Design and fabrication of multivalent Gal-containing quantum dots and study of its interactions with asialoglycoprotein receptor (ASGP-R)

Article abstract of DOI:10.1016/j.tetlet.2010.06.002

Multivalent lactose (Lac-QDs)- and galactose (Gal-QDs)- coated CdSeS-ZnS core-shell quantum dots (QDs) were prepared. The formula of the glyco-QDs was determined by nuclear magnetic resonance (NMR) and inductively coupled plasma-optical emission spectrometry (ICP-OES). The uptake of the Gal-containing glyco-QDs by HepG2 cells was investigated. Flow cytometry (FCM) and fluorescence microscopy analysis indicated that the uptake is receptor mediated and selective. The prepared multivalent glyco-QDs could be used to mimic the oligosaccharides in the study of hepatic endocytosis. Furthermore, this type of glyco-QDs can be used as a useful fluorescent probe in cell imaging and analysis of carbohydrate-protein interactions.

Full text of DOI:10.1016/j.tetlet.2010.06.002

Tetrahedron Letters 51 (2010) 4182–4185
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Design and fabrication of multivalent Gal-containing quantum dots and
study of its interactions with asialoglycoprotein receptor (ASGP-R)
a,
Yang Yang a, Yue-Tao Zhao a, Ting-Ting Yan b, Min Yu c, Yin-Lin Sha c, Zhi-Hui Zhao b, , Zhong-Jun Li
*
*
a State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, No. 38 Xueyuan Road, Beijing 100191, China
b College of Life Science, Nanjing Normal University, No. 1 Xianxia Road, Nanjing 210046, China
c Single-molecule and Nanobiology Laboratory, Department of Biophysics, School of Basic Medical Sciences and Biomed-X Center, Peking University,
No. 38 Xueyuan Road, Beijing 100191, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Multivalent lactose (Lac-QDs)- and galactose (Gal-QDs)- coated CdSeS-ZnS core-shell quantum dots
(QDs) were prepared. The formula of the glyco-QDs was determined by nuclear magnetic resonance
(NMR) and inductively coupled plasma-optical emission spectrometry (ICP-OES). The uptake of the
Gal-containing glyco-QDs by HepG2 cells was investigated. Flow cytometry (FCM) and fluorescence
microscopy analysis indicated that the uptake is receptor mediated and selective. The prepared multiva-
lent glyco-QDs could be used to mimic the oligosaccharides in the study of hepatic endocytosis. Further-
more, this type of glyco-QDs can be used as a useful fluorescent probe in cell imaging and analysis of
carbohydrate–protein interactions.
Received 17 March 2010
Revised 19 May 2010
Accepted 1 June 2010
Available online 9 June 2010
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
can be easily bleached and are not suitable for long time
observation.
The asialoglycoprotein receptor (ASGP-R) on hepatocytes plays
a critical role in the clearance of desialylated proteins from the ser-
um.1,2 It is a C-type (Ca2+-dependent) lectin and is responsible for
galactose (Gal) or N-acetylgalactosamine (GalNAc) terminal glyco-
proteins from the circulation by receptor-mediated endocytosis.3
Due to its high specificity, predominant expression on hepatocytes,
and important roles in receptor-mediated endocytosis, the ASGP-R
has been validated as a potential target for drug and gene delivery
to the liver.4
In addition to its preference for sugars (GalNAc ꢀ Gal),
the binding to the ASGP-R strongly depends on the valency of
the ligand (tetraantennary > triantennary ꢀ biantennary ꢀ mono-
antennary galactosides).5 As a result, most efforts for the synthesis
of high affinity ligands were focused on multivalent ligands, con-
taining primarily GalNAc but also Gal or lactose (Lac) as terminal
recognition elements.6 But some research efforts were also direc-
ted toward the discovery of potent monovalent ligands.7 The syn-
Recently, the use of oligosaccharide quantum dots (glyco-QDs)
to mimic the polysaccharides on the surface of the cell and as a
fluorescence probe opens new opportunities for studying the car-
bohydrate–protein interactions.8,14,15 Seeberger and co-workers9
have shown that Gal-capped PEGylated (PEG2000) QDs are prefer-
entially taken up via asialoglycoprotein receptor (ASGP-R)-medi-
ated endocytosis in vitro. Moreover, they demonstrated in the
mouse model that QDs capped with D-mannose and D-galactos-
amine are sequestered specifically by the liver. PEG2000 was intro-
duced to prevent cytotoxicity and increase the water solubility.
However, the preparation of oligosaccharides with a PEG-linker is
complex and needs multi-steps synthesis. Moreover, the character-
ization of the glyco-QDs is also difficult.
We previously reported a convenient fabrication technique for
glyco-QDs, in which 1-thiol sugar without a spacer was used as a
functional assembly molecule to coat the QD surface.10 Moreover,
we have developed a strategy using Nuclear Magnetic Resonance
(NMR) and Inductively Coupled Plasma-Optical Emission Spec-
trometry (ICP-OES) to analyze the coated polysaccharide molecules
and the inner structure of the core and shell of the glyco-QDs. The
biological assay showed that the lectin binding property of the
polysaccharide on the glyco-QDs is maintained and this strategy
can also dramatically enhance their binding activity through cluster
effect.11 This type of glyco-QDs is not toxic to human cancer cells
(Hela cell) and normal cells (Human Umbilical Vein Endothelial
Cells) even at high concentrations of 0.5 mg/ml and 0.05 mg/ml.
thesis of the multivalent oligosaccharides needs
a series of
conversion of the protecting groups and purification by chroma-
tography. Furthermore, the bioassay is often through fluorescence
labeling, but the organic dyes (e.g., Alexa FluorÒ 488 and FITC) used
* Corresponding authors. Tel.: +86 10 8280 1714; fax: +86 01 8280 5496 (Z.-J.L.).
E-mail addresses: zhaozhihui_1964@yahoo.com.cn (Z.-H. Zhao), zjli@bjmu.
edu.cn (Z.-J. Li).
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.06.002

Y. Yang et al. / Tetrahedron Letters 51 (2010) 4182–4185
4183
Herein we also used ternary core/shell CdSeS/ZnS QDs to con-
2.4. Preparation of Gal-QDs and Lac-QDs
struct multivalent galactose-containing derivatives Lac-QDs (1)
and Gal-QDs (2). Although GalNAc has stronger binding affinity
to ASGP-R than galactose, most researches used lactose and galact-
ose because of their availability. Using NMR and ICP-OES, we deter-
mined that about 157 galactose or 150 lactose were coated on the
QDs. The selective uptake of glyco-QDs by HepG2 and its interac-
tion with the ASGP-R were investigated using Flow cytometry
(FCM) and fluorescence microscopy. The experiments showed that
the galactose-containing glyco-QDs can selectively bind to HepG2,
and the agglutination of the glyco-QDs after endocytosis could be
clearly visualized under a microscope.
The exchange of TOPO/OA-capped QDs with 3 and 4 were car-
ried out by phase transfer reaction between chloroform phase
and aqueous phase of phosphate-buffered saline (PBS) (Scheme
1). QDs (10 mg) were dispersed in chloroform (10 ml), and
200 mg 1-thiol sugar was dissolved in PBS and then added in the
reaction system. The mixture was stirred at room temperature un-
til the luminescent QDs were fully transferred from the chloroform
phase in the bottom to the upper PBS layer. The upper aqueous
layer was purified with Sephadex G-70 and lyophilized to give a
yellow powder as the final product.
2.5. Determination of the weight of Gal and Lac on QDs
2. Experimental
1H NMR of Lac-QDs 1 (400 MHz, 5 mg in D2O with 1
lL acetoni-
2.1. Materials and methods
trile as internal standard): d 1.94 (s, 8.37H, CH3CN); 3.12–4.08 (m,
12H); 4.33 (d, 1H, J = 8 Hz, H-10); 4.48 (d, 1H, J = 10 Hz, H-1). LacS
was calculated to be 48.7% of the total weight.
All starting materials, reagents, and solvents were obtained
from commercial suppliers and used as supplied without further
purification. NMR spectra were recorded on a Bruker AMX-400
(400 MHz) or JEOL-300 (300 MHz) spectrometer. Inductively Cou-
pled Plasma-Optical Emission Spectrometry (ICP-OES) was re-
corded on
a Thermo-iCAP 6000 spectrometer. Nikon E200
microscope was used to study the adhesion and receptor-mediated
endocytosis. The uptake of glyco-QDs was measured by Flow
Cytometry (Guava EasyCyte Mini System) and analyzed with the
Guava ExpressPro software. Hepatocellular carcinoma cell line
(HepG2) was obtained from Institute of Biochemistry and Cell Biol-
ogy, the Chinese Academy of Sciences (Shanghai, China).
2.2. Preparation of QDs
Ternarycore/shell CdSeS/ZnS QDs capped with trioctylphosphine
oxide(TOPO)andoleicacid(OA)wereprepared according toourpre-
vious report with minor adjustment of the amount of Se.10 Green
(kem = 534 nm) QDs were synthesized and characterized by ICP-
OES. Transmission Electron Microscopy (TEM) revealed that the
QDs have a diameter of 4.5 0.5 nm (see Supplementary data).
2.3. Preparation of GalSH and LacSH
1-Thiol sugar LacSH (3) and GalSH (4) were synthesized with
three steps from lactose10 and galactose12 (Scheme 1). No purifica-
tion was necessary for each step (Supplementary data).
Figure 1. 1H NMR of 1 and 2.
O
AcO
O
O
AcO
HO
a
b
SH
Br
OH
O
O
O
O
O
S
S
S
O
S
S
S
O
HO
c
S
CdSeS
SH
O
ZnS
CdSeS
ZnS
3 LacSH
4 GalSH
1 Lac-QDs
2 Gal-QDs
TOPO/OA
OH
OH
OH
OH
OH
O
O
O
O
HO
HO
O
=
OH
OH
HO
OH
Gal
Lac
Scheme 1. Synthesis of GalSH and LacSH. Reagents and conditions: (a) AcBr/MeOH,
Figure 2. Uptake of LacSH- and GalSH-coated glyco-QDs by HepG2 cells. PBS was
Ac2O/AcOH, 95%; (b) (i) NH2CSNH2; (ii) Na2S2O5, 50%; (c) NaOMe, MeOH, 98%.
added to the HepG2 cells as negative control.

4184
Y. Yang et al. / Tetrahedron Letters 51 (2010) 4182–4185
Figure 3. HepG2 cells were incubated for 0.5 h with 40 lM of Lac-QDs or Gal-QDs. (a), (b) Lac-QDs bind to HepG2; (c), (d) Gal-QDs bind to HepG2. HepG2 cells were
incubated for 4 h with 40 lM of Lac-QDs or Gal-QDs. (e), (f) Lac-QDs taken up by HepG2; (g), (h) Gal-QDs taken up by HepG2.
Figure 4. HepG2 cells were incubated for 12 h with 40 lM Lac-QDs or Gal-QDs. (a), (b) Endocytosis of Lac-QDs by HepG2; (c), (d) endocytosis of Gal-QDs by HepG2.
1H NMR of Gal-QDs 2 (400 MHz, 5 mg in D2O with 1
trile as internal standard): d 1.95 (s, 6.38H, CH3CN); 3.34–3.89 (m,
6H); 4.44 (t, 1H, J = 8.4 Hz, H-1). GalS was calculated to be 35.3% of
the total weight.
lL acetoni-
effective at disrupting protein/protein interactions.13 Particle-
based displays of multiple ligands can create a high local concen-
tration of binding molecules (called statistical effect). Conse-
quently, binding equilibrium between a surface-bound ligand
and receptor favors the formation of ligand–receptor pairs (called
receptor clustering effect).
2.6. Uptake of glyco-QDs by HepG2 cells in vitro
Our previous work showed that the removal of the linker did
not affect the binding property of the glyco-QDs. On the contrary,
the directly coated glyco-QDs showed higher affinity to its receptor
than that coated via a PEG (n = 3) linker. Based on these principles,
we design our multivalent glyco-QDs 1 and 2 as a fluorescent
probe to investigate their interactions with ASGP-R.
2.6.1. Flow cytometry
HepG2 cells were cultivated in DMEM culture medium. Cells
were collected at the concentration of 105/ml. 1 and 2 (40
lM)
were added to the cells. After incubation for 0.5 h, 2 h, and 4 h,
the cells were washed three times with cold PBS. The uptake of gly-
co-QDs was measured by flow cytometry.
3.2. Determination of the number of sugar molecules on QDs
2.6.2. Fluorescence microscopy
After the washing step, the cells were transferred onto glass
slides for bright field images and fluorescent imaging.
Traditionally the amount of the carbohydrate on nanoparticles is
chemically analyzed by phenol–sulfuric acid method.9,14 However,
the procedure is complex and the result is strongly affected by the
heating time and the duration of the experiment. To quantitatively
determine the weight of sugar on QDs surface, we developed a facile
strategy using NMR. Acetonitrile was used as the internal standards
3. Results and discussion
3.1. Design of the glyco-QDs
(IS). By peak integration we calculated that about 6.8
or 9 mol GalSH was loaded onto the QDs surface. Together with
the ICP-OES and the calculated size of the QDs and 1-thiol sugar,
lmol of LacSH
It has been shown that nanoparticles that are comparable in
size to proteins and coated with protein-binding ligands may be
l

Y. Yang et al. / Tetrahedron Letters 51 (2010) 4182–4185
4185
we calculated that the formula of 1 was [Cd171Se12S356-(ZnS)9]@
(LacS)150 and 2 was [Cd171Se12S356-(ZnS)9]@(GalS)157. The number
of the sugar molecules on the QDs surface was in agreement with
the previous reports for the carbohydrate-encapsulated 5 nm diam-
eter QDs15 and 6 nm gold nanoparticles.16
vesicles was clearly visualized under a fluorescence microscope.
QDs represent a valuable carrier to assemble sugars to exhibit
maximal multivalency effects. The convenient synthesis and char-
acterization of the glyco-QDs allow the study of various carbohy-
drate–protein interactions.
1H NMR of 1 and 2 was basically similar to that of LacSH and
GalSH. Sometimes, the anomeric hydrogen of the glyco-QDs be-
came multiple peaks, because of the instability of nanocrystals
coated with hydrophilic thiols (Fig. 1).17 The luminescence emis-
sion wavelength of the glyco-QDs was around 534 nm (30 nm, full
width at half height, Supplementary data).
Acknowledgments
This work was financially supported by the Natural Science
Foundation of China (Grant No. 90713004, 20973008), the State
New Drug Innovation (Grant No. 2009ZX09103-044), and the State
Key Laboratory of Natural and Biomimetic Drugs and Peking
University.
3.3. Uptake of glyco-QDs by HepG2 cells in vitro
Two types of cells, Hela cells that do not express the ASGP-R18
and HepG2 cells, were used to study the association (binding and
uptake) with the glyco-QDs. At first the ASGP-R-mediated uptake
of compounds 1 and 2 was quantitatively evaluated by flow cytom-
etry. The cellular uptake of the glyco-QDs was evaluated by com-
paring the shift in median intensity of fluorescence (MFI)
between untreated cells (background fluorescence) and treated
cells. The MFI of cells incubated with compounds 1 and 2 at hours
ranging from 0.5 to 4 h indicated the uptake of the glyco-QDs into
HepG2 cells, but no uptake by Hela cells was observed (data not
shown) (Fig. 2).
Fluorescence microscopy was used to assess the binding of 1
and 2 with the cell surface. Binding of 1 and 2 with HepG2 cells
was observed after 0.5 h (Fig. 3). To determine if endocytosis of 1
and 2 was involved, cells after 4 h and 12 h treatment were also
observed. It is clear that 1 and 2 were taken into the cells through
endocytosis after 12 h (Fig. 4). It was observed under the bright
field that the glyco-QDs were agglutinating in the cells after
endocytosis.
A number of studies have indicated that ASGP-R-based recogni-
tion is highly dependent on spatial presentation. In other words,
the Gal residues connected by flexible spacers with appropriate
lengths are needed. It is important to note that these results are
based on small molecules, in which the sugar density is limited
by the conjugation points of the linker. In our study, we used nano-
particle as the fluorescent anchor, which allows the sugar mole-
cules to be more densely coated on the surface of the glyco-QDs.
Because the exact mechanism of the interactions of multivalent
glyco-QDs with proteins is not yet clear, we further reasoned that
the sugar molecules on the glyco-QDs might be assembled in dif-
ferent orientations, which can provide a variety of spatial presen-
tations for the interactions with ASGP-R.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2010.06.002.
References and notes
1. Spiess, M. Biochemistry 1990, 29, 10009–10018.
2. Drickamer, K.; Taylor, M. E. Annu. Rev. Cell Biol. 1993, 9, 237–264.
3. Lee, R. T.; Lee, Y. C. Biochemistry 1986, 25, 6835–6841.
4. (a) Wu, J.; Nantz, M. H.; Zern, M. A. Front. Biosci. 2002, 7, d717–725; (b)
Nishikawa, M. Biol. Pharm. Bull. 2005, 28, 195–200; (c) Wilson, J. M.; Grossman,
M.; Wu, C. H.; Chowdhury, N. R.; Wu, C. Y.; Chowdhury, J. R. J. Biol. Chem. 1992,
267, 963–967.
5. Rensen, P. C. N.; Sliedregt, L. A. J. M.; Ferns, M.; Kieviet, E.; van Rossenberg, S. M.
W.; van Leeuwen, S. H.; van Berkel, T. J. C.; Biessen, E. A. L. J. Biol. Chem. 2001,
276, 37577–37584.
6. (a) Biessen, E. A.; Beuting, D. M.; Vietsch, H.; Bijsterbosch, M. K.; Van Berkel, T. J.
J. Hepatol. 1994, 21, 806–815; (b) Biessen, E. A.; Vietsch, H.; Rump, E. T.; Fluiter,
K.; Kuiper, J.; Bijsterbosch, M. K.; van Berkel, T. J. Biochem. J. 1999, 340, 783–
792; (c) Khorev, O.; Stokmaier, D.; Schwardt, O.; Cutting, B.; Ernst, B. Bioorg.
Med. Chem. 2008, 16, 5216–5231.
7. (a) Stokmaier, D.; Khorev, O.; Cutting, B.; Born, R.; Ricklin, D.; Ernst, T. O. G.;
Böni, F.; Schwingruber, K.; Gentner, M.; Wittwer, M.; Spreafico, M.; Vedani, A.;
Rabbani, S.; Schwardt, O.; Ernst, B. Bioorg. Med. Chem. 2009, 17, 7254–7264; (b)
Wong, T. C.; Townsend, R. R.; Lee, Y. C. Carbohydr. Res. 1987, 170, 27–46.
8. (a) de la Fuente, J. M.; Penades, S. Tetrahedron: Asymmetry 2005, 16, 387–391;
(b) de la Fuente, J. M.; Penades, S. Biochim. Biophys. Acta 2006, 1760, 636–651;
(c) Mukhopadhyay, B.; Martins, M. B.; Karamanska, R.; Russell, D. A.; Field, R. A.
Tetrahedron Lett. 2009, 50, 886–889; (d) Kikkeri, R.; Laurino, P.; Odedra, A.;
Seeberger, P. H. Angew. Chem., Int. Ed. 2010, 49, 2054–2057.
9. Kikkeri, R.; Lepenies, B.; Adibekian, A.; Laurino, P.; Seeberger, P. H. J. Am. Chem.
Soc. 2009, 131, 2110–2112.
10. Yu, M.; Yang, Y.; Han, R. C.; Zheng, Q.; Wang, L. J.; Hong, Y. K.; Li, Z. J.; Sha, Y. L.
Langmuir 2010, 26, 8534–8539.
11. Yang, Y.; Yu, M.; Yan, T. T.; Zhao, Z. H.; Sha, Y. L.; Li, Z. J. Bioorg. Med. Chem.
2010. doi:10.1016/j.bmc.2010.05.046.
12. Matta, K. L.; Johnson, E. A. Z.; Girotra, R. N.; Barlow, J. J. Carbohydr. Res. 1973, 30,
414–417.
13. Bowman, M. C.; Ballard, T. E.; Ackerson, C. J.; Feldheim, D. L.; Margolis, D. M.;
Melander, C. J. Am. Chem. Soc. 2008, 130, 6896–6897.
14. (a) Babu, P.; Sinha, S.; Surolia, A. Bioconjugate Chem. 2007, 18, 146–151; (b)
Robinson, A.; Fang, J. M.; Chou, P. T.; Liao, K. W.; Chu, R. M.; Lee, S. J.
ChemBioChem 2005, 6, 1899–1905.
4. Conclusion
15. Sun, X. L.; Cui, W.; Haller, C.; Chaikof, E. L. ChemBioChem 2004, 5, 1593–1596.
16. Lin, C. C.; Yeh, Y. C.; Yang, C. Y.; Chen, C. L.; Chen, G. F.; Chen, C. C.; Wu, Y. C. J.
Am. Chem. Soc. 2002, 124, 3508–3509.
17. Aldana, J.; Wang, Y. A.; Peng, X. G. J. Am. Chem. Soc. 2001, 123, 8844–8850.
18. Spiess, M.; Lodish, H. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 6465–6469.
In this study we prepared fluorescent and multivalent Gal-con-
taining glyco-QDs as ligands for the ASGP-R. Using flow cytometry,
we have shown that compounds 1 and 2 exhibit selective uptake
via the ASGP-R by HepG2 cells. The formation of distinct endocytic