Abstract
New copolymers of ε-caprolactone with three hydroxy-fatty acids, 12-hydroxy stearic acid, 16-hydroxyhexadecanoic acid and ricinoleic acid, were synthesized by catalytic polyesterification. The reactions were carried out in solvent-free systems and in organic solvents as well, using tin(II) 2-ethylhexanoate as catalyst, at different temperatures and molar ratios of the comonomers. Cyclic and linear polymeric products with medium molar weight of about 2000 Da have been synthesized and their chemical structures were confirmed by FT-IR, NMR and MALDI-TOF MS analysis. The synthesis parameters were optimized and the ε-caprolactone/hydroxy acid molar ratio was set as 5:1, according to mass spectrometry results. The biodegradability of the newly synthesized polymers was studied in the presence of Candida antarctica B lipase in phosphate buffer solutions (pH=7.4), at 37°C. The weight-loss profile emphasized the degradation of the 16-hydroxyhexadecanoic acid based polymer samples at more than 50% of their initial weight in 18 days of incubation in the presence of the lipase. The composition of the degradation products was assessed using the GC-MS technique and displayed residues of the comonomers moieties.
Introduction
Polymers based on natural raw materials have attracted a great deal of attention due to their potential applications in the biomedical field and their biodegradability. Among their applications, these polymers stand out for their efficiency as drug delivery systems and for the development of different medical devices and tissue engineering. In this regard, poly(ε-caprolactone) (PCL) is widely used in biomedical applications because of its biocompatibility, permeability and good mechanical properties [1], [2], [3], [4]. Moreover, the use of PCL for implants and surgical absorbable sutures has been approved by the Food and Drug Administration [5]. The permeability of polymers of ε-caprolactone was comparable to silicone rubber and 104 times higher compared to poly(lactic acid) [6].
Up to now the properties ε-caprolactone were modified by using different comonomers such as sugars/polysaccharides, ethylene glycol, methacrylates, etc. [7]. Hydroxy-fatty acids (HFA) are other possible candidates as co-substrates, being among the most spread natural compounds. Hydroxy-fatty acids (HFA) are natural occurring compounds which have been intensively investigated for their applications in cosmetics, paints, lubricants, food industry, medicine as well as intermediates in the synthesis of fine chemicals and pharmaceuticals [8], [9]. For example, 12-hydroxystearic acid can be used as thickening agent in the field of lubricants [10], ricinoleic acid obtained from castor oil finds applications in soaps and as viscosity controller for chocolate and as emulsifier in margarine [11], [12] while 16-hydroxyhexadecanoic acid originating from cutin and suberin [13] is commonly used as surfactant [14], [15].
Polyesters based on hydroxy-fatty acids derived from vegetable oils, called estolides, have many potential applications due to their biodegradability, biocompatibility and higher thermal oxidative stability compared to the vegetable oils [16]. However, they also have several disadvantages as inappropriate thermal properties and relatively low molecular weights.
Degradation of plastics by microorganisms and enzymes is essential for the reduction of the amount of synthetic plastic waste in the environment [17], as well as for appropriate utilization of polymeric materials in biomedical applications [18]. The hydrolytic degradation and erosion of solid polymers have been studied by several groups and depends on diverse factors including the relation between the rate of water/enzyme diffusion into the polymer, the rate of chain cleavage by water ions/enzymes, etc. The rate of water diffusion into a solid polymer is strongly influenced by a number of structural parameters, such as its porosity, crystallinity, hydrophobicity and size of the sample [19]. The PCL biodegradation involves the hydrolytic cleavage of the ester groups. Several enzymatic hydrolysis tests were reported [20], [21], but utilization of Novozyme 435 for degradation studies of PCL copolymers was mentioned only in a few studies.
In this respect, our research was focused on the chemical synthesis of new polyesters derived from ε-caprolactone and different hydroxy-fatty acids, with controlled biodegradability and biocompatibility. From our knowledge, these compounds have been not previously synthesized and characterized. The synthesis of the polymers has been carried out at different temperatures, using tin (II)-2-ethylhexanoate (SnOct2) as catalyst and different molar ratios between the comonomers. The structures of the resulted polymers were confirmed by MALDI-TOF MS which revealed appropriate molar weights and good polydispersity. Their biodegradability was evaluated in enzymatic environment using lipases, the most important enzymes which can hydrolyze ester linkages, at 37°C and pH=7.4. The results were well correlated with the theoretical models provided by the HyperChem software.
Materials and methods
Materials
ε-Caprolactone (ECL), 12-hydroxystearic acid (12HSA) (99%), 16-hydroxyhexadecanoic acid (16HHDA) (98%), ricinoleic acid (RCA) (98%), tin (II)-2-ethylhexanoate (SnOct2), trans-2-[3-(4-t-butyl-phenyl)-2-methyl-2-propenylidene]malononitrile (DCTB), potassium trifluoroacetate (KTFA), bis(trimethylsilyl)trifluoroacetamide-trimethylchlorosilane (BSTFA+TMCS=99:1), tetrahydrofuran (THF) (>99%) and toluene (>99%) were purchased from Sigma Aldrich. Immobilized Candida antarctica lipase B on acrylic resin (Novozyme 435) was from Novozymes.
Methods
Polyester synthesis
The appropriate amount of hydroxy-fatty acid (16HHDA, 12HSA and RCA) was added to ECL at different molar ratios. At low ECL:HFA molar ratio (1:1 and 2:1), 1 mL toluene was added as reaction medium. The reactions were started by addition of SnOct2 and were carried out at different temperatures in the range 90–115°C, at 400 rpm for 24 h. The reaction mixtures were dissolved in THF, dried at 25°C, resulting a white solid, which was further analyzed by FT-IR, MALDI-TOF MS and NMR techniques.
Enzymatic degradation
The samples were formulated as pellets with about 1.3 cm diameter and 1 mm thickness using a 7 tones manual hydraulic press. Each sample was placed in an individual flask and incubated at 37°C in phosphate buffer solution (PBS, pH=7.4) containing 0.02% NaN3 (as anti-mildew and antibacterial agent to avoid bacterial proliferation) and 0.6 mg/mL Novozyme 435. Samples were taken out, gently wiped with filter paper, dried at room temperature in a desiccator for 24 h and weighted at different time intervals [22], [23]. The degradation rate was assessed by the weight-loss, which was determined by the following formula for each sample:
where W0 is the initial weight of each sample and Wt is the weight of the sample at time t. Control experiments were carried out for each sample by incubation in buffer solution in the absence of the enzyme. The degradation products were extracted 3 times with 500 μL dichloromethane and their composition was assessed using GC-MS and MALDI-TOF MS techniques.
All synthesis and degradation experiments were made at least in duplicate and the MALDI-TOF MS and GC-MS analyses in duplicate, as well. The difference between the individual data for the same experiment was in all cases less than 3%, therefore only the mean values are provided in the tables and figures, without specifying the standard deviations.
Characterization of the reaction products
MALDI-TOF MS analysis was carried out for composition analysis of polymers, using an UltrafleXextreme Bruker spectrometer with FlexControl and FlexAnalysis software packages for acquisition and processing of the data (BrukerDaltonics, Germany), at an acceleration voltage of 25 kV, using DCTB as matrix and KTFA as ionization agent. The sample preparation and analysis was performed as previously described by our group [16]. The number average molecular weight (Mn), weight average molecular weight (Mw) and the polydispersity index (PDI) have been calculated as described elsewhere [24].
Fourier Transform Infrared (FT-IR) spectra of the samples were obtained in attenuated total reflectance (ATR) mode on a Bruker Vertex 70 (Bruker Daltonik GmbH, Germany) spectrometer equipped with a Platinium ATR, Bruker Diamond Type A225/Q. Spectra were collected in the range 4000–400 cm−1 with a resolution of 4 cm−1 and with 64 co-added scans.
GC-MS analysis was performed using a Thermo Analytic GC-MS system composed from Trace 1310 gas chromatograph, MS module and TriPlus RSH autosampler. The HFA conversion/formation was determined after derivatization with BSTFA+TMCS (99:1), at 2:1 reagent:sample ratio (w/w), for 1 h at 95°C, as previously described [25]. Hexadecane was used as internal standard. The analysis conditions for hydroxy-fatty acid analysis have been previously described [16].
NMR spectra of the isolated product were recorded on a Bruker AVANCE III spectrometer operating at 500.0 MHz (1H) and 125.0 MHz (13C). The samples were dissolved in tetrahydrofurane-d8 and the chemical shifts δ are given in ppm from TMS.
Computational methods
Quantum chemical molecular simulations were carried out by using MM+ and PM3 [26] methods implemented in HyperChem software [27]. All the structures were created with HyperChem package and were first geometrically optimized with molecular mechanics MM+ method. The obtained optimized structures were used in the subsequent geometry optimization calculations performed with the semi-empirical PM3/RHF method. The Polak–Ribiere conjugate gradient algorithm [28] was used with a RMS gradient of 0.01 kcal/mol.
Results and discussion
In this work, we investigated three commercially available hydroxy fatty acids (HFA):12-hydroxystearic acid (12HSA, obtained by hydrogenation of castor oil), 16-hydroxyhexadecanoic acid (16HHDA, an ω-hydroxy derivative of palmitic acid) and ricinoleic acid (RCA, the main component of castor oil) as co-substrates with ε-caprolactone for the synthesis of novel polyesters with potential pharmaceutical, medical or cosmetic use.
The possible reaction products of 16HHDA and ECL reactions are depicted in Fig. 1, as an example of the polymerization reaction which can occur. They could be linear and cyclic copolymers with different number of 16HHDAmoieties in the backbone. However, together with these products of interest, ECL homopolymers and estolides could be also formed as secondary reaction products.
Reaction scheme of synthesis of copolymers and homopolymers from ε-caprolactone and 16-hydroxyhexadecanoic acid.
The formation of the desired copolymers was demonstrated based on MALDI-TOF MS analysis. In Fig. 2 a typically MALDI-TOF MS spectrum of the synthesized 16HHDA-ECL copolymer (at 5:1 molar ratio) is presented. The molecular peak at about m/z=1630.8 Da is assigned to the K+ adduct of the polymeric chain containing two 16HHDA and nine ECL moieties. We can also notice in the same spectrum the formation of polymeric chains with higher number of ECL moieties, showing a Gaussian distribution, at unit ratios between the 16HHDA and ECL comonomers of 2:10; 2:11; 2:12; 2:13 and 2:14, respectively.
MALDI-TOF MS spectrum of the copolymers synthesized from ECL and 16HHDA using SnOct2 as catalyst (ECL:16HHDA molar ratio 5:1).
In a similar manner, the other two hydroxy-fatty acids, 12HSA and RCA yielded the proposed copolymers containing one or more HFA moieties inserted in the PCL backbone. Examples of MALDI-TOF MS spectra of such polycondensation products are presented in the Supplementary material (Fig. S1 and Fig. S2).
Influence of the ECL/hydroxy-fatty acid molar ratio and temperature on the molecular weight and composition of the copolymer
The influence of the molar ratio and temperature was studied in order to favor the formation of a higher amount of copolymer. For this reason, ECL/HFA molar ratios between 1:2 and 10:1 were investigated at two reaction temperatures, 90°C and 115°C. The 90°C temperature value was choose regarding the results previously reported by Dai et al. when poly(4-hydroxyl-ε-caprolactone-co-ε-caprolactone)-g-poly(L-lactide) compounds have been synthesized [7]. The reactions were started by adding SnOct2 on the pre-solubilized hydroxy-fatty acid and ECL. The average molecular weights and the linear/cyclic copolymer and homopolymer content of each product were calculated based on the MALDI-TOF MS spectra and are presented in Tables 1–3. Generally, an increase of the average molecular weight with increasing ECL content in the reaction mixture was observed, due to the higher reactivity of the 7-membered ring caprolactone compared to the C16/C18 hydrophobic hydroxy-fatty acids with linear chain.
Influence of the molar ratio and temperature on the composition of 16HHDA_ECL copolymer.
| ECL/16HDDA molar ratio | Mn (Da) | Mw (Da) | PDI | Composition of the product |
DPmax | |||
|---|---|---|---|---|---|---|---|---|
| LC (%) | CC (%) | LH (%) | CH (%) | |||||
| Reaction temperature: 90°C | ||||||||
| 1:2 | 814 | 875 | 1.07 | 89.9 | 1.7 | 6.6 | 1.9 | 11 |
| 1:1 | 944 | 1023 | 1.08 | 85.5 | 6.2 | 4.4 | 3.9 | 17 |
| 2:1 | 1184 | 1338 | 1.13 | 83.3 | 6.3 | 6.5 | 3.9 | 23 |
| 5:1 | 1301 | 1445 | 1.11 | 88.2 | 0.0 | 10.8 | 0.99 | 23 |
| 10:1 | 1611 | 1729 | 1.07 | 96.9 | 0.0 | 3.1 | 0.0 | 18 |
| Reaction temperature: 115°C | ||||||||
| 1:2 | 742 | 823 | 1.11 | 59.9 | 37.6 | 2.6 | 0.0 | 8 |
| 1:1 | 752 | 830 | 1.10 | 94.8 | 2.3 | 3.0 | 0.0 | 8 |
| 2:1 | 1047 | 1276 | 1.22 | 66.3 | 11.3 | 14.2 | 8.2 | 10 |
| 5:1 | 1334 | 1571 | 1.18 | 78.5 | 3.7 | 14.4 | 3.3 | 27 |
| 10:1 | 795 | 961 | 1.21 | 69.6 | 0.0 | 0.0 | 30.4 | 10 |
LC, Linear copolymer; CC, cyclic copolymer; LH, linear homopolymer; CH, cyclic homopolymer; DPmax, maximal degree of polymerization (of the copolymer).
Influence of the molar ratio and temperature on the composition of 12HSA_ECL copolymer.
| ECL/12HSA molar ratio | Mn (Da) | Mw (Da) | PDI | Composition of the product |
DPmax | |||
|---|---|---|---|---|---|---|---|---|
| LC (%) | CC (%) | LH (%) | CH (%) | |||||
| Reaction temperature: 90°C | ||||||||
| 1:2 | 985 | 1069 | 1.09 | 70.6 | 10.4 | 13.1 | 5.9 | 16 |
| 1:1 | 1872 | 2265 | 1.21 | 54.9 | 15.4 | 22.3 | 7.4 | 23 |
| 2:1 | 1409 | 1560 | 1.11 | 55.3 | 10.3 | 21.7 | 12.8 | 26 |
| 5:1 | 1737 | 2206 | 1.27 | 59.5 | 10.3 | 21.8 | 8.5 | 34 |
| 10:1 | 2000 | 2375 | 1.19 | 46.1 | 8.5 | 22.1 | 23.4 | 37 |
| Reaction temperature: 115°C | ||||||||
| 1:2 | 1376 | 1585 | 1.15 | 60.1 | 29.5 | 9.2 | 1.3 | 23 |
| 1:1 | 1534 | 1760 | 1.15 | 68.4 | 25.6 | 5.5 | 0.5 | 24 |
| 2:1 | 1321 | 1524 | 1.15 | 70.4 | 17.7 | 8.7 | 3.2 | 22 |
| 5:1 | 1562 | 1838 | 1.18 | 56.0 | 20.1 | 10.7 | 13.2 | 27 |
| 10:1 | 1834 | 2013 | 1.10 | 67.6 | 17.6 | 10.7 | 4.0 | 26 |
LC, Linear copolymer; CC, cyclic copolymer; LH, linear homopolymer; CH, cyclic homopolymer; DPmax, maximal degree of polymerization (of the copolymer).
Influence of the molar ratio and temperature on the composition of RCA_ECL copolymer.
| ECL/RCA Molar ratio | Mn (Da) | Mw (Da) | PDI | Composition of the product |
DPmax | |||
|---|---|---|---|---|---|---|---|---|
| LC (%) | CC (%) | LH (%) | CH (%) | |||||
| Reaction temperature: 90°C | ||||||||
| 1:2 | 873 | 936 | 1.07 | 46.0 | 14.2 | 31.9 | 7.7 | 12 |
| 1:1 | 822 | 874 | 1.06 | 47.7 | 1.0 | 48.0 | 3.3 | 11 |
| 2:1 | 1258 | 1363 | 1.08 | 64.8 | 2.8 | 26.6 | 5.8 | 16 |
| 5:1 | 1853 | 2144 | 1.16 | 65.0 | 3.4 | 19.6 | 12.0 | 31 |
| 10:1 | 1252 | 1447 | 1.16 | 43.1 | 0.0 | 48.8 | 8.1 | 17 |
| Reaction temperature: 115°C | ||||||||
| 1:2 | 1048 | 1163 | 1.11 | 71.7 | 4.8 | 23.5 | 0.0 | 13 |
| 1:1 | 1356 | 1519 | 1.12 | 66.1 | 12.9 | 9.6 | 11.4 | 23 |
| 2:1 | 1191 | 1286 | 1.08 | 81.5 | 5.3 | 4.0 | 9.2 | 18 |
| 5:1 | 1310 | 1502 | 1.15 | 71.6 | 1.3 | 21.3 | 5.8 | 17 |
| 10:1 | 1080 | 1225 | 1.13 | 52.0 | 0.0 | 37.3 | 10.8 | 15 |
LC, Linear copolymer; CC, cyclic copolymer; LH, linear homopolymer; CH, cyclic homopolymer; DPmax, maximal degree of polymerization (of the copolymer).
In the case of ECL and 16HHDA copolymers (Table 1) the highest average molecular weight was registered at ECL:16HHDA molar ratio of 10:1. The relative content of the copolymer was higher than 95% and the medium molecular weight of the isolated polymer product was above 1600 Da. The increase of the temperature had no positive effect on the average molecular weight (regardless to the molar ratio) and maximal polymerization degree. Surprisingly, at the highest ECL:16HHDA molar ratio of 10:1 the cyclic polymers were not synthesized at 90°C, resulting the highest content in linear copolymer, but the polymerization degree was significantly lower than at lower ECL excess. Increasing the temperature from 90°C to 115°C, at the same 10:1 molar ratio more than 30% cyclic homopolymer was synthesized. When 12HSA was used as substrate (Table 2), the same decrease of average molecular weight and polymerization degree was observed at higher temperature. The medium molecular weight showed at the whole an increasing tendency with the increasing of the ECL/HFA molar ratio, but the MALDI-TOF spectra revealed the formation of the reaction product with only one HFA unit in the polymer chain, as major compound (data not shown in Table 2). The linear copolymer content of the synthesized products was in the range of 55–70%. Thus, in this case controlling the formation of products with higher content of linear copolymers was not possible by tuning the molar ratio and/or the temperature. When the comonomer was ricinoleic acid (Table 3), the highest average molar weight of the copolymer was obtained at 5:1 molar ratio, more than 2000 Da at 90°C and about 1500 at 115°C. Unlike in the case of the other two HFA, higher temperature favored a higher linear copolymer content of the product. At 115°C this content was above 80%, while at 90°C it could not reach more than 65%.
Although a general rule cannot be stated, it is obvious that the careful selection of molar ratio and temperature allows for every HFA the synthesis of a copolymer with appropriate composition for the designed application.
Structural analysis of the products
The FT-IR absorption bands of the polymeric products (Figure S3, Supplementary material) confirm the ester formation, by shifting the band assigned to the carbonyl group stretching vibration at about 1720 cm−1. The vibration bands from 2930 cm−1 to 2860 cm−1 can be assigned to the methylene groups of the polymeric chain. The C–O ester linkage exhibits a stretching vibration band at about 1180 cm−1.
The existence of the ester carbonyl group in the structure of copolymers was more reliably demonstrated based on 2D-NMR analysis. For the 16HHDA_ECLproduct the 1H-13C HMBC 2D-NMR (Fig. 3) revealed the formation of the carbonyl ester group by the distance coupling (3J, over 3 bonds), between the ester carbonyl group (C=O) from 173.2 ppm with the ester protons (O–CH2) from 4.01 ppm. Moreover, it can be observed the distance coupling between the carbon atom from the ester group and the methylene protons from α and β of the hydroxy-fatty acid moiety [2.32 ppm (–CH2–CH2–C=O) and 61 ppm (–CH2–CH2–C=O)].
The 1H-13C HMBC spectra of the 16HHDA_ECL copolymer, synthesized at 5:1 ECL:16HHDA molar ratio.
Enzymatic degradation by lipase-catalyzed hydrolysis
The advantages of PCL as drug delivery carriers, particularly for small drug molecules, have been explored by several groups due to its biocompatibility, high permeability and inability to generate an acid environment compared to other polymers such as polylactide or polyglycolide [29], [30]. Moreover, the slower degradation rates of PCL make it suitable for long-term delivery system for a period of more than 1 year [31]. Determination of the degradation rate of the polymer is a first step for the selection of materials for pharmaceutical formulations, allowing a preliminary evaluation of the concentration of low molecular chains present in the tissue/media. In this regard, lipase-catalyzed degradation of the synthesized copolymer products was evaluated, compared to PCL.
The samples selected for the degradation studies were the products obtained by polycondensation of ECL with 12HSA and 16HHDA, at 5:1 molar ratio and 90°C, as presented earlier. They were assessed by enzymatic incubation at 37°C at pH=7.4 (buffered aqueous solution) for 32 days, in the presence of 0.02% sodium azide and commercial Novozyme 435 lipase. For each type of product control pellets have been incubated in the same conditions, in the absence of the enzyme, to ensure that the weight loss occurs only due to the hydrolysis reaction catalyzed by the enzyme.
The weight loss profiles, depicted in Fig. 4, indicate that the presence of the HFA in the molecule induce a decrease of the polyester degradation rate. Compared to 12HSA_ECL the enzymatic degradation rate of 16HHDA_ECL copolymer was higher, more than 65% of its weight being lost in about 30 days, due to the linear structure of the 16HHDA compared with the C6 branched structure present in the 12HSA_ECL molecules. These results are in concordance with previously reported data for other types of ECL based copolymers, when the decrease of the degradation rate with the increase of the content of hydrophobic (butyrolactone [32]) and even hydrophilic (L-lactide [33], polyethylene glycol [34]) residues was observed.
Weight-loss profiles of PCL, 16HHDA_ECL and 12HSA_ECL, during the enzymatic degradation at pH=7.4 (phosphate buffer) catalyzed by lipase (Novozyme 435, 0.6 mg/mL), at 37°C.
The enzyme acts as hydrolysis agent of the ester bounds formed by polycondensation, reverting the polymer into the raw materials, hydroxy-fatty acids and caprolactone. The composition of the degradation products was investigated by GC-MS, after 3 consecutive extractions with dichloromethane. The resulted chromatograms (an example is given in Fig. S4, Supplementary material) showed the presence of the hydroxy-fatty acids, clearly indicating that the desired biodegradation took place as expected.
Quantum chemical calculations of the most stable copolymer structures
For this study, polyester model structures of 10 units each, formed from ECL and the same HFA used in the synthesis part (16HHDA, 12HSA, and RCA) were selected. The calculations were performed for the homopolymers (HP) with 10 ε-caprolactone units and 10 HFA units each, as well as for the linear copolymers (LC) and cyclic copolymers (CC) consisted from 9, 8, and 7CPL units and 1, 2, and 3HFA units, respectively, taking into consideration all possible isomers (178 isomers for each HFA). One of the possible structures, containing eight ECL and two 16HHDA units is depicted in Fig. 5. The stability of the polymers, as well as the most energetically favored binding positions were evaluated based on the computed enthalpies of formation.
Optimized structure of the LC-(8)ECL-(2)16HHDA linear copolymer, formed from eight ECL and two 16HHDA units (the numbers in brackets indicate the number of units from each comonomer).
All evaluated structures appear to be stable, having enthalpies of formation in the range from –1008.27 kcal/mol (for cyclic HP-(10)ECL, the least stable) to –1713.49 kcal/mol (for linear HP-(10)12HSA, the most stable) for homopolymers and –1058.35 kcal/mol (for cyclic CP-(9)ECL-16HHDA, the least stable) to –1268.95 kcal/mol (linear CP-(7)ECL-(3)12HSA, the most stable) for copolymers (the numbers in brackets indicate the number of units from each comonomer).
For all linear and cyclic copolymer structures, the stability increases in the order ECL_RCA<ECL_16HHDA<ECL_12HSA and with the increasing number of HFA units (Fig. 6). In the case of homopolymers, the stability increases in the succession ECL (–1071.63 kcal/mol)<RCA (–1458.18 kcal/mol)<16HHDA (–1615.60 kcal/mol)<12HSA (–1713.49 kcal/mol), for both linear and cyclic structures (the above data are given for the linear structures).
Enthalpies of formation ΔH (kcal/mol) of the linear copolymers (a) and cyclic copolymers (b) of ECL and the hydroxy fatty acids 16HHDA, 12HSA and RCA, respectively. The presented data were calculated for structures containing 1, 2, or 3 HFA units in the molecule.
These findings agree with the experimental data collected using MALDI-TOF MS (Tables 1–3), showing that the main reaction products in all cases were the linear copolymers, also found to be the most stable ones. Moreover, these results indicate that the linear copolymers are more stable than the cyclic ones with the same number of hydroxy fatty acid units (Fig. 7). The explanation could be that cyclic structures are more geometrically constrained. The same remark is valid for the homopolymers as well, the cyclic structures being less stable than the linear ones.
Comparative enthalpies of formation for linear and cyclic copolymers containing structural units of 16HHDA (a); 12HSA (b); RCA (c).
According to the quantum chemical calculations, the most stable structure among the linear copolymers having a degree of polymerization of 10 are those with three 12HSA moieties in the copolymer structure (Fig. 7). The real product represents, logically, a mixture of copolymers and homopolymers with different degree of polymerization. As demonstrated earlier, the molar ratio of the starting comonomers has an important influence on the composition of the copolymer product and a higher ECL amount in the starting reaction mixture leads obviously to formation of copolymerization products with higher relative ECL content. However, based on the MALDI-TOF MS spectra it was possible to estimate the relative amount of this copolymer, related to the total amount of linear and cyclic copolymers with DP of 10. The obtained value was 49.11%, (the MALDI-TOF spectrum of the 16HHDA_ECL copolymer synthesized, at 1:2 molar ratio, used for this calculation is presented in Fig. S5, Supplementary material) and confirms the quantum chemical calculations.
Based on their computed enthalpies of formation, the most stable polyesters within each series are those with a HFA unit in position 1 of the chain, having the carboxyl group involved in the ester bond. As example, in the case of the linear copolymers containing one HFA structural unit, the ΔH differences between the mentioned copolymers with the HFA unit in position 1 of the chain and their isomers with the same HFA unit in another position (calculated as average value) are 34.55 kcal/mol for (9)ECL_(1)16HHDA, 35.69 kcal/mol for (9)ECL_(1)12HSA, and 37.92 kcal/mol for (9)ECL_(1)RCA.
The results obtained from the enzymatic degradation of the copolymers also endorse the theoretical calculations, since ECL_12HSA suffers a slower enzymatic degradation than ECL_16HHDA. This fact confirms that ECL_12HSA copolymers are more stable than ECL_16HHDA, which agrees with the stability evaluation based on their computed enthalpies of formation.
Conclusions
New copolyesters of ε-caprolactone with three different hydroxy-fatty acids, 12-hydroxy stearic acid, 16-hydroxyhexadecanoic acid and ricinoleic acid, have been synthesized using SnOct2 as catalyst. Using the MALDI-TOF MS technique, it was possible to determine the average molar weight of the synthesized copolymers. The reaction parameters (molar ratio of the comonomers and temperature) were optimized, the best results being registered at ECL:RCA and ECL:16HHDA molar ratio 5:1 and 90°C, leading to m/z of about 2200 Da and maximal degree of polymerization higher than 30. The copolymers were characterized by FT-IR and NMR, confirming the formation of the desired polymeric structures.
The biodegradability of the copolymers was investigated in aqueous phosphate buffer at pH=7.4, and 37°C, in the presence of a commercial lipase. The insertion of HFA units leads to slower degradation of these compounds during the lipase-catalyzed hydrolysis, suggesting that their behavior in biological fluids could be also controlled by fine-tuning the structure of the copolymers.
Article note
A collection of invited papers based on presentations at the 16th International Conference on Polymers and Organic Chemistry (POC-16), Hersonissos (near Heraklion), Crete, Greece, 13–16 June 2016.
Acknowledgments
This work was performed through the Partnerships in priority areas – PN II program, developed with the support of UEFISCDI, project no. PN-II-PT-PCCA-2013-4-0734. Part of this research was done in the Center of Genomic Medicine from the “Victor Babes” University of Medicine and Pharmacy of Timisoara, Seventh Framework Programme (POSCCE Project ID: 1854, code SMIS: 48749, contract 677/09.04.2015).
References
[1] T. K. Dash, V. B. Konkimalla. J. Control. Release158, 15 (2012).10.1016/j.jconrel.2011.09.064Search in Google Scholar PubMed
[2] Z. Li, B. H. Tan. Mater. Sci. Eng. C45, 620 (2014).Search in Google Scholar
[3] I. Manavitehrani, A. Fathi, H. Badr, S. Daly, A. N. Shirazi, F. Dehghani. Polymers8, 20 (2016).10.3390/polym8010020Search in Google Scholar PubMed PubMed Central
[4] Y. Li, Y. Wang, J. Ye, J. Yuan, Y. Xiao. Mater. Sci. Eng. C68, 177 (2016).10.1016/j.msec.2016.05.117Search in Google Scholar PubMed
[5] B. Azimi, P. Nourpanah, M. Rabiee, S. Arbab. J. Eng. Fiber. Fabr.9, 74 (2014).10.1177/155892501400900309Search in Google Scholar
[6] C. G. Pitt, A. R. Jeffcoat, R. A. Zweidinger, A. Schindler. J. Biomed. Mat. Res.13, 497 (1979).10.1002/jbm.820130313Search in Google Scholar PubMed
[7] W. Dai, J. Zhu, A. Shangguan, M. Lang. Eur. Polym. J.45, 1659 (2009).10.1016/j.eurpolymj.2009.03.010Search in Google Scholar
[8] H. M. Kim, H. R. Kim, C. T. Hou, B. S. Kim. J. Am. Oil Chem. Soc.87, 1451 (2010).10.1007/s11746-010-1634-6Search in Google Scholar
[9] I. Martin-Arjol, M. Busquets, A. Manresa. Process Biochem. 48, 224 (2013).10.1016/j.procbio.2012.12.006Search in Google Scholar
[10] B. H. Carter. in Chemistry and Technology of Lubricants, R. M. Mortier, M. F. Fox, S. T. Orszulik (Eds.), pp. 240–243, Springer, New York (1992).Search in Google Scholar
[11] G. L. Hasenhuettl. in Food Emulsifiers and Their Applications, G. L. Hasenhuettl, R. W. Hartel (Eds.), pp. 11–37, Springer, Wisconsin (2008).10.1007/978-0-387-75284-6_2Search in Google Scholar
[12] D. G. Hayes, V. K. Mannam, R. Ye, H. Zhao, S. Ortega, M. C. Montiel. Polymers4, 1037 (2012).10.3390/polym4021037Search in Google Scholar
[13] D. Arrieta-Baez, M. Cruz-Carrillo, M. B. Gómez-Patiño, L. G. Zepeda-Vallejo. Molecules16, 4923 (2011).10.3390/molecules16064923Search in Google Scholar
[14] M. Svensson. in Surfactants from Renewable Resources, M. Kjellin, I. Johansson (Eds.), pp. 1–19, John Wiley & Sons, Ltd, Great Britain (2010).Search in Google Scholar
[15] R. Kim, D. K. Oh. Biotechnol. Adv.31, 1473 (2013).10.1016/j.biotechadv.2013.07.004Search in Google Scholar
[16] A. Todea, L. G. Otten, A. E. Frissen, I. W. C. E. Arends, F. Peter, C. G. Boeriu. Pure Appl. Chem.87, 51 (2015).10.1515/pac-2014-0716Search in Google Scholar
[17] Y. Tokiwa, B. P. Calabia, C. U. Ugwu. S. Aiba. Int. J. Mol. Sci.10, 3722 (2009).10.3390/ijms10093722Search in Google Scholar
[18] Y. Ikada, H. Tsuji. Macromol. Rapid Commun.21, 117 (2000).10.1002/(SICI)1521-3927(20000201)21:3<117::AID-MARC117>3.3.CO;2-OSearch in Google Scholar
[19] A. Kulkarni, J. Reiche, J. Hartmann, K. Krantz, A. Lendlein. Eur. J. Pharm. Biopharm.68, 46 (2008).10.1016/j.ejpb.2007.05.021Search in Google Scholar
[20] H. Peng, J. Ling, J. Liu, N. Zhu, X. Ni, Z. Shen. Polym. Degrad. Stab.95, 643 (2010).10.1016/j.polymdegradstab.2009.12.005Search in Google Scholar
[21] E. Ozsagiroglu, B. Iyisan, Y. Avcibasi Guvenilir. Ekoloji22, 90 (2013).10.5053/ekoloji.2013.8611Search in Google Scholar
[22] G. P. Sailema-Palate, A. Vidaurre, A. F. Campillo, I. Castilla-Cortázar. Polym. Degrad. Stab.130, 118 (2016).10.1016/j.polymdegradstab.2016.06.005Search in Google Scholar
[23] S. Hermanová, R. Bálková, S. Voběrková, I. Chamradová, J. Omelková, L. Richtera, L. Mravcová, J. Jančář. J. Appl. Polym. Sci.127, 4726 (2013).10.1002/app.38078Search in Google Scholar
[24] H. J. Rader, W. Schrepp. ActaPolym.49, 272 (1998).10.1002/(SICI)1521-4044(199806)49:6<272::AID-APOL272>3.3.CO;2-TSearch in Google Scholar
[25] J. A. Hudson, C. A. M. MacKenzie, K. N. Joblin. Appl. Microbiol. Biotechnol.44, 1 (1995).10.1007/BF00164472Search in Google Scholar
[26] J. J. P. Stewart. J. Comput. Chem.10, 209 (1989).10.1002/jcc.540100208Search in Google Scholar
[27] HyperChem (TM) 8.0.6 for Windows, Hypercube, Inc., Gainesville, Florida 32601, USA.Search in Google Scholar
[28] E. Polak, G. Ribiére. RAIRO – Oper. Res.16, 35 (1969).Search in Google Scholar
[29] D. Hernán Pérez de la Ossa, A. Ligresti, M. E. Gil-Alegre, M. R. Aberturas, J. Molpeceres, V. Di Marzo, A. I. Torres Suarez. J. Control. Release161, 927 (2012).10.1016/j.jconrel.2012.05.003Search in Google Scholar
[30] A. L. Sisson, D. Ekinci, A. Lendlein. Polymer54, 4333 (2013).10.1016/j.polymer.2013.04.045Search in Google Scholar
[31] V. R. Sinha, K. Bansal, R. Kaushik, R. Kumria, A. Trehan. Int. J. Pharma.278, 1 (2004).10.1016/j.ijpharm.2004.01.044Search in Google Scholar
[32] S. Li, M. Pignol, F. Gasc, M. Vert. Macromolecules37, 9798 (2004).10.1021/ma0489422Search in Google Scholar
[33] Z. Zhao, L. Yang, Y. Hu, Y. He, J. Wei, S. Li. Polym. Degrad. Stab.92, 1769 (2007).10.1016/j.polymdegradstab.2007.07.012Search in Google Scholar
[34] S. Zhou, X. Deng, H. Yang. Biomaterials24, 3563 (2003).10.1016/S0142-9612(03)00207-2Search in Google Scholar
Supplemental Material
The online version of this article (DOI: https://doi.org/10.1515/pac-2016-0920) offers supplementary material, available to authorized users.
©2016 IUPAC & De Gruyter. This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. For more information, please visit: http://creativecommons.org/licenses/by-nc-nd/4.0/
