Polyanhydride Copolymers Research Articles

Polyanhydride nanoparticles stabilize pancreatic cancer antigen MUC4β.

Pancreatic cancer (PC) is one of the most lethal malignancies and represents an increasing and challenging threat, especially with an aging population. The identification of immunogenic PC-specific upregulated antigens and an enhanced understanding of the immunosuppressive tumor microenvironment have provided opportunities to enable the immune system to recognize cancer cells. Due to its differential upregulation and functional role in PC, the transmembrane mucin MUC4 is an attractive target for immunotherapy. In the current study we characterized the antigen stability, antigenicity and release kinetics of a MUC4β-nanovaccine to guide further optimization and, in vivo evaluation. Amphiphilic polyanhydride copolymers based on 20 mol % 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane and 80 mol % 1,6-bis(p-carboxyphenoxy)hexane were used to synthesize nanoparticles. Structurally stable MUC4β protein was released from the particles in a sustained manner and characterized by gel electrophoresis and fluorescence spectroscopy. Modest levels of protein degradation were observed upon release. The released protein was also analyzed by MUC4β-specific monoclonal antibodies using ELISA and showed no significant loss of epitope availability. Further, mice immunized with multiple formulations of combination vaccines containing MUC4β-loaded nanoparticles generated MUC4β-specific antibody responses. These results indicate that polyanhydride nanoparticles are viable MUC4β vaccine carriers, laying the foundation for evaluation of this platform for PC immunotherapy.

Synthesis and Characterization of Rapidly Degrading Polyanhydrides as Vaccine Adjuvants.

There is a currently a need to develop adjuvants that are best suited to simultaneously enhance immune responses, induce immunologic memory, improve patient compliance (i.e., reduce doses and inflammation), and provide vaccine shelf stability for stockpiling and global deployment to challenging environments. Biodegradable polyanhydrides have been investigated extensively to overcome such challenges. It has been shown that controlling copolymer composition can result in chemistry-dependent immunomodulatory capabilities. These studies have revealed that copolymers rich in sebacic acid (SA) are highly internalized by antigen presenting cells and confer improved shelf stability of encapsulated proteins, while copolymers rich in 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) also exhibit enhanced internalization by and activation of antigen presenting cells (APCs), in addition to providing superior retention of protein stability following encapsulation and release. However, to date, CPTEG:SA copolymers have not been synthesized and described. In this work, we hypothesized that new copolymers composed of CPTEG and SA would combine the advantages of both monomers in terms of enhanced thermal properties, maintaining antigenicity of encapsulated proteins following nanoparticle synthesis, and superior cellular internalization and activation by APCs, demonstrated by the upregulation of costimulatory markers CD80, CD86, and CD40, as well as the secretion of proinflammatory cytokines IL-6, IL-1β, and TNF-α. Herein, we describe the synthesis and design of novel CPTEG:SA nanoparticles with improved thermal properties, payload stability, and internalization by antigen presenting cells for applications in vaccine delivery. The performance of these new CPTEG:SA formulations was compared to that of traditional polyanhydride copolymers.

Recent Advances in Polyanhydride Based Biomaterials.

This review focusses on recent developments of polyanhydrides, a class of degradable synthetic biopolymers. Polyanhydrides have been used as carriers for controlled delivery of drugs. A polyanhydride copolymer of carboxyphenoxy propane and sebacic acid has been used in Gliadel brain tumor implants for the controlled delivery of carmustine or bis-chloroethylnitrosourea. They are easy and inexpensive to synthesize (especially scale up). However, polyanhydrides possess a short shelf-life. Hydrolytic cleavage and anhydride interchanges lower their molecular weights during storage. One of the highlights in recent developments of polyanhydride chemistry is the discovery of alternating copolymers having extended shelf-life. Other highlights include their applications in biomedical electronics, vaccine delivery, and nano/micro particulate delivery systems. This review examines approaches for polyanhydride synthesis followed by their recent developments in biomedical applications.

In vitro degradation study of polyanhydride copolymers / surface grafted hydroxyapatite composites for bone tissue application

Poly (1,6-bis- (p-carboxyphenoxy hexane)-co- (sebacic anhydride)) (PANH), is a polyanhydride copolymer which has good biocompatibility, and degrades to non-toxic products with a predictable rate of degradation. Nano-sized hydroxyapatite (HAP) is a well-known biomaterial which has been applied in bone regeneration due to its osteoconductivity. However, nano-sized HAP has poor colloidal stability which leads to agglomeration when incorporated into polymeric composites. In this work we describe the surface grafting of poly(ɛ-caprolactone) to HAP (PCL-gHAP) to improve the interfacial adhesion and dispersion of HAP particles in a PANH matrix to form a composite material. The use of scanning electron microscopy-backscattered electron (SEM-BSE) detector and the combination of focused ion beam (FIB)/ transmission electron microscopy (TEM) provided a powerful approach for observing the dispersion of HAP particles in the polymer matrix. We show that surface modification of HAP with PCL improved the homogeneity of the dispersion of HAP particles in the composites and affected the composite morphology during hydrolytic degradation. Composites with high HAP content displayed high compressive strength and a fast rate of degradation. The PCL-gHAP/PANH composites showed superior maintenance of mechanical properties compared with HAP/PANH composites during degradation. A preliminary in vivo study on rat calvaria repair, demonstrated the superior performance of PCL-gHAP/PANH composites. The results suggest that the newly developed PCL-gHAP/PANH composite materials have great potential of serving as a new substrate for bone tissue engineering.

The effect of polyanhydride chemistry in particle-based cancer vaccines on the magnitude of the anti-tumor immune response.

Compared to soluble cancer vaccine formulations, tumor antigens encapsulated in biodegradable polymeric particles have been shown to sustain antigen release and provide long-term protection against tumor challenge by improving the immune response towards the antigen. Treatment of mice with cancer vaccines based on different polyanhydride copolymers encapsulating OVA resulted in stimulation of tumor-specific immune responses with different magnitudes. This clearly indicates that polyanhydride chemistry plays a substantial role in stimulating the immune response. Vaccination with 20:80 CPTEG:CPH/OVA, the most hydrophobic formulation, stimulated the strongest cellular and humoral immune responses and provided the longest survival outcome without adding any other adjuvant. The most important finding in this study is that the copolymer composition of polyanhydride particle-based vaccines can have a direct effect on the magnitude of the antitumor immune response and should be selected carefully in order to achieve optimal cancer vaccine efficacy.

Redox-responsive polyanhydride micelles for cancer therapy

Biodegradable polyanhydrides possess unique features like those that they can predominantly undergo surface erosion, and the payloads can be released by a steady speed. However, there is little work that has been published to describe the polyanhydride micelles with redox-responsiveness as a nanocarrier for drug delivery. In this study, we develop one type of new amphiphilic polyanhydride copolymer containing disulfide bonds between the hydrophilic and hydrophobic segments. The copolymer can self-assemble into stable micelles with well-defined core–shell structure and a uniform size distribution with an average diameter of 69 nm. The disassembly behaviors of the micelles triggered by glutathione are evaluated from the changes of the micellar size, morphology and molecular weight. An approximate zero-order in vitro drug release mode with a fast speed can be achieved in a reducing and acid environment similar with that of tumor cells. In vitro cytotoxicity analysis demonstrate that the Cur-loaded micelles are of great efficiency in inhibiting the growth of cancer cells due to the rapidly intracellular delivery of therapeutic agent. Both the qualitative and quantitative results of the antitumor activity in 4T1 tumor-bearing BALB/c mice reveal that the redox-responsive micelles have a more significant therapeutic effect to artificial solid tumor compared to the redox-insensitive micelles. This study provides a new insight into the biomedical application of polyanhydrides in drug delivery.

Polyanhydride nanoparticle vaccine platform delays tumor growth (P2050)

Abstract Immunotherapies designed to direct the immune system to elimate malignancies is a growing area of research aimed at providing a safe and effective alternative and/or adjunct therapy to chemotherapeutic and surgical interventions. These approaches require the development of immunization regimens that induce efficacious cell-mediated immunity directed at antigens expressed by tumor cells. Particles consisting of polyanhydride copolymers based on 1,6-bis(p-carboxyphenoxy) hexane (CPH), and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctante (CPTEG) have been shown to enhance dendritic cell activation, which in turn induces activation and proliferation of antigen-specific CD4+ and CD8+ T cells in vitro. We demonstrate that immunization of mice with a polyanhydride nanoparticle-based vaccine platform induced a potent immune response to a specific tumor-associated model antigen in order to control tumor progression in vivo. Specifically, we challenge immunized mice with a transgenic tumor cell line, expressing ovalbumin (Ova) as a target antigen, to determine if our vaccination strategy is effective. Mice immunized once with a regimen consisting of soluble Ova and Ova encapsulated in 20:80 CPTEG:CPH nanoparticles exhibited delayed tumor growth during the course of our study when compared to mice immunized with soluble Ova adjuvanted with Alum, soluble Ova alone, or PBS. Mice that receive the nanoparticle formulation also have improved survival rates when compared other formulations.

Polyanhydride copolymer and bioceramic composites as bone substitutes

Background/PurposeDurable mechanical strength and biocompatibility are the two major requirements for osteogenic scaffolds. Polyanhydrides are a class of biodegradable polymers characterized by anhydride bonds that connect repeating units of the polymer backbone chain. Hydroxyapatite (HAP) is the main component of human bone and is a good osteoinductive factor that promotes bone mineralization. This work validates the combination of polyanhydrides and HAP for biomedical application. MethodsPolyanhydride copolymers were fabricated from sebacic acid (SA) and 1,6-bis(p-carboxyphenoxy)hexane (CPH). HAP was surface-modified by polycaprolactone (PCL), and testing tablets were made using different ratios of copolymers and surface-grafted HAP (g-HAP). Degradation tests were performed to evaluate mechanical strength, pH, and weight loss. Biocompatibility was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and live/dead stain test. Cell affinity was measured using scanning electronic microscopy (SEM). ResultsThe favorable surface erosion property of polyanhydrides prevented marked changes in the mechanical properties over time. In addition, the degradation byproducts of the copolymer did not cause a serious decline in pH and were less harmful to the cells. g-HAP increased cell affinity for the polymer surface. ConclusionThe research team synthesized polyanhydride/g-HAP composites with high mechanical strength, slow degradation, and excellent biocompatibility. The result showed that a CPH/SA ratio of 7:3 in combination with 10 wt% g-HAP was optimal as bone substitute.

Characterizing the antitumor response in mice treated with antigen-loaded polyanhydride microparticles

Delivery of vaccine antigens with an appropriate adjuvant can trigger potential immune responses against cancer leading to reduced tumor growth and improved survival. In this study, various formulations of a bioerodible amphiphilic polyanhydride copolymer based on 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and 1,6-bis(p-carboxyphenoxy) hexane (CPH) with inherent adjuvant properties were evaluated for antigen-loading properties, immunogenicity and antitumor activity. Mice were vaccinated with 50:50 CPTEG:CPH microparticles encapsulating a model tumor antigen, ovalbumin (OVA), in combination with the Toll-like receptor-9 agonist, CpG oligonucleotide 1826 (CpG ODN). Mice treated with OVA-encapsulated CPTEG:CPH particles elicited the highest CD8+ T cell responses on days 14 and 20 when compared to other treatment groups. This treatment group also displayed the most delayed tumor progression and the most extended survival times. Particles encapsulating OVA and CpG ODN generated the highest anti-OVA IgG1 antibody responses in mice but these mice did not show significant tumor protection. These results suggest that antigen-loaded CPTEG:CPH microparticles can stimulate antigen-specific cellular responses and could therefore potentially be used to promote antitumor responses in cancer patients.

Preparation and Characterization of Biodegradable Electrospun Polyanhydride Nano/Microfibers

The biodegradable polyanhydride copolymers P(CPP-SA) composed of p-carboxyhenoxy propane (CPP) and sebacic acid (SA) at weight ratios of 20:80, 35:65 and 50:50 were polymerized by a melt polycondensation process without catalyst. The copolymers were characterized by fourier transform infrared spectroscopy (FT-IR), 1H-nuclear magnetic resonance (1H-NMR), Ubbelohde viscometer, differential scanning calorimetry (DSC) and wide angle X-ray powder-diffraction (XRD). P(CPP-SA) nano/microfibers were the first time to be fabricated by electrospinning. The copolymers hold an excellent fibre-forming performance and the diameter range of 80-3,200 nm can be obtained. The in vitro degradation of the polyanhydride copolymers was evaluated in form of the nano/microfibers by investigating the change of fibrous morphology, weight loss and pH change of degradation medium. The experimental results showed that degradation rate was fast in the fist day and slow in the following period, furthermore the degradation rate decreased with the increase of the content of CPP in copolymers. Therefore, the electrospun polyanhydride nano/microfibers exhibited strong potential as drug delivery vehicle and tissue engineering scaffold.

Novel, High Throughput Method to Study in Vitro Protein Release from Polymer Nanospheres

Controlled delivery of therapeutic protein drugs using biodegradable polymer carriers is a desired characteristic that enables effective, application-specific therapy and treatment. Previous studies have focused on protein delivery from polymers using conventional "one-sample-at-a-time" techniques, which are time-consuming and costly. In addition, many therapeutic proteins are in limited supply and are expensive, so it is desirable to reduce sample size for design and development of delivery devices. We have developed a rapid, high throughput technique based on a highly sensitive fluorescence-based assay to detect and quantify protein released from polyanhydrides while utilizing relatively small amounts of protein (approximately 40 microg). These studies focused on the release of a model protein, Texas Red conjugated bovine serum albumin, from polyanhydride copolymers based on sebacic acid (SA) and 1,6-bis(p-carboxyphenoxy)hexane (CPH). The protein release profiles were assessed simultaneously to investigate the effect of polymer device geometry (nanospheres vs films), polymer chemistry, and pH of the release medium. The results indicated that the nanosphere geometry, SA-rich chemistries, and neutral pH release medium led to a more rapid release of the protein compared to the film geometry, CPH-rich chemistries, and acidic pH release medium, respectively. This high throughput fluorescence-based method can be readily extended to study release kinetics for other proteins and polymer systems.

Polymer Chemistry Influences Monocytic Uptake of Polyanhydride Nanospheres

To demonstrate that polyanhydride copolymer chemistry affects the uptake and intracellular compartmentalization of nanospheres by THP-1 human monocytic cells. Polyanhydride nanospheres were prepared by an anti-solvent nanoprecipitation technique. Morphology and particle diameter were confirmed via scanning election microscopy and quasi-elastic light scattering, respectively. The effects of varying polymer chemistry on nanosphere and fluorescently labeled protein uptake by THP-1 cells were monitored by laser scanning confocal microscopy. Polyanhydride nanoparticles composed of poly(sebacic anhydride) (SA), and 20:80 and 50:50 copolymers of 1,6-bis-(p-carboxyphenoxy)hexane (CPH) anhydride and SA were fabricated with similar spherical morphology and particle diameter (200 to 800 nm). Exposure of the nanospheres to THP-1 monocytes showed that poly(SA) and 20:80 CPH:SA nanospheres were readily internalized whereas 50:50 CPH:SA nanospheres had limited uptake. The chemistries also differentially enhanced the uptake of a red fluorescent protein-labeled antigen. Nanosphere and antigen uptake by monocytes can be directly correlated to the chemistry of the nanosphere. These results demonstrate the importance of choosing polyanhydride chemistries that facilitate enhanced interactions with antigen presenting cells that are necessary in the initiation of efficacious immune responses.

Synthesis and characterization of novel poly(sebacic anhydride- co-Pluronic F68/F127) biopolymeric microspheres for the controlled release of nifedipine

Amphiphilic block copolymers composed of prepoly(sebacic anhydride) and Pluronic-F68/F127 have been synthesized in different molar compositions via melt-polycondensation reaction. Poly(sebacic anhydride- co-PLF68/PLF127) thus formed was characterized by Fourier transform infrared spectroscopy (FTIR), gel permeation chromatography (GPC) and nuclear magnetic resonance spectroscopic (NMR) techniques. The amphiphilic block copolymers were used to prepare microspheres and to encapsulate nifedipine (NFD) by the solvent evaporation technique. Differential scanning calorimetry (DSC) was used to confirm the incorporation of Pluronic into polyanhydrides, while X-ray diffraction (XRD) was performed on the drug-loaded microspheres to investigate the crystalline nature of the drug after encapsulation. Scanning electron micrograph (SEM) pictures indicated spherical nature of the microspheres. Microspheres obtained were in the size range of 10–50 μm as measured by the laser particle size analyzer. In vitro release studies of NFD from poly(sebacic anhydride- co-Pluronic-F68/F127) microspheres performed in pH 7.4 phosphate buffer indicated sustained release rates of NFD at higher amounts of Pluronic in polyanhydride copolymers; there was no significant difference obtained between the release patterns of NFD from Pluronic-F68 and Pluronic-F127 copolyanhydride microspheres when same amount of Pluronics were used.

Parallel Synthesis and High Throughput Dissolution Testing of Biodegradable Polyanhydride Copolymers

We have demonstrated that polycondensation reactions can be carried out in a combinatorial fashion and that the polymer library can be screened at high throughput using a rapid prototyping technique to fabricate multiwell substrates. A linearly varying compositional library of 100 different biodegradable polyanhydride random copolymers that are promising carriers for controlled drug delivery was designed, fabricated, and characterized by IR microscopy within a few hours. The polyanhydride copolymer library was based on 1,6-bis(p-carboxyphenoxy)hexane (CPH) and sebacic anhydride (SA) and was characterized with infrared microspectroscopy to determine the composition within each well. Since degradation and release rates depend on copolymer composition, we also developed new high-throughput methods to investigate drug release from this library of copolymers by designing specific wells for each task. A subset of this library was chosen, and a substrate was designed and fabricated to enable the synthesis and monitoring of dye dissolution from a range of polyanhydride copolymers in a parallel fashion using a CCD camera. Multisample substrates were fabricated with a novel rapid prototyping method that consists of an organic solvent-resistant array of 10 x 10 microwells of 2-muL volume each. The libraries were deposited with a custom-built liquid dispensing system consisting of a series of computer-controlled volume-dispensing pumps and XYZ motion stages. The parallel dye dissolution study displayed a decreasing rate of release with increasing CPH content. This result agrees with previously published data for dye release from poly(CPH-co-SA) copolymers. The methodology described in this work is amenable to numerous applications in the arenas of high-throughput polymer synthesis and characterization.

Synthesis and characterization of novel polyanhydrides with tailored erosion mechanisms

We have designed a new synthesis route to create polyanhydrides based on monomers that contain hydrophilic entities within highly hydrophobic backbones. The method results in polyanhydrides that can be easily processed into drug-containing tablets. The synthesis, characterization, and erosion studies of polyanhydride copolymers based on 1,6-bis(p-carboxyphenoxy)hexane (CPH), which is highly hydrophobic, and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG), which has hydrophilic oligomeric ethylene glycol segments in the monomer unit, was performed using a combination of molecular spectroscopy, thermal analysis, gravimetry, and scanning electron microscopy. The studies demonstrate that by increasing the CPH content in the CPTEG:CPH copolymers, the erosion of the system can be tailored from bulk-eroding to surface-eroding mechanism. These systems have promise as protein carriers.

Molecular Description of Erosion Phenomena in Biodegradable Polymers

A new model for the erosion kinetics of semicrystalline surface-erodible homopolymers and copolymers is presented. The model is derived for a class of surface-erodible polyanhydride copolymers, with the goal of describing erosion in terms of fundamental, elementary processes. This model is based on an accurate description of copolymer microstructure and can thereby account for the heterogeneous erosion due to microphase separation and crystallinity. In addition to accurately predicting the overall erosion profile and the release of individual monomer species, several key phenomena that occur during erosion are described. These include precipitation of slightly soluble degradation products inside the pores of the erosion zone and pH changes during erosion due to dissolution of acidic monomers and the consequent changes in monomer solubility. This model also motivates future experiments to investigate predicted phenomena such as the effects due to local changes in pH and degradation rate constants for cryst...

Morphology of polyanhydride copolymers: Time‐resolved small‐angle X‐ray scattering studies of crystallization

AbstractSynchrotron small‐angle X‐ray scattering (SAXS) was used to study the isothermal crystallization kinetics of a family of polyanhydride copolymers consisting of 1,6‐bis(p‐carboxyphenoxy)hexane and sebacic acid monomers. In situ SAXS experiments permitted the direct observation of the crystallization kinetics. The structural parameters (the long period, lamellar thickness, and degree of crystallinity) were obtained from Lorentz‐corrected intensity profiles, one‐dimensional correlation functions, and interface distribution functions to form a comprehensive picture of the crystal morphology. The combination of these three analyses provided information not only on the lamellar dimensions but also on the polydispersity (nonuniformity) of these dimensions. Where possible, the crystallization kinetics were interpreted with a modified version of the Avrami equation. The results can be used to perform the rational design of controlled‐drug‐release formulations because crystallinity affects drug‐release kinetics. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 43: 463–477, 2005

The Effect of Storage Temperatures on the In Vitro Properties of a Polyanhydride Implant Containing Gentamicin

ABSTRACTSeptacin® is a biodegradable sustained-release implant containing 20% (w/w) gentamicin sulfate. The matrix of the implant is a polyanhydride copolymer composed of erucic acid dimer (EAD) and sebacic acid (SA) in a one-to-one weight ratio. The effect of storage temperatures (−15°C and 25°C) on the stability of Septacin® was evaluated with respect to gentamicin potency, copolymer molecular weight, and in vitro drug release. The drug in polymer matrix was stable for at least 12 months when stored at 25°C, but the molecular weight of the copolymer declined rapidly at this temperature. At −15°C, there was no change in the molecular weight of the copolymer. However, the placebo (copolymer without gentamicin) exhibited a significant drop in copolymer molecular weight at both temperatures. The drug release profiles showed no change for samples stored at −15°C for the duration of this study, while the release of drug slowed down significantly for samples stored at 25°C for longer than one month. A pronounced difference in the morphology of the −15°C samples and the 25°C samples was observed during the in vitro dissolution test; cracking of the −15°C samples was evident, but the 25°C samples remained intact.

Characterization of chemical structure and morphology of eroding polyanhydride copolymers by liquid-state and solid-state [formula omitted] n.m.r.

An n.m.r. investigation of the chemical and morphological changes occurring during erosion in pH 7.4 phosphate buffer at 37°C of melt-fabricated polyanhydride devices formed from copolymers of 1,3-bis( p-carboxyphenoxy)propane (CPP) and sebacic acid (SA) is reported. From sequence distributions determined using solution-state 1 H n.m.r. spectroscopy, the following order of anhydride bond lability was determined: SA–SA≈SA–CPP≫CPP–CPP, leading to preferential hydrolysis of aliphatic blocks within the copolymer chain. Solid-state 1 H and 2 H n.m.r. spectroscopy were used to investigate morphological changes occurring during erosion. Both indicated significant absorption of mobile water after ca. 10 h. Subtle variations in the symmetry of the narrow signals confirmed the changes in chemical composition apparent from high resolution 1 H n.m.r. analysis. In the initial stages, there was an increase in crystallinity attributed to preferential erosion of amorphous regions.

Degradation of double-walled polymer microspheres of PLLA and P(CPP : SA)20 : 80. I. In vitro degradation

Double-walled (DW) polymer microspheres with a core of poly(1,3-bis-( p-carboxyphenoxy propane)-co-(sebacic anhydride)20 : 80 (P(CPP : SA)20 : 80) and an external coat of poly(L-lactic acid) (PLLA) were degraded for up to 6 months in vitro. The effects of hydrolytic degradation on the polymers were studied by Fourier-transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), gel permeation chromatography (GPC) and scanning electron microscopy (SEM). The inner core of the polyanhydride copolymer (P(CPP : SA)20 : 80) degraded first, breaking down into oligomers during the first week and then into monomers which were trapped by the outer shell of PLLA, crystallized, and remained in the core for the duration of the study. As expected, the PLLA coat degraded at a slower rate due to the ester bonds between lactic acid monomers being more hydrolytically resistant than the anhydride bonds. The PLLA in the DW microspheres decreased from its original molecular weight ( M w) of 24,000 g mol -1 to approximately 5,000 g mol -1 over the 6 month study. The melting temperature of the PLLA decreased more than 30°C during the study, but no changes were observed in the FTIR spectra. The 6 month samples were very brittle, showing the concomitant drop in mechanical strength with the decrease in M w.