Smart Fe3O4@ZnO Core-Shell Nanophotosensitizers Potential for Combined Chemo and Photodynamic Skin Cancer Therapy Controlled by UVA Radiation

Abstract

Purpose
Photodynamic therapy (PDT) is a non-invasive therapeutic modality that is used for several types of cancer and involves three essential elements (light, photosensitizer (PS), and oxygen). However, clinical PS is limited by the low yield of reactive oxygen species (ROS) and a long retention time. Therefore, developing a low-cost PS that can significantly increase ROS yield in a short time is of utmost importance.

Methods
In this study, brusatol (Bru) was loaded on the surface of ultraviolet A (UVA)-responsive zinc oxide (ZnO)-coated magnetic nanoparticles (Fe3O4@ZnO-Bru). The PS was well characterized by transmission electron microscopy (TEM), Fourier Transform infrared spectroscopy (FTIR), a superconducting quantum interference device, and dynamic light scattering (DLS). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and Hoechst staining were used to determine the inhibitory effect of Fe3O4@ZnO-Bru on squamous cell carcinoma cells (SCC) with or without UVA radiation. Intracellular ROS levels and expression of the Nrf2 signaling pathway were also determined.

Results
FTIR showed that Bru was successfully loaded on Fe3O4@ZnO. Fe3O4@ZnO-Bru was superparamagnetic, and the zeta potential was 8.86 ± 0.77 mV. The Bru release behavior was controlled by UVA. Fe3O4@ZnO-Bru with UVA irradiation induced an increase of 48% ROS productivity compared to Fe3O4@ZnO-Bru without UVA irradiation, resulting in a strong inhibitory effect on SCC. Furthermore, Fe3O4@ZnO-Bru nanocomposites (Fe3O4@ZnO-Bru NCs) had nearly no toxic effect on healthy cells without UVA radiation. The released Bru could significantly inhibit the Nrf2 signaling pathway to reduce the activity of scavenging excess ROS in SCC.

Conclusion
In this study, Fe3O4@ZnO-Bru was successfully synthesized. PDT was combined with photochemotherapy, which exhibited a higher inhibitory effect on SCC. It can be inferred that Fe3O4@ZnO-Bru holds great potential for skin SCC therapy.

Full text

ORIGINAL RESEARCH
Smart Fe
3
O
4
@ZnO Core-Shell
Nanophotosensitizers Potential for Combined
Chemo and Photodynamic Skin Cancer Therapy
Controlled by UVA Radiation
Qian Ren
1
, Caixia Yi
2
, Jun Pan
1
, Xin Sun
2
, Xiao Huang
2,3
1
Key Laboratory for Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing,
People’s Republic of China;
2
School of Sports and Health Science, Tongren University, Tongren, People’s Republic of China;
3
School of Physical
Education, Guangxi University of Science and Technology, Guangxi, People’s Republic of China
Correspondence: Jun Pan; Xiao Huang, Tel/Fax +86023-65102507, Email panj@cqu.edu.cn; humphrey8531@hotmail.com
Purpose: Photodynamic therapy (PDT) is a non-invasive therapeutic modality that is used for several types of cancer and involves
three essential elements (light, photosensitizer (PS), and oxygen). However, clinical PS is limited by the low yield of reactive oxygen
species (ROS) and a long retention time. Therefore, developing a low-cost PS that can signicantly increase ROS yield in a short time
is of utmost importance.
Methods: In this study, brusatol (Bru) was loaded on the surface of ultraviolet A (UVA)-responsive zinc oxide (ZnO)-coated magnetic
nanoparticles (Fe
3
O
4
@ZnO-Bru). The PS was well characterized by transmission electron microscopy (TEM), Fourier Transform
infrared spectroscopy (FTIR), a superconducting quantum interference device, and dynamic light scattering (DLS). 3-(4,5-Dimethyl-
2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and Hoechst staining were used to determine the inhibitory effect of Fe
3
O
4
@ZnO-Bru on squamous cell carcinoma cells (SCC) with or without UVA radiation. Intracellular ROS levels and expression of the
Nrf2 signaling pathway were also determined.
Results: FTIR showed that Bru was successfully loaded on Fe
3
O
4
@ZnO. Fe
3
O
4
@ZnO-Bru was superparamagnetic, and the zeta potential
was 8.86 ± 0.77 mV. The Bru release behavior was controlled by UVA. Fe
3
O
4
@ZnO-Bru with UVA irradiation induced an increase of 48%
ROS productivity compared to Fe
3
O
4
@ZnO-Bru without UVA irradiation, resulting in a strong inhibitory effect on SCC. Furthermore,
Fe
3
O
4
@ZnO-Bru nanocomposites (Fe
3
O
4
@ZnO-Bru NCs) had nearly no toxic effect on healthy cells without UVA radiation. The released
Bru could signicantly inhibit the Nrf2 signaling pathway to reduce the activity of scavenging excess ROS in SCC.
Conclusion: In this study, Fe
3
O
4
@ZnO-Bru was successfully synthesized. PDT was combined with photochemotherapy, which
exhibited a higher inhibitory effect on SCC. It can be inferred that Fe
3
O
4
@ZnO-Bru holds great potential for skin SCC therapy.
Keywords: UVA-triggered chemotherapy, photodynamic therapy, reactive oxygen species, magnetic targeting
Introduction
Cutaneous squamous cell carcinoma (SCC), the second most common type of skin cancer, is a metastatic cancer that
originates from squamous cells located in the suprabasal epidermis.
1
Although chemotherapy is widely used in the
treatment of skin cancer,
2
it often causes a weakened immune system, signicant side effects, and low drug efcacy due
to the poor targeting ability of chemotherapeutics.
3
Therefore, it is important to improve the targeting ability of
chemotherapeutics to improve their treatment effect and reduce side effects.
4
Photodynamic therapy (PDT) as
a minimally invasive treatment modality
5
possesses important characteristics including selective localized treatment,
target specic selectivity, and fewer side effects.
6
In this study, photosensitizer (PS) was administered and subsequently
activated by illumination with exciting light at the target site. The activated PS reacts with available oxygen to form
reactive oxygen species (ROS), which induce tumor cell death and vascular shutdown. The therapeutic effect of PDT
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depends on the generation of a sufcient amount of ROS.
7
In addition, after intravenous injection of PS, patients need to
stay in a dark environment until the PS is eliminated from the body because the eyes of patients are sensitive to indoor
bright light or sunlight and their skin is readily sunburned, swollen, and blistered when exposed to bright light for a short
period of time.
8
Furthermore, damage to healthy tissues as well as cutaneous photosensitivity can occur. Therefore,
increasing the amount of ROS as well as the targeting ability of PS are effective means to improve the efcacy of PDT.
Zinc oxide (ZnO) is a common metal oxide that is non-toxic and of low cost.
9
Its cellular toxicity could be activated
by UVA irradiation, which enables ZnO to be a potential PS in clinical applications.
10
Indeed, when semplice ZnO
nanoparticles were exposed to UVA light, they exhibited some toxicity to several types of cancer cells.
11,12
However, the
amount of ROS was erratic, due to the different sizes of ZnO nanoparticles, and did not meet clinical requirements.
Fortunately, ZnO nanoparticles not only exhibit ultra-high drug-loading efciency but also high-performance intracellular
delivery of chemotherapeutics.
13
Chemotherapeutics can couple with the photo-toxicity of ZnO to induce cell death.
Furthermore, in our previous studies,
14,15
we found that ZnO nanoparticles were UVA-responsive and could transfer from
a hydrophobic state to a hydrophilic state to accurately control the release of loaded drugs. In this way, it is easy to realize
combined chemo and PDT controlled by UVA radiation. Bru is a quassinoid isolated from the Brucea javanica plant and
has extensive pharmacological activities, such as anti-inammatory and antitumor activities.
16
Recently, it was reported
that Bru is a potent inhibitor of Nrf2 activation, thereby ultimately leading to tumor growth inhibition and ameliorated
chemoresistance.
17
Bru regulated the Nrf2 signaling pathway by reducing the level of Nrf2 factor in cancer cells, and
weakened the antioxidant ability of cells, resulting in increasing the accumulation of intracellular ROS.
18
Herein, we
intended to employ ZnO-coated iron oxide core-shell nanocomposites (Fe
3
O
4
@ZnO NCs) to carry Bru as a novel PS.
This PS not only owns favorable magnetic targeting property but also intelligently controls the release of Bru under the
irradiation of UVA to show a synergistic effect combined with the photo-toxicity of ZnO (Figure 1). We hypothesize that
this PS shows synergistic photo-toxicity to improve the therapeutic effect and targeting ability to reduce side effects.
Figure 1 The illustration of the preparation and inhibitory mechanism Fe
3
O
4
@ZnO-Bru. Such novel PS is easily to realize combined chemo and photodynamic therapy
controlled by UVA radiation.
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Therefore, in this study, we synthesized and characterized Fe
3
O
4
@ZnO-Bru and studied their UVA-controlled drug
release behavior. The anti-cancer effect and its inhibitory mechanism of Fe
3
O
4
@ZnO-Bru for combined chemo and PDT
were investigated. Moreover, the biocompatibility of Fe
3
O
4
@ZnO-Bru was evaluated.
Materials and Methods
Materials
FeCl
3
•6H
2
O (AR, Kelong, China), FeCl
2
•4H
2
O (AR, Damao, China), ethanol, and acetone (AR, Chuandong, China),
zinc acetate dihydrate (Zn(Ac)
2
•2H
2
O)(AR, Gracia, China), phosphate-buffered saline (PBS), penicillin–streptomycin
(PS) (Dingguo, China), fetal bovine serum (FBS) (NTC, Logan, USA), trypsin (Hyclone, Logan, USA), RPMI 1640
(Gibco, New York, USA) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT, Solarbio,
Beijing, China) were used as received unless otherwise noted. Hoechst staining kit (C0003) and a reactive oxygen
species (ROS) assay kit (S0033) were purchased from Beyotime Biotechnology (Shanghai, China). RIPA lysis buffer,
SDS lysis buffer, phenylmethylsulfonyl uoride, and a BCA protein assay kit (P0010) used for Western blot analysis
were purchased from Beyotime Biotechnology (Shanghai, China). Nrf2 (#12121), NQO1 (#3187), HO-1 (#43966),
GSTP1 (#3369), and NF-κB (#8242) primary antibodies were obtained from Cell Signaling Technology (USA). GAPDH
antibody (BM1623) was purchased from Wuhan Boster Biological Technology (Wuhan, China). Brusatol was purchased
from Chengdu Desite Biotechnology (Chengdu, China). For all experiments, ultrapure deionized water (Millipore,
Massachusetts, USA) was used. Immortalized human skin keratinocytes (HaCaT cells) and squamous carcinoma cells
(SCC) were received as a valuable gift from Prof. Li Zhong, and human umbilical vein endothelial cells (HUVECs) were
kindly provided by Prof. Kaiyong Cai in our afliation. All cell lines were conducted under the approval of the Ethics
Committee of Chongqing University.
Optimizing the Adsorption of Brusatol
Fe
3
O
4
cores were synthesized by a co-precipitation method. Briey, under the protection of N
2
, FeCl
3
·6H
2
O and FeCl
2
·4H
2
O were reacted in an HCl aqueous solution and then in a NaOH solution to generate Fe
3
O
4
. Next, Zn(Ac)
2
·2H
2
O was added, and
Fe
3
O
4
@ZnO was obtained by an oil bath at 160 °C for 1.5 h. This method is detailed in our previously published article.
19
Various masses of previously synthesized Fe
3
O
4
@ZnO NCs (W: W=100:1, 50:1, 10:1, 1:1, 1:10, 1:50, 1:100, Fe
3
O
4
@ZnO: Bru)
fully dispersed in an aqueous solution, then added into the acetone containing a corresponding amount of Bru, respectively. Then,
the solution was ultrasonically shocked for 1h. After centrifugation at 30,000 rpm for 10 min, the concentration of Bru in the
supernatant was determined by measuring the absorbance at 590 nm using a UV-VIS spectrophotometer. Accordingly, the
adsorption rate (AR) and drug loading capacity (DLC) of Bru were calculated referring to the standard curve of Bru in an aqueous
solution as follows:
AR %ð Þ ¼ ðQtQsÞ=Qt�100% (1)
LC %ð Þ ¼ ðQtQsÞ=QNCs �100% (2)
Q
t
refers to the raw weight of Bru used for adsorption; Q
s
refers to the weight of Bru in the supernatant, and Q
NCs
refers
to the raw weight of Fe
3
O
4
@ZnO used for Bru loading. Experiments were performed in triplicate.
Characterization
The morphology of Fe
3
O
4
@ZnO NCs and Fe
3
O
4
@ZnO-Bru NCs was observed by transmission electron microscope
(TEM, FEI Tecnai G2 F20, Oregon, USA). Fourier transform infrared spectroscopy (FTIR) spectra of Fe
3
O
4
@ZnO NCs
and Fe
3
O
4
@ZnO-Bru NCs were determined by a Fourier transform infrared spectrometer (Nicolet iS5, Thermo Fisher,
Massachusetts, USA). The magnetization value was determined by a superconducting quantum interference device
(MPMS-XL-7, Quantum Design, San Diego, USA). The zeta potential was measured by analyzing 0.1 g of Fe
3
O
4
@ZnO/Fe
3
O
4
@ZnO-Bru NCs in 10 mL of water using the Zetasizer Nano ZS (ZetasizerNano S90, Malvern, UK). All
samples were fully dispersed before zeta potential measurement.
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UVA-Controlled Bru Release Behavior
Fe
3
O
4
@ZnO-Bru NCs with an optimal AR were used for a UVA-controlled drug release study. Fe
3
O
4
@ZnO-Bru NCs
were spread evenly on glass slides and were placed in a darkroom equipped with an UVA light therapy system
(carnation-58, Lifotronic, 365 nm) at 37 °C. A total of ve experimental groups (three samples per group) were prepared.
Fe
3
O
4
@ZnO-Bru NCs were exposed to UVA radiation. At dened time points (1, 2, 4, 6, 8 h), one group of Fe
3
O
4
@ZnO-Bru NCs was removed and slightly washed with 1 mL of ethanol. The concentration of Bru in ethanol, which was
the released Bru from Fe
3
O
4
@ZnO-Bru NCs, was determined by ultraviolet spectrophotometry and represented the
release behavior of Bru. Furthermore, Fe
3
O
4
@ZnO-Bru NCs without UVA radiation served as a control. Experiments
were performed in triplicate.
Cell Culture
HaCaT, HUVEC and SCC cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 U/mL of
penicillin, and 150 U/mL of streptomycin. Cells were incubated in a humidied cell incubation chamber at 37°C with 5%
CO
2
. Cells in the exponential growth phase were used for experiments.
Cytotoxicity on Normal Cells
HaCaT and HUVEC cells (3000 per well) were seeded in 96-well plates, grown to 50% conuence, then Fe
3
O
4
@ZnO-Bru
NCs suspension was added at dened concentrations (0.005, 0.01 and 0.05μg/mL). After 2 h, cells were exposed to 100 kJ/
m
2
UVA irradiation. A UVA light therapy system (carnation-58, Lifotronic) 365nm (peak) spectrum lamp was used to
irradiate the cells following a standard procedure, while non-irradiated cells were used as a background control (control = 0
kJ/m
2
). Cells were incubated for another 24 h or 48 h, respectively, then washed with PBS. Subsequently, 90 μL fresh RPMI
1640 medium and 10 μL MTT solution were added to each well followed by incubation at 37°C for 4 h. Next, 110 μL
DMSO was added to each well, and the 96-well plate was placed on a shaker at a low speed for 10 min to fully dissolve the
crystals. The optical density of the Formazan solution was read on an enzyme-labeled instrument (iMark
TM
Microplate
Reader, Bio-rad680, California, USA) to represent the cellular viability. Untreated cells were used as a positive control to
set the cellular viability to 100%. Six parallel samples were prepared for each experiment.
To determine the status of cell survival, a Hoechst staining kit was used. In brief, HaCaT and HUVEC cells were
plated in 35-mm cell culture dishes (2×10
4
per dish). After 24 h or 48 h of incubation, the culture solution was discarded,
and 1.0 mL of xation solution (C0003-1, Beyotime, Shanghai, China) was added for 30 min. After removal of the
xative, cells were washed 3 times with PBS on a shaker. HaCaT and HUVEC cells were stained with 0.5 mL Hoechst
33258 dye solution for 20 min, washed three times with PBS solution and observed by inverted uorescence microscopy
(Olympus IX71, Tokyo, Japan).
Inhibitory Effect on Cancer Cells Under UVA Radiation
After overnight incubation of SCC, Fe
3
O
4
@ZnO-Bru NCs, Bru, and Fe
3
O
4
@ZnO NCs at dened concentrations (the
concentration of Fe
3
O
4
@ZnO-Bru NCs added was 0.005, 0.01, 0.05μg/mL, Bru and Fe
3
O
4
@ZnO NCs were added
according to the DLC) were added into the culture medium. After 2 h, cells were exposed to 100 kJ/m
2
UVA irradiation
(Fe
3
O
4
@ZnO+UVA). Then, cells were incubated for another 24 h or 48 h, respectively. Cells were washed with PBS;
then, 90 μL fresh cell culture medium and 10 μL MTT solution were added to each well, followed by incubation at 37°C
for 4 h. DMSO (110 μL) was added to each well, and the 96-well plate was placed on a shaker at a low speed for 10 min
to fully dissolve the crystals. The optical density of the Formazan solution was read on an enzyme-labeled instrument
(iMark
TM
Microplate Reader, Bio-Rad Model 680, California, USA) to represent the cellular viability. Untreated cells
served as the positive control to set the cellular viability to 100%. Six parallel samples were prepared for each
experiment.
To determine the status of cell survival, the Hoechst staining kit was used. After 24 h or 48 h of incubation, the
culture solution was discarded, and 1.0 mL of xation solution was added for 30 min. Then, the xative was removed,
and cells were washed 3 times with PBS on a shaker. SCC cells were stained with 0.5 mL Hoechst 33,258 dye solution
for 20 min, washed three times with PBS, and observed by inverted uorescence microscopy.
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Detection of Intracellular Oxygen Radicals
The production of ROS in solution was routinely detected by a ROS assay kit according to the manufacturer’s instructions.
In brief, SCC cells were seeded in 35-mm cell culture dishes at a density of 1×10
5
cells per dish and incubated at 37°C
overnight. Next, Fe
3
O
4
@ZnO NCs, Bru, and Fe
3
O
4
@ZnO-Bru NCs were added at different concentrations (see section
Inhibitory effect on cancer cells under UVA radiation). After 2 h, cells were treated with 100 kJ/m
2
UVA irradiation. After
being incubated for another 24 h or 48 h, 10 μM of 2′,7′-dichlorodihydrouorescein diacetate (DCFH-DA) was added to the
cells. After 30 min, cells were washed with PBS and collected. The uorescence intensities of the cells, which indicated
their ROS levels, were recorded by ow cytometry (CytoFLEX, Beckman, Florida, USA) at excitation and emission
wavelengths of 488 and 525 nm, respectively. Three parallel samples were prepared for each experiment.
Western Blot Analysis
The treatment method of SCC cells was similar to the method described in section Inhibitory effect on cancer cells under
UVA radiation. After incubation for 24 h, cells were lysed in RIPA lysis buffer. To determine the various proteins, cell
lysates were prepared using extraction with SDS lysis buffer in the presence of 1 mM phenylmethylsulfonyl uoride
(PMSF). The protein concentration was measured by a BCA protein assay kit (P0010, Beyotime, Shanghai, China), and
10 μg protein per lane was separated by SDS-PAGE on an electrophoresis apparatus (PowerPac
TM
Basic, Bio-rad,
California, USA) and transferred to polyvinylidene diuoride membranes. After transfer, membranes were blocked with
5% nonfat milk in Tris-buffered saline tween (TBST, T1086, Solarbio, Beijing, China) for 2 h at room temperature and
incubated with respective primary antibodies (Nrf2, HO-1, GSTP1, NQO1, NF-κB, GAPDH) overnight at 4°C.
Membranes were washed three times with 1×TBST and incubated with horseradish peroxidase-conjugated secondary
antibodies. The electrophoresis instrument was purchased from Bio-rad (PowerPac
TM
Basic, California, USA). Signals
were recorded by ECL reagent (#34095, Thermo Scientic, Massachusetts, USA) and visualized by AZURE Biosystems
(c300, California, USA).
Xenograft Assay in Nude Mice
Athymic nude (nu/nu) mice were obtained from the Chongqing Medical University (Chongqing, China). All animal
experiments were conducted under the Animal Management Rules of the Ministry of Health of the People’s Republic of
China (Document No. 55, 2001). In brief, 4-week-old male mice were injected with SCC cells (3×10
6
cells) in the right ank
and into the subdermal space. Once tumors reached a mean volume of 30–50 mm
3
, mice were randomly allocated into two
groups and treated with either PBS (control group) or nanocomposites with UVA (100kJ/m
2
) every other day for 16 days.
Statistical Analysis
Statistical analyses were performed using GraphPad Prism 8.0 for Windows (La Jolla, CA, USA). The results are
presented as the mean ± SEM. The signicance of all experiments was determined by one-way ANOVA plus Bonferroni
posttest analysis (p<0.05).
Results and Discussion
Preparation and characterization of Fe
3
O
4
@ZnO-Bru NCs
TEM conrmed the core-shell structure of Fe
3
O
4
@ZnO NCs, which was analogously square and monodisperse (Figure 2A).
After Bru loading, no obvious changes were observed in morphology and size (Figure 2B). The size of the Fe
3
O
4
@ZnO NCs and
Fe
3
O
4
@ZnO-Bru NCs was in the range of 7–10 nm as determined by electron microscopy. The FTIR results of Fe
3
O
4
@ZnO
NCs and Fe
3
O
4
@ZnO-Bru NCs are shown in Figure 2C. Compared to the spectrum of Fe
3
O
4
@ZnO NCs, the adsorption at
1740.30 cm
–1
and 1682.24 cm
–1
showed the existence of –COO– and C=O, respectively. Several intense bands are present
around 2954.48 cm
–1
, resulting from the increment of –CH
3
. The adsorption at 1295.87 cm
–1
indicated the existence of –OH. All
FTIR results displayed that Bru was successfully loaded on the surface of Fe
3
O
4
@ZnO NCs. The magnetization curves of Fe
3
O
4
@ZnO NCs and Fe
3
O
4
@ZnO-Bru NCs demonstrated that they are superparamagnetic because there was no hysteresis and both
their remanence and coercivity were zero (Figure 2D). Despite the saturation of Fe
3
O
4
@ZnO-Bru NCs decreased to 33.56
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emu•g
–1
due to the loading of Bru in comparison with the 41.50 emu•g
–1
of Fe
3
O
4
@ZnO, their magnetic response was not
inuenced because they were gathered together within 5 min under an external magnetic eld (Figure 2D insert), which indicated
a great magnetic response ability. In consideration of other nano biomaterials based on Fe
3
O
4
@ZnO,
20,21
it can be inferred that
Fe
3
O
4
@ZnO-Bru NCs had good magnetic separating and targeting properties. They, therefore, have the potential of magnetic
targeting. In addition, the zeta potential changed from −2.43 ± 0.21 mV (Fe
3
O
4
@ZnO NCs) to 8.86 ± 0.77 mV (Fe
3
O
4
@ZnO-
Bru NCs) (Figure 2E). The drug-loaded nanocomposites were positively charged, and because the outside of the cell membrane
also has a positive charge, it is not easily adsorbed by cells, and can only be localized to cancer cells through magnetic targeting.
Taken together, these observations indicated that Fe
3
O
4
@ZnO-Bru NCs could be localized by an externally applied magnetic
eld in a desired region, such as the SCC area to realize a targeting function and decrease side effects in healthy tissues.
22
UVA-Controlled Drug Release
Realizing UVA-controlled drug release is key in designing our Fe
3
O
4
@ZnO-Bru NCs to realize combined chemotherapy
and PDT. Thus, the UVA-controlled Bru release behavior was studied. A UVA light therapy system 365 nm spectrum
lamp was used. Under the initial UVA radiation for 1 h, 82.96±1.82% of Bru was released from Fe
3
O
4
@ZnO-Bru NCs,
while nearly no Bru was released without UVA irradiation (Figure 3).
In our previous studies, we found that the surface of ZnO could release the adsorbed drug in a controlled manner via
UVA irradiation: Fe
3
O
4
@ZnO NCs could intelligently control and release the loaded drug depending on the conversion
Figure 2 Characterization of Fe
3
O
4
@ZnO NCs and Fe
3
O
4
@ZnO-Bru NCs. (A and B) TEM images of Fe
3
O
4
@ZnO NCs (A) and Fe
3
O
4
@ZnO-Bru NCs (B). Scale bar:
50 nm. Insert: TEM image with a larger magnication, scale bar: 20 nm; (C) FTIR of Fe
3
O
4
@ZnO NCs and Fe
3
O
4
@ZnO-Bru NCs; (D) Magnetic hysteresis loops of Fe
3
O
4
@ZnO NCs and Fe
3
O
4
@ZnO-Bru NCs. The saturation of Fe
3
O
4
@ZnO-Bru slightly decrease compared with Fe
3
O
4
@ZnO but is still superparamagnetic, indicating that
our drug delivery systems have the potential to be directed to a certain area by externally applied magnetism. Insert: The NCs solution was changed from stable to
aggregated state under the magnetic eld; (E) Zeta potential.
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of hydrophilic/hydrophobic of ZnO shell under UVA irradiation.
15,23
In the absence of UVA irradiation, the drug was still
adsorbed on ZnO shell and not released from the carrier. In recent years, ZnO nanoparticles have been preferable drug
delivery carriers.
24,25
Light-responsive drug delivery systems have attracted more interest due to the controllable stimuli.
The trigger is easily steerable in emitted energy and range of exposure. Based on this, ZnO was chosen as the appropriate
carrier to prepare UVA-responsive drug delivery systems. In this study, Fe
3
O
4
@ZnO NCs were used for Bru loading and
UVA-controlled release. Moreover, it is very possible to realize combined chemotherapy and PDT for enhanced treatment
of SCC using Fe
3
O
4
@ZnO-Bru NCs.
Cytotoxicity of Fe
3
O
4
@ZnO-Bru on HaCaT Cells and HUVEC
To evaluate the biocompatibility of Fe
3
O
4
@ZnO-Bru NCs, their cytotoxicity on HaCaT cells and HUVEC was
determined. The relative viability of HaCaT and HUVEC cells was measured by conducting MTT assays in the presence
of Fe
3
O
4
@ZnO-Bru NCs at concentrations of 0.005, 0.01, and 0.05 mg/mL. When the cells were co-cultured with Fe
3
O
4
@ZnO-Bru NCs after 24 h or 48 h, Fe
3
O
4
@ZnO and Fe
3
O
4
@ZnO-Bru NCs did not display obvious toxicity on HaCaT
cells and HUVEC (Figure 4). The cellular viability was above 80% in all the experimental groups, which was deemed to
Figure 3 The UVA-controlled release behavior of Fe
3
O
4
@ZnO-Bru NCs.
Figure 4 The cytotoxicity of Fe
3
O
4
@ZnO-Bru NCs on HaCaT and HUVEC cells with different concentrations.
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have nearly no toxicity to HaCaT cells and HUVEC. Accordingly, we believe our Fe
3
O
4
@ZnO-Bru NCs would be safe
drug delivery systems for cancer treatment with few side effects.
Figure 5 shows the morphology of HaCaT cells (Figure 5A) and HUVEC (Figure 5B) cells treated with Fe
3
O
4
@ZnO-
Bru NCs at a concentration of 0, 0.005, 0.01 and 0.05 mg/mL. When Fe
3
O
4
@ZnO-Bru NCs were cultured with HaCaT
cells and HUVEC after 24 or 48 h, the morphology of the cells was not affected compared to untreated cells, thereby
indicating that HaCaT cells and HUVEC grew exuberantly with Fe
3
O
4
@ZnO-Bru NCs.
Furthermore, Hoechst staining was used to determine the status of cell survival. The results of the ratio of viable cells
are shown in Figure 6. The ratio of all groups was approximately 85%, which was consistent with the cytotoxicity results.
Figure S1 shows the uorescence images of HaCaT cells (Figure S1A) and HUVEC (Figure S1B) treated with Fe
3
O
4
@ZnO-Bru NCs after Hoechst 33,342 staining. After being co-cultured with Fe
3
O
4
@ZnO-Bru NCs for 24 or 48 h,
HaCaT cells and HUVEC emitted bright blue uorescence, indicating that HaCaT cells and HUVEC grew exuberantly
under these conditions. These results are in accordance with the cell morphology results. Thus, the results demonstrated
good biocompatibility of Fe
3
O
4
@ZnO-Bru NCs.
Biocompatibility is a key concern of biomaterials for their applications in the clinic. In this study, the cytotoxicity
assay was used to evaluate the potential application of Fe
3
O
4
@ZnO-Bru NCs on the skin. In this study, the MTT assay,
cell morphology, and Hoechst staining were carried out for cytotoxicity evaluation. The results showed that Fe
3
O
4
@ZnO-Bru NCs had little cytotoxicity. After 48 h of co-incubation with Fe
3
O
4
@ZnO-Bru NCs, the cell viability was still
above 82%. Fe
3
O
4
@ZnO-Bru NCs exhibit better performance in the cell survival rate compared with other nanoparticles,
such as TiO
2
NPs, incubation with HaCaT cells for 24 h with the concentration of 0.005 mg/mL, cellular viability was
less than 75%.
26
In addition, incubation of 0.01 mg/mL SiO
2
nanoparticles with HaCaT cells for 24 h resulted in
Figure 5 Cellular morphology images. HaCaT cells (A) and HUVEC cells (B) were treated with Fe
3
O
4
@ZnO-Bru NCs for 24 or 48 h with the concentration of 0 mg/mL,
0.005 mg/mL, 0.01 mg/mL and 0.05 mg/mL.
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a cellular viability of about 80%, which was similar to the cell viability of our 0.01 mg/mL nanocomposites when
incubated for 48 h.
27
Overnight (24 h) viability decreased less than 50% at an iron concentration of 1 nM for both citrate-
IONP (citrate-Iron oxide nanoparticles) and dextran-IONP (dextran-Iron oxide nanoparticles).
28
Thus, our Fe
3
O
4
@ZnO-
Bru NCs have a toxicity that is similar to that of low-toxic superparamagnetic iron oxide nanoparticles (SPIONs), which,
when incubated with HUVEC cells for 24 h at a concentration of 37.5 μg/mL and 75 μg/mL, resulted in a cell viability of
80%.
29
These ndings show that our nanocomposites have good biocompatibility.
Inhibition of Fe
3
O
4
@ZnO-Bru to SCC Cells in vitro
To evaluate the effectiveness of chemotherapy combined with PDT of Fe
3
O
4
@ZnO-Bru NCs, the inuence of Fe
3
O
4
@ZnO-Bru NCs on the growth of SCC cells was investigated after UVA radiation. The inhibition rate of the 0.05 mg/mL
Fe
3
O
4
@ZnO-Bru+UVA group was 60% at 48 h, while that of the 0.05 mg/mL Fe
3
O
4
@ZnO-Bru group was 14%
(Figure 7). It was found that the inhibition rate of the Fe
3
O
4
@ZnO-Bru+UVA group was up to 4.2-fold greater compared
to that of the Fe
3
O
4
@ZnO-Bru group. The same dose of Fe
3
O
4
@ZnO had no obvious toxicity to SCC cells after UVA
light irradiation, and the cell viability was higher than 75% (Figure S2). Except for the minor effects of UVA radiation on
Figure 6 The ratio of viable cells. HaCaT cells (A) and HUVEC cells (B) were treated with Fe
3
O
4
@ZnO-Bru NCs for 24 or 48 hours with the concentrations of 0 mg/mL,
0.005 mg/mL, 0.01 mg/mL and 0.05 mg/mL.
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the cellular viability (inhibition rate <2.3%
30
), the results conrm the excellent combined therapeutic effect of Fe
3
O
4
@ZnO-Bru NCs.
Figure 8 shows the morphology images of SCC cells with different treatments after incubation for 24 h (Figure 8A) or
48 h (Figure 8B). Many SCC cells did not adhere to the plate and other cells gathered into masses in Fe
3
O
4
@ZnO-Bru
+UVA group. This phenomenon was much more obvious in Fe
3
O
4
@ZnO-Bru+UVA group treated with 0.05 mg/mL
concentration.
The viable cell ratio of SCC cells is shown in Figure 9. The ratio of live cells in the Fe
3
O
4
@ZnO and Fe
3
O
4
@ZnO-
Bru groups were both above 90%, and the proportion of live cells in the Bru and Fe
3
O
4
@ZnO-Bru+UVA groups was
decreased. In the Fe
3
O
4
@ZnO-Bru+UVA group, live cells accounted for about 50% (p < 0.01 vs Fe
3
O
4
@ZnO groups).
Figure S3 shows the Hoechst stained images of SCC cells after different treatments and incubation for 24 h (Figure S3A)
or 48 h (Figure S3B). The data revealed that more cell death was induced when SCC cells were treated with Bru and
Fe
3
O
4
@ZnO-Bru+UVA. In fact, some dead cells were washed away during Hoechst staining. Thus, the ratio of live cells
in the Bru and Fe
3
O
4
@ZnO-Bru+UVA groups should be lower. These results proved that the combination of chemother-
apy and PDT has an additive inhibitory action on SCC cells.
Accumulation of Intracellular ROS Induced by Combination Therapy
Suppression of the Nrf2 signaling pathway causes an increase in ROS and subsequently results in cell death.
31
Therefore,
we determined the level of ROS accumulation by SCC cells exposed to Fe
3
O
4
@ZnO-Bru+UVA. The data showed that
Bru and Fe
3
O
4
@ZnO-Bru+UVA treatment led to a signicant increase in ROS in SCC cells (Figure 10A and B). UVA
irradiation causes the photosensitizer ZnO to induce cells to produce ROS, and Bru, which has released Fe
3
O
4
@ZnO-Bru
NCs, causes cells to produce ROS. As a result, the Fe
3
O
4
@ZnO-Bru+UVA group has the highest ROS level.
ROS are closely related to the growth and death of cancer cells.
32,33
ROS can induce DNA mutations and genomic
instability, and accelerate the proliferation, immune tolerance as well as metastasis of cancer cells.
34,35
In addition, high
ROS levels enhance cellular oxidative stress, which is detrimental to DNA, proteins, and lipids and thereby causes cell
Figure 7 The treatment of Fe
3
O
4
@ZnO, Fe
3
O
4
@ZnO-Bru, Bru and Fe
3
O
4
@ZnO-Bru+UVA to SCC cells. Data are presented as mean ± SD. **p<0.01.
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death.
36,37
Ample studies have shown that inducing cells to produce high concentrations of ROS is a treatment strategy
for PDT.
38,39
The Fe
3
O
4
@ZnO-Bru+UVA prepared in this study not only uses ZnO to produce ROS, but also allows Bru
to help reduce the ability of cells to remove ROS, which leads to producing more ROS. Therefore, the reason why many
cells in this study, is by signicantly increasing the level of ROS. Thus, photochemotherapy plus PDT is more conducive
to the treatment of skin SCC.
Figure 8 The morphology images of a Fe
3
O
4
@ZnO, b Fe
3
O
4
@ZnO-Bru, c Bru, and d Fe
3
O
4
@ZnO-Bru+UVA treated SCC cells, with the concentrations of 0.005 mg/mL
(a
1
, b
1
, c
1
, d
1
, a
4
, b
4
, c
4
and d
4
), 0.01 mg/mL (a
2
, b
2
, c
2
, d
2
, a
5
, b
5
, c
5
and d
5
) and 0.05 mg/mL (a
3
, b
3
, c
3
, d
3
, a
6
, b
6
, c
6
and d
6
) after incubation for 24 h (A) or 48 h (B).
Figure 9 The ratio of viable cells to the total number of cells in the Hoechst image. SCC cells were treated with Fe
3
O
4
@ZnO, Fe
3
O
4
@ZnO-Bru, Bru and Fe
3
O
4
@ZnO-
Bru NCs for 24 and 48 hours with the concentration of 0.005 mg/mL, 0.01 mg/mL and 0.05 mg/mL. Data are presented as mean ± SD. **p<0.01.
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Figure 10 Fe
3
O
4
@ZnO-Bru NCs responsed to UVA irradiation and increases intracellular ROS level in SCC. SCC cells were treated with Fe
3
O
4
@ZnO, Fe
3
O
4
@ZnO-Bru,
Bru, or Fe
3
O
4
@ZnO-Bru+UVA for 24 h (A) or 48 h (B), and ow cytometry was used to analyze the level of ROS in cells. Bar graphic representation of the uorescence
intensity upon different treatments relative to control were shown. Data are presented as mean ± SD. **p<0.01.
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Fe
3
O
4
@ZnO-Bru NCs Inhibit the Nrf2 Signaling Pathway in SCCs
To conrm whether Fe
3
O
4
@ZnO-Bru inhibit the Nrf2 pathway in SCC cells, whole cell lysates were prepared and
protein expression levels were determined by Western blot analysis after cells were treated with Fe
3
O
4
@ZnO-Bru,
Bru, or Fe
3
O
4
@ZnO-Bru+UVA. After incubation for 24 h, the Fe
3
O
4
@ZnO-Bru+UVA group showed a reduction in
protein levels of Nrf2, HO-1, GSTP1, NQO1 and NF-κB compared to the Fe
3
O
4
@ZnO-Bru group in SCC cells
(Figure 11A and B). Thus, these results suggested that Bru enhanced the antitumor effect of PDT through inhibiting
the Nrf2/HO-1 signaling pathway in SCC cells.
In recent years, Nrf2 has been deemed an important and promising target in cancer therapy and many efforts
have been made to seek therapeutic strategies directed to block the Nrf2 antioxidant pathway.
40
Bru, as a unique
Nrf2 inhibitor, has been shown to regress tumor burden through inhibiting the Nrf2 signaling in several tumor
models.
41,42
More importantly, Bru exhibits the potential as an adjuvant drug to enhance the efcacy of chemother-
apeutics, such as gemcitabine or cisplatin in pancreatic cancer or lung cancer.
31
In this study, we found that Bru was
a potent antitumor compound against SCC cancer cells assisting with PDT. Bru inhibited the Nrf2 pathway, resulting
in the inability to clear ROS in time. It caused the therapeutic effect to be enhanced. Notably, the inhibitory effect of
Bru combined with PDT on the Nrf2/HO-1 signaling pathway in SCC was identied as a novel mechanism,
suggesting a therapeutic advantage for the use of Bru in cancer therapy. Furthermore, the anticancer drug Bru can
reduce the level of Nrf2 in SCC cells to negatively regulate the Nrf2 signaling pathway and weaken the antioxidant
function of the cells. Therefore, the intracellular ROS level has been accumulated, which may cause greater damage
to cancer cells. Thus, using Fe
3
O
4
@ZnO-Bru NCs to combine chemotherapy with PDT controlled by UVA radiation
shows a better therapeutic effect compared with chemotherapy or PDT therapy alone.
Combination Treatment Inhibited Tumor Growth in Nude
To explore the effect of combined chemo and photodynamic cotreatment on tumor growth in vivo, SCC xenografts were
grown in Balb/c nude as a heterotopic tumor model. To form a tumor, Balb/c nude were injected with SCC cells (3×10
6
per injection site). After 10 days, the tumor volumes reached of 30–50 mm
3
and nanocomposites with UVA (100 kJ/m
2
)
were administered to the tumor site every other day for 16 days. Mice were covered with silver paper, and the tumors
were irradiated using a UVA lamp. A signicant inhibition of tumor growth in vivo was observed after combination
treatment (Figure 12) compared with the control group.
Figure 11 The Fe
3
O
4
@ZnO-Bru+UVA potently inhibited the activation of Nrf2/HO-1 pathways. (A) SCC cells were treated with Fe
3
O
4
@ZnO-Bru, Bru or Fe
3
O
4
@ZnO-
Bru+UVA for 24 h. The changes in Nrf2/HO-1 signaling pathways were monitored by Western blotting. (B) Densitometric analysis was performed on the Western blotting.
The levels of Nrf2, HO-1, GSTP1, NQO1 and NF-κB were quantied by using the software Image J. Data are expressed as the mean ± SD of three independent experiments.
**p<0.01.
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Conclusion
In this study, Fe
3
O
4
@ZnO-Bru NPs were successfully synthesized. They show good magnetic targeting performance and
biocompatibility. Moreover, their drug release behavior is UVA-responsive, which is easy to realize with combined
chemo and dynamic therapy. The combined therapeutic effect for skin cancer treatment is much more efcient compared
to chemotherapy or PDT. In addition, it was found that Fe
3
O
4
@ZnO-Bru could suppress activation of the Nrf2-mediated
signaling pathway to decrease the ROS scavenging ability of SCC cells. Therefore, the photo-toxicity of Fe
3
O
4
@ZnO-
Bru increased to perform a more satisfying therapeutic efcacy. Overall, Fe
3
O
4
@ZnO-Bru have great potential in clinical
use for the treatment of cutaneous SCC.
Acknowledgments
This study was supported by the National Natural Science Foundation of China (81860324, 31470902), High-level
Innovative Talents in Guizhou Province (2018-2016-023), Fundamental Research Funds for the Central Universities of
China (2020CDCGSW051, 2018CDPTCG0001/46).
Disclosure
The authors report no conicts of interest in this work.
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