th P uz of I , Ira Article history: Keywords: Biodegradable poly(ether-urethane-urea)s PTMG 3,6-diisobutyl-2,5-diketopiperazine (DIBDKP) was prepared from L-Leucine with good yield. Then a new as environmental friendly polymers as well. In addition in response to public concern about the effects of plastics wastes in the envi- ronment and their damaging effects on animals and birds, it has become a widely accepted opinion that (bio)degradable polymers have a well-grounded job in solving waste problem [5,6]. amino acid based chain extender in hard segment can increase its degradability. Skarja and Woodhouse [16] reported an enzyme mediated and hydrolytic in-vitro degradation of a series of PUs containing a novel amino acid based degradable chain extender (DCE) (phenylalanine diester chain extender). They found that PUs containing these chain extenders were more susceptible to enzyme mediated degradation but not buffer-mediated hydrolysis. Diketopiperazines are the smallest cyclic peptides which are commonly biosynthesized fromamino acids by different organisms. * Corresponding author. Tel.: þ98 311 7932709; fax: þ98 311 6689732. E-mail addresses: Frafi
[email protected], Frafi
[email protected] Contents lists available at Polymer Degradat ev Polymer Degradation and Stability 97 (2012) 72e80 (F. Rafiemanzelat). Polyurethanes (PU) are present in many aspects of modern life. They represent a class of polymers that have found a widespread use in the medical, automotive and industrial fields. Polyurethanes can be found in products such as furniture, coatings, adhesives, constructional materials, fibers, paints, elastomers and synthetic skins [1,2]. A diisocyanate, a chain extender and a macrodiol (or polyol) react to form linear, segmented copolymers consisting of alternating hard segment (HS) and soft segment (SS) [3]. Currently there is a great interest on the development of biodegradable polymers suitable for a variety of biomedical appli- cations, such as temporary scaffolds or matrices for controlled drug release [4]. A large number of degradable polymers have been used important classes of degradable polymers are poly(a-hydroxy acid) s, poly(a-amino acid)s and their different block copolymers and synthetic peptide based polymers [6e8]. The synthesis of biodegradable PUs is a relatively recent issue in PU chemistry. It is well known that polyester-based PUs are much more susceptible to biodegradation than PUs derived from poly- ether diols. PUs obtained by reacting diisocyanates with poly(- caprolactone)diols of various molecular weights were treated with microorganisms and enzymes. There was a correlation between flexibility and biodegradability [9e12]. It has been shown that the HS due to urethane bonds generally is much less susceptible than ester bonds of the SS to degradation [13e15]. Incorporation of Diketopiperazine Cell-toxicity 1. Introduction 0141-3910/$ e see front matter � 2011 Elsevier Ltd. doi:10.1016/j.polymdegradstab.2011.10.009 poly(ether-urethane-urea)s (PEUU)s. These multiblock copolymers are biodegradable and thermally stable. Some structural characterization and physical properties of these polymers before and after degradation in soil, river water and sludge are reported. The environmental degradation of the polymer films was investigated by SEM, FT-IR, TGA, DSC, GPC and XRD techniques. A significant rate of degradation was occurred in PEU samples under riverwater and sludge condition. The polymeric filmswere not toxic to Escherichia coli (Gram negative), Staphylococcus aureus and Micrococcus (Gram positive) bacteria and showed good biofilm formation on polymer surface. Our results show that hard segment degraded selectively as much as soft segment and these polymers are susceptible to degradation in soil and water. � 2011 Elsevier Ltd. All rights reserved. Degradable polymers are generally achieved by incorporating labile moieties susceptible to degradation into the polymer chain. Some Accepted 10 October 2011 Available online 19 October 2011 DIBDKP with 4,4-methylene-bis-(4-phenylisocyanate) (MDI). Prepolymer reacted with poly(tetra- methylene glycol) (PTMG) with molecular weight of 1000 (PTMG-1000) to obtain a series of new Received 20 September 2011 class of biodegradable poly(ether-urethane)s (PEUs)was synthesized by the pre-polymerization reaction of Synthesis and characterization of poly(e 3,6-diisobutyl-2,5-diketopiperazine and in environment Fatemeh Rafiemanzelat a,*, Abolfazl Fathollahi Zono aOrganic Polymer Chemistry Research Laboratory, Department of Chemistry, University bDepartment of Biology, Faculty of Sciences, P.O. Box 117, University of Isfahan, Isfahan a r t i c l e i n f o a b s t r a c t journal homepage: www.els All rights reserved. er-urethane)s derived from TMG and study of their degradability a, Giti Emtiazi b sfahan, Isfahan, 81746-73441, Islamic Republic of Iran n SciVerse ScienceDirect ion and Stability ier .com/locate/polydegstab rada Cyclic dipeptides are extensively obtained by extraction from natural sources, and may also be easily synthesized. 2,5- diketopiperazines attracted attention due to their biological prop- erties [17e20]. They have also activities as anti-tumour, antiviral, antifungal, and antibacterial [18,21e27]. Some of the chemical properties of 2,5-diketopiperazines are very interesting for medic- inal chemistry, such as resistance to proteolysis, mimicking of peptidic pharmacophoric groups, substituent group stereochem- istry, conformational rigidity, and donor and acceptor groups for hydrogen bonding, which favors interactionswith biological targets [28,29]. It is expected that these cyclopeptides as important precursors prepared from amino acids may be good candidates for preparation of degradable polymers. In recent years, degradable polyester-based PUs [30e34], were studiedmore. PEUs showgoodbiocompatibility, hydrolytic stability, and good low temperature properties but their degradation rate is comparatively slow. In order to combine aforementioned properties and increase degradation rate of PEUs, in this study a new kind of (bio)degradable PEUs containing ecofriendly, degradable segments using a peptide basedmonomer was prepared. PEUs were prepared via the reaction of DIBDKP as a peptide based monomer incorpo- rating into hard segment, MDI and PTMG. The obtained polymers were characterized by 1H NMR, FT-IR, DSC, and TGA methods, and the degradation behavior of these (bio)degradable copolymers was investigated. 2. Experimental 2.1. Materials All chemicals were purchased from Fluka Chemical Co. (Buchs, Switzerland), Aldrich Chemical Co. (Milwaukee,WI), Riedel-deHaen AG (Seelze, Germany) and Merck Chemical Co. MDI (Aldrich, 98%) was used without further purification. PTMG-1000 was purchased from Merck and was dried under vacuum at 80 �C for 8 h. N,N- dimethyl formamide (DMF),1-methyl-2-pyrrolidone (NMP) (Merck, 99.5%) were distilled under reduced pressure over BaO (Aldrich, 97%). Dibutyltin dilurate (DBTDL) (Merck, 97%) and (S)-(þ)-Leucine (Merck, 99%) were used as received. Ethylene glycol (EG) (Merck, 99%) was distilled under reduced pressure over CaO (Merck, 97%). 2.2. Instruments and measurements Proton nuclear magnetic resonance 1H NMR (400 MHz) spectra were recorded on a Bruker, Avance 400 instrument in dimethyl sulfoxide (DMSO-d6) at room temperature (RT). Multipilicities of proton resonance were designated as singlet (s), doublet (d), doublet of doublet (dd), multiplet (m), and broad (br). FT-IR spectra were recorded on a Jasco FT-IR spectrophotometer. Spectra of solids were carried out using KBr pellets. Vibrational transition frequen- cies are reported in wave number (cm�1). Band intensities are assigned as weak (w), medium (m), shoulder (sh), strong (s), broad (br), stretching (st.) and bending (bend). Inherent viscosities were measured by a standard procedure using a Cannon-Fensk Routine Viscometer. Thermal Gravimetric Analysis (TGA) data for polymers were taken on a Mettler-Toledo TG-50 Thermal Analyzer under N2 atmosphere at heating rate of 20 �C/min. The initial and peak temperatures were read at the beginning and in the middle of the decomposition step obtained by TGA curve. Differential Scanning Calorimetery (DSC) data for polymers were recorded on a DSC-30/S instrument under N2 atmosphere. Glass transition temperatures (Tg) were read at the middle of the transition in the heat capacity taken from heating DSC traces. A sample was first scanned from room temperature to 200 �C and maintained for 1 min followed by F. Rafiemanzelat et al. / Polymer Deg quenching to �100 �C at a cooling rate of 10 �C/min, and then a second heating scanwas used to measure sample’s Tg of soft (Tgs) or hard segment (Tgh). A heating rate of 10 �C/min was applied to all samples. Scanning Electron Microscopy (SEM) study was per- formed with a field emission EM XL30 Philips Scanning Electron Microscope, after samples were metallized with gold. Wide angle X-ray diffraction measurements (WAXS) were carried out with a Bruker, D8advance XRD Diffractometer using a graphite mono- chromatized Cu Karadiation (40 kV; 40 mA). The number (Mn) and weight average molecular weight (Mw) and polydispersity index (PDI) of the polymer samples were determined using a gel permeation chromatography system (Manager 5000- Knauer-GPC). DMF was used as eluent at a flow rate of 0.5 mL min�1 at RT. Monodispersed polystyrene standards were used to obtain a cali- bration curve. 2.3. Synthesis of peptide based monomer 3,6-diisobutyl-2,5-diketopiperazine (DIBDKP) was prepared according to the reported procedure [35] via cyclization reaction of L-leucine (1) in dried EG at 180 �C for 10 h and recrystallization from hot ethanolewater (5/1). DIBDKP was dried under vacuum at 80 �C for 6 h to give diketopiperazine (2) in 54% yield. FT-IR Peaks (cm�1) (KBr pellet): 3317 (m) NH st., 3199 (s, br) NH st., 3091 (s) NH st., 3056(s) NH st., 2956 (s) CH st., 2923 (s) CH st., 2871 (s) CH st., 1681 (m) C]O (amide I) (non-H bonded) st., 1630 (s) C]O (amide I) (H bonded) st., 1530 (m) CeN stþNH bend (amide II),1512 (w) CeN st. þ NH bend (amide II), 1455 (s) CeN st., 1407 (m), 1386 (m), 1368 (m), 1347 (m), 1324 (s), 1261 (w) CeN st þ NH bend, 1234 (w), 1172 (w), 1142 (m), 1122 (m), 1093 (m), 899 (m), 822 (m, br), 805 (m, br) NH bend., 766 (m), 487 (m). 1H NMR peaks, DMSO-d6 at RT, d ppm: 8.01 (br, 2H, NH), 4.53 (m, 2H, CH ring), 1.81 (m, 2H, CH side chain), 1.77 (m, 4H, CH2 side chain), 1.03 (m, 12H, CH3 side chain). CHN analysis: Calculated: C% ¼ 63.7, N% ¼ 12.4, H% ¼ 9.7. Found: C % ¼ 63.61, N% ¼ 11.74, H% ¼ 9.53. 2.4. Synthesis of PEUUs block copolymers A general procedure for the preparation of PEUUs by pre- polymerization method is as follow: Into a dried 25 mL round bottom flask with an addition inlet, occupied with drying tube and N2 balloon, DIBDKP (2) (0.2 g, 8.67 � 10�4 mol) was dissolved in 0.15 mL of NMP 1% (NMP containing 1% w/w LiCl) at 120 �C then MDI (3) (0.443 g, 17.34 � 10�4 mol) was added. The mixture was heated at 120 �C for 10 min. The solution was cooled to 80 �C, and was stirred between 80e90 �C for 2 h, between 90e100 �C for 2 h and at 120 �C for 1 h. During this period appropriate amounts of NMP 1% were added upon viscosity build-up of the reaction mixture. Then it was cooled to 50 �C, and a solution of PTMG-1000 (5) (0.884 g, 8.67 � 10�4 mol) in 0.3 mL of NMP 1% was added. The temperature was gradually increased up to 80 �C during 1 h. The reaction mixture was stirred at 80 �C for 4 h, and at 100 �C for 6 h and NMP 1% was also added. The total solid content of the reaction mixture was kept at 50%W/V. Then the viscous solution of reaction mixture was poured into 15 mL of water. After vigorous grinding and stirring in water, the precipitated polymer was isolated by filtration. Additional purification was applied by re-dissolving and re-precipitation of polymer in DMF and water, respectively. The precipitated polymer was collected by filtration, dried at 80 �C for 6 h under vacuum to give 1.191 g (78%) of P1. Viscosity of the resulting polymer was 0.51 dL/g. The FT-IR (Fig. 1) and 1H NMR (Fig. 2) spectra were consistent with the assigned structure. FT-IR Peaks (cm�1): 3479 (m, br) NH st., 3321 (m, br) NH st., 3150 (m) NH st., 3052 (m) CH aromatic st., 2938 (m) CH st., 2856 (m) CH st., 2795 (m) CH st., 1772 (w, sh) C]O urethane st., 1705 (m, sh) C]O tion and Stability 97 (2012) 72e80 73 urethane-amide st., 1672 (s) C¼O urea st., 1599 (m) C]C st., 1520 (m) CeN st. þNH bend, 1509 (s) CeC st., 1460 (m) C]C st., 1416 (m) CeN st., 1400 (m) CH bend, 1385 (m), 1370 (w), 1347 (w), 1313 (m), 1236 (m) CeN st.þNH bend,1203 (m),1201 (w), 1180 (w), 1144 (w), 1117 (m) CeOeC ether st., 1042 (m) O]CeOeC st., 1018 (w), 917 (w), 850 (w), 808(w) NH bend, 764 (w) O]CeO, 663 (w), 509 (w), 482 (w). 1H NMR peaks, DMSO-d6 at RT, d ppm: 0.91 (d, CH3(1), 6H), 1.5 (t, CH2(2), 2H), 1.8 (m, CH(3), 1H), 2.7 (m, CH2(4), 4H), 2.71 (m, CH2(5)), 2.9 (t, CH2(6), 2H), 3.8 (t, CH2(7), 2H), 3.9 (s, CH2(8), 2H), 4.4 (t, CH(9), 1H), 7.1 (CH(10), 4H), 7.4 (CH(11), 4H), 7.9 (br, NH), 8.15(br, NH), 8.6 (br, NH). months. Specimens withdrawn from the containers at regular intervals washed with distilled water and dried under vacuum at 50 �C. Then they were thoroughly characterized with various methods including the determination of weight changes, functional group content (FT-IR), molecular weight (GPC), thermal (DSC, TGA), and morphological (SEM, XRD) properties. 2.5.2. Degradation test in river water PEUU filmwas immersed in riverwater in one container together with a reference sample (an empty inert plastic tea bag). The water was obtained from Zayand-e- Rood River in Isfahan. The tests samples were allowed to stand at room temperature for 4 months. Every 15 days the film was removed from container washed with distilled water, dried under reduced pressure, and weighted. 2.5.3. Degradation test in activated sludge Degradation test in activated sludgewas carried out in a manner similar to the procedure described above by using an activated sludge instead of river water. The activated sludge was obtained from Municipal waste water in Isfahan. The container containing immersed film was allowed to stand at room temperature for 4 months. Every 15 days thefilmwas removed fromcontainerwashed with distilled water, dried under reduced pressure, and weighted. 2.5.4. Soil burial degradation test PEUU filmwas buried in soil enriched with plant fertilizer in one Fig. 1. FT-IR spectra of PEUU (KBr). F. Rafiemanzelat et al. / Polymer Degradation and Stability 97 (2012) 72e8074 2.5. Degradation study 2.5.1. Film preparation PEUU films were cast fromNMP solution of polymers intoTeflon moulds. The films were dried in vacuum at 50 �C prior to the experiments and cut into strips to dimensions of 10 mm � 10 mm � 0.2 mm. Polymer mass was determined using a Mettler Toledo AB204-S Classic balance. Samples were stored in plastic bags in a desiccator prior to in-vitro degradation tests then immersed or buried in selected environment using tea bag method. Polymer samples were stored in river water, soil, and sludge for more than 4 Fig. 2. 1H NMR (400 MHz) spectru container together with a reference sample (an empty inert plastic tea bag). The relative humiditywas kept ca. 80% byMunicipal water. The container was allowed to stand at room temperature for 4 months. Every 15 days the filmwas removed from containerwashed with distilled water, dried under reduced pressure, and weighted. 2.5.5. Mass loss measurement Every 15 days PEUU films were removed from their container and were placed in water to remove environmental impurities. Then placed under nitrogen and subsequently vacuum for 24 h until a constant weight was reached. Post-degradation weight was m of PEUU in DMSO-d6 at RT. measured and mass loss of each polymer sample was obtained using the following formula: Mass lossð%Þ ¼ ðMt1 �Mt2=Mt1Þ � 100 (1) whereMt1 is the pre-degraded dry weight of the polymer andMt2 is the dry weight of the sample after degradation, each time. 2.5.6. Study of toxicity effect of polymer film on alive microorganism cells The toxicity effect of polymer was studied on Gram negative and the final pH was 7.2} supplemented with PEUU. All slides were thoroughly cleaned with sterile de-ionized water and sterilized with 70% ethanol, sonicated for 5 min, and rinsed in sterile de- ionized water three times. After 24e72 h growth of bacteria was investigated under microscope. 2.5.7. Investigation of the effect of polymer film as nutrition source for bacteria Some selected bacteria grew in test tubes that have a medium comprised a basal medium {0.2% KH2PO4; 0.7% K2HPO4; 0.1% (NH4)2SO4; 0.01% MgSO4$7H2O; 0.0001% ZnSO4$7H2O; 0.00001% CuSO4$7H2O; 0.001% FeSO4$7H2O; 0.0002% MnSO4$H2O; the final pH was 7.2} supplemented with PEUU as the only carbon source for bacteria growth. The test tubes were incubated at 37 �C. After 24e72 h and after one week, the growth of bacteria was investi- gated under microscope. 2.5.8. Water uptake experiments Water uptake studies were conducted by immersing solution- cast polymer films (0.05 g) into 25 ml of de-ionized water kept for 1, and 4 days in an incubator at 37 �C. The samples were removed fromwater at predetermined time intervals, wiped gently with filter paper, and weighed with an analytical balance. The sample mass change resulting from the water uptake (expressed as a percentage) was calculated according to the following formula: C NH2 CO2H CH2 H CH CH3H3C HOCH2CH2OH CH2CH(CH3)2 N N OO R H R H H H R= (1) (2) Scheme 1. Preparationof3,6-diisobutyl-2,5-diketopiperazine (DIBDKP) fromL-leucine (1). F. Rafiemanzelat et al. / Polymer Degradation and Stability 97 (2012) 72e80 75 positive bacteria. For this, some selected bacteria were grown in Petri dishes on Nutrient agar. Once the growth medium in the Petri dish was inoculated with the desired bacteria, the plates were incubated at 37 �C in the presence of small pieces of polymer films (small discs with a diameter of 5 mm). Nutrient agar-based growth medium typically contains a carbon source for bacterial growth, such as glucose and various salts which generally provide essential elements such as magnesium, nitrogen, phosphorus, and sulfur to allow the bacteria to synthesize protein, nucleic acid, and etc. The medium comprised a basal medium {0.2% KH2PO4; 0.7% K2HPO4; 0.1% (NH4)2SO4; 0.01% MgSO4$7H2O; 0.0001% ZnSO4$7H2O; 0.00001% CuSO4$7H2O; 0.001% FeSO4$7H2O; 0.0002% MnSO4$H2O; N N OO R H R H H H CH2OCN O (2) + 2 (3) H O C H2 OH C N N OO R H R H C C N H N H O ArCH2A ArH2CArOCN AN H COH2COH2C O m m (5) 4 44 Scheme 2. Preparation of PEUU by two step polymeri Water uptake% ¼ ðmw �md=mdÞ � 100 (2) where md and mw are the masses of dry and wet samples, respectively. 3. Results and discussion 3.1. Monomer and polymer synthesis DIBDKP as an amino acid based monomer was prepared via heating L-leucine(1) in dried EG and recrystallization from hot ethanol (Scheme 1). Then DIBDKP as a preformed amide containing NCO r NH C O N N O O R H R H C N H O ArCH2Ar NCO Ar C BC C N H ArCH2Ar NH C ON H rH2CAr OOO B NMP/Catalyst n' n (4) (6) zation reaction of MDI, DIBDKP and PTMG-1000. F. Rafiemanzelat et al. / Polymer Degradation and Stability 97 (2012) 72e8076 heterocyclic monomer was reacted with MDI diisocyanate (3) to establish NCO terminated hard segment containing urea linkage (4). The chemical structure and purity of the resulting compound (DIBDKP) were confirmed with FT-IR, NMR, CHN, melting point measurement and thin layer chromatography (TLC). The PEUUmultiblock copolymers were synthesized according to Scheme 2 by the two-step method. The effect of polymerization conditions such as reaction time and temperature, reaction solvent, reaction catalysts and soft segment length on the viscosities and yields of the resulting PEUUswas studied in our previous work [36]. Our studies showed these polymers based on polyether polyols and cyclopeptide were hydrolysable. In additions it was shown that soft segment length, composition and morphology of PEUUs play an important role on the degradability of the polymers. Thus investi- gation of the physical, chemical and degradation properties of these PEUUs with respect to their compositionwould be important in the further considerations about their environmental degradation and proper applications as degradable materials. We continue our studies on these kinds of polyether based PUs to investigate the Fig. 3. The percentage weight loss of PEUU vs. exposure time during soil burial, river water and activated sludge degradation tests. effect of changes in soft segment type on the physical and degra- dation properties of these PEUUs. Here we have presented part of our studies on PEUUs based on PTMG as soft segment and represent their properties before and after degradation tests. 3.2. In-vitro degradation tests; study of changes in PEUUs properties 3.2.1. Weight loss study Degradation tests were carried out in accordance with methods outlined in experimental section. The following data represent the percentage of mass loss vs. times during exposure to different Fig. 4. Scanning electron micrographs of PEUU surface before (A) and after expo environments (Fig. 3). Degradation curves of polymer samples show relatively the same behavior. In the first step, after an initial induction period for about 5e10 days, the weight loss percentage values of PEUUs increased with exposure time during the first 30 days. This could be due to degradation of amorphous regions of polymer samples. Then curves show slow degradation rate after 30 days up to 90 days (second degradation step). Slow degradation rate during step 2 may be due to crystalline phase in PEUUs. After 90 days, degradation curves during step (3) continued with a decreased slop which may be due to inter-connected hard segment region. Percentage weight loss values of PEUUs increased with exposure time and varied in the range of 8e16%, 14e18%, and 16e20% during degradation steps 1e3, respectively. The hydro- philicity of PEUU allows microorganisms to penetrate the polymer matrix and induce bulk erosion of PEUUs based on PTMG -MDI- 2,5-diketopiperazine. Aqueous media such as river water and sludge show higher degradation rate. This can be due to their fluid character and easy penetration of water together with organisms into the polymer bulk. Traditionally, it is believed that amorphous phase is more susceptible than crystalline phase to degradation. It was shown that the degradation of PUs and their copolymers depends on their chemical structure, molecular weight, composition and morphology of polymers, soft segment or hard segment length, phase concentration and cystallinity. Usually PU hard segments provide materials with extra strength due to hydrogen bonding involving urethane and polar groups. Thus hard segment generally degrades slower than soft segment. However, a crystalline soft segment degrades slower than an amorphous soft segment. Thus it Fig. 5. WAXS spectra of PEUU before (a) and after (b) exposure to Soil for four months. can be said that degradation rate can be in the order of: amorphous region of soft segment > crystalline region of soft segment > hard segment. It can be said that ether, urethane and peptide linkages in soft and hard segments of polymer chains may be the points that are susceptible to in-vitro degradation and depending on their inter-chain cohesiveness they are attacked bymicroorganisms with different rate. sure to River Water (B), Activated Sludge (C), Soil (D) at RT for four months. F. Rafiemanzelat et al. / Polymer Degradation and Stability 97 (2012) 72e80 77 3.2.2. Scanning electron microscopy study (SEM) Samples were examined under a SEM before and after exposure to degradation environments. It can be seen that under SEM, polymer surface show more holes and damaging splits after degradation for 4 months (Fig. 4). Fig. 4 provides evidences for progress of degradation process through polymer surface in different degradation environments. 3.2.3. WAXS studies WAXSmeasurementswere carried out for PEUUs before and after degradation tests. The percentage of crystallinity was calculated Fig. 6. DSC trace of PEUU at heating rate of 10 �C/min in N2 before (a) and after (b) exposure to soil for four months. throughgraphical integration of the diffracted intensity data in the 2q range 5e60� and subtraction of the amorphous scattering band intensity. Diffraction patterns for PEUU based PTMGeMDIeDIBDKP show three main crystalline regions A, B and C. Fig. 5 presents and comparesWAXS spectra of PEUU before (a) and after (b) exposure to soil for four months. WAXS of PEUU before exposure to soil shows three crystal reflection patterns A, B and C with maximum at about 2q¼ 6.5,19 and 22�, respectively,whileWAXSof PEUUafter exposure to soil shows two reflection patterns A and B. It is suggested that the crystalline phase which develops at A region is associated with crystallization of MDIeDIBDKP hard segment. Crystalline phase which develops at B region can be due to hard segment togetherwith Table 1 Thermal stability and thermal properties data of PEUU before and after exposure to soil Char yield%h T10 (�C)g T5 (�C)f T after before after before after before a 16 12 365 347 352 290 1 ( a Polymer sample based on PTMG1000eMDIeDIBDKP. b Glass transition temperature of soft segment before and after degradation. c Glass transition temperature of hard segment and/or melting temperature of crystal d Data were read at the middle of the transition taken from the heating DSC traces at e Data range were read at the beginning and at the end of the transition taken from t f Temperature at which 5% weight loss was recorded by TGA at heating rate of 20 �C/ g Temperature at which 10% weight loss was recorded by TGA at heating rate of 20 �C h Percentage of weight residue at 600 �C recorded by TGA at heating rate of 20 �C/min PTMG including MDIePTMG, and MDIeDIBDKPePTMG crystalline regions. Crystalline phase which develops at C region can be associ- ated with soft segment PTMG-PTMG crystalline region. Comparing with curve (a), in curve (b) the region A shows a drastic decrease in percentageof crystallinity, the regionB is changed toabroaddiffusion scattering (amorphous halo) with maximum at 2q ¼ 6.5 and 21� respectively and the region C is vanished. Diffraction patterns ob- tained for PEUU before exposure to soil show 9.8%, 31.6% and 8.4% of crystallinity at A, B and C regions, respectively. Diffraction patterns obtained for PEUU after exposure to soil show 2.3% crystallinity at A region, B region has changed to an amorphous pattern and C region has been removed. As it was mentioned before, hard segments generally degrade slower than soft segments andamorphousphase is more susceptible than crystalline phase to degradation. Interestingly comparing the percentage of crystallinity of polymers before and after exposure to soil shows that the total percentage of crystallinity of polymers inA, B andC regions decreased afterdegradation test and some crystalline regions in polymer has been disappeared giving us an idea about degradation of crystalline phase together with amor- phous phase. It can be inferred that not only physical washing out of polymer chains and hydrolytic attack play a role for polymer degra- dation but also a selective degradation preferably has been occurred in crystalline regions of polymers affecting both soft and hard Fig. 7. Comparing TGA thermograms of PEUU before and after exposure to soil for four months under N2 atmosphere at heating rate of 20 �C/min. segments. River water and sludge degradation tests also show the same results to some extent. This could be related to the effect of microorganisms which are present in these environments. Although slower than amorphous soft segment, crystalline hard segment has been affected by physical, chemical and biological degradation agents. for four months. gh (Tm) (�C)c Tgs (�C)b Polymera fter before after before 75d 170d 52d 50d P-PTMG 160e210)e (150e200)e 32e80e 35e70e line microdomains of hard segment before and after degradation. heating rate of 10 �C/min. he heating DSC traces at heating rate of 10 �C/min. min in N2 before and after degradation. /min in N2 before and after degradation. in N2 before and after degradation. ΔTg ¼ Tgh � Tgs of PEUU are higher than those of PEUU before degradation test (Table 1). The above mentioned transitions of PEUU after test show higher depression than those of PEUU before test. The results indicate that the residual sample show more inter-chain interactions due to higher remaining hard segment contents than soft segment because of faster degradation and washing out of soft segment and because of remaining higher molecular weight chains more than short chains (GPC confirm this result). DSC results show that Tgh of PEUU after test is higher than Tgh of the respective PEUU before test. This can be due to themore contribution of hard segment domainswhich decreases chainflexibility and consequently increases Tg. TGA thermogram of PEUU after degradation test show increasing of its initial thermal stability and char yield % as well (Fig. 7). This confirmed the above mentioned conclusions about increasing contribution of hard segment content, more inter-chain interactions and average molecular weight of residual polymer in F. Rafiemanzelat et al. / Polymer Degradation and Stability 97 (2012) 72e8078 Our previous study showed that the percentage of crystallinity of polymers increased after hydrolytic degradation test giving us an idea about washing out amorphous phase more than crystalline phase. This conclusion was confirmed by thermal studies which showed increasing of char residue for each degraded polymer sample due to increasing their hard segment contents [36]. Ob- tained results in current study confirm that both soft and hard segments, amorphous as well as crystalline regions have been degraded. Decreasing polymer crystallinity after tests shows the Fig. 8. Comparing FT-IR spectra of PEUU before (a) and after (b) soil burial degradation test for four months. effects of biological and environmental agents in addition to hydrolytic, chemical and physical degrading agents on polymer degradation. 3.2.4. Thermal study Thermal properties of PEUU before and after degradation tests were also evaluated with TGA and DSC techniques. Fig. 6 presents and compares thermal behavior of selected PEUU samples before and after soil burial degradation test. It can be seen that after four months exposure to soil Tgs, Tgh (Tm) and Fig. 9. Light microscopy of PEUUs films after exposure to the E. coli and Staphylococcus au aureus, B: polymer in E. coli cell culture plates). comparison with PEUU sample before test. Resulting data obtained from other PEUU specimens exposed to river water and sludge are to some extent similar. DSC and TGA data of PEUUs show that these polymers are thermally stable and processable. Their T5% and T10% are about 300 and 370 �C, respectively and their Tg and Tm are below their decomposition temperatures. 3.2.5. FT-IR studies Fig. 8 shows the FT-IR spectra of PEUU before and after soil burial degradation test. After four months exposure, the spectra show reduction, elimination or changes in absorption bands of NeH, C]O, CeN, CeO, CeH aliphatic and aromatic bonds at 3400, 3100, 2930, 1672, 1446, and 1100 cm�1 as well as changes in the finger print area (below 900 cm�1) of FT-IR spectra of polymer sample. This indicates that the chemical structure of the polymer changed considerably after degradation, mainly due to the hydro- lytic and enzymatic degradation of polymer chains. These indicate possible breaks or changes of the urethane, ether and peptide groups. 3.2.6. Study of toxicity and nutrition effect of polymer films on microorganism cells The toxicity and nutrition effects of copolymers films were evaluated by incubating the Escherichia coli, Staphylococcus aureus and Micrococcus bacteria in the presence of copolymer films over a period of 24e72 h or 7 days at 37 �C. First, formation of inhibition zone of growth on Nutrient agar- based medium in the presence of copolymer films was studied. reus Bacteria after a period of 24 h in cell culture plate (A: polymer in Staphylococcus Fig. 10. SEM micrographs of biofilm formation of bacteria on polymer surface after 7 F. Rafiemanzelat et al. / Polymer Degrada The aim of this experiment was to determine the potential toxi- cological hazard of the copolymer films on bacteria. The copolymer films do not show toxicity against above bacteria. The absence of inhibition zone and dead bacteria around prepared polymeric films showed nontoxic behavior of this polymer. Fig. 9 shows the poly- mer films in contact with the bacteria cells. On agar surface, toxic components in the sample discs are able to diffuse into the culture medium, forming a concentration gradient and adversely affecting cells at varying distances from the sample disc. As shown in the Fig. 9, there is not any inhibition zone around the PEUU samples. Second test included study of growth of these bacteria in a growth medium comprising a basal medium and copolymer films as the only carbon source for bacterial growth. Fig. 10 shows biofilm formation on polymer surface by Micrococcus. After initial attach- days of incubation at 30 �C and 60% relative humidity. ment of microorganisms to surfaces of polymer, their growth can form extensive biofilms, and damage to the materials. Fig. 11. GPC diagrams of PEUU before (A) (Mw ¼ 11.485 � 103, Mn ¼ 8.800 � 103) and after degradation in soil for four months (B) (Mw ¼ 15.904 � 103, Mn ¼ 11.400 � 103). 3.2.7. Water uptake study As these PEUUs are designed to perform and degrade in bio- logical environments, the amount of penetrated water molecules into the bulk of polymers plays an important role on their hydro- lytic and microbial degradation. Data of water uptake percent as a function of time show that the equilibrium water uptake increases with increasing time and reaches constant value after 72 h. This behavior is due to the new hydrogen bonding interaction between water molecules and PTMG segments or polar hard segments of PEUU. Water uptake percentages were 37.9% and 57.47% after 24 h and 72 h, respectively. 3.2.8. GPC analysis The weight average molecular weight ð �MwÞ, number average molecular weight ð �MnÞ and polydispersity index (PDI) of the PEUU samples were determined using a gel permeation chromatography system before and after degradation test. A typical GPC chro- matograph of PEUU sample shows to some extent a bi-modal peak (a, bec) in GPC chromatograph before degradation test, corre- sponding to molecular weights of about 9000, 7000e3000 (Fig. 11- A). The GPC profile after degradation shows a multi-modal profile resulting in increasing PDI which comprises of at least four different components (d, e, f, g) corresponding to molecular weights of about 15 000,13 000, 7000 and 2000 (Fig.11-B). It can be inferred from the multi-modal GPC profile, PEUU typically degraded within four months breaking down into shorter frag- ments (f, g). Thus PEUU backbone consists of the hydrolytically and biologically degradable bonds. However, GPC peak profile of PEUU after four months exposure to soil shows a shift to higher average molecular weights comprising of hydrolytically and biologically resistant components which can be related to crystalline regions of polymer residue (Fig.11-B, e, d). Also it can be inferred that very low molecular weight of polymer chains resulting from chain braking have been washed out and eliminated from polymer bulk. This has been confirmed by TGA and DSC results which showed increasing of thermal stability and thermal transition temperatures. 4. Conclusion Poly(ether-urethane)s were synthesized through the reaction of PTMG-1000, MDI and a 2,5-diketopiperazine as peptide based hard segment in NMP. In-vitro degradation tests were carried out in soil, river water and activated sludge. The changes in physical and morphological properties of PEUUs and their percentage weight loss were significant. DSC, XRD, FT-IR and SEM data proved that the chemical structure and morphological feature of PEUUs changed after environmental degradation tests. XRD study confirms selec- tive degradation of hard segments and crystalline domains by microorganisms. The SEM micrographs of degraded polymer films showed large number of small holes, cracks, cavities and surface irregularities indicating that the surface of polymerwas attacked by the hydrolytic and biological agents. 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Synthesis and characterization of poly(ether-urethane)s derived from 3,6-diisobutyl-2,5-diketopiperazine and PTMG and study ... 1 Introduction 2 Experimental 2.1 Materials 2.2 Instruments and measurements 2.3 Synthesis of peptide based monomer 2.4 Synthesis of PEUUs block copolymers 2.5 Degradation study 2.5.1 Film preparation 2.5.2 Degradation test in river water 2.5.3 Degradation test in activated sludge 2.5.4 Soil burial degradation test 2.5.5 Mass loss measurement 2.5.6 Study of toxicity effect of polymer film on alive microorganism cells 2.5.7 Investigation of the effect of polymer film as nutrition source for bacteria 2.5.8 Water uptake experiments 3 Results and discussion 3.1 Monomer and polymer synthesis 3.2 In-vitro degradation tests; study of changes in PEUUs properties 3.2.1 Weight loss study 3.2.2 Scanning electron microscopy study (SEM) 3.2.3 WAXS studies 3.2.4 Thermal study 3.2.5 FT-IR studies 3.2.6 Study of toxicity and nutrition effect of polymer films on microorganism cells 3.2.7 Water uptake study 3.2.8 GPC analysis 4 Conclusion Acknowledgements References