Multiplexed screening of cellular uptake of gold nanoparticles using laser desorption/ionization mass spectrometry

Article abstract of DOI:10.1021/ja805392f

Gold nanoparticles (AuNPs) are highly promising candidates as drug delivery agents into cells of interest. We describe for the first time the multiplexed analysis of nanoparticle uptake by cells using mass spectrometry. We demonstrate that the cellular up

Full text of DOI:10.1021/ja805392f

Published on Web 10/01/2008
Multiplexed Screening of Cellular Uptake of Gold
Nanoparticles Using Laser Desorption/Ionization Mass
Spectrometry
Zheng-Jiang Zhu, Partha S. Ghosh, Oscar R. Miranda, Richard W. Vachet,* and
Vincent M. Rotello*
Department of Chemistry, UniVersity of Massachusetts, Amherst, Massachusetts 01003
Received February 7, 2008; E-mail: rotello@chem.umass.edu (V.M.R.); rwvachet@chem.umass.edu (R.W.V.)
Abstract: Gold nanoparticles (AuNPs) are highly promising candidates as drug delivery agents into cells
of interest. We describe for the first time the multiplexed analysis of nanoparticle uptake by cells using
mass spectrometry. We demonstrate that the cellular uptake of functionalized gold nanoparticles with cationic
or neutral surface ligands can be readily determined using laser desorption/ionization mass spectrometry
of cell lysates. The surface ligands have “mass barcodes” that allow different nanoparticles to be
simultaneously identified and quantified at levels as low as 30 pmol. Using this method, we find that subtle
changes to AuNP surface functionalities can lead to measurable changes in cellular uptake propensities.
Introduction
Therapeutic nanocarriers carry drugs, genes, and/or imaging
fluorescence dyes that can be “read out” by a fluorescence
spectrometer. Simultaneous screening of the cellular uptake
of multiple particles with different surface functional groups,
however, is a challenge for existing approaches. Here, we
describe a new “mass barcoding” technique for monitoring the
cellular uptake of multiple functionalized gold nanoparticles
2
1
-4
agents into cells and tissues of interest.
Various materials,
5
6
such as polymeric micelles, mesoporous silica nanorods,
7
8-10
carbon nanotubes, and nanoparticles,
have been used as
therapeutic nanocarriers, as well as probes for following
intracellular processes. Effective use of nanoparticles as carriers
and intracellular probes requires the ability to monitor these
particles in cells, and several techniques are available for this
purpose. These techniques can be classified into two groups:
(
(
AuNPs) by using laser desorption/ionization mass spectrometry
LDI-MS).
Nanoparticles have been used in mass spectrometric analyses
primarily to facilitate the laser desorption/ionization of com-
1
4
pounds of interest. Tanaka et al. showed that cobalt particles
(
1) label-free imaging techniques such as luminescent quantum
1
0,11
12
(∼30 nm) suspended in glycerol facilitated the ionization of
dots imaging,
atomic force microscopy (AFM), and
1
5
16-22
23
24,25
1
3
proteins. Subsequently, Ag, Au,
C, and Si
nano-
transmission electron microscopy (TEM) and (2) barcoding
techniques such as those that encode nanoparticles with
materials have been demonstrated as LDI-MS matrixes with
different degrees of success. Meanwhile, some mass spectro-
metric work has also been devoted to the analysis of nanopar-
ticles themselves. For pure samples of gold nanoparticles,
(
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10.1021/ja805392f CCC: $40.75

2008 American Chemical Society
J. AM. CHEM. SOC. 2008, 130, 14139–14143 9 14139

A R T I C L E S
Zhu et al.
the chemical and physical parameters that influence AuNP
uptake by cells. To the best of our knowledge, this is the first
report of multiplexed analysis of cellular uptake of functional-
ized AuNPs with LDI-MS.
Results and Discussion
To explore the viability of mass barcodes for determining
the uptake efficiency of AuNPs, we cultured monkey kidney
cells (COS-1) with AuNPs (500 nM) for 6 h (Figure 2a). This
4
0
concentration of AuNPs is nontoxic to the cells, and after 6 h
of incubation, no cell morphology changes are observed. The
cells were washed three times with cold phosphate-buffered
saline (PBS) to remove extra AuNPs that were not taken up by
the cells. Before the cells were lysed, TEM was used to verify
the cellular uptake of AuNPs. As shown for a single cell (Figure
Figure 1. Structural representation of the AuNPs. The mass/charge ratios
(
m/z) attached to each AuNP act as mass barcodes used for identification
of the AuNPs.
2
8,29
LDI-MS
have been used to measure the compositions of
surface functionalities and atom numbers in the NP core.
In this study, we have tagged AuNPs with readily ionizable
surface functionalities, i.e., “mass barcodes” (shown in Figure
2
b), multiple gold nanoparticles are trapped in vesicles (most
likely via endosomal entrapment) in the cytoplasm, which is in
13,41,42
agreement with results by other groups.
It is possible that
1
). These alkanethiolate cationic or neutral monolayers are
some AuNPs have escaped from the vesicles, as NP escape
4
2,43
barriers between the nanoparticle core and the environment,
effectively protecting and stabilizing the gold cluster core in
biological environments.
of these monolayers dictates interfacial interactions between cells
and AuNPs, thus governing cellular uptake of AuNPs.
Upon laser irradiation of these AuNPs, mass barcodes of
alkanethiolate monolayers rather than the AuNPs themselves
can be simply read out by LDI-MS, thus providing characteristic
peaks for identifying AuNPs (e.g., Figure S1 in the Supporting
Information, LDI-MS spectrum of AuNP 1). Ionization of
alkanethiolate monolayers on flat gold surfaces has also been
depends on the NP surface functionalities,
but direct
evidence for this was not found in the TEM images. After the
cells were lysed, the AuNPs taken up by the cells were then
collected as part of the precipitate after centrifugation of the
lysate. The precipitate was subjected to LDI-MS, and spectra
similar to that shown in Figure 3a were obtained. Figure 3a
illustrates the characteristic peaks that are observed for AuNP
1. The spectrum of AuNP 1 includes an ion at m/z 422, which
3
0-32
Moreover, the chemical nature
3
3-35
+
corresponds to the molecular ion (M ) of the ligand attached
to AuNP 1. The spectrum also includes ions at m/z 388, which
+
corresponds to [M - H S] , m/z 197 and 394, which correspond
2
36,37
38,39
+
+
observed using LDI
and matrix-assisted LDI (MALDI).
to Au and Au , respectively, and m/z 184, which corresponds
2
The surface ligands on the AuNPs are primarily detected because
the NP core efficiently absorbs the laser light (337 nm), and
this energy is readily transferred to desorb and ionize the surface
ligands, probably via a mechanism similar to that proposed by
Tanaka. In this work, we investigate a potential advantage of
such a mass barcoding approachsthe ability to simultaneously
analyze many different functionalized AuNPs. We predicted that
each functionalized AuNP could be identified by its unique mass
barcode. Furthermore, we explore this LDI strategy for direct
analyses of AuNPs taken up by cells. Such a multiplexed
screening of AuNPs could be very valuable for rapidly assessing
to the headgroup fragment of one of the most abundant lipids
in animal cell membranessphosphatidylcholine (PC). Moreover,
a series of peaks spaced by 14 Da from m/z 262 to m/z 360
indicate successive losses of CH units from the alkyl chains
2
1
4
of the surface ligands. Each of the other AuNPs in this study
had similar mass spectral patterns, with each AuNP featuring
different m/z ratios allowing identification (Figure S2 in the
Supporting Information).
The different mass barcodes of each AuNP facilitate multi-
plexed screening by LDI-MS. After cells are simultaneously
exposed to two different types of AuNPs, LDI-MS analysis of
the resulting cell lysate indicates that both AuNPs can be readily
identified. For example, in Figure 3b, diagnostic molecular ions
at m/z 422 and m/z 436 indicate the cellular uptake of AuNP 1
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and AuNP 2, respectively. Other characteristic peaks, such as
(
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+
[
M - H2S] (m/z 388 for AuNP 1 and m/z 402 for AuNP 2),
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28, 6036–6037.
also assist the identification of two AuNPs.
(
(
(
30) Templeton, A. C.; Wuelfing, W. P.; Murray, R. W. Acc. Chem. Res.
Quantifying the relative cellular uptake of these two different
AuNPs should be possible by comparing the ion abundances
of their respective molecular ion peaks. To do so, however, the
relative ionization efficiencies of the ligands in cell lysates must
be considered. The ionization efficiencies of the surface ligands
were investigated under two sets of control conditions. First,
equal molar amounts of AuNP 1 and AuNP 2 (300 pmol each)
were directly analyzed by LDI-MS, indicating that the ligands
2
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1
4140 J. AM. CHEM. SOC. 9 VOL. 130, NO. 43, 2008

Multiplexed Screening of Cellular Uptake of AuNPs
A R T I C L E S
Figure 2. (a) Schematic illustration of the analysis of the AuNPs in cell lysates by LDI-MS. (b) TEM images of cellular uptake of AuNP 1.
Table 1. LDI-MS Relative Quantification of Cellular Uptake of
AuNPs 1-5
determined separately (see the Experimental Section for details).
The relative uptake amounts for each AuNP (compared to AuNP
1) as determined by ICP-MS (Figure 4 and Table S1 in the
Supporting Information) are very similar to the LDI-MS data,
indicating that LDI-MS can reliably provide the relative
quantities of each AuNP taken up by COS-1 cells. Interestingly,
the similarity of the ICP-MS data, which were acquired after
uptake of individual AuNPs, and the LDI-MS data, which were
acquired after uptake of two different AuNPs, indicates that
interactions between the AuNPs in the cell uptake experiments
are minimal.
mass
barcodes (m/z) control ratio
experimental
b
ratio
a
c
relative ratio
AuNP 2/AuNP 1
AuNP 3/AuNP 1
AuNP 4/AuNP 1
AuNP 5/AuNP 1
436/422
492/422
465/422
369/388
1.41 ( 0.09 1.70 ( 0.14 1.21 ( 0.13
0.72 ( 0.08 0.40 ( 0.10 0.50 ( 0.20
0.19 ( 0.05 0.21 ( 0.03 1.10 ( 0.30
0.26 ( 0.05 0.09 ( 0.02 0.34 ( 0.09
a
A 300 pmol sample of each AuNP was mixed with 300 pmol of
b
AuNP 1 in lysed COS-1 cells and subjected to LDI-MS analysis.
A
3
00 pmol sample of each AuNP was mixed with 300 pmol of AuNP 1
and then cultured with living COS-1 cells. These cells were then
c
washed, lysed, and analyzed by LDI-MS. Relative ratios are generated
Taken as a whole, the quantitative data in Figure 4 indicate
that AuNP 2 and AuNP 4 are more readily taken up by COS-1
cells than AuNP 3 and AuNP 5. The exact mechanism by which
the AuNPs are taken up by the cells is probably complex and
is still an active area of investigation. Nonetheless, several
factors have been recognized to govern the cellular uptake
by comparing experimental ratios with control ratios.
on AuNP 2 are ionized more readily as indicated by a molecular
ion abundance ratio (AuNP 2/AuNP 1) of 1.79 ( 0.04. Second,
equal molar amounts of AuNP 1 and AuNP 2 (300 pmol each)
were mixed with cell lysates and analyzed by LDI-MS, and
again, the surface ligands on AuNP 2 are more readily ionized,
giving rise to a molecular ion abundance ratio (AuNP 2/AuNP
1
3,41
13
efficiency of nanoparticles, such as size,
properties.
shape, and surface
Because the cell membrane is negatively
charged, positively charged nanoparticles are generally found
1
2,33-35
1
) of 1.41 ( 0.09 (Table 1 and Figure S3a in the Supporting
3
3,35
to have higher uptake efficiencies.
Therefore, it is perhaps
Information). Because the data from the second set of experi-
ments are more comparable to the cell uptake experiments, the
ion abundance ratio of 1.41 can be used to determine the relative
quantity of the AuNPs taken up by the cells. After correction
of the experimentally observed ratio of 1.70 ( 0.14 (Figure 3b
and Table 1) by the ratio observed from the analysis of the cell
lysates, the cellular uptake of AuNP 2 was determined to be
not surprising that AuNP 5 with its neutral surface is less readily
3
3,35
taken up by the cells than the cationic AuNPs 1-4.
taken up more efficiently than AuNP 5, the four cationic NPs
AuNP 1 through AuNP 4) all exhibit slightly different uptake
While
(
efficiencies. From this limited set of AuNPs, it is difficult to
conclude what other factors control uptake, but surface ligand
hydrophobicity might be important. AuNP 3 is the most
hydrophobic of the cationic AuNPs, and it is least efficiently
taken up by the COS-1 cells. Obviously, more work is needed
to better understand the effect of hydrophobicity on AuNP cell
uptake, and this mass barcode approach along with LDI-MS
detection will be helpful in this regard.
1
.21 ((0.13) times greater than that of AuNP 1. A similar
analysis can be done for each of the other AuNPs in comparison
to AuNP 1 (Figure 4 and Table 1).
The relative uptake amounts of each AuNP provided by the
LDI-MS data was validated using inductively coupled plasma
mass spectrometry (ICP-MS). ICP-MS provides high sensitivity
13
and robustness for elemental analysis, but it lacks the capacity
to identify AuNPs with different surface functionalities. There-
fore, the cellular uptake amounts of each AuNP had to be
To ensure that the results in Table 1 and Figure 4 do not
simply reflect different AuNP stabilities in a cellular environ-
ment, the AuNPs were kept in cell lysate for different periods
J. AM. CHEM. SOC. 9 VOL. 130, NO. 43, 2008 14141

A R T I C L E S
Zhu et al.
Figure 5. (a) Multiplexed LDI mass spectrum of COS-1 cell lysate with
the four cationic AuNPs 1-4. m/z 422, m/z 436, m/z 492, and m/z 465
correspond to AuNP 1, AuNP 2, AuNP 3, and AuNP 4, respectively. The
symbol key is the same as in Figure 3. (b) Relative amounts of AuNPs
1-4 obtained from LDI-MS. The AuNP amounts are normalized to that of
AuNP 1.
Figure 3. LDI mass spectrum of COS-1 cell lysate after uptake of (a)
AuNP 1 and (b) AuNP 1 and AuNP 2. m/z 422 and m/z 436 correspond to
+
the molecular ion (M ) of the ligands attached to AuNP 1 and AuNP 2,
respectively. Symbol key: *, Au+ (m/z 197); @, Au2+ (m/z 394); #, the
molecular ion corresponding to the headgroup fragment of phosphatidyl-
choline (m/z 184).
in cell cultures could be readily detected. Figure S5 (Supporting
Information) illustrates this for experiments with AuNP 1. The
characteristic peak for AuNP 1 is readily apparent, m/z 422,
with a signal-to-noise ratio of 75. With such a good signal, we
feel that the AuNPs could be detected even when present at
even lower amounts.
A key advantage of the LDI-MS measurements over the ICP-
MS measurements or other measurements is the ability to
simultaneously identify and quantitate the uptake of multiple
AuNPs. To demonstrate the advantages of such a multiplexed
analysis, COS-1 cells were cultured with four cationic AuNPs
(
AuNPs 1-4) (300 pmol each), and the resulting cellular
contents were analyzed by LDI-MS (Figure 5a). Diagnostic
molecular ion peaks at m/z 422, m/z 436, m/z 492, and m/z 465
indicate the presence of AuNP 1, AuNP 2, AuNP 3, and AuNP
4, respectively. The relative quantities of each AuNP (Figure
Figure 4. Relative quantities of the AuNPs in COS-1 cell lysates
determined by LDI-MS and ICP-MS. In both cases, the AuNP amounts
are normalized to that of AuNP 1.
5
b and Table S2 in the Supporting Information) indicate that
AuNP 2 is the most readily taken up by the cells, while AuNP
3 is the least readily taken up, quite similar to the results in
Figure 4. The slight differences might be explained by the
greater total concentrations of AuNPs present in the experiments
giving rise to Figure 4. These greater concentrations may have
increased the cell uptake competition between the AuNPs,
causing AuNP 4, for example, to be taken up less readily.
of time before analysis by LDI-MS. As an example, equal
amounts of AuNP 1 and AuNP 2 (300 pmol each) were
incubated with cell lysate for different amounts of time, and
the relative ratios between the two nanoparticles were found to
remain steady for up to 24 h (Figure S4 in the Supporting
Information), which indicates that these two nanoparticles have
essentially the same stability in a cellular environment.
We also investigated the sensitivity of our LDI-MS approach
by culturing COS-1 cells with differing amounts of AuNPs. We
found that AuNPs present at levels as low as 30 pmol (50 nM)
Conclusion
We have described the use of AuNPs with mass barcodes
for the multiplexed screening of AuNP cellular uptake. We
1
4142 J. AM. CHEM. SOC. 9 VOL. 130, NO. 43, 2008

Multiplexed Screening of Cellular Uptake of AuNPs
A R T I C L E S
demonstrate that the relative quantities of four different AuNPs
taken up by cells can be simultaneously determined using LDI-
MS with this approach. We also find that the cellular uptake of
the functionalized AuNPs is dependent on the NP surface
functionality, suggesting that differential cellular uptake and
specific cell targeting might be possible if the appropriate surface
functionalities are chosen. In the future, we plan to use this
technique to evaluate differential AuNP uptake by different cell
states (e.g., normal and diseased). A future improvement will
be to combine the LDI-MS approach with subcellular fraction-
ation. This combination should be able to identify the intracel-
lular targets for the AuNPs. On the whole, this LDI-MS
technique has great potential for both the development of AuNP-
based delivery vectors and probing transport of nanomaterials
in vitro and in vivo.
the sensitivity experiments (Figure S5 in the Supporting Informa-
tion) were performed on a Waters Micromass M@LDI L/R mass
spectrometer. The Bruker Reflex III is equipped with a 337 nm
nitrogen laser, a 1.0 m flight tube, and a stainless steel sample target.
All mass spectra were acquired in reflectron mode using a voltage
of 16 kV. All reported spectra represent an average of 50 shots
acquired at 90% laser power. The accelerating voltage was set to
2
0 kV. The Waters instrument was operated in positive reflection
mode. The pulse voltage was set to 2400 V, the source to 15 000
V, and MCP to 1850 V. Matrix suppression delay was set to m/z
1
7
00. The spectrum represents an average of 50 shots acquired at
0% high laser power.
LDI-MS Sample Preparation and Measurements. The lysed
cells were centrifuged at 14 000 rpm for 10 min. AuNPs precipitated
together with the cell lysates were washed with 60% acetonitrile/
40% water, applied directly to a stainless steel target, and allowed
to dry. The dry samples were washed with 60% acetonitrile/40%
water and then allowed to dry before LDI analysis. Each AuNP
experiment was performed in triplicate, and at least five spots of
each replicate were measured by LDI-MS.
Experimental Section
All chemicals were obtained from Sigma-Aldrich (St. Louis, MO)
unless otherwise noted.
AuNP 1-5 Synthesis. The Brust-Schiffrin two-phase synthesis
ICP-MS Instrumentation. All ICP-MS measurements were
performed on a Perkin-Elmer Elan 6100. Operating conditions of
the ICP-MS are listed below: rf power, 1200 W; plasma Ar flow
rate, 15 L/min; nebulizer Ar flow rate, 0.96 L/min; isotopes
3
1,44
method
was used for synthesis of AuNPs with core diameters
around 2 nm (see Figure S6 in the Supporting Information). After
that, the Murray place-exchange method was used to obtain
3
0,32
functionalized AuNPs 1-5
for details).
(see the Supporting Information
197
103
monitored, Au and Rh (as an internal standard); dwell time,
5
0 ms; nebulizer, cross-flow; spray chamber, Scott.
Cell Culture and Cellular Uptake of AuNPs. Monkey kidney
COS-1 cells (75 000 cells/well) were grown on a 24-well plate in
ICP-MS Sample Preparation and Measurements. Each AuNP
was incubated with COS-1 cells separately as described above
(Table S3 in the Supporting Information). After incubation and
lysing of the cells, the resulting cell lysate was digested overnight
high-glucose Dulbecco’s modified Eagle’s medium (DMEM;
glucose (4.5 g L-1)) containing 4-(2-hydroxyethyl)-1-pipera-
zineethanesulfonic acid (HEPES) buffer (pH 7.4, 25 mM) supple-
mented with fetal bovine serum (FBS; 10%). Cultures were
maintained at 37 °C under a humidified condition with 5% CO .
2
3 2 2
using 3 mL of HNO and 1 mL of H O . On the next day, 3 mL of
aqua regia was added, and then the sample was allowed to react
for another 1-2 h. Aqua regia is highly corrosiVe and must be
used with extreme caution! A hot plate (∼100 °C) was used to
reduce the above digested solution to 1-2 mL. The concentrated
sample solution was then diluted to 100 mL with deionized water,
After 24 h of plating, the cells were washed once with cold PBS,
and the solutions of nanoparticles (Table S3 in the Supporting
Information) were added. Following 6 h of incubation, the cells
were washed three times with PBS to remove extra nanoparticles
and lysed for 15 min with a lysis buffer according to the kit from
GENLANTIS. A quick freeze/thaw cycle (freeze for 2 h at -20
1
03
and aqua regia and a
Rh internal standard solution were also
added. The final AuNP sample solution contained 5% aqua regia
1
03
and 10 ppb
Rh. The AuNP sample solution was measured by
°
C and then thaw at room temperature) was performed to improve
lysis. The lysed cells were then prepared for LDI-MS or ICP-MS
analyses. For screening of four cationic nanoparticles (AuNPs 1-4)
simultaneously, the COS-1 cells were plated on a six-well plate
with 75 000 × 4.8 cells/well. The cells were treated in a manner
similar to that mentioned above. A mixture of four AuNPs (Table
S3 in the Supporting Information) was added to each well and
incubated for 6 h.
Control Experiments for Quantification. COS-1 cells were first
lysed, and then the solutions of nanoparticles in PBS only (Table
S4 in the Supporting Information) were added to each well and
kept in the incubator for 6 h. The amount of nanoparticles and
sample preparations were the same as described above except that
no medium was added.
ICP-MS under the operating conditions described above. Cell uptake
experiments with each AuNP were repeated 3 times, and each
replicate was measured 10 times by ICP-MS. A series of gold
standard solutions (20, 10, 5, 2, 1, 0.5, 0.2, and 0 ppb) were prepared
before each experiment. Each gold standard solution contained 5%
aqua regia and 10 ppb Rh. Each standard solution was measured
10 times by ICP-MS using the operating conditions described above.
The resulting calibration line was used to determine the gold amount
taken up by the cells in each sample. A ∼100 ppm solution of
dithiothreitol was used to wash the instrument between analyses
to facilitate gold removal.
103
Acknowledgment. This work is supported by the Office of
Naval Research under Award No. N000140510501 (R.W.V. and
V.M.R.), the NIH (Grant GM077173, V.M.R.), and the NSF Center
for Hierarchical Manufacturing (Grant DMI-0531171). We thank
Prof. Julian F. Tyson and Dr. Elena Dovova for access to and
assistance with the ICP-MS instrumentation and Dale A. Callaham
for help with TEM imaging.
Cell TEM. Cells were treated with AuNP 1 (1 µM NP in a 600
µL solution per well in a 24-well plate) and washed with PBS buffer
after 6 h. Then the cells were removed from the plate by
trypsinization and centrifuged to collect the pellet. The pellet was
fixed with 2% gluteraldehyde for 30 min and postfixed with 1%
4 4
OsO for 1 h. OsO is highly poisonous and must be used with
extreme caution! Following agarose (1.5%) enrobing, Spurr’s resin
embedding, and ultrathin (50 nm) sectioning, the samples were
stained with 2% aqueous uranyl acetate and 25 mg/mL lead citrate
and imaged with a JEOL 100S microscope.
LDI-MS Instrumentation. Most of the LDI-MS analyses were
done on a Bruker Reflex III time-of-flight mass spectrometer. Only
Supporting Information Available: Description of the syn-
thesis of the gold nanoparticles, more detailed experimental
protocols, and additional LDI-MS and ICP-MS data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(
44) Brust, M.; Walker, M.; Bethell, D.; Schiffrin, D. J.; Whyman, R.
J. Chem. Soc., Chem. Commun. 1994, 801–802.
JA805392F
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