Importance of sialic acid residues illuminated by live animal imaging using phosphorylcholine self-assembled monolayer-coated quantum dots

Article abstract of DOI:10.1021/ja111201c

Glycans are expected to be one of the potential signal molecules for controlling drug targeting/delivery or long-term circulation of biopharmaceuticals. However, the effect of the carbohydrates of artificially glycosylated derivatives on in vivo dynamic d

Full text of DOI:10.1021/ja111201c

ARTICLE
pubs.acs.org/JACS
Importance of Sialic Acid Residues Illuminated by Live Animal Imaging
Using Phosphorylcholine Self-Assembled Monolayer-Coated
Quantum Dots
Tatsuya Ohyanagi,† Noriko Nagahori,† Ken Shimawaki,† Hiroshi Hinou,† Tadashi Yamashita,† Akira Sasaki,†
Takashi Jin,‡ Toshihiko Iwanaga,§ Masataka Kinjo,† and Shin-Ichiro Nishimura†
†Field of Drug Discovery Research, Faculty of Advanced Life Science, Hokkaido University, Sapporo, 001-0021, Japan
‡WPI Immunology Frontier Research Center, Osaka University, Yamada-oka 1-3, Suita, Osaka 565-0871, Japan
§Department of Anatomy, Hokkaido University Graduate School of Medicine, Sapporo, 060-8638, Japan
S Supporting Information
b
ABSTRACT: Glycans are expected to be one of the potential signal
molecules for controlling drug targeting/delivery or long-term circula-
tion of biopharmaceuticals. However, the effect of the carbohydrates of
artificially glycosylated derivatives on in vivo dynamic distribution
profiles after intravenous injection of model animals remains unclear
due to the lack of standardized methodology and a suitable platform. We
report herein an efficient and versatile method for the preparation of
multifunctional quantum dots (QDs) displaying common synthetic
glycosides with excellent solubility and long-term stability in aqueous
solution without loss of quantum yields. Combined use of an aminooxy-terminated thiol derivative, 11,110-dithio bis[undec-11-yl
12-(aminooxyacetyl)amino hexa(ethyleneglycol)], and a phosphorylcholine derivative, 11-mercaptoundecylphosphorylcholine,
provided QDs with novel functions for the chemical ligation of ketone-functionalized compounds and the prevention of nonspecific
protein adsorption concurrently. In vivo near-infrared (NIR) fluorescence imaging of phosphorylcholine self-assembled monolayer
(SAM)-coated QDs displaying various simple sugars (glyco-PC-QDs) after administration into the tail vein of the mouse revealed
that distinct long-term delocalization over 2 h can be achieved in cases of QDs modified with R-sialic acid residue (Neu5Ac-PC-
QDs) and control PC-QDs, while QDs bearing other common sugars, such as R-glucose (Glc-PC-QDs), R-mannose (Man-PC-
QDs), R-fucose (Fuc-PC-QDs), lactose (Lac-PC-QDs), β-glucuronic acid (GlcA-PC-QDs), N-acetyl-β-D-glucosamine (GlcNAc-
PC-QDs), and N-acetyl-β-D-galactosamine (GalNAc-PC-QDs) residues, accumulated rapidly (5ꢀ10 min) in the liver. Sequential
enzymatic modifications of GlcNAc-PC-QDs gave Galβ1,4GlcNAc-PC-QDs (LacNAc-PC-QDs), Galβ1,4(FucR1,3)GlcNAc-
PC-QDs (Lex-PC-QDs), Neu5AcR2,3Galβ1,4GlcNAc-PC-QDs (sialyl LacNAc-PC-QDs), and Neu5AcR2,3Galβ1,4(FucR1,3)-
GlcNAc-PC-QDs (sialyl Lex-PC-QDs) in quantitative yield as monitored by direct matrix-assisted laser desorption ionization time-
of-flight mass spectrometry analyses. Live animal imaging uncovered for the first time that Lex-PC-QDs also distributed rapidly in
the liver after intravenous injection and almost quenched over 1 h in similar profiles to those of LacNAc-PC-QDs and Lac-PC-QDs.
On the other hand, sialyl LacNAc-PC-QDs and sialyl Lex-PC-QDs were still retained stably in the whole body after 2 h, while they
showed significantly different in vivo dynamics in the tissue distribution, suggesting that structure/sequence of the neighboring
sugar residues in the individual sialyl oligosaccharides might influence the final organ-specific distribution. The present results clearly
visualize the evidence of an essential role of the terminal sialic acid residue(s) for achieving prolonged in vivo lifetime and
biodistribution of various glyco-PC-QDs as a novel class of functional platforms for nanomaterial-based drug targeting/delivery.
A standardized protocol using multifunctional PC-QDs should facilitate live animal imaging of ligand-displayed QDs using versatile
NIR fluorescence photometry without influence of size-dependent accumulation/excretion pathway for nanoparticles (e.g., viruses)
>10 nm in hydrodynamic diameter by the liver.
’ INTRODUCTION
et al. developed for the first time a protocol for the construction
of magnetic resonance imaging (MRI)-visible high Fe-content
glyconanoparticles having E-/P-selectin ligand carbohydrate that
allows presymptomatic in vivo imaging of rat brain disease
Recent successful approaches of noninvasive imaging for
probing in vivo dynamics of glycans and glycoconjugates have
provided valuable information on tissue distribution of individual
glycosylated compounds, such as small molecular glycans, nat-
ural and neoglycoproteins, sugar-modified liposomes, and mag-
netite-based nanoparticles having glycans.1 For example, Davis
Received: December 23, 2010
Published: July 08, 2011
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models.1c Fukase et al. also reported an efficient method for the
synthesis of glycoclusters labeled by 68Ga-1,4,7,10-tetraazacyclo-
dodecane-1,4,7,10-tetraacetic acid (68Ga-DOTA) and showed
that the use of 68Ga-DOTA labeling of dendrimer-type N-
glycoclusters made noninvasive imaging of the in vivo dynamics
and organ-specific accumulation of various N-glycans by positron
emission tomography (PET) possible.1f It is believed that
accumulation of such important data sets of the dynamic
biodistribution profiles for individual glycosylated derivatives
would become beneficial for further drug discovery research as
well as the development of highly sensitive diagnostic methodol-
ogy based on MRI and PET.
scaffold materials, and hydrophobic photosensitive probes as
well as protein core structures in native glycoproteins. It is well-
known that glycans usually exhibit relatively weak affinity with
their partner molecules, such as enzymes and cell surface
receptors, even though specificity of the interaction may be very
high.2 To investigate the functional role of carbohydrate itself for
controlling glycoprotein circulation and distribution in vivo, it
seems likely that a novel class of simple glycoprotein model, in
which aglycon-scaffold should entail general globular protein-like
structure/property and the potential to prevent nonspecific
interaction with other biomolecules, cells, tissues, or artificial
materials surfaces, would be used.
Our attention is now directed toward the influence of scaffold
materials to the in vivo dynamics and organ-specific accumula-
tion of the attached glycans. It should be noted that evaluating
the effect of carbohydrate moiety of various natural/non-natural
glycoconjugates on biodistribution and its lifetime appears to be
difficult, independent from the influence by the structure/
property of aglycon (nonglycan) moieties, notably artificial
Gold nanoparticles carrying carbohydrates are one of the
simplest and most versatile glycoprotein models for probing and
investigating functional roles of glycans in vitro using atomic force
microscopy (AFM), transmission electron microscopy (TEM), or
surface plasmon resonance (SPR).3 Since it has been documented
by in vivo imaging that gold nanoparticles exhibit a higher adsorp-
tion than iodine as a X-ray contrast agent with less bone and tissue
interference, achieving better contrast with lower X-ray dose,4
noninvasive imaging of the biodistribution of glycan-conjugated
gold nanoparticles may become possible in the near future. On the
other hand, quantum dots (QDs) are fluorescence semiconductor
nanoparticles that have unique optical properties, including narrow
band and size-dependent luminescence with broad absorption,
long-term photo stability, and resistance to photobleaching.5 An
attractive application of QDs is the living cells/animals imaging by
Figure 1. Chemical structures of phosphorylcholine-type monothiol
ligand (1) and aminooxy-functionalized monothiol anchor group (2).
Figure 2. Preparation and characterization of PC-QDs. (a) General protocol of the ligand exchange reaction from TOPO-QDs to PC-QDs. (b) The
progress of the ligand exchange reaction: PC-QDs were dissolved in water phase after the ligand exchanging. (c) Photograph of PC-QDs of various
emission wavelengths. (d) Direct MALDI-TOFMS analysis of PC-QDs (SeTe/CdS, λem = 800 nm).
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Figure 3. Characterization of PC-QDs and PEG-QDs by FCS. Fluorescence fluctuations of (a) PC-QDs and (b) PEG-QDs in water. The
autocorrelation curves were fitted by using one component diffusion model. (c) Fluorescence spectra of QDs coated by TOPO in chloroform,
PC-SH, and PEG-SH17 in water. The fluorescence spectra were measured by using an excitation at 488 nm, and the quantum yield was estimated by
absolute photoluminescence quantum yield measurement. PEG-QDs were prepared by the same procedure for the preparation of PC-QDs using PEG-
SH17 instead of PC-SH.
simple fluorescent measurements,6 which is possible due to success-
ful surface modifications with various biomolecules, such as DNA,7
peptides,8 proteins,9 antibodies,10 and carbohydrates.11 As a scaffold
to display carbohydrates and investigate their functions, advantages
of QDs are summarized as follows: (a) QDs can be detected and
monitored in vivo by simple fluorescent photometric analysis
without any special and expensive equipment. (b) Multiple carbo-
hydrates can be displayed on a single QD, and the carbohydrate
density on the single QD can be readily controlled. Thus enhanced
affinity with target molecules is expected as a result of the glycoside
cluster effect.2 (c) The QDs range in size from several and to dozens
of nanometers, the same level as typical folded proteins (∼10 nm in
diameter),3c is suitable for designing a new class of glycoprotein
models. It is therefore expected that the QDs will exhibit behavior
and dynamics similar to common globular proteins distributed in
living cells and animals.6e,i Since inorganic, metal-containing QDs
are generally synthesized in organic solvents by coating with
hydrophobic ligands, such as trioctylphosphine oxide (TOPO),5,6
it is obvious that surface modification of QDs is needed to improve
solubility and stability in common physiological conditions. Thus
far, many water-soluble QDs have been developed by coating with a
variety of thiols and copolymers based on poly(ethylene glycols)
(PEG)-like structures.6e Recently, Seeberger et al. developed a
convenient method to prepare different sugar-capped PEGylated
QDs that can be used for in vitro imaging and in vivo liver targeting,
in which paraffin sections of mouse livers were prepared for the
visualization of QDs by fluorescence microscopy.1e However, it
should be noted that the nanoparticle hydrodynamic diameter is a
crucial design parameter in the development of potential diagnostic
and therapeutic agents. Organic coatings often result in a significant
increase of the final hydrodynamic diameter. In fact, QDs > 10 nm
in hydrodynamic diameter were proved to be accumulated by the
liver designed specifically to capture and eliminate nanoparticles,
while smaller QDs < 5.5 nm resulted in rapid renal excretion.6i
There is no versatile QDs platform mimicking typical folded
proteins (∼10 nm in diameter) to have satisfactory functions for
both displaying glycans and reducing nonspecific interactions with
abundant serum and cellular proteins without loss of quantum yield
of the original QDs. Here we communicate a standardized protocol
for the construction of an ideal glycoprotein model, namely
phosphorylcholine self-assembled monolayers (SAMs)12-coated
QDs displaying various glycans (glyco-PC-QDs), exhibiting excel-
lent water solubility, long-term stability in the stock solution, pH
resistance, and a capacity to prevent nonspecific protein adsorption.
’ RESULTS AND DISCUSSION
QDs Coated by Phosphorylcholine SAMs. It is well-known
that surface modification by thiol tethering or copolymers containing
PEG-like structures is one of the most commonly used methods for
the solubilization of QDs.5,6 However, we thought that the use of
phospholipid-like derivatives is a potential alternative to PEGylation
because phosphatidylcholine is the most abundant and indispensa-
ble lipid component of stable biomembranes. We hypothesized that
combined use of a simple monothiol, 11-mercaptoundecylpho-
sphorylcholine (PC-SH, 1),12c and an aminooxy-terminated thiol
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(CdSeTe/CdS, λem = 800 nm)15 proceeded smoothly and gave
PC-QDs showing excellent solubility toward water (Figure 2b
and c). The surface modification with PC-SH (1) was directly
confirmed by MALDI-TOFMS (Figure 2d), where the peaks at
m/z 370.0 and 737.3 are simply assigned as PC-SH and disulfide
form (PCꢀSꢀSꢀPC). Solubility toward water and the fluores-
cence intensity of PC-QDs was evaluated by fluorescence
correlation spectroscopy (FCS)15,16 in comparison with those
of simple PEG-QDs. As shown in Figure 3, the fluores-
cence fluctuation of PC-QDs was proved to be rapid and an
ideal profile in the fitting curves with single-component diffusion
model (Figure 3a), suggesting that PC-QDs are well dispersed in
water, while the results of PEG-QDs showed an evidence of the
formation of serious aggregation shortly after PEGylation of QDs
surface (Figure 3b). The diffusion coefficient and the hydro-
dynamic diameter of PC-QDs prepared from SeTe/CdS-type
QDs were estimated to be 18.2 μm2 sꢀ1 and 11.7 ( 1.5 nm,
based on the value of diffusion coefficient using 20 nm when
fluospheres fluorescent beads (D = 10.4 μm2 sꢀ1) were used as a
standard material. On the contrary, PEG-QDs did not show any
satisfactory fitting profile with neither the one- nor the two-
component diffusion model, suggesting the importance of am-
photeric nature of the PC-QDs for preventing aggregation of
QDs capped with monothiol derivatives. It was also revealed by
absolute photoluminescence quantum yield measurement
that quantum yield of PC-QDs in water is identical to that of
TOPO-QDs in CHCl3 (quantum yield = 0.47), while the
quantum yield of PEG-QDs in H2O was reduced to be 0.1 after
PEG-SH [(1-mercaptoundec-11-yl)hexa(ethylene glycol)]17
coating (Figure 3c). Given the facts that the quantum yields of
QDs generally appear to decrease by the ligand exchange
reaction with general thiol compounds,10b,18 long-alkyl mono-
thiol derivatives having both amphoteric and amphipathic prop-
erties might be essential to retain high fluorescence quantum
yields and to prevent aggregation due to nonspecific interactions.
It was reported that QDs capped by zwitterionic ligands, such as
cysteine (Cys)6h and dihydrolipoic acid modified by sulfobetaine
(DHLA-SB),6l preserved QD optical properties and excellent
stability as well as good resistance to pH variations, while Cys-
QDs quickly aggregated after a few days.
Merit of the use of compound 1 is evident because PC-QDs
showed prominently long-term stability in water at 4 °C and
excellent resistance to an extensive range of pH (pH 2ꢀ14) as
compared with those of PEG-QDs (Figure 4a and b). Surpris-
ingly, PC-QDs were stable for at least 6 months, and the loss of
fluorescence intensity was only 20% even at pH of 3. These
favorable characteristics of PC-QDs are quite similar to those
observed for DHLA-SB-QDs capped by a dithiol anchor group.6l
This suggests clearly that a balanced charge of zwitterions and a
minimized dipole of monothiol 1 are essential for the strong
resistance of phosphorylcholine SAMs formed on QDs from
aggregation due to their strong hydration capacity through
electrostatic interaction.12c Moreover, it was also demonstrated
that monothiol anchor can stabilize QDs surface efficiently when
such suitable amphoteric nature was incorporated into the
hydrophilic head as well as good molecular packing of the
hydrophobic linker moiety.
Figure 4. Long-term stability and pH resistance. (a) Fluorescence
intensity per molecule was measured and estimated by FCS for the
solutions of PC-QDs and PEG-QDs stored in water at 4 °C in the dark
for 6 months. (b) The fluorescence intensity measured by spectro-
photometer and photograph under UV irradiation at 365 nm of PC-QDs
and PEG-QDs in aqueous buffer solutions at various pHs.
derivative, 11,110-dithio bis[undec-11-yl 12-(aminooxyacetyl)amino
hexa(ethyleneglycol)] (ao-SH, 2),13 should provide QDs with
promising characteristics, such as much higher solubility, stability,
and ability to display multiple ligands as well as biomembrane-
mimetic surface properties (Figure 1). Jiang et al. revealed that
zwitterions with a balanced charge and minimized dipole are crucial
for the strong resistance of phosphorylcholine SAMs formed on a
gold-coated chip to protein adsorption due to their strong hydration
capacity through electrostatic interaction.12c Phosphorylcholine
SAMs on magnetic beads12d could also be applied for stable
supporting materials to immobilize reusable engineered glycosyl-
transferases. In addition, our previous study demonstrated that
simple glycans and glycosphingolipid derivatives enriched by glyco-
blotting reaction with ao-SH (2) on gold nanoparticles can be
directly ionized from Au surfaces under general matrix-assisted laser
desorption ionization (MALDI) condition.13,14 Taking the charac-
teristics of the two monothiol derivatives 1 and 2 into account, the
present protocol seems to be advantageous for the construction of a
variety of compound library displayed on the QD surfaces in a
controlled density because the structure (quality) and the quantity
of molecules displayed on QDs might be determined concurrently
by direct matrix-assisted laser desorption ionization time-of-flight
mass spectrometry (MALDI-TOFMS) without any purification or
pretreatment procedure.
To assess the feasibility of PC-SH (1) as a novel coating
material for improving the properties of QDs surface, various PC-
QDs were prepared by ligand exchange reaction of PC-SH with
TOPO-coated QDs (Figure 2a). Reaction of compound 1 with
commercially available TOPO-coated QDs with diameters in a
range from 5.8 to 9.3 nm (CdSe/ZnS, λem = 545, 565, 585, 605,
and 655 nm) and synthetic TOPO-coated QDs of 11.7 nm
Construction of Glyco-PC-QDs as Glycoprotein Models.
General glycans released from naturally occurring glycoconju-
gates such as N- and O-glycans of glycoproteins and glyco-
sphingolipid derivatives with oligosaccharide head groups can be
directly captured by “glycoblotting”,19 a promising method for
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Figure 5. The preparation of glyco-PC-QDs. (a) chemical structures of p-(4-oxopentanamido)-phenyl glycosides 3aꢀh. (b) A simple protocol for the
preparation of glyco-PC-QDs by the reaction of ao-PC-QDs with glycosides 3aꢀh. (c) Glycoblotting of 3a by ao-PC-QDs monitored by MALDI-
TOFMS. (d) MALDI-TOFMS of various glyco-PC-QDs. MALDI-TOFMS were measured at positive mode, while Neu5Ac-PC-QDs needed negative
mode due to carboxyl groups.
chemoselective ligation of any reducing sugars or compounds
bearing aldehyde/ketone by use of the aminooxy/hydrazide-
functionalized materials.13,19 To display general carbohydrates
and synthetic glycosides efficiently on the surface of QDs in
combination with phosphorylcholine SAMs, we selected an
aminooxy-functionalized ao-SH (2)13 containing 11-mercap-
toundecyl moiety, an alkanethiol anchor group of PC-SH (1),
in order to obtain an appropriate molecular packing structure of
SAMs on QDs. As expected, QDs surface could be coated
efficiently by the mixed SAMs of ao-SH (2) and PC-SH (1) at
an arbitrary ratio and afforded a novel class of multifunctional
QDs (ao-PC-QDs) as characterized by direct MALDI-TOFMS
and FCS.
Glycoblotting of free sugars by the aminooxy-functional group
induces generally ring-opening reaction at the reducing end of any
carbohydrates and gives a mixture of E/Z-isomers during stable
oxime bond formation.19 To control the stereochemistry at an
anomeric carbon of the reducing end of oligosaccharides even in
cases of simple monosaccharides, we decided to employ versatile p-
nitrophenyl glycosides as key starting materials. Various p-nitro-
phenyl glycosides readily prepared by conventional synthetic
methods were converted into p-(4-oxopentanamido)-phenyl gly-
cosides 3aꢀh in high yields (Figure 5a and Scheme S1, Supporting
Information). Reactions of glycosides 3aꢀh with ao-PC-QDs
(ao-SH/PC-SH = 2/8) proceeded smoothly in acetic acid buffer
(pH of 4.0) for 30 min at room temperature to afford glyco-PC-QDs
carrying β-Glc (Glc-PC-QDs), R-Man (Man-PC-QDs), R-Fuc
(Fuc-PC-QDs), β-GlcA (GlcA-PC-QDs), β-GlcNAc (GlcNAc-
PC-QDs), β-GalNAc (GalNAc-PC-QDs), β-Lac (Lac-PC-QDs),
and R-Neu5Ac (Neu5Ac-PC-QDs), respectively (Figure 5b). As
shown in Figure 5c, the signal at m/z 908.7 corresponding to
aoꢀSꢀSꢀPC disappeared completely instead of the advent of new
signal at m/z 1259.6 due to a heterodisulfide bearing Glc residue,
indicating successful oxime bond formation between ao-PC-QDs
and ketone-functionalized glycoside 3a. Similarly, all other products
derived by the reactions of 3bꢀh with ao-PC-QDs were also
characterized as signals corresponding to the heterodisulfides by
MALDI-TOFMS (Figure 5d). As shown in Figure 6, the GlcNAc
residues on the GlcNAc-PC-QDs were quantified simply by
1
measuring H NMR spectrum of QDs directly. No unreacted
aminooxy functional group (H2NꢀOꢀCH2ꢀ at 4.17 ppm) was
detected after glycoblotting with compound 3e (Figure 6b), and
the product generated by treating the GlcNAc-PC-QDs with I2 was
also fully characterized as the reasonable composition (Figure 6c).
These results clearly indicate that the ratio of glycan/PC on the QD
surface can be controlled by that of ao-SH/PC-SH of the ao-PC-
QDs. As indicated by the TEM views in Figure 6d and e, the
diameters of ao-PC-QDs and GlcNAc-PC-QDs were proved to be
a range from 7 nm (short side) to10 nm (long side), and the results
were in good agreement with those estimated by FCS (∼11 nm).
In our previous study,14 it was demonstrated that simple sugar
residues displayed on the surface of gold nanoparticles (3ꢀ8 nm
in diameter) can be further modified by some glycosyltrans-
ferases in the presence of sugar nucleotides. Here we examined
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suggesting that the excellent dispersibility and fluorescence
property of PC-QDs/ao-PC-QDs were not changed during sugar
elongation reactions of GlcNAc-PC-QDs (3c) by sequential
treatment with three glycosyltransferases.
Live Animal NIR Fluorescence Imaging by Means of Glyco-
PC-QDs. Glyco-PC-QDs (CdSeTe/CdS) derived from NIR
ao-PC-QDs with an excitation of 710 nm and an emission of
800 nm were employed for live animal imaging in order to reduce
nonspecific photoabsorption by biomolecules.15,21 Initially, we
tested the real-time fluorescent monitoring of Lac-PC-QDs (3g)
and Neu5Ac-PC-QDs (3h) in comparison with PC-QDs as a
control after injection into the vein of mouse tail (Figure 8a).
Choi et al. reported that QDs of 5.5 nm or less drained from urine
immediately and that those of 5.5 nm or more accumulated in the
liver,6i even though cysteine6h was employed as a zwitterionic
component in addition to DHLA, cysteamine, and DHLA-PEG.
Therefore, it seemed that PC-QDs may distribute in the liver
according to this size effect of QDs, since NIR PC-QDs with ca.
11.7 nm in diameter were tested for the present live animal
imaging. In addition, it was also considered that Lac-PC-QDs
should accumulate in the liver quickly due to the hepatic
asialoglycoprotein receptor22 responsible for the specific inter-
action with terminal β-Gal residues of various oligosaccharides,
while Neu5Ac-PC-QDs might show a different organ distribu-
tion profile due to the effect of sialic acids. As anticipated, it was
demonstrated that QDs bearing lactose 3g began to accumulate
in the liver immediately after administration. However, PC-QDs
and Neu5Ac-PC-QDs did not distribute in any specific organ,
indicating that nonfouling behavior observed in PC-QDs is due
to the different mechanism from the resistance to nonspecific
protein adsorption by QDs displaying cysteine6h having amino
and carboxyl groups. Highly packing structure of monothiol
phosphorylcholine-SAMs may provide QDs surface with specific
nonfouling nature by strong hydration capacity through electro-
static interaction. This characteristic of PC-QDs was not influ-
enced by modification with Neu5Ac residues, while lactose
moiety in Lac-PC-QDs (3g) drastically altered PC-QDs as
liver-directed QDs. Interestingly, a photograph taken 10 min
after the injection of other glyco-PC-QDs (3aꢀf) revealed that
PC-QDs having Glc, Man, Fuc, GlcNAc, and GalNAc also
accumulated immediately in the liver, while GlcA-PC-QDs
(3d) did not (Figure 8b). Surgery confirmed that the entire liver
strongly fluoresced with Glc-PC-QDs and GlcNAc-PC-QDs,
and the bottom edge of the liver weakly fluoresced with GlcA-
PC-QDs (data not shown). These results clearly showed that
distinct long-term delocalization over 2 h is observed only in
cases of QDs modified with R-sialic acid (Neu5Ac-PC-QDs)
and control PC-QDs, while QDs bearing other popular neutral
sugars were accumulated rapidly (5ꢀ10 min) in the liver.
This trend was preliminarily demonstrated by photographs of
fluoresced organs isolated from tested mice injected by three
typical materials, PC-QDs, Lac-PC-QDs, and Neu5Ac-PC-QDs
(Figure 8c). It should also be noted that β-glucuronic acid (GlcA-
PC-QDs) appeared to show a different metabolic profile when
compared with other glyco-PC-QDs.
Figure 6. Characterization of Glyco-PC-QDs by 1H NMR and TEM.
(a) ao-PC-QDs, (b) GlcNAc-PC-QDs, (c) the product generated by
treating GlcNAc-PC-QDs with I2. TEM views of (d) ao-PC-QDs and
(e) GlcNAc-PC-QDs. See also Supporting Information.
the conversion of a simple glyco-PC-QDs (3c) into more
complicated glyco-PC-QDs bearing LacNAc (4), Lex (5), sialyl
LacNAc (6), and sialyl Lex (7) by treating with recombinant
β1,4GalT, R1,3FucT, and R2,3Neu5Ac in the presence of suited
sugar nucleotides under a general condition of enzymatic
synthesis20 (Figure 7a). Surprisingly, all reactions proceeded
smoothly and afforded target derivatives in quantitative yields,
respectively, as monitored by MALDI-TOFMS shown in
Figure 7b. Given the fact that globular protein-like dendrimers
having spherical molecular shape and size (∼10 nm in diameter)
are ideal polymer supports in the fully automated enzymatic
glycan synthesis,20d glyco-PC-QDs 3c and 4ꢀ6 appear to
become a convenient glycoprotein-like model. These results
clearly indicate that a strategy based on enzymatic sugar elonga-
tion of chemically synthesized simple glyco-PC-QDs allows for
the construction of highly complicated glycan-conjugated PC-
QDs library.
Considering that PC-QDs were proved to be an ideal scaffold
material mimicking general globular proteins having specific
nonfouling surface, our interest was next focused on the insight
into functional roles of the terminal sialic acid residues involving
in the characteristic oligosaccharide moieties, such as sialyl
Lewis antigens known as important core structures for possible
ligands of mammalian lectins, such as selectins.23 In vivo NIR
As indicated in Figure 7c, ao-PC-QDs, GlcNAc-PC-QDs (3c),
and sialyl Lex-PC-QDs (7) exhibited good fitting curves with
a single-component diffusion model of the FCS analyses,
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Figure 7. Enzymatic sugar extension of GlcNAc-PC-QDs. (a) Synthetic route of fourglyco-PC-QDs by means of three recombinant glycosyltransferases
and sugar nucleotides. (b) Monitoring the processes of enzymatic modifications by direct MALDI-TOFMS. (c) Fluorescence fluctuations of ao-PC-QDs,
GlcNAc-PC-QDs (3c), and sialyl Lex-PC-QDs (7) in water. The autocorrelation curves were fitted by using an one component diffusion model.
fluorescence imaging using PC-QDs displaying LacNAc (4), Lex
(5), sialyl LacNAc (6), and sialyl Lex (7) uncovered for the first
time that Lex-PC-QDs (5) are also accumulated rapidly (5ꢀ30
min) in the liver after intravenous injection and almost quenched
over 1 h in a similar profile to that of LacNAc-PC-QDs (4)
(Figure 9a). On the other hand, sialyl LacNAc-PC-QDs (6) and
sialyl Lex-PC-QDs (7) were still retained stably in the body after
2 h while they exhibited significantly different in vivo dynamics in
the tissue distribution observed by dissection at 2 h after
injection. As indicated in Figure 9b, sialyl LacNAc-PC-QDs
(6) appeared to locate spleen or intestine, and sialyl Lex-PC-
QDs (7) did not localize in any organs, while Lex-PC-QDs (5)
distributed specifically in the liver. These results suggest that
structure/sequence of the neighboring sugar residues in the
individual sialylated oligosaccharides might influence signifi-
cantly the organ-specific distribution after long-term circulation.
Disappearance of fluorescence of these glyco-PC-QDs after
administration might be due to the disintegration in the body
without any leaking into urinary bladder or the urine. Metabo-
lism and excretion pathway of PC-QDs and individual sialic acid-
containing glyco-PC-QDs after injection into mice remains
unclear, while there was no significant change in cell viability
tested by using A549 cells between PC-QDs and GlcNAc-PC-
QDs (Figure S4, Supporting Information).
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Figure 8. Live animal imaging of glyco-PC-QDs. (a) Imaging of PC-QDs, Lac-PC-QDs (3g), and Neu5Ac-PC-QDs (3h) after injection of 100 pmole of
glyco-PC-QDs. (b) Pictures taken 10 min after administration of QDs carrying 3aꢀh compared with PC-QDs. The mice were imaged using Lumazone
equipped with Photometrics Cascade II EM CCD camera. QDs were excited with 710 nm, and emission filter was 800/12 nm bandpass filter.
(c) Photographs of major organs isolated from three tested mice (PC-QDs, Lac-PC-QDs, and Nue5Ac-PC-QDs) at 2 h after administration.
’ CONCLUSION
Further enzymatic modifications of simple glyco-PC-QDs pro-
vided QDs having more complicated Lewis antigen-related oligo-
saccharides. It was demonstrated that a nonfouling characteristic
of highly stable PC-QDs makes in vivo direct monitoring of
glycan-specific interaction and distribution of glyco-PC-QDs after
injection into the vein of mouse tail possible. Live animal NIR
fluorescence imaging of glyco-PC-QDs revealed the importance
of the terminal sialic acid residues for achieving prolonged
in vivo lifetime24 without size-dependent liver localization of
nanoparticles.6i Advantage of the present approach is clear because
We established a standardized procedure for the preparation of
PC-QDs, highly sensitive, stable, and nonfouling QDs coated by
phosphorylcholine SAMs by means of a simple monothiol,
11-mercaptoundecylphosphorylcholine (PC-SH, 1). Combined
use of 1 with an aminooxy-terminated thiol derivative, 11,110-dithio
bis[undec-11-yl 12-(aminooxyacetyl)amino hexa(ethyleneglycol)]
(ao-SH, 2), allowed for the construction of an ideal glycoprotein
model, namely glyco-PC-QDs, through the glycoblotting-based
chemical ligation with ketone-functionalized synthetic glycosides.
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Figure 9. Live animal imaging of glyco-PC-QDs carrying Lewis antigen-related oligosaccharides. (a) Time course after injection of 100 pmole of glyco-
PC-QDs (4ꢀ7). (b) Distribution of glyco-PC-QDs carrying Lex (5), sialyl LacNAc (6), and sialyl Lex (7) uncovered preliminarily by dissection at 2 h
after administration.
ao-PC-QDs are a novel class of convenient and versatile scaffold for
displaying a variety of compounds having reactive ketone/aldehyde
as well as reducing sugars and synthetic glycosides used in this
study. Considering the impact of current and emerging nanoma-
terials systems for the development of practical drug delivery,25 the
present strategy would enable entirely novel classes of therapeutics.
’ ACKNOWLEDGMENT
This work was supported partly by a program “Innovation
COE program for future drug discovery and medical care” from
the Ministry of Education, Culture, Science, and Technology,
Japan.
’ REFERENCES
’ ASSOCIATED CONTENT
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preparation of PC-QDs, ao-PC-QDs, glyco-PC-QDs, synthesis
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refs 24a and 24d. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
shin@glyco.sci.hokudai.ac.jp.
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