COATING FOR A DEVICE

Abstract

The present invention relates to a coating for a device, wherein the coating comprises a polymeric film, wherein the polymeric film comprises a polymerisation product formed from a polymerisation solution comprising dopamine, or a salt thereof, and at least one amino acid, or a salt thereof; and a metallic layer formed on the polymeric film.

Claims

1. A coating for a device, wherein the coating comprises a polymeric film, wherein the polymeric film comprises a polymerisation product formed from a polymerisation solution comprising dopamine, or a salt thereof, and at least one amino acid, or a salt thereof; and a metallic layer formed on the polymeric film.

2. A coated device comprising a device, and the coating according to claim 1 on at least a part of a surface of the device.

3. A method of forming a coating on a device, wherein the method comprises: a. exposing at least a part of a surface of the device to a polymerisation solution comprising dopamine, or a salt thereof, and at least one amino acid, or a salt thereof; b. polymerising the polymerisation solution so as to form a polymeric film on the at least a part of a surface of the device; and c. exposing the polymeric film to a solution comprising metallic ions so as to form a metallic layer on the polymeric film.

4. The coating according to claim 1, the coated device according to claim 2, or the method according to claim 3, wherein the at least one amino acid comprises at least one amino acid selected from the list of lysine, histidine, glycine, serine, arginine, leucine, asparagine, glutamic acid, alanine, tyrosine and proline.

5. The coating according to claim 1 or claim 4, the coated device according to claim 2 or claim 4, or the method of claim 3 or claim 4, wherein the at least one amino acid comprises at least one of lysine and glycine.

6. The coating according to any one of claim 1, 4 or 5, the coated device according to any one of claim 2, 4 or 5, or the method of any one of claims 3-5, wherein the at least one amino acid comprises lysine.

7. The coating according to any one of claim 1 or 4-6, the coated device according to any one of claim 2 or 4-6, or the method of any one of claims 3-6, wherein the metallic layer is continuous.

8. The coating according to any one of claim 1 or 4-7, the coated device according to any one of claim 2 or 4-7, or the method of any one of claims 3-7, wherein the metallic layer is present in an amount of 0.2 mg/cm.sup.2 or greater.

9. The coating according to any one of claim 1 or 4-8, the coated device according to any one of claim 2 or 4-8, or the method of any one of claims 3-8, wherein the metallic layer comprises silver.

10. The coating according to any one of claim 1 or 4-9, the coated device according to any one of claim 2 or 4-9, or the method of any one of claims 3-9, wherein the metallic layer has a surface roughness, Ra, of greater than or equal to 20 nm and/or a surface roughness, Rq, of greater than or equal to 25 nm.

11. The coating according to any one of claim 1 or 4-10, the coated device according to any one of claim 2 or 4-10, or the method of any one of claims 3-10, wherein the metallic layer has a water contact angle of greater than or equal to 100°.

12. The coating according to any one of claim 1 or 4-11, the coated device according to any one of claim 2 or 4-11, or the method according to any one of claims 3-11, wherein the pH of the polymerisation solution is between 7 and 12.

13. The coated device according to any one of claim 2 or 4-12, or the method of any one of claims 3-12, wherein the at least part of the surface of the device is formed from a polymer.

14. The coated device according to claim 13, or the method according to claim 13, wherein the at least part of the surface of the device is formed from a silicone polymer or polyurethane.

15. A coated device obtainable by the method any one of claims 3-14.

Description

[0063] FIG. 1. An embodiment of the method of forming a coating on a device of the present invention.

[0064] FIG. 2. (A) Silver quantification normalized by surface on silicone samples as a function of lysine concentration in the polymerisation solution, and (B) Water contact angle of silver surface on silicone samples as function of lysine concentration in the polymerisation solution. Error bars indicate standard deviation (SD).

[0065] FIG. 3. FE-SEM microscopy images and confocal 3D reconstruction of surface roughness of silicone coated with metallic silver surface immobilized on polydopamine (PDA)/Lysine coatings for different lysine concentrations a) PDA only b) lysine 0.2 mg/mL c) lysine 3 mg/mL d) lysine 10 mg/mL.

[0066] FIG. 4. Roughness values (Ra and Rq) of silver coated samples with different lysine concentrations in polymerisation solution.

[0067] FIG. 5. Quantification of adhered bacteria on polydimethylsiloxane (PDMS) samples coated with different lysine concentrations. Adhesion test were performed with Gram-positive bacteria Staphylococcus aureus (MRSA) (A) and Gram-negative bacteria Pseudomonas aeruginosa (PAO1) (B). All samples were compared with PDMS uncoated as a reference of bacterial colonization. Error bars indicate SD, * indicates “significant” with P<0.005, ** indicates “very significant” with P<0.001 and n.s. indicates “not significant”.

[0068] FIG. 6. End point value of optical density after 72 hours of bacterial growth curves. The bacteria (PAO1 or MRSA) were exposed to different samples in order to evaluate possible antibacterial effect. The samples evaluated were PDMS as negative control and metallic layers of the invention with different lysine concentrations. Error bars indicate SD.

[0069] FIG. 7. Roughness values (Ra and Rq) of silver coated samples for different mixtures of glycine/lysine concentrations in polymerization solution.

[0070] FIG. 8. Quantification of adhered bacteria on PDMS samples coated with coatings of the invention wherein the polymeric film is formed from a polymeric solution having different glycine concentrations. Adhesion test were performed with Gram-positive bacteria Staphylococcus aureus (MRSA) (A) and Gram-negative bacteria Pseudomonas aeruginosa (PAO1) (B). All samples were compared with PDMS uncoated as a reference of bacterial colonization. Error bars indicate SD, * indicates “significant” with P<0.005, ** indicates “very significant” with P<0.001 and n.s. indicates “not significant”.

[0071] FIG. 9. Bacteria adhesion quantification on a urinary Foley catheter after 15 days in vivo. The Foley catheters studied were a Degania© regular 2 ways Foley catheter as a control, the same catheter with an inner Bacteriophobic metallic layer (Tractivus) and a Bactiguard© Foley catheter from BARD.

[0072] FIG. 10. Bacteria quantification (CFU) of tracheal stent in vivo performed on a mini pig. Bacteria was quantified by bronchial washes after each period of 15 days and on the surface of the stent at the end of the study.

[0073] FIG. 11. Hemolysis test to evaluate the behavior of red blood cells in contact with the metallic layers of the invention.

[0074] FIG. 12. The water contact angle (WCA) of metallic layers of the invention comprising glycine.

[0075] FIG. 13. Confocal microscopy images of metallic silver layer on a silicone substrate without and with thickness measurements (A and B, respectively). Thickness measurements revealed a metallic layer with a thickness of near 1 μm.

[0076] FIG. 14. Representation of different levels of wettability of a droplet of water on a surface depending on the contact angle. Low wettability: Poor interaction substrate—water (θ>90°) indicating hydrophobicity, standard wettability (θ<90°) and completely wet (θ˜0°).

EXAMPLE 1—PDMS SUBSTRATE PREPARATION

[0077] A polydimethylsiloxane (PDMS) substrate was prepared by mixing the two components of a Sylgard™ 184 Silicone Elastomer kit (ref 2085925) in a 10:1 (silicone elastomer:curing agent) proportion and then spreading the mixture with a paint applicator to obtain a 500 μm thick film. The film was incubated for 10 min at 150° C. and, afterwards, it was cut into circles of 10 mm diameter. The substrate circles were washed and stored in aqueous solution of 70% v/v ethanol.

EXAMPLE 2—PDA COATING

[0078] A PDA solution was prepared by adding 0.121 g of dopamine hydrochloride (ref H852, Sigma Aldrich®) and the corresponding amount of Lysine and/or Glysine into 100 mL of 50 mM Tris buffer solution (ref Sigma Aldrich®). The pH of the solution was adjusted to basic (pH>8, preferable 10) to allow optimized self-polymerization of PDA. A PDMS film from example 1 was immersed in the dopamine solution for 6 hours at room temperature. Freshly coated membranes were rinsed with MilliQ to eliminate the excess of PDA.

EXAMPLE 3—SILVER COATING

[0079] PDA-coated samples from example 2 were immersed into a Tollens' reagent to perform the metallic coating. The Tollens' reagent was prepared by adding 1.70 g of silver nitrate (ref, Sigma Aldrich®) to 100 mL of MilliQ water. Then, a sufficient amount of 15% (v/v) aqueous ammonia solution was added under stirring to precipitate silver oxides and re-dissolve the formed silver precipitates. PDMS films were immersed on the Tollens' solution for 1.5 hours at 80° C. temperature. Freshly coated membranes were rinsed with MilliQ to remove excess PDA.

EXAMPLE 4—SURFACE ROUGHNESS AND HYDROPHOBICITY STUDIES

[0080] The inventors found that the presence of at least one amino acid in the polymerisation solution during the oxidative polymerisation of dopamine increases the silver reduction process and therefore allows more silver to be deposited on the polymeric film (see FIG. 2A). This higher amount of silver makes the micro-nano structure of the metallic layer more pronounced and adjusts the hydrophobic nature of the metallic layer (see FIG. 2B). Both the amount of silver deposited and the hydrophobicity increase with the concentration of lysine in the polymerisation solution.

[0081] Without being confined to theory, it is believed that this increase in hydrophobicity is caused by the morphological changes to the micro-nano roughness of the surface of the metallic layer. FIG. 3 shows FE-SEM microscopy images and confocal 3D reconstructions of the surface roughness of silicone coated with metallic layers of the invention with varying concentrations of lysine in the polymerisation solution.

[0082] When no lysine is added to polymerization media, the sample had a flat surface with a roughness of Ra=20 nm, which is considered a low value for roughness on the nano-scale. When the lysine concentration was increased to 0.2 mg/mL the formation of few sharp structures on the surface of the sample with a height of 5 μm was observed (FIG. 3B). Higher concentration values of lysine resulted in the formation of new structures, revealing a squared-shape pattern on the silver coating (FIGS. 3C and 3D). This pattern built has two regions of roughness: an initial nano-roughness observed for zero and near-zero amino acid concentrations and a micro-roughness observed due to the formation of the sharp structures at higher amino acid concentrations. Values of the arithmetical and quadratic mean roughness (Ra and Rq) against the concentration of lysine are presented on FIGS. 4A and 4B respectively. The values Ra and Rq confirm the results of the FESEM and confocal images (FIG. 3). i.e. increasing the amino acid concentration in the polymerisation solution increases the level of roughness of the obtained metallic layer. The presence of two orders of roughness is thought to play a critical role in increasing the hydrophobicity and bacteria resistant properties of the substrate.

[0083] Metallic layers of the invention were also prepared from a polymerisation solution comprising glycine and from a polymerisation solution comprising glycine and lysine. The water contact angle of the surface of the resulting metallic layers is are provided in FIG. 12.

EXAMPLE 5—FESEM AND CONFOCAL MICROSCOPY

[0084] Silver coated PDMS films of example 3 were dried under a compressed air stream and the surface morphology of the samples was studied with a field emission scanning electron microscope (Zeiss Merlin, FESEM). The surface roughness was evaluated by confocal microscopy and interferometry (Leica DCM 3D 3.3.2). Confocal images of a sample section were used to measure the metallic coating thickness. To do this, thin layers of few millimeters (preferably 3 mm) were cut from the main sample using a scalpel to obtain coupons. The coupons were placed on a sample holder support using double side adhesive tape and evaluated using confocal microscopy as shown in FIG. 13. Then, an image processing software (LeicaMap 6.2) was used to obtain different measurements of the film thickness, obtaining an average near 1 μm.

[0085] From the lengths of the studied surface (mm) values, and the depth of the surface (μm) values, the arithmetic surface roughness (Ra) and the Quadratic surface roughness (Rq) could be determined. Ra is determined by the following equation:

[00001]$\mathrm{Ra}=\frac{1}{lb}\underset{0}{\overset{lb}{.Math.}}.Math.Z\left(x\right).Math.$

[0086] wherein x is the length of the studied region, Z(x) is the depth of the studied region and lb is the number of measurements performed. The results are expressed in the length units of the Z axis.

[0087] Rq is determined by the following equation:

[00002]$\mathrm{Rq}=\sqrt{\frac{1}{lb}\underset{0}{\overset{lb}{.Math.}}{Z}^2\left(x\right)}$

[0088] wherein x is the length of the studied region, Z.sup.2(x) is the square of the depth of the studied region and lb is the number of measurements performed. The results are expressed in the length unit of the Z-axis.

[0089] Ra and Rq values for the samples of the present invention were determined with an lb value of 15 and an x value of about 15 μm.

EXAMPLE 6—INDUCTIVELY COUPLED PLASMA

[0090] Inductively coupled plasma mass spectrometry analysis (ICP) was used to quantify the amount of metallic silver present in the coatings of the present invention. Silver coated PDMS films of the invention were immersed in 5 mL of MilliQ water for at least 1 day. After immersion, a 1 mL sample of the water was taken and stored at 4° C. until it is run on CP-OES Perkin Elmer Avio 500 to determine the amount of silver in it. From the amount of silver in the 1 mL sample, the total amount of silver in the silver coated PDMS film can be determined. ICP is a type of mass spectrometry that uses an inductively coupled plasma to ionize the sample. It atomizes the sample and creates atomic and small polyatomic ions, in this case silver ions, which are then detected

EXAMPLE 7—BACTERIAL ADHESION STUDY

[0091] Metallic layers of the invention were tested in a bacterial adhesion study with both gram-positive bacteria (Staphylococcus aureus—MRSA) and gram-negative bacteria (Pseudomonas aeruginosa—PAO1). In both of these studies, silicone based samples of polydimethylsiloxane (PDMS) were used as negative controls.

[0092] In relation to gram-positive bacteria, FIGS. 5A and 8A demonstrate that the presence of an amino acid in the polymerisation solution results in a reduction in bacterial adhesion of up to two orders of magnitude. In relation to gram-negative bacteria, FIGS. 5B and 8B demonstrate that the presence of an amino acid in the polymerisation solution results in a reduction in bacterial adhesion. Further, FIG. 5B demonstrates that higher concentrations of amino acid in the polymerisation solution can result in a more bacteriophobic metallic layer.

EXAMPLE 8—ANTIBACTERIAL EFFECT STUDIES

[0093] The metallic layers of the invention were tested in a bacterial growth assay to confirm that the reduction in CFU shown in the bacterial adhesion study can be attributed to a greater bacteriophobic effect. FIG. 6 shows the optical density values of bacteria after 72 hours growth of a bacteria inoculum exposed to samples of metallic layers of the invention.

[0094] After 72 hours, there were no observed differences in the optical density of either gram-positive bacteria (MRSA) or gram-negative bacteria (PAO1) for any of the tested concentrations of amino acid in the polymerisation solution. Nor was there a difference in optical density between metallic layers of the invention relative to the negative control (PDMS). This confirmed that the reduction in CFU shown in the bacteria adhesion study can be attributed to a more bacteriophobic coating resisting colonisation of bacteria.

EXAMPLE 9—URINARY CATHETER WITH BACTERIOPHOBIC COATING

[0095] As application proof of concept of the invention, a regular Foley urinary catheter, purchased from Degania Medical©, was coated with Bacteriophobic metallic layer in the inside. The bacteriophobic behaviour of the coated catheter was evaluated during an in vivo test performed in a regular pig as animal model. The coated catheter (Tractivus), the regular catheter (Control) and an Antibacterial catheter (BARD) were implanted for 15 days in groups of 6 pigs to observe the amount of bacteria attached in the device at endpoint. The results shown in FIG. 9 reveals that bacterial adhesion was reduced by one order of magnitude during the test, even when compared to the antibacterial catheter.

EXAMPLE 10—TRACHEAL STENT WITH BACTERIOPHOBIC COATING

[0096] To evaluate the effectiveness of the invention, the bacteriophobic metallic layer of the invention was applied on a silicone tracheal stent in order to quantify biofilm formation during an in vivo test. The in vivo test was performed using mini pig as the animal model, where a tracheal stent was implanted to a mini pig trachea for 30 days. At day 15 and at the endpoint (30 days), a bronchial wash of the stent was performed using a flexible bronchoscope to collect the fluids from bronchial wash. FIG. 10 shows the bacteria quantification of the bronchial washes and the bacteria immobilized on the surface of the explanted device after 30 days. One order of magnitude less of bacteria and no biofilm was observed on the surface of the tracheal stents with the bacteriophobic metallic layer of the invention.

EXAMPLE 11—CENTRAL VENOUS CATHETER

[0097] To evaluate suitability of the invention for implementation in a central venous catheter (CVC), specifically if the metallic coating presents and effect on red blood cells, haemolysis tests were carried out to confirm that the invention does not cause haemolysis when in contact with red blood cells (FIG. 11). Haemolysis tests were performed by introducing blood from a healthy donor to a CVC coated with the metallic coating of the invention for a period of 30 minutes and 24 hours. The results demonstrate that the haemolysis levels present in red blood cells are below 2%. These low haemolysis values indicate that the invention can be used to avoid bacterial infection in devices that have to be implanted in the circulatory system.

[0098] The following list of embodiments forms part of the description [0099] 1. A coating for a device, wherein the coating comprises [0100] a polymeric film, wherein the polymeric film comprises a polymerisation product formed from a polymerisation solution comprising dopamine, or a salt thereof, and at least one amino acid, or a salt thereof; and [0101] a metallic layer formed on the polymeric film. [0102] 2. A coated device comprising [0103] a device, and [0104] the coating according to embodiment 1 on at least a part of a surface of the device. [0105] 3. A method of forming a coating on a device, wherein the method comprises: [0106] a. exposing at least a part of a surface of the device to a polymerisation solution comprising dopamine, or a salt thereof, and at least one amino acid, or a salt thereof; [0107] b. polymerising the polymerisation solution so as to form a polymeric film on the at least a part of a surface of the device; and [0108] c. exposing the polymeric film to a solution comprising metallic ions so as to form a metallic layer on the polymeric film. [0109] 4. The coating according to embodiment 1, the coated device according to embodiment 2, or the method according to embodiment 3, wherein the at least one amino acid comprises at least one amino acid selected from the list of lysine, histidine, glycine, serine, arginine, leucine, asparagine, glutamic acid, alanine, tyrosine and proline. [0110] 5. The coating according to embodiment 1 or embodiment 4, the coated device according to embodiment 2 or embodiment 4, or the method of embodiment 3 or embodiment 4, wherein the at least one amino acid comprises at least one of lysine and glycine. [0111] 6. The coating according to any one of embodiments 1, 4 or 5, the coated device according to any one of embodiments 2, 4 or 5, or the method of any one of embodiments 3-5, wherein the at least one amino acid comprises lysine. [0112] 7. The coating according to any one of embodiments 1 or 4-6, the coated device according to any one of embodiments 2 or 4-6, or the method of any one of embodiments 3-6, wherein the coating further comprises a cover layer, wherein the cover layer is formed on the metallic layer. [0113] 8. The coating according to embodiment 7, the coated device according to embodiment 7, or the method according to embodiment 7, wherein the cover layer comprises a polymer selected from polyvinyl alcohol, polyurethane, polymers from the acrylates family, or a silicone polymer. [0114] 9. The coating according to embodiment 7 or embodiment 8, the coated device according to embodiment 7 or embodiment 8, or the method according to embodiment 7 or embodiment 8, wherein the cover layer is water soluble. [0115] 10. The coating according to any one of embodiments 1 or 4-9, the coated device according to any one of embodiments 2 or 4-9, or the method of any one of embodiments 3-9, wherein the polymeric film has a thickness of less than or equal to 1000 nm. [0116] 11. The coating according to any one of embodiments 1 or 4-10, the coated device according to any one of embodiments 2 or 4-10, or the method of any one of embodiments 3-10, wherein the metallic layer is continuous. [0117] 12. The coating according to any one of embodiments 1 or 4-11, the coated device according to any one of embodiments 2 or 4-11, or the method of any one of embodiments 3-11, wherein the metallic layer is present in an amount of 0.2 mg/cm.sup.2 or greater. [0118] 13. The coating according to any one of embodiments 1 or 4-12, the coated device according to any one of embodiments 2 or 4-12, or the method of any one of embodiments 3-12, wherein the metallic layer comprises silver. [0119] 14. The coating according to any one of embodiments 1 or 4-13, the coated device according to any one of embodiments 2 or 4-13, or the method of any one of embodiments 3-13, wherein the metallic layer has a surface roughness, Ra, of greater than or equal to 20 nm and/or a surface roughness, Rq, of greater than or equal to 25 nm. [0120] 15. The coating according to any one of embodiments 1 or 4-14, the coated device according to any one of embodiments 2 or 4-14, or the method of any one of embodiments 3-14, wherein the metallic layer has a water contact angle of greater than or equal to 100°. [0121] 16. The coating according to any one of embodiments 1 or 4-15, the coated device according to any one of embodiments 2 or 4-15, or the method of any one of embodiments 3-15, wherein the metallic layer has a surface roughness, Ra, of greater than or equal to 50 nm. [0122] 17. The coating according to any one of embodiments 1 or 4-16, the coated device according to any one of embodiments 2 or 4-16, or the method of any one of embodiments 3-16, wherein the metallic layer has a surface roughness, Rq, of greater than or equal to 50 nm. [0123] 18. The coating according to any one of embodiments 1 or 4-17, the coated device according to any one of embodiments 2 or 4-17, or the method of any one of embodiments 3-17, wherein the metallic layer has a surface roughness, Ra, of greater than or equal to 100 nm. [0124] 19. The coating according to any one of embodiments 1 or 4-18, the coated device according to any one of embodiments 2 or 4-18, or the method of any one of embodiments 3-18, wherein the metallic layer has a surface roughness, Rq, of greater than or equal to 100 nm. [0125] 20. The coating according to any one of embodiments 1 or 4-19, the coated device according to any one of embodiments 2 or 4-19, or the method according to any one of embodiments 3-19, wherein the pH of the polymerisation solution is between 7 and 12. [0126] 21. The coating according to any one of embodiments 1 or 4-20, the coated device according to any one of embodiments 2 or 4-20, or the method according to any one of embodiments 3-20, wherein the concentration of the at least one amino acid or a salt thereof in the polymerisation solution is greater than or equal to 0.0001 mg/mL and less than or equal to 10 mg/mL, or wherein the concentration of the at least one amino acid or a salt thereof in the polymerisation solution is greater than or equal to 0.001 mg/mL and less than or equal to 10 mg/mL. [0127] 22. The coating according to any one of embodiments 1 or 4-21, the coated device according to any one of embodiments 2 or 4-21, or the method according to any one of embodiments 3-21, wherein the concentration of the at least one amino acid or a salt thereof in the polymerisation solution is greater than or equal to 0.001 mg/mL. [0128] 23. The coated device according to any one of embodiments 2 or 4-22, or the method of any one of embodiments 3-22, wherein the at least part of the surface of the device is flexible. [0129] 24. The coated device according to any one of embodiments 2 or 4-23, or the method of any one of embodiments 3-23, wherein the at least part of the surface of the device is formed from a polymer. [0130] 25. The coated device according to embodiment 24, or the method according to embodiment 24, wherein the at least part of the surface of the device is formed from a silicone polymer or polyurethane. [0131] 26. The coated device of embodiment 25, or the method according to embodiment 25, wherein the at least part of the surface of the device if formed from a polydimethylsiloxane. [0132] 27. The method according to any one of embodiments 3-26, wherein the solution comprising metallic ions is Tollens' reagent. [0133] 28. A coated device obtainable by the method any one of embodiments 3-27.