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Hydrolytic and enzymatic degradation of flexible polymer networks comprising fatty acid derivatives
Polymer Degradation and Stability 120 (2015) 368e376
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Polymer Degradation and Stability journal homepage: www.elsevier.com/locate/polydegstab
Hydrolytic and enzymatic degradation of flexible polymer networks comprising fatty acid derivatives Je˛ drzej Skrobot, Wojciech Ignaczak, Miroslawa El Fray* The West Pomeranian University of Technology, Szczecin, Department of Biomaterials and Microbiological Technologies, Al. Piastow 45, 71-311 Szczecin, Poland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 March 2015 Received in revised form 18 July 2015 Accepted 23 July 2015 Available online 29 July 2015
In this study a degradation process of flexible polymer networks fabricated from telechelic macromonomers comprising methacrylic functionalities and dimer fatty acid derivatives were discussed. Hydrolytic degradation in simulated body fluid (SBF) as well as enzymatic degradation with a lipase of six different systems comprising ester, anhydride and urethane bonds was investigated. In parallel, in order to make a comparison with other analogous polymeric systems, reference materials were also considered. The degradation process was monitored by determining the weight loss and water uptake, FT-IR analysis and changes in pH of degradation medium as a function of time. A rapid progress of degradation of the network containing anhydride bonds was observed. Furthermore, polymer networks showed low water absorption from 10 to 2 wt.%. Polymer networks did not release acidic degradation products in an amount that causes acidification of the medium except the anhydride network. The IR analysis of characteristic bands showed degradation of specific bonds leading to formation of possible hydrolysis products of these networks such as poly(methacrylic acid), macromonomer precursor molecules and derivatives of the macromonomers having terminal carboxylic and hydroxyl groups. We demonstrated that the referred polymeric networks undergo a controlled gradual degradation in the presence of lipase. This study provides an example of how the susceptibility of cross-linked polymers to degradation can be tailored by varying their molecular structure, depending on the needs. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Polymer network Cross-linked polymers Fatty acid Hydrolytic degradation Enzymatic degradation
1. Introduction Sustainable and balanced development involve, in the largest number of areas of human activity, the replacement of petrochemical raw materials by using materials from renewable resources [1e3]. The group of renewable materials includes fatty acids, in the form of triglycerides which are a component of oils present in the seeds of plants such as canola, corn, soybeans, flax, sunflower [4]. Fatty acids and their derivatives are often used for the preparation of polymeric bio-based materials. The industrial applications of fatty acids include binders and film-forming agents in coatings and paints, hydraulic fluids, lubricants, inks, rubbers [5e9]. Due to the natural origin and the associated biocompatibility potential fatty acids are also used in materials contacting with living tissue. The latest scientific literature abounds with reports on biomaterials containing derivatives of fatty acids [10e13]. Effects of
* Corresponding author. E-mail address: [email protected] (M. El Fray). http://dx.doi.org/10.1016/j.polymdegradstab.2015.07.022 0141-3910/© 2015 Elsevier Ltd. All rights reserved.
the introduction of fatty acids in the structure of (bio)materials include: increasing the hydrophobicity [14], improving the flexibility and elastic recovery after deformation [15,16], lowering the glass transition temperature and melting point, as well as reducing the degree of crystallinity [17e19], or improving the resistance to oxidative degradation and heat resistance (stability strongly depends on the degree of unsaturation) [20e22]. One very important aspect of using biomaterials for in vivo applications is that they should persist in a robust state long enough to allow the formation of new tissue or to serve as an artificial lifelong replacement. In both cases, as well as for its successful implementation in applications such as tissue engineering scaffolds, hydrolytic degradation is of crucial importance [23]. There are three types of polymer chain breakdown, that lead to molecular mass reduction. These include the processes of destruction, depolymerization and degradation. In the process of destruction low molecular weight products other than the monomer are released. It can be caused by physical factors such as heat, high energy radiation or chemicals. Depolymerization in contrary to the polymerization, leads to thermal decomposition of macromolecules [24,25].
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Degradation is in turn the process of disintegration of the polymer chain into shorter oligomeric units under different physical agents (radiation, heat, stress), or chemicals. Aliphatic polyesters such as PLA, PLGA copolymers [26], PCL are widely known and used in tissue engineering. A lot of efforts have been done to investigate this group of polymers. FDA approval has been granted to various medical devices made of these polyesters [27]. Nevertheless, applications of these crystalline polyesters might be limited due to relatively slow degradation and resorption, as compared with other aliphatic polyesters [28]. Therefore, a large number of research contributed to the exploration and synthesis of new materials able to comprise an artificial substitute in tissue engineering has been performed [29e33]. A significant step forward in the field of aliphatic polyesters for medical application has been done creating copolymers of commercial polyesters with materials from biocompatible renewable resources such as fatty acids [34,35]. Introducing fatty acid moieties influences the degradation profile of such multiblock copolymers. The changes are associated with specific molecular structure. Hydrolysis of ester bonds in such copolymers causes fas less acidification of the environment as compared to other polyesters. This effect is associated with the lower concentration of ester bonds in the polymeric chain [36]. In this aspect great attention is paid to cross-linked polymers, especially photopolymerizable systems [37,38]. Recently, we developed telechelic macromonomers with a,u-dihydroxy, a,udicarboxy or a,u-diamine functionalities and used such “cores” to build up different structures via simple organic chemistry [39]. Our primary goal was to obtain unique photocurable macromonomers with fatty acid derivatives, that are able to form flexible polymer networks with potential in soft tissue engineering. In this paper, we present a hydrolytic and enzymatic degradation study of polymer networks fabricated from telechelic macromonomers. The networks under consideration contain one or more moieties from among anhydride, ester and urethane groups. All of these groups may undergo hydrolytic degradation, and the mechanisms of this process is shown in Fig. 1. The degradation process was monitored by determining the weight loss and water uptake, FT-IR analysis and changes in medium pH as a function of degradation time. 2. Experimental 2.1. Materials and methods A series of 9 materials: six telechelic macromonomers containing fatty acid moieties, one telechelic macromonomer synthesized without fatty acid derivatives, namely from poly[(di(ethylene glycol) adipate] containing urethane groups (PDEGA-UR), and 2
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commercially available reference materials e poly(caprolactone) (PCL), PURASORB® PC12 (Purac Biomaterials, Gorinchem, the Netherlands), Mn ¼ 124 kDa and poly(D,L-lactide-glycolide) 50:50, Resomer® RG 509 S (Evonik Industries AG, Essen, Germany), were used for degradation studies. Six telechelic macromonomers were prepared according to procedure described in Ref. [39]. The structural formulae of macromonomers comprising ester, anhydride and urethane bonds is shown in Fig. 2. The dimerized fatty acid fragment was marked as grey dot. Possible structures of the fatty acid compound are illustrated in Fig. 3. Each macromonomer was terminated with reactive methacrylic groups. The molecular masses as well as dispersity index of the macromonomers were determined by GPC as reported in Ref. [39], and are given in Table 1. The cross-linked polymers were prepared according to the following procedure. The solution of photoinitiator, Irgacure 819 (BASF, Ludwigshafen, Germany) in methylene chloride was added to the appropriate amount of macromonomer in order to achieve a desired macromonomer:photoinitiator weight ratio. Unless otherwise stated, the ratio was 50:1. After dissolution of the macromonomer, the viscous solution was poured into a Petri dish, and then methylene chloride was evaporated under reduced pressure. Then, the composition was irradiated with UV light with a narrow spectral range and maximum intensity at the wavelength lmax of 365 nm in an air atmosphere. A flashlight Labino® UVG2 was used as a light source. The intensity of the radiation was controlled with the help of radiometer AktiPrint (Technigraf GmbH) and was 20 mW/cm2. The exposure time was 200 s. 2.2. Estimation of the cross-linking density Two methods were used to estimate the cross-linking density of the polymer networks from telechelic macromonomers, namely gel fraction measurement and differential photo-calorimetry (DPC). Gel fraction was measured in dichloromethane which was found to be great solvent for the macromonomers. The cross-linked samples were refluxed for 1 h. After that the samples were dried under vacuum to constant mass. The gel fraction GF was calculated as the ratio of mass after drying to initial mass of the samples. DPC measurements were performed on Q100 differential calorimeter (TA Instruments) equipped with UV Omnicure 2000 module. The macromonomer was polymerized in air and the content of initiator (Irgacure 819) was 2 wt. %. The degree of conversion C was calculated from the formula:
C¼
M$DH DHp $n
(1)
where M is the molecular weight of the macromonomer, DH is the enthalpy of reaction, DHp is the standard enthalpy of methacrylate polymerization equal to 58.6 kJ/mol [40] and n is the number of reactive groups per molecule. 2.3. Degradation of polymer networks
Fig. 1. Simplified hydrolytic degradation mechanisms of: ester, anhydride and urethane bonds.
In order to investigate stability of the obtained polymer networks, hydrolytic and enzymatic degradation has been performed. Hydrolytic degradation was carried out for all prepared materials, and for enzymatic degradation two cross-linked polymers, namely P1838-DMA and P1838-UR, were selected. The degradation process was carried out in polystyrene microplates. The polymer discs used in this study were cut using manual die cutter from polymer films cross-linked as described above. They were of 6 mm in diameter and weighted 15e20 mg. Each disc was immersed in 2 cm3 of the
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Fig. 2. The chemical structures of synthesized macromonomers.
Fig. 3. Examples of the possible structures of dimerized linoleic acid. The degree of unsaturation is low. On average, one double bond occurs in more than five molecules.
Table 1 GPC results of synthesized macromonomers. DI e dispersity index (adopted from Ref. [39]).
P1009-DMA P1838-DMA P2033-DMA P1074-UR P2033-UR P1838-UR
Mn [g/mol]
DI
1910 5960 1040 1210 2550 12,560
1.22 1.39 1.05 1.09 1.32 1.21
medium. Each data point was an average of 4 samples. For examining the changes of degradation medium's pH, additional samples containing 0.1 g of material in 10 cm3 of medium were prepared. During the test samples have been incubated and shaken in Heidolph incubator. Shaking frequency was set at 0.33 Hz. Poly (ε-caprolactone) (PCL) was selected as a reference material for enzymatic degradation study. This polymer was chosen because it is a widely used biomaterial and has a hydrophobic character, similarly to the tested networks. In the study of the pH changes of the medium a further reference material, namely a poly(D,L-lactideco-glycolide) copolymer (PDLA) was selected. The degradation process for such copolymers is widely described the literature where great attention is given to environmental acidification induced by degradation products [41,42].
Hydrolytic degradation was carried out for 49 days at 37 C in simulated body fluid (SBF), prepared according to of the protocol for “corrected SBF” given in Ref. [43], that was buffered to pH 7.25. Samples for analysis were taken every seven days. Enzymatic degradation was induced by lipase from Pseudomonas cepacia (Sigma Aldrich, Poznan, Poland). A solution of enzyme in Dulbecco's PBS buffer (Sigma Aldrich, Poznan, Poland) at 25 units/ml (pH 7.25) was prepared. Sodium azide (POCh, Gliwice, Poland) was added to prevent the growth of bacteria. Degradation was carried out at 37 C for 22 days and samples were taken for analysis at every 24, 48 or 96 h. In order to preserve the enzyme activity, degradation medium was changed every 48 h. 2.4. Analytical methods The weight loss and water uptake were determined gravimetrically. Samples before and after degradation were weighed with an accuracy of 0.01 mg. After being removed from the medium the discs were washed thoroughly with distilled water, droplets were wiped with cloth and samples were weighed. Then the discs were dried at 37 C under a pressure of <10 mmHg for 7 days and weighed again. The weight of the discs before degradation was denoted as m0, the mass of wet discs as mw, while the mass of the discs after drying as md. The water uptake, P was calculated using the following formula:
P¼
mw md $100%; md
(2)
The weight loss was calculated from Formula (3):
D¼
m0 md $100% m0
(3)
The changes in the chemical structure of polymer networks before and after degradation were evaluated by the ATR-FTIR analysis (Thermo Nicolet Nexus). The pH of degradation medium was measured using a Schott HandyLab 12 apparatus equipped with an InLab MicroPro Mettler Toledo electrode.
J. Skrobot et al. / Polymer Degradation and Stability 120 (2015) 368e376 Table 2 Gel fraction (GF) of polymer networks and the degrees of conversion (C) for the telechelic macromonomers calculated from DPC.
P1009-DMA P1838-DMA P2033-DMA P1074-UR P2033-UR P1838-UR
GF
C [%]
0.38 0.94 1 0.99 0.99 0.91
80.9 100 72.9 68.0 63.2 100
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3. Results and discussion 3.1. Cross-linking density of polymer networks The gel fraction values of polymer networks and the degrees of conversion for the telechelic macromonomers calculated from DPC are presented in Table 2. The low gel fraction of the anhydride network can be explained based on the reported tendency of this type of compounds to undergo rapid disproportionation and scission of chains even under mild conditions [44]. Thus the network structure broke and the polymer became soluble. Gel fraction of all other networks is close to 1 which indicates that, even though the conversion varied between the materials, there were very few macromonomer molecules that were not linked to the network. 3.2. In vitro hydrolytic degradation in SBF
Fig. 4. The mass loss during the hydrolytic degradation in SBF.
Figs. 4e5 show the weight loss, and water absorption of crosslinked polymers after hydrolytic degradation. The appearance of samples after 49 days of incubation is shown in Fig. 6. A rapid degradation of the network containing anhydride bonds (P1009DMA) was observed. Already after the first week of the test the mass loss was close to 80%, and the water uptake was more than 300%. Similar susceptibility to hydrolytic degradation showed polyanhydride networks designed from photocrosslinkable multimethacrylate monomers [45,46]. In the case of other networks, a slight weight loss after 7 days was observed and it remained constant during the degradation process. Such loss may be attributed to the uncross-linked top layer rapidly washed off from the polymer surface. Most likely, this layer is washed off in the first days of testing. There was a slight increase in water absorption with time. The highest water absorption (up to 10 wt.%) after seven weeks exhibited P1074-UR and P2033-DMA. The cross-linked polymers
Fig. 5. Water absorption during the hydrolytic degradation in SBF. Fig. 7. Changes in pH medium during the hydrolytic degradation in SBF.
Fig. 6. The samples after 49 days of hydrolytic degradation in SBF (P1009-DMA after 21 days).
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Fig. 8. Changes in the chemical structure of polymer networks during hydrolytic degradation in SBF.
P1838-UR and P2033-UR absorb water the least (2 wt.%). These results can be related to Henry et al. work on slowly degrading biodegradable polyester-urethane, where degradation process was
monitored by changes in mechanical performance of such copolymer [47]. Mondal et al. proved in their degradation study high susceptibility to hydrolysis of segmented polyurethanes exhibiting
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Fig. 9. Infrared spectrum of the salt mixture used for preparation of SBF. Fig. 11. Mass loss during the enzymatic degradation.
mass loss up to 60 wt.% after 7 weeks of incubation [32]. Liu et al. investigated materials with totally different degradation profile. They investigated polymers containing fatty acids derivatives, phosphoesters cross-linked with vegetable oils (PVOs), where material degraded rapidly and completely within 4 days [12]. The generally very low water uptake is strongly correlated to the gel fraction of polymer networks and shows no clear relation to the degree of monomer conversion. Slow hydrolytic degradation of dimer fatty acid containing networks closely correlates with the results of the analysis of pH medium (Fig. 7). The degradation products of the anhydride network (P1009eDMA) caused considerable acidification of the medium after seven days of incubation. Other networks did not release acidic degradation products in an amount that causes acidification of the medium. A slight decrease in pH to a level of 7.06 after 49 days for PDEGA-UR network was observed. The acidic degradation products of the copolymer of glycolide and lactide caused a decrease in pH to about 3 after seven weeks which closely correlates with literature reports [48]. Infrared spectra (Fig. 8, Fig. 14) show the changes in the chemical structure of cross-linked polymers after hydrolytic degradation. The most significant changes were observed for the P1009-DMA network. After 7 days of degradation a broad absorption band in the range of 2500e3500 cm1 contributed to OH groups linked to carboxyl groups appeared. A band at 1016 cm1 corresponding to stretching CO vibration in anhydride groups disappeared as well as did a double band at 1813 and 1743 cm1, replaced by a single sharp carbonyl band at 1706 cm1 of carboxyl groups. These changes indicate a very intense degradation of the network as a result of cleavage of the anhydride bonds according to the mechanism shown in Fig. 1. In FT-IR spectra of P1074-UR network, a noticeable changes in
the bands corresponding to the vibrations in the ester groups were noticed. In that spectra a double overlapping band around 1700e1720 cm1 was noticed. The maximum at 1700 cm1 corresponds to stretching vibration of C]O urethane groups, and the one at 1720 cm1 to analogous vibration in the ester group. With the progress of degradation, the urethane band intensity increases in comparison with intensity of the ester band, which proves that the degradation of the ester groups occurred and urethane bonds were relatively stable during the study. Similar changes were observed in the spectra of P1074-UR network between wavenumbers 1140 to 1160 cm1, corresponding to stretching CO vibration in ester (1159 cm1) and urethane (1141 cm1) groups. Changes shown in Fig. 8 indicate that degradation of the ester bonds occurred. Also in the spectra of the P2033-UR network a decrease in intensity of the band associated with ester bonds (1717e1718 cm1) is observed with respect to the urethane band intensity (1679 cm1). Such relationship is an evidence of slow scission of ester groups. The infrared spectrum of P2033-DMA cross-linked polymer after seven weeks of hydrolytic degradation in SBF showed a broad band between 3200 and 3600 cm1 assigned to stretching vibration of hydroxyl groups and indicating breakdown of ester bonds. A slight shift in bands maximum at 1721 and 1163 cm1 towards 1725 and 1150 cm1, respectively also underlines changes in ester groups. The same observation in FT-IR spectra can be found in the work of Pamuła et al. on PLA and PLGA polymers [49]. In the spectra of samples after degradation the band at 1638 cm1 corresponding to unreacted double bonds disappeared. This can be attributed to washing off the surface layer which was mentioned in the above discussion on mass loss.
Fig. 10. Degradation mechanisms of the considered polymer networks. The bonds marked in red belong to the polymethacrylate chain (solid line), the bonds situated within macromonomer molecules (dotted lines) are marked in blue. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 12. Water absorption during the enzymatic degradation.
The spectrum of P2033-DMA network after seven weeks of degradation shows numerous additional bands marked by the arrows (Fig. 8). As it turned out, after the spectra analysis of the salt mixture used for SBF preparation (Fig. 9), these bands were derived from salts, which were not washed out from the sample during the washing step. IR spectra of the reference material PDEGA-UR before and after the study are identical and therefore indicate no changes in the chemical structure of the cross-linked polymer during 49 days in SBF. Infrared spectra of P1838-UR and P1838-DMA networks (Fig. 14) before and after hydrolytic degradation tests showed no significant differences, which may indicate the breakdown of the polymer structure. The possible mechanism by which the hydrolytic degradation of considered networks can occur are shown in Fig. 10. The products of the hydrolysis of these cross-linked polymers can be poly(methacrylic acid), free dimers of fatty namely Pripol 2033, Pripol 1838 and Pripol 1009, from which the macromonomers were synthesized), and derivatives of the macromonomers, i.e. fatty acid derivatives having terminal amino and hydroxyl groups (for networks containing urethane bonds, i.e. P1074-UR, P2033-UR, P1838-UR and PDEGA-UR). 3.3. In vitro enzymatic degradation
Fig. 13. The samples after 22 days of enzymatic degradation in lipase.
Figs. 11 and 12 show the mass loss and water absorption of selected polymer networks during the enzymatic degradation catalysed by lipase. The macroscopic appearance of samples after 22 days of study is shown in Fig. 13. There is a clear difference in the enzymatic degradation of PCL and cross-linked fatty acid derivatives. While the reference material is highly susceptible to degradation by lipase, the weight loss of polymer networks was increasing very slowly, and after 22 days reached 3.5% for urethaneester network and 4% for the ester network. The values are in line with these for hydrolytic degradation. The water absorption of polymer networks increased from less
Fig. 14. Changes in chemical structure during the hydrolytic and enzymatic degradation.
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than 1% after 24 h of degradation to 7% for the ester network and to 4.2% for the urethane-ester network after 22 days. After same period of time during hydrolytic degradation, the mass loss was ca. 4% and ca. 2%, respectively, and did not show a trend to increase. This constant increase in water absorption in the presence of lipase is most probably caused by a decrease in network density. This loosening of the network allows more and more water to migrate inside the structure. These results and the upward trend of the curves in Fig. 11 demonstrate a slight susceptibility of the studied cross-linked polymers to enzymatic degradation in the presence of the lipase. Analysis of the P1838-UR network spectra (Fig. 14) pointed out scission of the ester groups. Before degradation the stretching vibration band of C]O was observed at 1733 cm1 as a single band with a clear maximum. However, after 22 days of the enzymatic degradation this band was split and new maximum at 1716 cm1 appeared. The lower wavenumber indicates a carbonyl group with higher degree of electron delocalization, in this case situated at a carboxyl group that was formed as a result of degradation of some of the ester bonds. The spectra of the P1838-DMA cross-linked polymer before and after enzymatic degradation did not show significant differences, indicating no changes in the chemical structure of the network in the presence of the lipase. 4. Conclusions In this study, a degradation process of flexible polymer networks fabricated from telechelic macromonomers comprising methacrylic functionalities and dimer fatty acid derivatives was investigated. The cross-linked polymer containing anhydride bonds showed the highest susceptibility to hydrolytic degradation in SBF. Other materials, i.e. esters and ester-urethanes, remained stable under the test conditions and demonstrated low mass loss and water absorption that remained relatively constant or increased very slightly after first week of study. Mass loss was up to 8 wt.% and water absorption up to 10 wt.% for these materials. The degradation products of the fast degrading anhydride (P1009eDMA) caused considerable acidification of the medium in comparison with other networks that did not release acidic degradation products in an amount that caused the medium pH drop. The infrared analysis of the polymers revealed the formation of new functional groups that derived from hydrolysis products of these networks such as poly(methacrylic acid), macromonomer precursor molecules and other derivatives of the of macromonomers having terminal carboxylic and hydroxyl groups. Infrared spectra of P1838-UR and P1838-DMA before and after hydrolytic degradation tests showed no significant differences, which indicated no changes in the polymer structure. There is a significant difference in the enzymatic degradation of PCL and of the polymeric networks. While the reference material was highly susceptible to degradation by lipase, the weight loss of polymeric networks comprising fatty acid increased very slowly, and after 22 days reached 3.5 wt.% for urethane-ester network and 4 wt.% for the ester network. Analysis of the P1838-UR network spectra confirmed the scission of the ester groups. P1838-DMA network spectra before and after enzymatic degradation did not show significant differences. All discussed results confirmed the usefulness of considered flexible polymer networks in biomedical applications where controlled slow degradation is a desirable feature. Acknowledgements This work has been partially supported by research project N507
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Contents lists available at ScienceDirect
Polymer Degradation and Stability journal homepage: www.elsevier.com/locate/polydegstab
Hydrolytic and enzymatic degradation of flexible polymer networks comprising fatty acid derivatives Je˛ drzej Skrobot, Wojciech Ignaczak, Miroslawa El Fray* The West Pomeranian University of Technology, Szczecin, Department of Biomaterials and Microbiological Technologies, Al. Piastow 45, 71-311 Szczecin, Poland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 March 2015 Received in revised form 18 July 2015 Accepted 23 July 2015 Available online 29 July 2015
In this study a degradation process of flexible polymer networks fabricated from telechelic macromonomers comprising methacrylic functionalities and dimer fatty acid derivatives were discussed. Hydrolytic degradation in simulated body fluid (SBF) as well as enzymatic degradation with a lipase of six different systems comprising ester, anhydride and urethane bonds was investigated. In parallel, in order to make a comparison with other analogous polymeric systems, reference materials were also considered. The degradation process was monitored by determining the weight loss and water uptake, FT-IR analysis and changes in pH of degradation medium as a function of time. A rapid progress of degradation of the network containing anhydride bonds was observed. Furthermore, polymer networks showed low water absorption from 10 to 2 wt.%. Polymer networks did not release acidic degradation products in an amount that causes acidification of the medium except the anhydride network. The IR analysis of characteristic bands showed degradation of specific bonds leading to formation of possible hydrolysis products of these networks such as poly(methacrylic acid), macromonomer precursor molecules and derivatives of the macromonomers having terminal carboxylic and hydroxyl groups. We demonstrated that the referred polymeric networks undergo a controlled gradual degradation in the presence of lipase. This study provides an example of how the susceptibility of cross-linked polymers to degradation can be tailored by varying their molecular structure, depending on the needs. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Polymer network Cross-linked polymers Fatty acid Hydrolytic degradation Enzymatic degradation
1. Introduction Sustainable and balanced development involve, in the largest number of areas of human activity, the replacement of petrochemical raw materials by using materials from renewable resources [1e3]. The group of renewable materials includes fatty acids, in the form of triglycerides which are a component of oils present in the seeds of plants such as canola, corn, soybeans, flax, sunflower [4]. Fatty acids and their derivatives are often used for the preparation of polymeric bio-based materials. The industrial applications of fatty acids include binders and film-forming agents in coatings and paints, hydraulic fluids, lubricants, inks, rubbers [5e9]. Due to the natural origin and the associated biocompatibility potential fatty acids are also used in materials contacting with living tissue. The latest scientific literature abounds with reports on biomaterials containing derivatives of fatty acids [10e13]. Effects of
* Corresponding author. E-mail address: [email protected] (M. El Fray). http://dx.doi.org/10.1016/j.polymdegradstab.2015.07.022 0141-3910/© 2015 Elsevier Ltd. All rights reserved.
the introduction of fatty acids in the structure of (bio)materials include: increasing the hydrophobicity [14], improving the flexibility and elastic recovery after deformation [15,16], lowering the glass transition temperature and melting point, as well as reducing the degree of crystallinity [17e19], or improving the resistance to oxidative degradation and heat resistance (stability strongly depends on the degree of unsaturation) [20e22]. One very important aspect of using biomaterials for in vivo applications is that they should persist in a robust state long enough to allow the formation of new tissue or to serve as an artificial lifelong replacement. In both cases, as well as for its successful implementation in applications such as tissue engineering scaffolds, hydrolytic degradation is of crucial importance [23]. There are three types of polymer chain breakdown, that lead to molecular mass reduction. These include the processes of destruction, depolymerization and degradation. In the process of destruction low molecular weight products other than the monomer are released. It can be caused by physical factors such as heat, high energy radiation or chemicals. Depolymerization in contrary to the polymerization, leads to thermal decomposition of macromolecules [24,25].
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Degradation is in turn the process of disintegration of the polymer chain into shorter oligomeric units under different physical agents (radiation, heat, stress), or chemicals. Aliphatic polyesters such as PLA, PLGA copolymers [26], PCL are widely known and used in tissue engineering. A lot of efforts have been done to investigate this group of polymers. FDA approval has been granted to various medical devices made of these polyesters [27]. Nevertheless, applications of these crystalline polyesters might be limited due to relatively slow degradation and resorption, as compared with other aliphatic polyesters [28]. Therefore, a large number of research contributed to the exploration and synthesis of new materials able to comprise an artificial substitute in tissue engineering has been performed [29e33]. A significant step forward in the field of aliphatic polyesters for medical application has been done creating copolymers of commercial polyesters with materials from biocompatible renewable resources such as fatty acids [34,35]. Introducing fatty acid moieties influences the degradation profile of such multiblock copolymers. The changes are associated with specific molecular structure. Hydrolysis of ester bonds in such copolymers causes fas less acidification of the environment as compared to other polyesters. This effect is associated with the lower concentration of ester bonds in the polymeric chain [36]. In this aspect great attention is paid to cross-linked polymers, especially photopolymerizable systems [37,38]. Recently, we developed telechelic macromonomers with a,u-dihydroxy, a,udicarboxy or a,u-diamine functionalities and used such “cores” to build up different structures via simple organic chemistry [39]. Our primary goal was to obtain unique photocurable macromonomers with fatty acid derivatives, that are able to form flexible polymer networks with potential in soft tissue engineering. In this paper, we present a hydrolytic and enzymatic degradation study of polymer networks fabricated from telechelic macromonomers. The networks under consideration contain one or more moieties from among anhydride, ester and urethane groups. All of these groups may undergo hydrolytic degradation, and the mechanisms of this process is shown in Fig. 1. The degradation process was monitored by determining the weight loss and water uptake, FT-IR analysis and changes in medium pH as a function of degradation time. 2. Experimental 2.1. Materials and methods A series of 9 materials: six telechelic macromonomers containing fatty acid moieties, one telechelic macromonomer synthesized without fatty acid derivatives, namely from poly[(di(ethylene glycol) adipate] containing urethane groups (PDEGA-UR), and 2
369
commercially available reference materials e poly(caprolactone) (PCL), PURASORB® PC12 (Purac Biomaterials, Gorinchem, the Netherlands), Mn ¼ 124 kDa and poly(D,L-lactide-glycolide) 50:50, Resomer® RG 509 S (Evonik Industries AG, Essen, Germany), were used for degradation studies. Six telechelic macromonomers were prepared according to procedure described in Ref. [39]. The structural formulae of macromonomers comprising ester, anhydride and urethane bonds is shown in Fig. 2. The dimerized fatty acid fragment was marked as grey dot. Possible structures of the fatty acid compound are illustrated in Fig. 3. Each macromonomer was terminated with reactive methacrylic groups. The molecular masses as well as dispersity index of the macromonomers were determined by GPC as reported in Ref. [39], and are given in Table 1. The cross-linked polymers were prepared according to the following procedure. The solution of photoinitiator, Irgacure 819 (BASF, Ludwigshafen, Germany) in methylene chloride was added to the appropriate amount of macromonomer in order to achieve a desired macromonomer:photoinitiator weight ratio. Unless otherwise stated, the ratio was 50:1. After dissolution of the macromonomer, the viscous solution was poured into a Petri dish, and then methylene chloride was evaporated under reduced pressure. Then, the composition was irradiated with UV light with a narrow spectral range and maximum intensity at the wavelength lmax of 365 nm in an air atmosphere. A flashlight Labino® UVG2 was used as a light source. The intensity of the radiation was controlled with the help of radiometer AktiPrint (Technigraf GmbH) and was 20 mW/cm2. The exposure time was 200 s. 2.2. Estimation of the cross-linking density Two methods were used to estimate the cross-linking density of the polymer networks from telechelic macromonomers, namely gel fraction measurement and differential photo-calorimetry (DPC). Gel fraction was measured in dichloromethane which was found to be great solvent for the macromonomers. The cross-linked samples were refluxed for 1 h. After that the samples were dried under vacuum to constant mass. The gel fraction GF was calculated as the ratio of mass after drying to initial mass of the samples. DPC measurements were performed on Q100 differential calorimeter (TA Instruments) equipped with UV Omnicure 2000 module. The macromonomer was polymerized in air and the content of initiator (Irgacure 819) was 2 wt. %. The degree of conversion C was calculated from the formula:
C¼
M$DH DHp $n
(1)
where M is the molecular weight of the macromonomer, DH is the enthalpy of reaction, DHp is the standard enthalpy of methacrylate polymerization equal to 58.6 kJ/mol [40] and n is the number of reactive groups per molecule. 2.3. Degradation of polymer networks
Fig. 1. Simplified hydrolytic degradation mechanisms of: ester, anhydride and urethane bonds.
In order to investigate stability of the obtained polymer networks, hydrolytic and enzymatic degradation has been performed. Hydrolytic degradation was carried out for all prepared materials, and for enzymatic degradation two cross-linked polymers, namely P1838-DMA and P1838-UR, were selected. The degradation process was carried out in polystyrene microplates. The polymer discs used in this study were cut using manual die cutter from polymer films cross-linked as described above. They were of 6 mm in diameter and weighted 15e20 mg. Each disc was immersed in 2 cm3 of the
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Fig. 2. The chemical structures of synthesized macromonomers.
Fig. 3. Examples of the possible structures of dimerized linoleic acid. The degree of unsaturation is low. On average, one double bond occurs in more than five molecules.
Table 1 GPC results of synthesized macromonomers. DI e dispersity index (adopted from Ref. [39]).
P1009-DMA P1838-DMA P2033-DMA P1074-UR P2033-UR P1838-UR
Mn [g/mol]
DI
1910 5960 1040 1210 2550 12,560
1.22 1.39 1.05 1.09 1.32 1.21
medium. Each data point was an average of 4 samples. For examining the changes of degradation medium's pH, additional samples containing 0.1 g of material in 10 cm3 of medium were prepared. During the test samples have been incubated and shaken in Heidolph incubator. Shaking frequency was set at 0.33 Hz. Poly (ε-caprolactone) (PCL) was selected as a reference material for enzymatic degradation study. This polymer was chosen because it is a widely used biomaterial and has a hydrophobic character, similarly to the tested networks. In the study of the pH changes of the medium a further reference material, namely a poly(D,L-lactideco-glycolide) copolymer (PDLA) was selected. The degradation process for such copolymers is widely described the literature where great attention is given to environmental acidification induced by degradation products [41,42].
Hydrolytic degradation was carried out for 49 days at 37 C in simulated body fluid (SBF), prepared according to of the protocol for “corrected SBF” given in Ref. [43], that was buffered to pH 7.25. Samples for analysis were taken every seven days. Enzymatic degradation was induced by lipase from Pseudomonas cepacia (Sigma Aldrich, Poznan, Poland). A solution of enzyme in Dulbecco's PBS buffer (Sigma Aldrich, Poznan, Poland) at 25 units/ml (pH 7.25) was prepared. Sodium azide (POCh, Gliwice, Poland) was added to prevent the growth of bacteria. Degradation was carried out at 37 C for 22 days and samples were taken for analysis at every 24, 48 or 96 h. In order to preserve the enzyme activity, degradation medium was changed every 48 h. 2.4. Analytical methods The weight loss and water uptake were determined gravimetrically. Samples before and after degradation were weighed with an accuracy of 0.01 mg. After being removed from the medium the discs were washed thoroughly with distilled water, droplets were wiped with cloth and samples were weighed. Then the discs were dried at 37 C under a pressure of <10 mmHg for 7 days and weighed again. The weight of the discs before degradation was denoted as m0, the mass of wet discs as mw, while the mass of the discs after drying as md. The water uptake, P was calculated using the following formula:
P¼
mw md $100%; md
(2)
The weight loss was calculated from Formula (3):
D¼
m0 md $100% m0
(3)
The changes in the chemical structure of polymer networks before and after degradation were evaluated by the ATR-FTIR analysis (Thermo Nicolet Nexus). The pH of degradation medium was measured using a Schott HandyLab 12 apparatus equipped with an InLab MicroPro Mettler Toledo electrode.
J. Skrobot et al. / Polymer Degradation and Stability 120 (2015) 368e376 Table 2 Gel fraction (GF) of polymer networks and the degrees of conversion (C) for the telechelic macromonomers calculated from DPC.
P1009-DMA P1838-DMA P2033-DMA P1074-UR P2033-UR P1838-UR
GF
C [%]
0.38 0.94 1 0.99 0.99 0.91
80.9 100 72.9 68.0 63.2 100
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3. Results and discussion 3.1. Cross-linking density of polymer networks The gel fraction values of polymer networks and the degrees of conversion for the telechelic macromonomers calculated from DPC are presented in Table 2. The low gel fraction of the anhydride network can be explained based on the reported tendency of this type of compounds to undergo rapid disproportionation and scission of chains even under mild conditions [44]. Thus the network structure broke and the polymer became soluble. Gel fraction of all other networks is close to 1 which indicates that, even though the conversion varied between the materials, there were very few macromonomer molecules that were not linked to the network. 3.2. In vitro hydrolytic degradation in SBF
Fig. 4. The mass loss during the hydrolytic degradation in SBF.
Figs. 4e5 show the weight loss, and water absorption of crosslinked polymers after hydrolytic degradation. The appearance of samples after 49 days of incubation is shown in Fig. 6. A rapid degradation of the network containing anhydride bonds (P1009DMA) was observed. Already after the first week of the test the mass loss was close to 80%, and the water uptake was more than 300%. Similar susceptibility to hydrolytic degradation showed polyanhydride networks designed from photocrosslinkable multimethacrylate monomers [45,46]. In the case of other networks, a slight weight loss after 7 days was observed and it remained constant during the degradation process. Such loss may be attributed to the uncross-linked top layer rapidly washed off from the polymer surface. Most likely, this layer is washed off in the first days of testing. There was a slight increase in water absorption with time. The highest water absorption (up to 10 wt.%) after seven weeks exhibited P1074-UR and P2033-DMA. The cross-linked polymers
Fig. 5. Water absorption during the hydrolytic degradation in SBF. Fig. 7. Changes in pH medium during the hydrolytic degradation in SBF.
Fig. 6. The samples after 49 days of hydrolytic degradation in SBF (P1009-DMA after 21 days).
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Fig. 8. Changes in the chemical structure of polymer networks during hydrolytic degradation in SBF.
P1838-UR and P2033-UR absorb water the least (2 wt.%). These results can be related to Henry et al. work on slowly degrading biodegradable polyester-urethane, where degradation process was
monitored by changes in mechanical performance of such copolymer [47]. Mondal et al. proved in their degradation study high susceptibility to hydrolysis of segmented polyurethanes exhibiting
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373
Fig. 9. Infrared spectrum of the salt mixture used for preparation of SBF. Fig. 11. Mass loss during the enzymatic degradation.
mass loss up to 60 wt.% after 7 weeks of incubation [32]. Liu et al. investigated materials with totally different degradation profile. They investigated polymers containing fatty acids derivatives, phosphoesters cross-linked with vegetable oils (PVOs), where material degraded rapidly and completely within 4 days [12]. The generally very low water uptake is strongly correlated to the gel fraction of polymer networks and shows no clear relation to the degree of monomer conversion. Slow hydrolytic degradation of dimer fatty acid containing networks closely correlates with the results of the analysis of pH medium (Fig. 7). The degradation products of the anhydride network (P1009eDMA) caused considerable acidification of the medium after seven days of incubation. Other networks did not release acidic degradation products in an amount that causes acidification of the medium. A slight decrease in pH to a level of 7.06 after 49 days for PDEGA-UR network was observed. The acidic degradation products of the copolymer of glycolide and lactide caused a decrease in pH to about 3 after seven weeks which closely correlates with literature reports [48]. Infrared spectra (Fig. 8, Fig. 14) show the changes in the chemical structure of cross-linked polymers after hydrolytic degradation. The most significant changes were observed for the P1009-DMA network. After 7 days of degradation a broad absorption band in the range of 2500e3500 cm1 contributed to OH groups linked to carboxyl groups appeared. A band at 1016 cm1 corresponding to stretching CO vibration in anhydride groups disappeared as well as did a double band at 1813 and 1743 cm1, replaced by a single sharp carbonyl band at 1706 cm1 of carboxyl groups. These changes indicate a very intense degradation of the network as a result of cleavage of the anhydride bonds according to the mechanism shown in Fig. 1. In FT-IR spectra of P1074-UR network, a noticeable changes in
the bands corresponding to the vibrations in the ester groups were noticed. In that spectra a double overlapping band around 1700e1720 cm1 was noticed. The maximum at 1700 cm1 corresponds to stretching vibration of C]O urethane groups, and the one at 1720 cm1 to analogous vibration in the ester group. With the progress of degradation, the urethane band intensity increases in comparison with intensity of the ester band, which proves that the degradation of the ester groups occurred and urethane bonds were relatively stable during the study. Similar changes were observed in the spectra of P1074-UR network between wavenumbers 1140 to 1160 cm1, corresponding to stretching CO vibration in ester (1159 cm1) and urethane (1141 cm1) groups. Changes shown in Fig. 8 indicate that degradation of the ester bonds occurred. Also in the spectra of the P2033-UR network a decrease in intensity of the band associated with ester bonds (1717e1718 cm1) is observed with respect to the urethane band intensity (1679 cm1). Such relationship is an evidence of slow scission of ester groups. The infrared spectrum of P2033-DMA cross-linked polymer after seven weeks of hydrolytic degradation in SBF showed a broad band between 3200 and 3600 cm1 assigned to stretching vibration of hydroxyl groups and indicating breakdown of ester bonds. A slight shift in bands maximum at 1721 and 1163 cm1 towards 1725 and 1150 cm1, respectively also underlines changes in ester groups. The same observation in FT-IR spectra can be found in the work of Pamuła et al. on PLA and PLGA polymers [49]. In the spectra of samples after degradation the band at 1638 cm1 corresponding to unreacted double bonds disappeared. This can be attributed to washing off the surface layer which was mentioned in the above discussion on mass loss.
Fig. 10. Degradation mechanisms of the considered polymer networks. The bonds marked in red belong to the polymethacrylate chain (solid line), the bonds situated within macromonomer molecules (dotted lines) are marked in blue. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 12. Water absorption during the enzymatic degradation.
The spectrum of P2033-DMA network after seven weeks of degradation shows numerous additional bands marked by the arrows (Fig. 8). As it turned out, after the spectra analysis of the salt mixture used for SBF preparation (Fig. 9), these bands were derived from salts, which were not washed out from the sample during the washing step. IR spectra of the reference material PDEGA-UR before and after the study are identical and therefore indicate no changes in the chemical structure of the cross-linked polymer during 49 days in SBF. Infrared spectra of P1838-UR and P1838-DMA networks (Fig. 14) before and after hydrolytic degradation tests showed no significant differences, which may indicate the breakdown of the polymer structure. The possible mechanism by which the hydrolytic degradation of considered networks can occur are shown in Fig. 10. The products of the hydrolysis of these cross-linked polymers can be poly(methacrylic acid), free dimers of fatty namely Pripol 2033, Pripol 1838 and Pripol 1009, from which the macromonomers were synthesized), and derivatives of the macromonomers, i.e. fatty acid derivatives having terminal amino and hydroxyl groups (for networks containing urethane bonds, i.e. P1074-UR, P2033-UR, P1838-UR and PDEGA-UR). 3.3. In vitro enzymatic degradation
Fig. 13. The samples after 22 days of enzymatic degradation in lipase.
Figs. 11 and 12 show the mass loss and water absorption of selected polymer networks during the enzymatic degradation catalysed by lipase. The macroscopic appearance of samples after 22 days of study is shown in Fig. 13. There is a clear difference in the enzymatic degradation of PCL and cross-linked fatty acid derivatives. While the reference material is highly susceptible to degradation by lipase, the weight loss of polymer networks was increasing very slowly, and after 22 days reached 3.5% for urethaneester network and 4% for the ester network. The values are in line with these for hydrolytic degradation. The water absorption of polymer networks increased from less
Fig. 14. Changes in chemical structure during the hydrolytic and enzymatic degradation.
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than 1% after 24 h of degradation to 7% for the ester network and to 4.2% for the urethane-ester network after 22 days. After same period of time during hydrolytic degradation, the mass loss was ca. 4% and ca. 2%, respectively, and did not show a trend to increase. This constant increase in water absorption in the presence of lipase is most probably caused by a decrease in network density. This loosening of the network allows more and more water to migrate inside the structure. These results and the upward trend of the curves in Fig. 11 demonstrate a slight susceptibility of the studied cross-linked polymers to enzymatic degradation in the presence of the lipase. Analysis of the P1838-UR network spectra (Fig. 14) pointed out scission of the ester groups. Before degradation the stretching vibration band of C]O was observed at 1733 cm1 as a single band with a clear maximum. However, after 22 days of the enzymatic degradation this band was split and new maximum at 1716 cm1 appeared. The lower wavenumber indicates a carbonyl group with higher degree of electron delocalization, in this case situated at a carboxyl group that was formed as a result of degradation of some of the ester bonds. The spectra of the P1838-DMA cross-linked polymer before and after enzymatic degradation did not show significant differences, indicating no changes in the chemical structure of the network in the presence of the lipase. 4. Conclusions In this study, a degradation process of flexible polymer networks fabricated from telechelic macromonomers comprising methacrylic functionalities and dimer fatty acid derivatives was investigated. The cross-linked polymer containing anhydride bonds showed the highest susceptibility to hydrolytic degradation in SBF. Other materials, i.e. esters and ester-urethanes, remained stable under the test conditions and demonstrated low mass loss and water absorption that remained relatively constant or increased very slightly after first week of study. Mass loss was up to 8 wt.% and water absorption up to 10 wt.% for these materials. The degradation products of the fast degrading anhydride (P1009eDMA) caused considerable acidification of the medium in comparison with other networks that did not release acidic degradation products in an amount that caused the medium pH drop. The infrared analysis of the polymers revealed the formation of new functional groups that derived from hydrolysis products of these networks such as poly(methacrylic acid), macromonomer precursor molecules and other derivatives of the of macromonomers having terminal carboxylic and hydroxyl groups. Infrared spectra of P1838-UR and P1838-DMA before and after hydrolytic degradation tests showed no significant differences, which indicated no changes in the polymer structure. There is a significant difference in the enzymatic degradation of PCL and of the polymeric networks. While the reference material was highly susceptible to degradation by lipase, the weight loss of polymeric networks comprising fatty acid increased very slowly, and after 22 days reached 3.5 wt.% for urethane-ester network and 4 wt.% for the ester network. Analysis of the P1838-UR network spectra confirmed the scission of the ester groups. P1838-DMA network spectra before and after enzymatic degradation did not show significant differences. All discussed results confirmed the usefulness of considered flexible polymer networks in biomedical applications where controlled slow degradation is a desirable feature. Acknowledgements This work has been partially supported by research project N507
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