Selective Integrin α5β1 Targeting through Spatially Constrained Multivalent DNA-Based Nanoparticles

Abstract

Targeting cells specifically based on receptor expression levels remains an area of active research to date. Selective binding of receptors cannot be achieved by increasing the individual binding strength, as this does not account for differing distributions of receptor density across healthy and diseased cells. Engaging receptors above a threshold concentration would be desirable in devising selective diagnostics. Integrins are prime target candidates as they are readily available on the cell surface and have been reported to be overexpressed in diseases. Insights into their spatial organization would therefore be advantageous to design selective targeting agents. Here, we investigated the effect of activation method on integrin α5β1 clustering by immunofluorescence and modeled the global neighbor distances with input from an immuno-staining assay and image processing of microscopy images. This data was used to engineer spatially-controlled DNA-scaffolded bivalent ligands, which we used to compare trends in spatial-selective binding observed across HUVEC, CHO and HeLa in resting versus activated conditions in confocal microscopy images. For HUVEC and CHO, the data demonstrated an improved selectivity and localisation of binding for smaller spacings ~7 nm and ~24 nm, in good agreement with the model. A deviation from the mode predictions for HeLa was observed, indicative of a clustered, instead of homogeneous, integrin organization. Our findings demonstrate how low-technology imaging methods can guide the design of spatially controlled ligands to selectively differentiate between cell type and integrin activation state.

Multivalent Pattern Recognition through Control of Nano-Spacing in Low-Valency Super-Selective Materials

“…The structure’s rigidity is related to sequence length (e.g., persistence length of the DNA double helix) as well as single- or double-stranded interactions . Finally, the complexity of DNA-based architectures ranges from low (a single helix) to very high, in the case of DNA-origami where hundreds of strands self-assemble into a macromolecular architecture of virtually any desired shape. ,,, Indeed, DNA-based nanomaterials have been used to explore the multivalent clustering of active cell surface receptors, and the importance of spatial organization was recently demonstrated in the case of apoptosis, , integrin targeting, and focal adhesion formation . The selective effect of patterns has been demonstrated in targeting of the dengue viral surface with aptamers organized in DNA star motifs .…”

Integration of functional peptides into nucleic acid-based nanostructures

“…Additionally, DNAscaffolded bivalent RGD ligands, displaying two RGD sequences in different spacings, were engineered to explore spatial distribution of integrin α5β1 on the cell surfaces of various cell lines. 123 A study carried out by Liu et al reported the generation and in vivo application of a DNA-structured cancer vaccine. The underlying nanostructures were composed of a DNA nanotube, which contained two types of toll-like receptor agonists (doublestranded RNA (dsRNA) and CpG DNA) along with a tumor antigen peptide (Fig.…”

“…Additionally, DNA-scaffolded bivalent RGD ligands, displaying two RGD sequences in different spacings, were engineered to explore spatial distribution of integrin α5β1 on the cell surfaces of various cell lines. 123…”

Balancing the Nanoscale Organization in Multivalent Materials for Functional Inhibition of the Programmed Death-1 Immune Checkpoint

“…In contrast to other available nanoparticles, DNA origami offers perfect control over size, shape, and surface properties guaranteeing uniform particle design. In addition to their inherent biocompatibility, the modulatory properties of nucleic acid-based systems allow the positioning of individual ligand with nanometer precision. − Benefiting from efforts on structural stabilization and surface characterization, the stability of DNA origami in biological environments as well as interactions with the biointerface are now well understood. − Together, these features make DNA origami a platform for controlled ligand presentation for receptor binding susceptible to nanoscale ligand presentation.…”