Orthogonally Functionalizable Polyurethane with Subsequent

Publication Date (Web): May 25, 2016 ... *E-mail: [email protected] (X.M.)., *E-mail: [email protected] (M.Y.). ... coimmobilization modifications of ...
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Orthogonally Functionalizable Polyurethane with Subsequent Modification with Heparin and Endothelium-Inducing Peptide Aiming for Vascular Reconstruction Jun Fang,†,⊥ Jialing Zhang,‡,⊥ Jun Du,§ Yanjun Pan,‡ Jing Shi,§ Yongxuan Peng,‡ Weiming Chen,† Liu Yuan,† Sang-Ho Ye,∥ William R. Wagner,∥ Meng Yin,*,‡ and Xiumei Mo*,† †

State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of Materials Science and Engineering, Donghua University, Shanghai 201620, China ‡ Department of Cardiothoracic Surgery, Shanghai Children’s Medical Center, Shanghai Jiaotong University School of Medicine, Shanghai 200127, China § Imaging Diagnosis Center, Shanghai Children’s Medical Center, Shanghai Jiaotong University School of Medicine, Shanghai 200127, China ∥ McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15219, United States S Supporting Information *

ABSTRACT: Surface coimmobilization modifications of blood-contacting devices with both antithrombogenic moieties and endothelium-inducing biomolecules may create a synergistic effect to improve their performance. However, it is difficult to perform covalent dual-functionalization with both biomolecules on the surface of normally used synthetic polymeric substrates. Herein, we developed and characterized an orthogonally functionalizable polymer, biodegradable elastic poly(ester urethane)urea with disulfide and amino groups (PUSN), which was further fabricated into electropun fibrous scaffolds and surface modified with heparin and endothelial progenitor cells (EPC) recruiting peptide (TPS). The modification effects were assessed through platelet adhesion, EPC, and HUVEC proliferation. Results showed the dual modified PUSN scaffolds demonstrated a synergistic effect of reduced platelet deposition and improved EPC proliferation in vitro study, and demonstrated their potential application in small diameter vascular regeneration. KEYWORDS: orthogonally functionalizable polymer, polyurethane, surface modification, endothelialization, vascular regeneration

1. INTRODUCTION Cardiovascular disease has become the leading cause of death worldwide.1 Cardiovascular devices such as vascular grafts, stents, and heart valves have been widely used to treat cardiovascular diseases. However, these devices are usually failed by the formation of thrombus and neointimal hyperplasia on the device surfaces, particularly, resulting in a great challenge for small diameter vascular repair in clinic.2 Over the past decade, vascular tissue engineering has provided some promising technologies for small diameter vascular reconstruction, including self-assembling cell sheets, as well as natural or synthetic scaffold-guided and decellularized-matrix approaches.3 Among them, biodegradable synthetic scaffolds without cell seeding are attractive for future clinic-scale applications, due to the fact that they can be available off-the-shelf, and possess broad affordability and availability.4,5 Surface modification with biofunctional molecules has been widely employed to improve the hemocompatibility and longterm patency of synthetic vascular grafts, by grafting hydrophilic poly(ethylene glycol), zwitterionic polymers, heparin, or other bioactive molecules to prevent thrombosis,2 or by grafting © XXXX American Chemical Society

specific peptides, antibodies, oligosaccharides, and aptamers to accelerate the in situ endothelialization.6 However, none of the modified small diameter grafts have achieved successful results in vivo only by preventing platelet response or by enhancing endothelialization, due to the fact that both of them are complex and synergistic pathological processes.7,8 To circumvent these obstacles, several researchers coimmobilized both antithrombogenic and endothelial cells (ECs) or endothelial progenitor cells (EPCs) preferential ligands on the surface of blood-contacting materials and demonstrated a synergistic effect of antithrombogenic properties as well as improved endothelialization.9−14 Particularly, EPCs have been accepted as an important cell source for in situ endothelialization due to the much higher proliferative potential than mature ECs.15,16 Chen et al.13 synthesized a phospholipid/peptide polymer with phosphorylcholine groups, EPC-specific TPS peptide (TPSLEQRTVYAK) and catechol groups, and anchored it Received: April 11, 2016 Accepted: May 25, 2016

A

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Figure 1. Synthesis route of poly(ester urethane)urea with pendant disulfide and amino groups (PUSN).

blood compatibility and proliferations of EPCs and HUVECs were evaluated in vitro.

onto titanium (Ti) surface to fabricate a biomimetic surface that reduced platelet adhesion and improved EPCs proliferation. Ji et al.14 immobilized poly(ethylene glycol) and TPS peptide with nonbiofouling property and EPC-binding capability onto the PCL surface via host−guest inclusion complexation. Generally, dual or multifunctionalized surfaces have been achieved by the combination of current commonly utilized strategies, including physical, covalent, and bioaffinity immobilization. Reactive groups are usually needed to be deposited on the surface of commonly utilized synthetic polymeric substrates (e.g., polyester and polyurethane) by a physical or chemical handling, however, which are often accompanied by some limitations and potential risks.17,18 Biodegradable polyurethane (PU), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), polycaprolactone (PCL), and their copolymers have been extensively investigated for vascular reconstruction.19−21 In particular, biodegradable polyurethanes are considered as promising base materials because of their diverse chemical structure, tunable physical properties (degradability, elasticity, compliance), and excellent biocompatibility.19 However, biodegradable PU small diameter vascular grafts are often failed by the acute thrombosis and neointimal hyperplasia, which warrant surface treatment of the PU graft before surgical implantation.22,23 Recently, our group developed a series of biodegradable PUs with pendant reactive groups for the subsequent biofunctionalization,24−26 these strategies could overcome the limitations by the activation treatment, while such polyurethanes with single reactive groups are difficult to perform dual or multi-covalent modifications with different bioactive molecules. Considering dual or multifunctionalization of blood-contacting grafts with both antithrombogenic and endotheliuminducing biomolecules may create a synergistic effect of antithrombogenic properties and in situ endothelialization to improve their blood compatibility. In this study, we developed and characterized an orthogonally functionalizable polymer, biodegradable elastic poly(ester urethane)urea with disulfide and amino groups (PUSN), which was further fabricated into electropun fibrous scaffolds with heparin and EPC recruiting peptide (TPS) modification. The effects of modification on

2. MATERIALS AND METHODS 2.1. Materials. Polycaprolactone diol (PCL diol, Mn = 2000 Da), N-Boc-serinol (97%), oxidized dithiothreitol (ODTT), hexamethylene diisocyanate (HDI), putrescine, stannous octotate (Sn(Oct)2), trifluoroacetic acid (TFA), tris(2-carboxyethyl)phosphine hydrochloride (TCEP), rhodamine B isothiocyanate, fluorescein diacetate 5-maleimide, heparin sodium salt (⩾150 U/mg), Nhydroxysuccinimide (NHS), N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC) and other reagents were purchased from Sigma-Aldrich and used as received, except where mentioned otherwise. Maleimide-TPSLEQRTVYAK (TPS-maleimide, M w = 1585 Da, 95% purity) was synthesized with a 6maleimidohexanoic acid linked on the N-terminus of TPS peptide through solid-phase peptide synthesis technology at GL Biochem Ltd. (Shanghai, China). Alexa Fluor 568 Phalloidin, and 4′,6-Diamidino-2phenylindole dihydrochloride (DAPI) were purchased from Thermo Fisher Scientific. PCL diol was dried in a vacuum oven at 60 °C overnight to remove residual water before synthesis. 2.2. Synthesis of Poly(ester urethane)urea with Disulfide and Amino Groups (PUSN). The polymer was synthesized by the traditional two-step solution polymerization with a further deprotection process of the Boc-protected amino groups (Figure 1), which was performed as previously described methods with slight modification.25,26 Briefly, the stoichiometry of mixed diols/HDI/putrescine was 1:2:1, where the mixed diols are equimolar ratio of PCL diol, O−DTT and N-Boc-serinol. In a typical example, PCL diol (12 g, 6 mM), O−DTT (0.91 g, 6 mM) and N-Boc-serinol (1.15 g, 6 mM) were dissolved in anhydrous dimethyl sulfoxide (DMSO) in a three-necked flask, then HDI (5.78 mL, 36 mM) was added dropwise under nitrogen protection with magnetic stirring, followed by the addition of Sn(Oct)2 (0.05 wt %) as a catalyst. The prepolymerization reaction was carried out for 3 h at 80 °C. Afterward, the reaction solution was cooled to room temperature, and putrescine (1.81 mL, 18 mmol) in DMSO solution was added dropwise, then the reaction of final 5% (w/ v) polymer solution continued for another 18 h at 40 °C. The resulting polymer was precipitated in deionized water (DI water), and immersed in isopropanol for further purification, then dried in a vacuum oven to obtain PUS-Boc (yield was 89%). The synthesized PUS-Boc was further dissolved in 5% (w/v) TFA/ chloroform (50/50) mixture for 1.5 h at room temperature to remove B

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Figure 2. Schematic representation of a PUSN fibrous scaffold functionalized with heparin and an EPC specific capture molecule. (A) PUSN fibrous scaffold was first modified with heparin by carboxyl/amino reaction and further modified with TPS-maleimide by (B) disulfide based click reaction. 2.5. Characterization of Polymer and Scaffolds. Polymer chemical structure was characterized by 1H nuclear magnetic resonance (1H NMR, Bruker 300 MHz, Biospin Co., Billerica, MA) using DMSO-d6 as a solvent. Thermal properties were measured by differential scanning calorimetry (DSC, DSC-60, Shimazu) with a scanning range of −100 to 200 °C at a rate of 10 °C/min under nitrogen flow, and the second cycle was recorded. The morphology of the fabricated electrospun scaffolds was examined by Digital Vacuum Scanning Electron Microscope (JSM-5600LV, Japan Electron Optical Laboratory) at the accelerating voltage of 15 kV after sputter-coating with gold. The fiber diameters were calculated with ImageJ 1.3 (National Institute of Health, U.S.A.) by measuring approximately 100 random fibers in the SEM micrographs. The surface hydrophilicity of electrospun sheets was studied by measuring the water contact angle (WCA) using a sessile drop method with distilled water (OCA40, Dataphysics, Germany). In vitro degradation of cast films and fibrous scaffolds was tested in PBS and lipase solutions as previously described.26 For uniaxial tensile testing, strips (30 × 10 mm2, n = 5) were cut from the PUSN cast films and ES sheets, and measured by universal material tester (H5K-S, Hounsfield, England) with a crosshead speed of 10 mm/min and a 50 N load cell at ambient temperature. 2.6. Characterization of Grafted Heparin and Peptide. The surface composition of PUSN fibrous scaffolds with or without modifications of heparin and peptide was analyzed by X-ray photoelectron spectroscopy (XPS, Physical Electronics PHI 5802 equipped with a monochromatic Al Kα source), acquired at 45° takeoff angles. To visualize the modification of PUSN fibrous scaffolds with heparin, PUSN, PUSN-Hep, and PUSN-Hep/TPS fibrous tubes were immersed with toluidine blue solution (0.005 wt % in 0.2% NaCl aqueous solution), and the pictures of tubes and their cross sections were taken by digital camera. And to visualize the modification of PUSN fibrous scaffolds with TPS peptide, the FITC-labeled TPSmaleimide was covalently immobilized onto PUSN-Hep fibers as described previously. Meanwhile, PUSN fibers and PUSN-Hep fibers physically immersed with the fluorescent peptide solution were used as control samples. The fibers were observed by fluorescence microscopy (Nikon Eclipse 80i, Japan). FITC fluorochrome was covalently linked to the TPS peptide via lysine. Briefly, TPS-maleimide (15 mg) was reacted with FITC (5.53 mg, 1.5 equiv) in 1.5 mL of DMSO for 24 h at room temperature in the dark. The product was purified by dialysis (MWCO = 1000 Da, Spectra/Por 6, Spectrum, U.S.A.) against DI water and dried by lyophilization. The density of immobilized heparin on the PUSN-Hep and PUSNHep/TPS fibrous scaffolds was measured by the toluidine blue assay.27

the Boc groups. After removing TFA and chloroform by rotary evaporation, the polymer was precipitated and neutralized in Na2CO3 aqueous solution (pH = 11.4) to remove residual TFA, then rinsed with DI water and isopropanol for further purification, and freezedried to obtain PUSN (yield was 92%). 2.3. Fabrication of Cast Films and Fibrous Scaffolds. Cast films (∼200 μm thick) were prepared with 8.0 w/v% PUSN in 1,1,1,6,6,6-hexafluoroisopropanol (HFIP) solution poured into a Teflon mold, followed by solvent evaporation in a fume hood, and further dried in a vacuum oven to remove residual solvent. The fibrous scaffolds were prepared by the electrospinning (ES). Briefly, a 15% (w/v) solution of PUSN in HFIP was fed at 1.5 mL/h through a stainless steel capillary (inner diameter = 0.56 mm) charged to 15 kV and located 15 cm over a stainless steel mandrel with 10 cm diameter for sheets (2.0 mm diameter for tubes). The mandrel was rotated at 100 rpm for sheets (1000 rpm for tubes) and reciprocally translated in the direction of the mandrel axis at a speed of 2 cm/s. All of the scaffolds were dried in a vacuum oven at room temperature to eliminate the solvent completely. 2.4. Surface Modification of Fibrous Scaffolds. Heparin was immobilized on the surface of PUSN scaffolds through carboxyl-amino condensation reaction to obtain heparin modified scaffolds (PUSNHep). Which were further modified with TPS peptide end-linked with maleimide via a disulfide-based click chemistry,26 to get heparin and peptide dual-modified scaffolds (PUSN-Hep/TPS) (Figure 2). The detailed modification procedures were as following: PUSN-Hep: The fibrous PUSN scaffolds (around 0.45 g) were first prewetted in 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution (0.1 M, pH 5.5) for 30 min at room temperature. Carboxylic acid groups of heparin (30 mg) were activated by using NHS (20 mg) and EDC (12 mg) in 10 mL MES buffer. After 10 min activation, the prewetted scaffolds were soaked in the activated heparin solution with gentle agitation for overnight at 4 °C. The scaffolds were then washed thoroughly with phosphate-buffered saline (PBS) followed by DI water for 24 h, and freeze-dried to obtain PUSN-Hep scaffolds. PUSN-Hep/TPS: The heparinized fibrous scaffolds were further click modified by TPS-maleimide. PUSN-Hep scaffolds were prewetted in DI water and reduced the disulfide bond (SS) by 20 mM TCEP solution in DI water for 1 h at room temperature with mild shaking. After removing the reduction solution, TPS-maleimide (1 mg/mL) solution in DI water was added for proceeding 2 h at room temperature and further overnight at 4 °C to ensure the reaction completed. The scaffolds were then washed thoroughly with PBS followed by DI water for 24 h, and freeze-dried to obtain PUSN-Hep/ TPS scaffolds. C

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Figure 3. 1H NMR spectra of PUS-Boc and PUSN; DMSO-d6 as solvent.

Figure 4. (A) Macrographic view of electrospun (ES) PUSN fibrous tube and (B and C) their surface and cross-sectional SEM images. PUSN fibrous scaffolds with heparin physical absorption were utilized as control groups, which were treated as the up-mentioned procedures for PUSN-Hep preparation except the heparin was inactivated. First, a series of heparin standard solutions (0−30 μg/mL) was prepared in PBS (pH = 7.4). Then, the heparin standard solutions (2.5 mL) or heparin grafted fibrous sheets with 2.5 mL PBS were reacted with 2.5 mL toluidine blue solution (0.005 wt % in 0.2% NaCl aqueous solution) in a centrifuge tube. The samples were mild shaken for 2h at 37 °C, followed by addition of 5 mL N-hexane to each centrifuge tube for another 15 min shaking, which led to phase separation of the mixture. Then, the upper organic layer was removed, and the absorbance of the aqueous layer was measured with a spectrophotometer at 630 nm. 2.7. Platelet Adhesion Test. Human venous blood was drawn from healthy adult donors in compliance with protocols approved by the Institutional Review Board for Human Investigations at the Shanghai Jiaotong University School of Medicine. Blood was drawn into plastic Vacutainer tubes containing 3.2% sodium citrate (2.7 mL, Becton Dickinson, U.S.A.) in a 9:1 ratio (by volume). Platelet-rich plasma (PRP, 2 × 107 platelets/mL) was obtained by centrifugation at 1200 rpm for 10 min at 25 °C. Disk samples (diameter = 1.4 cm) were sterilized with 70% ethanol immersion for 2 h, and rinsed with PBS three times, then placed into 24-well tissue culture plate with PRP (500 μL/well). After 2 h incubation at 37 °C with mild shaking, the samples were gently rinsed with PBS to wash away nonattached platelets. Then, the platelets deposited on the surface were fixed in 4% paraformaldehyde, and dehydrated with gradient alcohol and dried at room temperature. Finally, the platelets adhered on the scaffolds were sputter-coated with gold for SEM observation. 2.8. Cell Culture of EPC and HUVEC. Modified and nonmodified PUSN sheets (diameter = 1.4 cm) were sterilized with 70% ethanol immersion for 2 h, and rinsed with PBS three times, then placed into 24-well tissue culture plate for the cell culture test, and tissue culture polystyrene plate (TCPS) was served as control.

Mouse bone marrow-derived endothelial progenitor cells (EPCs) were isolated and identified after 7 days of culture as our previously described.26 EPCs were seeded upon the sheets with concentration of 1 × 104 cells/well in 500 μL of endothelial basal medium-2 supplemented with EGM-2MV single aliquots (Lonza) and 1% penicillin/streptomycin solution, and cultured at 37 °C with 5% CO2 humidified atmosphere. The culture medium was exchanged every 2 days. The proliferation of EPCs on the substrates was quantified on days 1, 3, and 5 by the MTT assay (Sigma, U.S.A.). In addition, cellular morphology and quantity was also examined by confocal laser scanning microscopy (Carl Zeiss LSM 700, Jena, Germany) after staining with Alexa Fluor 568 phalloidin and DAPI. In the study of endothelial cells, human umbilical vein endothelial cells (HUVECs) proliferation and morphology analysis was performed as the same protocol as afore-mentioned for EPCs, except the cell culture medium was changed to Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solution. 2.9. Statistical Analysis. All data were expressed as mean ± standard deviation. The data were analyzed by one-way ANOVA, followed by Tukey’s test for the evaluation of specific differences with Origin Pro 8, and p < 0.05 was considered statistically significant difference.

3. RESULTS 3.1. Chemical and Mechanical Properties of Polymer and Fibrous Scaffolds. The chemical structures of polymers were confirmed by 1H NMR analysis (Figure 3). PUS-Boc showed a strong signal at 1.38 ppm assigned to methyl protons in the Boc groups, and disappeared completely in PUSN after deprotection of amino groups with TFA solution. In addition, a weak specific chemical signal at 4.72 ppm (OCHCHO) assigned to O−DTT units appeared in the spectra, confirming the existence of a cyclic disulfide structure in the PUSN D

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Figure 5. Physical properties of PUSN polymer. (A) DSC analysis of PUSN (second cycle). (B) Typical stress−strain curves of PUSN cast film and ES fibrous sheet. (C) Mass remaining for PUSN cast film and ES fibrous sheet after degradation in PBS and lipase solutions.

Table 1. Summarized Mechanical Properties of PUSN Cast Film and ES Fibrous Sheet PUSN

strain at break (%)

strength at break (MPa)

initial modulus (MPa)

cast film ES sheet

580 ± 40 170 ± 20

27 ± 3 19 ± 3

45 ± 2 15 ± 1

Figure 6. XPS spectra of the original and modified PUSN fibrous scaffolds. (A) C 1s; (B) N 1s; (C) O 1s; and (D) S 2p.

E

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ACS Applied Materials & Interfaces Table 2. Surface Composition (%) of the Original and Modified PUSN Fibrous Scaffolds Analyzed by XPS samples

C 1s

O 1s

N 1s

S 2p

N 1s/O 1s

PUSN PUSN-Hep PUSN-Hep/TPS

72.3 ± 1.5 71.8 ± 2.1 70.7 ± 2.0

21.9 ± 0.6 22.6 ± 0.9 23.0 ± 0.5

4.7 ± 0.5 4.6 ± 0.7 5.00 ± 0.7

0.85 ± 0.09 0.91 ± 0.25 0.75 ± 0.17

0.215 0.205 0.217

Figure 7. Functionalization of fibrous scaffolds was confirmed by the analysis of staining, morphology and hydrophilicity. (A and B) Toluidine blue staining of fibrous conduits and corresponding cross sections. (C) The density of immobilized heparin on fibrous scaffolds by toluidine blue assay. PUSN without and with physical absorption of heparin was utilized as controls, n = 3, *p < 0.05. (D−F) Fluorescent staining assay with FITC labeled TPS-maleimide modification, (D) PUSN fibers and (E) PUSN-Hep fibers were physical immersed in FITC labeled TPS-maleimide solution; (F) PUSN-Hep fibers were further with FITC labeled TPS-maleimide click modification. (G−I) SEM image of original and modified PUSN sheets, and water contact angle was inserted in each lower right corner, (G) PUSN; (H) PUSN-Hep; and (I) PUSN-Hep/TPS.

PUSN conduit were given in Table S1. PUSN fibrous grafts revealed the burst pressure over than 7600 mmHg, the burst pressure was the upper-most limitation measured by our device, with a compliance value of 1.75 (%/100 mmHg). Polymer degradation was evaluated in PBS and lipase solutions at 37 °C (Figure 5C). PUSN showed minimal degradation in PBS over the 8 week period, whereas significantly higher mass loss occurred in lipase solution (p < 0.05). In particular, PUSN cast samples were degraded significantly faster than electrospun samples in lipase solution. After 8 weeks of degradation in lipase solution, the mass remaining was 60% for cast samples, and 93% for electrospun samples. 3.2. Functionalization of Fibrous Scaffolds. Before the biofunctionalization, two fluorescent dyes (rhodamine B isothiocyanate and fluorescein diacetate 5-maleimide) were used to visually demonstrate the availability of orthogonal modification of PUSN fibers based on amino and disulfide groups (Figure S1). The fluorescent images showed that the

polymer. Thus, the polyurethane with dual pendant of amino and cyclic disulfide groups was successfully synthesized. The developed PUSN polymer can be fabricated into fibrous scaffolds by electrospinning (Figure 4). The tube with the diameter of 2 mm showed a smooth surface and the scaffolds possessed fiber diameters ranging from 300 to 700 nm. Physical properties of PUSN polymer and scaffolds are shown in Figure 5. The DSC curve showed the thermal properties of PUSN (Figure 5A), which exhibited a glass transition temperature (Tg) at −57 °C and a melting peak (Tm) for the soft segment at 31 °C. Representative tensile stress− strain curves of the PUSN cast films and ES sheets are shown in Figure 5B. And the mechanical properties of PUSN cast film and ES fibrous sheet were summarized in Table 1. PUSN cast film was flexible at room temperature and showed the strain at break of 580 ± 40%, strength at break of 27 ± 3 MPa and initial modulus of 45 ± 2 MPa. The corresponding data of ES PUSN sheets were 170 ± 20%, 19 ± 3 MPa, and 15 ± 1 MPa, respectively. The physical and mechanical properties of fibrous F

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Figure 8. SEM images of human platelet rich blood adhesion on (A) PUSN, (B) PUSN-Hep, and (C) PUSN-Hep/TPS fibrous sheets, as indicated by red arrows.

Figure 9. (A) MTT assay of EPCs proliferation on fibrous sheets. TCPS was utilized as a control; *p < 0.05. (B−E) Confocal laser scanning micrographs showing 3 days EPC cultured on fibrous sheets with DAPI (blue) and phalloidin (red) staining, (B) PUSN; (C) PUSN-Hep; (D) PUSN-Hep/TPS; and (E) TCPS.

of heparin with 0.3 ± 0.09 μg.mm−3 was observed on primary PUSN fibrous scaffold with physical absorption of heparin. FITC labeled TPS was used for the further modification of PUSN-Hep fibers (Figure 7D−F). No signal or weak signal was detected for primary PUSN fibers and PUSN-Hep fibers (Figure 7D, E), which were immersed in the fluorescent TPS solution. While PUSN-Hep fibers with FITC labeled TPS modification appeared strong green fluorescence (Figure 7F), illustrating TPS peptide can be further clicked on the fibers after heparin modification. By the comprehensive analysis, heparin and heparin/peptide were successfully immobilized on the PUSN fibrous scaffolds. The morphology of primary and modified PUSN sheets was shown by SEM images (Figure 7G−I). After modification, some fibers were bonded together, and the diameter of fibers was increased from 468 ± 128 nm for PUSN to 756 ± 180 nm for PUSN-Hep and 803 ± 217 nm for PUSN-Hep/TPS. The inserted pictures of the water contact angle of three fibrous sheets showed the PUSN sheets changed from hydrophobic to hydrophilic with either modification. It was 127° for PUSN, while became undetectable after modifications. 3.3. Blood Compatibility. SEM pictures showed platelets adhesion on surfaces of fibrous sheets following 2 h incubation of human platelet rich plasma (Figure 8). A large number of platelets deposited onto PUSN membrane. In contrast, platelet deposition onto the PUSN-Hep and PUSN-Hep/TPS sheets was markedly reduced, with sparse deposition of individual platelets observed. Thus, heparin modification could limit platelet adhesion, and this effect was not interfered with further TPS modification. This result was further confirmed by the anticoagulant assay, which demonstrated that the optical density (OD) values of free hemoglobin were closing between

fibers were dual marked by red and green fluorescence, while no signal for the control immersed with rhodamine B and fluorescein solution, thus confirmed the strategy for orthogonal covalent modifications could be performed under the mild conditions. The surface modification with heparin and peptide was identified by XPS (Figure 6) and reflected by the change of surface compositions (Table 2). The N 1s (399.4 eV) peak for urethane or urea nitrogen (NHCO) and free NH2 decreased after heparin modification, while increased with further TPS modification, this due to the content of nitrogen is lower for heparin, while higher for peptide. In addition, the O 1s peak of PUSN showed CO (533 eV) signals corresponding to amide structure was higher than CO (532 eV) signals corresponding to diethylene glycol initiators of the PCL soft segment, while CO signals became stronger after heparin modification, and both increased with further TPS modification, this is due to more CO structure in the heparin, while both structures are higher for TPS peptide. Further, new S 2p signals at approximately 168.5 eV showed in the PUSN-Hep spectra, which attributing to the OSO3 and NSO3 moieties in heparin. The functionalization of fibrous scaffolds was further confirmed by the staining analysis, morphology, and hydrophilicity changes (Figure 7). Toluidine blue staining (Figure 7A, B) showed the color of PUSN-Hep and PUSN-HEP/TPS sheets changed from white for primary PUSN sheet to purple after heparin modification, confirmed the immobilization of heparin on the sheets. The grafting concentration of heparin on fibrous sheets was also quantified by the toluidine blue assay (Figure 7C). The density of heparin on the fibrous scaffolds was 0.76 ± 0.06 μg·mm−3 for PUSN-Hep and 0.78 ± 0.08 μg· mm−3 for PUSN-Hep/TPS. In contrast, a relatively low amount G

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Figure 10. (A) MTT assay of HUVECs proliferation on fibrous sheets. TCPS was utilized as a control; *p < 0.05. (B−E) Confocal laser scanning micrographs showing 3 days HUVEC cultured on fibrous sheets with DAPI (blue) and phalloidin (red) staining, (B) PUSN; (C) PUSN-Hep; (D) PU-SN-Hep/TPS; and (E) TCPS.

surface covalent functionalization, including oxygen plasma,28 ultraviolet irradiation (UV)29 or hydroxylation,30 and activating urethane or urea hard segments with diisocyanate,31 bromoalkylation.32 However, some disadvantages were generated by these methods, such a plasma etching process alters both surface chemistry and surface morphology with an unpredictable way, in addition, because of the limited plasma penetration, this method can only be used for two-dimensional (2D) films or very thin 3D structures.33 Otherwise, the chemical activation method needs too harsh reaction conditions (high temperature) and complex attachment processes, and existing potential side reactions and toxicity are associated with chemical linkage.18 Recently, we have developed a series of polyurethane with pendant carboxyl, amino, and disulfide groups for the subsequent functionalization in a more controllable and uniform way,24−26 these strategies should be preferred to overcome the limitations by activation treatment. In this study, we further designed the orthogonally functionalizable polyurethane (PUSN) to perform subsequent dual or multimodifications without an activation process, this is totally different from previously mentioned dual or multifunctionalization strategies. The chemical structure of the developed PUSN with the existence of both functional groups was confirmed by NMR and XPS. People may worry about the ester bond breaking of the polymer during the deprotection of amino groups by TFA solution, while that seems not to have happened in our results and those of the other study.24,34 This could also be reflected by the uniaxial tensile properties of PUSN cast films and processed electrospun sheets (Table 1), which were close to the common PEUU without pendant functional groups.35,36 Improved compliance between the vessel and the synthetic graft is considered to be critical to reduce intimal hyperplasia and improve graft patency. The physical and mechanical properties of the fibrous PUSN conduit are given in Table S1. PUSN fibrous scaffolds presented that the initial modulus of 15 ± 1 MPa is approaching that of native human blood vessels (saphenous vein: 23.7 MPa, left internal mammary artery: 16.8 MPa, femoral artery: 9−12 MPa), the compliance value of 1.75 (%/100 mmHg) is approaching that of the human saphenous vein of 0.7−1.5 (%/ 100 mmHg).4,5 Furthermore, the compliance and burst pressure of electrospun PU grafts can be tuned to closely match native vessels by altering PU chemistry and fibrous microarchitecture.37 The biodegradability of synthetic polymeric scaffold is critical for their applications in regenerative medicine. Polyester-based polyurethane is an

PUSN-Hep and PUSN-Hep/TPS, while both were higher than that of the original samples (Figure S2). 3.4. Proliferation of EPC and HUVEC. The in vitro adhesion and proliferation of EPCs and HUVECs on the PUSN, PUSN-Hep, and PUSN-Hep/TPS sheets were evaluated at days 1, 3, and 5 culture time points (Figures 9A and 10A). Similar EPCs proliferation was shown on the pristine PUSN and PUSN-Hep, while the highest quantity of EPC attachment was observed on the surface of PUSN-Hep/TPS samples during 5 days culture period. Therefore, the TPS functionalized surface demonstrated enhanced attachment toward EPCs. In the case of endothelial cells, PUSN-Hep/ TPS was found to have much more HUVECs than other surfaces after the first day of culture, while the quantities of cell attachment on various surfaces were similar without significant differences at days 3 and 5. The results demonstrate that both functionalized surfaces showed excellent biocompatibility for endothelial cell proliferation. The morphologies of both cells cultured on three scaffolds at day 3 are shown in Figures 9B−E and 10B−E. The adherent EPC and HUVEC on the fiber scaffolds showed the obvious spreading process other than flat TCPS, although, it was hard to find the obvious morphological differences between original and modified PUSN scaffolds.

4. DISCUSSION Autograft veins and arteries are the preferred vascular substitutes for the surgical treatment of diseased small-diameter vessels, but they are hampered by the limited availability and suitability of donor tissue.3 Recently, biodegradable synthetic vascular graft has become a promising approach for small vascular regeneration, especially a preferred choice for younger patients. However, many challenges still have to be overcome for the synthetic materials by finetuning the inner surface properties, with the tethering of carefully chosen biomolecules, to synergistically preventing thrombogenicity and improving endothelialization. Typically, such covalent dual or multifunctionalization strategies can be achieved by three main steps: (1) bringing moieties onto inert substrate, and (2) introducing a spacer layer acting as both an antithrombogenic background and a chemical linker for (3) further immobilizing endothelium-inducing biomolecules.9,10,12 Our previous studies have shown that biodegradable polyurethane constructs warrant surface treatment before surgical implantation.22,23 The polyurethane substrates can be activated by physical or chemical treatment for the further H

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are necessary for in vivo endothelialization. We found nonmodified PUSN and heparin modified samples showed a relatively close adhesion and proliferation for both kinds of cells, while the proliferation rate of EPC was significantly enhanced with further TPS peptide modification (Figures 9 and 10). The results indicated that both modified scaffolds showed reduced platelet adhesion compared to primary scaffolds, while the dual modified PUSN scaffolds demonstrated a synergistic effect of reduced platelet deposition and improved EPC proliferation in vitro study. Therefore, further study for the modified PUSN grafts would be worth implementing in vivo model. It is worth noting that numerous antithrombosis and EPCspecific bioactive molecules can be carefully chosen and considered in the surface orthogonal functionalization of PUSN scaffolds for designing optimal vascular substitutes. Otherwise, the orthogonally functionalizable platform also has potential in various other biomedical applications by grafting specific molecules through carboxyl-amino condensation reaction and disulfide-based click chemistry.

important class of hydrolytically and enzymatically degradable polymer.19 The current study demonstrated that the developed PUSN could be degraded in water and lipase solution. We also found that PUSN cast samples were degraded faster than electrospun samples in lipase solution, which may be due to the process of electrospinning leading to molecular-level orientation within nanofibers and more amount of hard segment distributed on the surface of fibers,38 thus preventing the fiber degradation from lipase. EPCs are a type of predifferentiated adult stem cells that have the potential to proliferate and differentiate into mature endothelial cells. They have been accepted as an important cell source for in situ endothelialization due to the much higher proliferative potential than mature ECs.15,16 While EPCs circulate in the bloodstream in relatively low abundance in normal, physiological conditions, so graft designs utilizing the accelerated endothelialization potential of these cells must incorporate methods of increased mobilization of EPCs. EPCspecific ligands, including growth factors (VEGF, SDF-1), oligonucleotide (DNA molecules), aptamers (nucleic acids), monoclonal antibodies (Anti-CD34 antibodies and kinase insert domain receptor (KDR)), peptides (Nap-FFGRGD), selectins, or magnetic molecules have been immobilized on cardiovascular devices.15,16,39 Among them, the TPS peptide showed high affinity and specificity to human EPCs in vitro studies.13,14,40 Furthermore, the antithrombogenic property of a blood-contacting surface is required as long as endothelialization has not been completed. Heparin is a widely used anticoagulant, and it is well-known that oral therapy with heparin exhibits undesirable side effects, such as hemorrhagic complications, heparin-induced thrombocytopenia, and/or low bioavailability, while heparin covalent modification could reduce the undesirable limitations of lifelong anticoagulation therapy.33 Therefore, heparin and TPS peptide were chosen for the dual-functionalization of PUSN fibrous scaffolds, expecting to endow the synergistic performances of antithrombosis and EPC capturing for in situ endothelialization under blood flow. Before the biofunctionalization, we confirmed that the strategy for orthogonal covalent modifications could be performed with two fluorescent dyes under the mild conditions (Figure S1). The heparin modification or heparin/TPS modification were confirmed by XPS (Figure 6) and further reflected by that PUSN-Hep and PUSN-Hep/TPS became purple with toluidine blue staining, while only the PUSN-Hep/ TPS showed strong green fluorescence with further FITC labeled TPS click modification (Figure 7). Successful modifications also can be supported by the morphology change and increased diameter of fibers, which are also reflected by the fibrous scaffolds changed from hydrophobic to hydrophilic (Figure 7G−I). The heparin density was the same between PUSN-Hep and PUSN-Hep/TPS, as both were over double the values of physically absorbed heparin on the PUSN scaffolds (Figure 7C). Although heparin can be physically absorbed onto the PUSN scaffolds, probably due to their mutual ionic bonding,41 while covalent conjugation with heparin showed an obviously reduced platelet adhesion and a higher hemoglobin concentration, suggesting a lower rate of clotting. Furthermore, the presence of TPS peptide did not hamper the heparin antithrombogenic properties (Figures 8 and S2). This result can be supported by the former report where TPS and PEG were immobilized on the PCL surface through host−guest assembly technology.14 The proliferations of EPC and HUVEC

5. CONCLUSIONS A novel orthogonally functionalizable polyurethane (PUSN) was developed for surface covalent cofunctionalization. The developed PUSN can be electrospun into fibrous grafts, which showed the matching mechanical compliance to the human native vessel. The scaffolds were successfully modified by heparin and heparin/peptide via covalent conjugation. Both the modified scaffolds showed reduced platelet adhesion compared to primary scaffolds, while heparin/TPS peptide modified scaffolds showed the best efficiency of EPC proliferation. Thus, the orthogonally functionalized PUSN grafts showed the potential application in small diameter vascular regeneration. Further, the orthogonally functionalizable polyurethane also could be applied in other biomedical fields by attaching different bioactive molecules.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b04289. Characterization and modification of PUSN vascular grafts with anticoagulant assay and corresponding results (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (X.M.). *E-mail: [email protected] (M.Y.). Author Contributions ⊥

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by National Nature Science Foundation of China (31271035, 31470941), National Research Foundation for the Doctoral Program of Higher Education of China (20130075110005), Science and Technology Commission of Shanghai Municipality (15JC1490100, 15441905100), and Light of Textile Project (J201404). This I

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research was also supported by National Natural Science Foundation of China (81271726), Shanghai Jiaotong University Biomedical Engineering (PolyU) Cross Research Fund Project (YG2012MS35), and Collaborative Innovation Center for Translational Medicine at Shanghai Jiao Tong University School of Medicine (TM201504).

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