Efficient Triplet-Triplet Annihilation-Based Upconversion for Nanoparticle Phototargeting

Article abstract of DOI:10.1021/acs.nanolett.5b01325

High-efficiency upconverted light would be a desirable stimulus for triggered drug delivery. Here we present a general strategy to achieve photoreactions based on triplet-triplet annihilation upconversion (TTA-UC) and F?rster resonance energy transfer (FRET). We designed PLA-PEG micellar nanoparticles containing in their cores hydrophobic photosensitizer and annihilator molecules which, when stimulated with green light, would undergo TTA-UC. The upconverted energy was then transferred by FRET to a hydrophobic photocleavable group (DEACM), also in the core. The DEACM was bonded to (and thus inactivated) the cell-binding peptide cyclo-(RGDfK), which was bound to the PLA-PEG chain. Cleavage of DEACM by FRET reactivated the PLA-PEG-bound peptide and allowed it to move from the particle core to the surface. TTA-UC followed by FRET allowed photocontrolled binding of cell adhesion with green light LED irradiation at low irradiance for short periods. These are attractive properties in phototriggered systems.

Full text of DOI:10.1021/acs.nanolett.5b01325

Letter
pubs.acs.org/NanoLett
Efficient Triplet−Triplet Annihilation-Based Upconversion for
Nanoparticle Phototargeting
Weiping Wang†,†,‡ Qian Liu†,†,‡ Changyou Zhan,†,‡ Aoune Barhoumi,†,‡ Tianshe Yang,§
Ryan G. Wylie,†,‡ Patrick A. Armstrong,†,‡ and Daniel S. Kohane*,†,‡
†Laboratory for Biomaterials and Drug Delivery, Department of Anesthesiology, Division of Critical Care Medicine, Boston
Children’s Hospital, Harvard Medical School, 300 Longwood Avenue, Boston, Massachusetts 02115, United States
‡David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Avenue,
Cambridge, Massachusetts 02139, United States
§Institute of Advanced Materials, School of Materials Science and Engineering, Nanjing University of Posts and Telecommunications,
9 Wenyuan Road, Nanjing 210023, People’s Republic of China
S
* Supporting Information
ABSTRACT: High-efficiency upconverted light would be a
desirable stimulus for triggered drug delivery. Here we present
a general strategy to achieve photoreactions based on triplet−
triplet annihilation upconversion (TTA-UC) and Forster
̈
resonance energy transfer (FRET). We designed PLA−PEG
micellar nanoparticles containing in their cores hydrophobic
photosensitizer and annihilator molecules which, when
stimulated with green light, would undergo TTA-UC. The
upconverted energy was then transferred by FRET to a
hydrophobic photocleavable group (DEACM), also in the
core. The DEACM was bonded to (and thus inactivated) the
cell-binding peptide cyclo-(RGDfK), which was bound to the
PLA−PEG chain. Cleavage of DEACM by FRET reactivated the PLA−PEG-bound peptide and allowed it to move from the
particle core to the surface. TTA-UC followed by FRET allowed photocontrolled binding of cell adhesion with green light LED
irradiation at low irradiance for short periods. These are attractive properties in phototriggered systems.
KEYWORDS: Caged ligands, photocleavage, photoresponsive, phototargeted, shielding, upconverting micelles
Triplet−triplet annihilation (TTA) is an upconversion
process which has been explored in nonbiomedical photo-
chemical reactions.18−22 It can be driven by low-power
noncoherent light sources (∼mW cm−2),17,23−26 which
enhances safety and is of practical and economic benefit. In
TTA-upconversion (TTA-UC; Figure 1a),17 a low-energy
photon is absorbed by a photosensitizer, which then undergoes
intersystem crossing (ISC) to form a more stable triplet state.
The triplet state energy of the photosensitizer is subsquently
transferred to a molecule that is thus excited to its triplet state.
Two such molecules in the triplet state can then combine their
energies through TTA to form one molecule in the singlet state
(with higher energy) and another in the ground state. (Those
molecules are termed annihilators because their interaction
annihilates the triplet state.) The molecule in the singlet state
can relax to the ground state, usually by emission of a higher-
energy photon. A key hypothesis of this work is that relaxation
timulus-responsiveness allows spatiotemporal control over
Snanocarrier targeting and drug release and can therefore
enhance therapy while minimizing side effects.1,2 We and others
have demonstrated the possibility of using light to control the
binding of nanocarriers to target cells (phototargeting).3−9
Conjugation of a photocleavable group (i.e., caging) deactivates
the binding activity of ligands on the nanocarrier surface.
Irradiation at the appropriate wavelength removes the caging
group and exposes the ligand, enabling binding to target cells.
Photocleavage requires short wavelength (high-energy) light,
which can cause phototoxicity.10,11 However, long-wavelength
(low-energy) light that is less toxic cannot trigger photo-
cleavage. Strategies for converting long-wavelength light into
short-wavelength light are of interest, because they may allow
photocleavage reactions with less phototoxicity. Two-photon
absorption12,13 and second hamonic generation14,15 are
examples of approaches that have been empolyed to trigger
UV-sensitive reactions with long-wavelength light. These
strategies require the use of high laser irradiances. Lantha-
nide-based upconversion16,17 requires lower irradiances but a
coherent light source is still needed.
Received: April 5, 2015
Revised: May 23, 2015
© XXXX American Chemical Society
A
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Figure 1. Schematic illustration of TTA-UC and FRET processes. (a) Jablonski diagram illustrating the mechanism of TTA-UC and FRET processes
discussed in the text. GS: the ground state. ISC: intersystem crossing. TTET: triplet−triplet energy transfer. TTA: triplet−triplet annihilation. FRET:
Forster resonance energy transfer. (b) Schematic of the phototriggering of the polymeric micellar nanoparticle by TTA-UC and FRET.
̈
to the ground state can also be achieved by Forster resonance
energy transfer (FRET).
distance between the donor and the acceptor, typically in the
range of 1−10 nm,35 this arrangement (PdOEP, DPA, and
DEACM being in the core) would allow TTA-UC energy to be
efficiently transferred to the DEACM through FRET (Figure
1), causing the removal of the hydrophobic caging group.
Uncaging will allow the hydrophilic peptide to return to the
micelle surface, allowing the binding of micelles to target cells.
Results and Discussion. Construction of Phototargeted
Polymeric Micelles. The photoresponsive cell-targeting portion
of the micellar nanoparticle, DEACM-caged cRGDfK (c[R]-
GDfK; Figure 2a), was synthesized and characterized. c[R]-
GDfK showed a broad UV−visible absorption spectrum with a
peak at 388 nm and a full width at half-maximum (fwhm) of 31
nm (Figure 2b) that overlapped with the TTA-UC emission
spectrum from DPA (annihilator/donor; Supporting Informa-
tion Figure 1), suggesting the possibility of FRET between
DPA (donor) and DEACM (acceptor). The DEACM group on
the peptide had a fluorescence emission peak at 493 nm (Figure
2b) when excited at 385 nm; this was used below to probe the
polarity of the environment surrounding DEACM. Irradiation
of c[R]GDfK with a 400 nm light-emitting diode (LED) at 50
mW cm−2 for 1 min resulted in cleavage of c[R]GDfK: on high-
performance liquid chromatography (HPLC; Supporting
Information Figure 2) the c[R]GDfK peak decreased and two
new peaks appeared that had the same elution times as free
cRGDfK and 7-diethylamino-4-hydroxymethylcoumarin
(DEACM−OH). The identity of the peaks was further
confirmed by mass spectrometry (Supporting Information
Figure 3). Approximately 86% of cleavage occurred within 30 s
of irradiation at 50 mW cm−2 (Figure 2c). At an irradiance as
low as 2.3 mW cm−2, ∼42% of c[R]GDfK was cleaved after 2
min of irradiation. The results demonstrate that the photo-
̈
Here we describe an approach whereby TTA-UC is coupled
with FRET to create an upconversion-based photoresponsive
nanoparticulate system. Incident long-wavelength light is
efficiently upconverted to high energy through TTA, and
transferred by FRET to a photocleavable group, triggering its
cleavage; some of the energy goes to emitting a high-energy
photon. Cleavage of that bond removes the photocleavable
(caging) group from a targeting ligand, restoring its binding
activity (Figure 1b).
We selected palladium octaethylporphyrin (PdOEP, excita-
tion = 532 nm) as the photosensitizer and 9,10-diphenylan-
thracene (DPA, emission = 400−500 nm) as the annihilator,
due to their high upconversion efficiency.27 PdOEP and DPA
were encapsulated in the hydrophobic core of a polymeric
micelle self-assembled from the block copolymer poly(D,L-lactic
acid)-poly(ethylene oxide) (PLA−PEG). The peptide cyclo-
(RGDfK) (cRGDfK) was conjugated on the PEG end as the
targeting group. cRGDfK was chosen because it binds
preferentially to αvβ3 integrin, which is overexpressed on
tumor cells and angiogenic endothelial cells during tumor
growth.28,29 Photocaging of this peptide with a 2-nitrobenzyl-
based group has been used to regulate cell adhesion with UV
light on solid surfaces in vitro and hydrogels in vivo.30,31 We
selected a coumarin-based group, (7-diethylaminocoumarin-4-
yl)methyl (DEACM), as the caging group, because of its high
photocleavage efficiency and relatively long absorption wave-
length32−34 (up to 455 nm, which overlaps with the emission
spectrum of DPA, enabling FRET35). We hypothesize that the
hydrophobicity of the DEACM would place the DEACM-caged
cRGDfK in the PLA core of the PLA−PEG micellar
nanoparticles. Because FRET efficiency also depends on the
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Figure 2. Photocleavage of c[R]GDfK. (a) Photocleavage of c[R]GDfK. DEACM−OH and intact cRGDfK peptide are released upon irradiation at
400 nm. (b) UV−visible absorption and fluorescence emission spectra of c[R]GDfK in PBS (pH 7.4). The excitation wavelength for the fluorescence
spectrum was 385 nm. (c) Photocleavage rate of c[R]GDfK in PBS, as determined by HPLC (detected at 390 nm), after continuous irradiation with
400 nm LED light at 2.3 mW cm−2 and 50 mW cm−2 (data are means SD; n = 4). a.u. = arbitrary units. The concentration of c[R]GDfK in all
samples was 50 μg mL−1.
cleavage reaction could generate intact cRGDfK peptide after
short periods of relatively low irradiances.
According to the proposed structural arrangement, irradi-
ation of NP-c[R]GDfK with 400 nm LED light would release
free DEACM−OH, which is more hydrophilic than the
conjugated DEACM,38 into the aqueous environment (Figure
3a). This was supported by a red shift and decrease in the
emission intensity of NP-c[R]GDfK solution upon irradiation
(Figure 3b). The red shift was attributable to the increased
polarity of DEACM’s environment and the decrease in intensity
to the quenching of fluorescence by water.37,39
To further demonstrate that DEACM was localized in the
hydrophobic core, DEACM-PLA-mPEG was synthesized
(Supporting Information Figure 6), which self-assembled into
micellar nanoparticles (NPDEACM; Supporting Information
Figures 7 and 8). Because the DEACM group was on the
hydrophobic end of the conjugate, it should be located in the
PLA core. The emission peak of NPDEACM at 464 nm in PBS
(Figure 3d) further supports the view that the DEACM in NP-
c[R]GDfK (Figure 3b; emission peak also at 464 nm) was
located in the hydrophobic PLA core. Moreover, the difference
in the emission peaks of DEACM (Figure 3d) from 464 nm
(for NPDEACM) to 494 nm (for DEACM−OH solution) is
consistent with the change in the polarity of DEACM’s
environment from the nonpolar PLA core to aqueous
conditions. These results indicate that DEACM is located in
the PLA core of NP-c[R]GDfK.
The c[R]GDfK was conjugated onto block copolymer PLA−
PEG to produce PLA−PEG-c[R]GDfK (Supporting Informa-
tion Figure 4). Phototargeted polymeric micelles were made by
the thin-film hydration method from PLA−PEG-c[R]GDfK
and PLA-methoxy PEG (mPEG) (1:4 weight ratio).36 The
resulting micellar nanoparticles, NP-c[R]GDfK, were disper-
sible in aqueous solution and had a hydrodynamic diameter of
33.0 nm (Supporting Information Figure 5).
We hypothesized that the hydrophobicity of DEACM would
cause it to localize in the PLA core of NP-c[R]GDfK (Figure
3a). That hypothesis was supported by the fact that the
fluorescence spectrum of NP-c[R]GDfK (maximum at 464 nm;
Figure 3b) was blue shifted in relation to that of free c[R]GDfK
in aqueous solution (maximum at 493 nm; Figure 2b),
suggesting a change in ambient polarity. This possibility was
supported by the finding that the fluorescence spectra of
c[R]GDfK showed a clear blue shift with decreasing solvent
polarity (Figure 3c). These data indicated that incorporation of
DEACM into NP-c[R]GDfK placed it in a less polar
environment than that of c[R]GDfK molecules in aqueous
solution,37 that is, that the DEACM was not on or in the
hydrophilic PEG shell of the micelle but that the PEG block
had looped around so that the hydrophobic DEACM38 was in
the less polar hydrophobic PLA core (Figure 3a).
The structure was investigated directly by proton nuclear
magnetic resonance (1H NMR) spectroscopy (Figure 3e). The
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Figure 3. Localization of c[R]GDfK in the hydrophobic core of NP-c[R]GDfK. (a) Schematic of light-triggered activation of c[R]GDfK on NP-
c[R]GDfK. (b) Fluorescence emission spectra of NP-c[R]GDfK and NP-c[R]GDfK irradiated for 1 min (50 mW cm−2, 400 nm) in PBS. (c)
Fluorescence emission spectra of c[R]GDfK in different solvents, including tetrahydrofuran (THF, polarity relative to water: 0.21), chloroform
(CHCl3, 0.26), dimethyl sulfoxide (DMSO, 0.44), ethanol (0.65), and water (H2O, 1.00). The arrow indicates the direction of decreasing solvent
polarity. The inset is the plot of the emission maximum (λmax) versus the relative polarity of solvents. (d) Fluorescence emission spectra of NPDEACM
(micelles with DEACM on the hydrophobic end of the block polymer) and DEACM−OH in PBS. In panels (c,d), spectra were normalized so that
their maximum intensities equaled 1. The excitation wavelength of all fluorescence measurements was 385 nm. (e) 1H NMR spectra of free cRGDfK
and different polymeric micelles in D2O. Irradiation was done with a 400 nm LED (50 mW cm−2, 1 min).
1H NMR spectrum of NP-cRGDfK, which was formed with
of NP-c[R]GDfK and that photocleavage would return it to the
surface. The absence of the characteristic peaks of DEACM−
OH in Figure 3e (NP-c[R]GDfK + 400 nm LED group) is
likely because the released DEACM−OH is present at too low
PLA−PEG-cRGDfK (Supporting Information Figure 9) and
PLA-mPEG (1:4 weight ratio), in D2O showed chemical shifts
of 7.25−7.45 ppm that were from the resonances of the phenyl
protons of cRGDfK (Figure 3e).40 Micelles formed with PLA-
mPEG only (termed plain NP) did not show those peaks. The
1H NMR spectrum of NP-c[R]GDfK also did not show those
peaks, presumably because of the restricted mobility of the
phenyl protons of cRGDfK within the PLA cores of the
micelles,40 where they were because of DEACM’s hydro-
phobicity. Irradiation at 400 nm resulted in the return of peaks
at the same positions as in NP-cRGDfK. These results confirm
that the phenylalanine in cRGDfK was located in the PLA core
1
concentration to be detected by H NMR. We cannot exclude
the possibility that some DCEAM−OH remained within the
particle core after irradiation.
Photocleavage Triggered by TTA-UC. The photosensitizer
PdOEP and the annihilator DPA were incorporated into PLA-
mPEG micellar nanoparticles (termed NPTTA) by simple
mixing during micelle formation. NPTTA produced upconver-
sion emission (Figure 4a) under irradiation at 532 nm
(commercially available green lasers; Supporting Information
Figure 10a). At low irradiances, the emission intensity of NPTTA
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Figure 4. Characterization of the FRET process in NPTTA-c[R]GDfK. (a) TTA-UC emission spectra of NPTTA and NPTTA-c[R]GDfK when excited
at 532 nm. Spectra were normalized so that their maximum intensities equaled 1. (b) Decay of fluorescence of DPA in NPTTA and NPTTA-c[R]GDfK
with excitation at 379 nm and emission at 410 nm. τ is fluorescence lifetime; τDA = lifetime in the presence of the acceptor DEACM; τD = lifetime in
the absence of the acceptor DEACM.
was proportional to the square of the irradiance and was linear
at high irradiances (Supporting Information Figure 10b), a
pattern characteristic of TTA-UC.41 The TTA-UC efficiency13
in NPTTA (see Methods in Supporting Information) was 3.8%
when irradiated at 532 nm and 150 mW cm−2. (Here,
calculation of UC efficiency included multiplication by a factor
of 2 to reflect the fact that emission of a single photon required
the absorption of two.)
energy transfer from DPA to DEACM cannot be ruled out,
FRET played the major part, given that transfer by reabsorption
is orders of magnitude less efficient than FRET.46
Photocleavage of DEACM from NPTTA-c[R]GDfK was
assessed by irradiating the micelles with a 530 nm LED in
PBS, separating the free DEACM−OH from the micelles by
centrifugal filtration (50 000 Da cutoff), and analyzing the
filtrate by HPLC. The filtrate showed a peak with the same
elution time as that of DEACM−OH and as the peak from the
filtrate of NPTTA-c[R]GDfK irradiated with a 400 nm LED
(Figure 5a). However, irradiation of a mixture of NPTTA and
free c[R]GDfK with a 530 nm LED did not cleave DEACM
from c[R]GDfK; only a peak with the same elution time as free
c[R]GDfK could be observed. These results show that the
photocleavage reaction occurring in NPTTA-c[R]GDfK was
mainly induced by TTA-UC through FRET. The time course
of photorelease of DEACM from NPTTA-c[R]GDfK under
continuous irradiation (Figure 5b), assessed by measuring the
fluorescence of the filtrates (Supporting Information Figure
13), showed that 5 min of irradiation released around 75% of
DEACM from NPTTA-c[R]GDfK. In contrast, the filtrate of the
nonirradiated NPTTA-c[R]GDfK showed relatively minimal
Phototargeted micellar nanoparticles containing PdOEP and
DPA (termed NPTTA-c[R]GDfK) with a hydrodynamic
diameter of 36.7 nm (Supporting Information Figure 11)
were produced by self-assembly of PLA−PEG-c[R]GDfK and
PLA-mPEG (1:4 weight ratio) together with PdOEP and DPA.
The analyses of TTA-UC emission spectra and fluorescence
lifetimes of DPA (Figure 4) were consistent with FRET
between DPA and DEACM in NPTTA-c[R]GDfK. When
irradiated with a 532 nm laser, the TTA-UC emission spectrum
of NPTTA-c[R]GDfK showed two peaks in the relatively long
wavelengths at 437 and 467 nm, that were not present in
NPTTA (Figure 4a), indicating that DEACM accepted the TTA-
UC energy and emitted fluorescence. The peak at 467 nm
could be attributed to the fluorescence of DEACM excited
through FRET, because the fluorescence maximum of DEACM
inside NP-c[R]GDfK is around 464 nm (Figure 3b). These
changes in the spectrum of NPTTA were not seen with a mixture
of NPTTA and free c[R]GDfK in solution (Supporting
Information Figure 12), indicating that FRET did not happen,
presumably because DEACM was far from the DPA (>10 nm)
due to the separation of the PEG (MW 3000) shell (around 10
nm in thickness43) and the free movement of c[R]GDfK
molecules in solution.
release. The photocleavage profile of DEACM from NPTTA
-
c[R]GDfK was confirmed by HPLC (Supporting Information
Figure 14).
When NP-c[R]GDfK containing the photosensitizer PdOEP
(but no DPA), termed NPPdOEP-c[R]GDfK, were irradiated at
530 nm, DEACM was not cleaved from the micelles
(Supporting Information Figure 15), which confirms that the
photosensitizer alone could not transfer its energy to DEACM
to cause photocleavage and 530 nm light irradiation could not
directly cause photocleavage too. DPA does not absorb at 530
nm (Supporting Information Figure 16), so DPA alone could
not have transferred the light energy to DEACM under
irradiation at 530 nm.
Cell Binding Triggered by TTA-UC. Phototriggered binding
of NPTTA-c[R]GDfK to cells by flow cytometry and confocal
microscopy was studied with micelles in which a hydrophilic
dye, Lissamine rhodamine B (LRB), was covalently bound to
the PLA−PEG copolymer (forming PLA−PEG-LRB; Support-
ing Information Figure 17). LRB is photostable under
irradiation with a 530 nm LED (150 mW cm−2) for at least
10 min (Supporting Information Figure 18).
In general, FRET reduces the fluorescence lifetime of donor
fluorophores.44,45 In the absence of the acceptor DEACM, the
fluorescence lifetime of the donor DPA(τD) in NPTTA was 5.99
0.04 ns (Figure 4b); in the presence of the acceptor
DEACM, the fluorescence lifetime of the donor DPA (τDA) in
NPTTA-c[R]GDfK was reduced to 3.00 0.02 ns, indicating the
existence of FRET from DPA to DEACM. The FRET efficiency
(E) was 49.9%, determined according to eq 135
τDA
τD
E = 1 −
(1)
where E indicates the percentage of excitation photons that
contribute to FRET. These results demonstrate the occurrence
of FRET in NPTTA-c[R]GDfK from DPA to DEACM, as
illustrated in Figure 1b. Although a role for reabsorption in the
Human umbilical vein endothelial cells (HUVECs) and
human glioblastoma (U87) cells, both of which express
integrins including αvβ3 integrin,47,48 were incubated for 30
E
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caging group prevented ligand-mediated micelle binding to
cells. Irradiation with a 530 nm LED (150 mW cm−2, 5 min)
increased cell binding of NPTTA-c[R]GDfK by 3.3-fold. Similar
results were obtained with U87 cells (Figure 6b). The results
indicate that the DEACM caging group was cleaved from
NPTTA-c[R]GDfK by TTA-UC energy, revealing the cRGDfK
on the micelle surface and allowing micelle binding to cells.
The cell-associated fluorescence of HUVECs and U87 cells
irradiated while incubated with NPTTA-c[R]GDfK was less than
that of cells incubated with NPTTA-cRGDfK (Figure 6). This
difference is attributable to the fact that both particle types
contain the same % (w/w) of PLA−PEG-c[R]GDfK or PLA−
PEG-cRGDfK but that with PLA−PEG-c[R]GDfK only 54.5%
of polymers bore the peptide while with PLA−PEG-cRGDfK
96.6% bore the peptide.
Light-controlled micelle binding was further confirmed by
confocal laser scanning microscopy (Supporting Information
Figure 19). Irradiation with 530 nm LED (150 mW cm−2, 5
min) induced cell binding and uptake of NPTTA-c[R]GDfK in
both HUVECs and U87 cells, while there was negligible
binding and uptake of NPTTA and nonirradiated NPTTA
c[R]GDfK.
-
We have demonstrated a phototriggered targeting system
using TTA-UC by loading a photosensitizer (PdOEP) and
annihilator (DPA) into PLA−PEG polymeric micelles function-
alized with DEACM-caged cRGDfK. Because of its hydro-
phobicity, the DEACM caging group was enclosed in the
hydrophobic PLA core, so that the distance between DPA
(donor) and DEACM (acceptor) was short, allowing FRET.
Cell binding of this nanoparticle system was enabled by a short
exposure (5 min) to a relatively low irradiance by a green light
LED at 150 mW cm−2. In contrast to other upconversion-based
approaches where coherent light is required, TTA-UC can be
triggered with noncoherent LED light. Given the limited tissue
penetration by the wavelength of light used here, this particular
TTA-UC system may be most relevant for applications where
light can readily reach its target, as in ocular drug delivery.
Research into methods to achieve excitation at longer
wavelengths is currently the focus of considerable research
interest.
Figure 5. Photocleavage of DEACM from NPTTA-c[R]GDfK by TTA-
UC. (a) HPLC traces (detected at 390 nm) demonstrating
photocleavage. For the bottom three traces, it was only the filtrates
of the samples (i.e., not the micelles) that were tested by HPLC. (b)
Cumulative fluorescent intensity (from integrated area under emission
spectra; arbitrary units) of DEACM−OH photoreleased from NPTTA
-
c[R]GDfK with 530 nm LED irradiation (150 mW cm−2) and in the
dark.
min with the following micellar nanoparticles containing 10%
PLA−PEG-LRB: NPTTA-cRGDfK, NPTTA, NPTTA-c[R]GDfK,
and NPTTA-c[R]GDfK, the latter only irradiated with a 530 nm
LED (150 mW cm−2, 5 min). Cell-associated LRB fluorescence
(a measure of particle binding) was measured by flow
ASSOCIATED CONTENT
■
cytometry (Figure 6). HUVECs incubated with NPTTA
-
S
* Supporting Information
cRGDfK exhibited 7.8-fold greater fluorescence than those
Additional information and figures. The Supporting Informa-
tion is available free of charge on the ACS Publications website
at DOI: 10.1021/acs.nanolett.5b01325.
exposed to ligand-missing NPTTA (Figure 6a), indicating the
ability of cRGDfK to target micelles to cells.49 NPTTA
-
c[R]GDfK exhibited little binding to cells, showing that the
Figure 6. Flow cytometric analysis of cell binding and uptake of micelles. (1) NPTTA, (2) NPTTA-cRGDfK, (3) NPTTA-c[R]GDfK, and (4) NPTTA
-
c[R]GDfK irradiated with a 530 nm LED (150 mW cm−2, 5 min) were incubated with HUVECs (a) or U87 cells (b) at 37 °C for 30 min. Cell
fluorescence was then measured by flow cytometry. Data are means SD (n = 4), *p < 0.001. All micelles were labeled with LRB.
F
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: daniel.kohane@childrens.harvard.edu.
(23) Liu, Q.; Yang, T.; Feng, W.; Li, F. J. Am. Chem. Soc. 2012, 134
(11), 5390−5397.
Author Contributions
W.W. and Q.L. contribute equally to this work. W.W., Q.L., and
D.S.K. designed the project. W.W., Q.L., C.Z., A.B., T.Y.,
R.G.W., and P.A. performed the experiments. W.W., Q.L., A.B.,
and D.S.K. analyzed and interpreted the data. W.W., Q.L. and
D.S.K. wrote the manuscript.
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Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
(29) Desgrosellier, J. S.; Cheresh, D. A. Nat. Rev. Cancer 2010, 10
(1), 9−22.
This work was supported by NIH Grant GM073626 (to
D.S.K.). We thank the Biopolymers & Proteomics Core Facility
at the Koch Institute, the Flow Cytometry Core Facility at
Dana-Farber Cancer Institute and the Harvard Medical School
EM Facility for technical support. We thank Dr. Lisa Cameron
at Dana-Farber Cancer Institute for her assistance with confocal
laser scanning microscopy.
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