Photopolymerized Cross-Linked Thiol–Ene Polyanhydrides: Erosion

Article pubs.acs.org/Biomac

Photopolymerized Cross-Linked Thiol−Ene Polyanhydrides: Erosion, Release, and Toxicity Studies Katie L. Poetz,† Halimatu S. Mohammed,† Brittany L. Snyder,† Garrett Liddil,† Damien S. K. Samways,‡ and Devon A. Shipp*,†,§ †

Department of Chemistry and Biomolecular Science, ‡Department of Biology §Center for Advanced Materials Processing Clarkson University, Potsdam, New York 13699-5810, United States S Supporting Information *

ABSTRACT: Several critical aspects of cross-linked polyanhydrides made using thiol−ene polymerization are reported, in particular the erosion, release, and solution properties, along with their cytotoxicity toward fibroblast cells. The monomers used to synthesize these polyanhydrides were 4-pentenoic anhydride and pentaerythritol tetrakis(3-mercaptopropionate). Techniques used to evaluate the erosion mechanism indicate a complex situation in which several phenomena, such as hydrolysis rates, local pH, water diffusion, and solubility, may be influencing the erosion process. The mass loss profile, the release rate of a hydrophilic dye, the rate of hydrolysis of the polyanhydride, the hydrolysis product solubility as a function of pH, average pKa and its cytotoxicity toward fibroblast cells were all determined. The solubility of the degradation product is low at pH values less than 6−7, and the average pKa was determined to be ∼5.3. The cytotoxicity of the polymer and the degradation product was found to be low, with cell viabilities of >97% for the various samples studied at concentrations of ∼1000−1500 ppm. These important parameters help determine the potential of the thiol−ene polyanhydrides in various biomedical applications. These polyanhydrides can be used as a delivery vehicle, and although the release profile qualitatively followed the mass loss profile for a hydrophilic dye, the release rate appears to be by both diffusion and mass loss mechanisms.

1. INTRODUCTION Degradable polymer systems have been widely studied since the 1960s when the first biomedical devices composed of such polymers were commercially produced. 1−4 These early polymers were typically polyesters and more often than not were composed of lactic acid and glycolic acid repeat units.5 The degradation of polyesters occurs through the hydrolysis of the ester linkage, thus, yielding products that contain acid groups.6−10 Other degradable polymer systems, such as polyorthoesters and polyanhydrides, also undergo hydrolysis and produce acid-containing degradation products.11−14 Regardless of the type of product formed, predictable degradation, cytocompatibility and biocompatibility are important for a host of applications, including drug release. Thus, much effort has been expended in the design and synthesis of new degradable polymers over the past few decades. Degradability is not the only important process in the mass loss of polymeric material as other processes such as dissolution may also be important. Thus, a better description of mass loss is erosion, which is mass loss from any process, including degradation from bond cleavage. Erosion may be described by two idealized mechanisms: bulk erosion or surface erosion.6,14,15 In many real systems, however, the erosion mechanism may be both or change from predominately one to the other.16,17 Bulk erosion of polymers is characterized by slow degradation and relatively fast water uptake such that mass loss occurs throughout the entire sample. A classic example of © 2014 American Chemical Society

materials that typically undergo such a homogeneous erosion mechanism are polyesters, for example, poly(glycolic acid)s and poly(glycolic acid-co-lactic acid)s.18 In contrast, surface eroding polymers exhibit a heterogeneous erosion mechanism in which hydrolysis occurs quickly at the surface while water diffusion into the sample is relatively slow. Examples of polymers that may undergo such a mechanism include polyanhydrides and polyorthoesters. We have recently developed a new method to synthesize photo-cross-linked polyanhydrides using thiol−ene polymerization (Scheme 1).19−21 These polymers appear to exhibit a surface erosion mechanism throughout most of the erosion process.22 The thiol−ene polymerization approach brings many benefits to polyanhydride synthesis compared to previous preparation methods that relied on condensation reactions, which required the removal of small byproducts, such as water12 or methacrylate-based chain polymerizations,7,23 which required complex monomer synthesis24 and resulted in heterogeneously cross-linked polyanhydrides with much less than 100% conversion of monomer to polymer. In contrast, thiol−ene polymerization allows for photoinitiation to be used, giving polymerization times of the order of minutes and temporal and spatial control and can be conducted at ambient Received: March 19, 2014 Revised: May 8, 2014 Published: May 21, 2014 2573

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Scheme 1. Thiol−Ene Polymerization Approach to Give Cross-Linked Polyanhydrides, Illustrated Using 4-Pentenoic Anhydride (PNA), Pentaerythritol Tetrakis(3-mercaptopropionate) (PETMP), and Subsequent Degradation via Hydrolysis

PETMP), 4-pentenoic acid (97%), 1-hydroxycyclohexyl phenyl ketone (99%), anhydrous chloroform, 4-hydroxybenzoic acid, and acid orange 8 (AO8). 2.2. Instrumentation. 1H (400 MHz) and 13C (100 MHz) NMR spectroscopy were performed on Bruker Avance 400 with a BBO probe. The UV light source for curing was an Oriel Instruments, Model 68811, 500 W mercury−xenon arc lamp (intensity ∼70 mW/ cm2 measured by a Dymax Corp. Accu-Cal-30 intensity meter). Ultraviolet−visible spectroscopy (UV−vis) was performed on Agilent 8453 spectrometer. The microscope used for images was an Olympus optical microscope. ATR-FTIR spectra were collected on a Bruker Vector 22 infrared instrument equipped with Pike Technologies ATR apparatus. A MIRacle ZnSe crystal was used. 2.3. Example Procedure for the Synthesis of Cross-Linked Polyanhydrides. A typical procedure for the synthesis of thiol−ene cross-linked polyanhydrides is as follows. 1-Hydroxycyclohexyl phenyl ketone (photoinitiator, 0.6 mg, 0.0029 mmol, 0.1 wt %) was weighed and placed into a vial. PNA (0.26 mL, 0.0014 mol) was transferred by a syringe into the vial, followed by PETMP (0.27 mL, 0.0007 mol). The monomers and initiator were mixed to establish a relatively homogeneous mixture and transferred to a polydimethylsiloxane (PDMS) mold (10 mm × 10 mm × 2 mm) and placed under the UV light for 15 min. For samples containing AO8, the AO8 was weighed (6 mg, 0.0165 mmol, 1 wt %) and placed into a scintillation vial with PETMP. The photoinitiator was placed into a separate vial, followed by the addition of PNA. Once the dye was dissolved, the monomers were transferred to a single vial and mixed until a relatively homogeneous mixture was established. The samples containing dye were cured for 30 min and washed in phosphate buffered saline (PBS) solution for 30 min prior to degradation (PBS solution was pH = 7.4). During this washing process, 5 mL aliquots were used and replenished every 3 min. 2.4. Erosion and Release Studies. 2.4.1. Degradation of CrossLinked Polyanhydrides in PBS Solution Containing 0.1 wt % Acid Orange 8. Polyanhydrides were synthesized by the aforementioned procedure. The initial mass of polyanhydride was recorded, and the sample placed into 100 mL PBS solution at pH = 7.4 containing 0.1 wt

temperatures without the need for significant water or oxygen exclusion.25−27 Thiol−ene polymerization also gives essentially 100% conversion of monomer functionality (thiols and enes), and there are many commercially available monomers. Thus, the new thiol−ene polymerization approach for the production of polyanhydrides is both facile and highly modular, utilizing the “click” chemistry characteristics of thiol−ene reactions, and has significant potential in a variety of areas but, in particular, biomedical devices and similar applications. Notwithstanding the significant benefits of synthesizing polyanhydrides using thiol−ene polymerization, there are several critical areas that have not been explored but need to be understood before these materials can be seriously considered for use in biomedical applications. For example, a complete analysis of degradation and erosion rates, ability to release drug/drug mimics, degradation product solubility and cytotoxicity of polymer and degradation product are important parameters that will have a bearing on the usability of these materials. Hence, the work described here focuses on the erosion, release and degradation product from the cross-linked polyanhydrides synthesized from thiol−ene polymerization. More specifically, we examine erosion kinetics and study release of a dye (acid orange 8, AO8). Additionally, several other important parameters are studied, including the rate of hydrolysis of the polyanhydride, the solubility as a function of pH, the average pKa of the degradation product, and the cytotoxicity of the polymer and degradation product.

2. EXPERIMENTAL SECTION 2.1. Materials and Methods. The following materials were purchased from Sigma-Aldrich and were used as received after being characterized by 1H NMR spectroscopy: 4-pentenoic anhydride (98%, PNA), pentaerythritol tetrakis(3-mercaptopropionate) (98%, 2574

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% AO8. Each hour, the mass of the polymer was recorded after removing excess water on the surface and buffer changed to maintain a pH of 7.4. After 4, 8, 10, 12, 14, and 16 h, cross-section images of the polymer were collected using an Olympus microscope. From these images, the depth of AO8 penetration from the surface was determined. 2.4.2. Example Procedure for the Degradation of Cross-Linked Polyanhydrides for ATR-IR Analysis. A polyanhydride sample was synthesized in a 1 cm3 Teflon mold and degraded in 100 mL of PBS solution at pH = 7.4. At 15, 30, and 60 h, the sample was removed from the buffer and the surface and interior were analyzed by ATRFTIR. These degradations were performed at room temperature. 2.4.3. Degradation of Cross-Linked Polyanhydrides With and Without AO8. A typical procedure for the degradation of thiol−ene cross-linked polyanhydrides is as follows. After curing, the sample was removed from the PDMS mold, the mass of polyanhydride recorded, and the sample placed into 100 mL of PBS solution at pH = 7.4 in a sealed vessel and the vessel placed into a 37 °C water bath for degradation. The mass of the polymer was recorded each hour after quickly removing any excess surface water; a portion of the buffer solution saved for UV−vis analysis and new buffer solution was added to the vessel containing the sample. This was repeated until the polymer had completely degraded. The percentage of the mass of the polymer remaining was determined from the mass reading taken each hour. The concentration of the dye was determined through UV−vis analysis at a wavelength of 495 nm using an extinction coefficient of 28.14 L mmol−1 cm−1 for the absorption of AO8. 2.5. Polyanhydride Hydrolysis Kinetics. A polyanhydride sample was synthesized in a PDMS mold (10 mm × 10 mm × 2 mm) and degraded at 37 °C in deionized water, with a starting pH 7.4. The mass of the polymer and pH of water were measured every 15 min for 5 h. 2.6. Cytotoxicity Studies. 2.6.1. Cell Culture. The in vitro cytotoxicity properties of PETMP/PNA polyanhydride and its degradation products were investigated using cultured human dermal fibroblast adult (HDFa) cells via the propidium iodide (PI) exclusion assay. HDFa cells (obtained from Dr. C. Woodworth, Clarkson University, NY) were cultured in RPMI 1640 growth medium supplemented with 10% fetal bovine serum and 1% penicillin− streptomycin (Gibco Life Technologies). Cell cultures were incubated in a humidified atmosphere with 5% CO2 at 37.0 °C. To access cytotoxicity of PETMP/PNA polyanhydride, we used the following three methods: (1) seeding cells onto polyanhydride-coated wells, (2) adding the in situ degraded polymer degradation product to otherwise healthy adherent cells proliferating in 6-well plates, and (3) adding a synthetic model degradation product of the polyanhydride to adherent cells proliferating in 6-well plates. For the first method (1), the polyanhydride substrate was formed by adding a monomer/polymeric mixture into each well of a 6-well plate at a concentration of approximately 1500, 6000, and 12000 ppm of material and cured under UV lamp (λ = 365 nm) for 30 min (1 μg/mL ∼ 1 ppm). Polymeric materials were then further sterilized under UV light in a closed and sterile cell culture cabinet for 1 h. To each well, 1.8 mL of fresh growth medium was added along with 200 μL of HDFa cell suspension, giving a cell density of 6.0 × 105 cells/well. For method (2), the polyanhydride was sterilized in the UVilluminated cell culture cabinet for 1 h. Growth medium (10 mL) was added to the polymer and allowed to degrade in situ at 37 °C for 48 h. Degradation product (either ∼1200, ∼6000, or ∼12000 ppm), 200 μL of HDFa cell suspension, and fresh medium were added to each well of a 6-well plate giving a cell density of 6.0 × 105 cells/well. For method (3), a model degradation compound (2) was synthesized (see Supporting Information). Approximately 1000 and 10000 ppm of the model degradation product was added to each well and sterilized under UV light for 1 h. Cells (6.0 × 105 cells/well) were seeded into 6-well plates with fresh medium containing the added model degradation compound. In control experiments, cells were seeded into wells in the absence of the polymer or degradation product as positive control samples. Negative controls consisted of high concentrations (>9000 ppm) of the polymer or degradation

materials. The viability results represent the mean with standard deviations of three or more assays (n > 3). For each method, cell viability was assessed via PI exclusion assay after 24 h of incubation using fluorescence microscopy. 2.6.2. Fluorescence Imaging. After incubation under the various treatment regimens, the cell medium was removed and cells washed with PBS containing 2 mM CaCl2 before being incubated at room temperature for 5 min with 1 mL of the PBS supplemented with the cell viability dye, 3 μM PI (Sigma-Aldrich, St. Louis, MO). The 6-well plate was transferred to the stage of a Nikon TE200 inverted epifluorescence microscope for imaging. Images were acquired using a Hamamatsu Photonics camera and PI staining detected by excitation with a mercury arc lamp with the appropriate fluorescence filters. For each well, three or more fields of view (FOVs) were captured through a 10× objective in order to ensure that at least 100 cells in total could be counted per well. For each FOV, one image was taken using light microscopy in order to identify the total number of cells present, and one image was taken using the PI fluorescence filter to count dead cells. Dead cells were identified by their red-stained nuclei, indicative of PI uptake. The number of dead cells was then taken as a percentage of the total in a given well, and the data from three or more experiments was pooled to produce a mean and standard deviation for cell viability under a given treatment condition.

3. RESULTS 3.1. Synthesis and Experiment Design. The polyanhydrides studied here were synthesized using thiol−ene photopolymerizations.19,20 Thiol−ene reactions have been classified as a “click” reaction and have been shown to be easy to perform, be orthogonal to various functionalities, and provide materials with a range of properties and, therefore, may be a good candidate for the synthesis of drug delivery vehicles or other biomaterials.25−27 Thiol−ene polymerizations occur via a step-growth mechanism, which leads to low molecular weight polymer being present throughout most of the polymerization until high conversion is achieved. This allows for the network structure to be more homogeneous relative to those formed by chain-growth polymerizations, such as found in radical polymerizations of dimethacrylates, which tend to have limited conversions and produce variable cross-link densities within the polymer network.28,29 Furthermore, because the anhydride group resides in the main chain of the polymer, degradation yields relatively low molecular weight products. Finally, and perhaps most importantly, these thiol−ene-based polyanhydrides exhibit one of the main attractions of polyanhydrides in that they undergo surface erosion. In this study we examined four aspects of the thiol−ene polyanhydrides: (1) the erosion and degradation process, (2) the release kinetics of a model compound (a dye, AO8), (3) the degradation product, and (4) the cytotoxicity of the polyanhydrides and degradation product in the presence of fibroblast cells. For all studies, the thiol−ene polyanhydrides were made with the thiol:ene functionality ratio at 1:1. The dye release studies we examined with a 1 wt % dye loading. These erosion studies were repeated six times, with the average and 95% confidence limits being reported in the figures. The solution conditions used were phosphate buffered saline (PBS) at a pH of 7.4 at 37 °C, unless otherwise noted. 3.2. Erosion and Dye Release Kinetics. The erosion (mass loss) and dye release kinetics for two polymer samples (with and without dye) are shown in Figure 1. These samples were ∼2 × 10 × 10 mm cuboids. For the erosion measurements, during the initial 8 h of degradation, the polymer samples exhibit a period where they undergo minor swelling and gain approximately 1% of the initial starting mass. 2575

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The period from 20 h until the polymer is completely eroded (12. The pKa of the carboxylic acids in the degradation product is an important factor in terms of the solubility of the degradation product, thus we followed the methods provided by Burns et al.,31 to determine the average pKa of the isolated tetraacid product (1). This was achieved by titrating the tetraacid in an acetonitrile/water cosolvent system at three different volume fractions of solvent: 35, 40, and 45% acetonitrile. Analysis of the data (see Figures SI-6−SI-10) yielded an average pKa of 5.3. This is comparable to the pKas of some common di and tri carboxylic acids; for example, succinic acid (4.19, 5.48),32 glutaric acid (4.34, 5.42),32 adipic acid (4.42, 5.41),32 and citric acid (3.09, 4.75, 5.41).33 The average pKa (∼5.3) determined in the current study correlates with our observations that the degradation product has limited solubility below pH values of ∼6 since the more ionized form of the degradation product will be more soluble in water than the nonionized form. Conversely, increasing the pH well into the basic region (>9) improves solubility greatly. 3.6. Cytotoxicity Studies. For a material to be useful in medical applications, for example, as a substrate for cell or tissue grafting or as a vehicle for delivery of therapeutic agents, it is essential that the material is nontoxic both in vitro and in vivo. Fibroblast cells have been recommended by ASTM and is used as a standard cell type for cytocompatibility testing of biomaterials.34,35 Thus, we used HDFa, a fibroblast cell type which is a known immortal and a standard cell for determining the in vitro cytotoxicity properties of the polyanhydrides.35,36 We completed the cytotoxicity studies using three methods: (1) culturing HDFa cells to PETMP/PNA polyanhydride coated surface; (2) growing cells in the presence of degradation product of PETMP/PNA polyanhydride degraded in situ (i.e., degraded within the cell growth medium); and (3) in the presences of the synthesized model degradation product (2). Cell viability was determined via the PI exclusion assay by

Figure 5. Composite fluorescence microscope images of HDFa cells (A) control cells (cells alone). HDFa cells treated with (B) ∼1000 ppm model degradation product, (C) ∼1200 ppm in situ degraded polyanhydride, and (D) ∼1500 ppm PETMP/PNA polyanhydride material (10× magnification).

When cells were exposed to the different materials, we observed 98.8 ± 1.13% cell viability, 97.2 ± 0.98% cell viability and 97.8 ± 1.4% cell viability for ∼1000 ppm model degradation product (Figure 5B), ∼1200 ppm degradation products from in situ degraded polymer (Figure 5C) and ∼1500 ppm polyanhydride (Figure 5D), respectively. Light microscopy revealed that the morphology of cells remained unchanged when exposed to tested samples (polymer, in situ degraded polymer and synthesized model degradation compound 2; Figure 5). Therefore, based on these tested conditions, both the polymer and its degradation products have very good cytocompatibility. To ensure that the assay works and to confirm these results, we tested two much higher concentrations of polymer (6000 and 12000 ppm) in the presence of HDFa cells (Figure 6). HDFa cells exposed to approximately 6000 and 12000 ppm of the PETMP/PNA polyanhydride exhibited clear signs of

Figure 6. Overlaid light microscopy and fluorescence images of HDFa cells exposed to (A) ∼6000 ppm PETMP/PNA polyanhydride with 53.0% ± 18% cell viability and (B) ∼12000 ppm PETMP/PNA polyanhydride showing 15.6 ± 2% cell viability. The red fluorescence shows the nuclei of dead cells stained with PI (10× magnification). 2578

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sample is exposed to the atmosphere during the ATR-FTIR measurement, it may also be that acid formation is occurring in the interior through hydrolysis or thioester formation. Since the latter should only occur if there are free thiols to react with the anhydride, which is not likely given these polymerizations reach very high (>99%) conversions, then it appears that some amount of water must be reaching the interior of the sample resulting in some hydrolysis, even though the dye penetration experiments do not indicate significant water diffusion into the sample at later times. The ATR-FTIR data and dye penetration data may be reconciled if the dye penetration technique is not sensitive enough to show the erosion front accurately. The rate of anhydride hydrolysis may also contribute to the complex erosion behavior. Anhydride hydrolysis is known to be base-catalyzed and acid-catalyzed, although not to the same degree as esters or amides.37 However, some mineral acids, because of salt effects, can even retard hydrolysis of anhydrides.38,39 Additionally, the mechanism for hydrolysis can change from bimolecular (so-called A2 mechanism, where A indicates nucleophilic attack at the acyl carbon by water) to unimolecular (A1, where the acylium ion forms first and is the rate-determining step), depending on water and acid concentration.40 Hence, the hydrolysis of anhydrides in the present system is not likely to be straightforward since parameters such as the water concentration gradient in the polymer matrix, carboxylic acid formation, and polarity and pH changes as the polymer degrades, may contribute to complex degradation and erosion processes. The idea that autocatalysis is a major factor in determining the erosion profile is also supported by the data we collected regarding the average pKa of the tetraacid degradation product (∼5.3), hydrolysis rates, solubility, and dye penetration (water diffusion). The average pKa of the degradation product is typical for what would be expected for a multicarboxylic acid compound. Its solubility is also unsurprisingly limited at low pH, when the acid groups are protonated, while at physiological pH (∼7.4) and above the degradation product is reasonably soluble because of the dominance of the carboxylate groups. What this may mean during the erosion process is that for complete dissolution of the degradation product the local pH should not be too low; it is possible that obtaining a sufficiently high pH to dissolve the degradation product takes some time to achieve since the local pH will initially be lowered because of the formation of the carboxylic acid groups and diffusion of buffered water to the newly hydrolyzed area may be limited. Crystallization of the hydrolyzed (or partly hydrolyzed) product may also be a complicating factor. This process is consistent with our observations that during erosion the outer layer of the polyanhydride samples consists of a non-crosslinked water insoluble material that can be easily wiped from the surface. The rate constant for hydrolysis within the thiol−ene polyanhydrides is also an important factor in determining the overall erosion rates, notwithstanding the complications produced by acid-catalysis or salts, as discussed above. The average hydrolysis rate constant (1.7 × 10−3 s−1 (0.104 min−1)) was obtained in nonbuffered systems at 37 °C and can be compared with both small molecule and other polymeric anhydride systems. As expected, small molecule anhydrides have larger hydrolysis rate constants, such as acetic anhydride, which has a hydrolysis rate constant of ∼2.6 × 10−3 s−1 at 25 °C (∼0.156 min−1).41,42 Hydrolysis of other polyanhydrides has been shown to be much slower than for small molecules.

toxicity, both in terms of cell morphology, as inspected under light microscopy, and failure to exclude PI. Indeed, cells exposed to 6000 ppm of the polyanhydride exhibited only 53.0 ± 18% viability, making this an approximate LD50 concentration (Figure 6A). Upon increasing the concentration of polymer to approximately 12000 ppm only 15.6 ± 2% cells remained viable (Figure 6B).

4. DISCUSSION In the following we discuss aspects of the polymer erosion, anhydride hydrolysis, the release of a drug/therapeutic mimic (AO8) and cytotoxicity of the polyanhydrides toward fibroblast cells. The aim is to provide an overall picture of how these materials may be used in potential applications within the biomedical field. 4.1. Erosion and Release. The overall erosion behavior of these polyanhydrides (in the form of 2 × 10 × 10 mm cuboids) is shown in Figure 1; namely, a small increase in weight over several hours and then fast erosion over several more hours is quite different to that observed in other polyanhydrides, which in many cases exhibit a linear mass loss over time.15 Cubes of different dimensions (such as 10 × 10 × 10 mm) show the same trends but different time scales (larger dimensions leading to longer erosion times). At first glance the complex erosion profile of the thiol−ene polyanydrides appears to be due to an autocatalytic process, similar to that normally observed in polyester degradation and erosion.5 However, a variety of factors such as solubility, water diffusion, pKa, and local pH may also play roles in determining the erosion profile, either as part of such autocatalysis or via other phenomena. This complex erosion profile is also expected to be symbiotic with the release profile of drugs and other therapeutics, and so correlating the release of a small molecule drug mimic with the erosion process may yield further insight into the erosion mechanism. The release of the AO8 dye from the thiol−ene polyanhydrides (Figure 1) can be segmented into timeframes that correspond to the different erosion regimes. The increased rate of release in regime III, corresponding to the increase in the rate of erosion, leads to the conclusion that erosion is a factor in determining the release of the AO8. However, the burst release seen at early times (regime I) must be due to diffusion, and our conclusions from this component of the work are that hydrophilic dye is released through both diffusion and mass loss mechanisms, with the dominant mechanism changing from diffusion to mass loss mechanism as the erosion process proceeds. The dye penetration experiments (Figure 2), in which we assume that the dye is indicative of water penetration/diffusion into the polymer matrix, are consistent with the initial stages of the erosion profile. This erosion front (where the water penetrates the outer layers) progresses into the sample without significant loss of polymer within first 10 h or so. After the 10 h mark, the erosion front becomes much smaller because the polymer degrades and the subsequent product dissolves. Eventually, the erosion front is hardly discernible because the rate of mass loss is larger than the rate of water penetration. The ATR-FTIR data (Figure 3) confirm the presence of carboxylic acid groups (∼1694 cm−1) at the surface of the degrading sample and anhydride groups (∼1817 cm−1) at both the surface and interior of the sample. There is no significant evidence of acid groups within the interior at early times (15 h), but there appears to be some at later times (30 and 60 h). While it is possible that these acid groups are formed when the 2579

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Scheme 2. Illustration of the Degradation and Erosion of Cross-Linked Thiol−Ene Based Polyanhydrides

4.2. Cytotoxicity. Finally, the cytotoxicity results show that these polyanhydride materials, and the degradation product, have very high cytocompatibility even at high concentrations. This is similar to other polyanhydrides and thiol−ene-based polymers that also show low toxicity.47 Other reports that have studied polyanhydrides48 or polymeric materials with similar chemistries have reported high cell viability,35,49−51 but the concentrations of the polymer/degradation product were at much lower concentrations52 compared to concentrations used in current studies. Thus, our data indicate a very good cytocompatibility. These low toxicity results suggest that these photo-cross-linked polyanhydrides can potentially be used in drug delivery and other biomedical applications. The toxicity of these materials is currently under further investigation in to better understand its potential in vivo behavior.

For example, variants of p(CPP-SA) are commonly studied semicrystalline polyanhydrides and typically degrade over days to years, depending on the composition of monomers used. Linhardt et al. measured that the hydrolysis rates for a 20:80 ratio of CPP/SA in p(CPP-SA) to be 0.1 mmol/h at 60 °C.43 It is apparent that the structure of the polymer is a major factor in dictating the rate of hydrolysis of the anhydride, in addition to the pH and solvent in which the polyanhydride is being degraded. Polymer morphology also is a major factor in the rate of hydrolysis; for example, the rate of hydrolysis will decrease if the polymer is semicrystalline. Overall, the hydrolysis rate constant found in the present work is smaller than small molecule anhydrides but much faster than the linear p(CPP-SA) polymers previously reported.43,44 The faster rate of hydrolysis of our amorphous polyanhydrides provides one explanation as to why they erode over much faster time scales (hours-to-days) compared with the p(CPP-SA) copolymers (days-to-months). This faster rate also means that the concentration flux of the degradation product will be much higher than the (CPP-SA) copolymers; thus, solubility and pH changes due to the formation of acid groups may have a greater impact on polymer degradation and thermomechanical properties. In summarizing the work focused on the degradation and erosion behavior of the thiol−ene polyanhydrides, one can conclude that erosion is not a simple process and involves several parameters, many of which are interrelated and potentially change throughout the erosion process. This complexity manifests itself in nonlinear erosion behavior (as evidenced in Figure 1). Scheme 2 illustrates the overall erosion process and presents a qualitative picture, but we are, unfortunately, unable to presently deduce a detailed and quantitative description of the erosion of these cross-linked thiol−ene polyanhydrides based on the data presented here. This is likely to be rectified with further investigation into degradation intermediates, and transport and solution phenomena (of water and products), ideally coupled with modeling. We note that a full understanding of degradation and erosion is still elusive even in the most widely studied degradable polymer systems based on PLGAs5,45,46 and other polyanhydrides.17 In the meantime, we can say that the thiol− ene polyanhydrides provide surface-erosion characteristics, as supported by FTIR and dye-penetration studies, with the degradation product being reasonably soluble under conditions where the pH > 6, and, as demonstrated by the dye release experiments, can be considered as a useful matrix for the delivery of therapeutics.

5. CONCLUSIONS We report here studies on the erosion, release, and degradation product from the cross-linked polyanhydrides synthesized from thiol−ene polymerization. In particular, the erosion and release kinetics of a dye were correlated with the erosion profiles, and erosion was studied by following the penetration of a watersoluble dye into the polyanhydrides and FTIR spectroscopy of the surface and interior of the polymers. Additionally, several other important parameters are studied, including the rate of hydrolysis of the polyanhydride, the solubility as a function of pH, the average pKa of the degradation product, and the cytotoxicity of the polymer and degradation product toward fibroblast cells. It was found that the polyanhydrides provide release of a hydrophilic dye that qualitatively follows the erosion profile. Based on weight loss measurements, the erosion mechanism appears to be dominated by surface erosion, but is more complicated than other surface-eroding polyanhydride systems in that the weight loss profiles are nonlinear. We believe that autocatalytic processes, perhaps as similarly observed in the degradation of polyesters, play a significant role in the erosion of these polyanhydrides. Further studies are currently underway in our lab to more fully elucidate the erosion mechanism. The degradation product solubility is low at pH values less than 6, and the average pKa was determined to be ∼5.3. The cytotoxicity of the polymer and the degradation product was found to be low, with cell viabilities of >97% for the various samples studied at concentrations of ∼1000−1500 ppm. This study allows us to conclude that thiol−ene based polyanhydrides may be used as a drug delivery vehicle and are essentially nontoxic toward fibroblast cells. Furthermore, as long as the pH of the solution containing the degradation 2580

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product from the thiol−ene polyanhydrides is kept high enough to ionize the carboxylic acid groups then the product is expected to remain soluble. In cases such as in in vivo applications, the bicarbonate buffering capacity of blood or other body fluids should be able to maintain such pH conditions and, furthermore, should be able to dilute the product so that cytotoxicity remains low. Thus, such thiol−ene based polyanhydrides are biocompatible and may find niche uses in the fields of drug delivery, tissue engineering, and related areas.

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

* Supporting Information S

Erosion profile of 10 × 10 × 10 mm cube, procedures and characterization (1H and 13C NMR, IR, mass spectrometry data) for the synthesis of model compounds, and pKa determination data. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author

*E-mail: [email protected]. Tel.: 315-268-2393. Fax: 315268-6610. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Department of Chemistry and Biomolecular Science, the Department of Biology, and the School of Arts and Sciences at Clarkson University for their support. We also acknowledge the Center for Advanced Materials Processing (CAMP), a New York State Center for Advanced Technology, at Clarkson University, for continued support and access to instrumentation. We thank Professors Craig Woodworth, Costel Darie, and Vladimir Privman, along with Dr. Izabela Sokolowska, Mr. Armand Ngounou, and Mr. Sergii Domansky, for their assistance and helpful discussions.

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REFERENCES

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