Control of Integrin Affinity by Confining RGD Peptides on Fluorescent Carbon Nanotubes

Integrins are transmembrane receptors that mediate cell-adhesion, signaling cascades and platelet-mediated blood clotting. Most integrins bind to the common short peptide Arg-Gly-Asp (RGD). The conformational freedom of the RGD motif determines how strong and to which integrins it binds. Here, we present a novel approach to tune binding constants by confining RGD peptide motifs via noncovalent adsorption of single-stranded DNA (ssDNA) anchors onto single-walled carbon nanotubes (SWCNTs). Semiconducting SWCNTs display fluorescence in the near-infrared (nIR) region and are versatile fluorescent building blocks for imaging and biosensing. The basic idea of this approach is that the DNA adsorbed on the SWCNT surface determines the conformational freedom of the RGD motif and affects binding affinities. The RGD motif was conjugated to different ssDNA sequences in both linear ssDNA-RGD and bridged ssDNA-RGD-ssDNA geometries. Molecular dynamics (MD) simulations show that the RGD motif in all the synthesized systems is mostly exposed to solvent and thus available for binding, but its flexibility depends on the exact geometry. The affinity for the human platelet integrin α_IIbβ₃ could be modulated up to 15-fold by changing the ssDNA sequence. IC₅₀ values varied from 309 nM for (C)₂₀-RGD/SWCNT hybrids to 29 nM for (GT)₁₅-RGD/SWCNT hybrids. When immobilized onto surface adhesion of epithelial cells increased 6-fold for (GT)₁₅-RGD/SWCNTs. (GT)₁₅-RGD/SWCNTs also increased the number of adhering human platelets by a factor of 4.8. Additionally, α_IIbβ₃ integrins on human platelets were labeled in the nIR by incubating them with these ssDNA-peptide/SWCNT hybrids. In summary, we show that ssDNA-peptide hybrid structures noncovalently adsorb onto SWCNTs and serve as recognition units for cell surface receptors such as integrins. The DNA sequence affects the overall RGD affinity, which is a versatile and straightforward approach to tune binding affinities. In combination with the nIR fluorescence properties of SWCNTs, these new hybrid materials promise many applications in integrin targeting and bioimaging.