Redox responsive paclitaxel dimer for programmed drug release and selectively killing cancer cells

Article abstract of DOI:10.1016/j.jcis.2020.07.086

Redox stimulus responsive drug delivery systems have been widely investigated and proved to be promising prospects for efficient cancer therapy due to the abnormal high level of reactive oxygen species and glutathione in tumor microenvironment. Herein, three paclitaxel dimers (named as PTX₂-R, R = S, Se and Te) bridged with alkyl sulfide, selenide or telluride are synthesized. These dimers can self-assemble into stable uniform nanoparticles (named as PTX₂-R NPs, R = S, Se and Te) with impressively high drug loading. As expected, sulfur/selenium/tellurium bonds exhibit different redox responsiveness, thereby affecting the drug release and cytotoxicity. Of note, tellurium bridged paclitaxel dimer shows ultra-sensitivity to hydrogen peroxide, which rapidly cleaves into two paclitaxel under the subsequent dithiothreitol stimulation. Our findings provide deep insight into the redox sensitivity of chalcogenide elements and offer the rational design strategies to biologically redox condition for programmed drug release.

Full text of DOI:10.1016/j.jcis.2020.07.086

Journal of Colloid and Interface Science 580 (2020) 785–793
Contents lists available at ScienceDirect
Journal of Colloid and Interface Science
journal homepage: www.elsevier.com/locate/jcis
Redox responsive paclitaxel dimer for programmed drug release
and selectively killing cancer cells
a,b,
Rui Xia a,b, Qing Pei a,b, Jian Wang a,b, Zhanfeng Wang c, Xiuli Hu a, , Zhigang Xie
⇑
⇑
a State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun, Jilin 130022,
PR China
b University of Science and Technology of China, Hefei 230026, PR China
c Department of Neurosurgery, China-Japan Union Hospital of Jilin University, Changchun, Jilin 130033, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
ꢀ
ꢀ
ꢀ
Three paclitaxel dimers with alkyl
sulfide, selenide or telluride are
synthesized.
Tellurium linked paclitaxel dimer
exhibits superior redox
responsiveness.
Tellurium linked dimer can
effectively release paclitaxel under
redox stimulation.
Tellurium linked paclitaxel dimer
exhibits selective killing cancer cells
ability.
a r t i c l e i n f o
a b s t r a c t
Article history:
Redox stimulus responsive drug delivery systems have been widely investigated and proved to be
promising prospects for efficient cancer therapy due to the abnormal high level of reactive oxygen species
and glutathione in tumor microenvironment. Herein, three paclitaxel dimers (named as PTX2-R, R = S, Se
and Te) bridged with alkyl sulfide, selenide or telluride are synthesized. These dimers can self-assemble
into stable uniform nanoparticles (named as PTX2-R NPs, R = S, Se and Te) with impressively high drug
loading. As expected, sulfur/selenium/tellurium bonds exhibit different redox responsiveness, thereby
affecting the drug release and cytotoxicity. Of note, tellurium bridged paclitaxel dimer shows ultra-
sensitivity to hydrogen peroxide, which rapidly cleaves into two paclitaxel under the subsequent dithio-
threitol stimulation. Our findings provide deep insight into the redox sensitivity of chalcogenide elements
and offer the rational design strategies to biologically redox condition for programmed drug release.
Ó 2020 Elsevier Inc. All rights reserved.
Received 23 April 2020
Revised 16 July 2020
Accepted 17 July 2020
Available online 21 July 2020
Keywords:
Chemotherapy
Redox stimulus responsive
Tellurium bridged paclitaxel
1. Introduction
Today, cancer is still a major problem disturbing mankind [1].
⇑
Corresponding authors at: State Key Laboratory of Polymer Physics and
Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of
Sciences, 5625 Renmin Street, Changchun, Jilin 130022, PR China (Z. Xie).
E-mail addresses: lily@ciac.ac.cn (X. Hu), xiez@ciac.ac.cn (Z. Xie).
People spend a lot to fight with cancer, but cancer mortality is still
increasing year by year. Despite the booming researches on new
cancer therapies, such as phototherapy [2] and immunotherapy
https://doi.org/10.1016/j.jcis.2020.07.086
0021-9797/Ó 2020 Elsevier Inc. All rights reserved.

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R. Xia et al. / Journal of Colloid and Interface Science 580 (2020) 785–793
[3], traditional chemotherapies still play important role on cancer
treatment [4]. However, the chemotherapies [5] are greatly limited
by the drug delivery efficiency [6] and the serious side effects [7].
To solve these problems, nano drug-delivery systems (DDS) with
high drug delivery efficiency have gained considerable attention
[8–11]. A widely accepted concept is that ideal DDS could keep
the cargoes from premature leakage during the delivery process
or turn the drug into an inactive state with excellent biocompati-
bility. While entering the tumor area, DDS will readily release
the payloads in responsive and controlled manner or effective
drugs release from the prodrug stimulated by tumor microenviron-
ment [12–17].
Compared with normal cells, tumor cells usually possess higher
level of reactive oxygen species (ROS) and glutathione (GSH), lead-
ing to different redox potential, which has attracted much atten-
tion for designing tumor specific responsive DDSs [18–22]. A
verity of ROS-sensitive moieties, such as thioketals, thioethers,
phenylboronic acid/ester, selenium, tellurium, peroxalate ester,
and GSH responsive disulfide groups have been selected and used
to design tumor redox heterogeneity-triggered DDSs [23–27].
Inspired by the redox sensitivity of sulfur bonds, selenium and tel-
lurium have also attracted increasing attention for higher sensitiv-
ity to ROS due to their lower electronegativity than sulfur [28–30].
In previous reports, several sulfur or selenium containing polymers
can respond to both ROS and GSH, whereby enhance the therapeu-
tic efficacy [31–34]. Some selenium containing polymers are able
to produce ROS and induce apoptosis of cancer cells [35–36]. Tel-
lurium containing chemical bonds have lower bond energies than
selenium bond, which endows it with more sensitivity to ROS
[37]. However, tellurium-based materials have been rarely investi-
gated, especially in the field of prodrugs or dimeric drugs [38–40].
Here, three paclitaxel (PTX) dimers [41–43] bridged with sulfur,
selenium, or tellurium with the same length of carbon chain are
synthesized by conjugating the corresponding diacid with pacli-
taxel, abbreviated as PTX2-S, PTX2-Se, PTX2-Te, respectively
(Scheme 1). The obtained dimeric prodrugs can self-assemble into
uniform nanoparticles (named as PTX2-R NPs, R = S, Se and Te) with
impressively high drug loading. The influence of sulfur/selenium/
tellurium bonds on the self-assembly, stability, ROS and GSH
induced drug release, cytotoxicity are compared systematically.
2. Materials and methods
2.1. Materials
Paclitaxel (>99%) was purchased from Dalian Meilun Biotech-
nology Co., Ltd. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride (EDCꢁHCl) (>98%), 4-dimethylaminopyridine
(DMAP) (>98%) were purchased from Energy Chemical Co., Ltd.
Selenium and Tellurium (>99.9%) powders were purchased from
Aladdin Co., Ltd. Sodium borohydride (>98%) were purchased from
Energy Chemical Co., Ltd. 6-bromohexanoic acid (>98%) were pur-
chased from Heowns Co., Ltd. Dithiothreitol (DTT) were purchased
from Aladdin Co., Ltd. Tubulin-Tracker Red were obtained from
Jiangsu KeyGEN Biotechnology Co., Ltd. Native lysis Buffer (RIPA)
was purchased from Solarbio.
2.2. Characterization
Transmission electron microscope (TEM) images were carried
out on a JEOL JEM–1011 (Japan) at the accelerating voltage of
100 kV. Bruker AV400 M was adopted to recorded proton nuclear
magnetic resonance (1H NMR) spectra at 25 °C. Malvern Zeta-
sizer Nano was used to determine size, size distribution. Dimers
detection were carried out on High performance liquid chromatog-
raphy (HPLC) (SHIMADAZU, Japan). The mass spectrum (MS) anal-
yses were performed on a LTQ ion trap mass spectrometer
Scheme 1. Schematic illustration of the sulfur, selenium and tellurium-bridged prodrug assemblies and the proposed redox dual-responsive drug release in tumor cells.

R. Xia et al. / Journal of Colloid and Interface Science 580 (2020) 785–793
787
(Finnigan, USA). Endocytosis test was carried out on confocal laser
scanning microscope (CLSM) (Zeiss LSM 700, Zurich, Switzerland).
1H), 1.68 (s, 3H), 1.63 – 1.59 (m, 3H), 1.34 (dt, J = 8.2, 7.0 Hz,
2H), 1.23 (s, 3H), 1.11 (d, J = 17.6 Hz, 3H). Electrospray ionization
(ESI)/MS for PTX2-Se: 1981.07 calculated, 2015.6 found, [M + Cl]-.
For PTX2-Te, 1H NMR (500 MHz, CDCl3) d 8.16 – 8.10 (m, 2H),
7.73 (d, J = 7.3 Hz, 2H), 7.61 (t, J = 7.4 Hz, 1H), 7.51 (dd, J = 14.9,
7.5 Hz, 3H), 7.45 – 7.31 (m, 7H), 6.89 (d, J = 9.2 Hz, 1H), 6.31 –
6.22 (m, 2H), 5.96 (dd, J = 9.2, 3.1 Hz, 1H), 5.68 (d, J = 7.1 Hz,
1H), 5.51 (d, J = 3.3 Hz, 1H), 4.97 (d, J = 8.3 Hz, 1H), 4.47 – 4.41
(m, 1H), 4.32 (d, J = 8.4 Hz, 1H), 4.20 (d, J = 8.5 Hz, 1H), 3.81 (d,
J = 7.0 Hz, 1H), 2.52 (dt, J = 9.7, 6.6 Hz, 3H), 2.45 (s, 3H), 2.43 –
2.32 (m, 3H), 2.22 (d, J = 5.5 Hz, 3H), 2.19 – 2.12 (m, 1H), 1.96 –
1.85 (m, 4H), 1.80 (d, J = 4.4 Hz, 1H), 1.72 – 1.64 (m, 5H), 1.59 –
1.54 (m, 1H), 1.32 (dt, J = 15.0, 7.4 Hz, 2H), 1.21 (d, J = 15.6 Hz,
3H), 1.11 (d, J = 18.2 Hz, 3H). Electrospray ionization (ESI)/MS for
PTX2-Te: 2029.71 calculated, 2065.7 found, [M + Cl]ꢂ.
2.3. Synthesis of materials
Synthesis of HOOCC5-SH and S[(CH2)5COOH]2. 6-bromohexanoic
acid (12.0 mmol, 2.34 g) and thiourea (17.7 mmol, 1.35 g) were dis-
solved in 30 mL ethanol. The mixture was refluxed at 85 °C under
nitrogen atmosphere for 20 h. Then ethanol was evaporated and
pH was adjusted to 2.0. Product was extracted with ethyl acetate
and dried with magnesium sulfate. After vacuum distillation, yel-
lowish liquid was obtained. 1H NMR (400 MHz, CDCl3) d 11.76 (s,
1H), 2.48 (q, J = 7.4 Hz, 2H), 2.32 (t, J = 7.4 Hz, 2H), 1.68 – 1.52
(m, 4H), 1.46 – 1.35 (m, 2H), 1.31 (t, J = 7.8 Hz, 1H).
The yellowish liquid was dissolved in sodium hydroxide solu-
tion (7.5 M, 25 mL). 6-bromohexanoic acid (18.8 mmol, 3.66 g)
was dissolved in water and dropwise added to the prepared solu-
tion at 0 °C. Then the reaction was kept at nitrogen atmosphere
at 45 °C and stirred for 24 h. After that, hydrochloric acid solution
was added and pH was adjusted to 1.0. The product was extracted
with ethyl acetate and dried with magnesium sulfate. White solid
was obtained. 1H NMR (500 MHz, DMSO) d 11.97 (s, 1H), 2.46 (t,
J = 7.3 Hz, 2H), 2.23 – 2.15 (m, 2H), 1.54 – 1.45 (m, 4H), 1.37 –
1.29 (m, 2H).
2.4. Preparation of nanoparticles (NPs)
3 mg of PTX2-S was dissolved in 3 mL of tetrahydrofuran. This
solution was added dropwise into 10 mL of pure water in ten min-
utes, then stirred for overnight to volatilize tetrahydrofuran. At last
the crude product was dialyzed against water with a 3500D dialy-
sis bag to removal residual tetrahydrofuran. PTX2-Se and PTX2-Te
NPs were prepared with the same method.
Synthesis of Se[(CH2)5COOH]2 and Te[(CH2)5COOH]2. These two
compounds were synthesized with a similar method. For the syn-
thesis of Te[(CH2)5COOH]2, tellurium powders (800 mg, 1 eq) were
dispersed in water, sodium borohydride (600 mg, 2.5 eq) were
added. The reaction was heated to 45 °C under nitrogen atmo-
sphere. The dark solution gradually turned to pink and at last chan-
2.5. DTT or H2O2-triggered PTX release
The three kinds of dimers (60
solutions (acetonitrile/water, v/v = 1/1, 500
DTT, 1 mM H2O2 or 100 M H2O2 at 37 °C. At different points in
time, 500 L acetonitrile was added and solution was centrifuged
at 14,000 r/min for 5 min. 20 L supernatant was detected by HPLC
lg) were separately dissolved in
l
L) containing 10 mM
l
ged to
a colorless transparent solution in 4 h. Then 6-
l
bromohexanoic acid (2.7 g, 2.2 eq) in tetrahydrofuran was added
and stirred overnight. After reaction, tetrahydrofuran was moved
away and pH was adjusted to 4.0. Precipitation was collected and
washed with water and chloroform. Light yellow powder was
obtained. 1H NMR (400 MHz, DMSO) d 11.96 (s, 1H), 2.59 (t,
J = 7.5 Hz, 2H), 2.19 (t, J = 7.3 Hz, 2H), 1.67 (dt, J = 14.9, 7.5 Hz,
2H), 1.51 (dt, J = 15.0, 7.3 Hz, 2H), 1.32 (dt, J = 14.8, 7.3 Hz, 2H).
Se[(CH2)5COOH]2 was synthesized in a similar way. 1H NMR
(400 MHz, DMSO) d 11.96 (s, 1H), 2.55 – 2.50 (m, 2H), 2.19 (t,
J = 7.3 Hz, 2H), 1.63 – 1.53 (m, 2H), 1.49 (dd, J = 15.1, 7.5 Hz,
2H), 1.38 – 1.29 (m, 2H).
l
to characterize the cleavage and hydrolysis of the dimers.
3. Results and discussion
3.1. Preparation and characterization of prodrug NPs
First three paclitaxel prodrug dimers were synthesized by using
similar approach. Three diacid intermediates R(C5COOH)2 (R = S, Se
or Te) were prepared and then conjugated with paclitaxel through
esterification reaction. Scheme S1 shows the synthetic routes of
sulfur/selenium/tellurium bond bridged diacid and the corre-
sponding paclitaxel dimers. The obtained PTX2-S, PTX2-Se, PTX2-
Te were purified via a silica gel column with high yields (>80%)
and their chemical structures were verified by nuclear magnetic
resonance hydrogen spectrum (1H NMR) (Figs. S1–7). The charac-
teristic peak of 20-hydroxyl group of PTX at 3.6 ppm disappeared,
verifying the completion of esterification reaction and the reaction
site was at the 20-hydroxyl group of PTX. The particular peak values
in mass spectrometry were consistent with that of theoretical cal-
culation (Figs. S8–10), further proved the successful synthesis of
these three dimers.
These obtained dimers could self-assemble into nanoparticles
in aqueous solution by one-step nanoprecipitation method [44].
The three kinds of nanoparticles (PTX2-R NPs, R = S, Se and Te) dis-
play homogeneous spherical structures, with diameters of
179.9 nm, 187.5 nm, and 204.9 nm, respectively, as measured by
transmission electron microscopy (Fig. 1A, Fig. S11) and dynamic
light scattering (DLS) (Fig. 1B). Detailed hydrodynamic size and
polydispersity index (PDI) of PTX2-S NPs, PTX2-Se NPs and PTX2-
Te NPs are shown in Table S1. As the prodrugs themselves act as
both the carriers and payloads, the drug loading capacity of
PTX2-R NPs is impressively high (>84 wt%). These three nanoparti-
cles may have comparable endocytosis capacity due to their
Synthesis of PTX2-S, PTX2-Se and PTX2-Te dimers. These three
kinds of dimers were synthesized by esterification. Specific steps
were referred in our previous work [49].
For PTX2-S, 1H NMR (400 MHz, CDCl3) d 8.18 – 8.10 (m, 2H),
7.77 – 7.70 (m, 2H), 7.61 (t, J = 7.4 Hz, 1H), 7.56 – 7.48 (m, 3H),
7.47 – 7.30 (m, 7H), 6.88 (d, J = 9.2 Hz, 1H), 6.30 (s, 1H), 6.25 (t,
J = 8.9 Hz, 1H), 5.96 (d, J = 9.2, 3.2 Hz, 1H), 5.68 (d, J = 7.1 Hz,
1H), 5.51 (d, J = 3.3 Hz, 1H), 4.98 (d, J = 7.9 Hz, 1H), 4.49 – 4.41
(m, 1H), 4.32 (d, J = 8.4 Hz, 1H), 4.20 (d, J = 8.3 Hz, 1H), 3.81 (d,
J = 7.0 Hz, 1H), 2.61 – 2.53 (m, 1H), 2.51 (d, J = 4.1 Hz, 1H), 2.45
(d, J = 4.2 Hz, 3H), 2.43 – 2.31 (m, 4H), 2.22 (s, 3H), 2.16 (dd,
J = 15.5, 8.8 Hz, 1H), 1.98 – 1.83 (m, 4H), 1.79 (s, 1H), 1.68 (s,
3H), 1.57 – 1.47 (m, 3H), 1.36 (dd, J = 15.6, 8.7 Hz, 2H), 1.23 (s,
3H), 1.13 (s, 3H). Electrospray ionization (ESI)/MS for PTX2-S:
1934.17 calculated, 1973.1 found, [M + K]+.
For PTX2-Se, 1H NMR (500 MHz, CDCl3) d 8.16 – 8.11 (m, 2H),
7.75 – 7.70 (m, 2H), 7.61 (t, J = 7.4 Hz, 1H), 7.50 (dt, J = 18.0,
9.2 Hz, 3H), 7.44 – 7.31 (m, 7H), 6.87 (d, J = 9.2 Hz, 1H), 6.30 (s,
1H), 6.25 (t, J = 8.6 Hz, 1H), 5.96 (dd, J = 9.2, 3.2 Hz, 1H), 5.68 (d,
J = 7.1 Hz, 1H), 5.51 (d, J = 3.3 Hz, 1H), 4.97 (d, J = 8.0 Hz, 1H),
4.47 – 4.41 (m, 1H), 4.32 (d, J = 8.4 Hz, 1H), 4.20 (d, J = 8.5 Hz,
1H), 3.81 (d, J = 7.0 Hz, 1H), 2.56 (dd, J = 15.9, 9.6, 6.6 Hz, 1H),
2.52 – 2.47 (m, 2H), 2.45 (s, 4H), 2.43 – 2.32 (m, 3H), 2.22 (s,
3H), 2.19 – 2.11 (m, 1H), 1.96 – 1.84 (m, 4H), 1.72 (d, J = 5.4 Hz,

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R. Xia et al. / Journal of Colloid and Interface Science 580 (2020) 785–793
Fig. 1. (A) TEM image of PTX2-Te NPs. (B) Size distribution of PTX2-R NPs. (C) Size changes of PTX2-R NPs in water for a week. (D) HPLC analysis of PTX2-R dimers.
similar structure and particle size. The colloidal stabilities of PTX2-
R NPs were investigated and the result proves that they are stable
in water for a week with a slight change of size and size distribu-
tion (Fig. 1C and Fig. S12). High performance liquid chromatogra-
phy (HPLC) was used to determine the content of PTX2-R NPs.
Results display that these dimers possess various peak elution
times (Fig. 1D).
bond. We guess that the largest atomic radius and weakest elec-
tronegativity endow tellurium with the lowest bond energy, lead-
ing to its superior oxidation sensitivity. This ultrasensitive
character has important potential in drug delivery. In previously
reported work [48], the oxidation of sulfur or selenium show excel-
lent hydrophilicity and could facilitate the hydrolysis of the adja-
cent ester bond and promote drug release. However, in the
present study, no apparent PTX was detected for all the three
dimers even after incubation with H2O2 for 24 h. It’s probably
because the five methylene between PTX and chalcogen signifi-
cantly reduce the interaction between sulfur, selenium or tel-
lurium atoms and the ester bonds. The chalcogen in PTX dimer
could only be oxidized by H2O2. After oxidation, the valence states
of sulfur, selenium or tellurium were elevated. So the individual
oxidative stimulation could not promote drug release or dimer
cleavage. But this kind of chemical structures may have potential
in solubility switchable materials.
Next, dithiothreitol (DTT, a prevailing simulating of GSH) was
employed to monitor the degradation of dimers under reduction
condition. As shown in Fig. 2D–F and Fig. S15, in the presence of
10 mM DTT, >95% of PTX2-Te degraded in 24 h, by contast, the
degradation rate of PTX2-S and PTX2-Se is less than 20%. Consider-
ing the extremely oxidizable nature of tellurium, we studied the
interaction between PTX2-Te and DTT in nitrogen atmosphere at
37 °C. As shown in Fig. S16, PTX2-Te only exhibits less than 20%
of degradation, meaning that PTX2-Te alone has very weak interac-
tion with DTT. Combining these experimental results, we propose
that PTX2-Te may be first oxidized to PTX2-TeO or PTX2-TeO2 in
the air at 37 °C, then the oxidation products easily react with the
thiol group of DTT, leading to the degradation of PTX2-Te
(Fig. 3A). To further prove this point, we tested the mass spectra
3.2. DTT and GSH triggered release of PTX and speculative mechanism
As previously reported, PTX dimer bridged with thioether linker
could dually respond to the tumor redox heterogeneity and gradu-
ally release PTX for chemotherapy [45–47]. We next evaluate the
change of PTX2-S, PTX2-Se, PTX2-Te dimers under the simulated
redox environmentꢁH2O2 and DTT were selected as oxidation and
reduction model triggers, respectively. HPLC was employed to
record the change of these dimers under redox stimulations. After
incubation with 1 mM H2O2 for different time points, sulfur/sele-
nium/tellurium bonds present in the dimers are gradually oxidized
to form hydrophilic sulfoxide, selenoxide and tellurium oxide
respectively (Fig. 2A–C). Especially for PTX2-Te dimer, it was
almost completely oxidized in an hour. Fig. S13 are the oxidation
rate curves of PTX2-S, PTX2-Se and PTX2-Te after incubated with
1 mM H2O2 for 24 h at 37 °C. We then explored the oxidation rate
of three dimers incubated with 100 mM H2O2 which was closer to
real cancer cell environment (Fig. S14). Compared with that incu-
bated in 1 mM H2O2 for 24 h at 37 °C, only 10% of PTX2-S and
50% of PTX2-Se dimers were oxidized while PTX2-Te is still com-
pletely oxidized in an hour. These results reveal that the oxidation
rate of dimers is related to the concentration of H2O2 and tellurium
bond is much more sensitive to H2O2 than sulfur and selenium

R. Xia et al. / Journal of Colloid and Interface Science 580 (2020) 785–793
789
Fig. 2. HPLC analysis of PTX2-S (A) PTX2-Se (B) and PTX2-Te (C) dimers in the presence of 1 mM H2O2 for 24 h at 37 °C. HPLC analysis of PTX2-S (D) PTX2-Se (E) and PTX2-Te (F)
dimers in the presence of 10 mM DTT for 24 h at 37 °C.
Fig. 3. (A) Proposed mechanism of the degradation of PTX2-Te dimer stimulated by H2O2 and DTT. (B) Mass spectrum of PTX2-Te with or without incubated with 10 mM DTT
for 24 h at 37 °C.
of these dimers after incubation with DTT for 24 h. According to
Figs. S17 and S18, no significant degradation was observed for
PTX2-S and PTX2-Se dimers. While the characteristic peak of
PTX2-Te almost disappeared in the mass spectrum (Fig. 3B) and
the molecular weight changed to 888.5 [PTX + Na]+, 986.5
[M + 2H]2+, 1002.5 [Mmonoxide + 2H]2+ and 1018.5 [Mdioxide + 2H]2+
(M represents PTXC6-SH). The result matches well with HPLC, con-
firming the formation of sulfoxide/sulphone and further proving

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R. Xia et al. / Journal of Colloid and Interface Science 580 (2020) 785–793
that the interaction between DTT and oxidized tellurium promotes
the release of PTX. All the above results show that PTX2-Te exhibits
higher oxidation sensitivity than PTX2-S and PTX2-Se, and its oxi-
dation products are more likely to be degraded under reductive
stimulation. Nevertheless, PTX2-S and PTX2-Se are not easily oxi-
dized in the air and the drug release ability is closely related to
the distance of chalcogen atom from the ester bond. All these char-
acteristics may make PTX2-Te an effective redox responsive dimer,
especially with methylene as little as possible.
we first need to investigate the biosafety of tellurium-containing
diacid. As shown in Fig. 5A, Hela cells showed normal proliferative
state and the cell viability was >95% even after incubation with
these three diacids for 48 h. So these diacids have negligible cyto-
toxicity and can be well applied in nanomedicine therapy. We then
tested the cytotoxicity of PTX2-R NPs against HeLa cells. In Fig. 5B
and C, PTX2-Te NPs showed higher cytotoxicity than PTX2-S NPs
and PTX2-Se NPs. The cell viability of PTX2-Te NPs group is less
than 16%, while it is >70% and 55% for the groups of PTX2-S NPs
and PTX2-Se NPs, at the same concentration of 12.5
lM. All these
results are consistent well with those of in vitro release experi-
ments. In summary, due to the ultrasensitive oxidative sensitivity
of PTX2-Te, ingested into Hela cells can be easily oxidized by high
intracellular ROS and further react with GSH to release PTX. But
this process is difficult for insensitive PTX2-S and PTX2-Se, so they
show unapparent cytotoxicity. It is worth mentioning that PTX2-Te
NPs showed comparable cytotoxicity to Taxol, meaning that PTX2-
Te could be cleaved into PTX to exert its cytotoxicity. Thus PTX2-Te
dimer maybe a potential effective prodrug. Furthermore, cells
incubated with PTX2-Te NPs for 48 h show obvious lower cell via-
bility than that for 24 h. This result may be due to different endo-
cytosis capacity of nanoparticles and the drug release process is
time-dependent. Normal cells (L929 cells) were also incubated
with NPs or Taxol as comparison. Similarly, the three diacids
showed no significant cytotoxicity against L929 cells (Fig. 5D). Dif-
ferently, PTX2-Te NPs show significantly lower cytotoxicity against
L929 cells than that against HeLa cells, at the same concentrations
of PTX2-Te NPs (Fig. 5B, C, E and F). However, Taxol maintained its
significant cytotoxicity to both L929 and HeLa cells. This result may
be due to the different level of redox substances in the two cell
lines. Additionally, cytotoxicity of Hela cells incubated with
PTX2-R NPs under hypoxic condition was explored (Fig. S22).
PTX2-Te NPs still showed the most superior cytotoxicity among
the three groups. It is worth mentioning that cytotoxicity of
PTX2-Te NPs under hypoxic condition can be comparable with that
3.3. Cellular uptake of PTX2-R NPs
To investigate cellular uptake of PTX2-R NPs, fluorescent dye
boron dipyrromethene (BDP) was employed as a marker. Molecular
structure of BDP is shown in Fig. S19. Fig. S20 shows the UV–Vis
absorption and fluorescence spectra of BDP in acetone. BDP exhibits
strong red fluorescence with maximum emission at 668 nm under
the excitation of 555 nm laser. After co-assembly with PTX2-S,
PTX2-Se, or PTX2-Te dimers, BDP was encapsulated into PTX2-R/
BDP NPs. Fig. S21 shows the particle size and polydispersity index
of PTX2-R/BDP NPs. PTX2-R/BDP NPs possess the similar sizes with
PTX2-R NPs. After incubation with HeLa cells for different time, the
red fluorescence of PTX2-R/BDP NPs was employed to monitor the
endocytosis process and the blue fluorescence of 40,6-diamidino-2-
phenylindole (DAPI) was used to localize the nucleus. As shown in
Fig. 4A, the internalization of PTX2-R/BDP NPs by HeLa cells was time
dependent. As time prolongs, the red fluorescence significantly
enhanced. It is worth mentioning that PTX2-R/BDP NPs showed little
difference in endocytosis, which may be due to their similar struc-
tures and sizes. Fig. 4B is the statistic of their average fluorescence
intensity. The effect of temperature on endocytosis was further
explored. As shown in Fig. 4C, cells incubated with PTX2-Te/BDP
NPs at 37 °C exhibit much stronger fluorescence than that at 4 °C,
indicating the endocytosis is energy-dependent.
under normal oxygen condition. In detail, at the dose of 12.5
lM,
3.4. In vitro cytotoxicity of PTX2-R NPs
the cell viability is 15.2% for normal oxygen group and 25.3% for
hypoxic group. This result further proves that tellurium based pro-
drug may have excellent redox responsiveness to endogenous
redox substances. Taking all into consideration, we believe that
Herein, Hela cells were employed to assess the cytotoxicity of
PTX2-R NPs by using MTT assays. Because tellurium-containing
compounds have been seldom investigated in cancer therapy, so
Fig. 4. Study on endocytosis of PTX2-R/BDP NPs (n = 3). (A) Endocytosis contrast of PTX2-R/BDP NPs at different time points. Scale bars, 20
comparison of PTX2-R/BDP NPs with different endocytosis time. (C) Comparison of endocytosis at 4 °C and 37 °C of PTX2-Te/BDP NPs. Scale bars, 20
l
m. (B) Fluorescence intensity
m.
l

R. Xia et al. / Journal of Colloid and Interface Science 580 (2020) 785–793
791
Fig. 5. In vitro cytotoxicity against HeLa cells and L929 cells (n = 4). Cytotoxicity of sulfur, selenium and tellurium based dihexanoic acid against Hela cells (A) and L929 cells
(D) after incubated for 48 h. Cytotoxicity of PTX2-S NPs, PTX2-Se NPs and PTX2-Te NPs against Hela cells after incubated for 24 h (B) and 48 h (C). Cytotoxicity of PTX2-S NPs,
PTX2-Se NPs and PTX2-Te NPs against L929 cells after incubated for 24 h (E) and 48 h (F).
Fig. 6. Fluorescence images of Calcein-AM and propidium iodide stained HeLa cells and CLSM images of microtubule. (A) Fluorescence images of Calcein-AM and propidium
iodide stained HeLa cells after treatment with three kinds of nanoparticles and Taxol for 48 h, respectively. Scale bars, 100
after incubation with PBS, PTX2-S NPs, PTX2-Se NPs, PTX2-Te NPs and Taxol for 12 h. Scale bars, 20 m.
lm. (B) CLSM images of microtubule of HeLa cells
l
tellurium containing dimer has a faster degradation rate at the
stimulation of overproduced ROS and GSH in tumor cells, leading
to selective cytotoxicity against tumor cells. In normal cells, rela-
tively low level of ROS and GSH limit the release of PTX from
dimers. However, Taxol has no redox response, its high toxicity
to normal cells usually would cause severe side effect. HeLa cell
extracts were utilized to further investigate the degradation of pro-
drugs. As shown in Fig. S23, after incubated with cell extracts for
24 h, only 3% of PTX2-Te dimers exists. As a comparison in
Fig. S25, 90% of PTX2-Te dimers still exist when incubated in phos-
phate buffer saline (PBS) for 24 h, meaning the rapid degradation of
PTX2-Te nanodrugs in cancer cell extracts. It is worth mentioning
that the elution time of PTX2-S and PTX2-Se are coincided with cell
extracts, so we failed to get the degradation data of PTX2-S and
PTX2-Se nanodrugs incubated with the extract. Figs. S24 and S26
are HPLC analysis of PTX2-S NPs, PTX2-Se NPs and PTX2-Te NPs
incubated with cell extracts or PBS. All the cytotoxicity tests above
proved that tellurium containing dimer have excellent redox
responsiveness and it shows highly selective cytotoxicity against
tumor cells, which may be effective to reduce the adverse side
effects.
3.5. Calcein-AM/PI studies and the immunostaining assays
Cell live/death staining experiment was conducted to further
verify the cytotoxicity of PTX2-R NPs against Hela cells. Compared
with the control groups, cells incubated with PTX2-Te NPs and
Taxol for 48 h showed the strongest red fluorescence from propid-

792
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4. Conclusions
In conclusion, three PTX dimers (PTX2-R, R = S, Se and Te) were
designed and synthesized using alkyl sulfide, selenide or telluride
as linkages and the corresponding nanoparticles (PTX2-R NPs,
R = S, Se and Te) were obtained. PTX2-R NPs possess robust stability
and high content of PTX, the drug content is far more than that of
other nanoparticle formulations [50,51]. As we hypothesized,
PTX2-Te dimer exhibits preferable redox dual-responsive capability
compared with sulfur and selenium-based paclitaxel dimers.
Uniquely, tellurium-based dimer can only work under the syner-
getic stimulation of ROS and GSH displaying a different mechanism
as reported [48]. Importantly, cell cytotoxicity test further reveals
that PTX2-Te NPs shows comparable cytotoxicity to Taxol and it dis-
plays high selective cytotoxicity against tumor cells, which is sel-
dom seen in literature [52]. Our work gives new insight into
tellurium-based redox responsive materials and may provide possi-
bilities for the development of hypersensitive DDS for cancer
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CRediT authorship contribution statement
Rui Xia: Methodology, Investigation, Software, Writing - origi-
nal draft. Qing Pei: Validation. Jian Wang: Validation. Zhanfeng
Wang: Resources. Xiuli Hu: Conceptualization, Resources, Supervi-
sion, Writing - review & editing, Data curation. Zhigang Xie:
Resources, Supervision, Writing - review & editing.
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Declaration of Competing Interest
[23] C. Luo, J. Sun, D. Liu, B. Sun, L. Miao, S. Musetti, J. Li, X. Han, Y. Du, L. Li, L.
Huang, Z. He, Self-assembled redox dual-responsive prodrug-nanosystem
formed by single thioether-bridged paclitaxel-fatty acid conjugate for cancer
chemotherapy, Nano Lett. 16 (2016) 5401–5408.
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
[24] B. Sun, C. Luo, X. Zhang, M. Guo, M. Sun, H. Yu, Q. Chen, W. Yang, M. Wang, S.
Zuo, P. Chen, Q. Kan, H. Zhang, Y. Wang, Z. He, J. Sun, Probing the impact of
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Acknowledgements
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This work was financially supported by the National Natural
Science Foundation of China (Project No. 51773197 and
51973214) and the Medical Health Industry Technology Project
of Jilin Province (20180311070YY).
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