PROCESS TO COAT A MEDICAL DEVICE SURFACE WITH PEPTIDE-BASED NANOPARTICLES

Abstract

A process of coating a medical device surface with peptide-based nanoparticles with antimicrobial and healing properties; a process to coat a polyurethane (PU) dressing with a cross-linkable polymer adhesive in which was immobilized LL37 peptide conjugated-gold (Au) nanoparticles (LL37NPs) suitable to be applied on wounds. by following the steps of: 1) preparation of medical device surface; 2) coating the surface with a cross-linkable polymer adhesive; 3) spreading of peptide-based nanoparticles over the surface coated with the photo cross-linkable polymer adhesive; 4) exposing the surface coated with the adhesive and the nanoparticles to UV light; 5) placing the surface in phosphate buffer to leach loosely bound nanoparticles. The process described herein may be employed in the production of wound dressings, bandages, PU catheters and medical tubings.

Claims

1. A process of coating a medical device surface comprising the steps of: preparing a medical device surface; coating the medical device surface with a photo cross-linkable polymer adhesive; immobilizing peptide-based nanoparticles over the top of the surface coated with the cross-linkable polymer adhesive after UV curing; exposing the surface coated with the cross-linkable polymer adhesive and peptide-based nanoparticles to an UV light source with wavelength of from 365 to 395 nm; placing the medical device surface in phosphate buffer at a pH between 6 and 7.5 to leach loosely bound nanoparticles.

2. The process of coating a medical device according to claim 1, wherein the medical device surface is placed in the phosphate buffer for 120 to 360 minutes.

3. The process of coating a medical device according claim 1, wherein the cross-linkable polymer adhesive has a viscosity between 3 and 300 cP.

4. The process of coating a medical device according to claim 1, wherein the medical device surface comprises a film selected from polyurethane (PU), polystyrene (PS), poly(ethylene terephthalate) (PET) and polycarbonate (PC).

5. The process of coating a medical device according to claim 1, wherein the medical device is a wound dressing comprising a polyurethane (PU) film.

6. The process of coating a medical device according to claim 1, wherein the cross-linkable polymer adhesive is a photo cross-linkable adhesive.

7. The process of coating a medical device according to claim 1, wherein the photo cross-linkable polymer adhesive is selected from acrylated epoxies, acrylated polyesters, vinyl ethers, N-vinyl compound and vinylpyrrolidone compounds.

8. The process of coating a medical device according to claim 1, wherein the cross-linkable polymer adhesive is a non-photo cross-linkable adhesive.

9. The process of coating a medical device according to claim 1, wherein the non-photo cross-linkable polymer adhesive is selected from the group consisting of dopamine, polyethylenimine, amino-propyltrimethoxy silane, polymer brushes containing trifluromethacrylate and 2hydroxyethyl methacrylate.

10. The process of coating a medical device according to claim 1, wherein the peptide-based nanoparticles are LL37 NPs, wherein said peptide is LL37 (SEQ ID NO:1).

11. The process of coating a medical device according to claim 1, wherein the LL37 NPs are solubilized in ethanol, acetone, and dimethoxy sulfoxide (DMSO).

12. The process of coating a medical device according to claim 1, wherein the distance between UV light source and the film should be between 6 to 8 cm in order to coat LL37NPs.

13. The process of coating a medical device according to claim 1, wherein the amount of polymer adhesive should be between 10 to 30 .Math.L per cm.sup.2 of film surface.

14. The process of coating a medical device according to claim 1, wherein the LL37 NPs have a concentration of 40 to 70 .Math.g NPs/cm.sup.2.

15. The process of coating a medical device according to claim 1, wherein the amount of peptide available on the surface of medical device should be between 13 to 23 .Math.g/cm.sup.2.

16. A medical device comprising a medical device surface, a photo cross-linkable polymer adhesive, a cross-linkable polymer adhesive and LL37 NPs, wherein said peptide is LL37 (SEQ ID NO: 1).

17. The medical device according to claim 16, wherein the cross-linkable polymer adhesive has a viscosity of between 3 and 300 cP.

18. The medical device according to claim 16, wherein the medical device surface comprises a film selected from polyurethane (PU), polystyrene (PS), poly(ethylene terephthalate) (PET) and polycarbonate (PC).

19. The medical device according to claim 16, wherein the medical device is a wound dressing comprising a polyurethane (PU) film.

20. The medical device according to claim 16, wherein the cross-linkable polymer adhesive is a photo cross-linkable polymer adhesive.

21. The medical device according to claim 16, wherein the photo cross-linkable polymer adhesive is selected from acrylated epoxies, acrylated polyesters, vinyl ethers, N-vinyl compound and vinylpyrrolidone compounds.

22. The medical device according to claim 16, wherein the cross-linkable polymer adhesive is a non-photo cross-linkable polymer adhesive.

23. The medical device according to claim 16, wherein the non-photo cross-linkable polymer adhesive is selected from dopamine, polyethylenimine, amino-propyltrimethoxy silane, polymer brushes containing trifluromethacrylate and 2hydroxyethyl methacrylate.

24. The medical device according to claim 16, wherein the LL37 NPs are immobilized on the top of the cross-linkable polymer adhesive coating the medical device.

25. The medical device according to claim 16, wherein, the medical device has a water contact angle lower than 60°.

26. The medical device according to claim 16, wherein the medical device surface comprises 10 to 30 .Math.L per cm.sup.2 of film surface.

27. The medical device according to claim 16, wherein the LL37 NPs have a concentration of 40 to 70 .Math.g NPs/cm.sup.2.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0077] For easier understanding of this application, figures are attached in the annex that represent the preferred forms of implementation which nevertheless are not intended to limit the technique disclosed herein.

[0078] FIG. 1 illustrates a schematic diagram represents coating of LLNPs on PU dressing and their antimicrobial and wound healing properties.

[0079] FIG. 2 shows dressing physicochemical characterization. (A) AFM image of PU-adhesive-LL37NPs dressing. (B) ICP-MS analyses of leached LL37NPs from PU-adhesive-LL37NPs dressings incubated in PBS. FTIR spectra (C), contact angle (D) and zeta potential (E) measurements of PU, PU-adhesive and PU-adhesive-LL37NPs dressings.

[0080] FIG. 3 shows Antimicrobial testing. (A) Schematic representation of the antimicrobial test. Approximately 500.000 bacteria suspended in 100 .Math.L of media (TSY or 10% human serum) were placed in a dressing (1 cm.sup.2) for 20 h and then plated in TSY agar for 24 h before counting the colonies. (B, C) Antimicrobial activity of different dressings against E.coli, P.aeruginosa and S.aureus incubated in PBS (pH 7.2) (B) or in 10% human serum (C). In B and C, results are average ± SD, n=5. Statistical analyses were performed by One-way ANOVA followed by a Tukey’s post-test, ****P < 0.0001.

[0081] FIG. 4 shows Bacteria morphology after contact with soluble or immobilized LL37 peptide. Height mode AFM images of E.coli: (A1) in suspension, (B1) in suspension in the presence of LL37 peptide (20 .Math.g/mL) (C1) adhered to PU-adhesive films and (D1) adhered to PU-adhesive-LL37NPs films. Figures from A2 to D2 show line profile images of corresponding height mode images.

[0082] FIG. 5 shows Bacteria resistance testing. (A) Schematic diagram showing experimental procedures being used for bacterial resistance assay. (B) Results of resistance assay with LL37NPs and chloramphenicol with E.coli and S.aureus. (C) Antimicrobial activity of leached silver from Acticoat dressings against E.coli and S.aureus. Acticoat dressing (1 cm.sup.2) was incubated in PBS (1 mL, pH 7.2) for 1 day to collect the leached product. ICP-MS analysis was performed to quantify the amount of Ag. (D) Result of resistance assay with the leached silver against E.coli and S.aureus. After 10.sup.th cycle, a higher concentration of leached silver was used to show that resistant bacteria may be killed by higher concentration of silver. In B, C and D, results are average ± SD, n=3.

[0083] FIG. 6 shows In vivo wound healing properties of PU-adhesive-LL37NPs. (A) Schematic representation of wound healing experiments performed in diabetic mice (db/db mice) using PU-adhesive-LL37NPs and PU dressings. (B) Images of wounds treated with PU or PU-adhesive-LL37NPs taken at different times during the healing process. (C) Quantification of wound area measured from the optical images. Results are average ± SEM (n=7 animals). Statistical analyses were performed by One-way ANOVA followed by a Tukey’s post-test, *P<0.01. (D) ICP-MS analysis of NPs present in wounds, skin around wounds and liver, which are leached from PU-adhesive-LL37NP dressings. Results are average ± SEM (n=6 animals). (E) ICP-MS analysis of LL37NPs present in PU-adhesive-LL37NP dressings applied on wounds for 6 and 14 days. Results are average ± SEM (n=6 animals). Statistical analyses were performed by One-way ANOVA followed by a Tukey’s post-test, *P<0.05 (F) H&E images of wounds at days 6 and 14 treated with PU-adhesive-LL37NPs and PU dressings. Scar bars in all images correspond to 100 .Math.m.

[0084] FIG. 7 shows In vivo wound healing properties of PU-adhesive-LL37NPs. (A) Schematic representation of wound healing experiments performed in diabetic mice using PU-adhesive-LL37NP and PU dressings. (B) Optical images of wounds taken at different times of healing process. (C) Quantification of wounds area measured from the optical images (n=7 animals; 2 wounds per animal). (D) ICP-MS analyses of LL37NPs present in wounds leached from PU-adhesive-LL37NP dressings. Results are average ± SEM (n=6 animals; 2 wounds per animal). Statistical analyses were performed by One-way ANOVA followed by a Tukey’s post-test, *P<0.01, **P<0.001. (E) H&E stained images of wounds treated with PU-adhesive-LL37NP and PU dressings. Scale bar corresponds to 100 .Math.m.

[0085] FIG. 8 shows In vivo wound healing mechanism mediated by PU-adhesive-LL37NP dressings: re-epithelization. (A, B) Immunofluorescence analyses of wounds at days 6 to show expression of keratin 14 and 5 after treatment with PU-adhesive-LL37NPs (A) and PU (B) dressings. (C) Quantification of fluorescence intensity, thickness of keratin 14 in wound slides and proliferative length as well as wound gaps at day 6. Results are average ± SEM (n=6 animals). Statistical analyses were performed by unpaired t-test, ****P<0.0001, **P<0.0016.

[0086] FIG. 9 shows In vivo wound healing mechanism mediated by PU-adhesive-LL37NP dressings: immunomodulation. (A, B) Quantification of M1 and M2 phenotype macrophage cells in wounds at days 6 (A) and 14 (B) treated with PU-adhesive-LL37NPs and PU dressings. (C, D) Immunofluorescence analyses of co-localization of M1 and M2 phenotype macrophage cells at day 6 in wounds treated with PU-adhesive-LL37NPs (D.1) or PU (D.2) dressings. Arrows show co-localization of M1 and M2 phenotypes in different cells. Results are average ± SEM (n=6 animals). (E) qRT-PCR analysis of TNF-α, IL6 and IL19 cytokines in wounds treated with PU-adhesive-LL37NPs and PU dressings. Results are average ± SEM (n=6). Statistical analyses were performed by One-way ANOVA followed by a Tukey’s post-test, *P<0.01, **P<0.001.

[0087] FIG. 10 shows Physicochemical characterization of LL37NPs and films coated with LL37NPs. (A) UV-vis spectrum of LL37NPs. (B) Representative TEM image of LL37NPs. (C) Quantification of particle size of LL37NPs (n=100) from TEM images. (D) Water contact angle images of PU, PUadhesive and PU-adhesive-LL37NP films (n=4, average ± SD).

[0088] FIG. 11 shows Antimicrobial activity of PU-adhesive-LL37NP films. (A) Antimicrobial activity of PU, polymer adhesive-PU and CureMat (20 .Math.g/cm2) against E.coli, P.aeruginosa and S.aureus in PBS. (B) Antimicrobial activity of leached solution from CureMat and PU dressings (C) ICP-MS analysis of leached silver (Ag) and gold (Au) from Acticoat and CureMat dressings respectively incubated in PBS for different time.

[0089] FIG. 12 shows Cytotoxicity of PU-adhesive-LL37NPs against skin cells. (A) ATP production in keratinocytes seeded on top of PU, PU-adhesive or PU-adhesive-LL37NP films for 4 or 24 h. As controls, cells were cultured in tissue culture poly(styrene) (TCPS) with and without LL37 peptide (20 .Math.g/mL). Results are average ± SEM (n=3). (B) ATP production in fibroblasts cultured in TCPS and exposed to extracts of PU-adhesive-LL37NPs and Acticoat dressings. Control represent fibroblast cells grown on TCPS. The area of acticoat was similar to PU-adhesive-LL37NPs and both dressings were incubated in PBS (pH 7.2) at room temperature for 24 h. Results are average ± SEM (n=8).

[0090] FIG. 13 shows quantification of fluorescence intensity, thickness of keratin 5 (K5) in wound sides, proliferative length and wound gaps of day 6 wounds. Results are average ± SEM (n=6). Statistical analyses were performed by unpaired t-test, ****P<0.0001, **P<0.0094, *P<0.0096.

[0091] FIG. 14 shows immunofluorescence analysis of M1 (A) M2 (B) phenotype macrophage cells in wound treated with PU-adhesive-LL37NPs for 6 days.

[0092] FIG. 15 shows immunofluorescence analysis of M1 (A) M2 (B) phenotype macrophage cells in wound treated with PU dressings for 6 day.

DESCRIPTION OF EMBODIMENTS

[0093] Now, preferred embodiments of the present application will be described in detail with reference to the annexed drawings. However, they are not intended to limit the scope of this application.

[0094] In the context of the present patent application, “medical device”, is understood to be a wound dressing, a bandage, medical tubings, PU catheters and PU implants whereas “medical device surface” or “medical surface” is understood to be the surface of such devices.

[0095] In the context of the present patent application, “cross-linkable polymer adhesive” is understood to be either a “photo cross-linkable polymer adhesive” polymerizes under the exposure of UV light or a “non-photo cross-linkable polymer adhesive” that polymerizes independently of light of exposure.

[0096] The cross-linkable polymer adhesives of the present application have a low viscosity, improved adhesion to film, maintain tensile strength of film and biocompatible to human cells. A low viscosity of resin means the resin which can easily spread on the PU film. The improved adhesion means resin can strongly adhere to film after UV curing and does not leach or stick to other surfaces.

[0097] The present patent application describes, as the main embodiment of the invention, a process of coating a medical device surface comprising the steps of: [0098] Preparation of a medical device surface; [0099] Coating the medical device surface with a photo cross-linkable polymer adhesive; [0100] Immobilization of peptide-based nanoparticles over the surface coated with the photo cross-linkable polymer adhesive; [0101] Exposing the surface coated with the cross-linkable polymer adhesive and peptide-based nanoparticles to UV light with range of 365 o 395 nm; [0102] Placing the medical device surface in phosphate buffer at a pH between 6 and 7.5 to leach loosely bound nanoparticles.

[0103] In one embodiment, the medical device surface is placed in the phosphate buffer for 120 to 360 min.

[0104] In one embodiment, the peptide-based nanoparticles are uniformly immobilized on the top of cross-linkable polymer adhesive coated medical devices after UV curing.

[0105] In one embodiment, the cross-linkable adhesive has viscosity between 3 and 300 cP.

[0106] In one embodiment, the UV light has a power of 100 W.

[0107] In one embodiment, the phosphate buffer has a molar concentration of 100 mM.

[0108] In one embodiment, the medical device surface comprises a film selected from polyurethane (PU), polystyrene (PS), poly(ethylene terephthalate) (PET) and polycarbonate (PC). In one preferable embodiment, the medical device is a wound dressing comprising a polyurethane (PU) film.

[0109] In one embodiment, the cross-linkable polymer adhesive is a photo cross-linkable adhesive.

[0110] In one preferable embodiment, the photo cross-linkable polymer adhesive comprise compounds selected from acrylated epoxies, acrylated polyesters, vinyl ethers, N-vinyl compound or vinylpyrrolidone compounds, wherein the said cross-linkable polymer adhesive polymerizes under the exposure of UV light.

[0111] In another embodiment, the cross-linkable polymer adhesive is a non-photo cross-linkable adhesive.

[0112] In one preferable embodiment, the non-photo cross-linkable adhesive is used to coat conjugated peptide-gold (Au) nanoparticles (LL37 NPs) on PU film wherein the said polymer adhesives are selected from dopamine, polyethylenimine, amino-propyltrimethoxy silane, polymer brushes containing trifluromethacrylate and 2hydroxyethyl methacrylate.

[0113] In one embodiment, the peptide-based nanoparticles are conjugated peptide-gold (Au) nanoparticles (LL37 NPs), wherein said peptide is LL37 (SEQ ID NO:1).

[0114] In one embodiment, the LL37 NPs are solubilized in ethanol, acetone, and dimethoxy sulfoxide (DMSO).

[0115] In one embodiment, LL37NPs are synthesized using LL37 peptide (0.1 to 0.25 mM) and HAuCl.sub.4 (0.5 to 1 mM) in the presence of HEPES buffer (pH 5 and 7.5).

[0116] In another embodiment, the distance between UV light source and the film should be between 6 to 8 cm in order to coat LL37NPs.

[0117] In another embodiment, the amount of cross-linkable polymer adhesive should be 10 to 30 .Math.L per cm.sup.2 of film surface in order to have a very thin layer.

[0118] In one embodiment, the LL37 NPs have a concentration of 40 to 70 .Math.g NPs/cm.sup.2, preferably 60 .Math.g NPs/cm.sup.2.

[0119] According to the preferable embodiment of the present application, it is understood that, with the currently described approach, only a very negligible amount of LL37NPs ranging from 0.1 to 2 .Math.g per cm.sup.2 of PU-adhesive-LL37NP films leaches in PBS (pH 7.2) or in the wound environment.

[0120] Also part of the present application is the medical device obtained from the process described above, wherein said medical device comprises a medical device surface, a cross-linkable polymer adhesive and LL37 NPs, wherein said peptide is LL37 (SEQ ID NO: 1).

[0121] In one embodiment, the medical device surface comprises a film selected from polyurethane (PU), polystyrene (PS), poly(ethylene terephthalate) (PET) and polycarbonate (PC).

[0122] In one preferable embodiment, the medical device is a wound dressing comprising a polyurethane (PU) film.

[0123] In one embodiment, the cross-linkable polymer adhesive is a photo cross-linkable polymer adhesive or a non-photo cross-linkable polymer adhesive.

[0124] In one embodiment, photo cross-linkable polymer adhesive is selected from acrylated epoxies, acrylated polyesters, vinyl ethers, N-vinyl compound and vinylpyrrolidone compounds.

[0125] In another embodiment, the non-photo cross-linkable adhesive is selected from dopamine, polyethylenimine, amino-propyltrimethoxy silane, polymer brushes containing trifluromethacrylate and 2hydroxyethyl methacrylate.

[0126] In one embodiment, the top layer of the medical device is coated with LL37 NPs.

[0127] In one embodiment, the medical device has a water contact angle lower than 60°.

[0128] In one embodiment, the medical device surface comprises 10 to 30 .Math.L per cm.sup.2 of film surface.

[0129] In one embodiment, the LL37 NPs have a concentration of 40 to 70 .Math.g NPs/cm.sup.2.

[0130] According to the preferable embodiment of the present application, the medical device, preferably a wound dressing, has a water contact angle lower than 60° wherein the said contact angle should be hydrophilic in order to maintain moist environment of wound.

[0131] According to the preferable embodiment of the present application, the medical device, preferably the PU-adhesive-LL37NP films of the application, kill Gram-positive and Gram-negative bacteria from 1 log to 4 log.

[0132] According to the preferable embodiment of the present application, the medical device, preferably the PU-adhesive-LL37NP films of the application, kills bacteria without inducing resistance in sub-MIC concentrations of the immobilized peptide.

[0133] According to the preferable embodiment of the present application, the medical device, preferably the PU-adhesive-LL37NP films of the application, promote rapid healing of diabetic wounds by the expression of keratin 14 and 5 along with transition of early macrophages (M1) to late macrophages (M2) in day 6 wounds.

[0134] This description is of course not in any way restricted to the forms of implementation presented herein and any person with an average knowledge of the area can provide many possibilities for modification thereof without departing from the general idea as defined by the claims. The preferred forms of implementation described above can obviously be combined with each other. The following claims further define the preferred forms of implementation.