DOI 10.1007/s11746-015-2611-x
ORIGINAL PAPER
Biodegradable Poly(ε‑Caprolactone)‑Based Graft Copolymers
Via Poly(Linoleic Acid): In Vitro Enzymatic Evaluation
Sema Allı · R. Seda Tıg˘lı Aydın · Abdülkadir Allı · Baki Hazer
Received: 3 November 2014 / Revised: 6 January 2015 / Accepted: 23 January 2015 / Published online: 13 February 2015 © AOCS 2015
rate. The molar ratio of [CL]/[Lina] dramatically decreased, from 21.3 to 8.4, after 30 days of incubation. Moreover, reduced PCL content in PLina-g-PSt-g-PCL copolymers decreased the degradation rate, probably due to the PSt enrichment within the structure, which blocks lipase con-tact with PCL units. Thus, copolymerization of PCL with PLina and PSt units leads to a controllable degradation pro-file, which encourages the use of these polymers as promis-ing biomaterials for tissue engineerpromis-ing applications.
Keywords Enzymatic degradation ·
Poly(ε-caprolactone) · Poly(linoleic acid) · Poly(styrene)
Introduction
Biodegradation is one of the main challenges of the prepa-ration of appropriate biomaterials for tissue engineering applications. Adjustable material properties lead to tailor biomaterials performance in terms of generating new tis-sue. With regard to successful tissue regeneration, degra-dation time of the biomaterials must be balanced with the time of new generated tissue. Thus, the material properties must be carefully considered to ensure proper biomate-rial choice [1, 2]. Poly(ε-caprolactone) (PCL), its copoly-mers, and its blends have been involved in various tissue engineering applications [3–7]. However, some basic prop-erties of PCL (i.e., hydrophilicity, high crystallinity and selectively enzymatic degradation time) limit the design of proper materials for several tissue engineering applications [1, 5]. In order to overcome these limitations, copolymers of PCL were used to tailor the properties of biomaterials [8]. Specific requirements for several applications of tis-sue engineering could be achieved by controlling the chain design and the microstructure [1, 6–9].
Abstract Well-defined graft copolymers based on
poly(ε-caprolactone) (PCL) via poly(linoleic acid) (PLina), are derived from soybean oil. Poly(linoleic poly(ε-caprolactone) (PLina-g-PCL) and poly(linoleic acid)-g-poly(styrene)-g-poly(ε-caprolactone) (PLina-g-PSt-g-PCL) were synthesized by ring-opening polymerization of
ε-caprolactone initiated by PLina and one-pot synthesis of
graft copolymers, and by ring-opening polymerization and free radical polymerization by using PLina, respectively. PLina-g-PCL, PCL3, and PLina-g-PSt-g-PCL4 copolymers containing 96.97, 75.04 and 80.34 mol% CL, respectively, have been investigated regarding their enzymatic degradation properties in the presence of Pseu-domonas lipase. In terms of weight loss, after 1 month, 51.5 % of PLina-g-PCL, 18.8 % of PLina-g-PSt-g-PCL3, and 38.4 % of PLina-g-PSt-g-PCL4 were degraded, leaving remaining copolymers with molecular weights of 16,140, 83,220 and 70,600 Da, respectively. Introducing the PLina unit into the copolymers greatly decreased the degradation
Electronic supplementary material The online version of this
article (doi:10.1007/s11746-015-2611-x) contains supplementary material, which is available to authorized users.
S. Allı · B. Hazer (*)
Department of Chemistry, Bülent Ecevit University, 67100 Zonguldak, Turkey
e-mail: bhazer2@yahoo.com R. S. Tıg˘lı Aydın (*)
Department of Biomedical Engineering, Bülent Ecevit University, 67100 Zonguldak, Turkey
e-mail: rseda.tigli@gmail.com A. Allı
Department of Chemistry, Düzce University, 81620 Düzce, Turkey
Soy bean oil, a renewable resource which contains linoleic acid (51 %), oleic acid (25 %), palmitic acid (11 %), linolenic acid (9 %), and stearic acid (4 %) residues [10], is a polyun-saturated plant oil (MW approx. 874) and is a suitable start-ing material for polymers because of its abundance and the rich chemistry that the triglyceride structure provides [10– 13]. In recent years, natural oils and derivatives used as raw materials for the preparation of polymers and copolymers based on unsaturated oils [14]. Polymeric oils can be syn-thesized by using macroperoxy initiators (polyunsaturated oil/oily acids) for obtaining block/graft copolymers via free radical polymerization [14–18]. Çakmaklı et al. [15] syn-thesized polymeric soybean oil-graft-methyl methacrylate (PSO-g-PMMA) by peroxidation, epoxidation and/or pere-poxidation of soybean oil. Allı and Hazer [16] reported the polymerization of auto-oxidized polymeric soybean oil with N-isopropyl acrylamide. In our previous study, Alli et al. [14] reported one-pot synthesis of poly(linoleic
acid)-g-poly(ε-caprolactone) (PLina-g-PCL) and poly(linoleic acid)-g-poly(styrene)-g-poly(ε-caprolactone)
(PLina-g-PSt-g-PCL) graft copolymers by ring opening polymerization of ε-caprolactone initiated from the carboxylic acid groups of PLina. Moreover, Miao et al. [17] reported a new class of biocompatible polyurethanes prepared from soybean-oil-based polyol and Aydın Tığlı et al. [18] prepared polymeric soybean oil-g-polystyrene (PSO-g-PS) membranes for suit-able use in biomedical applications. Synthesis of these mate-rials has prompted research into the production of polymeric oil-based biomaterials for tissue regeneration because of the biocompatibility of those oil-based polymers [15, 17, 18].
Since biodegradation facilitates the formation of new tis-sue, controlling the degradation parameters of biomaterials is required for successful tissue repair. The method for in vitro degradation of biodegradable biomaterials is generally assessed by using buffer solutions which leads very long degradation period for some biomaterials like PCL (more than 1 year) [19]. Since the period is very long, characteri-zation of the biomaterials from synthesis to the practical application of will be very long; therefore, enzymatic deg-radation methods were suggested which takes the advan-tage of degradation rate comparison determined from data between buffer solutions and in vivo [19]. Lipase, which is secreted by macrophages, has been reported to degrade of PCL and its copolymers [20, 21]. Previously, it had been reported that Pseudomonas lipase was the most effective type of lipase on PCL films in comparison to porcine pan-creatic lipase and candida cylindracea lipase [19].
In the present study, the PCL homopolymer and
PLina-g-PCL and PLina-g-PSt-g-PCL copolymers were prepared. Then, in vitro enzymatic degradation behavior of these polymers was evaluated in the presence of Pseudomonas lipase with regard to a potential biomaterial candidate within the scope of tissue regeneration.
Materials and Methods
Linoleic acid (cis–cis-9-12-octadecadienoic acid) was obtained from Fluka (Steinheim, Germany), and used as received (purity: 99 %). ε-caprolactone (ε-CL) was (Aldrich, Germany) dried over anhydrous CaSO4 and then
fractionally distilled prior to use. Styrene (S) (Aldrich, Ger-many) was purified extensively by washing with 10 wt% aqueous NaOH solution, drying over anhydrous CaCl2 overnight, and distilling over CaH2 under reduced pressure
prior to use. Lipase (from Pseudomonas cepacia, 30 units/ mg) and sodium azide were purchased from Sigma–Aldrich (Germany) and used as received. All other chemicals were of analytical grade and used without further purification. Polymerization of ε-CL and Linoleic Acid Autooxidation
ε-CL was polymerized via ring opening polymerization
(ROP) according to the procedure written below. The cata-lyst, 0.031 g of tin(II) 2-ethylhexanoate or tin(II) octoate (Sn(Oc)2), and the monomer, 2 g of ε-CL, were charged into a flame-dried Schlenk flask. Then argon was intro-duced through a needle into the tube for about 3 min to expel the air. The flask was placed in an oil bath preheated at 110 °C for 24 h and the reaction was finalized by adding cold chloroform and the catalyst was filtered out. The crude polymer was dissolved in chloroform and poured into excess petroleum ether to precipitate the polymer. Then, the precipitated polymer was dried under a vacuum at 40 °C for 24 h. Polymeric linoleic acid (PLina) was obtained through autooxidation of linoleic acid as previously reported [16]. Briefly, 10 g of linoleic acid (Lina) spread out in a Petri dish (diameter = 16 cm) was exposed to sunlight in the air at room temperature. A pale-yellow, viscous liquid PLina was obtained after 3 months of autooxidation. Then sam-ples were isolated by the chloroform extraction. Peroxide analysis of the PLina fraction was carried out by a reflux of a mixture of 2-propanol (50 ml)/acetic acid (10 ml)/satu-rated aqueous solution of KI (1 ml) and 0.1 g of PLina for 10 min and titrating the released iodine against thiosulfate solution, according to a procedure reported before [11]. Polymerization of PLina-g-PCL Copolymers
PLina-g-PCL graft copolymers were synthesized by ROP of ε-caprolactone initiated from the carboxylic acid groups of PLina [14]. In brief, 0.5 g of Plina and 4.0 g of ε-CL was introduced into a catalyst (Sn(Oc)2) loaded Schlenk flask under argon (catalyst/monomer: 1/100). Then, polymeriza-tion was allowed to proceed at 110 °C for 24 h and reacpolymeriza-tion was stopped by adding cold chloroform. The catalyst was filtered out; the crude polymer was dissolved in chloroform and poured into excess petroleum ether to precipitate the
copolymer. Then, the precipitated copolymer was dried under a vacuum at 40 °C for 24 h.
Polymerization of PLina-g-PSt-g-PCL Copolymers PLina-g-PSt-g-PCL copolymers were synthesized by a one-pot polymerization procedure where PLina peroxides initi-ated vinyl monomer, at the same time with ε-caprolactone initiated from the carboxylic acid groups of PLina via ROP [14]. In brief, 0.50 g of PLina and the catalyst were charged into a flame-dried Schlenk flask. Then 2.0 g of styrene and 3.0 or 4.0 g of ε-caprolactone were added into the Schlenk flask under an argon atmosphere. Then, polymerization was processed at 110 °C for 5 h. The crude copolymer was dis-solved in tetrahydrofurane (THF) and poured into excess petroleum ether to precipitate the copolymer. Then, the pre-cipitated polymer was dried at 40 °C for 24 h.
Polymer Characterization
Nuclear magnetic resonance (1H NMR) spectra of PLina,
PCL, PLina-g-PCL and PLina-g-PSt-g-PCL were recorded in CDCl3 at 17 °C with a tetramethylsilane internal stand-ard using a 400 MHz/54 mm Ultra Shield Plus NMR (Burker, Ultra long hold time). The molecular weight of the polymeric samples was determined by gel permea-tion chromatography (GPC) with a Waters model 6000A solvent delivery system with a model 401 refractive index detector and a mode 730 data module and with two Ultras-tyragel linear columns in series. Chloroform was used in the elution at a flow rate of 1.0 mL/min. A calibration curve was generated with polystyrene standards.
Enzymatic Degradation
In vitro degradation studies of homo PCL, PLina-g-PCL and PLina-g-PSt-g-PCL copolymers were carried out enzy-matically in the presence of Pseudomonas lipase according to a procedure reported previously [1]. Dry polymer sam-ples (20 mg) were immersed in capped bottles containing 3 mL phosphate buffer solution (PBS) (pH 7.4), 0.02 % sodium azide (NaN3) as the bacteriostatic agent and 0.1 %
lipase (from P. cepacia, 30 units/mg). Samples were incu-bated at 37 °C on a shaker and the buffer was refreshed every 24 h to maintain the initial activity of the lipase. At predetermined time intervals, three parallel samples were withdrawn from the degradation medium, rinsed with dis-tilled water three times and then dried to constant weight in a vacuum at room temperature. Weight loss was determined gravimetrically by comparing the dry weight remaining at a specific time (W) with the initial weight (W0) and degrada-tion was determined as the percentage of weight loss (WL) according to Eq. (1).
Degraded polymers were also characterized by 1H-NMR
and GPC analysis as described before.
Results and Discussion
Polymeric Linoleic Acid and Graft Polymerization
PLina, which was successfully synthesized by autooxida-tion of Lina, has been found to be useful for incorporat-ing hydrophobic and biodegradable oil-sourced polymers into graft copolymer structures [14]. In our previous stud-ies, PLina was used as a macroperoxy initiator within the free radical polymerization of selected vinyl monomers [22] and ring opening polymerization of ε-caprolactone via carboxylic groups [14]. PLina, the macroperoxy initiator, was autoxidized for 3 months. The molar mass of 1,870 Da (PDI = 1.49) was obtained in quantitative yields of GPC results and peroxygen content of the PLina was found to be 1.10 wt%, which have also been reported before [14]. In this study, the soluble part of the autooxidized linoleic acid was used as the macroperoxidic initiator in the ROP of ε-caprolactone and one-pot polymerization of styrene and ε-caprolactone for the synthesis of PLina-g-PCL and PLina-g-PSt-g-PCL, respectively, as described before [14]. Figure 1a describes the synthesis of PLina-g-PCL and PLina-g-PSt-g-PCL polymers. 1H-NMR spectra of PLina
and PCL polymers are shown in Fig. 1b and c, respectively. The characteristic peaks of PLina and PCL were marked on the spectrum which confirms the autooxidation of Lina and polymerization of ε-caprolactone. As seen from the 1
H-NMR spectrum of PLina (Fig. 1b), polyunsaturated fatty acids have the signals of –CH–O– oxide groups formed by the autooxidation at the chemical shifts between 3.4 and 3.8 ppm [22]. The peaks at 4.06 (peak 1), 2.31 (peak 5), 1.60–1.70 (peaks 2, 4), and 1.35–1.43 (peak 3) were assigned to –OCH2, –COCH2, –CH2(4 H) and –CH2(2 H) segments of PCL, respectively [23]. Figure 1d–f clearly demonstrates 1H-NMR spectra of the graft copolymer
samples (g-PCL and g-PSt-g-PCL).
PLina-g-PSt-g-PCL3 (Fig. 1e) and PLina-g-PSt-g-PCL4 (Fig. 1f) graft copolymers were coded according to their initial PCL amounts during polymerization (PLina-g-PSt-g-PCL3 and PLina-g-PSt-g-PCL4 stands for 3.0 and 4.0 gr ε-CL, respectively). Characteristic peaks of the related segments which were 0.9 ppm (–CH3 of fatty acid macroperoxide-PLina), 4.1 ppm (–CH2–O–poly(ε-caprolactone) and 6.5– 7.1 ppm (the phenyl protons in polystyrene) confirmed the structure of copolymers (Fig. 1d–f). Table 1 presents polymerization conditions and polymer analysis results as well as molecular weights and polydispersity of the (1)
polymers from GPC analysis. Lina, CL, and St contents in mol% were calculated from the peak areas of the methyl protons in Lina (0.9 ppm), phenyl protons in styrene (6.5– 7.1 ppm) and (–CH2–O–) protons in caprolactone segments
(4.1 ppm). CL contents (mol%) in g-PCL,
PLina-g-PSt-g-PCL3 and PLina-g-PSt-g-PCL4 copolymers were calculated as 96.97, 75.04 and 80.34 %.
Enzymatic Degradation and Mechanism
PCL, which is a well-known aliphatic polyester, slowly degrades over several years [24] if it is not within any bac-terial environment. In fact, in vivo, macrophages, which are activated after implantation of a foreign material dur-ing cellular infiltration, are able to secrete various enzymes
Fig. 1 a Scheme of PLina-g-PCL and PLina-g-PSt-g-PCL graft copolymer synthesis, 1H-NMR spectra of b PLina, c PCL, d PLina-g-PCL, e
Table 1 Results and conditions of autooxidation of Lina, ROP of ε-CL, and ROP of ε-CL with PLina and one-pot synthesis of
PLina-g-PSt-g-PCL graft copolymers
Catalyst (tin(II)-ethyl hexanoate)/monomer: 1/100
a Calculated from 1H NMR
Polymer ε-CL (g) PLina (g) St (g) Amount of polymer (%) Linaa (mol%) Sta (mol%) CLa (mol%) M nGPC (kDa) MwGPC (kDa) PDI PLina – 10.01 – 99.4 100 – – 1.87 2.80 1.49 PCL 2.00 – – 84.9 – – 100 18.11 23.87 1.32 PLina-g-PCL 4.00 0.50 – 72.13 3.03 – 96.97 14.86 36.73 2.47 PLina-g-PSt-g-PCL3 3.00 0.50 2.00 61.82 3.16 21.78 75.04 63.97 140.43 2.19 PLina-g-PSt-g-PCL4 4.00 0.50 2.00 56.15 3.57 16.09 80.34 70.95 141.49 2.20
Table 2 Degradation properties
of homo PCL, PLina-g-PCL and PLina-g-PSt-g-PCL copolymers
a The molar ratio were
determined by integrals of the peaks of Lina (0.9 ppm), St (6.5–7.1 ppm) and CL (4.1 ppm) units in 1H NMR
spectra
Time (day) Weight loss (%) MnGPC (kDa) MwGPC (kDa) PDI PCL 0 0 18.11 23.87 1.32 1 4.2 15.33 24.42 1.59 2 15.2 14.87 23.73 1.60 3 22.0 14.34 25.02 1.75 4 35.0 13.53 25.23 1.87 5 45.0 12.20 22.71 1.86 6 100 0 0 –
Time (day) Weight loss (%) MnGPC (kDa) MwGPC (kDa) PDI [CL]/[Lina]a
PLina-g-PCL 0 0 14.86 36.73 2.47 21.3 1 4.5 11.54 24.61 2.13 20.6 3 12.2 10.65 24.60 2.31 16.0 5 18.2 10.38 23.26 2.24 14.9 18 47.7 9.49 19.66 2.07 12.6 30 51.5 8.38 16.14 1.93 8.4
Time (day) Weight loss (%) MnGPC (kDa) MwGPC (kDa) PDI [CL3]/[St]/[Lina]a
PLina-g-PSt-g-PCL3 0 0 63.97 140.43 2.19 0.46 1 4.3 62.27 119.26 1.92 0.45 3 6.3 60.15 118.48 1.97 0.42 5 10.5 57.91 95.46 1.65 0.38 18 15.5 54.60 83.74 1.55 0.37 30 18.8 52.51 83.22 1.59 0.35
Time (day) Weight loss (%) MnGPC (kDa) MwGPC (kDa) PDI [CL4]/[St]/[Lina]a
PLina-g-PSt-g-PCL4 0 0 70.95 141.49 2.20 0.67 1 4.2 69.56 109.72 1.58 0.65 3 11.2 63.03 86.55 1.37 0.52 5 18.3 61.33 85.14 1.38 0.50 18 25.4 55.29 80.74 1.46 0.49 30 38.4 51.53 70.60 1.37 0.49
including lipase [25]. Pseudomonas lipase, which is known for its ability to catalyze the degradation of PCL [26], has been reported for the degradation of PCL and its copoly-mers via hydrolysis and dissolution [1, 20, 21, 26, 27]. The enzymatic degradation of PCL by using Pseudomonas lipase, which is an endoenzyme, proceeds by two stages. First, a random scission of the ester linkage of the PCL chain in amorphous regions is done after the adsorption of the enzyme. Then, endoenzyme attacks on the crystal-line regions by its active site [28]. In this study in vitro degradation of PCL-based copolymers in the presence of Pseudomonas lipase was investigated with regard to their di and tri blocked grafted structure compositions via PLina, obtained from one of the most abundant renewable resource.
Enzymatic Degradation of PCL
Table 2 demonstrates the degradation properties of homo PCL, PLina-g-PCL and PLina-g-PSt-g-PCL copolymers. Homo PCL, which is polymerized via ROP with molecular weights of 18,110 Da (Mn) and 23,870 Da (Mw), degraded completely at the end of 6 days (Table 2). Amounts of per-centage weight losses of degraded PCL were calculated and degradation kinetics of PCL was demonstrated in Fig. 2.
Results from GPC analysis (Fig. 3a) indicated the molecu-lar weight decrement during the incubation period (Table 2; Fig. 2). Previous studies reported that PCL was enzymati-cally degraded completely in 4 days [29]; thus, the results agreed with the literature.
Enzymatic Degradation of PLina-g-PCL Copolymers Controlling degradation kinetics of biomedical materials is critical when designing and optimizing their use as a bio-material in vivo. Predictable degradation kinetics would be helpful for specific applications (i.e., skin tissue vs bone tissue regeneration) since tissue regeneration and host response are directly affected by the degradation process. During the enzymatic hydrolysis of polymers, the adsorp-tion and rate of hydrolysis reacadsorp-tion are affected by the phys-icochemical properties of polymer (i.e., molecular weight, chemical composition, crystallinity, surface area) [30]. Moreover, the enzymatic degradation rates can be changed by the chemical modification of polymers (crosslinking, removal, or introduction of chemical groups in the polymer chain) [8, 30]. The enzymatic degradation of PLina-g-PCL copolymers was investigated and weight loss data were cal-culated after selected degradation times (Table 2). As dem-onstrated in Fig. 2 and Table 2, after the first day, a sharp
Fig. 2 Degradation behaviors of PCL, PLina-g-PCL and PLina-g-PSt-g-PCL copolymers. Filled squares MnGPC (kDa), filled diamonds relative
Fig. 3 GPC diagrams of PCL, PLina-g-PCL and PLina-g-PSt-g-PCL copolymers before and after degradation at selected time intervals (1, 3, 5,
decrease in weight loss was seen similar to homo PCL, however, after the first 2–3 days PLina-g-PCL copolymers degrade much slower than homo PCL. After 5 days only 18.2 % weight loss was recorded for PLina-g-PCL while weight loss for PCL reached 45 %. At the end of 30 days, 51.5 % weight loss was determined for PLina-g-PCL while
PCL was degraded completely after 6 days. Results from GPC analysis indicated the decrease in molecular weight with time (Fig. 3; Table 2). Mn and Mw of degraded PLina-g-PCL after 30 days were recorded as 8.375 and 16.142 kDa, respectively (Table 2). Figure 4a demonstrated composition of PLina-g-PCL after degradation of 30 days by 1H-NMR
spectra. As seen in Fig. 4, the peaks at 0.9 and 4.1 ppm lead to Lina and CL units. The integrals of peaks correspond-ing to Lina units (0.9 ppm) and CL units (4.1 ppm) were compared, and the ratios of [CL]/[Lina] were calculated (Table 2) in order to evaluate the enzymatic degradation characteristics of the copolymer. Figure 4 indicated that lipase did not have any proper effect on the Lina peaks at 0.9 ppm; however, the peak areas corresponding to CL at 4.1 ppm greatly changed as time passed during the degra-dation process, indicating that PCL units were degraded within this time frame. After the first day, a sharp decrease in the [CL]/[Lina] ratio was recorded (Supplementary material 1) which is in accordance with the GPC results. Although molecular weights of PLina-g-PCL and PCL were Mn:14.860 kDa; Mw:36.731 kDa and Mn:18.110 kDa;
Mw:23.870 kDa, respectively, degradation characteristics of
those were revealed as very different from each other. This discrepancy is probably due to the copolymer-ized structure of the PCL-based polymer. Several stud-ies confirmed that degradation characteristics were greatly influenced by the chemical modification of polymers (i.e., crosslinking, copolymers) [30–32]. He et al. [31] reported weight loss data of copolymers of γ-butyrolactone and
ε-caprolactone degraded by Pseudomonas lipase and
indi-cated a decreasing degradation rate with increase of the
γ-butyrolactone contents. Darwis et al. [32] reported that
an increment of the cross-linking density which reduced the enzymatic degradation rate of cross-linked PCL sam-ples. Another factor effecting the enzymatic degradation of polymers is the crystallinity of the structure. Previous studies indicated that enzymatic degradation takes place first at amorphous regions, prior to the degradation of the crystalline regions [28]. Cho et al. [28] reported degradation characteristics of PCL/poly(styrene-co-acrylonitrile) (SAN) by Pseudomonas lipase. According to their study, the non-biodegradable SAN content increases at the surface of PCL/ SAN films and prevents the lipase from attacking the bio-degradable PCL chains. Thus, a similar result can be con-cluded for PLina-g-PCL copolymers. PLina units may prob-ably prevent the enzyme attacking amorphous PCL units and lipase could only degrade the crystalline phase slowly. Enzymatic Degradation of PLina-g-PSt-g-PCL
Copolymers
The enzymatic degradation of PLina-g-PSt-g-PCL copol-ymers with different PCL contents was investigated and
Fig. 4 1H NMR spectra of a PLina-g-PCL, b PLina-g-PSt-g-PCL3, c
weight loss data were calculated during the degradation process at various time intervals (Table 2). As demon-strated in Table 2, on the first day of degradation, a similar weight loss was observed for all polymers (PCL,
PLina-g-PCL and PLina-g-PSt-g-PCL copolymers). In 5 days of the incubation period, the degradation rates of
PLina-g-PCL and PLina-g-PSt-g-PCL4 were similar (Fig. 2) and at the 5th day, the weight losses were calculated as 18.2 % and 18.3 %, respectively (Table 2). After 5 days, the degradation rates of PLina-g-PSt-g-PCL4 was reduced compared to PLina-g-PCL and at the end of 30 days, the weight losses of PLina-g-PCL and PLina-g-PSt-g-PCL4 were calculated as 51.5 and 38.4 %, respectively (Table 2). These results suggest and confirm the fact that copolym-erization greatly affects the degradation which may hap-pen due to the change of crystallinity of the structure as discussed before. Moreover, molecular weight of
PLina-g-PCL was remarkably smaller than PLina-g-PSt-g-PCL4 (Fig. 3), which may also affect the degradation behaviors. A sharp decrease of weight losses for PLina-g-PSt-g-PCL3 was determined when compared to PLina-g-PSt-g-PCL4 (Fig. 2). After 30 days, weight losses were calculated as 18.8 and 38.4 % for g-PSt-g-PCL3 and
PLina-g-PSt-g-PCL4, respectively. GPC analysis results indi-cated molecular weight decreased with time (Fig. 3, Table 2). Mn and Mw of degraded PLina-g-PSt-g-PCL3
and PLina-g-PSt-g-PCL4 after 30 days were recorded as
Mn: 52.510 kDa; Mw: 83.220 kDa and Mn: 51.530 kDa;
Mw: 70.60 kDa, respectively (Table 2). The integrals of peaks corresponding to Lina units (0.9 ppm), CL units (4.1 ppm) and St units (6.5–7.1) (Fig. 4b, c) were com-pared and the ratios of [CL3]/[St]/[Lina] and [CL4]/[St]/ [Lina] were calculated and presented in Table 2. [CL3]/ [St]/[Lina] ratios were smaller than [CL4]/[St]/[Lina] which clearly indicates that PLina-g-PSt-g-PCL4 degrades faster than PLina-g-PSt-g-PCL3. Although molecular weights of PLina-g-PSt-g-PCL3 and PLina-g-PSt-g-PCL4 are not very different from each other, the reduction of degradation for PLina-g-PSt-g-PCL3 with low PCL con-tent may be due to the inaccessibility of the amorphous PCL with higher non-biodegradable PSt content, which can prevent Lipase attack to the structure. These results were in accordance with the literature [28]. Cho et al. [28] reported that low PCL content lowers the degradation rates of PCL/SAN blends and suggested that the enzyme could not reach PCL in blends.
Conclusions
This study demonstrated the enzymatic degradation behav-iors of homo PCL- and PCL-based graft copolymers (PLina-g-PCL and PLina-g-PSt-g-PCL copolymers) in the
presence of Pseudomonas lipase. Results from quantita-tive degradation studies with GPC and 1H-NMR analysis
concluded that copolymers degraded considerably more slowly than homo PCL. Moreover, at the end of 30 days, weight loses of PLina-g-PCL, PLina-g-PSt-g-PCL3 and PLina-g-PSt-g-PCL4 copolymers were determined as 51.5, 18.8 and 38.4 %, respectively. These experimental results suggest that copolymers from renewable resources like PLina as well as the composition of copolymerized PCL content play an important role in the enzymatic degrada-tion of PCL-based polymers. However, it is noteworthy that the addition of PSt also prolonged the degradation of the copolymers due to the non-degradable property of PSt. Moreover, in terms of considering weight loss of PCL con-tent during the degradation period (Table 2), copolymers demonstrated slower degradation rates (CL mol% is 2.5 and 1.4 times reduced for PLina-g-PCL and
PLina-g-PSt-g-PCL copolymers, respectively) due to the fact that PLina and PSt may hinder the solubility of the attached PCL unit in the degradation medium. Therefore, it can be concluded that by tailoring these properties (the chemical modifica-tion of polymers, the structure and the composimodifica-tion) gives biomaterials a chance to be used for several applications of tissue engineering. Thus, the copolymers studied here can be evaluated as potential biomaterial candidates under the scope of tissue regeneration. However, it should be noted that in vivo biocompatibility studies should be conducted and in vivo degradation properties of these polymers should be highlighted before launching them as a biomaterials for several uses.
Acknowledgments This work was supported financially by the
Turkish Scientific Research Council (Grants Numbers: 110T884, 211T016) and Bülent Ecevit University Research Fund (Grant Num-ber: 2012-17-21-03).
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