Hydrolytic degradation and protein release studies of thermogelling polyurethane copolymers consisting of poly[(R)-3-hydroxybutyrate], poly(ethylene glycol), and poly(propylene glycol)

This paper reports the hydrolytic degradation and protein release studies for a series of newly synthesized thermogelling tri-component multi-block poly(ether ester urethane)s consisting of poly[(R)-3-hydroxybutyrate] (PHB), poly(propylene glycol) (PPG), and poly(ethylene glycol) (PEG). The poly(PEG/PPG/PHB urethane) copolymer hydrogels were hydrolytically degraded in phosphate buffer at pH 7.4 and 37 degrees C for a period of up to 6 months. The mass loss profiles of the copolymer hydrogels were obtained. The hydrogel residues at different time periods of hydrolysis were visualized by scanning electron microscopy, which exhibited increasing porosity with time of hydrolysis. The degradation products in the buffer were characterized by GPC, (1)H NMR, MALDI-TOF, and TGA. The results showed that the ester backbone bonds of the PHB segments were broken by random chain scission, resulting in a decrease in the molecular weight. In addition, the constituents of degradation products were found to be 3-hydroxybutyric acid monomer and oligomers of various lengths (n=1-5). The protein release profiles of the copolymer hydrogels were obtained using BSA as model protein. The results showed that the release rate was controllable by varying the composition of the poly(ether ester urethane)s or by adjusting the concentration of the copolymer in the hydrogels. Finally, we studied the correlation between the protein release characteristics of the hydrogels and their hydrolytic degradation. This is the first example that such a correlation has been attempted for a biodegradable thermogelling copolymer system.     

Improving hydrophilicity,mechanical properties and biocompatibility of poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyvalerate] through blending with poly[(R)-3-hydroxybutyrate]-alt-poly(ethylene oxide)

Natural source poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyvalerate] (PHBV) with a low hydroxyvalerate (HV) content (～8 wt.%) was modified by blending it with poly[(R)-3-hydroxybutyrate]-alt-poly(ethylene oxide) (HE) alternating block copolymer. We hypothesized that the adjoining PHB segments could improve the miscibility of the poly(ethylene oxide) segments of HE with the PHBV matrix and therefore improve the physical properties of the PHBV/HE blends. A differential scanning calorimetry study revealed the improved miscibility of PEO segments of HE characterized by the interference of the crystallization of PHBV. The decrease in water contact angle and the increase in equilibrium water uptake of the PHBV/HE blends indicated that both the surface and bulk hydrophilicity of PHBV could be improved through blending HE. The mechanical properties of the hydrated PHBV/HE blends were assessed by measuring their tensile strength. In contrast to the hydrated natural source PHBV, which failed in a brittle manner, the hydrated PHBV/HE blends were ductile. Their strain at break increased with increasing HE content, reaching a maximum of 394% at an HE content of 15 wt.%. The excellent integrity of the PHBV/HE blends in water is attributed to the strong affinity between the PHB segments of HE and the PHBV matrix. Platelet adhesion on the film surface of the PHBV/HE blends was investigated in vitro to evaluate their blood compatibility. The results demonstrated that the PHBV/HE blends effectively resisted the adhesion of platelets due to the anchored PEO segments from HE on the film surface.     

Characterization, biodegradability and blood compatibility of poly[(R)-3-hydroxybutyrate] based poly(ester-urethane)s

Poly(ester-urethane)s (PUs) were synthesized using hexamethylene diisocyanate (HDI) or toluene diisocyanate (TDI) to join short chains (M(n) = 2000) of poly(R-3-hydroxybutyrate) (PHB) diols and poly(epsilon-caprolactone) (PCL) diols with different feed ratios under different reaction conditions. The multiblock copolymers were characterized by nuclear magnetic resonance spectrometer (NMR), gel permeation chromatography (GPC), differential scanning calorimetry (DSC), thermogravimetric analyses (TGA), X-ray diffraction (XRD), and scanning electron microscope (SEM). XRD spectra and second DSC heat thermograms of the multiblock copolymers revealed that the crystallization of both PHB and PCL segments was mutually restricted, and, especially, the PCL segment limited the cold crystallization of the PHB segment. The SEM of platelet adhesion experiments showed that the hemocompatibility was affected to some extent by the chain flexibility of the polymers. Hydrolysis studies demonstrated that the hydrolytic degradation of PUs was generated from the scission of their ester bonds or/and urethane bonds. Simultaneously, the rate of ester bond scission was determined to some extent by the crystallization degree, which was further affected by the configuration of polymer chains. These highly elastic multiblock copolymers combining hemocompatibility and biodegradability may be developed into blood contact implant materials for biomedical applications.     

Synthesis and water-swelling of thermo-responsive poly(ester urethane)s containing poly(epsilon-caprolactone), poly(ethylene glycol) and poly(propylene glycol)

Thermo-responsive multiblock poly(ester urethane)s comprising poly(epsilon-caprolactone) (PCL), poly(ethylene glycol) (PEG), and poly(propylene glycol) (PPG) segments were synthesized. The copolymers were characterized by GPC, NMR, FTIR, XRD, DSC and TGA. Water-swelling analysis carried out at different temperatures revealed that the bulk hydrophilicity of the copolymers could be controlled either by adjusting the composition of the copolymer or by changing the temperature of the environment. These thermo-responsive copolymer films formed highly swollen hydrogel-like materials when soaked in cold water and shrank when soaked in warm water. The changes are reversible. The mechanical properties of the copolymer films were assessed by tensile strength measurement. These copolymers were ductile when compared to PCL homopolymers. Young's modulus and the stress at break increased with increasing PCL content, whereas the strain at break increased with increasing PEG content. The results of the cytotoxicity tests based on the ISO 10993-5 protocol demonstrated that the copolymers were non-cytotoxic and could be potentially used in biomedical applications.     

Structure of poly[(ethylene glycol)-(diethylene glycol) terephthalate] as determined by chemical degradation

The influence of preliminary thermal treatment and diethylene glycol (DEG) content on the rate of degradation, DEG amount in the degradation products, WAXS, relative viscosity, and density of the understructed poly(ethylene terephthalate) (PET) is studied upon aminolysis with hydrazine hydrate at 60 and 85°C. The results indicate that (i) the aminolysis is a two-stage process, corresponding to the degradation of the amorphous and crystalline regions and (ii) the phase boundary is not sharp. A model is proposed, describing the comonomer distribution in the semicrystalline copolyesters. The comonomer units are concentrated mainly in the amorphous regions. The effect of degradation conditions like temperature, concentration, duration, particle size, deposition of low molecular products on the polymer surface, etc. is also discussed.     

Biodegradable thermogelling poly(ester urethane)s consisting of poly(lactic acid)--thermodynamics of micellization and hydrolytic degradation

Multiblock poly(ether ester urethane)s comprising of poly(lactic acid) (PLA), poly(ethylene glycol) (PEG), and poly(propylene glycol) (PPG) segments were synthesized, and their aqueous solutions exhibited thermogelling behavior at critical gelation concentrations (CGC) ranging from 7 to 9 wt%. The chemical structures and molecular characteristics of the copolymers were studied by GPC, 1H NMR, 13C NMR and FTIR. The thermal stability of the poly(PEG/PPG/PLA urethane)s was studied by thermogravimetry analysis (TGA), and the PLA contents were calculated based on the thermal degradation profile. The results were in good agreement with those obtained from the 1H NMR measurements. The critical micellization concentration (CMC) of these water-soluble poly(ether ester urethane)s was determined at different temperatures using a dye solubilization method. The thermodynamic parameters for micelle formation were calculated, indicating that the process is largely entropy-driven. Interestingly, it appears that there exists a requirement for the system to possess a minimum gain in entropy before the thermogelling effect can be observed. Dilute copolymer solutions showed a lower critical solution temperature (LCST) behavior similar to pNIPAM dissolved in aqueous solutions. The thermogels hydrolytically degraded to polymer fragments corresponding to the constituent segment blocks within 3 months.     

Alternative block polyurethanes based on poly(3-hydroxybutyrate-co-4-hydroxybutyrate) and poly(ethylene glycol)

A series of amphiphilic alternative block polyurethane copolymers based on poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3/4HB) and poly(ethylene glycol) (PEG) were synthesized by a coupling reaction between P3/4HB-diol and PEG-diisocyanate, with different 3HB, 4HB, PEG compositions and segment lengths. Stannous octanoate was used as catalyst. The chemical structure, alternative block arrangement, molecular weight and distribution were systematically characterized by FTIR, ¹H NMR, GPC and composition analysis. The thermal property was studied by DSC and TGA. Platelet adhesion study revealed that the alternative block polyurethanes possess excellent hemocompatibility. CCK-8 assay illuminated that the non-toxic block polyurethanes maintain rat aortic smooth muscle cells (RaSMCs) good viability. The in-vitro degradation of the copolymers in PBS buffer solution and in lipase buffer medium was investigated. Results showed that the copolymer films exhibit different degradation patterns in different media from surface erosion to diffusion bulk collapsing. The synthetic methodology for the alternative block polyurethanes provides a way to control the exact structure of the biomaterials and tailor the properties to subtle requirements.     

Thermodynamics of depolymerization of biotechnological poly[(R)-3-hydroxybutyrate] with formation of (R,R,R)-4,8,12-trimethyl-1,5,9-trioxadodeca-2,6,10-trione and of its polymerization with ring-opening to highly isotactic poly[(R)-3-hydroxybutyrate] from 0 to 500 K at standard pressure

In adiabatic vacuum and dynamic calorimeters the temperature dependence of the heat capacity C of (R,R,R-4,8,12-trimethyl-1,5,9-trioxadodeca-2,6,10-trione a twelve-membered cyclic trilactone), biotechnological poly[(R)-3-hydroxybutyrate] and highly isotactic poly[(R)-3-hydroxybutyrate] was studied between 5 K and 500 K; temperatures and enthalpies of melting of the above mentioned substances were measured. In a calorimeter with a static bomb and an isothermal shield the energy of combustion of the same substances was measured. From the results the thermodynamic functions C (T), H⁰(T) ? H⁰ (0), S⁰ (T), G⁰ (T) ? H⁰ (0) were calculated in the range of 0 K to 500 K and thermochemical parameters ΔH, ΔH, ΔS, ΔG were estimated at T = 298,15 K and standard pressure. The thermodynamic parameters of depolymerization of the biotechnological polymer to the 12-membered trilactone ΔH, ΔS ΔG and of the polymerization of the monomer formed in the highly isotactic poly[(R)-3-hydroxybutyrate] ΔH, ΔS, ΔG were calculated for 0 K to 500 K.     

Telechelic diols from poly[(R)-3-hydroxybutyric acid] and poly{[(R)-3-hydroxybutyric acid]-co-[(R)-3-hydroxyvaleric acid]}

We describe the preparation of telechelic OH-terminated poly[(R)-3-hydroxybutyric acid] (PHB) and poly{[(R)-3-hydroxybutyric acid]-co-[(R)-3-hydroxyvaleric acid]} (PHB/HV), on a semi-preparative scale, by a transesterification procedure from the high-molecular weight polymers. The oligomers have well-defined reactive end groups and are well suited for the preparation of high-molecular-weight block copolymers by chain extension.     

In vitro cytotoxicity, hemolysis assay, and biodegradation behavior of biodegradable poly(3-hydroxybutyrate)-poly(ethylene glycol)-poly(3-hydroxybutyrate) nanoparticles as potential drug carriers

Nanoparticles based on amorphous poly(3-hydroxybutyrate)-poly(ethylene glycol)-poly(3-hydroxybutyrate) (PHB-PEG-PHB) are potential drug delivery vehicles, and so their cytotoxicity and hemolysis assay were investigated in vitro using two kinds of animal cells. The PHB-PEG-PHB nanoparticles showed excellent biocompatibility and had no cytotoxicity on animal cells, even when the concentrations of the PHB-PEG-PHB nanoparticle dispersions were increased to 120 microg/mL. Moreover, no hemolysis was detected with the PHB-PEG-PHB nanoparticles, suggesting that the PHB-PEG-PHB nanoparticles were obviously much hemocompatible for drug delivery applications. In the presence of intracellular enzyme esterase, the biocompatible PHB-PEG-PHB nanoparticles might be hydrolyzed, and their biodegradable behavior was monitored by the fluorescence spectrum and the pH meter. The initial biodegradation rate of the PHB-PEG-PHB nanoparticles was closely related to the enzymatic amount and the PHB block length. Compared with that obtained from the fluorescence determination, the initial biodegradation rate from pH measurement was faster. The biodegraded products mainly consisted of 3HB monomer and dimer, which were the metabolites present in the body.     

Biodegradable nanoparticles of amphiphilic triblock copolymers based on poly(3-hydroxybutyrate) and poly(ethylene glycol) as drug carriers

New amorphous amphiphilic triblock copolymers of poly(3-hydroxybutyrate)-poly(ethylene glycol)-poly(3-hydroxybutyrate) (PHB-PEG-PHB) were synthesized using the ring-opening copolymerization of beta-butyrolactone monomer. They were characterized by fluorescence, SEM and (1)H NMR. These triblock copolymers can form biodegradable nanoparticles with core-shell structure in aqueous solution. Comparing to the poly(ethylene oxide)-PHB-poly(ethylene oxide) (PEO-PHB-PEO) copolymers, these nanoparticles exhibited much smaller critical micelle concentrations and better drug loading properties, which indicated that the nanoparticles were very suitable for delivery carriers of hydrophobic drugs. The drug release profile monitored by fluorescence showed that the release of pyrene from the PHB-PEG-PHB nanoparticles exhibited the second-order exponential decay behavior. The initial biodegradation rate of the PHB-PEG-PHB nanoparticles was related to the enzyme amount, the initial concentrations of nanoparticle dispersions and the PHB block length. The biodegraded products detected by (1)H NMR contained 3HB monomer, dimer and minor trimer, which were safe to the body.     

Synthesis of poly(ethylene glycol)-modified urethane acrylates and their soap-free emulsification

In order to prepare the soap-free emulsion of urethane acrylates, poly(ethylene glycol)-modified urethane acrylates (PMUA), containing poly(oxyethylene) chains as terminal groups, were synthesized by reaction of poly(ethylene glycol) (PEG) with residual isocyanate groups. The size of the droplets of PMUA emulsions decreases as the molar ratio of PEG to 2-hydroxyethyl methacrylate (2-HEMA) increases. The interfacial activity of PMUA was confirmed by the measurement of the adsorption isotherm at the water/benzene interface. The chain length of poly(oxyethylene) and the type of polyol (PTMG, PPG) affects significantly the size of the droplets and the stability of the PMUA emulsions.     

Controlled release of paclitaxel from biodegradable unsaturated poly(ester amide)s/poly(ethylene glycol) diacrylate hydrogels

Biodegradable hydrogels (FPBe-G) were synthesized by the photopolymerization of two precursors: FPBe, a fumurate-based unsaturated poly(ester amide) (UPEA), and poly(ethylene glycol) diacrylate (PEG-DA). Depending on the feed ratio of these two precursors, the resultant FPBe-G hydrogels showed different crosslinking levels of network structure, mesh sizes (xi) and matrix morphology. When a lipophilic drug, paclitaxel, was preloaded into FPBe-G hydrogels, the two-month drug-release kinetics from FPBe-G hydrogels in both pure PBS buffer and alpha-chymotrypsin media were measured. The paclitaxel-preloaded FPBe-G hydrogels in a alpha-chymotrypsin solution had significantly faster drug release rate than the corresponding hydrogels in a pure PBS buffer due to an enzyme catalyzed biodegradation of FPBe-G hydrogels. Sustained paclitaxel releases over a two-month period without initial burst release were also achieved by using hydrogels having certain feed ratios of hydrogel precursors. These paclitaxel release data correlated well with the molecular mesh size (xi), molecular weight between cross-links (M(c)) and matrix morphological structure of FPBe-G hydrogels.     

Controlled release of paclitaxel from biodegradable unsaturated poly(ester amide)s/poly(ethylene glycol) diacrylate hydrogels

Biodegradable hydrogels (FPBe-G) were synthesized by the photopolymerization of two precursors: FPBe, a fumurate-based unsaturated poly(ester amide) (UPEA), and poly(ethylene glycol) diacrylate (PEG-DA). Depending on the feed ratio of these two precursors, the resultant FPBe-G hydrogels showed different crosslinking levels of network structure, mesh sizes (ξ) and matrix morphology. When a lipophilic drug, paclitaxel, was preloaded into FPBe-G hydrogels, the two-month drug-release kinetics from FPBe-G hydrogels in both pure PBS buffer and α-chymotrypsin media were measured. The paclitaxel-preloaded FPBe-G hydrogels in a α-chymotrypsin solution had significantly faster drug release rate than the corresponding hydrogels in a pure PBS buffer due to an enzyme catalyzed biodegradation of FPBe-G hydrogels. Sustained paclitaxel releases over a two-month period without initial burst release were also achieved by using hydrogels having certain feed ratios of hydrogel precursors. These paclitaxel release data correlated well with the molecular mesh size (ξ), molecular weight between cross-links (M _c) and matrix morphological structure of FPBe-G hydrogels.     

In vivo and in vitro degradation of poly(ether ester) block copolymers based on poly(ethylene glycol) and poly(butylene terephthalate)

Two in vivo degradation studies were performed on segmented poly(ether ester)s based on polyethylene glycol (PEG) and poly(butylene terephthalate) (PBT) (PEOT/PBT). In a first series of experiments, the in vivo degradation of melt-pressed discs of different copolymer compositions were followed up for 24 weeks after subcutaneous implantation in rats. The second series of experiments aimed to simulate long-term in vivo degradation. For this, PEOT/PBT samples were pre-degraded in phosphate buffer saline (PBS) at 100 degrees C and subsequently implanted. In both series, explanted materials were characterized by intrinsic viscosity measurements, mass loss, proton nuclear magnetic resonance spectroscopy (1H-NMR) and differential scanning calorimetry (DSC). In both studies the copolymer with the higher PEO content degraded the fastest, although all materials degraded relatively slowly. To determine the nature of the degradation products formed during hydrolysis of the copolymers, 1000 PEOT71PBT29 (a copolymer based on PEG with a molecular weight of 1000 g/mol and 71 wt% of PEO-containing soft segments) was degraded in vitro at 100 degrees C in phosphate buffer saline (PBS) during 14 days. The degradation products present in PBS were analyzed by 1H-NMR and high performance liquid chromatography/mass spectroscopy (HPLC/MS). These degradation products consisted of a fraction with high contents of PEO that was soluble in PBS and a PEOT/PBT fraction that was insoluble at room temperature. From the different in vitro and in vivo degradation experiments performed, it can be concluded that PEOT/PBT degradation is a slow process and generates insoluble polymeric residues with high PBT contents.     

Functionalization of poly(ethylene glycol) and monomethoxy-poly(ethylene glycol)

Simple methods are described for the substitution of poly(ethylene glycol) and monomethoxy-poly(ethylene glycol) substitution. Affinity ligands, coenzymes, or enzymes can be covalently attached to the substitution product or they can be used as liquid ionexchangers.     

Improvement of the cytocompatibility of electrospun poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyvalerate] mats by Ecoflex

Poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyvalerate] (PHBV) is a nature-derived polyester with potential application in tissue engineering scaffolds. However, PHBV is associated with disadvantages including high brittleness, slow degradation, high hydrophobicity, and unsatisfactory biocompatibility. In this study, we sought to improve the properties of PHBV by blending it with Ecoflex, a synthetic biopolyester with a high flexibility, fast degradation, and comparatively higher hydrophilicity. PHBV was codissolved with Ecoflex in dichloromethane at different mass ratios (PHBV/Ecoflex: 100/0, 70/30, 50/50, and 30/70) and electrospun into mats. Compared with the pure PHBV mat, the Ecoflex-containing mats showed decreased contact angles with phosphate-buffered saline (PBS), accelerated weight loss in PBS, and increased strain at break with increasing Ecoflex mass ratios. In vitro cell culture also showed significantly improved adhesion and proliferation of human bone marrow stroma cells with the introduction of Ecoflex. Blending PHBV with Ecoflex is a simple and effective method to improve the chemical, mechanical, and biological properties of PHBV simultaneously and thereby to expedite its application in tissue engineering. To our knowledge, this is the first report showing the biocompatibility of Ecoflex-containing materials with human cells.     

Surface modification using silanated poly(ethylene glycol)s

Surface-grafted poly(ethylene glycol) (PEG) molecules are known to prevent protein adsorption to the surface. The protein-repulsive property of PEG molecules are maximized by covalent grafting. We have synthesized silanated monomethoxy-PEG (m-PEG) for covalent grafting of PEG to surfaces with oxide layers. Two different trialkoxysilylated PEGs were synthesized and characterized. The first trialkoxysilylated PEG was prepared by direct coupling of m-PEG with 3-isocyanatopropyltriethoxysilane through a urethane bond (silanated PEG I). The other silanated PEG (silanated PEG II) containing a long hydrophobic domain between PEG and a silane domain was prepared by reacting m-PEG with 1,6-diisocyanatohexane and 10-undecen-1-ol in sequence before silylation with 3-mercaptopropyl trimethoxysilane. Silanated PEGs I and II were grafted onto glass, a model surface used in our study. The PEG-grafted glass surfaces were characterized by contact angle, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). Although contact angle did not change much as the bulk concentration of silanated PEG used for grafting increased from 0.1 to 20 mg/ml for both PEGs I and II, the surface atomic concentrations from XPS measurements showed successful PEG grafting. Surface PEG grafting increased concentration of surface carbon but decreased silicone concentration. The high resolution C1s spectra showed higher ether carbon with lower hydrocarbon compositions for the PEG-grafted surfaces compared to the control surface. AFM images showed that more PEG molecules were grafted onto the surface as the bulk concentration used for grafting was increased. AFM images of the dried surfaces showed that the surfaces were not completely covered by PEG molecules. After hydration, however, the surface appears to be covered completely probably due to the hydration of the grafted PEG chains. Glass surfaces modified with silanated PEGs reduced fibrinogen adsorption by more than 95% as compared with the control surface. Silanated PEGs provides a simple method for PEG grafting to the surface containing oxide layers.     

Preparation of poly(ethylene glycol) grafted poly(3-hydroxyalkanoate) networks

Poly(3-hydroxyalkanoate)-g-poly(ethylene glycol) crosslinked graft copolymers are described. Poly(3-hydroxyalkanoate)s containing double bonds in the side chain (PHA-DB) were obtained by co-feeding Pseudomonas oleovorans with a mixture of nonanoic acid and anchovy (hamci) oily acid (in weight ratios of 50/50 and 70/30). PHA-DB was thermally grafted with a polyazoester synthesized by the reaction of poly(ethylene glycol) with MW of 400 (PEG-400) and 4,4′-azobis(4-cyanopentanoyl chloride). Sol-gel analysis and spectrometric and thermal characterization of the networks are reported.     

Poly(ether/ester)s based on poly(butylene terephthalate) and poly(ethylene glycol), 1. Poly(ether/ester)s with various polyether: polyester ratios

A series of poly(ether/ester)s 1 based on poly(butylene terephthalate)

1 Systematic IUPAC nomenclature: poly(oxytetramethyleneoxyterephthaloyl).

and poly(ethylene glycol) is synthesized. ¹H and ¹³C nuclear magnetic resonance, infrared and differential scanning calorimetry (DSC) measurements prove a block structure of the prepared copolymers. The DSC curves suggest the existence of a three-phase morphology — two amorphous phases for both polyether and polyester segments, and a crystalline phase for the polyester segments. Small angle X-ray scattering (SAXS) measurements lead to the same conclusion. The degree of crystallinity assigned to the polyester fraction differs insignificantly and is similar to that of the homopolymer. SAXS data show approximately the same long spacing for all crystallizable copolymers. The tensile parameters of the studied polymers are similar to those of available commercial products based on poly(butylene terephthalate) and poly(tetrahydrofuran).