Binding affinity and kinetic analysis of targeted small molecule-modified nanoparticles

Abstract

Nanoparticles bearing surface-conjugated targeting ligands are increasingly being explored for a variety of biomedical applications. The multivalent conjugation of targeting ligands on the surface of nanoparticles is presumed to enhance binding to the desired target. However, given the complexities inherent in the interactions of nanoparticle surfaces with proteins, and the structural diversity of nanoparticle scaffolds and targeting ligands, our understanding of how conjugation of targeting ligands affects nanoparticle binding remains incomplete. Here, we use surface plasmon resonance (SPR) to directly and quantitatively study the affinity and binding kinetics of nanoparticles that display small molecules conjugated to their surface. We studied the interaction between a single protein target and a structurally related series of targeting ligands whose intrinsic affinity varies over a 4500-fold range and performed SPR at protein densities that reflect endogenous receptor densities. We report that even weak small molecule targeting ligands can significantly enhance target-specific avidity (by up to 4 orders of magnitude) through multivalent interactions and also observe a much broader range of kinetic effects than has been previously reported. Quantitative measurement of how the affinity and kinetics of nanoparticle binding vary as a function of different surface conjugations is a rapid, generalizable approach to nanoparticle characterization that can inform the design of nanoparticles for biomedical applications.

Figure 1

Specific protein-nanoparticle binding documented in SPR sensorgrams. A. Nanoparticle intermediate 3 does not bind to a control surface of GST. B. Targeted nanoparticle 4a(17) does not bind to a GST surface. C. Sensorgrams for targeted nanoparticle 4a(17) at different immobilization densities of FKBP12-GST. Significant binding occurs; appreciable dissociation only occurs at the lowest protein density tested. D. Representative sensorgram for a free small molecule (1e) flowed over an FKBP12-GST surface. Inset: Depiction of calculating K_D from steady state affinity studies. E. Representative sensorgram for multivalent nanoparticle 4e(5). In figure parts D. and E., red curves depict experimental data at different analyte concentrations; fitted curves modeled to describe a 1:1 binding event are overlaid in black.

Figure 2

Rate maps summarizing binding affinity and kinetics. Different combinations of k_a and k_d that result in the same K_D are indicated by dashed lines. Data for free ligands is depicted by open symbols; for each ligand, the corresponding nanoparticles are depicted by a solid symbol of the same shape, with conjugation valency listed in parentheses next to the solid symbol. Valency of 8(nd) was not determined.

Figure 3

Relative contribution of monovalent vs. bivalent reaction terms to nanoparticle binding as a function of the intrinsic K_D of the free ligand. A. As the intrinsic affinity of the free ligand becomes weaker, K_D1/K_D2 increases (R² = 0.57). B. No correlation between k_a1/k_a2 and intrinsic affinity of the free ligand. C. As the intrinsic affinity of the free ligand becomes weaker, k_d1/k_d2 increases (R² = 0.76).

Scheme 1

Conjugation of small molecules to nanoparticles. A. Conjugation of a series of synthetic derivatives of FK506 (1) by sulfhydryl exchange. B. Conjugation of a small molecule that binds aurora A kinase (6) by sulfhydryl exchange and Huisgen 1,3-dipolar cycloaddition.