AAPS PharmSciTech ( # 2018)

DOI: 10.1208/s12249-018-0969-4

Research Article

In Vitro Characterization and Evaluation of the Cytotoxicity Effects of Nisin

and Nisin-Loaded PLA-PEG-PLA Nanoparticles on Gastrointestinal (AGS
and KYSE-30), Hepatic (HepG2) and Blood (K562) Cancer Cell Lines

Fariba Goudarzi,1,3 Asadollah Asadi,1 Maryam Afsharpour,2 and Robab Hassanvand Jamadi1

Received 26 September 2017; accepted 30 January 2018

Abstract. The aim of this study was an in vitro evaluation and comparison of the cytotoxic
effects of free nisin and nisin-loaded PLA-PEG-PLA nanoparticles on gastrointestinal (AGS
and KYSE-30), hepatic (HepG2), and blood (K562) cancer cell lines. To create this novel
anti-cancer drug delivery system, the nanoparticles were synthesized and then loaded with
nisin. Subsequently, their biocompatibility, ability to enter cells, and physicochemical
properties, including formation, size, and shape, were studied using hemolysis, fluorescein
isothiocyanate (FITC), Fourier transform infrared (FTIR) spectroscopy, dynamic light
scattering (DLS), and scanning electron microscopy (SEM), respectively. Then, its loading
efficiency and release kinetics were examined to assess the potential impact of this
formulation for the nanoparticle carrier candidacy. The cytotoxicities of nisin and nisin-
loaded nanoparticles were evaluated by using the MTT and Neutral Red (NR) uptake assays.
Detections of the apoptotic cells were done via Ethidium Bromide (EB)/Acridine Orange
(AO) staining. The FTIR spectra, SEM images, and DLS graph confirmed the formations of
the nanoparticles and nisin-loaded nanoparticles with spherical, distinct, and smooth surfaces
and average sizes of 100 and 200 nm, respectively. The loading efficiency of the latter
nanoparticles was about 85–90%. The hemolysis test represented their non-cytotoxicities and
the FITC images indicated their entrance inside the cells. An increase in the percentage of
apoptotic cells was observed through EB/AO staining. These results demonstrated that nisin
had a cytotoxic effect on AGS, KYSE-30, HepG2, and K562 cancer cell lines, while the
cytotoxicity of nisin-loaded nanoparticles was more than that of the free nisin.
KEY WORDS: nisin; PLA-PEG-PLA Co-polymeric nanoparticles; cancer cell line; cytotoxic effect;
apoptosis.

INTRODUCTION that create a stable relationship between PLA-PEG-PLA

Nowadays, different methods are employed to deliver the
effective and safe therapeutic agents to human cancer cells.
Targeting the right material is important when designing such
drug delivery systems [1]. For this purpose, nanoparticles and
micelles are utilized for the investigation of drug delivery [2].
Pharmaceutical nanotechnology relates to the use of nanoscale
agents, such as active and biocompatible micellar systems,
nanoparticles, and conjugates [3]. Poly (lactic acid) (PLA) is
the hydrophobic core of micelles, which provides an environ-
ment for the integration of hydrophobic drugs, and poly
(ethylene glycol) (PEG) makes up their hydrophilic outer shells
1
Department of Biology, Faculty of Science, University of
Mohaghegh Ardabili, Daneshgah St, Ardabil, 11367-56199, Iran.
2
Department of Inorganic Chemistry, Chemistry and Chemical
Engineering Research Center of Iran, Tehran, 14335-186, Iran.
3
To whom correspondence should be addressed. (e–mail:
Faribagoodarzi@yahoo.com)
nanoparticles and the external environment [4]. The previous
reports indicated that PLA-PEG-PLA can be introduced as a
novel drug delivery carrier due to its good stability, high
encapsulation efficiency, good release profile, biodegradability,
and biocompatibility [5–8]. Lantibiotics are a class of
bacteriocins that have lanthionine and methyl-lanthionine
amino acids in their structures. A subset of lantibiotics is
stretched and flexible with positively charged peptides like nisin,
which create some pores in the membranes of the target
bacterial species [9]. Nisin is a water-soluble poly-cyclic peptide
with 34 amino acids produced by gram-positive bacteria
Lactococcus lactis, which can be obtained from the culture
medium. This peptide has an amphipathic structure with a
charge of + 4. The N-terminal of the peptide is more hydropho-
bic than its C-terminal. Nisin is a non-toxic peptide that is
quickly inactivated by the digestive enzymes. For this reason, in
1969, Food and Agriculture Organization (FAO) and World
Health Organization (WHO) allowed its use as a preservative in
food industry. In recent years, many researches have been done

1530-9932/18/0000-0001/0 # 2018 American Association of Pharmaceutical Scientists


on the various biological activities and therapeutic effects of
nisin and some studies have provided scientific evidence on its
anti-cancer effects [10–12].
Gastrointestinal cancers associated with hereditary and
environmental factors have high worldwide prevalence
[13–16]. Almost 50% of the gastrointestinal cancers are
related to gastric cancer [17], which is the second cause of
death from cancer in the world [18, 19]. Today, gastric cancer
treatment remains a major challenge [20, 21]. Esophageal
cancer is another common cancer in the world with a high
offensive power [22]. Despite some progress in the treatment,
esophageal cancer has still remained as a challenge for
researchers [14, 22–24]. In many parts of the world, liver
cancer has high prevalence, while its treatment currently has
been associated with the relatively low effects of the related
drugs [13, 25, 26]. Leukemia is a progressive disease and
malignant hematopoietic tissues in the body like K562 human
chronic myeloid leukemia (CML) cell line [27, 28].
Due to the numerous biological effects of nisin and lack
of a comprehensive study on its functional mechanism in
cancer cell lines, we tried to formulate a biocompatible PLA-
PEG-PLA nanoparticles in the present work. After loading
nisin onto the nanoparticles, their physical characteristics
were determined. The cytotoxic effects of free nisin and the
nisin-loaded nanoparticles on AGS, KYSE-30, HepG2, and
K562 cancer cell lines were evaluated and compared by MTT
and Neutral Red (NR) uptake assays. Also, their cytotoxic
effects on the Vero cell line were assessed to determine their
non-specific responses on non-tumor cell lines.
MATERIALS AND METHODS
Materials
L-lactic acid, antimony trioxide (Sb2O3), dichlorometh-
ane (DCM), toluene, polyethylene glycol (PEG Mn = 2000),
acetic acid, ethanol, comassie brilliant blue, ethyl acetate,
nisin, Neutral Red, Acridine Orange, MTT, and dimethyl
sulfoxide were purchased from Merck Co (Germany).
Fluorescein isothiocyanate, stannous octoate, and Drabkin’s
Reagent were purchased from Sigma-Aldrich Co (USA).
RPMI-1640, fetal bovine serum, and trypsin-EDTA were
purchased from Gibco Co (UK). All cancer cell lines
including human esophageal squamous cell carcinoma
(KYSE-30), human hepatocellular carcinoma (HepG2), hu-
man gastric carcinoma (AGS), K562, and Vero cell line
(kidney epithelial cells of the normal adult African green
monkey) were provided from Pasteur Institute of Iran Cell
Bank (Tehran, Iran). Before use, all medium and reagents
were prepared.
Methods
Preparation of PLA-PEG-PLA Copolymer
PLA-PEG-PLA copolymer was synthesized by respec-
tively using 45.5% PLA, 9% PEG, and 45.5% PLA with a
molecular weight of 88,000 through the following procedure:
First, PEG was dried under vacuum at 40°C overnight.
Then, toluene was dried and stored together with metal sodium
in a bottle. Finally, antimony trioxide (Sb2O3) was utilized to
Goudarzi et al.
catalyze L-lactide synthesis using L-lactic acid. The two steps of
oligomerization at 180–200°C and depolymerization and dimer-
ization at 250°C were followed in this process. L-lactide
recrystallization from ethyl acetate was done three times. Then,
the product was dried under vacuum at 40°C overnight (Fig. 1).
Afterwards, 6.5 g of the purified L-lactide was poured into a
100 mL round-bottom flask containing 1 g of pre-dried PEG
2000 and protected by nitrogen. 0.15% (w/w) of L-lactide and
stannous octoate dissolved in dry toluene was added to the
flask. Then, the mixture was heated to 130°C for 12 h (Fig. 1).
The final products were repeatedly dissolved into dichloro-
methane to purify them. Subsequently, they were precipitated
by the addition of cold ethanol to later examine their
structures. Ultimately, the segregated polymers were dried
under vacuum at 40°C for 24 h [29].
PLA-PEG-PLA Nanoparticle Formation
PLA-PEG-PLA copolymer (20 mg) was dissolved in
dimethylformamide (DMF) (1 mL), and the solution was added
slowly into 20 mL stirring deionized water. After the polymer
addition, the water turned into cloudy, and the aqueous
emulsion was stirred for 4 h. Dialysis method was used to
eliminate DMF. Briefly, the micellar solution was transferred
into a dialysis bag and dialyzed in a water bath. The bath water
was changed at certain intervals. Required concentrations were
ready by dilution of the stock solutions [29].
Preparation of Nisin-Loaded PLA-PEG-PLA Nanoparticles
Nisin is an amphipathic polypeptide with a charge of +
4 at physiological pH due to having lysine amino acid. Thus,
in acidic conditions (pH < 7.4), it has the most activity and
highest solubility. For this reason, to load nisin onto the
nanoparticles, it was dissolved in HCl (0.02 N) to prepare
10 mg/mL of its solution. 1 mL of nisin solution was added to
10 mL of PLA-PEG-PLA nanoparticle solution (100 mg/mL).
The two solutions were mixed and stirred for 2 h. The loaded
nanoparticles were gained through an ultrafiltration tech-
nique (Amicon, Ultracel-100 K, 100-kDa cutoff). Then, the
nisin-loaded nanoparticles were washed with DMF and
centrifuged at 8000×g rpm at 4°C for 10 min to remove the
untreated polymeric nanoparticles. After that, the nanoparti-
cles were thoroughly washed with deionized water to remove
any unreacted nisin.
Nanoparticle Characterization
Morphology of the PLA-PEG-PLA Nanoparticles. The
surface morphology of the polymeric nanoparticles, such as
aggregation phenomenon and shape, were studied via scan-
ning electron microscopy (SEM). To prepare the samples for
SEM, the nanoparticle suspensions were mounted on alumi-
num stabs and plating-coated under vacuum. Then, the
samples of the nanoparticle suspensions were examined
through SEM (LEO 1430 VP, UK).
Size Determination of the Nanoparticles by Dynamic
Light Scattering. Before measurement, 0.1 mg/L and 0.01 g/L of
PLA-PEG-PLA and the nisin-loaded nanoparticles were dissolved
in DMF and phosphate-buffered saline (PBS) after sonication,
Evaluation of Cytotoxicity Effects of Nisin on Cancer Cell Lines

Fig. 1. Synthesis of L-lactide and PLA-PEG2000-PLA triblock copolymers

respectively. To perform a dynamic light scattering (DLS)
measurement, the size distributions and average sizes of the
nanoparticles and the nisin-loaded nanoparticles were determined
at a wavelength of 633 nm at 25°C by a light scattering
spectrometer (Zetasizer 3000 HS, Malvern Instruments, UK).
media) for the experiment. A 12-kDa MWCO membrane was
employed for nisin to be able to pass through its pores. The
membrane was at the interface between the solution contain-
ing the nisin-loaded nanoparticles (upper part of the tube)

Zeta Potential Measurements. To assess the colloidal stabil-

ity, the surface charges of the PLA-PEG-PLA and nisin-loaded
nanoparticles were determined by zeta potential analyzer (Malvern
Instruments, UK). Before the analysis, 1.5 mg/mL of each type of
the nanoparticles was dispersed in PBS at a pH of 7.4. The
dispersion was sonicated in a bath-type ultrasonicator for 1 min.

Fourier Transform Infrared Spectroscopy Analysis

(FTIR). Fourier transform infrared (FTIR; RXI, Perkin
Elmer, UK) was applied to analyze PLA-PEG-PLA powder,
nisin powder, and the powder of nisin-loaded PLA-PEG-PLA
nanoparticles. The specimens were blended with dry KBr at a
ratio of 2% (w/w). Then, KBr discs were produced under
10,000 psi hydraulic pressure. FTIR spectra were recorded on
each KBr disc at 4 mm/s with a resolution of 2 cm over a
wavenumber range of 400–4000 cm−1. The experiment was
repeated three times for each sample [30].

Loading Efficiency of the Nanoparticles. The dispersions

of the nisin-loaded nanoparticles were centrifuged at 11,000×g
through a 100-kDa molecular weight cutoff (MWCO) ultrafil-
tration at 25°C for 10 min. The protein amount in the
supernatant was determined by Bradford protein assay [30,
31]. The experiment was repeated three times. The nisin-loading
efficiency was calculated using the following equation:

Nisin total−Nisin supernatant

Loading Efficiency ¼
Nisin total

Evaluation Release of Nisin from the PLA-PEG-PLA

Nanoparticles. The three tubes were filled with 5 mL of PBS Fig. 2. a SEM image of PLA-PEG-PLA nanoparticles; b SEM image
solution (1 mg/mL) at the pH values of 4, 7.4, and 8.5 (release of the nisin-loaded PLA-PEG-PLA nanoparticles

and the release media of PBS (lower part of the tube). The
release media were constantly stirred at 300 rpm by a
magnetic stirring bar to prohibit the formation of an unstirred
water layer between the outer solution and the membrane.
The released amount of nisin from the nanoparticles was
determined by sampling the outer solution at the periodic
intervals of 0, 15, 30, 60, 120, 240, 300, 360, 420, 520, 560, 600,
700, 800, 900, and 1000 min at 37°C [30, 32].
Hemolysis Evaluation of Nanoparticles. A common test
for assessing the biocompatibility of nanoparticles is to
determine their hemolytic properties. In the hemolysis assay,
Drabkin’s Reagents are employed to convert the released
hemoglobin by damaged cells into red-colored
cyanmethemoglobin. Any forms of hemoglobin can be
involved in a reaction with Drabkin’s solution, except for
sulfhemoglobin, which is a pigment with a very low concen-
tration normally appearing in the blood. The mentioned
solution is prepared by mixing its dry reagent with a
surfactant. This is done by oxidizing hemoglobin and its
derivatives (except for sulfhemoglobin) into methemoglobin
in the presence of alkaline potassium ferricyanide.
Cyanmethemoglobin, which has maximum absorption at
540 nm, is formed through the reaction between potassium
cyanide and methemoglobin.
In this assay, the nanoparticle samples (1 mL) were added
to a 2% erythrocyte suspension (1 mL) at the concentrations of
2.5, 5, 10, and 20 mg/mL. Distilled water and PBS were
considered as the positive and negative controls, respectively.
The reaction was allowed to occur at 25°C and continue for 3 h.
Then, the centrifugation of erythrocyte suspension at 2000 rpm
was followed for 10 min to remove the undamaged red blood
cells (RBCs) and the nanoparticles. To detect the supernatant of
erythrocyte suspension containing cyanmethemoglobin at
540 nm and thus specify the hemolytic ratio, a Sinco UV/Vis
spectrophotometer (Model S4100, Korea) was applied. The
Fig. 3. Release diagram of nisin from the nanoparticles at different pH and times
Goudarzi et al.
standard non-hematotoxicity grade was obtained since the
hemolytic percentage was lower than 5%. The hemolysis
percentage was measured via the following equation [33–35]:
Hemolysis Ratio
Optical Density supernatant−Optical Density Negative Control
¼
Optical Density Positive Control−Optical Density Negative Control
 100
The data were totally obtained from the three experi-
ments and expressed as mean ± SD (SD = standard
deviation) (n = 3).
Evaluation of Nanoparticles’ Ability to Entrance in
Cells. To detect the uptake and internalization of PLA-
PEG-PLA nanoparticles into the cancer cell lines, the
nanoparticles were labeled with FITC. The FITC-loaded
nanoparticles were prepared by direct dissolution method
[36]. Briefly, FITC together with an appropriate amount of
the nanoparticle was dissolved in PBS, and after 30 min of
sonication, the solution was maintained at room temperature
for further 30 min. The unloaded FITC was removed by
centrifugation and washed twice in PBS. After 4 h of
treatment, the outer medium of the cells labeled with FITC
nanoparticles were removed, the cells were washed in PBS,
fixed with 150 mL of a fixation solution (0.2% glutaraldehyde
and 2% formaldehyde in PBS) for 10 min, and then evaluated
via fluorescence microscopy. The cells treated with no FITC-
loaded nanoparticles were considered as the control group.
Cell Culture and Treatment. The cells were cultured in
RPMI-1640 medium with 10% fetal bovine serum (FBS), 100
unit/mL of penicillin, and 100 μg/mL of streptomycin. Then, they
were incubated under an atmosphere of 5% CO2 and 95% air at
37°C. They were removed by a solution of 0.25 (w/v) trypsin and
Evaluation of Cytotoxicity Effects of Nisin on Cancer Cell Lines
0.02 (w/v) ethylenediaminetetraacetic acid after 1 week. To
investigate the cytotoxicity impacts of the nanoparticles, nisin
and nisin-loaded nanoparticles, they were placed in 96-well
culture plates at a density of 1 × 104 cells per well and incubated
for 24 h. After removing the medium (200 μL), the cells were
treated with a FBS-free medium at 0, 15, 30, 40, 60, 75, 150, 250,
350, and 450 μM concentrations of the free nisin and nisin-loaded
nanoparticles. It should be noted that nisin concentrations in the
nisin-loaded nanoparticles were considered to be equal to the
free nisin concentrations. 0, 2.5, 5, 10 and 20 mg/mL of the
nanoparticle concentrations were affected in each cell line. After
the intervals of 24, 48, and 72 h, the cell viability was determined
via the NR uptake and MTT assays. The wells not treated with
the nanoparticles, free nisin, and nisin-loaded nanoparticles were
used as the control groups. The experiments were done in
triplicate and the averages of the results were reported as mean ±
SD (n = 3). The statistical significance of the results was evaluated
by Student’s t test. P < 0.05 was considered significant.
MTT Assay. After removing the medium and 4 h before
the end of each treatment period, 20 μL of 2.5 mg/mL MTT
Fig. 4. FTIR spectra of: a PLA-PEG-PLA nanoparticles; b nisin-loaded PLA-
PEG-PLA nanoparticles
and 180 μL of FBS-free medium were added to each well. To
complete the incubation time, the plates were incubated for
further 4 h. Then, after removing the supernatants, 200 μL of
DMSO was added to each well. The plates were shaken for
10 min. The absorbance of each well was measured at 570 nm
on an ELISA plate reader (ELX808 Biotek, USA). The wells
without any cells were used as blanks [37–40]. The assay
was repeated three times and the average of the results was
expressed as mean ± SD (n = 3).
Neutral Red Uptake Assay. This method was used to
assess the number of live cells in the culture medium. It was
based on the ability of living cells to accumulate NR within
their lysosomes. To prepare an NR stock solution of 0.05%,
1 g of NR was added to 200 mL of PBS and filtered through a
0.45-μm filter. For preparing the fixative solution, 1 mL of
glacial acetic acid was added to 99 mL of 50% ethanol
solution. Three hours before finishing each incubation time
(24, 48, and 72 h), the medium was removed from each well
and 180 μL of FBS-free medium and 20 μL of NR 0.05%
stock solution were immediately added to each well to be
 Goudarzi et al.

subsequently incubated for 3 h. After completing the The nisin-loaded nanoparticles had a good size for circulating
incubation time, the supernatants were removed and each in the blood stream and passive targeting for a long time.
well was washed twice with PBS at 37°C. To elute the NR of
the cells into the solution and fix them, 150 μL of the fixative Size Distribution of Nanoparticles
solution was added to each well. The plates were shaken for
5 min and the absorbance measurement of each well on the The DLS results showed the average size of 100 and
plate reader was done at 540 nm [39, 40]. The assay was 200 nm for PLA-PEG-PLA and nisin-loaded nanoparticles,
repeated three times and the average of the results was respectively.
expressed as mean ± SD (n = 3).
Zeta Potential Measurements
Ethidium Bromide/Acridine Orange Staining for Apoptotic
Cells. Many biological studies require apoptosis analysis. How- Zeta potential measurements revealed that PLA-PEG-
ever, the current methods of measuring apoptosis have many PLA and nisin-loaded nanoparticles had the surface charges
limitations, including unwanted cellular damage, inability to gather of − 28.90 and − 22.63 mV, respectively. The lower zeta
only apoptotic and necrotic cells, inability to measure cell life-span, potential of PLA-PEG-PLA nanoparticles could be related
and misdiagnosis. To overcome these problems, we applied to PEG arrangement on the nanoparticle surface.
Ethidium Bromide/Acridine Orange (EB/AO) staining. Cancer
cells were cultured at a density of 1 × 103 cells per well in 96-well
plates and incubated for 24 h to be then treated with different Evaluation of Released Nisin from the Nanoparticles and
concentrations of free nisin and nisin-loaded nanoparticles within a Loading Efficiency
range of IC50 values obtained for each cell line. The plates were
centrifuged at 1000 rpm at 4°C for 5 min. 10 µL of EB/AO dye mix The release of an encapsulated protein from the particles
(100 μg/mL of EB and 100 μg/mL of AO) was prepared in PBS includes the swelling and weakening of the nanoparticle
and then added to each well. The cells were viewed and counted compact structure. The protein and peptide molecules were
using an inverted fluorescence microscope (IX71, Olympus, entrapped at sub-layers of the particles under hydrolytic
Japan). The green uptaking AO fluorescence determined the live
cells. The live and dead apoptotic cells associated with dense
chromatin fractions were stained with AO and EB, respectively, so
as to create apoptotic bodies. The necrotic cells were uniformly
labeled with EB [40–42]. The tests were performed three times and
at least 100 cells were counted each time. The results were
reported as mean ± SD (n = 3).

Statistical Analysis

The concentration value for 50% inhibition of cell

growth (IC50) was calculated using GraphPad Prism soft-
ware. All the experiments were repeated at least three times
and measured at least in triplicate. The results were expressed
as mean ± SD. Analysis of the statistical significance was done
by Student’s t test. The differences between the experimental
groups were regarded to be significant at a P value of less
than 0.05 (P < 0.0 5).

RESULTS

Microscopic Image Evaluation

Figure 2 displays the SEM images of PLA-PEG-PLA

nanoparticles (Fig. 2a) and nisin-loaded nanoparticles
(Fig. 2b). The images portray the formation of spherical
micelles with distinct and smooth surfaces of 50–100 and 50–
250 nm particle sizes for the nanoparticle and nisin-loaded
nanoparticles, respectively. As shown by the results, the
polymeric nanoparticles size is enhanced after nisin loading. Fig. 5. a Hemolytic diagram of PLA-PEG-PLA nanoparticle. b
As a big molecule, nisin could increase the diameters when Macroscopic image of hemolysis test for PLA-PEG-PLA polymeric
immobilized on the surfaces of the polymeric nanoparticles nanoparticles: A, negative control; B, 2.5 mg/mL; C, 5 mg/mL; D,
and subsequently decrease their surface smoothness (Fig. 2b). 10 mg/mL; E, 20 mg/mL; F, positive control
Evaluation of Cytotoxicity Effects of Nisin on Cancer Cell Lines
activities then spread and finally separated from the particle
structure.
Nisin cumulative releases from the nanocapsules at the
pH values of 4, 7.4, and 8.5 are exhibited in Fig. 3. The lower
values of pH demonstrated faster releases of nisin (pH 4 >
7.4 > 8.5). The cumulative release of nisin was 95% at a pH of
4 within 240 min, while no significant augmentation was
observed at a later time. At the pH values of 7.4 and 8.5, nisin
releases reached a balance within 420 and 600 min and its
cumulative releases were 90 and 75%, respectively. Nearly
85% of nisin molecules were transitionally released 240, 420,
and 600 min after initiation. This amount was found to be
much higher than those of the small drug molecules (almost
30%) released from other polymeric nanoparticles at the
early stages [43]. Assessment of the loading efficiency (85–
90%) was done through the equation mentioned in BLoading
Efficiency of the Nanoparticles^ section.
FTIR Evaluation
To evaluate the interaction of the various ingredients of the
nanoparticles to form the polymeric and nisin-loaded nanopar-
ticles, a study was done on the electrolyte interactions (Fig. 4). In
the polymeric nanoparticles structure (Fig. 4a), the bands
presented at around 2927 and 2890 cm−1 were related to the
vibrations of C-H bond. C=O bond stretching vibration could be
seen within the area of 1790 cm−1. The bands observed within
the regions of 1350–1450 and 1090–1200 cm−1 were related to C-
Fig. 6. The fluorescence microscopy images of cell lines treated with nanoparticle-FITC: a AGS; b KYSE-30; c HepG2; d
K562
H and C-O bending vibrations, respectively, which showed the
formation of PLA-PEG-PLA polymeric nanoparticles.
In the nisin-loaded nanoparticle structure (Fig. 4b), the
absorptions of the bonds in 2999 and 2949 cm−1 were related to
the vibrations of C-H, which was dislocated compared to the
polymeric nanoparticles structure. The absorption of the bond
observed in 1758 cm−1 was related to the stretching vibration of
the bond of C=O, which had been moved to a lower frequency
region. The bond stretching vibration of amide carbonyl in nisin
at about 1620 cm−1 was shifted to a lower frequency region. This
shift confirmed the link between nisin and the nanoparticles. The
absorption bond in the regions of 1450 and 1500 cm−1 were
related to C-N and N-H bonds in nisin, respectively. C-O
vibrations were seen to be slightly displaced in comparison with
the polymeric nanoparticles spectra. C=O bond of the polymeric
nanoparticle in the nisin-loaded nanoparticles was weaker. The
absorption of the bond in 3400 cm−1 was related to O-H and N-H
stretching vibrations, which were much smaller than those of
nisin. This indicated the possibility of these functional groups of
nisin bonding to C=O of the polymeric nanoparticles. The
physicochemical interactions could corroborate the evidence of
nisin presence inside PLA-PEG-PLA nanoparticles.
Evaluation of Hemolysis
As shown in Fig. 5, PLA-PEG-PLA polymeric nanopar-
ticles at a concentration range of 2.5–20 mg/mL demonstrated
a hemolysis percentage of less than 5%, which was similar to
 Goudarzi et al.

that of the negative control. Significant differences (P < 0.05)
were observed between all our polymeric nanoparticles
solutions as compared to the positive control, which was set
as 100% hemolysis. Hence, a negligible hemolysis capacity of
PLA-PEG-PLA co-polymeric nanoparticles was achieved and
the nanoparticles were thus deemed as biocompatible.
Ability of Entrance PLA-PEG-PLA Nanoparticle into Cells
The fluorescence microscopy images of the cells treated
with FITC-loaded nanoparticles revealed that the nanoparti-
cles was able to enter the AGS, KYSE-30, HepG2, and K562
cell lines and deliver intracellular FITC. The control cells
treated with non-labeled nanoparticles did not display any
fluorescence. Figure 6a–d are illustrations of the AGS,
KYSE-30, HepG2, and K562 cell lines treated with FITC-
loaded nanoparticles, respectively.
show any cytotoxic effect as shown in Fig. 7a, which was a
sign of the polymer biocompatibility as one of the main
essential characteristics for its use as a vector. The results of
the viability assays of nisin and nisin-loaded nanoparticles
indicated that nisin had a cytotoxic effect on the cancer cell
lines after 24, 48, and 72 h, while this effect was dependent on
time and concentration (Fig. 7b–e). Nisin had the greatest
cytotoxic effect on AGS cell line. The IC50 values of the free
nisin and nisin-loaded nanoparticles for the cancer cell lines
are listed in Table I. These values indicated that the nisin-
loaded nanoparticles had a higher cytotoxic effect on the cell
lines compared to the free nisin (P < 0.05 vs. control). As
shown in Fig. 7f, nanoparticle, nisin, and nisin-loaded
nanoparticles have no cytotoxic effects on Vero (non-tumor)
cell line at the studied concentrations.
Morphological Changes of Cancer Cell Lines

Evaluation of Cytotoxic Effect To investigate the effects of the free nisin and nisin-
loaded PLA-PEG-PLA nanoparticles on the morphologies of
The treatments induced in the cancer cell lines by the AGS, KYSE-30, HepG2, and K562 cell lines, the cells were
nanoparticles were indicative that these nanoparticles did not treated for 24, 48, and 72 h. The microscopic images

Fig. 7. a MTT assay of cytotoxic activity of PLA-PEG-PLA nanoparticle on AGS, KYSE-

30, HepG2, K562, and Vero cell lines; MTT assay of cytotoxic activity of nisin and nisin-
loaded nanoparticles on b AGS, c KYSE-30, d HepG2, e K562, and f Vero cell lines after
24, 48, and 72 h compared to normal (control), respectively. P < 0.0 5 vs. control
Evaluation of Cytotoxicity Effects of Nisin on Cancer Cell Lines

Table I. IC50 Values of (a) Nisin (b) Nisin-loaded Nanoparticles, on presence of apoptotic bodies (Fig. 9b). The control cells
AGS, KYSE-30, HepG2, and K562 Cell Lines After 24, 48, and 72 h showed a normal appearance (Fig. 9a). After the cells were
Determined by MTT, Neutral Red Assays; Data are Presented as further treated with the nisin and nisin-loaded nanoparticles,
Mean ± SD (n = 3), P < 0.0 5 vs. Control a significant increase occurred to the number of apoptotic
cells in the cancer cell lines.
(a) Cell line IC50 by MTT IC50 by Neutral Total IC50 (μM)
(time) assay (μM) Red assay (μM)
AGS 24 h 227 ± 2 237 ± 3 232 ± 3 DISCUSSION
AGS 48 h 143 ± 3 152 ± 2 147 ± 2
AGS 72 h 62 ± 3 60 ± 3 61 ± 3 Many peptides and proteins with biological activities have
KYSE-30 24 h 405 ± 4 413 ± 4 409 ± 4
been introduced as therapeutic (particularly anti-cancer) agents.
KYSE-30 48 h 321 ± 2 347 ± 2 334 ± 2
Applications of pharmacological agents have some limitations,
KYSE-30 72 h 131 ± 6 129 ± 3 130 ± 5
HepG2 24 h 147 ± 5 143 ± 5 145 ± 5 including their rapid removal of blood flow through kidney
HepG2 48 h 154 ± 5 146 ± 5 150 ± 5 filtration, enzymatic degradation induced by the reticuloendothe-
HepG2 72 h 97 ± 4 94 ± 2 95 ± 3 lial system, and accumulation in the non-target tissues and organs.
K562 24 h 287 ± 4 273 ± 4 280 ± 4 These anti-cancer drugs have been designed to kill cancer cells, but
K562 48 h 239 ± 3 235 ± 2 237 ± 3 drug release may occur inside the healthy organs and tissues and
K562 72 h 157 ± 6 136 ± 3 146 ± 5 subsequently cause severe side effects in some cases. Some of these
(b) Cell IC50 by MTT IC50 by Neutral Total IC50 (μM) peptides or proteins follow the intracellular targets since they need
line (time) assay (μM) Red assay (μM) to enter into the cells to perform their functions, while transpor-
AGS 24 h 98 ± 4 90 ± 4 94 ± 4 tation of these biomacromolecules through the cell membrane is
AGS 48 h 65 ± 3 72 ± 2 69 ± 3 another limitation [44]. Many of these restrictions can be overcome
AGS 72 h 37 ± 3 32 ± 5 34 ± 4 by the use of nanoparticles. Nanocarriers with encapsulated
KYSE-30 24 h 345 ± 4 339 ± 4 342 ± 4
pharmaceutical agents avoid any contacts with the biological
KYSE-30 48 h 250 ± 5 255 ± 3 252 ± 4
environment before they can reach the diseased tissues. Thus,
KYSE-30 72 h 79 ± 5 83 ± 5 81 ± 5
HepG2 24 h 134 ± 4 141 ± 2 137 ± 3 they not only protect the detection of drugs via the immune system,
HepG2 48 h 121 ± 2 119 ± 4 120 ± 3 but also increase their sizes by making a complex with them so that
HepG2 72 h 87 ± 5 84 ± 4 85 ± 5 they cannot be rapidly removed through kidney filtration. Loading
K562 24 h 240 ± 4 248 ± 4 244 ± 4 proteins and biomacromolecules onto nanocarriers not only
K562 48 h 143 ± 6 145 ± 2 144 ± 4 enhances their stabilities and prolongs their circulation time, but
K562 72 h 124 ± 3 158 ± 3 141 ± 3 also causes drug molecules to be efficiently released into the
cytoplasm, and endosomal escape [44]. The results of our study
revealed that PLA-PEG-PLA polymeric nanoparticles were
represented the enhanced unusual shapes of the cells and capable of forming distinct spherical micelles with smooth surfaces.
cytotoxicity and growth-inhibitory effects with the increasing The mean sizes of the nanoparticles and nisin-loaded nanoparticles
concentrations of the nisin and nisin-loaded nanoparticles. were 100 and 200 nm, respectively. In the FTIR diagrams, some
The cytotoxicity effects on the cells were greater at the variations in the absorption bands could be observed before and
concentrations of 250, 350, and 450 μM and decreased cell after loading nisin onto the nanoparticles. These changes could be
densities. The cytotoxic effects were higher with the time resulted from the physicochemical interactions between the
intervals of longer than 72 h. The effects of the free nisin and nanoparticles and nisin to form nisin-loaded nanoparticles [45].
nisin-loaded nanoparticles on the cells in the concentration The loading efficiency of nisin onto the nanoparticles was 85–90%,
and time-dependent manners were confirmed through the which was acceptable.
comparison of the images obtained from the three experi- As shown by the results, nisin release was related to the pH
mental time periods as they had caused cell deformation and of the release medium in a way that higher amounts of nisin
reduced cell density. Moreover, further increases in the were released at a lower pH. The medium penetrated into the
concentrations and durations of the treatments led to more nanoparticles and dissolved the encapsulated nisin. Therefore,
reductions in the intercellular connections (Fig. 8). the major factor determining the drug release from the
nanoparticles was its solubilization rate in the release medium.
Nisin had the highest solubility in the pH range of 2–5.
Ethidium Bromide/Acridine Orange Staining for Apoptosis Accordingly, a pH of 4 caused its further solubilization and
Cells separation into the release medium. Its initial release might be
caused by the fast dehydration of the hydrophilic nature of the
The morphological changes on the cancer cell lines were PEG segment of the nanoparticles [30, 46].
assessed by EB/AO staining and analyzed under a fluorescent Hemolysis test proved the biocompatibility of the
microscope (Table II and Fig. 9). The results revealed nanoparticles. Also based on non-cytotoxicity of the nano-
morphological changes to all the cancer cell lines like particles, it could be concluded that the synthesized nanopar-
chromatin condensation and fragmentation and apoptotic ticles in this study were biocompatible. The FITC-labeled
body formation occurred at the different nisin and nisin- nanoparticles successfully entered into the cells and were thus
loaded nanoparticle concentrations (IC50 values) of 235, 410, capable of delivering nisin to them.
145, 280 μM and 95, 345, 140, 245 μM for AGS, KYSE-30, Nisin had a cytotoxic effect on gastrointestinal, hepatic, and
HepG2, and K562 cell lines after 24 h treatments, respec- K562 cell lines and induced apoptosis. Vero cell line as a normal
tively. These alterations were taken as evidence for the cell line was observed not to be influenced by nisin cytotoxicity at
 Goudarzi et al.

Fig. 8. The invert microscopy images of AGS cell line treatment with different concentrations of nisin after 72 h

micromolar concentrations. This finding was in line with that of
Kordell and Sahl’s study [47]. As more receptors on the cellular
membrane surfaces are developed by malignant cells, various
biological substances like nisin can be well impelled to attach to
them [48, 49]. After nisin loading, zeta potential was enhanced.
Nisin has a positive charge, thus the surface charge of nisin-
loaded nanoparticles was more positive than PLA-PEG-PLA
nanoparticles (− 28.90 < − 22.63 mV). This is considered as the
main factor that provides good stability of nanoparticles. The
importance of an electrostatic charge is that it causes a better
release and higher load capacity. On the other hand, since a
nanoparticle structure protects nisin against any unwanted
interactions with other molecules and maintains its activity until
it reaches the target, nisin-loaded nanoparticles can provide more
efficiency than free nisin [50]. Bench and colleagues found that
nisin encapsulation within the liposome can provide a powerful

Table II. Apoptosis Induction of Nisin and Nisin-loaded Nanoparticles on AGS, KYSE-30, HepG2, and K562 Cell Lines, Data are Expressed
as Percentage of Cells, Data are Presented as Mean ± SD (n = 3), P < 0.0 5 vs. Control

Cell line Live cells (%) Apoptotic cells (%) Necrotic cells (%)

AGS control 98 ± 2 2 ± 2 1 ± 1
AGS treated with nisin 51 ± 2 46 ± 2 4 ± 1
AGS treated with nisin-loaded nanoparticle 45 ± 2 51 ± 4 4 ± 1
KYSE-30 control 95 ± 2 4 ± 1 1 ± 0
KYSE-30 treated with nisin 66 ± 3 30 ± 1 4 ± 1
KYSE-30 treated with nisin-loaded nanoparticle 56 ± 3 40 ± 3 4 ± 2
HepG2 control 98 ± 1 1 ± 2 1 ± 1
HepG2 treated with nisin 59 ± 7 38 ± 4 2 ± 1
HepG2 treated with nisin-loaded nanoparticle 52 ± 3 45 ± 4 3 ± 2
K562 control 94 ± 5 4 ± 5 1 ± 0
K562 treated with nisin 62 ± 5 34 ± 2 4 ± 1
K562 treated with nisin-loaded nanoparticle 55 ± 4 40 ± 1 5 ± 1
Evaluation of Cytotoxicity Effects of Nisin on Cancer Cell Lines
Fig. 9. Apoptosis induction of nisin on HepG2 cell line. a Normal untreated cells, b cells treated with nisin (145 μM). The
histogram indicates that after treating with: c nisin (145 μM according to IC50 value for HepG2 after 24 h) and d nisin-
loaded nanoparticles (140 μM according to IC50 value for HepG2 after 24 h) there was a significant increase of apoptotic
cells in cancer cells
tool to improve its stability and inhibitory activity [46, 51]. Our
research suggested that the cytotoxicity of nisin and encapsulated
nisin was dependent on the concentration and time factors as its
cytotoxic effect was enhanced by increasing the concentrations
and durations of the treatments. The results of our study were
almost similar to those obtained by Bedge and colleagues, who
examined nisin effect on a variety of lymphoma cells.
Nisin induced apoptosis on the studied cancer cell lines.
This was verified by the morphological changes detected
through EB/AO staining. After comparing the results of the
MTT and NR uptake assays for the free nisin and nisin-
loaded nanoparticles, a significant reduction was seen in the
IC50 values of the latter substance. Therefore, we could
conclude that the nisin-loaded nanoparticles were more
effective on the cell growth reduction compared to free nisin.
AGS cell were the most sensitive cell line to nisin cytotoxic
effects. Although there are few reports on its effects,
especially those of its encapsulation form, on cancer cells,
our research was found to be in line with Shaowen, Nam, and
Preet’s studies. Shaowen investigated nisin encapsulation into
PLA nanoparticles and showed the uniform dispersion of
hydrophilic nisin in the matrix of a hydrophobic polymeric
nanoparticles in an aqueous solution and its subsequent
adsorption onto the particle surface [52]. In his investigation,
Nam assessed the therapeutic potential of nisin for treating
head and neck squamous cell carcinoma (HNSCC) and
observed reduced HNSCC cell proliferation to be induced
by cell cycle arrest and preferential apoptosis [53]. The anti-
cancer effect of nisin and doxorubicin (DOX) mixture on
mouse skin carcinogenesis was studied by Preet et al. in vivo
[54]. This finding further delineated the underlying anti-
cancer mechanism of nisin-DOX additive against skin
carcinogenesis in a murine model in vivo. Additionally, its
in vitro mechanism against HaCaT cell lines [55] was
demonstrated in another study. This is the first study to
show that nisin treatment of AGS, KYSE-30, HepG2, and
K562 cancer cell lines leads to their apoptosis. These results
proved nisin application as a potentially new approach to
treating these cell lines. Moreover, its use in a clinical setting
can be facilitated by the fact that it is currently utilized for
food preservation since being safe for human consumption.
Nisin encapsulation into PLA-PEG-PLA nanoparticles can
provide a promising technology by delivering therapeutic
peptides to target cells with higher performance. A more
stable system was exhibited by PLA-PEG-PLA as a
polymeric nanoparticle compared to liposome encapsulation.
However, the potentiality of this approach to delivering
peptides should be further investigated by developing
fundamental research on nanoparticles in the field of
medical sciences in the future.

CONCLUSION
Nisin hydrophobic side shows a more tendency to attach to
a hydrophilic substrate and its hydrophilic side represents a
further tendency towards a hydrophilic substrate [56]. This
property provides a simple efficient option for enhancing nisin
incorporation into polymeric nanoparticles. Its incorporation
into polymer particles provides better protection of active
substances like nisin and their sustained release [52]. Nisin is
decomposed by proteases in the digestive system, while it is
unstable at physiological pH. Thus, its application to clinical
practice would be difficult. This was the first study to report the
cytotoxic effects of nisin and its encapsulated forms on the
gastrointestinal, hepatic, and K562 cell lines. Accordingly,
application of nisin as a harmless peptide for anti-tumor
purposes can be promising in the future. In addition to its
reduced IC50 values when it is encapsulated with biocompatible
polymeric nanoparticles and decreased doses, nisin could herald
the emergence of a new generation of anti-cancer drugs with
minimal side effects in comparison with the conventional
chemotherapeutic agents. Based on this study, we can conclude
that the synthesized PLA-PEG-PLA polymeric nanoparticles
are biocompatible and capable of forming nanosized micelles to
transfer peptide drugs. Hence, it can be used as a good candidate
for transporting such drugs in the release systems. Taking into
consideration all the concentrations obtained by the laboratory
studies in this research, more investigations are required to
probably generalize them to those usable in clinical trials.
Nonetheless, further research on nisin and its encapsulated
forms, especially on animal tumor models, is needed to
determine their anti-tumor activities and approved applications
in cancer chemotherapy in vivo.
ACKNOWLEDGEMENTS
This work was supported by funding from University of
Mohaghegh Ardabili and we are greatly appreciated of
Ardabil University of Medical Science and University of
Tehran for support this reasearch.
COMPLIANCE WITH ETHICAL STANDARDS
Conflict of Interest All authors have read and approved this
version of the article and we have no conflicts of interest.
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