Impact of structurally modifying hyaluronic acid on CD44 interaction

Abstract

CD44 is a widely-distributed type I transmembrane glycoprotein that binds hyaluronic acid (HA) in most cell types, including primary tumor cells and cancer-initiating cells and has roles in cell migration, cell-cell, and cell-matrix adhesion. HA-derived conjugates and nanoparticles that target the CD44 receptor on cells have been reported for targeted delivery of therapeutics and imaging agents. Altering crucial interactions of HA with CD44 active sites holds significant importance in modulating targeting ability of hyaluronic acid to other cancer types that do not express the CD44 receptor or minimizing the interaction with CD44⁺ cells that are not target cells. The approach adopted here was deacetylation of the N-acetyl group and selective sulfation on the C6-OH on the HA polymer, which form critical interactions with the CD44 active site. Major interactions identified by molecular modeling were confirmed to be hydrogen bonding of the C6-OH with Tyr109 and hydrophobic interaction of the N-acetyl group with Tyr46, 83 and Ile 92. Modified HA was synthesized and characterized and its interactions were assessed by in vitro and molecular modeling approaches. In vitro techniques included flow cytometry and fluorescence polarization, while in silico approaches included docking and binding calculations by a MM-PBSA approach. These studies indicated that while both deacetylation and sulfation of HA individually decrease CD44 interaction, both chemical modifications are required to minimize interaction with CD44⁺ cells. The results of this study represent the first step to effective retargeting of HA-derived NPs for imaging and drug delivery.

Figure 1

Interactions of {β1→3} N-acetylglucosamine (GlcNAc) and {β1→4} glucuronic acid (GlcUA) repeat unit of hyaluronic acid with crucial amino acids in the murine CD44 active site.

Figure 2

FTIR spectra of native and modified 10 kDa HA.

Figure 3

Zeta potential of HA and modified HA.

Figure 4

CD44 expression studies. Flow-cytometry histograms showing the expression of CD44 receptor (left plot) and the ability for HA to block HA-FITC binding (right plot) of CD44⁺ (A) MDA-MB-231, (B) RKO, (C) PC-3 and CD44⁻ (D) LNCaP cell lines. HA competitively inhibited HA-FITC binding in CD44+ cell lines, while CD44- cell lines had lower HA-FITC signal and no difference in signal after competition with HA.

Figure 5

Competition binding assay using flow cytometry analysis. Binding of HA-FITC (20 μg/ml) for 1 hr in the presence of modified HA and native HA determined in (A) MDA-MB-231, (B) RKO, (C) PC-3, and (D) LNCaP cancer cell lines. Negative control comprised of incubation of cells with unlabeled HA while positive control cells were incubated only with HA-FITC. The left column indicates fluorescence intensities obtained after competition binding of modified HA polymers during flow analysis, while the right column indicates the normalized fluorescence intensities based on the obtained negative and positive fluorescence intensities. Data are shown as mean ± S.D., * p<0.05, ** p<0.01, *** p<0.001; One-way ANOVA with Tukey multiple comparisons test.

Figure 6

(A) Binding curve isotherm of HA-FITC (50 nM) to CD44-Fc protein (130 nM-72 μM). K_d value obtained for the conjugate = 21 μM. Values are expressed as mean ± s.d of n=3 (B) Effect of increasing concentration of unlabeled derivatives of HA (64nM- 1000μM) and constant CD44 concentration of 23.4 μM competing with pre-incubated HA-FITC (50 nM).

Figure 7

Docking energy calculated by Autodock software of HA and modified HA derivatives with CD44 indicate that each of the modifications had a lower binding energy compared to native HA with CD44. Graphs show mean ± SD (n=10), **** p < 0.0001; One-way ANOVA with Tukey multiple comparisons test.

Scheme 1

Synthesis of deacetylated, sulfated, and deacetylated and sulfated HA. i: ion exchange with TBA in H₂O; ii: hydrazine, hydrazine sulfate, DMF; and iii: sulfur trioxide pyridine, DMF.