Chitosan-based hydrogel for magnetic particle coating

Journal Pre-proof Chitosan-based hydrogel for magnetic particle coating

Vanessa A. de Pereira, Irisvan S. Ribeiro, Haroldo C.B. Paula, Regina C.M. de Paula, Rubem Luis Sommer, Ruben Jesus Sanchez Rodriguez, Flavia O.M.S. Abreu PII:

S1381-5148(19)31020-X

DOI:

https://doi.org/10.1016/j.reactfunctpolym.2019.104431

Reference:

REACT 104431

To appear in:

Reactive and Functional Polymers

Received date:

27 September 2019

Revised date:

13 November 2019

Accepted date:

15 November 2019

Please cite this article as: V.A. de Pereira, I.S. Ribeiro, H.C.B. Paula, et al., Chitosanbased hydrogel for magnetic particle coating, Reactive and Functional Polymers (2018), https://doi.org/10.1016/j.reactfunctpolym.2019.104431

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© 2018 Published by Elsevier.

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Chitosan-based hydrogel for magnetic particle coating Vanessa de A. Pereiraa, Irisvan S. Ribeirob , Haroldo C. B. Paulaa, , Regina C. M. de Paulab*, Rubem Luis Sommerc, Ruben Jesus Sanchez Rodriguezd , Flavia O.M.S. Abreue a

Post-Graduation in Chemistry Program, Building 940, Federal University of Ceará,

Fortaleza, 60455-760, Ceará, Brazil b

Department of Organic and Inorganic Chemistry, Federal University of Ceará, UFC,

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c

f

Fortaleza-CE, Brazil,

Brazilian Center for Physical Research- Centro Brasileiro de Pesquisas Físicas, Rio de

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Janeiro - RJ - Brazil d

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Estate University of North Fluminense-, Universidade Estadual do Norte Fluminense,

e

Pr

Rio de Janeiro –RJ- Brazil

Analytical and Environmental Chemistry Laboratory, Post-Graduation in Natural

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Science Program, Estate University of Ceará, Fortaleza-CE, Brazil.

ABSTRACT Magnetic

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Regina C. M. de Paula

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* Corresponding author: [email protected]

particles

coated

with

chitosan-based

hydrogel

were

prepared

and

characterized by infrared spectroscopy, thermal analysis, atomic force microscopy (AFM), energy dispersive X-ray (EDX), wide angle X-ray diffraction (WAXD) and by vibrating sample magnetometry. A polyelectrolyte complex of chitosan (CH) and Sterculia striata gum (CHG) was employed as a coating. FT-IR analysis of the hydrogel obtained revealed the presence of main characteristic bands of CH, CHG and magnetite (Fe3 O 4 ). The thermograms showed a moderate efficiency of magnetite incorporation (26.0 %) in the hydrogel, as well as evidence of interactions between functional groups

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of magnetite and biopolymers. Patterns of X-ray diffraction showed the presence of magnetite in the hydrogel with saturation magnetization of 36.5 emu g-1 and crystal average size of 20.8 nm. The hydrogel was found to present a negatively charged outer surface. EDX and AFM revealed that Fe3 O4 nanoparticles were aggregated, forming large clusters. The CH /CHG hydrogel was shown to be suitable for magnetite coating, enabling its future application such as a drug carrier and a water remediation agent.

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Keywords: chitosan; hydrogel; Sterculia striata gum; magnetic particle 1. Introduction

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Magnetic particles have broad applications in many areas such as in materials

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science, chemistry, medicine, and environment. In recent years, such particles have attracted much attention due to their biological applications such as hyperthermia in

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tumors [1,2] as carriers guided by a magnetic field [3-5], in diagnostic imaging, in form of contrasts in nuclear magnetic resonance [6,7] and in bovine serum albumin

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separation processes [8]. These aforesaid applications have major advantages over conventional techniques such as its non-invasive character and non-use of radiation

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during analysis. In industrial biotechnology, magnetic particles have been used for

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enzyme immobilization such as galactosidase and phospholipase [9,10], and in biocatalysis and biosensors [11-13]. Aiming at a likely application, magnetic particles must be coated with materials that allow their solubilization in a given medium (usually water), forming so called ferrofluids. Natural polymers such as chitosan, alginate, carboxymethylated dextran, pullulan, cellulose derivative, and cyclodextrin have been used for ferrofluid coatings due to the fact that they have desirable characteristics such as biodegradability and biocompatibility [14-17]. Coating occurs through the formation of micro-, nanoparticles or hydrogels which usually behave like conventional fluids [18] despite the fact that they contain magnetic particles in their core. Coating has been carried

out

[3,16,27,28],

employing

alginate

[15,19-23],

chitosan

[8,9,11,24-26],

dextran

carboxymethylcellulose [29], cyclodextrin [17] and polyacrylic acid

[30,31]. Particle size, magnetic properties, surface charge, nature of the coating material and stability in aqueous medium are usually reported as parameters / characteristics of obtained products which need optimization.

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Some of these substances exhibit high contents of carboxylic groups and charge density and therefore have been reported [32,33] as being suitable for coupling to magnetite molecules for use in magnetic imaging, since for this application it is desirable that the polymer coating possesses negative charges, which would enable magnetic stability against aggregation, in addition to the improving resolution of contrasts in magnetic resonance imaging. Belonging to the same family of karaya gum (Sterculia urens), the Sterculia striata gum (CHG), commonly named “chichá”, which is extracted from the Sterculia

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striata trees, is a polyanion [34] possessing main chains containing high content of

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uronic acid (40%), besides galactose (20 %), rhamnose (25 %), xylose (5 %), and acetyl groups (10 %). Moreover, its structure and physicochemical properties are similar to

likely

applications

such

as

microfluids

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those of commercial karaya gum. CHG gelling and swelling capabilities points out to devices

[35].

Dilute solutions of this

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polysaccharide yield viscous gels which have been used for adsorption of enzymes on

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silicon wafers [36]. CHG forms a polyelectrolyte complex with chitosan which enables drug encapsulation such as chloroquine [37]. The Sterculia striata tree is abundant in Brazil, particularly in “cerrado” of Northeastern region.

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Similar regional gums such as the cashew gum (Anacardium occcidentale) and

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the angico gum (Anadenanthera macrocarpa) have been employed for the formation of hydrogels with chitosan, resulting in polyelectrolyte complexes [38-41] with large

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potential application in pharmacy, medicine and the environment. Due to the abovementioned CHG properties, i. e., high content of carboxylic groups and the ability to form gels, along with the fact that CHG has a large application potential, this work aimed at the preparation of chitosan (CH) and chichá gum (CHG) hydrogels containing magnetite and their characterization by infrared spectroscopy (FT-IR), AFM, EDX, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), vibrating sample magnetometry (VSM) and WAXD. To the best of authors’ knowledge, this is the first time that CHG has been used for magnetite stabilization.

2. Experimental 2.1. Materials Chitosan was duly obtained from a local company (82 % deacetylation degree, Mv = 1.8 x105 g mol-1 ), while the chichá gum (molar mass Mw = 4.18 x 106 g mol-1 ),

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was extracted from Sterculia striata trees in Ceará, Brazil and isolated as previously reported [34]. Ferric chloride FeCl3 and ferrous sulphate FeSO 4 (Dinamica) and sodium hydroxide (Vetec) were used as they were being received. 2.2. Hydrogel preparation Magnetic nanoparticles were synthesized by the modified method of ferrous and ferric ions co-precipitation via alkaline hydrolysis [11,15,21,42] employing chitosan and chichá gum as coating materials, with ratios of magnetite (Mag) to the polymeric matrix CHCHG (Mag: CHCHG) of 1:1. The CH: CHG ratio was 4:1 (w/w),

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corresponding to a charge density of n+/n- = 10 (protonated amine groups/carboxylate

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groups). This said charge ratio has been employed for polyelectrolyte synthesis, yielding complexes that have small particle size and better pH stability [38-40].

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A 0.4 % CH solution was prepared by dissolving 0.4 g of chitosan in 100 mL of 1 % acetic acid. Hence a 0.4 % (m/v) CHG solution was added dropwise, leaving the clear stirring for an additional 30

minutes.

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mixture

After completion of CH/CHG

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polyelectrolyte complex formation, to this solution was added 25 mL of solutions of FeCl3 .6H2 O and FeSO 4 .4 H2 O, at concentrations of 4.64 and 2.4 %, respectively and the mixture left stirring for 15 minutes. The magnetite was duly obtained by adding a 25

precipitate.

The

aforementioned

system was

maintained

under nitrogen

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brown

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% (w/v) solution of sodium hydroxide until pH 10 and observing the formation of a

atmosphere and a magnetic separation was then carried out by using a magnet, followed

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by an extensive washing of the hydrogel with distilled water until a neutral pH. The hydrogel obtained was then dialyzed against distilled water through a Millipore membrane (pore size 14 kDa) and thereafter freeze dried. A sample of pure magnetite was prepared according to the above described procedure, however, without the addition of biopolymers. 2.3. Physicochemical characterization The infrared spectra of CH, CHG, CH/CHG blank and hydrogel samples were obtained using KBr pellets with the samples scanned in the region between 400 and 4000

cm-1

in

the

FT-IR

unit

Shimadzu

spectrophotometer

(Model 8300).

The thermal properties of the CH, CHG and CH/CHG hydrogel were evaluated by thermogravimetric analysis (TGA) using a Shimadzu equipment (Japan), model TGA50, under nitrogen atmosphere, using a heating rate of 10 °C min-1 , and the temperature range from 25 to 800 °C, and by Differential Scanning Calorimetry (DSC) through a

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Shimadzu Calorimeter, Model DSC-50 (Japan) with a heating rate of 10 °C min-1 , from 25 °C to 500 °C. Wide angle X-ray diffraction (WAXD) measurements were made on a PANalytical X’Pert Pro MPD instrument, where powder forms of the CH, CHG and CH/CHG hydrogel samples were exposed to Cu radiation, with increments of 1°/min and scanned over a 2Ɵ range of 3° to 40°. The hydrogel magnetization at room temperature was measured using a vibrating sample magnetometer (VSM) OXFORD, Model 3001, operating at 1.6 Tesla, using the

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external field of 2 kOe.

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Aiming at determination of the hydrogel surface charge, zeta potential samples was analyzed using a Malvern Zetasizer Nano, Model ZS 3600. Samples were placed in

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distilled water and sonicated for 2 minutes prior to being analyzed. The hydrodynamic diameter was measured by dynamic light scattering with laser wavelength of 633 nm

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and a fixed scattering angle of 173°. Particle size was measured considering the particle

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as spherical like. Each sample was measured 3 times (n=3). The CH/CHG hydrogel sample was analyzed by EDX in a Bruker AXS

3. Results and Discussion

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3.1. FTIR

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Microanalysis instrument, GmbH, Germany.

Fig. 1 shows the FTI-IR spectra of Mag, CH, CHG and CH/CHG samples. In the

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spectrum of pure magnetite, Fe3 O4 (Mag), it can be observed the main absorption bands at 584 cm-1 , attributed to vibrations of the Fe-O bonds and vibrations of free or adsorbed water at 1627 and 3404 cm-1 . These stated bands are in good agreement with data duly reported in literature for dextran [16] coated Fe3 O4 ; chitosan coated magnetic particle [41] and magnetite stabilized using oleic acid and Tween 80 surfactant [42]. Chitosan was found to present its major bands at 1633 cm-1 (amide I), 1540 cm-1 (amide II), 1070 cm-1 (C-O-C group), as well as at 1735 cm-1 due to carbonyl of acetyl groups, in full agreement with literature data [39,43]. Higher wavenumber values for amide II band (at 1594 cm-1 ) and for C-O-C group (at 1089 cm-1 ) were reported by Jaiswal et al. [44] No acetyl bands at 1735 cm-1 was observed, likely due to the fact that chitosan

used

in

their

work

had

a

higher

deacetylation

degree

(95%).

CHG shows strong bands at 1724 cm-1 due to carbonyl of uronic acids and acetyls groups, while the carboxylate groups arise at 1651 and 1436 cm-1 . Hydroxyl groups

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appear at 3431 cm-1 and C-H groups at 2922 cm-1 . C-O-C anomeric carbon bonds are present at 1145 cm-1 . Similar data was previously reported by Brito et al. [34] whereby these authors have been employed CHG from same said region and it is therefore likely to exhibit similar composition and properties. However, it may be noticed that by the formation of chitosan/chicha gum polyelectrolyte complex (bare CH/CHG hydrogel), occurs the disappearance of band at 1724 cm-1 and formation of a new band at 1633 cm-1 , while CH amide II band shifts to 1595 cm-1 . This feature has also been reported for a chitosan/ cashew gum

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polyelectrolyte complex, which was shown [38] to exhibit chemical stability at pH

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range 2-10.

In the absorption spectrum of Mag: CHCHG 1:1 hydrogel, a broad and strong

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band is observed at 3431 cm-1 and attributed to bonds stretches of -OH and -NH groups. This band enlargement and the relative increase in relation to spectrum of bare CHCHG

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is likely to due to Fe complex formation with CH. At 1633 cm-1 appears chitosan amide

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I band and amide II [25,44] is overlapped with carboxylate form of CHG uronic acids, resulting in a broad band at 1375 cm-1 , pointing out to likely interactions of Fe, CH and CHG. The absorption band at 1069 cm-1 is attributed to the asymmetrical stretching of

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the C-O-C linkages present in both polysaccharides, whereas the vibration at 1729 cm-1

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is related to the stretching of the carbonyl groups of the uronic acid and acetyl groups of CHG [34]. It worth notice that CH band at 1540 cm-1 disappears, pointing out to the fact

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the protonated chitosan amino group has been involved in complex formation with magnetite. Moreover, it was noticed a shift to 600 cm-1 in the Mag absorption band at 584 cm-1 , characteristic of the vibration of Fe-O bonds. Similar data was obtained for carboxymethylated cellulose and pullulan [16], crosslinked chitosan [43] and dextran, whereby it is shown that the polysaccharide was successfully coated on the surface of iron oxide particles through Van der Waals forces, hydrogen bond and electrostatic interactions [16]. Fig. 1. 3.2. Thermal Analysis Fig. 2 show thermal data of CH, CHG, Mag, blank and Mag: CHCHG 1:1 hydrogel samples. The sample Mag presents only one event due to moisture evaporation (8.7%) in the temperature range investigated, in full agreement with reported data for magnetite [9,42,45]. The decomposition of the polysaccharide structures (CH and

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CHG), was found to happen in an event around 290 °C and another above 400 °C for CH, and in two major events in the range of 270 -473 °C, for CHG, in good agreement with previously reported data [34]. A blank sample (CH/CHG hydrogel without Fe3 O4 ) exhibits two

main events,

namely water evaporation (12.8

%) and

polymer

decompositions (64.0 %) at 275 °C, the later being a clear evidence of CH and CHG complex formation, otherwise two events would have being observed, due to parent molecules degradations. Mag: CHCHG 1:1 sample exhibits a different decomposition pattern, most likely owing to magnetite-biopolymers interactions. The existence of three

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major thermal events for Mag: CHCHG 1:1 sample can be observed. The first event

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occurred at temperature range from 25.0 to 80.4 °C with a weight loss of 10.8 % and can be attributed to the elimination of adsorbed and bound water molecules. The second

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event happens in the range of 256.4 - 347.3 °C with a mass loss of 21.8 % corresponding to major biopolymers degradations and the third event is in the range

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621-667 °C, due to carbonization, with mass loss of 18.5 %. The complete

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decomposition of the Mag: CHCHG 1:1 sample leads to a residue of 48.8%. Taking into consideration that blank the sample leaves a residue of 23.0 % at 700 °C, it can be inferred that magnetite content in Mag: CHCHG 1:1 sample amount to 26.0 %.

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Dextran coated magnetic particles [3] in a 1:1 ratio to magnetite with a coating

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efficiency of 45.1%, exhibited two thermal events, namely, water evaporation and polymer decomposition, the latter beginning at 300 °C. It can be seen that magnetic by biopolymers such as dextran [16,30], chitosan [9,17,25],

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particles coated

carboxymethyl starch [46], alginate [15,19,21] and CHG exhibit similar decomposition patterns, although their complex formations and decomposition temperatures may be different. Hence, it can be inferred that Mag: CHCHG 1:1 sample seems to be more stable than dextran magnetic particle, although the later has a higher Fe 3 O 4 content. This is likely to be due to the fact that CHG has more carboxylate groups (from uronic acids) available for magnetite stabilization. Fig. 2. Data of DSC analysis, shown in Fig. 3 presents the endothermic (melting) and exothermic (decomposition) transitions for CH, CHG, Mag: CHCHG 1:1, blank CH/CHG and pure magnetite, Mag, where the endothermic events are related to the evaporation of residual water and the exothermic decompositions are due to the biopolymer degradations. CH decomposes in two exothermic stages (probably due to

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the rupture of its acetylated and deacetylated polymeric units) at higher temperatures (287 and 460 °C) than those reported by Singh et al. [46] CH thermal properties have been found to depend on its molar mass and degree of acetylation: high molar mass and degree of acetylation lead to a more stable biopolymer, in good agreement with data shown herewith. CHG also decomposes in two events, however at lower temperatures (274 and 418 °C), in good agreement with the trend exhibited by other biopolymers such as cashew gum [38], whose decomposition temperatures were at 248 °C and 309 °C. CHCHG blank sample presents an endothermic transition at 175.0 °C due to bound

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water elimination and a single major exothermic peak at 284.8 °C due to CH/CHG

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complex formation, providing further evidence of biopolymer interactions. It is worth notice that CH presented higher melting temperature than CHG, indicating that water is

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more tightly bound to CH molecule than to CHG, likely because the latter is a more hydrophilic polymer and water is released easier. Mag sample exhibits a single

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endothermic peak at 93 °C due to residual water elimination. The sample Mag: CHCHG

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1:1 shows decomposition pattern similar to that of CHCHG blank sample, presenting endothermic transitions at 171 °C (major) and 460 °C (minor), being the latter attributed to Mag: matrix complex come apart, providing evidence of magnetite linking to

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CHCHG 1:1 hydrogel. It is worth notice that neither the parent polymers nor magnetite

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exhibit such endothermic transition. The interactions of water with species that makes up the matrix are responsible for water retention capacity, causing the displacement of

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the transition peaks. This feature was also reported by Ghaemy and Naseri [8] for chitosan crosslinked nanohydrogels. Fig. 3.

3.3. WAXD and VSM

Fig. 4 shows the X-ray pattern of CH/CHG hydrogel coated magnetite. It can be seen that the Fe3 O4 sample shows major peaks characteristic of magnetite diffraction located in 2θ= 35.3, 41.5 , 50.5 , 59.7 , 67.4 and 74.5

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corresponding to crystal planes

(220), (311), (400), (511), (440) and (533) respectively. After coating with the hydrogel, these peaks become broad and decrease in intensity. This seems to indicate that the Fe3 O4 coating did not lead to a phase change. These data are in good agreement with figures reported for dextran [3], alginate [15], chitosan/gelatin complexes [47], chitosanstarch derivative nanocomposites [45], and carboxymethyl cellulose [29], which exhibit similar diffraction peaks and crystal planes, indicating that the cubic spinel structure of

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Fe3 O4 is present in the obtained samples. The peak at the 2 theta = 74.5

o

was also

observed by Kim et al. [48] and Ma et al. [15]. Fig. 4. CH/CHG hydrogel exhibits significant shifts in the signals at 2θ in comparison to bare Fe3 O 4 probably due to interactions between magnetite groups and biopolymers CH and CHG. Chitosan and CHG diffractograms show low 2 θ values in the range 15-25° (data not shown), which were attributed to the crystalline regions formed by hydrogen bonds of amino and hydroxyl groups, being typical of amorphous polymers. The

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average size of the crystals was calculated using Sherrer's equation [3,30] and the peak

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width at (311), resulting in a value of 20.8 nm. The curve of the saturation magnetization is shown in Fig. 5, where it can be seen that the hydrogel coated sample

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has a value of 36.5 emu g-1 , which is indicative of typical superparamagnetic behavior. This low value was expected, since the coverage of magnetite with polymers results in a

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decrease of its saturation magnetization. Bare magnetite (Mag sample) presented Ms =

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76.0 emu g-1 (calculated from thermogravimetric date). Tudorachi and Chiriac [45] reported a low value for Ms (3.04 emu g-1 ) for a matrix based on carboxymethyl starchg-polylactic acid, using a in situ magnetite synthesis. Dextran coated magnetic

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nanoparticles presented saturation magnetization of 54.8 emu g-1 , for a 45.1% magnetite

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content [3], while Fe3 O4 coated with carboxymethyl cellulose [29] exhibit Ms as low as 13.9 emu g-1 , alginate embedded magnetite exhibited similar value [19] and chitosan

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coating [49] resulted in Ms = 25.2 emu g-1 , for a magnetite content of 48.3 %. In summary, Ms was revealed to be dependent on magnetite content as well as on the nature of polymer coating; comparing to CH coatings reported by Kalkan et al. [49] and nanocomposites reported by Tudorachi and Chiriac [45], Mag CHCHG 1:1 sample yielded higher Ms value, at a lower magnetite content. Fig. 5. 3.4. AFM and EDX Mag: CHCHG 1:1 sample was analyzed by AFM. It is observed in Fig. 6 (a, b, c and d) that the magnetic particles seem to have spheroidal morphologies, whereby agglomerated areas are predominating. These particles have a profile of bimodal average particle size, with an average size of 65.0 ± 5.0 nm for smaller particles and 100.0 ± 5.0 nm for larger particles. Since crystal sizes are much smaller than that data, this seems to point out to fact that magnetite is located within aggregated particles.

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These results are in good agreement with the literature, whereas Ma et al. [15] reported similar data for alginate and Wotschadlo et al. [16] presented particles having particles size of 229

nm, for carboxymethylated dextran. Nanostructured polysaccharide

complexes of chitosan and arabic gum were also investigated by Coelho et al. [50] where the nanoparticles of arabic gum/chitosan with a 1:1 ratio presented sizes from 200 to 250 nm. Fig. 6. The EDX spectrum presents peaks (Fig. 7) corresponding to the elements making

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up the true composition of the sample being analyzed. For Mag: CHCHG 1:1 sample,

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the Fe content was found to be 23.5 %, very close to the value obtained from TGA. Moreover, EDX mapping revealed that magnetite is uniformly distributed on hydrogel

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surface, nevertheless, presenting aggregation regions. Fig. 7.

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Aiming at clarifying the structure of the complex formed, samples with different

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ratios of magnetite and matrix Mag: CHCHG = 2:1 and 1:2 were prepared and analyzed in relation to their zeta potentials (Fig. 8). It was observed that the zeta potential of the blank sample (no Fe3 O4 ) is + 36.7 mV, while the samples Mag: CHCHG with ratios 1:1,

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1:2 and 2:1 showed zeta potential of -9.20 mV, -4.75 mV and - 17.5 mV, respectively.

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As it can be seen, increasing the Fe3 O4 insertion in the hydrogel resulted in a more negative zeta potential, which means that the positive charges of the bare hydrogel,

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assigned to protonated amino groups (in excess, at a charge ratio of n+/n- = 10) were neutralized by those negative ones of Mag (zeta potential -11.2 mV), therefore leaving the excess of negative charges on hydrogel surface. Fig. 8.

As the medium is aqueous, the tightly packed Fe3 O 4 molecules are not soluble; hence they ought to be coated with the hydrogel hydrophilic matrix, which is achieved by a molecular rearrangement where CHG carboxylate groups are likely to be located at the hydrogel surface, as it can be seen in Scheme 1. This feature was also observed by Hajdu et al. [51], whereby citric acid and humic acid were found to be adsorbed on Fe– OH surface sites. Furthermore, the decrease in saturation magnetization of coated magnetite corroborated the aforementioned assumption. As reported by Tombácz et al. [52] the amphoteric magnetite character allows for the fact that it is positively charged at pH lower than 7.9, bearing negative charges above that particular value. The authors

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have also pointed out that macromolecular organic acids would provide better particle stability to the said system. In this sense, uronic acids present in the polyelectrolyte CHCHG

complex

seems

to

contribute

for

magnetite

stabilization,

under

the

experimental conditions of this study. Cellulose derivative, carboxymethylated dextran and pullulan covering magnetite [16] showed negative zeta potentials (from -35 mV to 54 mV), while pure magnetite showed zeta potential of +44 mV, at pH 4. Huang and Tang [53] reported negative potentials for polystyrene coated magnetite at the whole pH range, thus providing clear evidence that in the event the polymer is neutral, all negative

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charges in aqueous medium are likely due to magnetite Fe-O atoms. A matrix based on

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carboxymethyl starch-g-polylactic acid was also reported to be acting as a shell for magnetite incorporation [45]. The negative charges at outer surface of Mag CHCHG 1:1

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sample points out to its potential application such as the removal of hazardous metal cations, i.e., Cd2+, Cu2+, Pb2+ and Hg2+. These stated metals have been adsorbed on gum

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kondagogu containing magnetite [54], an Indian polysaccharide whose structure is

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similar to the CHG. Although no zeta potential analysis has been reported, the gum was found to exhibit good adsorption capability. On the other hand, coating Fe3 O4 with a polycation and a polyanion allows the coupling of drugs to the magnetic particle and its

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further usage in drug delivery, as reported for dextran/chitosan magnetic particles

4. Conclusion

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Scheme 1

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loaded with doxorubicin, an anticancer drug [55].

Magnetic nanoparticles were successfully coated with CH/CHG hydrogel, showing a high magnetite content and a good saturation magnetization, as well as a hysteresis behavior. Particle size was 20.8 nm, however in the form of large clusters, as revealed by AFM and EDX. The presence of the major functional groups of CH, CHG and Fe3 O4 as well as their likely interactions, were evidenced by FT-IR-infrared spectroscopy. Thermal analysis revealed the main events of degradations of the sample constituents, along with indication of Fe-O / CH interactions, which were responsible for the higher than the parent molecules (CH and CHG) thermal stability exhibited by the hydrogel. The pattern of X-ray diffraction of the samples showed characteristic peaks of magnetite, which were broad, due to the coating process. The zeta potential data revealed that hydrogel surface was negatively charged, likely due to uronic acids of

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CHG which ultimately promoted magnetite stabilization. The aforementioned data revealed that the hydrogel obtained is a likely candidate to the usage as a release device for drugs and in water remediation, such as for the heavy metal adsorption.

Acknowledgments

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CNPQ, CAPES and FUNCAP for financial support; Prof. Dr. J. M. J. Sassaki, coordinator for the X-rays Laboratory , Physics Department of the Federal University of Ceará; Institute for Research, Development and Innovation- IPDI, State of Ceará Government; Biomass Technology Laboratory, EMBRAPA –Fortaleza, Rede INOMAT (National Institute for Science, Technology and Innovation on Functional Complex Materials). DECLARATION OF CONFLICT OF INTEREST

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[8] M. Ghaemy, M. Naseri, Synthesis of chitosan networks: Swelling, drug release, and magnetically assisted BSA separation using Fe3 O4 nanoparticles, Carbohydr. Polym. 90 (2012) 1265-1272. [9] C. Pan, B. Hu, W. Li, Y. Sun, H. Ye, X. Zeng, Novel and efficient method for immobilization and stabilization of β-d-galactosidase by covalent attachment onto magnetic Fe3O4–chitosan nanoparticles, J. Mol. Catal. B: Enzym. 61 (2009) 208-215. [10] Y. Qu, L. Sun, X. Li, S. Zhou, Q. Zhang, L. Sun, D. Yu, L. Jiang, B. Tian, Enzymatic degumming of soybean oil with magnetic immobilized phospholipase A2, LWT - Food Sci. Technol. 73 (2016) 290-295. [11] A.F.A.A. Melo, R.A.S. Luz, R.M. Iost, I.L. Nantes, F. N. Crespilho, Highly stable magnetite modified with chitosan, ferrocene and enzyme for application in magnetoswitchable bioelectrocatalysis, J. Braz. Chem. Soc. 24 (2013) 285-294. [12] R.A. Fernandes, A. Luísa, D. Silva, A.P.M. Tavares, A. M.R.B. Xavier, EDTA-Cu (II) chelating magnetic nanoparticles as a support for laccase immobilization, Chemical Eng. Sci. 158 (2017) 599–605. [13] A. Kumar, G. D. Park, S.K.S. Patel, S. Kondaveeti, S. Otari, M.Z. Anwar, V. C. Kalia, Y. Singh, S.C. Kim, B. K. Cho, J.H. Sohn, D.R. Kim, Y.C. Kang, J.K. Lee, SiO 2 microparticles with carbon nanotube-derived mesopores as an efficient support for enzyme immobilization, Chemical Eng. J. 359 (2019) 1252–1264. [14] S. Khoramian, M. Saeidifar, A. Zamanian, A.A. Saboury, Synthesis and characterization of biocompatible ferrofluid based on magnetite nanoparticles and its effect on immunoglobulin G as an immune protein, J. Mol. Liq. 273 (2019) 326-338. [15] H. Ma, X. Qi, W. Maitani, T. Nagai, Preparation and characterization of superparamagnetic iron oxide nanoparticles stabilized by alginate, Int. J. Pharm. 333 (2007) 177-186. [16] J. Wotschadlo, T. Liebert, T. Heinze, K. Wagner, M. Schnabelrauch, S. Dutz, R. Muller, F. Steiniger, M. Schwalbe, T.C. Kroll, K. Hoffken, N. Buske, J. H. Clement, Magnetic nanoparticles coated with carboxymethylated polysaccharide shellsinteraction with human cells, J. Magn. Magn. Mater. 321 (2009) 1469-1473. [17] A. Bocanegra-Diaz, N.D.S. Mohallem, R.D. Sinisterra, Preparation of a ferrofluid using cyclodextrin and magnetite J. Braz. Chem. Soc. 14 (2003) 936-941. [18] J.N. Nygaard, S.P. Strand, K.M. Vårum, K.I. Draget, C.T. Nordgård, Chitosan: Gels and Interfacial Properties, Polymers. 7 (2015) 552-559. [19] M. Srivastava, J. Singh, M. Yashpal, D.K. Gupta, R.K. Mishra, S. Tripathi, A.K. Ojha, Synthesis of superparamagnetic bare Fe 3 O4 nanostructures and core/shell (Fe3 O 4 /alginate) nanocomposites, Carbohydr. Polym, 89 (2012) 821-829. [20] V. Ivanova, P. Petrova, J. Hristov, Application in the ethanol fermentation of immobilized yeast cells in matrix of alginate/magnetic nanoparticles, on chitosanmagnetite microparticles and cellulose-coated magnetic nanoparticles, Int. Rev. Chem. Eng. 3 (2011) 289-299. [21] L.E. Wenger, G.M. Tsoi, P.P. Vaishnava, U. Senaratne, E.C. Buc, R. Naik, V.M. Naik, Magnetic properties ofγ-fe2o3nanoparticles precipitated in alginate hydrogels, IEEE Trans. Magn. 44 (2008) 2760-2763. [22] M.M. Abrougui, M.T. L. Lopez, J.D.G. Duran, Mechanical properties of magnetic gels containing rod-like composite particles, Philos. Trans. R. Soc. A. 337, (2019) 20180218. [23] A. Teleki, F.L. Haufe, A.M. Hirt, S.E. Pratsinis, G. A. Sotiriou, Highly scalable production of uniformly-coated superparamagnetic nanoparticles for triggered drug release from alginate hydrogels, RSC Adv. 6 (2016) 21503–21510.

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[24] G. Lawrie, I. Keen, B. Drew, A. Chandler-Temple, L. Rintoul, P. Fredericks, L. Grondahl, Interactions between Alginate and Chitosan Biopolymers Characterized Using FTIR and XPS, Biomacromolecules. 8 (2007) 2533-2541. [25] H. Nagahama, H. Maeda, T. Kashiki, R. Jayakumar, T. Furuike, H. Tamura, Preparation and characterization of novel chitosan/gelatin membranes using chitosan hydrogel, Carbohydr. Polym. 76 (2009) 255-260. [26] T. Miyazaki, A. Iwanaga, Y. Shirosaki, M. Kawashita, In situ synthesis of magnetic iron oxide nanoparticles in chitosan hydrogels as a reaction field: Effect of cross-linking density, Colloids Surf. B. 179 (2019) 334-339. [27] H. Su, X. Han, L. He, L. Deng, K. Yu, H. Jiang, C. Wu, Q. Jia, S. Shan, Synthesis and characterization of magnetic dextran nanogel doped with iron oxide nanoparticles as magnetic resonance imaging probe, Int. J. Biol. Macroml. 128 (2019) 768–774. [28] M. Eid, A. Mansour, Preparation and magnetic investigation of magnetic nanoparticles entrapped hydrogels and its possible use as radiation shield, J. Inorg. Organomet. Polym. Mater. 23 (2013) 1255–1265. [29] J.F.L. Martınez, E.R. Melo, V.G. Gonzalez, C.G. Salazar, A.T. Castro, S.S. Guzman, Synthesis and characterization of a magnetic hybrid material consisting of iron oxide in a carboxymethyl cellulose matrix, J. Appl. Polym. Sci. 127 (2013) 2325-2331. [30]. R.Y. Hong, T.T. Pan, Y.P. Han, H.Z. Li, J. Ding, S. Han, Magnetic field synthesis of Fe3O4 nanoparticles used as a precursor of ferrofluids, J. Magn. Magn. Mater. 310 (2007) 37-47. [31] A.M. Szekeres, I.Y. Tóth, R.A. Bauer, J. Mihály, I. Zupkó, E. Tombácz, Enhanced stability of polyacrylate-coated magnetite nanoparticles in biorelevant media, Colloids Surf. B. 94 (2012) 242-249. [32] A.J. Hajdu, E. Tombacz, I. Banyai, M. Babos, A. Palko, Carboxylated magnetic nanoparticles as MRI contrast agents: Relaxation measurements at different field strengths, J. Magn. Magn. Mater. 324 (2012) 3173-3180. [33] A. Hajdú, A.M. Szekeres, I.Y. Tóth, R.A. Bauer, J. Mihály, I. Zupkó, E. Tombácz, Enhanced stability of polyacrylate-coated magnetite nanoparticles in biorelevant media, Colloids Surf. B. 94 (2012) 242-249. [34] A.C.F. Brito, D.A. Silva, R.C.M. De Paula, J.P.A. Feitosa, Sterculia striata exudate polysaccharide: characterization, rheological properties and comparison with Sterculia urens (karaya) polysaccharide, Polym. Int. 53 (2004) 1025-1032. [35] A.A. Oladipo, M. Gazi. Hydroxyl-enhancedmagnetic chitosan microbeads for boron adsorption: parameter optimization and selectivity in saline water, React. Funct. Polym. 109 (2016) 23–32. [36] A.F. Dario, R.C.M. De Paula, H.C.B. Paula, J.P.A. Feitosa, D.F.S. Petri, Effect of solvent on the adsorption behavior and on the surface properties of Sterculia striata polysaccharide, Carbohydr. Polym. 81 (2010) 284-290. [37] G.A. Magalhães Jr., E. Moura Neto, V.G. Sombra, A. R. Richter, C.M.W.S. Abreu, J.P.A. Feitosa, H.C.B. Paula, F.M. Goycoolea, R.C.M. De Paula, Chitosan/sterculia striata polysaccharides nanocomplex as a potential chloroquine drug release device, Int. J. Biol. Macromol. 88 (2016) 244–253. [38] H.C.B. Paula, F.J.S. Gomes, R.C.M. De Paula, Swelling studies of chitosan/cashew nut gum physical gels, Carbohydr. Polym. 48 (2002) 313-318. [39] H.C.B. Paula, R.C.M. De Paula, S.K.F. Bezerra, Swelling and release kinetics of larvicide-containing chitosan/cashew gum beads, J. Appl. Polym. Sci. 102 (2006) 395400.

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[40] J.S. Maciel, H.C.B. Paula, M.A.R. Miranda, J M.J. Sassaki, R.C.M. De Paula, Reacetylated Chitosan/Cashew Gum Gel: Preliminary study for potential utilization as drug release matrix, J. Appl. Polym. Sci. 99 (2006) 326-334. [41] M.A. Oliveira, P.C. Ciarlini, J. P. A. Feitosa, R.C.M. De Paula, H.C.B. Paula, Chitosan/“angico” gum nanoparticles: synthesis and characterization, Mater. Sci. Eng. C. 29 (2009) 448-451. [42] Y.M. Wang, X. Cao, G.H. Liu, R.Y. Hong, Y.M. Chen, X.F. Chen, B. Xu, D.G. Wei, Synthesis of Fe3 O4 magnetic fluid used for magnetic resonance imaging and hyperthermia, J. Magn. Magn. Mater. 323 (2011) 2953-2959. [43] H.Y. Zhu, R. Jiang, L. Xiao, W. Li, A novel magnetically separable γFe2O3/crosslinked chitosan adsorbent: Preparation, characterization and adsorption application for removal of hazardous azo dye, J. Hazard. Mater. 179 (2010) 251-257. [44] M.K. Jaiswal, R. Banerjee, P. Pradhan, D. Bahadur, Thermal behavior of magnetically modalized poly(N-isopropylacrylamide)-chitosan based nanohydrogel, Colloids Surf. B. 81 (2010) 185-194. [45] N. Tudorachi, A. Chiriac, Obtaining of new magnetic nanocomposites based on modified polysaccharide, Carbohydr. Polym, 98 (2013) 451-459. [46] J. Singh, M. Srivastava, J. Dutta, P.K. Dutta, Preparation and properties of hybrid monodispersed magnetic α-Fe2O3 based chitosan nanocomposite film for industrial and biomedical applications, Int. J. Biol. Macromol. 48 (2011) 170-176. [47] M. Chamundeeswari, V. Senthil, M. Kanagavel, S.M. Chandramohan, T.P. Sastry, Preparation and characterization of nanobiocomposites containing iron nanoparticles prepared from blood and coated with chitosan and gelatina, Mater. Res. Bull. 46 (2011) 901-904. [48] E.H. Kim, H.S. Lee, B.K. Kwak, B.K. Kim, Synthesis of ferrofluid with magnetic nanoparticles by sonochemical method for MRI contrast agent, J. Magn. Magn. Mater. 289 (2005) 328-330. [49] N. A. Kalkan, S. Aksoy, E.A. Aksoy, N. Hasirci, Preparation of chitosan-coated magnetite nanoparticles and application for immobilization of laccase, J. Appl. Polym. Sci. 123 (2012) 707-716. [50] S. Coelho, S.M. Flores, J.L.T. Herrera, M.A.N. Coelho, M.C. Pereira, S. Rocha, Nanostructure of polysaccharide complexes, J. Colloid Interface Sci. 363 (2011) 450455. [51] A. Hajdu, E. Illes, E. Tombacz, I. Borbath, Surface charging, polyanionic coating and colloid stability of magnetite nanoparticles, Colloids Surf. A. 347 (2009) 104-108. [52] E. Tombácz, I.Y. Tóth, D. Nesztor, E. Illés, A. Hajdú, M. Szekeres, L. Vékás, Adsorption of organic acids on magnetite nanoparticles, pH-dependent colloidal stability and salt tolerance, Colloids Surf. A. 435 (2013) 91-96. [53] Z. Huang, F.J. Tang, Preparation, structure, and magnetic properties of polystyrene coated by Fe3 O4 nanoparticles, Colloid Interface Sci. 275 (2004) 142-147. [54] P. Saravanan, V.T.P. Vinod, B. Sreedhar, R.B. Sashidhar, Gum kondagogu modified magnetic nano-adsorbent: An efficient protocol for removal of various toxic metal ions, Mater. Sci. Eng. C. 32 (2012) 581-586. [55] Sahu, S. K.; Maiti, S.; Pramanik, A.; Ghosh, S. K.; Pramanik, P. Controlling the thickness of polymeric shell on magnetic nanoparticles loaded with doxorubicin for targeted delivery and MRI contrast agent, Carbohydr. Polym. 87 (2012) 2593-2604. Figure captions

Fig. 1. Infrared spectroscopy of Mag, CH, CHG, CHCG and Mag: CHCHG 1:1 samples.

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Fig. 2. TGA (A) and DTG (B) analysis of Mag, CH, CHG, CHCG and Mag: CHCHG 1:1 samples. Fig. 3. Differential Scanning Calorimetry of (A) CH and CHG samples; (B) Mag, CHCG and Mag: CHCHG 1:1 samples. Fig. 4. X-ray diffraction of Mag and Mag: CHCHG 1:1 samples Fig. 5. Saturation magnetization of Mag: CHCHG 1:1 sample. Fig. 6. Atomic Force Microscopy images for Mag: CHCHG 1:1 sample: 2D topography, showing morphology within (a) 5 µm2 of area and (b) 2.5 µm2 of area; 3D topography

f

within (c) 5 µm2 of area and (d) 2.5 µm2 of area, showing the nanoparticle distribution

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patterns. Fig. 7. EDX of Mag: CHCHG 1:1 sample.

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Fig. 8. Zeta potential data of Mag, CHCHG blank, Mag : CHCHG 1:1, 2:1 and 1:2

e-

samples.

Scheme 1. Proposed scheme for Mag: CHCHG 1:1 sample.

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Chitosan-based hydrogel for magnetic particle coating Vanessa de A. Pereiraa, Irisvan S. Ribeirob , Haroldo C. B. Paulaa, , Regina C. M. de

al

Paulab*, Rubem Luis Sommerc, Ruben Jesus Sanchez Rodriguezd , Flavia O.M.S.

rn

Abreue a

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Post-Graduation in Chemistry Program, Building 940, Federal University of Ceará,

Fortaleza, 60455-760, Ceará, Brazil b

Department of Organic and Inorganic Chemistry, Federal University of Ceará, UFC,

Fortaleza-CE, Brazil, c

Brazilian Center for Physical Research- Centro Brasileiro de Pesquisas Físicas, Rio de

Janeiro - RJ - Brazil d

Estate University of North Fluminense-, Universidade Estadual do Norte Fluminense,

Rio de Janeiro –RJ- Brazil e

Analytical and Environmental Chemistry Laboratory, Post-Graduation in Natural

Science Program, Estate University of Ceará, Fortaleza-CE, Brazil. * Corresponding author: [email protected]

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Regina C. M. de Paula ABSTRACT Magnetic

particles

coated

with

chitosan-based

hydrogel

were

prepared

and

characterized by infrared spectroscopy, thermal analysis, atomic force microscopy (AFM), energy dispersive X-ray (EDX), wide angle X-ray diffraction (WAXD) and by vibrating sample magnetometry. A polyelectrolyte complex of chitosan (CH) and

oo

f

Sterculia striata gum (CHG) was employed as a coating. FT-IR analysis of the hydrogel obtained revealed the presence of main characteristic bands of CH, CHG and magnetite

pr

(Fe3 O 4 ). The thermograms showed a moderate efficiency of magnetite incorporation

e-

(26.0 %) in the hydrogel, as well as evidence of interactions between functional groups of magnetite and biopolymers. Patterns of X-ray diffraction showed the presence of

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magnetite in the hydrogel with saturation magnetization of 36.5 emu g-1 and crystal

al

average size of 20.8 nm. The hydrogel was found to present a negatively charged outer

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surface. EDX and AFM revealed that Fe3 O4 nanoparticles were aggregated, forming large clusters. The CH /CHG hydrogel was shown to be suitable for magnetite coating,

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enabling its future application such as a drug carrier and a water remediation agent. Keywords: chitosan; hydrogel; Sterculia striata gum; magnetic particle 1. Introduction

Magnetic particles have broad applications in many areas such as in materials science, chemistry, medicine, and environment. In recent years, such particles have attracted much attention due to their biological applications such as hyperthermia in tumors [1,2] as carriers guided by a magnetic field [3-5], in diagnostic imaging, in form of contrasts in nuclear magnetic resonance [6,7] and in bovine serum albumin separation processes [8]. These aforesaid applications have major advantages over conventional techniques such as its non-invasive character and non-use of radiation during analysis. In industrial biotechnology, magnetic particles have been used for

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enzyme immobilization such as galactosidase and phospholipase [9,10], and in biocatalysis and biosensors [11-13]. Aiming at a likely application, magnetic particles must be coated with materials that allow their solubilization in a given medium (usually water), forming so called ferrofluids. Natural polymers such as chitosan, alginate, carboxymethylated dextran, pullulan, cellulose derivative, and cyclodextrin have been used for ferrofluid coatings due to the fact that they have desirable characteristics such as biodegradability and biocompatibility [14-17]. Coating occurs through the formation of micro-, nanoparticles or hydrogels which usually behave like conventional fluids [18]

out

[3,16,27,28],

employing

alginate

[15,19-23],

chitosan

[8,9,11,24-26],

oo

carried

f

despite the fact that they contain magnetic particles in their core. Coating has been dextran

carboxymethylcellulose [29], cyclodextrin [17] and polyacrylic acid

pr

[30,31]. Particle size, magnetic properties, surface charge, nature of the coating material and stability in aqueous medium are usually reported as parameters / characteristics of

e-

obtained products which need optimization.

Pr

Some of these substances exhibit high contents of carboxylic groups and charge density and therefore have been reported [32,33] as being suitable for coupling to magnetite molecules for use in magnetic imaging, since for this application it is

al

desirable that the polymer coating possesses negative charges, which would enable

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magnetic stability against aggregation, in addition to the improving resolution of contrasts in magnetic resonance imaging.

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Belonging to the same family of karaya gum (Sterculia urens), the Sterculia striata gum (CHG), commonly named “chichá”, which is extracted from the Sterculia striata trees, is a polyanion [34] possessing main chains containing high content of uronic acid (40%), besides galactose (20 %), rhamnose (25 %), xylose (5 %), and acetyl groups (10 %). Moreover, its structure and physicochemical properties are similar to those of commercial karaya gum. CHG gelling and swelling capabilities points out to likely

applications

such

as

microfluids

devices

[35].

Dilute solutions of this

polysaccharide yield viscous gels which have been used for adsorption of enzymes on silicon wafers [36]. CHG forms a polyelectrolyte complex with chitosan which enables drug encapsulation such as chloroquine [37]. The Sterculia striata tree is abundant in Brazil, particularly in “cerrado” of Northeastern region. Similar regional gums such as the cashew gum (Anacardium occcidentale) and the angico gum (Anadenanthera macrocarpa) have been employed for the formation of

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hydrogels with chitosan, resulting in polyelectrolyte complexes [38-41] with large potential application in pharmacy, medicine and the environment. Due to the abovementioned CHG properties, i. e., high content of carboxylic groups and the ability to form gels, along with the fact that CHG has a large application potential, this work aimed at the preparation of chitosan (CH) and chichá gum (CHG) hydrogels containing magnetite and their characterization by infrared spectroscopy (FT-IR), AFM, EDX, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), vibrating sample magnetometry (VSM) and WAXD. To the best of authors’ knowledge, this is

oo

f

the first time that CHG has been used for magnetite stabilization.

2. Experimental

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2.1. Materials

Chitosan was duly obtained from a local company (82 % deacetylation degree,

e-

Mv = 1.8 x105 g mol-1 ), while the chichá gum (molar mass Mw = 4.18 x 106 g mol-1 ),

Pr

was extracted from Sterculia striata trees in Ceará, Brazil and isolated as previously reported [34]. Ferric chloride FeCl3 and ferrous sulphate FeSO 4 (Dinamica) and sodium 2.2. Hydrogel preparation

al

hydroxide (Vetec) were used as they were being received.

rn

Magnetic nanoparticles were synthesized by the modified method of ferrous and ferric ions co-precipitation via alkaline hydrolysis [11,15,21,42] employing chitosan

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and chichá gum as coating materials, with ratios of magnetite (Mag) to the polymeric matrix CHCHG (Mag: CHCHG) of 1:1. The CH: CHG ratio was 4:1 (w/w), corresponding to a charge density of n+/n- = 10 (protonated amine groups/carboxylate groups). This said charge ratio has been employed for polyelectrolyte synthesis, yielding complexes that have small particle size and better pH stability [38-40]. A 0.4 % CH solution was prepared by dissolving 0.4 g of chitosan in 100 mL of 1 % acetic acid. Hence a 0.4 % (m/v) CHG solution was added dropwise, leaving the clear mixture

stirring for an additional 30

minutes.

After completion of CH/CHG

polyelectrolyte complex formation, to this solution was added 25 mL of solutions of FeCl3 .6H2 O and FeSO 4 .4 H2 O, at concentrations of 4.64 and 2.4 %, respectively and the mixture left stirring for 15 minutes. The magnetite was duly obtained by adding a 25 % (w/v) solution of sodium hydroxide until pH 10 and observing the formation of a brown

precipitate.

The

aforementioned

system was

maintained

under nitrogen

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atmosphere and a magnetic separation was then carried out by using a magnet, followed by an extensive washing of the hydrogel with distilled water until a neutral pH. The hydrogel obtained was then dialyzed against distilled water through a Millipore membrane (pore size 14 kDa) and thereafter freeze dried. A sample of pure magnetite was prepared according to the above described procedure, however, without the addition of biopolymers. 2.3. Physicochemical characterization The infrared spectra of CH, CHG, CH/CHG blank and hydrogel samples were cm-1

in

the

FT-IR

unit

Shimadzu

spectrophotometer

oo

4000

f

obtained using KBr pellets with the samples scanned in the region between 400 and (Model 8300).

The thermal properties of the CH, CHG and CH/CHG hydrogel were evaluated by

pr

thermogravimetric analysis (TGA) using a Shimadzu equipment (Japan), model TGA50, under nitrogen atmosphere, using a heating rate of 10 °C min-1 , and the temperature

e-

range from 25 to 800 °C, and by Differential Scanning Calorimetry (DSC) through a

Pr

Shimadzu Calorimeter, Model DSC-50 (Japan) with a heating rate of 10 °C min-1 , from 25 °C to 500 °C.

Wide angle X-ray diffraction (WAXD) measurements were made on a

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PANalytical X’Pert Pro MPD instrument, where powder forms of the CH, CHG and

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CH/CHG hydrogel samples were exposed to Cu radiation, with increments of 1°/min and scanned over a 2Ɵ range of 3° to 40°.

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The hydrogel magnetization at room temperature was measured using a vibrating sample magnetometer (VSM) OXFORD, Model 3001, operating at 1.6 Tesla, using the external field of 2 kOe.

Aiming at determination of the hydrogel surface charge, zeta potential samples was analyzed using a Malvern Zetasizer Nano, Model ZS 3600. Samples were placed in distilled water and sonicated for 2 minutes prior to being analyzed. The hydrodynamic diameter was measured by dynamic light scattering with laser wavelength of 633 nm and a fixed scattering angle of 173°. Particle size was measured considering the particle as spherical like. Each sample was measured 3 times (n=3). The CH/CHG hydrogel sample was analyzed by EDX in a Bruker AXS Microanalysis instrument, GmbH, Germany. 3. Results and Discussion 3.1. FTIR

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Fig. 1 shows the FTI-IR spectra of Mag, CH, CHG and CH/CHG samples. In the spectrum of pure magnetite, Fe3 O4 (Mag), it can be observed the main absorption bands at 584 cm-1 , attributed to vibrations of the Fe-O bonds and vibrations of free or adsorbed water at 1627 and 3404 cm-1 . These stated bands are in good agreement with data duly reported in literature for dextran [16] coated Fe3 O4 ; chitosan coated magnetic particle [41] and magnetite stabilized using oleic acid and Tween 80 surfactant [42]. Chitosan was found to present its major bands at 1633 cm-1 (amide I), 1540 cm-1 (amide II), 1070 cm-1 (C-O-C group), as well as at 1735 cm-1 due to carbonyl of acetyl

f

groups, in full agreement with literature data [39,43]. Higher wavenumber values for

oo

amide II band (at 1594 cm-1 ) and for C-O-C group (at 1089 cm-1 ) were reported by Jaiswal et al. [44] No acetyl bands at 1735 cm-1 was observed, likely due to the fact that used

in

their

work

had

a

higher

deacetylation

pr

chitosan

degree

(95%).

CHG shows strong bands at 1724 cm-1 due to carbonyl of uronic acids and acetyls

e-

groups, while the carboxylate groups arise at 1651 and 1436 cm-1 . Hydroxyl groups

Pr

appear at 3431 cm-1 and C-H groups at 2922 cm-1 . C-O-C anomeric carbon bonds are present at 1145 cm-1 . Similar data was previously reported by Brito et al. [34] whereby these authors have been employed CHG from same said region and it is therefore likely

al

to exhibit similar composition and properties.

rn

However, it may be noticed that by the formation of chitosan/chicha gum polyelectrolyte complex (bare CH/CHG hydrogel), occurs the disappearance of band at

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1724 cm-1 and formation of a new band at 1633 cm-1 , while CH amide II band shifts to 1595 cm-1 . This feature has also been reported for a chitosan/ cashew gum polyelectrolyte complex, which was shown [38] to exhibit chemical stability at pH range 2-10.

In the absorption spectrum of Mag: CHCHG 1:1 hydrogel, a broad and strong band is observed at 3431 cm-1 and attributed to bonds stretches of -OH and -NH groups. This band enlargement and the relative increase in relation to spectrum of bare CHCHG is likely to due to Fe complex formation with CH. At 1633 cm-1 appears chitosan amide I band and amide II [25,44] is overlapped with carboxylate form of CHG uronic acids, resulting in a broad band at 1375 cm-1 , pointing out to likely interactions of Fe, CH and CHG. The absorption band at 1069 cm-1 is attributed to the asymmetrical stretching of the C-O-C linkages present in both polysaccharides, whereas the vibration at 1729 cm-1 is related to the stretching of the carbonyl groups of the uronic acid and acetyl groups of

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CHG [34]. It worth notice that CH band at 1540 cm-1 disappears, pointing out to the fact the protonated chitosan amino group has been involved in complex formation with magnetite. Moreover, it was noticed a shift to 600 cm-1 in the Mag absorption band at 584 cm-1 , characteristic of the vibration of Fe-O bonds. Similar data was obtained for carboxymethylated cellulose and pullulan [16], crosslinked chitosan [43] and dextran, whereby it is shown that the polysaccharide was successfully coated on the surface of iron oxide particles through Van der Waals forces, hydrogen bond and electrostatic interactions [16].

f

Fig. 1.

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3.2. Thermal Analysis

Fig. 2 show thermal data of CH, CHG, Mag, blank and Mag: CHCHG 1:1

pr

hydrogel samples. The sample Mag presents only one event due to moisture evaporation (8.7%) in the temperature range investigated, in full agreement with reported data for

e-

magnetite [9,42,45]. The decomposition of the polysaccharide structures (CH and

Pr

CHG), was found to happen in an event around 290 °C and another above 400 °C for CH, and in two major events in the range of 270 -473 °C, for CHG, in good agreement with previously reported data [34]. A blank sample (CH/CHG hydrogel without Fe3 O4 ) main events,

namely water evaporation (12.8

al

exhibits two

%) and

polymer

rn

decompositions (64.0 %) at 275 °C, the later being a clear evidence of CH and CHG complex formation, otherwise two events would have being observed, due to parent

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molecules degradations. Mag: CHCHG 1:1 sample exhibits a different decomposition pattern, most likely owing to magnetite-biopolymers interactions. The existence of three major thermal events for Mag: CHCHG 1:1 sample can be observed. The first event occurred at temperature range from 25.0 to 80.4 °C with a weight loss of 10.8 % and can be attributed to the elimination of adsorbed and bound water molecules. The second event happens in the range of 256.4 - 347.3 °C with a mass loss of 21.8 % corresponding to major biopolymers degradations and the third event is in the range 621-667 °C, due to carbonization, with mass loss of 18.5 %. The complete decomposition of the Mag: CHCHG 1:1 sample leads to a residue of 48.8%. Taking into consideration that blank the sample leaves a residue of 23.0 % at 700 °C, it can be inferred that magnetite content in Mag: CHCHG 1:1 sample amount to 26.0 %. Dextran coated magnetic particles [3] in a 1:1 ratio to magnetite with a coating efficiency of 45.1%, exhibited two thermal events, namely, water evaporation and

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polymer decomposition, the latter beginning at 300 °C. It can be seen that magnetic particles coated

by biopolymers such as dextran [16,30], chitosan [9,17,25],

carboxymethyl starch [46], alginate [15,19,21] and CHG exhibit similar decomposition patterns, although their complex formations and decomposition temperatures may be different. Hence, it can be inferred that Mag: CHCHG 1:1 sample seems to be more stable than dextran magnetic particle, although the later has a higher Fe 3 O 4 content. This is likely to be due to the fact that CHG has more carboxylate groups (from uronic acids) available for magnetite stabilization.

f

Fig. 2.

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Data of DSC analysis, shown in Fig. 3 presents the endothermic (melting) and exothermic (decomposition) transitions for CH, CHG, Mag: CHCHG 1:1, blank

pr

CH/CHG and pure magnetite, Mag, where the endothermic events are related to the evaporation of residual water and the exothermic decompositions are due to the

e-

biopolymer degradations. CH decomposes in two exothermic stages (probably due to

Pr

the rupture of its acetylated and deacetylated polymeric units) at higher temperatures (287 and 460 °C) than those reported by Singh et al. [46] CH thermal properties have been found to depend on its molar mass and degree of acetylation: high molar mass and

al

degree of acetylation lead to a more stable biopolymer, in good agreement with data

rn

shown herewith. CHG also decomposes in two events, however at lower temperatures (274 and 418 °C), in good agreement with the trend exhibited by other biopolymers

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such as cashew gum [38], whose decomposition temperatures were at 248 °C and 309 °C. CHCHG blank sample presents an endothermic transition at 175.0 °C due to bound water elimination and a single major exothermic peak at 284.8 °C due to CH/CHG complex formation, providing further evidence of biopolymer interactions. It is worth notice that CH presented higher melting temperature than CHG, indicating that water is more tightly bound to CH molecule than to CHG, likely because the latter is a more hydrophilic polymer and water is released easier. Mag sample exhibits a single endothermic peak at 93 °C due to residual water elimination. The sample Mag: CHCHG 1:1 shows decomposition pattern similar to that of CHCHG blank sample, presenting endothermic transitions at 171 °C (major) and 460 °C (minor), being the latter attributed to Mag: matrix complex come apart, providing evidence of magnetite linking to CHCHG 1:1 hydrogel. It is worth notice that neither the parent polymers nor magnetite exhibit such endothermic transition. The interactions of water with species that makes

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up the matrix are responsible for water retention capacity, causing the displacement of the transition peaks. This feature was also reported by Ghaemy and Naseri [8] for chitosan crosslinked nanohydrogels. Fig. 3. 3.3. WAXD and VSM Fig. 4 shows the X-ray pattern of CH/CHG hydrogel coated magnetite. It can be seen that the Fe3 O4 sample shows major peaks characteristic of magnetite diffraction located in 2θ= 35.3, 41.5 , 50.5 , 59.7 , 67.4 and 74.5

o

corresponding to crystal planes

f

(220), (311), (400), (511), (440) and (533) respectively. After coating with the hydrogel,

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these peaks become broad and decrease in intensity. This seems to indicate that the Fe3 O4 coating did not lead to a phase change. These data are in good agreement with

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figures reported for dextran [3], alginate [15], chitosan/gelatin complexes [47], chitosanstarch derivative nanocomposites [45], and carboxymethyl cellulose [29], which exhibit

e-

similar diffraction peaks and crystal planes, indicating that the cubic spinel structure of o

was also

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Fe3 O4 is present in the obtained samples. The peak at the 2 theta = 74.5 observed by Kim et al. [48] and Ma et al. [15]. Fig. 4.

al

CH/CHG hydrogel exhibits significant shifts in the signals at 2θ in comparison to

rn

bare Fe3 O 4 probably due to interactions between magnetite groups and biopolymers CH and CHG. Chitosan and CHG diffractograms show low 2 θ values in the range 15-25°

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(data not shown), which were attributed to the crystalline regions formed by hydrogen bonds of amino and hydroxyl groups, being typical of amorphous polymers. The average size of the crystals was calculated using Sherrer's equation [3,30] and the peak width at (311), resulting in a value of 20.8 nm. The curve of the saturation magnetization is shown in Fig. 5, where it can be seen that the hydrogel coated sample has a value of 36.5 emu g-1 , which is indicative of typical superparamagnetic behavior. This low value was expected, since the coverage of magnetite with polymers results in a decrease of its saturation magnetization. Bare magnetite (Mag sample) presented Ms = 76.0 emu g-1 (calculated from thermogravimetric date). Tudorachi and Chiriac [45] reported a low value for Ms (3.04 emu g-1 ) for a matrix based on carboxymethyl starchg-polylactic acid, using a in situ magnetite synthesis. Dextran coated magnetic nanoparticles presented saturation magnetization of 54.8 emu g-1 , for a 45.1% magnetite content [3], while Fe3 O4 coated with carboxymethyl cellulose [29] exhibit Ms as low as

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13.9 emu g-1 , alginate embedded magnetite exhibited similar value [19] and chitosan coating [49] resulted in Ms = 25.2 emu g-1 , for a magnetite content of 48.3 %. In summary, Ms was revealed to be dependent on magnetite content as well as on the nature of polymer coating; comparing to CH coatings reported by Kalkan et al. [49] and nanocomposites reported by Tudorachi and Chiriac [45], Mag CHCHG 1:1 sample yielded higher Ms value, at a lower magnetite content. Fig. 5. 3.4. AFM and EDX

f

Mag: CHCHG 1:1 sample was analyzed by AFM. It is observed in Fig. 6 (a, b, c

oo

and d) that the magnetic particles seem to have spheroidal morphologies, whereby agglomerated areas are predominating. These particles have a profile of bimodal

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average particle size, with an average size of 65.0 ± 5.0 nm for smaller particles and 100.0 ± 5.0 nm for larger particles. Since crystal sizes are much smaller than that data,

e-

this seems to point out to fact that magnetite is located within aggregated particles.

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These results are in good agreement with the literature, whereas Ma et al. [15] reported similar data for alginate and Wotschadlo et al. [16] presented particles having particles size of 229

nm, for carboxymethylated dextran. Nanostructured polysaccharide

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complexes of chitosan and arabic gum were also investigated by Coelho et al. [50]

Fig. 6.

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200 to 250 nm.

rn

where the nanoparticles of arabic gum/chitosan with a 1:1 ratio presented sizes from

The EDX spectrum presents peaks (Fig. 7) corresponding to the elements making up the true composition of the sample being analyzed. For Mag: CHCHG 1:1 sample, the Fe content was found to be 23.5 %, very close to the value obtained from TGA. Moreover, EDX mapping revealed that magnetite is uniformly distributed on hydrogel surface, nevertheless, presenting aggregation regions. Fig. 7. Aiming at clarifying the structure of the complex formed, samples with different ratios of magnetite and matrix Mag: CHCHG = 2:1 and 1:2 were prepared and analyzed in relation to their zeta potentials (Fig. 8). It was observed that the zeta potential of the blank sample (no Fe3 O4 ) is + 36.7 mV, while the samples Mag: CHCHG with ratios 1:1, 1:2 and 2:1 showed zeta potential of -9.20 mV, -4.75 mV and - 17.5 mV, respectively. As it can be seen, increasing the Fe3 O4 insertion in the hydrogel resulted in a more

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negative zeta potential, which means that the positive charges of the bare hydrogel, assigned to protonated amino groups (in excess, at a charge ratio of n+/n- = 10) were neutralized by those negative ones of Mag (zeta potential -11.2 mV), therefore leaving the excess of negative charges on hydrogel surface. Fig. 8. As the medium is aqueous, the tightly packed Fe3 O 4 molecules are not soluble; hence they ought to be coated with the hydrogel hydrophilic matrix, which is achieved by a molecular rearrangement where CHG carboxylate groups are likely to be located at

f

the hydrogel surface, as it can be seen in Scheme 1. This feature was also observed by

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Hajdu et al. [51], whereby citric acid and humic acid were found to be adsorbed on Fe– OH surface sites. Furthermore, the decrease in saturation magnetization of coated

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magnetite corroborated the aforementioned assumption. As reported by Tombácz et al. [52] the amphoteric magnetite character allows for the fact that it is positively charged

e-

at pH lower than 7.9, bearing negative charges above that particular value. The authors

Pr

have also pointed out that macromolecular organic acids would provide better particle stability to the said system. In this sense, uronic acids present in the polyelectrolyte CHCHG

complex

seems

to

contribute

for

magnetite

stabilization,

under

the

al

experimental conditions of this study. Cellulose derivative, carboxymethylated dextran

rn

and pullulan covering magnetite [16] showed negative zeta potentials (from -35 mV to 54 mV), while pure magnetite showed zeta potential of +44 mV, at pH 4. Huang and

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Tang [53] reported negative potentials for polystyrene coated magnetite at the whole pH range, thus providing clear evidence that in the event the polymer is neutral, all negative charges in aqueous medium are likely due to magnetite Fe-O atoms. A matrix based on carboxymethyl starch-g-polylactic acid was also reported to be acting as a shell for magnetite incorporation [45]. The negative charges at outer surface of Mag CHCHG 1:1 sample points out to its potential application such as the removal of hazardous metal cations, i.e., Cd2+, Cu2+, Pb2+ and Hg2+. These stated metals have been adsorbed on gum kondagogu containing magnetite [54], an Indian polysaccharide whose structure is similar to the CHG. Although no zeta potential analysis has been reported, the gum was found to exhibit good adsorption capability. On the other hand, coating Fe3 O4 with a polycation and a polyanion allows the coupling of drugs to the magnetic particle and its further usage in drug delivery, as reported for dextran/chitosan magnetic particles loaded with doxorubicin, an anticancer drug [55].

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Scheme 1 4. Conclusion

Magnetic nanoparticles were successfully coated with CH/CHG hydrogel, showing a high magnetite content and a good saturation magnetization, as well as a hysteresis behavior. Particle size was 20.8 nm, however in the form of large clusters, as revealed by AFM and EDX. The presence of the major functional groups of CH, CHG and Fe3 O4 as well as their likely interactions, were evidenced by FT-IR-infrared

f

spectroscopy. Thermal analysis revealed the main events of degradations of the sample

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constituents, along with indication of Fe-O / CH interactions, which were responsible for the higher than the parent molecules (CH and CHG) thermal stability exhibited by

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the hydrogel. The pattern of X-ray diffraction of the samples showed characteristic peaks of magnetite, which were broad, due to the coating process. The zeta potential

e-

data revealed that hydrogel surface was negatively charged, likely due to uronic acids of

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CHG which ultimately promoted magnetite stabilization. The aforementioned data revealed that the hydrogel obtained is a likely candidate to the usage as a release device

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Acknowledgments

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for drugs and in water remediation, such as for the heavy metal adsorption.

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CNPQ, CAPES and FUNCAP for financial support; Prof. Dr. J. M. J. Sassaki, coordinator for the X-rays Laboratory , Physics Department of the Federal University of Ceará; Institute for Research, Development and Innovation- IPDI, State of Ceará Government; Biomass Technology Laboratory, EMBRAPA –Fortaleza, Rede INOMAT (National Institute for Science, Technology and Innovation on Functional Complex Materials).

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Figure captions

Fig. 1. Infrared spectroscopy of Mag, CH, CHG, CHCG and Mag: CHCHG 1:1 samples.

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Fig. 2. TGA (A) and DTG (B) analysis of Mag, CH, CHG, CHCG and Mag: CHCHG

Pr

1:1 samples.

Fig. 3. Differential Scanning Calorimetry of (A) CH and CHG samples; (B) Mag, CHCG and Mag: CHCHG 1:1 samples.

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Fig. 4. X-ray diffraction of Mag and Mag: CHCHG 1:1 samples

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Fig. 5. Saturation magnetization of Mag: CHCHG 1:1 sample. Fig. 6. Atomic Force Microscopy images for Mag: CHCHG 1:1 sample: 2D topography,

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showing morphology within (a) 5 µm2 of area and (b) 2.5 µm2 of area; 3D topography within (c) 5 µm2 of area and (d) 2.5 µm2 of area, showing the nanoparticle distribution patterns.

Fig. 7. EDX of Mag: CHCHG 1:1 sample. Fig. 8. Zeta potential data of Mag, CHCHG blank, Mag : CHCHG 1:1, 2:1 and 1:2 samples. Scheme 1. Proposed scheme for Mag: CHCHG 1:1 sample.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8