A LUBRICIOUS, THERAPEUTIC AND ABRASION-RESISTANT COATING FOR DEVICES AND METHODS FOR PRODUCING AND USING THEREOF

Abstract

There is provided herein a method of coating a polyurethane surface of an insertable medical device, the method comprising obtaining an insertable medical device or a part thereof comprising a polyurethane surface; performing a direct thiolization of the polyurethane surface to produce thiolated polyurethane surface comprising free thiol groups, the direct thiolization comprises a direct reaction between a secondary amine of the polyurethane surface and ethylene sulphide (ES) to form a covalent bond between the amine and the free thiol group; and reacting the thiolated polyurethane surface with a therapeutic/antithrombogenic compound having a vinyl/methacrylate functional group through thiol-ene click reaction, to produce an insertable medical device coated with a therapeutic/antithrombogenic and abrasion (delamination)-resistant coating.

Claims

1.-25. (canceled)

26. A method of coating a polyurethane surface of an insertable medical device, the method comprising: obtaining an insertable medical device or a part thereof comprising a polyurethane surface; performing a direct thiolization of the polyurethane surface to produce thiolated polyurethane surface comprising free thiol groups, the direct thiolization comprises a direct reaction between a secondary amine of the polyurethane surface and ethylene sulphide (ES) to form a covalent bond between the amine and the free thiol group; reacting the thiolated polyurethane surface with a therapeutic/antithrombogenic compound having a vinyl/methacrylate functional group through thiol-ene click reaction, to produce an insertable medical device coated with a therapeutic/antithrombogenic and abrasion (delamination)-resistant coating.

27. The method of claim 26, wherein the direct thiolization of the polyurethane surface to produce thiolated polyurethane surface is devoid of a pre-treatment of the polyurethane surface.

28. The method of claim 26, wherein the direct thiolization of the polyurethane surface to produce thiolated polyurethane surface is devoid of plasma pre-treatment, chemical pre-treatment, flame pre-treatment, corona pre-treatment or any combination thereof, of the polyurethane surface.

29. The method of claim 26, wherein the therapeutic/antithrombogenic compound having a vinyl/methacrylate functional group comprises zwitterionic methacrylate.

30. The method of claim 26, wherein the zwitterionic methacrylate comprises sulfobetaine methacrylate, phosphorylcholine methacrylate or a combination thereof.

31. The method of claim 29, wherein the zwitterionic methacrylate comprises 2-methacryloyloxylethyl phosphorylcholine (MPC) and wherein the coated polyurethane surface is (PU-S-MPC).

32. The method of claim 26, wherein the therapeutic/antithrombogenic compound having a vinyl/methacrylate functional group comprises Linalool, Limonene, Citral or any combination thereof.

33. An insertable medical device having a polyurethane surface coated according to the method of claim 26.

34. A method for preparing a stock product for use as a coating material for coating a polyurethane surface of an insertable medical device, the method comprising: obtaining a thiolated polyethyleneimine (PEI-SH); and reacting the thiolated polyethyleneimine (PEI-SH) with a therapeutic/antithrombogenic compound having a vinyl/methacrylate functional group through thiol-ene click reaction to produce a stock product comprising polyethyleneimine-thiol-therapeutic/antithrombogenic compound conjugate having free primary and/or secondary amines capable of binding to an activated surface of the insertable medical device.

35. The method of claim 34, wherein the therapeutic/antithrombogenic compound having a vinyl/methacrylate functional groups comprises zwitterionic methacrylate.

36. The method of claim 35, wherein the zwitterionic methacrylate comprises sulfobetaine methacrylate, phosphorylcholine methacrylate or a combination thereof.

37. The method of claim 35, wherein the zwitterionic methacrylate comprises 2-methacryloyloxylethyl phosphorylcholine (MPC) and wherein the stock product comprises polyethyleneimine-thiol-MPC (PEI-S-MPC) conjugate.

38. The method of claim 34, wherein the therapeutic/antithrombogenic compound having a vinyl/methacrylate functional groups comprises Linalool, Limonene, Citral or any combination thereof.

39. The method of claim 34, wherein obtaining the thiolated-polyethyleneimine (PEI-SH) comprising reacting polyethyleneimine (PEI) with ethylene sulphide (ES), halogen-alkyi thiol, cysteine, bromopyridine thiol, bromobenzoxazole thiol, chloropyridine thiol, halobenzo thiazole thiol, chloropyrimidine thiol, halo-phenyl thiazole thiol or any combination thereof.

40. The method of claim 34, wherein the polyethyleneimine (PEI) and/or the thiolated polyethyleneimine (PEI-SH) comprises brunched polyethyleneimine (bPEI) and/or thiolated-brunched polyethyleneimine (bPEI-SH), respectively.

41. A method for preparing polyethyleneimine-thiol-2-methacryloyloxylethyl phosphorylcholine (PEI-S-MPC) for use as a stock product for coating a surface of an insertable medical device, the method comprising: obtaining a thiolated polyethyleneimine (PEI-SH); and reacting the thiolated polyethyleneimine (PEI-SH) with 2-methacryloyloxylethyl phosphorylcholine (MPC) through thiol-ene click reaction to produce brunched polyethyleneimine-thiol-2-methacryloyloxylethyl phosphorylcholine (PEI-S-MPC) having free primary and/or secondary amines capable of binding to an activated surface of the insertable medical device.

42. The method of claim 41, wherein obtaining the thiolated-polyethyleneimine (PEI-SH) comprising reacting polyethyleneimine (PEI) with ethylene sulphide (ES), halogen-alkyi thiol, cysteine, bromopyridine thiol, bromobenzoxazole thiol, chloropyridine thiol, halobenzo thiazole thiol, chloropyrimidine thiol, halo-phenyl thiazole thiol or any combination thereof.

43. A stock product for use in coating an activated polyurethane surface of an insertable medical device, the stock product prepared according to claim 34.

44. A method of coating a polyurethane surface of an insertable medical device, the method comprising: obtaining an insertable medical device or a part thereof comprising a functionalized polyurethane surface having free isocyanate groups; reacting the functionalized polyurethane surface with the stock product prepared according to the method of claim 34, the stock product comprising a conjugate of polyethyleneimine-thiol-therapeutic/antithrombogenic compound having free primary and/or secondary amines capable of binding to the free isocyanate groups of the polyurethane surface.

45. The method of claim 44, wherein the polyurethane surface is functionalized using diisocyanate substance to produce.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0099] Some embodiments of the disclosure are described herein with reference to the accompanying figures. The description, together with the figures, makes apparent to a person having ordinary skill in the art how some embodiments may be practiced. The figures are for the purpose of illustrative description and no attempt is made to show structural details of an embodiment in more detail than is necessary for a fundamental understanding of the disclosure. For the sake of clarity, some objects depicted in the figures are not to scale.

[0100] In the Figures:

[0101] FIG. 1 shows an example of a scheme for the preparation of PEI-S-MPC, in accordance with some embodiments;

[0102] FIG. 2 shows an ATR-FTIR spectroscopy of thiolated bPEI and untreated bPEI, according to some embodiments;

[0103] FIG. 3 shows a UV-VIS analysis of bPEI-SH using Ellman's reagent, according to some embodiments;

[0104] FIG. 4 schematically depicts a mechanism of Ellman's reagent for the detection of thiol groups, according to some embodiments;

[0105] FIG. 5 shows a calibration curve for the quantification of free thiol groups using cysteine, according to some embodiments;

[0106] FIG. 6 shows the IR spectroscopy of the end product bPEI-S-MPC (prepared according the scheme of FIG. 1), according to some embodiments;

[0107] FIG. 7 shows two vials, the vial on the right contains the end ‘stock product’ (bPEI-S-MPC), which is soluble in water, whereas the vial on the left contains bPEI-SH, which is insoluble in water, according to some embodiments;

[0108] FIG. 8 shows a reaction scheme between Urethane and isocyanate functional group, according to some embodiments;

[0109] FIG. 9 shows a reaction scheme between PU and TSC, according to some embodiments;

[0110] FIG. 10 shows an ATR-FTIR spectrum of PU-TSC compared to untreated PU surface and TSC reagent, according to some embodiments;

[0111] FIG. 11 shows a scheme of the peak areas that was analyzed for the optimization of the reaction of PU with isocyanate functional group, according to some embodiments;

[0112] FIG. 12 shows the influence of the duration (left graph) and the temperature (right graph) of the reaction on the quantity of conjugated molecules according to the integration ratio factor A1157/A1527, according to some embodiments;

[0113] FIG. 13 shows a scheme of an application of the ‘stock product’ (bPEI-S-MPC) on PU surface through functionalization of the PU surface, according to some embodiments;

[0114] FIG. 14 shows a scheme of a functionalization of PU surface using hexamethylene diisocyanate (HDI) producing PU-HDI, according to some embodiments;

[0115] FIG. 15 shows a scheme of a functionalization of PU surface using L-lysine diicosyanate (lysine-D) producing PU-lysine-D, according to some embodiments;

[0116] FIG. 16 shows an ATR-FTIR spectrum of PU-HDI (produced in a process according to FIG. 14), according to some embodiments;

[0117] FIG. 17 shows an ATR-FTIR spectrum of PU-lysine-D (produced in a process according to FIG. 15), according to some embodiments;

[0118] FIG. 18 shows a scheme of an application of bPEI on PU surface through functionalization with HDI, according to some embodiments;

[0119] FIG. 19 shows a scheme of the sample series that was made for the characterization of coated PU surfaces, according to some embodiments;

[0120] FIG. 20 shows blood-agar petri dishes containing the sample series shown in FIG. 19 and a control dish, that went through JIS Z2801:200 test for antimicrobial properties, according to some embodiments;

[0121] FIG. 21 shows COF of the sample series shown in FIG. 19, on PMMA in the presence of PBS, according to some embodiments;

[0122] FIG. 22 shows the hemolysis ratio of PU-HDI-bPEI-S-MPC, compared to an uncoated PU surface and a commercial hydrogel coat, which can be found in medical use today, according to some embodiments;

[0123] FIG. 23 shows a scheme of direct thiolation of PU surface, according to some embodiments;

[0124] FIG. 24 shows a UV-VIS absorbance curve of Ellman's reagent decomposition products after the exposure to thiolated PU surface (PU-SH), according to some embodiments; and

[0125] FIG. 25 shows fluorescent microscopy of fluorescein-O-methacrylate grafted PU-SH (A) compared to a reference (B), according to some embodiments.

DETAILED DESCRIPTION

[0126] The principles, uses and implementations of the teachings herein may be better understood with reference to the accompanying description and figures. Upon perusal of the description and figures present herein, one skilled in the art will be able to implement the teachings herein without undue effort or experimentation.

[0127] Materials and Methods

[0128] All chemicals were purchased and used without further purification. Polyethyleneimine (PEI branched, Mw 800 by LS), ethylene sulfide (ES, 98%), 2-methacryloyloxyethyl phosphorylcholine (MPC 97%, ≤100 ppm MEHQ as inhibitor), 2,2-dimethoxy-2-phenylacetophenone (DMPA 99%), Fluorescein-O-methacrylate (95%), hexamethylene diisocyanate (HDI≥99% by GC), dibutyltin dilaurate (DBTDL, 95%) were all purchased from Sigma-Aldrich, IL. 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB, 97%), N-acetyl-L-cysteine (C.sub.5H.sub.9NO.sub.3S, 98+%), L-lysine ethyl ester diisocyanate (97%) were all purchased from Alfa Aesar. Toluene AR-b, ethanol absolute (dehydrated) AR-b, methanol AR-b, diethylether AR were all purchased from Bio-Lab. Biomedical grade of PU was generously donated by Lubrizol.

[0129] Synthesis of the Lubricious Antimicrobial Coating Complex (Stock Product)

[0130] bPEI-SH was synthesized through ring opening of ES. 6 gr of bPEI were dissolved in a mixture of toluene; ethanol solution (9:1 ratio, respectively) in a 100 ml round flask. The solution was refluxed under nitrogen atmosphere for 15 min, then 400 μl of ES were added dropwise over 1 min. The reaction was refluxed for another 2 hr, following the removal of the solvent mixture by evaporation under reduced pressure. The thiolation of bPEI was analyzed using IR spectroscopy, UV-VIS spectroscopy and fluorescent microscopy.

[0131] bPEI-S-MPC was synthesized through thiol-ene click reaction. 800 mg of MPC and 20 mg of DMPA were dissolved in 4.5 ml of methanol and were added to 2 g of bPEI-SH. The mixture was radiated under UV light (20-watt, 365 nm, Analytik Jena US) for 40 min. The solvent was removed by rotary evaporator under reduced pressure. The product was analyzed using IR spectroscopy and elemental analysis.

[0132] IR Spectroscopy

[0133] Absorption spectrum was obtained using Fourier-transform infrared spectroscopy spectrometer (Bruker, Germany), with attenuated total reflection method (ATR-FTIR). Using OPUS software, 100 scan signals were provided for each sample and the average resolution of the measurement was adjusted to 2 cm.sup.−1.

[0134] UV-VIS Spectroscopy

[0135] DTNB, also called Ellman's reagent, can be used for the detection of free thiol groups using UV-VIS spectroscopy. DTNB reacts with a free sulfhydryl groups to yield a disulfide molecule and 2-nitro-5-thiobezoic acid (TNB). Elevated absorption in the range of 412 nm is associated with the presence of TNB, which indicates for the presence of thiol free groups in a tested sample.

[0136] The absorbance was detected using a UV-VIS spectrometer (UV-1650PC, Shimadzu Corporation, Japan). 4.6 mg/ml of bPEI-SH were dissolved in distilled water for the measurement. As a reference, 4.6 mg/ml of bPEI were dissolved in distilled water.

[0137] Ellman's reagent protocol enabled the quantification of thiols, based on molar absorptivity of a standard concentration of thiols using cysteine.

[0138] Preparation of PU Surfaces

[0139] The samples to be coated were prepared by solvent casting onto glass petri dishes. PU resins were dissolved in THF in a concentration of 2% w/t. Air plasma was applied on glass petri dishes (90 mm in diameter) for 5 min, following by casting of 15 ml of 2% w/t PU solution (in THF). After ambient evaporation of the solvent, the casted dishes were dried overnight at 55° C. and held under vacuum.

[0140] Coating Application on PU Surfaces

[0141] PU surfaces were functionalized using diisocyanate substances. 5% v/v of HDI and 0.25% v/v of DBTDL were added to 15 ml of toluene and the solution was added over a PU solvent casted petri dish. The reaction was conducted for 60 min at 70° C. under orbital spinning of 50 rpm.

[0142] The application of the coating complex onto functionalized surfaces was conducted in the same reaction procedure. 1 gr of bPEI/bPEI-SH/bPEI-S-MPC and 0.25% v/v of DBTDL were added to toluene and the solution was spread over a functionalized surface. The reaction was conducted for 60 min at 70° C. under orbital spinning of 50 rpm.

[0143] The coated surfaces were analyzed using elemental analysis. The coefficient of friction (COF) of coated PU was measured in PBS. Fibrinogen absorption assay and antimicrobial tests were performed on coated PU surfaces.

[0144] X-Ray Photoelectron Spectroscopy (XPS)

[0145] The X-ray Photoelectron Spectroscopy (XPS) measurements were performed on a Kratos Axis Ultra X-ray photoelectron spectrometer (Karatos Analytical Ltd., Manchester, UK). High resolution XPS spectra were acquired with monochromatic Al Ka X-ray radiation source (1,486.6 eV) with 90° takeoff angle (normal to the analyzer). The pressure in the chamber was 2.Math.10-9 Torr. The high-resolution XPS spectra were collected for with pass energy 20 eV and step 0.1 eV. Data analyses were performed using Casa XPS (Casa Software Ltd.) and Vision data processing program (Kratos Analytical Ltd.).

[0146] Elemental Analysis

[0147] Determination of atomic percent of the coating complex was performed using elemental analysis. C, N, H and O percentage was measured using the Thermo Flash 2000 CHN-O Elemental Analyzer. This system uses a simultaneous flash combustion method (950-1060° C.) for CHN and pyrolysis of oxygen to convert the sample elements to simple gases. The gases are detected as a function of their thermal conductivity. The determination of S, P percentage is done using the Anton Paar Microwave Induced Oxigen Combustion (MIC) for the decomposition of organic samples and by Ion chromatography analysis using a Dionex IC system.

[0148] Coefficient of Friction (COF) Measurements

[0149] The COF of coated surfaces had been measured in PBS. For this purpose, a bath was constructed from PMMA to fit the standard apparatus to perform a standard COF test according to ASTM 1894. The bath was filled with 30 ml of PBS and each sample was tested for 25 cycles. After 10 and 20 cycles, 1.5 ml of the test liquid were collected to further evaluation of particulates. After 25 cycles, the remained PBS had been collected to an empty vial. The tested surfaces were analyzed using scanning electron microscopy (SEM). The PBS was analyzed using particle size analyzer to identify and measure particles and was seen under tunneling electron microscopy (TEM) to check for amorphous particles.

[0150] To evaluate the influence of the coating on the COF values of the surfaces, a standard measurement had been conducted, according to some adjustments

[0151] Antimicrobial Test

[0152] The antimicrobial activity of the coating was evaluated using the JIS Z2801:2000 test. The tested samples (5 mm in diameter) were incubated with E. coli bacteria for 24 hours at 37° C. in a humid atmosphere. Then, the samples were sonicated to detach all bacteria on the surface. The sonicated liquid was cultured on blood-agar petri dishes, following by another overnight incubation at 37° C. in a humid atmosphere. The antimicrobial activity of each sample was determined by the number of colony forming units developed over the culture petri dishes.

[0153] Hemolysis Test

[0154] The degree of hemolysis was evaluated as follows: each sample (5 mm in diameter) was soaked in 160 μl of PBS at 37° C. for 30 min. 510 μl of fresh blood from healthy pigs (containing 6% v/v of 20 mg/ml of potassium oxalate) were added to each sample and the samples were incubated at 37° C. for another 60 min. then, the samples were centrifuged at 1350 rpm for 5 min. the absorbance of the supernatant solution was measured using a plate reader at a wavelength of 545 nm. The hemolytic ratio (HR) was calculated by the following equation: HR(%)=(A.sub.s−A.sub.nc)/(A.sub.pc−A.sub.nc), were A.sub.s is the obtained absorbance of the tested sample, A.sub.nc and A.sub.pc are the absorbance of the negative control (0.02% v/v of diluted blood in PBS) and positive control (0.02% v/v of diluted blood in DI water), respectively.

EXAMPLES

[0155] Synthesis of bPEI-SH

[0156] Reference is now made to FIG. 1, which shows an ATR-FTIR spectroscopy of thiolated bPEI and untreated bPEI, according to some embodiments.

[0157] Both peaks at 670 cm−1 and 2523 cm−1 are known for common frequencies of thiol stretch. This could be an indication for the presence of thiols in the bPEI. However, detecting thiols in IR spectroscopy can be misleading. Although thiols can be considered as analogs of the equivalent oxygenated compounds, C—S—H and C—S stretching vibrations give rise to weak absorptions in the IR spectrum. Thus, UV-VIS analysis was performed to support the IR findings.

[0158] Reference is now made to FIG. 3, which shows a UV-VIS analysis of bPEI-SH using Ellman's reagent, according to some embodiments and to FIG. 4, which schematically depicts a mechanism of Ellman's reagent for the detection of thiol groups, according to some embodiments.

[0159] Following a standard protocol for using the Ellman's reagent, thiol concertation in an unknown sample can be expressed by Equation 1, when c is the concentration of thiols in the sample, A is the absorbance at 412 nm, b is the size of the spectrophotometric cuvette in cm and E is the molar absorptivity at 412 nm.

[00001]$\mathrm{Equation}1.\mathrm{The}\mathrm{concentration}\mathrm{of}\mathrm{thiol}\mathrm{free}\mathrm{groups}$$C=\frac{A}{\mathrm{bE}}$

[0160] Reference is now made to FIG. 5, which shows a calibration curve for the quantification of free thiol groups using cysteine. The accuracy was found to be R.sup.2=0.9982 and the equation is y=1.5824x−0.0135

[0161] Thiol-ene Click for the Conjugation of MPC

[0162] Reference is now made to FIG. 6, which shows the IR spectroscopy of the end product bPEI-S-MPC (prepared according the scheme of FIG. 1), according to some embodiments. The peaks at 1708 cm.sup.−1, 1634 cm.sup.−1 and 951 cm.sup.−1 correspond to carbonyl functional group, alkene double bond and (N.sup.+(CH.sub.3).sub.3) group, respectively, which are present in MPC molecule. The peak at 1233 cm.sup.−1 correspond to the phosphonate group. All of these peaks could be found on the end product bPEI-S-MPC as well.

[0163] Table 1 shows the atomic percent distribution that was obtained from elemental analysis. After the thilation of bPEI, 3.27% of the detected atoms was found to be sulfur. Phosphorous was detected only in the end ‘stock product’ and its atomic percentage was 3.03%. Visually, there was no significant change between bPEI-SH and bPEI-S-MPC. However, the end ‘stock product’ (bPEI-S-MPC) is soluble in water, where bPEI-SH is insoluble in water, as could be seen in FIG. 7.

TABLE-US-00001 TABLE 1 Elemental analysis results of the ‘stock product’ (prepared according the scheme of FIG. 1): bPEI bPEI-SH bPEI-S-MPC C % 52.37 49.67 47.91 H % 10.49 10.64 9.19 N % 34.36 30.46 23.06 S % 0 3.27 2.17 O % 2.78 5.24 15.28 P % 0 0 3.03

[0164] As disclosed herein, in accordance with some embodiments, the stock product may be prepared as a preliminary step for the coating, thus significantly simplifies the coating application itself.

[0165] Functionalization of the Surface of Polyurethane

[0166] To observe a strong covalent bond between the ‘stock product’ (PEI-S-MPC, e.g., bPEI-S-MPC) and the surface of PU, a third party, diisocyanate molecule, was used. Urethane linkage, which can be found in the backbone of PU, consist of a secondary amine. The reaction between a secondary amine group and isocyanate functional group results in the formation of a substituted urea linkage, as shown in FIG. 8. This reaction occurs at 70° C., using toluene as the solvent and dibutyltin dilaurate (DBTDL) to catalyze the reaction.

[0167] Model Reaction with Toluenesulfonyl Isocyanate (TSC)

[0168] TSC molecule was used to model the reaction between isocyanate end group and the secondary amine that is found in urethane linkage. TSC consist of a sulfonyl group, which can facilitate the analysis of the product. FIG. 9 shows a scheme of a reaction between PU and TSC. The reaction was analyzed using ATR-FTIR and XPS.

[0169] FIG. 10 shows ATR-FTIR absorption of untreated PU, TSC and the treated surface PU-TSC, according to some embodiments. The peak at 1157 cm.sup.−1 stands for the absorption of the sulfonyl group. This peak could be found on the treated PU surface, indicating the presence of sulfonyl groups after the treatment. Additionally, the peak at 2222 cm.sup.−1, which represents the isocyanate group could not be detected on the treated PU surface. These two findings indicate for the binding of TSC on PU surfaces. Moreover, the results of XPS analysis, which are given in Table 2, confirms the presence of sulfur atoms on the treated PU surfaces.

TABLE-US-00002 TABLE 2 The atomic content on the surface of PU-TSC compared to PU: Atomic content [%] C N O S PU neat 73.72 1.58 22.08 0 PU-TSC 69.88 4.41 22.91 1.47

[0170] Optimization of the Reaction Conditions

[0171] Reference is now made to FIG. 11, which shows a scheme of the peak areas that was analyzed for the optimization of the reaction of PU with isocyanate functional group, according to some embodiments. The integration of desired peaks in ATR-FTIR spectrum was used as a quantitative method to optimize the conditions of the reaction. The peak area of the sulfonyl group at 1157 cm.sup.−1 (A1157) was divided by the peak area of a reference absorption peak in PU spectrum, 1527 cm−1 (A1527). The ration between the peaks was taken as the comparison factor. The parameters that were optimized were the duration and the temperature of the reaction.

[0172] Reference is now made to FIG. 12, which shows the influence of the duration (left graph) and the temperature (right graph) of the reaction on the quantity of conjugated molecules according to the integration ratio factor A1157/A1527, according to some embodiments. It was found that the ideal treated PU surface is observed in a temperature of 70° C. for 60 min.

[0173] Diisocyanates as Efficient Mediators

[0174] Diisocyanate may be used, in accordance with some embodiments, as coating mediator enables the simplification of the coating application on PU surfaces. As the ‘stock product’, PEI-S-MPC/bPEI-S-MPC, consists of free amine end-groups, it can bind to a free isocyanate group that can be found on PU surfaces using the same reaction as the functionalization step. The scheme of the reaction is shown in FIG. 13, which shows a scheme of the application of the ‘stock product’ (bPEI-S-MPC) on PU surface through functionalization of the PU surface, according to some embodiments. As seen in FIG. 13, the PU surface is functionalized using diisocyanate substance. One isocyanate group is covalently attached to the surface while the other isocyanate group is free and available for further reaction. Then, the covalent attachment of the ‘stock product’ to the isocyanate free groups of the treated PU takes place via free primary and/or secondary amines which present in the stock product, resulting in a urea bond.

[0175] Hexamethylene diisocyanate (HDI) and L-lysine diicosyanate (lysine-D) were substituted on PU surfaces. Reference is now made to FIG. 14, which shows a scheme of a functionalization of PU surface using hexamethylene diisocyanate (HDI), according to some embodiments and to FIG. 15, which shows a scheme of a functionalization of PU surface using L-lysine diicosyanate (lysine-D), according to some embodiments. Lysine is an amino acid that is found in human proteins. The use of lysine-D can facilitate the FDA approve of the coating. The treated PU surfaces with both HDI and lysine-D were analyzed using ATR-FTIR analysis. FIG. 16 shows an ATR-FTIR spectrum of PU-HDI (produced in a process according to FIG. 14), according to some embodiments, and FIG. 17 shows an ATR-FTIR spectrum of PU-lysine-D (produced in a process according to FIG. 15), according to some embodiments.

[0176] The Application of the Coating Complex on PU Films

[0177] A proof of concept for the application of the coating was made through the conjugation of bPEI on functionalized PU surface (PU-HDI), to produce PU-HDI-bPEI as shown in FIG. 18, according to some embodiments.

[0178] XPS analysis was performed for PU surface, which was coated with bPEI (PU-HDI-bPEI, produced according to FIG. 18) and was compared to untreated PU surface and activated PU surface (PU-HDI). The results are listed in Table 3 below. First, it could be seen that neat PU surface consists of 24 fold Oxygen atoms compared to Nitrogen. O/N ratio decreased dramatically after surface treatment, means that the neat PU surface composition was shielded with a different layer. N/C ratio increased after the addition of isocyanate groups and the conjugation of bPEI. Nitrogen atoms content increased in 4 fold on PU-HDI-bPEI compared to a neat PU surface. This fact confirms the presence of bPEI on the surface, since bPEI is reach in Nitrogen atoms.

TABLE-US-00003 TABLE 3 The atomic content of PU-HDI-bPEI as detected using XPS analysis Atomic content [%] C N O O/N N/C PU neat 71.49 2.05 50.17 24.47 0.03 PU-HDI 69.61 12.07 15.64 1.3 0.17 PU-HDI- 71.29 71.29 18.28 2.1 0.12 bPEI

[0179] A series of samples were made to characterize the PU coating that have been developed. FIG. 19 shows a scheme of this sample series, according to some embodiments. All of the samples were coated using HDI as the surface diisocyanate activator. As a control, a sample of neat PU surface was exposed to the procedure conditions without the reactants, i.e. the solvent, temperature, initiator and duration of the procedure.

[0180] Antimicrobial Test

[0181] Reference is now made to FIG. 20, which shows blood-agar petri dishes containing the sample series shown in FIG. 19, which went through JIS Z2801:200 test for antimicrobial properties. As a control, a sample of neat PU surface was exposed to the procedure conditions without the reactants, i.e. The solvent, temperature, initiator and duration of the procedure.

[0182] It can be seen that PU-HDI-bPEI-SH and PU-HDI-bPEI-S-MPC show good antimicrobial results—colony-forming unit (CFU)—0, compared to the control (CFU>200), PU-HDI (CFU>200) and compared to PU-HDI-bPEI (CFU˜50).

[0183] Coefficient of Friction (COF)

[0184] Reference is now made to FIG. 21, which shows COF of the sample series shown in FIG. 19, on PMMA in the presence of PBS. It can be seen that PU-HDI-bPEI-S-MPC shows improved lubrication properties compared to the other samples.

[0185] Hemolysis Ratio

[0186] Reference is now made to FIG. 22, which shows hemolysis ratio of PU-HDI-bPEI-S-MPC compared to a hydrogel coat which can be found in medical use today. As a control, hemolysis ratio was calculated for neat PU surface, according to some embodiments.

[0187] It can be seen that PU-HDI-bPEI-S-MPC shows improved (lower) hemolysis ratio compared to the other samples.

[0188] Direct Thiolation of Polyurethane Using Ethylene Sulfide (ES)

[0189] As disclosed hereinabove, there is provided herein, in accordance with additional or alternative embodiments, a method for the conjugation of a therapeutic/antithrombogenic compound (such as MPC) onto PU surfaces. FIG. 23, shows a scheme of direct thiolation of PU surface, utilizing ES, according to some embodiments. This approach may be used for direct conjugation via thiol-based reactions, such as thiol-ene with allyl or methacrylate bearing molecules (such as MPC), isocyanate bearing molecules, epoxides and the like.

[0190] Experimental

[0191] PU surface was soaked in a solvent mixture of toluene and ethanol (9:1, respectively). After 10 min of reflux under nitrogen atmosphere, 500 μl of ES were added dropwise over 90 sec. the reaction was refluxed for another 1.5 hours following three washing steps in excess of toluene for 15 min to remove all unreacted ES molecules. The modified PU surface was analyzed using UV-VIS spectrophotometer, elemental analysis and immunofluorescence. Similar experiments are performed using other toluene and ethanol ratios, such as 8:2, respectively and in toluene 100%, with addition of 100 μl-2 ml of ES, e.g., dropwise over 10-120 sec. The reaction is refluxed for 10 min-2 hours.

[0192] UV-VIS Analysis

[0193] Ellman's reagent was used to detect free thiol groups on treated (thiolated) PU surface. FIG. 24 shows the UV-VIS absorbance curve of Ellman's reagent decomposition products after the exposure to thiolated PU surface (PU-SH), according to some embodiments. Following the standard protocol for using the Ellman's reagent, the absorbance at a wavelength of 412 nm approve the presence of free thiol groups on the PU-SH surface.

[0194] Elemental Analysis

[0195] Table 2 shows the atomic percent distribution that was obtained from elemental analysis. After the thiolation of PU surface, 0.73% of the detected atoms was found to be sulfur. Visually, there was no significant change between PU and PU-SH.

TABLE-US-00004 TABLE 2 The atomic content of PU and PU-SH as was observed by elemental analysis: Atomic content [%] PU Neat PUSH C 67.16 65.64 H 8.41 8.43 N 4.19 5.28 S 0.1 0.73 O 19.33 19.14

[0196] Fluorescent Probe

[0197] Fluorescein-O-methacrylate was used as a fluorescent probe for reactive thiol groups. Thiol-ene click reaction was performed to conjugate reactive thiols with methacrylate groups. The reaction occurred in methanol using DMPA as the initiator and the observed product is called PU-S-Fluorescein. As a reference, the same procedure took place without the addition of DMPA. FIG. 25 shows fluorescent microscopy of fluorescein-O-methacrylate grafted PU-SH (A) compared to a reference (B), according to some embodiments. A significantly strong fluorescent signal was obtained from the fluorescein-O-methacrylate grafted PU-SH, compared to the reference. the fluorescent signal assures the presence of fluorescein group on the surface of the fluorescein-O-methacrylate grafted PU-SH sample. The reference sample was not fluorescent. These finings eliminate the possibility of physical absorption of the fluorescein-O-methacrylate, proving the covalent bond of fluorescein-O-methacrylate to the thiolated PU surface.

[0198] Ex-Vivo Thrombogenic Protocol

[0199] Samples (Nitinol discs, 5 mm in diameter, coated or uncoated) are being placed on the bottom of a 50 ml PTFE flask. 8 ml of fresh blood from healthy pigs is casted onto the samples and the flasks are gently shaken at 50 rpm for 3 hours.

[0200] Fixation of the samples occurs in 4% formaldehyde in PBS, following by dehydration of the samples using washing with elevated concentration of ethanol and the surfaces are being examined using scanning electron microscopy (SEM).

[0201] In Vivo Thrombogenic Evaluation

[0202] Through the right femoral artery of healthy rabbits, the samples (Nitinol wires, coated or uncoated) are administered to abdominal aorta for 3 hours.

[0203] The segment of abdominal aorta is then removed from the animal, its content is emptied into a petri dish containing 50 ml of a 0.9% saline solution, and the contents of the dish is photographed and examined for the presence of clots on the device.

[0204] The morbidity and mortality of the animals is examined daily. The thrombogenic potential is evaluated using SEM as follows:

[0205] 0—no clot

[0206] 1—Few macroscopic standards of fibrin

[0207] 2—Several small thrombi

[0208] 3—Two or more large thrombi

[0209] 4—A single thrombus forming a cast of the isolated segment.

[0210] In the description and claims of the application, the words “include” and “have”, and forms thereof, are not limited to members in a list with which the words may be associated.

[0211] As used herein, the term “about” may be used to specify a value of a quantity or parameter (e.g. the length of an element) to within a continuous range of values in the neighborhood of (and including) a given (stated) value. According to some embodiments, “about” may specify the value of a parameter to be between 80% and 120% of the given value. According to some embodiments, “about” may specify the value of a parameter to be between 90% and 110% of the given value. According to some embodiments, “about” may specify the value of a parameter to be between 95% and 105% of the given value.

[0212] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination or as suitable in any other described embodiment of the disclosure. No feature described in the context of an embodiment is to be considered an essential feature of that embodiment, unless explicitly specified as such.

[0213] Although steps of methods according to some embodiments may be described in a specific sequence, methods of the disclosure may include some or all of the described steps carried out in a different order. A method of the disclosure may include a few of the steps described or all of the steps described. No particular step in a disclosed method is to be considered an essential step of that method, unless explicitly specified as such.

[0214] Although the disclosure is described in conjunction with specific embodiments thereof, it is evident that numerous alternatives, modifications and variations that are apparent to those skilled in the art may exist. Accordingly, the disclosure embraces all such alternatives, modifications and variations that fall within the scope of the appended claims. It is to be understood that the disclosure is not necessarily limited in its application to the details of construction and the arrangement of the components and/or methods set forth herein. Other embodiments may be practiced, and an embodiment may be carried out in various ways.

[0215] The phraseology and terminology employed herein are for descriptive purpose and should not be regarded as limiting. Citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the disclosure. Section headings are used herein to ease understanding of the specification and should not be construed as necessarily limiting.

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