfolding extension of aptamer ARC1172 under a constant loading rate of pulling force using single molecule force measurements. Using the same technique, we also characterized the rupture force between aptamer ARC 1172 and VWF A1 under a constant loading rate as an indicator of the binding affinity between the two biomolecules.  

The construction of the SMORES material was achieved by conjugating thiolated ARC1172 aptamer molecules with 1-μm-diameter amine polystyrene beads using an amine-to-sulfhydryl crosslinker. (Fig. 2). The crosslinker contains NHS-ester and maleimide reactive groups at opposite ends of a short spacer arm to conjugate the aptamer to the amine beads. Conjugation was confirmed by TIRF microscopy and conjugates were purified by centrifugation, to precipitate the beads and wash them from excess, unbound molecules.

To investigate flow modulation of the structure and function of the aptamer-based construct, we optimized a protocol to immobilize the SMORES conjugate onto the surface of a micro-channel. Then, A1 was injected into the channel to bind with the aptamer-bead conjugate. A1 was labelled with quantum dots to enabled imaging of the aptamer-A1 interaction and shear response using total internal reflection fluorescence (TIRF) microscopy. Finally, variable flow conditions were applied to the conjugate-ligand pair using a syringe pump to examine the aptamer’s ability to release its ligand A1 at a critical shear rate.

Using single molecule force measurements based on an optical tweezers setup, over 300 unfolding events were recorded from aptamer ARC1172. The unfolding results were fitted into a worm-like chain (WLC) model (Fig. 3), which yielded a persistence length of 0.31 nm and a contour length of 45.10 nm. Similarly, 700 binding rupture events were captured under different pulling speeds from 50 to 500 nm/s. Fitting the data into the Bell Evans equation yielded a dissociation rate in the absence of force (k0) of 0.0089 s^-1 (Fig. 4). This indicates that aptamer ARC1172 and the A1 domain have high affinity.

 Construction of the shear-sensitive conjugate was accomplished successfully and immobilized in a microfluidic channel (Fig. 2).  After incubating with A1, we used TIRF to observe the co-localization of quantum dot labeled-A1 and the microbead-bound aptamers (Fig.5). By utilizing a syringe pump in conjunction with a bovine serum albumin-based buffer, we administered flow rates ranging from 10 to 100 ml/min into the flow channel. Subsequently, we collected the flow-through after each flow condition and determined the fluorescence intensity of quantum dots labelled A1. This enabled us to establish the critical flow rate at which the aptamer unfolds and releases A1, which initial data showed it to be around 50ml/min. (Fig. 5d).

Here we present a nanomaterial-based therapeutic system that mimics the natural shear-responsive function of VWF. To our knowledge, this is one of the first attempts to design single-molecule based biomaterials responsive to shear stimulation.  Compared with the various types of nanomaterials that have been used in shear-sensitive drug delivery systems in literature, including liposomes, polymeric nanoparticles, and micelles, single-molecule flow sensors better mimic the structure and function of circulating coagulation factors, thus are expected to have more accurate spatial and temporal control of the delivered functions.⁷

       In conclusion, the constructed SMORES material is promising to deliver bio-functions on a single molecular level, which is difficult to achieve using conventional drug carriers.  Ongoing research is focused on demonstrate the function of the release cargo and construct other flow sensitive conjugate for other applications.

The authors would like to acknowledge the generous support of the National Science Foundation (2004475) and National Institutes of Health (1R01HL151663). The authors would like to also acknowledge Professor Nathan Wittenberg for their assistance in TIRF microscopy and Dr. kip Kuttner for providing platelets samples.

1.         Frosen, J.; Cebral, J.; Robertson, A. M.; Aoki, T., Flow-induced, inflammation-mediated arterial wall remodeling in the formation and progression of intracranial aneurysms. Neurosurg. Focus 2019, 47 (1), 13.

2.         Rana, A.; Westein, E.; Niego, B.; Hagemeyer, C. E., Shear-Dependent Platelet Aggregation: Mechanisms and Therapeutic Opportunities. Front. Cardiovasc. Med. 2019, 6, 21.

3.         Trumbore, C. N., Shear-induced amyloid formation of IDPs in the brain. In Dancing Protein Clouds: Intrinsically Disordered Proteins in Health and Disease, Pt A, Uversky, V. N., Ed. Elsevier Academic Press Inc: San Diego, 2019; Vol. 166, pp 225-309.

4.         Petho, Z.; Najder, K.; Bulk, E.; Schwab, A., Mechanosensitive ion channels push cancer progression. Cell Calcium 2019, 80, 79-90.

5.         Zhang, X. H.; Halvorsen, K.; Zhang, C. Z.; Wong, W. P.; Springer, T. A., Mechanoenzymatic Cleavage of the Ultralarge Vascular Protein von Willebrand Factor. Science 2009, 324 (5932), 1330-1334.

6.         Smith, D. E.; Babcock, H. P.; Chu, S., Single-polymer dynamics in steady shear flow. Science 1999, 283 (5408), 1724-1727.

7.  Wang, Y. et al. (2021) “Recent developments in nanomaterial‐based shear‐Sensitive Drug Delivery Systems,” Advanced Healthcare Materials, 10(13), p. 2002196. Available at: https://doi.org/10.1002/adhm.202002196.