Enzymatic Ring-opening Polymerization Research Articles

Application of ultrasound-assisted compression and 3D-printing semi-solid extrusion techniques to the development of sustained-release drug delivery systems based on a novel biodegradable aliphatic copolyester

The purpose of this study was to investigate the ability of two advanced hot-processing technologies, Ultrasound-Assisted Compression (USAC) and 3D-printing Semi-solid Extrusion (SSE), to manufacture sustained-release drug delivery systems based on a novel biodegradable aliphatic copolyester. The copolymer was synthesized from ω-pentadecalactone (PDL), 1,4-cyclohexanedimethanol (CHDM) and dimethyl succinate (DMS) (monomer ratio PDL/CHDM/DMS 70/30/30) by enzymatic ring opening polymerization and two-step melt polycondensation processes and has a random microstructure, a high molecular weight (107,100 g mol−1) and a relatively low melting point (∼65 °C). The antibacterial agent metronidazole (MTZ) was chosen as model drug and binary physical mixtures copolymer:drug were prepared at 90:10 and 70:30 w/w ratios. Thermal analysis studies evidenced that the formulations could be processed below their degradation temperatures. Drug delivery devices with dense and meshed structures were manufactured using USAC and SSE techniques, respectively, with USAC devices exhibiting more reproducible physical properties than the SSE systems. Powder X-ray diffraction and scanning electron microscopy studies showed a partial sintering of the copolyester during USAC processing while MTZ remained mostly crystalline. In contrast, the copolymer melted and the drug underwent some amorphization when processed using SSE. In vitro drug release studies in phosphate buffer (pH 6.8) showed that, after an initial burst release of metronidazole, USAC and SSE devices exhibited a prolonged and/or sustained drug release over 20 days. The initial burst release was dependent on the manufacturing technique and the drug/polymer ratio, being minimized for SSE devices containing 10 wt% MTZ. The whole drug release profiles fitted well to the Peppas-Sahlin model, being drug diffusion the predominant release mechanism. After the burst release, the sustained release period of USAC and SSE devices containing 10 wt% MTZ showed a good fit to the zero-order kinetic model, with faster drug release from the SSE devices due to the larger surface area of their meshed structure. In conclusion, this work demonstrates that processing the aliphatic copolyester by both USAC and SSE technologies provides an attractive strategy to manufacture biodegradable drug delivery systems for long-term release of metronidazole.

Synthesis of Functionalized Triblock Copolyesters Derived from Lactic Acid and Macrolactones for Bone Tissue Regeneration.

Synthetic and functional grafts are a great alternative to conventional grafts. They can provide a physical support and the precise signaling for cells to heal damaged tissues. In this study, a novel RGD peptide end-functionalized poly(ethylene glycol)-b-poly(lactic acid)-b-poly(globalide)-b-poly(lactic acid)-b-poly(ethylene glycol) (RGD-PEG-PLA-PGl-PLA-PEG-RGD) is synthetized and used to prepare functional scaffolds. The PGl inner block is obtained by enzymatic ring-opening polymerization of globalide. The outer PLA blocks are obtained by ring-opening polymerization of both, l-lactide or a racemic mixture, initiated by the α-ω-telechelic polymacrolactone. The presence of PGl inner block enhances the toughness of PLA-based scaffolds, with an increase of the elongation at break up to 300% when the longer block of PGl is used. PLA-PGl-PLA copolymer is coupled with α-ω-telechelic PEG diacids by esterification reaction. PEGylation provides hydrophilic scaffolds as the contact angle is reduced from 114° to 74.8°. That difference improves the contact between the scaffolds and the culture media. Moreover, the scaffolds are functionalized with RGD peptides at the surface significantly enhancing the adhesion and proliferation of bone marrow-derived primary mesenchymal stem cells and MC3T3-E1 cell lines in vitro. These results place this multifunctional polymer as a great candidate for the preparation of temporary grafts.

Fungal-Lactobacteria Consortia and Enzymatic Catalysis for Polylactic Acid Production.

Polylactic acid (PLA) is the main biobased plastic manufactured on an industrial scale. This polymer is synthetized by chemical methods, and there is a strong demand for the implementation of clean technologies. This work focuses on the microbial fermentation of agro-industrial waste rich in starch for the production of lactic acid (LA) in a consolidated bioprocess, followed by the enzymatic synthesis of PLA. Lactic acid bacteria (LAB) and the fungus Rhizopus oryzae were evaluated as natural LA producers in pure cultures or in fungal-lactobacteria co-cultures formed by an LAB and a fungus selected for its metabolic capacity to degrade starch and to form consortia with LAB. Microbial interaction was analyzed by scanning electron microscopy and biofilm production was quantified. The results show that the fungus Talaromyces amestolkiae and Lactiplantibacillus plantarum M9MG6-B2 establish a cooperative relationship to exploit the sugars from polysaccharides provided as carbon sources. Addition of the quorum sensing molecule dodecanol induced LA metabolism of the consortium and resulted in improved cooperation, producing 99% of the maximum theoretical yield of LA production from glucose and 65% from starch. Finally, l-PLA oligomers (up to 19-LA units) and polymers (greater than 5 kDa) were synthetized by LA polycondensation and enzymatic ring-opening polymerization catalyzed by the non-commercial lipase OPEr, naturally produced by the fungus Ophiostoma piceae.

A road map on synthetic strategies and applications of biodegradable polymers.

Biodegradable polymers have emerged as fascinating materials due to their non-toxicity, environmentally benign nature and good mechanical strength. The toxic effects of non-biodegradable plastics paved way for the development of sustainable and biodegradable polymers. The engineering of biodegradable polymers employing various strategies like radical ring opening polymerization, enzymatic ring opening polymerization, anionic ring opening polymerization, photo-initiated radical polymerization, chemoenzymatic method, enzymatic polymerization, ring opening polymerization and coordinative ring opening polymerization have been discussed in this review. The application of biodegradable polymeric nanoparticles in the biomedical field and cosmetic industry is considered to be an emerging field of interest. However, this review mainly highlights the applications of selected biodegradable polymers like polylactic acid, poly(ε-caprolactone), polyethylene glycol, polyhydroxyalkanoates, poly(lactide-co-glycolide) and polytrimethyl carbonate in various fields like agriculture, biomedical, biosensing, food packaging, automobiles, wastewater treatment, textile and hygiene, cosmetics and electronic devices.

Enzymatic PCL-grafting to NH2-end grouped silica and development of microspheres for pH-stimulated release of a hydrophobic model drug

This study set out to evaluate novel PCL-based silica containing nanohybrids as the polymer matrix in a hydrophobic drug-loaded microsphere system. Nanohybrids were synthesized by PCL-grafting to NH2-end grouped silica by in situ enzymatic ring opening polymerization of ε-caprolactone. Molecular weight and monomer conversion, PCL grafting percentage, thermal properties and crystallinity of the nanohybrids were determined by 1H NMR, TGA, DSC and XRD. Synthesized nanohybrids had low crystallinity percentage (32 and 39 %) and molecular weight (4800 and 8700 g/mol), promising for controlled drug release applications. The nanohybrids were used for fabrication of trans-chalcone-loaded microspheres by O/W single emulsion solvent evaporation. Mean particle diameter of the microspheres were between 15 and 30 µm. The result of release studies showed that optimum microsphere formulations (AP4 and A2, respectively) had 61 and 64 % encapsulation efficiency. One of the more significant findings to emerge from this investigation is that TC release was extended to 16 and 37 days, in a controlled manner. TC release was significantly enhanced in acidic pH media (pH 3.6 and 5.6) indicating pH-dependent release from nanohybrid microspheres; releasing 80–100 % of the loaded drug in 4–14 days. Drug/polymer interactions and molecular structures were investigated by FT-IR spectroscopy and DSC analysis. According to the results obtained, enzymatically synthesized nanohybrids have potential for pH-dependent release of the model drug, trans-chalcone.

The Effect of Solvent Hydrophilicity on the Enzymatic Ring-Opening Polymerization of L-Lactide by Candida rugosa Lipase.

Contradictions have been reported on the effect of organic solvents, especially toluene, on enzymatic ring-opening polymerization (eROP) of L-lactide. Studies have shown that log P, a common measure of hydrophilicity, affects enzyme activity. This study examines the effect of solvents with various log P values on the eROP of L-lactide, performed using Candida rugosa lipase (CRL). N,N-dimethylacetamide (DMA), 1,2-dimethoxybenzene, 1,4-dimethoxybenzene, diphenyl ether, and dodecane were used as the organic solvents. The eROP in ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]) was also conducted to compare its performance with the organic solvents. The results show that [BMIM][PF6]-mediated eROP gave better conversion and molecular weight than the organic solvent-mediated eROP. In this study, the effects of solvents hydrophilicity are discussed, including the possibility of hexafluorophosphate ion ([PF6]−) hydrolysis to occur.

Synthesis of polyglobalide by enzymatic ring opening polymerization using pressurized fluids

Here, the synthesis of polyglobalide (PGl) by enzymatic ring-opening polymerization (e-ROP) is investigated, using pressurized carbon dioxide (CO2), pressurized CO2+ dichloromethane (DCM), and pressurized propane as solvents. Particularly, the effects of phase equilibrium on the course of e-ROP and PGl final properties are discussed. The partition coefficients of CO2, DCM, propane, globalide and PGl were calculated with help of thermodynamic models, providing proper understanding of monomer partitioning in the reaction system. Reactions performed in pure CO2 resulted in monomer conversions of 100%. Besides, when only one liquid phase was present inside the reactor, PGl samples presented low polydispersities and high average molecular weights. When carried out in CO2+ DCM, e-ROP resulted in lower monomer conversions and PGl samples with higher polydispersities and lower average molecular weights. Finally, reactions carried out in pressurized propane (200 bar) produced PGl samples with the highest average molecular weights among the analyzed products.

Microreactor-based chemo-enzymatic ROP-ROMP platform for continuous flow synthesis of bottlebrush polymers

Microreactor-based enzymatic ring-opening polymerization (ROP) and metal-catalytic ring-opening metathesis polymerization (ROMP) cascade reaction platform was developed for graft through synthesis of bottlebrush polymers. Single and integrated microreactor system were assembled for the evaluation of ROP, ROMP, and ROP-ROMP, respectively. By applying a continuous flow chemo-enzymatic ROP-ROMP strategy, a series of well-defined poly(norbornene)-graft-poly(ε-caprolactone) (PNB-g-PCL) bottlebrush polymers were successfully prepared with fast kinetics, exemption of macromonomer isolation and purification, the absence of macromonomer residue in the product, controlled molecular weight and narrow distribution. This work would provide inspiration for the chemo-enzymatic synthesis, microflow chemistry, and bottlebrush polymers.

Amphiphilic Pentablock Copolymers Prepared from Pluronic and ε-Caprolactone by Enzymatic Ring Opening Polymerization.

Amphiphilic copolymers are appealing materials because of their interesting architecture and tunable properties. In view of their application in the biomedical field, the preparation of these materials should avoid the use of toxic compounds as catalysts. Therefore, enzymatic catalysis is a suitable alternative to common synthetic routes. Pentablock copolymers (CUC) were synthesized with high yields by ring-opening polymerization of ε-caprolactone (ε-CL) initiated by Pluronic (EPE) and catalyzed by Candida antarctica lipase B enzyme. The variables to study the structure–property relationship were EPEs’ molecular weight and molar ratios between ε-CL monomer and EPE macro-initiator (M/In). The obtained copolymers were chemically characterized, the molecular weight determined, and morphologies evaluated. The results suggest an interaction between the reaction time and M/In variables. There was a correlation between the differential scanning calorimetry data with those of X-ray diffraction (WAXD). The length of the central block of CUC copolymers may have an important role in the crystal formation. WAXD analyses indicated that a micro-phase separation takes place in all the prepared copolymers. Preliminary cytotoxicity experiments on the extracts of the polymer confirmed that these materials are nontoxic.

Enzymatic Synthesis and Antimicrobial Activity of Oligomer Analogues of Medicinal Biopolymers from Comfrey and Other Species of the Boraginaceae Family

This study reports the first enzymatic synthesis leading to several oligomer analogues of poly[3-(3,4-dihydroxyphenyl)glyceric acid]. This biopolymer, extracted from plants of the Boraginaceae family has shown a wide spectrum of pharmacological properties, including antimicrobial activity. Enzymatic ring opening polymerization of 2-methoxycarbonyl-3-(3,4-dibenzyloxyphenyl)oxirane (MDBPO) using lipase from Candida rugosa leads to formation of poly[2-methoxycarbonyl-3-(3,4-dibenzyloxyphenyl)oxirane] (PMDBPO), with a degree of polymerization up to 5. Catalytic debenzylation of PMDBPO using H2 on Pd/C yields poly[2-methoxycarbonyl-3-(3,4-dihydroxyphenyl)oxirane] (PMDHPO) without loss in molecular mass. Antibacterial assessment of natural polyethers from different species of Boraginaceae family Symhytum asperum, S. caucasicum, S. grandiflorum, Anchusa italica, Cynoglossum officinale, and synthetic polymers, poly[2-methoxycarbonyl-3-(3,4-dimethoxyphenyl)oxirane (PMDMPO) and PMDHPO, reveals that only the synthetic analogue produced in this study (PMDHPO) exhibits a promising antimicrobial activity against pathogenic strains S.aureus ATCC 25923 and E.coli ATCC 25922 the minimum inhibitory concentration (MIC) being 100 µg/mL.

Polylactones synthesis by enzymatic ring opening polymerization in flow

Polycondensation in batch is the classical way to produce polyesters. However, this method requires harsh conditions to reach high conversions and drive the equilibrium of the reaction forward. In order to produce controlled polymers in a greener way, we worked on the enzymatic ring opening polymerization (e-ROP) of lactones using the CALB lipase immobilized on porous beads (Novozym® 435) as a catalyst and 3-phenyl-1-propanol (PP) as initiator in a flow tubular reactor made of fluorinated ethylene propylene (FEP) tubing with i.d = 1.55 mm. In a short residence time of 214 s ε-caprolactone (CL) was fully polymerized (conv = 100 %) with a fair dispersity (Đ = 1.30). Polymerization of δ-valerolactone (VL) (conv = 93 %, Đ = 1.27) as well as copolymerization of CL and VL mixtures (conv up to 88 %, Đ = 1.24) behaved similarly. By alternating the lactones, a block-polymer was obtained. As expected, primary alcohols were more efficient than secondary ones for initiating the e-ROP of lactones. Very interestingly, using cholesterol as initiator induced the formation of cyclic polylactones only. In addition, we showed the importance of the residence time as at a long time the formed polymer was depolymerized by the enzyme. We demonstrated also that the enzyme may be reused more than ten times with maintaining its high efficiency. The whole study shows that the use of tubular reactor for performing e-ROP improves its control as shown by the low Đ values and decreases the residence time needed to reach high conversions compared to similar reactions in batch.

Synthesis of bilayer films from regenerated cellulose nanofibers and poly(globalide) for skin tissue engineering applications

Commercial cell-based skin regenerative products are highly expensive, carry the risk of rejection and require a long cell culture period to manufacture. This work describes the synthesis of bilayer films from poly(globalide) (PGl) and regenerated cellulose nanofibers (rCNFs) and their use as a cell-free scaffold to support keratinocyte attachment and proliferation. The method is simple, eco-friendly (as the cellulose precursor is obtained from agricultural waste) and of low cost. The rCNFs were produced by acid hydrolysis and PGl was obtained via enzymatic ring-opening polymerization. The bilayer films were synthesized by layer-by-layer casting at ambient temperature. All the films showed a well-defined interface between PGl and cellulose. The produced rCNF/PGl bilayer films showed cell metabolic activity far superior in comparison with pristine PGl regarding the keratinocyte growth, which illustrates the potential use of these materials in skin tissue engineering.

One-pot three-step enzymatic ROP in situ to form polycaprolactone from cyclohexanone: Optimizing and kinetic modeling

A one-pot ring-opening polymerization (ROP) to form poly ε-caprolactone (PCL) from cyclohexanone (CHN) mediated by immobilized T. laibacchii lipase was proposed. The optimal conditions by using response surface methodology (RSM) were established as follows: the supplement of lipase, 23.55 mg, the reaction temperature, 59.4 °C and the molar ratio of urea hydrogen peroxide (UHP) to CHN, 1.31:1, under which the yield of PCL and number-average molar mass (Mn) at a high CHN concentration of 1.25 M were 96.5% and 2912, respectively. The one-pot ROP consists three steps: first, ethyl acetate (EtOAc) reacts with UHP to generate peracetic acid catalyzed by lipase; then, CHN is lactonized to ε-caprolactone (CL) with peracetic acid in situ generated in nonenzymatic, and acetic acid formed reacts with UHP to form peracetic acid; finally, CL suffers from enzymatic ROP to in situ generate PCL. There are three factors contributing to the high yield: cross-linked immobilized lipase employed, the supplement of lipase added in step 3 of ROP and optimal conditions adopted. The initial concentration of UHP was reduced from 2.5 M to 1.625 M owing to acetic acid reacting with UHP to yield peracetic acid which can help obtain a high yield of PCL. A kinetic model for this one-pot three-step enzymatic ROP reaction was developed, which fitted the observed data well. The enzymatic reaction mediated by lipase may obey the ping-pong mechanism with substrate inhibition of UHP and acetic acid, while the nonenzymatic reaction may obey a power law. Using the kinetic model, the contribution of the reaction of UHP with EtOAc and acetic acid to the yield of peracetic acid can be distinguished, contributing to better comprehend the reaction process.

“Water-like” ammonium-based ionic liquids for lipase activation and enzymatic polymerization

Novel dual-functionalized ammonium-based hydrophobic ionic liquids (ILs) have been synthesized by mimicking the water structure to carry both ether group (hydrogen-bond acceptor) and tert-alcohol group (hydrogen-bond donor). These ammonium-type ionic solvents exhibit the advantage of lower viscosities (as low as 129 mPa s at 30 °C) than the imidazolium analogue (∼300 mPa s at 30 °C); more importantly, these “water-like” media are highly compatible with immobilized Candida antarctica lipase B (Novozym 435) in terms of producing high transesterification activities (1.5-fold higher than that in tert-butanol, and slightly higher than that in diisopropyl ether) and higher thermal stability than that in tert-butanol. To demonstrate the utilization of this new solvent system, enzymatic ring-opening polymerization (ROP) of ε-caprolactone using these “water-like” ILs as co-solvents was carried out to synthesize polyesters, achieving high molecular mass Mw (up to 18,000 Da) and high yields (up to 74 %).

Dual-Enzyme-Catalyzed Synthesis of Enantiocomplementary Polyesters.

Herein, a series of enantiocomplementary polyesters with either (S)- or (R)-configurations were successfully prepared by applying a dual-enzyme biocatalytic system. In the step of Baeyer-Villiger oxidation, cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 (CHMOAcineto) was engineered rationally to tailor the enantiopreference of mutants, providing (S)- and (R)-lactones, respectively, with high optical purities (up to 99% ee) as polymeric precursors. By subsequent enzymatic ring-opening polymerization of the enantiopure monomers, enantiocomplementary polyesters with high molecular weight (up to 21.8 kDa Mn) were synthesized by lipase CALB/MML. Our research offers an environmentally friendly synthesis route for the production of optically pure lactones and chiral polyesters, which are of particular significance for their application in organic syntheis or biomedical materials.

Photopolymerization of enzymatically synthesized methacrylated poly(caprolactone) with poly(ethylene glycol) macromonomer

Polycaprolactone (PCL) based α,ω-methacrylated macromonomer (DMPCL) was synthesized via enzymatic ring-opening polymerization (eROP) by using Novozyme 435 as the enzyme immobilized catalyst. DMPCL was further photopolymerized with monofunctional poly(ethylene glycol) methyl ether methacrylate (PEGMA-950) macromonomer and trimethylolpropane triacrylate (TMPTA) as tri-functionalized crosslinking agent in glass vials when CHCl3 was the solvent and Irgacure 819 was the photoinitiator. Ultraviolet (UV) Light Emitting Diode (LED) bulbs enabled photoinduced reactions at room temperature with low heat generation and high reaction efficiency. The obtained gels were characterized with Fourier Transform Infrared Spectroscopy (FT-IR) and Differential Scanning Calorimetry (DSC). DMPCL participated as an effective crosslinking agent in the photopolymerization of PEGMA-950. Combined usage of DMPCL and PEGMA-950 resulted in significantly more effective polymerization than the separate photopolymerizations of these macromonomers.

Synthesis of Aromatic–Aliphatic Polyesters by Enzymatic Ring Opening Polymerization of Cyclic Oligoesters and their Cyclodepolymerization for a Circular Economy

Cyclic oligomers of hexamethylene furanoate and hexamethylene terephthalate were obtained from 1,6-hexanediol and the corresponding methyl esters using Candida antarctica lipase B (CALB) enzyme catalyst. HPLC, MALDI-TOF MS, and NMR analyses showed that mixtures composed from cyclic dimer up to heptamer were obtained almost quantitatively. Subsequently, these cycles were polymerized by ring opening polymerization (ROP) mediated by CALB to obtain semicrystalline polymers. In addition, we demonstrated that the polymers obtained from the ROP process could be transformed into cyclic oligomers in high yields using enzymatic cyclodepolymerization, thereby recycling the polymer in a circular biosynthetic path.

Novel green route towards polyesters-based resin by photopolymerization of star polymers

Bio-based star-shaped poly(e-caprolactone)s (S-PCL) derived from sugar-based D-sorbitol as an initiator were obtained via solvent-free enzymatic ring-opening polymerization (eROP). The star S-PCL were converted into UV-curable maleates by employing maleic anhydride for subsequent crosslinking with tri(ethylene glycol) divinyl ether (DVE-3) in the presence of Darocur 1173 as a radical photoinitiator. The kinetics of the UV-induced radical copolymerization was monitored by real-time Fourier-Transform InfraRed (FTIR) spectroscopy, which revealed that the star S-PCL maleate/divinyl ether system was not scavenged by molecular oxygen (donor/acceptor polymerization). The UV-crosslinking reaction was fast (~10 s) to reach near quantitative conversions. The S-PCL maleate / divinyl ether liquid formulation cast on glass substrates successfully gave films upon UV-crosslinking. The thermal properties of the polymer films and their precursor polymers were characterized by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). Finally, the crosslinked polymer film demonstrated promising adhesive properties on steel, aluminum and glass substrates.

Optimization and modelling of enzymatic polymerization of ε-caprolactone to polycaprolactone using Candida Antartica Lipase B with response surface methodology and artificial neural network

Recently enzymatic catalysts have replaced organic and organometallic catalysts in the synthesis of bio-resorbable polymers. Enzymatic polymerization is considered as an alternative to conventional polymerization as they are less toxic, environmental friendly and can operate under mild conditions. In this research, the enzymatic ring-opening polymerization (e-ROP) of e-caprolactone (e-CL) using Candida Antartica Lipase B (CALB) as catalyst to produce the Polycaprolactone. Two modelling techniques namely response surface methodology (RSM) and artificial neural network (ANN) have been used in this work. RSM is used to optimize the parameters and to develop a model of the process. ANN is used to develop the model to predict the results obtained from the experiment. The parameters involved are time, reaction temperature, mixing speed and enzyme-solvent ratio. The experimental result is Polydispersity index (PDI) of the polymer. The experimental data obtained was adequately fitted into second-order polynomial models. Simulation was done using artificial neural network model developed with Mean absolute error (MAD) value of 1.65 in comparison with MAD value of 7.4 for RSM. The Regression value (R2) values of RSM and ANN were found to be 0.96 and 0.93 respectively. The predictive models were validated experimentally and were found to be in agreement with the experimental values.

Synthesis of polyvalerolactone via enzymatic ring opening polymerization in different reaction media

The objective of this work was to fit together the starch extraction from Pachyrhizus ahipa roots and the recovery of the proteins present in these storage organs, making an improved use of this novel raw material. The replacement of water by buffer PO4−3/NaCl as solvent in the first extraction steps improved protein extraction without lowering the starch yield. The starches obtained from the traditional and the proposed methods exhibited some differences in appearance and technological and thermal properties, which were endorsed to the adjustment in the methodology of extraction rather than to the use of buffer as solvent. Thus, P. ahipa starch obtaining procedure could be coupled to protein extraction with a minimum change in the methodology. This innovation did not significantly shift the characteristics of the starch obtained and allowed to obtain a protein yield of 135.7 mg BSA equivalent protein/100 g of fresh roots.