Physicochemical, Morphological, and Cytotoxic Properties of Brazilian Jackfruit (Artocarpus heterophyllus) Starch Scaffold Loaded with Silver ...
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Journal of
Functional
Biomaterials
Article
Physicochemical, Morphological, and Cytotoxic Properties of
Brazilian Jackfruit (Artocarpus heterophyllus) Starch Scaffold
Loaded with Silver Nanoparticles
José Filipe Bacalhau Rodrigues * , Valeriano Soares Azevedo, Rebeca Peixoto Medeiros,
Gislaine Bezerra de Carvalho Barreto, Maria Roberta de Oliveira Pinto , Marcus Vinicius Lia Fook
and Maziar Montazerian *
Academic Unit of Materials Science and Engineering, Federal University of Campina Grande,
Campina Grande 58429-140, PB, Brazil
* Correspondence: filipe.rodrigues@certbio.ufcg.edu.br (J.F.B.R.); maziar_montaz@yahoo.com (M.M.);
Tel.: +55-83-991-284-865 (J.F.B.R.)
Abstract: Due to the physical, thermal, and biological properties of silver nanoparticles (AgNPs), as
well as the biocompatibility and environmental safety of the naturally occurring polymeric compo-
nent, polysaccharide-based composites containing AgNPs are a promising choice for the development
of biomaterials. Starch is a low-cost, non-toxic, biocompatible, and tissue-healing natural polymer.
The application of starch in various forms and its combination with metallic nanoparticles have
contributed to the advancement of biomaterials. Few investigations into jackfruit starch with silver
nanoparticle biocomposites exist. This research intends to explore the physicochemical, morphologi-
cal, and cytotoxic properties of a Brazilian jackfruit starch-based scaffold loaded with AgNPs. The
AgNPs were synthesized by chemical reduction and the scaffold was produced by gelatinization. X-
ray diffraction (XRD), differential scanning calorimetry (DSC), scanning electron microscopy coupled
Citation: Rodrigues, J.F.B.; Azevedo, with energy-dispersive spectroscopy (SEM-EDS), and Fourier-transform infrared spectroscopy (FTIR)
V.S.; Medeiros, R.P.; Barreto, G.B.d.C.; were used to study the scaffold. The findings supported the development of stable, monodispersed,
Pinto, M.R.d.O.; Fook, M.V.L.;
and triangular AgNPs. XRD and EDS analyses demonstrated the incorporation of silver nanoparticles.
Montazerian, M. Physicochemical,
AgNPs could alter the scaffold’s crystallinity, roughness, and thermal stability without affecting
Morphological, and Cytotoxic
its chemistry or physics. Triangular anisotropic AgNPs exhibited no toxicity against L929 cells at
Properties of Brazilian Jackfruit
concentrations ranging from 6.25 × 10−5 to 1 × 10−3 mol·L−1 , implying that the scaffolds might
(Artocarpus heterophyllus) Starch
Scaffold Loaded with Silver
have had no adverse effects on the cells. The scaffolds prepared with jackfruit starch showed greater
Nanoparticles. J. Funct. Biomater. crystallinity and thermal stability, and absence of toxicity after the incorporation of triangular AgNPs.
2023, 14, 143. https://doi.org/ These findings indicate that jackfruit is a promising starch source for developing biomaterials.
10.3390/jfb14030143
Keywords: jackfruit; silver; nanoparticles; scaffold; biomaterials
Academic Editors: Kai Yang
and Elisa Boanini
Received: 25 October 2022
Revised: 23 November 2022 1. Introduction
Accepted: 6 December 2022
Scaffolds are three-dimensional, porous structures that facilitate tissue growth/remodeling
Published: 3 March 2023
by supporting human cell adhesion, proliferation, differentiation, and orientation in a sta-
ble environment. They are commonly employed in biomedical tissue engineering and are
composed of biocompatible and biodegradable materials that allow for the incorporation and
Copyright: © 2023 by the authors. transport of medicines and biological components [1–4].
Licensee MDPI, Basel, Switzerland. Scaffolds composed of metals, ceramics, polymers, and composites are used in biomed-
This article is an open access article ical tissue engineering [5]. Composite scaffolds are produced by mixing two or more mate-
distributed under the terms and rials, each with its own set of desirable properties. The tensile and compressive mechanical
conditions of the Creative Commons strengths of composite polymeric scaffolds, for example, are significantly higher than those
Attribution (CC BY) license (https:// of polymeric scaffolds [6]. As some are produced from biodegradable polymers, such as
creativecommons.org/licenses/by/ cellulose, chitosan, and starch, they are not only more durable, but also less toxic [6,7].
4.0/).
J. Funct. Biomater. 2023, 14, 143. https://doi.org/10.3390/jfb14030143 https://www.mdpi.com/journal/jfb
J. Funct. Biomater. 2023, 14, 143 2 of 15
Starch is one of the main biopolymers present in nature [8]. It is an abundant, low-cost,
biodegradable, biocompatible, and natural polymer widely used in fabricating scaffolds [9].
Amylose (glucose units joined by α-1,4-glycosidic bonds) and amylopectin (glucose units
linked by glycosidic bonds at the α-1,4 and α-1,6 carbons) chains make up starch’s molecular
structure [10]. Starch, like other polysaccharides and proteins, is a thermoplastic polymer
composed of linear chains linked by weak bonds and is capable of being processed into
membranes [11], gels [12], nanofibers [13], microparticles [14], nanoparticles [15], and
scaffolds [16]. It can also encapsulate pharmaceuticals [16] and metallic nanoparticles [15]
that bond to the long polymeric chains and hydroxyl groups of molecular structures.
Silver nanoparticles (AgNPs) stand out among metallic nanoparticles due to their
distinctive physical, chemical, and biological features, particularly their anti-bacterial and
antifungal activities [17], simple production, low cost [18], high conductivity, chemical
stability, and catalytic activity [19]. They can be obtained with different morphologies, such
as spheres, triangles, and rods; however, triangles are more biologically active [20,21].
AgNPs have a wide range of applications in biomedicine and are commonly coupled
with biomaterials to provide bactericidal effects [22]. By attaching to peptidoglycans, Ag-
NPs can permeate bacterial membranes, causing structural alterations, increased membrane
permeability, and, eventually, death [23]. AgNPs can also interact with bacterial proteins
through this mechanism, inhibiting DNA replication and bacterial growth [24].
AgNPs are used to improve the physical, chemical, and biological characteristics of
biomaterials. A variety of studies have linked enhanced porosity, improved thermal and
mechanical properties, increased water vapor absorption, anti-bacterial activity aggrega-
tion, and other biological aspects of biomaterials to the incorporation of AgNPs (see Table 1).
Most of these studies used AgNPs with spherical forms due to their rapid synthesis and
simplicity of acquisition [25] and employed more traditional polymer matrices, such as chi-
tosan, collagen, cellulose, and alginate. Although the advanced properties of biomaterials
loaded with spherical AgNPs produced from these biopolymers are well recognized, less is
known about the impact that AgNPs of different morphologies can have on them, as well
as their behavior in biomaterials produced from other polymer matrices, such as starch.
A few different types of starches can be utilized to produce scaffolds and biomaterials.
They are derived from many plant sources and have varying compositions and properties that
are determined by the plants’ growing circumstances, area, harvest season, and climate [26].
Commercial starches produced from wheat, corn, potato, rice, and cassava have already been
extensively explored and are widely exploited in industry [27] as films [8], hydrogels [28],
micro/nanoparticles [29,30], composites [31], and scaffolds of starches extracted from these
sources. For this reason, searching for novel forms of local starch sources, especially in Brazil,
would be critical, given their importance and the differences in their qualities.
Table 1. Physical and biological properties of scaffolds, sponges, mats, and fibers loaded with silver
nanoparticles prepared from starch and other polymers.
Material AgNPs Morphology Physical Properties Biological Properties Ref.
Highly interconnected porous Absence of toxicity against human
Potato starch/PVA nanofibrous structure, relevant thermal fibroblast cells and
Spherical [32]
mats loaded with AgNPs stability, and fast release AgNPs imparted anti-bacterial
of AgNPs activity against E. coli and S. aureus
Bactericidal activity against E. coli
and S. aureus; absence of toxicity
Carboxymethyl chitosan/oxidized
Spherical Improved thermal properties and ability to promote the growth [33]
starch sponges loaded with AgNPs
of L929 fibroblast cells; faster
wound repair healing
Reduced hydrophilicity and Bactericidal activity against E. coli;
Potato starch nanofibrous mats improved mechanical properties absence of toxicity against L929
Spherical [34]
loaded with AgNPs with increased tensile strength fibroblasts cells up to AgNPs
and reduced deformation concentration of 2.5 mg.mL-1
J. Funct. Biomater. 2023, 14, 143 3 of 15
Table 1. Cont.
Material AgNPs Morphology Physical Properties Biological Properties Ref.
Anti-bacterial activity against
Hybrid Ag
E. coli and S. aureus; promoted
nanoparticles/polyoxometalate–
Highly porous wound healing; good
polydopamine nano-flowers Spherical [35]
interconnected structure biocompatibility with L929 cells
Loaded chitosan/gelatin
and human umbilical vein
hydrogel scaffolds
endothelial cells (HUVEC)
Highly porous interconnected
Anti-bacterial activity against
structure; improved mechanical
Chitosan/Ag E. coli and S. aureus;
Spherical properties and good water vapor [36]
nanocomposite sponges non-cytotoxicity (cell viability
transmission properties (in the
values were greater than 90%)
range of 2000–2400 g/m2 /d)
Cellulose-based Unexpected influence on water
scaffolds containing absorption and reduction in Anti-bacterial activity against
Spherical [37]
orange essential oil and mechanical strength B. subtilis and E. coli
silver nanoparticles and elongation
Silver nanoparticles significantly Anti-bacterial activity against
increased the thermal stability E. coli and S. aureus with 0.2 mM
3D cellulose nanofiber scaffolds
Spherical and mechanical properties, and AgNPs content; low toxicity at [38]
decorated with silver nanoparticles
reduced the scaffolds’ expansion concentrations of 0.2 and
process and swelling ratio 1 mM AgNPs
In vitro cytocompatible and
3D hybrid scaffold based on non-genotoxic in human gingival
collagen, chondroitin 4-sulfate, fibroblast cultures;
Microstructure with high
and fibronectin, functionalized Spherical biocompatibility with chick [39]
number of interconnected pores
with silver embryos CAM; antimicrobial
nanoparticles activity against F. nucleatum and
P. gingivalis
Significantly enhanced cell
Poly(ε-caprolactone) Increased crystallinity and
proliferation of C2C12 mouse
nanocomposite fiber scaffolds Wires thermal properties, and retarded [40]
myoblast cells before and after
loaded with silver nanowires enzymatic biodegradation
electrical stimulations
Effective anti-bacterial and
Chitosan/Bletilla striata Preserved porous interconnected anti-biofilm properties both
polysaccharide composite scaffold Spherical structure and improved in vitro and in vivo against MRSA; [41]
loaded with silver nanoparticles mechanical properties potential to promote cell
proliferation and angiogenesis
Jackfruit (Artocarpus heterophyllus) provides a different type of starch, showing po-
tential among starch sources. The high amylose concentration [26], low gelatinization
temperature [42], and small gelatinization enthalpy change [43] of jackfruit starch make it
a promising starch resource. However, jackfruit starch’s potential applications have not
been fully studied by the scientific community; a limited number of studies have focused
on this material, making more investigation into its potential role in the development of
biomaterials imperative.
In this study, a jackfruit (Artocarpus heterophyllus) starch-based scaffold loaded with
triangular AgNPs was synthesized and tested for its physicochemical, thermal, morpholog-
ical, and cytotoxic properties. The production of such scaffolds utilizing readily accessible,
inexpensive, and domestic starch sources was another primary objective of this study.
2. Materials and Methods
2.1. Chemicals
The jackfruit used for starch extraction was obtained from the local market of Campina
Grande, Paraiba, Brazil. All chemical reagents were of analytical grade. Silver nitrate
(AgNO3 , purity 99.0%), tribasic sodium citrate dihydrate (Na3 C6 H5 O7 ·2H2 O, purity 99.0%),
and hydrogen peroxide (H2 O2 , 35%) were purchased from Neon. Sodium borohydride
(NaBH4 , purity ≥96%) and glycerol (C3 H8 O3 , purity ≥99%) were obtained from Sigma-
Aldrich. The aqueous solutions were prepared with ultrapure water (18.2 mΩ·cm), obtained
J. Funct. Biomater. 2023, 14, 143 4 of 15
from a GEHAKA Master System MS2000 System. The L929 Mouse Fibroblast Cell Line
(ATCC NCTC clone 929) was acquired from the Rio de Janeiro Cell Bank, Brazil.
2.2. Starch Extraction from Jackfruit Seed Endocarp
The starch extraction method was adopted from Perez, et al. [44]. In the first stage,
jackfruit seeds were washed, peeled, and crushed in a blender until a uniform, thick, and
homogenous mass was obtained, adding water in a 1:4 (m/v) ratio. The paste was filtered
through organza bags (100 mesh). The filtered starch suspension was decanted for 24 h
in a refrigerated atmosphere at 5 ◦ C. The floating portion was removed, and the starch
suspended in water was again decanted. This suspension and settling procedure was
repeated until a white starch color was acquired. Following this, the starch was lyophilized
(for 48 h) and sieved to 200 mesh.
2.3. Synthesis of AgNPs
AgNPs were synthesized through the chemical reduction method described by Zhang,
et al. [45]. Initially, 30 mL of ultrapure water was transferred to a beaker and magnetic
stirring (500 rpm) was performed at room temperature (25 ◦ C). The system was then filled
with 30 µL of silver nitrate (0.1 mol·L−1 ), 1.5 mL of sodium citrate (0.9 mmol·L−1 ), 60 µL of
hydrogen peroxide (35%), and 200 µL of sodium borohydride (90 mmol·L−1 ). After adding
sodium borohydride, the magnetic stirring was increased to 1150 rpm for 3 min, the period
required for forming AgNPs. Our earlier study [46] provides more information on the
effects of mixing intervals and variations in the volume and concentration of NaBH4 and
H2 O2 on the size, dispersion, and stability of AgNPs.
J. Funct. Biomater. 2023, 14, x FOR PEER REVIEW 5 of 16
2.4. Synthesis of Jackfruit Starch Scaffold
The starch scaffold was obtained according to the methodology proposed by Perez,
et al. [47]. Amounts of 7.5 g of starch and 2.5 mL of glycerol were first added to a beaker
85 °C. The starch solution was then cooled to room temperature (25 °C), transferred to
containing 250 mL of ultrapure water while magnetic stirring (300 rpm) was performed
Petri dishes, and frozen (24 h), defrosted (2 h), frozen (24 h), and lyophilized (72 h).
at 85 ◦ C. The starch solution was then cooled to room temperature (25 ◦ C), transferred to
Petri dishes, and frozen (24 h), defrosted (2 h), frozen (24 h), and lyophilized (72 h).
2.5. Synthesis of Starch–AgNPs Scaffold
The starch–AgNPs
2.5. Synthesis scaffold
of Starch–AgNPs was developed using the same process as the jackfruit
Scaffold
starchThe
scaffold, with a few
starch–AgNPs modifications.
scaffold After cooling
was developed to room
using the sametemperature,
process as the 12.5 mL of
jackfruit
the AgNP (a volume fraction of 5% to the starch solution) solution (1 × 10
starch scaffold, with a few modifications. After cooling to room temperature, 12.5 mL of −3 mol·L−1) was
added,
the AgNP followed by freezing
(a volume (245%
fraction of h),todefrosting
the starch(2 h), freezing
solution) (24 h),
solution (1 × and
10−lyophilizing (72
3 mol·L−1 ) was
h). As the AgNPs were homogenized in the starch solution, this resulted in a
added, followed by freezing (24 h), defrosting (2 h), freezing (24 h), and lyophilizing (72 h).uniformly
AgNP-decorated
As the AgNPs were scaffold. Figure 1inillustrates
homogenized the starchthe steps followed
solution, during
this resulted in athe scaffold’s
uniformly syn-
AgNP-
thesis.
decorated scaffold. Figure 1 illustrates the steps followed during the scaffold’s synthesis.
Figure
Figure 1.
1. Schematic
Schematic representation
representation of
of starch–AgNPs scaffold’s synthesis.
starch–AgNPs scaffold’s synthesis.
2.6. Characterization
2.6. Characterization of
of the
the Silver
Silver Nanoparticles
Nanoparticles (AgNPs)
(AgNPs)
A UV-Vis
A UV-Vis spectrum
spectrum model MB-102 (Bomem-Michelson,
model MB-102 (Bomem-Michelson, Vanier,
Vanier, Canada)
Canada) was used to
was used to
collect AgNPs spectra and confirm its synthesis. The scan was performed in the wavelength
collect AgNPs spectra and confirm its synthesis. The scan was performed in the wave-
length range of 250–1100 nm. The spectrums were collected using quartz cuvettes with a
10 mm optical path.
The size, polydispersity, and stability of AgNPs are properties that directly influence
their biological properties [48]. They were determined using dynamic light scattering
J. Funct. Biomater. 2023, 14, 143 5 of 15
range of 250–1100 nm. The spectrums were collected using quartz cuvettes with a 10 mm
optical path.
The size, polydispersity, and stability of AgNPs are properties that directly influence
their biological properties [48]. They were determined using dynamic light scattering
(DLS) and zeta potential (PZ) techniques. The analyses were carried out on a Brookhaven
ZetaPals (Brookhaven Instruments, New York, USA). The tests were performed at room
temperature without diluting the samples, with a scattering angle of 90◦ , laser wavelength
of 632.8 nm (He-Ne), average viscosity of 0.887 mPa·s, and refractive index of 1.330. All
measurements were taken three times.
The AgNPs’ morphology was investigated using a field-emission scanning electron
microscope (FE-SEM) model S4700EI (Hitachi, Chiyoda, Japan) operating at a voltage of
15 kV. AgNPs were diluted in ultrapure water at a ratio of 1:10 and coated with platinum.
2.7. Scaffolds Characterization
When developing a biomaterial that contains metallic nanoparticles, it is very impor-
tant to check that the particles have been incorporated. Therefore, X-ray diffraction (XRD)
was used to prove the inclusion of AgNPs in the starch–AgNPs composite and to verify
the crystallinity of the starch. The experiment was carried out on a X-ray diffractometer
model XRD-7000 (Shimadzu, Kyoto, Japan) with CuKα radiation (1.5418), 40 kV, and 30 mA
current in the interval of 10–80◦ and a resolution of 2◦ /min. The General System Analyzer
Structure (GSAS II) program was utilized for Rietveld refinement.
The type of interaction that exists between a composite matrix and its filler provides
information on how easily this filler will be released from the biomaterial. Thus, FTIR
spectroscopy was employed to identify the vibration bands of starch and to assess the
interaction between starch and AgNPs. The analysis was carried out on a Spectrum
400 FT-IR (Perkin Elmer, Waltham, MA, USA) device in the range of 4000–650 cm−1 with
a resolution of 4 cm−1 in the diffuse reflectance mode for 32 scans at room temperature
(25 ◦ C) using an attenuated total reflectance (ATR) accessory equipped with a zinc selenium
(ZnSe) crystal.
To analyze the existence of AgNPs in the scaffold, evaluate morphological changes
following AgNPs incorporation, and identify the starch microstructure, scanning electron
microscopy (SEM) was conducted on a SEM microscope model TM-1000 (Hitachi, Chiyoda,
Japan) coupled with energy-dispersive X-ray spectroscopy (EDS) was performed. All
images were taken from uncoated samples fixed on an aluminum alloy sample holder at
a 15 kV accelerating voltage, 1 mm depth of focus, 30 nm resolution, low vacuum, and
variable pressure (1 to 270 Pa). Under the same conditions, EDS analyses were performed
on an EDS Quantax 50 XFlash (Bruker, Billerica, MA, USA). The EDS spectra were collected
at 3 different points of the starch–AgNPs scaffolds to detect the AgNPs. Image processing
was carried out using the Quantax 50 program.
The thermal stability of a biomaterial provides information on the conditions of
use, storage, and application [49]. The thermal stability of the starch and starch–AgNPs
scaffolds was evaluated using differential scanning calorimetry (DSC). A DSC, model
8500 (PerkinElmer, Waltham, USA), was employed in a temperature range of 25–300 ◦ C
with a heating rate of 10 ◦ C/min under a nitrogen atmosphere and with a flow rate of
20 mL/min. an alumina crucible and a sample mass of 3.00 ± 0.05 mg were used.
2.8. Cytotoxicity Assessment
Cytotoxicity tests determine whether a biomaterial is safe or not [50]. The cytotoxicity
assessment of the AgNPs, starch scaffold, and starch–AgNPs scaffold was performed
according to the direct contact method described in ISO 10993-5:2009 [50] and ISO 10993-
12:1998 [51]. Initially, 0.5 g of scaffold was weighed and sterilized for 30 min via UV
radiation. Next, the extracts were prepared by submerging the scaffolds in 1.25 mL of PBS
solution (0.05 M) for 24 h. Then, 100 µL/well of L929 cell suspension was seeded in 96-well
plates and incubated for 24 h at 37 ◦ C ± 1 ◦ C under a 5% ± 1% CO2 atmosphere. After
J. Funct. Biomater. 2023, 14, 143 6 of 15
cultivation, 50 µL of the medium extracted from the samples was added and the cells were
incubated for another 24 h.
For the AgNPs, five dilutions (1/1, 1/2, 1/4, 1/8, and 1/16) were performed to
obtain final plaque concentrations ranging from 1 × 10−3 to 6.25 × 10−5 mol·mL−1 . The
method used for determining the concentration of AgNPs was solvent evaporation, which
is well-known and widely used in the literature. In references [52,53], the researchers
employed thermal gravimetric analysis to determine the weight of AgNPs and other
metallic nanoparticles. Then, 100 µL/well of L929 cells suspension was seeded in 96-well
plates and incubated for 24 h at 37 ◦ C ± 1 ◦ C under a 5% ± 1% CO2 atmosphere. Amounts
of 20 µL of each of these dilutions were added to 96-well plates and the cells were incubated
for another 24 h. The cytotoxicity assay was conducted following the MTT method.
After the incubation time, the culture medium was removed and 100 µL of MTT
solution (5 mg·mL−1 ) was added; the plates were then incubated under the same condi-
tions for another 4 h. The cells were then treated with 100 µL of DMSO to dissolve the
J. Funct. Biomater. 2023, 14, x FOR PEER REVIEW crystals. The plates were read following the optical density method on a 7Victor
formazan of 16
X3 microplate reader (PerkinElmer, Waltham, USA) at 570 nm with 650 nm reference filters.
Latex sheets were used as the positive control and high-density polyethylene (HDPE) as
triangular nanoparticles
the negative control. [54], and the third band with maximum absorption at 744 nm
resembles the plasmonic surface characteristic of the resonance band of almost perfectly
3. Results and Discussion
triangular nanoparticles [54,55]. According to Mie’s hypothesis [56], anisotropic particles
3.1. Characterization of the Silver Nanoparticles (AgNPs)
should be present because they have three absorption bands [55]. Furthermore, the colloi-
dal solution of AgNPs
The UV-Vis acquired
spectrum a blue
of the AgNPscolor after synthesis,
colloidal solutionwhich
is showncanin
beFigure
attributed
2A. to the
Three
plasmonic excitation of the surface of triangular-shaped nanoparticles (nanoplates) [57].
absorption bands were found in the UV-Vis analysis. According to the Schatz calculation,
the first
The band at 333 nm
nanoparticles incorresponds
the DLS result to (Inset
out-of-plane
of Figurequadrupole
2A) had anresonance, theofsecond
average size 33.27
nm and a polydispersity index (PDI) of 0.205, which is typical of monodisperseof
shoulder-shaped band around 465 nm indicates dipole resonances characteristic triangu-
solutions
lar nanoparticles
(0.300), which have [54], and ranging
values the thirdfrom
band0 with maximum
to 1. As a result,absorption
the smalleratthe
744 nm resembles
value, the more
the plasmonic surface characteristic of the resonance band of almost perfectly
monodisperse the colloid [58]. The PZ findings revealed AgNPs with a surface charge of triangular
nanoparticles
−33.39 [54,55]. According
mV, confirming stable AgNPsto Mie’s hypothesis
production [57].[56],
The anisotropic particles should
FE-SEM micrographs (Figuresbe
present because they have three absorption bands [55]. Furthermore, the colloidal
2B–D) show the formation of nano-sized, anisotropic, and triangular monodisperse silver solution
of AgNPsThese
particles. acquired a blue
results are color after synthesis,
in agreement with thosewhich can beby
observed attributed to the plasmonic
others [59,60].
excitation of the surface of triangular-shaped nanoparticles (nanoplates) [57].
Figure
Figure 2.
2. (A)
(A) UV-Vis
UV-Vis spectrum
spectrum of the AgNPs
of the AgNPs colloidal
colloidal solution.
solution. Inset:
Inset: Size
Size distribution
distribution of
of AgNPs
AgNPs
measured by the DLS. (B–D) FE-SEM micrograph showing the formation of nano-sized, anisotropic,
measured by the DLS. (B–D) FE-SEM micrograph showing the formation of nano-sized, anisotropic,
triangular, and monodisperse silver particles.
triangular, and monodisperse silver particles.
3.2. Morphological Characterization
SEM micrographs of the jackfruit starch granules, starch scaffold, and starch–AgNPs
scaffold are shown in Figure 3A–F. The starch granules (Figure 3A,B) had a rounded and
J. Funct. Biomater. 2023, 14, 143 7 of 15
The nanoparticles in the DLS result (Inset of Figure 2A) had an average size of 33.27 nm
and a polydispersity index (PDI) of 0.205, which is typical of monodisperse solutions
(0.300), which have values ranging from 0 to 1. As a result, the smaller the value, the
more monodisperse the colloid [58]. The PZ findings revealed AgNPs with a surface
charge of −33.39 mV, confirming stable AgNPs production [57]. The FE-SEM micrographs
(Figure
J. Funct. Biomater. 2022, 13, x FOR PEER 2B–D) show the formation of nano-sized, anisotropic, and triangular monodisperse
REVIEW 8 of 16
silver particles. These results are in agreement with those observed by others [59,60].
3.2. Morphological Characterization
incorporation of metallic
SEM micrographs nanoparticles
of the jackfruit starchinto the starch
granules, polymer
starch scaffold,matrix was shown to mod
and starch–AgNPs
ify the interactions
scaffold are shown in between
Figure 3A–F.starch
The and
starchglycerol
granules[68,69],
(Figure increasing
3A,B) had a its compactness
rounded and and
roughness.
irregular bell shape ranging from 4–6 µm. Starch extracted from Brazilian jackfruit is
TheofEDS
typically this analysis revealed
size and form the presence
[61], whereas ofstarch
jackfruit AgNPs in the from
extracted scaffold
Thai,(Figure 3G). Starch
Malaysian,
or
wasChinese jackfruit
associated is different
with levels ofin 59.4%
size andcarbon
shape [26].
and The variations
33.2% oxygen. in granule
The Fe morphology
content was linked
are influenced
to the samplebyholder,
differences in cultivars
which and environmental
was composed factors [62]
of an aluminum andwith
alloy affectiron.
starch’s
The silver
functional characteristics [26]. The small grains and bell-shaped morphology of jackfruit
ions in the nanoparticles were responsible for the 2.0% silver content. The findings of Li
starch developed for usage in the food and health industries contribute to its increased
et al. [70] and Vaidhyanathan, et al. [71] are consistent with these results.
swelling capacity and viscosity.
Figure3.3. SEM
Figure SEM micrographs
micrographsofof(A,B)
(A,B)jackfruit
jackfruitstarch granules;
starch (C,D)
granules; (C,D)cross-sections of the
cross-sections starch
of the starch scaf
scaffold
fold andand (E,F)
(E,F) starch–AgNPsscaffold,
starch–AgNPs scaffold, and
and(G)
(G)EDS
EDSanalysis of the
analysis starch–AgNPs
of the starch–AgNPsscaffold.
scaffold.
3.3. Phase Analysis
Figure 4 depicts the XRD pattern of the jackfruit starch, starch scaffold, and starch–
AgNPs scaffold. According to Figure 3B, the starch scaffold exhibited amorphous polymerJ. Funct. Biomater. 2023, 14, 143 8 of 15
The SEM micrographs of the starch scaffold and starch–AgNPs scaffold shown in
Figure 3C–F revealed that both scaffolds had a porous structure created by well-defined
pores arranged with high interconnectivity. Water vaporization is one possible mechanism
that results in the formation of this porous structure. When water is removed, as in
the lyophilization process, a large amount of porosity is created [63]. For the functional
properties of starch, this same type of interconnected porous structure could also be
observed in scaffolds prepared with starch extracted from rice [64], a source of starch that
J. Funct. Biomater. 2023, 14, x FOR PEER
hasREVIEW
a similar size and morphology to starch extracted from jackfruit [26]. This implies 9 of 16
that
the interconnected porous structure presented by the jackfruit starch scaffolds is influenced
by the morphology and size of the starch grains extracted from the jackfruit.
For thepeaks
diffraction starch–AgNPs
positionedscaffold (Figure
at 15.86°, 3E,F),
17.38°, it appears
21,04°, 22.94°,that adding
23.88°, and AgNPs roughened
28.88°, confirming
the surface of the scaffold, facilitating cell attachment and proliferation
the type-A crystalline structure A [72], which, as other authors have pointed out, [65,66]. Previous
is con-
studies have reported a relationship between the incorporation
sistent with the crystalline structure of jackfruit starch [73,74]. of nanoparticles and increased
total porosity of polymeric
These peaks and ceramic
in the scaffold werecomposite
identical scaffolds
to the peaks[67]. in
Herein, the incorporation
the starch diffractogram of
metallic nanoparticles into the starch polymer matrix was shown to modify the interactions
(Figure 4A), although with amorphous characteristics and lower intensity, suggesting a
between starch and glycerol [68,69], increasing its compactness and roughness.
disappearance of the crystalline structure of type-A jackfruit starch after processing. Stud-
The EDS analysis revealed the presence of AgNPs in the scaffold (Figure 3G). Starch
ies indicate that plasticized starch tends to form a V-type crystalline structure, which was
was associated with levels of 59.4% carbon and 33.2% oxygen. The Fe content was linked to
observed by the appearance of a slight shoulder around 17.00° and 19.18° (Figure 4B) [75].
the sample holder, which was composed of an aluminum alloy with iron. The silver ions in
In manufacturing starch scaffolds, glycerol was used, which acts as a plasticizer [76].
the nanoparticles were responsible for the 2.0% silver content. The findings of Li, et al. [70]
Therefore, it is suggested that, during processing, there was a change in the crystalline
and Vaidhyanathan, et al. [71] are consistent with these results.
structure of starch through its interaction with glycerol.
AfterAnalysis
3.3. Phase adding AgNPs to the scaffold (Figure 4C), the crystallinity increased to 33.88%,
representing a 16.93% increase over the scaffold without AgNPs. It has been shown that
Figure 4 depicts the XRD pattern of the jackfruit starch, starch scaffold, and starch–
the presence of AgNPs leads to a higher degree of crystallinity in polyimide films [77].
AgNPs scaffold. According to Figure 3B, the starch scaffold exhibited amorphous polymer
The starch–AgNPs scaffold exhibited four diffraction peaks at 37.80°, 44.02°, 64.36°, and
structures with a crystallinity of 16.95% according to the Rietveld refinement; there were
77.50°, which
diffraction corresponded
peaks positionedto atplanes
15.86◦ ,(111),
17.38(200),
◦ , 21,04(220), and◦ ,(311)
◦ , 22.94 23.88and correlated
◦ , and with the
28.88◦ , confirm-
ing the type-A crystalline structure A [72], which, as other authors have pointed out,the
face-centered cubic structure of metallic silver (JCPDS file No. 03-0921). This validated is
inclusion of
consistent AgNPs
with in the scaffold
the crystalline [78]. of jackfruit starch [73,74].
structure
Figure 4.
Figure 4. XRD
XRD patterns
patterns of
of the
the (A)
(A)jackfruit
jackfruitstarch,
starch,(B)
(B)starch
starchscaffold,
scaffold,and
and(C)
(C)starch–AgNPs
starch–AgNPsscaffold.
scaf-
fold.
These peaks in the scaffold were identical to the peaks in the starch diffractogram
3.4. Scaffold
(Figure 4A),Chemistry
although with amorphous characteristics and lower intensity, suggesting a
disappearance
The FTIR of the crystalline
spectra structure
of the starch, ofscaffold,
starch type-A jackfruit starch after processing.
and starch–AgNPs scaffold areStudies
shown
indicate that plasticized starch tends to form a V-type crystalline structure, which was
in Figure 5. The vibration bands in the jackfruit starch spectrum (Figure 5A) represent
observed by the appearance of a slight shoulder around 17.00◦ and 19.18◦ (Figure 4B) [75].
features of the molecular deformations present in the starch molecules. The stretching de-
formation of –OH is responsible for the band located at 3400 cm−1 and angular deformation
of the band at 1650 cm−1 [79]. The symmetrical stretching of –C–H is represented by the
vibration band at 2926 and asymmetrical stretches at 2897 cm−1. The C–O–H bonds are
represented by the bands at 1460–1400 cm−1 [80]. Absorptions at 1340 and 1024 cm−1 areJ. Funct. Biomater. 2023, 14, 143 9 of 15
In manufacturing starch scaffolds, glycerol was used, which acts as a plasticizer [76].
Therefore, it is suggested that, during processing, there was a change in the crystalline
structure of starch through its interaction with glycerol.
After adding AgNPs to the scaffold (Figure 4C), the crystallinity increased to 33.88%,
representing a 16.93% increase over the scaffold without AgNPs. It has been shown that
the presence of AgNPs leads to a higher degree of crystallinity in polyimide films [77].
The starch–AgNPs scaffold exhibited four diffraction peaks at 37.80◦ , 44.02◦ , 64.36◦ , and
77.50◦ , which corresponded to planes (111), (200), (220), and (311) and correlated with the
face-centered cubic structure of metallic silver (JCPDS file No. 03-0921). This validated the
inclusion of AgNPs in the scaffold [78].
3.4. Scaffold Chemistry
The FTIR spectra of the starch, starch scaffold, and starch–AgNPs scaffold are shown in
Figure 5. The vibration bands in the jackfruit starch spectrum (Figure 5A) represent features
of the molecular deformations present in the starch molecules. The stretching deformation
of –OH
J. Funct. Biomater. 2023, 14, x FOR PEER REVIEWis responsible for the band located at 3400 cm−1 and angular deformation10ofofthe 16
band at 1650 cm−1 [79]. The symmetrical stretching of –C–H is represented by the vibration
band at 2926 and asymmetrical stretches at 2897 cm−1 . The C–O–H bonds are represented
by the bands at 1460–1400 cm−1 [80]. Absorptions at 1340 and 1024 cm−1 are due to –C–OH
groupThe FTIR spectraVibration
deformations. of the starch
modes(Figure 5B) and
associated withstarch–AgNPs
–C–C–H bonds(Figure
were5C) scaffolds
detected at
matched the jackfruit starch
− 1 spectrum very well. Such behavior shows that
1418, 1205, and 1080 cm , whereas C–O, C–O–C, and C–C stretches corresponded to the the interaction
of the1107,
1153, AgNPsand with
933 cmthe
−1 starch
bands,was
whichphysical, ratherofthan
were typical chemicalmodes
the vibration [82,83].associated
This suggests
with
pyranose rings found in natural polysaccharides [81]. All of these bands could bedata),
that, while AgNPs were present in the scaffold (as shown by the XRD and EDS they
related to
did not chemically
jackfruit starch bonds.interact with the starch’s polymer chains.
Figure5.
Figure 5. FTIR
FTIR spectra
spectraof
ofthe
the(A)
(A)jackfruit
jackfruitstarch,
starch,(B)
(B)starch
starchscaffold,
scaffold,and
and(C)
(C)starch–AgNPs
starch–AgNPsscaffold.
scaf-
fold.
3.5. Thermal Analysis
Figure 6 depicts the thermograms of the starch and starch–AgNPs scaffolds, which
display three endothermic peaks. The first referred to starch gelatinization, which had aJ. Funct. Biomater. 2023, 14, 143 10 of 15
The FTIR spectra of the starch (Figure 5B) and starch–AgNPs (Figure 5C) scaffolds
matched the jackfruit starch spectrum very well. Such behavior shows that the interaction
of the AgNPs with the starch was physical, rather than chemical [82,83]. This suggests that,
while AgNPs were present in the scaffold (as shown by the XRD and EDS data), they did
not chemically interact with the starch’s polymer chains.
3.5. Thermal Analysis
Figure 6 depicts the thermograms of the starch and starch–AgNPs scaffolds, which
display three endothermic peaks. The first referred to starch gelatinization, which had a
gelatinization temperature of 81.3 ◦ C in the starch scaffold and 71.3 ◦ C in the starch–AgNPs
scaffold
J. Funct. Biomater. 2023, 14, x FOR PEER REVIEW[42,84]; the second peak, at temperatures of 110.2 ◦ C and 105.8 ◦ C in the11starch
of 16
scaffold and starch–AgNPs scaffold, could be attributed to the gelatinization of the amylose
present in the starch, which was complexed by lipids [85,86]. The melting temperature (Tm ,
third peak) of the starch increased from 146.3 ◦ C to 185.2 ◦ C with AgNPs incorporation.
whenpeak
This AgNPs
was were
causedincluded in gelatin
by the melting films [91] and
of crystalline sugar
starch palmthat
domains starch
werebiocomposites
reorganized
[89].
during retrogradation [87].
Figure 6.
Figure 6. DSC
DSC analysis
analysis of
of (A)
(A) starch
starch scaffold
scaffold and
and (B)
(B) starch–AgNPs
starch–AgNPs scaffold.
scaffold.
3.6. Cytotoxicity
The results Analysis
revealed that, after incorporating AgNPs, the gelatinization temperatures
of starch and amylose decreased while the Tm rose. There is evidence that AgNPs and other
According to Figure 7A, it was observed that the tested dilutions of the AgNPs solu-
metallic nanoparticles have a greater effect on the Tm than they do on changing the initial
tion presented cytotoxicity values in the range of 99 to 86%. The highest dilution 1/16,
degradation temperatures, as was the case for−5the gelatinization temperatures [83,88]. The
corresponding to a concentration of 6.25 × 10 mol·L−1, showed a cell viability of 99%. By
increase in Tm could be explained by the fact that AgNPs are more thermally stable [83,89].
gradually increasing the concentration of AgNPs, a slow, but proportional, reduction in
Shameli, et al. [90] reported similar results, observing an 18% increase in the enthalpy of
cell viability was noted. This was due to the AgNPs causing apoptosis and cell death [92].
starch–AgNPs composite films. The thermal stability was similarly enhanced when AgNPs
However, even in the sample with the highest concentration of 1/1 (1 × 10−3 mol·L−1), a cell
were included in gelatin films [91] and sugar palm starch biocomposites [89].
viability of 86% was observed, which, according to ISO 10993-5, indicates the absence of
toxicity
3.6. [50]. Therefore,
Cytotoxicity Analysis triangular anisotropic AgNPs with a diameter of ~30 nm in the
concentration range of 6.25 × 10−5 to 1 × 10−3 mol·L−1 were not toxic against L929 fibroblast
According to Figure 7A, it was observed that the tested dilutions of the AgNPs
cells.
solution presented cytotoxicity values in the range of 99 to 86%. The highest dilution 1/16,
Figure 7B to
corresponding demonstrates that of
a concentration the6.25
cell×viability
10−5 mol values of both scaffolds,
·L−1 , showed one without
a cell viability of 99%.
AgNPs and one with, were 80.2% and 80.3%, respectively. Additionally, adding
By gradually increasing the concentration of AgNPs, a slow, but proportional, reduction AgNPsin
did not result in any reduction in the viability of the cells, suggesting that AgNPs did not
have any cytotoxic effects. The veracity of the data was confirmed by the fact that the neg-
ative control showed a hundred-percent cell proliferation. According to ISO 10993-5, if the
material has a value that is lower than 70%, it is considered to have a toxic effect [50]. Both
scaffolds exhibited cell survival values higher than 70%, demonstrating that the jackfruitJ. Funct. Biomater. 2023, 14, 143 11 of 15
cell viability was noted. This was due to the AgNPs causing apoptosis and cell death [92].
However, even in the sample with the highest concentration of 1/1 (1 × 10−3 mol·L−1 ), a
cell viability of 86% was observed, which, according to ISO 10993-5, indicates the absence
J. Funct. Biomater. 2023, 14, x FOR PEER
ofREVIEW
toxicity [50]. Therefore, triangular anisotropic AgNPs with a diameter of ~3012nm of 16
in
the concentration range of 6.25 × 10−5 to 1 × 10−3 mol·L−1 were not toxic against L929
fibroblast cells.
Figure
Figure 7.
7. Cytotoxicity
Cytotoxicity of AgNPs dilutions
of AgNPs dilutions (A)
(A)and
andstarch
starchscaffold
scaffoldand
and starch–AgNPs
starch–AgNPs scaffold
scaffold (B)(B)
ob-
obtained by the MTT method.
tained by the MTT method.
4. Conclusions
Figure 7B demonstrates that the cell viability values of both scaffolds, one without
AgNPsIn this
and study, wewere
one with, investigated
80.2% andthe impact
80.3%, of incorporating
respectively. silver adding
Additionally, nanoparticles
AgNPs
(AgNPs) into a Brazilian jackfruit (Artocarpus heterophyllus) starch-based scaffold by
did not result in any reduction in the viability of the cells, suggesting that AgNPs didana-
not
have any cytotoxic effects. The veracity of the data was confirmed by
lyzing its chemical, physical, thermal, and morphological characteristics. The chemical re-the fact that the
duction technique yielded monodisperse, isotropic, and stable AgNPs with a size of 33.27if
negative control showed a hundred-percent cell proliferation. According to ISO 10993-5,
the material
nm. has aofvalue
The presence AgNPs thatinisthe
lower than
starch 70%, itwas
scaffold is considered
verified byto EDShave a toxic
and XRD effect [50].
analyses.
Both scaffolds exhibited cell survival values higher than 70%, demonstrating
The FTIR spectra of the jackfruit starch and starch–AgNPs scaffolds were identical, sug- that the
jackfruit
gesting starch
that scaffold loaded
the interaction of AgNPswith AgNPs waswas
with starch not purely
toxic tophysical.
L929 cells,
SEM which suggests
revealed the
scaffolds to have a very porous and three-dimensional structure. AgNPs inclusionsuch
that it is appropriate for application as a biomaterial in treating various conditions, main-as
healing wounds.
tained the scaffold’s porosity while increasing its surface roughness and crystallinity,
which are all conducive to enhancing biological responses. In conclusion, the crystallinity,
4. Conclusions
roughness, and melting temperature of the jackfruit starch scaffold were all improved by
In this study, we investigated the impact of incorporating silver nanoparticles (AgNPs)
the addition of AgNPs. The scaffold could be developed using AgNPs to better survive
into a Brazilian jackfruit (Artocarpus heterophyllus) starch-based scaffold by analyzing its
temperature changes and demonstrate stronger biological interactions. The L929 cell sur-
chemical, physical, thermal, and morphological characteristics. The chemical reduction
vival rate was greater than 70% for AgNPs at concentrations of 6.25 × 10−5 to 1 × 10−3 mol·L-
technique yielded monodisperse, isotropic, and stable AgNPs with a size of 33.27 nm. The
1 and for both scaffolds, confirming that triangular anisotropic AgNPs with a 30 nm diam-
presence of AgNPs in the starch scaffold was verified by EDS and XRD analyses. The FTIR
eter and the scaffold loaded with AgNPs were non-toxic to L929 cells and clinically rele-
spectra of the jackfruit starch and starch–AgNPs scaffolds were identical, suggesting that
vant for treating wounds, for example. These findings help to fill a gap in our understand-
the interaction of AgNPs with starch was purely physical. SEM revealed the scaffolds to
ing of the toxicity and impacts of triangular anisotropic AgNPs on the physicochemical
have a very porous and three-dimensional structure. AgNPs inclusion maintained the
and biological properties of polymeric matrices. They have implications for a wide range
scaffold’s porosity while increasing its surface roughness and crystallinity, which are all
of starch-based scaffoldings
conducive to enhancing and highlight
biological responses.theInpotential
conclusion,usethe
of crystallinity,
jackfruit starch in bio-
roughness,
material
and meltingproduction. The next
temperature of thestep wouldstarch
jackfruit be using this approach
scaffold to modifyby
were all improved thethebiological
addition
and mechanical properties of scaffolds by including AgNPs.
of AgNPs. The scaffold could be developed using AgNPs to better survive temperature
changes and demonstrate stronger biological interactions. The L929 cell survival rate was
Author Contributions: Conceptualization, data curation, investigation, methodology and writing—
greater than 70% for AgNPs at concentrations of 6.25 × 10−5 to 1 × 10−3 mol·L−1 and
original draft, J.F.B.R.; Funding acquisition, V.S.A.; Data curation and visualization, R.P.M.; Inves-
for both scaffolds, confirming that triangular anisotropic AgNPs with a 30 nm diameter
tigation, data curation, and methodology, G.B.d.C.B.; Project administration and supervision,
and the scaffold
M.R.d.O.P.; Resources,loaded with Visualization
M.V.L.F.; AgNPs wereand non-toxic to L929 and
writing—review cellsediting,
and clinically
M.M. Allrelevant
authors
for treating
have read andwounds,
agreed to for
the example. These findings
published version help to fill a gap in our understanding
of the manuscript.
of the toxicity and impacts of triangular anisotropic AgNPs on the physicochemical and
Funding: This research received no external funding.
biological properties of polymeric matrices. They have implications for a wide range of
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors would like to acknowledge BioRender for allowing us to create theJ. Funct. Biomater. 2023, 14, 143 12 of 15
starch-based scaffoldings and highlight the potential use of jackfruit starch in biomaterial
production. The next step would be using this approach to modify the biological and
mechanical properties of scaffolds by including AgNPs.
Author Contributions: Conceptualization, data curation, investigation, methodology and writing—
original draft, J.F.B.R.; Funding acquisition, V.S.A.; Data curation and visualization, R.P.M.; Investiga-
tion, data curation, and methodology, G.B.d.C.B.; Project administration and supervision, M.R.d.O.P.;
Resources, M.V.L.F.; Visualization and writing—review and editing, M.M. All authors have read and
agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors would like to acknowledge BioRender for allowing us to create
the Graphical Abstract and Figure 1, the Coordination for the Improvement of Higher Education
Personnel (CAPES), the National Council for Scientific and Technological Development (CNPq), the
Northeast Laboratory for Evaluation and Development of Biomaterials (CERTBIO), and the Federal
University of Campina Grande (UFCG).
Conflicts of Interest: The authors declare no conflict of interest.
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