- Email: [email protected]
starch blends and cytotoxicity assays
Materials Science and Engineering C 31 (2011) 443–451
Contents lists available at ScienceDirect
Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m s e c
Effect of starch type on miscibility in poly(ethylene oxide) (PEO)/starch blends and cytotoxicity assays A.G.B. Pereira a, A.T. Paulino c, C.V. Nakamura b, E.A. Britta b, A.F. Rubira a, E.C. Muniz a,⁎ a
Grupo de Materiais Poliméricos e Compósitos, GMPC, Departamento de Química Universidade Estadual de Maringá, Av. Colombo 5790, 87020-900, Maringá, Paraná, Brazil Laboratório de Inovação Tecnológica no Desenvolvimento de Fármacos e Cosméticos, DBS, Departamento de Análises Clínicas. Universidade Estadual de Maringá, Av. Colombo 5790-87020-900, Maringá, Paraná, Brazil c Departamento de Engenharia de Sistemas Químicos, Faculdade de Engenharia Química, Universidade Estadual de Campinas, Av. Albert Einstein 500, Campinas, São Paulo, Brazil b
a r t i c l e
i n f o
Article history: Received 11 June 2010 Received in revised form 10 September 2010 Accepted 8 November 2010 Available online 9 December 2010 Keywords: PEO/starch blends Miscibility WAXS Raman and IR spectroscopy Cytotoxicity
a b s t r a c t This study reports results on the miscibility of polymer blends based on PEO and different starches (unmodified, cationic, and hydrophobic) and their respective cytotoxicity. Films of PEO/starch blends at different weight ratios (95/05, 90/10, 80/20, 70/30, 65/35, and 60/40), as well as films of pure PEO as control, were prepared by casting methodology. Several techniques, such as SEM, WAXS, FTIR, and FT-Raman spectroscopy were used in this study for evaluating blend miscibility. The results revealed that the miscibility of such blends is dependent on the type of starch used. Regarding the PEO/unmodified starch blends, it was concluded that the system is miscible in the ratio range from 90/10 to 65/35. Although the PEO/hydrophobic starch blends are miscible in all the studied range, blends of PEO and cationic starch are immiscible, regardless the blend ratio. The different samples presented distinct cytotoxic behaviors. PEO and hydrophobic starch presented no relevant toxicity (CC50/72 N 2.5 mg/mL). Otherwise, the cationic starch was the most harmful for the cells. The blends presented cytotoxicity values between those of PEO and cationic starch. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Due to some attractive properties, such as biodegradability [1,2], biocompatibility [3], and the relatively low cost [2], starch has been extensively studied as a promising candidate for many biomedical applications, including artificial skin [4], drug delivery systems [5–7], scaffolds for cell culture in tissue engineering [8,9], bone cements [10], implant and filling agents [3], and contact lenses [11]. In general, besides possessing unsatisfactory mechanical properties, polysaccharides, such as starch, are water-soluble and degrade at relatively low temperatures [12]. For these reasons, chemical and physical modifications (grafting and blending) of starch are often required for most uses of starch-based materials [13–15]. The chemical modification of starch generally occurs by the grafting of functional groups, mainly by the substitution of the hydrogen atoms of the hydroxyl groups [16]. Poly(ethylene oxide) (PEO), a semi-crystalline synthetic polymer, has the general molecular structure (CH2CH2O)n. Due to its biocompatibility and low toxicity, the FDA has approved its use in many biomedical devices [16], including drug delivery systems [17,18], tissue replacement and scaffolds [19–21], and surface coatings for the inhibition of protein and/or cell adsorption [22–26]. The development of blends based on PEO and starch enables one to obtain interesting materials for biomedical application, because both ⁎ Corresponding author. Fax: +55 44 3261 4125. E-mail address: [email protected] (E.C. Muniz). 0928-4931/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2010.11.004
polymers are biodegradable and biocompatible. Furthermore, the presence of PEO may improve the poor mechanical properties of the polysaccharide and provide a semi-crystalline structure to the final material, which may be attractive in cell culturing [27,28]. In vitro cytotoxicity tests represent the first stage in the evaluation of biocompatibility of a potential biomedical material. Several assays are described on the literature, and most of them are based on the cell death or other negative effects on cellular functions [29–31]. In order to receive the approval on the tests, the material should not be harmful to cells. Therefore, the use of cell culturing techniques allows to verify if the material induces some undesirable effects and its potential use in biomedical devices. In this work, the miscibility of unmodified, cationic, and hydrophobic starches and PEO was investigated through several techniques, and the cytotoxicity of blends was evaluated by the viability of VERO cells. 2. Experimental 2.1. Materials PEO (Aldrich 18,199-4, Mv: 200 kg mol− 1), unmodified cassava starch (Inpal S.A. Ind. Química, Brazil), and modified maize starches (Lorenz Company, Brazil) were used without further purification. Commercial starch samples were kindly donated by the producers. The molar masses of the starches, determined by GPC/SEC as described in the next section, are: unmodified — 5.4 × 103 kg mol− 1,
444
A.G.B. Pereira et al. / Materials Science and Engineering C 31 (2011) 443–451
cationic — 12 × 103 kg mol− 1 (5.8 mol% with quaternarium ammonium salts substituted in hydroxyl groups) and hydrophobic — 3.7 × 103 kg mol− 1 (9.4 mol% ethyl groups substituted in hydroxyl groups). 1H NMR and 13C NMR were used for the determination of degree of substitution and chemical characterization of both modified starches (see Figures S1A-B, Supplementary data). 2.2. Sample preparation PEO and PEO/starch blend films (95/05, 90/10, 80/20, 70/30, 65/35, and 60/40 wt.%) were obtained as follows: for each blend ratio, the desired amount of starch was solubilized in distilled water at 80 °C for gelatinization; after the cooling of the starch solution, the needed quantity of PEO was added to form a final aqueous solution of 5 w/v-%. In each case, the final solution was almost transparent, showing the solubilization of polymers in that condition. Therefore, films of the different blends were obtained by casting of the respective solution at room temperature. The films were dried for three days under ambient conditions, followed by 24 h under vacuum at room temperature (ca. 25 °C). The dried films were approximately 120 μm thick. 2.3. Characterization 2.3.1. Evaluation of miscibility Wide-angle X-ray scattering (WAXS) patterns were obtained using a Shimadzu XRD-6000 diffractometer (40 kV, 30 mA, CuKα) in the diffraction angle (2θ) range from 3° to 60° at a rate of 3°min− 1. The morphologies of the blends were analyzed through scanning electronic microscopy (SEM) using a Shimadzu apparatus model SS-550 operating at 30 kV. The samples were gold covered before analysis. Raman spectra were measured in a Renishaw Microscope Raman-RM 2000. Spectra were recorded in the 200–1000 cm− 1 range at two different sites of each sample. An objective lens with 50× magnification and a diode laser emitting at 788 nm as the excitation source were used. For FTIR analysis, the films of PEO/starch blend films were cast beforehand and placed between KBr crystals. Infrared spectra were obtained at two different sites of each sample using a FTIR Shimadzu AIM 8800 apparatus equipped with an optical microscope. 2.3.2. Evaluation of the molar masses of starches by GPC/SEC Aqueous solution of each starch type (0.5 w/v-%) was prepared under 24-h stirring at 80 °C. Next, each solution was diluted with distilled water to a final concentration of 0.25 (w/v-%) and then filtered using cotton padding. The molar masses of the starches were determined via gel permeation chromatography/size permeation chromatography (GPC/SEC) in a Shimadzu apparatus at room temperature using an Ultrahydrogel Linear (7.8 × 300 mm) chromatographic column and a flow rate of 0.5 mL min− 1. Differential refractometer was used as a detector. The calibration curve was constructed using Pullulan solutions with molar masses ranging from 103 to 105 g mol− 1 as references. Aqueous solution of NaNO3 (0.1 mol L− 1) was used as eluent. The straight line of molar mass (M) as a function of elution volume obtained was fitted with the following equation: log (M) = −0.9273 Ve + 12.55, where M is the standard Pullulan averaged Mn, and Ve is the eluted volume. The distribution profiles of the starches are shown in Figure S2 (Supplementary data). 2.3.3. Cytotoxicity assays VERO (kidney epithelial cells extracted from an African green monkey) cell viability was measured by the sulforhodamine B method with some modifications, as previously reported [32,33]. A suspension of 2.5 × 105 cells mL− 1 in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% of fetal bovine serum and 50 μg/mL of gentamicin was added in 96-well plates, and then allowed to grow as a monolayer for 24 h at 37 °C in 35 v/v-% CO2/air mixture. After this time, solutions with different concentrations (2.5%, 1.25%, 0.625% and
0.313 w/v-%) of PEO, starch, and blends of PEO/starch (unmodified, cationic and hydrophobic) were added to the wells, and the plate was incubated for 72 h under the same conditions described above. Cell control was also performed without the addition of sample (PEO/ starch blends). The cells were fixed in 10% trichloroacetic acid for 1 h, washed four times with distilled water, and then dried at room temperature. A solution of 4% sulforhodamine B was added to each well, and the plate was kept protected from light for 30 min. Then, the wells were washed four times with 1% acetic acid aqueous solution before adding an aliquot of 150 μL of Tris-base (10 mM). The absorbance was read at 530 nm in a microplate spectrophotometer (Bio Tek-Power Wave XS). Data were calculated as the percentage of inhibition. The concentration of 50% cellular toxicity (CC50) was defined as the concentration which reduced the optical density reading of treated cells at 530 nm (OD530) to 50% of the value of untreated cells. Triplicates were performed for all assays. 3. Results and discussion 3.1. Miscibility of PEO/starch blends through WAXS analysis Fig. 1 presents the WAXS patterns for pure PEO and PEO/ unmodified starch blends. The presence of well-resolved peaks in the diffraction patterns of PEO in the range from 2θ = 16° to 30° is characteristic of its crystalline structures. The (7/2) helical crystalline geometry of PEO, whose structure completes two turns around an axis every seven repeating units, due to trans (C-C-O-C), trans (C-O-C-C), and gauche (O-C-C-O) conformational arrangements, is appropriately described in the literature [34]. It is also important to emphasize that unmodified, cationic, and hydrophobic starches were considered amorphous, as their WAXS profiles did not present any peak, but rather a halo curve, as shown in Figure S3 (Supplementary data). It should be noticed that the strong peaks at 19.2° and 23.3°, according to Bragg's law, correspond to interplanar distances of 0.46 and 0.38 nm, respectively [35]. The peak at 2θ = 19.2° is related to the crystallographic plane (120), and the peak at 2θ = 23.3° may correspond to several planes: (032), (132), (112), (212), (004), and (124) [36]. The peak observed at 2θ = 29.4° in Fig. 1 is barely discussed in the literature. This work shows that this peak is very sensitive to polymer interactions. It is also important to point out that this peak is not present in the WAXS pattern of some PEO/starch blends. Consequently, it was of great importance for evaluating the influence of starch on blend miscibility and on the crystalline structure of PEO in PEO/starch blends.
Fig. 1. WAXS patterns for pure PEO and different PEO/unmodified starch blends.
A.G.B. Pereira et al. / Materials Science and Engineering C 31 (2011) 443–451
Fig. 2 presents the regions in Fig. 1 where the peaks at 2θ = 19.2° (2a) and 23.3° (2b) appear. Some peak shifts at 2θ = 19.2° and 2θ = 23.3°, related to the WAXS pattern of pure PEO, can be observed for several blend ratios. Considering that the peak at 2θ = 29.4° is not present in the WAXS patterns of PEO/unmodified starch blends, except in the 95/05 blend, for which it presented very low intensity (Fig. 1), and based on the information extracted from Fig. 2, it was possible to infer that the unmodified starch induces some changes in the crystalline structure of PEO, possibly due to interactions among the polymer chains. However, due to the presence of the peak at 2θ = 29.4° in the WAXS patterns of pure PEO (with a medium intensity) and in the pattern of the 95/05 PEO/unmodified blend (though with a very low intensity), it is also possible to infer that PEO crystallizes almost as a pure component in the 95/05 PEO/unmodified blend. Thus, in this blend ratio, PEO does not interact with the unmodified starch, or if it does, it does differently from other blends in this system. Table 1 presents values for the ratio between the areas of two characteristic diffraction peaks of PEO, area peak 2/area peak 1, in which peak 1 represents 2θ = 19.2°, and peak 2 corresponds to 2θ = 23.3°. It could be observed that for 95/05 and 60/40 blends the value of such ratio remained constant and equal to pure PEO. This fact
Fig. 2. WAXS patterns for pure PEO and different PEO/unmodified starch blends.
445
Table 1 Area ratios of peaks from WAXS profiles of PEO and PEO/unmodified starch blends. Blend Ratio (PEO/unmodified starch) 100/0 95/05 90/10 80/20 70/30 65/35 60/40 a
Ratio Area peak 2/area peak1a 3.1 3.1 3.2 3.9 2.9 3.0 3.1
Peak 1 (2θ = 19,2°); peak 2 (2θ = 23.3°).
is a strong indicative that unmodified starch is predominantly dispersed on the amorphous phase of PEO, for that blend ratios. For the other blend ratios slight shifts on the areas ratio were verified. This fact it might be related to miscibility of starch and PEO, as could be demonstrated in the following sections. Fig. 3 shows the WAXS patterns of PEO/cationic starch blends (3a) and PEO/hydrophobic starch blends (3b). The WAXS pattern of pure PEO is included for the sake of comparison.
Fig. 3. WAXS patterns obtained for pure PEO, PEO/cationic starch, and PEO/hydrophobic starch blends.
446
A.G.B. Pereira et al. / Materials Science and Engineering C 31 (2011) 443–451
The regions of Fig. 3A and B where the peak at 2θ=29.4° appears are magnified in Fig. 4A and B, respectively. For the PEO/cationic starch blends (Fig. 4A), it can be seen that although the intensity of the peak at 2θ=29.4° decreased as the amount of starch was increased, it was still present up to the 70/30 blend ratio. This finding suggests that the attractive forces between of the PEO and cationic starch chains do not exist, or, if they do, they are much weaker than those of pure polymer chains. Therefore, because the peak at 29.4° is present in WAXS profiles of PEO and cationic starch blends, it was inferred that the crystalline structure of PEO is not significantly changed by the presence of the cationic starch. In blends with over 30% cationic starch, the cationic starch chains may be entrapped within the amorphous regions of PEO, leading to distortions and defects in the crystalline arrangements due to the high starch concentration, and consequently, to the disappearance of the peak at 2θ=29.4°. The peak at 2θ = 29.4° was not observed for the blends constituted of PEO and hydrophobic starch, even for small quantities of starch. Based on this fact, it is reasonable to infer that the absence of the peak may be due to attractive interactions between PEO and hydrophobic starch chains. 3.2. Miscibility of PEO/starch blends through analysis of morphology by SEM
In this work, this was done by direct observation of SEM micrographs. Fig. 5 presents the micrographs of pure PEO and PEO/unmodified starch blends. By comparison of the micrographs from different blend ratios, it was possible to observe that their morphologies are very dissimilar. However, small amounts of unmodified starch (at ratio 95/05, for instance) basically do not affect the blend morphology, as compared to the morphology of pure PEO. This evidences the lack of interaction between both polymers and explains why the characteristic PEO spherulite structure remains in that blend, as previously published [37]. Higher amounts of unmodified starch in blends induce significant alteration in morphology as compared to the 95/05 blend. For instance, the characteristic morphology of pure PEO was not observed for blend ratios ranging from 90/10 to 70/30. In these blends, the density of interaction is enough to prevent the system from being completely immiscible. However, two phases similar to those of the individual components were observed in the morphologies of the 65/35 and 60/40 blend ratios. Thus, these blends were considered to be immiscible systems. Fig. 6 shows the SEM micrographs of the PEO/cationic starch and PEO/hydrophobic starch blends. In the PEO/cationic starch system, the spherulitic structure of PEO was preserved, despite the increasing amount of cationic starch. On the
Miscibility, immiscibility, and some other system characteristics, such as roughness and defects, can be evaluated through morphology analysis.
Fig. 4. WAXS patterns obtained for PEO/cationic starch and PEO/hydrophobic starch blends in the range of 2θ = 28° to 32°.
Fig. 5. SEM images of pure PEO, unmodified starch, and PEO/unmodified starch blends.
A.G.B. Pereira et al. / Materials Science and Engineering C 31 (2011) 443–451
447
Fig. 6. SEM images of PEO/cationic starch and PEO/hydrophobic starch blends.
other hand, the PEO/hydrophobic starch blends presented the opposite tendency. Morphologies distinct from that of pure PEO were obtained even using small amounts of hydrophobic starch. The difference in such behaviors can be clarified when the micrographs obtained for 90/10 and 65/35 blend ratios of each system are compared. For the PEO/cationic starch, the 90/10 and 65/35 blend ratios presented morphologies very similar to that of pure PEO, but quite different from the morphologies of the respective PEO/hydrophobic starch blends. Therefore, the characteristic PEO spherulites were not observed in the PEO/hydrophobic
starch blends, suggesting that the interaction between PEO and hydrophobic starch is enough for providing miscibility. 3.3. Miscibility of PEO/starch blends through FTIR coupled with optical microscope The use of FTIR spectroscopy in the characterization of polymer blends has been widely reported in the literature [38]. It allows identifying and quantifying specific intra- and inter-polymer chain
Fig. 7. FTIR spectra of PEO, unmodified starch, and two distinct sites of the 95/05 PEO/unmodified starch blend.
448
A.G.B. Pereira et al. / Materials Science and Engineering C 31 (2011) 443–451
Fig. 8. FTIR spectra of PEO, unmodified starch, and two distinct sites of the 90/10 PEO/unmodified starch blend.
molecular interactions. The use of FTIR-optical microscopy helps such a characterization because it allows the analysis of specific regions of the sample. In the case of polymer blends, FTIR-optical microscopy also allows the evaluation of miscibility based on the spectra obtained at specific chosen blend sites [39]. It is worth emphasizing that although the starches (unmodified, cationic, and hydrophobic) are chemically different, their FTIR spectra are not significantly different (Supplementary data, Figure S4). This suggests that although the degree of modification is small, it is enough to result in different behaviors when blended with PEO, as discussed before. The main differences in the FTIR of the starches used include CO-C and C-O-H vibrational modes (in the range of 1300–950 cm− 1). In this work, the vibrational modes of PEO and of starch in the spectra in Figs. 7–9 were assigned based on the data in Table 2 [40,41].
Fig. 7 presents the FTIR spectra of PEO, of unmodified starch, and of 95/05 PEO/unmodified starch blend obtained at distinct sites (1 and 2). It can be verified that the spectra of sites 1 and 2 are quite different with regard to the C-O-C and C-O-H vibrational modes of starch (ca. 1000 cm− 1), mainly because the band is clearly present in the FITR spectrum obtained at site 1 of 95/05 but not in the FTIR spectrum at site 2 of this blend. These results reinforce the point of view that at the ratio of 95/05, the PEO/unmodified starch blend is heterogeneous at the FTIR depth (2–5 μm) [42], indicating its immiscibility, as already proposed [37]. Fig. 8 shows the FTIR spectra of PEO, of unmodified starch and of the 90/10 PEO/unmodified starch blend obtained at two distinct sites (1 and 2). For this blend, the FTIR spectra of both sites are very similar, presenting a band at ca. 1000 cm− 1 which was attributed to the C-O-C and C-O-H
Fig. 9. FTIR spectra of PEO, unmodified starch, and two distinct sites at the 90/10 PEO/unmodified starch blends in the wave number range of 3500 to 2500 cm− 1.
A.G.B. Pereira et al. / Materials Science and Engineering C 31 (2011) 443–451
449
Table 2 FTIR vibrational modes for PEO and starch [40,41].
PEO
STARCH
Vibrational modes
Wave number (cm− 1)
O–H C–H C–O–C O–H C–H C–C, C–O and C–O–H
3490 2800–2935 1250–950 3600–3000 2930 1200–900
vibrational modes of the unmodified starch, and a broad band at ca. 2000 cm− 1, which was attributed to the asymmetric stretching of PEO. The C-H stretching for PEO and the unmodified starch that appears at ca. 2880–2890 cm− 1 are also present in the FITR of the 90/10 blend obtained at sites 1 and 2, as presented in the Fig. 9. The FTIR spectra obtained at sites 1 and 2 show a broader band related to band coalescence, which can be attributed to the C-H stretching of PEO (2879 cm− 1) and the unmodified starch (2921 cm− 1). Based on the FTIR spectra, the main features observed are that site 1 is richer in starch than site 2, while the band assigned to PEO in FTIR spectrum obtained at site 2 is weaker, considering that this blend (90/10) is nine-fold richer (in weight) in PEO than in starch. The similarity of the FTIR spectra obtained at sites 1 and 2 indicates that the blend 90/10 PEO/unmodified starch is somewhat chemically homogeneous, which confirms the miscibility of this blend ratio suggested by WAXS and SEM results. The FTIR spectra of PEO/cationic starch blends (Supplementary data, Figure S5) do not show any evidence of either interaction between the polymers, or system miscibility in the whole range of studied blend ratios. This can be noted because the FTIR spectrum obtained at a given site of the sample is very dissimilar to that obtained at another site of the same sample. On the other hand, for the PEO/hydrophobic starch blends, the FTIR spectra (Supplementary data, Figure S6) are quite similar, regardless of the site chosen. The results presented in this section are in agreement with WAXS and SEM data for PEO/cationic starch and PEO/hydrophobic starch, as discussed above. 3.4. Miscibility of PEO/starch blends through Raman imaging In this work, more attention was paid to Raman spectra in the range of 100 to 1000 cm− 1. This range contains signals attributed to the main vibrations of PEO groups (C-C-O and C-O-C bending; C-C and C-O rotation). If oxygen atoms such as those of the ether groups in PEO interact through hydrogen bonds, those vibrations must be affected, leading to shifting of the peaks and changing their intensities. In this work, Raman spectra were obtained at two distinct sites of PEO/unmodified starch blend at ratios 95/05, 80/20, and 60/40. One of these sites was the center of the spherulite, while the other was outside of the center of the spherulite, as shown in Fig. 10. For the pure PEO and unmodified starch, the Raman spectra were obtained at random sites of the samples. Fig. 11 gives the Raman spectra obtained in the center of the spherulites for the ratios 95/05, 80/20, and 60/40 of PEO/unmodified starch. For comparison, Raman spectra of PEO and unmodified starch are also included in the Fig. 11. Three interesting events are observed in the spectrum of the 80/20 blend ratio: i) widening of the main PEO peaks; ii) shifting of the peak assigned to PEO from 844 cm− 1 to 847 cm− 1; iii) appearance of new bands at 680 and at 770 cm− 1. This set of results gives enough information to infer that, at this blend ratio, PEO and the unmodified starch interact at the center of the spherulite. The presence of starch at the center of the spherulites was assigned based on the presence of the characteristic starch band at 480 cm− 1 in
Fig. 10. Micrograph showing the sample sites where the Raman spectra were obtained. The red spot is the center of the spherulite and the black spot is relative to the body of the spherulite. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
spectra obtained using PEO/unmodified starch blends at 95/05 and 60/40 ratios. Well-resolved peaks of the individual polymers were observed in the Raman spectra of such blends, and no shifting or changing in intensity was noticed. Therefore, it is suggested that there is a lack of interaction between the PEO and the unmodified starch at the center of the spherulites for the 95/05 and 60/40 blend ratios. The Raman spectra obtained out of the center of the spherulites of 95/05, 80/20, and 60/40 PEO/unmodified starch blends are presented in Fig. 12. For the 60/40 blend, the C-C stretching band at ca. 480 cm− 1 of starch is present, but with a lower intensity than that observed at the center of the spherulites (Fig. 11). Other peaks remained unchanged when compared with those in the spectra of pure polymers, indicating the absence of interaction between PEO and unmodified starch. For the 80/20 blend ratio, important changes were verified: the C-C stretching of starch shifted from 480 cm− 1 to 484 cm− 1, and the C-O-C bending of PEO shifted from 535 cm− 1 to 538 cm− 1. For the 95/ 05 blend, new bands appeared at 680 cm− 1 and 770 cm− 1, followed by the disappearance of the band at 480 cm− 1. Based on Raman spectral data, it can be concluded that PEO/ unmodified starch is immiscible at ratios of 95/05 and 60/40, while the system with the 80/20 composition is miscible. These results match those obtained by SEM, WAXS, and FITR-microscopy.
Fig. 11. Raman spectra obtained at the center of the spherulites for different compositions of PEO/unmodified starch blends (laser λ = 788 nm).
450
A.G.B. Pereira et al. / Materials Science and Engineering C 31 (2011) 443–451 Table 3 Cytotoxicity, growth inhibition (CC50/72 h) of samples against VERO cells.
Fig. 12. Raman spectra of the spherulite bodies for different compositions of the PEO/ unmodified starch blends (laser λ = 788 nm).
Sample
CC50/72h (mg/mL)
Poly(ethylene oxide) (PEO) Unmodified starch Cationic starch Hydrophobic starch 80/20 PEO/unmodified starch 80/20 PEO/cationic starch 80/20 PEO/hydrophobic starch
N 2.5 N/D* 0.49 ± 0.31 N 2.5 1.1 ± 0.98 0.93 ± 0.67 0.62 ± 0.25
It is worth mentioning that the analyses were performed two weeks after the sample preparation. It is possible that degradation had already begun at the time of analysis. As ammonium groups were grafted onto cationic starch, the explanation for its highest toxicity against VERO cells is that when the degradation process starts, some ammonia molecules, which are very harmful for the cells, are released into the medium, thus increasing the toxicity of the sample. 4. Conclusions
3.5. Mechanisms of interaction between PEO and the starches The PEO/unmodified starch system is partially miscible, as described above. When the amount of starch in the blend is small (ratio 95/05), there are preferentially intra-chain interactions. When the amount of unmodified starch is increased (90/10 to 70/30), inter-chain interactions gradually presented an important role to miscibility, probably due to H-bonds between hydroxyl groups in starch and oxygen atoms in PEO. Increasing the amount of starch even more, the stability is established by intra-chain rather than inter-chain interactions. For blends of PEO/cationic starch, the cationic groups grafted onto starch may interact at intra-molecular level. In the case of blends of PEO with hydrophobic starch, the hydrophilic H-bond interactions between hydroxyl groups of starch and oxygen atoms of the PEO's ether groups and interactions between the hydrophobic chain segments of PEO (ethyl groups) and of hydrophobic starch were also responsible for the miscibility of the PEO/hydrophobic starch system. Figure S7 (Supplementary data) depicts the probable interactions between PEO and the starches. The miscibility behavior of the different blend systems was explained based mainly on the chemical differences of starches. However, another important factor that it might have contributed to those behaviors was the molar masses of starches. It seems that the increasing molar mass of starch decreases the miscibility with PEO, taking into account that PEO/hydrophobic starch was totally miscible, PEO/unmodified starch was partially miscible and PEO/cationic starch was totally immiscible. It must be emphasized that when lyophilized directly from the aqueous solutions, PEO/unmodified starch blends at ratios of 80/20 and 65/35 presented interesting morphologies (Supplementary data, Figure S8), differing from those obtained by casting, indicating that miscibility also depends on the film production process. These morphologies are potentially suitable for use in tissue scaffolding. 3.6. Cytotoxicity assays of PEO/starch blends through VERO cells culturing The 50% cytotoxic concentrations after 72 h of incubation (CC50/72) of samples on VERO cells are shown in Table 3. The lowest cytotoxicity was observed for PEO and hydrophobic starch, which presented CC 50/72h N 2.5 mg mL − 1 , whereas for the blends containing unmodified and cationic starches, the CC50/72h were very close (1.1 and 0.9, respectively). The most harmful sample for the cells was the cationic starch, with CC50/72h = 0.49 mg mL− 1. The CC50/72h of unmodified starch was not determined due to sample contamination.
The miscibility of PEO-based blends with different starches (unmodified, cationic, and hydrophobic) was evaluated via FTIR and Raman spectroscopies, WAXS, and SEM techniques. The results revealed that the miscibility of a given system depends on the blend composition. Comparison of the different PEO/starch blends showed that in these systems miscibility depends on the type of starch. PEO/unmodified starch blends obtained by casting are miscible in the composition range from 90/10 to 65/35, since the characteristic morphology of PEO disappeared and the diffraction peak at 2θ = 29.4° was absent. These findings were attributed to the molecular interactions between the oxygen atoms of PEO and hydroxyl groups of the unmodified starch. For the other blend ratios, the unchangeable morphology of PEO/unmodified starch blends in relation to those of the individual polymers indicates the lack of interaction between the polymers. By the use of FTIR spectroscopy, it was possible to verify the presence of two phases in the 95/05 ratio blend. Its WAXS patterns presented the peak at 2θ = 29.4°, which was not observed on the WAXS pattern of the 80/20 ratio blend. Raman spectroscopy showed a lack of interactions between PEO and starch at blend ratios of 95/05 and 60/40. All studied PEO/cationic starch blend ratios cast films were immiscible. On the other hand, there is strong evidence of miscibility across the entire studied blend ratio range for the PEO/hydrophobic starch system cast films. The difference in miscibility found for the investigated systems in this work was attributed to the chemical modification of starch. Despite the fact that starches were modified only to a small degree, those modifications were enough to provoke significant changes in miscibility. The different molar masses may also have contributed to the changes in miscibility; however, this was not investigated in depth in this work. It was seen that the miscibility in PEO/unmodified 80/20 and 65/35 ratio blends also depends on the film production process. The cytotoxicity assays were performed using VERO cells. The different samples presented distinct cytotoxic behaviors. Pure PEO and hydrophobic starch presented the least relevant toxic effects quantified as CC50/72 N 2.5 mg/mL, which is a considerably high value; otherwise the cationic starch was the most harmful for the cells. The blends presented intermediate cytotoxicity, between PEO and cationic starch values. Therefore, the PEO/hydrophobic starch system could be suitable for cell culturing and tissue engineering. Acknowledgements AGBP thanks CAPES (Brazil) for the Master's scholarship. ECM thanks CNPq (Brazil, Proc. 308611/2006-3) for the financial support.
A.G.B. Pereira et al. / Materials Science and Engineering C 31 (2011) 443–451
Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10.1016/j.msec.2010.11.004. References [1] G. Scott, Polym Degrad Stab 68 (2000) 1–7. [2] X.L. Wang, K.K. Yang, Y.Z. Wang, D.Y. Wang, Z. Yang, Acta Mater 52 (2004) 4899–4905. [3] J.F. Mano, R.A. Sousa, L.F. Boesel, N.M. Neves, R.L. Reis, Compos Sci Technol 64 (2004) 789–817. [4] P. Kunal, A.K. Banthia, D.K. Majumdar, Afr J Biomed Res 9 (2006) 23–29. [5] A.P. Vieira, P. Ferreira, J.F.J. Coelho, M.H. Gil, Int J Biol Macromol 43 (2008) 325–332. [6] G.F. Mehyar, Z. Liu, J.H. Han, Carbohydr Polym 74 (2008) 92–98. [7] M. Sadeghi, H. Hoosseinzadeh, J Bioact Compat Polym 23 (2008) 381–404. [8] A.J. Salgado, O.P. Coutinho, R.L. Reis, Tissue Eng 10 (2004) 465–474. [9] S.C. Mendes, R.L. Reis, Y.P. Bovell, A.M. Cunha, C.A. Blitterswijk, J.D. Bruijn, Biomaterials 22 (2001) 2057–2064. [10] L. Boesel, R.L. Reis, Biomaterials 27 (2006) 5627–5633. [11] I. M. Katz, (Merck & Co). U. S. Patent 4,287,175, September 1, 1981. [12] B. Hoffmann, E. Volkmer, A. Kokott, M. Weber, S. Hamisch, M. Schieker, et al., J Mater Chem 17 (2007) 4028–4033. [13] J.F. Mano, D. Koniarova, R.L. Reis, J Mater Sci Mater M 14 (2003) 127–135. [14] X. Fu, X. Chen, R. Wen, X. He, X. Shang, Z. Liao, et al., J Polym Res 14 (2007) 297–304. [15] S. Godbole, S. Gote, M. Latkar, T. Chakrabarti, Bioresour Technol 86 (2003) 33–37. [16] P.X. Wang, X.L. Wu, X.D. Hua, X. Kun, T. Ying, D.X. Bing, et al., Carbohydr Res 344 (2009) 851–855. [17] K.Y. Lee, S.H. Yuk, Prog Polym Sci 32 (2007) 669–697. [18] M. Nagata, Y. Yamamoto, React Funct Polym 68 (2008) 915–921. [19] S. Salmaso, A. Semenzato, S. Bersani, P. Matricardi, F. Rossi, P. Caliceti, Int J Pharm 345 (2007) 42–50.
[20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42]
451
Y.C. Kuo, I.N. Ku, Biomacromolecules 9 (2008) 2662–2669. J. Reignier, M.A. Huneaul, Polymer 47 (2006) 4703–4717. Q. Hou, D.W. Grijpma, J. Feijen, Biomaterials 24 (2003) 1937–1947. D. Bozukova, C. Pagnoulle, M.C.P. Gillet, S. Desbief, R. Lazzaroni, N. Ruth, et al., Biomacromolecules 8 (2007) 2379–2387. H. Chen, Z. Zhang, Y. Chen, M.A. Brook, H. Sheardown, Biomaterials 26 (2005) 2391–2399. L.D. Unsworth, H. Sheardown, J.L. Brash, Biomaterials 26 (2005) 5927–5933. J.G. Archambault, J.L. Brash, Colloids Surf B Biointerfaces 33 (2004) 111–120. R.S. Lima, K.A. Khor, H. Li, P. Cheang, B.R. Marple, Mater Sci Eng A 396 (2005) 181–185. K. Balani, R. Anderson, T. Laha, M. Andara, J. Tercero, E. Crumpler, et al., Biomaterials 28 (2007) 618–624. S.C. Mendes, R.L. Reis, Y.P. Bovell, A.M. Cunha, C.A. van Blitterswijk, J.D. Bruijn, Biomaterials 22 (2001) 2057–2064. E. Cer, O.A. Gurpinar, M.A. Onur, A. Tuncel, J. Biomed, Mater Res B 80B (2007) 406–414. X. Gao, Y. Zhou, G. Ma, S. Shi, D. Yang, F. Lu, et al., Carbohydr Polym 79 (2010) 507–512. P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica, et al., Cancer Inst 82 (1990) 1107–1112. I.C. Filho, D.A.G. Cortez, T.U. Nakamura, C.V. Nakamura, B.P.D. Filho, Phytomedicine 15 (2008) 202–208. Y. Takahashi, H. Tadokoro, Macromolecules 6 (1973) 672–675. Y. Deng, J.B. Dixon, G.N. White, Colloid Polym Sci 248 (2006) 347–356. C.M. Burba, R. Frech, B. Grady, Electrochim Acta 53 (2007) 1548–1555. A.G.B. Pereira, R.F. Gouveia, G.M. Carvalho, A.F. Rubira, E.C. Muniz, Mater Sci Eng C 29 (2009) 499–504. Y. He, B. Zhu, Y. Inoue, Prog Polym Sci 29 (2004) 1021–1051. R.J. Meier, Chem Soc Rev 34 (2005) 743–752. S. Ramesh, T.F. Yuen, C.J. Shen, Spectrochim Acta A 69 (2008) 670–675. R. Kizil, J. Irudayaraj, K. Seetharaman, J Agr Food Chem 50 (2002) 3912–3918. L.A. Utracki, Polymer Alloys and Blends: Thermodynamic and Rheology, Hanser Publishers, Munich, 19898 pp 356.
Contents lists available at ScienceDirect
Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m s e c
Effect of starch type on miscibility in poly(ethylene oxide) (PEO)/starch blends and cytotoxicity assays A.G.B. Pereira a, A.T. Paulino c, C.V. Nakamura b, E.A. Britta b, A.F. Rubira a, E.C. Muniz a,⁎ a
Grupo de Materiais Poliméricos e Compósitos, GMPC, Departamento de Química Universidade Estadual de Maringá, Av. Colombo 5790, 87020-900, Maringá, Paraná, Brazil Laboratório de Inovação Tecnológica no Desenvolvimento de Fármacos e Cosméticos, DBS, Departamento de Análises Clínicas. Universidade Estadual de Maringá, Av. Colombo 5790-87020-900, Maringá, Paraná, Brazil c Departamento de Engenharia de Sistemas Químicos, Faculdade de Engenharia Química, Universidade Estadual de Campinas, Av. Albert Einstein 500, Campinas, São Paulo, Brazil b
a r t i c l e
i n f o
Article history: Received 11 June 2010 Received in revised form 10 September 2010 Accepted 8 November 2010 Available online 9 December 2010 Keywords: PEO/starch blends Miscibility WAXS Raman and IR spectroscopy Cytotoxicity
a b s t r a c t This study reports results on the miscibility of polymer blends based on PEO and different starches (unmodified, cationic, and hydrophobic) and their respective cytotoxicity. Films of PEO/starch blends at different weight ratios (95/05, 90/10, 80/20, 70/30, 65/35, and 60/40), as well as films of pure PEO as control, were prepared by casting methodology. Several techniques, such as SEM, WAXS, FTIR, and FT-Raman spectroscopy were used in this study for evaluating blend miscibility. The results revealed that the miscibility of such blends is dependent on the type of starch used. Regarding the PEO/unmodified starch blends, it was concluded that the system is miscible in the ratio range from 90/10 to 65/35. Although the PEO/hydrophobic starch blends are miscible in all the studied range, blends of PEO and cationic starch are immiscible, regardless the blend ratio. The different samples presented distinct cytotoxic behaviors. PEO and hydrophobic starch presented no relevant toxicity (CC50/72 N 2.5 mg/mL). Otherwise, the cationic starch was the most harmful for the cells. The blends presented cytotoxicity values between those of PEO and cationic starch. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Due to some attractive properties, such as biodegradability [1,2], biocompatibility [3], and the relatively low cost [2], starch has been extensively studied as a promising candidate for many biomedical applications, including artificial skin [4], drug delivery systems [5–7], scaffolds for cell culture in tissue engineering [8,9], bone cements [10], implant and filling agents [3], and contact lenses [11]. In general, besides possessing unsatisfactory mechanical properties, polysaccharides, such as starch, are water-soluble and degrade at relatively low temperatures [12]. For these reasons, chemical and physical modifications (grafting and blending) of starch are often required for most uses of starch-based materials [13–15]. The chemical modification of starch generally occurs by the grafting of functional groups, mainly by the substitution of the hydrogen atoms of the hydroxyl groups [16]. Poly(ethylene oxide) (PEO), a semi-crystalline synthetic polymer, has the general molecular structure (CH2CH2O)n. Due to its biocompatibility and low toxicity, the FDA has approved its use in many biomedical devices [16], including drug delivery systems [17,18], tissue replacement and scaffolds [19–21], and surface coatings for the inhibition of protein and/or cell adsorption [22–26]. The development of blends based on PEO and starch enables one to obtain interesting materials for biomedical application, because both ⁎ Corresponding author. Fax: +55 44 3261 4125. E-mail address: [email protected] (E.C. Muniz). 0928-4931/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2010.11.004
polymers are biodegradable and biocompatible. Furthermore, the presence of PEO may improve the poor mechanical properties of the polysaccharide and provide a semi-crystalline structure to the final material, which may be attractive in cell culturing [27,28]. In vitro cytotoxicity tests represent the first stage in the evaluation of biocompatibility of a potential biomedical material. Several assays are described on the literature, and most of them are based on the cell death or other negative effects on cellular functions [29–31]. In order to receive the approval on the tests, the material should not be harmful to cells. Therefore, the use of cell culturing techniques allows to verify if the material induces some undesirable effects and its potential use in biomedical devices. In this work, the miscibility of unmodified, cationic, and hydrophobic starches and PEO was investigated through several techniques, and the cytotoxicity of blends was evaluated by the viability of VERO cells. 2. Experimental 2.1. Materials PEO (Aldrich 18,199-4, Mv: 200 kg mol− 1), unmodified cassava starch (Inpal S.A. Ind. Química, Brazil), and modified maize starches (Lorenz Company, Brazil) were used without further purification. Commercial starch samples were kindly donated by the producers. The molar masses of the starches, determined by GPC/SEC as described in the next section, are: unmodified — 5.4 × 103 kg mol− 1,
444
A.G.B. Pereira et al. / Materials Science and Engineering C 31 (2011) 443–451
cationic — 12 × 103 kg mol− 1 (5.8 mol% with quaternarium ammonium salts substituted in hydroxyl groups) and hydrophobic — 3.7 × 103 kg mol− 1 (9.4 mol% ethyl groups substituted in hydroxyl groups). 1H NMR and 13C NMR were used for the determination of degree of substitution and chemical characterization of both modified starches (see Figures S1A-B, Supplementary data). 2.2. Sample preparation PEO and PEO/starch blend films (95/05, 90/10, 80/20, 70/30, 65/35, and 60/40 wt.%) were obtained as follows: for each blend ratio, the desired amount of starch was solubilized in distilled water at 80 °C for gelatinization; after the cooling of the starch solution, the needed quantity of PEO was added to form a final aqueous solution of 5 w/v-%. In each case, the final solution was almost transparent, showing the solubilization of polymers in that condition. Therefore, films of the different blends were obtained by casting of the respective solution at room temperature. The films were dried for three days under ambient conditions, followed by 24 h under vacuum at room temperature (ca. 25 °C). The dried films were approximately 120 μm thick. 2.3. Characterization 2.3.1. Evaluation of miscibility Wide-angle X-ray scattering (WAXS) patterns were obtained using a Shimadzu XRD-6000 diffractometer (40 kV, 30 mA, CuKα) in the diffraction angle (2θ) range from 3° to 60° at a rate of 3°min− 1. The morphologies of the blends were analyzed through scanning electronic microscopy (SEM) using a Shimadzu apparatus model SS-550 operating at 30 kV. The samples were gold covered before analysis. Raman spectra were measured in a Renishaw Microscope Raman-RM 2000. Spectra were recorded in the 200–1000 cm− 1 range at two different sites of each sample. An objective lens with 50× magnification and a diode laser emitting at 788 nm as the excitation source were used. For FTIR analysis, the films of PEO/starch blend films were cast beforehand and placed between KBr crystals. Infrared spectra were obtained at two different sites of each sample using a FTIR Shimadzu AIM 8800 apparatus equipped with an optical microscope. 2.3.2. Evaluation of the molar masses of starches by GPC/SEC Aqueous solution of each starch type (0.5 w/v-%) was prepared under 24-h stirring at 80 °C. Next, each solution was diluted with distilled water to a final concentration of 0.25 (w/v-%) and then filtered using cotton padding. The molar masses of the starches were determined via gel permeation chromatography/size permeation chromatography (GPC/SEC) in a Shimadzu apparatus at room temperature using an Ultrahydrogel Linear (7.8 × 300 mm) chromatographic column and a flow rate of 0.5 mL min− 1. Differential refractometer was used as a detector. The calibration curve was constructed using Pullulan solutions with molar masses ranging from 103 to 105 g mol− 1 as references. Aqueous solution of NaNO3 (0.1 mol L− 1) was used as eluent. The straight line of molar mass (M) as a function of elution volume obtained was fitted with the following equation: log (M) = −0.9273 Ve + 12.55, where M is the standard Pullulan averaged Mn, and Ve is the eluted volume. The distribution profiles of the starches are shown in Figure S2 (Supplementary data). 2.3.3. Cytotoxicity assays VERO (kidney epithelial cells extracted from an African green monkey) cell viability was measured by the sulforhodamine B method with some modifications, as previously reported [32,33]. A suspension of 2.5 × 105 cells mL− 1 in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% of fetal bovine serum and 50 μg/mL of gentamicin was added in 96-well plates, and then allowed to grow as a monolayer for 24 h at 37 °C in 35 v/v-% CO2/air mixture. After this time, solutions with different concentrations (2.5%, 1.25%, 0.625% and
0.313 w/v-%) of PEO, starch, and blends of PEO/starch (unmodified, cationic and hydrophobic) were added to the wells, and the plate was incubated for 72 h under the same conditions described above. Cell control was also performed without the addition of sample (PEO/ starch blends). The cells were fixed in 10% trichloroacetic acid for 1 h, washed four times with distilled water, and then dried at room temperature. A solution of 4% sulforhodamine B was added to each well, and the plate was kept protected from light for 30 min. Then, the wells were washed four times with 1% acetic acid aqueous solution before adding an aliquot of 150 μL of Tris-base (10 mM). The absorbance was read at 530 nm in a microplate spectrophotometer (Bio Tek-Power Wave XS). Data were calculated as the percentage of inhibition. The concentration of 50% cellular toxicity (CC50) was defined as the concentration which reduced the optical density reading of treated cells at 530 nm (OD530) to 50% of the value of untreated cells. Triplicates were performed for all assays. 3. Results and discussion 3.1. Miscibility of PEO/starch blends through WAXS analysis Fig. 1 presents the WAXS patterns for pure PEO and PEO/ unmodified starch blends. The presence of well-resolved peaks in the diffraction patterns of PEO in the range from 2θ = 16° to 30° is characteristic of its crystalline structures. The (7/2) helical crystalline geometry of PEO, whose structure completes two turns around an axis every seven repeating units, due to trans (C-C-O-C), trans (C-O-C-C), and gauche (O-C-C-O) conformational arrangements, is appropriately described in the literature [34]. It is also important to emphasize that unmodified, cationic, and hydrophobic starches were considered amorphous, as their WAXS profiles did not present any peak, but rather a halo curve, as shown in Figure S3 (Supplementary data). It should be noticed that the strong peaks at 19.2° and 23.3°, according to Bragg's law, correspond to interplanar distances of 0.46 and 0.38 nm, respectively [35]. The peak at 2θ = 19.2° is related to the crystallographic plane (120), and the peak at 2θ = 23.3° may correspond to several planes: (032), (132), (112), (212), (004), and (124) [36]. The peak observed at 2θ = 29.4° in Fig. 1 is barely discussed in the literature. This work shows that this peak is very sensitive to polymer interactions. It is also important to point out that this peak is not present in the WAXS pattern of some PEO/starch blends. Consequently, it was of great importance for evaluating the influence of starch on blend miscibility and on the crystalline structure of PEO in PEO/starch blends.
Fig. 1. WAXS patterns for pure PEO and different PEO/unmodified starch blends.
A.G.B. Pereira et al. / Materials Science and Engineering C 31 (2011) 443–451
Fig. 2 presents the regions in Fig. 1 where the peaks at 2θ = 19.2° (2a) and 23.3° (2b) appear. Some peak shifts at 2θ = 19.2° and 2θ = 23.3°, related to the WAXS pattern of pure PEO, can be observed for several blend ratios. Considering that the peak at 2θ = 29.4° is not present in the WAXS patterns of PEO/unmodified starch blends, except in the 95/05 blend, for which it presented very low intensity (Fig. 1), and based on the information extracted from Fig. 2, it was possible to infer that the unmodified starch induces some changes in the crystalline structure of PEO, possibly due to interactions among the polymer chains. However, due to the presence of the peak at 2θ = 29.4° in the WAXS patterns of pure PEO (with a medium intensity) and in the pattern of the 95/05 PEO/unmodified blend (though with a very low intensity), it is also possible to infer that PEO crystallizes almost as a pure component in the 95/05 PEO/unmodified blend. Thus, in this blend ratio, PEO does not interact with the unmodified starch, or if it does, it does differently from other blends in this system. Table 1 presents values for the ratio between the areas of two characteristic diffraction peaks of PEO, area peak 2/area peak 1, in which peak 1 represents 2θ = 19.2°, and peak 2 corresponds to 2θ = 23.3°. It could be observed that for 95/05 and 60/40 blends the value of such ratio remained constant and equal to pure PEO. This fact
Fig. 2. WAXS patterns for pure PEO and different PEO/unmodified starch blends.
445
Table 1 Area ratios of peaks from WAXS profiles of PEO and PEO/unmodified starch blends. Blend Ratio (PEO/unmodified starch) 100/0 95/05 90/10 80/20 70/30 65/35 60/40 a
Ratio Area peak 2/area peak1a 3.1 3.1 3.2 3.9 2.9 3.0 3.1
Peak 1 (2θ = 19,2°); peak 2 (2θ = 23.3°).
is a strong indicative that unmodified starch is predominantly dispersed on the amorphous phase of PEO, for that blend ratios. For the other blend ratios slight shifts on the areas ratio were verified. This fact it might be related to miscibility of starch and PEO, as could be demonstrated in the following sections. Fig. 3 shows the WAXS patterns of PEO/cationic starch blends (3a) and PEO/hydrophobic starch blends (3b). The WAXS pattern of pure PEO is included for the sake of comparison.
Fig. 3. WAXS patterns obtained for pure PEO, PEO/cationic starch, and PEO/hydrophobic starch blends.
446
A.G.B. Pereira et al. / Materials Science and Engineering C 31 (2011) 443–451
The regions of Fig. 3A and B where the peak at 2θ=29.4° appears are magnified in Fig. 4A and B, respectively. For the PEO/cationic starch blends (Fig. 4A), it can be seen that although the intensity of the peak at 2θ=29.4° decreased as the amount of starch was increased, it was still present up to the 70/30 blend ratio. This finding suggests that the attractive forces between of the PEO and cationic starch chains do not exist, or, if they do, they are much weaker than those of pure polymer chains. Therefore, because the peak at 29.4° is present in WAXS profiles of PEO and cationic starch blends, it was inferred that the crystalline structure of PEO is not significantly changed by the presence of the cationic starch. In blends with over 30% cationic starch, the cationic starch chains may be entrapped within the amorphous regions of PEO, leading to distortions and defects in the crystalline arrangements due to the high starch concentration, and consequently, to the disappearance of the peak at 2θ=29.4°. The peak at 2θ = 29.4° was not observed for the blends constituted of PEO and hydrophobic starch, even for small quantities of starch. Based on this fact, it is reasonable to infer that the absence of the peak may be due to attractive interactions between PEO and hydrophobic starch chains. 3.2. Miscibility of PEO/starch blends through analysis of morphology by SEM
In this work, this was done by direct observation of SEM micrographs. Fig. 5 presents the micrographs of pure PEO and PEO/unmodified starch blends. By comparison of the micrographs from different blend ratios, it was possible to observe that their morphologies are very dissimilar. However, small amounts of unmodified starch (at ratio 95/05, for instance) basically do not affect the blend morphology, as compared to the morphology of pure PEO. This evidences the lack of interaction between both polymers and explains why the characteristic PEO spherulite structure remains in that blend, as previously published [37]. Higher amounts of unmodified starch in blends induce significant alteration in morphology as compared to the 95/05 blend. For instance, the characteristic morphology of pure PEO was not observed for blend ratios ranging from 90/10 to 70/30. In these blends, the density of interaction is enough to prevent the system from being completely immiscible. However, two phases similar to those of the individual components were observed in the morphologies of the 65/35 and 60/40 blend ratios. Thus, these blends were considered to be immiscible systems. Fig. 6 shows the SEM micrographs of the PEO/cationic starch and PEO/hydrophobic starch blends. In the PEO/cationic starch system, the spherulitic structure of PEO was preserved, despite the increasing amount of cationic starch. On the
Miscibility, immiscibility, and some other system characteristics, such as roughness and defects, can be evaluated through morphology analysis.
Fig. 4. WAXS patterns obtained for PEO/cationic starch and PEO/hydrophobic starch blends in the range of 2θ = 28° to 32°.
Fig. 5. SEM images of pure PEO, unmodified starch, and PEO/unmodified starch blends.
A.G.B. Pereira et al. / Materials Science and Engineering C 31 (2011) 443–451
447
Fig. 6. SEM images of PEO/cationic starch and PEO/hydrophobic starch blends.
other hand, the PEO/hydrophobic starch blends presented the opposite tendency. Morphologies distinct from that of pure PEO were obtained even using small amounts of hydrophobic starch. The difference in such behaviors can be clarified when the micrographs obtained for 90/10 and 65/35 blend ratios of each system are compared. For the PEO/cationic starch, the 90/10 and 65/35 blend ratios presented morphologies very similar to that of pure PEO, but quite different from the morphologies of the respective PEO/hydrophobic starch blends. Therefore, the characteristic PEO spherulites were not observed in the PEO/hydrophobic
starch blends, suggesting that the interaction between PEO and hydrophobic starch is enough for providing miscibility. 3.3. Miscibility of PEO/starch blends through FTIR coupled with optical microscope The use of FTIR spectroscopy in the characterization of polymer blends has been widely reported in the literature [38]. It allows identifying and quantifying specific intra- and inter-polymer chain
Fig. 7. FTIR spectra of PEO, unmodified starch, and two distinct sites of the 95/05 PEO/unmodified starch blend.
448
A.G.B. Pereira et al. / Materials Science and Engineering C 31 (2011) 443–451
Fig. 8. FTIR spectra of PEO, unmodified starch, and two distinct sites of the 90/10 PEO/unmodified starch blend.
molecular interactions. The use of FTIR-optical microscopy helps such a characterization because it allows the analysis of specific regions of the sample. In the case of polymer blends, FTIR-optical microscopy also allows the evaluation of miscibility based on the spectra obtained at specific chosen blend sites [39]. It is worth emphasizing that although the starches (unmodified, cationic, and hydrophobic) are chemically different, their FTIR spectra are not significantly different (Supplementary data, Figure S4). This suggests that although the degree of modification is small, it is enough to result in different behaviors when blended with PEO, as discussed before. The main differences in the FTIR of the starches used include CO-C and C-O-H vibrational modes (in the range of 1300–950 cm− 1). In this work, the vibrational modes of PEO and of starch in the spectra in Figs. 7–9 were assigned based on the data in Table 2 [40,41].
Fig. 7 presents the FTIR spectra of PEO, of unmodified starch, and of 95/05 PEO/unmodified starch blend obtained at distinct sites (1 and 2). It can be verified that the spectra of sites 1 and 2 are quite different with regard to the C-O-C and C-O-H vibrational modes of starch (ca. 1000 cm− 1), mainly because the band is clearly present in the FITR spectrum obtained at site 1 of 95/05 but not in the FTIR spectrum at site 2 of this blend. These results reinforce the point of view that at the ratio of 95/05, the PEO/unmodified starch blend is heterogeneous at the FTIR depth (2–5 μm) [42], indicating its immiscibility, as already proposed [37]. Fig. 8 shows the FTIR spectra of PEO, of unmodified starch and of the 90/10 PEO/unmodified starch blend obtained at two distinct sites (1 and 2). For this blend, the FTIR spectra of both sites are very similar, presenting a band at ca. 1000 cm− 1 which was attributed to the C-O-C and C-O-H
Fig. 9. FTIR spectra of PEO, unmodified starch, and two distinct sites at the 90/10 PEO/unmodified starch blends in the wave number range of 3500 to 2500 cm− 1.
A.G.B. Pereira et al. / Materials Science and Engineering C 31 (2011) 443–451
449
Table 2 FTIR vibrational modes for PEO and starch [40,41].
PEO
STARCH
Vibrational modes
Wave number (cm− 1)
O–H C–H C–O–C O–H C–H C–C, C–O and C–O–H
3490 2800–2935 1250–950 3600–3000 2930 1200–900
vibrational modes of the unmodified starch, and a broad band at ca. 2000 cm− 1, which was attributed to the asymmetric stretching of PEO. The C-H stretching for PEO and the unmodified starch that appears at ca. 2880–2890 cm− 1 are also present in the FITR of the 90/10 blend obtained at sites 1 and 2, as presented in the Fig. 9. The FTIR spectra obtained at sites 1 and 2 show a broader band related to band coalescence, which can be attributed to the C-H stretching of PEO (2879 cm− 1) and the unmodified starch (2921 cm− 1). Based on the FTIR spectra, the main features observed are that site 1 is richer in starch than site 2, while the band assigned to PEO in FTIR spectrum obtained at site 2 is weaker, considering that this blend (90/10) is nine-fold richer (in weight) in PEO than in starch. The similarity of the FTIR spectra obtained at sites 1 and 2 indicates that the blend 90/10 PEO/unmodified starch is somewhat chemically homogeneous, which confirms the miscibility of this blend ratio suggested by WAXS and SEM results. The FTIR spectra of PEO/cationic starch blends (Supplementary data, Figure S5) do not show any evidence of either interaction between the polymers, or system miscibility in the whole range of studied blend ratios. This can be noted because the FTIR spectrum obtained at a given site of the sample is very dissimilar to that obtained at another site of the same sample. On the other hand, for the PEO/hydrophobic starch blends, the FTIR spectra (Supplementary data, Figure S6) are quite similar, regardless of the site chosen. The results presented in this section are in agreement with WAXS and SEM data for PEO/cationic starch and PEO/hydrophobic starch, as discussed above. 3.4. Miscibility of PEO/starch blends through Raman imaging In this work, more attention was paid to Raman spectra in the range of 100 to 1000 cm− 1. This range contains signals attributed to the main vibrations of PEO groups (C-C-O and C-O-C bending; C-C and C-O rotation). If oxygen atoms such as those of the ether groups in PEO interact through hydrogen bonds, those vibrations must be affected, leading to shifting of the peaks and changing their intensities. In this work, Raman spectra were obtained at two distinct sites of PEO/unmodified starch blend at ratios 95/05, 80/20, and 60/40. One of these sites was the center of the spherulite, while the other was outside of the center of the spherulite, as shown in Fig. 10. For the pure PEO and unmodified starch, the Raman spectra were obtained at random sites of the samples. Fig. 11 gives the Raman spectra obtained in the center of the spherulites for the ratios 95/05, 80/20, and 60/40 of PEO/unmodified starch. For comparison, Raman spectra of PEO and unmodified starch are also included in the Fig. 11. Three interesting events are observed in the spectrum of the 80/20 blend ratio: i) widening of the main PEO peaks; ii) shifting of the peak assigned to PEO from 844 cm− 1 to 847 cm− 1; iii) appearance of new bands at 680 and at 770 cm− 1. This set of results gives enough information to infer that, at this blend ratio, PEO and the unmodified starch interact at the center of the spherulite. The presence of starch at the center of the spherulites was assigned based on the presence of the characteristic starch band at 480 cm− 1 in
Fig. 10. Micrograph showing the sample sites where the Raman spectra were obtained. The red spot is the center of the spherulite and the black spot is relative to the body of the spherulite. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
spectra obtained using PEO/unmodified starch blends at 95/05 and 60/40 ratios. Well-resolved peaks of the individual polymers were observed in the Raman spectra of such blends, and no shifting or changing in intensity was noticed. Therefore, it is suggested that there is a lack of interaction between the PEO and the unmodified starch at the center of the spherulites for the 95/05 and 60/40 blend ratios. The Raman spectra obtained out of the center of the spherulites of 95/05, 80/20, and 60/40 PEO/unmodified starch blends are presented in Fig. 12. For the 60/40 blend, the C-C stretching band at ca. 480 cm− 1 of starch is present, but with a lower intensity than that observed at the center of the spherulites (Fig. 11). Other peaks remained unchanged when compared with those in the spectra of pure polymers, indicating the absence of interaction between PEO and unmodified starch. For the 80/20 blend ratio, important changes were verified: the C-C stretching of starch shifted from 480 cm− 1 to 484 cm− 1, and the C-O-C bending of PEO shifted from 535 cm− 1 to 538 cm− 1. For the 95/ 05 blend, new bands appeared at 680 cm− 1 and 770 cm− 1, followed by the disappearance of the band at 480 cm− 1. Based on Raman spectral data, it can be concluded that PEO/ unmodified starch is immiscible at ratios of 95/05 and 60/40, while the system with the 80/20 composition is miscible. These results match those obtained by SEM, WAXS, and FITR-microscopy.
Fig. 11. Raman spectra obtained at the center of the spherulites for different compositions of PEO/unmodified starch blends (laser λ = 788 nm).
450
A.G.B. Pereira et al. / Materials Science and Engineering C 31 (2011) 443–451 Table 3 Cytotoxicity, growth inhibition (CC50/72 h) of samples against VERO cells.
Fig. 12. Raman spectra of the spherulite bodies for different compositions of the PEO/ unmodified starch blends (laser λ = 788 nm).
Sample
CC50/72h (mg/mL)
Poly(ethylene oxide) (PEO) Unmodified starch Cationic starch Hydrophobic starch 80/20 PEO/unmodified starch 80/20 PEO/cationic starch 80/20 PEO/hydrophobic starch
N 2.5 N/D* 0.49 ± 0.31 N 2.5 1.1 ± 0.98 0.93 ± 0.67 0.62 ± 0.25
It is worth mentioning that the analyses were performed two weeks after the sample preparation. It is possible that degradation had already begun at the time of analysis. As ammonium groups were grafted onto cationic starch, the explanation for its highest toxicity against VERO cells is that when the degradation process starts, some ammonia molecules, which are very harmful for the cells, are released into the medium, thus increasing the toxicity of the sample. 4. Conclusions
3.5. Mechanisms of interaction between PEO and the starches The PEO/unmodified starch system is partially miscible, as described above. When the amount of starch in the blend is small (ratio 95/05), there are preferentially intra-chain interactions. When the amount of unmodified starch is increased (90/10 to 70/30), inter-chain interactions gradually presented an important role to miscibility, probably due to H-bonds between hydroxyl groups in starch and oxygen atoms in PEO. Increasing the amount of starch even more, the stability is established by intra-chain rather than inter-chain interactions. For blends of PEO/cationic starch, the cationic groups grafted onto starch may interact at intra-molecular level. In the case of blends of PEO with hydrophobic starch, the hydrophilic H-bond interactions between hydroxyl groups of starch and oxygen atoms of the PEO's ether groups and interactions between the hydrophobic chain segments of PEO (ethyl groups) and of hydrophobic starch were also responsible for the miscibility of the PEO/hydrophobic starch system. Figure S7 (Supplementary data) depicts the probable interactions between PEO and the starches. The miscibility behavior of the different blend systems was explained based mainly on the chemical differences of starches. However, another important factor that it might have contributed to those behaviors was the molar masses of starches. It seems that the increasing molar mass of starch decreases the miscibility with PEO, taking into account that PEO/hydrophobic starch was totally miscible, PEO/unmodified starch was partially miscible and PEO/cationic starch was totally immiscible. It must be emphasized that when lyophilized directly from the aqueous solutions, PEO/unmodified starch blends at ratios of 80/20 and 65/35 presented interesting morphologies (Supplementary data, Figure S8), differing from those obtained by casting, indicating that miscibility also depends on the film production process. These morphologies are potentially suitable for use in tissue scaffolding. 3.6. Cytotoxicity assays of PEO/starch blends through VERO cells culturing The 50% cytotoxic concentrations after 72 h of incubation (CC50/72) of samples on VERO cells are shown in Table 3. The lowest cytotoxicity was observed for PEO and hydrophobic starch, which presented CC 50/72h N 2.5 mg mL − 1 , whereas for the blends containing unmodified and cationic starches, the CC50/72h were very close (1.1 and 0.9, respectively). The most harmful sample for the cells was the cationic starch, with CC50/72h = 0.49 mg mL− 1. The CC50/72h of unmodified starch was not determined due to sample contamination.
The miscibility of PEO-based blends with different starches (unmodified, cationic, and hydrophobic) was evaluated via FTIR and Raman spectroscopies, WAXS, and SEM techniques. The results revealed that the miscibility of a given system depends on the blend composition. Comparison of the different PEO/starch blends showed that in these systems miscibility depends on the type of starch. PEO/unmodified starch blends obtained by casting are miscible in the composition range from 90/10 to 65/35, since the characteristic morphology of PEO disappeared and the diffraction peak at 2θ = 29.4° was absent. These findings were attributed to the molecular interactions between the oxygen atoms of PEO and hydroxyl groups of the unmodified starch. For the other blend ratios, the unchangeable morphology of PEO/unmodified starch blends in relation to those of the individual polymers indicates the lack of interaction between the polymers. By the use of FTIR spectroscopy, it was possible to verify the presence of two phases in the 95/05 ratio blend. Its WAXS patterns presented the peak at 2θ = 29.4°, which was not observed on the WAXS pattern of the 80/20 ratio blend. Raman spectroscopy showed a lack of interactions between PEO and starch at blend ratios of 95/05 and 60/40. All studied PEO/cationic starch blend ratios cast films were immiscible. On the other hand, there is strong evidence of miscibility across the entire studied blend ratio range for the PEO/hydrophobic starch system cast films. The difference in miscibility found for the investigated systems in this work was attributed to the chemical modification of starch. Despite the fact that starches were modified only to a small degree, those modifications were enough to provoke significant changes in miscibility. The different molar masses may also have contributed to the changes in miscibility; however, this was not investigated in depth in this work. It was seen that the miscibility in PEO/unmodified 80/20 and 65/35 ratio blends also depends on the film production process. The cytotoxicity assays were performed using VERO cells. The different samples presented distinct cytotoxic behaviors. Pure PEO and hydrophobic starch presented the least relevant toxic effects quantified as CC50/72 N 2.5 mg/mL, which is a considerably high value; otherwise the cationic starch was the most harmful for the cells. The blends presented intermediate cytotoxicity, between PEO and cationic starch values. Therefore, the PEO/hydrophobic starch system could be suitable for cell culturing and tissue engineering. Acknowledgements AGBP thanks CAPES (Brazil) for the Master's scholarship. ECM thanks CNPq (Brazil, Proc. 308611/2006-3) for the financial support.
A.G.B. Pereira et al. / Materials Science and Engineering C 31 (2011) 443–451
Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10.1016/j.msec.2010.11.004. References [1] G. Scott, Polym Degrad Stab 68 (2000) 1–7. [2] X.L. Wang, K.K. Yang, Y.Z. Wang, D.Y. Wang, Z. Yang, Acta Mater 52 (2004) 4899–4905. [3] J.F. Mano, R.A. Sousa, L.F. Boesel, N.M. Neves, R.L. Reis, Compos Sci Technol 64 (2004) 789–817. [4] P. Kunal, A.K. Banthia, D.K. Majumdar, Afr J Biomed Res 9 (2006) 23–29. [5] A.P. Vieira, P. Ferreira, J.F.J. Coelho, M.H. Gil, Int J Biol Macromol 43 (2008) 325–332. [6] G.F. Mehyar, Z. Liu, J.H. Han, Carbohydr Polym 74 (2008) 92–98. [7] M. Sadeghi, H. Hoosseinzadeh, J Bioact Compat Polym 23 (2008) 381–404. [8] A.J. Salgado, O.P. Coutinho, R.L. Reis, Tissue Eng 10 (2004) 465–474. [9] S.C. Mendes, R.L. Reis, Y.P. Bovell, A.M. Cunha, C.A. Blitterswijk, J.D. Bruijn, Biomaterials 22 (2001) 2057–2064. [10] L. Boesel, R.L. Reis, Biomaterials 27 (2006) 5627–5633. [11] I. M. Katz, (Merck & Co). U. S. Patent 4,287,175, September 1, 1981. [12] B. Hoffmann, E. Volkmer, A. Kokott, M. Weber, S. Hamisch, M. Schieker, et al., J Mater Chem 17 (2007) 4028–4033. [13] J.F. Mano, D. Koniarova, R.L. Reis, J Mater Sci Mater M 14 (2003) 127–135. [14] X. Fu, X. Chen, R. Wen, X. He, X. Shang, Z. Liao, et al., J Polym Res 14 (2007) 297–304. [15] S. Godbole, S. Gote, M. Latkar, T. Chakrabarti, Bioresour Technol 86 (2003) 33–37. [16] P.X. Wang, X.L. Wu, X.D. Hua, X. Kun, T. Ying, D.X. Bing, et al., Carbohydr Res 344 (2009) 851–855. [17] K.Y. Lee, S.H. Yuk, Prog Polym Sci 32 (2007) 669–697. [18] M. Nagata, Y. Yamamoto, React Funct Polym 68 (2008) 915–921. [19] S. Salmaso, A. Semenzato, S. Bersani, P. Matricardi, F. Rossi, P. Caliceti, Int J Pharm 345 (2007) 42–50.
[20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42]
451
Y.C. Kuo, I.N. Ku, Biomacromolecules 9 (2008) 2662–2669. J. Reignier, M.A. Huneaul, Polymer 47 (2006) 4703–4717. Q. Hou, D.W. Grijpma, J. Feijen, Biomaterials 24 (2003) 1937–1947. D. Bozukova, C. Pagnoulle, M.C.P. Gillet, S. Desbief, R. Lazzaroni, N. Ruth, et al., Biomacromolecules 8 (2007) 2379–2387. H. Chen, Z. Zhang, Y. Chen, M.A. Brook, H. Sheardown, Biomaterials 26 (2005) 2391–2399. L.D. Unsworth, H. Sheardown, J.L. Brash, Biomaterials 26 (2005) 5927–5933. J.G. Archambault, J.L. Brash, Colloids Surf B Biointerfaces 33 (2004) 111–120. R.S. Lima, K.A. Khor, H. Li, P. Cheang, B.R. Marple, Mater Sci Eng A 396 (2005) 181–185. K. Balani, R. Anderson, T. Laha, M. Andara, J. Tercero, E. Crumpler, et al., Biomaterials 28 (2007) 618–624. S.C. Mendes, R.L. Reis, Y.P. Bovell, A.M. Cunha, C.A. van Blitterswijk, J.D. Bruijn, Biomaterials 22 (2001) 2057–2064. E. Cer, O.A. Gurpinar, M.A. Onur, A. Tuncel, J. Biomed, Mater Res B 80B (2007) 406–414. X. Gao, Y. Zhou, G. Ma, S. Shi, D. Yang, F. Lu, et al., Carbohydr Polym 79 (2010) 507–512. P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica, et al., Cancer Inst 82 (1990) 1107–1112. I.C. Filho, D.A.G. Cortez, T.U. Nakamura, C.V. Nakamura, B.P.D. Filho, Phytomedicine 15 (2008) 202–208. Y. Takahashi, H. Tadokoro, Macromolecules 6 (1973) 672–675. Y. Deng, J.B. Dixon, G.N. White, Colloid Polym Sci 248 (2006) 347–356. C.M. Burba, R. Frech, B. Grady, Electrochim Acta 53 (2007) 1548–1555. A.G.B. Pereira, R.F. Gouveia, G.M. Carvalho, A.F. Rubira, E.C. Muniz, Mater Sci Eng C 29 (2009) 499–504. Y. He, B. Zhu, Y. Inoue, Prog Polym Sci 29 (2004) 1021–1051. R.J. Meier, Chem Soc Rev 34 (2005) 743–752. S. Ramesh, T.F. Yuen, C.J. Shen, Spectrochim Acta A 69 (2008) 670–675. R. Kizil, J. Irudayaraj, K. Seetharaman, J Agr Food Chem 50 (2002) 3912–3918. L.A. Utracki, Polymer Alloys and Blends: Thermodynamic and Rheology, Hanser Publishers, Munich, 19898 pp 356.