NOVEL COMPOSITIONS AND USES THEREOF

Abstract

The present invention provides a composition comprising: (a) collagen VI or a polypeptide comprising or consisting of an amino acid sequence derived from collagen type VI or a fragment, variant, fusion or derivative thereof; and (b) polylysine. Also provided are pharmaceutical compositions and kits comprising the composition of the invention. Related aspects provide medical devices, implants, wound care products and materials for use in the same associated with the composition of the invention, and methods of their preparation. Also provided are methods and uses of the composition in the treatment and/or prevention of microbial infections and in wound care, and a method of killing microorganisms in vitro.

Claims

1. A composition comprising: (a) collagen type VI, or a polypeptide comprising or consisting of an amino acid sequence derived from collagen type VI, or a fragment, variant, fusion or derivative thereof, or a fusion of said fragment, variant of derivative thereof; and (b) polylysine.

2. A composition according to claim 1, wherein the polylysine is poly-L-lysine.

3. A composition according to any one of claim 1 or 2, wherein the collagen type VI or polypeptide, fragment, variant, fusion or derivative is capable of killing or attenuating the growth of microorganisms.

4. A composition according to claim 3 wherein the microorganisms are selected from the group consisting of bacteria, mycoplasmas, yeasts, fungi and viruses.

5. A composition according to any one of the preceding claims wherein the collagen type VI or polypeptide is capable of binding to the membrane of the microorganism.

6. A composition according to any one of the preceding claims wherein the collagen type VI or polypeptide is capable of causing membrane disruption of the microorganisms.

7. A composition according to any one of the preceding claims which is capable of promoting wound closure.

8. A composition according to any one of the preceding claims, wherein the collagen type VI or polypeptide is capable of exhibiting an antimicrobial effect greater than or equal to that of LL-37.

9. A composition according to any one of the preceding claims wherein the microorganisms are Gram-positive or Gram-negative bacteria.

10. A composition according to claim 9, wherein the microorganisms are selected from the group consisting of: Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, group A Streptococcus (e.g. Streptococcus pyogenes), group B Streptococcus (e.g. Streptococcus agalactiae), group C Streptococcus (e.g. Streptococcus dysgalactiae), group D Streptococcus (e.g. Enterococcus faecalis), group F Streptococcus (e.g. Streptococcus anginosus), group G Streptococcus (e.g. Streptococcus dysgalactiae equisimilis), alpha-hemolytic Streptococcus (e.g. Streptococcus viridans, Streptococcus pneumoniae), Streptococcus bovis, Streptococcus mitis, Streptococcus anginosus, Streptococcus sanguinis, Streptococcus suis, Streptococcus mutans, Moraxella catarrhalis, Non-typeable Haemophilus influenzae (NTHi), Haemophilus influenzae b (Hib), Actinomyces naeslundii, Fusobacterium nucleatum, Prevotella intermedia, Klebsiella pneumoniae, Enterococcus cloacae, Enterococcus faecalis, Staphylococcus epidermidis, multi-resistant Pseudomonas aeruginosa (MRPA), multi-resistant Staphylococcus aureus (MRSA), multi-resistant Escherichia coli (MREC), multi-resistant Staphylococcus epidermidis (MRSE) and multi-resistant Klebsiella pneumoniae (MRKP).

11. A composition according to any one of the preceding claims wherein the microorganisms are bacteria which are resistant to one or more conventional antibiotic agents.

12. A composition according to claim 11 wherein the microorganism is selected from the group consisting of: multidrug-resistant Staphylococcus aureus (MRSA), multidrug-resistant Pseudomonas aeruginosa (MRPA), multi-resistant Escherichia coli (MREC), multi-resistant Staphylococcus epidermidis (MRSE) and multi-resistant Klebsiella pneumoniae (MRKP).

13. A composition according to any one of the preceding claims wherein the collagen type VI or polypeptide is substantially non-toxic to mammalian cells.

14. A composition according to any one of the preceding claims wherein the collagen type VI or polypeptide is capable of exerting an anti-endotoxic effect.

15. A composition according to any one of the preceding claims wherein the polypeptide is derived from a von Willebrand Factor type A domain.

16. A composition according to any one of the preceding claims wherein the polypeptide is or is derived from the α1, α2 and/or α3 chain of collagen type VI.

17. A composition according to claim 16 wherein the polypeptide is or is derived from the α3 chain of collagen type VI.

18. A composition according to any one of the preceding claims wherein the polypeptide is or is derived from the N2, N3 or C1 domain of the α3 chain of collagen type VI.

19. A composition according to any one of the preceding claims wherein the collagen type VI or polypeptide has a net positive charge.

20. A composition according to claim 19 wherein the charge on the collagen type VI or polypeptide ranges from between +2 to +9.

21. A composition according to any one of the preceding claims wherein the collagen type VI or polypeptide has at least 30% hydrophobic residues.

22. A composition according to any one of the preceding claims comprising or consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 23, and fragments, variants, fusions or derivatives thereof, and fusions of said fragments, variants and derivatives thereof, which retain an antimicrobial activity of any one of SEQ ID NOs:1 to 23.

23. A composition according to claim 22 comprising or consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5: TABLE-US-00003 [SEQ ID NO: 1] “GVR28”: GVRPDGFAHIRDFVSRIVRRLNIGPSKV [SEQ ID NO: 2] “FYL25”: FYLKTYRSQAPVLDAIRRLRLRGGS [SEQ ID NO: 3] “FFL25”: FFLKDFSTKRQIIDAINKVVYKGGR [SEQ ID NO: 4] “VTT30”: VTTEIRFADSKRKSVLLDKIKNLQVALTSK [SEQ ID NO: 5] “SFV33”: SFVARNTFKRVRNGFLMRKVAVFFSNTPTRASP and fragments, variants, fusions or derivatives thereof, and fusions of said fragments, variants and derivatives thereof, which retain an antimicrobial activity of any one of SEQ ID NOs:1 to 5.

24. A composition according to claim 23 comprising or consisting of the amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5.

25. A composition according to any one of the preceding claims wherein the polypeptide comprises or consists of a variant of the amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 23.

26. A composition according to claim 25 wherein the variant has at least 50% identity with the amino acid sequence amino acid sequence of any one of SEQ ID NOs: 1 to 23, for example at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity.

27. A composition according to any one of the preceding claims wherein the polypeptide is between 10 and 200 amino acids in length, for example between 10 and 150, 15 and 100, 15 and 50, 20 and 40 or 25 and 35 amino acids in length.

28. A composition according to claim 27 wherein the polypeptide is at least 20 amino acids in length.

29. A composition according to any one of the preceding claims wherein the collagen type VI or polypeptide, or fragment, variant, fusion or derivative thereof, comprises one or more amino acids that are modified or derivatised.

30. A composition according to claim 29 wherein the one or more amino acids are modified or derivatised by PEGylation, amidation, esterification, acylation, acetylation and/or alkylation.

31. A composition according to any one of the preceding claims wherein the collagen type VI or polypeptide is a recombinant polypeptide.

32. A composition according to any one of claims 1 to 31, wherein the polylysine is polymerized at the ε carbon position of the lysine monomer units.

33. A composition according to any one of claims 1 to 32, wherein the polylysine has a molecular weight in the range 30,000 Da to 300,000 Da, preferably wherein the polylysine has a molecular weight of 30,000 Da to 70,000 Da.

34. A composition according to any one of claims 1 to 33 wherein the polylysine is made up of between 200 and 2054 monomer units of lysine polymerized together, preferably wherein the polylysine is made up of between 200 and 480 monomer units of lysine polymerized together.

35. A pharmaceutical composition comprising a composition according to any one of claims 1 to 34 together with a pharmaceutically acceptable excipient, diluent, carrier, buffer or adjuvant.

36. A medical device, implant, wound care product, or material for use in the same, which is coated, impregnated, admixed or otherwise associated with a composition or pharmaceutical composition according to any one of the preceding claims.

37. A medical device, implant, wound care product, or material for use in the same according to claim 36, which is coated with a composition according to any one of claims 1 to 34 or pharmaceutical composition according to claim 35.

38. A medical device, implant, wound care product, or material for use in the same, according to any one of claim 36 or 37 wherein the device, implant, wound care product, or material is for use in by-pass surgery, extracorporeal circulation, wound care and/or dialysis.

39. A medical device, implant, wound care product, or material for use in the same, according to any one of claims 36 to 38 wherein the composition is coated, painted, sprayed or otherwise applied to a suture, prosthesis, implant, wound dressing, catheter, lens, skin graft, skin substitute, fibrin glue or bandage.

40. A medical device, implant, wound care product, or material for use in the same, according to any one of claims 36 to 39 comprising or consisting of a polymer, metal, metal oxide and/or ceramic.

41. A medical device, implant, wound care product, or material for use in the same according to claim 40, comprising or consisting of a metal, wherein the metal is titanium or a titanium alloy.

42. A medical device, implant, wound care product, or material for use in the same according to any one of claims 36 to 41, wherein the device, implant, wound care product, or material is for use in the same is selected from the group consisting of: a joint prosthesis, an orthopaedic device, a bone replacement device, a bone fixation device, a bone plate, a stem of an artificial hip joint, an artificial organ, a spinal rod, a maxillofacial plate, a stent graft, a percutaneous device, and a pacemaker.

43. A medical device, implant, wound care product, or material for use in the same according to any one of claims 36 to 42 wherein the percentage binding of collagen type VI or the polypeptide of the composition to the medical device, implant, wound care product, or material for use in the same is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, or at least 86.8%.

44. A medical device, implant, wound care product, or material for use in the same, according to any one of claims 36 to 43 wherein the percentage binding of collagen type VI or the polypeptide of the composition to the medical device, implant, wound care product, or material for use in the same is greater than the percentage binding achieved by a composition that does not contain polylysine, for example; at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or at least 109% greater.

45. A method of preparing a medical device, implant, wound care product or material for use in the same, comprising the step of coating, impregnating, admixing or otherwise associating the medical device, implant, wound care product, or material for use in the same with the composition or pharmaceutical composition according to any one of claims 1 to 35.

46. A method of preparing a medical device, implant, wound care product or material for use in the same comprising the following steps: (i) preparing a composition comprising collagen type VI or a polypeptide as defined by any one of claims 1 and 3 to 31 and polylysine as defined by any one of claims 1 to 2 and 32 to 34; and (ii) coating, impregnating, admixing or otherwise associating the medical device, implant, wound care product, or material for use in the same with the composition prepared in step (i).

47. A method of preparing a medical device, implant, wound care product or material for use in the same comprising the following steps: (i) coating, impregnating, admixing or otherwise associating the medical device, implant, wound care product, or material for use in the same with polylysine as defined in any one of claims 1 to 2 and 32 to 34; and (ii) coating, impregnating, admixing or otherwise associating the medical device, implant, wound care product, or material for use in the same produced in step (i) with collagen type VI or a polypeptide as defined in any one of claims 1 and 3 to 32.

48. A kit comprising: (i) a composition according to any one of claims 1 to 34 or a pharmaceutical composition according to claim 35 or a medical device, implant, wound care product, or material for use in the same according to any one of claims 36 to 44, and (ii) instructions for use.

49. A kit comprising: (i) a collagen type VI or polypeptide as defined in any one of claims 1 and 3 to 31; (ii) polylysine as defined in any one of claims 1 to 2 and 32 to 34; and (iii) instructions for use.

50. A composition according to any one of claims 1 to 34 or a pharmaceutical composition as defined in claim 35 for use in medicine.

51. A composition according to any one of claims 1 to 34 or a pharmaceutical composition as defined in claim 35 for use in the curative and/or prophylactic treatment of microbial infections.

52. A composition or pharmaceutical composition for use according to claim 51 wherein the microbial infection is a systemic infection.

53. A composition or pharmaceutical composition for use according to claim 51 or 52 wherein the microbial infection is resistant to one or more conventional antibiotic agents.

54. A composition or pharmaceutical composition for use according to any one of claims 51 to 53 wherein the microbial infection is caused by a microorganism selected from the group consisting of: Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, group A Streptococcus (e.g. Streptococcus pyogenes), group B Streptococcus (e.g. Streptococcus agalactiae), group C Streptococcus (e.g. Streptococcus dysgalactiae), group D Streptococcus (e.g. Entero-coccus faecalis), group F Streptococcus (e.g. Streptococcus anginosus), group G Streptococcus (e.g. Streptococcus dysgalactiae equisimilis), alpha-hemolytic Streptococcus (e.g. Streptococcus viridans, Streptococcus pneumoniae), Streptococcus bovis, Streptococcus mitis, Streptococcus anginosus, Streptococcus sanguinis, Streptococcus suis, Streptococcus mutans, Moraxella catarrhalis, Non-typeable Haemophilus influenzae (NTHi), Haemophilus influenzae b (Hib), Actinomyces naeslundii, Fusobacterium nucleatum, Prevotella intermedia, Klebsiella pneumoniae, Enterococcus cloacae, Enterococcus faecalis, Staphylococcus epidermidis, multi-resistant Pseudomonas aeruginosa (MRPA), and multi-resistant Staphylococcus aureus (MRSA), multi-resistant Escherichia coli (MREC), multi-resistant Staphylococcus epidermidis (MRSE) and multi-resistant Klebsiella pneumoniae (MRKP).

55. A composition or pharmaceutical composition for use according to any one of claims 51 to 54 wherein the microbial infection is caused by a microorganism selected from the group consisting of: multidrug-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa (MRPA).

56. A composition or pharmaceutical composition for use according to any one of claims 50 to 55 in combination with one or more additional antimicrobial agents.

57. A composition or pharmaceutical composition for use according to claim 56 wherein the one or more additional antimicrobial agent is selected from the group consisting of: antimicrobial polypeptides and antibiotics.

58. A composition as defined in any one of claims 1 to 34 or a pharmaceutical composition as defined in claim 35 for use in wound care.

59. Use of a composition as defined in any one of claims 1 to 34 or a pharmaceutical composition as defined in claim 35 in the manufacture of a medicament for the treatment of microbial infections.

60. Use of a composition as defined in any one of claims 1 to 34 or a pharmaceutical composition as defined in claim 35 in the manufacture of a medicament for the treatment of wounds.

61. A method of treating an individual with a microbial infection, the method comprising the step of administering to an individual in need thereof an effective amount of a composition as defined in any one of claims 1 to 34 or pharmaceutical composition as defined in claim 35.

62. A method of treating a wound in an individual, the method comprising the step of administering to an individual in need thereof an effective amount of a composition as defined in any one of claims 1 to 34 or a pharmaceutical composition as defined in claim 35.

63. A method for killing microorganisms in vitro comprising contacting the microorganisms with a composition as described in any one of claims 1 to 34 or a pharmaceutical composition as defined in claim 35.

64. A composition or pharmaceutical composition for use according to claims 50 to 58, a use according to claim 59 or 60, or a method according to claims 61 to 63, wherein the composition or pharmaceutical composition is coated or impregnated onto, or admixed or otherwise associated with, a medical device, implant, wound care product, or material for use in the same.

65. A composition substantially as described herein with reference to the description and figures.

66. A medical implant or device, or biomaterial for use in the same, substantially as described herein with reference to the description and figures.

67. Use of a composition substantially as described herein with reference to the description and figures.

68. A method for treating or preventing infection substantially as described herein with reference to the description and figures.

69. A method for treating wounds substantially as described herein with reference to the description and figures.

Description

DESCRIPTION OF THE FIGURES

[0288] FIG. 1: Quantitative evaluation of the coating efficiency of collagen I scaffolds with native collagen VI fibrils and other biomolecules. Binding of native collagen VI microfibrils to collagen I scaffolds with 1 mm (thin biodegradable scaffold), or 2 mm (thick biodegradable scaffold) thickness, respectively, was assessed by 131-iodine assays as indicated in the Figure. The coating efficiency with and without precoating with PLL was compared to the cathelicidin peptide LL-37, and the host defence peptides and proteins NAT26, HKH20, GGL27, α defensins 3-6 and β defensins 1-4. Coating with collagen VI microfibrils is considerably enhanced with PLL as an intermediate layer as compared to the controls.

[0289] FIG. 2: Quantitative evaluation of the coating efficiency of collagen I scaffolds with different collagen VI peptides. Binding of native collagen VI microfibrils to collagen I scaffolds with 1 mm (thin biodegradable scaffold), or 2 mm (thick biodegradable scaffold) thickness, respectively, was assessed by 131-iodine assays as indicated in the Figure. The coating efficiency with and without precoating with PLL was compared to the different collagen VI alpha-chains, and the bioactive collagen VI peptides GVR28, FYL25, FFL25, VTT30, and SFV33. Coating with the collagen VI alpha-3 chain and its derived peptides is considerably enhanced with PLL as an intermediate layer as compared to the other parts of the collagen VI molecule.

[0290] FIG. 3: Quantitative evaluation of the coating efficiency of titanium surfaces with native collagen VI fibrils and other biomolecules. Binding of native collagen VI microfibrils to titanium surfaces was assessed by 131-iodine assays as indicated in FIG. 3. The coating efficiency with and without precoating with PLL was compared to the cathelicidin peptide LL-37, and the host defence peptides and proteins NAT26, HKH20, GGL27, α defensins 3-6 and β defensins 1-4. Coating with collagen VI microfibrils is considerably enhanced with PLL as an intermediate layer compared to the controls.

[0291] FIG. 4: Quantitative evaluation of the coating efficiency of titanium surfaces with different collagen VI peptides. Binding of native collagen VI microfibrils to titanium surfaces was assessed by 131-iodine assays as indicated in FIG. 4. The coating efficiency with and without precoating with PLL was compared to the different collagen VI alpha-chains, and the bioactive collagen VI peptides GVR28, FYL25, FFL25, VTT30, and SFV33. Coating with the collagen VI alpha-3 chain and its derived peptides is considerably enhanced with PLL as an intermediate layer compared to the other parts of the collagen VI molecule.

[0292] FIG. 5: Quantitative evaluation of the Gram-negative bacterial killing efficiency on titanium surfaces coated with different collagen VI peptides. Killing of Pseudomonas aeruginosa bacteria on the different titanium surfaces was assessed by scanning electron microscopy assays. The killing efficiency with and without precoating with PLL was compared to the different collagen VI alpha-chains, and the bioactive collagen VI peptides GVR28, FYL25, FFL25, VTT30, and SFV33. Killing by the collagen VI alpha-3 chain and its derived peptide SFV33 is considerably enhanced with PLL as an intermediate layer as compared to the other parts of the collagen VI molecule.

[0293] FIG. 6: Quantitative evaluation of the Gram-positive bacterial killing efficiency on titanium surfaces coated with different collagen VI peptides. Killing of Staphylococcus aureus bacteria on the different titanium surfaces was assessed by scanning electron microscopy assays. The killing efficiency with and without precoating with PLL was compared to the different collagen VI alpha-chains, and the bioactive collagen VI peptides GVR28, FYL25, FFL25, VTT30, and SFV33. Killing by the collagen VI alpha-3 chain and its derived peptide SFV33 is considerably enhanced with PLL as an intermediate layer as compared to the other parts of the collagen VI molecule.

[0294] FIG. 7: Quantitative evaluation of the fibroblast adherence efficiency on titanium surfaces coated with different collagen VI peptides. Adherence of skin fibroblasts on the different titanium surfaces was assessed by scanning electron microscopy assays. The cell adherence efficiency with and without precoating with PLL was compared to the different collagen VI alpha-chains, and the bioactive collagen VI peptides GVR28, FYL25, FFL25, VTT30, and SFV33. Fibroblast adherence mediated by the collagen VI alpha-3 chain and its derived peptides GVR28, FFL25 and VTT30 is considerably enhanced with PLL as an intermediate layer as compared to the other parts of the collagen VI molecule.

[0295] FIG. 8: Quantitative evaluation of the keratinocyte adherence efficiency on titanium surfaces coated with different collagen VI peptides. Adherence of skin keratinocytes on the different titanium surfaces was assessed by scanning electron microscopy assays. The cell adherence efficiency with and without precoating with PLL was compared to the different collagen VI alpha-chains, and the bioactive collagen VI peptides GVR28, FYL25, FFL25, VTT30, and SFV33. Keratinocytes adherence mediated by the collagen VI alpha-3 chain and its derived peptides GVR28, FFL25 and VTT30 is considerably enhanced with PLL as an intermediate layer as an intermediate layer as compared to the other parts of the collagen VI molecule.

[0296] FIG. 9: Quantitative evaluation of the Gram-negative bacterial killing efficiency on collagen I scaffolds coated with different collagen VI peptides. Killing of Pseudomonas aeruginosa bacteria on collagen I scaffolds with 1 mm (thin biodegradable scaffold), or 2 mm (thick biodegradable scaffold) thickness, respectively, was assessed by scanning electron microscopy assays. The killing efficiency with and without precoating with PLL was compared to the different collagen VI alpha-chains, and the bioactive collagen VI peptides GVR28, FYL25, FFL25, VTT30, and SFV33. Killing by the collagen VI alpha-3 chain and its derived peptide SFV33 is considerably enhanced with PLL as an intermediate layer as compared to the other parts of the collagen VI molecule.

[0297] FIG. 10: Quantitative evaluation of the Gram-positive bacterial killing efficiency on collagen I scaffolds coated with different collagen VI peptides. Killing of Staphylococcus aureus bacteria on collagen I scaffolds with 1 mm (thin biodegradable scaffold), or 2 mm (thick biodegradable scaffold) thickness, respectively, was assessed by scanning electron microscopy assays. The killing efficiency with and without precoating with PLL was compared to the different collagen VI alpha-chains, and the bioactive collagen VI peptides GVR28, FYL25, FFL25, VTT30, and SFV33. Killing by the collagen VI alpha-3 chain and its derived peptide SFV33 is considerably enhanced with PLL as an intermediate layer as compared to the other parts of the collagen VI molecule.

[0298] FIG. 11: Quantitative evaluation of the fibroblast adherence efficiency on collagen I scaffolds coated with different collagen VI peptides. Adherence of skin fibroblasts on the different collagen I scaffolds was assessed by scanning electron microscopy assays. The cell adherence efficiency with and without precoating with PLL was compared to the different collagen VI alpha-chains, and the bioactive collagen VI peptides GVR28, FYL25, FFL25, VTT30, and SFV33. Fibroblast adherence mediated by the collagen VI alpha-3 chain and its derived peptides GVR28, FFL25 and VTT30 is considerably enhanced with PLL as an intermediate layer as compared to the other parts of the collagen VI molecule.

[0299] FIG. 12: Quantitative evaluation of the keratinocyte adherence efficiency on collagen I scaffolds surfaces coated with different collagen VI peptides. Adherence of skin keratinocytes on the different collagen I scaffolds was assessed by scanning electron microscopy assays. The cell adherence efficiency with and without precoating with PLL was compared to the different collagen VI alpha-chains, and the bioactive collagen VI peptides GVR28, FYL25, FFL25, VTT30, and SFV33. Keratinocytes adherence mediated by the collagen VI alpha-3 chain and its derived peptides GVR28, FFL25 and VTT30 is considerably enhanced with PLL as an intermediate as compared to the other parts of the collagen VI molecule.

EXAMPLES

Example 1

Improved Binding of Collagen VI to a Solid Surface

[0300] Introduction

[0301] Biomaterials are placed internally to maintain or replace human body functions. They are constructed of various combinations of metal alloys, ceramics, polymers, or biopolymers due to their excellent mechanical properties, corrosion resistance, and biocompatibility. Wound matrices come in a variety of materials including natural polymers and synthetic polymers manufactured into various forms, such as foams, films, hydrocolloids, hydrogels, sponges, membranes, skin substitutes, electro spun micro- and nanofibers. Bioactive wound matrices deliver substances active in wound healing either by delivery of bioactive compounds or by being constructed from materials having endogenous activity. The healing success rate is highly determined by cellular and physiological processes taking place at the host-biomaterial interface during wound healing. Specifically, adverse host response processes often lead to chronic inflammation and encapsulation that preclude the performance of the biomaterial. Hence, it is important to design appropriate wound treatment strategies with the ability to work actively with wound properties such as tissues and cells to enhance healing. Also, patients often suffer from severe infections at the implant site, precluding the normal wound healing process. Usually, harmless bacteria like Staphylococci, Pseudomonas, or Streptococci can infiltrate the damaged tissue in the fresh wound. Here they may develop a high pathogenic potential and establish persistent infections, severely compromising biomaterial function. Therefore, new strategies including bioactive wound healing promoting biomaterial surfaces with antimicrobial effects can be beneficial for the patient. In particular, coating strategies, which allow loading a given biomaterial surface with a maximum quantity of bioactive biomolecules, are crucial for premium biomaterial function and biocompatibility.

[0302] To achieve this goal, the inventors investigated the effect of modifying titanium surfaces with Poly-L-Lysine (PLL) on the coating density of collagen VI and different peptides derived from its sequence. They were compared to a number of host defence molecules and peptides. Collagen VI and peptides thereof were immobilized to different collagen I-based biodegradable scaffold model discs on intermediate layers of poly-L-lysine (PLL) and exhibited an unexpected, particularly high coating efficiency, resembling native in vivo connective tissue structures.

[0303] In summary, these data show that all tested combinations of collagen VI/collagen VI peptide/PLL surface modifications exhibit particularly high coating efficiencies on the biomaterial surface. They may thus promote early and intermediate cellular events on the host-biomaterial interface at a particular high rate as compared to other biomolecules. Thus, collagen VI and its derived peptides, bound to an intermediate layer of PLL, may be considered biologically appropriate for premium biocompatibility and tissue integration of the bioactive host-biomaterial interface. In particular, they may protect the wound bed against local infections and the patient against systemic infections after biomaterial insertion and during the initial steps of wound closure and wound healing. This treatment strategy will be beneficial for the wound environment, with the potential to promote improved wound repair and reduce abnormal scar formation.

[0304] Therefore, in the present study, the inventors explored whether the use of PLL, together with native collagen VI-derived biomolecules, can promote superior coating density on the surface of different commercially available collagen scaffolds. Here, we describe for the first time that the use of polylysine such as PLL greatly enhances the surface coating of biological collagen scaffolds with native bioactive collagen VI molecules. This effect will result in a pronounced potential to provide a versatile, multifunctional, and appropriate extracellular environment, able to actively contrast the onset of infections and inflammation, while promoting tissue regeneration and scar remodeling, and consequently deliver the desired enhancement in biocompatibility.

[0305] Materials and Methods

[0306] Materials—MDS Collagen was obtained from MedSkin Solutions Dr. Suwelack A G (MDS). 1 mm thickness refers to thin, and 2 mm thickness refers to thick collagen scaffold, respectively. c-poly-L-lysine hydrobromide (PLL, 30 000-150 000 g/mol) was purchased from Sigma-Aldrich, St. Louis, USA. Collagen VI and its derived peptides were prepared as described in [18]. LL-37 was a kind gift of Dr. Artur Schmidtchen, University of Lund. NAT26, HKH20, GGL27 were kind gifts of Dr. Inga-Maria Frick, University of Lund. α defensins 3-6 and β defensins 1-4 were kind gifts of Dr. Arne Egesten, University of Lund. Proteins were radiolabeled with 131-iodine according to standard protocols prior to coating assays on collagen scaffold surfaces.

[0307] Biomaterial surface coating with PLL—collagen scaffold discs with a diameter of 5 mm and thickness of 2 mm (thick scaffold) and 1 mm (thin scaffold), respectively, were punched out from a larger sheet (approximately 10 cm×10 cm). Prior to collagen coating, 150 μl poly-L-lysine hydrobromide solution (0.2 mg/ml) were applied on the collagen scaffold discs by incubation at 60° C. for 2 h. Afterwards the discs were washed twice in distilled water to remove unbound PLL, air-dried and stored at room temperature.

[0308] Biomaterial surface coating with bioactive collagen VI molecules—collagen scaffold discs, after prior treatment with PLL, were put into 24-well cell culture plates (TPP, Trasadingen, Switzerland). They were coated by incubation with 150 μl bovine collagen VI microfibrils (concentration 15 μM), or with 150 μl collagen type VI peptides (concentration 3 μM), or with 150 ml LL-37, NAT26, HKH20, GGL27, α defensins 3-6 and β defensins 1-4 at 4° C. for 2 h, followed by rinsing with distilled water and air-drying. The coating efficiencies of the different biomolecules were assessed by determining the ratio between bound and free 131-iodine radioactivity associated with collagen scaffolds by determining radioactivity as cpm values in a γ-counter.

[0309] Results

[0310] PLL Mediates Superior Coating Efficiency of Collagen Type VI Microfibrils on Collagen Scaffolds.

[0311] In order to assess possible effects of PLL on collagen type VI coating, collagen I scaffolds were pretreated with PLL. Coating with the cathelicidin peptide LL-37, and the host defence peptides and proteins NAT26, HKH20, GGL27, α defensins 3-6 and β defensins 1-4 served as controls. The results from 131-iodine assays show binding of the different proteins to collagen I scaffolds (FIG. 1). Pre-treatment of the collagen I scaffolds with PLL significantly enhanced the adherence of collagen VI microfibrils as compared to the controls (FIG. 1). Notably, the coating efficiency of collagen VI was considerably enhanced as compared to the previous gold standard human antimicrobial peptide LL-37.

[0312] The Collagen Type VI Binding Properties are Associated with the Alpha-3 Chain and the Bioactive Peptides Derived From it.

[0313] For a more detailed understanding of the underlying coating mechanism collagen I scaffolds were incubated with different collagen type VI-derived peptides in the presence or absence of PLL and assessed by 131-iodine radioactivity. FIG. 2 depicts the adherence of these molecules. In the presence of PLL superior coating of collagen I scaffolds with the collagen type VI alpha-3 chain, and the bioactive collagen VI peptides GVR28, FYL25, FFL25, VTT30, and SFV33 were observed.

[0314] Taken together, the data presented in FIGS. 1 and 2 show that collagen type VI and/or parts of the collagen VI alpha-3 chain strongly bind to collagen I scaffolds/biomaterials in the presence of PLL. This effect of PLL was considerably less pronounced for the other tested proteins. These effects are expected to result in superior wound healing properties of biomaterials coated with peptides derived from the collagen VI together with polylysine. This coating strategy will be beneficial for the wound environment, with the potential to promote improved wound repair and reduce abnormal scar formation.

Example 2

Improved Binding of Collagen Vi to a Titanium Surface

[0315] The inventors also investigated the effects of PLL on the binding of collagen VI directly to titanium surfaces.

[0316] Materials and methods

[0317] Materials—Titanium discs with a diameter and thickness of 5 mm and 0.25 mm, respectively, and with a maximum average roughness (Ra) of 0.8 μm, were punched out from a commercially available titanium foil (Alfa Aesar GmbH & Co. KG, Karlsruhe, Germany). ε-poly-L-lysine hydrobromide (PLL, 30,000-150,000 g/mol) was purchased from Sigma-Aldrich, St. Louis, USA. Collagen VI and its derived peptides were prepared as described in [18]. LL-37 was a kind gift of Dr. Artur Schmidtchen, University of Lund. NAT26, HKH20, GGL27 were kind gifts of Dr. Inga-Maria Frick, University of Lund. α defensins 3-6 and β defensins 1-4 were kind gifts of Dr. Arne Egesten, University of Lund. Proteins were radiolabeled with 131-iodine according to standard protocols prior to coating assays on collagen scaffold surfaces.

[0318] Biomaterial surface coating with PLL—Titanium discs were degreased with chloroform and 97% ethanol, followed by rinsing with distilled water, and air-drying at room temperature. Subsequently, prior to collagen coating, 150 μl poly-L-lysine hydrobromide (70,000-150,000 g/mol, Sigma-Aldrich, St. Louis, USA, 0.2 mg/ml) were applied on desired titanium discs by incubation at 60° C. for 2 hours. Afterwards, the titanium was washed twice in distilled water to remove unbound PLL, air-dried and stored at room temperature.

[0319] Biomaterial surface coating with bioactive collagen VI molecules—Titanium discs, after prior treatment with PLL, were put into 24-well cell culture plates (TPP, Trasadingen, Switzerland). They were coated by incubation with 150 μl collagen type VI peptides (concentration 3 μM), at 4° C. for 2 hours, followed by rinsing with distilled water and air-drying. The coating efficiencies of the different biomolecules were assessed by determining the ratio between bound and free 131-iodine radioactivity associated with titanium discs by determining radioactivity as cpm values in a γ-counter.

[0320] Results

[0321] PLL Mediates Superior Coating Efficiency of Collagen Type VI Microfibrils on Titanium Surfaces.

[0322] In order to assess possible effects of PLL on collagen type VI coating, titanium surfaces were pre-treated with PLL. Coating with the cathelicidin peptide LL-37, and the host defence peptides and proteins NAT26, HKH20, GGL27, α defensins 3-6 and β defensins 1-4 served as controls. The results from 131-iodine assays show binding of the different proteins to titanium surfaces (FIG. 3). Pre-treatment of the titanium surface with PLL significantly enhanced the adherence of collagen VI microfibrils as compared to the controls (FIG. 3). Notably, the coating efficiency of collagen VI was considerably enhanced as compared to the previous gold standard human antimicrobial peptide LL-37.

[0323] The Collagen Type VI Binding Properties are Associated with the Alpha-3 Chain and the Bioactive Peptides Derived from it.

[0324] For a more detailed understanding of the underlying coating mechanism, titanium surfaces were also incubated with different collagen type VI-derived peptides in the presence or absence of PLL and assessed by 131-iodine radioactivity. FIG. 4 depicts the adherence of these molecules. In the presence of PLL superior coating of collagen I scaffolds with the collagen type VI alpha-3 chain, and the bioactive collagen VI peptides GVR28, FYL25, FFL25, VTT30, and SFV33 were observed.

[0325] Taken together, the data presented in FIGS. 3 and 4 show that collagen type VI and/or parts of the collagen VI alpha-3 chain also strongly bind to titanium surfaces in the presence of PLL. This effect of PLL was considerably less pronounced for the other tested proteins. These effects are expected to result in superior properties of titanium biomaterials coated with peptides derived from the collagen VI together with polylysine. This coating strategy will be beneficial for the use of titanium prosthesis, with the potential to promote improved wound repair and improved soft tissue integration of the prosthesis, as well as reduce infection risk due to accelerated wound closure at the host-titanium interface.

Example 3

Improved Bacterial Killing

[0326] Gram-negative and gram-positive bacterial killing efficiency of collagen I scaffolds and titanium surfaces coated with different collagen VI peptides with and without PLL was also investigated.

[0327] Collagen I scaffolds and titanium surfaces were coated with collagen VI microfibrils, each of collagen VI alpha 1-3 chains and the bioactive collagen VI peptides GVR28, FYL25, FFL25, VTT30, and SFV33 as described in Examples 1 and 2 above, respectively.

[0328] Killing efficiency of the gram-negative Pseudomonas aeruginosa bacteria and the gram-positive Staphylococcus aureus were assessed by scanning electron microscopy. For killing efficiency and scanning electron microscopy experiments, titanium scaffold discs with a size of 5 mm and a thickness of 0.25 mm, and collagen I scaffold discs with a size of 5 mm and a thickness of 1 mm (thin scaffold) or 2 mm (thick scaffold), respectively, were punched out and coated with PLL as described in Examples 1 and 2 above. They were subsequently incubated with 500 μL of bacterial suspension (5×10.sup.8 cfu/ml bacteria) and incubated for 2 hours at 37° C. in a humid atmosphere containing 5% CO.sub.2.

[0329] The discs were then prepared for scanning electron microscopy. In short, samples were incubated overnight at 4° C. with fixation buffer (0.15M sodium cacodylate, pH 7.4, containing 2.5% v/v glutaraldehyde) and subsequently washed with cacodylate buffer (0.15M sodium cacodylate, pH 7.4), followed by a standard dehydration series with ethanol-water mixtures. Specimens were then dried in liquid CO.sub.2, using ethanol as an intermediate solvent. Samples were mounted on aluminium discs and coated with 20 nm gold/palladium. Finally, samples were investigated with an XL 30 FEG scanning electron microscope and images were processed by AnalySIS ITEM software. Viable and non-viable bacteria were directly identified and quantified under in the scanning electron microscope due to their structural differences.

[0330] FIG. 5 (titanium surface) and FIG. 9 (collagen I scaffolds) demonstrate that killing of Pseudomonas aeruginosa is considerably enhanced by the collagen VI alpha-3 chain and its derived peptide SFV33 when PLL is used as an intermediate layer, compared to when PLL is not present, and compared to other parts of the collagen VI molecule.

[0331] FIG. 6 (titanium surface) and FIG. 10 (collagen I scaffolds) demonstrate that killing of Staphylococcus aureus is considerably enhanced by the collagen VI alpha-3 chain and its derived peptide SFV33 when PLL is used as an intermediate layer, compared to when PLL is not present, and compared to other parts of the collagen VI molecule.

[0332] Taken together, these results demonstrate the ability of surfaces (both titanium surfaces and collagen I scaffolds) with enhanced collagen VI binding to achieve high levels of bacterial killing, and that this effect is applicable to both gram-positive and gram-negative bacteria. This confirms that the anti-microbial properties of collagen VI translate effectively to situations where collagen VI peptides are bound to surfaces utilising PLL.

Example 4

Improved Skin Cell Adherence During Wound Healing

[0333] Adherence of fibroblasts and keratinocytes to titanium surfaces coated with collagen VI peptides with and without polylysine was also investigated.

[0334] Collagen I scaffolds and titanium surfaces were coated with collagen VI microfibrils, each of collagen VI alpha 1-3 chains and the bioactive collagen VI peptides GVR28, FYL25, FFL25, VTT30, and SFV33 as described in Examples 1 and 2 above.

[0335] Adherence of fibroblasts or keratinocytes was assessed by scanning electron microscopy.

[0336] Cells and culture conditions—Keratinocytes (Human Epidermal Keratinocytes, adult (HEKa), C-005-5C, Gibco) and fibroblasts (Human Dermal Fibroblasts, adult (HDFa), C0135C, Gibco) were purchased from Thermo Fisher Scientific, Waltham, USA. Cryopreserved cells were thawed according to the manufacturer's protocol and seeded on three 75 mL tissue culture flasks, each containing 15 mL of keratinocyte basal medium (KBM; KBM Gold, Lonza Group AG, Basel, Switzerland). The medium was supplemented with transferrin, recombinant human epidermal growth factor (rhEGF), bovine pituitary extract (BPE), antibiotics (Gentamycin: GA-1000), insulin, epinephrine and hydrocortisone (PeproTech, New Jersey, USA). For fibroblasts, complete growth medium consisting of Dulbecco's modified eagle medium (D-MEM) with 100 mM sodium pyruvate (PAA Laboratories, Pasching, Austria), supplemented with 10% fetal bovine serum (FBS) (PAA Laboratories, Pasching, Austria) and 2 mM L-glutamine (Invitrogen, Carlsbad, USA), was used. Medium was changed every day and substituted with additional rhEGF at a final dilution of 1:1000. The cells were incubated for seven days until they reached confluency. The cells were harvested by trypsinization (TrypLE™ Select (1x), Life Technologies Corporation, Carlsbad, USA) and transferred into freezing medium, containing 1% BSA (Sigma Aldrich, St. Louis, USA) and 10% DMSO (Sigma-Aldrich, St. Louis, USA). 1 mL aliquots of the cell suspension were transferred into cryotubes. The cryotubes were transferred to the freezer at −80° C. for 24 hours and then finally stored in liquid nitrogen until further use.

[0337] Cell attachment—Cells were gently thawed and counted in a hemocytometer using trypan blue (Fisher Scientific, Waltham, USA) to estimate the number of live/dead cells. They were finally diluted in appropriate media to final cell concentrations of 16700 viable cells/mL. 300 μL cell solution were added on top of punched out titanium or collagen I scaffold samples, followed by incubation at 37° C. for 1 hour. Growth medium was removed and the cells were gently washed twice with PBS. The cells were then fixed by the addition of 4% formaldehyde in PBS, followed by incubation at 4° C. for 20 minutes. Finally, cells were rinsed three times in PBS and stored in 3 mL PBS until further evaluation by scanning electron microscopy.

[0338] FIG. 7 (titanium surface) and FIG. 11 (collagen I scaffolds) demonstrate that fibroblast adherence (measured in cells/mm.sup.2) for the collagen VI alpha-3 chain and its derived peptides GVR28, FFL25 and VTT30 is considerably enhanced with PLL as an intermediate layer as compared to the other parts of the collagen VI molecule, and compared to when PLL is not used.

[0339] FIG. 8 (titanium surface) and FIG. 12 (collagen I scaffolds) demonstrate that keratinocyte adherence for collagen VI alpha-3 chain and its derived peptides GVR28, FFL25 and VTT30 is considerably enhanced with PLL as an intermediate layer as compared to the other parts of the collagen VI molecule, and compared to when PLL is not used.

[0340] Taken together, these results demonstrate the ability of surfaces (both titanium surfaces and collagen I scaffolds) coated with collagen VI alpha-3 chains and peptides derived from it to adhere to skin cells, for example keratinocytes and fibroblasts, with increased efficiency compared to surfaces bound to other parts of collagen VI, or surfaces bounds to collagen VI in the absence of PLL. This confirms the ability of collagen VI and its derived peptides to retain the ability to recruit skin cells when bound to surfaces in the presence of PLL, which will be beneficial in promoting the process of skin healing.

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