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Cite this: DOI: 10.1039/c9bm01472a
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Responsive agarose hydrogel incorporated with
natural humic acid and MnO2 nanoparticles for
effective relief of tumor hypoxia and enhanced
photo-induced tumor therapy†
Mengmeng Hou,‡a,b Weiwei Liu,‡c Lei Zhang,d Leiyang Zhang,a Zhigang Xu,
Yang Cao,*c Yuejun Kang a,b and Peng Xue *a,b
a,b
In spite of widespread applications of nano-photosensitizers, poor tumor penetration and severe
hypoxia in the tumor microenvironment (TME) always result in an undesirable therapeutic outcome of
photodynamic therapy (PDT). Herein, a biocompatible agarose-based hydrogel incorporated with
sodium humate (SH), manganese oxide (MnO2) and chlorin e6 (Ce6) was synthesized as agarose@SH/
MnO2/Ce6 through a “co-trapped” strategy during a sol–gel process and employed for combined
photothermal therapy (PTT) and enhanced PDT. NIR-induced local hyperthermia is responsible for not
only activating Ce6 release, but also triggering the catalytic decomposition of H2O2 mediated by MnO2
to relieve hypoxia. Such a hybrid hydrogel can realize deep tissue penetration through intratumoral
injection, and exhibit remarkable tumor-site retention. Moreover, programmed laser irradiation led to an
extremely high tumor growth inhibition rate of 93.8% in virtue of enhanced PTT/PDT. In addition, ultraReceived 12th September 2019,
Accepted 4th November 2019
low systemic toxicity caused by the hybrid hydrogel was further demonstrated in vivo. This reliable and
DOI: 10.1039/c9bm01472a
eco-friendly hydrogel paves the way for the development of smart gel-based biomaterials, which
respond to both exogenous and endogenous stimuli, towards the management of cancer and other
rsc.li/biomaterials-science
major diseases.
Introduction
Cancer, as one of the most complicated and intractable diseases, has been seriously threatening human health and
causing a huge reduction of life expectancy, attributed to the
high morbidity and mortality rate in the past few decades.1–3
Surgery is the most commonly used approach to treat cancer
by the manual removal of tumorous tissue.4 However, this
therapeutic method is only valid for eliminating well-defined
and primary tumors on non-vital organs or tissues. Moreover,
a
Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest
University), Ministry of Education, School of Materials and Energy, Southwest
University, Chongqing 400715, China. E-mail: xuepeng@swu.edu.cn
b
Chongqing Engineering Research Center for Micro-Nano Biomedical Materials and
Devices, Chongqing 400715, China
c
Chongqing Key Laboratory of Ultrasound Molecular Imaging, Institute of Ultrasound
Imaging, Second Affiliated Hospital, Chongqing Medical University, Chongqing,
400010, China. E-mail: yangcao@cqmu.edu.cn
d
Institute of Sericulture and System Biology, Southwest University, Chongqing
400716, China
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c9bm01472a
‡ These authors contributed equally to this work.
This journal is © The Royal Society of Chemistry 2019
potential bleeding and infections may impair patient compliance during surgery, and the rate of treatment failure is relatively high owing to the incidence of relapse.5,6 To reduce the
high recurrence rate and incomplete eradication of solid
tumors via surgery, chemotherapy and radiotherapy have been
implemented as adjuvant treatment modalities.7,8 However,
severe complications and adverse side effects always emerge in
the process of these therapies owing to the limited selectivity
and specificity of chemotherapeutic drugs and radioactive
exposure. In this respect, there is an urgent need for developing promising therapeutic modalities with decent tumor specificity, high treatment efficacy and minimal adverse effects for
clinical applications.
Currently, tumor-targeted drug delivery techniques allow a
desirable kinetics of drug release specifically to the tumor
region with extended periods of time, which can effectively
overcome the above-mentioned shortcomings of traditional
methods.9,10 To date, many drug delivery systems have been
successfully developed for the purpose of localized drug delivery, such as dendrimers,11 micelles,12 liposomes,13 nano/
microparticles,14 hydrogel,15 etc. Among them, injectable
hydrogel is highly promising for intratumoral administration
of both hydrophilic and hydrophobic autitumor drugs, which
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not only realizes sustained and controllable drug release but
also minimizes side effects caused by systemic exposure.15–20
Polymeric hydrogel can be flexibly designed with high biocompatibility and degradability, benefiting the activity maintenance of the encapsulated drug. The release of the pharmaceutical ingredient from hydrogel can be activated by
diffusion, swelling or environmental stimuli.21,22 Having been
approved as a biocompatible polysaccharide by the US Food
and Drug Administration (FDA), agarose is a purified linear
and neutral galactan hydrocolloid extracted from agar or agarbearing marine algae.23 Low melting point (LMP) agarose
melts at 65.5 °C and sol-to-gel transition is initiated during the
cooling process at a temperature below 25 °C. LMP agarose
hydrogel has displayed enormous potential for on-demand
localized drug administration, which can be precisely regulated by varying the ambient temperature.24–26 Therefore, functionalized LMP agarose hydrogel is anticipated to be rationally
designed and developed for multimodal tumor therapy
through one single injection, thereby avoiding the insufficient
bioavailability of drugs and improving therapeutic outcome
with high patient compliance.
Photothermal therapy (PTT) has been extensively studied
as a minimally invasive tumor ablation strategy, in which the
destruction of cancerous tissue is achieved based on local
hyperthermia mediated by photothermal agents (PTAs)
under near-infrared (NIR) light irradiation.27–30 Taking
advantage of the simplicity, minimal invasiveness and high
spatiotemporal precision, PTT can effectively improve the
therapeutic outcome compared to traditional treatment
approaches.29 Apart from eradicating tumor through thermal
shock, light-responsive PTAs also serve as potential candidates for triggering on-demand drug release by simply modulating the stimuli.31,32 In particular, light-induced phase
transition of drug-loaded hydrogel has received increasing
attention for controlled drug delivery, which can be accurately adjusted by varying the operating parameters of the
light source, such as laser wavelength, output power and
irradiation time.33–35 Thus far, a good variety of PTAs have
been developed to attain satisfactory PTT performance.36
However, the majority of them still possess limitations for
clinical usage, such as complex synthesis, low biosafety and
non-degradability. Humic acid (HA), as an organic substance
extracted from biochemical humification of plant and animal
matter, has recently attracted increasing interest for both
PTT and photoacoustic (PA) imaging, attributed to its outstanding photothermal conversion capacity.37,38 Potential
biohazard caused by HA is minimal towards a good variety of
living organisms.39 Unlike nanoagents liable to aggregate in
the non-aqueous phase, small molecules of HA are extremely
prone to be uniformly dispersed when being encapsulated
into hydrogel, which facilitates the maintenance of their
photothermal stability and the homogeneous heat generation
triggered by laser irradiation. Sodium humate (SH), as a
sodium salt derivative, displays an improved solubility compared to HA while retaining the equivalent photothermal
property of HA.
Biomater. Sci.
Biomaterials Science
Meanwhile, photodynamic therapy (PDT) has been identified as an effective approach for cancer treatment both in preclinical trials and clinical applications, by utilizing the oxygen
reactive species (ROS) produced from oxygen molecules in the
presence of photosensitizers (PS) under light excitation.40,41
Chlorin e6 (Ce6), as a second generation of PS, has been commonly used for PDT owing to its high ROS quantum yield and
low dark toxicity.42 In another aspect, intrinsic hypoxia inside
the solid tumor has been regarded as one of the undesirable
characteristics that promote tumor progression and metastasis, which originates from abnormal cell proliferation, aberrant vasculature, and dysfunctional lymphatic system.43,44
Thereby, hypoxia-associated resistance often occurs as a result
of insufficient oxygen supply during the process of PDT, which
severely compromises therapeutic efficacy during the treatment of large solid tumors.45–47 To modulate the hypoxic
tumor microenvironment (TME), manganese dioxide (MnO2)
incorporated nanostructures have exhibited high selectivity
and specificity towards a sustained production of O2 from the
decomposition of high-level endogenous H2O2 inside the
tumor, which has been verified to be effective for the amelioration of hypoxia and favorable for enhanced PDT of the
tumor.48–50
Herein, we innovatively proposed a “co-trapped” approach
by simultaneously encapsulating SH, Ce6 and MnO2 nanoparticles (NPs) into LMP agarose, and the as-synthesized
agarose@SH/MnO2/Ce6 hybrid hydrogel was successfully
applied for enhanced PTT/PDT through an effective relief of
tumor hypoxia (Fig. 1). The resulting hybrid hydrogel as an
injectable material into the diseased lesion exhibits superiorities for tumor therapy: first, this hydrogel with acceptable biocompatibility and biodegradability can be introduced into the
solid tumor, especially into the innermost region, through
precise injection. Second, permeation of Ce6 and MnO2 NPs
from the hydrogel matrix into the ambient environment was
sustainable subject to the softening and hydrolysis of the
agarose hydrogel, which can also be accelerated by the generation of local hyperthermia. Third, the hydrogel itself can be
used for PTT, as SH acts as a light absorber that converts light
into thermal energy under NIR light irradiation. Moreover,
tumor hypoxia is intended to be effectively attenuated through
oxygen generation from H2O2 decomposition catalyzed by
MnO2. Thereafter, a tremendous enhancement of PDT is
achieved upon the hydrogel being exposed to a 660 nm laser.
The potential biohazard effects and clearance by the immune
system are reduced to a minimal level, thanks to the remarkable retention of the hydrogel within the tumorous area but
without entering the circulatory system, eventually achieving
the objective of “one injection, multiple therapies”.
Experimental section
Materials
Humic acid sodium salt (sodium humate, SH), ultrapure LMP
agarose, hydrogen peroxide (H2O2, 30%), dimethyl sulfoxide
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Fig. 1 Schematic diagram of the synthesis process and working principle of the agarose@SH/MnO2/Ce6 hydrogel. Effective tumor inhibition was
accomplished through enhanced photo-induced tumor therapy on the basis of the relief of tumor hypoxia.
(DMSO) and 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, 98%) were purchased from Shanghai
Aladdin Bio-Chem Technology Co., Ltd (Shanghai, China).
Potassium permanganate, 1,3-diphenyl-isobenzofuran (DPBF)
and oleic acid (technical grade, 90%) were purchased from
Sigma-Aldrich (MO, USA). Dulbecco’s modified Eagle’s
medium (DMEM), penicillin–streptomycin (10 000 U mL−1),
fetal bovine serum (FBS), TrypLE™ Express Enzyme (1×), phosphate-buffered saline (PBS, 10×), 4′,6-diamidino-2-phenylindole (DAPI), calcein AM and propidium iodide (PI) were
acquired from Thermo Fisher Scientific (MA, USA). 2,7Dichlorofluorescein diacetate (DCFH-DA) was obtained from
GEN-VIEW Scientific Inc. (CA, USA). Chlorin e6 (Ce6) was supplied by Frontier Scientific, Inc. (UT, USA). Hematoxylin and
eosin (H&E) staining kit, one step TUNEL apoptosis assay kit,
Ki67 cell proliferation kit, HIF-1α monoclonal antibody and
Rat TNF-α ELISA Kit were purchased from Beyotime
Biotechnology (Shanghai, China). All cell line types were
obtained from the Cell Bank of Type Culture Collection of the
Chinese Academy of Sciences (Shanghai, China). Female
BALB/c mice and KM mice (6 weeks, ∼20 g) were obtained
from Chongqing Teng Xin Bill Experimental Animal Sales Co.,
Ltd.
at ambient temperature for 24 h until the formation of a dark
brown product. The final product was harvested after rinsing
with DI water and ethanol at least three times to completely
remove the residual reactants. Finally, the as-synthesized
MnO2 NPs were dried under vacuum at 60 °C overnight for
further use.
Preparation of the hybrid hydrogel
SH (500 mg) was dissolved in DI water (60 mL), and insoluble
residues were eliminated through centrifugation at 12 000 rpm
for 30 min. The collected supernatant was dialyzed for at least
3 days for purification. Then, the obtained solution containing
SH was immediately frozen in liquid nitrogen and further dehydrated in a vacuum to harvest the black SH powder. To constitute the agarose@SH/MnO2/Ce6 hydrogel, LMP agarose
(100 mg), MnO2 NPs (10 mg), purified SH powder (10 mg) and
Ce6 (2 mg) were firstly dissolved in DI water (10 mL) to form a
heterogeneous mixture. Then, a uniform dispersion was
attained by melting the abovementioned mixture in a microwave oven. Finally, the agarose@SH/MnO2/Ce6 hydrogel was
obtained subsequent to the previous dispersion being cooled
down to room temperature. The composition of the as-synthesized agarose-based hybrid hydrogel remained constant for
the follow-up studies if not otherwise stated.
Synthesis of MnO2 NPs
Honeycomb MnO2 NPs were synthesized through a typical
reduction process.47,48 Briefly, KMnO4 (0.25 g) was dissolved in
DI water (125 mL), and the solution was stirred at room temperature for 0.5 h. Then, oleic acid (2.8 mL) was introduced to
form a steady emulsion, which was subsequently maintained
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Characterization of the hybrid hydrogel
A digital image of the agarose@SH/MnO2/Ce6 hydrogel was
acquired by using a Nikon D810 digital camera. The element
composition and morphology of MnO2 NPs were analyzed
using JSM-7800F field emission scanning electron microscopy
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with energy dispersive X-ray spectroscopy (FESEM-EDX). X-ray
diffraction (XRD) pattern of MnO2 NPs was recorded using an
XRD-7000 X-ray diffractometer. The UV-vis-NIR absorption
spectrum of MnO2 NPs was acquired using a UV-1800 UV-vis
spectrophotometer. The dynamic shear rheological properties
of the hybrid hydrogel were evaluated under shear conditions
at a frequency of 1 Hz using a DHR-1 temperature-controlled
rheometer. Briefly, a plate was lowered onto the sample with a
nominal gap of ∼1 mm to the facing specimen holder.
Temperature-sweep dynamic shear test was conducted in a
temperature range from 30 to 60 °C with a ramp rate of 3 °C
min−1.
Biomaterials Science
weight). Then, the swelling ratio of the hydrogel was determined in accordance with formula (1).
Swelling ratio ð%Þ ¼ ðW 1 W 0 Þ=W 0 100%
ð1Þ
To characterize hydrogel degradation, the agarose@SH/
MnO2/Ce6 hydrogel was collected from the medium at a predesigned time point and was immediately freeze-dried in liquid
nitrogen, and its weight was subsequently measured as W2.
The degradation rate is reflected by the weight loss percentage
of the hydrogel after the drying process, which was calculated
as formula (2).
Degradation rate ð%Þ ¼ ðW 0 W 2 Þ=W 0 100%
ð2Þ
Photothermal property of the hybrid hydrogel
To investigate the photothermal conversion capacity of the
agarose@SH/MnO2/Ce6 hydrogel, a quartz tube containing
1 mL hybrid hydrogel at various equivalent SH concentrations
was irradiated using a NIR laser (808 nm, 1.5 W cm−2) for
10 min. The local temperature was dynamically monitored by
using a digital thermometer, and real-time infrared thermal
images were recorded using an infrared thermal imager
(TiS55, Fluke, USA). Photothermal stability of the hybrid hydrogel was analyzed by periodic NIR irradiation for four cycles
(laser on for 10 min per cycle), and the temperature variation
was digitally measured using the same thermometer.
NIR-triggered drug release in vitro
1 mL agarose@SH/MnO2/Ce6 hydrogel was firstly prepared in
the bottom of a cuvette via a standard gelatinization process.
Afterwards, 1 mL 1 × PBS was added into the same cuvette
upon hydrogel formation. Then, the hybrid hydrogel was irradiated using a NIR laser (808 nm, 1.5 W cm−2) for a predesigned time period, and the released Ce6 in the supernatant
was determined through fluorescence spectrophotometry.
Specifically, the laser was switched on for 5 min and subsequently switched off for another 5 min during 60 min of
treatment. Then, a liquid sample (200 µL) was obtained from
the releasing system every 5 min, and the system was replenished with fresh medium at an equivalent volume. The
amount of released Ce6 was quantified based on a standard
curve of fluorescence intensity vs. standard Ce6 concentration
(λex: 425 nm, λem: 660 nm) using a SPARK 10M microplate
reader. In the meantime, the temperature change of the hybrid
hydrogel was continuously monitored using an infrared
thermal imager (TiS55, Fluke, USA). On the other hand, the
release of MnO2 NPs from the hybrid hydrogel was measured
by using inductively coupled plasma mass spectrometry
(ICP-MS; XSeriesII, Thermo Scientific).
Swelling and degradation behavior
To understand the swelling behavior, 1 mL of agarose@SH/
MnO2/Ce6 hydrogel was immersed in 40 mL of 1 × PBS ( pH =
7.4 or 6.5) at 37 °C or 60 °C. The initial weight of the hybrid
hydrogel was measured as W0. At a predetermined time point,
the hydrogel was taken out and weighed as W1 (hydrated
Biomater. Sci.
Oxygen generation efficiency in vitro
O2 generation capacity of the agarose@SH/MnO2/Ce6 hydrogel
was determined in a closed chamber coupled with an oxygen
probe from a JPB-607A portable digital LCD dissolved oxygen
meter. Briefly, the hydrogel (1 mL) was immersed in deoxygenated 1 × PBS (10 mL) containing 100 mM H2O2, and NIR
laser irradiation (808 nm, 1.5 W cm−2) was applied where
applicable. The dissolved O2 level was monitored at 1 min
intervals.
Cellular uptake of Ce6 in vitro
Cellular internalization efficiency of Ce6 was studied through
both laser scanning confocal microscopy (LSCM) and flow
cytometry. Specifically, 4T1 cells (initial seeding density: 1 ×
105 cells per well) were cultured in a 12-well plate at 37 °C overnight. After that, the cells were exposed to the agarose@SH/
MnO2/Ce6 hydrogel (100 µL), which was immediately irradiated using a NIR laser (808 nm, 1.5 W cm−2) for 5 min. After
another incubation for 0.5 or 2 h, the cells were fixed and
stained with DAPI (1 µg mL−1) for 5 min, followed by examination under an LSM 800 confocal microscope. In another
aspect, flow cytometry was conducted to quantitatively evaluate
the cellular uptake of Ce6. After various treatments, the cells
were resuspended in PBS and Ce6 fluorescence emission from
individual cells was tracked using a NovoCyte flow cytometer.
Data collected from the acquisition system were analyzed
using the software FlowJo v10 (FlowJo, LLC, USA).
Light-induced ROS generation in vitro
DPBF, as a fluorescent probe, was firstly used to evaluate the
ROS generation efficiency of the agarose@SH/MnO2/
Ce6 hydrogel during the photodynamic process. Briefly, 1 mL
hybrid hydrogel was prepared in the bottom of a cuvette, followed by adding 1 mL PBS into the container. Thereafter, the
hydrogel was exposed to a NIR laser (808 nm, 1.5 W cm−2) for
10 min. 1 mL of the supernatant was transferred into a new
cuvette, and 5 µL of DPBF (1 mg mL−1, dissolved in DMSO)
was added into the previous solution. Then, the mixture was
irradiated using an optical laser (660 nm, 1 W cm−2) for
10 min, during which UV-vis absorption spectra were recorded
at a time interval of 2 min. The decreasing rate of optical
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absorption intensity at 417 nm was calculated to reflect in situ
ROS generation efficiency.
DCFH-DA, as a cell-permeable fluorogenic probe, was utilized to measure intracellular ROS generation. Briefly, 4T1 cells
were incubated in a 12-well plate (initial seeding density: 1 × 105
cells per well) overnight. Afterwards, the culture medium was
displaced with 1 mL of the supernatant as previously mentioned
in the DPBF assay. After incubation for 2 h, the cells were subjected to optical laser irradiation (660 nm, 1 W cm−2) for
10 min with or without H2O2 (100 µM). Subsequently, the cells
were thoroughly rinsed with 1 × PBS, followed by staining with
DCFH-DA (10 µM) for 1 h and DAPI (1 µg mL−1) for 5 min. In
the end, fluorescence images of the stained cells were obtained
using LSCM (LSM800, Zeiss, Germany).
Biocompatibility of the hybrid hydrogel in vitro
L929 fibroblasts and human umbilical vein endothelial cells
(HUVECs) were utilized to assess the biocompatibility of the
hybrid hydrogel. Specifically, a certain category of cells was cultured in a 96-well plate (initial seeding density: 1 × 104 cells
per well) overnight. After discarding the previous medium,
fresh culture medium (200 µL) containing the hybrid hydrogel
at various concentrations was replenished into each well, followed by another incubation for 24 h. Next, the cells were
rinsed with 1 × PBS, and were further treated with 200 µL MTT
(0.5 mg mL−1). After 4 h of incubation, the supernatant was
then discarded and DMSO (200 µL) was introduced into each
well. Finally, cell viability was quantified based on the
measurement of optical density (OD) (λ: 490 nm and 630 nm)
using a SPARK 10 M microplate reader as indicated by formula
(3).
Cell viability ð%Þ ¼
Tumor model establishment in vivo
All animal studies complied with the guidelines and policies
of the National Guide for Care and Use of Laboratory Animals
(China), and were approved by the Institutional Animal Care
and Use Committee (IACUC) of Southwest University.
Xenografted tumors were built on female BALB/c mice (6
weeks, ∼20 g each) by subcutaneous inoculation with 4T1 cells
(1 × 106 in 100 μL saline) onto their flank dorsal region upon
anesthetization with isoflurane. Afterwards, all the mice were
continuously bred for ∼10 days until the tumor size reached
up to ∼150 mm3. The dimension of the tumors was manually
measured using calipers, and the tumor volume was calculated
according to the following formula (4).
Tumor volume ¼ ðlongest diameterÞ ðshortest diameterÞ2
OD490 nm sample OD630 nm sample
OD490 nm blank OD630 nm blank
100%:
660 nm laser illumination (1.5 W cm−2) was switched on for
10 min, followed by another 2 h of incubation. Afterwards, the
supernatant was discarded and the cells were gently rinsed
with 1 × PBS at least three times. Then, cell staining was
carried out by incubating the cells with a mixture of calcein
AM (1 µg mL−1) and PI (1 µg mL−1) for 15 min, and the cells
were then examined under a fluorescence microscope (IX73,
Olympus, Japan). On the other hand, quantitative cell viability
was measured using the standard MTT cell viability assay.
Tumor cells in a 96-well tissue culture plate (seeding density of
1 × 104 cells per well) were cultured at 37 °C overnight. Then,
50 µL of diversified hydrogel was introduced into each well,
and the total volume of the medium was kept constant at
200 µL per well. Thereafter, the cells received the same treatments as mentioned above with live/dead cell staining.
Complying with a typical MTT protocol, cell viability in each
well was calculated based on formula (3).
ð3Þ
0:5:
ð4Þ
Cytotoxicity of the hybrid hydrogel in vitro
Photoacoustic/fluorescence (PA/FL) imaging in vivo
The murine mammary carcinoma cell line 4T1 and the human
cervical carcinoma cell line HeLa were utilized for investigating
the hybrid hydrogel-induced cytotoxicity in vitro. Specifically,
tumor cells in a 12-well tissue culture plate (seeding density: 1
× 105 cells per well) were cultured at 37 °C overnight. Then,
200 µL of diversified hydrogel was introduced into each well,
and the total volume of the medium was kept constant at 1 mL
per well. Afterwards, the cells were subjected to the following
treatments: DMEM (group 1), 808 nm + 660 nm laser (group
2), 100 µM H2O2 (group 3), agarose@SH + 808 nm laser (group
4), agarose@Ce6 + 660 nm laser (group 5), agarose@SH/Ce6 +
808 nm + 660 nm laser (group 6), agarose@SH/MnO2/Ce6
(group 7), agarose@SH/MnO2/Ce6 + 808 nm + 660 nm laser
(group 8), and agarose@SH/MnO2/Ce6 + H2O2 + 808 nm +
660 nm laser (group 9). The H2O2 concentration was set at
100 µM for groups 3 and 9. For laser irradiation groups,
808 nm laser illumination (1.5 W cm−2) was performed for
10 min, followed by a further 2 h of incubation. Subsequently,
To demonstrate the PA imaging property, the agarose@SH/
MnO2/Ce6 hydrogel was firstly prepared at various SH concentrations (31.25, 62.5, 125, 250, 500 μg mL−1). Then, PA images
were acquired in vitro using a Vevo LAZR photoacoustic
imaging system (λex = 725 nm, VisualSonics Inc., Canada) and
quantitatively analyzed. In another aspect, tumor bearing
BALB/c mice were treated with 50 µL of hybrid hydrogel
through intratumoral injection, and PA imaging of the tumor
region was performed at 1 h post-injection using the abovementioned PA imaging platform. For fluorescence imaging
in vivo, tumor bearing BALB/c mice were intratumorally administered with the hybrid hydrogel or free Ce6 (equivalent Ce6
concentration: 0.5 mg kg−1). At 0, 2, 6, 24, 72 and 168 h postinjection, fluorescence images of the mice were recorded using
a Fusion imaging system (λex = 630 nm, λem = 670 nm, Fusion
FX7 Spectra, VILBER, France). In the meantime, the mice were
euthanized to collect the tumors and major organs for ex vivo
imaging via the same Fusion imaging system.
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Biomaterials Science
Tumor inhibition in vivo
Results and discussion
BALB/c mice bearing 4T1 tumors were randomly allocated into
10 groups (n = 4 for each group): (1) saline, (2) laser (808 nm +
660 nm); (3) agarose@Ce6; (4) agarose@Ce6 + 660 nm; (5)
agarose@SH; (6) agarose@SH + 808 nm; (7) agarose@SH/Ce6;
(8) agarose@SH/Ce6 + 808 nm + 660 nm; (9) agarose@SH/
MnO2/Ce6; and (10) agarose@SH/MnO2/Ce6 + 808 nm +
660 nm. The mice in all groups were intratumorally injected
with 50 µL hydrogel, followed by gelatinization for 1 h. Next,
the tumor region was irradiated by NIR laser irradiation
(808 nm, 1.5 W cm−2) for 10 min, and thermographic mapping
of the mouse was concurrently performed using a thermal
imaging camera (Ti55, Fluke, USA). One hour later, the tumor
site was further exposed to an optical laser (660 nm, 1 W cm−2)
for 10 min where applicable. Tumor volume and mouse body
weight were continuously monitored for two weeks post-treatment. The tumor growth inhibition (TGI) rate was calculated
on the basis of formula (5)
Characterization of MnO2 NPs
TGI ¼ ð1 V T =V c Þ 100%
ð5Þ
where Vc represents the average tumor volume of the saline
group, and VT stands for the tumor volume in the treatment
groups on a specific evaluation day.
On day 14, mice from all groups were sacrificed to harvest
tumors. After being weighed, solid tumors were fixed with
10% formalin and paraffin-embedded, followed by slicing into
thin sections (thickness: 5 µm) that were further stained with
H&E, TUNEL, Ki67 and HIF-1α antibody according to the manufacturer’s protocol. All histological sections were observed
under an IX73 fluorescence microscope.
Biosafety evaluation
On day 14 post-treatment, mice from all groups were sacrificed
to collect the vital organs. All these tissues were sliced into sections (thickness: 5 µm), which were further stained with H&E
to evaluate the morphological features of necrosis. Moreover,
routine blood tests were performed on KM mice subjected to
an intratumoral injection of the hybrid hydrogel (50 µL) into
the flank dorsal region, and whole blood (200 µL) was collected from the orbital venous plexus at day 0, 1, 7 and 14
post-treatment. The major index of blood was assessed using a
hematology analyzer (BC-2600Vet, Mindray, China). Potential
systemic inflammation after intratumoral injection of the
hybrid hydrogel (50 µL) was explored by quantifying the TNF-α
(an inflammatory marker) level in the peripheral blood. The
supernatant containing blood serum was collected by centrifugation of whole blood (100 µL) at 3000 rpm for 10 min, after
which the TNF-α level was determined using the Rat TNF-α
ELISA Kit.
Statistical analysis
All data were presented as mean ± SD using one-way analysis
of variance (ANOVA) using OriginPro 8.5 software (OriginLab,
MA, USA). A p-value less than 0.05 (*p < 0.05, n = 4) was considered to be statistically significant.
Biomater. Sci.
The synthesis of MnO2 NPs was performed via a simple soft
chemistry through the reduction of KMnO4 at ambient temperature. The morphology and structure of MnO2 NPs were elucidated by SEM imaging (Fig. 2a). Clearly, MnO2 NPs exhibited
a honeycomb structure with a diameter of ∼97 nm, which
could be self-assembled from MnO2 nanoplatelets as revealed
in the previous studies.51,52 To evaluate the elemental composition of MnO2 NPs, FESEM-EDX analysis was performed on a
single particle. An intense signal corresponding to Mn and O
was detected as evidenced by elemental mapping, suggesting
their main chemical composition (Fig. S1†). Moreover, the
crystallographic phase of MnO2 NPs was determined by XRD
(Fig. 2b). Strong peaks were recorded at 2θ = 12.3, 24.3, 36.6,
and 65.7°, which could be indexed to the (001), (002), (100)
and (110) planes of birnessite-type MnO2, respectively.53
Additionally, the FT-IR spectrum of MnO2 NPs is illustrated in
Fig. S2.† Characteristic peaks at 578 and 519 cm−1 are assigned
to Mn–O stretching vibrations, and infrared bands at 3450 and
1640 cm−1 are ascribed to O–H vibrations. The bands at 1421
and 2935 cm−1 are indexed to the C–H bending or stretching
vibrations of oleic acid.54 Furthermore, compared to the
KMnO4 precursor, MnO2 NPs displayed a broad absorption
band ranging from 250 to 500 nm, which corresponds well
with the optical properties of these reported MnO2 nanomaterials (Fig. S3†).55 The hydrodynamic size of MnO2 NPs
was measured as 110 nm with a polydispersity index (PDI) of
0.125, manifesting the formation of a uniform dispersion
(Fig. 2c). In another aspect, decent aqueous dispersity, high
performance of photothermal conversion and good biocompatibility of SH have been demonstrated in previous studies.26
Characterization of the hybrid hydrogel
High homogeneity agarose@SH/MnO2/Ce6 hydrogel is significantly dependent on a thorough stirring of the corresponding
liquid mixture at a temperature above the melting point of
LMP agarose. SH, Ce6 and MnO2 NPs can be uniformly encapsulated into the cross-linked agarose matrix during the gelatinization process. The morphology of the as-developed hybrid
hydrogel is revealed by the SEM image in Fig. 2d. Obviously,
MnO2 NPs were effectively decorated onto the surface of the
agarose matrix, and the loose structure of the hybrid hydrogel
was incredibly favorable for liquid adsorption and follow-up
dissolution. The FT-IR spectra of the hybrid hydrogel and its
components are exhibited in Fig. 2e. Distinct peaks of 2890
and 1150 cm−1 from Ce6 denote the stretching vibration of the
C–H and C–O–C groups, respectively.56 The FT-IR spectra of
SH confirm the presence of numerous chemical groups,
including hydroxyl, carboxyl and vinyl groups.26 As for agarose,
a broad absorption band at 3400 cm−1 is associated with O–H
stretching. Characteristic bands at 773, 894, and 932 cm−1 are
ascribed to the 3,6-anhydro-β-galactose skeletal bending. The
absorption peak at 1072 cm−1 is attributed to the deformation
mode of C–O.57 The FT-IR spectrum of the hybrid hydrogel dis-
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Fig. 2 Characterization of MnO2 NPs and the hybrid hydrogel. (a) FESEM image of MnO2 NPs; (b) XRD pattern of MnO2 NPs; (c) hydrated diameter
of MnO2 NPs measured by DLS; (d) FESEM image of the cross-section of the hybrid hydrogel; (e) FT-IR spectra of agarose, SH, MnO2, Ce6 and the
hybrid hydrogel; (f ) images of the mixture containing agarose, SH, Ce6 and MnO2 NPs before and after gelatinization; (g) temperature elevation of
the hybrid hydrogel with various equivalent concentrations of SH under NIR laser irradiation (808 nm, 1.5 W cm−2) for 10 min; (h) temperature
change of the hybrid hydrogel under four cycles of NIR laser irradiation (laser on: 10 min, laser off: 10 min per cycle); (i) thermographic mapping of
the cuvette containing various hybrid hydrogels after NIR laser irradiation for 0–10 min.
played all representative bands of agarose, Ce6, SH and MnO2
NPs, verifying a successful preparation of the final product.
Moreover, the hybrid hydrogel exhibited a black color, which
was in accordance with the precursor solution before gelatinization (Fig. 2f ).
Photothermal response of the hybrid hydrogel
At the beginning, we evaluated the photothermal property of
purified SH upon NIR laser irradiation for 10 min (Fig. S4a†).
As expected, the temperature increase of the SH solution was
positively correlated to the agent concentration and laser
irradiation time. In particular, the temperature of SH (1 mg
mL−1) was increased from the initial 25 °C to 65.2 °C after
10 min of laser irradiation, suggesting a high performance of
SH-mediated photothermal conversion. In parallel, the
response of the hybrid hydrogel toward NIR laser illumination
was similarly explored at various equivalent SH concentrations
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(Fig. 2g). Likewise, intense hyperthermia generation was effectively triggered by light irradiation, which is adequate for
damaging tumor cells through the destruction of intracellular
proteins and genetic materials. Interestingly, an even higher
temperature increase was found in the hybrid hydrogel compared to free SH upon receiving laser irradiation for the same
period, which could be interpreted as slower heat dissipation
from the hydrogel attributed to attenuated liquid evaporation
(Fig. S4b†). To investigate the photothermal stability of the
hybrid hydrogel, periodic NIR laser irradiation was conducted
for four cycles. There was no significant decrease of the peak
temperature in each cycle, implying a satisfactory stability for
light-induced PTT (Fig. 2h). Infrared thermography of the
hybrid hydrogel was further performed to dynamically
monitor the temperature during laser irradiation, and the data
exhibited good agreement with the measurements using a
digital thermometer (Fig. 2i).
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In vitro drug release
A facile route was proposed to investigate Ce6 release from the
hybrid hydrogel, ingeniously avoiding the use of traditional
dialysis bags. Briefly, the solidified hydrogel was formed in the
bottom of a cuvette, and the drug content in the liquid supernatant was quantified after various treatments (Fig. 3a). We
firstly evaluated the role that temperature played in regulating
drug release. A more rapid Ce6 release was found at a higher
temperature of 60 °C, in comparison with that at 37 °C under
physiological conditions (Fig. 3b). Hyperthermia-responsive
drug release can be ascribed to enhanced hydrogel hydrolysis
and drug diffusion as indicated in the previous studies.24–26
Inspired by the positive correlation between temperature and
drug dissolution, we further explored the effect of laser
irradiation on Ce6 release. The fluorescence spectrum of the
supernatant in each cuvette (λex = 405 nm) was measured after
a specific period of NIR irradiation (808 nm, 1.5 W cm−2). A
sustained Ce6 release was observed in the process of laser
irradiation for 60 min, as evidenced by a time-dependent
increase of fluorescence emission intensity at 662 nm, implying a good opportunity to use NIR light as an exogenous
stimulus to regulate Ce6 diffusion (Fig. 3c, S5†). In addition,
the release of MnO2 NPs can also be accelerated under NIR
laser irradiation in comparison with the group without any
treatment (Fig. S6a†).
To further investigate the NIR-triggered unloading of the
drug cargo, the hybrid hydrogel was periodically irradiated
using a NIR laser in an “ON/OFF” fashion (5 min on and
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5 min off per cycle). The temperature of the hybrid hydrogel
rapidly rose to ∼50 °C after laser irradiation for 5 min, caused
by the high performance of SH in photothermal conversion
(Fig. 3d). Moreover, Ce6 release was considerably accelerated
in the course of laser irradiation compared to that without any
treatment (Fig. 3d, S6b†). Moreover, the profile of temperature
variation and responsive Ce6 release was not remarkably
changed during all five irradiation cycles, implying the potential for recycling in practical applications. To be more specific,
the average Ce6 release rate during five consecutive ON/OFF
laser irradiation cycles was calculated as shown in Fig. 3e. As
anticipated, laser irradiation could effectively initiate a more
rapid Ce6 release from the hybrid hydrogel. Notably, a relatively higher release rate in the first irradiation cycle could be
ascribed to the dissolution of Ce6 on the surface or in the
external layer of the hydrogel. By contrast, a moderate drug
release was observed in the following irradiation cycles, owing
to slower drug dissolution from the interior hydrogel. In
another aspect, re-gelatinization of the hydrogel instantly
occurred when switching off the laser, thereby restraining the
encapsulated cargoes from releasing.
To elucidate in depth the underlying mechanism of Ce6
release, various hybrid hydrogels were fabricated by varying
the concentrations of SH, DOX and LMP agarose. Obviously,
cumulative Ce6 release was evidenced to have a positive correlation with the loading amount of SH and Ce6 in the hydrogel.
A larger amount of encapsulated SH may render a stronger
local hyperthermia during the same period, which resulted in
faster drug release from the hydrogel (Fig. S7a and b†).
Fig. 3 Drug release property: (a) schematic diagram of the setup for investigating NIR light-triggered drug diffusion; (b) in vitro Ce6 release from
the hybrid hydrogel under different ambient temperatures of 37 °C or 60 °C; (c) fluorescence spectra of the supernatant containing Ce6 subjected
to NIR laser irradiation for different time periods (λex = 405 nm); (d) temperature elevation and concurrent cumulative Ce6 release under cyclic NIR
laser irradiation (laser on: 5 min, laser off: 5 min per cycle); (e) average Ce6 release rate in five laser irradiation cycles; (f ) oscillatory shear rheology
(G’ and G’’) of the hybrid hydrogel (1% agarose) displayed in the temperature-dependent modulus.
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Furthermore, the hybrid hydrogel prepared from a higher proportion of agarose content may lower the Ce6 release rate,
which can be interpreted as the blockage of the Ce6 release
pathway in a more densely crosslinked network (Fig. S7c†). As a
result, follow-up hydrolysis and degradation could also be
impeded when subjected to laser irradiation. Apart from the
abovementioned factors related to the ingredient composition
of the hybrid hydrogel, Ce6 release can also be accelerated by
increasing the laser power density, implying that higher NIR
laser power may facilitate a rapid disruption of the compact
polymeric matrix and further trigger an accelerated dissolution
of entrapped Ce6 (Fig. S7d†). To sum up, there exists appealing
potential to customize the Ce6 release behavior from the hybrid
hydrogel via the optimization of its chemical composition and
the adjustment of the operating parameter of the NIR laser.
Rheological property and in vitro degradation
To investigate the thermoresponsive behavior of the hybrid
hydrogel developed from 1% agarose, the rheological property
was characterized through temperature sweeping analysis,
where the change in storage modulus (G′) and loss modulus
(G″) was recorded over a temperature ranging from 30 °C to
60 °C (Fig. 3f). Both G′ and G″ values decreased upon heating,
suggesting a typical reduction of noncovalent crosslinking with
increasing temperature. An intersection of G′ and G″ curves was
found at a temperature of ∼60 °C, indicating a gel–sol transition
above the physiological temperature. The macromolecular chain
disentanglement was dynamically slow as G′ was always higher
than G″ before phase transformation. We assume that the
agarose concentration may play an important role in determining its thermoresponsive behavior, and thus temperature swelling curves of the hybrid hydrogel with 0.5% and 2% agarose
were determined for comparison (Fig. S8†). G″ was considerably
larger than G′ for the gel prepared from 0.5% agarose, explicitly
implying a liquid form of the mixture. Conversely, G″ was an
order of magnitude lower than G′ for 2% agarose hydrogel,
manifesting its existence in solid state. Hence, the concentration of agarose for hybrid hydrogel synthesis was optimized
as 1% for the following studies both in vitro and in vivo. The
degradability of the hydrogel was assessed by calculating the
swelling ratio and weight loss of the hybrid hydrogel in 1 × PBS.
An elevated swelling ratio of the hydrogel was found at a relatively high incubation temperature (60 °C), further leading to an
accelerated hydrolysis of the hydrogel as indicated by a more
rapid weight loss under the same conditions (Fig. S9†).
Thermal-induced acceleration of degradation can be ascribed to
the rapid hydrolysis of long polysaccharide chains and subsequent slow-paced hydrolysis of the fragmented chains into
soluble oligosaccharides.58 In another aspect, we found that the
pH value of the incubating medium exhibited an insignificant
impact on the swelling ratio and weight loss of the hybrid
hydrogel.
Cellular uptake of the released Ce6 in vitro
Effective uptake and intracellular internalization of Ce6
released from the hybrid hydrogel are highly desirable for
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Paper
enhanced PDT because the as-generated ROS directly induces
damage to the mitochondrial respiratory chain. In this study,
4T1 cells were exploited to study the cellular uptake of Ce6 by
tracking its green fluorescence using LSCM and flow cytometry
(Fig. 4). Obviously, a typical time-dependent cellular uptake of
Ce6 was evidenced by a higher fluorescence intensity when the
incubation time was increased from 0.5 to 2 h. Notably, NIR
laser irradiation further increased the intracellular localization
of Ce6, in sharp contrast to the control group in the absence of
irradiation (Fig. 4a). Light-enhanced cellular uptake of Ce6 can
be interpreted as a higher level of available Ce6 in the culture
medium, attributed to an accelerated drug release from the
hybrid hydrogel under local hyperthermia as previously verified. Besides, mild local hyperthermia may also facilitate the
cellular uptake of small drug molecules by an increase in the
fluidity of the cell membrane as demonstrated in the previous
studies.59,60 Quantitative results obtained from flow cytometry
similarly indicated a NIR-enhanced cellular uptake of Ce6, and
the uptake efficiency notably reached up to 72.88% in the
group of “2 h plus laser” (Fig. 4b–d).
H2O2 decomposition and enhanced ROS generation in vitro
MnO2 has been recognized as a prestigious inorganic catalyst
that can activate the decomposition of H2O2 into O2, which is
known to relieve hypoxia and improve the outcome of PDT. In
this respect, the O2 generation performance was evaluated by
measuring the level of dissolved O2 after incubating the hybrid
hydrogel in H2O2 solution (Fig. 5a). We found that the MnO2incorporated hydrogel effectively initiated a rapid O2 production in the first 15 min of incubation compared to other
control groups. Subsequent O2 increase was not remarkable,
which could be explained as the saturation of dissolved
oxygen. Furthermore, a more rapid O2 generation was observed
in the group of hybrid hydrogel plus NIR laser irradiation. We
deduce that the laser-activated acceleration of gel hydrolysis
may introduce more H2O2 into the polymeric matrix, which
simultaneously reacted with the incorporated MnO2 NPs to
lead to O2 generation.
Oxygen in the triplet state can be converted into singlet
oxygen (1O2), a category of cytotoxic ROS to destroy tumor
cells, during the light-induced photodynamic process. The 1O2
producing ability mediated by the hybrid hydrogel was firstly
evaluated using a DPBF probe, which is a non-selective probe
for the detection and quantitative determination of ROS. As
shown in Fig. 5b, H2O2 alone can render a certain degree of
DPBF absorption decrease at 417 nm. In sharp contrast, a
more remarkable absorbance decrease was observed in both
hydrogel groups plus NIR laser irradiation, verifying that
effective NIR-triggered release of the Ce6 photosensitizer is a
prerequisite for efficient follow-up ROS generation. Notably,
the highest amount of ROS was achieved in the hybrid hydrogel with MnO2 NP incorporation, on account of the amelioration of the dissolved O2 level through catalytic decomposition
of H2O2. Alternatively, the intracellular 1O2 yield mediated by
the hydrogel was analyzed using a DCFH-DA probe, which can
be oxidized by ROS to generate a highly fluorescent substance
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Fig. 4 Intracellular internalization of Ce6 released from the hybrid hydrogel subjected to NIR laser irradiation (“L”) in vitro: (a) LSCM imaging and
flow cytometry analysis including (b) dot plots, (c) cell count vs. log of normalized fluorescence at 660 nm and (d) mean intensity of fluorescence
emission, after 4T1 tumor cells were subjected to various treatment regimens (scale bar: 50 µm).
DCF (Fig. 5c). Compared to the groups in the absence of NIR
irradiation, there was a vivid green fluorescence signal representing ROS upon the hydrogel receiving NIR irradiation,
suggesting that hyperthermia-induced hydrogel degradation is
essential for Ce6 release to trigger PDT. Likewise, the strongest
ROS generation was distinctly found in the group of hybrid
hydrogel plus NIR laser, which agreed well with the previous
findings of the 1O2 yield in the solution system. To quantitatively study the DCF fluorescence inside the cells, flow cytometry was performed and the results were in accordance with
fluorescence microscopy (Fig. S10†).
Biomater. Sci.
In vitro cytotoxicity of the hybrid hydrogel
Satisfactory biocompatibility is critical for the successful application of biomaterials. SH alone would cause negligible cell
death, which has been explicated in our previous study.26
Thereby, the cytotoxicity of MnO2 NPs and the hybrid hydrogel
was evaluated on normal somatic cells, including L929 fibroblasts and HUVECs, via the MTT cell viability assay. After incubation with MnO2 NPs and the hybrid hydrogel for 24 h, the
percentage of live cells was both above 90% even at a concentration as high as 2 mg mL−1, manifesting a good biocompat-
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Fig. 5 H2O2 decomposition and enhanced ROS generation in vitro. (a) Simultaneous dissolved O2 level in 1 × PBS after various treatments (808 nm
laser power: 1.5 W cm−2, H2O2: 100 µM); (b) DPBF consumption after 660 nm laser irradiation (1 W cm−2) for 10 min on the basis of the corresponding treatments in (a); (c) fluorescence imaging of 4T1 cells after various treatments and subsequent irradiation using an optical laser (660 nm, 1
W cm−2) for 10 min (H2O2: 100 µM, scale bar: 50 µm).
ibility for clinical use (Fig. 6b and c). We thereby measured the
cytotoxicity of the hybrid hydrogel with 4T1 murine breast
cancer cells. The standard live and dead cell fluorescence
staining indicated that hybrid hydrogel plus 808 and 660 nm
laser (groups 8 and 9) induced the most outstanding tumor
cell destruction, as illustrated by the strong red fluorescence
signal in the PI channel (Fig. 6a). It is worth noting that a
more severe cell ablation was observed in the presence of
H2O2, which can be ascribed to enhanced ROS production at a
higher level of oxygen. Quantitative cytotoxicity induced by the
hybrid hydrogel was further measured by the MTT assay
(Fig. 6d). The data present a similar trend with the corresponding observations in previous fluorescence microscopy. In
sharp contrast to the control groups, more than 85% of the
tumor cells were eradicated in group 9, implying a remarkable
tumor ablation effect through enhanced PTT/PDT in vitro.
In vivo retention of the hybrid hydrogel
In vivo retention of the hybrid hydrogel was firstly evaluated
through PA imaging. A previous study has proved that dynamic
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contrast-enhanced PA imaging can be realized on stimuliresponsive photothermal hydrogel.61 Therefore, we assume
that PA imaging with high resolution and deep tissue penetration could be achieved by the as-synthesized hybrid hydrogel featured with high photothermal conversion performance.
To evaluate the PA properties of the hybrid hydrogel, the
amplitudes of the PA signal at a hydrogel concentration
ranging from 25 to 500 μg mL−1 were measured in vitro
(Fig. 7a). An intense PA signal was produced under laser excitation, and the signal intensity (average value within the pseudocolor region) strictly followed a concentration-dependent
pattern. We thereby explored hydrogel retention after intratumoral injection by collecting the PA signal in the tumor area.
After 1 h post-injection, a detectable PA signal was locally
recorded, which was in striking contrast to the blank control
group (Fig. 7b and c). These results clearly indicated that the
hybrid hydrogel can be successfully administered with
good retention through intratumoral injection, and may also
benefit high performance and long-term PA monitoring
in vivo.
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Fig. 6 In vitro biocompatibility and cytotoxicity of the hybrid hydrogel. (a) Fluorescence images of 4T1 cells stained with calcein AM and PI after
receiving various treatment recipes (scale bar: 200 µm); viability of HUVECs and L929 cells after treatment with (b) MnO2 NPs or (c) the hybrid hydrogel for 24 h; (d) viability of 4T1 and HeLa cells after diverse treatments measured by the MTT assay (*p < 0.05 between two groups). Group 1: DMEM,
group 2: 808 nm + 660 nm laser, group 3: H2O2 (100 µM), group 4: agarose@SH + 808 nm laser, group 5: agarose@Ce6 + 660 nm laser, group 6:
agarose@SH/Ce6 + 808 nm + 660 nm laser, group 7: agarose@SH/MnO2/Ce6, group 8: agarose@SH/MnO2/Ce6 + 808 nm + 660 nm laser, group 9:
agarose@SH/MnO2/Ce6 + H2O2 + 808 nm + 660 nm laser.
In addition, biodistribution of Ce6 after intratumoral injection of the hybrid hydrogel was characterized by tracking Ce6
fluorescence using an animal imaging system, and free Ce6injected mice served as the control group (Fig. 7d and e). Free
Ce6 exhibited instant tumoral accumulation and rapidly
diffused throughout the mouse body within 6 h owing to
blood circulation. Moreover, a rapid decay of Ce6 fluorescence
within the tumor region was initiated at 2 h post-injection,
suggesting a poor retention at the injection site. By contrast,
the fluorescence signal of Ce6 was gradually intensified in the
tumorous tissue within 24 h after hydrogel administration,
which can be attributed to its long retention in the tumor area
and a sustained cargo release from the hydrogel (Fig. S11†).
In vivo antitumor effect
The antitumor effect of the hybrid hydrogel was evaluated on
BALB/c mice bearing 4T1 tumors subjected to diverse treatments. After intratumoral injection of various therapeutic
agents and allowing for 1 h of gelatinization, the NIR-induced
photothermal effect on the mouse body was dynamically monitored by thermographic imaging (Fig. 8a and b). A dramatic
temperature increase was observed in all hydrogel groups after
5 min of NIR irradiation, indicating that the incorporation of
SH can efficiently facilitate the conversion of light energy into
local hyperthermia. Moreover, the final temperature exceeded
50 °C, which is higher than the threshold of 48 °C for effica-
Biomater. Sci.
cious PTT.62 Then, the mice were randomly assigned into ten
groups. The tumor volume in each group was dynamically
measured using a caliper for 14 days after various treatments
(Fig. 7c). Compared to the saline group, single-modal treatment of PDT (group 4) or PTT (group 6) led to a moderate
tumor inhibition effect after 14 days. By contrast, predominant
tumor inhibition with a TGI of 65.8% was observed in the
mice administered with agarose@SH/Ce6 plus laser irradiation
(group 8), owing to a strong combined PTT/PDT effect.
Furthermore, the most effective tumor eradication with a TGI
as high as 93.8% was achieved upon laser irradiation mediated
by the agarose@SH/MnO2/Ce6 hydrogel (group 10). We speculate that the component of MnO2 was responsible for the amelioration of hypoxia in the tumor microenvironment, which
further resulted in improved PDT in vivo. Such tumor variation
was also verified by the smallest anatomical size (Fig. 7d) and
average weight of the dissected tumors among all groups at
day 14 post-treatment (Fig. 7e). In addition, the weight loss of
mice was not distinct regardless of any treatment, suggesting
minimal adverse effects caused by these drug formulations
(Fig. 7f ).
Histological analysis and biosafety evaluation
Tumor tissue destruction was evaluated by immunostaining of
the excised tumor sections using anti-HIF-α, H&E, Ki67 and
fluorometric TUNEL assays (Fig. 9). Hypoxia-inducible factor-
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Fig. 7 In vivo retention of the hybrid hydrogel characterized by PA and fluorescence imaging. (a) PA signal intensity and the corresponding PA
images as a function of the concentration of the hybrid hydrogel; (b) PA images of the tumor region at 1 h after intratumoral (i.t.) injection or without
any treatment; (c) average PA signal intensity in the tumor region corresponding to (b); (d) fluorescence images of BALB/c mice bearing 4T1 tumors
at various time points after intratumoral injection of free Ce6 or hybrid hydrogel (equivalent Ce6 concentration: 0.5 mg kg−1); (e) ex vivo fluorescence images of the tumor and major organs dissected at 24 h post-injection.
Fig. 8 In vivo antitumor effect on BALB/c mice bearing 4T1 tumors. (a) Thermographic images of mice during NIR laser irradiation (808 nm, 1.5 W
cm−2) at the tumor site for 5 min after various treatments; (b) change of the peak temperature in the tumor region over time during NIR irradiation
corresponding to (a); (c) tumor volume variation within 14 days after various treatments (**p < 0.01 as compared to other groups); (d) digital image
of the tumors collected at day 14 after different treatments; (e) average weight of the dissected tumor at day 14 corresponding to (d) (*p < 0.05 as
compared to other groups); (f ) variation of the mouse body weight within 14 days after various treatments. Group 1: saline, group 2: laser (808 nm +
660 nm); group 3: agarose@Ce6; group 4: agarose@Ce6 + 660 nm; group 5: agarose@SH; group 6: agarose@SH + 808 nm; group 7: agarose@SH/
Ce6; group 8: agarose@SH/Ce6 + 808 nm + 660 nm; group 9: agarose@SH/MnO2/Ce6; group 10: agarose@SH/MnO2/Ce6 + 808 nm + 660 nm.
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Fig. 9 Histological analyses of dissected tumor sections via immunostaining, including (a) anti-HIF-α fluorescence staining, (b) H&E staining; (c)
Ki67 staining and (d) fluorometric TUNEL assays. Scale bars: 100 µm.
1α (HIF-α) immunofluorescence staining was firstly used to
evaluate the oxygen level inside the tumoral tissues. In contrast to other treatment groups, Nile red fluorescence was not
observed from the tumor slices in group 10 (hybrid hydrogel +
dual-laser), indicating an effective local hypoxia amelioration
thanks to MnO2-mediated catalytic conversion of endogenous
H2O2 into O2 (Fig. 9a). Such oxygenation in the tumor microenvironment is particularly favorable for attenuation of
hypoxia-associated resistance during PDT. Moreover, typical
pyknosis, karyorrhexis and karyolysis were further observed in
the H&E stained slices from group 10, whereas intact mem-
Biomater. Sci.
brane and nucleus were almost retained in the tumor cells
from other control groups (Fig. 9b). Likewise, tumor cells
expressing a high level of Ki67 were dramatically decreased in
group 10, manifesting a significant weakening of cell proliferation (Fig. 9c). In the meantime, TUNEL-positive tumor cells
considerably increased in the same group, implying a potent
tumor killing effect by inducing cell apoptosis and necrosis.
These histopathological findings verified a strong therapeutic
outcome against 4T1 solid tumors through photo-activated
therapy mediated by the hybrid hydrogel. The potential systemic toxicity of the hybrid hydrogel was assessed through
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blood routine test and histopathological analysis. The complete blood counts and other key hematology parameters of
mice were not dramatically changed in two weeks after
administration (Fig. S12†). Moreover, there were no apparent histological abnormality and lesion at the cellular or
tissue level, as revealed by H&E stained slices of the major
organs at day 14 post-treatment, which implied minimal
systemic toxicity or adverse effect for in vivo applications
(Fig. S13†). To further assess the immunogenicity of the
hybrid hydrogel, the level of TNF-α in the blood serum of
mice was determined by ELISA (Fig. S14†). The data fluctuation was insignificant during 14 days after intratumoral
injection, indicating a weak immunogenicity induced by
the hybrid hydrogel.
Genetic alterations of tumor cells always lead to hyperplasia, uncontrolled proliferation, resistance to apoptosis and
anaerobic glycolysis, which further produce a unique TME of
hypoxia, oxidative stress and acidosis. In particular, aberrant
vascularization and a poor blood supply generally result in permanent or transient hypoxia in the extracellular matrix of the
tumor tissue.63 Unfortunately, hypoxia would cause an undesirable resistance to medical therapies, such as radiotherapy,
chemotherapy and PDT. Perfluorocarbon-based oxygen shuttles have been extensively used to transport oxygen into the
tumor. However, this strategy suffers from limited oxygen
delivery efficiency, particularly toward the deep tumor region
that is far away from the intratumoral vessels.64 Alternatively,
MnO2 nanostructures can react with the high level of intrinsic
H2O2 to sustainably produce O2, in order to enhance the
efficacy of PDT, and the catalysis includes the following
reactions:
2MnO2 þ H2 O2 ¼ 2MnOOH þ O2
ð6Þ
2MnOOH þ 4Hþ þ H2 O2 ¼ 2Mn2þ þ 4H2 O þ O2
ð7Þ
2MnOOH þ 2Hþ ¼ MnO2 þ Mn2þ þ 2H2 O:
ð8Þ
In this study, we have synthesized a category of honeycomb
MnO2 NPs, which were used to relieve local hypoxia. This synthesis process is rapid through a series of mild reactions.
Moreover, MnO2 NPs exhibited a high aqueous dispersity,
which guarantees their uniform distribution within the
hydrogel. After being encapsulated into the hydrogel, it is not
essential to release MnO2 NPs from the gel matrix and
perform their catalytic functions. In fact, interstitial fluid
containing a high level of H2O2 can rapidly infuse into the
hydrogel to react with MnO2 NPs during the hydrolysis
process. Subsequently, the generated O2 can be released from
the hydrogel and further diffuse into the tumor cells to
increase the intracellular O2 level. On the other hand, we also
demonstrated the release of Ce6 from the hydrogel with or
without NIR laser activation. In the meantime, a highly
efficient cellular uptake of Ce6 and the resultant high level
ROS production were verified in these oxygenated tumor
cells. It is worth noting that extracellular ROS generation may
also cause the small-molecule oxidants to induce cell apopto-
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sis and inhibit cell proliferation, in the case of Ce6 molecules
that are present in the ECM without intracellular
internalization.65
In another aspect, the SH-laden agarose-based hydrogel was
responsive to NIR light to produce local hyperthermia, which
not only initiated local PTT but also triggered effective Ce6
release. Simultaneously, thermal-induced sol–gel phase transition also activated the rapid hydrolysis of hydrogel, which
facilitated the introduction of interstitial fluid into the gel
matrix to initiate oxygen generation. Thanks to the high controllability and good tumor-site retention of such smart hydrogel, systemic toxicity and local inflammation can be significantly prevented after administration as evidenced by comprehensive histopathological analysis. These results suggest a
good opportunity of developing agarose-based hydrogel platforms for highly effective tumor treatments, which respond to
both endogenous and exogenous factors.
Conclusion
In summary, an injectable agarose-based hydrogel was successfully prepared for enhanced photo-induced tumor therapy
through the effective relief of hypoxia. Such a hybrid hydrogel
exhibited good biocompatibility, high performance in photothermal conversion and light-triggered cargo release behavior.
On account of the hydrolysis of the hydrogel under local
hyperthermia, the incorporated MnO2 can effectively ameliorate hypoxia through catalytic decomposition of H2O2, which
is produced in a high amount in the TME. Not only an
increase of cellular internalization of Ce6 and elevation of the
local O2 level, but also an efficacious PTT effect were achieved
under NIR activation in vitro. Subsequently, enhanced PDT was
achieved under 660 nm laser irradiation owing to the improvement of local oxygenation. Remarkably, merely single intratumoral injection of the hybrid hydrogel can produce a significant tumor inhibition effect under light activation
in vivo, without causing a pathological lesion in the
primary organs. Overall, the injectable SH/MnO2/Ce6
incorporated agarose hydrogel offers new opportunities for
tumor microenvironment modulation, and inspires the development of novel hydrogel-based strategies towards clinical
applications.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This project was supported by the Technology Innovation and
Application Demonstration Grant of Chongqing (cstc2018jscxmsybX0078) and the National Natural Science Foundation of
China (51703186, 31671037).
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