Preparation and characterization of biodegradable and hemocompatible copolymers

Reactive and Functional Polymers 146 (2020) 104373

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Preparation and characterization of biodegradable and hemocompatible copolymers

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Ji Hoon Park, Seung Hun Park, Joon Yeong Park, Hyeon Jin Ju, Yun Bae Ji, Jae Ho Kim, ⁎ Byoung Hyun Min, Moon Suk Kim Department of Molecular Science and Technology, Ajou University, Suwon 443-759, Republic of Korea

A R T I C LE I N FO

A B S T R A C T

Keywords: Polyesters Biodegradation Hemocompatibility Anti-thrombosis Functionalization

In this study, we developed biodegradable polyesters with hemocompatibility and anti-thrombotic functions. First, we synthesized 3-benzyloxymethyl-6-methyl-1,4-dioxane-2,5-dione (LA-Bz). The ring opening polymerization of ε-caprolactone (CL), L-lactide (LA), and LA-Bz monomers provided poly(ε-caprolactone-ran-L-lactide-ran-3-benzyloxymethyl lactide) (PCLA-Bz) copolymers. Poly(ε-caprolactone-ran-L-lactide) (PCLA) was prepared as a control biodegradable copolymer. Subsequent deprotective benzyl reactions of PCLA-Bz and additional reactions with glutaric anhydride yielded PCLA copolymers with COOH pendant groups (PCLACOOH). Afterward, methoxypolyethylene glycol (PCLA-MPEG) or heparin (PCLA-heparin) as an anti-thrombotic group was introduced in the pendant position of PCLA. The in vitro degradation and mechanical properties of PCLA, PCLA-COOH, PCLA-MPEG, and PCLA-heparin were examined over 8 weeks. In the hemocompatibility testing, PCLA-COOH, PCLA-MPEG, and PCLA-heparin exhibited hemocompatibility with little adherence of platelets. In addition, PCLA-heparin exhibited significantly reduced platelet adhesion and enhanced blood stability and thrombin inactivation. These results show that the introduction of anti-thrombotic groups in the pendant position of PCLA represents a useful approach to prepare biodegradable and hemocompatible copolymers.

1. Introduction Stenosis and thrombosis are often the causes of vascular disease, and serious and potentially lethal conditions [1]. Stenosis is usually caused by the accumulation of fat, cholesterol, and other substances over time, while thrombosis refers to the formation of blood clots in blood vessels, mainly by the aggregation of platelets and fibrin. Among several biomedical devices used in the treatment of vascular disease in the early days, bare metal stents (BMSs) were used to constrict stenosis and thrombosis [2], but they caused other side effects such as restenosis and thrombosis [3]. To overcome these, drug-eluting stents have been developed where drugs such as immunosuppressants and cell proliferation inhibitors are coated on the surface of the metal stents [4]. Drug-eluting stents have been well and widely utilized until now, but the metal stents remaining in the body after drug release result in long-term thrombotic problems [5–8]. Recently, the concept of a biodegradable stent, such as Igaki-Tamai stents made of polylactide [9] and REVA biodegradable stents made of polycarbonate [10], has been introduced to overcome the problems associated with metal stents. The aliphatic polyesters of Igaki-Tamai and ⁎

REVA stents are gradually biodegradable in a blood vessel over time [11–13]. Meanwhile, biocompatibility and hemocompatibility can be improved by the functional modification of the stent surface. Mostly, this is easily performed by electric discharge, plasma treatment, or chemical treatment [14–17]. However, the modification of Igaki-Tamai and REVA stents by these methods can induce the deformation of the stent owing to the breaking of the aliphatic polyester segment. However, an aliphatic polyester segment with a functional group in the pendant position as a biomaterial can be easily used for the biocompatible and hemocompatible modification of a biodegradable stent. Numerous anti-thrombotic groups have been introduced to improve biocompatibility and hemocompatibility by introducing hydrophilic or charged groups onto the stent surface. Among these, hydrophilic polyethylene glycol (PEG) group and heparin have become the most successful anti-thrombotic groups and are commercially available [18–23]. Recently, we prepared 3-benzyloxymethyl-6-methyl-1,4-dioxane2,5-dione (LA-Bz) and then poly(ε-caprolactone-ran-L-lactide-ran-3benzyloxymethyl lactide) (PCLA-Bz) copolymers by ring opening

Corresponding author. E-mail address: [email protected] (M.S. Kim).

https://doi.org/10.1016/j.reactfunctpolym.2019.104373 Received 16 July 2019; Received in revised form 24 September 2019; Accepted 27 September 2019 Available online 31 October 2019 1381-5148/ © 2019 Elsevier B.V. All rights reserved.

Reactive and Functional Polymers 146 (2020) 104373

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O

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O CH3O CH2CH2

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Sn(Oct)2, 130oC, 24h

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Fig. 1. Preparation of PCLA, PCLA-Bz, PCLA-OH, PCLA-NH2 PCLA-COOH, PCLA-MPEG, and PCLA-heparin copolymers.

polymerization of ε-caprolactone (CL), L-lactide (LA), and LA-Bz monomers [24]. Among several ratios of CL/[LA + LA-Bz], we chose the molecular ratio of 50 / [45 + 5] in this work because the poly(εcaprolactone-ran-L-lactide) (PCLA) at a CL/LA ratio of 50/50 showed elasticity in the previous studies [25,26]. A benzyloxymethyl group in the pendant position of the PCLA-Bz copolymers can act as functional group to introduce an anti-thrombotic group. Thus, in this work, PCLA-Bz copolymers were derivatized with three anti-thrombotic groups: a carboxylic group (PCLA-COOH) as a single negative-charge group, a heparin group (PCLA-heparin) as a multiple negative-charge group, and a hydrophilic group (methoxypolyethylene glycol: PCLA-MPEG) (Fig. 1). Therefore, the objective of this study was to answer the following questions: (1) Can PCLA copolymers with anti-thrombotic groups at the pendant position be prepared as biodegradable and hemocompatible anti-thrombotic films? (2) Do the prepared PCLA copolymers with anti-thrombotic groups degrade over time? (3) Can the mechanical properties of the prepared PCLA copolymers with anti-thrombotic groups be monitored over time? (4) Which anti-thrombotic groups introduced into the PCLA copolymer provide suitable hemocompatibility? Elucidation of these issues will have an important impact on the feasible development of biodegradable PCLA copolymers with good hemocompatibility and anti-thrombotic functions.

number-average molecular weight of MPEG = 750 g/mol), stannous octoate (Sn(Oct)2), ε-caprolactone (CL), heparin sodium, p-nitrophenyl chloroformate, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC), N-hydroxysuccinimide (NHS), dimethylformamide (DMF), palladium, tetrahydrofuran (THF), diamino butane, Tris buffer, polyethylene glycol (PEO-6000, Mn = 6000 g/mol), bovine serum albumin (BSA), NaCl, glutaric anhydride, acetic acid, trimethylamine (TEA), toluidine blue, anti-thrombin III, and S-2238 thrombin substrate were purchased from Sigma-Aldrich (St. Louis, MO, USA). CL was distilled over CaH2 under reduced pressure, while L-lactide (LA; Boehringer Ingelheim, Blanquefort, France) was recrystallized twice in ethyl acetate. Sodium citrate buffer was purchased from Yakuri Pure Chemicals (Kyoto, Japan) and single donor human whole blood was purchased from Innovative Research (Novi, MI, USA).

2.2. Characterization Proton nuclear magnetic resonance (1HNMR) spectra of the PCLA, PCLA-Bz, PCLA-COOH, PCLA-MPEG, and PCLA-heparin copolymers were measured using a Varian Mercury Plus 400 system, with chloroform (CDCl3) as an internal standard in the presence of tetramethylsilane (TMS). The molecular weight distribution of each copolymer was measured using a YL-Clarity gel permeation chromatography (GPC) system (YL9170 RI detector) with three columns (Shodex K-802, K-803, and K-804 polystyrene gel columns) at 40 °C, with calibration using polystyrene and using CHCl3 as an eluent at a flow rate of 1.0 mL/ min.

2. Materials and methods 2.1. Materials Methoxypolyethylene glycol (MPEG) and MPEG-NH2 (with a 2

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2.3. Synthesis of prepared poly (ε-caprolactone-ran-L-lactide) (PCLA) copolymer

resulting solution was concentrated by rotary evaporation and dried under a vacuum.

The PCLA diblock copolymer (Mn = 50,000 g/mol) with a CL/LA ratio of 50/50 was prepared as reported previously [26].

2.8. Synthesis of the PCLA-NH2 copolymer The process to yield the PCLA-NH2 copolymer was as follows. PCLAOH (2 g, 0.00042 mmol: OH concentration) was dissolved in toluene (60 mL) and introduced into a flask. The solution of the PCLA-OH copolymer was distilled azeotropically to remove the included water, and toluene was then distilled off to give a final volume of 30 mL. 4Nitrophenyl chloroformate (0.061 g, 0.335 mmol) and TEA (0.1 mL) were added to the PCLA-OH solution and stirred for 24 h at room temperature under nitrogen. The reaction mixture was filtered and washed with toluene, followed by pouring into a mixture of n-hexane and ethyl ether (v/v = 4/1) to precipitate the copolymer, which was then separated from the supernatant by decantation. The obtained copolymer was dissolved in CH2Cl2 and filtered. The copolymer solution was concentrated by a rotary evaporator and dried in vacuo to give a PCLA-OH copolymer with an oxy-nitrophenoxy end group. Diamino butane (2.31 g, 3.57 mmol) and THF (20 mL) were added to the PCLAOH copolymer with the oxy-nitrophenoxy group in THF (35 mL), and the solution was stirred for 24 h at room temperature under nitrogen. The obtained polymer was dissolved in CH2Cl2 and then filtered. The copolymer solution was concentrated using a rotary evaporator and dried in a vacuum to give PCLA-NH2. Element analysis; calculated values: C, 58.8; H, 8.8; N, 1.5; measured values: C, 59.6; H, 8.9; N, 1.3.

2.4. Synthesis of prepared poly (ε-caprolactone-ran-L-lactide-ran-3benzyloxymethyl lactide) (PCLA-Bz) copolymers To prepare the PCLA-Bz copolymers (Mn = 50,000 g/mol), all glassware was dried by heating in a vacuum and was handled under a dry nitrogen stream. 3-Benzyloxymethyl-6-methyl-1,4-dioxane-2,5dione (LA-Bz) was prepared using a previously reported method [18]. The polymerization procedure of the PCLA-Bz copolymer with a ratio of the PCL/PLA/PLA-Bz of 50/45/5 was as follows. The solution of MPEG (0.012 g, 0.16 mmol) and toluene (150 mL) was distilled azeotropically to remove the included water in the original MPEG, after which around 50 mL of toluene was distilled off. CL (4.25 g, 2.95 mmol), LA (3.05 g, 2.67 mmol), and LA-Bz (0.745 g, 0.298 mmol) were first added, followed by 0.1 mL of a 0.1 M solution of Sn(Oct)2 in dried toluene, to the MPEG solution under a nitrogen atmosphere at room temperature. After polymerization for 48 h at 130 °C, the reaction solution was poured into a mixture of n-hexane and ethyl ether (v/v = 4/1) to precipitate the PCLA-Bz copolymer of 500 k (g/mol); this was filtered and dried under vacuum to yield a slight yellow copolymer. A 1H NMR spectrum of the prepared PCLA-Bz copolymer was used to determine the molecular weight and ratio of the PCL, PLA, and PLABz segments by comparing the total methylene protons in PCL at δ = 2.3 ppm, the methane proton signal of PLA at δ = 5.2 ppm, and the phenyl proton signals of PLA-Bz at δ = 7.2–7.4 ppm with the total methyl protons in MPEG at δ = 3.38 ppm as a standard at 750 g/mol.

2.9. Synthesis of the PCLA-heparin copolymer The PCLA-NH2 copolymer (1 g, 0.00026 mmol: NH2 concentration), EDC (0.05 g, 0.336 mmol) and NHS (0.038 g, 0.336 mmol) were added to a heparin sodium (500 mg)/DMF solution (30 mL). After the reaction mixture was stirred at room temperature for 24 h, it was poured into a mixture of ethyl ether and n-hexane (v/v = 1/1) to precipitate the polymer, which was separated from the supernatant by decantation, dissolved in CH2Cl2, and filtered. The resulting solution was concentrated by rotary evaporation and dried under vacuum. Element analysis; measured values for heparin: C, 20.4; H, 3.8; N, 1.8; S, 9.9; for the PCLA-heparin copolymer: C, 50.5; H, 5.6; N, 0.7; S, 3.9.

2.5. Synthesis of the PCLA-OH copolymer The process to yield the PCLA-OH copolymer was as follows. PCLABz copolymer (6 g, 0.00168 mmol: benzyl concentration) was dissolved in anhydrous THF (200 mL), and then 10% w/w (1.86 g) of Pd/C (Palladium, 10 wt% dry basis on activated carbon 50% water w/w, Degussa type E101 NE/W) was added. The suspension was stirred under a hydrogen atmosphere for 16 h. The catalyst was removed by filtration over a Celite filter. The copolymer solution was concentrated by rotary evaporation and dried in a vacuum.

2.10. Preparation of the PCLA, PCLA-COOH, PCLA-MPEG, and PCLAheparin films The copolymer films were prepared using solvent casting. The PCLA, PCLA-COOH, PCLA-MPEG, and PCLA-heparin copolymers were dissolved at a concentration of 20 wt% in CH2Cl2. The solutions were cast onto polyethylene films and allowed to dry slowly at 10 °C for 4 days. The copolymer films were then dried in a vacuum oven at room temperature for 2 days. The copolymer films were cut into disks with a diameter of 16 mm and a thickness of 200 μm for in vitro degradation testing, hemolysis analysis, anti-platelet adhesion, and thrombin deactivation analysis [27].

2.6. Synthesis of the PCLA-COOH copolymer PCLA-OH (2 g, 0.00042 mmol: OH concentration) was dissolved in toluene (60 mL) and introduced into a flask. The solution of the PCLAOH copolymer was distilled azeotropically to remove the included water, and toluene was then distilled off to give a final volume of 30 mL. Glutaric anhydride (5.74 mg, 0.0504 mmol) and acetic acid (1.5 mL) were added to the PCLA-OH solution, and the mixture was stirred at 110 °C for 24 h. The reaction mixture was poured into a mixture of n-hexane, ethyl ether, and methanol (v/v/v = 8/1.5/0.5) to precipitate the copolymer, which was separated from the supernatant by decantation, dissolved in CH2Cl2, and filtered. The resulting solution was concentrated by rotary evaporation and dried under a vacuum.

2.11. In vitro degradation testing Each disk (diameter of 16 mm and thickness of 200 μm) was immersed in 10 mL of 1× phosphate-buffered saline (PBS, pH = 7.4) in a vial (20 mL) and incubated at 37 °C with shaking at 100 rpm for 1 day and 2, 4, 6, and 8 weeks. At the predetermined time intervals, vials were retrieved and freeze-dried for 3 days, and immediately afterward, the number-average molecular weight change (%) was measured using the GPC system. The extent of relative degradation of the copolymers was calculated by the ratio of molecular weights determined using the GPC system during the experimental period and on the initial day. The in vitro half-life degradation of the copolymers was determined as the time taken to degrade the molecular weight to half of the original

2.7. Synthesis of the PCLA-MPEG copolymer The PCLA-COOH copolymer (1 g, 0.00028 mmol: COOH concentration), EDC (0.05 g, 0.336 mmol), and NHS (0.038 g, 0.336 mmol) were added to an MPEG-NH2 (16.8 mg, 0.0244 mmol)/MC solution (50 mL). After the reaction mixture was stirred at room temperature for 24 h, it was poured into a mixture of ethyl ether and n-hexane (v/ v = 1/1) to precipitate the polymer, which was separated from the supernatant by decantation, dissolved in CH2Cl2, and filtered. The 3

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Fig. 2. 1H NMR spectra of PCLA-Bz, PCLA-OH and PCLA-NH2 copolymers. TMS (tetramethylsilane).

citrate (3.8%) to prevent clotting. To prepare platelet-rich plasma, the blood was centrifuged at 1200 rpm for 5 min, after which the supernatant was collected and centrifuged at 3500 rpm for 10 min. The collected platelet-rich plasma was used in the following experiment.

molecular weight. 2.12. Mechanical testing of the PCLA copolymer films under in vitro degradation Films of PCLA, PCLA-COOH, PCLA-MPEG, and PCLA-heparin were prepared in a dog-bone shape. The specimens were made with a thickness of 0.5 mm, a total area of 15 × 50 mm2, and a middle section of 5 × 10 mm2. In vitro degradation was allowed to proceed for predefined times (1 day and 2, 4, 6, and 8 weeks) before measuring the mechanical properties with a Universal Testing Machine (H5KT, Tinius Olsen, Horsham, PA, USA) at a cross-head speed in the vertical direction of 3 mm/min strain velocity at room temperature by using 50 N load cells until the specimen broke.

2.14. Hemolysis analysis Hemolysis experiments were performed to confirm the blood stability of the PCLA, PCLA-COOH, PCLA-MPEG, and PCLA-heparin copolymers. The experiment was divided into the following three groups. The experimental group comprised the PCLA, PCLA-COOH, PCLAMPEG, and PCLA-heparin copolymers prepared in 10 mL NaCl, the (−) control group was 20 mL NaCl, and the (+) control group was 10 mL NaCl +10 mL distilled water. NaCl was used for erythrocyte hemolysis by osmotic pressure. After 30 min of reaction at room temperature, 0.2 mL of platelet-rich plasma was added to each group, which were then allowed to react for a further 60 min. After centrifugation at 2000 rpm for 5 min, 0.2 mL of supernatant was taken and its absorbance

2.13. Preparation of platelets Single donor human whole blood was added with 10% sodium 4

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Fig. 3. 1H NMR spectra of the PCLA-COOH, PCLA-MPEG, and PCLA-heparin copolymers. TMS (tetramethylsilane).

Fig. 4. In vitro degradation properties of the PCLA, PCLA-COOH, PCLA-MPEG, and PCLA-heparin copolymers: (a) gel permeation chromatography (GPC) curves after degradation up to 8 weeks and (b) plots of degradation ratio versus time.

70 mU/mL were mixed in Tris buffer (0.5 M, pH 8.4), 1 g/L PEO-6000, 1 g/L BSA, and 150 mM NaCl. The PCLA-heparin was then treated by mixture solution. Thrombin (150 μL, 1.2 U/mL) was then added and the reaction was allowed to proceed at room temperature for 10 min before being terminated by the addition of 50 μL of acetic acid (20%). The absorbance of a 200-μL sample of the reaction solution was determined at 405 nm using a spectrometer.

measured at 540 nm using a multi-plate reader. 2.15. Anti-platelet adhesion Platelets (1 × 105) were applied to heparinized copolymer films and reacted at room temperature for 3 h. The specimens were fixed with 2.5% glutaraldehyde for 2 h and then dehydrated sequentially with 40, 60, 80, and 90% ethanol. Optical images of the platelets on the copolymer films were observed using an Eclipse TS 100 microscope (Nikon, Japan).

2.17. Toluidine blue assay The stability of PCLA-heparin was analyzed by determining the amount of heparin derivative using a toluidine blue assay. For the stability testing, each PCLA-heparin was immersed in 2 mL of PBS solution and incubated at 37 °C with shaking at 100 rpm. At the

2.16. Thrombin deactivation analysis S-2238 (a thrombin substrate) at 0.2 mg/mL and anti-thrombin III at 5

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Fig. 5. Mechanical properties of the (a) PCLA, (b) PCLA-COOH, (c) PCLA-MPEG, and (d) PCLA-heparin films tested from 1 day (d) to 8 weeks (w).

2.18. Statistical analysis Hemolysis and thrombin deactivation analyses values for the PCLA, PCLA-COOH, PCLA-MPEG, and PCLA-heparin films were recorded in independent experiments (n = 4 for each data point). The results were analyzed via one-way analysis of variance (ANOVA) using the Prism 3.0 software package (GraphPad Software Inc., San Diego, CA, USA); pvalues less than 0.01 were considered statistically significant. 3. Results and discussion 3.1. Preparation of the PCLA-COOH, PCLA-MPEG, and PCLA-heparin copolymers First, an LA-Bz monomer having a benzyloxymethyl group was synthesized by using O-benzyl-L-serine. Next, copolymerization at a ratio of CL/LA/LA-Bz of 50/45/5 was performed using MPEG as an initiator in the presence of Sn(Oct)2 at 130 °C for 24 h. Slightly yellow copolymers in an almost quantitative yield were obtained after precipitation. The PCLA-Bz copolymer exhibited characteristic 1H NMR peaks of PCL, PLA, and PLA-Bz (Fig. 2) and its molecular weight (Mn = 50,000 g/mol) and ratio determined from a comparison of the proton signals were in agreement with those calculated from the feed ratio of CL (2.3 ppm), LA (5.2 ppm), and LA-Bz (7.2–7.4 ppm) to MPEG. PCLA-OH was prepared by the quantitative deprotection of the benzyl moiety of PCLA-Bz using Pd/C. Glutaric anhydride and diamino butane were introduced into PCLAOH to give PCLA-COOH and PCLA-NH2, with a carboxylic acid group and an amine group in the pendant position, respectively, in quantitative yield. Finally, an MPEG-NH2 was introduced into PCLA-COOH to give PCLA-MPEG and a heparin was introduced into PCLA-NH2 to give PCLA-heparin. The structures of both were confirmed by a 1H NMR (Fig. 3) and elemental analysis.

Fig. 6. Hemocompatibility of the BMS, PCLA, PCLA-COOH, PCLA-MPEG, and PCLA-heparin copolymers determined using hemolysis testing.

predefined time points, each specimen was removed from the PBS solution, and the amount of heparin derivative remaining on the PCLAheparin copolymer was determined using a toluidine blue colorimetric method. Samples were placed in 5 mL vials and dissolved in 1 mL of DMF, after which 0.5 mL of toluidine blue solution (0.01 M) was added and the samples were incubated at room temperature for 30 min. Next, 3 mL of n-hexane was added and incubation was continued for 20 min. After phase separation, the optical density of the aqueous phase was measured using a multi-plate reader at a wavelength of 630 nm. The amount of heparin-toluidine blue complex was calculated from a standard curve prepared using known concentrations of heparin.

6

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Fig. 7. PCLA, PCLA-COOH, PCLA-MPEG, and PCLA-heparin copolymer films without (a) and with (b) platelet adhesion during in vitro degradation (scale bars represent 200 μm).

degradation, showing the fastest decomposition compared with other copolymers. The degradation of the copolymers exhibited a dependence on the hydrophilicity of the pendant group. These results show that although the degradation rates of the PCLA, PCLA-COOH, PCLA-MPEG, and PCLA-heparin were slightly different, they would be degraded over time by the biological activity in the blood. 3.3. Mechanical properties of the PCLA, PCLA-COOH, PCLA-MPEG, and PCLA-heparin copolymers A biodegradable stent is required to maintain its mechanical properties in the blood vessels until the blood vessels' function recovers, thus the stress-strain of the PCLA, PCLA-COOH, PCLA-MPEG, and PCLAheparin copolymers were measured to compare their mechanical properties (Fig. 5). The tensile strength decreased gradually with the degree of degradation and showed a similar tendency to decrease as the in vitro degradation results. In all groups, the tensile strength was maintained for 6 weeks but reduced significantly after 8 weeks regardless of the decrease in molecular weight. This reduction in tensile strength appears to have been caused by the formation of cracks and pores in the specimens after 8 weeks in PBS. The PCLA-heparin group, whose molecular weight decreased by 50% when degraded for 8 weeks, retained 80% of its initial tensile strength when strained to 20%. These results show that the PCLA, PCLA-COOH, PCLA-MPEG, and PCLA-heparin copolymers are biodegradable but retain their mechanical properties for up to 6 weeks and can withstand 20% deformation after 8 weeks. Collectively, the copolymers prepared in this work would be expected to maintain their mechanical properties in a blood vessel for more than 6 weeks.

Fig. 8. Anticoagulant activity of the PCLA-heparin copolymer during in vitro degradation.

3.2. In vitro degradation of the PCLA, PCLA-COOH, PCLA-MPEG, and PCLA-heparin copolymers Biodegradation is necessary to evaluate a biodegradable stent remaining in the body. Thus, the in vitro degradation behavior of the PCLA, PCLA-COOH, PCLA-MPEG, and PCLA-heparin films was examined in PBS at 37 °C. After 8 weeks of immersion in PBS, we determined their molecular weights using GPC. Fig. 4 shows the GPCdetermined change in molecular weight of the PCLA, PCLA-COOH, PCLA-MPEG, and PCLA-heparin films during in vitro degradation for 8 weeks. The degradation of the copolymers caused a molecular weight decrease, which resulted in a gradual increase in the retention time through the GPC column. Fig. 4b shows the in vitro degradation ratio of the copolymers. The molecular weight on day 0 (before degradation) was 100%. The rate of degradation was PCLA-heparin > PCLAMPEG > PCLA-COOH > PCLA. In the case of the PCLA-heparin copolymer, its molecular weight decreased by 50% during 8 weeks of in vitro

3.4. Hemocompatibility testing of the PCLA, PCLA-COOH, PCLA-MPEG, and PCLA-heparin copolymers Hemolysis was evaluated to examine the hemocompatibility of the PCLA, PCLA-COOH, PCLA-MPEG, and PCLA-heparin copolymers, with a BMS used as a control. Fig. 6 shows that the BMS induced more than 13% hemolysis. Meanwhile, PCLA, PCLA-COOH, and PCLA-MPEG 7

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Fig. 9. Toluidine blue assay of the PCLA-heparin copolymer during in vitro degradation: (a) changing of the heparin-toluidine blue complex color and (b) a graph showing the loss of heparin from the PCLA-heparin copolymer calculated using a heparin standard curve. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.6. Thrombin inactivation properties of the PCLA and PCLA-heparin copolymers

induced approximately 8% hemolysis, indicating that they are more hemocompatible than the BMS. Differences in the COOH negative charge and the hydrophilic MPEG side chain appear to have little effect on hemolysis. However, PCLA-heparin with multiple negative charges showed high hemocompatibility, indicated by a hemolysis score of less than 3%. Even though the biodegradation occurred over time and became lower, the PCLA-heparin had the highest hemocompatibility compared with the other groups during the 8 weeks of in vitro degradation, showing that it had higher hemocompatibility than PCLA, PCLA-COOH, and PCLA-MPEG. Collectively, it appears that the anticoagulant property of heparin on PCLA-heparin reduced the hemolysis of PCLA through stabilization of the erythrocytes.

Because PCLA-heparin exhibited high hemocompatibility and almost no adherence of platelets in previous results, we chose it for the next anticoagulant experiment and used PCLA and BMS as the control. Fig. 8 shows the degree of activation of anticoagulation of BMS, PCLA, and PCLA-heparin over time. The PCLA-heparin copolymer showed anticoagulant activity that gradually decreased as a function of incubation time and remained after 8 weeks. By contrast, the BMS and PCLA without heparin showed no anticoagulant activity during the 8 weeks. The blood clotting process begins with the conversion of prothrombin to thrombin. Anti-thrombin III inhibits this process and heparin is known to increase the activity of anti-thrombin III. Thus, we confirmed that the PCLA-heparin copolymer could inactivate thrombin during blood coagulation.

3.5. Platelet adhesion of the PCLA, PCLA-COOH, PCLA-MPEG, and PCLAheparin copolymers

3.7. Toluidine blue assay of the PCLA-heparin copolymer Thrombosis occurs when biomaterial comes into contact with human blood, which results in plasma proteins being adsorbed and platelets aggregating, and inhibiting platelets from adhering to the surface of the stent would help to prevent thrombosis. Thus, the PCLA, PCLA-COOH, PCLA-MPEG, and PCLA-heparin films were treated with platelet-rich plasma and we observed the adherence of platelets over 8 weeks. The PCLA, PCLA-COOH, PCLA-MPEG, and PCLA-heparin films showed smooth surfaces in SEM. Meanwhile, a large number of platelets were observed on the PCLA film (Fig. 7 and Supporting Information Fig. S1–4). There were little or no platelets at day 1 on all films. PCLA showed a few platelets at 2 weeks but a large number of platelets were observed from 4 weeks. The adhered platelets on the PCLA-COOH films were only observed after 6 weeks and the films showed significantly fewer platelets compared with PCLA. This result indicates that the negative charge on the surface reduced the adhesion of plasma proteins and platelets. The PCLA-MPEG films showed only a few small platelets even at 8 weeks, which might reflect the steric stabilization and chain mobility effects of the hydrophilic MPEG chain. The PCLA-heparin films did not attract platelets until 8 weeks because of the multiple negative charges of heparin and its anticoagulation function. The results show that PCLA copolymer with antithrombotic groups of COOH, MPEG, and heparin could function as antiplatelet adhesion and anti-thrombotic factors. Thus, the introduction of these anti-thrombotic groups into the PCLA copolymers could prevent thrombosis by inhibiting the adherence of plasma proteins and platelets on the PCLA copolymer films.

For the PCLA-heparin copolymer to be continuously anti-thrombotic in blood vessels, the introduced anti-thrombotic heparin factor should be stably bound and the concentration of the heparin should be maintained constantly. To confirm the stability of heparin introduced into the PCLA-heparin copolymer, the residual amount of heparin in the PCLA-heparin copolymer over time was confirmed using the toluidine blue assay (Fig. 9). At day 1, the PCLA-heparin copolymer contained around 4 wt% heparin, which decreased gradually as a function of incubation time and reached around 50% of the initial content after 8 weeks. The amount of remaining heparin in the copolymer varied according to the biodegradation rate of PCLA. Because the heparin was immobilized chemically on the PCLA copolymer, the decrease in heparin was probably affected by the biodegradation of the polyester PCLA segment in the PCLA-heparin copolymer. These results suggest that the anti-thrombotic factor introduced into the PCLA was comparatively stable and would exhibit its anti-thrombotic properties for more than 8 weeks. 4. Conclusions In the present study, we prepared PCLA copolymers with antithrombotic groups to investigate their feasibility as biodegradable antithrombotic films. These copolymers exhibited gradual biodegradability, maintained good mechanical properties during biodegradation, and exhibited hemocompatibility. Especially, the PCLA-heparin copolymer exhibited heparin-related functions such as high hemocompatibility, inactivation of thrombin, and inhibition of platelet adsorption. These 8

Reactive and Functional Polymers 146 (2020) 104373

J.H. Park, et al.

results indicated that PCLA copolymers with anti-thrombotic groups could serve as suitable stent materials as well as vascular implant materials, and further experiments are currently underway to investigate the efficacy of the PCLA copolymers with anti-thrombotic groups in animal models.

[5] [6] [7] [8]

Declaration of Competing Interest

[9]

The authors declare no competing interests.

[10] [11] [12]

Acknowledgment

[13]

This study was supported by a grant from Creative Materials Discovery Program through the National Research Foundation (2019M3D1A1078938), Priority Research Centers Program (2019R1A6A1A11051471) funded by the National Research Foundation of Korea (NRF) and from the Korea Health Technology R&D Project (HI17C2191) through the Korea Health Industry Development Institute funded by the Ministry of Health & Welfare.

[14] [15] [16] [17] [18] [19] [20] [21]

Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.reactfunctpolym.2019.104373.

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