VASCULAR STENT WITH ANTITHROMBOTIC PROPERTIES

Description

Technical field

[0001] The present invention belongs to the field of medical devices and more particularly to that of vascular endoprostheses.

[0002] The invention relates to a vascular stent, deployed or non-deployed, the surface of which is covered by a film comprising at least one protein, to a method for covering the surface of a vascular stent with a film comprising at least one protein and to a device for implementing the method according to the invention. State of the art

[0003] A stent (also called a "stent") is a device, most often metallic, meshed and tubular, that can be placed in a human or animal cavity to dilate a stenosis or occlusion and keep the cavity open. Stents are mainly used in arteries, more rarely in veins.

[0004] Endoprostheses are mainly used in vascular pathologies (cardiology and endovascular neurosurgery) but can also be used in the urethra, trachea, esophagus or bile ducts.

[0005] Since the stent is a material foreign to the human or animal body into which it is inserted, it is a device conducive to the formation of a clot or thrombus. When a stent is placed, for example in an artery, this step must be followed by drug treatment intended to prevent the appearance of clots. This treatment is essential, for at least several weeks after the stent is placed, until it is naturally covered by the cells of the inner wall of the artery. This process is called "endothelialization".

[0006] Historically, drug treatment consisted of low-dose aspirin combined with ticlopidine (CAS No. 55142-85-3). This treatment has evolved and the drug combination now includes aspirin and another antiplatelet agent such as clopidogrel (CAS No. 113665-84-2), prasugrel (CAS No. 150322-43-3) or ticagrelor (CAS No. 274693-27-5).

[0007] There are two main types of vascular endoprostheses or "stents": so-called "bare" stents and so-called "active" or drug-active stents. Active stents are also called drug-eluting arterial stents.

[0008] Active stents are generally distinguished from bare metal stents by the integration of an antiproliferative substance into a biodegradable polymer coated on the metal mesh, which reduces the risk of delayed restenosis, due to the thickening of the vascular wall, by blocking the proliferation of smooth muscle cells in the vessel wall. This phenomenon is particularly critical in coronary pathology because the vessel treated by the stent is heavily remodeled by an atherosclerotic lesion and highly inflammatory. Unfortunately, the substances diffused by the active stent, which are antimitotic, delay the endothelialization process, which relies on the multiplication of circulating endothelial progenitors responsible for covering the stent. This leaves the active stent and the vessel wall in contact with it partially uncovered, which induces a significant risk of thrombosis in the treated area.Endothelialization of the stent mesh is thus incomplete and delayed, and clot formation remains a potential hazard, justifying powerful antiplatelet therapy for a prolonged period of 3 to 12 months depending on the case in coronary pathology. Active stents are therefore not an ideal solution since they prolong the period at risk of thrombosis and require treatment with a high risk of systemic bleeding for a long period.

[0009] Furthermore, active stents cannot be used in neurosurgery in the case of an ischemic stroke or a ruptured aneurysm, because they would delay the healing of the vessel that has just been treated.

[0010] There is therefore a real need for vascular stents that overcome these defects, drawbacks and obstacles of the prior art, by preventing the activation of hemostasis, in particular of platelets in contact with the stent without affecting the adhesion of circulating endothelial progenitors which, by differentiating and multiplying, will lead to the restoration of the luminal endothelial cover in contact with the blood.

[0011] WO 95 / 29647 A2 describes the preparation of a stent coated with a collagen or albumin film by electrodeposition. Statement of the invention

[0012] It is to the applicants' credit that they have developed a new type of vascular endoprosthesis, overcoming both the defects and disadvantages previously cited of bare or active vascular endoprostheses (bare stents or active stents).

[0013] The vascular stents according to the invention thus have several advantages, compared to vascular stents known in the state of the art and in particular have antithrombotic properties, without having the disadvantage of limiting the endothelialization process, even partially. Once endothelialized, the wall and the stent become less thrombogenic and the antiplatelet treatment can be reduced, with fewer side effects.

[0014] The vascular endoprostheses according to the invention thus combine the advantages of bare stents and active stents, without having their disadvantages.

[0015] The vascular stents according to the invention may further possess antibacterial and / or anti-inflammatory properties.

[0016] The invention relates to a vascular stent, deployed or non-deployed, the surface of which is covered by a film comprising at least one protein of interest having been subjected to an electric field having a periodic structure.

[0017] In the context of the invention, the term "proteins" or "proteins of interest" means proteins chosen from the group comprising one or more blood plasma proteins (such as, for example, albumin, immunoglobulins (antibodies, essentially IgG), fibrinogen, alpha-1-antitrypsin, alpha-2 macroglobulin, transferrin, lipoproteins (essentially HDL and LDL)) and / or one or more synthetic biological macromolecules making it possible, after the application of a pulsed electric field, to obtain a film of at least one protein strongly adhered to the surface of the stent and having antithrombotic properties without preventing endothelialization. Preferably, the at least one protein of interest may be albumin or a mixture of blood plasma proteins, comprising albumin.

[0018] Advantageously, the at least one protein of interest is soluble at a pH between 3 and 12.

[0019] The film comprising the at least one protein of interest according to the invention is a film of protein(s) (for example plasma) which can be deposited by applying a pulsed electric field to a solution comprising at least one protein of interest modified (for example destructured) or not by applying the electric field. Unlike a conventional deposition method, without applying an electric field, such as for example described in WO2017 / 004598, the at least one protein of interest having been subjected to the electric field covers the endoprosthesis in a durable manner and with demonstrated antithrombotic properties (see examples below).

[0020] Advantageously, the film comprising at least one protein of interest can be distributed homogeneously on the surface of the vascular stent. The film comprising at least one protein of interest has a thickness which can vary depending on the nature of the protein and the duration of exposure to the electric field.

[0021] The thickness of the film comprising the at least one protein of interest may be greater than or equal to 30 Angstroms, and is preferably homogeneous on the surface of the stent. The protein concentration on the surface of the vascular stent may be greater than or equal to 2 µg.cm -2< .

[0022] Advantageously, the vascular endoprosthesis (or stent) according to the invention may be any commercially available bare vascular endoprosthesis (or bare stent) which has then been treated to form the film of at least one protein of interest. It is a medical device, most often metallic, meshed and tubular, intended to be inserted into a natural human or animal cavity to keep it open. The vascular endoprosthesis may be made of various materials such as, for example, metal alloys, silicone and polymers. Preferably, the vascular endoprosthesis according to the invention is metallic and comprises one or more metal alloy(s). For example, the metal alloy(s) may be chosen from the group comprising alloys of the stainless steel, nickel / titanium (such as, for example, nitinol), tantalum, cobalt / chromium, platinum / chromium type, alloys optionally comprising magnesium, and mixtures thereof.

[0023] Advantageously, the vascular stent according to the invention can be deployed or non-deployed. Generally, the stent is non-deployed when it is kept in the original micro-catheter (in which it is marketed), before implantation. The stent is said to be deployed when part or all of it is no longer in the micro-catheter used to place it and the mesh constituting it is partially or totally deployed outside the original micro-catheter. When a vascular stent is deployed entirely in the open air (without constraints), it can have a diameter ranging from 2 mm to 60 mm depending on the type of stent, for example from 2 mm to 10 mm for a carotid stent, from 2 mm to 5 mm for an intracranial stent, from 18 mm to 46 mm for an aortic vascular stent.A stent graft can have a length ranging from 10 mm to 200 mm, for example from 10 mm to 70 mm for a carotid stent, from 10 mm to 35 mm for an intracranial stent or up to 170 mm in the case of an aortic stent graft.

[0024] Advantageously, the vascular endoprosthesis according to the invention can be: a cardiac stent (often more rigid than an intracranial stent), bare or comprising for example a biodegradable carrier such as salicylic acid, polylactic acid (PLLA) or magnesium. The stent according to the invention may further comprise a coating of polymer containing a drug and / or different materials (such as antibodies, carbon, etc.), a self-expanding cardiac stent or on an expandable balloon (pre-mounted on a balloon), a bare intracranial stent with loose or dense mesh (Flow Diverter), a self-expanding intracranial, carotid or peripheral stent.

[0025] The vascular endoprosthesis can, for example, be a stent intended to be placed in the venous system (nitinol alloy; diameter 10 - 20 mm; length 40 - 160 mm) or an implant such as a vena cava filter (nitinol alloy, stainless steel or cobalt-chromium; diameter 25 - 40 mm; length up to 50 mm).

[0026] The antithrombotic properties of the vascular stents according to the invention can be observed for an extended period of time, ranging from one week to several months. For example, the antithrombotic properties can be observed for a period of time greater than or equal to 3 months, greater than or equal to 6 months, or even greater than or equal to 9 or 12 months.

[0027] The invention also relates to a method for covering the surface of a vascular stent, deployed or non-deployed, with a film of at least one protein of interest, comprising the steps of: bringing the vascular stent into contact with an aqueous solution comprising at least one protein of interest, applying an electric field, having a periodic structure, generated by an electrode system preferably comprising at least one first electrode, a dielectric and at least one second electrode, said dielectric electrically insulating the stent and the at least one second electrode from the at least one first electrode, and covering the surface of the stent with the film of at least one protein of interest.

[0028] The at least one first electrode is insulated from the stent by a dielectric. The dielectric insulates the at least one first electrode from the vascular stent and from the at least one second electrode. There is no electric current flowing between the electrodes due to the presence of the dielectric (insulator). This results in the generation of a pulsed electric field which will cause the process of formation of the film of at least one protein of interest.

[0029] Advantageously, when implementing the method according to the invention, the vascular stent can be deployed (or partially deployed, a part of the stent remaining embedded in the original sheath or the microcatheter used to place it). In deployed form, the surface is more accessible and facilitates the placement of the first electrode.

[0030] Advantageously, the aqueous solution comprises one or more proteins of interest as defined above. The concentration of protein(s) in the aqueous solution may be greater than or equal to 0.1 mg / ml. For example, when the protein is albumin, the concentration may be between 5 and 50 mg / ml when the aqueous solution is a buffered solution comprising phosphate buffered saline (PBS) or between 30 and 50 mg / ml when the aqueous solution is blood plasma. The method according to the invention may be implemented in a solution comprising one or more types of proteins. The aqueous solution comprising at least one protein may be, for example, blood plasma, a buffered solution comprising a buffer such as, for example, PBS (phosphate buffered saline) or distilled salt water (NaCl). The aqueous solution may thus be any aqueous solution that does not degrade the proteins of interest dissolved therein.

[0031] Advantageously, the aqueous solution comprising at least one protein of interest can be animal or human blood plasma extracted directly from the patient (autologous transplant). This solution offers the advantage of treating the vascular endoprosthesis just before its implantation with a plasma that greatly limits the risk of patient rejection.

[0032] Advantageously, the pH of the aqueous solution comprising at least one protein of interest has a value such that the at least one protein is soluble during the implementation of the method. The aqueous solution is preferably homogeneous. The pH of the aqueous solution can thus be between 3 and 12, preferably between 6 and 9 and even more preferably between 7 and 8. The pH of the aqueous solution is for example 7.4.

[0033] The "isoelectric point" (IP) of a protein is the pH at which the overall electrical charge of the protein is neutral. At its IP, the protein has an equal number of positive and negative charges and there is no protein transport under an electric field. If the pH is higher than the IP, then the overall charge of the protein is negative; otherwise, it is positive. For example, the pH of human blood is regulated around 7.4, and albumin having a IP of 4.8, the total charge of albumin in human blood is negative (7.4 > 4.8). Preferably, in the method according to the invention, the pH of the aqueous solution may be higher or lower than the IP of the protein(s) forming the film of at least one protein of interest.

[0034] When implementing the method according to the invention, the aqueous solution comprising at least one protein of interest may be mobile or static, preferably static, relative to the vascular stent. Preferably, the stent is immersed in the solution comprising at least one protein of interest, so that its entire surface can be covered by the protein film when implementing the method.

[0035] Advantageously, the electric field is generated by a voltage signal applied to the electrode system. The electric field can thus be generated by energizing the at least one first electrode, insulated by the dielectric. The amplitude of the voltage signal can be in a range from 1 V to 50 kV. Preferably, the amplitude is in a range from 5 kV to 40 kV, and even more preferably from 10 kV to 30 kV. The applied voltage can be positive and / or negative, preferably positive.

[0036] The voltage signal (and therefore the resulting electric field) has a periodic structure. It can be characterized by its period Tp (duration of the repeating pattern) and within a period by the duration Tv where the voltage is non-zero. The ratio Tv / Tp defines the duty cycle of the periodic signal. The pattern that repeats over each period can be piecewise continuous (e.g., a pulse), sinusoidal, triangular, or sawtooth. For example, the voltage signal is a pulse with a duration Tv of 5.10 -7< s that repeats every 0.01 s (this is a piecewise continuous periodic signal with a frequency of 100 Hz and a duty cycle of 5.10 -5< ). The frequency of the signal can thus have a value in an interval from 0.1 Hz to 100 kHz, preferably from 1 Hz to 1 kHz, and the duty cycle a value in an interval from 5.10 -8< to 1 (1 corresponds to a rectified or unrectified alternating signal), preferably from 5.10 -6< to 5.10 -3<.

[0037] Advantageously, the electric field may be generated for a duration greater than or equal to 10 seconds, preferably greater than or equal to 5 minutes and even more preferably greater than or equal to 10 or 20 minutes. The duration may be increased or decreased depending on the type of protein, the protein concentration in the aqueous solution and / or the type of vascular stent (e.g. the material).

[0038] Advantageously, the voltage is applied to the at least one first electrode, the at least one second electrode being connected to ground. The voltage may also be applied to the at least one second electrode, the at least one first electrode being connected to ground. The stent may also serve as an electrode if it is metallic and separated from the second electrode by a dielectric.

[0039] Advantageously, the method according to the invention can be carried out under sterile or non-sterile conditions. The method can further comprise a sterilization step. According to the invention, any sterilization method suitable for sterilizing vascular stents can be implemented. The sterilization step does not alter the antithrombotic properties of the vascular stent according to the invention.

[0040] Advantageously, when implementing the method according to the invention, the endoprosthesis is positioned in a receiving element, preferably of tubular shape. The method according to the invention may further comprise a step of heparinization of said receiving element.

[0041] The invention further relates to a vascular endoprosthesis capable of being obtained by the method according to the invention.

[0042] The invention further relates to a device for implementing the method according to the invention comprising: at least one first electrode, at least one second electrode, at least one dielectric insulating the first electrode from the second electrode, a receiving element capable of containing the vascular stent, said receiving element having a shape adapted to the shape of the vascular stent, said receiving element being identical to or different from the dielectric, preferably in the form of a dielectric tube (for example made of PVC) and, a source of electrical energy connected to the electrodes and a voltage regulator making it possible to regulate the voltage on the at least one first electrode and the at least one second electrode in order to generate an electric field having a periodic structure.

[0043] Advantageously, the device for implementing the method of the invention comprises a tubular-shaped receiving element intended to receive the vascular stent, preferably cylindrical, comprising an inlet for the aqueous solution comprising at least one protein and an outlet, said device being provided with an electrode system comprising at least one first electrode, a dielectric and at least one second electrode as well as a source of electrical energy connected to the electrodes and a voltage regulator making it possible to regulate the voltage on the at least one first electrode and the at least one second electrode in order to generate the electric field, said at least one first electrode being insulated from the interior of the receiving element by said dielectric.

[0044] Advantageously, the at least one first electrode is covered by the dielectric. In the context of the invention, a dielectric is understood to mean a substance or material that does not have free electrons capable of carrying an electric current, but which can be polarized by an electric field. The dielectric is positioned so as to insulate the at least one first electrode from the vascular stent and from the second electrode, thus making it possible, when the voltage is applied in the first electrode, to generate an electric field (the second electrode being connected to ground). The dielectric can be made of any insulating material. For example, the dielectric can be PVC, parylene, glass, Teflon, ceramic, epoxy resin, Kapton or polymethacrylate. The dielectric can in certain cases be the original microcatheter in which the vascular stent is stored before its implantation.The dielectric can have a thickness ranging from 10 µm to 10 mm. When the stent is positioned in the device, the thickness of the dielectric is defined as the distance between the metal part of the electrode and the stent, the different elements being in contact.

[0045] Advantageously, the voltage for generating an electric field can also be applied to the second electrode, the first electrode being connected, in this configuration, to ground. The stent, if metallic, can also serve as an electrode.

[0046] Advantageously, the receiving element can be any support allowing the vascular endoprosthesis to be held during the implementation of the method and in particular, the original micro-catheter. It is tubular in shape and can be, for example, a PVC tube (or any of the dielectrics cited as examples above) into which the vascular endoprosthesis is introduced.

[0047] THE Figures 1a and 1b represent a device 7 according to the invention in which a deployed endoprosthesis 5 is positioned. The first electrode 1 covered by the dielectric 4 is positioned between the receiving element 3 (which may be insulating) and the endoprosthesis 5 (see Figure 1b in section). The second electrode 2 is located inside the receiving element 3 at a distance from the endoprosthesis in an interval ranging from 5 to 15 cm, preferably 8 to 12 cm and even more preferably 10 cm. The aqueous solution comprising at least one protein is intended to circulate in the cavity 6 of the receiving element 3. The generation of the pulsed electric field between the two electrodes leads to the formation of the film of at least one protein on the surface of the endoprosthesis which is in direct contact with the aqueous solution.

[0048] Advantageously, the receiving element and the dielectric can be identical or different. For example, the dielectric can be a PVC tube in which the vascular stent will be placed during the implementation of the method (see figure 2 ). In this configuration, the thickness of the dielectric corresponds to the thickness of the receiving element which can be a PVC tube (or any of the dielectrics cited as examples above).

[0049] There figure 2represents a device 17 according to the invention in which a stent 15, deployed or not deployed, is positioned. The first electrode 11 is located on the outer surface of the receiving element 13 which acts as a dielectric in this configuration. The second electrode 12 is located inside the receiving element 13 at a distance from the stent in a range from 5 to 15 cm, preferably 8 to 12 cm and even more preferably 8 cm. The aqueous solution comprising at least one protein is intended to circulate in the cavity 16 of the receiving element 13. The generation of the pulsed electric field between the two electrodes leads to the formation of the film of at least one protein on the surface of the stent which is in contact with the aqueous solution. In this configuration, the first electrode may be a so-called annular electrode or a plurality of electrodes positioned on the external surface of the receiving element.The second electrode may be a simple electrical wire connected to ground or an annular electrode positioned inside or outside the receiving element 13.

[0050] Advantageously, the receiving element may be the original microcatheter of the stent. In this example, the method is implemented on a non-deployed stent.

[0051] Advantageously, when the stent intrinsically, partially or totally, has dielectric properties or when it comprises one or more dielectric material(s), the method according to the invention can be implemented directly by positioning the electrode system on the vascular stent. The vascular stent is brought into contact with the aqueous solution comprising at least one protein. In this configuration, the vascular stent plays the role of the dielectric. The electric field applied by the electrode system is made possible by the insulating properties of the vascular stent.

[0052] Advantageously, when the stent is metallic, it can serve as a grounded or energized electrode. In this configuration, electrode 2 or 12 may no longer be necessary.

[0053] Advantageously, the ends of the receiving element can be closed during the implementation of the method, the aqueous solution comprising at least one protein is then immobile or stagnant during the implementation of the method.

[0054] The invention also relates to a kit comprising a device according to the invention, an aqueous solution comprising at least one protein as defined above and optionally a vascular stent.

[0055] The invention also relates to a use of the device or kit according to the invention for covering vascular endoprostheses with a film of at least one protein of interest.

[0056] Other advantages may still become apparent to those skilled in the art upon reading the examples below, illustrated by the attached figures, given for illustrative and non-limiting purposes. Brief description of the figures

[0057] THE Figures 1a and 1brepresent a device according to the invention in which the receiving element and the dielectric are different and comprising a vascular endoprosthesis. The figure 2 represents a device according to the invention in which the receiving element and the dielectric are identical and comprising a vascular endoprosthesis. The figure 3 represents a comparison of the visual and scanning electron microscopy (SEM) appearance of (a) stent C, untreated after its extraction from the PVC tube and (b) stent A, treated according to the invention, 14 days after its treatment. Figures 3c and 3d show the surface condition of the untreated C and treated A stents respectively. figure 4represents the impact on the platelet count of the blood after passage through the Chandler Loop comprising a stent A or C, as described in Example 2. (a) Blood extracted from the tubing after passage through the Chandler Loop and in the absence of a vascular stent; (b) Blood extracted from the tubing after passage through the Chandler Loop and in the presence of a vascular stent C, untreated; (c) Blood extracted from the tubing after passage through the Chandler Loop and in the presence of a vascular stent A, according to the invention. Figure 5 represents (a) the surface of an untreated vascular stent C, and (b) the surface of a vascular stent A, according to the invention, observed in the focal plane of an epi fluorescent microscope after labeling of the proteins using an NHS Alexa 488 probe. The figure 6represents an example of an electrical signal (voltage in volts as a function of time in seconds) which can be applied to the electrode system of the device according to the invention, making it possible to generate the electric field according to the method of the invention. The Figure 7 (A) represents a comparison of the proteins released by a stent A, treated according to the invention (T) or C, untreated (NT), after chemical treatment and before / after sonication then analyzed by SDS PAGE electrophoresis. (B) represents the quantification by densitometry of the protein bands separated by SDS PAGE electrophoresis. The figure 8 represents a scanning electron microscopy image showing colonization by human endothelial cells (HUVEC) of an untreated stent C (control) (a) and of an stent A, treated according to the invention (b), after 5 days of static culture. Magnification of 90x and 500x for the zoom window. (Scale: 500µm). The figure 9represents a comparison of leukocyte adhesion to an untreated stent and an stent treated according to the invention after 1 hour of rotation of control blood in the Chandler Loop. (A) represents the quantity of free white blood cells in the blood after the Chandler Loop, (B) is a scanning microscopy image illustrating the appearance of white blood cells interacting with the surface of an untreated stent C (left), or an stent A, treated according to the invention (right). Magnification 4700x. (Scale: 10µm). EXAMPLES Example 1a: Preparation of a vascular endoprosthesis according to the invention (Endoprosthesis A)

[0058] A first high-voltage electrode, which is a conductive wire covered with a dielectric (this electrode is a micro-guide used in neurosurgery), is inserted into a PVC tube. The metal core of the electrode has a diameter of 170 µm and is covered with a thickness of parylene (dielectric) of 50 µm (this leads to a total external diameter of the micro-guide of 270 µm).

[0059] A nitinol flow diverter stent (registered trademark Silk) is deployed in the transparent PVC tube with an internal diameter of 3.7 mm and an external diameter of 6 mm. The inner wall of the PVC tube is heparinized, that is, it is incubated with heparin, a powerful anticoagulant (it prevents the formation of fibrin) which will cover the entire surface of the PVC tube and prevent the activation of circulating cells which could mask the effect of the stent treatment.

[0060] The first electrode (microguide) is held between the stent and the inner wall of the PVC tube. The length of the PVC tube is 20 cm. The length of the deployed stent is about 4 cm and one end of the stent is about 2 cm from one end of the PVC tube.

[0061] At the other end of the PVC tube, a conductive wire (second ground electrode) is placed in the PVC tube. The distance between the conductive wire and the stent is approximately 10 cm. The PVC tube is filled with blood plasma (or PBS containing albumin) and both ends of the PVC tube are clamped to prevent the plasma from flowing out.

[0062] The ends of the wires of the first and second electrodes remain accessible outside the PVC tube over a length of several centimeters.

[0063] The end of the conductive wire is connected to ground and the metal core of the high-voltage electrode to a voltage supply.

[0064] Positive voltage pulses of 10 kV amplitude, 500 ns duration at a frequency of 100 Hz are applied for 20 minutes.

[0065] We obtain the treated endoprosthesis A. Example 1b: Preparation of a vascular endoprosthesis according to the invention (Endoprosthesis B)

[0066] The nitinol flow diverter stent (registered trademark Silk+: Blat Extrusion Monmorency, France) is placed in a transparent PVC tube with an internal diameter of 1.6 mm and an external diameter of 2.4 mm. The length of the PVC tube is 20 cm. The length of the deployed stent is about 2.5 cm and one of its ends is at a distance of about 2 cm from one of the edges of the PVC tube.

[0067] The outer surface of the PVC tube is surrounded by a metallic electrode (copper tape) about 1 cm wide. The width of the tape partially covers the stent, which is separated from the conductive electrode by the dielectric thickness of the PVC tube. At the other end of the PVC tube, a conductive wire is placed in the PVC tube. The distance between the conductive wire and the stent is about 8 cm.

[0068] The PVC tube is filled with blood plasma (or PBS containing albumin) and both ends of the PVC tube are clamped to prevent the plasma from flowing out. The end of the conductive wire is accessible outside the PVC tube for a length of several cm.

[0069] The end of the conductive wire is connected to ground and the external electrode (the metal tape) to a voltage supply.

[0070] Positive voltage pulses of 10 kV amplitude, 500 ns duration at a frequency of 100 Hz are applied for 20 min.

[0071] We obtain endoprosthesis B.

[0072] In the following, endoprosthesis C will be referred to as an untreated control endoprosthesis, which is not part of the invention. Example 2: Results

[0073] After the treatment, the plasma in contact with the endoprosthesis A, treated according to the invention, of example 1a is replaced by human blood (it is poured into the same PVC tubing that was used for the treatment). The whole is then placed in a Chandler Loop System ® device (industriedesign, ebo kunze) to reproduce the rheological conditions of circulation in a blood vessel (the PVC tube in the form of a torus is rotated in a water bath at 37°C so that the blood is set in motion in the tube as it would be in an artery). An untreated endoprosthesis C is prepared under the same operating conditions but without being subjected to the electric field. After 1 hour of rotation in the Chandler Loop, stents A or C are removed from the PVC tube. Blood is collected for analysis and platelet count. Figure 3 (a)shows the visual and scanning electron microscopy (SEM) appearance of untreated stent C after its extraction from the PVC tube. The surface of the stent (see white arrows) is the site of platelet thrombus formation and fibrin threads are visible between its meshes. Figure 3 (b) shows the appearance of endoprosthesis A, treated according to the invention, 14 days after its treatment. No platelet thrombus or fibrin filaments are observed in the same model. The Figure 3 (b) thus shows that the effects last at least 14 days after treatment with the vascular stent.

[0074] The various tests carried out show that the effect lasts at least up to 6 months.

[0075] Thus, when the endoprosthesis is simply placed in contact with human plasma (without the application of the electric field), a thick deposit of proteins is observed filling the asperities of the nitinol ( Figure 3 (c)), whereas the protein film(s) induced by the application of the electric field is significantly thinner ( Figure 3 (d) ). The scales of the nitinol alloy on the surface of stent A are still visible, whereas they are no longer visible on the untreated stent C.

[0076] There figure 4 shows that stent A, treated according to the invention, has an impact on the blood platelet count. In the three cases presented (a), (b) and (c), the platelet count in the blood is measured after 1 hour of rotation in the Chandler Loop. It is noted that the platelet count in the blood is lowered in the case of stent C, untreated, following their interaction with the stent ( Figure 4b ) whereas in the presence of an endoprosthesis A, treated according to the invention ( Figure 4c ), we find the level of free platelets observed in the absence of a vascular endoprosthesis ( Figure 4a ).

[0077] Blood protein labeling was performed using a probe (NHS Alexa 488) that binds covalently to the -NH2 terminal portion of proteins. This allows proteins to be visualized by epifluorescent microscopy. Figure 5 shows a comparison between an untreated stent that has been in contact with human blood and stent A, treated according to the invention, that has been in contact with human blood for a period of 2 hours in the Chandler Loop. The untreated stent C of the Figure 5 (a) exhibits high and relatively heterogeneous fluorescence in the focal plane of the microscope due to platelet adhesion and the forming fibrin network (thrombosis). The stent treated according to Example 1 shown in the Figure 5 (b)exhibits homogeneous fluorescence in the focal plane of the microscope only on the surface of the stent (homogeneous thickness) indicating the presence of the film of at least one protein present on the surface of the endoprosthesis A, treated according to the invention. Example 3: Identification and quantification of proteins deposited on the endoprosthesis

[0078] The stent was subjected to different treatment conditions (untreated and treated according to the invention), then incubated in 300 µl of Laemmli buffer (4% SDS, 10% DTT, 20% glycerol, 0.004% bromophenol blue, 0.125M TRIS HCL; pH = 6.8) for 10 minutes at 90°C followed by 1 min with stirring. The stents were recovered and then soaked again in the same volume of Laemmli buffer but this time they were sonicated by an ultrasound cycle for 40 seconds at a frequency of 40kHz. A volume of 40 µl of the suspension was then analyzed by SDS PAGE electrophoresis.

[0079] After migration, the proteins will be revealed for staining with Imperial stain (BIORAD) and the area corresponding to albumin (majority protein observed) is shown in the Figure 7A

[0080] Unlike the untreated (NT) stent, the treated (T) stent requires a sonication step so that the deposited protein film can be recovered and analyzed, as shown in Figures 7A and 7B This result demonstrates that the protein film deposited during the treatment of the treated stent is very strongly attached to the surface of the stent, which correlates with the observation of the persistence of the treatment effect of the stent over a long period (6 months at 4°C). Example 4 : static cell culture endothelialization test

[0081] After treatment with the electric field, the stent A, treated according to the invention, was removed from the PVC tube and placed for 5 days in a culture medium containing human endothelial cells (HUVEC) in suspension. The images obtained by scanning electron microscopy of the stents show that the treatment does not prevent the endothelial cells from colonizing the surface of the stent in static conditions. The black spots observed on the figure 8 (a and b) correspond to adhered endothelial cells (some of which are indicated by arrows). The images of the figure 8 clearly show that endothelial cells adhere to the surface and that the coverage rate at 5 days is identical for an untreated C endoprosthesis (a) as for an A endoprosthesis, treated according to the invention (b). Example 5 : effect of treatment on leukocyte recruitment

[0082] It is observed that the treatment of the endoprosthesis according to the invention also has an impact on the recruitment of circulating leukocytes after 1 hour of rotation in the Chandler Loop, as shown in Figure 9A. When the stent is untreated, the white blood cell count in the blood after 1 hour of rotation is lowered, which is not the case when the stent is treated. In addition, observation under a scanning electron microscope ( Figure 9B ) shows that the leukocytes are very spread out on the untreated stent (a sign of strong adhesion and cellular activation). On the contrary, the rare leukocytes that interact with the stent treated according to the invention keep their rounded shape (their attachment to the surface of the stent is therefore fragile).

[0083] This result demonstrates a beneficial impact of stent treatment on the processes of recruitment and activation of leukocytes, which can reduce the inflammation aspect generally observed during stent placement. List of references

[0084] [1] WO2017 / 004598

Claims

1. Vascular stent, deployed or non-deployed, whose surface is coated by a film comprising at least one protein of interest which has been subjected to an electric field having a periodic structure.

2. A vascular stent according to the preceding claim, wherein the at least one protein of interest is selected from the group comprising blood plasma proteins, preferably albumin, and a synthetic biological macromolecule, and mixtures thereof.

3. A vascular stent according to any of the preceding claims in which the concentration of protein of interest on the surface of the stent is greater than or equal to 2 µg / cm2.

4. A stent according to any of the preceding claims, wherein the stent comprises one or more alloys selected from the group consisting of stainless steel, nickel / titanium, tantalum, cobalt / chromium, platinum / chromium alloys, alloys optionally including magnesium, and mixtures thereof.

5. A process of coating the surface of a vascular stent, deployed or non-deployed, with a film of at least one protein of interest, comprising the steps of : - bringing the vascular stent into contact with an aqueous solution comprising at least one protein of interest, - application of an electric field, having a periodic structure, generated by a system of electrodes comprising at least one first electrode, a dielectric and at least one second electrode, said dielectric electrically isolating the stent and the at least one second electrode from the at least one first electrode, and - coating of the surface of the stent with the film of at least one protein of interest.

6. A process according to the preceding claim, in which the concentration of protein of interest in the aqueous solution is greater than or equal to 0.1 mg / ml.

7. A process according to any of claims 5 to 6, wherein the protein is selected from the group comprising blood plasma proteins, preferably albumin, and a synthetic biological macromolecule, and mixtures thereof.

8. A process according to any one of claims 5 to 7, wherein the electric field is generated by a voltage signal applied to the system of electrodes having an amplitude ranging from 0.1 kV to 50 kV, preferably from 5 kV to 40 kV, even more preferably from 10 kV to 30 kV, having a duty cycle ranging from 5.10-8 to 1, preferably from 5.10-6 to 5.10-3, and having a frequency ranging from 0.1 Hz to 100 kHz, preferably from 1 Hz to 1 kHz.

9. A process according to any one of claims 5 to 8, in which the electric field is applied for a duration greater than or equal to 10 seconds, preferably greater than or equal to 5 minutes and even more preferably greater than or equal to 10 minutes.

10. A device for carrying out the process according to any of claims 5 to 9, comprising : - at least one first electrode, - at least one second electrode, - at least one dielectric isolating the first electrode from the second electrode, - a receiving element capable of containing the vascular stent, said receiving element having a shape adapted to the shape of the vascular stent, said receiving element being identical or different from the dielectric, - a source of electrical energy connected to the electrodes and a voltage regulator for regulating the voltage on the at least one first electrode and the at least one second electrode in order to generate the electric field having a periodic structure.

11. A kit comprising a device according to claim 10, an aqueous solution comprising at least one protein as defined in claim 7, and optionally a vascular stent.

12. Use of the device or kit according to any one of claims 10 to 11 for coating a vascular stent with a film of at least one protein of interest.