Journal Pre-proof

Anti-recurrence/metastasis and chemosensitization therapy with thioredoxin

reductase-interfering drug delivery system

Jichun Yang, Shuojiong Pan, Shiqian Gao, Yiheng Dai, Huaping Xu

PII: S0142-9612(20)30300-8
DOI: https://doi.org/10.1016/j.biomaterials.2020.120054
Reference: JBMT 120054

To appear in: Biomaterials

Received Date: 23 December 2019

Revised Date: 24 March 2020
Accepted Date: 11 April 2020

Please cite this article as: Yang J, Pan S, Gao S, Dai Y, Xu H, Anti-recurrence/metastasis and
chemosensitization therapy with thioredoxin reductase-interfering drug delivery system, Biomaterials
(2020), doi: https://doi.org/10.1016/j.biomaterials.2020.120054.

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.

© 2020 Published by Elsevier Ltd.

H.P. Xu and J.C. Yang: Conceptualization, Methodology. J.C. Yang:

Writing-Original Draft, Investigation, Formal Analysis. S.J. Pan, S.Q. Gao, Y.H. Dai:

Validation. H.P. Xu: Writing-Reviewing and Editing, Supervision.

Anti-recurrence/metastasis and chemosensitization therapy with

thioredoxin reductase-interfering drug delivery system

Jichun Yang,a,b Shuojiong Pan,a Shiqian Gao,a,b Yiheng Dai,a and Huaping Xu a,*

a
Key Laboratory of Organic Optoelectronics and Molecular Engineering, Department

of Chemistry, Tsinghua University, Beijing 100084, China.

b
Tsinghua-Peking Joint Center for Life Sciences, Beijing 100084, China.

* Corresponding author.

E-mail: xuhuaping@mail.tsinghua.edu.cn
ABSTRACT

Thioredoxin reductase (TrxR) is an essential mammalian enzyme that possesses a

selenocysteine active site. TrxR is overexpressed in many malignant tumors and has a

close relationship with apoptosis, drug resistance, recurrence and metastasis of tumors.

Recently, TrxR has emerged as a promising target for anticancer therapy. Herein, we

developed a TrxR-interfering drug delivery system (DDS) based on

RGD-PEG-PUSeSe-PEG-RGD self-assembling micelles for imaging-guided

gemcitabine (GEM) chemosensitization and anti-recurrence/metastasis therapy. The

diselenide-containing micelles were degraded in response to TrxR stimuli for GEM

releasing. In the meantime, the dissociated polymers’ chain segments targeted the

active site of TrxR via Se-Se/Se-S dynamic reactions for activity inhibition. This

inhibition by the micelles not only provided chemosensitization, but reduced tumor

recurrence/metastasis risk via the induction of residual tumor cell apoptosis by

triggering ROS production post-chemotherapy. In this work, we took the

transformation between Se-containing dynamic covalent bonds developed by our

group from in vitro to in vivo, which furthered the knowledge on the biochemistry of

selenium and provided aspects to develop new TrxR inhibitors. Overall, the

TrxR-interfering DDS combined excellent antitumor effects for primary solid tumors

with the inhibition of tumor recurrence/metastasis during post-treatment care,

providing new perspectives for efficient cancer therapy.

KEYWORDS

Thioredoxin reductase; Drug delivery system; Dynamic covalent chemistry;

Chemosensitization; Anti-recurrence/metastasis

Scheme 1. Schematic illustration of TrxR-responsive DDS for ROS-mediated

chemosensitization and anti-recurrence/metastasis therapy based on TrxR activity

inhibition.

1. Introduction

Although conventional small molecule drugs are limited by side effects, low solubility,

short circulation times, drug resistance and poor tumor specificity, chemotherapy

remains the most commonly administered cancer therapy [1-3]. Accordingly, the

encapsulation of drugs in stimuli-responsive nanocarriers has been proposed as a

strategy to overcome these issues. Drug delivery systems (DDS) with

stimuli-responsive properties can accurately release drugs in cancer cells to improve

treatment efficiency and reduce side effects. [4-9]. Amongst the DDS, selenium

(Se)-containing polymers are regarded as promising candidates due to their

stimuli-responsive properties, efficient drug delivery capacity, and unique

biofunctions [10-12]. To-date, only limited Se-containing polymers have been

successfully used in drug delivery systems due to unsatisfactory stability and poor

solubility [13]. The design of DDS with Se-containing polymers with

delicately-designed structures are critical to their broad application in biomedicine

fields.

A further challenge of chemotherapy is tumor recurrence and metastasis that occurs

following the ablation of solid tumors [14-16], accounting for more than 90% of

cancer-related deaths [17]. Developing effective anti-recurrence/metastasis strategies

with high biocompatibility can significantly improve survival rates.

Thioredoxin reductase (TrxR) is a NADPH-dependent selenocysteine

(Sec)-containing enzyme with a unique C-terminal-Gly-Cys-Sec-Gly active site for

extensive substrates, which is found overexpressed in many cancer cells and plays a

key role in regulating intracellular redox balance [18,19]. Additionally, TrxR is

closely related to proliferation, invasion, apoptosis, drug resistance, recurrence and

metastasis of tumors [20,21]. Hence, TrxR has emerged as a valuable target for tumor

recurrence/metastasis and anticancer drug development [22]. Tumor

recurrence/metastasis can be prevented by inhibiting TrxR [23], but combination

treatments with synergistic effects are required for the treatment of solid tumors [24].

The design of simple platforms that integrate the therapeutic effects of primary solid

tumors with anti-recurrence/metastasis capacity by inhibiting TrxR activity have

emerged as a hotspot in nanomedicine fields.

In this study, we proposed a TrxR-interfering DDS based on diselenide-containing

micelles with 5, 10 (15)-bis (4-aminophenyl)-15, 20-diphenylporphyrin (Por) as an

imaging-guided agent for GEM chemosensitization and anti-recurrence/metastasis

therapy (Scheme 1). GEM was rapidly released following TrxR stimulation and the

dissociated diselenide-containing micelles targeted the active site of TrxR for activity

inhibition via Se-Se/Se-S dynamic reactions. The Se-containing dynamic covalent

bond metathesis revealed by our group was applied from in vitro to in vivo [25],

which not only furthered the knowledge on the biochemistry of selenium, but

provided new perspectives for the development of TrxR inhibitors for cancer

therapeutics. In vivo analysis showed that the diselenide-containing DDS had

antitumor effects on primary solid tumors due to the chemosensitization of GEM

through TrxR inhibition. More importantly, tumor recurrence and metastasis was also

obviously suppressed via the inhibition of TrxR activity to induce residual tumor cell

apoptosis by triggering ROS production. This work successfully combined the ability

to eliminate primary solid tumors with post-treatment care in a single

TrxR-interfering DDS based on diselenide-containing polymers. This reveals new

avenues for comprehensive chemotherapy.

2. Materials and methods

2.1. Materials

Selenium powder, sodium borohydride and Tolylene-2,4-diisolyanate (TDI, 98%)

were purchased from Aladdin Chemical Company, China. Poly (ethylene glycol)

(PEG) monomethyl ether (Mw = 5000) was purchased from Sigma-Aldrich.

3-bromo-1-propanol (97%) and gemcitabine (GEM) were obtained from J&K

Scientific. Meso-tetraphenyl porphyrin (TPP) was supplied by 9 Ding Chemistry.

Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS) and

penicillin/streptomycin were obtained from Gibco, USA. Recombinant human

thioredoxin reductase was supplied by AmyJet Scientific Inc., China. TrxR activity

assay kit, -SH content assay kit and glutathione (GSH) content assay kit were

purchased from Solarbio Science & Technology Company. Radio

immunoprecipitation assay (RIPA) lysis buffer, reactive oxygen species (ROS) assay

kit, mitochondrial membrane potential assay kit, bicinchoninic acid (BCA) protein

assay kit and cell counting kit-8 (CCK-8) were purchased from Beyotime

Biotechnology Company. Tissue ROS test kit was obtained from BestBio Company.

Primary antibody and second antibody used in western blot analysis were obtained

from Abcam Company, USA. All of the other chemical reagents, organic solvents and

biological reagents used in this work were purchased from commercial sources, and

all materials for polyurethane synthesis were dried under vacuum at 90 °C overnight

before use.

2.2. Characterizations

1 77
H NMR and Se NMR were obtained with a JEOL JNM-ECA 400 (400 MHz).
Fourier transform infrared (FTIR) spectra were recorded on a Bruker IFS 66v/s

spectrometer (Ettlingen, Germany) using KBr substrates. The gel permeation

chromatography (GPC) was measured with a Waters 2414 Index Detector. TEM

images were taken on a transmission electron microscopy with an accelerating voltage

of 200 kV (TEM, JEM-2010HR, Japan). For dynamic light scattering (DLS)

measurement, the hydrodynamic sizes of the micelles were measured using a

Zetasizer Nano-ZS (Malvern, England). The UV-vis absorption and fluorescence

properties of the micelles were charactered by a UV-2450-visible spectrophotometer

(Shimadzu, Japan) and a Hitachi F-7000 (Tokyo, Japan) spectrofluorometer,

respectively. X-ray photoelectron spectrum (XPS) was performed on an Axis Ultra

DLD with nonmonochromatized Al Kα radiation. A high performance liquid

chromatography (Agilent technologies co., Ltd, USA) was selected to determine the

cumulative release of GEM from micelles@GEM at pH 7.4 and 5.0 treated with and

without 2.25 nM TrxR. The contents of Se in major organs of mice were detected by

an ELAN DRC-e ICP Mass Spectroscopy (ICP-MS, Perkin-Elmer, USA).

2.3. Preparation of diselenide-containing monomers (HO-H6C3-Se-Se-C3H6-OH)

The diselenide-containing monomers were synthesized according to our previous

report with slight modification [13]. Typically, 1.65 g selenium powder and 0.76 g

sodium borohydride were dissolved in 30 mL ultrapure water with an ice bath. After

degassing with nitrogen for 5 min, 2.85 g 3-bromo-1-propanol was dissolved in 30

mL THF and added into the mixture. The reaction was sealed and kept at 50 °C

overnight under magnetic stirring. Finally, the obtained product was purified by
column chromatography using ethyl acetate (EA) for elution. 1H NMR (400 MHz,

77
CDCl3) δ (ppm): 3.76 (t, 2H), 3.04 (t, 2H), 2.03 (m, 2H). Se NMR (600 MHz,

CDCl3) δ (ppm): 300.74.

2.4. Preparation of 5, 10 (15)-bis (4-aminophenyl)-15, 20-diphenylporphyrin (Por)

Por was obtained as previous report [26]. 200 mg TPP and 183 mg NaNO2 were

dissolved in 10 mL trifluoroacetic acid (TFA). After 90 s stirring at room temperature,

100 mL ultrapure water was slowly added to terminate the reaction, and the solution

was further extracted with dichloromethane (DCM). NaHCO3 power was used to

remove excess TFA in DMC. Then the product was purified by column

chromatography with DCM/petroleum ether (PE) (1/1) as elution. After evaporation

of the solvent, the product and 800 mg SnCl2 was dissolved into 50 mL concentrated

HCl. After processing at 65 °C for 4 h, 100 mL ice water was poured into the mixture

to terminate the reaction, then the solution was neutralized with ammonium hydroxide

until pH=8. The resulting solution was extracted with DCM and purified by column

chromatography to obtain Por. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.95 (d, 4H),

8.78 (d, 4H), 8.22 (dt, 4H), 7.86 (m, 10H), 7.01 (dd, 4H), 5.60 (d, 4H), -2.80 (d, 2H).

2.5. Preparation of RGD-PEG5k-OH

RGD (arginine-glycine-aspartic acid) is the binding sequence for the integrin receptor

family that is highly expressed in many cancer cells [27]. It is widely used in the

modification of DDS for targeted drug delivery [28-30]. RGD-PEG5k-OH was

prepared through the reaction between sulfhydryl and maleimide. 1500 mg

Mal-PEG5k-OH and 71 mg RGD-SH peptide were dissolved in 15 mL PBS (pH=7.4),

and the mixture was stirred at room temperature with the protection of N2 for 48 h.

Then the solution was transferred to a dialysis bag (MWCO 5000) and dialyzed

against deionized water for two days. The HO-PEG-RGD was collected as white

powder after freeze-drying.

2.6. Preparation of RGD-PEG-PUSeSe-PEG-RGD polymer

The RGD-PEG-PUSeSe-PEG-RGD polymer was synthesized according to our

previous report with slight modification [13]. Initially, 287 mg HOC3SeSeC3OH, 129

mg Por and 193 µL TDI were dissolved in 10 mL THF and sealed in 50 mL flask.

Then the mixture was degassed by N2 for 30 min. After reacting at 50 °C for 12 h

under magnetic stirring, 1000 mg RGD-PEG5k-OH was dissolved in 5 mL THF and

injected into the flask. The reaction was further kept at 50 °C overnight under the

protection of N2. Finally, THF was evaporated under vacuum, and the product was

washed with deionized water and ethanol for several times.

RGD-PEG-PUSeSe-PEG-RGD was obtained after drying with vacuum oven.

2.7. Assembly of the micelles

The micelles were prepared through the amphiphilic nature of

RGD-PEG-PUSeSe-PEG-RGD. 10 mg RGD-PEG-PUSeSe-PEG-RGD co-polymer

was dissolved in 1 mL DMF, then the solution of 10 mg mL-1 was added dropwise to

8 mL ultra-pure water under sonication. After dialyzing with ultra-pure water for 48 h,

the solution was filled to 10 mL to obtain an aggregate solution at a concentration of 1

mg mL-1 for further use.

2.8. Preparation of HO-H22C11-Se-S-Ph

HO-H22C11-Se-S-Ph was synthesized as we reported previously [31]. 0.25 g

HOC11SeSeC11OH and 0.11 g PhSSPh were dissolved in 6 ml chloroform and sealed

in a 10 ml quartz bottle. After the irradiation of 254 nm UV light (15 flux) for 6 h, the

product was purified by column chromatography using PE/ethyl acetate (4/1) as the

eluent. HO-H22C11-Se-S-Ph was obtained after drying overnight in the vacuum oven

at 40 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 7.15-7.60 (m, 5H), 3.64 (t, 2H), 2.94

(t, 2H), 1.78-1.21 (m, 18H). 77Se NMR (600 MHz, CDCl3) δ (ppm): 427.42.

2.9. Drug loading and release profiles from the micelles

Micelles@GEM was prepared in the same processes as preparing the micelles, except

that 10 mg GEM was dissolved in 1 mL DMF with 10 mg

RGD-PEG-PUSeSe-PEG-RGD simultaneously. Micelles@GEM was dialyzed against

ultra-pure water for 48 h to remove the unloaded GEM. The concentration of GEM in

the dialysate was determined by HPLC, and the drug loading efficiency (LE) was

calculated according to the following equation (1).

ℎ
% = × 100% 1
ℎ
The drug release experiment of micelles@GEM was performed with a dynamic

dialysis method. 2 mL micelles@GEM solution was sealed in a dialysis bag (MWCO

2000 Da). Then the dialysis bag was immersed in 10 mL PBS (pH = 5.0 and 7.4) with

and without 2.25 nM TrxR. At defined time periods, 50 μL of release media was taken

out to measure the concentration of GEM by HPLC. The release efficiency (RE) is

calculated based on the following equation (2).

ℎ
% = × 100% 2
ℎ
2.10. Cell culture

Both A549 cells and H1299 cells were cultured at 37 °C and 5% CO2 in Dulbecco’s

Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS)

and 1% penicillin-streptomycin. The medium was replaced every day, and the cells

were digested by trypsin and redispersed in a fresh culture medium before plating.

2.11. Cellular uptake

To study the effect of the incubation concentrations on cellular uptake behavior, A549

cells were seeded in confocal dishes at a density of 2 × 105 cells per dish and in 6-well

plates at a density of 1 × 106 cells per well, respectively. Cells were incubated with the

micelles of different concentrations for 24 h. Por fluorescence was measured with an

LSM 710 scanning microscope and flow cytometer.

To study the effect of the incubation time on cellular uptake behavior, A549 cells were

seeded in confocal dishes at a density of 2 × 105 cells per dish and in 6-well plates at a

density of 1 × 106 cells per well, respectively. Cells were incubated with 150 µg mL-1

micelles for different time periods and Por fluorescence in A549 cells was measured

on an LSM 710 scanning microscope and flow cytometer.

2.12. Cytotoxicity assays

For in vitro cytotoxicity evaluation, A549 and H1299 cells were seeded in 96-well

plates at a density of 5 × 103 per well and incubated with various drug formulations of

different concentrations for 24 h. Then the cell viabilities were assessed with standard

CCK-8 assay (Dojindo, Japan) according to the manufacturer's instructions.

2.13. Determination of cellular TrxR activity, thiol and GSH contents

The effects of the micelles on TrxR activity, thiol and GSH contents were studied

using a DTNB-thioredoxin reductase assay kit, a DTNB-thiols assay kit and a

DTNB-GSH assay kit purchased from Beijing Solarbio Science and Technology Co.,

Ltd.

2.14. Western blotting

A549 cells were seeded in a 6-well plate at a density of 1 × 106 per well and incubated

overnight. After being treated with the micelles of different concentrations for 12 h

and 24 h, the cells were washed with PBS for three times and lysed with RIPA lysis

buffer on an ice bath. The total protein concentrations in the cell lysates were

quantified by BCA method. 20 µL protein samples were separated with 8% and 15%

SDS-PAGE. After electrophoresis, separated proteins were transferred to a

polyvinylidene difluoride (PVDF) membrane and blocked with 5% non-fat milk in

Tris-Buffered-Saline with Tween (TBST) for 1 h. The PVDF membrane was

incubated with particular primary antibodies against TrxR/Trx and Tubulin in 5%

non-fat milk overnight at 4 °C. Thereafter, the membrane was washed with TBST for

three times and incubated for 1 h at room temperature with horseradish peroxidase

(HPR)-conjugated secondary antibodies. The membrane was washed with TBST for

three times and treated with chemiluminescent HRP substrate. The protein expression

levels were observed by Azure biosystem C400, USA.

2.15. In vitro migration assays

A549 cells were seeded in a 6-well plate at a density of 1 × 106 cells per well and

cultivated overnight. Then, the media were replaced with DMEM containing 3% FBS
for 6 h as starvation treatment. Afterwards, the cells were scratched with pipette tips

and co-incubated with the micelles of different concentrations for 24 h with new

DMEM. Finally, the cells were stained with DAPI, and the photographs of migrated

cells were taken with an Olympus inverted microscope.

2.16. ROS assays

Intracellular ROS detection was measured by staining cells with the fluorescent probe

2′, 7′-dichloro fluorescein diacetate (DCFH-DA). A549 and H1299 cells were seeded

in confocal dishes at a density of 2 × 105 cells per dish and treated with the micelles

of different concentrations for 24 h. After staining with 10 mM DCFH-DA at 37 °C

for 30 min, the cells were washed with PBS three times and fixed with 1 mL of 4%

paraformaldehyde. The fluorescence intensity of the cells was measured on an LSM

710 scanning microscope. Furthermore, ROS levels in the tumor tissues were detected

with tissue ROS detection kit obtained from BestBio Company.

2.17. Mitochondrial membrane potential

The depolarization of the mitochondria membranes was examined by CLSM and flow

cytometry using JC-1 as specific probe. A549 cells were seeded in confocal dishes at

a density of 2 × 105 cells per dish. After incubation with the micelles of different

concentrations for 24, the A549 cells were stained with 1 mL JC-1 dye working

solution at 37°C for 30 min and washed with PBS for three times. The red

fluorescence of aggregated JC-1 and the green fluorescence of monomeric JC-1 were

observed on an LSM 710 scanning microscope. The rates of aggregated JC-1 and

monomeric JC-1 were analyzed by flow cytometry instrument.

2.18. In vivo fluorescent imaging and biodistribution of the micelles

After intravenous injection of 200 μL PBS, free Por and the micelles with/without RGD

modification (1 mg mL-1) into the mice, the fluorescence images at different time

periods were recorded on an IVIS lumina II in vivo spectrum imaging system at an

excitation of 580 nm and an emission of 720 nm.

To investigate the biodistribution of the micelles in vivo, the mice were sacrificed at

24 h and 48 h after intravenous injection of 200 μL 1 mg mL-1 the micelles into the

mice. The tumors and main organs including heart, liver, spleen, lung, kidney,

intestine and stomach were dissected and digested, and the contents of Se were

determined by ICP-MS to monitor the biodistribution of the micelles.

2.19. In vivo antitumor efficiency evaluation

To study the antitumor efficacy of micelles@GEM in vivo, nude mice (6-7 weeks)

with subcutaneous A549 cancer xenografts were selected as a model. When the tumor

volumes reached ~100 mm3, the A549 tumor-bearing mice were randomized into four

groups (4 mice/group) and treated with intravenous injection of 200 μL PBS, the

micelles, GEM and micelles@GEM (the concentrations of GEM and the micelles

were approximate 1 mg mL-1), respectively. The treatments were repeated every 3

days within 21 days. The body weights and tumor sizes of the mice were also

recorded every 3 days. The tumor sizes were measured according to the following

equation: Tumor volume = (tumor length) × (tumor width)2/2. The mice were

sacrificed in 21 days treatments, and the solid tumors were excised for weighing and

hematoxylin and eosin (H&E) analysis. H&E stained images of the main organs of the
mice were used to investigate the biocompatibility of the treatments in vivo. All

animal experiments were performed in compliance with the People’s Republic of

China national standard (GB/T 16886.6-1997) and approved by the Institutional

Animal Care and Use Committee (IACUC) at Tsinghua University.

2.20. In vivo anti-recurrence and anti-lung metastasis efficacies evaluation

In vivo local recurrence and lung metastasis mice models were established to evaluate

the anti-recurrence/metastasis efficacy of the micelles by TrxR activity inhibition. For

anti-local recurrence efficacy study, 5 × 106 A549 cells treated with and without 150

μg mL-1 the micelles for 24 h were injected into the right flank of the mice (3

mice/group) to simulate local residual tumor cells after treatments. The tumor

progressions with time were monitored within 30 days.

For anti-lung metastasis efficacy study, 1 × 106 A549 cells treated with and without

150 μg mL-1 the micelles for 24 h were intravenously injected into the mice (3

mice/group) to simulate disseminated tumor cells after treatments. On the 30th day of

the injection, the mice were sacrificed. The lungs of mice were harvested and fixed in

Bouin's fluid to evaluate the anti-lung metastasis ability of the micelles. H&E staining

images and the weights of the excised lungs of the mice were also recorded to further

demonstrate the anti-lung metastasis efficacy of the micelles by TrxR activity

inhibition.

2.21. Blood biochemistry analysis

Blood biochemistry analysis (n = 3) was used to validate the biocompatibility of the

treatments. After 21 days treatments, the plasma of the mice of all groups were
collected. ALT, AST, ALB, ALP, UREA, CR, CK and LD levels in the blood were

determined and provided by the hospital of Tsinghua University.

2.22. Statistical analysis

All the experiments were conducted in at least triplicate and repeated three times.

Data are expressed as the mean ± standard deviation (SD). Statistical analysis was

performed using Student’s t test, and results of p<0.05 were considered statistically

significant (*p<0.05, **p<0.01, ***p<0.001).

3. Results and discussion

3.1. Synthesis of the TrxR-responsive copolymers RGD-PEG-PUSeSe-PEG-RGD

with tumor targeting and imaging capabilities

The synthesis and characterization of diselenide-containing monomers, Por, and

RGD-PEG were outlined in Supporting Information (Fig. S1). The

RGD-PEG-PUSeSe-PEG-RGD imaging copolymer was prepared via the stepwise

polymerization of toluene diisocyanate (TDI) with diselenide-containing diols as

monomers, and Por as imaging-guided agent according to our previous work with

slight modifications [13]. RGD-PEG monomethyl ethers were applied to terminate the

active ends. 1H NMR and gel permeation chromatography (GPC) measurements

illustrated the successful preparation of RGD-PEG-PUSeSe-PEG-RGD block

copolymers with molecular weights of 84.5 k (Mn) and 25.5 k (Mw), respectively

(Fig. 1a and Fig. 1b). The difference of molecular weights calculated by 1H NMR and

GPC was attributed to the different conformations of block copolymers compared to

the linear polystyrene reference when detected by GPC.

3.2. Preparation and validation of the micelles

Emulsification method was used to prepare the micelles due to the amphiphilic

self-assembly properties of RGD-PEG-PUSeSe-PEG-RGD [13]. The morphology and

stability of the micelles were characterized by transmission electron microscopy

(TEM) and dynamic laser light scattering (DLS) measurements. TEM images and

DLS revealed that the spherical micelles (ca. 80 nm) had relatively narrow size

distributions and were well dispersed in water (Fig. 1c). A high stable hydration

radius of the micelles after soaking in distilled water, phosphate-buffered saline (PBS),

10% fetal bovine serum (FBS) and complete DMEM were observed over 7 days,

suggestive of their potential for in vitro and in vivo therapeutic applications (Fig. 1d).

The UV-vis absorption and fluorescence spectra of the micelles and free Por were

recorded to further verify the possibility of the micelles as imaging-guided agents (Fig.

S2a and Fig. S2b). The micelles had similar spectral patterns with free Por, indicating

that the imaging capabilities of Por were well retained after conjugation in the

micelles. Furthermore, minimal levels of photobleaching were observed in the

micelles after repeated cycles of illumination (Fig. S2c and Fig. S2d), which

contributed to the stabilization effects of the micelles. These results demonstrated the

favorable optical properties of the micelles for imaging-guided cancer therapy.

Fig. 1. (a) 1H NMR spectra and (b) GPC plots of RGD-PEG-PUSeSe-PEG-RGD. (c)

TEM images of the self-assembled micelles (inset: size distributions from dynamic

light scattering). (d) Size distributions of the micelles after soaking in distilled water,

phosphate-buffered saline (PBS), 10% fetal bovine serum (FBS) and complete

DMEM for 7 days (Inset: images of the dispersed micelle solution).

3.3. Cellular uptake assays

The internalization of the micelles is significant for the effectiveness of cancer

therapy. Therefore, the cellular uptake behaviors of RGD-PEG-PUSeSe-PEG-RGD

micelles were assessed on TrxR overexpressing A549 cells with confocal laser

scanning microscope (CLSM) and flow cytometry by observing the red fluorescence
signal of Por. As shown in Fig. 2a and Fig. S3-S5, intracellular fluorescent intensities

increased with concentration and time, indicating that the micelles were continuously

internalized. PEG-PUSeSe-PEG micelles without RGD modification exhibited lower

intracellular uptake efficiency compared with RGD-PEG-PUSeSe-PEG-RGD

micelles at 150 µg mL-1 after 24 h incubation (Fig. 2a), validating the cancer cell

targeting ability of the micelles by grafting RGD successfully. Furthermore, the

lysosomes and nuclei of the A549 cells were labeled with Lyso tracker and DAPI to

evaluate the intracellular localization of the micelles (Fig. S4). Micelles with bright

Por fluorescence accumulated in the lysosomes over time, with no obvious red

fluorescence signals observed in the cell nuclei. These results suggested that the

micelles were internalized via the endo/lysosomal system and do not target the cell

nuclei.
Fig. 2. (a) Confocal images of A549 cells after incubation with the micelles of

different concentrations for 24 h. (b) Release profiles of GEM from micelles@GEM

at pH 7.4 and 5.0 treated with and without 2.25 nM TrxR. (c) TEM images and (d)

size distributions of the micelles after incubation with 2.25 nM TrxR from 0 to 24 h.

(e) XPS spectra of Se in the micelles before and after treatment with 2.25 nM TrxR

for 24 h. All data here are expressed as mean ± S.D. of triplicates.

3.4. In vitro drug loading and TrxR-responsive release properties.

GEM, one of the most effective chemotherapy drugs used for lung cancer in clinical

[32-35], was selected to investigate drug loading and TrxR-responsive release

behaviors of the micelles. The drug-loading efficiency (DLE) and drug-loading

capacity (DLC) of GEM were calculated as 85.5% and 46.1% based on HPLC

methods, indicating that the micelles were effective for GEM delivery. High rates of

DLE and DLC of GEM were achieved via hydrophobic interactions and the high

encapsulation ability of the micelles.

TrxR-responsive GEM release profiles of the micelles were studied under different

pH conditions as well as with and without 2.25 nM TrxR. As shown in Fig. 2b, in the

absence of TrxR (stimulating the very low levels of TrxR in normal tissue cells), only

1.7% and 5.2% of GEM were released after 48 h at pH 7.4 and pH 5.0, revealing the

micelles to be stable under different pH conditions. However, 2.25 nM TrxR

(stimulating high expression levels of TrxR in cancer cells) [21] led the releasing rate

of GEM significantly increase to 47.7% and 53.9% in 10 h at pH 7.4 and pH 5.0,

respectively. It was also deserved to mention that the not very high GEM release
amount was due to π-π stacking and hydrogen-bond interactions between GEM and

the micelles fragments. [36-39]. The sensitive responsive property to TrxR of the

micelles provided a smart DDS for “on-demand” tumor chemotherapy with high

biocompatibility.

Reactions between the micelles and 2.25 nM TrxR were recorded by TEM, DLS and

X-ray photoelectron spectroscopy (XPS) to visualize the GEM release mechanisms.

As observed in Fig. 2c, the micelles became adhesive and gradually degraded with the

interaction time of TrxR. Unstable hydrodynamic sizes and peaks of larger sizes were

also observed following DLS measurements (Fig. 2d). Furthermore, the binding

energy of Se 3d in the micelles decreased from 55.8 to 54.9 eV, indicating the

cleavage of diselenide bonds and the reduction of selenium by TrxR (Fig. 2e) [13, 40].

TEM, DLS and XPS analysis were consistent with the rapid release of GEM from

micelles@GEM, demonstrating that the TrxR-responsive DDS had been successfully

prepared. Further details on the interaction processes between the micelles and TrxR

will be discussed in subsequent sections.

3.5. In vitro cytotoxicity assays

To evaluate the feasibility of the TrxR-responsive micelles as a DDS for cancer

therapy, cell counting kit-8 (CCK-8) viability assays were performed on A549 cells

after co-incubation with the micelles, free GEM and micelles@GEM for 24 h (Fig.

3a). All three groups showed concentration-dependent cancer cell death. The survival

rates of the single-micelle-treated group was approximately 80% at 150 μg mL-1. The

slight toxicity of the micelles occurred due to elevated levels of reactive oxygen
species (ROS) in A549 cells caused by the inhibition of TrxR activity. In comparison

to free GEM treatment, the micelles@GEM group exhibited higher levels of

cytotoxicity at the same GEM concentrations that extended beyond the predicted

additive effects of the micelles and free GEM (Fig. S6). The predicted additive effects

were calculated by multiplying the cell survival rates of the micelles and free GEM.

This remarkable anticancer efficacy of micelles@GEM was attributed to high levels

of drug delivery, and to the synergistic effects of GEM chemotherapy and

chemosensitization following TrxR inhibition with the micelles.

3.6. TrxR inhibition by the micelles and cell migration assays

TrxR activity is closely associated with the evolution, proliferation, metastasis and

drug resistance of tumors, which is significant for anti-recurrence/metastasis cancer

therapy. TrxR activity inhibition induced by the micelles was therefore investigated

using standard DTNB [5,5-dithio-bis-(2-nitrobenzoic acid)] assay (DTNB can be

reduced to TNB by TrxR with characteristic absorption peak at 412 nm) [41]. The

micelles exhibited favorable inhibition effects on the activity of 2.25 nM TrxR in a

concentration- and time-dependent manner outside the cells (Fig. S7). TrxR activity in

A549 cells also gradually decreased with increasing concentrations of the micelles

after 24 h of co-incubation (Fig. 3b). All the results of extracellular and intracellular

demonstrated the capacity of Se-containing micelles as an inhibitor of TrxR activity.

Moreover, western blot analysis showed that the micelles inhibited TrxR activity in

A549 cells without down-regulating TrxR or Trx expression (Fig. 3c). These data

suggested that the intracellular TrxR/Trx system remained intact after the micelles
treatment, consistent with previous studies [42].

Fig. 3. (a) Cell viability of A549 cells after incubation with the micelles, free GEM

and micelles@GEM for 24 h. [For free GEM and micelles@GEM-treated groups, the

caption of X axial (Concentrations) presents the concentrations of GEM. For

micelles-treated group, the caption of X axial (Concentrations) indicates the same

concentrations of the micelles in micelles and micelles@GEM-treated groups]. (b)

TrxR activity, (c) TrxR and Trx expression levels, (d) migration analysis, (e) thiol

concentrations, (f) fluorescence images of DCFH-DA and (g) changes in membrane

potentials of A549 cells after incubation with the different concentrations of micelles

for 24 h. All data are the mean ± SD of three independent experiments.

Scratch assays were performed to evaluate the inhibition efficiency of the micelles on

A549 cell migration. As shown in Fig. 3d and Fig. S8, the migration rates of A549

cells treated with the micelles for 24 h were strongly suppressed in a

concentration-dependent manner, highlighting the potential of the micelles to prevent

the recurrence and metastasis of tumors in vivo. The suppression of tumor cell

proliferation and invasion by the micelles was through the inhibition of TrxR activity

that induced cells apoptosis by triggering ROS production. This will be discussed in

later sections.

3.7. ROS-mediated apoptosis and chemosensitization

TrxR regulates intracellular redox balance by reducing disulfides to thiols [43]. We

therefore assessed the concentrations of thiols and ROS production in A549 cells after

24 h of co-incubation with the micelles to provide an in-depth insight into the

interaction between the micelles and TrxR. In addition to reduced TrxR activity,

decreased thiol concentrations and enhanced ROS generation were observed in A549

cells with the concentrations of the micelles increasing from 0 to 150 μg mL-1,

providing evidence that TrxR activity inhibition could elevate the intracellular

oxidative stress (Fig. 3e and Fig. 3f). GSH is another reduced regulation molecule in

cells in addition to the thioredoxin antioxidant defense system. We therefore analyzed

the contents of GSH in A549 cells after treatment with the micelles for 24 h. As

shown in Fig. S9, a marked increase in ROS levels in A549 cells were related to the

depletion of cellular GSH. On one hand, the accumulation of ROS was accompanied

by the dysfunction of mitochondrial membrane potential which triggered cell

apoptosis (Fig. 3g and Fig. S10), suggesting slight cytotoxicity of the micelles. On the

other hand, the accumulation of ROS increased the sensitivity of cancer cells to GEM

promoting chemotherapeutic efficacy [44-45]. These data explain the higher

cytotoxicity of the micelles@GEM compared to the additive effects of the micelles

and free GEM via CCK analysis. This highlights a new treatment strategy for

Se-containing DDS as a ROS-mediated chemosensitizer through TrxR inhibition.

To further confirm the ROS-mediated apoptosis and chemosensitization induced by

Se-containing micelles, assays were performed in a second lung cancer cell line

H1299, in which the expression of TrxR is ~50% lower than that of A549 cells (Fig.

S11). As shown in Fig. S12, the ability of the micelles to regulate TrxR activity, thiol

concentration, GSH concentration, and ROS levels in H1299 were weaker than those

of A549 cells due to the low expression levels of TrxR. Thus, H1299 cells were less

susceptible to the micelles compared to A549 cells (Fig. S13), which verified the

ROS-mediated apoptosis induced by the micelles. These results demonstrated that the

suppression of cell viability and proliferation by the micelles was related to the

inhibition of TrxR activity. Interesting, H1299 cells were more sensitive to free GEM

compared to A549 cells (Fig. S14a), whilst the micelles@GEM showed higher

cytotoxicity to A549 cells than H1299 cells (Fig. S14b). We speculated that was
contributed to the higher TrxR activity inhibition efficiency by the micelles leading to

higher ROS accumulation in A549 cells, and increased sensitivity to chemotherapy.

These results highlight the chemosensitization effects induced by the micelles, which

were consistent with previous studies [46-48].

3.8. Mechanism of TrxR inhibition by Se-containing micelles

The active site of TrxR is a redox-active selenolthiol/selenenylsulfide formed from

cysteine-selenocysteine with an open C-terminal sequence of -Gly-Cys-Sec-Gly

[49,50]. We therefore synthesized a small compound (HOC3SeSPh) with a Se-S

structure according to our previous studies [31] to simulate TrxR (characteristics of

the compound are outlined in Fig. S15) and used the diselenide monomer (HOC3Se)2

to simulate the micelles to investigate the mechanism of TrxR inhibition. The

diselenide monomer was incubated with NADPH-reduced Se-S for 2 hours at 37 ℃ to

mimic microenvironment in vivo. ESI-MS spectra were constructed to provide

information on the reaction products. The molecular ion peak of m/z 391.1 and 249.0

were ascribed to the molecular formula of [HOC3SeSeC11OH2]+ and [PhSSeC3OH2]+,

respectively, indicating dynamic metathesis reactions between the diselenide

monomer and Se-S compound (Fig. 4a) [31]. Thus, in combination with previous

studies [18,51], the process of the micelles for TrxR activity inhibition was speculated

to be that, the cysteine-selenocysteine active site in TrxR was reduced to reductive

selenol and thiol under NADPH interaction firstly. The Se-Se bonds with

reduction-responsive in the micelles then underwent cleavage in the presence of the

reductive selenol and thiol of TrxR for drug release. In the meantime, the reduced
diselenide bonds in the micelles formed new Se-Se/Se-S bonds between TrxR and the

micelles through dynamic covalent bond metathesis reactions (reaction process

illustrated in Fig. 4b). This targeted binding of the micelles to the

cysteine-selenocysteine active site in TrxR led to TrxR activity inhibition. The

Se-Se/Se-S dynamic covalent bonds metathesis revealed by our group also verified

these speculations [25,31]. In this study, we applied Se-Se/Se-S dynamic covalent

bonds metathesis revealed by our group from in vitro to in vivo, that not only enriched

the knowledge of biochemistry of selenium, but provided new perspectives to develop

TrxR inhibitors for cancer therapy based on diselenide metathesis reactions.

Fig. 4. (a) ESI-MS spectrum demonstrating dynamic metathesis reactions between the

diselenide monomer and Se-S compound. (b) Proposed reaction process of the

micelles targeted to the active site of TrxR.

3.9. In vivo fluorescent imaging and biodistribution

The capacity of RGD-modified micelles for fluorescence imaging-guided tumor

chemotherapy was evaluated on A549 tumor-bearing mice. After intravenous

injection with PBS, free Por and the micelles with/without RGD modification,

fluorescence signals were gathered at 4, 8, 24, 36, and 48 h, respectively (Fig. 5a).

The fluorescence intensity of the tumors treated with RGD-modified micelles

increased over time, peaking at 24 h post-injection, illustrating a sustained

accumulation of RGD-modified micelles in the tumors and the potential of

RGD-modified micelles as a fluorescence imaging agent. Furthermore, compared to

free Por and micelles lacking RGD modification, the RGD-modified micelles

exhibited a longer circulation time and improved tumor-targeting ability due to the

enhanced permeability and retention (EPR) effect and the modification with RGD.

To evaluate the tissue distribution of RGD-modified micelles, tumors and major

organs of the mice were harvested 24 h post-injection and imaged ex vivo (Fig. 5a).

Peak fluorescence signals were also observed in the tumors of RGD-modified

micelles-treated group, which mimicked those of the imaging experiments in vivo,

indicating the tumor-targeting properties of RGD-modified micelles for imaging and

GEM delivery. The biodistribution of RGD-modified micelles was also determined by

ICP-MS at 24/48 h post-injection (Fig. 5b). This was consistent with the fluorescence

imaging data. The higher contents of Se in the liver, the stomach and the intestine

highlighted the metabolism of the micelles in the liver and their subsequent excretion

through the digestive tract, decreasing the side effects of chemotherapy. These data

suggest that the RGD-modified micelles have a high capacity as a DDS for

imaging-guided chemotherapy.
3.10. In vivo targeted chemotherapy and TrxR activity inhibition for synergistic

therapy

To verify the synergic effects of target chemotherapy and TrxR activity inhibition, the

antitumor effect of micelles@GEM was estimated on xenograft A549 tumor-bearing

mice. As shown in Fig. 5c, tumor growth of the micelles-treated group was slightly

suppressed compared to the saline group, which was attributed to the decreased

proliferation ability of tumor cells induced by TrxR activity inhibition. Moreover, the

micelles@GEM group showed notably reduced tumor volumes compared to the free

GEM group. These results were in agreement with the cytotoxicity data, and

suggested that the micelles@GEM achieved a combined therapeutic effect with

targeted GEM chemotherapy and the chemosensitization induced by TrxR activity

inhibition. We comprehensively analyzed the mean tumor weight and the levels of

necrosis in the dissected tumors through immunohistochemical analysis. The

micelles@GEM-treated group showed the most potent antitumor effects amongst all

treatments (Fig. 5d-f and Fig. S16). Furthermore, ROS levels in the tumor tissues after

different groups treatments were also detected with tissue ROS test kit according to

the manufacturer’s instructions (BestBio Company). Micelles@GEM markedly raised

ROS level in the tumor tissues (Fig. S17). This illustrated the effectiveness of

ROS-mediated therapy via TrxR activity inhibition, which was consistent with that of

cell experiment.
Fig. 5. (a) Fluorescence images of the mice and dissected organs recorded at an

excitation of 580 nm and an emission of 720 nm after injection of PBS, free Por and

the micelles with/without RGD modification through the tail vein for different time

periods. (b) Biodistribution of RGD-modified micelles in the major organs of mice

(heart, liver, spleen, lung, kidney, intestine, stomach and tumor). (c) Tumor volumes,

(d) images of excised tumors and (e) tumor weights after different treatments with

PBS, the micelles, free GEM and micelles@GEM for 21 days. A549 tumor-bearing

mice were randomized into four groups (4 mice/group). (f) H&E stained images of

tumor slices from different groups after 21 days treatments.

The biocompatibility of the micelles@GEM was further investigated by

hematoxylin-eosin histologic (H&E) analysis (Fig. S18) and serum biochemical tests
[alanine aminotransferase (ALT), aspartate transaminase (AST), albumin (ALB)

alkaline phosphatase (ALP), urea, creatinine (CR), creatine kinase (CK), Lactate

dehydrogenase (LD)] after 21 days treatments (Fig. S19). There were no apparent

changes in the physiological morphology of the major organs (heart, liver, spleen,

lung, kidney, intestine, stomach) of the mice in each of these treatment groups.

Analysis of the biochemical indicators suggested that targeted GEM delivery and

TrxR-responsive GEM release could reduce the side effects of chemotherapy (Fig.

S19). Additionally, the body weights of mice in the micelles@GEM group did not

decrease with prolonged exposure times (Fig. S20). These data suggested that

TrxR-responsive Se-containing DDS was applicable for in vivo applications.

3.11. Anti-recurrence and anti-lung metastasis capability

The ability to reduce tumor recurrence and metastasis can improve the survival rates

of patients after treatment. Given the known association between TrxR activity and

the invasion, recurrence and metastasis of tumors, anti-recurrence and anti-lung

metastasis experiments were performed to investigate the ability to suppress tumor

recurrence and metastasis through TrxR-interfering DDS (Fig. 6a). For the local

cancer recurrence inhibition study, A549 cells treated with and without the micelles

for 24 h were injected into the flank of the mice to simulate local residual tumor cell

growth. Tumor progression over time was then monitored. As shown in Fig. 6b and

Fig. 6c, the tumors of mice injected with the none-treated A549 cells (average tumor

volume: 383.3 mm3) were larger than those of the micelles-treated group (average

tumor volume: 8.3 mm3). The dramatic differences in tumor growth indicated a lower
cancer recurrence capacity of the micelles-treated A549 cells due to the inhibition of

TrxR activity [23]. Furthermore, A549 cells treated with and without the micelles for

24 h were intravenously injected into the mice to simulate disseminated tumor cells.

The lungs of the mice were then harvested and fixed in Bouin's fluid to evaluate the

anti-lung metastasis ability of the micelles. Compared to the micelles-treated group,

larger tumor nodules were excised from the lungs of untreated groups (Fig. 6d). The

average lung weight of untreated mice (575.2 mg) were ≥ 3-fold higher than those of

the micelles-treated group (154.4 mg), due to increased metastasis of the tumors in the

lungs (Fig. 6e). Reduced levels of metastasis in the micelle treated group were also

observed through H&E staining (Fig. 6f). Both anti-recurrence and anti-lung

metastasis experiments demonstrated that our innovative Se-containing DDS not only

exhibited more efficacious therapeutic effects for solid tumors, but prevented tumor

recurrence and metastasis through the induction of residual tumor cell apoptosis

through TrxR inhibition.

Fig. 6. (a) Schematic of the anti-recurrence/metastasis studies performed with the

diselenide-containing micelles. For anti-recurrence studies, 5 × 106 A549 cells treated

with and without the micelles for 24 h were injected into the flank of the mice (3

mice/group) to simulate local residual tumor cells. For anti-metastasis studies, 1 × 106

A549 cells treated with and without the micelles for 24 h were intravenously injected

into the mice (3 mice/group) to simulate disseminated tumor cells. (b) Images and (c)

tumor volumes of the mice after the local injection of A549 cells treated with and

without the micelles. (d) Lung images, (e) weights and (f) H&E staining of the mice

intravenously injected with the micelles-treated A549 cells and none-treated A549

cells, respectively.

4. Conclusions

In summary, a TrxR-responsive chemosensitization and anti-recurrence/metastasis

DDS has been constructed based on diselenide-containing micelles. The novel

diselenide-containing nanoplatform was used for the targeted delivery of GEM into

the tumors, and the micelles could be rapidly disassembled by TrxR for GEM release.

The dissociated micelle segments could bind to the active site of TrxR for activity

inhibition via Se-Se and Se-S dynamic reactions. The inhibition of TrxR activity by

the micelles not only disrupted intracellular redox balance for chemosensitization

effects, but prevented the recurrence and metastasis of tumors via the induction of

residual tumor cell apoptosis by triggering ROS production after the elimination of

primary solid tumors. This work provides new perspectives for the development of

TrxR inhibitors for cancer therapy and demonstrates a promising avenue of

diselenide-containing micelles for application in the clinical, through combining

excellent antitumor effects for primary solid tumors with post-treatment care.

Acknowledgments

This work was financially supported by the National Basic Research Program of

China (2018YFA0208900), the National Natural Science Foundation of China

(21734006), China National Funds for Distinguished Young Scientists (21425416),

the Foundation for Innovative Research Groups of the National Natural Science

Foundation of China (21821001), the China Postdoctoral Science Foundation

(043200000).

Conflict of interests:

The authors declare no competing financial interest.

Data availability

The data generated during the current study are available from the corresponding
author on reasonable request.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://xxx

References

[1] G. Housman, S. Byler, S. Heerboth, K. Lapinska, M. Longacre, N. Snyder, S.

Sarkar, Drug resistance in cancer: an overview, Cancers 6 (2014) 1769-1792.

[2] M. Karimi, P.S. Zangabad, F. Mehdizadeh, H. Malekzad, A. Ghasemi, S. Bahrami,

H. Zare, M. Moghoofei, A. Hekmatmanesh, M.R. Hamblin, Nanocaged platforms:

Modification, drug delivery and nanotoxicity. Opening synthetic cages to release the

tiger, Nanoscale 9 (2017) 1356-1392.

[3] R. Duncan, The dawning era of polymer therapeutics, Nat. Rev. Drug Discov. 2

(2003) 347-360.

[4] C.D.H. Alarcon, S. Pennadam, C. Alexander, Stimuli responsive polymers for

biomedical applications, Chem. Soc. Rev. 34 (2005) 276-285.

[5] J.C. Yang, Y. Chen, Y.H. Li, X.B. Yin, Magnetic resonance imaging-guided

multi-drug chemotherapy and photothermal synergistic therapy with pH and

NIR-stimulation release, ACS Appl. Mater. Interfaces 9 (2017) 22278-22288.

[6] J. Zhu, M.X. Qiao, Q. Wang, Y.Q. Ye, S. Ba, J.J. Ma, H.Y. Hu, X.L. Zhao, D.W.

Chen, Dual-responsive polyplexes with enhanced disassembly and endosomal escape

for efficient delivery of siRNA, Biomaterials 162 (2018) 47-59.

[7] Y. Liang, W.X. Gao, X.Y. Peng, X. Deng, C.Z. Sun, H.Y. Wu, B. He, Near

infrared light responsive hybrid nanoparticles for synergistic therapy, Biomaterials

100 (2016) 76-90.

[8] C.X. Yue, Y.M. Yang, J. Song, G. Alfranca, C.L. Zhang, Q. Zhang, T. Yin, F. Pan,

J.M. Fuente, D.X. Cui, Mitochondria-targeting near-infrared light-triggered

thermosensitive liposomes for localized photothermal and photodynamic ablation of

tumors combined with chemotherapy, Nanoscale 9 (2017) 11103-11118.

[9] F.F. Xia, W.X. Hou, C.L. Zhang, X. Zhi, J. Cheng, J.M. Fuente, J. Song, D.X. Cui,

pH-responsive gold nanoclusters-based nanoprobes for lung cancer targeted

near-infrared fluorescence imaging and chemo-photodynamic therapy, Acta

Biomaterialia 68 (2018) 308-319.

[10] J.H. Xia, T.Y. Li, C.J. Lu, H.P. Xu, Selenium-containing polymers: Perspectives

toward diverse applications in both adaptive and biomedical materials,

Macromolecules 51 (2018) 7435-7455.

[11] W. Cao, L. Wang, H.P. Xu, Selenium/tellurium containing polymer materials in

nanobiotechnology, Nano Today 10 (2015) 717-736.

[12] H.P. Xu, W. Cao, X. Zhang, Selenium-containing polymers: Promising

biomaterials for controlled release and enzyme mimics, Acc. Chem. Res. 46 (2013)

1647-1658.

[13] N. Ma, Y. Li, H.P. Xu, Z.Q. Wang, X. Zhang, Dual redox responsive assemblies

formed from diselenide block copolymers, J. Am. Chem. Soc. 132 (2010) 442-443.

[14] T. Holler, J. Theriault, R.J. Payne, J. Clark, S. Eski, J.L. Freeman, Prognostic

factors in patients with multiple recurrences of well-differentiated thyroid carcinoma,

J. Oncol. 2009 (2009) 650340.

[15] Y. Kaneda, Therapeutic strategies for controlling the metastasis and recurrence

of cancers: Contribution of drug delivery technologies, Adv. Drug Deliver Rev. 64

(2012) 707-709.

[16] Y.Q. Qi, H. Min, A. Mujeeb, Y.L. Zhang, X.X. Han, X. Zhao, G.J. Anderson, Y.

Zhao, G.J. Nie, Injectable hexapeptide hydrogel for localized chemotherapy prevents

breast cancer recurrence, ACS Appl. Mater. Interfaces 10 (2018) 6972-6981.

[17] Z.Y. Zhang, G.Z. Kuang, S. Zong, S. Liu, H.H. Xiao, X.S. Chen, Sandwich-like

fibers/sponge composite combining chemotherapy and hemostasis for efficient

postoperative prevention of tumor recurrence and metastasis, Adv. Mater. 30 (2018)

30 1803217.

[18] L.W. Zhang, D.Z. Duan, Y.P. Liu, C.P. Ge, X.M. Cui, J.Y. Sun, J.G. Fang,

Highly selective off-on fluorescent probe for imaging thioredoxin reductase in living

cells, J. Am. Chem. Soc. 136 (2014) 226-233.

[19] A. Tuladhar, K.S. Rein, Manumycin A is a potent inhibitor of mammalian

thioredoxin reductase℃1 (TrxR-1), ACS Med. Chem. Lett. 9 (2018) 318-322.

[20] C. Fan, W. Zheng, X. Fu, X. Li, Y.S. Wong, T. Chen, Enhancement of

auranofin-induced lung cancer cell apoptosis by selenocystine, a natural inhibitor of

TrxR1 in vitro and in vivo, Cell Death Dis. 5 (2014) e1191.

[21] F. Zhao, J. Yan, S.J. Deng, L.X. Lan, F. He, B. Kuang, H.H. Zeng, A thioredoxin

reductase inhibitor induces growth inhibition and apoptosis in five cultured human

carcinoma cell lines, Cancer Lett. 236 (2006) 46-53.

[22] P. Wipf, S.M. Lynch, G. Powis, A. Birminghamb, E.E. Englund, Synthesis and
biological activity of prodrug inhibitors of the thioredoxin-thioredoxin reductase

system, Org. Biomol. Chem. 3 (2005) 3880-3882.

[23] M.H. Yoo, X.M. Xu, B.A. Carlson, V.N. Gladyshev, D.L. Hatfield, Thioredoxin

reductase 1 deficiency reverses tumor phenotype and tumorigenicity of lung

carcinoma cells, J. Biol. Chem 281 (2006) 13005-13008.

[24] M.P. Purohit, N.K. Verma, A.K. Kar, A. Singh, D. Ghosh, S. Patnaik, Inhibition

of thioredoxin reductase by targeted selenopolymeric nanocarriers synergizes the

therapeutic efficacy of doxorubicin in MCF7 human breast cancer cells, ACS Appl.

Mater. Interfaces 9 (2017) 36493-36512.

[25] S.B. Ji, W. Cao, Y. Yu, H.P. Xu, Dynamic diselenide bonds: Exchange reaction

induced by visible light without catalysis, Angew. Chem. Int. Ed. 53 (2014)

6781-6785.

[26] L. Raymond, J. Laurent, R.F. Frank, H.V.M. Graça, M.S. Kevin, Synthesis and

reactions of meso-(p-nitrophenyl) porphyrins, Tetrahedron 60 (2004) 2757-2763.

[27] M. Viale, R.Tosto, V. Giglio, G. Pappalardo, V. Oliveri, I. Maric, M.A. Mariggiò,

G. Vecchio, Cyclodextrin polymers decorated with RGD peptide as delivery systems

for targeted anti-cancer chemotherapy, Invest. New Drug. 37 (2019) 771-778.

[28] H.T. Ruan, X.S. Chen, C. Xie, B.B. Li, M. Ying, Y. Liu, M.F. Zhang, X.S. Zhang,

C.Y. Zhan, W.Y. Lu, W.Y. Lu, Stapled RGD peptide enables glioma-targeted drug

delivery by overcoming multiple barriers, ACS Appl. Mater. Interfaces 9 (2017)

17745-17756.

[29] P. Loyer, W. Bedhouche, Z.W. Huang, S. Cammas-Marion, Degradable and

biocompatible nanoparticles decorated with cyclic RGD peptide for efficient drug

delivery to hepatoma cells in vitro, Int. J. Med. Pharm. 454 (2013) 727-737.

[30] Y. Sun, C. Kang, F. Liu, Y. Zhou, L. Luo, H.Z. Qiao, RGD peptide-based target

drug delivery of doxorubicin nanomedicine, Drug Develop. Res. 78 (2017) 283-291.

[31] F.Q. Fan, S.B. Ji, C.X. Sun, C. Liu, Y. Yu, Y. Fu, H.P. Xu,

Wavelength-controlled dynamic metathesis: A light-driven exchange reaction

between disulfide and diselenide bonds, Angew. Chem. Int. Ed. 57 (2018)

16426-16430.

[32] L. Toschi, G. Finocchiaro, S. Bartolini, V. Gioia, F. Cappuzzo, Role of

gemcitabine in cancer therapy, Future Oncol. 1 (2005) 7-17.

[33] A. Gaudin, E. Song, A.R. King, J.K. Saucier-Sawyer, R. Bindra, D. Desmaële, P.

Couvreur, W.M. Saltzman, PEGylated squalenoyl-gemcitabine nanoparticles for the

treatment of glioblastoma, Biomaterials 105 (2016) 136-144.

[34] S.T. Tuccia, A. Kheirolomoom, E.S. Ingham, L.M. Mahakian, S.M. Tam, J.

Foiret, N.E. Hubbard, A.D. Borowsky, M. Baikoghli, R.H. Cheng, K.W. Ferrara,

Tumor-specific delivery of gemcitabine with activatable liposomes, J. Control.

Release 309 (2019) 277-288.

[35] N. Soni, N. Soni, H. Pandey, R. Maheshwari, P. Kesharwani, R.K. Tekade,

Augmented delivery of gemcitabine in lung cancer cells exploring mannose anchored

solid lipid nanoparticles, J. Colloid Interf. Sci. 481 (2016) 107-116.

[36] S.S. Su, Y.P. Ding, Y.Y. Li, Y. Wu, G.J. Nie, Integration of photothermal therapy

and synergistic chemotherapy by a porphyrin self-assembled micelle confers

chemosensitivity in triple-negative breast cancer, Biomaterials 80 (2016) 169-178.

[37] Y.Y. Zhang, D. Yang, H.Z. Chen, W.Q. Lim, F.S.Z. Phua, G.H. An, P.P. Yang, Y.L.

Zhao, Reduction-sensitive fluorescence enhanced polymeric prodrug nanoparticles for

combinational photothermal-chemotherapy, Biomaterials 163 (2018) 14-24.

[38] G.L. Yang, J, Tian, C. Chen, D.W. Jiang, Y.D. Xue, C.C. Wang, Y. Gao, W.A.

Zhang, An oxygen self-sufficient NIR-responsive nanosystem for enhanced PDT and

chemotherapy against hypoxic tumors, Chem. Sci. 10 (2019) 5766-5772.

[39] J.K. Yao, S.S. Kang, J. Zhang, J. Du, Z. Zhang, M. Li, Amphiphilic near-infrared

conjugated polymer for photothermal and chemo combination therapy, ACS Biomater.

Sci. Eng. 3 (2017) 2230-2234.

[40] B.X. Zhang, Y.X. Liu, X.M. Li, J.Q. Xu, J.G. Fang, Small molecules to target the

selenoprotein thioredoxin reductase, Chem. Asian J. 13 (2018) 3593-3600.

[41] D.Z. Duan, B.X. Zhang, J. Yao, Y.P. Liu, J.Y. Sun, C.P. Ge, S.J. Peng, J.G. Fang,

Gambogic acid induces apoptosis in hepatocellular carcinoma SMMC-7721 cells by

targeting cytosolic thioredoxin reductase, Free Radical Bio. Med. 69 (2014) 15-25.

[42] L.H. Wang, Z.Y. Yang, J.N. Fu, H.W. Yin, K. Xiong, Q. Tan, H.W. Jin, J. Li, T.Y.

Wang, W.C. Tang, J. Yin, G.X. Cai, M. Liu, S. Kehr, K. Becker, H.H. Zeng, Ethaselen:

A potent mammalian thioredoxin reductase 1 inhibitor and novel organoselenium

anticancer agent, Free Radical Bio. Med. 52 (2012) 898-908.

[43] A.S. Freitas, A.S. Prestes, C. Wagner, J.H. Sudati, D. Alves, L.O. Porciúncula,

I.J. Kade, J.B.T. Rocha, Reduction of diphenyl diselenide and analogs by mammalian

thioredoxin reductase is independent of their gluthathione peroxidase-like activity: A

possible novel pathway for their antioxidant activity, Molecules 15 (2010) 7699-7714.

[44] Z.H. Song, Y.Z. Chang, H.H. Xie, X.F. Yu, P.K. Chu, T.F. Chen, Decorated

ultrathin bismuth selenide nanosheets as targeted theranostic agents for in vivo

imaging guided cancer radiation therapy, NPG Asia Mater. 9 (2017) e439.

[45] W.J. Ge, K.M. Zhao, X.W. Wang, H.Y. Li, M. Yu, M.M. He, X.T. Xue, Y.F.

Zhu, C. Zhang, Y.W. Cheng, S.J. Jiang, Y. Hu, iASPP is an antioxidative factor and

drives cancer growth and drug resistance by competing with Nrf2 for Keap1 binding,

Cancer Cell 32 (2017) 561-573.

[46] M. Nishimoto, M. Sakaue, S. Hara, Short-interfering RNA-mediated silencing of

thioredoxin reductase 1 alters the sensitivity of HeLa cells toward cadmium, Biol.

Pharm. Bull. 29 (2006) 543-546.

[47] L. Gan, X.L. Yang, Q. Liu, H.B. Xu, Inhibitory effects of thioredoxin reductase

antisense RNA on the growth of human hepatocellular carcinoma cells, J. Cell.

Biochem. 96 (2005) 653-664.

[48] H.H. Zeng, L.H. Wang, Targeting thioredoxin reductase: Anticancer agents and

chemopreventive compounds, Med. Chem. 6 (2010) 286-297.

[49] L.W. Zhong, E.S.J. Arnér, A. Holmgren, Structure and mechanism of

mammalian thioredoxin reductase: The active site is a redox-active

selenolthiol/selenenylsulfide formed from the conserved cysteine-selenocysteine

sequence, Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 5854-5859.

[50] J. Lu, E.H. Chew, A. Holmgren, Targeting thioredoxin reductase is a basis for

cancer therapy by arsenic trioxide, Proc. Natl. Acad. Sci. U.S.A. 104 (2007)
12288-12293.

[51] S.B. Ji, H.E. Mard, M. Smet, W. Dehaen, H.P. Xu, Selenium containing

macrocycles: Transformation between Se-N/Se-S/Se-Se bonds, Sci. China Chem. 60

(2017) 1191-1196.
Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: