Reactive and Functional Polymers 154 (2020) 104694

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Reactive and Functional Polymers

journal homepage: www.elsevier.com/locate/react

Effects of histidine modification of chitosan microparticles on metal ion T

adsorption
Marcella T. Maiaa, Débora N. Senab, Guilherme B. Calaisc, Francisco Murilo T. Lunab,
⁎
Marisa M. Beppuc, Rodrigo S. Vieirab,
a
Solid-Biological Interface Group (SolBIN), Departamento de Física, Universidade Federal do Ceará, Campus do Pici, 60.455-760 Fortaleza-CE, Brazil
b
Grupo de Pesquisa em Separações por Adsorção (GPSA), Departamento de Engenharia Química, Universidade Federal do Ceará, Campus do Pici, 60.455-760 Fortaleza-
CE, Brazil
c
Faculdade de Engenharia Química, Universidade Estadual de Campinas, Av. Albert Einstein 500, 13083-852 Campinas-SP, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: Imbalances in metal ions (Cu2+, Fe3+, and Zn2+) augment oxidative stress, and can lead to amyloid-β (Aβ)
Histidine peptide aggregation, a phenomenon that is associated with Alzheimer's disease (AD) progression. As such, the
Chitosan use of particles that can cross the blood-brain barrier (BBB) and chelate metals could potentially provide a
Metal ions therapy to restore ionic balance in AD. Herein, we functionalized chitosan microparticles (CM) with histidine (an
Adsorption
amino acid that contains an imidazole group, therefore mimicking Aβ) to chelate Cu2+, Fe3+, and Zn2+. CM
Amyloid-β
were prepared by spray-drying and and functionalized with histidine (CM-Histidine). The CM, before and after
functionalization, and interacting with metal ions were characterized by scanning electron microscopy with
energy dispersive spectroscopy, x-ray diffraction and potentiometric titration. The adsorption capacity of CM-
Histidine increased for Cu2+ and decreased for Zn2+ and Fe3+, when compared to unmodified CM. However,
the adsorption kinetics for Cu2+ ions was lower for CM-histidine, than for CM, and the reverse was observed for
Zn2+ and Fe3+. External mass resistance was the major factor that defined the mass transfer mechanism onto the
active sites of CM-histidine. Diffusional effects were more relevant for Zn2+ and Fe3+ in the sorption process
than for Cu2+.

1. Introduction concentration, catalyzing oxidation reactions. Histidine residues from

Alzheimer's disease (AD) is a difficult-to-diagnose neurodegenera-
tive and progressive disease that attacks the brain and commonly ap-
pears in patients aged 60–70 years. The most accepted hypothesis to
explain AD is the formation of insoluble plaques composed of β-amyloid
(Aβ) and tangles, and hyperphosphorylated tau [1]. According to one of
these hypotheses, the central event in the disease's pathogenesis is an
imbalance between Aβ production and clearance, with increased Aβ
production in familial disease and decreased Aβ clearance in sporadic
disease [2]. Oxidative stress, disruption of metal homeostasis, in-
flammation, metabolic disturbances, and the accumulation of un-
folded/misfolded proteins are among the cellular changes that finally
lead to programmed cell death [3]. Oxidative damage, influenced by
metal ions, is probably a significant contributor since some of them
(Fe2+, Cu2+, Zn2+, and Al3+) exist in a significant quantity in AD brain
tissue, stimulating free radical formation and contributing to neuron
degeneration [4,5]. In AD brain tissue, there is an increase in metal
Aβ, containing amino, imidazole and carboxyl groups, have been con-
sidered the main sites where the metal ions interact with Aβ. A number
of studies have demonstrated the elevated presence of Fe2+, Cu2+ [6],
and Zn2+, in the plaques of patients with advanced AD [7–9].
Chelation therapy has emerged as a strategy to mitigate AD symp-
toms, while slowing down its progression, as opposed to the current
FDA-approved drugs, such as acetylcholinesterase inhibitors (done-
pezil, galantamine, and rivastigmine) and N-methyl-D-aspartic acid
(memantine), which relieve its cognitive effects [6,10]. Ideally, pre-
venting the metal ions from interacting with Aβ and depositing on it, or
inhibiting them from catalyzing the synthesis of ROS, could reduce this
degenerative process. Desferrioxamine (Fe3+), clioquinol (Zn2+ and
Cu2+), trientine (Cu2+), and D-penicillamine (Cu2+) [11] are chelating
agents that have been used with such purposes. Recent approaches have
included 8-hydroxyquinoline and beta-aminopyridine hybrids [12,13].
Other chelators do not penetrate the blood-brain barrier (BBB), or are
toxic (i.e., ethylenediaminetetraacetic acid, which has been used for

⁎
Corresponding author at: Universidade Federal do Ceará, Departamento de Engenharia Química, bl 709, Campus do Pici – Fortaleza, Ceará, Brazil.
E-mail address: rodrigo@gpsa.ufc.br (R.S. Vieira).

https://doi.org/10.1016/j.reactfunctpolym.2020.104694
Received 5 May 2020; Received in revised form 14 July 2020; Accepted 17 July 2020
Available online 21 July 2020
1381-5148/ © 2020 Elsevier B.V. All rights reserved.
M.T. Maia, et al.
Fig. 1. CM production obtained by spray-drying process.
heavy metal ion poisoning and has a non-specific target) [14].
Chelating nanoparticles have been proved as safe and effective
therapeutic options, crossing the BBB and sequestering metal ions [10].
These nanoparticles present reduced toxicity, improved distribution
and therapeutic efficacy. They lower quantity of brain metal ions and
target Aβ/metal ion interactions [15]. These matrices have attracted
considerable attention as drug delivery systems because they present
the ability to deliver a wide range of drugs for controlled drug release
and site-specific drug targeting [16]. Natural polymers have been used
as a template matrix for the delivery of different drugs for AD treatment
[17]. Chitosan has some important properties such as biodegradability,
low toxicity and biocompatibility, which make it a promising material
for biomedical and pharmaceutical applications. This biopolymer has
amino and hydroxyl groups in its structure, which are highly reactive
and can easily interact with metal ions or with the Aβ peptide. Chitosan
oligosaccharides have also been used as an antioxidant protective agent
for NT2 neurons against cell death and it could prevent Aβ formation
[18–20].
The physicochemical features of this cationic polysaccharide de-
pend on its molecular weight and deacetylation degree (DD). From this
polysaccharide, chitosan-based materials such as polymer, films, na-
noparticles and microparticles can be easily obtained [21]. Among the
techniques to produce these microparticles, spray-drying is the simplest
one, as it is easy to scale, rapid, and reproducible. This technique allows
the generation of microparticles with specific characteristics (e.g., size
and shape) [22]; however, fumed silica particles may be required to
improve flow and compressibility features, as well as to protect the
chitosan from drying stress and favor the production of chitosan mi-
crospheres (CM) [23]. As a result, the silica particles can aid in en-
hancing the physical properties of CM obtained, contributing to further
solid pharmaceutical development [24]. This method has been ex-
tensively used for drug-delivery. However, chitosan microparticles are
prone to swelling in an aqueous medium since the amino radicals in the
C2 position of the glucosamine monomer favors chitosan solubility,
especially under acidic conditions. Crosslinking agents, such as glutar-
aldehyde or epichlorohydrin, are normally used to confer more stability
to its structure [25].
Studies have reported on transition metal ion removal using chit-
osan. However, most of these focused on addressing environmental
2
Reactive and Functional Polymers 154 (2020) 104694
issues, such as contaminated water. Eser et al. used functionalized
chitosan with histidine for adsorbing Ni2+ [26]. Similarly, Beppu et al.
[27] and Ngah et al. [28] used chitosan for Cu2+ adsorption. Recently,
Mahl et al. [29] showed the potential of functionalizing this biopolymer
with the following amino acids, l-glutamic acid, taurine, and l-me-
thionine for adsorbing the same metal ion. Becker et al. [30] synthe-
sized other chitosan derivatives for removing Ni2+, Zn2+ [31], and
Cd2+ from environmental waters. Additionally, Vieira and Beppu
[32,33] reported on Hg2+ removal. Another approach is to used chit-
osan doped with iron, or zinc-based particles to improve their capacity
to adsorb heavy metals [34,35].
Although many studies have previously reported on the interaction
of Cu2+ with chitosan, and a few have employed chitosan functiona-
lized with histidine, to the best of our knowledge, none of these studies
have explored the equilibrium and kinetic adsorption behavior of the
metal ions, Fe3+ and Zn2+, together with the chitosan-histidine system
or for chelation therapy purposes. In the present study, the ability of CM
to chelate Cu2+, Fe3+, and Zn2+ ions, before and after functionaliza-
tion with histidine (which has imidazole functionalities and amino
groups), was evaluated. The amino acid, histidine, was used to mimic
the Aβ peptide and add new active sites. Therefore, the amino acid may
have a synergic/antagonist effect on CM.
2. Experimental section
2.1. CM production by spray-drying
For CM production, a 1% (w.v−1) chitosan solution, with a medium
molecular weight (467 kDa; Sigma-Aldrich), was prepared in 5%
(v.v−1) acetic acid. This solution was diluted in ethanol (50%) to obtain
a final chitosan concentration of 0.1% (w.v−1). Ethanol was used to
reduce the viscosity of the medium, since it showed to be a limiting
factor in the spray-drying process. Initially, a defined amount of fumed
silica (Aerosil®, Evonik, Germany) was mixed with the 0.1% (w.v−1)
chitosan solution to achieve 0.03% (w.v−1) of silica particles in the
medium. Silica addition is important to improve the flow and com-
pressibility characteristics, protect the CM from drying stress and
achieve the expected particle size and thus favor further solid dosage
formulation. The final solution was atomized in a Spray-dryer model
M.T. Maia, et al.
LM MSD 1.0 (Labmaq Brazil LTDA, Brazil) as presented in Fig. 1. The
atomization was carried out with a two-fluid 1.0 mm pneumatic nozzle
and operated in concurrent flow. The following conditions were fixed to
the drying process: inlet air temperature, 140 °C; feed flow rate,
0.36 L.h−1; airflow rate, 35 L.h−1 and outlet air temperature, 112 °C.
The dried powder was collected and stored in a desiccator at room
temperature until analysis and adsorption experiments.
2.2. Functionalization of CM with histidine
The functionalization of CM with histidine (CM-Histidine) was
performed using the method suggested by Sano and Murase [36].
Firstly, EPI and histidine were mixed in an equimolar proportion. To
achieve this proportion, 0.78 mL of an EPI solution were added to about
20 mL of a solution containing NaOH (2 mol.L−1) and 1.30 g of histi-
dine. The reaction occurred for 4 h at 60 °C. After this reaction time, the
system was cooled down to 0 °C, followed by the addition of 4 g of
NaOH under constant agitation. In a second step, CM (6 g) was added to
this solution, and the functionalization was conducted at 65 °C for 16 h
under constant stirring. After this, the CM was extensively washed with
distilled water until reaching neutral pH and stored in ultrapure water
at 4 °C. The concentration of histidine in the particle was not quantified
since it is not possible to determine the amount of the amino acid re-
liably, as in addition to interacting covalently it may also be adsorbed at
the surface of CM. Fig. 2 shows a schematic reaction for CM functio-
nalization, following the mechanisms proposed by Adhikari et al. [37].
2.3. Characterization of metal-chitosan-histidine microspheres
CM, before and after functionalization with histidine and metal
addition, were characterized by scanning electron microscopy (SEM)
with energy dispersive spectroscopy (EDS), X-ray diffraction (XRD), and
potentiometric titration to better understand the changes in mor-
phology, crystallinity and charge during the metal-chitosan, chitosan-
histidine, and metal-chitosan-histidine interactions.
2.3.1. Scanning electron microscopy with energy dispersive spectroscopy
The samples were dried and covered with a thin layer of gold
(10 nm) using a sputter coater (SCD 050 – Baltec, Liechtenstein) and
were observed using the Leo440i (England) scanning electron micro-
scope (20 kV). EDS was performed after the adsorption with metal ions
to obtain elemental semi-quantitative and semi-qualitative data.
2.3.2. X-ray diffraction
X-ray diffraction spectra were obtained using a X'Pert PW3050
Philips, with scanning scope of 5 o – 90 o, step size of 0.02 o and
scanning speed of 2 o min−1, using Cu Ka radiation
(λ = 1.5406 × 10−10 m).
2.3.3. Potentiometric titration
The amount of free amino groups was quantified for CM and CM-
Histidine samples. For each compound, 200 mg were mixed with 40 mL
Fig. 2. Representation of CM functionalization with histidine.
3
Reactive and Functional Polymers 154 (2020) 104694
of HCl solution (0.05 mol.L−1), allowing enough time (24 h) to charge
all protonate binding groups. The resulting solution was titrated using a
NaOH solution (0.1 mol.L−1) and its millivolt potential of the resulting
solution was measured for the different basic volume added, obtaining
a titration curve [38]. For CM, the DD (in %) of chitosan was calculated
by Eq. 1 and the amount of ionizable groups (mainly amino groups)
available in the samples (CNH2, mol.g−1) was calculated by Eq. 2. For
CM-Histidine, however, only the quantity of ionizable groups was ob-
tained. This was because there is no chemical formula that reliably
represents the CM-Histidine material and, therefore, the molecular
weight of the monomeric unit, in contrast to the CM, in which there is
only chitosan:
MNaOH ∗ (V2 − V1 ) ∗ 161
DD = ∗ 100
m2 (1)
MNaOH ∗ (V2 − V1 )
CNH2 =
m2 (2)
−1
in which MNaOH is the molarity of the NaOH solution (mol.L ); V1 and
V2 are, respectively, the volume (L) of NaOH used to neutralize the
excess of HCl and the protonated chitosan sample; 161 is the molecular
weight of the monomeric unit of chitosan (g.mol−1); m2 is the mass (g)
of the chitosan or chitosan/histidine samples.
2.4. Batch experiments and modeling
Solutions containing metal ions were prepared by dissolving their
corresponding salts, copper, iron, and zinc nitrate (Cu(NO3)2, Fe(NO3)3,
Zn(NO3)2, respectively) in Milli-Q Millipore water with a resistivity of
18.2 MΩ cm. The pH values were fixed at 5.0, in accordance with the
diagrams of metal species distribution (Fig. S1), as a function of pH,
simulated using HYDRA (Hydrochemical Equilibrium-Constant
Database) software [39]. These salts were chosen to avoid the presence
of their metal species at the selected pH. Use of these salts at higher pH
values was not viable since the diagrams showed that Cu2+ and Zn2+
would precipitate as oxides or hydroxides at (Fig. S1). Moreover,
chitosan is substantially soluble at low pH values due to further pro-
tonation of its amino groups [40]. The stirring rate was 45 rpm for all
the experiments and the pH was adjusted using NaOH solution
(0.1 mol.L−1). The metal ion adsorption experiments were conducted
for CM, CM-Histidine and histidine to quantify the effect of histidine
and chitosan in their adsorption, coupled and individually. Previous
experiments from this research group regarding the conditions de-
scribed showed that 160 min was sufficient contact time to reach
equilibrium.
2.4.1. Equilibrium measurements and modeling
Equilibrium adsorption data were obtained by soaking 20 mg of CM,
CM-Histidine or pure histidine with 4 mL of ionic solutions (Cu2+,
Fe2+, and Zn2+) with initial concentrations varying from 0.1 to
15 mmol.L−1, at pH 5.0, 25 °C and under stirring of 45 rpm (TECHNAL,
TE-165, Brazil). The samples were maintained under agitation until
reaching the equilibrium. The metal ion concentration in the
M.T. Maia, et al. Reactive and Functional Polymers 154 (2020) 104694

supernatant was analyzed withdrawing aliquots at fixed time intervals ∂Cp

r = 0, =0
using atomic absorption spectroscopy with an air-acetylene flame ∂r (8)
(Perkin Elmer AA Analyst 100). Cu2+, Fe2+ and Zn2+ were analyzed at
Boundary 2:
the following wavelengths: 324.7, 248.2, and 213.9 nm, respectively.
The amount of metal ions adsorbed at equilibrium was calculated ∂Cp ∂q
r = R, Dp + ρp Ds = kf (Cb − Cp)
using Eq. 3: ∂r ∂r (9)

VS ∗ (C0 − Ceq ) Differential mass balance in the liquid phase:

q=
MA (3)
−1
where q (mmol·g ) is the adsorbate concentration in the adsorbent at
equilibrium; Co (mmol·L−1) represents the initial metal ion concentra-
tion in solution; Ceq (mmol·L−1) is the metal ion equilibrium con-
centration; VS (L) is the volume of the solution; and MA (g) is the mass
of the adsorbent.
The equilibrium data for metal ions adsorbed onto CM, CM-
Histidine and histidine systems have been described by Langmuir and
Sips models. The Langmuir equation is more commonly used, and
considers a monolayer and homogeneous adsorption, but does not as-
sume lateral interaction and steric hindrance between the adsorbate
molecules. The Sips equation has three parameters and combines the
Freundlich and Langmuir equations, and is used for multilayer and non-
homogeneous adsorption. Eqs. 4 and 5 display the Langmuir and Sips
models, respectively [41,42].
K ∗ Ceq
q = qm
1 + K ∗ Ceq (4)
ns
Ks ∗ Ceq
q = qms ns
1 + Ks Ceq (5)
qm and qms (mmol·g−1) are the Langmuir and Sips maximum adsorption
capacity, K and Ks (L·mmol −1) are the Langmuir and Sips equilibrium
constant and ns is the heterogeneity parameter. The difference between
the Sips and Langmuir equations is related to the parameter ns. If the
value is greater than unity, the system is more heterogeneous.
∂Cb −3Vs kf
= (Cb − Cp)
∂t Rp ∗ Vl (10)
Initial conditions:
t = 0, Cb = C0, Cp (r ) = 0, q (r ) = 0 (11)
where Cb is the bulk liquid phase concentration, Cp is the intra-particle
concentration and q is the amount of adsorbed ions per gram of ad-
sorbent calculated from the Eqs. 4 and 5, εp is the particle porosity, Rp is
the particle radius, Dp is the pore diffusion coefficient, Ds is the surface
diffusion coefficient, kf is the film mass transfer coefficient, r is the
radial coordinate, Vs is the adsorbent volume, Vl is the liquid phase
volume, ρs is the reverse of porosity, ρp is adsorbent density, and t is the
time. An estimation of the external mass transfer coefficient (kf) was
used, as proposed by Furusawa and Smith [44] for liquid batch ad-
sorption.
The mathematical model was numerically solved using the gPROMS
commercial simulator. The mathematical systems are composed of
systems of partial differential and algebraic equations (DAE). The
temporal and radial domains are discretized using a third-order or-
thogonal collocation method in finite elements (OCFEM), with six
sections and three placing spots per section. The estimate of pore and
surface diffusion coefficients was performed using the heteroscedastic
estimation method included in the computational package, gPROMS
[45].
The root mean squared error (RMSE) was used to evaluate the fit-
ting of the model to the experimental data, as shown in Eq. 12.

1 N
RMSE =
N
∗ ∑i =1 (CS − CE )2 (12)
2.4.2. Kinetic measurements and modeling
Kinetic adsorption experiments were conducted by soaking 20 mg of where CS is the concentration estimated from the model, CE is the ex-
CM, CM-Histidine or pure histidine with 4 mL of a metal solution, with perimental concentration and N is the number of measured values. A
an initial concentration of 3.15 mmol.L−1, 3.58 mmol.L−1, and minimal RMSE value indicates a better model prediction of the ex-
3.06 mmol.L−1 for Cu2+, Fe2+, and Zn2+, respectively, at pH 5.0, at perimental data.
25 °C and under stirring at 45 rpm (TECHNAL, TE-165, Brazil).The
samples were analyzed withdrawing aliquots at fixed time intervals to
3. Results and discussion
measure the metal ion concentration in the supernatant by atomic ab-
sorption spectroscopy with air-acetylene flame (Perkin Elmer AA Analyst
3.1. Microsphere characterization
100). The samples adsorption capacity was calculated by considering
the variation of the metal ions solution concentration.
3.1.1. Scanning electron microscopy with energy dispersive spectroscopy
The morphology of the dried CM, as obtained by SEM, is shown in
2.4.2.1. Diffusion models and parameter estimations. The estimation of Fig. 3A. Superficially, CM did not present porosity, displaying a dense
the mass transfer parameters in batch adsorption kinetics was structure, with a diameter range of ca. 1.0 to 10.0 μm. The presence of
performed using the Pore-Surface Diffusion Model (PSDM) [43]. The some small particles on CM can be seen; these are probably the silica
model assumes a spherical particle of adsorbent in a finite bath, particles that were used as a drying agent during the CM production
containing a component to be adsorbed. The PSDM equations may be process. These results are in accordance with the study of Oliveira et al.
described as shown in Eqs. 6 and 10 (solid phase and liquid phase [46], who produced CM crosslinked with glyoxal by spray-drying and
balances), using appropriate initial and boundary conditions (Eqs. 7, 8, obtained a diameter range from 0.9 to 6.7 μm.
9, and 11): An important particle parameter to consider is permeability across
Differential mass balance in the solid phase: the BBB. According to Kreuter [47], nanoparticles made of natural or
synthetic polymers ranging from 10 to 1000 nm are a potential tool to
∂Cp ∂q 1 ∂ ⎡ 2 ⎛ ∂Cp ∂q
εp + ρs = 2. r Dp
⎜ + ρp Ds ⎞ ⎤
⎟
transport drugs across this area. Despite the higher diameter achieved
∂t ∂t r ∂r ⎢
⎣ ⎝ ∂r ∂r ⎠ ⎥
⎦ (6) in this study, the microparticles are still significant to understand the
ability of histidine, when coupled to CM, to provide metal ion ad-
Initial condition: sorption and, therefore, to promote AD remission. These findings may
t = 0, Cp = 0 and q = 0 (7) lead to the development of other histidine-based nanosystems able to
cross the BBB to prevent metal ions from depositing in the Aβ ag-
Boundary 1: gregates and thereby misfolding, in turn decreasing oxidative stress.

4
M.T. Maia, et al.
Fig. 3. Scanning electron microscopy images of surfaces of spray-dried CM (A,
B1 and B2), CM-Histidine (C1 and C2), CM-Histidine/Cu2+ (D1 and D2).
Compounds from B to D were immersed in an aqueous solution at pH 5.
After the immersion in aqueous solution, the CM acquired a certain
roughness at the surface and exhibited a filamentous structure. This
happened, probably, as a result of the protonation of amino groups
(-NH3+), which increased chitosan solubility and may have allowed
interactions between the microspheres (as seen in Fig. 3B1 and B2);
however, the CM may still be seen. To enhance matrix stability and
covalently bind the amino acid to CM, EPI was used as a crosslinking
agent during the functionalization of CM with histidine. However, the
micrographs of the sample surfaces showed that, possibly, this was not
enough to completely avoid the likelihood of swelling (Fig. 3C1 and
C2), since a polymeric network was still present in CM-Histidine, which
became more porous [48]. A similar situation was observed after the
adsorption of metal ions on the CM-Histidine surface. However, in this
case, the matrix appeared to be less organized (Fig. 3D1 and D2). The
surface maps provided by EDS analysis proved the presence of ad-
sorbate in post-adsorption samples (Fig. S2), in which luminous dots
could be visualized. The EDS graphs (Fig. S3) show the peaks that
correspond to the presence of Cu2+, Fe3+, and Zn2+ in their respective
system samples. The analysis indicates that metal ions may have in-
teracted with active functional groups on chitosan or histidine. Both CM
and CM-Histidine were capable of adsorbing the ions, but the amount
and distribution of the dots varied according to the metal ion element
(Fig. S2).
3.1.2. Potentiometric titration
The results from the potentiometric titration of CM and CM-histi-
dine are displayed in Fig. 4. This figure depicts two inflections; the first
5
Reactive and Functional Polymers 154 (2020) 104694
is related to the NaOH consumption to neutralize the HCl excess, and
the second one results from the neutralization of the protonated amino
groups on the chitosan chain. With regard to the CM, the difference in
these volumes can be used to calculate its DD. To easily obtain this
difference, the first derivative was applied in the potentiometric curve
to highlight the NaOH volume used to reach each inflection point (local
minimum points in the curves), which were subtracted. The DD ob-
tained by Eq. 1 was 84.31%.
For both CM and CM-histidine, the amount of their available io-
nizable groups can be obtained by Eq. 2. These are functional groups
that were protonated by the HCl added in the sample during this
methodology, and are composed mainly by amino groups. Similarly, the
NaOH volume consumed to neutralize the protonated amino groups can
also be applied in this other equation, this time, to calculate the molar
quantity of these groups per mass of adsorbent (CNH2). These ratios
were 5.09 mmol.g−1 and 5.78 mmol.g−1, to CM and CM histidine,
respectively. A higher quantity was observed for the latter, which
suggests that the number of amino groups increased after chitosan
functionalization by the amino acid (as seen in Fig. 4). According to the
chitosan-histidine functionalization reaction equation (Fig. 2), the same
number of primary amino groups should be available before and after
the modification. Therefore, the increase in these groups was un-
expected, and was generated, possibly, due to the functionalization
process, during which chitosan was submerged in a hot NaOH solution,
a medium similar to that used to induce the deacetylation process.
Additionally, part of the histidine added might have been adsorbed on
the matrix, without the formation of a covalent bond, which could have
increased the amount of free primary amino groups.
3.1.3. X-ray diffraction
Fig. 5 presents the X-ray diffractograms for CM, CM-metal and CM-
Histidine-metal. The results show some differences in the CM crystal-
linity after histidine coupling and after the contact of CM-Histidine with
the metal ions. According to the literature, the XRD pattern for chitosan
shows distinct peaks for the scattering angle 2θ equal 10.6 and 20.4o
[42]. CM presented a modified pattern in comparison to raw chitosan,
one characteristic peak at 19.2 o and a less intense one between 40 and
50°, indicating some chitosan crystallinity. Chitosan is a semi-amor-
phous material, which presents low crystallinity. The production of the
material in microsphere shape by spray drying, and without a coagu-
lating step with NaOH, removed the halo usually observed at 10° in
chitosan membranes and CM [40,49]. This indicates a lower long-range
organization of the material, provoked by the faster nature of the
process used, compared to other production processes, and due the fact
that it did not use NaOH as a coagulating agent. The CM-metal and CM-
Histidine-metal systems presented very similar diffractograms, in which
analogous peaks (2θ = 19.2 o) were observed with a larger reduction in
the intensity compared to pristine CM, indicating a decrease in struc-
ture crystallinity during adsorption. Some silica particles remained
among the CM after the spray-drying process, as observed in the SEM
analysis. Silica is an amorphous material known to present broad peaks
at 2θ angle ca. to 0 o and 22 o, however, these could not be observed
due to a possible overlapping of the chitosan peak (20.4 o) and the silica
peak (ca. 22 o) [50,51]. Additionally, the XRD diffractogram was not
performed at low angles.
3.2. Equilibrium and kinetic adsorption
Before performing adsorption experiments, metal speciation dia-
grams and pH solution parameters were analyzed to determine the best
experimental conditions. These parameters were simulated using
HYDRA (Hydrochemical Equilibrium-Constant Database) software
[39]. Simulations using metallic sulfate or nitrate salts were carried out;
in sulfated salts at pHs of lower than 5.0, a large number of non-dis-
sociated species was observed, which could contribute to metallic
compound precipitation. As such, due to metal speciation (Fig. S1),
M.T. Maia, et al.
Fig. 4. Potentiometric titration of CM and CM-Histidine.
Fig. 5. XRD diffractograms for CM, Cu/CM and Cu/CM-Histidine.
nitrate salts at pH 5.0 were the best choice of metal salt for adsorption
experiments.
3.2.1. Equilibrium adsorption
In this study, adsorption models were used to describe the equili-
brium adsorption data to better understand the metal adsorption phe-
nomena. All data are described by the Sips and Langmuir models, where
the former provides the better description. Fig. 6 shows the isotherm
curves described by the Sips and Langmuir models, with their para-
meters listed in Table 1.
In general, metal cation adsorption on chitosan increases with so-
lution pH. At low pH values, amino group protonation occurs and NH3+
generates an electrostatic repulsion of metallic ions, decreasing the
adsorption capacity of these species [52]. On the other hand, the in-
crease in solution pH, generates hydroxides from metallic ions. For the
metals used in this study (Fig. S1), at pH lower than 5.0 there is metal
precipitation from ionic species to metallic.
The Sips model provided the best correlation with the experimental
data (Table 1). Both models (Langmuir and Sips) showed an increase in
the affinity constant for the CM-Histidine systems containing Zn2+ or
Fe3+ and the reverse for the interaction with Cu2+, when compared to
the unmodified CM. In addition, an increase in the adsorption capacity
of CM-Histidine system for Cu2+ was observed, whilst a decrease for
the ions of the other metals occurred. The adsorption capacity of CM
followed this order: Cu2+ < Zn2+ < Fe3+, while for CM-Histidine:
Fe3+ < Zn2+ < Cu2+.
The adsorption behavior can be explained by the nature of these
cations, such as ion charge, ionic ratio, hydration energy and the hy-
drolysis constant of these elements [53]. An ion with larger ionic radius
6
Reactive and Functional Polymers 154 (2020) 104694
(Zn2+ > Cu2+ > Fe3+) probably has less access to the adsorbent's
pores. The lower the energy needed for the dehydration process, the
more easily adsorption occurs (Fe3+ < Cu2+ < Zn2+). However, the
rate of hydrolysis of metal/hydrated cation increases with the charge
and decreases with the radius. Consequently, lower hydrolysis constant
value of (Fe3+ < Cu2+ < Zn2+) means greater stability in solution,
making it more difficult to achieve the balance in the sorption/deso-
rption process [54,55].
Metal ion chelation with chitosan in acidic solution usually occurs
by electrostatic attraction between anionic metal complexes with its
protonated amine groups. Free metal ions form complexes with chit-
osan through the nitrogen of the amino group, due to their free elec-
tronic doublet, as well as through the oxygens of the OH groups in the
C3 positions of the glucosamine rings by ion-exchange interaction.
Compared to histidine, chitosan possesses fewer groups available for
this interaction, as only its amino groups interconnect with the metal
ions with significant strength. Additionally, chitosan acetyl groups
hinder the adsorption, since they occupy a NH2 site. At an experimental
pH of 5, histidine's carboxyl group is deprotonated, which allows the
molecule to interact with metal ions via COO− groups; it is also able to
interact with its R-NH2 and, in a less intense manner, via the imidazole
ring. However, the metal ions can suffer competition from protons,
limiting their interaction with imidazole functions [27].
For Cu2+, the CM-Histidine shows an intermediary behavior, de-
monstrating an increase in adsorption capacity, when compared to
pristine chitosan, but also an affinity decrease. It also shows the op-
posite behavior when compared to pure histidine, whereby the amino
acid molecule in solution, naturally, has more freedom to chelate the
ion than do those coupled to chitosan. As such, the interaction of the
CM with these species may impart attractive forces to the metal ions
from several directions [52]. Studies demonstrate that Cu2+ usually
interacts with more than one histidine molecule simultaneously
[6,56,57]. The extensive adsorption of Cu2+ ions by histidine may
make the charge of the complex molecule much more positive, pro-
voking repulsion among the ions and a consequent affinity decrease for
CM-Histidine (Table 1). When coupled, histidine has its primary amine
transformed into a secondary amine during the modifying process,
which decreases its interaction with the adsorbate. However, its other
groups maintain their interaction level, together with those of chitosan.
These factors can lead to a significantly greater adsorption capacity for
histidine than for the same weight of CM, or CM-Histidine.
As to Fe3+ [58] and Zn2+ [59], previous studies have shown that
they interact with Aβ peptide via imidazole groups on histidine,
forming bridges and that the interaction affinity and stability depend on
the medium's pH. For Fe3+, chitosan modification neither significantly
affect the adsorption capacity nor the affinity of the adsorbent for Fe3+.
M.T. Maia, et al. Reactive and Functional Polymers 154 (2020) 104694

Fig. 6. Adsorption isotherms of metal ions (Cu2+, Fe3+, and Zn2+) for CM, CM-Histidine, and Histidine.

This could lead us to the assumption that the main groups responsible
for capturing iron are the same as those of pristine chitosan, or that they
interact with the metal ions with similar intensities. Zhou et al. [58]
showed that histidine is capable of complexing with Fe3+ by its car-
boxyl group and by its sp2 hybridized nitrogen atom, stabilizing their
interaction. Chitosan is also capable of binding to this ion by its amino
groups [60].
With regard to Zn2+, Kyzas et al. [61] suggested that this ion con-
nects to chitosan by a bridge, in which it is the middle atom, sur-
rounded on each side by one or more amino groups of a chitosan chain.
They point out that some studies suggest a pendant-like connection, in
which just one amino group is involved. These authors demonstrated
that OH− groups also interact with the metal, especially in the absence
of stronger nitrogen-based groups and deprotonated carboxyl, and re-
ported that the optimal pH for zinc adsorption by chitosan is ca. 5,
which may justify its higher adsorption by the biopolymer.
Moreover, according to the Sips model, the first binding sites could
be occupied more strongly than the following could, as the adsorption
energy is exponentially reduced at the end of the process. In addition,
values of ns > 1 for all adsorbents were obtained, indicating attractive
force due to lateral interactions and the heterogeneous nature of the
adsorption [62]. As such, steric effects might be occurring in our sys-
tems, as has been observed for uranyl adsorption into Mg(OH)2 mi-
nerals, by Ou et al. [63], who showed that this adsorption affected the
orientation of the -OH groups in the vicinity, making it difficult to
adsorb more uranyl in the adjacent regions and restricting the mono-
layer adsorption capacity. These parameter values corroborate with our
previous hypothesis, in which each following addition reduced the
metal ion-adsorbent affinity and, therefore, the apparent antagonism
between affinity and adsorption capacity.
As a buffer could influence adsorption, this substance was avoided,
which might have led to a fluctuation in the pH of each metal solution

Table 1
Parameters of the Langmuir and Sips equations for equilibrium experiments.
Sorbate/Adsorbent Langmuir equation Sips equation

qm (mmol.g−1) K (L.mmol−1) R2 qms (mmol.g−1) Ks (L.mmol−1) ns R2

Zn/Chitosan 2.877 3.766 0.853 2.451 29.571 2.148 0.926

Zn/Chitosan-Histidine 2.346 4.331 0.837 2.037 36.102 2.170 0.941
Zn/Histidine 1.550 1.720 0.902 1.176 23.610 3.086 0.988
Fe/Chitosan 2.899 3.471 0.835 2.502 13.219 1.750 0.862
Fe/Chitosan-Histidine 2.328 4.637 0.872 2.044 29.065 1.985 0.952
Fe/Histidine 2.243 4.495 0.878 1.972 27.487 1.953 0.951
Cu/Chitosan 2.368 3.852 0.896 2.031 21.428 1.951 0.963
Cu/Chitosan-Histidine 5.637 1.571 0.951 4.577 4.726 1.755 0.984
Cu/Histidine 5.987 0.918 0.953 4.198 3.468 2.061 0.98

7
M.T. Maia, et al.
throughout the adsorption experiments. The dissolution of different
metal salts decreases the solution's pH distinctly, and their adsorption
provokes the opposite effect. Therefore, the solution's pH might have
varied differently, affecting the charge distribution in the solution and,
therefore, the metal ion adsorption, on a different scale [27].
Mahl et al. showed that chitosan competes with histidine for Cu2+
adsorption, which may interfere in Aβ aggregation [52]. Furthermore,
M. Hoernke et al. [8] investigated the role of Zn2+ and Cu2+ in Aβ
aggregation, using a model peptide. The authors showed that the in-
teraction of these metal ions with Aβ peptide depends on the element
and its geometry in the binding sites. Zn2+ complexation is closely
related to histidine residues, leading to a rapid aggregation, while Cu2+
interacts with other sites along the peptide backbone in addition to
histidine groups, forming multiple coordination forms. Herein, we show
that, for Cu2+, CM-Histidine presented an intermediary behavior in
which a higher adsorptive potential and a lower affinity were observed.
The superior adsorption capacity, as a result of the presence of histi-
dine, compared to unmodified CM demonstrates the potential of CM-
Histidine to mitigate self-aggregation and ROS production, competing
with the ability of Aβ to adsorb metal ions, reducing the Cu2+ available
in the blood and thus preventing them from interacting with the pep-
tide. In contrast, for Zn2+ and Fe3+, CM-Histidine demonstrated a good
performance in the adsorption process, although this was inferior to
that of CM. CM functionalization increased the adsorbent-adsorbate
affinity of the microparticles, also indicating a capability to compete
with the histidine binding sites in Aβ and slow down AD progression by
driving conformational changes of Aβ, in turn reducing oxidative da-
mage.
3.2.2. Kinetic data and parameter estimation
Adsorption models usually consider that adsorption kinetics are
only controlled by the adsorption rate of the adsorbate on the surface of
the adsorbent, ignoring other factors. However, the mechanism of solid-
liquid mass transfer are controlled by chemical adsorption, intraparticle
diffusion (pore volume and surface diffusion), and/or external mass
transfer on active sites. The kinetic data were adjusted using the PSDM,
which constitutes a diffusional mass transfer model [43]. Batch kinetics
experiments, showing the concentration profile (C/Co) versus time, are
presented in Fig. 7.
The kinetic rate was higher for CM-Histidine than for CM regarding
Zn2+ and Fe3+ adsorption, and the opposite was observed for Cu2+
ions. This probably occurs due to the strongest interaction of Fe3+/
Zn2+ with the active sites of the CM-Histidine system, suggesting that
new functionalities increased the attractive forces and the interaction,
which were determinant in the rapid uptake and formation of metal
complexes. For Cu2+, both lower affinity and removal adsorption rate
were obtained for CM-Histidine, indicating that steric effects had an
important role on this, protonated amino groups led to repulsive in-
teractions and other sites in CM-Histidine were relevant for them to
interact actively and form complexes.
The RMSE values in Table 2 indicated a good agreement between
the experimental and predicted data. The model parameters are related
to the nature of the system, regarding pore and surface diffusivity,
adsorbent weight, density, liquid volume and initial solute concentra-
tion. The parameters kL, Ds, and Dp correspond to external transport,
surface diffusion, and pore volume diffusion mechanisms, respectively
[64,65]. Lower values were obtained for Dp, Ds, and Kf with Cu2+/CM-
Histidine and higher values of these parameters for Zn2+/CM-Histidine
and Fe3+/CM-Histidine. External mass resistance was the major lim-
iting phenomenon for the adsorption rate. As the stirring speed used in
this study was 45 rpm, these parameters should not be ignored, as both
bulk and film diffusion steps become insignificant in protocols that use
stirring speeds as high as 400 rpm [29]. For the Cu2+ system, the CM-
Histidine provided new groups for interacting with Cu2+, increasing
the adsorption capacity. However, this material did not provide a
higher chelating rate via diffusional effects. On the other hand, the
8
Reactive and Functional Polymers 154 (2020) 104694
functionalization with histidine for Zn2+ adsorption showed that the
pore diffusion contributed more to intraparticle diffusion than the
surface diffusion for this system, leading to a higher interaction and
chelating rate. With regard to the Fe3+ systems, the addition of histi-
dine to the structure resulted in a higher interaction and adsorbate
removal rate via diffusional effects. Its ionic radius and lower energy of
dehydration might have favored the rapid sorption process.
The kinetic results are aligned with those obtained with the equi-
librium analysis. For Cu2+, the lower affinity for the CM-Histidine af-
fects the kinetic rate, promoting a gradual removal of the metal ion by
the adsorbent. We showed that the intermediary behavior of CM-
Histidine contributes to an increased adsorption capacity, when com-
pared to pristine CM. With regard to their use in AD, CM-Histidine-
based chelating materials could increase Cu2+ removal from the
medium, competing with the adsorptive capability of Aβ sites. In con-
trast, Fe3+ and Zn2+ removal may occur differently, but induce a si-
milar effect; CM-Histidine could rapidly adsorb the ions from the
medium, preventing them from interacting with the peptide and from
supporting this degenerative process. For those two ions, the modified
chitosan also presented a stronger affinity for the ions than pure histi-
dine does. As a result, the material may remove these ions from the
peptide, and hence favor conformational changes in the Aβ that could
decrease the continuous cycle that leads to oxidative stress.
4. Conclusions
We chose to functionalize chitosan with the amino acid histidine to
mimic the residue by which transition metals mainly interact with Aβ
peptide. The treatment increased the adsorption of Cu2+ ions, while
decreasing its affinity. For other metals ions (Fe3+ and Zn2+), the re-
verse was observed. New chelating sites and the highest number of
ionizable amino groups can contribute differently, according to the
nature of the metal ions and the possible coordination complexes that
may be formed. Data obtained by equilibrium and kinetic adsorption
studies, as well as the characterization of CM and CM-Histidine, showed
the potential of CM-Histidine-based materials to hinder AD advance-
ment. This possibility arises since they could compete with Aβ for the
metal ions by removing these ions from the medium, leading to a re-
duction of the interaction of the ions with the peptide. For both Fe3+
and Zn2+, CM-Histidine binding sites offer greater affinity, when
compared to both pure chitosan and histidine, suggesting that they
might be more strongly attracted by this material than by Aβ sites,
which may lead to peptide plaque dissolution. With regard to Cu2+, the
intermediary behavior of the modified biopolymer may favor a greater
Cu2+ removal. The development of CM-Histidine-based chelating
agents might aid in a reduction of Aβ plaque deposition in the brain,
thereby decreasing oxidative stress. Future studies using molecular
dynamics can help understand the structural and energetic aspects in-
volved in metal ions adsorption onto the chitosan interface, with and
without histidine.
Declaration of Competing Interest
The authors declare no conflicts of interest.
Acknowledgments
The authors acknowledge Coordination for the Improvement of
Higher Education Personnel (CAPES: Procad 88882.151600/2017-01),
and National Council for Scientific and Technological Development
(CNPq) for the financial support to conduct this project.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
M.T. Maia, et al. Reactive and Functional Polymers 154 (2020) 104694

Fig. 7. Kinetic adsorption results achieved for CM and CM-Histidine with metal.

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