Nano-Assemblies from J-Aggregated Dyes: A Stimuli-Responsive Tool Applicable to Living Systems

Article abstract of DOI:10.1021/jacs.8b10396

Controlling the packing arrangements of dyes is a facile way of tuning their photophysical and/or photochemical properties, thus enabling new sensing mechanisms for photofunctional tools. Here, we present a general and robust strategy toward water-stable

Full text of DOI:10.1021/jacs.8b10396

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Article
Nano-Assemblies from J-Aggregated Dyes: A Stimuli-
Responsive Tool Applicable to Living Systems
Meihui Su, Shuoxin Li, Hao Zhang, Junqing Zhang, Haoliang Chen, and Changhua Li
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b10396 • Publication Date (Web): 14 Dec 2018
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Nano-Assemblies from J-Aggregated Dyes: A Stimuli-Responsive
Tool Applicable to Living Systems
Meihui Su , Shuoxin Li , Hao Zhang , Junqing Zhang , Haoliang Chen , and Changhua Li1,2,3*
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State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin 300071, P. R. China. College of Phar-
3
macy, Nankai University, Tianjin 300071, P. R. China. Key Laboratory of Functional Polymer Materials of Ministry of Ed-
ucation, Nankai University, Tianjin 300071, P. R. China.
ABSTRACT: Controlling the packing arrangements of dyes is a facile way of tuning their photophysical and/or photochemical
properties, thus enabling new sensing mechanisms for photofunctional tools. Here, we present a general and robust strategy toward
water-stable J-aggregated dye templated nano-assemblies by incorporating an amphiphilic diblock copolymer and a stimuli-respon-
sive dye as the only two building components. An iodo-substituted boron dipyrromethene (BODIPY) was adopted as a template to
direct the self-assembly of poly(ethylene glycol)-block-polycaprolactone (PEG-PCL), forming a core-shell nanoplate with slip-
stacked BODIPYs as core surrounded by hydrophilic PEG shell. The self-assembled nanoplate is stable in cell culture medium and
possesses a built-in stimuli-responsiveness that arises from BODIPY bearing meso-carboxylate protecting group, which is efficiently
removed upon treatment with peroxynitrite. The resulting negative charges lead to rearrangement of dyes from J-stacking to non-
stacking, which activates photoinduced singlet oxygen production from the nano-assemblies. The stimuli-activatable photosensitivity
has been exploited for specific photodynamic ablation of activated RAW 264.7 cells with excessive endogenous peroxynitrite. In
light of the generality of the sensing mechanism, the concept described herein will significantly expands the palette of design princi-
ples to develop diverse photofunctional tools for biological research and clinical needs.
association, BD-PGMe forms J-aggregate and serves as template
■
INTRODUCTION
Dye assemblies with high packing orders possess novel opti-
to direct the self-assembly of P
plate of BD-PGMe/P with J-aggregated BD-PGMe as core sur-
rounded by P shell. Whereas for the P with longer PCL seg-
8
, leading to a core-shell nano-
8
cal or electronic functionalities relative to individual dye mole-
cules.
would be a facile way of tuning photophysical and/or photo-
chemical properties of dyes and thereby enabling new design
principles for photofunctional tools including fluorogenic
1-22
8
n
Thus, controlling the packing arrangement of dyes
ments (e.g., P80) possessing strong hydrophobic interactions
with one another, they tend to form micelles and encapsulate
BD-PGMe inside the hydrophobic core in a random packing ar-
8
rangement. Upon incubation of BD-PGMe/P nanoplates with
23-31
32-34
probes,
photosensitizers,
ratiometric photoacoustic probes,
or activatable
peroxynitrite, a fast rearrangement of BODIPY dyes from
highly ordered J-stacking to non-stacking was established, with
a structural reorganization also taking place in which nanoplates
were converted into nanorods. As discussed below, capability
in generating singlet oxygen upon photoirradiation, i.e., photo-
3
5-45
among others. However, dye assemblies
with sensing capabilities or activatable photofunctions toward
biorelated species in living systems based on stimuli-triggered
dye rearrangement face a demanding set of performance re-
quirements, including stability in cell culture media, rapid re-
sponse to specific species at acceptable low concentrations, and
chemical flexibility (available for a diverse range of biorelated
species). Herein, we present a versatile and robust strategy to-
ward water-stable J-aggregated dye doped nano-assemblies
with photophysical responses toward external stimuli through
dye-templated self-assembly of amphiphilic copolymers. The
mechanism of stimuli-responsive photophysical changes is
based on the dye rearrangement within nano-assemblies driven
by the stimuli-triggered hydrophobic to ionic conversion of
dyes.
8
sensitivity, of BD-PGMe/P nanoplates, was inhibited in the
slip-stacked arrangement and could be selectively switched on
in response to peroxynitrite, owing to the dye rearrangement.
Distinguished from most conventional optical sensing mecha-
46,47
nisms including Förster resonance energy transfer (FRET),
48
photo-induced electron transfer (PeT), and intramolecular
49
charge transfer (ICT), to name a few, the sensing mechanism
presented here is based on the chemical structural change in-
duced dye rearrangement, thus expanding the palette of design
principles to develop diverse photofunctional tools for biologi-
cal research and clinical needs by adopting various dyes with
desired photofunctions as self-assembly templates. Moreover,
the synthesis of carboxyl-caged dyes is flexible, thus allowing
the customization of dye-templates with sensitivity toward a
wide range of biorelated species of interest via introducing spe-
cific triggering motifs.
A representative slip-stacked dye containing nano-assembly
was engineered by incorporating a peroxynitrite-responsive
iodo-substituted BODIPY dye (BD-PGMe; Figure 1a) and an
amphiphilic diblock copolymer poly(ethylene glycol)-block-
polycaprolactone (PEG-PCL
building components. As summarized in Figure 1c, for P
short hydrophobic PCL segment (e.g., P ) possessing weak self-
n
, denoted as P
n
; Figure 1b) as two
n
with
8
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Figure 1. Representation of templated self-assembly and stimuli-triggered structure rearrangement, and chemical structures of two building compo-
nents. (a) Chemical structures of three carboxyl-caged BODIPY dyes (BD-PGMe, BD-PGEt, and BD-PGBu), and stimuli-triggered deprotection
chemistry. (b) Synthesis of amphiphilic diblock copolymer PEG-PCL (P ) with different lengths of hydrophobic PCL segments. (c) Schematic
n n
depiction of engineering water-stable nanoplate with slip-stacked dyes as core surrounded by hydrophilic polymeric shell through dye templated
self-assembly by incorporating an amphiphilic diblock copolymer and a hydrophobic dye as the only two building components. Stimuli-triggered
shape transformation of nano-assemblies and concomitant rearrangement of dyes from J-stacking to non-stacking resulted in remarkable photophys-
ical and/or photochemical changes.
9
to those of other 2,6-iodo-substituted BODIPY dyes. Upon in-
■
RESULTS AND DISCUSSION
Core-shell nanoplate with J-aggregated dyes as core sta-
creasing the water content in the DMSO solution to a critical
point (i.e., 30% water in DMSO, v/v), an explicit absorption
band emerged at ca. 808 nm, indicating the formation of J-ag-
bilized by polymeric shell. A key to this new type of self-as-
sembled core-shell nanoplates with J-aggregated dyes as cores
lies in the molecular design of hydrophobic BODIPY dyes, BD-
6,9
gregates of BD-PGMe (Figures 2a and S1). Although lack of
hydrophilic groups, the J-aggregates formed in DMSO/water at
higher water fractions (e.g., 90%) were demonstrated to be sta-
ble (Fig. S2). To explore such J-aggregates for potential appli-
cations in living systems, we propose to establish a platform for
preparing water-based J-aggregates doped nano-assemblies via
dye-assembly templated self-assembly of amphiphilic diblock
copolymers (Figure 1c). Having no pendent group on the main
chain, biocompatible amphiphilic diblock copolymer PEG-
n
PGMe (Figure 1a), and amphiphilic diblock copolymer P (Fig-
ure 1b). BD-PGMe bears a large benzyl ester moiety at the meso-
position and two heavy iodine atoms at the 2,6-positions, which
may be effective in preventing close face-to-face p-stacking
9
,50-53
while facilitating J-stacking of BODIPY dyes.
Moreover,
the meso-ester groups of BD-PGMe are designed with the capa-
bility of stimuli-triggered hydrophobic ester to ionic carbox-
ylate conversion, with the aim of providing an additional feature
in actuating rearrangement of slip-stacked BODIPY dyes (Fig-
ure 1a). In organic solvents, such as DMSO, BD-PGMe dis-
played an absorption band ranging from 550 to 780 nm with an
absorption maximum (lmax) at 708 nm (Figure 2a), comparable
PCL
ference of polymer on the packing arrangement and/or rear-
rangement of BODIPY dyes. Moreover, P with different hy-
n n
(P ) was selected as building block to minimize the inter-
n
drophobic PCL lengths could be prepared in a facile fashion by
controlling the feed ratio of the ring-opening polymerization
8
(Figure 1b). In this work, P , P50, and P80 with degrees of
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Figure 2. BODIPY dyes templated self-assemblies and their photophysical properties. (a) UV-visible absorption spectra of BD-PGMe (4.0 µM) in
DMSO/water mixture with different water fraction (fwater) from 0 to 90% (vol %). (b) UV-visible absorption spectra of the aqueous solutions of BD-
PGMe/P
(
2
8
, BD-PGMe/P50, and BD-PGMe/P80 nano-assemblies. Conc. 4.0 µM BD-PGMe; 0.2 g/L P
steel blue) and BD-PGMe/P nano-assemblies (green) determined by DLS. (d) TEM image and (e) AFM image of BD-PGMe/P
.0% dye content. (f) Size distribution of aqueous solutions of P80 (steel blue) and BD-PGMe/P80 nano-assemblies (green) determined by DLS. (g)
n
. (c) Size distribution of aqueous solutions of P
8
8
8
nano-assemblies at
TEM image of BD-PGMe/P80 nano-assemblies at 2.0% dye content; inset shows a high magnification TEM image of the region marked with a red
rectangle. (h) UV-visible absorption spectra of aqueous solutions of BD-PGMe/P (0.4 g/L P ) with different dye contents (0.1-3.4%). (i) UV-visible
absorption spectra of the aqueous solutions of BD-PGMe/P , BD-PGEt/P , and BD-PGBu/P nano-assemblies. Conc. 4.0 µM BD-PG ; 0.2 g/L P
8
8
8
8
8
R
8
.
polymerization (DP) of PCL segments of 8, 50, and 80, respec-
tively, were prepared.
has a significant impact on the dye arrangement (Figure S5).
The variations in the absorption spectra suggest subtle differ-
ences in the arrangement of BD-PGMe dyes, as well as the mi-
croscopic structures of three BD-PGMe/P nano-assemblies. We
n
then focused our attention to gain insights into the microscopic
structures of the species generated from the self-assembly of
The nano-assemblies of BD-PGMe/P
assembly using BD-PGMe and P (P , P50, and P80) as two build-
ing components were prepared by slowly adding water to an
acetone solution of BD-PGMe and P over a period of 10 min to
yield a final concentration of 0.4 g/L P and 8.0 µM BD-PGMe
with dye content of 2.0%. Photophysical properties of the three
different BD-PGMe/P nano-assemblies were first investigated.
n
through templated self-
n
8
n
BD-PGMe and P
ous solutions of P
celle concentrations (CMC) of 0.48, 0.06, and 0.02 g/L, respec-
tively (Table 1). The relative high CMC of P was ascribed to
n
. Surface tensiometer measurements of aque-
n
8
, P50, and P80 at 25 °C produced critical mi-
n
8
Gratifyingly, remarkable bathochromic shifted absorption
bands with lmax at ca. 788 nm were observed for all three of the
solutions, indicating the formation of J-aggregates (Figure 2b).
its short hydrophobic PCL block, resulting in little driving force
for micelle formation. Dynamic light scattering (DLS) measure-
ments of aqueous solutions of P
vealed intensity-average hydrodynamic diameters of 8, 45, and
0 nm, respectively (Table 1, Figures 2c, 2f, and S6), indicating
that P was present as a unimer, whereas P50 and P80 formed
micelles at the concentration used.
8
, P50, and P80 at 0.1 g/L re-
However, differences in the absorption spectra of BD-PGMe/P
BD-PGMe/P50, and BD-PGMe/P80 solutions were observed. Re-
garding the BD-PGMe/P solution, the monomer band at 708 nm
8
,
6
8
8
almost disappeared, whereas for the other two solutions, resid-
ual monomer bands could still be observed to some extent. The
J
fraction of J-aggregated BD-PGMe dyes (α ) was estimated
from the apparent absorption coefficients (see Supporting In-
formation), suggesting that the length of hydrophobic segments
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Table 1. Self-assembly properties of P
BD-PGMe dyes
n
in water with or without
cessive P chains that interfere with the p-p stacking and hydro-
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phobic interaction between BD-PGMe dyes. Upon increasing
dye content, the J-band at 788 nm emerged with the decrease of
monomer band, indicating the formation of J-aggregates (Fig-
a
b
b
Sample
CMC / g/L
Size / nm
PDI
b
P
P
P
8
0.48
8.38
0.18
0.07
0.15
/
8
ure 2h). However, during the preparation of BD-PGMe/P nano-
b
50
80
0.06
44.21
assemblies with dye content up to 4.0%, precipitation of dye
aggregates was observed, and the actual dye content of the fil-
tered solution was corrected to 3.4%, which is the maximum
b
0.02
58.29
c
BD-PGMe/P
8
/
/
/
~200
b
dye content of BD-PGMe/P
aggregated BD-PGMe dyes could be well stabilized by P
8
nano-assemblies for which the J-
. A
BD-PGMe/P50
BD-PGMe/P80
59.71
0.13
0.19
b
8
60.84
plausible rationale for the instability of J-aggregates at high dye
content is that the J-aggregates of BODIPY dyes should be sta-
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a
b
Determined by surface tension analysis. Determined by dynamic light
c
scattering (DLS) analysis. The length of the nanoplates was determined by
atomic force microscopy (AFM) measurement. The results represent the av-
erage of three independent experiments.
bilized by the surrounding hydrophilic PEG chains of P
are insufficient in the case of dye content beyond 3.4%. A plot
of fraction of J-aggregated dyes (α ) versus dye content sug-
8
, which
J
We deduced that the weak self-association of P
could allow for dye assembly templated self-assembly in the
presence of BD-PGMe dyes through hydrophobic interaction be-
8 8
tween BD-PGMe and P (Figure 1c). Compared with unimer P ,
the presence of BD-PGMe dyes induced an explicit increase in
hydrodynamic diameters (Figure 2c), suggesting the formation
8
in water
gested that BD-PGMe dyes in the slip-stacked arrangement were
maximal with dye content in the range of 1.0-2.0% (Figure S10).
Considering that the alkyl chains of BODIPY dyes might affect
the intermolecular packing mode and self-assembly, BD-PGEt
and BD-PGBu with ethyl and n-butyl chains, respectively, were
synthesized (Figure 1a). Upon increasing alkyl chain length
of BD-PGMe/P
8
nano-assemblies. The morphology and size of
from methyl (BD-PGMe) to ethyl (BD-PG ) and n-butyl (BD-
Et
the nano-assemblies was investigated by transmission electron
microscopy (TEM) and atomic force microscopy (AFM), and
elliptical nanoplates with length over 200 nm and thickness ap-
proximately 10 nm were observed (Figures 2d and 2e). On the
basis of DLS, TEM, and AFM results, together with UV-visible
absorption studies (Figure 2b), we reasoned that BD-PGMe dyes
were assembled in a slipped fashion in water and that the
PG ), significant changes in absorption spectra of BD-PG /P
8
Bu
R
nano-assemblies were observed (Figure 2i). Closer examination
revealed that the fraction of J-aggregated dyes (α ) decreased in
J
the order of BD-PGMe > BD-PGEt > BD-PGBu (Figure S11),
presumably because long alkyl chains would interfere with p-p
stacking interactions between dyes and enhance hydrophobic
interactions between dyes and polymers. Collectively, the
above results revealed that the J-aggregate templated self-as-
sembly mechanism is governed by the subtle balance of non-
covalent interactions including the self-association of PCL
chains, p-p stacking and hydrophobic interactions between
BODIPY dyes, and the hydrophobic interaction between PCL
chains and BODIPY dyes.
formed J-aggregates templated the self-assembly of P
to the formation of BD-PGMe/P nanoplates via hydrophobic in-
teraction between the PCL blocks of P and J-aggregated BD-
PGMe dyes as illustrated in Figure 1c. The formed J-aggregates
doped BD-PGMe/P nanoplates were quite stable to dilution
8
, leading
8
8
8
with negligible change in UV-visible absorption spectra follow-
ing five 2-fold serial dilutions with phosphate buffered saline
(PBS; Figure S7). The nano-assemblies were also demonstrated
to be very stable in cell culture medium, or in saline with a so-
dium chloride concentration of 9.0% (w/v), which is 10 times
that of physiological saline (0.90%, w/v; Figures S8 and S9). In
the case of BD-PGMe/P80, the strong hydrophobic interaction be-
tween PCL segments was more favorable to the formation of
micelles with dye aggregates entrapped within the hydrophobic
core (Figure 1c). The size of BD-PGMe/P80 aggregates was
slightly increased by 2.5 nm compared to that of P80 alone (Ta-
ble 1 and Figure 2f), indicating little impact of BD-PGMe dyes
on the self-assembly of P80. TEM images revealed discrete na-
noparticles with a size of approximately 70 nm for BD-
PGMe/P80, as shown in Figure 2g. At higher magnification,
darkened dots with sizes of 3-5 nm were observed throughout
the nanoparticle due to localized aggregated BD-PGMe dyes (in-
set, Figure 2g); both monomer and J-bands were displayed (Fig-
ure 2b), demonstrating that BD-PGMe dyes were entrapped
within the hydrophobic core in both slip- and non-stacked ar-
rangements.
Stimuli-triggered dye rearrangement within nano-assem-
blies. Having established that stable core-shell BD-PGMe/P
noplates with J-aggregated BD-PGMe dyes as cores can be read-
ily prepared in water by mixing P and BD-PGMe following a
8
na-
8
templated self-assembly strategy, developing useful responsive
outputs from the water-based J-aggregates would enable addi-
tional features and functions that promote promising applica-
tions in living systems. Since the caged-ester group at the meso-
position of BD-PGMe is capable of stimuli-triggered hydropho-
bic ester-to-ionic carboxylate conversion via self-immolative
31
chemistry, according to our previous report (Figure 1a), it
could be anticipated that the generated negative charges of BD-
PGMe units would disrupt or remodel the packing arrangement
of BD-PGMe aggregates (Figure 1c), thereby inducing the pho-
tophysical and/or photochemical changes of the entire nano-as-
sembly. Possessing arylboronate as a protecting group, BD-
PGMe could be converted to meso-COOH-substituted BD-
-
- 54
COO Me in the presence of peroxynitrite (OONO ), as evi-
denced by a remarkable blueshift in the UV-visible absorption
spectra (Figure 3a). HPLC analysis and high resolution mass
spectrometry (HRMS) of the assay solution further confirmed
To assess the impact of the dye content on the templated self-
assembly, several BD-PGMe/P
ferent dye contents from 0.1% to 3.4% were prepared. With re-
gard to BD-PGMe/P with low dye content (e.g., 0.1%), only a
8
solutions (0.4 g/L P
8
) with dif-
-
the complete conversion from BD-PGMe to BD-COO Me in the
-
presence of OONO as shown in Figure 3b and Figure S57, re-
8
spectively. These preliminary results suggested that the iodo-
substituents at the 2 and 6 positions did not affect the stimuli-
responsive feature of meso-ester-BODIPY dyes.
monomer band was observed in the UV-visible absorption spec-
trum (Figure 2h), suggesting that BD-PGMe dyes existed in a
non-stacked arrangement, which should be attributed to the ex-
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Figure 3. Changes in photophysical property and morphology of BD-PGMe/P
version. (a) UV-visible absorption spectra of BD-PGMe (4.0 µM) in CH CN/H O (10/1, v/v) upon incubation with OONO at different concentrations
0-1 equiv) for 1 min at 37 C. (b) HPLC chromatograms of BD-PGMe without treatment of OONO (top); with OONO (1 equiv) treatment for 1
nanoplates upon stimuli-triggered “BD-PGMe to BD-COO-Me” con-
8
-
3
2
o
-
-
(
o
min at 37 C (middle); and BD-COOHMe only (bottom). (c) UV-visible absorption spectra of the aqueous solution of BD-PGMe/P
µM BD-PGMe) upon incubation with OONO (5 equiv) for varying time intervals (0-10 min) at 37 C. (d) UV-visible absorption response of BD-
8
(0.1 g/L P
8
; 2.0
-
o
-
-
PGMe/P
other species); inset shows optical photographs of BD-PGMe/P
TEM image, and (g) AFM image of BD-PGMe/P nano-assemblies (2.0% dye content) upon incubation with OONO (5 equiv) for 30 min at 37 C.
Inset of (3e) shows a high magnification cryo-TEM image of the region marked with a red rectangle. (h) UV-visible absorption spectra of aqueous
8 8
(0.1 g/L P ; 2.0 µM BD-PGMe) towards various ROSs and some physiological nucleophiles (5 equiv for ClO and OONO ; 100 equiv for
-
8
solutions before and after OONO (5 equiv) treatment. (e) Cryo-TEM image, (f)
-
o
8
-
o
8 8
solutions of BD-PGMe/P (0.4 g/L P ) with different BD-PGMe contents (0.5-3.4%) upon incubation with OONO (5 equiv) for 30 min at 37 C. (i)
UV-visible absorption spectra of two-fold serially diluted solutions of BD-PGMe/P
-
8
(0.4 g/L P
8
; 3.4% dye content) upon incubation with OONO (5
o
equiv) for 30 min at 37 C.
-
We further investigated the OONO -triggered ester-to-car-
physiological nucleophiles (Figures 3d and S12). Notably, in
our previous report, the arylboronate group of a water-soluble
BODIPY-based probe (P-HP-FR, similar to BD-PGMe) was
3
1
boxylate conversion of BD-PGMe/P
8
nanoplates in the aqueous
milieu. As expected, upon addition of OONO (5 equiv), J-ag-
-
gregated BD-PGMe dyes were converted into non-stacked BD-
2 2
completely cleaved after 150 min incubation with H O (200
equiv) in aqueous solution. However, no appreciable change in
UV-visible absorption spectra was observed before 90 min in-
-
COO Me within 10 min, as evidenced from the emergence of a
-
new absorption band of BD-COO Me at approximately 655 nm
and the gradual attenuation of the J-band at 788 nm (Figure 3c).
cubation of BD-PGMe/P
ure S13a). The deprotection was not yet finished upon incuba-
tion with a higher concentration of H (5000 equiv) over 150
8 2 2
nanoplates with H O (200 equiv; Fig-
Concomitantly, the color of the BD-PGMe/P
obviously, as shown in the inset of Figure 3d. Compared with
BD-PGMe monomers in CH CN/H O (10/1, v/v) mixture (Fig-
ure 3a), OONO -actuated ester-to-carboxylate conversion of the
BD-PGMe/P nanoplates was slower. We proposed that dense
8
solutions changed
2 2
O
3
2
min (Figure S13b). The slower reaction rate of J-aggregated
BD-PGMe within nano-assemblies further demonstrated that
dense packing of dyes would slow down the ester-to-carbox-
ylate conversion rate. The J-band of BODIPY dyes disappeared
-
8
slip-packing of BD-PGMe dyes within nano-assemblies should
be responsible for the slower ester-to-carboxylate conversion. It
-
after OONO -triggered hydrophobic BD-PGMe to ionic BD-
-
-
was also demonstrated that the BD-PGMe/P
highly selective for OONO over other ROSs and several typical
8
nanoplates are
COO Me transition (Figure 3c), whereas the BD-COO Me dye
-
was not observed in the effluent following 24 hours of dialysis
ACS Paragon Plus Environment

Journal of the American Chemical Society
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-
of BD-COO Me/P
shown), indicating that BD-COO Me and P
8
solution via HPLC monitoring (data not Stimuli-activatable nano-photosensitizers for specific
-
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4
5
6
7
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still remained in the
photodynamic therapy. Benefiting from their excellent envi-
ronmental stability, large extinction coefficients, facile modifi-
cations, and low dark toxicities, iodo-substituted BODIPY de-
8
form of nano-aggregates as also confirmed by DLS measure-
ment (Figure S14).
rivatives are emerging as ideal photosensitizers (PSs) to convert
Cryogenic transmission electron microscopy (cryo-TEM)
1
molecular oxygen to singlet oxygen ( O
2
) upon light irradiation
was adopted to characterize the in-solution structures of the BD-
9
,55-58
-
for photodynamic therapy (PDT).
However, low selectiv-
COO Me/P
8
nano-assemblies, and bundled nanorods of ~180 nm
ity of the photosensitizers currently available for clinical PDT
causes nonspecific photodamage to nearby healthy tissues, as
in length were observed (Figure 3e). In TEM and AFM meas-
urements, similar bundled nanorods were observed, possessing
the same shape but longer length as those observed by cryo-
TEM (Figures 3f and 3g). Taking the same nanostructure mor-
phologies into consideration, the longer size observed in TEM
and AFM images should be attributed to drying of the nano-
assemblies during sample preparation. To understand more
about the formation mechanism of the unusual bundled nano-
59
well as prolonged skin injury. Targeted activation enables the
activatable photosensitizers to distinguish healthy from dis-
eased cells, reducing nonspecific damage to adjacent healthy
0
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39-42
cells.
Since the slip-stacked BODIPY dye templated BD-
PGMe/P
8
nanoplates can act as an aqueous reservoir of PS and
exhibt dramatic spectroscopic responses to external stimuli, the
potential exists to selectively modulate the photosensitivity of
rods, we prepared nano-assemblies of BD-COOHMe/P
8
(de-
s
BD-PGMe/P nanoplates in response to specific stimuli. A wa-
8
noted as BD-COOHMe/P
and P
cedure as that used for BD-PGMe/P
8
) via self-assembly of BD-COOHMe
ter-soluble 1,3-diphenylisobenzofuran derivative (QDPBF) was
8
directly in aqueous solution according to the same pro-
preparation. The morphol-
nano-assemblies was investigated by
TEM, and nanoparticles rather than nanorods were observed
1
60
employed as an indicator to evaluate the O
ligible change in QDPBF absorption was observed within the
aqueous solution of BD-PGMe/P (0.05 g/L P , 1.0 µM BD-
2
generation. Neg-
8
s
ogy of BD-COOHMe/P
8
8
8
-
PGMe) either in darkness (Figure S22), or upon light irradiation
(
Figure S15), indicating that bundled nanorods of BD-COO
might evolve from BD-PGMe/P nanoplates in the pres-
ence of peroxynitrite. As for BD-PGMe/P50 and BD-PGMe/P80
2
at 655 nm (5 mW/cm ; Figure 4a), indicating that the photosen-
Me/P
8
8
1
sitivity of BD-PGMe/P
8
nanoplates in producing O
2
was well
-
-
suppressed. Encouragingly, the quenched photosensitivity of
nano-assemblies, OONO -actuated BD-PGMe to BD-COO Me
conversions were faster than those of BD-PGMe/P (Figures 3c,
nano
BD-PGMe/P
8
nanoplates (hereafter denoted as
PS-PG) is a
8
-
-
desirable feature as it meets the criteria for the off state of an
activatable photosensitizer.
S16, and S17). The converted BD-COO Me/P50 and BD-COO
Me/P80 remained in the form of micellar structures owing to
strong hydrophobic interactions between PCL segments with
-
-
Gratifyingly, for the OONO (5.0 µM) converted BD-COO
-
nano
slight increment in size due to the hydrophilic BD-COO Me dyes
Me/P
8
solution (hereafter denoted as
PS), significant de-
(
Figure S18).
We further investigated OONO -actuated transformation be-
haviors of various BD-PGMe/P (0.4 g/L P ) solutions with BD-
crease in QDPBF absorption was observed under light irradia-
2
-
tion at 655 nm (5 mW/cm ) as shown in Figure 4b, demonstrat-
1
ing an effective O
2
generation. The change in absorbance of
8
8
QDPBF (ΔAbs) at 412 nm was recorded at 10-s intervals to
PGMe loading content from 0.5% to 3.4%, wherein BD-PGMe
dyes adopted a slip-stacked arrangement (Figure 2h). With re-
1
evaluate the photosensitivity with respect to producing O
2
. In
nano
comparison, the ΔAbs of
than that of
PS increased much more rapidly
8
gard to BD-PGMe/P nano-assemblies with dye content below
nano
nano
PS-PG (Figure 4c). The ΔAbs of
PS within
2
.0%, only the monomer band at 655 nm was observed in UV-
-
-
90s is comparable to that of BD-COO Me monomers and 15.9-
visible absorption spectra after OONO (5 equiv) treatment,
nano
-
-
fold greater than that of
PS-PG (Figure 4d), suggesting an
confirming that all OONO -converted BD-COO Me dyes were
dispersed in a non-stacked form (Figure 3h). When the dye con-
tent increased to 3.4%, another absorption band emerged at 740
“off-on” response in the photoinduced singlet oxygen genera-
tion process, thus demonstrating the effectiveness of the slip-
stacked dye arrangement in photosensitivity quenching and the
-
nm after OONO (5 equiv) incubation, which should be ascribed
-
stimuli-triggered restoring process. For quantitative evaluation,
to the J-aggregates of BD-COO Me dyes (Figure 3h). The
1
-
relative O
2
quantum yield (Φ
Δ
) was determined by using meth-
change in the UV-visible absorption spectra of the OONO -
nano
-
ylene blue (MB) as a reference, and the Φ values of
PS-PG in aqueous media were calculated as 0.49 and 0.03,
respectively (Figure 4d). Thus, it is clear that OONO at rela-
PS and
converted BD-COO Me/P
8
(3.4% dye content) solution was
D
nano
negligible following four 2-fold serial dilutions with PBS (Fig-
-
-
ure S19), demonstrating that the BD-COO Me J-aggregates were
tively low concentration is capable of converting a J-aggre-
stable in water even after 1/16 dilution and thus should be lo-
nano
-
gated-photosensitizers doped nanoplate ( PS-PG) with in-
cated within the BD-COO Me/P
8
nano-assemblies. Moreover,
(3.4% dye content) solu-
hibited photosensitivity into an active nano-photosensitizer
two-fold serially diluted BD-PGMe/P
8
nano
1
(
2
PS) that generates O when photoirradiated within the ther-
tions (i.e., 1/2, 1/4, and 1/8 dilutions) were prepared and treated
-
apeutic window (in this case at 655 nm).
with OONO (5 equiv). Both monomer and aggregate bands
nano
-
were observed in UV-visible absorption spectra with the ratio
of two absorption bands comparable to that of the original solu-
tion (Figure 3i), suggesting that the dye rearrangement occurred
To gain insights into the quenching mechanism of
the photosensitivities of BD-PGMe and BD-COO
PS-PG,
Me
in a
DMSO/water (8/2, v/v) mixture were evaluated, wherein
-
within the nano-assemblies. Having established that OONO at
BODIPY dyes (1.0 µM) dissolved as monomers (Figures 2a and
-
low concentration could induce a rapid rearrangement of J-
stacked BODIPY dyes within nano-assemblies in water, with
remarkable photophysical changes, we then sought to apply our
insights to the development of new stimuli-sensitive photofunc-
tional tools applicable to living systems.
S20). As shown in Figure 4d, ΔAbs of BD-COO upon pho-
Me
2
toirradiation (655 nm, 5 mW/cm ) for 90 s was 4.6-fold greater
than that of BD-PGMe, probably due to the difference in elec-
3
1
tron-withdrawing capability of their meso-substituents. The
nano
ΔAbs of slip-stacked BD-PGMe
(
PS-PG) upon photoirradi-
ation was found to be approximately 25% that of the BD-PGMe
monomer, suggesting an inhibition of photosensitivity induced
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0
Figure 4. Inhibition and stimuli-triggered recovery of the photosensitivity of BODIPYs in nano-assemblies. (a,b) UV-visible absorption spectra of
-
QDPBF upon light irradiation in the presence of (a) untreated or (b) OONO -treated solution of BD-PGMe/P
8
for varying time intervals (0-90 s) at
o
3
(
7 C. (c) Plots of change in absorbance (ΔAbs) of QDPBF at 412 nm upon light irradiation for different time intervals in the presence of untreated
-
8
green) or OONO -treated (steel blue) solution of BD-PGMe/P at 37 °C. (d) ΔAbs of QDPBF at 412 nm upon light irradiation for 90 s in the presence
-
of BD-PGMe/P
maximum (λabs) and O
8
(steel blue) without or with OONO pretreatment, or BODIPY monomers (BD-PGMe or BD-COO-Me; green). Data of absorption
1
2
quantum yields (Φ
Δ
) are listed. (e) ΔAbs of QDPBF at 412 nm upon light irradiation for 60 s in the presence of BD-PGMe
(
9
green) or BD-PGMe/P
0 s (f) in the presence of BD-PGMe/P
of BD-PGMe/P , BD-PGEt/P , or BD-PGBu/P
BD-PGMe) with different BD-PGMe contents (0.5-3.4%) without (green) or with (red) OONO pretreatment. Light source used in above experiments
8
(steel blue) at different concentrations of BD-PGMe (1.0-8.0 µM). (f-h) ΔAbs of QDPBF at 412 nm upon light irradiation for
-
8
, BD-PGMe/P50, or BD-PGMe/P80 without (steel blue) or with (green) OONO pretreatment, (g) in the presence
-
8
8
8 8
without (steel blue) or with (green) OONO pretreatment, (h) in the presence of BD-PGMe/P (1.0 µM
-
2
is a laser (655 nm, 5 mW/cm ). Conc. 1.0 µM BD-PG
R
; 0.05 g/L P
n
unless otherwise specified. As for (c-h), results represent the average of three
independent experiments, and error bars represent mean ± SD.
by intermolecular J-stacking (Figure 4d). In addition, ΔAbs of
BD-PGMe monomer upon photoirradiation was found to be in-
confirmed by the exclusively monomeric bands in UV-visible
absorption spectra (Figures S16 and S21) all present strong pho-
tosensitivities comparable to that of PS (Figures 4f, 4g, S23b,
nano
creased with dye concentration in the range of 1.0-8.0 µM,
nano
while for slip-stacked BD-PGMe
(
PS-PG), ΔAbs was almost
and S24b).
irrespective of dye concentration (Figure 4e), indicating that the
We also evaluated the photosensitivities of BD-PGMe/P
lutions with different dye contents from 0.1% to 3.4% (Figures
h and S25). Regarding BD-PGMe/P solutions without OONO
8
incubation, the photosensitivities were well suppressed, and the
8
so-
nano
photosensitivity of
PS-PG at 655 nm was completely sup-
pressed as a result of the J-stacking arrangement of BD-PGMe
dyes. Further studies on the photosensitivities of various BD-
-
4
PGMe/P
n
(BD-PGMe/P50, BD-PGMe/P80; Figures 4f and S23a)
efficiency of inhibition was dependent on the fractions of J-ag-
and BD-PG /P (BD-PGEt/P , BD-PGBu/P ; Figures 4g and
R
8
8
8
-
gregated dyes (α
ity of the stimuli-converted BD-COO Me/P
nificantly enhanced, as shown in Figure 4h. The photosensitiv-
J
). Upon treatment with OONO , photosensitiv-
S24a) nano-assemblies indicated that the efficiency of photo-
-
8
solutions was sig-
sensitivity inhibition increased with the fractions of J-aggre-
-
gated BODIPY dyes (α
J
). Meanwhile, OONO pretreated sam-
-
8
ities of BD-COO Me/P solutions with dye contents in the range
-
-
-
ples (i.e., BD-COO Me/P50, BD-COO Me/P80, BD-COO Et/P
8
,
-
of 0.1-2.0% were comparable to that of BD-COO Me monomer
-
and BD-COO Bu/P
8
) with non-stacked dye arrangements as
-
owing to the non-stacked arrangement of BD-COO Me dyes. As
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1
Figure 5. Intracellular O
2
2
production and cell viability assay. (a) Intracellular singlet oxygen ( O
2
) production using O
2
probe DCF-DA. RAW
64.7 cells were treated with nanoPS-PG for 30 min, and incubated with SIN-1 at different concentrations (first column: without SIN-1; second-
fourth columns: 250, 500, 1000 µM SIN-1; last column: 1000 µM SIN-1 and 100 µM minocycline) for 2 h, then stained with 10.0 µM DCF-DA for
20 min. The cells were then subjected to light irradiation for 5 min and imaged. Scale bar is 100 µm. (b) Normalized green channel emission intensities
quantified from fluorescence images in (a). (c) Live/Dead staining images of normal (top two lines) and activated (bottom two lines) RAW 264.7
nano
nano
cells. Cells were treated with different samples (first column: blank; second column:
PS-PG; third column:
PS and 100 µM minocycline) for 2 h, and then subjected to light irradiation (second and last lines) or without
light irradiation (top and third lines) for 5 min. After treatment, the cells were stained with 1.0 µM Calcein AM (live staining, green) and 6.0 µM PI
dead staining, red) for 20 min. Scale bar is 50 µm. (d) Cell viability of normal (top) or activated (bottom) RAW 264.7 cells treated at different
PS-PG and 100 µM minocycline;
nano
nano
fourth column:
PS; last column:
(
conditions determined by MTT assay. Error bars represent mean ± SD (n = 5). Light source used in above experiments is a red LED lamp (620-660
2
nano
nano
nm, 15 mW/cm ).
PS-PG and
PS containing 1.0 µM BODIPY dyes were used in the above experiments.
-
nano
for BD-COO Me/P
8
solutions with dye contents beyond 2.0%,
their photosensitivities were remarkably decreased due to the
partial BD-COO Me J-aggregates located in the BD-COO Me/P
nano-assemblies (Figure 3h), further demonstrating that the
highly ordered J-stacking of dyes resulted in the inhibition of
photosensitivity. Taken together, it was established that the
photosensitivity of BODIPY dyes was governed by the dye ar-
rangement within the nano-assemblies. Since BD-PGMe/P
noplates with 2.0% dye content (denoted as
sensitizers ( PS-PG) after internalization, dichlorofluores-
cein diacetate (DCF-DA) was chosen as an oxidant-sensitive
fluorescent probe to detect the formed singlet oxygen ( O
which rapidly oxidize DCF-DA into highly fluorescent dichlor-
ofluorescein (DCF).
irradiation (620-660 nm, 15 mW/cm ), RAW 264.7 cells treated
2
PS-PG and then DCF-DA showed negligible O gen-
eration, which was reflected by the unobservable fluorescence
intensity of DCF. A remarkable increase of DCF fluorescence
was observed in cells pretreated with SIN-1 (OONO donor),
and the enhancement in the fluorescence intensity was propor-
tional to the dose of SIN-1 (250-1000 µM), suggesting that ex-
ogenous OONO apparently activated the potential nano-photo-
sensitizers (Figures 5a and 5b). In contrast, the fluorescence sig-
nal related to DCF was significantly decreased by pretreating
the cells with the OONO scavenger minocycline, further ver-
ifying that exogenous OONO is the primary reason for intra-
cellular activation of
-
-
1
8
2
),
6
1,62
As shown in Figure 5a, upon LED light
2
nano
1
with
8
na-
nano
PS-PG) exhib-
−
ited the largest off/on enhancement ratio, this sample was used
in the following biological studies.
−
RAW 264.7 cells pretreated with or without SIN-1 (OONO
donor ) were incubated with
which the cells were washed. Upon imaging, fluorescence of
PS-PG was observed within cells indicating effective inter-
nalization (Figures S26a and S26b). To assess the effectiveness
of intracellular OONO in activating our quenched nano-photo-
-
47
nano
PS-PG for 2 h, following
nano
−
47
−
-
nano
PS-PG. It should be noted that the
abovementioned RAW 264.7 cells in different experimental
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Journal of the American Chemical Society
groups all showed negligible fluorescence of DCF before pho-
toirradiation (Figure S27), demonstrating that SIN-1 and its
generated OONO did not interfere with the O detection.
2
8
stacked dyes. Upon incubation of BD-PGMe/P nano-assem-
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blies with peroxynitrite at relatively low concentration, a rapid
rearrangement of BODIPY dyes was established, leading to
photosensitivity restoration. Since the mechanisms of stimuli-
responsive photophysical and/or photochemical changes are
based on the chemical structure transformation-induced dye re-
arrangement, they are not limited to the design of activatable
nano-photosensitizers, but instead significantly expands the
palette of design principles for photofunctional tools applicable
to living systems, such as fluorogenic probes and ratiometric
photoacoustic probes, among others, which could be prepared
by facilely replacing the BODIPY-based photosensitizers
adopted herein with other fluorophores or photoacoustic dyes
as functional template dyes on demand. Moreover, the synthesis
of carboxyl-caged dyes is flexible, thus allowing customization
of diverse dye-templates sensitive to a broad range of biorelated
species of interest via the introduction of specific triggering mo-
tifs. We envision that the scope and generality of slip-stacked
dyes templated nano-assemblies with photophysical responses
toward external stimuli would span far beyond the seminal ex-
ample presented here.
-
1
Encouraged by the above observations, the potential of
nano
−
PS-PG in response to endogenous OONO was evaluated in
−
RAW 264.7 macrophages, which are known to release OONO
upon stimulation with lipopolysaccharide (LPS)/interferon-γ
6
3
(
IFN-γ). After demonstrating effective internalization of
nano
PS-PG into activated RAW 264.7 cells (Figure S26c), nor-
mal or activated RAW 264.7 macrophages (upon preincubation
with LPS/IFN-γ for 20 h) were incubated with
nano
PS-PG, then
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5
6
7
8
9
0
2
subjected to light irradiation (620-660 nm, 15 mW/cm ) for 5
min. After the light irradiation, cells were incubated at 37 °C (4
h for Live/Dead assay; 12 h for MTT assay), and the cell viabil-
ity was assessed by using the Live/Dead cell viability assay with
propidium iodide (PI) and Calcein AM (Figures 5c and S28), as
well as MTT assay (Figure 5d). Only the activated RAW 264.7
nano
cells pretreated with
PS-PG were predominantly stained
with PI, indicating selective photo-induction of cell death. Cell
nano
viability was positively correlated with
PS-PG concentra-
tion and light irradiation dose (Figure S29a), but little cytotoxi-
city was observed among cells without light irradiation or nor-
nano
mal RAW 264.7 cells treated with
PS-PG (Figures 5c, 5d,
−
and S29). Addition of OONO scavenger minocycline to the
medium resulted in recovery of the viability of cells treated with
PS-PG (Figures 5c, 5d, and S28), supporting the view that
endogenous OONO selectively triggered the conversion of
■ ASSOCIATED CONTENT
nano
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website.
−
nano
nano
PS-PG into
PS with an “off-on” activation of the photo-
sensitivity, and thus induced cytotoxicity upon light irradiation.
For further reference to confirm the mechanism, we also pre-
Experimental details, biological studies, supplementary fig-
ures, NMR and HRMS spectra (PDF)
nano
nano
-
pared
PS by incubation of
PS-PG with OONO , which
has been proven to possess strong photosensitivity. After incu-
nano
■ AUTHOR INFORMATION
bating RAW 264.7 cells with
PS, strong emissions were ob-
nano
served within cells, suggesting the internalization of
PS into
Corresponding Author
*chli@nankai.edu.cn
Notes
both normal and activated cells (Figure S30). Live/Dead stain-
ing and MTT assay clearly showed that both normal and acti-
nano
vated RAW 264.7 cells treated with
PS were completely
2
The authors declare no competing financial interest.
killed upon light irradiation (620-660 nm, 15 mW/cm , 5 min),
no matter whether or not they were pretreated with minocycline;
meanwhile, cells without light irradiation appeared to suffer lit-
tle damage (Figures 5c and 5d). Collectively, the above results
■
ACKNOWLEDGMENT
Financial support from National Natural Science Foundation of
China (NSFC) projects (Grant no. 51673101 and 81601590), Nat-
ural Science Foundation of Tianjin of China (Grant no.
15JCZDJC65800), and the Fundamental Research Funds for Cen-
tral Universities (China) is gratefully acknowledged.
nano
-
suggest that
PS-PG could serve as an endogenous OONO
−
activatable photosensitizer that targets cells or tissues with ex-
-
cessive peroxynitrite. Since OONO is known to be overpro-
duced in atherosclerotic plaque-associated activated macro-
64-66 nano
phages,
PS-PG could be used as a promising activatable
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