Efficient Delivery of Quantum Dots into the Cytosol of Cells Using Cell-Penetrating Poly(disulfide)s

Article abstract of DOI:10.1021/jacs.7b02952

Quantum dots (QDs) are extremely bright, photostable, nanometer particles broadly used to investigate single molecule dynamics in vitro. However, the use of QDs in vivo to investigate single molecule dynamics is impaired by the absence of an efficient way

Full text of DOI:10.1021/jacs.7b02952

Communication
pubs.acs.org/JACS
Efficient Delivery of Quantum Dots into the Cytosol of Cells Using
Cell-Penetrating Poly(disulfide)s
Emmanuel Derivery,*,§,‡ Eline Bartolami,†,‡ Stefan Matile,*,† and Marcos Gonzalez-Gaitan*,†
†School of Chemistry and Biochemistry, National Centre of Competence in Research (NCCR) Chemical Biology, University of
Geneva, Quai Ernest Ansermet 30, CH-1211 Geneva, Switzerland
§MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, United
Kingdom
S
* Supporting Information
ABSTRACT: Quantum dots (QDs) are extremely bright,
photostable, nanometer particles broadly used to inves-
tigate single molecule dynamics in vitro. However, the use
of QDs in vivo to investigate single molecule dynamics is
impaired by the absence of an efficient way to chemically
deliver them into the cytosol of cells. Indeed, current
methods (using cell-penetrating peptides for instance)
provide very low yields: QDs stay at the plasma membrane
or are trapped in endosomes. Here, we introduce a
technology based on cell-penetrating poly(disulfide)s that
solves this problem: we deliver about 70 QDs per cell, and
90% appear to freely diffuse in the cytosol. Furthermore,
these QDs can be functionalized, carrying GFP or anti-
GFP nanobodies for instance. Our technology thus paves
the way toward single molecule imaging in cells and living
animals, allowing to probe biophysical properties of the
cytosol.
Figure 1. (A) Structure of the CPDs used in this study (n = 49). (B)
TEM micrographs of streptavidin-functionalized QDs without (left) or
after incubation with CPDs (right; CPD/QD = 62:1, 1 h, 4 °C),
followed by negative staining. (C) Left panel: Live S2 cells incubated
nderstanding how tissue and cellular behavior emerges
Ufrom single molecule dynamics is a major endeavor of for 1 h with 3.33 nM CPD-QD complexes (62:1). Single confocal
plane, n = 197 QDs. Middle panel: Time projection of a movie of the
cell shown in the left panel. Right panel: Same time projection, color-
coded according to frame number. (D) Cell incubated for 1 h with
6.66 nM QDs. Time projection as in C. Dash: cell outlines; scale bars:
10 nm (B), 5 μm (C,D).
modern cell biology. This faces a unique challenge because
single molecule dynamics occurs at very short time scales
(milliseconds), whereas cell and tissue dynamics occur at time
scales of minutes or even hours. Meeting this challenge requires
the ability to label single proteins in cells with bright yet
photostable dyes, in order to maintain good localization
precision over hours despite the small exposure time. In this
context, quantum dots (QDs) are of particular interest because
these nanometer-scaled particles are bright, photostable and
amenable to functionalization.1
Though numerous studies have elegantly used QDs to
investigate transmembrane protein dynamics at the plasma
membrane,2,3 their use to investigate single molecule dynamics
in the cytoplasm has suffered from the lack of an efficient
cytosolic delivery technique. Until today, physical methods such
as microinjection,4 electroporation5 or osmotic shock6 remain
the most efficient way to introduce QDs in the cytosol.
Chemical approaches to reliably deliver QDs in the cytosol do
not exist, despite multiple efforts focusing mostly on cell-
penetrating peptides (CPPs).7−12 Here, we show that cell-
penetrating poly(disulfide)s13 (CPDs, Figure 1A) can meet this
challenge. CPDs have recently been introduced to overcome
the two main limitations of CPPs: endosomal capture and
toxicity.9,12 CPDs achieve this through thiol-mediated
uptake14,15 and reductive depolymerization upon arrival in
the cytosol, respectively.
In this study, we bound biotinylated CPDs16,17 to
streptavidin-coated QDs (CdSe core with ZnS shell, providing
20−40 biotin binding sites according to the manufacturer). We
also replaced the original CF dye16 by the red TAMRA (Figure
1A), leaving the green imaging channel free for further QD
functionalization and colocalization experiments (see below).
To optimize the uptake efficiency, long CPDs with n = 49 were
prepared (Mw = 21.3 kDa, PDI = 1.09; see Supporting
Information for full synthesis details and characterization,
Schemes S1−S5 and Figures S1−S9). Upon incubation with an
excess of these CPDs, QD diameter increased from d = 14.2
Received: March 24, 2017
© XXXX American Chemical Society
A
DOI: 10.1021/jacs.7b02952
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Journal of the American Chemical Society
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0.8 to d = 18.3 0.8 nm (transmission electron microscopy, n
= 10, Figure 1B). The surface coverage of QDs with CPDs is
complete and uniform (Figure 1B).
At low nanomolar concentrations, CPD-QD complexes were
efficiently delivered into cultured Drosophila S2 cells (Figures
1C, S10). CPD-mediated QD delivery was not restricted to a
particular cell state or phase because QDs were found in all
cells under these conditions (n = 69 cells). On average, the
number of internalized QDs per cell was 66 9 (mean SEM,
n = 36 cells). Most internalized QDs were very motile and
excluded from the nucleus (Figures 1C, S10, Movie S1).
Dose response experiments suggested that a 2-fold molar
excess of CPD over QDs was sufficient for efficient delivery
(Figure S11), implying that only few CPD molecules are
required per QD, if we assume homogeneous streptavidin-
functionalization of the QD population. In addition, neither
QDs by themselves (Figure 1D), QDs incubated with biotin-
free CPDs (Figure S12), nor QDs incubated with biotinylated
CPDs in the presence of excess biotin (Figure S12) did
efficiently enter into cells, but rather remained trapped in
endosomal compartments. This suggests that cytosolic delivery
requires specific interactions with CPDs, at least for
streptavidin-coated QDs.
We then checked that QDs were indeed delivered into the
cytosol using three independent methods (see below): (i)
delivered QDs have a diffusive motion with a diffusion
coefficient too high for endosomes (mean square displacement
(MSD) analysis), (ii) QDs have low colocalization with
endosomal markers and (iii) most QDs are found beyond the
plasma membrane imaged by TIRFM (total internal reflection
fluorescence microscopy).
We first characterized the motion of the QDs delivered into
cells. An object of the size of a QD (∼15 nm in diameter)
moving freely in the cytosol would undergo Brownian motion
with a fast diffusion coefficient, but would not perform long-
range, processive, directed motility mediated by molecular
motors.6 On the other hand, endosomes (in which QDs could
be trapped) are larger objects and therefore move with a lower
diffusion coefficient; however, when engaging microtubule
tracks through molecular motors, they show faster, processive,
directed motility.18 We automatically tracked the motion of
thousands of delivered QDs and performed an MSD analysis
(see Supporting Information for details). We observed three
types of behavior: (i) short-range motion (“diffusive events”;
Figure 2A, 87% of n = 10 127 tracks in N = 8 cells), (ii) long-
range motility (“processive events”; Figure 2C; only four tracks
found in our extended N = 27 cells data set) and (iii)
immobility.
Short-range motility tracks correspond to subdiffusive events
because their MSD as a function of delay time is described by a
power law function19 (R2 = 0.99; α = 0.648 0.012, D = 0.111
0.001 μm2/s, Figure 2B). Importantly, the diffusion
coefficient of these QDs is 2 orders of magnitude higher than
that reported for endosomes,18 but in the range of QDs
delivered to the cytosol by electroporation5 or osmotic shock.6
On the other hand, the four “long-range motility” tracks
correspond to events of processive motility (Figure 2C) as their
MSD curve is described by a quadratic function (R2 = 0.9999,
Figure 2D). This behavior is reminiscent of endosome motility
driven by molecular motors. Consistently, the mean velocity, v
Figure 2. (A) Examples of short-range motion tracks of CPD-
delivered QDs. (B) Quantitative analysis of short-range tracks. Blue
line (solid): Weighted mean square displacement (MSD) as a function
of delay time of short-range tracks (n = 8856 tracks, lighter area:
SEM). Dashed black line: power law fit reflecting subdiffusion (R2 =
0.99). Dashed red line: linear fit of the first five points of the curve (R2
= 0.99). (C) Example of a long-range motility track (<0.1% of total
tracks). (D) Quantitative analysis of long-range tracks. Solid red line:
weighted MSD as a function of delay time of long-range motility tracks
(n = 4 tracks, lighter area: SEM). Dashed red line: parabolic fit
(reflecting directed motility combined with diffusion, R2 = 0.9999).
(E) Snapshot (top) and time projection (bottom) of S2 cells
incubated with CPD-QDs (green) and fluorescently labeled dextran
(red). (F, G) Snapshot (left) and time projection (right) of CPD-QDs
imaged by epifluorescence, which illuminates the entire cells (F) and
TIRF microscopy, which only illuminates the ventral plasma
membrane (G). CPD/QD = 62:1, scale bars: 1 μm (A, C), 5 μm
(E−G).
characteristics incompatible with endosome motility, but
consistent with subdiffusive, Brownian motion in the cytosol.
Second, we studied the localization of CPD-delivered QDs
with respect to endosomes using endosomal markers. We
combined CPD-mediated QD uptake with a long chase of
fluorescent dextran, which labels both early and late endosomal
compartments (Figures 2E, S13, Movie S2). We found a low
colocalization (10
1%; N = 36 cells, see Supporting
Information), confirming that most QDs are indeed not in
endosomes. Analysis of dual color movies further confirmed
our automated tracking analysis: QDs nearly never colocalize
with dextran-positive endosomes, which moved processively
(Figure 2E, S13; time projection, red arrows). In contrast, the
few QDs that did colocalize with dextran were mostly immobile
(Figure 2E, S13; blue arrows). These immobile QDs (∼13% of
the tracks in our automated analysis) may thus correspond to
QDs trapped in large immobile compartments (and/or abortive
endocytosis events, see below).
= 0.366
0.002 μm/s, is in the range of motor-driven
Third, we confirmed that most QDs are found deep in the
cytosol using TIRFM. TIRFM images only the first 100 nm of
the ventral plasma membrane and excludes deeper optical
endosome motility in cells.18,20 Altogether, this suggests that
the vast majority of CPD-delivered QDs display motion
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DOI: 10.1021/jacs.7b02952
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Communication
planes within the cytoplasm, whereas epifluorescence illumi-
nates the whole cell. Though a minority of QDs was immobile
at the plasma membrane (appearing in both epifluorescence
and TIRFM fields), most QDs are diffusive and appear in the
epifluorescence field but not in the TIRF field (Figure 2F,G,
Movie S3). This indicates that most of the QDs uptaken by the
cell are deep into the cytosol and do not correspond to QDs
bound to the plasma membrane. Importantly, we verified using
four mammalian cell lines that the CPD-mediated cytosolic
delivery of QDs is not restricted to insect cells (Figures S14−
S15, Movie S4).
We then checked whether the remaining biotin-binding sites
of QDs, which were not used for CPD binding, could be used
to deliver proteins bound to the QDs. GFP-CPD-QDs were
prepared by incubation of streptavidin-QDs with purified
biotinylated GFP (3:1) then with CPDs (63:1, Figure 3A).
aggregates of GFP-positive QDs in the cytosol (Figures 3E,
S18). This suggests that GBP bound to the QD retains its GFP-
binding properties in the cytosol. GFPs bound to the QDs also
retain their fluorescence (Figures 3B−E and S16−S18).
Therefore, CPDs allow the delivery of proteins in their native,
functional state (Figure 3D).
The cytosolic delivery of particles and proteins is an area of
intense investigation due to its potential applications: in clinics,
it is expected to allow therapeutic antibodies to access cytosolic
targets, whereas in life science research, it can be used to image
and manipulate single molecules inside cultured cells or
animals. In this context, the cytosolic delivery of functionalized
QDs has emerged as one of the most persistent challenges. In
this report, we demonstrate that biotinylated CPDs deliver
streptavidin-coated QDs efficiently (about 70 per cell), and that
most of these QDs (about 90%) reach the cytosol without
being trapped in intracellular compartments or at the plasma
membrane (Figures 1, 2). Because just a few CPD molecules
are sufficient for QD delivery, some binding sites on the QDs
remain free for further interfacing with other proteins (Figure
3). Those proteins stay functional upon delivery.
In this report, we determined unambiguously the cytosolic
location of the CPD-delivered QDs. This was motivated by
previous reports using CPPs, which showed that they do not
reach the cytosol and remain trapped in endosomes.1,7,8,11
Although CPP architecture is similar to the present CPD-QDs,
they enter the endosome network by macropinocytosis; CPP-
QD filled endosomes then move along microtubules toward the
microtubule-organizing center, only to turn around and move
outward into filopodia to be expelled by vesicle shedding.11
The efficient cytosolic delivery by CPDs originates from the
combination of two independent uptake mechanisms involving
counterion activators (as with CPPs), and dynamic covalent
disulfide exchange with thiols on cell surfaces.12 Accordingly,
CPD uptake is insensitive to endocytosis inhibitors, weakly
depending on temperature and strongly depending on thiol-
mediated uptake inhibitors.13,17 The fact that only few CPDs
per QD are sufficient for delivery is in agreement with
counterion-mediated direct translocation through nonleaky
micellar defects. Their dynamic adjustment to the substrate
and their ability to self-repair after translocation further
contributes to low toxicity. Furthermore, destruction of CPDs
upon entry into the cytosol ensures irreversibility and
minimizes toxicity.12,13 CPDs conjugated to other small
fluorophores have indeed been confirmed to be much less
toxic than CPPs.13 Though extensive toxicity studies of CPD-
QDs have not yet been carried out, the low toxicity of CPDs
conferred by their intracellular degradation is characteristic of
this delivery platform. Importantly, long-term toxicity, which is
inherent to Cd release from QDs, is unrelated to the topic of
this study.1−8
Figure 3. (A) Sequential functionalization of QDs with GFP or GFP-
nanobodies and CPDs. (B) Single confocal plane of a live S2 cell
incubated for 1 h with 6.8 nM GFP-CPD-QDs (3:63:1). Arrowheads:
GFP-positive QDs. (C) Snapshots of a movie of the cell displayed in
panel B. Note the continuous colocalization between QD and GFP
signals. Occasional partial colocalization reflects the slow acquisition of
the two channels compared to the fast diffusive motion of QDs. (D)
Large cytosolic QD assemblies occur upon sequential delivery of GFP-
CPD-QDs and GBP-CPD-QDs (GBP: Anti-GFP nanobody). (E)
Image of live S2 cell incubated for 1 h with 6.66 nM GFP-CPD-QDs
(3:63:1), followed by 6.66 nM GBP-CPD-QDs (11:61:1). Images
correspond to maximum intensity z-projections. Note the cytosolic
aggregates of QDs. Dash: cell outlines. Scale bars: 5 μm (1 μm in
insets).
Upon incubation with cells, many internalized GFP-positive
QDs were observed (Figures 3B,C, S16, S17). Their motility
properties were similar to the delivered CPD-QDs (Figures 3C,
S19A,B; D = 0.103 0.001 μm2/s, 95% confidence interval, n =
7453 tracks in N = 7 cells). Delivery of CPD-QDs with
biotinylated anti-GFP nanobodies21 gave similar results (GBP:
GFP binding peptide, R2 = 0.999, D = 0.131 0.001 μm2/s, α
= 0.840 0.004, 95% confidence interval, n = 4169 tracks in N
= 3 cells for GBP-QDs; Figures 3D, S19C). Thus,
functionalization of QDs with GFP or GBP did not affect
their efficient cytosolic delivery by CPDs.
The delivery technology introduced here will offer a new
avenue for biologists to address the biochemical and biophysical
properties of proteins in intact cells, and also within animals.
The high number of QDs delivered per cell and the efficiency
of delivery across the cell population allows one to follow
thousands of single molecules in thousands of cells, yielding
precise statistics in time and space. This will be particularly
useful for the study of intrinsically stochastic molecular
machines, such as molecular motors, thereby generalizing to
tissues or whole animals the very elegant approach recently
described by the Kapitein lab using electroporation.5b
Sequential delivery of GFP-functionalized QDs followed by
GBP-functionalized QDs caused the appearance of large
C
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(11) Ruan, G.; Agrawal, A.; Marcus, A. I.; Nie, S. J. Am. Chem. Soc.
2007, 129, 14759−14766.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.7b02952.
■
S
(12) Gasparini, G.; Bang, E.-K.; Montenegro, J.; Matile, S. Chem.
Commun. 2015, 51, 10389−10402.
(13) Gasparini, G.; Bang, E.-K.; Molinard, G.; Tulumello, D. V.;
Ward, S.; Kelley, S. O.; Roux, A.; Sakai, N.; Matile, S. J. Am. Chem. Soc.
2014, 136, 6069−6074.
Movies as described in text (ZIP)
Detailed experimental procedures (PDF)
(14) Chuard, N.; Gasparini, G.; Moreau, D.; Lorcher, S.; Palivan, C.;
̈
Meier, W.; Sakai, N.; Matile, S. Angew. Chem., Int. Ed. 2017, 56, 2947−
AUTHOR INFORMATION
2950.
■
Corresponding Authors
*marcos.gonzalez@unige.ch
*stefan.matile@unige.ch
*derivery@mrc-lmb.cam.ac.uk
ORCID
Emmanuel Derivery: 0000-0003-3927-5944
Stefan Matile: 0000-0002-8537-8349
(15) (a) Aubry, S.; Burlina, F.; Dupont, E.; Delaroche, D.; Joliot, A.;
Lavielle, S.; Chassaing, G.; Sagan, S. FASEB J. 2009, 23, 2956−2967.
(b) Oupicky, D.; Li, J. Macromol. Biosci. 2014, 14, 908−922.
́
(16) Gasparini, G.; Matile, S. Chem. Commun. 2015, 51, 17160−
17162.
(17) Fu, J.; Yu, C.; Li, L.; Yao, S. Q. J. Am. Chem. Soc. 2015, 137,
12153−12160.
(18) Derivery, E.; Seum, C.; Daeden, A.; Louber
́
y, S.; Holtzer, L.;
Julicher, F.; Gonzalez-Gaitan, M. Nature 2015, 528, 280−285.
̈
Author Contributions
(19) Metzler, R.; Klafter, J. Phys. Rep. 2000, 339, 1−77.
‡E.D. and E.B. contributed equally.
(20) Soppina, V.; Verhey, K. J. Mol. Biol. Cell 2014, 25, 2161−2170.
(21) Rothbauer, U.; Zolghadr, K.; Muyldermans, S.; Schepers, A.;
Cardoso, M. C.; Leonhardt, H. Mol. Cell. Proteomics 2008, 7, 282−289.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Nicolas Chiaruttini for help with EM, Paola Morelli
for assistance with synthesis, the NMR, MS and bioimaging
platforms for services, and the University of Geneva, the NCCR
Chemical Biology and the Swiss NSF for financial support.
M.G.-G. is further supported by the SystemsX (epiPhysX),
ERC (Sara and Morphogen) and Polish-Swiss programs, S.M.
by the NCCR Molecular Systems Engineering and E.D. by the
Medical Research Council (file reference number
MC_UP_1201/13) and HFSP (LTF and CDA).
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DOI: 10.1021/jacs.7b02952
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