P s F R a S b a A R R 1 A A K H P C T R X 1 b m e a a w o s i c 2 b c r t ( 0 d International Journal of Pharmaceutics 423 (2012) 281– 288 Contents lists available at SciVerse ScienceDirect International Journal of Pharmaceutics jo ur nal homep a ge: www.elsev ier .com/ locate / i jpharm hysical properties of pectin–high amylose starch mixtures cross-linked with odium trimetaphosphate ernanda M. Carbinattoa, Ana Dóris de Castroa, Beatriz S.F. Curya, Alviclér Magalhãesb, aul C. Evangelistaa,∗ Faculdade de Ciências Farmacêuticas, Departamento de Fármacos e Medicamentos, Universidade Estadual Paulista – UNESP, Rod. Araraquara-Jaú, km 1, CEP14801-902, Araraquara, P, Brazil Instituto de Química, Universidade Estadual de Campinas – UNICAMP, Cidade Universitária Zeferino Vaz, s/n, CP 6154, CEP 13083-970, Campinas, SP, Brazil r t i c l e i n f o rticle history: eceived 9 August 2011 eceived in revised form 4 November 2011 ccepted 28 November 2011 vailable online 9 December 2011 a b s t r a c t Pectin–high amylose starch mixtures (1:4; 1:1; 4:1) were cross-linked at different degrees and charac- terized by rheological, thermal, X-ray diffraction and NMR analyses. For comparison, samples without cross-linker addition were also prepared and characterized. Although all samples behaved as gels, the results evidenced that the phosphorylation reaction promotes the network strengthening, resulting in covalent gels (highest critical stress, G′ and recovery %). Likewise, cross-linked samples presented the highest thermal stability. However, alkaline treatment without cross-linker allowed a structural reorga- nization of samples, as they also behaved as covalent gels, but weaker than those gels from cross-linked eywords: igh amylose starch ectin ross-linking hermal analysis heology -ray diffraction samples, and presented higher thermal stability than the physical mixtures. X-ray diffractograms also evidenced the occurrence of physical and chemical modifications due to the cross-linking process and indicated that samples without cross-linker underwent some structural reorganization, resulting in a decrease of crystallinity. The chemical shift of resonance signals corroborates the occurrence of structural modifications by both alkaline treatment and cross-linking reaction. . Introduction Polysaccharides are natural polymers well known for their iocompatibility and biodegradability, which make them raw aterials of great interest for the design of controlled drug deliv- ry systems. Starch is one of the most abundant available polymers nd can be obtained from a variety of sources. It is constituted by mylose, representing the linear fraction of the macromolecule, hile amylopectin is the highly branched fraction. Among vari- us commercially available starches, high amylose starch (modified tarch containing 70% of amylose) has been reported as possessing mproved properties for controlled drug delivery purposes when ompared to conventional starch (Rioux et al., 2002; Onofre et al., 009). Additionally, the modification of high amylose starch can be can e done by reaction of its hydroxyl groups (esterification, etherifi- ation and oxidation) and its properties, such as solubility, swelling ate, rheological behavior, gel and film formation, and biodegrada- ion rate, can be modified (Rioux et al., 2002). Cross-linking reaction ∗ Corresponding author. Tel.: +55 16 33016976; fax: +55 16 33016960. E-mail addresses: revangel@fcfar.unesp.br, raulrasec@yahoo.com.br R.C. Evangelista). 378-5173/$ – see front matter © 2011 Elsevier B.V. All rights reserved. oi:10.1016/j.ijpharm.2011.11.042 © 2011 Elsevier B.V. All rights reserved. has been shown to be a key technique to change the properties of starches and can be achieved by adding intra- and inter-molecular bonds randomly distributed in the starch granules (Singh et al., 2007). Sodium trimetaphosphate (STMP), monosodium phosphate, sodium tripolyphosphate, epichlorohydrin, phosphoryl chloride and vinyl chloride are the main agents used to cross-link food grade starches (Woo and Seib, 1997; Wattanchant et al., 2003). A high content of amylose combined to physical and chemical modifications of this material results, for example, in products with higher viscosity and in granules that are more resistant against swelling (Richardson et al., 2000; Van Hung et al., 2006). Many researches have demonstrated the successful use of high amylose starches cross-linked by different chemicals, such as epichlorohydrin and STMP, in the development of controlled drug delivery systems (Lenaerts et al., 1991; Fang et al., 2008; Cury et al., 2009a, 2009b; Li et al., 2009; O’Brien et al., 2009). Pectins are a family of complex polysaccharides constituted mainly by linearly linked �-(1–4)-d-galacturonic acid residues par- tially esterified with methanol. The degree of methoxylation (DM) is used to classify pectins as high methoxyl pectins (DM > 50) and low methoxyl pectins (DM < 50) (Sakai et al., 1993; Thakur et al., 1997; Ghaffari et al., 2007; Lutz et al., 2009). They are widely used in the pharmaceutical industry to compose hydrophilic matrices in dx.doi.org/10.1016/j.ijpharm.2011.11.042 http://www.sciencedirect.com/science/journal/03785173 http://www.elsevier.com/locate/ijpharm mailto:revangel@fcfar.unesp.br mailto:raulrasec@yahoo.com.br dx.doi.org/10.1016/j.ijpharm.2011.11.042 2 urnal o W l s m s a t F e c b 2 2 K b ( w h d v w ( B 2 d a 1 m a t 4 t s w T a a s i l 4 2 s m 1 3 ( s m c u b 82 F.M. Carbinatto et al. / International Jo ral controlled release dosage forms (Sungthongieen et al., 2004; ei et al., 2006). Recently, mixtures of conventional starch and pectin cross- inked by STMP were evaluated for food applications and exhibited uperior mechanical properties in comparison to the isolated poly- ers (Khondkar et al., 2007). Considering that the cross-linked mixtures of high amylose tarch/pectin represent a promising material for use in the food nd pharmaceutical field, the aim of this work is to contribute to he understanding of the structural characteristics of this material. or this purpose, pectin and high amylose starch mixed at differ- nt ratios were cross-linked to different degrees, by varying the ross-linking reaction conditions. The products were characterized y rheological, thermal, X-ray diffraction and NMR analyses. . Materials and methods .1. Materials Pectin (type LM-506CS, batch:S74431) was provided by CP elko (Copenhagen, Denmark), high amylose starch (Hylon VII, atch:HA9140) was obtained from National Starch & Chemical New Jersey, EUA), sodium trimetaphosphate (batch:112K1365) as purchased from Sigma–Aldrich Co. (St. Louis, USA), sodium ydroxide (batch: 6 11648) was supplied by Grupo Química (Rio e Janeiro, Brazil), 37% hydrochloric acid (batch: 29957) was pro- ided by Quimis (Diadema, Brazil), ethyl alcohol (batch:127698) as obtained from Synth (Diadema, Brazil), and nimesulide batch:NM/3680308) was provided by Henrifarma (São Paulo, razil). .2. Cross-linking of polymers Cross-linking of polymers was performed by the method escribed by Cury et al. (2008), with some adaptations. High mylose starch and pectin mixed at different mass ratios (1:4, :1, and 4:1) were cross-linked with STMP (30% of the polymer ass) at room temperature. Different degrees of cross-linking were chieved by varying the base (NaOH) concentration (2% and 4%) and he pectin/high amylose starch/NaOH/STMP contact time (1, 2 and h). After the desired mixing time, all samples were treated with he adequate amount of 1 mol L−1 HCl in order to set the pH at 6. The olids were separated by vacuum filtration and washed repeatedly ith ethanol of different concentrations (85 ◦GL, 65 ◦GL and 96 ◦GL). he final product was dried at room temperature, pulverized nd sieved (sieve opening = 0.97 mm). The samples were labeled ccording to the polymer mixture ratio (pectin:high amylose)–base trength–cross-linking reaction time. The physical mixtures were ndicated by the prefix PMP-HA and the samples without cross- inker by the suffix W. For instance, 14-4-2 means 1:4 polymer ratio, % NaOH and 2 h of cross-linking reaction. .3. Rheological measurements The polymers aqueous dispersions (5%) were prepared under tirring for 48 h and their dynamic viscoelastic properties were easured by using a controlled stress rheometer (Haake Rheostress , Gebruder Haake, Germany) equipped with two parallel-plates of 5 mm diameter and a gap of 200 �m. A circulating water bath Haake C25P, Germany) for sample temperature control and a oftware (Rheowin 2.94) for data acquisition were also used. All easurements were carried out in triplicate, within the linear vis- oelastic range, at 37 ◦C. Small deformation oscillatory experiments were conducted by sing three steps of rheological measurements: (1) stress sweeps etween 0.1 and 100 Pa at constant frequency (1 Hz) to determine of Pharmaceutics 423 (2012) 281– 288 the linear viscoelastic region and the maximum deformation attain- able by a sample, (2) frequency sweeps (0.6–623 rad/s) at a constant stress (5 Pa) to obtain mechanical spectra by recording the dynamic moduli G′ and G′′ as a function of frequency, (3) creep/recovery tests at constant stress (5 Pa). In this assay the stress was applied instantly and kept by 300 s. After removing stress, compliance was measured during further 300 s. 2.4. Thermal analysis The thermogravimetric analysis (TG), differential thermogravi- metric analysis (DTG) and differential thermal analysis (DTA) of samples 11-4-2, 14-4-2, 11-4-2W, 14-4-2W, PMP-HA 11 and PMP- HA 14, were performed on TA Instruments (SDT 600) (New Castle, DE, USA). The instrument was calibrated with indium and an empty pan was used as reference. Samples (10 mg) were accurately weighed in coated alumina pans and heated from 25 to 1200 ◦C at 10 ◦C/min under nitrogen atmosphere. 2.5. X-ray diffraction measurements The X-ray diffraction analysis of pectin, high amylose starch, PMP-HA 11 and of samples 11-4-2, 14-4-2, 11-4-2W and 14-4- 2W were performed on a X-ray diffractometer (Siemens® – Model D5000; Germany), using nickel-filtered Cu K� radiation (tube oper- ating at 40 kV and 30 mA). The scanning regions were collected from 4 to 60◦ (2�) in step size of 0.05 (2�). 2.6. NMR analysis The solid state NMR analyses were carried out on a Bruker Avance III (Germany) spectrometer at 400.13 MHz to 1H, and 100.62 MHz to 13C. All the chemical shifts were referenced to TSP- 4 (sodium trimethylsilyl propionate), the contact time for CP/MAS (cross-polarization/magic angle spinning) spectrum was 4 ms, and the samples were measured at 4 mm ZrO rotor spinning at 10 kHz. 3. Results and discussion 3.1. Rheological measurements Dynamic testing is commonly used to provide a more explicit and detailed characterization of a gel structure (O’Brien et al., 2009). Results of stress sweep are shown in Fig. 1. The samples containing the highest pectin proportion presented the lowest critical stress values (6.4–22.10 Pa), indicating that they present the weakest gel structures. The fact that samples 1:1 and 1:4 (pectin:high amylose starch ratio) have supported higher critical stress (100 Pa) before they begin to flow suggests that these sam- ples are systems with a more organized structure and a more rigid network (Durairaj et al., 2009; O’Brien et al., 2009). Besides, the highest values for critical stress presented by cross-linked samples indicate that the phosphorylation reaction of the cross-linking pro- cess strengthened the gel network, as suggested by O’Brien et al. (2009) in a study about starch phosphates. The frequency sweep (0.6–623 rad/s) at constant stress (5 Pa) was performed in order to obtain a mechanical spectrum of the samples and to analyze the frequency dependence of G′ and G′′ moduli. Such assay can provide useful information about the gel structure and it can be used to determine the difference between entangled networks, covalently cross-linked materials and physical gels (Clark and Ross-Murphy, 1987; Doucet et al., 2001; Khondkar et al., 2007). The mechanical spectra of samples 11-4-1, 11-4-2 and 11-4-4 are shown in Fig. 2, which demonstrates the behavioral trend fol- lowed by all the samples studied, indicating a viscoelastic nature with prevalence of elastic behavior because G′ � G′′ within the F.M. Carbinatto et al. / International Journal of Pharmaceutics 423 (2012) 281– 288 283 s of sa w ( s s a p s 2 s t b B G w A o p 2 t l i F the fluency phase and Jmin is the last point of the recovery phase (Ghannam, 2004). Fig. 1. Stress sweep hole frequency range and G′ is almost frequency independent O’Brien et al., 2009). A more detailed data analysis by linear regres- ion of the frequency sweep results allows to observe that the amples 1:1 and 1:4 were more frequency independent (r2 = 0.999 nd 0.994, respectively) and presented higher G′ values than sam- le 4:1 (r2 = 0.948). These findings are in agreement with critical tress values previously presented. Besides, the reaction conditions h and 4% of base strength seem to contribute to building systems tructurally more organized and to enhance the elastic character of he systems. The degree of frequency dependence can also be evaluated y parameters established by a “power law” (Ramkumar and hattacharya, 1996; Khondkar et al., 2007): ′ = AωB (1) here G′ is the storage modulus, ω is the oscillation frequency and is a constant. The B value can be related to both the strength and the structure f the gel, B = 0 indicating a covalent gel while B > 0 suggesting a hysical gel (Hsu et al., 2000; Yoneya et al., 2003; Khondkar et al., 007). The low values of B (Table 1) for all samples should be related o strong gels structures. Concerning to samples W, synthesized without adding cross- inking agent, both the critical stress, G′, and B values were nvariably lower than those for samples cross-linked with SMTP, ig. 2. Mechanical spectra (5 Pa; 37 ◦C) of samples 11-4-1, 11-4-2 and 11-4-4. mples (1 Hz, 37 ◦C). evidencing the network strengthening resulted from the phospho- rylation reaction. In the “creep and recovery” tests the samples were submit- ted to constant stress (5 Pa) during 300 s to evaluate the material deformation. Subsequently, the stress was removed to determine the recovery at the same time (300 s). All samples studied fol- lowed the same trend presented by samples 11-4-1, 11-4-2 and 11-4-4 (Fig. 3), evidencing their viscoelastic characteristic. The % recovery of all samples was calculated by Eq. (2) and it can be noted that samples containing higher proportions of pectin (4:1 pectin:high amylose starch ratio) showed lower recovery ability (47–68% recovery) than those prepared with higher proportions of high amylose starch; 1:1 and 1:4 pectin:high amylose starch ratio; that presented 67–102% and 56–107%, respectively, indicat- ing, again, that the former presents a weaker gel structure. %R = [ Jmax − Jmin Jmax ] × 100 (2) where %R is the recovery percentage, Jmax is the last point on Table 1 Values of B exponent of the samples (5 Pa; 37 ◦C). Samples B exponent 41-2-1 0.0260 41-2-2 0.0137 41-2-4 0.0253 41-4-1 0.0195 41-4-2 0.0137 41-4-4 0.0131 11-2-1 0.0145 11-2-2 0.0133 11-2-4 0.0138 11-4-1 0.0142 11-4-2 0.0116 11-4-4 0.0132 14-2-1 0.0140 14-2-2 0.0122 14-2-4 0.0126 14-4-1 0.0117 14-4-2 0.0102 14-4-4 0.0119 41-4-4W 0.0211 11-4-4W 0.0178 14-4-4W 0.0161 284 F.M. Carbinatto et al. / International Journal F 4 3 t e r G linked samples presented a third degradation stage, in which about ig. 3. Compliance versus time in creep and recovery tests at 37 ◦C for samples 11- -1, 11-4-2, 11-4-4. .2. Thermal analysis The TG/DTG profiles presented in Fig. 4 show a first degrada- ◦ ion stage between 40 and 110 C, which is related to moisture vaporation, according to previous studies on other polysaccha- ides (Einhorn-Stoll et al., 2007; Ghaffari et al., 2007; Shi and unasekaran, 2008). For cross-linked samples, after this initial Fig. 4. Thermogravimetri of Pharmaceutics 423 (2012) 281– 288 peak, an additional sharp peak between 110 ◦C and 150 ◦C can be observed, which is related to chemically bonded water (Godeck et al., 2001; Shi and Gunasekaran, 2008). The second and main decomposition stage of all samples can be observed between 200 ◦C and 400 ◦C, which corresponds to 28–37%, 51–56% and 60–67% of mass loss (cross-linked sample, sample without cross-linker and physical mixture, respectively). These results evidence the higher thermal stability of samples sub- mitted to cross-linking by phosphorylation and also demonstrate that the alkaline treatment of the polymer results in a structural reorganization, which promotes some increasing of thermal stabil- ity in relation to untreated samples. A more detailed analysis of the results allows observing that physical mixtures exhibit two peaks in this main stage. The first one, occurring at temperatures above 210 ◦C, can be attributed to depolymerization of pectin chains (Einhorn-Stoll et al., 2007; Ghaffari et al., 2007; Shi and Gunasekaran, 2008) and the sec- ond, at temperatures higher than 244 ◦C, is related to high amylose starch decomposition (Massicote et al., 2008), since it pre- vails in samples containing a higher proportion of this polymer (PMP-HA 14). Confirming previous results from rheological analysis, the pres- ence of only one peak at 200–400 ◦C indicates that the alkaline treatment caused structural reorganization. However, only cross- 30% of residual mass is degraded at temperatures above 900 ◦C, evi- dencing the higher thermal stability of these samples in relation to the others. c (TG/DTG) curves. F.M. Carbinatto et al. / International Journal of Pharmaceutics 423 (2012) 281– 288 285 urves a L 2 t a 3 F a s s l b ( 1 s d e o s Fig. 5. DTA c DTA profiles (Fig. 5) show an initial endothermic peak at temper- tures below 200 ◦C, corresponding to loss of adsorbed moisture. arge exothermic peaks can be observed at temperatures between 00 and 450 ◦C, which must be related to thermal depolymeriza- ion of pectin chains (Shi and Gunasekaran, 2008). These findings re in agreement with TG/DTG results. .3. X-ray diffraction measurements The X-ray diffraction patterns of all samples are presented in ig. 6. High amylose starch is a semi-crystalline polymer that can dopt different crystalline structures (A, B, C and V). The A, B and C tructures exhibit a packed double helix conformation while the V tructure is related to a single helix conformation, as result of amy- ose complexation with others components, such as water, iodine, utanol and fatty acids (Rioux et al., 2002). The X-ray diffractogram of the high amylose starch sample HYLON VII) shows characteristic peaks of B structure near to 17◦, 9◦, 23◦ and 25◦ (2�). The pectin X-ray diffractogram presents a eries of intense peaks at 12.7◦, 18.42◦, 28.22◦ and 40.14◦ (2�), emonstrating the crystalline behavior of this polymer (Mishra t al., 2008). The physical mixture exhibit crystalline peaks typical f pectin combined with an amorphous halo peak of high amylose tarch. of samples. From the X-ray diffractogram of samples treated in alkaline medium (Fig. 6), one can observe that the peaks at 19◦ (2�) and about 28◦ (2�), which are characteristic of B structure of high amy- lose starch and pectin, respectively, are not present. Instead, a new broader region between 16 and 24◦ (2�) is observed, which can be attributed to a decrease of the crystallinity degree (Rioux et al., 2002). This behavior suggests that the alkaline treatment of such samples promotes a structural reorganization that results in a decay of the degree of crystallinity. The cross-linked samples show (Fig. 6) new predominant peaks at 29◦ and 30◦ (2�), as well as reduction of intensity or even the dis- appearance of specific peaks of the original polymers. In this way, one characteristic peak of high amylose starch at 19◦ (2�) almost disappears, while another sharp peak at about 24◦ (2�) becomes evident and a new peak at about 15◦ (2�) can be observed (Ispas- Szabo et al., 2000). The changes observed in X-ray diffraction of cross-linked samples demonstrate the occurrence of physical and chemical modifications, indicating alterations on the tridimensional network attributed to the cross-linking process. 3.4. NMR analysis 13C NMR spectra of pectin, high amylose starch, 11-4-2W and 11-4-2 showed some structural differences between the samples 286 F.M. Carbinatto et al. / International Journal of Pharmaceutics 423 (2012) 281– 288 tion p s ( 7 a w a a o 2 r ( Fig. 6. X-ray diffrac ynthesized with STMP and those prepared without this reagent Fig. 7). The spectrum of pectin presents predominant signals around 0–82 ppm, which, according to Westerlund et al. (1991), can be ttributed to galacturonic ring carbons (C2–C5) and the same peaks ere displaced or even absent in the spectra of samples 11-4-2W nd 11-4-2. A signal in 53.27 ppm appears only in pectin spectrum nd it can be related to methyl-ester group close to carboxyl groups f pectin (Westereng et al., 2007). The major signal presented in high amylose starch and 11-4- W spectra (Fig. 7) occurs around 71.5 ppm, which must include the esonance signals of C2 (73.8 ppm), C3 (75.4 ppm) and C5 (72.6 ppm) Gil and Geraldes, 1987; Thérien-Aubin et al., 2007). atterns of samples. The spectra of samples 11-4-2 and 11-4-2W exhibit interesting differences in the region of resonance of carbonyl group (carboxyl and ester carbonyl) around 173–177 ppm, which must be related to DE of pectin (Westerlund et al., 1991). While for pectin the resonance of carbonyl group occurred between 170.3 and 175.4 ppm, the sample 11-4-2W presents decreased intensity of these signals and their displacement to the left side. Besides, only two signals were observed at 174.8 and 176 ppm, a feature that can be related to a reduction on DE (Westerlund et al., 1991). On the other hand, in spectra of sample 11-4-2, no characteristic peaks of carbonyl groups were observed, indicating that STMP caused a different structural rearrangement due to the presence of phosphate linkages. F.M. Carbinatto et al. / International Journal of Pharmaceutics 423 (2012) 281– 288 287 spec 4 s a e g s q r t m s p a n p s l t c m A A F a w 2 Fig. 7. 13C NMR . Conclusions All samples studied behaved as covalent gels, since they pre- ented G′ higher than G′′ within the whole frequency range nd these parameters were almost frequency independent. How- ver, cross-linked samples showed properties of the strongest els, indicating that the phosphorylation reaction promotes the trengthening of the network, resulting in higher and more fre- uency independent G′ values, as well as higher critical stress and ecovery %. Likewise, the increase of high amylose starch ratio in he polymer mixtures contributes for the gel strengthening. Ther- al stability of cross-linked samples was higher than that of the amples without cross-linker, which, in turn, was higher than of hysical mixtures. X-ray diffractograms evidenced that physical nd chemical modifications took place along the tridimensional etwork due not only to cross-linking process. When compared to hysical mixtures, structural changes are observed also in samples ubmitted to the reaction conditions, but without added cross- inker. 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Physical properties of pectin–high amylose starch mixtures cross-linked with sodium trimetaphosphate 1 Introduction 2 Materials and methods 2.1 Materials 2.2 Cross-linking of polymers 2.3 Rheological measurements 2.4 Thermal analysis 2.5 X-ray diffraction measurements 2.6 NMR analysis 3 Results and discussion 3.1 Rheological measurements 3.2 Thermal analysis 3.3 X-ray diffraction measurements 3.4 NMR analysis 4 Conclusions Acknowledgments References