PEG-ylated cationic CdSe/ZnS QDs as an efficient intracellular labeling agent

Article abstract of DOI:10.1039/b801317a

Quantum dots (QDs) have size-tunable optical properties, such as photostability, strong photoluminescence and a large Stokes-shift, which make it possible to adapt them to various biological applications. In many cases, surface modification of QDs by carboxylate ligands has been extensively studied. However, there have been few applications on QDs modified with a potential cationic ligand such as amine. In this study, we synthesized robust amine-functionalized QDs and modified their surface with poly(ethylene glycol) for long-term stability. These QDs showed an excellent stability over a broad pH-range and remarkable internalization efficiency into living cells. the Owner Societies.

Full text of DOI:10.1039/b801317a

View Article Online / Journal Homepage / Table of Contents for this issue
Physical Chemistry Chemical Physics
www.rsc.org/pccp
Volume 10 | Number 13 | 7 April 2008 | Pages 1713–1828
ISSN 1463-9076
PERSPECTIVE
Kaltsoyannis and Plane
COVER ARTICLE
Quantum chemical calculations on a
selection of iodine-containing species
(IO, OIO, INO , (IO) , I O , I O and I O )
Song et al.
PEG-ylated cationic CdSe/ZnS QDs as
3
2
2
3
2
4
2
5
an efficient intracellular labeling agent of importance in the atmosphere
1463-9076(2008)10:13;1-4

COMMUNICATION
www.rsc.org/pccp | Physical Chemistry Chemical Physics
PEG-ylated cationic CdSe/ZnS QDs as an efficient intracellular labeling
agentw
Junghan Lee, Junwon Kim, Eunjung Park, Shineun Jo and Rita Song*
Received 23rd January 2008, Accepted 1st February 2008
First published as an Advance Article on the web 18th February 2008
DOI: 10.1039/b801317a
Quantum dots (QDs) have size-tunable optical properties, such
as photostability, strong photoluminescence and a large Stokes-
shift, which make it possible to adapt them to various biological
applications. In many cases, surface modification of QDs by
carboxylate ligands has been extensively studied. However,
there have been few applications on QDs modified with a
potential cationic ligand such as amine. In this study, we
synthesized robust amine-functionalized QDs and modified their
surface with poly(ethylene glycol) for long-term stability. These
QDs showed an excellent stability over a broad pH-range and
remarkable internalization efficiency into living cells.
times worse due to their instability under the critical micelle
concentration (CMC). As a result, fundamental studies (for
¨
example, on the Forster Resonance Energy Transfer (FRET),
a process that occurs within the range o10 nm between the
donor and acceptor fluorophores) are definitely limited in QD-
1
6
mediated systems. The most common laboratory method
used for the preparation of water-soluble QDs is ligand
1
7
exchange, using a thiol-containing ligand. The major draw-
back of this method is the decrease of quantum yield (QY),
which usually drops to the range of 5–10% after the ligand
exchange reaction. In addition, these QDs lack long-term
1
8
stability and show strong quenching in acidic media. How-
ever, this method is quite simple and provides QDs that are
small in size. Ligand exchange using dihydrolipoic acid
Introduction
(
DHLA) has been used to prepare relatively stable, water-
soluble QDs by formation of a chelate via bidentate thiol
1
ligands.
The synthesis of quantum dots (QDs) has greatly progressed
during the past two decades, using organometallic precursors
at high temperature to obtain monodisperse and highly lumi-
nescent nanomaterials, especially in the case of CdSe/ZnS
9–21
However, these DHLA modified QDs also show a severe
decrease in photoluminescence and their conjugation reactions
with several biomolecules are sometimes unsuccessful due to
the formation of aggregates. Moreover, these QDs often lead
to significant non-specific binding with cell membrane. On the
other hand, amine-functionalized small QDs are not easily
available because of their severe aggregation in neutral solu-
tions and the strong quenching in acidic media. Therefore, the
need to obtain aggregation-free QDs retaining their initial
individual size is still strongly required in order to gain access
to FRET studies and to reduce non-specific binding with
biomolecules. In this paper, the syntheses of water soluble
QDs functionalized with amine containing multidentate
ligands originating from DHLA are described, and their
properties and stabilities are also presented. These QDs were
found to exhibit high quantum yields and remarkable stability
under harsh environmental conditions, for example, even at
pH 2.
1
–2
core/shell nanocrystals. From recent studies on their biolo-
gical and medical applications, hydrophilic QDs have shown
3
–9
extraordinary potential as excellent cellular labeling agents.
Since hydrophilic CdSe or CdTe QDs and their conjugates
1
0–13,18
were successfully prepared,
extensive efforts have been
focused to introduce a variety of functionalities on the surface
of QDs to render them water-soluble and to increase their
versatility for responding to various biological and biomedical
needs. However, the generation of water-soluble and biocom-
patible QDs, which maintain high photoluminescence (PL)
and stability in turbid biological fluids, still remains as a great
challenge. Such approaches include the use of polymer coat-
1
4,15
ing, micelle formation and ligand exchange methods.
Although polymer coating has been considered as a most
efficient capping method because the QDs prepared by this
method exhibit high stability and high photoluminescence, it is
well known that these QDs are rather large (410 nm) and it is
difficult to control their size. The use of QD micelles also
suffers from the same limitations, and their stability is some-
Experimental
Synthesis of CdSe/ZnS QDs and DEDEA ligands
Nano/Bio Chemistry Laboratory, Institut Pasteur Korea (IP-K), 39-1
Hawolgok-dong Seongbuk-gu, 136-791 Seoul, Korea.
E-mail: rsong@ip-korea.org; Fax: +82 (0)31 546 6710;
Tel: +82 (0)31 546 6701
Starting core/shell CdSe/ZnS nanocrystals were prepared by a
22
previously reported method with a slight modification. The
0
capping ligand
3
(DEDEA; DHLA-2,2 -(ethylenedioxy)
w Electronic supplementary information (ESI) available: Stability test
of QDs in various buffer solutions, fluorescence image of QDs in
borate buffer solution, stability in different concentration of salt,
hydrodynamic sizes, gel image of oligopeptide conjugated QDs and
fluorescence images of cells stained by different types of QDs. See
DOI: 10.1039/b801317a
bis(ethylamine)) was synthesized from the pentafluorophe-
0
nol-activated thioctic acid 1, using 2,2 -(ethylenedioxy)-
bis(ethylamine) as a hydrophilic linker bearing the amine
functionality (Scheme 1). This ligand 3 was made as a tri-
fluoroacetate (TFA) salt 4 to confer water-solubility and to
This journal is ꢀc the Owner Societies 2008
Phys. Chem. Chem. Phys., 2008, 10, 1739–1742 | 1739

Oligopeptide conjugation
In addition to the solubility and stability test of QDs in
aqueous solution, QDs 6 were reacted with an oligopeptide.
First of all, the N-terminal amine group of the oligopeptide,
Gly-Gly-His-His-His-His-His-His, was previously protected
with an acetyl group, and then the C-terminal carboxyl group
was conjugated with QDs 6 (n = 110) by the EDC coupling
method. 30 ml of QDs 6 solution (17 mM) and 10 ml of oligopep-
tide solution (10 mM) were mixed, then EDC (100 eq.)–
NHS (25 eq.) was added. After the reaction for 2 h at room
temperature, excess oligopeptides were removed by 30 K
centrifugation.
Cell preparation and staining
1
0 000–20 000 Hela cells were cultured in 200 ml DMEM
Dulbecco’s Modified of Eagle’s Medium with 10% fetal
bovine serum, 1% penicillin–streptomycin) per well in a
6 well plate in a humidified environment (37 1C, 5% CO2)
(
9
for 48 h. After incubation, 20 ml of QD solution (5 mM) was
added to the each well and the cell nuclei were stained by 2 ml
of DAPI (2-(4-amidinophenyl)-6-indolecarbamidine di-
ꢁ1
hydrochloride, 5 mg ml ) for 5 min. And then, the cells were
observed using confocal microscope (LSM 5, Carl Zeiss).
Results and discussion
After the ligand exchange reaction, the QY of QDs 5 was
B50% of original value. Using the same reaction conditions,
the QY of aminoethanethiol (AET) capped QDs, previously
reported amine-functionalized QD, was reported to be below
1
0% of original value. The stabilities of these two water-
soluble QDs were compared under the same conditions. The
differences were clearly demonstrated after 24 h, as shown in
Fig. 1. All AET capped QDs were only stable in distilled
water, precipitated at a pH between 3 and 8, accompanied by
complete fluorescence quenching (Fig. 1(a)).
Scheme 1 Synthetic scheme of DHLA-derived dimercaptoamine
ligand and PEG-ylated cationic QDs.
generate a positive charge to induce electrostatic repulsion
between the nanoparticles.
The DEDEA capped QDs were stable in all media tested
and, surprisingly, maintained their photoluminescence even at
pH 2 (see the ESIw). Their stability in acidic media seems to be
due to the strong coordination of the bidentate DHLA ligand
onto the surface of the ZnS shell and the positive charge from
appended ammonium cation at the terminal, which induces
electrostatic repulsion between the nanoparticles. In addition,
the penetration of oxygen should be decreased due to presence
Ligand exchange process
To 100 ml of hydrophobic CdSe/ZnS QD530 (PLmax 530 nm,
QY B70%) dissolved in 2 ml chloroform 0.05 M methanolic
solution of ligand 4 was added until the particles flocculated.
After the flocculation, QDs were spun down by centrifuge and
the supernatant was removed. After washing several times
with methanol to remove the excess ligands, QDs were
suspended in deionized water and filtered using a 0.2 mm
syringe filter.
PEG-ylation of DEDEA modified QD
QDs 5 were further modified by reacting with poly(ethylene
glycol) (PEG) to increase their stability in various aqueous
media. Different amounts of N-hydroxysuccimide (NHS) ac-
tivated PEG2000 and 5000 were reacted with QDs 5 to form a
stable amide bond. The ratio of QD and PEG was variable
from 1 : 10 to 1 : 500. After conjugation reaction for 3 h, excess
PEG was removed by 30 K centrifugation and these reactions
were analyzed by gel electrophoresis.
Fig.
1 Fluorescence images of (a) AET@QDs and (b) DE-
DEA@QDs. In water (i), 10 mM glycine buffer (pH 3.0) (ii), 10 mM
citrate–phosphate buffer (pH 5.0) (iii), 10 mM phosphate buffer (pH
6.0) (iv), 10 mM PBS buffer (pH 7.4) (v), 10 mM borate buffer (pH 8.0)
(vi), and 0.1 M NaCl solution (vii).
1
740 | Phys. Chem. Chem. Phys., 2008, 10, 1739–1742
This journal is ꢀc the Owner Societies 2008

of the large counter anion, TFA, and longer linker ligand than
AET or DHLA. The presence and bulkiness of TFA seems to
contribute to the stability of QDs because the QDs formed
most likely form inter-hydrogen bonding with the neighboring
nanoparticles. In addition to their pH stability, the behavior of
QDs 6 was investigated in aqueous solutions with various ionic
strengths. The photoluminescence properties of two sets of
QDs dispersed in water were monitored with an increasing
concentration of NaCl from 0 to 1 M. Over the concentration
range of NaCl used, little or no change in the PL intensity was
observed (see the ESIw). These results are very promising, as
these QDs will be used for in vitro and in vivo studies, where
the ionic concentration is known to be high. Moreover, the
sizes of QDs 5 and QDs 6 used in this study were less than
10 nm, (see the ESIw) these materials have a potential to
analyze the certain biological events by measuring FRET.
The specific interaction between 6ꢂHis and Ni–NTA has
been widely used in biological applications for the purification
ꢁ
ꢁ
with other anions such as Cl or CH COO didn’t exhibit
3
similar stabilities. Although DEDEA-capped QDs are stable
at pH 3, their stability is slightly limited in the presence of a
salt or high buffer concentrations, and under neutral or weakly
basic conditions. However, this unexpected acid-resistant
photoluminescence prompted us to render these QDs more
stable in different media. QDs 5 were further modified by
reacting with poly(ethylene glycol) (PEG) to increase their
stability in various aqueous media. Different amounts of
N-hydroxysuccimide (NHS) activated PEG2000 and 5000
were reacted with QDs 5 to form stable amide bond. Pegylated
QDs 6 exhibited persistent stability in 50 mM borate buffer
solution (pH 8.5) (see the ESIw).
2
4
and separation of proteins. To explore the potential of QDs
with recognizing group for biological target, these 6ꢂHis
modified QDs 7 were incubated with Ni–NTA agarose beads
at room temperature, and the unbound QDs were washed out
with 10 mM PBS. Fluorescence images were taken with a
confocal microscope and are shown in Fig. 3. This result
indicated that 6ꢂHis were correctly bound to QDs, and
demonstrated the intrinsic binding affinity to Ni–NTA. 6ꢂHis
tagged QDs were unbounded from the bead after washing with
1 M imidazole solution in PBS.
Fig. 2(a) clearly shows the different migration profiles of the
pegylated QDs depending on the PEG molecular weight and
conjugation ratios. The DEDEA capped QDs (lane (i)) have
maximum positive charges. The higher the PEG reaction ratio,
the greater is the importance of the charge coverage for
becoming neutral, which results in the delayed moving of the
conjugates. The coverage of the charges on the QDs conju-
gated with PEG5000 was demonstrated to be greater than
2
3
those with PEG2000.
DEDEA–PEG QDs (QD : PEG5000 = 1 : 500) are fairly
stable and well dispersed without any trace of aggregates even
in trifluoroacetic acid solution. (Fig. 2(b) and (c)) Under these
conditions, the fluorescence of both of AET- and AET-PEG
QDs were completely quenched. QDs PEG-ylated with 500 eq.
of PEG5000 were remarkably stable over the entire range of
pHs tested (pH 2–9). All PEG-ylated QDs bearing the DE-
DEA ligand showed good stability in both HCl and TFA
aqueous solutions (pH B2). However, these QDs, with the
exception of the 500 eq. of PEG5000 conjugated case, showed
some tendency to precipitate at pH 8.5 after 24 h. Under basic
conditions the ammonium groups are deprotonated, which
Finally, the studies were conducted to evaluate the entrance
capabilities of QDs into HeLa cells. Positively charged nano-
particles have been known to facilitate penetration into the
2
5
cell. Fig. 4 shows the remarkable entrance efficiency of QDs
6 into the cells. The nucleus was co-stained with DAPI to give
a clear contrast. QDs 6 were endocytosed very quickly into the
cells in 10 min (Fig. 4(a)). The transport efficiency of QDs 6
was compared to that of the previously well-known DHLA-
QDs (see the ESIw) and QDs 5 (Fig. 4(b)). DHLA-capped QDs
mostly bound to extracellular membranes and QDs 5 without
PEG showed a strong tendency towards aggregation in the cell
culture media. QDs 6 were also conjugated to antibody (anti-
human W6/32), and showed excellent stability and binding
efficiency to EBV infected B cells (see the ESIw).
In conclusion, we have demonstrated the synthesis and
application of PEG-ylated cationic QDs 6 as an efficient cell
Fig. 2 (a) Gel images of QDs. DEDEA@QDs (i), DEDEA–
PEG2000 QDs (QD : PEG2000, 1 : 50 (ii), 1 : 250 (iii), 1 : 500 (iv)),
and DEDEA–PEG5000 QDs (QD : PEG5000, 1 : 50 (v), 1 : 250 (vi), 1 :
5
00 (vii)). Fluorescence images of (b) AET@QDs and (c) DE-
DEA@QDs. QDs (i), QD : PEG2000 (1 : 50 (ii), 1 : 250 (iii), 1 : 500
iv)) and QD : PEG5000 (1 : 50 (v), 1 : 250 (vi), 1 : 500 (vii)) in TFA
(
Fig. 3 Confocal images of Ni–NTA agarose beads incubated with (a)
QDs 6 and (b) QDs 7.
solution.
This journal is ꢀc the Owner Societies 2008
Phys. Chem. Chem. Phys., 2008, 10, 1739–1742 | 1741

5
6
7
Z. Zhelev, H. Ohba and R. Bakalova, J. Am. Chem. Soc., 2006,
28, 6324.
Y. Xiao, W. G. Telford, J. C. Ball, L. E. Locascio and P. E. Barker,
Nat. Methods, 2005, 2, 723.
M. Stroh, J. P. Zimmer, D. G. Duda, T. S. Levchenko,
K. S. Cohen, E. B. Brown, D. T. Scadden, V. P. Torchilin, M.
G. Bawendi, D. Fukumura and R. K. Jain, Nat. Med., 2005, 11,
1
6
78.
8
9
R. L. Ornberg, T. F. Harper and H. Liu, Nat. Methods, 2005, 2, 79.
X. Michalet, F. F. Pinaud, L. A. Bentolia, J. M. Tsay, S. Doose, J.
J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir and S. Weiss,
Science, 2005, 307, 538.
1
0 M. Bruchez, Jr, M. Moronne, P. Gin, S. Weiss and A. P.
Alivisatos, Science, 1998, 281, 2013.
1
1
1 W. C. W. Chan and S. Nie, Science, 1998, 281, 2016.
2 A. L. Rogach, T. Franzl, T. A. Klar, J. Feldmann, N. Gaponik, V.
¨
Lesnyak, A. Shavel, A. Eychmuller, Y. P. Rakovich and J. F.
Donegan, J. Phys. Chem. C, 2007, 111, 14628.
1
3 I. Nabiev, S. Mitchell, A. Davies, Y. Williams, D. Kelleher, R.
Moore, Y. K. Gun’ko, S. Byrne, Y. P. Rakovich, J. F. Donegan,
A. Sukhanova, J. Conroy, D. Cottell, N. Gaponik, A. Rogach and
Y. Volkov, Nano Lett., 2007, 7, 3452.
Fig. 4 HeLa cell image stained with; (a) QDs 6, (b) QDs 5.
labeling agent. The most interesting property of this nanopar-
ticle is that the fluorescence is virtually unaffected by harsh
environmental conditions, even at pH 2 or at a very high salt
concentration. These QDs are water-soluble, aggregation free,
stable for several months, and were found to be internalized
very easily through the cell membrane. All of these features
allow us to access a universal intracellular labeling agent.
1
4 B. Dubertret, P. Skourides, D. J. Norris, V. Noireaux, A. H.
Brivanlou and A. Libchaber, Science, 2002, 298, 1759.
15 X. Wu, H. Liu, J. Liu, K. N. Haley, J. A. Treadway, J. P. Larson,
N. Ge, F. Peale and M. P. Bruchez, Nat. Biotechnol., 2003, 21, 41.
6 A. R. Clapp, I. L. Medintz, J. M. Mauro, B. R. Fisher, M. G.
1
Bawendi and H. Mattoussi, J. Am. Chem. Soc., 2004, 126, 301.
17 R. Gill, I. Willner, I. Shweky and U. Banin, J. Phys. Chem. B,
2005, 109, 23715.
1
8 K. Boldt, O. T. Bruns, N. Gaponik and A. Eychm u+ ller, J. Phys.
Chem. B, 2006, 110, 1959.
Acknowledgements
19 S. F. Wuister, I. Swart, F. van Driel, S. G. Hickey and C. Donega,
Nano Lett., 2003, 3, 503.
20 B. H. Huang and D. A. Tomalia, J. Lumin., 2005, 111, 215.
This work was supported by the Korean Ministry of Science
and Technology (MOST).
2
2
2
1 H. T. Uyeda, I. L. Medintz, J. K. Jaiswal, S. M. Simon and H.
Mattoussi, J. Am. Chem. Soc., 2005, 127, 3870.
2 Y. A. Yang, H. Wu, K. R. Williams and Y. C. Cao, Angew. Chem.,
Int. Ed., 2005, 44, 6712.
3 The gel images obtained with conventional TBE buffer (pH 8) were
impossible to analyze due to the different charge states of the QDs
at this pH. Therefore, acetic acid buffer (pH 6) was used as an
elution buffer to assure all protonation of amine functionalities.
Notes and references
1
B. O. Dabbousi, J. Rodriguez-Viejo, F. V. Mikulec, J. R. Heine, H.
Mattoussi, R. Ober, K. F. Jensen and M. G. Bawendi, J. Phys.
Chem. B, 1997, 101, 9463.
2
3
4
Z. A. Peng and X. Peng, J. Am. Chem. Soc., 2001, 123, 183.
J. V. Frangioni, Nat. Biotechnol., 2006, 24, 326.
A. Fu, W. Gu, B. Boussert, K. Koski, D. Gerion, L. Manna, M. L.
Gros, C. A. Larabell and A. P. Alivisatos, Nano Lett., 2007, 7, 179.
24 E. G. Guignet, R. Hovius and H. Vogel, Nat. Biotechnol., 2004, 22,
440.
25 H. T. Song, J. S. Choi, Y. M. Huh, S. J. Kim, Y. W. Jun, J. S. Suh
and J. W. Choi, J. Am. Chem. Soc., 2005, 127, 9992.
1
742 | Phys. Chem. Chem. Phys., 2008, 10, 1739–1742
This journal is ꢀc the Owner Societies 2008