Avidity modulation of folate-targeted multivalent dendrimers for evaluating biophysical models of cancer targeting nanoparticles

Abstract

We investigated two types of generation 5 polyamidoamine (PAMAM) dendrimers, each conjugated stochastically with a mean number of 5 or 10 methotrexate (MTX) ligands per dendrimer (G5-MTX5, G5-MTX10), for their binding to surface-immobilized folate binding protein (FBP) as a function of receptor density. The binding study was performed under flow by surface plasmon resonance spectroscopy. Two multivalent models were examined to simulate binding of the dendrimer to the receptor surface, showing that at relatively high receptor density, both dendrimer conjugates exhibit high avidity. However, upon reducing the receptor density by a factor of 3 and 13 relative to the high density level, the avidity of the lower-valent G5-MTX5 decreases by up to several orders of magnitude (KD = nM to μM), whereas the avidity of G5-MTX10 remains largely unaffected regardless of the density variation. Notably, on the 13-fold reduced FBP surface, G5-MTX5 displays binding kinetics similar to that of monovalent methotrexate, which is patently different from the still tight binding of the higher-valent G5-MTX10. Thus, the binding analysis demonstrates that avidity displayed by multivalent MTX conjugates varies in response to the receptor density and can be modulated for achieving tighter, more specific binding to the higher receptor density by modulation of ligand valency. We believe this study provides experimental evidence supportive of the mechanistic hypothesis of multivalent NP uptake to a cancer cell over a healthy cell where the diseased cell expresses the folate receptor at higher density.

Figure 1

Central hypothesis for folate-targeted nanoparticle (NP) delivery in cancer. It is based on a biophysical mechanism in which a folate-conjugated multivalent dendrimer NP adsorbs more strongly to the malignant cell surface expressing folate receptor (FAR) in a higher density (B) than to the normal cell (A).

Figure 2

Structure of folic acid, and methotrexate, and a fifth generation (G5) PAMAM dendrimer conjugates G5-MTX_n and G5-5T₃-MTX₁₀.

Figure 3

Time-dependent uptake of G5-5T₃-MTX₁₀ in FAR-positive KB cancer cells measured by flow cytometry (A) and confocal fluorescence microscopy (B). The blue and the red fluorescence represent the nuclei stained with DAPI and the cytoplasm with 5TAMRA (5T) attached to dendrimer, respectively. Inset (bottom right; 20 hr)

Figure 4

Representative dose-dependent SPR sensograms of G5-MTX_n (n = 0, 5, 10) in high FBP density. Each sensorgram is corrected against the reference sensorgram: ΔRU = RU₁ (Fc1) − RU₂ (Fc2).

Figure 5

Effect of receptor density on the binding kinetics of G5-MTX_n (n = 5, 10). (A, B) Each plot shows average traces of sensorgrams (n = 3) obtained from the dendrimer injection at the variable level of FBP density. (C) A plot of fractional desorption of G5-MTX_n in response to variation in FBP density (n = 3; mean ± SD).

Figure 6

Models for adsorption of G5-MTX_n to surface immobilized folate binding protein (FBP) in a flow cell: surface conversion (A), dual Langmuir (B).

Figure 7

Normalized mass of G5-MTX_n adsorbed to the folate binding protein (FBP) surface and fraction of species B as a function of receptor density(A) Each ΔRU value (t = 175 s) was normalized relative to the value of G5-MTX₁₀ at the high FBP surface per identical concentration, which is arbitrarily taken as 100% (n = 3; mean ± SD). (B) Fraction of species B at the end of an association phase refers to the value extracted by the simulation in a surface conversion model (Table 1): Fraction B = Mass B ÷ (Mass A + Mass B).