Hydrolytic Degradation of Ricinoleic-Sebacic-Ester-Anhydride

Apr 23, 2005 - bonds and significant release of sebacic acid (SA). The remaining oligoesters of SA and ricinoleic acid. (RA) degraded into shorter oli...
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Biomacromolecules 2005, 6, 1877-1884

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Hydrolytic Degradation of Ricinoleic-Sebacic-Ester-Anhydride Copolymers Michal Y. Krasko and Abraham J. Domb*,† Department of Medicinal Chemistry and Natural Products, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, 91120 Jerusalem, Israel Received December 5, 2004; Revised Manuscript Received March 7, 2005

The degradation process of poly(ricinoleic-co-sebacic-ester-anhydride)s in buffer solution was investigated by following the composition of the degradation products released into the degradation medium and the degraded polymer. The first week of degradation was characterized by the hydrolysis of the anhydride bonds and significant release of sebacic acid (SA). The remaining oligoesters of SA and ricinoleic acid (RA) degraded into shorter oligoesters composed of RA ester dimers, trimers, and tetramers as well as dimers of RA-SA. To confirm and determine the hydrolytic behavior of the degradation products, short oligoesters of sebacic and ricinoleic acid were synthesized and degraded. It was established that during the hydrolysis under physiological conditions the degradation products have a composition and water absorption similar to those of the synthesized oligoesters. Introduction Biodegradable polyanhydrides and polyesters are useful materials for controlled drug delivery.1-3 They have a hydrophobic backbone with hydrolytically labile anhydride and/or ester linkages and can be controlled by manipulation of the polymer composition. Our laboratory has reported on the synthesis of poly(ester-anhydride)s,4 derived from poly(sebacic acid) (PSA) and ricinoleic acid (RA) p(SA-RA). The main characteristics of these p(SA-RA)s are their simple one-pot preparation, biodegradability, and biocompatibility. P(SA-RA)s with up to 60% of RA are solids at room temperature and the polymer having 70% w/w of RA or more are pasty at room temperature. These polymers are useful biodegradable carriers for the controlled release of anticancer agents.5 In the process of degradation under physiological conditions, the polymers continuously change their composition. The hydrolysis causes a process of polymer chain cleavage and therefore a drop in the molecular weight and release of water soluble products from the polymer matrix to the degradation medium. Since p(SA-RA)s are intended for use as drug carriers in the human body, their content should be determined at each time point of their degradation. This study focused on two main issues: composition of the polymer matrix during the hydrolysis and composition of the degradation products. Experimental Section Materials. Ricinoleic acid, (85% pure; Fluka; Buchs; Switzerland); ricinoleic acid (99% pure; ICN Biomedicals; * Correspondence author. Phone: 972-2-6757573. Fax: 972-2-6757629. E-mail: [email protected]. † Lionel Jacobson Chair in Medicinal Chemistry. Affiliated with the David R. Bloom Center for Pharmacy, and the Alex Grass Center for Synthesis and Drug Design at The Hebrew University of Jerusalem.

Eschwege; Germany), sebacic acid (99% pure; SigmaAldrich; Israel) was recrystallized from hot ethanol, and sebacoyl chloride (97% pure; Fluka; Buchs; Switzerland), 2-chlorotrityl chloride resin (100-200 mesh) (Patras, Greece), N,N-diisopropylethylamine (99.5%, Sigma, Israel), trifluoroacetic acid, and acetic anhydride (Biolab; Jerusalem; Israel) were used in this study. All solvents, which were used for analytical tests were HPLC grade (Biolab; Jerusalem; Israel) and were used without further purification. Other common solvents were analytical grade and were used without further purification. Instrumentation. Infrared (IR) spectroscopy (Vector 22 FTIR, Bruker, Germany) was performed on prepolymer, polymer samples, and hydrolyzed samples cast on NaCl plates from dichloromethane solution. Thermal analyses were determined on a Metler TA 4000-DSC differential scanning calorimeter, calibrated with zink and indium standards, at a heating rate of 10 °C/min (typical sample weight was 10 mg) and on a Stuart Scientific Melting point SMP1 heater. Molecular weights of the polymers were estimated on a gelpermeation chromotography (GPC) system consisting of a Waters 1515 isocratic HPLC pump with a Waters 2410 refractive index (RI) detector and a Rheodyne (Coatati, CA) injection valve with 20-µL loop (Waters Ma). Samples with molecular weights above 4000 Da were eluted with CHCl3 through a linear Styragel HR2 column (Waters; 7.8 × 300 mm) at a flow rate of 1 mL/min, and samples with molecular weights below 4000 Da were eluted with CHCl3 through a linear Styragel HR1 THF (Waters; 7.8 × 300 mm) column. The molecular weights were determined relative to polystyrene standards (Polyscience, Warrington, PA) with a molecular weight range of 120-4500 for the Styragel HR1 THF column and 4500-18 000 for the Styragel HR2 column using a Breeze computer program. 1H NMR spectra (CDCl3) were obtained on a Varian 300-MHz spectrometer in 5 mm

10.1021/bm049228v CCC: $30.25 © 2005 American Chemical Society Published on Web 04/23/2005

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Scheme 1. Synthesis of RA-SA Dimer on Solid Phase Platform

o.d. tubes. CDCl3 containing tetramethylsilane served as the solvent and shift reference. SA and RA concentrations in buffer solutions were determined by an HPLC (HewlettPackard, Waldbronn, Germany) system composed of an HP 1100 pump, HP 1050 UV detector, and HP ChemStation data analysis program using a C18 reverse-phase column (LichroCart 250-4, Lichrospher 100, 5 µm). A mixture of acetonitrile:DDW:H3PO4 65:35:0.1 v/v/v at a flow rate of 1.4 mL/min was used as an eluent for the determination of RA with UV detection at 210 nm. A mixture of acetonitrile: DDW:H3PO4 10:90:0.1 v/v with a gradient from 10 to 30% acetonitrile at a flow rate of 1.4 mL/min was used as an eluent for the determination of SA with UV detection at 210 nm. Mass-spectroscopy analyses were performed on Electronspray Finnigan LCQDUO (Thermo Quest) apparatus. Ethanol served as solvent. Polymer Synthesis. A series of poly(sebacic-co-ricinoleicester-anhydride)s were synthesized as described elswere.4,5 Technical RA (RA, 85% pure) was purified by the potassium salt extraction method.6 The copolymers were prepared by transesterification, followed by polycondensation. Briefly, p(SA-RA) with 5:5 w/w ratio was prepared by mixing 5 g of PSA (Mw g 40 000 Da) with 5 g of purified RA (98% pure) at 120 °C for about 3 h. The transesterification was stopped when the molecular weight reached its lowest constant value. Acetic anhydride was added to the reaction flask at a 1:1 w/v ratio. The solution was refluxed at 140 °C for 30 min, filtered, cooled, and evaporated to dryness. The polycondensation was performed at 170 °C, under a vacuum of 0.1 mmHg for 2-4 h with continuous stirring. The polymerization was terminated when the molecular weight reached its highest constant value. Oligomer Synthesis. Preparation of RA Oligomers. A total of 10 g of RA was dissolved in 120 mL of toluene containing a catalytic amount of p-toluenesulfonic acid (pTSA) in a 250 mL round-bottomed flask equipped with a

Dean-Stark arm and refluxed for about 4 h. After solvent removal, the yellowish oil was purified by chromatography on a silica gel column using a mixture of hexane:ethyl acetate:acetic acid at 8:3:0.1 v/v/v ratio as eluent.7 Oligomers with an average molecular weight in the range of 1000 Da were obtained. Oligomers with an average molecular weight in the range of 3000 Da were similarly prepared, but after solvent removal from the reaction flask, it was connected to a vacuum line and stirred overnight at 100 °C. According to GPC analysis no peak with molecular weight in the range of 300 was present, which means that no free RA was left in the product. Preparation of RA-SA Dimer. RA-SA dimer was synthesized on a solid-phase platform, 2-chlorotrityl chloride resin (100-200 mesh) as shown in Scheme 1. RA (250 µmol, 75 mg) and of N,N-diisopropylethylamine (750 µmol, 400 µL) were dissolved in 1 mL of CH2Cl2. The solution was added to the synthesis vial equipped with a sinter glass filter, and 100 µmol of the resin were added to the solution. The reaction vial was shaken for 24 h at room temperature, then the solvent with unreacted RA was filtered out, and the solid phase was washed twice with CH2Cl2. Sebacoyl chloride (250 µmol, 60 mg) and N,N-diisopropylethylamine (750 µmol, 400 µL) were dissolved in 1 mL of CH2Cl2. The solution was added to the reaction vial and shaken for another 24 h at room temperature. After this period, the solvent with unreacted sebacoyl chloride was filtered out, and the solid phase was washed twice with CH2Cl2. The dimer was disconnected from the solid phase by the addition of 1 mL of 0.1% v/v triflouroacetic acid (TFA) solution in CH2Cl2. The product was filtered out and washed with DDW in order to hydrolyze the chloride end of the RA-SA dimer. The organic phase was acidified to pH 4 with HCl, washed with DDW, dried, and evaporated to dryness. The sample was divided in two parts, half was dissolved in chloroform for

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Degradation of p(SA-RA)

Figure 1. IR spectrum of p(SA-RA) 3:7 w/w. The typical anhydride peaks are 1732 and 1823 cm-1. IR spectroscopy was performed on polymer samples cast on NaCl plates from dichloromethane solution.

NMR, GPC, and IR analysis and half was dissolved in ethanol for mass-spectroscopy analysis. In Vitro Hydrolytic Degradation of the Polymers. To study the SA release from the polymers, samples (200 mg, diameter∼5 mm) of p(SA-RA)s with 5:5, 4:6, 3:7, 2:8, and 1:9 w/w ratios were incubated in 50 mL of buffer phosphate solution pH 7.4, 0.1 M at 37 °C, with orbital shaking (100 rpm). The buffer solution was replaced every 48 h to avoid SA saturation (SA solubility in water is about 1 mg/mL) and turbidity of the solution. All experiments were done in duplicate. Samples of the polymer were immersed in buffer solution at 37 °C as described above. Samples were withdrawn from the buffer after 24, 48, and 72 h of incubation. A total of 1.5 mL of the buffer solution was extracted with 2 mL of CHCl3, dried on MgSO4, filtered, and concentrated to ∼250 µL. At the same time points, samples were taken from the polymers, rinsed with water, and lyophilized. The samples were analyzed by GPC. The degradation of polymers was followed by weight loss and GPC analysis for three months. Some of the buffer solutions from this study were hydrolyzed in 1 M NaOH, acidified to pH ∼ 4.5 and analyzed by HPLC, IR, and NMR. The composition of the degraded polymers at different time points was determined by 1H NMR. Typically, samples of 20 mg were diluted with 1 mL of CDCl3 and immediately analyzed. In Vitro Degradation of RA Oligomers. RA oligomers of Mn in the range of 1000 and 3000 were incubated in 50 mL buffer phosphate solution pH 7.4, 0.1 M at 37 °C, with orbital shaking (100 rpm). The degradation was followed by weight loss, decrease in molecular weight and release of RA. At each time point, 1.5 mL of the solution was taken out and analyzed by HPLC. At the same time, a sample was taken from the oligomer, rinsed with water, and lyophilized. The buffer solution was replaced every 48 h for the first

Table 1. Physical Properties of the Polymers poly(SA-RA)saw/w

Mw (Da)b

Mn (Da)b

mp (°C)c

∆H (J/gr)c

1:9 2:8 3:7 4:6 5:5

2000 4500 7000 32000 42000

1600 3500 5000 11000 15000

-11.2 26.6 35.6 51 54.5

2.2 9.4 20.9 21.9 93.4

a Polymers were prepared by transesterification of PSA (initial Mw of 37,000) with ricinoleic acid and anhydride re-polymerization. The SA-RA ratios are w/w. b Weight average molecular weight (Mw) and number average molecular weight (Mn) were determined by GPC. c Tm and ∆H were recorded by DSC scanning at 10 °C/min.

two weeks and then once a week. No turbidity was noticed throughout the study. All experiments were done in duplicate. To measure solubility of short oligomers of RA in the dissolution medium, a mixture of dimer, trimer, and tetramer of RA were dissolved in buffer phosphate at 37 °C. Their solubility was analyzed by HPLC. Results and Discussion Polymer Synthesis. P(SA-RA)s with 1:9, 2:8, 3:7, 4:6, and 5:5 w/w ratios were used. Polymers containing 70% or more RA were pasty at room temperature and were found suitable for injections.5 Polymers containing 60% or less of RA were solid at room temperature. The physical data of these polymers are summarized in Table 1. The infrared spectrum of p(SA-RA) 3:7 w/w is shown in Figure 1. All polymers showed two anhydride peaks at 1732 and 1817 cm-1. The peak at 1732 cm-1 may also be referenced to the ester bonds. No free acid peaks at 1700 cm-1 (CdO) and 3500 cm-1 (O-H) were present.1H NMR spectrum of the same polymer is shown in Figure 2. 1H NMR spectroscopy confirmed the insertion of the RA into the PSA chain by the disappearance of the CH-OH peak at 3.613 ppm and appearance of the CH-O-CO peak at 4.853 ppm.

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Figure 2. 1H NMR spectrum of p(SA-RA) 3:7 w/w. Obtained in 5 mm diameter tubes. CDCl3 containing tetramethylsilane served as solvent and shift reference.

Figure 3. Hydrolysis of p(SA-RA)s monitored by weight loss of the degraded polymer. Hydrolysis was conducted in 0.1M phosphate buffer (pH 7.4) at 37 °C. At each time point, the remaining polymer was dried and weighted. In some systems, the degradation solution was turbid indicting fragmentation of the polymer.

In Vitro Hydrolytic Degradation of the Polymers. The composition of p(SA-RA)s during hydrolysis under physiological conditions in vitro was analyzed. In addition to the standard analysis of weight loss, change in molecular weight, and IR spectroscopy of the polymer mass during degradation, the degradation of the polymers was followed by monitoring SA and RA release to the buffer solution and the composition of the polymers and their degradation products by 1H NMR, mass-spectroscopy, HPLC, and GPC. Weight Loss Analysis. The weight loss study was performed in order to determine the end point for degradation of each polymer. The weight loss during three months is summarized in Figure 3. It can be seen that p(SA-RA) 5:5 was less stable toward the hydrolysis and fully degraded in two months. The polymers that contained 70% of RA and more were more stable and fully degraded in three months.

Figure 4. Release of sebacic acid (SA) from p(SA-RA)s during 54 days of hydrolysis. SA release profile was conducted in 0.1M phosphate buffer (pH 7.4) at 37 °C. The SA content in solution was determined by HPLC.

IR Analysis. During degradation, the polymers were analyzed by IR spectroscopy to indicate decreasing of the anhydride peak at 1815 cm-1 and increasing of the carboxylic acid peak at 1700 cm-1. It took one week for the disappearance of the anhydride peaks. After this period, the spectra show no anhydride peaks, a small acid peak and a large ester peak at 1736 cm-1, which means that the anhydride sequences of SA were hydrolyzed and there are no anhydride segments left. HPLC Analysis. SA released from the polymeric matrixes was studied (Figure 4). SA was released within one week from the polymers. The fact that not 100% SA was released during the experiment is due to SA-RA oligoesters that gradually degrade to water soluble SA-RA. To confirm the presence of SA in the remaining polymer matrix, samples after 30 days of incubation were dissolved in ethanol, hydrolyzed with 1 M NaOH, acidified to pH 4 and analyzed

Degradation of p(SA-RA)

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Figure 5. Profile of hydrolyzed degradation products of p(SA-RA) 3:7 on HPLC after 1 month of degradation. The degradation conditions were as described in Figure 3. (a) Analysis of buffer solution, note that the peak of ricinoleic acid at 8.7 min is absent. (b) Chromatogram of the further hydrolyzed buffer solution with 1 M NaOH. Note that the degradation products were converted into ricinoleic acid.

by HPLC for SA content. The amount of SA that was found in the polymer complemented the expected total SA. At the beginning of the study, it was assumed that the p(SA-RA)s degradation could be monitored also by RA release; however, HPLC analysis of the buffer solutions found that there was no RA release from the polymer. The spectra showed some different peaks during the acquisition time but no expected peak at 8.7 min (Figure 5a). When the samples were hydrolyzed by 1 M NaOH, the expected peak of RA at 8.7 min appeared (Figure 5b). Alternatively, when a spike of RA was added to some samples, a peak at 8.7 min was shown. The area of the peak fitted the amount of added RA. These results indicate that other RA containing species (RA-RA, RA-SA, RA-RA-RA, RA-RA-RA-RA) are released to the water and being removed when periodically replacing the degradation solution. GPC Analysis. Molecular weight decrease of p(SA-RA)s was monitored by GPC during the three months of degradation (Figure 6, parts a and b, the first 4 days, and part c, next three months). Typical chromatograms are represented in Figure 7a for p(SA-RA)3:7. It was found that after ∼1.5 month of incubation in buffer solution the polymers degraded to oligomers with an average molecular weight of 1000 Da (Table 2). The molecular weight remained constant for the three months of the study. The study was terminated when the weight loss of the polymers (that resembled oil drops on the walls of the vials) was undetectable. Typical GPC chromatograms taken during p(SA-RA)s degradation show peaks at molecular weights of approximately 480 and 560

Table 2. Oligomer Composition vs Time of p(SA-RA) 3:7 during Hydrolysis as Determined by GPC Molecular Weight Fractions molecular weight (Da)a peak (oligomer) 500 600 1200 1500 2400 3200 7500 average molecular weight (Da)

0 day (%)

12

16 72 5000

2 days (%)

17 days (%)

38 days (%)

60 days (%)

90 days (%)

45

48

48

55

52

52

30

12 36 24 28

1395

1536

1230

1210

1210

5 20 45

a Polymer with initial M ) 5000 was exposed to 0.1 phosphate buffer, n pH 7.4. At each time point (upper row of the table), the sample of the degraded polymer was analyzed by GPC to determine Mn and the percentage of polymer fraction having a certain Mn (left column of the table).

Da, which are attributed to SA-RA and RA dimer, respectively. Other common peaks of a Mw of ∼800 and ∼1100 Da attributed to the trimer and tetramer of RA, respectively (Table 3). This data was confirmed by mass-spectroscopy analyses that are presented further. 1 H NMR Analysis. The change in SA:RA ratio during the degradation inside the polymer matrix was monitored by 1H NMR. SA has its characteristic peaks at 1.302 ppm (4CH2 in the middle of the chain), 1.632 ppm (OOC-CH2CH2), and 2.424 ppm (OOC-CH2-CH2). The peak at 2.424 ppm is no longer detectable after 120 h which fits the more

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Figure 7. GPC chromatograms of (a) P(SA-RA) 3:7 (Mw ) 7000) and (b) RA3000 (Mw ) 3000) during 4 weeks of degradation. The degradation conditions were as described in Figure 3. The molecular weight change was determined by GPC. Each chromatogram represents the molecular weight distribution of the remaining polymer sample at certain time point.

Figure 6. Molecular weight change during 4 days of hydrolysis of p(SA-RA)s. (a) p(SA-RA) with w/w ratios 1:9, 2:8, and 3:7. (b) p(SARA)s with w/w ratios 4:6 and 5:5. (c) Molecular weight change during hydrolysis of p(SA-RA)s for three months. The hydrolysis conditions were as described in Figure 3.

sensitive IR analysis that showed the anhydride bond disappears after one week. In the case of poly(sebacic-coricinoleic-ester-anhydride) the peak at 1.632 ppm is overlapped by the peaks of RA. The peak at 1.302 overlaps with the peak of the 6CH2 in the middle of the RA chain. This peak represents 20 protons for RA:SA at a ratio of 12:8. The peak integration can be expressed as: integration ) 12Y + 8X, where Y and X represent the contribution of RA and SA protons to the total integration at 1.302 ppm, respectively. The contribution of 1H of RA is known from the peak at 4.8 ppm (CH-COO), which is unique to the RA proton adjacent to the ester group. The degraded polymers were analyzed by 1H NMR in order to establish SA:RA ratio during the hydrolysis. The results are summarized in Figure 8 for p(SA-RA)5:5. The plot shows that in the first 24 h there is no difference in the SA:RA ratio followed by a sharp drop in the SA content due to its fast release. According to 1H NMR, after two weeks, there is very little SA in the

Figure 8. Hydrolysis of the p(SA-RA) 5:5 w/w monitored by SA:RA ratio with time. Hydrolysis was conducted as described in Figure 3. At each time point, the remaining polymer was dried and analyzed by NMR. The SA:RA ratio was determined by the ratio between the characteristic RA peak at 4.8 ppm and characteristic SA peak at 1.3 ppm.

system. This simple method lacks accuracy, probably because of peak overlap. RA Oligomers. RA based oligomers with average molecular weight of 1000 and 3000 Da were synthesized and used as models to investigate p(SA-RA) degradation, since the degradation products of p(SA-RA)s seemed to be composed of oligomers of similar molecular weights. The goal was to compare the degradation of synthesized oligoesters of RA with the degradation products of p(SA-RA)s. Their identical hydrolitical behavior indicates the similarity of their structures and prooves that after some period of incubation under physiological conditions p(SA-RA)s are composed mostly of oligoesters of RA. In Vitro Hydrolytic Degradation of the RA Oligomers. The RA oligomers were incubated under the same conditions as p(SA-RA)s. When these colorless and clear oil oligoesters were incubated in buffer phosphate for 24 h, they became white and not transparent. The oil droplet stuck to the walls of the vial as a semisolid substance. The degradation was monitored by molecular weight loss, weight loss, and water absorption.

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Degradation of p(SA-RA) Table 3. Structures of Oligomers Estimated on GPC and MS Analysis

Table 4. Molecular Weight Composition vs Time of Poly(RA) 3000 during Hydrolysis molecular weight (Da)a fraction 900 1100 1500 1800 2100 2700 3300 average molecular weight (Da)

0 week (%) (initial polymer) 3 7 8

1 week (%)

2 weeks (%)

7 9 9

22 46 13

75

19

3213

1412

3 weeks (%) 26 34

12 82 2982

1310

a Oligomer with initial M ) 3000 was exposed to 0.1 phosphate buffer, n pH 7.4. At each time point (upper row of the table), the sample of the degraded oligomer was analyzed by GPC to determine Mn and the percentage of oligomer fraction having a certain Mn (left column of the table).

GPC analysis shows that the molecular weight distribution of the hydrolyzed samples of RA 3000 was very much alike the degradation product of p(SA-RA)s with PSA:RA w/w ratios of 1:9, 2:8, and 3:7 during one month of degradation (Tables 4 and 2 and Figure 7, parts a and c). It should be noted that, in the case of PSA:RA w/w ratios of 4:6 and 5:5, there was a lower chance for the formation of RA-RA sequences during the transesterification due to the high percentage of PSA. The molecular weight of RA 1000 almost was not changed for three weeks (Figure 9). The molecular weight of p(SA-RA)s at one time point before the degradation was completed resembled very much the molecular weight distribution of RA 1000 (Figures 6c and 10). Therefore, it can be concluded that the final products of the p(SA-RA)s degradation are short oligomers of RA that slowly degrade to water soluble RA oligomers.

Figure 9. molecular Figure 3. remaining

Hydrolysis of oligomers of ricinoleic acid monitored by weight loss. Hydrolysis was conducted as described in The molecular weight change was determined on the polymer sample by GPC.

Figure 10. Hydrolysis of oligomers of ricinoleic acid monitored by weight loss. The hydrolysis conditions were as described in Figure 3.

It can be seen that the weight loss of RA 1000 is relatively slow (only 17% in three weeks; Figure 10). Degradation of RA 3000 is faster (33% in three weeks; Figure 10), but it slows down thereafter similar to the degradation behavior of RA 1000.

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Scheme 2. Degradation Process of P(SA-RA)n in Vitroa

a First step: degradation of the anhydride bonds and release of the sebacic acid (SA). Second step: degradation of the ester bonds and release of short oligoesters of ricinoleic acid (RA) and SA. Third step: dissolution or dispersion of dimer and trimer degradation products.

Some mass-spectroscopy analyses (MS) were also performed during hydrolysis. MS of RA 1000 shows peaks at 578 and 858, which are attributed to RA dimer (Mw ) 578.9) and trimer (Mw ) 859). MS of p(SA-RA) 2:8 after 12 days of incubation also shows these peaks, which indicates the presence of dimers and trimers of RA in the polymer sample. Solubility of the tetramer of RA in buffer phosphate at 37 °C is 5.8 mg/mL and the solubility of a mixture of dimer and trimer of RA is 10.2 mg/mL. RA-SA Dimer. Characterization of RA-SA Dimer. MS spectra show a main peak at 481, which fit the calculated Mw of the dimer (482) and GPC analysis. 1H NMR and IR spectroscopy also confirmed the ester structure of the dimer by showing disappearance of free OH of RA and the appearance of the ester bond. According to the mass-spectroscopy analysis, the RA-SA dimer represented a peak of 481. The MS peak of 481 was found at the degradation products of p(SA-RA) 2:8 after 12 and 17 days of degradation, which indicated the presence of RA-SA dimer in the sample. Conclusions The in vitro degradation process of poly(ester-anhydride)s made from RA and SA progresses in two stages. In the first stage, which last for about one week, the anhydride bonds are fully degraded, releasing the SA units conjugated in both

of their carboxylic acids by anhydride bonds. In the next step, the remaining RA-RA or RA-SA oligoesters degrade into shorter RA ester dimers, trimers, and tetramers as well as dimers of RA-SA, which are dissolved in the degradation aqueous medium. The process is summarized in Scheme 2. Oligoesters of RA and SA showed to similarly degrade under physiological conditions to the polymer samples. Acknowledgment. This work is dedicated to the memory of Avra’am Ben Dov. References and Notes (1) Langer, R. Acc. Chem. Res. 2000, 33, 94. (2) Domb, A. J.; Elmalak, O.; Shastry, V. R.; Ta-Shma, Z.; Masters, D. M.; Ringel, I.; Teomim, D.; Langer, R. In Handbook of Biodegradable Polymers; Domb, A. J., Kost, J., Weisman, D. M., Eds.; Harwood Academic Publishers: Chichester, U.K. 1997; p 135. (3) Stephens, D.; Li, L.; Robinson, D.; Chen, S.; Chang, H. C.; Liu, R. M.; Tian, Y. Q.; Ginsberg, E. J.; Gao, X. Y.; Stultz, T. J. J. Controlled Release 2000, 63, 305-317. (4) Krasko, M. Y.; Shikanov, A.; Ezra, A.; Domb, A. J. J. Polym. Sci. A 2003, 41, 1059-1069. (5) Shikanov, A.; Vaisman, B.; Krasko, M. Y.; Nyska, A.; Domb, A. J. J. Biomed. Mater. Res. A 2004, 69A (1), 1015-1022. (6) Borsotti, G.; Guglienlmetti, G.; Spera, S.; Battistel, E. Tetrahedron 2001, 57, 10219-10227. (7) Teomim, D.; Ma¨der, K.; Bentolila, A.; Magora, A.; Domb, A. J. Biomacromolecules 2001, 2, 1015-1022.

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