International Journal of Biological Macromolecules 216 (2022) 520–527

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International Journal of Biological Macromolecules

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

Sulfated xyloglucan-based magnetic nanocomposite for preliminary

evaluation of theranostic potential
Aiêrta Cristina Carrá da Silva a, Raimundo Rafael de Almeida b, Cristine Soares Vidal a,
João Francisco Câmara Neto a, Alexandre Carreira da Cruz Sousa a,
Fabián Nicolás Araneda Martínez c, Daniel Pascoalino Pinheiro d, Sarah Leyenne Alves Sales d,
Cláudia Pessoa d, Juliano Casagrande Denardin c, Selene Maia de Morais e,
Nágila Maria Pontes Silva Ricardo a, *
a
Laboratory of Polymers and Materials Innovation, Department of Organic and Inorganic Chemistry, Sciences Center, Federal University of Ceará, Campus of Pici, Zip
Code 60440-760 Fortaleza, CE, Brazil
b
Federal Institute of Education, Science and Technology of Ceará, Campus Camocim, Zip Code 62400-000 Camocim, CE, Brazil
c
University of Santiago of Chile and Cedenna, USACH-CEDENNA, Department of Physics, Zip Code 9170124 Santiago, Chile
d
Laboratory of Experimental Oncology, Center for Research and Drug Development, Federal University of Ceará, Zip Code 60430-275 Fortaleza, CE, Brazil
e
Laboratory of Natural Products, Science and Technology Center, Ceará State University, Campus of Itaperi, Zip Code 60714-903 Fortaleza, CE, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Chemically sulfated xyloglucan (S_XLG) was applied to encapsulate hydrophilic quercetin sulfate (Q_SO3) into
Sulfated xyloglucan superparamagnetic nanocomposites (MNSXQ_SO3) synthesized via interfacial polyaddition reaction. This nano­
Multifunctional magnetic nanocomposite platform was evaluated concerning in vitro antitumor activity, drug release profile, and magnetic behavior under
Targeted drug delivery
a magnetic field. MNSXQ_SO3 exhibited antitumor activity against four human tumor cell lines: leukemia HL-60
(IC50 = 1.58 ± 0.54 μg mL− 1), glioblastoma SNB-19 (IC50 = 8.77 ± 0.36 μg mL− 1), colorectal carcinoma HCT-
116 (IC50 = 8.69 ± 0.21 μg mL− 1) and prostate PC3 (IC50 = 17.42 ± 3.57 μg mL− 1). Additionally, MNSXQ_SO3
was able to delay the release of Q_SO3 (76 %) compared to the free Q_SO3 (100 %), after 48 h of drug delivery
assay, revealing a release profile compatible with the Korsmeyer-Peppas kinetic model. These results represent a
relevant progress in the production of multifunctional hybrid nanocarriers, providing a versatile nanocomposite
with potential for targeted drug delivery and theranostic applications.

1. Introduction carbohydrate backbone structure/composition of sulfated poly­

The development of nanocomposites based on natural polymers
(polysaccharides) has been highlighted in different medical areas
because they are widely available, biocompatible, biodegradable,
nontoxic and flexible to chemical modification [1]. The sulfation reac­
tion of polysaccharides provides steric hindrance and electrostatic
repulsion between the biopolymer chains, improving the water solubi­
lity in relation to their naturally occurring counterpart. Moreover, the
sulfated group causes flexion and extension of the polysaccharide
chains, resulting in the alteration of polysaccharides biological proper­
ties [2].
These biological activities depend on the molecular weight (Mw), the
position of sulfate group, the degree of sulfation (DS) and the
saccharide [3,4]. However, some advantages in pharmacokinetic prop­
erties have been reported to chemically sulfated polysaccharides, when
compared to natural sulfated polysaccharides, such as the differences of
in vivo degradability, allowing their application in researches based on
biomaterials and drug delivery vehicles [5].
Xyloglucan (XLG) is a high Mw neutral hemicellulose obtained from
the Tamarindus indica Linn. seeds [6] composed by a main chain linked
to β-1,4-D-glucopyranosyl residues, partially replaced by α-1,6-D-xylo­
pyranosyl units, which may or may not have β-1,2-galactopyranosyl
monomers as branch [7]. The large amount of hydroxyl groups in these
monomers units can become sites of sulfation reaction. Previously
published works show that chemically sulfated xyloglucan have anti­
tumor activity in vitro against human liver cancer cell line (HepG2),

* Corresponding author.
E-mail address: naricard@ufc.br (N.M.P.S. Ricardo).

https://doi.org/10.1016/j.ijbiomac.2022.06.197
Received 22 February 2022; Received in revised form 28 June 2022; Accepted 29 June 2022
Available online 6 July 2022
0141-8130/© 2022 Elsevier B.V. All rights reserved.
A.C.C. da Silva et al.
while the unmodified polysaccharide has no anticancer effect [8].
Recently, superparamagnetic iron oxide nanoparticles (SPIONs)
have been demonstrated a great potential for diagnosis and therapy in a
number of cancer diseases, due to their various advantages such as
biocompatibility, imaging contrast ability, improved tissue penetration,
and superparamagnetic properties [9]. Additionally, SPIONs can be used
on the heating of malignant cells, which are more susceptible to heat
than normal cells, when SPIONs are under the action of an alternating
magnetic field. This technique (magnetic hyperthermia) has become an
alternative therapy to treat cancer and to reduce side effects caused by
traditional radiotherapy and chemotherapy [10].
According to Ahmadian et al. [11], several nano-based strategies
have been testified and approved for treatment and diagnosis of
different types of cancer. For instance, Resovist®, Cliavist®, Feridex®,
Endorem™, Feraheme®, Rienso®, Lumirem®, GastroMARK® and
Abdoscam® are SPIONs clinically approved, which have reached the
market for diagnosis by magnetic resonance imaging or therapeutic
application of iron supplementation in anemia [12]. However, SPIONs
are technological drivers of innovation and have been utilized in image-
guided drug delivery, theranostic tissue engineering, targeted delivery
and controlled release of drugs to specific sites [12,13].
Quercetin (3,3′ , 4′ , 5, 7-pentahydroxiflavone) is a flavonoid widely
found in vegetables, fruits, teas and wines [14], which can be conjugated
with SPIONs [15] or entrapped in magnetic nanocomposites [16] in
order to obtain targeted drug delivery systems. This compound has
several pharmacologic effects, such as antitumor and antioxidant
properties, that can help in the treatment of cancer and other diseases
[17]. However, quercetin has poor water solubility [18], low bioavail­
ability [19], rapid elimination from body, fast metabolism and can be
enzymatically degraded [20]. The sulfation reaction of this flavonoid
Fig. 1. Schematic representation for the obtention of MNSXQ_SO3.
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International Journal of Biological Macromolecules 216 (2022) 520–527
can change these characteristics and improve the therapeutic potential
of this biomolecule in clinical applications by the increase of its water
solubility.
For these reasons, we performed in this work the synthesis and
characterization of nanocomposites based on magnetic sulfated xylo­
glucan (S_XLG) loaded within sulfate quercetin (Q_SO3), obtaining a
multifunctional nanocontainer with potential for biomedical applica­
tions, mainly the treatment of cancer by targeting the delivery of hy­
drophilic drugs.
2. Experimental details
2.1. Synthesis of MNSXQ_SO3
Magnetic nanocapsules of sulfated xyloglucan loaded with hydro­
philic quercetin sulfate (MNSXQ_SO3) were synthesized through the
water-in-oil miniemulsion technique (Fig. 1), followed by interfacial
polymerization reaction using tolylene-2,4-diisocyanate (TDI) as cross­
linker agent [21].
Firstly, the aqueous phase (AP), composed by 4 mL of S_XLG 1 % (w/
w), 20 mg of Q_SO3 and 12 mg (330 μL) of SPIONs coated with poly­
acrylic acid sodium salt (Fe3O4@PAAS), was dripped into the organic
phase (OP), which was formed by 14.5 g of cyclohexane and 160 mg of
polyglycerol polyricinoleate (surfactant). This pre-emulsion was soni­
cated in an ultrasound (Branson Sonifier W-450-Digital and a ½” tip) at
70 % amplitude during 3 min (20 s pulse; 10 s pause), while cooling the
mixture with an ice bath to obtain a nanoemulsion. Finally, 42.5 mg of
TDI was dripped slowly into the obtained nanoemulsion for the inter­
facial polymerization reaction to occur. After 24 h of reaction,
MNSXQ_SO3 was redispersed in SDS solution 0.1 %.
A.C.C. da Silva et al.
All materials were described in Supplementary information, as well
as the experimental procedures of obtention and characterization of
S_XLG (% S = 4.19; Mw = 156.850 kDa) and Q_SO3 (% S = 13.41).
2.2. Characterization of MNSXQ_SO3
Fourier Transform Infrared spectroscopy was performed using a
Shimadzu IRTracer-100 Spectrophotometer (Model 8300) in the region
between 400 and 4000 cm− 1 [22]. The dried sample was prepared in the
form of KBr tablets and analyzed.
The stability study of MNSXQ_SO3 was determined by Dynamic Light
Scattering (DLS) measurements, using a Zetasizer Nano Series Malvern
W-ZS90. After preparation, MNSXQ_SO3 was stored at 25 ◦ C for 2
months. The sample was diluted 50 times with ultrapure water and
analyzed in an optical grade polystyrene cuvette at 25 ◦ C [16]. The
polydispersity index (PdI), hydrodynamic diameter average (DH) and
zeta potential (Zp) were determined immediately after MNSXQ_SO3
preparation (Day 1) and at predetermined time intervals (7, 14, 21, 28
and 60 days).
The morphology of MNSXQ_SO3 was evaluated by scanning electron
microscopy (SEM-FEG model Quanta 450, FEI Company) and trans­
mission electron microscopy (TEM-Hitachi®HT7700), according to the
methodology previously reported [23]. The distribution curve of parti­
cle size was generated by manual measurement of 200 particles, using
the software ImageJ [24].
The X-ray powder diffraction (XRD) of the synthesized sample was
collected using a PANalytical (X'pert Pro MPD) X-ray powder diffrac­
tometer operating in Bragg-Brentano reflection geometry with Co-Kα
radiation (40 kV, 40 mA) over the range 20◦ < 2θ < 100◦ and scan speed
of 0.013◦ /s [25].
The magnetic properties of MNSXQ_SO3 were investigated using a
homemade Vibrating Sample Magnetometer (VSM), operating at 300 K
with a magnetic field range from − 10 to 10 kOe [25]. The magnetometer
was calibrated using Yttrium Iron Garnet Sphere as standard reference
material (NIST-National Institute of Standards and Technology). For the
sample measurement, the magnetic moment obtained for each applied
field was normalized by the sample mass.
2.3. Release study of Q_SO3
The in vitro release profile studies were carried out for quercetin
sulfate in its free (Q_SO3) and encapsulated forms (MNSXQ_SO3). The
experimental procedure was performed according to the previously
published study [25]. Briefly, a dual-compartment system separated by
a dialysis tubing cellulose membrane (molecular weight cut off = 12
kDa) with donor (1.5 mL of MNSXQ_SO3 or Q_SO3) and acceptor com­
partments (50 mL, phosphate buffered saline-PBS, pH 7.4) was main­
tained under sink conditions with constant magnetic stirring. Aliquots
(1 mL) of the samples were collected from the acceptor compartment at
predetermined time intervals and analyzed by UV–Vis Spectrophotom­
eter (ThermoScientific®, model: Genesis 10S) at 265 nm. Q_SO3 was
quantified using a regression equation generated from the standard
calibration curve obtained from dissolving Q_SO3 in PBS solution at the
concentrations of 10 to 200 mg mL− 1. To evaluate the kinetic mecha­
nism of Q_SO3, the data obtained from this release assay were fitted to
the main mathematical models, as zero and first-order, Higuchi and
Korsmeyer-Peppas. The amount of Q_SO3 encapsulated (%EE) in
MNSXQ_SO3 sample was also determined by UV-VIS spectrometry using
an experimental procedure described in literature [25].
2.4. In vitro antitumor activity of the samples
The colorimetric MTT assay [26] was employed to determine the in
vitro cytotoxicity activity of Q, Q_SO3 and MNSXQ_SO3 against four
human tumor cell lines: SNB-19 (glioblastoma), HCT-116 (colon carci­
noma), HL-60 (leukemia) and PC3 (prostate). More details of the
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International Journal of Biological Macromolecules 216 (2022) 520–527
experimental procedure were described in Supplementary information.
2.5. In vitro antioxidant activity of Q and Q_SO3 by DPPH radical
scavenging experiment
The antioxidant potential of Q and Q_SO3 was determined by the
ability to scavenge the 2,2-Diphenyl-1-picrylhydrazyl (DPPH•) free
radical, using previously reported method [27]. The experimental pro­
cedure was described in Supplementary information.
3. Results and discussion
3.1. Characterization of MNSXQ_SO3
Fourier Transform Infrared (FT-IR) spectroscopy analysis was carried
out to confirm the formation of the nanocapsule polymeric shell. FT-IR
spectrum of MNSXQ_SO3 was depicted in Fig. 2a and displayed char­
acteristic bands of urea and urethane groups formed by the successfully
interfacial polyaddition reaction between OH groups of polysaccharide
and NCO groups of crosslinker agent (TDI). Absorptions bands at 1734
cm− 1 and 1643 cm− 1 are signed to the carbonyl stretching vibration of
urethane and urea groups, respectively. The crosslinked polymer shell is
also evidenced by the presence of N–H amide bending bands at 1550
cm− 1, which are related to urethane group. The flat signal at 2275 cm− 1
indicates the total incorporation of NCO groups from TDI into nano­
capsule shell by their both sides reaction with sulfated xyloglucan
[23,28,29]. The broad band at 3304 cm− 1 is reported to the stretching
vibration of O–H groups of glucan backbone, while the bands at 2926
cm− 1 and 2850 cm− 1 are attributed to C–H stretching of alkanes [30].
The bands at 1600 cm− 1 and 1230 cm− 1 are characteristic of the C– –C
stretching vibrations from aromatic systems and C– – O stretching [31],
respectively, suggesting the incorporation of sulfated quercetin into
nanocapsules. In addition, the band at 1420 cm− 1 corresponds to C–O
stretching vibration [32], while 1138 cm− 1 and 1053 cm− 1 indicate the
stretching vibration of pyranose [33]. The band at 813 cm− 1 is charac­
teristic of the symmetric stretching vibration of C-O-S [33], while the
band at 621 cm− 1 is related to Fe–O crystal lattice vibrations,
evidencing the presence of Fe3O4@PAAS into nanoproduct [34].
MNSXQ_SO3 sample was analyzed by DLS technique to determine the
polydispersity index (PdI), hydrodynamic diameter average (DH) and
zeta potential (Zp) values of nanocapsules at predetermined time in­
tervals (1, 7, 14, 21, 28, 60 days), as shown in Fig. 2b. The PdI (0.293 ±
0.052), DH (274.7 ± 2.2 nm) and Zp (− 28.7 ± 0.9 mV) values obtained
immediately after the preparation of MNSXQ_SO3 classify this sample as
a polydispersed particle (0.1 < PdI < 0.4) and moderately stable
dispersed system (− 20 < Zp < − 30 mV), that can be employed in
intravenous administration because are unable to cause embolism (DH
< 300 nm) [31,35,36].
Although MNSXQ_SO3 is classified as a moderately stable dispersed
system, the DLS analyses performed after the sample preparation reveal
no drastic changes in the PdI, DH and Zp values (Fig. 2b) during the
storage period at room temperature. Additionally, MNSXQ_SO3
remained as a transparent liquid without irreversible precipitation or
coagulation during the stability study.
The surface morphology and particle size of MNSXQ_SO3 were
observed by scanning electron microscopy (SEM) and transmission
electron microscopy (TEM), respectively. Fig. 3 displays the SEM mi­
crographs of MNSXQ_SO3, where the sample appears as deflated balls,
due to the drying and vacuum effects of SEM measurements, confirming
the core-shell morphology of nanocapsule [23].
TEM images of MNSXQ_SO3 sample are depicted at Fig. 4, showing
black dots over the gray dots, that are identified as the presence of
SPIONs (Fe3O4@PAAS) in the nanocapsules structure [37]. Black dots
were not seen deposited on the surface of carbon-coated copper grid,
evidencing the total encapsulation of magnetic nanoparticles into
sulfated xyloglucan nanocapsules [25]. The distribution curve particle
A.C.C. da Silva et al. International Journal of Biological Macromolecules 216 (2022) 520–527

Fig. 2. (a) FTIR spectrum and (b) DLS measurements of MNSXQ_SO3 sample.

Fig. 3. SEM micrographs of MNSXQ_SO3 sample.

Fig. 4. TEM micrographs and particle size distribution histogram of (a) Fe3O4@PAAS and (b) MNSXQ_SO3.

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size of magnetic nanoparticles (Fig. 4a) and nanocapsules (Fig. 4b) were
obtained by Log-normal fit of the size histogram. The values of 8 ± 2 nm
and 126 ± 27 nm were determined as mean particle size of magnetic
nanoparticles and nanocapsules, respectively. These results are similar
to those in previously published works [38,39].
The X-ray diffractogram (XRD) of MNSXQ_SO3 was displayed in
Fig. 5a, showing characteristics of amorphous structures with no sharp
peaks. A peak at approximately 2 θ = 20◦ can be seen in the XRD of the
nanocarrier, similar to those reported by Arruda et al. (2015) for xylo­
glucan obtained from Tamarind seeds. Although the XRD of Fe3O4@­
PAAS (Fig. 5b) reveals a pattern compatible with magnetite (JCPDS card
# 19-0629) phase [40], the typical crystallinity peaks of Fe3O4@PAAS
are not exhibited in the XRD of MNSXQ_SO3 due to the successful
encapsulation of the ferrofluid [41].
Fig. 6a shows the magnetization versus magnetic field dependence at
300 K, measured with a Vibrating Sample Magnetometer (VSM).
MNSXQ_SO3 sample reveals a superparamagnetic behavior with a
saturation magnetization (Ms) value of 4.8 emu/g, due the presence of
Fe3O4@PAAS into nanocapsules. This result is within the saturation
magnetization range published for magnetic nanocomposite based on
polymeric capsules [42]. The magnitude of Ms. is responsible for
theranostic properties, as the heating efficiency and magnetic respon­
siveness for nanocarrier guidance [43]. Finally, a small hysteresis loop is
shown in the low-field data (inset at Fig. 6a).
3.2. Release study of Q_SO3
The release profile of hydrophilic quercetin sulfate (Q_SO3) free and
loaded in magnetic nanocapsules based on sulfated xyloglucan
(MNSXQ_SO3) is displayed in Fig. 4b. The release assay was conducted in
PBS medium (pH 7.4) for 48 h, because the free Q_SO3 reached the
complete amount diffusion (100 %) at this time, using a dual-
compartment system separated by a cellulose membrane with a molec­
ular exclusion pore of 12 kDa [25]. During the first 3 h of the experi­
ment, about 43 % of free Q_SO3 was diffused from donor to receptor
compartment, while 35 % of this bioactive was released from
MNSXQ_SO3. After 48 h of experiment, the amount of free Q_SO3
released (100 %) is greater than the amount of Q_SO3 delivered by
MNSXQ_SO3 (76 %). Probably, the addition of sulfated groups to the
polysaccharide chains decreases the number of hydroxyls available for
the crosslinking reaction. Consequently, the nanocomposite of sulfated
xyloglucan exhibits a lower degree of reticulation, and it does not entrap
Q_SO3 strongly enough during the release study. De Almeida et al. have
already published similar results, where they observed the faster release
profile of mangiferin from sulfated polymeric matrix than from natural
polymeric matrix [44].
The kinetic mechanism of Q_SO3 release from MNSXQ_SO3 was
evaluated fitting the data obtained above to various mathematical
Fig. 5. XRD of (a) MNSXQ_SO3 and (b) Fe3O4@PAAS.
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International Journal of Biological Macromolecules 216 (2022) 520–527
models: zero-order, first-order, Higuchi and Korsmeyer-Peppas. Table 1
shows the results after applying the mathematical models of kinetic
study for the sample, indicating a release behavior compatible with
Korsmeyer-Peppas kinetic model (R2 = 0.9737). In this case, the release
of Q_SO3 from MNSXQ_SO3 is in accordance with the Fickian diffusion
(n = 0.4233) of Q_SO3 from nanocapsules. Literature data also report
that Korsmeyer-Peppas is the most commonly described semi-empirical
drug release model for polyurethane nanocarriers obtained by the cross-
linking reaction [45–47].
3.3. In vitro antitumor activity of the samples
The MTT assay was conducted to investigate cytotoxicity of
MNSXQ_SO3, Q_SO3 and quercetin (Q), as drug control, against four
cancer cell lines (Table 2). The results obtained in this experiment are
expressed in IC50 values (μg mL− 1), illustrated in Fig. 7, and correspond
to the mean ± SEM of at least three independent experiments performed
in triplicate.
According to Table 2, Quercetin (Q) showed lower IC50 values when
compared to Q_SO3, meaning that (Q) has a greater antitumor activity
than its sulfated derivative (Q_SO3). Sulfated flavonoids have interesting
properties for clinical preparations, as the advantage of being water-
soluble. However, these sulfated derivatives may or may not magnify
or not the biological activities in relation to the non-sulfated compound
[48].
Table 2 shows that Quercetin (Q) had greater antitumor activity
against HL-60, followed by HCT-116 and finally by SNB-19, with IC50
values of 2.04 μg mL− 1, 3.86 μg mL− 1 and 11.27 μg mL− 1, respectively.
The antitumor effect of Q_SO3 was similarly ranked as HL-60 > HCT-
116 > SNB-19 > PC3, with IC50 values of 28.99 μg mL− 1, 64.94 μg mL− 1,
79.78 μg mL− 1 and 91.76 μg mL− 1, respectively. According to literature
data, the greater antitumor effect of quercetin compared to its sulfated
derivative (quercetin-3′ -sulfate) was also previously reported in human
breast cancer MCF-7 cells. Both compounds led to a cell-cycle arrest at
the S phase and decreasing in the number of cells at G0/G1 and G2/M.
Afterwards, the flavonoids induced cells apoptosis, mediated by the
generation of intracellular reactive oxygen species (ROS) [49].
According to Mateus et al. (2018), the production of intracellular
ROS by quercetin occurs due a cellular redox imbalance caused by the
capacity of this flavonoid to be highly susceptible to autoxidation. Thus,
the treatment with molecules that induce oxidative stress, as quercetin
and its sulfated derivative, provides a significant dose-dependent in­
crease of intracellular ROS in tumor cells, pushing them to their toxic
threshold, which can led to cell apoptosis through the mitochondrial
pathway [49–51].
Literature data report that the increasing in intracellular ROS
induced by quercetin can be mainly related to 3′ -OH, 4′ -OH groups on
the B ring and 7-OH on the A ring. Then, the synthesis of quercetin
A.C.C. da Silva et al. International Journal of Biological Macromolecules 216 (2022) 520–527

Fig. 6. (a) Hysteresis loops of MNSXQ_SO3 sample with a zoom in low-field region inset and the (b) mean (±s.d.) percentage of free Q_SO3 (green line) and Q_SO3
encapsulated (pink line) in MNSXQ_SO3 sample. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of
this article.)

sulfate with substitutions at these positions would decrease this effect on

Table 1
the sulfated derivative in relation to the pattern compound [49,52].
Curve fitting analyses of kinetic studies obtained for Q_SO3 released from
In the present work, the chemical structure of Q_SO3 had three hy­
MNSXQ_SO3 in PBS medium (pH 7.4).
droxyls (5-OH, 3′ -OH and 4′ -OH) replaced by sulfate groups. Probably,
Sample Correlation coefficient (R2) Release
the absence of the –OH group in those positions in Q_SO3 decreased its
exponent (n)
Zero- First Higuchi Korsmeyer- cytotoxic activity when compared to Q, as seen in Table 2 and Fig. 5. To
order order Peppas avoid the quercetin sulfation reaction at those sites, a benzylation re­
Free Q_SO3 0.8995 0.5726 0.9826 0.9789 0.0716 action could be carried out to protect the 3′ , 4′ and 7 hydroxyl groups of
MNSXQ_SO3 0.9697 0.9544 0.9708 0.9737 0.4233 this compound [53]. Thereby, the sulfation reaction proposed in this
work could only form compounds with sulfate groups at positions 3 or 5.
According to Jang and Kang [53], the protective groups must be
Table 2 removed later by dissolution of the synthesized product in ethanol/THF
Antiproliferative activity of Q, Q_SO3 and MNSXQ_SO3 in four tumor cell lines (1:1, v/v), followed by the addition of Pd/C under stirring for 12 h.
evaluated by the MTT assay after 72 h treatment. Additionally, the results obtained in this research indicate that the
MTT (IC50 μg mL− 1) – 72 h encapsulation of Q_SO3 into magnetic nanocapsules based on sulfated
xyloglucan (MNSXQ_SO3) improves the antitumor activity of the free
Samples Cell lines
bioactive (Q_SO3) against all the four tested tumor cell lines. This result
SNB-19 HCT-116 HL-60 PC3
was similar to the one published by Huang et al., where the authors
(glioblastoma) (colorectal) (leukemia) (prostate)
demonstrated an increase of the doxorubicin inhibitory effects on the
Q 11.27 ± 2.76 3.86 ± 1.69 2.04 ± 1.02 n.d.a proliferation of human breast cancer (MCF-7) cells, when this bioactive
Q_SO3 79.78 ± 11.69 64.94 ± 5.89 28.99 ± 4.92 91.76 ±
13.85
was incorporated into polyurethane nanoparticles [54].
MNSXQ_SO3 8.77 ± 0.36 8.69 ± 0.21 1.58 ± 0.54 17.76 ± Nanocarriers based on polyurethane linkage are biocompatible and
3.42 demonstrate no significant cell toxicity [28,55]. Previous works re­
a
n.d.: not determined.
ported the biocompatibility of polyurethane nanoparticles, which was
assessed by HEK293 cells from the human embryonic kidney, demon­
strating a cell viability >75 % [45].
Although in vivo experiments have revealed an inflammatory state in
Swiss albino mice when the animals were treated with polyurethane
nanoparticles [56], in our previous work, polyurethane nanocomposites
based on starch provided no deaths in the in vivo acute toxicity assay
(zebrafish model) [47]. For those reasons, in vivo toxicity assays of
MNSXQ_SO3 will also be further performed to be included in future
studies.
According to results showed in Table 2, MNSXQ_SO3 exhibited better
antitumor activity against HL-60 (leukemia, IC50 = 1.58 μg mL− 1), fol­
lowed by SNB-9 (glioblastoma, IC50 = 8.77 μg mL− 1), HCT-116 (colo­
rectal, IC50 = 8.69 μg mL− 1), and PC3 (prostate, IC50 = 17.76 μg mL− 1).
Li et al. (2021) synthesized gold nanoparticles based on hyaluronic acid,
polyethylene glycol, and adipic dihydrazide for drug delivery of doxo­
rubicin. This nanocomposite shows better antitumor activity against
HCT-116 (IC50 = 54.6 μg mL− 1) and HepG2 (IC50 = 67.3 μg mL− 1) cells
Fig. 7. Antiproliferative activity of Q, Q_SO3 and MNSXQ_SO3 in four tumor [57].
cell lines evaluated by the MTT assay after 72 h treatment. Data are presented Data literature report that among the nanocomposites applied in
in IC50 values (μg mL− 1) and correspond to the mean ± SEM (n = 3). therapeutic approaches, polymeric nanoparticles are widely used for in

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vitro and in vivo experiments, due to their acceptable efficacy as a useful
carrier for targeted delivery of anticancer drugs [11]. The presence of
magnetic nanoparticles provides a great potential for diagnosis and
therapy of several types of cancer [9]. Thus, the combination of these
two nanocarrier can improve the theranostic potential of the nanoplat­
form. In the present work we developed hybrid nanocarriers
(MNSXQ_SO3) and performed preliminary experiments to determine the
in vitro antitumor activity of this nanoplatform, highlighting the po­
tential of MNSXQ_SO3 for cancer treatment and encouraging further
investigation about the nanocomposite.
3.4. In vitro antioxidant activity of Q and Q_SO3 by DPPH radical
scavenging experiment
DPPH• is a stable free radical applied to estimate the free radical
scavenging activities of compounds in a relatively short time [8]. Sig­
nificant differences were obtained for Q and Q_SO3. The antioxidant
capacity of Q was greater than Q_SO3, showing IC50 values of 6.03 ±
0.41 μg mL− 1 (**p < 0.01) and 628.64 ± 52.84 μg mL− 1 (values
expressed as the mean ± SEM, n = 6), respectively. As expected, quer­
cetin has well known view scavenging capacity, while the derivatization
of quercetin hydroxyl groups significantly reduces the antioxidant
ability [58]. Compounds with antioxidant properties are very important
to health body because they can accept free radicals and prevent many
diseases [14].
4. Conclusion
Quercetin sulfate (Q_SO3) was evaluated against four tumor cell
lines, using quercetin (Q) as control. Q_SO3 had its antitumor activity
decreased, compared to Q, due the presence of sulfate groups in the
chemical structure of Q_SO3. Despite the lower cytotoxicity of Q_SO3, it
is important to highlight that the sulfation reaction increases solubility,
which might be an advantage for further in vivo studies. Q_SO3 also
shows lower antioxidant properties than Q. Sulfated xyloglucan (S_XLG)
was employed as a polymeric matrix of magnetic nanocomposites to
encapsulate quercetin sulfate (MNSXQ_SO3). The nanocarriers were
produced via interfacial polyaddition reaction through inverse mini­
emulsion technique, forming nanocapsules with a core-shell
morphology, superparamagnetic characteristics, good colloidal stabil­
ity and able to entrap hydrophilic bioactives as Q_SO3. MNSXQ_SO3
delayed the Q_SO3 delivery, when compared to the free Q_SO3,
emphasizing the ability of the sulfated polysaccharide to modify the
release profile of bioactives. Aditionally, MNSXQ_SO3 showed better
antitumor activity than Q_SO3. Synthesis and study of new magnetic
nanocomposites might reveal the promising candidates for future
application in theranostic area as drug delivery of antitumor drugs or
diagnostic and treatment of diseases.
Acknowledgments
This work received financial support in part by Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) –
Finance Code 001, Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq) and Fundação Cearense de Apoio ao Desenvolvi­
mento Científico e Tecnológico (FUNCAP). The authors would like to
thank for the research grants of N.M.P.S.R. (CNPq Project No 309795/
2021-4), A.C.C.S. (CNPq Project No 140256/2017-2) and R.R.A. (FUN­
CAP Project No 88887.162539/2017-00). We also acknowledge the
analyses and data acquisition provided by Northeastern Center for the
Application and use of Nuclear Magnetic Resonance (CENAUREM-UFC),
Brazilian Agricultural Research Corporation (EMBRAPA), Central Ana­
lítica-UFC/CT-INFRA/MCTI-SISNANO/CAPES, Fondecyt 1200782 and
CEDENNA AFB180001 (USACH).
526
International Journal of Biological Macromolecules 216 (2022) 520–527
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijbiomac.2022.06.197.
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