Gold Nanoparticles Conjugated with Glycopeptides for Lectin Detection and Imaging on Cell Surface.

BACKGROUND

Lectins are carbohydrate binding proteins and related to various biological events and diseases including virus infection and cancer metastasis. In particular, galactose-binding lectins have attracted attention as targets for drug delivery and cancer markers. We, previously, demonstrated that sugar-modified peptides (glycopeptides) were useful ligands for the detection and characterization of lectins compared to the sugar unit alone. Gold nanoparticles (GNPs) conjugated with mannose-modified glycopeptides were useful in detection of concanavalin A, a mannose binding lectin.

OBJECTIVES

The main objective of this study was to expand our glycopeptide-GNP conjugates for detection and imaging of galactose-binding lectins.

METHODS

Four galactose-modified peptides (glycopeptides) were synthesized by Fmoc-based solid peptide synthesis method. Synthesized glycopeptides were conjugated with PEG-coated GNPs using thiol-maleimide chemistry. The interaction between glycopeptide-GNPs (GP/GNPs) (0.5 nM) and RCA120, a galactose binding lectin, (0.5-1000 nM) was evaluated by mesuring absorption spectra of GNPs. The inhibition experiment in the interaction between GP/GNPs (0.5 nM) and RCA120 (100 nM) was performed in the presence of 60 mM α- methyl mannose or 60 mM lactose. HepG2 and MCF7 cells were placed on 22×22 mm cover slip in 6 well cell culture plates (2×105 cells / well) and cultured overnight at 37°C under 5% CO2 condition. 1 mL of GP/GNPs (0.2 nM) were added in each well and incubated for 18 h at 37°C under 5% CO2 condition. After incubation, cells were washed twice with PBS and fixed with 4% paraformaldehyde solution. The cover slips were coated with 90% glycerol and sealed to slide glass. Dark-field images based on elastic light scattering were taken using a Nikon microscope (TieU) with an immersion dark field condenser.

RESULTS

In the titration experiment of RCA120, GP/GNPs showed a decrease of absorbance according to the addition of RCA120, suggesting that the aggregation of GP/GNPs is induced through the binding to RCA120. The EC50 values of AA(Gal)/GNP, WF(Gal)/GNP, TS(Gal)/GNP and ED(Gal)/ GNP were estimated as 66.2 nM, 43.2 nM, 38.6 nM and 104.4 nM, respectively. TS(Gal)/GNP showed the lowest EC50 value among GP/GNPs. RCA120 has several binding sites for the galactose, and there are hydrophilic amino acids (Thr24, Glu26, Gln35, Asn42 and Asp44) around one of galactose binding sites. This result indicates that the hydrogen bonds between these amino acids and Thr/Ser residues of TS(Gal) contribute to the efficient aggregation of TS(Gal)/GNP. Next, inhibition experiments in the aggregation of WF(Gal)/GNP with RCA120 revealed that lactose inhibits the WF(Gal)/GNP binding with RCA120, but α-methyl mannose does not, and that WF(Gal)/GNP selectively interacts with RCA120 and forms the aggregate. Finally, a galactose binding protein on the surface of HepG2 cells was successfully visualized by using GP/GNPs as optical probes.

CONCLUSION

Our results demonstrated that GP/GNPs could detect RCA120 by the selective binding and the aggregation formation. Furthermore, a galactose binding protein on the surface of HepG2 cells is successfully visualized using WF(Gal)/GNP as an optical probe. Thus, GNPs conjugated with glycopeptides will be useful probes for the selective detection and imaging of lectins.