Characterization of the Fluorescent Spectra and Intensity of Dabigatran and Dabigatran Etexilate: Application to HPLC Analysis with Fluorescent Detection†.

There is considerable interest in dabigatran etexilate (Pradaxa) and its major metabolite, dabigatran, which has been shown to be an important inhibitor of thrombin and clotting. In this study, the fluorescent excitation and emission spectra of dabigatran and dabigatran etexilate were characterized. In addition, a ultra performance liquid chromatography (UPLC) and high performance liquid chromatography (HPLC) method using fluorescent detection was developed for the analysis of dabigatran. Dabigatran and dabigatran etexilate were found to have excitation and emission maxima of 310 and 375 nm and 335 and 400 nm, respectively. UPLC analysis of dabigatran standards and plasma dabigatran samples were analyzed on a reversed phase C-18 column with methanol-water (70:30, v/v) as the mobile phase. The lower limit of quantitation for dabigatran was 10.0 ng/mL for both the standards and plasma samples. Standard curves were linear from 10.0 to 1000.0 ng/mL (R2 = 0.995). Within-day coefficient of variations of the fluorometric method at 50.0, 100.0 and 500.0 ng/mL were 1.38%, 4.83% and 2.31%, respectively. The intense fluorescent properties of dabigatran permit the sensitive and specific UPLC or HPLC fluorescent analysis of dabigatran.

Characterization of the Fluorescent Spectra and Intensities of Various Lignans: Application to HPLC Analysis with Fluorescent Detection†.

There is considerable interest in dietary lignans since they have been shown to have antioxidant, estrogenic and lipid-lowering activity in humans. In this study, the fluorescent excitation and emission spectra of seven lignans were characterized and their relative fluorescent intensities compared. The lignans were found to have similar excitation (286.6 ± 2.5 nm, X ± SD) and emission (320.1 ± 6.4 nm) maxima; however, their fluorescence intensities on a molar basis decreased in the following order: asarinin, sesamin, sesamolin, seco-isolariciresinol, seco-isolariciresinol diglucoside and matairesinol. Enterolactone, a mammalian lignan conversion product, and sesamol, an antioxidant found in sesame oil, also exhibited significant fluorescence excitation and emission intensities. A high-performance liquid chromatographic method using photodiode array (PDA) and fluorescent detection was developed for the analysis of the individual lignans. Analysis was performed on a reversed phase C-18 column with methanol-water (70:30, v/v) as the mobile phase. With fluorescent detection, the limits of quantitation (LOQ) was 0.1 ng or 2.82 nmol for sesamin and asarinin, 2.70 nmol for sesamolin, 2.76 nmol for seco-isolariciresinol, 1.45 nmol for seco-isolariciresinol diglucoside, 2.79 nmol for matairesinol and 0.5 ng or 1.67 nmol for enterolactone. With PDA detection, the LOQ was a 1000-fold less sensitive than with fluorescent detection.

Mechanisms of tissue uptake and retention in zotarolimus-coated balloon therapy.

BACKGROUND: Drug-coated balloons are increasingly used for peripheral vascular disease, and, yet, mechanisms of tissue uptake and retention remain poorly characterized. Most systems to date have used paclitaxel, touting its propensity to associate with various excipients that can optimize its transfer and retention. We examined zotarolimus pharmacokinetics. METHODS AND RESULTS: Animal studies, bench-top experiments, and computational modeling were integrated to quantify arterial distribution after zotarolimus-coated balloon use. Drug diffusivity and binding parameters for use in computational modeling were estimated from the kinetics of zotarolimus uptake into excised porcine femoral artery specimens immersed in radiolabeled drug solutions. Like paclitaxel, zotarolimus exhibited high partitioning into the arterial wall. Exposure of intimal tissue to drug revealed differential distribution patterns, with zotarolimus concentration decreasing with transmural depth as opposed to the multiple peaks displayed by paclitaxel. Drug release kinetics was measured by inflating zotarolimus-coated balloons in whole blood. In vivo drug uptake in swine arteries increased with inflation time but not with balloon size. Simulations coupling transmural diffusion and reversible binding to tissue proteins predicted arterial distribution that correlated with in vivo uptake. Diffusion governed drug distribution soon after balloon expansion, but binding determined drug retention. CONCLUSIONS: A large bolus of zotarolimus releases during balloon inflation, some of which pervades the tissue, and a fraction of the remaining drug adheres to the tissue-lumen interface. As a result, the duration of delivery modulates tissue uptake where diffusion and reversible binding to tissue proteins determine drug transport and retention, respectively.

Comparative assessment of transient exposure of paclitaxel or zotarolimus on in vitro vascular cell death, proliferation, migration, and proinflammatory biomarker expression.

Both paclitaxel and zotarolimus are currently employed in vascular interventional therapies, such as drug-eluting stents, and are under investigation for use in other novel drug-device combination products. Paclitaxel is a microtubule-stabilizing compound with potent antiproliferative properties and antimigration effects, whereas zotarolimus is a potent mammalian target of rapamycin inhibitor with antiproliferative and antiinflammatory properties. This study was intended to compare paclitaxel and zotarolimus for intravascular applications in which drug exposure time may be reduced, such as in drug-coated balloons. These applications are generally aimed at reducing neointimal hyperplasia by limiting smooth muscle cell (SMC) proliferation and inflammatory cell recruitment, while minimally interfering with vessel reendothelialization after balloon denudation. In the cellular models described in this study, transient exposure of zotarolimus resulted in the sustained inhibition of SMC proliferation and reduced endothelial cell (EC) proinflammatory cytokine expression, while not affecting EC migration and viability. Transient exposure of paclitaxel inhibited SMC proliferation, EC migration, and overall cell viability, with no effect on expression of the proinflammatory biomarkers studied.

Vascular response to zotarolimus-coated balloons in injured superficial femoral arteries of the familial hypercholesterolemic Swine.

BACKGROUND: Drug-coated balloons are rapidly emerging as a therapeutic alternative for the interventional treatment of peripheral vascular disease. The purpose of this study was to test the hypothesis that an angioplasty balloon coated with the mTOR inhibitor zotarolimus (ZCB) would inhibit neointimal hyperplasia in a novel injury-based superficial femoral artery model in the familial hypercholesterolemic swine. METHODS AND RESULTS: A total of 44 familial hypercholesterolemic swine were included (12 designated to study tissue pharmacokinetics and 32 to study safety and efficacy). Fogarty balloon denudation was performed in all superficial femoral artery segments, followed by balloon angioplasty. In the pharmacokinetic study, a total of 24 ZCBs (300 µg/cm(2)) were used. Zotarolimus was detected in arterial tissue at 5 minutes (162 ng/mg of tissue), 24 hours (5.9 ng/mg of tissue), and 28 days (0.007 ng/mg of tissue) after ZCB inflation. In the safety and efficacy study, superficial femoral artery segments were randomized to either high-dose (600 µg/cm(2), n=16), low-dose (300 µg/cm(2), n=16), or paired uncoated balloons (high-dose ZCB control, n=16; low-dose ZCB control, n=16). At 28 days, the percentage of angiographic stenosis was similar among all tested groups. Histological analysis demonstrated a reduction in neointimal formation in both ZCB groups compared with controls (high-dose ZCB 44% reduction, P=0.007; low-dose ZCB 22% reduction, P=0.08). There was no evidence of delayed arterial healing or vascular toxicity in any of the ZCB groups. CONCLUSIONS: The single delivery of zotarolimus via coated balloon is feasible, and therapeutic levels are maintained up to 28 days. The ZCB technology appears to be effective in the reduction of neointimal proliferation in the superficial femoral artery of the familial hypercholesterolemic swine.

In vivo performance of a phospholipid-coated bioerodable elastomeric graft for small-diameter vascular applications.

There remains a great need for vascular substitutes for small-diameter applications. The use of an elastomeric biodegradable material, enabling acute antithrombogenicity and long-term in vivo remodeling, could be beneficial for this purpose. Conduits (1.3 mm internal diameter) were obtained by electrospinning biodegradable poly(ester urethane)urea (PEUU), and by luminally immobilizing a non-thrombogenic, 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymer. Platelet adhesion was characterized in vitro after contact with ovine blood. The conduits were implanted as aortic interposition grafts in the rat for 4, 8, 12, and 24 weeks. Surface treatment resulted in a 10-fold decrease in platelet adhesion compared to untreated material. Patency at 8 weeks was 92% for the coated grafts compared to 40% for the non-coated grafts. Histology at 8 and 12 weeks demonstrated formation of cellularized neotissue consisting of aligned collagen and elastin. The lumen of the grafts was confluent with cells qualitatively aligned in the direction of blood flow. Immunohistochemistry suggested the presence of smooth muscle cells in the medial layer of the neotissue and endothelial cells lining the lumen. Mechanically, the grafts were less compliant than rat aortas prior to implantation (4.5 ± 2.0 × 10(-4) mmHg(-1) vs. 14.2 ± 1.1 × 10(-4) mmHg(-1) , respectively), then after 4 weeks in vivo they approximated native values, but subsequently became stiffer again at later time points. The novel coated grafts exhibited promising antithrombogenic and mechanical properties for small-diameter arterial revascularization. Further evaluation in vivo will be required to demonstrate complete remodeling of the graft into a native-like artery.

A bilayered elastomeric scaffold for tissue engineering of small diameter vascular grafts.

A major barrier to the development of a clinically useful small diameter tissue engineered vascular graft (TEVG) is the scaffold component. Scaffold requirements include matching the mechanical and structural properties with those of native vessels and optimizing the microenvironment to foster cell integration, adhesion and growth. We have developed a small diameter, bilayered, biodegradable, elastomeric scaffold based on a synthetic, biodegradable elastomer. The scaffold incorporates a highly porous inner layer, allowing cell integration and growth, and an external, fibrous reinforcing layer deposited by electrospinning. Scaffold morphology and mechanical properties were assessed, quantified and compared with those of native vessels. Scaffolds were then seeded with adult stem cells using a rotational vacuum seeding device to obtain a TEVG, cultured under dynamic conditions for 7 days and evaluated for cellularity. The scaffold showed firm integration of the two polymeric layers with no delamination. Mechanical properties were physiologically consistent, showing anisotropy, an elastic modulus (1.4 + or - 0.4 MPa) and an ultimate tensile stress (8.3 + or - 1.7 MPa) comparable with native vessels. The compliance and suture retention forces were 4.6 + or - 0.5 x 10(-4) mmHg(-1) and 3.4 + or - 0.3N, respectively. Seeding resulted in a rapid, uniform, bulk integration of cells, with a seeding efficiency of 92 + or - 1%. The scaffolds maintained a high level of cellular density throughout dynamic culture. This approach, combining artery-like mechanical properties and a rapid and efficient cellularization, might contribute to the future clinical translation of TEVGs.

Transient elastic support for vein grafts using a constricting microfibrillar polymer wrap.

Arterial vein grafts (AVGs) often fail due to intimal hyperplasia, thrombosis, or accelerated atherosclerosis. Various approaches have been proposed to address AVG failure, including delivery of temporary mechanical support, many of which could be facilitated by perivascular placement of a biodegradable polymer wrap. The purpose of this work was to demonstrate that a polymer wrap can be applied to vein segments without compromising viability/function, and to demonstrate one potential application, i.e., gradually imposing the mid-wall circumferential wall stress (CWS) in wrapped veins exposed to arterial levels of pressure. Poly(ester urethane)urea, collagen, and elastin were combined in solution, and then electrospun onto freshly-excised porcine internal jugular vein segments. Tissue viability was assessed via Live/Dead staining for necrosis, and vasomotor challenge with epinephrine and sodium nitroprusside for functionality. Wrapped vein segments were also perfused for 24h within an ex vivo vascular perfusion system under arterial conditions (pressure = 120/80 mmHg; flow = 100 mL/min), and CWS was calculated every hour. Our results showed that the electrospinning process had no deleterious effects on tissue viability, and that the mid-wall CWS vs. time profile could be dictated through the composition and degradation of the electrospun wrap. This may have important clinical applications by enabling the engineering of an improved AVG.

Hybrid nanofibrous scaffolds from electrospinning of a synthetic biodegradable elastomer and urinary bladder matrix.

Synthetic materials can be electrospun into submicron or nanofibrous scaffolds to mimic extracellular matrix (ECM) scale and architecture with reproducible composition and adaptable mechanical properties. However, these materials lack the bioactivity present in natural ECM. ECM-derived scaffolds contain bioactive molecules that exert in vivo mimicking effects as applied for soft tissue engineering, yet do not possess the same flexibility in mechanical property control as some synthetics. The objective of the present study was to combine the controllable properties of a synthetic, biodegradable elastomer with the inherent bioactivity of an ECM derived scaffold. A hybrid electrospun scaffold composed of a biodegradable poly(ester-urethane)urea (PEUU) and a porcine ECM scaffold (urinary bladder matrix, UBM) was fabricated and characterized for its bioactive and physical properties both in vitro and in vivo. Increasing amounts of PEUU led to linear increases in both tensile strength and breaking strain while UBM incorporation led to increased in vitro smooth muscle cell adhesion and proliferation and in vitro mass loss. Subcutaneous implantation of the hybrid scaffolds resulted in increased scaffold degradation and a large cellular infiltrate when compared with electrospun PEUU alone. Electrospun UBM/PEUU combined the attractive bioactivity and mechanical features of its individual components to result in scaffolds with considerable potential for soft tissue engineering applications.

Generating elastic, biodegradable polyurethane/poly(lactide-co-glycolide) fibrous sheets with controlled antibiotic release via two-stream electrospinning.

Damage control laparotomy is commonly applied to prevent compartment syndrome following trauma but is associated with new risks to the tissue, including infection. To address the need for biomaterials to improve abdominal laparotomy management, we fabricated an elastic, fibrous composite sheet with two distinct submicrometer fiber populations: biodegradable poly(ester urethane) urea (PEUU) and poly(lactide-co-glycolide) (PLGA), where the PLGA was loaded with the antibiotic tetracycline hydrochloride (PLGA-tet). A two-stream electrospinning setup was developed to create a uniform blend of PEUU and PLGA-tet fibers. Composite sheets were flexible with breaking strains exceeding 200%, tensile strengths of 5-7 MPa, and high suture retention capacity. The blending of PEUU fibers markedly reduced the shrinkage ratio observed for PLGA-tet sheets in buffer from 50% to 15%, while imparting elastomeric properties to the composites. Antibacterial activity was maintained for composite sheets following incubation in buffer for 7 days at 37 degrees C. In vivo studies demonstrated prevention of abscess formation in a contaminated rat abdominal wall model with the implanted material. These results demonstrate the benefits derivable from a two-stream electrospinning approach wherein mechanical and controlled-release properties are contributed by independent fiber populations and the applicability of this composite material to abdominal wall closure.

Biodegradable elastomeric scaffolds with basic fibroblast growth factor release.

Scaffolds that better approximate the mechanical properties of cardiovascular and other soft tissues might provide a more appropriate mechanical environment for tissue development or healing in vivo. An ability to induce local angiogenesis by controlled release of an angiogenic factor, such as basic fibroblast growth factor (bFGF), from a biodegradable scaffold with mechanical properties more closely approximating soft tissue could find application in a variety of settings. Toward this end biodegradable poly(ester urethane)urea (PEUU) scaffolds loaded with bFGF were fabricated by thermally induced phase separation. Scaffold morphology, mechanical properties, release kinetics, hydrolytic degradation and bioactivity of the released bFGF were assessed. The scaffolds had inter-connected pores with porosities of 90% or greater and pore sizes ranging from 34-173 microm. Scaffolds had tensile strengths of 0.25-2.8 MPa and elongations at break of 81-443%. Incorporation of heparin into the scaffold increased the initial burst release of bFGF, while the initial bFGF loading content did not change release kinetics significantly. The released bFGF remained bioactive over 21 days as assessed by smooth muscle mitogenicity. Scaffolds loaded with bFGF showed slightly higher degradation rates than unloaded control scaffolds. Smooth muscle cells seeded into the scaffolds with bFGF showed higher cell densities than for control scaffolds after 7 days of culture. The bFGF-releasing PEUU scaffolds thus exhibited a combination of mechanical properties and bioactivity that might be attractive for use in cardiovascular and other soft tissue applications.

Fabrication of cell microintegrated blood vessel constructs through electrohydrodynamic atomization.

Biodegradable synthetic matrices that resemble the size scale, architecture and mechanical properties of the native extracellular matrix (ECM) can be fabricated through electrospinning. Tubular conduits may also be fabricated with properties appropriate for vascular tissue engineering. Achieving substantial cellular infiltration within the electrospun matrix in vitro remains time consuming and challenging. This difficulty was overcome by electrospraying smooth muscle cells (SMCs) concurrently with electrospinning of a biodegradable, elastomeric poly(ester urethane) urea (PEUU) small-diameter conduit. Constructs were cultured statically or in spinner flasks. Hematoxylin and eosin (H&E) staining demonstrated qualitatively uniform SMCs integration radially and circumferentially within the conduit after initial static culture. In comparison with static culture, samples cultured in spinner flasks indicated 2.4 times more viable cells present from MTT and significantly larger numbers of SMCs spread within the electrospun fiber networks by H&E image analysis. Conduits were strong and flexible with mechanical behaviors that mimicked those of native arteries, including static compliance of 1.6+/-0.5 x 10(-3)mmHg(-1), dynamic compliance of 8.7+/-1.8 x 10(-4)mmHg(-1), burst strengths of 1750+/-220 mmHg, and suture retention. This method to rapidly and efficiently integrate cells into a strong, compliant biodegradable tubular matrix represents a significant achievement as a tissue engineering approach for blood vessel replacement.

Development of composite porous scaffolds based on collagen and biodegradable poly(ester urethane)urea.

Our objective in this work was to develop a flexible, biodegradable scaffold for cell transplantation that would incorporate a synthetic component for strength and flexibility and type I collagen for enzymatic lability and cytocompatibility. A biodegradable poly(ester urethane)urea was synthesized from poly(caprolactone), 1,4-diisocyanatobutane, and putrescine. Using a thermally induced phase separation process, porous scaffolds were created from a mixture containing this polyurethane and 0%, 10%, 20%, or 30% type I collagen. The resulting scaffolds were found to have open, interconnected pores (from 7 to >100 um) and porosities from 58% to 86% depending on the polyurethane/collagen ratio. The scaffolds were also flexible with breaking strains of 82-443% and tensile strengths of 0.97-4.11 MPa depending on preparation conditions. Scaffold degradation was significantly increased when collagenase was introduced into an incubating buffer in a manner that was dependent on the mass fraction of collagen present in the scaffold. Mass losses could be varied from 15% to 59% over 8 weeks. When culturing umbilical artery smooth muscle cells on these scaffolds higher cell numbers were observed over a 4-week culture period in scaffolds containing collagen. In summary, a strong and flexible scaffold system has been developed that can degrade by both hydrolysis and collagenase degradation pathways, as well as support cell growth. This scaffold possesses properties that would make it attractive for future use in soft tissue applications where such mechanical and biological features would be advantageous.

A seeding device for tissue engineered tubular structures.

One of the challenges in the tissue engineering of tubular tissues and organs is the efficient seeding of porous scaffolds with the desired cell type and density in a short period of time, without affecting cell viability. Though different seeding techniques have been investigated, a fast, reproducible, and efficient bulk seeding method with uniform cellular distribution has yet to be reported. In this paper, a novel seeding device utilizing the synergistic effects of vacuum, centrifugal force and flow has been developed and analyzed. The device allows porous tubular scaffolds to be uniformly bulk seeded as well as luminally surface-seeded with cells. Porous tubular polymer scaffolds were bulk and surface-seeded with cell suspensions, and cell viability and seeding efficiency were subsequently assessed. A rigorous quantitative analysis of shear stresses acting on the cells during the seeding process, and of cell location within the scaffolds following seeding was also performed. Our results showed that the scaffolds were uniformly seeded along the longitudinal and circumferential directions within the tube wall without affecting cell viability or exposing them to excessive shear stresses.

Microintegrating smooth muscle cells into a biodegradable, elastomeric fiber matrix.

Electrospinning permits fabrication of biodegradable elastomers into matrices that can resemble the scale and mechanical behavior of the native extracellular matrix. However, achieving high-cellular density and infiltration with this technique remains challenging and time consuming. We have overcome this limitation by electrospraying vascular smooth muscle cells (SMCs) concurrently with electrospinning a biodegradable, elastomeric poly(ester urethane)urea (PEUU). Trypan blue staining revealed no significant decrease in cell viability from the fabrication process and electrosprayed SMCs spread and proliferated similar to control unprocessed SMCs. The resulting SMC microintegrated PEUU constructs were cultured under static conditions or transmural perfusion. Higher cell numbers resulted with perfusion culture with 131% and 98% more viable cells versus static culture at days 4 and 7 (p<0.05). Fluorescent imaging and hematoxylin and eosin staining further illustrated high cell densities integrated between the elastomeric fibers after perfusion culture. SMC microintegrated PEUU was strong, flexible and anisotropic with tensile strengths ranging from 2.0 to 6.5 MPa and breaking strains from 850 to 1,700% dependent on the material axis. The ability to microintegrate smooth muscle or other cell types into a biodegradable elastomer fiber matrix embodies a novel tissue engineering approach that could be applied to fabricate high cell density elastic tissue mimetics, blood vessels or other cardiovascular tissues.

Fabrication of biodegradable elastomeric scaffolds with sub-micron morphologies.

The native extracellular matrix (ECM) of elastic tissues is strong and flexible and supports cell adhesion and enzymatic matrix remodeling. In an attempt to convey these ECM properties to a synthetic scaffold appropriate for soft tissue engineering applications, a biodegradable, elastomeric poly(ester urethane)urea (PEUU) was combined with type I collagen at various ratios (2.5, 5, 10, 20, 50, 60, 70, 80, and 90 wt% collagen) and electrospun to construct elastic matrices. Randomly orientated fibers in the electrospun matrices ranged in diameter from 100-900 nm, dependent on initial polymer concentration. Picrosirius red staining of matrices and CD spectroscopy of released collagen confirmed collagen incorporation and preservation of collagen structure at the higher collagen mass fractions. Matrices were strong and distensible possessing strengths of 2-13 MPa with breaking strains of 160-280% even with low PEUU content. Collagen incorporation significantly enhanced smooth muscle cell adhesion onto electrospun scaffolds. An approach has been demonstrated that mimics elastic extracellular matrices by using a synthetic component to provide mechanical function together with a biomacromolecule, collagen. Such matrices may find application in engineering soft tissue.