Targeted cellular uptake and siRNA silencing by quantum-dot nanoparticles coated with β-cyclodextrin coupled to amino acids

Article abstract of DOI:10.1002/chem.201003523

Quantum dots (QDs) have the potential to serve as photostable beacons to track siRNA delivery, which is fast becoming an attractive approach to probe gene function in cells. In this paper, we synthesized QD nanoparticles coated with β-cyclodextrin (β-CD)

Full text of DOI:10.1002/chem.201003523

FULL PAPER
DOI: 10.1002/chem.201003523
Targeted Cellular Uptake and siRNA Silencing by Quantum-Dot
Nanoparticles Coated with b-Cyclodextrin Coupled to Amino Acids
[
a]
[a]
[b]
[a]
[b]
Mei-Xia Zhao, Jin-Ming Li, Lingyan Du, Cai-Ping Tan, Qing Xia,*
[
a, b]
[a]
Zong-Wan Mao,*
and Liang-Nian Ji
Abstract: Quantum dots (QDs) have
the potential to serve as photostable
beacons to track siRNA delivery,
which is fast becoming an attractive ap-
proach to probe gene function in cells.
In this paper, we synthesized QD nano-
particles coated with b-cyclodextrin (b-
CD) coupled to amino acids with dif-
ferent surface charges (positive, nega-
tive, and neutral) through direct
ligand-exchange reactions and used
them to deliver siRNA. We found that
these QDs are diffluent in biological
buffer with high colloidal stability and
have strong optical emission properties
similar to those of tri-n-octylphosphine
oxide (TOPO)-coated QDs and also
(12.5 ns for l-His-b-CD-coated CdSe/
ZnSe QDs). The results of in vitro cy-
totoxicity and internalization of these
modified QDs in normal and cancer
cells showed that the b-CD coupled to
amino acid outlayers greatly improved
the biocompatibility of QDs, and con-
ferred with lower cytotoxicity even at
very high concentration. In particular,
the l-His-b-CD-coated CdSe/ZnSe
QDs presented lower cytotoxicity to
these cells (CC50 value is 180.6Æ
Transmission
electron
microscope
(TEM) images showed that the QDs
were localized in vesicles in the cyto-
plasm of the cells. Furthermore, com-
pared with existing transfection agents,
gene-silencing efficiency of the modi-
fied QDs was slightly improved for
HPV18 E6 gene in HeLa cells by gel
electrophoresis analysis. Finally, the
unique optical properties of QDs allow
visible imaging of siRNA delivery in
live cells. Taken together, our study not
only provides new insights into the
mechanisms of amino acid mediated
delivery, but also greatly facilities the
monitoring of gene-silencing studies.
À1
3.4 mgmL in ECV-304 cells for 48 h).
Keywords: cellular uptake · gene
delivery · nanoparticles · quantum
dots · siRNA
have
a
long fluorescence lifetime
[
4]
Introduction
ZnSe QDs. However, this approach does not efficiently
[5]
stabilize nanoparticles. After the ligand exchange, the
quantum yields (QYs) of the QDs drops significantly in
biological buffers or culture media and the QDs become
highly toxic to living cells. Therefore, several alternative
coating strategies have been developed, such as the use of
[6]
Quantum dots (QDs) have emerged as an attractive candi-
date for delivering various payloads into cells, such as small
drug molecules or large biomolecules (e.g., DNA and
siRNA ), owing to their good biocompatibility, monodisper-
sity, ready functionalization, size-tunable emission spectra,
broad absorption profiles, and superior photostability. The
most frequently used anchoring groups reactive to the sur-
face of QDs are thiol (-SH), which can be exchanged with
tri-n-octylphosphine oxide (TOPO) at the surface of CdSe/
[1]
[7]
[2]
[8]
[9]
[10]
[11]
peptides,
polyelectrolytes,
polysaccharides,
avidin
[3]
and QD-encapsulating amphiphilic molecules. However,
these methods have the disadvantages of relatively large
coating thickness, leading to large probe sizes (e.g., more
than 15 nm) and the difficulty to monovalently label biomol-
ecules. To reduce these disadvantages, the carboxylic acid
[12]
group can be further coupled with various biomolecules.
Previous studies have used cell-penetrating peptides, such as
arginine, and twin-arginine translocation (Tat) methods to
[
a] M.-X. Zhao, J.-M. Li, Dr. C.-P. Tan, Prof. Z.-W. Mao, Prof. L.-N. Ji
MOE Key Laboratory of Bioinorganic and Synthetic Chemistry
School of Chemistry and Chemical Engineering
Sun Yat-Sen University, Guangzhou 510275 (P. R. China)
Fax : (+86)20-8411-2245
[13]
deliver QDs into living cells, but the delivery mechanism
and the behavior of intracellular QDs are still under debate.
In recent years, small interfering RNA (siRNA) has
emerged as a promising tool for biological research and
E-mail: cesmzw@mail.sysu.edu.cn
[14]
[
b] L. Du, Dr. Q. Xia, Prof. Z.-W. Mao
holds a great potential for treatment of human diseases.
State Key Laboratory of Natural and Biomimetic Drug
Department of Chemical Biology, School of Pharmaceutical Science
Peking University, Beijing 100191 (P. R. China)
Fax : (+86)10-8280-5611
Although siRNA is a powerful gene regulation agent, its
therapeutic application is limited, mainly due to poor cellu-
lar uptake and accelerated degradation in biological
fluids. Some siRNA carrier complexes are not amenable
to either systemic delivery, because of their relatively large
size, rapid uptake by macrophages, or targeted delivery, or
[15]
E-mail: xqing@bjmu.edu.cn
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201003523.
Chem. Eur. J. 2011, 17, 5171 – 5179
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
5171

Scheme 1. Schematic illustration of the formation of the QD nanoparticles coated with b-CD coupled to amino acids and QD-siRNA complexes. (R =
l-His, l-Trp, l-Phe, and l-Cys residues.)
targeted delivery resulting from their ubiquitous internaliza-
[16]
[17]
tion. Size is a key parameter for in vivo applications:
small-sized nanoparticles are removed from the cell faster
than large-sized nanoparticles. Moreover, natural polysac-
charides b-cyclodextrin (b-CD) has good application in the
drug delivery systems due to their biocompatibility, low tox-
icity, antibacterial and antifungal activity, biodegradability,
[18]
and lack of foreign body response.
Here we reported a new class of multifunctional nanopar-
ticles conjugates of compatible size (d=4–5 nm) with even-
tual systemic delivery upon a coated QD core for siRNA
(
Scheme 1). We used these carboxyl groups in the b-CD
coupled to amino acid ligands as the anchoring groups to re-
place the organic alkylamine ligands-coated on CdSe/ZnSe
core-shell nanocrystals, as well as the anchoring groups to
link other functional molecules. The b-CD coupled to amino
acids not only improves the solubility and stability of the
coated QDs in biological buffer or cell culture media and
keeps the long fluorescence lifetime, as well simultaneously
reducing the cellular toxicity. We used QD nanoparticles
coated with b-CD coupled to amino acids as a model system
to examine the cellular uptake and intracellular transport of
QDs in living cells. As a result, TEM images provided evi-
dence that the QDs were localized in vesicles in the cyto-
plasm of the cells. Compared with current siRNA delivery
reagents, such as siPort NeoFX, and HiPerFect, the gene-si-
lencing activity of the QDs was slightly improved for
HPV18 E6 gene in HeLa cells. In addition, the QDs should
also provide a bright and stable fluorescent signal for intra-
cellular siRNA imaging. These self-tracking siRNA delivery
QDs would greatly facilitate monitoring gene-silencing ac-
tivities in vivo in future.
Figure 1. A) UV/Vis and B) photoluminescence spectra of a) l-His-b-
CD-coated CdSe/ZnSe QDs, b) l-Trp-b-CD-coated CdSe/ZnSe QDs, c)
l-Phe-b-CD-coated CdSe/ZnSe QDs, and d) l-Cys-b-CD-coated CdSe/
ZnSe QDs in PBS solution (pH 7.4), (Ex: 460 nm). Each sample was ex-
cited at the wavelength with an absorbance value of 0.1.
ing that the modified QDs maintained the optical properties
of the original QDs in biological buffer.
Results and Discussion
The QD nanoparticles were further characterized by
using fluorescence spectroscopy. Figure 1B shows the fluo-
rescence spectra of the QDs in biological buffer. The fluo-
rescence peak is red-shifted relative to the absorption onset
of these original QD nanoparticles. The broad nature of the
fluorescence band and the red-shift suggest that the emis-
sion arises due to the recombination of amino acids in the
surface of QDs. The QYs of these QDs were measured rela-
tive to the value of rhodamine B (QY=89%, EtOH) as a
criterion at room temperature, which were 69.3Æ2.2, 37.6Æ
3.4, 30.4Æ3.8, and 10.6Æ4.1% (Table 1), which are higher
Synthesis and characterization of QD nanoparticles and
QD-siRNA complexes: Scheme 1 shows the reaction
scheme for the formation of QD nanoparticles coated with
b-CD coupled to amino acids. In this system, we used the
carboxyl groups in the b-CD coupled to amino acid ligands
as the anchoring groups to replace the organic alkylamine
ligand coated on CdSe/ZnSe core-shell nanocrystals. Fig-
ure 1A shows the UV/Vis spectra of different coated QD
nanoparticles. There was no difference in the position or
width of absorbance bands from hydrophobic QDs, suggest-
[19]
than reported ones.
Its perhaps because the positive
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Gene Delivery
FULL PAPER
Table 1. Summary of the optical properties of QD nanoparticles coated
with b-CD coupled to amino acids.
with a negatively charged cell surface (Scheme 1). To inves-
tigate the interaction of siRNA with QDs, we labeled
siRNA molecules with FITC dye (green) and mixed the
siRNA with red l-His-b-CD-coated CdSe/ZnSe QDs at the
Samples
F [%]
Estimated
t
av
PL intensities in
size [nm] [ns] cells
[24]
l-His-b-CD-coated
CdSe/ZnSe
l-Trp-b-CD-coated
CdSe/ZnSe
l-Phe-b-CD-coated
CdSe/ZnSe
l-Cys-b-CD-coated
CdSe/ZnSe
69.3Æ2.2 ꢀ4.6
37.6Æ3.4 ꢀ4.5
30.4Æ3.8 ꢀ4.7
10.6Æ4.1 ꢀ4.8
12.5 30.5Æ1.4
9.6 22.7Æ2.1
9.2 15.1Æ1.8
5.7 10.3Æ2.5
ratio of 1:1. As shown in the fluorescence spectra (Fig-
charge located on the surface will prevent aggregation of
the QDs in buffer solution by strong repellent forces among
[20]
particles.
The shape and size of QDs were determined by using
electron microscopy. The TEM images in Figure 2 demon-
strated that the QDs were well monodispersed and uniform
in biological buffer with spherical shape and compact size of
Figure 3. Fluorescence spectra of QD-siRNA complexes and fluorescence
decay curves for QDs coated with b-CD coupled to amino acids. A)
Fluorescence spectra of QD-siRNA complexes. siRNA and QDs show
characteristic emission maxima at approximately a) 520 nm and b)
6
10 nm when measured separately. c) When siRNA molecules and QDs
Figure 2. TEM images of A) CdSe/ZnSe QDs (in hexane), B) l-His-b-
CD-coated CdSe/ZnSe QDs, C) l-Trp-b-CD-coated CdSe/ZnSe QDs,
D) l-Phe-b-CD-coated CdSe/ZnSe QDs, E) l-Cys-b-CD-coated CdSe/
ZnSe QDs in PBS solution, and F) l-His-b-CD-coated CdSe/ZnSe QDs-
siRNA in diethylpyrocarbonate (DEPC)-treated water. All scale bars are
are mixed together, they bind to each other quickly indicated by the fluo-
rescence drop of siRNA-FITC due to energy transfer. B) Fluorescence
decay curves for four amino acid-modified b-CD-coated CdSe/ZnSe QDs
in PBS solution (pH 7.4), one labeling ratio was used. Solid lines repre-
sent the fit curves using a mono-exponential function as described in the
text.
2
0 nm.
4
–5 nm in diameter. When bound to siRNA (Figure 2F), the
size of the nanoparticle complexes further increases to 5.8Æ
.0 nm, suggesting that QDs remain mainly single with
ure 3A), the fluorescence intensity of the FITC-labeled
siRNA decreases due to fluorescence resonance energy
transfer (FRET), indicating that siRNA molecules can be
immobilized onto the surface of QDs. In addition, the inter-
action of siRNA with QDs can be characterized by zeta-po-
tential measurements. Focused on the siRNA/QD ratio of
1:1, the zeta-potential measurements show that l-His-b-CD-
coated CdSe/ZnSe QDs has a zeta-potential value of
22.9 mV before siRNA binding, and it reduces to 17.8 mV
after siRNA binding because negatively charged siRNA par-
tially neutralizes the positive charge on the QD surface.
1
siRNA on the surface. This size is comparatively smaller
than those of QDs ligand exchanged with peptide-derivat-
ized coating, which allows the access of QDs to crowded
regions (e.g., cells). So the compact size of single particles is
highly desirable, because large particles enter cells at a
much slower rate and can be eliminated quickly by the re-
ticuloendothelial systems (RES) system in vivo.
In addition, the surface charge of the amino acid modi-
fied, b-CD-coated QDs is highly positive, which allows im-
mobilization of negatively charged siRNA and interaction
[21]
[22]
[23]
Chem. Eur. J. 2011, 17, 5171 – 5179
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
5173

Z.-W. Mao, Q. Xia et al.
Conjugate stability of the QD nanoparticles coated with b-
CD coupled to amino acids: In addition, the carboxyl bind-
ing motif employed in amino acids resulted in an aqueous
solution that remained stable under ambient conditions for
at least five months, suggesting that the original aliphatic
protons of TOPO-coated QDs disappeared after ligand ex-
change with b-CD coupled to amino acids and that the
amino acid protons from the complex are bound to the QDs
surface. To confirm the above notion, we performed fluores-
cence lifetime measurements to monitor the dynamics of
these QDs photoexcited carriers. We evaluated the photolu-
minescence (PL) lifetime of the QD nanoparticles coated
with b-CD coupled to amino acids in phosphate buffered
saline (PBS) solutions by using frequency domain spectros-
copy. Figure 3B shows the fluorescence decay profiles for
the four types of QDs. The fluorescence decay profile con-
structed from photons in the maximum intensity range is de-
scribed by a mono-exponential decay function. We found
that these QDs exhibited the fluctuations of lifetime similar
to the original CdSe/ZnSe QDs (see Figure S1 in the Sup-
porting Information). The apparent lifetime of the four QDs
ranged from 5.7 to 12.5 ns (Table 1). These values are in
agreement with those reported in previous studies in which
concentration) of the four amino acid modified b-CD-
coated CdSe/ZnSe QDs by the MTT assay (MTT=3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide salt)
over 48 h, indicating that the QDs with positive or neutral
charged amino acids had a lower cytotoxicity in cells. In
contrast, the cytotoxicity of l-Cys-b-CD-coated CdSe/ZnSe
QDs with negative charge was higher due to the presence of
thiol (-SH). Interestingly, the cytotoxicity of the QDs was
lower in normal cells than in the cancer cells. Since CdSe
II
QDs may slowly release the toxic Cd ions into solution, the
particles should be inert for a short time for any in vitro ap-
II
plication. The release of Cd from the particle surface can
be reduced by employing core/shell particles or coating of
the particles with biomolecules or a polymer. These results
indicated that the QDs we developed here can reduce the
II
release of toxic Cd ions from QDs.
Evaluation of conjugate cell internalization of functionalized
QDs by flow cytometry: To quantify the cellular binding
and uptake of the QDs, we measured the transfection effi-
ciency by flow cytometry. Figure 4 shows the flow cytometry
results in the HeLa cells incubated with QD nanoparticles
coated with b-CD coupled to amino acids and the control.
Single-color histograms of each sample, plotted as counts
versus log of red intensity, show a distribution of fluores-
cence intensities. Based on the flow cytometry values and
the average fluorescence intensity of cells, the QDs coated
by b-CD coupled to amino acids evidently resulted in a con-
[25]
the values depend on the QD microenvironment. In addi-
tion to other merits, the long lifetime provides a strong ad-
vantage of using QDs as intracellular imaging agents to dis-
criminate labeled ones from intrinsic cellular components.
Cell viability studies: A key question is whether QDs, as a
new siRNA carrier, are toxic to cells. This is a particularly
important issue when the core nanoparticle is a semiconduc-
tor QD, because it contains cadmium, and the biological ap-
plicability of the QDs is mainly based on their minimal ef-
[
12,26]
fects on cellular function and viability.
The toxicity of
II
the QDs not only depends on the concentration of free Cd
ions, but also on whether the particles are ingested by the
[27]
cells and where they are stored. The main characteristics
of QDs, such as small size and large surface area, may be re-
sponsible for the interactions that could result in their toxi-
cological effects. To directly evaluate the toxicity of the QD
nanoparticles coated with b-CD coupled to amino acids, we
monitored the viability of ECV-304, SH-SY5Y, and HeLa
À1
cells. Different doses (5, 10, 20, 50, 100 and 200 mgmL ) of
a series of the QDs were used for in vitro cytotoxicity tests
with cells to examine whether the QDs damage the cells or
not. Figure S2 in the Supporting Information shows the
spectra. Table 2 shows the CC50 values (50% cytotoxicity
Table 2. Summary of the cytotoxicity of QD nanoparticles coated with b-
CD coupled to amino acids.
À1
Samples
CC50 [mgmL
SH-SY5Y
]
Figure 4. Quantitative flow cytometry results on fluorescence intensities
of A) l-His-b-CD-coated CdSe/ZnSe QDs, B) l-Trp-b-CD-coated CdSe/
ZnSe QDs, l-Phe-b-CD-coated CdSe/ZnSe QDs, and D) l-Cys-b-CD-
coated CdSe/ZnSe QDs in HeLa cells. In the experiment, ten thousand
cells were collected in each measurement. The columns, from left to
right, represent autofluorescence, cells treated by amino acid-modified b-
ECV-304
HeLa
L-His-b-CD-coated CdSe/ZnSe
l-Trp-b-CD-coated CdSe/ZnSe
l-Phe-b-CD-coated CdSe/ZnSe
l-Cys-b-CD-coated CdSe/ZnSe
180.6Æ3.4
175.2Æ5.6
146.5Æ4.1
92.3Æ5.2
162.4Æ5.1
131.2Æ4.5
109.3Æ6.2
79.6Æ4.3
124.4Æ3.1
87.5Æ5.1
76.3Æ3.4
51.6Æ4.3
À1
CD-coated CdSe/ZnSe QDs at 50 mgmL , at 378C for 6 h.
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Gene Delivery
FULL PAPER
siderable increase in the intracellular delivery of QDs. The
average fluorescence intensity values are shown in Table 1.
Compared with the control, the fluorescence intensity of the
QD-labeled l-Cys-b-CD with negative charge slightly in-
creased in HeLa cells. In contrast, l-Trp-b-CD-coated and l-
Phe-b-CD-coated QDs showed specific uptake in cells, with
coated QDs were found in multiple pinosomes in the cyto-
plasm and did not enter the nucleus of the cells; the other
two kinds of QDs were found in large aggregated groups
the cytoplasm (without multiple vacuoles). It is apparent
that the mechanism of intracellular delivery of l-His- and l-
Trp-coated QDs is different from that of l-Phe- and l-Cys-
coated ones. This is perhaps because the positive charge of
the amino acid resulted in a considerable increase in the in-
tracellular delivery of QDs. Moreover, the morphology of
QDs did not form a linear nanoparticle chain in the cell; in-
stead, the nanoparticles were in a tangled, clumped arrange-
ment. Our finding is consistent with the confocal microscopy
results that arginine-conjugated QDs primarily localized
6–9-fold greater uptake relative to the autofluorescence of
the cells. Furthermore, l-His-b-CD-coated QDs with posi-
tive charge showed a markedly greater uptake than other
coated QDs in HeLa cells at the same concentrations. Based
on the flow cytometry values, the positive charge of amino
acid led to a considerable increase in the intracellular deliv-
ery of QDs. This is probably because the positive charge of
amino acids on the surface of QDs would prevent aggrega-
tion of the QDs in buffer solution by strong repellent forces
[13c]
within the cytoplasm.
Meanwhile, other cell internaliza-
tion mechanisms may have governed the aggregation of the
[
20]
[29]
among the particles. However, thiol groups on the surface
nanostructures, such as assistance from cellular proteins.
of the QD-labeled l-Cys-b-CD with negative charge can sig-
[
6]
nificantly quench the PL.
In vitro HPV18 E6 gene-silencing of QD-siRNA complexes:
It is known that localization into cytoplasm is the critical
prerequisite for siRNA carriers to improve siRNA silencing
efficiency. To evaluate the silencing efficiency of siRNA de-
livered by QD nanoparticles coated with b-CD coupled to
amino acids, we examined the suppression of an endogenous
protein, the HPV18 E6 gene, in HeLa cells. QD-based gene
carriers were applied to the cells in 10% serum-containing
media as well as conventional cationic carriers, such as
siPort, NeoFX, and HiPerFect (using the same siRNA con-
centration and the optimized amount for each transfection
reagent) in order to fairly compare the gene inhibition effi-
ciencies of different carriers. E6 is a positive regulator of
caveolin-1 gene transcription and protein expression. The
siRNA transfection experiment was designed in the same
way as the plasmid transfection experiment. We examined
the down-regulation of the HPV18 E6 protein expression by
gel electrophoresis analysis (Figure 6). Compared with gene-
silencing efficiencies of QDs with the commercial transfec-
tion reagents, we found that the level of E6 gene expression
was reduced to 70.2Æ2.6% by l-His-b-CD-coated CdSe/
ZnSe QDs—which was comparable to that of siPort NeoFX
Selectivity localization of the functionalized QDs in vitro:
The interactions between QDs and the cells establish a
series of nanoparticles biological interfaces that could lead
to intracellular uptake and have biocompatible or bioad-
[28]
verse outcomes.
Cellular signaling pathways may be af-
fected when QDs interact with cell surface receptors or with
intracellular proteins. The evaluation of cell uptake and in-
tracellular localization of QDs is crucial to understand the
mechanistic mechanism of their biological impact. Despite
the charge distributions on the surface of the QD nanoparti-
cles coated with b-CD coupled to amino acids, TEM images
showed that HeLa cells were able to be internalized within
[1a]
6
h of treatment (Figure 5). However, l-His- and l-Trp-
(
69.3Æ4.7%) and HiPerFect (68.3Æ3.2%) transfection re-
agents—to 65.7Æ3.9% by l-Trp-b-CD-coated QDs, and to
3
4.6Æ1.7% by l-Phe-b-CD-coated QDs. However, l-Cys-b-
CD-coated QDs exhibited a much lower gene inhibition ef-
ficiency of 17.1Æ3.6%. It is known that in the presence of
l-Cys, negative charges often reduce gene-silencing effects
due to nonspecific protein adsorption on the negatively
[30]
charged surface of the complexes. Thus, these results sug-
gest that the charge on the surface of QDs coated with b-
CD coupled to amino acids plays an important role in deliv-
ery siRNA. When complexes with siRNA form, QDs
remain single, and the small sizes facilitate their diffusion
and entry into cells. After the nanoparticles are endocy-
tosed, both the amino and carboxylic groups on the QD sur-
face play important roles in endosome escape. At low pH
values, amine groups are protonated, and at high pH values,
they will be deprotonated. Therefore, they can quickly neu-
tralize excess protons in the endosome. The osmotic pres-
Figure 5. TEM images of cell uptake of l-His-b-CD-coated A) CdSe/
ZnSe QDs, B) l-Trp-b-CD-coated CdSe/ZnSe QDs, C) l-Phe-b-CD-
coated CdSe/ZnSe QDs, and D) l-Cys-b-CD-coated CdSe/ZnSe QDs in
the cytoplasm of the HeLa cells. Their cellular locations are indicated by
yellow arrows. HeLa cells incubated with amino acid-modified QDs for
6
h at 378C. Yellow arrows indicate an example of the location of QDs
uptake in cells.
Chem. Eur. J. 2011, 17, 5171 – 5179
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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5175

Z.-W. Mao, Q. Xia et al.
cient transport across the plasma membrane. During this
period, the l-His-b-CD-coated CdSe/ZnSe QDs fluores-
cence (red) is evident, but that of the siRNA-FITC (green)
is weaker, indicating that siRNA and QDs are associated
with each other and FITC is decreased due to FRET. After
3
h incubation, the signal increased in the green channel
[2a]
show that the membranes. This observation will help over-
come the limitations of current methods and develop new
materials for siRNA delivery.
Conclusion
In summary, we prepared a series of CdSe/ZnSe-labeled b-
CD coupled to amino acids through direct ligand-exchange
reactions and used them as a new class of appropriate size
scaffolds for designing multifunctional nanoparticles. We
have slightly improved gene-silencing efficiency, but remark-
ably reduced cellular toxicity. The nanocrystal surface prop-
erties can be manipulated by using various amino acid coat-
ings. These QDs are very soluble and stable in biological
buffer with high colloidal stability and strong optical emis-
sion properties (e.g., size, charge, stability) that allow them
to label and to target membrane vesicles. The flow cytome-
try analysis enabled us to quantitatively evaluate the fluo-
rescence intensity uptake from the QD complexes. In addi-
tion, TEM images showed that the modified QDs were ac-
cumulated in vesicles in the cytoplasm of the cells. The posi-
tive charge of amino acid resulted in a considerable increase
in the intracellular delivery of QDs. In addition, the QDs
should also provide a bright and stable fluorescent signal for
intracellular siRNA imaging. These findings suggest that the
QD nanoparticles coated with b-CD coupled to amino acids
are simple to prepare, safe, and they significantly improve
the siRNA delivery in vitro. It is necessary to further study
the detailed mechanism of the synergy between amino acid
and QDs in order to design better gene transfer carriers.
Figure 6. Comparison of gene-silencing efficiencies between QDs and
commercial transfection reagents by gel electrophoresis analysis. The re-
duced level of HPV18 E6 protein expression was reduced to 70.2Æ2.6%
by l-His-b-CD-coated CdSe/ZnSe QDs, to 65.7Æ3.9% by l-Trp-b-CD-
coated CdSe/ZnSe QDs, to 34.6Æ1.7% by l-Phe-b-CD-coated CdSe/
ZnSe QDs, to 17.1Æ3.6% by l-Cys-b-CD-coated CdSe/ZnSe QDs, to
6
9.3Æ4.7% by siPort NeoFX, and to 68.3Æ3.2% by HiPerFect transfec-
tion reagents. The quantitative values were obtained from the gel electro-
phoresis analysis (inset). HeLa cells incubated with amino acid-modified
QDs-siRNA for 6 h at 378C. Among all the QDs, the gene-silencing abili-
ty of the l-His-b-CD-coated CdSe/ZnSe QDs was slightly improved com-
pared with the commonly used two transfection agents.
sure building along this proton buffering process will even-
tually rupture the endosomes, a process known as “proton
[31]
sponge effect”.
Cellular uptake and transport of QD-siRNA complexes:
Owing to the intrinsic fluorescence of QDs, the intracellular
behavior of QD-siRNA complexes is visible by the confocal
microscopy. The confocal microscopy images (Figure 7)
shows that the QD-siRNA complexes enter and accumulate
inside cells after incubating with cells for 1 h, suggesting effi-
Experimental Section
Materials and reagents: All chemicals unless indicated were obtained
from Sigma Aldrich and used as received. Fetal bovine serum (FBS),
Dulbecco minimum essential medium (DMEM) medium and siRNA
were purchased from Invitrogen Corporation. All organic solvents were
purchased from EM Sciences. b-Cyclodextrin (b-CD), triethanolamine, l-
histidine (l-His), l-tryptophan (l-Trp), l-phenylalanine (l-Phe) and l-
cysteine (l-Cys) were obtained from Shanghai Chemical Factory, China.
b-CD was recrystallized thrice from water and dried in vacuo for 12 h at
9
58C before use. Analytical grade pyridine was predried by refluxing
over NaOH pellets for 24 h. All solutions were prepared with twice-dis-
tilled water, deoxygenated by bubbling highly pure nitrogen for 15 min
and maintained under nitrogen atmosphere during measurements at
room temperature.
Figure 7. The fluorescence imaging of QD-siRNA complexes and their
transport in living HeLa cells. QD-siRNA entered cells after they were
added into the cell culture for less than 1 h. The green fluorescence from
FITC-labeled siRNA started to increase at incubation time of 3 h, indi-
cating siRNA separation from QDs. And the siRNA was distributed
evenly in cytoplasm, suggesting efficient endosomal escape.
Synthesis of b-CD coupled to amino acids: The syntheses of b-CD cou-
pled to amino acids were essentially as described by Liu and co-work-
[
32]
ers, but with some slight modifications. Briefly, mono ACHUTNGRENNUG[ 6-O-(p-toluene-
sulfonyl)]-b-cyclodextrin and a series of amino acid (such as l-His, l-Trp,
l-Phe, and l-Cys) were dissolved in water containing triethanolamine.
[
13c]
More details are available in our published report.
5176
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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 5171 – 5179

Gene Delivery
FULL PAPER
Data for l-His-b-CD: Yield 25%; MS: m/z: 1272.3 [M+H] (see Fig-
Cell culture general: The ECV-304, SH-SY5Y and HeLa cells were main-
ure S3 in the Supporting Information); elemental analysis calcd (%) for
tained in DMEM medium supplemented with 10% (v/v) heat-inactivated
À1
l-His-b-CD·6H
.69.
2
O: C 40.71, H 6.62, N 2.97; found: C 40.80, H 6.95, N
FBS and 1% (v/v) penicillin/streptomycin (100 UmL penicillin and
À1
2
100 mgmL streptomycin). These cells were allowed to grow in a mono-
layer in a tissue culture flask incubated at 378C, gassed with 5% CO
and held at 90% relative humidity.
2
Data for l-Trp-b-CD: Yield 16%; MS: m/z: 1321.3 [M+H] (see Fig-
ure S3 in the Supporting Information); elemental analysis calcd (%) for
l-Trp-b-CD·6H
.99.
2
O: C 44.54, H 6.49, N 1.96; found: C 44.28, H 6.28, N
Cell viability experiments: The cytotoxicity of the series of CdSe/ZnSe
QD nanoparticles coated with b-CD coupled to amino acids was evaluat-
ed in ECV-304, SH-SY5Y and HeLa cells by a modified MTT assay. At
3–4 days after seeding, the cells were counted by hemocytometer and
seeded into a 96-well cell-culture plate at a cell density of 5ꢁ10 cells per
2
well and then incubated for 24 h at 378C under 5% CO . Then the
1
Data for l-Phe-b-CD: Yield 47%; MS: m/z: 1282.3 [M+H] (see Fig-
ure S3 in the Supporting Information); elemental analysis calcd (%) for
4
l-Phe-b-CD·8H
.77.
2
O: C 42.95, H 6.71, N 0.98; found: C 42.92, H 6.81, N
0
growth medium was replaced with DMEM medium/10% FBS containing
different concentrations of QDs. The preparation of QD stock solutions
was as followed. All QDs complexes were dissolved in dimethylsulfoxide
Data for l-Cys-b-CD: Yield 65%; MS: m/z: 1236.2 [M+H] (see Fig-
ure S3 in the Supporting Information); elemental analysis calcd (%) for
l-Cys-b-CD·8H
.215.
Synthesis of CdSe/ZnSe QDs: CdSe/ZnSe core/shell QDs were synthe-
2
O: C 39.10, H 6.64, N 1.01; found: C 39.45, H 6.71, N
(
DMSO) at different concentrations. Then 1 mL of different concentra-
1
tion samples were added into 99 mL of DMEM medium to obtain final
À1
concentrations of 5, 10, 20, 50, 100, and 200 mgmL of QDs in the
[
33]
sized based on previous method have reported, but with some slight
modifications. In short, TOPO-coated CdSe QDs were synthesized in a
one-pot synthesis using CdO, Se, ODE, stearic acid, ODA and TOPO.
The ZnSe shells were added using zinc oxide. CdSe/ZnSe core/shell QDs
synthesized using this procedure were found to show high crystallinity
medium. Each sample was prepared in triplicate. Following a 48 h incu-
À1
bation period, MTT (20 mL of 2.5 mgmL in 0.01m sterilized PBS,
pH 7.4) was added to each well, and the plates were incubated for 4 h at
3
78C under 5% CO
2
. The medium was then removed and DMSO
(
100 mL) was added to the plates and shaken to dissolve the formazan
(
9
see Figure S4 in the Supporting Information), quantum yields of 80–
0%, and narrow emission spectra (fwhm ~25 nm), and narrow size dis-
products. Tecan Infinite M200 monochromator-based multifunction mi-
croplate reader was used to measure the OD570 of each well with back-
ground subtraction at 690 nm. The cell survival rate in the control wells
without the QDs was considered as 100% cell survival. A cytotoxic con-
centration (CC50) was defined as the concentration of compound that re-
duced the absorbance of the control samples by 50%.
tributions (ꢀ3.5 nm).
Synthesis of QD nanoparticles coated with b-CD coupled to amino acids
(
surface ligands exchange): QD nanoparticles coated with b-CD coupled
to amino acids were made by surface modification of the QDs with the
b-CD coupled to amino acids. The synthesis was essentially as described
[
13c]
Flow cytometric analysis of the binding of QDs with live cells: HeLa
cells were grown in DMEM medium containing 10% FBS at 378C and in
in our previous reported.
In short, the QDs complexes were prepared
in a mixture solution of CdSe/ZnSe QDs in hexane and b-CD coupled to
amino acids in water by ultrasonic method. The reaction mixture was al-
lowed to an extraction procedure was used to purify the nanocrystals
from side products and unreacted precursors. The purified nanocrystal
complexes could be dissolved in water or other various aqueous media
and confirmed by UV/Vis, photoluminescence (PL) spectroscopy and
fluorescence (FL) lifetime.
a 5% CO
2
atmosphere. The cells were trypsinized, counted, and adjusted
5
À1
to 1ꢁ10 cellsmL and 3 mL added per plate. Stock solutions of QDs
were diluted using DMEM medium and added to the plate in 20 mL
À1
quantities (c=50 mgmL ). After incubation for 6 h, cells were thorough-
ly washed with PBS in order to eliminate QDs that were not internalized.
The cells were trypsinised with trypsin and centrifugated in PBS buffer.
Then the QDs bound cells were harvested and single cell suspension in
Apparatus: UV/Vis absorption spectra were acquired with a Perkin–
Elmer Lambda-850 UV-vis spectrometer (American, Perkin–Elmer Co.).
Photoluminescence (PL) and photoluminescence excitation (PLE) were
measured using a Perkin–Elmer Ls55 luminescence spectrometer (Amer-
ican, Perkin–Elmer Co.). PL quantum yields (QYs) of the samples was
determined using organic dyes with known QY as the standard, following
0
.5 mL buffer was prepared and was subjected to flow cytrometric analy-
sis. A flow cytometer (Coulter Co. USA) was used to measure the fluo-
rescence intensity with excitation at 488 nm. The monitor gate of 25 mm
was set to accommodate the effective size of live cells. The signal along
the x-axis indicates the fluorescence intensity, and the height along the y-
axis indicates the corresponding number (counts) of species showing the
fluorescence.
[
34]
a previously reported method. The PL QY data reported in this work
were obtained using Rhodamine B (RhB) as the standard (QY=89%).
The transmission electron micrographs (TEM) images of the products
were taken on a JEOL JEM-200CX transmission electron microscope,
employing an accelerating voltage of 200 kV. Powder X-ray diffraction
Preparation of cells for transmission electron microscopy (TEM): HeLa
cells were grown in DMEM medium containing 10% FBS at 378C and in
a 5% CO
2
atmosphere. The cells were trypsinised, counted, and adjusted
5
À1
to 1ꢁ10 cellsmL and 3 mL added per plate. Stock solutions of QDs
(
XRD) of the products were carried out on a Shimadzu XRD-6000 X-ray
coated with b-CD coupled to amino acids were diluted using DMEM
diffractometer equipped with CuKa radiation (l=0.154060 nm), employ-
À1
À1
medium and added to the plate in 20 mL quantities (c=50 mgmL ).
ing a scanning rate of 0.02 s and 2q ranges from 15–708.The zeta poten-
After incubation for 6 h, cells were thoroughly washed with PBS in order
to eliminate QDs that were not internalized. Cell processing for TEM
analysis was carried out in situ, without displacement from the culture
dish. Cells were fixed in a 0.1m PBS solution containing 2.5% gluteralde-
hyde and 4% paraformaldehyde for 1 h. They were then rinsed with
tial of QDs was measured on a Zetaplus/90plus Zeta Potential Analyzer
(
Brookhaven, Holtsville, NY).
Fluorescence lifetime measurements: Fluorescence (FL) lifetimes were
measured using a Combined Fluorescence Lifetime and Steady State
Spectrometer (Edinburgh Instruments LTD, England). Data were collect-
ed at room temperature using a quartz cuvette (1 cm optical path) filled
with 3 mL of QD nanoparticles coated with b-CD coupled to amino
acids in buffer solution and 405 nm excitation. Control spectra from solu-
tions containing only MBP-dye were subtracted from the composite
signal to account for direct excitation. Spectral deconvolution of the
steady-state data was performed using a custom algorithm in MATLAB
which considered the measured composite signal as the linear combina-
tion (superposition) of known QD signals. Using standard regression
analysis, a best-fit curve was found consisting of representative fractional
contributions of each QD PL signal. To compare these decays we calcu-
lated the average PL times (tav) that describe the mean time taken for a
0
.1m PBS and postfixed in 1% osmium tetroxide solution (extremely
toxic; use caution) for 1 h, rinsed with distilled water, stained with 0.5%
uranyl acetate for 1 h, dehydrated in a graded series of ethanol (30, 60,
7
0, 90, and 100%), and embedded in epoxy resin. The resin was polymer-
ized at 608C for 48 h. Ultrathin sections (50–75 nm) obtained with an
LKB ultramicrotome were stained with 2% aqueous uranyl acetate and
2
% aqueous lead citrate and imaged under a 120 kV FEI Tecnai Spirit
TEM.
Gene-silencing efficiency evaluated by agarose gel electrophoresis: For
gene-silencing experiments, HeLa cells were plated in 24-well plates at
5
1ꢁ10 cells per well and maintained for 24 h. Five micrograms of siRNA
[
35]
photon to be emitted using the reported method.
was used to formulate complexes with l-His-b-CD-coated CdSe/ZnSe
Chem. Eur. J. 2011, 17, 5171 – 5179
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
5177

Z.-W. Mao, Q. Xia et al.
QDs, l-Trp-b-CD-coated CdSe/ZnSe QDs, l-Phe-b-CD-coated CdSe/
ZnSe QDs, l-Cys-b-CD-coated CdSe/ZnSe QDs, siPort NeoFX, and Hi-
PerFect. After 15 min incubation with the different gene carriers, HeLa
cells were transfected for 2 h. After the transfection, the media were re-
placed with fresh serum-containing media and incubated for 6 h. QD-
siRNA complexes were prepared by mixing the suspension of QD nano-
particles coated with b-CD coupled to amino acids with siRNA (diluted
in the siRNA buffer) to achieve a siRNA/QDs ratio of 1:1, which is de-
termined to be the optimal ratio in the current study. Immediately before
transfection, 500 mL of optimized DMEM medium were added to QD-
siRNA complexes and siPort, NeoFX, and HiPerFect transfection re-
agents and were mixed by pipetting. The QD-siRNA complexes diluted
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Received: December 7, 2010
Published online: April 4, 2011
3
Chem. Eur. J. 2011, 17, 5171 – 5179
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
5179