Fabrication of redox-responsive Bi(mPEG-PLGA)-Se2 micelles for doxorubicin delivery

Article abstract of DOI:10.1016/j.ijpharm.2019.118486

Stimuli-responsive polymeric nanostructures have emerged as potential drug carriers for cancer therapy. Herein, we synthesized redox-responsive diselenide bond containing amphiphilic polymer, Bi(mPEG-PLGA)-Se₂ from mPEG-PLGA and 3,3′-diselanediyldipropanoic acid (DSeDPA) using DCC/DMAP as coupling agents. Due to its amphiphilic nature, Bi(mPEG-PLGA)-Se₂ self-assembled in to stable micelles in aqueous solution with a hydrodynamic size of 123.9 ± 0.85 nm. The Bi(mPEG-PLGA)-Se₂ micelles exhibited DOX-loading content (DLC) of 6.61 wt% and encapsulation efficiency (EE) of 54.9%. The DOX-loaded Bi(mPEG-PLGA)-Se₂ micelles released 73.94% and 69.54% of their cargo within 72 h upon treatment with 6 mM GSH and 0.1% H₂O₂, respectively, at pH 7.4 and 37 °C. The MTT assay results demonstrated that Bi(mPEG-PLGA)-Se₂ was devoid of any inherent toxicity and the DOX-loaded micelles showed pronounced antitumor activities against HeLa cells, 44.46% of cells were viable at maximum dose of 7.5 μg/mL. The cellular uptake experiment further confirmed the internalization of DOX-loaded Bi(mPEG-PLGA)-Se₂ micelles and endowed redox stimuli triggered drug release in cytosol and nuclei of cancer cells. Overall, the results suggested that the smart, biocompatible Bi(mPEG-PLGA)-Se₂ copolymer could serve as potential drug delivery biomaterial for the controlled release of hydrophobic drugs in cancer cells.

Full text of DOI:10.1016/j.ijpharm.2019.118486

Accepted Manuscript
Fabrication of redox-responsive Bi(mPEG-PLGA)-Se micelles for doxorubicin
2
delivery
Yihenew Simegniew Birhan, Balkew Zewge Hailemeskel, Tefera Worku
Mekonnen, Endiries Yibru Hanurry, Haile Fentahun Darge, Abegaz Tizazu
Andrgie, Hsiao-Ying Chou, Juin-Yih Lai, Ging-Ho-Hsiue, Hsieh-Chih Tsai
PII:
S0378-5173(19)30528-9
DOI:
https://doi.org/10.1016/j.ijpharm.2019.118486
Article Number:
Reference:
118486
IJP 118486
To appear in:
International Journal of Pharmaceutics
Received Date:
Revised Date:
Accepted Date:
8 May 2019
15 June 2019
28 June 2019
Please cite this article as: Y.S. Birhan, B.Z. Hailemeskel, T.W. Mekonnen, E.Y. Hanurry, H.F. Darge, A.T. Andrgie,
H-Y. Chou, J-Y. Lai, Ging-Ho-Hsiue, H-C. Tsai, Fabrication of redox-responsive Bi(mPEG-PLGA)-Se micelles
2
for doxorubicin delivery, International Journal of Pharmaceutics (2019), doi: https://doi.org/10.1016/j.ijpharm.
2
019.118486
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Fabrication of redox-responsive Bi(mPEG-PLGA)-Se micelles for doxorubicin delivery
2
a
a
a
Yihenew Simegniew Birhan , Balkew Zewge Hailemeskel , Tefera Worku Mekonnen , Endiries
a
a
a
a
Yibru Hanurry , Haile Fentahun Darge , Abegaz Tizazu Andrgie , Hsiao-Ying Chou , Juin-Yih
Laia,b,c, Ging-Ho-Hsiue* , Hsieh-Chih Tsai*
,d
,a,b
a. Graduate Institute of Applied Science and Technology, National Taiwan University of
Science and technology, Taipei 106, Taiwan, ROC.
b. Advanced Membrane Materials Center, National Taiwan University of Science and
technology, Taipei 106, Taiwan, ROC.
c. R&D Center for Membrane technology, Chung Yuan Christian University, Chungli, Taoyuan
3
20, Taiwan, ROC.
d. Department of Chemical Engineering, National Chung Hsing University, Taichung, 402,
Taiwan, ROC.
[
*] To whom correspondence and reprint requests should be addressed.
Prof. Hsieh-Chih Tsai, E-mail: h.c.tsai@mail.ntust.edu.tw , Tel: +886-2-27303625, Fax: +886-2-
7303733.
Prof. Ging-Ho Hsiue, E-mail: ghhsiue@mx.nthu.edu.tw
2
1

Abstract
Stimuli-responsive polymeric nanostructures have emerged as potential drug carriers for cancer
therapy. Herein, we synthesized redox-responsive diselenide bond containing amphiphilic
polymer, Bi(mPEG-PLGA)-Se2 from mPEG-PLGA and 3,3'-diselanediyldipropanoic acid
(DSeDPA) using DCC/DMAP as coupling agents. Due to its amphiphilic nature, Bi(mPEG-
PLGA)-Se self-assembled in to stable micelles in aqueous solution with a hydrodynamic size of
2
1
6
23.9 ± 0.85 nm. The Bi(mPEG-PLGA)-Se micelles exhibited DOX-loading content (DLC) of
2
.61 wt% and encapsulation efficiency (EE) of 54.9%. The DOX-loaded Bi(mPEG-PLGA)-Se2
micelles released 73.94% and 69.54% of their cargo within 72 h upon treatment with 6 mM GSH
and 0.1% H O , respectively, at pH 7.4 and 37 ℃. The MTT assay results demonstrated that
2
2
Bi(mPEG-PLGA)-Se was devoid of any inherent toxicity and the DOX-loaded micelles showed
2
pronounced antitumor activities against HeLa cells, 44.46% of cells were viable at maximum
dose of 7.5 µg/mL. The cellular uptake experiment further confirmed the internalization of
DOX-loaded Bi(mPEG-PLGA)-Se micelles and endowed redox stimuli triggered drug release in
2
cytosol and nuclei of cancer cells. Overall, the results suggested that the smart, biocompatible
Bi(mPEG-PLGA)-Se copolymer could serve as potential drug delivery biomaterial for the
2
controlled release of hydrophobic drugs in cancer cells.
Keywords: Diselenide bond, Redox-responsive, Polymeric micelles, Anticancer drug delivery
2

1
. Introduction
Chemotherapy is still the main stream treatment modality for different forms of cancer. However,
unfavorable pharmacokinetic properties including poor solubility, slow absorption and non-
selective biodistribution limit the clinical outcomes of chemotherapeutic agents resulting in
insufficient therapeutic efficacy, problem of drug resistance and dose-limiting toxicities (Zhou et
al., 2017; Zhuang et al., 2018). The advancement of nanotechnology revitalized and brought
many chemotherapeutic drugs from shelf to life. Recently, nanoscale polymeric biomaterials
have gained significant consideration as drug delivery vehicles owing to their potential to
modulate the pharmacokinetic and pharmacodynamic profiles of conventional anticancer drugs,
selective accumulation in tumor tissue through the enhanced permeability and retention (EPR)
effect and ability to use unique cancer features (acidic pH, altered redox potential, overexpressed
proteins and enzymes) as stimuli to release their cargo in the biological milieu of target tissue
(Lee et al., 2017; Li et al., 2016; Ma et al., 2015; Wang et al., 2017; Yang et al., 2018; Zhou et
al., 2017). For instance, the obvious difference in reduced glutathione (GSH) concentration
between reducing intracellular (approximately 2-10 mM) and mildly oxidizing extracellular
space (approximately 2-20 µM) of tumor cell is the driving force for the design of redox
responsive drug delivery systems that are stable enough outside the cell and disassemble and
release their payload only when internalized in the cytosol and the cell nucleus (Chen et al.,
2
016). Moreover, redox response is exceedingly fast in the presence of high concentration of
GSH in the tumor or intracellular environments. Taking this in to account, disulfide (Maiti et al.,
018; Pandey et al., 2019; Peng et al., 2019; Zhang et al., 2018; Zhao et al., 2018) and diselenide
Deepagan et al., 2016; Hailemeskel et al., 2018; Sun et al., 2017; Sun et al., 2018; Wei et al.,
2
(
2
017; Zeng et al., 2015; Zhai et al., 2017) linked polymeric carriers, which respond to the
3

intracellular redox signals of tumor cells have been fabricated for the targeted delivery and
controlled release of anticancer drugs, resulting in enhanced antitumor efficacy.
-1
-1
The weak and dynamic Se-Se bond (172 kJ mol ) as compared to S-S (240 kJ mol ) bond
coupled with its large atomic radius (120 pm) and weak electronegativity, makes the Se-Se bond
more redox responsive than S-S bond in and around cancer environments (Cao et al., 2014;
Zhang et al., 2017). Furthermore, the Se-Se bond has a unique property that enables it to be
oxidized to seleninic acid (-SeOOH) via oxidants and reduced to selenol (-SeH) via reducers
(Wang et al., 2014). Due to the aforementioned comparative advantages over disulfide bond,
researchers focused on the design and synthesis of diselenide-linked polymeric biomaterials as
redox sensitive drug and gene carriers (Cheng et al., 2012; Li et al., 2015; Liu et al., 2012; Sun et
al., 2013; Yue et al., 2014). Overall, they followed different design approaches in the fabrication
process; based on the position and number of the Se-Se bond: mono-cleavable diselenide bond at
the interface between copolymers (Luan et al., 2015b) or on the core of block copolymers (Zeng
et al., 2015), multi-cleavable Se-Se groups on hyperbranched polychain (Liu et al., 2012) or
positioned on main chain (Hailemeskel et al., 2018) and sidechains (Zhai et al., 2017); with
regard to the type of nanostructure formed: polymeric nanoparticles (Cheng et al., 2012),
hydrogels (Sun et al., 2018; Zhang et al., 2017), nanogels (Cheng et al., 2016), micelles
(Deepagan et al., 2016; Zhai et al., 2017), etc had been prepared. Recently, the synthesis of
diselenide rich and redox responsive PEG-b-PBSe block copolymer which undergo visible light
induced cross-linking to form stable aggregates for camptothecin (CPT) and doxorubicin (DOX)
co-delivery was reported (Zhai et al., 2017). They concluded that facile, visible light-induced
diselenide metathesis enabled to form physiologically stable, core cross-linked micelle
possessing extended blood circulation with accelerated dual drug release in tumor’s
4

microenvironments. In addition, selenol bearing triblock polymer capable of forming in situ
diselenide shell cross-linked micelle was successfully synthesized from PEG and polypeptides
(Deepagan et al., 2016). The micelles maintained their structural integrity even in the presence of
destabilizing agents. Upon exposure to reactive oxygen species (ROS), the DOX-loaded micelles
undergo rapid release of DOX because the hydrophobic diselenide bond was cleaved into
hydrophilic selenic acid derivatives. After systemic administration into tumor bearing mice, the
cross-linked micelles delivered significantly more drugs to tumors (3.73-fold higher amount
compared with free drug) and effectively suppresses tumor growth. In general, diselenide linked
polymeric micelles presented notable sensetivity towards redox environments with pronounced
serum stability than disulfide bonds containing structural analogues (Wang et al., 2014; Zhang et
al., 2019), yet only limited research has been conducted sofar.
Amphiphilic block copolymers comprised of hydrophobic and hydrophilic segments are widely
used for the preparation of different nanocarriers because of their self-assembling property in
aqueous solutions (Chen et al., 2017; Cheng et al., 2016; Chu et al., 2016). PEG and PLGA are
the most commonly utilized polymers for drug delivery applications, partly because the former
suppresses opsonization phenomenon, enhances in vivo circulation time and facilitates cellular
uptake through EPR effect; the latter can undergo complete biodegradation and hence eliminated
from the body with greater ease (Avgoustakis et al., 2003; Chen et al., 2017; Zhang et al., 2014a).
The PEG-PLGA block copolymers are widely used in biomedicine, as both PEG and PLGA
segments are approved by FDA for clinical use (Wilkosz et al., 2018; Zhang et al., 2014a).
However, the relatively low DLC compromised the theraputic applications of PEG-PLGA as
nanocarrier for hydrophilic and hydrophobic drugs (Avgoustakis et al., 2002; Liu et al., 2014;
Song et al., 2011; Yoo and Park, 2001). In fact, DLC and hydrodynamic diameter depend on the
5

architecture of the PEG-PLGA copolymer used to prepare the nanoparticle. For that reason,
extending the hydrophobic PLGA segment (Chen et al., 2017; Gohy, 2005; Wilkosz et al., 2018)
or blending the copolymer with PLGA improve the DLC of PEG-PLGA nanoparticles (Xu et al.,
2
015). Still, increasing the PLGA segment make the core of the nanoparticle extreemly
hydophobic and compact which strongly diminish the drug releasing rate significantly
Avgoustakis et al., 2002). Thus, fabrication of stimuli-responsive PEG-PLGA block polymeric
(
nanoparticle with optimum DLC, particle size and reasonable drug releasing behavior is still in
great demand. In this study, diselenide linkage containing low molecular weight block
copolymer Bi(mPEG-PLGA)-Se was designed and synthesized as part of our endavor to
2
fabricate smart polymeric nanocarriers with finest DLC and sensible drug releasing behavior by
conjugating mPEG-PLGA with DSeDPA using DCC/DMAP coupling reaction. Due to the
incorporation of hydrophobic diselenide bond, Bi(mPEG-PLGA)-Se self-assembled into stable
2
micelles in aqueous solution with unimodal size distribution. The size, morphology, critical
micelle concentration, redox responsive cummulative drug release behavior of the micelle was
assessed. In vitro cytotoxicity of blank micelles, cell viability of DOX-loaded Bi(mPEG-PLGA)-
Se micelle and its cellular uptake against HeLa and HaCaT cells were assessed. The results
2
indicated that the synthesized material has the potential to serve as a controlled releasing drug
carrier.
2
. Materials and methods
2
.1. Materials
Methoxyl poly(ethylene glycol) (mPEG) with molecular weight ~750, stannous 2-ethylhexanoate
(
Sn(Oct) , 92.5~100%), N,N'-dicyclohexylcarbodiimide (DCC, 99%), 4-dimethylaminopyridine
2
(DMAP, 99%), 3-chloropropanoic acid (98%), selenium powder (~100 mesh, 99.5%),
6

dichloromethane (DCM), dimethyl sulfoxide (DMSO), anhydrous tetrahydrofuran (THF)
99.9%), pyrene (98%), triethylamine (99.5%), diethyl ether, ethyl acetate (99.8%),
(
paraformaldehyde (97%), doxorubicin hydrochloride (DOX·HCl) were products of Sigma-
Aldrich. Glycolide (GA) and D,L-Lactide (LA) were purchased from Jinan Daigang Biomaterial
Co., Ltd (China). Dulbecco’s modified Eagle’s medium (DMEM), 3-[4,5-dimethylthiazol-2-yl]-
2
,5-diphenyltetrazolium bromide (MTT), fetal bovine serum (FBS), penicillin-streptomycin,
trypsin, and Dulbecco’s phosphate buffered saline (PBS) were obtained from GIBCO Invitrogen
Corp. Unless otherwise specified, reagents were used as received without further purification.
2
.2. Synthesis of 3,3'-Diselanediyldipropanoic acid (DSeDPA)
The redox responsive moiety, DSeDPA was synthesized by following previous reports with
some modifications (Cheng et al., 2012). Briefly, selenium powder (0.79 g, 10 mmol) in 3 mL of
water was added to a three-necked flask under nitrogen atmosphere. NaBH (0.76 g, 20 mmol)
4
dissolved in 8 mL of cold water was injected dropwise in to the selenium suspension. The
reaction mixture was stirred at room temperature and reacted until colorless for complete
dissolution of the selenium powder. After 30 minutes, equivalent amount of selenium powder
was added to the reaction mixture and heated to 105 ℃ for 20 minutes until reddish brown
solution was formed. Then, 5 mL of aqueous 3-chloropropanoic acid (2.2 g, 20 mmol) was added
to the reddish brown solution, and the resulting mixture was stirred overnight at room
temperature under nitrogen atmosphere (Scheme 1). The mixture was further stirred for 4 hours
upon exposure to the atmosphere and was filtered to remove the unreacted species such as
disodium diselenide (Na Se ). Then, the pH of the yellow supernatant was adjusted to 3-4 using
2
2
1
M HCl solution and extracted twice with ethyl acetate. Finally, the combined organic layers
7

were washed with water, dried with anhydrous MgSO and DSeDPA was collected by removing
4
the ethyl acetate using rotary evaporator.
2
.3. Synthesis of mPEG-PLGA diblock copolymer
The diblock copolymer, mPEG-PLGA was synthesized by ring opening copolymerization of LA
and GA using mPEG as macro-initiator as described in previous publications with slight
modifications (Chen et al., 2014). In short, mPEG (7.5 g, 10 mmol) was added in to 500 mL
three-neck flask and dried under vacuum at 120 ℃ for 3 h. After cooling the system to 90 ℃, LA
(11.53 g, 80 mmol) and GA (1.98 g, 26 mmol) were added under argon atmosphere and then
heated under reduced pressure at 90 ℃ for 30 minutes to remove the residual moisture. Next, 18
mg of Sn(Oct) was injected in to the reaction mixture as a catalyst and continuously stirred for
2
1
2 h under nitrogen atmosphere by maintaining the reaction temperature at 150 ℃. The obtained
crude products were dissolved in DCM and precipitated in diethyl ether at -20 ℃ for 24 h. After
decanting the supernatant, the residual solvent was removed under vacuum to obtain pure
mPEG-PLGA copolymer.
2
.4. Synthesis of Bi(mPEG-PLGA)-Se conjugate
2
Bi(mPEG-PLGA)-Se conjugate was synthesized by coupling DSeDPA with mPEG-PLGA in
2
the presence of DCC and DMAP (Luan et al., 2015a). In brief, mPEG-PLGA (9.35 g) was
dissolved in anhydrous THF. Then, DSeDPA (0.57 g) and DMAP (0.17 g) were added in to the
polymer solution under argon atmosphere. DCC (1.14 g) was dissolved in THF and injected in to
ice-cooled mixture. The reaction was stirred in ice bath for 4 h and then for additional 48 h at
room temperature under nitrogen atmosphere. After the completion of the reaction, the mixture
was filtered and the solvent was removed by rotary evaporation. The synthesized product was
purified by dissolving crude products in to DCM and then precipitated in diethyl ether. The
8

residual solvents were removed after drying in vacuum and the product is stored at 10 ℃ until
further use.
2
.5. Characterization
1
13
The H NMR and C NMR spectra of the synthesized compounds were recorded using Varian
Unity-600 NMR spectrometer (Varian, Inc., Palo Alto, CA, USA) at 600 MHz. All the samples
(
DSeDPA, mPEG-PLGA and Bi(mPEG-PLGA)-Se ) were dissolved in DMSO-d and analyzed
2
6
using the NMR spectrometer. The Raman peaks of Se-Se and Se-C in DSeDPA and Bi(mPEG-
PLGA)-Se were investigated by placing small amount of sample in silicon sample holder and
2
-1
the Raman spectra was scanned between 150 and 3500 cm using JASCO NRS-5100 Laser
Raman spectrometer. The molecular weights and polydispersities of mPEG-PLGA and
Bi(mPEG-PLGA)-Se were determined by Advanced Polymer Chromatogram (APC) system.
2
Samples containing 2 mg/mL of mPEG-PLGA and Bi(mPEG-PLGA)-Se were prepared using
2
THF as a solvent and measurements were done at 45 ℃ via ACQUITY Advanced Polymer
Chromatography (APC™) with a flow rate of 0.8 mL/min.
The particle size, ζ-potential of the micelles were determined by using dynamic light scattering
(DLS) instrument. In brief, a suitable amount of samples was dispersed in ultra-distilled water,
filtered with 0.45 µm pore sized filter and sonicated for 2 min prior to measurement. For each
parameter, triplicate measurements were performed and the results were expressed as mean ± SD.
The morphology of the Bi(mPEG-PLGA)-Se micelles were assessed from the images taken
2
using field emission scanning electron microscope (FESEM) (JSM-6500F, JEOL) and the major
elemental composition was acquired by energy dispersive X-ray spectroscopy (FESEM-EDS)
operated at 10.0 kV. Here, 2 mg of sample was dissolved in distilled water, dropped on the
silicon substrate and air dried for 48 h, then the SEM image and SEM-EDS data were recorded
9

after the sample was coated with platinum for 10 min. In addition, UV-vis spectrophotometer
JASCO V-730) and Fluorescence spectrophotometer (JASCO V-330) were used to determine
the absorbance and emissions spectra of samples respectively.
.6. Bi(mPEG-PLGA)-Se micelle formation and drug loading
(
2
2
Self-assembled Bi(mPEG-PLGA)-Se micelles were prepared using sonication and aqueous
2
dialysis (Zhang et al., 2019). Briefly, 20 mg of Bi(mPEG-PLGA)-Se was dissolved in a mixture
2
of 3 mL DMSO and 1 mL of distilled water followed by probe sonication for 2 minutes. The
resulting mixture was added to 10 mL water and stirred vigorously for 12 h. Then, the DMSO
was removed by dialysis using a dialysis membrane (MWCO 1000 Da) against deionized water
for 24 h, during which water was exchanged at 4 h intervals. The resulting micellar solution was
stored at 10 ℃ for further use and characterization. The DOX-loaded Bi(mPEG-PLGA)-Se2
micelles were prepared following a similar procedure. In this case, 2 mg DOX·HCl was
dissolved in 2 mL of DMSO and 5 µL of trimethylamine (TEA) was added and stirred for 5 min
in dark environment (to neutralize DOX·HCl and form free/unprotonated DOX). Then, it was
mixed with 20 mg of Bi(mPEG-PLGA)-Se in 3 mL DMSO solution and probe sonicated for 2
2
minutes. Finally, the mixture was added dropwise to 15 mL water with vigorous stirring. After
2
4 h, the residual DMSO and unbound DOX were removed by dialyzing against deionized water
MWCO 1000 Da) for 36 h. The DOX-loaded micellar solution were collected and stored at
0℃ for further characterization and application. The amount of DOX encapsulated was then
(
1
evaluated using UV-vis (JASCO V-730) spectrophotometer at 499 nm. The EE and DLC of the
micelles were calculated using the following equation:
Weight of DOX in the micelles
EE (%) =
× 100
Weight of DOX fed initially
1
0

Weight of DOX in the micelles
DLC (%) =
× 100
Weight of DOX ‒ loaded micelles
2
.7. Determination of critical micelle concentration (CMC)
Above the critical concentration, amphiphilic block copolymers self-associate in aqueous
environment to form stable micelles (Chaudhuri et al., 2009) and disassemble into free
surfactants when diluted at concentration below the CMC. This limits the in vivo bioavailability
of injected hydrophobic drugs encapsulated in micellar systems (Lu et al., 2018). Due to its
amphiphilic nature, aqueous Bi(mPEG-PLGA)-Se solution self-aggregate into micelle where the
2
mPEG portion forms the corona and the PLGA-Se-Se-PLGA segment constitutes the core of the
micelle. In this study, the CMC of Bi(mPEG-PLGA)-Se was determined by following
2
previously reported procedures (Song et al., 2011) with slight modifications. The Bi(mPEG-
PLGA)-Se copolymer was precisely weighted and dissolved in distilled water at 4-10 ℃ and the
2
stoke solution was incubated at 4 ℃ for 24 h. Then, different serial concentrations of Bi(mPEG-
-3
-4
-4
-5
-5
-6
-6
-7
PLGA)-Se copolymer (1 × 10 , 5 × 10 , 1 × 10 , 5 × 10 , 1 × 10 , 5 × 10 , 1 × 10 , 5 × 10 ,
2
-7
-8
1
× 10 , 5 × 10 ) were prepared by diluting the stoke solution with redistilled water. 100 µL of 2
-4
×
10 M solution of pyrene (in acetone) was placed in to a series of amber glass vials and
acetone was evaporated under dark conditions. The Bi(mPEG-PLGA)-Se solution with the
2
aforementioned concentrations were added in to the respective vials to give a final pyrene
-6
concentration of 2 × 10 M. All the solutions were sonicated and equilibrated in water bath at 60
for 1 h, stored overnight at room temperature prior to measurement. The CMC of Bi(mPEG-
℃
PLGA)-Se solutions were determined by fluorescent spectroscopy using pyrene as fluorescent
2
probe. The emission spectra of the solutions were recorded from 350 nm to 440 nm by using a
fluorescence spectrophotometer with an excitation wavelength of 334 nm and the slit width for
1
1

both excitation and emission were 2.5 nm. The intensity ratio of the first peak at 372 nm and the
third peak at 383 nm (I372/I383) in the pyrene emission spectra were plotted as a function of
polymer concentration (log C). The CMC value was determined from the intersection of the
tangent to the curve at the inflection with the horizontal tangent through the points of low
concentration.
2
.8. Redox responsive drug release study
The in vitro release profile of DOX from Bi(mPEG-PLGA)-Se micelles was studied using
2
dialysis method. In brief, 1 mL solution containing DOX-loaded micelles was placed in to a
dialysis tubes with a molecular weight cutoff 1000 Da and immersed into separate vials
containing 10 mL PBS, 0.1% H O in PBS and 6 mM GSH in PBS. The pH and temperature of
2
2
each releasing media was maintained at 7.4 and 37 ℃, respectively, to mimic the human
physiological environment. To quantify the rate of drug release, 2 mL aliquots from the dialysate
were taken at predetermined time intervals (1, 2, 3, 4, 6, 12, 24, 48 and 72 h). Then, the
concentration of released drug molecule in the dialysate was measured at 499 nm (absorption
peak of DOX) using UV-vis spectrometry. To keep the total volume of the solution constant, 2
mL of freshly prepared PBS buffer solution (with the same pH and H O /GSH concentration)
2
2
was added after each sampling. The cumulative release of DOX was calculated as the total
percentage of DOX molecule released across the dialysis membrane over time using a pre-
established calibration curve of DOX. Cumulative drug release was calculated as (Hailemeskel et
al., 2018):
(
Absorbance ± intercept)
Slope
Concentration of drug (mg/mL) =
2
.9. In vitro cytotoxicity and cell viability study
1
2

The cytotoxicity of Bi(mPEG-PLGA)-Se micelles was investigated according to previously
2
reported method (Addisu et al., 2018; Andrgie et al., 2019; Mekuria et al., 2018). Briefly, HaCaT
and HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10% fetal bovine serum (FBS), 1% penicillin, 1% glutamine, and 1% sodium pyruvate at 37
4
℃
and 5% CO . The cells were seeded into a 96-well plate at a density of 1 × 10 cells/well in
2
complete DMEM and maintained in a humidified incubator at 37 ℃ under 5% CO for 24 h.
2
Different concentrations of Bi(mPEG-PLGA)-Se (10, 20, 500, 100, 200 and 400 µg/mL) were
2
added with fresh culture media in each well and incubated at 37 ℃ under 5% CO . After 24 h
2
incubation, the culture medium was aspirated and replaced by 100 µL fresh medium and 20 µL
MTT solution with a final concentration of 5 mg/mL and cultured for another 4 h at 37 ℃ for the
formation of formazan dye. Then, the medium was replaced with 100 µL of DMSO in order to
dissolve the formazan crystals. To estimate the proportion of viable cells in each well,
absorbance was measured at 570 nm using an enzyme-linked immunosorbent assay (ELISA)
reader (Multiskan FC, Skanlt, Software 3.0.0.64 RE). Similarly, the cell viability of free DOX
and DOX-loaded Bi(mPEG-PLGA)-Se on HaCaT and HeLa cells were investigated using the
2
same protocol (at DOX concentration of 0.25, 0.5, 1.0, 2.5, 5 and 7.5 µg/mL). The results were
quantified as the percentage of viable cells after treatment compared to a control of untreated
cells. The cell viability was expressed using the following formula:
Absorbance of test cells
Cell viability (%) =
× 100
Absorbance of control cells
2
.10. Cellular uptake study
The cellular uptake and intracellular drug release profile of DOX-loaded Bi(mPEG-PLGA)-Se2
micelles in HaCaT and HeLa cell lines were tracked by fluorescence microscope. The cells were
1
3

grown in DMEM supplemented with 10% fetal bovine serum (FBS), 1% penicillin, 1%
glutamine, and 1% sodium pyruvate at 37 ℃ and 5% CO . In order to visualize the cellular
2
uptake of DOX-loaded Bi(mPEG-PLGA)-Se , both HaCaT and HeLa cells were seeded into a 35
2
5
mm glass-bottom culture dish (MatTek Corporation) at a density of 2 × 10 cells/well in
complete DMEM and maintained in a humidified incubator at 37 ℃ under 5% CO for 24 h.
2
Then, the cells were co-incubated with free DOX and DOX-loaded Bi(mPEG-PLGA)-Se2
micelles at an equivalent DOX concentration of 3 µg/mL for 2, 4, and 6 h. Subsequently, the
culture medium was removed and cells were carefully washed with PBS three times followed by
staining with 300 nm of DAPI for 15 minutes to visualize the nuclei of HaCaT and HeLa cells as
per the standard protocol provided of the supplier. The cells were again rinsed twice with PBS
buffer and fixed with 4% paraformaldehyde solution (pH = 7.4). Finally, cellular images were
captured (using iRiSTM Digital Imaging System from Logos Biosystems) at excitation
wavelength 480 nm (red) and 340 nm (blue) to screen the release and localization DOX.
3
. Results and discussion
3
.1. Synthesis of DSeDPA, mPEG-PLGA and Bi(mPEG-PLGA)-Se2
Recently, different polymeric micelles have been synthesized as carriers of chemotherapeutic
agents due to their ability to enhance therapeutic efficacy of their payload in the region of
interest (Deepagan et al., 2016; Martínez-Jothar et al., 2019; Wei et al., 2017). In this study,
redox responsive polymeric micelle was designed and fabricated by linking two mPEG-PLGA
moieties by DSeDPA. The synthesized Bi(mPEG-PLGA)-Se2 could self-assembled into
noncovalent aggregates due to the amphiphilic nature of mPEG-PLGA-Se-Se-PLGA-mPEG
(Scheme 2). Moreover, the micelles would respond to the intracellular redox stimuli and release
the encapsulated DOX in cancer environment due to the conversion of diselenide linkage in to
1
4

selenol (-SeH) and seleninic acid (-SeOOH) via GSH and H O , respectively (Wang et al., 2014).
2
2
As shown in Scheme 1, the redox-labile linker DSeDPA was synthesized by reacting Na Se
2
2
with 3-chloropropanoic acid. First, the Na Se was successfully synthesized when selenium
2
2
powder is treated with NaBH (1:1 mole ratio). Then, the reddish brown product was reacted
4
with 3-chloropropanoic acid to form DSeDPA. The yield and the purity of DSeDPA was
controlled by pH adjustments. The pH of the 3-cholorpropanioic acid was changed to 8.0 prior to
reaction to minimize the formation of undesired products (H Se ) and unreacted DSeDPA was
2
2
removed when the pH is adjusted to 3-4 by 1M HCl (Cheng et al., 2012).
The amphiphilic block copolymer, mPEG-PLGA was synthesized by ring opening
copolymerization of LA and GA using mPEG as macro-initiator in nitrogen environment. Then,
the carboxyl groups of DSeDPA were activated by DCC and DMAP at 0 ℃ in anhydrous THF
and coupled with mPEG-PLGA through ester bond. The synthesis of DSeDPA and Bi(mPEG-
1
13
1
PLGA)-Se was validated by H NMR, C NMR and Raman spectroscopic techniques. In the H
2
NMR of DSeDPA, the methylene protons adjacent to the diselenide bond and the carboxylic
group appeared at δ 2.70 and δ 3.05 ppm respectively (Figure S1A). Similarly, the peaks for Se-
Se and Se-C stretching vibrations appeared with slight shift in their positions in the Raman
spectra (Figure S1B) were assigned to the Se-Se and Se-C bonds, respectively. All these
confirmed the successful synthesis of DSeDPA and were in agreement with previous reports
1
(
Cheng et al., 2012; Hailemeskel et al., 2018). In the H NMR spectrum of mPEG-PLGA (Figure
S1C), the peaks at 3.42 ppm (CH O-), between 3.51 ppm and 4.25 ppm (-OCH CH O-) were
3
2
2
attributed to the protons of mPEG. The integrated proton peaks at δ 1.46 ppm and δ 5.20 ppm
belonged to the LA repeating units (-CH(CH )O-). Moreover, the appearance of characteristic
3
peak at δ 4.86 ppm was assigned to the methylene protons of GA repeating units (-CH O-)
2
1
5

verified the synthesis of mPEG-PLGA (Wang et al., 2011). As shown in Figure 1A, in addition
to the characteristics peaks of mPEG-PLGA, new peaks appeared at δ 2.86 and δ 3.10 ppm
which belonged to the protons of Bi(mPEG-PLGA)-Se adjacent to Se-Se bond (-CH -Se-Se-
2
2
CH -) and carbonyl group (–Se-CH CH CO-), respectively (Ma et al., 2018). Apparently, the Se-
2
2
2
Se and Se-C bond stretching vibrations were also observed in the Raman spectrum of Bi(mPEG-
-1
-1
PLGA)-Se (Figure 1B) at 253 cm and 288 cm respectively, which verified the successful
2
1
3
conjugation of DSeDPA and mPEG-PLGA. The C NMR spectral data shown in Figure S2A-C
further confirmed the successful synthesis of the Bi(mPEG-PLGA)-Se copolymer.
2
According to molecular weight measurement by gel permeation chromatography (Advanced
Polymer Chromatography, APC™), the number average molecular weight of mPEG-PLGA and
Bi(mPEG-PLGA)-Se was found to be 2175 g/mol and 4534 g/mol, respectively with unimodal
2
distributions (Table 1). As expected, the molecular weight of Bi(mPEG-PLGA)-Se was two-
2
fold higher than the molecular weight of the mPEG-PLGA, which is consistent with the result
1
obtained from the H NMR data. Moreover, the higher molecular weight Bi(mPEG-PLGA)-Se
2
was eluted first and exhibited relatively small retention time as compared to lower molecular
weight mPEG-PLGA (Figure 1C). These results clearly asserted the successful conjugation of
mPEG-PLGA with DSeDPA via DCC/DMPA chemistry.
3
.2. Preparation and characterization of Bi(mPEG-PLGA)-Se micelles
2
PEG and PLGA copolymers, approved by FDA for human use, are getting significant attention
for biomedical applications due to their biodegradable and biocompatible properties (Chu et al.,
2
016). PEG-coated nanoparticles or micelles with PEG corona were frequently used as
intravenous delivery vehicles for hydrophobic drugs, which could increase the blood circulation
time and reduce liver accumulation (Chen et al., 2017). For efficient anticancer activity, the
1
6

blank and DOX-loaded Bi(mPEG-PLGA)-Se micelles should be small enough to escape
2
detection and destruction by the reticuloendothelial system (RES) (Nagheh et al., 2017). In this
study, the size and surface charge of the micelles were characterized by ζ-potential analyzer with
DLS capability. Table 2 summarized the average particle size and zeta potential of the micelles.
The bank Bi(mPEG-PLGA)-Se micelles showed an average particle size of 123.9 ± 085 nm
2
with a unimodal particle distribution (PDI = 0.26 ± 0.014) and its ζ-potential was -2.67 ± 1.8 mV.
The uniformity in the average size of the micelles for multiple measurements may be attributed
to the presence of mPEG head capable of maintaining constant size for relatively longer
incubation time (Avgoustakis et al., 2003). Moreover, the presence of diselenide bond in the
hydrophobic core could promote and stabilize the self-assembly of Bi(mPEG-PLGA)-Se2
micelles by balancing the competition between intermolecular forces (Wang et al., 2018). As
expected, the ζ-potential value of the blank micelles was closer to neutral due to the mPEG
corona which covered the surface charges (Avgoustakis et al., 2003; Xu et al., 2015). The DOX-
loaded Bi(mPEG-PLGA)-Se micelles demonstrated relatively larger hydrodynamic diameter
2
(
156 ± 6.5 nm) than blank micelles, which might be attributed to the insertion of hydrophobic
DOX in the core making the amphiphilic copolymers form interweaved structure (Zhang et al.,
019). The morphology of the Bi(mPEG-PLGA)-Se micelles were examined by Scanning
2
2
Electron Microscopy (SEM) (Figure 2A). The image revealed that the micelles were dispersed as
individual particles with a well-defined spherical structure with uniform size, around 100 nm. As
evaporation of water molecules happened during sample preparation procedure for SEM
scanning, it is inevitable that the hydrodynamic diameter of micelles in SEM image was slightly
smaller than the DLS result (Table 2).
3
.3. Critical micelle concentration (CMC) of copolymer
1
7

The fluorescence intensity of pyrene senses the polarity of solvents in its vicinity. This tendency
of pyrene is widely used for the determination of CMC. Pyrene shows five major vibrational
fluorescence bands (372, 379, 383, 388 and 393), which vary depending on the polarity of
nearby solvent (Chakraborty et al., 2011). The ratio of the fluorescence intensities of the first and
third vibronic bands of pyrene (I1/I3) is indicative of the local environment and increases
characteristically with increasing polarity of the probe environment (Chaudhuri et al., 2009;
Piñeiro et al., 2015). In this study, self-assembled Bi(mPEG-PLGA)-Se micelles were formed
2
by sonication and aqueous dialysis. At lower concentrations, pyrene sensed the local aqueous
environment (Figure 3A). Consequently, the ratio of the first and the third vibronic band (I1/I3)
was relatively higher. As the concentration of Bi(mPEG-PLGA)-Se increased and reached the
2
CMC level, I1/I3 became low. This is because the hydrophobic pyrene molecule tend to
accumulate more strongly in the core of the micelle than water and feel the less polar
surrounding environment (Chakraborty et al., 2011). As shown in Figure 3B, the CMC of the
-5
Bi(mPEG-PLGA)-Se copolymer at room temperature was found to be 1.39 × 10 g/mL. In
2
addition, we determined the CMC of mPEG-PLGA under the same experimental conditions as a
control group (Figure S2A) and it was 2.6-fold (3.55 × 10-5 g/mL) higher than CMC of
Bi(mPEG-PLGA)-Se . The relatively lower CMC value of Bi(mPEG-PLGA)-Se may be
2
2
attributed to the presence of hydrophobic diselenide linkage in the core of the micelles which
could play pivotal role in self-assembling and thereby improve micellar stability. The result
indicated that Bi(mPEG-PLGA)-Se micelles are more stable than low molecular weight
2
surfactants and can maintain its integrity in dilute solutions (Debele et al., 2017).
3
.4. Stability and redox responsiveness of micelles
1
8

The in vitro redox responsive nature of Bi(mPEG-PLGA)-Se micelles were investigated by
2
measuring the hydrodynamic diameter after 2, 3, 4 and 6 h treatment with 0.1% H O and 6 mM
2
2
GSH. As shown in Figure S2B, the size of the H O and GSH treated micelles significantly
2
2
increased from 123.9 ± 0.85 to 451.8 ± 21.46 and 480.53 ± 65.1, respectively within 6 h
incubation time. The result clearly confirmed the redox responsive nature of the micelles and
highlighted their importance in the transport and controlled release of drugs in cancer cells. The
increase in hydrodynamic diameter of the micelles may be attributed to the oxidation and
reduction of diselenide bond accompanied by phase change in the core of the micelles (Wang et
al., 2014). This in turn triggered lose micellar cores and gradual increase in size of the Bi(mPEG-
PLGA)-Se micelles. Furthermore, low molecular weight mPEG shell could have contributed for
2
time dependent micellar size increment due to its minimal shielding effect towards H O and
2
2
GSH (Sun et al., 2017). Hence, the stimuli could have accessed the core of the micelle with great
ease.
The interaction between polymeric micelles and serum proteins is of great importance for the
circulation time and in vivo anticancer activities of payloads (Xu et al., 2015). In this study,
Bi(mPEG-PLGA)-Se micelles were incubated in 40 mg/ml Bovine Serum Albumin (BSA) in
2
PBS (pH = 7.4) and the change in size was monitored at different time points. After 72 h
incubation, the hydrodynamic size of the blank and DOX-loaded Bi(mPEG-PLGA)-Se micelles
2
was found to be 126.49 ± 1.94 and 159.25 ± 2.94, respectively. There was no significant
difference in the hydrodynamic diameter between BSA treated and untreated micelles (Figure
S2C). The result suggested that the Bi(mPEG-PLGA)-Se micelles have good stability in serum
2
protein. By virtue of its “stealth” property, the mPEG corona could have contributed for the
1
9

observed stability of the blank and DOX-loaded Bi(mPEG-PLGA)-Se micelles (Avgoustakis et
2
al., 2003; Hu et al., 2018; Xu et al., 2015).
3
.5. Drug loading behavior of Bi(mPEG-PLGA)-Se micelles
2
Most hydrophobic drugs are characterized by precipitation and degradation in the blood stream
before reaching their target, and thus cause severe side-effects (Dutta et al., 2009). To alleviate
the problem of toxicity, minimize the frequency of drug administration and enhance therapeutic
efficacy of hydrophobic drugs, various delivery systems have been developed (Wang et al.,
2
011). Over the past few decades, there has been tremendous effort to establish PEG-PLGA
based nanostructures as potential drug delivery vehicles for chemotherapeutic agents
Gholizadeh et al., 2018; Hu and Zhang, 2010; Klippstein et al., 2015; Nagheh et al., 2017; Song
(
et al., 2011; Yang et al., 2016; Zhang et al., 2014b). In Bi(mPEG-PLGA)-Se , by virtue of the
2
mPEG and PLGA-Se-Se-PLGA segments having different solubility, there would be micro-
phase separation which occur locally. Consequently, the micelle core is spontaneously formed
(
Deepagan et al., 2016). The physical entrapment of DOX in Bi(mPEG-PLGA)-Se micelles
2
believed to takes place mainly through hydrophobic interaction between the core of the micelles
and the drug molecules. Moreover, DLC and EE are the contributing factor for the therapeutic
efficacy of drug-loaded micelles. In this study, sonication followed by aqueous dialysis were
used to load DOX in to the inner core of Bi(mPEG-PLGA)-Se micelles. According to the
2
absorbance measurement (Figure S3), the DLC and EE of DOX-loaded micelles were
approximately 6.61 wt% and 54.9% respectively. Compared to previous PEG-PLGA based
nanoparticles, improved drug loading content was observed in Bi(mPEG-PLGA)-Se micelles
2
(Avgoustakis et al., 2002; Liu et al., 2014; Song et al., 2011; Yoo and Park, 2001). This might
2
0

partly correspond to the hydrophobic diselenide linkage in the core of the micelle that can form
strong hydrophobic interaction with DOX molecules.
3
.6. Drug releasing trends of DOX-loaded Bi(mPEG-PLGA)-Se2 micelles
For effective anticancer activity of chemotherapeutic agents, the drug releasing behavior of
nanoparticles (strongly linked with the nature of the stimuli and sites of action) is equally
important as their DLC. Upon treatment of hydrophobic diselenide bonds with GSH and H O ,
2
2
hydrophilic reduction (-SeH) and oxidation (-SeOOH) products are formed. Consequently, the
nanoparticles undergoe phase change and disassemble in to its parts (Sun et al., 2017). This
process has been implicated in the redox responsive release of payloads from diselenide
containing nanostructures. Based on this fact, DOX release from DOX-loaded Bi(mPEG-
PLGA)-Se micelles was evaluated using PBS buffer (pH 7.4) alone and in the presence of 6 mM
2
GSH and 0.1% H O at 37℃, which mimicked the intracellular redox environment. The release
2
2
of DOX was measured at predetermined times intervals for 72 h. As shown in Figure 4, the
overall DOX release was higher in the presence of GSH and H O as compared to free PBS
2
2
buffer. This could be endorsed to the redox stimuli responsive nature of diselenide linkages in
Bi(mPEG-PLGA)-Se leading to micellar destabilization followed by gradual DOX release from
2
the core of Bi(mPEG-PLGA)-Se micelles. For the first 4 h, fast DOX release was observed in
2
all scenarios (28.91% in PBS, 56.54% 6 mM in GSH and 50.02% in 0.1% H O releasing media).
2
2
During drug loading procedure, the DOX enters the hydrophobic compartments and even in the
interfacial region between the hydrophobic and hydrophilic chains. This might be responsible for
the initial burst DOX release observed in the study (Ostacolo et al., 2010; Zhang et al., 2014a).
Approximately 74% and 69% of DOX were released after 72 h incubation in the presence of
GSH and H O respectively, which clearly indicated that the Bi(mPEG-PLGA)-Se micelles
2
2
2
2
1

were redox sensitive. Apart from this, the observed drug releasing behavior might be due to low
molecular weight of mPEG employed in micellar architecture that make its hydrophobic part
easily accessible for intracellular GSH and H O (Sun et al., 2017). The incomplete drug
2
2
releasing behavior of DOX-loaded Bi(mPEG-PLGA)-Se micelles might be attributed to the
2
hydrophobic interaction between DOX molecules and the hydrophobic PLGA segment (Chen et
al., 2017), which requires days to weeks for release.
3
.7. In vitro cytotoxicity study
Kinetic stability and biocompatibility of micelles are vital to achieve a steady state concentration
of drugs in the bloodstream for sufficiently longer period of time (Hoffman, 2008). It has been
reported in literature that free PEG-PLGA nanoparticles had no cytotoxic effect on cells
(
Koopaei et al., 2014). To assess the safety of blank Bi(mPEG-PLGA)-Se micelles for drug
2
delivery applications, MTT dye reduction assay was implemented to quantify the in vitro cell
viability on both HaCaT and HeLa cell lines at different concentrations (10, 20, 50, 100, 200 and
4
00 µg/mL). After 24 h of incubation, more than 84% of both HaCaT and HeLa cells were viable
against blank Bi(mPEG-PLGA)-Se micelles (Figure 5A). The result revealed that Bi(mPEG-
2
PLGA)-Se micelles were safe and there was no any observed toxicity towards HeLa and HaCaT
2
cells even at higher doses (400 µg/mL). The diselenide linked copolymer established excellent
biocompatibility towards HeLa and HaCaT cell lines, which could be attributed to the safety
profiles of each constituent polymers, mPEG and PLGA (Hu and Zhang, 2010). Moreover, the
Se-Se bond can also be metabolized and removed from the body without causing any harm (Xu
et al., 2013), which further assured the biocompatibility of the Bi(mPEG-PLGA)-Se micelles.
2
3
.8. In vitro cell viability study of DOX-loaded micelles
2
2

In this study, the in vitro anticancer efficiency of free DOX and DOX-loaded micelles (with 0.25,
.5, 1.0, 2.5, 5.0 and 7.5 µg/mL equivalent DOX concentrations) were investigated against HeLa
0
and HaCaT cells after 24 h of incubation. As shown in Figure 5B, DOX-loaded micelles induced
pronounced antitumor effect on HeLa cells (only 44.97% cells were viable) at maximum dose of
7
.5 µg/mL of DOX concentration. On the other hand, relatively weak cytotoxic effect (84.28%)
was observed in HaCaT cells (Figure 5C). The observed differences in antitumor effect was due
to the high level of GSH and H O in HeLa cells that stimulate the release DOX from the core of
2
2
the micelles. Thus, gradual release and accumulation of DOX was accompanied by translocation
in to the nucleus of cells and thereby cell death. As expected, the diminished antitumor effect of
the DOX-loaded Bi(mPEG-PLGA)-Se micelles towards normal cells as compared to HeLa cells
2
was associated with the relatively lower intracellular level of GSH and H O in HaCaT cell lines
2
2
(4- to 10-fold lower than HeLa cells). Due to this fact, most of the DOX-loaded micelles will
remain intact inside normal cells and do not cause any significant antitumor activity as observed
in this study. Consistent with previously reported DOX-loaded micelles (Chen et al., 2016; Hu et
al., 2018), DOX-loaded Bi(mPEG-PLGA)-Se micelles showed lower inhibition efficiency (IC
2
50
=
4.25 µg/mL) as compared to free DOX (IC = 0.59 µg/mL) at equivalent DOX concentrations
50
(26.13% viability for HeLa cells). This was due the pronounced initial inhibition efficiency of
free DOX with full exposure to cells in 24 h (Hu et al., 2018) and the incomplete redox triggered
DOX release, approximately 69% in 24 h (Figure 4) could have lowered the efficacy of DOX-
loaded Bi(mPEG-PLGA)-Se micelles in HeLa cell line (Zhuang et al., 2018). The result is
2
consistent with previous reports (Hailemeskel et al., 2019).
3
.9. Cellular uptake of DOX-loaded Bi(mPEG-PLGA)-Se micelles
2
2
3

Efficient accumulation and/or internalization of drug carriers is a prerequisite for controlled
release and pharmacological effectiveness of drugs in cancer tissues. To investigate the passive
accumulation of DOX-loaded Bi(mPEG-PLGA)-Se micelles inside cancer cells, a time
2
dependent cellular uptake study was conducted on HaCaT and HeLa cell lines. The inherent
fluorescence intensity of DOX was used to monitor cellular accumulation and release
qualitatively. Figure 6 showed fluorescent microscope images of HeLa and HaCaT cells treated
with 3 µg/mL free DOX and DOX-loaded Bi(mPEG-PLGA)-Se micelles for 2 h, 4 h and 6 h of
2
incubation. The release of DOX from the micelles and subsequent translocation into the nuclei of
cancer cells were clearly observed from the merged images of red fluorescence of DOX and blue
fluorescence of DAPI. As shown in the Figure 6A, for 2 h incubation with DOX-loaded micelles,
relatively weak fluorescence intensity of DOX was observed in the cytoplasm and nuclei of
HeLa cells. The fluorescent intensity of DOX was significantly increased both in the cytoplasm
and nuclei in a time dependent manner (for 4 h and 6 h incubation) confirming the internalization
of DOX-loaded micelles and intracellular drug release. On the other hand, there was no visible
fluorescent intensity of DOX in HaCaT cells incubated with DOX-loaded micelles, especially for
2
h and 4 h treatments (Figure 6B). When the time of incubation was increased to 6 h, very little
DOX fluorescent signal was observed in HaCaT cells which confirmed the cellular
internalization of DOX-loaded Bi(mPEG-PLGA)-Se micelles. The observed intense red
2
fluorescent intensity in HeLa cells (as compared to HaCaT cells) could be ascribed to the high
level of intracellular GSH and H O that could cleave the redox-sensitive diselenide bond and
2
2
trigger DOX release from the core of DOX-loaded Bi(mPEG-PLGA)-Se micelles. Both free
2
DOX treated HeLa cells (Figure 6C) and HaCaT cells (Figure 6D), showed pronounced red
fluorescent intensity as compared to cells treated with DOX-loaded micelles. Particularly for
2
4

HeLa cells, it is strongly associated with the difference in cellular internalization mechanisms
followed by free DOX and DOX-loaded micelles; free DOX is thought to be internalized mainly
through fast passive diffusion (Tacar et al., 2013). Because of this notable red fluorescent was
observed strating from 2 h treatment. In comparison with free DOX, the cellular uptake of DOX-
loaded Bi(mPEG-PLGA)-Se micelles were slow and only insignificant red fluorescent was
2
observed at 2 h. As the treatment time increased to 4 h and 6 h more DOX-loaded micelle
entered in to the cells resulting in release of DOX molecules and intense red fluorescent intensity
inside the cell, including the nuclei (Zhuang et al., 2018). The in vitro anticancer activities of
DOX-loaded micelles observed in the MTT assay (Figure 5B) was in agreement with the cellular
uptake result, suggesting efficient internalization and gradual redox responsive release of DOX
from Bi(mPEG-PLGA)-Se micelles occurred in HeLa cells.
2
4
. Conclusion
In summary, a redox responsive diselenide linked Bi(mPEG-PLGA)-Se2 micelle was
successfully prepared as a delivery vehicle for DOX and its controlled release in cancer tissue
environment. The amphiphilic Bi(mPEG-PLGA)-Se copolymer was synthesized by coupling
2
mPEG-PLGA segments with the redox labile linker, DSeDPA, via DCC/DMAP click chemistry.
The conjugate was capable of forming stable micelles and shown to have good DLC and
relatively lower critical micelle concentrations. Upon exposure to redox environment, the DOX-
loaded Bi(mPEG-PLGA)-Se micelle released its cargo in a rapid and controlled manner due to
2
the cleavage of diselenide bonds. The initial burst release was accompanied by sustained release
of DOX for 72 h from the micellar core. The in vitro cytotoxicity study confirmed that
Bi(mPEG-PLGA)-Se was devoid of any intrinsic toxicities towards HaCaT and HeLa cells.
2
Moreover, the in vitro cell viability study revealed that the DOX-loaded micelle significantly
2
5

decreased the cancer cell load within 24 h at 7.5 µg/mL DOX concentration. The fluorescent
images of HeLa cells further proved the cellular internalization of DOX-loaded Bi(mPEG-PLGA)-
Se micelles and redox stimuli triggered release of DOX in a time dependent manner. In
2
conclusion, the fabrication of redox stimuli responsive Bi(mPEG-PLGA)-Se micelle highlighted
2
the potential use of mPEG-PLGA based block polymers in the design of safe, stable and smart
drug carriers for hydrophobic anticancer drugs.
Acknowledgment
The authors would like to thank the Ministry of Science and Technology, Taiwan (MOST105-
2
221-E-011-133-MY3 and 1052221-E-011-151-MY3) for providing financial support.
Declaration of interest
Authors declare that there are no competing interests.
2
6

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