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org/Biomac Article

DEAE/Catechol−Chitosan Conjugates as Bioactive Polymers:

Synthesis, Characterization, and Potential Applications
Francisco J. Caro-León,* María Luisa López-Donaire, Roberto Vázquez, Miguel Huerta-Madroñal,
Jaime Lizardi-Mendoza, Waldo Manuel Argüelles-Monal, Daniel Fernández-Quiroz,
Luis García-Fernández, Julio San Roman, Blanca Vázquez-Lasa, Pedro García, and Maria Rosa Aguilar
Cite This: https://doi.org/10.1021/acs.biomac.2c01012 Read Online
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sı Supporting Information

ABSTRACT: This work provides the first description of the

synthesis and characterization of water-soluble chitosan (Cs)
derivatives based on the conjugation of both diethylaminoethyl
(DEAE) and catechol groups onto the Cs backbone (Cs−DC) in
Downloaded via CSIC on January 9, 2023 at 13:41:02 (UTC).

order to obtain a Cs derivative with antioxidant and antimicrobial

properties. The degree of substitution [DS (%)] was 35.46% for
DEAE and 2.53% for catechol, determined by spectroscopy.
Changes in the molecular packing due to the incorporation of
both pendant groups were described by X-ray diffraction and
thermogravimetric analysis. For Cs, the crystallinity index was
59.46% and the maximum decomposition rate appeared at 309.3
°C, while for Cs−DC, the values corresponded to 16.98% and
236.4 °C, respectively. The incorporation of DEAE and catechol
groups also increases the solubility of the polymer at pH > 7 without harming the antimicrobial activity displayed by the unmodified
polymer. The catecholic derivatives increase the radical scavenging activity in terms of the half-maximum effective concentration
(EC50). An EC50 of 1.20 μg/mL was found for neat hydrocaffeic acid (HCA) solution, while for chitosan−catechol (Cs−Ca) and
Cs−DC solutions, concentrations equivalent to free HCA of 0.33 and 0.41 μg/mL were required, respectively. Cell culture results
show that all Cs derivatives have low cytotoxicity, and Cs−DC showed the ability to reduce the activity of reactive oxygen species by
40% at concentrations as low as 4 μg/mL. Polymeric nanoparticles of Cs derivatives with a hydrodynamic diameter (Dh) of around
200 nm, unimodal size distributions, and a negative ζ-potential were obtained by ionotropic gelation and coated with hyaluronic acid
in aqueous suspension, providing the multifunctional nanoparticles with higher stability and a narrower size distribution.

■ INTRODUCTION
Some natural materials like polysaccharides have demonstrated
to be pertinent for their use in several areas due to their
biocompatibility, stability, and chemical and structural
characteristics.1 Chitosan (Cs) is a linear amino-polysacchar-
ide, whose chains are formed by 2-acetamide-2-deoxy-D-
glucopyranose and 2-amino-2-deoxy-D-glucopyranose (N-
acetyl glucosamine and glucosamine, respectively) units, linked
by β (1 → 4) bonds.2 As a polysaccharide, Cs possesses a
unique polycationic character due to the amino groups
distributed on its chain. The interest in this biopolymer and
its derivatives has increased over the recent years as a result of
a number of exceptional bioactivities and functional properties,
such as antioxidant, anticancer, anti-inflammatory, and
antimicrobial activities that have fostered its application in
the pharmaceutical industry.3−7 At pH = 6.2, half of the amino
groups of Cs are already protonated, and at pH < 6.2, more
than half of the amino groups also do so. It is known that the
Cs solubility is influenced by both its chemical composition
and molecular weight. Most of the Cs samples are insoluble at
physiological pH, which greatly affect its biological activity.
Some exceptions are Cs samples with a degree of acetylation
close to 50% that could be dissolved in neutral water or Cs
oligomers, with a degree of polymerization <10 that are also
soluble at pH about 7, even if they exhibit residual acetylation.
Another strategy to overcome this limitation has been to
conjugate appropriate moieties and functional groups onto the
Cs backbone.8,9 The reactivity of Cs depends on three reactive
groups: a free amino one and both primary and secondary
hydroxyl groups at C6 and C3. Among the most common
chemical modifications are acylation, quaternization, carbox-
ymethylation, and N-succinylation, and the reactions more
Received: August 16, 2022
Revised: December 16, 2022

© XXXX American Chemical Society https://doi.org/10.1021/acs.biomac.2c01012

A Biomacromolecules XXXX, XXX, XXX−XXX
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Scheme 1. Synthetic Procedure of Cs−DC
frequently used are Schiff base formation and graft
copolymerization.10−15
As secondary metabolites, polyphenols play important roles
in many different functions within plants.16,17 Polyphenols can
be divided into four types based on the amount of phenol rings
they have and the elements that bind the rings together, which
are phenolic acids, lignans, flavonoids, and stilbenes.18 Their
outstanding biological properties have substantially increased
interest in the study of these compounds, including their
antibacterial,19 antioxidant,20 antiviral,21 anti-inflammatory,22
and antitumor activities.23 Polyphenols are also known for
their good water solubility in addition to their widely studied
bioactivities. Consequently, polyphenols are considered
candidates to produce water-soluble Cs derivatives. Various
polyphenols such as gallic acid,24 ferulic acid,25 catechin,26
epigallocatechin gallate,27 eugenol,28 quercetin,29 caffeic,30 and
hydrocaffeic acid31 have already been introduced into the Cs
structure by using several methods that include free radical-
induced grafting reaction, activated ester-mediated modifica-
tion, and an enzyme-mediated method.32 Recently, Huerta-
Madroñal et al. in 2021 synthesized a water-soluble conjugate
based on Cs and rosmarinic acid, which has shown a threefold
increase in radical scavenger activity (RSA) against the 2,2-
diphenyl-1-picrylhydrazyl (DPPH) free radical and improved
antimicrobial properties over free polyphenol and unmodified
Cs. In addition, the conjugates showed a considerable increase
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in the antioxidant, anti-inflammatory, and photoprotective
activities in fibroblasts, keratinocytes, and macrophages.33
Since Cs and its derivatives have their primary amino groups
protonated at a low pH (below their pKa value), making Cs a
water-soluble cationic polyelectrolyte, a spontaneous associa-
tion occurs between it and negatively charged polyions to
generate polyelectrolyte complexes, which exhibit remarkable
physicochemical properties while preserving the biocompatible
characteristics.34−37 To favor the formation of these com-
plexes, Cs derivatives incorporating functional groups such as
diethylaminoethyl (DEAE) have been produced in order to
improve the aqueous solubility and to generate secondary and
tertiary amino groups in the polysaccharide structure, resulting
in materials with greater colloidal stability.38 These character-
istics have been reported in various research studies, in which
polyelectrolyte complexes based on chitosan−DEAE (Cs−
DEAE) have been obtained with RNA, DNA, or hyaluronic
acid (HA).39−42 In previous work, our group conjugated
DEAE moieties to Cs polymeric chains. Based on that
incorporation, the production of polymeric nanoparticles
(NPs) in aqueous suspension with specific affinity for the
choline-binding lysin Cpl-711 with activity against Streptococ-
cus pneumoniae (S. pneumoniae) was made possible. In addition
to their application as an antimicrobial agent, the NPs of Cs−
DEAE were more stable than the materials produced with Cs
without derivatization since the specific interaction between
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Cpl-711 and DEAE greatly reduced the aggregation in
suspension.43
This work details the synthesis and characterization of
polymeric conjugates based on Cs incorporating both DEAE
and catechol functional units. To our knowledge, no such
material has been reported to date. We hypothesized that the
conjugation of both functional groups would modify the
chemical and structural characteristics of the polysaccharide,
which would improve its properties, such as antioxidant
activity and water solubility, and expand the range of
applications while maintaining its biocompatibility. Besides,
developed conjugated polymers can generate nanoparticulate
materials in aqueous suspension with potential applications in
the biomedical field.
■ EXPERIMENTAL SECTION
Materials. Cs 90/200 was provided by Chitoscience (Halle,
Germany) with a viscosity of 151−350 mPas (1% in 1% acetic acid,
20 °C) and a degree of deacetylation of 87.6−92.5% provided by the
supplier and verified by the 1H nuclear magnetic resonance (1H-
NMR) study. 3-(3,4-Dihydroxyphenyl)propionic acid, 98+% (hydro-
caffeic acid, HCA), was provided by Alfa Aesar. N-(3-dimethylami-
nopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), 2-chloro-
N,N-diethylethylamine hydrochloride (DEAE-Cl), sodium tripoly-
phosphate (TPP), DPPH, CH3COOH, HCl, NaOH, CH2Cl2, DCl,
D2O (99.9 atom % D), and Dulbecco’s phosphate-buffered saline
(PBS) were purchased from Merck KGaA (Darmstadt, Germany) and
utilized without additional purification. HA with a molecular weight of
200−300 kDa was supplied by Bioiberica (Barcelona, Spain).
Synthesis of Cs Conjugates. Cs−DEAE was produced by
reacting Cs with DEAE-Cl at a basic pH, as reported previously.43
The comparison of the 1H-NMR spectra of Cs and Cs−DEAE
confirmed the conjugation of the polysaccharide as indicated in the
assignment of signals in Figure S1. The degree of substitution (mole
fraction of the substituent per Cs monomeric unit) [DS (%)] of
DEAE was obtained from the 1H-NMR spectrum, taking into
consideration the resonance signal of the methyl protons of DEAE
moieties at 1.68 ppm in relation to the signal of the anomeric protons
of the unsubstituted and substituted glucosamine residues of the
polysaccharide at 5.26 and 5.42 ppm, respectively,42 giving a value of
35.46 ± 3.98% based on the following equation
DS (%) = (I1.68/6[I5.26 + I5.42]) × 100 (1)
Chitosan−catechol (Cs−Ca) and chitosan−DEAE−catechol (Cs−
DC) conjugates were synthesized utilizing standard EDC chemistry as
shown in Scheme 1.
The reaction procedure was performed at room temperature,
minimizing light exposure to avoid phenol oxidation. Briefly, 62 mg
(0.385 mmol monosaccharide residue) of Cs or Cs−DEAE were
dissolved in 5.4 mL of HCl 1 N using magnetic stirring. After the
complete dissolution of the polysaccharide, the pH was adjusted to
5.5 with NaOH 0.2 N, and 7.5 mg (0.041 mmol) of HCA dissolved in
0.32 mL of deionized water (DW) were added. Then, 18.3 mg (0.118
mmol) of EDC dissolved in 5.34 mL of DW/ethanol mix (1:1, v/v)
was added dropwise in darkness. At room temperature, the reaction
mixture was vigorously stirred for 3 h, maintaining the pH of the
medium at 5.5. The reaction product was dialyzed against DW for 2
days in dark conditions and recovered by freeze-drying. The products
had a white, cotton-like appearance and were stored at 4 °C. Solutions
of 5 mg of each catecholic conjugate (Cs−Ca or Cs−DC) in 1 mL of
D2O/DCl mix (50:1 v/v) were characterized by 1H-NMR at 70 °C in
Varian Mercury equipment working at 400 MHz (Palo Alto, USA).
For comparison purposes, the 1H-NMR spectra of Cs and Cs−DEAE
were also obtained under the same conditions. The DS (%) of
catechol was quantified by UV spectrophotometry in a 2 mg/mL
solution of each catecholic polysaccharide in CH3COOH (0.1% v/v)
by using a calibration curve of HCA solutions with a known
concentration, measured at λ = 280 nm in a NanoDrop One (Thermo
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Fisher Scientific, Waltham, USA). The calibration curve showed
linearity between 0.004 and 0.061 mg/mL (y = 16.818x + 0.0072, r2 =
0.9992) (Figure S2). Measurements were carried out in triplicate.
Physicochemical and Structural Characterization of the Cs
Derivatives. The physicochemical and structural properties of freeze-
dried Cs, Cs−DEAE, Cs−Ca, and Cs−DC were studied by different
methods. All samples were characterized by attenuated total
reflection−Fourier-transform infrared (ATR−FTIR), and the spectra
were obtained in the mid-infrared absorbance region (4000−400
cm−1) utilizing a PerkinElmer spectrometer (Spectrum One,
Waltham, USA) equipped with an ATR accessory with a resolution
of 4 cm−1, using 32 scans. X-ray diffraction (XRD) tests were done on
a Bruker D8 ADVANCE diffractometer (Billerica, USA) with Cu Kα
radiation. The operating voltage and current were 45 kV and 100 mH,
respectively, and the angle range (2θ) was scanned from 2 to 40° at a
step size of 0.02°. The following equation was used to determine the
crystallinity index [CrI (%)] from the appropriate XRD diffractogram
CrI (%) = ((I110 Iam)/I110) × 100 (2)
where Iam is the intensity of the amorphous diffraction at 2θ = 16° and
I110 is the highest intensity of the plane 110 at 2θ = 20°.
Thermogravimetric analysis (TGA) of the samples was performed
on a Q500 thermogravimetric analyzer (TA Instruments, New Castle,
USA) to study how the stability of the materials changes with
temperature, heating from 20 °C up to 800 °C at 10 °C/min under a
nitrogen flow (90 mL/min).
Solubility Study of Cs Derivatives as a Function of pH. 1.0 M
buffer solutions were prepared dissolving the following reactants in
100 mL of DW: for pH 2.0, 13.61 g of calcium phosphate dibasic; for
pH 3.0, 7.61 g of glycolic acid; for pH 4.0 and pH 5.0, 15.80 g of
calcium acetate hydrate and 7.71 g of ammonium acetate; for pH 6.2
and pH 7.5, 14.20 g of sodium phosphate dibasic; for pH 8.3, 22.82 g
of potassium phosphate dibasic trihydrate; and for pH 9.4 and 11.3,
13.82 g of potassium carbonate. The pH of the solutions was adjusted
to 2.0, 3.0, 4.0, 5.0, 6.2, 7.5, 8.3, 9.4, and 11.3, using HCl 1 N and
NaOH 1 N and then diluted to 0.2 M with DW. Stock solutions of Cs,
Cs−DEAE, Cs−Ca, and Cs−DC (10 mg/mL) in CH3COOH (1.0%
v/v) were 10-fold diluted in the respective buffer solution for UV
measurement. Turbidity of the Cs, Cs−DEAE, Cs−Ca and Cs−DC
solutions was determined by the scattered value measured at 600 nm,
immediately after vigorously shaking the samples using a vortex.
Radical Scavenger Activity. The RSA of Cs, Cs−DEAE, Cs−Ca,
and Cs−DC conjugates was determined by measuring the
decolorization of the DPPH free radical, according to the method
reported by Ren et al. in 2013 and slightly modified.44 For
comparison, the RSA of HCA was also measured. Stock solutions
of HCA (3.6 μg/mL) and Cs derivatives (1000 μg/mL) were
obtained in CH3COOH (0.1%, pH = 4) and successively diluted.
Serial dilutions with various concentrations of free HCA and
polysaccharide were evaluated. Briefly, 100 μL of DPPH ethanolic
solution (0.125 mM) was added to 100 μL of the sample (Cs, Cs
derivative, or free HCA). The mixture reacted under magnetic stirring
for 15 min at room temperature, avoiding light exposure. Afterwards,
the absorbance was measured at 515 nm against a blank (100 μL of
CH3COOH 0.1% and 100 μL of DPPH solution) with a Multi-
Detection Microplate Reader Synergy HT (BioTek Instruments;
Vermont, USA). The RSA was obtained using the following equation
RSA = ((A 0 (A1 A 2))/A 0) × 100 (3)
where A0 corresponds to the absorbance of the blank, A1 corresponds
to the absorbance of the sample, and A2 corresponds to the
absorbance of the sample under the same conditions as A1 but using
ethanol instead of DPPH solution. Results appear as mean ± standard
deviation (SD) (n = 16).
Bacterial Strains, Media, Growth Conditions, and Anti-
microbial Assay of Conjugates. The bacterial strains used in this
research are detailed in Table S1. All Gram-negative bacteria [
Pseudomonas aeruginosa, Acinetobacter pitti, Acinetobacter baumannii
(A. baumannii), Escherichia coli, and Klebsiella pneumoniae (K.
pneumoniae)] were grown in lysogeny broth (LB, NZYTech) at 37
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Table 1. Sample Codes of All Conjugates, Cs/DEAE and Cs/EDC/HCA Feed Molar Ratios × 103, DS (%) Values of DEAE
and Catechol (Obtained by 1H-NMR and UV Spectrophotometry (λ = 280 nm), Respectively) and the Degree of Acetylation
(Obtained by 1H-NMR)
sample Cs/DEAE feed molar ratio × 103 DEAE DS (%) Cs/EDC/HCA feed molar ratio × 103
Cs
Cs−DEAE 17.99/39.57 35.46 ± 3.98
Cs−Ca
Cs−DC 17.99/39.57 35.46 ± 3.98
°C with rotary shaking at 200 rpm. Staphylococcus aureus (S. aureus)
and Streptococcus pyogenes (S. pyogenes) strains were cultured in Brain
Heart Infusion (BHI, Condalab) at 37 °C without shaking. S.
pneumoniae strains were grown in C medium adjusted at pH 8.045 and
supplemented with 0.08% yeast extract without shaking. All Gram-
positive bacteria were cultured in blood agar plates, while Gram-
negatives were grown in LB agar.
Antimicrobial activity assays were conducted by incubating resting
bacteria cell suspensions in 20 mM sodium phosphate buffer, pH 6.0,
150 mM NaCl at 37 °C for 2 h, and 50 μg/mL of each polymer
solution equivalent volume of water for the controls. Resting cell
suspensions were produced by harvesting bacteria at mid-to-late
exponential phase growth as determined by turbidimetry. Cell
cultures were centrifuged at 3000g for 10 min at 4 °C, and the
pelleted cells were resuspended in 0.5× volume of the buffer as
mentioned above and plated onto a 96-well plate (100 μL per well).
Then, another 100 μL of the same buffer with 2× the desired final
concentration of the polymer to be tested (or the equivalent volume
of water) was added, and the plate was incubated at the indicated
conditions. Finally, viable cell counts were obtained by plating
triplicate 10 μL samples of 10-fold serial dilutions at the end of the
experiment.
Biocompatibility and ROS Assay of Conjugates. Cell Culture.
Due to the poor solubility of Cs and Cs−Ca in DMEM cell-culture
medium, experiments on murine macrophage (RAW 264.7) culture
were carried out only with solutions of Cs−DEAE and Cs−DC. RAW
264.7 cell line was purchased from Merck (Merck KGaA, Darmstadt,
Germany). Cells were kept over permissive conditions in high-glucose
DMEM supplemented with 2% L -glutamine (Merck KGaA,
Darmstadt, Germany), Penicillin-G (Merck KGaA, Darmstadt,
Germany), and 10% fetal bovine serum (Gibco, Waltham, USA) at
37 °C in a humidified incubator with 5% of CO2. In vitro cell culture
experiments were performed with a polymeric solution of Cs−DEAE
and Cs−DC samples diluted at different concentrations in DMEM (4,
6, 8,10, 20, and 40 μg/mL) at 37 °C after sterilization by UV
exposure.
Cytotoxicity Assay. The cytotoxicity of the Cs−DEAE and Cs−DC
solutions was determined by the Alamar Blue (AB) test. RAW 264.7
cells were seeded in 96 well-plates under permissive conditions at
200,000 live cells/mL (100 μL per well). After 24 h, cells were treated
with either fresh DMEM (as positive control) or the corresponding
Cs derivative (Cs−DEAE or Cs−DC) sample at various concen-
trations. After 24 h of exposure, cellular viability was evaluated by
using the AB Reagent (Invitrogen). Absorbance at 570 nm was
measured with the Multi-Detection Microplate Reader. The
percentage of cell viability [CV (%)] was expressed in reference to
the positive control. All results are presented as mean ± SD (n = 16).
ROS Assay. The capacity of Cs−DEAE and Cs−DC to reduce
reactive oxygen species (ROS) activity was evaluated as follows: RAW
264.7 cells were seeded in 96 well-plates at 150,000 live cells/mL and
kept in a medium for 24 h. Afterward, cells were washed with PBS and
incubated for 24 h with 100 μL of Cs−DEAE or Cs−DC solution at
different concentrations and 100 μL of DMEM with (samples) or
without (negative control) the addition of lipopolysaccharide (LPS)
(5 μg/mL). Then, the medium was replaced, and the cells were
washed again with PBS. In addition, previously prepared dichlor-
odihydrofluorescein diacetate solution in dimethyl sulfoxide was
diluted to 40 μM with PBS, and 100 μL was poured into each well.
Cells were incubated at 37 °C for 45 min, carefully avoiding light
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HCA DS (%) degree of acetylation (%)
4.7
4.3
3.85/0.41/1.18 2.61 ± 0.15 4.0
3.85/0.41/1.18 2.53 ± 0.11 5.7
exposure. Cells were rewashed, and 100 μL of PBS was added.
Fluorescence was measured in the microplate reader at an excitation
of 485−490 nm and emission of 520−550 nm. ROS production (%)
was expressed with respect to the positive control, equivalent to 100%
of ROS activity (cells treated with LPS without adding Cs−DEAE or
Cs−DC). All results are provided as mean ± SD (n = 16). For
intracellular ROS imaging, RAW 264.7 cells were immediately
observed with a standard fluorescence microscope after the treatment
with the Cs derivatives.
Preparation and Characterization of NPs of Cs Derivatives.
Polymeric NPs of Cs, Cs−DEAE, Cs−Ca, and Cs−DC (Cs NPs, Cs−
DEAE NPs, Cs−Ca NPs, and Cs−DC NPs, respectively) were
produced by the ionotropic gelation approach with slight
modifications.46 Briefly, Cs derivatives were dissolved (1.0 mg/mL)
in CH3COOH 0.1% v/v at pH 4.0 separately. To 3 mL of the polymer
solution, 31.8 μL of Tween 80 were added, and the mixture was
heated at 45 °C for 2 h. Afterward, 0.3 mL of CH2Cl2 was added to
the Cs derivative solution under magnetic stirring at 1200 rpm for 15
min. Polymeric NPs were formed upon mixing different amounts of
TPP aqueous solution (1 mg/mL) into the polymeric phase at room
temperature under magnetic stirring (950 rpm) for 10 min. In order
to decrease the polydispersity and increase the stability of the NPs,
different amounts of a HA aqueous solution (0.5 mg/mL) were added
dropwise to the NP suspension under magnetic stirring (600 rpm),
resulting in HA-coated NPs.
The hydrodynamic diameter (Dh) and polydispersity index (PDI)
of all NPs were determined by dynamic light scattering (DLS) at 25
°C using a Malvern Zetasizer Nano ZS (Malvern Instruments Ltd.,
Worcestershire, United Kingdom) instrument with a scattering angle
of 173° and equipped with a 4 mW He−Ne laser (λ = 633 nm). Zeta
(ζ)-potential was measured by laser Doppler electrophoresis using the
Zetasizer Nano ZS. The morphology of the HA-coated NPs was
examined by field emission-scanning electron microscopy (FE-SEM)
with a Hitachi SU8000 TED (Tokyo, Japan) cold-mission FE-SEM
microscope, functioning with an accelerating voltage between 15 and
25 kV. To accomplish this, dispersion samples were dropped
independently onto a copper grid coated with a formvar membrane,
allowed to stand, dried, and coated with Au/Pd (60:40).
■ RESULTS AND DISCUSSION
Synthesis and Structural Characterization of Cs
Conjugate Derivatives. Cs conjugates bearing HCA and
DEAE moieties, separately or together, were successfully
prepared as described in the Experimental Section. Codes and
composition of Cs derivatives are shown in Table 1.
The DEAE functional group was incorporated by
nucleophilic substitution into the C-2 amino group of the
glucopyranose rings of Cs, raising the pH of the reaction
medium to 8.0.47 The conjugation reaction was carried out via
deacetylated units as was proved by determining the degree of
acetylation values of the conjugates and native samples. These
values were all of them circa 5%, the value for the native Cs
and are similar to those provided by the supplier.
Cs−Ca and Cs−DC conjugates were synthesized using EDC
coupling chemistry by forming amide bonds between a primary
amine group in Cs or Cs−DEAE and a carboxylic acid group in
HCA.48 Since the EDC chemistry is more propitious for the
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Figure 1. 1H-NMR spectra of Cs−Ca and Cs−DC dissolved in a D2O/DCl mix (50:1 v/v) at 70 °C.
non-protonated amino groups, the conjugation took place at
pH 5.5 to prevent the precipitation of the Cs (pKa = 6.2−6.4)
and limit amino protonation.49 It is important to note that the
incorporation of specific chemical groups can increase the
polymer’s solubility at a higher pH range compared to the
unmodified Cs. Remarkably, the benefit of DEAE groups
depends on its tertiary amine, whose pKa (∼10.0) allows
complete solubilization of the polysaccharide in neutral
aqueous media.50 However, HCA conjugation was carried
out at the same pH (pH = 5.5) for Cs and Cs−DEAE
polymers to standardize the reaction conditions. The synthesis
of catecholic derivatives was verified by using either 1H-NMR
or a UV−vis spectrophotometer. 1H-NMR spectra of Cs−Ca
and Cs−DC (Figure 1) exhibited the characteristic pattern of
Cs, that is, a multiplet between 3.52 and 4.23 ppm, associated
with the protons of the polysaccharide ring (a−e) and a singlet
at 5.26 ppm, corresponding to the resonance signal of the
anomeric proton of the DEAE-unsubstituted glucosamine
residues (f).51,52 In addition, the presence of the DEAE in the
Cs−DC structure was demonstrated by the appearance of the
characteristic bands of the methylene protons at 3.62 and 3.90
ppm (g and h, respectively), and the methyl protons of the
DEAE moieties (i), as well as the band attributed to the
anomeric proton of the DEAE-substituted glucosamine
residues (j). Moreover, a multiplet between 7.01 and 7.19
ppm, attributed to the aromatic protons of the catechol group,
is also observed in both Cs−Ca and Cs−DC spectra (k−m),
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confirming the incorporation of the catechol moieties.53,54 The
DS (%) of catechol was calculated by UV spectrophotometry,
giving values of 2.61 ± 0.15 and 2.53% ± 0.11 for Cs−Ca and
Cs−DC, respectively.
Physicochemical and Structural Characterization of
the Cs Derivatives. The ATR−FTIR spectrum of Cs (Figure
S3) presented numerous overlapped regions from 1200 to 890
cm−1, and the distinctive bands of the polysaccharide in the
range of 3356−1322 cm−1 were also observed; which is
consistent with previous reports.55 In addition to the
characteristic bands of Cs, the Cs−DEAE spectrum exhibited
an adsorption band attributed to the C−H bending of the
methyl groups of the DEAE at 1453 cm−1, demonstrating that
substitution occurs.43 The conjugation of Cs with HCA was
confirmed in the spectrum of Cs−Ca by the appearance of a
band related to the aromatic ring at 1519 cm−1, which
corresponds to the C−C aromatic stretching.56 Furthermore,
the Cs−DC spectrum showed the characteristic bands of both
DEAE and catechol groups, demonstrating that both
conjugations took place.
The XRD analysis of Cs and the Cs derivatives is shown in
Figure 2. Several crystalline polymorphs have been determined
for Cs samples, including annealed, tendon, eightfold right-
handed forms, form I, form II, 1−2, and L-2.57 The tendon is a
most frequently observed as a crystalline polymorph, and it has
a proposed orthorhombic unit having the following dimen-
sions: a = 8.95, b = 16.97, and c (fiber axis) = 10.34 Å. In this
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Figure 2. XRD diffractograms of Cs, Cs−DEAE, Cs−Ca, and Cs−DC.
Table 2. Main Results of TGA and DTG of Cs and Different Cs Derivatives
TGA
1st step (°C) 2nd step (°C)
Cs 90−120 290−340
Cs−DEAE 90−120 250−320
Cs−Ca 90−120 200−300
Cs−DC 90−120 200−300
form, the Cs molecule has a helical symmetry 21 stabilized by
hydrogen bonds (O3··O5), and eight molecules of water are
incorporated in the unit cell.58,59 The Cs diffractogram showed
diffraction peaks at 2θ = 9.87 and 19.96°, attributed to the
equatorial (020) and (110) of the microcrystalline reflections,
respectively.60 This diffraction pattern revealed hydrated
polymorphism with a 020 reflection at 2θ = 10°, a
characteristic attribute of the tendon form.61 The prominent
diffractive peaks of the Cs−DEAE diffractogram were centered
at 10.37 and 20.17°, clearly less intense than those of Cs,
indicating that intra and inter-chain interactions are remarkably
affected. The intensity of the peaks decreased even more with
the addition of catechol groups to the polymeric chains,
indicating an essential reduction in the semi-crystalline form or
the formation of an amorphous state.62 The diffraction peaks
of the equatorial (020) and (110) of the Cs−Ca diffractogram
were centered at 13.66 and 21.44°, respectively. For the Cs−
DC, the maximum corresponding to (020) practically
disappears, with only a broad peak remaining at 21.37°. The
volume of the aromatic groups incorporated into the polymer
chains results in more significant disruptions of the crystalline
regions. Such effect can be appreciated considering that the
CrI (%) values were 59.46, 51.86, 19.20, and 16.98% for Cs,
Cs−DEAE, Cs−Ca, and Cs−DC, respectively. More consid-
erable reductions in the CrI (%) indicate that the
incorporation of voluminous groups prevents the polysacchar-
pubs.acs.org/Biomac Article
DTG
3rd step (°C) residual mass (%) Tmaximum decomposition rate (°C)
350- 31.3 309.3
330- 29.8 301.1
310- 31.6 248.2
310- 26.6 236.4
ide chains from being densely packed, which produces the
breaking of hydrogen bonds between them.63
TGA results of all conjugate polymers along with those of Cs
are shown in Figure S4A,B and Table 2.
Cs showed three main steps; the first one goes up to 120 °C
and is attributed to moisture evaporation, producing 6% of the
mass loss. The second step is related to thermal depolymeriza-
tion and the dehydration of the polysaccharide rings, with a
maximum decomposition rate close to 300 °C and 43% mass
loss associated. The last step starts around 350 °C and
corresponds to secondary thermolysis, leaving around 31% of
the residual mass.64,65 The thermograms of the conjugates
showed three characteristic thermal events of the Cs; however,
the conjugation of DEAE and catechol groups reduces the
thermal stability of the corresponding conjugate polymers
compared to the unmodified one. Specifically, for Cs−DEAE,
the broad mass loss starts approximately at 250 °C, which is a
lower temperature compared to Cs. This is a result of
alterations in the crystalline structure, particularly by the loss of
hydrogen bonds due to the incorporation of DEAE groups
onto the polymeric backbone.66 However, the reduction in
thermal stability is more evident with the incorporation of the
larger catechol side groups. According to previous reports, the
packing pattern of the polysaccharide may be obstructed by the
grafting of phenolic compounds onto Cs chains, resulting in a
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Figure 3. (A) Turbidity of Cs, Cs−DEAE, Cs−Ca, and Cs−DC solutions at different pH conditions and (B) photographs of solutions of Cs and
Cs derivatives at pH = 11.3.
Figure 4. RSA of Cs, Cs−DEAE, Cs−Ca, Cs−DC (A), and free HCA (B) vs concentration.
decrease in thermal stability.67 At 800 °C, the residual char
yields of Cs and the three Cs derivatives were similar.
Differential thermogravimetric (DTG) curves provided
information on the maximum decomposition rate of the
samples. The incorporation of DEAE and catechol residues in
Cs−DC has the greatest effect on thermal stability, reducing it
more markedly compared to the other Cs derivatives.
Solubility Study of Cs Derivatives as a Function of
pH. The turbidimetric measurements at 600 nm for Cs, Cs−
DEAE, Cs−Ca, and Cs−DC solutions and their macroscopic
appearance at pH = 11.3 are shown in Figure 3A,B,
respectively. Many publications describe the ability of Cs to
form true solutions in acidic conditions. However, its low
solubility to form aqueous solutions at neutral pH has
significantly limited its application as a functional biomate-
rial.68 As can be observed in Figure 3A, at pH above 6.0, the
turbidity of the Cs solution increases to maximum values
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around 0.38. Figure 3B shows that the precipitate of Cs had
the appearance of white powder. For Cs−Ca, the absorbance
at 600 nm increases to 0.27 at pH = 7.5, reaching a value of
0.30 at pH = 11.3. The physical characteristics of the Cs−Ca
precipitate were distinct from those observed for Cs. The Cs−
Ca precipitate was significantly reddish, large, swollen, and
sticky. This gel-like precipitate is perhaps the result of
oxidation of the catechol groups to quinones at a high pH,
followed by their chemical conjugation with amine groups.69
The absorbance measurements at 600 nm for Cs−DEAE
showed values close to 0.0 in pH conditions under 8.3, without
the appearance of precipitates until the solution was adjusted
to pH = 11.3 (absorbance 600 nm = 0.20). The highest water
solubility of Cs−DEAE is attributable to the contribution of
the pKa ∼ 10.0 of the tertiary amino group of DEAE.49 On the
other hand, it is observed that the Cs−DC solutions only
slightly increase their absorbance at 600 nm to ∼0.06, even at
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Biomacromolecules
Figure 5. Antimicrobial activity of solutions of Cs and its derivatives against pathogenic bacteria (A,B). Viable cell counts of different bacterial
suspensions either untreated or treated with 50 μg/mL of the dissolved polymers for 2 h at 37 °C. A two-way ANOVA followed by all-against-all
multiple comparisons using Tukey’s test was conducted to detect a significant difference between the different conditions within each strain. Results
denoted by different letters are significantly different from each other (with a significance level of α = 0.05).
pH = 11.3. It was impossible to observe any visible precipitate
in the tube. The solubility of Cs−DC could be related to the
direct and indirect contributions of the conjugated DEAE and
catechol groups. First, the incorporated groups have pKa values
higher than those of Cs, which positively increases the effective
pKa of the conjugate polymer, contributing to its enhanced
solubility in an aqueous medium. Second, the chemical
modifications break the intra- and inter-hydrogen bonds of
Cs, which reduces the rigidity of the molecular arrangement,
improving the solubility, as has been previously described.70
Radical Scavenger Activity. The basis for the DPPH
assay is the ability of the DPPH free radical to receive an
electron or hydrogen radical from an antioxidant molecule in
order to become stable. DPPH solution has an absorption
maximum of 515 nm, presenting a deep purple color.
Antioxidants cause its color to turn yellow and its absorbance
to decrease.71 In this work, the antioxidant capacity in terms of
RSA of Cs and its Cs derivatives was first determined against
concentration (Figure 4A) and contrasted with that of free
HCA (Figure 4B), whose RSA has been previously
reported.72,73 As observed, Cs exerted no antioxidant capacity
at the tested concentrations. In addition, Cs−DEAE showed
minimum RSA at the same concentrations, as has been
previously reported.74 On the other hand, Cs−Ca and Cs−DC
showed a high concentration-dependent RSA, indicating that
the higher the catechol content, the higher the antioxidant
potential. The RSA of the catecholic derivatives is associated
with the nature of HCA as a polyphenol, having the second
hydroxyl group capable of quenching the free radicals in the
ortho or para position.75 Hence, our results agree with others’
reported for HCA-modified polymers, with an RSA of 89% for
concentrations equal to or greater than 25 μg/mL.76 In
addition, it is possible to conclude that HCA conjugation onto
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Cs or Cs−DEAE backbones notably increases the antioxidant
activity of the catecholic polysaccharides in terms of their half-
maximum effective concentration (EC50). For the solution of
free HCA, an EC50 of 1.20 μg/mL was quantified, while for
Cs−Ca and Cs−DC solutions, concentrations equivalent to
free HCA of 0.33 and 0.41 μg/mL were required, respectively.
Therefore, it is possible to confirm that the catecholic
conjugates present an antioxidant activity significantly higher
than that of free HCA.
Antimicrobial Activity of Cs Conjugates. Regarding
antimicrobial activity (Figure 5), in general, Cs and all of its
derivatives displayed a bactericidal effect on all the tested
pathogenic bacterial strains. The range of activity was between
1 and 6 log units of killing (using a 50 μg/mL treatment),
although there was a clear strain dependency. It can be said
that the conjugation of DEAE and catechol groups onto the Cs
backbone does not diminish the antimicrobial properties of the
polysaccharide, although none of the conjugates can be
considered a better antimicrobial agent. Nevertheless, in
some specific cases, conjugation had a positive effect on the
magnitude of the killing activity, for example, Cs−DEAE had a
more significant action on the K. pneumoniae or A. baumannii
(AB-RYC-2) isolates tested than any other polymer (Figure
5A), or Cs−Ca seemed better suited against the S. aureus and
S. pyogenes strains (Figure 5B). However, any other general
trends cannot be stressed enough due to the limited amount of
bacterial strains tested and the observed variability in the
killing outcomes. Such variability indicates that the anti-
bacterial mechanism of Cs and its derivatives depends on
specific features of the corresponding bacteria, which are most
probably related to the composition and architecture of the
bacterial surface. Cs exhibits pH-dependent antimicrobial
activity owing to the presence of copious amino groups on
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Biomacromolecules
Figure 6. Cell viability on RAW 264.7 exposed to Cs−DEAE or Cs−DC (A), ROS assay (B), and (C−F) intracellular ROS production of RAW
264.7. The figures include the mean, SD (n = 24), and the ANOVA results (*p < 0.05 statistically significant difference between untreated and
either control or treated cells and #p < 0.05 between control and the two Cs derivative-treated cells).
its macromolecular chain. The positively charged groups
interact more favorably with the cell membrane surface,
which is negatively charged due to the presence of teichoic
acids or LPS in Gram-positive and Gram-negative bacteria,
respectively.77−79 The specific bactericidal activity of Cs and its
derivatives has been the subject of debate since some authors
have stated that the Gram-negative bacteria are more
susceptible than the Gram-positive species against the Cs
molecule, but in other research, an inverse behavior has been
reported.80 As a general conclusion, although, while Cs
conjugation rendered polymers with more suitable properties
(solubility and antioxidant activity), it did not come at the cost
of losing antimicrobial activity, and this activity was even
increased against some of the particular strains tested.
Cellular Viability and Activity Against ROS. The
cytotoxicity of Cs−DEAE and Cs−DC was determined in
RAW 264.7 culture under ISO 10993-5:2009, and the results
are shown in Figure 6A. As observed, Cs−DEAE and Cs−DC
solutions with concentrations in the range of 10−20 μg/mL
showed CV (%) over 70%, confirming their biocompatibility
according to the mentioned standard. However, solutions with
a concentration of 40 μg/mL proved to be toxic for the cell
line. Although the biological properties of catechol groups,
such as mucoadhesive or antioxidant capacity are highly
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attractive, previous studies have revealed that these groups are
likewise recognized to be cytotoxic as they oxidize into
semiquinones and reactive quinones, generating ROS that are
directly associated with DNA damage, that is, oxidized
nucleotides, hyper-recombination, and single and double-
strand breaks.81,82 Furthermore, studies of cytotoxicity of Cs
derivatives functionalized with catechol in RAW 264.7,33 L929,
and NIH3T369 cell lines have shown that the DS (%) could
have a direct effect on the cytotoxicity of the materials because
high conjugation degrees could contribute to reducing the
intrinsic solubility of the polymer.69 However, the results
reported in this work do not show significant differences
between the cytotoxicity of Cs−DEAE and Cs−DC, which
allows us to conclude that the presence of catechol moieties
does not contribute to decreasing the biocompatibility of Cs
derivatives in the RAW 264.7 cell line.
Cs and its catecholic derivatives have shown antioxidant
capacity, increasing cell viability and reducing oxidative stress
in cells exposed to ROS.83−85 In order to study the antioxidant
capacity of Cs−DEAE and Cs−DC in stimulated RAW 264.7
cells, polymeric solutions were evaluated in terms of
attenuation of LPS-induced ROS effects. As shown in Figure
6B, both Cs derivatives were able to reduce the ROS activity.
However, Cs−DC solutions decreased the activity by 40% at
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Biomacromolecules
Figure 7. Hydrodynamic properties and ζ-potential of the uncoated and HA-coated Cs derivative NPs (A). Size distribution of uncoated (B) and
HA-coated Cs derivative NPs (C).
concentrations as low as 4 μg/mL, representing a 25% more
reduction compared to that observed for Cs−DEAE at the
same concentration. The analysis of the fluorescence
microscopy images allowed the observation of intracellular
ROS production in cell culture. Cells incubated with LPS
without Cs derivatives were taken as having 100% ROS
production (positive control, Figure 6C). As a result of basal
cellular metabolism, a specific fluorescence signal is emitted in
non-LPS-exposed cells (negative control, Figure 6D). When
cells were exposed to LPS in the presence of Cs−DEAE
(Figure 6E) and Cs−DC (Figure 6F) at a concentration of 10
μg/mL, the level of intracellular ROS generation was
comparable to the negative control. Based on these results, it
is possible to establish that the incorporation of catechol
groups significantly improves the ability of Cs−DC to reduce
the effect of ROS release. However, Cs−DEAE also presented
antioxidant capacity in the cell medium. In this context, it is
important to point out that, although neither Cs nor Cs−
DEAE showed significant RSA against the DPPH radical in all
the tested concentrations, it has been reported in the literature
that both materials may have some antioxidant capacity. Je &
Kim, 2006, obtained six kinds of amino-derivatized Cs from 90
and 50% deacetylated polysaccharides, including Cs−DEAE.
According to studies using 2,2′-azino-bis(3-ethylbenzothiazo-
line-6-sulfonic acid) and peroxidase, Cs−DEAE from 90%
deacetylated Cs exhibited strong H2O2 scavenging activity.86
On the other hand, Wen et al. in 2013 showed that Cs and Cs
NPs generated protective effects against H2O2-induced
oxidative damage in RAW264.7 cells, with a suggested
mechanism based on the attenuation of lipid peroxidation
and an enhancement of endogenous antioxidant preserva-
tion.84 Although these antecedents would ratify the antioxidant
activity of Cs and Cs−DEAE, future experiments are necessary
to specifically explain the contribution of the DEAE groups or
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the degree of acetylation of the polysaccharide in such activity
in cell culture.
Preparation and Characterization of NPs of Cs−
derivatives. Ionotropic gelation has been used frequently to
obtain Cs NPs based on the interaction between the anionic
phosphate groups of TPP molecules and the cationic amino
groups of the polysaccharide.87 For this reason, NPs of the
conjugates developed here were fabricated by applying this
approach. The physical−chemical characteristics of the
obtained NPs are summarized in Figure 7A. The availability
of the amino groups to produce an opalescent suspension
determines the correct concentration of TPP. Thus, the TPP/
polysaccharide ratio used showed the following order: Cs−DC
NPs > Cs−DEAE NPs > Cs NPs > Cs−Ca NPs. The
measurements of Dh showed similar results for the four NP
systems. However, significant differences could be observed for
the PDI. Cs−Ca NPs presented the highest value, which is
attributed to the production of gel-like precipitates, resulting
from the chemical cross-linking upon the oxidation of the
catechol moieties.70 In addition, the PDI of Cs NPs was higher
than 0.290, while Cs−DEAE NPs and Cs−DC NPs had PDI
lower than 0.130. This behavior can be attributed to the
grafting of DEAE moieties onto the Cs backbone, which
generates secondary and tertiary amino groups in the
polysaccharide structure. This chemical modification favors
the ability to condense negatively charged molecules,
improving the colloidal stability of the NPs.38 All the NPs
showed positive values of the ζ-potential, as has been
previously reported for similar materials.88−90 However, its
value can be affected by the conjugation of DEAE and
catechol. NPs with the presence of DEAE had lower ζ-
potential values than those of Cs NPs and Cs−Ca NPs.
Previous research has shown that the tertiary amines of Cs−
DEAE have ethyl groups that augment the hydrophobic nature
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Biomacromolecules
of the molecule, giving it amphiphilic behavior in aqueous
medium and a propensity to self-aggregation at a concentration
of about 0.1 g/L. Hence, we theorize that the decrease in the ζ-
potential values of Cs−DEAE NPs and Cs−DC NPs with
respect to Cs NPs and Cs−Ca NPs is related to the orientation
of the DEAE moieties toward the internal structures of the
NPs in water suspension, substantially reducing their
contribution to the surface charge.91
An alternative strategy to reduce the PDI is to coat the
nanomaterials with HA. Previous research has reported the
production of polyelectrolytic complexes based on the
interaction between Cs or its derivatives and negatively
charged HA.39,92 The ζ-potential measurements confirmed
the coating since all the materials had negative values. As
expected, the required HA/NPs ratio to produce the
complexes was higher for the Cs−DC NPs, owing to the
unavailability of their amino groups. The HA-coated NPs
showed higher Dh than non-coated NPs due to the presence of
the HA. The PDI of the HA-coated Cs NPs and Cs−Ca NPs
was significantly reduced compared to the uncoated systems.
Figure 7B shows the size distributions of the uncoated Cs
derivative NPs, in which a monomodal size distribution can be
seen for the Cs−DEAE NPs and Cs−DC NPs, while for the Cs
NPs and Cs−Ca NPs, it is possible to observe more than one
main population with different Dh. As shown in Figure 7C, all
the HA-coated NPs had unimodal-type distributions with
reduced PDI.
The morphology of HA-coated NPs was examined by FE-
SEM. According to the results, all the coated nanomaterials
showed a quasi-spherical shape, with sizes between 40 and 100
nm (Figure S5A−D). The differences with respect to the DLS
results may be related to the drying involved in processing
samples for SEM analysis.93
■ CONCLUSIONS
In this work, the synthesis and characterization of polymeric
conjugates based on Cs incorporating both DEAE and catechol
groups are first reported. The functionalization of Cs was
accomplished satisfactorily, as confirmed by the physicochem-
ical and structural characterization by 1H-NMR, ATR−FTIR,
UV spectrophotometry, TGA, and XRD analyses. The Cs
derivative conjugated with two functional groups remarkably
increased its solubility at pH > 7. The catecholic conjugates
showed a notable improvement in the RSA of the DPPH
radical compared to the other derivatives and free HCA.
Studies in different bacterial strains showed that the
antimicrobial properties of the polysaccharides were dependent
on the particular tested strains and that derivatization did not
contribute to reducing their activity. Cs derivatives were
biocompatible at concentrations in the range of 10−20 μg/mL,
and the conjugates significantly reduced the cellular damage
caused by LPS-induced ROS activity in the RAW 264.7
macrophage cell line. Cs derivative-based NPs were obtained
by the ionotropic gelation method. The coating of the NPs
with HA could be demonstrated by obtaining materials with a
negative surface charge, which allowed their polydispersity to
be reduced compared to the uncoated ones. Overall, the results
envision that the Cs derivative conjugates, especially those
containing both DEAE and catechol groups, have high
potential as biomaterials with antioxidant and antimicrobial
properties. In addition, the nanomaterials obtained with these
derivatives could be good candidates as drug delivery systems
for different biotechnological and biomedical applications.
pubs.acs.org/Biomac Article
■
*
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.biomac.2c01012.
1
H-NMR spectra of Cs and Cs−DEAE dissolved in a
D2O/DCl mix; calibration graph of HCA solutions at
different concentration; ATR−FTIR spectra of Cs, Cs−
DEAE, Cs−Ca, and Cs−DC; thermograms and DTG of
Cs, Cs−DEAE, Cs−Ca, and Cs−DC; strains used in
antimicrobial assays; and FE-SEM images of HA-coated
Cs NPs, Cs−DEAE NPs, Ca−Ca NPs, and Cs−DC NPs
(PDF).
■ AUTHOR INFORMATION
Corresponding Author
Francisco J. Caro-León − Instituto de Ciencia y Tecnología de
Polímeros (ICTP), CSIC, 28006 Madrid, Spain;
Biopolymers Research Group, Centro de Investigación en
Alimentación y Desarrollo A. C. (CIAD), 83304 Hermosillo,
México; orcid.org/0000-0001-8027-1502;
Email: javiercaroleon@gmail.com
Authors
María Luisa López-Donaire − Instituto de Ciencia y
Tecnología de Polímeros (ICTP), CSIC, 28006 Madrid,
Spain
Roberto Vázquez − Centro de Investigaciones Biológicas
Margarita Salas (CIB-CSIC), 28040 Madrid, Spain;
Networking Biomedical Research Centre in Respiratory
Diseases, CIBERES, 28029 Madrid, Spain; orcid.org/
0000-0002-7919-552X
Miguel Huerta-Madroñal − Instituto de Ciencia y Tecnología
de Polímeros (ICTP), CSIC, 28006 Madrid, Spain; CIBER
de Bioingeniería, Biomateriales y Nanomedicina, Instituto de
Salud Carlos III, 28029 Madrid, Spain
Jaime Lizardi-Mendoza − Biopolymers Research Group,
Centro de Investigación en Alimentación y Desarrollo A. C.
(CIAD), 83304 Hermosillo, México; orcid.org/0000-
0003-4636-4371
Waldo Manuel Argüelles-Monal − Biopolymers Research
Group, Centro de Investigación en Alimentación y Desarrollo
A. C. (CIAD), 83304 Hermosillo, México; orcid.org/
0000-0002-8356-1123
Daniel Fernández-Quiroz − Department of Chemical
Engineering and Metallurgy, Universidad de Sonora, 83000
Hermosillo, México
Luis García-Fernández − Instituto de Ciencia y Tecnología de
Polímeros (ICTP), CSIC, 28006 Madrid, Spain; CIBER de
Bioingeniería, Biomateriales y Nanomedicina, Instituto de
Salud Carlos III, 28029 Madrid, Spain; orcid.org/0000-
0002-4179-2556
Julio San Roman − Instituto de Ciencia y Tecnología de
Polímeros (ICTP), CSIC, 28006 Madrid, Spain; CIBER de
Bioingeniería, Biomateriales y Nanomedicina, Instituto de
Salud Carlos III, 28029 Madrid, Spain
Blanca Vázquez-Lasa − Instituto de Ciencia y Tecnología de
Polímeros (ICTP), CSIC, 28006 Madrid, Spain; CIBER de
Bioingeniería, Biomateriales y Nanomedicina, Instituto de
Salud Carlos III, 28029 Madrid, Spain
Pedro García − Centro de Investigaciones Biológicas
Margarita Salas (CIB-CSIC), 28040 Madrid, Spain;
K https://doi.org/10.1021/acs.biomac.2c01012
Biomacromolecules XXXX, XXX, XXX−XXX
Biomacromolecules
Networking Biomedical Research Centre in Respiratory
Diseases, CIBERES, 28029 Madrid, Spain
Maria Rosa Aguilar − Instituto de Ciencia y Tecnología de
Polímeros (ICTP), CSIC, 28006 Madrid, Spain; CIBER de
Bioingeniería, Biomateriales y Nanomedicina, Instituto de
Salud Carlos III, 28029 Madrid, Spain; orcid.org/0000-
0001-7395-5754
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.biomac.2c01012
Author Contributions
F.J.C.-L.: conceptualization, methodology, formal analysis,
investigation, writing�original draft, writing�review &
editing, visualization. M.L.L.-D.: methodology, investigation,
writing�review & editing. R.V.: methodology, investigation,
writing�review & editing. M.H.-M.: methodology, inves-
tigation, writing�review & editing. J.L.-M.: investigation.
W.M.A.-M.: investigation. D.F.-Q.: investigation, writing�
review & editing, visualization. L.G.-F.: investigation. J.S.R.:
conceptualization, methodology, writing�review & editing,
supervision, funding acquisition. B.V.-L.: conceptualization,
methodology, writing�review & editing, supervision, project
administration, funding acquisition. P.G.: conceptualization,
methodology, writing�review & editing. M.R.A.: conceptual-
ization, methodology, writing�review & editing, supervision,
project administration, funding acquisition.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This work was funded by the Spanish Ministry of Science and
Innovation (PID2020-114086RB-100). F.J.C.-L. acknowledges
financial support from CONACyT (México) through the
scholarship ‘Apoyo para estancias postdoctorales en el
extranjero vinculadas a la consolidación de grupos de
investigación y fortalecimiento del Posgrado nacional’.
M.R.A. and B.V.-L. are members of the SusPlast platform
from CSIC. This research work was performed in the
framework of the Nanomedicine CSIC HUB (ref
202180E048). The authors thank R.A.R. from (ICTP),
CSIC, for assistance in the biological experiments.
■
1
ABBREVIATIONS
H-NMR, 1H nuclear magnetic resonance; AB, Alamar Blue; A.
baumannii, Acinetobacter baumannii; ATR−FTIR, attenuated
total reflection−Fourier transform infrared; CrI (%), crystal-
linity index; Cs, chitosan; Cs−Ca, chitosan−catechol; Cs−DC,
chitosan−DEAE−catechol; Cs−DEAE, chitosan−DEAE; Cs
NPs, nanoparticles of Cs; Cs−Ca NPs, nanoparticles of Cs−
Ca; Cs−DC NPs, nanoparticles of Cs−DC; Cs−DEAE NPs,
nanoparticles of Cs−DEAE; CV (%), percentage of cell
viability; DEAE, diethylaminoethyl; DEAE-Cl, 2-chloro-N,N-
diethylamine hydrochloride; Dh, hydrodynamic diameter; DLS,
dynamic light scattering; DPPH, 2,2-diphenyl-1-picrylhydrazyl;
DTG, differential thermogravimetric; DW, deionized water;
EC50, half-maximum effective concentration; EDC, N-(3-
dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride;
FE-SEM, field emission-scanning electron microscopy; HA,
hyaluronic acid; HCA, hydrocaffeic acid; K. pneumoniae,
Klebsiella pneumoniae; LPS, lipopolysaccharide; NPs, nano-
particles; PBS, sodium phosphate buffer; PDI, polydispersity
index; ROS, reactive oxygen species; RSA, radical scavenger
pubs.acs.org/Biomac Article
activity; S. aureus, Staphylococcus aureus; S. pneumoniae,
Streptococcus pneumoniae; S. pyogenes, Streptococcus pyogenes;
TGA, thermogravimetric analysis; TPP, sodium tripolyphos-
phate; XRD, X-ray diffraction
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