Characterization of multivalent lactose quantum dots and its application in carbohydrate–protein interactions study and cell imaging

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Abstract

We have previously reported a facile and convenient method for the preparation of a new type of lactose-CdSeS/ZnS quantum dots conjugates (Lac-QDs) that exhibit biocompatibility, noncytotoxicity and specificity to leukocytes. In order to further study the carbohydrate–protein interactions, a series of Lac-QDs with different lactose densities and a PEGylated (n = 3) lactose-QDs conjugate (LacPEG-QDs) with more flexible sugar ligands were prepared. The amount of the sugar molecules on QDs can be determined by NMR, which was in agreement with the results from TGA determination. The formula of the conjugates was determined with ICP-OES. The interactions between the conjugated QDs and the PNA protein were measured using SPR, which revealed that higher lactose density favored binding affinity under the same concentration, and Lac-QDs exhibit higher affinity than LacPEG-QDs. We further used a solid phase assay to assess the anti-adhesion activity of Lac-QDs and LacPEG-QDs on the cell level. The results showed that Lac-QDs had stronger activity in preventing THP1 from adhering to HUVEC than LacPEG-QDs, which was consistent with the SPR results. We reasoned that decrease in the conformational entropy induced by appropriate restriction of sugar flexibility could enhance the binding affinity of glyco-QDs, which implies that entropy change may be the main contributor to the interaction between high valent glyco-QDs and protein. The fabrication of lactose on QDs provides a fluorescent multivalent carbohydrate probe that can be used as mimics of glycoprotein for the study of carbohydrate–protein interactions and cell imaging.

Introduction

Carbohydrate–protein interactions are important in a wide variety of physiological and pathological processes, such as cell-adhesion, cellular signalling, and inflammation, as well as infections induced by viral and bacterial agents.1, 2, 3, 4 There are several lines of evidences showing that typical monomeric carbohydrate–protein interactions are weak and nonspecific.5 Carbohydrate–protein binding interactions have dissociation constants in the mM range and different carbohydrate ligands have similar affinities for the same protein receptor. Cells overcome this paradox to mediate specific cellular processes by the strategy of polyvalency, namely ‘cluster-glycoside effect’.6 It is believed that protein receptors that produce highly specific cellular responses contain clustered multiple carbohydrate-binding sites. Because of the complexity of this interaction, simplified model systems are usually used to study multivalent carbohydrate–protein interactions. In recent years, a number of spherical or linear arrays of oligosaccharides have been designed for mimicking the multivalent displaying of oligosaccharide ligands and studying the carbohydrates involved in bio-recognitions in natural process.7, 8, 9, 10, 11

Due to the similar sizes of nanoparticles (NPs) and most glycoproteins, the oligosaccharide ligands assembled on the NPs could achieve the specific geometry and orientation that the sugar moieties display on the surface of glycoproteins. Also, in this nanotechnology method, NPs can be used as the vector or probe for bio-recognition studies, considering their specific physical properties.12 It is known that quantum dots (QDs) exhibit unique optical properties, such as broad absorption, sharp luminescent emission, high quantum efficiency, and high photostability. Taking advantage of QDs to template oligosaccharide clustering assemblies has been implemented by some groups in recent years, which offers an opportunity for developing fluorescent carbohydrate biolabels.13, 14, 15, 16, 17, 18

Previous studies demonstrated that the interaction between leukocytes and endothelial cells plays an important role in the pathogenesis of shock.19 Some glycosyl moieties are directly involved in the interactions between adhesion molecules expressed on the surface of leukocytes and endothelial cells. We have demonstrated that tetra- and divalent lactose derivatives Gu-4, 6 and An-2, 7 (Fig. 1) function as antagonists against CD11b on leukocytes. They inhibit the adhesion of leukocytes to microvessel endothelial cells during shock, while monovalent lactose does not.20

Based on these findings, we designed and fabricated the polyvalent lactosyl QDs (Lac-QDs) for specific labeling of live leukocytes. We also studied the cytotoxicity and photostabilities of Lac-QDs against a wide pH range (4.0–9.0) and reductive glutathione (GSH). The results showed that the Lac-QDs display good colloidal stability and low cytotoxicity.21 In this paper, we further prepared a series of Lac-QDs with different lactose densities and a PEGylated (n = 3) lactose-QDs conjugate (LacPEG-QDs). With Nuclear Magnetism Resonance (NMR), Thermogravimetric Analysis (TGA) and Inductively Coupled Plasma-Optical Emission Spectrometer (ICP-OES), we analyzed the structure of the QDs and glyco-QDs. Carbohydrate–protein interactions were investigated using Surface Plasmon Resonance (SPR) and Solid Phase Assay. Finally we showed that Lac-QDs with no linker have the highest binding affinity in carbohydrate–protein interactions. The glyco-QDs could be applied as a fluorescent probe in cell imaging and carbohydrate–protein interaction study.

Section snippets

Design and synthesis of the glyco-QDs

Ternary core/shell CdSeS/ZnS QDs was employed as the nanoscale platform to construct the multivalent lactosyl QDs. Unlike duplicate QDs (CdSe, CdS, CdTe), the emission wavelength of ternary QDs could be adjusted by the amount of Se, without changing the size of QDs.22, 23 Orange (λem = 564 nm) and green (λem = 506 nm) QDs were synthesized and analyzed by ICP-OES. The results showed that the orange QDs have a higher percentage of Se (11%) than the green one (0.8%, SI). Transmission Electron Microscopy

Conclusion

We have developed a convenient approach to regulate the density of the lactose on QDs by adding another thiol ligand 2-mercaptoethanol and a new strategy to determinate the amount of lactose on the QDs using NMR, which has several advantages over traditional methods. The NMR results are in good agreement with that obtained by TGA.

The biological assay showed that oligosaccharides coated on QDs surface can dramatically enhance their binding activity through cluster effect. The results also

General methods

All starting materials, reagents and solvents were obtained from commercial suppliers and used as supplied without further purification. NMR spectra were recorded on a JEOL-300 (300 MHz) instrument or a Bruker AMX-400 (400 MHz) instrument. Thermo gravimetric analysis (TGA) was carried out on a Q50SDT TA instrument and the samples were burned in nitrogen at a constant heating rate of 10 °C/min from 25 to 950 °C. Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES) was recorded on

Acknowledgments

This work was financially supported by the Natural Science Foundation of China (Grant No. 90713004), the State New Drug Innovation (Grant No. 2009ZX09103-044) and the State Key Laboratory of Natural and Biomimetic Drugs of Peking University.

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