Applied Surface Science 449 (2018) 15–22

Contents lists available at ScienceDirect

Applied Surface Science

journal homepage: www.elsevier.com/locate/apsusc

Synthesis and characterization of diselenide linked poly(ethylene

glycol) nanogel as multi-responsive drug carrier
Balkew Zewge Hailemeskel a , Kefyalew Dagnew Addisu a , Adhimoorthy Prasannan a ,
Shewaye Lakew Mekuria a , Chen-Yu Kao b , Hsieh-Chih Tsai a,∗
a
Graduate Institute of Applied Science and Technology, National Taiwan University of Science and Technology, Section 4, #43, Keelung Road, Taipei 106,
Taiwan, ROC
b
Graduate Institute of Biomedical Engineering, National Taiwan University of Science and Technology, Section 4, #43, Keelung Road, Taipei 106, Taiwan,
ROC

a r t i c l e i n f o a b s t r a c t

Article history: Diselenide containing polymeric-nanoparticles considered as stimuli-responsive due to their low bond
Received 28 July 2017 energy of Se Se linkage with stimuli-sensitive properties Se Se bond. Recently, diselenide bond con-
Received in revised form 3 December 2017 taining amphiphilic block copolymers based nanoparticles become emerging materials as drug carrier.
Accepted 7 December 2017
In this work, biocompatible, redox and gamma (␥)-ray sensitive diselenide-linked polymeric-nanogel
Available online 9 December 2017
(PEGSeSe)n was developed from alpha, omega-Bis-Bromo poly(ethylene glycol) (Br-PEG-Br) for anti-
cancer drug carrier. A stimuli-responsive anti-cancer drug, doxorubicin (Dox) releasing of the nanogel
Keywords:
was evaluated using an ultraviolet-visible spectrophotometer. The drug loading efficiency of the nanogel
Selenium
Cancer
was found to be 32.7%. Redox-stimuli-drug release study was conducted in the presence of 0.1% (w/w)
Polymer hydrogen peroxide (H2 O2 ), and 1 mg/mL of glutathione (GSH) in phosphate-buffered saline (PBS) pH
Nanogel of 7.4 at 37 ◦ C, as well as 5 Gy ␥-ray was applied for understand their radiation responsive release of
Drug Dox. The drug release profiles indicated that fast release were observed during the initial few hours and
maximum release 54% were shown after 48 h and 52.5% after 24 h in the presence of GSH and H2 O2
respectively, and gradually drug release were decreased as the time increases. Interestingly, ␥-ray irra-
diation drug release also showed that maximum of 46% the drug released within 12 h after irradiation.
Based on the obtained results, it can be concluded that the synthesized nanogel could be a promising
redox and radiation-sensitive nanocarrier biomaterial for high loading amount of anticancer drug and
could be used for anticancer drugs delivery in redox environment and a low dose of ␥-ray radiation.
© 2017 Published by Elsevier B.V.

1. Introduction bonds, hexatomic ring formed via Diels−Alder cycloaddition reac-

Selenium is an essential microelement that plays a crucial role
in human beings [1]. Selenium-containing compounds have shown
great potential application including therapeutic agents in cancer
[2], and the synthesis of organic compounds [3]. Moreover, it is an
important part of the antioxidant enzyme that can control the high
level of free radicals which can harm cells and results in the devel-
opment of different chronic diseases [4–6]. Recently, diselenide
bond containing polymeric biomaterials have attracted great atten-
tion due to their dynamic covalent bond nature, various physical
and chemical stimuli-responsive properties [4,7]. Dynamic cova-
lent bonds such as disulfide bonds, acylhydrazone bonds, imine
∗ Corresponding author.
E-mail address: h.c.tsai@mail.ntust.edu.tw (H.-C. Tsai).
tion, and Se X bonds (Se Se, Se N) are reversibly formed covalent
bonds having the properties of cleavable, reform of the bond or
metathesis under a certain conditions which make them hav-
ing wide application in self-healing materials, responsive systems,
and fabricating polymers [8–10]. Due to the weaker electroneg-
ativity and the bigger atomic size of selenium than sulfur, the
bond energies of Se Se (172 kJ mol−1 ) and C Se (244 kJ mol−1 )
are lower than the bond energies of S S (240 kJ mol−1 ) and C S
(272 kJ mol−1 ).The lower bond energy of Se Se and C Se bonds
make them stimuli responsive to mild condition [8,11]. There-
fore, selenium-containing polymers are among of the appropriate
candidates for stimuli-responsive polymers like sulfur-containing
polymers.
As for stimuli-responsive polymer, the synthesis of differ-
ent selenium containing amphiphilic copolymer structures such
as side-chain selenium containing polymers [12], main-chain

https://doi.org/10.1016/j.apsusc.2017.12.058
0169-4332/© 2017 Published by Elsevier B.V.
16 B.Z. Hailemeskel et al. / Applied Surface Science 449 (2018) 15–22
monoselenide containing polymers [13,14], main chain-diselenide
containing polymers [15], hyperbranched polydiselenide polymers
[16], hyperbranched polyselenide polymers [17],and dendrimers
[18] were reported. For biocompatible and biodegradable stimuli-
responsive aggregates, irradiation is among various stimuli that
have been used widely because of it require no addition chem-
icals and its convenience of operation. Near infrared (NIR) and
ultraviolet-visible (UV/vis) light have been used in drug deliv-
ery system to destroy the responsive aggregates and release the
encapsulated functional molecules [19–21]. Xu et al. reported that
metathesis reaction undergoes between two different diselenide
containing symmetric compounds (R1SeSeR1 and R2SeSeR2) to
form an asymmetric product (R1SeSeR2) with irradiation of visi-
ble light in the absence of any catalysts [9,22]. Yang et al. reported
␥-radiation could induce gel–sol transition and distraction of the
network structure of hydrogel based on a diselenide-containing
amphiphilic polymer [23].The disassembly of selenium-containing
polymeric aggregates in response to changes of stimuli under
appropriate physiological conditions with their promising applica-
tion for controlled drug release and synthetic enzyme mimics were
investigated [9,24]. The stimuli-responsive nanogel drug deliv-
ery system realizes that the encapsulated drugs would not be
released from the drug carrier systems before reaching the disease
foci (“zero premature release”) in response to either internal local
microenvironment difference or external stimuli [25].
In the biological system, redox potential is one of the stan-
dard sensitive stimuli that results from the significant variation in
the concentration of reduced GSH between the intracellular(∼2 −
10mM) and extracellular matrix (∼2 − 10M) compartment. Fur-
thermore, tumor tissues have at least four-fold higher GSH
concentration over that of the normal tissues and are even higher
in multidrug-resistant cancer cells so that drug release behaviors in
the cytoplasmic matrix in response to redox stimuli become effec-
tive [26–29]. Also, researches have shown that tumor cells typically
have higher levels of reactive oxygen species (ROS) such as H2 O2 ,
hydroxyl radical (HO• ), hydroperoxy radical (• HO2 ) and superox-
ide anion radical than normal cells [30,31]. The differences in ROS
levels between the tumor cells and the normal cells are because of
dysregulation of redox balance in tumor cells that take place when
intracellular production of ROS increases or levels of antioxidant
agents become reduced [2,32,33]. The presence of higher concen-
tration of Glutathione, higher levels of (ROS) in the tumor cell and
redox stimuli responsiveness of the diselenide bond have been used
to propose the diselenide containing biocompatible polymers for
redox-responsive drug carriers for cancer treatment.
Xu et al. reported the synthesis of a dual-redox-responsive
diselenide-containing multi-cleavable polymer micelles and
redox-responsive disassembly of the aggregate which promoted
the release of encapsulated molecules by the addition of reductants
or oxidants [14,24,34]. Zhang et al. reported low dose of ␥-radiation
(5 Gy) which is equivalent to the radiation dose applied during a
single radiotherapy treatment can destroy anticancer drug loaded
diselenide-containing block co-polymers aggregates in aqueous
solution and the release of drug [35]. Thus, the release of the
encapsulated drugs indicated that diselenide bond containing
aggregates have potential application for the combination of
chemotherapy and radiotherapy.
Although there are several works on the synthesis of stimuli-
responsive diselenide containing nanocarrier aggregates, many of
them were fabricated from the hydrophilic/hydrophobic polymer
combination. In this present study, redox and ␥-ray respon-
sive diselenide linkage containing nanocarrier aggregate has been
synthesized only from Br-PEG-Br (MW 6000 Da) and disodium dis-
elenide (Na2 Se2 ) in aqueous solution as depicted Scheme 1.The
formation of spherical aggregates most possibly due to the cross-
linking of selenium atom with its neighboring selenium and the
hydrophobic diselenide bond surrounded with the hydrophilic part
polyethylene glycol which can be used as an anti-cancer drug car-
rier in a controlled manner.
In this work, reductant (GSH), oxidant (H2 O2 ) and a low dose of
␥-radiation (5 Gy) were used to destroy the drug-loaded aggregates
formed by diselenide-containing polyethylene glycol in aqueous
solution. The encapsulated drug has been released based on the
sensitive diselenide bonds. Therefore, this study demonstrates that
diselenide linkage containing poly ethylene glycol aggregate is
potentially multi stimuli-responsive biomaterial for the use of anti-
cancer drug carrier.
2. Experimental
2.1. Materials
Br-PEG-Br (MW = 6kDa), Sodium borohydride (NaBH4 , 99%),
gray selenium powder (Se), H2 O2 , (35% w/w), GSH, 3-[4,
5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide
(MTT), D2 O and dimethyl sulfoxide (DMSO) were purchased
from Sigma-Aldrich. Cellulose dialysis membrane (MWCO: 1 and
6–8 kDa) were purchased from CelluSept T1 (Braine l Alleud,
Belgium). Sterilized phosphate-buffered saline (PBS), Dulbecco’s
modified eagle medium (DMEM), sodium pyruvate, fetal bovine
serum (FBS), trypsin and L-glutamine were purchased from Gibco
(Carlsbad, CA). Doxorubicin hydrochloride (Dox.HCl) was pur-
chased from Cayman Chemical Co., Ltd. Double distilled water
was used throughout the experiment. All other chemicals and
reagents were of analytical grades, were purchased from commer-
cial sources.
2.2. Preparation of disodium diselenide (Na2 Se2 )
An aqueous solution of Na2 Se2 was prepared by the reaction
of gray Selenium powder and sodium borohydride according to an
earlier reported procedure [12,36]. 70 mg (0.89 mmol) of gray sele-
nium powder was added to 4 mL of aqueous solution containing
70 mg (1.89 mmol) sodium borohydride at room temperature with
stirring under nitrogen atmosphere. The initial reaction occurred
vigorously with the release of H2 gas within 5 min, and then an
additional 70 mg (0.89 mmol) of selenium powder was added. The
reaction mixture was then warmed with stirring for 10 min to com-
plete the dissolution and reaction of the selenium. Finally, a reddish
aqueous solution of Na2 Se2 was formed.
2.3. Preparation of diselenide linked polyethylene glycol
(PEGSeSe)n nanogel
Diselenide linked polyethylene glycol polymer was prepared
through substitution reaction of a good leaving group containing
Br-PEG-Br with Na2 Se2 . Briefly, 100 mg (0.017 mmol) of Br-PEG-Br,
MW = 6 kDa was dissolved in 5 mL of double distilled water, and
excess amount (2 mL) of the synthesized solution of Na2 Se2 was
injected under nitrogen. The reaction was carried out at a tempera-
ture of 50 ◦ C in a water bath for 12 h. Then the solution was dialyzed
for 72 h with the dialysis membrane molecular weight cutoff of
6000–8000 against double distilled water within 4 h exchanging
interval. Finally, (PEGSeSe)n solution was filtered with a syringe fil-
ter pore size of 0.45 ␮m and lyophilized to obtain the sponge-like
(PEGSeSe)n nanogel product which was stored for further use.
2.4. Characterization
X-ray photo- electron spectroscopy (XPS) measurements were
carried out on a British VG Scientific ESCALAB 250 spectrome-
ter fitted with XR5M monochromatic X-ray Gun. Raman spectra
 B.Z. Hailemeskel et al. / Applied Surface Science 449 (2018) 15–22
Scheme 1. The synthesis route of diselenide linked polyethylene glycol (PEGSeSe)n nanogel.
of the sample were collected by JASCO NRS-5100 Laser Raman
Spectrometer. The amount of selenium in the (PEGSeSe)n poly-
meric nanogel was quantified by Inductively Coupled Plasma Mass
Spectrometry(ICP-MS). X-ray diffraction (XRD) patterns of Na2 Se2 ,
Br-PEG-Br and (PEGSeSe)n polymer were performed on a Siemens
type-F X-ray diffractometer with a Ni-filtered Cu KR radiation
source (ì) 1.54 Å) The supplied voltage and current were 30 kV
and 20 mA, respectively. The diffraction intensities were mea-
sured every 0.1◦ from (2␪) 5◦ to 80◦ at a rate of (2␪) 3◦ )/min. The
Particle size, Zeta potential at different pH and particle size distri-
bution of (PEGSeSe)n nanogel were determined by using dynamic
light scattering (DLS) instrument. A suitable amount of samples
were dispersed in ultra-distilled water, filtered with 0.45 ␮m pore
sized filter and sonicated for 2 min. Triplicate measurements were
performed for each analysis, and the results were expressed as
mean ± SD. To investigate the morphology of the nanogel, images
were taken using a field-emission scanning electron microscope
(FESEM) (JSM-6500F, JEOL) and the major elemental composition
was obtained by energy dispersive X-ray spectroscopy (EDS) FE-
SEM-EDS operated at 10.0 kV. In brief, 2 mg sample of (PEGSeSe)n
was dissolved in distilled water and dropped on the silicon sub-
strate followed by air-dried for 48 h and then the SEM image and
SEM-EDS observation at selected points were obtained after the
sample coated with platinum for 10 min. To study the porosity of
the nanogel, the nanogel was characterized by Cryo Transmission
Electron Microscope (Cryo-TEM, JEM-2010).Sample preparation for
cryo-TEM analysis involved dropping dilute aqueous solution of the
(PEGSeSe)n nanogel on TEM grid followed by air-dried.
2.5. Cytotoxicity of diselenide linked polyethylene glycol
(PEGSeSe)n nanogel
The cytotoxic effects of (PEGSeSe)n nanogel in vitro was tested
using HeLa cell. Plates were incubated for 24 h in saturated humid
conditions at 5% CO2 and 37 ◦ C. Spent HeLa cells were grown in
DMEM supplemented with 10% FBS, 1% penicillin, 1% glutamine,
and 1% sodium pyruvate at 37 ◦ C and 5% CO2 . A density of 8.6 × 105
cells per well were seeded into 96-well plate in culture medium
and kept in a humidified incubator of 5% CO2 at 37 ◦ C. After 24 h
incubation, the medium was discarded and replaced with 150 ␮L
fresh culture medium and 50 ␮L of five serial dilutions of polyethy-
lene glycol diselenide nanogels (0.2, 0.4, 0.8, 1.6 and 3.2 mg mL−1 )
and incubated again for 24 h at 37 ◦ C.
In the same way, the medium removed and 20 ␮L of 5 mg/mL
MTT solutions with fresh medium was added to each well and incu-
bated for 3 h at 37 ◦ C. Cells treated only with medium were used as a
control for all tests. And 20 ␮L of 5 mg/mL MTT solutions was added
to each well after the medium was removed, and incubated for 3 h
at 37 ◦ C. The medium was removed, and 50 ␮L of DMSO was added
to dissolve the formazan crystals, and absorbance was measured at
a reference wavelength of 570 nm and test wavelength of 450 nm
17
using an enzyme-linked immunosorbent test (ELISA) reader (Power
Wave XS, BioTek, Winooski, VT). Cell viability was calculated as
[37].
Absorbance test cells
Cell viability (%) = × 100%
Absorbance controlled cells
2.6. Drug loading methodology
Dox loading process was done by following previously reported
drug loading procedure [38].10 mg of (PEG-SeSe)n nanogel was dis-
solved in 10 mL of PBS under constant stirring and filtered with
0.45 micro liter to make the solution contained uniform nanogel
(Scheme 2). Then 1.5 mg of Dox was dissolved in 5 mL of DMSO
and neutralized with 5 ␮L triethylamine. The two solutions were
mixed stirred vigorously overnight. Unloaded Dox and DMSO were
removed by dialysis with a dialysis tubes molecular weight cutoff
of 6000–8000 against PBS for 36 h. The amount of loaded Dox was
then evaluated by using UV–vis (JASCO V-730) spectrophotome-
ter at 499 nm. This value was then compared to the amount of Dox
added for loading initially. The drug entrapment efficiency (EE) was
calculated according to the following formula:
The amount of Dox in nanogel
(EE) = × 100
Initial amount of Dox used for loading
2.7. Redox-responsive Dox release
Drug release from the nanogel was performed by dialysis
method. In brief, 1 mL solution containing Dox-loaded nanogel was
transferred into a dialysis tubes with a molecular weight cut off
1000 and immersed into in a vial containing 10 mL of PBS (buffer,
pH 7.4) with 0.1% H2 O2 and 1 mg mL−1 of GSH separately at a tem-
perature of 37 ◦ C to mimic the human physiological environment as
shown (Scheme 2). To detect the rate of drug release, 2 mL aliquots
of from the dialysate were taken at certain predetermined time
intervals. Then the concentration of drug molecule in the dialysate
was measured at 499 nm absorption peak of Dox using UV–vis spec-
trometry. As a control, the same way of the experiment was studied
using only PBS solution at the same condition. To keep the total vol-
ume of the solution constant, 2 mL of freshly prepared PBS buffer
solution with the same pH value was added after each sampling.
The cumulative release of Dox is calculated as the total percentage
of Dox molecule released across the dialysis membrane over time.
Cumulative drug release calculated as [39].
(absorbance ± intercept)
Concentartion of drug (mg/mL) =
slop
18 B.Z. Hailemeskel et al. / Applied Surface Science 449 (2018) 15–22
Scheme 2. Drug loading and stimuli-responsive drug release of (PEGSeSe)n nanogel.
Amount of drug released (mg/mL)
= Concentration × dialysate volume
Cumulative percent release (%)
(Volume of the sample withdrawn (mL)
= × P (t − 1) + Pt
Volume of dialysate
where Pt = percentage release at time t
where P (t − 1) = percentage release previous to  t
2.8. -Ray responsive Dox release
Radiation response experiment was conducted at Taipei Wan
Fang Hospital using ELEKTA Synergy instrument. A dose of 5 Gy
␥-ray was irradiated on 2 mL solution of Dox-loaded nanogel, (Gy
refers to the absorption of one joule of radiation energy per kilo-
gram of matter). Immediately after exposure of ␥-radiation, 1 mL
solution of ␥-ray irradiated Dox-loaded nanogel of was transferred
into a dialysis tube and Dox releasing experiment was performed
by following the same procedure used for redox-responsive drug
release mentioned above.
3. Results and discussion
3.1. Preparation and characterization of (PEGSeSe)n polymeric
nanogel
The surface and near-surface atomic composition of the syn-
thesized (PEGSeSe)n polymeric nanogel was investigated by XPS
measurement. As the XPS survey spectrum of the (PEGSeSe)n poly-
meric nanogel detected, C, O and Se elements with binding energies
of C1s, O1s and Se 3d were appeared at about 285.2 eV, 532.2 eV
and 54.4 eV respectively (Fig. S1) [40,41]. The selenium spectrum
was also used to confirm that the (PEGSeSe)n nanogel was cross-
linked by diselenide bonds. Binding energy of the selenium element
(3d5) was detected at 55.6 eV through the XPS measurement [42].
The obtained polymeric nanogel XPS spectrum was exhibited two
Fig. 1. XPS spectra of Se in the (PEGSeSe)n polymeric nanogel.
peaks around 54.4 and 55.1 eV (Fig. 1), which indicated the bind-
ing energies of C Se and Se Se bonds respectively, and is good
agreement with previously reported values [42,43]. Thus, the XPS
result revealed that the PEG molecules were bonded to selenium
through the C Se bond, with presence of Se Se bond in the synthe-
sized (PEGSeSe)n nanogel. The Raman spectrum of the sample was
collected to confirm whether polyethylene glycol molecules were
linked or not by diselenide bonds. As depicted in Fig. 2, the two
closely spaced Raman bands that appeared at 288, and 281 cm−1
were generated from (C Se) bond stretching vibration and the
intense band at 253 cm−1 arose from Se Se bond stretching vibra-
tion, which has an excellent agreement with previously reported
value [44]. Therefore, the result of Raman spectrum also confirmed
that the PEG molecules were well linked with diselenide bonds
and (PEGSeSe)n was synthesized. Moreover, Inductively Coupled
Plasma Mass Spectrometry (ICP-MS) result were used to quantify
the amount of selenium, and is confirmed that the synthesized
(PEGSeSe)n nanogel contains 13.5 ppm of selenium. XRD diagrams
were determined to survey the changes of the microstructure
among Na2 Se2 , Br-PEG-Br and (PEGSeSe)n and the results were
shown in Fig. 3. The diffraction curve of Na2 Se2 (Fig. 3(a)) exhibited
several typical crystal peaks at 2␪ equals 23.4◦ , 29.6◦ , 41.2◦ , 43.6◦ ,
45.3◦ , 51.6◦ and 55.9◦ . Peaks at 2␪ = 19.2◦ , 23.3◦ and 26.7◦ were the
 B.Z. Hailemeskel et al. / Applied Surface Science 449 (2018) 15–22
Fig. 2. Raman spectra of the synthesized (PEGSeSe)n nanogel.
Fig. 3. XRD patterns of (a) Na2 Se2 , (b) Br-PEG-Br and (c) (PEGSeSe)n polymer.
characteristic diffraction of Br-PEG-Br (Fig. 3(b)), which is repre-
sentative for the crystal form of brominated PEG [45]. The obtained
(PEGSeSe)n polymeric nanogel (Fig. 3(c)), showed the diffraction
peaks were allocated for the typical peaks of both Na2 Se2 and Br-
PEG-Br. However, some of the characteristic peaks of Na2 Se2 at
2␪ = 41.2◦ , 51.6◦ and 55.9◦ were disappeared, which are attribute to
the crystalline structure of Na2 Se2 , and were disturbed after chemi-
cally reacted with Br-PEG-Br. Therefore, the above results indicated
Fig. 4. (a) Average particle size and distribution and (b) zeta potential vs. pH of the nanogel.
19
that the bromine from Br-PEG-Br had been substituted by dise-
lenide bond linkage which proved that (PEGSeSe)n was successfully
synthesized.
In order to understand the particle size, particle distribution and
zeta potential at different pH of (PEGSeSe)n nanogel, DLS mea-
surement were employed. DLS results in Fig. 4(a) confirms that
(PEGSeSe)n aggregates possessed an average hydrodynamic diam-
eter of about 145.4 ± 16 nm. Surface charge of the nanogel were
obtained by the zeta potential vs. pH analysis and presented in
Fig. 4(b). In the lower pH region, surface charge of the obtained
nanogel may exhibited more acidic form of Se such as Seleninic acid
(RSeOOH, pKa = 4.2) and Selenols (RSeH, pKa = 5.7). The material
exhibited relatively a higher negative zeta potential approximately
−50.3 mV at pH of 7, which revealed that Se-Se and Selenenic acid
(R-SeOH) may exhibited.While increase the pH, surface charge of
the nanogel also decreasing because of reformation of Seleninic
acid. Surface charge of the obtained nanogels may be reversible
charges and retains critical surface charge on the physiological pH
range.
To investigate the stimuli-responsive of aggregation of
(PEGSeSe)n polymeric nanogel, redox stimuli reagents (GSH and
H2 O2 ) and ␥-radiation were used. To this end, 1 mg/mL GSH and
0.1% H2 O2 were added to the solution of (PEGSeSe)n aggregates, and
a dose of 5 Gy ␥-radiation was applied. DLS results (Table 1) showed
that the swelled of (PEGSeSe)n aggregates were obtained with the
adding of GSH and H2 O2 , and due to irradiation of ␥-ray, the size of
(PEGSeSe)n aggregates increased to 260.1 ± 20 nm, 203.6 ± 31 nm,
and 177.6 ± 12 nm respectively.
Moreover, to study the formation of polymeric nanogel, the
morphology of (PEGSeSe)n nanogels were observed by FESEM. The
FESEM image revealed that the spherical aggregated structures
were formed with uniformly distributed as shown in Fig. 5(a).
Furthermore, the elemental composition analysis of (PEGSeSe)n
nanogels was conducted using energy dispersive X-ray spec-
troscopy (EDS), and the result confirmed that presence of carbon,
oxygen, and selenium as shown in (Fig. S2), which supports the
insertion of all three major components. FESEM observation (Fig.
S3(a)) also showed that the particles become irregular shape which
indicated that the aggregates start to disintegrate after H2 O2 was
added and almost collapsed due to the addition of GSH as shown
in Fig. S3(b). The porosity of the nanogel was studied with Cryo-
TEM observation. The Cryo-TEM image is shown in Fig. 5(b).The
image shows that the nanogel consists of a network of tiny par-
tially aligned regions (white part of the black sphere in the figure)
which confirm that the nanogel has porosity characteristics.
20 B.Z. Hailemeskel et al. / Applied Surface Science 449 (2018) 15–22

Table 1
The particle size before and after applying stimuli and zeta potential of the nanoparticle. The data are presented as mean ± SD, were n = 3.

Nanogel Sample Size of the particle Zeta Potential (mV) Size of the particle after applying stimuli (nm)
(nm)
0.1% H2 O2 0.1 mg mL-1 GSH 5 Gy ␥-ray

(PEGSeSe)n 145.4 ± 16 −50.3 ± 4 203.6 ± 31 260.1 ± 20 177.6 ± 12

Fig. 5. (a). The SEM image of the nanogel, (b) cryo-TEM image of the nanogel.

Fig. 6. Cytotoxicity of (PEGSeSe)n nanogel at different concentrations. The data are Fig. 7. UV–vis spectra of Dox-loaded nanogel.
expressed as mean ± SD, where n = 3.

3.3. Drug loading

3.2. Cytotoxicity test
Even though selenium-containing polymers have received great
consideration as stimuli-responsive biomaterials for drug delivery
system, the toxicity of the selenium-containing biomaterials is still
an important factor that should be considered and needs to be eval-
uated [35,46]. To examine the toxicity of (PEGSeSe)n polymeric
nanogel, HeLa cells were used as a model to study the cytotox-
icity of (PEGSeSe)n nanogel. From Fig. 6, it can be seen that, at
high concentrations, such as 0.8 mg mL−1 and above, the nanogel
had somewhat higher inhibitory effect on the HeLa cells. How-
ever, 72% of the cells were kept growing for 24 h after adding the
nanogel, and relatively lower concentrations such as 0.2 mg mL−1
and 0.4 mg mL−1 , the (PEGSeSe)n nanogels showed less toxicity to
the HeLa cells and above 90% of the HeLa cells were growing. Thus,
the above results indicate that the cytotoxicity of (PEGSeSe)n poly-
meric nanogel was getting lower as the concentrations decrease
and may have an insignificant effect on cells at the concentration
of micronutrient level.
A Dox solution at the concentration of 0.1 mg/mL in DMSO was
used for loading into the nanogel which has a characteristic absorp-
tion maximum at 499 nm. The loaded Dox was purified with dialysis
membrane molecular weight cutoff of 6000–8000 against PBS, and
its concentration was determined from UV–vis spectrum and the
calibration curve of Dox as shown in Fig. 7 and Fig. S4. The con-
centration of loaded Dox was found to be 0.033 mg/mL, and the
encapsulation efficiency was evaluated about 32.7%.
3.4. Redox-responsive drug release
The redox-responsive drug release study was conducted with
0.1% H2 O2 and 0.1 mg/mL of GSH in PBS buffer pH of 7.4 at 37 ◦ C
temperature. When the drug-loaded diselenide containing poly-
meric nanogel interact with the release medium, the diselenide
bond in the nanogel cleaved by the redox stimuli (H2 O2 and GSH)
and the loaded Dox is stimulate to release into the medium. As
shown in Fig. 8(a), relatively fast release was observed during the
 B.Z. Hailemeskel et al. / Applied Surface Science 449 (2018) 15–22
Fig. 8. (a) Redox responsive drug release, (b) ␥-ray responsive drug release.
initial few hours and maximum release 54% was shown after 48 h
and 52.5% after 24 h in the presence of GSH and H2 O2 respectively.
Whereas relatively lower drug release 35.6% was observed in the
absence of GSH and H2 O2 after 72 h.
3.5. -Ray responsive Dox release
Under exposure to radiation, water can generate oxidative
species such as H2 O2 , HO• , and • HO2 , with concentrations pro-
portional to the applied radiation doses [35]. It is reasonable that
the irradiated ␥-ray generated oxidative species in the aqueous
solution of drug loaded (PEGSeSe)n nanogel. Consequently ␥-ray
responsive drug release has a similar mechanism with oxidative
drug release. In this study, the ␥-radiation responsive of Dox release
from the (PEGSeSe)n nanogel was monitored by absorbance mea-
surements, and the released percentages were evaluated by the
comparison of the amount of loaded Dox and the released amount
after dialysis. As seen from Fig. 8(b), the loaded Dox molecules were
released from aggregates due to a dose of 5 Gy ␥-ray irradiation,
the time of dialysis represents the releasing time after irradia-
tion of ␥-ray. The maximal release 46.4% was recorded about 12 h
dialysis. As described in the experimental section, the Dox-loaded
nanogel aggregates were prepared by dialysis against phosphate
buffer to remove the unloaded Dox molecules, and thus, the aggre-
gate showed stability in PBS. However, the drug was released 33%
without irradiation of ␥-ray within 12 h dialysis.
4. Conclusions
In this study, we have demonstrated an effective target anti-
cancer drug carrier preparation through dual stimuli of redox and
␥-ray responsive diselenide containing nanogel (PEGSeSe)n from
Br-PEG-Br and Na2 Se2 to achieve site-selective drug release. The
synthesized (PEGSeSe)n nanogel was characterized by various ana-
lytical methods (XPS, Raman, ICP-MS, XRD, DLS, and SEM).The
site-selective drug release was accomplished through stimuli-
sensitive diselenide bonds. As a model anti-cancer drug, Dox was
loaded into the spherical aggregates, and its encapsulation effi-
ciency of the synthesized material was 32.7%.
The release study showed that Dox was relatively stayed within
the aggregates in the absence of GSH or H2 O2 under physiological
conditions, and only 35.6% was the drug was released in the absence
of GSH and H2 O2 after 72 h. Also, the drug release profile indi-
cated that less drug release had been seen with no ␥-radiation was
applied. However, the release of Dox was considerably increased
to 54% after by addition of 0.1 g/mL GSH within 48 h and 52.5% in
21
the presence of 0.1% H2 O2 (w/w) in 72 h. After low irradiation dose
(5 Gy) of ␥-ray, 46.4% of the loaded drug was released in 12 h dial-
ysis.Therefore, this work gives an investigation about diselenide
linkage containing PEG-based multi stimuli-responsive drug deliv-
ery system, and the results confirmed that (PEGSeSe)n nanogel
showing multi-responsive drug release property, which can be con-
sidered as a promising biomaterial for cancer therapy.
Acknowledgement
The authors would like to thank the Ministry of Science and
Technology, Taiwan (MOST105-2221-E-011-133-MY3 and 105-
2221-E-011-151-MY3) for providing financial support.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in
the online version, at doi:https://doi.org/10.1016/j.apsusc.2017.12.
058
References
[1] H. Zeng, Selenium as an essential micronutrient: roles in cell cycle and
apoptosis, Molecules 14 (2009) 1263.
[2] A.P. Fernandes, V. Gandin, Selenium compounds as therapeutic agents in
cancer, Biochim. Biophys. Acta 1850 (2015) 1642–1660.
[3] T. Wirth, Chiral selenium compounds in organic synthesis, Tetrahedron 55
(1999) 1–28.
[4] X. Cheng, Y. Jin, R. Qi, W. Fan, H. Li, X. Sun, S. Lai, Dual pH and
oxidation-responsive Nanogels crosslinked by diselenide bonds for controlled
drug delivery, Polymer 101 (2016) 370–378.
[5] M. Roman, P. Jitaru, C. Barbante, Selenium biochemistry and its role for
human health, Metallomics 6 (2014) 25–54.
[6] A.V. Lobanov, D.L. Hatfield, V.N. Gladyshev, Eukaryotic selenoproteins and
selenoproteomes, Biochim. Biophys. Acta (BBA) Gen. Subj. 1790 (2009)
1424–1428.
[7] Y. Yi, H. Xu, L. Wang, W. Cao, X. Zhang, A new dynamic covalent bond of Se-N:
towards controlled self-assembly and disassembly, Chemistry 19 (2013)
9506–9510.
[8] S. Ji, J. Xia, H. Xu, dynamic chemistry of selenium: Se–N and Se–Se dynamic
covalent bonds in polymeric systems, ACS Macro Lett. 5 (2016) 78–82.
[9] J. Xia, S. Ji, H. Xu, Diselenide covalent chemistry at the interface: stabilizing an
asymmetric diselenide-containing polymer via micelle formation, Polym.
Chem. 7 (2016) 6708–6713.
[10] S. Ji, W. Cao, Y. Yu, H. Xu, Dynamic diselenide bonds: exchange reaction
induced by visible light without catalysis, Angew. Chem. Int. Ed. 53 (2014)
6781–6785.
[11] W. Zhang, W. Lin, Q. Pei, X. Hu, Z. Xie, X. Jing, Redox-hypersensitive organic
nanoparticles for selective treatment of cancer cells, Chem. Mater. 28 (2016)
4440–4446.
[12] H. Ren, Y. Wu, N. Ma, H. Xu, X. Zhang, Side-chain selenium-containing
amphiphilic block copolymers: redox-controlled self-assembly and
disassembly, Soft Matter 8 (2012) 1460–1466.
22 B.Z. Hailemeskel et al. / Applied Surface Science 449 (2018) 15–22
[13] W. Cao, Y. Li, Y. Yi, S. Ji, L. Zeng, Z. Sun, H. Xu, Coordination-responsive
selenium-containing polymer micelles for controlled drug release, Chem. Sci.
3 (2012) 3403.
[14] N. Ma, Y. Li, H. Ren, H. Xu, Z. Li, X. Zhang, Selenium-containing block
copolymers and their oxidation-responsive aggregates, Polym. Chem. 1
(2010) 1609.
[15] P. Han, S. Li, W. Cao, Y. Li, Z. Sun, Z. Wang, H. Xu, Red light responsive
diselenide-containing block copolymer micelles, J. Mater. Chem. B 1 (2013)
740–743.
[16] J. Liu, Y. Pang, J. Chen, P. Huang, W. Huang, X. Zhu, D. Yan, Hyperbranched
polydiselenide as a self assembling broad spectrum anticancer agent,
Biomaterials 33 (2012) 7765–7774.
[17] H. Xu, J. Gao, Y. Wang, Z. Wang, M. Smet, W. Dehaen, X. Zhang,
Hyperbranched polyselenides as glutathione peroxidase mimics, Chem.
Commun. (Camb.) (2006) 796–798.
[18] X. Zhang, H. Xu, Z. Dong, Y. Wang, J. Liu, J. Shen, Highly efficient
dendrimer-based mimic of glutathione peroxidase, J. Am. Chem. Soc. 126
(2004) 10556–10557.
[19] A.P. Goodwin, J.L. Mynar, Y. Ma, G.R. Fleming, J.M. Fréchet, Synthetic micelle
sensitive to IR light via a two-photon process, J. Am. Chem. Soc. 127 (2005)
9952–9953.
[20] J. Jiang, X. Tong, Y. Zhao, A new design for light-breakable polymer micelles, J.
Am. Chem. Soc. 127 (2005) 8290–8291.
[21] H. Ren, Y. Wu, Y. Li, W. Cao, Z. Sun, H. Xu, X. Zhang, Visible-light-induced
disruption of diselenide-containing layer-by-layer films: toward combination
of chemotherapy and photodynamic therapy, Small 9 (2013) 3981–3986.
[22] S. Ji, W. Cao, Y. Yu, H. Xu, Visible-light-induced self-healing
diselenide-containing polyurethane elastomer, Adv. Mater. 27 (2015)
7740–7745.
[23] W. Cao, X. Zhang, X. Miao, Z. Yang, H. Xu, Gamma ray-responsive
supramolecular hydrogel based on a diselenide-containing polymer and a
peptide, Angew. Chem. Int. Ed. Engl. 52 (2013) 6233–6237.
[24] H. Xu, W. Cao, X. Zhang, Selenium-containing polymers: promising
biomaterials for controlled release and enzyme mimics, Acc. Chem. Res. 46
(2013) 1647–1658.
[25] F. Tang, L. Li, D. Chen, Mesoporous silica nanoparticles: synthesis,
biocompatibility and drug delivery, Adv. Mater. 24 (2012) 1504–1534.
[26] A.L. Ortega, S. Mena, J.M. Estrela, Glutathione in cancer cell death, Cancers
(Basel) 3 (2011) 1285–1310.
[27] Q.-H. Zhou, J. Lin, L.-D. Li, L. Shang, Biodegradable micelles self-assembled
from miktoarm star block copolymers for MTX delivery, Colloid. Polym. Sci.
293 (2015) 2291–2300.
[28] L. Shang, Q. Wang, K. Chen, J. Qu, J. Lin, J.-b. Luo, Q. Zhou, Preparation of
polydopamine based redox-sensitive magnetic nanoparticles for doxorubicin
delivery and MRI detection, J. Bioresour. Bioprod. 2 (2017) 67–72.
[29] A. Singh, T.-H. Tran, M.M. Amiji, Redox-Responsive Nano-Delivery Systems for
Cancer Therapy, 2016, pp. 255–269.
[30] G.T. Wondrak, Redox-directed cancer therapeutics: molecular mechanisms
and opportunities, Antioxid. Redox Signal. 11 (2009) 3013–3069.
[31] G.Y. Liou, P. Storz, Reactive oxygen species in cancer, Free Radic. Res. 44
(2010) 479–496.
[32] N. Ballatori, S.M. Krance, S. Notenboom, S. Shi, K. Tieu, C.L. Hammond,
Glutathione dysregulation and the etiology and progression of human
diseases, Biol. Chem. 390 (2009) 191–214.
[33] A.J. Montero, J. Jassem, Cellular redox pathways as a therapeutic target in the
treatment of cancer, Drugs 71 (2011) 1385–1396.
[34] N. Ma, Y. Li, H. Xu, Z. Wang, X. Zhang, Dual redox responsive assemblies
formed from diselenide Block copolymers, J. Am. Chem. Soc. 132 (2010)
442–443.
[35] N. Ma, H. Xu, L. An, J. Li, Z. Sun, X. Zhang, Radiation-sensitive diselenide block
co-polymer micellar aggregates: toward the combination of radiotherapy and
chemotherapy, Langmuir 27 (2011) 5874–5878.
[36] T. Sun, Y. Jin, R. Qi, S. Peng, B. Fan, Oxidation responsive mono-cleavable
amphiphilic di-block polymer micelles labeled with a single diselenide,
Polym. Chem. 4 (2013) 4017.
[37] W. Wu, W. Yao, X. Wang, C. Xie, J. Zhang, X. Jiang, Bioreducible heparin-based
nanogel drug delivery system, Biomaterials 39 (2015) 260–268.
[38] H. Zou, W. Yuan, Temperature- and redox-responsive magnetic complex
micelles for controlled drug release, J. Mater. Chem. B 3 (2015) 260–269.
[39] A.R. Chandrasekaran, C.Y. Jia, C.S. Theng, T. Muniandy, S. Muralidharan, S.A.
Dhanaraj, Invitro studies and evaluation of metformin marketed
tablets-Malaysia, J. Appl. Pharm. Sci. 01 (05) (2011) 214–217.
[40] T. Çolakoğlu, M. Parlak, Structural characterization of polycrystalline
Ag–In–Se thin films deposited by e-beam technique, Appl. Surf. Sci. 254
(2008) 1569–1577.
[41] G. Zaiats, D. Yanover, R. Vaxenburg, J. Tilchin, A. Sashchiuk, E. Lifshitz, PbSe
based colloidal core/shell heterostructures for optoelectronic applications,
Materials 7 (2014) 7243–7275.
[42] C. Li, W. Huang, L. Zhou, P. Huang, Y. Pang, X. Zhu, D. Yan, PEGylated
poly(diselenide-phosphate) nanogel as efficient self-delivery nanomedicine
for cancer therapy, Polym. Chem. 6 (2015) 6498–6508.
[43] K. Keiji, T. Hideyuki, K. Koichi, I. Isao, NMR and XPS studies of some
diselenocarbamates. Bond Switch in Bis(dimethylselenocarbamoyl)
triselenide, Bull. Chem. Soc. Jpn. 59 (1986) 1741–1746.
[44] K. Helios, A. Pietraszko, W. Zierkiewicz, H. Wójtowicz, D. Michalska, The
crystal structure, infrared, Raman and density functional studies of
bis(2-aminophenyl) diselenide, Polyhedron 30 (2011) 2466–2472.
[45] M. Chen, C. Gao, S. Lü, Y. Chen, M. Liu, Dual redox-triggered shell-sheddable
micelles self-assembled from mPEGylated starch conjugates for rapid drug
release, RSC Adv. 6 (2016) 9164–9174.
[46] C.W. Nogueira, G. Zeni, J.B.T. Rocha, Organoselenium and organotellurium
compounds: toxicology and pharmacology, Chem. Rev. 104 (2004)
6255–6286.