Single-Molecule Force Probing of RGD-Binding Integrins on Pancreatic Cancer Cells

Abstract

Integrin-targeting arginine–glycine–aspartic acid (RGD)-based nanocarriers have been widely used for tumor imaging, monitoring of tumor development, and delivery of anticancer drugs. However, the thermodynamics of an RGD–integrin formation and dissociation associated with binding dynamics, affinity, and stability remains unclear. Here, we probed the binding strength of the binary complex to live pancreatic cancer cells using single-molecule binding force spectroscopy methods, in which RGD peptides were functionalized on a force probe tip through poly­(ethylene glycol) (PEG)-based bifunctional linker molecules. While the density of integrin αV receptors on the cell surface varies more than twofold from cell line to cell line, the individual RGD–integrin complexes exhibited a cell type-independent, monovalent bond strength. The load-dependent bond strength of multivalent RGD–integrin interactions scaled sublinearly with increasing bond number, consistent with the noncooperative, parallel bond model. Furthermore, the multivalent bonds ruptured sequentially either by one or in multiples, and the force strength was comparable to the synchronous rupture force. Comparison of energy landscapes of the bond number revealed a substantial decrease of kinetic off-rates for multivalent bonds, along with the increased width of the potential well and the increased potential barrier height between bound and unbound states, enhancing the stability of the multivalent bonds between them.

Single-molecule scale quantification reveals interactions underlying protein–protein interface: from forces to non-covalent bonds

“…In our experiments, most of the unbinding events exhibit single synchronous ruptures, and less than 1% of the bonds are ruptured sequentially. As previously described, 46 asynchronous rupture may be attributed to intrinsic mechanical or geometric constraints in pulling the complex. In this case, the breaking of multiple-bonds is considered a single apparent bond (SAB).…”

“…Our data support these observations since multiple-peaks in the bond unbinding force distribution correspond to the number of binding pairs. 45,46 The multivalent BAX/Bcl-2 bonds require greater unbinding forces than single-bond ruptures. In support of the hypothesis of multiple bond ruptures, we extended the single-bond model to a multiple-bond system consisting of N pairs of proteins and quantified the mechanical features of multivalency relationship between BAX/Bcl-2.…”

Modified Bovine Milk Exosomes for Doxorubicin Delivery to Triple-Negative Breast Cancer Cells

“…The iRGD peptide interacts with α v β 3 integrin and NRP-1, both upregulated on cancer cells and facilitates targeting and penetration of the exosomes (illustrated in Figure ). − To visualize the iRGD peptide integrated into the exosomes’ lipid bilayer, DSPE-PEG 5000 -FITC was incorporated into iHRX (CF-iHRX) and exosomes. The surface of the slides were coated with the α v β 3 integrin allowing for iRGD peptide to attach to the surface.…”

Two molecule force spectroscopy on ligand–receptor interactions

“…However, in biological processes, such as cell adhesion, multiple ligand-receptor pairs (which can be the same or different) are often involved and can be arranged in parallel, exhibiting multivalent ligand binding/unbinding. [24][25][26][27][28][29] Probing the rupture of such parallel multiple ligand-receptor pairs (or multivalent ligand-receptor interactions) is thus critical for comprehending the unbinding mechanisms of these systems in a biologically relevant context, and for discerning whether such multiple ligand-receptor pairs behave independently or synergistically.…”

“…Despite these theoretical progresses, rigorously testing these predictions has been challenging. Previous efforts 24,25,28,37 relied on the uncontrolled formation of multiple ligand-receptors during SMFS unbinding experiments. In a typical SMFS unbinding experiment, ligand-receptor pairs form while the AFM tip is brought into contact with the substrate.…”