Biodegradability and biocompatibility of copoly(butylene sebacate-co-terephthalate)s

1        Nina Heidarzadeh1, Mehdi Rafizadeh1*, Faramarz Afshar Taromi1, Luís J. del Valle2, Lourdes Franco2, Jordi Puiggalí2* 1Department of Polymer Engineering and Color Technology, Amirkabir University of Technology, PO Box 15875-441, Tehran, IRAN 2Departament d'Enginyeria Química, Universitat Politècnica de Catalunya, Av. Diagonal 647, Barcelona E-08028, SPAIN Correspondence to: J. Puiggalí (E-mail: [email protected]) and M. Rafizadeh (E-mail: [email protected].ir) BIODEGRADABILITY AND BIOCOMPATIBILITY OF COPOLY(BUTYLENE SEBACATE-coTEREPHTHALATE)S 2  ABSTRACT In the present study poly(butylene sebacate-co-terephthalate)s having different compositions were synthesized with a high yield and a random distribution by thermal transesterification of poly (butylene sebacate) and poly (butylene terephthalate) homopolymers. The copolymer with the highest comonomer ratio was the least crystalline sample, although the melting peaks corresponding to both, sebacate-and terephthalate-rich phases were still observable in calorimetric heating runs. This copolymer was associated with interesting thermal and mechanical properties, as the maximum melting point was higher than 100 ºC and the storage modulus was also high (i.e. 1.1 × 109 N/m2 and 1.7 108 N/m2 were determined just before and after the main glass transition temperature of -12 ºC). As all studied samples were thermally stable up to temperatures clearly higher than the fusion temperature, they could be easily processed. Increasing the terephthalate content of the copolymers resulted in higher hydrophobicity, which had a minor influence on cell adhesion and proliferation of both fibroblast-like and epithelial-like cells. Hydrolytic and enzymatic degradability were assessed and the effect of composition and crystallinity on the degradation rate was investigated. Molecular weight measurements during exposure to a hydrolytic media indicated a first order kinetic mechanism during the initial stages of degradation before reaching a limiting molecular size, which was indicative of solubilization. The most amorphous sample appears as a highly promising biodegradable material among the studied samples since it This sample not only showed the fastest a significant weight loss during exposure to all selected degradation media, but also exhibited good performance and properties that were comparable to those characteristic of polyethylene. Keywords: Poly(butylene terephthalate), poly(butylene sebacate), copolyesters, biocompatibility, hydrolytic degradation, enzymatic degradation. 3  INTRODUCTION Development of bioplastics seems to be a promising strategy to solve environmental problems due to waste accumulation and even the depletion of fossil resources. Thereby, producing biodegradable polymers from renewable resources is currently among the main goals of the society. In fact, the production of bioplastics (i.e. biodegradable or bio-based materials) has been estimated to exceed 2 million tons by 2016 [1]. Despite the great deal of interests towards biodegradable polymers, it is rather difficult to find a material with all desired specifications for application as a commodity material (i.e. low cost, easy synthesis and processing, good thermal and mechanical properties, reasonable degradation rate under both hydrolytic and enzymatic media and even in some cases low cytotoxicity). Polyesters can be generally considered as the main family of biodegradable polymers, a few of which show a good combination of properties. Polyglycolide, polylactide and poly (butylene succinate) (PBS) are the most common commercial homopolyesters. In fact, PBS is a biodegradable thermoplastic with thermal and mechanical properties closely comparable to those of the widely-used polyethylene and polypropylene [2-5]. It is well-known that biodegradability is influenced by multiple factors including surface wettability, crystallinity and chain flexibility [6]. Therefore, different strategies can be applied to control the degradation rate of a given polymer such as PBS. In this case, copolymerization, blending, formation of composites and even annealing processes have been considered [7-11]. Incorporation of aromatic units into an aliphatic polyester chain can greatly affect the mechanical properties while keeping the degradability. Recently aliphatic-aromatic polyesters have attracted a great deal of scientific attention. In particular poly butylene succinate-coterephthalate) (PBST), poly ethylene succinate-co-terephthalate) (PEST) and poly(butylene 4  adipate-co-terephthalate) (PBAT) have been produced in an industrial scale, being also investigated by different academic/research groups. In fact, PBAT has been commercialized under the trademarks of Ecoflex, Origo-Bi (previously Eastar Bio), EndPol G, Skygreen SG300 and Fepol by BASF, Novamont (previously Eastman), Samsung, SK Chemicals and Far Eastern, respectively [12], and PBST has been commercialized as Green Ecopet by Teijin [12]. Studies concerning aliphatic-aromatic copolyesters based on longer dicarboxylic acid units are surprisingly scarce. Nevertheless, Jaisankar et al. [13, 14] reported the biodegradability of suberic and sebacic acid derivatives, with a lower degradation rate compared to that related to adipate derivatives. Differences were mainly attributed to the higher crystallinity of the sebacate copolymers. The main goal of the present work was to prepare copolyesters based on 1,4-butanediol and sebacic and terephthalic acids so as to obtain materials with adequate thermal and mechanical properties as well as a good hydrolytic and enzymatic degradability. Furthermore, materials appear also interesting since 1,4 butanediol and sebacic acid can be obtained from bio-based feedstocks. Specifically, sebacic acid can be prepared by alkali fusion at high temperatures and alkali concentrations of ricinoleic acid. Unfortunately, Green synthesis of terephthalic acid has also been is still not commercial despite its previously reported. Therefore, production at a laboratory scale from isobutene or limonene, methyl coumalate, methyl pyruvate, furfural and p-cymene have been carried out [15-20]. In fact, production of a fully biobased polyethylene terephthalate from terephthalic acid derived from isobutene was launched in 2012 [21]. EXPERIMENTAL SECTION Materials 5  Terephthalic acid (TA) was supplied by Shahid Toundgoyan Petrochemical Complex (Mahshar, Iran). 1,4-Butanediol (BDO) and sebacic acid (SeA), were bought from Daejung Chemical & Metal Co. Ltd. (Shiheung, Korea). Titanium tetrabutoxide (TBT) as polycondensation catalyst was purchased from Merck Co., (Darmstadt, Germany). African green monkey kidney fibroblast-like (COS-7) and epithelial-like Madin-Darby Canine Kidney (MDCK) cells (ATCC, Manassas, VA, USA) were used in this work. Fetal bovine serum (FBS) was purchased from Gibco (Thermo Fisher Scientific Inc., Alcobendas, Spain). Lipases from porcine pancreas and Pseudomonas cepacia were purchased from Sigma-Aldrich (Barcelona, Spain). Polymerization The three selected copolymers (named as PBSeT with x denoting the molar ratio of sebacic acid units with respect to the total dicarboxylic content) were synthesized following a twostep melt polycondensation procedure based on a final transesterification between prepolymers of each dicarboxylic unit (i.e. terephthalic acid or sebacic acid) as shown in Figure 1. a) Esterification of the selected dicarboxylic unit was carried out using an excess amount of BDO (i.e. 1.7:1 [OH]:[COOH]). The reaction mixture was first stirred for 30 min at 140 ºC and under a pressure of 3-3.5 bars. A flow of N2 was provided to keep the required pressure while an electric condenser allowed separating the condensed water and the excess of diol. Reaction temperature was then increased to 200-215 ºC and 220 ºC, for sebacic and terephthalic acid derivatives, respectively. The reaction was stopped when no more water was recovered (approximately after 180 min). Reaction conversion was evaluated by weighting the recovered water at regular time intervals. b) Transesterification between the appropriate mixtures of prepolymers (i.e. 0.3:0.7, 0.5:0.5 and 0.7:0.3 molar ratios between sebacate and terephthalate derivatives) was 6  carried out using TBT as catalyst (1.4 mmol for 1 mol of dicarboxylic acid). Firstly, reaction mixture was kept for 10 min at 200 ºC and 1.5-2 bars and finally temperature was increased up to 250 ºC while applying vacuum (20 mbar). The reaction was stopped when the mixer torque reached a maximum value (approximately after 150 min). Copolymers were dissolved in 1,1,1,3,3,3,-hexafluroisopropanol (HFIP), filtered and subsequently precipitated in water and finally washed several times with water, methanol and ether; and dried in a vacuum desiccator. Measurements Molecular weights were estimated using size exclusion chromatography (GPC) through a liquid chromatograph (Shimadzu, model LC-8A, Tokyo, Japan) equipped with an Empower computer program (Waters, Milford, MA USA). A PL HFIP gel column (Polymer Lab) and a refractive index detector (Shimadzu RID-10A, Tokyo, Japan)) were utilized. The polymer was dissolved and eluted in HFIP containing CF3COONa (0.05 M) at a flow rate of 1 mL/min (injected volume of 100 μL, sample concentration of 2.0 mg/mL). The number and weight average molecular weights were calculated using polymethyl methacrylate standards. Infrared absorption spectra were recorded in the 4000-600 cm-1 range with a Fourier Transform FTIR 4100 Jasco spectrometer (Tokyo, Japan) dotted with a Specac model MKII Golden Gate attenuated total reflection (ATR) cell. 1H-NMR spectra were acquired via a Bruker AMX-300 spectrometer (Bremen, Germany) operating at 300.1 MHz. Chemical shifts were calibrated using tetramethylsilane as an internal standard. A mixture (1:1 v/v) of deuterated chloroform and trifluoroacetic acid was used as the solvent. Calorimetric data were obtained by differential scanning calorimetry with a TA Instruments Q100 series (New Castle, DE, USA) with Tzero technology and equipped with a 7  refrigerated cooling system (RCS). Experiments were conducted under a flow of dry nitrogen with a sample weight of approximately 5 mg and calibration was performed with indium. A first heating run (20 ºC/min) was performed to determine melting temperature and enthalpy, whereas a cooling run (10 ºC/min) was carried out after keeping the sample in the melt state for 3 minutes to erase the thermal history and to obtain crystallization data. Finally, a second heating run (20 ºC/min) was performed to characterize the melt crystallized sample. Thermal degradation was studied at a heating rate of 10 ºC/min with around 5 mg samples in a Q50 thermogravimetric analyzer (TGA) of TA Instruments (New Castle, DE, USA) and under a flow of dry nitrogen. Test temperatures ranged from 50 to 600 ºC. A TA Instruments DMA Q800 (New Castle, DE, USA) was used to study the dynamicmechanical properties of the materials. Prismatic rectangular samples (ca 10 × 12 × 1.3 mm3) were analyzed in single-cantilever mode at 1 Hz and 10 μm strain amplitude at 3 °C/min from -100 to 100 °C. X-ray diffraction patterns were acquired using a Bruker D8 Advance model (Bruker, Karlsruhe, Germany) with Cu Kalpha radiation (lambda = 0.1542 nm) and the geometry of Bragg–Brentano, theta–2theta. A one-dimensional Lynx Eye detector (Bruker, Karlsruhe, Germany) was employed. Deconvolution of diffraction peaks was performed with the Peak Fit v4 program by Jandel Scientific Software (San Rafael, CA, USA). Contact angles (CA) were measured at room temperature via sessile drops using an OCA15 plus Contact Angle Microscope (Dataphysics Instruments GmbH, Filderstadcity, Germany) and SCA20 software. Contact angle values of the right and left sides of distilled water and fetal bovine serum (FBS) drops were measured and averaged. Measurements were performed 10 s after the drop (0.5 µL) was deposited on the sample surface. All CA data were obtained by average of six measurements on different surface locations. 8  Degradation studies Thermally molded films were employed for different degradation studies. Polymers were heated at 10 ºC above their melting point for 2 min by means of a hydraulic press equipped with heating plates and a temperature controller (Graseby Specac, Kent, England). Pressure was progressively increased to 3-4 bars. Polymer films with a thickness of approximately 150 m were recovered after cooling the mold to room temperature and subsequently cut to the desired size. In vitro hydrolytic degradation assays were carried out in a pH 7.4 phosphate buffer (19.268 g of Na2HPO4.12H2O and 1.796 g of KH2PO4 in 1L of deionized water) at 37 ºC and under the accelerated conditions provided by raising temperature to 70 ºC. Samples (2×2 cm2 square pieces) were kept under orbital shaking in bottles filled with 20 mL of the degradation medium and sodium azide (0.03 wt-%) to prevent microbial growth for the selected exposure times. The samples were then thoroughly rinsed with distilled water, dried to constant weight under vacuum and stored over P4O10 before analysis. Degradation studies were performed in triplicate and the given data were reported according to the average values. The enzymatic studies were carried out with lipases from porcine pancreas (30–90 U/mg) and Pseudomonas cepacia (≥ 40 U/mg) using 3 replicates. All samples (1×1 cm2 square pieces) were exposed to 2 mL of phosphate buffer (pH 7.4) containing the enzyme (100 mg/L) along with sodium azide (0.03% w/v) and calcium chloride (5 mM). Solutions were renewed every week to prevent enzymatic activity loss. Samples were extracted, washed and dried as previously described. Scanning electron microscopy (SEM) was employed to examine the morphology of films after different times of exposure to the selected degradation media. Carbon coating was accomplished with a Mitec k950 Sputter Coater (fitted with a film thickness monitor k150x) (Quorum Technologies Ltd., West Sussex, UK). SEM micrographs were obtained 9  SEM micrographs were obtained with a Focus Ion Beam Zeiss Neon 40 instrument (Carl Zeiss, Oberkochen, Germany). Weight retention (Wr) of the specimens was determined by the percentage ratio of weight after degradation (Wd) to the initial weight before degradation (W0):   /  100 (1) Cell adhesion and proliferation assays COS-7 and MDCK cells were cultured in Dulbecco's modified Eagle medium (DMEM) as previously reported [22]. Square pieces (10 × 10 × 0.15 mm3) of hot-pressed films were placed and fixed in each well of a 24-well culture plate with a small drop of silicone (Silbione® MED ADH 4300 RTV, Bluestar Silicones France SAS, Lyon, France). They were then sterilized by UVradiation in a laminar flux cabinet for 15 min. The samples were stabilized for 24 h in 1 mL of medium under culture conditions. For the cell adhesion and proliferation assays, aliquots of 50–100 μL containing 5 × 104 and 2× 104 cells, respectively, were seeded onto the films in each well containing 1 mL of medium and incubated for 24 h (adhesion assay) or 7 days (proliferation assay). Samples were evaluated by the standard adhesion and proliferation methods based on a simple modification of the ISO10993-5:2009 standard test that describes the appropriate methodology to assess the in vitro cytotoxicity of the medical devices. Finally, the cellular viability on materials was evaluated through the MTT method [22]. This colorimetric assay was evaluated by means of a microplate reader (EZ Read 400 Research, Biochrom, Cambridge, UK).The study was carried out using five replicates and the results were averaged. Samples with adhered and grown cells on the mats were fixed with 16  with the increased content of sebacic acid units (Figure 9b). In contrast, a weight loss lower than 5% and 10% was determined after 60 and 300 days of degradation at 37 ºC (Figure 9a), being difficult to establish a clear differences between the different studied samples. 1H NMR spectra of samples exposed to the hydrolytic medium at 70 ºC (Figure 9c) clearly indicated that degradation mainly proceeded through the cleavage of ester groups involving sebacate units. Specifically, the relative intensity of the peak corresponding to the SeBSe sequence clearly diminished after 60 days of exposure and practically disappeared after 300 days. Note that this feature was clearly observed for the sebacate rich sample (PBSeT-70). Relative intensities of TBSe and SeBT peaks also decreased during degradation as expected from the preferred degradation of aliphatic ester linkages. In the same sense the TBT became clearly predominant in the residual product. NMR spectra showed also the appearance of well-defined triplets around 4.00 ppm that should be attributed to -CH2OH terminal groups. It is also remarkable that only slight differences were found between PBSeT-70 and PBSeT-50 samples despite the significantly different aliphatic content. Logically, degradation was lower for PBSeT-50 (i.e. weight losses of 78% with respect to 88% were found after 300 days of exposure). This small difference suggests that degradation became slightly hindered when SeBSe sequences were arranged into crystalline and less accessible domains as it is the case of PBSeT-70. Clear differences on the degradability have been found depending on the composition, being PBSeT-70 the most degradable sample. Figure 11 show the WAXD profiles of PBSeT copolymers from the films degraded for 60 and 300 days. The crystallized PBSeT-30 has the Bragg peaks at values of the scattering vector (q = 2/d) of 6.708, 11.490, 12.326, 14.610, 16.576, 17.758, 20.653 and 22.117 nm-1. Two amorphous halos at q = 14.321 and 20.896 nm-1 are required to reproduce the observed WAXD intensity. This WAXD profiles of PBSeT-30 was similar to the WAXD profiles in the heating process of PBT 17  crystallized at 188.2 ºC and it correspond to the alpha-form. However, the increase of sebacic acid units to 50% and 70% change the WAXD profiles with two Bragg peaks with high intensity at q = 14.875 and 17.260 nm-1 for PBSeT-50, and q = 14.813 and 17.114 nm-1 for PBSeT-70. However, the WAXD profiles changed during the degradation for 60 and 300 days; and finally, the WAXD profiles were similars at the end of the degradation on the 300 days. In this sense, the crystallinity of PBSeT samples were calculated as about 50% for initial samples, and as 60% and 70% at 60 and 300 days of hydrolitic degradation using a mode accelerated at 70 ºC (Figure 10). Crystallinity plays a determinant role such that the more amorphous PBSeT-50 sample shows the highest degradability despite having an intermediate composition. Results concerning the copolymers richer on terephthalate and sebacate units were somehow contradictory and reflected the inaccuracies that could be committed when only weight loss measurements were considered. In this way, as opposed to data obtained from NMR spectra, PBSeT-30 seems to be more degradable than PBSeT-70 (i.e. the weight lost levels after 60 days of exposure were 31% and 16%, respectively) (Figure 9b). Such discrepancy may be due to the different solubility of the degradation fragments. Molecular weight measurements of samples exposed to the hydrolytic medium showed a quick degradation process since only seven days were required for a decrease more than 50% of the initial number and weight average molecular weights. In this case, the slowest decrease was observed for the PBSeT-30 sample, in agreement with the expected behaviour contradicting again the conclusions from remaining weight measurements. A similar rate for the molecular weight decrease was detected for PBSeT-50 and PBSeT-70 samples, suggesting that chain breakages could take place in the lamellar folding surface causing a decrease in the molecular weight but not a remarkable change in the remaining weight since crystal entities were practically insoluble. 18  It is assumed that hydrolytic degradation of polyesters in the early stages can be simulated according to a first order mechanism that lead to an exposure time dependence of molecular weight given by equation 5 [31,32]: M = M0 e-kt (5) where M is the molecular weight at time t, M0 is the initial molecular weight and k the kinetic constant. Figure 11 shows a good fit between experimental and simulated data when the exposure time was low and a considerable deviation at times higher than 7 days. It is clear that molecular weight tended to asymptotic values that should be related with the molecular size required to get soluble fragments. Consequently, the first order approximation did not make sense at higher exposure times. Values of kinetic constants allowed quantifying the degradation rates and showed clear differences between PBSeT-30 and the other two copolymers (i.e. 0.18 days-1 in front of 0.34-0.40 days-1 from number average data and 0.20 days-1 in front of 0.270.30 days-1 from weight average data). SEM micrographs (Figure 12) clearly showed a degradation process that started through the formation of cracks on the film surface and the subsequent development of deep craters as a result of the hydrolysis of the inner material and the release of degradation products. Surfaces of exposed materials were clearly different from both the smooth texture of the initial samples as detected at low magnification and the slightly granular texture observed at high magnification. Although the initial texture of the three copolyesters was similar, clear differences could be observed between them after exposure to the hydrolytic medium for 60 days. Thus, the surface of PBSeT-30 after degradation was characterized by the presence of a limited number of holes with a reduced size while abundant, large sized and depth holes were developed in the surface of both, PBSeT-50 and PBSeT-70 samples. It is also worth noting 19  that at high magnifications the surface of these materials became rough due to the formation of multiple striations. Enzymatic degradation Copolyesters were also susceptible to the enzymatic attack of lipases as shown in Figure 13. The degradation rate was moderate but progressed steadily over the exposure time, resulted in weight losses between 8% and 16% after 60 days of exposure to a porcine lipase medium and between 14% and 25% after exposure for the same period to a Pseudomonas cepacia lipase medium. The nature of the lipase had therefore an influence on degradability, suggesting a different fitting of the active sites of the enzyme with the polymer chains. Nevertheless, the trends observed using the more aggressive enzymatic medium (Pseudomonas cepacia) indicated a higher degradation for PBSeT-50 and lower degradation for PBSeT-30 and PBSeT-70. The degradability is enhanced by the high amorphous content as it is well-known that the enzymatic attack hardly occurs in closely-packed crystalline regions. Por ello, todas las muestras estudiadas con una cristalinidad inicial muy similar (cercana al 50%) deberían mostrar la misma degradación enzimática. Por otro lado, were similar to those found for the hydrolytic degradation which resulted in higher and lower degradation rates for PBSeT-50 and PBSeT-30 samples, respectively. In the first case, degradability was enhanced by the high amorphous content as it is well-known that the enzymatic attack hardly occurs in closely-packed crystalline regions. In the second case, the slow degradation of the terephthalate-rich copolymer arises from the lower specificity of selected enzymes towards aromatic polyester substrates, the high crystallinity and the high 20  stiffness of the aromatic rich chains which hinders the location of ester groups in the active sites of the selected enzyme [33]. Morphological observations (Figure 12) of the film surfaces of samples exposed to enzymatic media demonstrated again the formation of craters and holes in an extension that was consistent with the weight loss measurements. In addition, surface roughness was clearly observed even at low magnification for the most degradable PBSeT-50 samples. CONCLUSIONS Thermal transesterification reactions between poly (butylene sebacate) and poly (butylene terephthalate) led to copolyesters with a random monomer distribution. These copolyesters were not cytotoxic as demonstrated by adhesion and proliferation of epithelial-like and fibroblast-like cell lines. The obtained aromatic-aliphatic copolyesters were thermally stable up to 320 ºC, and no significant differences were observed between samples with different composition. The copolymer constituted by a similar ratio of sebacate and terephthalate units (PBSeT-50) had the highest amorphous content. Nevertheless, crystalline domains with melting temperatures above 100 ºC were detected when samples were precipitated from solution or were slowly cooled from the melt state. The aromatic content together with the relatively high melting temperature led to materials with good mechanical properties and specifically with storage modulus values around 108 N/m2 at temperatures close to the fusion. Samples were both hydrolytically and enzymatically degradable, with a higher degradation rate for PBSeT-50 as a clear evidence of a predominant influence of crystallinity on degradability. In summary, PBSeT-50 appears to be a very interesting biocompatible material due to its high degradability and its mechanical and thermal properties comparable to those exhibited by polyethylene. 21  Acknowledgements. Authors are in debt to supports from MINECO and FEDER (MAT2015-69547-R), the Generalitat de Catalunya (2014SGR188). 22  REFERENCES [1] <http://www.european-bioplastics.org/>, 2014. [2] Fujimaki T. 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[33] Nagata M, Kiyotsukuri T, Ibuki H, Tsutsumi N, Sakai W, Synthesis and enzymatic degradation of regular network aliphatic polyesters, React Funct Polym 1996;30(1):165-171. doi:10.1016/13815148(95)00107-7 25  FIGURE CAPTIONS Figure 1. Synthesis scheme for the different studied copolyesters. Figure 2. FTIR spectrum of PBSeT-70 sample. The inset compares the methylene bands of the three copolymers when spectra were normalized with the intensity of the C=O band. Figure 3. 1H NMR spectrum of PBSeT-70 . The inset shows a magnification of the 4.70-4.25 ppm region where the OCH2 sequence sensitive signals appear. Figure 4. DSC traces corresponding to the first heating run of PBSeT-30 (a), PBSeT-50 (b) and PBSeT-70 (c) as-synthesized samples. The cooling run from the melt state of PBSeT-70 (d) and the subsequent heating run (e) are also given to show the three run protocol followed to characterize the samples as summarized in Table 3. Figure 5. Storage modulus (E’) and loss tangent curves of PBSeT-30 (solid lines), PBSeT-50 (dotted lines) and PBSeT-70 (dashed lines) copolyesters. Inset shows the small peak around 60 ºC that was observed in the loss tangent curve of PBSeT-30. Figure 6. TGA and DTGA curves comparing the thermal degradation of the three PBSeT-30 (black lines), PBSeT-50 (blue lines) and PBSeT-70 (red lines) copolyesters. Figure 7. Contact angle measurements for water (a) and fetal bovine serum (FBS) (b) onto films of PBSeT-30, PBSeT-50 and PBSeT-70. In the chart-box, the boxes represent the percentile 25%-75%, the bars represent the range 5%-95%, (x) indicate1% and 99% of the distribution, and (+) indicate the maximum and minimal values of the data. Tukey test, p<0.05 vs. PBSeT-30 (†), and vs PBSeT-70 (‡). Figure 8. SEM micrographs showing epitelial-like MDCK cells adhered on films of PBSeT30 (a), PBSeT-50 (b) and PBSeT-70 (c); arrows and asterisks point out the formation of 32  Figure 1 Heidarzadeh et al. 33  Figure 2 Heidarzadeh et al. cm-1 1000200030004000 Transmittance (%) 0 20 40 60 80 100 1712 1283 729 1220 1179 1079 2930 2850 PBSeT-30 PBSeT-50 PBSeT-70 34  Figure 3 Heidarzadeh et al. ppm 2468 8.15 4.65 4.60 2.43 2.06 1.92 1.79 1.64 1.32 1.00 2.06 0.37 1.35 1.37 2.10 4.16 3.00 0.39 0.65 0.75 1.21 4.33 4.38 T-B-Se Se-B-T Se-B-Se T-B-T B: OCH 2 CH 2 CH 2 CH 2 O HH HH B: OCH2CH2CH2CH2O B: OCH2CH2CH2CH2O T-B-T T-B-Se Se-B-T + Se-B-Se T-B-T T-B-Se Se-B-T Se-B-Se T: C6H4 COCH2CH2CH2CH2 COCH2CH2 COCH2 35  Figure 4 Heidarzadeh et al. -60 -20 20 60 100 140 180 220 Endotherm Temperature (ºC) Tm = 163 ºC Hm = 34 J/g Tm = 106 ºC Hm = 8 J/g Tm = 89 ºC Hm = 2 J/g Tm = 47 ºC Hm = 38 J/g Tm = 45 ºC Hm = 39 J/g Tm = 89 ºC Hm = 3 J/g T g = -39 ºC Tm = 42 ºC Hm = 9 J/g Tc = 24 ºC Hc = 34 J/g a) b) c) d) e) 36  Figure 5 Heidarzadeh et al. Temperature ( o C) -100 -80 -60 -40 -20 0 20 40 Storage Modulus (MPa) 500 1500 2500 3500 4500 0 1000 2000 3000 4000 5000 Tan Delta 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 40 50 60 70 80 37  Figure 6 Heidarzadeh et al.  Temperature (oC) 100 200 300 400 500 600 Weight (%) -20 0 20 40 60 80 100 Deriv. Weight (oC/%) -0.5 0.0 0.5 1.0 1.5 2.0 2.5 400 410 420 430 440 450 460 38  a) b) PBSeT-30 PBSeT-50 PBSeT-70 40 60 80 100 † † Contact Angle (  ) Water PBSeT-30 PBSeT-50 PBSeT-70 40 60 80 100 †,‡ FBS Contact Angle (  ) † Figure 7 Heidarzadeh et al. 39  Figure 8 Heidarzadeh et al. 40  300 days 60 days Initial PBSeT-30 ppm Se-B-T T-B-T Se-B-Se T-B-Se PBSeT-50 ppm Se-B-T T-B-T Se-B-Se T-B-Se PBSeT-70 ppm Se-B-Se Se-B-T T-B-Se T-B-T c) a) b) 0 50 100 150 200 250 300 0 20 40 60 80 100 Remaining weight (%) Time (days) PBSeT-30 PBSeT-50 PBSeT-70 0 50 100 150 200 250 300 0 20 40 60 80 100 PBSeT-30 PBSeT-50 PBSeT-70 Remaining weight (%) Time (days) Figure 9 Heidarzadeh et al. 41  Figure 10 Heidarzadeh et al. 300 days 60 days Initial PBSe T -70 Intensity (a.u.) q(nm -1 ) 525201510 0.772 nm 0.553 nm 0.507 nm 0.424 nm 0.367 nm 0.302 nm PBSe T -30 Intensity (a.u.) q(nm -1 ) 525201510 0.547 nm 0.510 nm 0.430 nm 0.937 nm 0.379 nm 0.304 nm 0.284 nm 0.354 nm