Enzyme-functionalized vascular grafts catalyze in-situ release of nitric oxide from exogenous NO prodrug

Journal of Controlled Release 210 (2015) 179–188

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Enzyme-functionalized vascular grafts catalyze in-situ release of nitric oxide from exogenous NO prodrug Zhihong Wang a,1, Yaxin Lu b,1, Kang Qin a, Yifan Wu a, Yingping Tian b, Jianing Wang a, Jimin Zhang a,c, Jingli Hou b, Yun Cui a, Kai Wang a, Jie Shen b, Qingbo Xu d, Deling Kong a,c, Qiang Zhao a,⁎ a State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials, Ministry of Education, Collaborative Innovation Center of Chemical Science and Engineering Tianjin, College of Life Sciences, Nankai University, Tianjin 300071, PR China b State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, Nankai University, Tianjin 300071, PR China c Institute of Biomedical Engineering, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300192, PR China d Cardiovascular Division, King's College London BHF Centre, London, United Kingdom

a r t i c l e

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Article history: Received 12 November 2014 Received in revised form 21 March 2015 Accepted 20 May 2015 Available online 22 May 2015 Keywords: Vascular grafts Nitric oxide Enzyme prodrug therapy (EPT) Controlled release Tissue regeneration

a b s t r a c t Nitric oxide (NO) is an important signaling molecule in cardiovascular system, and the sustained release of NO by endothelial cells plays a vital role in maintaining patency and homeostasis. In contrast, lack of endogenous NO in artificial blood vessel is believed to be the main cause of thrombus formation. In this study, enzyme prodrug therapy (EPT) technique was employed to construct a functional vascular graft by immobilization of galactosidase on the graft surface. The enzyme-functionalized grafts exhibited excellent catalytic property in decomposition of the exogenously administrated NO prodrug. Localized and on-demand release of NO was demonstrated by in vitro release assay and fluorescent probe tracing in an ex vivo model. The immobilized enzyme retained catalytic property even after subcutaneous implantation of the grafts for one month. The functional vascular grafts were implanted into the rat abdominal aorta with a 1-month monitoring period. Results showed effective inhibition of thrombus formation in vivo and enhancement of vascular tissue regeneration and remodeling on the grafts. Thus, we create an enzyme-functionalized vascular graft that can catalyze prodrug to release NO locally and sustainably, indicating that this approach may be useful to develop new cell-free vascular grafts for treatment of vascular diseases. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Vascular bypass surgery has proven to be a successful strategy to treat vascular diseases, including cardiovascular and periphery diseases. Due to the shortage of autologous vein graft, artificial blood vessels become indispensable and have received increasing attention [1–3]. Up to now, the commercialized vascular grafts often act merely as physical replacement without any bioactivity or biofunctions. Most of the related literatures mainly focused on material surface modification in order to enhance hemocompatibility [4–6] and (or) promote cell adhesion and growth [2,7,8]. In clinics, patients who need vascular replacement often suffer from chronic diseases such as atherosclerosis, diabetes, hypertension, and hypercholesterolemia, [9]. Therefore, the patency of the implanted grafts is far from satisfactory because of early as well as middle (or) late restenosis, which are caused by thrombosis and intimal hyperplasia [10]. ⁎ Corresponding author at: Institute of Molecular Biology, Nankai University, Tianjin 300071, PR China. E-mail address: [email protected] (Q. Zhao). 1 Co-first authors.

http://dx.doi.org/10.1016/j.jconrel.2015.05.283 0168-3659/© 2015 Elsevier B.V. All rights reserved.

Nitric oxide (NO) is an important signaling molecule in the cardiovascular system. NO secreted by the endothelium holds important physiological functions, including inhibiting platelet adhesion and aggregation, vascular smooth muscle cell (VSMC) proliferation [11], and promoting vasodilation. All of these functions make the endothelium an important physiological barrier to maintain the patency and homeostasis of blood vessels [12]. Hence, NO donors with different chemical structures and releasing profile have been developed as drug for treatment of cardiovascular diseases, such as, diazeniumdiolates (NONOates), S-nitrosothiols, in addition to the commonly used organic nitrates in clinics [9]. However, due to the unstable nature of NONOates, that is, spontaneous decomposition under thermal, acidic, or physiological conditions, a glycosylated NO drug was synthesized which is highly stable and only decomposes to release NO under the catalysis of glycosidases [13]. In most cases, NO donor molecules were loaded into biomaterials by physical blending or chemical binding for preparation of cardiovascular devices (such as vascular grafts, or stents) with localized NO-releasing property [14–21]. Our group has previously prepared polysaccharideand peptide-hydrogel based NO-releasing biomaterials by conjugation of glycosylated NO donors. The two biomaterials showed significant

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effect in treatment of mouse hindlimb ischemia and skin injury [22,23]. Even so, the disadvantage of this drug-loading system is the limited NO donor reservoir unlike the healthy endothelium which could sustainably produce NO at a low rate (10−10 mol/min/cm−2) but with high cumulative amount [24]. Enzyme prodrug therapy (EPT) is a versatile technique that allows synthesis of drugs at the site of action when prodrugs were systemically administrated [25–27]. In addition to targeted and localized drug delivery, the advantage of EPT also includes the fine tune of drug dosage, duration, and administration [28]. In the present study, we took the advantage of EPT technique in the fabrication of functional vascular grafts. First, galactosidase was immobilized onto the vascular grafts through the affinity binding between avidin and biotin. Then, the grafts were transplanted into the rat abdominal aorta. When a glycosylated NO prodrug was administrated through tail vein injection, the prodrug circulated and contacted the enzyme-immobilized vascular graft. The enzyme could catalyze the decomposition of NO prodrug to release NO locally (Scheme 1a). Promoted endothelialization and tissue regeneration were observed in our study owing to the localized and on-demand NO release. To our knowledge, it is the first report of such functional vascular grafts with in-situ catalysis of NO release based on the EPT technique. 2. Experimental section 2.1. Materials and reagents Poly(ε-caprolactone) (PCL) (Mn = 80,000) and poly(ε-caprolactone) diol (Mn = 2000), were purchased from Sigma-Aldrich. N-(3-

(dimethylamino) propyl)-N-ethylcarbodiimide hydrochloride (EDC · HCl, 99%), N-hydroxysuccinimide (NHS), D-(+)-Biotin (98 + %), and L-ascorbic acid sodium salt (99%) were obtained from Alfa Aesar. Galactosidase (8.9 units/mg) and avidin were also purchased from Sigma. DAF-FM DA (3-Amino, 4-aminomethyl-2′, 7′-difluorescein, diacetate) was the product of Beyotime Institute of Biotechnology (China). Copper (II) sulfate pentahydrate (CuSO4·5H2O, 98%) and other reagents were purchased from Tianjin sixth Reagent Company. All other reagents unless otherwise noted were used as received without further purification. Wistar rats (male, weight 280–320 g) were purchased from the Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, China).

2.2. Preparation of vascular grafts by electrospinning The fibrous membranes and tubular grafts were fabricated by electrospinning using a setup previously described [29]. Briefly, high molecular weight PCL was mixed with PCL-N3 at blending ratio of 8/2 (w/w). The mixture was dissolved in 10 mL of methylene chloride and methanol (5:1, v/v) mixture, and stirred sufficiently to obtain homogeneous solution (25 w/v%). The tubular grafts were fabricated by using a stainless steel rod (OD = 2 mm) as a collector with the following processing parameters: needle tip–collector distance = 11 cm, flow rate = 8 mL/h and voltage = 11 kV. The as-prepared grafts were dried in a vacuum at room temperature for more than 3 days in order to remove the residual solvents sufficiently. The details for the synthesis of PCL2000-N3 were shown in supporting information (Scheme S1, and Figs. S1–2).

A Enzyme

NO

NO prodrug

NO prodrug

N3

B

N3

PCL fiber

Click reaction

B

Avidin

PCL fiber

B B

NO probe

B

B

Biotin-galactosidase

PCL fiber

B

NO probe

B

Biotin

Avidin

B

PCL fiber

Galactosidase

Scheme 1. (A) Illustration for the enzyme immobilized on the vascular grafts to catalyze the decomposition of exogenously administrated NO-prodrug to release NO. (B) Pathway for the surface enzyme-functionalization of PCL vascular grafts.

Z. Wang et al. / Journal of Controlled Release 210 (2015) 179–188

2.3. Functionalization of PCL vascular grafts Biotins were grafted onto the surface of electrospun PCL through the click reaction between azide groups and alkynyl-biotin (Biotin-C ≡ CH) in dimethyl formamide (DMF)/water (1/2 in volume) under the catalysis of CuSO4/L-ascorbic acid sodium (1:10 in mole ratio) [30]. The concentration of Biotin-C ≡ CH was 2 mg/mL, and the mixture was maintained at 37 °C for 24 h with gentle shaking. The specimen was rinsed with deionized water and dried at room temperature. Then, the galactosidase was immobilized onto the biotinylated PCL surface by the aid of affinity interaction between biotin and avidin [31]. In detail, PCL–biotin was put into avidin solution of 0.5 mg/mL and incubated at room temperature for 4 h. Then, the specimen was removed and washed by deionized water. Then it was put into biotinylated βgalactosidase solution of 1 mg/mL and maintained at room temperature with gentle shaking for 2 h. Finally, the functionalized specimen was washed with deionized water and dried at room temperature in a vacuum. The details for the synthesis of Biotin-C ≡ CH, biotinylated β-galactosidase, and DAF-avidin were shown in supporting information (Scheme S2–3). 2.4. Characterization of the functionalized vascular grafts The surface morphology of electrospun PCL was observed by using a scanning electron microscope (SEM, Quanta 200, Czech) with an accelerating voltage of 15 kV. Samples were gold coated before SEM analysis. The surface chemistry was characterized by using Fourier transform infrared spectroscopy (FT-IR) at single attenuated total reflectance (ATR) mode (Bio-Rad FTS 6000 spectrometer). The spectral resolution is 8 cm− 1. The surface contact angle of PCL film was measured by the sessile drop method with a Harke-SPCA goniometer (Beijing, China). The samples were adhered on glass slice by double-face adhesive tape and put onto the sample stage. The images were recorded continuously at room temperature. The enzyme immobilized on the surface of vascular grafts was quantified by normalizing the catalytic activity with that of free enzyme using O-nitrophenyl-β-galactopyraniside (ONPG) as the substrate. In brief, 1 mL of ONPG solution (0.5 mg/mL) was catalyzed by addition of different amounts of β-galactosidase. After reaction at 37 °C for 1 h, the decomposed product O-nitrophenol (ONP) was quantified by measuring the optical density (OD) at wavelength of 415 nm using an iMarkTM microplate reader (BIO-RAD). The liner relationship between enzyme quantity and OD value was thus established. At the same time, ONPG was also catalyzed by the addition of enzymefunctionalized PCL graft (1 cm in length) under the same condition, therefore the relative amount of enzyme immobilized on the graft was determined through the working curve. Tensile stress–strain curves for the electrospun tubular grafts were obtained by using an Instron universal tensile tester (model 5865). The tubular grafts with 20 mm in length and about 0.4 mm in thickness were utilized for the measurement. The distance between the two grips was set as 10 mm. Tensile test was performed at ambient temperature with crosshead speed of 10 mm/s. Each test was repeated on 3 specimens and the mean value as well as standard deviation (SD) were reported. 2.5. In vitro NO release In vitro catalysis property of galactosidase-immobilized PCL was determined by Griess kit assay. In brief, 0.5 mg of NO prodrug was dissolved in 1 mL of PBS buffer (pH 7.4), and enzyme-functionalized PCL graft (1.0 cm in length) was put into the prodrug solution. Fetal bovine serum (2%) was added in the control without graft. At each pre-determined time interval, 50 μL of diluted supernatant was transferred into a 96-well plate, then 50 μL Griess I and 50 μL Griess II were added thereafter. The azo compound with purple color was formed

181

and the absorbance was measured at wavelength of 540 nm using an iMarkTM microplate reader (BIO-RAD). 2.6. Arteriovenous shunt assay Rat arteriovenous shunt (AV-shunt) assay was performed using the NO-probe immobilized vascular grafts according to a previous report [10]. All the animal protocols have been approved by the Animal Care Committee of Nankai University. Rats were anesthetized by intraperitoneal injection of 10% (w/v) chloral hydrate. A loop consisting of sample fragments was connected as a shunt system. After disinfection by 70% (v/v) ethanol for 30 min, and heparization (50 IU/mL) for 2 h, the whole extracorporeal circulation was established using 24-G indwelling needles to connect the abdominal aorta and superficial vein, and maintained for 1 h. At the end, the circuit was perfused with physiological saline, the luminal surface of the grafts was observed under the confocal laser scanning microscope (CLSM, ZEISS LSM710). 2.7. In vivo implantation Rats were anesthetized by intraperitoneal injection of chloral hydrate (300 mg/kg). Heparin (100 units/kg) was administered for anticoagulation through tail vein before surgery. A midline laparotomy incision was made, and the abdominal aorta was isolated, clamped, and transected. The enzyme-functionalized vascular grafts (2.0 mm in inner diameter and 1.0 cm in length) were anastomosed between the renal arteries and the aorta–iliac bifurcation in an end-to-end way using interrupted 9-0 monofilament nylon sutures. Finally, the blood flow was restored, and the incision was closed with 3-0 nylon sutures. The rats were divided into two groups after the implantation surgery. The experimental animals received injection of NO-prodrug through the tail vein every two days. The control animals only received injection of saline. Animals were followed up to 7, 14 and 30 days before sacrifice. No anticoagulation drug was administered to the rats after surgery. 2.8. Assessment of vascular function The physiological functions of the explanted grafts at 1 month were assessed by wire myography [3]. In brief, the grafts were dissected, and the connective and fat tissues was cleaned. The rings of 3 mm in length were obtained and bathed in the standard Krebs buffer (composition in mM: NaCl, 118.4; KCl, 4.7; CaCl2, 2.5; MgSO4, 1.2; KH2PO4, 1.2; NaHCO3, 25; dextrose, 11.1; pH 7.4) at 37 °C and gassed with carbogenic mixture (95% O2 and 5% CO2). All preparations were stabilized under a resting tension of 2.00 g for 1 h with the buffer changed every 15 min. The functionality of the neo-endothelium was confirmed by the relaxation by acetylcholine (Ach, 10 μM) in pre-constricted segments by adrenaline (AD, 1 μM). Isometric forces were recorded with force transducers connected to a PowerLab/870 Eight-channel 100 kHz A/D converter (AD Instruments, Sydney, Australia). Results were obtained from the explants from 3 animals in each group. 2.9. Histology analysis At the determined time points, the grafts were explanted and processed for histochemical analysis according to previous report [3]. For SEM observation, explants were fixed with 2.5% glutaraldehyde overnight, and dehydrated using graded ethanol. Samples were mounted onto aluminum stubs, sputter-coated with gold, and finally observed under SEM. To examine platelet adhesion, the samples were stained with mepacrine solution (10 mM) for 90 min, the stained platelets were observed under CLSM. The number of adhered platelets was quantitatively counted from six randomly selected fields for statistical analysis. For histology analysis, the explants were fixed by 4% paraformaldehyde, dehydrated using 30% sucrose solution for one day. After embedded in OCT, the explants were cryosectioned to 6 μm in thickness,

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and stained with hematoxylin and eosin (H&E). Images were acquired under a microscope (Nikon Eclipse TE2000-U Kanagawa, Japan) and analyzed by Nikon NIS Elements software. In immunofluorescent staining, the cryosections were fixed by cold acetone for 10 min, air-dried, and washed once by PBS. The slides were blocked with 5% normal goat serum (Zhongshan Golden bridge Biotechnology, China) for 45 min at 4 °C. For intracellular antigens staining, 0.1% Triton-PBS was used to permeate the specimen before incubation with serum. The slides were incubated with rabbit anti-von Willebrand factor (vWF, Dako, USA. 1:200 in PBS) overnight at 4 °C, which was followed by washing with PBS five times. Then, the slides were incubated with secondary antibody, Alex Fluo 488 goat antirabbit IgG (1:200, Invitrogen) for 2 h at room temperature. The nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI) (Dapi Fluoromount G, Southern Biotech, England). Images were taken by using a fluorescence microscope (Zeiss Axio Imager Z1, Germany). 3. Results and discussion 3.1. Structure and properties of the enzyme-functionalized vascular grafts The tubular grafts with an inner diameter of 2 mm were prepared by electrospinning of poly(ε-caprolactone) [3]. To introduce reacting sites for further surface functionalization, low molecular weight PCLs double-end-terminated with azide groups were mixed with PCL at a weight ratio of 1:4 [32,33]. This method resulted in high density of azide group on the surface of electrospun fiber due to electrostatic attraction forces during the electrospinning process [32]. The microstructure of electrospun grafts was analyzed by SEM. The fiber morphology was well-defined with a diameter of 3.61 ± 1.61 μm and pore size of ~ 30 μm, which has been shown to be favorable for cell infiltration during in vivo regeneration [3]. The fiber distribution is homogeneous and suitable for use as vascular grafts (Fig. 1). In general,

the fiber morphology of enzyme-functionalized PCL grafts was similar to those without functionalization. The enzyme-functionalization of electrospun vascular grafts was performed by two steps. First, the biotin was grafted onto the surface through the click reaction by using alkynyl-biotin. Then, the enzyme galactosidase was immobilized by the strong affinity interaction between biotin and avidin (Kd ~ 10− 15 M, Scheme 1b). The functionalization procedure is mild and environmentally friendly to preserve the bioactivity of enzyme [34]. The surface chemistry of functionalized PCL surface was characterized by ATR-FTIR. An evident peak at 2096 cm− 1 was identified on the spectrum of PCL/PCL-N3, which is attributed to the stretching of azide group. After the click reaction with alkynyl-biotin, this peak disappeared, indicating the complete substitution of azide group (Fig. 2A). The β-galactosidase immobilized on the graft was quantified by normalizing the catalytic activity to that of free enzyme, and it was equal to 0.018 mg per graft (1 cm in length). The interaction between biotin and avidin has been confirmed by fluorescent imaging when FITC-avidin was utilized (Fig. S3). After functionalization, the surface hydrophilic/hydrophobic property was evaluated by measuring the static water contact angles (Fig. 2B). The surface of electrospun PCL-N3 showed high level of hydrophobicity with a contact angle of 97.2° after 120 s. After grafting of biotin, the surface turned hydrophilic significantly with the contact angle reduced to about 48.0°. No detectable difference could be discerned after the affinity interaction with avidin. The surface hydrophilicity was further enhanced when the biotinylated galactosidase was immobilized, with contact angle decreased to 31.2°. Such surface functionalization modified the hydrophobic profile of polyester PCL, which was capable of improving cell affinity to the biomaterial matrix [35]. The mechanical properties of the functionalized vascular grafts were evaluated by tensile test (Fig. 2C). In general, the stress–strain curve of enzyme-functionalized PCL was similar to that of pure PCL, both of

A

B

C

D

Fig. 1. PCL vascular grafts (ID = 2 mm) after enzyme functionalization (A), and cross-section (B), inner surface (C) and outer surface (D) of the graft observed by SEM. Inserts show magnification of the image (B). Scale bar is 250 μm.

Z. Wang et al. / Journal of Controlled Release 210 (2015) 179–188

Transmittance (a.u.)

A

(Table 1), which were still sufficient to satisfy the requirement of artificial blood vessel. The burst pressure was 2853 ± 234 mm Hg, which was similar to that of mammary artery and higher than that of saphenous vein clinically utilized.

PCL-Biotin

3.2. In vitro catalytic property

PCL-N3 Azide group -1 (2096 cm )

4000

3000

2000

1000 -1

Wavenumber (cm )

B

100

Contact angle (Deg)

183

PCL-N3 80

PCL-Biotin 60

PCL-Biotin-Avidin

40

The catalytic performance of enzyme immobilized on the vascular grafts was first evaluated by in vitro assay in PBS buffer. The galactosidase first hydrolyzes the glucosidic bond, thus the un-protected NONOate decomposes spontaneously to release NO [22]. The releasing profile of NO was similar to that catalyzed by the free enzyme in terms of releasing kinetics and cumulative releasing ratio, which confirmed the catalytic activity of the immobilized enzyme on the vascular grafts (Fig. 3A). In contrast, no release of NO could be detected in the absence of catalyst even in the presence of serum, indicating the high stability of NO prodrug (Gal-NONOate). In vivo stability of the immobilized enzyme was assessed by subcutaneous implantation of the vascular grafts for 3, 14, and 30 days (Fig. 3B). The explanted vascular grafts still demonstrated effective catalytic property, although the catalytic efficiency decreased to some extent, which was ascribed to the macrophage-mediated degradation and elimination. These results suggest that the enzyme activity could be preserved within the tissue environment in the body for at least 1 month, an important time window for the regeneration of endothelium. 3.3. Ex-vivo catalytic property

PCL-Biotin-Avidin-Enzyme 20 0

20

40

60

80

100

120

Time (Second) 6

C Stress (MPa)

5

PCL-Enzyme graft

4 3 2

PCL graft

1 0 0

200

400

600

800

Strain (%) Fig. 2. Surface chemistry properties characterized by ATR-FTIR (A), surface hydrophilic/ hydrophobic performance characterized by static water contact angle measurement (B) of PCL surface before and after enzyme-functionalization, and representative stress– strain plot of tensile test (C).

which demonstrating high level of toughness. The addition of low molecular PCL-N3 moderately reduced the mechanical properties (including tensile strength, Young's modulus and elongation at break)

Table 1 Mechanical properties measured by tensile test. Mechanical properties

PCL grafts

PCL-enzyme grafts

Young's modulus (MPa) Stress at max (MPa) Strain at break (%) Burst pressure (mm Hg)

9.73 ± 0.61 5.44 ± 0.72 759.89 ± 11.09 2368 ± 136

8.68 ± 2.50 4.10 ± 0.76 494.67 ± 34.64 2853 ± 234

An arteriovenous shunt (AV-shunt) assay was utilized to obtain further insight into the catalytic property of immobilized enzyme under the actual blood flow in animal models. An NO probe, diaminofluoresceins (DAFs), was immobilized onto the vascular graft to trace the released NO in a real-time manner [36]. Rats implanted with enzyme-functionalized vascular grafts were injected with NO prodrug or saline through the tail vein before establishment of circulation (Fig. 4). After 1 h of circulation, the explanted specimens of vascular grafts showed evident green fluorescence in contrast to the control groups (without NO probe or NO prodrug), which verifies the in-situ (localized) release of NO by the catalysis of immobilized enzyme on the implanted vascular grafts. In contrast, endogenous NO secreted by endothelium failed to generate fluorescence on the implanted vascular grafts without supplying NO prodrug (Fig. 4D) due to the limited diffusion distance of nitric oxide (~ 100 μm) [9]. In addition, the avidin-functionalized vascular graft (without enzyme immobilization) was also tested in the presence of NO probe. No release of nitric oxide could be identified by the probe, which also precludes the possibility of decomposition of NO prodrug catalyzed by avidin or endogenous enzyme (Fig. S4). 3.4. In vivo implantation The enzyme-functionalized vascular grafts were further evaluated by in vivo implantation in a rat abdominal artery replacement model. As shown in Fig. 5, the artery between the infrarenal abdominal aortic and the aorta–iliac bifurcation was replaced by the vascular graft (2.0 mm in inner diameter, and 1.0 cm in length) by an end-to-end anastomosis pattern. After implantation, the rats were administrated with NO prodrug or saline every two days until the grafts were harvested at days 7, 14, and 30 respectively. After 2 weeks, all the grafts in the NO group (n = 7) were patent, however, one was occluded in the control group (n = 5). At 1 month, there was one occlusion in each group (n = 5). Further analysis by stereomicroscopy revealed that the surface of the experimental group was white and clean without formation of obvious thrombus at day 7. However, in control group, the surface is red due to

A

2.0

Cumulative releae amount (µmol)

Z. Wang et al. / Journal of Controlled Release 210 (2015) 179–188

Cumulative releae amount (µmol)

184

PCL-Enzyme graft

1.5

Free enzyme (0.005mg/mL) 1.0

0.5

Without enzyme 0.0 0

10

20

30

40

50

60

70

B

2.0

0 day

1.5

1.0

3 days

30 days 0.5

14 days 0.0 0

5

10

Time (hour)

15

20

25

Time (hour)

Fig. 3. Catalytic property of enzyme-functionalized PCL grafts compared with free enzyme (A), and catalytic property of the specimens after subcutaneous implantation for different time periods (B), measured by in vitro Griess kit assay (n = 3). Data are expressed as the mean ± standard deviation (SD).

the infiltration of red cells (Fig. 5B) [37]. Similar differences were also observed at day 14, and some microthrombi were evident on the surface of control group (arrow indicated). SEM revealed that the graft surface in the NO group was mostly bare fibers with significant less platelet adhesion than that in the control group at day 7 (Fig. 5D). The platelets adhered on the surface were further stained with mepacrine and examined using a fluorescent microscope. Quantification results again revealed that platelet adhesion was markedly reduced in the experimental group than in the control group at day 7 (Fig. 5G). However, the luminal fibrous surfaces were fully covered in both groups at day 14. No platelet could be detected at day 14 in both groups from the fluorescent images (Fig. S5). These data indicate that sustained release of NO can inhibit platelet adhesion and aggregation during the early stage, which is one of the physiological functions of NO released by the endothelium of the blood vessel, and is also very important to reduce thrombus formation and early restenosis in the vascular grafts. The tissue regeneration within the vascular grafts was analyzed by H&E staining. As shown in Fig. 6 A–D, a layer of neo-tissue was present

A

on the lumen of vascular graft in the experimental group at days 14 and 30. Quantitative analysis (Fig. 6E and F) demonstrated that both the coverage ratio and the thickness of the neo-tissue were dramatically increased in the experimental group (81.1 ± 11.8% and 22.8 ± 5.8 μm) when compared to the control group (37.9 ± 11.8% and 8.9 ± 4.7 μm). After 1 month, the grafts were almost 100% covered by newly regenerated tissues in both groups, and the thickness of the neo-tissue was also very close to each other (60.3 ± 25.4 μm vs. 65.4 ± 28.6 μm). It means that the sustained NO release promotes the tissue regeneration at early stage, and the tissue remodeling and physiological function are more important aspects to be considered for longer time period. Immunostaining of vWF, an endothelial marker, revealed higher percentage of endothelial coverage (dotted line) in the experimental group at day 14 (Fig. 7), indicating rapid endothelialization. Importantly, similar to the native endothelium at the anastomotic sites (Fig. S6), the grafts of experimental group exhibited a thin vWF+ layer, which was previously suggested to be the mature and quiescent endothelium

OH

B

OH OO

HO OH

O

N

N + N

Fluorescence immison

Galactosidase NH2

N

H2N

NH

N

2 NO CONH

CONH

O2 O

C

D

O

O-

E

O

O

O-

F

Fig. 4. AV-shunt assay for enzyme-functionalized vascular grafts by using NO-probe to trace the in-situ NO release: Illustration of AV-shunt assay (A); Schematic pathway for the reaction of NO with the NO-probe to produce fluorescence (B); The luminal surface of NO probe-immobilized graft with administration of the NO prodrug after 1 hour circulation observed by a stereomicroscope (C); and the corresponding CLSM images for fluorescence tracing (D). Control groups are graft without NO probe immobilization after administrated with prodrug, (−) probe (+) prodrug (E), and NO probe-immobilized graft without NO prodrug administration, (+) probe (−) prodrug (F). Scale bar is 50 μm.

Z. Wang et al. / Journal of Controlled Release 210 (2015) 179–188

A

185

B1

B2

C1

C2

Abdominal aorta

Graft

Abdominal aorta

D1

D2

E1

E2

F1

F2

G

Fig. 5. Implantation of enzyme-functionalized vascular grafts by replacing a part of the rat abdominal aorta (A), and the luminal surface of explanted vascular grafts at day 7 (B1, B2) and day 14 (C1, C2) without (B1, C1) and with (B2, C2) administrated NO-prodrug observed by a stereomicroscope. SEM images show the platelet adhesion and endothelialization of the enzyme-functionalized PCL grafts without (D1, E1) and with NO-prodrug administration (D2, E2) over days 7 (D1, D2) and 14 (E1, E2). CLSM images show the mepacrine-stained platelets adhered on explanted grafts over day 7 without (F1) and with NO-prodrug administration (F2), and the correlative statistical analysis (G). Scale bar is 50 μm. Data are expressed as mean ± SEM.

[38]. In contrast, the control grafts showed a thick layer consisting of diffused vWF+ cells (indicated by arrow), which were endocytosed platelets by macrophages in early inflammatory reaction, cannot be identified as true endothelium [39]. After 1 month, the lumen of the graft was almost fully covered by the endothelium in both groups (Fig. 7C and D). The physiological function of regenerated endothelium was further evaluated by wire myograph. In general, two explanted grafts in NO group showed detectable physiological response (2/3), however, none was detected in the control group (0/3) (Fig. 8). The regenerated graft demonstrated certain degree of vasoconstriction to adrenaline (AD) (tension N 5). More importantly, the contraction could be subsequently relaxed due to the stimuli of acetylcholine (Ach) (%), which demonstrating the vasodilation function of regenerated endothelium. Therefore, as an important signaling molecule, NO plays a vital role in maintaining the short-term patency of the vascular grafts through reducing platelet adhesion, aggregation, and thrombus formation. More importantly, sustained release of NO created a beneficial extracellular microenvironment to favor the vascular tissue regeneration and remodeling to achieve the physiological function of the blood vessel, which is very important to maintain of the long-term patency. Creating a functional vasculature represents one of the most fundamental challenges in tissue engineering, and most notable successes so far have been in thin or avascular structures such as the skin, bladder

and cartilage [40]. Hence, various strategies have been exploited for the construction of 3D vasculature in the engineered tissues, including optimization of scaffold structure [41,42], delivery of angiogenic factors [43], and novel fabrication technique [44,45]. It has been suggested that NO, as a small signaling molecule, plays a vital role in angiogenesis by mediating VEGF-induced mobilization of endothelial progenitor cells (EPCs) to the sites of vascular injury [46,47]. Indeed, we showed that delivery of NO effectively enhanced vascularization in diabetic mice with hind-limb ischemia [22]. Enhanced vascularization was also beneficial for wound healing in mice [23]. Controlled delivery of NO holds the advantages over the other above-mentioned strategies in terms of feasibility and cost. Therefore, the enzyme prodrug therapy (EPT) technique for NO delivery developed in this study can be extended to the research of other types of tissue engineering (including bone, myocardial tissue, etc.) to regenerate vascularized tissues or organs with complex 3-D structure in the future. 4. Conclusion In summary, a new and functional small-diameter vascular graft with enzyme prodrug therapeutic property was prepared in this study. The galactosidase immobilized on the grafts effectively catalyzed the decomposition of NO prodrug (Gal-NONOate), and enabled localized and on-demand NO release. The immobilized enzyme can retain

Z. Wang et al. / Journal of Controlled Release 210 (2015) 179–188

A

* P < 0.05

B

E

*

*

100

% of neo-tissue covered the lumen

186

80 60 40 20 0

D

C

*

F

*

100µm

100µm

The average thcikness of the neotissue (µm)

th th ks ks /2w l/1mon up/2w /1mon trol o p Con Contro NO gr O grou N

90 60 30 20 10 0

th th ks ks /2w l/1mon up/2w /1mon trol o p Con Contro NO gr O grou N

Fig. 6. Histological analysis of enzyme-functionalized vascular grafts explanted at days 14 (A, B) and 30 (C, D) by H&E staining. (A, C) NO group; (B, D) Control group. The corresponding statistical analysis of coverage ratio (E) and thickness (F) of the neo-tissue. Asterisk (*) indicated the lumen of the graft, and the dark arrow referred to the neo-tissue. Data are expressed as mean ± SEM.

A

* 500µm

B

* 500µm

C

* 500µm

D

* 500µm

Fig. 7. Endothelialization in the enzyme-functionalized vascular grafts explanted at days 14 (A, B) and 30 (C, D). Longitudinal section was immunostained by vwf antibody (Green) to identify endothelial cells. Cell nuclei in the immunofluorescent micrographs were counterstained by DAPI (Blue). (A, C) NO group; (B, D) Control group. Graft lumen is indicated by *, and anastomotic site of the graft is indicated by arrowhead. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

A

Relaxation of Ach (%)

Z. Wang et al. / Journal of Controlled Release 210 (2015) 179–188

D

B

187

40 30

E

20 10 0

th th rta on on ao ve /1m /1m i l t p o u Na ntr gro Co NO

C

Fig. 8. The physiological functions of the vascular grafts explanted at day 30 were assessed by wire myography. The representative curve of the graft with (A) and without (B) detectable response to AD and Ach, as well as the curve of the native aorta (C). Contractile response of the grafts to AD (D), and relaxation response to Ach (E). Data are expressed as mean ± SEM.

their catalytic activity in vivo for up to 1 month. This enzymefunctionalized strategy is superior to the traditional NO drug-loaded delivery system which is limited by the capacity of NO reservoir. In vivo implantation experiment revealed that the sustained release of NO markedly inhibited platelet adhesion and thrombus formation. More importantly, localized NO release improved tissue regeneration, remodeling, as well as physiological function. We also observed enhanced endothelialization, which is critical for the small-diameter vascular grafts to maintain long-term patency. Acknowledgments The work was financially supported by NSFC projects (No. 81171478, 81371699, 2127212, and 31400833), Science & Technology Project of Tianjin of China (No. 12JCQNJC09300), Program for Changjiang Scholars and Innovative Research Team in University (No. IRT13023), China Postdoctoral Science Foundation (No. 2014M560183). FP7-PEOPLE-IRSES project ABREM is also acknowledged.

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Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jconrel.2015.05.283.

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