Journal of Molecular Structure 1245 (2021) 131040

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Journal of Molecular Structure

journal homepage: www.elsevier.com/locate/molstr

Salicylic acid-loaded chitosan nanoparticles (SA/CTS NPs) for breast

cancer targeting: Synthesis, characterization and controlled release
kinetics
Mehdi Hassanpour a,b,¥, Hessam Jafari c,¥, Sina Sharifi d, Jafar Rezaie e,∗,
Zohreh Mehri Lighvan f, Gholam Reza Mahdavinia c, Gholamreza Gohari g, Ali Akbari e,∗
a
Department of Clinical Biochemistry and Laboratory Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
b
Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
c
Polymer Research Laboratory, Department of Chemistry, Faculty of Science, University of Maragheh, Maragheh, 55181-83111, Iran
d
Disruptive Technology Laboratory, Massachusetts Eye and Ear and Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School,
Boston, MA, USA
e
Solid Tumor Research Center, Cellular and Molecular Medicine Institute, Urmia University of Medical Sciences, Urmia, Iran.
f
Department of Polymer Processing, Iran Polymer and Petrochemical Institute, P.O. Box 14965-115, Tehran, Iran
g
Department of Horticulture, Faculty of Agriculture, University of Maragheh, Maragheh, Iran

a r t i c l e i n f o a b s t r a c t

Article history: The present study aimed to develop an easy controlled release system for salicylic acid (SA) through
Received 28 April 2021 ionotropic gelation of chitosan (CTS) using tripolyphosphate (TPP) as cross-linker. The synthesized
Revised 6 June 2021
nanoparticles (SA/CTS NPs) were characterized for their physicochemical properties by various techniques
Accepted 3 July 2021
including Fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), scan-
Available online 15 July 2021
ning electron microscopy (SEM) with an energy-dispersive X-ray (EDX) spectroscopy, X-ray photoelectron
Keywords: spectroscopy (XPS) and X-ray diffraction (XRD). A maximum encapsulation efficiency (EE) of 84% was ob-
Biomaterials tained. The obtained material not only was tested as drug delivery system in neutral (pH 7.4) and acidic
Salicylic acid medium (pH 5.5) but also the kinetic release behavior of SA from the CTS NPs was investigated based
Breast cancer on two models namely Korsmeyer-Peppas and Higuchi. These data demonstrated that the drug release
Nanoparticles mechanism governed by Korsmeyer-Peppas model. Finally, the cytotoxic effects of free SA and SA/CTS NPs
Drug delivery
were evaluated in-vitro against breast cancer (MDA-MB-231) cell lines by MTT assay. Results showed that
SA/CTS NPs were more cytotoxic than free SA. This work suggested the potential of SA/CTS NPs system
as an effective anticancer system.
© 2021 Elsevier B.V. All rights reserved.

1. Introduction
Breast cancer is the most leading cancer type in females with
high rate of mortality in almost all countries worldwide [1–3].
Antitumor chemotherapeutics compounds when administrated by
conventional drug delivery ways face several problems such as
non-specificity, high toxicity, and chemoresistance [4]. In the past
two decades, the reduction of bulk particles to the nanoscale
known as nanotechnology [5] has emerged as a promising strategy
in drug delivery system to overcome aforementioned problems [6].
On the other hand, when these nanoscale materials act as nanocar-
riers not only improve the efficiency and bioavailability of anti-
∗
Corresponding author.
E-mail address: akbari.a@umsu.ac.ir (A. Akbari).
¥
These authors worked equally
tumor compounds, but also target the site of tumor specifically
[7,8]. Therefore, alternative and complementary therapies are of-
ten appealing due to the most tumor cells are aggressive and resis-
tant to therapies [9]. Recently, nanoparticle-mediated drug delivery
systems have gained massive attentions for targeting the drug to
the cancer cells through cell signaling pathways. Till now, different
nanoparticles (NPs) such as solid lipid NPs, nanotubes, nanoshells,
nanoballs, nanorods, polymeric NPs, nanopillars and many more
have been reported as efficient and tunable devices in drug de-
livery systems [10–12]. Among them, polysaccharide-based poly-
meric NPs exhibited some advantageous in terms of biocompati-
bility and biodegradability and were used extensively for biomedi-
cal applications [13,14]. Chitosan (CTS) as an abundant natural lin-
ear polysaccharide not only could be extracted from the exoskele-
ton of insects, fungi and arthropods but also may be chemically
synthesized by the deacetylation of chitin [15–17]. The nontoxic,

https://doi.org/10.1016/j.molstruc.2021.131040
0022-2860/© 2021 Elsevier B.V. All rights reserved.
M. Hassanpour, H. Jafari, S. Sharifi et al.
biocompatibility, biodegradation properties of CTS and the exis-
tence of a high density of positive charge in acidic media related
to the amine groups on its backbone lead to increase massive at-
tention to the applications of CTS-based NPs in the biomedical and
pharmaceutical field [18–20]. Typically, CTS NPs are synthesized by
using various cross-linking agents such as polyaspartic acid, glu-
taraldehyde and tripolyphosphate (TPP) [21–23]. It should be high-
lighted that the ionic gelation is the most investigated method and
extensively used for preparing CTS NPs. In this method, cationic
CTS could interact with multivalent polyanions leading to form
CTS NPs under simple and mild conditions [24,25]. Because of its
fast gelling ability and environmentally friendly property, TPP is
the most studied polyanion that used in ionic gelation method
[26]. CTS-TPP NPs have been previously proved to be exceptional
carriers throughout encapsulation of various drugs and nutrients
such as curcumin [27], quercetin [28], tea catechins [29], essen-
tial oil [30] and ascorbic acid [31]. Very recently, we used CTS NPs
in the presence of hydroxypropyl methylcellulose as an efficient
pH-sensitive drug delivery system for melatonin release to MDA-
MB 231 breast cancer cells [32]. Salicylic acid (SA), colorless crys-
talline mono-hydroxybenzoic acid, is an important phenolic com-
pound and typically utilized as plant hormones in agricultural ap-
plications [33]. However, its ability to reduce fevers and pains has
been demonstrated previously [34,35] and the anti-cancer activity
of SA as chemotherapeutic drug was studied by Kutlu and cowork-
ers towards A549 human lung adenocarcinoma cell [36]. In this
study, the simple ionic gelation method was used to prepare CTS
NPs containing desired amount of SA (SA/CTS NPs with EE = 84%).
Both SA release profile and release kinetics from CTS NPs (in neu-
tral and acidic medium) were studied. To our knowledge, the cyto-
toxic effect of SA encapsulated CTS NPs against human breast can-
cer MDA-MB-231 cell line in vitro has not previously investigated
and this study is the first report toward this aim.
2. Experimental
2.1. Materials and instruments
All chemicals such as salicylic acid, chitosan (Molecular weight
of 310–375 kDa, 75–85% deacetylated) and Sodium Tripolyphos-
phate (TPP) were obtained from Sigma-Aldrich (USA). Other chem-
icals used in this project were all of analytical grade and no pu-
rification was applied. Human breast cancer cell line MDA-MB-231
(Pasteur Institute, Iran) were grown in Dulbecco’s modified Ea-
gle’s medium (DMEM, Gibco) containing 10% fetal bovine serum
(DMEM 10% FBS) (Gibco) and 1% penicillin/streptomycin (P/S). Cells
were kept in a humidified incubator of 5% CO2 at 37 °C. To treat
cells with Salicylic acid (SA) and SA loaded Chitosan (SA/CTS NPs),
cells were co-cultured with different concentrations of either SA or
SA/CTS NPs (10,20,40,80,160,320,640 ppm) over a period of 48 h.
Non-treated cells were considered as a control group. Furthermore,
the half maximal inhibitory concentration (IC50) of both SA and
SA/CTS NPs for 48 h of post treatment were analyzed by Graph
pad software ver.8. All experiments were performed in triplicate.
The existence of functional groups of SA/CTS NPs was studied us-
ing Fourier transform infrared (FT-IR) spectra (Bruker 113V FT-IR
spectrometer). Freeze-dried and powdered samples were mixed
with dried KBr (1:99) and the data was collected from 500–
3500 cm−1 wave numbers with 1 cm−1 resolution. For the crys-
tal phase investigation, Powder X-ray diffraction (XRD) pattern of
samples was obtained on a Siemens D-500 X-ray diffractometer
(λ = 1.54 Å (CuKα ), voltage of 35 kV). TEM image of SA/CTS NPs
was recorded with the help of transmission electron microscopy
(TEM; Philips CM10 operating at 60 kV tension). The morphol-
ogy and the elemental composition were evaluated using scanning
2
Journal of Molecular Structure 1245 (2021) 131040
electron microscopy/energy dispersive X-ray instrument (SEM/EDS,
VEGAII, XMU, Czech Republic).
2.2. Synthesis of SA-loaded CTS NPs (SA/CTS NPs)
Briefly, in 50 ml round bottom flask containing acetic acid
(0.5%) in 10 ml distilled water, 0.5 g of CTS and 0.1 g of SA (0.05%)
were dissolved and stirred at 100 rpm overnight. Finally, the pre-
pared stock TPP solution (0.1 g of TPP in 10 ml) was added to
CTS/SA solution slowly. After stirring for 120 min, final solution
was centrifuged at 12,0 0 0 rpm for 60 min. Resulted SA/CTS NPs
were freeze-dried and kept in the sealed vial for the next usages.
Moreover, the supernatant was collected in order to calculate the
amount of un-loaded SA by measuring its absorbance at 297 nm
using spectrophotometer. Finally, standard plot was utilized to cal-
culate the concentration of SA and EE% was obtained using follow-
ing equation:
T otal concent rat ion o f SA − unloaded SA
EE% = × 100
T otal concent rat ion o f SA
2.3. In vitro cytotoxicity assay and Statistical analysis
The cells were cultured in 96 well culture plates (10 0 0 0 cells
/well) and incubated in CO2 incubator. Next day, supernatants were
discarded and replaced with treatment medium. Then, at respec-
tive time point (48 h), 100 μl MTT solution (5 mg/ml) was added
into each well and cells were kept in CO2 incubator in the dark
for 4 h. Further, supernatant was discarded and 100 μl DMSO was
added to dissolve formazan crystals formed by the cells in each
well. The absorbance was read at 570 nm using a microplate reader
(BioTek). The cell viability was expressed as percentage (%) of con-
trol.
The viability of cells was calculated using the following equa-
tion:
V iabilit y(% ) = (ODamount o f t reatedcel l s)/(ODamount o f cont rol cel l s) × 100
In vitro cytotoxic activity was assessed by the calculation of
the inhibitory concentration of either SA or SA/CTS NPs required
to cause 50% of cells death recognized as the half maximal in-
hibitory concentration (IC50). IC50 were calculated by Graph pad
software ver.8. Results were analyzed by SPSS 16.0 software (SPSS
Inc., Chicago, IL) and expressed as mean ± SD from three indepen-
dent experiments carried out in triplicate. One-way Analysis of
Variance (ANOVA) and Tukey post hoc test were used to determine
the significance of differences between groups with significances of
P < 0.05.
3. Results and discussion
3.1. Preparation and characterization of SA/CTS NPs
Fig. 1 illustrates schematic diagrams for preparation of SA/CTS
NPs through ionic gelation method due to opposite charges of CTS
and TPP leading to successful encapsulation of SA. Moreover, the
interaction of TPP and carboxylic acid group of SA with primary
amine of CTS can be clearly seen from Fig 1.
The chemical composition of samples was elucidated by FTIR
analysis in Fig 2A. A strong peak at the 3428 cm−1 corresponds
to N-H and O-H stretching, as well as the intramolecular hydro-
gen bonds. This peak is sharper in the CTS NPs indicating that the
hydrogen bonding is enhanced [37]. The peaks around 2921 cm−1
and 2861 cm−1 can be attributed to C-H symmetric and asymmet-
ric stretching, respectively. Moreover, the presence of residual N-
acetyl groups was confirmed by the peaks at around 1656 cm−1
(C=O stretching of amide I) and 1596 cm−1 (NH2 ) groups, re-
spectively. These peaks shift hypsochromically to 1612 cm−1 and
M. Hassanpour, H. Jafari, S. Sharifi et al.
Fig 1. General procedure for SA/CTS NPs synthesis.
1537 cm−1 in the FTIR spectra of SA/CTS NPs which caused by the
interaction between NH3+ groups of CTS and phosphate groups
of TPP [37, 38]. This interaction is also supported by the de-
crease of amide I (1612 cm−1 ) peak intensity in SA/CTS NPs com-
pared to CTS. The CH2 bending and CH3 symmetrical deforma-
tions were confirmed by the presence of bands at around 1429 and
1373 cm−1 , respectively [39]. In addition, FTIR spectrum of SA/CTS
NPs showed the characteristic peaks of TPP at 1215 cm−1 (stretch-
ing vibration of P=O), 1160 cm−1 (symmetric stretching vibrations
in the O−P=O group), 1080 cm−1 (symmetric stretching vibrations
of the PO3 group) and 888 cm−1 (asymmetric stretching vibration
of the P−O −P bridge) [40, 41].. According to the Fig 2B, the XRD
patterns of CTS exhibited typical broad peak at 2θ of 20°. After en-
capsulation of SA into the CTS NPs, some intense peaks in the XRD
pattern of SA/CTS NPs at 2θ of 11°, 17°, 23° and 28° [42] related to
the SA could be seen in which suggested the crystalline nature of
SA in the synthesized SA/CTS NPs.
Surface morphology and elemental composition of SA/CTS NPs
were investigated using SEM and EDX analysis, respectively. From
Fig 3A, SA/CTS NPs illustrated slightly rough surface with the
spherical particles. Moreover, the existence of C, O, N and P ele-
ments in the EDX analysis indicated successful formation of NPs by
ionic gelation in the presence of TPP and the SA encapsulation in
the CTS NPs structure (Fig 3B). In addition, data obtained from XPS
analysis displayed CTS and TPP as C, O, N and P in NPs (Fig 3C).
Fig 2. (A) FTIR spectra of CTS, SA, SA/CTS NPs and (B) XRD patterns of CTS and SA/CTS NPs.
3
Journal of Molecular Structure 1245 (2021) 131040
TEM image of SA/CTS NPs exhibited spherical shapes with the av-
erage size about 170–180 nm (Fig 3D and 3E).
3.2. In-vitro SA loading/ release studies and kinetic modeling
Standard calibration curve was used to calculate the SA encap-
sulation efficiency (EE %) value on CTS NPs (Fig 4A). The maximum
EE% of SA was found to be 84%. Fig 4B showed the time-dependent
release profiles of the SA/CTS NPs at two different pH (5.5 and 7.4)
at 37 °C. As it could be seen, the release of SA from CTS NPs is pro-
longed and sensitive to the pH of the medium. In both medium,
the initial fast release was obtained in the first 5 h possibly be-
cause of the SA-located at the surface of CTS NPs, and during this
period approximately 18% and 35% of the SA was released from CTS
NPs in pH of 5.5 and 7.4, respectively. After 22 h, nearly, 68% and
31% drug release were observed from the CTS NPs in pH of 5.5 and
7.4, respectively. Obtained results clearly demonstrated that SA/CTS
NPs have remarkably prolonged persistent and higher amount of
release at pH 5.5 than pH 7.4, which made it promising drug de-
livery system for cancer therapy.
Korsmeyer-Peppas and Higuchi kinetic models was applied to
prove and explain the mechanism of SA release from CTS NPs (Eq.
1, Eq. 2, Fig 4C and 4D) [43].
Mt/M0 = KKP t n (Eq. 1)
F = KH × t 1/2 (Eq. 2)
Mt showed the amount of drug release at time t. F is the frac-
tion of drug release at time t. KKP and KH are the rate constants
of Korsmeyer-Peppas and Higuchi kinetic models, respectively. The
data extracted from Korsmeyer-Peppas model such as the kinetic
constant (K) and the characteristic index of the liberation (n) could
provide important information regarding the mechanism of drug
release from CTS NPs. Generally, when n < 0.45, the drug release
mechanism is related to the Fick’s diffusion. When 0.45 < n < 0.89,
the non- Fick’s diffusion mechanism was occurred and the drug
release mechanism is controlled by non- Fick’s diffusion. Finally,
when n ˃ 0.89 the drug release mechanism belongs to the skele-
ton erosion [44].
Fig 4C and 4D showed the linear fit of Korsmeyer-Peppas and
Higuchi kinetic models to in-vitro experimental SA drug release re-
sults, respectively. Moreover, the calculated kinetic parameters for
M. Hassanpour, H. Jafari, S. Sharifi et al. Journal of Molecular Structure 1245 (2021) 131040

Fig 3. (A, B and C) SEM-EDX and XPS analysis of SA/CTS NPs, (D and E) TEM image and size distribution of SA/CTS NPs.

Fig 4. (A and B) standard calibration curve and SA release profiles in two different pH of 5.5 and 7.4. (C and D) Kinetic models for SA release from CTS NPs for varying pH
conditions: (C) Korsmeyer-Peppas model and (D) Higuchi model.

4
M. Hassanpour, H. Jafari, S. Sharifi et al.
Fig 5. In-vitro cell viability of MDA-MB-231 cell after incubation with (A) free SA and (B) SA/CTS NPs for 48h in different concentrations.
Table 1
The calculated kinetic parameters of salicylic acid release according to Higuchi
and Korsmeyer-Peppas models.
Models Higuchi model Korsmeyer-Peppas model
k r2 n k
SA/CTS NPs pH = 5.5 0.850 0.95 0.467 1.043
pH = 7.4 1.443 0.93 0.641 0.623
studied models were summarized in Table 1. Based on the corre-
lation coefficients, r2 , the release of SA from SA/CTS NPs in pH 5.5
and 7.4 seems to follow Korsmeyer-Peppas model. Based on the
data from Table 1, the value of ‘n’ is 0.467 and 0.641 at pH of 5.5
and 7.4, respectively which revealed the non-Fickian transport of
SA from CTs NPs to in vitro solutions.
3.3. Cellular cytotoxicity studies using MTT assay
After successful preparation and characterization of SA/CTS NPs,
the ability of the SA/CTS NPs to release SA and induce cell cytotox-
icity was investigated in MDA-MB-231 breast cancer cell line by
MTT assay (Fig 5 A and 5B). The results showed that both free SA
and SA/CTS NPs had a dose-dependent toxicity toward the viabil-
ity of MDA-MB-231 breast cancer cell after 48 h incubation. Fur-
thermore, our results demonstrated that IC50 value for free SA and
SA/CTS NPs was 513 ppm and 445 ppm, respectively. Furthermore,
cell viability for drug-loaded CTS NPs is relatively lower than free
SA. These results show that CTS NPs can facilitate the delivery of
SA into the cancer cells with good efficiency leading to high cyto-
toxic effects towards MDA-MB-231 cells in comparison to free SA
over a period of 48 h.
4. Conclusion
Today, design and fabrication of smart drug delivery systems
are known as one of the most promising objects. Chitosan (CTS)
nanoparticles have been extensively investigated for the encapsu-
lation of numerous compounds such as drugs and nutrients. In
this work, salicylic acid (SA) has been effectively loaded into CTS
NPs through simple ionic gelation method in the presence of TPP
(SA/CTS NPs) and characterized using various techniques. Optical
microscopy observations showed that the synthesized SA/CTS NPs
had spherical shapes with the average size about 170–180 nm.
The sustained release of SA from CTS NPs was investigated in pH
5.5 and 7.4. Obtained results showed that the maximum cumula-
tive SA release rate was 68% and 31% in pH 5.5 and pH 7.4, re-
spectively. The kinetic release mechanism governed by Korsmeyer-
Journal of Molecular Structure 1245 (2021) 131040
Peppas model. For the first time, the toxicity effects of synthe-
sized sample were evaluated towards MDA-MB-231 breast cancer
cell line by MTT assay. The results of this investigation show the
potential of SA encapsulated CTS NPs as anti-cancer drug delivery
r2
system towards various human breast cancer cell lines.
0.99
0.98
Declaration of Competing Interest
The authors report no declaration of interest.
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