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Bioartificial materials based on blends of dextran and poly(vinyl alcohol-co-acrylic acid)
EUROPEAN POLYMER JOURNAL
European Polymer Journal 41 (2005) 3004–3010
www.elsevier.com/locate/europolj
Bioartificial materials based on blends of dextran and poly(vinyl alcohol-co-acrylic acid) N. Barbani a, F. Bertoni a,*, G. Ciardelli a, C. Cristallini b, D. Silvestri a, M.L. Coluccio a, P. Giusti a a
Department of Chemical Engineering, Industrial Chemistry and Materials Science, University of Pisa, via Diotisalvi 2, 56126 Pisa, Italy b Institute for the Composite and Biomedical Materials of the CNR, Section of Pisa, 56124 Pisa, Italy Received 11 January 2005; received in revised form 3 June 2005; accepted 5 June 2005 Available online 26 July 2005
Abstract Bioartificial polymeric materials based on blends of dextran and poly(vinyl alcohol-co-acrylic acid) P(VA-co-AA) were prepared in the form of films and characterised to evaluate the miscibility of the natural component with the synthetic one. The idea of this work was to compatibilise PVA and dextran by introducing carboxylic groups along the PVA chains. The copolymer was synthesised and characterised in our laboratories. The results evidenced that the copolymer had an appropriate molecular weight and the content of PAA in the copolymer was 45% (weight). Then, films with different composition ratios were prepared by solution casting and analysed by differential scanning calorimetry (DSC), scanning electron microscopy (SEM), infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA), chemical imaging analysis and mechanical tests. The results obtained indicated that the introduction of carboxylic groups along the PVA chains had a positive effect on the miscibility degree of the synthetic component with the biological one. 2005 Elsevier Ltd. All rights reserved. Keywords: Dextran; Poly(vinyl alcohol) (PVA); Bioartificial; Blends; Poly(acrylic acid) (PAA)
1. Introduction The combination of natural and synthetic macromolecules has become of increasing interest for the great potential of these blends in the close related fields of bioengineering, biomaterials and biotechnologies. The comprehension of their interactions and an increase in their reciprocal material compatibility is therefore a
* Corresponding author. Tel.: +39 050 511277; fax: +39 050 511266. E-mail address: [email protected] (F. Bertoni).
crucial point in order to develop polymer blends suitable to the perspective applications [1–3]. Polymeric materials are widely used for biomedical applications, but, in the past, synthetic and natural polymers have been used separately as potential biomaterials. Their interactions with living tissue remain the major problem to be solved. The success of synthetic polymers as a biomaterial mainly relies on their wide range of mechanical properties and transformation processes that allow a variety of different shapes to be easily obtained at low production costs. On the contrary, biological polymers present good biocompatibility but their mechanical properties are
0014-3057/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.eurpolymj.2005.06.010
N. Barbani et al. / European Polymer Journal 41 (2005) 3004–3010
often poor; the necessity of preserving biological properties complicates their processability and increases their production or recovery costs. Our research group designed new materials based on blends of biological and synthetic polymers with the final objective of producing new processable polymeric materials that hopefully possess both good mechanical properties and biocompatibility [4]. Such materials are prepared through radical polymerisation of unsaturated monomers in the presence of biomolecules (structural and functional proteins, polysaccharides) as a template [5] or by simple mixing or blending. This class of materials is called Ôbioartificial polymeric materialsÕ. Poly(vinyl alcohol) (PVA) is mainly used in aqueous solution and is commercially prepared by hydrolysis of poly(vinyl acetate) (PVAc). Its solubility in water depends on its degree of polymerisation and degree of hydrolysis; it exhibits excellent chemical resistance and complete biodegradability [6]. Poly(acrylic acid) (PAA) has been investigated for a variety of membrane applications in which its environmentally responsive behaviour is of interest [7,8]. Dextran belongs to the group of polysaccharides of D-glucose found in yeast and bacteria which shows enzymatic degradation behaviour and relatively good biocompatibility. It is widely under investigation as a polymeric carrier, both as a macromolecular prodrug and in the form of a hydrogel [9]. According to the recent results obtained in our laboratories [10], the possibility of realising, by solution casting, bioartificial materials from blends between dextran and a copolymer of P(VA-co-AA) has been investigated. In fact, previous studies carried out on dextran/PAA blends pointed out the presence of significant interactions between two components but their mechanical behaviour and their compatibility with natural tissues were rather poor. Researches related to dextran/PVA blends showed, instead, good mechanical and biocompatibility properties but a clear incompatibility between the two components [11]. The aim of the present work focuses on an attempt to compatibilise PVA and dextran by introducing carboxylic groups along the PVA chains in order to induce the miscibility with dextran for further application in biomedical or packaging fields. The miscibility degree was investigated using DSC, SEM, TGA, IR.
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Acrylic acid (Aldrich) and Vinyl acetate (Aldrich) were purified by distillation under vacuum to eliminate the inhibitor. Potassium persulphate, used as initiator, was also purchased from Aldrich (99% K2S2O8, 50–100 ppm Rb) and used as received. Reagent-grade water was obtained using a Waters Millipore purifying system. 2.2. Synthesis of the copolymer A solution of 150 ml H2O, 45.5 ml vinyl acetate, 5 ml 5% K2S2O8 and 1 g PVA (88% hydrolysed) was stirred continuously in a three necked flask. The solution was treated under reflux for 10 min. A burette solution was prepared containing 50 ml H2O, 21 ml acrylic acid and 5% K2S2O8. Then, 15 ml of the burette solution was added to the flask to start copolymerisation. The rest of the burette solution was added slowly in 15–30 min. Reflux followed for 4 h. The solution was cooled down to room temperature and the resulting white precipitate was filtered, rinsed and dried. The copolymer was repeatedly extracted with warm water until the unreacted acrylic acid and the homopolymer were totally removed. The obtained product was dried and then redissolved in tetrahydrofuran (THF) (c = 3%). The copolymer was separated by precipitation with chloroform and the remaining solution contained poly(vinyl acetate). The separated copolymer was washed with methanol, rinsed and dried. The poly(vinyl acetate-co-acrylic acid) ester groups were hydrolysed to hydroxyl groups. A flask solution containing 10 g of the copolymer and 200 ml CH3OH was heated up to 40 C and the mixture was refluxed for 30 min. A burette solution containing 5 g CH3ONa and 50 ml CH3OH was prepared. Initially 15 ml of burette solution was added to the flask, the rest was added slowly in 15–30 min. The poly(Na acrylate-co-vinyl alcohol) was washed with methanol to neutral pH and dried. In order to obtain the acid form of the copolymer it was dissolved with warm water (40 C) and dialysed, then precipitated with 0.1 M hydrochloric acid [12,13]. 2.3. Copolymer characterisation
2. Experimental 2.1. Materials Dextran (Mw = 76,900) produced by Leuconostoc mesenteroides (average molecular weight = 77,000) was provided by Sigma Chemical Co.
2.3.1. Gel permeation chromatography (GPC) This analysis was performed using a 600E WATERS equipped with differential refractomer (RI 410 Waters and RV 486 Waters) to investigate the average molecular weight and the molecular weight distribution of the copolymer (column: linear ultra hydrogel(WATER); flow rate: 0.5 ml/min; injection volume:
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N. Barbani et al. / European Polymer Journal 41 (2005) 3004–3010
2.5. Methods
Table 1 GPC of the copolymer Retention time (min)
% Area
Mn (Da)
Mw (Da)
18
100
200,000
900,000
2.5.1. Differential scanning calorimetry (DSC) The thermal behaviour of the dextran/P(VA-co-AA) films were studied by a Perkin–Elmer DSC 7, using aluminium pans. Two consecutive scans were carried out on each sample, at scan rate of 10 and 20 C/min respectively.
4.5 4
y = 0.0582x - 0.1051 2 R = 0.98
R(1700/1090)
3.5
2.5.2. Scanning electron microscopy (SEM) A morphological analysis was carried out using a Jeol JSM 5600 LV scanning electron microscope. The cross-section was analysed after sputter-coating with gold.
3 2.5 2 1.5 1 0.5 0
0
10
20
30
40
50
60
70
80
%PAA (weight)
Fig. 1. Calibration curve for the determination of the carboxylic groups content in the copolymer.
50 ll, eluent: 0.02 M sodium phosphate buffer). Peak area in RI detector was monitored and compared with the standard curve of the peaks areas obtained from standard PVA solutions. The results are shown in Table 1. A calibration curve using infrared spectroscopy was built to determine the quantity of the carboxylic groups in the copolymer (Fig. 1). Blends of PVA and PAA at different ratios were prepared using 1% aqueous solutions of the two polymers. Films were cast by pouring mixtures into Petri dishes and placing them at 37 C in an oven. A 1% aqueous solution of the copolymer was prepared and a film was cast in the same way as described above. Samples of these films were analysed using a FT-IR 1600 (Perkin– Elmer). The ratio between the carboxylic groups band (1716 cm 1) and the OH stretching band (1094 cm 1) was taken as an estimate of the content of poly(acrylic acid) in the blends. Measuring this ratio for the copolymer we calculated that the amount of poly(acrylic acid) was 45% in weight. 2.4. Film preparation Dextran/P(VA-co-AA) mixtures with 0:100, 30:70, 50:50, 70:30, 100:0 (w/w) ratios were prepared using 1% aqueous solutions of the two polymers. Films were obtained by pouring the mixtures into Petri dishes and placed in an oven at 37 C. The copolymer used for the preparation of the blends was partially acidified to maintain stability of the polysaccharide.
2.5.3. Infrared spectroscopy (IR) Infrared spectra were recorded on FT-IR 1600 Perkin–Elmer spectrophotometer and at least 16 scans were signal averaged. 2.5.4. Thermogravimetric analysis (TGA) A thermogravimetric analyser TGA 6 Perkin–Elmer operating under nitrogen flow with ceramic pans was used. 2.5.5. Chemical imaging Infrared chemical imaging was carried out using a Perkin–Elmer Spectrum Spotlight 350. Spectra were collected directly from the sample surface at a pixel size of either 6.25 · 6.25 or 25 · 25 lm. Samples were simply stepped below a linear detector array building an image in which each pixel corresponds to a high-quality spectrum and by using Spotlight software chemical differences between pixels were highlighted images showing the distribution of the components. 2.5.6. Mechanical tests Mechanical tensile tests were performed by an Instron model 5542 equipped with a 5 N cell operated at a velocity of 1 mm/min. Rectangular specimens 15 mm wide and at least 120 mm long were cut from films and mounted between the grips of the machine. A stress– strain curve was plotted based on the apparent stress r (MPa) and strain (e)% values were determined by dividing the load value with the initial cross-sectional area of each test specimen and the deformation values with the initial specimen height respectively. Each test trial consisted of three replicate measurements.
3. Results and discussion 3.1. Infrared spectroscopy Infrared spectroscopy investigation was used in several cases to identify intermolecular interaction in
N. Barbani et al. / European Polymer Journal 41 (2005) 3004–3010
polymer blends [14]. The FT-IR spectra of the copolymer after partial acidification showed the presence of typical absorption bands for PVA and PAA pendant groups: OH stretching absorption at 3300–3400 cm 1, CH stretching at 2942 cm 1, primary alcohol stretching at 1091 cm 1, carboxylic groups stretching at 1717 cm 1, carboxylate groups stretching at 1560 cm 1 and CO stretching at 1262 cm 1 and 1091 cm 1. In the FT-IR spectrum of dextran, the typical asymmetrical C–O–C stretching of the ring at 1150– 1085 cm 1 was evident. In the spectra of the blends, all the absorption bands related to both components were present. The spectra analysed revealed that no significant shifts occurred at the values of the bands at 1700 cm 1 and 1235 cm 1. Instead, the absorption bands related to C–O–C of the polysaccharideÕs ring shifted to higher frequency in blends with a higher content of the copolymer and a significant lowering of the intensity of the bands at 1262 cm 1 and at 1091 cm 1 related to the alcoholic C–O was evident. This result suggests the formation of intermolecular interaction between the biological component and the synthetic one. In particular, the shifts of these values are due to the interactions between the oxygen ether band in the polysaccharide ring and the hydrogen of the copolymer alcoholic groups. 3.2. Thermogravimetric analysis The analysis of the copolymer showed a four stage degradation process. An initial loss of about 8% weight occurred at 30–150 C and was related both to the loss of non-bound water and to the formation of intra and intermolecular anhydrides. A second weight loss of about 7% occurred in the range 200–280 C and it was associated to the decarboxylation process of intra and inter molecular anhydrides formed during the previous dehydration stage. A third weight loss occurring within 320–420 C was related to the dehydration of the OH groups of the copolymer with the formation of polyenes. To demonstrate these events a thin film of the copolymer was heated up to 380 C in the same conditions of TGA analysis. The FT-IR analysis of this film showed the disappearance of the 1717 cm 1 band related to –COOH groups and the presence of a large band between 1500 and 1650 cm 1 related to [(C@C)–C@O] polyenes stretching. Finally the loss of weight within 400–500 C was related to the full degradation of the macromolecules. The degradation process of dextran was characterised by an initial weight loss of 10% which occurred within 30–150 C related to the evaporation of the water contained in the sample and a second more evident
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weight loss at about 300–500 C related to the full degradation of the biological polymer. The degradation process of dextran/copolymer blends showed intermediate trends between those of the two pure components: they were characterised by an initial weight loss of 10–13% which occurred within 30–150 C and a second more evident weight loss at about 250–400 C. In the DGA blend curves, a peak was present at about 380 C corresponding to the degradation of dextran. On this peak a little shoulder between 350 and 450 C was detectable, which can be related to the third weight loss in the copolymer due to the formation of polyene after loss of water from OH groups. The reduction of the degradation event confirms the presence of H-bonding interaction occurring between the free OH groups in the copolymer and the functional groups of dextran. 3.3. Differential scanning calorimetry The determination of the glass transition temperature represents a very useful tool to evaluate the miscibility of a polymer blend. A miscible polymer blend shows a single Tg which is different and in general, intermediate, between those of the single components. A depression of the melting point is often observed for the crystalline component in the blend with an amorphous polymer. This phenomenon is explained in terms of interactions occurring between the two components [15]. The DSC curve of dextran showed an initial event at about 225 C related to its glass transition (Tg). The copolymer evidenced a glass transition at about 75 C and a second endothermic event at about 215 C related to the melting of the crystalline fraction. In the case of the blend dextran/P(VA-co-AA) 30/70 the DSC curves showed three thermal phenomena related to the pure components. For the dextran/P(VA-co-AA) 50/50 and 70/30 blends the DSC curves evidenced a single glass transition, the values of Tg decreasing as the copolymer content increased. The melting point was not detectable by increasing the dextran content in the blends (Table 2). The presence of a single Tg and the lack of the melting processes related to the copolymer confirmed the presence of interactions occurring between the copolymer and dextran which hinders the crystallisation of the copolymer. 3.4. Morphological and chemical imaging analysis Micrographies of polymers showed a compact structure characterised by a longitudinal arrangement for the copolymer and a transversal one for dextran (Fig. 2).
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N. Barbani et al. / European Polymer Journal 41 (2005) 3004–3010
Table 2 DSC analysis: temperature of glass transition (Tg) and melting temperature (Tm) of dextran, copolymer and their blends Dextran/P(VA-co-AA) 0/100 30/70 50/50 70/30 100/0 Tg (C) Tm (C)
P(VA-co-AA) Dextran
76 215
75 204 218
– 207 –
– 213 –
225
These morphologies reflect the plastic properties of the copolymer and to the brittleness of dextran. In the dextran/P(VA-co-AA) 70/30, 50/50, 30/70 blends SEM images showed a globular structure entrapped in a dense matrix. The number of globules decreased as the amount of dextran in the blends increased as a result of the bigger dispersion of the natural component in the blends (Fig. 3). We compared these micrographies with SEM images of dextran/PVA and dextran/PAA homopolymers blends (Fig. 4). For the dextran/PVA blend a separation phase between the two components was evident. In the
case of dextran/PAA blend the homogeneous structure confirmed the good miscibility of the polymers. Therefore, the introduction of carboxylic groups along PVA chains resulted in a positive effect on the miscibility of dextran/P(VA-co-AA) blends. A chemical imaging analysis was performed in order to obtain more accurate information on the homogeneity of the blend. Fig. 5 illustrates the chemical map of the dextran/P(VA-co-AA) 50/50 blend film (a) and the correlation spectrum (b) was the most representative of the sample. The correlation with spectrum (b) of other points in the film (map c) gave correlation values very close indicating a good chemical homogeneity of the film analysed. 3.5. Mechanical tests The characteristic stress–strain curves of copolymer and dextran/P(VA-co-AA) blends 30/70 and 50/50 are shown in Fig. 6. The test was not performed on blends with a higher content of dextran because their behaviour was too brittle and they were not suitable for this
Fig. 2. SEM images of P(VA-co-AA) (A) and dextran (B) (cross-section).
Fig. 3. SEM images of dextran/P(VA-co-AA) 30/70 (A), dextran/P(VA-co-AA) 50/50 (B), dextran/P(VA-co-AA) 70/30 (C) (crosssection).
N. Barbani et al. / European Polymer Journal 41 (2005) 3004–3010
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Fig. 4. SEM images of dextran/PVA 30/70 (A) and dextran/PAA 40/60 (B) (cross-section).
Fig. 5. Chemical map (a), correlation spectrum (b) and correlation map (c) of dextran/P(VA-co-AA) 50/50.
50 45 40
a
stress (MPa)
35 30 25
b
20 15 10 5
c
0 0
2
4
6
8
10
12
14
strain (%)
Fig. 6. Stress–strain curves for the copolymer (a), dextran/P(VA-co-AA) 30/70 (b), dextran/P(VA-co-AA) 50/50 (c).
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analysis. In fact, the blend films appeared to be more brittle as the dextran content increased. The curves corresponding to copolymer and dextran/P(VA-co-AA) 30/ 70 showed an initial elastic region where the samples evidenced typical Hookean elasticity, followed by typical plastic behaviour. The shape of the curve changed significantly for the dextran/P(VA-co-AA) 50/50 blend in which the material did not exhibit the plastic area before the break point. The elongation at break point decreased increasing the dextran content in the blends because the natural component showed low mechanical properties especially in the percentage elongation. Therefore, dextran generated defects in the specimen obtaining a brittle material. In particular, the dextran/P(VA-co-AA) 30/70 curve displayed a double slope in the linear portion of the elastic region probably due to a non homogenous behaviour of the sample. 4. Conclusions Bioartificial polymeric materials based on dextran and a copolymer containing 45% (in weight) of PAA were prepared in the form of films and their properties were investigated to evaluate the miscibility degree of the two components. The infrared spectroscopy showed the presence of intermolecular interactions (H-bonding) between dextran and P(VA-co-AA) that promote the possible miscibility of the two components. The occurrence of interactions between the two polymers was confirmed by thermal analysis that evidenced a depression of the melting point associated with a reduction of crystallinity of the copolymer in the blends. Also the morphological analysis proved these results. Therefore, bioartificial polymeric materials obtained from the blending of synthetic and natural polymers can provide new materials with enhanced properties. Furthermore, this system is expected to be ideal to realize drug delivery systems in which an environmentally responsive behavior (pH-sensitive) [16,17] is involved due to the presence of ionisable groups introduced along PVA chains. Further investigations using this bioartificial system as well as mechanical properties and degradation behavior will provide interesting information as to extend this class of materials to the production of environmentally friendly plastic materials for food packaging.
References [1] Giusti P, Lazzeri L, Barbani N, Lelli L, De Petris S, Cascone MG. Blends of natural and synthetic polymers: a new route to novel biomaterials. Macromol Symp 1994;78: 285–97. [2] Giusti P, Ciardelli F, Pracella M, Mercogliano R, Barbani N. Maui (Hawaii): 1st Pacific Polymer Federation. 1989. [3] Giusti P, Lazzeri L. Leningrado (USSR): 2nd joint Soviet Union Italy meeting on polymer science. 1991. [4] Ciardelli F, Giusti P, Mercogliano R, Pracella M. Gargnano (Italy): 1st Soviet Union–Italy polymer meeting. 1989. [5] Cristallini C, Barbani N, Giusti P, Lazzeri L, Cascone MG, Ciardelli G. Polymerisation onto biological templates, a new way to obtain bioartificial polymeric materials. Macromol Chem Phys 2001;202:1874–83. [6] Hoffman AS. Hydrogels for biomedical applications. Adv Drug Delivery Rev 2002;54:3–12. [7] Gudeman LF, Peppas NA. pH-Sensitive membranes from poly(vinyl alcohol)/poly(acrylic acid) interpenetrating networks. J Membr Sci 1995;107:239–48. [8] Gudeman LF, Peppas NA. Preparation and characterization of pH-sensitive, interpenetrating networks of poly(vinyl alcohol) and poly(acrylic acid). J Appl Polym Sci 1995;55:919–28. [9] Larsen C. Dextran prodrugs-structure and stability in relation to therapeutic activity. Adv Drug Rev 1989;3: 103–54. [10] Cascone MG, Polacco G, Lazzeri L, Barbani N. Dextran/ poly(acrylic acid) mixtures as miscible blends. J Appl Polym Sci 1997;66:2089–94. [11] Cascone MG, Barbani N, Cristallini C, Giusti P, Lazzeri L. Bioartificial polymeric materials based on polysaccharides. J Biomat Sci—Polym Edit 2001;12:267–81. [12] United States Patent: 4, 505, 998. [13] Dimitriu S, Popa M, Petrovan S, Staicu R. Bioactive polymers LVII. A novel drug delivery system based on 5nitro-2-furaldehyde-semicarbazone and poly (acrylic acidco-vinyl alcohol). Br Polym J 1989;21: 295–301. [14] Daniliuc L, DeKesel C, David C. Intermolecular interactions in blends of poly(vinyl alcohol) with poly(acrylic acid)-1. FTIR and DSC studies. Eur Polym J 1992;28(11): 1365–71. [15] Olabisi O, Robeson LM, Shaw MT. Polymer–polymer miscibility. New York: Academic Press; 1979. [16] Qiu Y, Park K. Environment-sensitive hydrogels for drug delivery. Adv Drug Delivery Rev 2001;53(3): 321–39. [17] Gudeman LF, Peppas NA. pH-Sensitive membranes from poly(vinyl alcohol)/poly(acrylic acid) interpenetrating networks. J Membr Sci 1995;107(3):239–48.
European Polymer Journal 41 (2005) 3004–3010
www.elsevier.com/locate/europolj
Bioartificial materials based on blends of dextran and poly(vinyl alcohol-co-acrylic acid) N. Barbani a, F. Bertoni a,*, G. Ciardelli a, C. Cristallini b, D. Silvestri a, M.L. Coluccio a, P. Giusti a a
Department of Chemical Engineering, Industrial Chemistry and Materials Science, University of Pisa, via Diotisalvi 2, 56126 Pisa, Italy b Institute for the Composite and Biomedical Materials of the CNR, Section of Pisa, 56124 Pisa, Italy Received 11 January 2005; received in revised form 3 June 2005; accepted 5 June 2005 Available online 26 July 2005
Abstract Bioartificial polymeric materials based on blends of dextran and poly(vinyl alcohol-co-acrylic acid) P(VA-co-AA) were prepared in the form of films and characterised to evaluate the miscibility of the natural component with the synthetic one. The idea of this work was to compatibilise PVA and dextran by introducing carboxylic groups along the PVA chains. The copolymer was synthesised and characterised in our laboratories. The results evidenced that the copolymer had an appropriate molecular weight and the content of PAA in the copolymer was 45% (weight). Then, films with different composition ratios were prepared by solution casting and analysed by differential scanning calorimetry (DSC), scanning electron microscopy (SEM), infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA), chemical imaging analysis and mechanical tests. The results obtained indicated that the introduction of carboxylic groups along the PVA chains had a positive effect on the miscibility degree of the synthetic component with the biological one. 2005 Elsevier Ltd. All rights reserved. Keywords: Dextran; Poly(vinyl alcohol) (PVA); Bioartificial; Blends; Poly(acrylic acid) (PAA)
1. Introduction The combination of natural and synthetic macromolecules has become of increasing interest for the great potential of these blends in the close related fields of bioengineering, biomaterials and biotechnologies. The comprehension of their interactions and an increase in their reciprocal material compatibility is therefore a
* Corresponding author. Tel.: +39 050 511277; fax: +39 050 511266. E-mail address: [email protected] (F. Bertoni).
crucial point in order to develop polymer blends suitable to the perspective applications [1–3]. Polymeric materials are widely used for biomedical applications, but, in the past, synthetic and natural polymers have been used separately as potential biomaterials. Their interactions with living tissue remain the major problem to be solved. The success of synthetic polymers as a biomaterial mainly relies on their wide range of mechanical properties and transformation processes that allow a variety of different shapes to be easily obtained at low production costs. On the contrary, biological polymers present good biocompatibility but their mechanical properties are
0014-3057/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.eurpolymj.2005.06.010
N. Barbani et al. / European Polymer Journal 41 (2005) 3004–3010
often poor; the necessity of preserving biological properties complicates their processability and increases their production or recovery costs. Our research group designed new materials based on blends of biological and synthetic polymers with the final objective of producing new processable polymeric materials that hopefully possess both good mechanical properties and biocompatibility [4]. Such materials are prepared through radical polymerisation of unsaturated monomers in the presence of biomolecules (structural and functional proteins, polysaccharides) as a template [5] or by simple mixing or blending. This class of materials is called Ôbioartificial polymeric materialsÕ. Poly(vinyl alcohol) (PVA) is mainly used in aqueous solution and is commercially prepared by hydrolysis of poly(vinyl acetate) (PVAc). Its solubility in water depends on its degree of polymerisation and degree of hydrolysis; it exhibits excellent chemical resistance and complete biodegradability [6]. Poly(acrylic acid) (PAA) has been investigated for a variety of membrane applications in which its environmentally responsive behaviour is of interest [7,8]. Dextran belongs to the group of polysaccharides of D-glucose found in yeast and bacteria which shows enzymatic degradation behaviour and relatively good biocompatibility. It is widely under investigation as a polymeric carrier, both as a macromolecular prodrug and in the form of a hydrogel [9]. According to the recent results obtained in our laboratories [10], the possibility of realising, by solution casting, bioartificial materials from blends between dextran and a copolymer of P(VA-co-AA) has been investigated. In fact, previous studies carried out on dextran/PAA blends pointed out the presence of significant interactions between two components but their mechanical behaviour and their compatibility with natural tissues were rather poor. Researches related to dextran/PVA blends showed, instead, good mechanical and biocompatibility properties but a clear incompatibility between the two components [11]. The aim of the present work focuses on an attempt to compatibilise PVA and dextran by introducing carboxylic groups along the PVA chains in order to induce the miscibility with dextran for further application in biomedical or packaging fields. The miscibility degree was investigated using DSC, SEM, TGA, IR.
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Acrylic acid (Aldrich) and Vinyl acetate (Aldrich) were purified by distillation under vacuum to eliminate the inhibitor. Potassium persulphate, used as initiator, was also purchased from Aldrich (99% K2S2O8, 50–100 ppm Rb) and used as received. Reagent-grade water was obtained using a Waters Millipore purifying system. 2.2. Synthesis of the copolymer A solution of 150 ml H2O, 45.5 ml vinyl acetate, 5 ml 5% K2S2O8 and 1 g PVA (88% hydrolysed) was stirred continuously in a three necked flask. The solution was treated under reflux for 10 min. A burette solution was prepared containing 50 ml H2O, 21 ml acrylic acid and 5% K2S2O8. Then, 15 ml of the burette solution was added to the flask to start copolymerisation. The rest of the burette solution was added slowly in 15–30 min. Reflux followed for 4 h. The solution was cooled down to room temperature and the resulting white precipitate was filtered, rinsed and dried. The copolymer was repeatedly extracted with warm water until the unreacted acrylic acid and the homopolymer were totally removed. The obtained product was dried and then redissolved in tetrahydrofuran (THF) (c = 3%). The copolymer was separated by precipitation with chloroform and the remaining solution contained poly(vinyl acetate). The separated copolymer was washed with methanol, rinsed and dried. The poly(vinyl acetate-co-acrylic acid) ester groups were hydrolysed to hydroxyl groups. A flask solution containing 10 g of the copolymer and 200 ml CH3OH was heated up to 40 C and the mixture was refluxed for 30 min. A burette solution containing 5 g CH3ONa and 50 ml CH3OH was prepared. Initially 15 ml of burette solution was added to the flask, the rest was added slowly in 15–30 min. The poly(Na acrylate-co-vinyl alcohol) was washed with methanol to neutral pH and dried. In order to obtain the acid form of the copolymer it was dissolved with warm water (40 C) and dialysed, then precipitated with 0.1 M hydrochloric acid [12,13]. 2.3. Copolymer characterisation
2. Experimental 2.1. Materials Dextran (Mw = 76,900) produced by Leuconostoc mesenteroides (average molecular weight = 77,000) was provided by Sigma Chemical Co.
2.3.1. Gel permeation chromatography (GPC) This analysis was performed using a 600E WATERS equipped with differential refractomer (RI 410 Waters and RV 486 Waters) to investigate the average molecular weight and the molecular weight distribution of the copolymer (column: linear ultra hydrogel(WATER); flow rate: 0.5 ml/min; injection volume:
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N. Barbani et al. / European Polymer Journal 41 (2005) 3004–3010
2.5. Methods
Table 1 GPC of the copolymer Retention time (min)
% Area
Mn (Da)
Mw (Da)
18
100
200,000
900,000
2.5.1. Differential scanning calorimetry (DSC) The thermal behaviour of the dextran/P(VA-co-AA) films were studied by a Perkin–Elmer DSC 7, using aluminium pans. Two consecutive scans were carried out on each sample, at scan rate of 10 and 20 C/min respectively.
4.5 4
y = 0.0582x - 0.1051 2 R = 0.98
R(1700/1090)
3.5
2.5.2. Scanning electron microscopy (SEM) A morphological analysis was carried out using a Jeol JSM 5600 LV scanning electron microscope. The cross-section was analysed after sputter-coating with gold.
3 2.5 2 1.5 1 0.5 0
0
10
20
30
40
50
60
70
80
%PAA (weight)
Fig. 1. Calibration curve for the determination of the carboxylic groups content in the copolymer.
50 ll, eluent: 0.02 M sodium phosphate buffer). Peak area in RI detector was monitored and compared with the standard curve of the peaks areas obtained from standard PVA solutions. The results are shown in Table 1. A calibration curve using infrared spectroscopy was built to determine the quantity of the carboxylic groups in the copolymer (Fig. 1). Blends of PVA and PAA at different ratios were prepared using 1% aqueous solutions of the two polymers. Films were cast by pouring mixtures into Petri dishes and placing them at 37 C in an oven. A 1% aqueous solution of the copolymer was prepared and a film was cast in the same way as described above. Samples of these films were analysed using a FT-IR 1600 (Perkin– Elmer). The ratio between the carboxylic groups band (1716 cm 1) and the OH stretching band (1094 cm 1) was taken as an estimate of the content of poly(acrylic acid) in the blends. Measuring this ratio for the copolymer we calculated that the amount of poly(acrylic acid) was 45% in weight. 2.4. Film preparation Dextran/P(VA-co-AA) mixtures with 0:100, 30:70, 50:50, 70:30, 100:0 (w/w) ratios were prepared using 1% aqueous solutions of the two polymers. Films were obtained by pouring the mixtures into Petri dishes and placed in an oven at 37 C. The copolymer used for the preparation of the blends was partially acidified to maintain stability of the polysaccharide.
2.5.3. Infrared spectroscopy (IR) Infrared spectra were recorded on FT-IR 1600 Perkin–Elmer spectrophotometer and at least 16 scans were signal averaged. 2.5.4. Thermogravimetric analysis (TGA) A thermogravimetric analyser TGA 6 Perkin–Elmer operating under nitrogen flow with ceramic pans was used. 2.5.5. Chemical imaging Infrared chemical imaging was carried out using a Perkin–Elmer Spectrum Spotlight 350. Spectra were collected directly from the sample surface at a pixel size of either 6.25 · 6.25 or 25 · 25 lm. Samples were simply stepped below a linear detector array building an image in which each pixel corresponds to a high-quality spectrum and by using Spotlight software chemical differences between pixels were highlighted images showing the distribution of the components. 2.5.6. Mechanical tests Mechanical tensile tests were performed by an Instron model 5542 equipped with a 5 N cell operated at a velocity of 1 mm/min. Rectangular specimens 15 mm wide and at least 120 mm long were cut from films and mounted between the grips of the machine. A stress– strain curve was plotted based on the apparent stress r (MPa) and strain (e)% values were determined by dividing the load value with the initial cross-sectional area of each test specimen and the deformation values with the initial specimen height respectively. Each test trial consisted of three replicate measurements.
3. Results and discussion 3.1. Infrared spectroscopy Infrared spectroscopy investigation was used in several cases to identify intermolecular interaction in
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polymer blends [14]. The FT-IR spectra of the copolymer after partial acidification showed the presence of typical absorption bands for PVA and PAA pendant groups: OH stretching absorption at 3300–3400 cm 1, CH stretching at 2942 cm 1, primary alcohol stretching at 1091 cm 1, carboxylic groups stretching at 1717 cm 1, carboxylate groups stretching at 1560 cm 1 and CO stretching at 1262 cm 1 and 1091 cm 1. In the FT-IR spectrum of dextran, the typical asymmetrical C–O–C stretching of the ring at 1150– 1085 cm 1 was evident. In the spectra of the blends, all the absorption bands related to both components were present. The spectra analysed revealed that no significant shifts occurred at the values of the bands at 1700 cm 1 and 1235 cm 1. Instead, the absorption bands related to C–O–C of the polysaccharideÕs ring shifted to higher frequency in blends with a higher content of the copolymer and a significant lowering of the intensity of the bands at 1262 cm 1 and at 1091 cm 1 related to the alcoholic C–O was evident. This result suggests the formation of intermolecular interaction between the biological component and the synthetic one. In particular, the shifts of these values are due to the interactions between the oxygen ether band in the polysaccharide ring and the hydrogen of the copolymer alcoholic groups. 3.2. Thermogravimetric analysis The analysis of the copolymer showed a four stage degradation process. An initial loss of about 8% weight occurred at 30–150 C and was related both to the loss of non-bound water and to the formation of intra and intermolecular anhydrides. A second weight loss of about 7% occurred in the range 200–280 C and it was associated to the decarboxylation process of intra and inter molecular anhydrides formed during the previous dehydration stage. A third weight loss occurring within 320–420 C was related to the dehydration of the OH groups of the copolymer with the formation of polyenes. To demonstrate these events a thin film of the copolymer was heated up to 380 C in the same conditions of TGA analysis. The FT-IR analysis of this film showed the disappearance of the 1717 cm 1 band related to –COOH groups and the presence of a large band between 1500 and 1650 cm 1 related to [(C@C)–C@O] polyenes stretching. Finally the loss of weight within 400–500 C was related to the full degradation of the macromolecules. The degradation process of dextran was characterised by an initial weight loss of 10% which occurred within 30–150 C related to the evaporation of the water contained in the sample and a second more evident
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weight loss at about 300–500 C related to the full degradation of the biological polymer. The degradation process of dextran/copolymer blends showed intermediate trends between those of the two pure components: they were characterised by an initial weight loss of 10–13% which occurred within 30–150 C and a second more evident weight loss at about 250–400 C. In the DGA blend curves, a peak was present at about 380 C corresponding to the degradation of dextran. On this peak a little shoulder between 350 and 450 C was detectable, which can be related to the third weight loss in the copolymer due to the formation of polyene after loss of water from OH groups. The reduction of the degradation event confirms the presence of H-bonding interaction occurring between the free OH groups in the copolymer and the functional groups of dextran. 3.3. Differential scanning calorimetry The determination of the glass transition temperature represents a very useful tool to evaluate the miscibility of a polymer blend. A miscible polymer blend shows a single Tg which is different and in general, intermediate, between those of the single components. A depression of the melting point is often observed for the crystalline component in the blend with an amorphous polymer. This phenomenon is explained in terms of interactions occurring between the two components [15]. The DSC curve of dextran showed an initial event at about 225 C related to its glass transition (Tg). The copolymer evidenced a glass transition at about 75 C and a second endothermic event at about 215 C related to the melting of the crystalline fraction. In the case of the blend dextran/P(VA-co-AA) 30/70 the DSC curves showed three thermal phenomena related to the pure components. For the dextran/P(VA-co-AA) 50/50 and 70/30 blends the DSC curves evidenced a single glass transition, the values of Tg decreasing as the copolymer content increased. The melting point was not detectable by increasing the dextran content in the blends (Table 2). The presence of a single Tg and the lack of the melting processes related to the copolymer confirmed the presence of interactions occurring between the copolymer and dextran which hinders the crystallisation of the copolymer. 3.4. Morphological and chemical imaging analysis Micrographies of polymers showed a compact structure characterised by a longitudinal arrangement for the copolymer and a transversal one for dextran (Fig. 2).
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Table 2 DSC analysis: temperature of glass transition (Tg) and melting temperature (Tm) of dextran, copolymer and their blends Dextran/P(VA-co-AA) 0/100 30/70 50/50 70/30 100/0 Tg (C) Tm (C)
P(VA-co-AA) Dextran
76 215
75 204 218
– 207 –
– 213 –
225
These morphologies reflect the plastic properties of the copolymer and to the brittleness of dextran. In the dextran/P(VA-co-AA) 70/30, 50/50, 30/70 blends SEM images showed a globular structure entrapped in a dense matrix. The number of globules decreased as the amount of dextran in the blends increased as a result of the bigger dispersion of the natural component in the blends (Fig. 3). We compared these micrographies with SEM images of dextran/PVA and dextran/PAA homopolymers blends (Fig. 4). For the dextran/PVA blend a separation phase between the two components was evident. In the
case of dextran/PAA blend the homogeneous structure confirmed the good miscibility of the polymers. Therefore, the introduction of carboxylic groups along PVA chains resulted in a positive effect on the miscibility of dextran/P(VA-co-AA) blends. A chemical imaging analysis was performed in order to obtain more accurate information on the homogeneity of the blend. Fig. 5 illustrates the chemical map of the dextran/P(VA-co-AA) 50/50 blend film (a) and the correlation spectrum (b) was the most representative of the sample. The correlation with spectrum (b) of other points in the film (map c) gave correlation values very close indicating a good chemical homogeneity of the film analysed. 3.5. Mechanical tests The characteristic stress–strain curves of copolymer and dextran/P(VA-co-AA) blends 30/70 and 50/50 are shown in Fig. 6. The test was not performed on blends with a higher content of dextran because their behaviour was too brittle and they were not suitable for this
Fig. 2. SEM images of P(VA-co-AA) (A) and dextran (B) (cross-section).
Fig. 3. SEM images of dextran/P(VA-co-AA) 30/70 (A), dextran/P(VA-co-AA) 50/50 (B), dextran/P(VA-co-AA) 70/30 (C) (crosssection).
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Fig. 4. SEM images of dextran/PVA 30/70 (A) and dextran/PAA 40/60 (B) (cross-section).
Fig. 5. Chemical map (a), correlation spectrum (b) and correlation map (c) of dextran/P(VA-co-AA) 50/50.
50 45 40
a
stress (MPa)
35 30 25
b
20 15 10 5
c
0 0
2
4
6
8
10
12
14
strain (%)
Fig. 6. Stress–strain curves for the copolymer (a), dextran/P(VA-co-AA) 30/70 (b), dextran/P(VA-co-AA) 50/50 (c).
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analysis. In fact, the blend films appeared to be more brittle as the dextran content increased. The curves corresponding to copolymer and dextran/P(VA-co-AA) 30/ 70 showed an initial elastic region where the samples evidenced typical Hookean elasticity, followed by typical plastic behaviour. The shape of the curve changed significantly for the dextran/P(VA-co-AA) 50/50 blend in which the material did not exhibit the plastic area before the break point. The elongation at break point decreased increasing the dextran content in the blends because the natural component showed low mechanical properties especially in the percentage elongation. Therefore, dextran generated defects in the specimen obtaining a brittle material. In particular, the dextran/P(VA-co-AA) 30/70 curve displayed a double slope in the linear portion of the elastic region probably due to a non homogenous behaviour of the sample. 4. Conclusions Bioartificial polymeric materials based on dextran and a copolymer containing 45% (in weight) of PAA were prepared in the form of films and their properties were investigated to evaluate the miscibility degree of the two components. The infrared spectroscopy showed the presence of intermolecular interactions (H-bonding) between dextran and P(VA-co-AA) that promote the possible miscibility of the two components. The occurrence of interactions between the two polymers was confirmed by thermal analysis that evidenced a depression of the melting point associated with a reduction of crystallinity of the copolymer in the blends. Also the morphological analysis proved these results. Therefore, bioartificial polymeric materials obtained from the blending of synthetic and natural polymers can provide new materials with enhanced properties. Furthermore, this system is expected to be ideal to realize drug delivery systems in which an environmentally responsive behavior (pH-sensitive) [16,17] is involved due to the presence of ionisable groups introduced along PVA chains. Further investigations using this bioartificial system as well as mechanical properties and degradation behavior will provide interesting information as to extend this class of materials to the production of environmentally friendly plastic materials for food packaging.
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