Molecular Biology Reports

https://doi.org/10.1007/s11033-020-05704-z

ORIGINAL ARTICLE

Hybrid of niosomes and bio‑synthesized selenium nanoparticles

as a novel approach in drug delivery for cancer treatment
Mahmoud Gharbavi1,2,3   · Behrooz Johari4   · Navid Mousazadeh4 · Bahareh Rahimi5   · Milad Parvinzad Leilan4   ·
Seyed Sadegh Eslami6 · Ali Sharafi3 

Received: 5 June 2020 / Accepted: 1 August 2020

© Springer Nature B.V. 2020

Abstract 
The current study intends to investigate a novel drug delivery system (DDS) based on niosomes structure (NISM) and bovine
serum albumin (BSA) which was formulated to BSA coated NISM (NISM-B). Also, selenium nanoparticles (SeNPs) have
been prepared by BSA mediated biosynthesis. Finally, the NISM-B was hybridized with SeNPs and was formulated as
NISM-B@SeNPs for drug delivery applications. Physicochemical properties of all samples were characterized by UV–Vis
spectroscopy, FT-IR, DLS, FESEM, and EDX techniques. The cytotoxicity of all samples against A549 cell line was assessed
by cell viability analysis and flow cytometry for apoptotic cells as well as RT-PCR for the expression of MDR-1, Bax, and
Bcl-2 genes. Besides, in vivo biocompatibility was performed by L ­ D50 assay to evaluate the acute toxicity. The proposed
formulation has a regular spherical shape and approximately narrow size distribution with proper zeta-potential values; the
proposed DDS revealed a good biocompatibility. The compound showed a significant cytotoxic effect against A549 cell
line. Although the Bax/Bcl-2 expression ratio was significantly in NISM-B@SeNPs- treated cancer cells, the expression of
MDR-1 was non-significantly lower in NISM-B@SeNPs-treated cancer cells. The obtained results suggest that the proposed
DDS presents a promising approach for drug delivery, co-delivery and multifunctional biomedicine applications.

Electronic supplementary material  The online version of this

article (https​://doi.org/10.1007/s1103​3-020-05704​-z) contains
supplementary material, which is available to authorized users.

3
* Behrooz Johari Zanjan Pharmaceutical Biotechnology Research Center,
dr.johari@zums.ac.ir Zanjan University of Medical Sciences, Zanjan, Iran
4
* Ali Sharafi Department of Medical Biotechnology, School of Medicine,
Sharafi.a@gmail.com Zanjan University of Medical Sciences, Zanjan, Iran
5
Navid Mousazadeh Department of Medical Biotechnology, Faculty of Allied
navidmusazadeh@zums.ac.ir Medicine, Iran University of Medical Sciences, Tehran, Iran
6
Seyed Sadegh Eslami Department of Genetics and Pathology, School of Medicine,
eslami.s@tak.iums.ac.ir Ardabil University of Medical Sciences, Ardabil, Iran
1
Student Research Committee, Zanjan University of Medical
Sciences, Zanjan, Iran
2
Department of Pharmaceutical Biomaterials, School
of Pharmacy, Zanjan University of Medical Sciences, Zanjan,
Iran

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 Molecular Biology Reports

Graphic abstract

Keywords  Albumin · Biosynthesis · BSA · Drug delivery · Selenium nanoparticles · Niosomes

Abbreviations RBC Red blood cells

BSA Bovine serum albumin RT Reverse-transcribed
NISM-B BSA coated NISM RESs Reticulo-endothelial systems
cDNA Complementary DNA SeNPs Selenium nanoparticles
DDS Drug delivery system UV–Vis Ultraviolet–Visible
DLS Dynamic light scattering ULV Unilamellar vesicles
FESEM Field emission scanning electron microscope
FT-IR Fourier-Transform Infrared
HAS Human serum albumin Introduction
LD50 Median lethal dose
MDR Multidrug resistance In recent years, vesicular nano-carrier’s systems such as non-
MLV Multilamellar vesicles ionic surfactant vesicles (niosomes) have attracted remark-
NISM Niosomes able attention in the pharmaceutical field due to their unique
KBr Potassium bromide properties. Although many studies have been focused on
PDI Polydispersity index improving the therapeutic drug efficacy along with decrease
PI Propidium iodide the side effects, only a few of them have been clinically

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Molecular Biology Reports
approved. The physicochemical characteristics of niosomes
are profoundly affected by their chemical composition [1, 2].
As such, niosomes with new functionalization and architec-
ture are continually explored. Our main goal in this regard is
to develop a feasible way to fabricate therapeutically useful
niosomal formulations [3].
Niosomes, which are fabricated with nonionic amphi-
philic surfactants in aqueous media by self-assembly tech-
nology, are usually structured in the form of multilamellar
vesicles (MLV) or unilamellar vesicles (ULV). Similar to
the liposomes and polymersomes, niosomes are gener-
ated by some input of energy; such as mechanical or heat,
were closed bilayers with internal hydrophilic cavities and
hydrophobic shells as the outer layers to accommodate the
therapeutic agents [4]. Since the niosomes are formed mostly
by non-ionic surfactants and cholesterol incorporation as
an excipient; they are more stable and cost-effective than
liposomes [5]. Numerous studies have shown that surface
modification of niosomes is much easier than liposomes due
to their appropriate stability [3].
As a previous result, human serum albumin (HSA) and
bovine serum albumin (BSA) have been widely used as coat-
ing agents in biomaterials and drug delivery systems [6, 7].
Besides, BSA has recently attracted much attention in the
fields of nanotechnology and pharmaceutical, because it is
biologically safe, biodegradable, hydrophilic, and then non-
toxic and non-immunogenic [8]. In the current study, BSA
coated niosomes are suggested as a novel strategy to make
niosomal formulation with high stability, more biocompat-
ibility, and versatile surface functionality.
Chemotherapy is one of the common strategies in the
cancer treatment; however it has many problems such as
their toxicity, gastrointestinal upset, bone marrow suppres-
sion, and most importantly, drug resistance [9]. Different
treatment strategies have been studied to overcome the drug
resistance and various side effects. Nanotechnology has
the potential to improve the side effects, drug efficacy, and
patient quality of life [10]. Various metal-based nanoparti-
cles have been examined to induce cancer cell cytotoxicity.
Lung cells are sensitive toward different triggers includ-
ing cytokines and oxidants that are secreted to their sites.
SeNPs are capable to produce oxidative tension releasing
ROS that capable to pose destruction to the DNA content
which could eventually induce programmed cell death via
activation of the apoptosis cascades [11]. Earlier research-
ers have reported that processing the cells with selenium
could considerably reduce the formaldehyde-stimulated
genotoxicity, oxidant–antioxidant system, and promoting
the AP-1 and NF-kB proteins in A549 cell line [12]. SeNPs
have been introduced as inorganic NPs in drug delivery for
cancer treatment. Numerous studies have shown that SeNPs
have the antioxidant activity, anticancer effects and redox
modulatory properties [13, 14].
SeNPs not only reduced DNA damage by a decreased
ratio of 8-hydroxy-2-deoxyguanosine and 2-deoxyguano-
sine, but also involved in DNA repair through cell signal-
ing pathways. Furthermore, they showed to significantly
induce apoptosis and necrosis in cancer cells, and inhibit
the growth of tumor cells [14]. Here, we focused on the
design and fabricate of a versatile nanostructure with
potentially co-delivery, multifunctional, and high effi-
ciency. SeNPs were bio-synthesized and hybridized with
niosomes nanocarrier, to introduce a new approach for
drug delivery in cancer treatment. Traditional synthesis
of SeNPs by chemically assisted elevates cellular toxic-
ity, accordingly this feature significantly limits their bio-
medical application [15]. Therefore, microbial synthesis
is used as an alternative method to fabricate SeNPs. Sev-
eral microorganisms are known for synthesizing SeNPs,
including Klebsiella pneumoniae [16], Shewanella sp.
HN-41 [17], and Pseudomonas alcaliphila [18]. Consider-
ing these facts, the bacteria may secrete endotoxin, result-
ing in contamination of the nanoparticle surface, which
limits their use in the medical field [19]. Thus, protein-
mediated synthesis offers a promising alternative for the
synthesis of SeNPs. In the current study, BSA was used
as a protein-mediated SeNPs synthesis, which may have
several advantages including low cost, non-toxic, and eco-
friendly synthesis method and will be used in the pharma-
ceutical and nanomedicine fields. SeNPs are located on the
surface of the final formulation (NISM-B@SeNPs), which
could lead to protecting the normal cells from toxic effects
or other side effects of chemotherapy via antioxidant or
protective effects of SeNPs [20]. The physicochemical
properties of the nanoparticles were characterized by
using FT-IR, UV, FESEM, EDX, and DLS Zetasizer. Fur-
thermore, the in vitro and in vivo cytotoxicity, as well as
apoptosis of the proposed DDS, were evaluated on the
A549 cell line. Finally, real-time PCR was performed for
Bax, Bcl-2, and MDR-1gene expression.
Materials and methods
Reagents and materials
Polyoxyethylene sorbitan monooleate (Tween 80- CAS.9005-
65-6), sorbitan monooleate (span80-CAS.1338-43-8),
BSA (CAS.9048-46-8), Cholesterol (CAS.57-88-5), were
obtained from Sobhan oncology pharmaceutical company
(Iran) and chloroform reagent was purchased from Sigma
Aldrich Company (Germany). Sodium selenite (Na2SeO3)
was purchased from Sigma-Aldrich (USA). All other chemi-
cals, solvents, and cell culture medium were obtained from
Emertat Chimi Company (Tehran- Iran).
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Methods
Biosynthesis of SeNPs
SeNPs were prepared by the redox reaction of sodium sel-
enite with BSA as a reducing agent, as previously described
by Kalimuthu Kalishwaralal et al. with some modifications
[21]. Briefly, 10 mg of sodium selenite was dissolved in
10 mL PBS (pH 7.4) with 200 mg BSA, as a reducing agent,
at 121 °C under vigorous stirring. After 45 min, the color
of the colloidal solution changed from colorless to orange-
red color. The colloidal solution was centrifuged at 700
rcf for 5 min in order to remove excess BSA. Finally, the
obtained colloidal solution was purified by dialysis using a
12-kDa molecular weight cut off against ultrapure water for
overnight to remove the excess sodium selenite and other
precursors.
Preparation of niosomes‑BSA (NISM‑B) hybridized SeNPs
(NISM‑B@SeNPs)
Niosomes were prepared by a thin-film hydration method
(handshaking method), as previously reported [22]. Briefly,
300  mg surfactant mixture (Tween 80 & Span 80), and
80 mg cholesterol were dissolved in chloroform in a round-
bottomed flask. The organic solvent was removed at 60 °C
under reduced pressure, using a rotary evaporator at 150
rcf to construct the thin lipid layer. Also, 100 mg BSA was
dissolved in 4 mL PBS under constant stirring (600 rcf) at
room temperature for 15 min. Then, 1-mL colloidal SeNPs
solution was loaded onto the BSA solution. Subsequently,
the last solution was slowly added into a thin lipid layer
and vigorously vortexed for one minute, followed by soni-
cation for five minutes with a bath Sonicator. The final for-
mulation NISM-B@SeNPs was prepared by sonication in a
water bath at 60 °C for 15 min.
Physicochemical characterization
The physicochemical properties of proposed DDS were
characterized by UV–Vis, FT-IR, DLS, and FESEM-EDX
techniques. The absorption spectrum of the proposed DDS
was examined using a UV–Vis spectrophotometer to deter-
mine the components of sodium selenite, SeNPs, and NISM
in the final formulation. FT-IR spectroscopy (Bruker, Ten-
sor 27, Biotage, Germany) was employed to determine the
chemical structure of BSA, NISM, NISM-B, SeNPs, and
NISM-B@SeNPs. The potassium bromide (KBr) disks were
prepared by mixing and mechanical grinding approximately
10% of a sample with 90% KBr and passed in the plate form
(pressure, 10 Ton).
DLS was used to determine the average particle size,
polydispersity index (PDI), and zeta-potential of the
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Molecular Biology Reports
proposed DDS by the Nano-Zeta sizer (Malvern Instruments,
Worcestershire, UK, model Nano ZS) apparatus. 0.5 mL
from each of the samples was diluted with two mL of deion-
ized water in a clean Malvern sample vial. DLS determined
the hydrodynamic diameter, PDI, and zeta-potential. The
rheological properties of free-NISM, NISM-B, and NISM-
B@SeNPs were determined using a viscometer (Brookfield
Viscometer LTD) [15]. In all of the experiments, 1-mL of
each sample was placed into the surface of the reading plate,
and the excess sample was removed. The data were collected
using Brookfield Viscometer LTD Software. Besides, the
stability of synthesized NPs (SeNPs & NISM-B@SeNPs)
was assayed by a DLS apparatus. The size of the samples
was monitored by DLS for more than 82 days. Synthesized
SeNPs were characterized by EDX elemental mapping to
identify the elemental distribution. Also, the FESEM tech-
nique was further used to describe the size and morphology
of the digestion DDS formulation. All samples were coated
by gold, and FESEM was operated at an acceleration voltage
of 15 kV a scale of 35 KX magnification (MIRA TESCAN,
Czech Republic).
In vitro cytotoxicity
MTT cytotoxicity assay was performed to determine the
anti-proliferation effects of NISM-B NPs, Sodium selenite,
and NISM-B@SeNPs against A549 lung cancer cell line.
Briefly, the cells were cultured overnight in 96-well plates
with a density of 7 × 103 cells per well.
They were incubated with complete growth medium
(200 µL) for attachment. One day after incubation, the cell
groups were treated with different concentrations (3–24 µg/
mL) of the test samples (NISM-B NPs, SeNPs, and NISM-
B@SeNPs). After 24 h of treatment, 20 µL of MTT solution
(5 mg/mL) was added, and after 4 h’ incubation, 100 µL of
DMSO was added to each well and absorbance measured at
570 nm wavelength. All assays were performed in triplicate.
Biocompatibility assay
Red blood cells (RBC) hemolysis refers to RBC membrane
damage, resulting in the release of hemoglobin into the
blood plasma. The RBC hemolysis that can cause anemia
(or worsening of anemia), jaundice, and other pathologi-
cal symptoms, it may become life threatening. As such, the
hemolytic degree of proposed DDS formulations is crucial to
justify their biocompatibility and clinical applications, which
was evaluated by a previously reported method (Dobrovol-
skaia et al., 2008). Healthy human blood was obtained from
Mousavi Hospital Blood Bank Center (Zanjan, Iran); it was
collected in tubes with heparin and centrifuged at 4000 rpm
for five minutes. The plasma was discarded, and RBCs were
washed three times with PBS. The purified RBCs were
Molecular Biology Reports
re-suspended in isotonic solution and it is diluted to 1:10 of
its initial concentrations. In this investigation, different con-
centrations of formulations (100–600 µg/mL), 1% Sodium
dodecyl sulfate (SDS), and 500 µL PBS were used as test
samples, positive control (100% Hemolysis) and negative
control (0% hemolysis), respectively. Next, a 200 µL aliquot
of RBC pellets was placed in a tube, to which 200 µL each
of test samples, positive control and negative control were
added and incubated in a shaking water bath at 37 °C for 4 h.
After incubation, samples were centrifuged at ambient
temperature for 10 min at 4000 rcf. To measure the hemo-
globin released into the incubation medium, 200 µL samples
from the supernatant were transferred to a 96-well plate and
the absorbance spectrophotometrically measured at 540 nm
by a plate reader. This process was repeated three times;
hemolysis% was calculated using the following formula:
Atreatedsample − Anegativecontrol
Hemolysis (%) = × 100 (1)
Apositivecontrol − Anegativecontrol
Apoptosis assay
A549 cell apoptosis was determined by using FITC-labeled
Annexin V/propidium iodide (PI) assay according to the
manufacturer’s instructions (Invitrogen Co. USA) [23].
First, the cells were seeded at a density of 8 × 104 cells per
well in 12-well plates and incubated in 5% ­CO2 at 37 °C for
24 h. A549 cells were treated at the same concentration of
all proposed DDS formulations for 24 h. After incubation
time, following the disposal of the cell culture medium, cells
were washed with PBS and added to 100 µL of Annexin-
V binding buffer and stained with Annexin-V FITC and PI
(Sigma–Aldrich) based on the manufacturer’s recommenda-
tions. Data analysis was conducted by flow cytometry (BD
Biosciences, San Jose, CA, USA) and FlowJo software (Tree
Star, Ashland, OR).
Real‑time PCR assay
A549 cells were plated in triplicate at a density of 3 × 105 per
well into 6-well plates, then incubated overnight with 6 µg/
mL of NISM-B@SeNPs and untreated cells, which were
considered as test sample and control, respectively. Total
cellular RNA was extracted by One Step-RNA extraction
kit (BIO BASIC INC, Canada) followed by DNase I diges-
tion (Thermo Fisher Scientific, UK), as recommended pro-
tocol. Next, the reverse-transcription (RT) enzyme was used
to synthesize the complementary DNA (cDNA) from total
RNA templates. Then, the synthesized cDNA was subjected
to real-time PCR to determine quantitative gene expression
on a light cycler apparatus (Roche Diagnostic). An initial
activation step at 95 °C for five minutes, followed by 45
cycles at 95 °C for 30 s, 60 °C for 15 s, and 72 °C for 30 s,
which were considered as denaturation step, annealing step,
and extension step, respectively. A melting curve analysis
was used to assess the amplification specificity of the prod-
ucts. All the experiments were performed in triplicate, and
the changes in gene expression were normalized by GAPDH
mRNA expression as the reference. Quantification of mRNA
fold change levels was determined by 2­ −∆∆Ct method. The
primer sequences of Bax, Bcl-2, and MDR-1 genes are pre-
sented in Table S1 (Supplementary file).
In vivo ­LD50 assay
The ­LD50 is the dose of a toxin required to kill half members
of the test population; it is thus a measure of the short-term
toxic potency (acute toxicity). Acute oral toxicity in the mice
was assessed 7 days after a single oral dose each of the pro-
posed DDS formulations to determine the immediate toxic
effect. Twelve BALB/c male mice, with an average weight
of 30–25 g, were used for this assay according to OECD
guidelines [24].
The ­LD50 assay was carried out for all formulations
(NISM-B NPs, and NISM-B@SeNPs) at 17.5–1750 mg/kg
concentrations, to determine the maximum non-toxic con-
centration of formulations. After administration, animals
were checked to examine any behavioral changes or signs
of toxicity. If all the animals survived after 24 h, the other
four animals were preserved at the highest dose. If these
animals survived too, the ­LD50 would be more than the dose
limit, and the test was stopped.
Statistical analysis
Statistical analysis of the data was performed by Graph-Pad
Prism 6 software. The experiment data were repeated at
least three times, and the data were stated as means ± SD.
One-way and two-way analysis of variance (ANOVA) was
applied as statistical tests. P < 0.05 was considered sig-
nificant; P < 0.05(*); P < 0.01(**); P < 0.001(***); and
P < 0.0001(****).
Results
Characterization of NISM‑B@SeNPs
The physicochemical properties of the nanocarrier system,
including optical properties, particle size, surface charge,
rheological behavior, and morphology, showing the primary
role in the clinical utilization of nanoparticles that are dis-
cussed below [25].
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UV–Vis and FT‑IR spectroscopy
As presented in Fig. 1a, the SeNPs have a UV absorbance
peak at 268 nm; while the sodium selenite has no absorbance
peak appeared in their spectra. Also, in the UV absorption
spectrum of the NISM NPs did not show any absorbance
peak, whereas the NISM-B@SeNPs were appeared the
absorbance peak with a maximum absorbance at 251 nm
(near the maximum absorbance of SeNPs; Fig. 1b).
As shown in Fig. 1c, the FT-IR spectrum of the NISM
NPs, BSA, and NISM-B NPs were compared with each other
to describe the successful synthesis of NISM-B NPs. The
characteristic absorption peaks of NISM NPs at approxi-
mately 1700 and 3400 cm−1 were attributed to a carboxylic
acid functional group (C=O) and hydroxy group, respec-
tively [26]. Besides, the characteristic absorption peaks
at 1680 (stretching vibrations (amide-I)) and 1500 cm−1
(N–H bending vibrations (amide-II)) corresponding to the
absorption peaks from BSA were observed. In compared
with BSA, the characteristic absorption peaks of NISM-B
NPs were found at approximately 3400 cm−1, 1700 cm−1,
and 1570–1400 cm−1.
Fig. 1  a UV–vis Spectra of selenium selenite & SeNPs, b UV–vis Spectra of SeNPs, NISM-B & NISM-B@SeNPs, c FT-IR spectra of BSA,
NISM & NISM-B NPs, d FT-IR spectra of SeNPs, NISM-B NPs & NISM-B@SeNPs
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Molecular Biology Reports
The functional groups of BSA were contained in NISM-
B NPs, which approved the successfully coated of BSA in
NISM-B NPs. Likewise, the FT-IR spectrum of the NISM-
B NPs, SeNPs, and NISM-B@SeNPs was compared with
each other to examine the successful synthesis of NISM-B@
SeNPs, as shown in Fig. 1d. The strong absorption peaks at
749, 1395, and 1580 cm−1 corresponding to the SeNPs are
observed. There is no strong absorption from NISM-B NPs,
at 749, 1395, and 1580 cm−1, but appeared in the FT-IR
spectrum of NISM-B@SeNPs. It can be concluded that the
hybridization of SeNPs in NISM-B NPs was successfully
approved.
Size and zeta‑potential
The hydrodynamic average size of all nano-formulations was
found approximately 118.63 ± 0.666 to 149.23 ± 3.164 nm
(mean ± SD), which was determined by DLS (Fig. 2a, b).
The NISM-B NPs and NISM-B@SeNPs formulations
were facilely synthesized with a polydispersity index (PDI)
around 0.2 and below 0.4, respectively (Fig. 2d).
Molecular Biology Reports

Fig. 2  a, c Hydrodynamic average particle size (nm), b, c Zeta-potential (mV) and d PDI evaluated for all samples

The PDI of NISM-B@SeNPs is significantly higher RSS

AIC = 2 × P + nlog (2)
than to compare to other groups. Additionally, the average n
zeta-potential of NISM NPs, NISM-B NPs, SeNPs, and
where P, n, and RSS represents, the number of parameters
NISM-B@SeNPs were observed to be − 16.03(± 2.12),
in the fitted model, the number of data set and residual sum
− 31.00(± 3.60), − 8.66(± 1.21), and − 36.00(± 2.26) mV,
of squares (RSS), respectively. The preferred model is the
respectively, as presented in Fig. 2(b & c; mean ± SD (mV).
one with the minimum AIC value among all models being
compared [28, 29]. The data obtained from all candidate
Rheological behavior and stability
models presented in Table S2 (supplementary file) and con-
firmed that all samples exhibited a non-Newtonian viscous
The rheological behavior of NPs formulation is a very
behavior. According to the AIC value, the Herschel-Bulkley
important parameter, which relates to the composition of
model characterized the NISM NPs, whereas the Power-
NPs [27]. The apparent viscosity (Pa) and shear stress (Pas)
Law/Ostwald model characterized the NISM-B NPs and
was observed as a function of shear rate (1/s) for proposed
NISM-B@SeNPs.
DDS formulations (NISM NPs, NISM-B NPs & NISM-B@
To examine all sample’s stability, the aggregation and
SeNPs), which were performed at room temperature and
integrity of the samples were described. As such, their aver-
atmospheric pressure ((Fig. S1a, b); supplementary file).
age size was measured as a function of incubation time was
Once the shear rate increased, the viscosity elevated strongly
monitored for 82 days. As presented in Fig. 4c, the Z-average
for all samples, and then, the decline of samples viscosity is
of all samples did not increase significantly throughout the
observed when the shear rate increased from 10 to 300 s−1,
measurement period ((Fig. S1c); supplementary file).
as expected. Moreover, when the shear stress reaches the
limit value (τ0), the steady-state shear stress is observed at
the shear rate ranging from 10 to 300 s−1. Morphological assay
Several models were employed to describe the rheological
behavior of formulations, including Newtonian, Herschel- The morphology and distribution size of SeNPs and NISM-
Bulkley, Bingham, and Power-Law/Ostwald. Candidate B@SeNPs were also described by FESEM ((Fig. 3a–d); sup-
models were then compared by calculation of the Akaike’s plementary file). All samples observed spherical and uni-
An Information Criterion (AIC) function, which is defined form in shape, with an average size of approximately 80 nm.
as following: The EDX profile demonstrated the absorption of strong Se

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Fig. 3  a, b The particle size histogram and FESEM image of SeNPs, c, d The particle size histogram and FESEM image of NISM-B@SeNPs, e
EDX analysis of SeNPs
signal (73.94% Wt.) along with some other elements, such
as sulfur (S) group ((Fig. 3e); supplementary file). The pres-
ence of sulfur (10% wt) in EDX spectra, clearly indicates
sulfur-containing albumin molecules bound to the surface
of the SeNPs [30].
In vitro experiments
Cell viability and biocompatibility assay
The MTT assay was performed at concentrations ranging
from 3 to 24 µg/mL. The viability was monitored after 24 h.
As presented in Fig. 4a, the NISM-B NPs had no signifi-
cant cytotoxic effect on the A549 cells at the concentrations
tested whereas, the SeNPs and NISM-B@SeNPs showed a
significant cytotoxic effect at concentrations up to 24 μg/
mL. The obtained data showed that cell toxicity is directly
equivalent to the samples concentration during a 24  h
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Molecular Biology Reports
period time. The cell inhibition capacity was significantly
improved, when the A549 cells were incubated with SeNPs
or NISM-B@SeNPs compared to the NISM NPs.
Besides, the hemolysis assay was performed at a range
of concentrations from 100 to 600 µg/mL and was done
at 540 nm with Eppendorf BioPhotometer. As presented
in Fig. 4b, the hemolytic activity of all samples (NISM
NPs, NISM-B NPs & NISM-B@SeNPs) at various con-
centrations was observed in the range of 5.86% ± 0.785 to
15.33% ± 0.567.
Apoptosis assay
The results of apoptosis assay on A549 cell line after treat-
ment with 6 µg/mL from NISM-B, SeNPs and NISM-B @
SeNPs samples showed that the induced apoptosis in the
cells are 5.9 ± 2.6, 23.18 ± 7.31, and 32.85 ± 9.35, respec-
tively. Induction of apoptosis was significant in two groups
Molecular Biology Reports
Fig. 4  a The cell viabili-
ties of the all samples on
A549 lung cancer cell line
after 24 h. b Percentage of
hemolysis induced by NISM,
NISM-B NPs & NISM-B@
SeNPs at various concentra-
tion and 37 °C, conditions.
Values with P < 0.01(**) and
P < 0.0001(****) were regarded
statistically meaningful
of SeNPs and NISM-B@SeNPs compared to the other two
groups (control and NISM-B). The effect of the final for-
mulation of NISM-B@SeNPs on increasing the apopto-
sis in comparison with the SeNPs group was significant,
which indicates the effective performance of the nanosystem
designed in this study (Fig. 5a, b).
Effect of NISM‑B@SeNPs on Bax, Bcl‑2 and MDR‑1
expression
Quantitative PCR was carried out to assess the effects of
six µg/mL of NISM-B@SeNPs on Bax, Bcl-2, and MDR-
1expersion levels in A549 cells. Whereas the Bax gene
expression was significantly elevated in A549 cells, which
were treated by NISM-B@SeNPs compared to control, but
Bcl-2 gene expression was significantly decreased under
treatment with NISM-B@SeNPs compared to control.
Furthermore, MDR-1 gene expressions were non-signif-
icantly decreased under treatment with NISM-B@SeNPs
compared to control, as presented in Fig.  5c. The ratio
of Bax/Bcl-2, as a suitable modulator of apoptosis, was
increased in NISM-B@SeNPs- treated A549 cells (Fig. 5d).
In vivo ­LD50 assay
The oral acute toxicity test (­ LD50) of all samples was per-
formed by mice oral administration at various concentrations
in the range of 17.5–1750 mg/kg [31]. The maximum safe
dose of a proposed system that can be given to mice was
calculated in the control and treatment groups. The isotonic
saline solution was used as an optional treatment for the
control group, and treatment groups orally received the sug-
gested system at the doses of 17.5, 175, and 1750 mg/kg,
considering body weight. None of the mice died during first
week of the treatment by using the proposed system.
Discussion
In the current study, NISM-B@SeNPs successfully fabri-
cated and characterized. As previously reported, BSA has
been widely used as the synthesis materials or bio-mineral-
izing agents for NPs [32]. The tertiary structure of BSA con-
tains multiple cysteine residues forming 17 disulfide bonds
to produce pattern-specific folding [33]. The protein is ther-
mally unfolded, when heated at 121 °C, resulted in further
promoting to break the disulfide bonds and then, formation
more –SH groups. Both –SH and hydroxyl groups in the
unfolded structure of BSA could be possessed a reduction
of aqueous selenium ions (Se (IV)) to metallic selenium (Se
(0)), and result in a color change from colorless to orange-
red color [34].
The NISM was synthesized with some modifica-
tion, where it was coated by BSA. The BSA with a nega-
tively charged protein at pH 7.4 has resulted in the formula-
tion (NISM-B NPs) with more stability and biocompatibility
[35]. Finally, the SeNPs were hybridized with NISM-B NPs
to open new approach in drug delivery for cancer treatment.
The present suggested DDS, highlighted by several
points: (I) Albumin in NPs structure promotes stability and
13

Fig. 5  a Live cell, Apoptotic and necrotic populations (%) with Con-
trol, NISM-B, SeNPs & NISM-B@SeNPs on A549 lung cancer cell
line were calculated from flow cytometry analysis, b Column graph
of apoptotic results analysis from all samples. c Bax, Bcl-2&MDR-1
genes expressions analysis of A549 cell line after treatment with
biocompatibility of DDS; (II) Albumin in NPs structure
leads to the creation of suitable conditions for facilitat-
ing the hybridization of SeNPs with BSA and preventing
the entire SeNPs from oxidizing; (III) Albumin in NPs
13
Molecular Biology Reports
NISM-B@SeNPs. d the Bax/Bcl-2 expression ratio of A549 cell
line after treatment with NISM-B@SeNPs. Values with P < 0.05(*),
P < 0.001(**), P < 0.001(***) and P < 0.0001(****) were regarded
statistically meaningful
structure resulted in an excellent surface for binding to
specific sites (target aim) or facile functionalization of
DDS; (IV) SeNPs, with antioxidant activity, is an emerging
Molecular Biology Reports
therapeutic strategy to protect healthy cells from the toxic
effects or the side effects of chemotherapy [20, 36].
The optical properties of all DDS formulations have
revealed that the SeNPs and NISM-B@SeNPs were charac-
terized with a maximum absorbance at 268 nm and 251 nm,
respectively [21]. Since the NISM NPs have not appeared
absorbance peak in their UV–Vis spectrum which indicates
that the SeNPs were successfully hybridized into NISN-B
NPs.
Physicochemical properties; such as shape, particle size,
PDI, and charge, play a crucial role in the development of
modern drug delivery systems and their clinical applications
[37]. The hydrodynamic average size of samples, which was
identified by DLS was approximately 118–149 nm, whereas
the average size of samples, determined by FESEM, was
approximately 80 nm. The larger hydrodynamic average
size, compared to that of FESEM, might be attributed to the
shell hydration in aqueous media.
However, nanoparticles with particle size smaller than
200 nm can avoid opsonization and increase the accumula-
tion of the anticancer drugs in tumor sites via enhanced per-
meation and retention effect. Zeta-potential of NPs system
is another critical parameter that it is applied to identify the
surface charge, which may affect the stability, pharmacoki-
netic and pharmaceutical properties, immunogenicity and
macrophage uptake, drug loading efficiency, drug or gene
delivery, targeting drug delivery or brain targeting, cellular
uptake, and multidrug resistance of NPs.
In general, the NPs with a higher value of the zeta poten-
tial had higher the surface charge of the NPs, which causes
the particles to become stable by preventing their aggrega-
tion. Also, zeta-potential values were significantly affected
by NPs compositions. The BSA coated NISM (NISM-
B NPs) increased the negative surface charge of the NPs
system from − 16.03 (± 2.1) to − 31.00 (± 3.6) mV, which
reflect the negative charges of BSA on the surface of NISN-
B-NPs. When SeNPs, with negative charge surface, were
hybridized into NISM-B NPs, the zeta-potential of NPs
were changed from − 31.00(± 3.6) to − 36(± 2.26) mV. It
can conclude the final DDS formulation was successfully
synthesized.
In the current study, we used MTT assay to evaluate the
anti-cancer effect of proposed DDS against A549 cancer
cell line. NISM-B@SeNPs, as final formulation, has excel-
lent toxicity against A549 cancer cell line, due to their good
properties and good design. The SeNPs have significant tox-
icity against A549 cell line. Besides, the NISM-B NPs have
not appeared toxic effects against A549 cells, which can be
concluded, the cell toxicity of proposed DDS (NISM-B@
SeNPs) observed owing to appropriated design and hybrid
of SeNPs into NISM-B NPs.
Several previous studies have reported similar results
as our study that the SeNPs could effectively induce the
cancer cell-apoptosis rate [38]. Xia et al. demonstrated that
the doxorubicin SeNPs coated were significantly increased
in the rate of A549 cell apoptosis compared to the doxoru-
bicin [39]. SeNPs Obtained result showed that in cell groups
treated with NISM-B@SeNPs, the apoptosis rate (early and
late stage) was significant compared with the other groups.
Conversely, the effect of NISM-B treatment on cell viability,
growth, and apoptosis rates was insignificant that indicat-
ing its good nanocarrier for drug delivery. Sanmartín et al.
demonstrated that the mechanism of apoptosis induction
of Se compounds is variable depending on their structure,
metabolism and type of cell line studied [40].
In spite of the fact that cellular could be independent from
P53, the type of apoptosis that happens following the DNA
destruction, often is controlled by BCL-2 family members
[41]. The members of BCL-2 family are categorized as cell
survival promoting members, including BCL-2, BCL-HL,
and MCL-1 and those that promote cell death; such as BAX,
BAK, and BCL-XS [42]. P53 is capable of modulating the
cellular susceptibility toward apoptosis via downregulat-
ing BCL-2 and upregulation of BAX, which are the major
controllers of the programmed cell death [43]. It has been
reported that the expression of BAX could trigger the apop-
tosis related cascades [44].
Cancer drug resistance is one of the most important fac-
tors affecting the outcome of chemotherapy [45]. The inhi-
bition of Multidrug resistances (MDRs) is one of the major
strategies to improve the effectiveness of chemotherapy
treatment [45].
Previously it has been indicated that main events in the
A549 cell drug resistance is the upregulation of MDR1 gene,
which is in charge of the expression of an efflux transporter
known as P-glycoprotein [46]. The results from the quanti-
tative PCR revealed that the NISM-B@SeNPs, as proposed
DDS, could be an excellent system to induce the expression
of the Bax gene as well as in suppressing the expression Bcl-
2 as an anti-apoptotic gene. Because of this, the Bax/Bcl-2
expression ratio, as the most critical factor to evaluate apop-
tosis induction, was significantly increased in NISM-B@
SeNPs-treated cells compared to the control, which is con-
firmed by the result of flow cytometry analysis of apoptosis
[47]. In the current study, the expression of the MDR-1gene
was examined as the most effective genes in drug resist-
ance. The MDR-1 gene expression was non-significantly
suppressed when A549 cells were treated with NISM-B@
SeNPs compared with the control group. The inhibition of
MDR efflux pumps located into the plasma membrane of
cancer cells could reduce the drug resistance to anticancer
agents [48].
The biocompatibility of the proposed system was carried
out by an in vitro hemolysis assay and in vivo L ­ D50 experi-
ment. The results confirmed that the proposed system pro-
vides good biocompatibility, and non-toxic for medicinal
13

applications. The main originality of the current study is
that SeNPs as inorganic nanoparticles were hybridized with
Niosomes as lipid nanoparticles.
Consequently, the SeNPs with antioxidant activity is an
emerging therapeutic strategy to protect healthy cells from
the toxic effects or the side effects of chemotherapy, which
represents a new approach for efficient cancer therapy [49,
50]. However, the current study has some limitations including
the in vivo study to an examination of the ROS formation with
proposed DDS (NISM-B@SeNPs) and requirement to verify
the curative effect of nanoparticles on tumor growth inhibition.
Conclusion
In conclusion, the physicochemical properties of the proposed
system confirmed that they were successfully synthesized.
The main originality of the current study is that SeNPs, as
inorganic nanoparticles, were hybridized with Niosomes as
lipid nanoparticles. Consequently, the SeNPs, with antioxidant
activity, is an emerging therapeutic strategy to protect healthy
cells from the toxic effects or the side effects of chemotherapy,
which represents a new approach for efficient cancer therapy. It
is worth noting that the NISM-B@SeNPs is a valid pharmaco-
logical tool for further in vitro and in vivo studies (in progress)
on conjugates of its system with drugs, drug candidates, target-
ing agent, and molecular probes.
Acknowledgments  This study was supported financially by Zanjan
University of Medical Sciences, Zanjan, Iran (Grant Number: A-12-
1244-11 & Ethical Code: IR.ZUMS.REC.1398.438).
Author contributions  MG: Methodology, Conceptualization, Software,
and Writing—original draft. BJ: Supervision, Project administration,
Writing—review and editing, and Final approved. NM: Methodology.
BR: Methodology. MPL: Methodology. SSE: Methodology. AS: Pro-
ject administration and Writing—review and editing.
Compliance with ethical standards  lization process. Braz J Microbiol 41(2):461–466
Conflict of interest Behrooz Johari received the grant from Zanjan
University of Medical Sciences. All the authors declare no financial
or commercial conflict of interests that could negatively influence the
study.
Informed consent  There was no human participant and consent was
not required.
Research involving human participants and/or animals  No human or
animal was involved in this study.
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