Colloids and Surfaces A 591 (2020) 124500

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Colloids and Surfaces A

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

Chitosan-coated magnetic iron oxide nanoparticles for DNA and rhEGF T

separation
Annia Gómez Péreza,*, Eduardo González-Martíneza, Carlos R. Díaz Águilab,
David A. González-Martínezc,d, Gustavo González Ruizd, Aymed García Artalejod,
Hernani Yee-Madeiraa,*
a
Instituto Politécnico Nacional – ESFM, Depto. De Física, U.P.A.L.M., San Pedro Zacatenco, 07738, CDMX, Mexico
b
Centro de Biomateriales, Universidad de La Habana, Avenida Universidad entre G y Ronda, Plaza de la Revolución, 10400, La Habana, Cuba
c
Facultad de Química, Universidad de La Habana, Zapata y G, Plaza de la Revolución, 10400, La Habana, Cuba
d
Centro de Inmunología Molecular, calle 216 esq. 15, Atabey, Playa, 11600, La Habana, Cuba

G R A P H I C A L A B S T R A C T

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

Keywords: The obtention and purification of DNA and recombinant proteins are critical steps in the biotech industries. In
chitosan-coated magnetic iron oxide this research, the use of chitosan-coated magnetic iron oxide nanoparticles as magnetic nano-adsorbent was
nanoparticles investigated. Iron oxide nanoparticles were obtained through a simple coprecipitation method. The spinel
DNA recovery structure of the nanoparticles was confirmed by X-Ray diffraction analysis. The particle size before (16 nm) and
Protein recovery
after chitosan coating (14 nm) was measured using scanning electron microscopy. Infrared spectroscopy and
thermogravimetric analysis measurements confirmed the presence of chitosan on the surface of magnetic na-
noparticles coated in a percentage of 11.24%. The Redlich-Peterson isotherm yielded the best fit for the DNA
experimental adsorption capacity and a maximum of 98 mg/g was obtained. The structural integrity of DNA,
after the elution process, was confirmed by agarose gel electrophoresis. An adsorption capacity of 440 mg/g for
rhEGF was found and the Langmuir-Freundlich isotherm showed the best fit for the experimental results. Finally,
SDS-PAGE and Western blot assays confirmed that the adsorption/desorption process did not affect the rhEGF
identity, thereby, suggesting that the biological activity was preserved.

⁎
Corresponding authors.
E-mail addresses: gpannia91@gmail.com (A. Gómez Pérez), hernaniyee@hotmail.com (H. Yee-Madeira).

https://doi.org/10.1016/j.colsurfa.2020.124500
Received 13 October 2019; Received in revised form 21 January 2020; Accepted 23 January 2020
Available online 23 January 2020
0927-7757/ © 2020 Elsevier B.V. All rights reserved.
A. Gómez Pérez, et al.
1. Introduction
The use of magnetic iron oxide nanoparticles (MNs) has become
exceedingly promising in nanobiotechnology. The MNs present mul-
tiple advantages, such as simple obtention, low cost [1,2] and easy
manipulation employing a magnetic field [3–5]. Among MNs applica-
tions are their use as biosensors [6–8], magnetic resonance imaging
(MRI) agent [9,10], drug delivery systems [11–14] and hyperthermia
[15,16].
The DNA/recombinant proteins process of separation and purifica-
tion is a critical step for biotechnology. There are several methods for
DNA separation and purification, including cetyl trimethyl ammonium
bromide (CTAB) separation, cesium bromide-ethidium chloride
method, extraction with phenol-chloroform, among others. However,
these methods are usually complicated and time-consuming. In addi-
tion, in several cases, organic solvents or toxic reagents are employed.
The procedure to obtain recombinant protein is also normally time-
consuming and complex. Hence, there has been a rapidly growing in-
terest in the use of magnetic nanoparticles for DNA/protein separation
[17–22]. The two most common methods of action of these systems are
through molecular recognition, where nanoparticles bind to specific
DNA bases or to amino acid residues of the protein, and through elec-
trostatic interactions [23]. Although there are several reports of dif-
ferent types of moieties able to develop charge over nanoparticles
surface, undoubtedly the most extended methodologies to develop po-
sitive charge on nanoparticles surfaces are the ones that use nitrogen
derivative modified nanoparticles [17,24–26], because most of the ni-
trogen derivative groups can easily become positively charged and in-
teract with DNA or proteins which are negatively charged (when pH is
above its isoelectric point).
Scientific literature commonly reports on the use of solvothermal
and thermal degradation methodologies as a means to obtain magnetic
nano-adsorbents, however, these methodologies use organic solvents,
which are expensive and sometimes complicated. In addition, the em-
ployed binding or elution buffers generally contain multiple compo-
nents, which are introduced into the solution containing the DNA or
recombinant protein. On the other hand, the molecules used for the
coating of MNs are sometimes not biocompatible, which limits their
possible uses to adsorption and desorption of DNA and proteins.
Therefore, it becomes impossible to use the system loaded with DNA or
protein for applications in living organisms such as gene therapies or
adjuvants. In that sense, chitosan is a biocompatible and biodegradable
polymer that has a polycationic character which confers high affinity
with therapeutic macromolecules (insulin, pADN, siRNA, heparin, etc.)
and antigens [27–29]. The pKa of its amino groups is around 6.5, so at a
pH lower than this value is positively charged which makes it a can-
didate for magnetic separation of DNA and proteins that have an iso-
electric point below 6.4, like the recombinant human epidermal growth
factor (rhEGF). The human epidermal growth factor (hEGF) plays a
major role in the proper function of the cell cycle, in fact, it is re-
sponsible to promote its initiation [30,31]. The rhEGF protein consists
of 53 amino acids and has a molecular weight of 6 kDa. The current
possibilities of obtaining recombinant hEGF have made it possible to
obtain stable formulations useful in several branches of medicine, like
in the case of Heberprot-P which is employed for diabetic foot ulcer
treatment, showing high wound healing capacity [32,33]. Another ex-
ample is the therapeutic vaccine against lung cancer CimaVax-EGF
[20].
In this work, we proposed to obtain chitosan-coated magnetic iron
oxide nanoparticles (ChMNs) by means of a simple methodology, such
as coprecipitation method, for its use as a magnetic nano-adsorbent in
the separation of DNA and rhEGF using a phosphate binding buffer.
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Colloids and Surfaces A 591 (2020) 124500
2. Experimental procedure
2.1. Materials
Chitosan (medium molecular weight, 75-85% deacetylated) and
ferrous chloride tetrahydrate (FeCl2·4H2O, PA) were purchased from
Sigma-Aldrich. Ammonium hydroxide solution (NH4OH, 28.4%), ferric
chloride hexahydrate (FeCl3·6H2O, 99.0%) and glacial acetic acid
(CH3COOH, 99.9%) were purchased from J.T. Baker and hydrochloric
acid (HCl, PA) from Fermont. Calf thymus DNA, potassium chloride
(KCl, 99.5%), sodium chloride (NaCl, 99.5%) and sodium hydroxide
(NaOH, 97.0%) were acquired from Merck. Potassium dihydrogen
phosphate (KH2PO4, 98.0%), disodium hydrogen phosphate (Na2HPO4,
98.0%) were obtained from Panreac and Tris-HCl buffer from Bio-Rad.
The rhEGF protein was obtained from the Center of Molecular
Immunology (CIM). All materials were used as received without further
treatment or purification.
2.2. Preparation of chitosan-coated magnetic iron oxide nanoparticles
To obtain the uncoated magnetic nanoparticles, 5 mL of FeCl2.4H2O
(2 mol/L) and 20 mL of FeCl3.6H2O (1 mol/L) solutions were mixed,
both prepared in HCl (2 mol/L). Later the mixture was dripped with a
Watson Marlow peristaltic pump at 48 rpm into a four-necked flask
containing 250 mL of NH4OH (0.7 mol/L). The reaction was kept in a
nitrogen atmosphere, under mechanical stirring (450 rpm) and in an
ultrasound bath for 25 min. Once the synthesis was completed, the
product was collected with a neodymium magnet, washed with deio-
nized water, and vacuum-dried.
In the next step, 0.125 g of medium molecular weight chitosan was
added into 50 mL of acetic acid 2% (v/v) and stirred until complete
dissolution. After that, 1 g of the dried nanoparticles was added to this
solution and mechanically stirred for 24 hours. Once the coating pro-
cess was concluded, the product was collected with a neodymium
magnet, washed with deionized water, and vacuum-dried.
For the Fourier transform infrared spectroscopy (FT-IR) study the
procedure was exactly the same except that the amounts of chitosan
added were 0.0625 g, 0.625 g and 1.250 g.
2.3. DNA adsorption/desorption experiments
Six Eppendorf tubes with 1 mL of known DNA concentrations (0.80,
0.62, 0.44, 0.30,0.22 and 0.13 mg/mL) were taken from a DNA stock
solution previously prepared in the binding buffer (phosphate buffer pH
= 4.8). Later, approximately 1.2 mg of magnetic nanoparticles were
added to DNA solutions and placed under mechanical agitation for 120
minutes. At the end of the stirring time, the magnetic nanoparticles
were collected with a neodymium magnet and the concentration of
residual DNA was measured (using a BioSpec-nano from Shimadzu
Biotech). The supernatant was disposed of, and the separated nano-
particles were washed once with ethanol. To each of the Eppendorf
tubes, 1 mL of elution buffer was added and placed in a thermomixer at
80 °C, for 10 minutes. Once this step was concluded, the nanoparticles
were separated again, and the concentration of the supernatant was
determined. Three analytical replicants were made for the adsorption
and the desorption process. The adsorption capacity of MNs was de-
termined at a DNA concentration of 0.44 mg/mL using the same pro-
cedure that was used for the ChMNs.
Three different elution phosphate buffers (pH 7, 8 and 9) were used
to determine the elution ratio and desorption in water was used as
control. The DNA adsorption capacity and elution ratio were calculated
according to the equations:
c (DNA) initial − c (DNA) residual
adsorptioncapacity = *Vol
m (MNs ) (1)
A. Gómez Pérez, et al.
m (DNA) desorbed
desorbed %= *100
m (DNA) adsorbed (2)
2.4. The rhEGF adsorption/desorption experiments
In this case, the methodology was similar to the DNA adsorption/
desorption process. Six Eppendorf tubes with 1 mL of known rhEGF
concentrations (1.25, 1.01, 0.84, 0.64, 0.46, and 0.28 mg/mL) were
taken from a rhEGF stock solution previously prepared in the binding
buffer (phosphate buffer pH = 4.8). After that, approximately 1.08 mg
of magnetic nanoparticles were added to rhEGF solutions and placed
under mechanical agitation for 120 minutes. Once the stirring was over,
the magnetic nanoparticles were collected with a neodymium magnet
and the concentration of the supernatant was measured (using a
BioSpec-nano from Shimadzu Biotech). The supernatant was disposed
of and 1 mL of elution buffer was added to the Eppendorf tubes with the
separated nanoparticles and placed in a thermomixer for 10 minutes.
Later the nanoparticles were separated again, and the concentration of
the supernatant was determined. The adsorption capacity of MNs was
determined at a rhEGF concentration of 0.84g/mL using the same
procedure that was used for the ChMNs.
Three different elution buffers were used to determine the elution
ratio: Tris-HCl of pH=8.8, NaCl (2 M) and KCl-HCl of pH=3.0, and
water was used as control. Three analytical replicants were made for
the adsorption and the desorption process. The rhEGF adsorption ca-
pacity and elution ratio were calculated with the same equations used
for DNA.
2.5. Characterization of chitosan-coated magnetic nanoparticles
FT-IR was carried out using an Equinox 55 spectrometer from
Bruker with an acquisition range between 400 and 4000 cm-1. The X-
ray diffraction measurements were performed in a D8 Advanced dif-
fractometer from Bruker using Cu-Kα (λ = 1.54183 Å) as incident ra-
diation. The range of measurement was between 10 and 80° with an
increment of 0.05 degrees and a scan speed of 5 seconds.
Thermogravimetric Analysis (TGA) was carried out in a TGA STA 409
PC Luxx from Netzsch. The samples were heated from 30 to 900 °C with
a heating rate of 20 K/min and an argon flux of 60 mL/min. The size
and morphology of nanoparticles were determined using Scanning
Electron Microscopy (SEM) with a JSM 7800 F microscope from JEOL.
For SEM determination the resulting powder was dispersed in ethanol
with sonication and a drop was placed in a carbon-copper grid and
dried over the night.
2.6. Characterization of the recovered DNA
The DNA obtained after the desorption process was characterized by
agarose gel electrophoresis. The agarose gel was prepared at 0.7%.
Once the desorption process was completed, 10 μL of the solutions were
injected in the gel and a simple electrophoresis procedure was carried
out. The DNA was labeled with ethidium bromide and revealed with UV
radiation. The same procedure was followed for the solutions obtained
with the three elution buffers.
2.7. Characterization of the recovered rhEGF
The rhEGF obtained after the desorption process was characterized
through sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) and by Western blot assay. Once the desorption process
was completed, the obtained solution was concentrated in a 3 kDa
Dispo-Biodialyzer by ultracentrifugation. Later, 20 μL of the solution
was injected in the electrophoresis gel, also the reduction buffer beta-
mercaptoethanol 4x and the SDS-PAGE procedure was carried out. The
protein bands were revealed using a silver-silver nitrate method. For
3
Colloids and Surfaces A 591 (2020) 124500
Western blot, rhEGF was electro-transferred from the electrophoresis
gel to a nitrocellulose membrane during 1 h. The membrane was
blocked with TBS1X/BSA 1% during 1 h, and then incubated with anti-
EGF antibody during 1 h at room temperature. The bands were visua-
lized using a peroxidase substrate solution. The same procedure was
followed for the solutions obtained with the three elution buffers. One
negative control was also injected into the gel. This control was pre-
pared by submitting the nanoparticles to the same procedure of rhEGF
adsorption and desorption (in this case in water) but without the pro-
tein.
3. Results and Discussions
3.1. Chitosan-coated magnetic nanoparticles characterization
3.1.1. X-Ray diffraction
The products obtained were based on the coprecipitation method.
The main chemical reaction that occurs during the synthesis is the
formation of magnetite (Eq. 3).
Fe2 +(aq) + 2Fe3 +(aq) + 8OH−(aq) = Fe3 O4 (s) + 4H2 O (3)
At the end of the synthesis, a black powder was obtained, suggesting
that the main reaction product was magnetite. The X-ray diffraction
pattern (XRDP) of uncoated magnetic nanoparticles and chitosan-
coated magnetic nanoparticles are shown in Fig. 1. The characteristic
spinel structure of magnetite was confirmed by taking into account the
main peaks observed at 18.3º, 30.2º, 35.6º, 43.3º, 53.6º, 57.25º, 62.9º
and 74.5º, which are indexed according to the (111), (220), (311),
(400), (422), (511), (440) and (533) hkl planes standard pattern of
magnetite JCPDS 19629.
The broad nature of the peaks in the XRDP suggests that the ob-
tained nanoparticles have a nanometric size. The average crystallite
size was determined by the Debye-Scherrer equation (Eq. 4):
kλ
D=
βcosθ (4)
where D is the crystallite size, k is the Debye-Scherrer constant
(0.89), λ is the used wavelength, β is the average width of the most
intense peak, and θ is the Bragg angle. The values obtained were 14 nm
for magnetic nanoparticles and 15 nm for ChMNs, thus confirming the
nanometric size of nanoparticles.
The cell parameters were calculated (Powdercell 2.4 program) and
it can be noticed that the value obtained for the coated sample is
smaller than for the uncoated MNs (8.3679 Å ChMNs and 8.3734 Å
MNs), according to T.M. Freire et al., [34], this contraction is due to the
Fig. 1. XRD patterns of the uncoated MNs and chitosan-coated magnetic na-
noparticles.
A. Gómez Pérez, et al. Colloids and Surfaces A 591 (2020) 124500

Fig. 2. SEM micrograph of (a) uncoated magnetic nanoparticles and (b) chitosan-coated magnetic nanoparticles with their corresponding nanoparticles diameter
histograms.

interaction of chitosan with the magnetite surface suggesting that the

coating process was effective.

3.1.2. Scanning electron microscopy

The morphology and histograms of uncoated and ChMNs are shown
in Fig. 2a–b. Micrographs obtained by SEM confirmed that both na-
noparticles samples present a spherical morphology and nanometric
size, with diameters of 16 ± 4 nm for uncoated magnetic nanoparticles
and 14 ± 4 nm for ChMNs. The fact that the diameters obtained by
means of the Debye-Scherer equation and the obtained by SEM are
almost the same suggests that the obtained nanoparticles are single
crystals.

3.1.3. Fourier transform infrared spectroscopy

Through FT-IR spectroscopy it was possible to confirm that the
coating with chitosan was effective. Fig. 3 shows the FT-IR spectra of
chitosan, uncoated magnetic nanoparticles and chitosan-coated mag-
netic nanoparticles. In the ChMNs curve, it is possible to observe several
bands that are present in the chitosan spectrum and that are not present
in the uncoated magnetic nanoparticles spectrum. For example, a band
attributable to OH bonding appears at 1410 cm-1, a band around 1377
cm-1 corresponds to CH3 bonding, and a more intense band related to
stretching vibration of the C-O-C bonds appears around 1060 cm-1. Also
around 1560 cm-1, it is possible to observe a band that could be at-
tributed to the bending of the NH group of amides. Additionally, three
more samples were studied in order to confirm that the observed bands
were due to chitosan molecules present on the surface of the nano-
particles. The first sample contains half of the chitosan used in the main
synthesis, the second the five-fold and the third ten-fold. As it can be
appreciated in Fig. S1 a) in the supporting information the intensity of
the main bands increases with the increase in the chitosan content. The
same trend is observed in the band's area for the four main signals (Fig.
S1 b) confirming that the coating process was effective.
On the other hand, several similar bands in the spectrums of un-
coated MNs and ChMNs appear. A broad band in the range of 3000-
3650 cm-1 is assigned to water present on nanoparticles surface, in the
Fig. 3. FT-IR spectra of chitosan, uncoated magnetic nanoparticles and chit-
osan-coated magnetic nanoparticles.
case of uncoated magnetic nanoparticles, and to stretching vibration of
OH and NH for ChMNs. Around 1630 cm-1 appears a band that can be
attributed to ammonia groups present on nanoparticles surface for
uncoated MNs and which corresponds to the combination of the
stretching vibration of C = O group of amides (amide I) and bending of
NH2 of amino moieties in the case of ChMNs. At 567 cm-1, it is possible
to observe a band, attributable in both cases, to the Fe-O deformation in
octahedral and tetrahedral position. Lastly, around 440 cm-1, there is a
band that can be attributed to the presence of maghemite. Taking this
band into account, and the fact that powder from the synthesis has a
black color, it is possible to conclude that there is a mixture of mag-
netite and maghemite present in both cases, as usually happens when
magnetic nanoparticles are obtained by the coprecipitation method
[35–37].

4
A. Gómez Pérez, et al. Colloids and Surfaces A 591 (2020) 124500

a summary of recently reported adsorption capacity for different ma-

Fig. 4. TGA curves of the uncoated magnetic nanoparticles and chitosan-coated
magnetic nanoparticles.
3.1.4. Thermogravimetric analysis
Thermograms of uncoated MNs and ChMNs are presented in Fig. 4.
In the thermogram of chitosan-coated magnetic nanoparticles can be
seen four fundamental thermal events, the first one occurs in the region
of 30-180 °C, which is due to the loss of water adsorbed on the material.
The second event occurs between 200-315 °C and is related to the
oxidation of the amino and hydroxyl groups of the polymer. The third
event can be seen between 315-500 °C and is related to the deacety-
lation of the acid groups formed previously by the oxidation of the OH
groups, the release of nitrogenous oxides due to the loss of previously
oxidized amines and the breakdown of glycosidic bonds. [34,38]. The
last event is seen between 500 and 700 °C due to the decomposition of
the polymer chain. A replicant of the TGA was conducted and the result
was alike (Fig. S2).
For uncoated MNs, an average weight loss of 5.14% was determined
between 30 and 900 °C, which is due to the loss of free and chemisorbed
water on the surface of the nanoparticles. On the other hand, in the case
of the chitosan-coated nanoparticles, the weight loss was 16.38%,
which is equivalent to a real coating of the chitosan layer of 11.24%.
3.2. DNA adsorption capacity and desorption ratio
Fig. 5 shows the DNA adsorption isotherms onto ChMNs. The values
of adsorption capacity obtained for each concentration were plotted
against the DNA equilibrium concentration (final concentration). This
led to an experimental maximum adsorption capacity of 98 mg of DNA
per g of coated magnetic nanoparticles. Wei Sheng et al. [24] presented
terials and a commercial kit used for DNA separation. The reported
values were between 61.88 mg/g and 385 mg/g in that sense, the ad-
sorption capacity obtained in this research falls in that range. Fur-
thermore, most of the authors use complicated and expensive synthesis
methods that require organic solvents. Moreover, in most cases, the
adsorption methodologies use a binding buffer with several compo-
nents. In this research. a simple coprecipitation methodology and a very
simple binding buffer (phosphate buffer) were employed.
There are different models of adjustments for the adsorption iso-
therms published in reported works, among which the best known are
the Langmuir (Eq. 5) and Freundlich (Eq. 6) models. On the other hand,
two of the most used three parameters isotherms are the Langmuir-
Freundlich (Eq. 7) and the Redlich-Peterson (Eq. 8) [39], which are
models that combine both, Langmuir and Freundlich, adsorption be-
haviors.
Qmax *KL*Ceq
Q=
1 + KL*Ceq (5)
1
Q = KF *Ceq n (6)
1
Qmax *KL − F *Ceq n
Q= 1
1+ KL − F *Ceq n (7)
KR*Ceq
Q=
1 + A*Ceq β (8)
Where: Q is the adsorption capacity, Qmax is the maximum adsorption
capacity, Ceq is the equilibrium concentration KL is the Langmuir's
equilibrium constant, KF is the Freundlich's constant, KL-F is the affinity
constant, KR and A are the Redlich-Peterson constant, β is the Redlich-
Peterson exponent, and n is the Freundlich's exponent.
The adjustments of the four models and the calculated parameters
are presented in Fig. 5 and Table 1. It is interesting the fact that the
values for n are far from 1, indicating that the adsorption sites are
energetically heterogeneous [40]. The Q max obtained with the Lang-
muir-Freundlich model (97 mg/g) is almost the same that the experi-
mental value (98 mg/g), however, the Redlich-Peterson model fit the
experimental values with an R2 value of the 0.97, while for Langmuir-
Freundlich is only 0.90. Therefore, is concluded that the model that best
fits the experimental values is the Redlich-Peterson isotherm.
The Langmuir isotherm assumes a monolayer model, also assumes
that once a molecule occupies a site, no further adsorption can take
place at that site [41]. These hypotheses correctly describe the ex-
perimental system, since the high electrostatic repulsion between the
DNA molecules does not allow the formation of multilayers. Moreover,
at a certain concentration of DNA, the material saturates and does show
no further adsorption, thus leading to Qmax. However, the Langmuir
model also suggests that the adsorption sites are energetically equiva-
lent. This hypothesis does not apply to the system in the study because
chitosan has a deacetylation percentage of 75–85 %. Meaning that there
are several units of N-acetyl-D-glucosamine randomly distributed on the
surface of the nanoparticles which cause the adsorption sites to be
energetically not equivalent. The advantage of the Freundlich model is

Table 1
Parameters of the adsorption isotherms for DNA adsorption capacity.
Isotherms Parameters

Qmax (mg/g) K (L/g)1/n A((L/g)β) n R2 β

Langmuir 108 18.73 – – 0.75 –

Freundlich – 0.11 – 0.17 0.50 –
Langmuir-Freundlich 97 1 049.55 – 2.56 0.90 –
Redlich-Peterson – 10.37 0.92 – 0.97 1.47
Fig. 5. DNA adsorption isotherm with Langmuir, Freundlich, Langmuir-
Experimental 98 – – – – –
Freundlich and Redlich-Peterson models fitting.

5
A. Gómez Pérez, et al. Colloids and Surfaces A 591 (2020) 124500

Fig. 6. DNA adsorption capacity and elution ratio using three different phos-
phate buffers and water.

that it assumes the energetic heterogeneity of the adsorption sites. [41]
Although, it has the disadvantage that it does not consider a saturation
concentration and therefore, in theory, the system does not have a
Qmax. The combination of these models in the Langmuir-Freundlich
and in the Redlich-Peterson isotherm takes the advantages of both and,
therefore, better fits the experimental model. The main difference be-
tween Langmuir-Freundlich and Redlich-Peterson is that the first re-
produce only systems with a high degree of heterogeneity while Red-
lich-Peterson is able of fitting a wider range of surfaces.
To determine the percentage of DNA desorption, the nanoparticles
were collected after the established adsorption time, washed with
ethanol, allowed to dry and placed in the elution buffer with stirring at
80 °C for 10 min. The same procedure was performed with three dif-
ferent elution buffers and also with water. The results obtained are
shown in Fig. 6.
The isoelectric point of chitosan is around 6.5, thereby, at pH above
this value, the chitosan coating of magnetic nanoparticles must lose its
positive charge. Then, the electrostatic attraction responsible for DNA
binding disappears, causing DNA strands to separate from the ChMNs.
In fact, in a previous study Chen et al. [42] demonstrated that in-
creasing the pH in the solution the overall charge in chitosan-coated
nanoparticles decreases. Therefore, is expected that increasing the pH
of the elution buffers the percentage of desorption also will increase. In
fact, the highest desorption percentage (46 %) was obtained with the
phosphate buffer at pH 9. Furthermore, the water only desorbed around
5 %, which is indicative that the buffers are responsible for the deso-
rption process. In that sense, Shan et al. [43] conducted a study for
amino-modified silica-coated magnetic nanoparticles where they de-
monstrated that by increasing the concentration of the phosphate buffer
up to 1 M, desorption can be obtained up to 96 %. However, the
methodology has the disadvantage that it takes 90 min to achieve this
elution ratio. The advantage of the developed methodology in this re-
search is that only 10 min is required for desorption. It would be in-
teresting to study the effect of buffer phosphate concentration at 10 min
of desorption time.
Fig. 7. Agarose gel image of desorbed DNA using three elution buffers. Lane 1:
DNA control solution, Lane 2, 3 and 5: desorbed DNA with the elution buffers
phosphate pH 9, 8 and 7 respectively.
corresponding to the eluted DNA, which are at the same level as the
band that contains the DNA control solution. However, in the bands
corresponding to the eluted DNA smear is observed, this could be due to
the presence of DNA degraded. Nevertheless, the smear is also present
in the band of the DNA control. Therefore, if there is any degradation it
is not because of the adsorption/desorption process if not it was already
present in the initial product. On the other hand, it can not be rejected
the idea that the presence of smearing is due to the injection of an
excess of DNA or also to a high amount of salts in the solutions. Con-
sidering the result obtained it can be affirmed that the structural in-
tegrity of DNA was not affected by the adsorption/desorption process
indicating that the eluted DNA can be used for future applications.
3.4. The rhEGF adsorption capacity and desorption ratio
A maximum adsorption capacity of 440 mg/g was obtained in the
thEGF adsorption experiment. The values of adsorption capacity ob-
tained for each sample were plotted versus the rhEGF equilibrium
concentration (final concentration) and the adjustments of the four
isotherms (Fig. 8) previously used in DNA adsorption were done.
Table 2 shows the values of the parameters calculated for each of
the different isotherm settings and the corresponding experimental
adsorption capacity obtained. As was expected the highest correlation

3.3. Characterization of desorbed DNA by electrophoresis

A very important step is to verify that the adsorption/desorption

process did not damage the structural integrity of DNA. In this sense,
agarose electrophoresis of the desorbed DNA was achieved (Fig. 7).
Lane 1 corresponds to the injected DNA control solution without
modification. From lane 2–4 were injected the solution of DNA ob-
tained after the desorption procedure with the three different elution
buffers. Lane 2 corresponds to phosphate buffer pH 9, lane 3 pH 8 and Fig. 8. The rhEGF adsorption isotherm with Langmuir, Freundlich, Langmuir-
lane 4 to pH 7. In the figure, it is possible to observe three bands Freundlich and Redlich-Peterson models fitting.

6
A. Gómez Pérez, et al.
Table 2
Parameters of the adsorption isotherms for rhEGF adsorption capacity.
Isotherms Parameters
β
Qmax (mg/g) K (L/g) 1/n
A((L/g) ) n R 2
Langmuir 523 8.45 – – 0.87
Freundlich – 0.49 – 0.08 0.72
Langmuir-Freundlich 441 324.71 – 2.56 0.99
Redlich-Peterson – 5.03 2.45 – 0.96
Experimental 440 – – – –
2
factors were obtained for Redlich-Peterson (R = 0.96) and Langmuir-
Freundlich (R2 = 0.99). Furthermore, for Langmuir-Freundlich iso-
therm, the Qmax calculated (441 mg/g) closely resembles the experi-
mental values (440 mg/g) and the R2 is higher. Therefore, it is con-
sidered that the adjustment that best reproduces the results obtained is
the Langmuir-Freundlich isotherm. The KL-F value is considered an in-
dication of the affinity between the adsorbent and the adsorbate.
Comparing the KL-F value of rhEGF with the obtained for DNA, it is
noticed that is almost three-fold smaller. A possible explanation is that
in the case of DNA the density of negative charge is higher than for
rhEGF which leads to a stronger interaction with ChMNs and a higher
KL-F value. This result could also a possible explanation of the fact that
the best fitting for the DNA adsorption is the Redlich-Peterson model
and for the rhEGF the Langmuir-Freundlich isotherm. As was mention
previously, the Redlich-Peterson can reproduce a wider interval of
surfaces than Langmuir-Freundlich. The high charge density of the DNA
could be less sensitive toward the charge inhomogeneities in the ChMNs
and therefore, interact with the surface in a more homogeneous way. In
the case of rhEGF, the density of charge is lower, then it would be more
sensitive to any charge change in the surfaces of the nanoparticles,
thereby, fitting better with the Langmuir-Freundlich isotherm.
To determine the rhEGF desorption ratio three different elution
buffers were used. A basic buffer of Tris−HCl (pH = 8.8), an acid
buffer HCl-KCl (pH = 3.0) and an ionic strength buffer (NaCl 2 M). The
highest percent of desorption was obtained with the acid buffer (Fig. 9).
This result can be explained if the driven forces that produce the re-
leasing of rhEGF from the ChMNs are considered in each buffer. For
example, in the case of Tris−HCl buffer, the pH is above 6.5, thus the
amino groups of the chitosan in SPIONs surface lose their positive
charge and the electrostatic interaction between the positively charged
ChMNs and negatively charged rhEGF disappears, releasing the protein.
However, the protein has amino acid residues that form hydrogen
bonds and also has Van der Waal interactions, with chitosan. These
interactions are strong enough to be responsible for remain protein over
the ChMNs surface. The objective of the ionic strength buffer is to shield
Fig. 9. EGF adsorption capacity and elution ratio using three different buffers
and water.
Colloids and Surfaces A 591 (2020) 124500
Table 3
Enhancement of the adsorption capacity of the MNs after chitosan coating.
Adsorption System Adsorption capacity (mg/g)
β DNA rhEGF
– MNs 11 ± 1 62 ± 6
– ChMNS 97 ± 4 440 ± 5
– Increment of Adsorption capacity 882 % 772 %
1.60
–
the protein and chitosan charges to achieve separation. but similar to
the previous case there are other interactions that cause part of the
protein to be retained. In the case of the acid buffer at pH 3, the amino
groups of the chitosan are positively charged, and the protein is below
its isoelectric point (about 4.6), so it is positively charged. Then the
electrostatic repulsions are strong enough to break the hydrogen bond
and Van der Wall interaction between protein and chitosan and lead to
93 % of the protein is desorbed. As for the DNA, the desorption of water
is low (around 14 %) compared whit the other three buffers.
The adsorption capacity of the MNS and ChMNs for DNA and rhEGF
can be observed in Table 3.
One of the main objectives of this work is to enhance the adsorption
capacity of the MNs using a chitosan coating. The previous table shows
the notable increment in the adsorption capacity of the ChMNs com-
pared with MNs, either for DNAor for rhEGF adsorption. Therefore, it
can affirmed that coating the nanoparticles with chitosan considerably
increase their ability to interact with biomolecules .
3.5. Characterization of desorbed rhEGF by electrophoresis and Western
Blot
In the biotechnology industry, it is extremely important to verify
that the structural integrity and biological activity of proteins do not
change during the process of obtention or purification, because they are
very sensitive to changes in pH, temperature, ionic strength, etc. The
structural integrity of the protein was confirmed through SDS-PAGE. In
the electrophoresis gel (Fig. 10a), lane 1 contains the molecular weight
standard, lane 2 a positive control of the pure, lane 3 a negative control
and the next three lanes contain the protein desorbed by the buffers:
Tris−HCl, NaCl and KCl−HCl, respectively. The bands of protein
desorbed with the three buffers are at the same level of control and
there is no presence of any residual bands. Moreover, the negative
control does not show any appreciable signal and the height of the
bands is in accordance with the molecular weight standard. Therefore,
it can be confirmed that the protein did not lose its structural integrity
after the adsorption/desorption process.
Fig. 10b is an image of the nitrocellulose membrane of Western blot.
Lane 1 contains the molecular weight pattern, lane 2 a positive control
of the pure protein, lane 3 the negative control and the next three lanes
contains the protein desorbed by the buffers Tris−HCl, NaCl and
KCl−HCl, respectively. The fact that the bands are registered by Wes-
tern blot indicates that at least a portion of the protein maintains its
biological activity. Moreover, the negative control does not present a
signal, indicating that the bands present in the membrane only because
of the recognition of the protein by the specific antibody. Therefore, the
adsorption/desorption process does not affect the identity of rhEGF and
presumably, the biological activity is preserved.
4. Conclusions
Through a simple synthesis methodology, a magnetic nano-ad-
sorbent of chitosan-coated magnetic iron oxide nanoparticles was ob-
tained. A maximum DNA adsorption capacity of 98 mg/g was attained
using an inexpensive phosphate buffer for 2 h of adsorption time. An
elution ratio of 46 % in a phosphate buffer at pH = 9.0 was achieved
within 10 min of desorption time. The best adjustment of the
7
A. Gómez Pérez, et al.
experimental data was obtained by the Redlich-Peterson isotherm.
Electrophoresis in agarose gel suggests that the adsorption/desorption
process does not damage the DNA structure. The possibility of applying
the system in the adsorption of rhEGF, with a maximum capacity of 440
mg/g was verified. An elution ratio of 93 % in a KCl−HCl buffer at pH
= 3.0, was obtained. The Langmuir-Freundlich isotherm produces the
best adjustment of the experimental data. SDS-PAGE confirmed that the
protein did not lose its structural integrity and Western blot suggests
that the biological activity after the adsorption/desorption experiments
was preserved.
CRediT authorship contribution statement
Annia Gómez Pérez: Methodology. Eduardo González-Martínez:
. Carlos R. Díaz Águila: . David A. González-Martínez: Methodology.
Gustavo González Ruiz: Methodology. Aymed García Artalejo:
Methodology. Hernani Yee-Madeira: Methodology.
Declaration of Competing Interest
Authors declare that there is not a conflict of interest regarding the
publication of this article
Acknowledgments
The authors acknowledge the experimental support provided by the
CNMN-IPN in the realization of the present work. Also, the authors
recognize the experimental support provided by the Centro de Estudios
Avanzados de Cuba (CEAC) and the scholarship granted by the
CONACyT to two of the students that were part of this research.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.colsurfa.2020.124500.
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