Light-Triggered Clustered Vesicles with Self-Supplied Oxygen and Tissue Penetrability for Photodynamic Therapy against Hypoxic Tumor

Article abstract of DOI:10.1002/adfm.201702108

Smart nanocarriers are of particular interest for highly effective photodynamic therapy (PDT) in the field of precision nanomedicine. Nevertheless, a critical challenge still remains in the exploration of potent PDT treatment against hypoxic tumor. Herein, light-triggered clustered polymeric vesicles for photoinduced hypoxic tumor ablation are demonstrated, which are able to deeply penetrate into the tumor and simultaneously afford oxygen supply upon light irradiation. Hydrogen peroxide (H₂O₂) and poly(amidoamine) dendrimer conjugating chlorin e6/cypate (CC-PAMAM) are coassembled with reactive-oxygen-species-responsive triblock copolymer into the polymeric vesicles. Upon 805 nm irradiation, the vesicles exhibit the light-triggered thermal effect that is able to decompose H₂O₂ into O₂, which distinctly ensures the alleviation of tumor hypoxia at tumor. Followed by 660 nm irradiation, the vesicles are rapidly destabilized through singlet oxygen-mediated cleavage of copolymer under light irradiation and thus allow the release of photoactive CC-PAMAM from the vesicular chambers, followed by their deep penetration in the poorly permeable tumor. Consequently, the light-triggered vesicles with both self-supplied oxygen and deep tissue penetrability achieve the total ablation of hypoxic hypopermeable pancreatic tumor through photodynamic damage. These findings represent a general and smart nanoplatform for effective photoinduced treatment against hypoxic tumor.

Full text of DOI:10.1002/adfm.201702108

FULL PAPER
Cancer Therapy
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Light-Triggered Clustered Vesicles with Self-Supplied
Oxygen and Tissue Penetrability for Photodynamic Therapy
against Hypoxic Tumor
Junjie Li, Kai Wei, Shuai Zuo, Yixuan Xu, Zengshi Zha, Wendong Ke, Huabing Chen,*
and Zhishen Ge*
to their multiple properties such as spati-
otemporally selective treatment, avoidance
Smart nanocarriers are of particular interest for highly effective photody-
namic therapy (PDT) in the field of precision nanomedicine. Nevertheless,
a critical challenge still remains in the exploration of potent PDT treatment
against hypoxic tumor. Herein, light-triggered clustered polymeric vesicles
for photoinduced hypoxic tumor ablation are demonstrated, which are able
to deeply penetrate into the tumor and simultaneously afford oxygen supply
of drug resistance, and minimal systemic
[
1]
adverse side effect. Several factors are
considered as essential prerequisites
for effective PDT including photosensi-
tizer, light, and oxygen. Fundamentally,
excited photosensitizer is able to trans-
form molecular oxygen into toxic reac-
upon light irradiation. Hydrogen peroxide (H O ) and poly(amidoamine)
2
2
dendrimer conjugating chlorin e6/cypate (CC-PAMAM) are coassembled with
reactive-oxygen-species-responsive triblock copolymer into the polymeric
vesicles. Upon 805 nm irradiation, the vesicles exhibit the light-triggered
thermal effect that is able to decompose H O into O , which distinctly
ensures the alleviation of tumor hypoxia at tumor. Followed by 660 nm irra-
diation, the vesicles are rapidly destabilized through singlet oxygen-mediated
cleavage of copolymer under light irradiation and thus allow the release of
photoactive CC-PAMAM from the vesicular chambers, followed by their deep
penetration in the poorly permeable tumor. Consequently, the light-triggered
vesicles with both self-supplied oxygen and deep tissue penetrability achieve
the total ablation of hypoxic hypopermeable pancreatic tumor through photo-
dynamic damage. These findings represent a general and smart nanoplat-
form for effective photoinduced treatment against hypoxic tumor.
tive oxygen species (ROS) such as singlet
1
oxygen ( O ) for achieving apoptosis-medi-
2
1a]
[
ated PDT. However, PDT treatment is
often compromised by several frequently
encountered concerns such as tumor
hypoxia, rapid oxygen depletion, as well
as limited penetration depth of photo-
2
2
2
[
1d,e,2]
sensitizers in tumor.
To date, several
strategies have been explored to clinically
overcome these drawbacks, including opti-
mization of PDT operation through alter-
[3]
nating cycles of light–dark irradiation,
and increase of oxygen concentration in
[
4]
tumor by hyperbaric oxygen. Neverthe-
less, it remains a critical challenge in the
exploration of nanocarriers that are able to
perform highly efficient PDT to effectively
ablate tumors, particularly hypoxic tumors.
Hypoxia as a hallmark of solid tumors significantly impairs
1
. Introduction
[
1c–e]
Photodynamic therapy (PDT) as a noninvasive therapeutic
modality has attracted great attention in recent years, owing
the anticancer efficiency of oxygen-dependent PDT.
continuous consumption of tissue oxygen during PDT often
The
[
3a,5]
leads to the aggravation and persistence of tumor hypoxia.
Moreover, the reoxygenation can often be limited by ROS-
J. J. Li, K. Wei, S. Zuo, Y. X. Xu, Z. S. Zha, W. D. Ke,
Prof. Z. S. Ge
CAS Key Laboratory of Soft Matter Chemistry
Department of Polymer Science and Engineering
University of Science and Technology of China
Hefei 230026, China
[5b,6]
induced destruction of the tumor vasculature as well.
Thus,
some approaches have been explored to afford reoxygenation,
such as catalytic decomposition of endogenous hydrogen per-
[
7]
oxide (H O ) by MnO nanoparticles/catalase at tumors, mod-
2
2
2
E-mail: gezs@ustc.edu.cn
ulation of tumor microenvironment by increasing blood vessel
Prof. H. B. Chen
[8]
density or ratio of dilated vessels, as well as perfluorocarbon-
Jiangsu Key Laboratory of Translational Research
and Therapy for Neuro-Psycho-Diseases
College of Pharmaceutical Sciences
Soochow University
Suzhou 215123, China
E-mail: chenhb@suda.edu.cn
[9]
containing nanoparticles as oxygen reservoir. Unfortunately,
low endogenous H O level in tumor tissue often causes insuf-
2
2
[
10]
ficient oxygen generation,
while exogenous oxygen is also
difficult to be delivered into tumor far away from blood ves-
sels. Furthermore, the limited penetration depth of photosen-
sitizers in heterogeneous tumors might further compromise
their PDT efficiency owing to their nonuniform distribution in
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/adfm.201702108.
[
7b,11]
DOI: 10.1002/adfm.201702108
tumors,
although various nanoparticles have been explored
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to facilitate deep penetration of nanocarriers into tumors
805 nm irradiation, the light-triggered thermal effect from the
vesicles decomposes encapsulated H O2 into O , which dis-
through multistage drug release or stimuli-responsive size
2
2
change.[
7b,12]
For instance, Shen and co-workers have demon-
tinctly facilitates the alleviation of tumor hypoxia at tumor site.
Followed by 660 nm irradiation, the copolymer is rapidly dis-
rupted through singlet oxygen mediated cleavage under light
irradiation, which allows the destabilization of vesicles and sub-
sequent release of CC-PAMAM from the vesicles in response to
light irradiation, followed by their deep penetration and subse-
quent ROS generation in the hypopermeable tumor. Thus, the
light-triggered clustered vesicles with both self-supplied oxygen
and deep tissue penetrability cause the total ablation of hypoxic
and hypopermeable pancreatic tumors.
strated “cluster-bomb”-like fusogenic lipid nanoparticles encap-
sulating drug-conjugated dendrimers with enhanced tumor
accumulation,[12a] which can release encapsulated dendrimers
in the tumor for subsequent penetration and drug release.
Hence, in view of primary barriers (e.g., tumor hypoxia and
poor tissue penetration) hampering efficient PDT, it is urgently
desired to explore high-performance vehicles that can alleviate
tumor hypoxia and simultaneously deliver photosensitizer into
deep tumor for potent PDT.
Previously, several types of polymeric micelles incorporating
cisplatin-conjugated poly(amidoamine) (PAMAM) dendrimers
were explored to achieve the multistage release behaviors
including initial release of encapsulated PAMAM and subse-
quent drug release from PAMAM for deep drug penetration in
the tumors.[ Herein, we engineered light-triggered, clustered
polymeric vesicles with self-supplied oxygen and tissue penetra-
bility for potent PDT against hypoxic tumor. Hydrogen peroxide
2
. Results and Discussion
2.1. Polymer Synthesis and Preparation of
CC-PAMAM-Clustered Vesicles
13]
It has been demonstrated that ROS from photosensitizers upon
light irradiation can trigger the cleavage of ROS-responsive
thioketal moiety.
(
H O ) and PAMAM dendrimer conjugating chlorin e6 (Ce6)/
2 2
[
14]
cypate (CC-PAMAM) are coassembled with ROS-responsive tri-
The amphiphilic ABA triblock copolymer
block copolymer P1 into polymeric vesicles (Scheme 1A). Upon
P1 consisting of both hydrophobic segment containing
Scheme 1. A) Schematic illustration of phototriggered clustered vesicles for potent PDT against hypoxic tumor, which are able to release CC-PAMAM
with tissue penetrability in response to light and simultaneously afford oxygen supply through photothermal decomposition of H O into O . B) Syn-
2
2
2
thetic routes of triblock copolymers P1 with ROS-responsive thioketal moiety and P2 without ROS-responsive moiety.
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ROS-responsive thioketal moiety and hydrophilic poly(ethylene
glycol) (PEG) at two sides was synthesized (Scheme 1B). Briefly,
the thiol-acrylate Michael addition reaction between excess
uniform spherical morphology with 155.2 ± 21.5 nm in diam-
eter from the TEM images, which was also consistent with
their hydrodynamic diameter of 187.5 ± 16.6 nm measured
by dynamic light scattering (DLS) (Figure 1C). The mem-
brane thickness was estimated to be 5.6 ± 1.5 nm as shown in
Figure 1A.
1
4
,6-hexanedithiol (M1) and thioketal-contained 8,8-dimethyl-
,12-dioxo-3,13-dioxa-7,9-dithiapentadecane-1,15-diyldiacrylate
(
M2) was employed to produce telechelic hydrophobic
[15]
polymer,
which was subsequently end-capped with PEG
methyl ether acrylate (PEG-acrylate) (number-average molecular
weight (M ), M = 2000) to produce ABA triblock copolymer
2.2. H O Retention in Vesicles, Light-Triggered Vesicle
Disruption and CC-PAMAM Release, Photothermal Effect,
Oxygen Generation, and ROS Production
n
n
2
2
(
Figures S1 and S2, Supporting Information). M1 was used in
excess to ensure the exposure of free sulfhydryl groups at the
end of telechelic polymer for the successful capping reaction
with PEG-acrylate. The specific excess amount of M1 compared
with M2 will determine the final molecular weight (MW). The
To evaluate H O2 retention inside HC@P1-Vesicle, we deter-
2
mined the retention behavior in their vesicular chamber using
theoretical M and degree of polymerization (DP) were adjusted
a membrane-impermeable H O -specific probe 6′-O-pentafluor-
n
2
2
[15]
[16]
via the feed monomer ratios of M1 and M2. The final poly-
mers were purified by dialysis in aqueous solution to remove
unreacted small molecules and PEG-acrylate. Gel permeation
chromatography (GPC) characterization indicated successful
removal of unreacted PEG-acrylate as evidenced by no shoul-
ders at the low MW position of P1 curve (Figure S2, Supporting
Information). As a control, the block copolymer P2 with compa-
rable MW and the corresponding hydrophobic segment without
ROS-responsive thioketal moiety was also synthesized.
obenzene sulfonyl-2′,7′-difluorofluorescein (BES-H O ).
It
2
2
indicates that less than 20% of H O was found to diffuse out
2
2
of HC@P1-Vesicle after 24 h incubation (Figure S6, Supporting
Information), indicating a slow leakage rate of H O . Such long-
2
2
term retention might result from the leakage-retarding eBsffect
of nitrogen-rich PAMAM on H O through the van der Waals
2
2
[
17]
interaction. In contrast, the subsequent exposure to one cycle
of 805/660 nm irradiation resulted in the fast release of H O
2
2
to over 80%. Presumably, one cycle of 805/660 nm irradiation
will induce disruption of the vesicles leading to quick release of
encapsulated H O . For comparison, single 805 nm irradiation
We further conjugated both Ce6 and cypate with PAMAM
dendrimers for Ce6/cypate-conjugated PAMAM dendrimers
2
2
(CC-PAMAM) through the amidation reaction between the
caused a small amount of H O release because only a short-
2 2
amino groups of PAMAM dendrimer (G5-PAMAM) and car-
boxyl groups of Ce6 and cypate, followed by the dialysis for
purification. The numbers of Ce6 and cypate molecules per
PAMAM were determined to be 10 and 8, respectively, based
on the UV–vis absorbance peaks at 405 and 778 nm, respec-
tively (Figure S3, Supporting Information). CC-PAMAM as the
photoactive agent were found to be well dispersed in aqueous
solution, as confirmed by their negligible size change com-
pared to free PAMAM (Figure S4, Supporting Information).
To construct H O and CC-PAMAM-loaded ROS-responsive
time irradiation (3 min) could not result in enough oxygen gen-
eration to dilate the vesicles remarkably.
To investigate the effect of light irradiation on the vesicles,
we first observed the morphology change of HC@P1-Vesicle
after light irradiation using TEM and SEM imaging. The results
showed irregular fragments with sheet structures after consecu-
tive 805 nm irradiation for 3 min and 660 nm irradiation for
10 min (805/660 nm irradiation) (Figure 1D,E). The significant
MW decrease and thioketal moiety cleavage of the copolymer in
HC@P1-Vesicle after one cycle of 805/660 nm irradiation were
2
2
1
P1 polymeric vesicles (HC@P1-Vesicle), block copolymer P1
Mn = 15 800, M /Mn = 1.35), CC-PAMAM, and H O2 were
further confirmed by GPC and H NMR as well, respectively
(
(Figures S7 and S8, Supporting Information). Notably, single
660 nm irradiation can also induce MW decrease and thioketal
moiety cleavage. Presumably, the dissolved oxygen in the
aqueous solution can allow the singlet oxygen generation upon
660 nm irradiation to disrupt the vesicles (Figures S7 and S8,
Supporting Information). However, the sole 805 nm irradiation
only had a negligible effect on the thioketal moieties and the
integrity of the vesicles (Figures S8 and S9, Supporting Informa-
tion). In addition, nonresponsive HC@P2-Vesicle as a contrast
showed no MW change after one cycle of 805/660 nm irradia-
tion (Figure S10, Supporting Information). Therefore, ROS-
responsive thioketal moiety of block copolymer P1 presumably
plays a critical role in the disruption of HC@P1-Vesicle.
w
2
dissolved in dimethylacetamide (DMA) and then mixed with
water for fabricating the clustered vesicles via self-assembly.
The critical micellization concentration of P1 was determined
to be as low as 9.7 × 10− mg mL , which ensured the self-
assembly of P1 in aqueous solution and maintained high sta-
bility after dilution (Figure S5, Supporting Information). The
self-assembled vesicles exhibited the drug loading levels of
3
−1
6
.1 and 3.2% for CC-PAMAM and H O , respectively, when
2 2
assembled from P1 (5 mg), CC-PAMAM (5 mg), and H O2
2
(
30% in 100 µL aqueous solution) to obtain the final vesicle
solution (5 mL). The CC-PAMAM-loaded P1 polymeric vesi-
cles without H O (C@P1-Vesicle) were employed as a control
2
2
without oxygen supply. In addition, H O and CC-PAMAM-
Accompanying the disruption of vesicles upon one cycle of
805/660 nm irradiation, the release of CC-PAMAM from the
vesicles was further measured using ultracentrifugation for
separation and analysis. Figure 1F shows over 80% of encapsu-
lated CC-PAMAM were released after one cycle of 805/660 nm
irradiation. However, under the exposure to sole 805 nm irradi-
ation for 3 min, only less than 5% CC-PAMAM were released,
possibly resulting from the good integrity of HC@P1-Vesicle
2
2
loaded P2 polymeric vesicles (HC@P2-Vesicle) were also fabri-
cated as the control without responsiveness. Transmission elec-
tron microscopy (TEM) images with clear contrast between the
periphery and the inner cores indicate the vesicular structure of
the self-assemblies (Figure 1A), which can also be confirmed by
the inset cracked nanoparticles in the scanning electron micro-
scope (SEM) images (Figure 1B). HC@P1-Vesicle exhibited a
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Figure 1. A) TEM and B) SEM images of HC@P1-Vesicle, respectively. The inset in (B) is the cracked vesicles during sample preparation. C) DLS meas-
−2
urement of HC@P1-Vesicle. D) TEM and E) SEM images of HC@P1-Vesicle after one cycle of 805/660 nm irradiation (805 nm, 1.0 W cm , 3 min;
−
2
6
60 nm, 0.1 W cm , 10 min), respectively. F) CC-PAMAM release from HC@P1-Vesicle after one cycle of 805/660 nm irradiation. Mean ± s.d., n = 3.
−1
−1
−1
−1
G) Temperature elevation (∆T) upon 805 nm irradiation at various cypate concentrations (a: 0 µg mL ; b: 5 µg mL ; c: 10 µg mL ; d: 20 µg mL ;
−1
−1
e: 50 µg mL ). H) Photothermal oxygen generation (∆DO, dissolved oxygen) upon 805 nm irradiation at various cypate concentrations (a: 0 µg mL ;
−1
−1
−1
−1
−1
b: 5 µg mL ; c: 10 µg mL ; d: 20 µg mL ; e: 50 µg mL ; f: 20 µg mL without light irradiation). I) Time-dependent fluorescence intensity of SOSG
−1
−1
(
SOSG FL) after reaction with singlet oxygen produced from HC@P1-Vesicle at 20 µg mL cypate (equivalent to 21.3 µg mL Ce6) under 805 nm irradia-
−2
−2
tion at 1.0 W cm for 10 min (805 nm), 660 nm irradiation at 0.1 W cm for 10 min (660 nm), or 660 nm irradiation for 10 min after 805 nm irradiation
for 3 min (805/660 nm). For 805/660 nm group, the SOSG signal curve was recorded from the beginning of 660 nm irradiation. Mean ± s.d., n = 3.
under short-time 805 nm light irradiation (Figure S9, Sup-
porting Information).
a dissolved oxygen (DO) meter. Distinctly, HC@P1-Vesicle had
a rapid temperature-induced conversion into O (Figure 1H),
2
To demonstrate the photothermal effect of HC@P1-Vesicle,
the temperature elevation was observed under 805 nm irradia-
tion for 3 min. Remarkably, the temperature of the solution was
rapidly increased within 3 min, and the temperature elevations
were dependent on the concentration of cypate (Figure 1G). To
evaluate the oxygen generation caused by thermal effect under
light irradiation, HC@P1-Vesicle suffered from 805 nm irradia-
tion for 3 min, and the produced oxygen was measured using
and oxygen generation also occurred in a cypate-concentration-
dependent manner.
To investigate ROS generation of HC@P1-Vesicle, singlet
oxygen sensor green (SOSG) as a highly selective singlet oxygen
indicator was employed to evaluate ROS production under
[
18]
660 or 805 nm irradiation. Figure 1I shows that only a little
singlet oxygen generation from HC@P1-Vesicle was observed
under 805 nm irradiation owing to a relatively low singlet
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1
[19]
oxygen ( O ) quantum yield of cypate. In contrast, 660 nm
singlet oxygen generation through the conversion of H O into
2
2
2
irradiation caused the production of abundant singlet oxygen
O under irradiation, instead of photothermal effect.
2
from Ce6 within HC@P1-Vesicle.[
1c]
Interestingly, HC@
To elucidate the mechanism of the photocytotoxicity of HC@
P1-Vesicle, we further examined the ROS level inside the cells
using ROS probe 2′,7′-dichlorofluorescin diacetate (DCFH-
P1-Vesicle further boosted singlet oxygen production under
05/660 nm. Presumably, disruption of the vesicles by 805/660
8
[
20]
nm facilitates singlet oxygen diffusion to the fluorescent probe
SOSG, i.e., SOSG traps a higher fraction of singlet oxygen
molecules. Moreover, improved oxygen concentration supplied
from thermal decomposition of H O may also contribute to
DA) (Figure 2C).
Distinctly, higher fluorescence intensity
was observed for the cells treated with HC@P1-Vesicle after
one cycle of 805/660 nm irradiation by confocal laser scanning
microscopy (CLSM), while the flow cytometry results reveal that
the percentage of ROS-positive cells caused by HC@P1-Vesicle
after one cycle of 805/660 nm irradiation was maximized to
97.3% (Figure 2C). In comparison, HC@P1-Vesicle caused
much lower ROS level and less ROS-positive cells (10.3%)
were observed in the absence of light irradiation. Additionally,
C@P1-Vesicle resulted in a moderate ROS level in cells after
one cycle of 805/660 nm irradiation. Possibly, higher ROS
level from HC@P1-Vesicle upon 805/660 nm irradiation was
likely ascribed to the release and diffusion of H O into cells
2
2
the more efficient singlet oxygen generation. Notably, HC@
P2-Vesicle can be detected to exhibit SOSG fluorescence inten-
sity increase upon 805/660 or 660 nm irradiation, but lower
elevated amount was observed as compared to HC@P1-Vesicle
(Figure S11, Supporting Information). This result indicates that
a small portion of produced singlet oxygen can diffuse out of
the vesicles and reacted with SOSG because HC@P2-Vesicle
was not disrupted upon 805/660 or 660 nm irradiation. Taken
together, HC@P1-Vesicle are able to distinctly generate oxygen
under 805 nm irradiation or produce abundant ROS under
2
2
as well as more singlet oxygen generation from CC-PAMAM
inside cells, which both contributed to photocytotoxicity of
HC@P1-Vesicle.
6
60 nm irradiation, thereby allowing more ROS generation
under 805/660 nm irradiation.
To evaluate the effect of intracellular ROS on the cells, we
examined DNA damage and oxidation of other cellular com-
ponents. The degree of DNA damage and level of lipid oxida-
tion were assessed using alkaline comet and lipid peroxidation
malondialdehyde (MDA) assays, respectively. A significantly
high ratio of comet tail (87.26%) and high level of lipid per-
oxidation product MDA (6.1 nmol mg protein) appeared
for the cells treated with HC@P1-Vesicle after one cycle
of 805/660 nm irradiation, indicating a severe cell damage
through ROS (Figure 2D,E). In contrast, both C@P1-Vesicle
with one cycle of 805/660 nm irradiation and HC@P1-Vesicle
without light irradiation exhibited lower cell damages. Although
HC@P2-Vesicle showed a little higher cytotoxicity and severer
cell damage upon one cycle of 805/660 nm irradiation as com-
pared to those without light irradiation or C@P2-Vesicle under
805/660 nm irradiation (Figure S12, Supporting Information),
the therapeutic efficacy was not significantly increased. There-
fore, we mainly employed HC@P1-Vesicle to perform the sub-
sequent in vivo studies.
2
.3. Cellular Uptake, Photothermal Damage, Photocytotoxicity,
and ROS Level
Positively charged CC-PAMAM released from HC@P1-Ves-
icle upon light irradiation were expected to be efficiently
internalized into the cells after the disruption of the vesicles.
To evaluate the efficiency of cellular uptake, the flow cytom-
etry analysis was performed to evaluate the internalization of
CC-PAMAM by human pancreatic BxPC-3 cancer cells. After
−
1
8
05/660 nm irradiation, the cellular uptake of CC-PAMAM
from HC@P1-Vesicle exhibited a fivefold increase as compared
to that without light irradiation (Figure 2A). In contrast, HC@
P2-Vesicle showed a similar cellular internalization regardless
of light irradiation (Figure S12A, Supporting Information).
Distinctly, the photoinduced release of positively charged CC-
PAMAM from HC@P1-Vesicle might account for their prefer-
able cellular uptake after irradiation.
The photothermal effect of HC@P1-Vesicle on their cell
viability under 805 nm irradiation was evaluated (Figure S13,
Supporting Information). HC@P1-Vesicle showed a negligible
influence on their cell viability upon 805 nm irradiation for
2.4. Penetration of CC-PAMAM from HC@P1-Vesicle at Tumor,
Tumor Accumulation, and Hypoxia Alleviation
3
min at a concentration of 4.7 µg mL−1 cypate. Even after five
cycles of irradiation, the cell viability was still more than 80%.
This weak influence of photothermal effect on cell viability
was presumably ascribed to both short irradiation time and
relatively low cypate concentration. Thus, HC@P1-Vesicle had
slight photothermal cell damage effect at 4.7 µg mL cypate.
The photocytotoxicity of HC@P1-Vesicle or C@P1-Vesicle
against BxPC-3 cells was further investigated with or without
one cycle of 805/660 nm irradiation (Figure 2B). Without light
irradiation, HC@P1-Vesicle exhibited no significant cytotox-
icity. However, HC@P1-Vesicle exhibited a significant cytotox-
To evaluate the penetration capacity of CC-PAMAM released
from HC@P1-Vesicle in the tumor, we first established multi-
cellular tumor spheroids (MCTS) of BxPC-3 cells. CC-PAMAM
that were released from HC@P1-Vesicle upon one cycle of
805/660 nm irradiation, distributed throughout the MCTS
mass with relatively uniform distribution (Figure 3A). In con-
trast, Ce6 fluorescence was observed only at the periphery of
MCTS treated with HC@P1-Vesicle without light irradiation,
confirming that released CC-PAMAM possessed an enhanced
penetration ability.
−
1
icity with Ce6-equivalent half maximal inhibitory concentration
(
To evaluate the in vivo accumulation of HC@P1-Vesicle,
BxPC-3 pancreatic tumor was selected as a model owing to
IC50) of ≈3.0 µg mL− upon one cycle of 805/660 nm irradia-
1
[
21]
tion, which displayed much more cytotoxicity as compared to
C@P1-Vesicle, presumably owing to H O release, and boosted
their hypovascular and hypopermeable features.
The in
vivo fluorescence imaging of BxPC-3 tumor-bearing mice was
2
2
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Figure 2. A) Cellular uptake of HC@P1-Vesicle (5 µg mL Ce6 and 4.7 µg mL−1 cypate) with or without one cycle of 805/660 nm irradiation (805 nm,
−1
3
min; 660 nm, 10 min) using flow cytometry. B) Ce6 concentration-dependent cytotoxicity of HC@P1-Vesicle or C@P1-Vesicle with or without one
cycle of 805/660 nm irradiation (805 nm, 3 min; 660 nm, 10 min). C) Intracellular ROS production of HC@P1-Vesicle or C@P1-Vesicle using ROS
probe DCFH-DA by CLSM and flow cytometry, respectively. D) DNA damage of HC@P1-Vesicle or C@P1-Vesicle using comet assay. White circles
indicate the original DNA sites. E) Lipid peroxidation of HC@P1-Vesicle or C@P1-Vesicle using Lipid Peroxidation MDA assay kit. Mean ± s.d., n = 4.
conducted to examine the accumulation of HC@P1-Vesicle
at the tumor. Remarkably, HC@P1-Vesicle were preferably
accumulated into the tumor tissues at 24 h postinjection as
compared to free CC-PAMAM (Figure S14, Supporting Infor-
mation). The passive targeting of HC@P1-Vesicle might be
responsible for their tumor accumulation due to their suit-
able size and neutralized PEG surface. Moreover, HC@
P1-Vesicle within the tumors were exposed to one cycle of
In contrast, HC@P1-Vesicle only resulted in the distribution
of CC-PAMAM around the blood vessels without light irradia-
tion (Figure 3B), indicating a poor ability to penetrate into the
tumor.
Furthermore, a hypoxia-specific probe (pimonidazole) was
[
22]
intravenously injected for imaging tumor hypoxia. Remark-
ably, hypovascular and poorly permeable BxPC-3 pancreatic
tumors exhibited a high hypoxia in the tumors, as evidenced by
the strong fluorescence intensity (Figure 3C). After treatment
with HC@P1-Vesicle under one cycle of 805/660 nm irra-
diation followed by 805 nm irradiation (805/660 nm–805 nm)
at 24 h postinjection, a significant hypoxia alleviation was
observed, as evidenced by weak fluorescence intensity and less
than 10% hypoxic areas. It was ascribed to the photothermal
8
05/660 nm irradiation at 24 h postinjection. At 2 h postir-
radiation, the tumors were excised and sectioned into 10 µm
thick slices, followed by CLSM observation. It indicates that
CC-PAMAM released from HC@P1-Vesicle with red fluores-
cence were deeply distributed into the tumor, suggesting a
uniform distribution of CC-PAMAM in the tumor (Figure 3B).
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Figure 3. A) Penetration profiles of CC-PAMAM after HC@P1-Vesicle treatment with or without 805/660 irradiation (805 nm, 3 min; 660 nm, 10 min)
in multicellular tumor spheroids. The imaging was conducted at 2 h postirradiation using CLSM. Z-stack scanning was performed from the periphery
to equatorial cross-section. The surface of the MCSs was defined as 0 µm. Scanning was performed from the periphery to equatorial cross-section.
B) Distribution of CC-PAMAM in the BxPC-3 tumor treated with HC@P1-Vesicle with or without 805/660 irradiation (805 nm, 3 min; 660 nm, 10 min)
at 24 h postinjection using CLSM immunofluorescence imaging, and their corresponding quantitative fluorescence intensity from blood vessels to
the deep tumor tissue. Mice were sacrificed at 2 h postirradiation; the tumors were excised and sectioned into 10 µm thick slices with a cryostat.
Blood vessels were stained with PECAM-1 (platelet and endothelial cell adhesion molecule 1) primary antibody and a CruzFluor 488 conjugated
secondary antibody (green). C) Hypoxia of BxPC-3 tumor treated with C@P1-Vesicle or HC@P1-Vesicle using a hypoxia-specific probe pimonidazole
(
hypoxyprobe-1 plus kit) after one cycle of 850/660 nm irradiation (805 nm, 3 min; 660 nm, 10 min) followed by another 3 min 805 nm irradiation
for reoxygenation (850/660 nm–805 nm) or without irradiation at 24 h postinjection. The quantification of hypoxia-positive areas was performed with
ImageJ. Mean ± s.d., n = 9.
decomposition of H O for reoxygenation in tumor. In contrast,
(Figure 3C). Reasonably, the photoinduced decomposition of
H O into oxygen accounts for the effective hypoxia alleviation.
Thus, HC@P1-Vesicle are able to facilitate the deep penetra-
tion of CC-PAMAM and also achieve the hypoxia alleviation in
poorly permeable tumors under proper light irradiation.
2
2
HC@P1-Vesicle with only 660 nm irradiation or C@P1-Ves-
icle with one cycle of 805/660 nm–805 nm irradiation exhib-
ited high fluorescence intensities with 57 and 68% hypoxic
areas, respectively, implying the distinct hypoxia at the tumors
2
2
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2
.5. Repeated Photothermal Temperature Increase,
for those suffering from only 660 nm irradiation (Figure 4F).
As a result, HC@P1-Vesicle are able to achieve light-triggered
hypoxia alleviation at tumor under repeated 805/660 nm
irradiation.
Oxygen Generation, and Hypoxia Alleviation
In clinical PDT operation, multiple light irradiations were fre-
quently performed for effective PDT efficacy. To evaluate the
repeated oxygen generation from HC@P1-Vesicle, we first con-
secutively performed multiple cycles of 805/660 nm irradiation
in the tubes (Figure S15, Supporting Information). Within at
least eight cycles of 805/660 nm irradiation, HC@P1-Vesicle
achieved the fast temperature increase when irradiated by
2.6. In Vivo PDT Efficacy
The excellent performances of HC@P1-Vesicle in the in vivo
delivery of H O and CC-PAMAM, photodynamic membrane
2
2
8
05 nm light for 3 min for oxygen generation, followed by effi-
disruption, photothermal O2 production, and CC-PAMAM
deep penetration in tumor encouraged us to examine thera-
peutic efficacy toward the hypovascular and hypopermeable
tumor BxPC-3 with severe hypoxia. The tumor-bearing mice
were treated with free Ce6 and HC@P1-Vesicle at the dose of
cient ROS production under 660 nm irradiation for 10 min.
Repeated short-time irradiation may not reduce the stability
of cypate significantly. Moreover, cypate-conjugated polymer
and cypate-loaded micelles can further improve the stability
of cypate.[ Hence, both cypate and Ce6 exhibit a good photo-
stability for repeated oxygen and ROS generations. Such a fea-
ture is highly advantageous for in vivo PDT treatment against
hypoxic tumor.
23]
−1
2.5 mg kg Ce6, respectively. The light irradiation was per-
formed at 24 h postinjection for five consecutive cycles on days
1 and 5, respectively, followed by tumor size measurement for
subsequent 24 d (Figure 5A,B). Phosphate buffered saline (PBS)
as a control had a significant tumor growth, while free Ce6 had
a similar tumor growth behavior under sole 660 irradiation as
compared to PBS, probably owing to its poor tumor accumula-
tion and limited ROS generation. HC@P1-Vesicle only exhib-
ited a relatively reduced tumor growth under 660 or 805 nm
irradiation as compared to PBS, possibly resulting from their
generated ROS or photothermal effect at tumor. Possibly, both
the hypoxia and oxygen depletion account for the slight PDT
efficacy, while the photothermal effect only causes mild hyper-
thermia (<45 °C) that leads to the mild photothermal therapy
(PTT) efficacy. Importantly, HC@P1-Vesicle exhibited the effec-
tive tumor ablation under 805/660 nm irradiation, indicating a
more preferable efficacy as compared to that under sole 660 or
805 nm irradiation (Figure 5A,B). Remarkably, HC@P1-Vesicle
primarily possess a potent PDT efficacy, reasonably owing to
the boosted ROS generation caused by deep CC-PAMAM pen-
etration and self-supplied oxygen under 805/660 nm irradia-
tion. Thus, HC@P1-Vesicle with both self-supplied oxygen and
deep tissue penetrability effectively achieve the total ablation
of hypoxic hypopermeable pancreatic tumor through boosted
photodynamic damage.
To confirm the therapeutic efficacy, histopathological
analysis of tumor sections was also performed on day 24.
The hematoxylin and eosin (H&E) staining exhibited severe
tumor destruction for 805/660 nm irradiation by injection
of HC@P1-Vesicle (Figure 5C). Terminal deoxynucleotidyl
transferase-mediated dUTP nick-end labeling (TUNEL) assay
further confirmed the presence of abundant apoptotic cells
(Figure 5C). However, for the groups with sole 660 or 805 nm
irradiation, low levels of tumor damage and cell apoptosis were
observed. It validates that the vesicles with deep penetration of
CC-PAMAM and self-supplied oxygen act as an effective nano-
platform for potent PDT treatment against poorly permeable
tumors.
We further investigated the in vivo photoinduced tem-
perature increase and O generation under multiple cycles of
8
at BxPC-3 tumors was recorded under 805/660 nm irradia-
tion for five cycles (Figure 4A). It shows that the temperature
was still increased to 43 °C even at the fifth cycle of 805 nm
irradiation (Figure 4B). On the other hand, the H O level in
the tumor can be observed by using the H O -specific probe
BES-H O after five cycles of 805/660 nm irradiation. The
BxPC-3 tumor exhibited a relatively low H O level as shown
by low fluorescence intensity in control group without injection
of HC@P1-Vesicle (Figure 4C). Apparently, upon one cycle of
2
05/660 nm irradiation. At 24 h postinjection, the temperature
2
2
2
2
[
24]
2
2
2
2
8
05/660 nm irradiation at 24 h postinjection of HC@P1-Ves-
icle, the H O level in the tumor was increased significantly
due to the disruption of vesicles resulted in 80% H O release.
2
2
2
2
With the multiple cycles of 805/660 nm irradiation, the tumor
tissues show a gradual decrease of H O contents to produce
oxygen through the photothermal effect, as indicated by the
decrease of fluorescence intensity in the tumor since the high
concentration of H O in tumor tissues will be decomposed and
produce oxygen (Figure 4C,D). After five cycles of 805/660 nm
irradiation, the H O concentration in the tumor still remained
a much higher level as compared to control group, thus ena-
bling sufficient oxygen supply via the photothermal decomposi-
tion of H O .
2
2
2
2
2
2
2
2
Moreover, the tumor hypoxia was investigated by the
[
22]
hypoxia-specific probe pimonidazole during irradiation. As
shown in Figure 4E, under only multiple cycles of 660 nm
irradiation, HC@P1-Vesicle caused severe hypoxia in the
tumor at 24 h postinjection as evidenced by high fluorescence
intensity, owing to the intrinsic tumor hypoxia and distinct
oxygen consumption of CC-PAMAM. To further validate the
hypothesis that photothermal decomposition of H O2 from
2
HC@P1-Vesicle under 805 nm irradiation could continuously
induce reoxygenation of hypoxic tumor, we performed 660 nm
irradiation followed by 805 nm irradiation (660/805 nm).
The consumed oxygen during 660 nm irradiation for PDT
in tumor was expected to be replenished. Notably, after five
cycles of 660/805 nm irradiation, HC@P1-Vesicle only had
less than 20% hypoxic areas as compared to more than 70%
3
. Conclusion
In summary, we have developed light-triggered, clustered
vesicles encapsulating CC-PAMAM and H O within vesicular
2
2
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Figure 4. A) Infrared thermograph and B) temperature elevation of BxPC-3 tumors treated with HC@P1-Vesicle at the dose of 2.35 mg kg−1 cypate during
50 nm irradiation with 805/660 nm irradiation for five cycles (each cycle contains 3 min 805 nm irradiation and subsequent 10 min 660 irradiation).
C) H O distribution in BxPC-3 tumor using an H O probe BES-H O and D) integrated optical density (IOD) of the H O probe after treatment with
8
2
2
2
2
2
2
2
2
HC@P1-Vesicle under five cycles of 805/660 nm irradiation (805 nm, 3 min; 660 nm, 10 min). E) Tumor hypoxia under multiple cycles of 660 nm irra-
diation (10 min irradiation and subsequent 3 min darkness) or five cycles of 660/805 nm irradiation (660 nm, 10 min; 805 nm, 3 min). F) Quantitative
hypoxia-positive area from (E) was acquired from ImageJ. Mean ± s.d., n = 9.
chamber for potent PDT treatment. Upon one cycle of
05/660 nm irradiation, the disruption of vesicle triggered
Meanwhile, released CC-PAMAM as a photoactive agent was
able to deeply penetrate into the tumors. Such vesicles with
self-supplied oxygen and deep penetration in this study lead
to the tumor ablation of the hypopermeable BxPC-3 without
any regrowth. Our proposed strategy represents a general and
effective approach for potent PDT against hypoxic tumor.
8
the rapid release of positively charged CC-PAMAM and H O2
2
molecules from vesicular chamber. The light-triggered O gen-
2
eration was achieved through the photothermal decomposition
of H O for facilitating subsequent tumor hypoxia alleviation.
2
2
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Figure 5. A) Picture of BxPC-3 tumor-bearing BALB/c nude mice on day 24 after treatment. The tumor sites are indicated by dashed circles. B) BxPC-3
tumor growth profiles of the mice treated with suppression. The multiple cycles of 660 nm irradiation (10 min light–3 min dark, total five cycles),
8
2
05 nm irradiation (3 min light–10 min dark, total five cycles), or 805/660 nm irradiation (805 nm, 3 min; 660 nm, 10 min, total five cycles) were applied
4 h postinjection of HC@P1-Vesicle at a Ce6-equivalent concentration of 2.5 mg kg . The treatments were performed on days 1 and 5 as the arrows
−
1
indicated. Mean ± s.d., n = 5. C) Histopathology analysis (H&E staining and TUNEL assay) of BxPC-3 tumor on day 24 of the treatment.
5
.94 mmol) were dissolved in 100 mL anhydrous dichloromethane
4
. Experimental Section
(
DCM) and stirred for 24 h at room temperature. The mixture was
Sample Preparation: Synthetic routes employed for the preparation of
filtered followed by silica gel column chromatography separation using
DCM/ethyl acetate (10:1, v/v). ROS-cleavable thioketal-containing
diacrylate (M2) was yielded as a transparent viscous liquid (3.35 g, yield:
37.7%). H NMR (300 MHz, δ, ppm, CDCl ; Figure S1B, Supporting
Information): 6.45 (d, 2H), 6.13 (q, 2H), 5.85 (d, 2H), 4.36 (s, 8H),
2.87 (t, 4H), 2.65 (t, 4H), 1.59 (s, 6H); C NMR (300 MHz, δ, ppm,
CDCl ; Figure S1C, Supporting Information), 171.2, 165.2, 131.3,
127.6, 62.2, 62.0, 56.1, 34.1, 30.6, 25.0 ppm; ESI-MS Calcd. for
(C H O S + H) : 449.12; Found: 449.18.
P1 and P2 are shown in Scheme 1B. Typical procedures are as follows.
Synthesis of ROS-Cleavable Diacrylate M2: Initially, anhydrous
1
3
-mercaptopropionic acid (5.31 g, 50 mmol) and anhydrous acetone
3
(
5.81 g, 100 mmol) were charged into a 25 mL two-necked flask,
1
3
followed by saturation with hydrogen chloride that was dried over
concentrated sulfuric acid. After stirring for 2 h at room temperature,
the flask was placed in a cooled ice-salt bath for product crystallization.
The crystals were filtered, followed by washing for several times using
cold hexane and cold deionized water alternately. After drying in a
vacuum oven overnight at room temperature, a snowy crystalline
product, 3,3′-(propane-2,2-diylbis(sulfanediyl))dipropionic acid, was
3
+
1
9
28
8 2
Synthesis of ROS-Responsive Amphiphilic Triblock Copolymer P1: The
block copolymer P1 was prepared via thiol-acrylate Michael addition
reaction.[ Briefly, 1,6-hexanedithiol (M1, 0.11 g, 0.75 mmol) dissolved
in degassed DMA (100 µL) and 0.5% dimethylphenylphosphine
15]
obtained (5.5 g, yield: 81.4%).[14a] 1H NMR (300 MHz, δ, ppm, CH DO;
3
Figure S1A, Supporting Information): 2.78 (t, 4H), 2.51 (t, 4H), 1.51
(Me PPh) solution in DMA (10 µL) were charged into a degassed
2
(
s, 6H). Then, ROS-cleavable 8,8-dimethyl-4,12-dioxo-3,13-dioxa-7,9-
miniature flask. The flask was placed in a cooled ice bath, followed by
addition of M2 (0.29 g, 0.65 mmol in 150 µL degassed DMA) through
a microsyringe. After 1 h, PEG-acrylate (0.3 g, 0.15 mmol) and 0.5 wt%
dithiapentadecane-1,15-diyldiacrylate M2 was synthesized through
dicyclohexylcarbodiimide (DCC) coupling reaction. Briefly, the crystalline
product (5 g, 19.8 mmol), 2-hydroxyethyl acrylate (6.9 g, 59.4 mmol),
DCC (12.3 g, 59.4 mmol), and 4-dimethylaminopyridine (0.73 g,
Me PPh solution in DMA (10 µL) were added into the reaction mixture
and stirred for another 2 h at room temperature. The reaction mixture
2
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was then precipitated into cold diethyl ether. The precipitate was
collected and dissolved in DCM, and the dissolution–precipitation
process was repeated twice. The obtained precipitate was then dispersed
into water and dialyzed using Millipore ultrafiltration (MW cutoff:
The temperature of the solution was monitored by an auto-recording
thermometer. The time-dependent oxygen generation was recorded
by D.O. Meter (WTW, Germany). For the detection of singlet oxygen
−
6
production, HC@P1-Vesicle or HC@P2-Vesicle with SOSG (10 × 10 m)
−
2
5
0 kDa). The solution was then lyophilized, affording the final product
were consecutively irradiated with an 805 nm laser (1 W cm ) for 3 min
−
2
P1 (0.44 g, yield: 88%) (Mn = 15 800, M /M = 1.35). Theoretical DP
and a 660 nm laser (0.1 W cm ) for 10 min, only 805 nm irradiation
for 10 min or 660 nm irradiation for 10 min. The time-dependent
fluorescence of oxidized SOSG was measured by F-4600 fluorescence
spectrophotometer.
w
n
was calculated according to equation: DP = (1 + m)/(1 − m), where
m = [M2]/[M1]. The triblock copolymers were characterized in detail by
1
H NMR and GPC (Figures S1 and S2, Supporting Information). GPC
traces showed the symmetrical unimodal peaks indicating the successful
ordered step growth propagation and subsequent capping reaction
Cellular Uptake and ROS Generation: The BxPC-3 cells were seeded at
a density of 1 × 10 cells per well into a 35 mm glass-bottom culture dish
(NEST, China) and incubated overnight in 2 mL RPMI-1640 medium
with 10% fetal bovine serum (FBS) at 37 °C with 5% CO2 humidified
atmosphere. The medium was replaced with fresh medium, followed
5
(
Figure S2B, Supporting Information). The final molecular weights could
1
be calculated from H NMR characterization through the integral ratio
of PEG (δ = 3.70-3.51) and the characteristic peaks of hydrophobic
segment (e.g., methyl of thioketal, δ = 1.65–1.51), which is in close
agreement with the theoretical values and GPC results. The actual
−
1
by addition of HC@P1-Vesicle or HC@P2-Vesicle solution (5 µg mL
−
1
−6
Ce6, 4.7 µg mL cypate, 520 × 10 m H O ). Then the cells were
consecutively irradiated with an 805 nm laser (1 W cm ) for 3 min and
a 660 nm laser (0.1 W cm ) for 10 min, followed by addition of ROS
2
2
1
−2
DP was determined to be 12 from H NMR (Figure S2A, Supporting
−
2
Information), while the theoretical DP was 14. According to similar
procedures, the block copolymer P2 with comparable MW without ROS-
responsive moieties was also synthesized.
−
6
probe (DCFH-DA) at the final concentration of 10 × 10 m. After 20 min,
the cells were washed three times with ice-cold PBS and imaged with
CLSM. For quantification of cellular uptake of Ce6 and ROS generation,
cells were detached by trypsin, harvested, and resuspended in PBS for
flow cytometry measurements. For flow cytometry, Ce6 was excited with
405 nm laser and emission wavelength was set at 670 nm. DCFH-DA
was excited with 488 nm laser and emission wavelength was set at
530 nm. Data were analyzed with Flowjo software.
Synthesis of Ce6 and Cypate-Conjugated PAMAM: Ce6 (0.23 g,
0
3
.38 mmol) and cypate (0.27 g, 0.38 mmol), 1-(3-dimethylaminopropyl)-
-ethylcarbodiimide hydrochloride (0.08 g, 0.4 mmol), and
N-hydroxysuccinimide (0.05 g, 0.4 mmol) were dissolved in dimethyl
sulfoxide (DMSO) (4 mL) and stirred for 30 min at room temperature.
Subsequently, G5-PAMAM (0.92 g, 0.032 mmol) in DMSO (4 mL) were
added. After stirring for 24 h, the reaction mixture was dispersed in water
and dialyzed against deionized water for 48 h using dialysis membrane
Cytotoxicity: The cytotoxicity of HC@P1-Vesicle or HC@P2-Vesicle
was evaluated via MTT assay. Briefly, BxPC-3 cells were seeded in 96-well
4
(
(
MW cutoff: 10 kDa). After lyophilization, a dark green solid was afforded
1.0 g, yield: 70.4%). The thin-layer chromatography method was used
plates at a density of 1 × 10 cells per well in 100 µL RPMI-1640 medium
with 10% FBS at 37 °C under 5% CO humidified atmosphere. After 24 h
2
to confirm no free Ce6 and cypate remained in the final CC-PAMAM
sample. The numbers of Ce6 and cypate per PAMAM molecule were
determined to be 10 and 8, respectively, via UV–vis absorbance peaks at
incubation, the medium was replaced with fresh RPMI-1640 medium
with 10% FBS, followed by addition of HC@P1-Vesicle, C@P1-Vesicle,
or HC@P2-Vesicle solution with varying equivalent amounts of Ce6
−
1
4
05 and 778 nm, respectively.
(0, 1, 3, or 5 µg mL ). Then the cells were consecutively irradiated with
2
−
−2
Preparation of HC@P1-Vesicle: The block copolymer, P1 (5 mg),
an 805 nm laser (1 W cm ) for 3 min and a 660 nm laser (0.1 W cm )
for 10 min. The medium was then removed and fresh culture medium
was added followed by another 24 h incubation. 3-(4,5-Dimethylthiazol-
CC-PAMAM (5 mg), and H O solution (30 wt%) (100 µL) were
2
2
dissolved in 500 µL DMA, followed by addition of 5 mL water at the
−
1
−1
constant rate of 4 mL h using an injection pump. CC-PAMAM and
H O unencapsulated into the vesicles were removed via Millipore
2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (20 µL, 5 mg mL
in PBS buffer) was added and incubated for 4 h. Subsequently, the
medium was removed and DMSO (200 µL) was added to dissolve
the formed purple formazan crystals. After shaking for 15 min,
the absorbance at 490 nm was measured by a microplate reader.
Penetration of CC-PAMAM in MCTS: BxPC-3 cell suspension (100 µL,
2
2
ultrafiltration (MW cutoff: 100 kDa). The final vesicle solutions were
−
1
volumed up to 5 mL with water (1 mg mL P1). The prepared HC@
P1-Vesicle was treated with Triton X-100, and then quantification of H O2
2
loaded in vesicle was conducted through measuring dissolved oxygen by
D.O. Meter (WTW, Germany) in the presence of catalase. CC-PAMAM
loaded in vesicle was also quantified via UV–vis spectroscopy
using standard curves. Drug loading content was calculated via the
formula: DLC =w(drug in vesicle)/w(total vesicle) × 100%. DLC of
CC-PAMAM and H O within the vesicles were determined to be 6.1
4
−1
3 × 10 cells mL RPMI-1640 medium) was added into 96-well spheroid
microplates with unique round well-bottom geometry, followed by
incubation at 37 °C with 5% CO humidified atmosphere for production
2
−
1
of MCTS. Then the MCTS were treated with HC@P1-Vesicle (5 µg mL
−
1
−6
Ce6, 4.7 µg mL cypate, 520 × 10 m H O ), followed by consecutive
2
2
2
2
−2
−
1
and 3.2%, respectively. Eventually, HC@P1-Vesicle (1 mg mL P1,
irradiation with an 805 nm laser (1 W cm ) for 3 min and a 660 nm
−
1
−1
−3
−2
1
0 µg mL Ce6, 9.4 µg mL cypate, 1.04 × 10 m H O ) was used for
subsequent experiments and the drug concentrations were controlled by
concentrating or diluting this vesicle solution.
laser (0.1 W cm ) for 10 min. At 4 h postirradiation, the spheroids were
2
2
carefully washed with cold PBS without agitation and observed with
CLSM.
Release Profiles of CC-PAMAM and H O from HC@P1-Vesicle: For
CC-PAMAM Penetration and Hypoxia Alleviation in Tumor: The animal
studies program of “Tumor Photodynamic Therapy” were carried
out according to the Regulations for the Administration of Affairs
Concerning Experimental Animals (Hefei, revised in June 2013). The
animal protocols used in this study were approved by The Institutional
Animal Use and Care Committee of University of Science and
Technology of China. Female BALB/c nude mice bearing BxPC-3 tumor
2
2
evaluation of phototriggered release of CC-PAMAM and H O2 from
2
−
1
HC@P1-Vesicle, the freshly prepared vesicle solution (0.5 mg mL P1,
−
1
−1
−6
5
µg mL Ce6, 4.7 µg mL cypate, 520 × 10 m H O ) was consecutively
2 2
2
−
irradiated with an 805 nm laser (1 W cm ) for 3 min and a 660 nm
−
2
laser (0.1 W cm ) for 10 min, followed by ultracentrifugation using a
Millipore ultrafiltration tube (MWCO: 100 kDa). The release profiles of
CC-PAMAM were quantified by UV–vis spectroscopy using standard
curves. The H O release was detected via the fluorescence intensity of
BES-H O probe (25 × 10 m). The release profiles of CC-PAMAM and
H O from HC@P1-Vesicle without light irradiation or single 805 nm
irradiation (1.0 W cm ) for 3 min were also measured as controls.
Photothermal Effect, Oxygen Generation, and ROS Production: HC@
P1-Vesicle (0.1 mL) with various cypate concentrations (0, 5, 10, 20,
and 50 µg mL ) were placed into the wells of a 96-well plate at room
temperature followed by irradiation with an 805 nm laser (1 W cm ).
3
(≈200 mm ) were administered intravenously with 200 µL of HC@
−
1
P1-Vesicle solution at Ce6-equivalent concentration of 2.5 mg kg . After
24 h postadministration, the tumors were consecutively irradiated with
2
2
−
6
2
2
−
2
−2
an 805 nm laser (1 W cm ) for 3 min and a 660 nm laser (0.1 W cm )
for 10 min. Then the mice were sacrificed at 2 h postirradiation, the
tumors were excised and sectioned into 10 µm thick slices with a
cryostat. The slices were briefly fixed with cold acetone, incubated with
PECAM-1 antibody, and then CruzFluor 488 conjugated secondary
antibody. Samples were observed by CLSM.
2
2
−
2
−
1
−
2
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As for evaluation of tumor hypoxia, after the tumors were consecutively
irradiated with a cycle of 805/660 nm followed by 805 nm irradiation
for 3 min for reoxygenation, a hypoxia-specific probe pimonidazole
Acknowledgements
The authors gratefully acknowledge financial support from the National
Natural Scientific Foundation of China (NNSFC) Project (21674104,
51473109, and 31422021), the Fundamental Research Funds for the
Central Universities (WK3450000002), and the Priority Academic
Program Development of Jiangsu Higher Education Institutions (PAPD).
(
hypoxyprobe-1 plus kit, Hypoxyprobe Inc.) at the dose of 60 mg kg−1
was intravenously injected into mice for covalent conjugation to thiol-
containing proteins in hypoxic tumor tissue. After 90 min, the tumors
were excised and sectioned into 10 µm thick slices with a cryostat. Then
tumor sections were incubated with mouse anti-pimonidazole antibody,
followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse
secondary antibody according to the manufacturer’s protocols. CLSM
observation was performed for imaging the hypoxia. The hypoxia area
was analyzed with ImageJ software.
Conflict of Interest
The authors declare no conflict of interest.
Repeated Oxygen Generation through Multiple Irradiation in
3
Tumor: Female BALB/c nude mice bearing BxPC-3 (≈200 mm ) were
administered intravenously with HC@P1-Vesicle (200 µL) at a Ce6-
−
1
equivalent concentration of 2.5 mg kg . After 24 h postinjection, the
tumors were consecutively and alternately irradiated with an 805 nm laser
Keywords
−
2
−2
(
1 W cm ) for 3 min and a 660 nm laser (0.1 W cm ) for 10 min. Five
hypoxic tumors, photodynamic therapy, singlet oxygen, vesicles
cycles of irradiation were performed. The temperature was monitored
by an FLIR thermal camera. For the detection of H O in tumor, 50 µL
2
2
−
6
Received: April 20, 2017
Revised: May 25, 2017
Published online:
of BES-H O probe (25 × 10 m) was injected intratumorally after each
2
2
cycle of 805/660 nm irradiation. After 30 min, the mice were sacrificed
followed by excision of the tumors. After sectioning the tumor into
1
0 µm thick slices, CLSM observation was performed. The fluorescence
emission of fluorescein decomposed from BES-H O was detected using
2
2
[
1] a) D. E. J. G. J. Dolmans, D. Fukumura, R. K. Jain, Nat. Rev. Cancer
003, 3, 380; b) C. K. Lim, J. Heo, S. Shin, K. Jeong, Y. H. Seo,
W. D. Jang, C. R. Park, S. Y. Park, S. Kim, I. C. Kwon, Cancer Lett.
the excitation of 488 nm and emission ranging from 500 to 550 nm.
Integrated optical density (IOD) of fluorescence was analyzed using
ImageJ. The fluorescence intensity was quantified by subtraction of the
background. The average fluorescence intensity in each mouse was
calculated on the basis of three images, and the fluorescence intensities
and standard errors were determined from three mice.
Tumor hypoxia during alternating irradiation was evaluated through
the hypoxia-specific probe, pimonidazole (Hypoxyprobe-1 plus kit,
Hypoxyprobe Inc.). Briefly, after each 660 nm irradiation for 10 min,
2
2013, 334, 176; c) S. S. Lucky, K. C. Soo, Y. Zhang, Chem. Rev. 2015,
115, 1990; d) W. Fan, P. Huang, X. Chen, Chem. Soc. Rev. 2016, 45,
6488; e) Z. Zhou, J. Song, L. Nie, X. Chen, Chem. Soc. Rev. 2016, 45,
6597; f) Z. P. Zhen, W. Tang, C. L. Guo, H. M. Chen, X. Lin, G. Liu,
B. W. Fei, X. Y. Chen, B. Q. Xu, J. Xie, ACS Nano 2013, 7, 6988;
g) N. Nishiyama, Y. Nakagishi, Y. Morimoto, P. S. Lai, K. Miyazaki,
K. Urano, S. Horie, M. Kumagai, S. Fukushima, Y. Cheng,
W. D. Jang, M. Kikuchi, K. Kataoka, J. Controlled Release 2009, 133,
245; h) H. B. Chen, L. Xiao, Y. Anraku, P. Mi, X. Y. Liu, H. Cabral,
A. Inoue, T. Nomoto, A. Kishimura, N. Nishiyama, K. Kataoka,
J. Am. Chem. Soc. 2014, 136, 157; i) D. Ling, W. Park, S. J. Park,
Y. Lu, K. S. Kim, M. J. Hackett, B. H. Kim, H. Yim, Y. S. Jeon,
K. Na, T. Hyeon, J. Am. Chem. Soc. 2014, 136, 5647; j) L. Cheng,
C. Wang, L. Z. Feng, K. Yang, Z. Liu, Chem. Rev. 2014, 114, 10869;
k) J. P. Celli, B. Q. Spring, I. Rizvi, C. L. Evans, K. S. Samkoe,
S. Verma, B. W. Pogue, T. Hasan, Chem. Rev. 2010, 110, 2795.
8
05 nm irradiation was performed for 3 min to reoxygenate. Five cycles
of 660/805 nm irradiation were performed. Subsequently, pimonidazole
was intravenously injected into mice. After 90 min, the tumors were
excised and sectioned into 10 µm thick slices with a cryostat. Then
tumor sections were incubated with mouse anti-pimonidazole antibody,
followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse
secondary antibody according to the manufacturer’s protocols. CLSM
observation was performed for imaging the hypoxia. The hypoxia area
was analyzed with ImageJ software.
Antitumor Efficacy: Female BALB/c nude mice bearing BxPC-3
3
(
≈50 mm ) were administered intravenously with 200 µL HC@P1-Vesicle
[
[
2] Z. Huang, H. P. Xu, A. D. Meyers, A. I. Musani, L. W. Wang, R. Tagg,
A. B. Barqawi, Y. K. Chen, Technol. Cancer Res. Treat. 2008, 7, 309.
3] a) T. M. Sitnik, J. A. Hampton, B. W. Henderson, Br. J. Cancer
−
1
solution at an equivalent Ce6 amount of 2.5 mg kg . The multiple
60 nm laser irradiation (10 min light-3 min dark, total five cycles),
6
multiple 805 nm laser irradiation (3 min light-10 min dark, total five
cycles), or alternating irradiation (660 nm, 10 min; 805 nm, 3 min, total
five cycles) was applied at 24 h postinjection. A second administration
and same irradiation treatment were performed at day 5 after the first
treatment. The tumor growth was monitored by measuring tumor size
with a digital vernier caliper. The individual tumor volume (V) was
1
998, 77, 1386; b) S. Iinuma, K. T. Schomacker, G. Wagnieres,
M. Rajadhyaksha, M. Bamberg, T. Momma, T. Hasan, Cancer Res.
999, 59, 6164.
1
[
4] a) A. Maier, U. Anegg, B. Fell, P. Rehak, B. Ratzenhofer, F. Tomaselli,
O. Sankin, H. Pinter, F. M. Smolle-Juttner, G. B. Friehs, Lasers Surg.
Med. 2000, 26, 308; b) D. J. H. Roberts, F. Cairnduff, B. Dixon,
S. B. Brown, Oncol. Rep. 1995, 2, 387; c) M. Jirsa, P. Pouckova,
J. Dolezal, J. Pospisil, M. Jirsa, Eur. J. Cancer 1991, 27, 109.
2
calculated according to the following formula: V = (L × W )/2, where
length L is the longer dimension of tumor and width W is the shorter
dimension perpendicular to length. Relative tumor volume was reported
as V/V0 (V0 was the initial tumor volume before PDT treatment). At
the end of treatment, the tumors were excised and sectioned into
[5] a) T. H. Foster, D. F. Hartley, M. G. Nichols, R. Hilf, Cancer Res. 1993,
53, 1249; b) W. Wang, L. T. Moriyama, V. S. Bagnato, Laser Phys.
Lett. 2013, 10, 023001; c) M. Krzykawska-Serda, J. M. Dabrowski,
L. G. Arnaut, M. Szczygiel, K. Urbanska, G. Stochel, M. Elas, Free
Radical Biol. Med. 2014, 73, 239; d) C. G. Qian, J. C. Yu, Y. L. Chen,
Q. Y. Hu, X. Z. Xiao, W. J. Sun, C. Wang, P. J. Feng, Q. D. Shen,
Z. Gu, Adv. Mater. 2016, 28, 3313.
1
0 µm thick slices with a cryostat for H&E staining according to the
manufacturer’s protocols. Apoptotic cells were identified using a TUNEL
assay kit according to the manufacturer’s protocols.
[
[
6] C. Abels, Photochem. Photobiol. Sci. 2004, 3, 765.
Supporting Information
7] a) H. C. Chen, J. W. Tian, W. J. He, Z. J. Guo, J. Am. Chem. Soc. 2015,
137, 1539; b) Q. Chen, L. Z. Feng, J. J. Liu, W. W. Zhu, Z. L. Dong,
Y. F. Wu, Z. Liu, Adv. Mater. 2016, 28, 7129; c) W. P. Fan, W. B. Bu,
Supporting Information is available from the Wiley Online Library or
from the author.
Adv. Funct. Mater. 2017, 1702108
1702108 (12 of 13)
© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.advancedsciencenews.com
www.afm-journal.de
B. Shen, Q. J. He, Z. W. Cui, Y. Y. Liu, X. P. Zheng, K. L. Zhao,
J. L. Shi, Adv. Mater. 2015, 27, 4155; d) W. Tang, Z. P. Zhen,
M. Z. Wang, H. Wang, Y. J. Chuang, W. Z. Zhang, G. D. Wang,
T. Todd, T. Cowger, H. M. Chen, L. Liu, Z. B. Li, J. Xie, Adv. Funct.
Mater. 2016, 26, 1757; e) W. W. Zhu, Z. L. Dong, T. T. Fu, J. J. Liu,
Q. Chen, Y. G. Li, R. Zhu, L. G. Xu, Z. Liu, Adv. Funct. Mater. 2016,
e) H. J. Li, J. Z. Du, J. Liu, X. J. Du, S. Shen, Y. H. Zhu, X. Y. Wang,
X. D. Ye, S. M. Nie, J. Wang, ACS Nano 2016, 10, 6753.
[14] a) Y. Y. Yuan, J. Liu, B. Liu, Angew. Chem., Int. Ed. 2014, 53, 7163;
b) X. Wang, G. Meng, S. Zhang, X. Liu, Sci. Rep. 2016, 6, 29911;
c) C. Tapeinos, A. Pandit, Adv. Mater. 2016, 28, 5553.
[15] K. Dan, S. Ghosh, Angew. Chem., Int. Ed. 2013, 52, 7300.
[16] H. Maeda, Y. Futkuyasu, S. Yoshida, M. Fukuda, K. Saeki,
H. Matsuno, Y. Yamauchi, K. Yoshida, K. Hirata, K. Miyamoto,
Angew. Chem., Int. Ed. 2004, 43, 2389.
2
6, 5490.
[
8] H. Gong, Y. Chao, J. Xiang, X. Han, G. S. Song, L. Z. Feng, J. J. Liu,
G. B. Yang, Q. Chen, Z. Liu, Nano Lett. 2016, 16, 2512.
[
9] a) X. Song, L. Feng, C. Liang, K. Yang, Z. Liu, Nano Lett. 2016, 16,
145; b) Y. H. Cheng, H. Cheng, C. X. Jiang, X. F. Qiu, K. K. Wang,
W. Huan, A. Yuan, J. H. Wu, Y. Q. Hu, Nat. Commun. 2015, 6, 8785.
[17] Z. Q. Li, X. L. Guo, J. J. Guan, Biomaterials 2012, 33, 5914.
[18] a) R. Franco, M. I. Panayiotidis, J. A. Cidlowski, J. Biol. Chem.
2007, 282, 30452; b) C. Flors, M. J. Fryer, J. Waring, B. Reeder,
U. Bechtold, P. M. Mullineaux, S. Nonell, M. T. Wilson, N. R. Baker,
J. Exp. Bot. 2006, 57, 1725.
6
[
[
[
10] S. Joshi-Barr, C. D. Lux, E. Mahmoud, A. Almutairi, Antioxid. Redox
Signaling 2014, 21, 730.
11] Y. Cheng, J. D. Meyers, A. M. Broome, M. E. Kenney, J. P. Basilion,
C. Burda, J. Am. Chem. Soc. 2011, 133, 2583.
12] a) Q. H. Sun, X. R. Sun, X. P. Ma, Z. X. Zhou, E. L. Jin, B. Zhang,
Y. Q. Shen, E. A. Van Kirk, W. J. Murdoch, J. R. Lott, T. P. Lodge,
M. Radosz, Y. L. Zhao, Adv. Mater. 2014, 26, 7615; b) R. Tong,
H. D. Hemmati, R. Langer, D. S. Kohane, J. Am. Chem. Soc.
[19] A. J. Gomes, L. O. Lunardi, J. M. Marchetti, C. N. Lunardi,
A. C. Tedesco, Photomed. Laser Surg. 2006, 24, 514.
[20] H. Wang, J. A. Joseph, Free Radical Biol. Med. 1999, 27,
612.
[21] H. Cabral, Y. Matsumoto, K. Mizuno, Q. Chen, M. Murakami,
M. Kimura, Y. Terada, M. R. Kano, K. Miyazono, M. Uesaka,
N. Nishiyama, K. Kataoka, Nat. Nanotechnol. 2011, 6, 815.
[22] M. A. Varia, D. P. Calkins-Adams, L. H. Rinker, A. S. Kennedy,
D. B. Novotny, W. C. Fowler, J. A. Raleigh, Gynecol. Oncol. 1998, 71,
270.
2
2
012, 134, 8848; c) S. Wang, P. Huang, X. Y. Chen, Adv. Mater.
016, 28, 7340; d) C. Wong, T. Stylianopoulos, J. A. Cui, J. Martin,
V. P. Chauhan, W. Jiang, Z. Popovic, R. K. Jain, M. G. Bawendi,
D. Fukumura, Proc. Natl. Acad. Sci. USA 2011, 108, 2426.
[
13] a) J. J. Li, Y. Han, Q. X. Chen, H. D. Shi, S. U. Rehman, M. Siddiq,
Z. S. Ge, S. Y. Liu, J. Mater. Chem. B 2014, 2, 1813; b) S. Sunoqrot,
J. Bugno, D. Lantvit, J. E. Burdette, S. Hong, J. Controlled Release
[23] a) Y. Han, J. J. Li, M. H. Zan, S. Z. Luo, Z. S. Ge, S. Y. Liu, Polym.
Chem. 2014, 5, 3707; b) M. Guo, H. J. Mao, Y. L. Li, A. J. Zhu, H. He,
H. Yang, Y. Y. Wang, X. Tian, C. C. Ge, Q. L. Peng, X. Y. Wang,
X. L. Yang, X. Y. Chen, G. Liu, H. B. Chen, Biomaterials 2014, 35,
4656.
2
014, 191, 115; c) J. J. Li, W. D. Ke, H. Li, Z. S. Zha, Y. Han, Z. S. Ge,
Adv. Healthcare Mater. 2015, 4, 2206; d) H. J. Li, J. Z. Du, X. J. Du,
C. F. Xu, C. Y. Sun, H. X. Wang, Z. T. Cao, X. Z. Yang, Y. H. Zhu,
S. M. Nie, J. Wang, Proc. Natl. Acad. Sci. USA 2016, 113, 4164;
[24] J. J. Li, W. D. Ke, L. Wang, M. M. Huang, W. Yin, P. Zhang,
Q. X. Chen, Z. S. Ge, J. Controlled Release 2016, 225, 64.
Adv. Funct. Mater. 2017, 1702108
1702108 (13 of 13)
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