Conjugation of transferrin to azide-modified cdse/zns core-shell quantum dots using cyclooctyne click chemistry

Full text of DOI:10.1002/anie.201202876

Angewandte
Chemie
DOI: 10.1002/anie.201202876
Quantum Dot Biolabeling
Conjugation of Transferrin to Azide-Modified CdSe/ZnS Core–Shell
Quantum Dots using Cyclooctyne Click Chemistry**
Christine Schieber,* Alessandra Bestetti, Jet Phey Lim, Anneke D. Ryan, Tich-Lam Nguyen,
Robert Eldridge, Anthony R. White, Paul A. Gleeson, Paul S. Donnelly,* Spencer J. Williams,*
and Paul Mulvaney*
[
10]
Quantum dots (QDs) are semiconductor nanocrystals with
unique optical properties that distinguish them from common
organic fluorophores. For example, they give size-dependent
emission spectra, high photoluminescence quantum yields,
and large molar extinction coefficients, which gives the
potential for single-molecule detection. They also have
broad absorption cross-sections, which enables simultaneous
excitation of mixed populations of QDs. Additionally, QDs
are more resistant to chemical degradation and are less
are often susceptible to hydrolysis and/or cross-linking. The
CuAAC reaction has been extensively used for the function-
alization of gold, silica, and iron oxide nanoparticles, as well
[11–14]
as carbon nanotubes.
Unfortunately, addition of cop-
per(I) to CdSe semiconductor QDs completely and irrever-
sibly quenches QD photoluminescence (see Supporting
An attractive alternative is the strain-
promoted azide–alkyne cycloaddition (SPAAC) reaction of
azides with strained cyclooctynes, which occurs rapidly and
[
15–17]
Information).
[
1]
[18,19]
affected by photo-bleaching than conventional dyes.
Many applications in diagnostics, biosensing, and biola-
does not require a copper catalyst.
Herein, we report a strategy to prepare azide-modified
QDs using a previously reported QD polymer-encapsulation
[
2]
beling could benefit from the optical properties of QDs. The
first step of any method is the preparation of water soluble
QDs, and various strategies have been developed to generate
[
20]
technique.
We have developed a modular and broadly
applicable conjugation strategy based on a bifunctional linker
(L) that enables incorporation of cyclooctyne groups onto the
metalloprotein transferrin and the SPAAC conjugation of the
cyclooctyne-modified transferrin to azide-modified QDs
(Figure 1). Finally, we demonstrate that the QD–protein
conjugates are biologically active by monitoring the uptake of
fluorescent QD–transferrin conjugates in transferrin-receptor
(TfR) expressing tumor cells.
[
3]
water-soluble nanoparticles. The attachment of biomole-
cules such as proteins, monoclonal antibodies, or enzymes to
water-soluble QDs is not trivial, and to date there is no
generic conjugation method that is broadly applicable, easily
implemented, and reproducible without compromising the
[4–6]
function of the QDs.
Among the most powerful biocon-
jugation reactions to emerge are a set of bioorthogonal
chemical reactions referred to as ꢀclickꢁ reactions. The
copper(I)-catalyzed azide-terminal alkyne cycloaddition
CdSe/ZnS core–shell QDs were synthesized in octadecene
using the SILAR (successive ion-layer adsorption reaction)
process. SILAR affords organic-soluble QDs with trioctyl-
[
7–9]
[21]
(
CuAAC) reaction
provides exquisite functional-group
selectivity and does not suffer the drawbacks of standard
coupling reactions (for example, oxime/hydrazone ligation,
esterification, and thiol-maleimide addition reactions) that
phosphine/trioctylphosphine oxide (TOP/TOPO) bound to
the nanocrystal surface. Azide-modified water-soluble QDs
were prepared from organic-soluble QDs in two steps (Fig-
ure 1a). Treatment of the QDs with an excess of low
molecular weight polystyrene-co-maleic anhydride polymer
[
*] Dr. C. Schieber, A. Bestetti, A. D. Ryan, Dr. T.-L. Nguyen, R. Eldridge,
Dr. P. S. Donnelly, Prof. S. J. Williams, Prof. P. Mulvaney
School of Chemistry and Bio21 Institute, University of Melbourne
Parkville, Vic 3010 (Australia)
(PSMA, MW 1700) results in polymer encapsulation of the
QD. Treatment of the PSMA-encapsulated QDs with a hydro-
philic amino poly(ethylene glycol) (amino-PEG), Jeffamine
M1000, results in aminolysis of the anhydride and sponta-
neous transfer to the aqueous phase. Controllable presenta-
tion of azide functional groups on the QD surface can be
achieved by blending various amounts of an azide-modified
H NÀPEGÀN into Jeffamine M1000. The resulting PEG-
E-mail: cschi@unimelb.edu.au
pauld@unimelb.edu.au
sjwill@unimelb.edu.au
mulvaney@unimelb.edu.au
Dr. J. P. Lim, Prof. P. A. Gleeson
Department of Biochemistry and Molecular Biology and Bio21
Institute, University of Melbourne
2
3
modified polymer-encapsulated QDs were colloidally stable
over a wide range of pH values and salt concentrations.
Functionalization of QDs with PEG minimizes nonspecific
binding to biological components, reduces cellular toxicity,
Parkville, Vic 3010 (Australia)
Dr. A. R. White
Department of Pathology, University of Melbourne
Parkville, Vic 3010 (Australia)
[
20,22–25]
and increases circulation times in vivo.
This phase-
transfer method allows the QD surface to be derivatized with
different functional groups in any desired ratio, and thus gives
control over the number and density of biomolecules that will
ultimately be attached. We denote the percentage of azide
groups on the QDs as QDx, such that QD0 corresponds to
[
**] This work was supported by the Faculty of Science, University of
Melbourne, and the Australian Research Council (ARC).
Supporting information for this article (experimental details) is
available on the WWW under http://dx.doi.org/10.1002/anie.
201202876.
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ate anion, iron-loaded trans-
ferrin (Fe Tf) binds to
2
a transferrin receptor and is
internalized into cells by
way of clathrin-mediated
endocytosis. Optimal condi-
tions for the formation of L-
Fe Tf were using a borate
2
buffer (0.5m, pH 9) incu-
bated for six hours at room
[
27]
temperature. The number
of linker molecules bound to
each Fe Tf was determined
2
by ESI-TOF-MS when 2:1
(
(
Figure 2b, center) and 4:1
Figure S2) molar ratios of
linker were added to the
protein, with peaks
observed corresponding to
, 1, 2, or 3 linker molecules
0
per protein. The reactivity
of the cyclooctyne func-
tional group was tested by
reaction of L-Fe Tf with 2-
2
azido-N-phenylacetamide
(MW= 176 Da).
A
mass
increase corresponding to
addition of one or more
molecules of the azide con-
firmed fast and efficient tri-
azole formation (Figure 2b,
bottom; and Figure S2).
Figure 1. a) Polymer encapsulation of CdSe/ZnS core–shell nanoparticles. b) QD conjugation strategy:
primary-amine coupling with ethyl squaramyl affords a squaramide conjugate between the linker L and Fe2-
transferrin (L-Fe Tf). Strain-promoted azide–alkyne cycloaddition of azide-modified QDs and L-Fe Tf yields
2
2
QD-L-Fe Tf.
2
To demonstrate that the
QDs without azide groups coated only with Jeffamine M1000,
azide groups on the azide-modified QDs are reactive to
and QD100 indicates QDs prepared using 100% H NÀPEGÀ solution-phase cyclooctyne groups, the linker molecule L was
2
N .
reacted with Alexa Fluor 594 cadaverine (A594). The result-
ing L-A594 conjugate was reacted with QDs functionalized
with different percentages of azide groups (QD0, QD1,
QD10, QD100). Unreacted L-A594 was removed by exhaus-
tive washing using spin filtration. Fluorescence spectra of
QD-linker-dye samples upon excitation at 400 nm show
FRET (Fçrster resonance energy transfer) between the
QDs and the dye, with the FRET signal increasing with
increasing azide loading on the QD (Figure 2c). This dem-
onstrates a successful SPAAC reaction between the QDs and
the linker and shows that the number of biomolecules
attached to the QDs can be tuned by controlling the
percentage of azide groups on the nanoparticle surface. A
control reaction between QD0 and L-A594 showed no FRET,
indicating that the conjugate does not form and confirming
the specificity of the SPAAC reaction.
3
The optical properties of the aqueous QDs were identical
to those of the original QDs measured in organic solvents,
indicating that no aggregation or surface degradation occurs
upon polymer encapsulation and phase transfer (Figure 2a,
left). The successful incorporation of azide groups was
confirmed using FTIR spectroscopy by monitoring the
À1
characteristic azide absorption band (n = 2100 cm ; Fig-
ure 2a, center). QDs were purified by size exclusion chroma-
tography to remove residual PSMA (Figure 2a, right).
A bifunctional linker (L) bearing a cyclooctyne functional
group and an electrophilic ethyl squaramate group was
synthesized (see Supporting Information). Bicyclo-
[
6.1.0]nonyne (BCN) was chosen as the cyclooctyne because
it is relatively simple to prepare, exhibits a high rate of
reaction, and yields only one regioisomeric triazole prod-
uct. The ethyl squaramyl group of L provides excellent
shelf stability, yet reacts rapidly with amino groups to give
proteins modified with pendent cyclooctynes, poised for
reaction with azide-functionalized QDs.
[
26]
[27]
Conjugation of QDs with transferrin was achieved by first
reacting Fe Tf with the linker L, and then the resulting L-
2
Fe Tf conjugate was added in tenfold excess to QD0, QD10,
2
and QD100. Samples were analyzed by gel electrophoresis
(Figure 2d). Because the QD surface is negatively charged, all
samples migrated from the negative to the positive pole and
their speed was determined by their charge and size. For
increasing density of azide groups on the QD surface, the
This bioconjugation strategy was applied to the iron-
binding glycoprotein transferrin (Fe Tf, MW= 80 kDa),
which is involved in cellular iron-acquisition. Upon apo-
transferrin binding two Fe cations and a synergistic carbon-
2
[28]
III
2
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Figure 2. a) Left: Absorption and emission spectra before (red) and after (blue) CHCl /water phase transfer of QDs. Center: Comparison of FTIR
3
spectra of H NÀPEGÀN and QDs functionalized with 0–100% azide groups. Right: Size-exclusion chromatogram of a crude QD sample (black)
2
3
and of the same sample after purification (red) measured at 220 nm. b) Top: Chemical formula of the triazole-conjugated Fe Tf. Center: ESI-TOF
2
MS of L-Fe Tf formed by treatment of Fe Tf with linker (L) at a 2:1 ratio. The difference in mass between the peaks is 475 Da (476 Da calculated).
2
2
Bottom: Reaction of azidophenylacetamide at a 1:1 ratio with the 2:1 complex of L-Fe Tf. c) Fluorescence spectra of QDx+L-A594 collected after
2
removal of excess dye. QDs emit at 543 nm and Alexa Fluor 594 emits at 617 nm. d) Agarose gel electrophoresis of QDs and QDx+Fe Tf under
2
UV lamp (left) and after staining with Coomassie blue (right).
difference in mobility between unconjugated and conjugated
hydrodynamic diameter and MW of a particle from its
sedimentation behavior. The samples showed a trend towards
higher sedimentation coefficients with increasing percentages
of azide, in agreement with the results from gel electro-
phoresis (Supporting Information, Figure S3), and confirmed
the specificity of the conjugation strategy, as well as the
success of the purification.
[
29]
samples increased. Because Fe Tf has a pI of 5.2–6.2 and is
2
negatively charged at the basic pH used for gel electro-
phoresis, the reduced mobility of the QDx-Fe Tf samples can
2
thus be ascribed to an increase in the hydrodynamic volume,
which indicates the success of the conjugation between QDs
and L-Fe Tf. Also, only the QD10-Fe Tf and QD100-Fe Tf
2
2
2
conjugates are stained by Coomassie blue, indicating the
The uptake of QD100-Fe Tf conjugates into HeLa cells
2
presence of conjugated protein, whereas for QD0 and Fe Tf,
the Coomassie stain appears only at the position of the free
protein.
was studied to demonstrate that the conjugates retained
biological activity, relative to Alexa Fluor 568 (A568)-labeled
Fe Tf. HeLa cells express TfRs and following uptake, Fe Tf is
2
2
2
QDx-Fe Tf conjugates were also characterized by analyt-
trafficked to the early endosomes. Following release of the
2
ical ultracentrifugation, which allows determination of the
iron atoms, apo-Tf is recycled to the plasma membrane. These
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studies involved binding of Fe Tf to cell-surface TfRs with the
2
cells on ice to retard endocytosis. Unbound Fe Tf was washed
2
off the cells and internalization of the receptor-bound Fe Tf
2
was initiated by warming the cells to 378C. Both A568-Fe Tf
2
and QD100-Fe Tf conjugates were internalized into HeLa
2
cells within 15-30 minutes (Figure 3). Control experiments
with unconjugated QD100 demonstrated that the binding was
Fe Tf-dependent (Figure 3, C1–3). Together these results
2
strongly suggest that QD100-Fe Tf binds to the TfR and is
2
internalized by receptor-mediated endocytosis. After incuba-
tion at 378C for 30 minutes, both the amount of uptake and
the intracellular distribution of QD100-Fe Tf and A568-Fe Tf
2
2
were similar. However, two hours after treatment, cells
incubated with A568-Fe Tf had significantly reduced intra-
2
cellular fluorescence than at 30 minutes, indicating that the
conjugate is trafficked out of the cell by known Fe Tf cellular-
2
efflux pathways (Figure 3, A2 and A3). In contrast, at two
hours in cells treated with QD100-Fe Tf, significant punctate
2
staining remained (Figure 3, B3), demonstrating that A568-
Fe Tf and QD100-Fe Tf have different rates of efflux.
2
2
To further investigate the localization of QD100-Fe Tf
2
after internalization, cells were stained for markers of
endocytic compartments. Early endosome antigen 1 (EEA1)
is a marker for early endosomes and CD63 for late endo-
Figure 3. Fe -transferrin uptake into HeLa cells using A568-Fe Tf (A1–
2
2
A3), QD100-Fe
Tf (B1–B3), and QD100 (C1–C3) for the indicated
2
times at 378C. Cells were fixed in 4% paraformaldehyde and nuclei
stained with DAPI. Images of QD100-Fe Tf and the corresponding
2
somes/lysosomes. After 15 minutes, QD100-Fe Tf showed
2
unconjugated QD100 control were taken at the same gain setting.
extensive co-localization with EEA1 but minimal co-local-
ization with CD63 (Figure 4a). Conversely, after two hours,
QD100-Fe Tf showed significant overlap with the lysosomal
cessful binding of QD100-Fe Tf to TfRs and internalization
2
2
marker CD63 (Figure 4b). These results demonstrate suc-
into cells, although the presence of the QDs appears to
Figure 4. a) QD100-Fe Tf uptake into HeLa cells were performed for 15 min at 378C. Cells were fixed in 4% paraformaldehyde and stained with
2
rabbit anti-EEA1 followed by A568-conjugated anti-rabbit IgG, and mouse anti-CD63 followed by A647-conjugated anti-mouse IgG. A,D) EEA1
(
green); B,E) QD100-Fe Tf (red); C,F) CD63 (blue). D–H) 2ꢀ magnification of the boxed region. G) overlay of (D–E); H) overlay of (E–F). Co-
2
localization is indicated by: yellow=QD100-Fe Tf +EEA1; magenta=QD100-Fe Tf +CD63. b) QD100-Fe Tf uptake into HeLa cells was performed
2
2
2
for 2 h at 378C. All colors and procedures are the same as in (a). Representative regions of overlap are indicated by arrows.
4
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2
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www.angewandte.org
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Angewandte
Communications
Communications
Quantum Dot Biolabeling
C. Schieber,* A. Bestetti, J. P. Lim,
A. D. Ryan, T.-L. Nguyen, R. Eldridge,
A. R. White, P. A. Gleeson,
P. S. Donnelly,* S. J. Williams,*
P. Mulvaney*
&&&& — &&&&
Conjugation of Transferrin to Azide-
Modified CdSe/ZnS Core–Shell Quantum
Dots using Cyclooctyne Click Chemistry
Twinkle twinkle quantum dot: Conjuga-
tion of biomolecules to azide-modified
quantum dots (QDs) through a bifunc-
tional linker, using strain-promoted
azide–alkyne cycloaddition with the QD
and a squaramide linkage to the biomol-
ecule (see scheme). Transferrin-conju-
gated QDs were internalized by trans-
ferrin-receptor expressing HeLa cells.
6
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