Carbohydrate Polymers 301 (2023) 120310

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

Microcapsules based on alginate and guar gum for co-delivery of

hydrophobic antitumor bioactives
Louhana M. Rebouças a, b, Alexandre C.C. Sousa a, b, Caroline G. Sampaio b, Larissa M.R. Silva c,
Pedro M.S. Costa d, Cláudia Pessoa d, Nilce V.G.P.S. Brasil a, 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 60440-900, Brazil
b
Federal Institute of Education, Science and Technology of Ceará, Fortaleza, CE 60410-426, Brazil
c
Department of Food Engineering, Federal University of Ceará, Fortaleza, CE 60356-000, Brazil
d
Laboratory of Experimental Oncology, Center for Research and Drug Development, Federal University of Ceará, Fortaleza, CE 60430-275, Brazil

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

Keywords: The main goal was the development of a polysaccharide microcapsule for anticancer application based on guar
Betulinic acid gum and sodium alginate for the controlled release of hesperidin and betulinic acid by spray drying technique.
Hesperidin The microcapsule showed an Encapsulation Efficiency of 98.15 ± 0.34 % for hesperidin and 99.76 ± 0.22 % for
Polysaccharide microcapsules
betulinic acid. In the release study, the Korsmeyer-Peppas mathematical model was identified as the most
Guar gum
Alginate
adequate to explain the observed release mechanism. In vivo tests were performed in zebrafish model, revealing
HL-60 that the microcapsules did not alter the locomotor activity and were not toxic within 96 h by oral administration,
suggesting their biological safety. In vitro cytotoxic activity against HL-60 cells confirmed an IC50 value of 2.52 ±
0.23 μg mL− 1 in 72 h. Additionally, a decrease in the cytotoxic activity of betulinic acid against L-929 (non-
tumor) cells was evidenced. Therefore, the microcapsules synthesized in this work represent a promising
formulation for anticancer applications.

1. Introduction protection of active components (Goh et al., 2012). The structure of

Polysaccharide microcapsules can promote the controlled release of
actives, allowing the carrying of hydrophobic actives encapsulated by
emulsions. After preparing the oil-based emulsion containing the ac­
tives, polymers and surfactants, it can then be sprayed in a hot chamber
through a process known as spray-drying (Martins et al., 2017).
Galactomannans can be used as an encapsulating polymer in mi­
crocapsules. These polymers consist of a skeleton formed by repeating
β-d-mannose units linked together by 1–4 bonds. The α-d-galactose units
are linked to the main chain by 1–6 type bonds. Among a hundred
legume species whose endosperm is formed by galactomannan, only
Ceratonia siliquia and Cyamopsis tetragonolobus have been widely culti­
vated for commercial purposes (Azero & Andrade, 1999). The poly­
saccharide obtained from Cyamopsis tetragonolobus is the guar gum.
Sodium alginate (NaC6H7O6)n is another polysaccharide used as an
encapsulant agent, derived from alginic acid, which is capable of
forming a highly versatile, biocompatible, non-toxic matrix for the
alginic acid consists of linear chains of β-D-mannuronic acid (M) resi­
dues joined by type (1 → 4) bonds and residues of its epimer, α-L-
guluronic acid (G), in various proportions. These residues are arranged
in the form of blocks of mannuronic (M) or guluronic (G) acids, linked in
such a way that the sequence of these residues in the molecule is
alternated. Alginate is mucoadhesive and can adhere to the intestinal
mucosa for prolonged periods of time. Studies have reported alginate
microspheres for the development of oral protein and drug delivery
systems (Qurrat-Ul-Ain et al., 2003). Other studies report therapeutic
efficacy and safety profiles of alginate comparable to those of omepra­
zole in patients with non-erosive gastroesophageal reflux (Saifullah
et al., 2018). Thus, alginate can be considered as one viable option
against heartburn associated with the use of aggressive drugs to the
stomach mucosa. In this context, the use of alginate in oral formulations
containing acidic actives can be explored as a smart strategy.
Betulinic acid (3-beta-hydroxy-lup-20(29)-en-28-oic acid) is a hy­
drophobic anticancer substance with selective action reported in the

* Corresponding author.
E-mail addresses: louhanar@gmail.com (L.M. Rebouças), alexandre.carreira@ifce.edu.br (A.C.C. Sousa), carolinesampaio@ifce.edu.br (C.G. Sampaio),
larissamrs@ufc.br (L.M.R. Silva), pmikaelcosta@alu.ufc.br (P.M.S. Costa), cpessoa@ufc.br (C. Pessoa), nilce@dqoi.ufc.br (N.V.G.P.S. Brasil), naricard@ufc.br
(N.M.P.S. Ricardo).

https://doi.org/10.1016/j.carbpol.2022.120310
Received 5 September 2022; Received in revised form 14 October 2022; Accepted 2 November 2022
Available online 8 November 2022
0144-8617/© 2022 Elsevier Ltd. All rights reserved.
L.M. Rebouças et al.
literature, considered highly promising as an antineoplastic bioactive
after the discovery of its induction of apoptosis in melanoma cell lines in
vitro and in vivo (Pisha et al., 1995). However, betulinic acid has some
limitations in terms of its low solubility (0.02 μg mL− 1) (Jäger et al.,
2007). As a way of circumventing this problem, new administration
strategies, such as nano and micro formulations, like polysaccharide
microcapsules, have been investigated (Csuk, 2014).
Anticancer actives for oral administration can be protected against
direct contact with stomach fluid through microencapsulation. Also, a
controlled release can decrease its cytotoxicity in non-tumor cells
(antitumor selectivity) (Rebouças et al., 2022). Recently, a decrease in
the cytotoxicity of encapsulated betulinic acid in comparison to the free
active was shown due to the sustained release promoted by nano­
encapsulation, which may suggest a reduction in side effects for treat­
ments based on encapsulated actives (Rebouças et al., 2022).
Additionally, the reduction of drug side effects was also shown in a
recent study about the strong association between slow release of met­
formin formulations and reduced gastrointestinal side effects (Tarry-
Adkins et al., 2021).
Besides the protection against stomachal degradation and the
reduction of side effects, the microencapsulation process also can
generate co-delivery systems in order to induce the possibility of syn­
ergistic effects. In this context, nanoparticles represent a useful platform
to promote drug co-delivery, due to their ability to simultaneously
deliver adequate doses of different drugs to individual cells across the
endothelial barrier, producing synergistic therapeutic effects, as well as,
suppressing the emergence of multidrug resistance (MDR) (Sindhwani
et al., 2020; Zeinali et al., 2020). For instance, there are reports indi­
cating that the association of the bioactive flavonoid hesperidin with the
chemotherapeutic chlorogenic acid and doxorubicin increases the anti­
tumor effect synergistically against MCF-7 (breast cancer) and Hela cells
(cervical carcinoma) (Majumdar & Srirangam, 2009; Kusharyanti, 2011;
Hsu et al., 2021). However, no study has investigated the co-encapsu­
lation of hesperidin and betulinic acid in polysaccharide microcapsules
or any other nano or micro system.
Therefore, our hypothesis is that microcapsules based on alginate
and guar gum could be synthesized in order to evaluate the synergistic
association between hesperidin and betulinic acid, as well as, its
simultaneous co-delivery and controlled release to enhance the anti­
cancer action and decrease the cytotoxicity against healthy cells. Thus,
the microencapsulation strategy proposed in this study has three ratio­
nales: (i) to protect the actives against direct contact with the stomach
fluid, (ii) to induce the promotion of mucus adhesion in the intestinal
epithelium and, (iii) to reduce the cytotoxicity for non-tumor cells
through sustained release.
2. Experimental sections
2.1. Materials
Sodium alginate PA (Mw = 4.56 × 105, M/G ratio = 1.36 and guar
gum PA (Mw = 11.2 × 106, M/G ratio = 1.60) were purchased from
Êxodo Científica (São Paulo, Brazil). Hesperidin 95 % and betulinic acid
98 % were purchased from Xi'an Quanao Biotech Co. (Xi'an, China). The
previously characterized flaxseed oil was obtained from another work at
the Laboratory of Polymers and Materials Innovation (LabPIM). Plur­
onic® F127 was purchased from Sigma-Aldrich (Germany). Chloroform
PA and dimethyl sulfoxide (DMSO) PA were purchased from Labsynth
(São Paulo, Brazil). HPLC-grade methanol and HPLC-grade acetonitrile
were purchased from Tedia (USA). MTT Formazan was purchased from
Merck Millipore (Germany). Ultrapure water was obtained from a Milli-
Q purifier.
2.2. Cell lines and culture
The cell line used in this study HL-60 (promyelocytic leukemia) was
2
Carbohydrate Polymers 301 (2023) 120310
provided by the National Cancer Institute (NCI, USA) and the non-tumor
cell line L929 (murine fibroblast) was provided by the BCRJ (Banco de
Células do Rio de Janeiro). Both were grown in their respective media,
Roswell Park Memorial Institute (RPMI) 1640 and Dulbecco's Modified
Eagle Medium (DMEM, Gibco®), supplemented with 10 % fetal bovine
serum (Gibco®) and 1 % antibiotic (Sigma-Aldrich Co, St. Louis, MO,
USA), incubated under 5 % CO2.
2.3. Synthesis and characterization of nanoemulsions
The nanoemulsions were synthesized to be used in the preparation of
microcapsules according to the amounts described in the supplementary
material. Initially, betulinic acid was solubilized in 4 mL of chloroform,
while hesperidin was separately solubilized in 4 mL of methanol. Then,
these two solutions were added to the flaxseed oil, homogenized, and
rotoevaporated at 40 ◦ C under reduced pressure until the chloroform
and methanol were completely removed. Subsequently, this solution
was added to an aqueous solution containing Pluronic® F127. The for­
mulations were then sonicated in a Branson Sonifier W-450D sonicator
(Teltow, Germany, Hielsher) with probe, 70 % amplitude and 100–103
W power, for 2 min in 12 cycles of 10 s on and 10 s off, in an ice bath
(Rebouças et al., 2022).
Size, polydispersity index (PdI) and zeta potential were determined
by dynamic light scattering (DLS) using a NanoZS® (Malvern In­
struments, Worcestershire, UK). Measurements were made at a 90◦
angle after dispersing 50 μL of the nanoemulsions in 5.0 mL of Milli-Q
water. All measurements were performed in triplicate at 25 ◦ C with
comparable conductivity to determine the zeta potential.
2.4. Obtaining microcapsules by spray-drying
Microcapsules based on nanoemulsion and polysaccharides were
obtained according to the amounts described in the supplementary
material. Initially, 200 mL of aqueous solution containing the poly­
saccharides was prepared under stirring at 500 rpm for 12 h. The other
components of the formulation were added and homogenized for
another 1 min in the same rotation in an ice bath. The microcapsules
were obtained after drying the solutions in a Mini Spray Dryer B-290
(Büchi). The inlet and outlet temperatures were 170 and 91 ◦ C,
respectively (Gallardo et al., 2013). The feed flow was 10 mL min− 1
(pumper rate 32 %) and the aspirator volume gas flow was 35 m3 h− 1
(100 % of max. Aspirator rate). No pulse nozzle cleaner and nozzle tip
diameter of 0.7 mm were used. These conditions were obtained after
drying optimization tests in order to ensure the highest yield and pre­
serve the drugs, obtaining a yield of 41 % on drying.
2.5. Morphology
The microcapsules and crystalline actives were fixed on metallic
stubs and sprayed with silver for microscopic evaluation of appearance
and morphology. Microcapsule morphology was observed using a
Quanta 400 FEG scanning electron microscope (SEM) at 5–6 kV (FEI,
Hillsboro, OR, America).
2.6. Encapsulation efficiency
The quantification of hesperidin and betulinic acid in the micro­
capsules was made by High Performance Liquid Chromatography
(HPLC) to obtain the value of the Encapsulation Efficiency (EE%). The
method for determining of encapsulation efficiency is in accordance
with Rehman et al. (2021) and the methods of quantification by HPLC is
in accordance to Saeidi et al. (2011) and Wójciak-Kosior et al. (2017),
been adapted and validated according to described in the supplementary
material.
L.M. Rebouças et al.
2.7. Fourier transform infrared spectroscopy (FTIR)
Infrared spectra were obtained in a Shimadzu IRTracer-100 model
spectrometer, in the wavenumber region from 4000 to 400 cm− 1. About
5 mg of each sample was mixed and pressed with KBr.
2.8. Thermal analysis by differential scanning calorimetry (DSC)
DSC analyses were performed on a Shimadzu DSC-Q50 equipment in
an inert atmosphere (N2, 50 mL min− 1). The program had a single run
from 25 to 350 ◦ C, at a heating rate of 10 ◦ C min− 1. The mass of each
sample was 10 mg.
2.9. X-ray diffraction
The microcapsule sample and the actives were analyzed using a D8
Advanced Bruker-AXS Diffractometer (Germany), equipped with a θ/θ
goniometer using a Cu Kα radiation source. The scan was performed in
the diffraction angle region (2θ) from 5◦ to 90◦ with a step size of 0.02◦ .
The samples were ground to a fine powder before analysis.
2.10. In vitro release kinetic study
The in vitro release kinetic study of co-encapsulated betulinic acid
and hesperidin was performed as described in the supplementary
material.
2.11. Non-clinical safety testing in zebrafish
The non-clinical safety study was evaluated using adult zebrafish
(Danio rerio) through tests of acute toxicity in 96 h and locomotor ac­
tivity (Open Field Test), following the methodology described in the
supplementary material. All experimental procedures were approved by
the Ethics Committee for the Use of Animals of the Federal University of
Ceará (CEUAP - UFC), under protocol number 1806202101 and meth­
odology based on the OECD protocols (1992) and EU Directive 2010/
63/EU.
2.12. MTT assay
The cytotoxic potential of betulinic acid, hesperidin, the combined
actives and the microcapsules and microcapsules controls were deter­
mined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay according to described in the supplementary
material (Ye et al., 2018). Cell viability at a single concentration of 500
μg mL− 1 and IC50 determined by the graphical and regression method
were calculated from the results obtained from the diluted samples.
2.13. Statistical analysis
Statistical evaluations of the results were analyzed by GraphPad
Prism 5.0. IC50 values are expressed as mean ± standard deviation (SD).
For comparison of several groups, analysis of variance (ANOVA) was
applied considering p < 0.05 as statistically significant.
3. Results and discussion
3.1. Characterization of nanoemulsions
The prepared nanoemulsions presented results of size smaller than
250 nm, zeta potential and polydispersity index (Pdi) characteristic of
stable systems. The Pdi provides information on the homogeneity of the
size distribution, according to Yakoubi et al. (2021). The NE-D (diam­
eter: 201 ± 5 nm, Pdi: 0.09 ± 0.02, zeta potential: − 29.4 ± 0.8 mV), NE-
B (diameter: 195 ± 7 nm, Pdi: 0.08 ± 0.03, zeta potential: − 26.9 ± 1.1
mV) and Control-NE (diameter: 179 ± 3 nm, Pdi: 0.09 ± 0.04, zeta
3
Carbohydrate Polymers 301 (2023) 120310
potential: − 35.7 ± 1.7 mV) nanoemulsions were narrowly mono­
dispersed while the NE-H (diameter: 238 ± 4 nm, Pdi: 0.21 ± 0.02, zeta
potential: − 24.1 ± 1.0 mV) nanoemulsion was moderately poly­
dispersed. The zeta potential can also be used as an indicator of stability.
When high molar mass surfactants like the one used in this work
(Pluronic F127) are used, which act mainly by steric stabilization, zeta
potential values of only 20 mV in modulus or much lower can provide
stability (Honary & Zahir, 2013). From the results, it was found that the
nanoemulsions presented desirable results for incorporation into the
polymeric solutions to be dried to obtain the microcapsules.
3.2. Morphology
The micrographs of the actives and microcapsule were obtained by
Scanning Electron Microscopy are shown in Fig. 1.
Through the images, the crystalline aspect of the active betulinic acid
and hesperidin can be identified. The MNED microcapsule presented an
approximate size of around 0.75 μm and 6 μm with a predominantly
spherical shape.
3.3. Encapsulation efficiency
The data obtained regarding the validation of the analysis method for
the quantification of drugs by HPLC are available in the supplementary
material.
From the powders that were obtained, the Encapsulation Efficiency
for each formulation was determined. The EE% is a useful tool to assess
the encapsulation process of assets. In Table 1, the values of Encapsu­
lation Efficiency are presented.
High values of EE% are observed for the microcapsules. This fact can
be explained by the total solubility of the hydrophobic actives in the
flaxseed oil, which can be confirmed through the micrographs obtained
by SEM (Scanning Electronic Microscopy). Rehman et al. (2021) found
EE% values similar (93.05 %) to the MNED microcapsule in microcap­
sules based on curcumin nanoemulsions (insoluble in water) encapsu­
lated in Borago officinalis seed oil using commercial gums Hicap 100 and
Purity Gum 2000.
3.4. Fourier transform infrared spectroscopy (FTIR)
To identify the components of the formulation or any chemical
interaction in the microcapsule, FTIR spectra were obtained as shown in
Fig. 2.
The spectra obtained from hesperidin and betulinic acid showed
typical profiles of their structures (Cîntă-Pînzaru et al., 2012; Balak­
rishnan et al., 2021). In the flaxseed oil spectrum, the intense charac­
teristic peak of the ester carbonyl stretching at 1745 cm− 1 is identified.
Analyzing the guar gum spectrum, the broad band at 3400 cm− 1 is
identified, corresponding to the stretching vibrations of the O–H bond
of the hydroxyl groups. The vibrations of the C–H bonds appear at 2920
cm− 1 (Rébufa et al., 2019).
The sodium alginate spectrum reveals a broad band at 3390 cm− 1
corresponding to the stretching of the O–H bond of the hydroxyl groups
present in the alginate polymer chain. The intense band observed at
1414 cm− 1 is correlated to the –COO– stretching. At 1032 cm− 1 an
intense and broad peak is identified due to the overlapping of the C–C
bonds. At 1604 cm− 1, an asymmetrical stretch peak of the carboxylate is
identified (Pilipenko et al., 2019). In the microcapsule spectrum, at 1943
and 2924 cm− 1, the characteristic signs of flaxseed oil can be identified.
The presence of guar gum in the composition is also visualized, being
possible to identify the presence of alginate by the signs referring to the
stretching of -COO- and linseed oil through the carbonyl sign, not being
possible to visualize the inherent signs referring to hesperidin and
betulinic acid because they are in a lower concentration in the compo­
sition. FTIR spectra of galactomannan microcapsules containing fluox­
etine synthesized by Josino et al. (2021) also did not show the active that
L.M. Rebouças et al.
Fig. 1. Micrographs obtained by SEM: (a) betulinic acid (b) hesperidin and (c) MNED microcapsule.
was in lesser amount and identified bands of galactomannan that was
found in greater quantity.
3.5. Thermal analysis by differential scanning calorimetry (DSC)
Differential Scanning Calorimetry (DSC) analyzes were performed to
understand the thermal behavior of the samples and to evidence the
encapsulation as shown in Fig. 2. The DSC curves of betulinic acid and
hesperidin are typical of crystalline material presenting an endothermic
peak at 316 ◦ C and 269 ◦ C corresponding to their melting points,
respectively. The guar gum DSC curve shows an 80 ◦ C event typical of
4
Carbohydrate Polymers 301 (2023) 120310
polysaccharides, also evidenced in the microcapsules. Analyzing the
thermogram of the microcapsule, an exothermic event initiated near
240 ◦ C is observed, characteristic of the decomposition of sodium algi­
nate (Abulateefeh & Taha, 2015). The events related to the melting point
in the thermograms of hesperidin (269 ◦ C) and pure betulinic acid
(316 ◦ C) disappeared in the MNED microcapsule, this fact shows that the
actives lost their crystalline structure, making it not possible to visualize
the event regarding the melting for this reason. This fact shows that the
actives were solubilized in the oil droplets that were dispersed in the
amorphous polymeric system, suggesting that they are encapsulated
(Josino et al., 2021).
L.M. Rebouças et al. Carbohydrate Polymers 301 (2023) 120310

Table 1
EE% of hesperidin and betulinic acid in the prepared microcapsules.
Microcapsule Encapsulation efficiency (EE%)

Hesperidin Betulinic acid

MNED 98.15 ± 0.34 c 99.76 ± 0.22 e

MNEH 99.53 ± 0.59 e –
MNEB – 99.23 ± 0.24 e

MNED: microcapsule containing hesperidin and betulinic acid based on nano­

emulsion, MNEB: microcapsule based on nanoemulsion containing betulinic
acid and MNEH: microcapsule based on nanoemulsion containing hesperidin.
*Different letters indicate significant difference with p < 0.05.

3.6. X-ray diffraction

X-ray diffractograms of hesperidin, betulinic acid and MNED mi­

crocapsules were obtained in order to assess crystallinity (Fig. 3).
Free betulinic acid and hesperidin were crystalline compounds while
guar gum, alginate substances and linseed oil were not. Therefore, it is
simple to assess the effect of encapsulation by measuring it as crystalline
properties with an XRD method. The diffractograms of the actives Fig. 3. X-ray diffractograms of actives and microcapsule.
showed structure characteristics with high crystallinity. In the MNED
microcapsule, intense peaks characteristic of crystallinity were not
identified, evidencing the amorphous state of the microstructured sys­
tem. These data confirm that hesperidin and betulinic acid are not in the
crystalline state in the microcapsule, as they were molecularly dispersed
in flaxseed oil and dispersed in the polymer matrix. This observation
suggests that the encapsulation method by spray drying the nano­
emulsion was quite satisfactory to encapsulate the hydrophobic actives
worked on in this study. Shu et al. (2006) when microencapsulating
lycopene with gelatin obtained the microcapsule diffractogram without
the presence of peaks related to crystalline lycopene, evidencing
encapsulation.

3.7. In vitro release kinetic study

For the in vitro release studies, the determination of the sink condi­
tion is important so that the solubility in the receptor medium does not
be a limiting factor for the diffusion of the active through the membrane.
The results of the solubility tests in the receiving media and the sink
condition for each asset are shown in the supplementary material.
According to Weng et al. (2020), DMSO can be used as a receptor
medium in drug delivery studies.
Fig. 4. Accumulated percentage of free and released actives in microcapsules.
Fig. 4 shows the data obtained from the accumulated percentage

Fig. 2. FTIR spectrum and DSC thermogram of the microcapsule and excipients.

5
L.M. Rebouças et al. Carbohydrate Polymers 301 (2023) 120310

released from the assets as a function of the time. Table 3

There is a slower and more controlled release of the actives in the Release kinetic parameters for betulinic acid and hesperidin in microcapsules.
microcapsules compared to the free actives. Table 2 shows the per­ Microcapsule Zero- First- Higuchi Korsmeyer-
centages accumulated in 48 h of release. order order model Peppas model
To study the release kinetics, data obtained from the in vitro drug R2 R2 R2 n R2
release studies were plotted in various kinetic models: zero order, first
MNED betulinic 0.9698 0.8722 0.9404 2.35 0.9709
order and Higuchi's model, available in the supplementary material. In acid
order to test the mathematical models of release, the value of the MNED hesperidin 0.9829 0.8288 0.9197 2.34 0.9555
determination coefficient R2 was calculated to find the model that best
suits the release, that is, the model with the highest value of R2 (Fou­
ladian et al., 2020). To evaluate the mechanism of drug release, data of
Table 4
drug release were plotted in the Korsmeyer-Peppas equation as log cu­ Results of acute toxicity tests of test samples (MNED and vehicle) against adult
mulative percentage of drug released versus log time, and the exponent n zebrafish.
was calculated through the slope of the straight line. From this model,
Sample Adult Zebrafish Mortality 96 h LC50 (μg mL− 1) / CI
the value of the diffusion exponent n was calculated. Table 3 shows the
values of R2 and n for the release of betulinic acid and hesperidin. NC C1 C2 C3

According to the R2 value, the model that best fits drug release in this
study would be the zero-order model. A zero-order delivery system
represents a controlled drug delivery that releases the drug at a constant
rate, which is desirable to keep drugs within the therapeutic range of
concentration. However, the zero-order model is more suitable for that
situation in which the release structure does not suffer disaggregation.
So, considering the polymeric microcapsule release structure for this
study, and considering the 0.9709 and 0.9555 values, we propose the
Korsmeyer-Peppas model as the more suitable mathematical model to
assess the release mechanism.
Diffusion exponent values ranged between 2.34 and 2.35, according
to Korsmeyer-Peppas, the type of release is Super Case II which is
characterized when n > 0.85 for particles with spherical geometry,
constituting an extreme form of transport (Tsirigotis-Maniecka et al.,
2021). According to this type of release mechanism, during the sorption
process, tension and relaxation of the polymer chains and swelling of the
external part occur. Furthermore, diffusion and dissolution occur
simultaneously. The Super Case II mechanism is predominant in poly­
mers that swell in an aqueous medium (Teimouri & Kasapis, 2022).
3.8. Zebrafish safety assessment and MTT assay
Currently, several studies have been using Danio rerio (zebrafish) as
an animal model to assess the non-clinical safety of new pharmaceutical
and food products. Moura et al. (2021) encapsulated essential oil of
Siparuna guianensis and found that the samples were considered safe
when exposed to zebrafish embryos. Coradini et al. (2021) evaluated the
safety of curcumin nanoparticles, where the authors found no change in
the locomotor activity of the animals, characterizing the samples eval­
uated as safe. The adult zebrafish was used as an animal model to assess
the acute toxicity of the MNED microcapsule. As a result, it was found
that all samples tested proved to be safe, as they were not toxic to
zebrafish within 96 h of analysis (Table 4).
In the evaluation of locomotor activity (Open Field Test), as a result,
no sample tested caused sedative effect and/or locomotor impairment of
the animals, as they presented locomotor activity significantly (p˃0.05)
similar between each sample group, as well as in relation to controls,
naive and vehicle as shown in Fig. 5.
The microcapsules anticancer efficacy was investigated for HL-60
Table 2
Percentages of actives released from microcapsules in 48 h of release under sink
condition.
Microcapsule % released accumulated in 48 h
MNED – betulinic acid 26.69 ± 0.93 a
MNED - hesperidin 30.35 ± 1.51 c
Free hesperidin 99.93 ± 0.71 d
Free betulinic acid 99.34 ± 0.82 d
Different letters indicate significant difference with p < 0.05.
MNED 0 0 0 0 >10
Vehicle 0 0 0 0 >10
NC - Negative control group: sterile distilled water. MNED – microcapsules
containing actives C1 – concentration 1 (2.5 μg mL-1; 20 μL); C2 – concentration
2 (5 μg mL-1; 20 μL); C3 concentration 3 (10 μg mL-1; 20 μL). Vehicle -
microcapsule without active in water. CL50-lethal concentration to kill 50 % of
adult zebrafish; CI – confidence interval.
cell line (promyelocytic leukemia) by the MTT test. Initially, the per­
centage of inhibition of cell growth in a single concentration was
determined and subsequently analyzed to determine the IC50. The re­
sults obtained from the percentage of inhibition of cell growth are shown
in Fig. 5.
In tests to obtain the IC50 in HL-60 cells (promyelocytic leukemia),
the results showed an increase in cytotoxicity with increasing concen­
tration. The IC50 values obtained in HL-60 and L-929 cells (non-tumor
murine fibroblasts) are shown in Table 5.
Free betulinic acid presented an IC50 of 0.41 ± 0.06 μg mL− 1, while it
associated with hesperidin presented an IC50 lower than 0.31 ± 0.01 μg
mL− 1. The hesperidin increased the cytotoxicity of betulinic acid on HL-
60 cells. In the same way, this fact was observed when comparing the
IC50 of MNEB (microcapsule containing only betulinic acid as active)
with IC50 of 5.11 ± 1.31 μg mL− 1 and MNED microcapsule (microcap­
sule with hesperidin and betulinic acid) with IC50 of 2.52 ± 0.23 μg
mL− 1. We did not find in the literature any study concerning the asso­
ciation of betulinic acid with hesperidin, however, there are reports
indicating that the association of hesperidin with the antitumor drugs
chlorogenic acid and doxorubicin, synergistically increase the antitumor
effect for MCF-7 cells (breast cancer) and Hela (cervical cancer)
(Majumdar & Srirangam, 2009; Kusharyanti, 2011; Hsu et al., 2021).
Korga et al. (2019) reported that hesperidin showed an effect on doxo­
rubicin toxicity in HepG2 (liver cancer) cells, while simultaneously
altering the expression of glycolytic pathway genes. Although separate
treatment with doxorubicin and hesperidin, in the work by Korga et al.
(2019), caused significant oxidative DNA damage, the simultaneous
administration of doxorubicin and hesperidin abolished this damage
and, simultaneously, increased the toxicity of doxorubicin against tumor
cells (Korga et al., 2019).
Currently, many studies aim to decrease the cytotoxic activity of
anticancer compounds for non-tumor cells and the encapsulation of
actives has sometimes shown an improvement in this aspect. It is
desirable that for the same concentration of active the cytotoxicity is
lower in non-tumor cells than in tumor cells in order to reduce the side
effects of chemotherapeutic agents. In the present work it was observed
that for the microcapsule MNED and MNEB, at the maximum concen­
tration tested against L-929 (non-tumor cells), the IC50 could not be
detected. On the other hand, for the HL-60 tumor cells, values of 2.52 ±
0.23 μg mL− 1 and 5.11 ± 1.31 μg mL− 1 were obtained, respectively,
showing that the microencapsulated form with the polysaccharides guar
gum and sodium alginate reduced the cytotoxicity of betulinic acid

6
L.M. Rebouças et al. Carbohydrate Polymers 301 (2023) 120310

Fig. 5. (a) Effect of test samples on locomotor activity of adult zebrafish (Danio rerio) in the Open Field Test (0–5 min). Naive - untreated animals. o.a. – oral
administration. MNED (microcapsule containing the actives). Vehicle – (microcapsule without actives) (20 μL; o.a.). (b) Percentage Inhibition of Cell Growth in HL-
60 Cells.

CRediT authorship contribution statement

Table 5
IC50 results in HL-60 and L-929 cells obtained by the MTT assay.
Louhana M. Rebouças: Conceptualization, Investigation, Method­
Samples IC50 / (μg mL− 1) ology, Formal analysis, Visualization, Writing – original draft. Alex­
HL-60 L-929 andre C.C. Sousa: Writing – review & editing. Caroline G. Sampaio:
AB 0.41 ± 0.06 a 158.42 ± 20.16 f Investigation. Larissa M.R. Silva: Formal analysis. M.S.C. Pedro:
HESP >500 >500 Investigation. Cláudia Pessoa: Investigation. Nilce V.G.P.S. Brasil:
AB-H 0.31 ± 0.01 b 118.73 ± 12.59 g Writing – review & editing, Conceptualization. Nágila M.P.S. Ricardo:
MNEH >500 >500 Conceptualization, Investigation, Writing – review & editing, Supervi­
MNEB 5.11 ± 1.31 c
sion, Project administration, Funding acquisition.
>500
Control-MNE >500 >500
MNED 2.52 ± 0.23 d >500
Dox (positive control) 0.02 ± 0.01 e 0.99 ± 0.08 h
Declaration of competing interest
Control-MNE: microcapsule without active. MNED: microcapsule containing
hesperidin and betulinic acid based on nanoemulsion. MNEB: nanoemulsion- The authors declare that they have no known competing financial
based microcapsule containing betulinic acid and MNEH: nanoemulsion-based interests or personal relationships that could have appeared to influence
microcapsule containing hesperidin. AB: free betulinic acid. AB-H: betulinic the work reported in this paper.
acid associated with hesperidin in the free form. Dox: doxorubicin and HESP:
free hesperidin. Different letters indicate significant difference with p < 0.05.
Data availability

against non-tumor cells. This fact can be correlated with the results of in Data will be made available on request.
vitro release already presented in this work, which show the slow and
controlled release of betulinic acid and hesperidin through the resis­
Acknowledgements
tance imposed by the polymeric network of the polysaccharides used,
preventing direct and immediate exposure of the active in higher con­
This study was supported in part by the Coordenação de Aperfei­
centrations in the tested cell lines.
çoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code
001 (CAPES/PROEX PROCESS: 23038.000509/2020-82) and Conselho
4. Conclusions
Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The
authors thank Central Analítica-UFC/CT-INFRA/MCTI-SISNANO/
Alginate and guar gum microcapsules were successfully synthesized
CAPES for the support. The corresponding author thanks CNPq, for the
for the simultaneous controlled release of betulinic acid and hesperidin
research grant (N.M.P.S.R – Project No. 309795/2021-4).
for anticancer application using the spray dryer nanoemulsion drying
technique. The microcapsules promoted the simultaneous controlled
Appendix A. Supplementary data
release of both actives due to the resistance imposed by the composition
of the polysaccharides guar gum and sodium alginate. Additionally, the
Supplementary data to this article can be found online at https://doi.
microcapsules presented cytotoxic activity against HL-60 cells (pro­
org/10.1016/j.carbpol.2022.120310.
myelocytic leukemia), improving the performance of betulinic acid by
association with hesperidin. With microencapsulation, it was achieved
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