Brief Article pubs.acs.org/jmc
Synthesis of Thrombolytic Sol−Gel Coatings: Toward DrugEntrapped Vascular Grafts Yulia Chapurina,† Vasiliy V. Vinogradov,† Alexander V. Vinogradov,† Vladimir E. Sobolev,† Ivan P. Dudanov,‡,§ and Vladimir V. Vinogradov*,† †
Laboratory of Solution Chemistry of Advanced Materials and Technologies, ITMO University, Kronverkskiy Prospekt 49, St. Petersburg, 197101, Russian Federation ‡ Petrozavodsk State University, Petrozavodsk 185910, Russian Federation § Regional Vascular Center, St. Petersburg, Russian Federation S Supporting Information *
ABSTRACT: As is evident from numerous investigations, drugeluting vascular grafts and stents have not solved the main problems associated with thrombosis and due to drug release only postpone their advance for a longer period. Here we point to a potential solution of this problem by developing thrombolytic sol−gel coatings which potentially could lead to drug-entrapped vascular grafts: urokinase-type plasminogen activator was entrapped within a porous alumina sol−gel film with a subsequent deposition on a polymer graft.
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INTRODUCTION Cardiovascular diseases are one of the main reasons for mortality in the developed world. Many methods for their treatment have been developed during last few decades, and most of them use surgical intervention including balloon angioplasty and coronary artery stent implantation. 1−3 However, the placement of vascular grafts and stents is associated with a number of problems such as restenosis and thrombosis, which occur in more than 30% of treated coronary arteries and often result in repeated surgical intervention.4 To date, a variety of techniques and different types of vascular grafts have been developed. Among them the most major advance became the appearance of drug-eluting grafts including stents. Coating for these grafts is designed in such a way that it can gradually release the drug into the vessel lumen, thereby providing a directional effect of the drug in the local area. As an example, the first drug-eluting stent was approved by FDA in 2003.5 Despite the emergence of new types of vascular grafts with drug-eluting coatings, the thrombosis and restenosis problems still persist.6 Slow drug release appeared to solve these problems only temporarily by postponing the effect for a certain period.7 A simple question is addressed in this work: why should drug elute from the vascular graft if it could be successfully entrapped with preserving activity? In our previous publications, we developed an alternative approach which deals with entrapment of a thrombolytic drug8 within a bioinert alumina matrix. As it was shown, tissue plasminogen activator not only preserves its activity in the © 2015 American Chemical Society
conversion of plasminogen to plasmin but also gets substantially stabilized, with no signs of losing its activity even after 30 days in Ringer’s solution. These results were made possible due to the concept developed in our works on entrapment within sol−gel alumina which, owing to its crystalline nature, preserves the protein structure to a far greater extent and does not get destroyed over time.10−13 Moreover, in contrast with many other sol−gel materials, alumina (namely boehmite) is biocompatible and has FDA approval as the most common adjuvant in vaccinology.9 We show here that urokinase-type plasminogen activator (uPA) can be successfully entrapped within alumina sol−gel films with preserved thrombolytic activity even when applied on the polymer vascular graft. Despite the fact that the sol−gel films were used for graft coating,14,15 there are some other issues which we address in this paper: (1) the first example of thrombolytic sol−gel coatings itself and on the vascular graft, (2) for this purpose, nanocrystalline boehmite was used, (3) the method of sol−gel entrapment as a means to produce a thrombolytic coating, (4) the obtained thrombolytic coatings are the only ones in the world that are potentially capable of operating for an indefinite time, and (5) a new mechanism for fixing a sol−gel coating using the Teflon fibers of polymer graft as reinforcing agents. Received: April 29, 2015 Published: July 22, 2015 6313
DOI: 10.1021/acs.jmedchem.5b00654 J. Med. Chem. 2015, 58, 6313−6317
Journal of Medicinal Chemistry
Brief Article
For the sake of brevity, representative and relevant data are presented here and additional characteristics of thrombolytic sol−gel films are collected in the Supporting Information. First of all, it should be noted that a sol−gel coating is composed of a densely packed crystalline boehmite nanorods with a size of 2 nm × 5 nm (for TEM images and XRD, see Figures 1S,2S). Boehmite nanorods formed during the sol−gel synthesis firmly hold uPA molecules after a condensation, yielding a porous framework with an average pore size of less than 3 nm (for the results of nitrogen physisorption, see Figure 3S). Despite the fact that the pore size for the alumina matrix is about 2−3 nm as defined by nitrogen physisorption, there are also bigger mesopores with diameter range from 14 to 40 nm as can be clearly seen from the HR SEM analysis (for the HR SEM results, see Figure 4S). The same bimodal porous structure we also observed previously8 on tissue plasminogen activator entrapped within alumina. Appearance of the coating applied on a polymer vascular graft is shown in Figure 1a. The thrombolysis process using
Figure 2. Thrombolysis process on a thrombolytic sol−gel film (uPA content is 5%). Structural changes in the clot immediately after applying on the substrate (a), after 55 (b), 130 (c), 160 (d), 205 (e), and after 420 min (f).
Table 1. Data of CloLA
pure alumina film uPA@alumina (2.5%) uPA@alumina (5%) uPA@alumina (7.5%) uPA@alumina (10%)
Figure 1. SEM image of the treated vascular graft with a thrombolytic sol−gel coating (a). Visualization of the thrombolysis process using a drug-entrapped vascular graft (b,c).
full lysis time, min (±15)
time for clot leakage from the implant, min (±10)
440 420 400 340
80 30 20 10
rather long, but the main goal of modern vascular grafts and stents is not destroying the already formed clot but preventing its formation in the parietal region. Indeed, the lysis time of a blood clot with entrapped enzymes is strongly reduced, but nevertheless they might cope with the objective of preventing its formation and growth. Moreover, we also believe that lysis time can be reduced by using more active enzymes but it is always compromise between stability, activity, and specific matrix−enzyme interactions. The next round of experiments on investigating the thrombolytic activity was performed on a polymer vascular graft treated with thrombolytic sol−gel coatings described above (see Experimental Section for details of treatment). For this purpose, we have used an 8 mm polymer graft based on polytetrafluoroethylene. In the modified uPA@alumina grafts, a clot was artificially formed and the time of its leakage was measured as described (see Experimental Section for details and Table 1). According to the obtained data, the time required to complete the leakage of the clot for polytetrafluoroethylene amounts from 10 to 80 min depending on the concentration of uPA. Thrombolysis was completely absent for the untreated grafts. Clots leaking from treated grafts had sufficiently dense cores and completely lysed edges that supports the concept of the parietal lysis. It is important to note that graft thrombosis is related with surface adsorption of plasma proteins and can be controlled by surface parameters.16 Despite the fact that we do not discuss here such an adsorption in detail, potentially it can occur especially after long-term exposition of the developed grafts in the real bloodstream. We believe that the treatment of the surface by polyethylene glycol or other masking and inert
drug-entrapped vascular grafts is schematically shown in Figure 1b,c. An alumina sol containing the enzyme molecules penetrates a few micrometers inside the graft. After drying and condensation, the sol is converted into a gel and the stent fibers (diameter range of 10−70 nm and more than few mm in length) serve as a kind of reinforcing agent, allowing firmly fixing of a thrombolytic film on its surface. Three questions were addressed here: can the uPA entrapped in a sol−gel alumina film dissolve a clot? What is the stability of this coating on a polymer vascular graft? How long does the thrombolytic effect last? To answer the first question, we have prepared transparent films on a glass (in order to monitor in a transmission mode) with following deposition of a clot (for details, see Experimental Section). As is evident from Figure 2, the thrombolysis mechanism starts immediately after the components come into contact. It is worth noting that thrombolysis was provided by totally entrapped uPA, which means that the process of forming plasmin takes place either via plasminogen migration along the matrix porous channels or directly at the interface. In our experiments, we have shown that the release of the enzyme does not occur if uPA content is less than 10%. When uPA content exceeds 10%, a partial release of uPA is observed, which was not studied in the experiments because it does not conform to the general concept of full entrapment. Details of clot lysis assay (CloLA) are studied depending on uPA content and shown in Table 1. According to the obtained data, the time required to complete thrombolysis amounts from 340 to 440 min depending on the concentration of uPA. It may seem 6314
DOI: 10.1021/acs.jmedchem.5b00654 J. Med. Chem. 2015, 58, 6313−6317
Journal of Medicinal Chemistry
Brief Article
coating has bimodal pore size distribution (Figures 3S,4S) due to which plasminogen easily penetrates through the channels of the matrix, on the one hand, and, on the other, interacts with uPA entrapped within pores with the smallest diameter. Thus, the parietal region constantly provides the concentration of plasmin which is necessary for lysis. Because the thrombolytic enzyme itself is completely entrapped within an inorganic matrix and does not lose its activity even after a prolonged exposure to concentrated Ringer’s solution, we can theoretically assume that this composition should become a significant competitor for modern drug-eluting vascular grafts and stents losing their activity with drug release, which essentially only postpones the problem, and does not solve it.
materials can subsequently minimize this effect and improve stent viability. Further investigations of treated uPA(5%)@alumina graft were carried out in Ringer’s solution with a salt concentration 10 times higher than it is usually used. First of all, it should be noted that alumina is one of the most stable oxides and practically insoluble even in a such strong ionic media as the human circulatory system.17,18 After a month of testing in Ringer’s concentrated solution, the surface of the implant remained practically unchanged (see. Figure 3).
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EXPERIMENTAL SECTION
Chemicals. Lyophilized human plasma and human thrombin (150 NIH units/mg) were obtained from “Kvik” LTD Company, Russia. Aluminum isopropoxide was obtained from Sigma-Aldrich. Purolase (recombinant prourokinase) 6 kU/mg with molecular weight 49.3 kDa was obtained from Russian Cardiology Research and Production Complex; vascular graft Tyvek Roll (8 mm diameter). Sol−Gel Synthesis of Alumina. Alumina sol was prepared as described in ref 10. In detail, 2.28 g of Al(C3H7O)3 was added to 50 mL of deionized water at 80 °C and a white precipitate was formed immediately. Before US treatment, the precipitate was kept at 80 °C under vigorous stirring for 15 min to complete the production of boehmite nanoparticles and to complete the evaporation of the 2propanol formed during hydrolysis. The final suspension was ultrasonically treated for 2 h. In 2 h, a viscous sol was formed. The resulting sol was cooled to room temperature. Mass concentration of the resulting alumina in the sol was 1.9%. Entrapment Procedure of the uPA within Sol−Gel Alumina Film. First, 11 mg of uPA was diluted in 1 mL of 0.05 M tris-HCl buffer solution (pH 7.4) to a concentration 66 kU/mL. For the entrapment of uPA, a mixture of 100 μL of freshly prepared alumina sol was then transferred to a 1.5 mL tube and then various volumes of as-made uPA solution were added (2.5%, 5%, 7.5%, and 10% uPA@ alumina composites were prepared according mass ratio of the components). Thrombolytic sol−gel films were formed by using Meyer rod (10 μm wire). For this aim, 50 μL of the final Biosol was deposited in a middle of a cover glass (2 cm × 2 cm) by a pipet with following Meyer rod coating. Meyer rod was rolled at least three times for film homogenization. Before analysis, the obtained thrombolytic sol−gel films were dried during 2 h in a vacuum desiccator. Drying during 2 h was enough for uPA to get entrapped within sol−gel alumina film and to leave activity at a high level. Final thickness of thrombolytic sol−gel coatings after Meyer deposition was amounted 150−200 nm. Deposition of Thrombolytic Sol−gel Coatings on Polymer Graft and Study of Thrombolytic Activity. The polymer vascular grafts were cut into 2 cm pieces. Internal surface of graft pieces was dip-coating treated (0.2 mm/s) with a mixture of 200 μL of the freshly prepared alumina sol and 66kU/mL uPA solution to produce 2.5%, 5%, 7.5%, and 10% of uPA@alumina films, respectively. Prior to the analysis, the treated stent pieces were dried in a vacuum desiccator for 3 h. The clots were formed from human plasma with known concentration of plasminogen and fibrinogen in each of the treated and untreated stents. For this aim, 10 mg of human plasma were solved in 1 mL of triple distilled water (plasminogen concentration, 102 μg/mL; fibrinogen concentration, 2.8 mg/mL) and then 0.05 mL of the plasma solution was mixed with 1 mL of 0.05 M tris-HCl buffer (pH 7.4). Thrombin solution was prepared by solving 1 mg of thrombin in 1.5 mL of 0.05 M tris-HCl buffer (pH 7.4). The clot was formed by mixing 0.2 mL of the final plasma solution in tris-HCl buffer, 0.2 mL of thrombin solution, and 0.1 mL of eosin−water solution (1 mg/mL) for staining. The clot mixture was immediately transferred in the middle part of the stent. After 5 min, a dense viscous
Figure 3. Surface and respective image mapping (Al and F atoms) of the polymer stent with thrombolytic sol−gel coating before (a,b,c) and after (d,e,f) a 30-day exposure of the stent to 10-fold Ringer’s solution.
As seen in Figure 3, exposure of the polymer stent with thrombolytic sol−gel coating during 30 days in 10-fold Ringer’s solution practically did not change the surface composition: it is mostly represented by the aluminum (due to the presence of alumina in the film) and fluorine atoms (due to the composition of the polymer stent), thus providing excellent stability. However, there are two potential ways to destroy thrombolytic sol−gel coating: dissolution of the matrix or destruction with the corresponding break-off. According to atomic absorption spectroscopy, the presence of the additional Al3+ ions in Ringer’s solution after the experiment was not observed. In contrast, as shown in Figure 5S, particles of two sizes (2 and 250 nm) were detected in the solution. The former is possibly due to individual alumina particles (having a similar size; Figure 1S), while the latter corresponds to aggregates. Formation of aggregates may be associated with a partial breakoff of fragments from the graft surface, however, their size is extremely small to block the vasculature19−21 and cause adverse reactions.13
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CONCLUSION In this study, we report designing a new class of grafts: drugentrapped vascular grafts which can potentially be applied to any type of surface. It is shown that introducing uPA into a porous sol−gel alumina matrix preserves the enzyme activity. Even after 30 days in concentrated Ringer’s solution, the activity of the coating remained unchanged and the matrix was not destroyed. Capability to dissolve a blood clot in the parietal region was clearly demonstrated by a polytetrafluoroethylene vascular graft. What are the unique features of the designed coatings and what is the mechanism of their action? We believe that the mechanism is similar to what we observed in our previous article.8 A thrombolytic sol−gel 6315
DOI: 10.1021/acs.jmedchem.5b00654 J. Med. Chem. 2015, 58, 6313−6317
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clot was formed inside each of the stents. Thrombolytic activity of coatings was measured as a time of clot leakage by dropping down and coloring the water. Figure 6S demonstrates the typical leakage of a clot from the polymer graft after respective lysis by thrombolytic sol−gel coating. Thrombolytic Activity of uPA@alumina Films. The activity of uPA@alumina films was studied under the optical microscope according to the scheme represented in Scheme 1. The clots were
Brief Article
AUTHOR INFORMATION
Corresponding Author
*Phone: +79218906773. E-mail: vinogradoff
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Russian Government, Ministry of Education. We are grateful to Prof. David Avnir (Hebrew University of Jerusalem) for useful advice and fruitful discussions on this project. We thank the Center for Nanoscience and Nanotechnology in Hebrew University for assistance in performing TEM and SEM experiments.
Scheme 1. Scheme for Thrombolytic Analysis of uTA@ alumina Films Carried out under the Optical Microscope
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ABBREVIATIONS USED uPA, urokinase-type plasminogen activator; CloLA, clot lysis assay; uPA@alumina, urokinase-type plasminogen activator entrapped within sol−gel alumina; NIH, normalized index of hemolysis; US, ultrasound
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formed as described above with following deposition on the thrombolytic sol−gel coatings formed on a cover glass. Transmission mode was used to see any morphological changes in clots during analysis. The thrombolysis was monitored as a function of time. The thickness of clots was controlled with a 10 μm copper foil clamped between glasses. The pictures were taken every 5 min with following analysis. Stability of uPA@alumina Coating in the Model Blood System. To test the stability of synthesized composite in a model blood system, 10-fold concentrated Ringer’s solution was used. Ringer’s solution has a similar ratio of salt concentrations to that typically found in the blood. For this aim, a 2 cm piece of treated polymer stent coated with 5% uPA@alumina was perfused with 100 mL of Ringer’s solution and kept under stirring for 30 days at a constant flow rate 0.03 m·s−1. The surface of coating after the test was compared with the initial by SEM analysis. Dynamic light scattering analysis was carried out to investigate the presence of particles in the solution after the test. Characterization Techniques. Specific surface areas, pore volumes, and pore size distributions were determined using the nitrogen adsorption−desorption method at 77 K (Quantachrome Nova 1200 series e). Surface areas were calculated using the BET equation. Pore volumes and pore size distributions were calculated using the BJH method. Prior to analysis, the sample was degassed for 24 h at room temperature. The crystal phase and crystallinity of the samples were studied by X-ray diffraction method (Bruker D8 Advance) using Cu Kα irradiation (λ = 1.54 Å), with samples being scanned along 2θ in the range of 4−75° at a speed of 0.5° per min. The samples for transmission electron microscopy (TEM) were obtained by dispersing a small probe in ethanol to form a homogeneous suspension. Then a suspension drop was coated on a copper mesh covered with carbon for TEM analysis (FEI Tecnai G2 F20, at an operating voltage of 200 kV). For scanning electron microscopy (SEM, ultrahigh resolution Magellan 400L electron microscope), the final suspension of the entrapped enzyme was coated on silicon wafer and fully dried under vacuum. Dynamic light scattering measurements were carried out using Compact Z Photocor instrument.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b00654. Additional characterization of thrombolytic sol−gel materials (PDF) 6316
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