Multifunctional surface modification of gold-stabilized nanoparticles by bioorthogonal reactions

Article abstract of DOI:10.1021/ja2012164

Nanocarriers that combine multiple properties in an all-in-one system hold great promise for drug delivery. The absence of technology to assemble highly functionalized devices has, however, hindered progress in nanomedicine. To address this deficiency, we have chemically synthesized poly(ethylene oxide)-β-poly(ε-caprolactone) (PEO-b-PCL) block polymers modified at the apolar PCL terminus with thioctic acid and at the polar PEO terminus with an acylhydrazide, amine, or azide moiety. The resulting block polymers were employed to prepare nanoparticles that have a gold core, an apolar polyester layer for drug loading, a polar PEO corona to provide biocompatibility, and three different types of surface reactive groups for surface functionalization. The acylhydrazide, amine, or azide moieties of the resulting nanoparticles could be reacted with high efficiencies with modules having a ketone, isocyanate, or active ester and alkyne function, respectively. To demonstrate proof of principle of the potential of multisurface functionalization, we prepared nanoparticles that have various combinations of an oligo-arginine peptide to facilitate cellular uptake, a histidine-rich peptide to escape from lysosomes, and an Alexa Fluor 488 tag for imaging purposes. It has been shown that uptake and subcellular localization of the nanoparticles can be controlled by multisurface modification. It is to be expected that the modular synthetic methodology provides unique opportunities to establish optimal configurations of nanocarriers for disease-specific drug delivery.

Full text of DOI:10.1021/ja2012164

ARTICLE
pubs.acs.org/JACS
Multifunctional Surface Modification of Gold-Stabilized
Nanoparticles by Bioorthogonal Reactions
Xiuru Li, Jun Guo, Jinkeng Asong, Margreet A. Wolfert, and Geert-Jan Boons*
Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, Georgia 30602, United States
S Supporting Information
b
ABSTRACT: Nanocarriers that combine multiple properties in
an all-in-one system hold great promise for drug delivery. The
absence of technology to assemble highly functionalized devices
has, however, hindered progress in nanomedicine. To address
this deficiency, we have chemically synthesized poly(ethylene
oxide)-β-poly(ε-caprolactone) (PEO-b-PCL) block polymers
modified at the apolar PCL terminus with thioctic acid and at
the polar PEO terminus with an acylhydrazide, amine, or azide moiety. The resulting block polymers were employed to prepare
nanoparticles that have a gold core, an apolar polyester layer for drug loading, a polar PEO corona to provide biocompatibility, and
three different types of surface reactive groups for surface functionalization. The acylhydrazide, amine, or azide moieties of the
resulting nanoparticles could be reacted with high efficiencies with modules having a ketone, isocyanate, or active ester and alkyne
function, respectively. To demonstrate proof of principle of the potential of multisurface functionalization, we prepared
nanoparticles that have various combinations of an oligo-arginine peptide to facilitate cellular uptake, a histidine-rich peptide to
escape from lysosomes, and an Alexa Fluor 488 tag for imaging purposes. It has been shown that uptake and subcellular localization
of the nanoparticles can be controlled by multisurface modification. It is to be expected that the modular synthetic methodology
provides unique opportunities to establish optimal configurations of nanocarriers for disease-specific drug delivery.
’ INTRODUCTION
moiety for covalent attachment to the gold core and at the PEO
terminus with acylhydrazides, amines, and azides for coupling
with functional modules having a ketone, isocyanate, or active
ester and alkyne, respectively. The gold core of the nanoparticles
was expected to provide stability during synthesis, ensure con-
trolled degradation after cellular uptake, and offer an opportunity
for imaging. Furthermore, the apolar polyester layer of the
nanoparticles offers a loading space for apolar drugs, and the
poly(ethylene oxide) (PEO) corona provides biocompatibility
and biological stealthiness.11 The synthetic strategy is highly
modular, and from a single nanoparticle platform, many different
combinations of surface modules can be attached, opening
important new avenues to establish optimal configuration for
targeted delivery of pharmaceuticals. As a proof of principle, we
have prepared and biologically evaluated nanoparticles loaded
with a cytotoxic drug and modified with various combinations of a
cell permeation peptide for improving cellular uptake, a histidine-
rich peptide for lysosomal escape, and a fluorescent label for
quantification and have demonstrated that by multisurface mod-
ification, uptake, and subcellular localization can be controlled.
Nanomaterials, such as liposomes, organomicelles, and gold
nanoparticles, are emerging as promising devices for drug and gene
delivery.1ꢀ4 These carriers can increase longevity of a drug in the
bloodstream, solubilizea hydrophobic drug, offer controlled release
by environmental sensitive or external stimuli, and accumulate in
solid tumors by enhanced permeability and retention effect.5 The
therapeutic efficiency of nanomaterials can be further improved by
surface functionalization by, for example, a tissue targeting ligand,6
a cell-penetrating molecule,7 or a signaling peptide for organelle
targeting.8 Moreover, therapeutic targeting can be combined with
imaging by attachment of an appropriate contrast agent.4
Multifunctional nanocarriers that combine several properties,
such as tissue targeting, cell entry, organelle-specific delivery, and
imaging, in an all-in-onesystem are expected to offera multimodal
approach for enhanced efficacy of many therapeutics and
diagnostics.9,10 A major hurdle in the developmentof such devices
is, however, a lack of robust chemical technology for the prepara-
tion of nanoparticles that can be modified at their surfaces with
various functional modules and possess other properties, such as
biological stealthiness, drugloading capacity, selective release, and
imaging capabilities. Herein, we describe a versatile chemical
approach for selective surface modification of nanoparticles using
three bioorthogonal coupling reactions. To implement the new
approach, nanoparticles were prepared that have a gold core
modified with a monolayer of poly(ethylene oxide)-β-poly(ε-
caprolactone) (PEO-b-PCL) block polymers. The block poly-
mers were modified at the PCL terminus with a thioctic acid
’ RESULTS AND DISCUSSION
Chemical Synthesis of Functionalized Block Polymers.
Amphiphilic block polymers composed of PEO-b-PCL assemble
Received: February 8, 2011
Published: June 16, 2011
r
2011 American Chemical Society
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Figure 1. Chemical synthesis of multifunctional gold-stabilized nanoparticles. The structure of block polymers modified at the apolar PCL end with
thioic acid moiety for attachment to a gold core and at the PEO terminus with a functional group for post synthesis modification (a). The chemical
synthesis of multifunctional nanoparticles by a modified Brust method (b). A cartoon of the architecture of the nanopaticles in aqueous solution and the
three sequential bioorthogonal reactions for surface modification (c). The nanoparticles have an inner gold core (yellow) that provides stabilization,
controlled degradation, and opportunity for imaging with TEM. The apolar PCL layer (green) provides a loading space for apolar compounds, and the
hydrophilic PEO shell (blue) offers biocompatibility. Three different surface functional groups are present for post-synthesis modification. First, the
acylhydrazides react selectively with ketones to give a hydrazone-linked modules. Next, the amines react with isocyanates or active ester at pH of 8ꢀ9 to
give thioureas or amides, respectively, and finally, azides are reacted with alkynes in the presence of Cu(I) to provide triazole-linked modules.
in water into polymeric micelles that can carry lipophilic drugs,
such as FK506 and dihydrotestosterone.3 It was envisaged that
polymeric micelles carrying acylhydrazide, amine, and azide at the
polar PEO terminus (compounds 2ꢀ4, Figure 1) will make it
possible to modify the surface in a sequential manner with
modules containing a ketone, isocyanate, or active ester and
alkyne, respectively. These coupling reactions have been em-
ployed in bioconjugation12 and were expected to be selective
when performed in an appropriate order. The additional use of a
PEO-b-PCL derivative, containing a terminal nonreactive methyl
ether (compound 1), will make it possible to control the density
of the various reactive functionalities at the nanoparticle surface.
Previously, it was found that an appropriate density of a surface
module may be important for optimal targeting efficiency.13
Furthermore, modification of the apolar PCL terminus of the
block polymers with thioctic acid should offer an opportunity to
attach the polymers in a controlled fashion to a gold nanoparticle
core.14,15 It was expected that the resulting gold-stabilized nano-
particles would be sufficiently stable to undergo several chemical
modifications without affecting the structural integrity of the
particles. In this respect, preliminary studies had shown that
micelles formed from N3-PEO-b-PCL block polymers disassem-
ble upon attachment of charged modules, such as arginine-rich
peptides. Furthermore, we found that even when neutral modules
were employed, N3-PEO-b-PCL micelles were not sufficiently
stable to undergo several chemical modification and purification
steps. In addition to providing stability, the gold core is expected
to facilitate controlled degradation of the particles after cell entry
by displacement of the gold-linked polymers by glutathione,16
which is present at high concentration in the intracellular
environment and provides opportunities for imaging.
Functionalized polymers 1 and 2 were prepared by a strategy in
which caprolactone was polymerized using methoxy- (5) and
azido-polyethylene glycol (8) (Mn ∼ 2000 Da) as the macro-
initiator to give block polymers 6 and 9, respectively, which have a
hydroxyl at the PCL terminus that provided an opportunity to
install selectively a thioctic acid moiety. Azido-polyethylene glycol
(8) was prepared by treatment of commercially available ethylene
glycol with tosyl chloride (1 equiv) in a mixture of dichloro-
methane and pyridine, followed by reaction of the crude product
with sodium azide in DMF at 80 °C. As expected, a small amount
of a diazido derivative and starting material was formed, which
could easily be removed by traditional silica gel column chroma-
tography to give 8 in an overall yield of 58%. The structure of 8
was confirmed by a combination of matrix-assisted laser desorp-
tion/ionization time-of-flight mass spectrometry (MALDI-TOF
1
MS), Fourier-transform infrared (FT-IR), and H NMR mea-
surements. Thus, a stretching vibration at ∼2098 cmꢀ1 in the FT-
IR spectrum of 8 indicated the presence of the azido group.
Furthermore, the 1H NMR spectrum showed that the protons of
the methylene moiety adjacent to the hydroxyl had an appropriate
relative integration, and the MALDI-TOF MS spectrum of 8
showed that all molecular ions had an increase of 25 mass units
compared to corresponding signals in the MS spectrum of 7,
indicating full conversion of a hydroxyl into an azido moiety
(Figures S1 and S2, Supporting Information).
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Scheme 1. Chemical Synthesis of Compounds 1ꢀ4a
a (a)Reagents and conditions: ε-caprolactone, SnOct, 130 °C; (b) thioctic acid, DCC, DMAP, Et3N, CH2Cl2; (c): TsCl, pyridine, CH2Cl2, ii) NaN3,
DMF, 80 °C; (d) Ph3P, THF, H2O; (e) 4-(N0-tert-butoxycarbonyl-hydrazino)-4-oxo-butyric acid, DCC, DMAP, CH2Cl2, ii) 20% TFA, CH2Cl2.
Table 1. Molecular Weight of Polymers 1ꢀ4
1H NMR
SEC
polymers
Mna (Da)
Mnb (Da)
PDIb
1
2
3
4
4700
5220
5200
5288
6070
7400
7420
7500
1.42
1.38
1.35
1.30
a The number-average molecular weight (Mn) was determined by
1H NMR. b Mn and polydispersity index (PDI) were measured by
SEC; PDI is the ratio of weight-average molecular weight Mw and Mn.
The difference in the molecular weight obtained by SEC and 1H NMR is
related to differences in the hydrodynamic volume of the polymer in
1
chloroform compared to polystyrene standards. H NMR provides a
more accurate method for average molecular weight determination.
Next, a ring-opening polymerization was performed by heat-
ing a mixture of caprolactone, a catalytic amount of tin(II)
2-ethylhexanoate (SnOct) and polyethylene glycol derivatives
5 and 8 as the macroinitiator in a sealed tube at 130 °C for 24 h17
to give, after purification by precipitation in hexane, block
polymers 6 and 9, respectively. The polymerization had resulted
in the formation of a hydroxyl at the polyester terminus, which
was used to selectively introduce a thioctic acid moiety. Thus,
treatment of 6 and 9 with thioctic acid in the presence of N,N0-
dicyclohexylcarbodiimide (DDC), 4-(dimethylamino) pyridine
(DMAP), and triethyl amine (Scheme 1) lead to the formation of
functionalized block polymers 1 and 2, respectively. FT-IR
confirmed the presence of the azido moiety (2098 cmꢀ1) of
Figure 2. Modules for surface modification and chemical composition
of nanoparticles. The chemical structures of compounds 10ꢀ15 used for
surface modification of the nanoparticles (a). The chemical composi-
tions of the various nanoparticles (b).
data demonstrate that the transformation of 2 into 3 and 4 does
not alter the molecular weight distribution, indicating that the
employed reaction conditions are compatible with the ester
groups of the PCL backbone.
Nanoparticle Preparation. Gold nanoparticles A covered
with amphiphilic block polymers (Figures 1 and 2), and having
three different surface reactive functional groups, were prepared
by the addition of LiBH4 to a vigorously stirred mixture of block
polymers 1ꢀ4 and HAuCl4 in anhydrous THF. The reducing
agent induced an immediate color change from yellow to deep
purple indicating that gold nanoparticles had been formed. After
a reaction time of 3 h, methanol was added to quench the excess
of LiBH4.14,15 It was found that the resulting surface-modified
gold nanoparticles were soluble in THF, which made it possible
to remove uncomplexed polymers and inorganic salts by dialyz-
ing against THF. As a control experiment, free polymer was
dialyzed against THF, and the absence of polymeric material in
the resulting solution confirmed that the dialysis procedure
efficiently removes uncomplexed material.
1
compound 2 (Figure S6, Supporting Information), and the H
NMR spectrum of 1 and 2 showed that the proton signals of the
methylene group adjacent to the SꢀS bond of the thioctate
moiety (3.05ꢀ3.20 ppm) had appropriate relative integrations
(compared to the terminal methoxy group) indicating fully
functionalized with a thioctic acid moiety.
Compound 2 was the starting material for the preparation of
amine 3 by reduction of the azido moiety with triphenyl phos-
phine in a mixture of THF and water. Acylhydrazide-modified 4
was obtained by acylation of the amine of 3 with 4-(N0-tert-
butoxycarbonyl-hydrazino)-4-oxo-butyric acid followed by re-
moval of the Boc protecting group using 20% TFA in DCM.
Analysis of the polymers 1ꢀ4 by 1H NMR and size exclusion
chromatography (SEC) gave a number average molecular weight
(Mn) of 4700ꢀ5288 Da and a PDI of 1.30ꢀ1.42 (Table 1). The
An aqueous solution of nanoparticles was obtained by partial
concentration of the organic solution, followed by the addition of
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water and the extensive dialysis against water. Dynamic light
scattering showed that the resulting nanoparticles have a narrow
size distribution and an average diameter of 33 ( 8 nm (Figure
S14, Supporting Information). Furthermore, transmission elec-
tron microscopy (TEM) images revealed that the gold core had an
average diameter of ∼5 nm, and a signal at 2098 cmꢀ1 in the IR
spectrum (stretching vibration of azide) confirmed the presence of
azido groups (Figures S9, S11, and S12, Supporting Information).
A similar procedure was employed to prepare nanoparticles B by
only employing block polymer 1. Thermogravimetric analyses
(TGA) of A and B gave a weight ratio of gold to organic matter of
6:94, which indicates that a significant percentage of polymers is
not chemically anchored to the gold core (Figure S16, Supporting
Information). Interestingly, aggregates of gold nanoparticles were
formedwhen a PCL-PEOblock polymer lackinga disulfidemoiety
was employed in the procedure for gold-stabilized nanoparticle
preparation. Thus, the thioctic acid moiety of the block polymers is
critical for the formation of stable nanoparticles. It is possible that
polymeric molecules that are not chemically anchored to the
surface of the gold core are held in place by polymers that are
covalently linked to the gold core. In this respect, PCL is
semicrystalline at ambient temperature,15 and thus it is likely that
covalently linked and unlinked material form a strong lattice
resulting in stable nanoparticles. However, the issue of higher
than expected ratio of polymer to gold is not fully resolved, as
dialysis against THF should have removed all unbound polymer,
yet TEM images (Figures S13, S15 and S17, Supporting In-
formation) show structural heterogeneity with some nanoparticles
appearing to lack a gold core.
To establish conditions for the selective conjugation, fucoside
10, mannoside 11, and galactoside 12, having a ketone, isocya-
nate, and alkyne moiety, respectively, were sequentially conju-
gated to the gold-stabilized nanoparticles A. Thus, ketone 10 was
added to an aqueous solution of nanoparticles A for reaction with
the surface acylhydrazides to form acylhydrazone linkages. After
a reaction time of 24 h, the pH was adjusted to ∼8 by adding
aqueous NaHCO3, and the isocyanate 11 was added for reaction
with the surface amines to form stable thioureas. After stirring for
an additional 24 h, the solution was dialyzed against water, and
then a Cu(I)-catalyzed reaction was performed between the
alkyne of 12 and the azides of the nanoparticles to form
triazoles,18 and after, dialysis trifunctionalized C was obtained
(Figure 2). Analysis by light scattering and TEM indicated no
significant change in size, and morphology had occurred during
the chemical transformations (Figure S15, Supporting In-
formation). Furthermore, quantitative sugars analysis by treat-
ment of the particles with TFA to hydrolyze glycosidic linkages
followed by monosaccharide analysis by high pH anion exchange
chromatography (HPAEC) showed that the fucoside, manno-
side, and galactoside had been incorporated with efficiencies of
91, 81, and 78%, respectively (Figure S18, Supporting In-
formation). When the same procedure was applied to nanopar-
ticles B, which do not carry surface reactive functional groups, no
monosaccharides were detected, confirming that the sugars were
attached to the particles by covalent bonds. TGA showed that
negligible amounts of polymeric material had been lost during
the chemical modification and purification steps (Figure S16,
Supporting Information) indicating that the employed coupling
procedures are compatible with the chemical nature of the
nanoparticles. Furthermore, the fact that the nucleophilic amine
and acylhydrazide moieties of B were modified in highly efficient
manners supports that these functional groups had remained
intact during the preparation of the nanoparticles and had, for
example, not reacted with the ester moieties of PCL.
Next, nanoparticles modified by Alexa Fluor 488 were prepared
(D, Figure 2), containing an additional oligo-arginine peptide (E)
or a histidine-rich peptide in addition to the latter two probes (F).
The fluorophore of the particles will make it possible to quantify
cellular uptake, the oligo-arginine peptide will facilitate cellular
uptake by endocytosis,19 and the histidine-rich peptide was
expected to promote escape of the particles from endosomes
and lysosomes.20 Thus, particles DꢀF were expected to differ in
cellular uptake and subcellular location. Succinimate ester mod-
ified Alexa Fluor probe 14 was selectively linked to the amines of
A after pretreatment with acetone to block the hydrazines to give
D. Nanoparticles D were the starting material for the preparation
of E by a Cu(I)-catalyzed reaction of its azides with the oligo-
arginine peptide 15 containing an N-terminal alkyne. Nanopar-
ticles F were synthesized by first modifying A with histidine-rich
peptide 13, which contains an aldehyde function and therefore
reacted selectively with the acylhydrazides. Next, the amines of
the resulting particles were reacted with 14 under the standard
conditions, and finally, the oligo-arginine peptide 15 was installed
by Cu(I) catalyzed alkyneꢀazide cycloaddition to give gold-
stabilized and multifunctional particles F. Quantitative amino
acid analysis and fluorescent intensity measurements showed that
the three probes had been incorporated with efficiencies ranging
from 55 to 80% (Figure 2b).
Biological Examinations. Having particles DꢀF at hand,
attention was focused on cellular uptake studies. HeLa cells were
exposed to different concentrations of the nanoparticles, and after
an incubation time of 5 and 24 h, cell lysates were analyzed for
fluorescence intensity. Inaddition, cells weretreated with trypsin21
prior to hydrolysis and fluorescent measurements to account for
the possible presence of cell surface absorbed material. As
expected, the presence of the oligo-arginine peptide of nanopar-
ticles E and F led to a significant increase in cellular uptake
compared to the treatment with unmodified particles A, and a
longer incubation time resulted in increased internalization
(Figures 3a and S20, Supporting Information). Furthermore, the
treatment with trypsin did not result in a significant reduction in
fluorescent intensity indicating that most of the particles had been
internalized by the cells. Surprisingly, the presence of the lysoso-
mal escape module of particles F (histidine-rich peptide) some-
what lowered the cellular uptake (compared to particles E that
only contain oligo-arginine moieties). Similar results were ob-
tained when MCF-7 cells were employed.
The intracellular localization of the particles was studied by
TEM, which can visualize the gold core of nanoparticles. HeLa
cells exposed to unfunctionalized D showed only a small number
of gold particles (Figure S22, Supporting Information), whereas
significant uptake was observed for E and F, which are modified
by a cell permeation peptide (Figures 3b and S23ꢀS25, Support-
ing Information). As designed, particles E and F were found in
different subcellular locations; E was mainly present in vesicular
structures, whereas F was predominantly found in the cytoplasm
and attached to the nuclear membrane. Confocal microscopy
also localized E in vesicular compartments, whereas F was found
at the periphery of the nucleus (Figures 3c and S26, Supporting
Information), and thus, the TEM and confocal microscopy
studies indicate that after an incubation time of 10 h, a significant
amount of polymer is still attached to the gold core.
Next, it was examined whether the gold-stabilized nanoparti-
cles can be loaded with a lipophilic drug and whether such a drug
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Figure 3. Cellular uptake and cytotoxicity of nanoparticles. HeLa cells were exposed to D (black), E (red), or F (green) at the indicated concentrations
of gold for 5 h. After cells were washed and lysed, fluorescence (absorbance 485 nm, emission 520 nm) was measured (solid lines/closed symbols).
Samples assessed for internalization were first treated with trypsin before washing and cells lyses (dashed lines/open symbols). Calibration curves of the
corresponding Alexa Fluor 488-conjugated gold particles were used to calculate uptake (Figure S19, Supporting Information). Data represent mean
values ( SD (n = 3) (a). TEM images of representative sections of HeLa cells that were incubated with E (I, II) and F (III, IV) at 100 μg mLꢀ1 gold for 10
h. The endosome is denoted as e and the cytosol as c. Images I and II indicate that nanoparticles E are internalized by cells, and the Au NPs were mainly
confined in endosomes (arrows e1ꢀ3). Only, a very small number of Au NPs were dispersed in the cytosol (arrows c1ꢀ3). Images III and IV show that
nanoparticles F are dispersed in the cytosol (arrow 1), filaments (arrow 2), lysosomes (arrow 3), and mitochondrion (arrow 4), and were especially
found around the nuclear membrane (arrow 5) and a few inside the nucleus (arrow 6) (b). Fluorescence images of cells incubated with gold particle
preparations DꢀF. HeLa cells were exposed to D (I), E (II), and F (III) at 30 μg mLꢀ1 gold for 10 h, and after washing, fixing, and staining of the nucleus
with the far-red-fluorescent dye TO-PRO-3 iodide, cells were imaged. Merged indicate that the images of cells labeled with Alexa Fluor (488 nm) and
TO-PRO iodide (633 nm) are merged and are shown in green and red, respectively (c). Cytotoxicity assessment of 7-Et-CPT-loaded gold nanoparticles
B and G. MCF-7 cells were incubated with 7-Et-CPT-loaded gold nanoparticles B (blue) and G (purple) at the indicated concentrations of 7-Et-CPT for
8 h. After replacement of the medium, incubation was continued for 3 days. Cell viability values were normalized for untreated control cells (100%). Data
represent mean values ( SD (n = 3) (d).
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A major objective of nanomedicine is the ability to combine in
a controlled manner multiple functions into a single nanoscale
entity.2ꢀ8,10 Although previous efforts have resulted in particles
with intriguing properties, they were endowed with a limited set
of functional properties.10,24 The gold-stabilized nanoparticles
described here have as a unique feature that they can be modified
with three different surface modules. Multisurface functionaliza-
tion will make it possible to target particles with great spatial
precision, which is expected to increase the selectivity and
potency of therapeutic cargo. For example, one of the modules
can target a specific cell type, another one can facilitate cellular
uptake, and a third module can direct the particles to a specific
subcellular location. As a proof of principle, we have demon-
strated that a combined use of a cell permeation and lysosomal
escape peptide results in delivery of particles in the cytoplasm.
Furthermore, it is to be expected that the specificity of delivery
can be enhanced by attachment of several ligands that target a
specific cell type. Surface modification post particle synthesis by
employing bioorthogonal reactive groups is more attractive than
random coupling processes,25 as it avoids unwanted dispersity.
The attraction of the use of a gold core is that it stabilizes the
nanoparticles during synthesis so that the various different
modules can be attached in a controlled manner without losing
the structural integrity of the particles. Furthermore, the gold
core ensures controlled degradation after cellular uptake26 and
offers an opportunity for imaging.27 The apolar polyester layer of
the gold-stabilized particle provides a functional loading space for
apolar drugs, and PEO corona offers biocompatibility and
biological stealthiness.11 The new synthetic approach makes it
also possible to control the surface density of targeting ligand,
which in turn is important to achieve optimal selectivity.13
Finally, the synthetic methodology described here is highly
modular, and for the first time, it is possible to prepare from a
single platform (e.g., particles B), a library of nanoparticles that is
modified by different surface entities. It is to be expected that
these unique features will make it possible to readily establish
optimal configurations of targeted nanoparticle delivery devices.
Figure 4. TEM images of gold nanoparticles B incubated in the
presence (a) or absence (b) of glutathione (10 mM) at 37 °C for 1 week.
can be released after cell entry to exhibit a pharmacological effect.
For this purpose, a DMSO solution of 7-ethyl-camptothecin (7-
Et-CPT), which is a lipophilic anticancer drug that inhibits
topoisomerase I,22 was added to unmodified (B) and particles
G derivatized with a cell permeation peptide. The resulting
preparation was dialyzed, and a loading of approximately 1.5%
(w/w of nanoparticles) corresponding to ∼70% encapsulation
efficiency was determined by fluorescence measurement of a
freeze-dried sample redissolved in DMSO. Unloaded particles
did not influence cell division of MCF-7 breast cancer cells even
at a relatively high concentration of gold (up to 100 μg Au mLꢀ1
,
Figure S21, Supporting Information), whereas the drug-loaded
nanoparticles exhibit dose-dependent cytotoxicity. Furthermore,
loaded particles G were more potent (IC50 = 0.25 μM of 7-Et-
CPT) than those of the unmodified and loaded particles B (IC50
= 1.5 μM). These results indicate that after cell entry, 7-Et-CPT
is made available for inhibition. The difference in cytotoxicity of
the unmodified and modified particles was not as large as
observed for cellular uptake, and this observation is probably
due to some leakage of drug from the nanoparticles before
cellular uptake. Indeed, it was found that over a period of 24 h,
approximately 15% of drug escapes from the particles when
dialyzed against water. Many drug delivery systems exhibit such a
property and possible remedies include covalent attachment of a
drug to the polymers of a nanoparticle preparation.23 As ex-
pected, free 7-Et-CPT (IC50 = 0.2 μM, Figure S21, Supporting
Information) is slightly more cytotoxic than when loaded in the
nanoparticles because it takes a considerable period of time for
the drug to escape the particles and be available for inhibiting
topoisomerase I. In a clinical setting, slow release may be
favorable property, and one of the limitations of cell culture
experiments is that it cannot mimic such time frames.
Finally, the influence of glutathione on the polymer layer of the
gold nanoparticles was examined. Treatment of nanoparticles B
in the presence of glutathione (10 mM) at 37 °C for 1 week
shifted the surface plasmon peak maximum (λmax) from 526 to
550 nm indicating formation of bulk gold during the agglomera-
tion process. As expected, no change in λmax was observed in the
absence of glutathione (Figure S27, Supporting Information).
TEM studies also confirmed that glutathione affects nanoparti-
cles B, and clusters of Au nanoparticles were formed after 24 h,
whereas large aggregates were observed at 72 h (Figures 4 and
S28, Supporting Information). Interestingly, these aggregates
had a similar morphology as observed for particles internalized by
cells. No change in morphology was observed in the absence of
glutathione after an incubation time of 3 weeks. The release of
7-Et-CPT in the presence of gluthatione was also examined,
however fluorescence quenching of this compound by glu-
tathione, complicated these efforts.
’ ASSOCIATED CONTENT
S
Supporting Information. Synthetic procedures and char-
b
acterization of polymers 1ꢀ4; procedures for the preparation of
nanoparticles; experimental procedures for nanoparticle charac-
terization including TEM, dynamic light scattering, ζ potential,
sugar, amino acid, and biotin quantification; general cell culture
protocols; cell-based assays including cellular uptake, TEM
imaging of nanoparticle uptake and confocal microscopy; pre-
paration and determination of 7-Et-CPT-loaded nanoparticles;
measurement of 7-Et-CPT cytotoxicity; and stability assessment
of nanoparticles in the presence of gluthathion. This material is
available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
gjboons@ccrc.uga.edu.
’ ACKNOWLEDGMENT
The authors wish to thank Dr. John Shields (Center for
Advanced Ultrastructural Research, University of Georgia) and
Mrs. Mary Ard (Electron Microscopy Laboratory, College of
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Journal of the American Chemical Society
ARTICLE
Veterinary Medicine, University of Georgia) for TEM assistance,
Prof. Yuesheng Li (Changchun Institute of Applied Chemistry,
Chinese Academy of Sciences) for the SEC and TGA measure-
ments, and Dr. Heather Flanagan-Steet for assistance with
confocal microscopy studies. This research was supported by
the National Cancer Institute of the U.S. National Institutes of
Health (grant no. R01 CA88986, G.-J.B.).
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