Avidity mechanism of dendrimer-folic acid conjugates

Abstract

Multivalent conjugation of folic acid has been employed to target cells overexpressing folate receptors. Such polymer conjugates have been previously demonstrated to have high avidity to folate binding protein. However, the lack of a monovalent folic acid-polymer material has prevented a full binding analysis of these conjugates, as multivalent binding mechanisms and polymer-mass mechanisms are convoluted in samples with broad distributions of folic acid-to-dendrimer ratios. In this work, the synthesis of a monovalent folic acid-dendrimer conjugate allowed the elucidation of the mechanism for increased binding between the folic acid-polymer conjugate and a folate binding protein surface. The increased avidity is due to a folate-keyed interaction between the dendrimer and protein surfaces that fits into the general framework of slow-onset, tight-binding mechanisms of ligand/protein interactions.

Figure 1

Distribution of conjugates resulting from a stochastic conjugation of 3 equiv of FA to 1 equiv of scaffold.

Figure 2

Proposed models for enhanced G5–FA binding to FBP. (a) Multivalent binding increases avidity with increasing valency. (b) Any multivalent binding (2 or more interactions) is irreversible, and monovalent binding is reversible. (c) FA “keys” the initial interaction between conjugate and FBP, which is followed by strong nonspecific interaction between the dendrimer and protein. C represents G5–FA_n conjugate, P is FBP, CP a complex between a conjugate and n ≥ 1 FBP. CP* is a tight complex formed by a conformation change in the polymer and the resulting polymer–protein interaction.

Figure 3

(a) Synthesis of PAMAM-COG conjugate. (b) Semiprep rp-HPLC isolation of PAMAM with 1, 2, 3, 4, or 5 COGs. (c) Isolated samples elute from rp-UPLC as a function of ligand-to-dendrimer ratio. (d) Scheme of G5-COG click reaction of γ-azide-FA.

Figure 4

SPR sensograms of conjugates (n = 1.0, red; n = 1.2, orange; n = 1.9, green; n = 2.7, blue) and controls (n = 0, gray; free FA, purple) on lower density chip. The color gradient represents concentration from low (light) to high (dark). Free FA samples were run at millimolar as opposed to micromolar concentrations to obtain adequate signal.

Figure 5

SPR sensograms of conjugates (n = 1.0, red; n = 1.2, orange; n = 1.9, green; n = 2.7, blue) and controls (n = 0, gray; free FA, purple) on higher density chip. The color gradient represents concentration from low (light) to high (dark).

Figure 6

Comparison of distributions in click reaction products vs theoretical stochastically conjugated products (purple bars) of the same average for ratios of (a) 1.0 (red bars), (b) 1.2 (orange bars), (c) 1.9 (green bars), and (d) 2.7 (blue bars).

Figure 7

Definition of fitting parameters.

Figure 8

Saturation of irreversible bound material (y°) as a function of FA concentration.