Ligand-decorated nanogels: Fast one-pot synthesis and cellular targeting

Article abstract of DOI:10.1021/bm300201x

Nanoscale vehicles for delivery have been of interest and extensively studied for two decades. However, the encapsulation stability of hydrophobic drug molecules in delivery vehicles and selective targeting these vehicles into disease cells are potential hurdles for efficient delivery systems. Here we demonstrate a simple and fast synthetic protocol of nanogels that shows high encapsulation stabilities. These nanogels can also be modified with various targeting ligands for active targeting. We show that the targeting nanogels (T-NGs), which are prepared within 2 h by a one-pot synthesis, exhibit very narrow size distributions and have the versatility of surface modification with cysteine-modified ligands including folic acid, cyclic arginine-glycine-aspartic acid (cRGD) peptide, and cell-penetrating peptide. T-NGs hold their payloads, undergo facilitated cell internalization by receptor-mediated uptake, and release their drug content inside cells due to the reducing intracellular environment. Selective cytotoxicity to cells, which have complementary receptors, is also demonstrated.

Full text of DOI:10.1021/bm300201x

Article
pubs.acs.org/Biomac
Ligand-Decorated Nanogels: Fast One-Pot Synthesis and Cellular
Targeting†
Ja-Hyoung Ryu,‡ Sean Bickerton, Jiaming Zhuang, and S. Thayumanavan*
Department of Chemistry, University of Massachusetts, 710 North Pleasant Street, Amherst, Massachusetts 01003, United States
S
* Supporting Information
ABSTRACT: Nanoscale vehicles for delivery have been of
interest and extensively studied for two decades. However, the
encapsulation stability of hydrophobic drug molecules in
delivery vehicles and selective targeting these vehicles into
disease cells are potential hurdles for efficient delivery systems.
Here we demonstrate a simple and fast synthetic protocol of
nanogels that shows high encapsulation stabilities. These
nanogels can also be modified with various targeting ligands
for active targeting. We show that the targeting nanogels (T-
NGs), which are prepared within 2 h by a one-pot synthesis,
exhibit very narrow size distributions and have the versatility of
surface modification with cysteine-modified ligands including
folic acid, cyclic arginine-glycine-aspartic acid (cRGD) peptide,
and cell-penetrating peptide. T-NGs hold their payloads, undergo facilitated cell internalization by receptor-mediated uptake, and
release their drug content inside cells due to the reducing intracellular environment. Selective cytotoxicity to cells, which have
complementary receptors, is also demonstrated.
carriers for targeting specific cell types. Additionally,
modification of the self-assembling molecules by this method
could alter their hydrophilic−lipophilic balance (HLB) and
introduce undesirable variations in assembly behavior, thus
requiring optimization on a case-by-case basis. A class of drug
delivery vehicle, which affords surface modification of chemi-
cally cross-linked nanocarriers with reactive functional groups
in a postassembly step can address these complications. In such
a system, the nanocarrier morphology is maintained during
modification, providing versatility in surface functionalization
without compromising the structural integrity of assembly
properties of the nanoscopic scaffold. It is additionally essential
that the nanocarrier hosts stably encapsulate guest/drug
molecules until they reach the site of the targeted cells.
We have recently reported on a cross-linked nanogel system
that can preferentially address these needs.17,18 In brief, this
system is generated from self-cross-linking polymers that
exploit disulfide exchange reactions to trap noncovalently
bound guest molecules by a lock-in strategy. The template
polymers, which form nanoscale aggregates in the aqueous
phase, contain pyridyl disulfide (PDS) units to serve as both the
lipophilic moieties and cross-linking functionalities. Initial
sequestration of hydrophobic guest molecules is achieved
within the lipophilic cores of these micelle-type polymer
aggregates. However, the aggregates themselves are very leaky,
INTRODUCTION
■
The decoration of nanoscopic objects with ligands is of great
interest because of its implications in a variety of areas,
including drug delivery and sensing.1−3 When such nanoscale
platforms also exhibit the propensity to act as carriers for
noncovalently loaded cargo, these implications are even greater.
For example, ligand-modified nanocarriers that are capable of
targeting specific cells hold great promise in therapeutic
applications, such as cancer chemotherapy.4,5 Targeting
mechanisms for nanocarriers in cancer chemotherapy can be
broadly classified into two categories, active and passive. Passive
targeting is based on the propensity of nanoscopic objects, with
10−200 nm sizes, to accumulate selectively in solid tumor
tissue due to the increased permeability of the tumor
vasculature and ineffective lymphatic drainage, which is
known as the enhanced permeation and retention (EPR)
effect.6,7 Active targeting is achieved by coating the nanocarrier
with ligands that exhibit high affinity for receptors that are
overexpressed on the cancer cell surface.8,9 While either of
these targeting approaches can be effective, the most successful
approach will likely be achieved by combining the key features
of both strategies.
Targeting ligands, such as folic acid, are widely studied as
components of active targeting systems in the field of drug
delivery.10−13 In many cases, these ligands are attached to the
hydrophilic ends of assembly forming amphiphilic molecules
(e.g., block copolymers).14−16 However, the installation of such
ligands onto these molecules requires complicated synthetic
steps, limiting the versatility of ligand functionalization to tailor
Received: February 6, 2012
Revised: March 22, 2012
Published: March 29, 2012
© 2012 American Chemical Society
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Figure 1. Nanogels are nonleaky but exhibit slow cellular internalizations; polymeric micelles exhibit rapid internalizations of lipophilic drug
molecules due to the leakage of free drug from the assembly interior; and T-NGs exhibit high encapsulation stability and fast cellular internalizations,
which has a great potential for passive and active targeting drug delivery.
The fluorescence spectra were obtained from a JASCO FP-6500
spectrofluorimeter. Transmission electron microscopy (TEM) images
were taken from JEOL 100CX at 100 KV.
permitting transient guest leakage into the solvent exterior and
transfer to surrounding hydrophobic compartments.19 Intra/
interchain disulfide cross-linking of the PDS functionalities that
are buried in the lipophilic interiors of these amphiphilic
nanoassemblies lock the drug molecules in the core, providing
enhanced noncovalent encapsulation stability. Whereas cross-
linking is observed to improve encapsulation stability over the
leaky amphiphilic polymer aggregates, it is noteworthy that the
cellular uptake efficiencies of the cross-linked polymer nanogels
are rather poor. However, this feature could be advantageous,
providing an opportunity to tailor the nanogels with surface
ligands that facilitate selective uptake by specific cells (Figure
1). In this Article, we report on a fast, one-pot synthesis of
ligand-decorated nanogels. The resulting delivery vehicles
exhibit high encapsulation stability to exploit passive targeting
mechanisms and can be easily modified at their surfaces with
various ligands for the active targeting of cell surface receptors.
We show that the targeted nanogels (T-NGs), which are
prepared within 2 h by a one-pot synthesis, exhibit very narrow
size distributions and can be simply prepared with cysteine-
modified ligands including folic acid, cyclic arginine-glycine-
aspartic acid (RGD) peptide, and cell-penetrating peptide. We
demonstrate that the T-NGs hold their payloads, undergo
facilitated cell internalization by receptor-mediated uptake, and
release their drug content inside cells due to cleavage of cross-
linked disulfide bonds by the reducing intracellular environ-
ment.20−23
Random Copolymer. A mixture of 2-cyano-2-propyl benzodi-
thioate (21.0 mg, 0.095 mmol), PDSEMA (860 mg, 3.37 mmol),
polyethylene glycol monomethyl ether methacrylate (620 mg, 1.31
mmol), and AIBN (5.0 mg, 0.031 mmol) was dissolved in THF (3
mL) and degassed by performing three freeze−pump−thaw cycles.
This reaction vessel was sealed and then put into a preheated oil bath
at 60 °C for 12 h. To remove unreactive monomers, we precipitated
the resultant mixture in cold diethylether (20 mL) to yield the random
copolymer as a waxy substance. GPC (THF) Mn: 23 K. PDI: 1.38. 1H
NMR (400 MHz, CDCl3) δ: 8.45, 7.66, 7.09, 4.20−4.06, 3.90−3.36,
3.01, 2.15−1.62, 1.43−0.86. The molar ratio between two blocks was
determined by integrating the methoxy proton in the poly(ethylene
glycol) unit and the aromatic proton in the pyridine and found to be
29%:71% (PEO:PDSEMA).
Cell-Penetrating Peptide CRRR. Peptide was synthesized on 2-
chloro-trityl chloride resin by solid-phase synthesis using standard
Fmoc methodology. In brief, coupling reaction was achieved by 3
equiv Fmoc protected amino acid, 3 equiv HATU, and 3 equiv DIPEA
in DMF and monitored by Kaiser Test. Fmoc protection group was
removed by 20% piperidine in DMF. Cleavage of peptide from resin
was performed in the presence of TFA/TIS/H2O/EDT mixture.
Then, cleavage mixture was precipitated in cold ether five times to
afford crude peptide. Peptide was used without further purification.
Yield: 80%. Peptide was characterized by 1H NMR and mass
spectrometry. (See the Supporting Information.)
Cysteine-Folic Acid. The ligand was prepared on cysteine
preloaded 2-chlorotrityl resin by solid-phase synthesis. The resin was
treated with 1.2 equiv folic acid and 1.2 equiv DIPEA in DMSO for 24
h. The coupling reaction was repeated twice. Cleavage of cysteine-folic
acid was done by treating the resin with TFA/TIS/H2O/EDT mixture.
Crude product was obtained by precipitation in cold ether five times.
Yield: 78%. Characterization was followed by 1H NMR and mass
spectrometry. (See the Supporting Information.)
T-NGs Preparation. The polymer (10 mg) was dissolved in water
(1 mL), and hydrophobic dyes (0.1 mg of DiO or DiI) or 0.5 mg
paclitaxel (PTX) in acetone (100 μL) was added and the mixture was
stirred for 6 h at room temperature, open to the atmosphere to allow
the organic solvent to evaporate. It was placed in a vessel preheated at
60 °C for 10 min; then, a measured amount of DTT was added. Then,
the mixture was stirred for 30 min to allow for cross-linking. To
modify the nanogels surface, we added the ligand (5 mg), CRRR,
cRGD, or cysteine-modified folic acid and then stirred the mixture for
another 1 h. The resulting nanogels were purified by filtration, and
unattached excess ligand was removed from the nanogel solution by
ultrafiltration (triplicate) using a membrane with a molecular weight
cutoff of 10 000 g/mol (Amicon Ultra cell-10K).
EXPERIMENTAL SECTION
■
General. 2,2′-Dithiodipyridine, 2-mercaptoethanol, polyethylene
glycol monomethyl ether methacrylate (MW 450), D,L-dithiothreitol
(DTT), 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbo-cyanine per-
chlorate (DiI) and 3,3′-dioctadecyloxacarbocyanine perchlorate
(DiO), folic acid, cyclo(Arg-Gly-Asp-D-Phe-Cys) peptide, 2-cyano-2-
propyl benzodithioate, and other conventional reagents were obtained
from commercial sources and were used as received, unless otherwise
mentioned. Polymers were synthesized using RAFT polymerization
and then purified by precipitation. Pyridyl disulfide ethyl methacrylate
(PDSEMA) was prepared using a previously reported procedure.24 1H
NMR spectra were recorded on a 400 MHz Bruker NMR
spectrometer using the residual proton resonance of the solvent as
the internal standard. Molecular weights of the polymers were
estimated by gel permeation chromatography (GPC) using PMMA
standard with a refractive index detector. Dynamic light scattering
(DLS) measurements were performed using a Malvern Nanozetasizer.
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Scheme 1. (a) Schematic Representation of the One-Pot Synthesis of T-NGs, (b) Chemical Structure of Polymer and T-NGs,
and (c) Cysteine-Containing Targeting Ligands
Figure 2. (a) Dynamic light scattering of polymer aggregates (10 mg/mL) at 60 °C and T-NG (10 mg/mL) at room temperature. UV absorbance of
pyridothione (b) during nanogel synthesis (0.04 mg/mL) and (c) ligand modification with CRRR (0.05 mg/mL). (d) Zeta potential of nanogel
(NG) (1 mg/mL) and NG-RRR (1 mg/mL). (e) AFM image of T-NG (2 μm × 2 μm).
Cross-Linking Density and Surface Modification. To
determine the cross-linking density, we first treated the polymer in
water with the requisite amount of DTT (20 mol % compared with
PDS groups) and stirred the mixture at 60 °C. UV−visible
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spectroscopic measurements were performed with samples of this
solution diluted 10 times. Once this was measured, we calculated the
amount of pyridothione based on its known molar extinction
coefficient (8.08 × 103 M−1 cm−1 at 343 nm).25 The percentage of
cross-linking was calculated by assuming that the formation of a single,
cross-linking disulfide bond would require cleavage of two PDS units
and produce two pyridothione molecules. The attachment of the
ligand was evaluated by further increase in pyridothione absorption.
Cell Culture. The cell viabilities of the nanogels were tested with
293T cells. 293T cells were cultured in T75 cell culture flasks using
Dulbecco’s modified Eagle medium/nutrient mixture F-12 (DMEM/
F12) with 10% fetal bovine serum (FBS) supplement. The cells were
seeded at 10 000 cells/well/200 μL in a 96-well plate and allowed to
grow for 24 h under incubation at 37 °C and 5% CO2. These cells
were then treated with nanogels of different concentrations and were
incubated for another 24 h. Cell viability was measured using the
Alamar Blue assay with each data point measured in triplicate.
Fluorescence measurements were made using the plate reader
SpectraMax M5 by setting the excitation wavelength at 560 nm and
monitoring emission at 590 nm on a black well plate. The toxicities of
PTX-encapsulated polymer aggregates, NG, and NG-RGD were tested
against the MCF7 and HeLa cell line. These cells were then treated
with the PTX-loaded polymer carrier solutions, all of 10 μM PTX
concentration, and were incubated for 12 h. After 12 h, the solution
was removed and replaced with fresh supplemented medium. The cells
were further incubated for 24 h, leading to a full incubation time of 36
h after sample addition. Cell death was measured by the Alamar Blue
assay in triplicate.
The cysteine-containing ligand, cysteine-triarginine (CRRR)
peptide, was added to this reaction mixture to modify the
surface and attachment of the ligand was evaluated by further
increase in pyridothione absorption. The peak became
saturated within 60 min, and it was calculated that 17 mol %
of total PDS is reacted with CRRR (Figure 2c). This calculation
led us to estimate that there were about 7000 ligands per T-
NG. (See the Supporting Information.) One can question
whether such a thiol-containing ligand will cleave the cross-
linking disulfide bonds, resulting in size change, nanogel
disassembly, and leakage of hydrophobic guest molecules
during ligand modification. We found that T-NG retained its
size from the polymer aggregate at high temperature (Figure
2a), and the absorption intensity of hydrophobic dye, 3,3′-
dioctadecyloxacarbocyanine (DiO), which is encapsulated prior
to cross-linking and surface modification, was not changed,
indicating that the nanogels were sufficiently stable to retain
their guest molecules during this functionalization period
(Figure 2c). We confirmed surface modification of the nanogels
by monitoring the change of surface charge. As shown in Figure
2d, the nanogels modified with CRRR (NG-RRR) showed
positive zeta potentials (+4 mV), whereas the nanogels showed
negative zeta potentials (−20 mV) before surface modification.
It is surprising that these nanogels exhibit negative zeta
potentials because their surface is decorated with neutral
poly(ethylene glycol) units. This is not well understood at this
time. However, we also note that this is apparently not unusual,
as similar observations have been previously reported.26−28 The
resulting T-NGs showed narrow size distribution. Atomic force
microscope (AFM) images reveal well-defined spherical
structures with very uniform size. The size is ∼50 nm in
diameter, which may be ideal for the passive targeting
applications.29 Additional ligands, including cysteine-modified
folic acid and cysteine-containing cyclic arginine-glycine-
aspartic acid (RGD) peptides, were also linked by this synthetic
method to make T-NGs, NG-FA, and NG-RGD, respectively.
(See the Supporting Information.)
Several features are noteworthy in the modified synthetic
method, relative to our prior report17,18 on the nanogel
synthesis: (i) The current method allows for obtaining these
nanogels in very short amount of time. (ii) It also allows for
decorating the nanogels with targeting ligands in a single pot,
and the precursor polymer is cross-linked into a nanogel and
decorated with targeting ligands in less than 2 h. (iii) The
resultant nanogels exhibit narrow polydispersity. (iv) These
nanogels also exhibit enhanced encapsulation stability (vide
infra).
Encapsulation Stability. To investigate the noncovalent
encapsulation stabilities of the T-NGs, we probed the dynamics
of guest interchange in the nanocarriers using a previously
reported fluorescence resonance energy transfer (FRET)
experiment.30 Two lipophilic FRET pair dye molecules, 3,3′-
dioctadecyloxacarbocyanine (DiO, donor) and 1,1′-dioctadecyl-
3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI, accept-
or), were independently encapsulated in the nanogels. In this
experiment, mixing of the prepared solutions will result in
either FRET development due to dye molecule interchange
among leaky nanocarriers or lack of FRET development due to
stable encapsulation of the dyes within the nanogel cores.
Whereas significant and rapid FRET evolution was observed in
the case of the polymer aggregates, the cross-linked nanogels
showed no significant peak shift, indicating their high
encapsulation stabilities (Figure 3). Note that these nanogels
Laser Scanning Confocal Microscopy. The laser confocal
experiment was performed with different cell lines. Each cell line
was cultured in T75 cell culture flask containing DMEM/F12 with
10% FBS supplement. The cells were seeded at 10 000 cells/100 μL in
coverslip-bottomed Petri dishes and allowed to grow for 1 day at 37
°C in a 5% CO2 incubator. The cells in 2 mL of culture medium were
treated with polymer aggregates, nanogels, or T-NGs (0.1 mg/mL)
containing dyes and incubated at 37 °C for different time intervals
before the cells were monitored by confocal microscopy.
RESULTS AND DISCUSSION
■
Preparation of T-NG. T-NGs were prepared through the
lock-in strategy using a random copolymer that contains
oligoethylene glycol (OEG) units (29%) and pyridyldisulfide
(PDS) moieties (71%) as side-chain functionalities (Scheme 1).
At elevated temperatures, the polymer formed larger aggregates
(42 nm in diameter) than at room temperature (24 nm in
diameter), likely due to intermolecular associations between the
polymer chains caused by the lower critical solution temper-
ature (LCST) behavior of the OEG units (Figure 2a). We
hypothesized that preparation at high temperatures would
facilitate the cross-linking reaction and that subsequent addition
of cysteine-containing ligands would enable a fast, one-pot T-
NG synthesis. The cross-linking and surface modification
reactions were observed to finish within a very short time
period. Figure 2b, which traces the production of the
pyridothione byproduct of disulfide bond formation during
nanogel synthesis, plateaus within 20 min. A deficient amount
of dithiothreitol (DTT) (20 mol % against the precursor PDS
groups) cleaved a corresponding amount of PDS function-
alities, generating free thiols that can then react with remaining
PDS functionalities in the polymer chain (both intra- and
interchain) to provide the cross-linked polymer nanogel. On
the basis of pyridothione release, the actual cross-linking
density was found to be 13 to 14% (39 mol % of PDS is
consumed), which is very close to the theoretically calculated
cross-linking density of 14%.
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contrast, the nanogels showed no emission, indicating that the
nanogels are not internalized in this short time period. This
result suggests that the dyes, from the leaky amphiphilic
polymeric aggregates, might be easily moved from the carrier to
cellular membrane. To confirm this, we performed in situ
FRET experiments with HeLa cells using the amphiphilic
aggregates in which the FRET pair DiO (green emission) and
DiI (red emission) were coencapsulated. In this case, if the
polymeric aggregates were internalized through the membrane,
then FRET (red emission, 585−615 nm spectral filter) would
be continually observed in the cytosolic interior within a short
time period. After 3 h of incubation, we found that green (no
FRET, 505−520 nm spectral filter) fluorescence is dominant
inside the cell by confocal microscopy (ex = 488 nm),
indicating that the two dyes are not close together within the
assembly core but that they have released from the micelles
(Figure 4c). This indicates that liphophilic guests loaded inside
micellar aggregates can be transferred into any cells, resulting in
nonspecific delivery. Therefore, high encapsulation stability is
essential to prevent premature, nontriggered release and
achieve selective delivery of the drug molecules to target cells.
Selective Cellular Internalization of T-NG. The cross-
linked nanogels show highly stable encapsulation, avoiding
undesirable drug leakage at off-target sites. However, the bare
nanogel also suffers from very slow internalization. Such slow
internalization seems to be a hurdle for drug delivery
application at first sight. However, it may be that the delayed
internalization of these nanogels poses an advantage in
achieving selective uptake through active targeting. In an
Figure 3. FRET evolutions from DiO and DiI encapsulated within (a)
polymer aggregates and (b) nanogels.
stably encapsulate these molecules even at relatively low cross-
link densities.
Cellular Internalization of Polymer Micelle and NG.
Next, we were interested in investigating the differences in
cellular internalization of the polymer aggregates, nanogels, and
T-NGs loaded with hydrophobic dye molecules. We hypothe-
sized that any leakiness of the nanocarriers would result in the
transport of their lipophilic cargo to surrounding cellular
membranes due to the hydrophobic interactions. This would
permit the cellular internalization of free dye prior to that of the
dye molecule’s nanocarrier. To test this, we added the polymer
aggregates and nanogels, containing DiO as a hydrophobic dye,
into three different cell cultures (293T, MCF-7, and HeLa cell
lines). The uptake was then monitored by tracing the dye’s
fluorescence using confocal microscopy (Figure 4a,b). In the
case of the polymer aggregates, intense green emission inside
the cells was observed within 6 h across all cell lines. In
Figure 4. Cell internalization of DiO which is encapsulated in polymer aggregates (a) and NG (b) with different cell lines (left: 293T, middle:
MCF7, right: HeLa, scale bar = 10 μm). (c) In situ cell internalization of hydrophobic dye by FRET (left: DiO channel, middle: DiI channel, right:
overlap of both channels). Absence of FRET inside the cell indicates that the dyes were simply transferred into the cells without polymer aggregates.
Each cell line was cultured in DMEM/F12 with 10% FBS supplement.
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Figure 5. Confocal microscopy images of T-NGs containing DiO with different cell lines: (a) 293T, (b) MCF7, (c) HeLa, and (d) SKOV3 (left:
NG-RRR, middle: NG-FA, right: NG-RGD, scale bar = 10 μm). Each cell line was cultured in DMEM/F12 with 10% FBS supplement.
attempt to promote selective and rapid internalization into
target cells, we decorated the surfaces of the slow-internalizing
nanogels with specific ligands. To probe the versatility of ligand
functionalization, we used three different ligands: CRRR,
CRGD, and cysteine-modified folic acid. RGD is the ligand
of the integrin αvβ3, which is involved in tumor angiogenesis
and metastasis and is overexpressed in many solid tumor types
such as breast, prostate, and ovarian cancers.31 Folic acid is one
of the most widely used ligands in targeted delivery research
because folate receptors (FR) are notably overexpressed on the
surfaces of many cancer cells such as ovarian, lung, and uterine
tumors.32 RRR can be considered to be a positive control in
this experiment, as this is a model cell-penetrating peptide and
is thus rapidly and nonselectively taken up by most cell types.
These arginine-rich peptides are considered to be mimics of the
well-studied TAT peptides, which are known for translocating
molecules and nanoscale objects across the cellular mem-
brane.33 The nanogels, which were modified with CRRR,
CRGD, and cysteine-modified folic acid, are referred to as NG-
RRR, NG-RGD, and NG-FA, respectively. To investigate target
selectivity, we explored cellular uptake in varied cell lines: 293T
kidney cells are normal cells used as a control, MCF7 is a
slightly FR-positive tumor cell line and is negative to RGD,
whereas SKOV3 and HeLa cells are positive to both ligands.
To see selective cellular internalization by receptor-mediated
endocytosis, ligand-decorated nanogels encapsulating DiO as a
model hydrophobic drug molecule were incubated with various
cell lines for 6 h and then qualitatively examined through
confocal microscopy (Figure 5). In the case of NG-RRR, high
green emission was observed inside cells in all cell lines within
just 2 h, suggesting that the nanogels were internalized rapidly
because of the highly positive cell-penetrating peptide, RRR. In
the case of NG-FA, the nanogels exhibit fast cellular uptake
with SKOV3, HeLa, and MCF7 cells in which FR is
overexpressed. However, the FR-negative 293T cells show
negligible cellular uptake of the folic-acid-decorated nanogel.
Similarly, with NG-RGD, HeLa and SKOV3 exhibit high
internalization, as these cells are considered to overexpress
integrin αvβ3. However, MCF7 and 293T cells, which are
negative to integrin, show little internalization. Taken together,
these results strongly support the selective internalization of the
various T-NGs through receptor-mediated endocytotic path-
ways. Therefore, this system has great potential as a targeted
drug delivery carrier.
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Selective Delivery of Chemotherapeutic Drug. Finally,
we were interested in investigating whether the observed target
selectivity would translate to differential cell kill efficiency of
these ligand-coated nanogels encapsulating a chemotherapeutic
drug molecule, PTX. The PTX-loaded polymeric aggregates
(polymer-PTX), NG (NG-PTX) and NG-RGD (NG-RGD-
PTX), were added to MCF7 cells and HeLa cells, and the
synthesizing the ligand-decorated nanogels, combined with the
intrinsic encapsulation stability and targeting capabilities of the
formed T-NGs, should open up new avenues in targeted drug
delivery for cross-linked polymer nanogels.
ASSOCIATED CONTENT
■
S
* Supporting Information
Spectral characterization data, turbidity experiment and plot of
the absorption maximum of pyridothione, plot of FRET ratio
versus time, UV−vis absorbance of T-NG, and in vitro toxicity
of polymer, empty nanogels, RGD-coated nanogels. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: thai@chem.umass.edu.
■
Present Address
‡Department of Chemistry and Center for Bio-Responsive
Assembly, Seoul National University, Seoul 151-747, Korea.
Figure 6. In vitro toxicity of PTX-loaded NG-RGD with MCF7 and
HeLa cells after 36 h of incubation. The loading amount of PTX is 5
wt % for polymer and NG. PTX (10 μM) is used for all experiments.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
extent of cell death was investigated after 36 h. As shown in
Figure 6, free PTX and polymer-PTX were highly toxic in both
cell lines. This supports our hypothesis that the polymeric
micellar aggregates have a leaky nature, which leads to free drug
being translocated through the cellular membrane. In contrast,
NG-PTX showed less toxicity, compared with both free-PTX
and polymer-PTX. This is consistent with the observed slow
internalization of nanogels, which should result in a reduced
amount of chemotherapeutic inside the cells. Remarkably, NG-
RGD-PTX showed different cell-killing efficiency in two
different cell lines. Whereas MCF7 showed similar toxicity
with NG-PTX, HeLa showed high toxicity that is similar to
free-PTX, indicative of the selective delivery of PTX to this cell
line. Together with the cellular internalization experiments, this
result demonstrates that NG-RGD is highly internalized into
the integrin receptor overexpressing HeLa cells, thus enhancing
the efficiency of chemotherapeutics by promoting selective drug
delivery.
This work was partially supported by DARPA and the NSF
Center for Hierarchical Manufacturing. We are also supported
by the NSF-MRSEC facility at the University of Massachusetts
for the infrastructure used in characterizations of the materials.
DEDICATION
■
†This paper is dedicated to Professor Jeffrey S. Moore on the
occasion of his 50th birthday.
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CONCLUSIONS
■
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In summary, we describe here a facile synthetic method for the
preparation of nanogels. We show that: (i) These nanogels can
be prepared in under 2 h from their polymeric precursor; the
reaction time includes the ligand decoration step. (ii) These
nanogels exhibit high encapsulation stability of lipophilic guest
molecules. (iii) The facile ligand functionalization possibility
can be utilized to decorate these nanogels with cell-targeting
ligands. (iv) Whereas the unfunctionalized nanogels are taken
up very poorly by various cells, the ligand-decorated nanogels
exhibit facilitated receptor-dependent cellular uptake, as
demonstrated by selective uptake of RGD- and folic-acid-
decorated nanogels by cells overexpressing integrin and FRs.
(v) Functionalization of the nanogels with cell-penetrating
peptides caused rapid nonspecific uptake by the cells,
independent of the receptor. (vi) The selective internalization
capability can be translated to delivering a chemotherapeutic
drug molecule specifically to a specific receptor-rich cell.
Overall, the reported versatile one-pot synthetic method for
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