Enzyme-catalyzed conversion of chemical structures on the surface of gold nanorods

Article abstract of DOI:10.1021/bc3005599

For developing metal nanoparticles of which surface chemical structures could be altered by enzymes in the cells, functional linkers caged by coumaric acids have been synthesized. Synthesized gold nanorod (GNR) conjugates possessing coumaric acid precursors underwent (porcine liver) esterase-catalyzed hydrolysis on their surface to afford GNRs coated with amino-functionalized polyethylene glycol and fluorescent coumarins as reporter molecules for monitoring the conversion. The chemical structural conversion on the GNR surfaces was successfully observed inside cells by fluorescence microscopy when GNR conjugates were incubated with tumor cells.

Full text of DOI:10.1021/bc3005599

Article
pubs.acs.org/bc
Enzyme-Catalyzed Conversion of Chemical Structures on the Surface
of Gold Nanorods
Eriko Kusaka, Takeo Ito,* Kazuhito Tanabe, and Sei-ichi Nishimoto
Department of Energy and Hydrocarbon Chemistry, Graduate School of Engineering, Kyoto University, Nishikyo-ku, Kyoto
615-8510, Japan
S
* Supporting Information
ABSTRACT: For developing metal nanoparticles of which
surface chemical structures could be altered by enzymes in the
cells, functional linkers caged by coumaric acids have been
synthesized. Synthesized gold nanorod (GNR) conjugates
possessing coumaric acid precursors underwent (porcine liver)
esterase-catalyzed hydrolysis on their surface to afford GNRs
coated with amino-functionalized polyethylene glycol and
fluorescent coumarins as reporter molecules for monitoring
the conversion. The chemical structural conversion on the
GNR surfaces was successfully observed inside cells by
fluorescence microscopy when GNR conjugates were
incubated with tumor cells.
pathways of nanoparticles is endocytosis. Nanoparticles present
in the extracellular fluid or deposited on the plasma membrane
are enclosed in vesicles of lipids and delivered to the endosomal
compartment, following the endosomes merging with lyso-
somes. In the lysosomal compartments the nanoparticles are
exposed to hydrolases capable of degrading organic molecules.
Cellular uptake, however, is not the only factor determining
intracellular concentration of nanoparticles: the relative abilities
of the host cells to remove the nanoparticles also decide their
ultimate fate. Although understanding the ensuing processes,
such as localization in the organelles and excretion from the
cells, is important for modulating cellular processes of the
engineered nanoparticles and for identifying their cytotoxic
effects,27 quantitative studies on their mechanisms are still
challenging.28−31 Especially, the effects of the surface charge
and chemical structure of nanoparticles on their excretion from
the host cells are not yet well explored. It has been reported
that engineered nanoparticles trap extracellular biomolecules
such as proteins on their surfaces depending on the chemical
structures and change their surface properties.15−18 Therefore,
molecular tools for determining surface properties of the
nanoparticles inside the cells would be useful for investigating
the intracellular processing mechanisms.
INTRODUCTION
■
Recent progress in nanoscience and technology is providing
great impetus for the development of new functional
nanomaterials that display physical and chemical properties
that are quite different from those of their bulk counterparts. In
particular, organic and inorganic nanoparticles have attracted
much interest from the viewpoint of biological applications
such as tools for bioimaging, diagnostics, and tissue- and cell-
specific drug delivery.1−10
Previous in vitro studies have demonstrated that synthetic
nanoparticles can be incorporated into living cells depending
on their size, shape, surface charge, and surface chemistry,
mainly via the endocytic pathway. It is thus important to
explore the effects of the surface properties of nanoparticles on
the possible interactions between nanoparticles and biomole-
cules, such as lipids and proteins that compose intracellular
structures, to understand the mechanisms of cellular uptake and
the following clearance from the cells. For example, as
evaluated by in vitro cytotoxicity assays, most engineered gold
nanoparticles are found to be less toxic or nontoxic toward
cultured cells.11−14 However, some reports indicated that the
cytotoxic effects are dependent on the surface properties of
gold nanoparticles, possibly because interactions between
nanoparticles and biomolecules (such as lipids or proteins)
could lead to the destruction of biological processes in
cells.15−18 Positively charged nanoparticles are generally taken
up well into the cells by electrostatic interaction because of the
negatively charged lipids of the plasma membrane.13,19−24
Despite the repellent interaction between negatively charged
nanoparticles and the cell membrane, some nanoparticles that
carry negatively charged functional groups on the surface can be
taken up into the cells.24−26 One of the major internalization
Here we report the development of metal nanoparticles
possessing functional linkers protected by esterase-sensitive
coumaric acids that can be enzymatically hydrolyzed to afford
functional groups on the surface of nanoparticles and, at the
same time, to form fluorescent coumarins as reporter molecules
Received: October 11, 2012
Revised: July 23, 2013
Published: July 27, 2013
© 2013 American Chemical Society
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of the conversion process (Figure 1). Because of the “masking”
effect of the protecting group, the variation in surface chemical
3). The mobile phase containing acetonitrile was buffered with
formic acid or acetic acid and delivered at a flow rate of 0.6 mL
min−1. The eluents were monitored by UV absorbance at 310
nm. 1H and 13C NMR spectra were recorded on a JEOL JNM-
AL 300, a JEOL EX 400, or a JEOL GSX 270 spectrometer.
The chemical shifts were expressed in ppm downfield from the
signals of solvents as internal standards. Fast atom bombard-
ment (FAB) mass spectrometry was recorded on a JMS-
SX102A (JEOL) mass spectrometer, using 3-nitrobenzyl
alcohol or polyethylene glycol (PEG) as a matrix. Electrospray
ionization mass spectrometry was carried out with a Bruker
Daltonics micro time-of-flight spectrometer. Fluorescence
spectra were obtained on a Shimadzu RF-5300PC spectro-
fluorophotometer. UV−visible absorption spectra were meas-
ured with a JASCO V-530 UV/vis spectrophotometer.
Organic Synthesis. Ethyl 3-(2-Hydroxy-4-methoxyphen-
yl)-(E)-2-propenoate (1).43,44 A mixture of 2-hydroxy-4-
methoxybenzaldehyde (1.08 g, 7.1 mmol) and ethyl-
(triphenylphosphoranylidene) acetate (5.03 g, 14.5 mmol) in
30 mL of CH2Cl2 was stirred at ambient temperature for 30
min. The mixture was washed with brine, dried over MgSO4,
and then concentrated under reduced pressure. Column
chromatography (30−50% EtOAc/hexanes) of the residue
gave the desired product 1 (1.62 g, quant.).
Figure 1. Esterase-catalyzed formation of GNRs possessing amino
linkers on the surface.
structures induces minimal effects on the internalization
process, but once the nanoparticles are taken up, esterase-
catalyzed hydrolysis induces structural conversion of the surface
linkers. Coumarin-based prodrug strategies were originally
developed by Wang’s groups,32−35 and similar prodrugs
possessing a “trimethyl lock”36−39 have been successfully
applied to drug delivery systems. In the present study, we
developed esterase-sensitive surface modifiers possessing
coumaric acid structures, which are applicable for under-
standing and modulating interaction between surface-modified
nanoparticles and biomolecules inside living cells. The
coumaric acid protecting groups at the termini of the surface
linkers enable estimation of the relative efficiency of the
structural conversion into the terminal amino groups on the
surface based on the fluorescence intensity of the released
coumarin by using high throughput fluorescence microplate
readers. The coumarin-forming precursors are almost non-
fluorescent, and thus a high signal-to-noise ratio is expected in
the present system, which is an advantage over the
fluorescence-quenching-based fluorophore−metal nanoparticle
conjugates, in which quenching efficiency usually depends on
the distance between the fluorophores and the nano-
particles.22,40−42
3-(2-Isobutyryloxy-4-methoxyphenyl)-(E)-2-propenoic
Acid (3). A solution of the ethyl ester 1 (1.40 g, 6.3 mmol) in
ethanol (30 mL), 5 M NaOH (2.5 mL), and water (30 mL)
was refluxed under an N2 atmosphere for 6 h. The dark solution
was cooled to room temperature and 6 M HCl was added until
the solution became acidic and a white precipitate appeared.
The solid was filtered, washed with CH2Cl2, and then dried
under reduced pressure. Carboxylic acid 2 (1.07 g, 88%) was
obtained as a white solid and used without further purification.
To a solution of 2 (499 mg) in dry THF (35 mL) was added
dropwise isobutyric anhydride (557 mg, 3.5 mmol), then
triethylamine (645 mg, 6.5 mmol) and 4-dimethylaminopyr-
idine (DMAP; 64 mg, 0.52 mmol) under an N2 atmosphere at
0 °C. After 10 min stirring at 0 °C, the solution was further
stirred at ambient temperature for 2 h. The resulting solution
was washed with water and then with diluted aqueous HCl (pH
∼4). The organic layer was dried over MgSO4 and filtered, and
the solvent was removed under reduced pressure. Column
chromatography (2% MeOH/CHCl3 + 1% CH3COOH) gave
the desired product 3 as a white solid (661 mg, 96%): 1H NMR
(400 MHz, CDCl3) δ 7.66 (d, J = 8.8 Hz, 1H), 7.64 (d, J = 16.1
Hz, 1H), 6.85 (dd, J = 2.4, 8.9 Hz, 1H), 6.66 (d, J = 2.4 Hz,
1H), 6.33 (d, J = 16.1, 1H), 3.80 (s, 3H), 2.89 (m, 1H), 1.34 (d,
J = 7.0 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 176.8, 170.3,
163.7, 152.2, 139.1, 129.5, 120.9, 118.6, 113.8, 109.4, 56.2, 35.3,
We synthesized gold nanorods (GNRs) with amino-func-
tional linkers protected by coumaric acids. We then successfully
quantified esterase-catalyzed conversion of the surface chemical
structures in living cells by monitoring fluorescence from the
generated coumarins. This approach can be applied for
quantitative evaluation of the surface structure conversion and
the subsequent alteration of the intracellular dynamics of metal
nanoparticles for designing new drug delivery nanocarriers.
+
19.3; HRMS (FAB+) m/z: Calcd for C14H17O5 : 265.1076 [M
+H]+, found 265.1071 [M+H]+.
3-(2-Isobutyryloxy-4-methoxyphenyl)-(Z)-2-propenoic
Acid (3′). An acetonitrile solution (15 mL) of the trans isomer
3 (52 mg, 0.2 mmol) in a quartz glass vial was exposed to UV
light (312 nm) from an FTI-20L transilluminator (Funakoshi)
at ambient temperature for 30 min. The solution was diluted
with ion-exchanged water (10 mL) and purified by HPLC using
35% acetonitrile/water containing 0.1% formic acid (3 mL/
min) as a mobile phase and the desired product was eluted at
46 min after the injection. Lyophilization of the isolated
fractions gave the cis isomer 3′ as a white solid (17 mg, 32%):
1H NMR (400 MHz, CDCl3) δ 7.60 (d, J = 8.6 Hz, 1H), 6.83
(d, J = 12.4 Hz, 1H), 6.73 (dd, J = 2.2, 8.6 Hz, 1H), 6.57 (d, J =
EXPERIMENTAL PROCEDURES
■
General Methods. Reagents were purchased from
commercial sources and were used without further purification.
Preparative HPLC was performed with a Tosoh LC system
(DP-8020 pumps, UV-8011 detector, AS-8020 auto sampler,
and FC-8000 fraction collector) equipped with a reversed-
phase column (5 μm, ϕ10 × 250 mm, Inertsil ODS-3, GL
Sciences). Analytical HPLC was carried out with a Shimadzu
10A HPLC system. Sample solutions were injected onto a
reversed-phase column (5 μm, ϕ4.6 × 250 mm, Inertsil ODS-
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2.4 Hz, 1H), 5.93 (d, J = 12.2, 1H), 3.78 (s, 3H), 2.78 (m, 1H),
1.28 (d, J = 6.8 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ
175.1, 170.3, 161.0, 149.6, 137.5, 131.6, 120.8, 120.4, 111.5,
107.5, 55.5, 34.1, 18.9; HRMS (FAB+) m/z: Calcd for
2,3,6,7-Tetrahydro-10-dimethyl-1H,5H,11H-[1]-
benzopyrano[6,7,8-ij]quinolizin-11-one (7). To a solution of
ethyl ester 4 (34.5 mg, 0.11 mmol) in ethanol (5 mL) was
added 0.1 M NaHCO3 (0.5 mL). The yellow solution in a
quartz glass vial was exposed to UV light (365 nm) from a
transilluminator at ambient temperature for 1 h. The resulting
fluorescent solution was evaporated under reduced pressure,
resuspended in water, and then extracted with CHCl3. The
organic layer was dried over MgSO4, filtered, and evaporated
under reduced pressure. A yellow solid was obtained as the
+
C14H17O5 : 265.1076 [M+H]+. Found 265.1078 [M+H]+.
Ethyl (E)-3-(8-Hydroxyjulolidine-9-yl)-2-methylpropenate
(4). Ethyl ester 4 was synthesized following the previously
reported procedure.45 A solution of 8-hydroxyjulolidine-9-
carboxaldehyde (435 mg, Tokyo Chemical Industry) and [1-
(ethoxycarbonyl)ethylidene]triphenylphosphorane (1.45 g,
Wako) in CH2Cl2 (5 mL) was stirred at 40 °C under an N2
atmosphere for 22 h. The resulting solution was cooled to
room temperature and dried under reduced pressure. The
crude product was purified by column chromatography (0−
25% EtOAc/hexanes) to afford 4 as a yellow solid (521 mg,
87%).
(E)-3-(8-Hydroxyjulolidine-9-yl)-2-methylpropenoic Acid
(5).45 A solution of ester 4 (401 mg) in ethanol (3 mL), 5
M NaOH (2 mL), and water (5 mL) was refluxed under a N2
atmosphere for 2 h. The solution was cooled to room
temperature and acidified with 6 M HCl. The resulting mixture
was extracted with CHCl3 and the organic layer was dried over
Na2SO4, filtered, and then dried under reduced pressure.
Column chromatography (0−4% MeOH/CHCl3) gave the
desired product 5 as a green solid (294 mg, 81%).
1
desired coumarin 7 (28.1 mg, 0.1 mmol, 90%): H NMR (400
MHz, CDCl3) δ 7.19 (s, 1H), 6.70 (s, 1H), 3.17−3.13 (m, 4H),
2.80 (t, 2H, J = 6.46 Hz), 2.67 (t, 2H, J = 6.34 Hz), 2.04 (s,
3H), 1.92−1.85 (m, 4H); 13C NMR (100 MHz, CDCl3) δ
163.6, 150.8, 144.8, 140.3, 124.0, 118.2, 117.5, 108.9, 106.6,
49.9, 49.6, 27.4, 21.6, 20.7, 20.4, 16.7; HRMS (FAB+) m/z:
Calcd for C16H18NO2: 256.1338 [M+H]+, found 256.1342 [M
+H]+.
Amino Linkers. Bifunctional PEG linkers possessing amino
and thiol groups on their termini (L2 and L4) were purchased
from Dojindo Laboratories and JenKem Technology USA,
respectively (Figure 2). Amino-PEG-thiol L3 was synthesized
(E)-3-(8-Isobutyryloxyjulolidine-9-yl)-2-methylpropenoic
Acid (6). To the carboxylic acid 5 (294 mg) in dry CH2Cl2 (6
mL) were added isobutyric anhydride (216 μL), DMAP (26
mg), and triethylamine (265 μL) under an N2 atmosphere, and
the solution was stirred at ambient temperature under an N2
atmosphere for 2 h. The resulting solution was washed with
water and then with diluted aqueous HCl (pH ∼4). The
organic layer was dried over MgSO4, filtered, and then
evaporated under reduced pressure. Column chromatography
(0−1% MeOH/CHCl3) gave the desired product 6 with a
small amount of isobutyric acid. For NMR measurement, the
mixture was further purified by HPLC to give 6 as a yellow
Figure 2. Chemical structures of the amino linkers (L1−4).
according to the synthetic procedure reported by Fiammengo’s
group.46 1,2-Dithia-3-(1-amino-n-pentyl)cyclopentane (L1)
was synthesized following the previously reported procedure47
with a slight modification. DL-α-Lipoamide (1.0 g) was
dissolved in 30 mL of dry THF. The solution was added to
BH3−THF (2.5 equiv, Nacalai) at ambient temperature. After
30 min stirring, the mixture was further refluxed under an N2
atmosphere for 24 h. The mixture was cooled to room
temperature, and 6 M HCl (5 mL) was added to the mixture.
The mixture was again refluxed for 1 h, and then the pH of the
solution was adjusted to 6−7 with 5 M aqueous NaOH, and the
resulting mixture was stirred at ambient temperature for 24 h.
After evaporation of the solvent, the reaction product was
extracted with THF, dried over MgSO4, filtered, and then
evaporated under reduced pressure. The residue was purified by
column chromatography using 5% MeOH/CH3Cl containing
1% triethylamine as eluent to afford the desired product L1
(0.345 g, 37%).
Coumaric Acid−Lipoamine Conjugate (3-L1). To a
solution of L1 in dry DMF (10 mL) were added the trans
coumaric acids 3 (104.8 mg, 0.40 mmol) and 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDC; 93.9
mg, 0.49 mmol) in 3 mL of DMF, and the mixture was stirred
at 4 °C for 10 min. After the addition of DMAP (6.1 mg, 0.05
mmol) and 1-hydroxybenzotriazole (HOBt; 75.0 mg, 0.49
mmol), the resulting mixture was stirred under N2 atmosphere
at 4 °C for 1 h, and then at 25 °C for 18 h. After evaporation of
the solvent, products dissolved in chloroform were washed with
saturated NaHCO3, saturated citric acid, and brine, succes-
sively. Organic layer was dried over MgSO4, filtered, and then
1
solid (276 mg, 75%): H NMR (400 MHz, CDCl3) δ 7.62 (s,
1H), 6.97 (s, 1H), 3.19−3.13 (m, 4H), 2.84−2.80 (m, 1H),
2.72 (t, J = 6.2 Hz, 2H), 2.50 (br, s, 2H), 2.07 (s, 3H), 1.96−
1.89 (m, 4H), 1.33 (d, 6H); 13C NMR (100 MHz, CDCl3) δ
174.8, 174.0, 146.6, 144.5, 135.5, 127.9, 123.7, 118.2, 115.1,
113.3, 49.8, 49.2, 34.3, 27.6, 21.7, 21.4, 21.0, 19.1, 13.9; HRMS
(FAB+) m/z: Calcd for C20H26NO4 : 344.1856 [M+H]+, found
+
344.1873 [M+H]+.
(Z)-3-(8-Isobutyryloxyjulolidine-9-yl)-2-methylpropenoic
Acid (6′). An acetonitrile solution (20 mL) of the trans isomer
6 (52.4 mg, 0.15 mmol) in a quartz glass vial was exposed to
UV light (365 nm) at 4 °C for 45 min. The solution was diluted
with water (6 mL) and methanol (8 mL), and then subjected to
HPLC purification (mobile phase: 70% acetonitrile/10 mM
triethylamine−acetic acid buffer (pH 7.0), 3 mL/min). Desired
product was eluted at 8−9 min after the injection.
Lyophilization of the isolated fractions gave the cis isomer 6′
as a white solid (12.6 mg, 24%): 1H NMR (400 MHz, CDCl3)
δ 6.73 (s, 1H), 6.46 (s, 1H), 3.13−3.09 (m, 4H), 2.83−2.81 (m,
1H), 2.67 (t, J = 6.4 Hz, 2H), 2.51 (s, br, 2H), 2.02 (d, J = 1.9
Hz, 3H), 1.94−1.91 (m, 4H), 1.30 (d, J = 6.9 Hz, 6H); 13C
NMR (100 MHz, CDCl3) δ 176.7, 170.9, 144.3, 144.1, 132.4,
129.4, 127.4, 119.3, 116.2, 112.7, 49.8, 49.1, 34.3, 27.2, 21.7,
21.3, 21.1, 20.9, 8.8; HRMS (FAB+) m/z: Calcd for
C20H25NO4: 343.1784 [M]+, found 343.1784 [M]+.
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Scheme 1. Synthesis of Coumaric Acid 3′
evaporated under reduced pressure. The residue was purified by
column chromatography using hexane/ethyl acetate (3:2) as
eluent to afford the desired product 3-L1 as a white solid (55.7
to remove excess linkers. Finally, surface-modified GNRs (L1−
L4@GNRs) were resuspended in 200 μL of deionized water.
To the GNR solution were added cis coumaric acids (3′ or 6′, 8
μmol) and EDC (10 μmol) in 200 μL of DMF or acetonitrile
and the mixture was stirred at 4 °C for 10 min. DMAP (1
μmol) and HOBt (10 μmol) were added to the mixture and
stirred at 4 °C for 1 h, and then at 25 °C for 12−18 h. The
mixture was centrifuged, decanted, and redispersed in DMF−
H2O (1:1) to remove unconjugated coumaric acids. The GNR
conjugates thus obtained were finally resuspended in 200 μL of
deionized water. Conjugation of the coumaric acids to the
1
mg, 31.4%): H NMR (300 MHz, CDCl3) δ 7.63 (d, J = 8.8
Hz, 1H), 7.63 (d, J = 15.5 Hz, 1H), 6.77 (dd, J = 2.4, 8.6 Hz,
1H), 6.59 (d, J = 2.6 Hz, 1H), 6.22 (d, J = 15.5, 1H), 5.54 (br,
1H), 3.79 (s, 3H), 3.54 (m, 1H), 3.34 (q, 2H), 3.12 (m, 2H),
2.87 (m, 1H), 2.44 (m, 1H), 1.89 (m, 1H), 1.68 (br, 4H), 1.4−
1.5 (m, 4H), 1.35 (d, J = 7.0 Hz, 6H); 13C NMR (75 MHz,
CDCl3) δ 175.3, 165.8, 161.4, 150.5, 133.9, 127.6, 120.2, 120.1,
112.7, 108.0, 56.2, 55.5, 40.2, 39.5, 38.4, 34.8, 34.2, 29.5, 28.9,
26.7, 19.0; HRMS (FAB+) m/z: Calcd for C22H31NO4S2:
438.1773 [M+H]+, found 438.1785 [M+H]+.
1
surface of GNRs was confirmed by H NNR (Supporting
Information).
Coumaric Acid−Lipoamine Conjugate (3′-L1). A chloro-
form solution (15 mL) of 3-L1 (39.3 mg, 0.09 mmol) in a
quartz glass vial was exposed to UV light (312 nm) at ambient
temperature for 0.5 h. Evaporation of the solvent gave the
Transmission Electron Microscopy (TEM). A drop of the
nanoparticle solution was allowed to air-dry onto an elastic-
carbon-coated copper grid (ELS-C10, Okenshoji, Japan). TEM
images were obtained with a JEOL JEM-1400 electron
microscope operating at an accelerating voltage of 120 kV.
Zeta Potential Measurement. Zeta potentials of the
surface modified GNRs were measured with a Malvern Nano-
ZS Zetasizer in disposable cuvettes. GNPs either in ethanol or
in water were sonicated before each measurement. Measure-
ments were carried out in triplicate, and the Smoluchowski
approximation was used to convert the electrophoretic mobility
to zeta potential.
Porcine Liver Esterase (PLE)-Catalyzed Hydrolysis.
Purified PLE (carboxylic-ester hydrolase; EC 3.1.1.1; E-2884)
was obtained from Sigma as a suspension in a 3.2 M
(NH4)2SO4 solution (pH 8). The suspension containing 1 ×
104 units of PLE was diluted to 0.7 mL with phosphate buffer
(0.05 M, pH 7.4) containing 2% DMSO. Then, 140 μL of the
prepared GNR conjugates (0.1−0.2 μM) was combined with
the above-mentioned buffer−PLE solution in a quartz cuvette.
During the reaction, the cuvette was incubated at 37 °C and
subjected to fluorescence measurement.
DNA Binding Assay. For preparation of the hydrolysate of
3′-L3@GNR, the conjugates were incubated at pH 10 for 1 h,
neutralized, and then centrifuged at 10,000 rpm for 15 min.
Then, 1 μg of plasmid DNA (ΦX174 RF I, New England
Biolabs) was added to 5 μL of the GNR conjugates (3′-L3@
GNRs, hydrolyzed 3′-L3@GNRs, or L3@GNRs, 3 nM) and
incubated at ambient temperature for 30 min. The solution was
diluted with 400 μL of 1.27 μM ethidium bromide (EB, Nippon
Gene) containing 20% DMF and subjected to fluorescence
measurement.
1
corresponding cis isomer 3′-L1 as a white solid (quant.): H
NMR (270 MHz, CDCl3) δ 7.17 (d, J = 8.4 Hz, 1H), 6.69 (dd,
J = 2.5, 8.6 Hz, 1H), 6.55 (d, J = 11.9 Hz, 1H), 6.50 (d, J = 2.5
Hz, 1H), 6.15 (br, 1H), 5.97 (d, J = 11.9, 1H), 3.75 (s, 3H),
3.44 (m, 1H), 3.09 (m, 2H), 3.02 (q, 2H), 2.75 (m, 1H), 2.37
(m, 1H), 1.81 (m, 1H), 1.71 (br, 4H), 1.4−1.5 (br, 4H), 1.24
(d, J = 6.8 Hz, 6H); 13C NMR (67.5 MHz, CDCl3) δ 176.3,
167.0, 160.8, 149.1, 130.9, 130.0, 128.7, 122.3, 112.1, 107.4,
56.5, 55.6, 40.2, 39.0, 38.4, 34.7, 34.1, 28.9, 28.7, 26.4, 19.0;
HRMS (FAB+) m/z: Calcd for C22H31NO4S2: 438.1773 [M
+H]+, found 438.1773 [M+H]+.
Synthesis of GNRs. GNRs were synthesized by the
photochemical method reported by Niidome et al.48 with a
slight modification. To an aqueous solution (12 mL) of
HAuCl4·3H2O (2 mM, Aldrich) and cetyltrimethylammonium
bromide (CTAB, 80 mM) were added cyclohexanone (100
μL), 0.15 M AgNO3 (50 μL), and 80 mM ascorbic acid (400
μL). The resulting colorless solution was exposed to UV light
(λ = 250−400 nm) at ambient temperature for 40 min. The
resulting dark solution was purified by repeating centrifugation
at 12,000 rpm (20 min) and washing with water, and then the
precipitates containing the CTAB-capped GNRs were
suspended in deionized water.
Preparation of GNR Conjugates. Typically, amino linkers
L1−L4 (20 mM, 200 μL) were added to a 200 μL solution of
CTAB-coated GNR (0.2−0.6 μM), which was then incubated
at 25 °C for 12−18 h. The GNR solution was then centrifuged
(10,000 rpm, 10 min), decanted, and redispersed in water twice
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Scheme 2. Synthesis of Coumaric Acid 6′
Bovine Serum Albumin (BSA) Binding Assay. Gold
nanoparticles (3′-L3@GNR or hydrolyzed 3′-L3@GNR, 0.03
nM) in PBS buffer (pH 7.0) containing 5% DMSO were
incubated with various concentrations (0.02−0.30 wt %) of
BSA (SIGMA, A4378) for 10 min at ambient temperature.
Absorption spectra were recorded in the wavelength range of
400−900 nm.
Cell Culture and Fluorescence Imaging. Human lung
cancer cells A549 (American Type Culture Collection) were
cultured in Dulbecco’s Modified Eagle Medium supplemented
with 10% fetal bovine serum and 1% penicillin/streptomycin at
37 °C in a humidified incubator with an atmosphere containing
5% CO2. Cells were plated in 96-well imaging microplates (5 ×
103 cells/well) and incubated for 24 h. Various concentrations
of GNR conjugates were added to the medium. The cells were
further incubated for 1−24 h, washed with fresh PBS buffer
three times, and then observed with a Keyence BZ-8100
fluorescence microscope using appropriate excitation and
emission filters. The fluorescence intensity was determined
with a Promega GloMax multi microplate reader (λex = 365 nm,
λem = 410−460 nm).
hydroxyl groups of 2 and 5 were protected with the isobutyryl
group by reaction with isobutyric anhydride; the carboxylic
acids 3 and 6 were obtained and were further converted to the
cis isomers 3′ and 6′ by UV irradiation. The cis isomers 3′ and
6′ were stable at −20 °C for at least a few months.
GNRs were prepared by the photochemical method
following the previously reported procedures with minor
modifications.48,49 Typically, the aqueous solution of Au(III)
ions containing AgCl, CTAB, acetone, and cyclohexanone was
treated with ascorbic acid, and the resulting colorless Au(I)
solution was then exposed to UV light through a cutoff filter (λ
= 240−400 nm) at room temperature. Replacing cyclohexane,
which was used in the original studies, with cyclohexanone
increased the reproducibility of the results. The dark-purple
solution was centrifuged and GNRs were collected as the
precipitate. Dispersion of GNRs in aqueous buffer solution
showed the characteristic plasmon absorption band at around
700 nm. Transmission electron microscope analysis of the
synthesized GNRs suggested their particle size was (36 4) ×
(11 2) nm (aspect ratio 3.3 0.4).
The surface of GNRs is easily modified by conjugation with
thiol compounds (L1−4) via the Au−S bond (Figure 2). To
avoid potential aggregation of GNRs during the assays, the
GNR surfaces were modified with heterobifunctional PEG
having a thiol group on one terminus and an amino group on
the other end (L2−4) for covalent attachment of the cis
coumaric acids 3′ and 6′. It has been demonstrated that PEG
coating of the surface of gold nanoparticles enhances
biocompatibility and dispersibility in aqueous solution.50 The
CTAB-capped GNRs prepared as above were incubated with
L1 or the PEG linkers L2−4, and then further conjugated with
3′ or 6′ in the presence of 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide, DMAP, and 1-hydroxybenzotriazole. Un-
reacted materials were removed by multiple centrifugation and
washing steps until cis coumaric acids were not detected in the
supernatant solution.
RESULTS AND DISCUSSION
■
Design and Synthesis of GNR Conjugates. Esterase-
sensitive coumaric acid structures for protecting amino groups
on the surface of GNRs were synthesized according to the
reaction pathways shown in Schemes 1 and 2. It has been
reported that photoisomerization of the trans coumaric acids
gives the corresponding cis isomers in acceptable yields.34
Therefore, we first attempted to protect the phenol hydroxyl
group of o-coumaric acids and then converted them into the cis
isomers by UV irradiation. As starting materials, we chose two
commercially available salicylic aldehyde derivatives, 8-hydrox-
yjulolidine-9-carboxaldehyde and 2-hydroxy-4-methoxybenzal-
dehyde. Wittig olefination of the aldehydes with triphenyl-
phosphorane in CH2Cl2 and the following alkali hydrolysis gave
the corresponding carboxylic acids 2 and 5 in good yields.43,44
We first attempted acetylation of the phenol hydroxyl group of
2 to obtain the corresponding acetyl esters. Photoisomerization
of the resulting carboxylic acid in acetonitrile was performed
with a UV light source (312 nm) until the trans to cis isomer
conversion reached ∼70%, and then the generated cis isomer
was purified by HPLC. However, the cis isomer possessing the
acetyl group was thermally hydrolyzed and transformed into 7-
methoxycoumain during the purification. Thus, the phenol
GNRs functionalized with 3′ via lipoamine L1 linker (3′-
L1@GNR) were immediately aggregated in aqueous solution.
By contrast, the coumaric acid−GNR conjugates possessing
PEG linkers (3′-L2@GNR, 3′-L3@GNR, 3′-L4@GNR, 6′-
L3@GNR, and 6′-L4@GNR) were dispersed in aqueous buffer
solution (pH 7.0) to some extent; in particular, the functional
PEG linker L4 improved their dispersibility. Figure 3 shows the
results of TEM observation of the synthesized 3′-L3@GNR.
The apparent size of the surface-modified GNRs as determined
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Figure 3. Transmission electron microscope image of 3′-L3@GNRs.
by transmission electron microscopy ((34 3 nm) × (11
2
nm)) was unchanged from that of unmodified GNRs, because
only the gold core is visible at the current acceleration voltage
(Supporting Information). The amino-PEG-modified GNRs
L2−4@GNR showed positive surface potentials of +29 to +32
mV; on the other hand, GNRs modified with the coumaric
acids showed somewhat neutralized surface potentials of +17 to
+22 mV (Table 1). As has been discussed earlier,51 a small
amount of residual CTAB on the surface of GNRs might be
attributable to the slightly positive potentials.
Table 1. Zeta Potential (mV) of the Surface Modified GNRs
Figure 4. Typical fluorescence spectra of (A) 3′-L1 and (B) 3′-L3@
GNR after incubation with PLE at 37 °C for the indicated periods.
(Insets) Time course of the PLE-catalyzed release of fluorescent
coumarins from (A) 3′-L1 (λex = 280 nm, λem = 390 nm) and (B) 3′-
L3@GNR (λex = 330 nm, λem = 390 nm).
end protection
nanoparticle
non
3′
6′
−
L2@GNR
L3@GNR
L4@GNR
+31.6 0.5
+31.4 2.1
+29.0 2.1
+17.6 2.1
+21.8 1.8
+18.3 0.4
+17.1 2.6
+17.2 0.6
free ligand 3′-L1, fluorescent coumarin formation was observed
for all of the conjugates, although the dispersibility of the
nanoparticles varied (Figure 4B). It has been suggested that
gold nanoparticles are quite efficient fluorescence quenchers;52
however, it is less likely that the emission from the released
coumarins is quenched by the GNR conjugates. In a separate
experiment, addition of the GNR conjugates to free coumarins
in aqueous buffer solutions showed no emission quenching,
probably because the surface PEG structures shield the gold
core from the free photoexcited coumarins. Table 2 shows
Hydrolysis and Lactonization of the Coumaric Acids
on the Surface of GNRs. To demonstrate enzyme-catalyzed
hydrolysis and lactonization of the coumaric acids by esterase,
PLE was used for the assays. PLE can recognize and hydrolyze
a wide variety of ester structures. Figure 4A shows time-
dependent fluorescence spectra obtained from the PLE-treated
coumaric acid−lipoamine conjugates (3′-L1, 10 μM) in
aqueous phosphate buffer containing 2% DMSO. Depending
on the incubation time, emission from the photoexcited 7-
methoxycoumarin increased; by contrast, only a slight increase
was observed for 3′-L1 untreated with PLE. The PLE-catalyzed
hydrolysis of 3′-L1 and the appearance of 7-methoxycoumarin
were also observed on the HPLC chromatograms (data not
shown). The apparent pseudofirst-order rate constant (kobs) for
the formation of 7-methoxycoumarin from 3′-L1 was
determined to be 3.7 × 10−4 s−1 (t1/2 = 32 min), which is
comparable to the value for a similar phenyl isobutylate.33 The
formation of coumarin should proceed via the following
multistep reactions: (1) recognition of the coumaric acid by
PLE, (2) hydrolytic release of the isobutyl group, and (3)
lactonization of the coumaric acids and release of the amines. It
is thus possible that conjugation of the coumaric acids to the
surface of GNRs affects the accessibility of PLE as well as the
rate of lactonization.
Table 2. Pseudo-First-Order Kinetic Rate Constants (kobs) of
the PLE-Catalyzed Release of Coumarin Derivatives from
the Surface-Modified GNRs
3′
6′
nanoparticle
k
obs/s−1
t1/2/min
kobs/s−1
t1/2/min
L2@GNR
L3@GNR
L4@GNR
4.6 × 10−5
3.6 × 10−5
1.8 × 10−4
250
320
65
−
−
49
34
2.4 × 10−4
3.4 × 10−4
kinetic data for the formation of PLE-catalyzed coumarin from
the GNR conjugates, as determined from the fluorescence
profiles. Under the present experimental conditions, the values
of kobs for coumarin formation from 3′-L4@GNR and 6′-L4@
GNR were larger than from 3′-L2@GNR, 3′-L3@GNR, and
6′-L3@GNR. Therefore, dispersibility of the GNR conjugates
might affect the accessibility of PLE to coumaric acids on the
surface of the GNR conjugates. Also, precursor 6′ is hydrolyzed
faster than 3′ suggesting that 6′ is a good substrate for
We next examined PLE-catalyzed hydrolysis of the coumaric
acid protecting group on the surface of synthesized GNRs. In
the present study, we prepared GNR conjugates having various
linker structures (3′-L2@GNR, 3′-L3@GNR, 3′-L4@GNR, 6′-
L3@GNR, and 6′-L4@GNR) to investigate the effects of linker
structures and length on PLE sensitivity. As in the case of the
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monitoring the conversion process on the surface of the GNR
conjugates. Based on the concentration of the GNR conjugates
and the fluorescence intensity obtained after completion of the
hydrolysis, it can be calculated that approximately 1.8 × 103
molecules of procoumarin 6′ are tethered on the surface of
single 6′-L4@GNR.
Esterase-catalyzed hydrolysis of the surface structure of the
GNR conjugates formed fluorescent coumarins and GNRs with
amino-PEG linkers as the counterparts. Alteration of the
surface chemical structures of GNRs would change the
interactions between nanoparticles and biomolecules in the
cells. It has been reported that metal nanoparticles coated with
amino-functional chemical structures interact with biomacro-
molecules, such as DNA and BSA.16,53 To confirm the
formation of amino residues on the surface of the synthesized
GNR conjugates, changes in the interactions between plasmid
DNA and the prepared GNR conjugates (3′-L3@GNR and 6′-
L3@GNR) were monitored using a fluorescent intercalator
displacement assay. Positively charged EB is a well-known
intercalator toward DNA. Upon intercalation between base
pairs in DNA, photoexcited EB shows intense fluorescence and
thus competitive binding of the GNRs to the DNA duplex
could be monitored as changes in fluorescence intensity.
As shown in Figure 5, addition of 3′-L3@GNR to ΦX174
plasmid DNA resulted in no changes in the fluorescence spectra
Figure 6. UV−vis absorption spectra of GNRs in the presence of
various concentrations of BSA (0.02−0.30%). (A) 3′-L3@GNRs in
PBS buffer (pH 7.0) containing 5% DMSO were incubated with BSA.
(B) Hydrolyzed 3′-L3@GNRs were resuspended in PBS buffer (pH
7.0) containing BSA and 5% DMSO. Absorption spectra were
normalized at 900 nm.
GNRs were not influenced by the addition of BSA. By contrast,
a hypsochromic shift of the surface plasmon band at around
700 nm was observed when BSA was added to the hydrolysate
of 3′-L3@GNR (expected to be converted to L3@GNR),
implying that the dispersibility of GNRs was changed as a result
of surface conversion and the subsequent BSA absorption on
the surface. Taken together with the fact that the shape and
peak position of the transverse surface plasmon band at around
520 nm were not much affected by the BSA addition, these
findings indicate that the Au(111) surface is crucial in the
interaction between BSA and L3@GNR.58
Figure 5. Fluorescence spectra of EB in H2O−DMF (4:1) as observed
upon excitation at 330 nm. (a) EB, 3′-L3@GNR, and DNA; (b) EB
and DNA; (c) EB, DNA, and L3@GNR; (d) EB, DNA, and
hydrolyzed 3′-L3@GNR; (e) EB alone.
As demonstrated above, we could observe deprotection of
amino groups as the formation of fluorescent coumarins.
Although DNA and BSA are not easily accessed by the
incorporated GNRs in the cells, it is highly likely that such
conversion of the surface chemical structures could alter
interactions between GNRs and other cytoplasmic proteins and
lipids
Cellular Uptake of GNR Conjugates and Their Surface
Structural Changes inside Living Cells. Previous studies on
cellular uptake of nonfluorescent metal nanoparticles were
conducted using dark-field microscopy and inductively coupled
plasma mass spectroscopy. Dark-field microscopy is useful for
directly observing metal nanoparticles within cells without any
pretreatment, but scattering light from subcellular organelles
sometimes makes quantifying the relative amounts of uptaken
nanoparticles difficult. However, inductively coupled plasma
mass spectroscopy is capable of detecting metal ion
concentration down to the parts-per-billion (ppb) level, and
it is thus suited for determining the number of metal
nanoparticles within cells, although some adequate pretreat-
ments, such as collection and homogenization of the cells, are
necessary prior to measurement. In principle, the GNR
of EB. By contrast, hydrolysate of 3′-L3@GNR suppressed the
fluorescence intensity, suggesting that amino-PEG-coated
GNRs (L3@GNR) generated as a result of the coumarin
release interacted with plasmid DNA, and either directly
inhibited the intercalation of EB into DNA base pairs or
induced conformational changes in the DNA duplex.
We further evaluated the formation of amino groups by
examining the affinity of the GNR conjugates to BSA before
and after the hydrolytic conversion of the surface. Recent
studies have demonstrated protein absorption on the surface of
manufactured nanoparticles depending on the size, charge
density, and other surface properties. Such binding can change
the original surface characteristics of nanoparticles and affect
the bioprocesses both inside and outside the living cells. In fact,
protein absorption on the surface altered the efficiency of
cellular uptake.16,54−56 UV-absorption spectra (λ = 400−900
nm) of the prepared GNR conjugates 3′-L3@GNR were
recorded in the presence of BSA (Figure 6). BSA does not
show intense absorption bands in that wavelength range.57 The
absorption bands corresponding to the surface plasmon of
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conjugates prepared in this study can form equal amounts of
surface-modified nanoparticles and fluorescent coumarin
derivatives, and thus relative amounts of nanoparticles with
specific surface structures can be estimated based on the
fluorescence intensity using fluorescence microscopy.
We first examined the cytotoxicity of the prepared GNRs
toward human lung adenocarcinoma epithelial cell line A549
using the WST method.59 Colorimetric measurements
indicated that, after 24 h of incubation, GNR conjugates
showed a cell viability of more than 80% (data not shown),
suggesting that the prepared nanoparticles could be used for in
vitro studies. GNR conjugates were incubated with A549 cells
and the subsequent coumarin release was observed with a
fluorescence microscope. We first examined 3′-L3@GNR to
demonstrate the surface conversion; however, excitation of the
nanoparticles with short-wavelength light illumination resulted
in cell death under the microscope during the observation. A
newly synthesized coumarin derivative 7 absorbs UV-light in
the wavelength range of 300−460 nm, and shows fluorescence
between 450 and 600 nm (Supporting Information). Therefore,
6′-L4@GNR was employed as an alternative, and the
microscopic images were obtained through a filter (460 nm
25 nm) by excitation at 360 nm 20 nm (Figure 7). A549
Figure 8. Quantification of fluorescence intensity (410−460 nm)
●
upon excitation at 365 nm. A549 cells were incubated ( ) alone or in
■
▲
the presence of either ( ) L4@GNRs or ( ) 6′-L4@GNRs (0.8 nM,
respectively) for the indicated periods.
lysosomes. From the time profiles of fluorescence intensity in
Figure 8, concentration of coumarin 7 released after 3 h
incubation was calculated to be 0.12 μM, which implies that 8%
of 6′ on the surface of 6′-L4@GNR were hydrolyzed and the
same amount of amine residues were generated. According to a
previous report, GNRs are internalized into A549 cells and
transferred to endosomes and lysosomes over several hours.60
Therefore, the rate-determining cellular uptake process should
be responsible for the observed slow release of coumarins.
Direct quantification of GNRs in living cells based on
fluorescence intensity is generally difficult, because the
quantum efficiency of such nanorods has been reported to be
very low,61 limiting the widespread use of the fluorescence
properties of GNRs in biosensing. Unfortunately, the enzyme-
catalyzed surface structure conversion and the concomitant
release of fluorophore seemed to be slower than we expected.
In view of the fact that kobs for the release of coumarin from free
3′-L1 is larger than those from GNR conjugates, it might be
possible to enhance the reactivity by designing a linker with
appropriate length.
We envision that the present approach would be applicable
for determining relative amounts of surface-modified gold
nanoparticles in living cells based on the fluorescence intensity
of the released coumarins. For example, it would be possible to
quantitatively investigate the effects of surface structures of
nanoparticles on the excretion from various types of cells based
on our strategy.
Figure 7. Fluorescence microscope images of A549 cells incubated
with (A) no additive, (B) coumarin 7 (1.0 μM), (C) L4@GNRs (1.1
nM), or (D) 6′-L4@GNRs (0.8 nM) for the indicated periods.
Cultured cells were photoexcited at 360 20 nm, and the fluorescence
images were obtained in the wavelength range of 460 25 nm.
CONCLUSIONS
■
We prepared gold nanoparticles whose surface was modified
with esterase-sensitive coumaric acids and evaluated the PLE-
catalyzed surface structure conversion of the particles
accompanied by the formation of fluorescent coumarins as
reporter molecules. PEG-coated GNRs protected by 6′ (6′-
L4@GNR) were used for the cell assay, because of their
stability in aqueous media and ease of observation with a
fluorescence microscope, and we successfully observed the
time-dependent surface conversion. This approach might
provide a potential strategy for investigating the effects of
surface chemical structures of nanoparticles on processes of
excretion from within the cells. Another application of our
nanoparticles would be for delivering nanoscale drugs into cells
even when the surfaces are unfavorably charged for internal-
ization. Alteration of the surface polarity could change the
interaction between nanoparticles and biomolecules, resulting
in the nanoparticles being accumulated in the cells and
cells treated with 6′-L4@GNR showed intense blue fluo-
rescence after a short period of incubation (∼0.5 h) and the
fluorescence from within the cells lasted over 6 h. By contrast,
when the coumarin derivative 7 was added to the cells, intense
blue fluorescence from within the cells was observed only a few
minutes after the addition. Also, when 6′-L4@GNR was treated
with the medium buffer, no increase of fluorescence was
observed (data not shown). The time-dependent surface
conversion of 6′-L4@GNR in A549 cells was monitored as
the formation of 7 by quantifying fluorescence intensity from
A549 cells cultured in 96-well imaging plates for desired periods
of time (Figure 8). These data imply that amino-PEG-coated
nanorods (L4@GNRs) were increasingly formed after 6 h
under the present conditions, most likely in endosomes or
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polymers (CGPs) and AIE-active fluorogens through triggered
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of the surface chemical structure on the exocytotic pathways of
the metal nanoparticles, we are currently investigating the fate
of gold nanoparticles after the surface conversion based on this
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR spectra of the conjugates, TEM micrograph, UV-
absorption and fluorescence spectra of 7, UV-absorption
maxima of the GNR conjugates. This material is available free
of charge via the Internet at http://pubs.acs.org.
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and Holl, M. M. B. (2007) Nanoparticle interaction with biological
membranes: does nanotechnology present a Janus face? Acc. Chem.
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interactions. Nano Today 3, 40−47.
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D. P., Hong, S., Mullen, D. G., DiMaggio, S. C., Som, A., Tew, G. N.,
Lopatin, A. N., Baker, J. R., Jr., Holl, M. M. B., and Orr, B. G. (2009)
Cationic nanoparticles induce nanoscale disruption in living cell
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AUTHOR INFORMATION
Corresponding Author
■
*Phone: +81-75-383-7054. Fax: +81-75-383-2504. E-mail:
takeoit@scl.kyoto-u.ac.jp.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors appreciate the measurement of FAB mass spectra
by Mr. Hiroshi Hatta, Kyoto University. This work was
supported by Hosokawa Powder Technology Foundation and
the Program for Fostering Regional Innovation “Kyoto
Environmental Nanotechnology Cluster” administered by the
Ministry of Education, Culture, Sports, Science, and Technol-
ogy (MEXT), Japan.
ABBREVIATIONS
■
GNR, gold nanorod; CTAB, cetyltrimethylammonium bro-
mide; EB, ethidium bromide; EDC, 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride; HOBt, 1-
hydroxybenzotriazole; DMAP, 4-dimethylaminopyridine
(19) Goodman, G. M., McCusker, C. D., Yilmaz, T., and Rotello, V.
M. (2004) Toxicity of gold nanoparticles functionalized with cationic
and anionic side chains. Bioconjugate Chem. 15, 897−900.
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Bhattacharya, R., Robertson, D., Rotello, V. M., Prakash, Y. S., and
Mukherjee, P. (2010) Effect of nanoparticle surface charge at the
plasma membrane and beyond. Nano Lett. 10, 2543−2548.
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cytotoxicity, and their relationship. ACS Nano 4, 5421−5429.
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