Paclitaxel and curcumin coadministration in novel cationic PEGylated niosomal formulations exhibit enhanced synergistic antitumor efficacy

Abstract

Background:
The systemic administration of cytotoxic chemotherapeutic agents for cancer treatment often has toxic side effects, limiting the usage dose. To increase chemotherapeutic efficacy while reducing toxic effects, a rational design for synergy-based drug regimens is essential. This study investigated the augmentation of therapeutic effectiveness with the co-administration of paclitaxel (PTX; an effective chemotherapeutic drug for breast cancer) and curcumin (CUR; a chemosensitizer) in an MCF-7 cell line.

Results:
We optimized niosome formulations in terms of surfactant and cholesterol content. Afterward, the novel cationic PEGylated niosomal formulations containing Tween-60: cholesterol:DOTAP:DSPE-mPEG (at 59.5:25.5:10:5) were designed and developed to serve as a model for better transfection efficiency and improved stability. The optimum formulations represented potential advantages, including extremely high entrapment efficiency (~ 100% for both therapeutic drug), spherical shape, smooth-surface morphology, suitable positive charge (zeta potential ~ + 15 mV for both CUR and PTX), sustained release, small diameter (~ 90 nm for both agents), desired stability, and augmented cellular uptake. Furthermore, the CUR and PTX kinetic release could be adequately fitted to the Higuchi model. A threefold and 3.6-fold reduction in CUR and PTX concentration was measured, respectively, when the CUR and PTX was administered in nano-niosome compared to free CUR and free PTX solutions in MCF-7 cells. When administered in nano-niosome formulations, the combination treatment of CUR and PTX was particularly effective in enhancing the cytotoxicity activity against MCF-7 cells.

Conclusions:
Most importantly, CUR and PTX, in both free form and niosomal forms, were determined to be less toxic on MCF-10A human normal cells in comparison to MCF-7 cells. The findings indicate that the combination therapy of PTX with CUR using the novel cationic PEGylated niosome delivery is a promising strategy for more effective breast cancer treatment.

Full text

Alemi et al. J Nanobiotechnol (2018) 16:28
https://doi.org/10.1186/s12951-018-0351-4
RESEARCH
Paclitaxel and curcumin
coadministration in novel cationic PEGylated
niosomal formulations exhibit enhanced
synergistic antitumor efficacy
Ashraf Alemi1, Javad Zavar Reza1,2*, Fateme Haghiralsadat3, Hossein Zarei Jaliani4, Mojtaba Haghi Karamallah2,
Seyed Ahmad Hosseini5 and Somayeh Haghi Karamallah6
Abstract
Background: The systemic administration of cytotoxic chemotherapeutic agents for cancer treatment often has
toxic side effects, limiting the usage dose. To increase chemotherapeutic efficacy while reducing toxic effects, a
rational design for synergy-based drug regimens is essential. This study investigated the augmentation of therapeutic
effectiveness with the co-administration of paclitaxel (PTX; an effective chemotherapeutic drug for breast cancer) and
curcumin (CUR; a chemosensitizer) in an MCF-7 cell line.
Results: We optimized niosome formulations in terms of surfactant and cholesterol content. Afterward, the novel
cationic PEGylated niosomal formulations containing Tween-60: cholesterol:DOTAP:DSPE-mPEG (at 59.5:25.5:10:5)
were designed and developed to serve as a model for better transfection efficiency and improved stability. The
optimum formulations represented potential advantages, including extremely high entrapment efficiency (~ 100%
for both therapeutic drug), spherical shape, smooth-surface morphology, suitable positive charge (zeta poten-
tial ~ + 15 mV for both CUR and PTX), sustained release, small diameter (~ 90 nm for both agents), desired stability,
and augmented cellular uptake. Furthermore, the CUR and PTX kinetic release could be adequately fitted to the Higu-
chi model. A threefold and 3.6-fold reduction in CUR and PTX concentration was measured, respectively, when the
CUR and PTX was administered in nano-niosome compared to free CUR and free PTX solutions in MCF-7 cells. When
administered in nano-niosome formulations, the combination treatment of CUR and PTX was particularly effective in
enhancing the cytotoxicity activity against MCF-7 cells.
Conclusions: Most importantly, CUR and PTX, in both free form and niosomal forms, were determined to be less
toxic on MCF-10A human normal cells in comparison to MCF-7 cells. The findings indicate that the combination
therapy of PTX with CUR using the novel cationic PEGylated niosome delivery is a promising strategy for more effec-
tive breast cancer treatment.
Keywords: Niosome, Paclitaxel, Curcumin, Combination therapy, Chemotherapy
© The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Open Access
Journal of Nanobiotechnology
*Correspondence: jzavar@ssu.ac.ir
1 Department of Clinical Biochemistry, Faculty of Medicine, Shahid
Sadoughi University of Medical Sciences, Yazd, Iran
Full list of author information is available at the end of the article
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Alemi et al. J Nanobiotechnol (2018) 16:28
Background
Chemotherapy is the standard treatment for various types
of cancers. However, chemotherapy is associated with
high systemic toxicity and low therapeutic effectiveness
[1]. Nanotechnology has revolutionized the diagnosis
and treatment of cancer [2]. A nano-sized drug delivery
system (DDS), or nanocarrier, is designed to deliver ther-
apeutic and/or diagnostic agents to their target sites [3].
Over recent decades, drug delivery systems using vesic-
ular carriers have attracted great interest because these
carriers provide high encapsulation efficiency, control
drug release, enhance drug solubility, carry both hydro-
philic and hydrophobic drugs, reduce side effects, pro-
long circulation in blood, and possess the ability to target
a specific area [4, 5]. Vesicles made of natural or synthetic
phospholipids are called liposomes, while transferosomes
are modified liposomal systems that, in addition to phos-
pholipids, contain a single chain surfactant as an edge
activator; ethosomes contain ethanol as an edge activator
instead of a single chain surfactant. Despite having some
advantages over conventional dosage forms, vesicular
carriers present many problems in practical applications,
such as high cost, the use of organic solvents for prepa-
ration, and a limited shelf life due to lipid rancidification
[6]. erefore, a continuous endeavor has been made
to find an alternative vesicular carrier. Niosomes meet
this requirement [7]. Niosomes, or non-ionic surfactant
vesicles, are unilateral or multilamellar spheroidal struc-
tures. Niosomes are preferred as an effective alternative
to conventional liposomes, as they offer several advan-
tages, including greater stability, lower cost, biodegra-
dability, biocompatibility, non-immunogenic, and low
toxicity, and they can be stored more easily for industrial
production in pharmaceutical applications [5, 8–12]. To
improve stability and circulation half-life, niosomes may
be coated with appropriate polymer coatings, such as
polyethylene glycol (PEG), creating PEGylated niosomes.
PEG coating also helps reduce systemic phagocytosis,
which results in prolonged systemic circulation, as well
as reduced toxicity profiles [13, 14]. Paclitaxel (PTX) is an
important antineoplastic drug, and it is isolated from the
bark of Taxus brevifolia. PTX demonstrates an effective
chemotherapeutic and cytotoxic activity against breast,
ovarian, colon, lung, prostate, and brain cancers. How-
ever, the wide therapeutic effects of PTX are limited due
to the low therapeutic index and poor water-solubility
[15, 16]. Curcumin (CUR) is a hydrophobic polyphe-
nol compound obtained from the rhizome of the plant
Curcuma longa. CUR exhibits various pharmacological
activities, such as anti-inflammatory, anti-oxidant, and
anti-tumor effects. Particularly, CUR has been demon-
strated to be highly effective against a variety of differ-
ent malignancies, including leukemia and lymphoma, as
well as colorectal, breast, lung, prostate, and pancreatic
carcinoma. However, the pharmacological application of
CUR has been impeded due to its extremely low aque-
ous solubility, instability, extremely poor bioavailability,
and high metabolic rate [17–19]. As a result, nanotech-
nology is considered one of the most significant methods
to design and develop various nano-carrier formulations
for curcumin and paclitaxel, such as polymeric micelles,
liposomes, self-assemblies, nanogels, niosome biode-
gradable microspheres, and cyclodextrin [18, 20, 21]. In
this study, we loaded both curcumin and paclitaxel into
cationic PEGylated niosomal formulations for enhanced
efficacy in MCF-7 human breast adenocarcinoma cells.
In addition to formulation design and optimization, we
have examined release profile, intracellular delivery, and
enhancement of cytotoxicity appears.
Results
The effect of surfactant:cholesterol ratio on CUR/PTX
niosome formulations
To specify the optimal formulation for attaining high
entrapment efficiency, controlled release (at 37°C and
pH 7.4), and small vesicle size, various niosomal CUR/
PTX formulations were evaluated (Table1). As shown in
Table1, cholesterol had a profound effect on CUR/PTX
entrapment efficiency in niosomes: by increasing the
amount of cholesterol content from 10% in formulation 1
(F1) to 30% in formulation 4 (F4), PTX/CUR entrapments
into nano-niosomes were constantly increased. However,
adding cholesterol from F1 to F4 decreased the percent-
age of CUR/PTX released over 12h. Furthermore, as can
be seen from the presented results, the mean diameter of
the niosomes increased with increasing the cholesterol
content (F1 → F5, Table 1). However, the addition of up
to 50% cholesterol to niosomes in F5 decreased niosomal
efficiency in trapping curcumin/paclitaxel compared to
the 30% cholesterol content in F4. Based on high entrap-
ment efficiency and sustained drug release, the F4 for-
mula has chosen as the formulation for further studies.
The effect of DSPE‑mPEG (2000) and DOTAP in niosomal
formulation
For attaining less aggregation, smaller niosomes, higher
entrapment efficiency, and improved stability, 5% PEG
was added to F4. According to Table 2, the F6 nioso-
mal formula containing 5% PEG showed higher drug
entrapment, smaller diameter, smaller Poly-Dispersity
Index (PDI), and lower drug release than the F4 for-
mula. Table2 shows the number of positive charge par-
ticles and the entrapment efficiency were increased by
adding 10% DOTAP to F6. However, vesicle size and
PDI declined with a 10% increase in the molar amount
of DOTAP. e obtained results showed the CUR/
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Alemi et al. J Nanobiotechnol (2018) 16:28
Table 1 Effect of the non-ionic surfactant Tween 60: cholesterol with various molar ratios on entrapment efficiency (EE %), size and % release (R) in CUR/PTX
loaded Niosomes
Code Mole Tween 60 (%) Mole cholesterol (%) EE (%) R (%) Size (nm) PDI Zeta potential (mV)
F1 90 10 EE % CUR = 52.24 ± 0.47
EE % PTX = 45.24 ± 0.12 R % CUR = 75.26 ± 0.42
R % PTX = 65.14 ± 0.32 Size CUR = 101.5 ± 0.12
Size PTX = 122.4 ± 0.46 PDI CUR = 0.281 ± 0.056
PDI PTX = 0.261 ± 0.056 Zeta CUR = − 20.34 ± 0.68
Zeta PTX = − 16.62 ± 0.47
F2 80 20 EE % CUR = 66.12 ± 0.86
EE % PTX = 61.74 ± 0.36 R % CUR = 69.11 ± 0.24
R % PTX = 56.12 ± 0.66 Size CUR = 107.5 ± 0.31
Size PTX = 131.24 ± 0.31 PDI CUR = 0.246 ± 0.12
PDI PTX = 0.236 ± 0.12 Zeta CUR = − 22.41 ± 0.75
Zeta PTX = − 19.08 ± 0.36
F3 75 25 EE % CUR = 81.24 ± 0.47
EE % PTX = 72.44 ± 0.63 R % CUR = 57.26 ± 0.11
R % PTX = 47.24 ± 0.36 Size CUR = 112.7 ± 0.64
Size PTX = 140.66 ± 0.72 PDI CUR = 0.224 ± 0.087
PDI PTX = 0.214 ± 0.087 Zeta CUR = − 21.38 ± 0.86
Zeta PTX = − 21.54 ± 0.44
F4 70 30 EE % CUR = 85.42 ± 0.11
EE % PTX = 81.37 ± 0.21 R % CUR = 46.11 ± 0.34
R % PTX = 39.22 ± 0.41 Size CUR = 118.7 ± 0.56
Size PTX = 149.32 ± 0.65 PDI CUR = 0.204 ± 0.062
PDI PTX = 0.194 ± 0.062 Zeta CUR = − 21.45 ± 0.42
Zeta PTX = − 19.56 ± 0.27
F5 50 50 EE % CUR = 71.24 ± 0.16
EE % PTX = 67.12 ± 0.47 R % CUR = 54.12 ± 0.22
R % PTX = 45.14 ± 0.32 Size CUR = 125.1 ± 0.44
Size PTX = 157.44 ± 0.66 PDI CUR = 0.214 ± 0.013
PDI PTX = 0.208 ± 0.013 Zeta CUR = − 24.16 ± 0.22
Zeta PTX = − 24.56 ± 0.42
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Table 2 Effect cationic phospholipid DOTAP and DSPE-mPEG (2000) on entrapment efficiency (EE %), size and % release (R) in CUR/PTX loaded Niosomes
Code Mole Tween
60 (%) Mole
cholesterol (%) Mole DOTAP
(%) Mole PEG
(%) EE (%) R (%) Size (nm) PDI Zeta potential (mV)
F6 64.4 27.6 0 5 EE % CUR = 91.22 ± 0.28
EE % PTX = 86.11 ± 0.66 R % CUR = 38.53 ± 0.18
R % PTX = 31.44 ± 0.16 Size CUR = 91.5 ± 0.25
Size PTX = 118.9 ± 0.31 PDI CUR = 0.179 ± 0.23
PDI PTX = 0.164 ± 0.31 Zeta CUR = − 20.99 ± 0.45
Zeta PTX = − 19.24 ± 0.44
F7 59.5 25.5 10 5 EE % CUR = 98.24 ± 0.11
EE % PTX = 98.79 ± 0.24 R % CUR = 33.11 ± 0.33
R % PTX = 20.08 ± 0.44 Size CUR = 85.4 ± 0.43
Size PTX = 111.6 ± 0.24 PDI CUR = 0.177 ± 0.17
PDI PTX = 0.158 ± 0.24 Zeta CUR = + 14.83 ± 0.21
Zeta PTX = + 16.17 ± 0.31
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Alemi et al. J Nanobiotechnol (2018) 16:28
PTX niosomal formulations containing Tween-60:
cholesterol:DOTAP:PEG with a 59.5:25.5:10:5 molar ratio
(F7) had the desired feature based on high entrapment
efficiency, sustained drug release, small diameter, and
improved transfection efficiency (Table2).
Physical characterization of niosomal vesicles
e internal structure of CUR/PTX niosomes was eval-
uated by cryogenic transmission electron microscopy
(Cryo-TEM). As illustrated in Fig. 1a, b, the optimum
formula of CUR/PTX niosomes was spherically shaped.
Furthermore, the niosomes structures’ rigid boundaries
were indicated. According to SEM photographs, the nio-
somal vesicles were found to be round with smooth sur-
faces (Fig.1c, d).
In‑vitro drug release study
Evaluation of invitro drug release was performed using
the dialysis method. e results of a 72-h release profile
of CUR and PTX from the optimum formulation (F7) in
PBS pH 7.4 at 37°C are displayed in Fig.2. After 72h,
29.93 and 28.16% of the loaded drugs were released for
CUR and PTX, respectively. e cumulative release pro-
file of CUR and PTX was apparently biphasic, with an
Fig. 1 Morphological assessment: a niosomal paclitaxel; b niosomal curcumin by cryogenic transmission electron microscopy (Cryo-TEM). Scan-
ning electron microscopy (SEM) of c curcumin niosome; and d paclitaxel niosome
In vitro Release Curve
Time (h)
Cumulative release%
020406
08
0
0
10
20
30
40
Curcumin
Paclitaxel
Fig. 2 The in vitro release profile of curcumin and paclitaxel from
niosomal optimum formula
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Alemi et al. J Nanobiotechnol (2018) 16:28
initial rapid release period followed by a slower release
phase.
Release kinetics modeling
Figure3 shows the CUR/PTX release data were analyzed
mathematically according to: zero-order, first-order,
Hixson–Crowell, and Higuchi’s equations. Table3 sum-
marizes the correlation coefficients (R2) calculated for
niosomal formulations. e results revealed that the
release of CUR and PTX from niosomal films is most
fitted to the Higuchi model, according to the higher cor-
relation coefficient.
Fourier transforms infrared (FTIR) spectral evaluation
To confirm the drug presence in CUR/PTX nano-nio-
some formulations, FTIR analysis was performed. Fig-
ure4a shows the FTIR spectrum of free paclitaxel. ere
were characteristic peaks in this spectrum: O–H stretch-
ing and N–H stretching in 2° amine at 3445cm−1, –CH3
asymmetric and symmetric stretching at 2923cm−1, con-
jugation of C=O with phenyl group at 1733cm−1, C–O
stretch at 1122cm−1, and the C–H out-of-plane bending
vibrations for monosubstituted rings in the paclitaxel
molecule in the region of 900–500cm−1.
Figure 4b demonstrates the FTIR spectrum of free
curcumin. e bands exhibited in this spectrum can
be assigned to: C–H stretching and O–H stretching at
3507cm−1, aromatic ring C=C stretching at 1506cm−1,
C=O stretch at 1152cm−1, and the C–H out-of-plane
bending vibrations for ortho-disubstituted rings in cur-
cumin molecule in the region of 800–600cm−1.
Fig. 3 Curcumin and paclitaxel comparative plots. a Zero order release kinetics; b first order release kinetics; c Higuchi (SQRT) release kinetics and d
Hixson–Crowell model
Table 3 Release kinetics data of CUR and PTX from the
niosomal optimum formulae
Regression coefficient (R2)
Formulation
code Zero order First order Higuchi
model Hixson–
Crowell
model
F7 (CUR) 0.9709 0.8492 0.9979 0.9002
F7 (PTX) 0.9686 0.882 0.9992 0.9161
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Fig. 4 FTIR spectra. a Free paclitaxel; b free curcumin; c blank noisome; d niosomal paclitaxel; e niosomal; curcumin; f comparison blank noisome
and niosomal paclitaxel; g comparison blank noisome and niosomal curcumin
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Alemi et al. J Nanobiotechnol (2018) 16:28
e FTIR pattern for blank niosome (Fig. 4c) dem-
onstrates various characteristic peaks of DOTAP,
Tween-60, cholesterol, and DSPE-mPEG in the range of
3500–1115cm−1. e band observed at 3435cm−1 was
assigned to cholesterol and Tween-60 (O–H stretching in
phenols and N–H stretching in 2°-amines). C–N stretch
and C–O stretch occur at 1148cm−1 and belonged to
DOTAP and Tween-60, respectively. e carbonyl group
exhibits a strong absorption band at 1642.15cm−1 due to
C–O stretching vibration in DSPE-mPEG, Tween-60, and
DOTAP. All peaks were repeated in the FTIR spectrum
of PTX/CUR niosome formulations. e niosomal pacli-
taxel FTIR spectrum (Fig. 1d) shows the out-of-plane
bending peaks in the range of 900–500cm−1, and it can
be used to assign mono-substitution on the paclitaxel
ring that confirms paclitaxel loading in the niosome for-
mulation. Furthermore, according to the niosomal cur-
cumin FTIR spectrum (Fig.1e), the out-of-plane bending
peaks in the 800–600cm−1 range can be utilized to allo-
cate ortho substitutions on the curcumin ring that cor-
roborates curcumin loading in the niosome formulation.
When compared to the blank noisome, the sharper band
in the 1600cm−1 region and the broader bands in the
3500cm−1 and 900–500cm−1 regions in the CUR/PTX
niosomal formulations (Fig.1f, g) affirm curcumin and
paclitaxel entrapment in the nano-niosomes.
Physical stability examination
To determine physical stability, the optimum formula-
tion of curcumin/paclitaxel-loaded niosomes, in terms
of encapsulation efficiency, vesicle size, PDI, and zeta
potential, were tested by storing them at 4°C. After stor-
age for 60days, the encapsulation efficiency, vesicle size,
PDI, and zeta potential of the optimized formulation (F7)
were not significantly changed from the freshly prepared
samples (p value < 0.05). ese results confirmed the sta-
bility of the F7 formula.
Cytotoxicity assays
IC50s for individual curcumin and paclitaxel on MCF‑7
and MCF‑10A cells
To determine the inhibitory effect of individual cur-
cumin and paclitaxel as a free form and as a niosomal
form on MCF-7 and MCF-10A cells, we first performed
Fig. 5 Inhibition of cell growth by curcumin (CUR) and paclitaxel (PTX) individual as a drug free form and drug niosomal form in MCF-7 and MCF-
10A cell. a Free CUR; b Free PTX; c Nio CUR; d Nio PTX for MCF-7 (filled square) and MCF-10A (filled triangle) cells
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Alemi et al. J Nanobiotechnol (2018) 16:28
dose–response experiments for curcumin and paclitaxel.
As indicated in Fig.5, individual treatments with the free
form and the niosomal form resulted in growth inhibition
of MCF-7 and MCF-10A cells in a dose-dependent pat-
tern. Table4 evaluates the IC50 values of these agents. e
IC50 values of free PTX solution and free CUR solution
was 13.54 and 44.60μgmL−1, respectively, against MCF-7
cells and 30.75 and 76.71μgmL−1, respectively, against
MCF-10A cells (Fig.5a, b). is revealed that MCF-10A
cells needed at least a ∼ 2.27-fold higher concentration
of PTX solution and a ∼ 1.7-fold higher concentration of
CUR solution to attain IC50 compared to their counter-
part MCF-7 cancer cells. As depicted in Fig.5c, d, nano-
niosomes were highly efficient in delivering the PTX and
CUR drugs to both MCF-7 and MCF-10A cells. A three-
fold and 3.6-fold reduction in CUR and PTX concentra-
tion were measured, respectively, when the CUR and PTX
were administered in nano-niosomes compared to free
CUR and free PTX solutions in MCF-7 cells. Similarly, the
CUR and PTX delivered in nano-niosomes to MCF-10A
cells demonstrated a 1.2- and 1.5-fold lowered concentra-
tion, respectively. ese results indicated that PTX and
CUR in free and niosomal forms had less cytotoxicity on
MCF-10A cells as a model for normal human mammary
epithelial cells. e IC50 concentrations were then utilized
to generate fixed ratios for subsequent combination exper-
iments and for the calculation of combination index (CI).
Growth inhibitory effects of paclitaxel in combination
with curcumin
To determine the synergistic antitumor effects of cur-
cumin and paclitaxel, we performed a combination study,
and the results are presented in Table 5. Figure 6a, b
showed the dose–response curves for MCF-7 and MCF-
10A cell lines exposed to paclitaxel and curcumin combi-
nation therapy. According to the results, curcumin could
significantly increase the cell growth inhibition of pacli-
taxel; in the presence of free CUR solution, the IC50 of free
PTX solution was diminished to ∼ 1.6-fold in MCF-7 cells
and ∼ 1.4-fold in MCF-10A cells. is combination ther-
apy regimen was significantly efficacious (p value < 0.05)
when the PTX and CUR was delivered in nano-niosome
formulations compared to a free solution (Table4). us,
the use of PTX and CUR together resulted in enhanced
therapeutic potential. Figure 6 also illustrates the com-
bination index analysis of the PTX and CUR interac-
tion in MCF-7 and MCF-10A cells. Values of CI < 1 were
obtained from the paclitaxel and curcumin combination
in both free forms and niosomal forms for MCF-7 and
MCF-10A cells, demonstrating that the two drugs interact
synergistically to inhibit cell growth (Fig.6c–f).
Nano‑niosomal CUR/PTX cellular uptake experiments
Cellular uptake experiments were performed to evaluate
the cellular uptake behavior of different CUR/PTX nio-
somal formulations in the following cells: MCF-7 cells
as a cancer cell model and MCF10A cells as a model for
normal human mammary epithelial cells. Figures 7, 8
and 9 illustrates the cellular uptake images of F6 and F7
CUR/PTX-loaded niosome formulations on MCF-7 and
MCF10A cell lines monitored by fluorescence micro-
scope. As depicted in Fig.7b, d, the MCF-7 cells treated
with the CUR/PTX F7 formula containing 10% DOTAP
showed greater green and cyan (blue–green) color inten-
sity compared to cells treated with CUR/PTX F6 formula
(without DOTAP, Fig.7a, c). By adding 10% DOTAP to
the F6 formula, the drug release, vesicle size, and polydis-
persity index decreased, while the transfection efficiency
was enhanced. Similarly, these results are observed in
MCF-10A cells (Fig.9a–d); however, the intensity of the
green and cyan color in these cells was much less than
in the MCF-7 cells. ese findings indicate that CUR/
Table 4 The IC50 values of paclitaxel, curcumin alone
and in combination on MCF-7 and MCF-10A cells, adminis-
tered in the forms of free drug and drug niosomal form
Data represents the mean ± SD
The values are shown as mean ± SD, n = 4
Treatment type IC50 values
On MCF‑7 cells On MCF‑10A cells
Free curcumin solution 44.60 ± 0.46 76.71 ± 0.45
Free paclitaxel solution 13.54 ± 0.28 30.75 ± 0.22
Free paclitaxel + free curcumin
solution 8.36 ± 0.38 22.26 ± 0.48
Curcumin niosome 14.90 ± 0.19 64.22 ± 0.36
Paclitaxel niosome 3.73 ± 0.29 20.54 ± 0.49
Paclitaxel niosome + curcumin
niosome 1.57 ± 0.1 8.89 ± 0.46
Table 5 Paclitaxel and curcumin combination index (CI) against MCF-7 and MCF-10A cells
CI < 1, synergistic; CI = 1, additive; CI > 1, antagonistic
Combination type MCF‑7 cells MCF‑10A cells
CI Interaction type CI Interaction type
Free paclitaxel + free curcumin solution 0.23 Synergistic 0.53 Synergistic
Paclitaxel niosome + curcumin niosome 0.35 Synergistic 0.53 Synergistic
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Alemi et al. J Nanobiotechnol (2018) 16:28
PTX-loaded niosome formulations entered healthy cells
much less than cancerous cells. ese results are consist-
ent with cytotoxicity experiments.
Apoptosis analysis
Apoptosis was measured by annexin V-fluorescein isothi-
ocyanate (FITC)/propidium iodide (PI) double staining
Fig. 6 Analysis of synergy between curcumin and paclitaxel for MCF-7 (filled triangle) and MCF-10A (filled square) cells. a Dose–response curve of
free CUR + Free PT X; b dose–response curve of Nio CUR + Nio PTX. CI values at different levels of growth inhibition effect (fraction affected, FA; c
Free CUR + Free PTX in MCF-7 cells; d Nio CUR + Nio PTX in MCF-7 cells; e Free CUR + Free PTX in MCF-10A cells, f Nio CUR + Nio PTX in MCF-10A
cell
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Alemi et al. J Nanobiotechnol (2018) 16:28
(Sigma-Aldrich, USA). MCF-7 cells were seeded in six-
well plates at a density of 1 × 105 cells per well. Apopto-
sis was induced by treating the cells with PTX and CUR,
either as single agents or as a PTX + CUR combination,
administered in aqueous solution or in nano-niosome
formulations at an IC50 concentration for each drug.
After 24h of incubation, the cells were detached using
0.25% trypsin/EDTA (Sigma-Aldrich, USA) and centri-
fuged at 1500rpm for 3min, after which the pellet was
resuspended in ice-cold, phosphate-buffered saline (PBS,
Fig. 7 Cellular uptake of F6 and F7 CUR/PTX loaded niosomes formulations on MCF-7cell line. MCF-7cell line [a1 F6 Nio CUR Nucleus, a2 F6 Nio
CUR, a3 F6 Nio CUR merged; b1 F7 Nio CUR Nucleus, b2 F7 Nio CUR, b3 F& Nio CUR merged; c1 F6 PTX CUR Nucleus, c2 F6 Nio PTX, c3 F6 Nio CUR
PTX; d1 F7 Nio PTX Nucleus, d2 F7 Nio PTX, d3 F& Nio PTX merged]
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Alemi et al. J Nanobiotechnol (2018) 16:28
Fig. 8 Cellular uptake of F6 and F7 CUR/PTX loaded niosomes formulations on MCF-7cell line. MCF-10A cell line [a1 F6 Nio CUR Nucleus, a2 F6 Nio
CUR, a3 F6 Nio CUR merged; b1 F7 Nio CUR Nucleus, b2 F7 Nio CUR, b3 F& Nio CUR merged; c1 F6 PTX CUR Nucleus, c2 F6 Nio PTX, c3 F6 Nio CUR
PTX; d1 F7 Nio PTX Nucleus, d2 F7 Nio PTX, d3 F& Nio PTX merged]
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Page 13 of 20
Alemi et al. J Nanobiotechnol (2018) 16:28
Fig. 9 Apoptosis assay using flow cytometry following the treatment of cells for 24 h. a Control; b free curcumin + free paclitaxel; c free curcumin;
d free paclitaxel; e niosomal curcumin; f niosomal paclitaxel; g niosomal curcumin + niosomal paclitaxel
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Page 14 of 20
Alemi et al. J Nanobiotechnol (2018) 16:28
pH 7.4). Annexin V-FITC solution (3µL) was added to
each cell suspension. In addition, 3 µL of propidium
iodide stock solution was added to the cells to identify
necrotic cells. After 30 min of incubation on ice, the
stained cells were analyzed by flow cytometry using the
BD FACSCalibur instrument. Cells that did not receive
any drug treatment served as the control.
Discussion
Plants have been employed as medicines for centu-
ries, and the usage of plant-derived chemicals has been
extended into anticancer drugs. Lately, chemotherapeutic
strategies have advanced to the utilization of combined
active compounds because they are believed to be more
active than a single agent. Hence, treatment effectiveness
could increase, and the toxic side effects may be reduced,
due to the extremely low use of drugs. Curcumin (diferu-
loylmethane), a yellow pigment isolated from the rhi-
zome of turmeric, has been reported to have an extensive
spectrum of pharmacological activities. Furthermore,
curcumin is currently involved in the early phase of a
clinical trial as a potential chemo-preventive agent [22,
23]. erefore, it is logical to evaluate whether curcumin,
as a new antiproliferative agent, can sensitize tumors
to the chemotherapeutic drug paclitaxel for breast can-
cer cells. Paclitaxel (PTX) has been used as an effective
chemotherapeutic drug for a wide range of tumors, such
as breast, lung, prostate, ovarian, and pancreatic cancers
[24, 25]. e CUR and PTX combination is a remark-
able anticancer drug therapy. PTX is a powerful micro-
tubule-stabilizing agent that commences cell cycle arrest,
while CUR attacks biologically by regulating several sig-
nal transduction pathways [26–28]. Despite these good
therapeutic effects, the wide therapeutic range of PTX
and CUR is limited due to poor aqueous solubility and
low therapeutic index. A promising approach for circum-
venting these issues is the use of a vesicular nanocarrier,
such as niosomes, which are an alternative to phospho-
lipid vesicles for the encapsulation of hydrophobic drugs
due to providing high encapsulation efficiency, biocom-
patibility, biodegradation, low preparation cost, and suffi-
cient stability, as well as being free from organic solvents
and offering easy storage [7]. In this study, we have devel-
oped a novel cationic PEGylated niosomal formulation
for encapsulating paclitaxel and curcumin. e vesicu-
lar systems were prepared from the nonionic surfactant
Tween-60, as a commercial surfactant, and all formula-
tions were compared in terms of entrapment efficiency,
drug release, vesicle size, and polydispersity index. Fur-
thermore, niosomes formulated without cholesterol
formed a gel, and only the addition of cholesterol was a
homogenous niosome obtained [29]. e hydrophilic–
lipophilic balance (HLB) of the nonionic surfactant, the
chemical structure of the components, and the criti-
cal packing parameter (CPP) are important in forming
bilayer vesicles instead of micelles. e HLB value of a
surfactant plays a key role in controlling the drug entrap-
ment efficiency of the vesicle it forms. A surfactant, such
as Tween-60, with an HLB value in the 14–17 range is
inappropriate for creating niosomes. For HLB > 6, cho-
lesterol must be added to the surfactant until forming a
bilayer vesicle. Also, the presence of cholesterol in the
formulation of niosomes is necessary for the physical
stability of these nano-sized vesicles (i.e., suppressing
the surfactant’s tendency to form aggregates, decreasing
drug leakage, vesicle size, and dispersion). is was pri-
marily ascribed to the increase in hydrophobicity (par-
ticularly with higher HLB surfactant molecules, such as
Tween-60) that augmented the structural affinity of the
bilayer membrane for CUR/PTX molecules [6, 11, 12,
30–32]. erefore, cholesterol is added to the formula-
tions as a membrane-stabilizing factor. As a result, by
increasing the amount of cholesterol content from 10
to 30%, PTX/CUR entrapments in nano-niosomes were
increased, while the percentage of CUR/PTX release was
decreased. Furthermore, the mean diameter of niosomes
increased with increasing the cholesterol content. How-
ever, the addition of cholesterol content to niosomes up
to 50% decreased niosomal efficiency in trapping cur-
cumin/paclitaxel compared to 30% cholesterol content.
is finding can be explained by the possible competi-
tion between curcumin and paclitaxel as lipophilic drugs
and the cholesterol incorporation into the niosomes. A
further increase in cholesterol tends to deposit between
the bilayers, excluding the drug from the niosomal bilay-
ers. Above a certain level of cholesterol, entrapment
efficiency decreased possibly due to a decrease in CPP
[6, 11, 12, 30–32]. Improving stability, increasing the
drug encapsulation, decreasing mean size diameter, and
reducing drug release is due to the presence of PEGyla-
tion in the niosomal formulations [33, 34]. erefore, 5%
PEG was added to the F4 formula. According to the find-
ings, the F6 niosomal formula demonstrated higher drug
entrapment, smaller diameter, smaller PDI, and lower
drug release than the F4 formula. Additionally, cationic
lipids added to the niosomal formulations enhanced the
niosomes’ physicochemical properties and the transfec-
tion efficiency. e addition of DOTAP decreased the
drug’s release and vesicle size due to a decline in the
cholesterol content. is effect also decreased the poly-
dispersity index, which is relevant to the further recipro-
cal repel force between the particles with the same sign
charge in the suspension system [35–38]. To hamper the
aggregation of vesicular systems, it is essential to intro-
duce a charge on the surface of the vesicle. A good indi-
cator for the size of this barrier is zeta potential. If all the
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Alemi et al. J Nanobiotechnol (2018) 16:28
particles possess large enough zeta potential, they pre-
sumably repel each other strongly enough that they will
not have the tendency to aggregate [39]. After storage for
60days, the presence of DOTAP and PEG in niosomal
formulations of CUR and PTX demonstrated no signifi-
cant changes when compared to freshly prepared sam-
ples in terms of encapsulation efficiency, vesicle size, PDI,
and zeta potential of the optimized formulation (F7).
is implies that the new F7 niosome formulation could
minimize problems associated with niosome instabil-
ity, including aggregation, fusion, and drug leakage. e
rate of drug release from a delivery system is a crucial
factor and must be appraised to attain an optimal system
with the desired drug-release profile. e invitro release
study was conducted to predict how a delivery system
may function under the ideal status, which might display
some indication of its in vivo efficiency. In-vitro drug
release demonstrated that the cumulative release profile
of CUR and PTX were apparently biphasic, with an initial
rapid release period followed by a slower release phase.
Because CUR and PTX are small molecules and the
permeability cut-off of the dialysis bag was 12kDa, the
released CUR and PTX poured easily from the bag. As a
result, neither the dialysis bag nor the drug size restricted
the drug’s release. e initial fast rate of release was
regulated by the diffuse mechanism (concentration gra-
dient of CUR/PTX between noisome and buffer), while
the later slow release resulted from the drug’s sustained
release from the inner layer [40–42].
e invitro release of CUR and PTX from the nioso-
mal formulation was assessed by fitting the cumulative
drug release into mathematical release models, which
are commonly applied to elucidate release kinetics and
to compare release profiles. e CUR/PTX niosomal
formulations followed the Higuchi model. ese find-
ings indicated that CUR and PTX molecules were dif-
fused in the niosome matrix and that there were no
possible interactions between the niosome components
and the drugs [5, 43–45]. In this study, we have investi-
gated the effect of PTX and CUR combination therapy,
in both free forms and niosomal forms, on MCF-7 cells
as a cancer cell model and MCF10A cells as a model for
normal human mammary epithelial cells (Tables4 and
5). e ratiometric combination of PTX and CUR sig-
nificantly suppressed the growth of MCF-7 cells. When
the free drugs were administered in nano-niosome for-
mulations, the cytotoxicity effects manifested even more.
e enhanced therapeutic activity achieved with the
combination therapy was ascribed to the P-glycopro-
tein (P-gp) downregulation and to the inhibition of the
NFκB pathway by CUR. Most importantly, CUR down-
regulates the NF-ĸB signaling pathways, thus inhibiting
cancer cell growth and inducing apoptosis. erefore,
CUR sensitizes cancer cells to increase the cancer cells’
response to anticancer drugs. Increasing the accumula-
tion of PTX within the cancer cell due to P-gp downregu-
lation can overcome the MDR phenomenon [1, 27, 46].
We observed a similar trend for MCF-10a cells. Never-
theless, as expected, CUR and PTX had fewer side effects
in both free form and niosomal form on MCF10A human
mammary epithelial cells. e cellular uptake experi-
ments were demonstrated by the addition of DOTAP,
which enhanced the transfection efficiency of the CUR/
PTX F7 formula; it is well known that cationic lipids
enhance the transfection efficiency of niosomal formula-
tions [35–38]. Quantitative apoptotic activity measure-
ments were made by flow cytometry analysis in PTX and
CUR treated cells. Statistically significant when apoptotic
activity of paclitaxel NanoNiosome formulation is com-
pared with free paclitaxel and curcumin NanoNiosome
formulation is compared with free curcumin solution in
MCF-7 cells (p < 0.05). In addition to these findings, flow
cytometry analysis also revealed that the apoptosis was
significantly greater with the combination therapy and
with drugs administered NanoNiosome formulations
at p < 0.05. ese results collaborate with the cell viabil-
ity experiment to affirm that NanoNiosomes were effec-
tive in delivering the PTX and CUR to the cells, and
combination therapy with PTX and CUR delivered in
NanoNiosome formulations indeed demonstrated higher
therapeutic efficacy in MCF-7 cells.
Conclusions
Our successful findings suggest novel cationic PEGylated
niosomal formulations for paclitaxel and curcumin co-
administration. e encapsulation efficiency of both
drugs was extremely successful. e drugs’ release pro-
file demonstrated burst release followed by a sustained
drug release for both agents. e combination of PTX (a
powerful anticancer drug) with CUR (an effective che-
mosensitizer), particularly in nano-niosome formula-
tions, can improve the therapeutic effectiveness of cancer
treatments. Our experimental evidence indicated that a
nanocarrier-based approach adopted for the delivery of
CUR/PTX combinations was efficient in battling cancer
cells invitro.
Methods
CUR/PTX niosomes preparation
We used the thin-film hydration method to prepare the
curcumin and paclitaxel-loaded niosomes [47]. Tween-
60 (DaeJung Chemicals & Metals, South Korea) and
cholesterol (Sigma-Aldrich, USA) were dissolved in
chloroform to obtain the different molar ratio molari-
ties (as illustrated in Table 1). PTX (Stragen, Switzer-
land) and CUR (Sigma-Aldrich, USA) were dissolved in
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Page 16 of 20
Alemi et al. J Nanobiotechnol (2018) 16:28
chloroform and added to the mixture of surfactant and
lipids. Fluorescent label Dil (Sigma, USA) was added to
the lipid phase at 0.1% mol for lipid staining to evaluate
cellular uptake. Niosomal formulations were screened
for particle size, controlled release, and high entrap-
ment efficiency parameters. After attaining optimized
synthetic conditions, the cationic lipid DOTAP (1,2-dio-
leoyl-3-trimethylammonium-propane, Sigma-Aldrich,
USA) and polyethylene glycol (Lipoid PE 18:0/18:0–
PEG2000, DSPE-mPEG 2000, Lipoid GmbH, Germany)
were added for improving stability and transfection effi-
ciency of the niosomal formulations. Organic solvent
was removed by rotary evaporator (Heidolph, Germany)
at 50°C until a thin-layered film formed. e dry lipid
films were hydrated by adding phosphate-buffered saline
(PBS, pH = 7.4) at 60°C for 60min to obtain the nioso-
mal suspensions. After hydration, the prepared vesicles
were sonicated for 30min using a microtip probe sonica-
tor (model UP200St, Hielscher Ultrasonics GmbH, Ger-
many) to reduce the vesicles’ mean size. ereafter, free
drugs (unloaded) were separated from niosomal vesicles
using a dialysis bag diffusion technique against PBS for
1 h at 4°C (MW = 12kDa, Sigma-Aldrich, USA) [48].
Drug-free niosomes were produced in a similar man-
ner without adding curcumin and paclitaxel. e dose of
both drugs was 0.5mgmL−1 for all formulations.
Analysis of encapsulation efficiency
To evaluate entrapment efficiency, spectroscopic meas-
urements were performed. e amounts of niosomal
encapsulated CUR and PTX were analyzed with a UV
spectro-photometer (model T80+, PG Instruments,
United Kingdom) at 429 and 236 nm (ʎmax), respec-
tively [7]. e encapsulation efficiency was determined as
follows:
Physical characterization of niosomal vesicles
e particle size distribution, zeta potential and Poly-
Dispersity Index (PDI) of the obtained niosomes were
measured by dynamic light scattering technique using
a ZetaPALS zeta potential and particle size analyzer
(Brookhaven Instruments, Holtsville, NY, USA). Scat-
tered light was detected at room temperature at an angle
of 90°, and the diluted samples in 1700µL of deionized
water (0.1 mg mL−1) were prepared and immediately
measured after preparation. All measurements were
carried out three times, and their mean values were cal-
culated. e internal structure of NanoNiosome formula-
tions was determined by cryogenic transmission electron
Encapsulation efficiency (%)
=The amount of CUR/
PTX encapsulated within niosomes
Total amount of CUR/PTX added
×100
microscopy (FEI Tecnai 20, type Sphera, Oregon, USA)
equipped with a LaB6 filament at 200 kV. A drop of
NanoNiosome solution was placed over a 200-mesh
Copper-coated TEM grids, and TEM measurement was
accomplished. Characterization of surface morphology of
Niosomes was evaluated using scanning electron micro-
scope (SEM. To prepare the sample used in SEM, a little
amount of the NanoNiosome solution dispersed in water
was placed on the mesh copper grid 400. en, the cop-
per grid was placed in an evacuated desiccator to evapo-
rate the solvent. Finally the samples were coated with
gold coater to make them conductive, followed by evalu-
ation of the surface morphology using SEM with 100W
power instrument (model KYKY-EM3200-30kV, China).
In‑vitro drug release study
e in vitro release of CUR/PTX from niosomes was
monitored using a dialysis bag (MW = 12 kDa) against
PBS (containing 2% Tween-20 to imitate a physiological
environment) for 72h at 37°C and 7.4 pH [42]. First, the
CUR/PTX niosome samples were suspended in a dialy-
sis tube, and the release of both drugs was evaluated in
10mL of PBS with continuous stirring. en, 2mL of
the sample was collected from the incubation medium at
specific time intervals and immediately substituted with
an equal volume of fresh PBS. e amount of CUR/PTX
released was determined using a UV–Vis spectrometer at
429 and 236nm, respectively.
Mathematical modeling of drug release kinetic
Cumulative percentages of the drug released from the
niosomes were calculated by the following Eq.(1):
where Mt and Mf are the cumulative amounts of drug
released at any time (t) and the final amounts of drug
released, respectively.
To determine the release kinetic, the release data were
fitted to the mathematical models by the linear regres-
sion analysis of Graph pad prism 6.0, as follows:
Zero-order rate equation:
where Qt is the amount of the remaining drug in the for-
mulation at time t; Q0 is the initial amount of drug in the
formulation; and K0 is the zero-order release constant.
First-order rate equation:
where C0 is the initial drug concentration; K is the first-
order release constant; and t is time.
(1)
Release
=
M
t
Mf
(2)
Qt
=
Q0
+
K0t
(3)
log C
=log C0−
K
t
2.303
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Alemi et al. J Nanobiotechnol (2018) 16:28
Higuchi’s model:
where Q is the amount of drug released in time t per unit
area, and KH is the Higuchi dissolution constant.
Hixson–Crowell model:
Q0 is the initial amount of the drug in the niosomes; Qt is
the cumulative amount of the drug released at time t; and
Ks is the Hixson–Crowell release constant.
Finally, the correlation coefficients’ values were com-
pared to determine the release model that best fits the
data [40, 42].
Fourier transforms infrared (FTIR) spectral evaluation
e samples’ functional group characterizations were
investigated using FTIR spectrometer (Model 8300, Shi-
madzu Corporation, Tokyo, Japan) for pure CUR, pure
PTX, blank noisome, niosomal-CUR, and niosomal-
PTX. For preparation, the samples were lyophilized as a
dry powder and mixed with potassium bromide (KBr).
en, the samples were placed in a hydraulic press to
form the pellets. e FTIR spectrum was scanned in the
wavelength range of 400–4000cm−1.
Physical stability examination
To determine the physical stability of niosomal cur-
cumin/paclitaxel during storage, the change in particle
size, zeta potential, PDI, and the remaining amount of
the drug in vesicle was assessed over 14-, 28-, and 60-day
intervals [9, 39].
Cell lines and culture conditions
Human breast cancer MCF-7 cells (the Iranian Bio-
logical Resource Center, Tehran, Iran) were cultured
in DMEM/F12 Ham’s mixture (InoClon, Iran) sup-
plemented with 2 mM GlutaMAX™-I (100X, Gibco,
USA), 10% FBS (Fetal Bovine Serum, Gibco, USA), and
1mgmL−1 penicillin/streptomycin (Gibco, USA). Non-
tumorigenic human breast epithelial cell line MCF-
10A (the Iranian Biological Resource Center, Tehran,
Iran) was grown in DMEM/F12 Ham’s mixture sup-
plemented with 2mM GlutaMAX™-I, 5% horse serum
(Gibco, USA), EGF (Epithelial growth factor, Sigma,
USA) 20ngmL−1, insulin 10 μg mL−1 (Sigma, USA),
hydrocortisone 0.5μgmL−1 (Sigma, USA), 100ngmL−1
cholera toxin (Sigma, USA), and 1mgmL−1 penicillin/
streptomycin. An MCF-10A cell line was used for com-
parison in all experiments.
(4)
Q
=
KHt1/2
(5)
Q1/3
0
−
Q1/3
t
=
Kst
Cytotoxicity assays
e cytotoxicity of various formulations was determined
by MTT (Sigma, USA) assay [49–51]. Briefly, MCF-7 and
MCF-10A cells were seeded in 96-well plates at 10,000
cells per well. Following attachment for 24h, the cells
were treated with 200μL fresh medium containing serial
dilutions of the different drug/niosome formulations:
free-PTX solution, free-CUR solution, free PTX + free
CUR physical mixture, niosomal CUR, niosomal PCT,
and the co-administration of niosomal CUR-niosomal
PTX. After incubation for 48h, 20μL MTT (5mgmL−1
in PBS) was added into each 96-well plate and incu-
bated for 3h at 37°C. Finally, the medium was carefully
removed, and 180μL of DMSO was added to each well
to dissolve the formazan crystals formed. Absorbance of
each well was recorded by EPOCH Microplate Spectro-
photometer (synergy HTX, BioTek, USA) at 570nm. e
cytotoxicity of the different formulations was expressed
as the Inhibitory Concentration (IC50) value, defined
as the drug concentration required for inhibiting cell
growth by 50% relative to the control. e IC50 values of
PTX and CUR as single drugs or in combination were
calculated using GraphPad Prism 6. e curcumin and
paclitaxel combination was appraised by calculating the
CI value using the CompuSyn software, with the method
utilized by Chou and Talalay:
where a is the PTX IC50 in combination with CUR at
concentration b; A is the PTX IC50 without CUR; and B
is the CUR IC50 in the absence of PTX. According to the
Chou and Talalay equation, when CI < 1, the interaction
between the two drugs is synergistic; when CI = 1, the
interaction between the two drugs is additive; and when
CI > 1, the two drugs are antagonistic [52–54].
Nano‑niosomal CUR/PTX cellular uptake experiments
MCF-7 and MCF-10A cells were seeded at a density of
2 × 105 cells per well in a 6-well plate and incubated for
24h to allow them to attach. e cells were then treated
with the different NioCUR and NioPTX formulations.
After 3 h of incubation, the cells were washed three
times with cold PBS and fixed with a 4% paraformalde-
hyde solution (Sigma, USA). en, the cells were stained
with DAPI (0.125 µg mL−1, ermo Fisher Scientific,
USA) and imaged with a fluorescence microscope (BX61,
Olympus, Japan) [48, 49, 51].
Apoptosis analysis
An annexin V-FITC/PI double staining assay was car-
ried out to confirm whether apoptosis was induced by
curcumin or paclitaxel alone or in combination when
(6)
CI
=
a
A
+
b
B
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Alemi et al. J Nanobiotechnol (2018) 16:28
administered in an aqueous solution and nano-niosome
formulation. e results in Fig.9 show quantitative apop-
totic activity in MCF-7 cells via apoptosis assay using
flow cytometry following the treatment of cells for 24h.
In apoptotic cells, the membrane phospholipid phos-
phatidylserine (PS) is translocated from the inner to the
outer surface of the plasma membrane, thereby expos-
ing PS to the external cellular environment. Annexin V
is a 35–36 kDa Ca2+-dependent phospholipid-binding
protein with high affinity for PS, and it binds to exposed
apoptotic cell-surface PS. Annexin V can be conjugated
to fluorochromes, such as FITC, while retaining its high
affinity for PS, thus serving as a sensitive probe for the
flow cytometric analysis of cells undergoing apoptosis.
Furthermore, propidium iodide (PI) is a fluorescent inter-
calating agent that can be used as a DNA stain in flow
cytometry. PI cannot pass the membrane of live cells
and apoptotic cells; however, it stains dead cells, mak-
ing it useful to differentiate necrotic, apoptotic, healthy,
and dead cells. In the scatter plot of double variable flow
cytometry, the Q4 quadrant (FITC−/PI−) shows liv-
ing cells; the Q2 quadrant (FITC+/PI+) stands for late
apoptotic cells; the Q3 quadrant (FITC+/PI−) represents
early apoptotic cells; and the Q1 quadrant (FITC−/PI+)
shows necrotic cells. e flow cytometry plots demon-
strate there was enhancement in cellular apoptosis in
MCF-7 cells when PTX and CUR were administered in
nano-niosome formulations as compared to free drugs
(p < 0.05). Furthermore, when PTX and CUR were co-
administered in nano-niosome formulations, there was a
significant increase in apoptosis (i.e., 15.27% early apop-
tosis in niosomal curcumin and 31.03% early apoptosis in
niosomal paclitaxel versus 49.79% early apoptosis in nio-
somal curcumin + niosomal paclitaxel, p < 0.05). ese
results are consistent with the growth inhibitory effects
of paclitaxel in combination with curcumin.
Statistical analysis
Statistical data analyses were performed via GraphPad
Prism 6 software and expressed as mean ± SD. A Student
t test was used when comparing two independent groups,
and an ANOVA test was used when comparing multiple
samples. A p value < 0.05 was considered significant.
Abbreviations
PTX: paclitaxel; CUR: curcumin; DDS: drug delivery system; PEG: polyethylene
glycol; EE: entrapment efficiency; Cryo-TEM: cryogenic transmission electron
microscopy; SEM: scanning electron microscopy; FTIR: Fourier transforms
infrared; CI: combination index.
Authors’ contributions
All authors had equal role in design, work, statistical analysis and manuscript
writing. All authors read and approved the final manuscript.
Author details
1 Department of Clinical Biochemistry, Faculty of Medicine, Shahid Sadoughi
University of Medical Sciences, Yazd, Iran. 2 Biotechnology Research Center,
International Campus, Shahid Sadoughi University of Medical Science, Yazd,
Iran. 3 Department of Life Science Engineering, Faculty of New Sciences &
Technologies, University of Tehran, Tehran, Iran. 4 Protein Engineering Labora-
tory, Department of Medical Genetics, School of Medicine, Shahid Sadoughi
University of Medical Sciences, Yazd, Iran. 5 Nutrition and Metabolic Diseases
Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz,
Iran. 6 Student Research Committee, Shiraz University of Medical Sciences,
Shiraz, Iran.
Acknowledgements
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analyzed during this study are included in this article.
Consent for publication
Not applicable.
Ethics approval and consent to participate
The manuscript was approved by the Shahid Sadoughi University of Medical
Sciences Internal Review Board. There are no human subjects or animals
involved in the study.
Funding
This study was financially supported by grant from the Shahid Sadoughi
University of Medical Sciences, Yazd, Iran.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional affiliations.
Received: 13 November 2017 Accepted: 13 March 2018
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