Clopidogrel eluting electrospun polyurethane/polyethylene glycol thromboresistant,hemocompatible nanofibrous scaffolds

Abstract

Biomaterials used as blood-contacting material must be hemocompatible and exhibit lower thrombotic potential while maintaining hemostasis and angiogenesis. With the aim of developing thromboresistant, hemocompatible nanofibrous scaffolds, polyurethane/polyethylene glycol scaffolds incorporated with 1, 5, and 10 wt% Clopidogrel were fabricated and evaluated for their physiochemical properties, biocompatibility, hemocompatibility, and antithrombotic potential. The results of physicochemical characterization revealed the fabrication of nanometer-sized scaffolds with smooth surfaces. The incorporation of both polyethylene glycol and Clopidogrel to polyurethane enhanced the hydrophilicity and water uptake potential of polyurethane/polyethylene glycol/Clopidogrel scaffolds. The dynamic mechanical analysis revealed the enhancement in mechanical strength of the polyurethane/polyethylene glycol scaffolds on incorporation of Clopidogrel. The polyurethane/polyethylene glycol/Clopidogrel scaffolds showed a tri-phasic drug release pattern. The results of hemocompatibility assessment demonstrated the excellent blood compatibility of the polyurethane/polyethylene glycol/Clopidogrel scaffolds, with the developed scaffolds exhibiting lower hemolysis, increased albumin and plasma protein adsorption while reduction in fibrinogen adsorption. Further, the platelet adhesion was highly suppressed and significant increase in coagulation period was observed for Clopidogrel incorporated scaffolds. The results of cell adhesion and cell viability substantiate the biocompatibility of the developed nanofibrous scaffolds with the HUVEC cell viability on polyurethane/polyethylene glycol, polyurethane/polyethylene glycol/Clopidogrel-1, 5, and 10% at day 7 found to be 12.35, 13.36, 14.85, and 4.18% higher as compared to polyurethane scaffolds, and the NIH/3T3 cell viability found to be 35.27, 70.82, 36.60, and 7.95% higher as compared to polyurethane scaffolds, respectively. Altogether the results of the study advocate the incorporation of Clopidogrel to the polyurethane/polyethylene glycol blend in order to fabricate scaffolds with appropriate antithrombotic property, hemocompatibility, and cell proliferation capacity and thus, might be successfully used as antithrombotic material for biomedical application.

Keywords

Hemocompatible electrospinning platelet adhesion cardiovascular coagulation

Introduction

Thrombosis is a significant, self-amplifying measure necessary for maintaining hemostasis.¹ It involves the accumulation of circulating platelets at the site of injury, and further activation of the coagulation cascade that produces thrombin and ultimately fibrin to stabilize the clot.¹ However, preventing thrombosis in the blood-contacting materials such as vascular graft, stents, heart valves, and vascularized tissues and organs is critical for their proper functioning.¹ A material to be successfully used for blood-contacting applications must be hemocompatible, and lower thrombosis on its surface while maintaining hemostasis and angiogenesis.^2,3

Synthetic biomaterials used as blood-contacting materials primarily encompass polymers and metals, or a combination of both.⁴ The physical, chemical, and biological properties of polymers are tunable, and thus their use as healthcare materials has evolved over the years.⁵ Electrospinning is a versatile technique used to generate polymeric fibrous scaffolds with fiber diameters ranging from micrometers to nanometers which could have a great potential to mimic the microenvironment of a natural extracellular matrix (ECM).^6–8 The use of nanofibers is a novel method for developing a physical barrier between the bloodstream and the tissue, offering a potential utility as a coating material for stents and other structural materials. The electrospun nanofibrous scaffolds have a higher surface area, high porosity, along with tunable mechanical properties. Nanofibers have been potentially used as drug delivery carriers. Moreover, fibers containing dual or multicomponent may be electrospun, exhibiting a set of properties that a single component fiber would not suffice.²

Thermoplastic polyurethanes (TPUs) belong to a class of the versatile polymer synthesized by using reaction of a polyol with a diisocyanate or a polymeric isocyanate group in the presence of suitable catalyst and additives.⁴ TPU has excellent mechanical properties such as elasticity, transparency, good processability⁴ and has been used for developing blood-contacting materials such as cardiovascular biomaterials, vascular grafts, hemodialysis membranes, blood storage containers, central venous catheters, IV bags, parts of joint prostheses, and various implants.^5,9,10 Moreover, they have also been used in coating stents, artificial heart, and vascular grafts.^11,12 The probability of TPU for thrombosis is similar to the other most commonly used materials such as expanded polytetrafluoroethylene and polyethylene terephthalate.^4,13 Although TPU is durable, its poor hydrophilicity, surface-induced thrombosis property, protein fouling, intrinsic antimicrobial properties, and cell affinity are a bottleneck for its biomedical applications as blood-contacting biomaterials.^9,14

Biomaterials may be confronted with adsorption of nonspecific proteins such as fibrinogen and immunoglobulin G (IgG) which may be an unfavorable event leading to pathological responses such as thrombosis, blood coagulation, and infections.^5,15 The surface properties of a material are known to dictate the biological response toward the material; thus, in order to address the multifaceted and self-enforcing characteristics of thrombosis and coagulation cascade, incorporation of biocompatible additives to the polymer may be explored to produce a antithrombotic surface.^1,16,17 Increasing the hydrophilicity of the material through the incorporation of a hydrophilic polymer may be one of the strategies to reduce the nonspecific protein adsorption on the material surface and thus, improving its biological properties. In this regard, polyethylene glycol (PEG) has been known as a biocompatible material having large number of hydrophilic groups on its surface¹⁶ and is often used as a hydrophilic polymer to create nonfouling materials resisting nonspecific protein adsorption which may be an unfavorable event promoting thrombosis, coagulation, and further infections.^{1,9,15,16,18,19} Wang et al.⁹ fabricated small diameter scaffolds using blends of polyurethane (PU) and PEG and reported hemocompatible PU/PEG co-electrospun blends with PEG content between 20 and 30 wt% supporting extensive endothelialization. Similar results have been reported by Karahaliloglu¹⁶ wherein they reported the fabrication of nanofibrous scaffold through a combination of PU and hydrophilic PEG as a potential substitute for small diameter vascular grafts. However, most in vivo studies related to PEG-modified implants have failed to prove the significant thromboresistant property of PEG-modified surfaces.^1,20,21 This might be because of the PEG layer getting modified by plasma proteins or because PEG layer may not be able to resist the adhesion of platelet aggregates.^1,20

In the clinical setting, patients are treated with systemic antithrombotic drugs such as anticoagulants and platelet inhibitors in order to prevent early graft occlusion.¹¹ Thus, incorporation of an anticoagulant provides an attractive choice in developing thromboresistant, hemocompatible nanofibrous scaffolds. With this regards, Clopidogrel, acetylsalicylic acid (aspirin), and dipyridamole are the gold standard for the secondary prevention of noncardioembolic ischemic stroke/transient ischemic attack.^11,22 The Clopidogrel versus Aspirin in Patients at Risk of Ischaemic Events trial has demonstrated marginal superiority for Clopidogrel over aspirin as a suitable antiplatelet drug.¹¹

Clopidogrel bisulfate is a second generation thienopyridine that prevents the formation of blood clots by irreversibly inhibiting the binding of an ADP to the platelet receptor P2Y12 and subsequent activation of glycoprotein GPIIb/IIIa complex, thereby inhibiting platelet aggregation. Due to favorable side effects and a comparatively rapid onset of action, Clopidogrel was preferred over Ticlopidine to prevent the occurrence of stent thrombosis in patients undergoing stenting.^23,24 Thus, we hypothesize that the incorporation of Clopidogrel in PU/PEG blend may provide an attractive strategy to develop a blood-contacting material with better thromboresistance and hemocompatibility.

In the current study, PU/PEG nanofibrous scaffolds incorporating Clopidogrel drug (1, 5, and 10 wt%) were fabricated using electrospinning technique. The concentration of PEG (20 wt%) in PU/PEG blend was selected on the basis of the previous study conducted by Wang et al.⁹ to prevent microphase separation of polymer. The scaffolds were evaluated for their physiochemical properties; morphological characterization of the nanofibrous scaffolds was carried out using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM). The elemental and chemical composition was determined by energy dispersive X-ray analysis (EDAX) and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) analysis. The surface hydrophilicity and mechanical properties were determined using contact angle (CA) analysis and dynamic mechanical analysis (DMA), respectively. The thermal properties were studied by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) analysis. The nature of scaffolds was determined using wide angle X-ray diffraction (WAXD) analysis and the Clopidogrel release from the scaffolds at pH 7.4 was determined. The hemocompatibility and the antithrombogenic properties of the nanofibrous scaffolds were evaluated by determining the hemolysis property, the adsorption of proteins such as bovine serum albumin (BSA), fibrinogen, and plasma protein followed by platelet adhesion and blood coagulation time (APTT and PT assay). Further, the in vitro biocompatibility of the scaffolds was evaluated by determining the cell adhesion, and cell viability of NIH/3T3 cells and HUVEC cells on the scaffolds.

Materials and methods

Materials

Clopidogrel bisulfate (Sigma Aldrich) (Figure 1), TPU, and Elastollan®1185-A-10 were obtained as a gift from Badische anilin-und Soda-Fabrik. PEG (Mw 6000 Da), tetrahydrofuran (THF), and dimethylformamide (DMF) were purchased from Loba Chemie. Rabbit brain calcified thromboplastin reagent (Uniplastin®) for PT test, rabbit brain cephaloplastin reagent (Liquicelin®-E), and calcium chloride 0.025 M recalcification solution for APTT test were procured from Tulip Diagnostics (P) Ltd, India. Bovine serum albumin, fibrinogen from human plasma, and Wright–Giemsa stain were purchased from Sigma Aldrich. All the chemicals were of the highest purity available.

Figure 1.

Structure of Clopidogrel bisulfate drawn using ChemDraw software.

Solution preparation for electrospinning

PU solution was obtained by dissolving PU granules at the concentration of 12% w/v in a solution of THF:DMF (70:30). PU/PEG blend solution was prepared by adding PEG at the final concentration of 20% w/w to the PU solution. Further, Clopidogrel (clop) was incorporated in PU/PEG blend solution at varying concentrations (1, 5, and 10% w/w of total polymer weight) in order to obtain solutions for spinning PU/PEG/Clopidogrel scaffolds.

Electrospinning nanofibers

Randomly oriented nanofibers of PU, PU/PEG, PU/PEG/Clop-1%, PU/PEG/Clop-5%, and PU/PEG/Clop-10% were fabricated using mono-axial electrospinning (Physic equipment, India). The electrospinning parameters were optimized in order to achieve beadless fiber morphology. The solution was ejected at the flow rate of 1 ml/h through a 24G needle placed at a distance of 20 cm from the rotating mandrel (speed <100 r/min) used to deposit nanofibers. The voltage was set at 20 kV and the fibers were electrospun at ambient temperature.

Physicochemical characterization of nanofiber

SEM

The morphological analysis of the nanofibrous scaffolds was performed using environmental SEM (E-SEM, Quanta 200 3D, FEI, USA). The diameter of the randomly selected nanofibers in SEM micrograph was measured using NIH Image J software and the histogram of the frequency distribution (n) of diameters of nanofiber (nm) was plotted.

TEM

The nanofibers were deposited on a carbon-coated copper grid (100 meshes × 3 mm) and TEM micrographs of the nanofiber were recorded using FEI-Technai-G2-T20 instrument operated at 200 keV.

Energy dispersive X-Ray analysis (EDAX)

EDAX analysis was performed to determine the elemental composition of the nanofibers. EDAX analysis was performed using the EDAX accessory equipped on the FEI-Tecnai-F20 TEM instrument operated at an accelerating voltage of 200 kV. The percentage elemental composition of C, N, O, S, and Cl was evaluated.

AFM

The surface roughness of nanofibrous scaffolds (1 cm²) was determined using AFM on a silicon wafer using a multimode scanning probe microscope equipped with a Nanoscope IV controller from Veeco Instrument Inc., Santa Barbara, CA. All the AFM measurements were done under ambient conditions to measure the root mean square roughness (Rq), using the tapping-mode AFM probes (model—Tap190Al purchased from Budget Sensors). For each sample, three locations with a surface area of 1 × 1 µm² and 500 × 500 nm² were imaged at a rate of 1 Hz and at a resolution of 512 × 512 pixels.

CA analysis

To study the hydrophilicity of developed material, the CA was measured using Kruss Drop Shape Analyzer. A drop of approximately 3 µl was placed on the scaffold and immediately the CA was measured using Kruss ADVANCE software. The measurements were carried out on randomly selected areas of at least three independent specimens and the average values were reported as mean ± SD.

Water uptake (WU) of nanofibers

Scaffolds were cut in 1 cm² (n = 3) and their dry weights were recorded. The scaffolds were soaked in milliQ water for 24 h at room temperature. Post incubation, the weights of the scaffolds were recorded immediately after removing from water and wiping with tissue paper. WU was calculated as WU (mg/cm²) = [(W2−W1)×S], where W1 and W2 represent the weights before and after soaking in water and S represents the surface area of the scaffold.^9,25

ATR-FTIR

FTIR spectra of nanofibrous scaffolds were recorded using a Perkin Elmer IR FTIR spectrometer, USA in the wavelength range of 500–4000 cm⁻¹ at a resolution of 4 cm⁻¹ and 32 scans per spectra in ATR mode.

TGA

TGA of nanofiber was conducted using a STA6000 Perkin-Elmer instrument in the range of 30–500°C at a rate of 10°C min⁻¹ under the nitrogen atmosphere.

DSC

DSC measurements of nanofiber were performed on a TA Q20 instrument at a heating rate of 10°C min⁻¹ under the nitrogen atmosphere. Typically, 4–5 mg of samples was placed in an aluminum pan, properly sealed, and scanned in the range of 0–250°C.

WAXD

The nanofibrous scaffolds were analyzed using WAXD in order to get more insights about the nature of the sample. The analysis was performed in the range of 2θ = 5–55° at a room temperature (25°C). The WAXD patterns of nanofiber sample were analyzed by Philips 1830 X-ray Diffractometry (Philips, Almelo, The Netherlands) using a Cu Kα source at a (λ = 1.5406 Å).

DMA

The mechanical strength of the nanofiber was measured using a dynamic mechanical analyzer (RSA3, TA Instruments, USA). The samples were cut into strips with dimensions of 3 × 1.4 cm² and then mounted onto the tensile grips. Each sample was tested five times to authenticate its normal stress–strain curves.

In vitro drug release study

The Clopidogrel drug release behavior from the nanofibrous scaffolds of PU/PEG/Clop-1%, Clop-5%, and Clop-10% was examined using UV–visible spectrophotometer at a wavelength of 220 nm. Each sample of the nanofibrous scaffold (n = 3) of weight 10 mg was immersed in 10 ml phosphate buffer saline (pH =7.4) and was then shaken at 120 r/min on a magnetic stirrer at 37°C. At different time intervals, 1 ml solution from the release medium was replaced with fresh PBS of the respective pH 7.4 and was used to evaluate the cumulative drug release.

In vitro hemocompatibility analysis

Hemolysis assay

The blood compatibility of the nanofibers was evaluated using hemolysis assay.²⁶ Human blood was obtained from a blood bank (Indian Serological Institute, Pune, India) and used as received. The red blood cells (RBCs) were collected by centrifugation of blood at 1200 r/min for 10 min. RBCs were washed twice with D-PBS and diluted at a ratio of 1:20 (v/v) using D-PBS. The scaffolds (1 cm², n = 3) were washed with D-PBS, placed in a 24-well plate; 1 ml diluted RBC solution was added per well and incubated at 37°C for 1 h. The sample was transferred to the centrifuge tube, centrifuged at 3000 r/min for 10 min, and the absorbance of the supernatant was recorded at 545 nm. PBS diluted blood was taken as negative control whereas 0.2% Triton-X-100 diluted blood was taken as positive control. The hemolysis percentage was estimated using the following equation.

Hemolysis % = [(OD value of Sample − OD value of Negative)/(OD value of Positive − OD value of Negative)] × 100

BSA/fibrinogen adsorption

Scaffolds (1 cm², n = 3) were placed in a 2 ml centrifuge tube. The scaffolds were equilibrated by incubating in 1 ml D-PBS/tube for 1 h at 37°C. One milliliter of BSA (1 mg/ml) or fibrinogen (1 mg/ml) was added per sample and incubated for 24 h at 37°C. Post incubation, the scaffolds were washed thrice with PBS and 2% w/v aqueous SDS solution was added. The tubes were kept on a shaker (200 r/min) for 2 h followed by bath sonication for 10 min. BCA assay of the protein lysate was performed as per manufacturer’s protocol and readings of protein adsorption were recorded at 562 nm.^27–29

Plasma protein adsorption

Platelet-poor plasma was procured from Indian Serological Institute, Pune, India. Plasma protein adsorption was determined by the method similar to the BSA or fibrinogen protein adsorption as mentioned above.

Platelet adhesion and activation

Platelets were obtained from Indian Serological Institute, Pune and maintained at 22–24°C on a rotator shaker until use. Scaffolds were placed in 24-well plates and 1 ml of platelets was added to each well and incubated at 37°C for 1 h. Platelets were aspirated from each well, loosely attached platelets on the scaffolds were removed by washing twice with 1× PBS. The adhered platelets were fixed on the scaffolds by adding 1 ml of 2.5% glutaraldehyde solution to each well and further incubating at 4°C for 4 h. Glutaraldehyde was removed and scaffolds were washed with PBS. Scaffolds were further stained by 1× Wright–Giemsa stain solution for 15 min, washed with PBS to remove excess stain, mounted on a clean glass slide, and observed under 20× phase contrast inverted microscope (Zeiss). The average number of adhered platelets was obtained from three photographs of different areas on the same sample. The platelet adhered scaffolds fixed with 2.5% glutaraldehyde were dehydrated by immersing in increasing concentration of ethanol in water (30–100%) for 30 min each. The platelet adhesion on the different scaffolds was observed using FE-SEM.^20,28

Activated partial thromboplastin time (APTT) assay

Scaffolds of PU, PU/PEG, PU/PEG/Clop-1%, PU/PEG/Clop-5%, and PU/PEG/Clop-10% were cut into 1 cm² (n = 3), gently washed with milliQ water and introduced into 48-well plates. The samples were soaked in D-PBS at 37°C for 30 min. One hundred microliters of platelet-poor plasma was introduced in the wells and incubated for 1 min at 37°C. One hundred microliters of rabbit brain cephaloplastin reagent was added, mixed, and incubated for 3 min at 37°C followed by addition of 100 µl of calcium chloride (CaCl₂). The mixture was stirred using the needle and the blood clot time was recorded using chronometer.³⁰

Prothrombin time (PT) assay

Scaffolds of PU, PU/PEG, PU/PEG/Clop-1%, PU/PEG/Clop-5%, and PU/PEG/Clop-10% were cut into 1 cm² (n = 3), gently washed with milliQ water and introduced into 48-well plates. The samples were soaked in D-PBS at 37°C for 30 min. One hundred microliters of platelet-poor plasma was introduced in the wells and incubated for 1 min at 37°C followed by addition of 100 µl of calcified thromboplastin reagent. The mixture was stirred using the needle and the time required until the blood clot was recorded using a chronometer.³⁰

In vitro biocompatibility assessment

Cell adhesion on nanofibrous scaffolds

The adhesion of NIH/3T3 cells, cultured on the scaffolds for 24 h was determined using confocal laser scanning microscopy (CLSM). Scaffolds were cut into1 cm², UV sterilized, and prewetted in DMEM media overnight. Cells were seeded on scaffolds and placed in 24-well culture plates at a density of 1 × 10⁵ cells/cm² and further incubated. On achieving respective time points, the cell grown scaffolds were stained by Alexa fluor 488 Phalloidin stain and Hoechst stain. Briefly, the scaffolds were washed with D-PBS and fixed by adding 200 µl of 4% paraformaldehyde (15 min incubation followed by washing with D-PBS). Cells were permeabilized using 200 µl of 0.1% Triton-X-100 (5 min incubation followed by washing with D-PBS) and stained for 20 min each with 200 µl of 50 µg/ml Alexa fluor 488 Phalloidin stain, and further with 200 µl of 1 µg/ml of Hoechst stain. The samples were kept at 4°C until the CLSM analysis was performed.

Cell viability on nanofibrous scaffolds

The viability of HUVEC cells and NIH/3T3 cells cultured on the scaffolds for one, three, and seven days was determined using MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide) assay. The HUVEC cells were cultured in HiEndo™ Endothelial cell expansion media (Himedia Biosciences) whereas NIH/3T3 cells were cultured in DMEM supplemented with FBS. To perform cell viability analysis, scaffolds were cut into1 cm², UV sterilized, and prewetted in the respective plain media overnight. HUVEC or NIH/3T3 cells were seeded on scaffolds placed in 24-well culture plates at a density of 1 × 10⁴ cells/cm² and further incubated. On achieving respective time points, the cell grown scaffolds were transferred to a fresh 24-well plate. Two hundred microliters of MTT (1 mg/ml) was added to each well and incubated at 37°C for 4 h in a humidified atmosphere of 5% CO₂. The purple formazan reaction product was dissolved by adding 200 µl dimethyl sulfoxide and shaking the plate for 10 min (200 r/min). The absorbance was recorded at 570 nm using a multi-plate reader (Eon, Biotek).

Statistical analysis

The results were expressed as the mean ± standard error of the mean (mean ± SEM). Statistical analysis was carried out using one-way ANOVA, and differences of P < 0.05 were considered to be statistically significant.

Results and discussion

Physicochemical characterization of nanofiber

SEM

E-SEM images of the fabricated nanofibrous scaffolds are shown in Figure 2; the histograms for the size distribution of nanofibers as analyzed by Image J software are shown in Figure 3. The results showed the fabrication of scaffolds with continuous, beadless morphology of the nanofibers. The blending of PU with 20 wt% PEG caused the reduction in the diameters of the PU/PEG nanofiber. This might be as a result of the improved conductivity properties of the blend solution. Similar trends were also observed for Clopidogrel-based nanofibers, wherein increasing concentration of Clopidogrel 1, 5, and 10 wt% resulted in decreased fiber diameters of the fabricated nanofibers. Nanofibers within the scaffolds showed a uniform size distribution and cylindrical morphology without surface deposition of Clopidogrel which shows uniform drug distribution. PU nanofibers showed rough surface morphology whereas addition of PEG and further Clopidogrel to PU or PU/PEG blend polymer solution resulted in smoother nanofiber surfaces as shown in FE-SEM (Figure 4).

Figure 2.

E-SEM micrographs for representative nanofibrous scaffolds: (a) PU, (b) PU/PEG, (c) PU/PEG/Clop-1%, (d) PU/PEG/Clop-5%, and (e) PU/PEG/Clop-10% with the scale bar of 10 µm.

Figure 3.

Histograms representing the size distribution of nanofibers as measured by Image J software: (a) PU, (b) PU/PEG, (c) PU/PEG/Clop-1%, (d) PU/PEG/Clop-5%, and (e) PU/PEG/Clop-10%.

Figure 4.

Representation of the surface morphology of nanofibrous scaffolds through FE-SEM micrographs for representative nanofibrous scaffolds: (a) PU, (b) PU/PEG, (c) PU/PEG/Clop-1%, (d) PU/PEG/Clop-5%, and (e) PU/PEG/Clop-10% with the scale bar of 200 nm.

TEM

The TEM image of the nanofiber as observed in Figure 5 shows the internal morphology of nanofibers. Incorporation of Clopidogrel within PU/PEG nanofibers was evident with the nanofibers showing visually smooth surfaces with no aggregation of the drug.

Figure 5.

TEM micrographs for representative nanofibrous scaffolds: (a) PU, (b) PU/PEG, (c) PU/PEG/Clop-1%, (d) PU/PEG/Clop-5%, and (e) PU/PEG/Clop-10% with the scale bar of 200 nm.

EDAX analysis

The results of EDAX analysis as shown in Table 1 confirmed the blending of PEG with PU as a result of an increase in elemental composition of oxygen in the PU/PEG scaffolds from 26.14% (PU) to 31.46% (PU/PEG). Further incorporation of Clopidogrel resulted in the emergence of peaks corresponding to sulfur and chlorine with their elemental composition increasing with the increasing drug content in the PU/PEG/Clop nanofibrous scaffolds.

Table 1.

Representation of the elemental composition of nanofibrous scaffolds analyzed by EDAX.

S no.	Sample	Elemental composition (wt%)
S no.	Sample	C	N	O	S	Cl
1	PU	69.71	3.55	26.74	–	–
2	PU/PEG	65.18	3.36	31.46	–	–
3	PU/PEG/Clop-1%	66.81	2.59	28.45	1.98	0.17
4	PU/PEG/Clop-5%	66.01	2.58	29.03	2.21	0.18
5	PU/PEG/Clop-10%	65.19	3.79	28.45	2.37	0.21

EDAX: energy dispersive X-ray analysis; PEG: polyethylene glycol; PU: polyurethane.

AFM

AFM helps to evaluate the surface roughness of nanofiber scaffolds.² AFM images of the nanofiber scaffolds analyzed in the tapping mode under dry condition are shown in Figure 6. Surface roughness parameter that is the root mean square parameter (Rq) was calculated for each nanofiber scaffold with similar surface area and represented in Figure 7. The Rq parameters for PU, PU/PEG, and PU/PEG/Clop-1, 5, and 10 wt% were as follows: 1678, 1331, 1289, 1018, and 876 nm, respectively. The results showed the order of surface roughness, which was as follows: PU > PU/PEG > Clop-1 wt% > Clop-5 wt% > Clop-10 wt%. Surface topography and surface roughness are critical factors affecting the hemocompatibility of materials.² Rough surfaces with higher surface area cause enhanced platelet adhesion, its spreading on the substrate and subsequent platelet activation.^2,31,32 ultimately leading to rapid blood coagulation.²

Figure 6.

Three-dimensional surface topography of the nanofibrous scaffolds using AFM.

Figure 7.

Root mean square roughness values representing the surface roughness parameter for nanofibrous scaffolds of PU, PU/PEG, and PU/PEG with varying concentrations of Clopidogrel. PEG: polyethylene glycol; PU: polyurethane.

CA analysis

Water CA analysis and WU analysis are commonly employed to characterize the scaffold surfaces for their relative hydrophobicity or hydrophilicity.⁹ Surface wettability is a critical factor affecting protein adsorption on a surface,⁵ which has a correlation with thromboresistance properties of a material. A scaffold with hydrophilic property is thought to inhibit nonspecific protein adsorption.^17,21 Enhancement in the average unfolding of proteins and increase in its spreading has been reported to occur on hydrophobic surfaces^5,17,33–35; in contrast, hydrophilic surfaces promote cell adhesion and proliferation, while resisting the undesired protein adsorption.⁹ Hydrophilic surfaces are strongly bound with water layer which is difficult to be displaced by adsorbed protein. In contrast, the proteins readily displace water from hydrophobic surfaces in order to get adsorbed.^5,17,36 It gets difficult to desorb the proteins from hydrophobic surfaces due to the presence of hydrophobic bonds and thus higher protein adsorption occurs on these surfaces; moreover, conformational changes in protein structures are also known to occur on hydrophobic surfaces. Altogether, this may promote undesirable events such as thrombus formation.⁹

Figure 8 illustrates the water CA of the fabricated scaffolds. PU exhibited a typical hydrophobic nature with CA value of 121.9 ± 8.59°. Addition of 20 wt% of PEG resulted in the decline in CA to 63.7 ± 3.48°, rendering a hydrophilic surface of the PU/PEG nanofibrous scaffolds. Similar results have been reported by Firkowska‐Boden et al.,⁵ wherein they observed a steady decline in CA of PU scaffolds from 110.9° to 75.7°, 63°, 54.9°, 38.3°, and 19° on varying the ratios of PEG (10, 20, 30, 40, and 50%) in PU/PEG blends. Addition of Clopidogrel further reduced the CA of the nanofibrous scaffolds. A concentration-dependent decline in CA was observed with the hydrophilic nature of the nanofibrous scaffolds increasing with increasing concentration of Clopidogrel. The CA for PU/PEG/Clopidogrel 1, 5, and 10% were found to be 58.41 ± 8.31°, 46.18 ± 3.26°, and 37.58 ± 4.02°, respectively.

Figure 8.

CA of representative nanofibrous scaffolds: (a) PU, (b) PU/PEG, (c) PU/PEG/Clop-1%, (d) PU/PEG/Clop-5%, (e) PU/PEG/Clop-10%, and (f) Mean CA value for nanofibrous scaffolds. PEG: polyethylene glycol; PU: polyurethane.

WU of nanofibers

The WU property of the scaffold facilitates waste removal, as well as the supply of nutrients.⁵ The WU capabilities of the fabricated scaffolds are mentioned in Figure 9. The WU potential of PU, PU/PEG, PU/PEG/Clop-1, 5, and 10% was found to be 10.1 ± 1.15, 12.76 ± 1.05, 12.5 ± 0.4, 12.76 ± 0.75, and 13.06 ± 1.18 mg/cm², respectively. The results of the WU studies correlated with the hydrophilicity of the polymer surfaces as observed by water CA analysis wherein the water retaining capabilities of the nanofibrous scaffolds enhanced with the increasing hydrophilicity. The swellings of the nanofiber scaffolds may be attributed to the H-bonding of groups present in PU/PEG with media in which it is subjected to WU.

Figure 9.

WUs by nanofibrous scaffolds of PU, PU/PEG, and PU/PEG with varying concentrations of Clopidogrel. PEG: polyethylene glycol; PU: polyurethane.

ATR-FTIR

ATR-FTIR spectra of PU, PU/PEG, PU/PEG/Clop-1, 5, and 10 wt% are shown in Figure 10. Peaks at 2936 and 2859 cm⁻¹ correspond to the asymmetric and symmetric stretch vibration of –CH₂ in PU. The 1246 cm⁻¹ peak was assigned to ester C–O–C stretching in the PU segment and stretch at 3328 cm⁻¹ corresponds to –NH of PU segment. Blending PU with PEG caused stretch at 1102 cm⁻¹ which attributed to the C–O–C stretching vibration of PEG. O–H bending vibration peaks of PEG at 1342 cm⁻¹ could also be observed in PU/PEG nanofiber. The significant weakness of the N–H stretching vibration peaks at 3328 cm⁻¹ and the shift of the C–O–C stretching vibration peaks from 1246 to 1260 cm⁻¹ of PU/PEG nanofiber indicated that there may be intermolecular forces or presence of hydrogen bond between PU and PEG. Further incorporation of Clopidogrel caused the emergence of a peak at 1749 cm⁻¹ which shows –C = O stretch of methyl ester, peak at 1156 cm⁻¹ corresponds to –C–O stretch, peak at 3106 cm⁻¹ corresponds to –C–H stretch and at 1496 cm⁻¹ to chlorophenyl ring stretch. The stretch at 1745 and 1532 cm⁻¹ corresponds to –C = O– and the C–N bonds, respectively.³⁷

Figure 10.

Representative ATR-FTIR spectra for Clopidogrel drug and nanofibrous scaffolds of PU, PU/PEG, and PU/PEG with varying concentrations of Clopidogrel. PEG: polyethylene glycol; PU: polyurethane.

TGA

TGA and DTG analysis evince thermal stability of the PU, PU/PEG, PU/PEG/Clop-1, 5, and 10 wt% as shown in Figure 11. The thermal degradation temperatures for PU, PU/PEG, PU/PEG/Clop-1, 5, and 10 wt% nanofibers were found to be 315, 320, 275, 262, and 241°C, respectively. Thermal stability order for nanofiber was found to be as follows: PU/PEG > PU > Clop-1% > Clop-5% > Clop-10% as per the parameters obtained from DTG as shown in Table 2.

Figure 11.

TGA of nanofibrous scaffolds of PU, PU/PEG, and PU/PEG with varying concentrations of Clopidogrel. PEG: polyethylene glycol; PU: polyurethane.

Table 2.

DTG analysis of nanofibrous scaffolds of PU, PU/PEG, and PU/PEG with varying concentrations of Clopidogrel.

Scaffold type	Onset (°C)
PU	315
PU/PEG	320
PU/PEG/Clop-1%	275
PU/PEG/Clop-5%	262
PU/PEG/Clop-10%	241

DTG: difference thermo-gravimetric analysis; PEG: polyethylene glycol; PU: polyurethane.

DSC

DSC thermogram as shown in Figure 12 shows sharp melting point T_m at 183°C which corresponds to the crystalline nature of Clopidogrel. PU does not show any transition in the graph; however, as mentioned in the previous reports, it has a T_g of −50°C. PU/PEG nanofiber shows melting point T_m at 59.6°C which may be attributed to PEG. However, incorporation of Clop-1, 5, and 10 wt% in PU/PEG caused melting point T_m of 59.2, 59.8, and 55.8°C without endothermic peak of Clopidogrel, which may be an indication of amorphous nature of Clopidogrel in nanofiber.

Figure 12.

DSC analyses of nanofibrous scaffolds of PU, PU/PEG, and PU/PEG with varying concentrations of Clopidogrel. PEG: polyethylene glycol; PU: polyurethane.

Wide-angle X-ray diffraction

The WAXD spectra of the nanofibrous scaffolds are shown in Figure 13. PU exhibited a Gaussian peak at 2θ = 20° which depicts its characteristic amorphous nature. On blending of PU with PEG, the peak at 2θ = 20° changes to sharp peaks at 2θ = 18° and 23° which are further retained in PU/PEG/Clop-1, 5, and 10 wt% with the same 2θ angle. This change may be attributed to the crystalline nature of PEG. Clopidogrel drug in pure form showed sharp Lorentzian peak between 2θ = 7–25° which are indicative of its crystalline nature. Absence of Lorentzian peak of Clopidogrel in nanofibers of PU/PEG/Clop-1, 5, and 10 wt% indicates Clopidogrel was present in an amorphous state and uniformly distributed in PU/PEG nanofibers; moreover, these results were supported by the results of DSC analysis.³⁸

Figure 13.

WAXD analyses of representative nanofibrous scaffolds of PU, PU/PEG, and PU/PEG with varying concentrations of Clopidogrel. PEG: polyethylene glycol; PU: polyurethane.

DMA

The mechanical properties of the fabricated nanofibrous scaffolds are depicted in Figure 14. From the results, it is evident that the addition of PEG to PU caused an increase in the mechanical strength of the PU/PEG nanofibrous scaffolds. The incorporation of Clopidogrel at the concentration of 1, 5, and 10 wt% within the PU/PEG nanofibrous scaffolds resulted in the decrease in diameter of nanofibers as a result of increased conductivity of the polymeric solution. The tensile properties and modulus shared an inverse relationship with the fiber diameter, with the tensile strength and modulus decreasing with increasing fiber diameter. The modulus and tensile strength were in the order as follows: Clop-10 wt% > Clop-5 wt% > Clop-1 wt% > PU/PE > PU. The degree of crystallinity and molecular orientation of fibers are enhanced with the increasing concentration of additives and reducing fiber diameter, resulting in improved mechanical strength and stiffness. The effect was evident due to the small fiber having higher crystallinity than larger fiber. The porosity and orientation of nanofiber might be the other possible factor responsible for the increased mechanical strength.³⁹

Figure 14.

DMA showing (a) stress–strain curves, (b) Young’s modulus, (c) tensile strength, and (d) break-point for nanofibrous scaffold of PU, PU/PEG, and PU/PEG with varying concentrations of Clopidogrel. PEG: polyethylene glycol; PU: polyurethane.

In vitro drug release study

Release of Clopidogrel from PU/PEG/Clop-1, 5, and 10 wt% nanofiber as presented in Figure 15 shows a tri-phasic release pattern. Drug release was seen to be significantly affected by the drug content in nanofiber. The amount of drug release was found to increase proportionally along with the increasing drug concentration in the nanofibers. The nanofiber showed sustained drug release up to 21 days without showing any burst release at the initial stage. On the 21st day, the release of Clopidogrel from PU/PEG/Clop-1, 5, and 10 wt% nanofibers was found to be 31, 70, and 100%, respectively. Depending on the release pattern necessary for the intended application, the respective Clopidogrel containing nanofiber can be further selected. The release fitted different release model such as Korsmeyer -Peppas, zero order, Higuchi, first order. The release rate exponent (n) according to Peppas model shows Fickian diffusion of release. Various parameters for different drug release kinetics model are shown in Table 3.

Figure 15.

Drug release kinetics from the nanofibrous scaffold of PU/PEG with varying concentrations of Clopidogrel. PEG: polyethylene glycol; PU: polyurethane.

Table 3.

Drug release kinetics model parameter.

Scaffold type	Higuchi Model	Zero order	First order	Korsmeyer–Peppas	n
Scaffold type	R²	R²	R²	R²	n
1% Clop	0.910	0.706	0.848	0.904	0.5
Equation	y = 5.2x − 2.8	y = 1.8x + 10	y = −0.0047x + 1.9	y = 0166x + 0.96
5% Clop	0.942	0.705	0.819	0.996	0.1
Equation	y = 7.7x − 7.0	y = 2.8x + 17	y = −0.02x + 1.009	y = 1.1x + 0.132
10% Clop	0.903	0.888	0.972	0.828	0.3
Equation	y = 5.2x − 16	y = 5.1x + 5.4	y = −0.081x + 2.1	y = 0.383x − 0.308

In vitro hemocompatibility analysis

Hemolysis assay

Hemolysis percentage may serve as a yardstick for determining the extent of RBCs broken by scaffold contacting with blood.^16,30 Hemolysis occurs when RBCs swell up to a critical bulk breaking up the cell membrane.⁵ The ADP released from the broken RBCs activates the platelets through a GTP binding protein. This ADP–platelet interaction may lead to a change in the shape of platelets and a decline in cAMP formation, causing platelet activation and further accelerating the clotting and thrombus formation.^2,5 An ideal vascular graft should be blood compatible in order to mimic the endothelium.¹⁶ The lower hemolysis percentage indicates better blood compatibility of the biomaterial.¹⁶ The hemolysis percentage values for different electrospun scaffolds are illustrated in Figure 16. All the electrospun scaffolds showed hemolysis percentage values < 2% and thus according to ASTMF 756-00-(2000) standards they can be used as blood-contacting biomaterials without causing the hemolysis of RBCs.^9,26,30,40 The hemolysis percentage value for pristine PU (1.85 ± 0.14%) was found to be higher as compared to that of PU/PEG (1.24 ± 0.018%), PU/PEG/Clop-1% (1.19 ± 0.07%), 5% (1.24 ± 0.12%), and 10% (1.29 ± 0.1%) containing scaffolds confirming that addition of PEG and further Clopidogrel renders fibers which are hemocompatible.

Figure 16.

Hemocompatibility analysis of PU, PU/PEG, and PU/PEG nanofibrous scaffolds with varying concentrations of Clopidogrel by hemolysis assay. Data are expressed as mean ± SEM (n = 3). Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001. PEG: polyethylene glycol; PU: polyurethane.

BSA/fibrinogen adsorption

It is believed that the adsorption of nonspecific plasma protein on biomaterial surface triggers subsequent platelet adhesion and activation, and plays a pivotal role in vivo toward the initiation of thrombus formation at the blood–material interfaces.^17,20,41,42 Albumin and fibrinogen are among the abundant plasma proteins maintaining their plasma concentration of 40 and 4 mg/ml, respectively. Thus, it is important to determine their adsorption on biomaterial surfaces.^5,17,43

The BSA adsorption on the nanofibrous scaffolds under the static condition is shown in Figure 17(a). Addition of PEG to PU caused 1.32% reduction in the adsorption of BSA on the PU/PEG scaffolds as determined by BCA assay. Incorporation of Clopidogrel increased the protein adsorption on the PU/PEG scaffolds, with Clop-1, 5, and 10% enhancing BSA adsorption by 4.42, 9.51, and 13.42%, respectively, as compared to PU nanofibers. Albumin is regarded as inert⁴⁰ or a passivating protein^43,44 toward the platelet adhesion and activation on biomaterial surface; moreover, it has been reported that if a material possesses hemocompatibility, it aids albumin adsorption which shields the activation of the coagulation cascade and further triggers the deposition of cell adhesive proteins.⁴⁰ Thus, our results suggest fabrication of a hemocompatible scaffold.

Figure 17.

Protein adsorption on PU, PU/PEG, and PU/PEG nanofibrous scaffolds with varying concentrations of Clopidogrel by BCA assay: (a) BSA adsorption and (b) fibrinogen adsorption. Data are expressed as mean ± SEM (n = 3). Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001. PEG: polyethylene glycol; PU: polyurethane.

The fibrinogen adsorption on the PU, PU/PEG scaffolds, under static condition, determined using the BCA assay is shown in Figure 17(b). The fibrinogen adsorption on PU nanofibrous scaffold was found to be the highest while the addition of PEG caused a sharp reduction in fibrinogen adsorption on PU/PEG surface by 32.84%. Further, incorporation of Clopidogrel had a limited effect on fibrinogen adsorption as compared to PU/PEG scaffolds, yet the PU/PEG/Clop-1, 5, and 10% scaffolds adsorbed 32.8, 29.28, and 29.76%, respectively, less fibrinogen as compared to PU scaffolds. Fibrinogen being a major protein mediating platelet adhesion on biomaterial surface promotes thrombus formation through binding of the platelet integrin receptor α_IIbβ₃ (GPIIb/IIIa), causing platelet immobilization and aggregation¹⁷; moreover, it stabilizes thrombi as fibrin polymer, which is a predominant structural component in blood clotting.^17,43–47 Fibrinogen has relatively low structural stability and upon adsorption it undergoes extensive surface rearrangement, ranging from native, through molten, to a fully denatured state,⁵ giving rise to an increased number of interaction sites between proteins and the material surface.^5,48 It has been reported that the structural deformation (spreading) of fibrinogen increases with material’s hydrophobicity.¹⁷ Our results corroborated this fact and showed a decrease in fibrinogen adsorption with an increase in hydrophilicity.

Plasma protein adsorption

Blood is often the first body fluid that comes in contact with the blood-contacting devices.¹⁷ The blood–material interaction leads to the capping of the material surface with plasma proteins,⁴⁰ which may catalyze, mediate, or moderate the subsequent biological response to the biomaterial.¹⁷

The plasma protein adsorption on the PU scaffolds was found to be the lowest and the incorporation of PEG and further different concentrations of Clopidogrel caused an increase in the adsorption of plasma proteins (Figure 18). The adsorption of plasma proteins on PU/PEG, PU/PEG/Clopidogrel-1, 5, and 10% were found to be 15.29, 30.29, 36.31, and 34.17%, respectively, higher as compared to pristine PU scaffolds.

Figure 18.

Plasma protein adsorption on PU, PU/PEG, and PU/PEG nanofibrous scaffolds with varying concentrations of Clopidogrel by BCA assay. Data are expressed as mean ± SEM (n = 3). Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001. PEG: polyethylene glycol; PU: polyurethane.

Adsorption of proteins to biomaterial involves competition of all proteins in the complex mixture for the available surface sites.⁴³ The surface properties of the biomaterials along with the intrinsic properties of the proteins (individual) in a solution such as its size, net charge, structure, and its stability are among the factors that may influence the composition and function of the adsorbed proteins on the implanted biomaterial.^5,17,49 And thus the increased concentration of plasma proteins on the developed scaffolds may be attributed to the adsorption of BSA which has the lowest size (65 kDa) and the highest concentration (40 mg/ml) among the plasma proteins. Our results follow the phenomenon of Vroman effect, which states that for a multiprotein system, proteins of smaller size, higher concentration, and higher diffusion rate primarily get adsorbed on the material surface and may be further displaced by larger molecules with higher affinity.^5,43,50–52

Platelet adhesion and activation

Platelet deposition on the PU, PU/PEG, and PU/PEG/Clop nanofibrous scaffold surface was evaluated using SEM micrograph (Figure 19), alternatively platelet deposition on the nanofibrous scaffolds was also studied by staining the platelets with Wright–Giemsa staining and visualizing under phase contrast inverted microscope (Figure 20). As visualized in SEM images, considerable amount of platelets with few filopodia were found to be deposited on the PU scaffolds, whereas the blending of PU with PEG caused a reduction in the platelet adhesion. The reduction in platelet adhesion on PU/PEG scaffolds is attributed to the increased hydrophilicity caused due to the blending of PU with PEG. Similar results were observed by Wang et al.,⁹ wherein they observed a reduction in platelet adhesion on blending PU with PEG (>20 wt%). Further, suppression of platelet adhesion on PU/PEG scaffold surface was observed upon the incorporation of 1, 5, and 10% Clopidogrel drug, which caused a concentration-dependent sharp reduction in platelet deposition. The results of Wright–Giemsa staining corroborated with the SEM results and showed the significant concentration (Clopidogrel) dependent reduction in platelet deposition and aggregation on PU/PEG/Clopidogrel nanofibrous scaffolds. The significant results observed in the Clopidogrel containing nanofibers may be attributed to (1) increased hydrophilicity and wettability of the scaffolds,^9,17,21 (2) smoother surface topography of the scaffolds,^2,5 (3) reduced surface area,^17,30 (4) anticoagulant property of Clopidogrel incorporated in PU/PEG,^23,24,37 and (5) reduced hemolysis and nonspecific protein adsorption on the developed materials.

Figure 19.

Platelet adhesion and activation on nanofibrous scaffolds: (a) PU, (b) PU/PEG, (c) PU/PEG/Clop-1%, (d) PU/PEG/Clop-5%, and (e) PU/PEG/Clop-10%, as determined by SEM micrograph.

Figure 20.

Platelet adhesion and activation on nanofibrous scaffolds: (a) PU, (b) PU/PEG, (c) PU/PEG/Clop-1%, (d) PU/PEG/Clop-5%, and (e) PU/PEG/Clop-10%, as determined by Giemsa staining.

APTT assay and PT assay

The process of thrombosis on blood-contacting material surface involves platelet-mediated reaction as well as coagulation of blood plasma.¹⁷ Thus, in order to prevent graft failure, the fabricated scaffold must possess thromboresistance and anticoagulant nature. The interaction of plasma proteins with the biomaterial surface triggers the coagulation cascade, leading to thrombus production and development of fibrin clot.¹⁷

The process of coagulation involves a series of self-amplifying zymogene enzyme conversion which is traditionally grouped as the intrinsic and extrinsic pathways.^17,53,54 Both intrinsic and extrinsic pathways are initiated separately; however, they further merge into a common pathway leading to thrombin F(IIa), which hydrolyzes fibrinogen into fibrin that assembles and causes plasma clotting.^17,54,55 The extrinsic pathway is responsible for hemostatic control and response to vascular injury whereas the intrinsic pathway has a slight physiological significance under normal conditions.¹⁷ The intrinsic pathway triggered by blood–material interaction is an important factor causing poor hemocompatibility of biomaterials.^17,56

The thromboresistance properties of the nanofibrous scaffolds were characterized by APTT and PT assessment, which are good indicators of intrinsic and extrinsic coagulation pathways, respectively. The APTT values of the electrospun PU/PEG scaffold incorporated with Clopidogrel showed prolonged blood clotting time with the increase in clotting time directly related to the amount of Clopidogrel incorporated (Figure 21(a)). The APTT values for PU, PU/PEG, PU/PEG/Clop-1, 5, and 10% were found to be 210.6 ± 5.8, 207.6 ± 12.11, 187 ± 20.23, 228 ± 8.32, and 261.3 ± 16.4 s, respectively. Similar results were obtained for PT assay (Figure 21(b)) wherein the coagulation time enhanced with the increasing concentration of Clopidogrel. The PT values for PU, PU/PEG, PU/PEG/Clop-1, 5, and 10% were found to be 25.9 ± 0.35, 26.18 ± 0.56, 30.12 ± 0.5, 31.38 ± 0.96, 34.15 ± 1.61 s, respectively.

Figure 21.

(a) APTT assay and (b) PT assay of PU, PU/PEG, and PU/PEG nanofibrous scaffolds with varying concentrations of Clopidogrel. PEG: polyethylene glycol; PU: polyurethane.

The APTT and PT results showed an increase in plasma clotting time which points out the enhanced anticoagulation nature of modified scaffolds by delaying both the extrinsic and intrinsic pathways as a result of the incorporation of Clopidogrel. Various parameters including surface wettability, surface chemistry, and surface area have been investigated for contact activation of coagulation cascade activation.¹⁷ The increase in blood clotting time of the modified scaffolds may be attributed to reduction in platelet adhesion and activation, and further, also due to (1) decreased fiber diameter with increasing drug content^14,30,31; similar results were reported by Jaganathan et al.³⁰ wherein they reported delay in blood clotting time for smaller diameter fibers, (2) smoother surfaces of nanofibers as observed by AFM,^2,5 (3) incorporation of Clopidogrel,²³ (4) increased hydrophilicity or wettability of the nanofibrous scaffolds,^9,17,21 and (5) presence of PEG.^9,21

In vitro biocompatibility of nanofibrous scaffolds

NIH/3T3 cells were cultured on the electrospun PU, PU/PEG, and PU/PEG/Clopidogrel nanofibrous scaffolds to investigate the cell adhesion property. Upon culturing for 24 h, the cell adhesion behavior was observed by CLSM (Figure 22). The cell attachment of the PU scaffolds was found to be relatively low, which might be due to the hydrophobic property exhibited by the PU scaffolds. The cells adhered and grew well on all the nanofibrous scaffolds containing PEG, which may be attributed to the physicochemical properties of the scaffold surface and the absorbed protein that might play a crucial role in cell behavior. Moreover, the incorporation of Clopidogrel did not induce cytotoxicity effects and had no adverse effect on the cell attachment on nanofibrous scaffolds, thus showcasing the biocompatibility of the PU/PEG/Clopidogrel scaffolds.

Figure 22.

Cell adhesions on nanofibrous scaffolds of (a) PU, (b) PU/PEG, (c) PU/PEG/Clop-1%, (d) PU/PEG/Clop-5%, and (e) PU/PEG/Clop-10% as evaluated by CLSM analysis.

Tissue engineered grafts are ought to possess excellent biocompatibility in order to support cell attachment and proliferation for their successful clinical application. The MTT assay measured the metabolic activity of the HUVEC cells and NIH/3T3 cells cultured on the scaffolds, which indicated their cell viability up to seven days of culture (Figure 23(a) and (b)). The addition of PEG to the PU enhanced the cell viability of HUVEC cells and NIH/3T3 cells on PU/PEG scaffolds at all the time points. At day 7, the PU/PEG scaffolds exhibited 35.27% higher NIH/3T3 cell viability and 12.35% higher HUVEC cell viability as compared to PU scaffolds. This might be attributed to the increase in hydrophilicity of the PU/PEG scaffolds which might have promoted the adhesion of proteins supporting cell proliferation. Further incorporation of Clopidogrel caused a concentration-dependent increase in the hydrophilicity of the scaffolds which further promoted the cell adhesion and proliferation on the PU/PEG/Clop-1% and PU/PEG/Clop-5% nanofibrous scaffolds. At day 7, the PU/PEG/Clop-1% and PU/PEG/Clop-5% nanofibrous scaffolds exhibited 70.82 and 36.60% higher NIH/3T3 cell viability, and 13.36 and 14.85% higher HUVEC cell viability as compared to PU scaffolds, respectively. However, further increase in Clopidogrel content caused an increase in hydrophilicity with CA < 40°. This may have caused reduction in protein absorption and consequently delayed the cell adhesion and proliferation on PU/PEG/Clop-10% scaffolds maintaining 7.95% higher NIH/3T3 cell viability and 4.18% higher HUVEC cell viability at day 7 as compared to PU scaffolds. The results thus highlight the biocompatibility of the PU/PEG/Clopidogrel nanofibrous scaffolds further pointing out the necessity of appropriate physicochemical properties of the nanofibrous scaffolds for supporting cell adhesion and proliferation.

Figure 23.

Cell viability analyses in PU, PU/PEG, and PU/PEG nanofibrous scaffolds with varying concentrations of Clopidogrel: (a) NIH/3T3 cell viability analysis and (b) HUVEC cells viability analysis. PEG: polyethylene glycol; PU: polyurethane.

Conclusion

In the current study, Clopidogrel was successfully incorporated into the PU/PEG polymeric blend solution in order to fabricate nanofibrous scaffolds by electrospinning technique. These nanofibers exhibited both hemocompatibility and antithrombogenic properties for its application as blood-contacting antithrombotic material. The electrospun PU/PEG/Clopidogrel scaffolds possessed nano-scale diameter fibers with a hydrophillic surface that might provide a three-dimensional environment mimicking the natural ECM. The PU/PEG/Clopidogrel scaffolds possessed smooth surfaces with enhanced hydrophilicity, better thermostability, excellent mechanical properties, and a tri-phasic drug release pattern. The hemolysis assay, protein adsorption assay under static condition, platelet adhesion test, and coagulation assay affirmed the enhanced blood compatibility of PU/PEG/Clopidogrel scaffolds as compared to the pristine PU scaffolds. Moreover, the results of cell adhesion and MTT assay point out the biocompatibility of the PU/PEG/Clop nanofibrous scaffolds further suggesting that the PU/PEG/Clop scaffolds with appropriate Clopidogrel content (≤5 wt%) would promote the cell viability and might be a promising substitute as a blood-contacting biomaterial. However, further studies at in vivo level are needed to ascertain the clinical applicability of the PU/PEG/Clopidogrel nanofibrous scaffolds as a biomaterial to be used for blood-contacting applications in the biomedical field.

Footnotes

Authors’ contribution

Ajinkya A Shitole and Prabhanjan S Giram contributed equally to the manuscript.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/ or publication of this article: Prabhanjan Giram gratefully acknowledges the University Grant Commission, Government of India, New Delhi, India, for the award of a research for his doctoral research fellowship. Ajinkya A Shitole and Piyush W Raut gratefully acknowledge Symbiosis Centre for Research and Innovation, Symbiosis International (Deemed University), Lavale, Pune, India for the award of their doctoral research fellowship.

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