Aspirin-loaded electrospun poly(Îµ-caprolactone) tubular scaffolds ...

J Mater Sci: Mater Med (2013) 24:523–532 DOI 10.1007/s10856-012-4803-3

Aspirin-loaded electrospun poly(e-caprolactone) tubular scaffolds: potential small-diameter vascular grafts for thrombosis prevention Costantino Del Gaudio • Enrico Ercolani • Pierluca Galloni Federico Santilli • Silvia Baiguera • Leonardo Polizzi • Alessandra Bianco

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Received: 20 June 2012 / Accepted: 19 October 2012 / Published online: 8 November 2012 Ó Springer Science+Business Media New York 2012

Abstract Thrombosis is the main cause of failure of small-diameter synthetic vascular grafts when used for by-pass procedures. The development of bioresorbable vascular scaffolds with localized and sustained intraluminal antithrombotic drug release could be considered a desirable improvement towards a valuable solution for this relevant clinical need. For this aim, we present the fabrication and characterization of aspirin-loaded electrospun poly(e-caprolactone) tubular scaffolds as a vascular drugdelivery graft. Three different drug concentrations were considered (i.e., 1, 5 or 10 % w/w). Although a fibrous structure was clearly observed for all the collected scaffolds, aspirin content was directly implied in the final microstructure leading to a bimodal fiber diameter distribution and fused fibers at crossing-points (5 or 10 % w/w). Mechanical response highlighted a direct relationship for modulus and stress at break with the aspirin content, while the elongation at break was not remarkably different for the investigated cases. The temporal drug release was strongly

C. Del Gaudio (&) E. Ercolani A. Bianco (&) Department of Industrial Engineering, University of Rome ‘‘Tor Vergata’’, INSTM Research Unit Roma Tor Vergata, Via del Politecnico 1, 00133 Rome, Italy e-mail: [email protected] A. Bianco e-mail: [email protected] P. Galloni F. Santilli Department of Chemical Science and Technology, University of Rome ‘‘Tor Vergata’’, Via della Ricerca Scientifica, 00133 Rome, Italy S. Baiguera L. Polizzi BIOAIRLab, European Center of Thoracic Research (CERT), University Hospital Careggi, Florence, Italy

dependent from the amount of loaded aspirin, reaching a steady state release after about 50 h. Finally, the adhesion assay confirmed the capability of the electrospun scaffolds to reduce platelet adhesion/aggregation onto aspirin loaded polymeric fibers. Aspirin-loaded electrospun tubular scaffold could represent a feasible candidate to develop a novel bioresorbable drug-releasing graft for small-diameter vessel replacements.

1 Introduction Each year cardiovascular diseases cause 17 million deaths worldwide, over 4.3 million deaths in Europe and over 2.0 million deaths in the European Union (EU), representing the main cause of the disease burden (illness and death) in Europe (23 %) and the second main cause in those EU countries with very low child and adult mortality (17 %) [1]. Moreover, even if large decrease in related cardiovascular disease death rates has been experienced in western countries, premature morbidity and mortality are expected to almost double in the next three decades from 85 million disability-adjusted life years (DALY) in 1990 to 140–160 million DALY in 2020, with about 80 % occurring in developing countries [2]. Vascular grafts are used for bypass procedures of coronary and peripheral arteries when percutaneous transluminal angioplasty or stenting procedures cannot assure long-term vessel patency [3]. For this aim, autologous arterial and venous grafts remain the best clinical option, though is known to be affected by several limitations mainly related to pre-existing vascular disease, prior surgery, limited length or poor quality [4, 5]. Commercially available biostable synthetic vascular grafts made of expanded polytetrafluoroethylene and polyethylene terephthalate (DacronÒ) have failed for small-diameter

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(\6 mm) vessel replacement due to an early graft occlusion [6], since thrombosis, together with intimal hyperplasia, remains one of the major causes of poor patency [7]. To prevent early graft occlusion, patients are treated with systemic antithrombotic drugs such as anticoagulants and platelets inhibitors [8]. Acetylsalicylic acid (ASA, aspirin), dipyridamole and clopidogrel are the gold standard for the secondary prevention of non-cardioembolic ischemic stroke/transient ischemic attack [9]. The CAPRIE trial (Clopidogrel vs. Aspirin in Patients at Risk of Ischaemic Events) has demonstrated slightly better results for clopidogrel compared to aspirin as a suitable antiplatelet drug. However, the costs per quality-adjusted life-year were too high and the evidence was insufficient to replace aspirin, which is the most frequently used antiplatelet drug for primary and secondary ischemic stroke prevention [10]. Aspirin mechanism of action lowers platelet aggregation through the irreversible acetylation of cyclooxygenase 1 (COX-1) and cyclooxygenase 2 (COX-2), which catalyze the conversion of arachidonic acid to prostaglandin H2 (PGH2), representing the direct antecedent of eicosanoids (prostaglandin, thromboxane A2 and prostacyclin) and playing a key role in the aggregation/coagulation process [11, 12]. Aspirin administration mainly relies on oral daily dosage for long period and its poor oral bioavailability (*40 %), gastrointestinal (GI) mucosa ulcer and massive GI hemorrhage are the major drawbacks associated to this therapy [13, 14]. Even though aspirin has an in vivo half-life of 13–20 min, after which is hydrolyzed to salicylic acid [15], its effect lasts for platelet lifetime (7–10 days) due to the irreversible inhibition of platelet COX and the impossibility for platelets (having no DNA) to synthesize new enzymes. However, platelet activation is not inhibited after the complete elimination of ASA from the circulation. The daily synthesis of new platelets is 2.5 9 107 cells/mL of blood [16], therefore, based on a daily aspirin administration, newly formed active platelets could reach nearly a 10 % level of the total platelet population in between dosages and it has been suggested that a small number of active platelets is sufficient to restore platelet function [15]. On this basis, a localized and sustained intra-luminal drug release could be a potential alternative when systemic side effects or low concentrations impair the actual therapeutic efficacy. To overcame these limitations, several drug delivery systems have been proposed, e.g. polymeric substrates for chemical immobilization of specific agents (i.e., vascular endothelial growth factor) [17], biodegradable coating for nondegradable vascular grafts [15, 18], biodegradable in situ gels [19], gel beads [8] and polymeric films [20]. In most cases, the release is obtained by diffusion of the drug at the surface of the polymeric scaffold in aqueous environments,

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J Mater Sci: Mater Med (2013) 24:523–532

being strictly correlated to the degradation rate of the polymer, the thickness and the structural morphology of the scaffold itself [21]. Several techniques can be considered for the production of drug-delivery systems, among them electrospinning represents an easy and cost-effective method to produce nano- or micro-fibrous polymeric scaffolds of desired shape and dimension. Fiber diameter, architecture, void size and interconnectivity, microstructural and mechanical properties can be properly tailored to mimic the natural extra-cellular matrix (ECM) of blood vessels and to affect the drug release mechanism from the selected material. Poly(e-caprolactone) (PCL) is a bioresorbable synthetic polymer widely used for cardiovascular applications [22, 23] and, due to its permeability to many drugs and slow biodegradation ([1 year), is a promising candidate for the development of tissue-engineered blood vessels, being suitable to effectively support the production of natural ECM and sustain a prolonged drug release [24]. Recent studies demonstrated that PCL is characterized by adequate mechanical properties for vascular grafts in the systemic circulation, showing better results than other commonly used polymers, such as polydioxanone, poly(L-lactide-co-e-caprolactone) and poly(lactic-co-glycolic acid) [3, 17, 25, 26]. Electrospun PCL tubular scaffolds loaded with paclitaxel have shown good patency up to 6 months in a rat model [6]. Although topical release of anti-proliferative drugs showed a reduction in neointimal formation, the correlated delayed endothelialization may cause thrombosis and occlusion in low-flow condition. Antithrombotic drugs may delay platelet adhesion and aggregation on the lumen of the grafts without interfering with the endothelialization process. In a recent study, ASA was immobilized by using a cross-linker on PCL films after chemical surface functionalization and no negative influence was observed for primary and secondary hemostasis [17]. In this regard, loading polymeric vascular grafts with ASA, an efficacious, relatively safe, widely available, inexpensive, and easy-to-use antiplatelet agent, could represent a novel, easy and time-saving way to improve hemocompatibility of polymeric vascular substitutes. Therefore, the purpose of this study was to fabricate smalldiameter aspirin-eluting electrospun PCL vessels as a potential model for vascular grafts aimed to inhibit thrombus formation and to evaluate their mechanical properties, in vitro drug release mechanism and platelet anti-adhesion properties as a function of the drug content. To the best of our knowledge this is the first report regarding the feasibility of electrospinning aspirin-loaded PCL tubular scaffolds resembling the natural ECM of blood vessels.


2 Materials and methods 2.1 Materials PCL (Mn = 70,000–90,000) and acetylsalicylic acid (ASA) were supplied by Sigma-Aldrich. Chloroform (CHCl3, analytical grade) was supplied by Carlo Erba Reagenti. Phosphate buffer solution (PBS, pH 7.4) tablets were purchased from Invitrogen Corporation. All materials and reagents were used as received. 2.2 Fabrication of drug/polymer electrospun tubular scaffolds PCL pellets were firstly dissolved in CHCl3 (20 % w/v), then aspirin was added to the polymeric solution for a final concentration of 1, 5 or 10 % w/w with respect to PCL. Resulting drug/polymer solutions were electrospun at room temperature through a blunt tip metallic needle (22G) at constant feed rate of 0.01 mL/min by means of a digital controlled infusion pump (KD Scientific, USA). A high voltage power supply (Spellman, USA) insured an applied voltage of 15 kV. Tubular scaffolds (ID = 5 mm) were collected onto a rotating aluminum mandrel (about 3000 rpm) located at 10 cm from the needle tip. All samples were vacuum dried for 48 h and stored in a desiccator. Based on the aspirin content, electrospun scaffolds were labeled PCL (neat), PCL-ASA1 (1 % w/w), PCL-ASA5 (5 % w/w) and PCL-ASA10 (10 % w/w). 2.3 Microstructural characterization of tubular scaffolds The microstructure was examined by means of scanning electronic microscopy (SEM) (Leo-Supra 35). All samples were sputter coated with gold for 2 min prior to their observation. The average fiber diameter was determined from SEM micrographs by measuring about 50 fibers randomly selected (ImageJ, NIH). 2.4 Mechanical characterization of tubular scaffolds Tests were performed following the standard ISO/DIN 7198 ‘‘Cardiovascular implants—Tubular vascular prostheses’’. Circumferential tensile properties were evaluated stretching electrospun rings cut out from the collected tubular scaffolds (n = 3 for each graft), the average length being 8.4 ± 1.1 mm. Rings were tested by means of a universal testing machine equipped with a 100 N load cell (Lloyd LRX). Two stainless steel rods (1 mm diameter) were placed through the lumen of the ring and fixed to two custom-made steel grips. Specimens were tested to rupture at 50 mm/min. Tensile modulus, tensile strength, referred

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to the nominal cross section area expressed as 2 9 length 9 thickness, elongation at break, and circumferential strength normalized to the sample length, as specified by the above mentioned standard, were calculated. 2.5 In vitro drug release Samples cut out from electrospun tubular scaffolds were weighed, immersed in 30 mL of PBS and kept into an incubator at 37 °C. Aliquots of release solution were withdrawn periodically for measurements and then added back to the solution. ASA and salicylic acid (SA) concentration in the release solution was measured by UV spectrophotometry (UV–Vis Spectrophotometer UV-2450, Shimadzu, Japan) at 267 and 296 nm, respectively. Extinction coefficient for each drug was determined from the slope of Lambert–Beer plot. ASA is hydrolyzed into SA and acetic acid in PBS [27]. The amount of ASA released from the samples was calculated from the SA concentration in the release solution (Eq. 1), where Vsolution represented the volume of the measured solution and MWx the molecular weight of ASA and SA, respectively. The percentage of the released drug was calculated as the ratio between the cumulative amount of ASA released in the solution and the ASA loaded into the electrospun sample (Eq. 2). ASAsolution ¼ ½SAsolution Vsolution ASA% ¼

MWASA MWSA

ð1Þ

ASAsolution 100 ASAsample

ð2Þ

Each experiment was carried out in triplicate. 2.6 Platelets concentrate obtainment Blood samples were obtained from umbilical cord at the time of delivery (90 mL). Within 24 h of being drawn, the citrate-anticoagulated blood was collected in 50 mL centrifuge tubes and centrifuged at 1,200 rpm for 10 min at 22 °C to softly sediment white and red blood cells in the lower fraction, while the platelet rich plasma (PRP) remained suspended. After centrifugation, the upper 25 % of the PRP was carefully collected using a pipette to limit contamination by other cellular elements and placed in a sterile tube. At each step a small aliquot of suspension was collected for platelet counting by using an automated counter (Micros60; Horiba-ABX, Rome, Italy). 2.7 Prosthesis seeding and fixation for SEM analysis Circular samples cut out from the grafts were placed in individual wells of 24-well tissue culture plate, held in

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place by a metal ring and sterilized by UV rays for 30 min. The samples were then incubated with 1 mL of PRP (400 9 106 platelet/mL) at 37 °C for 2 and 6 h. After each contact time, samples were rinsed twice with PBS and fixed with 3 % (v/v) glutaraldehyde (Merck, Darmstadt, Germany) in a solution of 0.1 M sodium cacodylate buffer (pH 7.2) (Prolabo, Paris, France). After rinsing, specimens were dehydrated through an ethanol gradient, critical point dried, sputter coated with gold and the morphological evaluation of platelets, retained on graft surface, was carried out by means of scanning electron microscopy. SEM micrographs (n = 4) were also used to count the adherent platelets [28]. 2.8 Statistical analysis Data are expressed as mean ± standard deviation. Statistical analysis was performed by means of non-parametric tests (Matlab, The Mathworks, Natick, MA, USA). Differences were checked in two steps: data were compared by using the Kruskal–Wallis nonparametric test; if significant differences were found, groups were compared by means of the Mann–Whitney U test. Significant level was set at P \ 0.05.

3 Results and discussion 3.1 Microstructure of electrospun tubular scaffold Neat and ASA-loaded PCL were electrospun onto a cylindrical collector to obtain tubular scaffolds. SEM micrographs showed a non-woven uniform deposition of fibers free of beads for each samples, the microstructure being strictly dependent to the aspirin content (Fig. 1). PCL, PCL-ASA5 and PCL-ASA10 revealed an architecture mainly composed of micrometric fibers associated to submicrometric fibers on the luminal side of the graft. This result can be explained considering the onset of a jet instability due to the electrostatic conditions experienced by the polymeric jet during the electrospinning. It can be assumed that a transient splaying phenomenon occurred as a consequence of the electric field established between the needle and the rotating target that caused the deposition of thin insulating fibers on the metallic collector, contributing therefore to modify the electrostatic conditions of the ongoing process and strongly limiting successive splaying occurrences. This observation was further supported by a control electrospinning session in which the rotating cylindrical target was replaced by a fixed circular one (10 cm diameter), the solely modified processing variable; in this case only micrometric PCL fibers were homogeneously and uniformly deposited (data not shown). A possible explanation for this result was previously

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proposed specifically considering the electrospinning of PCL. Secondary jets originating from the primary one can be the result of the onset of static undulations on the surface of the conducting fluid jet. Such undulating surfaces can become unstable at the sites of the highest local surface curvature, thus leading to the emanation of secondary branches [29]. Similarly, an initial oscillatory bi-stable mode rather than the bending instability, generally occurring during electrospinning, can induce a bimodal fiber diameter distribution, as observed for electrospun PCL [30]. PCL-ASA5 and PCL-ASA10 were affected by the same morphological characteristics which can be further related to the high ASA content. Taking a closer look at the electrospun fibers of these two cases, a partial fusion among overlapped fibers due to an incomplete solvent evaporation was observed (Fig. 1c, d). The presence of ASA in the starting polymeric solution can contribute to modify the characteristics of the solvent, leading to the deposition of wet fibers that melt together at fiber crossing points resulting in an overall different morphology with respect to PCL and PCL-ASA1. Interestingly, PCL-ASA1 did not show the presence of sub-micrometric fibers, suggesting that the electrospun solution was not subjected to secondary phenomena that led to a bimodal fiber distribution. Compared to PCL-ASA5 and PCL-ASA10, this result seemed strictly related to the aspirin content indicating a stabilizing effect when the drug is added in low concentration. This consideration was confirmed by electrospinning a polymeric solution containing ASA at 0.5 % w/w, only homogeneous and micrometric fibers were observed (data not shown). 3.2 Mechanical properties of electrospun tubular scaffolds Circumferential mechanical properties are summarized in Table 1. An overall increase of circumferential tensile modulus and stress at break were observed in relation to ASA content in the electrospun fibers. Loading the polymeric solution with a higher percentage of ASA led to morphological changes of electrospun fibers, which in turn increased the mechanical strength and modulus of the tubular scaffolds. Mechanical behavior of electrospun fibrous materials is generally characterized by a two-stage mechanism, the initial fiber rearrangement along the load direction governed by fiber packing density is followed by a specific response that depends on the chemical and physical properties of the polymer and fiber dimensions [31–33]. In a previous study, SEM investigation carried out after tensile tests revealed a complete re-orientation of electrospun micrometric PCL fibers along the direction of the load followed by multiple fiber necking [34]. Referring


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Fig. 1 SEM micrographs of electrospun aspirin loaded tubular grafts: a PCL, b PCL-ASA1, c PCL-ASA5, d PCL-ASA10. Scale bar 10 lm

to the acquired results, it is reasonable to assume that the presence of melted fibers at crossing-points in PCL-ASA5 and PCL-ASA10 hampered fiber re-orientation during the first stage of tensile deformation, resulting in higher values of tensile modulus with respect to PCL and PCL-ASA1. Moreover, the collected nanofibers at higher ASA concentration could affect the second stage deformation, since sub-microfibrous structures possess higher mechanical properties than microfibrous ones. Statistically significant differences (P \ 0.05) in circumferential elongation at break for electrospun grafts were calculated only for

PCL-ASA10 with respect to PCL and PCL-ASA5 and for PCL-ASA1 with respect to PCL-ASA5. In this investigation, we demonstrated that loading PCL with ASA increases mechanical characteristics of electrospun grafts. Circumferential tensile modulus and stress at break values for each ASA content resulted similar to those previously reported for electrospun synthetic small-diameter vascular grafts [3, 35]. However, considering the potential vascular applications of the proposed polymeric constructs, mechanical properties of electrospun tubular scaffolds should match those of human native vessels.

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Table 1 Mechanical and morphological characteristics of aspirin loaded electrospun tubular scaffolds E (MPa)

rmax (MPa)

emax (%)

Fmax/2L (N/mm)

Fiber diameter (lm)

Graft thickness (lm)

PCL

2.9 ± 0.1

1.8 ± 0.2

660 ± 90

1.4 ± 0.3

3.7 ± 0.3

760 ± 80

PCL-ASA1

2.4 ± 0.2§

2.7 ± 0.7§

840 ± 120§

2.2 ± 0.9

3.4 ± 0.4

800 ± 150

PCL-ASA5

6.4 ± 0.3°

5.4 ± 0.9°

550 ± 60^

2.8 ± 0.8§

3.6 ± 0.5

510 ± 70

#

§

3.5 ± 0.3

530 ± 80

PCL-ASA10

13 ± 2*

9 ± 2*

900 ± 20

4.8 ± 1.5

* P \ 0.05 with respect to PCL, PCL-ASA1 and PCL-ASA5 ° P \ 0.05 with respect to PCL and PCL-ASA1 § # ^

P \ 0.05 with respect to PCL P \ 0.05 with respect to PCL and PCL-ASA5 P \ 0.05 with respect to PCL-ASA1 Table 2 Fitting parameters of the power law model (Eq. 3) for aspirin release from the electrospun tubular scaffolds k

n

PCL-ASA1

12 ± 4

0.35 ± 0.08

PCL-ASA5

5.9 ± 1.0

0.63 ± 0.09

PCL-ASA10

5.1 ± 0.7

0.74 ± 0.03

2

R was in the range 0.98–0.99 for all the investigated cases

Mt ¼ ktn M1 Fig. 2 Temporal aspirin release from the electrospun tubular grafts

Circumferential stress at break values measured for PCL, PCL-ASA1 and PCL-ASA5 were comparable to those of native saphenous vein (3.01 ± 0.91 MPa) [36], whereas scaffolds loaded with 10 % of ASA were characterized by higher values. 3.3 In vitro drug release Continuous localized administration of aspirin to the site of graft implantation could overcome the limitations associated to the short half-life of aspirin in the body. In vitro release experiments were performed in an incubator at 37 °C. The cumulative release profiles of ASA from the electrospun vascular grafts as the released fraction with respect to the total drug content within the fibers are shown in Fig. 2. The ASA release study from electrospun PCL fibers was carried out over a period of 1 week. In this study, drug diffusion was considered the predominant mechanism for drug release from the polymeric matrix, since no significant evidence was reported for PCL degradation in such a short period of time [37]. Drug release rate from PCL depends on the method of preparation, shape and geometry of the scaffold, and type and drug content in the matrix. Ritger and Peppas [38] proposed the general solute release equation from non-swellable polymeric matrix (Eq. 3):

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ð3Þ

where Mt/M? is the fractional release of drug at time t, k is a constant incorporating structural and geometric characteristics of the device, and n is the diffusional exponent indicative of the transport mechanism. According to Ritger and Peppas, Eq. (3) was applied to the first 60 % of the measured fractional release. Assuming the electrospun scaffolds as non-swellable polymeric thin films, the diffusional exponent should be comprised in the range 0.5–1, the lower limit representing the Fickian diffusion, the upper limit the zero-order release and the values in between the anomalous (non-Fickian) transport. The computed fitting parameters are summarized in Table 2. The diffusional exponent n increased with the ASA content within the polymeric fibers, indicating an anomalous transport for PCL-ASA5 and PCL-ASA10 and a specific behavior for PCL-ASA1, since a lower value than 0.5 was computed for n. In this regard, the here presented results need to be critically discussed and eventual limitations of the assumed model should be properly addressed. Firstly, electrospun scaffold can be regarded as a polymeric thin film only from a macroscopic point of view, being indeed composed of micrometric fibers randomly arranged that realize a threedimensional structure comprising large voids among the fibers themselves. The resulting architecture, that is specific for the three investigated cases (Fig. 1), can directly influence the mechanism of drug release. Secondly, the complex structure of the scaffolds can prevent the fibers to be uniformly soaked, at least in a short-time period, thus


leading to a non-homogeneous ASA release in the aqueous environment. Reasonably, fibers in the central region of the scaffold did not experience the same experimental conditions of those collected on the outer layers. Moreover, it can be also added that even each fiber was not uniformly soaked, since only a portion of the lateral surface was directly exposed to the external environment while the rest laid on the underneath fibers. Thirdly, and related to the previous consideration, PCL is a hydrophobic polymer and this characteristic could further affect the penetration of PBS through the voids among the fibers, contributing to enhance the non-homogeneous experimental conditions in the whole open-pore structure, at least in a short-time period. Surprisingly, ASA release from PCL-ASA1 was characterized by a diffusional exponent n below the lower limit of the Fickian diffusion, as predicted by the thin film model, that therefore showed its limitations in this case. Even if the fitting procedure gave valuable mathematical results, as confirmed by the R2 values, a constraint might be introduced suggesting that the power law can only give limited insight into the exact release mechanism of the drug, as similarly reported for hydroxypropyl methylcellulose [39]. This concern is also supported by comparing the k values summarized in Table 2. PCL-ASA5 and PCLASA10 were characterized by similar values of the rate constant, differently from PCL-ASA1 which was the highest one. Considering the definition of this parameter, the reported occurrence can be partially explained by the morphological structure of the electrospun scaffold, which is unique compared to the other two cases, i.e. absence of sub-micrometric fibers and fused fibers at crossing-points. However, these features can be hardly regarded as adequate to determine the different behavior of PCL-ASA1 compared to those of PCL-ASA5 and PCL-ASA10. The acquired results could suggest a fast release in the first hours, most probably from the outer fibers, followed by a slower rate release from the inner layers of the scaffold. The measured behavior for PCL-ASA1 can be representative of a burst/diffusion release from the electrospun fibers [40], reasonably explaining the limitation of the power law model that could be overcome by introducing a constant value in its formulation representative of the burst effect [41]. In a recent study, Wulf et al. [17] described the release kinetics of ASA immobilized on modified PCL surface via cross-linking. They observed a burst release of ASA up to 90 % within the first 12 h, due to the fast hydrolytic cleavage of ester bonds between ASA and the cross-linker. In comparison, we reported a slower ASA release from the fibers attributed to an anomalous diffusional mechanism, with an incomplete drug release up to 1 week that suggests a positive long-term release of remaining ASA. Loading

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Fig. 3 Adherent plateletes on the electrospun tubular grafts at the two considered time points. *P \ 0.05 with respect to PCL, PCLASA1, PCL-ASA5

ASA directly into the PCL solution to be electrospun could represent an easier and faster way to fabricate shelf-ready vascular prosthesis with respect to multi-step surface immobilization processes. 3.4 Platelet adhesion assay Platelets play a critical role in the initiation and propagation of the coagulation cascade, when blood comes into contact with a foreign surface. Deposited platelets may change their morphology, influenced by the surface properties or by the proteins absorbed onto the surface. This change in morphology may then lead to further activation. In order to evaluate this possible response, electrospun grafts were incubated with PRP to assess their capability to inhibit platelet adhesion. SEM investigation revealed an increased platelet deposition after 2 h on PCL-ASA5 and PCLAASA10 with respect to PCL, while a decreasing trend was registered after 6 h, inversely related to the drug content. These observations were numerically quantified in Fig. 3. The absence of a monotonic response acquired at the first time step (2 h), as one might expect, can be explained combining the releasing characteristics of the drug with the morphology of the collected fibers. As previously discussed, PCL-ASA1 showed a burst release in the first hours that contributed to lower the platelet deposition compared to neat PCL. On the other hand, this behavior was not verified increasing the aspirin content, probably due (i) to the slower release with respect to the previous case and (ii) to the nonhomogeneous fiber architecture that lead to an exposed surface with adverse characteristics, e.g. rough texture and/ or locally increased area due to overlapping fibers, that can represent further available sites for the platelets to adhere. In this regard PCL-ASA5 showed the worst response, suggesting the role of the morphological features of the scaffold in the short period, even if loaded with a drug percentage five

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Fig. 4 Platelet adhesion on the electrospun tubular grafts after 6 h: a PCL, b PCL-ASA1, c PCL-ASA5, d PCL-ASA10. Scale bar 1 lm

times higher. A mitigated reaction was observed for PCLASA10, being probably counterbalanced by the higher aspirin content within the fibers. Conversely, on a longer time period (6 h) a monotonic response was registered, PCLASA10 induced the lowest adhesion (P \ 0.05 with respect to the other investigated cases). Concerning the morphology (Fig. 4), adherent platelets on PCL appeared to have protrusions (an early pseudopodial shape, Fig. 4a) that indicated their activation by contact with the microfibers. This occurrence was not so evident on aspirin-loaded grafts (Fig. 4b–d), suggesting that the added drug can contribute to enhance blood compatibility, even at low content. Different approaches were previously tested to evaluate the platelet adhesion on electrospun tubular grafts. Blending poly(ester urethane)urea with increasing contents of poly(2-methacryloyloxyethyl phosphorylcholine-comethacryloyloxyethyl butylurethane) lowered the platelet deposition on the collected fibers, this effect being most likely due to the presentation of 2-methacryloyloxyethyl phosphorylcholine moieties on the polymer blend surface [42]. Hashi et al. [3] conjugated hirudin to the poly(L-lactic acid) microfibers through an intermediate linker of poly(ethylene glycol) (PEG). Platelet adhesion and aggravation were reduced on the PEG surface, while hirudinPEG did not decrease platelet adhesion further, suggesting that the resistance to platelet adhesion could be attributed to the PEG layer.

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Our approach is based on the addition of an effective and largely used antiplatelet drug into a cheap and suitable bioresorbable polymer for vascular tissue engineering applications. The here reported results demonstrated the potential effect in inhibiting platelet adhesion, suggesting the in vivo translation into animal models to test the actual capability of the investigated grafts to prevent thrombus formation.

4 Conclusions Early graft occlusion due to thrombosis remains the major cause of poor patency of synthetic small-diameter vascular grafts. Aspirin is the most common antiplatelet drug used for primary and secondary prevention of vascular diseases. However, drawbacks associated to long-term oral administration have highlighted the need of a localized intraluminal drug release to overcome the systemic side effects of aspirin. In this study we explored the feasibility of electrospun tubular bioresorbable scaffolds as drug delivery system for aspirin release. Novel ASA-loaded PCL fibrous scaffolds were successfully fabricated by electrospinning. Limited influence on fiber mats was observed loading PCL with ASA, showing a different fibers arrangement only for higher drug concentrations. The collected fibrous structure affected the mechanical properties, proving our findings to


be in accordance, for each ASA concentration, with those of native vessels and previously reported synthetic electrospun grafts. A sustained release of ASA from the fibers was demonstrated over one week, being characterized by an anomalous (non-Fickian) transport as the values of the diffusional coefficient n increased with the drug content within the fibers. Moreover, platelet adhesion was significantly reduced by the ASA released, thus demonstrating the effectiveness of the proposed approach. Hence, ASA-loaded PCL electrospun tubular scaffolds could represent a feasible candidate as a novel bioresorbable drug-releasing synthetic graft for small-diameter vessel replacements.

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13. 14. 15.

16. 17.

18. Acknowledgments The authors thank the Cord Blood Bank, Department of Hematology, University Hospital Careggi, Florence, for kindly providing the cord blood samples. CDG acknowledges the Italian Interuniversity Consortium on Materials Science and Technology (INSTM) for the financial support of the scientific activity, Co-funded Grant ‘‘Progettazione, realizzazione e caratterizzazione funzionale di scaffold polimerici elettrofilati per l’ingegneria del tessuto cardiovascolare’’.

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