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Monoether-tagged Biodegradable Polycarbonate Preventing Platelet Adhesion and Demonstrating Vascular Cell Adhesion: A Promising Material for Resorbable Vascular Grafts and Stents Kazuki Fukushima, Yuto Inoue, Yuta Haga, Takayuki Ota, Kota Honda, Chikako Sato, and Masaru Tanaka Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01210 • Publication Date (Web): 03 Oct 2017 Downloaded from http://pubs.acs.org on October 10, 2017
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Monoether-tagged Biodegradable Polycarbonate Preventing Platelet Adhesion and Demonstrating Vascular Cell Adhesion: A Promising Material for Resorbable Vascular Grafts and Stents Kazuki Fukushima,*,†,‡,‖ Yuto Inoue,‡ Yuta Haga,† Takayuki Ota,‡ Kota Honda,‡ Chikako Sato,‡ Masaru Tanaka,*§,‖ †
Graduate School of Organic Materials Science, Yamagata University, 4-3-16 Jonan, Yonezawa,
Yamagata 992-8510, Japan ‡
Graduate School of Science and Engineering, Yamagata University, Yamagata 992-8510, Japan
§
Institute for Materials Chemistry and Engineering, Kyushu University, 6-1 Kasugakoen,
Kasuga, Fukuoka 816-8580, Japan ‖
Frontier Center for Organic Materials, Yamagata University, Yamagata 992-8510, Japan
KEYWORDS. blood compatible; biodegradable polymer; functional biomaterial; platelet adhesion; polycarbonate
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ABSTRACT.
We
developed
a
biodegradable
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polycarbonate
that
demonstrates
antithrombogenicity and vascular cell adhesion via organocatalytic ring-opening polymerization of a trimethylene carbonate (TMC) analog bearing a methoxy group. The monoether-tagged polycarbonate demonstrates a platelet adhesion property, 93% and 89% lower than poly(ethylene terephthalate) and polyTMC, respectively. In contrast, vascular cell adhesion properties of the polycarbonate are comparable to those controls, indicating a potential for selective cell adhesion properties. This difference in the cell adhesion property is well associated with surface hydration, which affects protein adsorption and denaturation. Fibrinogen is slightly denatured on the monoether-tagged polycarbonate, whereas fibronectin is highly activated to expose the RGD motif for favorable vascular cell adhesion. The surface hydration, mainly induced by the methoxy side chain, also contributes in slowing the enzymatic degradation. Consequently, the polycarbonate exhibits decent blood compatibility, vascular cell adhesion properties, and biodegradability, which is promising for applications in resorbable vascular grafts and stents.
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1. INTRODUCTION Aliphatic polycarbonates, represented by poly(trimethylene carbonate) (PTMC), have recently attracted increasing attention as a soft biomaterial owing to their unique biodegradation behavior including no acid generation and surface erosion.1 Many PTMC-based biomaterials have recently been investigated for applications in a broad range of implantable and/or resorbable devices. Chemically functionalized PTMC analogs with different pendant groups are derived from their corresponding cyclic monomers, demonstrating a diverse alteration of physicochemical properties.2-6 However, most of these analogs have been designed to be used as solutions, such as in drug/gene delivery, hydrogels, and antimicrobials.7-12 In contrast, composites of PTMC and copolymers including trimethylene carbonate (TMC), have been studied for bulk applications such as scaffolds for bone, cartilage, and nerve regeneration.13-15 In addition, applications in blood vessels and as vascular stents are a targeted area of PTMC-based materials, because the inherent flexible property of PTMC is well suited for the required task.16-18 However, as with other hydrophobic biodegradable polymers that require concomitant administration of anticoagulants and antiplatelets for applications in blood contacting devices, blood compatibility, in particular ability to suppress platelet adhesion, is indispensable for the PTMC-based materials. We have previously reported that no synthetic biodegradable polymers possessing high antithrombotic properties have been developed.19 A few (meth)acrylate polymers exhibit excellent blood compatibility. The most well-known of these is poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC).20, 21 which demonstrates high bioinert properties and does not interact with proteins and cells such as platelets. Since PMPC is water-soluble, a copolymer containing 70 mol% of butyl methacrylate called PMB is widely used in bulk applications and surface modification of medical devices such as conventional
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artificial blood vessels and artificial joints.22-25 Furthermore, phosphorylcholine (PC) side chains appear to be promising in improving blood compatibility, and polyurethanes incorporating the PC side chains also demonstrate excellent blood compatibility.26, 27 However, due to technical issues, biodegradable polyesters and polycarbonates with the PC side chains are yet to be reported. Alternatively, several efforts have been made to tag bioinert oligo- or poly(ethylene glycol) (OEG, PEG) to PTMC and biodegradable polyester platforms.28-33 Most of the attempts were to develop biodegradable PEG alternatives and thermoresponsive polymers inspired by the OEG/PEG-tagged (meth)acrylate polymers.34-36. However, their platelet adhesion property has never been discussed. Poly(2-methoxyethyl acrylate) (PMEA), which has the fewest ethylene glycol side chain units, is also known as a blood compatible polymer that exhibits little platelet adhesion.37, 38 PMEA is insoluble in water and is currently used as a coating in oxygenators. In this study, we designed a PTMC analog that incorporated the 2-methoxyethoxycarbonyl group side chain structure of PMEA. Several approaches to prepare PTMC analogs with the alkoxy carbonyl side chains from 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) have been reported.39-42 We chose this side chain structure, because the cyclic carbonate with the 2methoxyethoxy carbonyl group (ME-MTC) is obtained in two steps, starting with the Fischer esterification and followed by ring-closing (Scheme 1). For artificial blood vessels that also serve as resorbable scaffolds for vascular tissue regeneration, vascular cell adhesion and proliferation on the materials are desirable. Recently, PMEA has been found to show a high affinity for vascular endothelial cell adhesion.43 This is another reason why we decided to synthesize the monoether-tagged PTMC analog, named as PMEMTC. Although artificial blood vessels pragmatically require various physicochemical properties, combining antithrombotic properties,
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vascular cell adhesion properties, and biodegradability into a single polymer would be great significance to the fundamentals of biomaterials science. Thus, we describe the synthesis, platelet adhesion, vascular cell adhesion, and enzymatic degradation of PMEMTC below.
Scheme 1. Outline of Synthesis and Biological Evaluation of a Monoether-tagged Polycarbonate
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2. EXPERIMENTAL SECTION 2.1. Materials and Methods. 2,2-bis(methylol)propionic acid (bis-MPA), 2-methoxyethanol, Amberyst-15®, 1-pyrenebutanol (PB), and lipase (≥ 100,000 U/g, from Thermomyces lanuginosus) were purchased from Sigma-Aldrich Japan (Tokyo, Japan).
Trimethylene
carbonate (TMC), (+)-sparteine (SP), and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) were purchased from Kanto Chemical (Tokyo, Japan). Triphosgene was purchased from Tokyo Chemical Industry (Tokyo, Japan). Dehydrated THF and CH2Cl2 (water content < 10 ppm) were supplied by a solvent supply system (Kanto Chemical). 1-(3,5-bis(trifluoromethyl)phenyl)-3cyclohexyl-2-thiourea (TU) was prepared as reported previously.44 DBU and SP were vacuum distilled over CaH2 and stored in a nitrogen-filled glove box. PB and TU were also dehydrated over CaH2 and stored in the glove box. Other chemicals were purchased from any of the distributors above and used as received unless otherwise stated. Phosphate buffered saline (PBS; pH 7.4) was prepared by dissolving 10 tablets of phosphate buffer salts (Takara Bio, Tokyo, Japan) in 1 L of ultrapure water. Glutaraldehyde solution (1%) was prepared by diluting 25% glutaraldehyde aqueous solution (Wako Pure Chemical) with PBS, as mentioned above. 1
H and 13C-NMR spectra were acquired on a JEOL 500 MHz JNM-ECX, operated at 500 and
125 MHz, respectively. Size exclusion chromatography (SEC) in THF was performed using an integrated SEC unit of Tosoh HLC-8220 chromatograph equipped with three TSK-gel columns connected in series (super AW5000, super AW4000, and super AW3000) and a refractive index (RI) detector at 30 ºC. It was calibrated with polystyrene standards (2500 to 1.1 × 106 g/mol) to obtain molecular weight and polydispersity. 2.2. Polymer Synthesis.
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2.2.1. Synthesis of 2-methoxyethyl-5-methyl-2-oxo-1,3-dioxane-5-carboxylate (ME-MTC). The monomer ME-MTC was synthesized according to previously reported procedure45 that was slightly modified. Briefly, a mixture of bis-MPA (45.0 g; 0.336 mol) and 2-methoxyethanol (225 ml; 2.87 mol) was refluxed at 90 °C for 45 h under a nitrogen atmosphere in the presence of Amberyst-15® (9.00 g) for Fischer esterification. After the resins were removed through cerite with a glass filter, the filtrate was evaporated. The residue was purified by column chromatography using gradient elution of a mixture of ethyl acetate and hexane (7 : 3 to 10 : 0) to provide 2-methoxyethyl 2,2-bis(hydroxymethyl)propionate (ME-MPA; 35.7 g; yield 53.8%). Next, the ME-MPA (25.1 g; 0.131 mol) was subjected to carbonylative ring-closing using triphosgene (19.5 g; 65.5 mmol) and pyridine (63.5 ml; 0.787 mol) in CH2Cl2 (400 ml) at −75 °C. The mixture was allowed to warm up to room temperature and stirred for 2 h. Afterward, a sat. NH4Cl aq. was added, and the organic layer was washed twice with a 1N HCl aq. (200 ml), followed by sat. NaHCO3 aq. (200 ml), brine (200 ml), and deionized water (200 ml). After being dried over MgSO4, the organic layer was evaporated and dried in vacuum at room temperature. The residue was purified via column chromatography using ethyl acetate as an eluent to give ME-MTC (11.0 g; yield 38.6%). 1H NMR (500MHz, CDCl3) : δ 4.68 (d, J = 11 Hz, 2H, CHaHbOCOO), 4.32 (t, J = 9.5 Hz, 2H, CH2CH2OCH3), 4.20 (d, J = 11 Hz, 2H, CHaHbOCOO), 3.57 (t, J = 4.8 Hz, 2H, CH2OCH3,), 3.33 (s, 3H, OCH3), 1.31 (s, 3H, CH3). 13
C{1H} NMR (125MHz, CDCl3) : δ 171.2, 147.6, 73.0, 70.1, 65.0, 59.0, 40.3, 17.6. 2.2.2. Typical ring-opening polymerization (ROP) of ME-MTC. In a nitrogen filled glove box,
ME-MTC (0.456 g; 2.09 mmol), PB (5.7 mg; 0.021 mmol), TU (7.8 mg; 0.021 mmol) and DBU (3.4 mg; 0.022 mmol) were stirred in dry CH2Cl2 (1.0 ml) at room temperature for 60 mins. After more than 95% monomer conversion was confirmed by 1H-NMR, a few drops of acetic
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anhydride (Ac2O) was added for termination and end-capping. The solution was then reprecipitated into cold 2-propanol at least twice, and the precipitates were collected via centrifugation and dried in vacuum to give a glassy solid (0.350 g; yield 76.7%). SEC: Mn 6,300 g/mol, ÐM 1.18. 1H-NMR (500MHz, CDCl3) : δ 4.30 (m, 6H, COOCH2), 3.58 (t, J = 4.8 Hz, 2H, CH2OCH3), 3.36 (s, 3H, OCH3), 1.27 (s, 3H, CH3). 2.3. Differential Scanning Calorimetry (DSC) Measurement of Polymers.
DSC
measurements were performed using a Hitachi High-Tech Science X-DSC7000 with a ramp of 5 °C/min under a nitrogen atmosphere. 3 to 5 mg of specimen was used. Hydrated samples were prepared by immersing in ultrapure water for at least 24 h before measurement. It was confirmed that there was no weight loss during the measurement. 2.4. Preparation of Polymer-Coated Substrates. The synthesized polymers were immersed in ultrapure water for 24 h to elute out water soluble impurities, as they may induce unwanted biological responses to cells and proteins. The polymers were dissolved in chloroform (0.2 wt/v%) and spin-coated on a PET sheet (φ 14 mm; thickness 125 µm; Mitsubishi Plastics, Tokyo, Japan). This protocol was repeated one more time. The coated substrates were dried overnight in air, followed by drying overnight in vacuo. Previously, it was confirmed that the surface of the PET was fully covered by the polymers with thickness of around 100 nm under this condition.46, 47 The coverage of the spin-coated polymers was also supported by changes in static contact angles as described below. PMEA, poly(2-methoxyethyl methacrylate) (PMEMA), and PMB were used as control polymers along with PTMC and PET. PMEA (Mn 22,000 g/mol, ÐM 2.8) and PMEMA (Mn 120,000 g/mol, ÐM 4.8) were synthesized by ordinary radical polymerization of 2-methoxyethyl acrylate and 2-methoxyethyl methacrylate using 2,2'azobisisobutyronitrile (AIBN) at 70 °C as described in another study.38 PMB (Mn 600,000 g/mol,
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ÐM 2.4) was provided by NOF (Tokyo, Japan). PTMC was synthesized by ROP of TMC using TU and DBU as previously reported in another study (Mn 22,000 g/mol, ÐM 1.10, also see Supporting Information).48 2.5. Static Contact Angle Measurement. For the prepared polymer substrates, static contact angles against water were measured via the sessile drop and captive bubble methods using a Kyowa Interface Science contact angle meter, DropMaster DM501-N1. The contact angles were read after 30 seconds of deposition of a water droplet (2 µl) and air bubble (2 µl). The measurement was carried out at three different points per substrate using five substrates for one polymer. 2.6. Platelet Adhesion. The polymer-coated substrates were cut into an 8 mm square and sterilized with ultra-violet (UV) light for at least 3 h before use. Human whole blood was purchased from BizCom Japan (Tokyo). Platelet-rich plasma (PRP) and platelet poor plasma (PPP) were obtained by centrifugation. The detailed procedure for preparation of PRP and PPP is described in the Supporting Information (see S1.6). The platelet-suspension plasma (PSP) containing 4 × 107 cells/cm2 of platelets was prepared by mixing the PRP with the PPP. Then, 200 µl of the PSP was placed on the polymer-coated substrates and incubated for 1 h at 37 °C. After washing with PBS, the substrate was immersed into 1% glutaraldehyde in PBS for 2 h at 37 °C to fix the adhered platelets. The fixed samples were observed by a scanning electron microscopy (SEM, VE-9800, KEYENCE, Tokyo, Japan). The number of adherent platelets on a polymer was visually counted in five randomly selected SEM images for each polymer substrate. Two substrates were used for each polymer in a single test, and the test was triplicated. The number of adherent platelets was quantified in relation to those on PET normalized as 100.
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2.7. Vascular Cell Culture.
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Human umbilical vein endothelial cells (HUVECs) were
purchased from Health Science Research Resources Bank (Osaka, Japan) and maintained in Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 (DMEM/F-12; Thermo Fischer Scientific, Waltham, MA, USA), supplemented with 20% fetal bovine serum (FBS; Biowest, Nuaillé, France), 10,000 U/ml of penicillin, 10,000 µg/ml of streptomycin (Thermo Fischer Scientific), 100 µg/ml of heparin sodium salt (Sigma, St. Louis, MO, USA), and 50 µg/ml of endothelial cell growth factor (ECGF; Sigma). The polymer-coated substrates were placed into 24-well polystyrene plates (AGC Techno Glass Co., LTD, Tokyo, Japan) and sterilized with UV light for at least 3 h. The medium was added to the polymer surface (500 µl/well) and incubated overnight at 37 °C, prior to cell culture. HUVECs were seeded onto polymer surfaces at 5.0 × 104 cells/cm2. After 1 h, 1 day, and 3 days of culture, cells were fixed with 4% paraformaldehyde and treated with immunofluorescent staining for observation by a confocal laser-scanning microscope (CLSM; FV1000, Olympus, Tokyo, Japan). Each specimen was analyzed at five different points taken at a 40-fold view to quantify the number of nuclei. Three substrates were used for each polymer in a single test, and the test was triplicated. 2.8. Evaluation of Protein Adsorption and Denaturation.
Protein adsorption on the
polymer surfaces was quantified by a micro-bicinchoninic acid (µBCA) assay. Bovine serum albumin (BSA; Sigma), human fibrinogen (Fg; Sigma), and human fibronectin (Fn; Merck Millipore, Darmstadt, Germany) were used in this study. Polymer surfaces were prepared by solvent casting in a 96-well polypropylene (PP) plate (Evergreen Scientific, Rancho Dominguez, CA, USA). At least five surfaces were prepared for each polymer. The polymer surfaces were pre-treated with PBS (50 µl) at 37 °C for 1 h prior to assays. 50 µl of protein solutions (0.1
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mg/ml in PBS) were added to each well, and the 96-well plate was incubated at 37 °C for 10 mins (during preliminary tests, we confirmed via a quartz crystal microbalance (QCM) that 10 mins was sufficient for proteins to be adsorbed onto polymer surfaces). Each well was rinsed with PBS and treated with 5 wt% of aqueous sodium dodecyl sulfate (SDS; 30 µl) and 0.1 N NaOH aq. (30 µl) at 37 °C for 2 h. The amount of proteins was determined using a µBCA protein assay kit (Thermo Fischer Scientific) by following the manufacturer’s instructions. The absorbance of the solution was measured at 562 nm by a microplate reader (BIO-RAD, Hercules, CA, USA). Three repetitions were performed for all polymers. The degree of denaturation of plasma proteins absorbed on the polymer surfaces was evaluated by enzyme-linked immunosorbent assay (ELISA). Polymer surfaces were prepared in a 96-well PP plate as described above. PPP (50 µl) was added to each well and incubated at 37 °C for 1 h. Next, the polymer surfaces were rinsed with PBS, and incubated with 50 µl of Blocking-One (Nacalai Tesque, Kyoto, Japan) at 37 °C for 30 mins. After removing Blocking-One, each polymer surface was treated with 50 µl of anti-fibrinogen γ’-CT (Merck) or anti-fibronectin antibody HFN 7.1 (Abcam, Cambridge, UK) at 37 °C for 2 h, followed by 50 µl of goat antimouse IgG (H+L) horseradish peroxidase conjugate (BIO-RAD) at 37 °C for 1 h. After washing with PBS, the surfaces were incubated with 100 µl of a 2,2'-azinobis(3-ethylbenzothiazoline-6sulfonic acid) (ABTS) substrate (Roche, Basel, Switzerland) at 37 °C for 1 h. The absorbance of the ABTS solution was measured at 405 nm by a microplate reader (BIO-RAD). Three repetitions were performed for all polymers. 2.9. Statistical Analysis. All the data is represented as mean ± standard deviation (SD). Statistical comparisons were done using Student’s t-test (two-tail comparisons) on Microsoft Excel 2010. In all tests, p < 0.05 was considered statistically significant.
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2.10. Biodegradability. Degradation behavior of the PMEMTC was monitored by QCM Affinix Q8 (Initium, Tokyo, Japan) via a parent frequency of 27 MHz, using a polymer-coated gold electrode on the crystal. The frequency shift was correlated to the mass change using the Sauerbrey equation (1 Hz corresponds to 0.62 ng/cm2). After the polymer-coated electrode was immersed in a PBS lipase solution, previously formulated as 2.3 × 106 U/L, for a predetermined period of time at 37°C, the polymer-coated electrode was rinsed with ultrapure water and dried in vacuum at room temperature. The frequency was monitored and the net mass change of the polymer on the electrode was calculated by subtracting the mass of the electrode measured initially. The measurement was triplicated at each point.
3. RESULTS AND DISCUSSION 3.1. Polymer Synthesis. ME-MTC was synthesized via a simple two step processes with an overall yield of 20.8%, and reasonable yields of PMEMTC was obtained after organocatalytic ROP (~80%). We employed the organic catalyst system previously reported for the ROP,48-50 to avoid toxic metal contaminants from catalysts and to enable controlled polymerization so as to yield originally targeted molecular weights, fidelity of the end group, and narrow molecular weight distribution. However, controlled polymerization using an alcohol initiator (1pyrenebutanol) was unsuccessful. Since ME-MTC was highly hygroscopic or difficult to dry, a trace amount of water in the monomer most likely acted as an additional initiator for the ROP, resulting in lower molecular weights than targeted (M/I = 100). In fact, ME-MTC could be polymerized in the absence of the initiator, and higher molecular weights were obtained when the monomer solution was dried over CaH2 prior to the addition of the catalysts. We tried to further purify ME-MTC by vacuum distillation, which was also unsuccessful due to the
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concomitant pyrolysis and thermal oligomerization. Next, we explored the optimum ROP condition with respect to amine catalysts (DBU or SP), use of the initiator 1-pyrenebutanol, and pre-drying of the monomer via CaH2. As summarized in Table S1, we found that the DBUcatalyzed ROP of ME-MTC treated with CaH2 in the absence of initiators afforded PMEMTC with the highest molecular weight of 26 k (Table S1, run 5). This polymer was used for further biological evaluation. 3.2. Polymer Characterization.
PMEMTC was amorphous and viscous under ambient
conditions. Glass transition of PMEMTC was observed around −20 °C via DSC measurement, with a variation of a few degrees depending on the molecular weight (Figure S5), g. PMEMTC was soluble in acetone, THF, CH2Cl2, DMF, methanol, and toluene, and insoluble in diethyl ether, hexane, water, ethanol, and 2-propanol, where the solubility remained unchanged regardless of the molecular weight. Thus, PMEMTC can be used to coat materials coming into contact with water and alcohol for sterilization.
Table 1. Static Contact Angles against Water and Glass Transition Temperature (Tg) for Polymers Tested in This Study (degree, mean ± SD, n = 3). Contact angles
Tg (°C)
Polymers
Sessile drop
Captive bubble
Dry
Hydrated
PET
72 ± 0.4
127 ± 3.1
76
-
PMEA
42 ± 0.9
135 ± 2.6
-35
-51
PMB
91 ± 0.3
161 ± 2.6
57
-100
PMEMTC
60 ± 0.8
132 ± 0.4
-14
-18
PTMC
77 ± 1.7
125 ± 2.7
-17
-20
PMEMA
70 ± 0.9
121 ± 1.9
27
24
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As summarized in Table 1, PMEMTC showed slightly hydrophilic surface properties when compared to PTMC and PMEMA. Note that the terminal groups of PMEMTC were acetylated to exclude the effect of the hydroxy terminal groups on the hydrophilicity of the polymer. Interestingly, the contact angle of a sessile drop on PMEMTC was almost 20 degrees higher than that on PMEA, while the contact angles of a captive bubble on both polymers were almost identical. This result implies that surface structure of PMEMTC was changed upon contacting water. The 20-degree difference in the contact angles of a sessile drop may be explained by the side chain density and the affinity of the carbonate backbone with the side methoxyethyl esters. PMEA tags the side chains more densely so that the surface is filled up by the polar ether groups, while PMEMTC has lesser side chain density and polar carbonic esters in the main chain. This may allow the side chains more miscibility with the backbone and prevent them from localizing at the surface in a dry state. Therefore, the surface state of PMEMTC, after coming into contact with water, is thermodynamically more stable, as shown by the similar captive bubble contact angle values as PMEA. 3.3. Platelet Adhesion. Human platelet adhesion on polymer substrates after 1 h incubation is shown in Figure 1. Activated platelets usually form pseudopodia that develop networks to capture red blood cells and leukocytes, resulting in clot formation. Many platelets adhered to PET and PTMC and most of them were activated (Figures 1a and 1e), while a few round platelets were observed to adhere to PMEMTC (Figure 1d) and PMEA (Figure 1b), which is favorable for antithrombotic materials.
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Figure 1. SEM images of adherent platelets on (a) PET, (b) PMEA, (c) PMB, (d) PMEMTC, (e) PTMC, and (f) PMEMA (bar scales are 10 µm). (g) Typical morphologies of each class of activated platelets (bar scales are 5 µm). (h) Number of adherent platelets on polymers normalized to the number of adherent platelets on PET (a.u., mean ± SD, n = 3).
We categorized the adherent platelets on the polymers into three types as shown in Figure 1g: type I is the native form maintaining a round shape; type II is the slightly activated form generating a few pseudopodia (typically < 3); and type III is the fully activated form with pseudopodia markedly more than type II. Next, the number of adherent platelets on each polymer was quantified (Figure 1h). The platelet adhesion property of PTMC was similar to that of PET. An apparent decrease of adherent platelets was found on PMEMA when compared to PET and PTMC. However, measurable amounts of activated platelets were observed (Figure 1f), and the level was still insufficient for antithrombotic devices. In contrast, the number of adherent platelets on PMEMTC was significantly less than that on PET, PTMC, and PMEMA, although the extent of the non-adhesive property of the platelets was still not comparable with that of
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PMEA and PMB. In addition, most of adherent platelets on PMEMTC were type I, indicating the bioinert surface property. Thus, the antithrombotic property of PMEMTC was observed to be quite high among the synthetic biodegradable polymers, and introducing the simple 2methoxyethoxycarbonyl side chain drastically suppressed the platelet adhesion property of PTMC. Moreover, comparison of PMEMA bearing the α-methyl group with PMEMTC implied that the carbonate backbone served as a positive factor in lowering platelet adhesion. 3.4. Adhesion Properties of Vascular Cells. Resorbable artificial blood vessels for vascular tissue regeneration require surface properties favorable for the adhesion of vascular cells. For successful vascular regeneration, endothelialization is essential because endothelial layers play an important role in preventing, not only platelet adhesion, but also the over-proliferation of smooth muscle cells comprising the tunica of blood vessels, which often cause restenosis after angioplasty and bypass graft treatment if left unchecked.51-53 Next, we examined the adhesion and proliferation of HUVECs on the polymers as represented in Figure 2. Round shaped cells were observed at 1 h of incubation. After 1- and 3-day incubations, elongated cells with explicit focal adhesion and thick actin fibers were confirmed on PMEMTC and other polymers (Figure 2a, see also Figure S6). These results indicate that HUVECs can sufficiently attach and grow on PMEMTC with no adverse effects. Adherent cells were hardly found on PMB at any time points of incubation, which is consistent with previous reports.21, 22, 43 Quantitative evaluation of HUVECs adhesion on each polymer was conducted by counting cell nuclei from the CLSM images. As shown in Figure 2b, few differences in cell numbers among the polymers were observed, except for PMB. In this study, the HUVECs observed on PMB were all quite small and circular (Figure S6). Thus, PMB was recognized as non-cell-adhesive as with previous studies, although the cell numbers in Figure 2b were not null. PMEA demonstrated
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slightly higher cell adhesion properties when compared to PMEMTC. Cell proliferation appeared moderate for all polymers, which may be attributed to technical issues such as detachment of cells during medium exchange and staining/washing procedures. However, the magnitude correlation of the number of HUVECs remained unchanged among the polymers in the other three tests implemented separately. Similar trends were observed for the adhesion properties of the aorta smooth muscle cells (AoSMCs) on the same polymers (Figures S7 and S8), where AoSMCs adhered and proliferated on all polymers except PMB. These results suggest that PMEMTC can potentially serve as scaffold materials and basal membrane alternatives for vascular cells with good antithrombogenicity.
Figure 2. (a) CLSM images of HUVECs on polymers at different time points of incubation (1 h and 1 day). Scale bars are 50 µm. (b) Number of HUVECs cultured for predetermined time periods on polymers. Data shown represents mean ± SD derived from three different specimens. * P < 0.05 and ** P < 0.01 relative to PMEMTC for 1 h. †P < 0.05 and
††
P < 0.01 relative to
PMEMTC for day 1. ‡‡P < 0.01 relative to PMEMTC for day 3.
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3.5. Enzymatic Degradation. Figure 3 shows weight loss of polymers in the presence of lipase as a function of treatment time based on the QCM experiments. The enzyme was reported to decompose PTMC,54 and in this study, 48% of PTMC was degraded by the 8 h treatment. Contrastingly, PMEMTC was only moderately degraded when compared to PTMC, showing a 12% weight loss after the 8 h treatment. This slow degradation profile is presumably attributed to the higher hydration property of PMEMTC when compared to PTMC, which is supported by the contact angle measurements (Table 1). Esterase-type enzymes, including lipase, are known to often recognize hydrophobic structures for the degradation reaction. In fact, decrease in enzyme accessibility was reported in PEGylated PTMC with increased surface hydrophilicity.55 In addition, PTMC is subject to surface erosion54 and therefore, surface properties are critical in enzymatic degradation of PTMC-based materials. As previously reported, in vivo degradation proceeds slower for lower molecular weight PTMC, possessing more hydroxyl terminals, resulting in higher hydration.54 This is also explained by the mitigated enzymatic recognition for the softened surface induced by hydration.56 Nonetheless, slow degradability is desirable for applications in vascular grafts and stents, which require relatively long-term stability until complete vascular tissue regeneration. Although the in vivo biodegradation of PTMC is known to usually proceed faster than the in vitro degradation,57,
58
the actual in vivo degradation of
PMEMTC would take quite long, considering that the test in this study was performed under accelerated conditions with a high enzyme concentration. Consequently, this slow degradability of PMEMTC may prevent regenerated vascular tissue from detaching from the polymer surface, which is beneficial for vascular regeneration.
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Figure 3. Mass change profiles of polycarbonates as a function of time in the presence of lipase. Each point contains mean ± SD from three independent trials.
3.6. Effects of Polymer Structure on Hydration and Platelet Adhesion. As demonstrated by the contact angle data (Table 1), PMEMTC becomes more hydrophilic than PTMC, indicating that the incorporation of the methoxy side chain contributes to the increased hydration and the subsequent low platelet adhesion. PMEMTC also possesses an α-methyl group on the side chain as with PMEMA that demonstrated high platelet adhesion and a relatively large sessile drop contact angle.59 Therefore, the improved antithrombotic property and hydration for the PMEMTC is possibly explained by the increased mobility of the methoxy side chains due to a less dense alignment and by the contribution of the carbonate backbone to concerted hydration. Tanaka has revealed the correlation between materials with low platelet adhesion and hydration, especially “intermediate water”, which is defined as the water loosely bound by polymer chains detectable by DSC.59 Therefore, we measured the DSC of hydrated PMEMTC (Figure 4).
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Figure 4. DSC charts of hydrated PMEMTC (water content: 4.9 wt.%). Arrows represent crystallization and melting of freezing interacting water.
For hydrated PMEA, distinct cold crystallization observed on the heating scan is recognized as the intermediate water (Figure S9).60 As a consequence, the hydrated PMEMTC did not show such an exotherm on the heating scan (Figure 4). However, a melting endotherm overlapped with free water, as highlighted by the arrow. Therefore, we presumed that this endotherm (below 0 ºC) would correspond to the melting of imperfect ice, resulting from interacting with the polymer chain, as with cryoscopy. Indeed, crystallization exotherms were observed well below 0 ºC during the cooling scan. The exotherm around −20 ºC has been previously confirmed as a crystallization of free water.19 Thus, we regarded the other exotherms below −40 ºC as freezing water weekly interacting with polymer structures, as highlighted by the arrows. We are still elucidating the similarities and differences between this type of water and intermediate water observed in PMEA. Nevertheless, it was observed that PMEMTC can form water to interact with polymer chains upon hydration, named as “freezing interacting water”, which seems to result from the methoxy side chain and the carbonate backbone.
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3.7. Understanding Different Cell Adhesion Behaviors between Platelets and Vascular Cells.
As mentioned above, hydration appeared to be associated with preventing platelet
adhesion. However, other factors contributing to the adhesion of platelets and cells needs to be considered. Mechanical properties of the material surface, such as viscoelasticity, are often found to be significant for cell behaviors.61 Viscoelasticity can be roughly estimated from the glass transition temperature (Tg) of polymers, which is usually decreased upon hydration. As shown in Table 1, the Tg of hydrated polymers were lower than those of dry polymers. However, we could not find a clear correlation between the Tg of hydrated polymers and platelet adhesion since the Tg of PMEMTC and PTMC were similar. This result also implies that the vast difference in molecular weights of the polymers (20 - 600 kDa) little impacted on to the platelet adhesion, because Tg is a parameter related to molecular mobility that depends on the molecular weight. In addition, the OH terminal groups of PMEMTC were acetylated to exclude the possibility of varying hydrophilicity by the molecular weight. Thus, we decided to focus on the surface hydration and structure of hydrated water, including intermediate water and freezing interacting water. Although some hydrated water may directly affect cell adhesion, it is usually mediated by serum proteins. Next, we evaluated the adsorption of each plasma protein via the µBCA assay and the extent of the adsorbed protein denaturation in human blood plasma via the ELISA assay. Figures 5a-c show the adsorption of the plasma proteins on the polymers. Polypropylene (PP) was used as a control for protein adhesive materials, instead of PET. PET is a suitable substrate for spin-coating and microscopic experiments due to its resistance to different organic solvents and transparency. However, it is difficult to cut PET sheets to fit into a 96-well plate usually used for protein assays and to prepare a cast film due to its solubility in common organic solvents. In addition, tissue culture polystyrene (TCPS), which is among the most common
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material used in biological tests, was deemed unsuitable for this study since chloroform was used for spin-coating and solution casting. Therefore, PP was employed as a control material. Out of the polymers tested, PMEA demonstrated the lowest adsorption level with respect to all proteins. Albumin, a major serum protein, was highly adsorbed on PP and PTMC, while less BSA was adsorbed on PMEMTC and PMEA (Figure 5a). Adsorption levels of Fg, a plasma protein involving platelet adhesion, on PMEMTC was significantly lower than that on PTMC and slightly lower than that on PP (Figure 5b). Accordingly, the low platelet adhesion on PMEMTC and PMEA could be explained by the low adsorption of their relevant proteins.
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Figure 5. (a-c) Adsorption of proteins on spin-coated polymers (mean ± SD, n = 3): (a) bovine serum albumin (BSA), (b) human fibrinogen (Fg), and (c) human fibronectin (Fn). (d, e) Extent of denaturation of adsorbed plasma proteins derived from human blood (mean ± SD, n = 4): (d) γ-chain of Fg, (e) RGD motif of Fn.
Sivaraman has previously reported that the extent of denaturation of the adsorbed proteins affects cell adhesion more directly than the proteins adsorbed on the materials.62 Therefore, the extent of denaturation of adsorbed Fg was estimated via ELISA to quantify the exposed γ-chain of the denatured Fg, which is a site that is associated with platelet adhesion. Figure 5d shows the result of the ELISA used to detect denatured Fg in the adsorbed plasma proteins on each polymer. Interestingly, the denaturation levels of the Fg adsorbed on PMEMTC is comparably as low as that on PMEA. In contrast, those for PP and PTMC were significantly high, indicating that PP and PTMC adsorb and denature proteins at a high level. PMEMTC showed a certain level of protein adsorption. Hence, low Fg denaturation levels appear to be more plausible explanation than low adsorption levels for the low platelet adhesion achieved on PMEMTC. Trends in the adhesion of HUVECs and AoSMCs on each polymer are difficult to explain based on the adsorption (Figure 5c) and denaturation (Figure 5e) of Fn associated with the integrin-mediated cell adhesion. Contrary to the high levels of HUVEC adhesion, PMEA showed the lowest levels of both adsorption and denaturation of Fn. In addition, PMEMTC represented the same level of HUVECs adhesion and a higher level of AoSMCs adhesion when compared to PTMC. However, the adsorption and denaturation of Fn on PMEMTC were lower than on PTMC. As previously reported in the case of PMEA, these discrepancies may stem from integrin-independent cell adhesion.63 As of this moment, we have not run the verification
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experiments to block integrin by using ethylenediaminetetraacetic acid (EDTA) during cell culture.64 However, considering the similar protein behaviors and the side chain structure when compared to PMEA, PMEMTC remains a possibility including the integrin-independent cell adhesion mechanism,. The protein adsorption property may be explained by hydration (or water absorption) and wettability (contact angles), whereas the denaturation and conformational change of adsorbed proteins cannot be explained as such, as detailed in Figures 5d and 5e. The reduced Fg denaturation was recently correlated to intermediate water and its amounts in PMEA and its analogs.65, 66 Although protein denaturation is believed to arise from electrostatic interaction with the material surface, such as hydrogen bonding and amphiphilic balance, it was also found to vary depending on the types of proteins. The magnitude of hydrogen bonding and the amount of the intermediate water in the hydrated PMEA could be inadequate to prevent denaturation of Fn. Thus, a high level of the RGD motif of Fn was detected on PMEA. Another aspect of hydration and intermediate water affecting polymer surface properties has been recently discovered by Murakami et al. by directly observing hydrated polymer surfaces using atomic force microscopy.67 They reported that blood compatible polymers such as PMEA exhibited nanometer-scale protrusions, which is believed as polymer-rich domain, at the polymer-water interfaces and that Fg tended to be locally adsorbed on the protrusions. The fineness of the nanoprotrusions appears to be associated with the existence and amount of intermediate water. If the freezing interacting water observed in hydrated PMEMTC (Figure 4) was considered to be equivalent to intermediate water, the protein denaturation properties of PMEMTC could be reasonably explained in a similar way. However, we deduce that differences between the intermediate water and freezing interacting water may influence the extent of vascular cell
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adhesion and Fn denaturation in PMEMTC and PMEA (Figures 2b and 5e). The role of the freezing interacting water may be extended to the interaction with lipase for the enzymatic degradation test, where PMEMTC exhibited a slower degradation profile than PTMC (Figure 3).
4. CONCLUSION We successfully synthesized a biodegradable polycarbonate with antithrombogenic and vascular cell adhesive properties by incorporating a monoether group at the side chain. This polycarbonate exhibited improved surface wettability and significantly lower platelet adhesion when compared to an unsubstituted biodegradable polycarbonate (PTMC). Its enzymatic degradation profile was moderate compared to that of PTMC, and it favored vascular cell adhesion and proliferation. In addition, the monoether-tagged polycarbonate was water insoluble, which was suitable for coating and bulk applications. The simple structure of the side chain offered additional advantages such as simplification of synthesis and lowering production costs. Although the adhesion properties of both platelets and vascular cells on PMEMTC are still not comparable with PMEA, this polymer demonstrates the best performance among synthetic biodegradable polymers that have been reported before. Moreover, further studies related to water and hydrated surfaces may lead to a new structural design with improved properties. Thus, this new polycarbonate appears to be promising as a candidate material in artificial resorbable blood vessels and stents.
ASSOCIATED CONTENT
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Supporting Information. The supporting information is available free of charge via the Internet at http://pubs.acs.org. Additional description for Experimental Section and data including NMR, DSC, CLSM images of vascular cells, and polymerization results. AUTHOR INFORMATION Corresponding Author *Phone: +81.238.263759. E-mail: fukushima@yz.yamagata-u.ac.jp (KF) *Phone: +81.92.802.6235. E-mail: masaru_tanaka@ms.ifoc.kyushu-u.ac.jp (MT) Author Contributions K.F. designed and supervised the research and wrote the paper; M.T. co-supervised the research; Y.I. performed all of the research; Y.H., T.O. and K.H. supported the experiments; C.S. supported biological experiments and analyzing data; All authors approved the final version of the paper. Notes There is no conflict of interest for this work.
ACKNOWLEDGMENT The authors acknowledge for the Center of Innovation, Frontier Center for Organic System Innovations, financially supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. KF thanks to JSPS KAKENHI Grant Number 25870078 for the financial support. MT acknowledges the Funding Program for Next-Generation World-Leading Researchers (NEXT Program) of MEXT.
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For Table of Contents Use Only
Monoether-tagged
Biodegradable
Polycarbonate
Preventing
Platelet
Adhesion and Demonstrating Vascular Cell Adhesion: A Promising Material for Resorbable Vascular Grafts and Stents Kazuki Fukushima,* Yuto Inoue, Yuta Haga, Takayuki Ota, Kota Honda, Chikako Sato, Masaru Tanaka*
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