The role of the lecithin addition in the properties and cytotoxic activity of chitosan and chondroitin sulfate nanoparticles containing curcumin

Carbohydrate Polymers 227 (2020) 115351

Contents lists available at ScienceDirect

Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

The role of the lecithin addition in the properties and cytotoxic activity of
chitosan and chondroitin sulfate nanoparticles containing curcumin

T

Katiúscia Vieira Jardima, Joseilma Luciana Neves Siqueirab, Sônia Nair Báob,
⁎
Marcelo Henrique Sousaa, Alexandre Luis Parizec,
a

Green Nanotechnology Group, Universidade de Brasília, Brasília, DF 72220-900, Brazil
Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, CampusUniversitário Darcy Ribeiro – Asa Norte, Brasília, DF 70910-900,
Brazil
c
Polimat, Grupo de Estudos em Materiais Poliméricos, Departamento de Qmica, Universidade Federal de Santa Catarina, Florianópolis, SC 88040-900, Brazil
b

A R T I C LE I N FO

A B S T R A C T

Keywords:
Chitosan
Chondroitin sulfate

Lecithin
Curcumin
Polymeric nanoparticles
Drug delivery

Surfactants have been used as a tool to improve the properties of polymeric nanoparticles (NPs) and to increase
the rate of hydrophobic drug release by means of these nanoparticles. In this context, this study evaluated the
effect of lecithin on the characteristics of chitosan (CHI) and chondroitin sulfate (CS) nanoparticles, when applied in curcumin (Curc) release. CHI/CS NPs and CHI/CS/Lecithin NPs were prepared by the ionic gelation
method, both as standards and containing curcumin. Simultaneous conductimetric and potentiometric titrations
were employed to optimize the interaction between the polymers. NPs with hydrodynamic diameter of ∼130 nm
and zeta potential of +60 mV were obtained and characterized by HRTEM; their pore size and surface area were
also analyzed by BET method, DLS, FTIR, XPS, and fluorescence spectroscopy techniques to assess morphological
and surface properties, stability and interaction between polymers and to quantify the loading of drugs. The final
characteristics of NPs were directly influenced by lecithin addition, exhibiting enhanced encapsulation efficiency
of curcumin (131.8 μg curcumin per mg CHI/CS/Lecithin/Curc NPs). The release of curcumin occurred gradually
through a two-stage process: diffusion-controlled dissolution and release of curcumin controlled by dissolution of
the polymer. However, the release of curcumin in buffer solution at pH 7.4 was achieved faster in CHI/CS/
Lecithin/Curc NPs than in CHI/CS/Curc NPs. in vitro cytotoxic activity evaluation of the curcumin was determined by the MTT assay, observing that free curcumin and curcumin nanoencapsulated in CHI/CS/Curc and
CHI/CS/Lecithin/Curc NPs reduced the viability of MCF-7 cells in the 72 h period (by 28.4, 36.0 and 30.7%,
P < 0.0001, respectively). These results indicate that CHI/CS/Lecithin NPs have more appropriate characteristics for encapsulation of curcumin.

1. Introduction
Curcumin (1,7bis (4-hydroxy-3-methoxyphenyl)-1,6-heptadiene3,5-dione) is a yellow solid, classified according to the
Biopharmaceutics Classification System (BCS) as a class II drug that is
poorly water-soluble but highly permeable. It is a phenolic compound
that has methoxy and phenol groups in its chemical composition, which
are responsible for its biological and pharmacological properties (Dai
et al., 2018). Its potential in the prevention and treatment of various
diseases, including cancer, has been extensively investigated in recent
years, since it has antiproliferative and pro-apoptotic effects against

several types of tumors, contributing mainly to the inhibition of tumor
growth (Calaf, Ponce-Cusi, & Carrión, 2018). Recently, Siddiqui et al.

⁎

(2018) showed that curcumin decreases the Warburg effect on several
cancer cells (H1299, MCF-7, HeLa and PC3). Similarly, DavatgaranTaghipour et al. (2017) presented experimental evidence and clinical
perspectives that polyphenols such as curcumin are potentially capable
of acting as chemopreventive and chemotherapeutic agents in different
types of cancer. In addition, the authors report that nanoformulations of
natural polyphenols as bioactive agents, including resveratrol, curcumin, quercetin, epigallocatechin-3-gallate, chrysin, baicalin, luteolin,
honokiol, silibinin and coumarin derivatives, in a dose-dependent
manner, result in prevention and treatment of cancer. However, Nelson,
Dahlin, Bisson, Graham, Pauli & Walters (2017) reported in their study
that although the activity and therapeutic utility of curcumin have increased the interest of scientists, no evidence of the therapeutic benefits

Corresponding author.
E-mail address: (A.L. Parize).

/>Received 11 February 2019; Received in revised form 4 July 2019; Accepted 18 September 2019
Available online 21 September 2019
0144-8617/ © 2019 Elsevier Ltd. All rights reserved.


Carbohydrate Polymers 227 (2020) 115351

K.V. Jardim, et al.

and enhanced efficiency in the encapsulation of drugs (Shin, Chung,
Kim, Joung, & Park, 2013; Yang, Dai, Sun, & Gao, 2018; Tsai, Chiu, Lin,

Chen, Huang, & Wang, 2011).
In this context, the objective of this study was to evaluate the effect
of adding lecithin on the characteristics of chitosan (CHI) and chondroitin sulfate (CS) nanoparticles, prepared by the ionic gelation
method, used for the controlled in vitro release of curcumin and to
improve its cytotoxic activity in human breast tumor cells (MCF-7).

of curcumin has been found. The authors claim that curcumin has
disadvantages as a candidate in the clinical setting, since it has low
solubility in aqueous solutions, high decomposition rate in neutral or
basic pH and susceptibility to photochemical degradation, which is also
reported in other studies (Chuah, Roberts, Billa, Abdullah, & Rosli,
2014; Lim et al., 2018). However, considering these controversies regarding the therapeutic efficacy of curcumin, Heger (2017) suggests
that the thousands of research papers and more than 120 clinical trials
performed with curcumin should not be discarded; it is particularly
worth further investigating its potential as a therapeutic agent. In this
context, several strategies have been evaluated to increase the biological activity of curcumin, mainly aiming for greater absorption and
availability to tissues (Akbar et al., 2018).
Several methods are described in the literature for improving the
solubility of curcumin, such as: impregnation (Parize et al., 2009), liposomes (Li et al., 2018), copolymers and nanoemulsions (Akbar et al.,
2018; Dai et al., 2018), chemical modifications in curcumin structure
(Mohamed, El-Shishtawy, Al-Bar, & Al-Najada, 2017), and association
in polymer nanoparticles (Jardim, Joanitti, Azevedo, & Parize, 2015),
among others. Polymeric nanoparticles appear as easy-to-prepare systems and increase the effectiveness of the treatment, due to the increased solubility and increased effectiveness of the drug. Recently,
nanoparticles formed from chitosan and chondroitin sulfate through
ionic polyelectrolytic complexation have been reported as a promising
alternative for the encapsulation and release of hydrophobic drugs,
such as curcumin, since it is a simple and reversible process (Umerska,
Corrigan, & Tajber, 2017). In addition, NPs formed through ionic crosslinking have the ability to protect the active substance against degradation and increase its bioavailability in a physiological environment (Tsai, Chen, Bai, & Chen, 2011). Chitosan can also be associated
with biocompatible surfactants, such as lecithin, which promotes improvements in the properties of the polymer network that is formed, as
well as an increase in the incorporation rate of the drug (Dammak &

Sobral, 2018; Şenyiğit et al., 2017; Terrón-Mejía et al., 2018).
Chitosan is a natural biopolymer obtained from the reaction of Ndeacetylation of chitin in alkaline medium. It is represented as a copolymer of 2-amine-2-deoxy-D-glucose and 2-acetamide-2-deoxy-Dglucose, linked by β-type glycosidic bonds (1,4). It has a wide range of
applications because it is biodegradable, biocompatible, bioadhesive
and non-toxic (Biswas, Chattopadhyay, Sen, & Saha, 2015; TerrónMejía et al., 2018). When dissolved in aqueous acid solutions, pH <
6.2 has a positive charge in the −NH3+ groups, which facilitates their
solvation in water and aggregation to polyanionic compounds, such as
chondroitin sulfate, forming polyelectrolyte complexes (PECs) (Şenyiğit
et al., 2017; Terrón-Mejía et al., 2018).
Chondroitin sulfate belongs to the family of glycosaminaglans
(GAGs), and it is characterized as an alternating copolymer of the
monomers β(1,4)-D-glucuronic acid and β(1,3)-N-acetyl-D- galactosamine, which may be sulfated at the C4 or C6 carbons. It has low toxicity,
biocompatibility and specific biodegradability (Krichen et al., 2018). In
addition, it can form polyelectrolyte complexes (PECs) through electrostatic interaction with positively charged substances, thus providing
an optimal strategy to maintain CS in the solid state for use as a drug
delivery system (Jardim et al., 2015; Gul et al., 2018; Tan, Selig, &
Abbaspourrad, 2018).
Nanoparticles based on PECs formed by biopolymers do not often
promote adequate dispersion, solubilization and bioavailability of
drugs such as curcumin. Thus, to promote greater stability and efficiency in the encapsulation of poorly soluble drugs, a coating with
biocompatible compounds, such as lecithin, is required (Sun et al.,
2015). Lecithin is a highly bioactive compound that consists of a glycerol backbone esterified with two fatty acids and a phosphate group,
endowing it with strong potential for use in the food and pharmaceutical industries as an emulsifier, nutrition enhancer and carrier (Pawar
& Babu, 2014). The nanoparticles prepared with lecithin and chitosan
showed higher bioavailability, mucoadhesive property, storage stability

2. Materials and methods
Chitosan (99% purity) (medium molecular weight) with a molecular
mass around 106 kg mol−1 and deacetylation degree ∼76.9% was
determined by conductimetric titration (Alvarenga, 2011). Chondroitin
4-sulfate sodium salt (99% purity), originating from bovine trachea and

curcumin (95% purity), originating from Curcuma Longa L., with purity
of 98%, were obtained from Sigma-Aldrich (St. Louis, MO, USA). Lecithin obtained from egg yolk (60% of L-α-Phosphatidylcholine – Sigma
Aldrich. Dulbecco's Modified Eagle Medium (DMEM) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) were obtained
from Life Technologies (USA), and the line of human breast tumor cells
(MCF-7) was obtained from the cell bank of Rio de Janeiro (BCRJ),
Brazil. The other reagents were of analytical grade and were used
without prior purification.
2.1. Sample elaboration
The logarithmic values of the dissociation constant (pKa = −logKa)
were determined for pure CHI and CS samples, by means of simultaneous potentiometric and conductimetric titrations as described by
Farris, Mora, Capretti, and Piergiovanni (2012), to optimize conditions
of interaction between the polymers during synthesis of NPs. In this
way, 50 mL of the CHI or CS solutions (0.1 wt%) were titrated with a
0.1 mol.L−1 HCl or NaOH solution, using a Metrohm 856 Conductivity
Module with a 5-ring conductivity measuring cell (c =0.7 cm−1 with
Pt1000) and a Metrohm 827 pH lab pHmeter. Before titration, the pH of
the CS and CHI solution were respectively adjusted to ∼2.0 and ∼7.0,
using HCl and NaOH solutions. Based on the pKa values, the speciation
diagram of the mole fractions of the surface sites as a function of pH
was constructed, thus establishing the ideal pH (5.5) for obtaining the
nanoparticles.
The synthesis of CHI/CS and CHI/CS/Lecithin NPs was performed
by the ionic gelation method (Fan, Yan, Xu, & Ni, 2012). For this,
homogeneous solutions of CHI (1.0 mg/mL) and CS (1.0 mg/mL) were
prepared in 0.1 mol.L−1 acetic acid solution and pH adjusted to ∼5.5.
To obtain the CHI/CS NPs, 150 mL of CS solution was added slowly to
100 mL of CHI solution. The CHI/CS/Lecithin NPs were obtained with
the addition of 5.0 mL of 3.5% (w/v) of lecithin ethanol solution to
100 mL of CHI solution, and then 150 mL of the chondroitin sulfate
solution was added slowly. The lecithin-chitosan ratio was set at 1:20

(w/w) in the nanoparticles. The formation of the NPs was conducted
under constant magnetic stirring for 40 min at 25 °C.
The encapsulation of curcumin into the CHI/CS NPs was carried out
by adding an ethanolic solution of curcumin to the CHI solution prior to
the interaction with the CS solution. The following synthetic methodology is the same as that described in the previous paragraph in relation to the production of the CHI/CS NPs based on a previous study by
Jardim et al. (2015). A small amount of curcumin (∼30 mg) was added,
and stabilized in 100 mL of the CHI solution, which was maintained at
pH 5.5. After the curcumin had come into contact with the chitosan
solutions, the 150 mL of CS solution at pH 5.5 was added slowly to the
solution containing CHI/Curc. The solutions were maintained under
constant magnetic stirring for 40 min at 25 °C, leading to the formation
of CHI/CS/Curc NPs. The incorporation of curcumin in the CHI/CS/
Lecithin NPs occurred prior to the addition of 5.0 mL of 3.5% (w/v) of
lecithin ethanol solution to 100 mL of CHI solution at pH 5.5 and then
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K.V. Jardim, et al.

were excited at 429 nm, and the emission spectra were recorded from
440 to 700 nm. The relative fluorescence intensities were measured at λ
= 540 nm and compared to a standard calibration curve. To construct
the calibration curve, a stock solution of 108 μmol.L−1 curcumin was
prepared in a 80:20 (v/v) mixture of 0.1 mol.L−1 solution of phosphate
buffer solution (pH 7.4):ethanol. From the stock solutions, the working
solutions were prepared at concentrations of 0.5 to 15 μmol.L−1 by
dilutions of the stock solution in the phosphate buffer solution (pH
7.4):ethanol solution. Each sample was assayed in triplicate, and the

results were expressed as the amount of curcumin (in μg) per mg of
nanoparticles. Similarly, the encapsulation efficiency (EE%) was calculated as the ratio between the amount of drug entrapped in the nanoparticles and the initial amount of curcumin used to prepare the
nanoparticle batch.

was added to curcumin (˜ 30 mg) and 150 mL of CS solution at pH 5.5.
The solutions were also maintained under constant magnetic stirring for
about 40 min at 25 °C, leading to the formation of CHI/CS/Lecithin/
Curc NPs.
2.2. Sample characterization
The morphology and size of the prepared NPs was evaluated by
high-resolution transmission electron microscopy (HRTEM) using a
JEOL JEM-2100 microscope equipped with EDS, Thermo scientific. For
HRTEM analysis, the colloidal suspensions obtained were diluted in
water in a ratio of 50 μL of the colloidal suspension of NPs to 100 μL of
Type 1 water. A small aliquot of the resulting sample dilution (3 μL) was
placed on a copper screen (400 mesh), covered with a carbon film and,
before the measurements were taken, a solution of phosphotungstic
acid (2.0% w/v) was applied to provide contrast for better visualization
of NPs under the microscope.
Dynamic light scattering (DLS) (Nano-Zetasizer-ZS, Malvern
Instruments) was used to determine the hydrodynamic diameter, the
polydispersity index (PDI) and the zeta potential of the NPs as a function of pH. For DLS analysis, the NPs were dispersed in water (about
0.01 wt%), sonicated for 10 min and pH adjusted with 0.1 mol.L−1 of
the HCl or NaOH solutions.
The surface area and pore volume distribution of the NPs were
measured using a mass of 0.2 g sample at 77 K in AUTOSORB-1
equipment with a free space of about 16 cm³, cold free space around 48
cm³, equilibrium interval of 10 s, no low pressure dose and automatic
degassing.
The stability of the nanoparticles was evaluated by monitoring the

hydrodynamic diameter and the zeta potential, using Dynamic light
scattering (DLS) equipment (Nano-Zetasizer-ZS, Malvern Instruments).
The samples were kept at 37 °C in the form of aqueous suspension, and
the measurements were performed in triplicate, using a diluted solution
(0.01 wt%) of the samples, during the period of 90 days. The interpretation of data was performed by cumulative analysis of the experimental correlation function, and hydrodynamic radius was calculated
from the computed diffusion coefficients using the Stokes–Einstein
equation (Eq. 2)

kT
Rh =
6πηDt

2.4. Curcumin release profile
The kinetics of curcumin release was performed by adapting the
methodology described by Parize et al. (2009). For this purpose, approximately 30 mg of curcumin-containing CHI/CS/Curc and CHI/CS/
Lecithin/Curc NPs were suspended in 30 mL of 0.1 mol.L−1 phosphate
buffer solution at pH 7.4. The samples were kept under constant stirring
(700 rpm) in a thermostat-controlled bath at 37.0 ± 0.1 °C. The analysis was conducted for 240 h by means of measurements at predetermined time intervals, where a 2 mL aliquot of the supernatant was
analyzed on a Fluorolog-TSPC (Horiba-Jovine Ivone) fluorimeter with
both slits of excitation, and emission monochromators were adjusted to
5.0 nm. The samples were excited at 429 nm, and the emission spectra
were recorded from 440 to 700 nm. The amount of curcumin released
was determined using the Curc calibration curves, which correlate the
fluorescence intensity with the known concentration of curcumin
(μmol/L) in the same solution where release kinetics was conducted.
The results obtained were presented as percentage of release of curcumin over time. The release mechanism was analyzed by adjusting the
release kinetics profiles, applying the Gallagher-Corrigan mathematical
model (Gallagher & Corrigan, 2000).
2.5. Biological tests
2.5.1. Evaluation of in vitro cytotoxic activity

For the cell culture, the human breast epithelial adenocarcinoma
cell line (MCF-7) was routinely maintained in cell culture flasks
(75 cm2) in an incubator (37 °C, 5% CO2 and 98% humidity) with 15 mL
of DMEM cell culture medium supplemented with 10% (v/v) fetal bovine serum (FBS, Life Technologies, USA) and 1% (v/v) antibiotic solution (100 U/mL penicillin – 100 μg/mL streptomycin, Life
Technologies, USA) and passaged every 3 or 4 days. For the passage or
preparation of the experiments, the cells were desalted with 1 mL of
0.25% Trypsin-EDTA (Life Technologies, USA) and then inactivated
with 5 mL DMEM supplemented with 10% FBS and 1% antibiotic solution.
Cell viability was determined by the 3,4,5-dimethylthiazol-2,5-biphenyl tetrazolium bromide (MTT) assay, where MCF-7 cells (5 × 103
cells/well in 200 μL of DMEM) were seeded on 96-well plates and allowed to attach overnight. Cells were then incubated with 200 μL/well
of DMEM containing different concentrations (10 to 40 μmol.L−1) of
CHI/CS and CHI/CS/Lecithin NPs (with or without curcumin) and free
curcumin at 37 °C (5% CO2). The pH of the samples was adjusted to 7.4
with NaOH (1.0 mol.L−1) before being seeded with the cells. After 24,
48 and 72 h of incubation, the treatment was withdrawn and then
150 μL of MTT solution (0.5 mg/mL in DMEM) was added in each well,
and incubation was carried out for 3 h at 37 °C (5% CO2). The culture
medium was then aspirated, and 200 μL of dimethyl sulfoxide was
added to dissolve the purple formazan crystals from viable cells. The
absorbance was determined using a spectrophotometer with a microplate reader at a wavelength of 595 nm (SpectraMax®, model M2,

(1)

where Dt is the diffusion coefficient, k is the Boltzmann constant, T is
the absolute temperature, η is the dynamic viscosity and Rh is the
particle diameter.
The FTIR spectra were recorded with KBr pellets in the region of
4000–400 cm−1 on a Varian FTIR spectrophotometer with a resolution
of 2 cm−1.
By means of X-ray photoelectron spectrometry (XPS), strains were

acquired in a SPECS SAGE HR 100 system spectrometer, with energy of
30 eV and 15 eV for analysis of the regions and of 285 eV for calibration
of the binding energies of the peak C 1s. The atomic percentage of the
elements present on the surface of the NPs under study and their possible interactions were determined.
2.3. Determination of curcumin loading
In the quantification of curcumin, performed by the filtration/centrifugation technique (Sun, Me, Tian, & Liu, 2007), the free active was
determined in the supernatant and the total active was measured after
the complete dissolution of the samples. To obtain the supernatant,
10 mg of nanoparticle-based samples were dispersed in 10 mL of an
80:20 (v/v) mixture of 0.1 mol.L−1 phosphate buffer solution (pH
7.4):ethanol. After filtering through 0.2 μm PTFE filter, an aliquot of the
supernatant was transferred to a quartz cell and analyzed in a Fluorolog-TSPC (Horiba-Jovine Ivone) fluorimeter. Both slits of excitation
and emission monochromators were adjusted to 5.0 nm. The samples
3


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K.V. Jardim, et al.

deprotonation of the −SO3H and −COOH groups, where the third zone
is initiated. In the third zone, there was a sharp increase in the conductivity due to the excess of OH− ions in the dispersion. For CS,
pKa1 = 2.5 (−SO3H) and pKa2 = 4.0 (−COOH) were estimated from
titration data as described by Campos, Tourinho, Da Silva, Lara, and
Depeyrot (2001).
Thus, taking into account the pKa values obtained in the titrations,
the molar fraction of polymer surface species was plotted against the
pH, as shown in Fig. 1C. In these speciation curves, it was verified that
CS presents three distinct superficial sites. In an extremely acidic
medium, protonated sulfonic and carboxylic groups (CS-SO3H−COOH)

are observed. With the progressive increase of pH, sequential deprotonation of the sulfonic and carboxylic groups occurs. Thus, the surface
of CS becomes negatively charged (−SO3− and −COO−), allowing
complexation with positively charged species in neutral and alkaline
pHs (Rodrigues, Cardoso, Da Costa, & Grenha, 2015). However, at these
pHs the CHI remains deprotonated, as shown in speciation curves. On
the other hand, in an acidic medium the CHI is positively charged,
because of the protonation of −NH2 groups (CHI−NH3+). Considering
these speciation diagrams, at a pH halfway between the pKa of CHI and
pK2 of CS (pH ∼5.5) most superficial sites of CHI will be positively
charged and CS will be negatively charged. Thus, the optimized interaction between CHI and CS can occur (Menegucci, Santos, Dias, Chaker,
& Sousa, 2015).
Also in Fig. 1C, we observe the dependence of the zeta potential as a
function of pH variation (2.0–12.0) of the CHI/CS NP dispersions. From
this curve, it was found that at pH < 7.0 zeta potential becomes positive and increases as pH decreases. This is associated with the protonation of amine groups of CHI. Above pH ∼7.0, an increasingly negative zeta potential was observed and associated with the
deprotonation of −SO3H and −COOH groups. This change in the zeta
potential of the NPs confirms that the polymers are pH-responsive, i.e.,
the degree of ionization is significantly altered by virtue of a variation
in the pH near the pKa value of their functional groups (Ganta,
Devalapally, Shahiwala, & Amiji, 2008).
This speciation study improved the ionic gelation method, favoring
the formation of NPs with a narrow distribution and hydrodynamic
diameter in the range of nanometers, as shown in Table 1. Besides, the
addition of lecithin resulted in the reduction of the hydrodynamic
diameter and the PDI of the NPs (Table 1). The reduction in the hydrodynamic diameter of the nanoparticles after addition of lecithin may
be related to the contribution of the attractive hydrophobic and electrostatic interactions that occur between the polymer and the surfactant. This effect may be associated with the less effective overlap of
electrostatic potentials around the polymer chain (Khan & Brettmann,
2019). In addition, the presence of the surfactant may cause changes in
the behavior of the polymer in solution, such as surfactant-induced
thickening, surfactant-induced swelling or compaction, surfactant-induced phase separation, among other effects (Silva, Antunes, Sousa,
Valente, & Pais, 2011). Banik, Hussain, Ramteke, Sharma, and Maji

(2012) also suggest that the surfactant may decrease the solubility of
chitosan, favoring the formation of small particles. On the other hand,
an increase in the zeta potential of lecithin-containing NPs was observed due to the amine headgroup of choline present in the surfactant
structure, which increases the positive charge density on the surface of
NPs (Cheng, Oh, Wang, Raghavan, & Tung, 2014).
The positive zeta potential with a relatively high modulus value (at
pH ∼5.5) efficiently promoted curcumin encapsulation (Table 1).
However, it was also observed that the amount of encapsulated curcumin was higher in CHI/CS/Lecithin/Curc NPs (131.8 μg/mg) than in
CHI/CS/Curc NPs (118.4 μg/mg). This occurs because the negatively
charged polar portion (phosphate groups) of the lecithin interacts with
the −NH3+ groups of CHI, leading to the preliminary formation of a
vesicle. The self-assembling of phospholipid with the polymer leaves
the fatty-acid positively-charged polar chains available to interact with
the hydrophobic part of curcumin. (Taner et al., 2014). In addition, the

Molecular Devices, USA). This absorbance reading is directly proportional to the number of live cells in the culture. The percentage of cell
viability was presented as the percentage of the absorbance of treated
cells to the absorbance of non-treated cells (100% × (absorbance of
treated cells / absorbance of non-treated).
2.5.2. Internalization study of samples in MCF-7 cells
For the analysis of sample internalization, 1 × 106 MCF-7 cells/well
were seeded on 6-well culture plates and, after adhesion, the cells were
exposed to 40 μmol.L−1 free curcumin, CHI/CS/Curc NPs and CHI/CS/
Lecithin/Curc NPs for 24 h. The desalted cells in microtubes were then
washed with PBS and fixed with Karnovsky's solution (2.0% glutaraldehyde, 2.0% paraformaldehyde, 5.0 mmol CaCl2, 3.0% sucrose buffered in 0.1 mol.L−1 sodium cacodylate, at pH 7.2) for 2 h at 4 °C. After
fixation, the cells were washed in the same buffer and post-fixed for
30 min in 1% osmium tetroxide, 0.8% potassium ferricyanide and
5.0 mmol CaCl2 in 0.1 mol.L−1 sodium cacodylate buffer. Cells were
washed twice with ultrapure water and then stained with 0.5% uranyl
acetate overnight at 4 °C. The samples were washed twice with ultrapure water and dehydrated in increasing acetone gradient (50–100%)

for 10 min each and included in Spurr resin. The ultrafine sections were
obtained with an ultra-microtome (Leica, UCT, AG, Vienna, Austria)
and analyzed in a JEOL JEM-2100 transmission electron microscope
equipped with EDS, Thermo scientific.
2.5.3. Statistical analysis
All results are from three independent experiments and expressed as
the mean ± SD. The difference between the effect of the treated
compound compared with the control values was verified by analysis of
variance (ANOVA) and Tukey's post hoc test using the program
GraphPad Prism® 5.0. The values that were significantly different from
the control at P < 0.05 are indicated in the Figures by an asterisk.
3. Results and discussion
Chitosan and chondroitin sulfate are polyfunctional polymers (with
−NH2 and −SO3H and −COOH groups, respectively) that can be ionized in aqueous medium, where chitosan is represented by CHI-NH3+/
CHI-NH2
and
chondroitin
sulfate
as
CS−COOH−SO3H/
CS−COO−−SO3−
in
their
protonated/deprotonated
forms.
Considering that ionic crosslinking is based on the attractive electrostatic interaction between the CHI and CS polymers, it will be governed
by the number of ionizable surface sites of the polymers, which depends
directly on the pH of the dispersions (Maldonado, Terán, & Guzmán,
2012). Thus, before the preparation of the nanoparticles, the speciation
profiles were constructed in order to determine the ideal pH to optimize

the attractive interaction between the surface of the CHI and the CS
polymers, by means of their pKa values obtained by simultaneous potentiometric and conductometric titrations (Fig. 1).
In the CHI titration (Fig. 1A) two distinct zones were observed. In
zone 1, before adding the titrant (HCl), the pH of the CHI dispersion is
∼7.2 and the −NH2 groups are expected to be substantially deprotonated/discharged. As the titrant is added, the protonation of NH2
groups occurs and CHI becomes positively charged. Conductivity of CHI
solution, low before adding the titrant, sharply increases at the
equivalence point (pH 4.7) as an excess of titrant is added (zone 2)
(Farris et al., 2012). Cross-linking conductometric and potentiometric
data, a pKa = 6.3 was estimated for CHI. In the titration curves of the
CS (Fig. 1B), three zones were observed. In the first zone, as the titrant
(NaOH) is added, a reduction in the conductivity is due to the neutralization of the excess of H3O+ ions (from HCl, used to adjust the
initial pH), reaching the first point of equivalence (pH ∼1.9). In the
second zone, the variation of conductivity is due to the deprotonation of
the sulfonic and carboxylic groups (i.e. [H3O+] increasing) and [Na+]
variation, from the titrant (Scordilis-Kelley & Osteryoung, 1996). Thus,
the second equivalence point (pH ∼7.4) was reached after the complete
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K.V. Jardim, et al.

Fig. 1. Potentiometric (●) and conductometric (○) titration: CHI (A) and CS (B). Speciation diagram of the surface of CHI ( CHI-NH3+ and CHI-NH2) and CS (
CS-SO3H−COOH, CS-SO3−−COOH and CS-SO3−−COO−) (C). Representation of the chemical interactions between CHI and CS in obtaining the NPs by ionic
gelation.

interfacial tension, resulting in the increase of the surface area and
consequently the reduction of the size of the nanoparticles (Wen, Wen,

Wei, Zushun, & Hong, 2012). This indicates that the selective cavities of
NPs were more exposed after the addition of lecithin, allowing a greater
encapsulation of curcumin. In addition, it was verified that the increase
in surface area is directly proportional to the increase in the mean
volume of pores; however, there is an inverse correlation between the
surface area, the hydrodynamic and pore diameters in all NPs
(Marturano et al., 2017). In the NPs containing curcumin, it was observed that the values of surface area and mean volume of pores were
relatively lower, when compared to the values obtained for NPs without
curcumin, indicating the encapsulation of the drug, which causes
changes in the characteristics of NPs (Silva, Fideles, & Fook, 2015).
In prolonged periods of storage, NPs of this kind of material tend to
form agglomerates (Kumari, Yadav, & Yadav, 2010). It is therefore a
great challenge to produce systems that keep their colloidal stability for

use of the lecithin in the internal phase increased the wettability of the
solid particles of the drug, facilitating its adsorption on the nanoparticles (Chen et al., 2016). After encapsulation of the curcumin, an
increase in the hydrodynamic diameter, the PDI and the zeta potential
of the NPs was observed, as shown in Table 1. This indicates the success
in the encapsulation of the drug, which causes changes in the characteristics of the NPs (Marturano, Cerruti, Giamberini, Tylkowski, &
Ambrogi, 2017).
From the adsorption/desorption isotherms of N2 obtained by BET
(data not shown) the presence of micropores (pore diameter < 20 Å) in
the NPs was confirmed (Sing et al., 1985). The formation of the micropores may be related to the cross-linked bonds established between
the biopolymers during the synthesis process (Lu, Le, Zhang, Huang, &
Chen, 2017). From the BET results (Table 1) it was also observed that
an increase occurred in the surface area of the NPs after addition of
lecithin surfactant. The presence of the surfactant reduces the

Table 1
Values for the size, PDI and zeta potential of the CHI/CS and CHI/CS/Lecithin and encapsulation efficiency (EE%) of curcumin.

Samples

CHI/CS
CHI/CS/Lecithin
CHI/CS/Curc
CHI/CS/Lecithin/
Curc
a

Size (nm)a

111.7
102.2
154.6
126.2

±
±
±
±

1,8
1,5
1.3
1.6

PDIa

0.299
0.224

0.391
0.339

Zeta potential
(mV)a

±
±
±
±

0.03
0.02
0.02
0.02

58.2
60.7
59.7
60.4

±
±
±
±

1.3
1.1
1.1
1.2

BET analyses

Curcumin Loading

Surface area
(m2/g)

Pore volume
(cm3/g)

Pore diameter
(Å)

Amount of curcumin
encapsulated (μg/mg)a

Encapsulation efficiency
(%EE)a

28.9
33.8
19.5
23.6

0.043
0.054
0.029
0.035

13.1
11.5
17.6
15.4

–
–
118.4 ± 0.1
131.8 ± 0.2

–
–
78.6 ± 0.4%
87.5 ± 0.5%

Mean ± SD, n = 3.
5


Carbohydrate Polymers 227 (2020) 115351

K.V. Jardim, et al.

Fig. 2. Stability of the NPs at 37 °C for the
period of 90 days in function of the hydrodynamic diameter (A) and Zeta potential (B)
for: (●) CHI/CS NPs; ( ) CHI/CS/Lecithin NPs;
( ) CHI/CS/Curc NPs and ( ) CHI/CS/Lecithin/
Curc NPs.

a long time. Taking this into account, the stability of NP suspensions

stored at 37 °C was monitored relative to the hydrodynamic diameter
and zeta potential during the 90-day period. The pH of samples was also
monitored in this period and varied from 5.5 to 6.0. The results obtained are shown in Fig. 2, where it can be verified that the zeta potential of NPs remained stable and positive, with a high modulus value
(> +40 mV), as expected at this pH, indicating that there are large
repulsive forces in the system, reducing the possibility of NP aggregation (Tsai, Chen et al., 2011). The CHI/CS/Lecithin NPs did not show
statistically significant variations in relation to the hydrodynamic diameter during the 90-day period. However, the CHI/CS NPs presented an
increase of approximately 30% in relation to the original hydrodynamic
diameter of the NPs over this period. These results show that the addition of the surfactant reduces the flocculation of the particles, giving
greater stability to the NPs. Similar results were obtained for NPs
containing curcumin.
As observed in the transmission electron micrograph displayed in
Fig. 3, the morphology of the NPs developed in this study corresponds
to a compact structure with a tendency to exhibit a spherical shape, as
has been described for many formulations of polysaccharide-based
nanoparticles prepared by polyelectrolyte complexation (Hu, Chiang,
Hong, & Yeh, 2012). However, the particle size observed with TEM
(∼35 nm) was smaller compared to the result determined by DLS. This
discrepancy can be most likely explained as the shrinking of the nanoparticles during the drying process prior to the TEM observation. The
inset of Fig. 3 (corresponding to the micrograph of CHI/CS/Lecithin/
Curc NPs) shows a compact lipid nucleus surrounded by a contrasting
layer of chitosan and chondroitin sulfate, confirming the presence of
the polymers as the outmost layer surrounding the curcumin–lecithin
complex, as the result of expected electrostatic interaction, based on
opposite charges. Similar results were observed by (Sonvico et al.,

Fig. 4. FTIR spectra for a) CHI; b) CS c) CHI/CS NPs; d) CHI/CS/Lecithin NPs,
e) CHI/CS/Curc NPs, f) CHI/CS/Lecithin/Curc NPs and g) Curcumin.

2006; Souza et al., 2014).
The FTIR spectra (Fig. 4) present the main bands of the chemical

groups present in the NPs and their possible interactions. In the spectrum of all the NPs a band at 1020 cm−1 assigned to the groups
NH3+−SO3− was observed, indicating the interaction between the
polymers (Jardim et al., 2015). In addition, the characteristic bands of
chitosan, such as 1377-1257 cm-1 relative to the C–N bond, 1153 cm−1
attributed to the CeOeC bond of β 1–4 glucose, and 1072-1029 cm−1
due to the angular deformation of the amine group, were observed in
the spectra of the NPs. The presence of chondroitin sulfate was also
confirmed in the spectra of the NPs by the observation of the bands:
1238-1060 cm−1and 856 cm−1 assigned to the S]O and CeOeS
bonds, respectively. In the CHI/CS and CHI/CS/Curc NPs spectra
(Fig. 4c and 4e) a shift was observed in the bands at 1658 cm−1 to
1639 cm−1 assigned to the amide I and at 1593 cm−1 to1559 cm−1
related to the angular deformation of the amine group. This last shift
indicates that the NH2 group in the NPs is in the form of NH3+
(Guilherme et al., 2010; Parize, Stulzer, Laranjeira, Brighente, & Souza,
2012).
In the spectra of the NPs containing curcumin (Fig. 4e and 4f) the
band shift at 1593 cm−1 to 1558 cm−1 relative to NH2 of CHI was
observed, indicating the interaction between the amine group of CHI
and the phenolic group of curcumin. The stretches at 3451 cm−1 assigned to the phenolic −OH group at 1620 cm−1 relative to the C]O
bond of the conjugated ketone were also observed at 1562-1420 cm−1
related to the C]C bond of the aromatic ring, at 1380 cm−1 referring to

Fig. 3. TEM images of CHI/CS/Lecithin/Curc NPs with different magnifications. Histogram of particle diameters is shown in lower-right inset.
6


Carbohydrate Polymers 227 (2020) 115351

K.V. Jardim, et al.

curcumin from the CHI/CS/Curc and CHI/CS/Lecithin/Curc NPs can be
seen, performed at 37 °C in phosphate buffer solution at pH 7.4 during
the 240 h period. In this study, pH 7.4 was used as a stimulus for the
release of curcumin, simulating physiological pH. At pH 7.4, the −NH2
groups of chitosan (pKa = 6.3) are not ionized, while the −COOH and
−SO3H groups of the chondroitin sulfate (pKa = 2.6 and 4.5) and
phenolic groups of curcumin (pKa = 8.3) are deprotonated. Thus, there
is an increase in the density of negative charges, resulting in an aniontype electrostatic repulsion between the −COOH and −SO3H groups of
the chondroitin sulfate and phenolic groups of curcumin. This electrostatic repulsion associated with the reduction of the interaction force
between CHI and CS destabilizes the NPs that are formed. As a consequence, the curcumin molecules acquire greater mobility, thus facilitating their release into the environment (Yang et al., 2010).
However, several mathematical models have been developed and
studied to understand the release behavior of drugs from release systems (Pal, Singh, Anis, Thakur, & Bhattacharya, 2013). In this study, the
mathematical model of Gallagher-Corrigan (Gallagher & Corrigan,
2000) (Eq. 2) was applied in order to elucidate the mechanism by which
curcumin is released from the NPs.

Table 2
Chemical composition of the surface (in % at) obtained by XPS for nanoparticles
and their constituents.
Samples

C (% at)

O (% at)

N (%
at)

S (% at)

P (% at)

O/C
(%)

CHI
CS
Curcumin
Lecithin
CHI/CS
CHI/CS/Lecithin
CHI/CS/Curc
CHI/CS/Lecithin/
Curc

54.5
50.2
58.1
52.0
52.2
47.3
41.2
35.6

39.7
41.8
41.9
42.3
40.7

43.1
52.7
55.4

5.8
4.9
–
2.3
4.5
5.4
3.7
4.9

–
3.1
–
–
2.6
2.2
2.4
2.0

–
–
–
3.4
–
2.0
–
2.1

0.7
0.8
0.7
0.8
0.7
0.9
1.3
1.6

the CH3 groups and at 1070 cm−1 referring to the CeOeC vibration of
the ether. These bands were also observed in the curcumin spectrum
(Fig. 4g) (Anitha et al., 2011; Jardim et al., 2015; Şenyiğit et al., 2017).
The presence of the band at 1593 cm−1 corresponding to the deformation of the amine group from CHI was not observed in Fig. 4d and
4f, indicating the interaction of the NH2 groups of CHI with the phosphate groups of lecithin in an acid medium. In the spectra of the lecithin-containing samples (Fig. 4d and 4f), the following bands were
observed: 3423 cm−1 and 3437 cm−1 were attributed to the stretches of
the amine group; 1705 cm−1 corresponded to the carbonyl of the fatty
acids present in lecithin, and the bands at 1053 cm−1 were related to
the vibration of the phosphate group (Şenyiğit et al., 2017).
The NPs were also analyzed by XPS to characterize their surface
chemically and to evaluate the possible interactions between the
polymers (Table 2). The composition obtained for CHI (54.5% C, 39.7%
O and 5.8% N) is in agreement with the data obtained in the XPS
analysis performed by Rodrigues, Da Costa, and Grenha (2012). The
elemental composition of CS was found (50.2% C, 41.8% O, 4.9% N and
3.1% S). In the NPs only the elements carbon (C), oxygen (O), nitrogen
(N) and sulfur (S) present in the chemical structures of their precursors
were identified, indicating the absence of contamination during the
synthesis process. However, in NPs containing lecithin, the presence of
phosphorus (P) and an increase in the atomic percentage of N was

observed, indicating the contribution of the surfactant in the NPs. The
encapsulation of curcumin in the samples was further confirmed based
on the increase in the O/C atomic mass ratio in the NPs containing
curcumin CHI/CS/Curc NPs (1.3%) and CHI/CS/Lecithin/Curc NPs
(1.6%) when compared to NPs without curcumin: CHC/CS NPs (0.7%)
and CHI/CS/Lecithin NPs (0.9%).
In Fig. 5, the general aspect of the in vitro release curves obtained for

e k2 t − k2 tm ⎤
ft = fB (1 − e−k1 t ) + (1 − fB ) ⎡
⎢
+
1
e k2 t − k2 tm ⎥
⎦
⎣

(2)

Where ft is the fraction of the drug released at time t, fB is the maximum
fraction of drug release, k1 is the release constant at the first stage, tm is
the maximum release time and k2 is the release constant during the
degradation of the polymer.
According to the mathematical model described by GallagherCorrigan the release of the drug from polymeric systems occurs in a
two-stage process. In the first stage (k1) a rapid release occurs due to the
dissolution of the drug molecules present on the surface of the polymer
matrix, and then slower release occurs due to the degradation of the
polymer matrix (k2) (Gallagher & Corrigan, 2000). Thus, based on the
Gallagher-Corrigan model, note that for the CHI/CS/Curc NPs, the values of k1 and k2 obtained were equal to 0.15 and 0.02, respectively. For
the CHI/CS/Lecithin/Curc NPs, the values of k1 and k2, obtained were:

0.26 and 0.007, respectively. The first stage reflects the dissolution of
curcumin in the medium, controlled by diffusion, observing thus a more
accelerated release of curcumin. In the second stage, the percentage of
release of the curcumin is slower, probably because it depends on the
dissolution of the polymer over time. The results clearly show the difference in the release profile of curcumin caused by the addition of
lecithin. Although release of the curcumin occurs gradually in both NPs,
the release percentage is higher for CHI/CS/Lecithin/Curc NPs in the
first stage (k1), which can be attributed to the higher mean pore volume
and the higher percentage of curcumin encapsulation in these nanoparticles (131.8 μg/mg = 87.5%), facilitating the diffusion of curcumin
to the medium.
The viability of the MCF-7 human breast tumor cells from free and
nanoencapsulated curcumin evaluated by the MTT assay (Fig. 6) was
significantly reduced (P < 0.0001), exhibiting dose-dependent cytotoxic activity, with increased concentration of curcumin from 10 to 40
μmol.L−1 at 24, 48 and 72 h of incubation.
When analyzing the effect of free curcumin, a decrease in cell viability was observed, by 50.0, 42.0 and 28.4% (P < 0.0001) when used
in the concentrations of 10, 20 and 40 μmol.L−1, respectively, in the
72 h period. In this same period, the addition of 10 μmol.L−1 of CHI/
CS/Curc and CHI/CS/Lecithin/Curc NPs reduced the viability of the
MCF-7 cells by 56.4 and 58.0% (P < 0.0001), respectively. When
adding 20 μmol.L−1 of CHI/CS/Curc and CHI/CS/Lecithin/Curc NPs
there was a reduction of 45.0 and 40.8% (P < 0.0001), respectively, in
the viability of the cells. With the addition of 40 μmol.L−1 of CHI/CS/
Curc and CHI/CS/Lecithin/Curc NPs, the cellular viability was reduced
to 36.0 and 30.7% (P < 0.0001), respectively, when compared to the
control group.

Fig. 5. Cumulative release of the curcumin from developed ( ) CHI/CS/Curc
NPs and ( ) CHI/CS/Lecithin/Curc NPs at 37 °C in phosphate buffer solution at
pH 7.4 for 240 h.
7

Carbohydrate Polymers 227 (2020) 115351

K.V. Jardim, et al.

Fig. 6. Percentage of cell viability of MCF-7 after 24 h, 48 h and 72 h of incubation. Viability assay by MTT. Ultrapure water was used as negative control.
Significantly different from the control: *P < 0.0001.

Fig. 7. Transmission electron micrographs obtained for MCF-7 cells treated with 40 μmol.L−1 of: (A) Control, (B) Curcumin and (C) CHI/CS/Lecithin/Curc NPs for
24 h with different magnifications. Abbreviations: Nucleus (N); Mitochondria (M); Autophagic vesicle (arrow); Aggregation chromatin (arrow head closed);
Apoptotic bodies (arrow head cast).

released through the CHI/CS/Lecithin/Curc NPs were internalized in
the cytoplasm of the MCF-7 cells, thus presenting signs of cytotoxicity.
The main ultrastructural changes in all treated groups were chromatin
aggregation, mitochondrial denaturation, autophagy vesicle and apoptotic body formation, as well as cytoplasmic compartments, swelling
and disappearance of mitochondrial cristae.
The therapeutic potential of curcumin as a cytotoxic agent has been
extensively studied in recent years. Several studies show that curcumin
has distinct cytotoxicity profiles, depending on the cellular tissue and
its concentration (Hanahan & Weinberg, 2011). In relation to human
breast cancer, studies carried out by Bayomi et al., 2013; Bozta et al.,
2013; Zhi-Dong et al., 2014 demonstrated that curcumin is an antiproliferative, cytotoxic and anti-metastatic agent. The authors also
evaluated the cytotoxic activity of free and encapsulated curcumin in
nanoparticles in this cell line, obtaining profiles of cellular viability
similar to those obtained in our study.

The most significant reduction in MCF-7 cell viability was observed
over the 72 h period due to the prolonged exposure of free and nanoencapsulated curcumin in the cells, since the release of curcumin

from the formulations occurred within the first 24 h of treatment. In
addition, increased inhibition in MCF-7 cell growth was observed for
CHI/CS/Lecithin/Curc NPs. Thus, this system represents a promising
candidate for drug carrier for the treatment of breast cancer, since it has
increased the therapeutic efficacy of curcumin.
In fact, the interaction with the biological medium of free and nanoentrapped curcumin is expected to be different, since the cellular uptake pathway of the nanoparticles is different from that of free drugs
(Ahn, Seo, Kim, & Lee, 2013). While the free curcumin has direct
contact with the cell, facilitating its diffusion into the membrane, the
nanoparticles containing curcumin penetrate the cell by endocytosis,
and the drug is liberated gradually to the medium, leading to a reduction in cytotoxic effect. However, it is noteworthy that nanoparticles
can be accumulated in tumor regions that exhibit abnormal vascularization and low lymphatic drainage (effect of improved permeability
and retention - EPR). When accumulating in the area of interest, these
nanoparticles should not only gradually release a high amount of the
drug at the target site, but also minimize side effects in normal tissues
and, as a result, decrease systemic toxicity (Hiroshi, Hideaki, & Jun,
2013).
In the control experiments performed to evaluate the toxicity of the
release system in the 24, 48 and 72 h periods (data not shown), it was
observed that the addition of CHI/CS and CHI/CS/Lecithin NPs without
the presence of curcumin did not alter the viability of the MCF-7 cells. It
was especially notable that they did not present toxicity to the cells,
indicating the biocompatibility of the nanoparticles.
Observations in the transmission electron microscope (Fig. 7)
showed that after 24 h of exposure the free curcumin and curcumin

4. Conclusions
The use of the ionic gelation technique together with the speciation
study favored obtaining NPs with low PDI, hydrodynamic diameter in
the nanometer range, positive zeta potential with high modulus value
(+60 mV), and spherical and heterogeneous morphology. However, it

was observed that the CHI/CS/Lecithin NPs presented better characteristics for encapsulation and release of curcumin. When lecithin
was added, the NPs presented higher colloidal stability at the conditioning temperature (37 °C) during the 90-day period. In addition, the
amount of curcumin encapsulated in CHI/CS/Lecithin/Curc NPs was
131.8 μg/mg, corresponding to 87.5 ± 0.5%, where their release occurred more rapidly, via a dissolution mechanism followed by slower
release, as a result of degradation of the polymers. In the cell viability
8


Carbohydrate Polymers 227 (2020) 115351

K.V. Jardim, et al.

assay, it was also observed that the presence of the surfactant promotes
a greater cytotoxic effect for curcumin encapsulated in NPs, presenting
a more significant reduction in the viability of human breast tumor cells
(MCF-7) when compared to the control group. This indicates that the
addition of lecithin is an excellent strategy to improve the properties of
the NPs, making them a highly promising in vitro encapsulation and
controlled release system for hydrophobic substances, such as curcumin.

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Acknowledgements
The authors gratefully acknowledge financial support from Brazilian
Agencies: Conselho Nacional de Desenvolvimento Cientớco e
Tecnolúgico (CNPq), Coordenaỗóo de Aperfeiỗoamento de Pessoal de
Nớvel Superior (CAPES), Fundaỗóo de Apoio Pesquisa do Distrito
Federal (FAPDF), Decanato de Pesquisa e Inovaỗóo (DPI-UnB),
Financiadora de Estudos e Projetos (FINEP) and Laboratúrio
Multiusuỏrio de Microscopia de Alta Resoluỗóo (LabMic-UFG) for TEM
measurements.
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