Heparin and zwitterion functionalized small-diameter vascular grafts for thrombogenesis prevention

2.1 Materials

PCL (Mw 45000) and N-(3-dimethylaminopropyl)-N′-ethyl-carbodiimide (EDC) were purchased from Macklin (China). Hexafluoroisopropanol (99.5%) and 1,6-hexanediamine were purchased from Aladdin (China). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and cell counting kit-8 (CCK-8) were purchased from FeiyuBio Co., Ltd. (China). Methanol and ethanol were purchased from Rhawn (China), and chloroform was purchased from SinoPharm (China). The micro-bicinchoninic acid (BCA) assay kit was purchased from CWBio (China), and the lactate dehydrogenase (LDH) activity assay kit was purchased from Solarbio (China). CD-1 mouse whole blood was purchased from SenBeiJia Biological Technology Co., Ltd. (China), and platelet-rich plasma (PRP) preparation solution was purchased from Leagene Biotechnology (China). Lipase was purchased from Bide Pharmatech Ltd.

2.2 Preparation of PCL/SBPU small-diameter artificial vascular grafts

SBPU synthesis commenced with the reaction of N-methyl diethanolamine (0.2 mol) and 1,3-propanesultone (0.2 mol) in dichloromethane under 2 h stirring, followed by 2-day crystallization. The resultant sulfobetaine diol (SBD) was vacuum-filtered, sequentially washed with dichloromethane/isopropanol, and dried for 48 h. Subsequently, SBD (0.01 mol) and methylene diphenyl diisocyanate (0.01 mol) were dissolved in DMSO (30 ml) under N₂ at 80℃. Catalyzed by dropwise addition of dibutyltin dilaurate, polymerization proceeded for 20 min before methanol precipitation and vacuum drying (50℃, 48 h) to yield SBPU. For graft fabrication, double-layered vascular grafts (5 mm) with varying SBPU content (0, 5, 10, 20 wt%) were electrospun: the inner layer (PCL/SBPU blend) at 18 kV, 20 cm collector distance, and 1 mL·h⁻¹ flow rate, while the outer layer (pure PCL) at 12 kV, 20 cm, and 1.5 mL·h⁻¹. These were designated as PCL, PCL/SBPU5, PCL/SBPU10, and PCL/SBPU20, respectively.

2.3 Heparin modification of PCL/SBPU small aperture artificial vascular grafts

All samples (PCL, PCL/SBPU5, PCL/SBPU10, PCL/SBPU20) first underwent amination by immersion in 0.43 g·mL⁻¹ 1,6-hexanediamine/isopropanol at 37℃. Subsequently, heparin (1 mg·mL⁻¹) was activated in 2-(N-morpholino)ethanesulfonic acid buffer (0.05 M, pH 5.0) containing 0.3 mol·L⁻¹ EDC and 0.15 mol·L⁻¹ N-hydroxysuccinimide (37℃, 30 min), followed by grafting the aminated samples in this solution for 24 h at 37℃. Finally, samples were rinsed thrice with deionized water and freeze-dried for 24 h (36,37), yielding heparin-modified small-diameter vascular grafts designated PCLH, PCLH/SBPU5, PCLH/SBPU10, and PCLH/SBPU20.

2.4 Characterization

The synthesized SBD was subjected to nuclear magnetic resonance (NMR) spectroscopy (Bruke, Switzerland). The samples with 1 w/v% concentration were prepared using deuterated dimethyl sulfoxide (DMSO-d ₆), and ¹H NMR spectra ranged from 0 to 10 ppm. The surface morphology of grafts was observed by field-emission scanning electron microscopy (JSM-6510, JEOL Ltd., Japan) with an accelerating voltage of 5 kV. The chemical structure of the grafts was analyzed using Fourier-transform infrared spectroscopy (FI-TR, Nicoiet IS, Yixiang Instrument Co., Ltd., China).

2.5 Water contact angle assay

The hydrophilicity of vascular grafts was evaluated using an optical contact angle instrument (OCA1SEC, DPT GMBH). Surface hydrophilicity of PCLH, PCLH/SBPU5, PCLH/SBPU10, and PCLH/SBPU20 was determined via static and dynamic contact angle methods. Samples were sectioned to expose the inner wall for measurement. Three distilled water droplets were placed at different surface locations to measure contact angles.

2.6 Analysis of mechanical properties

The tensile properties of PCLH, PCLH/SBPU5, PCLH/SBPU10, and PCLH/SBPU20 were evaluated using an Instron 5969 universal testing system. Specimens measured 5 mm in diameter and 5 cm in length, with a gauge length of 3 cm and a crosshead speed of 50 mm·min⁻¹. Each sample was stretched until fracture, recording both the stress–strain curve and fracture strength.

2.7 Degradation assay

Four specimens each of PCLH, PCLH/SBPU5, PCLH/SBPU10, and PCLH/SBPU20 were dried to constant weight. Each sample was sectioned to 1 cm length, weighed, numbered sequentially, and the pre-degradation weight recorded as W ₀. Specimens were immersed in phosphate-buffered saline (PBS) containing 200 U·mL⁻¹ lipase, retrieved at intervals, rinsed thrice with deionized water, dried to constant weight, and reweighed to obtain degraded mass (W₁). Weight loss rates were calculated using Eq. 1 (38):

2.8 Platelet adsorption assay

Samples were sectioned into 1 cm × 1 cm pieces, placed in 24-well plates, disinfected with 70% ethanol and UV light for 10 min, and hydrated in PBS at 37℃ for 1 h. The solution was then removed; 1 mL of platelet solution was added to each well and incubated at 37℃ for 2 h. After removing the platelet solution and washing with PBS three times, 2% Triton X-100 solution was added, followed by incubation at 37℃ for 15 min. Absorbance was finally measured at 450 nm using a microplate reader (39,40).

2.9 Protein adsorption assay

Samples were sectioned into 1 cm × 1 cm pieces and placed in 24-well plates. After adding 1 mL of PBS to each well and equilibrating at room temperature for 2 h, PBS was aspirated. Then, 1 mL of either 2 mg·mL⁻¹ bovine serum albumin (BSA) or 0.1 mg·mL⁻¹ fibrinogen (Fg) solution was added per well and incubated with shaking at 37℃ for 1 h. Following protein solution aspiration and PBS addition, samples were rinsed via repeated pipetting (three cycles). Subsequently, 1 mL of 2 wt% SDS solution was added to each well and incubated with shaking at 37℃ for 2 h. Then, 0.1 mL aliquots of SDS solution were transferred from each well to a 96-well plate. After adding BCA solution to each well, the foil-wrapped plate was incubated at 37℃ for 1–2 h. Absorbance was finally measured at 562 nm using a microplate reader (41,42).

2.10 Coagulation assay

Samples were sectioned into circular sheets measuring 14 mm in diameter. For each experimental group, three replicate specimens were prepared and positioned in 24-well plates, with glass slides designated as positive controls. Each well received 100 μL of whole blood supplemented with 20 μL CaCl_₂ (0.2 mol·L⁻¹), followed by incubation at 37℃. After 1 h, the test materials were removed; 2.5 mL deionized water was introduced and incubated at 37℃ for 5 min. The resulting supernatant was transferred to centrifuge tubes, homogenized by vortex mixing at room temperature for 15 min, and the absorbance was measured at 540 nm. Elevated absorbance values directly indicate increased hemoglobin concentration in the supernatant, demonstrating greater quantities of free blood cells and correspondingly reduced clotted cell populations. This inverse relationship signifies slower coagulation kinetics. Consequently, higher supernatant absorbance correlates with elevated coagulation indices, reflecting superior anticoagulant properties of the biomaterial (43). Blood clotting index (BCI) is calculated according to the following Eq. 2:

where the absorbance value of the sample under test is denoted as I _t, and I _w is the absorbance value of the mixed solution obtained by 2.5 mL deionized water and 100 μL whole blood (44).

2.11 Cytotoxicity assay

Samples were sectioned to dimensions of 1 cm × 3 cm, sterilized via UV irradiation for 24 h, and subsequently immersed in PBS for ≥24 h to eliminate residual contaminants. L929 fibroblast cells were maintained in DMEM supplemented with 10% FBS at 37℃ under 5% CO₂. Sterilized graft samples (ethanol-immersed and UV-irradiated) were incubated in serum-free DMEM at an extraction ratio of 3 cm²·mL⁻¹ for 24 h at 37℃. The resulting extracts were then applied to cell monolayers for 24 h exposure. Cell viability was quantified via CCK-8 assay, measuring absorbance at 450 nm with 10% DMSO and plain DMEM serving as positive/negative controls, respectively (45,46). The cell viability was calculated according to the following Eq. 3:

2.12 Statistical analysis

All the experiments were done independently in three replicates, and the data were stated as mean value ± standard deviation. The one-way analysis of variance (ANOVA) and Tukey’s multiple comparison tests were used to analyze the significance of differences between experimental groups. A value of p* < 0.05 and p** < 0.01 was considered to be statistically significant. In this study, the analysis of tensile strength and cell viability data was conducted using ANOVA.

3.1 Nuclear magnetic resonance test (¹H NMR)

To confirm the successful synthesis of SBD, the chemical structure of the artificial vascular material was systematically characterized by NMR analysis using DMSO-d₆ as the solvent (Figure 1). Resonances at 3.82 ppm were assigned to the urethane linkage, while those spanning 2.1–4.6 ppm corresponded to the methyl and methylene protons of the SB moiety. Collectively, these findings confirm the successful synthesis of SBPU.

Figure 1

¹H NMR spectra of SBD.

3.2 Analysis of infrared spectral analysis

FT-IR spectra of the scaffolds are presented in Figure 2. All samples exhibited characteristic absorption peaks at approximately 1,720 and 1,171 cm⁻¹, corresponding to C═O and C–O–C stretching vibrations, respectively (28,47). A distinctive peak at ∼2,950 cm⁻¹, characteristic of PCL, was observed in all cases, confirming retention of PCL’s chemical integrity following incorporation of SBPU and heparin. In this study, heparin was covalently immobilized onto the PCL/SBPU matrix to provide sustained anticoagulant functionality while avoiding burst release issues inherent to physical blending methods. Characteristic heparin absorption peaks appeared at 3,300–3,500 cm⁻¹ (N–H and O–H stretching vibrations) (37,44). All four spectra displayed defined peaks at 3,265 cm⁻¹, verifying successful heparin grafting onto sample surfaces. Additionally, SBD spectra showed broad bands within 3,200–3,500 cm⁻¹ (O–H stretching) (39).

Figure 2

(a) FT-IR spectra of PCLH, PCLH/SBPU5, PCLH/SBPU10, PCLH/SBPU20, (b) enlarged FT-IR spectra of PCLH, PCLH/SBPU5, PCLH/SBPU10, PCLH/SBPU20.

3.3 Analysis of scanning electron microscope (SEM)

The micromorphology and diameter distributions of PCLH, PCLH/SBPU5, PCLH/SBPU10, and PCLH/SBPU20 samples are presented in Figure 3. Panels (a) through (d), respectively, display SEM images of these four material variants. Notably, SBPU incorporation induces significant alterations in fiber surface smoothness, with the magnitude of this effect being concentration-dependent. Corresponding fiber diameter distributions are shown in panels (e)–(h), revealing that increasing SBPU content progressively broadens the diameter distribution profile.

Figure 3

(a)–(d) Scanning electron micrographs of PCLH, PCLH/SBPU5, PCLH/SBPU10, PCLH/SBPU20, respectively. (e)–(h) Corresponding diameter distributions of PCLH, PCLH/SBPU5, PCLH/SBPU10, PCLH/SBPU20.

3.4 Analysis of water contact angle assay

The hydrophilic properties and wettability of vascular grafts constitute critical determinants influencing cellular adhesion, infiltration, and proliferation (48). Dynamic contact angle analysis quantifies material hydrophilicity through temporal measurements from initial liquid contact to complete wetting. As shown in Figure 4, heparin-modified grafts demonstrated significantly enhanced hydrophilicity, achieving complete liquid wetting within 0.43 s (49). The blending of PCL with SBPU revealed composition-dependent wettability characteristics. Incorporation of SBPU prolonged wetting duration, most notably in the 10 wt% PCLH-SBPU composite which exhibited the longest wetting time (4.79 s). This effect may be attributed to surface roughness modifications induced by SBPU addition that temporarily reduced hydrophilicity. In contrast, at 20 wt% SBPU loading, complete wetting time decreased significantly to 1.2 s. This reversal phenomenon stems from increased density of hydrophilic functional groups (ester linkages C═O and aldehyde groups ‒CHO) within the graft matrix at higher SBPU concentrations, enhancing surface hydrophilicity through strengthened polar interactions with aqueous media.

Figure 4

Contact angle projections of PCLH, PCLH/SBPU5, PCLH/SBPU10, and PCLH/SBPU20.

3.5 Analysis of mechanical properties

Uniaxial tensile testing evaluated the mechanical properties of vascular grafts to ensure adequate post-implantation mechanical stability (50,51). Figure 5 demonstrates that fracture strengths of SBPU-incorporated samples (3.17, 3.79, 4.49 MPa) significantly exceeded the PCLH control (1.72 MPa). This indicates that SBPU enhances tensile strength proportionally to its concentration. The mechanical improvement likely stems from structural differences between SBPU and PCLH molecules, where SBPU’s integration into PCLH chains modifies material behavior. Furthermore, increasing SBPU content correspondingly elevated strain tolerance, demonstrating concentration-dependent ductility enhancement.

Figure 5

Tensile stress–strain curves of PCLH, PCLH/SBPU5, PCLH/SBPU10, and PCLH/SBPU20.

3.6 Analysis of degradation assay

Enzymatic degradation was evaluated in lipase-containing PBS (200 U·mL⁻¹). Figure 6 displays time-dependent weight loss profiles for PCLH, PCLH/SBPU5, PCLH/SBPU10, and PCLH/SBPU20. PCLH underwent rapid degradation with >95% mass loss within 3 days, indicating accelerated enzymatic hydrolysis that potentially compromises cellular integration in vascular grafts. Conversely, SBPU-incorporated composites exhibited significantly retarded degradation kinetics. After initial degradation, PCLH/SBPU5, PCLH/SBPU10, and PCLH/SBPU20 reached weight loss equilibrium, demonstrating an inverse correlation between residual mass and SBPU content. Notably, PCLH/SBPU20 achieved only 15% total mass loss, confirming composition-dependent degradation resistance.

Figure 6

Weight loss rate of four groups of samples PCLH, PCLH/SBPU5, PCLH/SBPU10, and PCLH/SBPU20.

This stabilization confirms SBPU effectively modulates PCLH degradation kinetics, enabling prolonged structural–mechanical stability essential for vascular graft performance. The underlying mechanism likely involves SBPU’s superior hydrolysis resistance compared to PCL, attributable to strengthened interchain bonding within the composite matrix. These modifications promote endothelial cell adhesion and angiogenesis by maintaining scaffold integrity, consequently supporting long-term patency.

3.7 Analysis of platelet adsorption assay

As shown in Figure 7, L-LDH activity values for SBPU-modified samples were consistently lower than the PCLH control group, indicating progressive enhancement of antiplatelet performance with increasing SBPU content. At 5 wt% SBPU loading, L-LDH activity remained comparable to PCLH. However, significant reductions occurred at 10 and 20 wt% concentrations, with PCLH/SBPU20 exhibiting the lowest activity (5.88 U·mL⁻¹). These results demonstrate superior antiplatelet properties in PCLH/SBPU10 and PCLH/SBPU20 composites. The underlying mechanism likely involves SBPU-mediated suppression of platelet adhesion and activation on the vascular graft surfaces.

Figure 7

L-LDH viability values for four groups of samples PCLH, PCLH/SBPU5, PCLH/SBPU10, and PCLH/SBPU20.

3.8 Analysis of protein adsorption assay

Protein adsorption experiments assessed vascular graft protein affinity to predict biocompatibility and potential immune responses in clinical applications (52). Figure 8 shows BSA and Fg adsorption profiles across the four sample groups. Results demonstrate reduced protein adsorption with SBPU incorporation compared to PCLH controls, showing progressive attenuation proportional to SBPU content. This concentration-dependent reduction confirms SBPU enhances the material’s anti-protein adsorption efficacy.

Figure 8

PCLH, PCLH/SBPU5, PCLH/SBPU10, and PCLH/SBPU20 four groups of samples (a) BSA protein adsorption and (b) Fg protein adsorption.

3.9 Analysis of coagulation assay

The coagulation index (BCI) at designated timepoints reflects material anticoagulant capacity. Figure 9 presents 1-h BCI results for four vascular graft groups. PCLH samples exhibited comparatively low BCI values after 1-h coagulation. With increasing SBPU content (5–20 wt%), the coagulation index demonstrated progressive elevation proportional to SBPU concentration. While PCLH/SBPU5 showed minimal BCI deviation from PCLH controls, PCLH/SBPU10 and PCLH/SBPU20 composites displayed significantly enhanced indices. This concentration-dependent enhancement confirms superior anticoagulant performance at higher SBPU loadings.

Figure 9

Histogram of coagulation indices for five groups of samples, positive control, PCLH, PCLH/SBPU5, PCLH/SBPU10, and PCLH/SBPU20.

3.10 Analysis of cytotoxicity assay

Figure 10 results confirm cell viability exceeding 80% for all PCLH, PCLH/SBPU5, PCLH/SBPU10, and PCLH/SBPU20 samples. This demonstrates that SBPU incorporation and heparin modification induce no cytotoxic effects in PCL small-diameter vascular grafts. All four graft variants exhibit inherent biocompatibility, consequently supporting unimpeded cellular proliferation.

Figure 10

Cell viability of four groups of samples PCLH, PCLH/SBPU5, PCLH/SBPU10, and PCLH/SBPU20.