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.

Institute of Macromolecular Chemistry Czech Academy of Sciences Heyrovský Sq 2 16206 Prague 6 Czech Republic

Institute of Microbiology Czech Academy of Sciences Vídeňská 1083 14220 Prague 4 Czech Republic

Laboratory for Biomaterials Institute for Biotechnology and Helmholtz Institute for Biomedical Engineering RWTH Aachen University Pauwelsstraße 20 52074 Aachen Germany

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Rabinovich GA, van Kooyk Y, Cobb BA. Glycobiology of immune responses. Ann N Y Acad Sci. 2012;1253:1–15. doi: 10.1111/j.1749-6632.2012.06492.x. PubMed DOI PMC

Mrázek H, Weignerová L, Bojarová P, Novák P, Vaněk O, Bezouška K. Carbohydrate synthesis and biosynthesis technologies for cracking of the glycan code: recent advances. Biotechnol Adv. 2013;31:17–37. doi: 10.1016/j.biotechadv.2012.03.008. PubMed DOI

Lundquist JJ, Toone EJ. The cluster glycoside effect. Chem Rev. 2002;102(555–57):8. PubMed

Bojarová P, Křen V. Sugared biomaterial binding lectins: achievements and perspectives. Biomater Sci. 2016;4:1142–1160. doi: 10.1039/C6BM00088F. PubMed DOI

Remzi Becer C. The glycopolymer code: synthesis of glycopolymers and multivalent carbohydrate-lectin interactions. Macromol Rapid Commun. 2012;33:742–752. doi: 10.1002/marc.201200055. PubMed DOI

Eissa M, Cameron NR. Glycopolymer conjugates. Adv Polym Sci. 2013;253(71–11):4.

Kolb HC, Finn MG, Sharpless KB. Click chemistry: diverse chemical function from a few good reactions. Angew Chem Int Ed. 2001;40:2004–2021. doi: 10.1002/1521-3773(20010601)40:11<2004::AID-ANIE2004>3.0.CO;2-5. PubMed DOI

Ulbrich K, Šubr V. Structural and chemical aspects of HPMA copolymers as drug carriers. Adv Drug Deliv Rev. 2010;62:150–166. doi: 10.1016/j.addr.2009.10.007. PubMed DOI

Kostková H, Schindler L, Kotrchová L, Kovář M, Šírová M, Kostka L, Etrych T. Star polymer-drug conjugates with pH-controlled drug release and carrier degradation. J. Nanomater. 2017;2017:8675435. doi: 10.1155/2017/8675435. DOI

Kunjachan S, Gremse F, Theek B, Koczera P, Pola R, Pechar M, Etrych T, Ulbrich K, Storm G, Kiessling F, Lammers T. Non-invasive optical imaging of nanomedicine biodistribution. ACS Nano. 2013;7:252–262. doi: 10.1021/nn303955n. PubMed DOI PMC

Koziolová E, Goelb S, Chytil P, Janoušková O, Barnhart TE, Cai W, Etrych T. A tumor-targeted polymer theranostics platform for positron emission tomography and fluorescence imaging. Nanoscale. 2017;9:10906–18. doi: 10.1039/C7NR03306K. PubMed DOI PMC

Quan L, Zhang Y, Crielaard B, Dusad A, Lele S, Rijcken C, Metselaar B, Kostková H, Etrych T, Ulbrich K, Hennink W, Storm G, Lammers T, Wang D. Nanomedicine for inflammatory arthritis: head-to-head comparison of glucocorticoid-containing polymers, micelles and liposomes. ACS Nano. 2014;8:458–466. doi: 10.1021/nn4048205. PubMed DOI PMC

Maeda H, Wu J, Sawa T, Matsumura Y, Hori K. Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review. J Control Release. 2000;65:271–284. doi: 10.1016/S0168-3659(99)00248-5. PubMed DOI

Hirabayashi J, Kasai K. The family of metazoan metal-independent beta-galactoside-binding lectins: structure, function and molecular evolution. Glycobiology. 1993;3:297–304. doi: 10.1093/glycob/3.4.297. PubMed DOI

Ebrahim H, Alalawi Z, Mirandola L, Rakhshanda R, Dahlbeck S, Nguyen D, Jenkins M, Grizzi F, Cobos E, Figueroa JA, Chiriva-Internati M. Galectins in cancer: carcinogenesis, diagnosis and therapy. Ann Transl Med. 2014;2:88. PubMed PMC

Bumba L, Laaf D, Spiwok V, Elling L, Křen V, Bojarová P. Poly-N-Acetyllactosamine neo-glycoproteins as nanomolar ligands of human galectin-3: binding kinetics and modeling. Int J Mol Sci. 2018;19:372. doi: 10.3390/ijms19020372. PubMed DOI PMC

Šimonová A, Kupper CE, Böcker S, Müller A, Hofbauerová K, Pelantová H, Elling L, Křen V, Bojarová P. Chemo-enzymatic synthesis of LacdiNAc dimers of varying length as novel galectin ligands. J Mol Catal B Enzym. 2014;101:47–55. doi: 10.1016/j.molcatb.2013.12.018. DOI

Laaf D, Bojarová P, Mikulová B, Pelantová H, Křen V, Elling L. Two-Step Enzymatic synthesis of β-d-N-acetylgalactosamine-(1 → 4)-d-N-acetylglucosamine (LacdiNAc) chitooligomers for deciphering galectin binding behavior. Adv Synth Catal. 2017;359:2101–2108. doi: 10.1002/adsc.201700331. DOI

Jin C, Kenny DT, Skoog EC, Padra M, Adamczyk B, Vitizeva V, Thorell A, Venkatakrishnan V, Lindén SK, Karlsson NG. Structural diversity of human gastric mucin glycans. Mol Cell Proteomics. 2017;16:743–758. doi: 10.1074/mcp.M117.067983. PubMed DOI

Hirano K, Matsuda A, Shirai T, Furukawa K. Expression of LacdiNAc groups on N-glycans among human tumors is complex. Biomed Res Int. 2014;2014:981627. doi: 10.1155/2014/981627. PubMed DOI PMC

Morris S, Ahmad N, André S, Kaltner H, Gabius HJ, Brenowitz M, Brewer F. Quaternary solution structures of galectins-1,-3, and -7. Glycobiology. 2004;14:293–300. doi: 10.1093/glycob/cwh029. PubMed DOI

Ahmad N, Gabius HJ, André S, Kaltner H, Sabesan S, Roy R, Liu B, Macaluso F, Brewer CF. Galectin-3 precipitates as a pentamer with synthetic multivalent carbohydrates and forms heterogeneous cross-linked complexes. J Biol Chem. 2004;279:10841–10847. doi: 10.1074/jbc.M312834200. PubMed DOI

Lepur A, Salomonsson E, Nilsson UJ, Leffler H. Ligand induced galectin-3 protein self-association. J Biol Chem. 2012;287:21751–217516. doi: 10.1074/jbc.C112.358002. PubMed DOI PMC

Halimi H, Rigato A, Byrne D, Ferracci G, Sebban-Kreuzer C, El Antak L, Guerlesquin F. Glycan dependence of Galectin-3 self-association properties. PLoS ONE. 2014;9:e111836. doi: 10.1371/journal.pone.0111836. PubMed DOI PMC

Öberg T, Leffler H, Nilsson UJ. Inhibition of galectins with small molecules. Chimia. 2011;65:18–23. doi: 10.2533/chimia.2011.18. PubMed DOI

Cumpstey I, Sundin A, Leffler H, Nilsson UJ. C2-symmetrical thiodigalactoside bis-benzamido derivatives as high-affinity inhibitors of galectin-3: efficient lectin inhibition through double arginine-arene interactions. Angew Chem Int Ed. 2005;44:5110–5112. doi: 10.1002/anie.200500627. PubMed DOI

Wang H, Huang W, Orwenyo J, Banerjee A, Vasta GR, Wang LX. Design and synthesis of glycoprotein-based multivalent glyco-ligands for influenza hemagglutinin and human galectin-3. Bioorg Med Chem. 2013;21:2037–2044. doi: 10.1016/j.bmc.2013.01.028. PubMed DOI PMC

Laaf D, Bojarová P, Pelantová H, Křen V, Elling L. Tailored multivalent neo-glycoproteins: synthesis, evaluation, and application of a library of galectin-3-binding glycan ligands. Bioconjug Chem. 2017;28:2832–2840. doi: 10.1021/acs.bioconjchem.7b00520. PubMed DOI

Blanchard H, Bum-Erdene K, Hugo MW. Inhibitors of galectins and implications for structure-based design of galectin-specific therapeutics. Aust J Chem. 2014;67:1763–1779. doi: 10.1071/CH14362. DOI

Böcker S, Laaf D, Elling L. Galectin binding to neo-glycoproteins: LacdiNAc conjugated BSA as ligand for human galectin-3. Biomolecules. 2015;5:1671–1696. doi: 10.3390/biom5031671. PubMed DOI PMC

David A, Kopečková P, Minko T, Rubinstein A, Kopeček J. Design of a multivalent galactoside ligand for selective targeting of HPMA copolymer–doxorubicin conjugates to human colon cancer cells. Eur J Cancer. 2004;40:148–157. doi: 10.1016/j.ejca.2003.07.001. PubMed DOI

Blanchard H, Yu X, Collins PM, Bum-Erdene K. Galectin-3 inhibitors: a patent review (2008–present) Expert Opin Ther Pat. 2014;24:1053–1065. doi: 10.1517/13543776.2014.947961. PubMed DOI

Bojarová P, Křen V. Glycosidases in carbohydrate synthesis: when organic chemistry falls short. Chimia. 2011;65:65–70. doi: 10.2533/chimia.2011.65. PubMed DOI

Slámová K, Krejzová J, Marhol P, Kalachova L, Kulik N, Pelantová H, Cvačka J, Křen V. Synthesis of derivatized chitooligomers using transglycosidases engineered from the fungal GH20 β-N-acetylhexosaminidase. Adv Synth Catal. 2015;357:1941–1950. doi: 10.1002/adsc.201500075. DOI

Etrych T, Mrkvan T, Chytil P, Koňák Č, Říhová B, Ulbrich K. N-(2-Hydroxypropyl)methacrylamide-based polymer conjugates with pH-controlled activation of doxorubicin. I. New synthesis, physicochemical characterization and preliminary biological evaluation. J Appl Polym Sci. 2008;109:3050–3061. doi: 10.1002/app.28466. DOI

Zhang H, Laaf D, Elling L, Pieters RJ. Thiodigalactoside-bovine serum albumin conjugates as high-potency inhibitors of galectin-3: an outstanding example of multivalent presentation of small molecule inhibitors. Bioconjug Chem. 2018;29:1266–1275. doi: 10.1021/acs.bioconjchem.8b00047. PubMed DOI PMC

Slámová K, Bojarová P, Gerstorferová D, Fliedrová B, Hofmeisterová J, Fiala M, Pompach P, Křen V. Sequencing, cloning and high-yield expression of a fungal β-N-acetylhexosaminidase in Pichia pastoris. Protein Express Purif. 2012;82:212–217. doi: 10.1016/j.pep.2012.01.004. PubMed DOI

Fialová P, Carmona AT, Robina I, Ettrich R, Sedmera P, Přikrylová V, Petrásková-Hušáková L, Křen V. Glycosyl azides—novel substrates for enzymatic transglycosylations. Tetrahedron Lett. 2005;46:8715–8718. doi: 10.1016/j.tetlet.2005.10.040. DOI

Bojarová P, Chytil P, Mikulová B, Bumba L, Konefał R, Pelantová H, Krejzová J, Slámová K, Petrásková L, Kotrchová L, Cvačka J, Etrych T, Křen V. Glycan-decorated HPMA copolymers as high-affinity lectin ligands. Polym Chem. 2017;8:2647–2658. doi: 10.1039/C7PY00271H. DOI

Šubr V, Ulbrich K. Synthesis and properties of new N-(2-hydroxypropyl) methacrylamide copolymers containing thiazolidine-2-thione reactive groups. React Funct Polym. 2006;66:1525–1538. doi: 10.1016/j.reactfunctpolym.2006.05.002. DOI

Chytil P, Etrych T, Kříž J, Šubr V, Ulbrich K. N-(2-Hydroxypropyl)methacrylamide-based polymer conjugates with pH-controlled activation of doxorubicin for cell-specific or passive tumour targeting. Synthesis by RAFT polymerisation and physicochemical characterization. Eur J Pharm Sci. 2010;41:473–482. doi: 10.1016/j.ejps.2010.08.003. PubMed DOI

Slámová K, Bojarová P. Engineered N-acetylhexosamine-active enzymes in glycoscience. Biochim Biophys Acta. 2017;1861:2070–2087. doi: 10.1016/j.bbagen.2017.03.019. PubMed DOI

Bojarová P, Křenek K, Kuzma M, Petrásková L, Bezouška K, Namdjou DJ, Elling L, Křen V. N-Acetylhexosamine triad in one molecule: chemoenzymatic introduction of 2-acetamido-2-deoxy-β-d-galactopyranosyluronic acid residue into a complex oligosaccharide. J. Mol. Catal. B Enzymatic. 2008;50:69–73. doi: 10.1016/j.molcatb.2007.09.002. DOI

Slámová K, Gažák R, Bojarová P, Kulik N, Ettrich R, Pelantová H, Sedmera P, Křen V. 4-Deoxy-substrates for β-N-acetylhexosaminidases: How to make use of its loose specificity. Glycobiology. 2010;20:1002–1009. doi: 10.1093/glycob/cwq058. PubMed DOI

Bojarová P, Slámová K, Křenek K, Gažák R, Kulik N, Ettrich R, Pelantová H, Kuzma M, Riva S, Adámek D, Bezouška K, Křen V. Charged hexosaminides as new substrates for β-N-acetylhexosaminidase-catalyzed synthesis of immunomodulatory disaccharides. Adv Synth Catal. 2011;353:2409–2420. doi: 10.1002/adsc.201100371. DOI

Bojarová P, Petrásková L, Ferrandi E, Monti D, Pelantová H, Kuzma M, Simerská P, Křen V. Glycosyl Azides—an alternative way to disaccharides. Adv Synth Catal. 2007;349:1514–1520. doi: 10.1002/adsc.200700028. DOI

Drozdová A, Bojarová P, Křenek K, Weignerová L, Henßen B, Elling L, Christensen H, Jensen HH, Pelantová H, Kuzma M, Bezouška K, Krupová M, Adámek D, Slámová K, Křen V. Enzymatic synthesis of dimeric glycomimetic ligands of NK cell activation receptors. Carbohydr Res. 2011;346:1599–1609. doi: 10.1016/j.carres.2011.04.043. PubMed DOI

Lactose-Functionalized Carbosilane Glycodendrimers Are Highly Potent Multivalent Ligands for Galectin-9 Binding: Increased Glycan Affinity to Galectins Correlates with Aggregation Behavior

Oligosaccharide Ligands of Galectin-4 and Its Subunits: Multivalency Scores Highly

Mutation Hotspot for Changing the Substrate Specificity of β-N-Acetylhexosaminidase: A Library of GlcNAcases

Octahedral Molybdenum Cluster-Based Nanomaterials for Potential Photodynamic Therapy

Cross-Linking Effects Dictate the Preference of Galectins to Bind LacNAc-Decorated HPMA Copolymers

Interaction between Galectin-3 and Integrins Mediates Cell-Matrix Adhesion in Endothelial Cells and Mesenchymal Stem Cells

Acceptor Specificity of β-N-Acetylhexosaminidase from Talaromyces flavus: A Rational Explanation