Received: 2 February 2024 Revised: 26 April 2024 Accepted: 10 May 2024
DOI: 10.1002/agt2.600
R E S E A R C H A R T I C L E
Spatiotemporal responsive hydrogel microspheres for the
treatment of gastric cancer
Li Wang1,2,3 Lu Fan2,3 Anne M. Filppula2 Yu Wang1,3 Luoran Shang4
Hongbo Zhang1,2,5
1Joint Centre of Translational Medicine, Wenzhou Key Laboratory of Interdiscipline and Translational Medicine, The First Affiliated Hospital of Wenzhou Medical University,
Wenzhou, China
2Pharmaceutical Sciences Laboratory, Åbo Akademi University, Turku, Finland
3Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Wenzhou Institute, University of Chinese Academy of Sciences, Wenzhou, Zhejiang,
China
4Shanghai Xuhui Central Hospital, Zhongshan-Xuhui Hospital, the Shanghai Key Laboratory of Medical Epigenetics, the International Co-laboratory of Medical Epigenetics and
Metabolism (Ministry of Science and Technology), Institutes of Biomedical Sciences, Fudan University, Shanghai, China
5Turku Bioscience Centre, University of Turku and Åbo Akademi University, Turku, Finland
Correspondence
Hongbo Zhang, Pharmaceutical Sciences
Laboratory, Åbo Akademi University, Turku
20520, Finland.
Email: hongbo.zhang@abo.fi
Luoran Shang, Institutes of Biomedical Sciences,
Fudan University, Shanghai 200032, China.
Email: luoranshang@fudan.edu.cn
Yu Wang, Wenzhou Institute, University of
Chinese Academy of Sciences, Wenzhou,
Zhejiang 325001, China.
Email: 1146681561@qq.com
Funding information
National Natural Science Foundation of China,
Grant/Award Number: 82372145; Research
Fellow, Grant/Award Number: 353146; Research
Project, Grant/Award Number: 347897; Solutions
for Health Profile, Grant/Award Number: 336355;
InFLAMES Flagship, Grant/Award Number:
337531; Finland China Food and Health
International Pilot project funded by Finnish
MInistry of Education and Culture
Abstract
The development of tumor drug microcarriers has attracted considerable interest
due to their distinctive therapeutic performances. Current attempts tend to elab-
orate on the micro/nano-structure design of the microcarriers to achieve multiple
drug delivery and spatiotemporal responsive features. Here, the desired hydrogel
microspheres are presented with spatiotemporal responsiveness for the treatment of
gastric cancer. The microspheres are generated based on inverse opals, their skele-
ton is fabricated by biofriendly hyaluronic acid methacrylate (HAMA) and gelatin
methacrylate (GelMA), and is then filled with a phase-changing hydrogel composed
of fish gelatin and agarose. Besides, the incorporated black phosphorus quantum dots
(BPQDs) within the filling hydrogel endow the microspheres with outstanding pho-
tothermal responsiveness. Two antitumor drugs, sorafenib (SOR) and doxorubicin
(DOX), are loaded in the skeleton and filling hydrogel, respectively. It is found that
the drugs show different release profiles upon near-infrared (NIR) irradiation, which
exerts distinct performances in a controlled manner. Through both in vitro and in
vivo experiments, it is demonstrated that such microspheres can significantly reduce
tumor cell viability and enhance the efficiency in treating gastric cancer, indicating
a promising stratagem in the field of drug delivery and tumor therapy.
K E Y W O R D S
combination therapy, gastric cancer, inverse opal, microcarriers, spatiotemporal responsiveness
1 INTRODUCTION
The increasing incidence of cancer has brought huge burden
to the current social medical system.[1–3] Tremendous efforts
are devoted to cancer treatment including drug therapy, radi-
ation therapy, and surgical resection.[4–7] Of these, drug ther-
apy remains an effective stratagem.[8,9] For instance, doxoru-
bicin (DOX), a common chemotherapeutic drug, has demon-
strated its efficacy against various types of cancers.[10–12]
Additionally, target-therapeutic drugs like sorafenib (SOR)
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original
work is properly cited.
© 2024 The Author(s). Aggregate published by SCUT, AIEI, and John Wiley & Sons Australia, Ltd.
are often used as a combinatory agent to overcome the lim-
itations of traditional monochemotherapy.[13–15] Although
such synergistic therapy shows great progress,[16,17] systemic
and frequent administration of the drugs may lead to severe
adverse effects, such as organ toxicity and anaphylaxis.[18,19]
To handle these, microcarriers, particularly those based
on photothermal materials such as black phosphorus, have
garnered considerable attention.[20] Their remarkable pho-
tothermal conversion capabilities, coupled with the use of
near-infrared irradiation, enable microcarriers to achieve
Aggregate. 2024;e600. wileyonlinelibrary.com/journal/agt2 1 of 9
https://doi.org/10.1002/agt2.600
2 of 9 AGGREGATE
F I G U R E 1 Scheme of spatiotemporal responsive hydrogel microspheres and their application for gastric cancer therapy. (A) Scheme of the skeleton
and secondary filling network. (B) Spatiotemporal responsive hydrogel microspheres applied in gastric cancer synergistic treatment. The continuous release of
SOR inhibited blood vessel growth. The fast release of DOX led to tumor cell apoptosis.
controlled drug release within tissues.[21,22] However, the
treatment cycle and duration of different drugs are not the
same, and it remains challenging to specifically control the
delivery of several drugs in a single system.[23–25] Therefore,
a novel combination chemotherapy strategy with spatiotem-
poral control of the release profile of different drugs is
urgently needed to achieve efficient tumor therapy.
Herein, we present an ideal dual drug delivery system
based on inverse opal microparticles (IOMPs) with spa-
tiotemporal responsiveness for gastric cancer therapy, as
schemed in Figure 1. IOMP is a material characterized by
a skeleton and regularly arranged 3D nanovoids.[26–29] The
skeleton of IOMPs typically features a connected hydrogel
network, while the nanopore spaces can be refilled with
a secondary material of different properties from those of
the skeleton.[30,31] Based on this, composite IOMPs can be
fabricated and serve as microcarriers for delivery of multi-
drugs.[32,33] Compared to microcarriers with a homogeneous
structure and component, the skeleton and filling material
of such composite IOMPs offer distinct compartments for
accommodating different drugs.[34] Additionally, the release
profile of each drug can be modulated by controlling the
physicochemical properties of the skeleton and secondary
filling material separately. Thus, it is conceivable to develop
an innovative drug delivery system based on IOMPs possess-
ing exceptional spatiotemporal responsiveness behaviors and
thus achieving two different drug release modes.
Herein, we fabricated the desirable IOMPs-based drug
delivery system with SOR loaded in the skeleton and DOX
encapsulated in a reversible phase-changing hydrogel,
which was further filled into the nanovoids. The IOMPs were
obtained by using a photocured hydrogel polymer to replicate
a photonic crystal template. The skeleton hydrogel mainly
consisted of hyaluronic acid methacrylate (HAMA), gelatin
methacrylate (GelMA), and SOR. Then, fish gelatin/agarose,
a reversible phase-changing hydrogel, served as the sec-
ondary material to fill in the IOMPs nanovoids, with DOX
incorporated. Notably, black phosphorus quantum dots
(BPQDs) were doped in the secondary filling material too.
Attributing to the efficient photothermal conversion ability
of BPQDs, the secondary hydrogel achieved a direct gel–sol
phase transition via near-infrared irradiation, which promoted
the release of DOX at the tumor site. In comparison, SOR
was released continuously from the IOMPs skeleton, thus
impeding angiogenesis and tumor growth. Utilizing such
characteristics, it has been verified that dual drug-loaded
composite IOMPs possessed the desired properties of on-
demand drugs release and enhanced the therapeutic efficiency
of gastric cancer. These results revealed that the spatiotem-
poral responsive hydrogel microspheres possessed valuable
application potential for drug delivery and tumor therapy.
2 RESULTS AND DISCUSSION
In a typical experiment, the spatiotemporal responsive
hydrogel microspheres were fabricated, as illustrated in
Figure 2A. Firstly, silica photonic crystals (PhCs) templates
were generated by self-assembly of silica nanoparticles using
droplet microfluidic technology. The hexagonal stacking of
nanoparticles gave the microspheres periodically arranged
nanoholes, and the photonic band gap effect caused by this
special structure endowed them with bright visible structural
color. As shown in Figure 2B, a series of derived micro-
spheres, including hybrid PhCs, inverse opal, and composite
microspheres, absolutely inherited this optical property,
which was attributed to their ordered structures. To further
explore this, scanning electronic microscopy (SEM) was used
to characterize the structural feature of these microspheres,
and it showed that the PhC template exhibited regular hexag-
onal packing structures both at the surface and in the interior
(Figure 2C-i). For hybrid PhCs, the polymer was completely
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AGGREGATE 3 of 9
F I G U R E 2 (A) Scheme of preparation of different microspheres. (i) photonic crystal template; (ii) hybrid PhCs microsphere; (iii) inverse opal micro-
sphere; (iv) composite microsphere. (B) Microscopic images of different microspheres. (C) SEM images of the structure of corresponding microspheres. Scale
bars are all 150 µm in (B) and 1500 nm in (i) of (C) and 500 nm in (ii), (iii), and (iv) of (C).
infiltrated in the nanochannels, which then acted as the main
skeleton of the inverse opal after the silica nanoparticles
were etched. In addition, owing to the connected porous
structure of inverse opal, the secondary filling materials were
evenly distributed in the void spaces, thus forming composite
microspheres of independent components with the skeleton
and filling material (Figure 2C).
In our next experiment, the inverse opal skeleton
of the composite microspheres was constructed using
hyaluronic acid methacrylate (HAMA) and gelatin methacry-
late (GelMA), and the filling hydrogel was fish gelatin
and agarose. To endow the composite microspheres with
photothermal functions, black phosphorus quantum dots
(BPQDs) were incorporated within the filling hydrogel.
BPQDs have attracted much interest in the biomedical field
because of their effective photothermal capability. Mean-
while, BPQDs possess good biocompatibility and biodegrad-
ability, often being used as additives to add into the system.
The image of the nanodots and their absorbance spectrum are
shown in Figure S1. Using a transmission electron micro-
scope (TEM), it was evident that such BPQDs were in
nanoscale with excellent monodispersity (Figure 3A).
The filling hydrogel was composed of a reversible phase-
changing hydrogel obtained from a combination of fish
gelatin and agarose. The phase transition temperature of fish
gelatin/agarose hydrogel with different ratios of agarose was
analyzed (Figure S2). The hydrogel composed of 18% fish
gelatin and 2% agarose exhibited a melting point at nearly
40.8◦C, which is suitable for applying in vivo. Meanwhile,
to study the photothermal conversion ability of the com-
posite microspheres with different BPQDs concentrations,
near-infrared (NIR) irradiation with different powers includ-
ing 0.8 W, 1.0 W, 1.2 W, and 1.4 W were applied. As shown
in Figure S3, the temperature curve was positively corre-
lated with the concentration of BPQDs and laser intensity.
When the concentration of BPQDs was 0.3 mg/mL and the
NIR laser intensity was 1.2 W, the temperature change of
the microspheres is an ideal profile for photothermal ther-
apy. In this situation, the time-dependent infrared thermal
images of the photothermal microspheres with NIR irradia-
tion were recorded in Figure S4. The cyclic heating curve
showed outstanding photothermal stability and recyclability
of the microspheres (Figure 3B). Besides, as shown in Figure
S5, this cyclic heating process did not have a significant
effect on the composing material of the inverse opal skeleton
(HAMA/GelMA), thus indicating a stable physical structure.
As the composite microspheres possessed different hydro-
gel components in the skeleton and filling hydrogel, various
drugs could be encapsulated in distinct parts to show different
release profiles. To observe the distribution of two drugs in
the skeleton and the secondary filling material, coumarin (as
a model molecule for SOR) and DOX were encapsulated in
the inverse opal skeleton and the phase-changing hydrogel,
respectively. The microspheres were characterized under a
confocal microscope. As exhibited in Figure 3C and Figure
S6, the drugs were loaded in the microsphere uniformly.
Due to the excellent photothermal conversion efficiency of
BPQDs and the adjusted melting point of the secondary
filling hydrogel, it underwent a solid-to-liquid phase transi-
tion under NIR irradiation, thus promoting the rapid release
of wrapped DOX (Figure 3E). In contrast, for coumarin
(encapsulated in the skeleton), its difference of release with
or without NIR irradiation was relatively less pronounced
compared with that of DOX (Figure 3D). Meanwhile, mul-
tiple cycles of NIR irradiation were performed to study the
photothermal controllable release of DOX. It was obvious
that the intervention of NIR greatly promoted the release of
drugs in the secondary filling material. Therefore, the drugs
showed different release profiles in one system. These results
indicated that the proposed composite microsphere system is
an ideal spatiotemporal responsive carrier for the on-demand
release of multiple drugs.
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4 of 9 AGGREGATE
F I G U R E 3 (A) TEM image of BPQDs. (B) Cyclic heating curve of the photothermal microspheres. (C) Images of one microsphere loaded with coumarin
(in skeleton) and DOX (in phase-changing hydrogel). (D) Release curve of coumarin encapsulated in the inverse opal skeleton for 24 h (inner) and 172 h (outer).
(E) Release curve of DOX encapsulated in the secondary filling material for 24 h (inner) and 172 h (outer). NIR irradiation was performed every 12 h. (F)
Photocontrolled release of DOX with regular NIR irradiation. Scale bar is 20 nm in (A).
Naturally derived polymer networks typically exhibit
excellent biocompatibility. Therefore, hydrogels developed
based on such substances have been widely applied in
biomedical engineering, including HAMA, GelMA, fish
gelatin, and agarose. The biocompatibility of the inverse opal
and filling hydrogel was tested, respectively, by co-culture
with NIH-3T3 cells. The results showed the similar cell sur-
vival rates of these two groups to that in the control group
(Figure 4A–D). Moreover, the blood compatibility test of
the materials was carried out. The hemolysis rate of both
the control group and the experimental group was less than
5%, which showed that the materials possessed excellent
blood compatibility (Figure 4E,F). Notably, biodegradability
is another important factor for evaluating microcarriers. As
shown in Figure S7, the naturally derived hydrogel polymer
could lose 80% of its original weight after a week of shaking.
These characteristics laid the foundation for the microspheres
to act as drug microcarriers.
To confirm the anti-tumor effects of the combined drug
delivery system, the SOR and DOX co-loaded composite
microspheres were co-cultured with MKN-45 gastric can-
cer cells, and the fluorescent images of cells were recorded
(Figure 4G). Then, the relative cell viability is also shown
in Figure S8. The viability of gastric cancer cells did not
exhibit an obvious effect in the presence of corresponding
materials without drug loading. However, when the drug was
loaded, the MKN-45 cell viability decreased significantly,
whether in groups treated with mono-drug or multi-drugs.
It is worth noting that the group treated with photothermal
microspheres plus NIR irradiation showed a certain effect in
killing tumor cells. Meanwhile, the use of drug-loaded com-
posite microspheres combined with NIR irradiation rapidly
killed tumor cells due to their photothermal effect and fast
release of chemotherapeutic DOX.
When applied in the body, it is vital to investigate the
location and degradation of foreign material. Hence, the
fluorescence-labeled composite microspheres were injected
into nude mice. The fluorescent images and degradation curve
are shown in Figure S9. To further verify the anti-tumor effect
of the spatiotemporal responsive hydrogel microspheres in
vivo, we first constructed a tumor-bearing model in nude
mice by subcutaneously injecting MKN-45 cells. The inter-
vening treatments were performed after the volume of tumor
reached to 100 mm3. The scheme of animal experiments
is shown in Figure 5A. Briefly, the nude mice were allo-
cated into eight groups at random, which received treatments
with PBS buffer (G I), drug-free composite microspheres
(G II), drug-free photothermal microspheres plus NIR irra-
diation (G III), microspheres with DOX loading (G IV),
microspheres with SOR loading (G V), free DOX and SOR
(G VI), microspheres with DOX and SOR loading (G VII),
and microspheres with DOX and SOR loading plus NIR
irradiation (G VIII). Firstly, we measured the photother-
mal properties of the composite microspheres in vivo. As
shown in Figure 5B, the time-dependent infrared thermal
images were recorded, which indicated that the microspheres
also maintain excellent photothermal properties even in
the body.
The tumor treatment period was 21 days, and the weight
changes of the mice were recorded every three days (Figure
S10). Tumor samples were cautiously collected after the mice
were euthanized (Figure 5C), and their volumes were esti-
mated. As exhibited in Figure 5D,E, the tumors in G I and
II grew bigger than those in other groups. Meanwhile, G
III treated with composite microspheres plus NIR-induced
photothermal therapy also inhibited the growth of tumors to
some extent. Compared to that in G VII, G VI showed rel-
atively inferior efficacy, which may be caused by the rapid
metabolism of free drugs. In particular, the dual drug-loaded
microsphere system possessed a better therapeutic effect than
single drug-loaded microspheres, and it showed the best anti-
tumor performance when combined with NIR irradiation.
This was attributed to the intricate structural features of the
composite microspheres, which subtly integrated two drug
delivery profiles into one system, enabling on-demand release
and synergistic therapy.
To further evaluate the histopathological changes in differ-
ent groups, H&E staining, TUNEL, and immunofluoresence
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AGGREGATE 5 of 9
F I G U R E 4 (A–C) Fluorescent images of 3T3 cell treated with different materials. The groups are including control, inverse opal, and secondary filling
hydrogel, respectively. (D) Statistical analysis of OD value in different groups. (E,F) Images of hemolysis test in groups by different treatments, including
deionized water (I), PBS (II), and leaching liquors of inverse opal material (III), secondary filling material (IV), and both inverse opal hydrogel and secondary
filling hydrogel (V). (G) Fluorescent images of MKN-45 cells with different treatments, including control (CG), microspheres without drugs (G I), NIR irradi-
ation (G II), drug-free microspheres+NIR (G III), microspheres with SOR encapsulation (G IV), microspheres with DOX encapsulation (G V), microspheres
with DOX and SOR encapsulation (G VI), and dual drugs-loaded microspheres+NIR (G VII). Scale bars are 20 µm in (A–C) and (G).
staining were carried out. As shown in Figure 6A, H&E
staining images exhibited that the treatment of the spatiotem-
poral responsive hydrogel microspheres plus NIR irradiation
led to obvious apoptosis of tumor cells. In contrast, tumor
cells in G I and II demonstrated a higher density and normal
volume, with no apparent shrinkage. Besides, the TUNEL
staining results indicated a significantly elevated apoptosis
rate in G VIII compared to the other groups (Figure 6B
and Figure S11a). On the other hand, sorafenib can hinder
tumor growth by inhibiting tumor neovascularization and cell
proliferation. As exhibited in Figure 6C and Figure S11b,
the experimental groups including G V–VIII (supplemented
with sorafenib) showed less CD31 expression. Furthermore,
no evident pathological damage was found in H&E staining
of Group VIII, indicating the ideal in vivo biosafety of the
microspheres (Figure S12). All these results indicated that
the proposed spatiotemporal responsive microspheres with
DOX and SOR loading possessed excellent potential for drug
delivery and gastric cancer therapy.
3 CONCLUSION
In summary, we have proposed a drug-delivery system based
on IOMPs with desirable spatiotemporal responsiveness for
gastric cancer therapy. The IOMPs-based composite micro-
spheres were composed of HAMA and GelMA skeleton,
with a secondary hydrogel filling hydrogel consisting of
fish gelatin and agarose. The microspheres integrated two
drug release modes including NIR-induced rapid release of
DOX as well as sustained release of SOR, thus realizing
the spatiotemporal responsive on-demand release of differ-
ent drugs in a single system. Given these properties, it was
demonstrated that the microspheres possessed the desired
properties of the on-demand release of drugs and enhanced
the efficiency of tumor therapy. These results indicated that
the present spatiotemporal responsive hydrogel microspheres
owned valuable application potentials for drug delivery and
tumor therapy.
4 EXPERIMENTAL SECTION
4.1 Materials and animals
Doxorubicin (DOX), sorafenib (SOR), methacrylic anhydride
(MA), gelatin, fish gelatin, agarose, coumarin, and chitosan
were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Hydrofluoric acid and n-hexadecane were brought from
Aladdin Industrial Corporation. HAMA and GelMA were
manufactured according to previous work. CCK-8 Assay Kit
purchased from Beyotime Biotechnology. Black Phosphorus
quantum dots were purchased from Xianfeng Nano Tech-
nology Co., Ltd. Water obtained from a purification system
(Milli-Q).
4.2 Construction of photonic crystal (PhCs)
template
The droplet microfluidic technology was employed to
construct PhCs templates. The concentration of the silica
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F I G U R E 5 (A) Flowchart of the use of the composite microspheres for tumor therapy. (B) Infrared thermal images of mice under NIR irradiation after
intratumor injection of photothermal microspheres. (C) Photographs of the mice in different groups and their tumor size as indicated. (D) Photographs of
tumors in each group at day 21. (E) Estimated relative tumor size of mice suffered from different treatments at day 21. Groups: PBS buffer (G I), composite
microspheres without drugs (G II), photothermal microspheres with NIR irradiation (G III), microspheres loaded with DOX (G IV), microspheres loaded with
SOR (G V), free DOX and SOR (G VI), microspheres loaded with DOX and SOR (G VII), and microspheres loaded with DOX and SOR plus NIR irradiation
(G VIII). Scale bars are 1 cm in (C).
F I G U R E 6 (A) H&E stained tumor sample images from each group. (B) TUNEL staining of tumor samples from each group. (C) Fluorescence images
of CD31 impression in tumor. Groups: PBS buffer (G I), composite microspheres without drugs (G II), photothermal microspheres with NIR irradiation (G
III), microspheres loaded with DOX (G IV), microspheres loaded with SOR (G V), free DOX and SOR (G VI), microspheres loaded with DOX and SOR (G
VII), and microspheres loaded with DOX and SOR plus NIR irradiation (G VIII). Scale bars are 100 µm in all.
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AGGREGATE 7 of 9
nanoparticles solution was initially adjusted to approximately
20% (w/v). Subsequently, in the production of emulsion
droplets, the silica aqueous solution was used as the internal
phase, and n-hexadecane containing 1% surfactant 2296
served as the external phase, both flowing in a microfluidic
device. Water-in-oil droplets were produced in the device,
which were then collected in a collection bath. The droplets
were then subjected to 75◦C for 10 h on a hot table, allowing
for the self-assembly of the nanoparticles through water
evaporation. Last, the PhCs microspheres were calcined at
800◦C for 6 h to enhance their mechanical strength.
4.3 Preparation of porous inverse opal
microparticles (IOMPs)
A pregel solution was prepared, primarily consisting of
0.1 g/mL HAMA, 0.04 g/mL GelMA, and 1% photoini-
tiator HMPP. Subsequently, the as-prepared PhCs template
particles were immersed with the pregel solution for approx-
imately 30 min, by which the capillary force facilitated
infiltration of the pregel into the nanoholes of the silica
nanoparticles. Polymerization of the pregel was then carried
out using UV light, by which the hybrid particles were
stripped from the gel. Such hybrid microparticles were sub-
jected to immersion in 4% hydrofluoric acid to remove the
silica nanoparticles. The IOMPs obtained were then washed
and cleaned for further use.
4.4 Regulation of melting point of
secondary filling hydrogel
The secondary filling material with different ratios of agarose
was measured to find the appropriate melting temperature.
Specifically, the total concentration of the two components
was kept at 20%; the concentration of agarose ranged from
0% to 3.5%, with increments of 0.5%.
4.5 Preparation of photothermal composite
microspheres
Based on the IOMPs, we mixed the IOMPs with the sec-
ondary filling material doped with BPQDs and made the
secondary filling material fully fill the hole of IOMPs under
the conditions of centrifugation or vacuum negative pressure.
To optimize the photothermal performance, we also studied
the effect of BPQDs concentration and near-infrared (NIR)
light power on the temperature rise of the microspheres. SEM
was used to characterize the four types of microspheres. It is
worth noting that because the hydrogel is prone to collapse
during the sample preparation process, resin was used here
for shooting.
4.6 Drug encapsulation and release
experiment in vitro
For drug encapsulation, the pregel of IOMP polymer was
doped with SOR for its encapsulation in the skeleton. Then,
DOX was wrapped into the phase-changing hydrogel to fill
the voids of the IOMPs under negative pressure. The blended
composition underwent cooling and re-solidification. Sub-
sequently, the resultant solid subject was immersed in a
buffer solution. Following this immersion, the dislodged
drug-loaded particles were gathered after a delicate crushing
process. To study this drug release behavior of the micro-
spheres, the fluorescence intensity of the microspheres was
measured. Coumarin was used as a substitute for SOR. The
microspheres underwent incubation in 1 mL of phosphate-
buffered saline (PBS) with gentle agitation at ambient
temperature. Collagenase was also added to the solution.
Subsequently, 100 µL of the buffer solution was extracted
from a 96-well plate at hourly intervals for fluorescence
intensity measurement. Meanwhile, an equal amount of
PBS was added back into the system. For short-term release
experiments, the group treated with drug-loaded micropar-
ticles, followed by NIR irradiation using a laser (808 nm,
1.2 W, 10 cm) for a duration of 5 min, underwent subsequent
cooling for a period of 10 min. The sampling procedure is
the same as above. For that in the long-term release profile,
NIR irradiation was performed on a daily basis.
4.7 Biocompatibility experiment
To assess the biocompatibility of materials, a CCK-8 test was
performed. In this section, we set up a control group and two
experimental groups, each group comprising three parallel
samples. Cells in the control group were solely treated with a
culture medium, those in experimental group I were cultured
with IMOPs, and those in experimental group II were treated
with a hydrogel consisting of fish gelatin and agarose. NIH
3T3 cells were seeded in a 24-well plate and co-incubated
with the respective groups for 24, 48, and 72 h in an incuba-
tor. After that, a portion of the culture medium was removed,
and CCK-8 reagent was added at a final concentration of
10%. The reaction was maintained for 2–4 h, and cellular
viability was assessed through the quantification of OD value
employing a microplate reader. Additionally, the cell viability
was assessed each day using cell fluorescent staining for all
experimental groups.
4.8 In vitro antitumor effects
MKN-45 cells were seeded in a 24-well plate, each well
seeded with 5 × 104 cells. The cells were allowed to attach for
one night. Then, different treatments were administered to the
MKN-45 cells, including a control group (CG), microparti-
cles without drugs (G I), NIR irradiation (G II), photothermal
microspheres combined with NIR (G III), microspheres
encapsulated with SOR (G IV), microspheres encapsulated
with DOX (G V), microspheres encapsulated with both DOX
and SOR (G VI), and dual drug-loaded microspheres com-
bined with NIR (G VII). The dosage of DOX and SOR were
set as 3 µg/mL and 4 µg/mL, respectively. The group treated
with NIR irradiation was irradiated with a laser (808 nm,
1.2 W, 10 cm) for 5 min. Subsequently, live/dead staining
and CCK8 assay were performed. Cell viability was assessed
through live/dead staining, which included the application of
Calcein-AM and propidium iodide (PI) to the cells. Subse-
quently, the stained cells were examined using a fluorescence
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8 of 9 AGGREGATE
microscope. In the CCK-8 test, cells were added with the
CCK-8 reagent for a duration of 2–4 h, and the absorbance
was quantified employing a microplate reader.
4.9 In vivo anti-tumor evaluation
Animal experiments were approved by Ethical Committee
of Wenzhou Institute, University of Chinese Academy of
Sciences (WIUCAS22062103). For the in vivo fluorescence
and degradation test, the fluorescence-labeled microspheres
were injected into nude mice. The fluorescence images were
shot every three days. For the in vivo anti-tumor test, subcu-
taneous injection of 1 × 106 MKN-45 cells was performed
under the leg of nude mice. Then, tumor growth was allowed
until the volume reached approximately 100 mm3 to ensure
the smallest difference in tumor volume. Next, the mice were
randomly assigned into eight groups (n = 5) as follows:
PBS buffer (G I), composite microspheres without drugs (G
II), photothermal microspheres with NIR irradiation (G III),
microspheres loaded with DOX (G IV), microspheres loaded
with SOR (G V), free DOX and SOR (G VI), microspheres
loaded with DOX and SOR (G VII), and microspheres
loaded with DOX and SOR plus NIR irradiation (G VIII). The
dosage of DOX and SOR were set as 15 mg/kg and 20 mg/kg,
respectively. The body weights were recorded every three
days, and euthanasia was performed after 21 days. The
principal organs and tumors were harvested and preserved in
4% (v/v) paraformaldehyde. Tumor samples were embedded
and sliced for subsequent staining procedures, including
H&E staining and terminal-deoxynucleotidyl transferase-
mediated nick end labeling (TUNEL) staining. Furthermore,
immunofluorescence staining for CD31 was performed to
evaluate the vascular distribution within the tumor tissues.
4.10 Statistical analysis
The results were presented as mean values accompanied
by standard deviations (SD). To assess the significance
of the findings, statistical analysis was conducted using
either the Student’s t-test or one-way ANOVA, followed
by Tukey’s post-hoc test, as appropriate. Significance lev-
els were denoted as follows: NS (not significant), *p < 0.05,
**p < 0.01, ***p < 0.001.
A U T H O R C O N T R I B U T I O N S
Li Wang: Methodology; data curation; writing—original
draft preparation. Lu Fan: Supervision; writing—reviewing
and editing. Anne M. Filppula: Writing—reviewing and
editing. Yu Wang: Conceptualization; writing—reviewing
and editing. Luoran Shang: Conceptualization; supervision;
writing—reviewing and editing. Hongbo Zhang: Funding
acquisition; supervision; writing—reviewing and editing.
AC K N O W L E D G M E N T S
The authors appreciate the financial support from the
National Natural Science Foundation of China (82372145),
the Research Fellow (Grant No. 353146), Research Project
(347897), Solutions for Health Profile (336355), InFLAMES
Flagship (337531) grants from Academy of Finland, and the
Finland China Food and Health International Pilot Project
funded by the Finnish Ministry of Education and Culture.
C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflicts of interest.
D ATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available
from the corresponding author upon reasonable request.
O R C I D
Li Wang https://orcid.org/0000-0001-5543-3725
Luoran Shang https://orcid.org/0000-0001-7458-9100
Hongbo Zhang https://orcid.org/0000-0002-1071-4416
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S U P P O R T I N G I N F O R M AT I O N
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Supporting Information section at the end of this article.
How to cite this article: L. Wang, L. Fan, A. M.
Filppula, Y. Wang, L. Shang, H. Zhang, Aggregate
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