Combinatorial Approach to Develop Tailored Biodegradable Poly

Oct 16, 2014 - Physical insights into salicylic acid release from poly(anhydrides). Queeny Dasgupta , Kaushik Chatterjee , Giridhar Madras. Phys. Chem...
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Combinatorial Approach to Develop Tailored Biodegradable Poly(xylitol dicarboxylate) Polyesters Queeny Dasgupta,† Kaushik Chatterjee,†,‡ and Giridhar Madras*,§ †

Bioengineering Program, ‡Department of Materials Engineering, and §Department of Chemical Engineering, Indian Institute of Science, Bangalore-560012, India ABSTRACT: The objective of this work was to develop a versatile strategy for preparing biodegradable polymers with tunable properties for biomedical applications. A family of xylitol-based cross-linked polyesters was synthesized by melt condensation. The effect of systematic variation of chain length of the diacid, stoichiometric ratio, and postpolymerization curing time on the physicochemical properties was characterized. The degradation rate decreased as the chain length of the diacid increased. The polyesters synthesized by this approach possess a diverse spectrum of degradation (ranging from ∼4 to 100% degradation in 7 days), mechanical strength (from 0.5 to ∼15 MPa) and controlled release properties. The degradation was a first-order process and the rate constant of degradation decreased linearly as the hydrophobicity of the polyester increased. In controlled release studies, the order of diffusion increased with chain length and curing time. The polymers were found to be cytocompatible and are thus suitable for possible use as biodegradable polymers. This work demonstrates that this particular combinatorial approach to polymer synthesis can be used to prepare biomaterials with independently tunable properties. polymers3 designed to eliminate the need for secondary medical procedures following the completion of the healing process. Thus, there is a need to develop techniques to independently tune mechanical properties, degradation rates,18 and release properties of resorbable biomaterials. Hydrolytically labile polyesters are a widely used class of polymers to prepare tissues 19 due to their excellent biocompatibility, biodegradability, and mechanical properties that can match those of tissues in vivo. Drugs can be loaded for controlled release.13,15,16 Further, they can be processed into porous, three-dimensional (3D) scaffolds.20,21 Polyesters are typically synthesized by the condensation polymerization of a dicarboxylic acid and a diol. These esterification reactions can be performed by catalyst-free melt condensation reactions. Commercially used polyesters such as polycaprolactone (PCL) and poly(lactic-co-glycolic acid) (PLGA) have a few disadvantages. PCL, for instance, has a very slow degradation rate and takes more than 24 months to degrade completely.28 The degradation rate can be mildly tuned by varying the molecular weight but these changes also influence the mechanical properties and release kinetics, which cannot be varied independently. Likewise, the degradation rate of PLGA depends on the lactide to glycolide ratio.29 Increased glycolide ratio results in PLGA with fast degradation but poor mechanical properties. Therefore, there is a need to develop

1. INTRODUCTION The field of biomaterials science has seen a significant growth in recent years. The new emerging biomaterials are typically bioresorbable materials that are useful in diverse applications such as tissue engineering,1 drug delivery,2 and wound healing.3 The primary role of tissue scaffolds is to provide a microenvironment4 to the cells that can act as facsimiles of the native tissue that is being regenerated. These biomaterials must be tailored to meet individual requirements of the target tissue. The mechanical stiffness (modulus) of the substrate is known to regulate the cell response and tissue regeneration.5−7 The stiffness of different tissues in the body vary widely.8 The modulus of the substrate, therefore, should closely match the varying moduli of the target tissues.9 Moreover, different kinds of progenitor and stem cells have individual capacities to proliferate and differentiate.10−12 Thus, the resorption of the scaffold should be tailored to match the rate of tissue growth.3 Furthermore, drugs and biomolecules13,14 are often incorporated in the scaffolds toward minimizing potential bacterial infection and inflammation at the site of the implant and to stimulate the seeded cells. The release of these agents must also be tuned for burst or sustained release, as desired by the application.2,15,16 Resorbable polymers are also being developed for controlled release of drugs and biomolecules for treating a variety of diseases.17 The physicochemical properties of the resorbable polymers applied either as a barrier or a reservoir for the drug determines its availability. Wound care products, such as sutures, patches, and so on, are prepared using such resorbable © 2014 American Chemical Society

Received: September 2, 2014 Revised: October 13, 2014 Published: October 16, 2014 4302

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Scheme 1. Schematic of Esterification of Succinic, Adipic, Suberic, and Sebacic Acid with Xylitol

2.2. Synthesis. Xylitol (polyol) and the dicarboxylic acid (succinic acid, adipic acid, suberic acid, and sebacic acid) were taken in a molar stoichiometric ratio of 1:1. These reagents were mixed in an evacuated round bottomed flask. Polymerization was accomplished by melt condensation at 150 °C with stirring under a nitrogen atmosphere for 2 h (Scheme 1). Due to its higher melting point, the reaction with succinic acid was performed at 180 °C. Subsequently, a liquid nitrogen trap connected to a vacuum line (−700 mmHg) was used for 14−24 h for continuous removal of the byproduct (water) of esterification to increase the prepolymer yield. The prepolymers were postpolymerized at 120 °C in a vacuum oven (at −700 mmHg) for 3 days and 14 days to yield cured polymers. The polymers will be referred to as PX2, PX4, PX6 and PX8 where P denotes Poly, X for xylitol, and the numbers 2, 4, 6, and 8 indicate succinate, adipate, suberate, and sebacate, respectively, in keeping with the number of −CH2 groups present in the diacids. Subsequently, the ratio of adipic acid and xylitol was changed only for PX4 in order to study the influence of stoichiometric ratio on polymer properties. Polyesters with molar ratios of xylitol/adipic acid at 4:1, 2:1, 1:2 and 1:8 were synthesized. The nomenclature used for these polymers are PX4-41, PX4-21, PX4-12, PX4-18, where the numbers after the hyphen indicate the ratio of xylitol/adipic acid. 2.3. Materials Characterization. Fourier transform infrared (FTIR) spectroscopy and proton- nuclear magnetic resonance (NMR) spectroscopy were used for chemical characterization, dynamic mechanical analysis (DMA) was used for mechanical characterization, differential scanning calorimetry (DSC) was used for thermal analysis and contact angle goniometry was used for surface water wettability studies. 2.3.1. FTIR Spectroscopy. PerkinElmer Frontier FT-NIR/MIR spectrometer was used in the Universal attenuated total reflectance (uATR-FTIR) mode for FTIR analysis of the polyesters. An average of 32 scans was taken with a resolution of 4 cm−1 over the range 4000− 650 cm−1. Polymer discs were placed directly on the sample holder without further processing. 2.3.2. NMR Spectroscopy. Approximately 4 mg of the polyester prepolymers was dissolved in 500 μL of d-6 DMSO (with 0.03% (v/v) tetramethylsilane as reference standard) at 80 °C to ensure greater solubility. 1H NMR spectra was recorded in a 400 MHz Bruker Avance NMR Spectrometer. 2.3.3. Dynamic Mechanical Analysis. Mechanical properties of the samples were characterized by dynamic mechanical analysis (TA Instruments, Q 800). All the 14 day cured samples of PX2, PX4, PX6, PX8, PX4-12, and PX4-18 were characterized. A shorter cured (3 day) sample of PX8 was also characterized in order to study the influence of curing time on the mechanical properties of the polyesters. The samples were analyzed by in an isothermal frequency sweep. Experimental parameters consisted of frequency sweeps from 0.1 to 1 Hz with amplitude of 15 μm and a preload of 0.01 N. A film/fiber tension clamp was used to measure the mechanical response of the samples under isothermal-strain sweep conditions with the sample held at 37 °C (physiological temperature) throughout the experimental run. 2.3.4. Differential Scanning Calorimetry. Thermal properties of the samples were obtained using a differential scanning calorimeter (DSC, TA Instruments Q 2000). All the samples were scanned within the temperature range of −50 to 150 °C with a temperature ramp of

combinatorial methods wherein properties can be tuned independently. In this work, we propose a combinatorial approach to tune the properties of biodegradable polyesters to tailor their use in different tissues. Such combinatorial strategies22 have been reported earlier for the synthesis of degradable biomaterials like polyarylates, and their structure−property relationships have been explained in detail.23,24 A wide variety of combinatorial approaches and high-throughput screening platforms have been proposed in recent years to optimize properties of biomaterials.25−27 The family of poly(xylitol dicarboxylates) developed utilizing this particular combinatorial approach by tuning three independent variables can offer significant advantages over traditional resorbable polyesters. As a model system, we have prepared and characterized cross-linked polymers prepared using xylitol as a polyol. Xylitol is an intermediate in carbohydrate metabolism. It is a polyol having five hydroxyl groups and is a nontoxic, U.S. FDA approved precursor for polyester synthesis. A few studies have been reported in recent years on xylitol-based polymers developed for implant applications.30,31 We prepared a family of polyesters by melt condensation of a series of naturally derived fatty acid metabolites. These diacids, succinic acid, adipic acid, suberic acid, and sebacic acid, are linear diacids with monotonically increasing chain lengths. We have prepared polyesters with different stoichiometric ratios. The curing time of the prepolymers formed by condensation reaction was also systematically varied. Thus, we have varied three independent parameters, namely, the chain length of the diacids, stoichiometric ratios, and curing time. It is important to vary these three different variables because the targeted properties (degradation, mechanical strength, and release) have been reported to be functions of the degree of cross-linking (governed by curing time and stoichiometric ratios of monomers) and the physicochemical properties of the monomer (like solubility, hydrophobicity, etc.). We demonstrate that this combinatorial approach generates a class of cross-linked polymeric biomaterials wherein the mechanical properties, degradation rates, and the release kinetics can be tuned independently.

2. EXPERIMENTAL SECTION 2.1. Materials. Dicarboxylic acids, namely, succinic acid, adipic acid, sebacic acid (all SRL laboratories), suberic acid, and xylitol (both Sigma-Aldrich, U.S.A.) were used for the synthesis of cross-linked polyesters. All the dicarboxylic acids were recrystallized in ethanol (Merck Millipore, India) and kept at 4 °C prior to use in order to remove organic impurities. The low temperature was maintained since these dicarboxylic acids have low solubility in ethanol at low temperatures. Solvents used at various stages of the work include chloroform, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), and acetone (all from S D Fine Chemicals, India). 4303

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10 °C/min. The samples were subjected to a heat−cool−heat temperature program in order to remove any changes in thermal history due to processing conditions. 2.3.5. Surface Water Wettability. Surface water wettability was characterized by measuring the water contact angle (Dataphysics). A 1 μL droplet of ultrapure water (Sartorius) was placed on drop-casted films of the polyesters and measurements were taken at 0, 5, and 10 s. The data are shown as mean ± standard deviation (SD) for three independent readings. 2.3.6. Measurement of Cross-Linking Density. Swelling−deswelling experiments were conducted with these polyesters. Discs (diameter 4.5 mm) of these polyesters were immersed in ethanol at 37 °C and allowed to swell until equilibrium was attained. The samples were weighed after swelling (Ws). Excess ethanol was removed and the samples were then kept in a 37 °C incubator for drying until constant weight was obtained. This is denoted as the dried polymer weight (Wd). The swelling ratio (S) was calculated by the following formula:

S=

Ws − Wd Wd

polymer. A 10% (w/w) dye loaded (RhB and RhBB) PX4 samples were also prepared in order to check if % dye loading affects the dye release profiles. Discs were prepared as previously described for degradation studies. 2.3.9. Cell Studies. Discs of 4.5 mm diameter and thickness of 1 mm were prepared (by curing the molten prepolymers after casting on petridishes as described previously for hydrolytic degradation) to assess cytocompatibility in vitro using HeLa cells (ATCC, U.S.A.). HeLa, a cervical cancer cell line, is widely used for testing cytocompatibility of biomedical polymers. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Life Technologies) supplemented with 10% fetal bovine serum (FBS, Gibco, Life Technologies) at 37 °C and 5% CO2. Prior to cell seeding, polymer discs were ethanol and UV sterilized for 30 min. These discs were then incubated for 12 h in sterile PBS followed by 12 h in complete culture medium to remove the unreacted prepolymers. Subsequently, the discs were dried and sterilized under UV for 1 h. A total of 500 cells were seeded on each disc placed in a 96-well plate in 150 μL of medium and cultured as above. Cell viability was assessed 4 days post seeding on these polyesters using WST-1 (water-soluble tetrazolium salts, Roche). Only viable cells produce the oxidoreductases that can react with the WST to produce a water-soluble formazan. A total of 10 μL of WST-1 was dissolved in 100 μL of complete culture medium. This was then added to each well containing polymer discs and kept at 37 °C in 5% CO2 for 3 h. The formation of formazan crystals was quantified by measuring absorbance at 440 nm in a well plate reader (Synergy HT, Biotek). A calibration curve was made by plotting absorbance for a known number of cells. Final cell numbers were calculated from the calibration curve. All measurements were performed for four independent polymer discs and the data is presented as mean ± standard deviation for n = 4. Statistically significant differences were determined from analysis of variance (ANOVA) with Tukey’s test for p < 0.05.

(1)

The solubility parameters for the different polyesters (δp) were calculated using group contribution technique and the solubility parameter for ethanol32 was taken as δs = 12.92 cm−3/2 cal1/2. The Flory interaction parameter (χsp) was calculated33 based on eq 2, where Vs is the molar volume of the solvent:

χsp = 0.34 +

Vs (δs − δp)2 RT

(2)

The degree of cross-linking was obtained using the Flory−Rehner equation:33 ⌀p ⎞−1 ⎛ ⎟ n = − [ln(1 − ⌀p) + ⌀p + χsp ⌀ 2p]V s−1⎜⌀1/3 p + ⎝ 2⎠

(3)

In eq 3, ϕp is the volume fraction of the polymer after swelling in ethanol and n is the number of chains in the polymer network that are cross-linked on either ends of the chain. These measurements were also performed on the varying ratios of PX4. Since the PX4-21 and PX4-41 samples are viscous, the measurement of cross-linking density was not possible by this technique. 2.3.7. Hydrolytic Degradation Studies. Samples for degradation studies were prepared by drop casting 7 mL of molten prepolymer on a 50 mm petridish to prepare ∼1 mm thick film. . This prepolymer was then cured for appropriate time duration (3 and 14 days). Discs of 4.5 mm diameter were punched out using a hand-held single puncher (Kangaro). These polyester discs were placed inside bags made of nylon mesh and immersed in 20 mL phosphate buffered saline (PBS) at pH 7.4 and 37 °C in an incubator shaker at 100 rpm. The bags were taken out from the incubator, dried and weighed at fixed intervals. The percentage weight loss of the 3 day cured and 14 day cured samples of PX2, PX4, PX6, and PX8 was recorded with respect to time and calculated based on the following equation: %weight loss =

Mo − M t × 100 Mo

3. RESULTS AND DISCUSSION The prepolymers were all white in color. The prepolymers dissolved slightly in ethanol, acetone, chloroform and to a greater extent in DMSO and N,N-DMF. The cured polyesters did not dissolve in any of the stated solvents. Determination of molecular weight by Gel permeation chromatography was not possible because of this insolubility. The estimated percentage yield was 65−70% for PX2, PX4, and PX6 and 85% for PX8. The yield was ∼80 and ∼65% for PX4-12 and PX4-18, respectively. Yield could not be calculated for PX4-21 and PX441 due to very less esterification. 3.1. FTIR Spectroscopy. Consistent with previously reported literature,34 FTIR spectra (Figure 1a) shows ester peaks at ∼1730 cm−1 for all the polyesters. A broad peak around 3400 cm−1 is observed due to the presence of free unreacted −OH groups of xylitol. The −CH2 bending vibrations are at 1465 cm−1 for all the esters. The peaks corresponding to asymmetric and symmetric −CH stretching are observed at 2926 and 2853 cm−1, respectively.35 These −CH stretching interactions become more prominent as the chain length of the dicarboxylic acid increases from PX2 to PX8. The −CH2 bending vibration, which is particularly a long chain bonding interaction, is observed around 720 cm−1, only in the case of PX6 and PX8. A number of weak C−C bond peaks are observed around 1200 cm−1. FTIR spectra (Figure 1b) were also recorded for the various esters as a function of curing time. Figure 1b presents the FTIR spectra of PX8. It is observed that the −COOH peak due to carboxylic acid group in the diacid (at 1715 cm−1) progressively shifts toward 1730 cm−1 as curing time increases and esterification proceeds. The broad peak due to −OH at 3400

(4)

In equation4, Mo is initial weight of the sample and Mt is the weight of the sample after hydrolytic degradation in PBS for a time period t. The effect of pH on the hydrolytic degradation rate was also investigated by following a similar procedure as above by subjecting PX4 to degrade in phosphate buffers of pH 3.2 (acidic), 7.4 (physiological pH), and 9.0 (alkaline). 2.3.8. Dye Release Studies. The polyester prepolymers were mixed with a hydrophilic (Rhodamine B, RhB, Sigma-Aldrich) and a hydrophobic (Rhodamine B base, RhBB, Sigma-Aldrich) dye. A 1% (w/w) of RhB or RhBB was mixed with the prepolymers and dissolved in N,N-DMF. The solvent was removed and the dye-prepolymer mixture was cured at 120 °C in a vacuum oven for 14 days for PX2, PX4, PX6, and PX8 as above. PX4 was also cured for 3 and 7 days in order to study the effect of curing on the release properties of the 4304

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displayed in the range between 1.3 and 2.5 ppm. The peaks of the monomers match those obtained in the polyesters. Increased shielding of the −CH2 groups occurs as the chain length of the dicarboxylic acid increases. Peaks due to the protons attached to Cγ,δ are observed at around 1.4 ppm in PX8. Deshielding occurs as chain length of dicarboxylic acid decreases from sebacic to succinic acid. As a result of the deshielding of the protons attached to Cγ in PX6, this peak shifts marginally to 1.5 ppm. This peak is absent in PX2 and PX4 due to the absence of Cγ,δ. The peak due to the protons attached to Cα occurs approximately at 2.5 ppm across all esters but a gradual shift is observed from 2.5 ppm in PX2 to 2.2 ppm in PX8. Only the representative spectra of PX4 and PX6 are shown in Figure 2. All other polyesters show similar spectra. 3.3. Differential Scanning Calorimetry. It is important to characterize the crystallinity and thermal properties of the polyesters. The DSC thermogram shows a considerable decrease in the glass transition temperature of the samples as the size of the dicarboxylic acid increases. PX2 shows a glass transition temperature (Tg) of 48 °C. Tg decreased monotonically with increase in chain length. PX4, PX6, and PX8 have Tg values of 17, 8, and 2 °C, respectively (Table 1). This increase in Tg with the decrease in the chain length of the acid may be attributed to the formation of more rigid networks in polymers prepared using diacids with shorter chains. The cross-links are present more closely in PX2 than in PX8. This proximity of cross-links considerably reduces the freedom of rotation of these bonds restricting chain movement in the case of PX2. This trend results in the glassy nature of the PX2 polymer at physiological temperature, thereby rendering it less desirable for use as a biomedical implant. All the other esters have Tg below body temperature. The increased cross-linking density (as discussed further below) with increase in the chain length of the diacid is most likely responsible for the corresponding decrease in Tg. The stoichiometric ratios of PX4 are varied and it is observed that PX4-12 shows a Tg of 38 °C. This increase in the Tg as compared to PX4 may be attributed to the functionality ratio (−OH to −COOH), which is closer to 1:1 in PX4-12 and, therefore, forms more rigid networks, making polymeric chain movement difficult near physiological temperature. As the concentration of adipic acid is increased further in PX4-18, the Tg obtained is 13 °C. This lowering of Tg as compared to PX412 maybe because excess of adipic acid decreases the esterification reaction and conversion to ester is not as high as in PX4-12. Consequently, the samples with a lower stoichiometric ratio of the dicarboxylic acid, namely PX4-21 and PX4-41 showed much lower Tg values of −11 and −22 °C. These samples remained in a sticky, viscous fluid-like state even after 14 days of curing. No melting or crystallization peaks were observed in any of the samples because of formation of completely cross-linked, amorphous polyesters. 3.4. Dynamic Mechanical Analysis. Data from DMA analysis are tabulated in Table 1. The storage modulus increases in the following order: PX4 < PX6 < PX8. DMA of PX2 was not performed because of its extremely brittle nature. When the stoichiometric ratio of adipic acid in PX4 is increased, the storage and loss moduli increase in case of PX4-12, as compared to PX4, indicating that greater cross-linking has led to an increase in the moduli of the material. The moduli of PX4-18, however, decrease in comparison with PX4-12. This is likely because in PX4-18 there is an excess of the diacid. When cured at 120 °C, a large amount of adipic acid is removed from

Figure 1. FTIR spectra of (a) PX2, PX4, PX6, and PX8, (b) shift in ester peak as a function of curing time, and (c) varying stoichiometry of PX4.

cm−1 also decreases substantially with curing time indicating that increasing number of −OH groups formed cross-links between the polymeric chains. Figure 1c compiles FTIR spectra of PX4 prepared by using the precursors at different stoichiometric ratios. It is observed that with an increase in the adipic acid: xylitol ratio, PX4-12 shows a very sharp peak at ∼1730 cm−1. PX4-18, however, displays a shoulder near 1730 cm−1, indicating a lower extent of esterification. PX4-41 and PX4-21 display prominent −OH peaks near 3400 cm−1 and no observable shift in the −COOH peak at 1715 cm−1. This indicates that no appreciable esterification has occurred in these combinations. The low −COOH to −OH ratio due to excess of xylitol has yielded negligible conversion by esterification even after 14 days of curing. 3.2. NMR Spectroscopy. All peaks obtained in the 1H NMR spectra match those reported previously in literature34,36 for PX8 (Figure 2). Peaks corresponding to protons from xylitol in the polymer chain are observed in the region between 3.4 and 5.5 ppm. The peaks from the dicarboxylic acids are 4305

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Figure 2. Representative 1H NMR spectra of (a) PX4 and (b) PX6. All other esters display similar spectra.

Table 1. Physical Properties of the Family of Polyesters Cured for 14 Days storage modulus (MPa) polyester

Tg (°C)

PX2 PX4 PX6 PX8 PX4-12 PX4-18 PX4-21 PX4-41

46 12 8 3 37 13 −11 −22

at 1 Hz 0.8 0.9 2.4 14.7 1.4

at 0.1 Hz 0.4 0.87 2.4 13.3 1.3

loss modulus (MPa) at 1 Hz 0.65 0.12 0.53 2.9 0.33

at 0.1 Hz 0.2 0.03 0.44 0.84 0.1

contact angle

number of cross-links

degradation (%) in 7 days

63 ± 2 95 ± 1 88 ± 2 95 ± 5 91 ± 4 72 ± 3 droplet spreads immediately droplet spreads immediately

82 97 132 144

100 58 27 20 4 30 degrades completely within 24 h degrades completely within 24 h

with an increase in the amount of adipic acid in PX4-12 and PX4-18, the hydrophobicity increases up to PX4-12 and then decreases in the case of PX4-18. This indicates that in PX4-12, fewer free −OH groups are present that can interact with water. In contrast, in PX4-18, a higher amount of adipic acid is lost while curing under vacuum, leading to larger number of free polar −OH groups. PX4-21 and PX4-41, however, show much greater water wettability as compared to PX4. This is primarily because the excess of −OH groups (from xylitol) makes the surface of these compounds exceedingly hydrophilic. This is consistent with the FTIR spectra (Figure 1c), which shows very prominent −OH stretching peaks for PX4-21 and PX4-41. 3.6. Measurement of Cross-Linking Density. The solubility parameters obtained from group contribution were 10.9, 10.4, 10.1, and 9.8 cm−3/2 cal1/2 for PX2, PX4, PX6, and PX8, respectively. The Flory interaction parameters (χsp) for the various ethanol-polyesters were obtained from eq 2 and found to be 0.64, 0.81, 0.89, and 1.02 for PX2, PX4, PX6 and PX8, respectively. The degree of cross-linking increases as the chain length of the diacid increases and values of n calculated from eq 3 are 82, 97, 132, and 144 for PX2, PX4, PX6 and PX8, respectively. There is, therefore, an approximately linear increase in the cross-linking density with an increase in the chain length of the diacid. This increased cross-linking density would lead to an increase in the mechanical strength and a

the polymer. This causes a decrease in the loss and storage moduli of the PX4-18 ester. To demonstrate the effect of curing time on mechanical properties of the polymers, DMA analysis of 3 day cured PX4, PX6, and PX8 were performed. The 3 day cured samples exhibit storage moduli of 0.30, 0.45, and 0.54 MPa at 1 Hz for PX4, PX6, and PX8, respectively. PX4-12 cured for 3 days displays much lower mechanical properties (storage modulus 8.4 MPa at 1 Hz) than its 14 day cured counterpart. Three day cured PX4-18 has a storage modulus of 0.8 MPa at 1 Hz. This may be attributed to the lower degree of cross-linking in the shorter cured polyesters. DMA of the samples with higher ratio of xylitol (PX4-41 and PX4-21) could not be performed because of their viscous and sticky nature. A disk/rectangular sample could not be formed for testing. Note that loss moduli are always lower than the storage moduli indicating that these polyesters are elastomeric in nature. Values of tan δ varied between 0.1 and 0.5 in all cases. 3.5. Surface Water Wettability. Water contact angle values for the polymers are between 60 and 80 °C, as shown in Table 1. Hydrophobicity increases from PX2 to PX8 due to the increased hydrophobicity of the dicarboxylic acids. Hydrophobicity increases as a function of aliphatic chain length and curing time. The samples are comparatively more hydrophilic at lower curing times. This is mainly due to the presence of free polar −OH groups. The variation of ratios of PX4 shows that, 4306

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Figure 3. Hydrolytic degradation profiles of 3 day and 14 day cured samples: (a) PX2, (b) PX4, (c) PX6, and (d) PX8.

decrease in degradation, as observed earlier.37 Similarly, in this study, PX6 and PX8 demonstrate higher mechanical properties and lower degradation (as discussed further below) as compared to PX2 and PX4. No significant swelling is observed in the case of PX4-12 and slight swelling occurred in PX4-18. Therefore, cross-linking density was not calculated for these systems. 3.7. Hydrolytic Degradation Studies. Degradation studies show an increase in the hydrolytic degradation rate from PX2 to PX8. PX2 degrades the fastest and almost completely in 7 days, whereas PX8 degrades the slowest and undergoes a net mass loss of ∼20% in 7 days (Figure 3). PX4 and PX6 show intermediate rates of degradation and exhibit mass losses of nearly 58% and 27%, respectively in 7 days. These results are consistent with the hydrophobicity of the dicarboxylic acid chains. Longer aliphatic chains are more hydrophobic than the shorter aliphatic chains, thereby, modulating the amount of water that can reach the vicinity of the hydrolytically labile ester bonds. This is corroborated from the cross-linking density measurement, which shows that the degree of cross-linking gradually increases from PX2 to PX8. As reported earlier, polyesters usually deposit dicarboxylic acids on the polymer surface during erosion. Succinic and adipic acid have higher solubility in water than suberic and sebacic acid.38,39 This accounts for the accumulation of large amount of dicarboxylic acid on the surface of PX6 and PX8 during degradation without being washed off, hence thwarting the entry of water and eventually slowing down degradation. The PX4-12 polyester, nevertheless, displays approximately 4% degradation in 7 days. The rationale behind this change in degradation rate could most likely be due to the increased cross-linking in PX4−12 as compared to PX4. As observed in the swelling experiment, there is no noticeable swelling in case of PX4-12. This shows that cross-linking density is much higher in PX4-12 as compared to PX4. This enhanced cross-linking

and more rigid network structure retards water penetration into the bulk of the polymer and, therefore, slows the rate of degradation. The degradation data is tabulated in Table 1. In case of the PX4−18 ester, 30% degradation takes place in 7 days. This is consistent with the swelling data, where only a slight swelling is observed in this case. This is lesser than that observed in PX4 (58%) but higher than that observed in PX412 (4%). This could be because excess of adipic acid lowers the rate of conversion to ester and, therefore, forms a less densely cross-linked network. The shorter cured samples of all the esters display a faster degradation rate, owing to their milder curing and resultant higher water penetration. These samples are essentially more hydrophilic compared to their 14 day cured counterparts. Under physiological pH conditions, a small decrease in pH is observed probably due to the release of unreacted acids into the media. Largest drop in pH is observed in the case of PX2 due to its high degradation rate and hydrophilicity. No noticeable drop in pH is observed in the cases of PX6 and PX8. The effect of pH is also very prominent in polyesters. Degradation can be either catalyzed in the presence of an acid or a base.18 In this case, the degradation of PX4 was studied under basic, acidic, and physiological pH conditions. The degradation rate is fastest in basic buffer, followed by PBS at pH 7.4 and least in the acidic buffer. In 7 days, PX4 degrades nearly 46% in acidic media, 58% in physiological pH. Under basic conditions (pH 9), however, it degrades completely within 48 h (Figure 4). The degradation of the polymer is decelerated by the presence of acidic groups in the degradation media. The reason behind this observation could most likely be the differential solubility of the dicarboxylic acids at different pH.36 They have the highest solubility in basic buffer, intermediate at pH 7.4 and significantly lower solubility in basic buffer. The rate of degradation in the acidic buffer decreases gradually because of the accumulation of diacids on the surface of the 4307

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⎛ M∗ − M ⎞ lim ⎟ = kt ln⎜ ⎝ M 0* − Mlim ⎠

A semilog plot of the variation of (M* − Mlim/M*0 − Mlim) with time is linear with a slope equal to the rate coefficient k and an intercept at 0 (Figure 6). The values of k (×10−3, h−1) obtained are 7.40, 6.57, 5.50, and 4.58 for PX2, PX4, PX6, and PX8, respectively. The value of k decreases linearly with increasing chain length of the dicarboxylic acids (Figure 7). From the degradation data of the varying ratios of PX4, k (×10−3, h−1) values obtained are 1.44 and 3.44 for PX4-12 and PX4-18, respectively. This is consistent with the mass loss data that shows that degradation slows down to a greater extent in PX4-12 as compared to PX4-18. For varying ratios of PX4, the value of k does not increase linearly with the increase in adipic acid content. It increases up to PX4-12 and then decreases for PX4-18. 3.8. Dye Release Studies. The dye release data follows the degradation profile. Dye release is fastest from PX2 and slows down as the chain length of the dicarboxylic acid increases. All of these polyesters are essentially hydrophobic in nature, as is evident from the water contact angle (Table 1), but the hydrophobic content increases as the aliphatic chain length of the diacid increases. The degradation rate influences the rate at which the dye is released from the polymer matrix. The dye concentration (by wt) was 1.0 ± 0.2%. The dye release profiles show a systematic decrease in the dye release rate from PX2 to PX8. RhB is released much faster than RhBB (Figure 8). This trend is as expected because RhBB is more hydrophobic than RhB. In 7 days, RhB is released about 88, 60, 25, and 19% by PX2, PX4, PX6, and PX8, respectively, whereas RhBB is released approximately 52% in PX2 and PX4 and nearly 14 and 7% in PX6 and PX8, respectively. It is observed that the shorter cured PX4 samples (cured for 3 days and 7 days) release dyes much faster than the samples cured for 14 days (Figure 9). PX4 cured for 3 days releases 100% of the RhB dye within 24 h, whereas nearly 100% dye is released in the case of 7 day cured PX4 in 7 days. A similar observation is made for the shorter cured samples of PX4, where 100% RhBB is released within 24 h for the 3 day cured sample. The 7 day cured sample, however, releases ∼88% of RhBB in 7 days. This may be explained by the increased degree of cross-linking with increased curing time. The increased dye loading percentage (10% w/w) does not influence the release profile and the dye release with respect to time is similar to that obtained above for the 1% dye loaded samples, as reported in literature.41 The dye release mechanism can be modeled using the semiempirical relation given by Korsmeyer-Peppas42 to determine the drug dissolution profile from polymeric systems. The relationship governing this release profile can be written as

Figure 4. Degradation profile of PX4 at acidic (pH 3.2), basic (pH 9.0), and physiological pH (7.4).

polymer. The relatively lower solubility of these diacids in acidic media causes the polymer to become more hydrophobic, thereby, hindering interaction of water molecules with the polyester. SEM micrographs of PX4 (Figure 5) show that the surface of the polymer did not develop any cracks within the first 2 days. Imaging the transverse section of the fractured disc shows that cracks appear in the inner bulk of the disc (Figure 5d). Likewise, sample dimensions do not decrease appreciably during the first 2 days of degradation, while weight drops considerably. Thereafter, the sample begins to develop surface cracks (Figure 5e) as degradation proceeds. The degradation data may be explained by following equation: −

dM ∗ = kM ∗n dt

(5)

In eq 5, k is the degradation rate constant and n is the order of the degradation process. M* = M/M0, M is the mass remaining after degradation for time t and M0 is the initial mass of the polymer. Thus, at initial time (t = 0), M = M0 and M* = 1. The variation of weight loss with time clearly shows that the mass loss saturates after a certain time in the case of PX4, PX6, and PX8. Subsequently, there is little or no loss in the mass of the polymer. FTIR data corresponding to these degraded samples display a nearly complete shift in the polyester peak from ∼1730 to 1715 cm−1. This shows that no more mass loss occurs in the polymer after all the ester bonds in the polyester break and M* reaches its limiting mass (Mlim). This value is obtained experimentally and is taken as the value obtained after 360 h of degradation. Mlim has been considered to be 1 in the case of PX2, since the polymer degrades completely within 7 days. Because of this limiting mass, eq 5 may be rewritten as −

dM ∗ = k(M ∗ − Mlim)n dt

(7)

Mt = kt n M∞

(6)

(8)

Mt is the dye released at time t, M∞ is the total amount of dye released, k is the rate constant, and n is the release exponent. Equation 8 may be rewritten as

The degradation depends on the degradation of the labile ester bonds due to the incoming water. Since water is present at a large excess as compared to the polymer, the degradation rate is only controlled by the concentration of ester bonds, concomitant with previously reported literature.40 When the degradation is a first order process, this expression is integrated to be obtain

⎛M ⎞ ln⎜ t ⎟ = ln(k) + n ln t ⎝ M∞ ⎠

(9)

The log−log plot of Mt/M∞ with time is linear (Figure 10). The value of n changes with the hydrophobicity of the 4308

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Figure 5. SEM micrographs of different stages of hydrolytic degradation in PX4. (a) Surface before degradation, (b) surface of 1 day degraded sample, (c) surface of 2 day degraded sample, (d) transverse section of PX4 after degradation for 2 days, (e) appearance of cracks on the surface of PX4 on day 4, and (f) transverse section of PX4 after degradation for 4 days.

Figure 6. Variation of mass with time for (a) PX2, PX4, PX6, and PX8 and (b) PX4, PX4-12, and PX4-18 based on eq 7. The error bars indicate standard deviation between three replicates.

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cases except for PX2, in which the polymer in itself starts degrading. In case of PX2, the polymer degrades completely in 7 days while the dye loaded disc degrades completely in 8 days and ∼90% in 7 days (Figure 8). When a water-soluble, hydrophilic dye is incorporated into a hydrophobic polymer matrix, the release of the dye is primarily regulated by diffusion. In contrast, in the case of hydrophobic dyes, the release is mainly dependent on the degradation/erosion of the polymer. Hydrophobic interactions between the dye−polymer being higher and water solubility being lower, the driving force for release is much less. This slowing down of degradation may be attributed to the pH of the medium, which becomes slightly acidic during the first few hours of dye release and consequently slows down PX2 degradation. The hydrophilic dye is observed to be released by the polymer matrix faster than the hydrophobic dye. This is because RhB interacts poorly with the hydrophobic polymer matrix as compared to RhBB. Maximum hydrophobic interaction is observed between RhBB and PX8, where only 7% of the dye is released within 7 days. This could be used as a highly effective sustained release drug delivery system. PX6 and PX8 can be used for delivery applications where the drug is to be released at a constant rate for prolonged periods like in cases of chronic infections and diseases. PX2 and PX4 can also find applications as drug delivery vehicles for suppressing immediate immune responses like inflammatory responses after scaffold implantation. 3.9. Cell Cytocompatibility Studies. Cell cytocompatibility results show that all the polyesters PX2, PX4, PX6, and PX8 are cytocompatible. Figure 12 plots the number of viable cells 4 days after seeding on the four polyesters. Cell viability in PX8 is significantly higher than on PX2, PX4, and PX6. PX8 showed an increase in cell number to approximately 13000 cells from the initial seeding density of 500 cells. PX2, PX4, and PX6 displayed comparable cell numbers and were not significantly different from each other, with nearly 6000−8000 cells/well in each case. Predominant surface erosion of the shorter chain polyesters (PX2 and PX4) within 3 days most likely results in reduced cell attachment to these esters as compared to PX8. Continuous removal of surface of the polymer could lead to sloughing of the initially attached cells, resulting in fewer numbers of viable cells at 4 days. When placed in an aqueous medium, succinic and adipic acid tend to dissolve rapidly, thereby decreasing the local pH. These environmental stresses could have contributed to lower cell viability on the PX2 and PX4 discs. Importantly, however, these results indicate that all the four polyesters are

Figure 7. Variation of the rate coefficient (k) for (a) PX2, PX4, PX6 and PX8 and (b) PX4, PX4−12 and PX4−18. Rate coefficients were obtained from the slope of Figure 6

polyesters. The values of n obtained for RhB are 0.41, 0.49, 0.72, and 0.79 for PX2, PX4, PX6, and PX8, respectively. It is observed that the value of n increases almost linearly with the increase in the chain length of the diacid (Figure 11). Similarly, the values of n determined for RhBB are 0.45, 0.47, 0.63, and 0.67 for PX2, PX4, PX6, and PX8, respectively. This proves that the release mechanism of the dye from the polymeric matrix depends on the hydrophobicity of the polymer. The value of k decreases gradually from PX2 to PX8 for both RhB and RhBB, as is seen in the dye release data. The values of k for RhB release are 10.7, 5.53, 0.676, and 0.374 h−n for PX2, PX4, PX6, and PX8, respectively, and those for RhBB release are 5.47, 5.35, 0.587, and 0.239 h−n for PX2, PX4, PX6, and PX8, respectively. This decrease in k from PX2 to PX8 for both dyes indicates that dye release is slower in the longer chain diacids as compared to PX2 and PX4. It may also be noted that k values decrease for RhBB release when compared with that of RhB for each of the polyesters. It is further observed that value of ln k decreases linearly with an increase in the chain length of the diacid accompanied by a corresponding linear increase in the n value for both RhB and RhBB. Dye/drug release is governed by a number of factors. These include solubility of the dye in dispersion media, uniformity in dispersion of dye in polymer matrix, nature of bonding interactions between dye and polymer. Release is also dependent on inherent polymer properties like hydrolytic degradation, extent of cross-linking,43 water diffusivity, and retention. The results obtained are consistent with the expected characteristics of the polymer and respective dyes. The release is faster initially but stabilizes within the first 24 h period in all

Figure 8. Dye release profiles of (a) rhodamine B and (b) rhodamine B base loaded PX2, PX4, PX6, and PX8 polymer discs. The error bars indicate the standard deviation between three replicates. 4310

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Figure 9. Dye release profiles of (a) rhodamine B and (b) rhodamine B base loaded PX4 cured for 3, 7, and 14 days.

Figure 10. Variation of dye release % with time for PX2, PX4, PX6, and PX8 for (a) rhodamine B and (b) rhodamine B base on a log−log plot based on eq 9. The error bars indicate standard deviation for three replicates.

Figure 11. Variation of logarithm of rate constant (lnk) and diffusion coefficient (n) with respect to increase in chain length of polyesters of PX2, PX4, PX6, and PX8 for (a) rhodamine B and (b) rhodamine B base. For the dye release data, k and n are obtained from the intercept and slope of Figure 10.

days (0.8 MPa) have comparable moduli but the former degrades significantly slower (22%) than the latter (58%) in 7 days. The 14 day cured PX8 and 3 day cured PX4-12 have similar degradation rates (∼20%), but significantly differ in their mechanical properties. PX8 has a storage modulus of 2.4 MPa whereas PX4-12 (3 day cured) has a storage modulus of 8.4 MPa. Similarly, although PX2-RhBB and PX4-RhBB have similar release profiles, their degradation rates are quite different (100% for PX2 and 58% for PX4 in 7 days). The polyesters reported herein exhibit mechanical properties (ranging from 0.5 to ∼15 MPa) that are comparable to those of soft tissues, like aorta (Young’s modulus: 2.0−6.5 MPa), articular cartilage (Young’s modulus: 2.1−11.8 MPa), and so on, and may be appropriate materials for such tissue implants. Tissue scaffolds have been fabricated from polymers with

nontoxic and can support cell attachment and proliferation. Thus, polyesters appear to be suited for possible biomedical applications requiring use of resorbable materials.

4. SUMMARY AND CONCLUSIONS The properties of the reported polyesters are summarized in Table 1. This clearly depicts that a wide spectrum of polymer properties can be obtained by this combinatorial approach for potential use as biomaterials. As shown in Scheme 2, in the present study, the degradation, mechanical properties and release profiles have been tuned independently by modulating three parameters (chain length of the diacid, the stoichiometric ratio of the diacid to the polyol and the curing time) to yield a versatile class of polymers unlike the traditional polymers. For example, 3 day cured PX8 (0.5 MPa) and PX4 cured for 14 4311

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knowledges Ramanujan fellowship from the Department of Science and Technology (DST), India. The authors would like to thank Anirvan Komath Majumdar for technical assistance.

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Figure 12. Number of viable cells after 4 days of seeding.

Scheme 2. Combinatorial Strategy To Tune PhysicoChemical Properties of Polyesters for Tissue-Specific Applications

similar properties, such as poly(glycerol sebacate) and citric acid elastomers.44,45 The structure−property correlation and surface properties of the polymer may further be understood and predicted by employing molecular modeling simulations, as is reported in literature.46−48 Thus, we demonstrate that this particular combinatorial approach to polymer synthesis can be leveraged to prepare optimally tailored biomaterials. Although we have used poly(xylitol dicarboxylates) as a model system here, this strategy could be extended to many other such classes of polymers used for biomedical applications.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Tel./Fax: +91-80-2293-2321. E-mail: [email protected]. ernet.in. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by Department of Biotechnology (DBT), India (BT/PR5977/MED/32/242/2012). K.C. ac4312

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