Nanopatterns of Surface-Bound EphrinB1 Produce Multivalent Ligand–Receptor Interactions That Tune EphB2 Receptor Clustering

Abstract

Here we present a nanostructured surface able to produce multivalent interactions between surface-bound ephrinB1 ligands and membrane EphB2 receptors. We created ephrinB1 nanopatterns of regular size (<30 nm in diameter) by using self-assembled diblock copolymers. Next, we used a statistically enhanced version of the Number and Brightness technique, which can discriminate-with molecular sensitivity-the oligomeric states of diffusive species to quantitatively track the EphB2 receptor oligomerization process in real time. The results indicate that a stimulation using randomly distributed surface-bound ligands was not sufficient to fully induce receptor aggregation. Conversely, when nanopatterned onto our substrates, the ligands effectively induced a strong receptor oligomerization. This presentation of ligands improved the clustering efficiency of conventional ligand delivery systems, as it required a 9-fold lower ligand surface coverage and included faster receptor clustering kinetics compared to traditional cross-linked ligands. In conclusion, nanostructured diblock copolymers constitute a novel strategy to induce multivalent ligand-receptor interactions leading to a stronger, faster, and more efficient receptor activation, thus providing a useful strategy to precisely tune and potentiate receptor responses. The efficiency of these materials at inducing cell responses can benefit applications such as the design of new bioactive materials and drug-delivery systems.

Emerging Applications of Nanotechnology for Controlling Cell‐Surface Receptor Clustering

“…Many interdisciplinary efforts have been made to control nanoscale molecular interactions at cellular boundaries.F or example,several technologies that enable the local control of the spatial distribution of cell surface receptors have been developed, such as molecular nanopatterns,m agnetic actuation, biopolymers,a nd DNAn anostructures. [18][19][20][21][22][23] Thef eatures of some important technologies to control the clustering of cell surface receptors are summarized in Table 1.…”

“…[35] In ar ecent work, the Martinez group developed an ovel technique,t ermed enhanced Number and Brightness (eN&B), to measure the kinetics of eph-receptor oligomerization in live cells. [18] Thee N&B technique can discriminate between the oligomeric states of eph-B2 receptors with molecular sensitivity and quantitatively track the eph-B2 receptor oligomerization process in real time.T heir results indicated that randomly distributed surface-bound ligands were not sufficient to fully induce receptor aggregation. However,the nanopatterned ligands could effectively induce strong receptor oligomerization [18] (Figure 2).…”

“…[18] Thee N&B technique can discriminate between the oligomeric states of eph-B2 receptors with molecular sensitivity and quantitatively track the eph-B2 receptor oligomerization process in real time.T heir results indicated that randomly distributed surface-bound ligands were not sufficient to fully induce receptor aggregation. However,the nanopatterned ligands could effectively induce strong receptor oligomerization [18] (Figure 2). Molecular nanopatterns provide au seful strategy to induce multivalent ligand-receptor interactions,l eading to stronger,f aster, and more efficient receptor activation for precise control of the receptor responses.H owever,t he nanopatterns are mainly permanent structures that are not applicable to the tunable and reversible control of cell surface receptors.F urthermore,t he nanopatterns cannot be in as olution phase,o rd irectly added to cell culture medium under standard cell culture conditions.T herefore,a lternate, more convenient, strategies are required to control the clustering of cell surface receptors.…”

“…Since eph-ephrin interactions are highly sensitive to multivalent binding processes,s everal strategies have been developed to control the nanoscale distribution of ephrin, which lead to amore efficient eph-receptor clustering and downstream signaling. [18,20] Thef ormation of receptor cluster complexes is not restricted to the integrin family or eph receptors.V arious cell surface receptors from different structural classes can assemble into multi-receptor complexes,triggering many biological functions,s uch as Tc ell receptors (TCR) for immune recognition, [30] methyl-accepting chemotaxis proteins (MCPs) for chemotaxis,a nd mGluR for neuronal signaling. [31] These dimeric or oligomeric complexes govern cellular responses with surprisingly high sensitivity,which provides ameans for controlling cellular signaling cascades through interaction between nanomaterial and cell surface receptors.…”

Direct Measurement of Through‐Bond Effects in Molecular Multivalent Interactions

“…Multivalent interactions are ubiquitous in biology,w hich involve the bindingo fm ultiple ligandso fab iological entity to multiple binding pocketso rr eceptors of at arget, for example, ap rotein,v irus, bacterium, or cell. [1][2][3][4] Multivalent interactions have an umber of benefits over monovalenti nteractions, such as i) possessing higherb inding affinity;i i) standing ab etter chance of providing higher selectivity in target recognition;i ii) having stronger binding properties from weak homo-o rh eterogeneous ligands. [5] Scientists take these benefits to design new therapeutic entities,e specially those concerned with the allostericm odulation of the receptor [6] and the repetitive epitopes of antibodies.…”