Research Article

Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX www.acsami.org

Acid-Triggered Nanoexpansion Polymeric Micelles for Enhanced

Photodynamic Therapy
Sheng Zhong,†,‡ Chao Chen,†,§ Guoliang Yang,‡ Yucheng Zhu,‡ Hongliang Cao,*,‡ Beijian Xu,‡
Yaoqin Luo,‡ Yun Gao,‡ and Weian Zhang*,‡
‡
Shanghai Key Laboratory of Functional Materials Chemistry, School of Materials Science and Engineering, and §State Key
Laboratory of Bioreactor Engineering, Biomedical Nanotechnology Center, School of Biotechnology, East China University of
Science and Technology, 130 Meilong Road, Shanghai 200237, China
*
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ABSTRACT: Photodynamic therapy (PDT) as a noninvasive and selective

treatment technology has presented great potential in cancer prevention and
precision medicine, but its therapeutic efficacy is still greatly inhibited by the
limitations of photosensitizers (PSs) in the microenvironment such as the
aggregation caused quenching (ACQ) of PSs. Herein, we proposed an “acid-
triggered nanoexpansion” method to further reduce the aggregation of photo-
sensitizers by constructing acetal-based polymeric micelles. A pH-responsive
amphiphilic block copolymer, POEGMA-b-[PTTMA-co-PTPPC6MA] was synthe-
sized by reversible addition−fragmentation chain transfer (RAFT) polymerization
and self-assembled into spherical micelles. In the normal physiological environ-
ment, the micelles were stable and had good biocompatibility. Upon entry into the
acidic microenvironment of the tumor, the acid-responsive hydrophobic 2, 4, 6-
trimethoxybenzaldehyde in the micelles hydrolyzed and generated a hydrophilic
diol moiety. Although the hydrophility of the micellar core was increased, the
assembled structure of block copolymers was not dissociated but expanded. The responsive expansion of the micelles could
allow the photosensitizers to well-disperse in the core, whereas more tumor-dissolved oxygen entered the micelles. This
phenomenon could provide a better nanoenvironment for photosensitizers to reduce the ACQ of the photosensitizers, leading
to more singlet oxygen (1O2) produced under the laser irradiation (650 nm). Both in vitro and in vivo studies have
demonstrated that the remarkable photodynamic therapeutic efficacy of acid-responsive micelles could be realized. Thus, the
acid-triggered nanoexpansion method might provide more possibilities to develop efficient platforms for treating cancers.
KEYWORDS: acid-triggered nanoexpansion, photodynamic therapy, porphyrin, RAFT polymerization,
aggregation caused quenching (ACQ)

■ INTRODUCTION
Photodynamic therapy (PDT) plays an important role in the
field of cancer treatment.1 The general process of photo-
dynamic therapy involves photosensitizers (PSs) activated
under light irradiation with a specific wavelength to generate a
large amount of reactive oxygen species (ROS), ultimately
causing irreversible damage of diseased tissues or cells.2−4 In
the past decades, singlet oxygen (1O2) has been widely studied
as a common ROS.5−10 It is well-known that amphiphilic block
copolymers have been widely used in drug delivery systems for
PDT because of their high drug-loading efficiency and more
controllable drug release.11−13 The small molecular photo-
sensitizers could be encapsulated in assembled micelles formed
from amphiphilic block copolymers or quantitatively covalently
conjugated to the polymer chain to form release system.14,15
Although porphyrins and their derivatives as mostly used PSs
can be well delivered to tumor sites by block copolymers, they
are still inhibited by some limitations in the tumor micro-
environment such as the ACQ of photosensitizers and the
hypoxic environment of tumors.14,16−20 Traditional PSs are
limited by π−π interaction and their hydrophobic character-
istics, which cause the quenching of the electronic excited
state, thereby resulting in the limited 1O2 quantum yield.21
Meanwhile, the inadequate oxygen in the tumor environment
will also limit the production of singlet oxygen.22,23 In order to
overcome these challenges, typically, metal−organic frame-
works (MOFs) were constructed to prevent the aggregation of
PSs,24 and a porphyrin/polyhedral oligomeric silsesquioxane
(POSS) alternating copolymer was also designed to decrease
the aggregation between porphyrin units.25 Additionally,
MnO2 was utilized to react with endogenous H2O2 in tumors
to produce O2 and further regulate hypoxic tumor micro-
environment.26
Recently, stimuli-responsive drug delivery systems (espe-
cially, endogenous stimuli such as redox species,27−30
enzymes,31−35 and pH gradient36) offer a great promise in
Received: July 18, 2019
Accepted: August 26, 2019

© XXXX American Chemical Society A DOI: 10.1021/acsami.9b12620

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Scheme 1. Schematic Representation of the Acid Response Process of PM Copolymer and the Formation of PM Micelles and
the Mechanism of Photodynamic Therapy
improving drug release efficiency, controlling drug release and
reducing side effects. It is known that the physiological pH in
normal tissues and blood in the human body is approximately
7.4, the intracellular endo/lysosomal pH ranges from 4.0 to
6.5, and the extracellular pH in tumor tissues is about 6.8.37−40
Thus, this pH gradient presented in the tumor microenviron-
ment offers a great potential for specific targeting of tumors
and controlled release drugs at tumor sites by designing some
specific pH-responsive moieties in release systems, which has
been widely reported in previous studies.41−44 The pH-
responsive nanocarriers cover a wide range of categories, e.g.,
the vehicle is inherently sensitive to pH,45 reversible cross-
linking via metal−ligand coordination,46 and pH-sensitive
linkers (e.g., hydrazone, acetal47). As a typical pH-responsive
moiety, cyclic benzylidene acetal has also been used in the
construction of drug delivery systems for cancer therapies. For
example, Zhao et al. synthesized a comblike amphiphilic
copolymer with a cyclic benzylidene acetal functionalized
skeleton, which could release curcumin in cells as an effective
nanocarrier because of its ability to trigger hydrophobic−
hydrophilic transition in acid condition.48 Wang et al.
constructed a pH-responsive amphiphilic diblock copolymer
containing a cyclobenzyl acetal, which self-assembled into well-
defined vesicles for the intracellular delivery of hydrophobic
drug Nile red and hydrophilic doxorubicin hydrochloride
(DOX·HCl).49 Zhong et al. synthesized an asymmetric triblock
copolymer based on a pH-responsive cyclic benzylidene acetal
moiety, which self-assembled into a “chimeric” polymeric
group for delivery and release of granzyme B apoptosis protein
and hydrophilic DOX·HCl.50 Similar to other typical acid-
responsive linkages such as hydrazine,51−53 acetal,54,55 oxime
bonds,56 and cis-acotinyl,57,58 the hydrophobic cyclobenzyli-
dene acetal structure rapidly cleaves under mild acidic
conditions to form new hydrophilic structures. In addition, it
has the following characteristics in a nanomedicine system: (i)
B DOI: 10.1021/acsami.9b12620
Research Article
polymers containing cyclic benzylidene acetals have good
biocompatibility; (ii) the cyclic benzylidene acetal contains a
hydrophobic aromatic ring which will help form micelles,
whereas most hydrophobic drugs contain a cyclic ring or an
aromatic ring, and the aromatic group has a strong π−π
interaction with these drug molecules, which would result in
higher drug-loading rates and drug-encapsulation rates; (iii)
the cyclic benzylidene acetal is rapidly cleaved into a polar diol
moiety under the acidic tumor microenvironment, resulting in
hydrophobic−hydrophilic transformation. It is worth noting
that the hydrophobic−hydrophilic transition resulted from the
cyclic benzylidene acetal in the acid environment would lead to
the swelling of the photosensitizer micelles, and further provide
the potential to alleviate the aggregation of photosensitizers
while to promote more oxygen into the micelles. To the best of
our knowledge, there is currently no report on the acid-
triggered nanoexpansion strategy based on acetal moieties for
enhance photodynamic therapy.
Herein, we design and construct an acetal-based polymeric
photosensitizer platform (Scheme 1) to enhance the efficacy of
photodynamic therapy by an “acid-triggered nanoexpansion”
method. The block copolymer POEGMA-b-[PTPPC6MA-co-
PTTMA] (PM) was synthesized through the copolymerization
of porphyrin-based momoner (TPPC6MA) and acetal-based
monomer (TTMA) by using hydrophilic poly(oligo(ethylene
glycol) methyl ether methacrylate) (POEGMA) as RAFT
chain transfer agent, and PM was further self-assembled into
spherical micelles. The block copolymer has the following
advantages: (i) the pH-sensitive acetal moieties of TTMA units
undergo a hydrophobic to hydrophilic transition in tumor
intracellular acidic microenvironment (pH 5.0−6.5) that
causes the nanoparticles to swell, thereby reducing the
aggregation of porphyrin units and providing a better
nanoenvironment for photosensitizers; (ii) the micelle
prepared from this block copolymer would have low
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Figure 1. (a) Acid-responsive process of the PM micelle. (b) UV/vis absorption spectra of PM copolymer in THF and PM micelles in water. (c)
Hydrolysis kinetics of PM micelles at different pH values (5.0, 6.8, and 7.4) by measuring the change in UV/vis absorbance at 292 nm. (d) UV/vis
absorption spectra of PM micelles in PBS (pH 5.0) at different times. (e−g) DLS size distribution of PM micelles (PBS, pH 5.0) at 0, 12, and 36 h;
data are given as the mean ± SD (n = 3); inset: TEM images of PM micelles; scale bar: 200 nm (e, f), 500 nm (g).

phototoxicity in a normal physiological environment and be to the higher molecular weight region in comparison with that
beneficial for blood circulation. of the POEGMA homopolymer. In addition, the synthesized

■ RESULTS AND DISCUSSION

Synthesis of Amphiphilic Block Copolymer (PM and
PC). In our previous work, we have synthesized porphyrin-
containing methyl acrylate monomer via three-step reactions
(Scheme S1, Figure S1).59 The acetal-based monomer, 5-
methyl-2-(2,4,6-trimethoxyphenyl)-[1,3]-5-dioxanylmethyl
methacrylate (TTMA), was synthesized according to a
previous report by Grinstaff (Scheme S2, Figure S2).60
Synthesis of amphiphilic block copolymers PC and PM is
through a two-step reaction. First, the hydrophilic block
POEMGA was synthesized using 4-cyano-4-(ethylsulfa-nylth-
iocarbonyl) sulfanyl pentanoic acid (CESPA) as a chain
transfer agent (CTA) (Scheme S3, Figure S3). The degree of
polymerization (DP) of the homopolymer POEGMA could be
calculated about 10. Then, POEGMA was further applied as
the macro-RAFT agent for the subsequent RAFT polymer-
ization of TPPC6MA to prepare amphiphilic block copolymer
POEGMA-b-PTPPC6MA (PC) (Scheme S4, Figure S4), and
POEGMA was also utilized for RAFT copolymerization of
TPPC6MA with TTMA to produce amphiphilic block
copolymers PM (Scheme S5, Figure S5). The results of
amphiphilic block copolymers are summarized in Table S1.
The DP of TPPC6MA (DP = 3) and TTMA (DP = 10) in the
block copolymers was calculated by comparing δ 3.36 ppm
(protons of OEGMA) to that δ2.78 ppm (protons of
TPPC6MA) and 6.09 ppm (protons of TTMA) (Figure S4).
Similarly, the DP of PC was also calculated as listed in Table
S1. The GPC curves of these polymers were shown in Figure
S6, and it could be seen that the traces of PC and PM shifted
C DOI: 10.1021/acsami.9b12620
polymers have a relatively narrow polydispersity index (PDI)
(Table S1), revealing that the RAFT polymerization was well-
controlled.
pH-Triggered Response of the Copolymer Micelles.
The self-assembly behavior of PC and PM was further studied,
and the micelles of these copolymer were prepared by a typical
nanoprecipitation method.61 Typically, pyrene is used as a
fluorescence probe to measure the critical micelle concen-
tration (CMC) of the amphiphilic copolymer PC and PM in
phosphate buffer saline (PBS). As a result, the CMC of PM
and PC could be measured to be 1.26 and 0.63 μg/mL,
respectively (Figure S7). The CMC of PC is smaller than that
of PM, since the hydrophobic chain of PC is longer than that
of PM. In addition, from the UV/vis absorption spectrum of
the assembled PM micelles (Figure 1b), it was observed that
the spectrum exhibited a certain degree of red shift, and the
absorption intensity at 650 nm was also slightly improved. The
self-assembled morphologies of PC and PM in PBS (pH 7.4)
were observed by transmission electron microscope (TEM)
(Figure S8), and the corresponding hydrodynamic diameters
(Dh) were 158 and 174 nm, as measured by dynamic light
scattering (DLS) (Figure S9a, S9b), respectively. These results
showed that these micelles had a similar Dh, which was
consistent with the result of TEM. As a result, it can be
observed that the typical core−shell spherical micelles (about
100 nm) are formed in Figure S8. Careful observation reveals
that there are some small black spots around the micelles in the
image. This may be because that the salt particles are formed
after the PBS solution was air-dried, and presented as small
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black spots in the TEM image when the micelles were
prepared and stored in PBS solution.
According to previous reports, the acetal moiety is relatively
stable in neutral or alkaline media but rapidly hydrolyzed in an
acidic environment.48,49,60 In this work, the acid-response
process of PM micelles is illustrated in Figure 1a. To determine
the extent of hydrolysis of the cyclobenzylidene acetal moieties
under various pH environments (pH 5.0, 6.8 and 7.4), the
absorbance at 292 nm (the characteristic absorbance of the
hydrolyzed small molecule, 2, 4, 6-trimethoxybenzaldehyde) in
UV/vis spectroscopy was recorded. Figure 1c shows that the
rate of hydrolysis of the acetal in the PM micelles is highly
dependent on the pH, e.g., the stronger the acidity of the
micelles, the faster the hydrolysis rate, which is consistent with
previous reports.48,49 Typically, PM micelles remained stable
for long time in the normal physiological environment (pH
7.4), and there was almost no hydrolysis. However, the
micelles exhibited a rapid structural change in the acidic
conditions, especially for the fast hydrolysis at pH 5.0.
Additionally, the hydrolysis degree of acetal moieties was
dependent on the time and it nearly completely hydrolyzed at
pH 5.0 after 24 h (Figure 1c). In contrast, the UV/vis
absorption intensity of PC micelles in PBS (pH 5.0) did not
change with time, indicating that the micelles did not have
acid-responsive characteristic (Figure S10).
Afterward, the size and morphological changes of the
micelles in response to the hydrolysis of the cyclobenzylidene
acetal were evaluated by DLS and TEM. As shown in Figure
1e, the Dh of the micelles (pH 5.0) gradually increased and the
unimodal distribution of the hydrodynamic diameter also
became broad with a small shoulder peak during the first 12 h,
indicating the occurrence of the hydrolysis and the micelles
were swollen. With the incubation time up to 36 h, it was
found that the size of the PM micelles became much larger,
and the hydrodynamic diameter changed from a single peak to
a bimodal distribution with a quite small peak. Correspond-
ingly, the polydispersity index increased sharply from 0.16 (0
h) to 0.52 (36 h), indicating the formation of hydrophilic diol
moieties. Furthermore, it could be seen that the size of PM
micelles remained constant after about 24 h. In contrast, the
Dh of PM polymeric micelles did not change significantly after
36 h of incubation in PBS (pH 7.4) (Figure S9a), because the
cyclic benzylidene acetal moiety is quite stable in the neutral.
The TEM images corresponding to the above processes were
shown in the insets of Figure 1e, 1f and 1g. The size of micelles
gradually increased with incubation time at pH 5.0 and most
micelles were swollen at 36 h, but their morphologies did not
change significantly. This means that the PM micelles were
only swollen but they still could keep spherical assembled
structures when the cyclic benzylidene acetal moieties were
hydrolyzed. Additionally, for the control samples of PC
micelles, they could remain stable in a weakly acidic
environment.
In addition, we explored the effect of the acetal structure on
the photophysical properties of micelles after hydrolysis. It
could be seen that there was no different UV/vis absorption
(300−700 nm) for two samples in PBS (pH 5.0) (Figure
S11b). However, the fluorescence intensity of PM micelles is
significantly improved with the hydrolysis time (Figure 2a).
Here, the hydrolysis of the acetal groups greatly improved the
hydrophilicity of the core, where the porphyrin units can be
well dispersed. For the control samples of PC, the fluorescence
intensity did not increase but reduce due to the self-
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Figure 2. Fluorescence emission spectra of (a) PM, (b) PC micelles
at different time intervals in a buffer solution (pH 5.0). (c) UV/vis
spectra at 420 nm after 10 s of irradiation of DBPF with PM micelles
in a buffer solution (pH 5.0 and 7.4) for different times (1 and 12 h).
(d) UV/vis spectra at 420 nm after 10 s of irradiation of DBPF with
PC micelles in a buffer solution (pH 5.0 and 7.4) for different times
(1 and 12 h).
aggregation of the hydrophobic porphyrin units in the micelles
(Figure 2b).62 In addition, PM below the CMC could not form
micelles, so it could be identified as broken micelles. To
compare the effects of the broken micelles and expanded
micelles on the photosensitizer ACQ under acidic environ-
ment, we measured the fluorescence intensity of PM in acidic
environments above CMC and below CMC, and compared the
fluorescence intensity of the same concentration of free
photosensitizers in organic solvent. When the concentration
of PM and free meso-tetraphenylporphyrin (TPP) was 4 ×
10−5 mg/mL (<CMC), the fluorescence intensity of TPP in
THF was significantly higher than that of PM in PBS (pH 5.0)
at 0 h. Furthermore, the fluorescence intensity of TPP in the
organic solvent remained stable, while the fluorescence
intensity of PM in PBS (pH 5.0) decreased with time (Figure
S13a), which was attributed to the fact that hydrophobic
porphyrins gradually aggregate in water to cause quenching.
When the concentration of PM and TPP was 0.002 mg/mL
(>CMC), the fluorescence intensity of TPP in THF was also
significantly higher than that of PM micelles in PBS (pH 5.0)
at 0 h. However, the fluorescence intensity of TPP in the
organic solvent remained stable after 12 h, and the
fluorescence intensity of PM in PBS (pH 5.0) increased with
time (Figure S13b). It could be seen from the above results
that the fluorescence intensity of the expanded PM micelles in
the acidic environment increased with time but the broken
micelles decreased, the fluorescence intensity of expanded PM
micelles in PBS (pH 5.0) would be close to that of the free
TPP in THF. It could be explained that the swollen PM
micelles reduced the ACQ of the photosensitizers and
enhanced the fluorescence intensity compared with the broken
micelles.
Singlet Oxygen (1O2) Generation. The yield of 1O2 is a
very important parameter for evaluating the efficacy of
photodynamic therapy. As a singlet oxygen scavenger, 1,3-
diphenylisobenzofuran (DPBF) was often used to detect the
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1
O2 produced by these micelles and the UV/vis absorption
spectrum of DPBF was monitored at intervals of 10 s with a
laser of 650 nm. The yield of singlet oxygen was evaluated by
observing the absorbance attenuation of up to 420 nm. It could
be seen that PM, PC and free TPP had a similar singlet oxygen
yield in THF (Figure S14). However, the singlet oxygen yield
of PM micelles increased with the increase of acidity in PBS
(pH 5.0 and 7.4), and their singlet oxygen yield in PBS (pH
5.0) was significantly higher than those in pH 7.4. The singlet
oxygen yield of PC did not change, because they are quite
stable at pH 5.0−7.4 (Figure 2d). At the same time, we also
studied the singlet oxygen yield of the micelle at the same pH
with different standing time. It was found that only the singlet
oxygen yield of PM micelles increased with the extension of
the standing time, further illustrating the acid-responsive
polymers could greatly promote singlet oxygen yield.
Cellular Uptake of Micelles. To evaluate intracellular
uptake of PC and PM micelles, the micelles were added to
HepG2 cells and incubated together for different time. The
fluorescence intensities of intracellular porphyrins of the two
samples were observed to increase with incubation time by
confocal laser scanning microscopy (CLSM) (Figure 3),
Figure 3. HepG2 cell uptake of PM and PC micelles at different times
(4 and 24 h). Scale bar: 50 μm.
demonstrating that the uptake of PC and PM micelles was
time-dependent. Furthermore, because the swelling of PM
micelles in the acidic environment of tumor cells reduced the
ACQ of porphyrins, the intensity of fluorescence of PM
micelles was stronger than that of PC micelles for the same
incubation time (Figure 3). To evaluate the effect of PM
micelles on the amount of ROS produced in cells, the probe
2′,7′-dichlorodihydrofluorescein diacetate (DCFHDA) was
utilized to reveal the level of ROS (1O2). It could be observed
from Figure 4a that there was almost no fluorescence in the
cells with PBS (pH 7.4) under the 650 nm laser irradiation, but
cells treated with PC and PM micelles showed significant
green fluorescence, demonstrating that ROS was produced
within cells treated by PC and PM micelles after laser
irradiation. Moreover, acid-responsive PM micelles could
generate significantly stronger fluorescence than PC micelles,
E DOI: 10.1021/acsami.9b12620
Research Article
which is consistent with the above results of the 1O2 detected
by DPBF.
On the basis of the above results, it is reasonable to propose
that hydrolysis of the acid-responsive cyclic benzyl acetal
structure in the weakly acidic microenvironment of the tumor
is beneficial for photodynamic therapy of the tumors. Thereby,
the antitumor efficacy of these micelles against HepG2 cells
was evaluated. First, the biocompatibility of PC and PM
micelles were evaluated by MTT assay. The experimental
results showed that the survival rate of HepG2 cells without
laser irradiation was higher than 90% (Figure 4b), indicating
that the two micelles have good biocompatibility. Then, we
evaluated the cell viability treated by the two micelles with
irradiation to determine the efficacy of PDT. As shown in
Figure 4c, the phototoxicity of two micelles was increased with
the concentration of porphyrin. When the concentration of
porphyrin in PM and PC micelles was higher than 25 μg/mL,
the phototoxicity of PM micelles was significantly higher than
that of PC. As we expected, the acid-responsive cyclic benzyl
acetal structure in the acidic tumor microenvironment could
effectively enhance the efficacy of PDT.
In Vivo Tumor Fluorescence Imaging and PDT
Effects. Encouraged by the improved antitumor efficacy of
PM micelles versus PC micelles exposed to laser irradiation in
HepG2 cells, the in vivo antitumor efficacy of these samples
was explored. The distribution of PM micelles at different time
was first determined by fluorescence imaging of tumors in
mice. As shown in Figure 4d, it could be clearly observed that
because of the enhanced permeability and retention (EPR)
effect, the porphyrin fluorescence signal of the PM micelles
increased over time in the tumor region. After 48 h of injection
of PM micelles into the tail vein of the mice, the mice were
euthanized, and tumors and organs were collected for
fluorescence imaging (Figure S15). It could be observed that
the porphyrin fluorescence signal in the normal organs was
significantly weaker than tumor, demonstrating that PM
micelles had an effective accumulation in the tumor region.
To study the phototherapeutic effect of each micelle in vivo,
we performed the following five treatment methods in the
HepG2 tumor-bearing nude mouse model: (a) PBS, (b) PC
micelles (no laser), (c) PC micelles with laser (PC micelles +
L), (d) PM micelles (no laser), and (e) PM micelles with laser
(PM micelles + L). On the basis of the results of previous in
vivo tumor fluorescence imaging, the tumors of mice were
irradiated with 650 nm laser for 30 min within 24 h after
administration. After treatment, the tumor size and body
weight of all mice were recorded every other day for 2 weeks.
As a result, the mice treated with PBS (pH 7.4) and the PC
and PM micelles groups without any illumination showed
similar maximum tumor growth. However, the size of the
tumors with irradiation was decreased and the photo-
therapeutic efficacy of PM micelles was better than that of
PC micelles (Figure 5a, b). Furthermore, representative
photographs of tumors excised from mice (Figure 5c)
demonstrated that the mice treated with PM micelles after
illumination could provide superior antitumor efficacy over PC
micelles. In addition, the body weight of all mice remained
stable during the treatment, indicating that these systemic
agents did not have major systemic toxicity and that mice grew
well (Figure 5d). Standard H&E staining was further
histologically applied to evaluate the therapeutic effect of
each treatment group on tumors. It was worth noting that the
H&E-stained tumor tissue of mice treated with PM micelles
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Figure 4. (a) CLSM images of intracellular 1O2 generation. Scale bar: 50 μm. (b) Cell viability of HepG2 cells with different concentration of
porphyrin without irradiation. Data are given as the mean ± SD (n = 5) (c) Cell viability of HepG2 cells with different concentration of porphyrin
after laser irradiation. Data are given as the mean ± SD (n = 5), *p > 0.05, **p < 0.05, and ***p < 0.001, determined by a Student’s t test. (d)
Fluorescence images of mice injected with PM micelles at different times.

under laser irradiation caused more severe tumor cell apoptosis
than PC micelles, further verifying that PM micelles containing
acid-responsive moieties had more excellent phototherapic
effects (Figure 5e). After 14 days of PDT, the histological
morphology of the main normal organs (i.e, heart, liver, spleen,
lung and kidney) of the mice in each treatment group was
studied by H&E staining. From the results of Figure S16, it
could be seen that there were no obvious tissue damage or
pathological change in the tissues and organs of the mice in
each treatment group, which proved that it had good
biocompatibility and future therapeutic prospects.
moiety is rapidly cleaved to form a polar diol, which undergoes
a hydrophobic to hydrophilic transition. The results of DLS
and TEM verified the acid-responsive expansion process of
micelles, which provides the possibility to reduce the
aggregation of the photosensitizers. Meanwhile, in vitro and
in vivo studies have been confirmed that the response
expansion of the micelles can make porphyrin produce more
singlet oxygen to enhance the antitumor effect, and the
fluorescence intensity of the photosensitizer is improved to
some extent. Therefore, the phenomenon that the micelles
expand under acidic conditions could develop new ideas for

■
simultaneously reducing the ACQ of the photosensitizers and
providing a better microenvironment for photosensitizers.

CONCLUSIONS
In summary, we have proposed a novel “acid-triggered
nanoexpansion” method for enhancing the efficiency of
photodynamic therapy. Spherical micelles have been obtained
by the self-assembly of the copolymer, POEGMA-b-
[PTPPC6MA-co-PTTMA]. After the micelles enter the acidic
microenvironment of the tumor, the cyclic benzylidene acetal
■
EXPERIMENTAL SECTION
Synthesis of Amphiphilic Block Copolymer, PM. Typically,
POEGMA (200 mg, 42.11 μmol), TPPC6MA (150 mg, 188.0 μmol),
TTMA (95 mg, 260.5 μmol), AIBN (1.82 mg, 11.1 μmol), and 3 mL
of THF were added into a dry polymerization tube. The mixture
solution was degassed and the polymerization tube then was sealed

F DOI: 10.1021/acsami.9b12620
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Figure 5. (a) Relative tumor volume of the mice after different
treatments (n = 4).(b) Tumor weights of mice after different
treatments on the 14th day (n = 4), *p > 0.05, **p < 0.05, and ***p <
0.001, determined by a Student’s t test. (c) Photographs of excised
tumors from tumor-bearing mice after received various treatments on
the 14th day (n = 4). (d) Body weight changes of mice after different
treatments (n = 4). (e) H&E- stained tumor after different treatments
on the 14th day (n = 4). Scale bar: 100 μm.
under vacuum. The polymerization tube was heated at 70 °C and
stirred for 48 h, and then precipitated three times with an excess of ice
diethyl ether. The product (181 mg, 35%) was characterized by 1H
NMR (Figure S4).
Self-Assembly of Block Copolymer (PM and PC). Typically,
10 mg of the copolymer was first dissolved in 10 mL of THF and
copolymer solution (1 mL) was then dropwise added into 4.0 mL of
PBS (10 mM, pH 7.4) over 1 h. After being further stirred for 1 h, the
micelles were formed in the mixture solution. In addition, the solution
was stirred overnight and the resulting solution was dialyzed against
PBS (pH 7.4) for 3 days to remove THF.
Cellular Uptake Assays. CLSM is used to detect the uptake of
drugs by HepG2 (liver hepatocellular carcinoma) cells. Similarly, cell
plating was first performed in a Petri dish, 10,000 cells per dish and 2
mL of medium was added for culture. After 24 h of incubation, PC
and PM micelles containing the drug (porphyrin) at a concentration
of 25 μg/mL were mixed with the medium and added to a 6-well plate
at 2 mL/well. After the solution was cultured 4 and 24 h, the medium
was removed and the HepG2 cells were washed 3 times with PBS (pH
7.4), and then 4% paraformaldehyde solution was added to the
culture dish for 20 min. Thereafter, the cells were washed again to
ensure that the excess paraformaldehyde was removed and DAPI was
added to the culture dish for nuclear staining for 3 min. Finally, excess
DAPI was removed and the HepG2 cells in the culture dish were
imaged using a laser confocal microscope.
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsami.9b12620.
G DOI: 10.1021/acsami.9b12620
Research Article
Experimental details and additional data (Figures S1−
S16, Schemes S1−S5, Table S1) (PDF)
■ AUTHOR INFORMATION
Corresponding Authors
*Email: caohl@ecust.edu.cn (H.C.).
*Email: wazhang@ecust.edu.cn (W.Z.).
ORCID
Hongliang Cao: 0000-0002-9342-7980
Weian Zhang: 0000-0002-1717-597X
Author Contributions
†
S.Z. and C.C. contributed equally to this work. The
manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This work was financially supported by the Shanghai Natural
Science Foundation (18ZR1408300) and the National Natural
Science Foundation of China (21574039 and 21875063).
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I DOI: 10.1021/acsami.9b12620
ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX