Diselenide–Pemetrexed Assemblies for Combined Cancer Immuno-, Radio-, and Chemotherapies

Article abstract of DOI:10.1002/anie.201914453

Immunotherapy has emerged as a promising new approach for cancer treatment. However, clinically available drugs have been limited until recently, and the antitumor efficacy of most cancer immunotherapies still needs to be improved. Herein, we develop diselenide–pemetrexed assemblies that combine natural killer (NK) cell-based cancer immunotherapy with radiotherapy and chemotherapy in a single system. The assemblies are prepared by co-assembly between pemetrexed and cytosine-containing diselenide through hydrogen bonds. Under γ-radiation, the hydrogen bonds are cleaved, resulting in the release of pemetrexed. At the same time, diselenide can be oxidized to seleninic acid, which suppresses the expression of human leukocyte antigen E (HLA-E) in cancer cells, thus activating the immune response of NK cells. In this way, cancer immunotherapy is combined with radiotherapy and chemotherapy, providing a new strategy for cancer treatment.

Full text of DOI:10.1002/anie.201914453

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Accepted Article
Title: Diselenide-Pemetrexed Assemblies for Combined Cancer
Immuno-, Radio-, and Chemotherapies
Authors: Tianyu Li, Shuojiong Pan, Shiqian Gao, Wentian Xiang,
Chenxing Sun, Wei Cao, and Huaping Xu
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201914453
Angew. Chem. 10.1002/ange.201914453
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Diselenide-Pemetrexed Assemblies for Combined Cancer
Immuno-, Radio-, and Chemotherapies
Tianyu Li,+ Shuojiong Pan,+ Shiqian Gao,* Wentian Xiang, Chenxing Sun, Wei Cao, and Huaping Xu*
Abstract: Immunotherapy has emerged as
a
promising new
immunotherapy in combination strategies, and these results have
been demonstrated in clinical trials.[9] In this regard, it would be
desirable if a combination of immune-, radio-, and chemotherapy
can be achieved. However, such combination strategies have
been seldom reported until recently.[10]
Nature killer (NK) cells represent a unique subset of immune
cells that are distinct from T cells and B cells. NK cells are
recognized based on their ability to induce tumour cell lysis
without prior sensitization.[11] However, several types of tumour
cells can overexpress human leukocyte antigen E (HLA-E) on
their membranes, which protects them from lysis by inhibiting the
activity of NK cells. Strategies that evoke the antitumour activity
of NK cells suggest the possibility of designing a NK cell-based
immunotherapy against human cancer, which would open a new
avenue in cancer immunotherapy.[12]
Selenium is the central component of selenoproteins, such
as glutathione peroxidases and thioredoxin reductases, which
play key roles in redox regulation of the human body.[13] An
inorganic selenium-containing compound selenite (SeO32-) has
been reported to suppress the expression of HLA-E on cancer
cells, thereby rendering them susceptible to NK cells.[14] Since
previous studies revealed that the high valence organic selenium-
containing compound seleninic acid exhibited antitumour activity
in a similar mechanism to that of selenite,[15] we hypothesize that
seleninic acid may also show cancer immunity through NK cell
activation. In addition, diselenides have been demonstrated to be
sensitive to γ-radiation.[16] Gamma-radiation can induce
production of reactive oxygen species (ROS) in water, which
oxidize diselenide bonds into seleninic acid. In this regard, cancer
immunotherapy can be combined with radiotherapy in a concise
system.
approach for cancer treatment. However, clinically available drugs
have been limited until recently, and the antitumor efficacy of most
cancer immunotherapies still needs to be improved. Herein, we
develop diselenide-pemetrexed assemblies that combine nature killer
(NK) cell-based cancer immunotherapy with radiotherapy and
chemotherapy in a single system. The assemblies are prepared via
co-assembly between pemetrexed and cytosine-containing diselenide
through hydrogen bonds. Under γ-radiation, the hydrogen bonds are
cleaved, resulting in the release of pemetrexed. At the same time,
diselenide can be oxidized to seleninic acid, which suppresses the
expression of human leukocyte antigen E (HLA-E) in cancer cells,
thus activating the immune response of NK cells. In this way, cancer
immunotherapy is combined with radiotherapy and chemotherapy,
which reveals a new strategy for combination therapy in cancer
treatment.
In recent years, cancer immunotherapy has been shown to hold
great promise in clinical treatment through modulation of tumour
clearance by targeting immune checkpoints.[1] Checkpoint
inhibitors that block negative regulatory immune pathways,
including CTAL-4 and PD-1 receptors, lead to extended survival
in patients with different types of cancers. Multiple FDA approvals
of checkpoint inhibitors were granted after 2011.[2] However,
except for the limited successful examples, most
immunotherapies have not achieved satisfactory antitumour
efficacy.[3]
A key approach to improving antitumour efficacy is to
combine cancer immunotherapy with other strategies, such as
radiotherapy and chemotherapy.[4] Radiotherapy is commonly
delivered with high-energy photons (γ-rays and x-rays) and has
been proven to be an effective cancer therapy.[5] The combination
of immunotherapy with radiotherapy was reported to enhance the
therapeutic outcome of single immunotherapy.[6] Irradiation of
tumour tissues can result in the activation of immune cells and
induce
a
long-term durable systemic immune response.[7]
Chemotherapy was also able to be combined with
immunotherapy in various types of cancer treatment.[8] Traditional
anticancer drugs, including cisplatin, doxorubicin, and
pemetrexed, can improve the antitumour efficacy of
[a]
T. Li, S. Pan, Dr. S. Gao, W. Xiang, C. Sun, Prof. H. Xu,
Key Lab of Organic Optoelectronics & Molecular Engineering,
Department of Chemistry, Tsinghua University,
Beijing, 100084, People’s Republic of China
E-mail: gaosq170@163.com, xuhuaping@mail.tsinghua.edu.cn
Dr. W. Cao, Department of Chemistry, Northwestern University,
2145 Sheridan Road, Evanston, IL, 60208, USA
[b]
[+]
These authors contributed equally to this work.
Scheme 1. Schematic illustration showed that diselenide-pemetrexed
assemblies combined cancer immunotherapy with radiotherapy and
chemotherapy.
Supporting information for this article is given via a link at the end of
the document.
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Herein we utilized pemetrexed,
a
clinically available
(Figure 1c). A peak at 3000 cm-1 appeared after self-assembly,
which corresponds to the -O-H bond in triple hydrogen bonds.
Then, the self-assembly behaviour of Pem/Se was
observed. Since Cyt-SeSe-Cyt is hydrophobic and pemetrexed is
hydrophilic, mixing these two compounds (molar ratio = 1: 2)
resulted in Pem/Se, which had amphiphilicity and formed self-
assemblies in water. Cyt-SeSe-Cyt could hardly dissolve in water,
but when added into the solution of pemetrexed, a transparent
solution of Pem/Se was obtained. The turbidity change suggested
the formation of self-assemblies in a macroscopic fashion (Figure
S3). Via transmission electron microscopy (TEM), spherical
assemblies with diameters of 47 ± 2 nm were observed (Figure
1d). The hydrodynamic diameters of the assemblies was further
determined via DLS, corresponding to those in TEM figures
(Figure S4). The assemblies were stable in cell culture medium
with FBS according to the DLS measurement (Figure S5).
chemotherapy drug, to self-assemble with cytosine-containing
diselenides (Cyt-SeSe-Cyt) through hydrogen bonds. The
prepared diselenide-pemetrexed (Pem/Se) self-assemblies
combined cancer immunotherapy with radiotherapy and
chemotherapy (Scheme 1). Under γ-radiation, diselenides were
transformed into seleninic acid, pemetrexed was released from
the assemblies, and combination therapy was consequently
achieved. Additionally, drugs in the form of nano-assemblies
possess enhanced stability and tumour targeting efficiency, which
further improves their antitumour efficacy and reduces their side
effects. The strategy based on selenium-containing assemblies
provides new insight into combination therapy and exhibits great
potential for cancer treatment.
Figure 2. Characterization of Pem/Se after γ-radiation. a) Schematic illustration
showing the structure change of Pem/Se under γ-radiation. b) ESI-MS spectrum
demonstrating the formation of Cyt-SeOOH. c) 77Se-NMR confirming the
formation of seleninic acid. d) FT-IR showing the cleavage of hydrogen bonds.
e) DLS determining the change of size and count rate under different dosages
of γ-radiation. f) TEM images without staining exhibiting the morphological
change of the Pem/Se assemblies. Scale bars represent 200 nm.
Figure 1. Synthesis and characterization of Pem/Se assemblies. a) Synthesis
of Pem/Se. b) ESI-MS showing the peak of Pem/Se as calculated. c) FT-IR
spectrum demonstrating the formation of hydrogen bonds. d) TEM image of
Pem/Se without staining. The scale bar represents 200 nm.
With the nano-assemblies in hand, we then studied the γ-
radiation response of the Pem/Se assemblies. Diselenide bonds
were cleaved and oxidized to seleninic acid by ROS generated
under γ-radiation (Figure 2a). The ESI-MS spectrum showed a
peak at m/z 544.08 (Figure 2b), which is the same as the
calculated molecular weight of cytosine-containing seleninic acid
(Cyt-SeOOH). The 77Se-NMR spectra exhibited a new peak at
1234 ppm, which was in the region of seleninic acid,
demonstrating the formation of Cyt-SeOOH (Figure 2c).
Additionally, in the 1H-NMR spectrum, a peak of seleninic acid at
2.95 ppm was observed (Figure S6a). XPS also suggested the
appearance of selenium at a higher oxidative state after γ-
radiation (Figure S6b).
Pem/Se was prepared via self-assembly between
pemetrexed and Cyt-SeSe-Cyt (Figure 1a). Cytosine in the
diselenide compound could form triple hydrogen bonds with
guanine in pemetrexed due to the principle of complementary
base pairing. First, the chemical structure of Pem/Se was
characterized by ESI-MS, 1H-NMR, 77Se-NMR, and FT-IR (Figure
S1, S2). The ESI-MS spectrum showed a molecular ion peak of
m/z 1829.47, which is the same as the calculated mass of Pem/Se
(Figure 1b). The shape of the isotopic peaks was also similar to
the calculated result, which confirmed the structure of Pem/Se.
The formation of hydrogen bonds was demonstrated by FT-IR
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Gamma radiation could also destroy the triple hydrogen
bonds in Pem/Se assemblies. The hydrogen bond cleavage
ability of γ-radiation has been reported before in DNA damage.[17]
In the FT-IR spectrum, the peak at 3000 cm-1 disappeared after
γ-radiation, suggesting the breakage of triple hydrogen bonds
(Figure 2d). The breakage of hydrogen bonds would lead to the
collapse of the Pem/Se assemblies; because the release of
pemetrexed, the hydrophilic group, caused a loss of amphiphilicity
in Pem/Se. The changes in the self-assembly behaviour were
determined by DLS. The sizes of the Pem/Se assemblies
dramatically increased after γ-radiation, which further increased
as the dosage of radiation was raised (Figure 2e). The
responsiveness to γ-radiation was also revealed by count rate
changes, which obviously decreased even under low dosage
radiation (5 Gy). The TEM images exhibited results corresponding
to the DLS measurement (Figure 2f). Under 5 Gy radiation, the
Pem/Se assemblies started to collapse and agglomerate. As the
dosage of radiation increased, fewer assemblies were observed
and increased in size. Therefore, γ-radiation not only cleaved the
diselenide bonds but also broke the hydrogen bonds, leading to
the release of pemetrexed and the disassembly of Pem/Se
assemblies.
levels of the expression of both TS and DHFR after Pem/Se
treatment, suggesting that pemetrexed in Pem/Se assemblies
could enter cancer cells and work as an anti-folate agent (Figure
3a). Under a γ-radiation of 5 Gy, Pem/Se assemblies induced
even lower expression level of DHFR, because γ-radiation
promoted the breakage of the triple hydrogen bonds. However,
pemetrexed itself did not show a strong ability to downregulate
the expression levels of TS and DHFR due to the difficulty of the
ionic small molecule to cross cell membranes.[19] Cell viability
assay exhibited higher anticancer activity of Pem/Se than
pemetrexed, corroborating the higher cellular uptake efficiency of
the assemblies (Figure S7). Compared with Cyt-SeSe-Cyt,
Pem/Se assemblies also showed much higher cellular uptake, as
revealed by intracellular Se concentration measurements (Figure
S8). Since Cyt-SeSe-Cyt could hardly dissolve in water, it had
difficulty crossing cell membranes, as indicated by the low
intracellular Se concentration.
The effect of γ-radiation was further studied with cell viability
tests. After a mild γ-radiation of 2 Gy or 5 Gy, Pem/Se showed
enhanced anticancer activity (Figure 3b). This result could be
explained by the cleavage of triple hydrogen bonds under γ-
radiation, which led to the release of pemetrexed. The flow
cytometry spectra further demonstrated the improved efficacy of
chemotherapy in combination with radiotherapy (Figure S9).
Treatment with Pem/Se resulted in increased levels of ROS, while
treatment with Pem/Se after γ-radiation resulted in even higher
level of ROS, which could induce apoptosis of cancer cells.
Figure 3. In vitro anticancer study of Pem/Se assemblies against MDA-MB-231
cells. a) Western blot demonstrating the down regulation of TS and DHFR. b)
Cell viability assay of Pem/Se at 72 h under different dosages of γ-radiation. c)
Western blot image showing the expression level of HLA-E. d) LDH assay
indicating the cell death rates of MDA-MB-231 cells co-cultured with NK92 cells.
ELISA results exhibiting the IFN-γ (e), TNF-α (f) levels produced by NK92 cells,
and IFN-γ levels produced by mouse NK cells. The dosage of γ-radiation was 5
Gy in all experiments. **P < 0.01; ***P < 0.001.
Figure 4. In vivo antitumour activity of the combination therapy. a) Tumour
growth curves of MDA-MB-231 xenograft tumour-bearing mice after an
intravenous injection of drugs and γ-radiation. Aspartate aminotransferase (b)
and creatine kinase (c) level in the blood of mice sacrificed at the end of the
experiment. d) Immunohistochemistry staining of tumour tissues showing HLA-
E and IFN-γ levels. e) Immunohistochemistry staining of lung tissues in
pulmonary metastasis mouse breast tumor model showing HLA-E and CD49b
levels. Scale bars represent 100 μm. The dosage of γ-radiation was 5 Gy in all
experiments.
The anticancer activity of Pem/Se against MDA-MB-231
cells was studied by in vitro experiments. The chemotherapy
efficacy of Pem/Se was explored by testing the activity of
pemetrexed. Pemetrexed is an anti-folate agent that exhibits
anticancer activity by inhibiting multiple folate-related enzymes,
such as thymidylate synthase (TS) and dihydrofolate reductase
(DHFR).[18] The anticancer activity of pemetrexed can be
analysed by determining the expression levels of TS and DHFR
in cancer cells. The western blotting results exhibited decreased
The cancer immunoactivity of selenite is involved in the
inhibition of the HLA-E protein on the membranes of cancer cells.
Therefore, expression of HLA-E under treatment was determined
by western blot (Figure 3c). MDA-MB-231 cells treated with
pemetrexed did not show any downregulation of HLA-E, even
under γ-radiation. However, the expression of HLA-E was
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inhibited after treatment with Pem/Se and was more strongly
inhibited after the combination treatment of Pem/Se and γ-
radiation. Pem/Se was slightly oxidized to seleninic acids by the
The improved immunoactivity of NK cells induced by the
combination therapy in the in vivo experiments was demonstrated
by immunohistochemistry staining. Tumour tissues of mice under
innate ROS in cancer cells and strongly oxidized under γ-radiation, different treatments were stained with anti HLA-E, anti IFN-γ, and
thus resulting in inhibition of HLA-E. To confirm the contribution of
seleninic acids, Pem/Se was first oxidized by H2O2 to form
seleninic acids and then used to treat cancer cells. The
expression of HLA-E was also inhibited in this group, confirming
the cancer immunoactivity of seleninic acids.
anti CD49b antibodies (Figure S14-16). Confocal laser scanning
microscopy (CLSM) showed that the combination therapy
inhibited the expression of HLA-E in tumour tissues (Figure 4d).
The upregulation of IFN-γ and CD49b was also exhibited, which
indicated the improved immunoactivity of NK cells in tumour
tissues. Combination of cancer radiotherapy and immunotherapy
always showed high efficacy to metastatic tumours due to the
abscopal effect. Combination therapies can induce systematic
immune response. Therefore, the activation of NK cells can be
observed in tumour metastases. The activation of NK cells in
tumour metastases induced by the combination therapy was
evaluated in a pulmonary metastasis mouse breast tumor model
(Figure 4e, S17, S18). The immunohistochemistry staining of lung
tissues demonstrated that the combination therapy dowregulated
HLA-E of metastatic tumours. And the downregulation of HLA-E
led to the activation of NK cells, which was exhibited by the
upregulation of CD49b and NK1.1. Lung tissues were originally
rich in NK cells, which was shown by the red fluorescence of
CD49b. However, the number of NK cells was reduced in tumour
metastases, indicated by high HLA-E levels and low CD49b levels.
The combination therapy induced both the downregulation of
HLA-E and the upregulation of CD49b. And the co-localization of
CD49b with NK1.1, another maker of NK cells, further confirmed
the activation of NK cells in vivo.
In conclusion, the combination of cancer immunotherapy
with radiotherapy and chemotherapy was achieved using
diselenide-pemetrexed assemblies. Under a γ-radiation of 5 Gy,
diselenide bonds could be cleaved to form seleninic acid, which
activated cancer immunity of NK cells via inhibiting the expression
of HLA-E protein. Pemetrexed was also released under γ-
radiation, which improved the anticancer activity. The
combination therapy exhibited good antitumour efficacy and low
side effects. The development of Pem/Se assemblies revealed
the great potential of selenium-containing compounds in cancer
immunotherapy and provide a novel strategy for combination
cancer therapy.
To demonstrate the efficacy of cancer immunotherapy,
MDA-MB-231 cells were co-cultured with NK92 cells. In the
lactate dehydrogenase (LDH) cytotoxicity assay, MDA-MB-231
cells treated with Pem/Se exhibited a much higher death rate than
those treated with pemetrexed (Figure 3d). The cell death rate
was further enhanced when Pem/Se was oxidized by γ-radiation
or H2O2. Interferon gamma (IFN-γ) is a cytokine produced by
immune cells in response to pathogens. NK92 cells have cancer
immunity by secreting IFN-γ, which is harmful to cancer cells.[20]
In addition, pemetrexed was reported to increase the IFN-γ
production of NK cells, providing an opportunity for combining
chemotherapy with immunotherapy.[21] The IFN-γ levels in the co-
culture environment were determined by ELISA (Figure 3e). The
level of IFN-γ was slightly enhanced when treated with Pem/Se,
while after radiation, the level of IFN-γ was strongly enhanced.
Tumour necrosis factor alpha (TNF-α) is also a cytokine related
to the cancer immunity of NK92 cells (Figure 3f). The increased
production of TNF-α after the combination treatment of Pem/Se
and γ-radiation further confirmed the efficacy of cancer
immunotherapy. Since the immune systems of mouse and human
are different, the efficacy of the combination therapy with mouse
NK cells was evaluated before in vivo experiments. When co-
cultured with mouse NK cells, MDA-MB-231 cells treated with
Pem/Se exhibited higher death rate than that in other groups,
which was demonstrated by LDH assay (Figure S10). The
increased ratio of mouse NK cells to MDA-MB-231 cells in co-
culturing also indicated the cancer immunity of mouse NK cells to
MDA-MB-231 cells. In addition, the ELISA results exhibited a
higher IFN-γ level produced by mouse NK cells under the
treatment of Pem/Se and γ-radiation (Figure 3g), which
suggested the combination therapy could be applied to mouse
models.
The in vivo antitumour activity of the combination therapy
was studied in MDA-MB-231 xenograft tumour-bearing BALB/c-
nude mice. Treatment started at day 1 when the tumour volume
reached 200 mm3. Pem/Se inhibited tumour growth on its own
(Figure 4a) and in combination with a γ-radiation of 5 Gy led to
smaller tumour volumes, demonstrating the higher antitumour
efficacy of the combination therapy. Pem/Se did not induce any
body weight loss of mice (Figure S11). Gamma-radiation caused
weight loss immediately after the treatment, but mice gradually
recovered after the treatment stopped. To further evaluate the
side effects, serum biochemical tests were performed on the
blood from sacrificed mice (Figure 4b, c, and S12). The markers
of liver function (aspartate transaminase, AST; alanine
aminotransferase, ALT) and heart function (creatine kinase, CK;
lactate dehydrogenase, LDH) were not elevated in mice treated
with Pem/Se or combination therapy. Moreover, the post-mortem
histopathology was analysed to study the changes in organ
morphology (Figure S13). No obvious morphological changes
were observed in combination therapy treated mice,
demonstrating the low side effects of the system.
Acknowledgements
This work was financially supported by the National Basic
Research Program of China (2018YFA0208900), China National
Funds for Distinguished Young Scientists (21425416), the
National Natural Science Foundation of China (21734006), and
the Funds for Creative Research Groups of China of the National
Natural Science Foundation of China (21821001). The authors
would like to thank Prof. Hang Yin (Tsinghua University, China)
and Prof. Guangjun Nie (National Center for Nanoscience and
Technology, China) for their help in some of the in vitro and in vivo
experiments.
Keywords: cancer immunotherapy• self-assembly• selenium•
gamma radiation• pemetrexed
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Entry for the Table of Contents (Please choose one layout)
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Diselenide-pemetrexed assemblies
were developed to combine cancer
immunotherapy with radiotherapy and
chemotherapy. Under γ-radiation,
diselenide was oxidized to seleninic
acid, which suppressed the expression
of HLA-E in cancer cells, thus
activating the immunoactivity of NK
cells. The chemotherapeutic drug-
pemetrexed could be simultaneously
released. Therefore, the combination
therapy was achieved.
Tianyu Li, Shuojiong Pan, Shiqian Gao,
Wentian Xiang, Chenxing Sun, Wei
Cao, and Huaping Xu*
Page No. – Page No.
Diselenide-Pemetrexed Assemblies
for Combined Cancer Immuno-,
Radio-, and Chemotherapies
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