Photoswitchable nanoparticles for triggered tissue penetration and drug delivery

Article abstract of DOI:10.1021/ja211888a

We report a novel nanoparticulate drug delivery system that undergoes reversible volume change from 150 to 40 nm upon phototriggering with UV light. The volume change of these monodisperse nanoparticles comprising spiropyran, which undergoes reversible photoisomerization, and PEGylated lipid enables repetitive dosing from a single administration and enhances tissue penetration. The photoswitching allows particles to fluoresce and release drugs inside cells when illuminated with UV light. The mechanism of the light-induced size switching and triggered-release is studied. These particles provide spatiotemporal control of drug release and enhanced tissue penetration, useful properties in many disease states including cancer.

Full text of DOI:10.1021/ja211888a

Article
pubs.acs.org/JACS
Photoswitchable Nanoparticles for Triggered Tissue Penetration and
Drug Delivery
Rong Tong,†,‡ Houman D. Hemmati,‡ Robert Langer,† and Daniel S. Kohane*,‡
†Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts
02139, United States
‡Laboratory for Biomaterials and Drug Delivery, Department of Anesthesiology, Division of Critical Care Medicine, Children’s
Hospital Boston, Harvard Medical School, 300 Longwood Avenue, Boston, Massachusetts 02115, United States
S
* Supporting Information
ABSTRACT: We report a novel nanoparticulate drug delivery
system that undergoes reversible volume change from 150 to
40 nm upon phototriggering with UV light. The volume
change of these monodisperse nanoparticles comprising
spiropyran, which undergoes reversible photoisomerization,
and PEGylated lipid enables repetitive dosing from a single
administration and enhances tissue penetration. The photo-
switching allows particles to fluoresce and release drugs inside
cells when illuminated with UV light. The mechanism of the
light-induced size switching and triggered-release is studied. These particles provide spatiotemporal control of drug release and
enhanced tissue penetration, useful properties in many disease states including cancer.
effects.11 Deep penetration of nanoparticles in tumors is
INTRODUCTION
Controlled release technology is expected to have a profound
■
necessary to enhance their therapeutic effect.12
impact in many medical fields including oncology.1 The
Another significant drawback of commercially available drug
delivery NPs is that drugs are released at a predetermined rate
incorporation of chemotherapeutic agents in nanoparticle
irrespective of patient needs or changing physiological
(NP) delivery vehicles has improved drug solubility, reduced
circumstances. A triggerable drug delivery system would allow
clearance, reduced drug resistance, and enhanced therapeutic
effectiveness.2 With controlled release NP systems, a single
repeated on-demand dosing that would be adaptable to the
patients’ regimen and allow multiple dosages from a single
dose can sustain drug levels within the desired therapeutic
administration.13 It might also help address the potential
importance of timing on therapeutic effect (“chrono-admin-
istration”) in the treatment of cancer,14 a concept that is
receiving burgeoning recognition, for example, the periodicity
of VEGF expression in breast cancer regulates tumor cancer
vascular permeability.15 Another clinical example of the
importance of timing is that periodic infusion of angiotensin
II via the tail vein can enhance macromolecular delivery into
tumors by overcoming the barrier of elevated interstitial fluid
pressure within tumors; no such increase of macromolecular
uptake occurs either by an acute or chronic increase in blood
pressure induced by angiotensin II.16 Furthermore, the
permeability of many tumor models varies with time and in
response to treatment, so that vascular pore sizes vary greatly,
resulting in heterogeneous NP extravasation and drug delivery
efficacy.5,17 On-demand drug release from NPs accumulated in
tumors could allow in situ chrono-administration, potentially
increasing drug retention in cancers, maximizing tumor killing
and minimizing metastatic spread.
range for long periods in various diseases (e.g., diabetes3 or
cancer4). Several nanoparticulate therapeutics, for example,
Doxil (∼100 nm PEGylated liposome loaded with doxorubicin)
and Abraxane (∼130 nm albumin-bound paclitaxel nano-
particles), have been approved by the FDA, and have shown
improved pharmacokinetics and reduced adverse effects
compared to their parent drugs.5 However, currently approved
nanomedicines provide modest survival benefits for patients,5,6
perhaps in part because of poor tumor penetration.
Nanoparticle size is one crucial determinant of accumulation
and penetration into tumor tissue.7 Nanoparticles with sub-100
nm sizes are optimal for the enhanced permeation and
retention (EPR) effect for passive tumor targeting.8 However,
physiological barriers, such as the dense interstitial matrixa
complex assembly of collagen, glycosaminoglycans, and
proteoglycanshinder the delivery of drugs throughout the
entire tumor.9 For example, Doxil (∼100 nm) is found trapped
near the tumor vasculature.10 Although the small size
(molecular weight = 544 Da) of doxorubicin released from
Doxil allows rapid diffusion, doxorubicin cannot migrate far
from the particles due to rapid uptake of doxorubicin by
perivascular cells, which results in heterogeneous therapeutic
Received: December 20, 2011
Published: March 5, 2012
© 2012 American Chemical Society
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Here, we have developed a photoswitching nanoparticulate
system that uses light as the remote means of triggering both
on-demand drug release and reversible changes in particle
volume to enhance tissue penetration.
hydroxy-5-nitrobenzaldehyde with substituted 2,3,3-trimethyl-
3H-indolium iodide (Figure S1a). NPs were initially prepared
by direct nanoprecipitation of SP alone (an extensively used
simple method for the preparation of NPs with therapeutic
agents embedded in the hydrophobic matrices).26 An
acetonitrile solution of SP-C9 (10 mg/mL) was nano-
precipitated into water (final acetonitrile/water = 1/40, v/v),
resulting in NP sizes of 198.1 2.5 nm with a polydispersity of
0.09 0.02, determined by dynamic light scattering (DLS, N =
5, Table S1).
Irradiation of the SP-C9 NPs with UV light (365 nm,
intensity ∼1 W/cm2, ∼ 3.1 × 10−6 einstein) led to
photoisomerization and the subsequent conversion of hydro-
phobic SP-C9 to amphiphilic MC-C9, and a change in the sizes
of the NPs. The irradiated NPs had a bimodal size distribution
(Figure S1b), with one peak at 39.6 3.0 nm (N = 5, 99.1% of
number population, determined by DLS; attributable to NPs
assembled by MC-C9), and another at 202.1 nm (0.9% of
number population; attributable to NPs formed with SP-C9).
After irradiation, the colorless NP solution became purple, with
a strong Vis absorption band characteristic of MC-C9
(maximum absorption wavelength λmax = 560 nm; Figure
S1c,d). Nanoprecipitation of a SP analogue with a shorter alkyl
chain, SP-C7, produced NPs that did not undergo a significant
size change upon UV irradiation (Table S1).
RESULTS AND DISCUSSION
■
Photochromic properties are controllable light-induced changes
in color or reversible photoexcited transformations between
two isomers.18 There has been intensive investigation of
photochromic materials for applications from sunglasses to
optically rewritable data storage,19 optical switching,20 and
chemical sensing.21 The photoswitchable NPs developed here
were composed of spiropyran (SP, a family of photochromic
molecules, Figure 1a,b) and lipids. SP consists of a nitro-
SP-C9 NPs formed in aqueous solution aggregated when
introduced into PBS (Table S1), presumably due to salt-
induced screening of electrostatic repulsive forces between
particles.27 In addition, the NPs had low actual drug loadings wt
% (loading wt % < 1%) and efficiencies (<13%; Table S2). The
loading efficiency did not increase in NPs made of SPs with a
longer alkyl chain (SP-C18, Table S2). Higher drug loading of
delivery vehicles is desirable for optimal therapeutic effect, to
enhance the potency of NPs that reach the tumors.28
Figure 1. (a) Structure and photoisomerization reaction between
spiropyran (SP) and merocyanine (MC). (b) Abbreviations for SP and
MC derivatives. (c) Scheme of photoswitching SP NPHs composed of
SP-C9 and DSPE-PEG. Yellow oval, SP molecule; blue line, the alkyl
chain (R) in SP; red, lipid part; green line, PEG. SP NPHs are
converted to MC NPHs (purple sphere: MC molecule) by UV light
irradiation; the reversible photoisomerization from MC NPHs to SP
NPHs happens in dark but is accelerated by visible light (500−600
nm).
To improve the stability and loading efficiencies of NPs while
maintaining the NPs’ photoswitching properties, we produced
hybrid SP/lipid-polyethylene glycol (PEG) NPs (termed NPHs;
Figure 1c) using a rapid ultrasonication method.29 An
acetonitrile solution of SP-C9 (1 mg/mL) was slowly added
into a 4 wt % ethanolic aqueous solution containing lecithin
and 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-
carboxy(polyethylene glycol)-5000 (DSPE-PEG, [SP-C9]/
[DSPE-PEG]/[lecithin] = 32/16/1), followed by addition of
water to adjust the organic/aqueous solution volume ratio to 1/
10. After sonication for 8 min and filtration of the organic
solvent, SP NPHs were obtained with an average hydrodynamic
diameter of 143.2 2.1 nm and a polydispersity of 0.03 0.01
(N = 5, Figure 2a). SP-C9 was not detected by HPLC in the
filtrate after repetitive washing of the NPHs by ultra-
centrifugation, indicating that SP-C9 was completely incorpo-
rated into NPs (Figure S2a,b). After UV illumination (30s,
∼100% conversion to MC), the absorption band of the NPHs
moved to a λmax at 551 nm (Figure 2b). As with the nonhybrid
NPs, UV irradiation of NPHs induced a size change (to 47.1
1.3 nm, polydispersity of 0.05 0.02, N = 5). These results
confirmed that both the photochromic properties of SP-C9 and
light-triggered size change were maintained in the SP NPHs.
MC NPH reverted to SP NPH in darkness or by Vis light,
with an accompanying increase in volume (Figure 2a).
Consequently, there could be inaccuracies in measuring MC
NPH size by relatively slow techniques such as DLS. To confirm
particle shrinkage after irradiation (Figure 2a), we produced
NPH containing MC−CN, a similar but relatively stable MC
benzopyran and an indoline moiety with orthogonal orientation
(Figure 1a). Both moieties absorb in the ultraviolet spectrum
independently.22 Ultraviolet light (UV, 365 nm) induces ring-
opening in the pyran to form merocyanine (MC, Figure 1a).
The nitrophenol and indoline chromophores are merged to
form one large planar π-system, leading to intense absorption in
the visible (Vis) spectral region (500−600 nm).23 The
zwitterionic MC form is less stable than the hydrophobic SP
form and undergoes spontaneous ring-closing back to SP in the
dark that is accelerated by photoexcitation of MC in the Vis
absorption band.18a The polarity or hydrophilicity changes of
SP molecules that accompany their photoisomerization have
been suggested to alter microenvironments within polymers
and supermolecular assemblies such as Langmuir−Blodgett
films, micelles, and liposomes.20b,24 We hypothesize that SP
isomerization upon irradiation would lead to hydrophilicity
changes which would switch the NPs’ physical assembly
properties and trigger drug release. Of note, micromolar
concentrations of SP derivatives are reported to have minimal
cytotoxicity in macrophages, gastric cells, and epithelial cells
after exposure for 72 h.25 These properties suggest that SP is a
suitable base material for light-responsive NPs for triggered
release.
Formulation of Photoswitchable NPs with Light-
Triggered Size Changes. SP derivatives bearing hydrophobic
alkyl chains (Figure 1a,b) were synthesized by coupling 2-
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in PBS (Figure S2c). The stability of NPHs was also evaluated
in serum by monitoring the absorbance change at 560 nm,
since nanoparticles cannot be accurately detected in dense
serum solutions by DLS.32 No significant aggregation was
observed over 4 h.
For eventual clinical translation, it has to be possible for NPs
to be stable during manufacturing, storage, and trans-
portation.33 SP NPHs were lyophilized for 48 h with bovine
serum albumin (BSA, NP/BSA = 1/15, w/w), a known
lyoprotectant reagent,34 then stored at −20 °C for over one
month. The subsequent reconstitution of lyophilized SP NPH
in PBS did not significantly change the NPH sizes and
photochromic properties (Figure S4). Lyophilization of SP
NPH in water (without albumin) led to micrometer-sized,
nondispersible aggregates upon reconstitution in PBS. Since
albumin is used clinically, this lyoprotection strategy may be
useful for potential translational of SP NPs.
To examine whether this formulation could be used to form
NPs containing a broad range of compounds, we tested the
ability to encapsulate rhodamine B, coumarin 6, cyanine 5
(Cy5), paclitaxel, docetaxel, proparacaine, and doxorubicin.
NPs with adjustable loadings up to 10 wt % (with relatively
high loading efficiencies) and low polydispersities were readily
obtained for all of the therapeutics (docetaxel, doxorubicin,
proparacaine) and dyes (Cy5, rhodamine B, coumarin 6) tested
(Table 1).
HeLa (cervical cancer cell), PC-3 (human prostate
carcinoma), and human umbilical vein endothelial cells
(HUVEC) were used to assess the cytotoxicity of SP NPHs.
Following 72 h of exposure to NPs, cell viability was
determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay.35 The SP NPH did not
cause significant cytotoxicity in either cell line except at
extremely high concentrations (Figure S5a). The EC50 values
(the concentrations at which cell viability was reduced by 50%,
determined by interpolation from the data in Figure S5a) for
the [SP-C9] in those NPHs were 9.53 mM for HUVEC (6.33
mg/mL NPHs), 7.01 mM for HeLa cells (4.66 mg/mL NPHs),
and 7.41 mM for PC-3 cells (4.92 mg/mL NPHs). In a 70 kg
adult, these EC50 values are approximately equivalent to 70 g/
dose (∼ 1 g/kg) assuming NPHs are restricted to the 14 L
extracellular fluid, or 25 g/dose (∼350 mg/kg) if the NPs are
restricted to the 5 L bloodstream, extremely high doses
compared to those used clinically with Doxil (dosage: 50 mg/
Figure 2. (a) Dynamic light scattering measurement of size changes of
SP-C9/DSPE-PEG/lecithin SP NPHs upon alternating UV (30 s) and
visible light (3 min) illumination. Inset: the solution of NPH before
and after UV irradiation. (b) Steady-state absorption spectra of NPH
([SP-C9] = 0.46 mM) and their corresponding isomerized MC NPH
(λmax = 551 nm) upon UV light irradiation.
quinoidal structure, which is a 1, 6-addition adduct of MC-C9
with trimethylsilyl cyanide (Figure S3).30 MC−CN NPHs were
59.4 nm in diameter, with a polydispersity of 0.04 (Figure S3c),
similar to the size of MC NPH produced from SP NPH by UV-
irradiation (47.2 nm with a polydispersity of 0.05, Figure 2a).
Direct nanoprecipitation of MC−CN resulted in 42.6 nm NPHs
with a polydispersity of 0.11 (Figure S3d), a result consistent
with the DLS measurements of MC NPHs.
The PEGylated lipid was designed to give NPHs a relatively
neutral surface charge for prolonged circulation and stabiliza-
tion.31 The ζ potential of SP NPH and MC NPH at pH 7.5 was
−6.25
0.31 mV and −5.12
0.12 mV, respectively. The
results indicated the similarly neutral charges of NPH before
and after irradiation. No aggregation was observed for over 4 h
a
Table 1. Characteristics of Photoswitching SP NPH
drug/dye
initial LD %
actual LD %
LD efficiency %
size (nm)
polydispersity
size-UV (nm)
polydispersity
Rhodamine B
Coumarin 6
Calcein
5
10
5
2.49 0.13
6.84 0.07
2.71 0.09
9.41 0.05
3.97 0.04
8.21 0.14
7.42 0.11
2.69 0.21
4.96 0.14
6.35 0.16
7.64 0.19
49.8
68.4
54.2
94.1
79.4
82.1
74.2
53.7
49.6
63.5
51.0
129.7 1.8
74.7 2.9
133.9 6.7
108.6 4.5
101.7 3.1
116.1 1.1
125.4 5.0
96.9 4.7
93.3 3.2
87.5 2.7
102.3 6.6
0.054
0.013
0.064
0.071
0.052
0.088
0.039
0.035
0.074
0.049
0.071
74.2 2.6
27.2 4.5
50.6 4.8
72.7 0.8
40.1 8.9
76.1 5.2
49.7 5.8
41.5 6.4
49.8 6.7
48.2 5.4
66.1 2.5
0.081
0.086
0.072
0.066
0.065
0.066
0.043
0.043
0.058
0.100
0.032
Cyanine 5
Paclitaxel
10
5
Paclitaxel
10
10
5
Docetaxel
Doxorubicin
Doxorubicin
Proparacaine
Proparacaine
10
10
15
a
Determined by DLS and HPLC. Abbreviations: LD, loading; size-UV, sizes of NPs treated by UV irradiation (N = 5). Data are means SD (N =
5).
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m2).36 The EC50 value for MC NPH in HeLa cells was 3.46 mg/
mL, similar to that for SP NPH (Figure S5b).
Repetitive Photoswitching and Light-Triggered Drug
Release Profiles of NPs. The repeatability of the photo-
switching property of NPH was evaluated by alternating cycles
of UV and Vis light. This modulation was fully reversible for at
least 4 continuous cycles (UV irradiation for 30 s and Vis light
for 3 min, Figure 3). However, the absorbance at the MC-C9
Figure 4. Release profiles in PBS for rhodamine B loaded in SP NPH
under different conditions: without irradation; with UV irradiation for
30 s at 0 h; with repetitive UV irradiation at 0, 3,and 6 h. The times of
irradiation are indicated by purple arrows. Data are means SD, N =
6.
particles will become diluted and fluoresces.38 SP NPH loaded
with calcein (2.7 wt %) were incubated with HeLa cells. After 4
h, the media containing NPHs was removed and the cells were
washed with PBS. Cells in medium were then illuminated by
UV (365 nm) for 2 s, left in darkness for 5 min, then imaged
(Figure S8). Strong fluorescence intensity with an emission
maximum at 510 nm was noted in the cells, indicating that the
calcein was released from NPs that had been taken up.
Illumination followed by imaging was repeated 5 times, during
which the fluorescence intensity gradually increased to
saturation (Figure S8a,b). Cells treated with same NPs but
without UV irradiation did not fluoresce, suggesting that the
UV triggered rapid calcein release and intracellular dispersal
from SP NPH. These results were validated by flow cytometry,
which showed a 24.7-fold increase in fluorescence intensity
after a 10 s UV treatment (Figure S8c).
Surface Functionalization of NPs. Nanoparticle ther-
apeutic effect can be enhanced and toxicity reduced by surface
modification with moieties that allow intracellular penetration
and/or targeting of specific tissues.39 To examine the potential
suitability of the NPH for targeted drug delivery, we formulated
NPs (NPM) composed of SP-C9 and a mixture of DSPE-
PEG3400-maleimido (DSPE-PEG-MAL) and DSPE-PEG in a 4/
1 ratio (w/w), 153.1 nm in diameter and with a polydispersity
of 0.09. A cell penetration peptide (Cpp) Cys-Tat (47−57)
(sequence: CYGRKKRRQRRR-NH2) was introduced onto SP
NPM loaded with Cy5 by reaction of the carboxyl-terminal Cys
of the peptide with the MAL on the NPM surface (NPs/Cpp =
100/1, w/w). The fluorescence intensity of HeLa cells
incubated with the resulting NPs (SP NPM-Cpp) for 30 min,
measured by flow cytometry, was 7.1 times higher than that of
cells treated with SP NPM lacking Cpp (N = 4, fluorescence
intensities of 1940 215 and 273 197, respectively; Figure
5a).
Figure 3. Reversible NPH photochromism (solid line, Abs:
absorbance) and size switching (dashed line) with alternating UV
(“UV”, 30 s) and visible light (“Vis”, 3 min) irradiation. The
modulation of NPH size and photochromism was fully reversible for at
least 4 cycles. Data are means SD, N = 4.
peak maximum decreased 43% after 4 cycles, and was
accompanied by a reduction in size from 143.2 to 98.7 nm in
the SP state (Figure 3). The decrease of absorbance after
repetitive irradiation could be due to photofatigue (the loss of
performance in photoisomerization)  a common property of
organic photochromic compounds.37 The absorption intensity
of MC in NPH at 551 nm faded at a rate dependent on the UV
(365 nm) irradiation time, and that antioxidant agents could
not eliminate the decrease in MC-C9 absorption, suggesting an
O2-independent fatigue mechanism for photofatigue in SP
NPHs (see Figure S6 and Scheme S1 and associated
discussion).
We hypothesized that the phototriggered shrinkage of NPHs
might induce drug release. In the absence of UV photo-
triggering, drugs (e.g., doxorubicin) and dyes (e.g., rhodamine
6B) loaded in SP NPH showed slow release in PBS that was
complete within 48−72 h (Figure 4, Figure S7). Upon UV
irradiation (30s), NPHs encapsulating rhodamine 6B (loading
wt% = 4.3%) released 29.3% of the loaded dye within 1 h as
determined by HPLC, while 7.2% was released in the same
period without UV irradiation. Of note, the release kinetics of
NPHs that had been triggered (Figure 4, blue line) eventually
slowed to a rate similar to that of NPHs that were not irradiated
(Figure 4, black line). This decrease in the release rate could be
explained by the majority of the MC-C9 in NPs spontaneously
converting back to SP-C9, resulting in NPs reassembled in their
original structure. In a separate group, UV triggering (30 s
irradiation) was conducted every 3 h for three cycles (Figure 4,
green line), with an increase in release at each event.
We compared the cytotoxicity of doxorubicin-loaded SP
NPM-Cpp (doxorubicin/SP NPM-Cpp) to that of SP NPM
without Cpp (doxorubicin/SP NPM). HeLa cells were
incubated with doxorubicin/SP NPM-Cpp or doxorubicin/SP
NPM for 2 h, then incubated in medium without NPs for a total
of 48 h; cell viability was measured by MTT assay. The
doxorubicin/SP NPM-Cpp were significantly more cytotoxic
UV-triggered release was demonstrated in cells by
fluorescence imaging of SP NPH loaded with calcein. Calcein
was selected because its fluorescence self-quenches while it is
entrapped inside particles, whereas calcein released from
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Figure 6. Normalized NIR fluorescence intensity vs distance profiles
for various formulations’ penetration into collagen gels over a period of
12 h (7.4 mg/mL): free ICG (black, diffusion coefficient = 3.59 1.94
× 10−7 cm2·s−1), ICG/SP NPH (red, diffusion coefficient = 7.65 1.63
× 10−7 cm2·s−1), ICG/SP NPH irradiated by UV light (blue, diffusion
coefficient = 2.24 0.42 × 10−6 cm2·s−1 at t = 0 for 10 s), and ICG/SP
NPH irradiated twice by UV (green, for 10 s each time, separated by 3
h,). Diffusion coefficient data are means SD, N = 4. The dashed
lines are theoretical curves fitting the intensity profiles using a one-
dimension diffusion model.
4.0 0.21 mm into the collagen gels, ICG/SP NPH penetrated
8.3
0.10 mm without UV triggering, and ICG/SP NPH
triggered by UV for 10 s penetrated 12.1 0.02 mm (N = 4, P
< 0.005 for irradiated ICG/SP NPH compared to free ICG and
unirradiated ICG/SP NPH). (The mechanical properties of
collagen barely change after 1 h irradiation at 254 nm UV light,
∼1.7 × 10−6 einstein.43) By fitting the fluorescence intensity of
ICG/SP NPH to a one-dimensional diffusion model, we
obtained an average diffusion coefficient of 2.24
0.42 ×
10−6 cm2·s−1 for UV-triggered ICG/SP NPH NPs (N = 4, P <
0.005 compared to free ICG), while the diffusion coefficient for
unirradiated ICG/SP NPH (7.65 1.63 × 10−7 cm2·s−1, N = 4)
was not statistically significantly different from that of free ICG
(3.59 1.94 × 10−7 cm2·s−1, N = 4, P = 0.064) compared to
unirradiated ICG/SP NPH (Figure 6). The relatively low
diffusion rate of free ICG in collagen gels compared to NPHs
might be partly due to the lipophilicity of ICG.44 Gel
penetration was further enhanced by increasing irradiation:
ICG/SP NPH irradiated twice (for 10 s each, separated by 3 h)
Figure 5. (a) Flow cytometric analysis of the internalization of Cy5 in
SP NPM-Cpp. Red line, untreated HeLa cells; green line, HeLa cells
treated with Cy5/SP NPM for 30 min; blue line, HeLa cells treated
with Cy5/SP NPM-Cpp for 30 min; (b) MTT assay to determine the
differential cytotoxicity of doxorubicin/SP NPM, and doxorubicin/SP
NPM. Data are means SD, N = 6, asterisks indicate P < 0.005.
than the doxorubicin/SP NPM (Figure 5b). These results
suggest that the SP NPM’s have the capacity to be
functionalized by a broad range of biomolecules (e.g.,
aptamers40 or other peptides41) to enhance drug delivery.
Light-Triggering Enhances Diffusion in Collagen
Matrices. As discussed above, the ability to penetrate tissue
could have a bearing on therapeutic effectiveness. We evaluated
whether the light- triggered size change could enhance diffusive
transport through a dense collagen gel at a concentration
penetrated 16.8
coefficient of 1.97
0.10 mm with an average diffusion
0.28 × 10−6 cm2·s−1 (calculated by
modified one dimension diffusion models; N = 4; Figure 6
green line). The fact that the diffusion coefficient of light-
triggered ICG/SP NPH was significantly larger than those for
nonirradiated ICG/SP NPH and free ICG (for both P < 0.005)
suggests that light-induced shrinkage might help deepen tissue
penetration of SP NPH and their payloads. That possibility is
supported by the observation that irradiation does not appear
to affect the other physicochemical properties of PEGylated
NPH (they have similar slightly negatively charged surfaces
before and after irradiation).
Enhanced Diffusion of Photoswitching NPs in the
Cornea. We assessed the potential for photoswitching SP NPH
to carry drugs across the cornea in a manner analogous to the
findings in collagen gels. Corneas are composed of 90−95 wt %
of dense collagens, rendering the delivery of drugs through the
(0.74%; 7.4 mg/mL12) similar to the 9.0
2.5 mg/mL of
interstitial matrix estimated for interstitial collagen in human
tumors (e.g., LS174T) and murine tumors (e.g., MCalV).10b,42
SP NPHs (1 mg/mL) loaded with 5 wt % indocyanine green
(ICG), a NIR dye, were placed in contact with collagen gels in
a horizontal capillary tube, then incubated for a further 12 h at
37 °C. A NIR imaging system was used to track particle
infiltration into the collagen (Figure 6). Free ICG penetrated
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cornea to the anterior chamber difficult. Particles containing
Cy5 (Cy5/SP NPH) were applied to fresh cadaveric porcine
corneas with or without UV light triggering for 1 min, and
incubated for 8 h. Gross examination of the corneas and NIR
scanning of Cy5 in corneal cross section demonstrated that the
diffusion of Cy5/SP NPH was markedly enhanced by UV light
triggering (Figure 7). Histologically, corneas treated with Cy5/
Although MC-C9 does not fluoresce in organic solvents (e.g.,
acetonitrile), we found that NPH could switch between
fluorescence (as MC-C9) and nonfluorescence (as SP-C9).
UV-irradiation of SP NPH in aqueous solution created MC
NPH (Figure 1c) with an ∼8-fold increase in red fluorescence
(600−800 nm) compared to MC-C9 in acetonitrile ([MC-C9]
= 0.20 mM for both acetonitrile solution and NPHs). The λmax
of MC NPH red-shifted by 32 to 672 nm compared to MC-C9
in acetonitrile (Figure S10a and associated discussion of
mechanism). The fluorescence exponential decay constant of
MC NPH (Figure S10b) was 1.44 × 10−4 s−1 at 672 nm (t1/2
=
4813 s), much slower than for free MC-C9 in acetonitrile
solution (t1/2 = 346 s). The intensity of the fluorescence and
the duration of the decay of that intensity for MC in NPH
would be sufficient for use in microscopic imaging, unlike free
MC.
The fluorescent photochromic properties of NPs could be
used to track them in biological studies (e.g., intracellular drug
delivery) with greater reliability than with simple fluorescence,
which can be confounded by interfering fluorophores or in vivo
autofluorescence.46c,47 In fact, NPs surface-modified with SP
have been utilized as light-triggerable fluorescent probes.24h
Here, we evaluated whether fluorescence switching of SP NPH
could be achieved in living cells in vitro in a HeLa cell line
(Figure 8a). We loaded Cy5 (emission max = 690 nm) into SP
Figure 7. Ex vivo study of Cy5/SP NPH penetration in porcine
corneas. (a) Fresh corneas after an 8-h treatment with Cy5/SP NPH
(with or without UV irradiation for 1 min) or Cy5. The green color
indicates the presence of Cy5 (a blue dye that becomes greenish in the
slightly yellow tissue of the eye); (b) near-infrared images of cross
sections of corneas tissues treated as in panel (a). The scale bar = 1
cm.
SP NPH and UV light were indistinguishable from untreated
controls under light microscopy, showing no tissue injury
(Figure S9). Since collagen is one of the major components of
the interstitial matrix, these results suggest the potential
usefulness of SP NPH for light-triggered drug delivery to
targeted tissues, for example, eyes and tumors. These results are
consistent with a recent report that polymeric micelles ∼30 nm
(close to MC NPHs sizes) showed enhanced tissue penetration
and potent antitumor activity in poorly permeable pancreatic
tumors.45 The histological findings, together with the benign
cytotoxicity (Figure S5) are consistent with a favorable safety
profile, but this remains to be validated by in vivo studies.
The wavelengths of the UV light we used for SP NPH
triggering might limit the application of this technology to areas
of the body that can be illuminated directly, for example, the
eyes and ears. Of note, the photochromic conversion of SP
could be potentially triggered at depths up to several
centimeters by near-infrared lasers using two-photon technol-
ogy (wavelength ∼ 720 nm), through soft tissues, bone, and
intact skull.46
Figure 8. Fluorescence images of the internalization of Cy5-containing
NPH by HeLa cells after 2 h incubation. (a) Nuclear staining with
DAPI (blue color). (b) NPHs were illuminated by UV for 2s then
imaged with 560 nm emission filters (green color); NPHs were seen to
be internalized. (c) The red color (emission at 700 nm) shows the
Cy5 loaded in the SP NPH. (d) The overlay of panels a−c. The orange
color demonstrated colocalization of SP NPH with Cy5. The scale bar
= 50 μm.
NPH since its emission spectrum would have little overlap with
that of MC NPH (emission max = 672 nm). Cy5-containing SP
NPHs were incubated with HeLa cells for 2 h in darkness then
exposed to UV illumination for 2 s, causing immediate
fluorescence attributable to MC NPH (Figure 8b). Fluorescence
microscopy (Figure 8) indicated that Cy5 (red color) and MC-
containing hybrid NPs (green color) were colocalized in HeLa
cells (orange color in Figure 8d).
Fluorescence of Photoswitching NPs. The possibility
that NPH could perform as fluorescent light-triggered imaging
probes was suggested by the fact that SP or nanoparticles
surface-modified with SP have been utilized as fluorescence
imaging probes in different microscopy techniques, including
optical lock-in detection (OLID),27 two-photon photoswitch-
ing, and imaging by noninvasive near-infrared (NIR) light.28
Mechanism of Photoswitching NPs. We propose the
following assembly model to explain the photoswitching of SP
NPH (Figure 9). The SP NPHs are composed of a hydrophilic
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to produce, and tolerated lyophilization, which may facilitate
potential clinical translation.28 The NPHs could achieve high
loadings with various drugs (chemotherapeutic, local anes-
thetics). The NPHs developed here could be adapted for a
range of applications, as they could be modified with functional
ligands. The phototriggering system could also be used to
enhance NPH tissue penetration, which might improve
antitumor efficacy, penetration into ocular tissue and across
the tympanic membrane. This is quite different from
conventional approaches, where external energy sources
enhance penetration by disrupting tissues.50
The photoswitchability is an attractive feature in that it can
allow fine spatiotemporal control of drug release: drug is
released at the irradiated site, during irradiation. This approach
also obviates the need for developing a specific ligand to the
tissue of interest. We have previously developed an analogous
approach to the same problem by decorating nanoparticles with
nonspecific ligands caged with photosensitive chemical
protecting groups; upon irradiation, the caging groups would
come off, allowing the nanoparticles to bind.41b These two
approaches and others13 could prove synergistic.
ASSOCIATED CONTENT
* Supporting Information
■
S
Figure 9. Proposed assembly states of reversible light-triggered SP
NPH size switching: SP NPH converted MC NPH upon irradiation
(solid arrow, i to ii); graduate transition (dash arrow, ii to iii to i) from
MC NPH to SP NPH in the dark, with the conversion of zwitterionic
MC-C9 to hydrophobic SP-C9 to cause the reassembly of NPH.
Yellow oval, SP molecule; blue line, the alkyl chain in SP; red, lipid
part; green line, PEG; and purple oval, MC molecule.
Experimental details, characterization of SP NPHs, in vitro
characterization, ex vivo histology, mechanism study. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
Daniel.Kohane@childrens.harvard.edu
■
PEG shell, beneath which are the hydrophobic alkyl chains of
the DSPE and the SP-C9 (Figure 9i). Given the reported
destabilization of monolayer surfactant films by SP,48 SP is
likely to perturb the alkyl chain packing inside the SP NPH,
causing the hydrophobic core to have a loose structure (Figure
9i) and increasing particle size. Upon irradiation, SP converts to
zwitterionic MC, that moves outward to relatively polar
microenvironments within the NPH, such as the phosphogly-
cerol moiety linking DSPE and PEG.49 The polar micro-
environment around MC in NPH is evidenced by the fact that
the λmax of MC in NPH (551 nm) is comparable to the λmax of
MC in polar solvents (Figure S11).49b (The effective dielectric
constant of the microenvironment of MC in NPH is ∼18, i.e., is
relatively polar; detailed discussion in Figure S11.) As MC
moves toward the more hydrophilic PEG layer of the NPH, it
moves away from the alkyl chains of the DSPE and lecithin,
allowing them to assemble tightly inside the hydrophobic cores;
in consequence, the NPH volume shrinks (Figure 9ii).
The NPH size will increase again once MC reverts to SP and
translocates into the hydrophobic core, perturbing the assembly
of the lipids. The alkyl chains of DSPE and lecithin may impede
the isomerization of MC to SP, as suggested by the fact that the
isomerization in NPH (λmax = 551 nm) was 12.2-fold slower
than that of free MC-C9 in acetonitrile (λmax = 560 nm, Figure
S12). This slowing of the isomerization from MC to SP has also
been observed in MC in polymeric films.18a
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The work was supported by a grant from Sanofi-Aventis, and
NIH (R21DC009986).
■
REFERENCES
■
(1) (a) Langer, R. Acc. Chem. Res. 1993, 26, 537. (b) Langer, R.;
Folkman, J. Nature 1976, 263, 797.
(2) (a) Gref, R.; Minamitake, Y.; Peracchia, M. T.; Trubetskoy, V.;
Torchilin, V.; Langer, R. Science 1994, 263, 1600. (b) Langer, R.
Nature 1998, 392, 5.
(3) Kost, J.; Langer, R. Adv. Drug Delivery Rev. 2001, 46, 125.
(4) Farokhzad, O. C.; Langer, R. ACS Nano 2009, 3, 16.
(5) Jain, R. K.; Stylianopoulos, T. Nat. Rev. Clin. Oncol. 2010, 7, 653.
(6) (a) O’Brien, M. E. R.; Wigler, N.; Inbar, M.; Rosso, R.; Grischke,
E.; Santoro, A.; Catane, R.; Kieback, D. G.; Tomczak, P.; Ackland, S.
P.; Orlandi, F.; Mellars, L.; Alland, L.; Tendler, C. Ann. Oncol. 2004,
15, 440. (b) Gradishar, W. J.; Tjulandin, S.; Davidson, N.; Shaw, H.;
Desai, N.; Bhar, P.; Hawkins, M.; O’Shaughnessy, J. J. Clin. Oncol.
2005, 23, 7794.
(7) Jiang, W.; Kim, B. Y. S.; Rutka, J. T.; Chan, W. C. W. Nat.
Nanotechnol. 2008, 3, 145.
(8) Perrault, S. D.; Walkey, C.; Jennings, T.; Fischer, H. C.; Chan, W.
C. W. Nano Lett. 2009, 9, 1909.
(9) Jain, R. K. J. Controlled Release 1998, 53, 49.
(10) (a) McKee, T. D.; Grandi, P.; Mok, W.; Alexandrakis, G.; Insin,
N.; Zimmer, J. P.; Bawendi, M. G.; Boucher, Y.; Breakefield, X. O.;
Jain, R. K. Cancer Res. 2006, 66, 2509. (b) Netti, P. A.; Berk, D. A.;
Swartz, M. A.; Grodzinsky, A. J.; Jain, R. K. Cancer Res. 2000, 60, 2497.
(c) Yuan, F.; Leunig, M.; Huang, S. K.; Berk, D. A.; Papahadjopoulos,
CONCLUSION
■
We have described photoswitchable NPHs that allow
spatiotemporal controlled release of drugs and enhanced tissue
penetration upon UV illumination. This formulation was simple
8854
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Journal of the American Chemical Society
Article
D.; Jain, R. K. Cancer Res. 1994, 54, 3352. (d) Campbell, R. B.;
Fukumura, D.; Brown, E. B.; Mazzola, L. M.; Izumi, Y.; Jain, R. K.;
Torchilin, V. P.; Munn, L. L. Cancer Res. 2002, 62, 6831.
(11) (a) Primeau, A. J.; Rendon, A.; Hedley, D.; Lilge, L.; Tannock, I.
F. Clin. Cancer Res. 2005, 11, 8782. (b) Ouar, Z.; Bens, M.; Vignes, C.;
Paulais, M.; Pringel, C.; Fleury, J.; Cluzeaud, F.; Lacave, R.;
Vandewalle, A. Biochem. J. 2003, 370, 185.
(12) Wong, C.; Stylianopoulos, T.; Cui, J. A.; Martin, J.; Chauhan, V.
P.; Jiang, W.; Popovic, Z.; Jain, R. K.; Bawendi, M. G.; Fukumura, D.
Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 2426.
(35) Mosmann, T. J. Immunol. Methods 1983, 65, 55.
(36) Draft Guidance on Doxorubicin Hydrochloride. http://www.
fda.gov/downloads/Drugs/GuidanceComplianceRegulatory
Information/Guidances/UCM199635.pdf.
(37) Baillet, G.; Giusti, G.; Guglielmetti, R. J. Photochem. Photobiol., A
1993, 70, 157.
(38) Patel, H.; Tscheka, C.; Heerklotz, H. Soft Matter 2009, 5, 2849.
(39) (a) Petros, R. A.; DeSimone, J. M. Nat. Rev. Drug Discovery
2010, 9, 615. (b) Davis, M. E.; Chen, Z.; Shin, D. M. Nat. Rev. Drug
Discovery 2008, 7, 771.
(40) (a) Farokhzad, O. C.; Cheng, J. J.; Teply, B. A.; Sherifi, I.; Jon,
S.; Kantoff, P. W.; Richie, J. P.; Langer, R. Proc. Natl. Acad. Sci. U.S.A.
2006, 103, 6315. (b) Cao, Z. H.; Tong, R.; Mishra, A.; Xu, W. C.;
Wong, G. C. L.; Cheng, J. J.; Lu, Y. Angew. Chem., Int. Ed. 2009, 48,
6494.
(13) Timko, B. P.; Dvir, T.; Kohane, D. S. Adv. Mater. 2010, 22,
4925.
(14) Youan, B.-B. C. Adv. Drug Delivery Rev. 2010, 62, 898.
(15) (a) You, S.; Li, W. Med. Hypotheses 2008, 71, 141. (b) Jain, R. K.
Nat. Med. 2001, 7, 987.
(41) (a) Chan, J. M.; Zhang, L. F.; Tong, R.; Ghosh, D.; Gao, W. W.;
Liao, G.; Yuet, K. P.; Gray, D.; Rhee, J. W.; Cheng, J. J.; Golomb, G.;
Libby, P.; Langer, R.; Farokhzad, O. C. Proc. Natl. Acad. Sci. U.S.A.
2010, 107, 2213. (b) Dvir, T.; Banghart, M. R.; Timko, B. P.; Langer,
R.; Kohane, D. S. Nano Lett. 2010, 10, 250.
(42) Ramanujan, S.; Pluen, A.; McKee, T. D.; Brown, E. B.; Boucher,
Y.; Jain, R. K. Biophys. J. 2002, 83, 1650.
(43) Sionkowska, A.; Wess, T. Int. J. Biol. Macromol. 2004, 34, 9.
(44) Vinegoni, C.; Botnaru, I.; Aikawa, E.; Calfon, M. A.; Iwamoto,
Y.; Folco, E. J.; Ntziachristos, V.; Weissleder, R.; Libby, P.; Jaffer, F. A.
Sci. Transl. Med. 2011, 3, 84ra45.
(16) (a) Netti, P. A.; Baxter, L. T.; Boucher, Y.; Skalak, R.; Jain, R. K.
Cancer Res. 1995, 55, 5451. (b) Netti, P. A.; Hamberg, L. M.; Babich, J.
W.; Kierstead, D.; Graham, W.; Hunter, G. J.; Wolf, G. L.; Fischman,
A.; Boucher, Y.; Jain, R. K. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 3137.
(17) (a) Yuan, F.; Chen, Y.; Dellian, M.; Safabakhsh, N.; Ferrara, N.;
Jain, R. K. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 14765. (b) Yuan, F.;
Dellian, M.; Fukumura, D.; Leunig, M.; Berk, D. A.; Torchilin, V. P.;
Jain, R. K. Cancer Res. 1995, 55, 3752.
(18) (a) Tamai, N.; Miyasaka, H. Chem. Rev. 2000, 100, 1875.
(b) Minkin, V. I. Chem. Rev. 2004, 104, 2751.
(19) Kawata, S.; Kawata, Y. Chem. Rev. 2000, 100, 1777.
(20) (a) Berkovic, G.; Krongauz, V.; Weiss, V. Chem. Rev. 2000, 100,
1741. (b) Vlassiouk, I.; Park, C. D.; Vail, S. A.; Gust, D.; Smirnov, S.
Nano Lett. 2006, 6, 1013.
(21) (a) Feringa, B. L.; Browne, W. R. Nat. Nanotechnol. 2008, 3,
383. (b) Shao, N.; Jin, J. Y.; Wang, H.; Zheng, J.; Yang, R. H.; Chan,
W. H.; Abliz, Z. J. Am. Chem. Soc. 2010, 132, 725.
(22) Tyer, N. W.; Becker, R. S. J. Am. Chem. Soc. 1970, 92, 1289.
(23) Heiligmanrim, R.; Hirshberg, Y.; Fischer, E. J. Phys. Chem. 1962,
66, 2465.
(24) (a) Matsumoto, M.; Nakazawa, T.; Mallia, V. A.; Tamaoki, N.;
Azumi, R.; Sakai, H.; Abe, M. J. Am. Chem. Soc. 2004, 126, 1006.
(b) Davis, D. A.; Hamilton, A.; Yang, J. L.; Cremar, L. D.; Van Gough,
D.; Potisek, S. L.; Ong, M. T.; Braun, P. V.; Martinez, T. J.; White, S.
R.; Moore, J. S.; Sottos, N. R. Nature 2009, 459, 68. (c) Cabrera, I.;
Shvartsman, F.; Veinberg, O.; Krongauz, V. Science 1984, 226, 341.
(d) Willner, I. Acc. Chem. Res. 1997, 30, 347. (e) Lee, H. I.; Wu, W.;
Oh, J. K.; Mueller, L.; Sherwood, G.; Peteanu, L.; Kowalewski, T.;
Matyjaszewski, K. Angew. Chem., Int. Ed. 2007, 46, 2453. (f) Osborne,
E. A.; Jarrett, B. R.; Tu, C. Q.; Louie, A. Y. J. Am. Chem. Soc. 2010, 132,
5934. (g) Zhu, M. Q.; Zhu, L. Y.; Han, J. J.; Wu, W. W.; Hurst, J. K.;
Li, A. D. Q. J. Am. Chem. Soc. 2006, 128, 4303. (h) Zhu, L. Y.; Zhu, M.
Q.; Hurst, J. K.; Li, A. D. Q. J. Am. Chem. Soc. 2005, 127, 8968.
(25) Movia, D.; Prina-Mello, A.; Volkov, Y.; Giordani, S. Chem. Res.
Toxicol. 2010, 23, 1459.
(45) Cabral, H.; Matsumoto, Y.; Mizuno, K.; Chen, Q.; Murakami,
M.; Kimura, M.; Terada, Y.; Kano, M. R.; Miyazono, K.; Uesaka, M.;
Nishiyama, N.; Kataoka, K. Nat. Nanotechnol. 2011, 6, 815.
(46) (a) Marriott, G.; Mao, S.; Sakata, T.; Ran, J.; Jackson, D. K.;
Petchprayoon, C.; Gomez, T. J.; Warp, E.; Tulyathan, O.; Aaron, H. L.;
Isacoff, E. Y.; Yan, Y. L. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 17789.
(b) Zhu, M. Q.; Zhang, G. F.; Li, C.; Aldred, M. P.; Chang, E.; Drezek,
R. A.; Li, A. D. Q. J. Am. Chem. Soc. 2011, 133, 365. (c) Ntziachristos,
V.; Ripoll, J.; Wang, L. V.; Weissleder, R. Nat. Biotechnol. 2005, 23,
313. (d) Srinivasan, S.; Pogue, B. W.; Jiang, S.; Dehghani, H.; Kogel,
C.; Soho, S.; Gibson, J. J.; Tosteson, T. D.; Poplack, S. P.; Paulsen, K.
D. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 12349. (e) Yoder, E. J.;
Kleinfeld, D. Microsc. Res. Tech. 2002, 56, 304.
(47) Zhu, L. Y.; Wu, W. W.; Zhu, M. Q.; Han, J. J.; Hurst, J. K.; Li, A.
D. Q. J. Am. Chem. Soc. 2007, 129, 3524.
(48) (a) Khairutdinov, R. F.; Hurst, J. K. Langmuir 2001, 17, 6881.
(b) Holden, D. A.; Ringsdorf, H.; Deblauwe, V.; Smets, G. J. Phys.
Chem. 1984, 88, 716. (c) Gruler, H.; Vilanove, R.; Rondelez, F. Phys.
Rev. Lett. 1980, 44, 590.
(49) (a) Mazeres, S.; Schram, V.; Tocanne, J. F.; Lopez, A. Biophys. J.
1996, 71, 327. (b) Khairutdinov, R. F.; Giertz, K.; Hurst, J. K.;
Voloshina, E. N.; Voloshin, N. A.; Minkin, V. I. J. Am. Chem. Soc. 1998,
120, 12707.
(50) (a) Hu, M.; Chen, J. Y.; Li, Z. Y.; Au, L.; Hartland, G. V.; Li, X.
D.; Marquez, M.; Xia, Y. N. Chem. Soc. Rev. 2006, 35, 1084. (b) Kong,
G.; Braun, R. D.; Dewhirst, M. W. Cancer Res. 2000, 60, 4440. (c) Jain,
P. K.; Lee, K. S.; El-Sayed, I. H.; El-Sayed, M. A. J. Phys. Chem. B 2006,
110, 7238.
(26) Cheng, J.; Teply, B. A.; Sherifi, I.; Sung, J.; Luther, G.; Gu, F. X.;
Levy-Nissenbaum, E.; Radovic-Moreno, A. F.; Langer, R.; Farokhzad,
O. C. Biomaterials 2007, 28, 869.
(27) Kjoniksen, A. L.; Joabsson, F.; Thuresson, K.; Nystrom, B. J.
Phys. Chem. B 1999, 103, 9818.
(28) Tong, R.; Yala, L.; Fan, T. M.; Cheng, J. Biomaterials 2010, 31,
3043.
(29) Fang, R. H.; Aryal, S.; Hu, C. M. J.; Zhang, L. F. Langmuir 2010,
26, 16958.
(30) Malatesta, V.; Neri, C.; Wis, M. L.; Montanari, L.; Millini, R. J.
Am. Chem. Soc. 1997, 119, 3451.
(31) Klibanov, A. L.; Maruyama, K.; Torchilin, V. P.; Huang, L. FEBS
Lett. 1990, 268, 235.
(32) Popielarski, S. R.; Pun, S. H.; Davis, M. E. Bioconjugate Chem.
2005, 16, 1063.
(33) Abdelwahed, W.; Degobert, G.; Stainmesse, S.; Fessi, H. Adv.
Drug Delivery Rev. 2006, 58, 1688.
(34) Tong, R.; Yala, L. D.; Fan, T. M.; Cheng, J. J. Biomaterials 2010,
31, 3043.
8855
dx.doi.org/10.1021/ja211888a | J. Am. Chem. Soc. 2012, 134, 8848−8855