Abstract
Photodynamic therapy (PDT) is a promising cancer treatment modality due
to its minimally invasive nature and spatiotemporal selectivity.
However, its effectiveness is substantially hindered by tumor hypoxia.
In this study, bismuth vanadate/molybdenum disulfide@hyaluronic acid
(BiVO[4]/MoS[2]@HA, BM@HA) nanoparticles were engineered to overcome
the challenges of tumor hypoxia in PDT. The formation of p–n
heterojunctions between MoS[2] and BiVO[4] facilitated electron
transfer from MoS[2] to BiVO[4], imparting BM@HA with photothermal
properties in the near-infrared (NIR) region and achieving an improved
photothermal efficiency of 51.9%. After 808-nm laser irradiation, the
electron transfers and the energy generated by photothermal effects
enhanced the separation of electron–hole pairs in BM@HA, leading to the
production of reactive oxygen species and the hydrolysis of oxygen.
Animal experiments revealed the strong tumor-targeting capability of
BM@HA, as shown by tumor photothermal imaging and in vivo small-animal
imaging. Following 808-nm laser irradiation, it enabled precise tumor
phototherapy by combining PDT with photothermal therapy. Furthermore,
proteomic analysis revealed that BM@HA + NIR may induce necroptosis of
tumor cells by activating peptidylprolyl isomerase D-related pathways.
In summary, the BM@HA photosensitizer facilitated NIR photocatalytic
oxygen hydrolysis, overcoming the hypoxia limitation in PDT. When
combined with photothermal therapy, it displayed improved antitumor
efficacy, offering a new strategy for the treatment of oral squamous
cell carcinoma.
Introduction
Oral cancer, encompassing malignancies originating within the oral
cavity, persists as a major global health burden due to its aggressive
biological behavior, elevated incidence rates, and unfavorable clinical
outcomes. According to the International Agency for Research on Cancer,
the incidence and mortality rates of oral cancer worldwide have been
rising steadily each year (Table [36]S1) [[37]1–[38]3]. In 2022, there
were a total of 389,485 new cases of oral cancer reported worldwide,
positioning it as the 16th most prevalent cancer. Additionally, there
were 188,230 fatalities attributed to this disease, ranking it as the
15th leading cause of cancer-related deaths globally [[39]2]. Oral
cancer represents a specific subtype of head and neck tumors, with more
than 90% of these cases being histologically categorized as oral
squamous cell carcinoma (OSCC). The prevention and treatment of OSCC
face severe challenges. Currently, the primary treatment approaches
involve surgical resection in combination with chemotherapy and
radiotherapy. Despite these interventions, the overall 5-year survival
rate remains below 50% [[40]4]. Furthermore, surgical resection can
result in tissue defects, functional impairments, and even
psychological disorders, markedly affecting the patients’ quality of
life. Therefore, there is an urgent need to develop more effective
treatment methods to improve both therapeutic effects and the overall
well-being of patients.
Photodynamic therapy (PDT) is a highly promising cancer treatment,
especially for superficial tumors, including skin cancer and oral
cancer. PDT offers various advantages, including spatiotemporal
selectivity, minimally invasive treatment, negligible drug resistance,
and minimal toxicity [[41]5]. PDT is particularly effective for OSCC,
as it preserves surrounding tissues and aesthetics. Furthermore, it
serves as the most effective palliative treatment for large or
inoperable tumors [[42]6]. PDT is classified into type I and type II
PDT, with type II being the primary form. Type II PDT requires an
oxygen-rich environment to be effective. However, the rapid
proliferation of tumors reduces oxygen, causing hypoxia within the
tumor, which is a common characteristic of the tumor microenvironment
[[43]7]. Hypoxia can activate hypoxia-inducible factor (HIF)-dependent
signaling pathways, leading to immune suppression and resistance to
apoptosis, promoting tumor cell proliferation, and contributing to
tumor invasion and metastasis [[44]8]. Thus, tumor hypoxia represents a
major barrier to the effectiveness of PDT.
To address this challenge, researchers have developed various PDT
strategies for hypoxic tumors, primarily focusing on 3 distinct aspects
[[45]9–[46]11]: (a) directly or indirectly increasing the O[2]
concentration within the tumor, such as using photosensitizers in
combination with hemoglobin or perfluorocarbons for O[2] delivery, or
generating in situ O[2] using peroxidases or MnO[2]; (b) developing
non-O[2]-dependent PDT strategies, such as type I PDT or
hypoxia-sensitive photosensitizers; and (c) combining PDT with other
non-O[2]-dependent treatments, such as photothermal therapy (PTT),
hypoxia-activated anticancer drugs, or immunotherapy. However, these
treatment strategies are still in the early stages of research and are
complex to implement. Hence, there is a critical need for the
development of more efficient and sustained oxygen supply methods to
improve the efficacy of PDT. The development of inorganic
nanophotosensitizers has emerged as an optimal approach due to their
ease of modification, making them versatile photosensitizers for both
PDT and PTT. PTT can moderately increase the local temperature of the
tumor, improving blood circulation and thereby increasing local O[2]
levels, which enhances the effectiveness of PDT. Meanwhile, PTT is a
non-O[2]-dependent treatment modality, and its combination with PDT can
markedly enhance the overall therapeutic effect [[47]12]. Further,
inspired by photocatalytic water oxidation, water is considered a
potential source for O[2] production owing to its abundance as an
endogenous substance in biological systems [[48]13]. Therefore, this
study developed inorganic nanophotosensitizers capable of near-infrared
(NIR) activation and photocatalytic water hydrolysis, enabling the
combined application of PDT and PTT for tumor treatment.
Bismuth vanadate (BiVO[4]) is an n-type semiconductor with a moderate
bandgap (2.3 to 2.5 eV), a suitable valence band (VB) potential (higher
than the 1.23 eV of H[2]O/O[2]), high photochemical stability, a low
cost, and low toxicity, all of which make it a highly promising
photocatalyst for the oxygen evolution reaction (OER); its typical
crystal structure is monoclinic scheelite [[49]14]. However, pure
BiVO[4] suffers from low photocatalytic water oxidation performance due
to the easy recombination of electron–hole pairs. To effectively
overcome this limitation, several modification strategies have been
developed, including doping, forming heterojunction composites, and
loading cocatalysts. For example, Ge et al. [[50]15] integrated
ultrathin (1.8-nm) FeOOH nanosheets (cocatalyst) with BiVO[4] via
electrostatic adsorption, resulting in an O[2] release rate that was
twice as high as that of pure BiVO[4] under visible light
photocatalysis. Wei et al. [[51]16] synthesized ZnO/BiVO[4] Z-type
heterojunctions by employing hydrothermal methods, demonstrating that
the O[2] release rate of the ZnO/BiVO[4] composite was significantly
higher than that of pure BiVO[4] under visible light. Yin et al.
[[52]17] prepared a novel butterfly-wing-shaped WO[3]/BiVO[4]
heterojunction using a one-step sol–gel method for photocatalytic water
splitting. They reported that the O[2] release after 5 h of light
irradiation was 7.6 times higher than that of pure BiVO[4]. In summary,
modified BiVO[4] demonstrated outstanding performance in photocatalytic
water splitting and O[2] evolution.
Molybdenum disulfide (MoS[2]) is a 2-dimensional semiconductor
nanomaterial having characteristics of both 2-dimensional materials and
semiconductors. As a cocatalyst, MoS[2] can modify semiconductor
photocatalysts by effectively suppressing electron–hole pair
recombination while increasing the number of catalytic sites, thereby
considerably enhancing the photocatalytic performance of the
semiconductors [[53]18]. Li et al. [[54]19] synthesized a
well-structured MoS[2]/γ-Fe[2]O[3]/graphene ternary heterojunction via
high-temperature calcination, which demonstrated a photocatalytic O[2]
release activity twice that of γ-Fe[2]O[3]/graphene under visible light
irradiation. Further research has led to the synthesis of a SnS/MoS[2]
Z-type heterojunction, revealing that the unsaturated S atoms at the
edges of the MoS[2] nanoribbons can provide active sites for the OER
[[55]20]. Moreover, it was first confirmed in 2013 that MoS[2] is an
efficient NIR photothermal agent [[56]21], demonstrating outstanding
biocompatibility and widespread application in PTT for tumor treatment.
Thus, it is postulated that combining MoS[2] with BiVO[4] can not only
enhance the photocatalytic OER activity of BiVO[4] but also extend the
absorption spectrum into the NIR range, imparting photothermal
properties to the composite material, thereby alleviating tumor hypoxia
and improving tumor treatment efficacy.
In this study, a BiVO[4]/MoS[2]@hyaluronic acid (HA) (BM@HA)
nanocomposite system with tumor-targeting features was synthesized,
combining the advantages of 3 materials: (a) The photocatalytic water
splitting and oxygen production characteristics of BiVO[4] are
anticipated to utilize the abundant water in living organisms. (b) The
combination of MoS[2] with BiVO[4] can markedly enhance the
photocatalytic performance of BiVO[4]. (c) MoS[2] endows the composite
material with outstanding photothermal performance and NIR absorption
potential, while a moderate temperature increase can accelerate tumor
blood flow and alleviate hypoxia. (d) HA provides the system with
tumor-targeting functionality. After irradiation with an 808-nm laser,
the effects of BM@HA on water oxidation and its combined PDT and PTT
therapeutic effects on OSCC were evaluated, offering new strategies for
targeted and efficient treatment of this cancer (Fig. [57]1).
Fig. 1.
Fig. 1.
[58]Open in a new tab
Schematic illustration of BiVO[4]/MoS[2]@hyaluronic acid (BM@HA)
photosensitive particles utilized in hypoxic tumor therapy. Upon
targeting the tumor, these nanoparticles exert therapeutic effects
through 3 primary mechanisms: (a) Photodynamic therapy (PDT) stimulates
the production of a substantial amount of reactive oxygen species (ROS;
·OH), which promotes an increase in peptidylprolyl isomerase D (PPID)
protein levels, leads to the opening of the mitochondrial permeability
transition pore (mPTP), and subsequently induces necroptosis in cells.
(b) The photocatalytic decomposition of water produces O[2], thereby
enhancing the efficacy of PDT. (c) Photothermal therapy (PTT) directly
induces cell death. HIF-1α, hypoxia-inducible factor 1-alpha.
Materials and Methods
Molecular dynamics simulations
Initially, a force field was constructed using a machine learning-based
approach in combination with the Vienna Ab initio Simulation Package
(VASP) based on first principles. Molecular dynamics simulations were
then performed using VASP. The calculation accuracy was set to
“Accurate”. The parameters were defined as EDIFF = 1 × 10^−4 eV and
EDIFFG = −1 × 10^−2 eV/Å, and the temperature remained constant at 300
K. A minimum vacuum thickness of 30 Å was maintained in all cases to
avoid interactions between adjacent layers. The binding energy E[b] was
defined as the total energy of the composite system minus the total
energies of each material, such as E[b(BM@HA)] = E[tot] − E[(MoS2)] −
E[(BiVO4)] − E[(HA)].
Synthesis of BiVO[4] nanoparticles
To a 250-ml 3-neck flask, 2 mmol of Bi(NO[3])[3]·5H[2]O, 20 ml of
octadecene, 4 ml of oleic acid, and 4 ml of oleylamine were added. The
mixture was vigorously stirred under a nitrogen atmosphere and heated
to 175 °C. Once the solution became completely cleared, the heating
mantle was removed, allowing the solution to naturally cool to 130 °C.
After that, the vanadium precursor solution (4 mmol of NH[4]VO[3]
dissolved in 20 ml of boiling water with stirring to ensure complete
dissolution) was quickly added to the mixture. The solution was then
stirred vigorously and maintained at 100 °C for 5 min and then
naturally cooled to room temperature, and the product was purified.
Next, 20 ml of ethanol was added to the reaction mixture, and the
mixture was thoroughly mixed. After allowing the mixture to settle, the
lower aqueous layer was discarded. The upper organic phase was then
treated with water (20 ml) followed by an additional 20 ml of ethanol,
ensuring thorough mixing. After standing, the lower aqueous layer was
removed again and the process was repeated twice. Finally, the product
was purified by washing 3 times with ethanol at 5,000 rpm for 5 min and
freeze-dried to yield the final product.
Synthesis of MoS[2] nanosheets
Ammonium molybdate (2 mmol) and 30 mmol of thiourea were dissolved in
38 ml of deionized water, and the mixture was stirred for 10 min,
followed by sonication for another 10 min. The mixture was placed in a
polytetrafluoroethylene autoclave and heated at 180 °C for 24 h. After
allowing it to cool to room temperature, the product was purified
through centrifugation with deionized water at 5,000 rpm for 5 min,
repeated 3 times. It was then centrifuged with ethanol under the same
conditions thrice and finally freeze-dried.
Synthesis of BM@HA
Initially, methyl methacrylate-modified HA (m-HA) was synthesized.
Briefly, 1.0 g of HA was dispersed in 50 ml of deionized water and
allowed to swell at 4 °C overnight, which was then added with 0.8 ml of
methacrylic anhydride, and the pH of the solution was adjusted to 8 to
9 using a 5 M NaOH solution. The reaction mixture was then stirred
continuously at 4 °C for 24 h to ensure complete modification.
Following this, the product was precipitated in acetone, and the
resulting solid was filtered, washed with ethanol, and air-dried.
Secondly, 15 mg of m-HA was completely dissolved in 100 ml of deionized
water and added with 100 μl of NaOH (0.1 M) and 500 mg of BiVO[4] and
MoS[2] (in different mass ratios). The mixture was then subjected to
ultrasonication at 4 °C for 30 min to ensure homogeneous dispersion of
the components. After that, the cross-linking agent
N,N′-methylenebisacrylamide (15 mg) and the photoinitiator Irgacure
2959 (0.1%, W:V) were added to the above prepared dispersion. The
polymerization of m-HA was initiated under ultraviolet (UV) irradiation
for 5 min, and the resulting product was separated by centrifugation
followed by purification with deionized water.
Characterization of materials
The morphology and size of the developed systems were analyzed using
transmission electron microscopy (TEM; Tecnai F30). The hydrodynamic
size and zeta potential of the samples was obtained using a Zetapals
analyzer. The composition of the samples was determined by Fourier
transform infrared spectroscopy (FTIR) using a Thermo Nicolet Nexus 670
attenuated total reflectance–infrared spectrometer, while UV absorption
spectra were recorded using a Genesys 10S UV–visible (UV–Vis)
spectrophotometer. X-ray diffraction (XRD) patterns were obtained using
a Rigaku Ultima IV diffractometer. X-ray photoelectron spectroscopy
(XPS) analysis was performed using a VG ESCALAB 220I-XL spectrometer.
Fluorescence spectra were recorded using a HORIBA FL-3 system. The data
were processed using the Origin software for accurate replotting and
interpretation.
Photothermal performance of BM@HA
BM@HA with different concentrations (0, 25, 50, 100, and 200 μg ml^−1)
was irradiated with an 808-nm laser at various power densities (0.5,
0.75, 1.0, and 1.5 W cm^−2) for 10 min. The solution temperature was
monitored using a German infrared thermal imager (testo 865), capturing
images at 60-s intervals. The photothermal conversion efficiency of
BM@HA was then calculated according to the following formulas:
[MATH: η=hsTmax−Tsurr−Q0I1−10−A808
:MATH]
(1)
[MATH: τs=mdCdhs :MATH]
(2)
[MATH: Q0=hsTmax,water−Tsurr
:MATH]
(3)
Detection of ROS and O[2] generation
Reactive oxygen species (ROS) generation was detected using
2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). DCFH-DA solution
(3 μl) was added to a 3-ml dispersion containing various samples (100
μg ml^−1, phosphate-buffered saline [PBS] pH 7.4). The mixture was
irradiated with an 808-nm laser (0.75 W cm^−2) for 5 min, followed by
incubation at 37 °C for 2 h. After centrifugation, the supernatant was
collected and analyzed using fluorescence spectroscopy.
O[2] generation was measured using a dissolved oxygen meter (JPBJ-608,
China). A mixture of PBS solution containing 0.03 mol l^−1 AgNO[3] and
the composite material was purged with N[2] and sealed with liquid
paraffin. After NIR irradiation with an 808-nm laser (0.75 W cm^−2) for
10 min, oxygen levels were measured using the dissolved oxygen meter.
In vitro cytotoxicity evaluation
L929 cells were seeded into 96-well plates at a density of 5,000
cells/well and cultured at 37 °C and 5% CO[2] for 12 h. Various
concentrations of 25BM@HA ranging from 0 to 200 μg ml^−1 were then
introduced, followed by an additional 24-h incubation. Cell viability
was determined using the standard Cell Counting Kit-8 (CCK-8) method
with absorbance recorded at 450 nm with a microplate reader.
Biocompatibility was further evaluated through a hemolysis analysis.
Mouse blood (0.5 ml) was centrifuged and washed 5 times with PBS to
obtain erythrocytes, which were then diluted 10-fold in PBS. The
diluted suspension (200 μl) was mixed with 800 μl of varying
concentrations of 25BM@HA PBS solution (0 to 400 μg ml^−1) or Triton
X-100 (0.025% in PBS) and incubated at 37 °C for 2 h. After
centrifugation, the absorbance of the supernatant was measured at 541
nm to assess hemolysis.
Detection of the cellular uptake of BM@HA
Fluorescein isothiocyanate (FITC)-labeled BM@HA was used for cellular
uptake detection. L929, Cal-27, SAS, and SCC9 cells were seeded at a
density of 2.0 × 10^4 cells/well into 24-well plates and cultured
overnight. The cells were then treated with 25BM@HA (fluorescently
labeled) for 6 h. After washing with PBS 3 times, the cells were
observed under a microscope and analyzed by flow cytometry.
The effect of BM@HA on tumor cell proliferation
Cal-27 and SAS cells were seeded into 96-well plates at a density of
5,000 cells per well and cultured overnight. The cells were treated
with varying concentrations of 25BM@HA (0 to 50 μg ml^−1) for 6 h,
followed by NIR laser irradiation (808 nm, 0.75 W cm^−2) for 5 min.
After an 18-h incubation, cell viability was evaluated using the CCK-8
assay by recording the absorbance at 450 nm to identify the most
effective concentration for further analysis. The hypoxia experiments
were conducted in a tri-gas incubator (1% O[2], 5% CO[2], 37 °C).
Live-cell staining using calcein-AM was conducted to evaluate the
impact of 25BM@HA on the viability of tumor cells. Cal-27 and SAS cells
were seeded at a density of 2.0 × 10^4 cells per well in a 24-well
plate. After 12 h of incubation, the cells were treated with or without
25BM@HA (50 μg ml^−1) for 6 h, followed by 5 min of irradiation with an
808-nm laser (0.75 W cm^−2) or no irradiation. The cells were assigned
to 4 groups: control group, NIR group, 25BM group, and 25BM + NIR
group. After 18 h of continued incubation, the culture medium was
removed, and the cells were washed twice with PBS followed by the
addition of 0.5 ml of diluted (1:1,000) calcein-AM working solution.
The cells were then incubated in the dark at 37 °C for 30 min and
observed under a fluorescence microscope.
Finally, the colony formation assay was performed to evaluate the
effect of 25BM@HA on tumor cell proliferation. Cal-27 and SAS cells
were seeded at a density of 500 cells per well into a 24-well plate.
Subsequently, the cells were treated with 25BM@HA for 6 h or left
untreated, followed by either 5 min of 808-nm laser irradiation (0.75 W
cm^−2) or no irradiation. The experimental setup consisted of 4 groups:
control, NIR group, 25BM group, and 25BM + NIR group. Following an 18-h
incubation, the culture medium was removed, and the cells were washed 3
times with PBS before adding fresh culture medium for a further 10 d.
After fixing the cells with 0.5 ml of methanol in the dark for 15 min,
methanol was removed, followed by staining with 0.5 ml of Giemsa stain
for 30 min. The cells were washed with flowing water, dried, and
photographed using a camera. The number of colonies with more than 50
cells was counted using the ImageJ software to calculate the numbers of
colonies.
Evaluation of intracellular ROS and O[2] levels
Intracellular ROS and O[2] levels were assessed using DCFH-DA and
tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dichloride
([Ru(dpp)[3]]Cl[2]) probes, respectively. Cal-27 and SAS cells were
seeded at a density of 2.0 × 10^4 cells per well into a 24-well plate
overnight. Afterward, the cells were treated with 25BM@HA for 6 h and
exposed to NIR laser irradiation (808 nm, 0.75 W cm^−2) for 5 min.
After 2 h of incubation, the cells were washed 3 times with PBS, and
the corresponding reagents were added according to the kit protocols.
The cells were washed 3 times with PBS, photographed under a
fluorescence microscope, and analyzed by flow cytometry.
Western blot
SAS cells were treated with or without 25BM@HA for 6 h, followed by 5
min of irradiation with an 808-nm laser (0.75 W cm^−2) or no
irradiation. The cells were categorized into 4 groups: control group,
NIR group, 25BM group, and 25BM + NIR group. Following further
incubation for 2 h, proteins were extracted using
radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific)
and quantified with a bicinchoninic acid assay kit (Beyotime
Biotechnology). Protein samples were separated by 10% or 12% sodium
dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to
polyvinylidene fluoride membranes using a semidry transfer apparatus.
The following primary antibodies were used: HIF-1α (1:1,000,
Servicebio, China), Nrf2 (1:500, Huabio, China), HO-1 (1:1,000,
Immunoway, USA), peptidylprolyl isomerase D (PPID; 1:500, Proteintech,
China), RIPK1 (1:1,000, Immunoway, USA), RIPK3 (1:1,000, Abcam, USA),
MLKL (1:1,000, Immunoway, USA), and β-actin (1:5,000, Immunoway, USA).
Protein bands were detected using enhanced chemiluminescence detection
reagents.
Evaluation of in vivo tumor phototherapy
Twenty-five male nude mice (4 to 6 weeks old) were purchased from SPF
(Beijing) Biotechnology Co., Ltd., and randomly divided into 5 groups
(n = 5): control group, NIR group, 25BM@HA group, and 25BM@HA + NIR
group. Five additional mice were used to assess the biodistribution and
photothermal imaging of 25BM@HA in vivo. After 1 week of the
acclimatization period, SAS cells (5.0 × 10^6/200 μl) were injected
into the right forelimb axilla of the mice to establish tumors.
When the tumor size reached 100 to 150 mm^3, in vivo thermal imaging of
25BM@HA was conducted using a thermal infrared imager. Mice were
intravenously injected with PBS (150 μl) containing 25BM@HA (10 mg
kg^−1), followed by irradiation with an 808-nm laser for 5 min at 0, 1,
6, 12, and 24 h postinjection. Thermal images and recordings were taken
at 60-s intervals, providing real-time monitoring of the temperature
changes. In vivo imaging was used to detect the biodistribution of
25BM@HA. Following intravenous injection of fluorescently labeled
25BM@HA (10 mg kg^−1), the mice were anesthetized at 6, 12, and 24 h.
The distribution of 25BM@HA in nude mice was then observed using a
versatile imaging system (Bio-Rad ChemiDoc MP). After euthanizing the
mice, heart, liver, spleen, lung, kidney, and tumor tissues were
harvested for imaging using the versatile imaging system. Fluorescence
intensity analysis was subsequently performed using the ImageJ
software.
Treatment was started when the tumor size reached approximately 80 mm^3
(day 0), with mice receiving intravenous injections of 150 μl of PBS,
either with or without 25BM@HA (10 mg kg^−1). After 12 h, the mice were
treated with an 808-nm laser (0.75 W cm^−2, 5 min) irradiation or no
irradiation. Treatment was administered every 2 d, with the tumor size
and mouse weight monitored throughout the study. After 2 weeks of
treatment, the mice were euthanized, and major organs (heart, liver,
spleen, lung, and kidney) along with tumor tissues were harvested for
histological analysis using hematoxylin and eosin (HE) staining.
Immunofluorescence was performed to detect HIF-1α expression in tumors,
while immunohistochemistry (IHC) was used to assess Ki-67 expression in
tumor tissues.
All animal experiments were conducted following the “Guide for the Care
and Use of Laboratory Animals” by the National Institutes of Health and
were approved by the Medical Ethics Committee of the School of
Stomatology, Lanzhou University (LZUKQ-2024-038).
Proteomic analysis
Tumor proteomics were evaluated by analyzing tissues derived from
treated nude mice, with 3 samples belonging to each of the control and
25BM@HA + NIR groups. The tissues were rapidly frozen in liquid
nitrogen and submitted to Shanghai Genechem Co., Ltd. for proteomic
analysis to identify differentially expressed proteins. Gene Ontology
(GO) annotation of the target protein set was performed using Blast2GO
(V1.4.4), with the GO database version go.obo (2019.07.01). For Kyoto
Encyclopedia of Genes and Genomes (KEGG) pathway annotation, the KOALA
(KEGG Orthology and Links Annotation, V3.0) software was employed to
perform comparisons against the KEGG GENES database. The target protein
sequences underwent KO classification, and pathway information
associated with these sequences was automatically obtained according to
KO classification. The KEGG database version number was KO_INFO_END.txt
(2024.06.03). Furthermore, Fisher’s exact test was applied to compare
the distribution of each GO term or KEGG pathway between the target
protein set and the overall protein set, determining the significance
level of the enrichment of a specific GO term or KEGG pathway.
Moreover, interaction networks, with direct or indirect relationships
to the target proteins, were searched in the String database using the
target protein ID. The resulting interaction network analysis was
visualized using the AnyChart software (V8.11.0.1934).
Statistical analysis
All experiments were repeated 3 times, and data were analyzed using t
tests through the SPSS software. All data are presented as mean ± SD. A
P value of <0.05 indicates a statistically significant difference: *P <
0.05, ** P < 0.01, and *** P < 0.001.
Results and Discussion
Synthesis and characterization of BM@HA
Molecular dynamics simulations were conducted using VASP to evaluate
the feasibility and stability of the composite system comprising
MoS[2], BiVO[4], and HA. These simulations calculated the interaction
forces between the 3 components (Fig. [59]2A). The binding energies of
BiVO[4]@HA, MoS[2]@HA, and BM@HA were determined to be −44.09, −5.29,
and −22.68 eV, respectively. All values were negative, indicating that
the 3 synthesized systems were relatively stable. Moreover, more
negative binding energy correlates with enhanced stability, providing a
theoretical basis for the follow-up experiments.
Fig. 2.
Fig. 2.
[60]Open in a new tab
Characterization of BM@HA. (A) Molecular dynamics simulations of
BiVO[4]@HA, MoS[2]@HA, and BM@HA. (B) Transmission electron microscopy
(TEM) of BiVO[4], MoS[2], and BM@HA and corresponding elementary
mapping of BM@HA (scale bar = 50 nm). (C) High-resolution TEM (HRTEM)
of BM@HA (scale bar = 5 nm, scale bar of the magnified image = 1 nm).
(D) X-ray diffraction (XRD) patterns of BiVO[4], MoS[2], and BM@HA. (E)
Fourier transform infrared spectroscopy (FTIR) of BM@HA. (F) The survey
x-ray photoelectron spectroscopy (XPS) spectrum of BM@HA. (G) XPS
spectra of Mo 3d in BM@HA and MoS[2]. (H) XPS spectra of Bi 4f in BM@HA
and BiVO[4].
MoS[2] and BiVO[4] nanoparticles were synthesized via hydrothermal and
solvothermal methods, respectively, and characterized using TEM, XRD,
and FTIR. TEM results showed that MoS[2] formed thin-layer nanosheets,
while BiVO[4] appeared as nanoparticles with diameters smaller than 10
nm (Fig. [61]2B and Fig. [62]S1A, B, D, and E). In pure BiVO[4], a
lattice fringe of 0.310 nm was observed (Fig. [63]S1C), corresponding
to the (121) phase of monoclinic BiVO[4] (JCPDS no. 14-0688). Pure
MoS[2] showed a lattice fringe of 0.615 nm (Fig. [64]S1F),
corresponding to the (002) plane of 2H MoS[2] (JCPDS no. 371492), which
was consistent with the XRD results (Fig. [65]2D). Further FTIR
analysis revealed that there was a typical Mo–S stretching vibration
peak at 436 cm^−1 for MoS[2] [[66]22], while the absorption band
appeared in the range of 700 to 900 cm^−1 was attributed to the
asymmetric stretching of the V–O bond present in BiVO[4] (Fig. [67]2E)
[[68]23]. MoS[2] and BiVO[4] were combined using a simple method and
modified with low-molecular-weight HA (20,000 Da) to synthesize BM@HA.
TEM results revealed that the final synthesized BM@HA displayed a
morphology similar to that of MoS[2] nanosheets, with BiVO[4]
nanoparticles attached to their surfaces (Fig. [69]2B and Fig.
[70]S1G). The hydrodynamic diameter of BM@HA was measured to be 217.34
± 12.23 nm (Fig. [71]S1H). The zeta potential of BM@HA was recorded at
−19.7 ± 5.01 mV (Fig. [72]S1H). Upon dispersion in water, no marked
agglomeration was observed after 24 h (Fig. [73]S1I), and a very small
amount of aggregation was observed after 7 d. Upon dispersion in PBS,
Dulbecco’s modified Eagle medium, and complete medium, similar results
were obtained. However, BiVO[4]/MoS[2] was dispersed in water, and
substantial aggregation was observed after 7 d (Fig. [74]S1J). The
results indicated that BM@HA exhibits excellent dispersibility and HA
could improve the dispersion of BiVO[4]/MoS[2]. High-resolution TEM
images of BM@HA revealed 2 lattice fringes with spacings of 0.615 and
0.254 nm, corresponding to the (002) planes of MoS[2] and BiVO[4],
respectively (Fig. [75]2C). The XRD results indicated that BM@HA
corresponds to the (002) and (121) planes of BiVO[4], as well as the
(002) plane of MoS[2] (Fig. [76]2D). Meanwhile, BM@HA nanoparticles
demonstrated a translucent outer layer, attributed to the HA layer
grafted on the surface (Fig. [77]2B). The elemental mapping images from
TEM revealed an even distribution of Bi, V, Mo, and S in BM@HA,
indicating that BiVO[4] was adhered to the surface of MoS[2]
nanosheets, contributing to the formation of a 3-dimensional structure.
These results confirmed the successful synthesis of BM@HA. Moreover,
the FTIR spectrum of BM@HA displayed a characteristic peak of Mo–S
stretching vibration at 436 cm^−1 and a V–O asymmetric stretching in
the 700 to 900 cm^−1 range. Meanwhile, the bands of BM@HA at 1,655 and
1,550 cm^−1 could be assigned to amide I and amide II, attributed to
the carbonyl stretching and N–H bending of HA, respectively
[[78]24,[79]25]. These results further validated the successful
synthesis of BM@HA.
XPS was employed to further investigate the formation and valence
states of the BM@HA nanocomposite. The analysis displayed the presence
of Bi, V, S, and Mo, as well as C and O, which may primarily originate
from organic modification (Fig. [80]2F). In the Bi 4f XPS spectrum of
BiVO[4] (Fig. [81]2H), binding energies of 159.11 and 164.41 eV were
assigned to Bi 4f[7/2] and Bi 4f[5/2], respectively, with a difference
of 5.3 eV, which was consistent with the presence of Bi^3+ [[82]26]. In
the Mo 3d XPS spectrum of MoS[2] (Fig. [83]2G), binding energies of
230.51 and 227.31 eV were assigned to Mo 3d[3/2] and Mo 3d[5/2],
respectively, with a binding energy difference of 3.2 eV, consistent
with Mo^6+ [[84]27]. On the other hand, after the combination of MoS[2]
and BiVO[4], the binding energies of Mo 3d[3/2] and Mo 3d[5/2]
increased, while those associated with Bi 4f[7/2] and Bi 4f[5/2] showed
a prominent decrease (Fig. [85]2G and H). The shift in binding energies
indicated the process of electron transfer from MoS[2] to BiVO[4] in
the BM@HA composite.
Optical properties of BM@HA
The MoS[2]@HA, 12.5BM@HA, 25BM@HA, 50BM@HA, and BiVO[4]@HA composites
were prepared by adjusting the BiVO[4] content to 0%, 12.5%, 25%, 50%,
and 100%, respectively. The UV–Vis absorption spectra revealed that all
samples showed prominent absorption in the NIR region, as shown in Fig.
[86]3A. At lower BiVO[4] doping levels, both 12.5BM@HA and 25BM@HA
showed enhanced absorption in the NIR region compared to MoS[2]@HA,
with the absorption intensity increasing as the BiVO[4] content
increased. The observed enhancement in absorption occurred because
doping introduced new energy levels within the semiconductor bandgap,
thereby enhancing light absorption. However, when the BiVO[4] content
reached 50%, the NIR absorption of 50BM@HA was found to be lower than
that of MoS[2]@HA, suggesting that optimal doping levels are essential
for maximizing absorption. To investigate the photothermal performance,
samples were dispersed in water at a concentration of 100 μg ml^−1 and
exposed to an 808-nm NIR laser (1 W cm^−2) for 10 min. Temperature
changes were then monitored, as shown in Fig. [87]3B. All samples
showed a temperature increase, aligning with the UV absorption
analysis. Among them, 25BM@HA demonstrated the highest absorption in
the NIR region (808 nm), resulting in the most substantial temperature
rise, reaching up to 42.9 °C. Furthermore, the photothermal capability
of 25BM@HA showed a positive correlation with both the laser
irradiation intensity (Fig. [88]3C) and the sample concentration (Fig.
[89]3D). Upon irradiation with an 808-nm NIR laser at a power density
of 0.75 W cm^−2 for 10 min, 25BM@HA demonstrated a
concentration-dependent temperature increase, ranging from 14.5 °C at
25 μg ml^−1 to 33.1 °C at 200 μg ml^−1, in contrast to water, which
showed a negligible rise of only 2.5 °C. These findings were further
validated through thermal imaging (Fig. [90]3E). Moreover, after 5
cycles of laser irradiation, 25BM@HA maintained a consistent
temperature rise without any sign of attenuation, highlighting its
exceptional photothermal stability (Fig. [91]3F). Furthermore, the
material demonstrated a high photothermal conversion efficiency of
51.9% (Fig. [92]S2). These results demonstrate that 25BM@HA possesses
outstanding photothermal tunability and stability, establishing its
potential as a promising photothermal agent for PTT application.
Fig. 3.
Fig. 3.
[93]Open in a new tab
Optical properties of BM@HA. (A) Ultraviolet–visible (UV–Vis)
absorption spectra of the samples. (B) Photothermal heating curves of
the aqueous dispersions of these samples (100 μg ml^−1) under 808-nm
laser irradiation (1 W cm^−2). (C) Photothermal heating curves of the
25BM@HA suspension at different laser powers (808 nm). (D) Photothermal
heating curves of the aqueous dispersions of 25BM@HA at different
concentrations under 808-nm laser irradiation (0.75 W cm^−2). (E)
Infrared thermal images of the aqueous dispersions of 25BM@HA at
different concentrations and irradiation times. (F) Photothermal
recycle curve of 25BM@HA. (G) Fluorescence spectra of
dichlorofluorescein diacetate (DCFH-DA) treated by different samples
(100 μg ml^−1) under near-infrared (NIR) irradiation (808 nm, 0.75 W
cm^−2). (H and I) Fluorescence spectra of DCFH-DA and 25BM@HA solution
under different conditions. (J) O[2] generation curves of 25BM@HA with
or without AgNO[3]. (K) O[2] generation photographic images of 25BM@HA
(without AgNO[3]). (L) Schematic illustration of the photocatalytic
mechanism of BiVO[4]/MoS[2] for generating O[2] and ROS. PBQ,
p-benzoquinone; TBA, tert-butanol; NHE, normal hydrogen electrode; CB,
conduction band; VB, valence band.
Afterward, the potential of BM@HA for application in PDT was
investigated. PDT is widely recognized for its mechanism of generating
ROS upon exposure to specific light irradiation. In this study, DCFH-DA
was used as a probe to assess the ROS generation capability after
808-nm (0.75 W cm^−2) NIR laser irradiation via fluorescence
spectroscopy. The oxidation of DCFH-DA by ROS produced
2′,7′-dichlorofluorescein, a compound that emits green fluorescence
with an intensity directly proportional to the ROS concentration. As
shown in Fig. [94]3G, all samples displayed marked fluorescence
emission at 522 nm after laser irradiation, confirming ROS generation.
The ROS production levels followed the order 25BM@HA > 12.5BM@HA >
50BM@HA > BiVO[4]@HA > MoS[2]@HA. 25BM@HA generated the most ROS after
laser irradiation, and this was time dependent (Fig. [95]3H). Given the
excellent photothermal performance and ROS generation capability of
25BM@HA, we selected 25BM@HA (or written as 25BM) for further
experiments. To determine the types of ROS, tert-butanol and
p-benzoquinone were used as ·OH and ·O[2]^− scavengers, respectively.
The results revealed that tert-butanol markedly reduced fluorescence
intensity, while p-benzoquinone had no marked effect (Fig. [96]3I),
indicating that ·OH was the primary reactive species, rather than
·O[2]^−. Moreover, the requirement for O[2] in ROS generation was
confirmed by the substantial decrease in fluorescence intensity when
argon was bubbled through to remove dissolved O[2] (Fig. [97]3I). The
decomposition of water by 25BM@HA to yield O[2] upon laser irradiation
was examined by measuring the dissolved oxygen levels in water using a
dissolved oxygen analyzer. Initially, the dissolved oxygen was removed
with argon, and after 10 min of 808-nm laser irradiation, the dissolved
oxygen levels increased by 1.4 mg l^−1 in 25BM@HA + AgNO[3] (electron
capture agent), while a 1.0 mg l^−1 increase was observed in the
absence of AgNO[3] (Fig. [98]3J). Moreover, the formation of numerous
bubbles in the solution was observed (Fig. [99]3K), confirming that
25BM@HA could decompose water to generate O[2] upon 808-nm laser
irradiation. The described process replenished the O[2] consumed during
ROS generation, highlighting its potential for PDT therapy.
Finally, to investigate the mechanism of ROS and O[2] generation, we
conducted UV–Vis diffuse reflection spectroscopy on the samples. The
results revealed a pronounced redshift in the spectrum of 25BM@HA,
which could be excited by NIR light (Fig. [100]S3A to C). According to
the formula of (αhν)^1/n = B(hν − E[g]), we calculated the bandgap of
the samples from the (αhν)^1/n–hν curve. The findings indicated that
the bandgaps of BiVO[4], MoS[2], and 25BM@HA were 2.54, 1.7, and 1.38
eV, respectively (Fig. [101]S3D to F). UV photoelectron spectroscopy
was employed to evaluate the VB of each sample; results showed that the
VB values were 2.67 eV for BiVO[4] and 1.17 eV for MoS[2] (Fig.
[102]S4). It could be calculated from the above results (E[CB] = E[VB]
− E[g]) that the conduction band (CB) of BiVO[4] was 0.13 eV and the VB
of MoS[2] was −0.53 eV. Based on their respective band structures,
BiVO[4] and MoS[2] could form an n–p heterojunction. Due to its smaller
bandgap, MoS[2] could first be excited under irradiation with an 808-nm
laser irradiation; this excitation generated electrons (e^−) in its CB,
which were subsequently transferred to the CB of BiVO[4]. This transfer
was corroborated by the results of XPS. Concurrently, owing to its
photothermal effect, MoS[2] enhanced thermal energy within the
composite material, facilitating further excitation of BiVO[4], and
this process generated holes (h^+) in its VB, which then migrate to the
VB of MoS[2]. It has been observed that O[2] can be produced through
reactions between photoexcited holes and water molecules (2H[2]O + 4h^+
→ O[2] + 4H^+, >1.23 V vs. normal hydrogen electrode [NHE]) [[103]28];
additionally, strongly oxidizing holes may directly react with water,
resulting in ·OH (h^+ + H[2]O → ·OH + H^+, >2.29 V vs. NHE) [[104]25].
Furthermore, electrons generated during this process can interact with
O[2], leading to ultra-oxygen free radical formation(O[2] + e^− →
·O[2]^−, <−0.33 V vs. NHE) [[105]29]. Specifically, the mechanism of
25BM@HA stimulating the generation of O[2] and ROS is shown in Fig.
[106]3L. 25BM@HA (1.38 eV) with a narrow bandgap could generate active
electrons and free holes in the CB and VB after 808-nm laser
irradiation. Notably the VB of BiVO[4] was positive enough (2.67 eV,
>2.29 V vs. NHE), and the free holes in VB could react with water to
produce O[2] and ·OH. Moreover, the CB of MoS[2] exhibited a
sufficiently negative potential (−0.53 eV, <−0.33 V vs. NHE), enabling
the free electrons in the CB to react with O[2] and form ·O[2]^−, and
then combined with H^+ to form ·OH. Therefore, the heterojunction
25BM@HA designed and synthesized in this study could react with water
to generate O[2] and ROS after NIR laser irradiation, overcoming tumor
hypoxia and exhibiting strong PDT performance.
The impact of 25BM@HA on OSCC cells
The biocompatibility of 25BM@HA was first assessed by evaluating the
viability of L929 cells using the CCK-8 assay. The results showed that
even at a concentration of 200 μg ml^−1, the viability of L929 cells
remained above 90% (Fig. [107]S5A), indicating the favorable cellular
compatibility of 25BM@HA. Furthermore, the blood compatibility of
25BM@HA was assessed using a hemolysis experiment carried out on mouse
red blood cells (Fig. [108]S5B). No noticeable hemolysis was observed
at a 400 μg ml^−1 concentration, suggesting that 25BM@HA has favorable
blood compatibility.
HA acts as a ligand for the CD44 protein, which is highly expressed on
the surface of various tumor cells, including OSCC cells [[109]30] and
breast cancer cells [[110]31]. HA-modified nanoparticles can target
tumors overexpressing CD44 [[111]32]. Therefore, in this study, HA was
selected as a specific targeting moiety for tumor cells. The Cal-27,
SAS, and SCC-9 OSCC cells, which express high levels of CD44 [[112]30],
were selected as the experimental models, and L929 cells, which express
low levels of CD44 [[113]33], served as the control, to explore the
uptake of 25BM@HA by OSCC cells. After incubating 50 μg ml^−1 25BM@HA
with these cells for 6 h, optical microscope observations revealed
distinct black aggregates in Cal-27, SAS, and SCC-9 cells, indicating
the uptake and accumulation of 25BM@HA, whereas almost no aggregation
was observed in L929 cells (Fig. [114]S6). Furthermore, 25BM@HA was
labeled with FITC, and fluorescence detection was performed using flow
cytometry. The results showed a significant increase in fluorescence in
Cal-27, SAS, and SCC-9 cells, while there was no significant
enhancement in fluorescence in L929 cells (Fig. [115]4A), confirming
the active targeting capability of 25BM@HA toward OSCC cells.
Fig. 4.
Fig. 4.
[116]Open in a new tab
The effect of 25BM@HA on oral squamous cell carcinoma (OSCC) cells. (A)
The cellular uptake behavior of 25BM@HA after incubation for 6 h was
determined by flow cytometry. (B) Viability of Cal-27 cells assessed by
Cell Counting Kit-8 (CCK-8; incubated with different concentrations of
25BM@HA). (C) Live staining of Cal-27 cells (scale bar = 100 μm). (D)
CCK-8 assay of Cal-27 cells incubated with 25BM@HA under hypoxic
conditions. (E) Colony formation assay of Cal-27 cells. (F and I)
Fluorescence images (scale bar = 100 μm) and flow cytometry of the
Cal-27 cells stained with DCFH-DA and
tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dichloride
([Ru(dpp)[3]]Cl[2]). (G and H) The HIF-1α, Nrf2, and HO-1 protein
expressions of Cal-27 cells were determined by western blot. *P < 0.05;
**P < 0.01; ***P < 0.001. FITC, fluorescein isothiocyanate.
After that, the antitumor activity of 25BM@HA was investigated. As
illustrated in Fig. [117]4B and Fig. [118]S7A, after stimulating tumor
cells (Cal-27 and SAS) with 25BM@HA (0 to 50 μg ml^−1) for 24 h without
NIR irradiation, the viability of Cal-27 and SAS cells remained above
80%. However, after 5 min of NIR laser irradiation (at 37 °C), 40 μg
ml^−1 25BM@HA significantly inhibited tumor cell growth, reducing the
cell viability of Cal-27 and SAS to 46.4% and 50.3%, respectively.
Moreover, the 50 μg ml^−1 concentration further decreased cell
viability to less than 20%. To eliminate the photothermal effect of
25BM@HA, analysis was conducted at a low temperature (4 °C). Under
these conditions, 40 μg ml^−1 25BM@HA showed no significant inhibition
of cell viability. However, 50 μg ml^−1 reduced the viability of Cal-27
and SAS to 45.3% and 46.7%, respectively, which was less effective
compared to the NIR laser irradiation group at 37 °C. The observed
findings indicated that the antitumor effect of 25BM@HA was caused by
the combination of PTT and PDT. Therefore, a concentration of 50 μg
ml^−1 was selected for subsequent experiments to further investigate
the combined therapeutic effects of PTT and PDT. Both live-cell
staining and colony formation assay confirmed that combined treatment
with 25BM@HA and laser significantly inhibited tumor cell proliferation
(Fig. [119]4C and E and Figs. [120]S7B to D and [121]S8).
Furthermore, the potential mechanisms through which 25BM@HA exerts its
antitumor effects were investigated. The generation of ROS, critical
for PDT treatment, was assessed in this study by using DCFH-DA to
detect intracellular ROS production. Compared to the 25BM@HA group, the
25BM@HA + NIR group exhibited stronger green fluorescence (Fig. [122]4F
and Fig. [123]S7E). This results were consistent with those of flow
cytometry (Fig. [124]4I and Fig. [125]S7F). These findings indicate
that NIR irradiation effectively stimulates the generation of
intracellular ROS. After PDT, elevated ROS levels trigger oxidative
stress, which induces cytotoxicity and DNA damage, ultimately resulting
in cell death [[126]34]. The Nrf2/HO-1 pathway plays an important role
in regulating oxidative stress, with Nrf2 serving as a key regulator of
the antioxidant response. Excessive ROS stimulation leads to an
increase in Nrf2 expression [[127]35]. Meanwhile, HO-1, a downstream
target protein of Nrf2, can be up-regulated in response to stress
conditions through Nrf2 activation [[128]36]. In the current study,
treating tumor cells with 25BM@HA + NIR resulted in a significant
increase in the expression levels of Nrf2 and HO-1 (Fig. [129]4G and H
and Fig. [130]S7H and I), indicating that 25BM@HA induces cell death by
stimulating oxidative stress response after exposure to 808-nm laser.
Cheng et al. [[131]37] prepared Bi[2]S[3]@Bi nanoparticles for the
treatment of hypoxic tumors, finding that after 808-nm laser
irradiation, the expression levels of Nrf2 and HO-1 proteins were
significantly increased, consistent with the findings observed in the
current study. Similarly, the expression of Nrf2 and HO-1 proteins
significantly increased after radiotherapy [[132]38]. However, the
Nrf2/HO-1 pathway has a dual role: serving as a critical regulatory
signal in the body’s antioxidant defense system while also being
involved in the response to oxidative stress. Elevated levels of Nrf2
and HO-1 may enhance the antioxidant potential of tumor cells, shifting
them from a pro-oxidative to an antioxidative state, thereby leading to
the resistance of cancer cells to radiotherapy, chemotherapy, and even
PDT [[133]39,[134]40]. A research study has shown that knocking down
Nrf2 or HO-1 expression increases the radiosensitivity of cancer cells
[[135]41], further supporting this perspective.
The generation of ROS depends on the availability of O[2] in the tumor
microenvironment. Sufficient oxygen supply can alleviate tumor hypoxia
and improve the effectiveness of PDT. The investigation focused on
whether NIR irradiation induces 25BM@HA to decompose water within tumor
cells to generate O[2]. For this purpose, [Ru(dpp)[3]]Cl[2] was used to
detect the O[2] levels in cells as its red fluorescence can be quenched
by O[2]. In the absence of 808-nm laser irradiation, the red
fluorescence intensity of the 25BM@HA group was similar to that of the
control group, indicating that the tumor cells were in a hypoxic state.
After 808-nm laser irradiation, the fluorescence intensity of the NIR
group was comparable to that of the control group, while the red
fluorescence of the 25BM@HA + NIR group was significantly quenched
(Fig. [136]4F and Fig. [137]S7E). The flow cytometry results supported
these findings (Fig. [138]4I and Fig. [139]S7G), suggesting that
25BM@HA has a good oxygen supply capacity and can alleviate hypoxia.
Furthermore, hypoxia, a hallmark of the tumor microenvironment in most
solid tumors, can activate the expression of HIF-1α. Hypoxia influences
tumor growth, metastasis, and treatment resistance by regulating
pathways associated with cell metabolism, oxidative stress,
angiogenesis, and immune evasion [[140]42]. HIF-1α is highly expressed
in various malignancies, such as breast cancer, esophageal cancer, and
OSCC, and has emerged as a key target for cancer therapy [[141]43].
Western blot results showed the expression of HIF-1α on both Cal-27 and
SAS cells, with its expression significantly reduced after treatment
with 25BM@HA + NIR (Fig. [142]4G and H and Fig. [143]S7H and I),
further indicating that 25BM@HA can supply oxygen and alleviate
hypoxia. It also suggests that 25BM@HA may inhibit tumor growth through
HIF-1α-related pathways. Interestingly, studies have indicated that
down-regulating Nrf2 expression in tumor cells can inhibit HIF-1α
accumulation under hypoxic conditions, thereby inhibiting
HIF-1α-dependent tumor angiogenesis and metabolic metastasis [[144]44].
Therefore, targeting Nrf2 in combination with phototherapy emerges as a
highly promising therapeutic strategy. It not only suppresses
antioxidant responses, promotes sustained ROS generation, and increases
the sensitivity of hypoxic tumors to PDT but also disrupts the HIF-1α
pathway, thereby synergistically enhancing the therapeutic effect.
Moreover, the antitumor effect of 25BM@HA combined with laser treatment
was evaluated on tumor cells (Cal-27) under hypoxic conditions. The
results showed that 25BM + NIR reduced the cell viability of Cal-27 to
37.4% (Fig. [145]4D) while simultaneously enhancing the generation of
ROS and O[2] (Fig. [146]4F and I). These results suggest that 25BM +
NIR can overcome the limitations posed by hypoxia in PDT and
demonstrate good antitumor effects.
In vivo antitumor effect of 25BM@HA
To further evaluate the antitumor effect of 25BM@HA, a tumor model was
developed in nude mice using SAS cells. The mice were randomly assigned
to 4 groups: control group, NIR group, 25BM group, and 25BM + NIR
group. Physiological saline and a saline solution of 25BM@HA were
administered via tail vein injection every 2 d, with a dose of 10 mg
kg^−1 per injection. The phototherapy groups were irradiated with an
808-nm laser for 5 min at a power density of 0.75 W cm^−2. Initially,
the temperature change in the tumors of the nude mice was recorded
using a photothermal imager after tail vein injection. The results
indicated that after 5 min of irradiation with the 808-nm laser, the
tumor temperature in the saline-injected group increased to less than
39 °C. However, after injecting 25BM@HA, the tumor temperature
displayed a prolonged increase, reaching 48 °C at 12 h postinjection,
before gradually decreasing by 24 h postinjection (Fig. [147]5A). This
indicates that 25BM@HA can effectively target and accumulate in the
tumor region following tail vein injection, with the highest
accumulation occurring at 12 h, followed by slow metabolism from the
body. The tumor-targeting and photothermal performance of 25BM@HA in
animals confirms its potential for tumor phototherapy. Following the
injection of FITC-modified 25BM@HA via the tail vein, in vivo imaging
discovered the strongest fluorescence intensity in the tumor area at 12
h postinjection (Fig. [148]5B). Based on this finding, 808-nm laser
irradiation was performed 12 h after the tail vein injection of
25BM@HA.
Fig. 5.
Fig. 5.
[149]Open in a new tab
In vivo tumor accumulation and phototherapy of 25BM@HA in
SAS-tumor-bearing mice. (A) In vivo thermal images of a tumor-bearing
mouse after injection with saline and 25BM@HA under 808-nm laser
irradiation (0.75 W cm^−2, 5 min). (B) In vivo distribution after an
injection with FITC-25BM@HA. (C) Body weight change of mice during 14 d
of different treatments. (D) Relative tumor volume change of mice
during 14 d of different treatments. (E) The photographs of tumors
dissected at 14-d treatment. (F) Hematoxylin and eosin (HE) staining
sections (scale bar = 100 μm), (G) immunohistochemistry (IHC) staining
of Ki-67 (scale bar = 100 μm), and (H) immunofluorescence (IF) staining
of the HIF-1α (scale bar = 50 μm) of tumor tissues after 14 d of
treatment. (I) HE-stained images of the heart, liver, spleen, lung, and
kidney from the different groups at 14-d treatment (scale bar = 50 μm).
After 2 weeks of treatment, the tumor volumes in the control, NIR, and
25BM groups grew significantly, expanding to approximately 5 times
their original size, while the tumor volume in the 25BM + NIR group
decreased significantly (Fig. [150]5D). Corresponding images visually
demonstrated the marked tumor suppression effect in the 25BM + NIR
group (Fig. [151]5E). HE staining results revealed extensive damage and
necrosis of tumor cells in the 25BM + NIR group (Fig. [152]5F). The
expression level of Ki-67, a commonly used biomarker for cell
proliferation, was evaluated. The IHC results showed that the
expression level of Ki-67 in the 25BM + NIR group was significantly
decreased (Fig. [153]5G and Fig. [154]S9), indicating the improved
antitumor performance of 25BM@HA. Immunofluorescence results further
revealed that the expression of HIF-1α in the 25BM + NIR group was
significantly lower compared to that in the control group (Fig. [155]5H
and Fig. [156]S10), consistent with the western blot in vitro results,
indicating that 25BM@HA retained its oxygen-supplying capability
in vivo, effectively alleviating tumor hypoxia following NIR laser
irradiation.
Moreover, in vivo fluorescence imaging results showed enhanced
fluorescence in the liver, spleen, and kidney regions following 25BM@HA
treatment (Fig. [157]5B), suggesting that the compound was metabolized
through these organs. HE staining analysis of the hearts, livers,
spleens, lungs, and kidneys of mice belonging to different treatment
groups showed no notable lesions (Fig. [158]5I). Furthermore, after 2
weeks of treatment, the body weight of the nude mice increased slightly
(Fig. [159]5C), further supporting that 25BM@HA had favorable biosafety
and was considered suitable for tumor phototherapy.
Mechanism of 25BM@HA in tumor phototherapy
To further explore the potential mechanism of 25BM@HA in tumor
phototherapy, proteomic analysis was carried out on the tumors of nude
mice after treatment. As shown in Fig. [160]6A, the 25BM + NIR group
displayed 39 differential proteins compared to the control group, with
7 up-regulated and 32 down-regulated proteins. A volcano plot and a
heat map were constructed based on the differentially expressed
proteins (Fig. [161]6B and C). Following this, Fisher’s exact test was
applied for the GO and KEGG pathway enrichment analysis of the
differential proteins. As shown in Fig. [162]6D and E, the 25BM@HA +
NIR treatment was primarily enriched in pathways associated with the
cell cycle and apoptosis/necrosis. The pathways include extracellular
matrix, intracellular organelles, misfolded RNA binding, positive
regulation of apoptosis, cellular response to UV-A, p53 signaling
pathway, TGF-β signaling pathway, and cellular senescence. These
findings align with previous transcriptomic analyses following PTT
[[163]45]. Particularly, in the GO enrichment analysis for “response to
UV-A”, only one differential protein, PPID, was identified. In the KEGG
pathway enrichment analysis, PPID was found to be enriched in pathways
associated with cellular senescence and necroptosis. Therefore, 25BM@HA
+ NIR may inhibit tumor growth by up-regulating PPID expression,
thereby inducing tumor cell senescence and necroptosis.
Fig. 6.
Fig. 6.
[164]Open in a new tab
Mechanism exploration of 25BM@HA in tumor phototherapy by proteomic
analysis. (A) The differentially expressed proteins in the control and
25BM + NIR groups. (B) The volcano map of identified differentially
expressed proteins in the control and 25BM + NIR groups. (C) Heat maps
displaying the differentially expressed proteins of different groups.
(D) Gene Ontology (GO) pathway enrichment analysis. (E) Kyoto
Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis.
(F) The PPID protein expression of SAS cells was determined by western
blot. (G) The PPID protein expression of tumor tissues was detected by
IHC (scale bar = 100 μm). (H and I) The RIPK1, RIPK1, and MLKL protein
expressions of SAS cells were determined by western blot. *P < 0.05;
**P < 0.01; ***P < 0.001. BP, Biological Process; MF, Molecular
Function; CC, Cellular Component.
PPID, also known as cyclophilin 40 or cyclophilin D, is a cyclophilin
protein located in the mitochondrial matrix. It serves as a gatekeeper
for mitochondria, regulating their function and integrity. PPID is a
critical regulator of cell death, while its increased phosphorylation
can trigger the opening of the mitochondrial permeability transition
pore (mPTP), thereby promoting necroptosis [[165]46]. Studies have
shown that compounds like bishonokiol A and
1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine can up-regulate
PPID expression by activating the RIP1/RIP3/MLKL pathway, thereby
promoting ROS production while facilitating mPTP-mediated tumor cell
necrosis [[166]47]. The activation of the PPID–mPTP axis represents a
key mechanism through which various antitumor drugs induce tumor cell
death [[167]46]. Similarly, bromocriptine can promote PPID
phosphorylation via the RIP3/MLKL pathway, promoting necrosis in
prolactinomas [[168]48]. Western blot of SAS cells (Fig. [169]6F and
Fig. [170]S11A) and IHC of the tumor model (Fig. [171]6G and Fig.
[172]S11B) showed that, compared to the control group, the 25BM + NIR
group had a significantly higher level of PPID protein expression. As
shown in Fig. [173]6H and I, the expression levels of RIPK1, RIPK3, and
MLKL proteins in SAS cells were significantly increased after treatment
with 25BM@HA combined with NIR laser, suggesting that 25BM + NIR can
promote necroptosis. Therefore, it is hypothesized that 25BM@HA + NIR
may trigger tumor cell necroptosis by activating the RIP1/RIP3/MLKL
pathway, leading to increased PPID expression. Further investigations
will be conducted to verify this pathway.
Interestingly, studies have demonstrated that inhibiting PPID
expression significantly weakens the toxicity of chemotherapy drugs on
tumors [[174]49], while the overexpression of PPID significantly
enhances cancer cell sensitivity to chemotherapy drugs and cytotoxicity
[[175]50]. Therefore, it can be inferred that overexpression of PPID
may reduce tumor cell resistance to phototherapy, and combining
treatments could better enhance the phototherapeutic effects of
nanomaterials.
Conclusion
In this study, we designed and synthesized the BM@HA semiconductor
photosensitizer, which, under NIR laser irradiation, can produce O[2],
relieve tumor hypoxia, and enhance PDT efficacy. Combined with PTT, it
achieved efficient treatment of OSCC. The formation of heterojunctions
and the high photothermal conversion ability of BM@HA facilitated the
effective separation of electron–hole pairs induced by NIR (808-nm
laser). Furthermore, it also maintained the high reduction and
oxidation ability of the separated charge carriers, promoting the
generation of O[2] and ROS. Surface modification of HA improved the
biocompatibility and tumor-targeting efficiency, resulting in
substantial tumor ablation when PDT and PTT were combined under 808-nm
laser irradiation. Proteomic analysis identified PPID as a key
differential protein, suggesting that 25BM@HA + NIR treatment may
induce tumor cell necroptosis by activating PPID-related pathways.
Inducing the expression of the key protein PPID in combination with
phototherapy appears to be a more effective therapeutic strategy. In
conclusion, the BM@HA semiconductor photosensitizer not only overcame
the challenges posed by tumor hypoxia in PDT but also displayed
improved anticancer activity when combined with PTT, highlighting its
considerable potential for the treatment of OSCC.
Acknowledgments