Dendrimer-mediated multivalent binding for the enhanced capture of tumor cells.

Multivalent binding, the simultaneous binding of multiple ligands to multiple receptors, has played a central role in a number of pathological processes, including the attachment of viral, parasitic, mycoplasmal, and bacterial pathogens. These biological activities have been extensively investigated to promote targeting of specific cell types, and biological multivalent inhibitors have yielded significant increases in binding avidities by one to nine orders of magnitude. In particular, nanoscale poly(amidoamine) (PAMAM) dendrimers have been reported to be an excellent mediator for facilitated multivalent effect owing to their capability to preorganize/orient ligands and the easy deformability of the polymer chains. We hypothesized that the advantages of enhanced binding avidity through the dendrimer-mediated multivalent effect could significantly improve detection of human diseaserelated rare cells (< 0.1% subpopulation), such as circulating tumor cells (CTCs) in peripheral blood. Given the extreme rareness of CTCs (as few as one out of a billion hematologic cells), a very sensitive, specific detection is obviously necessary to achieve clinically significant CTC detection. Many efforts to increase sensitivity of CTC devices have been reported, which are mostly based upon engineering, such as topographical modifications and chaotic mixer fluidics. Herein, we have investigated a new approach to exploit naturally occurring processes using nanotechnology, that is, biomimetic nanotechnology. To create a highly sensitive surface utilizing the multivalent effect, we have employed seventh-generation (G7) PAMAM dendrimers and the antiepithelial cell adhesion molecule (aEpCAM), as illustrated in Figure 1. Note that aEpCAM is one of the most commonly used CTC capturing agents, as EpCAM is often expressed by CTCs but not by normal hematological cells. G7 PAMAM dendrimers were chosen owing to their adequate size (8–10 nm in diameter) and number of surface functional groups (512 theoretically) to accommodate multiple aEpCAM (around 5.5 nm in diameter of Fc region) per dendrimer, thereby enabling multivalent binding. Furthermore, another physiological process cell rolling mediated by E-selectin, mimicking the initial CTC recruiting process to the endothelia, has been also implemented to our device to further enhance surface sensitivity and specificity towards tumor cells. To investigate the dendrimer-mediated multivalent binding, we directly measured the binding behaviors of the G7aEpCAM conjugates using surface plasmon resonance (SPR). G7 PAMAM dendrimers were carboxylated and conjugated with aEpCAM, which was confirmed by H NMR and size/zeta potential analyses (for details, see the Supporting Information). The UVanalysis revealed that 2.8 and 4.9 aEpCAM molecules were conjugated per dendrimer, resulting in G7-(aEpCAM)2.8 and G7-(aEpCAM)4.9, respectively. The binding parameters of the G7-aEpCAM conjugates to EpCAM-immobilized sensor chips were recorded and compared to those of free aEpCAM. The carboxylated G7 PAMAM dendrimers without aEpCAM showed no nonspecific binding, assuring that the observed binding events of the G7-aEpCAM conjugates are the result of specific EpCAM-aEpCAM interactions. The SPR sensorgrams (Supporting Information, Figure S5) were used to obtain the quantitative binding kinetic parameters, such as association rate constant (ka) and dissociation rate constant (kd; Table 1). Dissociation constants (KD) were calculated from the measured ka and kd (KD= kd/ka= 1/KA), where a lower value of KD corresponds to a stronger binding strength. As listed in Table 1, the dendrimer conjugates show significantly lower KD values than free aEpCAM. The Figure 1. Illustration and fluorescence images of tumor cell capture on surfaces using aEpCAM immobilized with a) dendrimers and b) linear poly(ethylene glycol).