Sialic Acid-Imprinted Fluorescent Core–Shell Particles for Selective Labeling of Cell Surface Glycans

Abstract

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 × 10(5) M(-1) in 2% water, 5.9 × 10(3) M(-1) in 98% water, B(max) ≈ 10 μmol g(-1)), whereas binding of the competitor glucuronic acid (GA) and other monosaccharides was considerably weaker (K (GA) = 1.8 × 10(3) 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.

Different expression levels of glycans on leukemic cells—a novel screening method with molecularly imprinted polymers (MIP) targeting sialic acid

“…This calls for the development of alternative glycan-specific probes and cell imaging technologies. Recent reports have shown that synthetic probes based on host guest chemistry [10] or molecular imprinting technology [11] can be used for glycan selective staining of cells and tissues. Using the former approach, Xu and colleagues developed fluorescent sensors based on two boronic acids linked to a functionalized peptide for detection of cell-surface cancer-associated glycans [10].…”

“…In flow cytometry and fluorescence microscopy investigations, the SA-MIP selectively targeted two different prostate cancer cell lines DU145 and PC3, as well as the T leukemia cell line Jurkat. The SA expression was verified with lectin-FITC [11]. …”

Artificial Biosensors: How Can Molecular Imprinting Mimic Biorecognition?

“…For example, NPs of MIP imprinted with melittin (a component of bee venom) injected into mouse protected the animal from the bee venom [77]. Moreover, NPs of SiO 2 coated with a sialic acid (SA)-imprinted MIP film bearing nitrobenzoxadiazole (an organic dye) moieties in the polymer matrix were prepared [45]. These NPs were used for fluorescent labeling cells with SA-terminated glycan motifs on their surface.…”

“…xx, No. yy 7 acid 16[36][37][38][39], 4-vinylphenylboronic acid 17[40][41][42][43][44][45], 3-acrylamidophenylboronic acid 18[46], and [ 1 9 _ T D $ D I F F ] 2,2 0 -bithiophene-5-boronic acid 19[47,48], as functional monomers. The boronic acid group reversibly forms esters with two adjacent hydroxyl groups of vicinal diols and catechols.…”

“…This strategy should be well suited for imprinting carbohydrates and polysaccharides in particular. Despite many examples of successful monosaccharide[36,37,[44][45][46][47] and glycoprotein[38][39][40][41][42][43] covalent imprinting, oligo-and polysaccharides appear to be more demanding and currently there are no published examples of the successful covalent imprinting of these saccharides and only a few examples of noncovalently imprinted oligo-and polysaccharides[49,50].MIPs for Aptamer-DNA Recognition Aptamers are short oligonucleotides or peptides complementary to target compounds of the same type. Typically, aptamer oligonucleotides are immobilized on a solid substrate to fabricate biosensors for the determination of the DNA or RNA of a complementary nucleotide sequence[51].…”