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

Abstract

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 αβ could be modulated up to 15-fold by changing the ssDNA sequence. IC values varied from 309 nM for (C)-RGD/SWCNT hybrids to 29 nM for (GT)-RGD/SWCNT hybrids. When immobilized onto surface adhesion of epithelial cells increased 6-fold for (GT)-RGD/SWCNTs. (GT)-RGD/SWCNTs also increased the number of adhering human platelets by a factor of 4.8. Additionally, αβ 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.

Effect of Adhesion and Substrate Elasticity on Neutrophil Extracellular Trap Formation

“…Another factor affecting cell adhesion is the available surface concentration of integrin ligands 21,36 . By using approaches from nanotechnology it is possible to control the exact distance and overall density of integrin ligands such as RGD and the Mac-1 ligand GPB1 or even link them to advanced near infrared fluorescent nanomaterials 27,42,43 . For neutrophils it has been shown that adhesion maturation and cellular functions such as spreading and migration depend on ligand density 27 .…”

Nanoröhren‐Nanobody‐Konjugate als zielgerichtete Sonden und Marker für die In‐vivo‐Nahinfrarot‐Bildgebung

“…Durch die große Verfügbarkeit von transgenen Organismen und Zellen mit GFP‐Fusionsproteinen ist GFP ist ein sehr vielseitiges Zielmolekül. Um die nIR‐Fluoreszenz der SWCNTs nicht zu zerstören, verwenden wir eine nicht‐kovalente Funktionalisierungsstrategie, obwohl Setaro et al. kürzlich auch eine kovalente Funktionalisierungsstrategie unter Erhaltung der nIR‐Fluoreszenz beschrieben haben .…”

Exfoliated near infrared fluorescent CaCuSi4O10 nanosheets with ultra-high photostability and brightness for biological imaging

“…nIR fluorescent nanomaterials include InAs quantum dots, lanthanide doped nanoparticles or semiconducting single-walled carbon nanotubes (SWCNTs) [8][9][10][11][12] . For example, SWCNTs have been used as building blocks for nIR imaging and as fluorescent sensors that detect small signaling molecules, proteins or lipids [13][14][15][16][17][18][19] . They can be chemically tailored and have been used to reveal spatiotemporal release patterns of neurotransmitters from single cells 2,15,[20][21][22] .…”

“…For example, SWCNTs have been used as building blocks for nIR imaging and as fluorescent sensors that detect small signaling molecules, proteins or lipids [13][14][15][16][17][18][19] . They can be chemically tailored and have been used to reveal spatiotemporal release patterns of neurotransmitters from single cells 2,15,[20][21][22] . However, most nIR fluorescent nanomaterials often have low quantum yields, lack biocompatibility or are restricted to certain emission/excitation wavelengths.…”