Chitosan-coated superparamagnetic iron oxide nanoparticles for DNA and rhEGF separation

Journal Pre-proof Chitosan-coated superparamagnetic iron oxide nanoparticles for DNA and rhEGF separation ´ ´ ´ Annia Gomez Perez, Eduardo Gonzalez-Mart´ ınez, Carlos R. D´ıaz ´ ´ ´ Aguila, David A. Gonzalez-Mart´ ınez, Gustavo Gonzalez Ruiz, Aymed Garc´ıa Artalejo, Hernani Yee-Madeira

PII:

S0927-7757(20)30093-5

DOI:

https://doi.org/10.1016/j.colsurfa.2020.124500

Reference:

COLSUA 124500

To appear in:

Colloids and Surfaces A: Physicochemical and Engineering Aspects

Received Date:

13 October 2019

Revised Date:

21 January 2020

Accepted Date:

23 January 2020

´ ´ ´ ´ Please cite this article as: Gomez Perez A, Gonzalez-Mart´ ınez E, D´ıaz Aguila CR, ´ ´ Gonzalez-Mart´ ınez DA, Gonzalez Ruiz G, Garc´ıa Artalejo A, Yee-Madeira H, Chitosan-coated superparamagnetic iron oxide nanoparticles for DNA and rhEGF separation, Colloids and Surfaces A: Physicochemical and Engineering Aspects (2020), doi: https://doi.org/10.1016/j.colsurfa.2020.124500

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Chitosan-coated superparamagnetic iron oxide nanoparticles for DNA and rhEGF separation Annia Gómez Péreza*, Eduardo González-Martíneza, Carlos R. Díaz Águilab, David A. González-Martínezc d, Gustavo González Ruizd, Aymed García Artalejod, Hernani YeeMadeiraa* a

Instituto Politécnico Nacional – ESFM, Depto. de Física, U.P.A.L.M., San Pedro Zacatenco,

07738, CDMX, México. b

Centro de Biomateriales, Universidad de La Habana, Avenida Universidad entre G y Ronda,

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Plaza de la Revolución, 10400, La Habana, Cuba. 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,

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Cuba.

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*Corresponding author

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Graphical abstract

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E-mail addresses: [email protected] (H. Yee), [email protected] (A. Gómez)

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Abstract The obtention and purification of DNA and recombinant proteins are critical steps in the biotech industries. In this research, the use of chitosan-coated superparamagnetic iron oxide nanoparticles as magnetic nano-adsorbent was investigated. Iron oxide nanoparticles were obtained through a simple coprecipitation method. The spinel structure of the nanoparticles was confirmed by X-Ray diffraction analysis. The particle size before (16 nm) and after chitosan coating (14 nm) was measured using scanning electron microscopy. Infrared spectroscopy and thermogravimetric analysis measurements confirmed the presence of

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chitosan on the surface of magnetic nanoparticles 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

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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

biological activity was preserved.

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adsorption/desorption process did not affect the rhEGF identity, thereby, suggesting that the

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Keywords: chitosan-coated superparamagnetic iron oxide nanoparticles; DNA recovery;

1. Introduction

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protein recovery

The use of superparamagnetic iron oxide nanoparticles (SPIONs) has become exceedingly

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promising in nanobiotechnology. The SPIONs present multiple advantages, such as simple obtention, low cost[1, 2] and easy manipulation employing a magnetic field[3-5]. Among

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SPIONs applications 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 purification 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 addition, in several cases, organic solvents or toxic reagents are employed. The procedure to obtain recombinant protein is also normally 2

time-consuming and complex. Hence, there has been a rapidly growing interest 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 electrostatic interactions[23]. Although there are several reports of different types of moieties able to develop charge over nanoparticles surface, undoubtedly the most extended methodologies to develop positive charge on nanoparticles surfaces are the ones that use nitrogen derivative modified nanoparticles,[17, 24-26] because most of the nitrogen derivative groups can easily become positively charged and interact with DNA or proteins which are negatively charged

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(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 employed binding or elution buffers generally contain multiple components, which are

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introduced into the solution containing the DNA or recombinant protein. On the other hand,

the molecules used for the coating of SPIONs are sometimes not biocompatible, which limits

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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

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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

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pKa of its amino groups is around 6.5, so at a pH lower than this value is positively charged which makes it a candidate for magnetic separation of DNA and proteins that have an isoelectric point below 6.4, like the recombinant human epidermal growth factor (rhEGF).

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The human epidermal growth factor (hEGF) plays a major role in the proper function of the cell cycle, in fact, it is responsible to promote its initiation[30, 31]. The rhEGF protein

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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 example is the therapeutic vaccine against lung cancer CimaVax-EGF[20]. In this work, we proposed to obtain chitosan-coated superparamagnetic iron oxide nanoparticles (ChMNs) by means of a simple methodology, such as coprecipitation method,

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for its use as a magnetic nano-adsorbent in the separation of DNA and rhEGF using a

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phosphate binding buffer.

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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%)

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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 superparamagnetic iron oxide nanoparticles

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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

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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

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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 deionized water, and vacuum-dried.

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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

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coating process was concluded, the product was collected with a neodymium magnet, washed with deionized water, and vacuum-dried.

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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 5

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 nanoparticles 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 SPIONs was determined at a DNA concentration of 0.44 mg/mL using the same procedure that was used for the ChMNs.

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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:

𝑚(𝐷𝑁𝐴)𝑑𝑒𝑠𝑜𝑟𝑏𝑒𝑑 ∗ 100 𝑚(𝐷𝑁𝐴)𝑎𝑑𝑠𝑜𝑟𝑏𝑒𝑑

Equation (1)

Equation (2)

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𝑑𝑒𝑠𝑜𝑟𝑏𝑒𝑑 % =

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𝜇𝑔 𝑐(𝐷𝑁𝐴)𝑖𝑛𝑖𝑡𝑖𝑎𝑙 − 𝑐(𝐷𝑁𝐴)𝑟𝑒𝑠𝑖𝑑𝑢𝑎𝑙 𝑐𝑎𝑝𝑎𝑐𝑖𝑡𝑦 ( ) = ∗ 𝑉𝑜𝑙 𝑚𝑔 𝑚(𝑀𝑁𝑠)

2.4. The rhEGF adsorption/desorption experiments

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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

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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

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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 BioSpecnano from Shimadzu Biotech). The supernatant was disposed of and 1 mL of elution buffer

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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 SPIONs was determined at a rhEGF concentration of 1.01 mg/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 6

replicants were made for the adsorption and the desorption process. The rhEGF adsorption capacity 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 diffractometer from Bruker using Cu-Kα (λ=1.54183 Å) as incident radiation. 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

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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 7800F 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.

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2.6. Characterization of the recovered DNA

The DNA obtained after the desorption process was characterized by agarose gel

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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

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UV radiation. The same procedure was followed for the solutions obtained with the three elution buffers.

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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

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desorption process was completed, the obtained solution was concentrated in a 3 kDa DispoBiodialyzer by ultracentrifugation. Later, 20 μL of the solution was injected in the

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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 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 visualized using a peroxidase substrate solution. The same procedure was followed for the solutions obtained with the three elution buffers. One negative control was

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also injected into the gel. This control was prepared by submitting the nanoparticles to the same procedure of rhEGF adsorption and desorption (in this case in water) but without the

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protein.

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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 (Equation 3). Fe2+ (aq) + 2Fe3+ (aq) + 8OH− (aq) = Fe3 O4 (s) + 4H2 O

(Equation 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

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nanoparticles and chitosan-coated magnetic nanoparticles are shown in Figure 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

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pattern of magnetite (JCPDS 19629)).

Figure 1. XRD patterns of the uncoated SPIONs and chitosan-coated SPIONs

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The broad nature of the peaks in the XRDP suggests that the obtained nanoparticles have a nanometric size. The average crystallite size was determined by the Debye-Scherrer equation

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(Equation 4):

𝐷=

𝑘𝜆 𝛽𝑐𝑜𝑠𝜃

Equation (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.

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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 SPIONs (8.3679 Å ChMNs and 8.3734 Å SPIONs), according to T.M. Freire et al.,[34], this contraction is due to the 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 Figure 2a-b. Micrographs obtained by SEM confirmed that both nanoparticles samples present a spherical morphology and nanometric size, with diameters of 16 ± 4 nm for uncoated magnetic

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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

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obtained nanoparticles are single crystals.

Figure 2. SEM micrograph of (a) uncoated SPIONs and (b) chitosan-coated SPIONs with their corresponding nanoparticles diameter histograms 3.1.3. Fourier transform infrared spectroscopy Through FT-IR spectroscopy it was possible to confirm that the coating with chitosan was effective. Figure 3 shows the FT-IR spectra of chitosan, uncoated magnetic nanoparticles and chitosan-coated magnetic 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 10

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 attributed 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 nanoparticles. The first sample contains half of the chitosan used in the main synthesis, the second the fivefold and the third ten-fold. As it can be appreciated in Figure S1 a) in the supporting information the intensity of the main bands increases with the increase in the chitosan content.

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The same trend is observed in the band's area for the four main signals ( Figure S1 b) confirming that the coating process was effective.

On the other hand, several similar bands in the spectrums of uncoated SPIONs and ChMNs appear. A broad band in the range of 3000-3650 cm-1 is assigned to water present on

nanoparticles surface, in the case of uncoated magnetic nanoparticles, and to stretching

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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 SPIONs and which

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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

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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

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synthesis has a black color, it is possible to conclude that there is a mixture of magnetite and maghemite present in both cases, as usually happens when magnetic nanoparticles are

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obtained by the coprecipitation method[35-37].

Figure 3. FT-IR spectra of chitosan, uncoated SPIONs and chitosan-coated SPIONs 11

3.1.4. Thermogravimetric analysis Thermograms of uncoated SPIONs and ChMNs are presented in Figure 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 ºC, which is due to the loss of water adsorbed on the material. The second event occurs between 200-315 ºC and is related to the oxidation of the amino and hydroxyl groups of the polymer. The third event can be seen between 315-500 ºC and is related to the deacetylation of the acid groups formed previously by the oxidation of the

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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 ºC due to the decomposition of the polymer chain. A replicant of the TGA was conducted and the result was alike (Figure S2).

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For uncoated SPIONs, an average weight loss of 5.14% was determined between 30 and 900 ºC, which is due to the loss of free and chemisorbed water on the surface of the nanoparticles.

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On the other hand, in the case of the chitosan-coated nanoparticles, the weight loss was

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16.38%, which is equivalent to a real coating of the chitosan layer of 11.24%.

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Figure 4. TGA curves of the uncoated SPIONs and chitosan-coated SPIONs 3.2. DNA adsorption capacity and desorption ratio Figure 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 a summary of recently reported adsorption capacity for different materials and a commercial kit 12

used for DNA separation. The reported values were between 61.88 mg/g and 385 mg/g in that sense, the adsorption capacity obtained in this research falls in that range. Furthermore, 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 components. 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 isotherms published in reported works, among which the best known are the Langmuir (Equation 5) and Freundlich (Equation 6) models. On the other hand, two of the most used three parameters isotherms are the

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Langmuir-Freundlich (Equation 7) and the Redlich-Peterson (Equation 8) [39], which are models that combine both, Langmuir and Freundlich, adsorption behaviors. 𝑄𝑚𝑎𝑥 ∗ 𝐾𝐿 ∗ 𝐶𝑒𝑞 1 + 𝐾𝐿 ∗ 𝐶𝑒𝑞

𝑄=

1⁄ 𝑛

Equation (6)

𝑄𝑚𝑎𝑥 ∗ 𝐾𝐿−𝐹 ∗ 𝐶𝑒𝑞 1 + 𝐾𝐿−𝐹 ∗ 𝐶𝑒𝑞 𝑄=

1⁄ 𝑛

1⁄ 𝑛

𝐾𝑅 ∗ 𝐶𝑒𝑞 1 + 𝐴 ∗ 𝐶𝑒𝑞 𝛽

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𝑄 = 𝐾𝐹 ∗ 𝐶𝑒𝑞

Equation (5)

Equation (7)

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𝑄=

Equation (8)

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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

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Redlich-Peterson exponent, and n is the Freundlich's exponent. The adjustments of the four models and the calculated parameters are presented in Figure 5

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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 Langmuir-Freundlich model (97 mg/g) is almost the same that the experimental value (98

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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.

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Figure 5. DNA adsorption isotherm with Langmuir, Freundlich, Langmuir-Freundlich and

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Redlich-Peterson models fitting.

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

Isotherms

K (L/g)1/n

A((L/g)β)

n

R2

β

Langmuir

108

18.73

-

-

0.75

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Freundlich

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0.11

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0.17

0.50

-

Langmuir-Freundlich

97

1 049.55

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2.56

0.90

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Redlich-Peterson

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Experimental

98

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Qmax (mg/g)

0.92

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0.97

1.47

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-

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10.37

The Langmuir isotherm assumes a monolayer model, also assumes that once a molecule

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occupies a site, no further adsorption can take place at that site[41]. These hypotheses correctly describe the experimental system, since the high electrostatic repulsion between the DNA molecules does not allow the formation of multilayers. Moreover, at a certain

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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

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energetically equivalent. 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 Nacetyl-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 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 14

the experimental model. The main difference between Langmuir-Freundlich and RedlichPeterson is that the first reproduce only systems with a high degree of heterogeneity while Redlich-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

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different elution buffers and also with water. The results obtained are shown in Figure 6.

Figure 6. DNA adsorption capacity and elution ratio using three different phosphate buffers

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and water.

The isoelectric point of chitosan is around 6.5, thereby, at pH above this value, the chitosan

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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 increasing the pH in

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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

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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 desorption process. In that sense, Shan et al.[43] conducted a

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study for amino-modified silica-coated magnetic nanoparticles where they demonstrated 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 research is that only 10 min is required for desorption. It would be interesting to study the effect of buffer phosphate concentration at 10 min of desorption time.

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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 (Figure 7). Lane 1 corresponds to the injected DNA control solution without modification. From lane 2 to 4 were injected the solution of DNA obtained after the desorption procedure with the three different elution buffers. Lane 2 corresponds to phosphate buffer pH 9, lane 3 pH 8 and lane 4 to pH 7. In the figure, it is possible to observe three bands corresponding to the eluted DNA, which are at the same level as the band that contains the

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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

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of an excess of DNA or also to a high amount of salts in the solutions. Considering the result obtained it can be affirmed that the structural integrity of DNA was not affected by the

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adsorption/desorption process indicating that the eluted DNA can be used for future

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applications.

Figure 7. Agarose gel image of desorbed DNA using three elution buffers. Lane 1: DNA control solution, Lane 2, 3 and 4: desorbed DNA with the elution buffers pH 9, 8 and 7 respectively 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 obtained for each sample were plotted versus 16

the rhEGF equilibrium concentration (final concentration) and the adjustments of the four isotherms (Figure 8) previously used in DNA adsorption were done.

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Figure 8. The rhEGF adsorption isotherm with Langmuir, Freundlich, Langmuir-Freundlich and Redlich-Peterson models fitting

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

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the highest correlation factors were obtained for Redlich-Peterson (R2=0.96) and Langmuir-

Freundlich (R2=0.99). Furthermore, for Langmuir-Freundlich isotherm, the Qmax calculated

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(441 mg/g) closely resembles the experimental values (440 mg/g) and the R2 is higher. Therefore, it is considered that the adjustment that best reproduces the results obtained is the

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Langmuir-Freundlich isotherm. The KL-F value is considered an indication 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

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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

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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

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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 LangmuirFreundlich isotherm.

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Table 2. Parameters of the adsorption isotherms for rhEGF adsorption capacity Parameters

Isotherms

K (L/g)1/n

A((L/g)β)

n

R2

β

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

1.60

Experimental

440

-

-

-

-

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Qmax (mg/g)

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 (Figure 9).

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This result can be explained if the driven forces that produce the releasing of rhEGF from the ChMNs are considered in each buffer. For example, in the case of Tris-HCl buffer, the pH is

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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

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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

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surface. The objective of the ionic strength buffer is to shield 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

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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

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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.

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Figure 9. EGF adsorption capacity and elution ratio using three different buffers and water

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The adsorption capacity of the SPIONS and ChMNs for DNA and rhEGF can be observed in Table 3.

Table 3. Enhancement of the adsorption capacity of the SPIONs after chitosan coating Adsorption capacity (mg/g) DNA

rhEGF

SPIONs

11±1

ChMNS

97±4

Increment of Adsorption capacity

882 %

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Adsorption System

62±6

440 ±5

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772%

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One of the main objectives of this work is to enhance the adsorption capacity of the SPIONs using a chitosan coating. The previous table shows the notable increment in the adsorption capacity of the ChMNs compared with SPIONs, either for DNA and for rhEGF adsorption.

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Therefore, we can affirm that through the chitosan coating of the nanoparticles the ability of them to interact biomolecules is considerably increased.

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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

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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 (Figure 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 19

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

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adsorption/desorption process.

Figure 10. Scanning photos of a) polyacrylamide gel electrophoresis of desorbed rhEGF;

Lane 1: molecular weight standard, Lane 2: rhEGF protein control solution, Lane 3 negative control solution Lane 4, 5 and 6: desorbed protein with the elution buffers Tris-HCl, NaCl and

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KCl-HCl, respectively. b) Western Blot of desorbed rhEGF; ; Lane 1: molecular weight

standard, Lane 2: rhEGF protein control solution, Lane 3 negative control solution Lane 4, 5

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and 6: desorbed protein with the elution buffers Tris-HCl, NaCl and KCl-HCl, respectively Figure 10b is an image of the nitrocellulose membrane of Western blot. Lane 1 contains the

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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 Western blot indicates

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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

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adsorption/desorption process does not affect the identity of rhEGF and presumably, the

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biological activity is preserved.

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4. Conclusions Through a simple synthesis methodology, a magnetic nano-adsorbent of chitosan-coated superparamagnetic iron oxide nanoparticles was obtained. A maximum DNA adsorption capacity of 98 mg/g was achieved using an inexpensive phosphate buffer for 2 hours of adsorption time. An elution ratio of 46% in a phosphate buffer at pH=9.0 was achieved within 10 minutes of desorption time. The best adjustment of the experimental data was achieved through 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

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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

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that the biological activity after the adsorption/desorption experiments was preserved.

5. Conflict of interest

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Authors declare that there is not a conflict of interest regarding the publication of this article 6. Acknowledgments

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realization of the present work.

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The authors acknowledge the experimental support provided by the CNMN-IPN in the

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6. References

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Legend of Figures and Tables

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Figure 1. XRD patterns of the uncoated SPIONs and chitosan-coated SPIONs Figure 2. SEM micrograph of (a) uncoated SPIONs and (b) chitosan-coated SPIONs with their corresponding nanoparticles diameter histograms

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Figure 3. FT-IR spectra of chitosan, uncoated SPIONs and chitosan-coated SPIONs Figure 4. TGA curves of the uncoated SPIONs and chitosan-coated SPIONs Figure 5. DNA adsorption isotherm with Langmuir, Freundlich, Langmuir-Freundlich and

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Redlich-Peterson models fitting.

Figure 6. DNA adsorption capacity and elution ratio using three different phosphate buffers

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and water.

Figure 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 Figure 8. The rhEGF adsorption isotherm with Langmuir, Freundlich, Langmuir-Freundlich and Redlich-Peterson models fitting Figure 9. EGF adsorption capacity and elution ratio using three different buffers and water

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Figure 10. Scanning photos of a) polyacrylamide gel electrophoresis of desorbed rhEGF; Lane 1: molecular weight standard, Lane 2: rhEGF protein control solution, Lane 3 negative control solution Lane 4, 5 and 6: desorbed protein with the elution buffers Tris-HCl, NaCl and KCl-HCl, respectively. b) Western Blot of desorbed rhEGF; ; Lane 1: molecular weight standard, Lane 2: rhEGF protein control solution, Lane 3 negative control solution Lane 4, 5 and 6: desorbed protein with the elution buffers Tris-HCl, NaCl and KCl-HCl, respectively Table 1. Parameters of the adsorption isotherms for DNA adsorption capacity Table 2. Parameters of the adsorption isotherms for rhEGF adsorption capacity

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Table 3. Enhancement of the adsorption capacity of the SPIONs after chitosan coating

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