Polyanhydrides of Castor OilâSebacic Acid for ... - ACS Publications

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Polyanhydrides of Castor Oil−Sebacic Acid for Controlled Release Applications Janeni Natarajan,† Shruti Rattan,‡ Utkarsh Singh,‡ Giridhar Madras,† and Kaushik Chatterjee*,‡ †

Centre for Nano Science and Engineering, ‡Department of Materials Engineering, Indian Institute of Science, Bangalore 560012, Karnataka, India S Supporting Information *

ABSTRACT: A family of high molecular weight castor oil (CO)-based biodegradable polyanhydrides was synthesized by a catalyst-free melt−condensation reaction between prepolymers of CO and sebacic acid (SA). The structure of the polymers was characterized by 1H NMR and Fourier transform infrared spectroscopy, which indicated the formation of the anhydride bond along the polymer backbone. Thermal analysis and X-ray diffraction confirmed the semicrystalline nature of the polymers. Incorporation of SA enhanced the crystallinity of the polymer. The hydrophobic nature of these polymers was revealed by contact angle goniometry. Water wettability decreased with increase in SA content. Compressive tests demonstrated a sharp increase in strength and decrease in ductility with increasing SA content. In vitro hydrolytic degradation studies indicated surfaceeroding behavior. The degradation rate decreased with an increase of SA content in the polymers because of increased crystallinity. The release studies of both hydrophobic and hydrophilic dyes followed zero-order kinetics. In vitro cell studies to assess the cytotoxicity of the polymer confirmed minimal toxicity of the degradation products. Thus, a family of CO-SA polyanhydrides have been synthesized and characterized for controlled release applications where the physical, mechanical, and degradation kinetics can be modulated by varying the weight fraction of the prepolymers.

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INTRODUCTION Several new classes of biodegradable polymers such as polyesters, polyorthoesters, polyanhydrides, polyurethanes, and polyphosphazenes have been developed for their potential use in biomedical applications including drug delivery, tissue engineering and regenerative medicine.1−5 Biodegradability is desirable as it obviates the need for surgical removal of these polymers once implanted in the human body. Furthermore, the polymer should not produce toxic degradation products and should not generate sustained inflammatory reaction in the body.6 Polyesters such as poly-L-lactic acid (PLLA), poly(glycolic acid) (PGA) and poly(lactic-co-glycolic acid) (PLGA), which are among the most widely used polymers for biomedical applications, undergo bulk erosion,7,8 and this makes it difficult to achieve controlled drug release. Controlled drug release can be achieved by the surface erosion of the polymer, wherein the polymer erodes layer by layer and can provide sustained zeroorder release of the drug. Poly(ortho-esters) can be made to undergo surface erosion, but only by incorporation of suitable additives.7 Polyanhydrides are considered to be the most promising class of polymeric biomaterials for drug delivery owing to their biocompatibility and the hydrolytically labile nature of the anhydride bond which readily splits to give nontoxic degradation products. Importantly, their surface-eroding behavior allows for controlled drug release.9,10 Polyanhydride based on 1,3-bis (p-carboxyphenoxy) propane (CPP) and sebacic acid (SA) has been shown to be an effective drug delivery carrier for treatment of brain parenchyma in rats.11 The copolymer has also been approved by the Food and Drug Administration (FDA) for clinical use in the form of Gliadel wafers. Polyanhydride implant composed of sebacic acid and © 2014 American Chemical Society

erucic acid dimer (Septacin) has been shown to be a potential drug delivery vehicle for gentamicin sulfate, which is used in the treatment of osteomylitis.12 “Self-delivering drugs” based on poly(anhydride-esters) developed by the Uhrich group hydrolytically degraded to give salicylic acid, an analgesic, and also an active metabolite generated upon the hydrolysis of Aspirin.13 Block copolymers composed of prepoly(sebacic anhydride) and Pluronic-F68/F127 prepared via a melt−polycondensation reaction were found to exhibit sustained drug release, and the release rate was dependent on the Pluronic content.14 In recent years, polyanhydride polymers have been developed to act as drug carriers in nanomedicine.15,16 Polymers have an added advantage of tailored degradation rates, from a few days to several years, accomplished by varying the ratio of the monomers that constitute the polymer to suit a particular application. Different compositions of CPP:SA copolymers show different degradation rates, which has led to their use in a variety of therapeutic applications.17 Zhai et al. synthesized a series of poly(sebacic acid-octadecanoic diacid) copolyanhydrides and showed that the rate of degradation decreased with the increase in octadecanoic diacid prepolymer content.18 Incorporation of fatty acids in the polymer backbone imparts a low melting point, flexibility, and hydrophobicity to the resulting polyanhydride, which helps in retaining the drug for an extended time period.19 Moreover, fatty acids are nontoxic, derived from natural resources, and are low in cost. Domb and Received: Revised: Accepted: Published: 7891

February 19, 2014 March 27, 2014 April 14, 2014 April 14, 2014 dx.doi.org/10.1021/ie500679u | Ind. Eng. Chem. Res. 2014, 53, 7891−7901

Industrial & Engineering Chemistry Research

Article

Scheme 1. Reaction Schematic for Polymerization of COMApp and SApp

family of polyanhydrides reported herein. Five polymers with different weight fractions (%) of CO and SA prepolymers (20, 33, 50, 66, and 80 wt % of SA prepolymer) were synthesized. The study reports on the synthesis, characterization, degradation kinetics, dye delivery, and in vitro cytocompatibility of the polymer. The weight fraction of the prepolymers was varied to tune the physical and mechanical properties to ultimately generate variations in degradation kinetics and dye release profiles.

co-workers have investigated polyanhydrides derived from fatty acids. For example, polymers (P(RAM:SUA) and P(RAS:SUA)) derived from fatty acid esters of ricinoleic acid (RA), maleic acid (MA), and succinic acid (SUA), lost 50% of their weight after 72 h of degradation.20 Poly(sebacic anhydride) terminated with different fatty acids was synthesized, and it was reported that the degradation times of these polymers increased in comparison to those of poly(sebacic anhydride) because of increased hydrophobicity of the polymer backbone.21,22 In this study, we have used castor oil (CO) and sebacic acid as the precursors for the synthesis of the polyanhydrides. CO, a vegetable oil, consists of triglycerides of fatty acids of which the most dominant is RA. It is a cheap and abundantly available renewable source and has been used in the synthesis of several biodegradable polymers.23−25 Some studies have been carried out with RA- and SA-based polymers.20,26 RA has to be extracted from CO, and the extraction process is difficult and tedious. CO is a readily available natural renewable resource and is comparatively cheaper. While the long aliphatic chains in CO impart hydrophobicity to the compound, the incorporation of SA will improve the hydrophilicity of the polymer. It has been observed that generally fatty acids are eliminated almost completely in the form of CO2 by β-Oxidation pathway in humans.27,28 SA, derived from CO, is also a nontoxic, easily available and low-cost compound which increases the hydrophilicity of the resulting polymer.9 It is the most commonly used raw material for the development of polyanhydrides for drug release applications.10,14,29 SA is eliminated from the body by conversion into acetyl and succinyl CoA that subsequently enter the Krebs cycle.27,30 Because of the nontoxicity of the two monomers, it is expected that the degradation products of the polymer will be eliminated by the body over a period of time. The degradation of poly(SA-RA) was studied in vivo, and the polymer degraded into SA and RA, which were further eliminated from the implantation site easily by the body.26 Therefore, we employed a simple melt condensation that does not use any cross-linkers, such as formaldehyde, gluteraldehyde, etc., and initiators, such as peroxides, aliphatic azo compounds, etc., which are reported to be carcinogenic and toxic in nature, or catalysts or solvents for the synthesis of a

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MATERIALS AND METHODS Materials. The monomers, CO, SA, and maleic anhydride (MA) were purchased from SD Fine Chemicals Ltd. (India). SA was recrystallized twice from ethanol before use. Acetic anhydride (AA) was purchased from Sigma-Aldrich (U.S.). Commercial grade solvents were distilled prior to use. All reactions were performed in oven-dried apparatus. Formaldehyde used in cell cultures was purchased from Merck chemicals (India). Preparation of Castor Oil Maleate. The esterification of CO (94 g, 0.1 mol) with MA (19.6 g, 0.2 mol) was carried out in a 250 mL single-neck round-bottom flask placed in a 110 °C oil bath. The flask was connected to a reflux condenser, and the reaction was carried out for 9 h with continuous stirring in dry nitrogen atmosphere. The orange-colored product, castor oil maleate (COMA), was allowed to cool at room temperature. It was then washed with distilled water four times to remove excess MA and maleic acid and dried over anhydrous magnesium sulfate (60% yield). The esterification reaction is depicted in Figure S1a of Supporting Information. Preparation of Prepolymers. COMA was refluxed with excess AA (1:5 w/v) for 2.5 h and evaporated to dryness to form viscous brown COMA prepolymer (COMApp) (Figure S1b of Supporting Information). Similarly, SA was refluxed with excess AA (1:5 w/v) and evaporated to dryness (Figure S1c of Supporting Information). Acetic anhydride was used to activate the maleate groups of COMA and acid group of SA. The viscous residue was dissolved in dichloromethane, and the product was precipitated in petroleum ether. The precipitate was filtered and vacuumdried at room temperature to obtain white powder of sebacic

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dx.doi.org/10.1021/ie500679u | Ind. Eng. Chem. Res. 2014, 53, 7891−7901


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prepared by compression molding for all the different polymers as described above. The hydrolysis studies were performed by immersing the samples in 20 mL of PBS (phosphate buffered saline) (pH 7.4; 37 °C) with constant shaking (100 rpm) in an incubator shaker (N-BIOTEK). At specified time points, the polymer disks were removed from the solution, rinsed with water, and vacuum-dried at room temperature until a constant weight was obtained. The buffer was replaced every 24 h to maintain perfect sink conditions. The degradation was evaluated by (a) monitoring the weight loss of the polymer and (b) percentage anhydride bond loss by IR spectroscopy and by (c) studying the changes in surface morphology of the polymer. The weight loss was measured by weighing the dried disks. The rate of erosion of the polymers was determined by measuring the change in dry weight of the polymer matrices at specific time points and using the following formula:

acid anhydride prepolymer (SApp). Both the prepolymers were stored under nitrogen atmosphere at −80 °C. Preparation of Polymers. The polyanhydrides were prepared by melt−condensation of the two prepolymers (COMApp and SApp), as described by Conix.31 Five polymers with different weight fractions were synthesized. In a typical experiment, different weight fractions of the prepolymers COMApp and SApp (20, 33, 50, 66, and 80 wt % SApp) were taken in a single-neck round-bottom flask and allowed to melt in a 180 °C oil bath in dry nitrogen atmosphere with continuous stirring (Scheme 1). When it reached 80 °C, high vacuum (10−2 mm of Hg) was applied to remove the acetic anhydride, formed as the byproduct, using a liquid nitrogen trap during the polymerization. After 15 min, the reaction mixture became highly viscous and the polymerization was considered complete. Characterization of Poly(COMApp-SApp). Fourier transform infrared (FTIR) spectroscopy (Jasco FTIR spectrum BX) was performed on the prepolymers and the polymer samples. Solid samples were pressed into KBr pellets, whereas the liquid samples (COMA, COMApp) were run neat for recording FTIR spectra. 1H NMR (proton nuclear magnetic resonance) spectra were obtained on a 400 MHz Bruker Avance NMR spectrometer by dissolving the samples in CDCl3 (tetramethylsilane used as reference). The molecular weights of the polymers were determined immediately after the polymerization through gel-permeation chromatographic (GPC) analysis by dissolving it in chloroform (1 g L−1). The GPC system consisted of a Waters 510 HPLC pump, Rheodyne 7725i injector, three size exclusion columns, and a differential refractometer (Water 2410). The eluent, THF, was pumped at a rate of 1 mL min−1. Thermal properties of the polymers were determined on a differential scanning calorimeter (DSC, Mettler Toledo DSC822e, U.S.). The polymer sample was heated from −80 to 150 °C at 10 °C min−1, maintained at 150 °C for 3 min, cooled from 150 to −80 °C at 10 °C min−1, and subsequently heated up to 150 °C and cooled until −80 °C for the second cycle at the same rate to remove any previous thermal history. X-ray diffraction pattern (XRD) of the polymer powders (∼100 mg) were recorded using a Philips X’pert diffractometer using Cu Kα radiation. Scanning was performed in the 2θ range of 5−40° for 20 min. The contact angles of the polymer surfaces were measured on a video-based optical contact angle measuring system equipped with a computer software using sessile drop method (OCA 15EC, Dataphysics). A drop (3 μL) of deionized water was placed at three different locations on the surface of a circular disk of the polymers (7 mm diameter × 1 mm thickness), and the values obtained at 5 s after the release of the drop were averaged. The mechanical characterization was carried out by compressive tests for all five polymers using an MTEST Quattro machine. The load cell used was 150 pounds. The displacement rate for all the samples was 0.2 mm min−1. Circular discs of the polymers was prepared (7 mm diameter × 1 mm thickness) by compression molding at 90 °C and a pressure of 5 psig for 1 min. After the samples were cooled to room temperature, the samples were cut in a rectangular shape using a sharp blade with the dimensions of around 3 mm × 2 mm × 1 mm of height, width, and thickness, respectively. In Vitro Hydrolytic Degradation of Polymers. Diskshaped specimens (7 mm diameter × 1 mm thickness) were

% weight loss =

Mo − M t × 100 Mo

(1)

where Mo is the original mass of the polymer device and Mt is the dry mass of the disks after degradation. The percentage anhydride bond loss for 50 wt % polymer was determined from the IR spectra. The areas under an asymmetric peak of anhydride which appears at around 1800 cm−1 and under an acid peak around 1700 cm−1 were determined. The values of the areas were normalized with respect to the area values of the peak, which appears around 1078 cm−1. Then the percentage anhydride loss was calculated using the formula ⎛ value of peak area at the specified time interval ⎞ ⎜1 − ⎟ × 100 value of initial peak area ⎝ ⎠ (2)

Scanning electron microscopy (SEM, ESEM Quanta 200, FEI) was used to observe the morphology of the degrading 50 wt % SA polymer. The specimens were coated with gold for 6 min using a sputter coater (JEOL, IFC-1100E Ion sputtering device) prior to imaging. To investigate the effect of pH on the degradation rate, the degradation of the 50 wt % polymer disks was also carried out in 20 mL of PBS buffer solutions at pH 3.2 and pH 10.2 (as described for the degradation studies carried out in pH 7.4). Similar experiments were carried out to study the change in degradation rate in different volumes of PBS (using identical 50 wt % polymer disks). In these cases also the weight loss was monitored with time replacing PBS solution every 24 h to maintain sink conditions. Drug Release of a Hydrophilic and a Hydrophobic Dye. A hydrophilic dye (Rhodamine B, RB) and a hydrophobic dye (Rhodamine B base, RBB) were chosen as model dyes to simulate drugs for release studies because the amount of these dyes present in the solution can be accurately measured by spectroscopic techniques. Chloroform was used as the solvent for the polymers and the dyes. Rhodamine B and Rhodamine B Base are both completely soluble in this solvent. 95 mg of the polymer was dissolved in 3 mL of chloroform to form a homogeneous solution. The dye (5 mg) was added to this solution (cosolution method) though the actual loading in the pellet would be around 1−2 mg. The solvent was evaporated by keeping the sample in a hot air oven at 60 °C. Discs (7 mm 7893



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Table 1. Physical Properties of the Polymers Synthesized in This Studya polymer

Mn (Da)

20 wt 33 wt 50 wt 66 wt 80 wt SApp

6.0 4.0 4.1 4.3 3.9 −

% % % % %

× × × × ×

103 105 105 105 105

contact angle (deg)

compressive modulus (MPa)

compressive strength (MPa)

strain at fracture (%)

Tc (±2 °C)

Tm (±2 °C)

Tg (±2 °C)

b 83 ± 2 68 ± 2 64 ± 1 c −

b 2 25 80 530 −

b 0.1 2.0 2.4 17.9 −

b 15.3 14.8 7.5 5.6 −

49 51 57 55 56 60

57 72 73 74 75 75

−56 −55 −54 −47 −48 −

a

Mn, Tc, Tm, and Tg represent number-average molecular weight, crystallization temperature, melting temperature, and glass transition temperature, respectively. bCould not be determined because difficult to mold. cCould not be measured reliably because of high surface roughness.

diameter × 1 mm thickness) of the polymer−dye mixture were prepared using compression molding, as described above. The discs were immersed in 20 mL of PBS (pH 7.4, 37 °C). The solution was continuously shaken at 100 rpm. PBS was changed once every 24 h because the medium becomes acidic as the polymer degrades. The acidic medium will decrease the dye release. This happens because the acidic conditions further reduce the ionization of SApp, causing a reduction in hydrolysis. 200 μL of the release medium of each disc was taken at specified time intervals for both the dyes, and the amount of dye released was estimated using a microplate reader (BioTek Synergy HT). The absorbance values were measured at 553 nm for both dyes. The concentrations of both dyes released were determined from the calibration curves. The cumulative dye release was calculated, and the dye release profiles were obtained for both dyes. Cytocompatibility of the Polymer. The cytocompatibility of the polymer was evaluated using MC3T3-E1 subclone 4 mouse osteoblast cells (ATCC, U.S.). MC3T3-E1 is a wellestablished osteoblast model and is one of many widely used lines for testing biocompatibility of materials for biomedical applications including drug delivery, implants, and tissue scaffolds.32,33 Cells were grown in T-25 flasks using α-minimum essential medium (α-MEM, Gibco, Life Technologies) with 10% (v/v) fetal bovine serum (Gibco, Life Technologies) and 10 μg mL−1 streptomycin and 10 U mL−1 penicillin (Sigma), and maintained in 5% CO2 incubator at 37 °C. The cells were harvested using 0.25% trypsin. Cells of 19th passage were used for all studies reported here. 2 × 103 cells were seeded in each well of a tissue culture grade polystyrene 96 well plate with 200 μL of complete culture medium and cells were allowed to attach for at least 24 h before testing of the toxicity of the polymer degradation products. Polymer discs were prepared as described above for degradation studies. Disks were sterilized (Anaprolene sterilizer) by ethylene oxide and placed individually in 50 mL falcon tubes containing 20 mL complete culture medium. The tubes were placed in a shaker incubator maintained at 37 °C and set at 100 rpm for 24 h periods. The “conditioned medium” containing the degradation products was replaced with fresh medium every 24 h up to 3 days mimicking the conditions of the hydrolytic degradation studies above. The medium in the wells containing the osteoblasts was then replaced by 200 μL of the conditioned medium to measure the effect of the degradation products on cells. Cell viability and morphology was evaluated 24 h after exposure to the conditioned media. Independent wells were used to assess the effect of the conditioned media of 1, 2, and 3 days degradation. In control wells, fresh culture medium was added to the cells in lieu of the conditioned media.

MTT assay was used to assess the cell viability after exposure to the conditioned medium. Three wells were used for each of the three discs (n = 3× 3= 9) whereas three additional wells (n = 3) served as controls. MTT reagent was prepared by dissolving (3−(4, 5−dimethyl thiazol−2-yl)−2,5−diphenyl tetrazolium bromide) in medium at 5 mg mL−1. This solution (20 μL) was added to each well and incubated at 5% CO2 and 37 °C for 4 h to provide sufficient time for the tetrazolium dye to get reduced to formazan crystals. The solution was replaced with 100 μL of DMSO to solubilize the formazan crystals, generating a characteristic purple color. The absorbance was measured at 570 nm using a microplate reader. Differences were considered statistically significant for p < 0.05 using Student’s t test. Additionally, cells in two wells treated with the conditioned medium from each disc were used to study cell morphology. Cells were fixed 24 h after exposure to the conditioned media using 3.7% formaldehyde for 15 min.

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RESULTS AND DISCUSSION Polymer Synthesis. CO contains triglycerides of fatty acids, and RA comprises 85−90% of the total fatty acid content.34 The hydroxyl groups present in each arm of the triglycerides are converted to carboxyl groups by esterification with maleic anhydride via a hydrolyzable ester bond, so that both carboxyl groups present in castor oil maleate and sebacic acid can be used for subsequent uniform acetylation with acetic anhydride and polymerization (Figure S1a of Supporting Information).35 Figure S1b,c of Supporting Information shows the reaction scheme for acetylation of COMA and SA. The monomers were refluxed with AA so that the carboxylic groups are activated for polymerization by converting them into more reactive functional groups. The polymerizations of COMApp and SApp for all weight percentages were carried out by a simple process of melt−condensation. The mechanism of polymerization is a conventional anhydride formation process where a nucleophile attacks the electrophilic carbonyl carbon and acetic anhydride is released as byproduct. Though CO has three hydroxyl moieties, as confirmed from 1H NMR (2.75 at around 3.6 ppm), which can potentially react, the OH group attached to the central carbon of the glycerol backbone does not react. This is also confirmed from 1H NMR (Figure S2c of Supporting Information) where the modified OH groups in the polymer appear around 6.3 ppm, which shows the value of 2. Hence, CO acts as a bifunctional monomer and the final polyanhydrides formed are linear polymers. Therefore, it is unlikely that the polymers were cross-linked. The polymerizations for all weight fractions were rapid and ended in 15 min. In contrast, it has been reported that the synthesis of other polyanhydrides takes 1 to 4 h for completion.20,21,36 The use of 7894



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all weight percentages. There is an increase in sum total of the peak integrals corresponding to SApp when SApp content was higher in the polymers. However, no increase in sum total of the peak integrals corresponding to COMApp was observed when its content in the polymers was increased (Figure S2e−i of Supporting Information). This could be because COMApp would not have reacted though its weight percentage was increased in the polymers. In the FTIR spectrum of COMA (shown in Figure S3a of Supporting Information) the band at 3520 cm−1 corresponding to the −OH groups reduced, whereas the band at 1643 cm−1 relating to the carbon double bonds increased in comparison with that of the spectrum of CO.37 The peaks corresponding to cyclic anhydride at 1779 and 1849 cm−1 were not observed in COMA, indicating almost complete esterification of MA with CO. There is no band around 1810 and 1740 cm−1, which corresponds to anhydride stretching in pure SA spectra.38 All the prepolymers (Figure S3b,c of Supporting Information) and the polymers (Figure S3d of Supporting Information) showed typical IR absorptions at about 2900 and 2850 cm−1(C−H stretching bands) and about 1810 and 1740 cm−1 (symmetrical and asymmetrical anhydride CO stretching bands), which confirmed the presence of anhydride bonds in the polymer backbone. DSC results showed (Table 1) that there is a steady increase in the cold crystallization temperature Tc, the melting temperature Tm, and the glass transition temperature Tg with the increase in content of SApp. The Tg of the polymers increased from −56 to −47 °C, and the crystalline and melting peaks became more intense and sharper when the weight fraction of SApp was higher in the polymers. Therefore, this indicates that the sebacic acid improves the crystallinity of the polymer, and as the polymers are well above its glass transition temperature, the polymer is rubbery at 25 °C. The XRD pattern of the polymers (Figure S4 of Supporting Information) confirmed that the crystalline nature of the polymers depends on the SApp content present in the polymers. The data showed sharper peaks when the content of SApp was increased in the polymer. SA is reported to have 66% crystallinity.35 It is characterized by five peaks at 9.4°, 19.7°, 21.1°, 23.53°, and 25.72° (2θ values), which match with the peak positions in the XRD of the polymers.39 XRD pattern of COMA was characterized by a peak at 19.99°. Hence, it confirmed the presence of both COMApp and SApp in all the polymers. A slight shift in peaks corresponding to SApp was observed in all the polymers, which could possibly be due to a change in the interplanar spacing. DSC and XRD revealed increasing crystallinity with an increase in SApp content. NMR also confirmed the increase in SApp content in 66 and 80 wt % polymers seen by the increase in peak integrals corresponding to SApp (1.0 to 2.5 ppm). Contact angle measurement is widely used to assess the surface wettability or hydrophobicity of a material.40 Hydrophobicity of the polymer backbone prevents water penetration into the bulk of the matrices, which may lead to surface erosion and enhanced stability of the drug incorporated in the polymer.41 The drop was allowed to equlibriate for 5 s. Because there were no significant differences in readings, zeroth and fifth second values are the same. The synthesized polyanhydride, 50 wt % polymer, had a contact angle of 68° ± 2°, which indicates that the polymer backbone is hydrophobic, possibly because of the long aliphatic chains of CO.42,43 This hydrophobicity may lead to slower degradation rates. The

a catalyst in the synthesis of biomaterials may lead to toxicity when implanted in the body.3 Thus, the polymerization reaction in this study yields polymers without potentially toxic residual agents. The plausible structure of the polymer is depicted in Scheme 1. The linear polymers were found to be soluble in chlorinated hydrocarbons such as dichloromethane and chloroform, indicating that the polymers were not cross-linked. All the synthesized polymers except 20 wt % had similar numberaverage molecular weight Mn (Table 1) and polydispersity of 1.7. The Mn of 20 wt % polymer is lower because COMApp would not have reacted even though its weight fractions increased in the polymer. Polymer Characterization. Figure S2a of Supporting Information presents the 1H NMR spectrum of CO. When the peak integral of the proton attached to hydroxyl (CH−OH) at 3.61 ppm is compared with the peak integral of the methylene protons of glycerol (CH2−O) in the range of 4.1− 4.3 ppm, it was determined that 2.7 mol of hydroxyl groups were available per triglyceride in a CO molecule. In the 1H NMR spectrum of COMA (Figure S2b of Supporting Information), the peak at 3.61 ppm reduced in intensity. A new peak was observed at 5.03 ppm, which represents the methine protons attached to the maleate groups, and the integral of this peak showed that 2.01 mol of hydroxyl groups were consumed in the esterification reaction. Hence, approximately 75% of the hydroxyl groups had reacted with MA.37 Two well-defined doublets in the range of 6.3−6.5 ppm corresponding to the olefinic protons (CHCH) of the MA units also appeared in the COMA spectrum. The peak at 3.61 was absent in the 1H NMR spectrum of COMApp (Figure S2c of Supporting Information) after COMA was acetylated with AA. The two doublets in the range of 6.3−6.5 ppm in the COMA spectrum changed to singlets, indicating a change in the neighboring environment of these protons because of acetylation of the carboxyl groups. In the 1H NMR spectrum of SApp (Figure S2d of Supporting Information), the chemical shifts at δ = 1.3, 1.6, 2.2, and 2.4 ppm correspond to the protons of the functional groups a, b, c, and d indicated in Figure S1c of Supporting Information.15 The estimated degree of polymerization n was calculated according to the following formula: n=

(sum of methylene proton integrals)/number of methylene protons integral per proton of end group

(3)

The 1H NMR spectrum of SApp (Figure S2d of Supporting Information) shows that the anhydride of SA is an oligomer with a degree of polymerization approximately equal to 8. The 1H NMR spectrum of the polymer, as shown in Figure S2g of Supporting Information, confirms that the polymerization between mixed anhydrides of COMA and SA had occurred. The chemical shifts at δ = 0.9, 4.1−4.3, 5−5.5, and 6.3−6.5 in the spectrum are characteristic of protons of the individual functional groups in COMA, and this indicates that the COMA unit is present in the copolymer. The sum total of the peak integrals in the range of 4−6.5 ppm remained the same in the spectra of COMApp and 50 wt % polymer. However, the sum total of the peak integrals in the range of 1− 2.5 ppm was much higher in the spectrum of the 50 wt % polymer as compared to that in COMApp, which indicates that the SApp repeat unit is present in the polymer structure. The peak positions for COMApp and SApp remained the same in 7895



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contact angle reduced as the SApp content increased in the polymer (Table 1). This is likely because the long aliphatic chains of the CO impart hydrophobicity, and this is reduced when the SApp content increases in the polymers. Contact angle measurements for 20 and 80 wt % could not be obtained because the surface of the discs was not uniform because the former was sticky and the latter was too brittle for compression molding. Several biodegradable polymers exhibited hydrophobicity, such as cross-linked polyanhydrides obtained by thiolene-polymerization were found to be hydrophobic in nature with a contact angle in the range of 82−92°.44 Poly(CPP-SA) system with higher content of CPP eroded very slowly because of the hydrophobic nature of the monomer CPP.9 An increase in the castor oil content in castor oil− mannitol−citric acid−sebacic acid based polyesters showed an increase in contact angle values.23 The degradation rate is often observed to depend on the mechanical strength of the polymer. Crystalline polymers are brittle in nature, and crystallinity reduces the degradation rate.45 Compressive tests revealed that the modulus for 50 wt % polymer was 25 MPa (Table 1 and Figure S5 of Supporting Information). Compressive test data also show that the modulus values increase with increase in SApp content. 80 wt % polymer showed the maximum value of modulus of 530 MPa. Carboxyphenoxy decanoate:para Carboxyphenoxy hexane anhydride-ester copolymer was observed to exhibit Young’s modulus of 244 kPa, and it showed changes ranging from 79 to 581 kPa with varying ratios of the prepolymers.46 The tensile strength of the CPP-SA system varied from 155 to 161 kg cm−2 as a function of CPP and SA content.47 In Vitro Hydrolytic Degradation. Polyanhydrides primarily degrade by passive hydrolysis because of the hydrolytically labile anhydride bond into water-soluble products, which can be easily eliminated from the body.48 It is well-known that the hydrolytically unstable nature of the anhydride bond and hydrophobicity causes polyanhydrides to undergo surface erosion which makes them well-suited for drug release applications.41 It has been shown that enzymes have no effect on the degradation rate.49 The rate of the degradation was modeled using the power law equation dM /dt = kM n

Figure 1. Degradation profiles of the polymers in 20 mL of PBS solution (pH 7.4). Inset shows modeling of the degradation profiles of the polymers.

poly(bis(p-carboxyphenoxy)propane anhydride) were found to degrade faster in basic medium.9,50,51 For 50 wt % polymer, the hydrolysis of the anhydride bond is base-catalyzed and the polymer eroded at an accelerated rate in basic buffer solution at (pH 10.2) with 77% weight loss within 48 h. It eroded negligibly in acidic buffer solution (pH 3.2), and the weight loss was less than 6% in 120 h. The degradation was likely suppressed because the acidic pH prevented SA in the polymer bulk from getting ionized and dissolving in the buffer; hence, it may have formed a barrier for further water intrusion.41 It was observed that the polymer disks became smaller in size with degradation time and the weight loss with time is linear (Figure 1), typical of surface-eroding polymers. Similar findings were reported for other polyanhydrides. For example, crosslinked polymer networks obtained from mixed anhydrides of SA with methacrylic acid degraded completely in 1 week, whereas those obtained from CPP lost 25% weight in 3 months.52 p(SA), p(CPP-SA) 20:80, p(CPP-SA) 50:50 displayed zero-order degradation kinetics.53 A series of poly(ester anhydride)s synthesized by the insertion of RA into poly(SA) lost around 60−75% weight during 350 h of degradation.54 The degradation for the synthesized polymers was very slow compared to that of RA-SA (1:1) based block-copolymers, which lost 50% weight within 72 h in PBS; a linear loss in weight was not observed.20 RA-SA (1:1) based random copolymers lost ∼50% weight within 96 h and showed a firstorder dry weight loss.35 Scanning electron micrographs for the surface of the 50 wt % polymer matrix at 0, 8, and 120 h of degradation are shown in Figure 2. Figure 2a shows the surface of the polymer prior to the degradation in PBS (magnification, 300×). Figure 2b shows the polymer surface after 8 h in PBS at the same magnification, and the development of small cracks on the surface was observed. The inset of Figure 2b shows the cross-section of the surface at a magnification of 6000× (scale bar, 5 μm), and small pores were seen, quite likely because of the penetration of water. Deep cracks were formed on the surface of the polymer after 120 h in PBS, as shown in Figure 2c (300×), and cracks can be seen progressing from the edge toward the bulk of the matrix (inset of Figure 2c) at a lower magnification of 80× (scale bar, 500 μm). As the polymer undergoes surface erosion,

(4)

In eq 4, M is the mass of the polymer at the time t, n the order of degradation, and k the rate constant. In all cases, the order of the degradation was zero; therefore, the variation of Mt/M0 with time is linear. Given Mt/M0 varies linearly with time with an intercept of unity, the values of k obtained were 7.3 × 10−3, 4.2 × 10−3, 3.5 × 10−3, 3.2 × 10−3, and 3.8 × 10−3 h−1 for 20, 33, 50, 66, and 80 wt % polymers, respectively. This indicates that the degradation of 50, 66, and 80 wt % polymers were similar, whereas the degradation of 33 wt % polymer was higher and that of 20 wt % polymer was much higher (Figure 1). For example, the 50 wt % polymer matrix eroded slowly in 20 mL of PBS buffer solution (pH 7.4) with 40% weight loss in 5 days, whereas the 20 wt % polymer matrix eroded faster with 88% weight loss after which the devices began to slowly disintegrate. As the COMApp content is increased, the polymer becomes more amorphous and prone to hydrolysis, resulting in a higher rate of degradation. It is essential to conduct degradation studies under different pH conditions. Polymers like PGA or PLGA degrade faster at acidic pH conditions. On the other hand, polymers such as 7896



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50% of the anhydride bonds after 48 h20, and a random copolymer prepared from the same monomers showed similar behavior.35 Dye Release. We selected one hydrophilic dye (RB) and one hydrophobic dye (RBB) to demonstrate the capability of loading and dye release from the polymer (Figure 3a,b).

Figure 2. SEM micrographs of degraded polymer (50 wt %) disks at 300× magnification: surface of the polymer matrix (a) initially (before degradation), (b) after 8 h in PBS, and (c) after 120 h in PBS. Scale bar is 100 μm in all images except insets, whose scale bars are 5 μm and 500 μm for b and c, respectively.

Figure 3. In vitro release of (a) hydrophilic RB dye and (b) hydrophobic RBB dye from the polymer discs in PBS at pH 7.4 and 37 °C. Insets show the fit of the data to the mathematical models.

Hydrophilic drugs such as 5-Fluorouracil56 and hydrophobic drugs such as BCNU (Carmustine)57 have been loaded in polyanhydrides and tested for drug delivery applications. The presence of tert-N+ and Cl− ions in RB makes it comparatively more hydrophilic than RBB. Dye release is usually controlled by factors such as dye dispersion in polymer, degradation of polymer, and diffusion of medium through polymer.58,59 Dye release profiles of both RB and RBB dyes show that there was no initial burst release but a controlled release from initial immersion of the polymer disc in PBS. Generally, polyanhydrides show two phases of release. There will be an initial burst release followed by a stabilized release. The initial burst release is most likely due to the fact that some of the dye is present on the surface and not encapsulated well inside the polymer.60 Therefore, when the polymer disc is immersed in the PBS medium, the dye is released from the polymer without significant polymer degradation and diffusion of the medium through the polymer. The release stabilizes after

the polymer degrades faster, which can lead to the formation of deep cracks resulting in higher water uptake by the polymer. In this case, the degradation of the polymer is faster than the water intake by the polymer.48 The anhydride peak intensity at 1812 cm−1 decreased with time, whereas the acid peak intensity at 1706 cm−1 increased with time in the IR spectra of the degraded 50 wt % polymer matrices as the anhydride bond undergoes cleavage to give carboxylic acids as degradation products. Percentage anhydride bond loss was determined by IR from the peak size ratio using the standard formula wherein the anhydride peak is normalized and the loss is calculated based on the reduction of this peak with time.55 The anhydride peaks at 1812 cm−1 diminished with time, and the acid peak at 1706 cm−1 intensified, with about 42% of the anhydride bonds lost after 8 h (shown in Figure S6a of Supporting Information) and about 79% of the anhydride bonds lost in 120 h (shown in Figure S6b of Supporting Information). RA-SA based block-co-polymers lost 7897



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controlled release, which is ideal and makes it unique from other polyanhydrides. Therefore, one can conclude that these polymers are suitable for localized anticancer drug delivery applications and both hydrophobic and hydrophilic drugs can be loaded into the polymers. Cytocompatibility Studies. MC3T3-E1 mouse osteoblasts were used to evaluate the cytocompatibility of the polymer. This is a well-established osteoblast model.62 The polymer discs were incubated in cell culture medium mimicking conditions of the degradation test. The viability and morphology of the cells exposed to the conditioned medium containing the degradation products were used as a test of biocompatibility of these polymers. For this study, 50 wt % polymer was chosen because there was a sustained dye release over a long period of time, as observed in Figure 3a,b. Further increase in SApp fraction in the polymer did not yield any significant difference in release profiles but increases the risk of cytotoxicity because of higher acid content of the degradation products. Cell viability was quantified using the MTT assay for cells exposed for 24 h to conditioned media collected at 1, 2, and 3 days (Figure 4). The absorbance value is proportional to the

some time because the dye that is encapsulated well is released. This release is governed by both the degradation of the polymer and the diffusion of the PBS inside the polymer. As the polymer starts degrading, there will be holes and channels created for the PBS to enter the polymer,58 facilitating the dye release from the polymer. The dye release can be modeled using power law which states that M t /M∞ = kt n

(5)

In eq 4, Mt is the amount of the dye released at that particular time, M∞ the total amount of the dye released, k the rate constant, t the time, and n the diffusion exponent indicative of the mechanism of transport of dye through the polymer. The dye release was zero-order for all the polymers, and the variation of the logarithm of Mt/M∞ with logarithm of time was linear with a slope of unity (n = 1), as shown in the insets of Figure 3a,b. The values of k were 3.5 × 10−5 and 3.1 × 10−5 min−1 for the release of RB and RBB, respectively, from the 20 wt % polymer. The 20 wt % polymer shows the fastest degradation, yet the drug release is relatively slower because of decreasing crystallinity and increasing hydrophobic nature of these polyanhydrides with increasing CO content. Because it undergoes surface erosion, the polymer degrades faster than the water uptake. The dye release, however, requires water uptake. Because of the hydrophobic nature of this polymer, the water uptake is slower in this polymer, resulting in slower dye release. The values of k for the release of RB and RBB from 33 wt % polymer were both ∼4.1 × 10−5 min−1. Similarly, the values of k were between 5.0 × 10−5 and 5.8 × 10−5 min−1 for the release of RB and RBB from the rest of the polymers. The value of unity for the diffusion coefficient n is indicative of the zerothorder dye transport through the polymer. RBB will have comparatively slower release than RB because it is a hydrophobic dye. This is observed in the case of other polyanhydrides where Langer and co-workers investigated the release of RBB from poly(fatty acid dimer: SA).60 They explained that the slower release is due to the lower solubility of the dye and its hydrophobic interactions with the polymer. Therefore, the interaction with the medium and the diffusion of the medium inside the polymer will be lower resulting in a lower rate of dye release. RB is a hydrophilic dye. Therefore, the interaction of the dye with the PBS is higher and faster release of the dye is expected. A similar trend was observed with 20 wt % polymer where SApp content in the polymer is lower. Because COMApp content is higher, the polymer is more amorphous and hydrophobic, which leads to a higher interaction of RBB with the polymer. Therefore, RB was released faster than RBB (Figure 3a,b) for this polymer. However, the release rates of both RB and RBB were similar in 33, 50, 66, and 80 wt % polymers. This could be because the solubilities of RB and RBB, which are 50 g L−1 and 1.25 g L−1, respectively, are considerably higher than the concentration released. Generally, controlled release is preferable in drug delivery applications as the burst release increases the concentration of the drug drastically in the blood which may cross the therapeutic limit, which is very toxic. For example, polyanhydrides such as p(fatty acid dimer: SA) and p(bis(carboxyphenoxy) alkane) were loaded with Acid Orange dye and nitroaniline in their matrices, respectively. They showed an initial burst release and then a constant release.60,61 The polymers synthesized herein showed no burst release but only a

Figure 4. Normalized cell viability determined by MTT assay for conditioned medium containing degradation products at 1, 2, and 3 days.

number of viable cells.63 The absorbance reading for the cells exposed to the conditioned medium was normalized to that of the controls (cells exposed to fresh medium) for that day. Figure 4 indicates that the cell viability at 1 day decreased statistically significantly to 70% of the control. At 2 days, the viability was 92% of the control but there was no statistically significant difference. At 3 days, the viability of the cells in the conditioned media was similar (not statistically different) to that of the control. The decrease in viability at 1 day is possibly due to the initial release of unreacted prepolymers from the polymer discs. Optical micrographs revealed that the cells maintained a characteristic spindle-shaped morphology after exposure to conditioned media (Figure 5a,b). These cells exhibited no discernible differences in morphology when compared to the controls at 1, 2, and 3 days. Taken together, these results indicate that the polymer and its degradation products are minimally cytotoxic.

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CONCLUSIONS A family of novel biodegradable polyanhydrides was synthesized from cheap, nontoxic, and renewable resources, CO and SA, using a catalyst-free melt−condensation technique. The polymers possessed different thermal properties with increase 7898



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oil (CO) (Figure S2a); 1H NMR spectrum of castor oil maleate (COMA) (Figure S2b); 1H NMR spectrum of castor oil maleate prepolymer (COMApp) (Figure S2c); 1H NMR spectrum of sebacic acid prepolymer (SApp) (Figure S2d); 1 H NMR spectrum of polymer (20 wt %) (Figure S2e); 1H NMR spectrum of polymer (33 wt %) (Figure S2f); 1H NMR spectrum of polymer (50 wt %) (Figure S2g); 1H NMR spectrum of polymer (66 wt %) (Figure S2h); 1H NMR spectrum of polymer (80 wt %) (Figure S2i); FTIR spectrum of COMA (Figure S3a); FTIR spectrum of COMApp (Figure S3b); FTIR spectrum of SApp (Figure S3c); FTIR spectra of the polymers with varying wt % of SA (Figure S3d); XRD spectra of COMApp, SApp, and polymers (Figure S4); stress− strain curve of polymers (33 wt %, 50 wt %, 66 wt %, 80 wt %) obtained by compressive tests (Figure S5); FTIR spectrum of degraded polymer matrix (50 wt %) after 8 h in PBS (Figure S6a); FTIR spectrum of degraded polymer matrix (50 wt %) after 120 h in PBS (Figure S6b). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: 091-80-22933408. Fax: 091-80-23600472. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by the Department of Biotechnology (DBT), India (BT/PR5977/MED/32/242/2012). K.C. was supported by the Ramanujan fellowship from the Department of Science and Technology (DST), India. We acknowledge Mr. I. Jarali for help with DSC thermograms and IR spectra, Mr. Shyam Sundar (Department of Mechanical Engineering) for contact angle measurements, Dr. Vibha (Department of Organic Chemistry) for her help with synthesis, and Mr. Hariprasad Gopalan (Department of Materials Engineering) for his help with compressive tests. We are grateful to the Department of Materials Engineering, SSCU, Organic Chemistry, SID, and NMR Research Centre of IISc for access to research facilities.

Figure 5. Bright-field optical micrographs of osteoblasts exposed to (a) conditioned medium at 1 day and (b) fresh growth media as control (magnification 20×; scale bar is 10 μm).

in melting temperature and crystallization temperature when SApp content was increased in the polymer. Mechanical tests showed that the polymers were brittle when SApp content was higher in the polymer. The polymers exhibited zero-order degradation and zero-order dye release profile. Polymers with higher SApp content degraded slowly because the crystallinity of the polymers increased. A linear degradation profile showed that it degraded by surface erosion. The dye delivery studies did not show burst release but a controlled linear release of the dyes. Except for the 20 wt % polymer, where the release of Rhodamine B was faster than that of Rhodamine B Base, the release was similar for both the dyes in the other polymers. The polymer was also found to be cytocompatible as determined by in vitro cell culture studies. Moreover, a systematic comparison performed here indicates that CO−50%SA offers optimal sustained release and cytocompatibility. This study indicates that a variety of polyanhydrides with different mechanical properties and degradation profiles can be synthesized by varying the prepolymer ratios.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Transesterification of castor oil with maleic anhydride to form castor oil maleate (COMA) (Figure S1a); acetylation of castor oil maleate to form castor oil maleate prepolymer (COMApp) (Figure S1b); acetylation of sebacic acid to form sebacic acid prepolymer (SApp) (Figure S1c); 1H NMR spectrum of castor 7899



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