Supramolecular polyelectrolyte complexes based on cyclodextrin-grafted chitosan and carrageenan for controlled drug release

Carbohydrate Polymers 245 (2020) 116592

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

Supramolecular polyelectrolyte complexes based on cyclodextrin-grafted
chitosan and carrageenan for controlled drug release

T

Thamasia F.S. Evangelistaa, George R.S. Andradeb,*, Keyte N.S. Nascimentoa,
Samuel B. dos Santosc, Maria de Fátima Costa Santosd, Caroline Da Ros Montes D'Ocae,
Charles dos S. Estevamc, Iara F. Gimenezf, Luís E. Almeidaa,*
a

Postgraduate Program in Materials Science and Engineering, Federal University of Sergipe, São Cristóvão, SE, Brazil
Postgraduate Program in Energy, Federal University of Espírito Santo, São Mateus, ES, Brazil
c
Department of Physiology, Federal University of Sergipe, São Cristóvão, SE, Brazil
d
Posgraduate Program of Chemistry, NMR Laboratory, Departament of Chemistry, Federal University of Paraná, Curitiba, PR, Brazil
e
NMR Laboratory, Departament of Chemistry, Federal University of Paraná, Curitiba, PR, Brazil
f
Department of Chemistry, Federal University of Sergipe, São Cristóvão, SE, Brazil
b

A R T I C LE I N FO

A B S T R A C T

Keywords:
Supramolecular polyelectrolyte complexes
Biopolymers
Controlled drug release
Silver sulfadiazine
Silver nanostructures

In the present study, supramolecular polyelectrolyte complexes (SPEC) based on a cyclodextrin-grafted chitosan
derivative and carrageenan were prepared and evaluated for controlled drug release. Samples were characterized by FTIR, SEM, and ζ-potential measurements, which confirmed the formation of the polymeric complex. The
phenolphthalein test confirmed the presence and availability of inclusion sites from the attached βCD. Silver
sulfadiazine was used as the model drug and the association with the SPEC was studied by FTIR and computational molecular modeling, using a semi-empirical method. DRS and TEM analyses have shown that Ag+ ions
from the drug were reduced to form metallic silver nanostructures. In vitro tests have shown a clear bacterial
activity toward Gram-positive bacteria Staphylococcus aureus and Enterococcus durans/hirae and Gram-negative
bacteria Klebsiella pneumoniae and Escherichia coli. Finally, this work shows that βCD-chitosan/carrageenan supramolecular polyelectrolyte complexes hold an expressive potential to be applied as a polymer-based system for
controlled drug release.

1. Introduction
The design of local drug release systems (LDRS) based on supramolecular polyelectrolyte complexes (SPECs) has become the subject of
fundamental research in the last decade. SPECs can be defined as threedimensional macromolecular structures constructed by associating oppositely charged polyelectrolytes in solution (Das & Tsianou, 2017).
The SPEC preparation is usually simple, feasible and performed under
mild conditions, allowing the final material to have various forms, including nano- and microparticles, membranes, tablets, gels, beads and
so on (Luo & Wang, 2014). Also, because of the intrinsic biocompatible
nature and the strong and reversible electrostatic interactions between
these polycations, the obtained transient structure avoids the use of

toxic cross-linkers (Liang et al., 2018), such as epichlorohydrin, allowing them to be used in humans. In this context, various natural
polymers can be used for preparing SPECs, including chitosan (CS) and
carrageenan (CRG).

Chitosan is a natural polysaccharide derived from the alkaline Ndeacetylation of chitin, a major structural component of arthropods and
crustacean shells. Due to its desirable properties, including high biocompatibility, nontoxicity, antifungal, and antimicrobial activities, this
cationic biopolymer can be used for a broad range of applications such
as food packaging films, drug carriers, wound dressings, polymeric
matrix for anchoring metal nanoparticles, and so on (Rezende et al.,
2010; Wu et al., 2018). On the other hand, carrageenan is a natural
sulfated polysaccharide isolated from red seaweeds (Rhodophyta) and

Abbreviations: βCD, β-cyclodextrin; CRG, carrageenan; CS, chitosan; DRS, diffuse reflectance spectroscopy; FTIR-ATR, Fourier transform infrared- attenuated total
reflectance; GTM, gentamicin; LDRS, local drug release system; MIC, minimum inhibitory concentration; NMR, nuclear magnetic resonance; PM3, parametric method
3; SPECs, supramolecular polyelectrolyte complexes; SPR, surface plasmon resonance; SSD, silver sulfadiazine; TEM, transmission electron microscopy; v/v %,
volume/volume percent; wt %, weight percent; XRD, X-ray diffraction; ZOI, zones of inhibition
⁎
Corresponding authors.
E-mail addresses: (G.R.S. Andrade), (L.E. Almeida).
/>Received 20 January 2020; Received in revised form 20 May 2020; Accepted 4 June 2020
Available online 11 June 2020
0144-8617/ © 2020 Elsevier Ltd. All rights reserved.


Carbohydrate Polymers 245 (2020) 116592

T.F.S. Evangelista, et al.

its chemical structure consists of a chain of alternating copolymer of βD-galactose and α-D-galactose linked through β-(1, 4) and α-(1, 3)
bonds. The presence of negatively charged sulfate groups in CRG allows
CRG polysaccharide to form a SPEC with CS. For instance, various
studies have addressed the production and characterization of SPECs
based on CS and CRG for various applications, including wastewater
purification (Liang et al., 2017), voltammetric glucose biosensor

(Rassas et al., 2019), gastroprotective agent (Volod’ko et al., 2014) and
controlled drug delivery (Mahdavinia, Karimi, Soltaniniya, &
Massoumi, 2019).
The use of these SPECs for encapsulating proteins and low molecular weight drugs has raised attention due to practical purposes.
However, despite the innumerable outstanding advantages, the association of polysaccharide-based SPECs with highly hydrophobic drugs is
still a challenge to be overcome. In Polymer Science, chemical modifications on biopolymer surface are an easy approach to provide new
functionalities for different applications. For example, Chen and Wang
(Chen & Wang, 2001) reported an easy procedure to modify CS with βcyclodextrin (βCD) for the controlled release of radioactive iodine.
Cyclodextrins are cyclic oligosaccharides built from 6, 7, and 8 glucose
units, respectively α-, β-, and γ-cyclodextrins, that are joined together
by α-1,4 bonds. Because of the presence and orientation of the hydroxyl
groups, these molecules are shaped like a truncated cone, with a hydrophilic outside part and a hydrophobic cavity. Thus, the most popular
and widely studied property of CDs is the ability to form inclusion
complexes with a wide variety of guest molecules based on hydrophobic interactions.
Host-guest inclusion complexes between βCD and various organic
molecules of biological interest were studied via experimental and
theoretical approaches (Zhang et al., 2019). For example, Lodagekar
and co-workers have shown that the formation of host-guest complexes
with cyclodextrins can significantly increase the solubility and dissolution of poorly soluble drugs as well as their pharmacokinetics
(Lodagekar et al., 2019). Various papers have shown the ability of cyclodextrin-based polymers to encapsulate small organic molecules for
drug delivery applications (El-Zeiny, Abukhadra, Sayed, Osman, &
Ahmed, 2020; Ghorpade, Yadav, & Dias, 2017; Kono & Teshirogi, 2015;
Tian, Hua, & Liu, 2020). For instance, Campos and co-workers reported
the use of β-cyclodextrin-grafted chitosan nanoparticles loaded with
volatile organic compounds for designing sustainable biopesticides
(Campos et al., 2018). In another work, Hardy and co-workers prepared
compact polyelectrolyte complexes based on βCD-functionalized chitosan/alginate for controlled release of anti-inflammatory drugs (Hardy
et al., 2018).
Herein, different compositions of SPECs based on βCD-grafted
chitosan and carrageenan were prepared via electrostatic interactions

between the negatively charged −SO3− groups of carrageenan and the
positively charged −NH3+ groups of chitosan for local drug delivery
system. These SPECs, as well as the isolated materials, were fully
characterized with FTIR, zeta potential analysis and SEM. Also, in order
to explore specific applications of the as-prepared SPECs as controlled
drug release systems, an inclusion complex with silver sulfadiazine was
prepared and characterized. Contributions to the understanding of the
interactions underlying the formation of the host-guest inclusion complex between the SPEC with silver sulfadiazine were provided by a
computational semi-empirical molecular modeling method. During the
adsorption of the drug, Ag+ ions from the drug were reduced and
metallic silver nanostructures were formed, as showed by DRS and TEM
analyses. To the best of our knowledge, this is the first work reporting
the design of SPECs based on βCD-grafted chitosan and carrageenan for
controlled release of an antibiotic drug and preparation of silver nanostructures. Finally, in vitro studies have shown a clear antibacterial
activity toward Gram-positive bacteria Staphylococcus aureus and
Enterococcus durans/hirae and Gram-negative bacteria Klebsiella pneumoniae and Escherichia coli.

2. Experimental section
2.1. Reagents
All the chemicals used in this work were analytical grade and used
without further purification: chitosan (CS, Mw =110 kDa, 84 % deacetylation degree, Sigma-Aldrich), β-cyclodextrin (βCD, C42H70O35,
Sigma-Aldrich), carrageenan (predominantly κ-carrageenan, CRG, Mw
=521 kDa, Sigma-Aldrich), p-toluenesulfonyl chloride (TsCl,
C7H7ClO2S, Sigma-Aldrich), phenolphthalein (PhP, C20H14O4, Aldrich),
sodium carbonate (Na2CO3, Dinâmica), silver sulfadiazine (SSD,
C10H9AgN4O2S, Aldrich), ethanol (C2H6O, Neon), acetic acid
(CH3COOH, Vetec), ethoxyethane ((C2H5)2O, Dinâmica), dimethylformamide (DMF, C3H7NO, Neon), Brian Heart Infusion (BHI,
Sigma-Aldrich), gentamicin (GTM, C21H43N5O7, Sigma-Aldrich), and
pyridine (C5H5N, anhydrous, 99.8 %, Sigma-Aldrich). All aqueous solutions were prepared using Milli-Q ultrapure water (resistivity around
18.2 MΩ cm at 25 °C).

2.2. Synthesis of mono-(6-O-p-toluenesulfonyl)-β-cyclodextrin
Prior to grafting βCD with chitosan, a monotosylated βCD derivative (6-OTs-βCD) was prepared by a classic method reported by Matsui
and Okimoto (Matsui & Okimoto, 1978). In a typical experiment, solutions of ρ-toluenesulfonyl chloride (30 mL, 6.7 mmol) and β-cyclodextrin (300 mL, 8.8 mmol), both in dry pyridine, were mixed, cooled
below 5 °C and stirred overnight. Then, the solvent was removed under
vacuum at 40 °C using a rotary evaporator (Fisatom 802) and 200 mL of
diethyl ether was added to the residue. The precipitate was collected,
successively recrystallized from water to obtain the pure monotosylated
derivative and dried in an oven at 80 °C for 48 h.
2.3. Preparation of βCD-CS
A method developed by Chen and Wang (Chen & Wang, 2001) was
used in this step. Initially, 3.0 g of powdered CS was swelled in 150 mL
of DMF and 20 mL of a mono-(6-O-p-toluenesulfonyl)-β-CD solution
(5.0 g dissolved in DMF) were slowly dropped into the CS solution. The
mixture was stirred at 140 rpm at 50 °C for 48 h, filtered and washed
with water. Finally, the powder was dried in an oven at 80 °C for 24 h.
A yield of 30 % was obtained.
2.4. Preparation of βCD-CS:CRG polyelectrolyte complexes
SPECs based on βCD-CS (or CS) and CRG were prepared by mixing
10 mL of each polymeric solution in acetic acid (2% v/v) at room
temperature, as following: (1) Initially, the polyion stock solutions were
prepared at the same concentration (0.181 g L−1) in 40 ml of acetic
acid solution; (2) 10 mL of the chitosan gel was added gradually to 10
mL of the carrageenan gel under constant magnetic stirring. In this
work, different proportions βCD-CS:CRG (or CS:CRG) were studied; (3)
After being kept for a given time (24, 48, 72, 96 and 120 h) under
magnetically stirring, a residual precipitate mass was collected by
centrifugation at 400 rpm for 10 min and dried in an oven at 45 °C for
12 h.
The practical yield was calculated by employing Equation 01:

SPEC yield (%) =

mSPEC
x 100
(mβCD−CS + mCRG )

(1)

where mβCD-CS, mCRG, and mSPEC are the initial masses of βCD-CS, CRG,
and the final mass of βCD-CS/CRG, respectively.
2.5. Evaluation of phenolphthalein inclusion
To evaluate if the cyclodextrin’s cavity in βCD-CS is available to
form inclusion complexes with organic molecules, an easy test using
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T.F.S. Evangelista, et al.

cultures was estimated by comparing the resulting suspension to the
McFarland standard.
For the antimicrobial disc diffusion susceptibility test, an adapted
methodology was used (Bauer, Kirby, Sherris, & Turck, 1966) which
follows the recommendations from the NCCLS (National Committee of
Clinical Laboratory Standards) guidelines (CLSI, 2019). In a typical
experiment, cultures of the microorganisms were transferred to 5 mL of
BHI agar and incubated overnight at 37 °C. The inoculum was standardized and compared to the standard 0.5 tube of McFarland. In this
work, Petri dishes containing 4 mm of culture medium Muller- Hinton
(pH 7.2–7.4) were used. Afterward, 6 mm filter discs were impregnated

with 20 μg mL−1 corresponding aqueous sample suspension and placed
onto the agar surface in each plate containing the microorganisms. All
the plates were incubated for 12 h at 37 °C. As control tests, the bacterial cells without any materials and with gentamicin (20 μg mL−1)
were carried out in the same manner. The diameters of the zones of
inhibition (ZOI) were measured. All of these experiments were replicated three times.
A microdilution method was used for determining the MIC values.
In a typical experiment, aliquots of 100 μL of the material stock solutions were each diluted in 2-fold serial dilutions in a 96-well plate, with
100 μL of TBS agar and a known amount of the bacteria suspension (108
cfu/mL). Then, the microplates were covered and incubated at 37°C for
24 h. The material concentrations varied in half fold according to a
standard protocol, ranging from 2000 to 7.81 μg mL−1 in all experiments.
An analysis of the variation of bactericidal activity over time (a
kinetic study) was also performed. Initially, a 1.5 × 108 CFU bacterial
solution in BHI was prepared and incubated for 18−24 h at 37 °C.
Then, 0.5 mL of the BHI was transferred to 3 tubes with 4.0 mL of the
Müller-Hinton broth: 0.5 mL of the as-prepared material suspension
was added to the first; 0.5 mL of sterilized water was added to the
second, as negative control; and 0.5 mL of gentamicin was added to the
third. Then all tubes were intubated in the oven at 37 °C and 100 μL of
this mixture were withdrawn after 3 h, 6 h, 12 h and 24 h. Finally, the
sample was seeded in petri dishes with Müller Hinton and the CFU
count was performed on the plate after 18−24 h. The results were
expressed in Log.

phenolphthalein was performed. The method used here was based on
previous works (Mohamed, Wilson, & Headley, 2010; Moreira,
Andrade, de Araujo, Kubota, & Gimenez, 2016) and it was chosen because of the strong affinity of phenolphthalein with βCD. Initially, a
3.75 mmol L−1 phenolphthalein solution was prepared in ethanol 94 %
(94 % ethanol:6 % water, v/v). This solution was diluted with Milli-Q
ultrapure water in a 1:10 proportion and the pH was adjusted to 10 by

adding a Na2CO3 aqueous solution (1 mol L−1). A known mass of βCDCS was added to 10 mL of the resulting phenolphthalein solution, followed by sonication for 4 h at room temperature (25 °C). Finally, the
suspensions were centrifuged at 4000 rpm for10 min and the absorbance of the supernatant read at 552 nm. Additionally, the same experiment was performed using natural CS, CRG, and βCD-CS:CRG
polyelectrolyte complex (4.25CRG:1.0βCD-CS). All the experiments
were performed in triplicate.
2.6. Complexation of silver sulfadiazine
Inclusion complexes based on the as-prepared SPEC with silver
sulfadiazine (SSD) were prepared by dispersing 0.6 g of the polymeric
matrix (4.25CRG:1.0βCD-CS) in 100 mL of ethanol, then 0.006 g of SSD
was slowly added to the reaction mixture and sonicated (40 kHz) for 30
min. After this step, the mixture was stirred for 24 h. The obtained
compound was filtered and dried in an oven for 24 h at 45 °C.
2.7. Molecular modeling/geometry optimization
The molecular modeling methodology was based on previous reports of our group (Borba et al., 2015; de Araújo et al., 2017). Initially,
the βCD structure built up based on the data from Cambridge Structural
Database (Allen, 2002), whereas the sulfadiazine ion structure was built
up using the molecular builder included in Cache Worksystem 6.1
(Fujitsu Ltd., Japan). All the isolated structures were initially optimized
employing the MM3 method (Allinger, Yuh, & Lii, 1989, p. 3), which is
implemented within the Cache Worksystem 6.1 software. Then, a complete geometry optimization without an geometric restriction with the
Parametric Method 3 (PM3), a semiempirical method implemented
within the MOPAC2007 program (Stewart, 1989), was employed. The
most energetically favorable structures of the isolated molecules (see
Fig. S1, Supporting Information) were used as starting structures to
construct the inclusion complexes.
The inclusion complex structures were constructed manually by
inserting the drug molecule from an end of the host molecule and no
geometry constraint was imposed during the optimization. Four different inclusion orientations were considered: (1) NH2-in, with the NH2
group pointing toward the narrower βCD rim, (2) NH2-out, having the
pyrimidine ring pointing toward the narrower rim, (3) NH2-c-in and (4)
NH2-c-out, with the drug molecule positioned in the center of the host

cavity. All the structures were initially optimized employing the MM3
method and then with PM3.

2.9. Characterization
The isolated polymers and SPEC samples were characterized by
various technics, as described below. For SEM-EDS, TEM, NMR, FTIR
and UV/visible spectroscopies, the chosen SPEC sample was the one
which presented the isoelectric point determined by ζ-potential study
(4.25CRG:1.0βCD-CS).
2.10. Net charge determination (ζ-potential)
For ζ-potential measurements, a Malvern Zetasizer NanoZS equipment was used. This instrument measures the electrophoretic mobility
of the sample and calculates the zeta potential using the Smoluchowski
expression. For this study, various SPEC compositions were prepared by
varying the βCD-CS/CRG ratio. The samples were prepared as described
before (see Section “Preparation of βCD-CS:CRG polyelectrolyte complexes”), where the interaction time was 96 h and pH around 4.5. Then,
1 mL of these samples was placed in a capillary cell (DTS 1070). All
measurements were performed at 25 ± 2 °C with the equilibration time
set to 5 min and without any salt addition or sample dilution.

2.8. Antibacterial activity assay
Antimicrobial activity of silver sulfadiazine (1.0 %, w/w) loaded
βCD-CS/CRG supramolecular polyelectrolyte complex was evaluated
using the Mueller-Hinton agar for disk diffusion susceptibility test and
by measuring the minimum inhibitory concentration (MIC) values.
Herein, Gram-positive bacteria Staphylococcus aureus (ATCC 25923)
and Enterococcus durans/hirae (SS1225/ IAL 03/10) and Gram-negative
bacteria Klebsiella pneumoniae (ATCC 700603) and Escherichia coli
(ATCC 25922) were used. All the bacterial strains used in this work
were donated by the National Institute for Quality Control in Health
(INCQS-Fiocruz). For bacteria growth, all apparatus and materials were

autoclaved and handled under sterile conditions during the experiments. All the bacteria strains were revived with Brain Heart Infusion
(BHI) agar at 35°C for 6 h. The density of bacterial cells in the liquid

2.11. FTIR spectroscopy
FTIR measurements were performed in order to characterize the
isolated materials (CS and CRG), the βCD-modified CS and the association between these biopolymers in SPECs. FTIR spectra for these
powdered materials were recorded on a VARIAN 640-IR FTIR
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T.F.S. Evangelista, et al.

Spectrometer using KBr pellets method. All the measurements were
performed in the wavenumber range 400–4000 cm−1 at a resolution of
4 cm−1 and 64 scans per sample.
2.12. UV/visible absorption spectroscopy
The UV–vis-NIR absorption spectra of samples in solid-state were
obtained by Diffuse Reflectance Spectroscopy (DRS) using an Ocean
Optics HR2000 spectrophotometer coupled to an integrating sphere. On
the other hand, the UV/vis spectra of samples in solution were measured using a Perkin Elmer Lambda 45 spectrophotometer.
2.13. SEM-EDS studies
The surface morphology of the as-prepared SPECs and the isolated
polymers was examined using a JSM-5700 scanning electron microscope (Jeol, Japan) operating at a voltage of 5 kV. Elemental analysis of
the samples by energy-dispersive X-ray spectroscopy (EDS) was performed at 5 kV using an accoupled EDS detector (Jeol/JCM5700). The
surface of the samples was coated with a thin layer of gold by vacuum
evaporation. The size distribution of the SPEC particles was obtained
using the software ImageJ64 by measuring the sizes of more than 100
particles.

Fig. 1. Zeta Potential (mV) for different compositions of βCD-CS/CRG SPECs
after 96 h under contact.

This study was performed by preparing SPECs with different polycation/polyanion mass mixing ratios. Initially, the ζ potential was measured for the isolated biopolymers. The ζ potential for CS was positive
(+92.72 mV), which is related to the positively charged amine groups
on its surface. After functionalizing this biopolymer with βCD, a decrease was observed in the ζ potential, which reached +48.83 mV. This
result is expected and confirms the reaction between CS with βCD,
which occurs by decreasing the number of free NH2 on the polymer
surface. The counterpart of the SPEC, CRG, presented a negative ζ potential value (−52.44 mV) due to the existence of negatively charged
sulfate groups. Fig. 1 shows the ζ potential versus different βCD-CS:CRG
compositions, differing the mass mixing ratio. From an exponential fit
applied to these data, it was observed that this SPEC system is characterized by the presence of two important zones. The first one goes
from point (I) to (II), where the ζ potential became significantly less
negative as the ratio βCD-CS/CRG varies from 0.12 (1:8, βCD-CS:CRG)
to 0.22 (1:4.5). The point (II) in Fig. 1 corresponds to the isoelectric
point, where ζ potential is zero. Above that point, a new zone (from
point (II) to (III)) is characterized by a charge switchover from negative
to positive values, indicating the excess of βCD-CS.
The excess of the polyanion or the polycation counterpart during the
preparation can afford the design of cationic or anionic SPECs, which
may occur via different mechanisms. The formation of cationic SPEC
particles can be described by the hydrophilic crown mechanism
(Volod’ko et al., 2018). This mechanism is based on the stabilization of
the system by unreacted amine groups from the polycation, which are
located outside the SPEC particles. On the other hand, as observed in
Fig. 1, anionic SPEC particles are formed when the βCD-CS:CRG ratio is
below 0.22 (point II). In this case, the excess of κ-CRG molecules enhances the negative charge density on the SPEC surface.

2.14. Transmission Electron Microscopy (TEM)

The samples were characterized by TEM using a Jeol JEM-1400 Plus
instrument operated at 120 kV. Samples were previously diluted in
water (1:10, v/v) and then deposited onto copper grids coated with
ultrathin carbon and formvar films.
2.15. NMR analysis
The samples (10 mg) were dissolved in D2O/DCl 2 % v/v solution
(600 μL) at room temperature. 1H NMR spectra were acquired on an
Bruker AVANCE II 400 NMR spectrometer (Bruker BioSpin
Corporation) equipped with a 5 mm multinuclear direct detection probe
with z-gradient, operating at 9.4 T observing the 1H nuclei at 400.13
MHz at 80 °C. All NMR spectra were acquired through pulse sequence
zg (Bruker library), 64 K data points, spectral width 14 ppm and 32 or
64 transients. The chemical shifts were expressed in relative to methyl
group of internal reference, TMSP-d4 at δH = 0.00.
3. Results and discussion
3.1. SPEC yield and net charge determination
Herein, SPEC materials based on a βCD-grafted CS and CRG were
prepared for controlled drug release. When the carrageenan solution is
dropped into the chitosan solution, instantaneous turbidity was observed due to the electrostatic attraction between positively charged
amine groups on CS and negatively sulfate groups on CRG. Depending
on the interaction time between the used biopolymers, different mass
yields can be achieved as a result of the kinetic formation of the SPEC,
which can involve nucleation, growth, and rearrangement steps
(Takahashi, Narayanan, & Sato, 2017). The yield of SPEC was calculated based on the complex mass obtained after the drying process and
the initial mass of the isolated biopolymers. Table S1 (see Supporting
Information) shows that the interaction between βCD-CS and CRG
produced the highest complex yield around 66.5 % after 96 h under
continuous stirring. After 120 h, the SPEC yield was very close to the
one found at 96 h, which suggests that the complex reached its highest
yield. This interaction time was used to prepare all the samples in this

work.
Zeta (ζ) potential was used to investigate the charge density of the
as-prepared SPECs and to determine the isoelectric point of this system.

3.2. Structural and molecular characterization of the isolated biopolymers
and SPECs
FTIR spectroscopy was used to explain the interaction between the
polyelectrolytic biopolymers. The functional groups of CS, βCD-CS,
CRG, and βCD-CS:CRG supramolecular polyelectrolytic complex were
studied using FTIR analysis, as shown in Fig. 2. The pure chitosan exhibited the following characteristic peaks: (1) a broadband at 3800 to
2985 cm−1, relative to OH asymmetric vibrations; (2) superimposed to
this large band, a contribution at 3360 cm−1 can be attributed to a
stretching mode of NH from a group located at the polysaccharide
chain’s glucose residue; (3) another peak was found at 2880 and 2930
cm−1, which is relative to CH groups; (4) a characteristic peak from
amide I at 1655 cm−1, (5) a band of -NH2 bending at 1590 cm−1; (6)
bands at 1420 cm−1 (CH2 deformation), 1383 (-CH3 symmetric
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some peaks are overlapped. However, the presence of a characteristic
peak at 944 cm−1, assigned to the α-pyranyl vibration of CD, and also
the absence of a characteristic peak of the benzene backbone from Ts
group (a peak at approximately 1605 cm−1) corroborates the reaction
between βCD and CS (Chen & Wang, 2001).
The FTIR spectrum of free carrageenan exhibited a broad vibration

band from OH moieties at 3450 cm−1, corresponding to OH stretching,
and bands around 2910 cm−1, which can be assigned to stretching
vibration of hydrocarbon groups (CH3 and CH2). Typical peaks of sulfate ester groups were found between 1210 and 1260 cm−1 (Dong
et al., 2018; Rhein-Knudsen, Ale, Ajalloueian, Yu, & Meyer, 2017). A
strong band at 930 cm−1 indicated the presence of 3,6-anhydro-D-galactose and a band at 840 cm−1 can be assigned to galactose-4-sulfate
(Roy & Rhim, 2019). The polycation and the polyanion macromolecules
interact mainly via electrostatic attraction, although inter-macromolecular interactions, including H-bonding, hydrophobic interactions,
dipole interactions, and van der Waals forces can also be involved. The
result of these interactions is the formation of a precipitate, the SPEC. In
Fig. 2, it is possible to find the characteristic bands from amide and
sulfate groups in the SPEC, confirming the presence of both biopolymers, although no significant changes have been observed in the band
positions (Carneiro et al., 2013).
The chemical structures of CS, βCD-CS, SPEC, SSD, and SSD/SPEC
were fully characterized by 1H NMR. For unmodified CS (see Fig. S3a,
Supporting Information), signals from anomeric hydrogens (H-1) can be
clearly identified at δH 4.94 (d, J =7.9 Hz) and 4.65 (d, J =7.3 Hz) for
glucosamine (D) and N-acetylglucosamine (A), respectively. Moreover,
the signal at δH 2.06 (s) can be attributed to hydrogens from methyl
group of N-acetylglucosamine (H3C-Ac) and the signal at δH 3.24 (H2)
of glucosamine (D). The multiplet proton signals at δH 4.03−3.55 were
attributed to the hydrogens 2−6. All assignment were characteristic of
CS and have also been previously reported (Auzély-Velty & Rinaudo,
2001; Lavertu et al., 2003). For determining the degree of acetylation
(DA%) of chitosan (CS), we used the integrals of the hydrogens from the
methyl group of N-acetylglucosamine (H3C-Ac) and hydrogen (H-2) of
glucosamine (D). The formula used was DA (%) = (ACH3/3. AH2) ×
100. The analysis showed that commercial CS presents an acetylation
degree of 16 % (Fig. S3), which is similar to the one described by the
supplier (15 %).
Fig. S3 (see Supporting Information) shows the 1H NMR of βCD-CS.

The degree of β-CD substitution on chitosan was determined as reported
by Venter et al. (Venter, Kotzé, Auzély-Velty, & Rinaudo, 2006), using
the following equation:

Fig. 2. FTIR spectra of the isolated components and SPECs.

deformation), and 1323 cm−1 (amide III and CH2 wagging); and (7)
bands related to the glycosidic ring at 1200−800 cm−1 (the glycosidic
linkage appears at 1156 cm−1). All these observations are in agreement
with previous studies (Liang et al., 2017, 2018).
The chemically active groups for CS are the free NH2 and OH
groups, both of which prone to be modified and involved in hydrogen,
hydrophobic or electrostatic bonds (Mohammed, Syeda, Wasan, &
Wasan, 2017). In this work, the CS surface was modified with 6-OTsβCD. This monotosylated βCD derivative is widely used as an intermediate for transforming the primary hydroxyl moiety of the βCD into
other functional groups. As observed in Fig. S2 (see Supporting Information), the reaction is processed with the removal of the leaving
group (OTs) and the direct attachment of βCD via NH2 group from CS.
The FTIR spectrum for βCD-CS is shown in Fig. 2. βCD and CS are both
carbohydrates, so they have some similar groups and, consequently,

DS (%) =

H1CD
x 100
H2'Chitosan

(2)

where, DS is the degree of substitution, H1CD is the integration value of
the H1 anomeric proton of βCD signal and H2’Chitosan is the integration value of the H2’ proton signal. Thus, the found value for DS was
18.45 %. Also, it was observed a signal related to the tosyl group at δH

Fig. 3. 1H NMR (D2O/DCl, 2 % v/v, 400 MHz, 80 °C) spectra: (a) CS/βCD (1), CRG (2) and SPEC (3). (b) SPEC (1), SDZ-Ag (2), SSD/SPEC (3).
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T.F.S. Evangelista, et al.

Fig. 4. SEM images of CS (A and B), β-CD (C and D), βCD-CS (E and F), CRG (G and H) and SPEC (I and J).

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T.F.S. Evangelista, et al.

2.4, which is absent in the 1H NMR spectra of SPEC (see Fig. 3). Its
narrow area suggests that it is merely a small remaining fraction from
the synthesis. The interaction between CS/β-CD with carrageenan
(CRG) was also studied by 1H NMR spectra (Tojo & Prado, 2003). As
observed in Fig. 3a, it is clean the change of the proton chemical shifts
for both biopolymer, especially for the H2’ proton signal and for the
chemical shifts from δH 5.5 to 4.5. According to Voron’ko et al. changes
like this for polyelectrolyte complexes indicate a change in the electrostatic interactions involving charged groups from the polyions
(Voron’ko, Derkach, Vovk, & Tolstoy, 2016).
The surface morphology of the isolated materials and the as-prepared SPEC was studied by SEM analysis. Fig. 4a and b show the surface
structure of commercial CS, which exhibited a flake-like structure with
a moderate degree of irregularity. βCD (Fig. 4c and d) presented a

three-dimensional block structure with an irregular shape. After the
functionalization of CS with βCD, the final polymer structure was
smoother than the initial CS, as observed in Fig. 4e and f. Commercial
CRG also presented an irregular morphology (see Fig. 4g and h). On the
other hand, the mixing of aqueous βCD-CS and CRG solutions leads to
the formation of a dense phase, which is observed in the SEM images as
well-defined SPEC sub-micro particles (Fig. 4i and j). The precipitation
of the SPEC as sub-micro particles fully confirms the electrostatic interactions between the oppositely charged macromolecules.
3.3. Evaluation of phenolphthalein inclusion

Fig. 5. Evaluation of phenolphthalein inclusion into different biopolymers.
Curve of [PhP]/[PhP]0 versus [Biopolymers], where [Biopolymers], [PhP]0,
and [PhP] are the concentration of the biopolymer, the initial and final concentrations of PhP, respectively.

Herein, the as-prepared SPECs were designed to act as drug delivery/release systems mainly by forming host-guest inclusion complexes. Thus, the first step is to evaluate the availability of inclusion
sites, which can be easily determined by absorbance changes of a suitable organic dye when in contact with these materials.
Phenolphthalein (PhP) is a phthalein dye generally used as a pH indicator because of its distinctive color change from colorless to pinkpurple in pH values above 8.4. When PhP is added to a CD aqueous
solution, a decrease of absorbance in the visible region (λmax =552 nm)
is observed, even in alkali solutions, due to a host-guest complex formation. This behavior was explained by Taguchi (Taguchi, 1986) in
terms of the transformation of PhP into its colorless lactonoid dianion
form within the host cavity. Thus, because of its high affinity for the CD
cavity, PhP has been used for proving the presence of inclusion sites
and, consequently, the existence of cyclodextrins (Akỗakoca Kumbasar,
Akduman, & ầay, 2014).
As observed in Fig. 5, when the SPEC containing βCD is added to a
PhP solution, a significant decrease of absorbance intensity at the absorption maxima (λ =552 nm) occurs, which 95 % of the absorbance
decrease for the lowest concentration of SPEC (1.0 mg/mL). This result
indicates the successful formation of supramolecular graft polymer
structures, as the βCD molecules in the SPEC surface are able to form
host-guest complexes with organic molecules. However, it is well

known that biopolymers and their derivatives are highly active towards
adsorption of dye molecules. This interaction can occur via physical
adsorption (when the drug is physically entrapped) or via a variety of
intermolecular attraction forces, which depends on the chemical nature
of all the components. Thus, a control test consisting of mixing the same
mass of CS, CRG and their complex (CS/CRG) with a PhP solution was
performed to evaluate the adsorption of the dye in the absence of βCD.
As observed in Fig. 5, the adsorption test demonstrated a small
contribution to the decrease of the PhP absorbance at 552 nm. As βCD
is absent in CS/CRG (as well as in bare CS and CRG), this result suggests
that there is interaction between PhP and the polymeric chain. Likewise, the same study was performed in the presence of the βCD-grafted
CS and the absorbance decrease was around 85 % for the highest
concentration of this polymer (6.0 mg/mL). When βCD-CS and SPEC
are compared, it is expected a higher decrease of PhP absorbance when
the SPEC is used, as PhP is able to interact not only by the formation of
inclusion complexes. Those tests evidence the presence of βCD in the as-

prepared SPEC and the possibility of preparing host-guest complexes.

3.4. Evaluation of SSD inclusion
Herein, SSD was incorporated into the as-prepared SPEC. SSD was
chosen as a model drug because it is an effective antibacterial agent
with a broad spectrum of activity against various bacterial strains, including P. aeruginosa and S. aureus. SSD is commonly used for topical
treatment of burn wounds, as the silver ions act both as a bacteriostatic
and as a bactericidal agent. The SPEC which presented neutral zeta
potential charge and higher stability in water was chosen as the drug
carrier vehicle. The incorporated amount of SSD in the SPEC was determined using UV–vis spectroscopy, by comparing the initial absorbance intensity at λ = 256 nm (AInitial = 1.7034) and final absorbance
after 24 h under contact with the SPEC (AFinal = 0.1790). So, it is
observed a decrease of 89.5 % of the absorbance intensity, which can be
related to the incorporated amount of the drug in the polymer matrix.

After adding a known amount of SSD to the SPEC suspension under
ultrasonic irradiation, the system color changes from pale yellow to
purple. In order to investigate this behavior, SSD/SPEC and the isolated
samples were characterized by DRS and TEM. As observed in Fig. 6, the
DRS spectrum of SSD/SPEC presents a shoulder at 360 nm related to the
guest drug. In addition, the SSD/SPEC spectrum presents a broad band
centered at 536 nm, which is absent in the bare biopolymers and SSD
spectra. The presence of this broadband can be related to the conversion of Ag+ ion to metallic Ag°.
According to Bastús et al., large metallic silver particles, prepared
using a mixture of two reducing agents (sodium citrate and tannic acid),
present bands around 500 nm, which can be related to the surface
plasmon resonance (SPR) (Bastỳs, Merkoỗi, Piella, & Puntes, 2014). SPR
for metallic silver particles can be understood as the collective oscillation of electrons in resonance with the frequency of the incident
electromagnetic radiation. In another work, Jiang and coworkers reported the preparation of anisotropic silver particles using a mix of
reducing agents, including citrate and sodium bis(2-ethylhexyl)sulfosuccinate (Jiang, Chen, Chen, Xiong, & Yu, 2011). In their study, the
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T.F.S. Evangelista, et al.

observed suggest the presence of interactions between components. For
instance, the two bands related to the –NH2 symmetric and asymmetric
stretching were shifted to longer wavenumbers (respectively 3426 and
3356 cm−1). SSD/SPEC was also characterized by 1H NMR. It is possible to observe in Fig. 3b the incorporation of aromatic hydrogens at
δH 8.60 to 7.00 from SSD (See Fig. S5, Supporting Information), confirming the presence of the drug in the SPEC matrix.
Finally, a preliminary in vitro release test using dialysis membranes
was performed in phosphate buffer to evaluate the role of the SPEC
during the release of the drug. Prior to this study, the molar extinction

coefficients (ε) of SSD was calculated in phosphate buffer using the
Beer-Lambert law. The absorption maxima of this drug in 262 nm
showed a good linear relationship to the concentration range (see Fig.
S6, Supporting Information). Thus, the calculated value for ε was 2.993
× 104 L mol−1 cm−1. This information is important to calculate the
released amount of the drug during the in vitro release studies. Fig. S7
(see Supporting Information) shows the drug release profile from pure
SSD and SSD/SPEC. It was observed that the burst release for pure SSD
occurs statistically at 5 h, when approximately 60 % of the drug was
released from the dialysis bag. After that, the drug concentration is
practically constant.
On the other hand, the release of SSD from SPEC showed a different
behavior when compared to the free drug. In this case, it was observed
an initial slow stage (0–3 h), which may be associated with the swelling
properties of the polymeric matrix (Ćirić et al., 2020). As soon as this
first swelling step is overcome, the release process starts and approximately 20 % of the drug was released in 5 h, when the release curve
reaches an intermediate plateau up to 15 h. After that, it is observed
another increase up to 48 h, when approximately 80 % of the drug is
released. Comparing both systems, the drug release rate for SSD/SPEC
was almost 10 times slower than pure SSD, suggesting that the incorporation of the drug on polymer matrix surface was essential to slow
the drug release process. Finally, the slower and continuous release rate
found for SSD/SPEC can be advantageous for the design of topical drug
release systems for local treatment, such as wound dressings.

Fig. 6. DRS spectrum of CRG, βCD-CS, and SSD/SPEC, SSD.

SPR band position ranged from ultraviolet to near-infrared and was
strongly dependent upon their shapes and sizes. The reduction of Ag+
ions can be performed via sonochemical methods in the presence of
biopolymers. For instance, Elsupikhe and coworkers showed a sonochemical synthesis of colloidal silver particles using κ-carrageenan

(Elsupikhe, Shameli, Ahmad, Ibrahim, & Zainudin, 2015). The mechanism for the reduction of Ag+ ions was proposed by the authors and
started with the generation of %H and O%H free radicals by ultrasonic
irradiation prior to the reduction of Ag+ ions to Ag°.
TEM images of SSD/SPEC fully confirm the presence of anisotropic
structures after the interaction between SSD and SPEC. As observed in
Fig. 7a and b, the sub-micro particles shape of the SPEC is preserved
after the inclusion of SSD. Also, it is observed the presence of metallic
silver nanostructures (in higher contrast) distributed in the polymer
matrix surface. However, the reduction process leads to the formation
of Ag nanoparticles with irregular morphologies and large size distribution. Fig. 7c shows the occurrence of larger silver nanocubes, nanospheres and other morphologies off the polymeric surface (other
images can be found in Fig. S4, Supporting Information). Thus, as expected the size distribution for this sample is large (see Fig. 7d), which
explains the broad SPR band found.
The SSD/SPEC inclusion complex was also characterized by FTIR
and 1HNMR. As seen in see Fig. 2, the free SSD exhibited characteristic
bands at 3392, 3344, 1630, 1551, 1501, 1418, 1228 and 1125 cm−1.
The peaks at 3392, 3344 and 1630 cm−1 are assigned to –NH2 symmetric and asymmetric stretching and NH2 bending, respectively. The
vibrational stretching of its phenyl structure conjugated to the NH2
group appears at 1551 cm−1. A band at 1500 cm−1 assigned to the
phenyl skeletal vibration was also observed. The peaks centered at 1228
and 1125 cm−1 are assigned to the asymmetrical stretching of the SeO
bonding. These results are in agreement with previous reports (Shao
et al., 2017). After the incorporation of the drug into the polymeric
particles, all the peaks related to SSD were found, proving the presence
of this drug in the as-prepared SPEC. However, some spectral changes

3.5. Theoretical study
Various non-covalent interactions, such as hydrogen bonding,
electrostatic forces, and π,π-stacking, can be the driving forces to the
formation and stabilization of a host-guest complex (Al-Jaber & BaniYaseen, 2019; Aree & Jongrungruangchok, 2018). Due to the nature of
these interactions, sometimes the analytical characterization of these

supramolecular species can be a difficult task. In this context, theoretical methods, such as semi-empirical PM3, are very useful tools for
studying the inclusion process. Various reports have shown the effective
use of PM3 to calculate some energetic parameters, including thermodynamic data, as well as the geometrical structure of these inclusion
complexes (de Araújo et al., 2017; Geng et al., 2018; Yang et al., 2018).
The isolated host and guest and the inclusion complexes structures were
fully optimized by PM3 without any symmetry constraints. Herein, the
chitosan or carrageenan structures were not calculated, as they do not
participate directly in the inclusion process. Fig. 8 shows the upper and
side views of all the inclusion complex optimized structures obtained by
energy minimization at the PM3 level of theory.
To quantify the interaction between these entities in the optimized
geometries, the binding energy (or complexation energy, ΔEcomplexation )
was calculated as follows:

ΔEcomplexation = EβCD−SSD − (EβCD + ESSD)

(3)

This equation considers the difference between the heat of formation of the complex (EβCD−SSD) and the heat of formation of the free
guest (ESSD ) and host (EβCD ) molecules. Thus, the magnitude of the
energy change is a sign of the driving force toward the inclusion
complex formation. As shown in Table 1, the binding energies for all the
inclusion complexes were negative, indicating that the complexation
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Carbohydrate Polymers 245 (2020) 116592

T.F.S. Evangelista, et al.

Fig. 7. TEM images and size distribution of SSD/SPEC.

antibacterial zone of inhibition for S. aureus (16 ± 3 mm) followed by
K. pneumoniae (13 ± 2 mm), E. coli (13 ± 2 mm) and E. durans/hirae
(12 ± 1 mm). These values are very close to the ones reported for
chitosan (Bhadra, Mitra, Das, Dey, & Mukherjee, 2011; Muthuchamy
et al., 2020; Nigam, Kumar, Dutta, Pei, & Ghosh, 2016), suggesting that,
even after the modification, the natural antibacterial property remains
active. This result is expected since the substitution reaction does not
consume all the amino groups, leaving the surface positively charged,
as observed before. The other counterpart of the SPEC, CRG, also did
not present a zone of inhibition after the contact with all the bacterial
strains. In fact, CRG is generally used as an antibacterial agent after the
incorporation of other materials (such as metal or semiconductor nanoparticles) to its structure or after chemical modifications (Zhu et al.,
2017).
Moderate in vitro antimicrobial activity of chitosan and βCD-CS
against drug-resistant bacterial pathogens has already been reported by
others (Ding et al., 2019; Verlee, Mincke, & Stevens, 2017). However,
the exact antibacterial mechanism is still not fully understood, as its
mode of action is significantly influenced by various factors, including
the type of microorganism, the molecular weight, the degree of deacetylation, ionic strength and pH (Shahid-ul-Islam & Butola, 2019). It is
usually accepted that the interaction is mostly electrostatic, occurring
between the positively charged amine groups of chitosan with the negatively charged molecules from the bacterial wall or plasma membrane (Perinelli et al., 2018). Then, the permeabilization of the antimicrobial agent into the cell surface leads to leakage of intracellular
constituents (such as of nucleic acids, proteins, low-molecular-weight
materials), disturbing the physiological activities of the microorganisms
and causing cell death.

processes are exothermic. The complexes having the NH2 group from
the drug molecule pointed toward the narrower βCD rim were found to
have the smaller complexation energy (approximately −19.45 kcal/

mol for both structures), while NH2-out and NH2-c-out complexes had
the highest complexation energy with βCD. The energy difference between these two orientations can reach 5.63 kcal/mol. It means that the
NH2-in orientation is energetically preferred over the complexes which
present the pyrimidine ring pointing toward the narrower rim of βCD. A
similar result was previously reported by our group for inclusion
complexes based on sulfadiazine and hydroxypropyl-β-cyclodextrin (de
Araújo et al., 2008), where the optimized NH2-in orientation was energetically favored (over the NH2-out conformation) and presented
protons from the aniline ring closer to the secondary face of the cyclic
oligosaccharide.
3.6. Antibacterial activity assays
The antimicrobial activities of the as-prepared SPEC compounds
towards Gram-positive bacteria Staphylococcus aureus (ATCC 25923)
and Enterococcus durans/hirae (SS1225/ IAL 03/10) and Gram-negative
bacteria Klebsiella pneumoniae (ATCC 700603) and Escherichia coli
(ATCC 25922) were investigated in this work. These bacterial strains
were chosen because they are nosocomial pathogens commonly responsible for biofilm-related infections. First, the initial screening for
the antimicrobial properties of the as-prepared SSD/SPEC, as well as
pure βCD-CS, SSD, and GTM, were evaluated by a disc diffusion
method, as presented in Table 2. As seen, pure βCD does not show any
antibacterial activity, while βCD-CS showed a clear activity on both
Gram-positive and Gram-negative bacteria, presenting the highest
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T.F.S. Evangelista, et al.

Fig. 8. Upper and side views of all SSD/SPEC inclusion complex optimized structures obtained by energy minimization at the PM3 level of theory.

10


Carbohydrate Polymers 245 (2020) 116592

T.F.S. Evangelista, et al.

concentration (MIC) values were found for SSD/SPEC and for the isolated SSD and GTM drugs (see Table 2). MIC can be defined as the
minimum concentration of the antibacterial agent, which exerts the
visible bacterial growth inhibition. Based on the 96-well microdilution
assay, it was found that both SSD/SPEC and the isolated SSD presented
MIC values of 60 and 32 μg/mL for E. coli and S. aureus, respectively,
showing that the antibacterial activity was not lost after the formation
of the inclusion complex. The MIC values for the antibacterial assay in
the presence of GTM were around 128 and 64 μg/mL for E. coli and S.
aureus, respectively, which is twice as higher in concentration than the
test with pure SSD or SSD/SPEC.

Table 1
Binding energies (kcal mol−1) obtained by PM3 for SSD/βCD
inclusion complexes in different conformations.
Sample

ΔEcomplexation (kcal mol−1)

NH2-in
NH2-out
NH2-c-in
NH2-c-out

−19.4483
−13.8147
−19.4403
−17.5297

Table 2
Zone of inhibition diameter of βCD-CS, βCD, CRG, SSD, GTM, and SSD/SPEC
and Minimum Inhibitory Concentration (MIC) of SSD, SSD/SPEC and GTM
against Gram-positive and Gram-negative bacterial strains.
Bacterial strains

4. Conclusions
In conclusion, this work showed a comprehensive description of
properties, characterization and in vitro applications of supramolecular
polyelectrolyte complexes based on a cyclodextrin-grafted chitosan
derivative and carrageenan. The formation of these SPECs was confirmed by FTIR, SEM, and ζ-potential measurements. Due to the presence of inclusion sites from the attached βCD, the as-prepared materials could be suitably applied as controlled drug release systems, using
silver sulfadiazine as the model drug. Also, DRS and TEM analyses have
shown the formation of metallic silver nanostructures via the reduction
of Ag+ ions from the drug, which can improve the bacterial activity of
the composite. In vitro tests revealed that the as-prepared SSD-SPECs
conjugated presented a clear bacterial activity toward Gram-positive
bacteria Staphylococcus aureus and Enterococcus durans/hirae and Gramnegative bacteria Klebsiella pneumoniae and Escherichia coli, which was
compared to gentamicin.

Zone of inhibition diameter (in mm)
βCD-CS

S. aureus
16 ± 3
E. durans/hirae

12 ± 1
K. pneumoniae
13 ± 2
E. coli
13 ± 2
MIC values (μg/mL)
S. aureus
–
E. coli
–

βCD

CRG

SSD

GTM

SSD/SPEC

0
0
0
0

0
0
0
0

25 ± 2
17 ± 1
20 ± 1
23 ± 2

18 ± 1
19 ± 2
20 ± 1
18 ± 2

18 ± 1
19 ± 1
18 ± 2
17 ± 2

–
–

–
–

32
64

64
128

32
64

According to the CLSI interpretive criteria for disk diffusion tests
(CLSI, 2019), when βCD-CS is used in our experimental condition, S.
aureus can be classified into the intermediate (I) category, since it
presented a zone diameter between 15–19 mm. Additionally, the other
bacterial strains studied here were resistant (R) to this polymer, since
all the zone diameters were below 14 mm. The intermediate category
implies clinical efficacy where the drug is concentrated in body sites.
Also, the I category is associated with clinical events when a higher
than normal dosage of a drug can be used. On the other hand, the R
category implies that the drug efficiency against the microorganism has
not been reliably shown in treatment studies.
Herein, SSD was incorporated to the as-prepared SPEC. This drug is
normally used as an external antibacterial agent, as in the treatment of
burns, and presents a broad-spectrum activity against both Gram-positive and Gram-negative organisms. As seen in Table 2, the isolated
drug presented a clear bacterial activity to all the microorganisms used
in this work. Despite being a well-known and widely used drug, the
precise antibacterial mechanism of the silver salt form of sulfadiazine is
not well-clarified among the scientific community and still opened to
debate. It is believed that the efficacy of SSD is related to the slow and
sustained delivery of silver ions, which directly attaches onto cell surfaces, damaging both cell envelope and bacterial genetic material
(Munhoz, Bernardo, Malafatti, Moreira, & Mattoso, 2019). According to
the CLSI interpretive criteria, S. aureus, K. pneumoniae and E. coli were
susceptible (S) to SSD, while E. shown/hirae can be classified into the
intermediate category. The S category implies that the microorganisms
were inhibited by the usually achievable concentrations of the drug,
resulting in likely clinical efficacy.
After the incorporation of this drug into the polymeric complex, the
values for the zone of inhibition diameter were very close to the isolated drug, proving the presence and the bacterial activity of SSD when
associated with the SPEC. Additionally, the presence of silver nanostructures (as found by DRS and TEM analyses) contributes to improving

the antibacterial activity because of the slow Ag+ leaching from metallic silver, as reported by Manna and coworkers (Manna, Goswami,
Shilpa, Sahu, & Rana, 2015). Also, these results were compared to
gentamicin, an aminoglycoside antibiotic widely used in various types
of bacterial infections. As observed in Table 2, GTM exhibited similar
results like the ones obtained when SSD/SPEC was used.
After the disc diffusion susceptibility test, the minimum inhibitory

CRediT authorship contribution statement
Thamasia F.S. Evangelista: Methodology, Project administration,
Investigation. George R.S. Andrade: Conceptualization, Methodology,
Writing - original draft, Writing - review & editing, Visualization,
Project administration, Investigation. Keyte N.S. Nascimento:
Investigation. Samuel B. dos Santos: Investigation. Caroline Da Ros
Montes D'Oca: Investigation. Charles dos S. Estevam: Investigation.
Iara F. Gimenez: Investigation. Luís E. Almeida: Conceptualization,
Methodology, Writing - original draft, Writing - review & editing,
Supervision, Funding acquisition, Investigation.
Declaration of Competing Interest
The authors declare no competing financial interest.
Acknowledgments
This work was financially supported by CNPq, Capes, Fapitec/SE
and FAPES/ES. G.R.S.A. received a postdoctoral scholarship from Capes
(PNPD/UFS).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
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