Antibacterial amphiphilic polymers based on enzymatically synthesized polycaprolactone

Amphiphilic polycaprolactone (PCL) based polymers were synthesized utilizing three different approaches including the combination of enzymatic ring-opening polymerization (eROP) either with photopolymerization and atom transfer radical polymerization (ATRP). The side reactions during the synthesis of methacrylate initiated eROP of ɛ-caprolactone (CL) was scrutinized to reveal the formation of different end-groups. Effect of reaction time initiator/lactone ratio and the synergetic effect of temperature and initiator/lactone ratio as 2-hydroxyethyl methacrylate (HEMA) was the nucleophilic initiator, Novozyme-435 (N435) was the immobilized lipase catalyst. α,ω-methacrylated PCL macromonomer was synthesized to have the benefit of the side reactions investigated at the first place, utilizing ethylene glycol dimethacrylate (EGDMA). Since, water acted as the nucleophilic initiator in the beginning of the reaction, EGDMA was degraded by N435 and tailored onto the ends of the PCL chains resulting in mixture of macromonomers comprising a combination of methacrylate, HEMA, and hydroxyl end-groups together with ethylene glycol (EG) moieties along the chain. The mixture of PCL chains were concisely called as dimethacrylated PCL or α,ω-methacrylated PCL (DMPCL). DMPCL was employed as a long crosslinking agent in the photopolymerizaton of poly(ethylene glycol) methyl ether methacrylate-950 (PEGMA-950) with the aid of trimethylolpropane triacrylate (TMPTA) and Irgacure-819 as the initiator under light emitting diod (LED) ultraviolet (UV) bulbs. To achieve amphiphilic copolymer of PCL and poly(ethylene glycol) (PEG) with another strategy, a macrophotoinitiator based on PCL was again synthesized via eROP when Irgacure-2959 was used as the nucleophilic initiator. Light-induced polymerization was again applied to reach PCL copolymers with butyl acrylate (BuA), benzyl acrylate (BzA), tetrahydrofurfuryl acrylate (THFA), 2,2,2-trifluoroethyl acrylate (TFEA), 2-hydroxyethyl acrylate (HEA) and poly(ethylene glycol) methyl ether acrylate-480 (PEGMEA-480). PEGMEA-480 and sulfobetaine methacrylate (SBMA) were also installed on enzymatically synthesized PCL to achieve AB and ABC type block copolymers via ATRP. Following the end-functionalization of enzymatically synthesized PCL with 2-bromoisobutyryl bromide, diblock copolymer of P(CL)n-b-P(PEGMEA)m and triblock copolymer of P(CL)n-b¬-P(PEGMEA)m-b-P(SMBA)k were achieved. The structures of the abovementioned polymers were analysed with 1H nuclear magnetic resonance (NMR), fourier transform infrared spectroscopy-attenuated total reflection (FTIR-ATR), differential scanning calorimetry (DSC) and size exclusion chromatography (SEC). The antibacterial properties of the polymers were investigated via zone inhibition test method. The side reactions during the synthesis of methacrylate initiated enzymatic ring-opening polymerization of ɛ-caprolactone (CL) was scrutinized. It was revealed that N435 has high activity enough to catalyze enzyme-monomer complex formation, initiation, and propagation, cleaving the ester bond of the hydroxyl bearing acrylate initiator at the same time. Therefore, it was revealed that it might be preferable to study at lower reaction temperatures to reduce the side reactions inside the sufficient activity range of N435 or other lipase types. It is obvious to finalize the eROP at low reaction times and conversions in order to obtain lower side reactions and comparable HEMA addition. Furthermore, the lower enzyme amount might be preferable in order to realise higher HEMA addition, lower methacrylate transfer, comparable total methacrylate end-groups, and minimum EG inside the chains. HEMA/CL ratio can also be chosen minimum as both of the side reactions seemed to be lower at both temperatures and enzyme amounts, still conserving the total methacrylate amount sufficient. In addition to these, higher molecular weight and polymerization yield of macromonomers can be obtained at lower initiator/lactone ratios. To synthesize α,ω-methacrylated macromonomer for the application as a crosslinking agent in UV polymerization, it was benefited from the previously investigated side-reactions during eROP of CL when an ester containing initiator presented in the reactor. The double methacrylation of the PCL chains was evaluated to be a successful route, since the one-pot synthesis comprised of the primary synthesis of PCL enzymatically, and subsequent tailoring of the methacrylate groups onto the chains provided that all the water molecules were removed from the reaction mixture with high vacuum for high a conversion time. Furthermore, the conversion values without the usage of either PEGMA-950 or DMPCL didn't give high conversion values of DMPCL and PEGMA-950. Therefore, the concurrent usage of DMPCL and PEGMA-950 in photopolymerization provided a synergetic influence for the conversion of both macromonomers with the help of TMPTA. The feed mole ratios 0.25, 0.50 and 0.75 for DMPCL (DMPCL/Total macromonomers) inside the photopolymerization mixture all gave high polymerization yields. Consequently, enzymatically polymerized dimethacrylated PCL macromonomer with average molecular weight of 4400 g/mole and 0.84 ratio of the total methacrylate end-groups was found to be an efficient crosslinking agent in photopolymerization and obtaining amphiphilic networks. It was noteworthy that the low Tg and Tm values of the amphiphilic networks revealed the rubbery and elastic behaviour of the materials. The enzymatic synthesis of PCL-based macrophotoinitiator was performed successfully. The macrophotoinitiator successfully photopolymerized the acrylate monomers: butyl acrylate, benzyl acrylate, tetrahydrofurfuryl acrylate, 2,2,2-trifluoroethyl acrylate, 2-hydroxyethyl acrylate and poly (ethylene glycol) methyl ether acrylate ~ 480 g/mole). Benzyl acrylate and tetrahydrofurfuryl acrylate had the highest conversion values. The block copolymer structure of poly(CL-b-BzA) and high conversion was confirmed with a single and broad Tg value with a high step decrease of the specific heat capacity. The relatively high dry polymer conversions for gel formation by using HEA and PEGMEA-480 monomers in photopolymerization also proved the efficiency of amphiphilic polymer synthesis via PCL based macrophotoinitiator. The low Tg value, thus the rubbery state, of the amphiphilic gels also made it possible for their usage in biomedical applications together with the biocompatible nature of the employed macromonomers. A chemoenzymatic route was utilized in order to synthesize amphiphilic diblock copolymer (P(CL)n-b¬-P(PEGMEA)m) and an amphiphilic triblock copolymer comprising zwitterionic monomer units (P(CL)n-b¬-P(PEGMEA)m-b-P(SMBA)k). It should be noted that the third polymer segment was installed by the synthesis of a methacrylate via ATRP onto an polyacrylate based diblock macroinitiator. Furthermore, the low solubility of the diblock copolymer in the reaction solvent ethanol/water mixture was also one of the challenging points of the third monomer addition. Another barrier for both PEGMEA-480 and SBMA ATRP polymerizations might be the steric hinderence of PEGMEA-480 brush segments together with the possible entaglements of the PEG chains. However, PEGMEA-480 conversion was calculated as 65% and SBMA was proven to be polymerized with diblock ATRP macroinitiator. Amphiphilic diblock copolymers comprised of PCL and PEG synthesized via photopolymerization and ATRP approaches both had antibacterial activity against S. aureus (Gram positive) and E. coli (Gram negative). The triblock copolymer, based on PCL, PEG and PSMBA was only antibacterial against S. aureus. However, neither of the polymers provided antifungal activity against A. niger. Antifouling feature assay might also be performed for the synthesized polymers, since composing antifouling surfaces is one of the candidate routes to obtain antibacterial activity due to their capability of inhibition of the protein based molecules which are employed by microorganisms to simplify their settlemt on surfaces. Protein adsorption test might be a method to investigate antifouling features. Consequently, enzymatic ring-opening polymerization in combination with photopolymerization or ATRP was found to be an approach to synthesize polymers performing antibacterial properties which might find applications in biomedical and marine uses.