Curcumin-loaded dual pH- and thermo-responsive magnetic microcarriers based on pectin maleate for drug delivery

Magnetic microgels with pH- and thermo-responsive properties were developed from the pectin maleate, N-isopropyl acrylamide, and Fe3O4 nanoparticles. The hybrid materials were characterized by infrared spectroscopy, scanning electron microscope coupled with X–ray energy dispersive spectroscopy, wide angle X–ray scattering, Zeta potential, and magnetization hysteresis measurements.

Carbohydrate Polymers 171 (2017) 259–266 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Curcumin-loaded dual pH- and thermo-responsive magnetic microcarriers based on pectin maleate for drug delivery Elizângela A.M.S Almeida a , Ismael C Bellettini d , Francielle P Garcia e , Maroanne T Farinácio a , Celso V Nakamura e , Adley F Rubira a , Alessandro F Martins b,c,∗ , Edvani C Muniz a,b a Grupo de Materiais Poliméricos e Compósitos (GMPC), Departamento de Qmica, Universidade Estadual de Maringá-UEM, 87020-900 Maringá-PR, Brasil Programa de Pós-graduac¸ão em Ciência e Engenharia de Materiais (PPGCEM), Universidade Tecnológica Federal Paraná (UTFPR), 86036-370 Londrina-PR, Brasil c Programa de Pós-graduac¸ão em Engenharia Ambiental (PPGEA), Universidade Tecnológica Federal Paraná (UTFPR), 86812-460 Apucarana-PR, Brasil d Departamento de Química, Universidade Federal de Santa Catarina–UFSC, 89065-300 Blumenau-SC, Brasil e Laboratório de Microbiologia Aplicada aos Produtos Naturais e Sintéticos, Departamento de Ciências Básicas da Saúde, Universidade Estadual de Maringá–UEM, 87020-900 Maringá-PR, Brasil b a r t i c l e i n f o Article history: Received 12 February 2017 Received in revised form 10 April 2017 Accepted May 2017 Available online 11 May 2017 Keywords: Pectin Magnetite Poly(N-isopropyl acrylamide) Curcumin Release a b s t r a c t Magnetic microgels with pH- and thermo-responsive properties were developed from the pectin maleate, N-isopropyl acrylamide, and Fe3 O4 nanoparticles The hybrid materials were characterized by infrared spectroscopy, scanning electron microscope coupled with X–ray energy dispersive spectroscopy, wide angle X–ray scattering, Zeta potential, and magnetization hysteresis measurements Curcumin (CUR) was loaded into the microgels, and release assays were carried out in simulated environments (SGF and SIF) at different conditions of temperature (25 or 37 ◦ C) A slow and sustainability CUR release was achieved under external magnetic field influence Loaded CUR displayed stability, bioavailability and greater solubility regarding free CUR Besides, the cytotoxicity assays showed that magnetic microgels without CUR could suppress the Caco-2 cells growth So, the pectin maleate, N-isopropyl acrylamide, and Fe3 O4 could be tailored to elicit hybrid-based materials with satisfactory application in the medical arena © 2017 Elsevier Ltd All rights reserved Introduction Curcumin (CUR) is widely applied in the biomedical and pharmaceutical fields owing to its anti-inflammatory capacity, and renowned potential to treat cystic fibrosis, Alzheimer’s disease and many cancer types (Maheshwari, Singh, Gaddipati, & Srimal, 2006) However, the clinical application of CUR is limited because of its low water solubility and weak oral bioavailability, as well as lack stability under neutral and mild alkaline conditions (Tang et al., 2010) These shortcomings should be overcome to achieve an efficient CUR approach Microencapsulation could be used to conceive CUR applicability, and many studies have been depicted the use of liposomes and polymeric composites (beads, particles, and films) ∗ Corresponding author at: Programa de Pós-graduac¸ão em Ciência e Engenharia de Materiais (PPGCEM), Universidade Tecnológica Federal Paraná (UTFPR), 86036-370 Londrina-PR, Brasil E-mail address: afmartins50@yahoo.com.br (A.F Martins) http://dx.doi.org/10.1016/j.carbpol.2017.05.034 0144-8617/© 2017 Elsevier Ltd All rights reserved to improve CUR efficacy (Chen et al., 2009; Li, Ahmed, Mehta, & Kurzrock, 2007; Martins et al., 2013; Shi et al., 2007) Polymeric materials often impart biocompatibility, biodegradability, and non-toxicity (Abolmaali, Tamaddon, & Dinarvand, 2013) to the composite materials Regarding the drug delivery approaches, polymeric composites act as protective matrices, repress non-specific interactions with biological molecules, foster controlled and sustained release, and enhance the bioavailability and stability of the loaded drug (Brewer, Coleman, & Lowman, 2011) Composite materials may increase the CUR solubility, decreasing its crystallinity because of the establishment of new interactions between polymer chains and CUR (Facchi et al., 2016) Smart polymeric devices are developed focusing responsiveness to an external magnetic field as well as changes in temperature and pH (Patra et al., 2015) These properties are mainly required for drug delivery purposes Biocompatible and superparamagnetic iron nanoparticles (SPIONs) have been highlighted due to their renowned magnetization property A magnetic-based material, containing a loaded drug can adjust the release rate under external 260 E.A.M.S Almeida et al / Carbohydrate Polymers 171 (2017) 259–266 Table Experimental conditions used to create the microparticles, loaded iron atom levels (g g−1 ) and Fe3 O4 encapsulation efficiencies (EE%) Samples NIPAAm wt.% Fe3 O4 wt.% EE(%) Fe (10−2 g g−1 ) AP-MA/PNIPAAm(10)-Fe3 O4 (1) AP-MA/PNIPAAm(2)-Fe3 O4 (10) AP-MA/PNIPAAm(10)-Fe3 O4 (10) AP-MA/PNIPAAm(2) 10 2.0 10 2.0 1.0 10 10 – 39 31 25 – 0.58 4.70 3.70 – magnetic field influence (Patra et al., 2015) Biopolymers should improve the SPIONs biocompatibility (Patel, Kumar, Jayawardana, Woodworth, & Yuya, 2014) and stability (Kim, Kim, Kim, & Lee, 2009), featuring potential for target delivery (Menegucci, Santos, Dias, Chaker, & Sousa, 2015), magnetic resonance imaging (MRI) (Song et al., 2015) and hyperthermia purposes (Kim et al., 2009) Overall, metallic nanoparticles (MNp) are covered by biopolymer layers or encapsulated into a polymeric matrix Polymer networks stabilize the MNp by steric hindrance, suppressing the aggregation due to the changes of pH and ionic strength (Tiwari, Mishra, Mishra, Arotiba, & Mamba, 2011) Temperature and pH-responsiveness are also paramount for acquisition of target delivery devices Poly(Nisopropyl acrylamide) (PNIPAAm) is a thermo-responsive polymer widely studied because contains a Lower Critical Solution Temperature (LCST) around 32 ◦ C, just below human body temperature Therefore, the PNIPAAm may be associated with polymeric systems to yield target delivery approaches with temperature sensibility (Jaiswal, Banerjee, Pradhan, & Bahadur, 2010) Pectin (AP) is a polysaccharide formed by galacturonic acid units, identified as a structural component of plant cell walls It is found in leaves, fruits, flowers, roots, and seeds Pectinbased hydrogels have been used in pharmacy because of the biocompatibility, biodegradability, non-toxicity and pH-responsive properties (Maxwell, Belshaw, Waldron, & Morris, 2012) Also, the AP decreases the blood cholesterol level (Brouns et al., 2012; Terpstra, Lapre, de Vries, & Beynen, 1998), reduces the glucose uptake (Kim, 2005), and supplies anti-tumor activity (Maxwell et al., 2012; Zhang, Xu, & Zhang, 2015) In a natural condition, galacturonic acid units over AP-based hydrogels are ionized, permitting repulsion among them, and hence swelling and release of drugs by diffusion So, these AP-systems may promote a satisfactory drug delivery in the colon place, favoring the uptake (Liu, Fishman, Kost, & Hicks, 2003 ; Wong, Colombo, & Sonvico, 2011) The development of magnetic materials with pH and thermal responses was proposed in this study The PNIPAAm grafted on the pectin maleate (AP–MA), and Fe3 O4 were associated to elicit microgels using oil/water emulsion (O/W) and poly(vinyl alcohol) (PVA) as a stabilizing agent The AP-MA synthesis, as well as its physicochemical properties, were previously reported in a recent paper published by our research group (Almeida et al., 2014) Hybrid materials were loaded with curcumin (CUR), and release tests assessed as a function of an external magnetic field at different conditions of pH and temperature This work will demonstrate that as-obtained microgels based on AP–MA/PNIPAAm/Fe3 O4 can kill Caco-2 cells and to provide a suitable matrix for CUR delivery In this case, AP–MA/PNIPAAm/Fe3 O4 composite may improve the solubility, stability, and bioavailability of the CUR Materials and methods 2.1 Materials Apple pectin (Mw = 2.9 × 105 g mol−1 ), sodium persulfate (98%), iron oxide (Fe3 O4 , 98%) of particle size up to 50 nm and curcumin (Turmeric, ≥65%) were purchased from Sigma-Aldrich (Brazil) Maleic anhydride (99%) and N,N,N’,N’–tetramethylethylenediamine (TEMED, 99%) were sup- plied by Vetec (Brazil) Poly(vinyl alcohol) (PVA, 88% hydrolyzed with Mw = 2.2 × 104 g mol−1 ) and N–isopropyl acrylamide (99%) were acquired from Across Organics (Brazil) Other reactants also utilized in this work, were of analytical grade and used as received 2.2 Pectin maleate/poly[N–isopropyl acrylamide]/Fe3 O4 microparticles synthesis Firstly, the AP-MA with a substitution degree of 24% was synthesized from apple pectin (Mw = 2.9 × 105 g mol−1 ) according to the previously procedure published by our research group (Almeida et al., 2014) Then, for microparticles synthesis, the aqueous phase (W) containing AP–MA (2.5 wt.%) and PVA (3.0 wt.%) was added to the oil phase (benzyl alcohol; BnOH) containing an appropriate Fe3 O4 content (Table 1), keeping the volume ratio at 1/3 (W/BnOH) The emulsion was prepared into ice bath under N2(g) from the sonication method (Hielscher Ultrasonic Processor, model UP200S) at 100% amplitude for Then, suitable quantities of NIPAAm (2.0 or 10 wt.%, Table 1), sodium persulfate (1.5 wt.%) and TEMED (1.0 wt.%) were added to the emulsion, maintained the sonication for more 15 The suspension containing the AP–MA/PNIPAAm–Fe3 O4 was precipitated in acetone, centrifuged, washed five times also with acetone, stocked under vacuum (24 h) and lyophilized (Mauricio, Guilherme, Kunita, Muniz, & Rubira, 2012) The experimental procedures are summarized in Table The microparticles will be called as AP–MA/PNIPAAm(x)–Fe3 O4 (y), where “x” and “y” correlate the NIPAAm and Fe3 O4 levels (%), respectively 2.3 Characterization Spectra of the AP-AM and AP-AM graphitized with PNIPAAm (Supplementary Material; Fig S1) were performed in a Varian Mercury Plus 300BB NMR spectrometer, operating at 300.06 MHz for H frequency 10 mg of the samples were dissolved in 1.0 mL D O, containing TMS as reference H NMR spectra were acquired at room temperature and the main acquisition parameters were as follows: pulse of 90◦ , recycle delay of 30 s and acquisition of 64 transients Measurements of Infrared Spectroscopy (FTIR) were recorded using a Fourier Transform Infrared Spectrophotometer (Shimadzu Scientific Instruments, Cary 630 Model), operating from 500 to 2000 cm−1 , at a resolution of cm−1 after 64 scans Scanning Electron Microscope (SEM) coupled with X–Ray Energy Dispersive Spectroscopy (EDS) was used to evaluate the morphology and to predict the material composition, using a Shimadzu apparatus (SS 550 model) Average diameters of the microparticles were assessed by SEM images using the software Size Meter© , version 1.1 The LCST was evaluated by Zeta potential measurements at different temperature conditions Microgel suspensions were carried out in a phosphate buffer solution (pH 7.0) at 1.0 mg mL−1 and placed in a Zetasizer Nano ZS apparatus at different temperatures as well The magnetization was estimated using a magnetometer (VSM), at 100 Hz frequency under 33 Oe s−1 field scan rate (25 ◦ C) The amount of iron into the microparticles was assessed by Flame Atomic Absorption Spectroscopy (FAAS), through a Varian AA–175 model (USA) Wide angle X–ray scattering (WAXS) profiles E.A.M.S Almeida et al / Carbohydrate Polymers 171 (2017) 259–266 261 Fig SEM images (left panel) and size distribution of the microparticles (right panel): (a, b and c) AP–MA/PNIPAAm(10)–Fe3 O4 (1), (d, e and f) AP–MA/PNIPAAm(2)–Fe3 O4 (10) and (g, h and i) AP–MA/PNIPAAm(10)–Fe3 O4 (10) (2␪ = 5–70◦ ) were obtained from a Shimadzu XRD–600 apparatus (Japan), equipped with a Ni-filtered Cu-K␣ radiation 2.4 Curcumin loading Curcumin (CUR; 1.0 mg) was solubilized in ethanol (100 mL) and, then 1.0 g AP–MA/PNIPAAm(10)–Fe3 O4 (1) was added into the CUR-solution (Martins et al., 2013) The suspension was kept under stirring for 20 h (25 ◦ C), avoiding light exposure After, the loaded microparticles were centrifuged, dried under vacuum (24 h) and lyophilized The entrapped CUR was assessed at ␭ = 430 nm, using an UV–vis spectrophotometer (Femto 800Xi model) from a standard curve ranging from 0.080 to 5.0 mg L−1 ; y = −9.9044 × 10−4 + 0.13384x (R2 = 0.999) 2.5 Curcumin release Different environments were used to evaluate the CUR release: simulated intestinal fluid (SIF; pH 6.8) and simulated gastric fluid (SGF; pH 1.2) (Bueno et al., 2015) The studies were performed in an USP type I dissolutor apparatus (Ethik Technology, 299–6TS model − Brazil) under mechanical stirring (40 rpm) at 25 ◦ C or 37 ◦ C at the presence or absence of the magnetic field (63.8 Oe) So, 0.10 g of dried AP–MA/PNIPAAm(10)–Fe3 O4 (1)/CUR was deposited in cellulose membranes (12 kDa size pore), containing SGF or SIF (30 mL) The system was stored in a sealed flask, containing 220 mL of SGF or SIF, avoiding the light exposure For assessing the magnetic field influence, an external magnetic field was created by a magnet placed outside the sealed flask containing the dialysis bag The released CUR was determined by UV–vis absorbance (␭ = 430) removing 3.0 mL aliquots at appropriate time intervals (Martins et al., 2013) The fraction of released CUR was evaluated from Eq (1) Relesead fraction = relesead amount × 100% encapsulated amount (1) 2.6 Cytotoxicity assay Cytotoxicity tests were assessed against VERO cells (kidney cells of African green monkey) and Caco-2 cells (human colonic adenocarcinoma cells), from the sulforhodamine B method (Almeida et al., 2014) The cells were cultured and maintained in Dulbecco’s ® modified Eagle’s medium (DMEM; Gibco , Grand Island, USA), supplemented with 10% heat-inactivated foetal bovine serum (FBS; ® Gibco ) and 50 ␮g mL−1 gentamycin, in an incubator at 37 ◦ C, with 5% CO2 and 95% relative humidity Cells were obtained at a density of 2.5 × 105 cells mL−1 after trypsinization and added to 96–well plate for 24 h at the same conditions described previously After the adherence, a fixed volume of each microparticle suspension at different concentrations (10, 100, 500 and 1000 ␮g mL−1 ) was added into the 96–well plate and incubated for 48 h The cellular toxicity (CC50 ) is defined when the sample level kills 50% of the treated cells regarding the control (untreated cells) Results and discussion 3.1 Characterization The methodology used to synthesize the microgels was based on inverse polymerization technique (Mauricio et al., 2012) H NMR analysis indicated that PNIPAAm was grafted in the AP–MA chains The resonance signals ranging from 6.5 to 6.2 ppm on AP–MA H NMR spectrum was attributed to the hydrogen atoms in vinylic 262 E.A.M.S Almeida et al / Carbohydrate Polymers 171 (2017) 259–266 Fig (a) FTIR spectra of the AP–MA/PNIPAAm(2), AP-MA and AP–MA/PNIPAAm(2)–Fe3 O4 (10) (left panel) (b and c) EDS spectra and WAXS profiles, respectively: (i) AP–MA/PNIPAAm(10)–Fe3 O4 (1), (ii) AP–MA/PNIPAAm(2)–Fe3 O4 (10), and (iii) AP–MA/PNIPAAm(10)–Fe3 O4 (10) (right panel) Fig (a) Paramagnetic response based on the hysteresis curves (b) Zeta potential measurements as a function of the temperature Sample codes: (i) AP–MA/PNIPAAm(10)–Fe3 O4 (1), (ii) AP–MA/PNIPAAm(2)–Fe3 O4 (10), and (iii) AP–MA/PNIPAAm(10)–Fe3 O4 (10) moieties (Supporting Material, Fig S1) These signals disappeared in the AP–MA/PNIPAAm H NMR spectrum, indicating that NIPAAm grafting on AP–MA was successful Such graphitization was also confirmed due to the NIPAAm signals in the AP–MA/PNIPAAm H NMR spectrum at 3.0 and 2.7 ppm ascribed to the hydrogen atoms on CH and CH2 , respectively (Supporting Material, Fig S1) (Leal, De Borggraeve, Encinas, Matsuhiro, & Muller, 2013) The ultrasound waves ensured the obtaining of multiple and stable oil emulsion seeking to create microparticles (Reis et al., 2011) The cavitation energy induced by the sonication led to the formation of rough, polydisperse and spherical microparticles with small fractures and deformities, due to the water randomly movements into the oil phase drops (Fig 1) (Reis et al., 2011) The average diameters of AP–MA/PNIPAAm(10)–Fe3 O4 (1), AP–MA/PNIPAAm(2)–Fe3 O4 (10) and AP–MA/PNIPAAm(10)–Fe3 O4 (10) were ca 10, 26, and 20 ␮m, respectively (Fig 1) The size and polydispersity of the microgels were raised when the magnetite level was also improved (from 1.0 up to 10 wt.%; Table 1), while the NIPAAm content did not change these features significantly The mild band at 600 cm−1 in the AP–MA/PNIPAAm(2)–Fe3 O4 (10) FTIR spectrum was assigned to Fe–O stretching due to the Fe3 O4 load (Fig 2a) (Mauricio et al., 2012) The band at 1740 cm−1 on AP–MA FTIR spectrum attributed to C O stretching of carboxylic acids was shifted for 1750 cm−1 in the AP–MA/PNIPAAm(2)–Fe3 O4 (10) spectrum, suggesting the establishment of new interactions (Mauricio et al., 2012) Overall, all the FTIR spectra predicted in Fig 2a were similar between them The magnetite stabilized the emulsion (da Silva et al., 2014; Fang et al., 2014) because microparticles based on AP–MA/PNIPAAm(2) was not obtained (Supporting Material, Fig S2) The Fe3 O4 may neutralize the excess of negative charges over the AP–MA chain segments by hindrance effects owing to the establishment of new bonds among COO− and Fe (Fang et al., 2014) The EDS spectra E.A.M.S Almeida et al / Carbohydrate Polymers 171 (2017) 259–266 263 Fig Fraction of released curcumin (%) from the AP–MA/PNIPAAm(10)–Fe3 O4 (1) at SIF and SGF under or not magnetic field influence: (a) at 25 and (b) at 37 ◦ C (c) Cytotoxicity effects against Caco-2 and VERO cells: (i) AP–MA/PNIPAAm(10)–Fe3 O4 (1), (ii) AP–MA/PNIPAAm(2)–Fe3 O4 (10) and (iii) AP–MA/PNIPAAm(10)–Fe3 O4 (10) Error bars represent the standard deviation of triplicates (n = 3) displayed the iron incidence in the microgels because of the peaks at 6.4 and 7.1 keV (Fig 2b) Even more, the WAXS profiles exhibited characteristic diffraction peaks assigned to the Fe3 O4 crystalline planes at 2␪ = 30◦ (220), 35◦ (311), 43◦ (400), 54◦ (422), 57◦ (511) and 63◦ (440) (Fig 2c) (Purushotham & Ramanujan, 2010) The AP–MA/PNIPAAm(10)–Fe3 O4 (1) WAXS pattern encompassed lowered diffraction peaks regarding the other samples due to the less level of loaded iron (0.58 × 10−2 g g−1 ; Table 1) Fig 3a displayed the paramagnetic behaviors of the microgels, and in Table was depicted the content of iron (g−1 ) loaded in the microparticles The magnetization of the AP–MA/PNIPAAm(10)–Fe3 O4 (1), AP–MA/PNIPAAm(2)–Fe3 O4 (10) and AP–MA/PNIPAAm(10)–Fe3 O4 (10) reached 0.68 ± 0.01, 6.20 ± 0.10 and 4.20 ± 0.05 emu g−1 , respectively Polymer networks should screen the iron atoms, suppressing the magnetic response (Fang et al., 2014) The AP–MA/PNIPAAm(2)–Fe3 O4 (10) achieved the highest magnetic response (6.20 ± 0.10 emu g−1 ) due to the large Fe3 O4 content (4.7 × 10−2 g g−1 ; Table 1) TGA analysis reinforced this fact since the AP–MA/PNIPAAm(2)–Fe3 O4 (10) imparted greater residual mass above 450 ◦ C, owing to the Fe3 O4 content (Support Information, Fig S3) The thermo-sensitive feature was evaluated by Zeta potential (␨) analysis at different temperature conditions, ranging from 25 to 50 ◦ C (Fig 3b) For all samples, the ␨ achieved −10 to −25 mV at pH 7.0 owing to the COO sites on AP-MA chains The ␨ values decreased at higher temperatures because the suspension was collapsed and conceived the LCST event (Pietsch et al., 2012) The LCST for AP–MA/PNIPAAm(2)–Fe3 O4 (10) was allowed at 37 ◦ C, whereas for AP–MA/PNIPAAm(10)–Fe3 O4 (10) and AP–MA/PNIPAAm(10)–Fe3 O4 (1) was close to 43 ◦ C The less PNIPAAm content grafted in AP–MA/PNIPAAm(2)–Fe3 O4 (10) lowered the LCST An LCST-type above the body temperature (≈37 ◦ C) can allow remarkable efficiency to the composite materials to act as drug delivery vehicles (Fang et al., 2014; Jaiswal et al., 2010) 3.2 Curcumin (CUR) release The AP-MA/PNIPAAm(10)-Fe3 O4 (1) sample was chosen as a drug carrier matrix due to its higher LCST (43 ◦ C) and smaller average size (≈10 ␮m) The CUR encapsulation efficiency (EE) reached 60% Release assays were assessed in simulated fluids (SIF or SGF) at 25 or 37 ◦ C, under (or not) magnetic field influence In SIF (25 ◦ C) and without magnetic field influence, the equilibrium was reached after 35 h when 50% CUR was released On the other hand, in the same condition, but under magnetic field influence, the equilibrium was permitted at 80 h, designing a slowed, modulated and sustained CUR release (90%) (Fig 4a) In SGF (25 ◦ C), the amount of CUR released was not higher than 10% for all evaluated conditions (Fig 4a) The exposition to the magnetic field may induce heat inside the AP–MA/PNIPAAm(10)-Fe3 O4 (1), decreasing the stability and hence enhancing CUR release rate (Kumar & Mohammad, 2011; Patra et al., 2015) At 37 ◦ C, the magnetic field did not significantly change the profile of CUR release (Fig 4b) In SIF (37 ◦ C), the released fraction achieved 95 and 80% in the presence or absence of magnetic field, respectively In SGF (37 ◦ C) the content of CUR released was lower, featuring 20% without and 6% with magnetic field influence 264 E.A.M.S Almeida et al / Carbohydrate Polymers 171 (2017) 259–266 Table Kinetic parameters (n, k, ␣, and kr ) obtained by application of Ritger-Peppas and Reis et al mathematical models on the CUR release curves evaluated in SIF Temperature (◦ C) Ritger–Peppas model Reis et al model R2 kr 25 25a 37 37a a n k R2 0.45 0.15 0.48 0.47 −1.17 −1.17 −0.93 −0.89 0.930 0.710 0.940 0.960 1.11 7.05 4.89 23.24 1st 2nd 1st 2nd 0.030 0.024 0.023 0.020 0.040 0.042 0.040 0.052 0.900 0.980 0.973 0.962 0.900 0.930 0.980 0.964 CUR release under magnetic field influence − 1st and 2nd refer to first- and second-order kinetics The amount of CUR released rose at 37 ◦ C due to the PNIPAAm The AP–MA/PNIPAAm(10)-Fe3 O4 (1) LCST was roughly 43 ◦ C, and release tests were carried out at 37 ◦ C (Fig 4b) These temperature conditions were close, and some PNIPAAm chains could become hydrophobic, causing polymer network collapses and enhancing the released level of CUR, especially in SIF (37 ◦ C) These features are also associated with hydrogel pH-responsiveness At pH 1.2 (SGF), all the carboxylic groups on AP–MA are entirely protonated, hindering the interactions with water molecules This fact suppressed the CUR release However, at pH 6.8 (SIF) the carboxylic sites are fully ionized ( COO− ), improving negative charge density, as predicted by ␨ measurements Then, the water molecules interact better with the hybrid material, favoring the CUR delivery Fe3 O4 nanoparticles cause tortuosity into a polymeric matrix, eliminating the burst release of CUR (da Silva et al., 2014) CUR comprises crystalline arrangement and has lack water solubility (≈11 ng mL−1 in a buffer solution at pH 5.0) (Martins et al., 2013) In this study, the solubility and stability of the loaded CUR were improved AP-MA/PNIPAAm(10)-Fe3 O4 (1) microparticles protected the CUR integrity allowing sustained release at pH close to physiological condition According to the assay performed at 25 ◦ C (SIF under magnetic field influence), the solubility of loaded CUR reached 216 ␮g mL−1 Besides, the CUR was not degraded at pH 6.8 (SIF), and its bioavailability was also enhanced These findings open new strategies for developing of CUR-based materials with application in the medical field 3.3 Transport mechanism The release of solutes from a flexible matrix may be described by diffusion transport (Ritger & Peppas, 1987) Ritger-Peppas proposed a semi-empirical mathematical model to explain the release mechanism of solutes (Eq (1) (Ritger & Peppas, 1987) Mt = kt n M∞ (2) Where Mt /M∞ refers to the solute fraction released in the desired time interval (t); k is the kinetic constant, and its value relies on the factors such as solvent, type and shape of the hydrogel, as well as depends on the experimental conditions In Eq (1), n represents the diffusion exponent being associated with the solute transport mechanism (Brazel & Peppas, 1999; Guilherme et al., 2010) and hydrogel geometry For n = 1, the mechanism is governed by a zero–order kinetic and the release rate rises with the time linearly and relies on the macromolecular relaxation When n is roughly 0.5, the release mechanism is controlled by Fickian diffusion; for n values ranging from 0.5 to 1.0, the anomalous transport takes place because of the simultaneous diffusion and macromolecular relaxation Regarding the spherical matrices, the n parameter can assume the values: 0.43 (Fickian diffusion), 0.43–0.85 (anomalous or non-Fickian), and 0.85 (zero–order kinetic) (Ritger & Peppas, 1987) The n and k parameters were assessed from the release curves performed in SIF (Table 2) For n values close to 0.5, the release was explained through non-Fickian transport (assays with fractions of released CUR at 80, 90 and 95%; Figs 4a–b) However, for the test with 50% of released CUR, the lower n value (0.15) showed that polymeric matrix/CUR set did not have affinity by the SIF (assay at 25 ◦ C in the absence of magnetic field) At the equilibrium condition, the released fraction (Fr ) reaches a maximum (Fmax ), and the transport mechanism can be treated as a diffusion-partition phenomenon (Reis, Guilherme, Rubira, & Muniz, 2007) For applying this model, the Fr and Fmax parameters were also assessed from the curves carried out in SIF, and the kinetic variables (kr ) predicted for first–order kinetic (Eq (3) and second–order kinetic (Eq (4)) (Reis et al., 2007) were measured as well (Table 2) The partition activity (␣ coefficient) is defined when the solute concentration reaches a constant value (Reis et al., 2007) For ␣ > 0, the solute diffusion takes place between the solvent and hydrogel phases, inferring better affinity between solute and solvent When ␣ = the solute release does not occur There is not any solute partition between the solvent and hydrogel phases (Reis et al., 2007) All studies showed ␣ > 0, indicating the diffusion-partition occurrence (Table 2) kr t = Fmax ln Kr t = ˛ ln Fmax Fmax − Fr Fr − 2Fr Fmax + Fmax Fmax − Fr (3) (4) The Ritger–Peppas model is valid for a time interval in which 60% solute is released (Ritger & Peppas, 1987), while the diffusionpartition model can predict the release curve profile entirely (Reis et al., 2007) The Ritger–Peppas model was adjusted better to the experimental outcomes performed at 37 ◦ C (R2 ≥ 0.940; Table and Fig S4) However, the diffusion-partition model is in good accordance with the results in the interval of 60–160 h (Fig S4 (e, f, g, and h)) (Reis et al., 2007) The release assays at 37 ◦ C were followed for a second-order kinetic (R2 ≥ 0.900; Table 2) and those at 25 ◦ C were predicted by a first-order kinetic (R2 ≥ 0.964; Table 2) All conditions carried out in SIF pinpointed low and positive kr values, explaining the sustained CUR release (Table 2) 3.4 Cytotoxicity assay Cell viability tests against the Caco–2 colon cancer and healthy VERO cells were evaluated from the microparticles without CUR (Fig 4c) The results indicated that AP–MA/PNIPAAm(10)–Fe3 O4 (1) imparted highest cytotoxic effect on the Caco–2 cells (65 ␮g mL−1 CC50 ), while AP–MA/PNIPAAm(2)–Fe3 O4 (10) and AP–MA/PNIPAAm(10)–Fe3 O4 (10) CC50 achieved 215 and 435 ␮g mL−1 , respectively The largest cytotoxicity found for AP–MA/PNIPAAm(10)–Fe3 O4 (1) towards Caco–2 cells could be attributed to the lowest loaded Fe3 O4 amount (Table 2) AP–MA (the pristine pectin maleate) possesses greater inhibitory activity (CC50 = 25 ␮g mL−1 ) for Caco–2 cells as previously shown in work published by our research group (Almeida et al., 2014) The raw AP CC50 reached 140 ␮g mL−1 Fe3 O4 oxide and PNIPAAm contents play significative role in the findings of cytotoxicity Higher levels E.A.M.S Almeida et al / Carbohydrate Polymers 171 (2017) 259–266 of Fe3 O4 and PNIPAAm could shield the AP-MA efficacy because the AP–MA/PNIPAAm(10)–Fe3 O4 (10) CC50 was improved for 435 ␮g mL−1 Reports suggest the antitumor activity of AP-MA was related to anhydride hydrolysis, and hence the formation of hydrogen–bonded fragments (Gao et al., 2016; Pascalau et al., 2016; Zhang et al., 2016) The Fe3 O4 /PNIPAAm excess may hinder the establishment of H–bonds among polymer networks, decreasing the antitumor activity (Almeida et al., 2014; Karakus, Yenidunya, Zengin, & Polat, 2011) For healthy VERO cells, the AP–MA/PNIPAAm(10)–Fe3 O4 (10), AP–MA/PNIPAAm(2)–Fe3 O4 (10) and AP–MA/PNIPAAm(10)–Fe3 O4 (1) CC50 were 370, 365 and 375 ␮g mL−1 , respectively So, all the microparticles were not cytotoxic towards VERO cells It was featured that AP–MA/PNIPAAm/Fe3 O4 set could be tailored to elicit cytocompatibility for Caco-2 cells, stability, and bioavailability for CUR, comprising an efficient device for CUR delivery The cytotoxicity of the AP–MA/PNIPAAm/Fe3 O4 /CUR composites were not assessed Many studies, including two papers published by our group, depicted that CUR-based composites could suppress the growth of cancerous cells (Martins et al., 2013; Pereira et al., 2013) Conclusions A new hybrid and magnetic material based on maleate pectin (AP–MA), PNIPAAm and Fe3 O4 was pH and thermal-responsive The curcumin (CUR) was loaded into the microparticles and release findings were favored in SIF Besides, the magnetic field influenced the tests carried out at 25 ◦ C Overall, the CUR release was slowly and sustainably The stability and bioavailability of the CUR were also improved, and cytotoxicity assays revealed that microparticles without CUR were cytocompatible for healthy VERO cells For diseased Caco-2 cells, the growth was suppressed mainly under AP–MA/PNIPAAm(10)–Fe3 O4 (1) presence (CC50 = 65 ␮g mL−1 ) The AP–MA/PNIPAAm/Fe3 O4 features can be useful to yield a potential carrier device for CUR delivery Acknowledgements E.A.M.S.A thanks the CNPq for her Master fellowship E.C M thanks to CNPq (Proc 400702/2012–6 and 308337/2013–1) and Nanobiotec (Proc 851/09) for financial supports The authors also acknowledge Professors: L.C Varanda (IQSC − USP), D.R Cornejo (IFSC − USP), I A dos Santos (DFI − UEM) and E Radovanovic (DQI − UEM) for their help during the developing of this work Appendix A Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.carbpol.2017.05 034 References Abolmaali, S S., Tamaddon, A M., & Dinarvand, R (2013) A review of therapeutic challenges and achievements of methotrexate delivery systems for treatment of cancer and rheumatoid arthritis Cancer Chemotherapy and Pharmacology, 71(5), 1115–1130 Almeida, E A M S., Facchi, S P., Martins, A F., Nocchi, S., 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(2015) Preparation and characterization of biofunctionalized chitosan/Fe3O4 magnetic nanoparticles for application in liver magnetic resonance imaging Journal of Magnetism and Magnetic Materials,... 2003 ; Wong, Colombo, & Sonvico, 2011) The development of magnetic materials with pH and thermal responses was proposed in this study The PNIPAAm grafted on the pectin maleate (AP–MA), and Fe3... (CUR), and release tests assessed as a function of an external magnetic field at different conditions of pH and temperature This work will demonstrate that as-obtained microgels based on AP–MA/PNIPAAm/Fe3