Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Research Paper Magnetic nanohydrogel obtained by miniemulsion polymerization of poly (acrylic acid) grafted onto derivatized dextran Rodolfo Debone Piazzaa,⁎, Eloiza da Silva Nunesb, Wesley Renato Vialib, Sebastião William da Silvac, Fermin Herrera Aragónc, José Antônio Huamaní Coaquirac, Paulo César de Moraisd, Rodrigo Fernando Costa Marquesa, Miguel Jafelicci Júniora a Laboratory of Magnetic Materials and Colloids, Departament of Physical Chemistry, Institute of Chemistry, São Paulo State University, Araraquara, SP, 14801-970, Brazil b Instituto Federal Goiano, Rio Verde, GO 75901-970, Brazil c Instituto de Física, Núcleo de Física Aplicada, Universidade de Brasília, Brasília, DF, 70910-900, Brazil d Anhui University, School of Chemistry and Chemical Engineering, Hefei 230601, China A R T I C L E I N F O Keywords: Derivatized dextran Nanohydrogels Iron oxide Miniemulsion polymerization A B S T R A C T This study describes the synthesis of magnetic nanohydrogels by miniemulsion polymerization technique. Dextran was derivatized by the glycidyl methacrylate to anchor vinyl groups on polysaccharides backbone, allowing its use as a macromonomer for miniemulsion polymerization, as confirmed by proton nuclear magnetic resonance spectroscopy (13C NMR). Magnetite nanoparticles were synthesized by coprecipitation, followed by air oxidation to maghemite. The results of X-ray diffractometry (XRD), Raman and transmission electron mi- croscopy (TEM) analysis showed that maghemite nanoparticles were obtained with a diameter of 5.27 nm. The entrapment of iron oxide nanoparticles in a dextran nanohydrogel matrix was confirmed by thermogravimetric analysis (TGA), scanning transmission electron microscopy (STEM) and Zeta potential data. The magnetic na- nohydrogels presented superparamagnetic behavior and were colloidal stable in physiological during 30 days. Our findings suggest that the synthesized magnetic nanohydrogel are potential candidates for use in drug de- livery systems due to its physicochemical and magnetic properties. 1. Introduction In the last decade, the use of polymers nanoparticles (Biswas, Kumari, Lakhani, &Ghosh, 2015; Karami, Sadighian, Rostamizadeh, Parsa, &Rezaee, 2016; Lu& Park, 2013; Mandal et al., 2013; Masood, 2015; Ta, Convertine, Reyes, Stayton, & Porter, 2010) (Easo&Mohanan, 2013; Hervault & Thanh, 2014; Laurent et al., 2008; Pankhurst, Thanh, Jones, &Dobson, 2009) as platform for bioactive molecules have attracted attention due to their potential in targeting tumor tissues through passive delivery via enhanced permeability retention (EPR) effect (Bertrand, Wu, Xu, Kamaly, & Farokhzad, 2014). Polymer nanoparticles show high col- loidal stability in addition to its versatility to retains bioactive molecules and delivery it when stimulated, increasing the biodistribution and avoiding premature drug delivery. (Ganguly, Chaturvedi, More, Nadagouda, &Aminabhavi, 2014; Hoare &Kohane, 2008; Peppas, 1997). Polysaccharides show some advantages over synthetic polymers once they are abundant and obtained from renewable sources (Coviello, Matricardi, Marianecci, & Alhaique, 2007). Polysaccharides are biocompatible, biodegradable, non-toxic and have free functional groups that can be used to modify their structure and/or anchor bioactive molecules, such as proteins, antibody and drugs (Dias, Hussain, Marcos, & Roque, 2011; Liu, Jiao, Wang, Zhou, & Zhang, 2008). Dextran is a suitable polysaccharide to prepare nanohydrogels and consists, predominantly, of α-1,6-glucosidic linkage, with some degree of branching in 1,3-linkage. The dextran-based hydrogel is ob- tained by derivatization of its structure with vinyl groups, which can be polymerized with acrylic acid to control the crosslinking degree and pH-responsive behaviour (Medeiros, Santos, Fessi, & Elaissari, 2011). The use of nanohydrogels as drug carrier allows a greater drug load by the circulatory system, avoiding the chemical and enzymatic degrada- tion before drug reach the targeted tissue (Ganguly et al., 2014; Iyer, Singh, Ganta, & Amiji, 2013; Liu et al., 2008; Wang et al., 2017). In addition to being capable to retain drug for a long circulation period, its desirable to guide the platform direct to the targeted site in the body, improving the therapy efficacy (Wassel, Grady, Kopke, & Dormer, 2007). To achieve this aim, nanohydrogels were supported onto http://dx.doi.org/10.1016/j.carbpol.2017.09.019 Received 26 May 2017; Received in revised form 23 August 2017; Accepted 6 September 2017 ⁎ Corresponding author. E-mail addresses: rodolfo.piazza@iq.unesp.br, rodolfo.piazza@gmail.com (R.D. Piazza). Carbohydrate Polymers 178 (2017) 378–385 Available online 07 September 2017 0144-8617/ © 2017 Elsevier Ltd. All rights reserved. MARK http://www.sciencedirect.com/science/journal/01448617 http://www.elsevier.com/locate/carbpol http://dx.doi.org/10.1016/j.carbpol.2017.09.019 http://dx.doi.org/10.1016/j.carbpol.2017.09.019 mailto:rodolfo.piazza@iq.unesp.br mailto:rodolfo.piazza@gmail.com http://dx.doi.org/10.1016/j.carbpol.2017.09.019 http://crossmark.crossref.org/dialog/?doi=10.1016/j.carbpol.2017.09.019&domain=pdf magnetic iron oxide nanoparticles, allowing it to be driven by an ex- ternal magnetic field to specific site. Iron oxide nanoparticles are also of great interest in biomedical ap- plications such as hyperthermia, magnetic resonance imaging and drug delivery, due to their properties such as superparamagnetism, high surface to volume area, biocompatibility, and nontoxicity. In order to avoid par- ticle agglomeration towards physiological conditions during use in the afore mentioned applications, the surface of superparamagnetic iron oxide nanoparticles (SPION) should be functionalized with suitable molecules such as carboxylic acids (Lattuada&Hatton, 2007; Petri-Fink, Chastellain, Juillerat-Jeanneret, Ferrari, &Hofmann, 2005; Turcheniuk, Tarasevych, Kukhar, Boukherroub, & Szunerits, 2013), aminoacids (Durmus et al., 2011; Gholami, Rasoul-amini, Ebrahiminezhad, Seradj, & Ghasemi, 2015), and polymers, (e.g., dextran, chitosan, poly(ethylene glycol), etc.) (Arruebo et al., 2007; Durmus et al., 2011). The main contribution of this investigation was the magnetic na- nohydrogels synthesis by miniemulsion polymerization. This poly- merization method shows particular features during the nucleation process, which results in nanoreactors formed by droplets with a lim- ited volume of reaction (Luo, Dai, & Chiu, 2009; Mittal, 2011). The size of the resultant magnetic nanohydrogels should be comprised between 50 and 500 nm (Mittal, 2011), being suitable to passively targeting to the tumor cells (Bertrand et al., 2014). The maghemite nanoparticle was surface functionalized with acrylic acid for further encapsulation with a polymeric matrix composed of derivatized dextran and acrylic acid. The magnetic nanohydrogels were obtained through vinyl poly- merization with different amounts of functionalized iron oxide. To the best of our knowledge, this is the first work describing the use of magnetic nanoparticles surface modified with derivatized dextran to be cross-linked in nanohydrogels using the miniemulsion polymerization instead of classical macroscopic hydrogels. 2. Materials and methods 2.1. Materials All chemicals were used as received. Iron(III) chloride hexahydrate (97%), Heptane (98.5%), and ammonium persulfate (APS) were pur- chased from Mallinckrodt Chemicals. Dimethyl sulfoxide (DMSO, 99%) and 2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS) were purchased from MERCK. Sodium hydroxide (NaOH, 97%) was purchased from Synth. Acrylic acid (AA, 97%) and 4-(dimethylamine) pyridine (DMAP, 99%) were purchased from Alfa Aesar. Iron(II) chloride tetrahydrate (99%), dextran (MW 40 kDa), glycidyl methacrylate (GMA, 97%), N,N′- methylenebisacrylamide (99%), sorbitan monooleate (Span®80), phos- phate buffered saline (0.01 mol L−1 phosphate, 0.135 mol L−1 NaCl and 0.002 mol L−1 KCl) buffer solution (PBS) were purchased from Sigma-Aldrich Brazil. 2.2. Dextran derivatization The dextran was modified through reaction with glycidyl metha- crylate as described by van Dijk-Wolthuis et al. (Hoof & Hennink, 1997; Steenbergenj, Bosch, & Hennint, 1995). The used molar ratio of dex- tran:GMA:DMAP was 1:0.5:0.25 with respect to 1.0 mol of glycosidic unit. Dextran (1.5 g) and DMAP (2.4 mmol) were dissolved in 30.0 mL of DMSO in a three-neck round bottom flask under N2 atmosphere and magnetic stirring. After complete dissolution, the system was heated up to 45 °C, then 4.8 mmol of GMA was injected into the flask, the tem- perature and stirring were kept for 24 h. The reaction was stopped by adding an equimolar amount of HCl to neutralize DMAP. The dextran modified polymer (Dex-GMA) was precipitate with acetone and dia- lyzed at 8 °C. The product was freeze-dried and a white powder was obtained. 2.3. Synthesis of SPION and functionalization with acrylic acid The synthesis of iron oxide nanoparticles was performed by co- precipitation of Fe2+ and Fe3+. A solution containing 0.04 mol of FeCl2·4H2O and 0.08 mol of FeCl3·6H2O dissolved in 500 mL of deio- nized water was added drop‐wise into 500 mL of 1.5 mol L−1 NaOH solution under mechanical stirring (2000 rpm) and constant bubbling on N2 gas at room temperature. A black precipitate formed instantly and after 20 min of reaction the solid was magnetically decanted and washed three times with deionized water. The SPION were suspended in water and the pH was adjusted to 3.5 with 1.0 mol L−1 HCl solution. The suspension was heated in a boiling water bath under constant magnetic stirring and air bubbling during 3 h. The reddish brown dis- persion was dialyzed against water for 7 days and stored for further use (Massart, 1981; Viali et al., 2010). The SPION concentration of 54.5 g L−1was obtained. The surface functionalization of maghemite nanoparticles with ac- rylic acid was performed by adding 2.0 mL of 0.10 mol L−1 acrylic acid solution in 3.0 mL of SPION suspension, at pH 4.0 (Nunes, Lemos, & Carneiro, 2013). The acrylic acid adsorption was performed under continuous stirring for 48 h at room temperature and then the free acrylic acid was removed by dialysis. The resulting dispersion was labeled as Magh-AA and used in further experiments. 2.4. Synthesis of magnetic nanohydrogels The magnetic nanohydrogels were synthesized by inverse mini- emulsion polymerization by using acrylic acid and derivatized dextran as monomers, in the ratio of 35% and 65% (w/w), respectively. A Span®80 solution in n-heptane (4% (w/w), 40 mL) and a solution con- taining Magh-AA (different amounts of 50, 100, and 150 mg), AA (100.0 mg), Dex-GMA macromonomer (200.0 mg), NaOH (1.12 mmol), MBA (0.097 mmol) and APS (0.22 mmol) dissolved in 3.0 mL of deio- nized water was homogenized in a Turrax stirrer at 20000 rpm for 2 min and then submitted to an ultrasound probe for 30 min, an ice bath was used to avoid initiation of polymerization. Afterward, 16 mg (0.15 mmol) of sodium bisulphite were added under ultra-sonication. Sodium bisulphite and ammonium persulfate act as pair redox to de- crease the temperature of thermodecomposition of initiators (Pohl & Rodriguez, 1981). The miniemulsion was transferred to a three- neck flask under magnetic stirring, purged for 15 min with Argon flux, and then heated up to 50 °C for 4 h. After cooling to room temperature, the magnetic nanohydrogels were removed by centrifugation at 10000 rpm and washed thrice with hexane. The particles were dis- persed in a 0.5% Tween®80 aqueous solution and dialyzed against water for 5 days. 2.5. Samples characterization XRD powder diffraction of the samples was recorded in the 2θ range of 10–80° using the Siemens D5005 system equipped with a Cu Kα radiation source. The XRD diffractograms were used to check the crystalline phase of the SPION-based material as well as to estimate the average crystallite size, the latter performed by using the Scherrer’s equation (Cullity, 1978). Raman scattering spectra were recorded at room temperature in a frequency range of 200–1000 cm−1 from a HORIBA Jobin Yvon model LabRAM HR micro Raman apparatus equipped with a 632.8 nm laser delivering 0.6 mW power. The size and morphology of maghemite nanoparticles were investigated by trans- mission electron microscopy (TEM). Low magnification was obtained using a JEOL 3010 TEM-HR operating at 300 kV. For TEM measure- ment, a drop of the sample dispersed in isopropanol was deposited on a copper grid covered with carbon film. The morphology and size of nanohydrogels were investigated by transmission scanning electron microscopy (STEM) in a FEI Inspect F50 microscope. For STEM ana- lysis, the samples were dispersed in isopropanol and deposited in a R.D. Piazza et al. Carbohydrate Polymers 178 (2017) 378–385 379 carbon coated copper grid. The images were obtained in secondary electrons, bright field and high-angle annular dark field modes (HA- ADF). The FT-IR measurements were carried out using a Bruker VERTEX 70 FT-IR spectrometer equipped with a diffuse reflectance infrared Fourier transform (DRIFT) collector accessory, using the system resolution set at 2 cm−1, while performing 256 scans. 1H NMR spectroscopy was carried out on a Varian INOVA 300 spectrometer measuring samples dissolved in deuterium oxide. Solid state 13C NMR measurements were carried out on Bruker Avance III HD 400WB spectrometer. The powders were packed into 4 mm rotors a spun at speeds of 10000 Hz, at fixed contact time of 2 ms. The deconvoluted spectral components were obtained using Voigt profile. Thermogravi- metric analyses (TGA) were carried out in STA 409C/CD system DTA- TGA from NETZSCH Instruments. Samples (15 mg) were analyzed from room temperature up to 800 °C under 50 mL min−1 air flow, using a heating rate of 10 °C min−1 to estimate the net weight of the SPION in the magnetic nanohydrogel. Hydrodynamic diameter and zeta potential of nanoparticle samples were measured using a Zetasizer Nanoseries ZSNano ZEN3600 from Malvern Instruments. Hydrodynamic diameter was measured by dynamic light scattering (DLS), which samples were dispersed in water to size distribution, in NaCl 1 mmol L−1 for pH de- pendence curve and in phosphate-buffered saline (PBS) or Tris buffer solution (pH 7.4) to colloidal stability measurments. For zeta potential measurements the samples were previously dispersed in NaCl 1 mmol L−1 solution. Magnetization measurements were performed in powder form, using a commercial Physical Property Measurement System (PPMS) model 6000 platform with the vibrating sample mag- netometer (VSM) module from Quantum Design. Hysteresis loops (M‐H curves) were recorded in the range of −20 to 20 kOe, at temperatures of 300 K. Zero-field-cooled (ZFC) and field-cooled (FC) curves were carried out in temperature range from −268 °C to 27 °C and applying a DC magnetic field of 30 Oe. 3. Results and discussion 3.1. Characterization of dextran macromonomer The polysaccharide dextran chains were modified by grafting me- thacrylate groups, through the reaction with GMA. The DMAP act as a Lewis base and induces polarization of hydroxyl groups of dextran, al- lowing grafting of methacrylate groups in the polymer backbone (Lo& Jiang, 2010). The dextran derivatization can occur by two me- chanisms: epoxy ring opening or transesterification, as show in Fig. S1 on supplementary information. In epoxy ring opening mechanism, the me- thylene carbon of the GMA undergoes a nucleophilic attack by hydroxyl groups of dextran, while the transesterification results in the attack at carbonyl ester of GMA. DMSO, an aprotic polar solvent, was used in reaction environment to avoid reactions of GMA with water (Hoof &Hennink, 1997; Steenbergenj et al., 1995). Both mechanisms results in the methacrylate groups grafted to dextran chain. The deriva- tization was evaluated by the 1H NMR spectroscopy (Fig. 1A) and solid state 13C NMR spectroscopy (Fig. 1B). From 1H NMR results, the signals between 3.10 ppm and 5.20 ppm corresponding to the dextran chain protons. The anomeric proton of the glucopyranosyl ring has the signal shifted from the others protons at 4.91 ppm. The peak at 5.20 ppm cor- responds to α-1-4 linkage among dextran units. The presence of metha- crylate groups was confirmed for Dex_GMA sample. The double doublet signals in 6.19 ppm and 5.70 ppm correspond to protons of vinyl group. The single peak of methyl protons is observed at 1.90 ppm. The degree of substitution (DS) of GMA on dextran can be calculated through 1H NMR spectrum, applying the equation DS= 100x/y, which x corresponds to the average integral of the vinyl doublets and y is the integral of anomeric proton plus 4% of α-1,4 linkages (Steenbergenj et al., 1995). The derivatization reaction presented a yield of 80.0% in mass of polymer and DS of 28.5%. Derivatized dextran act as macromonomer, allowing further polymerization process. The 13C NMR spectra of dextran sample is show in Fig. 1B. The peak at 98.09 ppm correspond to anomeric carbon (C1), while the carbons (C2–C5) connected to hydroxyl groups show a signal at 72.15 ppm. The carbon (C6) from eCH2 group of glycoside unit is assign to 65.46 ppm (Seymour, Knapp, & Bishop, 1976). The derivatized sample show ad- ditional peaks besides from pure dextran, which correspond to carbon atom from carbonyl group at 168.72 ppm. The peaks at 136.28 and 128.26 are attributed to the carbons from vinyl groups, respectively, while the signal at 18.58 is due to CH2-Ch group. Moreover, it is pos- sible noted two peaks at 97.88 and 98.38 ppm on derivatized sample near to anomeric carbon instead of only one signal. This shift indicated that the hydroxyl groups of C1 participated in the derivatization reac- tion (Zhang et al., 2014). The peak at 74–65 ppm was deconvoluted to Dex and Dex GMA samples, as can be seen in Fig. S2, which result in shift of peaks positions, indicating that the derivatization reaction also occurs by the hydroxyls bound to these carbons. 3.2. Characterization of SPION functionalized with acrylic acid The black precipitate of magnetite was obtained through the addi- tion of sodium hydroxide to a solution of ferric and ferrous chloride, in a molar ratio of 2:1. The aqueous suspension of the magnetite was Fig. 1. (A) 1H NMR spectra of dextran, glycidyl methacrylate (GMA) and dextran deri- vatized. (B) Solid state 13C NMR spectra of dextran and dextran derivatized. R.D. Piazza et al. Carbohydrate Polymers 178 (2017) 378–385 380 directly oxidized by aeration to form a brownish suspension of ma- ghemite. XRD analysis (Fig. S3(A)) of iron oxide sample Magh was indexed in the inverse spinel structure (Fd3m), in agreement with the protocols used to produce the superparamagnetic iron oxide SPION‐based mate- rials. The identification of structure of Magh sample was based on the Brag Peak Position of (1 1 1), (2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1) and (4 4 0) indices. According to the International Centre for Diffraction Data (JCPDS Card N° 39-1346), the features exhibited by Magh sample can be assigned to the maghemite structure. Broadening of the X-rays diffraction peaks is an indicative of the nanocrystalline nature of the synthesized powder (Cullity, 1978). The average X-rays crystallite diameter (DXRD) calculated by Scherrer’s equation was 6.2 nm and is in good agreement with electron microscopy data. The Raman spectrum of SPION samples is shown in Fig. S3(B) from 200 to 1000 cm−1 which vibrational modes are associated with ma- ghemite crystal structure. Typical Raman spectrum of maghemite is characterized by three main broad features, while magnetite shows only one feature broad structure, attributed to the A1g vibrational mode (Jubb & Allen, 2010). The Magh spectrum showed three bands at 345 cm−1 (Eg), 501 cm−1 (T2g), and 671 cm−1 (A1g) assigned to modes associated with tetrahedral iron sites, and one at 718 cm−1 assigned to the octahedral iron sites (Soler et al., 2011). Transmission electron microscopy was performed in order to access the Magh sample average particle size and morphology. It can be ob- served in Fig. 2(A) the TEM image of synthesized SPIONs with nearly spherical shape. The Fig. 2A inset shows particle size histogram of sample Magh obtained from the TEM micrographs. This data was fitted to a log-normal distribution and results in average particle diameter (DTEM) of 5.27 ± 0.05 nm and polydispersity index (PDI) of 0.21 ± 0.01. The size distribution was also measurement by dynamic light scattering for Magh bare sample, which result in average hydro- dynamic diameter of 48.50 ± 40 nm and PDI of 0.23 ± 0.01, as show in Fig. 2(B). This value is higher than shown in TEM images due to electric double layer, which is involved during DLS measurement (Easo &Mohanan, 2013). The DRIFT analysis was used to confirm the functionalization of Magh nanoparticles by acrylic acid. As show in Fig. S4, the DRIFT spectrum illustrates the characteristics infrared absorption bands of Magh bare and Magh-AA. The bands at 580 and 430 cm−1 correspond to FeeO stretching vibration modes. The 3430 and 1600 cm−1 bands at the Magh bare can be assigned to eOeH stretching and bending vi- brations, respectively, due to surface hydroxyl groups and water mo- lecules adsorbed on the SPION surface (Cornell & Schwertmann, 2003; Nakamoto, 1970). The surface modification through acrylic acid addi- tion was confirmed by the bands present at 1630 cm−1 assigned to carboxylate asymmetric and at 1434 cm−1 to symmetric stretching vi- brations. The weak band at 2911 cm−1 correspond to methine stretching of acrylic acid. It was not possible to assign the C]C stretching of the vinyl group because it shows weak absorption at 1670–1640 cm−1, the same region for hydroxyl groups of SPION sur- face (Pavia, Lampman, & Kriz, 2001). 3.3. Characterization of magnetic nanohydrogels The magnetic nanohydrogels were obtained through inverse mini- emulsion polymerization. In miniemulsion polymerization the droplet nucleation is the dominant mechanism of particle formation, which the monomer droplet being considered as a template for nanohydrogel formation, i.e. the size of nanohydrogel should be similar to the initial monomer droplet size (Asua, 2002; Gyergyek, Makovec, Mertelj, Huskić, & Drofenik, 2010; Luo et al., 2009). The dynamic light scat- tering technique was used to evaluate the nanohydrogels size dis- tribution. The z-average sizes are summarized in Table S1 (size dis- tribution profile is showed in Fig. S5). The hydrodynamic diameter distribution of nanohydrogels is in the range between 100 and 400 nm. It can be seen that size distribution of monomer droplet, before poly- merization step, was comparable to polymer nanohydrogels sizes. These results suggest that nanohydrogels are formed in a miniemulsion polymerization process. According to FTIR spectra showed in Fig. 3(A), the stretching vi- brations of FeeO bond were observed in the same wavenumber of Magh bare sample. Added to SPION absorptions bands, the character- istics of nanohydrogel correspond to the asymmetric and symmetric stretching at 2923 cm−1 and 2852 cm−1, respectively, that are assigned to CeH vibrations mode of dextran. The bands at 1463 cm−1 and 1353 cm−1 were attributed to methylene and methyl bending absorp- tions. The polysaccharides feature of CeOeC correspond to 1100 cm−1 stretching. The band exhibited at 1739 cm−1 is attributed to carbonyl group of derivatized dextran and PAA (Pavia et al., 2001). Fig. 3(B) shows the XRD pattern of magnetic nanohydrogels. The presence of main diffraction peaks of magnetite, according to JCPDS Card N° 39-1346, confirm that any structural change occurs during polymerization step, excluding the formation of other types of iron oxides. The width of diffraction peaks was broadened for magnetic nanohydrogels samples if compared with Magh bare sample in Fig. S4(A). Fig. 4 shows the macroscopic and scanning transmission electron microscopy (STEM) images of the magnetic nanohydrogels. Column (A) Fig. 2. (A) TEM micrography of Magh bare sample. The insert show particle size histo- gram, where vertical bars represent the experimental data whereas the solid line results from the curve fitting of the data using the log‐normal distribution function. (B) Average hydrodynamic diameter distribution of Magh bare sample from DLS measurements. R.D. Piazza et al. Carbohydrate Polymers 178 (2017) 378–385 381 show the suspended magnetic nanohydrogels in water in the absence of magnetic field, while in column (B) the samples were attracted to the magnet. The secondary electron image (column C) shows that the na- nohydrogels particles have aggregated in a globules form. These ag- gregates were formed during drying process of nanohydrogels suspen- sion. The encapsulation and distribution of iron oxide inside dextran- based nanohydrogels was evidenced by high-angle annular dark-field (HAADAF) and bright field images (Column D and E). The magnetic nanoparticles correspond to the black areas in the bright field image and to the bright areas in the HAADAF image. The amount of SPION encapsulated by nanohydrogels was eval- uated by TGA, in the range of 25–800 °C, as showed in Fig. S6. The Magh-bare sample shows only one step of 9.23% weight loss assigned to adsorbed water up to 130 °C. For magnetic nanohydrogels samples, two steps of weight loss were observed. The first step was associated with adsorbed water weight loss, in agreement with the Magh-bare sample. The second step, which starts at 165 °C and ends at 340 °C, was due to the decomposition of polymeric chains (Carp et al., 2009; Juríková, Csach, Koneracká, Kubov, & Kop, 2012). Table 1 shows the weight loss attributed to each TGA event, the iron oxide residue and the mass ratio of SPION per polymer content. The weight ratio among magnetic core content and nanohydrogels increased from 0.056 (sample Magh_Dex_50) to 0.113 (sample Magh_Dex_150). The results show that the sample Magh_Dex_100 reached the limit of encapsulation of SPION in dextran by inverse miniemulsion polymerization technique. The magnetic properties of the samples were measured using mag- netic hysteresis loop curves in the± 20000 Oe window, at 27 °C, as showed in Fig. 5(A). The saturation magnetization values were Fig. 3. (A) DRIFT spectra and (B) XRD patterns of magnetic nanohydrogel. Fig. 4. Digital and STEM images of sample Magh_Dex_50 (upper) and Magh_Dex_150 (bottom): (A) Suspended magnetic nanohydrogel in absence of magnetic field, (B) in presence of magnetic field, (C) secondary electron image, (D) HAADAF and (E) bright field images. Table 1 Summary of TGA data analysis on evaluation of SPION encapsulation. SPION Weight loss (%) SPION residue (%) SPION/polymer Step I Step II Magh‐bare 9.23 – 87.04 – Magh_Dex_50 11.39 83,9 4.71 0.056 Magh_Dex_100 9.14 81,8 9.06 0.110 Magh_Dex_150 9.84 81,0 9.16 0.113 R.D. Piazza et al. Carbohydrate Polymers 178 (2017) 378–385 382 normalized to the mass of iron oxide using TGA data (Medford et al., 2014). According to literature, the values of saturation magnetization for maghemite bulk were 83.5 emu g−1 (Cullity and Graham, 2009), while the Magh-bare sample this value decreased to 28.9 emu g−1. Decrease in saturation magnetization values of SPION with respect to the saturation magnetization of bulk counterparts is often observed in nanoparticles and is attributed to the surface contribution of spin canting, surface disorder, stoichiometric deviation, cation distribution (Kodama, 1999) and adsorbed layer species (Zhang, Su, Wen, & Li, 2008). For Magh_Dex_50, Magh_Dex_100 and Magh_Dex_150 samples the values of saturation magnetization were 32.6 emu g−1, 23.9 emu g−1 and 28.9 emu g−1, respectively. Magnetic nanohydrogel based on P(NIPPAm-co-AAc) synthesized by (Chou, Shih, Tsai, Chiu, & Lue, 2012) and (Fan, Li, Wu, Li, &Wu, 2011) showed different values of saturation magnetization, whether compared with our nano- particles, mainly due to the size of SPION which influence the magnetic properties (Iida, Takayanagi, Nakanishi, & Osaka, 2007). However, the SPION content is increased using miniemulsion polymerization method, which result in increased the sample magnetization. The superparamagnetic behavior is a desired magnetic property to use magnetic nanoparticle as drug delivery device. On insert of Fig. 5(A), it can be seen that samples show no coercivity and remanence values when the applied magnetic field was removed, suggesting that the nanoparticles showed superparamagnetic behaviour at 27.0 °C (Chou et al., 2012; Dou, Zhang, Jian, & Gu, 2010). To confirm that, zero field cooled (ZFC) and field cooled (FC) curves were obtained for all samples, as shown in Fig. 5(B). As observed, all samples show features consistent with a superpamagnetic behavior; i. e., a maximum in the ZFC trace and irreversible behavior between both traces below that maximum. Moreover, the position of the maximum (Tm) shows a de- pendence with the amount of coating. For the Magh bare sample the maximum is located at −178 °C and that maximum is shifted to lower temperature for the magnetic nanohydrogel samples. This result strongly suggests that the particle–particle magnetic interactions are relatively stronger for the bare sample and those interactions become weaker as the amount of coating is increased. 3.4. Colloidal stability Nanohydrogels were evaluated through zeta potential measure- ments in pH-dependence curve. The Magh-bare and magnetic nanohy- drogels samples were measured in the constant ionic strength of 1 mmol L−1 NaCl, as showed in Fig. 6 (upper). The amphoteric features of SPION are due to ionization of surface hydroxyl groups. Thus, the adsorption or desorption of protons have a pH dependence. In acid medium, the surface is protonated and the zeta potential value is po- sitive. On the other hand, the zeta potential is negative when the sur- face is deprotonated in basic solution. The isoelectric point (IEP) for Magh-bare was 8.2, which is in accordance with literature reports (Cornell & Schwertmann, 2003; Hajdú et al.,2012). The IEP for Magh AA sample was shift to 6.0. The magnetic nanohydrogels samples Fig. 5. (A) Magnetization versus applied field curves for Magh bare nanoparticles and magnetic nanohydrogels. The experimental data were normalized with respect to the iron oxide mass. The insert shows hysteresis loops near zero. (B) Zero-field-cooled (ZFC) and field-cooled (FC) curves as function of the temperature obtained with a DC magnetic field of H= 30 Oe. Fig. 6. pH dependence of zeta potential (upper) and pH dependence of hydrodynamic diameter (bottom) for Magh bare, Magh AA nanoparticles and magnetic nanohydrogels. R.D. Piazza et al. Carbohydrate Polymers 178 (2017) 378–385 383 Magh_Dex_50, Magh_Dex_100, and Magh_Dex_150 do not reach the IEP along of the pH range studied and show minimum zeta potential values at pH 2.0 of −0.16 mV, −0.47 mV and −1.04 mV, respectively. This behavior can be attributed to the carboxylic acid groups arising from the acrylic acid moieties present in the polymeric matrix and the sulfate groups from APS shear layer (Eissa et al., 2013). The zeta potential values were constants for magnetic nanohydrogels samples above pH 6.5. Fig. 6 (bottom) shows the hydrodynamic diameter in the pH range from 2.0 to 10.0, measured at a constant ionic strength of 1 mmol L-1 NaCl. The hydrodynamic diameter of Magh bare sample increase above pH 5.0. Although the potential zeta value indicate stability at this pH, no electrostatic or steric repulsion were predicted to this sample. Moreover, attractive dipolar magnetic force act in this sample, hin- dering the aggregates dispersion, until the instrument reached the limit of measurement (5 μm). To Magh AA sample, an increase in hydro- dynamic diameter was observed on isoelectric region, however, due to steric stabilization promoted by acrylic acid, the aggregation is not strong and the hydrodynamic diameter is restored. The magnetic na- nohydrogels samples show an increase in the average size near pH 3.0, which causes particle coagulation due to decrease in the electrostatic repulsion between nanohydrogels caused by protonation of the car- boxylate groups from the polymeric chains, as indicated in zeta po- tential curve (Fig. 6 upper). In fact, the magnetic nanohydrogels exhibit good colloidal stability and was observed no variation in hydrodynamic diameter over a range of pH between 6.5–10.0 where the zeta potential reaches constant values above −25.0 mV. In this pH range, the elec- trostatic repulsion is maximized due to complete deprotonation of the carboxylate groups. The colloidal stability of the magnetic nanohydrogels was evaluated in buffer solution of tris.HCl and PBS, in pH 7.4. The hydrodynamic diameters were measured during a 30 days period, as showed in Table 2. The magnetic nanohydrogels were stabilized by steric repul- sion promoted by the polymeric chains composed of dextran and the polyacrylate moieties. Thus, the magnetic nanohydrogels were stable over 30 days, once the hydrodynamic diameters did not show any sig- nificant increase over this period, except the sample Magh_Dex_150, in which the hydrodynamic diameter has increased to above 500 nm for both buffers solutions after the sample dispersion. This result may be related to the formation of aggregates after dispersion in buffer solu- tion. After a period of 8 days, the aggregates sedimented and hydro- dynamic diameter of the remained dispersed nanoparticles reach a steady state to 248.9 nm for tris.HCl and 218.5 nm for PBS. 4. Conclusions Magnetic nanohydrogels based on grafting polymerization of acrylic acid and derivatized dextran were obtained by encapsulating SPION through inverse miniemulsion technique. XRD, Raman and TEM results corroborate with the formation of maghemite by coprecipitation method. These magnetic nanoparticles show spherical morphology, with diameter is 5.27 ± 0.05 nm. Dextran macromonomer was deri- vatized by grafting with methacrylate groups on polysaccharide back- bone as confirmed by 1H NMR and solid state 13C NMR. Magnetic nanoparticles with desirable superparamagnetic behavior for biome- dical applications were encapsulated by dextran nanohydrogels. The samples Magh_Dex_50 and Magh_Dex_100 resulted in stable dispersion in buffer solutions at physiological pH indicating its colloidal stability. 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