Biocompatible glyconanomaterials based on HPMA-copolymer for specific targeting of galectin-3

Abstract

Background: Galectin-3 (Gal-3) is a promising target in cancer therapy with a high therapeutic potential due to its abundant localization within the tumor tissue and its involvement in tumor development and proliferation. Potential clinical application of Gal-3-targeted inhibitors is often complicated by their insufficient selectivity or low biocompatibility. Nanomaterials based on N-(2-hydroxypropyl)methacrylamide (HPMA) nanocarrier are attractive for in vivo application due to their good water solubility and lack of toxicity and immunogenicity. Their conjugation with tailored carbohydrate ligands can yield specific glyconanomaterials applicable for targeting biomedicinally relevant lectins like Gal-3.

Results: In the present study we describe the synthesis and the structure-affinity relationship study of novel Gal-3-targeted glyconanomaterials, based on hydrophilic HPMA nanocarriers. HPMA nanocarriers decorated with varying amounts of Gal-3 specific epitope GalNAcβ1,4GlcNAc (LacdiNAc) were analyzed in a competitive ELISA-type assay and their binding kinetics was described by surface plasmon resonance. We showed the impact of various linker types and epitope distribution on the binding affinity to Gal-3. The synthesis of specific functionalized LacdiNAc epitopes was accomplished under the catalysis by mutant β-N-acetylhexosaminidases. The glycans were conjugated to statistic HPMA copolymer precursors through diverse linkers in a defined pattern and density using Cu(I)-catalyzed azide-alkyne cycloaddition. The resulting water-soluble and structurally flexible synthetic glyconanomaterials exhibited affinity to Gal-3 in low μM range.

Conclusions: The results of this study reveal the relation between the linker structure, glycan distribution and the affinity of the glycopolymer nanomaterial to Gal-3. They pave the way to specific biomedicinal glyconanomaterials that target Gal-3 as a therapeutic goal in cancerogenesis and other disorders.

Keywords: Carbohydrate; ELISA; Galectin-3; Glyconanomaterial; HPMA copolymer; Surface plasmon resonance.

Scheme 1

Enzymatic syntheses of LacdiNAc standard (5) and of functionalized disaccharides 6 and 7. LacdiNAc (5) was prepared from donor 1 and GlcNAc (2) under the catalysis by Tyr470His mutant of the β-N-acetylhexosaminidase from Talaromyces flavus in (i), McIlvaine buffer pH 5.0/acetonitrile, 9/1, v/v, 45 °C, 4 h; disaccharide 6 was prepared from donor 1 and acceptor 3 under the catalysis by Tyr470His mutant of the β-N-acetylhexosaminidase from Talaromyces flavus in (ii), McIlvaine buffer pH 5.0/acetonitrile, 9/1, v/v, 45 °C, 5 h; disaccharide 7 was prepared from donor 1 and acceptor 4 using the same enzyme in (iii), McIlvaine buffer pH 5.0/acetonitrile, 9/1, v/v, 45 °C, 6.5 h

Scheme 2

a Synthesis of copolymers 13–18 and glyconanomaterials 24–37; b Synthesis of copolymer 23 and glyconanomaterials 38 and 39

Scheme 3

Structures of glyconanomaterials 24–39. The contents of LacdiNAc disaccharide in the glyconanomaterials are given in Table 2

Fig. 1

Competitive inhibition of Gal-3 binding to ASF by multivalent glyconanomaterials 24–39 as determined by ELISA. The following sample dose response inhibition curves are shown. a Inhibition by monovalent disaccharides lactose, LacNAc and LacdiNAc, by glyconanomaterials 30, 39 (two best inhibitors in the series), and 34 (the worst inhibitor in the series). The sigmoidal curves yield the values of IC₅₀ for the respective compounds. b Inhibition by glyconanomaterials 30, 31, and 32 (structural motif LacdiNAc-N-triazole). Here, the concentrations are calculated for the active substance of the glycomaterial—LacdiNAc glycan—and thus the curves yield the values of IC₅₀ per glycan. The higher the density of glycans on the polymer backbone, the lower the inhibitory potency per glycan. c Inhibition by glyconanomaterials 24, 29, 34 and 35 (containing various linkers with a comparable molar content of LacdiNAc). Here again, the curves yield the values of IC₅₀ per glycan. The type of attachment to the polymer backbone predestined the inhibitory potency per glycan

Fig. 2

SPR analysis of kinetics of the interactions between the immobilized Gal-3-AVI and glyconanomaterials 24, 26, 28 (carrying the LacdiNAc-OEt-triazole motif), 30, 31 (the LacdiNAc-N-triazole motif), 36, 37 (the LacdiNAc-triazole-phenyl motif) 38, 39 (the (LacdiNAc-triazole)₂-phenyl motif). Each of the sensograms represents “one-shot kinetics” data obtained with two-fold dilutions (10–0.625 µM) of the respective glyconanomaterial