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absorption curve in Figure 2d, after Au-TiO NDs and Au-
Au NRs, Au-TiO NDs, and Au-TiO @ZnS generally showed
2 2
2
TiO @ZnS were subjected to X-rays (1 Gy, 50 kV, 5 min), the
significant concentration- and time-dependent cytotoxicity
2
absorption of methylene blue at the maximum absorption
wavelength of 665 nm in the solution had significantly
decreased for both materials. More importantly, after stop-
ping X-ray irradiation for 10 minutes, the absorption of the
towards cancer cells. Among them, Au-TiO @ZnS displayed
the most outstanding killing ability.
2
The mechanism of Au-TiO @ZnSꢀs excellent cancer cell
2
killing ability was further explored. It was found that the
temperature of the solution changes only 1.9 K after 5 mi-
nutes of X-ray (1 Gy, 50 kV) irradiation. The possibility of the
fact that local hyperthermia leading to cell death caused by
high-energy X-rays interacting with the material could be
ruled out (Figure S9).From the results of the Annexin V-PI
early apoptosis detection assay, it can be inferred that the
exposure of MC38 cells to X-ray (50 kV, 5 min) after 12 h of
Au-TiO @ZnS solution at 665 nm showed a further drop
2
(
Figure 2e), whereas the absorption curves of the other
control groups (Figure S7) did not change further. Mean-
while, gas chromatograph was used to analyze the H2
generation efficiency of Au NRs, Au-TiO -cover, Au-TiO2
2
NDs and Au-TiO @ZnS. Compared with the H generation
2
2
efficiency of Au NRs and Au-TiO -cover with zero output,
2
that of Au-TiO NDs and Au-TiO @ZnS were as high as
co-incubation with Au-TiO @ZnS mainly led to cell death
2
2
2
À1 À1
À1 À1
1
01.6 mmolg h , 120.2 mmolg h , respectively. (Fig-
caused by apoptosis (Figure 3b). The cell cycle results
indicated that after X-ray irradiation, the G2-M cycle of
MC38 cells was blocked to inhibit cell proliferation (Fig-
ure S10). The results of the TUNEL experiment showed that
a great number of DNA fragments appeared in the X-ray
group, which are represented by bright green FITC fluores-
ure S8) This result indicates the significant effect of long
afterglow materials on H production after stopping X-ray
2
irradiation.
To further study the mechanism of Au-TiO @ZnS cata-
2
lyzed H generation under X-ray excitation and long after-
2
glow excitation, the conduction band (CB) and valence band
cence in Figure 3c, especially in the Au-TiO @ZnS co-
2
(
VB) of amorphous TiO were calculated by first-principles
incubation group. Thus, the primary DNA damage was
attributed to the ROS generated during the radiotherapy
process. Here, the ROS probe, 2,7-dichlorodihydrofluores-
cein diacetate (DCFH-DA), was used to detect the ROS
content, which was proportional to the green fluorescence
detected in the cells incubated with different materials after
X-ray irradiation (Figure 3d). The green fluorescence of the
2
and compared with the theoretical values for anatase, which is
considered the most active phase for photocatalytic H2
production. Figure 2 f shows that the band gap of amorphous
TiO was 2.97 eV, smaller than that of anatase (3.20 eV),
which means that amorphous TiO has a wider spectrum of
[
19]
[14]
2
2
absorption from ultraviolet light to visible light. In addition,
amorphous TiO can more easily lose electrons to catalyze H2
solution corresponding to Au-TiO @ZnS under X-ray irradi-
2
2
production, due to its more negative conduction band energy
ation was greatly increased compared with that of the control
group. This indicates that under the condition of X-ray
0
+
level compared with the H electrode potential (E (H /H )).
2
2
Then, the Schottky barrier at the metal-semiconductor inter-
irradiation and Au-TiO @ZnS co-incubation, the cells pro-
2
face of Au NRs-TiO was also simulated by first principles
duced a large amount of ROS. It is well-known that free
radicals can cause cell lipid peroxidation damage. Therefore,
liposome peroxidation assay was used to verify the degree of
lipid peroxidation damage to the MC38 cell membrane after
2
(
Figure 2g). The contact between Au NRs and amorphous
TiO was an ohmic contact because F > F . An anti-blocking
2
m
s
layer was produced at the contact interface, which had
minimal impact on charge transfer. Moreover, holes flowed
from the metal into the semiconductor, forming a hole
potential well on the surface of the semiconductor. This is
consistent with the direction of electrons flowing from TiO2
into Au NRs under X-ray irradiation, which slows down the
electron-hole recombination efficiency of Au NRs during the
H therapy and radiotherapy. The degree of damage was
2
positively correlated with the measured concentration of
MDA. Through the establishment of MDA standard curve
(Figures S11, S12). Under X-ray irradiation (1 Gy, 50 kV,
5 min), the lipid peroxidation level of the Au-TiO @ZnS
2
group was 5.1 times higher than that of the X-ray group
(Figure 3e). The cell proliferation ability after different
treatments was also measured through cell cloning experi-
ments. As shown in Figure 3 f, S13 the cell cloning rate of the
H catalytic reduction process to greatly improve the catalytic
2
H production capacity. Under the irradiation of afterglow
2
light in the visible light region, the electron transmission path
between Au NRs and amorphous TiO mainly comes from
Au-TiO @ZnS co-incubation group after X-ray irradiation
2
2
thermionic emission due to SPR, which can cross the Schottky
barrier and inject hot electron beams from Au NRs into the
was almost 0, which was one-thousandth of the control group.
Then, the relative expression of apoptosis inducing factor
(AIF), adenosine 5’-monophosphate-activated protein kinase
(AMPK) and caspase-3 was characterized by Western blot-
conduction band of TiO . As a result, reduction and H2
2
generation reaction occurs on TiO , while leaving holes for
2
oxidation reaction on Au NRs.
ting to study the effect of the combined action of H and
2
The above results verify the ability of X-ray excitation to
radiotherapy on the apoptosis pathway (Figure 3g). AIF and
AMPK related signaling pathways are related factors of non-
release H gas and the radio-sensitization effect of Au NRs.
2
[
8b]
Hence, it is worthwhile to conduct in vitro cytotoxicity studies
on MC38 cells, which will be used to establish an orthotopic
liver cancer model. It can be seen from Figure 3a that in the
absence of X-ray radiation, neither ZnS:Cu,Co-A, Au NRs,
Au-TiO NDs, nor Au-TiO @ZnS exhibited significant tox-
caspase-dependent apoptosis. The levels of AIF decreased
after treatment with hydrogen-rich water (HRW) which was
prepared by continuously bubbling H2 with a purity of
99.999% for 5 min by inserting a sterile needle into the
culture medium. while AIF levels significantly increased after
X-ray irradiation (50 kV, 5 min) in both the groups with or
2
2
icity. However, once exposed to X-ray (1 Gy, 50 kV, 5 min),
Angew. Chem. Int. Ed. 2021, 60, 2 – 10
ꢀ 2021 Wiley-VCH GmbH
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