RU2721280C1 - Method for creating antithrombogenic coating on polymer vascular prostheses - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine and discloses a method for creating an antithrombogenic coating on the inner and outer surfaces of a polymer prosthesis of a blood vessel. Method involves modifying the external and/or internal surface of the implant by forming a hydrophilic carbon layer on the outer and inner surfaces of the polymer prosthesis of the vessel by ion-plasma implantation with subsequent creation on the carbon surface of a solid protein coating (albumin, fibrinogen or tropoelastin), modification of the outer surface is performed by bombardment with high-energy nitrogen ions, creation of a solid protein coating on the carbonized surfaces of the prosthesis is performed by immersion into a phosphate-saline buffer solution with protein.

EFFECT: invention can be used to create on the surface of a polymer prosthesis of an active bio- and hemocompatible stable layer with low thrombogenicity and for accelerated endothelization along the entire inner and outer surfaces of the prosthesis by applying a continuous protein coating on the prosthesis.

9 cl, 6 ex

Description

The invention relates to medicine, namely to the development of new materials and coatings for prostheses of blood vessels, as well as for vascular catheters. Using the method proposed in the invention allows to reduce coagulation of blood cells and the formation of a blood clot on the surface of polymer prostheses directly in contact with blood used in cardiovascular surgery. This method allows you to change the physico-chemical properties of the surface of the polymers, which makes it possible to reduce the time of proliferation of endothelial cells on the inner surface of the prostheses of blood vessels.

The level of technology.

In vascular surgery, the use of implants made of polymeric materials is widespread: polyurethane, polytetrafluoroethylene (PTFE), including expanded, polyethylene terephthalate (lavsan), polycaprolactone, polyglycolide, nylon and other polyamides. Their use causes the risk of vascular thrombosis and thromboembolic syndrome and, as a result, a decrease in blood flow patency. Such negative side effects occur especially often when using vessels of small diameter, less than 4 mm.

It is important to note that in 89-99% of cases of the use of prostheses from lavsan and PTFE in the short period of use (up to one month) there were no problems with their patency [Bokeria L.A., Temrezov MB, Kovalenko V.I., Surgical treatment of patients with atherothrombotic lesions of the arteries of the lower extremities - the choice of transplant for femoral-popliteal bypass surgery // Annals of surgery. 2010. - No. 2. - S. 5-8.] [Devine S., McCollum S. Heparin-bonded Dacron or polytetrafluoroethylene for femoropopliteal bypass grafting: A multicenter Trial. J Vasc Surg 2004; 40; 924-931]. However, with the long-term use of implants from these types of materials, the statistics deteriorate, the patency of the prostheses is violated due to their thrombosis. After 3 years of use of these implants, replacement is required in 31% of cases, and after 5 years in 61% of cases [Klinkert P, Post PN, Breslau PJ, et al. Saphenous vein versus PTFE for above-knee femoropopliteal bypass: a review of the literature. Eur J Vasc Endovasc Surg 2004; 27: 357-362], [Rychlik I.J. et al. A meta-analysis to compare Dacron versus polytetrafluroethylene grafts for above knee femoropopliteal arterybypass. // J Vasc Surg. 2014; 60; 2; 506-515].

A large contribution to the process of thrombosis is made by the structure and physicochemical properties of the surface of the materials used. They affect the activation of the compliment system, cell adhesion and, primarily, protein adsorption. These phenomena together trigger the cascading mechanisms of thrombosis reactions [Biocompatible materials: Textbook / Ed. IN AND. Sevastyanova, M.P. Kirpichnikova - M: MIA, 2011].

To reduce the process of thrombosis in the postoperative period, the surface of the implant is modified in various ways. The most promising are plasma methods of processing materials. They form various functional groups on the surface of implants, change the surface morphology, its wettability, optical properties and electrical conductivity.

There is a method of endothelization of prosthetic blood vessels (RF patent No. 2659704, priority 03/31/2017, IPC A61L 27/04, A61L 31/08, A61L 33/02, published on July 3, 2018 Bull. No. 19). The method includes plasma-chemical surface modification with the application of a metal-containing coating. This method allows to obtain a biochemically and hemocompatible coating on the external and internal surfaces of vascular prostheses, characterized by low thrombogenicity and high stability. In vitro studies were conducted on the subject of cell adhesion and cell culture growth on a coated surface according to the claims. The disadvantage of this invention is the inability to create a continuous coating on the inner and outer surfaces of the prosthesis.

This disadvantage will lead to the fact that on surfaces where there is no coating, the adsorption of proteins from blood plasma will be carried out, as on the surface of the original polymer material. Using this method can delay the process of thrombosis, but not solve the problems of developing thromboresistant coatings and materials.

Thus, the inconsistency of the resulting coating will provide conditions for: launching the activation mechanisms of the compliment system; fibrin deposits; coagulation of blood cells on the surface of the prosthesis, which ultimately reduces the effectiveness of this method of surface treatment as a means of counteracting the process of thrombosis. As the disadvantages of the proposed method it should also be noted that:

- there are no results of its tests in vitro with whole or heparinized blood;

- its effectiveness is not confirmed by the results of in vivo experiments.

All this casts doubt on the advantage of the material processed using this method compared to untreated materials.

This method is adopted as a prototype.

Disclosure of the invention.

The objective of the invention is a method for creating on the surface of polymeric prostheses of veins and arteries an active carbon coating capable of covalently binding protein molecules to it while maintaining their conformation and activity.

The technical result of this invention is the creation on the surface of a polymeric prosthesis of a vessel of an active bio- and hemocompatible stable layer with low thrombogenicity, accelerated endothelization over the entire internal and external surfaces of the prosthesis due to the application of a continuous protein coating on them.

The technical result is achieved due to the formation of a hydrophilic carbon layer of optimal thickness and activity on the outer and inner surfaces of the polymeric vessel prosthesis by ion-plasma implantation followed by the application of a protein layer on the carbon surface that retains its molecular conformation, biological activity and adhesive properties.

The method of creating an antithrombogenic coating on polymeric vascular prostheses, on the surface of a polymeric prosthesis of a blood vessel, is carried out as follows: modification of the external and / or internal surface of the implant is carried out using the method of ion-plasma implantation, bombardment by high-energy nitrogen ions of 5-20 keV with a pulse duration of 10 -40 microseconds, a dose of ions 5 * 10 ¹⁴ -10 ¹⁶ ions per cm ² . Modification of the inner surface is carried out using a system comprising a cone nozzle inside a vacuum reactor and an electrode located around the vessel prosthesis being treated. Nitrogen gas is supplied to the reactor through the nozzle and the cavity of the polymeric vessel prosthesis. After modification of the external and / or internal surfaces of the prosthesis, a continuous protein coating is created on its active carbonized surfaces by immersing the prosthesis in a phosphate-saline buffer solution with protein.

The invention is illustrated by the following examples of specific performance. The intervals of parameter values in the examples are established experimentally.

Example 1. The treatment of the external surface of the prosthesis by the method of ion-plasma implantation (mode 1).

The vascular prosthesis is a rectilinear polymer tube. To process the external surface of the prosthesis by ion-plasma implantation, the tube is placed on a metal plate (holder) that is closed by a metal mesh and connected to a high-voltage power source. The plate is placed in a vacuum reactor, from which air is pumped out using an oil-free scroll pump and a turbomolecular pump to a pressure of 10 ^-5 Torr. Then the reactor is filled with high purity nitrogen (99.999%).

A pressure of 2 mTorr is maintained in a nitrogen-filled reactor by pumping excess gas. To create a plasma inside the reactor, a high-frequency pulse generator with a frequency of 13.75 MHz and an output power of 100 W is used, which, through the matching unit, supplies pulsed voltage to the antenna located outside the glass part of the reactor.

The holder is supplied with a pulsed high voltage from a low-frequency high-voltage pulse generator. Due to this, acceleration and implantation of ions on the external surface of the polymer prosthesis located on the holder occurs. The average ion flux per sample is set so as to exclude overheating of the sample under the longest processing conditions. The measured temperature of the holder does not exceed 40 ° C for 1 hour of treatment. The voltage value is 5-20 kV, the pulse duration is 10-40 microseconds. Front rise of the pulse 1 μs. The pulse repetition rate is 50 Hz. The average current was 1.4-1.5 mA.

Example 2. The treatment of the external surface of the implant by the method of ion-plasma implantation (mode 2).

To process the external surface of the prosthesis by ion-plasma implantation, polymer tubes are placed on a metal plate (holder), which is connected to a high-voltage power supply, by a metal plate closed by a metal mesh. The plate is placed in a vacuum reactor, from which air is pumped out using an oil-free scroll pump and a turbomolecular pump to a pressure of 10 ^-5 Torr. Then the reactor is filled with high purity nitrogen (99.999%). A pressure of 4-5 mTorr is maintained.

The holder is pulsed high voltage from a high voltage pulse generator. The average ion flux per sample is set so as to prevent overheating of the sample under the longest processing conditions. The measured temperature of the holder does not exceed 40 ° C for 1 hour of treatment. The voltage value is 5-20 kV, the pulse duration is 10-40 microseconds. The creation of plasma occurs under the influence of an electric field formed by a low-frequency pulse generator. Acceleration of ions and their implantation on the outer surface of the polymer prosthesis located on the holder occurs due to the low-frequency pulse generator. Front rise of the pulse of 0.1-1 μs. The pulse repetition rate is 100 Hz. The average current is 5.5-6 mA with an average charge and discharge current in the absence of a barrier discharge of 3.8 mA.

The ion irradiation dose (fluence) for both treatment modes (examples 1 and 2) of the outer surface of the polymer prosthesis was determined by measuring the UV absorption spectra of polyethylene and comparing them with the spectra of polyethylene treated on an ion implant with a known ion flux density [A. Kondyurin, M. Bilek, Ion Beam Treatment of Polymers. Application aspects from medicine to space, Second Edition, Elsevier, Oxford, 2014].

To obtain the desired structure and activity of the carbon layer on the surface of the polymers, it is necessary to carry out processing with a radiation dose of 5 * 10 ¹⁴ to 10 ¹⁶ ions per cm ² .

According to a specific ion flux density, polyurethane implants for laboratory animals were treated with an irradiation dose of 1e16 ions per square centimeter (ions / cm ² ).

In the above-described treatment scheme, the ion flow is directed perpendicular to the flat surface of the holder, on which the straight prosthetic tubes lie horizontally. Due to this, in one of the above cycle, only part of the outer surface of the tube is processed. To process the entire external surface of the implant, the complete treatment cycle is repeated three times. In this case, after the first and second cycle, the tubes rotate about 120 degrees in one direction relative to their axes.

Example 3. The treatment of the inner surface of the prosthesis of the vessel by the method of ion-plasma implantation.

Vascular prostheses can have different diameters from 1 to 10 mm, as well as bifurcations, so for such medical implants it is almost impossible to invert the external to the internal surface without damaging the carbon layer. To process the inner surface of such prosthetic vessels, a system is used that includes a cone nozzle inside the vacuum reactor and an electrode located around the vessel prosthesis being treated. The base of the cone nozzle is connected via a polymer pipe to the gas inlet in the reactor. The nozzle outlet is guided into the cavity of the polymeric vessel prosthesis. A gap of 1-2 mm is created between the nozzle and the hole of the polymer prosthesis. Air is pumped out of the vacuum reactor to a pressure of 1e-5 Torr. Next, a stream of high purity nitrogen gas (99.999%) through the nozzle is fed into the reactor. The flow rate is selected so that the pressure at the inlet of the reactor is 1-2 Torr. In this case, the pressure in the reactor with constant pumping of excess gas by vacuum pumps is not more than 4 mTorr. High voltage pulses of 5-15 kV with a duration of 10-40 microseconds with a pulse repetition rate of 50-100 Hz are fed to the electrode. The processing of the inner surface is carried out by obtaining a barrier discharge between a vacuum reactor with zero potential and a high-voltage electrode located around the processed polymer prosthesis of the vessel.

The processing period of the inner surface of the prosthetic vessels with an outer diameter of 2 mm and a length of 7-10 cm from polyurethane for experiments on laboratory animals was 10 minutes.

Example 4. The study of changes in the molecular structure of the inner and outer surfaces of the prosthetic vessel as a result of ion-plasma implantation.

The carbon nanolayer formed by the method of ion-plasma implantation on the inner and outer surfaces of the polymer, in particular in the case of polyurethane, was studied using IR spectroscopy. The spectra were recorded on a Fourier-IR spectrometer with a spectral microscope. According to the IR MNE spectra, vibrations of the hydroxyl group in the vicinity of 3370 cm ^-1 , double bonds C = O, C = C and C = N in the interval 1750-1600 cm ^-1 , the amine group in the region of 1534 cm ^{-1 were} observed. The appearance of oxygen-containing groups is associated with surface oxidation during interaction with the external environment after depressurization of a vacuum reactor. This phenomenon is largely due to the presence of unpaired electrons at the carbon atom in the surface nanolayer, which is confirmed by the results of EPR spectroscopy.

Example 5. The creation of a continuous protein coating and evaluation of protein adsorption on the inner and outer surfaces of the polymeric vessel prosthesis.

After ion-plasma treatment, polymer implants were immersed in a phosphate-saline buffer solution with protein. Solutions of various proteins — albumin, fibrinogen, tropoelastin, and others — were used. Such treatment with a solution made it possible to form a continuous layer of protein molecules on the external and internal surfaces of the prostheses. The concentration of the solution was 100 μg / ml, the exposure of the implant samples in the solution was 12 hours. After removal from the solution, the implant was dried. For comparative analysis, samples that were not processed by ion-plasma implantation were subjected to the same treatment with a protein solution.

Next, the adsorption of proteins on the surface of the implants was evaluated by IR MNPVO spectroscopy. In the IR spectra, the lines of Amide 1 and Amide 2 were analyzed. A comparative analysis was made of the intensity of the spectral lines of polymer samples (in the particular case of polyurethane), processed and not processed by the method of ion-plasma implantation. It was found that the intensity of the protein lines in the spectrum of the treated polymer is several times higher than in the spectrum of the untreated. The intensity of the protein lines on the processed polymer prostheses was close to the spectrum of a monolayer coating of protein molecules. For untreated implant samples, an intensity analysis showed inconsistency of the coating.

To assess the adhesion properties of the protein layer, the protein was washed from the surface of polymer implants treated and not processed by the ion-plasma method. For this, samples of polymer prostheses with a protein layer were placed in a 2% sodium dodecyl sulfate solution, kept for 1 hour at a temperature of 70 ° C and washed three times in deionized water. After the samples were dried for 24 hours at a temperature of 23-25 ° C without dust. Next, the IR MNE spectra were recorded and the intensity of the lines of the Amide 1 and Amid 2 protein was analyzed, as described above. It was found that after washing, the intensity of the lines of the spectrum of the protein on the processed samples decreased, but still corresponded to the spectrum of the monolayer coating of protein molecules. The intensity of the lines of the spectrum of the protein in untreated samples after washing significantly decreased.

The comparative analysis made it possible to establish higher adhesion properties of the protein layer on the surface of the implant treated with the ion-plasma implantation method compared to the untreated implant. This result confirms the validity of the assumption that ion-plasma implantation leads to the formation of unpaired electrons on carbon atoms in the surface layer. This, in turn, allows the formation of covalent bonds of the modified surface of the implant with protein molecules, which explains the increased adhesive properties. The results of these experiments allow us to conclude that adsorbed protein molecules on the hydrophilic carbon surfaces of a vessel prosthesis cannot be washed off with body fluids, for example, blood plasma.

Example 6. Evaluation of thrombosis in the cavities of polymer prosthetic vessels after implantation in a blood vessel of laboratory animals.

In order to evaluate the process of thrombus formation on the inner surfaces of polymer implants untreated and processed by the method of ion-plasma implantation, in particular in the case of polyurethane, operations were carried out to introduce a vessel prosthesis into the bloodstream of the rabbit carotid artery.

At the time of the operation, anticoagulant therapy was carried out: heparin - 0.05 ml and atropine 1% - 0.1 ml. The implant placement operation was performed under general anesthesia using the Zoletil preparation. After shaving the surgical site and cutting the soft tissues into the left carotid artery, on which 2 vascular clips were applied to stop blood flow, implants were inserted with forceps. For stitching the vascular incision, suture material PROLEN No. 007 was used and sulfacrylate adhesive was additionally applied. To close the soft tissue wound, suture material “Vikril” 4/0 was used. After the operation, the animals were injected subcutaneously with an isotonic sodium chloride solution of 20 ml and intramuscularly with furosemide 0.5 ml, dexamethasone 0.5 ml, sulfocamphocaine 0.5 ml and ceftriaxone 100 mg. The operation time was no more than 1 hour. In the postoperative period, blood-thinning drugs were not used. The animals were monitored daily. According to the monitoring results in animals, no violations were detected.

After 7 days of exposure, euthanasia was applied to the animals. Then, the vessel with the implant and surrounding soft tissues was excised using the surgical method and immersed in formalin. Then, a standard technique for fixing the test tissues with a vessel prosthesis in paraffin blocks was carried out for further histological studies. On the microtome, tissue sections were made in paraffin blocks. The obtained sections were stained with hematoxylin, eosin and stained according to Zerbino. Then, with the help of microscopy, the histological analysis of the sections was completed.

As a result of the analysis, a typical pattern of thrombosis on the surface of a foreign body that comes in contact with blood was observed for a group of animals with untreated polymer implants. The implant cavity was characterized by large blood clots, weak proliferation of intima, and slow formation of a slit-like cavity.

For the experimental group of animals with vascular prostheses processed by the ion-plasma method and coated with a protein layer, a more optimistic morphological picture was observed.

First, thrombi of relatively small sizes formed in the internal cavities of the implants. Secondly, from the intima side, endothelial proliferation and the replacement of thrombotic masses with connective tissue were observed.

From a comparison of the results obtained by the thrombus formation process for the control and experimental groups of animals, it follows that the experimental group is characterized by the presence of larger blood flow cavities, accelerated blood clots and endothelization of blood cavities and vessel prosthesis. The size of the blood flow cavities in the experimental group is 2.5 times the size in the control. These data are obtained by counting the restored blood flow channels during microphotography processing. This result is statistically significant, the p-value is 0.0275, the null hypothesis about the randomness of the results is rejected.

Claims (9)

1. A method of creating an antithrombogenic coating on polymeric vascular prostheses, on the surface of a polymeric prosthesis of a blood vessel, wherein the polymeric vascular prosthesis is made of polyurethane, the method includes modifying the outer and / or inner surfaces of the implant by forming a hydrophilic vessel on the outer and / or inner surfaces of the polymeric prosthesis the carbon layer by ion-plasma implantation followed by the creation of a continuous protein coating on the carbon surface, while the protein is selected from albumin, fibrinogen or tropoelastin, and the outer surface is modified by bombarding with high-energy nitrogen ions of 5-20 keV with a pulse duration of 10-40 microseconds, the dose of ions 5⋅10 ¹⁴ -10 ¹⁶ ions per cm ² ; modification of the inner surface is carried out using a system comprising a cone nozzle inside a vacuum reactor and an electrode located around the vessel prosthesis being processed, nitrogen gas is supplied to the reactor through the nozzle and the cavity of the polymer vessel prosthesis; the creation of a continuous protein coating on the carbon surfaces of the prosthesis is carried out by immersion in phosphate-saline buffer solution with protein. 2. The method according to p. 1, in which the full cycle of processing the outer surface by the method of ion-plasma implantation is performed sequentially 3 times, each time the prosthesis is rotated by 120 degrees. 3. The method according to p. 1, in which the ion-plasma implantation is carried out when creating a plasma using a high-frequency generator with a frequency of 13.75 MHz, and the implantation of nitrogen ions in the sample using a low-frequency generator. 4. The method according to p. 1, in which the ion-plasma implantation of nitrogen ions into the surface of the prosthesis with the creation of plasma is carried out using a low-frequency generator. 5. The method according to p. 3, in which ion-plasma implantation with nitrogen is carried out while maintaining a constant pressure of 2 mTorr and low-frequency generator mode parameters: pulse rise front 1 μs, frequency 50 Hz and average current 1.4-1.5 mA . 6. The method according to p. 4, in which ion-plasma implantation with nitrogen is carried out while maintaining a variable pressure at a level of 4-5 mTorr and low-frequency generator mode parameters: pulse rise front 0.1-1 μs, frequency 100 Hz and average current 5, 5-6 mA. 7. The method according to p. 1, in which the flow rate of nitrogen gas supplied through the nozzle is determined so that the pressure at the outlet of the reactor is 1-2 Torr, while the pressure in the reactor is maintained at a level of not more than 4 mTorr, per electrode provide high voltage pulses of 5-15 kV with a duration of 10-40 microseconds and a pulse repetition rate of 50-100 Hz. 8. The method according to p. 1, in which the concentration of the solution is 100 μg / ml. 9. The method according to p. 8, in which the exposure time of the prosthesis in solution is 12 hours.