Alma Mater Studiorum – Università di Bologna DOTTORATO DI RICERCA IN CHIMICA INDUSTRIALE Ciclo XXV Settore Concorsuale di afferenza: 03/B2 Settore Scientifico disciplinare: CHIM/07 Novel etheroatom containing aliphatic polyesters for biomedical and environmental applications Presentata da: Matteo Gigli Coordinatore Dottorato: Relatore: Chiar.mo Prof. Fabrizio Cavani Chiar.ma Prof.ssa Nadia Lotti Correlatore: Chiar.mo Prof. Andrea Munari Esame finale anno 2013 Abstract Biodegradable polymers for short time applications have attracted much interest all over the world. The reason behind this growing interest is the incompatibility of the polymeric wastes with the environment where they are disposed after usage. Synthetic aliphatic polyesters represent one of the most economically competitive biodegradable polymers. In addition, they gained considerable attention as they combine biodegradability and biocompatibility with interesting physical and chemical properties. In this framework, the present research work focused on the modification by reactive blending and polycondensation of two different aliphatic polyesters, namely poly(butylene succinate) (PBS) and poly(butylene 1,4-cyclohexanedicarboxylate) (PBCE). Both are characterized by good thermal properties, but their mechanical characteristics do not fit the requirements for applications in which high flexibility is requested and, moreover, both show slow biodegradation rate. With the aim of developing new materials with improved characteristics with respect to the parent homopolymers, novel etheroatom containing PBS and PBCE-based fully aliphatic polyesters and copolyesters have been therefore synthesized and carefully characterized. The introduction of oxygen or sulphur atoms along the polymer chains, by acting on chemical composition or molecular architecture, tailored solid-state properties and biodegradation rate: type and amount of comonomeric units and sequence distribution deeply affected the material final properties owing, among all, to the hydrophobic/hydrophilic ratio and to the different ability of the polymer to crystallize. The versatility of the synthesized copolymers has been well proved: as a matter of fact these polymers can be exploited both for biomedical and ecological applications. Feasibility of 3D electrospun scaffolds has been investigated, biocompatibility studies and controlled release of a model molecule showed good responses. As regards ecological applications, barrier properties and eco-toxicological assessments have been conducted with outstanding results. Finally, the ability of the novel polyesters to undergo both hydrolytic and enzymatic degradation has been demonstrated under physiological and environmental conditions. Table of Contents 1. Introduction 1 1.1 Aliphatic polyesters 5 1.1.1 Synthesis 6 1.1.1.1 Polycondensation 7 1.1.1.2 Ring-opening polymerization 12 1.1.2 Blending 15 1.1.2.1 Reactive blending 18 1.1.3 Physical properties 19 1.1.4 Degradation 20 1.1.4.1 Chemical hydrolysis 21 1.1.4.2 Enzymatic hydrolysis 23 1.1.4.3 Factors influencing hydrolysis 24 1.2 Copolymers 26 1.2.1 Random copolymers 27 1.2.2 Block copolymers 30 1.3 Biomedical applications 32 1.3.1 Tissue engineering 33 1.3.1.1 Electrospinning 37 1.3.2 Controlled drug release 39 1.3.3 Polymers used in biomedical applications 44 1.4 Environmental applications 47 1.4.1 Packaging 49 1.4.1.1 Starch-based polymers 51 1.4.1.2 Polyesters 51 1.4.2 Agricultural applications 55 2. Aim of the work 59 3. Materials and Methods 63 3.1 Materials 64 3.2 Synthesis of homopolymers 64 3.3 Synthesis of copolymers 66 3.3.1 Polycondensation 66 3.3.2 Reactive blending 67 3.4 Film preparation 69 3.5 Scaffold fabrication 69 3.6 Molecular characterization 70 3.6.1 Nuclear magnetic resonance (NMR) 70 3.6.2 Gel permeation chromatography (GPC) 71 3.7 Thermal characterization 71 3.7.1 Thermogravimetric analysis (TGA) 71 3.7.2 Differential scanning calorimetry (DSC) 72 3.8 Wide-angle X-ray measurements (WAXD) 72 3.9 Mechanical characterization 73 3.10 Surface wettability 73 3.11 Hydrolytic degradation tests 73 3.12 Enzymatic degradation tests 73 3.12.1 Opacity assay 74 3.12.2 Attenuated total reflectance infrared spectroscopy (ATRIR) 75 3.13 Soil burial experiments 75 3.14 Composting 76 3.15 Film/scaffold weight loss analyses 76 3.16 Scanning electron microscopy (SEM) 76 3.17 Barrier properties evaluation 77 3.18 Ecotoxicity assessment 77 3.19 Biocompatibility evaluation 78 3.19.1 P(BCEmBDGn) biocompatibility studies 78 3.19.1.1 Cell culture 78 3.19.1.2 Cell viability to materials 79 3.19.1.3 Confocal Laser Scanning Microscopy (CLSM) 79 3.19.2 P(BCEmTECEn) biocompatibility studies 79 3.19.2.1 Cell culture 79 3.19.2.2 MTT assay 80 3.19.3 PBS and P(BS80BDG20) biocompatibility studies 80 3.19.3.1 Cell culture 80 3.19.3.2 Cell Viability Assay 80 3.20 In vitro FITC release experiment 80 4. Results and discussion 83 4.1 Enzymatic hydrolysis studies on PBS PDGS and PBS PTDGS block copolymers m n m n 84 4.1.1 Synthesis and characterization of the polymers 84 4.1.2 Screening of the degrading hydrolytic enzymes 88 4.1.3 Optimization and selection of the biodegradation test conditions 89 4.1.4 Biodegradation studies 90 4.1.5 Conclusions 97 4.2 Environmentally friendly PBS-based copolyesters containing PEG-like subunit: effect of block length on solid-state properties and enzymatic degradation 97 4.2.1 Synthesis and molecular characterization of the polymers 98 4.2.2 Thermal properties and crystallization ability 100 4.2.3 Mechanical characterization and wettability behaviour 107 4.2.3 Enzymatic degradation 109 4.2.4 Comparison between PBSTES and PBSPDGS copolymers 113 4.2.5 Conclusions 114 4.3 Synthesis and characterization of novel PBS-based copolyesters designed as potential candidates for soft tissue engineering 115 4.3.1 PBS and PBDG homopolymers characterization 115 4.3.2 PBSPBDGt copolymers synthesis and molecular characterization 117 4.3.3 PBSPBDGt copolymers thermal characterization 120 4.3.4 PBSPBDGt copolymers mechanical characterization 129 4.3.4 Conclusions 130 4.4 Macromolecular design of novel sulphur-containing copolyesters with promising mechanical properties for soft tissue engineering 131 4.4.1 PBTDG homopolymer characterization 131 4.4.2 Solution cast blends 132 4.4.3 PBSPBTDGt block copolymer synthesis and molecular characterization 134 4.4.4 PBSPBTDGt copolymers thermal characterization 137 4.4.5 PBSPBTDGt copolymers mechanical characterization 143 4.4.6 Electrospinning of PBSPBDG and PBSPBTDG copolymers 145 4.4.7 Hydrolytic degradation 147 4.4.8 Cell morphology and viability 152 4.5 Novel random copolyesters of poly(butylene succinate) containing ether-linkages 154 4.5.1 P(BSxBDGy) synthesis and molecular characterization 154 4.5.2 P(BSxBDGy) thermal characterization 155 4.5.3 P(BSxBDGy) copolymers mechanical characterization 163 4.5.4 Electrospinning of PBS and P(BS80BDG20) 164 4.5.5 Characterization of PBS and P(BS80BDG20) scaffolds 165 4.5.6 Hydrolytic degradation 167 4.5.7 Biocompatibility assay 169 4.6 Random copolyesters based on poly(butylene 1,4-cyclohexanedicarboxylate) containing ether-oxygen atoms 170 4.6.1 Synthesis, molecular and thermal characterization 171 4.6.2 Mechanical characterization 181 4.6.3 Barrier properties 182 4.6.4 Enzymatic and hydrolytic degradation studies 185 4.6.5 In vitro fluorescein isothiocyanate (FITC) release 189 4.6.6 Biocompatibility assay 191 4.7 Random PBCE-based copolyesters containing PEG-like subunit 194 4.7.1 Synthesis, molecular and thermal characterization 194 4.7.2 Mechanical characterization 201 4.7.3 Barrier properties 202 4.7.4 Soil burial and composting studies 205 4.7.5 Ecotoxicity assessment 207 4.7.6 Electrospinning of P(BCEmTECEn) copolymers 208 4.7.7 Hydrolytic degradation 209 4.7.8 Biocompatibility 210 5. Conclusions 213 References a Publications g Scientific contributions to national and international congresses h List of Abbreviations 2-CE: 2-chloroethane A: crystalline area of the diffraction pattern c A: initial activity i A: total area of the diffraction profile t ATRIR: attenuated total reflectance infrared spectroscopy b: degree of randomness BD: 1,4-butanediol BL: γ-butyrolactone CI: crystallinity index CL: ε-caprolactone CLSM: confocal laser scanning microscopy D: diffusion coefficient D: distance between collector plate and syringe in the electrospinning apparatus DCM: dichloromethane DDS: drug delivery system DEG: diethylene glycol DGA: diglycolic acid DMAC: dimethylacetamide DMCED: dimethylcyclohexane-1,4-dicarboxylate DMEM: dulbecco’s modified eagle medium DMS: dimethylsuccinate DP : number average polymerization degree n DP : weight average polymerization degree w DSC: differential scanning calorimetry ECM: extracellular matrix EEE: electrical and electronic equipment ES: electrospinning; electrospun FBS: fetal bovine serum FITC: fluorescein isothiocyanate GA: glycolic acid GI: germination index GPC: gel permeation chromatography GTR: gas transmission rate HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HFIP: hexafluoro-2-propanol k: kinetic constant K : equilibrium constant of condensation C LA: lactic acid LDPE: low density polyethylene LLA: L,L-dilactide MD: microdomains M : average number molecular weight n MTT: 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide M : average mass molecular weight w n : molar fraction i NMR: nuclear magnetic resonance PA: polyamide PBCE: poly(butylene cyclohexanedicarboxylate) P(BCExBDGy): poly(butylene cyclohexanedicarboxylate/diglycolate)s P(BCExTECEy): poly(butylene/triethylene cyclohexanedicarboxylate)s PBDG: poly(butylene diglicolate) PBS: poly(butylene succinate) PBSA: poly(butylene succinate/adipate) P(BSxBDGy): poly(butylene succinate/diglycolate)s PBT: poly(butylene terephthalate) PBTDG: poly(butylene thiodiglicolate) PCL: poly(ε-caprolactone) PDGS: poly(diethyleneglycol succinate) PDI: polydispersity index PDLA: poly(D-lactic acid) PDLLA: poly(D,L-lactic acid) PE: polyethylene PEG: poly(ethylene glycol) PET: poly(ethylene terephthalate) PGA: poly(glycolic acid) PHAs: polyhydroxyalkanoates PHB: poly(3-hydroxybutyrate) PLA: poly (lactic acid) PLGA: poly(lactide-co-glycolide) PLLA: poly(L-lactic acid) PP: polypropylene PS: polystyrene PTDGS: poly(thiodiethyleneglycol succinate) PTECE: poly(triethyleneglycol cyclohexanedicarboxylate) PTES: poly(triethyleneglycol succinate) PVC: polyvinylchloride R : solvent (or drug) diffusion rate diff R : polyesterification rate p R : polymer chain relaxation rate relax RH: relative humidity ROP: ring-opening polymerization S: solubility SEM: scanning electron microscopy TDEG: thiodiethylene glycol TDGA: thiodiglycolic acid TEG: triethylene glycol TFE: 2,2,2-trifluoroethanol TGA: thermogravimetric analysis TRITC: tetramethylrhodamine isothiocyanate T : temperature corresponding to 5% weigh loss 5% T: crystallization temperature c T : cold crystallization temperature cc T : glass transition temperature g T : melting temperature m T : max weight loss rate temperature max TMS: tetramethylsilane t : time lag L T : order-disorder transition temperature ODT WAXD: wide angle X-ray diffraction WCA: water contact angle w: weight fraction i X: crystallinity degree c c : specific heat increment p H: heat of crystallization c H : heat of fusion m ρ: density