In Vivo Degradation and Elimination of Injectable Ricinoleic Acid

Mar 26, 2013 - Moran Haim-Zada , Arijit Basu , Tal Hagigit , Ron Schlinger , Michael ... Janeni Natarajan , Shruti Rattan , Utkarsh Singh , Giridhar M...
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In Vivo Degradation and Elimination of Injectable Ricinoleic AcidBased Poly(ester-anhydride) Boris Vaisman, Diana E. Ickowicz, Ester Abtew, Moran Haim-Zada, Ariella Shikanov, and Abraham J. Domb* Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, 91120 Jerusalem, Israel ABSTRACT: The in vivo degradation and elimination after subcutaneous implantation of injectable p(SA-RA) 3:7 copolymer in rats, followed by characterization of the polymer matrix composition during hydrolysis and erosion, is reported. Major chemical changes were observed during the first few days post implantation, the anhydride bonds hydrolyzed along with about 45% weight loss and a significant decrease in the molecular weight. 1H NMR spectral analysis was used to determine the structures and content of ricinoleic acid containing oligomeric chains present in the degraded polymer. The polymer degrades into ester oligomers of 2−4 ricinoleic acid units which further degrade to ricinoleic acid, a natural fatty acid. The polymer hydrolytic degradation process fit the in vitro degradation process.

1. INTRODUCTION Biodegradable polyanhydrides and polyesters are useful materials for controlled drug delivery.1−4 They have a hydrophobic backbone with hydrolytically labile anhydride and ester linkages and can be controlled by manipulation of the polymer composition.3−7 Fatty acids are good candidates for the preparation of biodegradable polymers because they are natural body components and they are hydrophobic; thus, they may retain an encapsulated drug when used as drug carriers.8 This laboratory has reported on the synthesis of biodegradable poly(ester-anhydride)s, derived from poly-(sebacic acid) (PSA) and ricinoleic acid (RA) p(SA-RA).9 Scheme 1 shows the

degradation under physiological conditions. The hydrolysis causes chain cleavage, a drop in the molecular weight, and the release of water-soluble polymer degradation products to the surrounding biological medium.5,6 The processes involves water penetration into the polymer bulk and/or interaction with the polymer surface, which is sometimes accompanied by swelling.5,8,18−20 The intrusion of water triggers chemical polymer degradation, leading to the formation of oligomers and monomers. Progressive degradation changes the microstructure of the polymer through the formation of pores in the bulk and disturbing surface structural continuity. Finally, oligomers and monomers are released by diffusion and surface erosion leading to the weight loss of polymer devices.5,21 Polymer erosion is far more complex than degradation, because it depends on many other processes, such as degradation, swelling, the dissolution and diffusion of oligomers and monomers, and morphological changes.6,22 Knowledge of the erosion mechanism is important for the development of a useful degradable polymer for tissue engineering and drug delivery.5,23,24 Moreover, polymer degradation products should be characterized to facilitate potential risk assessment.1 Previous research conducted in this laboratory has focused on the p(SA-RA)s degradation process in vitro.25 It included investigation of the composition of the polymer matrix during hydrolysis and composition of the degradation products. The in vitro degradation studies allow monitoring of both the chemical changes occurring inside the polymer bulk8,26 and the degradation product accumulated in the degradation medium.22,25 On the other hand, biological systems represent an open experimental model in which degradation products are released from the matrix of the

Scheme 1. General Structure of p(SA-RA) Copolymers

general structure of p(SA-RA) copolymers, which can be prepared at different weight ratio. These polymers have been used as drug carriers, particularly as liquid polymers that can be injected into a patient. The drug containing formulations of injectable poly(sebacic acid-co-ricinoleic acid) 3:7 (p(SA-RA) 3:7) copolymer, loaded with cis-platinum and paclitaxel, showed promising results for localized antitumor agents delivery.9−12 The same polymer was used for the delivery of gentamicin sulfate for the treatment of osteomeylitis,13 controlled release of protein and peptide drugs,14 and delivery of local anesthetics.15 Biodegradable polymers are polymers that degrade after implantation into nontoxic products, which are then eliminated from the body or metabolized therein.16,17 The polymers continuously change their composition in the process of © XXXX American Chemical Society

Received: January 30, 2013 Revised: March 19, 2013

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degraded polymer into surrounding extracellular fluid, distributed to body tissues, and eventually excreted from the body or metabolized.27,28 The aim of the current research is to study degradation and elimination from the implantation site of injectable p(SA-RA) 3:7 copolymer. This goal has been accomplished by characterization of the polymer matrix composition during hydrolysis and erosion in vivo at different points of time.

Approval Number: OPRR-A01-5011) reviewed our application for the animal study and found it compatible with the standards for care and use of laboratory animals (ethics committee research number: MD104.12-3, May 26, 2005). The animals were anesthetized with 85% ketamine (Ketaset, 100 mg/mL, Fort Dodge) and 15% xylazine (20 mg/mL, Biob, France) administrated IP at a dose of 1.2 mL/1 kg body weight. Euthanasia was accomplished by IP administration of pentobarbitone sodium, 200 mg/mL (Pental, CST, Israel), at a dose of 1 mL/1.5 kg body weight. 2.5. Implantation Technique. Subcutaneous (SC) implantation of the polymer in the posteriolateral flank of SD rats was performed according to the procedure described previously.30 Briefly, the skin above the implantation sites was shaved and disinfected with 70% ethyl alcohol prior to injection. The pasty polymer was implanted into fully anesthetized rats by injection using a sterile 22-G needle (Artsana S.P.A., Italy) from prefilled Fortuna Optima 1 mL luer-lock glass syringes (Bein Z.M., Tel-Aviv, Israel). Each rat received a total of 500 μL (equivalent to 500 mg) of p(SA-RA) 3:7. The polymer was delivered to two implantation sites in each rat; two SC injections of 250 μL each were made on the opposite dorsal sides of the animal. 2.6. Sample Processing. Three animals were sacrificed randomly at each scheduled point of time (3, 7, 14, 21, 28, 35, and 42 days postimplantation). Implants were collected, separated from surrounding tissues, and lyophilized. The weight of the samples was recorded before and after lyophilization. The lyophilized samples were analyzed by GPC, HPLC, IR, 1H NMR, and mass spectroscopy. The comonomer composition of the remaining polymer matrix was determined by HPLC after hydrolysis. The samples were hydrolyzed with 1 M NaOH, acidified with HCl to pH 2, and extracted with CHCl3; then the solvent was removed by evaporation, and the hydrolyzed samples were dissolved in ethanol.25

2. EXPERIMENTAL SECTION 2.1. Materials. Castor oil European Pharmacopoeia (Eur Ph) was obtained from Florish (Haifa, Israel); ricninoleic acid (99%) was purchased from ICN Biomedicals Inc. (OH, U.S.A.); sebacic acid (99%) was obtained from Sigma-Aldrich Ltd. (Rehovot, Israel); and acetic anhydride was purchased from BioLab (Jerusalem, Israel). All solvents used for the 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. 2.2. Instrumentation. Infrared spectroscopy (2000 FTIR; PerkinElmer) was performed on polymer samples and on degraded samples cast onto NaCl plates from dichloromethane solution.10 Molecular weights of the polymers were estimated using a gel permeation chromatography (GPC) system consisting of a Waters 1515 isocratic HPLC pump with a Waters 2410 refractive index detector and a Rheodyne (Coatati, CA) injection valve with a 20 μL loop (Waters, Milford, MA). Samples with molecular weights below 4000 Da were eluted with CHCl3 through a linear Styragel HR1 THF (Waters; 7.8 mm I.D. × 300 mm) column and samples with molecular weights above 4000 Da were eluted with CHCl3 through linear Styragel HR2 and HR4 columns (Waters; 7.8 mm I.D. × 300 mm) at a flow rate of 1 mL/min. The molecular weights were determined relative to polystyrene standards (Polyscience, Warrington, PA) using a Breeze computer program.10,25 1H NMR spectra were obtained on a Varian 300 MHz spectrometer in 5 mm o.d. tubes. CDCl3 containing tetramethylsilane served as the solvent and shift reference. SA and RA content were determined by HPLC. HPLC-UV was carried out using an HPLC system consisting of an HP 1050 quaternary pump, an HP 1050 autosampler with 200 μL loop, and an HP 1050 Photodiode Array Detector (PDA) coupled with HP ChemStation for LC 3D Systems indented for data processing (Agilent Technologies, Palo Alto, U.S.A.). Separation was performed on C18 RP analytical columns (LichroCart 250−4, Lichrospher 100, 5 μm), which were protected with a C18 RP guard column (LichroCart 4−4, Lichrospher 100, 5 μm). A mixture of 70% acetonitrile/30% water/0.2% phosphoric acid at a flow rate of 1.0 mL/min was used as an eluent for the determination of RA with UV detection at 205 nm.29 A mixture of 10% acetonitrile/90% water/0.1% phosphoric acid 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 205 nm.25 Mass spectroscopy analysis was performed on a Finnigan LCQ DUO mass spectrometer (TermoQuest, San Jose, CA, U.S.A.) running on a negative ion mode.25,29 Ethanol served as the solvent. 2.3. Polymer Synthesis. The poly(ester-anhydride) copolymer of SA and RA in a 3:7 weight to weight ratio (p(SA-RA) 3:7) was synthesized as previously described by transesterification followed by anhydride melt condensation.9,25 SA was used as supplied by SigmaAldrich without any additional purification. RA (>98%) was isolated from castor oil by fractional precipitation using the salt-solubility methods.29 The synthesized polymer was characterized by GPC, IR spectroscopy, and 1H NMR spectroscopy, employing the instrumentation described above. 2.4. Animals, Anesthesia, and Euthanasia. A total of 21 inbred female Sprague−Dawley (SD) rats (Harlan, Jerusalem, Israel) were obtained from a specific pathogen-free (SPF) colony and kept under SPF conditions in a controlled environment, as described previously.30 The animals were treated in accordance with local laws and National Institute of Health (NIH) Guidelines for Animal Protection. The ethics committee at the Hebrew University in Jerusalem (NIH

3. RESULTS 3.1. Weight Loss, Comonomer Release, and Water Uptake Analysis. The polymer weight loss in vivo was about 45% after the first three days, followed by a significantly slower degradation phase (Figure 1). During the 42 days of degradation in vivo, more than 90% of the administrated polymer was eliminated from the implantation site.

Figure 1. In vivo hydrolytic degradation and elimination from the implantation site of the p(SA-RA)3:7 copolymer monitored by weight loss. Polymer samples were implanted SC in the posteriolateral flank of SD rats. At each time point, the remaining polymer was excided, dried, and weighed. Data are expressed as the mean and standard deviation of six implants.

The comonomer composition of p(SA-RA) 3:7 copolymer during hydrolysis and elimination from the implantation site was analyzed. HPLC analysis of the hydrolyzed samples revealed that during the first three days postimplantation both the SA and RA comonomer content of the polymer matrix decreased, however the release of the SA comonomer was B

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higher (Figure 2). The comonomer content of the polymer degraded with time is given in Figure 3. The plot shows that,

Figure 4. Water uptake by the matrix of p(SA-RA) 3:7 copolymer degraded in vivo. At each time point, the remaining polymer matrix was excided from the implantation site, the weight was recorded, and the samples were lyophilized. The difference between sample weight before and after lyophilization was taken as a water uptake of the degraded polymer matrix. Data are expressed as the mean and standard deviation of six implants.

Figure 2. Release of RA and SA from p(SA-RA)3:7 copolymer during 42 days of SC implantation in rats. The aliquot of each sample was hydrolyzed with 1 M NaOH, acidified with HCl to pH 2, extracted with CHCl3, the solvent was evaporated, and the hydrolyzed samples were dissolved in ethanol and analyzed by HPLC. Data are expressed as the mean and standard deviation of six implants.

characteristic anhydride peak of the blank polymer at 1815 cm−1 (Figure 5a) disappeared, and the strong signal of carboxylic acid peak at 1705 cm−1 was recorded (Figure 5b). At this point of time the peak at 1732 cm−1 is attributed to the ester bonds stretching vibrations only. This means that the anhydride linkages were hydrolyzed, and no anhydride segments remained in the system, which now consisted of degradation products of the oligoester structure. Another notable difference between the time zero spectra and the spectra obtained for the samples after 3 days of degradation in vivo is the emergence of the carboxylate peak in vicinity of 1550 cm−1 (Figure 5a,b). This indicates that some of the material is being salted by the components of extracellular liquid. The salting process was enhanced at day 7 postimplantation, and it was evidenced by intensifying the peak of carboxylate ion, which was accompanied by decreasing the intensity of carboxylic acid peak (Figure 5b,c). The pattern of IR spectra established at day 7 postimplantation remained stable until the end of the experiment (Figure 5c−e). Finally, at all examined points of time a broad band due to hydrogen bonding (2500−3600 cm−1) was observed as a copolymer degrade (Figure 5b−e). This finding supplies additional evidence of a hydrolytic process, since hydrogen bonding in the studied case is possible only due to formation of free carboxylic acid groups and free secondary alcohol groups of RA comonomer. Nevertheless, the differential interpretation of this region of IR spectra was limited, because the dependence of the intensity and the shape of these peaks on molecular orientation in the cast film and also due to the actual overlapping of the wavenumber ranges. Therefore, the question on the contribution of the degradation products bearing RA moieties with free hydroxyl group at the CH3-end of their chains to the total mass of degraded polymer was attributed in 1 H NMR analyses. At each time point, the remaining polymer was excided from implantation site, dried and dissolved in dichloromethane. IR spectroscopy was performed on polymer samples cast on NaCl plates from the solution in dichloromethane. The peaks of anhydride are double peaks at 1740 and 1815 cm−1; the saturated acyclic aliphatic esters peak is at 1732 cm−1, which overlapped with the anhydride peak; the carboxylic acid peak is at 1700−1725 cm−1; and the carboxylate ion peak is at 1550−1610 cm−1.

Figure 3. Content of RA and SA in the matrix of degraded p(SA-RA) 3:7 during 42 days of SC implantation in rats. The aliquot of each sample was hydrolyzed with 1 M NaOH, acidified with HCl to pH 2, extracted with CHCl3, the solvent was evaporated, and the hydrolyzed samples were dissolved in ethanol and analyzed by HPLC. Data are expressed as the mean and standard deviation of six implants.

despite the initial rapid release of SA, it continued to be an integral part of the remaining polymer matrix until the end of the experiment. Over the last two weeks of the experiment, SA content was below 10%, indicating that during this period of time the system consisted mainly of RA. The water uptake by the degraded polymer was estimated using a gravimetric approach. During the first 3 days, the excided samples contained 34.2 ± 3.2% of water, but after one week of degradation in vivo, the water uptake raised in the samples up to 116.8 ± 14.9% (Figure 4). It remained at the level of 100% with a slight increase to the end of the study. 3.2. IR Analysis. The peaks of main interest in the IR spectroscopy analysis of p(SA-RA) 3:7 were the typical signals originated from carbonyl stretching vibrations. This included double anhydride peaks at 1815 and 1740 cm−1, the saturated acyclic aliphatic esters peak at 1732 cm−1 (which overlapped with the anhydride peak, as well as the carboxylic acid peak at 1700 cm−1), and the carboxylate peak at 1550 cm−1. IR analysis revealed that the most significant changes in the chemical structure of the studied polymer occurred during the first week after implantation. Three days postimplantation, the C

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Figure 6. Molecular weight changes during the hydrolytic degradation of the p(SA-RA) 3:7 copolymer in vivo monitored by GPC. At each point in time the remaining polymer was excided from implantation site, dried, and dissolved in CHCl3. Data are expressed as the mean and standard deviation of six implants.

and SA comonomers was confirmed by HPLC, while MS analysis was used to estimate the structure of RA and SA short esters, such as dimers, trimers, and tetramers (Table 1). The water solubility of these degradation products has been reported previously.25 3.4. 1H NMR Analysis. The comparison of the 1H NMR spectra of RA comonomers and the polymer confirmed that CH−OH peak at 3.618 ppm characteristic for RA (Figure 7a) disappeared and the peak of CH−O−CO at 4.872 ppm appeared as a resulted of esterification (Figure 7b). This shift is Table 1. Structures of Oligomers Estimated on GPC and MS Analysis

Figure 5. In vivo hydrolytic degradation of the p(SA-RA) 3:7 copolymer monitored by IR spectroscopy.

3.3. GPC and MS Analysis. Results of molecular weight measurements of degraded p(SA-RA) 3:7 copolymer are presented in Figure 6. The data show a rapid decrease of the average molecular weight (Mw) of the studied polymer over the first 3 days after implantation. The initial decrease in Mw is followed by a very slow degradation phase, which keeps the Mw at about 1900 Da throughout the remaining period of the experiment. The average molecular number weight (Mn) has a similar pattern of the rapid initial decrease followed by a plateau. GPC analysis also indicated presence of low amounts of comonomers and their short esters in the degraded polymer matrix at all examined points in time. The presence of the RA

a

Theoretically calculated. bThe respective mass obtained after direct injection into the mass spectrophotometer running on a negative ion mode, which corresponds to the deprotonated molecule [M − (H)n]−, and matches the oligomer Mw; n = 1 for RA esters, and n = 2 for RA and SA coesters. cThe respective average molecular number weight (Mn) obtained by GPC analysis that indicates the presence of the corresponding oligomer. D

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Figure 7. 1H NMR spectra of RA comonomer (a), p(SA-RA) 3:7 copolymer (b), and the enlargement of the 1H NMR spectrum of degraded polymer sample illustrating the principle of the calculation of CH−OH:CH−O-CO ratio (c). Spectra were obtained in 5 mm diameter tubes. CDCl3 containing tetramethylsilane served as solvent and shift reference.

a consequence of deshielding effect of electronegative carbonyl groups of RA and SA that create ester bonds with hydroxyl group at C12 in the RA skeleton. Randomized hydrolytic degradation of the polymer may occur at different points of its backbone. The hydrolysis of the hydrolytically liable linkages results in the liberation of RA comonomers or oligoesters of RA having free hydroxyl group at the CH3-end of their chains (Table 1). These degradation products may be retarded in the matrix of degraded polymer or extracted by the surrounding extracellular fluid and consequently eliminated via physiological drainage to circulating system. The CH−OH/CH−O−CO ratio determined for the degraded polymer matrix is dependent not only on the kinetics of degradation product elimination from the implantation site, but also on the structure of the oligomeric chains composing the degraded polymer matrix and the pattern of their degradation. Therefore, the determination of the content of RA moieties possessing free hydroxyl group in

the total bulk of RA in the degraded polymer matrix may be a useful tool for interpretation of the structure of oligomeric chains and the elucidation of the pattern of their degradation. The data obtained in 1H NMR experiments was used to calculate the content of RA moieties possessing free hydroxyl group in the total bulk of RA present in the polymer matrix. The following equation was used:

∫ [H(C12)free OH]/∑ ∫ H(C12)× 100% where ∫ [H(C12)_free OH] is an integral of the characteristic peak of CH−OH at 3.6 ppm of RA moiety bearing free hydroxyl group and ∑∫ H(C12) is a sum of integrals of both peaks CH−OH at 3.6 ppm and the characteristic peak of CH− O−CO at 4.8 ppm of RA moiety possessing esterified hydroxyl group (Figure 7c). The results are summarized in Figure 8 and show that the degradation process is characterized by the appearance of the RA moieties bearing free groups, however E

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composing the matrix of degraded polymer. This was done under the assumption that every oligomeric chain has a free hydroxyl group at the CH3-end. The calculation was performed using the following equation:

1/n × 100% where n is the average oligomer length calculated as Mn/ (Mw(RA) × w%(RA)/100% + Mw(SA) × w%(SA)/100), where Mn is the average molecular number weight (Figure 6), Mw(RA) and Mw(SA) are the respective molecular weights of RA and SA, and w%(RA) and w%(SA) are the respective RA and SA content in the degraded polymer matrix (Figure 3). It can be clearly seen from the data presented in Figure 8 that the population of the oligoesters possessing free hydroxyl grouping at the CH3-end is 5−14 times lower than the theoretical maximal value, which takes into account all existing chains. In other words, the main part of the oligoester chains does not possess free hydroxyl grouping at the CH3-end. Moreover, these data also indicate that polymer degradation products having a high CH−OH/CH−O−CO ratio as well free RA comonomer are effectively extracted from the degraded polymer matrix. Combining this finding with the data regarding the comonomer composition of the degraded polymer (Figure

Figure 8. Experimentally observed and theoretically maximal content of RA moieties possessing free hydroxyl group in the total pool of RA in the degraded polymer matrix.

their content did not exceed 5% w/w of the total content of RA in the matrix of the degraded polymer. No statistically significant differences were observed in their contribution to the total pool of RA moieties in the bulk of degraded polymer with time (p > 0.05, Kruskal−Wallis Test). The theoretical maximal content of RA moieties possessing free hydroxyl group was also calculated (Figure 8) to utilize the results for interpretation of the structure of oligomeric chains Scheme 2. Degradation Process of p(SA-RA) 3:7 In Vivo

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copy revealed that anhydride linkages of the studied polymer have been already split by water in a biological environment over the first three days of the degradation (Figure 5). This process was accompanied by a respective sharp decrease in the weight-average molecular weight (Mw) of the polymer (Figure 6) and the initial rapid weight loss (Figure 1). The rapid decrease of the Mw of the polymer was followed by a plateau associated with relatively more stable oligoester chains (Figures 5 and 6) that slowly degrade and eliminate from the implantation site (Figures 1 and 2). The information obtained by the various characterization methods employed in this study reveals some interesting insights regarding the degradation mode of p(SA-RA) 3:7 copolymer and elimination kinetics of the polymer degradation products from the implantation site. When polyanhydrides degrade in an aqueous environment, the anhydride bonds are cleaved into two carboxylic acid end groups.1,2 Polar carboxylic acid end groups are responsible for the water attraction and retention inside the matrix of a degraded polymer as a result of hydrogen bonding.26 Despite the low water uptake by the third day (Figure 4), degradation of anhydride bonds was completed at this moment (Figure 5b). The low water uptake at the first point of time examined might be explained by its utilization for hydrolysis of anhydride bonds.5 Water uptake has been raised in the polymer matrix with time (Figure 4) because it was facilitated by leaching the degradation products (Figure 1), which leads to an increase of the polymer matrix porosity, as well as by salting carboxylic acid end groups (Figure 5b−e) that evaluate polarity inside the pores.6,26 The formation of free carboxylic acid end groups is not only responsible for device porosity maintenance, but also might ensure further hydrolytic degradation of the polymeric device.5,6 1H NMR experiments revealed that after the rapid hydrolytic phase (Figure 6) the degraded polymer matrix consists mainly of oligoester chains possessing free carboxylic acid end groups and lacking free hydroxyl end groups (Figure 8). On the basis of this information, the structure of the oligoester chains became clearer. It was also found that the low molecular weight degradation products were effectively eliminated from the implantation site (Figures 6 and 8). Interestingly, the same data may have additional interpretations allowing even better understanding of the p(SA-RA) 3:7 degradation mode. The low abundance of free hydroxyl groups at the CH3-end of oligomeric chains also indicates that hydrolysis of long oligomers occurs preferably at or in vicinity of free carboxylic acid end groups. If hydrolysis will take place indiscriminately over the entire length of the long oligomers, splitting the esters bonds in the middle of the chain or at the CH3-end should lead to an increase in the content of RA moieties possessing free hydroxyl group. However, the abundance of the RA moieties having free hydroxyl group at the CH3-ends remained relatively low and stable during the degradation (Figure 8). The data obtained revealed that the in vivo degradation process of the p(SA-RA) 3:7 copolymer progresses in two stages (Scheme 2). In the first and more rapid stage, the anhydride bonds are fully degraded, releasing the SA units conjugated in both of their carboxylic acid groups by anhydride bonds, as well as shorter soluble esters of RA and SA possessing free carboxylic groups at both ends, such as dimers, trimers, and tetramers (HO-OC-R-CO-(O-R′-CO)t-OH, where t = 1−3). In the next step, the remaining oligoesters built of SA and RA degrade into shorter RA ester dimers, trimers and tetramers

3), the presence of free carboxylic groups in the oligoester structure (Figure 5), and the acetylation of the hydroxyl group of RA moiety at the CH3-end of the polymer chain (Scheme 1) during its synthesis,25 it is possible to elucidate the structure of the oligomeric chains, which are the main constituents of the degraded polymer matrix. They should be (1) oligomers having free carboxyl group at both ends, such as HO-(OC-R′-O)l-OCR-CO-(O-R′-CO)m-OH and HO-OC-R-CO-(O-R′-CO)k-OH, and (2) oligomers containing acetylated hydroxyl group at the CH3-end and free carboxyl group at the C-end, such as CH3CO-(O- R′-CO)n-OH, where R′ is ricinoleic acid residue and R is sebacic acid residue (Scheme 2). Similarly, taking into account the data presented in Figure 6 and Figure 8, as well as the data obtained by MS analysis (Table 1), it can be concluded that oligomers and low molecular weight components possessing free hydroxyl group at their CH3-end are (1) long and short RA oligoesters, HO-R′-CO-(O-R′-CO)r(s)-OH, and (2) RA comonomer, HO-R′-CO-OH. The experimentally observed content was calculated on the basis of the data obtained in 1H NMR experiments using the following equation: Content of RA free OH, (%) = ∫ [H(C12) _free OH]/∑∫ H(C12) × 100%, where ∫ [H(C12)_free OH] is the integral of the characteristic peak of CH−OH at 3.6 ppm of RA moiety having free OH- group and ∑∫ H(C12) is a sum of the integrals of both peaks CH−OH at 3.6 ppm and the characteristic peak of CH−O−CO at 4.8 ppm of RA moiety possessing esterified OH-group (Figure 7). The theoretical maximum content was calculated assuming that each oligomer possesses free OH-group at the CH3-end of its chain. This calculation was performed by employing the following equation: 1/n × 100%, where n is average oligomers length calculated as Mn/(Mw(RA) × w%(RA)/100% + Mw(SA) × w %(SA)/100); where Mn is the average molecular number weight (Figure 6); Mw(RA) and Mw(SA) are the respective molecular weights of RA and SA; and w%(RA) and w%(SA) are the respective RA and SA contents in the degraded polymer matrix (Figure 3). Data are expressed as the mean and standard deviation of six implants.

4. DISCUSSION In this work, the degradation in vivo and elimination from the implantation site of the injectable p(SA-RA) 3:7 copolymer was studied. For most biodegradable materials, especially artificial polymers, passive hydrolysis is the most important mode of degradation.6,16 There are several factors that influence the velocity of this reaction: the most important are the type of chemical bond, pH, copolymer composition, and water uptake.6,26 The type of bond within the polymer backbone primarily determines the rate of hydrolysis.1,6,20 Several classifications for ranking the reactivity exist; these are either based on hydrolysis kinetics data for polymers or are extrapolated from low-molecular weight compounds containing the same functional group.5,21 Anhydride- and ortho-ester bonds are the most reactive, followed by esters and amides.2,6 Such rankings must be viewed, however, with circumspection. Reactivity can change tremendously upon catalysis or by altering the chemical neighborhood of the functional group through steric, electronic effects, copolymer composition, and so on.5,23,26 The p(SA-RA) 3:7 copolymer studied in this work belongs to a class of biodegradable synthetic matrices containing both types of hydrolytically labile linkages, that is, ester bonds and anhydride bonds along the polymer backbone.8,9 IR spectrosG

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(HO-R′-CO-(O-R′-CO)s−OH, where s = 1−3), shorter esters of RA and SA (described above, as well as RA and SA comonomers, which are extracted by the surrounding extracellular fluid from the degraded polymer matrix and eventually eliminated from the implantation site. This step also includes the formation of long RA esters (HO-R′-CO-(O-R′CO)r-OH, where r > 3) as an intermediate step before degradation into shorter and soluble degradation products. The results obtained in this study show that the in vivo degradation pattern of p(SA-RA) 3:7 correlates well with that observed under in vitro conditions.10,25 The main features of the degradation pattern were the same: rapid hydrolysis of the anhydride bonds, which was reflected in the respective sharp weight loss and decrease in the Mw of the polymer, followed by a slow degradation phase in which the Mw was kept stable throughout the period of the experiment. The pattern of low molecular weight degradation products was also very similar. These findings indicate that the degradation mode of p(SA-RA) 3:7 is determined by its chemical structure and is not impacted by the biological environment. Considering erosion kinetics, it should be understood that polymer disappearance from the implantation site was slower under in vivo conditions compared with the weight loss rate observed in vitro. It was reported in particular that during hydrolytic degradation in vitro p(SA-RA) 3:7 completely eroded over 4 days, resulting in 100% weight loss.25 On the other hand, it was found that 42 days under in vivo conditions are required until 90% of the polymer is hydrolyzed and eliminated from the implantation site (Figure 1). The slower weight loss observed under in vivo conditions could not be associated with the fibrous capsule developed around the implant as a part of normal tissue response to the polymer.30 A very thick and dense fibrous capsule may disturb the diffusion from the polymeric implant to the surrounding interstitial fluid.31 However, the low molecular weight degradation products of p(SA-RA) 3:7 copolymer does not accumulate at the implantation site as described above. The observed discrepancy may be explained by disintegration of the degraded matrix of soft polymers occurring in vitro under vigorous shaking.10,32 This leads to the formation and dispersion of oily drops or oily globules composed of fragmentations of the polymer, including entrapped low molecular weight degradation products.32 Such disintegration was evidenced by turbidity of the degradation medium during performance of this particular in vitro degradation study.25 Consequently, a loss of the part of degradation products during replacing the degradation medium takes place. The results obtained in another study in which in vitro degradation of the same polymer was conducted under quite different conditions (without vigorous shaking) confirm the finding of this research project by showing that the complete hydrolytic degradation of p(SA-RA) 3:7 occurred over 45 days.10 Furthermore, reports in scientific literature indicate that RA oligoesters dispersed in vitro in the form of larger oily drops or oily globules are less susceptible to hydrolytic degradation, and thus, their formation might result in delayed degradation kinetics of the studied polymer and its degradation intermediates in comparison with in vivo conditions.32 This might explain why traces of RA comonomer were found in the degraded polymer matrix and not observed under in vitro conditions.25

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CONCLUSION This study has shown that the p(SA-RA) 3:7 copolymer is biodegradable in vivo and that the pattern of its hydrolytic degradation in vivo correlates well with that observed under in vitro conditions. The degradation process was fully characterized by determining the structures of oligomeric degradation products throughout the degradation process in vivo. First step: degradation of anhydrides bonds and release of the sebacic acid (SA) and soluble shorter esters of ricinoleic acid (RA) and SA, such as dimers, trimers, and tetramers (HOOC-R-CO-(O-R′-CO)t-OH, where t = 1−3). Second step: degradation of the ester bonds and release of short oligoesters of RA and SA described above, RA and SA comonomers, and short oligoesters of RA, such as dimers, trimers and tetramers (HO-R′-CO-(O-R′-CO)s-OH, where s = 1−3), which are extracted from the degraded polymer matrix and eliminated from the implantation site. The second step also includes the intermediate formation of long RA esters (HO-R′-CO-(O-R′CO)r-OH, where r > 3) before their degradation into shorter and soluble degradation products.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 972-2-675-7573. Fax: 972-2-675-7076. E-mail: avid@ ekmd.huji.ac.il. Notes

The authors declare no competing financial interest.

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REFERENCES

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