Biomacromolecules 2008, 9, 1195–1199
1195
Polysaccharide-Based Hydrogels: Preparation, Characterization, and Drug Interaction Behaviour Eunice F. S. Vieira,*,† Antonio R. Cestari,† Claudio Airoldi,‡ and Watson Loh‡ Laboratory of Materials and Calorimetry, Departamento de Química/CCET, Universidade Federal de Sergipe, 49100-000, São Cristóvão, Brazil, and Instituto de Química, Universidade Estadual de Campinas, 13084-971, Campinas, Brazil Received October 31, 2007; Revised Manuscript Received January 11, 2008
Oxidized alginate (ADA) and oxidized alginate blended with chitosan (ADA-Chit) were prepared in the presence of borax and CaCl2, and their interactions with an antifolate drug, pyrimethamine (PYR), have been investigated. Tablets with a mean diameter of 1.2 ( 0.06 cm were produced and drug interactions were performed in dimethyl sulfoxide (DMSO) using isothermal titration calorimetry (ITC). From ITC responses, the enthalpy changes of interaction PYR/materials, ∆intH, have been determined and were found to be -11.73 ( 0.517 kJ mol-1 for ADA and -4.86 ( 0.156 kJ mol-1 for ADA-Chit. The PYR encapsulation of approximately 75% was achieved for both materials, as measured by UV spectrometer.
1. Introduction Alginate and chitosan are naturally occurring biopolymers that are finding widespread biomedical applications, as they are biodegradable and biocompatible.1–5 Nowadays, there is a growing interest in the production and use of new materials from renewable sources, and thus, natural polymers are replacing synthetic polymers in different areas. Alginate is a linear (unbranched) polysaccharide that is produced by brown algae (Laminarales)andsomebacteria(AzotobacterVinelandii,Pseudomonas aeruginosa among others). The monomers are residues of 4-linked, D-mannuronic acid (M) and its 5-epimer, 4-linked R-Lguluronic acid (G), as shown in Figure 1. Chitosan, an N-deacetylated derivative of chitin, is a linear copolymer polysaccharide consisting of β-(1–4)-linked 2-amino-2-deoxyD-glucose (D-glucosamine) and 2-acetamido-2-deoxy-D-glucose (N-acetyl-D-glucosamine) units (Figure 2). In recent years, biodegradable natural polymers are becoming increasingly important in the design of controlled-release drug delivery systems. Control-release is a method by which active chemicals are made available to a specified target at a rate and duration to accomplish an intended effect.6 The many favorable characteristics of alginate and chitosan may aid in their utility as potential delivery vehicles for drugs. In the present study, alginate has been combined with chitosan to overcome the drawbacks of the former and to combine the good characteristics of both polysaccharides. Chitosan is known to increase the mechanical strength of alginate particles1 whose carboxylic groups induce therapeutic activity of many drugs.7 It has recently been reported that periodate oxidized alginates are highly susceptible to biodegradation,8 therefore, have potential to be used in a number of biomedical applications wherein biocompatibility and biodegradability are important criteria. A previous study has shown that oxidized alginates could also function as potential nontoxic and biodegradable cross-linking agents for proteins in the preparation of hydrogels.9 In the present investigation, efforts have been made to design appropriately functionalized, biocompatible, and biodegradable * Corresponding author. E-mail:
[email protected]. † Universidade Federal de Sergipe. ‡ Universidade Estadual de Campinas.
Figure 1. Chemical structure of an alginate fragment (. . .GGMM. . .).
Figure 2. Chemical structure of chitosan.
Figure 3. Structure of pyrimethamine.
polymeric systems. A method of alginate modification by oxidation with sodium periodate and subsequent Schiff’s base formation is reported. It is shown that the aldehyde groups on the surface of 2,3-dialdehyde alginate may be used for attachment of the amino-containing chitosan, thus creating hydrogel with interconnecting pores for diffusion of drugs. This study assesses the interaction of the antifolate drug pyrimethamine (Figure 3) with the hydrogels of oxidized alginate (ADA) and oxidized alginate blended with chitosan (ADA-Chit), using isothermal titration calorimetry (ITC). To the best of our knowledge, there is no literature report focusing on the interaction processes of antifolate drugs with biomaterials evaluated using isothermal calorimetry. Hence, this is an inedited
10.1021/bm7011983 CCC: $40.75 2008 American Chemical Society Published on Web 03/18/2008
1196
Biomacromolecules, Vol. 9, No. 4, 2008
and important study, because drug-hydrogel interactions can have considerable importance in optimizing drug delivery. For example, the drug release profiles of a polymeric matrix system for controlled drug release have been directly correlated to the interactions of drugs with polymers.10
2. Experimental Section 2.1. Materials. Sodium alginate, sodium tetraborate decahydrate, pyrimethamine (PYR, greater than 99% purity), and phosphate-buffered saline (PBS, pH 7.4) were purchased from Sigma (U.S.A.). Chitosan with deacetylation degree of 85% and Mv (viscosity-average molecular weight) of 3.22 × 105 g/mol was a generous gift of Primex Ingredients A.S. (Norway). Sodium periodate and ethylene glycol were obtained from Aldrich and used as received. Dimethyl sulfoxide (DMSO) was purchased from Merck. All other reagents were of analytical or equivalent grade. Double-distilled water was employed throughout. 2.2. Preparation of the 2,3-Dialdehyde Alginate in the Presence of Borax (ADA). Sodium alginate solution (3% w/v, 200 mL of double-distilled water + 50 mL of ethanol) was mixed after adding an amount of sodium periodate (30% oxidation degree of the uronic acid units11) in the dark at room temperature to obtain 2,3dialdehyde alginate. The reaction was stopped after 24 h by the addition of ethylene glycol (5 mL) to reduce the excess periodate.12 The reaction mixture was stirred for 2 h at room temperature. The degree of oxidation was followed by determining the concentration of unreacted periodate by iodometry.13 After reaction, the solution was dialyzed against doubledistilled water with several changes of water until the dialyzate was periodate free. Complete removal of periodate was ensured by testing the dialyzate for the absence of turbidity or precipitate with an aqueous solution of silver nitrate.9 The gel ADA was prepared by dissolving 10 mL of dialyzate 2,3-dialdehyde alginate in an equal volume of 0.1 mol/L borax solution and adding to 25 mL of CaCl2 at the concentration of 0.4% (w/v). The mixture was magnetically stirred for 5 min and then left overnight. The gel was collected by centrifugation, washed thoroughly with water, freeze-dried, and stored in a refrigerator at 4 °C. 2.3. Preparation of the Alginate-Chitosan Hydrogel (ADA-Chit). A chitosan solution was prepared by stirring a dispersion of chitosan (3.0 g) in 100 mL of 2% (v/v) aqueous acetic solution for 1 h at room temperature. For the preparation of ADA-Chit, 10 mL of dialyzate 2,3dialdehyde alginate was dissolved in 10 mL of 0.1 mol/L borax solution, which was added to 20 mL of the chitosan solution, followed by the addition of 25 mL of CaCl2 at the concentration of 0.4% (w/v). After 30 min, the gel was removed and washed with water. The ADA-Chit hydrogel thus formed was freeze-dried and then stored at 4 °C. 2.4. Characterization. 2.4.1. FTIR Spectroscopy. Infrared spectra were recorded as % transmittance using a FTIR Bomem MB-Series, model B1000, ABB-Bomem at a resolution of 4 cm-1. Samples were pressed as KBr pellets using a hydraulic press Carver. 2.4.2. ThermograVimetric Analysis. Thermogravimetric analysis (TG and DTG) of the materials were made using masses of about 10 mg, under nitrogen atmosphere from 25 to 600 °C, in a TGA 2050 Thermogravimetric Analyzer, from TA Instruments. 2.4.3. Scanning Electron Microscopy (SEM) Analysis. The surfaces of freeze-dried ADA and ADA-Chit were morphologically observed by SEM (Jeol-JSM-6360LV) at a voltage of 20 kV. The samples were previously coated with gold/palladium under vacuum by sputtering using a BAL-TEC apparatus. The electron micrographs were scanned at 150–2000 magnification. 2.4.4. Swelling Studies. Tablets of ADA and ADA-Chit were prepared from the freeze-dried hydrogels and pressed with a Carver hydraulic press using a force of 5.0 kN for 10s. The weight of the tablets was 285 ( 11 mg and their diameter was 1.2 ( 0.06 cm. The tablets were soaked in 5 mL of PBS, and they were kept in a water bath at 37 °C. The tablets were withdrawn at different periods intervals, carefully dried with filter paper and weighed immediately. The
Vieira et al. experiment was repeated until the weight varied less than 0.5%. Experiments were performed in duplicate for each hydrogel. 2.5. Isothermal Titration Calorimetry (ITC). ITC experiments were performed at 298.15 K using a Thermal Activity Monitor (TAM 2277, Thermometric AB, Sweden), which is a heat conduction calorimeter and is controlled by a computerized software DIGITAM. For ITC measurements, three tablets of ADA or ADA-Chit, prepared as described in Section 2.4.4, were placed in a stainless steel vessel containing 2.5 mL of DMSO. Measurements of heats attributed to the interaction drug/hydrogel, Qint, consisted of injecting 10 µL aliquots of 0.08 mol L-1 pyrimethamine solution (prepared in DMSO) into the suspension of ADA or ADA-Chit through a syringe fitted with a gold cannula driven by a computer-controlled pump at intervals of 60 min. Heats of dilution/mixing were determined by injecting aliquots (10 µL) of the pyrimethamine solution into 2.5 mL of pure DMSO. The output signal was collected as power, P, versus time, t, integrated and quantified by the amount of adsorbed drug to give the enthalpy change of interaction, ∆intH. Thus, after each titration experiment (i.e., after 15 additions of 10 µL of the drug solution), the drug equilibrium concentration, Ceq, was determined in the supernatant and, thus, the drug amount that interacts, nint. The nint value was determined by recording the absorbance value of the equilibrium concentration at the wavelength of maximum absorbance 280 nm, on a Beckman UV–vis spectrophotometer, DU 640 B (USA). Data presented are the average of a minimum of two replicate titrations.
3. Results and Discussion Sodium alginate was partially oxidized to a theoretical extent of 30% and the degree of oxidation, defined as the percentage of oxidized urinate groups in the alginate, was determined to be 28% as measured by the consumption of sodium periodate. The periodate ion cleaves the C2-C3 linkage by an oxidation reaction, leading to the formation of a dialdehyde. Compared with the activation of hydroxyl or carboxyl groups directly available on the alginate backbone, the introduction of aldehydic groups offers an advantage because they are more reactive than hydroxyl ones.14 It is known that due to intramolecular hemiacetal formation, an oxidation limit of about 44% occurs in alginates, although subsequent reduction of the hemiacetals allows further oxidation.15 Here we show that the gelation of 2,3-dialdehyde alginate with CaCl2 or chitosan/CaCl2 is possible in the presence of borax (Figure 4), which is considered to be a nontoxic agent and has a long history of medical use.13 We believe that the gel formation is due to the ability of borax to complex with hydroxyl groups of the alginate,16 as well as the ability of the polysaccharide to form gels by interacting with the divalent cation Ca2+. It is known that monovalent cations ions and Mg2+ do not induce gelation, while other divalent cations can be used to both produce and stabilize alginate gels.17 In the case of ADA-Chit, besides the complexation with hydroxyl groups of both alginate and chitosan, the borax facilitates the Schiff’s base formation between the amino groups of chitosan and the available aldehyde groups of 2,3-dialdehyde alginate. Because the pH of the medium is alkaline, we believe that electrostatic interactions between the anionic alginate and cationic chitosan should be weak. 3.1. Characterization. 3.1.1. FTIR Spectroscopy. The FTIR spectra of sodium alginate, chitosan, ADA, and ADA-Chit are shown in Figure 5. The spectrum of sodium alginate (Figure 5a) shows the characteristic absorption bands of its polysaccharide structure,18,19 bands around 1320 cm-1 (C-O stretching), 1130 cm-1 (C-C stretching), 1020 cm-1 (C-O-C stretching), and 950 cm-1 (C-O stretching). The absorption bands at 1619 and 1417 cm-1 are assigned to asymmetric and
Polysaccharide-Based Hydrogels
Biomacromolecules, Vol. 9, No. 4, 2008
1197
Figure 6. TG and DTG curves for (a) alginate, (b) ADA, (c) chitosan, and (d) ADA-Chit.
Figure 4. Preparation of ADA and ADA-Chit hydrogels.
Figure 5. FTIR spectra of sodium alginate (a), ADA (b), chitosan (c), and ADA-Chit (d).
symmetric stretching peaks of the carboxylate salt groups. The FTIR spectrum of ADA in Figure 5b shows the chemical structure difference due to reaction with sodium periodate. The reaction was followed by the appearance of the aldehyde symmetric vibrational band at 1733 cm-1. This band is weak and, in some cases, is not detected due to hemiacetal formation of free aldehydes groups.11 In addition, the strong and broad peak centered around 3440 cm-1 became much narrower, indicating the ability of borax to complex with hydroxyl groups of the polysaccharide. Characteristic absorption bands are shown in the spectrum of chitosan (Figure 5c). The bands at 1657 cm-1 (CdO of -NH-CdO stretching), 1595 cm-1 (N-H bending), 1378 cm-1 (-CH2 bending), and 1085 and 1033 cm-1 (C-OH stretching) are characteristics of its saccharide structure.18,20 In the spectrum of ADA-Chit (Figure 5d), the absence of a band at 1733 cm-1 clearly indicates the formation of the Schiff’s base involving the -NH2 groups of chitosan and the free aldehyde groups of the alginate dialdehyde. Probably, the absorption due to CdN stretching, which is weak in aliphatic Schiff’s base, is masked by amide I.21 The presence of chitosan and alginate bands in the FTIR spectrum of ADA-Chit confirms the formation of a new polymeric material.
3.1.2. ThermograVimetric Analysis. TG and differential thermogravimetric (DTG) curves for alginate, ADA, chitosan, and ADA-Chit are shown in Figure 6. For alginate, different kinds of interactions can be identified:22 the first stage with a weight loss of 6% is assigned to free water and water linked through hydrogen bonds, which are released in the 45–100 °C region and reaches a maximum at 65 °C; The second point of a weight loss of 39%, up to 165 °C, corresponds to the release of water more tightly bound through polar interactions with the carboxylate groups. For chitosan, weight loss takes place in two stages. The first one starts at 39.5 °C and reaches a maximum at 61.5 °C with a weight loss of 8%. The second stage starts at 230 °C and reaches a maximum at 303 °C with a weight loss of 48%. These results are in agreement with those in the literature.23 The first stage is assigned to the loss of water, and the second one corresponds to the decomposition (thermal and oxidative) of chitosan and vaporization and elimination of volatile products. It is known that pyrolysis of polysaccharides starts by a random split of the glycosidic bonds, followed by further decomposition forming acetic and butyric acids and a series of lower fatty acids, where C2, C3, and C6 predominate.24 Variations on the peak area and position related to water loss are expected to reflect physical and molecular changes caused by the modification of the polysaccharies.25 Examination of Figure 6b,d reveals that there are differences in peak area and position, indicating that ADA and ADA-Chit differ in their water holding capacity and in the strength of water-polymer interaction. Such changes might clearly indicate the formation of distinct hydrogels. The second stage of degradation displayed in Figure 6b,d shows peaks with maxima at 221 °C and at 243 °C for ADA and ADAChit, respectively, indicating a decrease in sample thermal stability when compared to pure alginate and chitosan. In the case of ADA-Chit, this behavior is probably due to the formation of the CdN bonds and is in agreement wit the study that has shown that biopolymeric Schiff bases are thermally less stable than unmodified chitosan.26 For ADA and ADA-Chit, new peaks with maxima at 359 °C for ADA and at 483 °C for ADA-Chit might be caused by the decomposition of the materials. 3.1.3. Scanning Electron Microscopy (SEM) Analysis. The morphology of the hydrogels was viewed using scanning electron microscopy, and two representative SEM micrographs are represented in Figure 7a,b. From the SEM analysis, both hydrogels exhibit rough surfaces, and the morphology is typified by interconnected pores in the matrix. Furthermore, the distinctions in the fine structures of the two gels are obvious. The SEM image of ADA (Figure 7a) obtained with 500 × magnification depicts a more homogeneous structure with average pore size of 10 µm. As shown in Figure 7b, the morphology of ADA-Chit exhibits many irregular and interlocked pores with pore sizes of around 50 µm revealing a more heterogeneous bulky gel with void spaces exhibiting cauliflowerlike morphology.
1198
Biomacromolecules, Vol. 9, No. 4, 2008
Vieira et al.
Figure 7. SEM micrographs of ADA (a) and ADA-Chit (b).
Figure 9. ITC responses for titration of ADA, ADA-Chit, and pure DMSO by the solution of pyrimethamine (0.8 mol L-1 in DMSO). Part (a) shows the row data and part (b) shows the data after integration of each of the peaks from each injection.
Figure 8. Swelling behaviour of ADA (filled bars) and ADA-Chit (striped bars) as functions of time.
3.1.4. Swelling BehaVior of the Hydrogels. Swelling behavior is another characteristic for the evaluation of hydrogels. The equilibrium weight swelling ratio (q) was experimentally determined using the following eq 127
q ) weight of swollen hydrogel (Ws)/ weight of dry hydrogel (Wd) (1) The swelling behavior as a function of time is shown in Figure 8. The percentage equilibrium water content or equilibrium hydration (H) of the hydrogel was calculated from the equation:14
H(%) ) [1 - 1/q]100
(2)
Equilibrium fluid content was found to be 55 and 38% for ADA and ADA-Chit, respectively. This lower water uptake ability for ADA-Chit is believed to be due to cross-linking predominantly due to Schiff’s base formation between the amino groups of chitosan and the aldehyde groups in 2,3-dialdehyde alginate. When ADA-Chit is soaked in pH 7.4 phosphate buffer (which is above the pKa values of polyguluronic and polymannuronic acids of alginate chains), the carboxylic groups may ionize to yield negatively charged -COO- groups, while chitosan chains possess deprotonated -NH2 groups and the interactions between them are strong enough to make a dense structure with minimum water uptake and no disintegration. 3.2. ITC Measurements. Because of the poor solubility of pyrimethamine, in the present work we use DMSO, which is a good solvent for many pharmaceutical products and is capable of penetrating cellular membranes without significant or permanent damage.27 The ITC responses observed upon titration of suspended ADA and ADA-Chit by pyrimethamine solution are shown in Figure 9. Each peak in Figure 9a represents the heat produced by the addition of 10 µL of the PYR solution into pure DMSO or into suspended tablets of ADA and ADA-Chit. Heat of dilution of pyrimethamine solution was found to be negligible, as can be
seen in Figure 9a. For the interactions PYR/ADA-Chit and PYR/ ADA, it is observed that the intensity of the peaks significantly decreases and further injections produce only small uniform peaks due to dilution of the pyrimethamine solution. Integration of the peaks over time results in the data shown in Figure 9b, where the number of millijoules in each peak is plotted against the volume of added titrant. From the net heat attributed to the interaction drug/hydrogel, Qint, and the drug amount that interacts, nint, both normalized for 1 g of material, the average enthalpy change of interaction, ∆intH, can be calculated by the following equation:
∆intH )
∑ Qint/nint
(3)
From the Ceq values, determined after the addition of 12 µmol of pyrimethamine, as described in Section 2.5, the nint values were found to be 9.09 ( 0.38 µmol for ADA and 8.98 ( 0.43 µmol for ADA-Chit, thus indicating that approximately 75% of the PYR added interacted with the materials. Despite the similar nint values for both hydrogels, the net heat in mJ attributed to the interaction drug/hydrogel shows significant difference. The values corresponding to the total peak areas of the ITC responses observed in Figure 9 (ΣQint) are -106.66 and -43.66 mJ for ADA and ADA-Chit, respectively. The interaction PYR/ADA involves enthalpy change more negative (-11.73 ( 0.517 kJ mol-1) than its interaction with ADA-Chit (-4.86 ( 0.156 kJ mol-1). Exothermicity observed for both interaction processes implies that possible solvation effect is not an important contribution.28 Although enthalpy changes might be predicted to be negative if ionic interactions are dominant, negative enthalpy values are usually observed for interactions involving covalent attachment.29,30 In this work, it is likely that contribution of substantial structural changes of the PYR to the ∆intH values are markedly observed. However, this contribution to the find result cannot be evaluated. Alginate forms a reticulated structure in contact with calcium ions and this network can entrap solutes. The higher ∆intH value for the interaction of PYR with ADA is probably due to the chemical attachment of the drug whose amino functions can enter into Schiff’s reaction with the aldehyde groups in ADA. This hypothesis finds support in the FTIR spectra of ADA after interaction with the drug (Figure 10). As can be seen, the spectrum of PYR/ADA shows characteristic absorption band of the drug and the absence of the band corresponding to
Polysaccharide-Based Hydrogels
Biomacromolecules, Vol. 9, No. 4, 2008
1199
Acknowledgment. Financial support and fellowships from the Brazilian Agencies CAPES and CNPq are gratefully acknowledged.
References and Notes
Figure 10. FTIR spectra of pyrimethamine and ADA after titration with pyrimethamine solution.
aldehyde groups. Notably, In the case of ADA-Chit, because the amines of chitosan reacted with the aldehydes of ADA to form imine bonds, and thus, the aldehyde groups are not available to interact with PYR, the drug could not be chemically attached to the hydrogel, but only held by physical entrapment within the polymer matrix. Thus the ∆intH value is smaller than that of ADA. For drugs not chemically conjugated to the matrix, the release mechanism may simply involve desorption of the adsorbed active agent, which may be released in a biologically active form.31 We expect these materials to be biodegradable, and this study a well-established approach to drug delivery applications.
Conclusions As a remarkable result of this work, we have described the successful preparation of two polysaccharide-based hydrogels, which were investigated for interaction with an antifolate drug. Results from isothermal titration calorimetry showed that 2,3dialdehyde alginate with borax (ADA) and ADA-Chit hydrogels are suitable for encapsulation of pyrimethamine. The behavior of the interacton is known to differ for both polysaccharidebased hydrogels. The chemical interaction between the drug and ADA is explored. Furthermore, it should also be noted that there is physical entrapment within ADA-Chit. To rationalize this observation, we note the absence of the band corresponding to aldehyde groups after interaction of the PYR with ADA. Because release profiles for controlled drug release have been directly correlated to the interactions of drugs with polymers, we think this study opens the way for novel potential applications of these materials as new delivery systems.
(1) De, S. J.; Robinson, D. J. Controlled Release 2003, 89, 101. (2) Eiselt, P.; Yeh, J.; Latwala, R. K.; Shea, L. D.; Mooney, D. J. Biomaterials 2000, 21, 1921. (3) Muzzarelli, R.; Baldassare, V.; Conti, F.; Ferrara, P.; Biagini, G.; Gazzanelli, G.; Vasi, V. Biomaterials 1988, 9, 247. (4) Muzzarelli, R. A. A. Cell. Mol. Life Sci. 1997, 53, 131. (5) Koga, D. In Chitin enzymology—Chitinase; Chen, R., Chen H. C., Eds; AdV. Chitin Sci. 1998; Vol. 3, pp 16–23. (6) Kenawy, El-R. Polym.-Plast. Technol. Eng. 2001, 40 (4), 437. (7) Jagur-Grodzinski, J. React. Funct. Polym. 1999, 39, 99. (8) Bouhadir, K. H.; Lee, K. Y.; Alsberg, E.; Damm, K. L.; Anderson, K. W.; Mooney, D. J. Biotechnol. Prog. 2001, 17, 945. (9) Balakrishnan, B.; Lesieur, S.; Labarre, D.; Jayakrishnan, A. Carbohydr. Res. 2005, 340, 1425. (10) Jenquin, M.R.; McGinity, J. W. Int. J. Pharm. 1994, 101, 23. (11) Kang, H. A.; Shin, M.; Yang, J. W. Polym. Bull. 2002, 47, 429. (12) Bouhadir, K. H.; Hausman, D. S.; Mooney, D. J. Polymer 1999, 40, 3575. (13) Balakrishnan, B.; Jayakrishnan, A. Biomaterials 2005, 26, 3941. (14) Laurienzo, P.; Malinconico, M.; Motta, A.; Vicinanza, A. Carbohydr. Polym. 2005, 62, 274. (15) Vold, I. M. N.; Kristiansen, K. A.; Bjorn, E.; Christensen, B. E. Biomacromolecules 2006, 7, 2136. (16) Strauss, G.; Kral, H. Biopolymers 1982, 21, 459. (17) Gombotz, W. R.; Wee, S. F. AdV. Drug DeliVery ReV. 1998, 31, 267. (18) Smitha, B.; Sridhar, S.; Khan, A. A. Eur. Polym. J. 2005, 41, 1859. (19) Kim, J. H.; Kim, J. H.; Jegal, J.; Lee, K. H. J. Membr. Sci. 2003, 213, 273. (20) Sun, S.; Wang, A. J. Hazard. Mater. B 2006, 131, 103. (21) Vieira, E. F. S.; Cestari, A. R.; Dias, F. S.; dos Santos, E. B. J. Colloid Interface Sci. 2005, 298, 42. (22) Russo, R.; Giuliani, A.; Immirzi, B.; Malinconico, M.; Romano, G. Macromol. Symp. 2004, 218, 241. (23) Qu, X.; Wirsén, A.; Albertsson, A. C. Polymer 2000, 41, 4841. (24) Nieto, J. M.; Peniche-Covas, C.; Padron, G. Thermochim. Acta 1991, 176, 63. (25) Neto, C. G. T.; Giacometti, J. A.; Job, A. E.; Ferreira, F. C.; Fonseca, J. L. C.; Pereira, M. R. Carbohydr. Polym. 2005, 62, 97. (26) Tirkistani, F. A. A. Polym. Degrad. Stab. 1998, 60, 67. (27) Jacob, S. W.; Bischel, M.; Herschler, R. J. Curr. Ther. Res. 1975, 6, 193. (28) Scott, M. J.; Jones, M. N. Colloids Surf. 2001, 182, 247. (29) Vieira, E. F. S.; Cestari, A. R.; Santos, E. B. J. Colloid Interface Sci. 2006, 298, 74. (30) Vieira, E. F. S.; Cestari, A. R.; Lopes, E. C. N.; Barreto, L. S.; Lázaro; G, S.; Almeida, L. E. React. Funct. Polym. 2007, 67, 820. (31) Jayakumar, R.; Reis, R. L.; Mano, J. F. J. Bioact. Compat. Polym. 2006, 21, 327.
BM7011983
