Smart liposomal drug delivery for treatment of oxidative stress model in human embryonic stem cell-derived retinal pigment epithelial cells

Article abstract of DOI:10.1016/j.ijpharm.2018.05.056

Oxidative stress has been implicated in the progression of age-related macular degeneration (AMD). Treatment with antioxidants seems to delay progression of AMD. In this study, we suggested an antioxidant delivery system based on redox-sensitive liposome composed of phospholipids and a diselenide centered alkyl chain. Dynamic light scattering assessment indicated that the liposomes had an average size of 140 nm with a polydispersity index below 0.2. The percentage of encapsulation efficiency of the liposomes was calculated by high-performance liquid chromatography. The carriers were loaded with N-acetyl cysteine as a model antioxidant drug. We demonstrated responsiveness of the nanocarrier and its efficiency in drug delivery in an oxidative stress model of human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells. The modeled cells treated with diselenide containing liposomes loaded with 10 mM NAC, showed a better therapeutic effect with a cell metabolic activity of 90%, which was significantly higher compared to insensitive liposomes or NAC treated groups (P < 0.05). In addition, the expression of oxidative-sensitive gene markers in diselenide containing liposomes groups were improved. Our results demonstrated fabricated smart liposomes opens new opportunity for targeted treatment of retinal degeneration.

Full text of DOI:10.1016/j.ijpharm.2018.05.056

Accepted Manuscript
Smart Liposomal Drug Delivery for Treatment of Oxidative Stress Model in
Human Embryonic Stem Cell-derived Retinal Pigment Epithelial Cells
Farnaz Behroozi, Mohammad-Jafar Abdkhodaie, Hamid Sadeghi Abandansari,
Leila Satarian, Mohammad Kazemi Ashtiani, Mahmoud Reza Jaafari, Hossein
Baharvand
PII:
S0378-5173(18)30347-8
https://doi.org/10.1016/j.ijpharm.2018.05.056
IJP 17527
DOI:
Reference:
International Journal of Pharmaceutics
To appear in:
Received Date:
Revised Date:
Accepted Date:
14 January 2018
18 April 2018
15 May 2018
Please cite this article as: F. Behroozi, M-J. Abdkhodaie, H.S. Abandansari, L. Satarian, M.K. Ashtiani, M.R. Jaafari,
H. Baharvand, Smart Liposomal Drug Delivery for Treatment of Oxidative Stress Model in Human Embryonic
Stem Cell-derived Retinal Pigment Epithelial Cells, International Journal of Pharmaceutics (2018), doi: https://
doi.org/10.1016/j.ijpharm.2018.05.056
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Smart Liposomal Drug Delivery for Treatment of Oxidative Stress Model in Human
Embryonic Stem Cell-derived Retinal Pigment Epithelial Cells
Farnaz Behroozi1, Mohammad-Jafar Abdkhodaie1, *, Hamid Sadeghi Abandansari2, Leila
Satarian3, Mohammad Kazemi Ashtiani2, Mahmoud Reza Jaafari4, 5,*, Hossein Baharvand3, 6,*
1. Department of Chemical and Petroleum Engineering, Sharif University of Technology,
Tehran, Iran.
2. Department of Cell Engineering, Cell Science Research Center, Royan Institute for Stem
Cell Biology and Technology, ACECR, Tehran, Iran.
3. Department of Stem Cells and Developmental Biology, Cell Science Research Center,
Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
4. Nanotechnology Research Center, Pharmaceutical Technology Institute, Mashhad
University of Medical Sciences, Mashhad, Iran.
5. Department of Pharmaceutical Nanotechnology, School of Pharmacy, Mashhad
University of Medical Sciences, Mashhad, Iran.
6. Department of Developmental Biology, University of Science and Culture, Tehran, Iran.
*Correspondence
Baharvand@Royaninstitute.org
and
abdmj@sharif.edu
and
JafariMR@mums.ac.ir
1

Abstract
Oxidative stress has been implicated in the progression of age-related macular degeneration
(AMD). Treatment with antioxidants seems to delay progression of AMD. In this study, we
suggested an antioxidant delivery system based on redox-sensitive liposome composed of
phospholipids and a diselenide centered alkyl chain. Dynamic light scattering assessment
indicated that the liposomes had an average size of 140 nm with a polydispersity index below
0.2. The percentage of encapsulation efficiency of the liposomes was calculated by high-
performance liquid chromatography. The carriers were loaded with N-acetyl cysteine as a model
antioxidant drug. We demonstrated responsiveness of the nanocarrier and its efficiency in drug
delivery in an oxidative stress model of human embryonic stem cell-derived retinal pigment
epithelial (hESC-RPE) cells. The modeled cells treated with diselenide containing liposomes
loaded with 10 mM NAC, showed a better therapeutic effect with a cell metabolic activity of
90%, which was significantly higher compared to insensitive liposomes or NAC treated groups
(P<0.05). In addition, the expression of oxidative-sensitive gene markers in diselenide containing
liposomes groups were improved. Our results demonstrated fabricated smart liposomes opens
new opportunity for targeted treatment of retinal degeneration.
Keywords: Redox sensitive, Liposome, Diselenide, Drug delivery, Human embryonic stem
cells, Retinal pigment epithelium cells, Age macular degeneration
2

1. Introduction
Age-related macular degeneration (AMD) accounts for 8.7% of blindness worldwide (Cai et al.,
2000). Its prevalence increases exponentially after the age of 70 (Damico et al., 2012). Elderly
people may suffer from loss of photoreceptors in the central retina, which is associated with
retinal pigment epithelium (RPE) apoptosis due to harmful oxidative stress and diminished
antioxidant defenses (Roth et al., 2004). There is currently no effective treatment for the most
common form of this disease (dry AMD). In recent years, human embryonic stem cells (hESCs)
providing a number of clinically attractive features, including the capacity for indefinite self-
renewal and the potential to differentiate into RPE in vitro [for review see (Parvini M, 2014)].
These hESC-derived RPE (hESC-RPE) cells are similar to native RPE in expression of markers,
transcriptome, and function (Parvini M, 2014). These hESC-RPE cells are a promising source for
the treatment of AMD diseases (Schwartz SD, 2015; Mandai M, 2017) and modeling of AMD
diseases for testing potential proposed drug and carriers (Kaczara P, 2010).
The invention of drug delivery systems has a tremendous impact on medicine, especially the
field of retinal drug delivery (Thrimawithana Thilini, 2011). Nanoparticle drug delivery systems
show potential for treatment of dry AMD in the posterior segment of the eye due to their
capability to overcome ocular barriers, enhance permeation, improve drug water solubility,
decrease frequent drug administration, protect administered drugs, and enable sustained, targeted
drug release (Delplace et al., 2015; Lin et al., 2015). The nano-based drug delivery systems such
as nanoemulsions, niosomes, dendrimers, nanoparticle loaded contact lenses (Hsu et al., 2014),
and liposomes have gained tremendous attention due to their ability to enhance bioavailability of
drug in different route of administration including systemic, topical, and injected drugs (Sahoo et
al., 2008). Among these, liposomes appear to be suitable candidates’ due to their ability to
3

encapsulate both hydrophobic and hydrophilic drugs, biocompatibility, enhancement of drug
retention, and localization. According to Hathout et al., multilamellar liposomes that contained
acetazolamide were more efficient than naked drug in lowering intraocular pressure (Hathout et
al., 2007). As reported by Shen and Tu, the ocular bioavailability of liposomal ganciclovir in
rabbits was 1.7-fold higher compared to naked drug (Shen and Tu, 2007). Products such as
Visudyne (Sharman et al., 1999) are used to treat choroidal neovascularization in wet AMD.
Smart liposomes, which release medications upon exposure to triggers, are of interest. Their
spatial and temporal specificity could provide better local control over the release at the
appropriate time and place (Torchilin, 2014). In AMD, a redox sensitive liposome could provide
targeted controlled delivery of the drug to the retina, enhance drug efficacy, and reduce drug off-
target. This was achieved by incorporation of reduction-sensitive lipid like conjugate into
liposome (Goldenbogen et al., 2011). Also, phospholipase-triggered drug release that form
liposomal carriers (Arouri et al., 2015; Davidsen et al., 2003), reduction triggered delivery with
disulfide conjugates of poly (ethylene glycol) and phospholipids (Zalipsky et al., 1999), and
redox-triggered liposomes built from a quinone-lipid conjugate have been reported (Farokhzad
and Langer, 2009). Inspired by the success of disulfide bonds, selenium (Se), an element listed in
the same family as S in the periodic table of elements (chalcogens), has recently attracted great
attention for their reduction responsiveness and their sensitivity to low concentrations of
oxidative agents. Se-higher sensitivity is due to weaker bond energy of the C-Se (244 kJ/mol)
and Se-Se bonds (172 kJ/mol) compared to the C-S (272 kJ/mol) and S-S (240 kJ/mol) bonds
(Song and Du, 2014). Despite numerous advantages, no work has reported the design of redox-
sensitive liposomes containing diselenide linkages.
In this study, we designed a novel redox liposome formulation based on incorporation of a
4

diselenide to treat oxidative stress model in hESC-derived RPE cells. These liposomes contained
an alkyl chain in combination with Egg L-α-phosphatidylcholine (Egg PC), as the membrane
destabilizing component and 1,2-dio-leoyl-sn-glycero-3-phosphoethanolamine (DOPE). The
diselenide containing liposome can disrupt at redox conditions and release its payload (Fig. 1).
Physicochemical and biological characteristics of liposome were investigated for particle size,
zeta potential, and morphology. furthurmore, oxidative-induced degradation, drug loading
capacity, drug release, in vitro cytotoxicity, and in vitro anti-oxidative effects of the carrier were
studied.
2. Materials and Methods
2.1. Materials
Egg L-α-phosphatidylcholine (Egg, Chicken), 1,2-dio-leoyl-sn-glycero-3-phosphoethanolamine
(DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and polycarbonate membrane
filter were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA) and used without
further purification. Tris(2-carboxyethyl)phosphine (TCEP), N,N'-dicyclohexylcarbodiimide
(DCC), EtOH, 10-bromodecanoic acid, 4-(Dimethylamino)pyridine (DMAP), magnesium sulfate
(MgSO4), L-cysteine, 5(6)-Carboxyfluorescein (dye content 90%), dimethyl sulfoxide (DMSO),
anhydrous tetrahydrofuran (THF), sodium borohydride (NaBH4), selenium (Se),
Dimethylformamide (DMF), streptavidin, HEPES buffer, sonic hedgehog (SHH), N-acetyl
cysteine (NAC), 2,7-dichlorodihydrofluorescein diacetate and anti-mouse IgG-peroxidase were
obtained from Sigma-Aldrich (Taufkirchen, Germany). Triton X-100 was purchased from Carl
Roth (Karlsruhe, Germany). Phosphate-buffered saline (PBS), trypsin/EDTA, L-glutamine (L-
Gln), DMEM/F12, knock-out serum, noggin, penicillin, and streptomycin were purchased Gibco
5

BRL (Rockville, MD, USA). Mouse anti-RPE65 was obtained from Abcam (ab13826,
Cambridge, MA, USA). Goat anti-mouse Alexa fluor 546 was purchased from Invitrogen
(Carlsbad, CA, USA). L-glutathione reduced (GSH), phosphotungstic acid, hydrogen peroxide
(H2O2) solution 30% (w/w), and methanol were purchased from Merck (Darmstadt, Germany).
Dialysis membrane 12 kDa MWCO was purchased from Spectrum (Houston, TX, USA). Nylon
membrane filter was obtained from Sartorius (Germany). 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTS) was purchased from Promega (Madison, WI, USA).
RNeasy Mini kit with on-column DNase I digestion was purchased from Qiagen (Germany).
High Capacity cDNA Reverse Transcription Kit was purchased from Takara Bio (Shiga, Japan).
Parafilm was purchased from Pechiney Plastic Packaging (Menasha, WI, USA). Matrigel was
purchased from Corning (New York, USA). Basic fibroblast growth factor (bFGF) was
purchased from Thermo Fisher Scientific (Boston, MA, USA). All other chemicals and solvents
were of analytical grade.
2.2. Preparation of smart liposomes
2.2.1. Synthesis of diselenide
We prepared diselenide according to a previously described method (Fig. 2A) (Rafique et al.,
2015). Briefly, elemental Se (0.157 g, 1.99 mmol) and NaBH4 (0.152 g, 3.98 mmol) were placed
in a two-neck round-bottom flask and mixed under nitrogen gas flow. The 98% EtOH (0.7 mL)
was added to this mixture until complete dissolution of Se and the formation of a white-gray
solid. Dimethylformamide (DMF) (4 mL) was then added to the stirring solution until a red-
brown color formed, followed by continuous addition of commercial 80% EtOH (0.45 mL) with
vigorous stirring until the end of gas production. Then, Se powder (0.157 g, 1.99 mmol) was
6

added to the solution and stirred until the powder completely dissolved, and we observed a clear
dark-red solution. We added 10-bromodecanoic acid (1 g, 3.98 mmol) to this solution, which
changed its color from red to yellow. After 24 h, the reaction was stopped by the addition of
water (12 mL). The reaction mixture was extracted with diethyl ether (3×10 mL), and the
extracted organic phase was washed with water (3×20 mL) and brine (15 mL). The organic phase
was dried over MgSO4 and filtered. The solvent was completely evaporated, leaving a yellow
1
powder as the product with the following specifications and HNMR results (Fig. 2B I,II) –
1
yield: 65%; H NMR (CDCl3, 400 MHz, δ): 1.22 (m, 20H, -CH2), 1.50 (m, 4H, -CH2CH2Se-),
1.6 (m, 4H, -CH2CH2COOH), 2.2 (t, 4H, -CH2Se-), 2.8 (t, 4H, -CH2COOH).
2.2.2. Liposome preparation
Liposomes were prepared by the thin lipid film hydration method, followed by extrusion and
dissolving the lipids (40 mM) in chloroform. Briefly, we prepared a dry lipid film, by rotary
evaporation of the chloroform solution, from Egg PC, diselenide (SeSe), and DOPE with the
following molar ratios: DOPE:SeSe:Egg PC (60:10:30) (Lip1), DOPE:SeSe:Egg PC (60:20:20)
(Lip2), DMPC:SeSe (80:20), and DMPC:SeSe (90:10). Afterwards, these combinations were
freeze-dried (FreeZone Freeze Dry System, Labconco) for at least 4 h to remove any traces of
the solvents. This dry lipid film was hydrated in PBS (pH 7.4) and a 4 mg/mL lipid
concentration. The hydrated mixture was vigorously vortexed for 30 min to generate
multilamellar vesicles from the dry film. These vesicles were extruded 11 times through a 200-
nm pore size polycarbonate membrane filter, then through a 100-nm pore size polycarbonate
membrane filter (Avanti Polar Lipids Inc., USA) to generate a uniform particle size. We
7

prepared two control groups, DOPE:Egg PC (60:40) and DMPC, by the above mentioned
protocol.
2.3. Preparation of N-acetyl cysteine (NAC) loaded liposome
Liposome groups loaded with NAC were prepared as described earlier. The lipid film was
hydrated with 2 mL of a 30 mg/mL NAC stock solution. The suspension was briefly sonicated,
then extruded 11 times through 200 and 100 nm pore size polycarbonate membrane filters.
Unloaded drug was separated from the liposomes by overnight dialysis against PBS (pH 7.4)
using a cellulose ester membrane (MWCO: 12 kDa). The final formulation was sterilized by
passage through a 0.45 µm syringe filter.
2.4. Preparation of carboxyfluorescein (CF) loaded liposome
First, we sought to determine the optimal formulation for the smart liposomes. We loaded
different formulations with a hydrophilic dye and studied their release behavior upon exposure to
H2O2. First, the dried lipid film was hydrated in PBS (pH 7.4) that contained 40 mM
carboxyfluorescein (CF) to form multilamellar liposomes. Subsequently, the liposomes were
extruded 11 times through a 200 nm, followed by a 100-nm pore size polycarbonate membrane
filter to obtain liposomes of a uniform particle size. The liposomes were sterilized by passage
through a 0.45 µm syringe filter.
2.5. Liposome size and zeta potential ()
The liposome size and size distribution were measured by dynamic light scattering (DLS) using a
Zeta Plus Instrument (Brookhaven Instrument Corporation, Holtsville, NY, USA). The zeta
8

potential of the formulations was measured by a zeta Phase Analysis Light Scattering (PALS)
system with an ultrasensitive zeta potential analyzer (Brookhaven Instruments, Holtsville, NY,
USA). The liposome suspension was diluted as needed with PBS to measure the size and with 10
mM of NaCl (pH 7.4) to determine the zeta potential. All size and zeta potential measurements
were performed in triplicate (Table 1).
2.6. Transmission electron microscopy (TEM)
We used negative staining of the liposomes to verify their size and morphology. The stained
samples were examined by a JEM-2000CX (JEOL Ltd., Japan) transmission electron microscope
that had an accelerating voltage of 15 kV. For the negative staining, we used a 400-mesh grid
(EM fine-grid F400, Nisshin EM Co., Tokyo, Japan) with a carbon support film (10-20 nm in
thickness), The grid was given a hydrophilic treatment. Samples were stained at room
temperature (RT). A drop of liposomal solution, distilled water, and 1% phosphotungstic acid
(Merck, Tokyo, Japan) was placed on Parafilm® (Pechiney Plastic Packaging Co., Menasha, WI,
USA). The grid was placed into the liposomal drop (30 seconds), then in a distilled water drop
for washing (10 seconds), and finally in a phosphotungstic acid drop for staining (10 seconds).
Any excess solution was removed with filter paper (Kodama et al., 2010) (Anthony C., 2005).
2.7. N-acetyl cysteine (NAC) assay
We used HPLC to measure the NAC concentration against a standard curve according to
previously reported procedure (Anthony C., 2005). A stock standard solution (1 mg/mL) was
prepared by dissolving 25.0 mg of NAC in 25.0 mL of the mobile phase by ultrasound for 30
min. From this solution, we prepared a 30 mg/mL working standard by adding 300 µL of the
9

stock standard solution to 10.0 mL of the mobile phase. In addition, the stock standard solution
was diluted with the mobile phase to give five standard solutions that contained varying
concentrations of NAC (10.0, 20.0, 30.0, 40.0, and 50.0 µg/mL) to be used in the linearity study.
All solutions were injected (n=3) into the HPLC system.
The HPLC method was performed on a Shimadzu LC system (Kyoto, Japan) equipped with a
214-nm detector and a 3.9 mm x 30 cm column that contained packing L1. The mobile phase
was composed of 0.05 M KH2PO4 and acetonitrile (95:5 v/v and pH adjusted to 3.0 with
phosphoric acid) pumped at a flow-rate of 1.0 mL/min, isocratic elution. The mobile phase was
filtered through a 0.45 µm nylon membrane filter (Sartorius, Germany) and degassed before use.
A 100 µL volume was injected and NAC was detected at 214 nm. All analyses were performed
at RT (25±1ºC).
We dissolved the liposomes in methanol in order to measure the loaded NAC. We quenched the
liposomes by using 50 µL liposome dissolved in 950 µL methanol and heated the solution at 67
℃ for 10 min. Loading of NAC in the liposomal formulation was determined before and after
purification by dialysis, and we calculated the percent of encapsulated NAC.
2.8. Carboxyfluorescein (CF) assay
Fluorescence measurements for CF were performed by an SLM-4000 spectrofluorometer (SLM
Instruments, Urbana, IL, USA). The excitation wavelength of CF at 488 nm (1 nm slit-width)
and emission wavelength at 530 nm were detected using a Corning 3-68 cutoff filter (Corning,
NY, USA). The fluorescence standard was 20 nM of CF. Lysis of the liposomes was performed
by addition of Triton X-100 detergent (final concentration: 0.5%).
10

2.9. N-acetyl cysteine (NAC) triggered release
In vitro release profiles of NAC from liposomes were investigated in two different buffers, PBS
(0.01 mol/L, pH 7.4) and 0.1 wt.% H2O2, by using a dialysis bag diffusion technique. Lip1
(DOPE:SeSe:Egg PC (60:10:30))+NAC, Lip2 (DOPE:SeSe:Egg PC (60:20:20))+NAC, and Egg
PC/DOPE were introduced into dialysis membrane tubing (MWCO: 12-14 kDa) and incubated in
25 mL of buffer at 37°C with stirring. At predetermined intervals, the samples were drawn from
each buffer outside the dialysis bag and replaced by an equal volume of the same medium. The
concentration of NAC in the solution was measured by HPLC.
2.10. Carboxyfluorescein (CF) triggered release
Ten µl of different CF loaded liposome formulations, DMPC and Egg PC/DOPE were incubated
at 37C with 100 µl of the freshly prepared H2O2 buffers for 10 min. Total concentrations of
H2O2 ranged from 0 to 800 µM. Aliquots (10 µl) were subsequently diluted in 1 ml HEPES
buffer (200 mM, pH 7.4) and the fluorescence was measured before and after addition of Triton
X-100.
2.11. Cell culture
Passage-30 human embryonic stem cells (hESCs, Royan H6) were cultured as described
(Baharvand et al., 2006). The hESCs were induced to neuroectoderm (NE) in induction medium
that contained DMEM/F12 medium, 5% KOSR, 0.1% B-27 supplement, 2% N-2 (Invitrogen;
which included recombinant insulin, human transferrin, sodium selenite, putrescine, and
progesterone), 1% NEAAs, 2 mM L-Gln, 0.1 mM β-ME, and 10 ng/mL bFGF, supplemented
with noggin (250 ng/mL, R&D) for 6 days (Zahabi et al., 2012). The cells were subsequently
11

grown without noggin for another 6 days in the same medium in the presence of bFGF (10
ng/ml) and decreasing concentrations of all-trans retinoic acid (RA, Sigma-Aldrich; 0.6 to 0.2
µM) to promote additional rosette formation. The medium was renewed every other day at these
stages. Then, cells were exposed to 25 ng/mL bFGF along with 25 ng/ mL SHH (Sigma Aldrich,
UK) in the absence of RA for an additional 6 days to give rise to what appeared to be neural tube
(NT) like structure-organized rosettes that included lumen. On day 18, the culture was
maintained in DMEM-F12 medium supplemented with 5% KOSR, 1% NEAAs, and 2 mM L-
Gln. Half of the medium was renewed every 3 days. We observed pigmented areas from day 25
that increased with further culturing at passage 0. RPE were expanded as passage 1 by 10 min
exposure to trypsin/EDTA. Purified RPE were seeded onto Matrigel-coated tissue culture plates
and expanded for 10-15 days until the desired density, at which point the appropriate phenotype
was achieved. For the phagocytosis assay, the RPE cells were incubated with 1 µm green
fluorescent latex beads (1.0 ×109 beads/ml; Polysciences, Inc., Warrington, PA, USA) for 24 h at
37°C, after which they were washed three times with PBS/Tween and viewed with an inverted
fluorescent microscope (X71 Olympus, Tokyo, Japan).
Immunofluorescence staining
We sought to characterize the hESC-RPE cells. Passage-1 hESC-RPE cells were fixed with 2%
paraformaldehyde in PBS for 10 min, permeabilized for 10 min in 0.1% NP-40 substitute
(Sigma-Aldrich) in PBS and blocked with 10% normal goat serum in PBS for 1 h, followed by
an overnight incubation with RPE65 antibody (ab13826, Abcam, Cambridge, MA, USA) at 4°C.
Cells were then washed 3 times for 15 min each time in 0.1% Tween/PBS, incubated with anti-
mouse Alexa-594 for 1 h at RT, washed 3 times for 15 min each in 0.1% Tween/PBS and
12

mounted using VectaShield with DAPI (Vector Laboratories, Burlingame, CA, USA). Stained
cells were examined under an inverted fluorescence microscope (X71 Olympus, Tokyo, Japan).
2.12. Oxidative stress modeling
We conducted the oxidative stress experiments as follows. hESC-RPE cell cultures that had been
passaged for 15 days were plated in RPE medium. After the cells reached 80% confluency, they
were treated with chemical oxidants for 24 h and 48 h. A stock solution of freshly prepared 0.1
wt. % H2O2 in deionized water was added to the medium to produce a range of final
concentrations (0 μΜ to 750 μΜ). The cells were exposed to these stock solutions for 24 h and
48 h in order to measure cell metabolic activity (Hanus et al., 2013).
2.13. Cytotoxicity assay
2.13.1. Cytotoxicity assay for H2O2 treated hESC-RPE cells
The MTS assay was performed according to the manufacturer's protocol. For H2O2 cytotoxicity
studies, hESC-RPE cell medium was replaced by either fresh medium or medium that contained
various concentrations of H2O2. The cells were incubated for 24 h and/or 48 h. After the
specified incubation period, the cells were washed three times with PBS. Then, MTS stock
solution (20 µL/well MTS and 100 µL/well cell media) was added to the cells. After an
additional 2 h incubation, we used a microplate reader (SpectraMax, M5, Sunnyvale, CA, USA)
to measure absorbance of the formazan at 490 nm. Measurements were obtained from three
replicated wells of the 96-well plate and used to calculate metabolic activity of the chemically-
treated cells.
13

2.13.2. Cytotoxicity assay for liposomes
We conducted NAC and liposome cytotoxicity studies on the hESC-RPE cells. First, the cells
were incubated with NAC, Egg PC/DOPE+NAC (5 mmol/L), Lip1+NAC (5 mmol/L),
Lip2+NAC (5 mmol/L), Lip1 (5 mmol/L), and Lip2 (5 mmol/L) for 24 h. After removal of the
media, the cells were washed and we performed the MTS assay as previously described.
2.13.3. Quantitative real-time PCR (qRT-PCR)
Total RNA was isolated from 3 replicates of the hESC-RPE treated cells by using TRIzol reagent
(Takara) and chloroform according to the manufacturer’s instructions. cDNA was produced by
reverse transcription with a Takara kit. Quantitative real-time PCR (qRT-PCR) was performed
on a 7900HT Sequence Detection System (Applied BioSystems, CA, USA) with a Fast SYBR
Green Master Mix (Applied Biosystems, CA, USA). The gene expression levels was normalized
to the reference gene GAPDH and analyzed by Prism software (Prism 6.01, GraphPad Prism)
using the comparative Ct method, 2-△△Ct. Supplementary Table 1 lists the primer sequences
(Applied Biosystems) for superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), and
catalase (CAT).
Intracellular ROS measurement assay was performed. hESC-RPE cells (1×104 cells/well) in 96-
well plates were incubated with 0 µM and 200 µM H2O2 for 2h at 37 °C. They were then treated
with 0 mM NAC ,10 mM NAC, Lip1/NAC and Lip 2/NAC. They were washed with PBS and
incubated for 20 min at 37 °C in PBS containing 20 μM 2,7-dichlorodihydrofluorescein diacetate
(Sigma Aldrich). Intracellular ROS production was measured on a fluorescence plate reader
with an excitation of 485 nm and emission of 528 nm. The experiments were performed in
triplicate and repeated three times.
14

2.14. Statistical analysis
Data were analyzed by GraphPad Prism version 6.01 (GraphPad Software, San Diego, CA,
USA). Average results are shown as mean±standard deviation (SD). Descriptive statistics, the
independent t-test, and post hoc test for one-way ANOVA were used for statistical analyses.
Alternative nonparametric tests of the above statistical tests were conducted. Statistical
significance was set at P<0.05.
3. Results and Discussion
3.1. Characterization of nanocarriers
In the first step, we synthesized diselenide by a reaction between 10-bromodecanoic acid and Se
1
powder in the presence of NaBH4. H-NMR confirmed the formation of bis(decanoic acid)-
diselenide by a shift in the peak at 3.40 ppm (–CH2-Br of 10-bromodecanoic acid) to 2.86 ppm
(–CH2-Se- of bis(decanoic acid)-diselenide) as seen in Figure 2B (I,II). FTIR spectrum analysis
further confirmed the synthesis by the presence of one band at 1734 cm-1, which was related to -
COO- band stretching vibrations in PCL and a broad band at 2500-3400 cm-1 that was assigned
to the terminal carboxyl groups of the diselenide segments (Fig. 2C).
We studied liposome release behavior in different concentrations of H2O2 to find the optimal
formulation. The two candidates were liposomes composed of DMPC and Egg PC/DOPE.
Carboxyl fluorescent release was assessed by fluorescence measurements with an SLM-4000
spectrofluorometer (SLM Instruments, Urbana, IL, USA) at an excitation wavelength of 488 nm
(1 nm slit-width) and an emission above 530 nm. The liposomes were exposed to varying
15

concentrations of H2O2 (0 to 800 µM) as well as Triton-X100. DMPC that lacked Se-Se bond
showed negligible release, which agreed with our expectations (Fig. 3A). DMPC formulations
that contained 10% and 20% diselenide did not show a significan difference in release when
exposed to the various concentrations of H2O2 (Fig. 3A). However, DMPC formulations that
contained 10% diselenide had no release at low concentrations of H2O2, which might be
attributed to the low permeability of the liposome structure. DMPC that contained 20%
diselenide released CF at a high concentration (800 µM H2O2). This observation indicated that
DMPC responded only at the high concentrations of H2O2. For our delivery purposes, a carrier
should release its load at low and high H2O2 concentrations covering both ends of the spectrum.
Based on the above results, DMPC based liposomes seem to be effective only at high
concentrations of H2O2, while, DOPE/Egg PC carriers show response at both low and high
concentrations of H2O2.
We observed no release with the Egg PC/DOPE formulations (Fig. 3A). However, the
DOPE/Egg PC that contained 10% diselenide had CF release was proportional to the
concentration of H2O2. (Fig. 3A). By increasing H2O2 concentration, the amount of CF release
increased as well. Interestingly, when we increased the diselenide concentration of the formula to
20%, we observed a sharper response of CF from our carrier after exposure to different
concentrations of H2O2 (Fig. 3A). The data implied that the Egg PC/DOPE seemed to be a better
candidate than DMPC since it was sensitive at low H2O2 concentrations compared to DMPC. In
addition, this carrier had a linear release response that was a function of the H2O2 concentration.
When we increased the diselenide concentration in the liposome bilayer for the Egg PC/DOPE,
we observed a sharper release. Therefore, we chose this formulation for the rest of the study.
16

The physical characteristics of the carriers were evaluated. The unloaded liposomes had a mean
diameter of less than 150 nm with a polydispersity index (PDI) below 0.2. All drug-loaded
liposomes had mean diameters less than 170 nm and good polydispersity (PDI<0.2). The
liposomes that did not contain diselenide had slightly negative zeta potentials; however, those
with diselenide had significantly more negative charges (Table 1). The TEM results showed
liposome with round morphology and size ranges of 100-140 nm, which is in accordance with
the size acquired by DLS (Figure 3 B (I, II)). The encapsulation efficiency (EE) of NAC loaded
liposomes was approximately 12%.
In this study, we prepared three different liposomal formulations, DOPE:SeSe:Egg PC (molar
ratio: 60:10:30) (Lip1); DOPE:SeSe:Egg PC (molar ratio: 60:20:20) (Lip2); and DOPE:Egg PC
(molar ratio: 60:40), that contained NAC with a lipid concentration of 40 mM. We observed no
significant differences in formulation sizes and EE (Table 1). A stability study conducted over 28
days showed that the liposomes ranged between 155  3.30 nm and 223.3  2.89 nm, with a PDI
of approximately less than 0.2. The DLS measurements of size and PDI of all groups showed
that the liposomes had suitable size and homogeneity. The surface charges were approximately -
3.51  0.50 mV for liposomes that lacked diselenide and approximately -20 mV for those with
diselenide. The negative surface charge was due to the presence of negatively charged
phospholipids, which would be ideal for intravitreal injections of drugs to the posterior segment
of the eye. The vitreous humor of the mammalian eye mainly consists of collagen, fibrils,
glycosaminoglycans (GAGs), hyaluronic acid (HA), and heparin sulfate (HS). Both HA and HS
carry negative charges. A charge dependent suppression on particle diffusion for positively
charged nano-carriers has been reported (Käsdorf et al., 2015). Therefore, the negatively charged
17

liposome would be a suitable carrier for drug delivery to the posterior segment of the eye,
particularly the retina, due to repulsive induced electrostatic forces (Käsdorf et al., 2015).
We examined the sensitivity of these liposomes by measuring the drug release at different redox
environments. Their trigger drug release in two different buffers: PBS (0.01 mol/L, pH 7.4) and
0.1 wt. % H2O2 were measured by dialysis bag diffusion technique. The release profiles of drug-
loaded liposomes that contained diselenide showed instant release of drug when exposed to 0.1
wt. % H2O2. Liposomes that lacked diselenide showed no release in these buffers. According to
the above results, the liposomes with diselenide showed a reduction in triggered release of their
load upon exposure to 0.1 wt. % H2O2, which was due to cleavage of the diselenide bond upon
exposure to the reducing agents. This would disturb the vesicle membrane and allow release due
to the formation of transient defects or structural rearrangements. The release of the loaded drug
from the liposome was negligible without the reducing agent (Fig. 4).
Our data showed that the reduction kinetics is a function of the amount of diselenide bond
present in the bilayer of the liposomes. Data from the first 15 min for liposomes with 20%
diselenide (Lip2) showed that more than 50% of the load released upon addition of 0.1 wt. %
H2O2. However, we observed 40% release of the drug from the liposomes that contained 10%
diselenide (Lip1). This finding indicated that the rate of release in liposomes with 20%
diselenide was slightly higher compared to 10% diselenide. This implied that the higher the
concentration of sensitive linkage, the quicker the system would respond to the reducing agent.
Release of drug from Egg PC/DOPE that lacked diselenide was negligible in the presence and
absence of the reducing agent.
3.2. In vitro studies
18

The hESCs were induced to RPE cells according to the protocol by Zahabi et al. Followed by 10-
15 days’ culture in passage 1, the monolayer of RPE cells were observed (Fig. 5A). After 10
days of passaging, the cells rearranged in a regular hexagonal mosaic, with sufficient confluency
to proceed with the oxidative stress modeling experiments (Fig. 5B). We performed the
phagocytosis which in known as a critical function of RPE cells. Fluorescent microscopy
analysis of the hESC-RPE cells after 24 h incubation with florescent latex beads demonstrated
that the cells had the potential for phagocytosis. (Fig. 5C). In addition, data from immunostaining
with anti-RPE65 implied that passage-1 RPE cells expressed significant levels of the mature
RPE cell marker (Fig. 5D-F).
3.2.1 Cytotoxicity assay of carriers
hESC-RPE cells at passage 1 were divided into 5 groups (control, NAC, Lip1+NAC,
Lip2+NAC, Egg PC/DOPE) for all experiments. We assessed the cytotoxicity of the Egg
PC/DOPE, Lip1, and Lip2 in the presence and absence of NAC. The MTS assay was used to
determine the effects of the hESC-RPE cells at total NAC concentrations that ranged from 0 to
10000 µM on healthy cells. Figure 5G shows that the DOPE/Egg PC liposome had a 15%
toxicity effect at concentrations that ranged from 3 to 10 M on hESC-RPE cells. This result was
similar to carriers that contained diselenide with or without NAC, which had a toxicity effect of
approximately 5%-10%. This negligible difference among the groups might be due to the
presence of diselenide and its therapeutic effects (Peter C. Raich, 2015; Jacek Młochowski,
2015). This negligible cytotoxic effect of the carriers indicated that liposomes could be suitable
candidates for drug delivery.
19

3.2.2 Cytotoxicity assay of oxidative stress modeled hESC-RPE cells
The hESC-RPE cells were modeled using varying concentrations of H2O2 (0 to 750 µM) in an
attempt to find the appropriate oxidative stress model to study the therapeutic effect of these
carriers. Therefore, we used the MTS assay to assess metabolic activity of cells exposed to H2O2
after 24 h and 48 h. Figure 5H shows that the control groups (not treated with H2O2) had more
than 99% metabolic activity. However, with increasing H2O2 concentration, the cell metabolic
activity dropped to approximately 80%. At the 200 µM concentration of H2O2, cell metabolic
activity decreased to approximately 35% in the 24 h and 50% in the 48 h treated groups
(P<0.01). Increased concentration and longer exposure time of H2O2 caused higher mortality.
Pretreatment with 500 µM of H2O2 caused a 50% decline in survival rate at 24 h, whereas there
was a 70 % drop during 48 h exposure to H2O2 (P<0.01). We observed almost 20% metabolic
activity at the 750 µM concentration of H2O2 for both 24 h and 48 h exposure time. Therefore,
the groups treated for 24 h with 200 and 500 µM H2O2 were suitable since they had less cell
death compared to the other groups. These levels of oxidative stress model different stages of
AMD, which also evaluates the sensitivity of our carrier at low levels of stress.
3.2.3 Therapeutic effect of smart liposomes on oxidative stress modeled hESC-RPE cells
Previous studies reported that 10 to 20 mM of NAC had therapeutic effects on reducing
oxidative stress in RPE cells (Gerona et al., 2010; Tate et al., 1995). For the therapeutic assay,
hESC-RPE cells pretreated overnight with 200 and 500 µM H2O2 were exposed to different
groups of carriers and naked drug (Fig. 6). The group pretreated with 200 µM of H2O2 had a cell
survival of 70%; the group pretreated with 500 µM had a cell survival of 50% (previously
reported). We intended to study the therapeutic effect and responsiveness of liposomes under
20

both reduction-oxidation conditions. The cells pretreated with 200 µM H2O2 were treated with
liposomes. We observed that both diselenide containing liposomes, Lip1/NAC and Lip2/NAC,
with 10 mM NAC, showed a better therapeutic effect with a cell metabolic activity of
approximately 90%. It was was significantly higher compared to control group or NAC treated
groups (P<0.05). We noted that for the 500 µM H2O2, the liposomes that contained 10%
diselenide had a cell metabolic activity of approximately 70% and the liposomes with 20%
diselenide had a cell metabolic activity of approximately 80%. Cell metabolic activity in both
groups were significantly higher compared to the control (P<0.05) and NAC treated groups
(P<0.01).
The data from the cells pretreated with 200 µM H2O2 showed that for low levels of oxidative
stress, the smart carriers had the potential to mitigate cell death due to oxidative responsiveness
of the liposomes. At higher levels of ROS, we observed that liposome treatments significantly
enhanced cell survival. This finding was particularly noted in groups that had the higher
concentration of diselenide (20%). This data indicated that the concentration of diselenide
present in the liposomes could be an important variable for more extreme levels of oxidative
stress, even though we observed satisfactory results for both groups of liposomes. On the other
hand, the protective effect of the liposomes in prevention of NAC damage when exposed to free
radicals might explain why smart liposomes showed a better therapeutic effect compared to NAC
alone.
3.2.4 Gene expression levels of oxidative-sensitive markers in treated hESC-RPE cells
Reactive oxygen species develop oxidative stress, which contributes to the pathogenesis of
AMD. RPE cells are considered a primary target in AMD. Enzymes such as SOD1, SOD2, and
21

CAT have key roles in protecting cells against oxidative stress and regulating ROS. Expressions
of these enzymes in hESC-RPE cells were detected by qRT-PCR.
We evaluated the therapeutic effectiveness and responsiveness of the smart liposomes at low
levels of oxidative stress by conducting a gene study test on cells treated with 200 µM of H2O2
rather than other groups of oxidative stress. After 24 h, the cells were treated with Lip2+NAC,
Lip1+NAC, and Egg/DOPE with an NAC concentration of 10 mM. We also included a non-
treated group and a group of healthy cells without any oxidative stress to find the treatment effect
of the smart delivery system in regulating generated ROS in RPE cells.
As shown in Figure 7, expression of SOD1 gene in the untreated cells was 5-fold lower than the
healthy cells, whereas the NAC treated cells was approximately 3.3-fold lower than the healthy
cells (P<0.01). In addition, the SOD2 gene in the untreated cells was approximately 2.5-fold
lower; for the NAC treated, they were approximately 2.5 and 2-fold lower than the healthy cells
(Fig. 7; P<0.01). However, cells treated with Lip2+NAC and Lip1+NAC showed the similar
expression levels for SOD1 and SOD2 to healthy cells. This finding indicated that cells induced
with oxidative stress maintained normal levels of SOD1 and SOD2 when treated with smart
liposomes loaded with NAC. The reason that naked drug had no significant treatment effect was
possibly due to its sensitivity and degradation in the oxidative stress regions; therefore, when
NAC is protected in a carrier like liposome it has better therapeutic effect. CAT expression of
modeled cells treated with Lip2+NAC and Lip1+NAC did not significantly differ from healthy
cells (Fig 7), whereas cells under oxidative stress that were untreated had a 2.2-fold increase and
those treated with 10 mM NAC showed a 2-fold increase compared to healthy cells (P<0.01).
Data from gene expressions indicated that NAC loaded smart liposomes seemed to effectively
deliver NAC to the modeled cells, whereas naked drug or liposomes that lacked diselenide
22

seemed to be unsuccessful. This agreed with our observation from the drug release data, which
implied that smart liposomes released their cargo upon exposure at the target site unlike
liposomes that lacked the sensitive bond. The liposomes protected NAC from degrading in the
oxidative stress regions.
In addition, treatment of hESC-RPE cells with H2O2 led to an increase in intracellular ROS
levels compared with untreated cells. The intensity of the mean oxidized DCF peak was
increased by 2.5-fold compared with controls after 200 μM H2O2 treatment in hESC-RPE cells.
However, treatment with Lip 1/NAC and Lip 2/NAC attenuated the ROS accumulation level to
the same value as intact cells, while treatment with naked NAC was as satisfying (Fig. S1).
4. Conclusion
The redox responsive liposome containing diselenide linkage was developed as an anti-oxidant
carrier on oxidative stress model of hESC-RPE cells (Fig. 1). Smart liposomes showed a
triggerable release of their content when exposed to oxidative stress regions while they had no
cytotoxicity on cells. The protective effect of NAC, as anti-oxidant, was greater in hESC-RPE
cells treated with smart liposomal NAC formulation compared to conventional methods. The
protecting effect of liposome for loaded drug and high level release of anti-oxidant in response to
oxidative stress, resulted in reducing ROS level and enhancing therapeutic effect. Taken
together, our data demonstrate hESC-RPE cells induced with oxidative stress treated with smart
liposomes loaded with NAC leads to increased anti-oxidant gene expression, resulting in RPE
cell death prevention. These results strongly suggest an essential role of oxidative responsive
liposomes, for delivering antioxidants to the diseased cells in order to boost cellular defense
system, in maintaining the redox homeostasis in RPE cells. The proposed carrier is promising for
23

localized drug delivery. Although it is noteworthy to mention that for in vivo studies there is the
concern for multiple intravitreal injections which cause side effects such as retinal detachment.
We are hoping that such smart carriers will reduce the number of required injections and the
caused side effects to a degree. We will also try topical administration of the liposomes in animal
studies to evaluate the ability of the carriers to reach the posterior segment. Also, studies have
demonstrated that the toxicity of diselenide depends on the type of species (rat or mice) and route
of administration (i.p or s.c) (Nogueira et al., 2002). On the other hand, these compounds have
been considered for potential use as pharmaceutical antioxidants, anti-inflammatory and
antinociceptive (Anderson et al., 1994; Schewe et al., 1995). There is a lack of toxicity studies of
Se organic forms which needs to be done. The mentioned above could be examined in future
studies. The proposed carrier is promising for localized drug delivery.
Acknowledgments
This work was supported by grants from Royan Institute, the Iran National Science Foundation
(INSF), and Iran Science Elites Federation to H. B.
Competing financial interests
The authors declare no competing financial interests.
24

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Figure legends
Figure 1. The schematic illustration of liposomal drug delivery effect on hESC-RPE and
mechanism of drug release from responsive liposome in redox environment.
1
Figure 2. Synthesis scheme of diselenide (A) H-NMR spectra of 10-bromodecanoic acid (I)
and diselenide (II)(B), and FTIR of diselenide (C).
Figure 3. Evaluation of the optimal formulation for liposomes. We measured the intensity of
released carboxyl fluorescence at 488 nm excitation and emission from the CF loaded liposomes
when exposed to different concentrations of H2O2 (0-800 µM). Two groups of liposomes (DMPC
and Egg PC/DOPE) with different concentrations of SeSe were studied. DMPC release,
DMPC/10% SeSe, DMPC/20% SeSe , Egg PC/DOPE, Egg PC/DOPE/10% SeSe, and Egg
PC/DOPE/20% SeSe (A). Transmission electron microscopy (TEM) images of negatively
stained liposomes (Egg PC/DOPE) at 8000x magnification on the left (I) and 31500x
magnification on the right (II) (B). Egg PC: Egg/L-α-phosphatidylcholine; DOPE: 1,2-dio-leoyl-
sn-glycero-3-phosphoethanolamine; DMPC: 1,2-dimyristoyl-sn-glycero-3-phosphocholine;
SeSe: Diselenide.
Figure 4. Release of N-acetyl cysteine (NAC) from Lip2 (Egg PC/DOPE/20% SeSe), Lip1 (Egg
PC/DOPE/10% SeSe), and Egg PC/DOPE when exposed to 0.1 wt. % H2O2 buffer. HPLC assay
was used to measure NAC release. Release from carriers that contained liposomes were
significantly higher than carriers without SeSe. Data presented as mean±SD. *: P<0.05; **:
28

P<0.01
Egg
PC:
Egg/L-α-phosphatidylcholine;
DOPE:
1,2-dio-leoyl-sn-glycero-
phosphoethanolamine; SeSe: Diselenide.
Figure 5. hESC-RPE cells characterization and cell metabolic activity assay. hESC-RPE cells
characterization and cell metabolic activity assay. Phase- contrast image of hESC- derived RPE,
10 days after passage 1(A) hESC-RPE recover its hexagonal morphology (B) the fluorescent
image showing phagocytosis of green fluorescent beads by hESC- derived pigmented cells (C).
Representative immunostaining images of hESC-RPE cells at passage 1 analyzed for typical
mature RPE protein; RPE65 (D). Cell metabolic activity of RPE cells when treated with different
concentrations of H2O2. Nuclei were counterstained with DAPI (D). Scale bar: 50 µm (A- C) and
100 µm (D-F). Effects of drug and carriers on hESC-RPE cells. MTS assay of Lip1, Lip2, and
Egg PC/DOPE [with or without N-acetyl cysteine (NAC)] on passage-1 hESC-RPE cells after 24
h. No significant toxicity of the carriers was observed (G). Cell metabolic activity of hESC-RPE
cells for oxidative stress modeling after exposure to different concentrations of H2O2 (0-750
µM). Significant difference was observed for the 200 µM and 500 µM groups (H). Student’s t-
test. *: P<0.05; **: P<0.01.
Figure 6. MTS assay of hESC-RPE cells. The cells were treated with different concentrations of
H2O2 (0 µM, 200 µM, and 500 µM) exposed to N-acetyl cysteine (NAC; 10 mM), Lip2/NAC
(Egg PC/DOPE/20% SeSe), and Lip1/NAC (Egg PC/DOPE/10% SeSe). Total concentration of
NAC was 10 mM. Values are the mean±standard deviation (SD). *: P<0.05; **: P<0.01; ***:
P<0.001. Data are written as the mean±SD. Egg PC: Egg/L-α-phosphatidylcholine; DOPE: 1,2-
dio-leoyl-sn-glycero-3-phosphoethanolamine; SeSe: Diselenide.
29