S p d C S A a b c d T e B a A R R 1 A A K C N G I E c 1 D a b C d e G P B h 0 Carbohydrate Polymers 157 (2017) 1695–1702 Contents lists available at ScienceDirect Carbohydrate Polymers j ourna l ho me page: www.elsev ier .com/ locate /carbpol ynthesis and factorial design applied to a novel chitosan/sodium olyphosphate nanoparticles via ionotropic gelation as an RGD elivery system harlene Priscila Kiilll a,∗, Hernane da Silva Barudb, Sílvia Helena Santagneli c, idney José Lima Ribeiroc, Amélia M. Silvad, Agnieszka Tercjake, Junkal Gutierreze, ndressa Maria Pironib, Maria Palmira Daflon Gremiãoa Univ. Estadual Paulista, Faculty of Pharmaceutical Sciences, Rod. Araraquara-Jau, Km 1, 14802-901, Araraquara, SP, Brazil Laboratório de Biopolímeros e Biomateriais (BioPolMat) – Centro Universitário de Araraquara (UNIARA), Araraquara, SP, Brazil Institute of Chemistry, UNESP – Univ. Estadual Paulista, Rua Prof. Francisco Degni, 55, 14800-900, Araraquara, SP, Brazil Department of Biology and Environment, Centre for Research and Technology of Agro Environmental and Biological Sciences (CITAB/UTAD), University of rás-os-Montes e Alto Douro (UTAD), P.O. Box 1013, 5001-801, Vila Real, Portugal Group ‘Materials + Technologies’ (GMT), Department of Chemical and Environmental Engineering, Engineering College of Gipuzkoa, University of the asque Country (UPV/EHU), Plaza Europa 1, 20018 Donostia-San Sebastián, Spain r t i c l e i n f o rticle history: eceived 20 July 2016 eceived in revised form 8 November 2016 ccepted 18 November 2016 vailable online 21 November 2016 eywords: hitosan a b s t r a c t Chitosan nanoparticles have been extensively studied for both drug and protein/peptide delivery. The aim of this study was to develop an optimized chitosan nanoparticle, by ionotropic gelation method, using 32 full factorial design with a novel polyanion, sodium polyphosphate, well known under the trade name Graham salt. The effects of these parameters on the particle size, zeta potential, and morphology and association efficiency were investigated. The optimized nanoparticles showed an estimated size of 166.20 ± 1.95 nm, a zeta potential of 38.7 ± 1.2 mV and an efficacy of association of 97.0 ± 2.4%. The Atomic Force Microscopy (AFM) and Scanning Electronic Microscopy (SEM) revealed spherical nanoparticles with uniform size. Molecular interactions among the components of the nanoparticles and peptide were anoparticles raham’s salt onic gelation xperimental factorial design RGDfV evaluated by Fourier Transform Infrared Spectra (FTIR) and Differential Scanning Calorimetry (DSC). The obtained results indicated that, the developed nanoparticles demonstrated high biocompatible, revealing no or low toxicity in the human cancer cell line (Caco-2). In conclusion, this work provides parameters that contribute to production of chitosan nanoparticles and sodium polyphosphate with desirable size, biocompatible and enabling successful use for protein/peptides delivery. © 2016 Elsevier Ltd. All rights reserved. . Introduction Chitosan (Ch) is a polysaccharide composed of �-(1-4)-linked -glucosamine and N-acetyl-d-glucosamine. In particular, Ch has dvantageous biological properties, such as relative non-toxicity, iodegradability, biocompatibility and bioadhesive characteristics. hitosan-based nanoparticles have been extensively studied for elivery of therapeutic proteins and peptides (1990; Fundueanu t al., 2004; Gavini et al., 2006; Gavini, Rassu, Sanna, Cossu, & iunchedi, 2005). Ch have positively charged surfaces in biologi- ∗ Corresponding author at: Faculdade de Ciências Farmacêuticas, Univ. Estadual aulista – UNESP, Rodovia Araraquara-Jau, Km 1, CEP, 14801-902, Araraquara, SP, razil. E-mail address: char kiill@yahoo.com.br (C.P. Kiilll). ttp://dx.doi.org/10.1016/j.carbpol.2016.11.053 144-8617/© 2016 Elsevier Ltd. All rights reserved. cal fluid, thus various macromolecular drugs containing negatively charged ones (e.g., peptides, proteins and genes) can be incorpo- rated by the electrostatic interactions for their delivery into the site of action (Calvo, Remuñán-López, Vila-Jato, & Alonso, 1997; Csaba, Koping-Hoggard, & Alonso, 2009; Sadeghi et al., 2008). Among the variety of methods developed to prepare Ch nanoparticles, ionotropic gelation technique has attracted atten- tion since nanoparticles obtained by this process are non-toxic, it requires mild control conditions without involving high tem- peratures and is organic solvent free. All previously mentioned characteristics are important to preserve the protein/peptide struc- ture and function during the nanoparticles formation (Chandra Hembram, Prabha, Chandra, Ahmed, & Nimesh, 2016; Chen, Zhang, & Huang, 2007). dx.doi.org/10.1016/j.carbpol.2016.11.053 http://www.sciencedirect.com/science/journal/01448617 http://www.elsevier.com/locate/carbpol http://crossmark.crossref.org/dialog/?doi=10.1016/j.carbpol.2016.11.053&domain=pdf mailto:char_kiill@yahoo.com.br dx.doi.org/10.1016/j.carbpol.2016.11.053 1 e Polym b g t m w p i c c P i t P v c s a c o d ( & X s e 2 2 t s t ( p c p d p t C 2 2 S S M c P A p C r 2 c a 3 ( 696 C.P. Kiilll et al. / Carbohydrat Ionotropic gelation technique is based on the ionic interactions etween the positively charged Ch and the negatively charged roups of polyanions, such as sodium tri-polyphosphate, which is he most extensively used ion crosslinking due to its non-toxic and ultivalent properties (Fan, Yan, Xu, & Ni, 2012). However, other ork proposed a novel polyanion, composed of long metaphos- hate chains, sodium polyphosphate. It is the only water soluble norganic polyphosphate able to spontaneously form nanoparti- les by electrostatic interactions between the long polyphosphate hains and the Ch (Casettari, Cespi, Palmieri, & Bonacucina, 2013; ickup et al., 2014). In this work, we choose a class of macromolecules called dis- ntegrins that comprise a group of low molecular weight proteins hat interact with integrin receptors on the surface of cancer cells. ertaining to a family of cysteine-rich proteins isolated from snake enoms, these peptides are known to inhibit cell-to-matrix and ell-to-cell interactions mediated by integrins. However, administration of therapeutic disintegrins is con- trained mainly by their low bioavailability and susceptibility to cidic and enzymatic degradation in biological fluids. In order to ircumvent problems related to drug stability and consequent loss f activity, researchers have begun exploring novel protein drug elivery systems, e.g., liposomes, microparticles and nanoparticles Andreani, Kiill et al., 2014; Andreani, de Souza et al., 2014; Callens Remon, 2000; Chen et al., 2007; Dudhani & Kosaraju, 2010; Liu, u, Jiang, & Yuan, 2012). Among them, polymeric nanoparticles, uch as chitosan nanoparticles, stand out as a promising deliv- ry strategy for therapeutic protein/peptides (Dudhani & Kosaraju, 010; Fundueanu et al., 2004; Gan, Wang, Cochrane, & McCarron, 005; Gavini et al., 2006). In the present study, we focused on development of chi- osan nanoparticles (Ch-NPs) using a novel crosslinking agent, odium polyphosphate (PP). The aim of this study was the prepara- ion and characterization of chitosan/polyphosphate nanoparticles Ch-PP-NPs) that represent promising carrier properties for roteins/peptides delivery. The developed chitosan nanoparti- les, prepared by the ionotropic gelation method using sodium olyphosphate as a crosslinking agent, were characterized by ifferent techniques to determinate the interaction between com- onents of prepared NPs and their size. The biocompatibility of he Ch-PP-NPs and cRGDfV-loaded Ch-PP-NPs were performed on aco-2 cell line. . Materials and methods .1. Materials Low molecular weight Chitosan (Ch) was purchased from igma-Aldrich (USA) with deacetylation degree of 75%–85%. odium polyphosphate and acetic acid were purchased from erck (Germany). Bicinchoninic acid (BCA) solution was pur- hased from Pierce (Thermo Scientific and Life Science Research roducts Rockford, IL USA). cRGDfV (Arginine-Glycine-Aspartic cid-d-Phenylalanine-Valine) disintegrin was kindly provided as a owder and were purchased from Mocell Biotech Limited (Shangai, hina). All other chemicals were at least reagent grade and used as eceived. Water used was of high purity (Milli-Q, Millipore). .2. Experimental factorial design Three different variables and their influence on the physico- hemical properties of chitosan nanoparticles were evaluated using 9 full factorial design composed of 2 variables which were set at -levels each. Independent variables were ratio PP (w/v) and Ch w/v). The established dependent variables were the mean parti- ers 157 (2017) 1695–1702 cle size (Z-Ave), polydispersity index (PdI) and zeta potential (ZP). The design required a total of 9 experiments. For each factor, the lower and higher values of the lower and upper levels were rep- resented by a lower level (−1), medium and a higher level (+1) sign, respectively (Table 1). These were chosen on the basis of the tested lower and upper values for each variable, according to pre- formulation studies and literature research (de Pinho Neves et al., 2014). A factorial design approach was applied to maximize the experimental efficiency requiring a minimum of experiments to optimize the chitosan nanoparticles produced by ionotropic gela- tion technique. The data were analyzed using the STATISTICA 10.0 (StafSoft, Inc.) software. 2.3. Development of the chitosan/polyphosphate nanoparticles The Ch-PP-NPs were prepared following the procedure described by Calvo et al. (1997). Ch solution (2 mg/mL; 3 mg/mL and 4.4.1 mg/mL) was prepared by dissolving Ch in a 0.75% (v/v) acetic acid solution (0.1 M) and leaving it under stirring for 24 h. The pH was adjusted to 4.4 with a 0.5 M sodium hydroxide solution and diluted in deionized water to the final desired concentrations. PP was dissolved in deionized water to a final concentration of 1 mg/mL. Then, the PP solution was added to the Ch solution drop- wise at different Ch/PP ratios under vigorous magnetic stirring at room temperature (Calvo et al., 1997). cRGDfV-loaded Ch-PP-NPs were prepared by dissolving the cRGDfV (1 mg/mL) in the PP solu- tion which was then added to Ch solution dropwise under magnetic stirring. The indicated nomenclature for produced nanoparticles has in account the quantity of Ch and of PP used in the respective formulation, and a complete list is shown in Table 2. 2.4. Physicochemical characterization 2.4.1. Mean particle size and zeta potential analysis Particles size (Z-Ave), polydispersity index (PdI) and zeta poten- tial (ZP) were determined by using a dynamic light scattering technique (Zetasizer model Nano ZS, Malvern Instruments, UK) with red laser of 633 nm. All samples were diluted with ultra puri- fiedwater to suitable concentration and analyzed in triplicate. ZP of nanoparticles andcRGDfV-loaded Ch-PP-NPs were also measured with the same instrument since it allows determining the elec- trophoretic mobility to assess the surface electrical charge of the nanoparticles. 2.4.2. Fourier Transmission Infrared Spectroscopy (FTIR) Ch-PP-NPs were separated from suspension and were dried by a freeze-dryer (Thermo ® – MicroModulyo115), then were gently mixed with a suitable amount of micronized KBr powder to pre- pare the KBr pellets. Infrared spectra were performed using a FTIR spectrometer (Shimadzu ® Europe – Prestige-21) with resolution of 4 cm−1 and 128 scans. 2.4.3. Differential scanning calorimetry (DSC) DSC analysis was carried out in a Mettler DSC apparatus (Mettler Toledo, Gieben, Switzerland). The instrument was calibrated with indium and zinc and DSC scans were recorded from 25 ◦C to 350 ◦C at a heating constant rate of the 10 ◦C/min under a nitrogen purge (50 mL min−1). The DSC parameters, including enthalpy and onset temperature, were calculated by the STARe Software. 2.4.4. Morphological characterization The morphological examination of the Ch-PP-NPs was deter- mined by scanning electron microscopy (SEM) (Jeol JSM 7500 F) and using atomic force microscopy (AFM). For SEM analysis the nanoparticles were prepared by placing a drop of colloid disper- sion containing nanoparticles on a sample holder and then coated C.P. Kiilll et al. / Carbohydrate Polymers 157 (2017) 1695–1702 1697 Table 1 Initial 32 full factorial design, providing the lower (−1), medium level (0) and upper (+1) level values for each variable. Dependent variables Lower level (-1) Medium level (0) Higher level (+1) Independent variables Chitosan% (w/v) 2.0 3.0 4.41 Z-Ave; PdI and ZP Polyphosphate% (w/v) 0.6 1.2 2.1 Table 2 Response values (Z-Ave, PdI and ZP) of the three factors depicted in Table 1 for the 9 experiment formulations. The influence of the Ch/PP concentration are showed below. Nanoparticles Variable Responses Ch%(w/v) PP%w/v) Ratio Ch:PP Z-Ave (nm)±SD PdI ± SD PZ ± SD Ch2PP6-NPs (−) 2.0 (−) 0.6 3.3:1 166.20 ± 1.95 0.283 ± 0.02 38.7 ± 1.20 Ch2PP12-NPs (−) 2.0 (0) 1.2 1.6:1 20.10 ± 2.95 0.364 ± 0.04 35.0 ± 2.30 Ch2PP21-NPs (−) 2.0 (+) 2.1 0.95:1 336.0 ± 44.0 0.417 ± 0.03 32.0 ± 3.10 Ch3PP6-NPs (0) 3.0 (−) 0.6 5:1 316.0 ± 14.9 0.288 ± 0.07 38.1 ± 1.32 Ch3PP12-NPs (0) 3.0 (0) 1.2 2.5:1 424.50 ± 21.4 0.433 ± 0.03 33.0 ± 2.10 Ch3PP21-NPs (0) 3.0 (+) 2.1 1.4:1 431.60 ± 8.79 0.466 ± 0.11 31.0 ± 1.45 Ch441PP6-NPs (+) 4.41 (−) 0.6 7.3:1 472.50 ± 9.79 0.314 ± 0.02 32.5 ± 2.54 Ch441PP12-NPs (+) 4.41 (0) 1.2 3.6:1 534.80 ± 10.1 0.489 ± 0.166 30.2 ± 2.40 Z w a s S s s w q o m i s 2 d c a t e w p t u 2 2 C t H m m a a A a p Ch441PP21-NPs (+) 4.41 (+) 2.1 2.1:1 -Ave = mean particle size; PdI = polydispersity index; ZP = zeta potential. ith a thin layer of carbon and examined by electron microscope t an intensity of 2.0 kV. In the case of AFM, all the nanoparticles were spin-coated onto ilicon wafer substrates using a spin-coater (Model P6700 from pecialty Coating Systems, Inc.) at 2000 rpm for 120 s. Prepared amples were analyzed by AFM operating in a tapping mode with a canning probe microscope Dimension ICON from Bruker equipped ith an integrated silicon tip/cantilever having a resonance fre- uency of 300 kHz. Scan rates ranged from 0.7 to 1.2 Hz s−1. In rder to obtain repeatable results, different regions of the speci- ens were scanned to choose representative AFM images. Taking nto account that obtained height and phase AFM images were very imilar, only AFM phase images are shown. .5. Association efficiency The amount of cRGDfV associated into the Ch-PP-NPs was etermined in the supernatant using the bicinchoninic acid (BCA) olorimetric assay (Sigma–Aldrich Co., St. Louis, Missouri). The mount of cRGDfV-loaded in the nanoparticles was calculated as he difference between the peptide loaded and the peptide recov- red in the supernatant. The supernatant of unloaded nanoparticles as used as a blank, in order to subtract the interference of the com- onents of loaded samples with the BCA. The assay was realized in riplicate for each batch. Association Efficiency (AE) was calculated sing the following equation: AE (%) = total amount of cRGDfV − free cRGDfV total amount of cRGDfV × 100 (1) .6. In vitro assay .6.1. Cell line The cells used in this work were obtained from American Type ulture Collection (ATCC − Rockville, Maryland, USA), namely, he human epithelial colorectal adenocarcinoma Caco-2 (ATCC ® - TB-37TM) cell line. The cells were maintained in appropriated edium (Dulbecco’s Modified Eagle Medium (DMEM), supple- ented with 10% (v/v) fetal bovine serum (FBS), 2 mM l-glutamine, nd antibiotics (50 U mL−1 penicillin, 100 �g mL−1 of streptomycin nd 0.25 �g/mL amphotericin B), all items obtained from Gibco, lfagene, Invitrogen, Portugal) at 37 ◦C, in an atmosphere of 5% CO2 nd controlled humidity. In this study, cells were used between assages 41 and 46. 562.40 ± 14.1 0.502 ± 0.02 29.5 ± 3.40 2.6.2. Measurement of Caco-2 cells viability uppon exposure to cRGDfV-loaded Ch-PP-NPs The cell viability assay used in this study consisted on the Alamar Blue ® assay. This method consists on the conversion of resazurin (Bluebond-Langner, Perkel, Goertzel, Nelson, & McGeary, 1990) into resorufin (pink/red), whose absorbance can be deter- mined spectrophotometrically (Bluebond-Langner et al., 1990). This method provides a quantitative measure of the number of viable cells that continuously convert resazurin to resorufin, and thus producing a quantitative measure of cell viability (or cyto- toxicity). After trypsin treatment, Caco-2 cells were counted and diluted in culture medium (at density of 105 cells/mL) and then were seeded in 96-well plates (100 �L/well). Then, 24 h after seeding, the culture media was removed and replaced by media containing Ch-PP-NPs, cRGDfV solution and cRGDfV-loaded Ch- PP-NPs were added at different concentrations (1, 5, 10, 15 and 20 �g/mL). Microplates were placed in the incubator, and cells were exposed for 24 h and 48 h at 37 ◦C and 5% CO2. The Alamar Blue assay was conducted according to the manufacturer’s protocol. The absorbance readings occurred about 4 h after Alamar Blue addition, at 570 and 620 nm using a Multiskan EX microplate reader (MTX Labsystems, USA). The percentage of Alamar Blue reduction was calculated according to the following equation: %AB reduction = (εox�2) (A�1) − (εox�1) (A�2) (εred�1) (A′�2) − (εred�2) (A′�1) × 100 In the formula, ε�2 and ε�1are constants representing the molar extinction coefficient of AB at 620 and 570 nm, respectively, in the oxidized (εox) and reduced (εred) forms. A�1 and A�2represent absorbance of test wells at 570 nm and 620 nm, respectively. A′�2 and A′�1 represent absorbance of negative control wells at 620 and 570 nm, respectively. 2.7. Statistical analysis Data were expressed as mean ± standard deviation, and were compared by analysis of variance (ANOVA), Tukey-Kramer’s and Dunnet’s test. Differences were considered statistically significant at p value < 0.05*. The program used was GraphPad Prism 5.00 (GraphPad Software, San Diego, USA). 1 e Polym 3 3 p o c a Z C t s o S G 2 f P n o b t t t p 3 o r t i H J 2 C C s i c o a t r m l i l ( ( c t i t a i a s o W p 698 C.P. Kiilll et al. / Carbohydrat . Results and discussion .1. Experimental factorial design, mean particle size and zeta otential analysis To analyze the dependent variables for the development of an ptimal and stable Ch-PP-NPs based on an ionotropic gelation pro- ess, a 32 full factorial design was employed to provide information bout the effects of the selected variables (Table 1) on Z-Ave, PdI and P. The results showed in Table 1 indicate that the ratio between h and PP are critical and this parameter controls the size dis- ribution of the Ch-PP-NPs. This analysis indicates that particle ize characteristics is very important due to its strong influence n the biological performance of the nanoparticles (Jain, Thakur, harma, Kush, & Jain, 2016; Jin et al., 2016; Joseph, Sangeetha, & omathi, 2016; Montha, Maneeprakorn, Buatong, Tang, & Pon-On, 016; Wang, Tong, Liu, Liu, & Li, 2015). The average size range of Ch-PP-NPs were from 166.2 ± 1.95 nm or Ch2PP6-NPs to 562.4 ± 14.1 nm for Ch441PP21-NPs, whereas dI ranged from 0.283 ± 0.02 to 0.502 ± 0.002 (Table 2). The anoparticles with lower concentration of Ch are smaller than that f other formulations, which may be due to the effective ratio etween Ch:PP generated by the electrostatic interaction between he negative charge of PP and the positive charge of Ch according o the factorial design experimental data. The other parameter of characterization of nanoparticles is he surface charge of the nanoparticles directly related to zeta otential. All Ch-PP-NPs are positively charged in the range from 0.2 to 38.7 mV, which was attributed to the positive charge f the Ch, as shown in Table 2. High positive values of ZP can eflected in luck of aggregation of investigated nanoparticles, and hereby stabilize their dispersion. Thus, according to previous stud- es (Barbi et al., 2015; Csaba et al., 2009; Fabregas et al., 2013; asanovic, Zehl, Reznicek, & Valenta, 2009; Huang & Yang, 2004; iang, Wu, Xu, Wang, & Zeng, 2011; Jonassen, Kjoniksen, & Hiorth, 012; Koukaras, Papadimitriou, Bikiaris, & Froudakis, 2012) for h-Tripolyphosphate nanoparticles, the Z-Ave and ZP obtained to h-PP-NPs are similar to that found in the literature. In order to evaluate the effect of Ch and PP concentration the urface response was plotted in Fig. 1. One can observed a gradual ncrease in the Z-Ave (Fig. 1) with an increase in the Ch and PP con- entration. For each of the 2 variables present in Fig. 1, the analysis f variance (ANOVA) (data not shown) showed that all the vari- bles have a significant effect (p-value < 0.05) on Z-Ave, however, he Ch concentration (>3.0%, w/v) was the most important factor esponsible for the increase in the nanoparticles size. This trend ay be explained by the fact that higher amounts of Ch may not ead to complete solubilization and thus resulting in an increase n the nanoparticles size or even aggregation. Nanoparticles with owest particle size (166.20 nm) were obtained for the lowest Ch 2.0%, w/v) and PP (0.6%, w/v) concentration. This explains the results depicted in the surface responses Fig. 1A), showing that the amount of Ch affects the Z-Ave. As shown in Fig. 1B, the surface respond showed that the PP oncentration have a higher influence on the PdI values. When he amount of PP increased from 0.6% w/v to 2.1% w/v, PdI values ncreased significantly (p-value = 0.002). These results confirmed hat the fraction of free primary amino groups of Ch decreased with n increase of the PP concentration (>1.2% w/v), indicating decrease n the cross-linking interactions between the amino groups of Ch nd the PP, as a consequence of a decrease of the cross-linking den- ity between Ch and PP leading an increase in the polydispersity f the nanoparticles (Csaba et al., 2009; Hu et al., 2008; Yang, Fu, ang, & He, 2009). Fig. 1C showed that Ch-PP-NPs developed had a higher and ositive ZP and the surface response graph shows the effect of ers 157 (2017) 1695–1702 variables on the ZP and was found to have statistical significance (p-value = 0.007) when the PP concentration increased. Since Z-Ave and PdI are the limiting factors, the aim of this factorial design was to optimize a formulation with appropriate physicochemical parameters for the incorporation of the pep- tide cRGDfV, as a model of therapeutic peptide. Thus, from the obtained results, an optimal Ch-PP-NPs was found to be composed of lower concentration of Ch (2.0% w/v) and lower concentration of PP (0.6% w/v). Ch2PP6-NPs were thus selected as the optimized nanoparticles formulation for peptide encapsulation. However, the other two Ch-PP-NPs, Ch2PP12-NPs and Ch2PP21-NPs will be char- acterized as a comparison to understand the influence in the increase the PP concentration on the nanoparticles formation, since this is the first time using a Graham salt to make Ch nanoparticles. 3.2. Morphological characterization The size and shape of Ch2PP6-NPs were examined by SEM and AFM. As can be clearly seen from both measurement the Ch2PP6- NPs showed a spherical shape with smooth surfaces (Fig. 2, left) and had a homogeneous particle size distribution in the range of 70–100 nm. The discrepancy in the size of Ch-PP NPs between DLS and SEM can be related to the fact that DLS measurement of Ch-PP NPs was performed in aqueous media and gives a hydrodynamic diameter of nanoparticles, while SEM and AFM gives diameter of nanoparticles in dry form. AFM phase images of Ch2PP6-NPs are shown in Fig. 2 (right). Similarly, to SEM results, nanoparticles had a spherical shape with the size lower than 100 nm homogeneously dispersed on the sil- ica wafer surface. Additionally, same aggregates of Ch2PP6-NPs were also distinguished. The aggregates can be a consequence of the sample-drying procedure, arising from the decrease in sol- vent volume surrounding the nanoparticles. The size of Ch-PP NPs obtained from AFM was slightly smaller than the equivalent size data obtained from DLS analysis probably as consequence of direct tip-nanoparticles interactions in the case of AFM. Deeper analy- sis of the AFM data indicated the average size of nanoparticles in the ranges of 30–50 nm (Fig. 2, right). de Moura et al. (2009) found similar results for Ch-Tripolyphosphate nanoparticles developed for the same concentrations as used in this study. Additionally, as visualized in high magnification AFM images, each analyzed nanoparticles was surrounded by softer material which appeared darker in AFM phase images. Thus every single nanoparticles was covered by softer materials with the size of around 5–10 nm. 3.3. Structural and thermal characterization Fig. 3 shows the FTIR spectra relative to the Ch (a), Ch2PP6-NPs at 0.6% w/v of PP (b), Ch2PP12-NPs at 1.2% w/v of PP (c), Ch2PP21-NPs at 2.1% w/v of PP (d) and PP (e). In the FTIR spectrum of Ch, we observe seven main bands which can be attributed to the O H and N H stretch at 3290 cm−1, C H stretch at 2872 cm−1, the amide I at 1660 cm−1, the N H bending from amine and amide II at 1593 cm−1, −CH2 bending at 1422 cm−1, a symmetrical deformation of CH3 groups at 1380 cm−1 and anti- symmetric stretch of C O C and C H stretch at 1156 cm−1. For pure NaPO3 compound, the data can be interpreted by considering previous publications about sodium phosphate (Brow, 2000; Brow, Tallant, Myers, & Phifer, 1995; Hudgens, Brow, Tallant, & Martin, 1998; Khawaja, Durrani, Al-Adel, Salim, & Hussain, 2016; Moustafa & El-Egili, 1998). The FTIR spectra displays bands at 1255 cm−1 and 1160 cm−1, being assigned, respectively, to the asymmetric and to the symmetric stretching vibration modes of (PO2) groups of metaphosphate units. In addition, bands at 850 cm−1 and 770 cm−1 are assigned to the asymmetric and symmetric stretching modes of C.P. Kiilll et al. / Carbohydrate Polymers 157 (2017) 1695–1702 1699 Fig. 1. Surface response chart of: (a) the effect of the concentration (w/v) of Ch and PP on the Z-Ave; (b) the effect of the concentration (w/v) of Ch and PP on the PdI. and (c) the effect of the concentration (w/v) of Ch and PP on the ZP. Fig. 2. Scanning electron microscopic images (a) Ch2PP6-NPs showing spherical nanoparticles within 160 nm size (left) and Atomic force microscopy topographic images of (b) Ch2PP6-NPs showing nanoparticles with 50 nm (right). 1700 C.P. Kiilll et al. / Carbohydrate Polymers 157 (2017) 1695–1702 F ( ( P f e t w n N P i 7 i T a c s w o s o i 7 m F Table 3 Peak temperatures and enthalpy changes in the DSC thermograms collected from CS, PP and nanoparticles obtained with a varying concentration of PP. Samples Onset/◦C Melting peak/◦C Enthalpy (�H)/ J/g−1 Ch 35.66 285.52 75.41 302.84 145.2 118.3 PP 300.6 340.54 81.27 Ch2PP6-NPs 39.04 239.50 80.72 249.67 225.6 26.34 Ch2PP12-NPs 38.82 236.26 80.34 242.77 206.2 44.57 Ch2PP21-NPs 38.78 83.49 206 ig. 3. FTIR spectra of Ch and of Ch-PP-NPs with various PP concentrations. (a) Ch; b) Ch2PP6-NPs, with PP at 0.6% (w/v); (c) Ch2PP12-NPs, with PP at 1.2% (w/v) and d) Ch2PP21-NPs, with PP at 2.1% (w/v) and PP (e). -O-P linkages, respectively, while the O P O bending vibration is ound at around 490 cm−1. The nature of the interaction and formation of nanoparticles was videnced through the FTIR data, when Ch is crosslinked with PP, he nanoparticle formation of Ch-PP-NPs occurs and this interaction as evidenced by amide I groups. The band is unfolds giving rise a ew band at 1674 cm−1. The same behavior was observed for the H bending from amine and amide I, shifting to 1551 cm−1and the CH2 bending, shifting to 1408 cm−1. However, the interaction of P with Ch in nanoparticles was not evident through FTIR data. The nteraction of Ch with PP was observed only at the range between 80 and 550 cm−1, this range is characteristic of symmetric stretch- ng modes of these linkages, �s(P–O–P) and O-P-O deformation. hese results are not clear if there is variation in the size of PP chain, s well as, the dependence on Ch-PP-NPs nanoparticles. Thus, the areful study by solid state NMR, using the 13C and 31P nuclei with tructural probe will confirm the interaction between PP and Ch, as ell as, the size of the polyphosphate chain in the future. The analysis of DSC results shows the influence of the increase n PP concentration on the Ch-PP-NPs formation. Table 3 presents a ummary of the temperature transitions and associated enthalpies f each Ch and PP melting peak of the pristine components and nvestigated nanoparticles produced by ionotropic gelation. As indicated in Table 3, Ch exhibits a sharp endothermic peak at 5 ◦C ascribed to the loss of water adsorbed by Ch and an exother- ic peak at 302 ◦C associated with the decomposition of the Ch. urthermore, the DSC thermogram of PP showed two endothermic 234.97 240.13 57.26 peaks associate to a glass transition peak at 271 ◦C and an exother- mic peak in 351 ◦C corresponding to peak assigned to phosphate chains crystallization process that occurs in conjunction with a condensation process (Guinesi & Cavalheiro, 2006; Kittur, Harish Prashanth, Udaya Sankar, & Tharanathan, 2002; Mucha & Pawlak, 2005; Neto et al., 2005). In the DSC thermograms of Ch2PP6-NPs, Ch2PP12-NPs, Ch2PP21-NPs, the exothermic peaks were shifted toward lower temperature than PP and Ch alone. The exothermic peaks for Ch2PP6-NPs appeared at 249 ◦C and at 242 ◦C for Ch2PP12-NPs and for Ch2PP21-NPs, respectively. These exothermic peaks appeared mainly due to decomposition and dissociation of Ch and PP hydrol- ysis, decreasing the thermal stability of the Ch-PP-NPs. The increase in the enthalpy of Ch2PP6-NPs, Ch2PP12-NPs and Ch2PP21-NPs are observed with an increased in phosphate content in the nanopar- ticles (Fig. 4). As evidenced from X-Ray Diffraction (XRD) results (data not shown), Ch-PP-NPs showed a pattern predominately amorphous, which corroborates with DSC curves. This finding suggests that PP is dispersed in the matrix of Ch-PP-NPs showing the amorphous nature. 4. Association efficiency and in vitro cytotoxicity assay Considering the results of factorial design, Ch2PP6-NPs was cho- sen as the best formulation to encapsulate de peptide cRGDfV, since it has the smallest particle size (166 nm) and the higher ZP value (38.7 mV). The incorporation of peptide in the Ch2PP6-NPs was achieved by dissolving protein in PP solutions. The peptide loading did not significantly change the size (233 nm), however it was possible to see an increase in the ZP of the Ch2PP6-NPs (46.5 mV) after peptide encapsulation. This fact indicates that cRGDfV was conveniently associated within the Ch-PP-NPs. The AE was 97.0%, similar results were found for Ch- Tripolyphosphate nanoparticles (Csaba et al., 2009; Dudhani & Kosaraju, 2010; Hashad, Ishak, Fahmy, Mansour, & Geneidi, 2016; Liu et al., 2012; Ragelle, Vanvarenberg, Vandermeulen, & Preat, 2016; Xu & Du, 2003). This result suggested that PP could be an option for the development of Ch-NPs with high AE instead of using only the trypoliphosphate. To assess the biocompatibility of Ch2PP6-NPs and cRGDfV- loaded-Ch2PP6-NPs, in vitro cell culture was performed and viability was accessed by the resazurin (Alamar Blue ® ) reduction assay in Caco-2 cell line. The results of cell viability were com- pared with those of non-exposed cells (control) and are shown in terms of% of control. The cell viability above 70% of the control is an indication of “no toxicity” or of a safe material, and only viabil- ity below 70% considers the material as toxic (Borges et al., 2006; Doktorovova, Souto, & Silva, 2014; Huang, Khor, & Lim, 2016). C.P. Kiilll et al. / Carbohydrate Polymers 157 (2017) 1695–1702 1701 F C a N 2 f p C c C o c d C i t d t t a d 5 C m Fig. 5. Cell viability of the cRGDfV-loaded in Ch2PP6-NPs by Alamar Blue® assay for cell proliferation activity in vitro in Caco-2 cells *p < 0.05. Cell viability assessment upon exposure of Caco-2 cells to Ch2PP6-NPs (left set of columns), cRGDfV solution (middle set of columns) and cRGDfV-loaded-Ch2PP6-NPs (left set of columns, cRGDfV- Ch2PP6-NPs), at different concentrations (as indicated ig. 4. DSC curves of Ch and of Ch-PP-NPs with different concentrations of PP. (a) h; (b) Ch2PP6-NPs, with PP at 0.6% (w/v); (c) Ch2PP12-NPs, with PP at 1.2% (w/v) nd (d) Ch2PP21-NPs, with PP at 2.1% (w/v) and PP (e). Fig. 5 compares the viability of Caco-2 after exposure to Ch2PP6- Ps, cRGDfV solution and cRGDfV-loaded-Ch2PP6-NPs (1, 5, 10, 15, 0 �g/mL) for 24 h and 48 h. No differences on the viability were ound between all the treatments, demonstrating that these com- ounds by themselves did not show any apparent toxicity to the aco-2 cell line even after long periods of incubation at the same oncentration. Cell viability assessment upon exposure of Caco-2 cells to h2PP6-NPs (left set of columns), cRGDfV solution (middle set f columns) and cRGDfV-loaded-Ch2PP6-NPs (left set of columns, RGDfV- Ch2PP6-NPs), at different concentrations (as indicated by ifferent bas pattern) for 24 h (left panel) and 48 h (right panel). ell viability was assessed by Alamar Blue assay and data is shown n percentage of control (non-exposed cells). These studies showed that Ch and PP are an attractive combina- ion for development of nanoparticles for protein/peptide delivery ue to various characteristics including its biocompatibility, low oxicity, and low immunogenicity. These results clearly indicate hat all materials don’t have any cytotoxic effect on Caco-2 cells nd they are highly nontoxic and biocompatible for protein/peptide elivery. . Conclusion Experimental factorial design enables to successfully formulate h-PP NPs with suitable physicochemical parameters requiring a inimum of experiments. The influence of the independent vari- by different bas pattern) for 24 h (left panel) and 48 h (right panel). Cell viability was assessed by Alamar Blue assay and data is shown in percentage of control (non- exposed cells). ables (i.e., concentration of Ch and PP) on the Z-Ave, PdI and ZP was analyzed and showed that the size of NPs are highly depen- dent on the Ch concentrations. Optimal parameters were obtained for formulation Ch2PP6-NPs composed of 2.0% (w/v) of Ch and 0.6% (w/v) of PP. For these conditions, the average size of particles were 166 nm, zeta potential was 38.7 mV. The presented SEM/AFM, FTIR and DSC analysis confirmed the Ch-PP-NPs formation by morphol- ogy evaluation, interaction between components of particles and thermal behavior, respectively. In the SEM/AFM studies, results showed a spherical shape of the Ch2PP6-NPs with a homogeneous distribution. FTIR and DSC analysis demonstrated the interaction and partial miscibility between Ch and PP indicating the forma- tion of the Ch-PP-NPs. Additionally, the cytotoxicity assay showed that Ch2PP6-NPs and cRGDfV-loaded Ch2-PP6-NPs did not affect, or affected on a low level, the cell viability, demonstrating none or very low toxicity in Caco-2 cell line, indicating that the developed nanoparticles are biocompatible and suitable for protein/peptide delivery systems. Acknowledgments This work was supported by FAPESP (number 2012-10174-3), Brazil, São Paulo. Moreover, we are grateful to the ‘Macrobehavior- Mesostructure-Nanotechnology’ SGIker unit of the UPV/EHU. We also acknowledge FEDER/COMPETE/POCI (POCI-01-0145- FEDER-006958) and FCT-Portuguese Foundation for Science and Technology (to AMS). References Andreani, T., Kiill, C. P., de Souza, A. L., Fangueiro, J. F., Fernandes, L., Doktorovova, S., . . . & Silva, A. M. (2014). Surface engineering of silica nanoparticles for oral insulin delivery: Characterization and cell toxicity studies. Colloids and Surfaces B: Biointerfaces, 123, 916–923. http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0005 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0005 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0005 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0005 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0005 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0005 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0005 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0005 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0005 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0005 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0005 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0005 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0005 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0005 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0005 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0005 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0005 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0005 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0005 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0005 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0005 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0005 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0005 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0005 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0005 1 e Polym A B B B B B C C C C C C d d D D F F F G G G G H properties of chitosan nanoparticles. International Journal of Pharmaceutics, 250(1), 215–226. 702 C.P. Kiilll et al. / Carbohydrat ndreani, T., de Souza, A. L., Kiill, C. P., Lorenzon, E. N., Fangueiro, J. F., Calpena, A. C., . . . & Souto, E. B. (2014). Preparation and characterization of PEG-coated silica nanoparticles for oral insulin delivery. International Journal of Pharmaceutics, 473(1–2), 627–635. arbi, M. D. S., Carvalho, F. C., Kiill, C. P., Da Silva Barud, H., Santagneli, S. H., Ribeiro, S. J. L., & Gremião, M. P. D. (2015). Preparation and characterization of chitosan nanoparticles for zidovudine nasal delivery? Journal of Nanoscience and Nanotechnology, 15(1), 865–874. luebond-Langner, M., Perkel, D., Goertzel, T., Nelson, K., & McGeary, J. (1990). Children’s knowledge of cancer and its treatment: Impact of an oncology camp experience? Journal of Pediatrics, 116(2), 207–213. orges, O., Cordeiro-da-Silva, A., Romeijn, S. G., Amidi, M., de Sousa, A., Borchard, G., & Junginger, H. E. (2006). Uptake studies in rat Peyer’s patches, cytotoxicity and release studies of alginate coated chitosan nanoparticles for mucosal vaccination. Journal of Controlled Release, 114(3), 348–358. row, R. K., Tallant, D. R., Myers, S. T., & Phifer, C. C. (1995). The short-range structure of zinc polyphosphate glass. Journal of Non-Crystalline Solids, 191(1–2), 45–55. row, R. K. (2000). Review: The structure of simple phosphate glasses. Journal of Non-Crystalline Solids, 263–264, 1–28. allens, C., & Remon, J. P. (2000). Evaluation of starch-maltodextrin-Carbopol 974 P mixtures for the nasal delivery of insulin in rabbits? Journal of Controlled Release, 66(2–3), 215–220. alvo, P., Remuñán-López, C., Vila-Jato, J. L., & Alonso, M. J. (1997). Novel hydrophilic chitosan-polyethylene oxide nanoparticles as protein carriers. Journal of Applied Polymer Science, 63(1), 125–132. asettari, L., Cespi, M., Palmieri, G. F., & Bonacucina, G. (2013). Characterization of the interaction between chitosan and inorganic sodium phosphates by means of rheological and optical microscopy studies. Carbohydrate Polymer, 91(2), 597–602. handra Hembram, K., Prabha, S., Chandra, R., Ahmed, B., & Nimesh, S. (2016). Advances and characterization of chitosan nanoparticles for therapeutics. Artif Cells Nanomed Biotechnol, 44(1), 305–314 (in preparation). hen, F., Zhang, Z. R., & Huang, Y. (2007). Evaluation and modification of N-trimethyl chitosan chloride nanoparticles as protein carriers? International Journal of Pharmaceutics, 336(1), 166–173. saba, N., Koping-Hoggard, M., & Alonso, M. J. (2009). Ionically crosslinked chitosan/tripolyphosphate nanoparticles for oligonucleotide and plasmid DNA delivery? International Journal of Pharmaceutics, 382(1–2), 205–214. e Moura, M. R., Aouada, F. A., Avena-Bustillos, R. J., McHugh, T. H., Krochta, J. M., & Mattoso, L. H. C. (2009). Improved barrier and mechanical properties of novel hydroxypropyl methylcellulose edible films with chitosan/tripolyphosphate nanoparticles? Journal of Food Engineering, 92(4), 448–453. e Pinho Neves, A. L., Milioli, C. C., Müller, L., Riella, H. G., Kuhnen, N. C., & Stulzer, H. K. (2014). Factorial design as tool in chitosan nanoparticles development by ionic gelation technique. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 445, 34–39. oktorovova, S., Souto, E. B., & Silva, A. M. (2014). Nanotoxicology applied to solid lipid nanoparticles and nanostructured lipid carriers −A systematic review of in vitro data? European Journal of Pharmaceutics and Biopharmaceutics, 87(1), 1–18. udhani, A. R., & Kosaraju, S. L. (2010). Bioadhesive chitosan nanoparticles: Preparation and characterization. Carbohydrate Polymers, 81(2), 243–251. abregas, A., Minarro, M., Garcia-Montoya, E., Perez-Lozano, P., Carrillo, C., Sarrate, R., . . . & Sune-Negre, J. M. (2013). Impact of physical parameters on particle size and reaction yield when using the ionic gelation method to obtain cationic polymeric chitosan-tripolyphosphate nanoparticles. International Journal of Pharmaceutics, 446(1–2), 199–204. an, W., Yan, W., Xu, Z., & Ni, H. (2012). Formation mechanism of monodisperse, low molecular weight chitosan nanoparticles by ionic gelation technique. Colloids and Surfaces B: Biointerfaces, 90, 21–27. undueanu, G., Constantin, M., Dalpiaz, A., Bortolotti, F., Cortesi, R., Ascenzi, P., & Menegatti, E. (2004). Preparation and characterization of starch/cyclodextrin bioadhesive microspheres as platform for nasal administration of Gabexate Mesylate (Foy) in allergic rhinitis treatment. Biomaterials, 25(1), 159–170. an, Q., Wang, T., Cochrane, C., & McCarron, P. (2005). Modulation of surface charge, particle size and morphological properties of chitosan-TPP nanoparticles intended for gene delivery. Colloids and Surfaces B: Biointerfaces, 44(2–3), 65–73. avini, E., Rassu, G., Sanna, V., Cossu, M., & Giunchedi, P. (2005). Mucoadhesive microspheres for nasal administration of an antiemetic drug, metoclopramide: In-vitro/ex-vivo studies. Journal of Pharmacy and Pharmacology, 57(3), 287–294. avini, E., Hegge, A. B., Rassu, G., Sanna, V., Testa, C., Pirisino, G., . . . & Giunchedi, P. (2006). Nasal administration of carbamazepine using chitosan microspheres: In vitro/in vivo studies. International Journal of Pharmaceutics, 307(1), 9–15. uinesi, L. S., & Cavalheiro, É. T. G. (2006). The use of DSC curves to determine the acetylation degree of chitin/chitosan samples. Thermochimica Acta, 444(2), 128–133. asanovic, A., Zehl, M., Reznicek, G., & Valenta, C. (2009). Chitosan-tripolyphosphate nanoparticles as a possible skin drug delivery ers 157 (2017) 1695–1702 system for aciclovir with enhanced stability. Journal of Pharmacy and Pharmacology, 61(12), 1609–1616. Hashad, R. A., Ishak, R. A., Fahmy, S., Mansour, S., & Geneidi, A. S. (2016). Chitosan-tripolyphosphate nanoparticles: Optimization of formulation parameters for improving process yield at a novel pH using artificial neural networks. International Journal of Biological Macromolecules, 86, 50–58. Hu, B., Pan, C., Sun, Y., Hou, Z., Ye, H., & Zeng, X. (2008). Optimization of fabrication parameters to produce chitosan-tripolyphosphate nanoparticles for delivery of tea catechins? Journal of Agricultural and Food Chemistry, 56(16), 7451–7458. Huang, H., & Yang, X. (2004). Synthesis of chitosan-stabilized gold nanoparticles in the absence/presence of tripolyphosphate. Biomacromolecules, 5(6), 2340–2346. Huang, M., Khor, E., & Lim, L.-Y. (2016). Uptake and cytotoxicity of chitosan molecules and nanoparticles: Effects of molecular weight and degree of deacetylation. Pharmaceutical Research, 21(2), 344–353. Hudgens, J. J., Brow, R. K., Tallant, D. R., & Martin, S. W. (1998). Raman spectroscopy study of the structure of lithium and sodium ultraphosphate glasses. Journal of Non-Crystalline Solids, 223(1–2), 21–31. Jain, A., Thakur, K., Sharma, G., Kush, P., & Jain, U. K. (2016). Fabrication, characterization and cytotoxicity studies of ionically cross-linked docetaxel loaded chitosan nanoparticles. Carbohydrate Polymers, 137, 65–74. Jiang, H., Wu, H., Xu, Y. L., Wang, J. Z., & Zeng, Y. (2011). Preparation of galactosylated chitosan/tripolyphosphate nanoparticles and application as a gene carrier for targeting SMMC7721 cells. Journal of Bioscience and Bioengineering, 111(6), 719–724. Jin, H., Pi, J., Yang, F., Wu, C., Cheng, X., Bai, H., . . . & Chen, Z. W. (2016). Ursolic acid-loaded chitosan nanoparticles induce potent anti-angiogenesis in tumor. Applied Microbiology and Biotechnology. Jonassen, H., Kjoniksen, A. L., & Hiorth, M. (2012). Stability of chitosan nanoparticles cross-linked with tripolyphosphate. Biomacromolecules, 13(11), 3747–3756. Joseph, J. J., Sangeetha, D., & Gomathi, T. (2016). Sunitinib loaded chitosan nanoparticles formulation and its evaluation. International Journal of Biological Macromolecules, 82, 952–958. Khawaja, E. E., Durrani, S. M. A., Al-Adel, F. F., Salim, M. A., & Hussain, M. S. (2016). X-ray photoelectron spectroscopy and Fourier transform-infrared studies of transition metal phosphate glasses. Journal of Materials Science, 30(1), 225–234. Kittur, F. S., Harish Prashanth, K. V., Udaya Sankar, K., & Tharanathan, R. N. (2002). Characterization of chitin, chitosan and their carboxymethyl derivatives by differential scanning calorimetry. Carbohydrate Polymers, 49(2), 185–193. Koukaras, E. N., Papadimitriou, S. A., Bikiaris, D. N., & Froudakis, G. E. (2012). Insight on the formation of chitosan nanoparticles through ionotropic gelation with tripolyphosphate? Molecular Pharmaceutics, 9(10), 2856–2862. Liu, G., Xu, D., Jiang, M., & Yuan, W. (2012). Preparation of bioactive interferon alpha-loaded polysaccharide nanoparticles using a new approach of temperature-induced water phase/water-phase emulsion. International Journal of Nanomedicine, 7, 4841–4848. Montha, W., Maneeprakorn, W., Buatong, N., Tang, I. M., & Pon-On, W. (2016). Synthesis of doxorubicin-PLGA loaded chitosan stabilized (Mn: Zn)Fe2O4 nanoparticles: Biological activity and pH-responsive drug release. Materials Science & Engineering C: Materials for Biological Applications, 59, 235–240. Moustafa, Y. M., & El-Egili, K. (1998). Infrared spectra of sodium phosphate glasses. Journal of Non-Crystalline Solids, 240(1–3), 144–153. Mucha, M., & Pawlak, A. (2005). Thermal analysis of chitosan and its blends. Thermochimica Acta, 427(1–2), 69–76. Neto, C. G. T., Giacometti, J. A., Job, A. E., Ferreira, F. C., Fonseca, J. L. C., & Pereira, M. R. (2005). Thermal analysis of chitosan based networks? Carbohydrate Polymers, 62(2), 97–103. Pickup, D. M., Newport, R. J., Barney, E. R., Kim, J. Y., Valappil, S. P., & Knowles, J. C. (2014). Characterisation of phosphate coacervates for potential biomedical applications. Journal of Biomaterials Applications, 28(8), 1226–1234. Ragelle, H., Vanvarenberg, K., Vandermeulen, G., & Preat, V. (2016). Chitosan nanoparticles for SiRNA delivery In vitro. Methods in Molecular Biology, 1364, 143–150. Sadeghi, A. M., Dorkoosh, F. A., Avadi, M. R., Saadat, P., Rafiee-Tehrani, M., & Junginger, H. E. (2008). Preparation, characterization and antibacterial activities of chitosan, N-trimethyl chitosan (TMC) and N-diethylmethyl chitosan (DEMC) nanoparticles loaded with insulin using both the ionotropic gelation and polyelectrolyte complexation methods. International Journal of Pharmaceutics, 355(1–2), 299–306. Wang, W., Tong, C. Y., Liu, B., Liu, X. Y., & Li, T. (2015). Preparation and functional characterization of tumor-targeted folic acid-chitosan conjugate nanoparticles loaded with mitoxantrone. Journal of Controlled Release, 213, e110–111. Xu, Y., & Du, Y. (2003). Effect of molecular structure of chitosan on protein delivery Yang, W., Fu, J., Wang, T., & He, N. (2009). Chitosan/sodium tripolyphosphate nanoparticles: Preparation, characterization and application as drug carrier. Journal of Biomedical Nanotechnology, 5(5), 591–595. http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0010 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0010 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0010 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0010 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0010 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0010 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0010 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0010 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0010 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0010 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0010 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0010 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0010 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0010 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0010 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0010 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0010 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0010 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0010 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0010 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0010 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0010 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0010 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0015 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0015 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0015 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0015 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0015 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0015 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0015 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0015 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0015 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0015 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0015 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0015 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0015 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0015 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0015 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0015 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0015 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0015 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0015 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0015 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0015 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0020 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0020 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0020 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0020 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0020 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0020 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0020 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0020 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0020 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0020 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0020 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0020 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0020 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0020 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0020 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0020 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0020 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0020 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0020 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0020 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0020 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0020 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0025 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0025 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0025 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0025 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0025 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0025 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0025 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0025 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0025 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0025 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0025 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0025 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0025 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0025 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0025 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0025 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0025 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0025 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0025 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0025 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0025 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0025 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0025 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0025 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0025 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0025 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0025 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0025 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0030 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0030 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0030 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0030 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0030 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0030 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0030 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0030 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0030 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0030 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0030 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0030 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0030 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0030 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0030 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0030 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0030 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0030 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0030 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0035 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0035 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0035 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0035 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0035 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0035 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0035 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0035 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0035 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0035 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0035 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0035 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0035 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0035 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0035 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0035 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0035 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0035 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0035 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0040 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0040 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0040 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0040 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0040 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0040 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0040 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0040 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0040 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0040 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0040 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0040 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0040 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0040 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0040 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0040 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0040 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0040 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0040 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0040 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0040 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0040 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0040 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0040 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0040 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0040 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0045 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0045 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0045 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0045 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0045 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0045 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0045 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0045 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0045 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0045 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0045 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0045 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0045 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0045 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0045 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0045 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0045 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0045 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0045 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0050 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0050 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0050 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0050 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0050 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0050 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0050 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0050 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0050 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0050 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0050 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0050 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0050 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0050 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0050 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0050 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0050 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0050 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0050 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0050 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0050 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0050 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0050 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0050 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0050 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0050 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0055 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0055 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0055 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0055 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0055 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0055 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0055 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0055 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0055 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0055 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0055 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0055 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0055 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0055 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0055 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0055 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0055 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0055 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0055 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0055 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0060 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0060 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0060 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0060 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0060 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0060 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0060 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0060 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0060 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0060 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0060 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0060 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0060 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0060 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0060 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0060 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0060 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0060 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0060 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0060 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0060 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0065 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0065 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0065 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0065 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0065 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0065 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0065 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0065 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0065 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0065 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0065 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0065 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0065 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0065 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0065 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0065 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0065 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0065 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0065 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0065 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0065 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0065 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0070 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0070 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0070 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0070 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0070 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0070 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0070 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0070 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0070 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0070 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0070 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0070 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0070 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0070 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0070 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0070 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0070 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0070 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0070 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0070 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0070 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0070 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0070 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0070 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0075 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0075 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0075 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0075 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0075 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0075 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0075 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0075 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0075 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0075 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0075 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0075 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0075 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0075 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0075 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0075 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0075 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0075 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0075 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0075 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0075 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0075 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0075 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0075 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0075 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0075 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0080 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0080 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0080 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0080 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0080 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0080 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0080 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0080 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0080 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0080 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0080 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0080 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0080 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0080 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0080 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0080 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0080 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0080 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0080 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0080 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0080 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0080 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0080 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0080 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0080 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0080 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0080 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0080 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0080 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0080 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0085 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0085 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0085 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0085 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0085 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0085 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0085 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0085 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0085 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0085 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0085 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0085 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0085 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0085 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0090 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0095 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0095 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0095 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0095 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0095 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0095 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0095 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0095 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0095 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0095 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0095 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0095 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0095 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0095 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0095 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0095 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0095 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0095 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0095 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0095 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0095 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0095 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0095 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0095 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0100 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0100 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0100 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0100 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0100 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0100 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0100 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0100 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0100 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0100 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0100 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0100 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0100 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0100 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0100 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0100 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0100 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0100 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0100 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0100 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0100 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0100 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0100 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0100 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0100 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0100 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0100 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0105 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0105 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0105 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0105 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0105 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0105 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0105 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0105 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0105 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0105 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0105 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0105 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0105 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0105 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0105 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0105 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0105 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0105 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0105 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0105 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0105 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0105 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0105 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0105 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0105 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0105 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0105 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0105 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0105 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0110 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0110 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0110 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0110 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0110 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0110 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0110 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0110 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0110 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0110 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0110 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0110 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0110 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0110 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0110 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0110 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0110 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0110 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0110 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0110 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0110 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0110 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0110 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0115 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0115 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0115 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0115 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0115 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0115 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0115 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0115 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0115 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0115 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0115 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0115 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0115 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0115 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0115 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0115 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0115 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0115 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0115 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0115 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0115 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0120 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0120 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0120 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0120 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0120 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0120 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0120 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0120 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0120 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0120 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0120 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0120 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0120 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0120 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0120 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0120 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0120 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0120 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0120 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0120 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0120 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0125 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0125 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0125 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0125 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0125 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0125 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0125 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0125 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0125 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0125 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0125 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0125 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0125 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0125 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0125 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0125 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0125 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0125 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0125 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0125 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0125 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0125 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0125 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0125 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0125 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0130 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0130 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0130 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0130 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0130 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0130 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0130 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0130 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0130 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0130 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0130 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0130 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0130 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0130 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0130 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0130 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0130 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0130 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0130 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0130 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0130 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0130 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0130 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0130 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0130 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0130 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0130 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0130 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0130 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0135 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0135 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0135 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0135 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0135 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0135 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0135 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0135 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0135 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0135 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0135 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0135 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0135 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0135 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0135 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0135 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0135 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0135 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0135 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0135 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0135 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0135 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0135 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0135 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0135 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0140 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0140 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0140 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0140 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0140 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0140 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0140 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0140 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0140 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0140 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0140 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0140 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0140 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0140 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0140 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0140 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0140 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0145 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0145 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0145 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0145 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0145 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0145 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0145 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0145 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0145 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0145 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0145 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0145 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0145 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0145 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0145 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0145 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0145 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0145 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0145 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0145 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0145 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0145 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0145 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0145 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0150 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0150 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0150 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0150 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0150 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0150 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0150 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0150 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0150 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0150 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0150 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0150 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0150 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0150 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0150 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0150 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0150 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0150 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0150 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0150 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0150 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0150 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0150 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0150 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0155 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0155 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0155 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0155 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0155 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0155 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0155 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0155 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0155 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0155 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0155 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0155 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0155 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0155 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0155 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0155 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0155 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0155 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0155 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0155 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0160 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0160 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0160 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0160 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0160 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0160 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0160 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0160 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0160 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0160 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0160 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0160 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0160 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0160 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0160 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0160 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0160 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0160 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0160 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0160 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0160 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0160 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0160 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0160 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0160 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0160 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0165 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0165 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0165 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0165 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0165 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0165 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0165 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0165 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0165 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0165 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0165 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0165 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0165 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0165 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0170 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0170 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0170 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0170 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0170 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0170 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0170 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0170 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0170 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0170 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0170 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0170 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0170 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0170 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0175 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0175 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0175 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0175 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0175 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0175 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0175 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0175 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0175 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0175 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0175 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0175 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0175 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0175 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0175 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0175 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0175 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0175 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0175 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0180 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0180 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0180 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0180 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0180 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0180 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0180 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0180 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0180 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0180 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0180 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0180 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0180 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0180 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0180 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0180 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0180 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0180 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0180 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0180 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0180 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0180 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0185 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0185 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0185 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0185 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0185 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0185 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0185 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0185 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0185 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0185 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0185 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0185 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0185 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0185 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0185 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0185 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0185 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0185 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0185 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0185 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0190 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0190 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0190 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0190 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0190 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0190 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0190 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0190 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0190 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0190 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0190 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0190 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0190 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0190 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0190 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0190 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0190 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0190 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0190 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0190 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0195 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0195 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0195 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0195 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0195 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0195 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0195 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0195 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0195 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0195 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0195 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0195 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0195 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0195 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0195 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0195 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0195 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0195 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0195 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0195 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0195 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0195 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0195 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0195 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0195 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref0195 http://refhub.elsevier.com/S0144-8617(16)31327-3/sbref