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Chitosan complex hydrogel
Journal of Molecular Liquids 156 (2010) 28–32
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Journal of Molecular Liquids j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m o l l i q
Kinetic study of Chondroitin Sulphate release from Chondroitin Sulphate/Chitosan complex hydrogel Juliana F. Piai, Laís C. Lopes, André R. Fajardo, Adley F. Rubira, Edvani C. Muniz ⁎ Grupo de Materiais Poliméricos e Compósitos, GMPC-Departamento de Química, Universidade Estadual de Maringá-Av. Colombo 5790-87020-900, Maringá, Paraná, Brazil
a r t i c l e
i n f o
Available online 2 June 2010 Keywords: Hydrogels Chondroitin Sulphate release device Mathematical models Diffusion Partition phenomenon
a b s t r a c t Hydrogels based on complexes of Chitosan/Chondroitin Sulphate (CT/CS) can be formed just by mixing and have been applied as devices for CS releasing. In the CT/CS complex investigated in this work, the release of CS occurs mainly at pHs higher than 6.5, the value of pKaCT, and the maximum fraction of CS released is close to 0.5. In this work, the release of CS was treated as a diffusional transport process and as a partition phenomenon in which a partitioning of solutes between the solvent phase and the hydrogel phase occurs. Mathematical models for predicting the in vitro CS release profile from CT/CS polymer network were used. The hydrogel showed to be pH-responsive and values of half-time for CS releasing are compatibles for the application of CT/CS hydrogels in pharmacological field as devices for CS controlled release. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Hydrogels synthesized from biodegradable polymers own greater biocompatibility than hydrogels synthesized from synthetic polymers [1–3]. Chitosan (CT) is a polysaccharide obtained from deacetylation of chitin, a natural mucopolysaccharide found in invertebrates such as crustaceans, fungi, insects, annelids and mollusks [4]. Nowadays, the CT has been applied in the biomedical and pharmaceutical field, for instance, as drugs carrier due to its biodegradability and nontoxicity properties that provide a desirable biocompatibility [4,5]. Due to its cationic nature and high charge density in acid environments, the CT forms insoluble-water complexes with anionic moieties such as chondroitin sulphate (CS) [6–9]. CS, a glycosaminoglycan extracted mainly from bovine aorta, is water-soluble biopolymer that has been widely used to treat arthritis-related diseases [10]. Also, it is an important component in protein complexes found in the extracellular matrix of conjunctive animal tissues. In connection tissue the CS forms networks with the collagen which allow CS to be responsible for the good mechanical properties of such connection tissues [11]. Hydrogels based on CT/CS complexes are widely applied as biomaterial [12,13]. Thus, CT/CS complex hydrogels with excess of CS (40% CT and 60% CS, in weight) can be prepared aiming at their application as drug release devices [8]. The prevision of overall release profile of solute from a given device allows for better evaluation of solute dosage [14], whereas any variation in administered amount can lead to undesirable side effects [15]. Several mathematical models
⁎ Corresponding author. Fax: + 55 44 3011 4125. E-mail address: [email protected] (E.C. Muniz). 0167-7322/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.molliq.2010.05.017
have been developed to describe the solute release profiles from polymer networks [16–19]. Among the models, there is the semiempirical one proposed by Peppas et al. [18] that describes the mechanism of transport of solutes from a flexible matrix as a diffusional transport process [16,17]. Thus, the diffusion model takes into account three driving forces that contribute to release phenomenon: the penetrant concentration gradient, the polymer stress gradient and the osmotic forces. In the case of nonswelling controlled delivery systems, the relaxation rate of the polymer is very slow in comparison to the water transport inside the hydrogel. Then, the transport mechanism in this type of systems follows Fickian diffusion. When the macromolecular chain relaxation is the dominating driving force, Case II transport is observed. However, in many swellingcontrolled delivery systems, anomalous transport mechanism has been observed, characterized by an intermediate Fickian diffusion and Case II transport [18]. Other mathematical model considers the release process as a partition phenomenon in which the partitioning of solutes occurs between the solvent and hydrogel phases [19]. The fraction of solute released, FR, is related to a release parameter designated partition activity (α) and such parameter determines the existence of partition phenomena. It may be used to express the physical chemical affinities of the solute between the solvent and hydrogel phases. This work investigates the capacity of CS to be released from the CT/CS complex hydrogels in water at several pH conditions. To foresee the profile of CS released, mathematic models [diffusional/chain relaxation transport process and partition phenomena] were used. After analyzing the data, it was verified that the releasing of CS occurs in a controlled way with a compatible velocity, so the CT/CS complex hydrogels are able to be applied as carrier for CS controlling release.
J.F. Piai et al. / Journal of Molecular Liquids 156 (2010) 28–32
2. Experimental 2.1. Materials Chitosan (CT), 15% acetylated (9012-76-4, Acros Organics, Belgium), MV: 400 × 103 g mol− 1 according to the method proposed by Mao et al. [20]. The intrinsic viscosity of CT in 2% HAc/0.2 mol L− 1 NaAc was measured using an Ubbelohde capillary viscometer (Model Cannon 100/E534) at 25.0 °C. The CT solution concentrations were adjusted based on the viscosity of the samples. Mark-Houwink constants K = 1.38 × 10− 5 and a = 0.85 were reported for chitosan with DD value of 85%. CS, lot B5B234-B.01, kindly supplied by Solabia (Maringá, Brazil) presented MV 22 × 103 g mol− 1, according to the method proposed by Wasteson [21]. Viscometry of CS was solution carried out in Ubbelohde capillary viscometer (Model Cannon 100/ E534) at 25.0 °C. An aqueous solution of 0.2 mol L− 1 sodium chloride, containing 1–5mg of polysaccharide/mL, was prepared. The K value is 5.0 × 10− 6 and the a value is 1.14 [21]. All reagents and products were used as received, without previous purification. 2.2. Procedures 2.2.1. CT/CS complex hydrogel formation Two solutions were prepared for the synthesis of hydrogel. To prepare the first solution, 1 g of CT was dissolved in 80 mL of an aqueous solution of acetic acid with 0.57 mol L− 1 HCl at 65 °C under constant stirring. For the second solution, the required amount of CS was dissolved in 20 mL of distilled water to conc. 25 wt.%. The CS solution was then slowly poured into solution CT under magnetic stirring at room temperature. The in situ pH where hydrogel was formed is 0.50. The suspension formed was stored for 24 h for sedimentation of hydrogel. Thus, precipitate was separated from the supernatant and purified by immersion in 500 ml of distilled water for 24 h. After immersion, the pH was adjusted to neutral condition using a requested volume of 1.0 mol L− 1 aqueous solution of NaOH. An aliquot of each supernatant was stored for later analysis. The obtained hydrogel showed good consistency and easy to handling. Therefore, the mechanical strength was qualitatively good. The swollen hydrogel was cut into small cubes (about 2 in. of tip) and dried at room temperature for 48 h. 2.2.2. Preparation of buffer solutions with constant ionic strength Buffer solutions with pHs ranging from 2 to 8 and conc. equal to 50 mmol L− 1 were prepared following the United States Pharmacopeia — National Formulary (USP30-NF25) [22]. Table 1 describes the amounts of feed solutions used for preparing the buffer solutions used in this work.
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PolyView and PolySep-GFC-P 6000 column chromatography, 300 × 7.8 mm (Phenomenex) were used. Buffer solutions with pH 2 (HCl/KCl), 6 and 8 (KH2PO4/NaOH) with conc. equal to 50 mmol L− 1 and constant ionic strength (0.1 mol L− 1) prepared with the amounts described in Table 1 were used as mobile phase flowing at 0.5 mL min−1. The eluted solution was monitored at λ = 210 nm, that is the maximum UV absorbance of CS. For quantifying the values of CT and CS in the hydrogel, a known volume of supernatant was collected after the hydrogel has been precipitated and after adjusting the pH to 2. Moreover, the desired volumes of supernatant were collected during the purification of hydrogel. All collected aliquots were analyzed by HPLC. The amounts of both CS and CT incorporated hydrogel were evaluated by comparing the remaining value of the CS hydrogel forming solution with the values found in both CS supernatant and purified water. 3. Results and discussion 3.1. In vitro release of CS from the CT/CS complex hydrogel The nominal composition of dry hydrogel used in this study is, in wt.%, CS 60% and CT 40%. Just after preparation, cubic samples of hydrogels were soaked in aqueous solutions of different pHs. The curves of swelling vs. immersion time presented maxima at pHs 6 and 8 (data published elsewhere [8]). In these pH conditions, the hydrogel swelled more and faster than at pH 2. The maxima were connected to the hydrogel mass loss, due to erosion process [23], which might have occurred by release mechanism of fraction of incorporated CS [8,9]. The amount of released CS was detected through HPCL technique. Fig. 1 presents the curves of timedependence fraction of released CS from CT/CS complex hydrogels at pHs 2, 6 and 8 buffers. It was observed that the samples immersed in pH 2 buffer presented lower level of CS releasing (0.15) than the samples immersed in pH 6 and 8 buffers (0.5). At pH 2 the ionization of sulfate groups is supposed to occur according to the following + reaction: -OSO3H ⇌ –OSO− 3 + H , (pKa = 2.60). The electrostatic interactions are established between CS-OSO− 3 and COOH groups and CT–NH+ 3 groups, besides the H-bonding between like and/or unlike polymer chains. Thus, the CS chains are released at pH 2.0 just until the hydrogel achieve the equivalent mol-ratio of 2/1 (CT/CS), i.e., the ratio stoichiometric [7]. At pH 8, (higher than pKaCT = 6.5 [24]), the CT amine groups remain neutral, while the sulfate (–OSO3H) (pKa = 2.60 [25]) and carboxylic (–COOH) (pKa = 4.57 [25]) groups of CS are negatively ionized. In view of the higher amount of CS in the CT/CS hydrogel with respect to CT (60 wt.% against 40 wt.%), the repulsion force between negatively charged groups at pH higher than the pKaCT is sufficient for the whole hydrogel to become more
2.2.3. In vitro release of CS from the CT/CS complex hydrogel After dry-CT/CS complex hydrogel cubes were weighed in advance and stored in 25 mL of buffer with pH 2, 6 or 8, with ionic strength constant at 37 °C. Aliquots of supernatant were collected at the desired time of immersion. The amount of CS released was quantified by HPLC technique. A Star workstation chromatography (Varian Inc. Scientific Instruments, USA) with a spectrophotometer detector based on the model of a diode array ProStar 350 and operated by software
Table 1 Amounts of solute and solvent used to prepare 1000 mL of buffer solutions with pH varying from 2 to 8. The KCl salt was used to adjust the ionic strength to 0.1 mol L− 1. pH
Solute
Solution (*) 0.5 mol L− 1 (mL)
HCl 0.2 mol L− 1 (mL)
NaOH 0.2 mol L− 1 (mL)
KCl (g)
2.00 6.00 8.00
KCl KH2PO4 KH2PO4
100 100 100
53.0 0 0
0 29 234
2.94 3.30 3.04
Fig. 1. Cumulative fraction of CS released as a function of immersion time in three different buffer solutions at 37 °C. Figure obtained from Ref. [8].
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J.F. Piai et al. / Journal of Molecular Liquids 156 (2010) 28–32
swollen and increase the CS release rate. Therefore, at pHs 6 and 8 the amounts of CS released are larger than that released at pH 2 and the equivalent mol-ratio CT/CS achieves 7/2 at these pHs against the molratio 2/1 at pH 2. 3.2. Consistency of hydrogel at several pHs In spite the CT/CS to be physical hydrogels formed in acidic medium, such hydrogel are relatively stables in basic medium. After releasing almost 50% of the initial used amount CS to form the hydrogel, the hydrogel presented good consistency and mechanical properties even after 200 h of immersion at different pHs. Fig. 2 shows the aspect of CT/CS hydrogel after 200 h of immersion in pHs 1 to 12. Morphological analysis of same material used in this work was published elsewhere [8]. It was pointed out in that paper the CT/CS hydrogels show spread pore sizes. Also, it was visualized by SEM the presence of a skin inside the pores of such materials [8]. 3.3. Study of transport mechanism of in vitro CS release from the CT/CS complex hydrogel The release process can be seen as a diffusional transport process and/or as a partition phenomenon in which the partitioning of solutes occurs between the solvent phase and the hydrogel. Ritger and Peppas [18] proposed a well know semi-empirical model that describes the mechanism of transport of solutes from a flexible matrix that is given by the equation Mt n = kt M∞
Table 2 Values of diffusional exponent (n) and constant (k) obtained by application of RitgerPeppas model in mediums with different pHs, at 37 °C. pH
n
k (10− 2)
R2
2 6 8
0.166 0.439 0.423
5.7 8.9 12.5
0.987 0.983 0.982
Fig. 1, the obtained values of n and k for the releasing of CS from CT/CS hydrogel are shown in Table 2. According to Table 2 the obtained values for n were lower than 0.5. So, it can be said that the diffusion mechanism of CS within the hydrogel to the solution prevails and mainly by Fickian diffusion at pHs 6 and 8 buffers. Given that k is a constant dependent on solvent/polymer system [18], the values obtained show that hydrogel exhibit a pH-sensitive property, with higher values of k at pH 6 and 8 buffers. By analyzing the profiles of CS releasing from CT/CS complex hydrogel at 37 °C, shown in Fig. 1, it can be accomplished the occurrence of equilibrium of release after around 200 h immersion time. It can be pointed out that when the equilibrium is reached, the release and absorption rates of solute by the hydrogel are equal and the fractional release (FR) attain a maximum value (Fmax) for a given condition. In this way, the CS released from hydrogel could be treated, as well as a phenomenon of diffusion, as a phenomenon of partition. The parameter that determines the occurrence of this phenomenon is α, which was calculated by means of Eq. (2) [19].
ð1Þ α=
where Mt/M∞ is the fractional drug release, t is the release time, k is a constant dependent on the solvent/polymer and n is the diffusional exponent that can be related to the drug transport mechanism [18]. Eq. (1) characterizes the solvent diffusion mechanism within the gel or the release mechanism of the solute. For blocks or thin film hydrogels, when n is around 0.5, the drug release mechanism would be controlled by Fickian diffusion [26]. When n = 1, the transport mechanism is described as Case II and the release kinetics of zero order occurs, i.e., the slow release of drug occurs from dosage forms that do not degrade and the amount of released moiety increases straightforward with time. This is associated with the matrix relaxation that, in the case of hydrogels, implies in a macromolecular relaxation. When the value of n is between 0.5 and 1, an anomalous transport is observed which result of contributions of diffusion and matrix relaxation [27]. After applying the Eq. (1) to the curves of
Fmax 1−Fmax
ð2Þ
Being that α expresses the physical–chemical affinity of the drug with hydrogel and with solvent phases. With α N 0, it is observed the diffusion of solute in solvent phase. The α value is dependent on a large number of variables such as temperature, pressure, pH, ionic strength, hydrogel composition, hydrogel geometry, degree of swelling of hydrogel, the chemical nature of solvent and drug, etc. Thus, the partition activity (α) can take an unlimited number of values [19]. With Fmax values obtained from the release curves performed at different pHs, as shown in Fig. 1, we obtained the values of α for the CT/CS complex hydrogel, which are presented in Table 3. Once determined the α values for each pH buffer, the respective kinetic constants of release (kR) could be calculated, assuming that release kinetics occurs according to second-order kinetics (Eq. (3)). Details for obtaining the Eq. (3) are given in ref [19]. The obtained values for
Fig. 2. Pictures of CT/CS swollen hydrogel after 200 h immersed in buffers of pH 1 to 12.
J.F. Piai et al. / Journal of Molecular Liquids 156 (2010) 28–32 Table 3 Partition coefficient (α) and release kinetics constant (kR) values for CS release from CT/ CS complex hydrogel in pHs 2, 6 and 8, at 37 °C, using Eq. (3). pH
α
kR (10− 3)
R2
2 6 8
0.177 1.020 0.958
5.8 33.2 54.0
0.999 0.998 0.999
the rate constant for CS releasing (kR) from the CT/CS hydrogel after applying the Eq. (3) are presented in Table 3. α F −2FR Fmax + Fmax × ln R = kR t 2 Fmax −FR
ð3Þ
The chemical nature of the solvent that is in contact with the hydrogel can affect both the release rate and the value of α as well [19]. This means that, when in contact with pH 6 and 8 buffers, the CT/ CS hydrogel has higher values of α if compared with that obtained at pH 2. In accordance with the predetermined conditions, higher values of α imply that the solute has more affinity for the solvent than for the device (3D matrix) in which it is inserted. This occurs because in media with pH close to or higher than the pKa of hydrogen sulfate and carboxylic acid groups, the sulfate and carboxylate groups on CS are in their ionized state and, consequently, increasing the electrostatic repulsion and results in a decreasing in the polymers chains entanglement and thus a greater CS diffusion outward solution. The pH of the gastrointestinal tract and transit time of food through the digestive tract are seen as strategic in the development of oral modified release drug [28]. In the stomach, low pHs (1–3.5) and the highest rates of gastric emptying (5 min–2 h) are two limiting factors for release and absorption of drugs. In the small intestine, the
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transit time ranges from 3 to 4 h, but, the intense activity of pancreatic enzymes limits the release of drug at this site. Thus, the colon has been widely suggested as the most appropriate place for the release of drugs [28]. The low activity of enzymes, the high pH (6–7.5) and large transit time (20–35 h) allow the integrity of the drug and a long period of residence of the therapeutic device in this region [28,29]. If the release kinetics of CS is characterized as second-order and reversible (Eq. (3)), the half-time release of drug (t1/2) can be determined using the partition model [19]: t1 = 2 =
α × ln ð3−2Fmax Þ 2kR
ð4Þ
being the t1/2 the required time for the drug concentration on solvent to reach 50% of Fmax. From the CS release profiles of studied hydrogel, the obtained values of t1/2 (hours) were equal to 15.24, 10.57 and 6.24 for pH 2, 6 and 8 buffers, respectively. This means that in media with high pH the time required for releasing of 50% of CS from the CT/CS complex hydrogel is smaller than in mediums with low pH values. This is compatible for application of CT/CS complex hydrogel in pharmacological field since that the hydrogel can protect the CS before their arrival in the colon due values of t1/2 are greater than digestive transit time in the stomach and small intestine than in the colon. The fraction of solute released at a given time t, FR, is related to the activity partition (α) and such relation is defined by Eq. (3) [19]. So, the value of FR at time t can be calculated for a system with a defined α (or Fmax) parameter. Based on this statement and on the respective values of Fmax, α and kR for a given solution with a specific pH in which the hydrogel is swelled, values of FR can be obtained through the use of Eq. (3). Curve of FR as a function of time was plotted for pHs buffer 2, 6 and 8 (Fig. 3). For comparison purposes, the curves obtained by
Fig. 3. Experimental and theoretical profiles (Eq. (1) e 3) obtained for fraction of CS released from CT/CS complex hydrogel at 37 °C as a function of time at pHs 2, 6 and 8 buffers.
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application of Ritger-Peppas model [18] (Eq. (1)) were superimposed in the same figures. The data generated from the model described by Eq. (3) are in accordance with the experimental data exhibited in the release profiles at pHs 6 and 8 buffers, as presented in Fig. 3. The values of R2 are given in Table 3 for the three investigated buffers (pHs 2, 6 and 8) and are higher than 0.998. The two models used for providing the CS released profile at a given condition of pH showed significant differences (compare Tables 2 and 3). Furthermore, the constant release obtained for the semi-empirical model, given by Eq. (1), showed higher values than those obtained in the model given by Eq. (3). It cannot have, at same time, a favorable comparison between the two models due some important factors. First, the semi-empirical model given by Eq. (1) predicts at maximum 60% of the releasing profile [18], while the model given by Eq. (3) predicts up to 100% [19]. Furthermore, both Fickian diffusion mechanism and molecular relaxation are seen in the model given by Eq. (3) as intrinsic parameters included in α and kR [19]. Eq. (3) shows that releasing of CS is both a diffusional-transport process as a partition phenomenon where the solute partitioning occurs between the phases of the solvent and hydrogel [19]. Analyzing the ratios between the rate constants of releasing evaluated by Eq. (3) with those same rate constants evaluated by Eq. (1), it becomes clear that at pH 6 and 8 buffers, the obtained results for that ratio were very almost similar, being 0.37 and 0.43 respectively. However, at pH 2 buffer such ratio is equal to 0.10 being, therefore, much lower than at pH 6 and 8 buffers. This implies that the differences in the CS release due to pH change are not weigh up in a same way for both models. As stated before, the prevision of overall profile solute release from a given device allows for better evaluation of solute dosage. In this way, the model given by Eqs. (2) and (3) can best explain the results obtained in this work, since that it provides 100% of solute released and shows release mechanisms of solutes. It can be seen both as a transport process and as a diffusion phenomenon of partition. Thus, with support of mathematical models, it was observed that the CT/CS complex hydrogel responds to changes of the pH of and the fraction of CS released is intensified at buffers of pH 6 and 8 than at pH 2 buffer. Since that CS is classified as a drug that produces symptomatic relief of protracted action, it can be inferred that the CT/CS complex hydrogel is an interesting device for CS release at specific physiological conditions where the pH is greater than the pKa of carboxylic and hydrogen sulfate groups on CS. 4. Conclusions In this work CT/CS complex hydrogel was prepared and the releasing of CS from such hydrogel was characterized and quantified by HPLC. It was found that the maximum of CS released fraction is close to 0.5 and that the process of CS chains release during the swelling process of the CT/CS complex hydrogel is driven by electrostatic repulsion among chains at circumstances in which the pH is greater than the pKa of carboxylic and hydrogen sulfate groups
on CS. The profile of CS released was evaluated using different mathematical models. The prediction of in vitro CS releasing from CT/ CS complex hydrogel can be made using mathematical models based on partitioning even the hydrogel presented different behaviors release by changing the pH. Both diffusion and partition phenomena occur when hydrogel is swelled in medium with different pH values. This result in releasing of CS that was intensified when the CT/CS complex hydrogel is swelled in media with higher pH values. The values of t1/2 (hours) equal to 15.24, 10.57 and 6.24 for pH 2, 6 and 8 buffers, respectively. They are compatible for application in pharmacological field since that the t1/2 values are higher than stomach and small intestine transit time. The interesting behavior demonstrated by this material allows its application how an effective device for CS release, mainly at pH ≥ 6.0 conditions. Acknowledgements All authors thank CNPq (Brazil) for the financial support. ARF thank CAPES (Brazil) for the doctoral fellowship. References [1] S.C. Chen, Y.C. Wu, F.L. Mi, H.W. Lin, L.C. Yu, H.W. Sung, J. Control. Release 96 (2004) 285. [2] J. Berger, M. Reist, J.M. Mayer, O. Felt, N.A. Peppas, R. Gurny, Eur. J. Pharm. Biopharm. 57 (2004) 19. [3] A. Nascimento, M.C. Laranjeira, V.T. Favere, A. Josue, J. Microencapsul. 18 (2001) 679. [4] M.N.V.R. Kumar, R.A.A. Muzzarelli, C. Muzzarelli, H. Sashiwa, A. Domb, J. Chem. Rev. 104 (2004) 6017. [5] D. Capitani, V. Crescenzi, A.A. de Angelis, A.L. Segre, Macromolecules 34 (2001) 4136. [6] S. Vasiliu, M. Popa, M. Rinaudo, Eur. Polym. J. 41 (2005) 923. [7] A. Denuziere, D. Ferrier, A. Domard, Carbohydr. Polym. 29 (1996) 317. [8] J.F. Piai, A.F. Rubira, E.C. Muniz, Acta Biomater. 5 (2009) 2601. [9] A.R. Fajardo, J.F. Piai, A.F. Rubira, E.C. Muniz, Carbohydr. Polym. 80 (2010) 934. [10] F. Richy, O. Bruyere, O. Ethgen, M. Cucherat, Y. Herotin, J.Y. Reginster, Arch. Intern. Med. 163 (2003) 1514. [11] Y.L. Chen, H.C. Chen, H.P. Lee, H.Y. Chan, Y.C. Hu, Biomaterials 27 (2006) 2222. [12] A. Denuziere, D. Ferrier, O. Damour, A. Domard, Biomaterials 19 (1998) 1275. [13] C. Peniche, M. Fernández, G. Rodríguez, J. Parra, J. Jimenez, A.L. Bravo, D. Gómez, J.S. Román, J. Mater. Sci. Mater. Med. 18 (2007) 1719. [14] J.F. Piai, M.R. Moura, A.F. Rubira, E.C. Muniz, Macromol. Symp. 266 (2008) 108. [15] D.C. Berry, P. Knapp, D.K. Raynor, Lancet 359 (2002) 853. [16] V.M.M. Lobo, A.J.M. Valente, A. Ya, G. Geuskens Polishchuk, J. Mol. Liq. 94 (2001) 179. [17] J. Siepmanna, N.A. Peppas, Adv. Drug Delivery Rev. 48 (2001) 139. [18] L. Serra, J. Doménech, N.A. Peppas, Biomaterials 27 (2006) 5440. [19] A.V. Reis, M.R. Guilherme, A.F. Rubira, E.C. Muniz, J. Colloid. Interfaces Sci. 310 (2007) 128. [20] S. Mao, X. Shuai, F. Unger, M. Simon, D. Bi, T. Kissel, Int. J. Pharm. 281 (2004) 45. [21] A. Wasteson, Biochem. J. 122 (1971) 477. [22] USP30-NF25, United States Pharmacopeial-National Formulary, The United Pharmacopeial Convention, Rockville, MD, U.S.A, , 2007. [23] K. Makino, R. Idenuma, H. Ohshima, Colloids Surf., B 8 (1996) 93. [24] S.P. Strand, K. Tommeraas, K.M. Varum, K. Ostgaard, Biomacromolecules 2 (2001) 1310. [25] B. Larsson, M. Nilsson, H. Tjalve, Biochem. Pharm. 30 (1981) 2963. [26] T. Higuchi, J. Pharm. Sci. 52 (1963) 1145. [27] N.A. Peppas, J.J. Sahlin, Int. J. Pharm. 57 (1989) 169. [28] A.C. Freire, F. Podczeck, J. Sousa, F. Veiga, Rev. Bras. Cienc. Farm. 42 (2006) 319. [29] D.R. Friend, Adv. Drug Delivery Rev. 57 (2005) 247.
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Journal of Molecular Liquids j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m o l l i q
Kinetic study of Chondroitin Sulphate release from Chondroitin Sulphate/Chitosan complex hydrogel Juliana F. Piai, Laís C. Lopes, André R. Fajardo, Adley F. Rubira, Edvani C. Muniz ⁎ Grupo de Materiais Poliméricos e Compósitos, GMPC-Departamento de Química, Universidade Estadual de Maringá-Av. Colombo 5790-87020-900, Maringá, Paraná, Brazil
a r t i c l e
i n f o
Available online 2 June 2010 Keywords: Hydrogels Chondroitin Sulphate release device Mathematical models Diffusion Partition phenomenon
a b s t r a c t Hydrogels based on complexes of Chitosan/Chondroitin Sulphate (CT/CS) can be formed just by mixing and have been applied as devices for CS releasing. In the CT/CS complex investigated in this work, the release of CS occurs mainly at pHs higher than 6.5, the value of pKaCT, and the maximum fraction of CS released is close to 0.5. In this work, the release of CS was treated as a diffusional transport process and as a partition phenomenon in which a partitioning of solutes between the solvent phase and the hydrogel phase occurs. Mathematical models for predicting the in vitro CS release profile from CT/CS polymer network were used. The hydrogel showed to be pH-responsive and values of half-time for CS releasing are compatibles for the application of CT/CS hydrogels in pharmacological field as devices for CS controlled release. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Hydrogels synthesized from biodegradable polymers own greater biocompatibility than hydrogels synthesized from synthetic polymers [1–3]. Chitosan (CT) is a polysaccharide obtained from deacetylation of chitin, a natural mucopolysaccharide found in invertebrates such as crustaceans, fungi, insects, annelids and mollusks [4]. Nowadays, the CT has been applied in the biomedical and pharmaceutical field, for instance, as drugs carrier due to its biodegradability and nontoxicity properties that provide a desirable biocompatibility [4,5]. Due to its cationic nature and high charge density in acid environments, the CT forms insoluble-water complexes with anionic moieties such as chondroitin sulphate (CS) [6–9]. CS, a glycosaminoglycan extracted mainly from bovine aorta, is water-soluble biopolymer that has been widely used to treat arthritis-related diseases [10]. Also, it is an important component in protein complexes found in the extracellular matrix of conjunctive animal tissues. In connection tissue the CS forms networks with the collagen which allow CS to be responsible for the good mechanical properties of such connection tissues [11]. Hydrogels based on CT/CS complexes are widely applied as biomaterial [12,13]. Thus, CT/CS complex hydrogels with excess of CS (40% CT and 60% CS, in weight) can be prepared aiming at their application as drug release devices [8]. The prevision of overall release profile of solute from a given device allows for better evaluation of solute dosage [14], whereas any variation in administered amount can lead to undesirable side effects [15]. Several mathematical models
⁎ Corresponding author. Fax: + 55 44 3011 4125. E-mail address: [email protected] (E.C. Muniz). 0167-7322/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.molliq.2010.05.017
have been developed to describe the solute release profiles from polymer networks [16–19]. Among the models, there is the semiempirical one proposed by Peppas et al. [18] that describes the mechanism of transport of solutes from a flexible matrix as a diffusional transport process [16,17]. Thus, the diffusion model takes into account three driving forces that contribute to release phenomenon: the penetrant concentration gradient, the polymer stress gradient and the osmotic forces. In the case of nonswelling controlled delivery systems, the relaxation rate of the polymer is very slow in comparison to the water transport inside the hydrogel. Then, the transport mechanism in this type of systems follows Fickian diffusion. When the macromolecular chain relaxation is the dominating driving force, Case II transport is observed. However, in many swellingcontrolled delivery systems, anomalous transport mechanism has been observed, characterized by an intermediate Fickian diffusion and Case II transport [18]. Other mathematical model considers the release process as a partition phenomenon in which the partitioning of solutes occurs between the solvent and hydrogel phases [19]. The fraction of solute released, FR, is related to a release parameter designated partition activity (α) and such parameter determines the existence of partition phenomena. It may be used to express the physical chemical affinities of the solute between the solvent and hydrogel phases. This work investigates the capacity of CS to be released from the CT/CS complex hydrogels in water at several pH conditions. To foresee the profile of CS released, mathematic models [diffusional/chain relaxation transport process and partition phenomena] were used. After analyzing the data, it was verified that the releasing of CS occurs in a controlled way with a compatible velocity, so the CT/CS complex hydrogels are able to be applied as carrier for CS controlling release.
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2. Experimental 2.1. Materials Chitosan (CT), 15% acetylated (9012-76-4, Acros Organics, Belgium), MV: 400 × 103 g mol− 1 according to the method proposed by Mao et al. [20]. The intrinsic viscosity of CT in 2% HAc/0.2 mol L− 1 NaAc was measured using an Ubbelohde capillary viscometer (Model Cannon 100/E534) at 25.0 °C. The CT solution concentrations were adjusted based on the viscosity of the samples. Mark-Houwink constants K = 1.38 × 10− 5 and a = 0.85 were reported for chitosan with DD value of 85%. CS, lot B5B234-B.01, kindly supplied by Solabia (Maringá, Brazil) presented MV 22 × 103 g mol− 1, according to the method proposed by Wasteson [21]. Viscometry of CS was solution carried out in Ubbelohde capillary viscometer (Model Cannon 100/ E534) at 25.0 °C. An aqueous solution of 0.2 mol L− 1 sodium chloride, containing 1–5mg of polysaccharide/mL, was prepared. The K value is 5.0 × 10− 6 and the a value is 1.14 [21]. All reagents and products were used as received, without previous purification. 2.2. Procedures 2.2.1. CT/CS complex hydrogel formation Two solutions were prepared for the synthesis of hydrogel. To prepare the first solution, 1 g of CT was dissolved in 80 mL of an aqueous solution of acetic acid with 0.57 mol L− 1 HCl at 65 °C under constant stirring. For the second solution, the required amount of CS was dissolved in 20 mL of distilled water to conc. 25 wt.%. The CS solution was then slowly poured into solution CT under magnetic stirring at room temperature. The in situ pH where hydrogel was formed is 0.50. The suspension formed was stored for 24 h for sedimentation of hydrogel. Thus, precipitate was separated from the supernatant and purified by immersion in 500 ml of distilled water for 24 h. After immersion, the pH was adjusted to neutral condition using a requested volume of 1.0 mol L− 1 aqueous solution of NaOH. An aliquot of each supernatant was stored for later analysis. The obtained hydrogel showed good consistency and easy to handling. Therefore, the mechanical strength was qualitatively good. The swollen hydrogel was cut into small cubes (about 2 in. of tip) and dried at room temperature for 48 h. 2.2.2. Preparation of buffer solutions with constant ionic strength Buffer solutions with pHs ranging from 2 to 8 and conc. equal to 50 mmol L− 1 were prepared following the United States Pharmacopeia — National Formulary (USP30-NF25) [22]. Table 1 describes the amounts of feed solutions used for preparing the buffer solutions used in this work.
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PolyView and PolySep-GFC-P 6000 column chromatography, 300 × 7.8 mm (Phenomenex) were used. Buffer solutions with pH 2 (HCl/KCl), 6 and 8 (KH2PO4/NaOH) with conc. equal to 50 mmol L− 1 and constant ionic strength (0.1 mol L− 1) prepared with the amounts described in Table 1 were used as mobile phase flowing at 0.5 mL min−1. The eluted solution was monitored at λ = 210 nm, that is the maximum UV absorbance of CS. For quantifying the values of CT and CS in the hydrogel, a known volume of supernatant was collected after the hydrogel has been precipitated and after adjusting the pH to 2. Moreover, the desired volumes of supernatant were collected during the purification of hydrogel. All collected aliquots were analyzed by HPLC. The amounts of both CS and CT incorporated hydrogel were evaluated by comparing the remaining value of the CS hydrogel forming solution with the values found in both CS supernatant and purified water. 3. Results and discussion 3.1. In vitro release of CS from the CT/CS complex hydrogel The nominal composition of dry hydrogel used in this study is, in wt.%, CS 60% and CT 40%. Just after preparation, cubic samples of hydrogels were soaked in aqueous solutions of different pHs. The curves of swelling vs. immersion time presented maxima at pHs 6 and 8 (data published elsewhere [8]). In these pH conditions, the hydrogel swelled more and faster than at pH 2. The maxima were connected to the hydrogel mass loss, due to erosion process [23], which might have occurred by release mechanism of fraction of incorporated CS [8,9]. The amount of released CS was detected through HPCL technique. Fig. 1 presents the curves of timedependence fraction of released CS from CT/CS complex hydrogels at pHs 2, 6 and 8 buffers. It was observed that the samples immersed in pH 2 buffer presented lower level of CS releasing (0.15) than the samples immersed in pH 6 and 8 buffers (0.5). At pH 2 the ionization of sulfate groups is supposed to occur according to the following + reaction: -OSO3H ⇌ –OSO− 3 + H , (pKa = 2.60). The electrostatic interactions are established between CS-OSO− 3 and COOH groups and CT–NH+ 3 groups, besides the H-bonding between like and/or unlike polymer chains. Thus, the CS chains are released at pH 2.0 just until the hydrogel achieve the equivalent mol-ratio of 2/1 (CT/CS), i.e., the ratio stoichiometric [7]. At pH 8, (higher than pKaCT = 6.5 [24]), the CT amine groups remain neutral, while the sulfate (–OSO3H) (pKa = 2.60 [25]) and carboxylic (–COOH) (pKa = 4.57 [25]) groups of CS are negatively ionized. In view of the higher amount of CS in the CT/CS hydrogel with respect to CT (60 wt.% against 40 wt.%), the repulsion force between negatively charged groups at pH higher than the pKaCT is sufficient for the whole hydrogel to become more
2.2.3. In vitro release of CS from the CT/CS complex hydrogel After dry-CT/CS complex hydrogel cubes were weighed in advance and stored in 25 mL of buffer with pH 2, 6 or 8, with ionic strength constant at 37 °C. Aliquots of supernatant were collected at the desired time of immersion. The amount of CS released was quantified by HPLC technique. A Star workstation chromatography (Varian Inc. Scientific Instruments, USA) with a spectrophotometer detector based on the model of a diode array ProStar 350 and operated by software
Table 1 Amounts of solute and solvent used to prepare 1000 mL of buffer solutions with pH varying from 2 to 8. The KCl salt was used to adjust the ionic strength to 0.1 mol L− 1. pH
Solute
Solution (*) 0.5 mol L− 1 (mL)
HCl 0.2 mol L− 1 (mL)
NaOH 0.2 mol L− 1 (mL)
KCl (g)
2.00 6.00 8.00
KCl KH2PO4 KH2PO4
100 100 100
53.0 0 0
0 29 234
2.94 3.30 3.04
Fig. 1. Cumulative fraction of CS released as a function of immersion time in three different buffer solutions at 37 °C. Figure obtained from Ref. [8].
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swollen and increase the CS release rate. Therefore, at pHs 6 and 8 the amounts of CS released are larger than that released at pH 2 and the equivalent mol-ratio CT/CS achieves 7/2 at these pHs against the molratio 2/1 at pH 2. 3.2. Consistency of hydrogel at several pHs In spite the CT/CS to be physical hydrogels formed in acidic medium, such hydrogel are relatively stables in basic medium. After releasing almost 50% of the initial used amount CS to form the hydrogel, the hydrogel presented good consistency and mechanical properties even after 200 h of immersion at different pHs. Fig. 2 shows the aspect of CT/CS hydrogel after 200 h of immersion in pHs 1 to 12. Morphological analysis of same material used in this work was published elsewhere [8]. It was pointed out in that paper the CT/CS hydrogels show spread pore sizes. Also, it was visualized by SEM the presence of a skin inside the pores of such materials [8]. 3.3. Study of transport mechanism of in vitro CS release from the CT/CS complex hydrogel The release process can be seen as a diffusional transport process and/or as a partition phenomenon in which the partitioning of solutes occurs between the solvent phase and the hydrogel. Ritger and Peppas [18] proposed a well know semi-empirical model that describes the mechanism of transport of solutes from a flexible matrix that is given by the equation Mt n = kt M∞
Table 2 Values of diffusional exponent (n) and constant (k) obtained by application of RitgerPeppas model in mediums with different pHs, at 37 °C. pH
n
k (10− 2)
R2
2 6 8
0.166 0.439 0.423
5.7 8.9 12.5
0.987 0.983 0.982
Fig. 1, the obtained values of n and k for the releasing of CS from CT/CS hydrogel are shown in Table 2. According to Table 2 the obtained values for n were lower than 0.5. So, it can be said that the diffusion mechanism of CS within the hydrogel to the solution prevails and mainly by Fickian diffusion at pHs 6 and 8 buffers. Given that k is a constant dependent on solvent/polymer system [18], the values obtained show that hydrogel exhibit a pH-sensitive property, with higher values of k at pH 6 and 8 buffers. By analyzing the profiles of CS releasing from CT/CS complex hydrogel at 37 °C, shown in Fig. 1, it can be accomplished the occurrence of equilibrium of release after around 200 h immersion time. It can be pointed out that when the equilibrium is reached, the release and absorption rates of solute by the hydrogel are equal and the fractional release (FR) attain a maximum value (Fmax) for a given condition. In this way, the CS released from hydrogel could be treated, as well as a phenomenon of diffusion, as a phenomenon of partition. The parameter that determines the occurrence of this phenomenon is α, which was calculated by means of Eq. (2) [19].
ð1Þ α=
where Mt/M∞ is the fractional drug release, t is the release time, k is a constant dependent on the solvent/polymer and n is the diffusional exponent that can be related to the drug transport mechanism [18]. Eq. (1) characterizes the solvent diffusion mechanism within the gel or the release mechanism of the solute. For blocks or thin film hydrogels, when n is around 0.5, the drug release mechanism would be controlled by Fickian diffusion [26]. When n = 1, the transport mechanism is described as Case II and the release kinetics of zero order occurs, i.e., the slow release of drug occurs from dosage forms that do not degrade and the amount of released moiety increases straightforward with time. This is associated with the matrix relaxation that, in the case of hydrogels, implies in a macromolecular relaxation. When the value of n is between 0.5 and 1, an anomalous transport is observed which result of contributions of diffusion and matrix relaxation [27]. After applying the Eq. (1) to the curves of
Fmax 1−Fmax
ð2Þ
Being that α expresses the physical–chemical affinity of the drug with hydrogel and with solvent phases. With α N 0, it is observed the diffusion of solute in solvent phase. The α value is dependent on a large number of variables such as temperature, pressure, pH, ionic strength, hydrogel composition, hydrogel geometry, degree of swelling of hydrogel, the chemical nature of solvent and drug, etc. Thus, the partition activity (α) can take an unlimited number of values [19]. With Fmax values obtained from the release curves performed at different pHs, as shown in Fig. 1, we obtained the values of α for the CT/CS complex hydrogel, which are presented in Table 3. Once determined the α values for each pH buffer, the respective kinetic constants of release (kR) could be calculated, assuming that release kinetics occurs according to second-order kinetics (Eq. (3)). Details for obtaining the Eq. (3) are given in ref [19]. The obtained values for
Fig. 2. Pictures of CT/CS swollen hydrogel after 200 h immersed in buffers of pH 1 to 12.
J.F. Piai et al. / Journal of Molecular Liquids 156 (2010) 28–32 Table 3 Partition coefficient (α) and release kinetics constant (kR) values for CS release from CT/ CS complex hydrogel in pHs 2, 6 and 8, at 37 °C, using Eq. (3). pH
α
kR (10− 3)
R2
2 6 8
0.177 1.020 0.958
5.8 33.2 54.0
0.999 0.998 0.999
the rate constant for CS releasing (kR) from the CT/CS hydrogel after applying the Eq. (3) are presented in Table 3. α F −2FR Fmax + Fmax × ln R = kR t 2 Fmax −FR
ð3Þ
The chemical nature of the solvent that is in contact with the hydrogel can affect both the release rate and the value of α as well [19]. This means that, when in contact with pH 6 and 8 buffers, the CT/ CS hydrogel has higher values of α if compared with that obtained at pH 2. In accordance with the predetermined conditions, higher values of α imply that the solute has more affinity for the solvent than for the device (3D matrix) in which it is inserted. This occurs because in media with pH close to or higher than the pKa of hydrogen sulfate and carboxylic acid groups, the sulfate and carboxylate groups on CS are in their ionized state and, consequently, increasing the electrostatic repulsion and results in a decreasing in the polymers chains entanglement and thus a greater CS diffusion outward solution. The pH of the gastrointestinal tract and transit time of food through the digestive tract are seen as strategic in the development of oral modified release drug [28]. In the stomach, low pHs (1–3.5) and the highest rates of gastric emptying (5 min–2 h) are two limiting factors for release and absorption of drugs. In the small intestine, the
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transit time ranges from 3 to 4 h, but, the intense activity of pancreatic enzymes limits the release of drug at this site. Thus, the colon has been widely suggested as the most appropriate place for the release of drugs [28]. The low activity of enzymes, the high pH (6–7.5) and large transit time (20–35 h) allow the integrity of the drug and a long period of residence of the therapeutic device in this region [28,29]. If the release kinetics of CS is characterized as second-order and reversible (Eq. (3)), the half-time release of drug (t1/2) can be determined using the partition model [19]: t1 = 2 =
α × ln ð3−2Fmax Þ 2kR
ð4Þ
being the t1/2 the required time for the drug concentration on solvent to reach 50% of Fmax. From the CS release profiles of studied hydrogel, the obtained values of t1/2 (hours) were equal to 15.24, 10.57 and 6.24 for pH 2, 6 and 8 buffers, respectively. This means that in media with high pH the time required for releasing of 50% of CS from the CT/CS complex hydrogel is smaller than in mediums with low pH values. This is compatible for application of CT/CS complex hydrogel in pharmacological field since that the hydrogel can protect the CS before their arrival in the colon due values of t1/2 are greater than digestive transit time in the stomach and small intestine than in the colon. The fraction of solute released at a given time t, FR, is related to the activity partition (α) and such relation is defined by Eq. (3) [19]. So, the value of FR at time t can be calculated for a system with a defined α (or Fmax) parameter. Based on this statement and on the respective values of Fmax, α and kR for a given solution with a specific pH in which the hydrogel is swelled, values of FR can be obtained through the use of Eq. (3). Curve of FR as a function of time was plotted for pHs buffer 2, 6 and 8 (Fig. 3). For comparison purposes, the curves obtained by
Fig. 3. Experimental and theoretical profiles (Eq. (1) e 3) obtained for fraction of CS released from CT/CS complex hydrogel at 37 °C as a function of time at pHs 2, 6 and 8 buffers.
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application of Ritger-Peppas model [18] (Eq. (1)) were superimposed in the same figures. The data generated from the model described by Eq. (3) are in accordance with the experimental data exhibited in the release profiles at pHs 6 and 8 buffers, as presented in Fig. 3. The values of R2 are given in Table 3 for the three investigated buffers (pHs 2, 6 and 8) and are higher than 0.998. The two models used for providing the CS released profile at a given condition of pH showed significant differences (compare Tables 2 and 3). Furthermore, the constant release obtained for the semi-empirical model, given by Eq. (1), showed higher values than those obtained in the model given by Eq. (3). It cannot have, at same time, a favorable comparison between the two models due some important factors. First, the semi-empirical model given by Eq. (1) predicts at maximum 60% of the releasing profile [18], while the model given by Eq. (3) predicts up to 100% [19]. Furthermore, both Fickian diffusion mechanism and molecular relaxation are seen in the model given by Eq. (3) as intrinsic parameters included in α and kR [19]. Eq. (3) shows that releasing of CS is both a diffusional-transport process as a partition phenomenon where the solute partitioning occurs between the phases of the solvent and hydrogel [19]. Analyzing the ratios between the rate constants of releasing evaluated by Eq. (3) with those same rate constants evaluated by Eq. (1), it becomes clear that at pH 6 and 8 buffers, the obtained results for that ratio were very almost similar, being 0.37 and 0.43 respectively. However, at pH 2 buffer such ratio is equal to 0.10 being, therefore, much lower than at pH 6 and 8 buffers. This implies that the differences in the CS release due to pH change are not weigh up in a same way for both models. As stated before, the prevision of overall profile solute release from a given device allows for better evaluation of solute dosage. In this way, the model given by Eqs. (2) and (3) can best explain the results obtained in this work, since that it provides 100% of solute released and shows release mechanisms of solutes. It can be seen both as a transport process and as a diffusion phenomenon of partition. Thus, with support of mathematical models, it was observed that the CT/CS complex hydrogel responds to changes of the pH of and the fraction of CS released is intensified at buffers of pH 6 and 8 than at pH 2 buffer. Since that CS is classified as a drug that produces symptomatic relief of protracted action, it can be inferred that the CT/CS complex hydrogel is an interesting device for CS release at specific physiological conditions where the pH is greater than the pKa of carboxylic and hydrogen sulfate groups on CS. 4. Conclusions In this work CT/CS complex hydrogel was prepared and the releasing of CS from such hydrogel was characterized and quantified by HPLC. It was found that the maximum of CS released fraction is close to 0.5 and that the process of CS chains release during the swelling process of the CT/CS complex hydrogel is driven by electrostatic repulsion among chains at circumstances in which the pH is greater than the pKa of carboxylic and hydrogen sulfate groups
on CS. The profile of CS released was evaluated using different mathematical models. The prediction of in vitro CS releasing from CT/ CS complex hydrogel can be made using mathematical models based on partitioning even the hydrogel presented different behaviors release by changing the pH. Both diffusion and partition phenomena occur when hydrogel is swelled in medium with different pH values. This result in releasing of CS that was intensified when the CT/CS complex hydrogel is swelled in media with higher pH values. The values of t1/2 (hours) equal to 15.24, 10.57 and 6.24 for pH 2, 6 and 8 buffers, respectively. They are compatible for application in pharmacological field since that the t1/2 values are higher than stomach and small intestine transit time. The interesting behavior demonstrated by this material allows its application how an effective device for CS release, mainly at pH ≥ 6.0 conditions. Acknowledgements All authors thank CNPq (Brazil) for the financial support. ARF thank CAPES (Brazil) for the doctoral fellowship. References [1] S.C. Chen, Y.C. Wu, F.L. Mi, H.W. Lin, L.C. Yu, H.W. Sung, J. Control. Release 96 (2004) 285. [2] J. Berger, M. Reist, J.M. Mayer, O. Felt, N.A. Peppas, R. Gurny, Eur. J. Pharm. Biopharm. 57 (2004) 19. [3] A. Nascimento, M.C. Laranjeira, V.T. Favere, A. Josue, J. Microencapsul. 18 (2001) 679. [4] M.N.V.R. Kumar, R.A.A. Muzzarelli, C. Muzzarelli, H. Sashiwa, A. Domb, J. Chem. Rev. 104 (2004) 6017. [5] D. Capitani, V. Crescenzi, A.A. de Angelis, A.L. Segre, Macromolecules 34 (2001) 4136. [6] S. Vasiliu, M. Popa, M. Rinaudo, Eur. Polym. J. 41 (2005) 923. [7] A. Denuziere, D. Ferrier, A. Domard, Carbohydr. Polym. 29 (1996) 317. [8] J.F. Piai, A.F. Rubira, E.C. Muniz, Acta Biomater. 5 (2009) 2601. [9] A.R. Fajardo, J.F. Piai, A.F. Rubira, E.C. Muniz, Carbohydr. Polym. 80 (2010) 934. [10] F. Richy, O. Bruyere, O. Ethgen, M. Cucherat, Y. Herotin, J.Y. Reginster, Arch. Intern. Med. 163 (2003) 1514. [11] Y.L. Chen, H.C. Chen, H.P. Lee, H.Y. Chan, Y.C. Hu, Biomaterials 27 (2006) 2222. [12] A. Denuziere, D. Ferrier, O. Damour, A. Domard, Biomaterials 19 (1998) 1275. [13] C. Peniche, M. Fernández, G. Rodríguez, J. Parra, J. Jimenez, A.L. Bravo, D. Gómez, J.S. Román, J. Mater. Sci. Mater. Med. 18 (2007) 1719. [14] J.F. Piai, M.R. Moura, A.F. Rubira, E.C. Muniz, Macromol. Symp. 266 (2008) 108. [15] D.C. Berry, P. Knapp, D.K. Raynor, Lancet 359 (2002) 853. [16] V.M.M. Lobo, A.J.M. Valente, A. Ya, G. Geuskens Polishchuk, J. Mol. Liq. 94 (2001) 179. [17] J. Siepmanna, N.A. Peppas, Adv. Drug Delivery Rev. 48 (2001) 139. [18] L. Serra, J. Doménech, N.A. Peppas, Biomaterials 27 (2006) 5440. [19] A.V. Reis, M.R. Guilherme, A.F. Rubira, E.C. Muniz, J. Colloid. Interfaces Sci. 310 (2007) 128. [20] S. Mao, X. Shuai, F. Unger, M. Simon, D. Bi, T. Kissel, Int. J. Pharm. 281 (2004) 45. [21] A. Wasteson, Biochem. J. 122 (1971) 477. [22] USP30-NF25, United States Pharmacopeial-National Formulary, The United Pharmacopeial Convention, Rockville, MD, U.S.A, , 2007. [23] K. Makino, R. Idenuma, H. Ohshima, Colloids Surf., B 8 (1996) 93. [24] S.P. Strand, K. Tommeraas, K.M. Varum, K. Ostgaard, Biomacromolecules 2 (2001) 1310. [25] B. Larsson, M. Nilsson, H. Tjalve, Biochem. Pharm. 30 (1981) 2963. [26] T. Higuchi, J. Pharm. Sci. 52 (1963) 1145. [27] N.A. Peppas, J.J. Sahlin, Int. J. Pharm. 57 (1989) 169. [28] A.C. Freire, F. Podczeck, J. Sousa, F. Veiga, Rev. Bras. Cienc. Farm. 42 (2006) 319. [29] D.R. Friend, Adv. Drug Delivery Rev. 57 (2005) 247.