VASCULAR STENT WITH ANTITHROMBOTIC PROPERTIES

Abstract

The present invention relates to a vascular stent, deployed or non-deployed, the surface of which is coated by a film comprising at least one protein, to a process for coating of the surface of a vascular stent with a film comprising at least one protein and to a device for carrying out the process according to the invention.

Claims

1. Vascular stent, deployed or non-deployed, comprising a surface coated by a film comprising at least one protein which has been subjected to an electric field.

2. A vascular stent according to claim 1, wherein the at least one protein comprises at least one of blood plasma proteins, a synthetic biological macromolecule, and mixtures thereof.

3. A vascular stent according to claim 1, wherein a concentration of the protein on the surface of the stent is greater than or equal to 2g/cm.sup.2.

4. A stent according to claim 1, 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 a surface of a vascular stent, deployed or non-deployed, with a film of at least one protein, comprising the steps of: bringing the vascular stent into contact with an aqueous solution comprising the protein, application of an electric field 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 the protein.

6. A process according to claim 5, wherein the concentration of the protein in the aqueous solution is greater than or equal to 0.1 mg/ml.

7. A process according to claim 5, wherein the protein is comprises at least one of blood plasma proteins, a synthetic biological macromolecule, and mixtures thereof.

8. A process according to claim 5, 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, having a duty cycle ranging from 5.10.sup.8 to 1, and having a frequency ranging from 0.1 Hz to 100 kHz.

9. A process according to claim 5, wherein the electric field is applied for a duration greater than or equal to 10 seconds.

10. A device for carrying out the process according to claim 5, 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 being identical or different from the dielectric.

11-12. (canceled)

13. A vascular stent according to claim 2, wherein the at least one protein comprises albumin.

14. A process according to claim 8, wherein the amplitude of the voltage signal ranges from 5 kV to 40 kV.

15. A process according to claim 8, wherein the voltage signal has a duty cycle ranging from 5.10.sup.6 to 5.10.sup.3.

16. A process according to claim 8, wherein the voltage signal has a frequency ranging from 1 Hz to 1 kHz.

17. A process according to claim 5, wherein the electric field is applied for a duration greater than or equal to 5 minutes.

18. A kit comprising: a device; an aqueous solution comprising a protein; and a vascular stent; wherein the device comprises: a first electrode, a second electrode, a dielectric isolating the first electrode from the second electrode, and a receiving element capable of containing the vascular stent, said receiving element being identical or different from the dielectric; wherein the protein comprises at least one of a blood plasma protein, a synthetic biological macromolecule, and mixtures thereof.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0067] FIGS. 1a and 1b represent a device according to the invention in which the receiving element and the dielectric are different and comprising a vascular stent.

[0068] FIG. 2 represents a device according to the invention in which the receiving element and the dielectric are identical and comprising a vascular stent.

[0069] FIG. 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. FIGS. 3c and 3d show the surface condition of the untreated stent C and the treated stent A, respectively.

[0070] FIG. 4 represents the impact on the platelet count of the blood after passing through the Chandler Loop with 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 stent; (b) Blood extracted from the tubing after passage through the Chandler Loop and in the presence of a stent C, untreated; (c) Blood extracted from the tubing after passage through the Chandler Loop and in the presence of a stent A, according to the invention.

[0071] FIG. 5 represents (a) the surface of an untreated stent C and (b) the surface of a stent A, according to the invention, observed in the focal plane of a fluorescent epi-microscope after protein labeling with an Alexa 488 NHS probe.

[0072] FIG. 6 represents an example of an electrical signal (voltage in volts as a function of time in seconds) that can be applied to the system of electrodes of the device according to the invention, allowing the electric field to be generated according to the process of the invention.

[0073] FIG. 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 and analysed by SDS PAGE electrophoresis. (B) represents the quantification by densitometry of the protein bands separated by SDS PAGE electrophoresis.

[0074] FIG. 8 represents a scanning electron microscopy image showing the colonization by human endothelial cells (HUVEC) of an untreated (control) stent C (a) and a treated stent A according to the invention (b) after 5 days of static culture. Magnification of 90 and 500 for the zoom window. (Scale: 500m).

[0075] FIG. 9 shows a comparison of leukocyte adhesion on an untreated stent and a stent treated according to the invention after 1 hour rotation of a control blood in the Chandler Loop. (A) represents the amount of free white blood cells in the blood after the Chandler Loop, (B) is a scanning microscopy image showing the appearance of white blood cells interacting with the surface of an untreated stent C (left) or A treated according to the invention (right). Magnification 4700. (Scale: 10m).

EXAMPLES

Example 1a

Preparation of a Vascular Stent According to the Invention (Stent A)

[0076] A first high-voltage electrode is inserted into a PVC tube, which is a conductive wire covered with a dielectric (this electrode is a micro-guide used in neurosurgery). The metal core of the electrode has a diameter of 170m and is covered with a 50m thickness of parylene (dielectric) (this leads to a total external diameter of the microguide of 270m).

[0077] A nitinol flow diverter stent (Silk registered trademark) 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 heparinised, i.e. it is incubated with heparin, a powerful anticoagulant (it prevents the formation of fibrin) that will cover the entire surface of the PVC tube and prevent the activation of circulating cells that could mask the effect of the stent treatment.

[0078] The first electrode (micro-guide) 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 expanded stent is about 4 cm and one end of the stent is about 2 cm from one end of the PVC tube.

[0079] At the other end of the PVC pipe, a lead wire (second ground electrode) is placed in the PVC pipe. The distance between the conductor wire and the stent is about 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.

[0080] The wire ends of the first and second electrodes remain accessible on the outside of the PVC pipe over a length of several centimetres.

[0081] The end of the conductor wire is connected to ground and the metal core of the high-voltage electrode is connected to a voltage supply.

[0082] Positive voltage pulses with an amplitude of 10 kV and a duration of 500 ns at a frequency of 100 Hz are applied for 20 minutes.

[0083] The treated stent A is obtained.

Example 1b: Preparation of a Vascular Stent According to the Invention (Stent B)

[0084] The nitinol flow diverter stent (Silk+registered trademark: 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 approximately 2.5 cm and one of its ends is at a distance of approximately 2 cm from one of the edges of the PVC tube.

[0085] The outer surface of the PVC tube is wrapped with a metal electrode (copper tape) about 1 cm wide. The width of the tape partly 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.

[0086] 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 conductor wire is accessible outside the PVC tube for a length of several cm.

[0087] The end of the conductor wire is connected to ground and the outer electrode (the metal tape) to a voltage supply.

[0088] Positive voltage pulses with an amplitude of 10 kV and a duration of 500 ns at a frequency of 100 Hz are applied for 20 min.

[0089] Stent B is Obtained.

[0090] In the following, stent C will refer to a control stent, untreated, not being part of the invention.

Example 2

Results

[0091] After treatment, the plasma in contact with the stent A, treated according to the invention, of example la is replaced by human blood (it is poured into the same PVC tubing used for the treatment). The assembly 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 stent 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, the stents A or C are removed from the PVC tube. Blood is collected for analysis and platelet count. FIG. 3 (a) shows the visual and scanning electron microscopy (SEM) appearance of the untreated stent C after removal 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 the stent mesh. FIG. 3 (b) shows the appearance of stent A, treated according to the invention, 14 days after treatment. No platelet thrombi or fibrin filaments are observed in the same pattern. FIG. 3 (b) thus shows that the effects persist at least 14 days after treatment of the stent.

[0092] The various tests carried out show that the effect lasts at least up to 6 months.

[0093] Thus, when the stent is simply brought into contact with human plasma (without the application of the electric field), a thick deposit of protein is observed which fills the nitinol asperities (FIG. 3 (c)), whereas the film of protein(s) induced by the application of the electric field is much thinner (FIG. 3 (d)). The scales of the nitinol alloy on the surface of the stent A are still visible, whereas they are no longer visible on the untreated stent C.

[0094] FIG. 4 shows that stent A, treated according to the invention, has an impact on platelet blood counts. 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 can be seen that the level of platelets in the blood is lowered in the case of stent C, untreated, as a result of their interaction with the stent (FIG. 4b), whereas in the presence of stent A, treated according to the invention (FIG. 4c), the level of free platelets observed in the absence of a vascular stent (FIG. 4a) is found.

[0095] The blood proteins were labelled using a probe (NHS Alexa 488) which binds covalently to the terminal NH2 part of the proteins. This enables the proteins to be visualised by fluorescent spike microscopy. FIG. 5 shows a comparison between an untreated stent which has been in contact with human blood and stent A, treated according to the invention, which has been in contact with human blood for a period of 2 hours in the Chandler Loop. The untreated stent C in FIG. 5 (a) shows a high and relatively heterogeneous fluorescence in the focal plane of the microscope due to platelet adhesion and the fibrin network being formed (thrombosis). The stent treated according to Example 1 shown in FIG. 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 stent A treated according to the invention.

Example 3

Identification and Quantification of Proteins Deposited on the Stent

[0096] The stent was subjected to different treatment conditions (untreated and treated according to the invention), then incubated in 300l 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. and then 1 min with stirring. The stents are recovered and then soaked again in the same volume of laemmli buffer but this time they are sonicated by an ultrasound cycle for 40 seconds at a frequency of 40 kHz. A 40l volume of the suspension is then analysed by SDS PAGE electrophoresis.

[0097] After migration, the proteins will be revealed for Imperial stain (BIORAD) and the area corresponding to albumin (majority protein observed) is shown in FIG. 7A.

[0098] Unlike the untreated (NT) stent, the treated (T) stent requires a sonication step so that the deposited protein film can be retrieved and analysed, as shown in FIGS. 7A and 7B. This result demonstrates that the protein film deposited during processing of the treated stent is very strongly adhered to the surface of the stent which correlates with the observation of the persistence of the effect of the stent treatment over a long period of time (6 months at 4 C.).

Example4

Endothelialization Test in Static Cell Culture

[0099] 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. Scanning electron microscopy images of the stent show that the treatment does not prevent the endothelial cells from colonizing the surface of the stent under static conditions. The black spots observed in FIG. 8 (a and b) correspond to adhered endothelial cells (some of which are indicated by arrows). The images in FIG. 8 clearly show that the endothelial cells adhere to the surface and that the coverage rate at 5 days is the same for an untreated stent C (a) as for a stent A treated according to the invention (b).

Example 5

Effect of Treatment on Leukocyte Recruitment

[0100] It can be seen that the treatment of the stent 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 FIG. 9A. When the stent is untreated, the white blood cell count after 1 hour of rotation is lowered, which is not the case when the stent is treated. In addition, observation with a scanning electron microscope (FIG. 9B) shows that the white blood cells are very spread out on the untreated stent (a sign of strong adhesion and cell 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).

[0101] This result demonstrates a beneficial impact of stent treatment on the recruitment and activation processes of leukocytes, which can reduce the inflammation generally observed during stent placement.

LIST OF REFERENCES

[0102] [1] WO2017/004598