Investigation into the Anticancer Activity and Apoptosis Induction of Brevinin-2R and Brevinin-2R-Conjugated PLA–PEG–PLA Nanoparticles and Strong Cell Cycle Arrest in AGS, HepG2 and KYSE-30 Cell Lines

Article abstract of DOI:10.1007/s10989-018-9772-z

Our study aims to establish a biocompatible nanostructure for the improved delivery of anticancer peptide, Brevinin-2R, to treat human gastric adenocarcinoma (AGS), human liver hepatocellular carcinoma (HepG2) and human squamous cell carcinoma (KYSE-30) cells. Poly(l-lactide)–poly(ethylene glycol)–poly(l-lactide) (PLA–PEG–PLA) nanoparticles were synthesized, obtained by a solvent evaporation method and characterized using scanning electron microscopy (SEM), FTIR and DLS; chemically-synthesized Brevinin-2R was encapsulated in micelles. In vitro release and cell uptake assay were conducted before cytotoxicity tests. Cell cycle analysis and apoptosis study were performed through flow cytometry and Annexin-V-FlOUS cell staining. PLA–PEG–PLA nanoparticles showed a narrow-size distribution with a zeta potential of ? 26.63 and a high cell internalization. Brevinin-2R-conjugated nanoparticles were spherical in shape with an increased surface charge of ? 21.90. For the first time, viability tests showed that Brevinin-2R-conjugated nanoparticles were more efficient than Brevinin-2R against cancer cells causing higher rates of cell cycle arrest and apoptosis induction. Our new findings demonstrate the potential of PLA–PEG–PLA nanoparticles to boost the anticancer effect and improve the delivery of Brevinin-2R. The study of Brevinin-2R-loaded nanoparticles indicated noticeable results in terms of novel cancer therapy. PLA–PEG–PLA nanoparticles can act as a biocompatible delivery platform to take the advantage of Brevinin-2R toward cancer cells. This is a novel study as the Brevinin-2R-conjugated nanoparticles and applied approaches have not been already reported.

Full text of DOI:10.1007/s10989-018-9772-z

International Journal of Peptide Research and Therapeutics
https://doi.org/10.1007/s10989-018-9772-z
Investigation into the Anticancer Activity and Apoptosis Induction
of Brevinin-2R and Brevinin-2R-Conjugated PLA–PEG–PLA
Nanoparticles and Strong Cell Cycle Arrest in AGS, HepG2 and KYSE-30
Cell Lines
Robab Hassanvand Jamadi1 · Asadollah Asadi1 · Hashem Yaghoubi2 · Fariba Goudarzi1
Accepted: 3 October 2018
© Springer Nature B.V. 2018
Abstract
Our study aims to establish a biocompatible nanostructure for the improved delivery of anticancer peptide, Brevinin-2R,
to treat human gastric adenocarcinoma (AGS), human liver hepatocellular carcinoma (HepG2) and human squamous cell
carcinoma (KYSE-30) cells. Poly(l-lactide)–poly(ethylene glycol)–poly(l-lactide) (PLA–PEG–PLA) nanoparticles were
synthesized, obtained by a solvent evaporation method and characterized using scanning electron microscopy (SEM), FTIR
and DLS; chemically-synthesized Brevinin-2R was encapsulated in micelles. In vitro release and cell uptake assay were
conducted before cytotoxicity tests. Cell cycle analysis and apoptosis study were performed through flow cytometry and
Annexin-V-FlOUS cell staining. PLA–PEG–PLA nanoparticles showed a narrow-size distribution with a zeta potential of
−26.63 and a high cell internalization. Brevinin-2R-conjugated nanoparticles were spherical in shape with an increased
surface charge of −21.90. For the first time, viability tests showed that Brevinin-2R-conjugated nanoparticles were more
efficient than Brevinin-2R against cancer cells causing higher rates of cell cycle arrest and apoptosis induction. Our new
findings demonstrate the potential of PLA–PEG–PLA nanoparticles to boost the anticancer effect and improve the deliv-
ery of Brevinin-2R. The study of Brevinin-2R-loaded nanoparticles indicated noticeable results in terms of novel cancer
therapy. PLA–PEG–PLA nanoparticles can act as a biocompatible delivery platform to take the advantage of Brevinin-2R
toward cancer cells. This is a novel study as the Brevinin-2R-conjugated nanoparticles and applied approaches have not
been already reported.
Keywords Apoptosis induction · Brevinin-2R-conjugated nanoparticle · Cell cycle arrest · Cell internalization · Drug
loading · Enhanced anticancer efficiency
Introduction
acid sequence of KLKNFAKGVAQSLLNKASCKLSGQC
(Ghodsi-Moghadam and Asoodeh 2018), is a synthesized
antimicrobial peptide that belongs to the Brevinin-2 family
(Ghavami et al. 2008). With 25 amino acid and a net charge
of +5, in spite of a majority of Brevinins, it shows a negligi-
ble hemolytic action toward red blood cells (Ghavami et al.
2008). This peptide shows antimicrobial activity against
gram-positive and gram-negative bacteria (Ghodsi-Mogh-
adam and Asoodeh 2018). Added to anti-bacterial activity
on reference strains of bacteria, this water-soluble peptide
has dramatically exhibited an anti-proliferating effect on a
broad spectrum of cancer cell lines (Asoodeh et al. 2013;
Ghavami et al. 2008), inhibiting the growth of Jurkat, BJAB,
HT29/219, SW742 and some other cell lines (Ghavami et al.
2008; Homayouni-Tabrizi et al. 2015; Mehrnejad et al.
Although progress in diagnosis and therapy has notice-
ably decreased the mortality of cancer, it is still a pervasive
problem (Perez-Tomas 2006). Cancer therapy using custom
chemical drugs and surgery is accompanied by toxicity and
severe side effects on normal tissues and cells (Hami et al.
2014a, b; Kakde et al. 2011). Brevinin-2R, with the amino
* Robab Hassanvand Jamadi
robab.hasanvand@yahoo.com
1
Department of Biology, Faculty of Sciences, University
of Mohaghegh Ardabili, Ardabil, Iran
2
Department of Biology, Ardabil Branch, Islamic Azad
University, Ardabil, Iran
Vol.:(0123456789)
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International Journal of Peptide Research and Therapeutics
2008).Thanks to its negligible hemolytic activity, Brevinin-
2R can be considered as a promising therapeutic agent
(Ghodsi-Moghadam and Asoodeh 2018).
7.4), cellular uptake, investigation into inhibitory effects of
Brevinin-2R and Brevinin-2R-conjugated nanoparticles on
AGS, HepG2 and KYSE-30 cell lines, cell cycle arrest and
apoptosis induction. PLA–PEG–PLA micelles, prepared
by a solvent evaporation method, were characterized for
particle size distribution, shape, structure, average size,
biocompatibility and cell internalization through scanning
electron microscopy (SEM), FTIR, DLS, hemolysis assay,
confocal laser scanning microscopy; the results revealed
that PLA–PEG–PLA triblock copolymer and Brevinin-2R
were successfully synthesized. PLA–PEG–PLA nanoparti-
cles were localized into selected cancer cells. For the first
time, we conducted the study on the in vitro cytotoxicity
of synthesized Brevinin-2R and Brevinin-2R-conjugated
micelles against the above mentioned cell lines using the
3-(4,5-dimethylthiaol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) and neutral red (NR) uptake assays. Results
revealed severe cytotoxic effect of Brevinin-2R-conjugated
nanoparticles compared to that of Brevinin-2R. Our fresh
evidence also elucidated the cell cycle arrest and apoptosis
induction in treated cancer cells.
Drug delivery systems such as micelle-like nanoparticles
(Chan et al. 2009; Licciardi et al. 2010) can reduce side
effects and dosages by improving targeted drug delivery
and increasing antitumor efficacy (Li et al. 2008; Venkatra-
man et al. 2005). Polymeric micelles are nano-sized carri-
ers (Yokoyama 2011) with amphiphilic block copolymers,
consisting of PLA–PEG block copolymers (Jelonek et al.
2016) that are usually prepared by self-assembly (Wora-
phatphadung et al. 2018). With a hydrophobic inner core
and hydrophilic corona, PLA–PEG–PLA nanoparticles can
encapsulate drugs. Also, some other advantageous properties
of these nanoparticles include high solubility, high stability,
superior biocompatibility, biodegradability (He et al. 2007),
controlled drug release, prolonged circulation in the blood-
stream, high drug loading, well suited size (20–200 nm),
selective accumulation in tumor sites and escaping the
reticuloendothelial system (RES), renal exclusion, tumor
targeting by improved permeability, etc. (Chau et al. 2006;
He et al. 2007; Hrubý et al. 2005; Kataoka et al. 2001; Mas-
sodi et al. 2010; Phan et al. 2016; Venkatraman et al. 2005).
Due to these properties, PLA–PEG–PLA nanoparticles
have been considered as effective delivery systems. Thus,
they can improve the antitumor efficacy and reduce toxicity
(Ghavami et al. 2008) that illustrate a high capacity to be
developed in drug delivery (Duda et al. 1998; Mehrnejad
et al. 2008; Woraphatphadung et al. 2018; Xiao et al. 2010;
Zhao et al. 2012). The PLA and PEG copolymerization can
raise loading efficiency (LE), decrease the burst effect, and
increase sustained drug release that is significantly related to
the controlled micelles’ degradation (Dell’Erba et al. 2001;
Xiao et al. 2010). Given the expectation of only 10–15%
of tumor cells being in the mitotic stage of the cell cycle
at any time, the prolonged drug release is highly beneficial
(Wolinsky et al. 2012).
Materials and Methods
Materials
The AGS, HepG2, KYSE-30 and human normal fibroblast
of gingiva (HGF1-PI1) cell lines were purchased from the
Pasteur institute, Iran; cell culture media and antibiotics
were purchased from GIBCO; toluene, acetone, methanol,
ethanol, acetic acid, dimethyl sulfoxide (DMSO), phosphate
buffered saline (PBS) (pH 7.4), MTT and NR dye were pre-
pared from Merck (Darmstadt, Germany); trypsin–EDTA,
lactic acid (LA), poly(ethylene glycol) (PEG) with a molecu-
lar weight of 2000, N,N-dimethyl formamide (DMF) and
fluorescein isothiocyanate (FITC) were prepared from
Sigma-Aldrich (USA). Distilled water was applied for all
experiments. The other chemicals and solvents were of ana-
lytical grade and employed without further purification.
Gastric cancer is the fourth most common malignant
neoplasm and the second leading cause of cancer-related
mortality worldwide (Li and Gao 2018). Despite decreas-
ing mortality related to gastric cancer, about 700,000 con-
firmed deaths have been reported around the globe, annually
(Jemal et al. 2011). Liver cancer is the third main cause
of cancer-related deaths all over the world, being responsi-
ble for about 9.1% of total deaths in 2012 (Nakagawa et al.
2018). Esophageal cancer is the 8th most common cancer in
the world and the 6th cause of cancer deaths. The esopha-
geal squamous cell carcinoma is the dominant histological
subtype of esophageal cancer globally, followed by adeno-
carcinoma (Allum et al. 2018; Middleton et al. 2018).
The aim of this study was to synthesize Brevinin-2R and
Brevinin-2R-conjugated polymeric micelles, in vitro evalu-
ation of release behavior in phosphate buffer solution (pH
Peptide Synthesis
Brevinin-2R was designed and manually synthesized
through solid phase strategies, using standard Fmoc chem-
istry, as previously described (Amaral et al. 2012; Hilpert
et al. 2008). Purification of peptide was performed on a C18
analytical reverse phase high pressure chromatography (RP-
HPLC) column (Pharmacia Biotech, US) with a linear gradi-
ent of acetonitrile in 0.1% TFA. Purified peptides were then
characterized through a mass spectrometer (LTQ orbitrap,
Thermo Electra, San Jose, CA) and C8 semi preparative
RP-HPLC.
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International Journal of Peptide Research and Therapeutics
diameter for the particles. All results were the average of
three different test runs.
Synthesis of PLA–PEG–PLA Triblock Copolymer
Ring-opening polymerization of the lactide was employed
for synthesizing the 44.5%PLA–11%PEG2000–44.5%PLA
tri-block copolymer in the presence of polyethylene glycol
(PEG) under optimized conditions. Briefly, l-lactide was
prepared from l-lactic acid, using antimony trioxide (Sb2O3)
as a catalyst. In order to eliminate the residual impurities in
the monomer, the synthesized l-lactide was re-crystallized
from ethyl acetate and vacuum dried at 40 °C, overnight
(High Vacuum Evaporation Systems, Model: ETS-160,
EDS-160, High Vacuum Technology Center-ACECR, Sharif
Branch, Iran). Afterward, 16 g of the vacuum-dried lactide
and 3 g of PEG2000 were dissolved in anhydrous toluene
with the protection of nitrogen. 200 mg stannous octoate
(Sn(Oct)2) was then added into the reaction flask as a cata-
lyst [0.15% (w/w), in toluene solution] (Fig. 1). Polymeri-
zation reaction was carried out at 130 °C under inert gas
atmosphere. After 12 h, solvent was evaporated and the
dried copolymer was repeatedly purified by dissolution in
dichloromethane and crystallization from cold ethanol, and
the obtained mixture was centrifuged, supernatant was then
removed and the pellet vacuum was dried at 45 °C for 48 h.
Biocompatibility of Nanoparticles
Freshly isolated human blood from a healthy donor was
washed three times with the same volume of PBS (pH 7.4),
centrifuged at 150×g for 5 min, and resuspended in PBS to
obtain a final concentration at 4% (v/v). Then various con-
centrations of copolymer were incubated with the washed
RBCs at 37 °C for 30 min. Red blood cells in PBS and 0.2%
Triton X-100 were considered as negative and positive con-
trols, respectively; after centrifugation (10 min, 900×g), the
supernatants were carefully transferred to a micro plate and
the release of hemoglobin was monitored at 540 nm through
micro-plate reader. HC50 value was taken as the mean con-
centration causing 50% hemolysis.
Preparation of Brevinin‑2R‑Loaded Polymeric
Micelles
Brevinin-2R-loading in PLA–PEG–PLA micelles was con-
ducted by a solvent evaporation method. In brief, an opti-
mized amount of Brevinin-2R was dissolved in distilled
water (1 mg/ml). Polymeric nanoparticles were dissolved in
acetone (1% W/W) (10 mg/ml). Afterward, 1 ml of peptide
solution was added drop-wise to concentration of 10 ml of
PLA–PEG–PLA solution in acetone under magnetic stirring
for 4 h at room temperature. Acetone was then evaporated
and the mixture was centrifuged at 3000 rpm for 10 min
to eliminate unloaded Brevinin-2R. The resulting pellet,
included stabilized Brevinin-2R-conjugated copolymeric
micelles, dispersed for following studies. The concentration
of free Brevinin-2R in supernatant was measured through
Bradford protein assay (Davies et al. 1997; Zohri et al.
2010). The samples were evaluated and the LE was deter-
mined through the equation below:
Size and Zeta Potential of Nanoparticles
The average particle size distribution and zeta potential of
blank micelles, as well as Brevinin-2R-conjugated micelles
were measured using dynamic light scattering, through a
90 PLUS Zeta potential analyzer (Brookhaven Instruments
Cooperation. New York, USA) at 25 °C with a scattering
angle of 90°. The measurement was conducted using a clear
disposable sizing cuvette which eventuate a hydrodynamic
(Brevinin − 2Rtotal) − (Brevinin − 2Rsupernatant
)
LE (%) =
× 100
Brevinin − 2Rtotal
Particle Morphology
The morphology and structural characteristics of nanopar-
ticles and Brevinin-2R-conjugated micelles were assessed
using SEM (LEO 1430 VP, UK) equipped with an Electron
Microscope Camera (Fanavari Khala Kahroba Co., Iran).
Diluted solutions of micelles and conjugates were placed
onto a glass slide and dried in vacuum. The samples were
mounted on metal stub plating and coated under the vacuum
prior to observation.
Fig. 1 Synthesis of l-lactide and PLA–PEG2000–PLA tri-block copol-
ymer
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International Journal of Peptide Research and Therapeutics
to a confocal laser scanning microscope (IX 71, Olympus,
Japan) equipped with a digital camera (DP 71, OLYMPUS).
Free FITC (1 µM) was used as the control.
FTIR Spectroscopy
The FTIR transmission spectra of PLA–PEG–PLA copoly-
mer and Brevinin-2R-loaded copolymer were collected on
a Perkin Elmer spectrum RXI spectrometer (Waltham, MA,
USA) using the KBr pellets in the wave number region of
400–4000 cm−1 under a resolution of 4 cm−1 and with a
scanning speed of 2 mm s−1 at 25 °C. FTIR was applied to
identify the functional groups involved in the formation of
the nanoparticles and investigate the interaction between the
PLA–PEG–PLA copolymer and Brevinin-2R in conjugates.
Cell Viability Assay
MTT Assay
To determine cytotoxic efficacy of nanoparticles, Brevinin-
2R and Brevinin-2R-loaded PLA–PEG–PLA micelles
in vitro, AGS, HepG2, KYSE-30 and HGF1-PI cells were
seeded in 96-well plates (5×103 cells/well), 24 h prior to the
cytotoxicity test. Then, various concentrations of nanopar-
ticles, Brevinin-2R and micelles conjugates in RPMI-1640
medium were added to the wells and further incubated for
24, 48, and 72 h. Then, 20 µl MTT (5 mg/ml) was added to
wells and cells were incubated for 4 h. After removing the
MTT solution, the cells were incubated with 150 µl DMSO
for 10 min to dissolve the formazan crystals formed only by
living cells. Thereafter, the absorbance of wells was deter-
mined using a microplate reader (Synergy HT, BioTek) at
540 nm. The IC50 values were calculated as the drug con-
centrations that inhibit cell growth by 50%.
In Vitro Release Study
In vitro release study of Brevinin-2R-loaded micelles was
conducted using the dialysis technique. 5 ml of suspen-
sion was enclosed in a pre-soaked dialysis bag (MW cutoff:
12 kDa) immersed in a diffusion cell containing 50 ml PBS
(pH 7.4) as a release medium, which was then incubated at
37 °C under constant shaking to avoid an unstirred water
layer formation at the membrane/release medium interface.
At regular time intervals, 1 ml samples were withdrawn for
analysis of the Brevinin-2R concentration and the release
medium was then replaced with an equal volume of fresh
PBS to maintain a sink condition. The concentration of
released Brevinin-2R was detected by UV/Vis spectropho-
tometer (Teif sanj pishro pajohesh, Iran) at 520 nm (11). The
release data have been shown as the amounts of released
drug relative to the total drug content in the conjugates. All
experiments were performed in triplicate.
Neutral Red Uptake Assay
The cytotoxicity of nanoparticles, Brevinin-2R and Brev-
inin-2R-loaded PLA–PEG–PLA micelles against AGS,
HepG2, KYSE-30 and HGF1-PI1 cell lines were determined
with NR assay. Briefly, cells were plated into a 96-well plate
(5×103 cells/well). After 24 h, cells were incubated for 24,
48, and 72 h with different concentrations of nanoparticles,
Brevinin-2R and Brevinin-2R-conjugated micelles. After
incubation time points, the medium was removed and a
medium containing NR (20 ml of 0.05% NR solution) was
added to wells and cells were then incubated for 3 h. After
removing the NR solution, the cells were rinsed with PBS
(37 °C) and 150 µl fixative solution (50% ethanol 96%, 49%
deionized water and 1% glacial acetic acid) was added to
wells; after 5 min of gentle shaking, the absorbance was
detected with a micro plate reader (Synergy HT, BioTek)
at 540 nm.
Cell Culture
The AGS, KYSE-30 and HepG2 and HGF1-PI1 cell lines
were cultured in RPMI-1640 and supplemented with 10%
fetal bovine serum (FBS), 100 unit/ml penicillin and 100 µg/
ml streptomycin. After incubation at 37 °C in a water satu-
rated atmosphere of 5% CO2, the cells were plated in micro
plates (around 1×104 cells per well) and went through an
overnight incubation.
Qualitative Cellular Uptake Study
Cell Cycle Arrest Analysis
In order to visualize cell internalization of copolymeric
micelles, AGS, KYSE-30 and HepG2 cancer cells were
seeded at a density of 4 × 105 cells/well on coverslips in
6-well plates for fluorescence microscopy until they formed
a monolayer. The medium was then eliminated and cells
were incubated with the FITC-conjugated micelles (weight
ratio 1:1). After 4 h of incubation, the cells were washed
three times with cold PBS and fixed with 4% paraformal-
dehyde in PBS for 15 min at room temperature and then
rinsed twice with fresh PBS. The fixed cells were subjected
Cell cycle distribution and DNA content of treated cells
with Brevinin-2R and Brevinin-2R-loaded nanoparticles
were evaluated through flow cytometry. AGS, HepG2 and
KYSE-30 cells were treated with Brevinin-2R and Brevinin-
2R-loaded nanoparticles for 48 h and then rinsed with PBS.
After fixing with ice cold ethanol for 2 h, cells were again
rinsed and after adding propidium-iodide staining solu-
tion (sigma) incubated for 40 min; afterward, cells were
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International Journal of Peptide Research and Therapeutics
evaluated by the flow cytometer (Partec, Germany) and then
Cell cycle profiles were analyzed using Flomax Software
(Partec, Germany).
than those of corresponding blank micelles. The enlarge-
ment of the particle size because of Brevinin-2R incorpora-
tion into the hydrophobic core might be attributed to inter-
actions between Brevinin-2R and copolymer, leading to the
variation in size. As a macromolecule, Brevinin-2R could
boost the diameter, however, Brevinin-2R-loaded nanopar-
ticles can easily enter tumor capillaries with pore cutoff size
ranging 200 nm to 1.2 µm. The conjugates prepared in this
study may also avoid the mononuclear phagocytic system
(MPS) and culminate in prolonged blood circulation (Hami
et al. 2014a; Hobbs et al. 1998; Phan et al. 2016).
Apoptosis Study by Annexin‑V‑FLOUS‑PI
AGS, KYSE-30 and HepG2 cells, treated with Brevinin-2R
and Brevinin-2R-loaded nanoparticles (48 h), were rinsed
with PBS and went through 20 min incubation with Annex-
inV FLOUS (20 ml) and PI (20 ml) (Roche) at 25 °C. After
staining, cells were rinsed and suspended into the incuba-
tion buffer and apoptotic cells were studied through flow
cytometry.
FTIR Analysis
Statistical Analysis
The polymerization of PLA–PEG2000–PLA triblock copoly-
mer and Brevinin-2R loading on micelles were characterized
by FTIR spectroscopy. To display the most important bands
of absorption, the second order derivatives of FTIR spec-
tra were determined using the spectrometer software OPUS
(Fig. 4). The spectrum analysis of triblock PLA–PEG–PLA
copolymer displayed characteristic vibrational modes
including stretching frequencies for asymmetric and sym-
metric motions; the band at 2899.25 cm−1 corresponded to
the C–H stretching vibration of –CH3 groups of the PEG.
The characteristic sharp and intense bands at 1760.68
and 1122.58 cm−1 confirm the presence of the carboxylic
ester (C=O in PLA) and ether (C–O–C from PEG block)
groups. The peak at 1456.79 cm−1 is attributed to a C–H
bond of the methyl group. Bending frequencies for the –CH3
symmetric have been identified at 1356.13 cm−1. Band of
C–C–O stretching of PEG chains appears around 952.98
and 838.85 cm−1. The FTIR spectrum of PLA–PEG–PLA
copolymer detected the presence of two constituent blocks
in polymer, indicating the ring opening polymerization of
lactide and formation of a PLA–PEG–PLA triblock copoly-
mer (Fig. 4A) (Asadi et al. 2011; Zhao et al. 2012).
All experiments were conducted repeatedly at least three
times in triplicate. Data were graphed and analyzed by
GraphPad Prism Software 7.03 using one-way analysis of
variance (ANOVA) and the Student’s t test and expressed
as the mean SD; P < 0.05 was considered statistically
significant.
Result and Discussion
Synthesis of Peptide
In order to mass confirmation, the synthesized peptide was
purified through HPLC (Fig. 2A) and then subjected to the
mass spectrometer. Mass analysis proved the expectation
and validate the mass ion of m/z 2634.63 for Brevinin-2R
(Fig. 2B).
Dynamic Light Scattering and Zeta Potential
DLS measurements illustrated the mean hydrodynamic
diameter of 48 nm and 100 nm for PLA–PEG2000–PLA
nanoparticles and Brevinin-2R-conjugated nanoparticles,
respectively. Also, zeta potential measurement displayed the
surface charge of −26.63 and −21.90 mV for blank nano-
particles and Brevinin-2R-loaded nanoparticles, respectively.
The smaller surface charge of blank micelles may be due to
the arrangement of PEG on the outer layer of nanoparticles.
FTIR spectrum of Brevinin-2R-loaded nanoparticles
(Fig. 4B), characterizing the potential interactions between
Brevinin-2R and nanoparticles, is accompanied by slight
shifts in absorption bands of amino and carboxylic groups,
and amide bonds due to the possible interaction of a carboxyl
group of the polymer with the amino group of Brevinin-2R;
this evidence can be taken into account as an indication for
the presence of Brevinin-2R into the nanoparticles.
The representative intensity at 3542.55 cm−1, related
to N–H and O–H stretching vibrations for the Brevinin-
2R-loaded nanoparticles, is due to the incorporation of
Brevinin-2R into the nanoparticles through probability of
physicochemical interactions. The peak at 3158.02 cm−1 is
thought to be the N–H bending vibration of amide A and
the peak at 2559.29 is ascribed to CH2 stretching vibration.
The absorption band at 1094.46 cm−1 is assigned to the C–O
stretching in the secondary hydroxyl group (characteristic
Particle Size Distribution and Morphology
of Nanoparticles
The obtained micrographs expose the formation of spherical
micelles as smooth and discrete spots with a size distribution
of 40–80 nm for nanoparticles (Fig. 3A) and 70–150 nm for
Brevinin-2R-loaded nanoparticles (Fig. 3B); hence the mean
diameters of the Brevinin-2R-loaded micelles were larger
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International Journal of Peptide Research and Therapeutics
Fig. 2 A Chromatography of crude peptide in C18 analytical RP-
HPLC column. Only encircled fraction was further purified by C8
reverse-phase semi-preparative columns. The inset depicts the chro-
matogram of the pure Brevinin-2R gained from analytical C8 reverse-
phase column. B MALDI-TOF mass spectral data confirmed the
expected molecular mass for Brevinin-2R. The spectrum was col-
lected in a scan range of 999–3012 m/z
Fig. 3 SEM imaging by
300 nm order of magnitude for:
nanoparticles (A) and Brevinin-
2R-conjugated PLA–PEG–PLA
micelles (B)
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International Journal of Peptide Research and Therapeutics
bending (amide IV and amide V). Accordingly, there is a
relocation for amide I band on account of some intermo-
lecular and electrostatic interactions between Brevinin-2R
and PLA–PEG–PLA. The peak at 3542.55 is ascribed to the
stretching vibration of –OH and –NH; FTIR data analysis
demonstrated efficient incorporation of Brevinin-2R in the
PLA–PEG–PLA micelles.
Biocompatibility Assay
The hemolytic activity of PLA–PEG–PLA copolymer
against human red blood cells was considered as a criterion
for toxicity to mammalian cells. According to our data, at a
concentration range of 2.5–80 mg/ml, the hemolytic activity
of PLA–PEG–PLA polymeric nanoparticle toward human
erythrocytes being measured at less than 5% (Fig. 5), indi-
cates low hemolytic activity (P<0.05), displaying its quali-
fication as a proper biocompatible drug delivery system.
Fig. 4 FT-IR spectra for PLA–PEG–PLA polymeric nanoparticles
(A) and Brevinin-2R-loaded PLA–PEG–PLA (B) in the region of
4000–400 cm−1
peak of –CH–OH). The amide I band is the most sensitive
and intense absorption band in the protein secondary struc-
tural components. Its frequency is found in 1636.62 cm−1;
it is associated with the C=O stretching vibrations of the
peptide bond (amide I) and is directly related to backbone
conformation and hydrogen bonding pattern. The peak at
1456.28 cm−1 is attributed to peptide side-chain –COO
and also polymer carboxyl that are overlapped. The peaks
at 698.84 and 630.80 are attributed to O=C–N and NH
Cell Uptake and Internalization
Fluorescence microscopy was utilized to visualize the effect
of PLA–PEG–PLA co-polymeric micelles on the cellular
uptake by the AGS, KYSE-30 and HepG2 cells. Taking
free FITC as a negative control, the fluorescence of FITC-
labeled co-polymeric micelles provided evidence for inter-
nalization of polymeric micelles; cytoplasmic distribution
Fig. 5 A Hemolytic activity
diagram for PLA–PEG–PLA
polymeric nanoparticles against
human erythrocytes. B Hemoly-
sis test for PLA–PEG–PLA
polymeric nanoparticles: (A)
positive control; (B) negative
control; (C) 2.5 mg/ml; (D)
5 mg/ml; (E) 10 mg/ml; (F)
20 mg/ml; (G) 40 mg/ml; (H)
80 mg/ml
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International Journal of Peptide Research and Therapeutics
Fig. 6 A Confocal microscopy
images of PLA–PEG–PLA co-
polymeric nano micelles uptake
by cancer cells after 4 h incuba-
tion with FITC-labeled polymer
vs. control FITC treated cells.
(b) AGS cell treated with FITC-
labeled polymer, (a) control;
(d) KYSE-30 cell treated with
FITC-labeled polymer, (c)
control; (f) HepG2 cell treated
with FITC-labeled polymer,
(e) control. B In vitro (PBS pH
7.4, 37 °C) release profile of
Brevinin-2R from the PLA–
PEG–PLA nanoparticles; each
point represents mean SD for
three samples and error bars
represent the SD (n=3)
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International Journal of Peptide Research and Therapeutics
of FITC-labeled micelles was indicated through strong
cell-associated green fluorescence (Fig. 6A). As expected,
there was no fluorescence in samples with free FITC
(Fig. 6A—a, c, e). The internalization of PLA–PEG–PLA
micelles into AGS, KYSE-30 and HepG2 cells has plausibly
come about by endocytosis. Penetrating plasma membrane
and translocating into the cytoplasm of the cancer cells
highlighted that the FITC-labeled nanoparticles did not
lead to disruption of cell membrane integrity (Fig. 6A—
b, d, f), indicating the feasibility of triblock micelles for
drug delivery systems. These results represented that as a
delivery platform, PLA–PEG–PLA nanoparticles could be
able to boost the efficacy of Brevinin-2R cellular uptake.
The Fig. 6A displays confocal optical images of the AGS,
KYSE-30 and HepG2 cancer cells after 4 h incubation with
PLA–PEG–PLA–FITC.
Based on former studies, PLA–PEG–PLA micelles are
highly effective in encapsulation and the sustained release
of therapeutic agents (Phan et al. 2016; Venkatraman et al.
2005).
Cell Viability Assay
In vitro cytotoxicity of blank nanoparticles, free Brevinin-
2R and Brevinin-2R-conjugated PLA–PEG–PLA micelles
was investigated on AGS, HepG2, KYSE-30 and HGF1-PI
cells by MTT and NR methods in the range of concentra-
tions tested using non-treated cells as the control. As shown
in Fig. 7A, blank nanoparticles did not show any cytotoxic
effect on treated cell lines, which indicated the biocompat-
ibility of nanoparticles as the defining characteristics for a
delivery system. On the other hand, both free Brevinin-2R
and Brevinin-2R-conjugated micelles dramatically decreased
cell viability of AGS, KYSE-30 and HepG2 cells in a time
and dose-dependent trend. However, the sensitivity of
the treated cells towards the cytotoxic effect of Brevinin-
2R-loaded nanoparticles was more than that of Brevinin-2R,
Fig. 7C–E. The IC50 values of the Brevinin-2R and Brevinin-
2R-loaded nanoparticles have been shown in Table 1. These
values highlighted that at the same time intervals, the Brev-
inin-2R-loaded nanoparticles had higher inhibitory effects
on the mentioned cancer cell lines compared to the Brevinin-
2R (P<0.05), which can be attributed to the increased cel-
lular uptake. Brevinin-2R-loaded micelles were efficiently
internalized by the AGS, HepG2 and KYSE-30 cells. These
results demonstrated that incorporation of Brevinin-2R into
PLA–PEG–PLA micelles maintained the anti-proliferation
effect of Brevinin-2R. The AGS cells showed higher sensi-
tivity towards Brevinin-2R and Brevinin-2R-loaded copoly-
mer than the two other cells. On the other hand, at the inves-
tigated concentrations, Brevinin-2R and Brevinin-2R-loaded
nanoparticles showed negligible cytotoxic effects on HGF1-
PI cell as a control non-tumor cell line Fig. 7B.
Loading Efficiency and In Vitro Drug Release
Mean Brevinin-2R entrapment efficiency from three inde-
pendent assessments was approximately 95%. This could
be due to intermolecular interactions. The in vitro release
of Brevinin-2R from PLA–PEG–PLA micelles was investi-
gated using the dialysis method in PBS (pH 7.4) as a release
medium to simulate the physiological condition. Brevinin-
2R is an amphiphilic peptide that can be dissolved in water.
Thus, the release profile would be comparable with a hydro-
philic drug such as 5-FU (Venkatraman et al. 2005). The
Brevinin-2R-conjugated micelles showed a biphasic release
profile (Fig. 6B); the vast majority of Brevinin-2Rs were
released from Brevinin-2R-loaded micelles during transi-
tional release in primary phase 2–8 days; after the primary
stage, the release reached a plateau; for a small percentage
of Brevinin-2R, release possibly has taken place in 12 days
caused by the gradual degradation of copolymer and diffu-
sion of Brevinin-2R entrapped inside the nanoparticles. The
cumulative release of Brevinin-2R from sub layers of nano-
particles after 8 days was 88.5%; almost all the entrapped
Brevinin-2R was released within 20 days in PBS medium.
It was found that micelles indicated a superior drug release
that is consistent with previous studies for 5-FU (Venkatra-
man et al. 2005). Copolymeric micelles, in part, prolonged
release time and increased inhibitory effect of Brevinin-2R.
Cell Cycle Analysis
Cell cycle analysis detected intense Sub G1 arrest in Brev-
inin-2R and Brevinin-2R-loaded nanoparticles treated
AGS, HepG2 and KYSE-30 cells compared to untreated
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International Journal of Peptide Research and Therapeutics
Fig. 7 A Viability test for treated AGS, KYSE-30, HepG2 and
HGF1-PI1 cell lines with PLA–PEG–PLA nanoparticles measured by
MTT assay; viability study of treated B HGF1-PI, C AGS, D HepG2
and E KYSE-30 cell lines with Brevinin-2R and Brevinin-2R-loaded
micelles at different concentrations measured through MTT, after 24,
48, and 72 h; each point represents mean SD, and error bars rep-
resent the SD (n=3). The differences in cytotoxicity between free
Brevinin-2R and Brevinin-2R-conjugated micelles are significant
(P<0.05)
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International Journal of Peptide Research and Therapeutics
Table 1 The average IC50 values for (a) Brevinin-2R, (b) Brevinin-
2R-loaded nanoparticles for treated AGS, HepG2 and KYSE-30 cell
lines after 24, 48, and 72 h measured through MTT and neutral red
uptake assays; data are as mean SD (n=3), P<0.0 5
nanoparticles were captured in Sub G1 stage of the cell
cycle and KYSE-30 rates were 13.30% and 38.21%, respec-
tively (P<0.05).
Cell line (time) IC50 by MTT IC50 by neutral Total IC50 (µg/ml)
Apoptosis Study
assay (µg/ml) red assay (µg/
ml)
Brevinin-2R induced apoptosis cascade in cancer cells.
Compared to Brevinin-2R treated cells, the higher rates of
apoptosis were witnessed in cells treated with Brevinin-
2R-conjugated nanoparticles (Fig. 9). Regarding AGS,
compared to the control, 16.44% and 27.66% of cells under
treatment with Brevinin-2R and Brevinin-2R-conjugated
nanoparticles undertook apoptosis, respectively. In the
interim, 13.99% and 25.03% of HepG2 cells saw apoptosis
in treated cells with Brevinin-2R and Brevinin-2R-conju-
gated nanoparticles. In comparison to 12.58% apoptotic
cells in KYSE-30 treated with Brevinin-2R, 21.55% of
cells were captured by apoptosis in treated cells with Brev-
inin-2R-conjugated nanoparticles (P < 0.05).
(a)
AGS 24 h
AGS 48 h
AGS 72 h
279.81 7.9 285.29 2.7
167.78 6.4 157.71 4.2
141.58 1.8 140.06 1.6
340.65 1.2 334.41 1.8
221.35 5.3 215.36 1.4
188.45 5.1 186.25 3.5
282.55 5.3
162.74 5.3
140.82 1.7
337.53 1.5
218.35 3.3
187.35 4.3
357.63 3.8
265.89 2.9
223.46 3.1
HepG2 24 h
HepG2 48 h
HepG2 72 h
KYSE-30 24 h 356.78 4.7 358.48 2.9
KYSE-30 48 h 266.33 1.4 265.45 4.5
KYSE-30 72 h 225.52
(b)
AGS 24 h
AGS 48 h
AGS 72 h
HepG2 24 h
HepG2 48 h
HepG2 72 h
2
221.4 4.2
114.44
5
105.17 5.1
109.80 5
89.21 2.6
66.84 2.2
145.12 0.7
79.23
61.48
142.5
4
1
3
84.22 3.3
64.16 1.6
143.81 1.8
109.53 1.8
Conclusions
112.03 1.04 107.03 2.6
82.76 0.6 81.03 1.4
81.89
1
There has been an increasing interest in the usage of
broad-spectrum, alternative natural anticancer agents,
such as drug peptides to address the problems related to
cancer therapy. Our findings showed that the synthesized
PLA–PEG–PLA copolymeric micelles, as biocompatible
carriers, increased the inhibitory effect of Brevinin-2R
on AGS, HepG2 and KYSE-30 cells. Furthermore, this
nanoparticle caused higher cell cycle arrest and apop-
tosis induced by Brevinin-2R-conjugated nanoparticles
compared to that of Brevinin-2R. In conclusion, through
protection from proteolytic decomposition and sus-
tained release behavior, leading to improved anticancer
activity (Zhang et al. 2017), PLA–PEG–PLA triblock
copolymer can act as a promising nano drug delivery
system for hydrophilic therapeutic agents such as Brev-
inin-2R. This is the first study on the anti-proliferative
effect of Brevinin-2R and Brevinin-2R incorporated into
PLA–PEG–PLA on gastric, hepatocellular and squamous
KYSE-30 24 h 199.75 3.3 197.74 3.7
KYSE-30 48 h 132.55 1.4 129.13 2.8
KYSE-30 72 h 115.76 1.6 109.19 1.8
198.74 3.5
130.84 2.1
112.47 1.7
control cells. Also, the percentages of cells in G2-M phase
dropped remarkably after 48 h of treatment. In comparison
with Brevinin-2R treated cells, higher percentages of cell
arrest were observed in cancer cells treated with Brevinin-
2R-conjugated nanoparticles. In AGS cell line, treated with
Brevinin-2R and Brevinin-2R-loaded nanoparticles com-
pared to untreated control cells, 22.58% and 48.77% of cells
were arrested in Sub G1 phase, respectively (Fig. 8). After
48 h of incubation, up to 18.70% and 43.30% of HepG2
cells treated with Brevinin-2R and Brevinin-2R-loaded
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International Journal of Peptide Research and Therapeutics
Fig. 8 After 48 h treatment with IC50 concentrations of Brevinin-2R
and Brevinin-2R conjugated PLA–PEG–PLA, cell cycle distributions
were analyzed by flow cytometry. Each value is a representative of
three independent experiments compared to the untreated same cells.
Data are as mean SD, P<0.05. In comparison with Brevinin-2R,
Brevinin-2R-loaded nanoparticles induced higher rates of Sub G1
Cell cycle arrest in treated cells
carcinoma cells. With reduced IC50 values, when conju-
gated with PLA–PEG–PLA nanoparticle as a biocompat-
ible delivery system, Brevinin-2R could be an alternative
anti-cancer therapeutic agent in lieu of chemotherapy
and its side effects. Additional studies will be required to
examine the possibilities of this potential delivery system
in vivo.
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International Journal of Peptide Research and Therapeutics
Fig. 9 A The effects of the Brevinin-2R and Brevinin-2R-conjugated
PLA–PEG–PLA on apoptosis induction in AGS, HepG2 and KYSE-
30 cell lines after 48 h treatment with IC50 concentrations for each
cell line were determined by AV/PI staining. Samples were analyzed
using a flow cytometry and representative dot plot is illustrated. Q1:
necrotic, AV−/PI+; Q2: late apoptotic, AV+/PI+; Q3: live, AV−/
PI−; Q4: early apoptotic, AV+ /PI−; the graphs are representative
of three independent experiments compared to untreated control cells.
Data are as mean SD, P<0.05. Compared to Brevinin-2R, Brev-
inin-2R-loaded nanoparticles induced higher rates of apoptosis in
treated cells. B Bar diagrams of cells which are positive for annexin-
V, PI and both annexin-V/PI
Acknowledgements This research was financially supported by the
University of Mohaghegh Ardabili, Ardabil, Iran. The authors would
like to acknowledge Jennifer Mason for proofreading the article. We
are also grateful to the contribution of Dr. Hossein Akbari from Arda-
bil Azad University and Mehdi Hassanvand Jamadi from University
of Shahid Beheshti to statistical analysis and critical reviewing of
manuscript.
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