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The expression of cell surface glycans terminating with sialic acid (SA) residues has been found to correlate with various disease states there among cancer. We here report a novel strategy for specific fluorescence labeling of such motifs. This is based on sialic acid-imprinted core–shell nanoparticles equipped with nitrobenzoxadiazole (NBD) fluorescent reporter groups allowing environmentally sensitive fluorescence detection at convenient excitation and emission wavelengths. Imprinting was achieved exploiting a hybrid approach combining reversible boronate ester formation between p-vinylphenylboronic acid and SA, the introduction of cationic amine functionalities, and the use of an NBD-appended urea-monomer as a binary hydrogen-bond donor targeting the SA carboxylic acid and OH functionalities. The monomers were grafted from 200 nm RAFT-modified silica core particles using ethylene glycol dimethacrylate (EGDMA) as cross-linker resulting in a shell thickness of ca. 10 nm. The particles displayed strong affinity for SA in methanol/water mixtures (K = 6.6 × 105 M-1 in 2% water, 5.9 × 103 M-1 in 98% water, Bmax ≈ 10 µmol g-1), whereas binding of the competitor glucuronic acid (GA) and other monosaccharides was considerably weaker (K (GA) = 1.8 × 103 M-1 in 98% water). In cell imaging experiments, the particles selectively stained different cell lines in correlation with the SA expression level. This was further verified by enzymatic cleavage of SA and by staining using a FITC labeled SA selective lectin.
In vitro cultured 3D models of CRC have been demonstrated to hold considerable worth in drug discovery, drug resistance analysis, and in studying cell-cell and cell-matrix interactions that occur in the tumor microenvironment. The 3D models resemble the in vivo physiological microenvironment by replicating the cell type composition and tissue architecture. Molecularly imprinted polymers (MIPs) have been investigated for use instead of antibodies against small nonimmunogenic structures, such as sialic acid (SA). Glyco-conjugates including SA are present on all cells, and often deregulated on cancer cells. Here, we present a novel approach for targeting and detecting colorectal cancer cells (CRC) by using in vitro cultured HT29 3D spheroids co-cultured in vitro with either fluorescent MIPs targeting SA, SA-MIPs, or the two lectins targeting SA, MAL I, and SNA. Both formaldehyde-fixed and viable HT29 3D spheroids with or without SA-MIPs were imaged in 3D by confocal microscopy. The results revealed a preserved cell morphology and viability of the HT29 3D spheroids co-cultured in vitro with SA-MIPs. However, the lectins MAL I and SNA targeting the alpha-2,3 or alpha-2,6 SA glycosidic linkages, respectively, affected the cell viability when co-cultured with the viable HT29 3D spheroids, and no living cells could be detected. Here, we have shown that the SA-MIPs could be used as a safe and low-cost diagnostic tool for targeting and detecting cancer cells in a physiologically relevant 3D cancer model in vitro.