Carbohydrate Polymers 264 (2021) 118017

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

Starch-based magnetic nanocomposite for targeted delivery of hydrophilic

bioactives as anticancer strategy
Alexandre C.C. Sousa a, b, Adolfo I.B. Romo c, Raimundo R. Almeida a, Aiêrta C.C. Silva a,
Lillian M.U. Fechine a, Débora H.A. Brito a, Rafael M. Freire d, e, Daniel P. Pinheiro f, Larissa M.
R. Silva g, Otília D.L. Pessoa c, Juliano C. Denardin d, Claudia Pessoa f, Nágila M.P.S. Ricardo a, *
a
Laboratory of Polymers and Materials Innovation, Department of Organic and Inorganic Chemistry, Federal University of Ceará, Fortaleza – CE, Zip Code 60440-900,
Brazil
b
Federal Institute of Education, Science and Technology of Ceará, Quixadá – CE, Zip Code 63902-580, Brazil
c
Department of Organic and Inorganic Chemistry, Federal University of Ceará, Fortaleza – CE, Zip Code 60440-900, Brazil
d
University of Santiago of Chile (USACH), Physics Department and CEDDENA, Santiago, Zip Code 9170-124, Chile
e
Institute of Applied Chemical Sciences, Universidad Autónoma de Chile, Zip Code 8910-060, Chile
f
Laboratory of Experimental Oncology, Center for Research and Drug Development, Federal University of Ceará, Fortaleza – CE, Zip Code 60430-275, Brazil
g
Department of Food Engineering, Federal University of Ceará, Fortaleza – CE, Zip Code 60356-000, Brazil

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

Keywords: Magnetic nanocomposites were synthesized for the targeted delivery of hydrophilic bioactives through guidance
SPIONs generated by a magnetic field. Superparamagnetic iron oxide nanoparticles (SPIONs) were used to generate
Magnetic nanocarriers hydroxyethyl starch magnetic nanocapsules (HES MNCs). This synthesis allowed the co-encapsulation of onco­
Drug delivery vehicles
calyxone A (onco A) and surface-modified magnetite nanoparticles (Fe3O4@citrate) into the same nanostructure.
Prolonged release
Nanocapsules
The synthesized nanocapsules exhibited a core-shell morphology, with an average diameter of 143 nm. This
Oncocalyxone A nanocomposite showed potential anticancer activity (IC50) against four human tumor cell lines: glioblastoma
SNB-19 (1.010 μgmL− 1), colon carcinoma HCT-116 (2.675 μgmL− 1), prostate PC3 (4.868 μgmL− 1), and leukemia
HL-60 (2.166 μgmL− 1). Additionally, in vivo toxicity and locomotor activity were evaluated in a zebrafish (Danio
rerio) model. The nanocomposite exhibited in vitro cytotoxicity, prolonged drug release profile and also
responded to an applied magnetic field, representing a versatile compound with perspectives for highest con­
centration of different hydrophilic bioactives in a target tissue through magnetic vectorization.

1. Introduction allow greater success in the administration of drugs in tumor tissues,

representing promising applications related to medicine and pharma­
Nanoparticle-based medicines, such as liposomal doxorubicin cology (Wang et al., 2019; Zhang et al., 2019).
(Doxil®) and albumin/paclitaxel nanoparticles (Abraxane®), have Superparamagnetic iron oxide nanoparticles (SPIONs) have attracted
already been adopted in clinical cases, while others candidates are being a lot of attention in biomedical applications due to their biocompati­
evaluated by the Food and Drug Administration (FDA) (Gao et al., bility, high potential for heat generation and ability to be manipulated
2021). Targeting agents (proteins, antibodies and small organic mole­ by an external magnetic field (Favela-Camacho et al., 2019). In this
cules) could be conjugated over the surface of nanocarriers for breast context, several studies aim to develop applications for SPIONs for
cancer treatments (Grewal et al., 2021). Also, the combination of cancer diagnosis and treatment (Zhao et al., 2019). For instance, iron
different nanotechnological applications, such as magnetic hyperther­ oxide nanoparticles have been used to elaborate nanocarrier to load
mia, magnetic vectorization and controlled drug release can be used to anti-cancer drug doxorubicin (Jia et al., 2021). Clinical trials have

* Corresponding author.
E-mail addresses: [email protected] (A.C.C. Sousa), [email protected] (A.I.B. Romo), [email protected] (R.R. Almeida),
[email protected] (A.C.C. Silva), [email protected] (L.M.U. Fechine), [email protected] (D.H.A. Brito), [email protected]
(R.M. Freire), [email protected] (D.P. Pinheiro), [email protected] (L.M.R. Silva), [email protected] (O.D.L. Pessoa), jcdenardin@gmail.
com (J.C. Denardin), [email protected] (C. Pessoa), [email protected] (N.M.P.S. Ricardo).

https://doi.org/10.1016/j.carbpol.2021.118017
Received 28 January 2021; Received in revised form 10 March 2021; Accepted 29 March 2021
Available online 2 April 2021
0144-8617/© 2021 Elsevier Ltd. All rights reserved.
A.C.C. Sousa et al. Carbohydrate Polymers 264 (2021) 118017

shown that increasing the tissue temperature, to around 42 ± 2 ◦ C, Chromatography (GPC) as described in the supporting information.
significantly enhances radiotherapy and chemotherapy effectiveness The FTIR spectrum were obtained using a Shimadzu IRTracer-100
(Xie et al., 2020). Thus, the combination of SPIONs with anticancer spectrophotometer, with scan amplitude from 400 to 4000 cm− 1. The
drugs, in the same nanostructure, creates a new field of therapeutic samples were dispersed in potassium bromide (KBr) tablets and the
possibilities that can combines heat generation (magnetic hyperther­ spectra were captured at room temperature by 64 scans. X-ray patterns
mia) with the targeting of bioactive agents to a specific tissue (de Moura of the synthesized nanoparticles were collected using a Bruker D2 Phaser
et al., 2020). diffractometer, controlled by a Diffract.
An efficient process of drug encapsulation has been demonstrated All the TEM measurements were performed using a Hitachi HT7700
through nanoemulsions, with droplets in the range of 50–500 nm stably TEM system in the Microscopy Laboratory of Centro para el Desarrollo
dispersed. This technique, called miniemulsion synthesis by probe ul­ de la Nanociencia y la Nanotecnología (CEDENNA) in the Universidad
trasound, allows the synthesis of polymeric nanocapsules with core-shell de Santiago de Chile, operating at an accelerating voltage of 120 keV.
morphology, allowing stabilization of drugs and other nanoparticles into The samples were deposited on a carbon-coated copper grid sample
nanocapsules (Baier et al., 2012; Callender et al., 2017; Paleos et al., holder (Formvar Carbon grids of 200 copper grids (F/C mesh 200 Cu)).
2017; Singh et al., 2017). The process also allows the synthesis of The histogram for particle size distribution was evaluated by measuring
nanocapsules with hydrophilic or hydrophobic nucleus, allowing the 200 particles randomly chosen by the ImageJ software.
encapsulation of several lipophilic or hydrophilic drugs (Landfester & The magnetic curves were obtained at Magnetism Laboratory of
Mailänder, 2013). University of Santiago, using a homemade vibrating sample magne­
Starch-based nanomaterials have attracted great interest due to their tometer at 300 K. The equipment was calibrated using a standard
good biocompatibility and their potential applications in drug delivery reference material (Yttrium Iron Garnet Sphere) from the National
carriers (nanospheres, micelles, nanogels and nanofibers) (Yu et al., Institute of Standards and Technology (NIST). After calibration, when all
2021). Hydroxyethyl starch (HES) is a starch derivative, whose hydroxyl the samples are centered in the correct saddle point position, the
groups at the C2 or C6 positions of each glucose unit are partially repeatability is similar to commercial VSMs (better than 2 %), the sen­
functionalized with hydroxyethyl groups (Kang et al., 2015). As a sibility is 1E− 5 emu, oscillation frequency is 37 Hz and amplitude is
medical agent, HES improves the transport of oxygen in organisms, 2 mm for such equipment. For all measurements, the magnetic moment
being commonly used for hypovolemia treatment, while some studies obtained for each applied field was normalized by the sample mass.
have reported HES nanocapsules as a biocompatible and biodegradable
drug delivery system (Baier et al., 2012; Kang et al., 2015). 2.3. Synthesis of SPIONs
Oncocalyxone A (onco A) is a quinone obtained from Auxemma
oncocalyx taub, a Brazilian northeast native tree. Onco A presents Citrate coated magnetite (Fe3O4@citrate, Fig. 1b) were synthesized
several pharmacological properties, such as: antiplatelet, leishmanici­ through two steps by a coprecipitation method (Favela-Camacho et al.,
dal, antimicrobial, analgesic, anti-inflammatory, hypoglycemic and 2019). In 250 mL of deionized water, 9.3 g of iron(II)chloride tetrahy­
antioxidant activity (Barreto et al., 2013; Tavares et al., 2019). In drate and 15 g of iron (III) chloride hexahydrate were dissolved. This
addition, onco A presents antitumor activity against three human tumor solution was stirred for 15 min at 50 ◦ C. Then, 30 mL of NH4OH 12 mol
cell lines: sarcoma 180, leukemia HL-60 and leukemia L1210 (Barreto L− 1 was added dropwise, resulting in a hydrophobic dark precipitate
et al., 2013; Tavares et al., 2019), which might generates several (Fe3O4). In the second step, 2.5 g of sodium citrate dihydrate was dis­
applications. solved in 20 mL of deionized water. The mixture was heated at 80 ◦ C for
Thus, the main goal of this work is to provide a versatile starch-based 1 h and the ammonia was evaporated. The magnetic precipitate was
magnetic nanocapsule with potential for magnetic targeting of hydro­ sedimented using a magnet. The supernatant was discarded for redis­
philic compounds, generating perspectives for highest concentration of persion of the solid in fresh solvent. The washing procedure was
hydrophilic drugs in a target tissue through magnetic vectorization. repeated with deionized water for five times. Finally, the Fe3O4@citrate
Onco A was the selected hydrophilic bioactive model for the develop­ nanoparticles were dried, dispersed in a minimal amount of deionized
ment of this work. water and stored at room temperature in a ferrofluid form.

2. Experimental sections 2.4. Synthesis of (onco A)-loaded HES MNCs

2.1. Chemicals The magnetic nanocomposite was prepared through an inverse

miniemulsion system (water in oil), inspired and adapted from previ­
Hydroxyethyl starch (HES) [≥ 99 %, molecular weight (Mw) ously published protocols (Baier et al., 2012; Kang et al., 2015). The
=260 kDa, degree of substitution (DS) = 0.15], Iron (II) chloride tetra­ organic phase (OP) was prepared by dissolving 50 mg of a lipophilic
hydrate (FeCl2.4H2O, ≥ 99.0 %), Iron (III) chloride hexahydrate emulsifier, polyglycerol polyricinoleate (PGPR) in 14.5 g of cyclo­
(FeCl3.6H2O, ≥ 99.0 %), sodium citrate dihydrate [HOC(COONa) hexane. This solution was homogenized using an ultrasound bath for
(CH2COONa)2. 2H2O, ≥ 99.0 %], tolylene-2,4-diisocyanate (TDI, ≥ 95.0 5 min and was separated in three different flasks (OP1 = 7.5 g,
%), sodium dodecyl sulfate (SDS, 99 %), phosphate buffered saline (PBS, OP2 = 5.0 g and OP3 = 2 g). Separately, the aqueous phase was prepared
pH 7.4), sodium nitrate (NaNO3, 99 %), potassium bromide (KBr, 99 %), through the solubilization (within 4 mL deionized water) of 4 mg of
deuterium oxide (99.9 atom % D) were purchased from Sigma-Aldrich® oncocalyxone A, 100 mg of HES and 40 μL of Fe3O4@citrate ferrofluid.
and used as received. Ammonium hydroxide (NH4OH, 30 %) and First, the OP1 was added dropwise to the aqueous phase at 1000 rpm for
cyclohexane (99 %) were purchased from Synth® and used as received. 30 min. This was followed by ultrasonication at 70 % of amplitude for
Polyglycerol polyricinoleate (PGPR Grindsted®) was donated by DuPont 3 min, with a pulse of 20 s on: 10 s off, using a Branson Sonifier
Danisco®. Distilled and ultrapure water (Milli-Q Advantage A-10 sys­ W-450-Digital and a ½” tip. Then, OP2 was added dropwise to the
tem, Millipore®) were used in this work. Onco A was isolated as a deep dispersed solution, repeating the same ultrasonication procedure. To the
red powder from the ethanol extract of the heartwood of A. oncocalyx, OP3, 50 mg of TDI was added, and this solution was added dropwise to
as previously described (Ferreira et al., 2008). the final formulated miniemulsion. The interfacial crosslink reaction
was carried out at 25 ◦ C for 24 h. For purification, the nanocapsules were
2.2. Apparatus dispersed in cyclohexane and centrifuged at 3000 rpm for 10 min. The
precipitate was redispersed in the same amount of fresh cyclohexane
The molecular weight for HES was determined by Gel Permeation and homogenized by vortex agitation. This process was repeated for 3

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A.C.C. Sousa et al. Carbohydrate Polymers 264 (2021) 118017

Fig. 1. TEM micrographs with particle size distribution histograms of a) uncoated Fe3O4. b) Fe3O4@citrate. c) (onco A)-loaded HES MNCs. (Red lines is Gaussian
distribution fit).

times. Finally, the (onco A)-loaded HES MNCs, dispersed in cyclo­ 2.7. In vitro and in vivo biological assays
hexane, were added slowly to an SDS (anionic surfactant) aqueous so­
lution (0.1 % w/w) and stirred for 24 h under 500 rpm at room The human tumor cell lines used in this study, SNB-19 (glioblas­
temperature, in an open vial to allow evaporation of the cyclohexane. toma), HCT-116 (colon carcinoma), PC3 (prostate) and HL-60 (leuke­
The final nanocomposite formulation resulted in a SDS aqueous mia), were provided by the National Cancer Institute (Bethesda, MD,
dispersion of (onco A)-loaded HES MNCs. USA). All the in vivo experimental procedures with zebrafish were
approved by the Animal Use Ethics Committee of the Federal University
of Ceará (CEUA No. 5639090320). Methodologies are described in the
2.5. Oncocalyxone A encapsulation efficiency (EE)
supporting information.
Previously published methods (Da Silva et al., 2020; dos Santos et al.,
3. Results and discussion
2020) were used to evaluate the bioactive encapsulation efficiency. The
(onco A)-loaded HES MNCs were centrifuged at 9000 rpm for 10 min
3.1. Nanocomposite characterizations
using a 3 kDa pore size ultracentrifugation filter to separate the loaded
magnetic nanocapsule from the free onco A. Then, in triplicate inde­
Fe3O4@citrate nanoparticles, synthesized by the coprecipitation
pendent assays, the non-magnetic filtered aqueous fraction was
method, were evaluated by X-ray diffraction (Supporting information,
analyzed by UV–vis spectroscopy. Thus, the onco A encapsulation effi­
Fig. S1), exhibiting XRD signals with sharp peaks that indicate the high
ciency was determined by the difference between the initial amount of
crystallinity of the synthesized magnetite nanoparticles. The peaks were
bioactive added to the HES MNCs formulation and the free amount
compared with the commercial patterns for iron faces, with peaks at
observed after the filtration process in the non-magnetic fraction, as
(111), (220), (311), (400), (422), (511) and (440), which can be
represented by the Eq. (1):
attributed to the crystal planes of a cubic spinel structure (ICSD number
EE(%) = [(M(total drug) − M(free drug) ] × 100 (1) 065339) (Hrdina et al., 2010; Li et al., 2017; Paredes-García et al.,
2013). No additional peaks were observed, revealing the absence of
where M(total drug) represents the total concentration of onco A added to secondary phases related to impurities or common oxidation processes
the HES MNCs formulation and M(free drug) represents the onco A in iron oxide nanoparticles, indicating a good efficiency of the copre­
unencapsulated fraction obtained in the non-magnetic filtered aqueous cipitation method used for SPIONs synthesis.
fraction. The molecular weight for HES (260 kDa) was determined by GPC
(Kilicarislan Ozkan et al., 2019) (Fig. S2). A degree of substitution (DS)
of 0.15 (SEq. 1) was determined by 13C-NMR (Chen et al., 2020; Cuenca
2.6. Release studies
et al., 2020) (Fig. S3).
FTIR was evaluated (Fig. S4b) in order to study the (onco A)-loaded
The release profile of oncocalyxone A from (onco A)-loaded HES
HES MNCs formation. The nanocapsule polymeric shell was generated
MNCs was evaluated using vertical Franz diffusion cells, adapted from
by the chemical reaction between the cross-linker TDI and some portion
previously protocols (De Almeida et al., 2017; Marto et al., 2018). A
of OH groups from HES (Scheme 1). This leads to the consumption of
dialysis membrane (permeation area: 1.75 cm2) having molecular
part of the OH groups from HES which can be observed by the decrease
weight cutoff about 12 kDa was fixed between the receptor and the
of the relative intensity of the band around 3400 cm− 1 (Fig. S4b, gray
donor compartment. 2 mL of the (onco A)-loaded HES MNC formulation
line) that is related to the asymmetric stretching vibration of OH groups
were added to the donor compartment. The receptor compartment was
(Torlopov et al., 2020). Additionally, the appearance of bands at
prepared with 14 mL of buffered phosphate solution (pH = 7.4). The
1740 cm− 1 (C– – O stretching of carbonyl groups) and 1546 cm− 1 (N–H
release assay was carried out, independently, for free onco A in the same
deformation) suggests the formation of urethane linkages (dos Santos
buffered phosphate solution. Samples were analyzed by UV–vis spec­
et al., 2020; Steinmacher et al., 2017; Zhang et al., 2017), and are not
troscopy. The release studies were evaluated in two independent ex­
observed in the unreacted HES molecules (Fig. S4b, black line). The
periments performed in triplicate.

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A.C.C. Sousa et al. Carbohydrate Polymers 264 (2021) 118017

Scheme 1. Representative illustration showing the formation of (onco A)-loaded nanocomposite. Images are out of scale.

intense band at 2920 cm-1 is characteristic of the presence of C–H (sp3) (Moghadam Ziabari et al., 2020) as observed in hysteresis loops by VSM
stretching (Taheri et al., 2014; Zhang et al., 2017). The peak at measurements at room temperature (Fig. 2). The organic surface coated
2280 cm− 1 indicates an uncompleted consumption of NCO groups from in the magnetite nanoparticles added to them a mass from citrate
TDI (Steinmacher et al., 2017; Taheri et al., 2014). Several bands present groups, however, did not lead to a significant change in the magnetic
in free onco A (Fig. S4b, pink line), such as C–H stretching around nanoparticles behavior (Fig. 2a). The saturation magnetization values
2850 cm− 1, C– – C and C–– O stretching vibrations at 1600 and 1230 cm-1, obtained for MNPs were 58.98 and 56.21 emu/g for Fe3O4 and
respectively (Hadj-Hamou et al., 2014), also were observed in (onco Fe3O4@citrate, respectively, which is in the range of values
A)-loaded HES MNCs (Fig. S4b, gray line), indicating the presence of (30–80 emu/g) reported for synthetic magnetite powders (Noval &
onco A in the synthesized magnetic nanocomposite. Universitaria, 2019).
TEM analysis of the synthesized uncoated Fe3O4 nanoparticles The magnetic nanocapsules showed a much lower magnetization
(Fig. 1a) showed an average size of 12 nm. However, with the addition (Fig. 2b) value than MNPs samples. This is expected due to the incor­
of the citrate groups (Fe3O4@citrate, Fig. 1b), the size distribution of the poration of the nonmagnetic organic materials on nanocapsule shell,
nanoparticles decreased to an average size of 10 nm. This decrease which reduces the percentage of magnetic mass in the sample, while
suggests that the citrate groups prevent small aggregations of the increasing the total mass (mass dilution effect). However, despite having
nanomaterial. In both cases, it was possible to observe a spherical reduced the sample magnetization by around 45 times, the nanocapsules
morphology profile and a polydisperse particle size. Fe3O4@citrate still maintained magnetic character, responding to an external magnetic
nanoparticles also exhibited a higher aqueous stability, due its organic vectorization.
coated that reduce the dipolar attraction between them (Barreto et al.,
2013). 3.2. Onco A release studies
It is possible to observe a well-defined core-shell morphology of the
synthesized (onco A)-loaded HES MNCs (Fig. 1c), with the SPIONs Measurements of the electronic spectrum of the drug were made at
(Fe3O4@citrate) inside the core, as darker spots, and the nanocapsule as different known concentrations (Fig. 3a) to generate a calibration curve
a large container. These structures were observed in different evaluated (Fig. 3b). According Eq. (1), the onco A encapsulation efficiency was EE
areas of the TEM images, presenting nanocapsules with an average = 92.43 %. Therefore, this high EE(%) suggest that the synthetic pro­
diameter of 143 nm (Fig. 1c). The core-shell morphology can be posed mechanism of inverse miniemulsion was effective for the co-
explained by the nature of the nanocapsule formation process, called encapsulation of onco A and magnetic nanoparticles as a hydrophilic
inverse miniemulsion technique. In this process, an interfacial poly­ drug releasing nanosystem.
merization reaction occurs at the interface of stable nanosized droplets The release behavior of onco A from (onco A)-loaded HES MNCs was
obtained through ultrasonication (Kang et al., 2015; Singh et al., 2017). evaluated for 72 h in PBS medium (Fig. S6a), using a vertical Franz
In the water-in-oil droplet interface, the reticulation agent (TDI) reacts diffusion cell equipped with a dialysis membrane. Then, the nano­
with the hydroxyl groups from HES, generating the nanocapsule capsules release behavior was compared with the onco A (free drug)
cross-linked polymeric shell by urethane linkages (Scheme 1) (dos diffusion behavior (Fig. 3c, black and blue lines). During the initial 6 h of
Santos et al., 2020; Kang et al., 2015). the experiment, about 65 % of the free onco A was diffused from donor
Additionally, the synthesized nanocapsules also were evaluated in to receptor compartment (Fig. 3c, blue line). In the same period, only 40
solution by dynamic light scattering (DLS) (Fig. S5), showing a hydro­ % of the drug was released from the nanocapsules (Fig. 3c, black line).
dynamic diameter average of 264 nm. This diameter average is related After 24 h, the release of onco A from nanocapsules is minimal, since a
with the Brownian fluctuations in solution and the hydrodynamic radius plateau of stabilization is reached around 80 % of released drug. The
(Soares et al., 2020), being higher than that observed by TEM, where the nanocapsule polymeric shell plays a significant role in the release pro­
nanocapsules were completely dried (Chamorro Rengifo et al., 2019; file, protecting the incorporated bioactive and prolonging the release
Zhang et al., 2019). process. In this context, a similar behavior was reported with other
The magnetic properties of magnetic nanoparticles (MNPs) are polymeric wall nanocapsule systems, where approximately 70 % of drug
highly influenced by the particle size and domain structure. A single- is released from the nanocarriers within 30 h of beginning the release
domain allows these MNPs to display a superparamagnetic behavior test (Mora-Huertas et al., 2010).
(no remaining magnetization after the action of a magnetic field) The release data were fitted to different kinetics model, obtaining an

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A.C.C. Sousa et al. Carbohydrate Polymers 264 (2021) 118017

Fig. 2. Magnetic hysteresis loops at 300 K for a) Fe3O4 (black line) and Fe3O4@Citrate (blue line). b) (onco A)-loaded HES MNCs (Red line). Inset in panels a) and b)
magnified view of the small field region from − 400 to 400 Oe.

Fig. 3. a) Electronic spectra of onco A at different concentrations in PBS 0.1 mol L− 1 (pH = 7.4) at 310 K. b) Plots of [onco A] vs absorbance at 282 nm (Calibration
Curve). c) Release profile of onco A (free drug), (onco A)-loaded HES MNC and remnant profile of (onco A)-loaded HES MNC in PBS 0.1 mol L− 1 (pH = 7.4) at 310 K.
d) Plots of onco A (%) vs Time between 1 to 6 h for released and remnant weight.

intermediate behavior between Higuchi (Fig. 3d) and Korsmeyer-Peppas planar surface or a spherical pellet having a homogeneous matrix), in
(Fig. S6d) model, showing R-squared 0.9760 and 0.9740 respectively. which the release rate is decreased with time (Higuchi, 1963). On the
However, the Higuchi model is more suitable for studying the release other hand, for HES-based nanocapsules release systems (Pereira et al.,
profile of solid drugs incorporated into a solid matrix (release from a 2020), the release behavior is affected by conformational changes,

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A.C.C. Sousa et al. Carbohydrate Polymers 264 (2021) 118017

swelling/deswelling or solubilization of the polymer chains of nano­

carriers, due to hydrolytic cleavage or by the introduction of ionizable
groups, which expands the volume, generating more porous to drug
release (Pereira et al., 2020; Pramanik et al., 2018), following
Korsmeyer-Peppas as the main suitable kinetics model.

3.3. In vitro and in vivo assays

The MTT assay was performed to investigate cytotoxicity and to

calculate IC50 values for free and encapsulated oncocalyxone A, against
four human tumor cell lines (Table 1). Data are represented in IC50
values (μgmL− 1) and correspond to the mean ± SD of at least four in­
dependent experiments performed in triplicate.
According to the results in Table 1, free onco A exhibited cytotoxicity
Fig. 4. Antiproliferative activity of free and encapsulated onco A in four tumor
for all tested tumor cell lines, showing the lowest IC50 values for HL-60
cell lines evaluated by the MTT assay after 72 h treatment. Data are presented in
cells. The cytotoxic activity was higher for (onco A)-loaded HES MNCs
IC50 values (μgmL− 1) and correspond to the mean ± SD of at least four inde­
when compared to onco A free drug and was even higher for the (onco pendent experiments performed in triplicate. * p < 0.05 compared to onco A
A)-loaded nanocapsules without SPIONs (system C). Consequently, the (free drug); ** p < 0.05 compared to (onco A)-loaded HES MNCs; and #
system C exhibited the greatest antitumor activity in all tested cell lines, p < 0.05 compared to HES MNCs (without onco A) by ANOVA followed by
which may suggest that the SPIONs incorporated into the nanocapsule Dunnett’s test.
seems to decrease the nanocomposite cytotoxicity (comparing systems B
and C). Despite that, the influence of SPIONs in the IC50 values is not treatment was included (Naive group). After 96 h, the number of dead
worrisome, once system B still exhibits low IC50 values and, conse­ fish in each group was recorded, and the lethal concentration capable of
quently, a great potential for anticancer applications. Therefore, the killing 50 % of the animals (LC50) was determined (Table 1). For all
main role of SPIONs in the synthesized nanocomposite is to respond to tested groups, no deaths were observed (Table 1), suggesting potential
an external magnetic field, generating perspectives for magnetic tar­ for in vivo applications.
geting of encapsulated drugs. The locomotor activity is an important indicator for the behavioral
Additionally, a blank control sample (system D) that consists of the analysis of drug affecting the central nervous system, evaluating
nanocomposite formulation without onco A was also evaluated. The whether the sample tested can cause locomotor impairment (Lira et al.,
system D also exhibited cytotoxicity, but with less potency than (onco 2020). The nanocomposite effects in the locomotor activity of adult
A)-loaded nanocapsules (system C), for all tested tumor cell lines. The zebrafish were evaluated (Fig. 5b). The results showed that, for the
cytotoxicity observed in system D (without onco A) might be explained control group, the SDS solution caused impact on the central nervous
by the reminiscent SDS concentration (0.1 % w/w) in the nano­ system of the animals. However, this impact was not increased by the
composite formulation, which may act as a detergent, disrupting the nanocomposite formulation (25, 50 and 75μgmL− 1) (Fig. 5c). Thus, the
cell membrane, leading to in vitro cell lysis and, consequently, cell decrease of the reminiscent SDS concentration in the final formulation
death (Avila-Calderón et al., 2020). Any residual TDI from the contributes for in vivo applications.
formulation process might also be contributing for the cytotoxicity,
although the final product polyurethane (in the crosslinked nano­ 4. Conclusions
capsule shell) is a biocompatible linkage (Barman et al., 2020).
Nonetheless, when the acute toxicity of this formulation was evaluated A magnetic nanocomposite with sizes in the range of 100–340 nm
by an in vivo toxicity assay (zebrafish model), no deaths were observed was successfully synthesized through an inverse miniemulsion
(Table 1), even in a much higher concentration (75 μgmL− 1) than those method. This nanocomposite has a well-defined core-shell
tested in the in vitro experiments. It emphasizes the formulation po­ morphology and superparamagnetic characteristics, which allows the
tential for further in vivo anticancer applications. The data from Table 1 co-encapsulation of a natural hydrophilic anticancer bioactive with
are illustrated in Fig. 4. SPIONs in the same nanostructure. The nanocomposite exhibited in
The nanocomposite in vivo toxicity was evaluated for the first-time vitro cytotoxicity against four human tumor cell lines and provided no
using a zebrafish model. The fishes (n = 8/group) were randomly deaths in the in vivo acute toxicity assay (zebrafish model), generating
selected and orally treated (Fig. 5a) with 20 μL of (onco A)-loaded HES perspectives for magnetic vectorization of anticancer drugs (Fig. 6).
MNCs formulations (25 μg onco A/mL or 50 μg/mL or 75 μg/mL) or The SPIONs (Fig. 6a) were incorporated in the nanocapsules (Fig. 6b)
control sample that contains only SDS solution (formulation vehicle 0.1 and these nanocapsules were capable to respond to an external
% w/w, without nanocomposite). A group of animals without any

Table 1
Antiproliferative activity of free and encapsulated onco A in four tumor cell lines evaluated by the MTT assay after 72 h treatment. Acute toxicity study (96 h) for (onco
A)-loaded HES MNC in adult Zebrafish model.
MTT (IC50 μgmL− 1) – 72 h Adult Zebrafish mortality
(μgmL− 1) – 96 h

Samples PC3 SNB-19 HCT-116 HL-60 Control 25 50 75 LC50

A onco A (free drug) 12.99 ± 2.127 30.26 ± 6.027 10.68 ± 1.415 3.259 ± 0.754
B (onco A)-loaded HES MNCs 4.868 ± 1.768* 1.010 ± 0.480* 2.675 ± 0.435* 2.166 ± 0.489* 0 0 0 0 >75
C (onco A)-loaded nanocapsules (without SPIONS) 2.504 ± 0.431* 0.708 ± 0.137* 1.452 ± 0.343*,** 1.235 ± 0.206*,#
D HES MNCs (without onco A) 4.765 ± 1.326* 0.949 ± 0.234* 2.245 ± 0.545* 3.557 ± 1.443
*
p < 0.05 compared to onco A (free drug) by ANOVA followed by Dunnett’s test.
**
p < 0.05 compared to (onco A)-loaded HES MNCs by ANOVA followed by Dunnett’s test.
#
p < 0.05 compared to HES MNCs (without onco A) by ANOVA followed by Dunnett’s test.

6
A.C.C. Sousa et al. Carbohydrate Polymers 264 (2021) 118017

Fig. 5. a) Oral treatment representation. b) Open Field Test representation. c) Effect of (onco A)-loaded HES MNCs in the locomotor activity of adult zebrafish (Danio
rerio) through the Open Field Test (0–5 min). Naive: untreated animals. Control: SDS solution 1 mg/mL (without nanocomposite) in distilled water (20 μL).

Fig. 6. Macroscopic view of a) SPIONs in ferrofluid form responding to an applied magnetic field. b) HES MNCs without magnetic field. c) HES MNCs with an
external magnetic field. d) (onco A)-loaded HES MNCs targeting onco A (anticancer drug) through an external magnetic field.

magnetic field (Fig. 6c). Thus, the nanocomposite synthesized in this Acknowledgements
work represents a smart perspective for drug delivery, since it can
transport hydrophilic drugs by magnetic vectorization (Fig. 6d). This study was financed in part by the Coordenação de Aperfeiçoa­
Further studies will be conducted to better investigate the nano­ mento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.
composite anticancer activity and biocompatibility, including in vivo PROEX 23038.000509/2020-82. N.M.P.S.Ricardo would like to thank
studies. CNPq for the financial support throughout the Project Nº 307837/2017-
3. We would like to thank the Bioinorganic Group of the Department of
CRediT authorship contribution statement Organic and Inorganic Chemistry of the Federal University of Ceará for
the UV-vis measurements. We also thank Prof. Dr. Yogeshvar N. Kalia of
Alexandre C.C. Sousa: Conceptualization, Investigation, Method­ University of Geneva for the encouragement and support for further
ology, Formal analysis, Visualization, Writing - original draft. Adolfo I. studies.
B. Romo: Investigation. Raimundo R. Almeida: Investigation. Aiêrta
C.C. Silva: Investigation. Lillian M.U. Fechine: Investigation. Débora Appendix A. Supplementary data
H.A. Brito: Formal analysis. Rafael M. Freire: Formal analysis. Daniel
P. Pinheiro: Formal analysis, Investigation. Larissa M.R. Silva: Formal Supplementary material related to this article can be found, in the
analysis. Otília D.L. Pessoa: Resources. Juliano C. Denardin: Formal online version, at doi:https://doi.org/10.1016/j.carbpol.2021.118017.
analysis, Funding acquisition. Claudia Pessoa: Formal analysis, Fund­
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